Catharanthus Roseus (L.) G. Don. AN IMPORTANT DRUG: IT'S APPLICATIONS AND

Aslam J et al.
/ Pharmacie Globale (IJCP) 2010, 4 (12)
Available online at www.pharmacie-globale.info ISSN 0976-8157
Review Article
PHARMACIE GLOBALE
INTERNATIONAL JOURNAL OF COMPREHENSIVE PHARMACY
Catharanthus roseus (L.) G. Don. AN IMPORTANT DRUG: IT’S APPLICATIONS AND

PRODUCTION
Junaid Aslam1,5*, Sheba Haque Khan4, Zahid Hameed Siddiqui1, Zohra Fatima1, Mehpara Maqsood1, Mukthar Ahmad Bhat6,
Sekh Abdul Nasim1, Abdul Ilah3, Iffat Zareen Ahmad2, Saeed Ahmad Khan5, Abdul Mujib1 and Maheshwar Prasad Sharma1
1Department of Botany, Jamia Hamdard (Hamdard University), New Delhi, India.
2Department of Biotechnology, Integral University, Dasauli, Kursi Road, Lucknow, Uttar Pradesh, India
3Department of Medical Technology, Omar Al Mukhtar University, Tobruk, Libya
4Department of Chemistry, M.J.P. Rohilkhand University, Uttar Pradesh, India
5Dubai Pharmacy College, Al Muhasinah 1, Dubai, United Arab Emirates
6University of Kashmir, Srinagar Kashmir, India
Received: 18 August 2010; Revised: 20 October 2010; Accepted: 29 October 2010; Available online: 1 November 2010
ABSTRACT
Catharanthus roseus is an important drug. Traditionally, different parts of it used in the treatments of various
diseases (viz. diabetes, menstrual regulators, hypertension, cancer and antigalactagogue etc.), in numbers of
countries (Australia, Brazil, China, Cook Island, Dominica, England, Europe, France, French Guina, India,
Jamaica, Kenya, Mexico, Mozambique, North Vietnam, Pakistan, Peru, Philippines, South Africa, South Vietnam,
Taiwan, Thailand, USA, Venda, Vietnam, West Indies etc.). Moreover, more than 130 alkaloids have been
isolated from different parts; amongst which two important alkaloids (Vinblastine and Vincristine used in
cancer treatment) present in very low concentrations. Keeping these views; researcher continuously using
different approaches to enhance the level of important alkaloid to meet the required demand. Thus, in
present communication various in vitro biotechnological and biochemical approaches (viz. effect of media
composition, plant growth regulators, pH of the culture medium, temperature, light, aeration, elicitors,
mutagenesis, high cell density culture, selection of superior cell lines, bioreactor and immobilization methods,
hairy root culture, somatic embryogenesis, biosynthesis of alkaloids, metabolic and genetic engineering in
alkaloids biosynthesis, coupling method for alkaloids biosynthesis, cellular compartmentation etc.) have
been used worldwide; which directly concerning with the in-vitro micropropagation and the enhancement
of important secondary metabolites present in different parts of Catharanthus and being used in the treatment
of various diseases.
Keywords: Catharanthus roseus, secondary metabolites, disease, in-vitro micropropagation.
INTRODUCTION
Catharanthus roseus is a renowned medicinal plant, lungs and other organs in acute and chronic leukemia.
belonging to the family Apocynaceae; and is a rich source Vincristine sulphate (sold as Oncovin) arrest mitosis in
of alkaloids, which are distributed in all parts of the plant. metaphase and is very effective for treating acute
The alkaloid content of C. roseus varies considerably in leukaemia in children and lymphocytic leukemia. It is also
various parts; the maximum being in the root bark which used against Hodgkin’s disease, Wilkins’s tumor,
ranges from 0.15 to 1.34 % and even up to 1.79 in some neuroblastoma and reticulum cell sarcoma. Today India is
strains.1 The plant contains about 130 alkaloids of the the third largest manufacture of Vinblastine and
indole group out of which 25 are dimeric in nature. Two Vincristine in the world and is exporting these alkaloids to
of the dimeric alkaloids vinblastine and vincristine European countries. High demand and low yield of these
mainly present in the aerial parts, have found extensive alkaloids in the plant has led to research for alternative
application in the treatment of human neoplasma. Among means for their production. Vinblastine is also modified
the monomeric alkaloids ajmalicine (raubacine) found in structurally to yield deacetyl vinblastine amide
the roots has been confirmed to have a broad application (Vindesine) introduced recently as Eldisine for use in the
in the treatment of circulatory diseases, especially in the treatment of acute lymphoid leukemia in children.
relief of obstruction of normal cerebral blood flow. Biochemical coupling of alkaloids Catharanthine and
Vinblastine sulphate (sold as Velban) is used particularly Vindoline to get dimeric compounds is also achieved.
to treat Hodgkin’s disease besides lymphocarcoma, Beside these, tissue culture technique is developed for the
choriocarcinoma, neuroblastoma, carcinoma of breast, development of these dimeric alkaloids.2 In the present
*Corresponding Author: communication a detailed application of C. roseus
Dr Junaid Aslam including traditional uses in various developed and
Department of Botany, Jamia Hamdard(Hamdard University),
New Delhi-110062, India. developing countries, pharmacological activities and
Email: [email protected] the application of various biotechnological tools viz.
1 Pharmacie Globale (IJCP), Vol. 01, Issue 04

Aslam J et al. / Pharmacie Globale (IJCP) 2010, 4 (12)
Optimization of Media Composition, Phytohormones, pH, (more than 100) with colour ranging from white to pink
Temperature, Light, Aeration, Elicitors, Mutagenesis, High to purple.
Cell Density Culture, Selection of Superior cell lines,
Bioreactors and Immobilization Methods, Hairy root TRADITIONAL USE OF C. roseus
culture, In Vitro Somatic embryogenesis, Biosynthesis of The plant has historically been used to treat a wide
alkaloids in Catharanthus, Metabolic and Genetic assortment of diseases. It was used as folk remedy for
Engineering in alkaloids biosynthesis, Coupling method diabetes in Europe for centuries.3 In India, juice from the
for Alkaloids biosynthesis, Cellular Compartmentation has leaves was used to treat wasp stings. In Hawaii, the plant
been applied for the enhancement of important was boiled to make a poultice to stop bleeding. In china, it
secondary metabolites present in different parts of was used as an astringent, diuretic and cough remedy.4
Catharanthus. In central and south America, it was used as a
TAXONOMY, HABIT AND HABITAT OF homemade cold remedy to ease luncongestion and
inflammation. Throughout the Caribbean, an extract from
Catharanthus roseus the flowers was used to make a solution to treat eye
Catharanthus roseus is an important medicinal plant, irritation and infections. It also had a reputation as magic
commonly known as Madagascar periwinkle. It is a plant, European thought it could ward off evil spirits, and
perennial, evergreen herb, 30-100 cm height that was the French referred to it as “violet of the sorcerers.”
originally native to the island of Madagascar. It has been Western researchers finally noticed the plant in 1950’s
widely cultivated for hundreds of year and can now be when they learn of a tea Jamaican were drinking to treat
found growing wild in most warm regions of the world. diabetes. They discovered that the plant contains a
The leaves are glossy, dark green (1-2 inch long), oblong – motherlode of useful alkaloids (130 in all at last count).
elliptic, acute, rounded apex; flowers fragrant, white to Some, such as catharanthine, leurosine sulphate,
pinkish purple in terminal or axillar y cymose clusters; lochnerine, tetrahydroalstonine, vindoline and vindolinine
follicle hairy, many seeded, 2-3 cm long; seeds oblong, lower blood sugar level, however, others act as
minute, black. The plant is commonly grown in gardens for haemostatics (arrest bleeding) and two others, vincristine
beddings, borders and for mass effect. It blooms througout and vinblastine have anticancerous properties. Periwinkle
the year and is propagated by seeds or cuttings. The bloom also contains the alkaloids reserpine and serpentine,
of natural wild plants are pale pink with a purple eye in which are powerful tranquilizers. Table 1. Summarized
the centre, but horticulturist has developed varieties traditional uses of the C. roseus in different countries.
Table 1 . Traditional Medicinal Uses of Catharanthus roseus in various developed and developing countries.
Country Used as
Australia Hot water extract of dried leaves is taken orally for menorrhagia, diabetes and extract of root bark is taken orally as
febrifuge146,147
Brazil The hot water extract of dried entire plant is taken orally by human for diabetes mellitus148,149.
China Hot water extract of the aerial parts is taken orally as a menstrual regulators.150, 151
Cook Island Decoction of dried leaves used orally to treat diabetes, hypertension and Cancer 152
Dominica Hot water extract of leaves is taken orally by pregnant woman to combat primary inertia in childbirth and the boiled
leaves are drink to treat diabetes153
England Hot water extract of dried entire plant is taken orally for the curing of diabetes154
Europe Decoction of dried leaves is taken orally for diabetes mellitus24.
France Hot water extract of entire plant is taken as an antigalactagogue 150.
French Guina Hot water extract of entire plant is taken orally as a cholagogue 155.
India The hot water extract of dried entire plant is taken orally by human for cancer. Hot water extract of dried leaves is taken orally to
Hodgkin’s disease. The root extract is taken orally for menorrhagia 151, 156.
Jamaica Hot water extract of dried leaves is taken orally for diabetes 158
Kenya Hot water extract of dried leaves is taken orally for diabetes158
Mexico Infusion of whole plant is taken orally for stomach problem 159
Mozambique Hot water extract of leaves is taken orally for diabetes and rheumatism and the root extract is taken orally as hypotensive and
febrifuge 160.
North Vietnam Hot water extract of the aerial parts is taken orally as a menstrual regulator 150, 151.
Pakistan Hot water extract of dried ovules is taken orally for diabetes161.
Peru Hot water extract of dried entire plant is taken orally by human adults for cancers, heart disease and leishmaniasis162.
Philippines Hot water extract of root is taken orally by pregnant women to produce abortion151, 163, 164.
South Africa Hot water extract of dried leaves is taken orally for menorrhagia and diabetes 146.
South Vietnam Hot water extract of the entire plant is taken orally by human adults as an antigalactagogue 150,151.
Taiwan Decoction of dried entire plant is used orally by human adults to treat diabetes mellitus164 and liver disease 165.
Thailand Hot water extract of dried entire plant is taken orally for diabetes166.
USA Hot water extract of leaves are smoked as a euphoriant 167.
Venda Water extract of dried root is taken orally for venereal disease 168.
Vietnam Hot water extract of dried aerial parts is taken orally as drug in Vietnamese traditional medicine, listed in Vitnamese
pharmacopoeia (1974 Edition) 169.
West Indies Hot water extract of leafy stems is taken orally for diabetes 170.
Antitumor Activity: Ethanol (70%) extract of leaves was active on Leuk-P3887. The plant contains about 130
administered intraperitoneally to female mice5, and prove alkaloids (Table 2) of the indole group, out of which 25 are
to be highly active on CA-Ehrlich ascites. Alkaloid fraction dimeric in nature. Total alkaloids of the entire plant
of dried leaves was also used externally was actives. administered to mice intraperitoneally at a dose of 10.0
Nineteen patients with either flat, verruca vulgaris, plantar mg/kg and orally at a 75.0 mg/kg were active on Leuk-
or genital warts were treated in this study. Six patients P15348.
had all warts disappears, seven had the majority of their Antispermatogenic Effect: Hot water extract of dried
warts disappears, five had 50% disappears and one leaves administered intraperitoneally to male mice at a
showed no response.6 Chloroform extract of leaves was dose of 0.2 ml/animal produced weak activity.9 At 10.0
mg/ animal, regressive changes in seminiferous tubules vindolinine,19-epi,N-oxide P Cell suspension culture
and Leydig cells, increased cholesterol in testes and fluorocarpamine, N -oxide M Plant extract, leaf
degeneration of all germinal elements.10 Total alkaloids of perividine U Plant extract
entire plants administered intraperitoneally to male rats isositsirikine, 19,20-cis-16
C
Plant extract, cell suspension
were active.11 (R)- culture
isositsirikine, 19,20-trans-16 Plant extract, cell suspension
C
Table 2. Alkaloids isolated from different parts of (R)- culture
Catharanthus roseus (largely based on Dictionary of isositsirikine, 19,20-trans-16 Plant extract, leaf, cell
C
(S)- suspension culture
Natural Products, version 8.2, 2000). minovincinine P Cell suspension culture
Alkaloids Class1 Extracted from
Plant extract, leaf, callus
β-carboline M Leaf sitsirikine C culture, cell suspension
tryptamine,N,N-dimethyl M Cell suspension culture culture, shoots
tryptamine,Nb-acetyl M Cell suspension culture Plant extract, leaf, root, callus
apparicine A Leaf, flower yohimbine C culture, cell suspension
ammocalline U Plant extract, root culture, hairy root
Plant extract, cell suspension Plant extract, leaf, root, callus
anthirine C
culture sitsirikine,dihydro- C culture, cell suspension
akuammicine S Plant extract, leaf, root, callus culture, cell suspension culture,culture
shoots
iochrovicine U Leaf perimivine C Plant extract, root
pericyclivine C Plant extract, leaf tabersonine,11-methoxy P Plant extract, flower
pleiocarpamine C Cell suspension culture almalicine, 7-hydroxy -
Plant extract, leaf, root, callus C Callus culture
cavincine C indolenine
culture, hair y root ajmalicine pseudo-indoxyl C Callus culture
iochnerine C Cell suspension culture akuammiline,10-
Callus culture, cell suspension C Callus culture
tubotaiwine A hydroxy- deacetyl
culture epimisiline,19(s) P Hairy root
rosicine P Leaf horhammericine P Cell suspension culture, shoots
Plant extract, leaf, flower, mitraphyllline C Flower, callus culture
catharanthine I seedlings, callus culture, cell vincoline P Plant extract, leaf
suspension culture, shoots vindolinine P Plant extract, leaf, cell suspen
Plant extract, leaf, seedlings, Plant extract, leaf, cell
tabersonine P seed, callus culture, cell vindolinine,19-epi P
suspension culture
suspension culture vincolidine U Plant extract, leaf
venalstonine P Root akuammine C Plant extract
akuammicine,12-hydroxy S Cell suspension culture Plant extract, leaf, cell
Plant extract, leaf, flower, lochnerinine S
suspension culture
perivine C root, callus culture, cell lochrovidine U Plant extract
suspension culture tabersonine,19-
vinervine S Cell suspension culture P Plant extract
hydroxy-11- methoxy
coronaridine I Flower iochrovine U Plant extract
vincadifformine I * vindolidine,O-deacetyl- P *
Callus culture, cell suspension Plant extract, cell suspension
cyclolochnerine,21-hydroxy C akuammiline C
culture, shoots, hairy root culture
Leaf, callus culture, cell horhammericine,11-methoxy P Cell suspension culture, shoots
Iochneridine S
suspension culture, hairy root vincarodine E Plant extract, leaf
alstonine C Root, callus culture vinosidine U Root
Leaf, root, seedlings Cell suspension culture, leaf,
vindoline,deacetoxy- P
seedlings
callus
tabersonine,19- Plant extract, leaf, cell
P
serpentine C acetoxy-11- hydroxy- suspension culture
culture,
vindoline,deacetyl- P Plant extract, leaf
cell suspension culture, iochnerinine U Leaf, root
shoots, ,hairy root tabersonine,19-acetoxy-11-
P Cell suspension culture
cathenamine C Plant extract methoxy
Callus culture, cell suspension cathovaline P Leaf
vallesiachotamine V vindolidine P Plant extract, flower
culture
Callus culture, cell suspension Cell suspension culture, shoots,
isovallesiachotamine V strictosidine lactam V
culture hairy root
Callus culture, cell suspension Plant extract, leaf, flower,
ajmalicine C vindoline P
culture seedlings, shoots
Plant extract, callus culture, akuammicine, xylosyloxy- S Cell suspension culture
ajmalicine,19-epi,3-iso C
cell suspension culture Plant extract, leaf. Root, seed,
Plant extract, callus culture strictosidine D callus culture, cell suspension
ajmalicine, 3-epi C culture
,cell suspension culture
akuammigine C Cell suspension culture bannucine P Plant extract, leaf
akuammiline, O-deacetyl C Leaf, callus culture leurosivine B Leaf
Plant extract, leaf, cell leurosine,17-deacetoxy- B Plant extract
Iochnericine P
suspension culture vinblastine,4-deacetoxy- B Plant extract, leaf
minovincine P Plant extract vinblastine, deacetyl- B Plant extract
preakuammicine S Seedlings vinsedine B,U Seed
rosamine U Leaf leurosinine B,U Plant extract
tabersonine,19-hydroxy P Cell suspension culture
vinsedicine B,U Seed
Plant extract, flower, root,
vinblastine,3’,4’-anhydro- B Leaf, shoots
callus culture, cell
tetrahydroalstonine C vingramine B Seed
suspension culture, shoots,
hairy root vinblastine,4’-deoxy- B Plant extract, leaf
Plant extract, cell suspension vinosidine B,U Plant extract
vindolinine, Nb-oxide P
culture vinblastine, N-demethyl- B Plant extract
vingrmine, methyl- B Seed a dose of 4000.0 mg/kg was active 65% inhibition was
catharanthamine B Plant extract, leaf noticed in Edema.20
leurosine B Plant extract, leaf, shoots
roseadine B Plant extract, leaf Antihyperchcholesterollemic Activity: Hot water extract
vincathicine B Plant extract, leaf of dried leaves were taken orally to rabbits was active.21
roseamine B,U Plant extract
Plant extract, leaf, flower,
Antidiuretic Activity: Alkaloid fraction of the entire plant
vinblastine B seedlings, cell suspension was given subcutaneously to male rats at a dose of 50.0
culture mg/kg was active.22
vinblastine,20’-epi- B Plant extract, leaf
catharicine B Plant extract, leaf, flower
Antimalerial Activity: Chloroform extract of root at a
catharine B Plant extract, leaf, shoot dose 400.0 mg/kg along with water extract at a dose 4.42
leurosine, 5’-oxo-* B Leaf gm/kg was administered orally to chicken produced weak
carosine B Plant extract, leaf, flower activity on Plasmodium gallinaceum.23
leurosine,N b’-oxide B Leaf
vinamidine B Plant extract, leaf Antihyperglycemic Activity: Dried leaves in the ration of
vincristine B Plant extract, leaf male in mice at a concentration of 6.25% of the diet for 28
leurosidine, N b-oxide B Plant extract days was inactive vs streptozotocin –induced
vinblastine,14’-hydroxy- B Plant extract hyperglycemia.24 Hot water extract of dried aerial parts
vinblastine, 15’hydroxy- B Plant extract administered intragastrically to dogs at a dose of 50.0
neoleurocristine B Plant extract, leaf
vindolidine B Plant extract
gm/kg (dry weight of plant) was inactive; a dose of 10.0
leurosinone B Leaf gm/kg in case of rabbits was active vs alloxan induced
neoleurosidine B Plant extract, leaf hyperglycemia.25 Water extract of fresh cells administered
neoleurosidine,N b-oxide B Plant extract, leaf intragastically to male rats was active vs streptozotocin
vindolicine B Plant extract, leaf induced hyperglycemia, ultimately 60% decreased in
ammorosine U Root blood sugar was observed.26
cathalanceine U Root
Leaf, root, cell suspension Antibacterial Activity: Benzene extract of dried flowers
cathindine C
culture at a concentration of 50% on agar plate was active on
Plant extract, leaf, root, callus Proteus, Pseudomonas, Shigella and Stphylococcus
cavincidine C culture, cell suspension
culture species, however, benzene extract of leaves at a
lochneririne U Leaf, root concentration of 50% on agar plate was active on Proteus,
maandrosine U Plant extract, root Pseudomonas, Salmonella, Shigella and Staphylococcus
perosine C
Plant extract, leaf, root, callus species.27 Ethanol (70%) extract of dried leaves on agar
culture
plate was active on Bacillus megaterium and
rovindine B,U Plant extract, leaf
vinaphamine B,U Plant extract, leaf
Staphylococcus albus and inactive on Bacillus cereus and
vinaspine B,U Plant extract, leaf Staphylococcus aureus.28 Total alkaloids of root at a
vincamicine B,U Plant extract, leaf concentration of 500.0 mcg/ml in broth culture were
1
Class (Biogenetic class according to [18]: A = aspidospermatan, B = inactive on E.coli, Salmonella lyphosa and Shigella
bisindole, C = corynanthean, D = vincosan, E = eburnan, I = ibogan, M dysenteries.14 Water extract of entire plant on agar plate at
= miscellaneous, P = plumeran, S = strychnan, V = vallesiachotaman, a concentration of 1:4 was inactive on Salmonella
U = unknown structure. paratyphi.22
Animutagenic Effect: Hot water extract of dried leaves Antiviral Activity: Water extract of callus tissue in cell
was active on red blood cells. A reduction in number of
culture was active on Tobacco Mosaic Virus.29
micro- nucleated polychromatic red blood cells caused by
various mutagens showed ant mutagenic effect.12 Cardiotonic Activity: Ethanol (70%) extract of leaf and
stem administered intravenously to rats at a dose of 120.0
Antifertility Activity: Methanol/water (1:1) extract of mg/kg was active.14
dried leaf and stem administered orally to male rats was
active.13 CNS Depressant Activity: Total activity of root was
administered intraperitoneally to rats at a dose of 120.0
Antihypertensive Activity: Total alkaloids of root mg/kg was active.14
administered intravenously to dogs at a dose 4.0 mg/kg
was active.14 Cytotoxic Activity: Alkaloid fraction of dried leaves in cell
culture was active on CA-9KB, ED50 0.0435 mcg/ml.30
Antifungal Activity: Acetone and water extracts of dried Chloroform extract and culture filtrate of callus tissue in
aerial parts at a concentration (50%) on agar plate was cell culture at dose of 50.0 gm (dry wt of plant) were
inactive on Neurospora crossa.15 Hot water extract of
active on Leuk-L12 10 culture, water extract.
dried leaves in broth culture was active on Trichophyton
mentagrophytes.16 Hot water extract of dried stem in ROLE OF BIOTECHNOLOGICAL APPROACHES
broth culture was active on T. mentagrophytes and IN CATHARANTHUS MICROPROPAGATION
weakly active on T. rubrum.17 Leaves and roots on agar
plate were active on Pythium aphanidermatum.18
AND ENHANCEMENT OF PHARMACEUTICAL-
LY ACTIVE COMPOUNDS BEING USED IN THE
Antimitotic Activity: Ethanol (70%) extract of leaves was
administered to female mice was active on CA-Fhrlich TREATMENT OF VARIOUS DISEASES
ascites vs induction of metaphase arrest in ascites cells. Due to the pharmaceutical importance and the low
Dosing was applied four days after tumor cell inoculation content in the plant of vinblastine and vincristine
ascetic samples removed 2, 4, 6 and 24 hours post Catharanthus roseus became an important model system
treatment.19 for biotechnological studies on plant secondary
metabolism. Researchers are focusing their attention to
Anti-Inflammatory Activity: Ethanol extract (95%) of enhance the alkaloids yield by various ways (chemically,
dried leaves was administered intraperitoneally to rats at enzymatically, synthetically or by cell culture method).

The plant cell can be cultured at large scale31, but the yield DNA gene integration were made in hairy root cultures of
of alkaloids production is too low and limits commercial C. roseus.
applications. In recent times, however, two strategies Phytohormones: The role of plant growth regulators in
have been commonly used for the enhancement of alkaloids production of C. roseus has been extensively
alkaloids. studied, but the response varies with genetic makeup of
a) In vitro cultivation of shoot via organogenesis and the used explant, type and quantity of phytohormones.33,50
somatic embryogenesis, callus or suspension by the The cytokinin applied exogenously either alone or in
optimization of media, phytohormones, temperature, combination with auxins to suspension cultured cells
pH, light, aeration etc. In addition, high cell density enhanced alkaloids accumulation in tumorous and non-
culture, elicitor’s treatment, mutagenesis, bioreactors tumorous cell lines.51,52 Enzyme peroxidase play a
and immobilization are also practiced to improve significant role in alkaloids biosynthesis, addition of 2,4-D
alkaloids yield. to the culture medium however, reduced the peroxidase
b) Genetic engineering and over expression of activity.53
biosynthetic rate limiting enzymes in alkaloid
biosynthesis pathways. An increase,54 in vindoline and catharanthine
concentration by using 0.1 mg/l BAP and 0.1 mg/l NAA
In-vitro STUDIES added MS medium had been reported. Exogenously
In tissue culture, the response of culture has been supplied cytokinin increased ajmalicine and serpentine
influenced by a number of factors which in turn regulate content in untransformed callus from cotyledons55. At the
alkaloids yield. Some of them are discussed in brief protein level it was shown that endogenously produced
Media Composition: The yield of alkaloids in suspension cytokinin did not mimic the effect of exogenously applied
culture is directly influenced by the surrounding cytokinin in Catharanthus,56 and they also noticed that
environmental conditions and genetic constitution of the the protein pattern of Ipt transgenic callus lines were
concerned plant material. Over the years efforts have insensitive to exogenously used cytokinin. A28 KD
been made in numbers for optimization of culture polypeptide and simultaneous Ajmalicine accumulation
media for better biomass and alkaloids production, was noted on omission of 2, 4-D in medium and by the use
some patents have also been filed.32-35 Carbon sources of NaCl treatments.57, 58
and inorganic compounds play a significant role in indole pH of Culture Medium: In-vitro biomass and alkaloid
alkaloid production. It was earlier reported that nitrogen production are directly influenced by the pH values of the
and phosphate both promoted growth but had an adverse medium; values with a range of 5.5-6.5 did not have much
effect on alkaloids yield36,37 The inhibitory effect of effect on alkaloids yield. The value 5.5 was found to be
nitrogen on alkaloid production has not always been optimum for serpentine production.59 It has been60
observed.38 The effect of nitrogen on alkaloids production reported that alkaloids produced by suspension culture
is dependent on carbon availability to the cells which were stored in vacuole and simultaneously storage
makes the carbon-to-nitrogen ratio (C/N ratio) an capacity changed as the changes of pH in the medium and
important factor to be taken into account. By the vacuole take place. Low and higher values of pH were
determination of the cellular C/N ratio,39 identified three used to release intracellular alkaloids into the culture
distinct growth phases: an active growth phase, an medium.61 It is quite known that the optimized value (5.5-
accumulation phase, and a biomass decline phase 5.8) occasionally fluctuates during culture time and
(endogenous metabolism). They also noticed that influences in-vitro responses including alkaloid yield.
phosphate (0.56 Mm), nitrate (12.97 Mm) and low
concentration of ammonia were beneficial for maximum Temperature: For in-vitro study temperature range from
growth and increased alkaloids production. Similarly 20-30 has been considered best for better biomass and
higher concentration of sucrose only enhanced biomass, growth of cultures, but contradictory information have
the optimized glucose (500Mm), ammonium and been reported about the alkaloids yield. Temperature in
phosphate (0- 12Mm) were previously used for higher low range had inhibitory62, stimulatory63, and no effect on
alkaloids yield.40 alkaloid yield. In the tested cell lines under different
temperature range (20,25,30°C), highest serpentine
Medium composition and day’s interval had direct effect production was64 recorded at 25°C and, no effect was
on induction and accumulation of indole alkaloids.41 A recorded at temperature 17,23 and 32°C while in hair y
medium added with 6% sucrose is favourable for both root culture low temperature enhanced alkaloid yield.65
biomass and alkaloids production in Catharanthus.42
Liquid medium with 3-6% maltose was also found to be Light: Light is an important factor for both ex vitro and in-
highly effective for production of somatic embryos.43 It vitro morphogenetic study. Its presence, absence, time
has been44 reported that agitated liquid media added with and intensity directly influence anabolic and catabolic
BAP (1.0 mg/l) was very productive for large-scale plant processes, particularly secondary metabolism.66,67 Most of
regeneration. Alteration in macro and micronutrient of the study of the effect of light was made on serpentine and
MS medium45 has also been used to promote growth and ajmalicine where serpentine content was directly related
subsequent alkaloid production.46 to the intensity of light in Catharanthus68, same was true
for vindoline69 and however, another alkaloid
Surface methodology47, has been used for the rapid catharanthine was decreased in the absence of light. But it
biomass growth and increase in ajmalicine production in has also been reported70, that light did not affect yield but
hairy root cultures. Similar results in cell suspension it affect the accumulation site. However, 15h per day
culture have been noticed.40 Hairy root culture is a unique exposure instead of 24 improved serpentine
system, often used for root specific indole alkaloids accumulation. Although, dark-grown culture was much
production48 Recently,49 have observed an increase in better in comparison to light grown, where serpentine
growth and terpenoids indole alkaloids (ajmalicine and and ajmalicine content were decreased (serpentine from
serpentine) yield when left and right termini-linked Ri T- 79%-14% and ajmalicine 78%-18%). Gradual transfer of
dark grown culture of Catharanthus towards the light culture increased catharanthine, serpentine and
increased serpentine content, however, continuous tryptamine production but was concentration dependent.
exposure of light decreased serpentine level.64 It has been At 25 ppm, catharanthine and ajmalicine were primarily
optimized that 12h light period54, for better callus growth accumulated, and at 50-75 ppm tryptamine accumulation
and alkaloid production, however, dark period more than was only noticed. Moreover, the effect of heavy metal was
12h decreased alkaloid contents. It has been found71, that studied87 where addition of copper (200µm) increased
an increased chloroplast number and enhanced total indole alkaloid accumulation which was correlated
chlorophyll accumulation in response to light influenced with decreased tryptamine concentration.
serpentine production. Besides, exposure of monochromic Several stress factors (fungal elicitor, vanadyl sulphate
light such as blue (450 nm) or red (670 nm) did not affect and potassium chloride) were used and it was found that
growth and alkaloid accumulation, showed constant the alkaloids accumulation was concentration
ajmalicine and serpentine synthesis which decreased dependent88 The optimal concentration (29,1.45 and 145
further under white light.54, 71 mg g-l dry weight) of fungal elicitor, vanadyl sulphate
Aeration: Different types of gases, mainly CO2 and and potassium chloride into medium increased alkaloids
ethylene, are usually evolved within the culture. In many accumulation, however, higher concentration had toxic
cases these gases reduce O2 level in close vessels, inhibit effects and resulted in the loss of cell viability. Two fold
plant culture growth and secondary metabolism. High increase in alkaloids yield was noticed added tryptophan,
dissolved oxygen and improved gaseous permeability at fungal elicitor and vanadyl sulphate to the culture
aerated condition stimulated secondary metabolism as production medium.89
observed by 72, when ajmalicine production was increased Exposure of 2,2-azobis dehydrochloride (AAPH, an
with high oxygen level. Improved oxidative metabolism at oxidative stress agent) and UVB irradiation to C. roseus
rich O2 level is believed to be the reason for better increased nicotinamide and trigolline content.90
product conversion. Aeration has been provided in Simultaneously phenylalanine ammonia lysate (PAL)
culture to influence the alkaloids synthesis and to make it activity was also increased. The increase in PAL activit y
more efficient modern stirring devices have been caused by 2µm AAPH was prevented by 0.1 mm 3-amino
employed along with traditional shake flask.73,64,74,76,77 benzomide, which is an inhibitor of poly (ADP-ribose)
Different types of fermenters have also been used; polymerase. This suggests that nicotinamide and its
shikonin and ginseng, the two important secondary metabolites function as signal transmitter in response to
metabolites have been commercially produced by the use the oxidative stress, since poly-polymerase has defensive
of fermenters. Several researchers78, 79 have suggested the metabolic functions. The level of vinblistine and leurosine
use of bioreactors in secondary metabolites production in increased in response to irradiation with near (370 nm)
plant cell culture of Catharanthus. An impeller with a ultraviolet light91, 92 in shoot culture of C. roseus; however,
speed of 100 rpm was most appropriate for the catharanthine and vindoline content were decreased.
accumulation of alkaloids; however, higher impeller speed Leaves were more sensitive to dimeric alkaloid
increased callus/suspension growth. The rate of accumulation in comparison to shoot, however,93 near
ajmalicine production was studied80 by using different ultraviolet’s irradiation in whole plant of C. roseus,
vessels including shake flask and bioreactors. He found accumulation of dimeric alkaloids was increased.
that biomass was not affected by different culture vessels;
however, ajmalicine production was decreased with over Yeast extract induces transcription of the biosynthetic
feeding of biomass in shake flask and fermentor. gene encoding strictosidine (STR) in cultured C. roseus
cells and alkalinization of the culture medium. The active
Elicitors: New groups of triggering factors which are principle from yeast extract was partially purified and
better known as elicitors have been reported to stimulate found to be of a proteinacious in nature.94 Age of culture is
the secondary metabolites.81 The substance used as very important factor for the elicitor’s to be effective34, 95;
elicitors may be of biotic and abiotic in origin. Biotic addition of elicitors is preferred after a few days of
elicitors include microbial filtrates (Yeast, Pythium and inoculation of the culture when the cells are rapidly
other fungal filtrate), while abiotic elicitors comprise of dividing.
simple inorganic and organic molecules (vanadyl
sulphate, oxalate, UV irradiation etc.). Mutagenesis: Mutagenesis plays a potent role in the
alteration of the genetic constitution which leads to
It has been reported82 that addition of Phytium produce new varieties. Penicillium is the most classic
aphanidermatum filtrate increased the accumulation of example, with many other successful cases. Process of
phenolic compounds instead of alkaloids production. mutagenesis in diploid plants is very complex.
Effect of different concentrations of Pythium vexans Mutagenesis enhance alkaloids yield but the route of
extract was studied by83, who had noticed that low elicitor biosynthesis and the necessary regulation procedure are
concentration increased serpentine production but no not elucidated yet clearly. Therefore, mutation at target
effect was on catharanthine yield. Addition of site in duplicate is really difficult. In spite of several
nicotinamide (8.2 mm) in C. roseus cell lines was used to limitations in this process, scientists in numbers have
enhance the anthocyanin accumulation.84 The extract of used mutagens.96 Some p-flurophynylalanine resistant cell
Pythium aphanidermatum in a hormone free cell lines lines of Nicotiana tabacum and N. glauca accumulated
responded well and induced enzymes {(TDC and higher level of phenolics.96 In case of Catharanthus, he
anthranilate synthase (AS)} which catalyse the noticed that a tryptophan analog resistant mutant
biosynthesis of several intermediates and subsequently accumulated catharanthine in both growth and
accumulated tryptamine.34 Several inorganic compounds production medium. Similarly several research groups
(sodium chloride, potassium chloride and sorbitol) had used x-rays where more serpentine was produced. Beside
also a positive effect on catharanthine accumulation.85 these examples, some successful reports are available in
The addition of vanadyl sulphate86 to cell suspension other group of crops where mutagenesis improved

metabolic accumulation. reported in Catharanthus.114 The advantage of SE is that

High Cell Density Culture: In order to increase secondary the initial cell populations can be used as a single cellular
metabolites production, high cell density culture feeding system and their genetic manipulation are easy and are
has been attempted with or without much success. Study similar to microorganisms.
on Catharanthus in relation to high cell density was found BIOSYNTHESIS OF ALKALOIDS IN C. roseus
to be unsuccessful. Ajmalicine production was very low Beside alkaloids, many other secondary metabolites have
when inoculam potential was increased to 2:8 from 1:9 been isolated from C. roseus, which include
mg/g.70 Moreover,97 low-density cultures increased monoterpenoids, glucosides (loganin, secologanin,
alkaloids yields. It has also been remarked72 that low deoxyloganin, dehydrologanin) steroids (catasteron,
oxygen level and inadequate nutrient uptake are among brassinolides), phenolics, flavaonoids and anthocyanins.
the possible causes for low metabolic accumulation Metabolites are in fact the end products of a complex
during high cell density culture. process comprising the involvement of several enzymes,
Selection of Superior Cell Lines: Isolation and selection of genes, regulatory genes and (transport through) intra-and
superior lines from the heterogeneous cell populations inter-cellular compartments. The TIA (terpenoids indole
help to improve the yield of alkaloids. These cells show alkaloids) are condensation products of two biosynthetic
genetic variability which was further diversified by the routes which require coordination of the amount of the
use of various mutagenic agents. Ajmalicine and intermediates supplied by both pathways. The
serpentine level were increased in Catharanthus by the biosynthesis of vinblastine requires the participation of at
selection of superior cell lines.98 least 35 intermediates, 30 enzymes, 30 biosynthetic
Bioreactor and Immobilization: In tissue culture, for agents, 2 regulatory genes and 7 intra and intercellular
alkaloids production research has been mainly focused on compartments.
suspension culture which requires a rotatory shaker. For The first study on the biosynthesis of alkaloids was
large-scale production, however, large size culture vessel performed at the end of the 1950s for Catharanthus.
fermenter/bioreactor is most important. In both types of Plants were grown in an atmosphere containing14 CO2
systems a stirring devices is provided for improved and after the extraction of alkaloids; many labeled
aeration.70,99,100 There are several important vessels fitted alkaloids have been detected by using column and paper
with compressors which provide filtered air. For plant chromatography. Among the isolated alkaloids vinblastine
culture growth and productivity, it is recommended and vincristine were found only in a very low quantity.
that bioreactors with low shear stress are much more Thereafter, to increase the level of vincristine and
suitable than those of high shear stress. Bioreactors with vinblastine, cell cultures of Catharanthus were used.
improved mechanical designs are regularly introduced in Biosynthesis of alkaloids by in vitro cell culture has the
bioreactors industry with innovated impeller which helps advantages to manipulate the physiological (rapid
to regulate shear agitation.101 growth, ease of precursor feeding, etc) and genetical
In Catharanthus, immobilization of plants cells has been process. During the biosynthesis of alkaloids of C roseus
suggested for better accumulation of terpenoids. various types of proteinaceous compounds have been
61,102,103,104 Immobilization not only maintains the cells reported in different biosynthetic pathways (Table 3).
viable for a longer period of time but also helps in Table 3. List of proteins isolated during the alkaloids
extracellular alkaloids accumulation. Alginate mediated biosynthesis in Catharanthus roseus.
Name Abbreviation References
immobilized cells enhanced the accumulation of
Proteins involved in regulation
tryptamide, ajmalicine and serpentine.98,105 The use of Octadecanoid-responsive
agar and agarose are found to be effective for long-term ORCA2 94
Catharanthus
maintenance of cells. In the last few years surface AP2-domain protein 2
Octadecanoid-responsive
ORCA3 172
immobilization has been proposed using different types of Catharanthus
AP2-Pdomain
Box Bindingprotein
Factor 3 CrBPF-1 173
matrices for large- scale production of alkaloids.106, 107 In
some other cases, negative influence of immobilization on Proteins from the indole pathway
cell was noticed;104 gel or matrices entrapment on anthranilate synthase ASA 134
polysaccharide sheet is employed in many plant systems chorismate mutase CM 134
and in Catharanthus it is fairly successful. isochorismate synthase ICS 174
Hairy Root Culture: Root contains a variety of secondary tryptophan decarboxylase TDS 174
metabolites which produce alkaloids. High rooting can be Proteins from the terpenoids pathway
1-deoxy-D-xylulose -5-phosphate
induced by genetic transformation using Agrobacterium DXR 175
reductoisomerase
rhizogenes. Induced roots grew with a faster rate in
2C-methyl-D-erythritol -2,4-
hormone free medium with high accumulation of cyclodiphosphate synthase
MECS 175
secondary metabolites in Catharanthus.66 In transgenic 1-deoxy-D-xylulose -5-phosphate
DXPS 176
Catharanthus root, a significant increase in ajmalicine and synthetase
catharanthine was noticed.49,108 Other groups used mevalonate kinase MK 177
various types of bioreactors/fermenters to improve the Phosphomevalonate kinase PMK 178
growth of hairy roots and then for better production of Proteins involved in secologanin biosynthesis
secondary metabolites.109, 110 geraniol 10-hydroxylase G10H 124
cytochrome P 450 reductase CPR 124
In-vitro Somatic Embryogenesis: Although somatic secologanin synthase SCS 179
embryogenesis (SE) has been reported in a wide variety Proteins involved in the biosynthesis of TIAs
of plant genera;111,112 in Catharanthus roseus it has been strictosidine synthase STR 180
reported for the first time.113 Earlier, a preliminarily study strictosidine β-D-glucosidase SGD 181
on plant regeneration from immature zygotic embryo was tabersonine 11-hydroxylase T11H 182

S-adenosyl-L-methionine : 16-
OMT 182 METABOLIC AND GENETIC ENGINEERING IN
hydroxytabersonine O-
methyltransferase ALKALOIDS BIOSYNTHESIS
S-adenosyl-L-methionine: 2,3-
In alkaloids biosynthesis the role of several enzymes have
dihydro-3-hydroxytabersonine-N- NMT 183
methyltransferase been discussed in Catharanthus roseus, a few of them have
desacetoxyvindoline-17-hydroxylase D17H 184 been purified, identified, characterized and their encoding
Acetyl CoA: 17-O-deacetylvindoline genes were also cloned (Table 4 ,5).
DAT 185
17- O-acetyltransferase The alkaloids biosynthesis is a very complex process arise
Acetyl CoA: minovincinine-O- from the precursor’s tryptamine and secologanin. These
MAT 186
acetyltransferase two precursors are derived from two different pathways.
Table 4. Metabolites from cell and tissue culture of C.roseus
S.N. Class Compound Reference
Tryptophan
1 Tryptamine, N, N-dimethyltryptamine, N-acetyltryptamine 187, 188
derivatives
2 Phenolics dehydrodiconiferyl glucosides, 2,3-dihydroxybenzoic acid 189, 82, 190, 191
3 Anthocyanins petunidine malvidin hirsutidin 189
Campesterol, sitosterol, stigmasterol, cholesterol, isofucosterol, 24-methylene-cholesterol,
4 Steroids 192, 193
brassinolide catasterone, α –amyrin, β- amyrin, ursolic acid
5 Fatty acids Palmitic acid, Oleic acid, Linoleic acid 188
Table 5.Enzymes involved in indole alkaloids biosynthesis in Catharanthus roseus

Enzymes Abbreviation Localization Reference
Glutamine synthetase GS Cytosol, Chloroplast 194
Anthranilate synthetase AS - 195
Geraniol 10- hydroxylase G10H Provaculor membranes 196
NADPH-cytochrome P-450 reductase --- Provaculor membranes 196, 197
SAM: loganic acid methyltransferase LAMT - 198
Trptophan decarboxylase TDC Cytosol 121, 199
Strictosidine synthase SS Vacuole 200, 201, 122
Strictosidine β- glucosidase SG Tonoplast 127
Geissoschizine -- - 202
Cathenamine -- - 203
Tetrahydroalastonin e synthase -- - 130
Peroxidase -- - 204
SAM: methoxy2,16-dihydro-16- hydrotabersonine-
N-methyltransferase NMT Vacuole Thylakoid membrance 183
desacetoxyvindoline-4-hydroxylase OHT Cytoplasm 205, 206

AcetylCoA: deacetylvindoline, dd 17-0-acetyltransferase DAT Cytoplasm 207
Tryptamine is formed by the enzyme tryptophan roseus. The native enzyme is a monomer, has a molecular
decarboxylase (TDC), which has been reviewed earlier weight 45KD with three isoforms.128
by various workers115,116,117 while the strictosidine Recently, attention has been paid on the regulation of
synthetase (SSS) helps in the coupling of tryptamine and mevalonate biosynthesis that terminates with the end
secologanin to produce strictosidine.118,119 The other product strictosidine. The encoding genes and enzymes of
enzymes such as geraniol 10-hydroxylase (G10H), different steps of mevalonate pathway have been
NADPH-cytochrome P-450 reductase, anthranilate elucidated.129 After the formation of strictosidine, first
synthetase (AS) have the similar TDC activities which are step of alkaloid biosynthesis is the removal of sugar
involved in the biosynthesis of indole alkaloids.120 The moiety from strictosidine to form an unstable
TDC enzyme has been purified from cell suspension aglycone. Two strictosidine β-glucosidases (SG) were
culture121 and ultimately the cDNA gene was partially purified and characterized from C.roseus cell
established.122 The cytochrome P450 enzyme, geraniol- cultures.130, 127
10-hydroxylase (G10H) and other enzymes have been
studied extensively from intact plant of C. roseus. By HPLC Feeding of terpenoids precursors to C. roseus cell
study123 and selection of a cell line with high G10H suspension cultures increased alkaloids production.131-133
activity;124 the enzyme was purified to homogeneity.123 Addition of trytophan (0.5 Mm) to Catharanthus cells
Based on the internal amino acid sequences obtained resulted in high intracellular levels of tryptamine and an
from the digested protein, gene was cloned and increase in STR activity but did not influence ajmalicine
functionally expressed in yeast. The enzyme belongs to accumulation much.134 As in other feedback inhibitions,
the CYP76B subfamily and is designated as CYP76B6. The product accumulation depend upon the product
activity of this enzyme was induced by treating the cells degradation and this phenomenon has been reported
with the cytochrome P450 inducer phenobarbital and in cell suspension culture of Catharanthus. It is now
decreased after treatment of the inhibitor known that the precursor for alkaloids (tryptophan to
ketoconazole.125 Besides, many other enzymes have tryptamide) was located in the cytosol whereas the
been identified and characterized that metabolize enzyme SSS has been localized in the vacuole.135
strictosidine, which after undergoing several COUPLING METHODS FOR ALKALOIDS
rearrangements produced cathenamine and
ajmalicine.126,127 An another important enzyme is
BIOSYNTHESIS
The bisindoles are derived from the coupling of vindoline
desacetoxyvindoline-4-hydroxylase (DAVH), active during
and catharanthine. Catharanthine is thought to be derived
vindoline biosynthesis, purified from intact plant of C.
from strictosidine via 4,21-dehydrogeissoschizine, alkaloids metabolism. This process of metabolism

stemmadenine and dehydrosecodine while vindoline is involves the participation of plant cell to separate the
derived from strictosidine via stemmadenine and enzyme from their substrates and end products. In this,
tabersonine. This pathway (transformation of tabersonine alkaloids biosynthesis requires three celluler
to vindoline) has got orderly six reactions.69,136 The compartments, namely vacuole, cytosol, and plastid.143
enzyme anhydrovinblastine synthase couples The transformation of tryptophan into tryptamine takes
catharanthine and vindoline to yield AVLB which was place in cytosol139,135, SSS in vacuoles.135,144 SG was tightly
purified and characterized from C. roseus leaves. This bound to the tonoplast boundary.135 Synthesis of
heme protein has a molecular weight of 45 KD and shows strictosidine takes place inside the vacuole which later
the peroxidase activity. During this enzymatic coupling transported to the cytoplasm where its glucose moiety
both the monomers were incubated at 30°C with cultured detached. Ajmalicine has the potentiality to move freely
C.roseus cells as enzymatic source at acidic pH (tris buffer across the cell membrane and accumulated into the
7.0). Only after 3 h the chemical reaction produced vacuoles here it converted into the serpentine by use of
vinblistine and anhydrovinblastine as major products peroxidases145, produced serpentine stored in vacuole
along with other dimeric alkaloids. Vindoline and and cannot pass the tonoplast. In cell suspension cultures
catharanthine were also nonenzymatically coupled to the alkaloid accumulation seems to be restricted to certain
dihydropyridinium intermediate (DHPI) under near- UV cells.61 Permeability of cell plays a potent role to release
light irradiation with a peak at 370 nm in the presence of plant products. There are several permeabilizing agents
flavin mononucleotide. Subsequently DHPI can be like DMSO and Triton X-100 are found to be very
reduced to anhydrovinblastin (AVLB) with an overall effective in Catharanthus cell culture. Besides, for the
yield of 50%, based on initial amount of vindoline. release of secondary products several other agents
Vinblastine content was further improved up to 50% by (chitosan, alginate beads, electroportion and ultra
using various compounds as stimulants.137 Similarly, sonication) have been used with or without cell viability
vincristine can be isolated from vinblastine by chemical in other groups of plants.
conversion. Two routes are employed; first route is the
isolation of N-deformyl-VCR which further converted The cell membrane with active uptake mechanism has
into vincristine by formylation. The second method also been noticed in Catharanthus. Most of the secondary
involves a formylation of the C.roseus extract in which products are generally accumulated intercellular,
conversion of N-deformyl – VLB to VCR takes place, after however several compounds such as taxol,
which the material is oxidized. In both cases vincristine anthraquinones are identified in the media which filtrate
was purified by column chromatography and then itself through membrane. For this extracellular product
sulfated (Lily medicinal information service, Netherland). secretion, addition of resin XAD-7 enhanced the product
adsorption in Cinchona33. The media provided with
It was also reported that MnCl2 and FMN/FAD stimulated amberlite type resin and XAD-7 resin adsorbed ajmalicine
coupling process. However, in the absence of C. roseus cell and catharanthine effectively in C. roseus.
suspension enzymes, ferric acid stimulated coupling
process. The production of vinblastine through enzymatic CONCLUSION
coupling pathway is thought to be highly efficient and Different pharmacological studies and the traditional used
likely to be used commercially very soon. proved the high medicinal properties of the Catharanthus;
Vindoline and bisindole alkaloids are accumulated only in which continuously being used in the treatments of
green tissue and not found in root and cell suspension numbers diseases. Various important alkaloid, mostly the
cultures.138 The developmental regulation of TDS, SSC and monomers were successfully identified in culture media
the enzymes involved in late steps of vindoline with the enhanced yields; however the commercial
biosynthesis has been studied extensively.69.139.140 In production is still far away. The main problem is due to
seedlings of C.roseus, expression of these enzymes was not the lack of optimization of cultural conditions and
under strong developmental control where enzymes several strategies leading with increased accumulation
activity were modulated by tissue specific or light of secondary metabolites. A detail studies are required to
dependent factors. The concentration of vindoline, know the proper enzyme functions at various levels,
catharanthine and 3,’4’-anhydrovinblastine (AVLB) are product membrane permeability and adsorption for
age dependent.141 Vinblastine increased as seedlings improvements towards achieving a viable economic
matured, reaching a steady concentration when the plants production methodology. In addition, over-expression of
become more than three months old. On an average, enzymes and the genetic modification could be very
whole seedlings, young plants and mature plants useful via organogenesis or somatic embryogenesis for
contained 7, 11.5 and 12-µg/g dry weight VLB, the production of desired levels of secondary metabolite.
respectively. After induction of shoot formation the VLB
contents increased rapidly to similar levels of in vitro ACKNOWLEDGEMENTS
seedlings.142
First author (Dr. Junaid Aslam) greatfully acknowledge
CELLULAR COMPARTMENTATION Professor S.A. Khan for their valuable suggestions during
Subcellular compartmentation plays an important role in the preparation of the manuscript.
REFERENCES
1. Singh VP, Jagdev RSD. Ajmalicine (raubacine); A 2. Shah MB, Chauhan G. Recent development of some
medicinally important alkaloid from Catharanthus natural products. In: Supplement to cultivation and
roseus (Vinca rosea). In supplement to cultivation utilization of medicinal plants. SS Honda and MK
and utilization of medicinal plants. SS Handa and MK Koul (eds). RRL, Jammu, pp 53-96.1996.
Kaul (eds) RRL, Jammu, pp. 199-206. 1996. 3. Swanston-Flatt, Day C Flatt PR, et al. Glycaemia

effects of traditional Europian plant treatments for animals. Indian J Physiol Pharmacol. 1992; 36 (4):
diabetes studies in normal and streptozotocin 291-292.
diabetic mice. Diabetes Res. 1989; 10(2): 69-73. 21. Asthana RB, Misra MK. Orally effective hypoglycemic
4. Farnsworth NR: The pharmacognosy of the agent from Vinca rosea. Indian J Biochem
periwinkles: Vinca and Catharanthus. Lloydia. 1961; Biophys.1979; 16: 30-35.
24(3):105-138. 22. Neagi NC, Bhatia MC. Biological investigation of
5. El-Merzabani MM, El-Aaser AA, Attia MA, et al. Vinca rosea. Indian J Pharma. 1956; 18: 73-76.
Screening system for Egyptian plants with potential 23. Spencer CF, Koniuszy FR. Rogers E.F. et al. Survay of
anti-tumor activity. Planta Med. 1979, 36: 150-155. plants for Antimalerial activity. Lloydia. 1949; 10:
6. Chattopadhyay SP, Das PK. Evaluation of Vinca rosea 145-174.
for the treatment of warts. Indian J Dermatol Venerol 24. Swanston-Flatt SK, Day C, Flatt PR, et al. Glycaemia
Leprol. 1990; 56 (2): 107-108. effects of traditional European plant treatments for
7. Cordell GA, Weiss SG, Farnsworth NR. Structure diabetes studies in normal and streptozotocin
elucidation and chemistry of Catharanthus alkaloids. diabetic mice. Diabetes Res. 1989; 10: 69-73.
XXX. Isolation and structure of vincarodine. J Org 25. Shorti DS, Kelkar M, Deshmukh VK, et al.
Chem. 1974; 39 (4): 431-434. Investigation of the hyperglycemic properties of
8. Johnson IS, Wright HF, Svoboda GH, et al. Antitumor Vinca rosea, Cassia Auriculata and Eugenia
principles derived from Vinca rosea 1. Jambolana. Indian J Med Res.1963; 51(3): 464-467.
Vincaleukblastine and leurosine. Cancer Res. 1960; 26. Benjamin BD, Kelkar SM, Pote MS, et al. Catharanthus
20: 1016. roseus cell culture: Growth, alkaloid synthesis and
9. Murugavel TA, Ruknudin ST, Akbarsha MA. antidiabetic activity. Phytother Res.1994; 8(3): 185-
Antifertility effect of Vinca rosea (Linn.) leaf extract 186.
on male albino mice – a sperm parametric study. 27. Rajas MCN, Cuellar MCA. Comparative
Curr Sci. 1989; 58(19): 1102- 1103. microbiological studies of the alkaloids of
10. Murugavel T, Akbarsha MA. Anti-spermatogenetic Catharanthus roseus and other related compounds.
effect of Vinca rosea Linn. Indian J Exp Biol. 1991; 29 Rev Cubana Farm. 1981; 15 (2):131-138.
(9): 810-812. 28. Ross SA, Megalla SE, Bishay DW, et al. Studies for
11. Joshi MS, Ambaye RY. Effect of alkaloids from Vinca determining antibiotic substances in some Egyption
rosea on Spermatogenesis in male rats. Indian J Exp Plants. Part 1. Screening for antimicrobial activity.
Biol.1968; 6: 256-257. Fitoterapia.1980; 51: 303-308.
12. Sharma OP, Makkar HPS, Dawra RK. Biochemical 29. Misawa M : Production of natural substances by
effects of the plant Lantana camara in guinea pig plant cell cultures described in japanese patents.
liver mitochondria. Toxicon. 1982; 20: 783-786. Plant Tissue Culture its Bio-Technol Appl Int Cong.
13. Anon: Antifertility studies on plants. Annual Report 1976; 17-26.
of the Director General – Indian Council Med Res. 30. El-Sayed A, Cordell GA. Catharanthus alkaloids
1979; 71-72. XXXIV. Catharanthamine, a new antitumor bisindole
14. Chopra IC, Jamwal KS, Chopra CL, et al. Preliminary alkaloid from Catharanthus roseus. J Nat Prod.1981;
pharmacological investigations of total alkaloids of 44: 289-293.
Lochnera rosea (Rattonjot). Indian J Med Res. 1959; 31. Verpoorte R, Van der Heijden R, Van Gulik WM, et al.
47: 40-43. Plant biotechnology for the production of alkaloids:
15. Kubas J: Investigation on known or potential present status and prospects. In: The alkaloids. A
antitumoral plants by means of microbiological test. Brossi, (ed.). Academic press, San Diego. Vol. 40, pp
Part III. Biological activity of some cultivated plant 1-187.1991.
species in Neurospora crassa test. Acta Biol Cracov 32. Van der Heijden R, Verpoorte R, Ten Hoopen HJG.
Ser Bot.1972; 15: 87-100. Cell and tissue cultures of Catharanthus roseus (L.)
16. Rai MK, Upadhyay S. Screening of medicinal plants of G.Don: a literature survey. Pl Cell Tiss Org Cult.1989;
Chhindwara district against Trichophyton 18:231-280.
mentagrophytes: A casual organism of Tineapedis. 33. Ganapathi G, Kagri F. Recent advances in indole
Hindustan Antibiot Bull.1988; 30 (1/2): 33-36. alkaloid production by Catharanthus roseus
17. Chile SK, Saraf M, Barde AK. Efficacy of Vinca rosea (Periwinkle). J Exp Bot.1990; 41: 259-267.
extract against human pathogenic strains of 34. Moreno PRH, Van der Heijden R, Verpoorte R.
Trichophyton rubrum Sab. Indian Drugs Pharm Ind. Cell and tissue cultures of Catharanthus roseus; a
1981; 16(1): 31-33. literature survey II. Updating from 1988-1993. Pl
18. Kulkarni R, Ravindra N. Resistance to Pythium Cell Tiss Org Cult. 1995; 42:1-25.
aphanidermatum in diploid and induced 35. Mujib A, Ilah A, Gandotra N, et al. In vitro application
autotetraploids of Catharanthus roseus. Planta Med. to improve alkaloid yield in Catharanthus roseus. - In:
1988; 54 (4): 356-359. Biotechnology and genetic engineering of Recent
19. El-Merzabani MM, El-Aaser AA, El-Duweini AK, et al. Progress in medicinal plants. J.N., Govil, P.A. Kumar,
A Bioassay of antimitotic alkaloids of Catharanthus and V.K. Singh, (eds.). Sci Tech Publication, Houston -
roseus, Planta Med.1979; 36: 87-90. USA. Vol. 4, pp 415-440., 2002.
20. Chattopadhyay RR, Banerjee RN, Sarkar SK, et al. 36. Knobloch KH, Berlin J. Influence of medium
Antiinflammatory and acute toxicity studies with composition on the formation of secondary
the leaves of Vinca rosea Linn. In experimental compounds in cell suspension cultures of
Catharanthus roseus (L.) G.Don. Z.Naturforsch. 1980;
35: 551-556. indoliques dans des suspension cellulaires de

37. Van Gulik WM, Ten Hoopen HJG, Heijnen JJ. A Catharanthus roseus G.Don. CR Acad Sci Par Ser III.
structured model describing carbon and phosphate 1989; 309: 453-458.
limited growth of Catharanthus roseus plant cell 52. Decendit A, Liu D, Ouelhazi L. et al. Cytokinin
suspensions in batch and chemostat cultures. enhanced accumulation of indole alkaloids in
Biotechnol Bioeng. 1993; 41: 771-780. Catharanthus roseus cell culture: The factors
38. Drapeau D, Blanch MW, Wike CR. Ajmalicine, affecting the cytokinin responses. Pl Cell Rep.1992;
serpentine and Catharanthine accumulation in 11: 400-403.
Catharanthus roseus bioreactor cultures. Pl Med. 53. Liman F, Chahed K, Ouelhazi N, et al.Phytohormone
1987a; 53: 373-376. regulation of isoperoxidase in Catharanthus roseus
39. Rho D, Andre G. Growth and stoichiometry of a suspension cultures. Phytochemistry.1998; 49(5):
Catharanthus roseus cell suspension culture grown 1219-1225.
under nitrogen-limiting conditions.Biotechnol 54. Hirata K, Houriuchi M, Ando T. et al. Vindoline
Bioeng. 1991; 38: 579-587. and catharanthine production in multiple shoot
40. Schlatmann JE, Ten Hoopen HJG, Heijnen JJ. cultures of Catharanthus roseus. J Ferment
Optimization of the medium composition for Bioener. 1990; 70(3): 193-195.
alkaloids production by Catharanthus roseus using 55. Garnier F, Carpin S, Label P, et al. Effect of cytokinin
statistical experimental designs. Med Fac Landbouw on alkaloids accumulation in periwinkle callus
Univ Gent. 1992; 57:1567-1569. cultures transformed with a light inducible ipt gene.
41. Junaid A, Mujib A, Nasim SA, et al. Screening of Pl Sci. 1996; 120(1): 47-55.
vincristine in ex vitro and in vitro developed somatic 56. Carpin S, Garnier F, Andreu F, et al. Changes of both
embryo derived Catharanthus roseus (L) G. Don) polypeptide pattern and sensitivity to cytokinin
plantlets. Scien Horticul. 2009; 119: 325-329. following transformation of periwinkle tissues with
42. Scragg AH, Ashton S, York A, et al.Growth of the isopentenyl transferase gene. Pl Physiol
Catharanthus roseus suspension from maximum Biochem.1997a; 35(8): 603-609.
biomass and alkaloids accumulation. Enzyme 57. Carpin S, Ouelhazi L, Filalim M, et al.The relationship
Microb Technol.1990; 12:292-298. between the accumulation of a 28 KD polyleptide
43. Junaid A, Bhatt MA, Mujib A, et al. Somatic embryo and that of indole alkaloids in Catharanthus roseus
proliferation maturation and germination in cell suspension cultures. J.Plan.Physiol. 199b7;
Catharanthus roseus. Pl Cell Tiss Org Cult. 2006; 84: 150(4): 452-457.
325-332. 58. Ouelhazi L, Filali M, Decendit A, et al. Differential
44. Mujib A, Das S, Dey S, et al. Influence of agitation in protein accumulation in zeatin-and 2,4-D-treated
in-vitro cultivation of Catharanthus roseus (L). G. Don cells of Catharanthus roseus: Correlation with indole
multiple shoot. Phytomorphology, 1995; 45(3-4): alkaloid biosynthesis.Pl Physiol Biochem. 1993;
239-245. 31(3): 421-431.
45. Murashige T, Skoog F. A revised medium for rapid 59. Doller G, Alfermann AW, Reinhard E. Production von
growth and bioassays with tobacco tissue cultures. indole alkaloiden in callus kulturen von
Physiol Planat.1962; 15: 473-497. Catharanthus roseus. Pl Med. 1976; 30:14-20.
46. Smith JL, Quesnel AA, Smart NJ, et. al. The 60. Neumann D, Kraus G, Heike M, et al. Indole alkaloid
development of single-stage growth and indole formation and storage in cell suspension cultures of
alkaloids production medium for Catharanthus C.roseus. Pl Med.1983; 48: 20-23.
roseus (L.) G. Don suspension cultures. Biotechnol 61. Asada M, Shuler ML. Stimulation of ajmalicine
Bioeng.1987b; 9:466-469. production and extraction from Catharanthus roseus:
47. Tuominen U, Toivonen L, Markkanen P, et al., Studies effect of adsorption in situ, elicitors and alginate
on the growth and cardenolide production of immobilization. Appl Microbial Biotechnol. 1989; 30:
Digitalis lanata tissue culture. Biotechnol 475-481.
Bioeng.1988; 33: 558-562. 62. Morris P: Regulation of product synthesis in cell
48. Toivonen L, Ojala M, Kauppinen V. Studies on the cultures of Catharanthus roseus. Effect of
optimization of growth and indole alkaloids temperature. Pl Cell Rep.1986a; 5: 427- 429.
production by hairy roots cultures of 63. Courtosis D, Guern J. Temperature of Catharanthus
Catharanthus roseus. Biotechnol Bioeng. 1989; 37: roseus cells cultivated in liquid medium. Pl Sci
673-680. Lett.1980; 17: 473-482.
49. Batra J, Dutta A, Singh D, et al., Growth and 64. Scragg AH, Creswell R, Ashton S, et al. Growth and
terpenoids indole alkaloid production in secondary product formation of a selected
Catharanthus roseus hairy root clones in relation to Catharanthus roseus cell line. Enzyme Microb
left-and right- termini-linked Ri T-DNA gene Technol.1 988b; 10: 532-536.
integration. Pl Cell Rep. 2005; 23: 148-154. 65. Toivonen L, Laakso S, Rosenqvist H. The effect of
50. Smith JI, Smart NJ, Kurz WGW, et al. The use of temperature on hairy root cultures of Catharanthus
organic and inorganic compounds to increase the roseus growth. Indole alkaloid accumulation and
accumulation of indole alkaloids in Catharanthus membrane lipid composition of cell suspension
roseus (L). G. Don cell suspension cultures. J Exp Bot. cultures. Pl Cell Rep.1992b; 11: 395-399.
1987a; 38: 1501-1506. 66. Seibert M, Kadkade PG. Environmental factors.
51. Kodja HP, Liu D, Merillon JM, et al. Stimulation par A.Light. In: Plant tissue culture as a source of
les cytokinines de I’ accumulation d’ alcaloides Biochemicals. E.J. Staba, (ed.). Boca Raton, Florida
CRC Press, pp 123-141.1980. of monoterpene indole alkaloids formation in

67. Morris P. Regulation of product synthesis in cell suspension cultures of C.roseus. J Pl Physiol. 1986;
cultures of Catharanthus roseus. Effect of 126(1): 11-22.
temperature. Pl Cell Rep. 1986a; 5: 427- 429. 82. Seitz HU, Eilert U, De Luca V, et al. Elicitor mediated
68. Lounasmaa M, Galambos J. Indole alkaloid induction of phenylalanine ammonia lyase and
production in Catharanthus roseus cell suspension tryptophan decarboxylase: accumulation of phenols
cultures .In: Progress in the Chemistry of Organic and indole alkaloids in cell suspension culture of
natural products W. Herz, H. Crisebach, G. W. Kirby, Catharanthus roseus. Pl Cell Tiss Org Cult.1989; 18:
and C.H. Tamm, (eds.). Springer-Verlag, Wien-New 71-78.
York. Vol. 55, pp 89-115.1989. 83. Nef C, Rio B, Chrestin H. Induction of Catharanthine
69. De Luca V, Balsevich J, Tyler RT, et al. Biosynthesis of synthesis and stimulation of major indole alkaloids
indole alkaloids: Developmental regulation of the production Catharanthus roseus cells under non-
biosynthetic pathway from tabersonine to vindoline growth altering treatment with Pythium vexons
in Catharanthus roseus. J Pl Physiol. 1986; 125: 147- extracts. Pl Cell Rep.1991; 10: 26-29.
156. 84. Berglund T, Ohlsson AB, Rydstrom J. Nicotinamide
70. Drapeau D, Blanch MW, Wike CR. Ajmalicine, increases glutathione and anthocyanins in tissue
serpentine and Catharanthine accumulation in culture of Catharanthus roseus. J Pl Physiol.1993a;
Catharanthus roseus bioreactor cultures. Pl Med. 141: 596-600.
1987a; 53: 373-376. 85. Smith JI, Smart NJ, Kurz WGW, et al. The use of
71. Loyola-Vargas VM, Mende-Zeel M, Monforte- organic and inorganic compounds to increase the
Gonzales, M. et al.Serpentine accumulation during accumulation of indole alkaloids in Catharanthus
greening in normal and tumor tissues of roseus (L). G. Don cell suspension cultures. J Exp Bot.
Catharanthus roseus. J Pl Physiol.1992; 140: 213- 1987a; 38: 1501-1506.
217. 86. Tallevi SG, DiCosmo F. Stimulation of indole alkaloid
72. Schlatmann JE, Moreno PRH, Ten Hoopen HJG, et al. content in vanadium- treated Catharanthus roseus
Effect of oxygen and nutrient limitation on suspension cultures. Planta Med.1988; 149-152.
Ajmalicine production and related enzyme activities 87. Backer-Royer C de, Vannereau A, Duret S, et al.
in high density cultures of Catharanthus roseus. Successful cryopreservation of suspension cells by
Biotechnol Bioeng. 1994; 44: 461-468. encapsulation dehydration. Pl Cell Tiss Org Cult.
73. Tom R, Jardin B, Chavarie C, et al. Effect of culture 1990; 43(3): 241-248.
process on alkaloid production by Catharanthus 88. Kargi F, Potts P. Effect of various stress factors on
roseus cells.II.Immobilized cultures. J Biotechnol. indole alkaloid formation by C.roseus (periwinkle)
1991b; 21: 21-42. cells. Enzyme Microb Technol. 1991; 13: 760-763.
74. Mohamed MTZ, Scragg AH. A comparision of the 89. Kargi F, Ganapathi B. Effect of precursor stimulating
growth and alkaloids production by suspension agents on formation of indole alkaloids by C.roseus in
and immobilized Catharanthus roseus cells in 61 a biofilm reactors. Enzyme Microb Technol. 1991;
and 71 bioreactors. Immob cell. 1990; 335-342. 13: 643-647.
75. Leckie F, Scragg AH, Cliffe KC. Effect of bioreactor 90. Berglund T, Kalbin G, Strid A, et al. UV-B and
design and agitator speed on the growth and oxidative stress induced increase in nicotinamide
alkaloids accumulation by cultures of Catharanthus and trigonalline and inhibition of defensive
roseus. Enzyme Microbiol Technol. 1991b; 13: 296- metabolism induction by poly (ADP-ribose)
305. polymerase inhibitors in plant tissue. FEBS - Letters.
76. Leckie F, Scragg AH, Cliffe KC. Effect of impeller 1996; 380(1-2): 188-193.
design and speed on the large-scale cultivation of 91. Hirata K, Horiuchi M, Ando T, et al. Effect of near-
suspension cultures of Catharanthus roseus. Enzyme ultraviolet light on alkaloid production in multiple
Microbiol Technol. 1991c; 13: 801-810. shoot cultures of Catharanthus roseus. Pl Med. 1991;
77. Lee CWT, Shuler ML. Different shake flask closure 57: 499-500.
alter gas phase composition and ajmalicine 92. Hirata K, Horiuchn M, Asada M, et al. Stimulation of
production in Catharanthus roseus cell suspensions. dimeric alkaloid production by near-ultraviolet
Biotech Techni. 1991; 5(3): 173-178. light in multiple shoot cultures of Catharanthus
78. Paynee GF, Paynee NN, Shuler ML. Bioreactor roseus. Ferm Bioeng. 1992; 74: 222-225.
considerations for secondary metabolite production 93. Hirata K, Asada M, Yatani M, et al. Effects of near-
from plant cell tissue culture; indole alkaloid from ultraviolet light on alkaloid production in
Catharanthus roseus. Biotechnol Bioenerg. 1988a; Catharanthus roseus plants. Pl Med. 1993; 59(1): 46-
31(9): 905-912. 50.
79. Paynee GF, Paynee NN, Shuler ML, et al. In situ 94. Menke FLH, Parchmann S, Mueller MJ, et al.
adsorption for enhanced alkaloid production by Involvement of the octadecanoid pathway and
Catharanthus roseus. Biotechnol Lett. 1988b; 10: protein phosphorylation in fungal elicitor-
102-201. induced expression of terpenoid indole alkaloid
80. Hoopen HJG, Ten Gulik WMVAN, Schlatmann JE, et biosynthetic genes in Catharanthus roseus. Pl Physio.
al.Ajmalicine production by cell cultures of 1999; (4): 1289-1286.
Catharanthus roseus: from shake flask to bioreactor. 95. Ramos-Valdivia AC, van der Heijden R, Verpoorte R.
Pl Cell Tiss Org Cul. 1994; 38(2-3): 85-91. Elicitor-mediated induction of anthraquinone
81. Eilert U, Constable F, Kurz WGW. Elicitor stimulation biosynthesis and regulation of isopentenyl
diphosphate isomerase and farnesyl diphosphate Dordrecht. Netherlands.1995.

synthase activities in cell suspension cultures of 112. Mujib A, Samaj J. Somatic embryogenesis. -Springer-
Cinchona robusta. Planta. 1997; 203: 155-161. Verlag - Berlin- Heidelberg, New –York. 2006.
96. Berlin J: Para-fluorophenylalanine resistant cell lines 113. Junaid A, Bhatt MA, Mujib A, et al.Somatic embryo
of tobacco.Z.Pflanzenphysiol.1982; 97: 309-311. proliferation maturation and germination in
97. Moreno PRH, Van der Heijden R, Verpoorte R. Effect Catharanthus roseus. Pl Cell Tiss Org Cult. 2006; 84:
of terpenoids precursor feeding and elicitation on 325-332.
formation of indole alkaloids in cell suspension 114. Kim SW, In DS, Choi PS, et al. Plant regeneration from
cultures of Catharanthus roseus. Pl Cell Rep.1993b; immature zygotic embryo derived embryogenic
12: 702-705. calluses and cell suspension cultures of Catharanthus
98. Zenk MH, EI-Shagi H, Arens H, et al.Formation of the roseus. Pl Cell Tiss Org Cult. 2004; 76: 131–135.
indole alkaloids serpentine and Ajmalicine in cell 115. Bentley R. The shikimate pathway - A metabolic tree
suspension culture of Catharanthus roseus. In: Plant with many branches. In: Critical Reviews in
Tissue Culture and its Biotechnological Application. Biochemistry and Molecular biology. G.D. Fasman,
W. Barz, E. Reinhard, and M.H. Zenk, (eds.).Springer (ed.). Vol. 25, pp 307-384.1990
Verlag, Berlin, pp 27-44.1977. 116. Poulsen C, Verpoorte R. Activities of chorismate
99. Kargi F, Rosenberg MZ. Plant cell bioreactors: utilizing enzymes and of enzymes involved in indole
present status and future trends. Biotechnol Prog. alkaloid biosynthesis in cell suspension cultures.
1987; 3: 1-8. Pl Physiol Biochem. 1992; 30: 105-113.
100. Scragg AH, Allan EJ, Leckie F. Effect of shear on the 117. Singh BK, Siehl DL, Connelly JA. Shikimate pathway:
viability of plant cell suspensions. Enzy Microbiol why does it mean so much for so many? In: Oxford
Technol. 1988a; 10: 361-367. surveys of Plant Molecular biology. Miflin, B.J. (ed.).
101. Joilicoer M, Chavarie C, Carreau PJ, et al. Vol. 7, 143-185. 1991,
Development of a helical ribbon impeller bioreactors 118. Madyastha KM, Coscia CJ. Enzymology of indole
for high density plant cell suspension cultures. alkaloid biosynthesis. Rec Adv Phytochem. 1979; 13:
Biotechnol Bioeng.1992; 39: 511-521. 85-129.
102. Kutney JP, Boulet CA, Choi LSL, et al. Alkaloid 119. Inouye H, Uesato S. Biosynthesis of iridoids and
production in Catharanthus roseus (L). G.Don cell secoiridoids. In: Progress in the chemistry of natural
cultures. XV. Synthesis of bisindole alkaloids by use products. W. Herz, H. Grisebach, G.W. Kirby, and C.H.
of immobilized enzyme system. Heterocycle. 1988a; Tamm, (eds.). Springer-Verlag, Wien-New York. Vol
27: 621-628. 50, pp 169-236. 1986
103. Hulst AC, Tramper J. Immobilized plant cells: a 120. Poulsen C, Bongaerts R, Verpoorte R. Purification
literature survey. Enzyme Microbiol Technol.1989; and characterization of anthranilate synthase from
11: 546-558. Catharanthus roseus. Eur J Biochem. 1993; 212(2):
104. Archambault J, Volesky B, Kurz WGM. Production of 431-440.
indole alkaloids by surface immobilized C. roseus 121. Pennings EJM, Van den Bosch RA, Van der Heijden
cells. Bioenggnering. 1990b; 35: 660-667. R, et al. Assay of strictosidine synthase from plant
105. Majerus F, Pareilleux A. Alkaloid accumulation in Ca- cells by high-performance liquid chromatography.
alginate entrapped cells of Catharanthus roseus using Anal Biochem. 1989a; 176: 412–415.
a limiting growth medium. Pl Cell Rept. 1986; 5(4): 122. Pasquali G, Goddijin OJM, De Wall A. et al.
302-305. Coordinated regulation of two indole alkaloid
106. Facchini PJ, Di Cosmo F, Radvanyi LG, et al. biosynthetic genes from Catharanthus roseus by
Adhesion of Catharanthus roseus cells to surfaces: auxin and elicitors. Pl Mol Biol. 1992; 18: 1121-1131.
effect of substrate hydrophobocity. Biotechno 123. Collu G, Bink HHJ, Moreno PRH, et al. Determination
Bioeng. 1988; 32: 935-938. of the activity of cytochrome p450 enzyme geraniol
107. Facchini PJ, Dicosmo F. Immobilization of cultured 10-hydroxylase in plants by high performance liquid
Catharanthus roseus cells using fiberglass chromatography. Phytochem Analy. 1999; 10 (6):
substratum. Appl Microbiol Biotechnol. 1990; 33:36- 314-318.
42. 124. Collu G, Unver N, Peltenburg-Looman AMG, et al.
108. Vazquez-Flota F, DeCarolis E, Alarco AM, et al. Geraniol 10-hydroxylase, a cytochrome P450
Molecular cloning and characterization of enzyme involved in terpenoid indole alkaloid
desacetoxyvindoline-4-hydroxylase, a 2- biosynthesis. FEBS Lett. 2001; 508: 215–220.
oxoglutarate dependent-dioxygenase involved in the 125. Contin A, Collu G, Van Der Heijden R, et al. The
biosynthesis of vindoline in Catharanthus roseus (L.) effects of phenobarbital and ketoconazole on the
G.Don. Pl Mol Biol. 1997; 34 (6): 935-948. alkaloid biosynthesis in Catharanthus roseus cell
109. Davidou E, Kan C, Quirion JC, et al. Epiall-oyohimbine suspension cultures. Pl Physiol Biochem. 1999; 37:
derivatives isolated from in-vitro hairy root cultures 139-144.
of Catharanthus trichophyllus. Phytochemistry. 1989; 126. Hemscheidt T, Zenk MH. Partial purification and
28: 1383-1387. characterization of a NADPH dependent
110. Nuutila AM. Bioreactor studies on hairy root tetrahydroalstonine synthase from Catharanthus
cultures of Catharanthus roseus. Composition of roseus cell suspension cultures. Pl Cell Rep. 1985; 4:
three bioreactors types. Biotechnol Tech. 1994; 8: 216-219.
61-66. 127. Stevens LH: Formation and conversion of
111. Thorpe TA: In vitro embryogenesis in plants. Kluwer, strictosidine in the biosynthesis of monoterpenoid
indole quinoline alkaloids. Ph.D. Thesis, Leiden Catharanthus roseus and effect of wounding on the
University (121 p).1994. process. Phytochemistry. 1991; 36: 1462-1463.
128. Carolis E-de, Luca V-de, Carolis E-de, et al. A novel 2- 142. Datta A, Srivastava PS. Variation in vinblastine
oxyglutarate dependent dioxygenase involved in production by Catharanthus roseus during in vivo
vindoline biosynthesis; characterization, purification and in vitro differentiation. Phytochemistry. 1997;
and kinetic properties. Pl Cell Tiss Org Cult. 1994; 38 46(1): 135-137.
(2-3): 188-191. 143. Meijer AH, Verpoorte R, Hoge JHC. Regulation of
129. Maldonado-Mendonza IE, Burnett RJ, Nessler CL. enzymes and genes involved in terpenoids indole
Nucleotide sequence of a cDNA encoding 3 hydroxy- alkaloid biosynthesis in Catharanthus roseus. J Pl Res.
3-methylglutaryl coenzyme A reductase from 1993d; 3:145-164.
Catharanthus roseus. Pl Physiol. 1992; 100: 1613- 144. McKnight TD, Bergey DR, Burnett RJ, et al.
1614. Expression of enzymatically active and correctly
130. Hemscheidt T, Zenk MH. Glucosidases involved in targeted strictosidine synthase in transgenic tobacco
indole alkaloid biosynthesis of Catharanthus roseus plants. Plant. 1991; 185: 148-152.
cell cultures. FEBS Lett. 1980; 110:187-191. 145. Blom TJM, Sierra MI, Van Vliet TB, et al. Uptake and
131. Moreno PRH. Van der Heijden R, Verpoorte R. accumulation of Ajmalicine into isolated vacuoles of
Effect of terpenoids precursor feeding and elicitation cultured cells of Catharanthus roseus (L). G. Don.
on formation of indole alkaloids in cell suspension and its conversion into serpentine. Planta. 1991;
cultures of Catharanthus roseus. Pl Cell Rep. 1993b; 183: 170-177.
12: 702-705. 146. Bhandari PR, Mukerji B. Lochnera rosea Linn Reichb.
132. Naudascher F, Doireau P, Thiersault M, et al. Gauhati Ayurvedic Coll Mag. 1959; 8: 1-4.
Influence de la disponibilite en precurseurs 147. Webb LJ: Guide to medicinal and poisonous plants of
accumulation alcaloidique dans les cellules de Queensland. CSIR Bull 232, Melbourne.1984.
Catharanthus roseus cultivees in vitro comparision 148. Brandao M, Botelho M, Krettli E. Antimalerial
entre suspensions en phase de crossence et Experimental Chemotherapy using Natural Products.
suspensions en phase stationale. Les. Colloq. de Cienc Cult. 1985; 37(7): 1152-1163.
FINRA. 1990; 5: 307-309.
149. De Mello JF: Plants in traditional medicine in Brazil. J
133. Facchini PJ, DiCosmo F. Secondary metabolites Ethnopharmacol. 1980; 2(1): 49-55.
biosynthesis in cultured cells of Catharanthus roseus
150. Farnsworth NR: The pharmacognosy of the
(L.) G. Don immobilized by adhesion of glass fibres.
periwinkles: Vinca and Catharanthus. Lloydia.1961;
Appl Microbiol Biotechnol. 1991; 35: 382-392.
24 (3): 105-138.
134. Bongaerts RJM, The chorismate branching point in
151. Virmani OP, Srivastava GN, Singh P. Catharanthus
Catharanthus roseus: Aspects of anthranilate
roseus – The tropical periwinkle. Indian Drugs.1978;
synthase regulation in relation to indole alkaloide
15: 231-252.
biosynthesis.Ph.D.thesis, Leiden University.1998.
152. Holdsworth DK: Traditional medicinal plants of
135. Stevens LH, Blom TJM, and Verpoorte R, Subcellular
Rarotonga, Cooks Islands Part I. Int J Crude Drug Res.
localization of tryptophan decarboxylase,
1990; 28(3): 209-218.
strictosidine synthase and strictosidine glucosidase
in suspension cultured cells of Catharanthus roseus 153. Hodge WH, Taylor D. The ethanobotany of the island
and Tabernaemontana. Pl cell Rep.1993; 12: 573- Caribes of Dominica. WEBBIA.1956; 12: 513-644.
576. 154. Thompson WAR: Herbs that heal. J. Roy Coll. Gen.
136. Balsevich J, De-Luca V, Kurz WGM. Altered alkaloid Pract.1976; 26: 365-370.
pattern in dark grown seedlings of Catharanthus 155. Luu C: Notes on the traditional pharmacopeia of
roseus. The isolation and characterization of 4- French Guyana. Plant Med Phytother. 1975; 9: 125-
desacetoxyvindoline: a novel indole alkaloid and 135.
proposed precursor of vindoline. Heterocycle. 1986; 156. ANON: Ayurvedic drug to fight cancer. Probe. 1985;
24: 2415-2421. 24(2): 234-236.
137. Bede JC, DiCosmo F. Enzymatic synthesis of α-3', 4’- 157. Farnsworth NR, Kaas CJ. An approach utilizing
anhydrovinblastine: optimization and information from traditional medicine to identify
immobilization. Pl Med. 1992; 58: 576-579. tumor-inhibiting plants. J. Ethanopharmacol. 1981;
138. Endo T, Goodbody A, Vikovic J. et.al. Alkaloid 3(1): 85-99.
production in root and shoot cultures of 158. Morrison EYSA, West M. Indian medicinal plants on
Catharanthus roseus. Pl Med. 1987; 53: 479-482. blood sugar levels in the dog. West Indian Med J.
139. De Luca V, Cutler AJ. Subcellular localization of 1982; 31: 194-197.
enzymes involved in indole alkaloid biosynthesis in 159. Johns T, Kokwaro JO, Kimanani EK. Herbal remedies
Catharanthus roseus. Pl Physiol. 1987; 85: 1099- of the Luo of Siaya District, Kenya. Establishing
1102. quantitative criteria for consensus. Econ Bot. 1990;
140. Fernandez JA, Oven TG, Kurz WGW, et al. 44(3): 369-381.
Immunological detector and quantitation of 160. Amico A: Medicinal plants of Southern Zambesia.
tryptophan decarboxylase in developing Fitoterapia. 1977; 48: 101-139.
Catharanthus roseus seedlings. Pl Physiol. 1989; 91: 161. Atta-Ur-Rahman: Some approaches to the study of
79-84. indigenous medicinal plants. Bull Islamic Med. 1982;
141. Naaranlahti T, Auriola S, Lapinjoki S. Growth related 2: 562-568.
dimensation of vindoline and Catharanthine in 162. Ramirez VR, Mostacero LJ, Garcia AE, et al. Vegetales
empleados en medicina tradicional Noreruana. 179. Irmler S, Schroder G, St-Pierre B, et al. Indole
Banco Agrario Del Peru and NACL. Univ Trujillo, alkaloid biosynthesis in Catharanthus roseus: new
Trujillo. Peru, 54pp. 1988. enzyme activities and identification of cytochrome
163. Zaguirre JC: Guide notes of bed-size preparations of P450 CYP72A1 as secologanin synthase. Plant J.
most common local (Philippines) medicinal 2000; 24: 797-804.
plants.1944. 180. Kutchan TM: Strictosidine: from alkaloid to enzyme
164. ANON:Description of the Philippines. Part I., Bureau to gene. Phytochemistry. 1993; 32: 493-506.
of Public Printing, Manila.1903. 181. Geerlings A, Ibanez MML, Memelinks J. et
165. Hsu FL, Cheng JT. Investigation in rats of the al.Molecular cloning and analysis of strictosidine
antihyperglycaemic effect of plant extracts used in beta-D-glucosidase, an enzyme in terpenoid indole
Taiwan for the treatment of diabetes mellitus. alkaloid biosynthesis in Catharanthus roseus. J Bio
Phytother Res. 1992; 6(2): 108-111. Chem. 2000; 275(5): 3051-3056.
166. Yang LL, Yen KY, Kiso Y, et al. Antihepatotoxic 182. St.Pierre B, Laflamme P, Alarco AM. et al. The
Actions of Formosan Plant Drugs. J Ethnopharmacol. terminal O- acetyltransferase involved in vindoline
1987; 19 (1): 103-110. biosynthesis defines a new class of proteins
167. Mueller-Oerlinghausen B, Nagmwathana W, responsible for coenzyme A-dependent acyltransfer.
Kanchanapee P. Investigation into Thai medicinal Plant J. 1998; 14: 703-709.
plants said to cure diabetes. J Med Assoc Thailand. 183. Dethier M, De Luca V. Partial purification of a N-
1971; 54: 105-111. methyltransferase involved in vindoline in
168. Siegel RK: Herbal intoxication. Pscyhoactive effects Catharanthus roseus. Phytochemistry. 1993; 32:
from herbal cigarettes, tea and capsules. J Amer Med 673–678.
Assoc. 1976; 236 (5): 473-476. 184. Vazquez-Flota F, DeCarolis E, Alarco AM, et al.
169. Arnold HJ, Gulumian M. Pharmacopea of traditional Molecular cloning and characterization of
medicine in Venda. J Ethnopharmacol. 1984; 12 (1): desacetoxyvindoline-4-hydroxylase, a 2-
35-74. oxoglutarate dependent - dioxygenase involved in
the biosynthesis of vindoline in Catharanthus roseus
170. Nguywen, Van D. List of simple drugs and medicinal
(L.) G. Don. Pl Mol Biol. 1997; 34 (6): 935-948.
plants of value in Vietnam. Proc Seminar of the use of
Medicinal Plants in Healthcare. Tokyo 13-17, 185. St-Pierre B, De Luca V. A Cytochrome P-450
September, WHO Regional Officer Manila.1977; 65- Monooxygenase Catalyzes the First Step in the
83. Conversion of Tabersonine to Vindoline in
Catharanthus roseus. Pl Physiol. 1995; 109; 131-139.
171. Ayensu ES: Medicinal plants of the West Indies.
Unpublished Manuscript 110pp. 1978. 186. Laflamme P, St-Pierre B, De Luca V. Molecular and
biochemical analysis of a Madagascar periwinkle
172. Van der Fits L, Memelink J. ORCA3, a jasmonate-
root-specific minovincinine-19-hydroxy-O-
responsive transcriptional regulator of plant
acetyltransferase. Pl Physiol. 2001; 125: 189-198.
primary and secondary metabolism. Science.2000;
289: 295-297. 187. Kurtz WGW, Chatson KB, Constable F, et al. Alkaloid
production in Catharanthus roseus cell cultures.
173. Van der Fits L, Zhang H, Menke FL, et al. A
Characterization of the 953 cell line. Helv Chim Acta.
Catharanthus roseus BPF-1 homologue interacts
1980; 63: 1891-1896.
with an elicitor-responsive region of the secondary
metabolite biosynthetic gene Str and is induced by 188. Lee SL, Reporter M, Hsu SN, et al. The lipid
elicitor via a JA-independent signal transduction composition and conversion of tryptamine to Nb-
pathway. Pl Mol Biol. 2000; 44: 123-124. acetyltryptamine in a Catharanthus roseus cell line
without indole alkaloids. J Indust Microbiol . 1988; 3:
174. Van Tegelen LJP, Moreno PRH, Croes AF, et al.
293-297.
Purification and cDNA cloning of isochorismate
synthase from elicited cell cultures of Catharanthus 189. Knobloch KH, Bast G, Berlin J. Medium and light
roseus. Pl Physiol. 1999; 119(2): 705-712. induced formation of serpentine and anthocyanins in
cell suspension cultures Catharanthus roseus.
175. Veau B, Courtois, Oudin A, et al. Cloning and
Phytochemistry. 1982; 21: 591-594.
expression of cDNA encoding two enzymes of the
MEP pathway in Catharanthus roseus. Acta Gene St 190. Lynn DG, Chen RH, Manning KS, et al. The structural
Expres. 2000; 1517 (1): 159-163. characterization of endogenous factors from Vinca
rosea crown gall tumors that promote cell division of
176. Chahed K, Oudin A, Guivarc’h N, et al. 1-Deoxy-D-
tobacco cells. Proc Nad Acad Sci USA. 1987; 84: 615-
xylulose 5-phosphate synthase from periwinkle:
619.
cDNA identification and induced gene expression in
terpenoids indole alkaloid-producing cells. Pl Physiol 191. Verpoorte R, Moreno PRH, Van der Heijden R.
Biochem. 2000; 38 (7-8): 559-566. Secondary metabolismin plants and plant cell
cultures of some terpenoids indole alkaloid
177. Schulte AE, van der Heijden R. Microplate enzyme-
producing plants. Abstr. XV International Botanical
coupled assays of mevalonate and
Congress, Yokohama. Japan. 1993.
phosphomevalonate kinase from Catharanthus
roseus suspension cultured cells. Analy 192. Duperon P, Allais JP, Dupaix AL. Composition of the
Biochem.1999; 269(2): 245-254. sterol content of tonoplast and microsomal fractions
isolated from Catharanthus roseus suspension-
178. Schulte AE, Van der Heijden R, Verpoorte R.
cultured cells. Pl Physiol Biochem. 1992; 30: 495-
Purification and characterization of
498.
phosphomevalonate kinase from Catharanthus
roseus. Phytochemistry.1999; 52(6): 975-983. 193. Park KH, Saimoto H, Nakagawa S, et al. Occurrence
of brassinolide and castasterone in crown gall Nucleotide sequence of a cDNA encoding the
cells of Catharanthus roseus. Agri Biol Chem. 1989; vacuolar protein strictosidine synthase from
53(3): 805-812. Catharanthus roseus. Nucl Acids Res. 1990; 18: 49-
194. Miranda HML, Loyola VVM. Purification and 39.
characterization of glutamine synthetase from leaves 202. Pfizner A, Stockigt J. Partial purification and
of Catharanthus roseus plants. Pl Physiol Biochem. characterization of geissoschizine dehydrogenase
1992; 30(5): 585-592. from suspension cultures of Catharanthus roseus.
195. Poulsen C, Bongaerts R, Verpoorte R. Purification Phytochemistry. 1982; 21: 1585-1588.
and characterization of anthranilate synthase from 203. Ruffer M, Kan-Fan C, Husson HP, et al. 4,1-
Catharanthus roseus. Eur J Biochem. 1993; 212(2): Dehydrogeissoschizine, an intermediate in
431-440. heteroyohimbine alkaloid biosynthesis. J Chem Soc
196. Meije AH, De Waal A, Verpoorte R. Purification of the Chem Commun. 1979; 1016-1018.
cytochrome P-450 enzyme geraniol 10-hydroxylase 204. Endo T, Goodbody A, Vikovic J, et al. Enzymes from
from cell cultures of Catharanthus roseus. J Chro. Catharanthus roseus cell suspension cultures that
1993a; 635(2): 237-249. couple vindoline and catharanthine to form 3′,4′-
197. Meijer AH, Souer E, Verpoorte R, et al. Isolation of anhydrovinblastine. Phytochemistry. 1988; 27:
cytochrome P- 450 cDNA clones from the higher 2147–2149.
plant Catharanthus roseus by a PCR strategy. Plant 205. De Carolis E, Chan F, Balsevich J, et al. Isolation and
Mol Bio. 1993c; 22(2): 379-383. characterization of a 2-oxoglutarate dependent
198. Madyastha KM, Coscia CJ. Enzymology of indole dioxygenase involved in the second-to-last step in
alkaloid biosynthesis. Rec Adv Phytochem. 1979; 13: vindoline biosynthesis. Pl Physiol. 1990; 94:1323–
85-129. 1329.
199. Pennings EJM, Verpoorte R, Goddijn OJM, et al. 206. De Carolis E, De Luca V. Purification,
Purification of tryptophan decarboxylase from a characterization, and kinetic analysis of a 2-
Catharanthus roseus cell suspension culture. J Chro. oxoglutarate -dependent dioxygenase involved in
1989 b; 483: 311-318. vindoline biosynthesis from Catharanthus roseus. J
200. Pfitzner U, Zenk MH. Homogeneous strictosidine Bio Chem. 1993; 268: 5504-5511.
synthase isoenzymes from cell suspension cultures 207. Powers R, Kurz WGW, De Luca V. Purification and
of Catharanthus roseus. Pl Med. 1989; 55(6): 525- characterization of acetyl-CoA: deacetylvindoline 4-
530. O-acetyltransferase from Catharanthus roseus. Arch
201. McKnight TD, Roesner CA, Devagupta R, et al. Biochem Biophys. 1990; 279: 370–376.

Catharanthus Roseus (L.) G. Don. AN IMPORTANT DRUG: IT'S APPLICATIONS AND

Uploaded by

Copyright:

Available Formats

Catharanthus Roseus (L.) G. Don. AN IMPORTANT DRUG: IT'S APPLICATIONS AND

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Catharanthus Roseus (L.) G. Don. AN IMPORTANT DRUG: IT'S APPLICATIONS AND

Uploaded by

Copyright:

Available Formats

Aslam J et al.

/ Pharmacie Globale (IJCP) 2010, 4 (12)

Available online at www.pharmacie-globale.info ISSN 0976-8157

Catharanthus roseus (L.) G. Don. AN IMPORTANT DRUG: IT’S APPLICATIONS AND

1 Pharmacie Globale (IJCP), Vol. 01, Issue 04

4 Pharmacie Globale (IJCP), Vol. 01, Issue 04

6 Pharmacie Globale (IJCP), Vol. 01, Issue 04

metabolic accumulation. reported in Catharanthus.114 The advantage of SE is that

7 Pharmacie Globale (IJCP), Vol. 01, Issue 04

Table 5.Enzymes involved in indole alkaloids biosynthesis in Catharanthus roseus

desacetoxyvindoline-4-hydroxylase OHT Cytoplasm 205, 206

from strictosidine via 4,21-dehydrogeissoschizine, alkaloids metabolism. This process of metabolism

9 Pharmacie Globale (IJCP), Vol. 01, Issue 04

35: 551-556. indoliques dans des suspension cellulaires de

CRC Press, pp 123-141.1980. of monoterpene indole alkaloids formation in

diphosphate isomerase and farnesyl diphosphate Dordrecht. Netherlands.1995.

16 Pharmacie Globale (IJCP), Vol. 01, Issue 04

You might also like