European Journal of Medicinal Plants

28(3): 1-21, 2019; Article no.EJMP.49995

ISSN: 2231-0894, NLM ID: 101583475

A Study on Phytochemical and Anticancer Activities

of Epiphytic Orchid Aerides odorata Lour.
Jhansi Katta1, Venkatesh Rampilla2 and S. M. Khasim1*
1
Department of Botany and Microbiology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur,
India.
2
Dr. L.H.R Government Degree College, Mylavaram, Krishna District, Andhra Pradesh, India.

Authors’ contributions

This work was carried out in collaboration among all authors. Author SMK designed the study.
Authors JK and VR performed the experimental analysis, wrote the protocol, wrote the first draft of the
manuscript, managed the analyses of the study and managed the literature searches. All authors read
and approved the final manuscript.

Article Information

DOI: 10.9734/EJMP/2019/v28i330135
Editor(s):
(1) Dr. Patrizia Diana, Professor, Department of Molecular and Biomolecular Sciences and Technologies, University of
Palermo, Palermo, Italy.
(2) Dr. Marcello Iriti, Professor, Plant Biology and Pathology, Department of Agricultural and Environmental Sciences, Milan
State University, Italy.
Reviewers:
(1) A. Ayeshamariam, Mohideen College, India.
(2) Mustafa Sevindik, Akdeniz University, Turkey.
(3) S. Murugesan, University of Madras, India.
Complete Peer review History: http://www.sdiarticle3.com/review-history/49995

Received 25 April 2019

Original Research Article Accepted 04 July 2019
Published 23 July 2019

ABSTRACT

Aim: The present study was carried out to evaluate the phytochemical composition and anticancer
activities of leaf extract of Aerides odorata Lour., a widely distributed epiphytic herb found in the
Eastern Ghats of Vizianagram district.
Methodology: The solvents like n-hexane, ethyl acetate and methanol were used to extract dried
leaf material of A. odorata. These extracts were analysed for phytochemical constituents by GC-MS
analysis and in vitro anticancer activity was done against two cancer cell lines (MCF-7 and HeLa
cell line) by using MTT assay.
Results: Preliminary phytochemical analysis revealed the presence of alkaloids, coumarins,
flavonoids, glycosides, phenols, and terpenoids. GC-MS analysis determines presence of 15
compounds in ethyl acetate and 14 compounds in methanol extracts respectively. Among
two extracts a total 13 compounds have anticancer activity. Both the solvent extracts exhibit
_____________________________________________________________________________________________________

*Corresponding author: E-mail: [email protected];

 Katta et al.; EJMP, 28(3): 1-21, 2019; Article no.EJMP.49995

significant cancer cell growth inhibition with IC50 value ranging between 26.211 µg/mL to 59.061
µg/mL.
Conclusion: Methanol about the best solvent and its activity. Our result showed A. odorata is a
promising source of anticancer drugs.

Keywords: GC-MS analysis; anticancer; Aerides odorata.

1. INTRODUCTION rotary evaporator (Buchi Labortech Ag, model l,

R-215) under reduced pressure. The
Orchids are one of the beautiful flowering plants concentrates of various solvent extracts were
and they are highly confined to ornamentation. In kept in the refrigerator at 4°C until use.
addition to ornamental, orchids have medicinal
value in folklore and traditional systems [1,2]. 2.1 Preliminary Phytochemical Screening
Current ethnobotanical studies on orchids
indicate that orchids have immense potential in The dried extract of various solvents hexane,
the treatment of various diseases [3,4] and chloroform, ethyl acetate and methanol were
Chinese first described medicinal uses of orchids preliminary screened by using standard
[5]. India is a harbour of orchids with 1331 procedures/tests [14,15,16,17].
species and 186 genera [6]. Among them 33 2.2 GC-MS Analysis
genera belonging to 66 species were distributed
mainly in the hilly areas of Andhra Pradesh. The GC-MS analysis of methanol and ethyl
About 10 species of orchids have been used acetate solvent extracts was injected to Agilent
ethnobotanically by tribals in different regions of 7890 A, GC system coupled with MS 5975. The
Andhra Pradesh to treat various diseases [7,8]. operating conditions of GC-MS set for analysis
A. odorata is widely distributed epiphytic herb were as follows: oven temperature was
found in the Eastern Ghats of Vizianagaram programmed from 50-150°C at 3C/min s. An
district. Ethno botanically A. odorata used to treat aliquot of 2 µL of the sample was injected and
various diseases such as chest pain and the carrier of inert helium gas at a constant flow
stomach disorder, skin disorders, tuberculosis, rate of 1mL/1 min. The electron ionization of
cuts and wounds, boils in ears and nose, sample components was carried out with
pneumonia, inflammations etc. in various regions ionization energy 70ev. The total running time
[2,9,10,11,12,13]. Many pharmacological was 55.3 minutes. National Institute of Standard
activities of these ethnomedicinal plants are due and Technology (NIST) Data Base Library 2.0
to natural phytochemical composition. version searched to compare structure of the
Phytochemical analysis of A. odorata may leads compounds. Compounds were identified based
to explore of new bioactive compounds. Hence, on the retention times and mass spectra of NIST
the present study was carried out to determine library. The name, molecular weight and
the phytochemical analysis and anticancer structure of the components of the test materials
efficiency of A. odorata leaf extracts. were ascertained.
2. METHODOLOGY 2.3 Anticancer Activity by MTT Assay
In present study fresh leaves of A. odorata were The two solvent extracts (Ethyl acetate and
collected from Vizianagaram District, Andhra Methanol) were tested for in vitro cytotoxicity
Pradesh. Plant was authenticated with voucher using MCF-7 and HeLa cell lines by MTT (3, 4 5-
number of ANUBH01211 and preserved at the Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium
herbarium of department of Botany, Acharya Bromide) assay. 100 mL of diluted leaf extract
Nagarjuna University, Guntur. The fresh healthy was added to 100 mL of media followed by the
5
leaves of A. odorata were air-dried under shade addition of cell lines (6X10 ) into 96 well micro-
º
at room temperature for fifteen days. The dried titer and incubated overnight at 37 C for 48 hrs.
material pulverized into a coarse powder by MTT was added after the incubation, precipitates
means of electrical grinder. The dried leaf were formed as a result of the reduction of the
powder of (250g) was extracted with Soxhlet MTT salt to chromophore formazan crystals by
apparatus with n-hexane, ethyl acetate and the cells with metabolically active mitochondria.
methanol solvents for about 12-15hr at room The optical density was measured at 570 nm on
temperature of 35-400C. Finally, crude extracts of a microplate reader. Dose response curve was
different solvents were concentrated in a vacuum used to calculate IC50 dose values [18].

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3. RESULTS shown in Table 3; Fig. 3. The compounds in the

methanol extract are 2-Naphthalenemethanol,
3.1 Phytochemical Analysis 1,2,3,4,4a,5,6,7-octahydro-α,α,4a,8-tetramethyl -
(Fig. 4A), (2R-cis)-, 2-Propen-1-ol, 3-(2,6,6-
Preliminary phytochemical screening of the trimethyl-1-cyclohexen-1-yl) (Fig. 4B), -, m-
different solvent extracts like hexane, ethyl Toluylaldehyde(Fig. 4C), Methyl (2E) - 3-phenyl -
acetate and methanol extract of leaves in A. 2-propeonate (Fig. 4D), 1,2,3-Propanetriol,
odorata revealed the presence of various diacetate (Fig. 4E), 5-Ethyl-2-methyl-2,3-
secondary metabolites such as alkaloids, dihydrofuran (Fig. 4F), cis-11-Eicosenoic acid
coumarins, flavonoids, glycosides, phenols, (Fig. 4G), Ethyl α-D-glucopyranoside (Fig. 4H),
steroids and terpenoids (Table 1). Gas 6-Isopropyl-3-methyl-1-cyclohex-2-enone (Fig.
chromatography and mass spectroscopy is an 4I), 3,7,11-Trimethyl-1,6,10-dodecatrien-3-ol
important technological tool used to identify (Fig. 4J), Erucic acid (Fig. 4K), (9Z,12Z)-
phytocompounds in plant species [19,20]. GC- Octadeca-9,12-dienoyl chloride (Fig. 4L),
MS analysis carried out based on the results of (2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-ol
preliminary phytochemical analysis. Methanolic (Fig. 4M) and 9,12,15-Octadecatrienoic acid,
and ethyl acetate extracts of A. odorata used for methyl ester (Fig. 4n).
the identification of bioactive compounds. GC-
MS analysis of ethyl acetate leaf fraction of A. 3.2 Anticancer Activity
odorata revealed the presence of 12 bioactive
compounds and 6 unknown compounds as Anticancer activity The MTT assay for
shown in Table 2; Fig. 1. From the results of GC- cytotoxicity of ethyl acetate and methanol
MS spectra compounds found in the ethyl extracts of A. odorata was carried out at five
acetate extract are 2-Methyl-5-(1,2,2-Trimethy different concentrations of 5, 10, 25, 50, 75 and
cyclopentyl)phenol (Fig. 2A), 1,3-Propanediol 100 μg/mL on two different cell lines MCF-7 and
(Fig. 2B), 1,2,3-Propanetriol, 1-acetate (Fig. 2C), HeLa (Plates 1 and 2; Plates 3 and 4). The
Butanamide (Fig. 2D), Phenyl(piperidin-3-yl) results of the cytotoxicity of A. odorata two
methanone (Fig. 2E), 4-Methyl-2-pentadecyl-1,3- solvent extracts on both the cell lines are shown
dioxane (Fig. 2F), 3,7,11,15-Tetramethyl-2- in Tables 4, 5. The data suggest that the
hexadecen-1-ol (Fig. 2G), β-Selinene (Fig. 2H), methanolic leaf extract of A. odorata showed
Longipinocarvone (Fig. 2I), (E)-5-Methylundec-4- more cytotoxicity as compared to the ethyl
ene (Fig. 2J), Methyl heptadecanoate (Fig. 2K), acetate extract on MCF-7 cell lines. The ethyl
Hexadecan-1-ol (Fig. 2L), Methyl 14- acetate extract of the A. odorata at the
methylpentadecanoate (Fig. 2M) 2-O-(2- concentration 100 μg/mL showed the highest
Ethylhexyl) 1-O-pentadecyl oxalate (Fig. 2N), growth inhibition 61.128% on MCF-7 cell lines as
Squalene (Fig. 2O), and three Unidentified compared to the methanol extract having
compounds. 60.69%. The recorded IC50 (50% of growth
inhibition) value for methanol extract was
The methanol crude extracts isolated from the 26.211µg/mL and 41.094µg/mL in ethyl acetate
leaves of A. odorata analyzed by using GC-MS extracts. It indicates that the methanol extract
had led to the identification of 14 different organic exhibit significant cytotoxicity effect on MCF-7
compounds and 4 unidentified compounds cell lines.

Table 1. Preliminary phytochemical screening of leaf extracts of A. odorata

Sl. no Phytochemicals Test name Hexane Ethyl acetate Methanol

1 Alkaloids Dragendorff’s test - + +
2 Coumarins Sodium hydroxide test - + +
3 Flavonoids Ferric chloride test - - +
4 Glycosides Anthrone test - - +
5 Phenolic compounds Phenol test - + -
6 Quinones H2SO4 test - + +
7 Resins Acetone H2O test - - -
8 Saponins Foam test - - -
9 Tannins Braemer’s test - - -
10 Steroids Salkowski test - + -
11 Terpenoids Salkowski test - + -
(+) = positive (present); (-) = negative (absent)

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Table 2. Bioactive compounds present in ethyl acetate extract of A. odorata by using GC-MS analysis

Sl. no R.T (min) Name of the compound Molecular Molecular Peak area % Biological activity
formula Mass (gm/mol)
1 4.0167 2-Methyl-5-(1,2,2-Trimethy C15H22O 218.34 0.56 Anticancer [21]
cyclopentyl)phenol
2 4.5167 1,3-Propanediol C3H8O2 76.095 7 -
3 5.8 1,2,3-Propanetriol, 1-acetate C5H10O4 134.131 1.74 Antibacterial [22]
4 6.1167 Butanamide C4H9NO 87.122 6.58 -
5 9.2667 Phenyl(piperidin-3-yl)methanone C12H15NO 189.258 4.76 Anticancer [23]
6 16.65 4-Methyl-2-pentadecyl-1,3-dioxane C20H40O2 312.538 0.64 Antibacterial and Antifungal [24]
7 19.99 3,7,11,15-Tetramethyl-2-hexadecen- C20H40O 296.539 2.72 Anticancer [25], antihelmintic and anti-
1-ol (Phytol) inflammatory [26]
8 20.0333 β-Selinene C15H24 204.357 6.93 Antioxidant and anti-inflammatory [27]
9 22.9833 Longipinocarvone C15H22O 218.34 2.03 -
10 31.2167 (E)-5-Methylundec-4-ene C12H24 168.324 1.69 Anticancer and Antitumor [26]
11 41.4167 Methyl heptadecanoate C18H36O2 284.484 2.8 Catechol-O-Methyl-Transferase
Inhibitor [26]
12 41.5003 Hexadecan-1-ol 1- C16H34O 242.447 14.72 Skin diseases [28]
13 47.9833 Methyl 14-methylpentadecanoate C17H34O2 270.457 4.63 Methyl guanidine inhibitor [26]
14 50.0607 2-O-(2-Ethylhexyl) 1-O-pentadecyl C25H48O4 412.655 1.55 Anticancer, Antitumour and Inhibit
oxalate production of tumour necrosis factor
[26]
15 58.2667 Squalene C30H50 410.73 2.15 Antibacterial, Antioxidant, pesticide,
Antitumour, anti-cancer, preventive,
Immunostimulent, Chemo preventive,
Lipoxygenase-inhibitor [29,30]
16 6.58 Unidentified compound 1 - 297.58 10.9500 -
17 4.76 Unidentified compound 2 - 344.08 14.4167 -
18 14.79 Unidentified compound 3 - 140.46 27.0667 -

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Table 3. Bioactive compounds present in methanolic extract of A. odarata by using GC-MS analysis

Sl. no R.T (min) Name of the compound Molecular Molecular Peak area % Biological activity
formula Mass
(gm/mol)
1 1.15 2-Naphthalenemethanol, C15H26O 222.372 6.9167 Antimicrobial [31]
1,2,3,4,4a,5,6,7-octahydro-α,α,4a,8-
tetramethyl-, (2R-cis)-
2 2.41 2-Propen-1-ol, 3-(2,6,6-trimethyl-1- C12H20O 180.291 8.15 -
cyclohexen-1-yl)-
3 2.3 m-Toluylaldehyde C17H34O2 270.45 12.6667 Anticancer and antidote [26]
4 1.21 Methyl (2E) - 3-phenyl - 2-propeonate C10H9DO2 162.188 15.4833 Anticancer, antitumour and Cytochrome-
P450-2E1-Inhibitor [26]
5 4.44 1,2,3-Propanetriol, diacetate C7H12O5 176.168 22.6667 Cellular narcotic and fragrance agent [32,
33]
6 17.11 5-Ethyl-2-methyl-2,3-dihydrofuran C7H12O 112.172 29.8 Methyl guanidine inhibitor [26]
7 4.17 cis-11-Eicosenoic acid C20H38O2 310.522 31.4833 Acidifier [26], Antimicrobial [34]
8 4.77 Ethyl α-D-glucopyranoside C8H16O6 208.21 34.8833 Hepatic and skin moisturizing effect [35];
Anticancer and alcohol dehydrogenase
inhibitor [26]
9 4.1 6-Isopropyl-3-methyl-1-cyclohex-2- C10H16O 152.237 35.3137 Antibacterial [36]
enone (piperitone)
10 6.45 3,7,11-Trimethyl-1,6,10-dodecatrien-3-ol C15H26O 222.372 38.75 Antimicrobial, antioxidant, anti-
(Nerolidol) nociceptive, anti-inflammatory and anti-
cancer [37]
11 6.53 Erucic acid C22H42O2 338.576 40.4167 Antibacterial [38]
12 2.48 (9Z,12Z)-Octadeca-9,12-dienoyl C18H31OCl 298.895 43.15 Antimicrobial [26]
chloride (Linoleoyl chloride)
13 12.32 (2E,6E)-3,7,11-trimethyldodeca-2,6,10- C15H26O 222.372 43.4833 Antifungal [39]; Anticancer and antitumour
trien-1-ol (farnesol ) [26]
14 4.47 9,12,15-Octadecatrienoic acid, methyl C19H32O2 292.463 55.9667 Anticancer, Antimicrobial, Antioxidant and
ester, (Z,Z,Z) Hyperchloesteralemic [40,41]
15 3.7500 Unidentified compound 1 - 158.74 6.43 -
16 10.5667 Unidentified compound 2 - 134.18 12.87 -
17 18.4167 Unidentified compound 3 - 276.38 4.47 -
18 25.6533 Unidentified compound 4 - 209.11 2.32 -

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Table 4. Cytotoxic properties of ethyl acetate extract of A. odorata on MCF -7 and HeLa cell
lines

Cell Concentration Absorbance at 570nm Average Average- % IC50

line (μg/mL) Blank Viability (μg
/mL)
MCF-7 100 0.792 0.794 0.796 0.794 0.787 38.241 41.094
75 0.889 0.891 0.893 0.891 0.884 42.954
50 0.993 0.995 0.997 0.995 0.988 48.007
25 1.105 1.107 1.109 1.107 1.1 53.45
10 1.161 1.163 1.165 1.163 1.156 56.171
5 1.185 1.187 1.188 1.186 1.179 57.288
Untreated 2.065 2.066 2.065 2.065 2.058 100
Blank 0.007 0.008 0.007 0.007 0
HeLa 100 0.803 0.805 0.807 0.805 0.8 41.928 59.061
75 0.891 0.893 0.895 0.893 0.888 46.54
50 0.975 0.977 0.978 0.976 0.971 50.891
25 1.08 1.082 1.084 1.082 1.077 56.446
10 1.162 1.164 1.165 1.163 1.158 60.691
5 1.196 1.197 1.199 1.197 1.192 62.473
Untreated 1.913 1.914 1.913 1.913 1.908 100
Blank 0.005 0.006 0.005 0.005 0

In the present study growth inhibition of HeLa cell The reported IC50 (50% of growth inhibition)
lines increase with a rise in concentration of A. value for methanol extract was 52.167µg/mL and
odorata leaf extract. The viability percentage of 59.061µg/ml in ethyl acetate extracts.
extract Cytotoxic
HeLa cell lines of ethyl acetate and methanol leaf effect of ethyl acetate and methanol leaf extract
extracts at concentration 100 µg/mLµg/m reduced on MCF-7 and HeLa cell lines were shown in
from 100% to 41.92% and 41.29% respectively. Figs. 5A and 5B; 6A and 6B.

Fig. 1. GC-MS
MS chromatogram of ethyl acetate leaf extract of A. odorata

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Fig. 2(A-D). Phytocompounds identified in ethyl acetate leaf extract of A. odorata

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Fig. 2(E-H). Phytocompounds identified in ethyl acetate leaf extract of A. odorata

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Fig. 2(I-L). Phytocompounds identified in ethyl acetate leaf extract of A. odorata

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Fig. 2(M-O). Phytocompounds identified in ethyl acetate leaf extract of A. odorata

Table 5. Cytotoxic properties of methanolic leaf extract of A. odorata on MCF -7and HeLa cell
lines

Cell Concentration Absorbance at 570 nm Average Average- % IC50 (μg

line (μg/mL) Blank Viability /mL)
MCF-7 100 0.814 0.816 0.818 0.816 0.809 39.31
75 0.871 0.873 0.875 0.873 0.866 42.079
50 0.922 0.924 0.925 0.923 0.916 44.509 26.211
25 0.995 0.997 0.998 0.996 0.989 48.056
10 1.068 1.07 1.072 1.07 1.063 51.652
5 1.176 1.178 1.179 1.177 1.17 56.851
Untreated 2.065 2.066 2.065 2.065 2.058 100
Blank 0.007 0.008 0.007 0.007 0
HeLa 100 0.791 0.793 0.795 0.793 0.788 41.299 52.167
75 0.85 0.852 0.854 0.852 0.847 44.392
50 0.963 0.965 0.967 0.965 0.96 50.314
25 1.036 1.038 1.039 1.037 1.032 54.088
10 1.105 1.107 1.109 1.107 1.102 57.756
5 1.181 1.183 1.185 1.183 1.178 61.74
Untreated 1.913 1.914 1.913 1.913 1.908 100
Blank 0.005 0.006 0.005 0.005 0

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Fig. 3. GC-MS
MS chromatogram of methanol leaf extract of A. odorata

Plate 1. Cytotoxic Properties of ethyl acetate extract on HeLa Cell Line

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Fig. 4(A-D). Phytocompounds identified in Methanol leaf extract of A. odorata

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Fig. 4(E-H). Phytocompounds identified in Methanol leaf extract of A. odorata

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Fig. 4(I-L). Phytocompounds identified in Methanol leaf extract of A. odorata

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Fig. 4(M-N). Phytocompounds identified in Methanol leaf extract of A. odorata

Plate 2. Cytotoxic Properties of Methanol extract on HeLa Cell Line

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100 100
y = -0.2133x + 62.582 y = -0.2036x + 60.591
80 R² = 0.9905 80 R² = 0.9695
% Viability

% Viability
60 60
40 40
20 20
0 0
0 20 40 60 80 100 0 20 40 60 80 100
A Concentration (µg/ml) B Concentration (µg/ml)

Fig. 5. A) Cytotoxic effect of ethyl acetate extract on HeLa Cell Line B) Cytotoxic effect of
Methanol extract on HeLa Cell Line

100 y = -0.2019x + 58.269 100 y = -0.1619x + 54.225

R² = 0.9995 R² = 0.8974
80 80
% Viability

% Viability
60 60
40 40
20 20
0 0
0 20 40 60 80 100 0 20 40 60 80 100
A Concentration (µg/ml) B Concentration (µg/ml)

Fig. 6. A) Cytotoxic effect of ethyl acetate extract on MCF-7 Cell Line B) Cytotoxic effect of
Methanol extract on MCF-7 Cell Line

4. DISCUSSION antihypertensive [21]. 3,7,11,15-Tetramethyl-2-

hexadecen-1-ol (phytol) is an unsaturated acyclic
The documentary evidences on orchid diterpenoid alkene alcohol and act as precursor
metabolites and extracts proved their efficiency of vitamin E. This compound has acute oral
over number of human ailments [42,43,44,45,46, cytotoxicity LD50 in rats > 5 g/kg [55]. 9,12,15-
47,48,49]. They also have a significant role in octadecatrienoic acid methyl ester is an
prevention of cancer and its treatment [50,51, unsaturated fatty acid ester which has been
52]. Phytochemical analysis of different organic shown to possess anticancer,
extracts of A. odorata contains fatty acids, hypocholesterolemic, antimicrobial and
secondary alcohols, diketones, esters and antioxidant activities [40,41]. Apart from this
phenols. These secondary metabolites may be other compounds reported in the present study
for various biological activities of medicinal such as Phenyl(piperidin-3-yl) methanone, β-
plants [53,54]. Most of the compounds identified Selinene, (E)-5-Methylundec-4-ene, 2-O-(2-
in ethyl acetate and methanol extracts of the Ethylhexyl) 1-O-pentadecyl oxalate, Squalene,
plant are biologically active (Tables 2 and 3). In m-Toluylaldehyde, Methyl (2E) - 3-phenyl - 2-
the present study a total of seven propeonate, Ethyl α-D-glucopyranoside, 3,7,11-
phytocompounds in ethyl acetate and six Trimethyl-1,6,10-dodecatrien-3-ol, (2E,6E)-
compounds in methanol extracts have anticancer 3,7,11-trimethyldodeca-2,6,10-trien-1-ol also
activity. 2-Methyl-5-(1,2,2-Trimethy cyclopentyl) posses anticancer properties. Squalene acts as a
phenol is also known as Xanthorrhizol. It has defence agent against certain pathogens causing
biological activities such as anticancer, human and animal diseases along with its
antimicrobial, anti-inflammatory, antioxidant and anticancer activity [56].

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Plate 3. Cytotoxic Properties of ethyl acetate extract on MCF -7 Cell Line

Plate 4. Cytotoxic Properties of Methanol extract on MCF -7 Cell Line

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Some compounds like 1,3 propanediol has a 3. Rosa M, Perz G. Orchids- A review of uses
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