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essential oil from whole fruits (0.8%) were tested
for antimicrobial potential. Table 1 shows the zone
of inhibition in mm of methanol extracts of rind
and fruits and essential oil of A. subulatum against
microorganisms used.
Methanol extract of rind of A. subulatum showed
remarkable antimicrobial activity against E. coli.
When compared with ciprofloxacin, it was found
equivalent in potency against S. aureus, whereas slight
inferior against remaining bacterial stains. Methanol
extract of fruits of A. subulatum was found to be
effective against S. aureus. It was found equivalent to
standard drug against B. pumilus and P. aerugenosa.
Essential oil isolated from whole fruits showed good
results against B. pumilus. It was found equivalent
to the standard used against S. epidermidis, P.
aerugenosa and S. cerevisiae. It was found that
methanol extracts of fruit and rind as well as essential
oil all possesses antifungal activity but less than
standard drug used.
It can be seen from the results obtained for MIC that
rind extract of A. subulatum is having lower values as
compared to fruit extract of A. subulatum in majority
of microorganisms. Methanol extract of rind was
showing exceptionally lower MIC in case of S. aureus
(10 μg/disc).
The results obtained in the present study revealed that
methanolic extracts of fruit and rind of A. subulatum
possessed broad spectrum antimicrobial activity. The
essential oil isolated from the whole fruits shows
good antimicrobial activity against microorganisms
used in the study.
ACKNOWLEDGEMENTS
Authors are grateful to Prof. (Dr.) S. S. Agrawal,
Director, Delhi Institute of Pharmaceutical Sciences
and Research, New Delhi for providing necessary
facilities to carry out the present study. Also, thanks
are due to Dr. K. C. Bhatt, National Bureau of Plant
and Genomic Research (NBPGR), New Delhi for
identifying and authenticating the herb.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
Anonymous, Wealth of India, Raw Materials, New Delhi: Publications
and Information Directorate; 1985. p. 227-9.
Kaskoos RA, Mir SR, Kapoor R, Ali M. Essential oil composition
of fruits of Amomum subulatum Roxb. J Essential Oil Bearing Plants
2008;11:184-7.
Gurudutt KN, Naik JP, Srinivas P, Ravindranath B. Volatile constituents
of large cardamom (Amomum subulatum Roxb). Flav Fragr J
1996;11:7-9.
Adegoke GO, Rao LJM, Shankaracharya NB, Jagmohan RL. A
comparison of the essential oils of Afromum daniellii and Amomum
subulatum. Flav Fragr J 1998;13:345-52.
Rout PK, Sahoo D, Jena KS, Rao YR. Analysis of the oil of large
cardamom (Amomum subulatum Roxb.) growing in Sikkim. J Essent
Oil Res 2003;15:265-6.
Gilani SR, Shahid I, Javed M, Mehmud S, Ahmed R. Antimicrobial
activities and physico-chemical properties of the essential oil from
Amomum subulatum. Int J Appl Chem 2006;2:81-6.
Sabulal B, Dan M, Pradeep NS, Valsamn RK, George V. Composition
and antimicrobial activity of essential oil from fruits of Amomum
subulatum. Acta Pharm 2006;56:473-80.
Kumar GP, Chaturvedi A. Antimicrobial activity of some medicinal
plants of Euphorbiaceae. Indian Drugs 2006;43:156-9.
Singh M, Govindarajan R, Nath V, Rawat A, Mehrotra S.
Antimicrobial, wound healing and antioxidant activity of Plagiochasma
appendiculatum Lehm. Et Lind. J Ethnopharmacol 2006;107:67-72.
Accepted 29 September 2010
Revised 16 June 2010
Received 7 January 2010
Indian J. Pharm. Sci., 2010, 72 (5): 657-659
In Vitro Anticancer Activity of the Root, Stem and Leaves
of Withania Somnifera against Various Human Cancer
Cell Lines
B. YADAV*, A. BAJAJ1, M. SAXENA AND A. K. SAXENA2
Advanced Materials and Processes Research Institute (CSIR), Bhopal-462 026, 1Botany Department, Motilal Vigyan
Mahavidhalaya, Bhopal-462 016, 2Indian Institute of Integrative Medicine (CSIR), Jammu Tawi-180 001, India
*Address for correspondence
E-mail: [email protected]
September - October 2010
Indian Journal of Pharmaceutical Sciences
659
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Yadav, et al.: In Vitro Anticancer Activity of Withania Somnifera
Withania somnifera Dunal know as Ashwagandha belong Solanaceae family. It is extensively used in most of the
Indian herbal pharmaceuticals and nutraceuticals. The current study, evaluate in vitro cytotoxicity in 50% ethanol
extract of root, stem and leaves of Withania somnifera against five human cancer cell lines of four different tissues
i.e. PC-3, DU-145 (prostrate), HCT-15 (colon), A-549 (lung) and IMR-32 (neuroblastoma). Root, stem and leaves
extracts showed cytotoxicity activity ranging 0-98% depending on the cell lines but maximum activity was found
in 50% ethanol extract of leaves of Withania somnifera. Ethanol extract of leaves obtained from treatments T2,
T3, T4 and T5 showed strong activity against PC-3 and HCT-15 with 80-98% growth inhibition, while the 50%
ethanol extract of leaves from T1 treatment showed a minimum of 39% and T3 treatment showed a maximum of
98% growth inhibition against HCT-15. This investigation is the first report of the anticancer activity in various
parts of Withania somnifera cultivated in fly ash amended soil.
Key words: Anticancer, cytotoxicity, fly ash, PC-3, HCT-15, prostrate, Withania somnifera
Cancer is one of the major human diseases and
causes large suffering and economic loss world-wide.
Chemotherapy is one of the methods of treating
cancer. However the chemotherapeutic drugs are
highly toxic and have devastating side effects. Various
new strategies are being developed to control and
treat several human cancers[1]. Over 60% of anticancer
drugs available in the market are of natural origin.
Natural products are also the lead molecules for
many of the drugs that are in use[2]. Therefore, the
phytochemicals present in several herbal products and
plants may have the potential to act as preventive or
therapeutic agents against various human cancer[1].
The increased popularity of herbal remedies for
cancer therapy perhaps can be attributed to the
belief that herbal drugs provide benefit over that of
allopathy medicines while being less toxic[3]. Since the
conventional therapies have devastating side effects,
there is a continuous need for search of new herbal
cures of cancer[4].
Withania somnifera Dunal (Ashwagandha) is
extensively used in most of the Indian herbal
pharmaceuticals and nutraceuticals[5]. It is an annual
herb growing in dry and arid soil as a wild plant[6]
and well described in Ayurveda, the ancient Indian
system of plant medicine for immunomodulation and
antiageing[7-8]. Thus, Withania somnifera also have
antiinflammatory[9] antitumour and radiosensitizing
actions [10-11] and analgesic activity [12]. However no
work was reported on in vitro studies of Withania
somnifera cultivated in different concentration of
fly ash amended soil. The present investigation was
taken up for evaluating the antiproliferative potential
possessed by the 50% ethanol extract of root, stem
and leaves of Withania somnifera against various
human cancer cell lines.
660
Ethanol GR Merck grade, RPMI-1640, fetal calf
serum, trypsin, PBS, tryphan blue, penicillin,
streptomycin, gentamycin, DMSO, sulphorhodamine,
paclitaxel (taxol), 5-fluorouracil, were obtained from
Sigma Chemical Co. USA and rest of the chemicals
were of high purity and obtained locally. Tissue
culture flasks and 96-Well cell culture plates were
obtained from NUNC, Germany.
The Withania somnifera Dunal plant was cultivated
in different concentration of fly ash amended soil
at Advanced Materials and Processes Research
Institute (CSIR), Bhopal, Madhya Pradesh, India. The
experiments were designed to have five treatments,
T1- control, T2- 5% fly ash, T3- 10% fly ash, T415% fly ash and T5- 20% fly ash. After completion of
cropping root, stem and leaves of Withania somnifera
were collected separately for each treatment and then
air dried.
The dried roots stem and leaves samples of Withania
somnifera Dunal were powdered separately and about
50 g of each powder was extracted with 50% ethyl
alcohol in 1:10 w/v ratio for 24 h. by separating
funnel. After many cycles, the desired compound
was extracted in flask. This extract was concentrated
using a Heidolph Rota Evaporator (Laboratra 4001,
Germany) and concentrate was oven dried at 500. The
root, stem and leaves crude extract were utilized as
test materials for in vitro anticancer activity testing.
Human cancer cell lines were procured from National
Cancer Institute, Frederick, USA. Cells were grown
in tissue culture flasks in complete growth medium
(RPMI-1640 medium with 2 mM glutamine, pH 7.4,
supplemented with 10% fetal calf serum, 100 μg/ml
Indian Journal of Pharmaceutical Sciences
September - October 2010
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streptomycin and 100 units/ml penicillin) in a carbon
dioxide incubator (37°, 5% CO2, 90% RH). The cells
at sub confluent stage were harvested from the flask
by treatment with trypsin (0.05% in PBS (pH 7.4)
containing 0.02% EDTA). Cells with viability of more
than 98% as determined by trypan blue exclusion
were used for determination of cytotoxicity. The
cell suspension of 1×105 cells/ml was prepared in
complete growth medium. Stock solutions (2×10-2 M)
of compounds were prepared in DMSO. The stock
solutions were serially diluted with complete growth
medium containing 50 μg/ml of gentamycin to obtain
working test solutions of required concentrations.
In vitro cytotoxicity against four human cancer cell
lines was determined[13] using 96-well tissue culture
plates. One hundred microlitres of cell suspension
was added to each well of the 96-well tissue culture
plate. The cells were allowed to grow in a carbon
dioxide incubator (37°, 5% CO2, 90% RH) for 24
h. Test materials in complete growth medium (100
μl) were added after 24 h of incubation to the wells
containing cell suspension. The plates were further
incubated for 48 h. The cell growth was stopped
by gently layering trichloroacetic acid (50%, 50 μl)
on top of the medium in all the wells. The plates
were incubated at 4 o for one hour to fix the cells
attached to the bottom of the wells. The liquid of all
the wells was gently pipetted out and discarded. The
plates were washed five times with distilled water
to remove trichloroacetic acid, growth medium low
molecular weight metabolites and serum proteins
and then air-dried. The plates were stained with
sulphorhodamine B dye (0.4 % in 1% acetic acid,
100 μl) for 30 min. The plates were washed five
times with 1% acetic acid and then air-dried[14]. The
adsorbed dye was dissolved in Tris-HCl Buffer (100
l, 0.01M, pH 10.4) and plates were gently stirred for
10 min on a mechanical stirrer. The optical density
(OD) was recorded on ELISA reader at 540 nm. The
cell growth was determined by subtracting mean OD
value of respective blank from the mean OD value
of experimental set. Percent growth in presence of
test material was calculated considering the growth
in absence of any test material as 100% and in turn
percent growth inhibition in presence of test material
was statistically calculated.
Samples were evaluated against five cell lines
representing four different tissues. These were PC-3
and DU-145 from prostrate, HCT-15 from colon,
A-549 from lung and IMR-32 of neuroblastoma
origin. All root samples showed cytotoxicity ranging
between 0-46% depending on the cell lines but
maximum cytotoxicity activity was found in T3 and
T5, which showed more than 46% and 32% growth
inhibition in A-549 and DU-145 (Table 1). Stem
samples showed cytotoxicity ranging between 4-64%.
Maximum cytotoxicity was found in T5 treatment
i.e. more than 71% growth inhibition against HCT-15
(colon). Stem extract of Withania somnifera grown in
fly ash-treated plot showed all most similar growth
inhibition against all cell lines, PC-3 and DU-145,
HCT-15, A-549 and IMR 032 (Table 2). The extract
of leaves showed cytotoxicity ranging between 2798%, which depended on the cell lines but maximum
cytotoxicity activity was found in T3 and T2 i.e.
more than 98% and 94% growth inhibition in HCT-15
(colon). Similarly the T4 and T5 treatments showed
greater growth inhibition against PC-3 and HCT-15,
while minimum cytotoxicity activity was found from
T1 treatment. Almost remarkable growth inhibition
was obtained from T3 treatment against all cell lines
studied (Table 3). The anticancer activity has been
previously reported in the Withania somnifera Dunal
against various human cell lines, but according to the
TABLE 1: IN VITRO CYTOTOXICITY AGAINST VARIOUS HUMAN CANCER CELL LINES IN 50% ETHANOL EXTRACT
OF ROOT
Samples
Root - T1
Root - T2
Root - T3
Root - T4
Root - T5
5- Fluorouracil
Adraimycin
Paclitaxel
Concentration
(μg/ml)
100
100
100
100
100
2 x 10-5M
1 x 10-5M
1 x 10-5M
September - October 2010
Prostrate
PC-3
16
12
11
20
0
52
DU-145
3
9
12
14
32
60
Cell line type
Colon
HCT-15
16
4
13
9
10
55
-
Indian Journal of Pharmaceutical Sciences
Lung
A-549
18
22
46
0
0
-
Neuroblastoma
IMR-32
13
10
19
10
13
661
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TABLE 2: IN VITRO CYTOTOXICITY AGAINST VARIOUS HUMAN CANCER CELL LINES IN 50% ETHANOL EXTRACT
OF STEM
Samples
Stem - T1
Stem - T2
Stem - T3
Stem - T4
Stem - T5
5- Fluorouracil
Adraimycin
Paclitaxel
Conc. μg/ml
100
100
100
100
100
2 x 10-5M
1 x 10-5M
1 x 10-5M
Cell line type
Colon
HCT-15
62
54
33
71
54
55
-
Prostrate
PC-3
DU-145
17
63
22
29
26
29
28
48
29
33
52
60
Lung
A-549
55
14
4
58
64
-
Neuroblastoma
IMR-32
36
38
35
52
40
-
TABLE 3: IN VITRO CYTOTOXICITY AGAINST VARIOUS HUMAN CANCER CELL LINES IN 50% ETHANOL EXTRACT
OF LEAVES
Samples
Leaves - T1
Leaves - T2
Leaves - T3
Leaves - T4
Leaves - T5
5- Fluorouracil
Adraimycin
Paclitaxel
Conc. μg/ml
100
100
100
100
100
2 x 10-5M
1 x 10-5M
1 x 10-5M
literature anticancer activity in root, stem and leaves
of Withania somnifera cultivated in fly ash amended
soil has been reported probably for the first time.
In this study, growth inhibitory activity of root, stem
and leaves of Withania somnifera using anticancer
drugs paclitaxel, adriamycin and 5-fluorouracil (5-Fu)
as positive controls was investigated against five
cell lines representing four different tissues, PC-3,
DU-145, HCT-15, A-549 and IMR-32. Although its
root is well known for its importance in Ayurveda but
reports on the medicinal activities of stem and leaves
are very few. Thus this study revealed the enormous
medicinal potential of stem and leaves of this plant.
This study gives support that Withania somnifera 50%
ethanol extract were highly cytotoxic to the human
cell lines studied.
Earlier studies indicated that only the root extract
of Withania somnifera as a potential source of new
molecules that can curtail cancer growth[15]. Withania
somnifera leaves have also been shown to inhibit
the growth of human cancer cell lines comparable
to that produced by adriamycin. The leaf extract
produced antiproliferative activity on NCI-H460
(lung), HCT-116 (colon), SF-268 (central nervous
662
Cell line type
Colon
HCT-15
39
94
98
86
83
55
-
Prostrate
PC-3
DU-145
32
28
88
70
80
70
85
70
86
27
52
60
Lung
A-549
49
53
47
42
54
-
Neuroblastoma
IMR-32
50
77
87
77
74
-
system) and MCF-7 (breast) human tumor cell lines.
The inhibitory concentrations obtained was 25.1±0.91
against colon cell line HCT-116[11], but in this study
leaf extracts from T2, T3, T4 and T5 treatments of
Withania somnifera cultivated in fly ash containing
soil had shown more than 80% inhibition against
PC-3 and HCT-15 cell lines. Further more this study
has reported growth inhibitory importance in Withania
somnifera against various human cancer cell lines i.e.
PC-3, DU-145, HCT-15, A-549 and IMR-32. Hence,
this study has revealed remarkable anticancer potential
in the leaves of Withania somnifera.
ACKNOWLEDGEMENTS
The authors thank Dr. Navin Chandra, Acting
Director, Advanced Materials and Processes Research
Institute (CSIR), Bhopal, India for the support and
permission to publish this paper. They also wish to
thank Ms. Monika Verma, IIIM, Jammu for samples
analysis.
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Modha J, Modha N. Role of Ayurveda in the Management of Cancer.
International Centre for Ayurveda Studies, Gujarat Ayurveda University,
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scavenging properties of twelve traditionally used Indian medicinal
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Sangwan RS, Chaurasiya ND, Misra LN, Lal P, Uniyal GC, Sharma
R, et al. Phytochemical variability in commercial herbal products
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Singh S, Kumar S. Withania somnifera: The Indian Ginseng
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Drug Manufacturer’s Association, Mumbai and Regional Research
Laboratory, Jammu-Tawi; 1998. p. 65-73.
Ray AB, Gupta M. Withasteroids, a growing group of naturally
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Al-Hindawi MK, Al-Khafaji SH, Abdul-Nabi MH. Anti-granuloma
activity of Iraqi Withania somnifera. J Ethnopharmacol 1992;37:113-6.
10. Devi PU. Withania somnifera Dunal (Ashwagandha): Potential
plant source of a promising drug for cancer chemotherapy and
radiosensitization. Indian J Exp Biol 1996;34:927-32.
11. Jayaprakasam B, Zhang Y, Seeram NP, Nair MG. Growth inhibition
of human tumor cell lines by withanolides from Withania somnifera
leaves. Life Sci 2003;74:125-32.
12. Twaij HA, Elisha EE, Khalid RM. Analgesic studies on some Iraqi
medicinal plants. Int. J Crude Res 1989;27:109-12.
13. Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica
D, et al. Feasibility of a high-flux anticancer drug screen using a
diverse panel of cultured human tumor cell lines. J Natl Cancer Inst
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14. Skehan P, Storeng R, Scudiero D, Monks A, Mc Mohan J, Vistica D,
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Accepted 30 September 2010
Revised 21 June 2010
Received 20 November 2009
Indian J. Pharm. Sci., 2010, 72 (5): 659-663
Design, Synthesis and Evaluation of Novel 1-(Substituted
Acetyl)-4-(10-Bromo-8-Chloro-5,6-Dihydro-11HBenzo[5,6]Cyclohepta[1,2-B]Pyridine-11-Ylidene)
Piperidines as Antitumor Agents and Farnesyl Protein
Transferase Inhibitors
P. S. GATNE*, C. L. VISWANATHAN*, PREMLATA K. AMBRE AND AARTI JUVEKAR1
Department of Pharmaceutical Chemistry, Bombay College of Pharmacy, Kalina, Santacruz, Mumbai-400 098,
1
Anticancer Drug Screening Facility (ACDSF), Tata Memorial Centre, Advanced Center for Treatment Research and
Education in Cancer (ACTREC), Kharghar, Navi Mumbai-410 210, India
Gatne, et al.: Dihydro-11H-Benzo[5,6]Cyclohepta[1,2-B]Pyridine-11-Ylidene)Piperidines as Antitumor Agents
Eight novel 1-(substituted acetyl)-4-(10-bromo-8-chloro-5,6-dihydro-11H-benzo[5,6] cyclohepta [1,2-b] pyridine11-ylidene)piperidines were designed by incorporating zinc binding groups to enhance activity. The designed
molecules were synthesized and were evaluated for antitumor activity in vitro in five cell lines and for farnesyl
protein transferase inhibition. Test compounds (6a-h) exhibited antitumor activity in most of the cell lines but were
less potent than adriamycin. Compound 6e was most active with IC50 values of <15 µM in two cell lines tested.
Test compounds also exhibited potent FPT inhibitory activity and 6c was most potent with IC50 value of <30 µM.
Key words: Anticancer agents, benzocycloheptapyridines, farnesyl protein transferase inhibitors
*Address for correspondence
E-mail: [email protected]
September - October 2010
Indian Journal of Pharmaceutical Sciences
663