H.N. Krishna Kumar and Jyoti Bala Chauhan
Department of Studies in Biotechnology, Microbiology & Biochemistry
Pooja Bhagavat Memorial Mahajana Education Centre, PG wing of SBRR Mahajana First
Grade College, K.R.S. Road, Metagalli, Mysore-570 016, Karnataka, India
In the present investigation Physalis minima a medicinally important plant of the family Solanaceae has been screened for anticancer activity. The results of preliminary phytochemical screening of the leaf extract of Physalis minima revealed the presence of flavonoids, alkaloids, tannins, saponins, steroids, cardiac glycosides, anthra quinones, reducing sugars and terpenoids. Determination of total phenolic contents revealed that methanolic leaf extract showed the highest amount of phenolic compounds (78.3 mg/g).
Evaluation of total flavonoid content revealed that methanolic leaf extract showed the highest amount of flavonoid (61.3 mg/g). Percentage cell viability of cell lines was carried out by using Trypan blue dye exclusion technique. It was found that the % viability of HeLa cell line & Hep 2 cell line are 80% & 71.8% respectively, which are most suitable to perform cytoxicity studies. The cytotoxicity study was carried out for methanolic leaf extract of
Physalis minima . The extract was screened for its cytotoxicity against HeLa & Hep
2 cell lines at different concentrations to determine the % growth inhibition by SRB assay and MTT assay. The percentage growth inhibition in SRB assay was found to be increasing with increasing concentration of test compounds against both the cell lines (HeLa and Hep
2
). The methanolic extract showed the strongest growth inhibitory effect on both HeLa and Hep
2 cell lines (68 and 58% respectively) (Table 2). In the present investigation, the leaf extract of
Physalis minima showed significant effects on the tumor inhibition activities against cell lines of HeLa and Hep
2
in MTT assay It was found that the percent growth inhibition increasing with increasing concentration on both the cell lines. The methanolic extract showed the strongest growth inhibitory effect on both HeLa and Hep
2 cell lines (85 and 73% respectively). In the present study, 8 flavonoid compounds showing anticancer activity have been isolated from the methanolic leaf extract of Physalis minima , of which one compound has been characterized by UV, MS and NMR spectroscopic analysis. The compound was identified as 5-methoxy-6, 7-methylenedioxy flavone.
___________________________________________________________________________
Among various diseases attributed to mortality in humans all over the world, cancer is a leading cause [1]. Plants have been a prime source of highly effective conventional drugs for the treatment of many forms of cancer. They have long history used in the treatment of cancer. Active constitutes of Catharanthus roseus, Angelica gigas, Podophyllum peltatum,
1

Taxus brevifolia, Podophyllum emodii, Ocrosia elliptica, and Campototheca acuminata have been used in the treatment of advanced stages of various malignancies [2]. There are various medicinal plants reported to have anti-cancer as well as anti-inflammatory activity in the
Ayurvedic system of medicine. Hartwell [3], in his review of plants used against cancer, lists more than 3000 plant species that have reportedly been used in the treatment of cancer. It is significant that over 60% of currently used anti-cancer agents are derived in one way or another from natural sources, including plants, marine organisms and micro-organisms.
Indeed, molecules derived from natural sources (so-called natural products) , including plants, marine organisms and micro-organisms, have played and continue to play a dominant role in the discovery of leads for the development of conventional drugs for the treatment of most human diseases. Plant-derived compounds have played an important role in the development of several clinically useful anti-cancer agents. These include vinblastine, vincristine, the camptothecin derivatives, topotecan and irinotecan, etoposide, derived from epipodophyllotoxin, and paclitaxel. Several promising new agents are in clinical development based on selective activity against cancer-related molecular targets, including flavopiridol and combretastin A4 phosphate. The search for anti-cancer agents from plant sources started in earnest in the 1950s with the discovery and development of the vinca alkaloids, vinblastine and vincristine, and the isolation of the cytotoxic podophyllotoxins.
These discoveries prompted the United States National Cancer Institute (NCI) to initiate an extensive plant collection program in 1960, focused mainly in temperate regions. This led to the discovery of many novel chemotypes showing a range of cytotoxic activities, including the taxanes and camptothecins. Currently chemotherapy is regarded as one of the most efficient cancer treatment aproach. Although chemotherapy significantly improves symptoms and the quality of life of patients with lung cancer, only modest increase in survival rate can be achieved. Faced with palliative care, many cancer patients use alternative medicines, including herbal therapies [4].
The Solanaceae or nightshades are an economically important family of flowering plants. The family ranges from annual and perennial herbs to vines, lianas, epiphytes, shrubs, and trees, and includes a number of important agricultural crops, medicinal plants, spices, weeds, and ornamentals. The solanaceae are known for their tropane alkaloids and for the steroidal lactones (withanolides). The principal tropane alkaloids, hyoscine and hyoscyamine
2

are reported in at least 15 genera such as Datura, Duboisia, Cyphanthera, Atropa and others.
Steroidal lactones are isolated from Acnistus, Datura, Lycium, Jaborosa, Nicandra, Physalis and Withania [5]. Members of the Solanaceae like Solanum nigrum, Physalis peruviana,
Capsicum fruitescence etc. have proven anti-cancer as well as anti-inflammatory activity.
They possesses medicinal properties like antimicrobial, anti-oxidant, cytotoxic properties, antiulcerogenic, and hepatoprotective activity. Solanum nigrum is a potential herbal alternative as anti-cancer agent and one of the active principles reported to be responsible for this action is Diosgenin [6].
Physalis minima Linn. of Solanaceae family is an annual herb having 0.5-1.5 m height with a very delicate purple-tinged stem and leaves. It is found throughout India and is reported as one of the important medicinal plants in Indian traditional system of medicines.
P. minima are traditionally used to cure various diseases. It is appetizing, diuretic, laxative and useful in inflammations, antigonorrhoeic, enlargement of the spleen and abdominal troubles [7]. Fruits and flowers are used in stomach pain and in constipation, Herb paste is applied in ear disorders [8]. Ripen fruits are used in gastric trouble [9]. The decoction of the whole plant is consumed by the Malay community in Malaysia as a remedy for cancer [10].
The limitations of the available cancer management modalities create an urgent need to screen and generate novel molecules. Despite, well-documented illustrations of phytochemicals being used for prevention and treatment of cancer, their importance in modern medicine remains underestimated. Plants are storehouse of pre-synthesized molecules that act as lead structures, which can be optimized for new drug development. In practice, a large number of cancer chemotherapeutic agents that are currently available in the market can be traced back to their plant source [11].
Hexane, Ethyl acetate, Acetone, Methanol, Chloroform, Sulphuric acid, Fehling’s solution (A and B), Ammonia, Aluminium chloride, Ferric chloride, Acetic acid, Mayer’s reagent, Draggendorff’s reagent, Folin-Ciocalteu reagent, Sodium carbonate, Gallic acid,
Sodium nitrite, Sodium hydroxide, were purchased from M/S Sisco Research laboratories,
Mumbai, India. Quercetin, Trypan Blue, Sulphorodamine B, Trichloroacetic acid, MTT [3-
(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide], DMSO (Dimethyl
3

sulfoxide), cisplatin, were purchased from Sigma (St. Louis, USA). All chemicals and solvents used were of analytical grade.
The medicinally important plant Physalis minima of the family Solanaceae for the present investigation was collected from different forest areas in Karnataka (Bangalore, Chikmagalur,
Karwar, Mysore, Shimoga, Tumkur and Uttarakannada district).
Different parts (stem, root and leaves) of the collected plant were washed thoroughly in water, shade dried for a week and ground to coarse powder in electric grinder separately. Hundred grams of each powder was extracted in soxhlet apparatus successively with solvents of increasing polarity viz., hexane, ethyl acetate, acetone and methanol separately. The soxhelation process was carried out until the solvent was found to be colorless. The extract was concentrated using a rotary flash evaporator. For aqueous extraction, 25 g of each sample was homogenized with 100 ml of distilled water separately and the homogenate was kept in a shaker at 40°C for 24 hrs.
The extract was filtered over Whatman No. 1 paper and lyophilized in lyophilizer.
The prepated extracts were studied for their phytoconstituents such as flavonoids, alkaloids, terpenoids, tannins, saponins, reducing sugars, steroids, anthraquinones and cadiac glycosides. Phytochemical screening were perfomed using standard procedures [12]
The total phenolic content of the extracts were estimated by a colorimetric assay, according to the method described by Singleton and Rossi [13] with slight modifications.
Briefly, 1 mL of each sample was mixed with 1 ml of Folin-Ciocalteu reagent. After 3 min, 1 mL of saturated Na
2
CO
3
solution was added to the mixture followed by the addition of 7 ml of distilled water. The mixture was kept in the dark for 90 min, after which the absorbance was read at 725 nm. The total phenol contents were determined using a standard curve prepared with gallic acid. The estimation of the phenolic compounds was carried out in triplicate. The results were mean ± standard deviations and expressed as milligram of gallic acid equivalent/g of extract.
The total flavonoid content of P. minima extracts was determined by the method described by Zhishen et al [14]. Briefly, 250 μl of each sample were mixed with 1 ml of
4

distilled water and subsequently with 150 μl of 150 g/l sodium nitrite solution. After 6 min,
75 μl of 100 g/l aluminium chloride solution was added, then the mixture was allowed to stand for a further 5 min before 1 ml of 40 g/l NaOH solution was added. The mixture was immediately made up to 2.5 ml with distilled water and mixed well. The absorbance of the mixture was then measured at 510 nm. Total flavonoid content was expressed as mg quercetin equivalent (QE)/g dried extract. Values presented are the average of three measurements.
Cell lines
Cell lines of different cancer type viz., HeLa and Hep
2
were collected from National
Centre for Cell Science, University of Pune Campus, Pune for anticancer activity study. The percentage viability of the cell line was carried out by using Trypan blue dye exclusion method. Antitumor potentials of prepared extracts against HeLa and Hep
2
cell lines were investigated using SRB and MTT assay.
Sulphorodamine B assay
The monolayer cell culture was trypsinized and the cell count was adjusted to 0.5-1.0 x 105 cells/ml using medium containing 10% new born sheep serum. To each well of the 96 well microtitre plate, 0.1ml of the diluted cell suspension (approximately 10,000 cells) was added. After 24 hours, when a partial monolayer was formed, the supernatant was flicked off, washed once and 100 μl of different test compound concentrations were added to the cells in microtitre plates. The plates were then incubated at 37oC for 72 hours in 5% CO2 incubator, microscopic examination was carried out, and observations recorded every 24 hours. After 72 hours, 25 μl of 50% trichloroacetic acid was added to the wells gently such that it forms a thin layer over the test compounds to form overall concentration 10%. The plates were incubated at 4oC for one hour. The plates were flicked and washed five times with tap water to remove traces of medium, sample and serum, and were then air-dried. The air-dried plates were stained with 100μl SRB and kept for 30 minutes at room temperature. The unbound dye was removed by rapidly washing four times with 1% acetic acid. The plates were then airdried. 100 μl of 10mM Tris base was then added to the wells to solubilise the dye. The plates were shaken vigorously for 5 minutes. The absorbance was measured using microplate
5

reader at a wavelength of 540nm22. The percentage growth inhibition was calculated using following formula
% cell inhibition= 100-[(At-Ab)/ (Ac-Ab)] X 100
Where, At= Absorbance value of test compound, Ab= Absorbance value of blank and
Ac=Absorbance value of control
MTT assay
In brief, the cells were seeded into 4 wells of a 96-well micro titer plate at 2 X 10 4 cells per well with 100 µl growth medium and then incubated for 24 hrs. at 37ºC under 5%
CO
2
. Later, the medium was removed while fresh growth medium containing prepared extracts at different concentrations (40, 80, 120, 160 and 200µg/ml) were added separately.
After 3 days of incubation at 37ºC under 5% CO
2
, the medium was removed while 0.1 mg/ml
MTT reagent was added and incubated for 5 hrs. at 37ºC. Then, the MTT reagent was removed and 100 µl of DMSO was added to each well. The absorbance was determined by
ELISA reader at 516 nm. The control well received only the media without extract. The conventional anticancer drug cisplatin was used as a positive control. The inhibition of cell growth was calculated as percent anticancer activity using the following formula:
[(Ac–As)/Ac]×100
Where Ac is the absorbance of the control and As is the absorbance of the sample. The results were mean ± standard deviations.
The compounds were isolated from the extract by using different chromatographic techniques (TLC, Column chromatography and HPLC). The compound will be characterized following the standard techniques (UV-visible, LC-MS, NMR spectroscopy).
Thin layer chromatography (TLC)
Phytoconstituents of the methonolic leaf extract of Physalis minima were separated using thin layer chromatography (TLC). Aliquots of 200µg of extracts were loaded on TLC plates (TLC F254; Merck, India). Different solvent systems of varying polarity were used to separate anticancer compounds such as
Colum chromatography
The column was packed in the form of slurry of silica gel (60-120 mesh,) and gradient elution was carried out by using different combinations of solvents (Methanol:
6

Hexane- 70:30, Methanol: Acetone- 20:80 and Acetone: Ethyl acetate- 60:40 ). The respective fractions were collected and concentrated by flash evaporation. Similar fractions were pooled after TLC analysis then assayed for anticancer activity. The isolated compounds were identified as flavonoids.
HPLC
The isolated bioactive compounds were tested for its purity using HPLC [LCsolution, ShimadzuTM, MAO 1527, USA with LC-UV-100 UV detector, A CAPCELL(C-
18) Column RP (18.5μm, 250
 4.0 mm),type MG 520μm, number AKAD/05245]. The mobile phase consisted of solvent mixtures (Acetonitrile: Water- 70:30) with a flow rate of
1.0ml/min. The injection volume was 20 μl, and UV detection was effected at 254nm.
UV-visible spectrometry
The UV-visible spectrum of the isolated compound in HPLC grade methanol was recorded by using Shimadzu 160A UV- visible spectrophotometer.
Liquid Chromatography-Mass Spectrometry (LC-MS)
The system consisted of a Hitachi L-6000 pump (Hitachi, Tokyo, Japan), a Rheodyne model 7125 injector with a 25 mL loop, and a 4.6 i.d. 325.0 mm Devosil C30 UG-5 column
(Nomura Chemical, Seto, Japan). LC was performed using an aqueous solution containing methanol and acetonitril as the mobile phase at a flow rate of 1 mL/min. The spectra were recorded on a TSQ 700 triple-quadrupole mass spectrometer (Finnigan MAT, San Jose, CA) equipped with an ESI source with an ICIS II data system in the positive ion mode using a spray voltage of 9.8 kV, at a source temperature of 90°C. The flow rate was 10 µL/min.
1 H and 13 C Nuclear Magnetic Resonance (NMR)
1 H and 13 C NMR spectra were recorded on a Bruker DRX-500 MHz spectrometer
(500.13MHz for 1H and 125 MHz 13C). A region from 0-12 ppm for 1H and 0-200 ppm for
13
C was employed. About 50 mg of the sample dissolved in DMSO was used for recording the spectra. Chemical shift values were expressed in parts per million relative to the internal tetramethylsilane standard.
Statistical analysis: All experiments were carried out in triplicates. Data were expressed as mean ± standard error. The significance of the study was assessed by analysis of variance (p
< 0.05) using computer program ANOVA, Excel.
7

Among the plants studied the Physalis minima showed very good result. Therefore, this plant has been selected for further studies. The results of preliminary phytochemical screening of the leaf extract of Physalis minima revealed the presence of flavonoids, alkaloids, tannins, saponins, steroids, cardiac glycosides, anthra quinones, reducing sugars and terpenoids when compared to stem and root (Table 1). The methanolic leaf extract showed maximum number of phytoconstituents when compared to other solvent extract.
Therefore, methanolic leaf extract has been selected for compound isolation and characterization.
Determination of total phenolic contents revealed that methanolic leaf extract showed the highest amount of phenolic compounds (78.3 mg/g) when compared to ethyl acetate (46.65 mg/g) and acetone extract (45.1 mg/g total phenolics). Hexane and aqueous extracts showed fewer amounts of phenolic compounds. Likewise, the amount of polyphenolic compound observed in stem and root extracts were less. Therefore, methanolic leaf extract has been selected for further studies. The phenolic concentration of the extract was expressed as milligram of gallic acid equivalents per gram of extract. The results of the present work strongly suggest that phenolic compounds are important components of this plant and some of their pharmacological effects could be attributed to the presence of these valuable constituents. Thus, the anticancer activity of the extract could be predicted from its total phenolic content. Phenolic compounds are a class of antioxidant agents, which act as free radical scavengers [15]. The interests in phenolic compounds, particularly flavonoids and tannins have considerably increased in recent years because of their broad spectrum of chemical and diverse biological properties which include the antioxidant effects [16].
Further, phenolic compounds are effective hydrogen donors which make them antioxidant
[17]. It is suggested that polyphenolic compounds have inhibitory effects on mutagenesis and carcinogenesis in humans, when up to 1.0 g daily ingested from a diet rich in fruits and vegetables [18]. In addition, it has been reported that phenolic compounds are associated with antioxidant activity and play a crucial role in stabilizing lipid peroxidation [19]. In our study, there is correlation between antioxidant activity and phenol content.
8

Evaluation of total flavonoid content revealed that methanolic leaf extract showed the highest amount of flavonoid (61.3 mg/g) when compared to ethyl acetate (22.1 mg/g) and acetone extract (32.6 mg/g). Hexane and aqueous extracts showed negligible amounts of flavonoids. Likewise, the amount of flavonoid observed in stem and root extracts were less.
Therefore, methanolic leaf extract has been selected for further studies. Total flavonoid content was expressed as mg quercetin equivalent (QE)/g dried extract.
Viability and characterization of cell lines
Percentage cell viability of cell lines was carried out by using Trypan blue dye exclusion technique. It was found that the % viability of HeLa cell line & Hep
2 cell line are
80% & 71.8% respectively, which are most suitable to perform cytoxicity studies.
Cytotoxicity activity
The cytotoxicity study was carried out for leaf extract of Physalis minima . The extract was screened for its cytotoxicity against HeLa & Hep
2 cell lines at different concentrations to determine the % growth inhibition by SRB assay and MTT assay.
Sulphorhodamine B (SRB) assay
Results are tabulated in Table 2. The percentage growth inhibition was found to be increasing with increasing concentration of test compounds against both the cell lines (HeLa and Hep 2 ). The methanolic extract showed the strongest growth inhibitory effect on both
HeLa and Hep 2 cell lines (68 and 58% respectively) whereas in comparison ethyl acetate and acetone extract demonstrated moderate cytotoxic effect with % inhibition of 51 & 47 for
HeLa and 58 & 50 for Hep
2 cell lines respectively (Table 2). In contrast hexane and aqueous extract did not show cytotoxic effect at any concentration. The result indicates that the methanolic leaf extract of Physalis minima has potential activity on both HeLa and Hep
2 cell lines. Therefore, methanolic leaf extract has been selected for the isolation of bioactive compound.
MTT assay
In the present investigation, the leaf extract of Physalis minima showed significant effects on the tumor inhibition activities against cell lines of HeLa and Hep
2
. It was found that the percent growth inhibition increasing with increasing concentration on both the cell
9

lines. The methanolic extract showed the strongest growth inhibitory effect on both HeLa and
Hep
2 cell lines( 85 and 73%) whereas, in comparison ethyl acetate and acetone extract demonstrated moderate cytotoxic effect with % inhibition of 58 & 65 for HeLa and 51 & 61 for Hep
2 cell lines respectively (Table 3). In contrast hexane and aqueous extract did not show cytotoxic effect at any concentration. The result indicates that the methanolic leaf extract of Physalis minima has potential activity on both HeLa and Hep
2 cell lines. Therefore, methanolic leaf extract has been selected for the isolation of bioactive compound. The chloroform extract of P. minima is reported to possess cytotoxic activities on NCI-H23
(human lung adenocarcinoma) cell line at dose- and timedependent manners. They exert programmed cell death in NCI-H23 cells with typical DNA ftagmentation, which is a biochemical Hallmark of apoptosis. It also produces apoptotic characteristics in the treated cells, including clumping and margination of chromatins, followed by convolution of the nuclear and budding of the cells to produce membrane-bound apoptotic bodies. An acute exposure to the extract produced a significant regulation of c-myc, caspase-3 and p53 mRNA expression in this cell line [20]. The chloroform extract showed anticancer activity against human T-47D breast carcinoma cells. The cytotoxic action of chloroform extract is reported to be due to induced apoptotic cell death via, p53, caspase-3 and c-myc-dependent pathways
[21]. Chloroform extract exhibited anticancer activity in human ovarian Caov-3 carcinoma.
Cytotoxicity of the extract was measured using the methylene blue assay. The mechanism of cell death was determined using four independent methods which revealed that the anticancer effect is due to a combination of apoptotic and autophagic cell death mechanisms on Caov-3 cells. The induction of these programmed cell deaths was mediated via, c-myc, p53 and caspase-3 dependent pathway [22].
Identification of the compound
In this study, 8 compounds showing anticancer activity have been isolated from the methanolic leaf extract of Physalis minima , of which one compound has been characterized by UV, MS and NMR spectroscopic analysis. The compound was identified as 5-methoxy-
6,7-methylenedioxy flavone. The UV spectrum of the compound showed maximum absorption at 280 nm. The mass spectrum of the bioactive compound showed the parent ion
(M
+
) at 296.1. The identity of the compound was deciphered from the NMR data analysis.
I
H NMR (δ 6.8, s, 1H, C-3 hydrogen) spectra indicated that it was a flavone. The presence of
10

a methylenedioxy group was suggested by the presence of a 2H signal at δ 6 (s) in its I
H
NMR and a
13 C signal at δ 103 (t) in its 13
C NMR spectra.
1
H NMR (CDCl
3
) δ 7.6-7.8 (m,
2H), 7.3-7.8 (m, 3H), 6.8 (s, IH), 6.7(s, IH), 6.3 (s, 2H), 4.2 (s, 3H).
13
C NMR (CDCl
3
): δ
177.8 (s, C=O, C-4), 160.5 (s, C-2), 154.7 (s, C-2), 152.7 (s, C- 5), 141.2 (s, C-7), 134.61 (s,
C-6), 131.4 (s, C-l'), 131.6 (d, C-4'), 128.6 (d, C-2'), 125.6 (d, C-3'), 112.6 (s.
C- 10), 108.3
(d, C- 3), 102 (t, O-CH
2
-O), 93.4 (d, C-8). 61.2 (q, OMe), 296 (M
+
34.5), 268 (55.6, M-CO),
250 (54.6), 222 (10.3), 237 (10.6), 194 (10.3), 166 (10), 164(96.5), 136 (33.6), 105 (17.7),
102 (62.3). Thus, it was characterized as 5- methoxy-6, 7- methylenedioxyflavone. The same has been reported in this plant [23]. The compound 5- methoxy-6, 7- methylenedioxyflavone isolated from Physalis minima leaves showed strong cytotoxic effect. The growth inhibition of methanolic leaf extract was found to be 85 and 73% for HeLa and Hep
2
cell lines in MTT assay. Whereas the comound 5- methoxy-6, 7- methylenedioxyflavone showed 79 and 70% of growth inhibition for HeLa and Hep
2 cell lines in MTT assay. The study clearly indicated that the characterized compound is a potent cytotoxic in nature.
Plant-derived compounds have played an important role in the development of several clinically useful anti-cancer agents. These include vinblastine, vincristine, the camptothecin derivatives, topotecan and irinotecan, etoposide, derived from epipodophyllotoxin, and paclitaxel. Several promising new agents are in clinical development based on selective activity against cancer-related molecular targets, including flavopiridol and combretastin A4 phosphate.
In conclusion, the isolated compound 5- methoxy-6, 7- methylenedioxyflavone could be regarded as promising drugs for cancer therapy, but the mechanisms of their anti-cancer activity and their toxicity should be further addressed. The results obtained in the in vitro models such as SRB assay and MTT assay clearly suggest that, the methanolic leaf extract of
Physalis minima showed strong cytotoxic activity when compared with different standards.
The results of this study showed that the extracts can be used as easily accessible source of natural anticancer agents and as a possible food supplement or in pharmaceutical industry.
The areal parts of Physalis minima could serve as a new source of natural anticancer agents with potential cytotoxic effects and related health benefits.
11

The financial assistance of University Grants Commission (UGC), Bangalore is gratefully acknowledged. The authors are thankful to Prof. C.K. Renukarya, Director, Pooja Bhagavat
Memorial Mahajana Post Graduate Centre, Mysore for providing necessary facilities to carry out the research work.
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13

Solve nts for extra ct
Hexa ne
Pla nt par ts
Ethyl acetat e
Aceto ne
Meth anol
Ro ot
Ste m
Ste m
Lea f
Ste m
Lea f
Ro ot
Lea f
Ro ot
Ste m
Lea f
Ro ot
Flavon oids
Alkal oids
--
--
--
++
++
--
++
++
++
++
++
++
--
--
--
--
++
++
--
++
--
--
++
--
Tann ins
Sapo nins
--
--
--
++
++
++
--
++
--
++
++
++
--
--
--
--
++
--
--
--
--
--
++
--
Stero ids
Cardi ac glycos ides
-- --
Anth ro quino nes
--
Redu cing sugar s
++
Terpen oids
--
--
--
++
++
++
++
++
++
++
++
++
++
--
--
++
++
--
--
++
--
++
++
--
--
--
--
--
--
--
--
--
--
--
++
--
--
--
--
--
++
--
--
--
--
--
--
++
++
++
++
++
++
++
++
++
14

Water Ste m
Lea f
Ro
--
++
--
--
--
--
--
--
--
--
++
++
-- -- ++ -- -- ++ ot
++ Presence of constituent
-- Absence of constituent
--
--
--
++
++
--
--
--
--
Solvents extract
Ethyl acetate
Acetone
Methanol for Concentration of HeLa cell line extract (µg/ml)
% inhibition
40 16 ±1.1
80
120
160
200
40
80
18 ±1.3
26 ±1.1
31 ±1.7
51 ±2.2
18 ±1.1
22 ±1.2
120
160
200
40
80
120
160
200
31 ±2.2
38 ±2.1
58 ±1.8
26 ±0.7
36 ±1.1
40 ±1.6
48 ±1.7
68 ±2.1
Hep 2 cell line
% inhibition
11 ±0.4
14 ±1.1
18 ±1.5
22 ±2.2
47 ±2.4
16 ±1.2
20 ±0.7
27 ±1.1
26 ±1.2
50 ±2.2
19 ±0.8
27 ±1.1
30 ±1.2
37 ±1.7
58 ±2.1
15

Solvents for Concentration of HeLa cell line Hep 2 cell line extract extract (µg/ml)
% inhibition % inhibition
Ethyl acetate
Acetone
Methanol
40
80
120
160
200
40
80
120
160
200
40
80
120
160
200
18 ±1.3
22 ±1.5
31 ±1.1
36 ±1.9
58 ±2.1
22 ±1.1
29 ±1.4
35 ±2.1
44 ±2.5
65 ±1.9
31 ±0.5
42 ±1.4
45 ±1.9
60 ±1.8
85 ±2.1
12 ±0.2
16 ±1.3
22 ±1.2
30 ±2.1
51 ±2.2
18 ±1.2
24 ±0.9
31 ±1.4
36 ±1.6
61 ±2.1
25 ±0.9
32 ±1.1
34 ±1.4
51 ±1.9
73 ±2.2
16