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Antioxidant activity of methanol extract of pandanus fascicularis Lam

Article  in  Pharmacologyonline · January 2011

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Pharmacologyonline 1: 833-841 (2011) Sanjeeva et al.

ATIOXIDAT ACTIVITY OF METHAOL EXTRACT OF

PADAUS FASCICULARIS Lam.

Sanjeeva1, Raveendra Kumar N1 , Padmalaxmi D1, Ananthbabu1,*Udupa A.L.2,

Nkem Ojeh2, Subir Gupta2, Rathnakar U. P3, Vijayalaxmi Patil3, Prabhath Kodancha G3, Deepa
Benegal4 ,Adarsh Benegal4, Sanjana R Rao5, Nisarga S5,Shubha H.V6 .
1
Shanthiram Medical College Nandyal , Andhra Pradesh, India.
2
FMS, UWI, Cavehill, Barbados. West Indies. BB11000
3
Kasturba Medical College, Mangalore-575001, Manipal University, Karnataka, India.
4
Vijayanagar Institute of Medical Sciences Bellary, Karnataka,India
5
Yennapoya Medical College,Mangalore-575018,Karnataka,India.
6
JSS Medical College Mysore,Karnataka,India.

* Corresponding author: Dr.A.L.Udupa,

Faculty of Medical Sciences, University of West Indies, P.B. # 64
Cave Hill Campus, University Drive, Barbados.BB 11000.
Phone:(+1246) 417 4690 (O); Fax: 438 9170
Email: [email protected]

Summary

Plants are widely used in traditional and folklore medicine. Use of medicinal plants and their
products are almost doubled over the last decade in developing countries and the present trend of
wide spread interest in alternative therapies is well known. Pandanus fascicularis is used in
traditional systems of medicine to treat varying conditions like rheumatism, fever, headache,
earache and also used as antispasmodic. No evidence of scientific study is available on this plant.
So the present study is aimed at investigating the invitro antioxidant activity of methanolic
extract of Pandanus fascicularis. Present study is aimed at investigating the invitro antioxidant
activity of methanolic extracts of leaves of Pandanus fascicularis(MEPF) by four different in-
vitro methods. The lipid peroxidation was assayed by estimating the thiobarbituric acid reactive
substances (TBARS) in different concentrations of MEPF on normal rat liver homogenates. The
reduced glutathione (GSH) was assayed in liver homogenates of different concentrations of
MEPF using the method of Ellman et.al. The nitric oxide (NO) scavenging activity and 1-1
Diphenyl,2-Picryl hydrazyl (DPPH) radical scavenging activity was measured using the methods
of Sreejayan et.al and Shimada et.al. respectively using spectrophotometer. Vitamin E and
normal saline were used as reference standard and control for all four invitro antioxidant
measurement assays. The results showed significant antioxidant activity of MEPF in all four in
vitro methods used in this study and the IC50,(the half maximal inhibitory concentration,
of an inhibitor that is required for 50% inhibition of antioxidant activity) of MEPF was
comparable to that of vitamin E, the reference standard compound used in this study.
It is concluded that the methanolic extract of leaves of Pandanus fascicularis has significant
antioxidant activity.

Key words: Pandanus fascicularis, antioxidant action, lipidperoxidation, reduced glutathione

nitric oxide scavenging ,diphenyl picryl hydrazyl radical scavenging.

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Introduction

Plants are widely used in various traditional and folklore systems of medicine. Consumption of
medicinal plants and their products has almost doubled over the last decade in developed
countries. At present the trend of wide spread interest in alternative therapies, is well known.
With the recent success of many plant derived drugs such as anticancer agent taxel and it’s
derivatives from Taxus baccata and Taxus brevifolia and antimalarial, artemisinin from the
Chinese wormwood Artemisia annua, the interest is growing.
The development of science and phytochemistry rejuvenated the hopes for remedies in
chronic diseases and this has generated new enthusiasm in the research work to develop herbal
medicine. WHO (World Health Organization) estimated that 80% of the population in
developing countries still relies on plant-based medicine for preliminary healthcare. Now efforts
are being made to develop herbal medicines in research institutes. Although India has the
tradition of alternative therapies like Ayurveda, Siddha and Unani, there are no procedures to test
the safety and efficacy of traditional remedies and to standardize their effective cure. For these
reasons we should increase our efforts in the area of medicinal plant research and should exploit
efficiently for the benefit of humanity.

Pandanus fascicularis Lam. (Synonyms: Pandanus tectorius, Pandanus odoratissimus Roxb,

Family: Pandanaceae). Vernacular names 1, 2: Sanskrit- ketaki, Hindi-Kura, Kewda, Ketki,
Gagandhul, Kannada-Tale mara, English-Screw pine) is distributed widely in coastal regions of
Indian subcontinent and Andaman Islands. The plant is a branched palm like shrub, up to 1-3 m
high, rarely erect, stem supported by aerial roots, leaves glaucous-green, 0.9-1.5m, ensiform,
long lanceolate, acuminate with three rows of prickles each on the margins and on midrib
beneath, spinescent. Male flowers in spikes enclosed in large, white fragrant spathes, female
flowers in solitary spadix. Syncarpium yellow or red, drups numerous, each consisting of 5-12
carpels; each carpel 5-12.5 cm long, turbinate and angular20. Propagation is by seeds and
vegetative method. Leaves, flowers, roots, fruits, spadices, bracts are used in leprosy, smallpox,
syphilis, scabies, pain, heat of body, diseases of heart and brain and leucoderma21. The tender
floral leaves are used to flavour cream, rice (giving a flavour similar to basmati rice) sherbets,
jellies and sometimes curries 2, 3. Oil from bracts is used for headache and
rheumatism and considered as stimulant and anti spasmodic3. Kewda attar or water prepared by
distillation of spadices is used to flavour sweets, syrups and soft drinks2.The flower is pungent,
bitter; improves complexion. The anthers are useful in pruritis, earache, headache, leucoderma,
eruptions, and diseases of the blood. Fruit is useful in relieving “vata”, “kapha” and urinary
discharge and is beneficial in leprosy3. A medicinal oil is prepared from the roots is considered
as diuretic, depurative and tonic3. Juice obtained from inflorescence is used for rheumatic
arthritis in veterinary medicine4. Kewda oil is used in ear ache, head ache, arthritis, debility,
depurative, giddiness, laxative, leprosy, rheumatism, small pox and spasms5.

The principle constituent of kewda oil responsible for the characteristic aroma of spadices is b-
phenyl ethyl methyl ether. 2-acetyl –1 pyrroline is a major volatile component in the tender floral
leaves or spathes. Blossoms yield 0.1-0.3% essential oil called kewda oil containing benzyl
benzoate, benzyl salicylate, benzyl acetate, benzyl alcohol, geraniol, linalool, linalyl acetate,
bromostyrene, guaiacol, phenyl ethyl alcohol, and aldehydes. Cirsilineol, n-triacontanol, β-

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sitosterol, β-sitostenone, stigmast-4-ene-3,6dione stigmasterol, campesterol, daucosterol, and

palmitic acid, stearic acid isolated from rhizomes2,6,7,8
Inflammatory diseases including different types of rheumatic diseases are very common
throughout the world. The greatest disadvantage in the potent synthetic drugs available at present
lies in their side effects, toxicity and reappearance of symptoms after discontinuation. Hence
search for new anti- rheumatic agents that retain the therapeutic efficacy and devoid of adverse
effects are justified.
So Pandanus fascicularis, a plant that was traditionally used for rheumatism with no scientific
proof claimed as yet on its leaf is investigated in this work. Inflammatory state is
associated with free radical formation and cell damage. The present study has been undertaken to
investigate and evaluate methanolic extract of leaves of Pandanus fascicularis for its free radical
scavenging potential. In this study after preliminary phytochemical screening of the extract, its in
vitro antioxidant activity were tested out using lipid peroxidation, reduced glutathione assay,
nitric oxide and DPPH scavenging activity and compared with that of vitamin E the standard
antioxidant compound.
Materials and Methods
Leaves of Pandanus fascicularis used for the investigation were collected from the east coast
road, Chennai-96, in the month of April 2004. The plant was identified and authenticated by
research officer (Pharmacognosy), Central Research Institute (Siddha), Arumbakkam, Chennai-
600106.
Preparation of methanol extract :
The leaves of Pandanus fascicularis were collected, coarsely powdered and was
successively extracted with methanol using Soxhlet extractor. The methanol extract of Pandanus
fascicularis (MEPF) was dried under reduced pressure using a rotary flash evaporator and it was
kept in the refrigeration. The percentage of yield of methanolic extract was 9%.

The methanol extract thus obtained was used for the preliminary phytochemical screening
and pharmacological studies such as for in-vitro antioxidant activity. The liver homogenate (3ml)
of normal rats was used for invitro lipid peroxidation(LPO) and reduced glutathione(GSH)
activity of MEPF after dissolving it in 3ml normal saline in varying concentrations (25µg-800
µg/ml). Lipid peroxidation (LPO) was assayed by estimating thiobarbituric acid reactive
substances(TABRS) and reduced glutathione (GSH) was assayed in the liver homogenates of
normal rats with different concentrations of methanol extract of Pandanus fascicularis
(MEPF).The experimental protocol was approved by the Institutional Animal Ethics Committee
(IAEC) , Reference No: IAEC-X-3 / CLBMCP / 2004-2005.

PRELIMIARY PHYTOCHEMICAL SCREEIG 9,10

The methanol extract of Pandanus fascicularis leaves was subjected to preliminary
phytochemical screening. This extract was tested for the presence of alkaloids, carbohydrates,
proteins, steroids,sterols,phenols,flavonoids,tannins,gums.mucilage,glycosides,saponins and
terpenes9,10..

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I-VITRO ATI OXIDAT STUDIES:

Lipid peroxidation (LPO)11

The degree of lipid peroxidation was assayed by estimating the thiobarbituric acid
reactive substances (TBARS). Different concentrations (25-800 µg/ml) of MEPF were added to
the normal rat liver homogenate(3ml). LPO was initiated by adding 100 µl 15 mM ferrous
sulphate solution to 3 ml of liver homogenate. After 30 min 0.5ml of this reaction mixture was
taken in a tube containing 1.5 ml of 10 % w/v trichloroacetic acid (TCA). After 10 min, tubes
were centrifuged and supernatant was separated and mixed with 1.5 ml of 0.67% thiobarbituric
acid (TBA) in 50% acetic acid. The mixture was heated in a boiling water bath at 85 °C for 30
min to complete the reaction. The pink coloured complex formed was measured at 535 nm in a
spectrophotometer. Vitamin-E was used as reference standard. The percentage inhibition of LPO
was calculated by comparing the results of the test with those of controls not treated with the
extracts as per following formula -Percentage inhibition = {(control-test) /(control)} X 100.

Reduced glutathione (GSH) assay12:

Liver homogenate(3ml) with different concentrations (25-800 µg/ml) of MEPF was
mixed with 0.5 ml of precipitating buffer (5% w/v TCA in 0.1 mM EDTA). The sample was
centrifuged at 2000 rpm for 10 min and the supernatant was mixed with 2.5 ml of 0.1 M
phosphate buffer (pH 8.0). The colour was developed by adding 100 µl of 0.01% DTNB.
Absorbance was noted at 412 nm using UV spectrophotometer. The percentage reduction was
calculated by comparing with control. Vitamin-E was used as reference standard.

itric oxide (O) scavenging activity13:

Nitric oxide scavenging activity was measured by using spectrophotometer. Sodium
nitroprusside (5 mM) in phosphate buffer saline was mixed with different concentrations of
MEPF (25-800 µg/ml) dissolved in normal saline and incubated at 25°C for 30 min. A control
without test compound but with equivalent amount of sodium nitroprusside was taken. After 30
min 1.5 ml of the incubation solution was removed and diluted with 1.5 ml of Griess reagent (1%
sulphanilamide, 2% phosphoric acid, and 0.1% napthyl ethylene diamine dihydrochloride). The
absorbance of the chromophore formed during diazotization of the nitrite with sulphanilamide
and subsequent coupling with napthyl ethylene diamine dihydrochloric acid was measured at 546
nm. Vitamin-E was used as reference standard.

1-1 Diphenyl, 2-picryl hydrazyl (DPPH) radical scavenging activity14:

DPPH scavenging activity was measured by spectrophotometric method. 0.1 mM
solution of DPPH was prepared in ethanol and 1 ml of this solution was added to 3 ml of
MEPF in normal saline at different concentrations (25-800 µg/ml). Equal amount of normal
saline was added to the control. The mixture was shaken well and incubated at room
temperature for 30 min. The absorbance was read at 517 nm using a spectrophotometer.
Vitamin-E was used as reference standard. All the assays were read at a particular nm using
spectrophotometer, UV –1601 Shimadzu

Statistical tests: In in-vitro experimental methods the percentage of inhibition and the IC50
value or the half maximal inhibitory concentration, representing the concentration of an
extract that is required to inhibit 50% of oxidant activity were calculated and compared with

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that of the standard antioxidant compound vitamin E and the control(normal saline) used in
this experiment. Results were presented as mean ± SEM and the correlation coefficient denoted
as “r”, indicating the potency of the test compound as compared to the standard. The
antioxidant activity is considered as significant if the “r” value is >0.5. The ‘p’ value is not
mentioned here (though it is p<0.001) since here it indicates the lesser activity than the
standard compound used.

Results
Preliminary phytochemical screening:
The results of preliminary phytochemical screening of the methanol extract of Pandanus
fascicularis leaves showed the presence of alkaloids, carbohydrates, phenols, steroids, sterols,
proteins and glycosides.

In vitro antioxidant methods:

Lipid peroxidation:
MEPF inhibited the ferrous sulphate induced lipid peroxidation in a concentration
dependent manner. The IC50 value of MEPF was found to be 669.23 µg/ml(r= 0.61). The IC50
value of vit-E was 411.3 µg/ml (Table - 1). The regression coeifficient “r” is the ratio between
vitamin E and MEPF indicating the potency of MEPF as compared to vitamin E.

Reduced glutathione assay.

MEPF inhibited the oxidation of reduced glutathione in a dose dependent manner. The
IC50 values of MEPF was found to be 697.1µg/ml (r= 0.59). The IC50 value of vit-E was 414.25
µg/ml (Table-1).

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Table 1: Effect of MEPF on Lipid peroxidation (LPO) and oxidation of GSH in rat
Liver homogenate (Mean ± SEM, n=6)

Drug (MEPF/Vit. E) Percent (%) inhibition Percent (%) inhibition

concentration (µg/ml)
Lipidperoxidation(LPO) in Oxydation of GSH in liver
liver homogenate homogenate
MEPF Vit. E MEPF Vit. E
2.67 ± 0.04
25 28.31 ± 0.05 4.25 ± 0.14 25.66 ± 0.16

50 10.47 ± 0.02 40.84 ±0.02 12.12 ±0.06 41.86 ± 0.01

22.56 ± 0.02
100 57.54 ±0.02 25.91 ±0.05 55.36 ± 0.08

35.45 ± 0.02
200 67.95 ±0.02 35.91 ±0.57 68.40 ± 0.12

400 44.49 ± 0.01 81.42 ±0.10 43.71 ±0.15 75.75 ± 0.08

800 59.77 ± 0.01 97.25 ±0.03 57.38 ±0.24 96.56 ± 0.12

IC50 669.23± 0.04 411.3± 0.14 697.1 ±0.11 414.25 ±0.15
r=Vit.E/MEPF 0.61 0.59

Table 2: Effect of MEPF on nitric oxide scavenging activity and free radical scavenging
activity by DPPH reduction action (Mean ± SEM, n=6)

Drug (MEPF/Vit. E) Percent (%)Inhibition Percent (%)Inhibition

Concentration (µg/ml)
Nitric oxide scavenging activity Free radical scavenging activity
by DPPH reduction
MEPF Vit. E MEPF Vit. E
8.27 ± 0.04
25 5.3 ± 0.04 24.20 ± 0.16 26.30 ± 0.09

50 12.3 ± 0.08 39.46 ± 0.02 17.55 ± 0.05 37.66 ± 0.02

20.4 ± 0.06 29.35 ± 0.07

100 50.82 ± 0.01 47.30 ± 0.03

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43.03 ±0.04
200 31.8 ±0.06 67.82 ± 0.01 60.60 ± 0.10

400 43.2 ± 0.08 81.05 ± 0.01 52.49 ± 0.50 79.19 ± 0.08

56.1 ± 0.02 66.98 ± 0.03

800 96.18 ± 0.01 98.77 ± 0.03

IC50 713.01± 0.07 415.89 ±0.02 597.19± 0.50 404.98 ± 0.18

r=Vit.E/MEPF 0.58 0.68

Discussion
Indigenous drug system can be a source of a variety of new drugs, which can provide
relief to pain, fever and inflammation but their claimed reputation has to be verified on a
scientific basis. The present investigation revealed that the extract of Pandanus fascicularis
leaves (MEPF) has a significant antioxidant activity in all the four invitro models used in this
study.
Recent studies suggest that inflammation and tissue damage are due to the liberation of
free radicals15. The free radicals have been implicated in the pathophysiology of various clinical
disorders including inflammation, acute hypertension and cancer etc.16. Normally endogenous
intracellular antioxidants protect the tissue from injury by free radicals17.Therefore development
of antioxidant drug could be beneficial as adjunct to anti-inflammatory therapy. Phytochemical
screening revealed the presence of phenols, which could be responsible for its anti-inflammatory
and antioxidant activity. Phenols are very important plant constituents because of their
scavenging ability due to their hydroxyl groups. The phenolic compounds may contribute
directly to antioxidant action.
The MEPF showed significant protection against ferrous sulphate induced LPO which
could cause by absence of ferryl perferyl complex. It is generally assumed that ability of the
plant phenolic compounds to chelate iron in LPO system is very important for their antioxidant
property. Therefore an attempt was made to determine the role of iron chelation, since the
inhibition of ferrous sulphate induced LPO could also be due to chelation of iron. So it can be
concluded from the present study, the extract offers protection against ferrous sulphate induced
LPO by either metal chelation or absence of ferryl perferyl complex, which is essential for
inhibition of LPO18. GSH is a non enzymic mode of defense against the free radicals. Thiols
especially cystein and glutathione are important in leukocyte functioning19.
The study on nitric oxide scavenging demonsrates that the methanol extract of Pandanus
facicularis is a potent scavenger of nitric oxide. NO generated from sodium nitroprusside reacts
with oxygen to form nitrite ions which can be estimated by the use of Griess reagent. Scavengers
of NO compete with oxygen leading to reduced production of NO13.
The free radical scavenging activity of the plant extract MEPF was evaluated based on
the ability to quench the synthetic DPPH. Because of the odd electrons DPPH shows a strong

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Pharmacologyonline 1: 833-841 (2011) Sanjeeva et al.

absorption band at 517 nm in visible spectrum. As this electron becomes paired off in presence
of free radical scavenger, the absorption vanishes and the resulting decolourisation is
stoichiometric with respect to the number of electrons taken up20. The bleaching of DPPH
absorption is representative of the capacity of the test compounds to scavenge free radicals
independently. The results revealed that the test compound /extract is an electron donor and
could react with free radicals to convert them to more stable products and terminate the radical
chain reaction. In the present study it can be concluded that the MEPF has significant antioxidant
activity. Since the antioxidants have been demonstrated to be useful in inflammatory disorders21
the claimed beneficial effects of Pandanus fascicularis in traditional medicine in various
rheumatic disorders could be due to its antioxidant activity. This enables us for further research
to find out the active principle responsible for the antioxidant action and its isolation to be used
in rheumatic disorders.

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