PHARMACOKINETIC STUDIES OF SOME SELECTED

ISOLATED COMPOUNDS OF HERBAL DRUGS

IN VARIOUS DOSAGE FORMS

Thesis submitted to

The Tamil Nadu Dr. M.G.R. Medical University, Chennai,

for the partial fulfillment of the requirements for the Award of Degree of

Doctor of Philosophy
In

Pharmacy
By
Vishnu Prasad, M.Pharm.

Under the guidance of

Dr. Malay K. Samanta., M. Pharm., Ph.D., AIC. 

July– 2011

Department of Pharmaceutics
J.S.S. College of Pharmacy
Ootacamund - 643001
Tamil Nadu, India.
 DECLARATION

I hereby declare that the thesis work entitled “Pharmacokinetic Studies of Some Selected
Isolated Compounds of Herbal Drugs in various Dosage Forms” submitted by me to The
Tamil Nadu Dr. M.G.R. Medical University, Chennai, India, for the partial fulfillment of
requirements for the award of Degree of Doctor of Philosophy in Pharmacy, is the result of my
original and independent work carried out at Department of Pharmaceutics, J.S.S. College of
Pharmacy, Ootacamund, India during the year 2008-2011, under the direct supervision of Dr. Malay
K. Samanta, M. Pharm, Ph.D., AIC. The work has not formed the basis for the award of any degree,
diploma, associate ship, fellowship, or any other similar title previously.

Vishnu Prasad
Research Scholar
Department of Pharmaceutics
J.S.S. College of Pharmacy
Ootacamund – 643001

Place: Ootacamund

Date:
 ACKNOWLEDGEMENTS

I take this opportunity with pride and immense pleasure to express my deep and affectionate
regards to my ever beloved Sir, Dr. Malay K. Samanta, M.Pharam., Ph.D.,AIC., Professor and
Head, Dept. of Pharmaceutical Biotechnology, JSS College of Pharmacy, Ootacamund, whose
exemplary guidance, innovative ideas, inspirations and continuous help was really unforgettable,
invaluable.

It is my pleasure in extending the deepest thanks to Dr. B. Suresh, Vice-chancellor, J.S.S.

University for his invaluable and constant encouragement throughout the progress of the work.

I wish to express my heartfelt gratitude to Dr. K. Elango, Principal (In-charge), J.S.S.

College of Pharmacy, Ooty for his valuable and constant suggestions during the tenure of the work
and support during the pharmacological part of my studies.

I am immensely thankful to Dr. K. Gowthamarajan, Professor and Head, Department of

Pharmaceutics and other faculties of the Department for their co-operation during the work.

It’s my pleasure to express my solely gratitude to Dr. P. Vijayan, Professor, Department of

Biotechnology, Dr. M. N. Satish Kumar, Professor and Head, Dept of Pharmacology for their
untiring support, encouragement and suggestions in completing this work successfully.

My sincere thanks to Dr. S.N. Meyyanathan, Head, Department of Pharmaceutical Analysis,

and Mr. Praveen Kumar, Mrs. Subashini, Mr. Rajesh, Mr. Sushanth Kumar Das and other
lecturers of J.S.S. College of Pharmacy for their continuous co-operation during every aspect of the
work.

I extend my thanks to Dr. M. J. Nanjan, Director of Research and PG studies, for his help
and encouragement.

I appreciate the consistent help of all the lab assistants of the J.S.S. College of Pharmacy,
Ooty, mainly Mr. Shivakumar, Mr. Mahadeva Swamy, Mr. Chinnaswamy, Mr. Pradeep, Mr.
Somesha, and Mr. Basavaraj.
 It’s my profound gratitude to Mrs. P. Mamtha and other college librarians for their active
help and co-operation.

I wish to acknowledge my co-research scholars Mr. Kalpesh, Mrs. Sangeetha, Mr.

Saurabh, Mr. Manjunath, Mr. Anil, Mr. Rizwan and others for their handful help throughout the
work.

My respectful thanks to chairman, Analysis and Technicians, Indian Institute of Sciences,

Bangalore for their immense help for the S.E.M. Studies.

I acknowledge the Management of the Institute with greet for providing the facilities, which
enabled me to do the work of this magnitude.

My feelings go beyond the limit of knowledge to express my salutation to my parents.

For their never ending blessings, selfless contribution, encouragement and constructive inspiration. I
also admire and adore my beloved wife, brothers and sister-in-laws and other relatives for their
constant moral support.

Above all my most revered charna sprasha and Pranams, respectful regards to “His Holiness
Jagadguru Sri Sri Shivarathri Deshikendra Mahaswamigalavaru of Sri Suttur Mutt, Mysore seeking
his blessing for my future life.

Vishnu Prasad

   
 

Sl.No Contents Page No.

1. Introduction 1-47

2. Literature Review 48-64

3. Aim & Objectives 65-66

4. Plan of Work 67-68

5. Botanical Informations 69-82

6. Drugs Profile 83-94

7. Materials and Instruments 95-96

8. Methodology 97-134

9. Results 135-237

10. Discussion 238-245

11. Summary 246-247

12. Conclusion 248

13. References 249-273

14. Annexure 274-277

 
 

CERTIFICATE

This is to certify that this dissertation work entitled “Pharmacokinetic Studies of Some
Selected Isolated Compounds of Herbal Drugs in various Dosage Forms” is a record of
research work, independently carried out by the candidate under my direct supervision. Mr. Vishnu
Prasad, M.Pharm., has conducted the study very sincerely, meticulously and methodically. I am
satisfied with the work of Mr. Vishnu Prasad, which was done at JSS College of Pharmacy,
Ootacamund, India, during the year 2008-2011, being submitted by him for the partial fulfillment of
the requirements for the award of degree of Doctor of Philosophy in Pharmacy, to The Tamil Nadu
Dr. M.G.R. Medical University, Chennai, India. I also certify that this thesis or any part, thereof has
not formed the basis for the award of any other research degree, of this or any other university,
previously.

Dr. Malay K. Samanta, M. Pharm, Ph.D., A.I.C.

Research Supervisor
Professor and Head
Dept. of Pharmaceutical Biotechnology

Place: Ootacamund
Date:
 

   

 
 
 

CERTIFICATE

This is to certify that this dissertation work entitled “Pharmacokinetic Studies of Some
Selected Isolated Compounds of Herbal Drugs in various Dosage Forms” is a record of
research work, independently carried out by the Mr. Vishnu Prasad, M.Pharm., at Department of
Pharmaceutics under direct supervision of Dr. Malay K. Samanta, M.Pharm., Ph.D., AIC.,
Professor and Head, Department of Pharmaceutical Biotechnology, J.S.S. College of Pharmacy,
Ootacamund, India, during the year 2008-2011. The thesis being submitted for the partial fulfillment
of the requirements for the award of degree of Doctor of Philosophy in Pharmacy, to The Tamil
Nadu Dr. M.G.R. Medical University, Chennai, India, has not previously formed the basis for the
award of any Degree, Diploma, Associate ship, Fellowship or any other similar title. I also certify
that the thesis represents the work done by the candidate and has not formed in part or fully, the basis
for the award of any other previous research degree.

Dr. K. Gowthamarajan, M. Pharm, Ph.D..

Professor and Head
Department of Pharmaceutics
Place: Ootacamund

Date :
 

 
 
 

CERTIFICATE

This is to certify that this dissertation work entitled “Pharmacokinetic Studies of Some
Selected Isolated Compounds of Herbal Drugs in various Dosage Forms” is a record of
research work, independently carried out by the Mr. Vishnu Prasad, M.Pharm., at Department of
Pharmaceutics under direct supervision of Dr. Malay K. Samanta, M.Pharm., Ph.D., AIC.,
Professor and Head, Department of Pharmaceutical Biotechnology, J.S.S. College of Pharmacy,
Ootacamund, India, during the year 2008-2011. The thesis being submitted for the partial fulfillment
of the requirements for the award of degree of Doctor of Philosophy in Pharmacy, to The Tamil
Nadu Dr. M.G.R. Medical University, Chennai, India, has not previously formed the basis for the
award of any Degree, Diploma, Associate ship, Fellowship or any other similar title. I also certify
that the thesis represents the work done by the candidate and has not formed in part or fully, the basis
for the award of any other previous research degree.

Dr. K. Elango, M. Pharm, Ph.D.

Principal, I/C

Place: Ootacamund
Date:

 
 Introduction

INTRODUCTION 
Herbal Medicine: Overview

Herbal medicine, also called botanical medicine or phytomedicine, refers to the use
of any plant seeds, berries, roots, leaves, barks or flowers for the medicinal
purposes. From the ancient days the conventional medicines and herbalism is
becoming more mainstream for the treatment of various chronic diseases.

Herbalism is a traditional medicinal or folk medicine practices based on the uses of

plants and plant extracts. Herbalism is also known as botanical medicine, medical
herbalism, herbal medicine, herbology, and phytotherapy. Sometimes the scope of
herbal medicine is extended to include fungi and bee products, as well as minerals,
shells and certain animal parts.

Many plants synthesize substances that are useful to the maintenance of health in
humans and other animals. These include aromatic substances, phenols or their
oxygen-substituted derivatives such as tannins. Many are secondary metabolites, of
which at least 12,000 have been isolated and still research work going for the
remaining. In many cases, these substances (particularly the alkaloids) serve as plant
defense mechanisms against predation by microorganisms, insects, and herbivores.
Many of the herbs and spices used by humans to seasonal foods yielding useful
medicinal compounds.

Herbs are staging a comeback and herbal ‘renaissance’ is happening all over the
globe. The herbal products today symbolize safety in contrast to the synthetics that
are regarded as unsafe to human and environment. Although herbs had been priced
for their medicinal, flavoring and aromatic qualities for centuries, the synthetic
products of the modern age surpassed their importance, for a while. However, the
blind dependence on synthetics is over and people are returning to the naturals with
hope of safety and security.

Traditional systems of medicine continue to be widely practiced on many accounts.

Population rise, inadequate supply of drugs, prohibitive cost of treatments, side
effects of several allopathic drugs and development of resistance to currently used
drugs for infectious diseases have led to increased emphasis on the use of plant
materials as a source of medicines for a wide variety of human ailments. Global
1 

 
 Introduction
estimates indicate that 80% of about 4 billion population cannot afford the products
of the Western Pharmaceutical Industry and have to rely upon the use of traditional
medicines which are mainly derived from plant material. This fact is well
documented in the inventory of medicinal plants, listing over 20,000 species. In
spite of the overwhelming influences and our dependence on modern medicine and
tremendous advances in synthetic drugs, large segments of the world population still
like drugs from plants. In many of the developing countries the use of plant drugs is
increasing because modern life saving drugs are beyond the reach of three quarters
of the third world’s population although many such countries spend 40-50% of their
total wealth on drugs and health care. As a part of the strategy to reduce the financial
burden on developing countries, it is obvious that an increased use of plant drugs
will be followed in the future.

India’s prospective in herbal medicine

Among ancient civilizations, India has been known to be rich repository of

medicinal plants. The forest in India is the principal repository of large number of
medicinal and aromatic plants, which are largely collected as raw materials for
manufacture of drugs and perfumery products. About 8,000 herbal remedies have
been codified in Ayurveda. The Rigveda (5000 BC) has recorded 67 medicinal
plants, Yajurveda 81 species, Atharvaveda (4500-2500 BC) 290 species, Charak
Samhita (700 BC) and Sushrut Samhita (200 BC) had described properties and uses
of 1100 and 1270 species respectively, in compounding of drugs and these are still
used in the classical formulations, in the Ayurvedic system of medicine.
Unfortunately, much of the ancient knowledge and many valuable plants are being
lost at an alarming rate. With the rapid depletion of forests, impairing the
availability of raw drugs, Ayurveda, like other systems of herbal medicines has
reached a very critical phase.

About 50% of the tropical forests, the treasure house of plant and animal diversity
have already been destroyed. In India, forest cover is disappearing at an annual rate
1.5mha/yr. What is left at present is only 8% as against a mandatory 33% of the
geographical area. Many valuable medicinal plants are under the verge of extinction.

2 

 
 Introduction
The Red Data Book of India has 427 entries of endangered species of which 28 are
considered extinct, 124 endangered, 81 vulnerable, 100 rare and 34 insufficiently
known species.

Ayurveda, Siddha, Unani and Folk (tribal) medicines are the major systems of
indigenous medicines. Among these systems, Ayurveda is most developed and
widely practised in India. Ayurveda dating back to 1500-800 BC has been an
integral part of Indian culture. The term comes from the Sanskrit root Au (life) and
Veda (knowledge). As the name implies it is not only the science of treatment of the
ill but covers the whole gamut of happy human life involving the physical,
metaphysical and the spiritual aspects.

Of the 2,50, 000 higher plant species on earth, more than 80,000 are medicinal. India
is one of the world’s 12 biodiversity centres with the presence of over 45000
different plant species. India’s diversity is unmatched due to the presence of 16
different agro-climatic zones, 10 vegetation zones, 25 biotic provinces and 426
biomes (habitats of specific species). Of these, about 15000-20000 plants have good
medicinal value. However, only 7000-7500 species are used for their medicinal
values by traditional communities.

Over three-quarters of the world population relies mainly on plants and plant
extracts for health care. More than 30% of the entire plant species, at one time or
other was used for medicinal purposes. It is estimated that world market for plant
derived drugs may account for about Rs.2,00,000 crores. Presently, Indian
contribution is less than Rs.2000 crores. Indian export of raw drugs has steadily
grown at 26% to Rs.165 crores in 1994-95 from Rs.130 crores in 1991-92. The
annual production of medicinal and aromatic plant’s raw material is worth about
Rs.200 crores. This is likely to touch US $1150 by the year 2000 and US $5 trillion
by 2050.1

WHO Definition of Herbal Medicine2

WHO has defined Herbal Medicine as a "Finished, labeled medicinal products that
contain as active ingredients aerial or underground parts of plants, or other plant
material, or combinations thereof whether in the crude state or as plant preparations.
3 

 
 Introduction
Plant material includes juices, gums, fatty oils, essential oils, and any other
substances of this nature.

Herbal medicines may contain excipients in addition to the active ingredients.

Medicines containing plant material combined with chemically defined active
substances, including chemically defined, isolated constituents of plants is not
considered to be herbal medicines. Exceptionally, in some countries herbal
medicines may also contain, by tradition, natural organic or inorganic active
ingredients which are not of plant origin.

In extreme cases, the therapeutic strategy may be derived solely from the traditional
use of the drug and is then comparable with the other traditional therapeutic
concepts mentioned above. Within the field of phytotherapy it is therefore
appropriate to draw further distinction between rational therapy, which is on a par
with treatment with synthetic chemical medicines, and other forms of therapy that
are derived from the traditional use of medicinal plants and preparations
manufactured from them. Whereas in “rational phytotherapy” the efficacy of the
products employed has been documented by appropriate pharmacological
investigations and clinical studies in patients, there are large numbers of
phytopharmaceuticals whose efficacy has not yet been established in that way3.

How do herbal medicines work? 4

Many conventional medicines originate from a single active ingredient of a plant.

Scientists can isolate this and produce it on a large scale in a laboratory. This is the
opposite of herbal medicines which may contain dozens of different ingredients.
Herbalists believe that all the elements are in balance within a plant and so it's
important to keep them together. The different components are made more or less
powerful depending on the others that are present.

Role of herbal medicine in modern human society

The use of herbs to treat disease is almost universal among non-industrialized

societies. Many of the pharmaceuticals currently available to physicians have a long
4 

 
 Introduction
history of use as herbal remedies, including opium, aspirin, digitalis and quinine. In
comparison, herbal medicines can be grown from seed or gathered from nature for
little or no cost. Herbal medicine is a major component in all traditional medicine
systems, and a common element in Ayurvedic, Homeopathic, Naturopathic,
Traditional Chinese medicine and Native American medicine.

Pharmacologists, microbiologists, botanists, and natural-products chemists are

searching for Phytochemicals and leads that could be developed for treatment of
various diseases. In fact, according to the World Health Organization, approximately
25% of modern drugs used in the United States have been derived from plants.

™ Three quarters of plants that provide active ingredients for prescription drugs
came to the attention of researchers because of their use in traditional
medicine.

™ Among the 120 active compounds currently isolated from higher plants and
widely used in modern medicine today, 80 percent show a positive correlation
between their modern therapeutic use and the traditional use of the plants
from which they are derived.

™ At least 7,000 medical compounds in the modern pharmacopoeia are derived

from plants.

Growing Demand for Herbal Drugs

The World Health Organization (WHO) has estimated the present demand for
medicinal plants is approximately US $14 billion per year. The demand for
medicinal plant-based raw materials is growing at the rate of 15 to 25% annually,
and according to an estimate of WHO, the demand for medicinal plants is likely to
increase more than US $5 trillion in 2050. In India, the medicinal plant-related trade
is estimated to be approximately US $1 billion per year.

Compared with well-defined synthetic drugs, herbal medicines exhibit some marked
differences, namely5.

™ The active principles are frequently unknown;

™ Standardization, stability and quality control are feasible but not easy;

™ The availability and quality of raw materials are frequently problematic;

5 

 
 Introduction
™ Well-controlled double-blind clinical and toxicological studies to prove their
efficacy and safety are rare.

™ Empirical use in folk medicine is a very important characteristic.

™ They have a wide range of therapeutic use and are suitable for chronic
treatments.

™ The occurrence of undesirable side effects seems to be less frequent with herbal
medicines, but well-controlled randomized clinical trials have revealed that they
also exist.

™ The phytomedicine market has grown at an expressive rate worldwide since

1985 (from 5 to 18% a year). Several important factors have contributed to the
growth of this worldwide phototherapeutic market, among which the following
may be mentioned:

™ Preference of consumers for natural therapies

™ Concern regarding undesirable side effects of modern medicines and the belief
that herbal drugs are free from side effects, since millions of people all over the
world have been using herbal medicines for thousands of years.

™ Great interest in alternative medicine:

™ Preference of populations for preventive medicine due to increasing population

age.

™ The belief that herbal medicines might be of effective benefit in the treatment of
certain diseases where conventional therapies and medicines have proven to be
inadequate.

™ Tendency towards self-medication.

™ Improvement in quality, proof of efficacy and safety of herbal medicines.

™ High cost of synthetic medicines.

6 

 
 Introduction
Biological background

Plants synthesize a bewildering variety of Phytochemical but most are derivatives of

a few biochemical motifs.

™ Alkaloids contain a ring with nitrogen. Many alkaloids have dramatic effects
on the central nervous system. Caffeine is an alkaloid that provides a mild
lift but the alkaloid in Datura cause severe intoxication and even health.

™ Phenolics contain phenol rings. The anthocyanins that give grapes their
purple color, the isoflavones, the phytoestrogens from soy and the tannins
that give tea its astringency are phenolics.

™ Terpenoids are built up from terpene building blocks. Each terpene consists
of two paired isoprenes. The names monoterpenes, sesquiterpenes,
diterpenes and triterpenes are based on the number of isoprene units. The
fragrance of rose and lavender is due to monoterpenes. The carotenoids
produce the red, yellow and orange colour of pumpkin, corn and tomatoes.

™ Glycoside consists of a glucose moiety attached to an aglycone. The

aglycone is a molecule that is bioactive in its free form but inert until the
glycoside bond is broken by water or enzymes.

The word drug itself comes from the Dutch word “drug” (via the French word
Drogue), which means ‘dried plant’.

Popularity of herbal medicine

The following are the some of the reasons for finding popularity of the herbal
medicines over synthetic drugs:

™ Rising cost of medical care.

™ As herbal medicines are from natural origin, they are believed to be free
from side effects.
™ Freedom from approaching various specialists.
™ Cure for many obstinate diseases.
™ Easy availability of drugs from natural sources.
™ Cure of diseases used by life style changes and social pathology.

7 

 
 Introduction
Advantages of plant based drugs6
™ Long history of use and better patient tolerance as well as public acceptance.
™ Renewable source.
™ Cultivation and processing environmental friendly.
™ Local availability, especially in developing countries.
™ Several important recent breakthroughs.
™ Plants constitute to be a major source of new lead generation.

Routes of administration
The exact composition of herbal product is influenced by the method of extraction.
A tisane will be rich in polar components because water is a polar solvent. Oil on the
other hand is a non-polar solvent and it will absorb non-polar compounds. Alcohol
lies somewhere in between. There are many forms in which herbs can be
administered, these include:
™ Tinctures (alcoholic extracts of herbs such as Echinacea extract.) usually
obtained by combining 100% pure ethanol (or a mixture of 100% ethanol with
water) with the herb. A completed tincture has an ethanol percentage of at least
40-60% (sometimes up to 90%).
™ Herbal wine and elixirs; these are alcoholic extract of herbs; usually with an
ethanol percentage of 12-38%. Herbal wine is a maceration of herbs in wine,
while an elixir is a maceration of herbs in spirits (e.g. vodka, grappa, …).
™ Tisanes (hot-water extracts of herb, such as chamomile).
™ Decoctions (long-term boiled extract of usually roots or bark).
™ Macerates (cold infusion of plants with high mucilage-content as sage, thyme)
Plants are chopped and added to cold water. They are then left to stand for 7 to
12 hours (depending on herb used). For most macerates 10 hours is used.
™ Vinegars (prepared as the same way as tinctures).
™ Topical:
™ Essential oils- applications of essential oil extracts, usually diluted in carrier
oil (many essential oils can burn the skin or are simply too high dose used
straight- diluting in olive oil or food grade oil can allow these to be used safely
as a topical).

8 

 
 Introduction
™ Salves, oils, balms, creams and lotions- Most topical applications are oil
extractions of herbs. Taking a food grade oil and soaking herbs in it for
anywhere from weeks to months allows certain phytochemicals to be extracted
into the oil. This oil can then be made into salves, creams, lotions, or simply
used as oil for topical application. Many massage oils, antibacterial salves and
wound healing compounds are made this way.
™ Poultices and compresses- One can also make a poultice or compress using
whole herb (or the appropriate part of the plant) usually crushed or dried and
re-hydrated with a small amount of water and then applied directly in a
bandage, cloth or just as is.
™ Whole herb consumption. This can occur in either dried form (herbal powder)
or fresh juice (fresh leaves and other plant parts.) Just as Hippocrates said “Let
food be thy medicine”, it has become clear that eating vegetables also easily
fits within this category of getting health through consumables (besides
medicinal herbs). All of the vitamins, minerals and antioxidants are
phytochemicals that we are accessing through our diet. There are clearly some
whole herbs consumed that are more powerful than others.
™ Syrups: extracts of herbs made with syrup or honey. Sixty five parts of sugar
are mixed with 35 parts of water and herb. The whole is then boiled and
macerated for three weeks.
™ Extracts: include liquid extracts, dry extracts and nebulisates. Liquid extracts
are liquids with a lower ethanol percentage than tinctures. They can (and are
usually) made by vacuum distilling tinctures. Dry extracts are extracts of
plants material which are evaporated into a dry mass. They can then be further
refined to a capsule or tablet. A nebulisate is a dry extract created by freeze
drying.
™ Inhalation as in aromatherapy can be used as a mood changing treatment, to
fight a sinus infection or cough, or to cleanse the skin on a deeper level (steam
rather than direct inhalation here).

9 

 
 Introduction
Criteria for the development of natural products6
Selection of herbs
Selection can be easily done based on recognised natural system of medicine by
referring to a well recognised validated and well accepted authentic books on natural
system of medicine where the safety and efficacy are time process. The most
important criteria for the selection of plant material are:
™ Evidence from indigenous material medica.
™ Evidence on the basis of data on experimental animals.
™ Chemotaxonomic or biogenetic theory evidence of presence novel
compounds.
In some cases these may vary from what guidelines have recommended. This is
because of the complexities involved in the problem of drug research. Secondly the
activity ascribed for a particular drug in the indigenous system may range many
more. Thirdly situations have been encountered in the evaluation of the crude
extracts. Lastly in some cases, the natural products may undergo changes after the
death of plant because of the enzymatic alteration or inactivation or during the work
up by chemists. The following factors would make it desirable that:
™ The protocol adopted in the selection of plants and their subsequent work up
as shown above has to be flexible.
™ An in-depth study of the indigenous system with particular reference to
original literature is necessary in many a time during translation.

Collection and identification of selected herbs

The following steps are involved in the effective collection and identification of
plants and documentation for further references.
Criteria for selection of species
™ Collection and processing which includes collection, documentation of field
data, chopping, drying, garbling, packing and transportation, voucher
samples, grinding and storage.
™ Identification and documentation.
™ Recollection of biologically active plants.

10 

 
 Introduction
Fractionation of the extracts
After preparation of the initial extract by the chemist, it should be subjected to
fractionation after demonstrating its activity. Fractionation can be done by following
steps.
™ Fractionation of the initial extract with a range of solvents with increasing
polarity, i.e. petroleum ether, benzene, chloroform and n-butanol.
™ Fractionation of the initial extract into acidic, neutral and basic components.
™ The initial extract could be crudely fractioned according to the molecular
weight of its components using a molecular sieve, for example sephadex.

Purification of the fractions

Purification of the fractions can be done by any one of the following
chromatographic methods:
™ Thin layer chromatography.
™ Column chromatography.
™ GLC and HPLC.
™ Ion exchange chromatography.

Role of Phytochemist after purification

™ Should select the potentially active plant.
™ Should reproduce the data available in indigenous material medica.
™ Should get the crude extract tested.
™ To get the activity into an organic solvent.
™ To separate the active compounds by one of the chromatographic technique
and link the activity with particular compound.
™ Purification of the active principle.
™ Elemental analysis, molecular weight and the physical constants of the pure
compound.
™ Elucidate structure by spectroscopic studies and chemical degradation
experiments.
™ Speculate on possible new compounds on the basis of biogenetic theory and
refine methods for isolation of these new compounds.
11 

 
 Introduction
™ Based on these novel structures, try to synthesize more active or less toxic
analogues by known chemical procedures.

Standardization of herbal drugs

The following are the parameters to be considered in standardization of herbal drugs
as well as its finished products.
™ Morphological evaluation
™ Microscopic evaluation.
™ Physical evaluations which include moisture content, viscosity, melting
point, solubility, optical rotation, refractive index, ash values, extractives,
volatile oil content, foreign organic matter.
™ Chemical evaluations which include identification of raw materials, finger
printing of materials, isolation of active constituents, detection of adulterants,
monitoring of aflotoxins, quantification of active constituents.

Introduction to pharmacokinetics7, 8, 9, 10, 11

Pharmacokinetic overview
Pharmacokinetics (in Greek: “pharmakon” meaning drug and “kinetikos” meaning
to do with motion, the study of the time dependency; sometimes abbreviated as
“PK”) is a branch of pharmacology dedicated to the determination of the fate of
substances administered externally to a living organism. In practice, this discipline is
applied mainly to drug substances, though in principle it concerns itself with all
manner of compounds ingested or otherwise delivered externally to an organism,
such as nutrients, metabolites, hormones, toxins, etc.

Pharmacokinetics is divided into several areas which include the extent and rate of
Absorption, Distribution, Metabolism and Excretion. This is commonly referred to
as the ADME scheme. However recent understanding about the drug-body
interactions brought about the inclusion of new term Liberation. Now
Pharmacokinetics can be better described as LADME.

   

12 

 
 Introduction
Liberation is the process of release of drug from the formulation.

™ Absorption is the process of a substance entering the body.

™ Distribution is the dispersion or dissemination of substances throughout the
fluids and tissues of the body.
™ Metabolism is the irreversible transformation of parent compounds into
daughter metabolites.
™ Excretion is the elimination of the substances from the body. In rare cases,
some drugs irreversibly accumulate in a tissue in the body.
Pharmacokinetics describes how the body affects a specific drug after
administration. Pharmacokinetic properties of drugs may be affected by elements
such as the site of administration and the concentration in which the drug is
administered. These may affect the absorption rate.

Pharmacokinetics in herbal medicine

One important issue which should underline much of the study of herbal
pharmacokinetics is that they are not usually directly introduced into blood stream,
oral or topical route of administration are preferred. This renders the study of
bioavailability of paramount importance or active constituents in plants.
Bioavailability can define as the degree of absorption of active substances into blood
stream after oral doses. Hence, bioavailability is also a factor of preparation, which
is used to deliver the dose of active substances. Conventional drugs intended for oral
dose are designed to have good bioavailability. In contrast, phytochemicals are of
natural origin and may exhibit unusual or poor bioavailability, which may be further
compounded to be the choice of dosage preparation. In the modern drug
development concept it is accepted that medicinal plants act at a chemical level in
the body so the knowledge of herbal pharmacokinetics is vital.

The bioavailability of a molecule depends on several factor which determine how

the molecule transverses the barrier of the gastrointestinal tract and survives into
blood stream. Particularly for evaluating the herbal pharmacokinetics the following
parameters need to be considered:

13 

 
 Introduction
™ Information to further assess the traditional and anecdotal uses of medicinal
plants and better information on which to base rational dosage.

™ A better interpretation of scientific information, particularly in vitro research or

in vivo studies where the active compounds are administered by injection. There
is an abundance of misinformation in the herbal literature related to excessive
extrapolation room of such studies, with no consideration of bioavailability.

™ A better appreciation of the safety and toxicity of plants and anticipation of

potential herb-drug interaction.

™ Supporting evidence for the synthetic nature of herbal medicine.

™ Ways to optimize the bioavailability and hence efficacy of herbal medicines.

™ The size of molecules: - large molecules still have some bioavailability (about
1% or less) which may due to pinocytosis.

™ The fat (lipid) solubility of the molecule: the more fat soluble, the better
bioavailability.

™ The water solubility of molecule makes it dissolve in the digestive juices and
then cross lipid membrane; i.e. purely water soluble molecules can be expected
to have poor bioavailability. Ionization of molecule usually means poor
bioavailability.

™ Specific factors related to crossing the gut wall, e.g. active transport.

™ Factor within the gut: interaction with food, stability in the gut, gastric emptying
etc.

™ Metabolism in the gut and first-pass metabolism in the liver.

™ Individual factors in the patients, including the influence of pathological factors.

™ The presence or absence of food may also influence the absorption and
bioavailability of plant constituents.

The study of herbal pharmacokinetics is a unique field, which is extraordinarily

complex for the reasons mentions below.

™ The chemical complexity of plant medicines and the potential interactions

between the constituents.
14 

 
 Introduction
™ The difference in bioavailability among the molecules of the compound.13

™ The active components are often not known, so the components in the plant
which should be studied can not be identified.

™ Herbal medicines are not designed for predictable pharmacokinetic

characteristics and in particular, natural compounds are often metabolized in the
digestive tract, i.e. they act like pro-drugs.

™ Often large polar molecules are involved, which might be expected to have poor
and unpredictable bioavailability.

Pharmacokinetics of herbal medicines is necessary to promote efficacy and safety of

herbal remedies and avoid adverse effects. Pharmacokinetic study on herbal
medicines is a challenge due to their complex composition, unknown active
constituents and low plasma concentrations of metabolites. Animal tests normally
are cheap and easy to perform, but the parameters derived from animal sometimes
can’t apply to human. Thus clinical pharmacokinetic parameters of herbal medicines
become an important evidence for rational herbal remedies. With increasing
knowledge of active compounds and availability of high sensitive and selective
analytical methods, data on clinical pharmacokinetics and clinical pharmacokinetics
and clinical drug interactions of certain herbs have been published.14

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 Introduction
Process of Absorption, Distribution Metabolism & Elimination
(Excretion) in the body

Figure - 1

Importance of Pharmacokinetics10

Pharmacokinetics data is important understanding interactions between herbs and

pharmaceuticals. Pharmacokinetic information about herbal remedies is not widely
available due to several factors including lack of studies, and inadequate reporting.
Herbalists have long studied the effect of herbal medicines on the body, but have
hitherto paid less attention to the effects of the body on herbal medicines. This
dichotomy expresses the distinction in classical pharmacology between
pharmacodynamics and pharmacokinetics. For a given dose of any herbal medicine,
its physiological (or that of its constituents) will be governed by the effective tissue
concentration of the remedy which in turn is determined by pharmacokinetic
parameters the absorption, distribution, metabolism and excretion of its various
components. Knowledge of herbal pharmacokinetics can provide valuable
information to aid practitioners in prescribing herbs safely and effectively. It may
also enable useful predictions to be made, for example regarding possible
interactions between herbal remedies and conventional pharmaceuticals. Drug-drug
interactions constitute the bulk of the conventional pharmacokinetic literature, but
currently herb-drug interactions are taking center stage, both in popular media and in

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 Introduction
terms of increasing physician awareness of the widespread and often undisclosed
use of herbal medicines by their patients, and the potential for significant
pharmacokinetic interaction between herbs and prescription pharmaceuticals. Useful
data about actual and potential interactions between pharmaceutical drugs comes
from various sources, including clinical trials, preclinical trials investigating adverse
effects, post licensing drug monitoring, controlled trials on healthy subjects required
for drug licensing, in vitro or in vivo studies on animals or human cell lines or
tissues, and Adverse Drug Reports (ADR’s). The situation for herb-drug interactions
data is very different, largely due to the different regulatory frameworks that govern
pharmaceuticals and herbs. In a recent review of the literature on herbal
pharmacokinetics, it was found very few human studies in the field; the available
studies covered only a handful of herbs, and most involved consumption by normal
or healthy volunteers. Given the chemical complexities of herbal remedies the use of
multiple herbs in herbal prescriptions and the many different parameters impacting
pharmacokinetics (especially individual factors such as age, genetic variation,
dietary habits etc.) together with the lack of commercial imperatives for conducting
such research, this lack of data is likely to continue for some time.

Parameters for assessment of Bioavailability9, 11

The important parameters to be considered in the evaluation of the blood level

curves following the oral administration of single dose are

™ The peak height concentration (cmax).

™ The time of peak concentration (tmax).

™ The area under the blood (or serum or plasma) concentration time curve (AUC).

™ Apparent volume of distribution (Vd).

™ Elimination half life (t1/2) and Elimination rate constant (keli).

™ Lag time (t0).

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 Introduction
Peak height

Peak height concentration is the cmax observed in the blood plasma or serum
following a dose of the drug, indicating a slope of zero, meaning the rates of
absorption and elimination are equal. For conventional dosage forms, such as tablets
or capsules, the cmax will usually occur at a single time, tmax. The amount of drug
usually expressed in terms of its concentration in relation to a specific volume of
blood, serum, or plasma.

Time of peak

This parameter reflects the rate of absorption from a formulation, which determines
the time needed for MEC (Minimum Effective Concentration) to be reached and
thus for the initiation of the desired effect. The rate of absorption also influences the
period over which the drug enters the blood stream and therefore affects the duration
that the drug is maintained in the blood.

Area under the curve

The AUC of a concentration-time plot is considered representative of the total

amount of drug absorbed into the circulation following the administration of single
dose of that drug. Equivalent doses of a drug when fully absorbed produce the same
AUC.

Apparent volume of distribution

The apparent volume of distribution for a drug is not a real volume of drug but
rather a hypothetical volume of fluid that would be rather required to dissolve the
total amount of drug at the same concentration as that found in the blood.

Elimination half life and Elimination rate constant

The elimination half life is the time the drug takes for the plasma drug concentration
(as well as the amount of the drug in the body) to fall by one half. The elimination
rate constant is the fractional rate of drug removal per unit time.

Lag time

Lag time is the time delay prior to the commencement of first-order drug absorption.

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 Introduction
Factors influencing bioavailability of oral drugs

™ Drug substance physiochemical properties:

Particle size, crystalline or amorphous form, salt form, hydration, lipid or water
solubility, pH and pKa.

™ Pharmaceutical ingredients:

Fillers, binders, coatings, disintegrating agents, lubricants, suspending agents,

surface active agents, flavoring agents, coloring agents, preservative agents,
stabilizing agents.

™ Dosage form characteristics:

Disintegration rates, dissolution time of drug in dosage form, product age and
storage conditions.

™ Physiologic factors and patient characteristics:

Gastric emptying time, intestinal transit time, gastrointestinal abnormality or

pathologic condition, gastric contents, other drugs, food, fluids, gastrointestinal
pH, drug metabolism.

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 Introduction
Oral Bioavailability and First-Pass Effects

Figure - 2

Absorption sites x; the hepatic portal vein p; the hepatic vein h; peripheral veins i,
j,k, l; peripheral arteries a; the mesenteric artery m; and arterial supply n to venous
effluent at j.

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 Introduction
TABLET AS A DOSAGE FORM
Tablets may be defined as solid dosage form containing drug substances with
or without suitable diluents and prepared either by compression or by moulding
methods. The British Pharmacopoeia defines tablets as being circular in shape with
either flat or convex faces and prepares by compression the medicament or mixture
of medicaments usually with added substances.

Tablets are now the most popular dosage forms accounting for 70% of all
pharmaceutical preparations produced (Michael, 1996).

They have been in wide spread use since the later part of the 19th century and
their popularity still continues. The term “compressed tablets” is believed to have
been used first by John Wiely and brother of Philadelphia. Tablets remain popular as
dosage forms because of the advantages afforded both to the manufacturers
(Simplicity and economy of preparation, Stability and convenience in packing,
shipping and dispensing) and the patient (e.g. Accuracy of dosage, compactness,
portability, blandness of taste and ease of administration).

The oral route of administration is the most important method of

administering drugs for systemic effects. Nevertheless, it is probable that at least
90% of all drugs used to produce systemic effects are administered by the oral route.
When a new drug is discovered one of the first questions, a pharmaceutical company
asked is whether or not the drug can be effectively administered for its intended
effect by the oral route. If a patient self-administration cannot be achieved the sales
of drug constitutes only a small fraction of what the market would be otherwise.

Although the basic mechanical approach for their manufacture has remains
the same, tablets technology has undergone great improvements. Compression
equipment continues to improve both the produced speed and the uniformity of
tablets compressed (Alfanso, 1990). Although it is possible to manufacture tablets in
a wide range of shapes, official tablets are defined as circular disc with either flats or
convex faces (Rawlins, 1984). Of the drugs that are administered orally, solid
dosage forms represent the preferred class of products.

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 Introduction
Distribution of drug after oral administration

Figure - 3

Following are the primary potential advantages of tablets

™ They are unit dosage forms and they offer the greatest capabilities of all
oral dosage form for the greatest precision and the least content
variability.
™ Their cost is the lowest of all dosage forms.
™ They are general the easiest and cheapest to package and ship of all oral
dosage forms.
™ They are lights and most compact of all oral dosage forms.
™ Product identification is potentially the simplest and cheapest.
™ They are better suited to large scale production than other unit oral
forms.
™ They have the best combined properties of chemical, mechanical and
microbiological stability of all oral forms.

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 Introduction
The disadvantage of tablets include the followings

™ Some drugs resist compression into dose compact owing to their amorphous
nature or fluculent low density characters.
™ Drugs with poor wetting, Slow dissolution properties, intermediate to large
dosage, optimum absorption or any combination of these features may be
difficult or impossible to formulate and manufacture as a tablet.
™ Bitter testing drugs, drugs with an objectionable order or drugs that are
sensitive to oxygen or atmoshfearic moisture may require coating. In such
cases, the capsule may offer the best and lowest cost approach.

In summery of the foregoing advantages and disadvantages of tablets in

comparison to other oral dosage forms, tablets to provide advantages to the
pharmacist, to the patients and to the physicians.

Different Types of Tablets

Tablet may be uncoated or coated. Uncoated tablets are chewable tablet,
effervescent, tablet, lozenge tablet, soluble tablet, and sublingual tablet. Coated
tablets are enteric coated tablet, film coated tablet, implant, sugar coated tablet, and
modified-release tablet. A broken section of a coated tablet shows a core which is
surrounded by a continuous layer of a different texture. The reasons for coating a
tablet are:

™ To protection of the active ingredients from air, moisture and light.

™ To mask the unpleasant tastes and odor.
™ To improve appearance.

According to drug release rate from the tablet (USP classification)

(a) Immediate-release tablet

The tablet is intended to be released rapidly after administration, or the tablet is

dissolved and administered as solution. It is the most common type and includes:

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 Introduction
Disintegrating tablet (conventional or plain tablet)

Disintegrating tablet is the most common type of tablets that is intended to be

swallowed and to release the drug in a relatively short time thereafter, by
disintegration and dissolution (fast and complete drug release in vivo). It includes
normally the following type of excipients; filler (with low dose drug), disintegrant,
binder, glidant, lubricant and antiadherent.

Tablet disintegration may be affected by

™ Choice of the excipients.

™ Production conditions during manufacture.
Conventional tablet may be single layer or multilayer.

Multilayer tablets are prepared by repeated compression of powders and are made
primarily to separate incompatible drugs from each other.

Chewable tablet

The tablet which is intended to be broken and chewed in between the teeth before
ingestion. Antacid and vitamin tablets are usually prepared as chewable tablets. It is
given to the children who have difficulty in swallowing and to the adults who dislike
swallowing.

Effervescent tablet

The tablet that contains acid substances and carbonate or hydrogen carbonate that
react rapidly in the presence of water to release carbon dioxide. Sodium bicarbonate,
citric acid and tartaric acid are added to the active ingredients to make the tablet
effervescent. This preparation makes the tablet palatable.

Lozenge tablet

The tablet that is intended to produce continuous effect on the mucous membrane of
the throat. There is no disintegrating agent. The quality of the binding agent is
increased so as to produce slow dissolution. Suitable sweetening (sugar), coloring
and flavoring agents must be include in this formulation. Gum is used to give
24 

 
 Introduction
strength and cohesiveness to the lozenge and facilitating slow release of the active
ingredient.

Soluble tablet

The tablet that dissolves completely in liquid to produce solution of definite

concentration. Mouth wash, gargle, skin lotion, douche; antibiotic, certain vitamins,
and aspirin are given in this formulation.

Sublingual tablet

The drug which is destroyed or inactivated within the gastrointestinal tract but can
be absorbed through the mucosal tissue of the oral cavity is usually given in this
formulation. The tablet is required to be placed below the tongue for the slow
release of drug. But for immediate effect some medicaments are formulated in such
a way to dissolve within 1 to 2 minutes. Nitroglycerin is prepared in this
formulation.

Enteric coated tablet

Some drugs are destroyed by gastric juice or causes irritation to the stomach. These
two factors can be overcome by coating the tablet with cellulose acetate phthalate.
This polymer is insoluble in gastric contents but readily dissolves in intestinal
contents. So there is delay in the disintegration of dosage form until it reaches the
small intestine. Like coated tablet, enteric coated tablet should be administered in
whole form Broken or crushed form of the enteric coated tablet causes destruction of
the drug by gastric juice or irritation to the stomach. Enteric coated tablet is
comparatively expensive.

Film coated tablet

The tablet that is covered with a thin layer or film of polymeric substance which
protects the drug from atmospheric conditions and mask the objectionable taste and
the odor of drug.

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 Introduction
Implant

A small tablet that is prepared for insertion under the skin by giving a small surgical
cut into the skin which is stitched after the insertion of the tablet. This tablet must be
sterile one. The drug used in this preparation is usually water insoluble and the tablet
provides a slow and continuous release of drug over prolonged period of time
ranging from 3 to 6 months or even more Contraceptive tablet is formulated as
implant.

Sugar coated tablet

The tablet that contains active ingredient(s) of unpleasant taste may be covered with
sugar to make it more palatable. This type of tablet should be administered in whole
form, otherwise the patient will experience the unpleasant taste of the active
ingredient.

(b) Modified-release tablet

Modified-release means that the escape of the drug from the tablet has been
modified in some way. Usually this is to slow the release of the drug so that the
medicine doesn't have to be taken too often and therefore improves compliance. The
other benefit from modifying release is that the drug release is controlled and there
are smaller peaks and troughs in blood levels therefore reducing the chance of peak
effects and increasing the likelihood of therapeutic effectiveness for longer periods
of time.

They have release features based on; time, course or location. They must be
swallowed intact.

Modified-released tablet is either uncoated or coated. This contains special additives

or prepared by special procedure which, separately or together, is intended to modify
the rate of release of the drug into the gastrointestinal tract. It prolongs the effect of
drug and also reduces the frequency of administration of drug. Several drugs are
available in modified release tablet such as indomethacin.

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 Introduction
There is another term popularly known as pill. Once the people’s idea was to use of
pill in every ill. Now days the term has been only used in contraceptive preparations
such as combination pill, minipill, and morning after pill.

Manufacture of the tabletting blend

In the tablet pressing process, the main guideline is to ensure that the appropriate
amount of active ingredient is in each tablet. Hence, all the ingredients should be
well-mixed. Powders that can be mixed well do not require granulation and can be
compressed into tablets through direct compression.

Direct Compression

In a dry direct compression method, the ingredients are simply dry-mixed and then
compressed. There is no granulation stage. It is essential that each component is
uniformly dispersed within the mixture. Any tendency for component segregation
must be minimized to assure that each tablet contains an accurate reproducible
dosage. In addition, the mixture must have certain flow characteristics to allow
accurate and convenient transport and must be cohesive when compressed. To
reduce segregation tendencies the particle size distribution, shape, and density of all
the ingredients should be similar. There are only a few substances available in forms
which could be compressed directly without further treatment. If these ingredient
characteristics are not present, then one of the granulation methods should probably
be used.

Wet granulation

Wet granulation is a process of using a liquid binder to lightly agglomerate the

powder mixture. The amount of liquid has to be properly controlled, as over-wetting
will cause the granules to be too hard and under-wetting will cause them to be too
soft and friable. Aqueous solutions have the advantage of being safer to deal with
than solvent-based systems.

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 Introduction

Dry granulation

Dry granulation processes create granules by light compaction of the powder blend
under low pressures. The compacts so-formed are broken up gently to produce
granules (agglomerates). This process is often used when the product to be
granulated is sensitive to moisture and heat. Dry granulation can be conducted on a
tablet press using slugging tooling or on a roll press called a roller compactor. Dry
granulation equipment offers a wide range of pressures to attain proper densification
and granule formation. Dry granulation is simpler than wet granulation, therefore the
cost is reduced. However, dry granulation often produces a higher percentage of fine
granules, which can compromise the quality or create yield problems for the tablet.
Dry granulation requires drugs or excipients with cohesive properties, and a 'dry
binder' may need to be added to the formulation to facilitate the formation of
granules.

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 Introduction
Process involved in the dissolution of a tablet before absorption

Figure - 4

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 Introduction

The Concept of Sustained Release

Probably the earliest work in the area of sustained drug delivery dosage
forms can be traced to the 1938 patent of Israel Lipowski. Ideally, a drug should
arrive rapidly at the site of action (receptor) in the optimum concentration, remain
for the desired time, be excluded from other sites, and be rapidly removed from the
site when indicated ie.e. the basic goal of the therapy is to achieve a steady state
blood level that is therapeutically effective and non-toxic for an extended period of
time. Generally, the time course of a dosage form (pharmacokinetics) in man is
considered to be controlled by the chemical structure of the drug. Decreasing the
rate of absorption and/or changing the dosage form provide a useful adjunct. When
it is not feasible or desirable to modify the drug compound on a molecular level,
often sought is a product that will require less frequent administration to obtain the
required biologic activity time profile; for example, a tablet that has the same
clinical effect when administered every twelve hours. In another instance, it may be
desirable to decrease the absorption rate in order to obtain a more acceptable clinical
response. The goal in designing sustained or controlled delivery systems is to reduce
frequency of dosing or to increase the effectiveness of the drug by localization at the
site of action, reducing the dose required, or providing uniform drug delivery.
Oral ingestion has long been the most convenient and commonly employed
route of drug delivery. Indeed, for sustained release systems, oral route of
administration has received most of the attention with respect to research on
physiological and drug constraints as well as design and testing of products. This is
because of the fact that there is more feasibility in dosage form design for oral route
than for parenteral or any other route. The design of oral sustained release delivery
systems is subject to several intercalated variables of considerable importance.
Among these are the type of delivery systems, the disease being treated, the patient,
and the length of therapy and the properties of the drug.

Potential Advantages of Sustained Drug Therapy

1. Patient compliance due to reduction in frequency of dosing.
2. Employ minimum drug.

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 Introduction
a. Minimize or eliminates local and systemic side effects.
b. Obtain less potentiation or reduction in drug activity with chronic Use.
c. Minimize drug accumulation with chronic dosing.
3. Improve efficacy in treatment.
a. Cure or control condition more promptly.
b. Improve control of condition i.e. reduce fluctuation in drug level.
c. Improve bioavailability of some drugs.
d. Make use of special effects, e.g. sustained release aspect for morning
relief of arthritis by dosing before bedtime.
4. Economy.

Sustained Release Matrix Tablets

One of the least complicated approaches to the manufacture of sustained,
release dosage forms is the direct compression of blends of drug, release retardant,
and additives to form a tablet in which drug is embedded in a matrix core of
retardant. Alternatively drug retardant blend may be granulated prior to
compression. Such tablets are called as matrix tablets. Three classes of release
retarding materials are used for the formulation of matrix tablets.

They include
1. Insoluble or ‘skeleton’ matrices.
2. Water insoluble, erodible matrices.
3. Hydrophilic matrices.
Tablets prepared from these materials are egested intact and not break apart
in the GI tract. The rate-limiting step in controlling the release of drug from these
formulations is liquid penetration into the matrix unless channeling agents are
included in the formulation to promote permeation of water into the matrix. This
allows drug dissolution and diffusion from the channels created in the matrix. In
these tablets; drug bioavailability is dependent on drug polymer-ratio. These forms
of matrix tablets are not useful if dose of drug is high or if the drug is insoluble in
water.

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 Introduction
Materials used as retardants in matrix tablets
Matrix Release Retarding Materials
Characteristics
Insoluble, inert Polyethylene
Polyvinyl Chloride
Methylacrylate-mthacrylate copolymer
Ethyl Cellulose
Insoluble, Carnauba wax
Erodible Stearyl alcohol
Stearic acid
Polyethylene glycol
Polyethylene glycol monostearate
Triglycerides
Hydrophillic Methyl cellulose
Hydroxyethy1 cellulose
Hydroxypropylmethy1 cellulose
Sodium carboxymethy1 cellulose
Carboxypolymethylene
Sodium alginate
Galactomannose
Table - 1

Drug interactions in the body

Figure - 5

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 Introduction
PELLETS (SPHEROIDS) AS A DOSAGE FORM

Pelletinization (Spheronization) is an agglomeration process that converts

fine powders or granules of bulk drug and excipients in small to free flowing
spherical or semispherical units referred to as pellets. These usually range in size
from 0.5-1.5 mm. Pellets (Spheroids) as a drug delivery system offer not only
therapeutic advantages such as less irritation of the gastro-intestinal tract and a
lowered risk of side effects due to dose dumping but also technological advantages
like better flow property, less friable dosage for narrow particle size distribution,
ease of coating and uniform packing. The reproducibility of the drug blood levels is
an additional advantage to the use of a pellet (Spheroids) formulation.

Advantages

™ Pellets (Spheroids) disperse freely in the stomach of GIT so they

invariably maximize drug absorption, reduce peak plasma
fluctuation, and minimize potential side effects without appreciably
lowering drug bioavailability.
™ Pellets (Spheroids) also reduce variation in gastric emptying rate and
overall transit time.
™ Drug is a cause upset of stomach so with the pellets (Spheroids), due
to large surface area fastest absorption is possible.
™ Pellets (Spheroids) reduced peak plasma fluctuation thus, intra and
inter-subject variability of plasma profile, which are common with
single unit regimens, are minimized.
™ Another advantage of pellets (Spheroids) over single unit dosage
forms is that high local concentration of bioactive agents.
™ When formulated as modified-release dosage forms, pellets
(Spheroids) are less susceptible to dose dumping then the reservoir-
type, single unit formulations.

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 Introduction
Methods of formulation of pellets (Spheroids)

1. Extrusion and spheronization: The most popular method of producing

pellets (Spheroids) is by the extrusion Spheronization technique. This
process was first reported by Reynolds (1970) and by Conine and Hadley
(1970).

It involves four steps

1. Granulation: preparation of the wet mass.

2. Extrusion: shaping the wet mass into cylinders.

3. Spheronisation: breaking up the extrudate and rounding of the particles into

spheres.

4. Drying of the pellets (Spheroids).

The preparation of spherical granules and pellets by extrusion and

spheronization is now more established method because of its advantages over other
methods.

Advantages of the extrusion and spheronization process

™ Easy of operation.
™ High throughput with low wastage.
™ Narrow particle size distribution.
™ Production of pellets (Spheroids) with low friability.
™ Production of pellets (Spheroids) that is suited for film coating.
™ More sustained as well as conventional release.

2. Spray drying process

In a spray-drying process, aqueous solution of core materials and hot

solution of polymer is atomized into hot air, the water then evaporates and the drug
solid is separated in the form of pellets (Spheroids), usually by air suspension.

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 Introduction

3. Spry congealing

In a spray congealing process, the solution or suspension of binder and the

drug is sprayed on to an inert core. The process also involves spraying of melted fats
and waxes from the top into a cold tower (spray-congealing) to form pellets
(Spheroids) due to the hardening of the molten droplets.

4. Fluidized bed technology

It is the most recent method for the production of pharmaceutical pellets by

means of the fluid-bed roto granulator or by the centrifugal granulator performing
the whole cycle in one closed system. The binder solution and the powder mix are
added at a fixed rate onto the plate of a kind of spheroniser so that, the particles are
stuck together and spheronised at the same time. The rotating plate is mounted in a
fluidized bed making it possible to dry the pellets (Spheroids) when they reach the
appropriate size because of the air blown through the small opening between the
rotating plate and the vertical wall of the fluidized bed.

Equipment used during an extrusion spheronisation cycle

A) Granulation

The first step of an extrusion-spheronisation cycle is the preparation of the

plastic mass. Different types of granulators are used to perform the mixing of the
powder blend and the granulation liquid. The three types are

™ Planetary mixer.
™ High shear or sigma blade.
™ Continuous granulator to prepare the wet powder mass.

During the granulation step the evaporation of the fluid phase should be
restricted to a minimum. This could especially be a problem with the high shear
mixers as they introduce a large amount of energy into the mass which is partly
transformed into heat. This rise in temperature will induce the evaporation of the
granulation liquid, thus influencing the extrusion behavior of the wet mass. Cooling
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 Introduction
of the granulation bowl might avoid this problem. Special feature of the granulation
step is the homogeneous distribution of the liquid phase throughout the granulated
mass.

B) Extrusion

The second step of the process is the shaping of the wet mass into long rods
during extrusion. Classes of extruders: screw, sieve and basket, roll, and ram
extruders. The screw extruder consists of one or two (twin-screw) archimedes
screws feeding the plastic mass to an axial or radial extrusion screen. In the sieve
and basket extruder, granulate is fed by a screw or by gravity into the extrusion
chamber, where a rotating or oscillating device pushes the plastic mass through the
screen. The difference between the sieve and basket extruders is similar to that
between the radial and axial screw extruders.

A third class of extruders is the roll extruders available in two different

types, first consisting of an extruder equipped with two contra rotating wheels of
which one or both are perforated. Using this type of extruder the mass is fed
between the two wheels and the extrudate is collected inside the extrusion wheels.
The second type of roll extruder has a perforated cylinder which rotates around one
or more rollers, discharging the material to the outside of the cylinder.

C) Spheronisatian

During spheronisation process the cylindrical rods i.e extrudate is dumped

onto the spinning plate of the spheroniser, called the friction plate, where the
extrudate is broken up into smaller spherical shaped particles with a length equal to
their diameter. According to Rowe (1985) those plastic cylinders are rounded due to
frictional forces. In the spheronisation process different stages can be distinguished
depending on the shape of the particles, i.e., starting from a cylinder over a cylinder
with rounded edges, dumbells and elliptical particles to eventually perfect spheres.
Another possible mechanism of pellet-formation is by twisting of the cylinders to
rounded edges, finally resulting in the breaking of the cylinders into two distinct
parts.

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 Introduction
D) Drying

The fourth and final step of the process is the drying of the pellets
(Spheroids). The pellets (Spheroids) can be dried at room temperature but the use of
micro-oven results in faster production of pellets (Spheroids).

Parameters influencing the final pellet (Spheroid) quality

A) The moisture content of the granulated mass

The moisture, necessary to give the powder masses its plasticity so that it can
be extruded and shaped afterwards, is an extremely important parameter in the
extrusion spheronisation process. It was shown that the moisture content can range
between a lower and an upper limit and still produce pellets of an acceptable quality.
If the moisture content is less than the lower limit, a lot of dust will be formed
during spheronisation resulting in a large yield of fines. Exceeding the range of the
moisture content leads to an over wetted mass and agglomeration of the individual
pellets (Spheroids) during spheronisation occurs due to. the excess of water at the
surface of the pellets (Spheroids).

B) The type of granulation liquid

In most cases water is used as the granulating liquid although the use of
alcohol or water/alcohol mixtures has also been reported .The effect of this
parameter was clearly shown by Milli and Schwartz (1990); a minimum of 5%of the
granulation liquid had to be the water in order to produce pellets when processing a
formulation of Avicel pH 101. By increasing the water content in the granulation
liquid lead to an increase in the hardness of the pellets (Spheroids). The increase in
the hardness was correlated with a slower in vitro release rate of drug.

C) The spheronisation speed

The spheroniser speed affects the particle size of the pellets (Spheroids). The
hardness, roundness, porosity, bulk and tapped densities, friability, flow rate and
surface structure of the pellets (Spheroids) were also influenced by a change in the
spheronisation speed. According to Rowe (1985), the spheronisation speed should
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 Introduction
be optimized to obtain the desired densification. A low spheronisation speed would
not provide sufficient densification to obtain perfect spheres, as opposed to a
spheronisation process at higher speed which could lead to agglomeration of the
individual pellets (Spheroids).

D) Drying method

The influence of the drying on the pellet (Spheroid) -quality was shown by
Bataille et al. (1993) comparing a formulation dried in a micro-oven or in an
ordinary oven, containing Avicel pH101 and lactose. The pellets (Spheroids) dried
in micro-oven differed from those dried in the ordinary oven as their surface was
rougher and those pellets (Spheroids) were more porous and of lesser hardness.

Evaluation of pellet (Spheroid)

In recent year, a number of oral solid dosage forms have been produced in the form
of coated pellets (Spheroids) that can be filled in to hard gelatin capsules or
compressed into tablets. The reproducibility of particle size distribution, surface
area, density, and hardness of pellets (Spheroids) in addition to reproducibility of
their morphological properties should become the criteria by which formulation,
equipment and process are selected.

A) Size determination

The size of pellets (Spheroids) can be determined using a verity of parameters like
particle size distribution, mean diameter, geometric mean diameter, geometric mean
diameter, interquartile range, mean particle width and length. Particle size analysis
in most cases is carried out by a simple sieve analysis.

B) Sphericity

One of the most important characteristics of a pellet (Spheroid) is its roundness.

Several methods exist to determine the roundness like visual inspection, Scanning
electron microscopy, confocal imaging etc...

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 Introduction
C) Friability and Hardness

It is necessary to attain acceptable hardness and friability of pellets

(Spheroids) that can withstand handling, shipping, storage and other processing,
such as coating of pellets (Spheroids) subjected to. Variation in the formulation and
/or process of pellets (Spheroids) as well as variability of raw material can
potentially result in significant variation in hardness or friability of pellets
(Spheroids). Hardness can be measured by using kah’l pellets (Spheroids) hardness
tester and friability by using friabilator.

D) Density

Density of pellets (Spheroids) can be affected by changes in the formulation

and/or processes or factor. The pellets (Spheroids) are filled into hard gelatin
capsules volumetrically, using automated capsule filling machines. Obviously if the
density of pellets (Spheroids) varies significantly from batch to batch, the potency of
the finished capsule will vary. Any significant variation in the density of pellets will
affect the batch size determination in the coating equipment.

The bulk density of pellets (Spheroids) can be measured by using a

automated tapper, while the true density of pellets (Spheroids) can be determined by
an air compression pycnometer or by solvent displacement method.

E) Porosity

Porosity of pellets (Spheroids) can affect the capillary action of dissolved drug from
the pellets (Spheroids). It also affects film deposition and formulation.

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 Introduction
HARD GELATIN CAPSULE

The hard gelatin capsule also referred to as the dry filled capsule (DFC),
consists of two sections, one slipping over the other thus completely surrounding the
drug formulation. These capsules are filled by introducing the powder material into
the longer end or body of capsule and then slipping on the cap. Hard gelatin capsules
contain 12-16% water. However, water content can vary depending on the storage
conditions when the humidity is low the capsule become brittle if stored at high
humidity the capsule becomes flaccid and lose their shape storage in high
temperature areas also can affect the quantity of hard gelatin capsules. Gelatin
capsules may protect hygroscopic material from atmospheric water vapor if stored in
suitable packing materials. Capsules are supplied in variety of sizes the hard empty
capsules are numbered from 000 the largest size, which can be swallowed, to 5 the
this may vary depending on the different densities of powdered drug materials.

The usual procedure to fill capsules is to mix the ingredient by triturating and
reducing them to a fine and uniform powder which is then placed on a paper and
flattened with a spatula so than the layer of the powder is not greater than about ⅓ rd
the length of the capsule which is been filled the cap is then removed from selected
capsule and held in the left hand, the body is pressed repeatedly into the powder unit
it is filled after which the cap is replaced and the capsule is weighed.

Numbers of manual and automatic capsule filling machines are available for
increasing the speed of capsule filling operation.

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 Introduction
HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

HPTLC17

High performance thin layer chromatography is a powerful tool for qualitative and
quantitative analysis of herbal drugs. It is a sophisticated and automated form of
Thin Layer Chromatography (TLC) and it uses the technique in a more optimized
way.

Advantages of HPTLC

HPTLC has more advantages than conventional TLC methods for separation of
herbal drugs. The advantages are

™ Multiple samples can be analyzed in one plate.

™ Shorter analysis time, as migration distance is almost half as compared to that of

classical TLC.

™ Increased detection sensitivity because of higher surface concentration of

sample.

™ Low signal to noise ratio and hence suitable for in situ quantitative analysis.

™ Better resolution and increased separation efficiency.

™ Cost and time efficiency.

™ Being an offline technique, HPTLC offers unsurpassed flexibility.

Factors influencing HPTLC analysis

The important factor in quantitative analysis is the extent in which the various
components of drugs are separated. This depends on the factors like type of
stationary phase, precoated plates, layer thickness of plates, binder in the layer,
mobile phase (solvent system), solvent purity, size of the developing chamber,
saturation of the developing chamber, sample level in the chamber, gradient, relative
humidity, temperature, and flow rate of solvent, separation distance, and mode of
development.
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 Introduction

Parameters involved in HPTLC analysis

HPTLC plates

HPTLC pre coated plates with different support materials (glass, aluminium, plastic)
and with different sorbent layers are available in different format and thickness by
various manufacturers. Usually plates with sorbent thickness of 100-250 µm are
used for the qualitative and quantitative analysis.

Sample preparation

Proper sample preparation is an important prerequisite for efficient HPTLC. The

sample preparation is to dissolve the sample in a suitable solvent with suitable
recovery of intact compounds of interest with a suitable concentration of analytes for
direct application on the plates.

Application of samples on the plate

Sample application is the most critical step for obtaining good resolution for
quantification. The application process should not damage the layer as damaged
layers in unevenly shaped spots. The samples are applied as bands using spray on
technique.

Mobile phase

The mobile phase should be selected according to the chemical nature of the
compound present in the sample and polarity of the solvents. The mobile phase
should be able to resolve maximum number of compounds present in the sample.

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 Introduction
Pre-conditioning (chamber saturation)

Chamber saturation has a pronounced influence on the separation profile. When the
plate is introduced into an unsaturated chamber, the solvent evaporates from the
plate mainly at the solvent front. Therefore, large quantity of the solvent will be
required for travelling a given distance, resulting in increased Rf values. If the plate
is placed in saturated chamber, it soon gets preloaded with solvent vapours, resulting
in optimum Rf values.

Chromatogram development

Ascending, descending and two dimensional and horizontal are the most common
modes of chromatographic development. Rectangular glass chamber, twin trough
chamber, horizontal development chamber and automated development chamber are
used for carrying out different types of chromatogram development.

Detection of spots

After development of the plates, the plates should be removed and dried. Then the
plates are observed under the UV light for the detection of spots.

Densitometric scanning

The developed plates should be scanned at a suitable wavelength for the quantitative
and qualitative analysis. Peak area and peak heights should be recorded from which,
unknown concentration of the samples could be determined.

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 Introduction
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

HPLC18

High Performance Liquid Chromatography (HPLC) is one mode of

chromatography; the most widely used analytical technique.

HPLC utilizes a liquid mobile phase to separate the components of a mixture. These
components (or analytes) are first dissolved in a solvent, and then forced to flow
through a chromatographic column under a high pressure. In the column, the
mixture is resolved into its components.

The interaction of the solute with mobile and stationary phases can be manipulated
through different choices of both solvents and stationary phases. As a result, HPLC
acquires a high degree of versatility not found in other chromatographic systems and
it has the ability to easily separate a wide variety of chemical mixtures.

Estimation of drugs in biological sample by HPLC

Most of the drugs in biological sample can be analyzed by HPLC method because of
several advantages like rapidity, specificity, accuracy, precision, ease of automation
and eliminates tedious extraction and isolation procedures.

There are different modes of separation in HPLC. They are normal phase mode,
reversed phase mode, reversed phase ion pair chromatography, ion exchange
chromatography, affinity chromatography and size exclusion chromatography (gel
permeation and gel filtration chromatography).

In normal phase mode, the nature of stationary phase is polar and the mobile phase
is non polar. In this technique, non polar compounds travel faster and are eluted first
because of the lower affinity between the non polar compounds and the stationary
phase. Polar compounds are retained for longer times and take more time to elute
because of their higher affinity with the stationary phase. Normal phase mode of
separation is, therefore, not generally used for pharmaceutical applications because
most of the drug molecules are polar in nature and hence take longer time to elute.

Reverse phase mode is the most popular mode for analytical and preparative
separations of compounds of interest in chemical, biological, pharmaceutical, food
and biomedical sciences. In this mode, the stationary phase is non polar hydrophobic
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 Introduction
packing with octyl or octa decyl functional group bonded to silica gel and the mobile
phase is a polar solvent. An aqueous mobile phase allows the use of secondary
solute chemical equilibrium (such as ionization control, ion suppression, ion
pairing and complexation) to control retention and selectivity. The polar compound
gets eluted first in this mode and non polar compounds are retained for longer time.
As most of the drugs and pharmaceuticals are polar in nature, they are not retained
for longer times and hence elute faster. The different columns used are octa decyl
silane (ODS) or C18, C8, C4 etc., (in the order of increasing polarity of the stationary
phase).

The various components of HPLC are pumps (solvent delivery system), mixing unit,
gradient controller and solvent degasser, injector (manual or auto) , guard column,
analytical columns, detectors, recorders and integrators. Recent models are
equipped with computer and software for data acquisition and processing. The
choice of the column should be made after a careful consideration of the mode of the
chromatographic technique.

The different types of detection used in HPLC methods based on

ultraviolet (UV), fluorescence, refractive index, mass spectrophotometric and
electrochemical detectors. In most cases, method development in
HPLC is carried out with UV detection using a variable wavelength
spectrophotometric detector or a diode array detector (DAD).

Methods for analyzing drugs in biological sample can be developed, provided one
has knowledge about the nature of the sample, namely, its molecular weight,
polarity, ionic character and the solubility parameter. An exact recipe for HPLC,
however, cannot be provided because method development involves considerable
trial and error procedures. The most difficult problem usually is where to start, what
type of column is worth trying with what kind of mobile phase. In general, one
begins with reverse phase chromatography, when the compounds are hydrophilic in
nature with many polar groups and are water soluble.

The organic phase concentration required for the mobile phase can be estimated by
gradient elution method. For aqueous sample mixtures, the best way to start is with
gradient reverse phase chromatography. Gradient can be started with 5-10 % organic
phase in the mobile phase and the organic phase concentration (acetonitrile or
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 Introduction
methanol) can be increased up to 100 % within 20-30 min. Separation can then be
optimized by changing the initial mobile phase composition and the slope of
gradient according to the chromatogram obtained from preliminary run. The initial
mobile phase composition can be estimated on the basis of where the compounds of
interest were eluted, namely, at what mobile phase composition.

Elution of drug molecules can be altered by changing the polarity of the mobile
phase. The elution strength of a mobile phase depends upon its polarity, the stronger
the polarity, higher is the elution. Ionic samples (acidic or basic) can be separated, if
they are present in undissociated form. Dissociation of ionic samples may be
suppressed by proper selection of pH.

The pH of the mobile phase has to be selected in such a way that the compounds are
not ionized. If the retention times are too short, the decrease of the organic phase
concentration in the mobile phase can be in steps of 5 %. If the retention times are
too long, an increase in 5 % steps of the organic phase concentration is needed.

Optimization can be started only after a reasonable chromatogram has been

obtained. A reasonable chromatogram means that all the compounds are detected by
more or less symmetrical peaks on the chromatogram. By a slight change of the
mobile phase composition, the shifting of the peaks can be expected. From a few
experimental measurements, the position of the peaks can be predicted within the
range of investigated changes. An optimized chromatogram is the one in which all
the peaks are symmetrical and are well separated in less run time.

Quantitative analysis in HPLC

Three methods are generally used for quantitative analysis. They are the external
standard method, the internal standard method and the standard addition method.

External standard method

The external standard method involves the use of a single standard or up to three
standard solutions. The peak area or the height of the sample and the standard used
are compared directly or the slope of the calibration curve based on standards that
contain known concentrations of the compounds of interest.

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 Introduction
Internal standard method

A widely used technique of quantification involves the addition of an internal

standard to compensate for various analytical errors. In this approach, a known
compound of a fixed concentration is added to the known amount of samples to give
separate peaks in the chromatograms, to compensate for the loss of the compounds
of interest during sample pre treatment steps. Any loss of the component of interest
will be accompanied by the loss of an equivalent fraction of internal standard. The
accuracy of this approach obviously depends on the structural equivalence of the
compounds of interest and the internal standard.

The requirements for an internal standard must give a completely resolved peak with
no interferences, elute close to the compound of interest, behave equivalent to the
compounds of interest for analysis like pre treatments, derivative formations, etc., be
added at a concentration that will produce a peak area or peak height ratio of
about unity with the compounds of interest, not be present in the original sample, be
stable, un reactive with sample components, column packing and the mobile phase
and be commercially available in high purity.

The internal standard should be added to the sample prior to sample preparation
procedure and homogenized with it. Response factor is used to determine the
concentration of a sample component in the original sample. The response factor
(Rf) is the ratio of peak areas of sample component (Ax) and the internal standard
(AISTD) obtained by injecting the same quantity.

Standard addition method

Standard addition method is used in many techniques in analytical chemistry. It is of

limited use in chromatography because of the difficulty of injecting accurately
known amounts of sample. A sample mixture is analysed for the analyte of interest
by adding a specified amount of this analyte to the sample, thus increasing its
concentration. The analysis is then repeated and the resulting increase in peak area
due to addition of the standard amount is noted. Hence, the concentration of the
analyte in the original sample may be calculated.

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 Literature Review

LITERATURE REVIEW

Hovhannisan A.S. et al. (2005)67 carried out the effect of Kan Jang extract on the
pharmacokinetics and pharmacodynamics of warfarin in rats with an aqueous solution of
Kan Jang at a dose of 17 mg/kg orally. During the first 6-7 hr. following administration,
and attained its maximum value (Cmax) earlier observed (Cmax) values were almost the
same (7.0870.46 and 7.5970.25mg/ml.).

Wangboonskul Jinda et al. (2006)68 was investigated Pharmacokinetics of

andrographolide in healthy six Thai male volunteers. Each was administered with 3 doses
(3,6 and 9 tablets) of A. paniculata tablet which is equivalent to 29.55, 59.10 and 88.65
mg of Andrographolide respectively. The concentration-time profile of andrographolide
in plasma was obtained from a volunteer taking the highest dose. The peak concentration
(Cmax) was 335.85ng/ml and reached the peak time (Tmax) at 1 hour. This would be
beneficial in the use of this plant and allow more confidence in their efficacy and safety.

Senthil Kumaran K. et al. (2002)69 developed a HPLC method for the estimation of
andrographolide in biological fluids. The mobile phase used was methanol: Water in the
ratio of 65.35 v/v at a flow rate of 1.0ml/min. The elutes were monitored at 223nm. The
absolute recovery of andrographolide ranged between 97.24% to 99.4% the inter-day
coefficient of variation was 0.4% to 1.4% and intra-day co-efficient of variation was
1.05% to 2.8%. The developed HPLC method for estimation of andrographolide in serum
in sensitive, reliable and accurate.

Lan Ding et al.(2007)70 has developed for determining andrographolide and

dehydroandrographolide in rabbit plasma. The UV detection was performed at 225nm.
The calibration curves showed excellent linear relationship (R=0.9993) over the
concentration range of 0.05-5.0 gmL-1. The limits of detection were 0.019gm L-1 for
andrographolide and 0.022 gmL-2 for dehydroandgrapholide. This method was
successfully applied to the plasma concentration-time curve study after oral
administration of Andrographis paniculata Nees extract in rabbit.

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 Literature Review

Gottumukkala Subbaraju V. et al. (2007)71 has carried out high performance liquid
chromatographic (HPLC) and high performance thin layer chromatographic (HPTLC)
methods for quantitative determination of andrographolide were developed and validated.
The methods showed satisfactory linearity, precision, good recovery and appropriate
limits of detection (LOD) and quantification (LOQ). The content of andrographolide was
determined and the results obtained by HPLC and HPTLC methods were in good
agreement. The methods developed are suitable for the quality control applications in
Andrographis paniculata plant, extracts and dosage forms.

Shariff Arshia et al. (2007)72 they converted the drug itself into a bitterless micropellet
The technique of ionotropic gelation of sodium alginate with calcium ions with
subsequent drug entrapment was employed. The micro pellets were finally prepared by
adding 2.5%w/v of sodium alginate into a 2%w/v solution of calcium chloride solution.
The so formed pellets were completely bitterless and released the andrographolide
preferably away from the stomach. In conclusion, andrographolide can be easily
converted to bitter less multiple unit dose oral delivery systems with good entrapment
efficiency and a maximum release of 86% by utilizing the technique of ionotropic
gelation.

Sankalia Jolly M. et al. (2007)73 developed a rapid, simple, and sensitive HPLC method
with UV for determination of Nateglinide (NTG) in rabbit plasma and validated the
process. The method was selective enough to estimate the pharmacokinetic parameters of
NTG with out any tedious sample clean-up after the oral administration of 15mg NTG to
white albino rabbits.

Jain D.C. et al. (1999)74 they reported reversed phase LC method using UV detector is
suitable for the analysis of hepatroprotective compounds in A paniculata extract and is
simple, rapid and precise. A good separation of all the three major diterpenoids has been
achieved and could be used for rapid screening of A. paniculata plants for their genotypic
quality assessment, drug analysis etc.

Akowuah G.A. et al. (2005)75 A rapid method based on HPTLC and RP-HPLC ith UV
detection for quantitative determination of two major ioactive compounds in

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 Literature Review

Andrographis paniculata, andrographolide and 14- deoxy-11,12 didehydroand

rographolide, is described. The recoveries of the two compounds were between 96.5-
99.0% by HPTLC method and 98.1-99.2% by HPLC assay. The relative standard
deviations of the two compounds ranged between 0.89-0.99 (intra-day) and 0.86-0.98
(inter-day) for the HPTLC method and 0.86-1.02 (intra-day) and 0.87-1.12 (inter-day) for
HPLC method. The ethods were used for routine analyses and to obtain relative amount
of the two compounds in the leaves of the plant cultivated in different locations of
Malaysia. The extracts and isolated compounds exhibited lipid peroxidation inhibition
and free radical activities.

Famei LI et al. (2006)76 described the application of chromatography, a modern

technolgy, for highly efficient separation and analysis in the research of therapeutic basis
matter and quality control of traditional Chinese medicines (TCM) , is summarized. A
new strategy with metabonomics is put forth to achieve integral study on the therapeutic
basis matter and the action mechanism of TCM and to reveal and control the
comprehensive quality of TCM. The result shown that modern chromatography and
hyphenated techniques are one of the important technique platforms in the quality control
of TCM.

Raina Archana P. et al. (2007)77 described a rapid, accurate and simple high
performance thin layer chromatography method for quantitative estimation of
andrographolide in Andrographis paniculata. Thirty accessions of Andrographis
paniculata were analyzed by this method. Results showed mean andrographolide content
ranged from 1.14% to 2.60% amongst these collection.

Gowrishanker B. et al. (1994)78 The genotoxic effects of two types of tannery effluent
(Raw-to-Wetblue and Wetblue-to-Finish) and the antigenotoxic property of a crude
extract of Phyllanthus amarus L. were evaluated using the root meristem of Vicia faba L.
as the in vivo test system. The root tip cells were exposed to the tannery effluents at
different concentrations for varying durations. Squash preparations were made following
Haematoxylin staining procedures. Cytological investigations revealed a duration- and
concentration-dependent decrease in mitotic frequency and an increase in chromosomal

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 Literature Review

irregularities. The root meristems pre-treated with effluents for 8 h (Raw-to-Wetblue) and
24 h (Wetblue-to-Finish) which caused the maximum incidence of mitotic anomalies,
were then exposed to the crude extract of Phyllanthus amarus (0.25, 0.5, 0.75 and 1%) to
study its efficacy modifying genetic damage. It was observed that the root meristems
post-treated with Phyllanthus amarus showed a significant reduction in the frequency of
chromosomal alterations. However, there was no significant variation in the mitotic
frequency. The study suggests that Phyllanthin, a principle of Phyllanthus amarus, is
antigenotoxic.

Umbare R.P. et al. (2009)79 Atherosclerosis, referred to as a “Silent Killer” is one of the
leading causes of death in the developed countries and is on the rise in developing like
India. Therefore therapists consider the treatment of hyperlipidemia to be one of the
major approaches towards decelerating the atherogenic process. Allopathic
hypolipidemic drugs are available at large in the market but the side effects and contra-
indications of these drugs have marred their popularity. Recently herbal hypolipidemics
have gained importance to fill the lacunae created by the allopathic drugs. The present
study has been carried out to evaluate the antihyperlipidemic effect of plant Phyllanthus
amarus Schumach against cholesterol diet induced hyperlipidemia in Wister rats. Hydro-
alcoholic extract of leaves of phyllanthus amarus Schumach (HAEPAS) was studied for
its in-vitro anti-hyperlipidemic potential using cholesterol diet induced hyperlipidemia
model in rats. The results of study indicated that HAEPAS possess significant
hypolipidemic activity at doses 300 and 500 mg/kg.

Jian You et al. (2005)80 developed a HPLC method for the analysis of germacrone in
rabbit plasma and found its application in pharmacokinetic study of germacrone after
administration of Zedoary turmeric oil and reported that germacrone exhibited a linear
pharmacokinetics after administration to rabbits over the dose range 3.2-9.6mg/ml.

Kadioglu yucel et al. (2005)81 developed method applied to pharmacokinetic study of

intravenous tramadol injection in rabbits. The extraction with ethy1 acetate yielded good
response. The extraction recovery of tramadol from plasma averaged 90.40% serum
plasma concentration after completion of the infusion. Tramadol concentration was

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 Literature Review

measured using reversed phase high performance liquid chromatography and

pharmacokinetics application with intravenous tramadol in rabbits revealed that tramadol
followed one compartment model. The HPLC method developed was simple, rapid
specific, sensitive and accurate for investigation of the pharmacokinetic parameters of
tramadol in rabbit plasma.

Bhargava V. K. et al. (1999)82 studied the effect of single and multiple doses of herbal
preparation trikatu, an ayurvedic prescription on the therapeutic effect of rifampicin. The
bioavailability and pharmacokinetics of rifampicin in rabbits (n=10) administered as a
single dose or in combination of trikatu was determined. The study had a cross over
design with washout period of 7 days. In the other study, six rabbits were administered
with a single dose of rifampicin before and after multiple doses of trikatu. In both studies,
blood sample were collected at different interval and assayed for rifampicin.

Beer De Jacques O. et al. (1984)83 developed HPLC method, applied in

pharmacokinetic study of antipyrine, administered by oral intubation and intravenous
injection to a rabbit at a dose of 12.5mg/kg. Sample were drawn into heparanized tubes
by vein puncture at various time interval for about 6 hrs after administration and
subjected for analysis.

Katayouna Javidnia et al. (2006)84 developed a high sensitive HPLC assay for plasma
analysis of a view 1,4-dihydropyridine (nitrimidodipine) to support the subsequent
preclinical development of the compound. Four adult male albino rabbits weighing 2.9-
3.7kg were used in this study. A single bolus intravenous dose of 1.5mg/kg of the
compoud dissolved in a mixture PEG400-ethenol-water (5:65:30 v/v/v) was administered
to each rabbit over a 1 min period. Blood sample were collected from marginal vein, at
different time interval. Plasma was separated by immediate centrifugation at room
temperature and was kept at -200c until analysis.

Taiwo Idowu A. et al. (2009)85 A study on haematological effects of aqueous extracts

from Phyllantus amarus and Xylopia aethiopica was investigated in albino rats. The
extracts from both plants caused a dose-dependent decrease in erythrocyte sedimentation
rate (ESR) with 400mg/kg of X. aethiopica causing the least ESR of 2.7±0.6mm/hr.

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 Literature Review

Significant increases were obtained in red blood cell (RBC) count especially with
100mg/kg of P. amarus and X. aethiopica that caused 5.6% and 7.8% increases in RBC
count respectively (P< 0.05). Similar pattern of result was obtained for packed cell
volume (PCV). P. amarus did not appear to affect haemoglobin concentration, but higher
values of HB concentration were obtained for X. aethiopica; the difference was,
however, not significantly different from the control (P>0.05).Total and differential count
studies showed significant increases in the number of circulating leucocytes and
neutrophils respectively especially with 100mg/kg of extracts (P<0.05). Assessment of
alanine aminotransferase (ALT) and aspartate aminotransferase (AST) gave significantly
higher values of ALT for P. amarus – treated rats (P < 0.05). It as therefore suggested
that while both plants can serve as immune boosters and blood tonics, there is need for
caution on excessive and prolonged consumption of P. amarus.

Klyushnichenko V.E. et al. (1995)86 evaluated the separation of model mixture of 6-

indole alkaloids by RP-HPLC and HPTLC on the normal and reversed phase Armsorb
supports respectively. The procedure was used for the analysis of alkaloids and its
derivatives in the extract of plant cell cultures and hair roots of Rauwolfia Serpentina and
Rauwolfia Vomitoria.

Ming Li San et al. (2008)87 designed method based on cloud point extraction (CPE) for
the determination of flurbiprofen (FP) in rat plasma after oral and transdermal
administration by high-performance liquid chromatography coupled with UV detection
(HPLC-UV). The extraction recoveries were more than 84.5% the accuracies were within
+3.8% , and the intra-and inter-day precisions were less than 10.1% in all cases, After
strict validation, the method indicated good performance in terms of reproducibility,
specificity, linerity, precision and accuracy, and it was successfully applied to the
pharmacokinetic study of flurbiprofen in rats after oral and transdermal administration.

Bhattaram Atul Venkatesh et al. (2002)88 reviewed pharmacokinetics and

bioavailability studies that have been conducted for some of the more important or
widely used phytopharmaceuticals e.g. hyperidum, ginkgo, Silybum, essential oil,
quercetin, aescin salix etc. Furthermore, various drug interactions are dicussed which

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shows that caution should be exercised when combining phytopharmaceuticals with

chemically derived active pharmaceutical ingredients.

Remon J.P. et al.(1996)89 showen that modification made to extruder design can affect
the quality of pellets and that the perforation method of the extrusion screen and the
perforation geometry are two factors to consider while optimizing a formulation for a
extrusion/spheronisation process.

Xin-Hua W. et al. (2001)90 Fifty-five patients with chronic viral hepatitis B were
randomly divided into two groups. Thirty patients were treated with Phyllanthus amarus
compound (PA Co) for three months in the treatment group, another 25 patients were
treated with domestic recombinant human interferon alpha-1b (IFN-alpha 1b) for three
months as controls. The total effective rate in the treatment group was 83.3%, showing no
significant difference from the control (p>0.05). The normalization rates of ALT, A/G
and SB in the treatment group were 73.3%, 80.0% and 78.2% respectively, which were
significantly higher than that in the control (p<0.05). The negative conversion rates of
HBeAg and HBV-DNA in the treatment group were 42.3% and 47.8%, showing no
significant difference from the control (p>0.005). It is indicated that PA Co has
remarkable effect for chronic viral hepatitis B in recovery of liver function and inhibition
of the replication of HBV.

Newton J.M. et al. (1995)91 studied the influence of spheronization process variable like
time, loan, speed of rotation and plate texture on the properties of size, shape and density
of granules. It was found too low speed produced no significant shape changes in the
extrudate, while too high speed resulted in a size reduction of particles. Overloading the
plate will require a longer time course of the process while under loading reduced the
efficiency interaction to provide rounding of the particles.

Vervaet C. et al. (1995)92 reviewed deals with the aspect of extrusion-spheronization

process. The different steps in the production process of pellets are described. In a second
part the parameters which can influence the pellet quality are discussed, finally an
overview of the methods available for analysis of quality of the pellets is given.

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Vonk P. et al. (1997)93 studied much other granulation process, different phases of
growth during high share pelletization such as nucleation, fragmentation, densification,
and exponential growth due to coalescence and break up during kneading phase. The
final pellet size was depending on the impeller speed and liquid content.

Vohra S.B. et al. (1990)94 an ethanol extract of Acorus Calamus rhizomes was screened
for CNS effects using a battery of 20 tests, in rats and mice. The extra exhibited a large
number of actions similar to alpha-asarone. (an active principle of A. Calamus) but
differed from the latter in serval other respects including the responses to electroshock,
apomorphine-and isolation induced aggressive behaviour.

Panchal G.M. et al. (1989)95 water soluble dried power of alcoholic extract of root and
rhizomes of Acorus Calamus Linn was used. The in vivo experments involved strychnine
convulsant activity in frogs, spontaneous motor activity and amphetamine hyperactivity
in mice, pentobarbitone sleeping time in rats and local anesthetic activity in guinea pigs
and rabbits. Plant extracts at 10, 20 mg/kg i.p. did not afford protection to strychnine,
(1,5, 2.5 mg/kg) induced convulsions and same effect was found on acetylcholine
induced concentrations of rectus muscle except that it inhibited caffeine citrate
concentration in frog.

Tsai T.H. et al. (1992)96 a simple and sensitive high performance liquid chromatography
(HPLC) method for the determination of asarone in rabbit plasma has been developed.
The method is rapid, easily reproduced, selective and sensitive. It was applied to
pharmacokinetic studies of asarone in rabbit, after 5, 10, or 20 mg/kg intravenous
administrations. Rapid distribution followed by a slower elimination phase was observed
from the plasma concentration time curve. The plasma disposition at each dose fitted well
to a two-compartment open model and the terminal disposition became much slower as
the dose was increased, suggesting a nonlinear dose-dependent plasma asarone
disposition.

Prinssen E.P. et al. (1999)97 examined the activity of 5-HT IA ligands to alter the effects
of neuroleptics in preclinical models for antipsychotic potential and extra pyramidal side-

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 Literature Review

effects and concluded that 5- HT 1 A agonists with intermediate or high, but now low,
intrinsic activity may abolish the extra pyramidal effects of neuroleptics.

Lawrence B.M. et al. (1997)98 studied the composition of a Acorus Calamus Oil,
Melaleuca alternifolia oil, pipe nigam oils, Narcissus species oils so as to report progress
in essential oils.

Yanyan Xu et al. (2009)99 A simple, sensitive and selective liquid chromatography–

electrospray mass spectrometric (LC–ESI-MS) method was developed and validated for
simultaneous determination of strychnine and brucine in rat plasma, using tacrine as the
internal standard (IS). Sample preparation involved a liquid–liquid extraction of the
analytes with n-hexane, dichloromethane and isopropanol (65:30:5, v/v/v) from 0.1 mL
of plasma. Chromatographic separation was carried out on a Waters C18 column using a
mobile phase of methanol–20 mM ammonium formate–formic acid (32:68:0.68, v/v/v).
Positive selected ion monitoring mode was used for detection of strychnine, brucine and
the IS at m/z 335.2, m/z 395.2 and m/z 199.2, respectively. Linearity was obtained over
the concentration range of 0.5–500 ng/mL for strychnine and 0.1–100 ng/mL for brucine.
The lower limit of quantification was 0.5 ng/mL and 0.1 ng/mL for strychnine and
brucine, respectively. The intra- and inter-day precision for both strychnine and brucine
was less than 7.74%, and accuracy ranged from −4.38% to 2.21% at all QC levels. The
method has been successfully applied to a pharmacokinetic study of processed Semen
Strychni after oral administration to rats.

Colombo R. et al. (2009)100 Phyllanthus niruri L., commonly known in Brazil as

'quebra-pedra', has long been used in the treatment of diverse diseases and especially
urolithiasis. The therapeutic effects of P. niruri are attributed to various compounds
present in the plant, including the hydrolysable tannin corilagin. In the present study,
high-performance liquid chromatography (HPLC-/PAD) profiles of leaves and
commercial extracts of P. niruri were examined and three compounds, found to be
present in all of the samples studied, were isolated by open column chromatography over
(C18)silica gel followed by preparative HPLC. These compounds were identified by
nuclear magnetic resonance as corilagin, rutin and ethyl 3,4,5-trihydroxybenzoate.

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Corilagin, which has been proposed as a phytochemical marker for P. niruri, was
employed as an external standard in the development and validation of a rapid and
efficient qualitative and quantitative HPLC assay for the analyte. The method may be
applied in the standardization of herbs and phytomedicines commercialized in Brazil as
quebra-pedra.

Bratati De et al. (1991)101 The common alkaloids present in Strychnos nux-vomica L.

were separated using normal-phase high-performance liquid chromatography.

Date B. B. et al. (1995)102 assessed the efficacy of “P-Tabs” in insomnia and irritability.
The efficacy of P-Tabs containing acorus calamus and other drugs have been determined
in 30 patients for insomnia and irritability. The treatment was given for 3 months and
drug was found to be effective in both types of patients and a good relief was observed in
more than half of the patients.

Vikneswaran Murugaiyah et al. (2007)103 A simple analytical method using HPLC with
fluorescence detection was developed for the simultaneous determination of four lignans,
phyllanthin (1), hypophyllanthin (2), phyltetralin (3) and niranthin (4) from Phyllanthus
niruri L. in plasma. The method recorded limits of detection for 1, 2, 3 and 4 as 1.22,
6.02, 0.61 and 1.22 ng/ml, respectivel, at a signal-to-noise ratio of 5:1 whereas their
limits of quantification were 4.88, 24.41, 4.88 and 9.76 ng/ml, respectively, at a signal-to-
noise ratio of 12:1. These values were comparable to those of other sensitive methods
such as gas chromatography–mass spectrometry (GC–MS), high-performance liquid
chromatography–MS (HPLC–MS) and HPLC–electrochemical detection (HPLC–ECD)
for the analysis of plasma lignans. A further advantage over known methods was its
simple protocol for sample preparation. The within-day and between-day accuracies for
the analysis of the four lignans were between 87.69 and 110.07% with precision values
below 10.51%. Their mean recoveries from extraction were between 91.39 and 114.67%.
The method was successfully applied in the pharmacokinetic study of lignans in rats.
Following intravenous administration, the lignans were eliminated slowly from the body
with a mean clearance of 0.04, 0.01, 0.03 and 0.02 l/kg h and a mean half-life of 3.56,
3.87, 3.35 and 4.40 h for 1, 2, 3 and 4, respectively. Their peak plasma concentration

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upon oral administration was 0.18, 0.56, 0.12 and 0.62 μg/ml, respectively, after 1 h.
However, their absorption was incomplete with a calculated absolute oral bioavailability
of 0.62, 1.52, 4.01 and 2.66% for 1, 2, 3 and 4, respectively.

Handa S.S. et al. (1994)104 reviewed a number of “Medhya Rasayana” drugs which are
supported to counteract the effect of mental stress by transquilizing action. This includes
acorus calamus and other plants.

Frank Notka et al. (2004)105 Phyllanthus amarus derived preparations were previously
shown to inhibit RT inhibitor-resistant HIV variants as efficiently as wild-type strains.
The drugs target different steps of the HIV life cycle, thereby presenting multiple
antiviral activities. Here we show that a water/alcohol extract blocks HIV-1 attachment
and the HIV-1 enzymes integrase, reverse transcriptase and protease to different degrees.
A gallotannin containing fraction and the isolated ellagitannins geraniin and corilagin
were shown to be the most potent mediators of these antiviral activities. The P. amarus
derived preparations blocked the interaction of HIV-1 gp120 with its primary cellular
receptor CD4 at 50% inhibitory concentrations of 2.65 (water/alcohol extract) to
0.48 μg/ml (geraniin). Inhibition was also evident for the HIV-1 enzymes integrase
(0.48–0.16 μg/ml), reverse transcriptase (8.17–2.53 μg/ml) and protease (21.80–
6.28 μg/ml). In order to prove the in vivo relevance of these biological activities, plant
material was administered orally to volunteers and a potent anti-HIV activity in blood
could be demonstrated. Sera at a final concentration of 5% reduced HIV replication by
more than 30%. These results support the conclusion that P. amarus has inhibitory effects
on HIV not only in vitro but also in vivo.

Dandiya P.C. et al. (1990)106 attempted to classify the CNS herbal drugs employed in
the traditional system of medicine on the basis of modern pharmacology. Besides other
drug classification includes acorus calamus.

Lolock R.A. et al. (1987)107 studied a brief description of various historical folk used for
acorus calamus rhizomes, differences in beta-asarone contents between American and
Europen or asian plants and spasmolytic activity in animal studies were discussed.

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Narayan J. et al. (1987)108 carried out clinical experience on compound ayurvedic

preparation on epilepsy. Six patients were tested with this preparation consisting of
acorus calamus and other herbs. By the end of 6 months treatment, 60% of the patients
were free of attack.

Menon M.K. et al. (1967)109 studied the mechanism of transquilizing action of asarone
from acorus calamus.

Srivastava Vandana et al. (2008)110 A sensitive, selective, and robust high-performance

TLC (HPTLC) method using chiral TLC plates for qualitative and quantitative analysis of
phyllanthin (A), hypophyllanthin (B), niranthin (C), and nirtetralin (D), the active lignans
of Phyllanthus species, was developed and validated. The effectiveness and role of
various stationary phases viz TLC silica gel 60F254, HPTLC silica gel 60F254, and
chiral TLC plates in the quantitation were evaluated. A precoated chiral TLC plate was
found suitable for the simultaneous analysis of four pharmacologically active lignans. For
achieving good separation, the optimized mobile phase of n-hexane/acetone/1,4-dioxane
(9:1:0.5 by volume) was used (Rf = 0.30, 0.36, 0.41, and 0.48 for compounds A, B, C,
and D, respectively). A densitometric determination of the above compounds was carried
out in reflection/absorption mode at 620 nm. Optimized chromatographic conditions
provide well-separated compact bands for the tested lignans. The calibration curves were
found linear in the concentration range of 100–500 ng/band. Recoveries of A– D were
99.98, 100.51, 99.22, and 98.74%, respectively. The method was validated according to
ICH guidelines. The method reported here is reproducible and applied for the quantitative
analysis of the above lignans in the leaves of four Phyllanthus species.

Tripathi Arvind K. et al. (2006)111 A simple, precise and rapid high-performance thin-
layer chromatographic method has been developed for the estimation of phyllanthin (1)
and hypophyllanthin (2), the important lignans of Phyllanthus species, especially
Phyllanthus amarus. Separation of 1 and 2 was carried out on silica gel 60 F254 layers
eluted with hexane:acetone:ethyl acetate (74:12:8), and the analytes were visualised
through colour development with vanillin in concentrated sulphuric acid and ethanol.
Scanning and quantification of spots was performed at 580 nm. Recoveries of 1 and 2

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were 98.7 and 97.3%, respectively. The method was validated and the peak purities and
limits of detection and quantification were determined.

Kukkar Rajiv et al. (2010)112 Rasa is important group of formulation used by Ayurvedic
Physicians to treat various types of diseases. Krimimudgara Rasa, as per Ayurvedic
literatures is used as an anthelmintic drug. It expels the worms present in cough and
faeces of human and gives relief from symptoms like indigestion, vomiting, fever,
sneezing and pain in stomach. Many manufactures are adding adulterants in Ayurvedic
formulations, which render them less effective and lack of phyto/biomarker(s)
responsible for their therapeutic efficacy. No method has been developed to check
quantity of phytomarker (s) so far. In the present study an attempt has been made to
develop a HPTLC method of quantitative estimation of marker compounds, embelin and
strychnine in laboratory prepared formulation (LF) and a marketed formulation (MF) of
Krimimudgara Rasa. The laboratory formulation (LF) was found to contain 0.254 % of
embelin and 0.080 % of strychnine while the market formulation (MF) shows 0.135 % of
embelin and 0.046 % of strychnine. The method develop was found to be accurate,
precise, reproducible & simple for quantitative estimation of phytomarkers i.e. embelin
and strychnine and can be recommended for routine analysis of formulation containing
such drugs.

Ranjitha Kumari B.D. et al. (2006)113 Reserpine is an important indole alkaloid that is
used to treat hypertension and various sychiatric diseases by acting as a tranquilizing
agent. In pharmaceutical industries, reserpine is in great demand. Chemical synthesis of
reserpine is costlier than extracting it from natural resources. So enhancing this alkaloid
in the already available system is a beneficial approach. Tryptophan is the starting
material in the biosynthesis of reserpine. Callus was induced from leaf explants of
Rauvolfia tetraphylla L. on MS medium supplemented with the combination of 9 μM 2,
4-D and 25, 50, 75 and 100 mg/l tryptophan. An increase in the reserpine content was
observed at 50 mg/l tryptophan than in other concentrations.

Yadav Narayan P. et al. (2008)114 In search of the effective and standardized

hepatoprotective combination therapy, silymarin and standardized extract of Phyllanthus

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amarus has been evaluated against CCl4-induced hepatotoxicity in rats. Eight groups of
rats were used. The animals of group A served as normal and were given only vehicle.
The animals of group B served as toxin control and were administered with CCl4 (50%
solution of CCl4 in liquid paraffin, 2 ml/kg b.w., intraperitoneally). The animals of groups
C–H received silymarin (100 mg/kg b.w.), Phyllanthus amarus aqueous extract
(100 mg/kg b.w.), Phyllanthus amarus ethanolic extract (100 mg/kg b.w.), silymarin
(50 mg/kg b.w.)+P. amarus aq. ext. (50 mg/kg b.w.), silymarin (50 mg/kg b.w.)+P.
amarus eth. ext. (50 mg/kg b.w.) and marketed formulation (M.F.) 5 ml/kg b.w. for 6
days orally as well as CCl4 (2 ml/kg b.w.) on 4th day intraperitoneally. The test materials
were found effective as hepatoprotective as evidenced by plasma and liver biochemical
parameters. The combination of silymarin and Phyllanthus amarus showed synergistic
effect for hepatoprotection and silymarin with ethanolic extract of P. amarus showed
better activity due to the higher concentration of phyllanthin in ethanolic extract in
comparison to aqueous extract of P. amarus as estimated by HPLC.

Hamrapurka Purnima et al. (2010)115 Phyllanthin, a characterizing compound present

in the plant Phyllanthus amarus is used as hepatoprotective, antiviral and hypoglycemic
drug. Phyllanthin was extracted from the plant by Soxhlet and supercritical-fluid
extraction and isolated by column chromatography. The compound was then
characterized by spectroscopic techniques. A simple, rapid, accurate, and specific
HPTLC method was established and validated for analysis. The proposed method can be
used successfully for routine analysis of plant material.

Singh A. K. et al. (2006)116 A method was developed for plant regeneration from
alginate-encapsulated shoot tips of Phyllanthus amarus. Shoot tips excised from in vitro
proliferated shoots were encapsulated in calcium alginate beads. The best gel
complexation was achieved using 3% sodium alginate and 75 mM CaCl2·2H2O.
Maximum percentage response for conversion of encapsulated shoot tips into plantlets
was 90% after 5 wk of culture on Murashige and Skoog (MS) medium without plant
growth regulator. The regrowth ability of encapsulated shoot tips was affected by the
concentration of sodium alginate, storage duration, and the presence or absence of MS
nutrients in calcium alginate beads. Plantlets with well-developed shoot and roots were

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transferred to pots containing an autoclaved mixture of soilrite and peat moss (1:1). The
conversion of encapsulated shoot tips into plantlets also occurred when calcium alginate
beads were directly sown in autoclaved soilrite moistened with 1/4-MS salts.
Encapsulation of vegetative propagules in calcium alginate beads can be used as an
alternative to synthetic seeds derived from somatic embryos.

Adeneye A.A. et al. (2006)117 The effect of the aqueous leaf and seed extracts of
Phyllanthus amarus at oral dose of 150, 300 and 600 mg/kg was investigated for their
antidiabetic and anti-lipidemic potentials. The extract produced a dose-dependent
decrease in the fasting plasma glucose and cholesterol, and reduction in weights in treated
mice. The results suggest that the extract could be enhancing peripheral utilization of
glucose but the mechanisms on how this works remain unclear.

Xukun Deng et al. (2006)118 Alkaloids are the main bioactive ingredients in nuxvomica
they are also responsible for the pharmacological and toxic properties possessed by
nuxvomica. In our previous study, 16 alkaloids have been separated and identified from
the crude nuxvomica, 80% of which are strychnine and brucine, as well as their
derivatives such as brucine N-oxide or isostrychnine.

Akbar Seema et al. (2010)119 Some plants used in Unani system of medicine are toxic,
even deadly poisonous. The drugs having such plants as their components are detoxified
before they are dispensed to the patients. One such drug, capsule Hudar, has Strychnos
nuxvomica L. (Azraqi) seeds as one of its components and is very effectively used to
elevate blood pressure. Ancient manuscripts describe many methods of its detoxification.
It has been found that the detoxification processes studied reduce the strychnine content,
as determined either by using uv-vis spectrophotometer or HPLC, present in Strychnos
nuxvomica seeds which is responsible for Strychnos nuxvomica toxicity. The decrease in
strychnine amount was best when the seeds were immersed for detoxification in excess of
water for 5 days, in milk for 2 days followed by their boiling in milk. Strychnine in small
amounts has been reported to give subjective feeling of stimulation.

Sharma Anupam et al. (1993)120 A reversed phase high performance liquid

chromatographic procedure for standardizing a popular hepatoprotective Indian herb

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 Literature Review

“Bhumyamlaki” (Phyllanthus amarus) on the basis of two of its bioactive lignans,

phyllanthin and hypophyllanthin, has been developed. The method has been found to be
sensitive, precise and to record more than 98% recovery of the two lignans. The leaves of
P. amarus were found to contain the highest amounts of phyllanthin (0.7% w/w) and
hypophyllanthin (0.3% w/w) as compared to the other parts of the plant.

Olajire Adegoke A. et al. (2007)121 The method is based on the aromatic ring coupling
of reserpine with 4-carboxyl-2, 6- dinitrobenzene diazonium ion with the consequent
formation of an azo adduct. Optimization of reaction conditions and validation were
carried out and the method applied to assay of reserpine in tablets. Reserpine coupled
readily with CDNBD and optimization of experimental conditions showed the reaction to
be completed in 10 min at room temperature. A 1:1 drug to reagent stoichiometric ratio
was obtained for the azo adduct formed. The adduct exhibited a bathochromic shift with
respect to the drug and pronounced hyperchromic shift with respect to the reagent.
Sample analyses were done using a colorimeter at 470 nm. The assays were linear and
reproducible over the concentration range of 2.25 - 24 μg/mL. The new method was
successfully applied in the assay of reserpine in tablets with a performance similar to the
official (USP) spectrophotometric method (p > 0.05). This method represents a profound
improvement on the previously reported colorimetric method for reserpine.

Pandey Ved Prakash et al. (2010)122 Rauwolfia serpentina (L.) Benth, from the family,
Apocynaceae, is an important medicinal plant due to the alkaloid content of its root. The
purpose of this study was to obtain roots directly from leaf explant using growth
regulators. The leaf explant was inoculated on MS (Murashige and Skoog) medium
supplemented with single and combinations of growth regulators. Root growth was also
observed on liquid MS medium and under dark conditions. The reserpine content of the
roots obtained was determined by HPLC.

Benjamin B.D. et al. (1994)123 Shoots selected from multiple shoot cultures of
Rauwolfia serpentina were infected with strains of Agrobacterium rhizogenes.
Agrobacterium rhizogenes caused tumorous growth at the site of infection which
eventually developed callus and hairy roots. The initial cultures, comprising transformed

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callus and hairy roots grown in hormone-free media, produced ajmaline (0.045% dry wt)
and serpentina (0.007% dry wt).

Gupta M. M. et al. (2007)124 High-performance thin-layer chromatography (HPTLC)

has been used for normal-phase separation of the components of hexane, chloroform,
methanol, and water extracts of Rauvolfia serpentina roots. Computerized densitometry
was used for two-dimensional spectrographic image analysis of the HPTLC plates. High
performance liquid chromatography (HPLC) was also used for reversed-phase separation
of these extracts. Different chromatograms of R. serpentina root extracts, obtained by use
of these techniques, revealed the presence of three marker indole alkaloids, ajmaline,
ajmalicine, and reserpine, in all four extracts. Use of chloroform resulted in most efficient
extraction of these three alkaloids. The results also showed that defatting with hexane
may result in loss of the alkaloids.

Goel M. K. et al. (2009)125 Rauwolfia serpentina holds an important position in the

pharmaceutical world because of its immense anti-hypertensive properties resulting from
the presence of reserpine in the oleoresin fraction of the roots. Poor seed viability, low
seed germination rate, and enormous genetic variability are the major constraints for the
commercial cultivation of R. serpentina through conventional mode. The present
optimized protocol offers an impeccable end to end method from the establishment of
aseptic cultures to in-vitro plantlet production employing semisolid as well liquid nutrient
culture medium and assessment of their genetic fidelity using polymerase chain reaction
based rapid amplification of polymorphic DNA analysis. In vitro shoots multiplied on
Murashige and Skoog basal liquid nutrients supplemented with benzo[a]pyrene (1.0
mg/L) and NAA (0.1 mg/L) and in-vitro rhizogenesis was observed in modified MS basal
nutrient containing NAA (1.0 mg/L) and 2% sucrose. In-vitro raised plants exhibited 90-
95% survival under glass house/field condition and 85% similarity in the plants
regenerated through this protocol. Field established plants were harvested and extraction
of indole alkaloid particularly reserpine, ajmaline and ajmalicine and their simultaneous
quantitation was performed using monolithic reverse phase high-performance liquid
chromatography (HPLC). 

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 Aim and Objectives

AIM AND OBJECTIVES

Pharmacokinetic data is important for understanding better bioavailability of

therapeutic molecules in biological systems. Pharmacokinetic data is compulsory for
launching any generic drugs into the market. But for herbal drugs, it is not mandatory as
on date. The extracts used in herbal preparation are always complex mixtures of many
compounds and concentration of single compound in final products is very less in
quantity per dose. Pharmacokinetics discipline is applied mainly to drug substances,
though in principle it concerns itself with all manner of compounds ingested or otherwise
delivered externally to an organism, such as nutrients, metabolites, hormones, toxins, etc.
Knowledge of herbal pharmacokinetics is needed to provide valuable information to aid
practitioners in prescribing herbs safely and effectively. Studies of isolated compounds of
herbal drugs are needed in order to safe use of the drugs. Pharmacokinetic study of those
isolated compounds will reveal the present scenario in the field of herbal drug
development, formulation and evaluation though sufficient but may not be optimum in
respect of the determination of the pharmacokinetic parameters of the herbal drugs. It is
essential and very much requirements to find out pharmacokinetic profiles of herbal
drugs for the better understanding of the efficacy, potency and safety mechanism of these
drugs. Therefore, it is essential to establish these areas by establishing modern and
efficient pharmacokinetic techniques. The immediate and future market requirements of
indigenous drugs depend upon the type and quality of products and authentic scientific
validated data in respect of plasma profiles, drug administration, distribution, metabolism
and elimination (ADME).

Hence, the studies will focus on formulation of oral tablets dosage form with the
extracts of Andrographis paniculata and strychnous nuxvomica containing
Andrographolide an Strychine as their therapeutic molecules; capsule dosage form with
the extacts of Acorus Calamus containing α and β Asarone as their therapeutic molecules;
Spheroids and pellets dosage form with the extracts of Phyllanthus amarus and
Rauwolfia serpentina containing Hypophyllanthin and Reserpine as their therapeutic

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 Aim and Objectives

molecules respectively and also with synthetic commercial compounds available in the
market.

The optimized formulations in the extract and synthetic compounds are then
subjected to pharmacokinetic studies in Albino Rabbits models to determine their
Pharmacokinetic parameters in two different dose levels. Pharmacokinetic parameters
studied determine the therapeutic efficacy and the dosage regimen of specified herbal
drug in order to avoid its side effects and toxicity. The results, which not only minimize
the cost but also increase the efficiency, safety, quality and better use of herbal drugs.

The dosage regimens of the selected herbal drugs are of great concern as the
physicians are not in a position to prescribe the right dose as no Pharmacokinetic data are
available. Therefore establishing the dosage regimen or dose schedule through
Pharmacokinetic profiles of isolated active molecules of the selected herbal drugs may
lead to better dose determination of these drugs. Thereby we can assure exact dosing
schedule, which may help to reduce the side effects, if any, and cost effective herbal
formulations are possible for better patients’ compliance.

Thereby these Pharmacokinetic studies of isolated therapeutic compounds from

selected herbal drugs have been designed.

66 
 
 Plan of Work

PLAN OF WORK

To attain the Aim and goal of the work was planned in following pattern.

Stage 1- Literature survey

All the available sources like text books, news papers, magazines, media,
journals, Internet, and CD – ROMS were browsed to gain a thorough knowledge on
contribution to the Topic.
Stage 2 – Botanical Information
The selected plant sources information.
Stage 3 – Preparation and Quantification of the extracts
The extracts of Andrographis paniculata, Acorus Calamus, Phyllanthus amarus,
Rauwolfia serpentina and Strychnous nuxvomica were prepared with various
extraction techniques and their quanitification were done through HPTLC.
Stage 4 – Pre-formulation
The section covered the development of UV and HPLC method development for
the selected drugs. The drug polymer compatibility with selected polymers by
transform infrared (FT-IR) peak matching technique was also determined for the
selected drugs.
Stage 5 – Formulation of matrix, conventional Tablets, pellets and Capsules
Matrix tablets were formulated for the extracts of Andrographis paniculata and
Capsules were formulated for the extracts of Acorus Calamus, Spheroids were
formulated for the extracts of Phyllanthus amarus, pellets were formulated for the
extract of Rauwolfia serpentina and conventional tablet were formulated for the extracts
of Strychnous nuxvomica
Stage 6 - Pre compression evaluations of the powders/ pellets (Spheroids)
Granules / powders were evaluated for bulk density, angle of repose, compressibility
index and drug content.

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 Plan of Work

Stage 7- Post compression evaluations of the tablets, pellets (Spheroids) and

capsules
The formulated tablets, Pellets (Spheroids) and Capsules were evaluated for average
weight, drug content, hardness, friability, disintegration and in vitro release studies.
Stage 8- Pharmacokinetic studies
The in vivo pharmacokinetic study was carried out below the following heading.
1. Study design This section deals with the selection of animal models, grouping
and administration of dose, collection of blood sample and separation of plasma.
2. Bio analytical method development and estimation
The method developed includes the chromatographic variables, namely pH,
solvent strength, solvent ratio, flow rate, addition of peak modifiers in mobile
phase, nature of the stationary phase and detection wavelength were studied and
optimized for the retention and separation of the drugs.

3. Determination of various pharmacokinetic parameters The various parameters

such as maximum plasma concentration (Cmax), time to reach maximum plasma
concenrtration (Tmax) and area under the curve (AUC0-24 and AUC0-∞), overall
elimination rate constant (ke) and lag time (t0) were calculated using PK1 and
PK2 software.
4. Determination of dosage form/ regimen The dosage form / regimen was
determined for the herbal extracts selected with the help of the pharmacokinetic
datas obtained.

Stage 9- Results In this section provides the practical conditions and the outcome of the
experimental procedures applied or studied.

Stage 10 – Discussion The discussion part provides and additional support with
pragmatic of hypothetical reasons beyond the experimental findings.

Stage 11- Summary and Conclusion This section outlines the techniques and outcome
of the entire project work.
 

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 Botanical Informations

BOTANICAL INFORMATIONS

ANDROGRAPHIS PANICULATA

Plate - 1
Botanical Name of Plant : Andrographis paniculata
Common name : Kalmegh, Kan jang, Kirayat, King of bitters,
Bhui nimb.
Family : Acanthaceae
Systematic position
Division : Angiosperms
Class : Dicotyledonae

Subclass : Gamopetalae

Series : Bicarpellatae

Order : Personales

Tribe : Justicieae

69 
 
 Botanical Informations

Family : Acanthaceae
Genus : Andrographis

Part used : Leaves

Dose : 85-300 mg. (oral twice-thrice a day)

Habitat : It is found in in Srilanka, chaina and throughout India, specifically In

Maharastra, Tamilnadu, Assam, Andhra pradesh and West Bengal.

Oragenoleptic identification:

Colour : Leaves dark green while flowers are rose coloured.

Odour : Odorless

Test : Intensely bitter

Size : leaves 7x2.5 cm, flowers 1.8 cm in length.

Macroscopic characters

Leaves-branches sharply quadrangular, often narrowly winged in the upper part. Leaves
simple, opposite, short, glabrous, slightly undulate, pale beneath, base tapering, main
nerves 4-6 pairs, slender, petioles 0-6 mm long.

Photochemistry

The major bioactive chemical constituents of Andrographis paniculata leaves contain a

diterpene lactone, Andrographolide as the major bio-active constituent. Other diterpene
lactones include neoandrographolide, andrographiside, 14-deoxy-11,12,-
didehydroandrographolide etc. flavonoids viz., oroxylin, wogonin, andrographidines
A,B,C,D,E,F.

Traditional uses

The herb is reported to possess astringent, anodyne, tonic and alexipharmic properties
and is helpful in dysentery, cholera, diabetes, influenza, bronchitis, piles, gonorrhoea,
hepatomegaly, skin disorders, fever and worm infestation. The plant is bitter, acrid,

70 
 
 Botanical Informations

cooling, laxative, vulnerary, antiperiodic, anti-inflammatory, expectorant, depurative,

digestive and stomachic. It is useful in burning sensation, wounds, ulcers, chronic
bronchitis, leprosy, pruritis, flatulence, colic and diarrhea.

Pharmacology

Andrographis Paniculata has been evaluated for different pharmacological activities.

Andrographis Paniculata has been reported for its potent hepatoprotective.
Andrographis paniculata has been reported to have antisecretory (antidiarrhoeal),
immuno stimulant, antimalarial, antifilarial activity. It is also reported to have anti cancer,
anti HIV, anti-inflammatory, hypotensive action. In addition, it has found to be effective
in myocardial infarction.

Marker constituents

Andrographolide, 14-deoxy-11, 12-didehydroandrographolide, Andrograpanin and

Neoandrographolide.

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 Botanical Informations

ACORUS CALAMUS

Plate - 2
Botanical name of the Plant : Acorus Calamus

Common name : Vacha, Sweet Flag, Rat Root.

Family : Araceae

Parts Used : Rhizomes

Related species : Acorus gramineus, Acorus americanus.

Description

Acorus Calamus, also known as sweet-flag, is a perennial herb with long creeping
and aromatic rhizomes or underground stems sprouting leaves. The flowering shoots of
the plant are supported by a large leaf-like structure called spathe. It has pale-green, small
flowers, in 5 to 10 cm long cylindric spikes and yellowish fruits. The dried rhizomes of
the plant consritute the drug Acorus Calamus, and is used in medicine.

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 Botanical Informations

The dry rhizomes of Acorus Calamus, contain a yellow aromatic oil. The
essential oil contains calamen, calamenos, calameori and asarone. Indian calameon oil
contains asarone, small amounts of sesquiterpenes and sesquiterpene alcohols. The odour
of the oil is ascribed to an unidentified constituent. The leaves contain oxalic acid and
calcium.

Uses

Healing Power and Curative Properties

The rhizomes are of great medicinal value. The root-stock of the plant is an
aromatic stimulant, bitter tonic and expectorant. It relieves flatulence, counteracts
spasmodic disorders and induces vomiting. It regulates menstural periods. It is also
laxative, diuretic and aphrodisiac.

Stomach Disorders

Acorus Calamus, gives relief to heavy stomach by relieving flatulence, colic and
increasing appetite. The burnt root mixed with some bland oil such as refined coconut oil
or a poultice of the root may be applied over the abdomen in treatment.

Diarrhoea and Dysentery

The drug is a time-tested remedy and is an ingredient in Ayurvedic medicines for

chronic diarrhoea. It is also effective in chronic dysentery, due to the presence of tannins.
Its infusion can be given to children suffering from these ailments.

Asthma

Acorus Calamus is highly beneficial in the treatment of asthma; it removes

catarrhal matter and phlegm from the bronchial tubes. About 65 centigrams of the herb is
taken every 2 or 3 hours in this condition.

73 
 
 Botanical Informations

Common Cold

The herb is also useful in treating common cold. A small piece of the rhizome is
roasted and powdered. A pinch of this powder taken with honey provides great relief. For
infants, the paste of Acorus Calamus, mixed in breast milk is touched on the baby's
tongue. Another convenient method of giving the medicine to infants is to apply a little
paste on the mother's nipples.

Whooping Cough

The powder of the roasted herb is an effective home remedy for children suffering
from whooping cough. A pinch of this powder can be given with honey. Being
antispasmodic, it prevents the severe bouts of coughing. For smaller children, the dose
must be proportionately smaller.

Intestinal Worms

Acorus Calamus is effective in expelling intestinal worms. The powdered root is

taken for this purpose.

Mouth Disorders

The herb is useful in treating mouth ulcers, coating on tongue and rawness, that is,
inflammation of the skin. A small piece of the herb should be rubbed on the tongue to
obtain relief.

Active Constituents

The drug (i.e. the rhizomes) contains monoterpene hydrocarbons, sequestrine

ketones (trans-or Alpha) Asarone (2,4,5-trimethoxy-1-propenylbenzene), and beta-
asarone (cis-isomer) contained in the roots essential oil (2-9%).

The asarone are MDA type compounds that are naturally occurring precursors of
TMA-2. The psychoactive constituents break down over time lessening potency until at a

74 
 
 Botanical Informations

year after harvest, the roots are considered worthless. Acorus calamus also contains a
bitter principle known as acorine.

The American variety has consistently tested free of the carcinogenic beta-
asarone. The asian varities do contain varying amounts of beta-asarone, and cause a more
esdate feeling when injected.

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 Botanical Informations

PHYLLANTHUS AMARUS

Plate - 3

Botanical name of the Plant : Phyllanthus amarus

Family : Euphorbiacea

Common name : Bahupatra (Sanskrit)

Tradition : Phyllanthus amarus is a traditional ayurvedic

herb used in southern India for the treatment

of jaundice.

Plant Constituents of Phyllanthus Amarus

Alkaloids, flavonoids, geraniin, tannin which is an in vitro anti-viral agent, lignans such
as hypophyllanthin, phyllanthin.

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 Botanical Informations

Action

Anti-viral [An agent that destroys viruses]

Hepatoprotective [Liver protector]
Hypoglycemic [Lowers blood sugar]

Medicinal Parts Used: Leaves or aerial parts may have a support role in Diabetes

Phyllanthus Amarus is also used for:

Liver Conditions

Hepatitis B
Jaundice
May have a support role in Diabetes

Viral Conditions

Acute Hepatitis
Chronic persistent hepatitis
Part of the Treatment for Chronic Active Hepatitis

77 
 
 Botanical Informations

RAUWOLFIA SERPENTINA

Plate -4

Botanical Name of plant : Rauwolfia Serpentina

Common name of plant : Snake Root, Serpentina Root and Indian Snake

Root.

Family : Apocyanaceae

Part used : Root

Dose : 100 to 150 mg. (oral twice daily)

Habitat : It is found in tropical Himalayas, Sikkim, North

Bihar, Assam and Deccan Peninsula.

Oraganoleptic identification

Colour : Root bark is grayish yellow to brown and wood pale

yellow.

Odour : Odorless

Taste : Bitter

Size : Roots are sub-cylindrical, slightly tapering and

tortuous.

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 Botanical Informations

Microscopic characters

The cork is made up of stratified cells followed by phelloderm of few rows of

parenchyma. Phloem is narrow, parenchymatus with small scattered sieve tissue.
Xylem vessels are elongated up to 350μ in length and 50μ in width and contain
simple or branched pits.

Chemical constituents

The powdered root (Rauwolfia Serpentina) generally contains 0.15 to 0.2% of active
alkaloid (reserpine and rescinnamine) by weight. Rauwolfia Serpentina is cultivated
for the medicinal use of its 30 alkaloids (particularly reserpine found in the root),
many used in treating hypertension. Besides reserpine, other alkaloids used in
hypertension and other cardiac disorders are ajmaline, rescinnamine, serpentinine,
sarpagine, deserpidine, and chandrine.

Uses

Rauwolfia Serpentina is antihypertensive in activity. Among the various alkaloids

of Rauwolfia Serpentina are reserpine, rescinnamine and Ajmalicine are clinically
important. Ajmalicine, though less in quantity has the uses in the treatment of
circulator diseases, in the relief of obstruction of normal cerebral blood flow. It is
used as an antidote to the bites of poisonous reptiles like snakes. It is also used
to treat dysentery and other painful affections of the intestinal canal.

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 Botanical Informations

STRYCHNOS NUX-VOMICA

Plate - 5

Botanical name of plant : Strychnos Nux-vomica

Family : Loganiaceae

Common name : Poison Nut, Semen strychnos, Quaker Buttons.

Part used : Dried ripe seeds

Habitat : India, In the Malay Archipelago.

Description

A medium-sized tree with a short, crooked, thick trunk, the wood is white hard,
close grained, durable and the root very bitter. Branches irregular, covered with a smooth
ash-coloured bark; young shoots deep green, shiny; leaves opposite, short stalked, oval,
shiny, smooth on both sides, about 4 inches long and 3 broad; flowers small, greeny-
white, funnel shape, in small terminal cymes, blooming in the cold season and having a

80 
 
 Botanical Informations

disagreeable smell. Fruit about the size of a large apple with a smooth hard rind or shell
which when ripe is a lovely orange colour, filled with a soft white jelly-like pulp
containing five seeds covered with a soft woolly-like substance, white and horny
internally. The seeds are removed when ripe, cleansed, dried and sorted; they are
exported from Cochin, Madras and other Indian ports. The seeds have the shape of
flattened disks densely covered with closely appressed satiny hairs, radiating from the
centre of the flattened sides and giving to the seeds a characteristic sheen; they are very
hard, with a dark grey horny endosperm in which the small embryo is embedded; no
odour but a very bitter taste.

Constituents

Strychnos Nux-vomica contains the alkaloids, Strychnine and Brucine, also traces of
strychnicine, and a glucoside Loganin, about 3 per cent fatty matter, caffeotannic acid
and a trace of copper. The pulp of the fruit contains about 5 per cent of loganin together
with the alkaloid strychnicine.

Medicinal Action and Uses

The properties of Strychnos Nux-vomica are substantially those of the alkaloid

Strychnine. The powdered seeds are employed in dyspepsia. The tincture of Strychnos
Nux-vomica is often used in mixtures - for its stimulant action on the gastro-intestinal
tract. In the mouth it acts as a bitter, increasing appetite; it stimulates peristalsis, in
chronic constipation due to atony of the bowel it is often combined with cascara and
other laxatives with good effects. Strychnine, the chief alkaloid constituent of the seeds,
also acts as a bitter, increasing the flow of gastric juice; it is rapidly absorbed as it reaches
the intestines, after which it exerts its characteristic effects upon the central nervous
system, the movements of respiration are deepened and quickened and the heart slowed
through excitation of the vagal centre. Strychnine is excreted very slowly and its action is
cumulative in any but small doses; it is much used as a gastric tonic in dyspepsia. Brucine
closely resembles strychnine in its action, but is slightly less poisonous, it paralyses the
peripheral motor nerves. It is said that the convulsive action characteristic of strychnine is

81 
 
 Botanical Informations

absent in brucine almost entirely. It is used in pruritis and as a local anodyne in

inflammations of the external ear.

Poisoning and Antidotes

In cases of poisoning by strychnine an emetic or the stomach pump should be

used at once and tannin or potassium permanganate given to render the strychnine
inactive. Violent convulsions should be controlled by administration of chloroform or
large doses of chloral or bromide. Urethane in large doses is considered an antidote.
Amyl nitrite is also useful owing to its rapid action during the convulsion, and in absence
of respiration 3 to 5 minims may be hypodermically injected.

82 
 
 Drugs Profile
 
DRUGS PROFILE

Andrographolide
Andrographolide (3 - (2 - (Decahydro - 6 -hydroxyl - 5 - (hydroxymethyl) - 5, 8a –
dimethyl - 2 - methylenenaphthyl)ethylidene) dihydro - 4 - hydroxyfuran - 2 (3H) - one)
is the active principle of Andrographis paniculata extracts. Andrographis paniculata
Nees (Family Acanthaceae) is available abundantly in India, Pakistan and Srilanka,
growing in hot and shade places. It is cultivated in certain parts of India, East and West
Indies and Mauritius. It is known by various names such as
Kalmegh, Kalupnath, Kiriat and Mahatila meaning the “King of bitters”. Andrographis
paniculata or kalmegh is one of the most widely used plants in Ayurvedic formulations.
Structure of Andrographolide

Figure - 6
Systematic Name : 3 - (2 - (Decahydro - 6 -hydroxyl - 5 - (hydroxymethyl) - 5,
8a – dimethyl - 2 - methylenenaphthyl)ethylidene) dihydro
4 - hydroxyfuran - 2 (3H) - one
Molecular Formula : C20H30O5
83 
 
 Drugs Profile
 
Molecular Weight : 350.45
Solubility in Water : Sparingly Soluble
Uses : Andrographolide has been used for liver complaints
and fever, and as an anti-inflammatory and
immunostimulant. In clinical trials, Andrographolide
has been studied for use as an immunostimulant
in upper respiratory tract infections and HIV
infection. The potential of andrographolide as an
anticancer agent is being investigated.

Dosages : Preparations containing 48-60 mg of the andrographolide

constituents, divided in to three or four daily doses.
Adverse Reactions : Headache, fatigue, rash, bitter and metallic taste, diarrhea,
pruritus, and decreased sex drive were reported in clinical
trial. HIV-positive participant experienced an
anaphylactic reaction.

84 
 
 Drugs Profile
 
α & β Asarone

The drug (i.e., the rhizomes) contains monoterpene hydrocarbons, Seqestrine

Ketones, Alpha (trans) and Beta (cis). The root contains volatile oil of 1.5 to 4.5%.
The oil contained 82% Asarone; other components of the oil are eugenol, Pinene,
Campheme and Caryophylollene.

Structure of Alpha Asarone

Figure - 7
Systematic Name : trans-2,4,5-Trimethoxypropenylbenzene
Molecular Formula : C12H16O3
Molecular Weight : 208.26
Solubility in Water : Insoluble
Uses : Alpha – asarone produces sedative effects. The
pharmacological property of the essential oil (spasmolytic)
and of Alpha-asorone (CNS Sedative) have been the
subjects of multiple studies. Other uses include intellect
promoting, nerve tonic, tranquilizing. It is useful in
vitiated condition of vata and kapha stomatopathy, colic,

85 
 
 Drugs Profile
 
flatulence, dyspepsia, helmentiasis, amenorrhoea,
dismenorrhoea, nephropathy, calculi, epilepsy, delirium,
amnetia, convulsion, depression and other mental disorder.
It is also used in constipation, to cure fevers, for asthma
and bronchitis, house-flies repellant, and hyperlipidemic.
Dosages : This is about 2µg/kg bw/day.
Adverse Reactions : Few side effects like heart, liver, kidney non-toxic, only
individual cases medication initially dizziness, nausea,
anorexia, fatigue, but were minor, to medication, to
disappear.  

Structure of Beta Asarone

Figure - 8

Systematic Name : cis-2,4,5-Trimethoxypropenylbenzene

Molecular Formula : C12H16O3

Molecular Weight : 208.26

Solubility in Water : Insoluble
Uses : Beta– asarone produces sedative effects. The
pharmacological property of the essential oil (spasmolytic)

86 
 
 Drugs Profile
 
and of Beta-asorone (CNS Sedative) have been the
subjects of multiple studies. Other uses include intellect
promoting, nerve tonic, tranquilizing. It is useful in
vitiated condition of vata and kapha stomatopathy, colic,
flatulence, dyspepsia, helmentiasis, amenorrhoea,
dismenorrhoea, nephropathy, calculi, epilepsy, delirium,
amnetia, convulsion, depression and other mental disorder.
It is also used in constipation, to cure fevers, for asthma
and bronchitis, house-flies repellant, and hyperlipidemic.
Dosages : 2µg/kg bw/day.

Adverse Reactions : Side effects like heart, liver, kidney non-toxic, only
individual cases medication initially dizziness, nausea,
anorexia, fatigue, but were minor, to medication, to
disappear.  

87 
 
 Drugs Profile
 
Hypophyllanthin

Hypophyllanthin are potent hepatoprotective lignans found in Phyllanthus

Amarus. (Family: Euphorbiacea). The genus includes more than 600 species of
shrubs, trees, and annual herbs distributed throughout the globe, many of which
are used medicinally in different countries such as P. emblica L., P. urinaria L., P.
reticulates in Indo-China, P. Amarus in Brazil and West Indies, P. elegans Wall,
P. urinaria in the Philippines. The plant is commonly known as ‘‘Bhuiamliki’’ in
India and ‘‘Look Tai Bai’’ in Thailand. P. Amarus is a small, erect, annual herb
that grows 30–60 cm in height mainly as a weed in both cultivated fields and
wastelands. It is a wellknown Ayurvedic plant used in folk remedy for jaundice
and other liver disorders.
Structure of Hypophyllanthin

Figure - 9

Systematic Name : r-1-(3,4-dimethoxyphenyl) -6-methoxy-t-2, c-3-

bimethaxymethyl-7-8-methylene dioxy-1234 tetra hydro
naphthalene.
Molecular Formula : C24H30O7
Molecular Weight : 430.40

88 
 
 Drugs Profile
 
Solubility in Water : Insoluble
Uses : Hypophyllanthin is used in Diabetes. It is also used in
Kidney and gall Bladder stones as cystitis, prostatitis,
venereal disesases and urinary tract infections.
Hypophyllanthin is also useful for liver conditions like
Hepatitis B, jaundice and Viral liver diseases including
acute hepatitis, chronic persistent hepatitis part of the
treatment for chronic active hepatitis. 

Dosages : 9.1 mg/kg bw/day.

Adverse Reactions : Hypophyllanthin showed adversre effects on the

hematological and serum biochemical parameters like
decrease in RBC count, packed cell volume, Hb
concentration and increase in WBC count apart from other
effects on liver, testis, kidney, weight loss and increase
frequency of urination.

89 
 
 Drugs Profile
P
 
Reserpin
ne
Reserp
pine is an inndole alkalooid antipsychhotic and anttihypertensivve drug thatt has
been used forr the control of high bloood pressure and
a for the relief
r of psycchotic sympttoms,
allthough becaause of the development
d t of better drrugs for thesee purposes and
a because of its
nuumerous sid
de-effects, it is rarely ussed today. The
T antihypeertensive acttions of reseerpine
arre a resullt of its ability to deplete caatecholaminees (among othermonoaamine
neurotransmittters) from peripheral sympatheticc nerve enddings. Thesee substances are
normally involved in conntrolling heaart rate, forcce of cardiaac contractioon and perippheral
reesistance.

Reserp
pine mediatted depletionn of monoam i the synapses is
mine neurotrransmitters in
often cited as
a evidence to the theoory that deppletion of the
t neurotraansmitters caauses
suubsequent deepression in humans (c.f. monoamiine hypothessis). Howevver, this claiim is
not without controversy;
c Some havee called reseerpine-induced depressioon a myth, while
w
otthers claim that teas maade out of the
t plant rooots containinng reserpine have a calm
ming,
seedative actio
on that can actually be coonsidered anntidepressantt.

Moreo
over, reserpiine has a perripheral actioon in many parts
p of the body,
b resultiing in
a preponderan
nce of the cholinergic part
p of the neervous systeem (GI tract,, smooth muuscles
vessels).

Stru
ucture of Reeserpine

Figure - 10
1

90 
 
 Drugs Profile
 
Systematic Name : methyl-11,17α-dimethoxy-18β-[(3,4,5-trimethoxybenzoyl)

oxy]-3β,20α-yohimban-16β-carboxylate

Molecular Formula : C33H40N2O9

Molecular Weight : 608.68

Solubility in Water : Insoluble

Uses : Reserpine is one of the few antihypertensive medications

that have been shown in randomized controlled trials to

reduce mortality: The Hypertension Detection and Follow-

up Program, the Veterans Administration Cooperative

Study Group in Anti-hypertensive Agents, and the Systolic

Hypertension in the Elderly Program.

Reserpine is rarely used in the management of

hypertension today. Reserpine is a second-line adjunct

agent for patients who are uncontrolled on a diuretic when

cost is an issue.

It is also used to treat symptoms of dyskinesia in patients

suffering from Huntington's disease.

Dosages : Originally, doses of 0.5 mg to 40 mg daily were used to treat

psychotic diseases. For adjunctive treatment, doses are
typically kept at or below 0.25 mg twice a day.

Adverse Reactions : At doses of less than 0.2 mg/day, reserpine has few side

effects, the most common of which is nasal congestion.

There has been much concern about reserpine causing

depression leading to suicide. However, this was reported in

uncontrolled studies using doses averaging 0.5 mg per day.

91 
 
 Drugs Profile
 
Reserpine can cause: nasal congestion, nausea,
vomiting, weight gain, gastric intolerance, gastric
ulceration (due to increased cholinergic activity in gastric
tissue and impaired mucosal quality), stomach cramps and
diarrhea are noted. The drug causes hypotension and
bradycardia and may worsen asthma. Congested nose and
erectile dysfunction are other consequences of alpha-
blockade. Depression can occur at any dose and may be
severe enough to lead to suicide. Other central effects are a
high incidence of drowsiness, dizziness, and nightmares.
Parkinsonism occurs in a dose dependent manner. General
weakness or fatigue is quite often encountered. High dose
studies in rodents found reserpine to cause fibroadenoma of
the breast and malignant tumors of the seminal vesicles
among others. Early suggestions that reserpine causes
breast cancer in women (risk approximately doubled) were
not confirmed. It may also cause hyperprolactinemia .

Reserpine passes into breast milk and is harmful to

breast-fed infants, and should therefore be avoided during
breastfeeding if possible.

92 
 
 Drugs Profile
P
 
Strychnine

hnine was thhe first alkaloid to be ideentified in plants of the genus Strychhnos,
Strych
Family Logan
niaceae. Stryychnos, nam
med by Carl Linnaeus
L in 1753, is a genus
g of trees and
cllimbing shru
ubs of the gentian
g ordeer. The gennus contains 196 variouus species and
a is
distributed th
hroughout the warm regiions of Asiaa (58 speciess), America (64 species)), and
A
Africa (75 species). The seeds
s and baark of many plants in thiis genus conntain the pow
werful
poison strych
hnine.

Strych
hnine was first disccovered byy French chemists Jooseph Biennaimé
C
Caventou d Pierre-Joseeph Pelletieer in 1818 in the Saiint-Ignatius’ bean. In some
and
Strychnos plaants a 9,10-ddimethoxy derivative
d off strychnine. The structuure of strychhnine
w first dettermined in 1946 by Sir
was S Robert Robinson
R andd in 1954 thhis alkaloidd was
syynthesized in
i a laboratoory by Robeert W. Wooodward. Thiss is one of the
t most fam
mous
syyntheses in
n the histoory of orgganic chemistry. Bothh chemists won the Nobel
N
prrize (Robinsson in 1947 and
a Woodward in 1965)).

Structure of Strrychnine

Figure - 11
1

93 
 
 Drugs Profile
 
Systematic Name : Strychnidin-10-one
Molecular Formula : C21H22N2O2
Molecular Weight : 334.41
Solubility in Water : Sparingly Soluble
Uses : Strychnine used in dyspepsia, while the tincture is used in
mixtures for its action on the gastro-intestinal tract. The bitter taste
increases appetite. It also stimulates peristalsis, in constipation, due
to "atony of the bowels." It is often combined with other laxatives
for it's good effects.
Strychnine, the main alkaloid constituent in the seeds, is also a
bitter, which increases the flow of gastric juice. It is quickly
absorbed in the intestines, after it exerts its characteristic effects on
the central nervous system. Strychnine is slowly excreted and its
actions are cumulative in small doses. It is used as a gastric tonic
dyspepsia.
Dosages : 1–2 mg/kg orally in humans.
Adverse Reactions : The ingestion of strychnine, symptoms of poisoning usually
happen within 15-60 minutes. People exposed to low or moderate
doses by any route could have; Agitation, Ability to be easily
startled, Apprehensio and Fear, Restlessness, Painful muscle
spasms, Fever, Kidney and Liver injury.
 

94 
 
 Materials and Instruments

 
MATERIALS AND INSTRUMENTS

LIST OF CHEMICAL USED

Andrographolide Biomax, India, Thane

Andrographis paniculata Local Market, Salem

α-β-Asarone Natural Remedies, Bangalore
Acorus Calamus Local Market, Salem
Hypophyllanthin Spic Limited, Chennai
Phyllanthus amarus Neyyor, Kanyakumari
Reserpine S.D. fine Chemical Ltd, Mumbai
Rauwolfia serpentina Local Market, Salem

Strychnine Spic Limited, Chennai

Strychnous nuxvomica Local Market, Salem
Cross Caramellose Signet Chemicals, Mumbai

Aerosil S.D. fine Chemical Ltd, Mumbai

Micro Crystalline Cellulose (AVICEL PH
Coloron Asia Pvt Ltd
101) MCC
Lactose (Monohydrate) S.D. fine Chemical Ltd, Mumbai
Starch 1500 Coloron Asia Pvt Ltd
Potassium Chloride S.D. fine Chemical Ltd, Mumbai
Talc S.D. fine Chemical Ltd, Mumbai
Magnesium Stearate S.D. fine Chemical Ltd, Mumbai
Potassium dihydrogen Phosphate S.D. fine Chemical Ltd, Mumbai
Ortho phosphoric acid S.D. fine Chemical Ltd, Mumbai
Sodium Hydroxide Pellets S.D. fine Chemical Ltd, Mumbai
Hydrochloric acid S.D. fine Chemical Ltd, Mumbai
Methanol HPLC grade S.D. fine Chemical Ltd, Mumbai
Actonitrile HPLC grade S.D. fine Chemical Ltd, Mumbai

Table- 2

95 
 
 Materials and Instruments

 
LIST OF EQUIPMENTS USED

EQUIPMENT MAKE
UV/ Visible Spectrophotometer Shimadz – 160 A, USA
Centrifuge Perkin – Elmer – 1600, USA
Vacuum Pump Toshnival, Chennai
Digital pH Meter Systronics, Mumbai
Electronic Balance Sartronics India, Bangalore
Tablet Friabilator Electro lab India, Mumbai
Dissolution Tester Electro lab India, Mumbai
Tablet disintegrator Electro lab India, Mumbai
Orbital Shaker Electro lab India, Mumbai
Rotary Compression Machine (10 Station) Rimek instruments, Ahmedabad
HPTLC CAMAG Linomat IV, Switzerland
HPLC Shimadzu – LC20AD, USA
Monsanto Hardness tester Cadmach Machineries, Ahamdaded
Extruder – RRE/EXT – 65/037 R.R. Enterprises, Thane
Spheronizer – RE/SPH- 150/010 R.R. Enterprises. Thane
Hitachi Scanning Electron Micro Scope,
Hitachi,Tokyo, Japan
Model S-3700N

Table - 3 

96 
 
 Materials and Instruments

 
MATERIALS AND INSTRUMENTS

LIST OF CHEMICAL USED

Andrographolide Biomax, India, Thane

Andrographis paniculata Local Market, Salem

α-β-Asarone Natural Remedies, Bangalore
Acorus Calamus Local Market, Salem
Hypophyllanthin Spic Limited, Chennai
Phyllanthus amarus Neyyor, Kanyakumari
Reserpine S.D. fine Chemical Ltd, Mumbai
Rauwolfia serpentina Local Market, Salem

Strychnine Spic Limited, Chennai

Strychnous nuxvomica Local Market, Salem
Cross Caramellose Signet Chemicals, Mumbai

Aerosil S.D. fine Chemical Ltd, Mumbai

Micro Crystalline Cellulose (AVICEL PH
Coloron Asia Pvt Ltd
101) MCC
Lactose (Monohydrate) S.D. fine Chemical Ltd, Mumbai
Starch 1500 Coloron Asia Pvt Ltd
Potassium Chloride S.D. fine Chemical Ltd, Mumbai
Talc S.D. fine Chemical Ltd, Mumbai
Magnesium Stearate S.D. fine Chemical Ltd, Mumbai
Potassium dihydrogen Phosphate S.D. fine Chemical Ltd, Mumbai
Ortho phosphoric acid S.D. fine Chemical Ltd, Mumbai
Sodium Hydroxide Pellets S.D. fine Chemical Ltd, Mumbai
Hydrochloric acid S.D. fine Chemical Ltd, Mumbai
Methanol HPLC grade S.D. fine Chemical Ltd, Mumbai
Actonitrile HPLC grade S.D. fine Chemical Ltd, Mumbai

Table- 2

95 
 
 Materials and Instruments

 
LIST OF EQUIPMENTS USED

EQUIPMENT MAKE
UV/ Visible Spectrophotometer Shimadz – 160 A, USA
Centrifuge Perkin – Elmer – 1600, USA
Vacuum Pump Toshnival, Chennai
Digital pH Meter Systronics, Mumbai
Electronic Balance Sartronics India, Bangalore
Tablet Friabilator Electro lab India, Mumbai
Dissolution Tester Electro lab India, Mumbai
Tablet disintegrator Electro lab India, Mumbai
Orbital Shaker Electro lab India, Mumbai
Rotary Compression Machine (10 Station) Rimek instruments, Ahmedabad
HPTLC CAMAG Linomat IV, Switzerland
HPLC Shimadzu – LC20AD, USA
Monsanto Hardness tester Cadmach Machineries, Ahamdaded
Extruder – RRE/EXT – 65/037 R.R. Enterprises, Thane
Spheronizer – RE/SPH- 150/010 R.R. Enterprises. Thane
Hitachi Scanning Electron Micro Scope,
Hitachi,Tokyo, Japan
Model S-3700N

Table - 3 

96 
 
 Methodology

METHODOLOGY

EXTRACTION AND QUANTIFICATION OF EXTRACTS THROUGH

HPTLC
Extraction of crude drugs19, 20

Extracts of all the individual drugs were obtained by maceration technique. Hydroalcohol
was used as the solvent, since it possess the optimum solubility characteristics required
for extraction. A known weight of the powdered drug was taken for extraction with water
and ethyl alcohol (50:50) ratio in a flat bottomed maceration flask and subjected to
maceration. The maceration flask was kept at room temperature protected from sunlight
and shaken several times daily. At the end of the 7th day, the extract was filtered,
concentrated and stored in a desiccator for further studies. The percentage yield of the
extracts obtained was calculated and reported.

Quantification of the extract by HPTLC

The extracts were quantified using standard drugs in the following stages.

a. Preparation of Samples

100mg of the selected extracts were weighed accurately and dissolved in 10ml of
methanol in a volumetric flask. The flask was shaken for 30 min, the solution filtered
through Whatman filter paper and the final volume was made up to 10 ml with methanol.

b. Preparation of Standard solutions

Accurately weighed 100 mg of extracts were dissolved in 10 ml of methanol.

From this 1.0 ml was diluted to 20ml methanol to produce 0.5 mg/ml. The standard drugs
solution was also prepared at a concentration of 0.5 mg/ml with methanol in the similar
manner and used for the analysis.

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 Methodology

c. Application of standard and sample solutions

For the application of the solutions, pre-coated plates of 4 x 10 cm size (Silica gel
60 F 254, E.MERC) were used. The standards and the sample solutions were applied on
different tracks of the plate. A thin band of 6mm width was applied using Linomat IV
(Automatic TLC applicator, CAMAG, Switzerland).

d. Chromatogram development and Densitometric scanning

With the help of the suitable solvent system selected for the quantification of the
selected extracts the plates were developed in the twin trough chamber. After the
development of the chromatogram, plates were taken out, dried using hair drier and
observed under UV light. The developed plates were then scanned using densitometer
followed by quantification of the extracts with reference to standard solution. The
suitable solvent system selected for the quantification of the selected active compounds in
their extracts and the wavelengths.

SOLVENT SYSTEM FOR THE HERBAL EXTRACTS FOR HPTLC

Sl.No. Extract Active Solvent System Wave
Compound length
Acetone:
Chloroform:
1. Andrographis paniculata Andrographolide 273 nm
Benzene
7:2:1(V/V/V)
Toluene: Ethyl
2. Acorus Calamus α-β-Asarone acetate 254 nm
93:7(V/V)
Hexane: Acetone:
3. Phyllanthus amarus Hypophyllanthin Ethyl acetate 235 nm
74:12:8 (V/V/V)
Toluene: Ethyl
acetate : De
4. Rauwolfia serpentina Reserpine 254 nm
ethylamine
7:2:1(V/V/V)
Toluene: Ethyl
acetate : De
5. Strychnous nuxvomica Strychnine 254 nm
ethylamine
7:2:1(V/V/V)
Table – 4

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 Methodology

PREFORMULATION STUDIES

Preformulation may be described as a phase of the research and development process

where the formulation scientist characterizes the physical, chemical and mechanical
properties of new drug substance, Preformulation, phase begins early in the discovery
process such that new chemical entities that enter the development process. During this
evaluation possible interaction with various inert ingredients intended for use in final
dosage form are also considered. In the present study, therefore, calibration of
Andrographis paniculata, Acorus Calamus, Phyllanthus amarus, Rauwolfia serpentina,
Strychnous nuxvomica and compatibility with the selected excipients using FTIR peak
matching were carried out. It was also done for the extracts.

1. Development of Calibration Curve for Andrographolide

A stock solution of Andrographolide was prepared by dissolving 100mg of drug
in 0.1N HCL pH 1.2 buffer. From this stock solution, 0,10, 20,30,40,50 µg/ml dilutions
were prepared using 0.1N HCL pH 1.2 buffer. The λmax 210nm of the drug was
determined by scanning one of the dilutions between 400 and 200 nm using UV-visible
spectrophotometer. At this wavelength, the absorbance of all the other solutions was
measured against a blank. Standard curve between concentration and absorbance was
plotted and the intercept (B) and slope (K) values were noted.

2. Development of Calibration Curve for α-β-Asarone

50 mg of α-β-Asarone was accurately weighed and transferred into a 50 ml
volumetric flask. Sufficient quantity of methanol was added to dissolve the drug and the
remainder of the volume was adjusted with methanol, to give a stock solution of 1 mg/ml
of α-β-Asarone. Standard solutions ranging from 1-5 µg /ml of α-β-Asarone were
prepared using methanol. The wavelength range of α-β-Asarone was scanned between
200-400 nm and the absorbances were noted using a Schimadzu 160 A
Spectrophotometer at 256 nm, using methanol as blank. Taking concentration on X-axis
and absorbances on Y-axis plotted calibration curve.

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 Methodology

3. Development of Calibration Curve for Hypophyllanthin 

A stock solution of Hypophyllanthin was prepared by dissolving 100 mg of drug

in 100ml of phosphate buffer of pH 7.2 (1mg/ml). From this stock solution, 0,1,2,3,4 and
5 µg/ml dilutions were prepared using the phosphate buffer of pH 7.2. The λmax 272nm

of the drug was determined by scanning one of the dilutions between 400 and 200 nm
using a UV-visible spectrophotometer. At this wavelength, the absorbance of all the other
solutions was measured against a blank. Standard curve between concentration and
absorbance was plotted and the intercept (B) and slope (K) values were noted.

4. Development of Calibration Curve for Reserpine 

A stock solution of the reserpine was prepared by dissolving 2mg of it in 25 ml of

0.1N glacial acetic acid of pH 3.2(80. µg/ml). Aliquote dilutions of this stock solution
was made with 0.1N glacial acetic acid to get the concentration of 4,8,12,16, and 20
µg/ml. The λmax of the drug was determined by scanning one of the dilutions between

200-400nm using a UV-visible spectrophotometer. A the determined λmax the

absorbance of all the other solutions were recorded against a blank. Standard curve was
obtained by plotting absorbance v/s concentration and the intercept (B) and slope (K)
values were noted.

5. Development of Calibration Curve for Strychnine

A stock solution of Strychnine was prepared by dissolving 100 mg of drug in 100 ml of

0.1N HCl buffer of pH 1.2 (1mg/ml). From this stock solution, 0,1,2,3,4,5 μg/ml
dilutions were prepared using the 0.1N HCl buffer of pH 1.2. The λmax 250nm of the drug
was determined by scanning one of the dilutions between 400 and 200 nm using a UV-
visible spectrophotometer. At this wavelength, the absorbance of all the other solutions
was measured against a blank. Standard curve between concentration and absorbance was
plotted and the intercept (B) and slope (K) values were noted.

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 Methodology

COMPATIBILITY STUDIES

Infrared spectra matching approach was used for detection of any possible chemical
interaction between the drug and the excipients. A physical mixture of the pure
drug/extract and excipients was prepared and mixed with suitable quantity of potassium
bromide. About 100 mg of this mixture was compressed to form a transparent pellet
using a hydraulic press at 15 tons pressure. It was scanned from 4000 to 400 cm-1 in a
perkin Elmer FTIR spectrophotometer. The IR spectrum of the physical mixture was
compared with those of pure drug/extract and excipients and matching was done to detect
any appearance or disappearance of peaks. The same procedure was applied for all the
selected drugs.

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 Methodology

FORMULATIONS AND EVALUATIONS OF TABLETS

Preparation of Matrix Tablets (Andrographolide)

Tablet formulations were prepared by wet granulation technique. The composition

of various formulations is taken. All the powder was passed through BSS – 80 mesh.
Required quantities of Andrographolide and polymer HPMC K100 were mixed in a
polybag, and mixer was passed through mess no 120 in cases of matrix tablets.
Granulation was done using a solution of PVP K30 in sufficient isopropy1 alcohol. After
enough cohesiveness was obtained, the mass was sieved through 10 mesh. The granules
were dried at 40ºC for 30 mins and then were sized by mesh no 16. Talc and magnesium
stearate were finally added as glidant and lubricant. The tablets were compressed (8 mm
diameter, standard concave punches) using a Rotary tablet compression machine (10
station, Rimek, Ahmedabad, India). Eight formulations were prepared by compressing
with the dose of 100 mg andrographolide as extract and 5 mg as pure form.

Prior the compression, eight different formulas, having different concentration of

HPMC K100 (0, 0, 20, 30, 40, 20, 30 & 40 respectively) the granules were evaluated for
drug release, angle of repose, bulk density and compressibility index.

Evaluation of Granules

Angle of Repose

The angle of repose of granules was determined by the funnel method. The
accurately weighed granules were taken in a funnel. The height of the funnel was adjusted
in such a way that the tip of the funnel just touched the apex of the heap of the granules.
The granules were allowed to flow through the funnel freely onto the surface. The
diameter of the powder cone was measured and angle of repose was calculated using the
following equation.

Tan θ = h/r

Where h and r are the height and radius of the powder cone.

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 Methodology

Bulk Density

Density is determined by liquid displacement method by pycnometer. Both loose

bulk density (LBD) and tapped bulk density (TBD) were determined. A quantity of 2 g of
powder from each formula, previously lightly shaken to break any agglomerates formed,
was introduced into a 10-ml measuring cylinder. After the initial volume was observed,
the cylinder was allowed to fall under its own weight onto a hard surface from the height
of 2.5 cm at 2-second intervals. LBD and TBD. were calculated using the following
formulas:

LBD = weight of the powder/volume of the packing

TBD = weight of the powder/tapped volume of the packing.

Hausner’s ratio

The Hausner’s ratio is another index of the flowability of the pellets. It is

calculated using the following formula,

Hausner’s Ratio = Vbefore/Vtapped

Hausner’s ratio closer to 1 indicates good flow properties.

compressibility Index

The compressibility index of the granules was determined by carr’s

compressibility index’s

Carr’s index (%) = [(TBD – LBD) x 100]/TBD

Total Porosity

Total porosity was determined by measuring the volume occupied by a selected

weight of a powder (Vbulk) and the true volume of granules (the space occupied by the

powder exclusive of spaces greater than the intermolecular space.

Porosity (%) = Vbulk – V/Vbulk x 100

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 Methodology

Drug Content

An accurately weighed amount of powdered andrographolide granules (100 mg)

was extracted with water and the solution was filtered through 0.45-µ membrane (Nunc,
New Delhi, India). The absorbance was measured at 225 nm after suit-able dilution.

Evaluation of Tablets

Thickness

The thickness of the tablets was determined using a thick-ness gauge (Mitutoyo,
New Delhi, India). Five tablets from each batch were used, and average values were
calculated.

Weight Variation Test

To study weight variation, 20 tablets of each formulation were weighed using an

electronic balance (Denver APX-100 Arvada, Colorado), and the test was performed
accord-ing to the official method.

Drug Content

Five tablets were weighed individually; the drug was extracted in water. The drug
content was determined as de-scribed above.

Hardness and Friability

For each formulation, the hardness and friability of 6 tablets were determined
using the Monsanto hardness tester (Cad-mach, Ahmedabad, India) and the Roche
friabilator (Camp-bell Electronics, Mumbai, India),respectively.

Determination of Viscosity

Viscosity of the aqueous polymeric solution (0.5, 1, 2% wt/vol) was determined

using the Brookefield viscometer (Brooke-field Engineering Laboratories, Stoughton,
MA).

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 Methodology

Swelling and Eroding Behavior

For the evaluation of a matrix tablets swelling and eroding behavior is very
essential. The mechanism of drug release from hydrophilic polymeric matrices involves
solvent penetration, hydration and swelling of the polymer, diffusion of the dissolved drug
in the matrix, and erosion of the gel layer. Initially, the diffusion coefficient of drug in the
dehydrated polymer matrix is low; it increases significantly as the polymer matrix imbibes
more and more water and forms a gel, as time progress. The hydration rate of the polymer
matrix, and thereby the gel formation and subsequent erosion depends significantly on
polymer proportion, viscosity, and to a lesser degree on polyme4r particle size. So
swelling and erosion studies were performed according to the method reported by Al-
Taani and Tashtoush, to understand the influence of swelling and erosion behavior on
drug release and also to determine the effect of polymer viscosity on swelling and erosion.
Matrix tablets were introduced into the dissolution apparatus under the standard set of
condition as specified for release rate studies. The tablets were removed using a small
basket, and the swollen weight of each tablet was determined. To determine matrix
erosion, swollen tablets were dried in a vacuum oven at 45-C to a constant weight.
Swelling (%) and erosion (%) were calculated according to the following formulas:

% Swelling ¼ S=R 100 ð4Þ

% Erosion ¼ ðT-RÞ=T 100 ð5Þ

Where, S is the weight of the matrix after swelling;

R is the weight of the eroded matrix; and

T is the initial weight of matrix.

In Vitro drug Release Studies

The In Vitro dissolution studies of the developed formulation were carried out using USP
apparatus type II (Electro lab, Mumbai, India) at 50 rpm. The dissolution medium
consisted of 900 ml of 0.1N HCl acidic buffer pH 1.2 from 0 to 12 hours for the
developed formulation maintained at 37°C ± 0.5°C. The drug release at different time
105 

 
 Methodology

intervals was measured by UV-visible spectrophotometer (Shimadzu) at λ max. It was

made clear that none of the ingredients used in the matrix formulations interfered with the
assay. The release studies were conducted in triplicate and the mean values were plotted
versus time.

Stability studies of the tablets

The international conference on Harmonization (ICH) Guidelines titled “Stability

testing of new drug substances and products” (QIA) describes the stability test
requirements for drug registration application in the European Union, Japan and United
States of America. ICH specifies the length of study and storage conditions.

™ Long-term testing: 25ºC ± 2ºC/60% RH ± 5% for 12 months.

™ Accelerated testing: 40ºC ± 2ºC/75% RH ± 5% for 6 months.

Stability studies for the present work carried out at 25ºC/60% RH and 40ºC/75% RH for
6 months and evaluated for their physical appearance and drug content at specified
intervals of time. The physical parameters of the dosage forms; were; evaluated at
different time intervals.

Formulation of Conventional Tablets (Strychnine)

Tablet formulation was prepared by direct compression technique. All the powders were
passed through BSS-80 mesh. Required quantities of Strychnine and excipients (120 #)
were mixed thoroughly. The tablets were compressed (12 mm diameter, standard concave
punches) using a Rotary tablet compression machine (10 station, Rimek, Ahmedabad,
India). Prior the compression, the powder mixture was evaluated for angle of repose, bulk
density and compressibility index, drug content.

Evaluation of Powder

Angle of Repose

The angle of repose of powder was determined by the funnel method. The accurately
weighed powder was taken in a funnel. The height of the funnel was adjusted in such a
way that the tip of the funnel just touched the apex of the heap of the powder. The

106 

 
 Methodology

powder was allowed to flow through the funnel freely onto the surface. The diameter of
the powder cone was measured and angle of repose was calculated using the following
equation

tan θ = h / r

where h and r are the height and radius of the powder cone.

Bulk Density

Both loose bulk density (LBD) and tapped bulk density (TBD) were determined. A
weighed quantity of powder, previously lightly shaken to break any agglomerates
formed, was introduced into a 50-mL measuring cylinder. After the initial volume was
observed, the cylinder was allowed to fall under its own weight onto a hard surface from
the height of 2.5 cm at 2-second intervals. The tapping was continued until no further
change in volume was noted. LBD and TBD were calculated using the following
formulas.

LBD = weight of the powder / volume of the packing

TBD = weight of the powder / tapped volume of the packing

Compressibility Index

The compressibility index of the powder was determined by Carr’s compressibility index.

Carr’s index (%) = [TBD-LBD] × 100/ TBD

Drug Content
An accurately weighed amount of mixture of Strychnine and excipients (100 mg) was
extracted with pH 1.2 (0.1N HCl) buffer and the solution was filtered through 0.45μ
membrane. The absorbance was measured at λ max after suitable dilution.

Evaluation of Tablets
Average weight
To study weight variation, 20 tablets of each formulation were weighed using an
electronic balance (Sartorius India, limited), and the test was performed according to the
U.S. pharmacopoeia.
107 

 
 Methodology

Drug Content

Five tablets were weighed individually, and the drug was extracted in pH 1.2 – 0.1N HCl
buffer. The drug content was determined as described above.

Hardness and Friability

The hardness and friability were determined using the Monsanto hardness tester
(Cadmach, Ahmedabad, India) and the friability testing apparatus (Indian equipments,
Mumbai, India), respectively.

In Vitro drug Release Studies

The In Vitro dissolution studies of the developed formulation were carried out using USP
apparatus type II (Electro lab, Mumbai, India) at 100 rpm. The dissolution medium
consisted of 900 ml of 0.1N HCl acidic buffer pH 1.2 from 0 to 1 hour for the developed
formulation maintained at 37°C ± 0.5°C. The drug release at different time intervals was
measured by UV-visible spectrophotometer (Shimadzu) at λ max. It was made clear that
none of the ingredients used in the tablets formulations interfered with the assay. The
release studies were conducted in triplicate and the mean values were plotted versus time.

Stability studies of the tablets

The international conference on Harmonization (ICH) Guidelines titled “Stability testing

of new drug substances and products” (QIA) describes the stability test requirements for
drug registration application in the European Union, Japan and United States of America.
ICH specifies the length of study and storage conditions.

™ Long-term testing: 25ºC ± 2ºC/60% RH ± 5% for 12 months.

™ Accelerated testing: 40ºC ± 2ºC/75% RH ± 5% for 6 months.

Stability studies for the present work carried out at 25ºC/60% RH and 40ºC/75%
RH for 6 months and evaluated for their physical appearance and drug content at
specified intervals of time. The physical parameters of the dosage forms ;were;
evaluated at different time intervals.

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 Methodology

FORMULATIONS AND EVALUATIONS OF PELLETS (SPHEROIDS)

Preparation of Pellets (Spheroids)

Micro Crystalline Cellulose & Lactose are the most frequently used excipient to aid
aqueous extrusion and pelletinization (spheroniztion) technique, because it forms a
plastic and cohesive mass upon wetting which are the most desired characteristics for
successful pelletinization (spheroniztion). Hence, a basic pellet (Spheroid) formulation
consisting of drug, MCC, croscarmallose and drug, lactose were used. Accurately
weighed quantity of the extract, MCC PH 101, Sodium CMC and extract, lactose in the
optimized ratio were mixed thoroughly and moistened with water to form a viscoelastic
mass (Rajendra et al, 1997). The prepared wet mass was extruded through a roller
extruder (RRE/EXT -65/037) and the extrudates were spheronized in a spheronizer
(RRE/SPH-150/010) at the optimized speed and rounded off in to spherical particles.
The pellets (Spheroids) were dried at 50ºC in a hot air oven for two hours and the dried
pellets (Spheroids) were stored in airtight container and utilized for further studies.

Process flow chart of the extrusion / Pelletinization (spheroniztion) process showing

process variable for each individual step.
Granulation 
Dry Mixing 
• Equipment type 
• Equipment Type  • Fluid type 
• Mixing time  • Fluid level 

Extrusion 
• Equipment Type 
• Die diameter 
• Die Length 

Pelletinization (spheroniztion) Drying 

• Equipment type  • Equipment type 
• Residence time  • Temperature 
• Time 
Figure - 12
109 

 
 Methodology

Evaluation of prepared pellets (Spheroids)

The prepared pellets (Spheroids) were evaluated for the following

™ Morphological characteristic
™ Bulk density
™ True density
™ Porosity Angle of Repose
™ Carr’s consolidation index
™ Friability
™ Drug content uniformity
™ In vitro dissolution study

Morphological characteristic of pellets (Spheroids)

Morphological characteristics of pellets (Spheroids) were observed by SEM.

Bulk density

The bulk density of prepared pellets (Spheroids) was measured by automated tapper. A
weighed amount of pellets (Spheroids) was introduced into a graduated measuring
cylinder. The cylinder was fixed on the bulk density apparatus and timer knob was set for
hundred tapping. After tapping the volume occupied by the pellets (Spheroids) was noted
as a final volume. Then the bulk density is calculated using the formula.

Wt. of sample (g)

Bulk density = ___________________________
Final volume (cc)

True density

True density of the prepared pellets (Spheroids) was determined by liquid

displacement method. The pellets (Spheroids) were taken in a specific gravity bottle

110 

 
 Methodology

containing solvent in which it is insoluble and the true density is calculated using the
formula.

Wt. of sample (W2-W1)

Bulk density = ______________________________________
Wt of solvent displaced by sample (W4-W2)

Where W 1 is weight of specific gravity bottle, W2 is weight of specific gravity

bottle and sample, W3 is weight of sample (W2 - W1) and W4 is weight of specific
gravity bottle with sample and solvent.

Porosity

Porosity is defined as the void volume to bulk volume. It is calculated by using the
formula.

Bulk density
Porosity = 1 - __________
True density

Angle of repose

The angle of repose of granules was determined by the funnel method. The
accurately weighed pellets (Spheroids) were taken in a funnel. The height of the funnel
was adjusted in such a -way that the tip of the funnel just touched the apex of the heap of
the pellets (Spheroids). The granules were allowed to flow through the funnel freely onto
the surface. The circumference of heap was drawn and the mid-point was located and its
radius was measured. The angle of repose was calculated by

Tan θ= h/r

Where h is height of pile, r is radius of the base of pile and θ is the angle of repose.

Carr’s consolidation index

Carr’s consolidation index can be calculated by following formula
Consolidation index (%) = [Tapped density - Fluff density] X 100 /tapped density

111 

 
 Methodology

Fluff density is a ratio of mass of pellets (Spheroids) to the fluff volume. Fluff volume is
the volume occupied by certain mass when gently poured in to measuring cylinder.
Tapped density is the ratio of mass of pellets (Spheroids) to the tapped volume. Tapped
volume is the volume occupied by the same mass of powder after a standard tapping of
measure.

Friability
About 5gm of the prepared pellets (Spheroids) were taken in a friabilator and was
allowed to rotate for 15 min at fixed rpm (25). The percentage of weight loss was studded
as friability.
Initial weight -final weight
% Friability = _________________________x 100
Initial weight

In Vitro Dissolution Testing

A pH 6.8 buffer was employed as the dissolution medium and the study was carried out
for 1hr, at 100RPM. 1 ml samples were withdrawn at pre determined time intervals and
replaced with an equivalent volume of the dissolution medium; each time. The samples
were diluted 10 times with pH 6.8 buffer and the absorbance noted at 257 nm using pH
6.8 as blank.

Stability studies of the pellets (Spheroids)

The formulated pellet dosage forms were packed in polyvinyl chloride (PVC) blisters and
taken for the study. Accelerated stability studies for theses samples were carried out for
six months. Three different temperature and humidity conditions, prescribed by the
International Conference on Harmonization (ICH) for Zone IV climatic zone were
employed namely;

™ 25ºC with 60% relative humidity (RH)

™ 40ºC with 75% relative humidity (RH)

The physical parameters of the dosage forms were evaluated at different period in
different storage conditions and the results were tabulated.
112 

 
 Methodology

FORMULATIONS AND EVALUATIONS OF CAPSULE

The encapsulation of medicinal agents remain a popular method for administering

drugs, as they are easily administer easily filled and masked test of the drug. In 1948

Murdock patented the two-piece hard gelatin capsule. Although development work has

been done since than on the preparation of capsule from methylcellulose and calcium

alginate, gelatin remain the primary composition material for manufacture of capsule

because of its unique properties.

In prescription practice the use of hard gelatin capsule permits a choice in

prescribing a single drug or a combination of drugs at the exact dosage level considered

best for the individual patient. This flexibility is an advantage over tablets. Also some

patient find it easier to swallow capsule than tablets, hence this preference has prompted

pharmaceutical manufacturers to market the product in capsule form even though the

product has already been produced in tablet form, while the industry prepares

approximately 75% of its solid dosage forms as compressed tablets, 23% as hard gelatin

capsule. Market survey have indicated a consume preference of 39.6% for tablet and

19.4% for hard gelatin capsule (Edward and Joseph, 1995).

Hard gelatin capsule

The hard gelatin capsule also referred to as the dry filled capsule (DFC),

consists of two sections, one slipping over the other thus completely surrounding the drug

formulation. These capsules are filled by introducing the powder material into the longer

end or body of capsule and then slipping on the cap. Hard gelatin capsules contain 12-

16% water. However, water content can vary depending on the storage conditions when

the humidity is low the capsule become brittle if stored at high humidity the capsule

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 Methodology

becomes flaccid and lose their shape storage in high temperature areas also can affect the

quantity of hard gelatin capsules. Gelatin capsules may protect hygroscopic material from

atmospheric water vapor if stored in suitable packing materials, (Brian, 1990). Capsules

are supplied in variety of sizes the hard empty capsules are numbered from 000 the

largest size, which can be swallowed, to 5th this may vary depending on the different

densities of powdered drug materials. (Jones, 1987).

The usual procedure to fill capsules is to mix the ingredient by triturating

and reducing them to a fine and uniform powder which is then placed on a- paper and

flattened with a spatula so that the layer of the powder is not greater than, about 1/3 rd the

length of the capsule which is been filled the cap is then removed from selected capsule

and held in the left hand, the body is Pressed repeatedly into the powder until it is filled

after which the cap is replaced and the capsule is weighed ( Van ,1991).

Numbers of manual and automatic capsule filling machines are available for increasing

the speed of capsule filling operation.

Filling of capsules

The three groups of capsule formulation were prepared one for extracts

and others for pure drugs. In case of extracts and the exicipients (1: 4), were mixed

thoroughly in the mortar and this mixture was filled into the hard gelatin capsules shells

(size "0") by usual manual filling procedure were as per pure drugs, were prepared using

the excipients in (1:4) ratio of drug excipients of BSS sieve no. 22 to fill into capsule

shells.

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 Methodology

EVALUATION OF CAPSULES

The evaluation of capsules, in general, follows

1) Drug content uniformity.

2) Weight variation.

3) Disintegration time.

4) In Vitro Dissolution profile

1) Drug content uniformity

In solid dosage forms uniform distribution of medicaments remains a problem.

The problem becomes more acute with potent medicaments administrated in low doses.

Hence, a number of capsules should be selected and assayed for drug content

individually. Pharmacopoeias specify 30 capsules out of which 10 should be assayed

individually in the first instance. Out of these at least 9 should be within -15% of average

and none should be beyond -25%.

2) Weight variation

This test was done by weighing 20 capsules individually, determining average

weight per capsule, and finding out weight variations of each capsule against the average

value -10 % variations are permitted. However, if the variations are beyond this limit net

weight of contents of each capsule should be determined, and compared with average net

weight. This will remove any doubt about the possible variation in the weights of

individual shells.

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 Methodology

3) Disintegration time

The test determines capsules disintegrate within a prescribed time when placed in

a liquid medium under the prescribed experimental conditions. The USP device for

capsule disintegration uses 6 glass tubes 3 inches long, open at the top and held against a

10-mesh screen at the bottom end of the basket rack assembly. To test the disintegrai on

time, the capsules were placed in each tube and the basket rack was positioned in a 1 liter

beaker containing water at 35+2°C, such that the dosage forms remain 2.5 cm below the

surface of the liquid on their upward movement and descend not closer than 2.5 cm from

the bottom of the beaker. A standard motor driven used to move the basket assembly up

and down through a distance of 5.6 cm at a frequency of 28 - 32 cycles/min. perforated

plastic disks may also be placed on top of the capsules and impart an abrasive action

(Gilbart 1991).

IN VITRO DISSOLUTION TESTING

Dissolution

The importance of dissolution rate on clinical performance of drugs and drug

delivery systems has long been recognized. It is the overwhelmingly important property

of dosage forms that contributes to the rate and content of drug availability to the body

and, as such, is deserving of the effort that has been put forth to develop dissolution

system that provide fundamental information on the dissolution process of many drugs

and chemicals as well as meaningful in-vitro dissolution system models that can be

correlated with some index of in-vivo performance.

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 Methodology

Need for dissolution testing:

The development and use of in vitro dissolution test models to evaluate and

describe dissolution and absorption in vivo serves the following purposes:

1) It is likely that the physicochemical properties existing in the model may be of

significance in the in vivo process once a successful model that adequately mimics the in

vivo situation has been developed. As a result, we may obtain a better understanding of

the in vivo environment by the design and operation of a well-characterized in-vitro

model.

2) Such model systems can be used to screen potential drugs and their associated

formulations for their dissolution and absorption characteristics. Meaningful quantitative

screening of the effect of formulation changes and structural modifications can readily be

undertaken once successful in vitro- in vivo correlations have been established by the use

of these models. Even in the absence of in-vivo data or in-vitro & in-vivo correlation,

strictly on the basis of in-vitro data alone, we can predict in a relative manner which form

of the drug or dosage form will result in optimization of a particular desired effect. This

does not necessarily mean accelerated dissolution or complete absorption will be

achieved. Frequently, a sustained effect may be desired; also, the focus of the drug action

may not be systemic, in which case transport of the drug out of the lumen of the

gastrointestinal tract is undesirable.

3) As indicated earlier, in vitro dissolution test systems can serve as quality control

procedures once the form of the drug and its formulation have been finalized. (Umesh V.

Banakar, 1992)

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 Methodology

Dissolution of dosage forms

Dissolution testing of dosage forms (whenever applicable) is considered one of

the most important quality control tools while assessing the efficacy of a product in vitro.

The process of dissolution of an active ingredient from solid pharmaceutical dosage

forms involves several intermediate physicochemical steps, such as wetting, swelling,

capillarity, solubility, and diffusion. Among the most significant factors that control the

process of dissolution are the type and nature of the dosage form within which the active

ingredient is contained.

Dissolution of Capsules

Capsules are still considered the second most popular form of presentation of

medicaments orally. Where all the ingredients are powders, a hard gelatin capsule is used.

The use of hard gelatin capsule is almost axiomatic in the preliminary pharmacologic

study of a new drug before any technological study is even contemplated.

A schematic representation via which a capsule undergoes dissolution and

presents its contents to the gastrointestinal medium is given in following process.

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 Methodology

Process flow chart of the Dissolution of Capsules

DISSOLUTION

DRUG IN CAPSULE DRUG IN CAPSULE MASS

OF CAPSULE SHELL

DRUG PARTICLES IN

SUSPENSION

DRUG IN SOLUTION

DRUG IN BLOOD

Figure - 13

It is apparent from the figure that capsules must have the capsule shell dissolved

before the contents are available to the gastrointestinal fluids for dissolution irrespective

of it being a hard or a soft gelatin capsule. While capsule mass might behave as a tablet,

if it is very compact it is more likely that the capsule contents will behave like a

suspension and distributive quickly into the gastrointestinal fluids. Additionally, since

capsules must allow the drug to go into solution for absorption to occur, the kinetics of

drug release from this dosage form can be critical to its therapeutic success.

Capsule contain not only the drug in solid form, but also other inert ingredients

that are necessary for the manufacture, as well as the physical and chemical stability, of

119 

 
 Methodology

the dosage form. Both the manufacturing process itself and the inert ingredients in the

final product can affect the dissolution rate of the drug from the dosage form.

Depending on the physicochemical characteristics of the drug to be placed in the

capsule shell, various other, ingredients are incorporated in the, formulation. These inert

excipients can influence the dissolution behavior, both rate and extent, of the drug. For

instance, it is important to have suitable diluents in a capsule dosage form of a poorly

soluble drug.

Dissolution Conditions and Sampling Times

1. Characterization of the dosage form over the full range of physiological pH

values is essential ex: pH 1.0, 4 .0, 6.8, and 7.4.

2. It is recommended that different agitation rates be used. This evaluation should

include the standard operating conditions of 50 rpm for the paddle and 100 rpm for the

basket. For solid dosage forms where particles result from disintegration, visual

observation of the dosage form is recommended to detect changes due to increased

agitation such as physical effects or changes in particle location and shape in the

dissolution vessel.

3. In general it is recommended that the media be confined to only aqueous system

rather than hydro-organic ex: Hydro alcoholic systems. For water insoluble drugs,

aqueous systems containing surfactants (Sodium Lauryl Sulphate) should first be

explored. For poorly soluble drugs where sink condition cannot be achieved with the

basket and paddle methods, the flow through apparatus may serve as an appropriate

alternative.

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 Methodology

4. At a minimum, at least three time points are recommended, but more are strongly

encouraged: a one-hour time point to assure that there is no dose dumping, a second time

point at about 50% dissolution, and a third time point at about 80% dissolution. However,

generally it is based to characterize the entire in vitro release profile.

In Vitro Dissolution Studies as Per USP XXIII Procedure

A pH 6.8 buffer was employed as the dissolution medium and the study was
carried out for 12 hrs, at 100 RPM using USP XXIII dissolution testing apparatus type II-
method. 1 ml samples were withdrawn at pre determined time intervals and replaced with
an equivalent volume of the dissolution medium; each time. The samples were diluted 10
times with pH 6.8 buffer and the absorbance noted at 257 nm using pH 6.8 as a blank on
UV-VIS SCHIMADZU 160 A Spectrophotometer.

Stability studies of the capsules

The international conference on Harmonization (ICH) Guidelines titled “Stability

testing of new drug substances and products” (QIA) describes the stability test
requirements for drug registration application in the European Union, Japan and United
States of America. ICH specifies the length of study and storage conditions.

™ Long-term testing: 25ºC ± 2ºC/60% RH ± 5% for 12 months.

™ Accelerated testing: 40ºC ± 2ºC/75% RH ± 5% for 6 months.

Stability studies for the present work carried out at 25ºC/60% RH and 40ºC/75%
RH for 6 months and evaluated for their physical appearance and drug content at
specified intervals of time. The physical parameters of the dosage forms were
evaluated at different time intervals.

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 Methodology

IN VIVO PHARMACOKINETIC STUDIES

As discussed earlier the pharmacokinetic studies are very important to determine the
dosage regimen which leads to give better therapeutic efficacy and safer to patients. For
these studies the animal model chosen was Albino Rabbits.

Animals

Five groups of healthy Albino rabbits weighing 1.5±2.0kg of either sex for each drug
with three rabbits in each group. Albino rabbits were obtained from the CPCSEA
(committee for the purpose of control and supervision of experiments on animal) /
institutional animal ethics committee (I.A.E.C.) PROPOSAL NO. JSSCP / IAEC /
PROJECTS / 01/2008-09. These animal were kept in environmentally controlled Animal
House, J.S.S. College of pharmacy, with the temperature maintained at 37°±2° and a 12 h
light dark cycle, lights on 0700-1900 h for at least one week before use with free access
to water other feed necessary to them. Eighteen hours before the experiments, food was
withheld.

Dose

Dose is calculated according to body weight of rabbit with the help of conversion factor.

Human dose x 0.07 (for 1.5 Kg of Albino rabbits)

Blood Sample Collection

Two milliliter blood sample was directly withdrawn from the ear vein of conscious
rabbits minimally restrained in a rabbit holder. Blood sample were collected at time
interval 0, 0.5,1,2,4,6,8,12,24 and 48 hrs after oral administration of drug. Three animals
were used for each dose tested.

Blood sample was transferred to a blood collection tube which contains sodium citrate
solution (11%) as anticoagulant. the blood sample was centrifuged at 2500 RPM for 15
min by using centrifuge. The resulting plasma were collected in a separate Eppendrof
tube and stored in deep freeze.
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 Methodology

Bio-Analytical method development and Estimation

Reverse phase HPLC method is the most popular mode for analytical and preparative
separations of the compounds in chemical, biological, pharmaceutical and food samples.
In reversed phase mode, the stationary phase is non polar and the mobile phase is polar.
The polar compounds gets eluted first in this mode and non polar compounds are retained
for longer time. In present study, methods for the estimation of the herbal drugs present
in the blood plasma samples were developed and validated. For the estimation of herbal
drugs in blood plasma, the chromatographic variables, namely pH, solvent strength,
solvent ratio, flow rate, addition of peak modifiers in mobile phase, nature of the
stationary phase, detection wavelength and internal standard were studied and optimized
for the separation and retention of the drug.

Preparation of Standard stock solution

50mg of Herbal drug was transferred into a 50ml standard flask and the volume was
made up with mobile phase. This solution was labeled and stored in a refrigerator below
8⁰c.

Preparation of working Standard stock solution

50ml of each 1, 2, 3, 4, 5 µg/ml of herbal drugs working standard solutions were prepared
using the standard stock solution and stored at -20±2⁰c until analysis.

Preparation of Analytical calibration curve samples

10ml of each 0.25, 0.375, 1.0, 1.75, 2.5, 3.5, 4.0, 5.0 µg/ml of the drugs were prepared
with (10µg/ml )or without internal standard.

Preparation of blank plasma

Blank plasma (0.5 ml) was transferred into 2.0 ml centrifuge tube and 0.1 ml of 10 µg/ml
internal standard solution, 0.1 ml of mobile phase and 0.3 ml of precipitating agent were

123 

 
 Methodology

added. The resulting solution was vortexed for 5 mins and centrifuged at 4000 rpm for 10
minutes. The supernatant layer was separated and analysed.

Preparation of Bio-analytical calibration curve samples

0.1 ml of 2.5, 3.75, 10, 17.5, 25, 35, 40, 50 µg/ml of drug solutions were transferred to
2.0 ml centrifuge tube respectively, to this 0.5 ml of plasma, 0.1 ml of internal standard
(100µg/ml), 0.3 ml of precipitating agent were added. The resulting solution was
vortexed for 5 mins and centrifuged at 4000 rpm for 10 minutes. The supernatant layer
was separated and analysed. The calibration curve was obtained.

Preparation of Plasma samples

Plasma samples (0.5ml) obtained from study subjects was transferred into 2.0 ml
centrifuge tube and 0.1 ml of internal standard (100µg/ml), 0.3 ml of precipitating agent
was added. The resulting solution was vortexed for 5 mins and centrifuged at 4000 rpm
for 10 minutes. The supernatant layer was separated and analysed.

Method of analysis

The standard solutions, calibration curve samples and plasma sample solutions were
injected with optimized chromatographic conditions and the chromatograms were
recorded. The quantification of the chromatogram was performed using peak area ratios
of the drug.

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 Methodology

ANDROGRAPHIS PANICULATA AND ANDROGRAPHOLIDE

CHROMATOGRAPHIC CONDITIONS

A Shimadzu® LC - 20AD HPLC, Software LC Version 1.23 was used for the analysis.

Stationary phase : Phenomenex GEMINI C18 (250 x 4.6 mm i.d., 5μ)

Mobile Phase : Methanol : phosphate buffer pH 4.5

Mobile phase ratio : 65:35 % v/v

Flow rate : 1.0 ml/min

Sample volume : 20μl using Rheodyne 7725i injector

Detection : 240 nm using SPD 20AD Diode Array Detector

The mobile phase was filtered through a 0.22μ membrane and degassed using

ultrasonicator. The experiments were carried out at room temperature of about 200C.

Preparation of Solutions for Estimation

1. Preparation of analytical calibration curve

10 mg of Andrographolide working standard was accurately transferred into a 10ml

volumetric flask and dissolved in phosphate buffer 50mM pH3.0 to give 1mg/ml solution
of Andrographolide. The solution was labeled and stored in a refrigerator below 8C.
Aliquot dilution of above stock solution was made to obtain the various working stock
solutions of concentration 100,200,300,400,and 500 ng/ml. The AUC estimated for
above working stock solutions was plotted verses concentration to get the calibration
curve.

125 

 
 Methodology

2. Preparation of blank plasma

Blank plasma (0.5ml) was transferred into 2.0ml centrifuge tube and 1ml of mobile phase
and 0.3ml of precipitating agent were added. The resulting solution was vortexed for 5
mins and centrifuged at 4000 rpm for 10 minutes. The supernatant layer was separated
and analysed.

3. Preparation of bioanalytical calibration curve samples

One ml of 100,200,300,400 and 500 ng/ml of Andrographolide solutions were

transformed to 2ml centrifuge tubes respectively and to this 0.5ml of plasma and 0.3ml of
precipitating agent were added. The resulting solution was vortexed for 5 mins and
centrifuged at 4000 rpm for 10 minutes. The supernatant layer was separated and
analyzed.

4. Preparation of plasma samples

Plasma samples (0.5ml) obtained from study subjects was transferred into 2.0ml
centrifuge tube and 0.3ml of precipitating agent was added. The resulting solution was
vortexed for 5 mins and centrifuged at 4000 rpm for 10 minutes. The supernatant layer
was separated and analysed.

5. Method of analysis
The standard solutions, calibration curve samples and plasma sample solutions were
injected with above chromatographic conditions and the chromatograms were recorded.

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 Methodology

ACORUS CALAMUS AND α-β ASARONE

CHROMATOGRAPHIC CONDITIONS

A Shimadzu® LC - 20AD HPLC, Software LC Version 1.23 was used for the analysis.
Stationary phase : Phenomenex GEMINI C18 (250 x 4.6 mm i.d., 5μ)
Mobile Phase : Acetonitrile- Triethylamine
Mobile phase ratio : 60: 40 % v/v
Flow rate : 1.0 ml/min
Sample volume : 20μl using Rheodyne 7725i injector
Detection : nm using SPD 20AD Diode Array Detector

The mobile phase was filtered through a 0.22μ membrane and degassed using
ultrasonicator. The experiments were carried out at room temperature.

Preparation of Solutions for Estimation

1. Preparation of α-β ASARONE standard stock solution
50mg of α-β Asarone was transferred into a 50ml standard flask and the volume was made
up with mobile phase. This solution was labeled and stored in a refrigerator below 8ºC.

2. Preparation of α-β ASARONE standard solution

50 ml of each 1,2,3,4,5 µg/ml of α-β Asarone working standard solutions were prepared
using the standard stock solution and stored at -20±2ºc until analysis.

3. Preparation of analytical calibration curve sample

10ml of each 0.25,0.375,1.0,1.75,2.5,3.5,4.0,5.0 µg/ml of α-β Asarone were prepared with
internal standard concentration of 10µg/ml.

127 

 
 Methodology

4. Preparation of blank plasma

Blank plasma (0.5 ml) was transferred into 2.0ml centrifuge tube and 0.1ml of 10 µg/ml
internal standard solution, 0.1ml of mobile phase and 0.3ml of precipitating agent were
added. The resulting solution was vortexed for 5 mins and centrifuged at 4000 rpm for 10
minutes. The supernatant layer was separated and analysed.

5. Preparation of bioanalytical calibration curve samples

0.1ml of 2.5, 3.75, 10, 17.5, 25, 35, 40, 50 µg/ml of α-β Asarone solutions were transferred
to 2.0ml centrifuge tube respectively, to this 0.5ml of plasma, 0.1ml of internal standard
(100µg/ml), 0.3ml of precipitating agent were added. The resulting solution was vortexed
for 5 mins and centrifuged at 4000 rpm for 10 minutes. The supernatant layer was
separated and analysed.

6. Preparation of plasma samples

Plasma samples (0.5ml) obtained from study subjects was transferred into 2.0ml centrifuge
tube and 0.1ml of internal standard (100µg/ml), 0.3ml of precipitating agent was added.
The resulting solution was vortexed for 5 mins and centrifuged at 4000 rpm for 10 minutes.
The supernatant layer was separated and analysed.

7. Method of analysis
The standard solutions, calibration curve samples and plasma sample solutions were
injected with above chromatographic conditions and the chromatograms were recorded.

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 Methodology

PHYLLANTHUS AMARUS AND HYPOPHYLLANTHIN

CHROMATOGRAPHIC CONDITIONS

A Shimadzu® LC - 20AD HPLC, Software LC Version 1.23 was used for the analysis.

Stationary phase : Phenomenex GEMINI C18 (250 x 4.6 mm i.d., 5μ)

Mobile Phase : Acetonitrile and Water Mobile
phase ratio : 75:25
Flow rate : 1.0 ml/min
Sample volume : 20μl using Rheodyne 7725i injector
Detection : 233 nm using SPD 20AD Diode Array Detector

The mobile phase was filtered through a 0.22μ membrane and degassed using
ultrasonicator. The experiments were carried out at room temperature.

Preparation of Solutions for Estimation

1. Preparation of analytical calibration curve
10mg of Hypophyllanthin working standard was accurately transferred into a 10ml
volumetric flask and dissolved in phosphate buffer 50 mM (pH3.0) to give 1mg/ml solution
of Hypophyllanthin. The solution was labeled and stored in a refrigerator below 8ºc.
Aliquot dilution of above stock solution was made to obtain the various working stock
solutions of concentration 10,20,30,40, and 50µg/ml. The AUC estimated for above
working stock solutions was plotted verses concentration to get the calibration curve.

2. Preparation of blank plasma

Blank plasma (0.5 ml) was transferred into 2.0ml centrifuge tube and 1ml of mobile phase
and 0.3ml of precipitating agent were added. The resulting solution was vortexed for 5
mins and centrifuged at 4000 rpm for 10 minutes. The supernatant layer was separated and
analysed.

129 

 
 Methodology

3. Preparation of bioanalytical calibration curve samples

One ml of 10,20,30,40 and 50 µg/ml of Hypophyllanthin solutions were transformed to 2ml
centrifuge tubes respectively and to this 0.5ml of plasma and 0.3ml of precipitating agent
were added. The resulting solution was vortexed for 5 mins and centrifuged at 4000 rpm
for 10 minutes. The supernatant layer was separated and analysed.

4. Preparation of plasma samples

Plasma samples (0.5ml) obtained from study subjects was transferred into 2.0ml centrifuge
tube and 0.3ml of precipitating agent was added. The resulting solution was vortexed for 5
mins and centrifuged at 4000 rpm for 10 minutes. The supernatant layer was separated and
analysed.

5. Method of analysis
The standard solutions, calibration curve samples and plasma sample solutions were
injected with above chromatographic conditions and the chromatograms were recorded.

130 

 
 Methodology

RAUWOLFIA SERPENTINA AND RESERPINE

CHROMATOGRAPHIC CONDITIONS
A Shimadzu® LC - 20AD HPLC, Software LC Version 1.23 was used for the analysis.
Stationary phase : Phenomenex GEMINI C18 (250 x 4.6 mm i.d., 5μ)
Mobile Phase : Acetonitrile: phosphate buffer pH 4.5
Mobile phase ratio : 55:45 % v/v
Flow rate : 1.0 ml/min
Sample volume : 20μl using Rheodyne 7725i injector
Detection : nm using SPD 20AD Diode Array Detector

The mobile phase was filtered through a 0.22μ membrane and degassed using
ultrasonicator. The experiments were carried out at room temperature.

Preparation of Solutions for Estimation

1. Preparation of Reserpine standard stock solution
50mg of Reserpine was transferred into a 50ml standard flask and the volume was made up
with mobile phase. This solution was labeled and stored in a refrigerator below 8ºC.

2. Preparation of Reserpine standard solution

50ml of each 10, 20, 30, 40, 50 µg/ml of Reserpine working standard solutions were
prepared using the standard stock solution and stored at 20±2ºC until analysis.

3. Preparation of analytical calibration curve samples

The calibration curve was developed using aliquot solutions of Reserpine ranging from 10-
50µg/ml.

4. Preparation of blank plasma

Blank plasma (0.5ml) was transferred into 2.0ml centrifuge tube and 0.1ml of mobile phase
and 0.3ml of precipitating agent were added. The resulting solution was vortexed for 5

131 

 
 Methodology

mins and centrifuged at 4000 rpm for 10 minutes. The supernatant layer was separated and
analysed.

5. Preparation of bioanalytical calibration curve samples

0.1ml of 10, 20, 30, 40, 50 µg/ml of Reserpine solutions were transferred to 2.0ml
centrifuge tube respectively, to this 0.5ml of plasma and 0.3ml of precipitating agent were
added. The resulting solution was vortexed for 5 mins and centrifuged at 4000 rpm for 10
minutes. The supernatant layer was separated and analysed.

6. Preparation of plasma samples

Plasma samples (0.5ml) obtained from study subjects was transferred into 2.0ml centrifuge
tube and 0.3ml of precipitating agent was added. The resulting solution was vortexed for 5
mins and centrifuged at 4000 rpm for 10 minutes. The supernatant layer was separated and
analysed.

7. Method of analysis
The standard solutions, calibration curve samples and plasma sample solutions were
injected with above chromatographic conditions and the chromatograms were recorded.

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 Methodology

STRYCHNOUS NUXVOMICA AND STRYCHNINE

CHROMATOGRAPHIC CONDITIONS
A Shimadzu® LC - 20AD HPLC, Software LC Version 1.23 was used for the analysis.
Stationary phase : Phenomenex GEMINI C18 (250 x 4.6 mm i.d., 5μ)
Mobile Phase : Acetonitrile and Methanol
Mobile phase ratio : 50:50
Flow rate : 0.65 ml/min
Sample volume : 20μl using Rheodyne 7725i injector
Detection : 254 nm using SPD 20AD Diode Array Detector

The mobile phase was filtered through a 0.22μ membrane and degassed using
ultrasonicator. The experiments were carried out at room temperature.

Preparation of Solutions for Estimation

1. Preparation of analytical calibration curve
10mg of Strychnine working standard was accurately transferred into a 10ml volumetric
flask and dissolved in water and Acetonitrile to give 1mg/ml solution of Strychnine. The
solution was labeled and stored in a refrigerator below 8ºC. Aliquot dilution of above stock
solution was made to obtain the various working stock solutions of concentration 10, 20,
30, 40, and 50 µg/ml. The AUC estimated for above working stock solutions was plotted
verses concentration to get the calibration curve.

2. Preparation of blank plasma

Blank plasma (0.5ml) was transferred into 2.0ml centrifuge tube and 1ml of mobile phase
and 0.3 ml of precipitating agent were added. The resulting solution was vortexed for 5
mins and centrifuged at 4000 rpm for 10 minutes. The supernatant layer was separated and
analysed.

3. Preparation of bioanalytical calibration curve samples

One ml of 10, 20, 30, 40 and 50 µg/ml of Strychnine solutions were transformed to 2ml
centrifuge tubes respectively and to this 0.5ml of plasma andf 0.3ml of precipitating agent

133 

 
 Methodology

were added. The resulting solution was vortexed for 5 mins and centrifuged at 4000 rpm for
10 minutes. The supernatant layer was separated and analysed.

4. Preparation of plasma samples

Plasma samples (0.5ml) obtained form study subjects was transferred into 2.0ml centrifuge
tube and 0.3ml of precipitating agent was added. The resulting solution was vortexed for 5
mins and centrifuged at 4000 rpm for 10 minutes. The supernatant layer was separated and
analysed.

5. Method of analysis
The standard solutions, calibration curve samples and plasma sample solutions were
injected with above chromatographic conditions and the chromatograms were recorded.

Pharmacokinetic analysis

Determination of various pharmacokinetic parameters

The plasma concentration obtained after oral administration of the tablet, pellets and
capsules formulations to rabbit models were subjected to pharmacokinetic analysis. The
various parameters such as maximum plasma concentration (Cmax); time to reach maximum
plasma concentration (Tmax) and area under the curve (AUC0-24 and AUC0-∞), and overall
elimination rate constant (ke) were calculated using PK1 and PK2 software.

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 Results

RESULTS

Quantification of Andrographolide, α- Asarone, β-Asarone, Hypophyllanthin,

Reserpine, Strychnine 

Quantification Analysis of compound separated by HPTLC can be carried by

estimating the area of Band detection and results showed that 100mg of
Andrographis paniculata, Acorus Calamus, Phyllanthus amarus, Rauwolfia
serpentina and Strychnous nuxvomica, extract contains 4.563mg, 5.1mg, 6.17 mg,
1.16mg, 0.345mg, & 10.0 mg of pure Andrographolide, α- Asarone, β-Asarone,
Hypophyllanthin, Reserpine and Strychnine respectively.

Andrographolide α-Asarone β-Asarone Hypophyllanthin Reserpine Strychnine

Sample
(%w/w) (%w/w) (%w/w) (%w/w) (%w/w) (%w/w)

Extract
4.563 mg 5.1 mg 6.17 mg 1.16 mg 0.345 mg 10.0 mg
(100mg)

Table -5

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 Results

Typical densitogram of Andrographis Paniculata

Wavelength : 273 nm [Rf] 
 

Figure- 14

Typical densitogram of Acorus Calamus

Wavelength : 254 nm  [Rf] 
 

Figure- 15

136 

 
 Results

Typical densitogram of Phyllanthus amarus

[Rf] 
Wavelength: 235 nm

Figure- 16

Typical densitogram of Rauwolfia Serpentina

Wavelength : 254 nm [Rf] 

Figure- 17

137 

 
 Results

Typical densitogram of Strychnous nuxvomica

 
 
AU 
 

[Rf]
Wavelength :254  nm 

Figure- 18

138 

 
 Resullts

Preformulaation Studiees: The calibbration curvve for Androographolide, α- Asaronee, β-

Asarone, Hy
ypophyllanth
hin, Reserpinne and Strycchnine

The Calibratioon Data for Andrograp

pholide

  Sl.N
No.  C
Concentrat
tion in µg/m
ml Abssorbance at 
210nm 
 

  1  1
10 0.108 

 
2  2
20 0.216 
 
3  3
30 0.319 
 

  4  4
40 0.416 
 
5  5
50 0.524 
 

Table - 6

Thee Calibration Curve forr Andrograp

pholide

Y=0
0.010x  +0.00
04 
R2 = 0.987  

C
Concentration
n (µg/ml)

Figure - 19
1

13
39 

 
 Results

The Calibration Data for α- Asarone

Sl.No.  Concentration in µg/ml Absorbance at 

256nm 

1  1.0 0.002 

2  2.0 0.0169 

3  3.0 0.040 

4  4.0 0.070 

5  5.0 0.0798 

Table - 7

The Calibration Curve for α- Asarone

Y=0.010x  +0.006 
R2 = 0.999  

Concentration (µg/ml)

Figure - 20

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 Results

The Calibration Data for β - Asarone

Sl.No.  Concentration in µg/ml Absorbance at 

256nm 

1  1.0 0.001 

2  2.0 0.0167 

3  3.0 0.038 

4  4.0 0.062 

5  5.0 0.0727 

Table – 8

The Calibration Curve for β - Asarone

Y=0.010x  +0.006 
R2 = 0.998  

Concentration (µg/ml)

Figure - 21

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 Results

The Calibration Data for Hypophyllanthin

Sl.No.  Concentration in µg/ml Absorbance at 

272nm 

1  1.0 0.054 

2  2.0 0.116 

3  3.0 0.162 

4  4.0 0.209 

5  5.0 0.268 

Table - 9

The Calibration Curve for Hypophyllanthin

Y=0.054x  +0.004 
R2 = 0.994  

Concentration (µg/ml)

Figure – 22

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 Results

The Calibration Data for Reserpine

Sl.No.  Concentration in µg/ml Absorbance at 

267.4nm 

1  4.0 0.098 

2  8.0 0.194 

3  12.0 0.298 

4  16.0 0.390 

5  20.0 0.513 

Table - 10

The Calibration Curve for Reserpine

Figure – 23

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 Results

The Calibration Data for Strychnine

Sl.No. Concentration in µg/ml Absorbance at 250nm

1 1 0.088

2 2 0.154

3 3 0.249

4 4 0.328

5 5 0.436

Table - 11

The Calibration Curve for Strychnine

Figure – 24

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 Results

IR Spectrum of Andrographis Paniculata

Figure – 25

IR Spectrum of Andrographis Paniculata and Excipients

Figure – 26

145 

 
 Results

IR Spectrum of Andrographolide

Figure – 27

IR Spectrum of Andrographolide and Excipients

Figure – 28

146 

 
 Results

IR Spectrum of Acorus Calamus 

Figure – 29

IR Spectrum of Acorus Calamus and Excipients 

Figure – 30

147 

 
 Results

IR Spectrum of α- Asarone

Figure – 31

IR Spectrum of α- Asarone and Excipients

Figure – 32

148 

 
 Results

IR Spectrum of β- Asarone

Figure – 33

IR Spectrum of β- Asarone and Excipients

Figure – 34

149 

 
 Results

IR Spectrum of Phyllanthus Amarus

Figure – 35

IR Spectrum of Phyllanthus Amarus and Excipients

Figure – 36

150 

 
 Results

IR Spectrum of Hypophyllanthin

Figure – 37

IR Spectrum of Hypophyllanthin and Excipients

Figure – 38

151 

 
 Results

IR Spectrum of Rauwolfia Serpentina

Figure – 39

IR Spectrum of Rauwolfia Serpentina and Excipients

Figure – 40

152 

 
 Results

IR Spectrum of Reserpine

Figure – 41

IR Spectrum of Reserpine and Excipients

Figure – 42

153 

 
 Results

IR Spectrum of Strychnous Nuxvomica

Figure – 43

IR Spectrum of Strychnous Nuxvomica and Excipients

Figure – 44

154 

 
 Results

IR Spectrum of Strychnine

Figure – 45

IR Spectrum of Strychnine and Excipients

Figure – 46

155 

 
 Results

CHARACTERISTIC IR PEAKS OF EXTRACTS, PURE DRUG, AND

EXCIPIENTS
Sl. Description Characteristic peaks (cm-1)
No
1. Andrographis 3828.01, 3531.02, 3297.68,2929.34, 2809.78, 2719.14,
Paniculata 1598.70, 1355.71, 1047.16, 773.315, 620.966, 487.902
2. Andrographis 3930.22,3858.86,3747.01,3525.24,3465.46,3342.03,3266.82,
Paniulata and 3212.83,3100.97,3815.56,2732.64,2339.23,2036.46,1592.91,
Excipients 1351.86,1029.80,888.095,719.318,615.181,493.688,430.048
3. Andrographolide 3828.01, 3531.02, 3297.68, 2929.34, 2809.78, 2717.14,
1598.70, 1355.71, 1047.16, 773.315, 620.966, 487.902
4. Andrographolide 3850.32, 3686.29, 3645.67, 3625.63, 2960.44, 2873.47,
and Excipents 2393, 2245.8, 1736, 1612, 1556, 1462.76, 1439.38, 1383.12,
1258.95, 1171.03, 1058.66, 1016.96, 934.28, 842.90, 805.39,
741.12, 714.18, 547.66, 520.56, 428.9
5. Acorus Calamus 3883.93, 3772.08, 3698.80, 3600.45, 3529.09, 3353.60,
3262.97, 3156.90, 2929.34, 2830.99, 2713.35, 1598.70,
1357.64, 1031.73, 965.601, 620.990, 553.470, 443.547
6. Acorus Calamus 3839.58, 3766.30, 3488.60, 3409.53, 3347.82, 3261.04,
and Excipients 2906.20, 2819.42, 2713.35, 2148.31, 1833.97, 1600.63,
1353.78, 1106.94, 1041.37, 773.315, 632.537, 536.114,
468.617
7. α- Asarone 3884.93, 3773.08, 3699.80, 3601.45, 3530.09, 3354.60,
3263.97, 3157.90, 2930.34, 2831.99, 2714.35, 1599.70,
1358.64, 1032.73, 966.601, 621.990, 554.470, 444.547
8. α- Asarone and 3800, 3742, 3650, 3340.06, 3196.48, 2959.42, 2875.09,
Excipients 2359.87, 2342.47, 2246.70, 1748.53, 1653.13, 1607.44,
1591.08, 1497.14, 1464.56,1437, 1414.15, 1380.77, 1255.69,
1177.16, 1059, 1017.44, 932.89, 842.90, 805.39, 741.12,
714.18, 547.66, 520.56, 428.9
9. β- Asarone 3883.93, 3772.08, 3698.80, 3600.45, 3529.09, 3353.60,
3262.96, 3156.88, 2929.32, 2829.96, 2712.32, 1598.71,
1356.60, 1030.72, 965.602, 620.960, 552.450, 443.540
10. β- Asarone and 3798, 3740, 3650, 3338.01, 3192.42, 2956.40, 2872.02,
Excipients 2358.80, 2340.45, 2246.70, 1746.52, 1652.12, 1606.42,
1590.08, 1496.12, 1464.56,1436, 1412.14, 1379.76, 1254.68,
1176.12, 1058, 1016.42, 930.81, 840.90, 804.30, 740.10,
712.15, 546.66, 518.50, 426.8
11. Phyllanthus 3963.00, 3856.93, 3814.51, 3747.01, 3417.17, 3328.53,
Amarus 3266.82, 2927.41, 2819.42, 2726.85, 1814.69, 1743.33,
1592.91, 1351.86, 1243.86, 1051.01, 929.521, 873.596,
784.886, 678.820, 607.467, 536.114, 474.403, 424.263
12. Phyllanthus 3937.93, 3876.22, 3814.51, 3766.30, 3698.80, 3575.38,
Amarus and 3529.09, 3471.24, 3372.89, 3307.32, 3249.47, 3193.54,
Excipients 2898.49, 2811.70, 2719.14, 2640.07, 2572.57, 2339.23,
2159.88, 2067.32, 1988.25, 1864.83, 1708.62, 1583.27,
1446.35, 1375.00, 1326.79, 1220.72, 1168.65, 1106.94,
1027.87, 873.596, 755.959, 696.177, 572.755, 453.190
156 

 
 Results

13. Hypophyllanthin 3839.58, 3741.23, 3502.10, 3421.10, 3191.61, 2925.48,

2825.20, 2713.35, 1598.70, 1353.78, 1052.94, 784.886,
688.463, 603.610, 538.042, 430.048
14. Hypophyllanthin 3902, 3858, 3804, 3748, 3682, 3630, 3590, 3472, 3318,
and Excipients 3274, 3126, 2972, 2942, 2876, 2486, 2362, 2248, 1866,
1722, 1590, 1564, 1442, 1340, 1310, 1240, 1214, 1196,
1166, 1064, 1024, 954.68, 920, 842.09, 754.53, 612,
573.24, 520, 482.
15. Rauwalfia 3978.43, 3866.58, 3814.51, 3741.23, 3464.46, 3382.53,
Serpentina 3316.96, 3128.61, 2991.05, 2917.77, 2807.85, 2713.35,
2572.57, 1594.84, 1452.14, 1353.78, 1211.08, 1052.94,
867.81, 767.530, 522.615, 476.331, 422.334
16. Rauwolfia 3949.50, 3841.51, 3772.08, 3617.80, 3532.95, 3440.38,
Sepentina and 3369.03, 3266.82, 3210.90, 2923.56, 2819.42, 2738.42,
Excipients 2535.93, 2474.22, 2283.30, 1594.84, 1351.86, 1249.65,
1064.51, 767.530, 696.177, 553.47, 499.473, 412.692
17. Reserpine 3839.58, 3741.23, 3502.10, 3421.10, 3191.61, 2925.48,
2825.20, 2713.35, 1598.70, 1353.78, 1052.94, 784.886,
688.463, 603.610, 538.042, 430.048
18. Reserpine and 3853.92, 3690.35, 3649.42, 3649.71, 3138, 2967.16,
Excipients 2877.11, 2730, 2486, 2344, 2245.05, 1748.75, 1617.12,
1560.21, 1470.60, 1440.79, 1385.27, 1371.48, 1344.89,
1248.93, 1172.88, 1104, 988.75, 971.33, 941.40, 912.86,
883.03, 804.50, 714.54, 548.54, 402.84
19. Strychnous 3840.58, 3742.23, 3503.10, 3422.10, 3192.61, 2926.48,
Nuxvomica 2826.20, 2714.35, 1599.70, 1354.78, 1053.94, 785.886,
689.463, 604.610, 539.042, 431.048
20. Strychnous 3964.93, 3833.79, 3764.37, 3625.52,
Nuxvomica and 355031,3473.17,3374.82, 3295.75, 3224.40, 3151.11,
Excipients 2929.34, 2821.35, 2713.35, 2659.36, 2535.93, 2485.79,
2412.51, 2323.80, 2221.59, 2111.67, 2036.46, 1974.75,
1803.12, 1612.20, 1452.14, 1349.93, 1224.58, 1120.44,
1035.59, 887.095, 775.244, 692.320, 620.966, 559.255,
468.617
21. Strychnine 3902.29, 3778.87, 3539.74, 3478.03, 3319.89, 2824.28,
2714.35, 2394.23, 1599.70, 1447.35, 1358.64, 1057.80,
770.458, 697.177, 610.396, 448.404.
22. Strychnine and 3858, 3822, 3800, 3734.66, 3472, 3319.17, 3270.67, 3130,
Excipients 2959.32, 2876.38, 2676.87, 2467.17, 2358, 2340, 2245.50,
1752.67, 1609.69, 1590, 1470.19, 1385.77, 1236, 1170,
1066.94, 989.05, 941.62, 906.17, 882.96, 842.08, 802.95,
790.07, 753.15, 678.85, 572.63, 522.40, 484.84, 457.06,
402.73
Table – 12

The IR spectra obtained were satisfactory with their characteristic absorption bands.
Similarly the physical mixtures also indicated the presence of characteristic peak of
the pure drugs / extracts and excipients.

157 

 
 Results

FORMULATION OF ANDROGRAPHIS PANICULATA MATRIX TABLETS

Andrographolide (200mg) Tablet Formulations

Drugs and % per tablet

Excipients

I II III IV V VI VII VIII

Pure Drug 5 - 5 5 5 - - -
Extract - 100 100 100 100
HPMC - - 20 30 40 20 30 40
K 100
PVP K30 - - 8 8 8 8 8 8
Mg. Stearate 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Talc - - qs. qs. qs. qs. qs. qs.
Starch qs. qs. - - - -  - -

Table – 13

158 

 
 Results

Properties of Granulations

Sl Parameter Batch code

No.
I /II III IV V VI VII VIII
1 Angle of Repose (0) 24.50 21.20 22.10 29.85 24.11 23.95 22.68
(n=5) +0.02 +0.02 +0.01 +0.02 +0.03 +0.01 +0.03

2 Loose Bulk Density 0.506 0.493 0.512 0.289 0.283 0.304 0.289
(g/cm3)(n=5) +0.02 +0.03 +0.04 +0.03 +0.03 +0.02 +0.04

3 Tapped bulk density 0.582 0.555 0.581 0.335 0.325 0.349 0.335
(g/cm3) (n=5) +0.04 +0.03 +0.02 +0.04 +0.06 +0.02 +0.03

4 Hausner’s ratio (n=5) 1.150 1.125 1.134 1.159 1.148 1.148 1.159
( %) +0.03 +0.04 +0.02 +0.03 +0.04 +0.03 +0.04

5 Compressibility index 13.08 11.25 11.82 13.75 12.95 12.92 13.45

(%) (n=5) +0.02 +0.03 +0.03 +0.02 +0.03 +0.04 +0.04
6 Drug content (%) (n=5) 98.55 95.53 96.51 97.54 96.79 98.55 97.62
+0.03 +0.04 +0.03 +0.02 +0.04 +0.02 +0.04
7 Total porosity (%) 27.43 26.97 26.92 37.03 37.61 34.27 37.03
(n=20) +0.03 +0.02 +0.03 +0.03 +0.04 +0.02 +0.04

Table – 14

n = Number of granules.

159 

 
 Results

Properties of Tablets

Sl Parameter Tablets
No.
I /II III IV V VI VII VIII
1 Average weight (mg) 198 201 200 199 199 200 2001
(n=20) +1.5 +1.8 +2.4 +1.5 +1.6 +1.8 +1.7

2 Drug content (%) (n=5) 99.34 98.35 98.35 99.09 97.78 98.20 96.69
+0.04 +0.05 +0.06 +0.02 +0.04 +0.05 +1.07

3 Hardness (kg/cm2) 4 6 6 7 5 5 6
(n=10) +0.5 +0.5 +0.5 +0.5 +0.5 +0.5 +0.5

4 Friability (%) (n=10) >0.5 <0.5 <0.5 <0.5 <0.5  <0.5  <0.5

5 Disintegration Time 3+2 12+3 14+2 16+3 11+3  14+2  15+2

(mins) (n=8)

Table – 15

n = Number of tablets.

160 

 
 Results

Invitro Release of Conventional Tablets

For Isolated Drug

Sl. No. Time Absorbance Concentration Isolated drug (I)

(min) (nm) (µg/ml)
Cumulative % drug
release
1 0 0 0 0
2 5 0.078 2.834 10.26
3 10 0.136 4.692 16.93
4 15 0.251 8.439 30.46
5 20 0.378 12.578 45.36
6 30 0.502 16.618 59.91
7 45 0.739 12.578 87.78
8 60 0.844 27.263 99.01

Table – 16

For Extract (Andrographis Paniculata)

Sl. No. Time (min) Absorbance Concentration Extracted drug

(nm) (µg/ml) (II)
Cumulative %
drug release
1 0 0 0 0
2 5 0.169 2.05 18.55
3 10 0.321 3.28 29.59
4 15 0.449 4.72 42.57
5 30 0.877 7.52 67.98
6 45 1.009 8.35 79.71
7 60 1.217 10.53 95.87

Table – 17

161 

 
 Results

In Vitro Release of Conventional Tablets

Figure - 47

In Vitro Release of Matrix Tablets

Sl.No. Time Cumulative % release

(Hrs)
Isolated drug Extracted drug
III IV V VI VII VIII
1 0 0 0 0 0 0 0
2 0.25 15.87 12.87 13.82 17.78 18.10 20.48
3 0.5 23.98 27.83 19.18 24.85 27.23 28.45
4 0.75 30.54 36.52 32.04 27.57 29.34 34.92
5 1 45.76 43.65 38.92 39.78 39.47 43.13
6 2 52.90 57.94 50.30 56.37 54.65 51.43
7 4 65.83 67.04 57.89 68.37 64.56 64.23
8 6 79.39 76.84 65.81 78.24 73.98 71.65
9 12 83.93 84.46 72.78 92.53 91.23 78.34
10 15 98.95 97.86 83.52 95.02 94.56 82.90

Table – 18

162 

 
 Results

In Vitro Release of Matrix Tablets

Figure - 48

163 

 
 Results

FORMULATIONS OF ACORUS CALAMUS CAPSULES

For Acorus Calamus extract and α- β-Asarone

Sl. No. Ingredients Formulation Formulation Formulation

I (500mg) II (500mg) III (500mg)

1 α- Asarone 100mg ---- ----

2 β - Asarone ----- 100mg ----

3. Acorus Calamus extract ---- ----- 100mg

4 Magnesium stearate 50mg ----- ----

5 Lactose 350mg 400mg 400mg

Table – 19

Evaluation of Granules

Sl. No. Formulations Granule/ Bulk density of Angle of

particle size granules/powder repose

(mm) (gm/cm3) (0)

1. Formulation F1 830+25.2 0.624 27.60

2. Formulation FII ---- 0.505 31.27

3. Formulation FIII ---- 0.531 25.30

Table – 20

164 

 
 Results

Evaluation of Capsules

Sl. Formulations Weight Drug content Disintegration Moisture

No. variation uniformity time (min) uptake

(mg) (%) (%)

Formulation
1. 500±9 98.90 6-7 0.075
F1

Formulation
2. 500±7 97.23 3-4 0.005
FII

Formulation
3. 500±9 99.30 3-4 0.004
FIII

Table – 21

165 

 
 Results

In Vitro Drug Release of Capsules

TIME OF FORMU- CONCENTRATION CUMULATIVE %
HOURS LATIONS IN(µg/ml) RELEASE
0.00 0.00 0.00 0.00
0.25 FI 1.55 14.01
FII 1.38 12.42
FIII AC(α):1.20, AC-(β): 1.18 AC(α):10.84, AC-(β): 10.80
0.50 FI 2.65 23.86
FII 2.29 20.63
FIII AC(α):1.99, AC-(β): 1.96 AC(α):17.96, AC-(β): 17.95
1.00 FI 3.90 35.13
FII 3.63 32.69
FIII AC(α):3.20, AC-(β): 3.18 AC(α):28.87, AC-(β): 28.84
1.5 FI 5.36 48.24
FII 4.93 44.38
FIII AC(α):4.39, AC-(β): 4.36 AC(α):39.56, AC-(β): 39.54
2.00 FI 6.75 60.80
FII 6.32 56.91
FIII AC(α):5.68, AC-(β): 5.66 AC(α):51.16, AC-(β): 51.14
3.00 FI 9.00 81.08
FII 8.53 76.80
FIII AC(α):7.58 , AC-(β): 7.55 AC(α):68.30, AC-(β): 68.27
4.00 FI 10.91 85.26
FII 10.68 83.16
FIII AC(α):9.45, AC-(β): 9.42 AC(α):80.10, AC-(β): 79.99
5.00 FI 10.92 86.16
FII 10.69 84.28
FIII AC(α):10.99, AC-(β): 10.97 AC(α):81.26, AC-(β): 80.88
6.00 FI 10.95 87.68
FII 10.87 85.86
FIII AC(α):11.06, AC-(β):11.05 AC(α):82.26, AC-(β):81.98
8.00 FI 10.95 89.56
FII 10.89 87.36
FIII AC(α):11.08, AC-(β): 11.06 AC(α):83.98, AC-(β):82.92
10.00 FI 10.97 92.19
FII 10.90 91.10
FIII AC(α):11.10, AC-(β): 11.09 AC(α):89.25, AC-(β):84.98
12.00 FI 10.98 98.12
FII 10.92 97.10
FIII AC(α):11.12, AC-(β): 11.10 AC(α):90.98, AC-(β): 90.56

Table – 22

AC – Acorus Calamus

166 

 
 Results

Cumulative percentage Drug release of Acorus Calamus

extract and α – β Asarone capsules

Figure - 49

167 

 
 Results

FORMULATIONS OF PHYLLANTHUS AMARUS SPHERIODS

Sl. No. Ingredients Formulation I (H1) Formulation II

(500mg) (H2) (500mg)

1 Hypophyllanthin ---- 100 mg

2 Phyllanthus Amarus Extract 100mg ----

2 Lactose 400mg 400mg

4 Starch Paste (10%) q.s q.s

Table – 23

Optimization of the Spheronization technique of Phyllanthus Amarus Spheroids

Drug: Residence Speed Description of Spheroids

Lactose time (min.) (rpm)
ratio
500 Brittle and rod shaped
1000 Brittle and rod shaped
80:20
15 1500 Brittle and rod shaped with fines
(H1)
2000 Brittle and rod shaped with fines
500 Rod and rope shaped
1000 Rod and rope shaped
60:40
15 1500 Rod and dumbbell
(H2)
2000 Ellipsoid and rope shaped with fines

Table – 24

Residence time refers to spheronization residence time and speed refers to disc
speed of the spheronizer.

168 

 
 Results

Evaluation of Spheroids

Weight Moisture
Sl. Particle size Bulk density Friability Disintegration
Formulations Variation uptake
No. (mm) (gm/cm3) (%) time (min)
(mg) (%)

1. Formulation I (H1) 1.32 ± 0.039 0.516 1.984 500 ± 7 5-7 0.004

2. Formulation II (H2) 1.53 ± 0.04 0.507 0.975 500 ± 9 6-9 0.008

Table – 25

169 

 
 Results

In Vitro Dissolution of Spheroids for Phyllanthus Amarus

TIME IN CONCENTRATION CUMULATIVE %

FORMULATIONS
Mins (µg/ml) RELEASE
H1 0.00 0.00
0.00
H2 0.00 0.00

H1 5.12 16.95
5.00
H2 5.46 13.41

H1 8.23 25.26
10.00
H2 7.59 24.10

H1 6.36 68.56
15.00
H2 6.27 59.15

H1 5.46 85.11
30.00
H2 5.63 74.68

H1 5.38 91.56
45.00
H2 5.34 85.10

H1 4.61 98.16
60.00
H2 4.74 90.97

Table – 26

170 

 
 Results

Cumulative Percentage Drug Release of Phyllanthus Amarus Extract and

Hypophyllanthin

Hypophyllanthin

Phyllanthus
Amarus
Extract

Figure - 50

171 

 
 Results

FORMULATION AND CHARACTERIZATION OF PELLETS RAUWOLFIA

SERPENTINA

Formulations of different batches of pellets containing 0.5 mg of reserpine with different

concentrations of Avicel pH 101

The required quantity of MCC as spheronization enhancer, other excipients and

reserpine were weighed and mixed. To this mixture sufficient quantity of water was added as a
granulating agent to get a wet mass. The solid blend mass was passed through the extruder
model RRE/EXT-65/037 to form extrudates. The formed extrudates were introduced into
spheroniser model RRE/SPH-150/010 to get spherical pellets by varying spheronization speed.

S1.No Ingredient (M1) (M2)

Formulation Formulation
(20mg) (20mg)

1. Reserpine  0.5mg (2.5%)  0.5mg(2.5%) 

2. Avicel pH 101  12mg(60%)  14mg(70%) 
3. Croscarmallose  0.3mg(1.5%)  0.3mg(1.5%) 
4. Lactose  7mg  5mg 
5. Magnesium stearate 0.2mg  0.2mg 

Table – 27

172 

 
 Results

Formulations of different batches of pellets containing Rauwolfia Serpentina root extract

containing Reserpine equivalent to 0.5mg with different concentrations of avicel pH101.

The required quantity of MCC as spheronization enhancer, other excipients and extract
of Rauwolfia serpentina root containing reserpine equivalent to 0.5mg were weighed and
mixed. To this mixture sufficient quantity of 1.25% w/v of Sodium CMC was added as
granulating agent to get a wet mass. The solid blend mass was passed through the extruder
model RRE/EXT-65/037 to form extrudates. The formed extrudates were introduced into
spheroniser model RRE/SPH-150/010 to get spherical pellets by varying spheronization speed.

S1.No Ingredient (C1) (C2)

Formulation Formulation
(800mg) (800mg)
1. R-serpentina extract 147mg (18.38%) 147mg (18.38%)

2. Avicel pH 101  560mg (70%) 600mg (75%) 

3. Na. CMC  l0mg (1.25%) l0mg (1.25%) 

4. Lactose  78.9mg 39mg 

5. Magnesium stearate 4mg 4mg 

Table – 28

173 

 
 Results

Optimization of the Pelletization Technique of Reserpine

Drug: Avicel pH Spheronization Residence Pellets description
101:croscarmallose (Pelletization) time (min)
ratio Speed (rpm)
900 Rod shape
100 Rod shape
1100 Rod shape
M1(60:20:20) 1300 33 Rod and Dumbbell shaped
80% pellets with more fines
1400

900 Rod shape

100 Rod shape
1100 Rod shape
M2(40:40:20) 1300 35 Rod and Dumbbell shaped
90%Round with less fines
1400

Table – 29

Optimization of the Pelletization Technique of Rauwolfia serpentina

Drug:Avicel pH Spheronization Residence Pellets description
101:Sodium CMC (Pelletization) time (min)
ratio Speed (rpm)
300 Rod shape
350 Rod shape
150 Rod shape
C1(60:20:20) 600 45 Rod and Dumbbell shaped
70% pellets with more fines
750
300 Rod shape
350 Rod shape
150 Rod shape
C2(40:40:20) 600 42 Rod and Dumbbell shaped
90%Round with less fines
750

Table – 30

Residence time refers to Pelletization residence time and speed refers to disc speed of the
spheronizer.

174 

 
 Results

Physiochemical properties of Reserpine Pellets

Properties M1 Batch M2 Batch

Bulk density (g/cm3) 0.6544 0.6344
True density (g/cm3) 0.6648 0.6542
Carr’ index (%) 4.28 4.71
Angle of repose (Degree) 24 21
Porosity (%) 0.0106 0.0303
Friability (%) 0.13 0.16
Drug content (%) 93.23 95.45

Table – 31

Physiochemical Properties of Rauwolfia serpentina Pellets

Properties C1 Batch C2 Batch

Bulk density (g/cm3) 0.644 0.61
True density (g/cm3) 0.6623 0.6742
Carr’ index (%) 4.76 4.68
Angle of repose (Degree) 22 25
Porosity (%) 0.0271 0.0128
Friability (%) 0.13 0.15
Drug content (%) 90.00 94.81

Table – 32

175 

 
 Results

In Vitro dissolution Profile of Reserpine Pellets

M1 Batch

Cumulative percentage
Sl.No Time interval (min)
release
1 0 0
2 5 7.91
3 10 19.53
4 15 40.56
5 30 61.08
6 45 82.10
7 60 94.56

Table – 33

M2 Batch

Cumulative percentage
Sl.No Time interval (min)
release
1 0 0
2 5 8.53
3 10 19.68
4 15 42.86
5 30 62.32
6 45 84.18
7 60 95.81

Table – 34

176 

 
 Results

In vitro dissolution profile of Rauwolfia Serpentina Pellets

C1 Batch

Sl.No Time interval (min) Cumulative percentage release

1 0 0
2 5 13.14
3 10 28.22
4 15 4051
5 30 58.90
6 45 72.60
7 60 91.95

Table – 35

C2 Batch

Cumulative percentage
Sl.No Time interval (min)
release
1 0 0

2 5 5.842

3 10 13.37

4 15 42.10

5 30 59.91

6 45 75.95

7 60 92.52

Table –36

177 

 
 Results

Cumulative Percentage Drug Release of Reserpine and Rauwolfia Serpentina Extract

(For M1-M2 Batch)

Time in Min

Figure - 51

(For C1-C2 Batch)

Time in Min
Time in Min 

Figure - 52

178 

 
 Results

FORMULATION OF TABLET OF STRYCHNOUS NUXVOMICA AND STRYCHNINE

Tablet formulation was prepared by direct compression technique. All the powders
were passed through BSS-80 mesh. Required quantities of Strychnine and excipients were
mixed thoroughly. The tablets were compressed (12 mm diameter, standard concave punches)
using of Rotary tablet compression machine (10 station, Rimek, Ahmedabad, India). Prior the
compression, the powder mixture was evaluated for angle of repose, bulk density and
compressibility index, drug content.

The formulation of Strychnous Nuxvomica extract and Strychnine tablets

Sl.No Ingredient Strychnine tablets Strychnous Nuxvomica

(500 mg) extract Tablets (500 mg)
1. Strychnine 325 mg -
2. Strychnous Nuxvomica - 325 mg
extract
3. MCC 150 mg 150mg
4. Cross carmellose 20mg 20mg
5. Aerosil 5 mg 5mg

Table – 37

Evaluation of powder and tablet dosage form

The powder mixture of drug and excipients were evaluated for their angle of repose, bulk
density, tapped density, compressibility, drug content and reported in the table. Then the
formulated tablet dosage forms were evaluated for average weight, hardness, friability,
disintegration, drug content and in vitro drug release and reported in the table. All the
parameters obtained denoted that the formulation possess ideal physiochemical and
pharmaceutical properties for the tablet dosage formulation.

179 

 
 Results

Evaluations for powder

Strychnous Nuxvomica
Sl.No Parameters Strychnine
extract
1 Angle of repose 0(degree) 28.2±0.753 33.47±0.619
2 Bulk density (g/cm3) 0.461±0.006 0.512±0.013
3 Tapped density (g/cm3) 0.530±0.008 0.632±0.022
4 Compressibility (%) 13.05±1.268 18.85±1.604
5 Drug content( %) 97.75±0.531 93.82±1.062

Table – 38

Evaluations for tablets

Strychnine Strychnous Nuxvomica

Sl.No Parameters
formulations extract formulation
1 Average weight (mg) 499.53±0.115 499.5±0.1
2 Hardness (Kg/cm2) 5.16±0.288 5.66±0.288

3 Friability (%) 0.286±0.041 0.18±0.02

4 Disintegration (Min) 3’58”±34” 13’38” ±1’6”

5 Drug Content (%) 96.27±0.509 92.15±0.740

Table – 39

180 

 
 R
Results

In vitro drrug release of tablets

Sl.No
o. Time in
i mins. %Cumullative drug rrelease

Strych
hnine Strych
hnous Nuxvoomica
formullation extraact formulaation
1 0 0 0.0
2 5 17.413±
±2.328 11.136±1.8700
3 10 26.866±
±1.633 220.913±1.4933
4 15 44.273±
±2.225 37.896±1.4899
5 3
30 64.793±
±1.580 661.54±3.241
6 4
45 86.22±
±1.059 775.29±3.4766
7 6
60 98.11±
±0.970 990.45±1.4200

Table – 40

mulative Perrcentage Drug Release of Strychnoous Nuxvomica Extract and Strych

Cum hnine

1
120

1
100
%Cumulative drug release 

80

Sttrychnine
60
Exxtract Strychn
nous
40 N
Nuxvomica

20

0
0 20 40 60 80
Tim
me(minutes)

Figure – 533

181 

 
 Results

S.E.M STUDIES

Scanning electron mircograph of Phyllanthus amarus Spheroids

Plate - 6

Scanning electron mircograph of Rauwolfia serpentina pellets

Plate - 7
182 

 
 Results

STABILITY STUDIES

The WHO defines the stability as the drug/formulation to retain the chemical, physical,
microbial and biopharmaceutical, properties within the specified limits throughout the shelf
life. The accelerated stability testing is defined as the studies designed to increase the rate of
chemical degradation and physical changes of a drug or product by using exaggerated storage
conditions as a part of the formal stability testing programme. Stability studies of the
formulated pure Andrographolide matrix tablets, α-β Asarone capsules, Hypophyllanthin
Spheroids, Reserpine pellets and Strychnine tablets and extract of Andrographis Paniculata
matrix tablets, Acorus Calamus capsules, Phyllanthus Amarus spheroids, Rauwolfia Serpentina
pellets and Strychnous Nuxvomica tablets were carried out by placing the samples at different
temperature (250C and 400C) and different relative humidity conditions (60% RH and 75%
RH).

Evaluations of stability studies of Andrographis Paniculata extract and pure

Andrographolide Matrix tablets stored at 250C / 60% RH. All values represent the mean
of three observations with SD.

Andrographis Paniculata Andrographolide

Parameters Sampling Interval (Month)
Sampling Interval (Month)
1 3 6 1 3 6
Physical
--- --- --- --- --- ---
Appearance
Hardness
5 5 5 5 5 5
(kg/cm2)
Weight
Variation --- --- --- --- --- ---
(mg)
Friability
--- --- --- --- --- ---
(%)
Drug 89.42 87.50 84.42 96.99 95.12 93.62
Content ± ± ± ± ± ±
(%) 0.65 0.73 0.66 0.47 0.73  0.49

Table – 41

--- = No significant change

183 

 
 Results

Evaluations of stability studies of Andrographis Paniculata extract and pure

Andrographolide Matrix tablets stored at 400C / 75% RH. All values represent the mean
of three observations with SD.

Andrographis Paniculata Andrographolide

Parameters Sampling Interval (Month) Sampling Interval (Month)
1 3 6 1 3 6
Physical
--- --- --- --- --- ---
Appearance
Hardness
5 5 5 5 5 5
(kg/cm2)
Weight
Variation --- --- --- --- --- ---
(mg)
Friability
--- --- --- --- --- ---
(%)
Drug 90.42 89.50 87.75 97.45 96.12 94.81
Content ± ± ± ± ± ±
(%) 0.48 0.96 1.79 0.83 0.44  0.59

Table –42

--- = No significant change

184 

 
 Results

Evaluations of stability studies of Acorus Calamus extract and pure α- Asarone capsules
stored at 250C / 60% RH. All values represent the mean of three observations with SD.

Acorus Calamus α- Asarone

Parameters Sampling Interval (Month) Sampling Interval (Month)
1 3 6 1 3 6
Physical
--- --- --- --- --- ---
Appearance

Appearance of Pale Pale Pale

Pale brown Pale brown Pale brown 
powder brown  brown  brown

Average weight
0.527 ± 0.527 ± 0.527 ± 0.528 ± 0.529± 0.530 ±
of capsule content
0.0200 0.0300 0.0260 0.0200 0.0300 0.0270
(gms)

Friability
1.29 1.29 1.28 1.30 1.31 1.28
(%)

Disintegration
time (min) 13.2 13.2 13.1 13.3 13.4 13.1
(%)

Drug content 0.620 ± 0.621 ± 0.620 ± 0.621 ± 0.622 ± 0.619 ±

%w/w 0.020 0.030 0.030 0.020 0.030 0.020

Table – 43

--- = No significant change

185 

 
 Results

Evaluations of stability studies of Acorus Calamus extract and pure α- Asarone capsules
stored at 400C / 75% RH. All values represent the mean of three observations with SD.

Acorus Calamus α- Asarone

Parameters Sampling Interval (Month) Sampling Interval (Month)
1 3 6 1 3 6

Physical
--- --- --- --- --- ---
Appearance

Appearance of Pale Pale Pale

Pale brown Pale brown Pale brown 
powder brown  brown  brown

Average weight
0.5275 ± 0.5280 ± 0.5283± 0.5280 ± 0.5284± 0.5281 ±
of capsule content
0.0200 0.0600 0.0300 0.0200 0.0600 0.0300
(gms)

Friability
1.29 1.30 1.31 1.30 1.31 1.28
(%)

Disintegration
time (min) 13.2 13.2 13.1 13.3 13.4 13.5
(%)

Drug content 0.620 ± 0.621 ± 0.615 ± 0.621 ± 0.622 ± 0.619 ±

%w/w 0.020 0.100 0.090 0.020 0.100 0.090

Table – 44

--- = No significant change

186 

 
 Results

Evaluations of stability studies of Acorus Calamus extract and pure β- Asarone capsules
stored at 250C / 60% RH. All values represent the mean of three observations with SD.

Acorus Calamus β- Asarone

Parameters Sampling Interval (Month) Sampling Interval (Month)
1 3 6 1 3 6
Physical
--- --- --- --- --- ---
Appearance

Appearance of Pale Pale Pale

Pale brown Pale brown Pale brown 
powder brown  brown  brown

Average weight
0.526± 0.526 ± 0.526 ± 0.527 ± 0.528± 0.530 ±
of capsule content
0.0200 0.0300 0.0280 0.0200 0.0300 0.0280
(gms)

Friability
1.28 1.27 1.28 1.29 1.30 1.28
(%)

Disintegration
time (min) 13.1 13.1 13.0 13.2 13.4 13.3
(%)

Drug content 0.619 ± 0.620 ± 0.619 ± 0.620 ± 0.621 ± 0.618 ±

%w/w 0.020 0.030 0.030 0.020 0.030 0.020

Table – 45

--- = No significant change

187 

 
 Results

Evaluations of stability studies of Acorus Calamus extract and pure β- Asarone capsules
stored at 400C / 75% RH. All values represent the mean of three observations with SD.

Acorus Calamus β- Asarone

Parameters Sampling Interval (Month) Sampling Interval (Month)
1 3 6 1 3 6

Physical
--- --- --- --- --- ---
Appearance

Appearance of Pale Pale Pale

Pale brown Pale brown Pale brown 
powder brown  brown  brown

Average weight
0.5274 ± 0.5279± 0.5284± 0.5279 ± 0.5283± 0.5282 ±
of capsule content
0.0200 0.0600 0.0300 0.0200 0.0600 0.0300
(gms)

Friability
1.28 1.29 1.30 1.29 1.30 1.29
(%)

Disintegration
time (min) 13.1 13.1 13.0 13.2 13.3 13.4
(%)

Drug content 0.618 ± 0.620 ± 0.614 ± 0.620 ± 0.621 ± 0.622 ±

%w/w 0.020 0.100 0.090 0.020 0.100 0.090

Table – 46

--- = No significant change

188 

 
 Results

Evaluations of stability of Phyllanthus Amarus extract and pure Hypophyllanthin

0
Spheroids stored at 25 C / 60% RH. All values represent the mean of three observations
with SD.

Parameters Phyllanthus Amarus Hypophyllanthin

Sampling Interval (Month) Sampling Interval (Month)
1 3 6 1 3 6
Physical
--- --- --- --- --- ---
Appearance

Appearance of Greenish Greenish Greenish Greenish Greenish Greenish

Spheroids Brown Brown Brown  Brown  Brown  Brown

Average weight
0.5532 ± 0.5532 ± 0.5534 ± 0.5533 ± 0.5533 ± 0.5535 ±
of Spheroid
0.0460 0.0100 0.0890 0.0461 0.0100 0.0890
content (gms)

Friability
0.67 0.68 0.66 0.68 0.69 0.68
(%)

Disintegration
time (min) 13.5 13.5 13.5 13.6 13.5 13.6
(%)

Drug content 6.21 ± 6.22 ± 6.23 ± 6.22 ± 6.23 ± 6.24 ±

%w/w 0.52 0.52 0.48 0.52 0.52 0.48

Table – 47

--- = No significant change

189 

 
 Results

Evaluations of stability studies of Phyllanthus Amarus extract and pure Hypophyllanthin

Spheroids stored at 400C / 75% RH. All values represent the mean of three observations
with SD.

Parameters Phyllanthus Amarus Hypophyllanthin

Sampling Interval (Month) Sampling Interval (Month)
1 3 6 1 3 6

Physical
--- --- --- --- --- ---
Appearance

Appearance of Greenish Greenish Greenish Greenish Greenish Greenish

Spheroids Brown Brown Brown  Brown  Brown  Brown

Average weight
0.5532 ± 0.5535 ± 0.5535 ± 0.5533 ± 0.5536± 0.5536 ±
of Spheroid
0.0460 0.0210 0.0320 0.0460 0.0210 0.0320
content (gms)

Friability
0.67 0.69 0.69 0.68 0.69 0.68
(%)

Disintegration
time (min) 13.5 13.4 13.3 13.6 13.5 13.4
(%)

Drug content 6.21 ± 6.22 ± 6.23 ± 6.22 ± 6.23 ± 6.24 ±

%w/w 0.52 0.52 0.48 0.52 0.52 0.48

Table – 48

--- = No significant change

190 

 
 Results

Evaluations of stability of Rauwolfia Serpentina extract and pure Reserpine pellets stored

at 250C / 60% RH. All values represent the mean of three observations with SD. 

Parameters Rauwolfia Serpentina Reserpine

Sampling Interval (Month) Sampling Interval (Month)
1 3 6 1 3 6

Physical
--- --- --- --- --- ---
Appearance

Appearance of Reddish Reddish Reddish Reddish Reddish

Reddish brown 
pellets brown brown brown  brown  brown

Average weight
0.5416 ± 0.5418 ± 0.5417 ± 0.5417 ± 0.5418 ± 0.5418 ±
of pellet content
0.0100 0.0260 0.0320 0.0100 0.0260 0.0320
(gms)

Friability
0.154 0.155 0.154 0.155 0.156 0.155
(%)

Disintegration
time (min) 15.0 15.1 15.0 15.1 15.2 15.1
(%)

Drug content 15.12 ± 15.11 ± 15.11± 15.13 ± 15.12 ± 15.12 ±

%w/w 0.960 0.280 0.540 0.960 0.280 0.540

Table – 49

--- = No significant change

191 

 
 Results

Evaluations of stability studies of Rauwolfia Serpentina extract and pure Reserpine

pellets stored at 400C / 75% RH. All values represent the mean of three observations

with SD.

Parameters Rauwolfia Serpentina Reserpine 

Sampling Interval (Month) Sampling Interval (Month)

1 3 6 1 3 6

Physical
--- --- --- --- --- ---
Appearance

Appearance of Reddish Reddish Reddish Reddish Reddish

Reddish brown 
pellets brown brown brown  brown  brown

Average weight
0.5416 ± 0.5420 ± 0.5422± 0.5417 ± 0.5421 ± 0.5423±
of pellet content
0.0100 0.0220 0.0340 0.0100 0.0220 0.0340
(gms)

Friability
0.154 0.156 0.158 0.155 0.157 0.159
(%)

Disintegration
time (min) 15.0 14.8 14.8 15.1 14.9 14.9
(%)

Drug content 15.12 ± 15.11 ± 15.10± 15.13 ± 15.12 ± 15.11±

%w/w 0.960 0.090 0.420 0.960 0.090 0.420

Table – 50

--- = No significant change

192 

 
 Results

Evaluations of stability studies of Strychnous Nuxvomica extract and pure Strychnine

tablets stored at 250C / 60% RH. All values represent the mean of three observations

with SD.

Strychnous Nuxvomica Strychnine

Parameters Sampling Interval (Month) Sampling Interval (Month)
1 3 6 1 3 6
Physical
--- --- --- --- --- ---
Appearance
Hardness
5 5 5 5 5 5
(kg/cm2)
Weight
Variation --- --- --- --- --- ---
(mg)
Friability
--- --- --- --- --- ---
(%)
91.94 90.57 89.42 97.11 96.24 94.10
Drug Content
± ± ± ± ± ±
(%)
1.45 1.94 1.48 1.46 1.56  1.73

Table – 51

--- = No significant change

193 

 
 Results

Evaluations of stability studies of Strychnous Nuxvomica extract and pure Strychnine

tablets stored at 400C / 75% RH. All values represent the mean of three observations

with SD.

Strychnous Nuxvomica Strychnine

Parameters Sampling Interval (Month) Sampling Interval (Month)
1 3 6 1 3 6
Physical
--- --- --- --- --- ---
Appearance
Hardness
5 5 5 5 5 5
(kg/cm2)
Weight
Variation --- --- --- --- --- ---
(mg)
Friability
--- --- --- --- --- ---
(%)

90.89 89.42 96.94 96.81 94.24 91.15

Drug Content
± ± ± ± ± ±
(%)
1.65 1.45 1.59 0.34  1.58  1.25 

Table – 52

--- = No significant change

194 

 
 Results

PHARMACOKINETIC STUDIES

Analytical calibration data for Andrographolide

Area under the

Sl.No. Concentration in (µg/ml)
curve (cm2)

1 100 180999

2 200 492889

3 300 780140

4 400 1055159

5 500 1501129

Table – 53

Analytical calibration curve for Andrographolide

Y=2832x  
R2=0.976

Figure – 54

195 

 
 Results

Bio analytical calibration data for Andrographolide

Sl.No. Concentration in (µg/ml) Area under the

curve (cm2)

1 100 20010

2 200 501908

3 300 769854

4 400 1050509

5 500 1405150

Table – 54

Bioanalytical calibration Curve for Andrographolide

Figure – 55

196 

 
 Results

Analytical calibration data for α-Asarone

Sl.No.  Concentration in  (µg/ml) Area under the curve

(cm2) 
1  10 79993 
2  20 150112 
3  30 221010 
4  40 281143 
5  50 350012 

Table – 55

Analytical calibration Curve for α- Asarone

 
AUC in (cm2) 

Concentration in (µg/ml) 

Figure – 56

197 

 
 Results

Bio-analytical calibration data for α—Asarone

Sl.No.  Concentration in (µg/ml) Area under the

curve (cm2) 
1  10 79050 
2  20 150111 
3  30 210215 
4  40 269909 
5  50 340123 

Table – 56

Bio-analytical calibration Curve for α—Asarone

AUC in (cm2) 

Concentration in (µg/ml) 
 

Figure – 57

198 

 
 Results

Analytical calibration data for β-Asarone

Sl.No.  Concentration in (µg/ml) Area under the curve

(cm2) 
1  10 79996 

2  20 150116 
3  30 221013 
4  40 281144 
5  50 350014 

Table – 57

Analytical calibration Curve for β – Asarone

Y=5781x  
R2=0.883 
AUC in (cm2) 

Concentration in (µg/ml) 

Figure – 58

199 

 
 Results

Bio-analytical calibration data for β --Asarone

Sl.No.  Concentration in (µg/ml) Area under the

curve (cm2) 
1  10 79056 

2  20 150115 
3  30 210218 
4  40 269912 
5  50 340125 

Table – 58

Bio-analytical calibration Curve for β –Asarone

Y=5828x  
R2=0.888 
AUC in (cm2) 

Concentration in (µg/ml) 

Figure – 59

200 

 
 Results

Analytical calibration data for Hypophyllanthin

Sl.No. Concentration in (µg/ml) Area under the curve

(cm2)

1 10 989991
2 20 1712511
3 30 2871522
4 40 3587512
5 50 4504512

Table – 59

Analytical calibration Curve for Hypophyllanthin

Y=10468x  
R2=0.991 

Figure – 60

201 

 
 Results

Bioanalytical calibration data for Hypophyllanthin

Sl.No. Concentration in (µg/ml) Area under the curve

(cm2)
1 10 969915
2 20 1611612
3 30 2672161
4 40 3379521
5 50 4201815

Table – 60

Bioanalytical calibration curve for Hypophyllanthin

Y=10221x  
R2=0.983 

Figure – 61

202 

 
 Results

Analytical calibration data for Reserpine

Sl.No.  Concentration in (µg/ml) Area under the curve

(cm2) 
1  10 177472 
2  20 257592 
3  40 492343 
4  80 1006520 
5  160 1790534 
    

Table – 61

Analytical calibration Curve for Reserpine

Y=14787x  
AUC in (cm2) 

R2=0.997 

Concentration in (µg/ml) 
                                                

Figure – 62

203 

 
 Results

Bioanalytical Calibration Data for Reserpine

Sl.No.  Concentration in  ( µg/ml) Area under the

curve (cm2) 

1  10 221846 
2  20 289418 
3  40 571147 
4  80 1139164 
5  160 2389168 

Table – 62

Bioanalytical Calibration Curve for Reserpine

 
AUC in (cm2) 

Y=18782x  
R2=0.992 

 
Concentration in (µg/ml) 
 
 
Figure –63
   

204 

 
 Results

Analytical calibration data for Strychnine

Sl.No.  Concentration in ( µg/ml) Area under the curve

(cm2) 
1  10 891721 
2  20 1664211 
3  30 2402131 
4  40 3301521 
5  50 4405411 
 

Table – 63

Analytical calibration Curve for Strychnine

Y= 8120x 
R2=0.998 
AUC in (cm2) 

Concentration in (µg/ml)

Figure – 64 

205 

 
 Results

Bioanalytical Calibration data for Strychnine

Sl.No.  Concentration in (µg/ml) Area under the curve

(cm2) 
1  10 860123 
2  20 1406123 
3  30 2204151 
4  40 3008120 
5  50 4001251 

Table – 64

Bioanalytical Calibration Curve for Strychnine

AUC in (cm2) 

Y= 80815x 
R2=0.889 

Concentration in (µg/ml)

Figure – 65

206 

 
 Results

Concentration profiles data of Andrographolide in plasma sample after oral

administration of pure Andrographolide formulation (Dose- 5.95 mg/kg)

Concentration in
Sl.No. Time in (hrs)
(ng/ml)
1 0 0
2 0.25 3.458
3 0.5 4.038
4 0.75 4.758
5 1.0 5.541
6 1.5 7.505
7 2 5.519
8 2.5 3.809
9 3 3.114
10 4 1.542
11 6 1.171
12 8 0.907
13 12 0.657

Table –65

Concentration profiles curve of Andrographolide in plasma sample after oral

administration of pure Andrographolide formulation (Dose- 5.95 mg/kg)

9
8
Concentration ( in ng/ml )

7
6
5
4
3
2
1
0
0 2 4 6 8 10 12 14
Time ( in hrs.)

Figure – 66

207 

 
 Results

Concentration profiles data of Andrographolide in plasma sample after oral

administration of pure Andrographolide formulation (Dose-21mg/kg)

Sl.No. Time in (hrs) Concentration in

(ng/ml)
1 0 0
2 0.25 3.734
3 0.5 4.299
4 0.75 5.408
5 1.0 6.068
6 1.5 8.160
7 2 5.802
8 2.5 4.850
9 3 3.624
10 4 1.579
11 6 1.230
12 8 0.987
13 12 0.911

Table – 66

Concentration profiles curve of Andrographolide in plasma sample after oral

administration of pure Andrographolide formulation (Dose-21mg/kg)

7
Concentration ( in ng/ml )

0
0 2 4 6 8 10 12 14
Time ( in hrs.)

Figure – 67

208 

 
 Results

Concentration profiles data of Andrographolide in plasma sample after oral

administration of Andrographis paniculata extract formulation (Dose - 130mg/ kg)

Sl.No. Time in (hrs) Concentration in

(ng/ml)
1 0 0
2 0.25 3.122
3 0.5 3.657
4 0.75 4.264
5 1.0 5.161
6 1.5 6.655
7 2 6.280
8 2.5 5.424
9 3 4.528
10 4 2.778
11 6 1.837
12 8 1.427
13 12 0.645

Table – 67

Concentration profiles curve of Andrographolide in plasma sample after oral

administration of Andrographis paniculata extract formulation (Dose - 130mg/ kg)

8
7
Concentration ( in ng/ml )

6
5
4
3
2
1
0
0 2 4 6 8 10 12 14
Time ( in hrs.)

Figure – 68

209 

 
 Results

Concentration profiles data of Andrographolide in plasma sample after oral

administration of Andrographis paniculata extract formulation
(Dose – 460.2mg/ kg)
Sl.No. Time in (hrs) Concentration in
(ng/ml)
1 0 0
2 0.25 2.974
3 0.5 3.871
4 0.75 4.539
5 1.0 5.476
6 1.5 7.357
7 2 6.320
8 2.5 5.975
9 3 5.205
10 4 3.293
11 6 2.055
12 8 1.410
13 12 0.992

Table – 68

Concentration profiles curve of Andrographolide in plasma sample after oral

administration of Andrographis paniculata extract formulation (Dose – 460.2mg/ kg)

8
7
Concentration ( in ng/ml )

6
5
4
3
2
1
0
0 2 4 6 8 10 12 14
Time ( in hrs.)

Figure – 69
210 

 
 Results

Concentration profiles data of α-Asarone in plasma sample after oral administration of

α- Asarone formulation (Dose – 3.5 mg/ kg)

Concentration in
Sl.No. Time in (hrs)
(ng/ml)
1 0.00 0.000000
2 0.25 0.523649
3 0.50 0.707223
4 0.75 0.550004
5 1.00 1.444824
6 2.00 2.256354
7 4.00 0.580754
8 6.00 0.573061
9 8.00 0.585358
10 12.00 0.378558
11 24.00 0.278528
12 48.00 0.098442

Table – 69

Concentration profiles curve of α-Asarone in plasma sample after oral administration of

α- Asarone formulation (Dose – 3.5 mg/ kg)
Concentration in (ng/ml) 

Figure – 70

211 

 
 Results

Concentration profiles data of β - Asarone in plasma sample after oral administration of

β - Asarone formulation (Dose – 3.5 mg/ kg)

Concentration in
Sl.No. Time in (hrs)
(ng/ml)
1 0.00 0.000000
2 0.25 0.280104
3 0.50 0.423273

4 0.75 1.213178
5 1.00 4.215536
6 2.00 4.091812
7 4.00 1.531693
8 6.00 1.185076
9 8.00 0.648747
10 12.00 0.444807
11 24.00 0.258024
12 48.00 0.147688

Table –70

Concentration profiles curve of β - Asarone in plasma sample after oral administration of

β - Asarone formulation (Dose – 3.5 mg/ kg)
Concentration in (ng/ml) 

Figure – 71
212 

 
 Results

Concentration profiles data of α -Asarone in plasma sample after oral administration of

Acorus Calamus extract formulation (Dose – 68.62mg/ kg)

Sl.No. Time in (hrs) Concentration in (ng/ml)

1 0.00 0.000000
2 0.25 2.157243
3 0.50 2.195086
4 0.75 2.251701
5 1.00 3.107011
6 2.00 5.483282
7 4.00 3.544540
8 6.00 3.402046
9 8.00 2.341266
10 12.00 1.824105
11 24.00 1.179075
12 48.00 0.468424

Table – 71

Concentration profiles curve of α -Asarone in plasma sample after oral administration of

Acorus Calamus extract formulation (Dose – 68.62mg/ kg)
Concentration in (ng/ml) 
 

Figure – 72

213 

 
 Results

Concentration profiles data of β -Asarone in plasma sample after oral administration of

Acorus Calamus extract formulation (Dose –56.72mg/ kg)

Concentration in
Sl.No. Time in (hrs)
(ng/ml)
1 0.00 0.000000
2 0.25 0.217487
3 0.50 8.563324
4 0.75 9.175556
5 1.00 10.47082
6 2.00 13.52670
7 4.00 13.11994
8 6.00 11.16883
9 8.00 10.17612
10 12.00 9.463674
11 24.00 7.798462
12 48.00 3.590367

Table – 72

Concentration profiles of β -Asarone in plasma sample after oral administration of

Acorus Calamus extract formulation (Dose –56.72mg/ kg)

 
Concentration in (ng/ml) 

Figure – 73
214 

 
 Results

Concentration profiles data of Hypophyllanthin in plasma sample after oral

administration of Hypophyllanthin formulation (Dose – 4.66mg/ kg)

Concentration in
Sl.No. Time in (hrs)
(ng/ml)
1 0 0
2 0.5 0.293452
3 1 1.183949
4 2 2.111762
5 3 2.712873
6 4 1.709515
7 5 0.935416
8 6 0.661756
9 12 0.312025
10 24 0.119831

Table – 73

Concentration profiles curve of Hypophyllanthin in plasma sample after oral

administration of Hypophyllanthin formulation (Dose – 4.66mg/ kg)

2.5
Concentration(µg/ml)
in (ng/ml) 

1.5
Concentration

0.5

0
0 5 10 15 20 25 30

Time(hours)

Figure – 74

215 

 
 Results

Concentration profiles data of Hypophyllanthin in plasma sample after oral

administration of Hypophyllanthin formulation (Dose – 46.6mg/ kg)

Concentration in
Sl.No. Time in (hrs)
(ng/ml)
1 0 0
2 0.5 0.338292
3 1 1.097662
4 2 1.640877
5 3 2.768013

6 4 1.640706

7 5 1.194946

8 6 1.05504

9 12 0.648731

10 24 0.158916

Table – 74

Concentration profiles of Hypophyllanthin in plasma sample after oral administration of

Hypophyllanthin formulation (Dose – 46.6mg/ kg)

3
Concentration in (ng/ml) 

2.5
Concentration(µg/ml)

1.5

0.5

0
0 5 10 15 20 25 30
Time(hours)

Figure – 75

216 

 
 Results

Concentration profiles data of Hypophyllanthin in plasma sample after oral

administration of Phyllanthus amarus formulation (Dose – 401.7mg/ kg)

Concentration in
Sl.No. Time in (hrs)
(ng/ml)
1 0 0
2 0.5 0.285633
3 1 1.184478
4 2 2.018361
5 3 2.595396

6 4 1.995921

7 5 0.807472

8 6 0.533373

9 12 0.183682

10 24 0.106233

Table – 75

Concentration profiles curve of Hypophyllanthin in plasma sample after oral

administration of Phyllanthus amarus formulation (Dose – 401.7mg/ kg)

2.5
in (ng/ml)  )
Concentration(µg/ml

1.5
Concentration

0.5

0
0 5 10 15 20 25 30
Time(hours)

Figure – 76

217 

 
 Results

Concentration profiles of Hypophyllanthin in plasma sample after oral administration of

Phyllanthus amarus formulation (Dose – 4017 mg/ kg)

Concentration in
Sl.No. Time in (hrs)
(ng/ml)
1 0 0
2 0.5 0.412941
3 1 1.450683
4 2 2.352798

5 3 3.250568

6 4 1.909495

7 5 1.498949

8 6 1.014825

9 12 0.367268
10 24 0.263886

Table – 76

Concentration profiles of Hypophyllanthin in plasma sample after oral administration of

Phyllanthus amarus formulation (Dose – 4017 mg/ kg)

3.5

3
Concentration(µg/ml)
Concentration in (ng/ml) 

2.5

1.5

0.5

0
0 5 10 15 20 25 30
Time(hours)

Figure – 77

218 

 
 Results

Concentration profiles data of Reserpine in plasma sample after oral administration of

Reserpine formulation (Dose – 0.0175 mg/ kg)

Concentration
Sl.No. Time in (hrs)
in (ng/ml)

1 0.00 0

2 0.30 0.561

3 1 2.216

4 2 2.431

5 4 1.617

6 6 1.261

7 8 0.861

8 12 0.426

9 24 0.369

Table – 77

Concentration profiles curve of Reserpine in plasma sample after oral administration of

Reserpine formulation (Dose – 0.0175 mg/ kg)

 
 
   
 
 
 
 
 
 
 
 

Figure – 78
   

219 

 
 Results

Concentration profiles data of Reserpine in plasma sample after oral administration of

Reserpine formulation (Dose – 0.035mg/ kg)

Concentration
Sl.No. Time in (hrs)
in (ng/ml)
1 0.00 0
2 0.30 2.212
3 1 3.168
4 2 3.891
5 4 3.762
6 6 2.361
7 8 1.891
8 12 1.369
9 24 0.596

Table – 78

Concentration profiles curve of Reserpine in plasma sample after oral administration of

Reserpine formulation (Dose – 0.035mg/ kg)

Figure – 79

220 

 
 Results

Concentration profiles data of Reserpine in plasma sample after oral administration of

Rauwolfia serpentina formulation (Dose – 5.0mg/ kg)

Concentration
Sl.No. Time in (hrs)
in (ng/ml)
1 0.00 0
2 0.30 0.461

3 1 1.490

4 2 1.990
5 4 1.689
6 6 1.102
7 8 0.451
8 12 0.443
9 24 0.312

Table – 79

Concentration profiles curve of Reserpine in plasma sample after oral administration of

Rauwolfia serpentina formulation (Dose – 5.0mg/ kg)

Figure – 80
221 

 
 Results

Concentration profiles data of Reserpine in plasma sample after oral administration of

Rauwolfia serpentina formulation (Dose – 10.0 mg/ kg)

Concentration
Sl.No. Time in (hrs)
in (ng/ml)
1 0.00 0
2 0.30 2.010
3 1 2.518
4 2 3.581
5 4 4.619
6 6 4.402
7 8 3.268
8 12 1.986
9 24 0.961

Table – 80

Concentration profiles curve of Reserpine in plasma sample after oral administration of

Rauwolfia serpentina formulation (Dose – 10.0 mg/ kg)

Figure – 81

222 

 
 Results

Concentration profiles data of Strychnine in plasma sample after oral administration of

Strychnine formulation (Dose – 0.051mg/ kg)

Concentration in
Sl.No. Time in (hrs)
(ng/ml)
1 0.00 0
2 0.30 0.512311
3 1 2.128161
4 2 2.412781
5 3 3.891211
6 4 3.121012
7 5 1.601431
8 6 1.234161
9 8 0.914561
10 12 0.501312
11 24 0.412052

Table – 81

Concentration profiles curve of Strychnine in plasma sample after oral administration of

Strychnine formulation (Dose – 0.051mg/ kg)

Figure – 82

223 

 
 Results

Concentration profiles data of Strychnine in plasma sample after oral administration of

Strychnine formulation (Dose – 0.1.49 mg/ kg)

Concentration
Sl.No. Time in (hrs)
in (ng/ml)
1 0.00 0
2 0.30 2.101812
3 1 2.121364
4 2 3.243617
5 3 5.613416
6 4 3.730131
7 5 3.531421
8 6 2.334151
9 8 1.934326
10 12 1.524316
11 24 0.953164

Table – 82

Concentration profiles curve of Strychnine in plasma sample after oral administration of

Strychnine formulation (Dose – 0.1.49 mg/ kg)

Figure – 83
224 

 
 Results

Concentration profiles data of Strychnine in plasma sample after oral administration of

Strychnous nuxvomica formulation (Dose – 0.51mg/ kg)

Concentration
Sl.No. Time in (hrs)
in (ng/ml)
1 0.00 0

2 0.30 0.453541
3 1 1.431426

4 2 1.843168
5 3 3.346986
6 4 2.401697
7 5 1.607812
8 6 1.116319
9 8 0.468971
10 12 0.447716
11 24 0.321629

Table – 83

Concentration profiles curve of Strychnine in plasma sample after oral administration of

Strychnous nuxvomica formulation (Dose – 0.51mg/ kg)

Figure – 84

225 

 
 Results

Concentration profiles data of Strychnine in plasma sample after oral administration of

Strychnous nuxvomica formulation (Dose – 1.49mg/ kg)

Concentration
Sl.No. Time in (hrs)
in (ng/ml)
1 0.00 0
2 0.30 1.990145
3 1 2.781652
4 2 3.589741
5 3 8.293871
6 4 9.857623
7 5 8.308317
8 6 6.972681
9 8 3.421496
10 12 1.987696
11 24 1.496785

Table – 84

Concentration profiles curve of Strychnine in plasma sample after oral administration of

Strychnous nuxvomica formulation (Dose – 1.49mg/ kg)

Figure – 85

226 

 
 Results

Pharmacokinetic Studies in Albino Rabbits

Plate - 8

227 

 
 Results

Typical chromatogram of standard solution of Andrographolide

 
Figure – 86
 

Typical chromatogram of sample solution of Andrographolide

Figure – 87

228 

 
 Results

Typical chromatogram of standard solution α- β asarone 

Figure – 88
 

Typical chromatogram of sample solution α- β asarone 

 
 
Figure – 89
229 

 
 Results

 
Typical chromatogram of standard solution of Hypophyllanthin

 
Figure – 90
 
Typical chromatogram of sample solution of Hypophyllanthin

Figure – 91

230 

 
 Results

Typical chromatogram of standard solution Reserpine

Figure – 92

Typical chromatogram of sample solution Reserpine

Figure – 93
231 

 
 Results

Typical chromatogram of standard solution of Strychnine

Figure – 94

Typical chromatogram of sample solution of Strychnine

Figure – 95

232 

 
 Results

PHARMACOKINETIC PARAMETERS OBTAINED FOR PURE (STANDARD) ANDROGRAPHOLIDE AND

EXTRACT OF ANDROGRAPHIS PANICULATA MATRIX TABLETS
Standard formulation Extract Formulation
Sl.No.
Parameters Dose: 5.95mg/kg
Dose:
Average Dose: 130mg/kg Dose: 460.2mg/kg Average
21mg/kg
1 Cmax (ng/ml) 24.48095 60.6788 42.579875 26.6357 54.215536 40.425618
2 tmax (h) 2 2 2 2 1.65 1.825
-1
3 keli (h ) 0.165757 0.1023185 0.13403775 0.127913 0.194619 0.161266
4 t1/2 (h) 5.18 6.21 5.695 5.41 6.87 6.14
5 AUC(0-48) 174.48768 342.4589 258.47329 134.5728 252.942 193.7574
(ng. h/ml)
6 AUC(0-∞) 231.5278 407.12459 319.326195 182.58288 286.987 234.78494
(ng. h/ml)
7 Lag Time to (h-1) No No No No No No

Table – 85

Cmax – Peak plasma concentration, tmax – Time to attain Cmax

Keli- Elimination rate constant, t1/2 – Half-Life, AUC-Area Under Curve, (to)- lag time

233 

 
 Results

PHARMACOKINETIC PARAMETERS OBTAINED FOR PURE (STANDARD) α- β ASARONE AND EXTRACT

OF ACORUS CALAMUS CAPSULES
For α-Asarone For β- Asarone

Standard Extract Formulation  Standard formulation Extract Formulation 

Sl.No.
Parameters formulation
Dose: 3.5mg/kg Dose: 68.62mg/kg Dose: 3.5mg/kg Dose: 56.72mg/kg
1 Cmax (ng/ml) 103267.6 14798.963 1191.24 1354.31
2 tmax (h) 12 12 12 12
3 keli (h-1) 0.387 0.236 0.724 0.227
4 t1/2 (h) 1.793 2.39 5.578 3.053
5 AUC(0-24)
980224.8 182500.39 14970.0 19104.2
(ng. h/ml)
6 AUC(0-∞)
982806.9 186166.53 16362.3 19495.7
(ng. h/ml)
7 Lag Time (to) (h-1) No No No No

Table - 86

Cmax – Peak plasma concentration, tmax – Time to attain Cmax

Keli- Elimination rate constant, t1/2 – Half-Life, AUC-Area Under Curve, (to)- lag time.
234 

 
 Results

PHARMACOKINETIC PARAMETERS OBTAINED FOR PURE (STANDARD) HYPOPHYLLANTHIN

AND EXTRACT OF PHYLLANTHUS AMARUS SPHEROIDS

Standard formulation Extract Formulation

Sl.No.
Parameters
Dose: 4.66mg/kg Dose: 46.6mg/kg Average Dose: 401.7mg/kg Dose: 4017 mg/kg Average

1 Cmax (ng/ml)
0.56 0.625 0.4852 0.5426
0.69 0.60
2 tmax (h) 1 1 1 1
1 1
-1
3 keli (h ) 0.149935 0.133947 0.154855 0.137503
0.117959 0.120151
4 t1/2 (h) 4.62 5.245 4.47 5.115
5.87 5.76
5 AUC(0-48)
(ng. h/ml) 2.727 3.01 2.8685 2.491 2.0269 2.25895

6 AUC(0-∞)
(ng. h/ml) 2.840383 3.338646 3.0895145 2.757332 2.097934 2.427633

7 Lag Time (to)

No No No No No No
(h-1)

Table- 87

Cmax – Peak plasma concentration, tmax – Time to attain Cmax

Keli- Elimination rate constant, t1/2 – Half-Life, AUC-Area Under Curve, (to)- lag time.
235 

 
 Results

PHARMACOKINETIC PARAMETERS OBTAINED FOR PURE (STANDARD) RESERPINE

AND EXTRACT OF RAUWOLFIA SERPENTINA PELLETS
Standard formulation Extract Formulation
Sl.No. Dose: Dose:
Parameters Dose: 0.0175mg/kg Dose: 0.035mg/kg Average Average
5mg/kg 10mg/kg
1 Cmax (ng/ml) 3.248 5.421 4.3345 0.94 4.85 2.895
2 tmax (h) 2 2 2 6 2 4
3 keli (h-1) 0.73222 0.07209 0.402155 0.026081 0.097476 0.0617785

4 t1/2 (h) 9.466363 9.615049 9.540706 26.57625 7.11093 16.84359

AUC(0-48)
5 22.5749 71.16785 46.871375 17.5315 37.3735 27.4525
(ng. h/ml)
AUC(0-∞)
6 29.62058 85.63591 57.628245 38.73432 40.70813 39.721225
(ng. h/ml)
Lag Time (to)
7 No No No No No No
(h-1)

Table - 88
Cmax – Peak plasma concentration, tmax – Time to attain Cmax
Keli- Elimination rate constant, t1/2 – Half-Life, AUC-Area Under Curve, (to)- lag time.

236 

 
 Results

PHARMACOKINETIC PARAMETERS OBTAINED FOR PURE (STANDARD) STRYCHNINE AND EXTRACT

OF STRYCHNOUS NUXVOMICA TABLETS

Sl.No. Standard formulation Extract Formulation

Dose: Dose: Dose: Dose: 1.49

Parameters Average Average
0.051mg/kg 0.149mg/kg 0.51mg/kg mg/kg
1 Cmax (ng/ml) 42.75 47.315 39.76 48.35 44.055
51.88
2 tmax (h) 1.5 1.5 1.5 1.5 .15
1.5
-1
3 keli (h ) 0.060084 0.0661845 0.052236 0.07369 0.062963
0.072285
4 t1/2 (h) 11.53 10.559553 13.2 9.40 11.3
9.589106
AUC(0-48)
5 334.81 341.05 337.93 284.69 328.81 306.75
(ng. h/ml)
6 AUC(0-∞) 612.8026 599.993
628.07 597.5352 559.91 579.9515
(ng. h/ml)
Lag Time (to)
7 No No No No No No
(h-1)

Table - 89
Cmax – Peak plasma concentration, tmax – Time to attain Cmax
Keli- Elimination rate constant, t1/2 – Half-Life, AUC-Area Under Curve, (to)- lag time. 
 
237 

 
 Discussion

Discussion
Extraction and Quantification

The botanical plants collected were subjected for extraction after diagnosis by the
experts of Department of Pharmacognosy, J.S.S. College of Pharmacy, Ooty.

Extracts of all the individual drugs were obtained by maceration technique.

Hydroalcohol was used as the solvent, since it possess the optimum solubility
characteristics required for extraction. A known weight of the powdered drug was taken
for extraction with water and ethyl alcohol (50:50) ratio in a flat bottomed maceration
flask and subjected to maceration. The maceration flask was kept at room temperature
protected from sunlight and shaken several times daily. At the end of the 7th day, the
extract was filtered, concentrated and stored in a desiccator for further studies. The
percentage yield of the extracts obtained was calculated and reported.

The solvent system consisting of Acetone: Chloroform: Benzene in the ratio 7:2:1
provided good separation for Andrographis Paniculata. The denistogram of sample
solution of Andrographis Paniculata was obtained at 273 nm. Using the proposed
method, the extract was found to have 4.563 mg% of Andrographolide.

The solvent system consisting of Toluene: Ethyl acetate in the ratio 93:7 provided
good separation for Acorus Calamus. The desnitogram of sample solution of Acrous
Calamus was obtained at 254nm. The extract were found to have about 5.1mg% and 6.17
mg% of α Asarone and β Asarone respectively.

Phyllanthus amarus in the extract was standardized using solvent system

consisting of Hexane: Acetone: Ethyl acetate in the ratio of 74:12:8 which provided good
separation for Phyllanthus amarus. The desnitogram of sample solution of Phyllanthus
amarus was obtained at 235nm. The extract was found to have 1.16mg% of
Hypophyllanthin.

238 
 
 Discussion

The solvent system consisting of Toluene: Ethyl acetate: Dethylamine in the ratio
7:2:1 provided good separation for Rauwolfia serpentina. The denistograms of standard
and sample solution of Rauwolfia serpentina was obtained at 254 nm. Using the proposed
method, the extract was found to have about 0.345mg% of Reserpine.

The solvent system consisting of Toluene: Ethyl acetate: Dethylamine in the ratio
7:2:1 provided good separation for Strychnous nuxvomica. The desnitogram of sample
solutions of Strychnous nuxvomica was obtained at 254 nm. Using the proposed method,
the extract was found to have about 10.0mg% of Strychnine.

Preformulation Studies

In preliminary study, the calibration for Andrographolide, α-β- Asarone,

Hypophyllanthin, Resepine and Strychnine was done with UV - spectrophotomatry and
HPLC analytical method were developed in order to estimate the same for future works.
The Bioanalytical HPLC curves were also developed for the selected drugs. The curves
obtained were linear with regression value almost equal to one indicating the increase in
concentration increased the absorbance and the AUC linearly. The HPLC method was
susceptible enough to measure the concentration of 100µg/ml solution further decrease in
concentration reported absence of drug peak in chromatogram which may be due to lack
of sensitivity. The retention time of the peak was adjusted to 6 to 7 mins to ensure the
absence of interference of the plasma peaks which was further confirmed by running the
blank plasma.

The spectral analysis by peak matching technique indicated that the physical
mixture of drug and the excipients exhibits the specific functional groups with almost
same chemical characteristics observed for pure drugs. The study suggests that the
excipients of choice lactose, starch 1500, HPMC and MCC used mostly in all the selected
drugs do not bring the molecular changes of the drug with alteration in its molecular
behavior. This was confirmed by the FTIR studies.

239 
 
 Discussion

Formulation optimization

Patients overwhelmingly prefer solid oral dosage over other drug formulations.
Hydrophilic matrix systems are among the most widely used means for controlled drug
delivery in solid oral dosage forms. Hydrophilic matrix systems have been proven for
over four decades. Matrix controlled release tablets are relatively simple systems that are
more forgiving of variations of ingredients, production methods, and end-use conditions
than coated controlled-release tablets and other systems. This results in more uniform
release profiles with a high resistance to drug dumping.

Matrix system are relatively easy to formulate. The performance of many

products is already well documented, providing a body of data to refer to and rely upon.
This helps speedy development work and can shorten approved times as well.

Matrix systems are easy to produce. Tablets are manufactured with existing,
conventional equipment and processing methods. This is true for almost any size tablet.
Whether it involves direct compression, dry granulation, or wet granulation. Matrix
systems are economical. Beyond the possibility of lower development costs and the use
of conventional production methods, the ingredients normally used are cost-effective.
Therefore wet compression technique was employed in development of Andrographolide
matrix tablets.

In α-β- Asarone capsules formulation were prepared one for extract and others for
pure drug. In case of pure drug and the exicipent were mixed thoroughly in the mortar
and this mixture was filled into the hard gelatin capsule shells (Size ‘0’) by usual manual
filling procedure where as extracts granules were prepared using the excipients and
passed through sieve no 22 to fill into capsule shells.

240 
 
 Discussion

Hypophyllanthin Spheroids and Reserpine pellets were prepared by the extrusion

and speronization technique. Different types of Spheroids and pellets are formulated and
particle size of Spheroids and pellets was determined by using scanning electron
microcopy.

Finally Strychnous nuxvomica and Strychnine conventional tablets were prepared

by the Direct Compression technique using conventional ingredients followed by
determing of tablets evaluations.

The starch 1500, sodium CMC acts as a binder, MCC Avicel PH101 acts as
spheronization (Pelletinization) enhancer, lactose as a filler cross caramellose as a
superdisintegrant, PVP K30 and HPMC acts as disintegrating Agent, talc and
magnesium stearate as glidant and lubricant, water as the granulating fluid were used in
the formulation. In the process of product development, some of the chemicals were,
omitted and added. The pure drug and the extract powder were mixed with excipients in
the geometrical pattern for 10min and to confirm the uniform distribution of the drug the
degree of mixing was carried out. The drug content were also found to be satisfactory in
case of pure drug and extract respectively. The further mixing let to demixing in case of
pure drug and no major change was seen in case of extract. The MCC was used as binder
cum diluents in the formulation as is widely used. The croscarmellose was used as the
superdisintegrant. The aerosil was used at concentration of 2% as it can nullify the use of
additional lubricant as it can serve both as glidant and lubricant. Various trails have been
done and only the optimized batch are taken.

Pre Compression and post compression evalutions

The angle of repose gives a qualitative assessment of internal and cohesive

frictional forces. All the batches had an angle of repose less than 300 indicating good flow
potential. The compressibility index measures the propensity of a powder to be
compressed. As such, they are measured of the relative importance of interparticulate
interactions. In a free-flowing powder, such interactions are generally less significant,

241 
 
 Discussion

and the bulk and tapped densities will be closer in value. For poorer flowing materials,
there are frequently greater interparticle interactions, and a greater difference are
reflected in the Compressibility index. As all compressibility values for the selected
drugs is less than 15% the blend produced the adequate flow and stable packing and the
bulk and true density values were also closer to each other. The drug content was
observed to be more than 90% in all the batches. But on comparison between the pure
form and the extract form the drug content was found to be slightly reduced in the extract
form.

The formulated tablets, Spheroids, pellets and capsules were evaluated for
average weight, hardness, friability, disintegration time, drug content, moisture uptake
and in vitro drug release. With the hardness of 4 - 6 Kgs /cm2 the other compression
parameters like average weight, friability, disintegration time, drug content, moisture
uptake were to the official limits.

For the determination of surface roughness and shape of the formulated Spheroids
and pellets the SEM was done. The SEM clearly showed spherical shape of Spheroids and
pellets, it was also observed that the Spheroids and pellets were porous in nature.

The in vitro release studies of Matrix tablets were performed for a period of 15
hrs. with their respective buffers. It showed the release profile of the drug from pure and
extract formulation i.e batch I and II which were in conventional form indicates that the
release of the drug was fast and within an hour it was a maximum release of 99.01% and
95.87% respectively. On the other side the matrix formulation of pure and extract form
were evaluated. An increase in concentration of HPMC did not significantly prolong the
durg release. Tablets III(batch) , IV(batch) and V(batch) showed 52.90%, 57.94% and
50.30% release of Andrographolide at the end of 2 hrs; and 83.93%, 84.46% and 72.78%
of drug at the end of 12 hrs release. From 6 to 15 hrs the matrix tablets slowly released
the drug, and at the end of 15hrs the drug release was 98.95%, 97.86% and 83.52% from
III, IV and V batch respectively.

242 
 
 Discussion

The in vitro release of capsule dosage form during 6 hrs study was found to be
87.68% , 85.86%, 82.36%, and 81.98 % of α Asarone, β Asarone, Acrous Calamus (α)
and Acrous Calamus (β) from pure and extracts forms respectively and at the end of 12
hrs the in vitro release of capsule dosage form is 98.12%, 97,10% , 90.98% and 90.56%
respectively.

The in vitro release of Hypophyllanthin Spheroids formulation carried out for 60

min and maximum release for Hypophyllanthin found to be 98.16% for drug formulation
and 90.97% for extract formulation.

The in vitro release of Reserpine pellets formulation carried out for 60 min and
maximum release for Reserpine found to be 94.56% and 95.81% (M1, M2) batch for
drug formulation and 91.95% and 92.52% for (C1, C2) batch for extract formulation.

The in vitro release of Strychnine tablets formulation carried out for 60 min and
maximum release for Strychnine found to be 98.11% for drug formulation and 94.45%
for extract formulation. So the release was comparatively low in the case of extract
formulations.

Stability studies

The stability of the pharmaceuticals remains one of the important criteria as it

should reach to the patient in an active form. As the deterioration of the product due to its
changes in physical, chemical and microbiological properties reduces the therapeutic
value with increased toxicity, 90% of the label claim is the minimum acceptable potency
level in general. Formulations in both extract and pure form were studied for stability for
a period of six months. The various parameters were tested at frequent intervals. The
results showed that there was no significant change in results of parameters even at the
end of 6 months on comparison to their day zero values. The formulations were termed to
be stable.

243 
 
 Discussion

Pharamacokinetic Studies

A single dose study, for two concentrations of formulated extract and pure drugs
were carried out in five groups for each drug, containing three rabbits in each group, for
each concentration of the formulations. The blood levels of drug were estimated by
validated HPLC method and PK parameters such as peak plasma concentration (Cmax),
time of peak concentration (tmax), area under the plasma concentration – time curve
(AUC0-24 and AUC0-∞), elimination rate constant (Keli), elimination half life (t1/2 ) and lag
time (t0). were calculated. The PK parameters for both the extract and the pure formulation
were, evaluated directly after oral administration of them into the stomach of Albino
rabbits.

The PK parameters were determined and their average for the two different doses
administered was calculated for the selected herbal drugs. The Cmax for all the
formulations were observed within 3 hrs which was significant at a level of P<0.05. The
formulations were found to posses no lag time (t0) when calculated using the “Method of
Residuals”. So from the obtained data, it can be considered that there is no delay in the
commencement of absorption of drug.

The study was, carried out to determine the dosage regimen for the selected
extracts through the obtained PK parameters. The frequency of dosing was determined
based upon the discussion such as the drugs with half life of 30 mins to 8 hrs can be
administered in every half life, but if the drug is having high therapeutic index it must be
administered once every1 to 3 half life or even less frequently. The drugs with 8-24 hrs
half life the most and convenient desirable form is one in which a dose is given every
half-life. For drugs greater than 24 hrs administration of the drug once daily is
convenient.8, 170

244 
 
 Discussion

In Andrographis paniculata extract formulation the t1/2 was found to be 6.14 hrs.
The results showed that the differences analysed were considered to be significant at a
level of P<0.05 which was found when different pharmacokinetic parameters of extract
formulation was compared with pure formulation.

The Acorus Calamus extract capsule gave a t1/2 of 2.39hrs for α – Asarone and
3.05hrs for β- Asarone. The results showed that the differences analysed were considered
to be significant at a level of P<0.05 which was found when different pharmacokinetic
parameters of extract formulation was compared with pure formulation.

The Phyllanthus amarus extract Spheroids gave a t1/2 of 5.11 hrs. The results
showed that the differences analysed were considered to be significant at a level of
P<0.05 which was found when different pharmacokinetic parameters of extract
formulation was compared with pure formulation.

The Rauwolfia serpentina extract pellets gave a t1/2 of 16.84 hrs. The results
showed that the differences analysed were considered to be significant at a level of
P<0.05 which was found when different pharmacokinetic parameters of extract
formulation was compared with pure formulation.

The Strychnous nuxvomica extract tablets gave a t1/2 of 11.3 hrs. The results
showed that the differences analysed were considered to be significant at a level of
P<0.05 which was found when different pharmacokinetic parameters of extract
formulation was compared with pure formulation.
 

245 
 
 Summary
 
Summary

The selected plants for the study such as Andrographis paniculata, Acorus
Calamus, Phyllanthus amarus, Rauwolfia serpentina and Strychnous nuxvomica were
extracted by maceration technique. Quantification analysis of compound separated by
HPTLC can be carried by estimating the area of Band detection and results showed that
100mg of Andrographis paniculata, Acorus Calamus, Phyllanthus amarus, Rauwolfia
serpentina and Strychnous nuxvomica, extract contains 4.563mg, 5.1mg, 6.17 mg,
1.16mg, 0.345mg, & 10.0 mg of pure Andrographolide, α- Asarone, β-Asarone,
Hypophyllanthin, Reserpine and Strychnine respectively.

The compatibility studies between the selected excipents and the pure
drugs/extracts were studied. The IR spectra obtained were satisfactory with their
characteristic absorption bands. Similarly the physical mixtures also indicated the
presence of characteristic peak of the pure drugs / extracts and excipients.

The formulated powders/granules were evaluated for their precompression

parameters such as angle of repose, bulk density , tapped density, compressibility, drug
content and their results were found to the satisfactory.

Andrographolide matrix tablets, α – β Asarone capsules, Hypophyllanthin

Spheroids, Reserpine pellets and strychinine conventional tablets were prepared by their
respective suitable techniques.

Then the formulated tablets, Spheroids and pellets, capsules were evaluated for
average weight, hardness, friability distintegration time, drug content, surface
charactertistic and invitro drug release. All the parameters obtained the tablets, Spheroids,
pellets & capsules formulation possessed ideal physiochemical and pharmaceutical
properties for the tablets, Spheroids, pellets, and capsules formulation.

Stability studies of the formulated pure and extract tablets, Spheroids, pellets and
capsules were carried out by placing the samples at 250C/60% RH and 400C/75% RH for
a period of six months. There was no significant change in physicochemical properties of

246 
 
 Summary
 
the tablets, Spheroids, Pellets & Capsules used in the study but only there was a decrease
in the drug content. Based on the results it can be concluded that the formulated tablet,
Spheroids pellets and capsules were stable at room temperature at different relative
humidities over a period of 6 months. Even though its stability is assured for six months,
further studies at different temperatures and humidity conditions are needed to be
established its shelf-life.

A single dose Pharmacokinetic study, for two concentrations of formulated

extracts and pure drugs were carried out in Albino rabbits, for each concentration of the
formulations and solvent control. The blood levels of drug were estimated by the
validated HPLC method and pharmacokinetic parameters such (Cmax), (tmax), (AUC0-24
and AUC0-∞), (Keli), (t1/2), and (to) were calculated. After determination of the PK
parameters the dosage regimen was predicted through their half life for the selected
herbal drugs.

Accordingly, Andrographalide in matrix tablet containing 100mg of

Andrographis paniculata extract can be administered 4 tablets per day in divided dosage.
(t1/2 :6.14 hrs)

For α-β asarone in capsule form containing 100mg of Acorus Calamus extract can
be administered 8 capsules per day in divided dosage. (t1/2 : 2.39 hrs and 3.05 hrs)

For Hypophyllanthin in Spheroids form containing 100mg of Phyllanthus amarus

extract can be administered 5 dosage units of Spheroids per day in divided dosage.
(t1/2 :5.11 hrs)

For Reserpine in pellets form containing 100mg of Rauwolfia serpentina extract

can be administered 1 dosage units of pellets per day in divided dosage. (t1/2 :16.84 hrs)

For Strychnine in conventional tablet containing 100mg of Strychnous nuxvomica

extract can be administered 2 tablets per day in divided dosage. (t1/2 :11.3 hrs) 

247 
 
 Conclusion

Conclusion

Oral dosage forms such as Tablet, Spheroids, Pellets and Capsule were prepared
with standard drug and herbal extract form. The dosage forms were evaluated for pre and
post formulation parameters. Based on the results, best batches were taken for in vivo
pharmacokinetic studies on rabbits. The pharmacokinetic data of Andrographolide,
α-β-Asarone, Hypophyllanthin, Reserpine and Strychnine revealed the various
parameters which are essential to project the exact dosage regimen of them in extract
formulation, indicating the therapeutic activity of the herbal extract of Andrographis
paniculata, Acorus Calamus, Phyllanthus amarus, Rauwolfia serpentina and Strychnous
nuxvomica. It is suggested that based on the calculated pharmacokinetic data of the
selected Herbal drugs should be formulated in various dosage forms for better dosage
regimen and thereby expected to deliver the better therapeutic profiles. This overall
guidelines will certainly be helpful for the herbal industry to prepare a suitable dosage
form with proper quantity of extracts.

Based on the above animal Pharmacokinetic data, we can get the idea for the
similar pharmacokinetic profiles in case of human subjects and is suggestd to determine
the pharamacokinetic profile on human volunteers and thereby can predict the actual
dosage schedule which may be helpful from manufacturers’ point of view and as well
accurate prescriptions by the physicians for better therapentic regimen of these herbal
drugs. 

248 
 
 References

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 Annexure

ANNEXURE – I

LIST OF ABBREVIATIONS
% - Percentage
0 - Degree
0C - Degree centigrade
± - Plus or Minus
µl - Microlitre
ACN - Acetonitrile
ADME - Absorption, Distribution, Metabolism and Elimination
AUC - Area under the curve
cc - Cubic centimeter
Cmax - Peak Plasma Concentration
Conc - Concentration.
e.g. - Example
etc. - Etcetera
fig. - Figure
FTIR - Fourier transform infra red
GI - Gastrointestinal
gm - Gram
HPLC - High Performance Liquid Chromatography
HPTLC - High Performance Thin Layer Chromatography
hr - Hour
hrs - Hours
i.e. - That is
i.v - Intravenous
IR - Infra red
MCC - Microcrystalline Cellulose
mcg or µg - Microgram
mcg/ml - Microgram per millilitre
MEC - Minimum Effective Concentration

274 
 
 Annexure

mg - Milligram
min - Minutes
ml - Millilitre
mm - Millimetre
MSC - Maximum Safety Concentration
N - Normality
Na CMC - Sodium Carboxy Methylcellulose
NDA - New Drug Application
ng - Nanogram
ng/ml - Nanogram per millilitre
nm - Nanometer
No. - Number
rpm - Rotations per minute
SEM - Scanning Electron Microscopy
Sl.No - Serial number
Std - Standard
t m ax - Time of Peak Concentration
v/v - Volume by Volume
Vol - Volume
w/v - Weight by Volume
W/W - Weight by weight
WHO - World Health Organization
wt - Weight
HClO4 - Perchloric Acid (Precipitating Agent)
KBr - Potassium bromide
Kg - Kilogram
P value - Probality factor
r2 - Regression coefficient
RH - Relative humidity
SD - Standard deviation

275 
 
 Annexure

TBD - Tapped Bulk Density

UV - Ultra Violet
HPMC - Hydroxy Propyl Methyl Cellulose
In vitro - Outside the body or in lab
In vivo - Inside the body
mM - Milli molar
λmax - Wavelength at which maximum absorption takes place
~ - Approximate Value
SR - Slow Release (Sustained Release)
CR - Control Release
USP/NF - United states pharmacopoeia/ National Formulary
Ka - Rate constant of Absorption

276 
 
 Annexure

ANNEXURE – II

277