GC-MS Analysis and In vitro Cytotoxicity Studies of Root Bark Exudates of Hardwickia binata Roxb.

Abstract

Hardwickia binata Roxb. (Fabaceae) root bark exudate has been traditionally used by tribes of Chitteri hills to cure breast cancer. The main objective of this study is to give a scientific validation to the traditional use of H. binata. The root bark exudates of H. binata was collected from Chitteri hills, shade dried, coarsely powdered and extracted with methanol using soxhlet apparatus. The Physico chemical properties, qualitative phytochemical studies, quantitative phytochemical studies and GC-MS analysis was carried out. The cytotoxic effect of methanol extract of H.binata root bark exudate was studied against cancer cell lines by MTT assay. The phytochemical studies indicated the presence of flavonoids, saponins, phenols and tannins. GC-MS study also revealed the presence of 22 organic compounds out of which 18 compounds were reported with various activities and five compounds reported to possess anticancer activity. In vitro cytotoxic activity on the cell lines showed more degree of inhibition against African Green Monkey Kidney Epithelial cells (Vero), Human cervical cancer cell lines (HeLa) and Human breast cancer cells (MCF-7).

Full text

American Journal of Phytomedicine and Clinical Therapeutics www.ajpct.org
Original Article
GC-MS Analysis and In vitro Cytotoxicity
Studies of Root Bark Exudates of
Hardwickia binata Roxb.
R. Prabakaran1, T. Senthil Kumar*2 and M.V. Rao3
1
Department of Botany, Vivekanandha College of Arts & Sciences for Women, Tiruchengode-637
205, Tamil Nadu
2
Department of Industry University Collaboration, Bharathidasan University, Tiruchirappalli-620
024, Tamil Nadu
3
Department of Plant Science, Bharathidasan University, Tiruchirappalli-620 024, Tamil Nadu
ABSTRACT
Hardwickia binata Roxb. (Fabaceae) root bark exudate has been
traditionally used by tribes of Chitteri hills to cure breast cancer. The
main objective of this study is to give a scientific validation to the
traditional use of H. binata. The root bark exudates of H. binata was
collected from Chitteri hills, shade dried, coarsely powdered and
extracted with methanol using soxhlet apparatus. The Physico
chemical properties, qualitative phytochemical studies, quantitative
phytochemical studies and GC-MS analysis was carried out. The
cytotoxic effect of methanol extract of H.binata root bark exudate
was studied against cancer cell lines by MTT assay. The
phytochemical studies indicated the presence of flavonoids, saponins,
phenols and tannins. GC-MS study also revealed the presence of 22
organic compounds out of which 18 compounds were reported with
various activities and five compounds reported to possess anticancer
activity. In vitro cytotoxic activity on the cell lines showed more
degree of inhibition against African Green Monkey Kidney Epithelial
cells (Vero), Human cervical cancer cell lines (HeLa) and Human
breast cancer cells (MCF-7).
Keywords: Anticancer, Cytotoxicity, Hardwickia binata,
Phytochemical, Cell lines.
INTRODUCTION
Cancer is a dreadful disease
characterized by the irregular proliferation
of the living cells1. Though chemotherapy is
now being generally used as a standard
treatment method, search for anticancer
agents from natural products has also been
increased over the years because plants
could exaggerate to diminish the toxicity
Address for
Correspondence
Department of
Industry University
Collaboration,
Bharathidasan
University,
Tiruchirappalli-620
024, Tamil Nadu
E-mail:
senthil2551964
@yahoo.co.in

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caused due to chemotherapy2-3. The plants
containing anticancer properties would
benevolently play a vital role in the
discovery of potential drugs for treating
cancer4. Phytochemicals have always been
sought after because of their inherent
potential to cure disease, as demonstrated by
ancient medicinal practices5-7. The present
study aimed to provide a scientific
validation to the traditional use of root bark
exudates of Hardwickia binata Roxb. by
Malayali tribes of Chitteri hills against
cancer.
Hardwickia binata Roxb. is a native
species of tropical south – Southern East
Asia. The synonyms are Hardwickia
trapeziformis R. Grah. and Harongana
madagascariensis Choisy. It grows in dry
and hot climate, characterised by a long
period of drought, low to moderate rainfall.
It is a deciduous, moderate to large-sized
tree, extremely hard, heavy and durable
timber. The wood is largely used for beams
and mine props, bridge and house
construction, agricultural implements, carts
and wooden wheels and railway sleepers.
Leaves are used as cattle fodder.
MATERIALS AND METHODS
Preparation of Extracts
The root bark exudates of H. binata
Roxb. were collected from the Chitteri,
Southern, Eastern Ghats, Tamil Nadu. Chitteri
hills. The Chitteri hills are situated towards
North East of Salem district within the
geographical limit of 78°51’10” - 78°32’40”
E, longitude and 11°55’14”-12 °4’48” N,
latitude and occupy an area of about 654.22
Km2. The average height of the Chitteri hills
is 3600ft. The plant was identified with the
help of local flora and they were authenticated
by taxonomists. The voucher specimens were
also submitted to Vivekanandha College
Herbarium (Ref. No. Angio-514). The
exudates were dried in shade and coarsely
powdered. The plant powder was sieved
through 40m sieve plate and the fine powder
was used for extraction. The root bark
exudates were extracted with Soxhlet
apparatus by a continuous hot percolation
process with various solvents such as
petroleum ether, chloroform, acetone, alcohol
and water, separately. After completion of
extraction, the extract was filtered and the
solvent was removed by evaporating in a
water bath. The extract was stored in
desiccators for further study.
Phyto chemical studies of Hardwickia binata
Roxb.
Physico-chemical studies, qualitative
and quantitative phytochemical tests were
carried out in the methanol extract of root
bark exudates of H. binata using standard
methods8-12
GC-MS analysis
The Gas chromatography–mass
spectroscopy (GC–MS) analysis affords the
advantage of identifying the chemical entities
present, which constitutes the chemical
picture of a plant (herbal) extract and by
which the complex mixtures can be resolved
into individual components. GC-MS analysis
of the methanol extract of H.binata was
performed using a Perkin–Elmer GC Clarus
500 system comprising an AOC-20i auto-
sampler and a Gas Chromatograph interfaced
to a Mass Spectrometer (GC-MS)13 equipped
with a Elite-5MS (5% diphenyl/95% dimethyl
polysiloxane) fused a capillary column (30 ×
0.25 µm ID × 0.25 µm df). For GC-MS
detection, an electron ionization system was
operated in the electron impact mode with
ionization energy of 70 eV. Helium gas
(99.999%) was used as a carrier gas at a
constant flow rate of 1ml/min, and an
injection volume of 2µl was employed (a split
ratio of 10:1). The injector temperature was
maintained at 250°C, the ion-source
temperature was 200°C, the oven temperature
was programmed from 110°C (isothermal for

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2min.), with an increase of 10°C/min. to
200°C, then 5°C/min. to 280°C, ending with a
9min isothermal at 280°C. Mass spectra were
taken at 70eV; a scan interval of 0.5s and
fragments from 45 to 450 Da. The solvent
delay was 0 to 2 min., and the total GC/MS
running time was 36min. The relative
percentage amount of each component was
calculated by comparing its average peak area
to the total areas. The mass-detector used in
this analysis was the Turbo-Mass Gold-
Perkin-Elmer, and the software adopted to
handle mass spectra and chromatograms was
the Turbo-Mass over-5.2.
Pharmacological studies on Hardwickia
binata
Cell culture has been used to screen
anticancer activity, since there is a clear
correlation between In vitro and In vivo
activities of potential chemotherapeutic
agents. There is a scientific justification for
cytotoxicity testing in tissue culture, since
animal models are in many ways inadequate
for predicting the effects of chemicals on
humans since there are many metabolic
differences between two. Cytotoxicity studies
involve the analysis of morphological damage
or inhibition of the zone of outgrowth induced
by the chemicals tested.
In vitro cytotoxicity studies
The Antiproliferative activity of root
extracts was tested by MTT [(3-(4,5-
dimethylthiazol-2yl)-2,5-diphenyltetrazo-
liumbromide] assay. MTT measures the
metabolic activity of viable cells. The assay
was nonradioactive and could be performed
entirely in a micro titer plate (MTP). It is
suitable for measuring cell proliferation, cell
viability and cytotoxicity. This method was
based on the principle that viable cells convert
MTT into a formazan14 an insoluble salt,
which is solubilised and quantified. Increase
in its concentration indicates the increased
number of viable cells. The absorbance
directly correlated with the viable cell
number.
RESULT AND DISCUSSION
The physico-chemical evaluation of
drugs (Table 1) is an important parameter in
detecting adulteration or improper handling of
drugs. The total ash value was 0.47g/dr.wt.,
acid insoluble ash was 0.27g/dr.wt., sulphated
ash was 1.6g/dr.wt. and water soluble ash was
0.37g/dr.wt. The extractive values are studied
with two solvents such as ethanol and water.
The extractive value of ethanol and water was
0.65g and 0.67g respectively. The loss on
drying crude drug of root barks exudates of
H. binata was 0.94g/g fr. wt. Qualitative and
quantitative analysis results are given in Table
2 & 3.
GC-MS studies
The GC-MS analysis led to
the identification of 22 compounds from the
gas chromatography fractions of the methanol
extract of H. binata. The active principles
with their retention time (RT), molecular
formula, molecular weight (MW) and
concentration (%) in the methanol extract are
presented in Table 4 and Fig. 2.
The components detected in methanol
extract were Methoxydi (1-pyrrolidinyl)
phosphine, an alkaloid, which is found at
retention time of 6.51and peak area was
0.02%; 1-tert-Butyl-3-(3-methoxyphenyl)-
bicyclo [1.1.1], a pentan, that is found at
retention time of 7.72 and peak area was
0.32%; Limonene dioxide is found at
retention time of 10.88 and peak area was
1.10; 2-[3-Cyclohexylaminopropylamino]
ethylthiophosphate is a sulphur compound
found at retention time of 11.86 and peak
area was 0.02%; and Pentanoic acid 2-(2-
hydroxy-2-methyl-4-phenylbut-3- an amino
compound is found at retention time of
12.10 and peak area was 2.03 %.

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Decanoic acid ethyl ester is a fatty
acid ester that is found at retention time of
12.70 and peak area was 17.55; Cis-9-
Hexadecenal is an aldehyde compound
which is found at retention time of 14.27 and
peak area was 58.68%; 4-Octadecenal is an
aldehyde compound that is found at retention
time of 14.34 and peak area was 35.67%; N-
[3-[6-Hydroxyhexyl]aminopropyl] aziridine
is an amino compound which is found at
retention time of 14.53 and peak area was
2.74%; 9,12,15-Octadecatrienoic acid methyl
ester (Z,Z,Z) which is a linolenic acid ester
that is found at retention time of 14.69 and
peak area was 3.91%; 9,12-Octadecadienoyl
chloride(Z,Z) which is a linoleic acid
compound that is found at retention time of
14.95 and peak area was 36.47%; 4-Hexenoic
acid 2-amino-6-hydroxy-4-methyl an amino
compound that is found at retention time of
15.33 and peak area was 0.05%; 9,12,15-
Octadecatrienoic acid ethyl ester, (Z,Z,Z) a
linolenic acid ester that is found at retention
time of 15.90 and peak area was 2.10%;
8,11,14-Eicosatrienoic acid, (Z,Z,Z) is an
unsaturated fatty acid is found at retention
time of 16.99 and peak area was2.61%;
5,8,11,14-Eicosatetraenoic acid ethyl ester
(all-Z) an unsaturated fatty acid ester is found
at retention time of 17.10 and peak area was
15.08%; 2H-Pyran-3-ol 2-ethoxy-3 4-
dihydro- acetate an alcoholic compound is
found at retention time of 17.12 and peak
area was 0.11%.
3-[N-[2-Diethylaminoethyl]-1-
cyclopentenylamino] propionitrile is a
nitrogen compound found at retention time of
18.05 and peak area was 0.03%;
Deoxyspergualin a nitrogen compound that is
found at retention time of 19.44 and peak
area was 0.06%; 1H-3a 7-Methanoazulene
octahydro-1,4,9,9-tetramethyl a nitrogen
compound is found at retention time of 19.66
and peak area was 1.38%; Benzoic acid 4-
nitro-1-methylethyl ester is an aromatic acid
compound found at retention time of 20.03
and peak area was 0.58%; Squalene a
triterpene which is found at retention time of
23.76 and peak area of was 4.78%. The peak
area ranged from 0.02 to 58.68 percentages
for reporting compounds.
The therapeutic activity of various
compounds was reported based on
Dr.Dukes’s phytochemical and
ethnobotanical database. Out of the 22
compounds identified from the extract, 18
compounds were reported to be active. Most
of the compounds have been reported to have
antimicrobial activity. Many of them have
been shown to be Hepato protective, Cardio
protective while some have anti-inflammatory
property also. Among the various bioactive
compounds present, the following five have
been identified and reported to have
anticancer properties. They are 9,12,15-
Octadecatrienoic acid methyl ester (Z,Z,Z)-),
9,12,15-Octadecatrienoic acid, Ethyl ester
(Z,Z,Z), 8,11,14-Eicosatrienoic acid (Z,Z,Z),
5,8,11,14-Eicosatetraenoic acid ethyl ester
(all-Z) and Squalene (Table 5).
In vitro anticancer studies
The cytotoxic potency of various
extracts was confirmed by In vitro
cytotoxicity assay methods against animal
cancer cell lines and human cancer cell lines.
Crude extracts of root bark exudates of H.
binata in petroleum ether, ethyl acetate,
chloroform, methanol and water was obtained
separately and they were tested for cytotoxic
activity in African green monkey Kidney
Epithelial Cells (Vero), Human Cervical
Cancer Cell Line (HeLa) and Human Breast
Cancer cells (MCF-7) by MTT assay. The
cell lines are treated with various extracts of
concentrations ranging from 32.25g/ml to
1000g/ml. The results in cell growth
inhibition by the various extracts and
concentrations are shown in Table 6 and Fig.
1.
In Human Breast Cancer cells (MCF-
7) IC50 value was 193.77g/ml of petroleum

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ether, 213.77g/ml of chloroform,
210.37g/ml of ethyl acetate, 234.74g/ml of
methanol and 282.28g/ml of water. In
Human Cervical Cancer Cell Line (HeLa)
IC50 value was 214g/ml of petroleum ether,
218.49 g/ml of chloroform, 241.41g/ml of
ethyl acetate, 282.1g/ml of methanol and
287.95 g/ml of water. In African green
monkey Kidney Epithelial Cells (Vero), IC50
value was 295.22 g/ml of petroleum ether,
299.69 g/ml of chloroform, 317.09 g/ml of
ethyl acetate, 429.7g/ml of methanol and
312.96 g/ml of water.
In MCF-7 and HeLa the cytotoxicity
activity was found to increase with the
polarity of the solvent, i.e. petroleum ether >
ethyl acetate > chloroform > methanol >
water. The extracts of different solvents have
shown cytotoxicity to a certain degree of
selectivity against different cell types. The
extracts shown a higher degree of inhibition
against the Human Breast Cancer cells (MCF-
7), Human Cervical Cancer Cell Line (HeLa)
in solvents like petroleum ether, ethyl acetate
and methanol. The activity may be because of
the metabolites present in the crude extracts,
which are active against the cell lines. The
aqueous extract exhibited only a weak activity
against the cell lines tested.
The phytochemical studies indicated
the presence of flavonoids, saponins, phenols
and tannins. Many such compounds are
known to possess potent antitumor properties
15. The extract of H. binata was found to be
rich in flavonoids and saponins. Flavonoids
have been found to possess anti-mutagenic
and anti-malignant effect 16-17. Moreover, they
have a chemo preventive role in cancer
through their effects on signal transduction in
cell proliferation and inhibition of
neovascularisation18. Saponins have been
found beneficial in the inhibition of tumour
angiogenesis by suppressing its inducer in the
epithelial cells of blood vessels and then on
adhering, invasion and metastasis of tumour
cells. They also exhibited antitumor effect by
cell cycle arrest19. The physiological function
of five compounds identified in GC-MS were
also reported with anticancer properties and
they were also reported in different plant
species by various authors20-22.
CONCLUSION
The findings of this study supported
the traditional knowledge of Malayali
traditional healers and also support folkloric
usage of this plant in treating breast cancer.
The present study is the first report on the
anticancer property of H. binata. This study
also opens avenues for pharmaceutical
researchers to develop a potential anticancer
drug.
ACKNOWLEDGEMENTS
We would like to thank Mr. S.
Kumaravel, Scientist, Indian Institute of Crop
Processing Technology, Thanjavur, for his
help in GC-MS studies. We also thank Amala
Cancer Research Centre, Thrissur, Kerala for
their help in In vitro cytotoxicity studies. We
express our gratitude to the traditional healers
of Malayali tribes for their information
regarding anti cancer ailment.
REFERENCES
1. Jemal A, Siegel R, Xu J, Ward E. Cancer
statistics, CA Cancer J Clin 2010; 60:277-
300.
2. Mehta RG, Murillo G, Naithani R, Peng X.
Cancer chemoprevention by natural
products: how far have we come Pharm Res
2010; 27: 950-61.
3. Desai AG, Qazi GN, Ganju RK, l. Medicinal
plants and cancer chemoprevention. Curr
Drug Metab 2008; 9: 581-591
4. Krishnaswamy K. Traditional Indian spices
and their health significance. Asia Pac J Clin
Nutr 2008; 17: 265-8.
5. Farnsworth NR .Screening plants for new
medicines. In Chapter 9 in Biodiversity.

Kumar et al________________________________________________ ISSN 2321 – 2748
AJPCT[2][6][2014]723-733
Edited by Wilson EO. Washington D.C:
National Academy Press; 1988.
6. Mann J. Natural products in cancer
chemotherapy: past, present and future. Nat
Rev Cancer 2002; 2:143–148.
7. Grabley S, Thiericke R. Bioactive agents
from natural sources: trends in discovery and
application. Adv Biochem Engin Biotechnol
1999; 64:104–154
8. Harbourne JB. Phytochemical methods -A
Guide to modern techniques of plant analysis
2
nd
edition. Chapman and Hall, London;
1984: 4-120.
9. Hebert E, Brain Ellery, Kenneth W. Text
book of practical pharmacognosy, Baiilere,
London; 1984:363.
10. Wagner H, Bladt S, Zgainski EM. Plant
drug analysis, Springer-Verlag, Berlin; 1984:
298-334.
11. Basset JJ, Denny JH, Jeffery J, Mendham.
Textbook of Quantitative in organic analysis,
4
th
edition. ELBS. Longman, Essex, OK;
1985:196.
12. Kokate CK, Parolit AP, Gokhale SB.
Pharmacognosy 1
st
Ed Niralli Prakasan,
Pune; 1980; 123.
13. McMaster, Christopher, McMaster, Marvin
C. GC/MS: a practical user's guide. New
York: Wiley 1998.
14. Altman FP. Tetrazolium salts and formazans.
Prog. Histochem. Cytochem. 1976; 9 (3): 1–
56.
15. Kintzios SE. Terrestrial plant derived
anticancer agents and plant species used in
anticancer research. Critical Reviews in Plant
Sciences 2006; 25: 79-113.
16. Brown JP. A review of the genetic effect of
occurring flavonoids, anthraquinones and
related compounds. Mutation Research
1980; 75: 243-77.
17. HiranoT, Oka K, Akiba M. Anti proliferative
effects of synthetic and naturally occurring
flavonoids on tumour cells of human breast
carcinoma cell line, ZR-75-1 Res Commun
Chem Pathol Pharmacol 1989; 64: 69-78.
18. Weber G, Shen F, Prajda N. Increased signal
transduction activity and down regulation in
human cancer cells. Anticancer Research
1996; 16: 3271-82.
19. Man S, Gao W, Zhang Y, Huang L, Liu.
Chemical study and medical applications of
saponins as anticancer agents. Fitoterapia
2010; 81: 703-14.
20. Zeeshan M, Rizvi SMD, Khan MS, Kumar
A. Isolation, partial purification and
evaluation of bioactive compounds from
leaves of Ageratum houstonianum. Excli
Journal 2012; 11:78-88.
21. Sheela D, Uthayakumari F. GC-MS analysis
of bioactive constituents from coastal sand
dune taxon – Sesuvium portulacastrum L.
Bioscience discovery 2013;4(1): 47-53
22. Vembarasi G,Velavan S, Mahadevan K.
Identification of bioactive components and
its biological activities of Asparagus
racemosus rhizomes through the gas
chromatography and mass spectrometry.
International Journal of Natural products
research 2013; 2(1): 17-19.

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Table 1. Preliminary phytochemical Screening of the various extracts of root bark exudates of
Hardwickia binata Roxb.
Table 2. Physico-chemical parameters of root bark exudates Hardwickia binata Roxb.
Table 3. Quantitative analysis of root bark exudates Hardwickia binata Roxb.
S. No Name of the Phytochemical content Values
1 Alkaloids 0.64 g /g dr/wt.
2 Glycosides 0.82 g /g dr/wt.
3 Saponins 0.74 g /g dr/wt.
4 Phenols 1.39 g /g dr/wt.
5 Flavonoids 1.08 g /g dr/wt.
S. No
Name of the
extract
Carbohydrates
Glycosides
Fixed oils & fats
Protein&
amino acids
Saponins
Tanins
Phytosterols
Alkaloids
Phenolic
compounds
Flavonoids
Gums&
Mucilages
1 Petroleum
Ether - - + - - - + - -
+
-
2 Chloroform - - - - - - + + - + -
3 Ethyl acetate + + - + - + + + - + -
4 Methanol + + + + + + + + + + -
5 Water + + - + + + - - + + -
S. No Treatment Values
1
Total ash
0.47 g
/g dr/wt.
2 Water soluble ash 0.37 g /g dr/wt.
3 Sulphated ash 1.60 g /g dr/wt.
4 Acid insoluble ash 0.27 g /g dr/wt.
5 Ethanol soluble extractive 0.65 g /g dr/wt.
6 Water soluble extractive 0.67 g /g dr/wt.
7 Loss of drying 0.94 g /g dr/wt.

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Table 4. Components identified in the root bark exudates of Hardwickia binata Roxb.
No. RT Name of the compound Molecular formula MW Peak
Area
1 6.51 Methoxydi(1-pyrrolidinyl)phosphine C9H19N2OP 202 0.02
2 7.72
1
-
tert
-
Butyl
-
3
-
(3
-
methoxyphenyl)
-
bicyclo[1.1.1]pentan C16H22O 230 0.32
3 10.88 Limonene dioxide C10H16O2 168 1.10
4 11.86
2
-
[3
-
Cyclohexylaminopropylamino]
Ethylthiophosphate C11H25N2O3PS 296 0.02
5 12.10
Pentanoic acid, 2
-
(2
-
hydroxy
-
2
-
methyl
-
4-phenylbut-3-ynyl)amino-4-methyl- C17H23NO3 289 2.03
6 12.70 Decanoic acid, ethyl ester C12H24O2 200 17.55
7 14.27 cis-9-Hexadecenal C16H30O 238 58.68
8 14.34 4-Octadecenal C18H34O 266 35.67
9 14.53 N-[3-[6-Hydroxyhexyl]aminopropyl]aziridine C11H24N2O 200 2.74
10 14.69
9,12,15
-
Octadecatrienoic acid,
methyl ester, (Z,Z,Z)- C19H32O2 292 3.91
11 14.95 9,12-Octadecadienoyl chloride, (Z,Z)- C18H31ClO 298 36.47
12 15.29 Octadecanoic acid, ethyl ester C20H40O2 312 14.69
13 15.33
4
-
Hexenoic acid, 2
-
amino
-
6
-
hydroxy
-
4-methyl- C7H13NO3 159 0.05
14 15.90 9,12,15-Octadecatrienoic acid, ethyl ester, (Z,Z,Z)- C20H34O2 306 2.10
15 16.99 8,11,14-Eicosatrienoic acid, (Z,Z,Z)- C20H34O2 306 2.61
16 17.10 5,8,11,14-Eicosatetraenoic acid, ethyl ester, (all-Z)- C22H36O2 332 15.08
17 17.12
2H
-
Pyran
-
3
-
ol, 2
-
ethoxy
-
3,4-dihydro-, acetate C9H14O4 186 0.11
18 18.05
3
-
[N
-
[2
-
Diethylaminoethyl]
-
1-cyclopentenylamino]propionitrile C14H25N3 235 0.03
19 19.44 Deoxyspergualin C17H37N7O3 387 0.06
20 19.66
1H
-
3a,7
-
Methanoazulene, octahydro
-
1,4,9,9-tetramethyl- C15H26 206 1.38
21 20.03 Benzoic acid, 4-nitro-, 1-methylethyl ester C10H11NO4 209 0.58
22 23.76 Squalene C30H50 410 4.78

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Table 5. Activity of Components identified in the root bark exudates of Hardwickia binata
Roxb.
S. No. Name of the compound Activity
1 Methoxydi(1-pyrrolidinyl) phosphine Antimicrobial, Anti-inflammatory
2
1
-
tert
-
Butyl
-
3
-
(3
-
methoxyphenyl)
-
bicyclo
[1.1.1] pentan Activity not reported
3 Limonene dioxide Fragrance compound
4
2
-
[3
-
Cyclohexylaminopropylamino]
Ethylthiophosphate Antimicrobial
5
Pentanoic acid, 2
-
(2
-
hydroxy
-
2
-
methyl
-
4
-
phenyl
but-3-ynyl)amino-4-methyl- Antimicrobial
6 Decanoic acid, ethyl ester Activity not reported
7 cis-9-Hexadecenal Antimicrobial, Anti-inflammatory
8 4-Octadecenal Antimicrobial, Anti-inflammatory
9 N-[3-[6-Hydroxyhexyl]aminopropyl] aziridine Antimicrobial
10 9,12,15-Octadecatrienoic acid,
methyl ester, (Z,Z,Z)-
Hypocholesterolemic, Nematicide Antiarthritic,
Hepatoprotective, Anti androgenic, Nematicide
5-Alpha reductase inhibitor, Antihistaminic
Anticoronary, Insectifuge, Antieczemic
Anticancer
11 9,12-Octadecadienoyl chloride, (Z,Z)-
Hypocholesterolemic,Nematicide
Antiarthritic,Hepatoprotective,Antiandrogenic,
Nematicide,5-Alphareductaseinhibitor,
Antihistaminic, Anticoronary, Insectifuge,
Antieczemic, Anticancer
12 Octadecanoic acid, ethyl ester Activity not reported
13 4-Hexenoic acid, 2-amino-6-hydroxy-4-methyl- Antimicrobial
14 9,12,15-Octadecatrienoic acid, ethyl ester,
(Z,Z,Z)
Hypocholesterolemic, Nematic
ide Antiarthritic,
Hepatoprotective, Anti androgenic ,Nematicide
5-Alpha reductase inhibitor, Antihistaminic
Anticoronary, Insectifuge, Antieczemic
Anticancer
15 8,11,14-Eicosatrienoic acid, (Z,Z,Z)-
Cardio protective, Hypocholesterolemic
Anticoronary ,Anticancer
16
5,8,11,14
-
Eicosatetraenoic acid, ethyl ester, (all
-
Z)
Cardio protective ,Hypocholesterolemic
Anticoronary, Anticancer
17 2H-Pyran-3-ol, 2-ethoxy-3,4-dihydro-, acetate Antimicrobial
18
3
-
[N
-
[2
-
Diethylaminoethyl]
-
1-cyclopentenylamino]propionitrile Antimicrobial
19 Deoxyspergualin Antimicrobial
20
1H
-
3a,7
-
Methanoazulene, octahydro
-
1,4,9,9-tetramethyl- Activity not reported

Kumar et al________________________________________________ ISSN 2321 – 2748
AJPCT[2][6][2014]723-733
21 Benzoic acid, 4-nitro-, 1-methylethyl ester Antimicrobial Preservative
22 Squalene
Antibacterial, Antioxidant,
A
ntitumor
,
Cancer preventive, Immunostimulant,
Chemo preventive, Lipoxygenase-inhibitor,
Pesticide, Diuretic
Table 6. IC50 of various extracts against normal and cancer cell lines
S. No Name of the
Extract
IC
50
(µg/ml)
Vero (African
green monkey
Kidney Epithelial
Cells)
HeLa (Human Cervical
Cancer Cell Line)
MCF-7 (Human
Breast Cancer Cell
Line)
1 Petroleum ether 295.22 214.00 193.77
2 Chloroform 299.69 218.49 213.27
3 Ethyl acetate 317.09 241.41 210.37
4 Methanol 429.7 282.1 234.74
5 Water 312.96 287.95 282.28

Kumar et al________________________________________________ ISSN 2321 – 2748
AJPCT[2][6][2014]723-733
Figure 1. IC
50
of various extracts against normal and cancer cell lines
2.06 4.06 6.06 8.06 10.06 12.06 14.06 16.06 18.06 20.06 22.06 24.06 26.06
Time
0
100
%
GCMS Analysis 928 Scan EI+
TIC
1.15e8
14.95
12.70
10.88 11.40
14.69
13.92
17.10
15.29
16.99
23.76
19.66
18.02 22.64
Figure 2. GC-MS Chromatogram of root bark exudates of Hardwickia binata Roxb.