comparative analysis of apoptotic activity from extracts...

Journal of Pharmacy Research Vol.8 Issue 12.December 2014

Priyanka Sharma et al. / Journal of Pharmacy Research 2014,8(12),1779-1784

1779-1784

Research ArticleISSN: 0974-6943

Available online throughwww.jpronline.info

*Corresponding author.Dr. Kumar Pranav NarayanAssistant Professor, Department of Biological SciencesBirla Institute of Technology and Science,Pilani, Hyderabad Campus,Hyderabad-500078, India

Comparative Analysis of Apoptotic Activity From Extracts ofSaponin-rich Indian Medicinal Plants

Priyanka Sharma#, Madhu Rani Bharati#, Mohit Dave, Suman Kapur, Kumar Pranav Narayan*Department of Biological Sciences, Birla Institute of Technology and Science, Pilani, Hyderabad Campus, Hyderabad – 500078, India

#The first two authors contributed equally to this work

Received on:21-05-2014; Revised on: 17-06-2014; Accepted on:25-07-2014

ABSTRACTNatural products have been regarded as an important source that could contribute to potential chemotherapeutic agents. In particular, plantderived compounds have gained importance in anticancer therapy. In our study, we investigated the inhibitory effects of different Indianmedicinal plant extracts (Bacopa monerri, Tribulus terrestris, Asparagus racemosus and Nelumbo nucifera) on cancer cells which are knownto be highly saponin producing. Saponins are a diverse group of compounds widely distributed in the plant kingdom, which are usuallycharacterized by their structure containing a steroidal or triterpenoid aglycone and one or more sugar chains. In the present investigation,methanolic and ethanolic saponin rich plant extracts were compared for its cytotoxic effect on cancer and normal cell lines. The extracts alsohave been shown to induce apoptosis and cell cycle arrest in cancer cells which implies its anticancer efficacy. Based on the current findings,it can be concluded that extracts from Nelumbia nucifera and Bacopa moneri have cytotoxic and apoptotic activity towards cancer cellswhich indicates the need of further investigation of these extracts for cancer therapeutics.

KEYWORDS: Natural products, chemotherapeutic agents, saponins, cytotoxic effect, apoptosis, cell cycle, cancer therapeutics

Type of study PercentageOccurrence

Cytotoxicity assay 77Cell cycle arrest studies 6Anti-invasive/ anti-angiogenic/ anti-metastatic activities 7Total mechanism of action (Multiple assays) 10

INTRODUCTIONNatural products are considered powerful sources of novel drug dis-covery and development. Medicinal plants are the richest bio-resourceof drugs of traditional systems of medicine, modern medicines,nutraceuticals, food supplements, pharmaceutical intermediates andchemical entities for synthetic drugs1,2; and interest in this area ofresearch has increased in all over countries. Natural remedies derivedfrom herbs3, food or raw materials are used by pharmaceutical andfood industries4. In recent years, antitumor drugs from plants havedrawn attention because of their low toxicities compared with con-ventional chemotherapy drugs5. Cancer is the main cause of deathworldwide and plant derived drugs, like vinblastine, vincristine, taxol,and camptothecin have improved the chemotherapy of somecancers6,7 . Many of these plant-derived anticancer agents have beendiscovered through large-scale screening programs8. During litera-ture survey, it was observed that majority of the work done (77% ofthe total work done in 2009-2013) on saponins has followed a charac-

teristic pattern of extraction of phytocompounds from plant sourceand testing its toxicity on cell lines but there are few reports on theapoptotic activity of these compounds and further cell cycle arreststudies.

Table 1. Summary of work done on saponins in the year 2009-2013

The present study was conducted to evaluate the anti-cancer activityof crude extracts from four different Indian medicinal plants with theobjective of testing the toxicity of the extracts on cancer cells and tostudy apoptosis for determining cell death and evaluation of effecton cell cycle.

MATERIALS & METHODS

ReagentsHPLC grade methanol and ethanol were purchased from Himedia



1779-1784

Laboratories, India. The cell culture medium and reagents, penicillin,streptomycin, kanamycin, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and Dimethyl Sulphoxide (DMSO, cellculture grade) were purchased from Sigma-Aldrich Inc. (St. Louis,MO, USA). Fetal bovine serum was purchased from Lonza.

Extraction of plant materialsFour plants, Bacopa monerri, Tribulus terrestris, Asparagusracemosus and Nelumbo nucifera known to be highly saponin pro-ducing were collected from different areas of Hyderabad, India.Soxhlet, which has been used for a long time, is a standard techniqueand the main reference for evaluating the performance of other solid–liquid extraction methods9. The leaves of Bacopa moneri, Tribulusterrestris, Asparagus racemosus and roots of Nelumbia nuciferawere separated and oven dried at 45 ºC overnight. The plants weregrounded into powder form using the grinder. Extraction using Soxhletapparatus with either 95% (v/v) ethanol or methanol as solvent for 40h was performed10.

Preparation of test materialsThe crude extracts were dissolved in dimethyl sulfoxide (DMSO) at500mg/ml as stock solutions which were then diluted with DMEM todesired concentrations. The final concentrations of the extract dilu-tions in culture were 10, 50, 75, 100 and 150, µg/mL. The final concen-tration of DMSO in each sample did not exceed 1% v/v, to keep thecytotoxicity of DMSO at less than 10%. Ethanolic extracts were namedas BME, TTE, STE and NNE and methanolic were named as BMM,TTM, STM, NNM for Bacopa moneri, Tribulus terrestris,Asparagus racemosus and roots of Nelumbia nucifera respectively.

Cell lines and cell culturesA549 (human lung cancer cell line), and CHO (chinese hamster ovarycells) were obtained from the National Centre for Cell Science (NCCS),Pune, India. Cells were cultured in DMEM (Dulbecco’s modified eaglemedium) supplemented with 10% (v/v) FBS, penicillin 50 mg/lt, strep-tomycin 50 mg/lt, kanamycin acid sulphate 100 mg/lt and 3.7gm/ltsodium bi-carbonate solution. The cells were maintained as monolay-ers in 25 cm2 plastic tissue culture flasks at 37°C in a humidifiedatmosphere containing 5% CO2 in air.

Cytotoxicity studiesThe MTT colorimetric assay was used to screen for cytotoxic activityof each plant extract. This colorimetric assay is based on the ability ofviable cells to reduce a soluble yellow tetrazolium salt [3-(4,5-di Me-thyl Thiazol-2-yl) 2,5-diphenyl Tetra-zolium bromide] (MTT) to a blueformazan crystal by mitochondrial succinate dehydrogenase activityof viable cells11. This test is a good index of mitochondrial activity

and, thus, of cell viability. The cells were seeded in 96-well plates at a

Cell cycle analysisCells were seeded at a density of 8x104cells/well in a 6-well plate andallowed to attach overnight. Cells were treated with 75µg/mL of eachplant extract for 18 hours. Cells were harvested, washed with 1X PBS,and fixed with 70% ethanol and kept at -200C for at least overnight.Cells were centrifuged to discard ethanol and re-suspended in 1XPBS. After centrifugation cells were suspended in 1ml of PI stainingsolution (0.05% triton X-100, 0.1mg/ml RNase A and 50µg/ml of PIfrom 50X stock solution of 2.5mg/ml) and kept in dark for 45 min at370C. After incubation cells were collected by centrifugation, washedwith 1X PBS, re-suspended in 1ml PBS, and analyzed using a flowcytometer (FACS Canto II, Becton- Dickinson, San Jose, CA, USA)and data were analyzed with FCS Express V3 software. A minimum of10,000 events were gated per sample.

Statistical analysisAll experiments were repeated three times and then data were shown

Quantification of Apoptosis StudiesThe annexin V-FITC- labeled apoptosis detection kit (Sigma) wasused to detect and quantify apoptosis by flow cytometry as permanufacturer’s protocol. In brief, cells (2×105cells/well) were seededin 6-well plates and cultured over night in 10% fetal bovine serummedia. After 16-18 h cells were either kept untreated or treated with75µg/mL of different plant extracts for 24 hours. Then the cells wereharvested and collected by centrifugation for 5 minutes at 1000 rpm.Cells were then resuspended at a density of 1 x 106 cells/ml in 1Xbinding buffer and stained simultaneously with FITC- labeled AnnexinV (25 ng/ml) and propidium iodide (50 ng/ml). Cells were analyzedusing a flow cytometer (FACS Canto II) and data were analyzed withFCS Express V3 software. A minimum of 10,000 events were gated persample.

Cell viability (%) = OD of treated cells/OD of control cell ×100.The results were generated from three independent experiments; eachexperiment was performed in triplicate.

hours. Untreated cultures and blank wells received 100 µL of DMEMmedium supplemented with 10% (v/v) FBS. After 24 hours, 10 µL ofthe MTT reagent was added to each well, and the plate was incu-bated for 4 hours at 37°C. The MTT solution was removed from thewells by aspiration. After removal of the medium, 0.1 ml of 1:1 DMSO:methanol was added to each well and plates were shaken. The absor-bance was measured at 550 nm in an automated plate reader. Thepercentage of cytotoxicity compared to the untreated cells was deter-mined with the equation:

density of 1x104 cells/well in 100 µL culture medium and allowed toattach overnight at 37°C. The cells were then incubated with 100 µLof extract at different concentrations of 10 µg/mL-150 µg/mL for 24



1779-1784

as means ± standard deviation (S.D) of three assays. Student t- testwas applied, and p<0.05 was considered as statistically significant.

RESULTS

MTT assay for cytotoxic studiesInitially, cytotoxic studies were carried out for dose optimization. Dif-ferent extracts were used in a range of 10µg/mL - 200µg/mL of mediafor A549 and CHO cell lines. A well marked increase in toxicity wasobserved between 10-100µg/mL, whereas after that cell viability wasnoticed to be almost negligible. The dose was optimized to be75µg/mL media and used as optimized dose for carrying out furtherstudies. Upon treatment of normal cells with these extracts, viabilityremained comparatively high to be around 80%. Also, not much dif-ference was observed in results obtained for ethanolic and methanolicextracts as shown in figure 1 and so, for further studies treatmentswere carried out with ethanolic extracts.

% c y t o t o x i c i t y o f e t h a n o l i c e x t r a c t s o n C H O a n d A 5 4 9 c e l ls

2 5

3 5

4 5

5 5

6 5

7 5

8 5

9 5

T T B M S T N N

C r u d e e t h a n o l i c p l a n t e x t r a c t s

% c

ytot

oxic

ity

C H O

A 5 4 9

% c y t o t o x i c i t y o f m e th a n o l i c e x t r a c t s o n C H O a n d A 5 4 9 c e lls

2 5

3 5

4 5

5 5

6 5

7 5

8 5

9 5

T T B M S T N N

C r u d e m e t h a n o l i c p l a n t e x t r a c t s

% c

ytot

oxic

ity

C H O

A 5 4 9

Fig 1 : Cytotoxic studies of methanolic and ethanolic extracts ofdifferent plants on A549 lung carcinoma (black bar) and non-can-cerous CHO (chinese hamster ovary, white bar) cells (A) ethanolicextracts (B) methanolic extracts

Apoptotic assayCell death was determined in the A549 cell line using FITC-conju-gated Annexin-V membrane staining and PI nuclear counterstaining.Cell populations were quantified by flow cytometry analysis after 18hours of treatment with 75µg/mL of each extract. In Fig 2, the percent-age of dead cells is relative to the total cell population, while thepercentage of apoptotic cells is presented with regard to the PI, ie,nondead cells. In this experiment, percentage of apoptotic cells wasfound to be high in those treated with NNE as compared to untreatedcells. Percent apoptosis was comparatively low in cells treated withBME and TTE. Negligible apoptosis was reported in STE treated cells(Table 2, Figure 2). While considering MTT data which has shown agood level of toxicity in cancerous cells of each treatment group, itcan be pointed out that the extract is toxic to the cells but no apoptosiswas observed in STE.

Fig 2: FACS images of A549 cells treated with 75µg/mL of differentethanolic extracts to detect and quantify apoptosis by flow cytometry



1779-1784

Table 2: Population in terms of percentage showing apoptotic cellsin A549 cells: Q1 indicates necrotic cells, Q2- apoptotic cells, Q3-healthy cells & Q4 showing early apoptotic cells

Plant extract % Apoptotic population quadrant wise

Q 1 Q 2 Q 3 Q 4

UT 3.1 3.0 93.4 0.5TTE 1.9 20.7 75.3 2.1STE 2.0 1.8 96.1 0.1BME 2.8 25.0 69.9 2.2NNE 2.5 34.3 59.6 3.6

Cell cycle analysisThe effect of different plant extracts on cell cycle was evaluated us-ing flow cytometric analysis. Human lung cancer cell line A549 wastreated with different extracts at a concentration of 75µg/ml for 24hours and were subjected to flow cytometric analysis after PropidiumIodide staining. These concentrations were chosen based on theresults from MTT assay.

Fig. 3: FACS images of A549 cells treated with 75µg/mL of differentethanolic extracts to detect and quantify phases of cell cycle by flowcytometry

Percentage population of S phase after treatment with differentextracts was remarkably increased. These results indicate that theextracts induce S phase arrest in A549 cell line. Increase in percentageof cells in pre-phase correlates well with the apoptosis data shown infigure 3.

DiscussionThere has been a long standing interest in the identification ofmedicinal plants and derived natural products for developing cancertherapeutics12 . Bachran et al. 2008 in their review of saponins playinga role in tumor therapy focused on certain groups of compounds,such as dioscins, saikosaponins, julibrosides, avicins, soy and ginsengsaponins, and on combinations of saponins and conventional anti-tumorigenic drugs13.

In the present study, we investigated the inhibitory effects of fourdifferent Indian medicinal plant extracts’ on cancer cells. Methanolicand ethanolic plant extracts were compared for its cytotoxic effect onlung cancer cell line, A549 and normal Chinese Hamster ovary (CHO)cells. The results indicated that the extracts caused growth inhibitionor cell death in concentration dependent manner. Optimal concentra-tion of the extracts was determined using MTT assay. The concentra-tion used for further experiments is 75µg/ml as the percent cytotoxic-ity was found to be close to 50. At the same concentration the cyto-toxic effect was found to be even more in cancer cell lines. Howeverwe could not observe any significant difference between methanolicand ethanolic extracts. Podolak et al., 2010 summarized the cytotoxicactivity of extracted saponins isolated from different plants14. Patil etal., 2014 reported the phytoextracts from Bacopa moneri to be activeagainst human breast cancer cell lines15. Prasad et al., 2008 also re-ported anticancer property of Bacopa monnieri (L.) Wettst3516. Thehigh steroid saponins levels in Tribulus terrestris and the data ontheir cytotoxic activity towards a number of tumor cell lines presumesa high antitumor potential of this medicinal plant17. In popular medi-cine, Nelumbium nucifera is used in the treatment of tissue inflamma-tion, cancer, skin diseases, leprosy and as a poison antidote18.Asparagus racemosus is well known for its anti tumor activity. In ourstudy, we have compared the extracts from these four medicinal plantsand tested its anti-cancer activity.

The occurrence and development of tumours is closely related to cellapoptosis. The potential to induce apoptosis has therefore becomean important topic in the study of antitumor drugs19. To prove thedata further, apoptosis and cell cycle analysis experiments werecarried out. Apoptotic data indicated that NNE has maximum apoptoticeffect on cancer cells. The genus Asparagus is rich source ofsaponins having apoptotic activity against cancerous cells but in



1779-1784

present investigation, Asparagus extract, STE has shown no apoptoticeffect on cancer cells. Extracts from Bacopa monnieri was able toinduce apoptosis as shown in Figure 2. Sivakrishna et al., 2005 iso-lated two triterpenoid glycosides along with 10 known saponins fromBacopa monnieri and delineated the chemical compositions ofBacosides A and B20. Ji et al., 2012 showed saponin extracts fromAsparagus racemosus inducing apoptosis on HepG2 cells21. We, how-ever, could not observe an inhibiting or apoptotic effect of the extractof Asparagus racemosus in our experimental model. This may de-pend on the method of extraction and different compounds that havebeen isolated with the extraction method used. Cell cycle was analysedusing propidium iodide method. Cell cycle analysis results correlatewell with apoptosis data. When comparing untreated and treatedcells, we observed a remarkable increase from 6.61 in S-phase of un-treated cells. Further studies are being carried out in our laboratory topurify and determine the effect of different groups present in thesecrude extracts.

CONCLUSIONIn this work, extracts of four different Indian medicinal plants werescreened for their anti-cancer and cytotoxic properties. The findingsof the present investigation demonstrate that the ethanolic extractscaused growth inhibition irrespective of the origin of the cell, how-ever we could observe high cytotoxicity (IC50 = 75µg/ml) in cancercells. Screening of the extracts revealed that though all the ethanolicextracts had shown cytotoxicity, BME and NNE could induce apoptosisin cancer cells. Also, these extracts have shown difference in phasesof cell cycle using Fluorescence activated cell sorter (FACS). Thepresent study indicates the need of phyto-extract screening for can-cer therapeutics from Indian medicinal plants.

ACKNOWLEDGEMENTThis work was supported by Research Initiation Grant, BITS Pilani,Hyderabad Campus. The authors would like to thank Dr. RajkumarBanerjee, Principal Scientist, Lipid Science and TechnologyDivision, Indian Institute of Chemical Technology (IICT), Hyderabadfor his immense support and suggestions during the work. Theauthors are grateful to Biomaterials group, IICT Hyderabad forgranting permission to use their laboratory facilities.

REFERENCES1. Ncube NS, Afolayan AJ, Okoh AI, Assessment techniques

of antimicrobial properties of natural compounds of plantorigin: current methods and future trends, African Journalof Biotechnology, 2008, 7 (12), 1797-1806.

2. Tiwari P, Kumar B, Kaur M, Kaur G, Kaur H, Review ofphytochemical screening and extraction process,

Internationale Pharmaceutica Sciencia, 2011, 1(1), 25-30.3. Faodun A, Herbal medicines in Africa-Distribution,

standardization and prospects, Research Journal ofPhytochemistry, 2010, 4, 154-161.

4. Pazhanisamy M, Ebenezer GAI, Phytochemical screening ofOrmocarpum cochinchinense Leaf extracts, Journal ofAcademia and Industrial Research, 2013, 2(5), 275-278.

5. Yu J, Ma Y, Drisko J, Chen Q, Antitumor Activitiesof Rauwolfia vomitoria extract and potentiation ofcarboplatin effects against ovarian cancer, CurrentTherapeutic Research, 2013, 75, 8-14.

6. Newman DJ, Cragg GM, Snader KM, Natural products assources of new drugs over the period 1981–2002, Journal ofNatural Products, 2003, 66, 1022–1037.

7. Debernardis D, Cimoli G, Parodi S, Russo P, Interactionsbetween taxol and camptothecin, Anticancer Drugs, 1996,7(5), 531-534.

8. Pezzuto JM, Plant-derived anticancer agents, Biochemicalpharmacology, 1997, 53(2), 121-133.

9. Wang L, Weller CL, Recent advances in extraction ofnutraceuticals from plants, Trends in Food Science &Technology, 2012, 17, 300-312.

10. Somchit MN, Reezal I, Elysha IN, Mutalib AR, In vitroantimicrobial activity of ethanol and water extracts of Cassiaalata, Journal of Ethnopharmacology, 2006, 84, 1-4.

11. Mosmann T, Rapid colorimetric assay for cellular growthand survival: application to proliferation and cytotoxicityassays, Journal of Immunological Methods, 1983, 65 (1–2),55–63.

12. George S, Bhalerao S V, Lidstone E A, Ahmad I S, Abbasi A,Cunningham B T, Watkin K L, Cytotoxicity screening ofBangladeshi medicinal plant extracts on pancreatic cancercells, BMC Complementary and Alternative Medicine, 2010,10,52-62.

13. Bachran C, Bachran S, Sutherland M, Bachran D, Fuchs H,Saponins in tumor therapy, Mini Reviews in MedicinalChemistry, 2008, 8, 575–584.

14. Podolak I, Galanty A, Sobolewska D, Saponins as cytotoxicagents: a review, Phytochemistry Reviews, 2010, 9, 425-474.

15. Patil A, Vadera K, Patil D, Phatak A, Juvekar A, Chandra N, Invitro anticancer activity and phytochemical analysis ofBacopa monnieri (L.) Wettst, International journal ofPharmaceutical Sciences and Research, 2014, 5(10), 4432-4438.

16. Prasad R, Bagade US, Pushpangandan P, Varma A, Bacopamonniera L.: Pharmacological aspects and case studyinvolving Piriformospora indica, International Journal ofIntegrative Biology, 2008, 3(2), 100-110.



1779-1784

17. Hu K, Yao X, Methyl protogracillin (NSC-698792): thespectrum of cytotoxicity against 60 human cancer cell linesin the National Cancer Institute’s anticancer drug screenpanel, Anticancer Drugs, 2001, 12(6), 541-547.

18. Liu CP, Tsai WJ, Lin YL, Liao JF, Chen CF, Kuo YC, (2004)The extracts from Nelumbo Nucifera suppress cell cycleprogression, cytokine genes expression, and cellproliferation in human peripheral blood mononuclear cells,Life Sciences, 2004, 75(6), 699-716.

19. Krysko DV, Vanden Berghe T, D’Herde K, Vandenabeele P,Apoptosis and necrosis: detection, discrimination andphagocytosis, Methods, 2008, 44, 205–221.

20. Sivaramakrishna C, Rao CV, Trimurtulu G, Vanisree M,Subbaraju GV, Triterpenoid glycosides from Bacopamonnieri, Phytochemistry, 2005, 66, 2719–2728.

21. Ji Y, Ji C, Yue L, Xu H, Saponins isolated from Asparagus induce apoptosis in human hepatoma cell line HepG2through a mitochondrial-mediated pathway, CurrentOncology, 2012, 19(2), eS1-S9.

Source of support: Nil, Conflict of interest: None Declared

comparative analysis of apoptotic activity from extracts...

Documents