Joseph mahimaidoss et al. / Journal of Pharmacy Research 2014,8(9),1223-1225 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info The anticancer activity of ethanolic extract of felina linn

Joseph M a *, Alex Ramani Vincent b, Charles A b a. Department of Chemistry, Dr.Navalar Nedunchezhiyan College of Engineering, Tholudur, Tamilnadu, India. b. Department of Chemistry, St.Joseph’s College(Autonomous), Tiruchirappalli-600 002, Tamilnadu, India.

Received on:14-06-2014; Revised on: 17-07-2014; Accepted on:31-08-2014

ABSTRACT Background: In the last decades, there is again a growing popularity of medicinal and derived products. Cancer is a class of diseases in which a cell or a group of cells display uncontrolled growth, invasion and sometimes metastasis. The anticancer activities of certain cleome genera have been studied. The present work deals with the anti cancer activity of the ethanolic extract of Cleome felina Linn. (C.felina). Methods: The anticancer activity against HepG2 (Human hepatocellular liver carcinoma cell line) cells was analysed using 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenylterttrazolium bromide (MTT) assay. Data were collected from triplicate separate experiments and the per- centage of C.felina extract induced cell growth inhibition was determined by comparing with DMSO treated control cells. Results :. The maximum inhibition of cell growth was observed at the concentration of 200 µg/ml of C.felina extract. Conclusion:The plant extract has potent to decrease the viability of HepG2 cells in a dose – dependent manner.

KEYWORDS: Cleome felina , Anticancer activity, MTT assay, HepG2 cells

1. INTRODUCTION are a small family of flowering plants in the order swiss albino mice and showed that plant has potent dose dependent , comprising about 300 species in 10 genera. These genera anticancer activity. Cleome arabica leaf extract has anticancer prop- were previously included in the family Capparaceae, but were raised erties in human cancer cells8. Venu Gopal et al (2012) have studied to a distinct family when DNA evidence suggested that the genera the antitumour activity of Cleome viscose9. The present work aims at included in it are more closely related to Brassicaceae than they are the anticancer of the C.felina. The medicinal plant – C. felina Linn is to Capparaceae. The APG II system allows for Cleomaceae to be chosen for the present study as they are medicinally very important. included in Brassicaceae1,2. The medicinal properties of number of C .felina Linn is widely distributed in Deccan districts of Madras Cleome species have been studied; Analgesic, anti-inflammatory and Presidency. It is an annual erect herb, 30-60 cm high, much branched, antipyretic activities of Cleome rutidosperma3. Leaf Paste of Cleome leaves 3-foliolate, leaflets 10-25 mm. long, obovalate,obtuse, equal- viscosa L. has wound healing property4. Whole plant Juice of Cleome ing or shorter than the pelioles. Flowers axilliary, solitary on long viscosa and Cleome ciliata has been used to treat ear infection, pedicels, 12-18 mm. long, purple or pink. Stamens about 30; 5,6 pain and deafness . filiform.Capsule 8 times long as broad, compressed, linear anlong, acute at both ends, striate, glabrous, seeds large, glabrous, Species like Cleome viscosa, Cleome gynandra and Cleome tubercled10. chelidonii have many medicinal applications, often in rubifacient and counter irritant preparations. They are also used for rheumatism 2. MATERIALS AND METHODS and even headache Some species like C. gynandra are eaten as vegetable in extreme conditions. Oil of Cleome seeds has insect 2.1. The Collection of plant Material repellent properties so used against tick. Seeds of Cleome chelidonii The leaves of C .felina were collected from Kolli Hills during 7 are used as condiment [Flora of India vol 2]. Bala et al. studied December 2010. The shade dried plant material was extracted with anticancer activity of methanolic extract of Cleome gynandra in 80% Ethanol. The extract was concentrated and subjected to anti-cancer activity . *Corresponding author. Joseph Mahimaidoss 2.2. Cell culture: Department of Chemistry, The HepG2 is a perpetual cell line (Human hepatocellular liver Dr.Navalar Nedunchezhiyan College of Engineering, carcinoma cell line) obtained from the National Center for Cell Sci- Tholudur, Tamilnadu, India ence (Pune,India) and grown in Dulbecco’s Modified Medium (DMEM) supplemented with 10% fetal bovine serum (FBS),penicillin

Journal of Pharmacy Research Vol.8 Issue 9. September 2014 1223-1225 Joseph mahimaidoss et al. / Journal of Pharmacy Research 2014,8(9),1223-1225 (100U/ml)/streptomycin (100µg/ml), 2 mM glutamine and 1 mM so- Table 1.Effect of C.felina on HepG2 cells death One-way ANOVA dium pyruvate. HepG2 cells were cultured as adherent monolayers and maintained at 37°C in a humidified atmosphere of 5% CO and 95 Concentration (µg/ml) Effect of C.felina on 2 HepG2 cells death % air in 100 % relative humidity. HepG2 cells were harvested after brief trypsinization. All cell cultures reagents were purchased from 25 4±0.6 e 50 8±1.8 d Himedia, Mumbai, India. 100 16±0.8 c 150 23±0.8 b 2. 3. Cell growth inhibition study using the MTT assay: 200 45±0.6 a Control 100 Cell growth inhibition of C.felina crude extracts were analysed us- ing the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl- tertrazolium bro- Means followed by the same letter do not differ significantly using LSD (P=0.05) mide (MTT) assay. Briefly, HepG2 cells were seeded in 96 well plates at a density of 6x103 cells per well. After treatment with 25, 50, 100, creased the total number of HepG2 cells and were accompanied by 150, 200µg/ml of C.felina plant crude extracts for 48 hours, 20µl MTT cell shrinkage, condensed nuclei, blebbing and shape changes (Fig (5 mg/ml) was added. Four hours later, 100 µl DMSO was added to 1). When the cells were stained with trypan blue, which can discrimi- each well to dissolve the resulting formazan crystals. Absorbance nate between apoptosis and necrosis, condensed nuclei, blebbing, was read at 490 nm using an enzyme-linked immunosorbent assay indicators of typical apoptosis manifestation were observed in plant reader (SpectraMax; Molecular Devices, Sunnyvale, CA). crude extracts treated cells.

Data were collected from triplicate separate experiments and the C. felina crude extract treated HepG2 cells exhibited more than 45 percentage of C. felina plant crude extracts induced cell growth increase in apoptosis and Investigation of the staining pattern indi- inhibition was determined by comparison to DMSO-treated control cells13. For statistical analysis, data were analyzed using analysis of Fig 1. Cytotoxic effect of C. felina plant crude extracts against HepG2 variance. Fisher’s least significant difference (LSD)11,12 at the 5% level was calculated using the statistical package for social science 120 (Version 12.0 for Windows, SPSS Inc.) 100

3. RESULTS: 80

60 3.1 Treatment of C.felina crude extracts inhibits the growth of HepG2 cells 40 Series1 HepG2 cells were treated with different concentrations of (25, 50, 20

100, 150 and 200µg/ml plant crude extracts and their growth was Percentage of viability monitored. C. felina plant crude extracts decreased the viability of 0 HepG2 cells in a dose-dependent manner. The maximum inhibition of control 25mg 50mg 100mg 150mg200mg cell growth was observed at the concentration of 200µg/ml (45% Concentration of C.felina extract (µg/ml with IC50) of C. felina crude extract. cated that the predominant cause of cell death in HepG2 was due to The decreased growth rates of HepG2 cells growth rates of HepG2 apoptosis. Based on the one-way ANOVA analysis compared the cells of about 4%, 8%, 16%, 23% and 45% observed at the C.felina means of C.felina crude extracts induces apoptosis of HepG2 cells plant crude extract concentration of 25, 50, 100 150 and 200µg/ml, significantly (P<0.05) in a concentration-dependent manner. respectively. All the concentrations used in the experiment decreased the cell viability significantly (P<0.05) in a concentration-dependent 3.3. Apoptotic effect of plant crude extracts on normal cells manner (Table 1). Apoptotic effect of plant crude extracts on normal cells were studied to test whether C.felina extract plant extract induced similar cell death 3.2. C. felina crude extracts induces apoptosis of HepG2 cells in normal cells. After 30 minutes of incubation with of C.felina extract Treatments with C.felina crude extracts (200µg/ml) effectively de- (200µg/ml) resulted only 16.5% cell death (Fig.2).

Journal of Pharmacy Research Vol.8 Issue 9. September 2014 1223-1225 Joseph mahimaidoss et al. / Journal of Pharmacy Research 2014,8(9),1223-1225

Fig.2). Cytotoxicity and apoptotic activity of C .felina crude extract 3. Bose et al. Analgesic, anti-inflammatory and antipyretic ac- against HepG2 (A) Controls cells; (B) C. felina treated HepG2 tivities of the ethanolic extract and its fractions of Cleome cells showing cytotoxicity and apoptotic activity rutidosperma Fitoterapia 78, (2007), 515–520.

4. S. Ignacimuthu et al. Ethnobotanical study of medicinal plants used by Paliyar tribals in Theni district of Tamil Nadu, India, Fitoterapia 79 (2008) 562–568. 5. R. Qureshi, G. Raza Bhatti Ethnobotany of plants used by the Thari people of Nara Desert, Pakistan Fitoterapia 79 (2008) 468–473. 6. Finn Sandberg, Premila Perera-Ivarsson a, Hesham Rushdey El-Seedi A Swedish collection of medicinal plants from Cameroon,Journal of Ethnopharmacology, 102, (2005)336– 343. 7. Bala, A., Kar, B., Haldar, P. K., Mazumder, U. K. and Bera, S.. (A) (B) Evaluation of anticancer activity of Cleome gynandra on 4. DISCUSSION Ehrlich’s Ascites Carcinoma treated mice. Journal of Large scale screening of plant crude extracts on cell culture is an Ethnopharmacology,129(1), 2010, 131-134. important initial step to determine their potential efficacy in clinical 8. Tigrine C, Bulzomi P, Leone S, Bouriche H, Kameli A, Marino application. Several reports have shown that plant crude extracts M. Cleome arabica leaf extract has anticancer properties in replicate at tumour sites under hypoxic conditions and stimulate the human cancer cells.Pharm Biol. 51(12), 2013,1508-14. host immune response and gene expression, leading to the inhibi- 9. Venu Gopal. Y, Ravindernath. A, Kalpana. G, Prabhakar Reddy tion of tumour growth. V, Antitumor activity of Cleome Viscosa against Ehrlich As- cites Carcinoma (EAC) in Swiss Albino Mice, International ACKNOWLEDGMENTS Journal of Phytopharmacy,Vol. 2 (2), Mar-Apr 2012, 51-55 The authors would like to thank Anna University, Chennai for the 10. Indian Medicinal Plants , Plate, Part-I, kirtikar & basu., help to carry out the anticancer activity studies. Mohan basu publications (1984). 11. Anthony JH (1986) The maximum familywise error rate of Fisher’s least significant difference test. J Am Stat Assoc REFERENCES 81:1000–1004. 1. Raghavan, R. S., Sharma, B.D. & N. P. Balakrishnan (Eds), 12. Fisher RA (1935) The Design of Experiments. University of Flora of India. Vol. 2. Botanical Survey of India, Howrah., Adelaide (1993), 248 – 335. 13. Mosmann T., 1983. Rapid colorimetric assay for cellular 2. Stevens, P. F. (2001). Angiosperm Phylogeny Website. Ver- growth and survival: application to proliferation and cyto- sion 9, June 2008.http://www.mobot.org/MOBOT/research/ toxicity assays. Journal of immunological methods 65: 55- APweb/ 63.

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

Journal of Pharmacy Research Vol.8 Issue 9. September 2014 1223-1225