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Withaferin A from cell cultures of Withania somnifera

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Ciddi Veeresham at Kakatiya University
Ciddi Veeresham
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Abstract

Suspension cultures of Withania somnifera cells were established and shown to produce withaferin A. The identification of withaferin A was done by TLC, UV absorption, HPLC and electron spray mass spectroscopy. These cultures could be strongly elicited by exposure to salacin. Addition of salacin at the concentration of 750 µM to the cultures in production medium enhanced production levels of withaferin A to 25±2.9 mg/l compared to 0.47±0.03 mg/l in unelicited controls. This report is the first to demonstrate withaferin A production in plant suspension cultures and provides prerequisites for commercial scale, controlled production of withaferin A.
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Withaferin A from Cell Cultures ofWithaferin A from Cell Cultures of
Withaferin A from Cell Cultures ofWithaferin A from Cell Cultures of
Withaferin A from Cell Cultures of Withania somnifera Withania somnifera
Withania somniferaWithania somnifera
Withania somnifera
VEERESHAM CIDDI*
Faculty of Pharmaceutical Sciences, Kakatiya University, Warangal–506 009, India.
Suspension cultures of Withania somnifera cells were established and shown to produce withaferin A. The
identification of withaferin A was done by TLC, UV absorption, HPLC and electron spray mass spectroscopy.
These cultures could be strongly elicited by exposure to salacin. Addition of salacin at the concentration of 750 µM
to the cultures in production medium enhanced production levels of withaferin A to 25±±
±±
±2.9 mg/l compared to
0.47±±
±±
±0.03 mg/l in unelicited controls. This report is the first to demonstrate withaferin A production in plant
suspension cultures and provides prerequisites for commercial scale, controlled production of withaferin A.
Withania somnifera Dunal, an erect evergreen shrub,
commonly called Ashwagandha (Sanskrit), grows
throughout India, Pakistan and Italy. The plant contains a
group of compounds known as withanolides. There is a
significant variation in the withanolide expressed from
plants grown in different geographical areas. Withanolides
are naturally occurring steroids built on ergostane
skeleton, in which C-22 and C-26 are appropriately
oxidized to form a d-lactone ring. Withaferin A has
antibacterial, anti-tumor, anti-inflammatory and
immunomodulatory properties1.
The commercial cultivation of Withania somnifera has two
major problems. The first problem is plant to plant
variation in quality and quantity of active constituents and
the second is the long gestation period (4-5 y) between
planting and harvesting. To enhance the commercial
prospects for the production of Withaferin A, an
alternative choice could be the use of plant cell cultures2.
Vitali et al.3 described a method for initiation of callus
from Withania somnifera (Israel) and withanolides
production from them. Roja et al.4 described a method for
induction of callus of Withania somnifera but the callus
failed to synthesize withanolides. However, multiple shoot
cultures produced withanolides. The transformed root
cultures of Withania somnifera produced withanolide-D5.
Banerjee et al.2 reported the hairy root cultures of
Withania somnifera able to produce Withaferin A.
However, commercial production would be much more
practical with suspension cultures which have not yet
*For correspondence
E-mail: ciddiveeresham@yahoo.co.in
been demonstrated to produce withanolides.
Laboratory scale studies for optimization of plant cell
suspension culture conditions is usually performed in glass
shake flasks. Six-well polystyrene plates have been used
for mammalian and insect cell culture for several years;
this technology has very limited application in the
culturing of plant cells6-7. Srinivasan et al.8 reported the
combined use of six-well plates and TLC for rapid
screening of Taxus sp cell suspension cultures for
taxane production. Their studies revealed that six-well
plates were superior to shake flasks for rapidly
predicting which conditions may be most effective for
growth and product formation. This communication reports
the establishment of suspension cultures of Withania
somnifera capable of production of Withaferin A and the
use of six well plates to test various elicitors for
enhancement of product formation.
MATERIALS AND METHODS
Callus initiation and maintenance:
The seeds of Withania somnifera were washed
thoroughly with water and allowed to germinate
aseptically on 3% sucrose-agar medium. The seeds
germinated after 3 w. The hypocotyl portions of 3-w old
seedlings were aseptically transferred onto MS medium9
containing 3% sucrose plus 2,4-D (1 mg/l) plus kinetin (0.2
mg/l). The callus induction was observed after 5 d and
callus was sufficiently well formed by 3 w so that it could
be transferred. The callus was maintained by subculturing
onto the half strength B5 medium10 with 3% sucrose plus
dicamba (2 mg/l) plus kinetin (1 mg/l) at 4 w intervals.
Indian Journal of Pharmaceutical Sciences July - August 2006 490
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Initiation and maintenance of suspension cell
cultures:
The suspension cultures were initiated by transferring
10% w/v of callus into half strength B5 medium
supplemented with dicamba (2 mg/l), kinetin (1 mg/l) and
glutamine (292 mg/l) and incubated at 25±2° at 120 rpm.
After one week the cultures were filtered aseptically
through a 100 size sieve to remove large cells. The
uniform suspension was filtered through Buchner funnel
and transferred to fresh medium using an inoculum of 5%
w/v and cultures were maintained by subculturing at 1 w
intervals.
Induction of product synthesis:
The cell cultures were filtered aseptically through a
Buchner funnel with vacuum and washed with medium.
Then the cells were transferred to production medium, ¼
B5 medium supplemented with 2,4-D (2 mg/l), zeatin (0.5
mg/l) and sucrose (5% w/v) in six-well plates or shake
flask in 20% w/w inoculum and incubated for 2 w.
Elicitor treatments:
The solutions of elicitors were prepared by dissolving in
a suitable solvent and added to the cultures before
incubation. The elicitors used were: salacin dissolved in
water; methyl jasmonate and arachidonic acid dissolved in
ethanol.
Extraction of withaferin A:
Cell culture samples were first filtered to obtain a cell
fraction and cell-free filtrate. The cell mass was weighed
to determine fresh weight. Cells were then frozen and
thawed and then extracted three times with three volumes
of dichloromethane. The combined dichloromethane
extract was evaporated and the residue was dissolved in
methanol and saved for analysis.
TLC analysis:
The methanolic extract was analyzed by using pre-coated
silica gel plates of 250 µm layer, UV 254 (Whatman Ltd,
Maidstone, Kent, England) by co-chromatography with
authentic sample of withaferin A. The plates were
developed in a solvent system of chloroform: methanol
(95:5) and detected using a vanillin-sulfuric acid reagent11.
HPLC analysis:
The HPLC analysis of extracts was carried out by
Phyton Inc, USA. The conditions of the analysis includes,
gradient method with water with 0.1% TFA (tri-fluoro
acetic acid) in Pump A, recovered acetonitrile (86%, 14%
water) with a 0.1% TFA in pump B, with a column of
Waters delta pack C-18, 150x4.6 mm, 4 µm with a guard
column. The flow rate of mobile phase was 0.85 ml/min.
RESULTS AND DISCUSSION
The growth index (GI) after 7 d was 1.8 (five
independent wells or n=5) for six well plates and 2.2
(n=3) for flask cultures. The growth index is the final
dry weight divided by the initial dry weight. The value of
GI changed little after day 7 (measured at day 10, day 15
and day 20 in replicate experiments) and was always in
the range of 1.8 to 2.2. Thus, on growth medium the
doubling time was approximately 7 d or less.
The production of withaferin A for cultures transferred to
production medium was confirmed by TLC analysis,
HPLC and ES mass spectra. With TLC analysis the Rf
values of presumptative withaferin A had the same value
as the authentic withaferin A; when derivatized with
vanillin sulfuric acid reagents both spots were blue. The
HPLC analysis gave the same retention time as the
standard (9.9 min) for a presumptive withaferin A peak.
HPLC was used for quantification. The extracts also had
an ES mass spectra signal the same as the standard with a
peak at 473.5. These measurements taken together
provide strong evidence for production of withaferin A
from these suspension cultures.
The kinetic pattern for production of withaferin A is
shown in fig. 1. Measurements from four independent
cultures were made at each time point. Total withaferin A
is reported. The cellular fraction was about 60% of the
total withaferin A with the remainder being in the
medium. The maximum value observed was 2.6±0.35 mg/l
on day 7 in the 6 well plate system. With the flasks the
maximum value 1.07±0.52 mg/l was also observed on day
7. In both cases the values of withaferin A decreased at
day 10 and 15. These data demonstrate that the same
production trend is observed in both methods of culture
confirming the suitability of 6-well plate cultures for use
in evaluating secondary metabolite production in plant
cell suspension cultures.
A strategy that has often proved successful in enhancing
secondary metabolite production from plant cell cultures
has been the use of elicitors7,12. Elicitors typically cause
the cells to activate their defense system through an
incompletely understood signal transduction system. The
use of methyl jasmonate (at 100 µM) and arachidonic acid
(1 mg/l) did not improve withaferin A production over
non-elicited controls. However, salacin was an effective
July - August 2006 Indian Journal of Pharmaceutical Sciences 491
3.5
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Withaferin A (mg/l)
3
2.5
2
1.5
1
0.5
0
0 7 10 15
Time (days)
0
5
10
15
20
25
30
0 250 500 750 1000
Salacin Conc (µM)
Withaferin A (mg /l)
Fig. 1: Comparison of time course of withaferin A production.
Comparison of time course of withaferin A production. in six
well plate (-♦♦
♦♦
♦-) and shake flask cultures (- �-).
elicitor of withaferin A production (fig. 2). With the
introduction of salacin on day 0 the amount of withaferin
A on day 15 was 25±2.9 mg/l with 750 µM salacin in flask
cultures (n=4) versus 0.47±0.03 mg/l for control flasks that
received no elicitor. Using a dose of 500 µM salacin
introduction on day 7 rather than day 0 improved day 15
withaferin A production to 21±2.6 mg/l versus 11±0.6 mg/l
for salacin addition on day 0. While withaferin A
production could be further optimized, these results
demonstrate that 50 fold enhancement using salacin as an
elicitor is possible.
Addition of the sterol inhibitor, chlorocholinechloride (at 1
mM) inhibited the production of withaferin A. This result
suggests that precursors enter through the acetate-
mevalonate pathway rather than through the non-
mevalonate pathway.
In summary, we have shown for the first time production
of withaferin A from suspension culture (using Withania
somnifera) and a strong positive response to elicitation by
salacin. These results provide a basis for controlled
production of withaferin A as a pharmaceutical.
ACKNOWLEDGEMENTS
The author is thankful to Dr. M. D. Kharya, Dr. H.S.
Gaur University, Sagar, M.P., India for providing the
seeds of Withania somnifera, Prof. M. L. Shuler, Paula
Miller and Dr. S. Y. Yoon for their help during the work,
Fig. 2: Response of W. somnifera suspension flask cultures to
elicitation with various concentrations of salicin.
Dr. V. Bringi, Phyton Inc. for analysis of the extracts,
Prof. Atta-ur-Rahman of HEJ Research institute, Karachi,
Pakistan for supplying authentic sample of Withaferin A
and to UNESCO for financial assistance.
REFERENCES
1. Archana, R. and Namasivayam, A., J. Ethnopharmacol., 1999, 64,
91.
2. Banerjee, S., Naqvi, A.A., Mandal, S. and Ahuja, P.S.,
Phytotherapy Res., 1994, 8, 452.
3. Vitali, G., Conte, L. and Nicoletti, M., Planta Med., 1996, 62, 287.
4. Roja, G., Heble, M.R. and Sipahimalani, A.T., Phytotherapy Res.,
1991, 5, 185.
5. Ray, S., Ghosh, B., Sen, S. and Jha S., Planta Med., 1996, 62, 571.
6. Schumacher, H.M., Gundlach, H., Fieldler, F. and Zenk, M.H., Plant
Cell Rep., 1987, 6, 410.
7. Gundlach, H., Mueller, M.J., Kutchan, T.M. and Zenk, M.H., Proc.
Natl. Acad. Sci. USA, 1992, 89, 2389.
8. Srinivasan, V., Roberts, S.C. and Shuler, M.L., Plant Cell Rep.,
1997, 16, 600.
9. Murashige, T. and Skoog, F., Physiol Plant., 1962,15, 473.
10. Gamborg, O.L., Miller, R.A. and Ojima, K., Exp. Cell Res.,
1968,50,151.
11. Wagner, H. and Bladt, S., Plant Drug Analysis – A Thin-Layer
Chromatography Atlas, Springer-Verlag, 1996.
12. Ciddi, V., Srinivasan, V. and Shuler, M.L., Biotechnol. Lett., 1995,
17,1343.
Accepted 25 July 2006
Revised 18 November 2005
Received 15 June 2005
Indian J. Pharm. Sci., 2006, 68 (4): 490-492
Indian Journal of Pharmaceutical Sciences July - August 2006 492
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Chapter
  • Oct 2022
The journey of drug discovery from plant natural products is practiced from the development of first medicine, morphine and still continuing in the modern era. The plant Withania somnifera (Ashwagandha), known as Indian Ginseng is one of the most reputed medicinal plants of Ayurveda, principally due to the presence of steroidal lactone, collectively known as withanolides. The withanolides are C-28 steroidal lactone triterpenoids having ergostane structure backbone. In this structure, six membered lactone ring was formed after the oxidation of C-22 and C-26 carbon atoms. Among various withanolides, withaferin A, withanolide A and withanone are the most abundant. The withanolides has been well studied for its antiserotogenic, addaptogenic, neuroprotective and anticancer activity in addition to widely known for their antioxidant, anti-inflammatory, immunomodulating, anti-stress and rejuvenating properties. The industrial demand of dried plant material with respect to withanolide drugs preparation from this species is increasing gradually which fail to meet the current ashwagandha global market by our conventional cultivation method due to a range of issues such as low seed viability and poor germination, biotic and abiotic environmental factors, soil ecological factors fail to maintain uniform withanolide synthesis, accumulate and moreover, these methods are time consuming and laborious. Under such circumstances, biotechnological advances must be adopted to increase the yield for the continuous supply of bioactive compounds. In vitro cell, tissue and organ culture could offer an alternative to field-grown plants for seasonal independent production of cellular biomass in a large scale which will deliver an adequate number of raw materials of uniform quality for pharmaceutical purposes. Other biotechnological methods are also involved such as the application of various elicitors, feeding of precursor molecules, genetic transformation particularly hairy root culture, genetic engineering specifically metabolic engineering and exploitation of bioreactor technology for quality production in huge quantity.
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Elicitation of benzophenanthridine alkaloid synthesis in Eschscholtzia cell cultures
Article
  • Dec 1987
Quaternary benzophenanthridine alkaloids (sanguinarine, chelerythrine, chelirubine, chelilutine and macarpine) are specifically induced by cell wall components of Penicillium and Saccharomyces in a colorless strain of Eschscholtzia californica cell suspension cultures. Classical elicitors such as the Phytophthora megasperma elicitor are inactive. The alkaloid synthesis is, however, strongly induced by certain polypeptide antibiotics. Out of 190 tested plant species the yeast elicitor provoked benzophenanthridine synthesis in 13 cultures. One of the branch point enzymes, namely the berberine bridge enzyme, catalysing the formation of (S)-scoulerine from (S)-reticuline, is strongly stimulated during the elicitation process. These results clearly demonstrate the induction of the benzophenanthridine biosynthetic pathway by microbial elicitors.
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Transformation of Withania somnifera (L) Dunal by Agrobacterium rhizogenes: Infectivity and phytochemical studies
Article
Three different strains of Agrobacterium rhizogenes, namely A4, LBA 9402 and LBA 9360 were used for infection to induce hairy root formation in W. somnifera. Strain specificity of the bacterium and frequency of transformation events were analysed. Significant differences were observed between the transformation ability of the different strains of Agrobacterium. The best response in terms of transformation ability and growth of the hairy roots was obtained with A. rhizogenes strain A4, followed by LBA 9402, whereas LBA 9360 strain failed to induce a transformation event. The production of withanolides with special reference to withaferin A was studied through HPLC in the A4 induced hairy root lines and their respective media at different growth phases (i.e. 4, 10 and 24 weeks). The presence of withaferin A in the media as well as in the hairy roots of 10-week-old cultures highlights the importance of the present study for the commercial prospects of this plant.
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Combined use of six-well polystyrene plates and thin layer chromatography for rapid development of optimal plant cell culture processes: Application to taxane production by Taxus sp.
Article
  • Jun 1997
TwoTaxus (T. chinensis andT. baccata) cell suspension cultures were used as a model system to demonstrate the similarities of biomass accumulation and secondary metabolite (taxane) production obtained from cultures in six-well polystyrene plates and glass shake flasks (25 ml and 125 ml). Interference from binding of taxanes in cell-free culture broth to the polystyrene plates was minimal with 85% of the paclitaxel (Taxol) and 100% of baccatin and 10-deacetyl-7-xylosyl-taxol remaining in the medium after 24 h beyond which no further binding was observed. A simple thin layer chromatography (TLC) procedure with a chloroform: acentonitrile (4:1) solvent system on silica gel was developed to simultaneously test up to 17 cultures for taxane production. The combination of six-well plate technology for experimentation and TLC for rapid taxane analysis can greatly accelerate the establishment of conditions for an optimalTaxus plant-cell culture process for taxane production.
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Antistressor effect of Withania Somnifera
Article
  • Feb 1999
  • J ETHNOPHARMACOL
Withania somnifera is an Indian medicinal plant used widely in the treatment of many clinical conditions in India. Its antistressor properties have been investigated in this study using adult Wistar strain albino rats and cold water swimming stress test. The results indicate that the drug treated animals show better stress tolerance.
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Withanolide Composition and in vitro Culture of Italian Withania somnifera
Article
  • Jul 1996
From Italian plants of Withania somnifera six withanolides were isolated, whose structures allowed us to assign the Italian race of W. somnifera to the Israel chemotype III. In vitro cultures of Italian W. somnifera under different conditions were obtained, as well as infection by agrobacterium rhizogenes. HPLC analysis of IN vitro derived tissues showed low contents of withanolides.
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Withanolide Production by Root Cultures of Withania somnifera Transformed with Agrobacterium rhizogenes
Article
  • Dec 1996
Transformed root cultures of Withania somnifera Dunal (Solanaceae) were obtained by infecting shoots cultured in vitro with Agrobacterium rhizogenes LBA 9402. They grew axenically in the absence of exogenous plant growth regulators in Murashige and Skoog's medium containing 3% (w/v) sucrose. The root cultures synthesized several withanolides of which withanolide D was isolated and identified. Transformed root (clone HR (1)) showed a growth index of 20.07 and a withanolide D yield of 0.30 mg g(-1) DW. The productivity of withanolide D in transformed roots (0.181 mg 1(-1) d(-1)) was higher than in untransformed root cultures (0.026 mg 1(-1) d(-1)).
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