Antioxidant and Antitumor Activity of Phyllanthus Emblica in Colon Cancer Cell Lines
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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 Phyllanthus 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 plant 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 Tannin (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, flavonoids, and protective activities of the various tannins 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 : Flowering plant targeting specific key transcription factors Class : Magnoliopsida like nuclear factor kappa B (NF-kappaB), Order : Malpighiales 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 polyphenols, garlic, onion, soybeans as well as minerals and is regarded as one of the medicinal plants. 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 191 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