ABSTRACT- The Human papillomavirus (HPV) is the single etiological factor in cervical cancer, contributing to neoplastic progression through the action of viral oncoproteins, mainly E6 and E7.Cervical cancer remains the second most common cancer in women worldwide with India as a major contributor to global burden with an annual incidence of 132,000 new cases and mortality rate of 74,000 deaths annually. In this study turmeric, neem, tulasi and ginger were selected as natural anticancer drugs. The objective of the study was to analyze the anticancer property of turmeric (Curcuma longa), neem (Azadirachta indica), tulasi (Occimum sanctum) and ginger (Zingiber officinale) on HeLa cells. Turmeric, neem, tulasi and ginger capsules (Himalaya’s Company) were used and aqueous and methanolic extracts of the turmeric, neem, tulasi and ginger were obtained using a soxhlet extraction. To check the efficacy of these drug MTT assay was performed, that determines % viability and/or cytotoxicity. IC50 of aqueous turmeric, neem, tulasi and ginger extracts in case of HeLa cells were 17.8, 22, 79.4, 27.86 respectively and in case of methanolic turmeric, neem, tulasi and ginger extracts 17, 7.35, 75.24 and 16.1 respectively. To confirm apoptosis as the sole reason behind cell death immunofluorescence based apoptosis assay was performed using TALI image based cytometer. The study has led to postulate hypothesis that natural drugs e.g. turmeric, neem, tulasi and ginger are potent anti-cancer compound that are capable of inhibiting the growth of immortal cells by apoptosis.
Key-words- Cervical cancer, Human papillomavirus (HPV), Oncoproteins E6 and E7, Natural compounds, HeLa cell line (adherent), Cell viability and MTT assay, Apoptosis assay

INTRODUCTION
Human papillomavirus (HPV) infection is found in more than 95% of cervical cancer. In India, cervical cancer is a leading cancer among women with annual incidence of about 130,000 cases and 70-75,000 deaths ^[1]. Expression of HPV E6 and E7 oncogenes are one of the high risk factor for the initiation and maintenance of cervical cancer. Its deregulated expression leads to disruption of normal cell cycle regulation, abrogation of apoptosis and genetic instability.
Furthermore, repression of E6 and/or E7 may induce cancer cells to undergo apoptosis or senescence ^[2-3]. In recent years, natural dietary agents have drawn a great deal of attention because of their potential ability to suppress cancers as well as reduce the risk of cancer development. There are more than one thousand species that have been found to possess significant anticancer properties ^[4-5]. The tremendous ability of natural products to act as effective scaffolds and bind bewildering types of protein domains and folding motifs makes them effective modulators of various cellular processes, contributing to immune responses, signal transduction, cell division and apoptosis ^[6-7]. The cytotoxic activity of natural compounds to cervical cancer cells in a concentration and time dependent manner were selectively more in HPV16 and HPV18 infected cells compared to non-HPV infected cells [11]. Natural compounds analogs have effective binding with different active sites on HPV16 E6 protein, ideal target for restoring the tumor suppressor function of p53 and thus allowing the apoptosis of infected cells ^[8-9].Therefore, proposed study aim to identify potential natural compounds and their derivatives targeting HPV16 & 18 on cervical cancer cell line (HeLa) using MTT cell proliferation assay. ^[10-12]

MATERIALS AND METHODS
Culturing Cell line and Media Conditions: The human cervical cancer cell lines (HeLa) were obtained from National Centre for Cell Sciences, Pune (NCCS), India. The cells were maintained in DMEM growth media with HEPES, 10% FBS, Non-essential amino acids, Sodium bi-carbonate and antibiotic cocktail (Penicillin, Streptomycin, Amphotericin-B (HIMEDIA Laboratories Pvt. Ltd.) in a humidified atmosphere of CO2 incubator at 37şC and 5% CO2 with 95% air. Cultured cells were replaced with fresh media after 2-3 days. When cells were reaching up to 90% confluences considered for proliferation assay. Cell viability was assessed by trypan blue dye exclusion assay. Percent viability was calculated by following formula:

               Cell viability assay = %viability = (viable cells / total cells) * 100

Extraction of Phytochemicals: Himalaya’s company capsules were used for selected natural compound’s material. About 10gm of the powdered sample of each compound were weighed into 150 ml of aqueous extract (AE) and methanol extract (ME) in a soxhlet apparatus separately. Vaporization of methanol and water were allowed until the extract appeared from color to colorless and the above process was repeated for several times, until the sufficient amount of extract is produced. Extracted samples were kept in to hot air oven overnight at 60°C and100°C, so that the methanol and water gets evaporated. The concentrated extract of each plant was stored at 4°C until when required for use.Yield of extraction was calculated using following formula:

                % Yield of extraction = Weight of final dried extract x 100 / Weight of initial powder taken for extraction

Table 1: Method and indication for phytochemicals- Phytochemical analysis were carried out in natural compounds to determine the presence or/and absence of alkaloids, flavonoids, glycosides, phenol, saponin and tannin.

S. No.	Test	Method	Indication
1.	Alkaloid	1gm of powder + 1.5% HCl + Few drops of Wagner"s Reagent	Brown precipitate indicated presence of Alkaloid
2.	Flavanoid	2gm of powder + 5ml of dilute ammonia solution + few ml of concentrated Sulfuric acid solution	Yellow color indicated presence of flavanoid
3.	Glycoside	1gm of powder + 1ml of Distilled Water + few ml of 1N Sodium Hydroxide solution	Yellow color indicated presence of Glycoside
4.	Phenol	2gm of powder + 2ml Distilled water + Few drops of 10% Ferric Chloride solution	Blue/Green Color indicated presence of phenol
5.	Saponin	1gm of powder + 5ml Distilled Water	Honey comb like froth indicated presence of saponin
6.	Tannin	Few gm of powder + few drops of 5% Ferric Chloride solution	Bluish black color indicated presence of tannin

Preparation of AE and ME Sample for MTT Assay: ME and AE were dissolved in methanol and PBS solution respectively to get a concentration of 1mg/10µl. From this, dilutions were prepared to obtain final drug concentration ranging from100µg to100ng.

MTT Assay Procedure: This colorimetric assay is based on the capacity of mitochondria succinate dehydrogenase enzymes in living cells to reduce the yellow water soluble substrate 3-(4, 5-dimethylthiazolyl) - 2, 5 - diphenyl- tetrazolium bromide (MTT) into an insoluble purple colored formazon product which is measured spectrophotometrically. This formazon production is directly proportional to the viable cell number and inversely proportional to the degree of cytotoxicity. HeLa cells were seeded in 96-well plates at a density of 1 × 104 cells/well in DMEM. After 24 hours of seeding at 37°C and 5% CO2 condition. Stock solutions of 1mg/10µl were prepared from each and every extracted sample. Samples were further diluted in a range of 100µg to 100ng. Aqueous and methanolic extracts were added in increasing concentration in the wells. After incubation, 20µl of MTT reagent was added into each well. Incubated for 4 hrs followed by monitoring using inverted microscope. Solubilizing reagent was added to solubilize the formazon crystals (Hi-Media kit based procedure). The resulting MTT products were determined by measuring the absorbance at 590nm using ELISA reader.IC50 values were calculated as the concentrations that show 50% inhibition of HeLa cells proliferation and it was calculated using following equation:
y = x²+x+1 where, y = % viability and x = log [conc.]

Apoptosis Assay Procedure: Exposed cells to drug were assessed for apoptosis and cells not exposed to test drug were considered as control.1X Annexin binding buffer was prepared by diluting the Component C in deionized water. Cells were re-suspended in 1XAnnexin binding buffer, so that there was at least 100µL of cells per individual assay at a concentration of approximately 5×105 to 5×106 cells/ml. To each 100µL of sample, 5µL of Component A was added and mixed well. The cell-annexin V mixture was incubated at room temperature in the dark for 20 minutes. The cells were centrifuged and re-suspended in 100µL of ABB.1µL of Tali® component was added to each 100µL sample and mixed well. Samples were incubated at room temperature in the dark for 1–5 minutes. 25µL of the stained cells were used for counting and further analysis (Invitrogen kit based).

RESULTS
Turmeric, neem, tulasi, ginger shows that they contain Alkaloid, Flavonoid, Phenolic, Tannin, Saponin and Glycoside.

Table-2: Phytochemical analysis of Turmeric, Neem, tulasi, ginger

S. No.	Phytochemicals	Turmeric	Neem	Tulasi	Ginger
1.	Alkaloid	+	+	+	+
2.	Flavonoid	+	+	+	+
3.	Tannins	-	+	+	-
4.	Saponins	-	+	+	-
5.	Phenolic	+	+	+	+
6.	Glycoside	-	+	+	+

Table-3: Dry weight (yields) of Aqueous and Methanolic Turmeric, Neem, Tulasi and Ginger extract

Sr. No.	Extracted sample	Final wt. of the aqueous extracted sample (gms)	Final wt. of the methanolic extracted sample (gms)
1	Turmeric	0.15	0.46
2	Neem	1.37	0.69
3	Tulasi	0.45	0.97
4	Ginger	0.43	0.52

Table-4: IC50 value of Turmeric, Neem, Tulasi and Ginger in case of HeLa cells

\

S. No.	Compound	Extraction	IC50 value of HeLa cells
1	Curcumin	Methanolic	17
		Aqueous	17.8
2	Ginger	Methanolic	16.01
		Aqueous	27.86
3	Neem	Methanolic	7.35
		Aqueous	22
4	Tulasi	Methanolic	75.24
		Aqueous	79.4

IC50 value of aqeous and methanolic turmeric, neem, tulasi and ginger extract on HeLa cells were as above. Different extracted drug concentrations were used and found decreasing cell viability with increasing drug concentration. Also curcumin, neem and tulasi and ginger aqueous extract show higher cytotoxicity value and among four compounds neem gave lower cytotoxicity value and higher viable cell counts.

Table-5: Cytotoxicity on HeLa cells by Aqueous extract of Turmeric

S.No.	Conc. (ug)	%Viablity	%Cytoxicity
1	Cell control	100	0
2	0.1	86	14
3	1	79	21
4	5	66	34
5	10	54	46
6	25	46	54
7	50	35	65
8	100	25	75

Figure 1: Cytotoxicity on HeLa cells by aqueous extract of turmeric

The cytotoxicity of aqueous turmeric extract on HeLa cells was maximum at a drug concentration of 100µg/ml i.e. 75% and lowest viability value found was 25% at 100µg/ml of drug concentration. Also obtained IC50 value was 17.8 µg /ml.

Apoptosis Assay Indication- Apoptosis assay for selected natural compounds on HeLa cells were as following. Green fluorescence determines % apoptosis.

For Turmeric: 1.44 x 10⁷cells/ml out of 1.45 x 10⁷cells/ml show green fluorescence (apoptosis) and 1.31 x 10⁵ cells/ml show no green fluorescence (no apoptosis). So the resulting output data indicates that, 99% of cell death was due to apoptosis.

For Neem: 2.68 x10⁶ cells/ml out of 2.68x10⁶ cells/ml show green fluorescence (apoptosis). So the resulting output data indicates that, 100% of cell death was due to apoptosis.

For Tulasi: 2.92 x10⁶ cells/ml out of 2.92x10⁶ cells/ml show green fluorescence (apoptosis). So the resulting output data indicates that, 100% of cell death was due to apoptosis.

For Ginger: >1.5 x10⁷ cells/ml out of, >1.5x 10⁷ cells/ml show green fluorescence (apoptosis). So the resulting output data indicates that, 100% of cell death was due to apoptosis.



Figure 2: Tali image based flow cytometer revealed 99% cell death due to apoptosis in case of turmeric

Immuno fluorescence Study for Validation/Confirmation: Apoptosis assay was carried out by Annexin-V and PI staining Cells exposed to 45mM concentration were stained using FITC labeled anti-Annexin V Ab and propidium iodide to stain nuclei. The images were captured using specific filters and in phase contrast mode for total cellularity estimation. Then the capture images were superimposed to confirm the apoptosis and necrotic cell death. Cells stained in green only shows apoptotic cell death where as cells stained with only red nuclei suggestive of necrotic cells. Cells stained with both green and red shows early apoptotic cells. However, unstained cells are live and with intact membrane.



Figure 3: Apoptic assay showed Annexin-v and PI stained cells of HeLa

DISCUSSION
HPV infection is closely associated with the development of more than 95% of cervical cancer. Natural compounds are gaining interest as potential cancer therapeutics including for the treatment of cervical cancer. The present need is to develop drugs that can be potentially target cancer cells by means of their inherent difference to normal cells [13]. The present study demonstrated the promising cytotoxic and anticancer activities of the aqueous and methanolic extract of turmeric, neem, tulasi and ginger against HeLa cell lines. Different extracts of the plant exhibited different activity on different cell lines. This selectivity could be due to the sensitivity of the cell line to the active compounds in the extract or to tissue specific response. Phytochemicals present in plants protect the cells from oxidative damage and are responsible for the death of cells [14-15]. In this study results of phytochemical analysis shows turmeric, neem, tulasi and ginger contain alkaloids, flavonoids, and glycosides, saponin, tannin and phenolics ^[16]. Different in vitro cytotoxicity assays with different endpoints have been employed for screening of potential natural compounds extract preparation for their anticancer activities. The most commonly used assays involve the use of dye stains that include MTT. This dye has some indicator properties allowing them to reveal ongoing cellular processes, providing indirect measure of mitochondria function ^[17-19].
In this study aqueous and methanolic extracts of turmeric, neem, tulasi and ginger were screened for its cytotoxicity at different concentrations to determine the IC50 value. A chart was plotted using the % cell viability in Y-axis and concentration of natural compounds extracts in X-axis. IC50 of aqueous turmeric, neem, tulasi and ginger extracts for HeLa cells were 17.8,22,79.4,27.86 respectively and in case of methanolic turmeric, neem, tulasi and ginger extracts 17,7.35,75.24 and 16.1 respectively. Also comparing our results with others data it is clearly indicate that curcumin and other natural products can be cytotoxic to cervical cancer cells in a concentration-dependent and time-dependent manner and It also induced apoptosis in cervical cancer cells ^[20-21]. The cytotoxic activity was selectively more in HPV16 and HPV18 infected cells compared to non-HPV infected cells ^[22-23]. Apoptosis assay was performed to confirm the cause of death of the cancer cells. In this study apoptosis assay result obtained 99% death of cells was due to apoptosis and remaining 1% of death might have been due to necrosis, or any other cell inhibiting factor. In present study 99% cell death in case of turmeric and 100% cell death obtained in case of neem, tulasi and ginger. The study has led to postulate hypothesis that turmeric, neem, tulasi and ginger are potent anti-cancer compound that are capable of inhibiting the growth of immortal HeLa cells by apoptosis ^[24-25].

CONCLUSIONS
In our study, we deeply believe that the cross killing occurred due to cytotoxic activity against the Cervical cancer- HeLa cell line. The results of the present study demonstrated the potent cytotoxic activity of the aqueous and methanolic extracts of turmeric, neem, tulasi and ginger and phytochemical constituents are the major com ponents which are responsible for the potential cytotoxic activity. Even though there was increase in the cell growth inhibition or cell viability decreased when concentration of drug extract was increased. To confirm apoptosis as the sole reason behind cell death immune fluorescence based apoptosis assay was performed using TALI image based cytometer. Further research also need for proving with other cancer models and human beings with also isolating the active principle of natural compounds. The anticancer property of selected natural compounds will provide useful information in the possible application in the prevention and treatment of cervical cancer.

REFERENCES

1. Arends M J et al, Aetiology, pathogenesis, and pathology of cervical neoplasia, 96 ClinPathol, 1998, 96-103.
2. Hengstermann A et al, Growth suppression induced by down regulation of E6-AP expression in human papilloma virus-positive cancer cell lines depends on p53, Journal of virology, 79, 2005, 9296–9300.
3. Al-Hazzaniand A, Alshatwi A, Catechin hydrate inhibits proliferation and mediates apoptosis of SiHa human cervical cancer cells, Food and chemical toxicology 49, 2011, 3281–3286.
4. Lin CL and Lin JK, Curcumin: a potential cancer chemo preventive agent through suppressing NF-?B Signaling, Journal of cancer molecules, 4(1), 2008, 11-16.
5. Bharti AC et al, Anti-human papilloma virus therapeutics: Facts & future, Indian J Med Res, 130, 2009, 296-310.
6. Patel JB and Patel PM, anticancer and cytotoxic potential of Triticum aestivum extract on HeLa cell line, International research journal of pharmacy, 2013, 103-105.
7. Senapathy GJ et al, The present scenario of cervical cancer control and HPV epidemiology in India: an outline, Asian pacific J Cancer Prev, 2012, 1107-1115.
8. Chowdhury MK et al, Leaf extracts of Azadirachta indica and Terminalia arjuna induce death of HeLa cells without DNA degradation, Dhaka univ. J. Pharm. Sci., 8(1), 2009, 75-79.
9. Shaikh F et al, Molecular screening of compounds to the predicted Protein protein interaction site of Rb1-E7 with p53-E6 in HPV, ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation, 8(13), 2012, 607-612.
10. Rahbari R et al., A novel L1 retrotransposon marker for HeLa cell line identification., Bio Techniques, 46 (4), 2009, 277–284.
11. Miguel LL, Anticancer and carcinogenic properties of curcumin: considerations for its clinical development as a cancer chemo-preventive and chemotherapeutic agent, Mol. Nutr. food res. 52, 2008, 103–127.
12. Sakarkar DM and Deshmukh VN., Ethno-pharmacological review of traditional medicinal plants for anticancer activity, International journal of Pharmtech research, 3, 1, 2011, 298-308
13. Kowti R et al, Antimicrobial activity of ethanol extract of leaf and flower of Spathodea campanulata P. Beauv, , 1(3), 2010, 691-698.
14. Castellsagu´e X et al., 2006, Worldwide human papillomavirus etiology of cervical adenocarcinoma and its cofactors: implications for screening and prevention. J Natl Cancer Inst; 98: 303–15.
15. Paavonen J, 2007, Human papillomavirus infection and the development of cervical cancer and related genital neoplasias, International journal of infectious diseases, 11, (Supplement 2), 3-9.
16. Shahedur Ret al., 2011, In-vitro antioxidant and anticancer activity of young Zingiber officinale against human breast carcinoma cell lines, BMC complementary and alternative Medicine, 11:76,1-7.
17. Yarden Y and Cald C., 2013, Basic cancer research is essential for the success of personalized medicine, European journal of cancer 49, 2619–2620.
18. Hingorani R et al, 2011, Detection of apoptosis using the BD Annexin V FITC Assay on the BD FACSVerse^™ System, BD Biosciences, 1-12.
19. Walters et al., Cytotoxic effects of curcumin on osteo sarcoma cell lines. Invest new drugs 26(4), 2008, 289-297.
20. Umashanke M and Srivastava S, Traditional indian herbal medicine used as antipyretic, antiulcer, anti-diabetic and anticancer: A review, International journal of research in pharmacy and chemistry, 1(4), 2011, 1152-1159.
21. Ekunwe et al., Potential cancer-fighting Ocimum gratissimum (OG) leaf extracts: increased anti-proliferation activity of partially purified fractions and their spectral fingerprints. Ethn Dis, 20 (1 Suppl 1), 2010, 1-12
22. Divya C and Pillai M, 2006, Anti-tumor action of curcumin in human papillomavirus associated cells involves down regulation of viral oncogenes, prevention of NFkB and AP-1translocation, and modulation of apoptosis. Mol Carcinog, 45(5): 320-332.
23. Jha ., Reversal of hyper methylation and reactivation of the RARbeta2 gene by natural compounds in cervical cancer cell lines, Folia Biol (Praha) 56(5): 2010, 195-200.
24. Joseph B and Nair VM, 2013, Ocimum Sanctum Linn. (Holy Basil): Pharmacology behind its anti-cancerous effect, Int. J Pharm Bio Sci; 4(2):556 – 575.
25. Patel J and Patel P, 2013, anticancer and cytotoxic potential of Triticum aestivum extract on HeLa cell line, International research journal of pharmacy, 103-105.
    
    

    International Journal of Life-Sciences Scientific Research (IJLSSR) Open Access Policy Authors/Contributors are responsible for originality, contents, correct references, and ethical issues. IJLSSR publishes all articles under Creative Commons Attribution- Non-Commercial 4.0 International License (CC BY-NC). https://creativecommons.org/licenses/by-nc/4.0/legalcode