Int.J.Curr.Microbiol.App.Sci (2013) 2(5): 189-195

ISSN: 2319-7706 Volume 2 Number 5 (2013) pp. 189-195 http://www.ijcmas.com

Original Research Article Antioxidant and Antitumor activity of emblica in colon cancer cell lines

D. Sumalatha*

Department of Biotechnology, Valliammal College for women, Chennai, Tamil Nadu, India. *Corresponding author e-mail: [email protected]

A B S T R A C T

The present study was designed to investigate the antioxidant and antitumor activity, of Phyllanthus emblica (fruit). Antioxidant potential of the edible was evaluated invitro by DPPH (1, 1 diphenyl 2 picrylhydrazyl) K e y w o r d s scavenging assay and FRAP assay method. The radical scavenging activity Phyto- of the extract was measured as decolourising activity followed by the chemicals; trapping of the unpaired electron of DPPH. The percentage decrease of antioxidant; DPPH standard solution was recorded 71.75% for Phyllanthus emblica cytotoxicity Phytochemical analysis revealed the presence of major phytocompounds assay; like alkaloids, flavanoids, protprocesseseins, sap oinvolvingnins and redoxTanni n enzymess. The c y andtoto xic Phyllanthus effect was determined against tbioenergeticshe cancer ce l l selectron lines H T - 2 transfer9 using t hande M T T emblica assay. In conclusion Phyllanthus emblica possess more potential cytotoxic activity against HT-29 cells lines .The result indicated that this plant extract could be an important dietary source with antioxidant & anticancer activities.

Introduction

Cancer is the abnormal growth of cells in exposure to a plethora of exogenous our bodies that can lead to death. Cancer chemicals (Rajkumar et al., 2011). cells usually invade and destroy normal Antioxidant may mediate their effect by cells. These cells are born due to directly reacting with ROS, quenching imbalance in the body and by correcting them or chelating the catalytic metal ions this imbalance , the cancer may be treated. (Sun et al., 2002). Antioxidants rich diets Free radical, one of the major cause for the can reduce oxidative damage to DNA, conversion of normal cell to cancerous thus preventing a critical step in the onset cells,are generated as a consequences of a of carcinogenesis and the impact of number of endogenous metabolic antioxidants on mutagenesis and

189 Int.J.Curr.Microbiol.App.Sci (2013) 2(5): 189-195 carcinogenesis has been well established diet has an important role in the etiology (Zhang et al.,2008;Meyskens et al., 2005). of colon cancer ,dietary chemoprevention Chemoprevention, a novel approach for received attention for colon cancer controlling cancer, involves the use of prevention. specific natural product or synthetic chemical agents to reverse, suppress or Phyllanthus emblica commonly known prevent premalignancy before the as gooseberry or aamla is a deciduous tree development of invasive cancer. Studies of phyllantaceae family. It has undergone on a wide spectrum of plant secondary preliminary research demonstrating in metabolites extractable as natural products vitro antiviral,antimicrobial (Saeed et al., from fruits, vegetables, teas, spices, and 2007) and anticancer activity (Ngam et traditional medicinal herbs show that these al., 2010). Phyllanthus emblica has high plant natural products can act as potent level storage of polysaccharides,total anti-inflammatory, antioxidant or protein and calcium(Suriyavathana and anticancer agents. Subha, 2011) It is an oxidant with free radical scavenging properties due to the The recent advances in genomics and presence of high level of superoxide metabolomics have enabled biologists to dismutage. It is rich in (gallicaid, better investigate the potential use of elagic acid, phyllemblis acid, emblicol) immunomodulatory natural products for (Anila et al., 2003). A wide range of treatment or control of various cancerous phytochemical components including diseases. The cancer preventive or terpenoids, alkaloids, , and protective activities of the various have been shown to posses useful immunomodulatory natural products lie in biological activities. their effects on cellular defenses including detoxifying and antioxidant enzyme Plant anatomy systems, and the induction of anti- inflammatory and antitumor or Kingdom : Plantae antimetastasis responses, often by Division : targeting specific key transcription factors Class : Magnoliopsida like nuclear factor kappa B (NF-kappaB), Order : activator protein (AP-1), signal Family : Phyllanthacae transducers and activators of transcription Tribe : Phyllantheae (STAT) and others (Arvindaram et al., 2 Subtribe : Fluegginae 010). Nutritive value There are numerous reports on cancer chemopreventive activity of dietary Amla is well known for its nutritional botanicals, includes cabbage, broccoli, qualities. It is rich in , garlic, onion, soybeans as well as minerals and is regarded as one of the medicinal . Several lead compounds, richest source of vitamin C (200 900 mg such as genistein (from soybeans), per 100 g of edible portion) (Jain et al., lycopene (from tomatoes) brassinin (from 2000; Bharthakur et al., 1993; Gopalan et cruciferous vegetables) sulforaphane (from al., 1991). Major components of asparagus) are in preclinical or clinical nutritional importance are reported in table trials for cancer chemoprevention since 1.

190 Int.J.Curr.Microbiol.App.Sci (2013) 2(5): 189-195

Table. 1 Nutritional Value of fruit of Phytochemical Screening Phyllanthus emblica (% or per 100g). The extracts was subjected to qualitative chemical investigation for the Chemical components Percentage identification of different phyto constituents like steroids, tannins, Fruits: Moisture 81.2% flavanoids Alkaloids, Saponins, and Protein 0.5% Anthraquinones (Prashant Tiwari et al., Fat 0.1% 2011). Mineral matter 0.7% Fibre 3.4% In- vitro Antioxidant Assay Carbohydrate 14.1% Bulk elements Mg/100g Net weight DPPH Radical Assay Calcium 0.05% Phosphorous 0.02 The ability of the extracts to Iron 1.2 mg/100g scavenge DPPH free radicals was Vitamin C 600mg/100g determined by the method (Gyamfi et al., 1999) with minor modification. The Nicotinic acid 0.2mg/100g controls contained all the reaction reagents

except the extract or positive control substance. Different concentration of the Materials and Methods tested sample was placed in a cuvette and 0.5ml of 100mm methanolic solution of Sample collection DPPH radical was added. Mixtures were vigorously shaken and left 30 min in the The samples of Phyllanthus emblica (fruit) dark at ambient temperature. The were collected in Chennai, Tamilnadu absorbance was then measured at 517nm. (India). The fruit and leaves were dried. Inhibition of DPPH radical was calculated as follows Preparation of Extract DPPH radical % = (1- Absorbance of test The plant material was shade dried with / Absorbance of control)/ X 100 occasional shifting and the powered with a mechanical grinder, passing through sieve FRAP assay and stored in a tight container. Then 25gms of air dried powder of plant were FRAP reagents was freshly prepared by continuously refluxed with ethanol at 45 C mixing 25ml acetate for 3hrs using soxhlet apparatus. The buffer(300mM,pH3.6),2.5ml 2,4,6-tris(2- mixtures were filtered. The filtrates were pyridyl)-S-triazine (TPTZ) solution(10mM evaporated using vacuum rotary TPTZ in 40mM/L HCL) and 2.5ml evaporator and air dried at 40°C. The FeCl3(20mM) water solution. Each stock solution of crude ethanolic extract sample (150µl) (0.5mg/ml) of dissolved in were prepared by diluting the dried methanol was added with 4.5ml of freshly extracts with 0.25% dimethyl sulphoxide prepared FRAP reagent & stirred and after (DMSO) solution to obtain a final 5 min, absorbance was measured at 593 concentration of 100mg/ml. nm using FRAP working solution as blank

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Int.J.Curr.Microbiol.App.Sci (2013) 2(5): 189-195

(Szollosi and Szollosi Varga, 2002).The incubated for another 4h. The supernatant relative activity of the sample was was then removed & replaced with 500µl compared with Ascorbic acid. FRAP value of DMSO to dissolve the resulting MTT was calculated using the formula formazan crystals followed by mixing and measuring the absorbance using FRAP value of sample (µM) = change in spectrophotometer at 590nm. absorbance of sample from 0-4 mins / / change in Absorbance of standard from 0- The cell viability % = 0.D of the sample/ 4 min X FRAP value of std 0 D of control / X 100 (Mossmann, 1983). (100mM) To determination the % of cytotoxicity, graphs were plotted with the % of cytotoxities against their respective In -vitro cytotoxicity assay concentration.

Cell culture and experiment design Results and Discussion

HT-29 cells are obtained from king s Preliminary phyto chemical screening Institute, Chennai. The cells were grown and maintained in a humified incubator at In recent years attention has been focused 37*C under 5% Co2 atmosphere in MEM on the antioxidant properties of plant (Minimal Essential Media) medium derived dietary constituents of food supplemented with TPVG and 10% Fetal (Gulein, 2006) The results of qualitative calf serum and (100 units/ml penicillin). screening of phytochemical components in For experiments, cells were plated in 48 Phyllanthus emblica, were listed in 4 well plates (at a density of1x10 cells/ ml). Table.2. Phyllanthus emblica showed After 25h incubation to allow cell positive for all the test except steroids attachment, the cells were treated with (Dhale and Mogle, 2011). fresh medium containing different concentration of Phyllanthus emblica , Table.2 Preliminary screening of dissolved in DMSO and incubated for phytochemical compounds in Phyllanthus 48hrs under same condition. Control emblica groups received the same amount of DMSO. Phyllanthus Phytochemicals Cytotoxicity Assay emblica Tannins + The MTT colorimetric assay which is based on the reduction of MTT [3-(4,5- Saponins + dimethyl]-2-thiazolyl)-2,5-diphenyl -2H Flavonoids + tetrazolium bromide] by mitochondrial Alkaloids + dehydrogenase to purple formazan product was used to assess the antiproliferative Proteins + action of, Phyllanthus emblica extracts in Steroid _ human HT-29 cells. At the end of 48hrs incubation, the medium in each plate was Anthraquinone + added by 200µl of MTT solution and

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Determination of Free radical the antioxidant effect of plant products is scavenging activity using DPPH method mainly due to radical scavenging activity of phenolic compounds such as DPPH test which is based on the ability of flavanoids,polyphenols,tannins, and DPPH a stable free radical, to decolourize phenolic terpenes (Rahman and Moon, in the presence of antioxidants, is a direct 2007). and reliable method for determining radical scavenging action. The DPPH radical contains an odd electron, which is FRAP responsible for the absorbance at 515- 517nm and also for a visible deep purple Frap values for investigated extract are colour. when DPPH accepts an electron shown in Figure. 2 .The reducing power of donated by an antioxidant compound, the the extract and the standard increased DPPH is decolorized which can be with the concentration. The reducing quantitatively measured from the changes power of Phyllanthus emblica was more in absorbance. Phyllanthus emblica extract pronounced.The reducing power of a showed high scavenging activity with compound was related to its electron 71.75% inhibition (Yadav et al., 2007) transfer ability and may, therefore serve as (Figure .1). an indicator of its potential antioxidant activity (Sanchez Moreno (2002). Figure.1 Free radical scavenging activity of different concentration of Phyllanthus Figure.2 Antioxidant activity measured by emblica and ascorbic acid by DPPH FRAP assay of Phyllanthus emblica. radicals.

Phyllanthus emblica posses antioxidant Anticancer activity and predominantly responsible for cytoprotective action in Non steriodal As a result of antioxidant activities antiinflammatory drug induced Phyllanthus emblica were selected for ulcer.(Ananya chatterjee et al., 2011). further investigation on anticancer .Additionally it has been determined that activities. The effect of ethanolic extracts on HT-29 cancer cell lines were evaluated

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Int.J.Curr.Microbiol.App.Sci (2013) 2(5): 189-195 by MTT assay. Curves of percentage study supports the anticancer property of viability of treated cells were plotted medicinal plants used in the traditional against extracts concentration. The Indian medicine system and further present study demonstrated the anticancer evaluation of the selected medicinal profile of Phyllanthus emblica effective plants for an effective anticancer drug with against HT-29 cells The effect of minimal side effects. Therefore Phyllanthus emblica,on Ht-29 cells is supplementing a balanced diet with given figure.3. It can be found that the Phyllanthus emblica fruit may have incubation of cancer cells with beneficial effect. Phyllanthus emblica, reduced the viability of these cells and the dead cells were significantly increased with high extract concentration. The ethanolic extract of References Phyllanthus emblica exhibited high cytotoxicity (88% ) (Syam et al ., 2011) . Ananya chatterjee., Subrata chattopadhyay Phyllanthus emblica showed the highest and Sandip K. Bandyopadhyay. 2011. activity and this was observed in the Biphasic effect of Phyllanthus emblica earlier work with the HepG2 cells (Syam Extract on NSAID- Induced ulcer: An et al ., 2011). Even at very low Antioxidant rail weaved with concentration Phyllanthus emblica showed Immunomodulaory effect. (20%) dead cells. In conclusion Complem.Alter. Medi. 1-13. Phyllanthus emblica possessed strong Anila, L., and Vijayalakshmi.N.R.2003. antioxidant activity and anticancer Antioxidant action of flavanoids from activity. Magnifera indica and Emblica officinalis in hypercholesterolemic Figure. 3 Antitumor activity of rats. Food. Chem. 83:569-574. Phyllanthus emblica on HT-29 colon Aravindaram, K., and Yang, N.S. 2010. cancer cell lines Anti-inflammatory plant natural products for cancer herapy. Planta. Med. 76(11):1103-17. Bharthakur, N.N., and Arnold, N.P. 1991. Chemical analysis of the emblica (Phyllanthus emblica L) and its potential as a good sources. Scientia. Horticult. 47:99-105. Dhale,D.A., and Mogle,U.P.2011. phytochemical screening and antibacterial activity of Phyllanthus emblica. Sci. Res. Report. 1(3):138- 142. Gopalan, C. , B.V.R. Sastri, and Balasubramaniam, S.C. 1991. Nutritive value of Indianfoods. NIN, The results of the present study support the Hyderabad, India. need of further studies to isolate Gulein, I., 2006. Antioxidant and potential anticancer drug with cancer cell- antiradical activities of L-carnitine. specific cytotoxicity. Additionally, the Life. Sci.78:803-811

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