A JOURNAL OF COMPOSITION THEORY ISSN : 0731-6755

Anticancer activity of reticulates ethanolic and chloroform extracts against liver cancer cell lines

M Deivayanai, J Vadivelu*, M K Vadivazhagi Department of Biochemistry, Sri akilandeswari Womens’s College, Wandiwash - 604408 *Corresponds to Dr J Vadivelu [email protected] Abstract Cancer is an alarming disease and quite lethal in nature in developed and developing nations. Many new therapeutic agents and therapies are available in the market but have some severe side effects on human beings’ organs. These therapeutic agents are quite costly and not easily available in some of the developing nations. Various scientific reports have shown that chemoprevention through naturally derived herbal and dietary phytochemicals is an innovative therapeutic tool against different cancer types. These herbal phytochemicals have shown their potential anticancer activity in both in vitro and in vivo studies. Further, many of them have been successfully proved for their chemopreventive property by inducing apoptosis equivalent to certain other chemical drugs without causing any side effects. The combinational role of herbal and dietary phytochemicals has proved to be very effective against cancer prevention. The present chapter summarised the effectiveness of herbal and dietary phytochemicals for chemoprevention and also highlighted their combinational role on various kinds of cancer.

Keywords: Extract, Anticancer, Liver Cancer, Phyllanthus reticulates

Introduction Anticancer Activity of Herbal Medicine Cancer is one the most alarming diseases which is mainly distinguished by irregular and uncontrollable proliferating activities of the cells. According to a report, mil- lions of people are dying from cancers (Jemal et al. 2011). Curcumin is mainly derived from the rhizome part of Curcuma longa which is known to have a polyphenolic nature (bis-α,β-

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unsaturated β-diketone, commonly called diferuloylmethane) and belongs to the Zingiberaceae family. (Kawamori et al. 1999). Genistein is a phytoestrogen in nature which is mainly classified and included under isoflavone group. It was first isolated from Genista tinctoria (also known as dyer’s broom) in 1899. Genistein is chemically known as 5,7-dihydroxy-3-(4- hydroxyphenyl)chromen-4- one It is helpful in supressing and treating cancers including prostate and breast cancers (Lampe et al. 2007). Resveratrol, an antioxidant having polyphenol in nature, is a well- studied phyto- chemical with an abundant potential in health regards due to its inherent properties including antioxidant, anticancer and the like. Studies on resveratrol have eluci- dated that it has the potential of inhibiting cancer progression (Lucie 2000). Epigallocatechin gallate (EGCG), commonly recognised as epigallocatechin-3-gallate, is a derivative of catechin and an ester of epigallocate- chin and gallic acid. EGCG is mostly found in white tea and green tea. The polyphe- nol EGCG possesses a powerful antioxidant activity compared to other polyphenols and helps in chemoprevention of cancers (Lambert and Elias 2010). Carnosol is a polyphenolic diterpene found in herbs such as rosemary (Rosmarinus officinalis) and mountain desert sage (Salvia pachy- phylla) and is being commercially used in food processing. Carnosol also has the potential to reduce risks of cancer. Carnosol was first extracted from mountain desert sage in 1942 (Johnson 2011). Silibinininhibits cancer cells ffectively. This compound is an active constituent of silymarin isolated from milk thistle plant (Silybum marianum) and leaves of artichoke (Cynara scolymus) . Silibinin is also well known for its , strong antioxidant activities (Singh and Agarwal 2009). Vernodalin, Gypenosides, Crocetin, Saffron is a spice and accumulated in the stigma of saffron flower. It is a major food colourant and has significant antitumour activity in different experimental models (Bakshi et al. 2010). The active potential of crocetin anticancer agent, is reported for hepatocellular carcinoma, lung carcinoma, pancreatic cancer cell line, skin carcinoma and several cell lines such as colon, breast and gastric cancer cell lines. (Afshari et al. 2008). In traditional culture medicinal plants are used all over the world and they are becoming increasingly popular in modern society as natural alternatives to synthetic chemicals (Verma et al.,2011). Phyllanthus reticulatus (Family – Euphorbiaceae) commonly known as pancoli or karineli, (Synonym: Kirganelia reticulata Poir.) The plant is widely present in Tropical Africa, Srilanka, South East Asia, China, Malaysia and throughout India, mostly in hedges or waste places. Fruiting and flowering season of plant is from July to March. Leaves of the plant contain tannic acid and gum. Fruit of the plant becomes purplish black when ripe.

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The leaves are used as diabetic and cooling medicine. It is reported to be used as diuretic, alternative, attenuant, astringent and as antidiarrhoel (Kirtikar & Basu,1991, Pullaiah, Nadkarni KM & Nadkarni AK ,2009). :

Domain : Eukaryota

Kingdom: Plantae

Claudus : Angiosperm

Order :

Family : Euphorbiaceae

Tribe : Phyllantheae

Genus : Phyllanthus

Species : P.reticulatus

Pharmacological review of Phyllanthus reticulatus were Antidiabetic activity: Petroleum ether and ethanolic extracts of leaves of the Phyllanthus reticulatus was found to have antidiabetic activity in tribal area. (Kumar et al.,2008, the leaves of Phyllanthus reticulatus were reported to have invitro antiplasmodial activity against chloroquine sensitive (K67) and chloroquine-resistant (ENT36) stains of plasmodium falciparum (Omulokoli et al.,1997).The aqueous extract of aerial parts of Phyllanthus reticulatus was shown to have hypocholesterolemic activity (Maruthappan & Sakthi,2010), The methanolic extract of stem bark of P. reticulatus shows antimicrobial activity against many disease cauinsg pathogens (Begum et al.,2006), Methanolic extract of stem bark of P. reticulatus was reported to have cytotoxic activity on A. salina (Das et al.,2008), Hepatoprotective activity of Phyllanthus

reticulatus was evaluated using ethanolic extract of aerial parts of the plant in CCL4 - induced liver damage (Shruthi et al.,2010), The methanol, chloroform, and hexane extract showed potential in vitro antibacterial activityagainst Staphylococcus aureus and Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhi (Rahmatullah et al.,2010), Antinociceptive and Anti-hyperglycemic activity ( Saha et al.,2007), Analgesic and Anti-inflammatory activity (Maruthappan & Sakthi Shree, 2010), Antioxidant activity (Das et al., 2011). The

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main objective of this study is collection of Phyllanthus reticulatus and prepration the ethaloic and chloroform extract of Phyllanthus reticulatus and finally study the anticancer activity of ethanolic and chloroform extract of Phyllanthus reticulatus Materials and methods Preparation of Phyllanthus reticulatus:

Leaves of Phyllanthus reticulatus were collected from Thiruvannamalai district of Tamilnadu, India during the months of September-December. Fresh leaves were dried & powdered with mechanical grinder and extracted with Ethanol and chloroform successively in Soxhlet apparatus. The concentrated crude extracts were lyophilized in to powder and used for study.

HepG2 cell line (Human Liver cancer cell line) A hardy cell line, HepG2 resists temperature, nutritional and environmental changes without loss of viability. It has supported growth of 10 of 14 arboviruses and measles virus, and it has been used for experimental studies of tumor production in rats, hamsters, mice, embryonated eggs and volunteer terminal cancer patients. Cell Viability test using MTT assay

The assay was performed to check the cell viability of liver cancer cell line (HepG2) in the given samples using MTT assay.

Sample Preparation:

The given sample was filtered using 0.2 micron syringe filter and used directly on cells.

Short Procedure:

The log phase cells (HepG2) were suspended in the complete DMEM media to make a final cell concentration of 2 x 103/ml cells. In a tissue-culture coated 96-well plate, 150 μl of these

cells were seeded and incubated at 37°C overnight supplemented with 5% CO2. After that, when cells were adhered to plate surface, the given samples was supplemented at different concentration to make the final volume of 200 μl. The concentrations used were 0µL, 20 µL, 40 µL, 60 µL, 80 µL and 100 µL. All the concentrations were added as triplicates along with control i.e. media without cells and cells without test samples. After 24h, 20 μl of MTT solution (5 mg/ml in PBS) was added to each well and incubated for 4h at 37°C. Then, 100 μl of DMSO (to solubilize the purple formazon) was added, incubated for 30 min, after which

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the absorbance at 570 nm was recorded with a microplate reader (Thermoscientific Multiskan FC). Growth was expressed as the percentage of cell viability.

% cell viability = Absorbance of sample (Average of triplicates) * 100 / Absorbance of control

Phytochemical analysis

1) Steroids and Terpenoids: 10mg of the extract was dissolved in chloroform. Few drops of

acetic anhydride were added followed by 1 ml of conc Sulphuric acid. Blue colour in

chloroform layer which changes to green shows the presence of steroids, whereas the

appearance of pink colour in chloroform layer shows the presence of terpenoids.

2) Alkaloids: 10mg of the extract was dissolved in conc HCL and filtered. A few drops of

solution are poured into the center of watch glass. Mayer reagent is added along the sides of

the watch glass with the help of a glass rod. Formation of a gelatinous white precipitate at the

junction of two liquid shows the presence of alkaloids.

3) Flavonoids: 10mg of the extract was dissolved in methanol. Magnesium turnings were

added into this followed by conc HCL. A magenta colour shows the presence of Flavonoids.

4) Coumarins: 10mg of the extract is dissolved in methanol and alcoholic KOH was added.

The appearance of yellow colour which decolorizes while adding conc HCL shows the

presence of Coumarin.

5) Saponins: Extract was dissolved in water and shaken well. Froth which last for a long time

shows the presence of saponins

6) Tannins: 10 mg of the extract was boiled with 1 ml water for 30 min. The extract is filtered

clear and to this 0.5 ml 2% gelatin was added. A curdy white precipitate indicates the

presence of tannin.

7) Phenolic compounds: Extract was dissolved in alcohol and 1 drop of neutral ferric chloride

was added to this. The intense colour indicates the presence of phenolic compound.

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8) Anthraquinone: To the extract Magnesium Acetate solution was added the pink colour

developed indicates the presence of Anthraquinone.

9) Quinone: Few mg of the substrate in alcohol is treated with sulphuric acid. The colour

developed indicates the presence of Quinone. 10) Catechin: Few mg of the substrate in

alcohol is treated with a few drops of Ehrlish reagent and a few drops of concentrated Hcl.

The pink colour developed indicates the presence of catechin.

Results and discussion

Figure 1: Phyllanthus reticulatus extraction Table: 1 Phytochemical screening Phyto compounds Ethanolic extract Chloroform extract Phyllanthus reticulatus Steroids + - Terpenoids - + Alkaloids - - Flavanoids - - Coumarins - - Saponnins - - Tannins + + Phenol + + Anthraquinone - - Quinone + +

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Catechin - -

The phyllanthus reticulatus hows many phyocompounds such as steroids, tannins, phenol and quinine in the ethanoic extract and terpinoids, tannins, phenol and quinine in the ethanoic extract in the chloroform extract. Phyllanthus reticulatus: Phyllanthus reticulatus is very common and widespread in India. It often grows in low altitudes. Ethnobotanical survey of these plants reveals anti-diabetic effects, although reliable research required and not yet been performed.

Anticancer activity of Phyllanthus reticulatus Ethanolic extract MTT reduction assay was performed to study mitochondrial/non mitochondrial dehydrogenase activity as a cytotoxic test for a variety of chemical compounds. The effect of extracts on the growth of HepG2 cells was assessed by the MTT assay. The percentage of viability is reducued in the cancer cell line due to the action of plant extract against cell lines. Table 1: Viablity of P. reticulatus ethanolic extract on liver cancer cell line Absorbance % cell viability Control 0.1 100 Standard 0.034 34 10 0.089 89 20 0.074 74 30 0.065 65

40 0.054 54 50 0.041 41

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Figure 2: Viablity of P. reticulatus ethanolic extract on liver cancer cell line The cell viability of the Phyllanthus reticulatus ethanolic extract is decreased while increasing the concentration of extat. The activity was 89 %, 74 %, 65 %, 54%, and 41 %, for the ethanolic extract for the concentration of 10, 20, 30, 40 and 50 respectively. From the present investigation, it is apparent that the phyllanthus reticulatus leaf extracts have the anticancer potential. This is revealed from their anti-proliferative, apoptotic and anti-migratory activity. Our experiments clearly demonstrate that the EENL exert more anti-cancer effect on breast cancer cells than MENL. (Bakshi et al., 2010). Table 2: Viablity of P. reticulatus chloroform extract on liver cancer cell line Absorbance % cell viability

Control 0.1 100

Standard 0.038 38

10 0.079 79

20 0.071 71

30 0.06 60

40 0.049 49

50 0.039 39

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Figure 3: Viablity of P. reticulatus chloroform extract on liver cancer cell line Differential cytotoxic effect of chloroform P reticulatus extract on liver cancer cell lines treated at varying concentrations (10–50 μg/mL), resulting in dose- and time-dependent growth inhibition. Regardless of recent advances in the prevention and detection of cancer and development of newer treatment modalities, cancer still as remains one of the most dreadful diseases due to the limitations of available treatment strategies. Research is under way to identify pharmacologically safe chemopreventive agents that can suppress the carcinogenesis process at various stages along with enhancing the therapeutic effects of conventional cancer therapy by tapping the potential of combinational approaches utilizing one or more synthetic or natural phytochemicals along with an effective drug such as chemotherapy (Sharma et al., 2017).

Conclusion These herbal and dietary phytochemicals derived from various sources showed their anticancer activity either alone or in combination and significantly enhanced the chemopreventive activity against can- cer cells, but these studies are very much limited. Thus, the future researches should be more focused on the combined evaluation of phytoconstituents for a better cure against different kinds of cancers.

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Kawamori T, Lubet R, Steele VE, Kelloff GJ, Kaskey RB, Rao CV, Reddy BS (1999) Chemopreventive effect of curcumin, a naturally occurring anti-inflammatory agent during the promoting/progression stages of colon cancer. Cancer Res 59:597–601 Lampe JW, Nishino Y, Ray RM, Wu C, Li W, Lin MG, Gao DL, Hu Y, Shannon J, Stalsberg H, Porter PL, Frankenfeld CL, Wahala K, Thomas DB (2007) Plasma isoflavones and fibrocystic breast conditions and breast cancer among women in Shanghai, China. Cancer Epidemiol Biomark Prev 16:2579–2586 Lucie F (2000) Biological effects of resveratol. Life Sci 66:663–673 Lambert JD, Elias RJ (2010) The antioxidant and pro-oxidant activities of green tea polyphenols:a role in cancer prevention. Arch Biochem Biophys 501:65–72 Johnson J (2011) Carnosol: a promising anti-cancer and anti-inflammatory agent. Cancer Lett 305:1–7 Singh RP, Agarwal C, Agarwal R (2003) Inositol hexaphosphate inhibits growth, and induces G1 arrest and apoptotic death of prostate carcinoma DU145: modulation of CDKICDK- cyclin and pRb-related protein-E2F complexes. Carcinogenesis 24:555–563 Bakshi H, Sam S, Rozati R (2010) DNA fragmentation and cell cycle arrest: a hallmark of apopto- sis induced by crocin from Kashmiri saffron in a human pancreatic cancer cell line. Asian Pac J Cancer Prev 11:675–679 Afshari JT, Brook A, Mousavi SH (2008) Study of cytotoxic and apoptogenic properties of saffron extract in human cancer cell lines. Food Chem Toxicol 46:3443–3447

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Maruthappan V and Sakthi KS: Effects of Phyllanthus reticulatus on lipid profile and oxidative stress in hypercholesterolemic albino rats. Indian Journal of Pharmacology 2010; 42 (6): 388-391. Begum T, Rahman SM and Rashid AM: Phytochemical and Biological Investigations of Phyllanthus reticulatus. Dhaka University Journal of Pharmaceutical Sciences 2006; 5 (1-2): 21-23. Das KB, Bepary S, Datta KB, Chowdhury Azad AAK, Ali SM and Rouf SS: Hepatoprotective activity of Phyllanthus reticulatus. Pakistan Journal of Pharmaceutical Sciences 2008; 21 (4): 333-337. Shruthi SD, Ramachandra YL, Padmalatha RS and Shettty VA: Antibacterial potential of leaf extracts from Kirganelia reticulate Baill. International Journal of Pharmaceutical Research and Development 2010; 2 (6): 0974-9446. Rahmatullah M, Ghosh KC, Almamun A, Hossain MT and Ahmed S: A pharmacological study on Antinociceptive and Anti-hyperglycemic effects of methanol extract of leaves of Phyllanthus reticulates Poir. In swiss albino mice. Advances in Natural and Applied Sciences 2010; 4 (3): 229-232. Saha A, Masud AM, Bachar CS, Kundu KJ, Datta KB, Nahar FL and Sarker DS: The analgesic and anti-inflammatory activities of extracts of Phyllanthus reticulatus in mice model. Pharmaceutical Biology 2007; 45 (5): 335-359. Das BP, Mohammad S, Pavel AM, Bhattacharjee R, Das B, Yasmin T, Akhter n and hannam JMA: Anti-hepatitis B viral activity of Phyllanthus reticulatus. Bangladesh Pharmaceutical Journal 2011; 14 (1): 11-14.

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