• Yogesh Kumar Pondicherry University
  • Latha Periyasamy Department of Biochemistry and Molecular Biology, Pondicherry University, Puducherry 605014, India


Objective: The present study was carried out to analyze the bioactive component of petroleum ether (PE) extract of Bixa orellana seed and its anticancer potential against B16F-10 melanoma cell line.

Methods: The presence of a bioactive component of PE extract was analyzed by GC-MS method. Cell viability was measured using MTT assay. Apoptotic inductive effect of the extract was evaluated by AO/EB staining; DAPI is staining and Annexin V/PI staining. DNA fragmentation analysis of the control and treated cell was carried out by agarose gel electrophoresis and comet assay. In vitro prevention of cell proliferation and migration was further estimated by colony forming assay and wound healing assay respectively.

Results: The cytotoxicity of the PE extract against B16F10 melanoma cell was dose and time dependent. The IC50 values for the B16F-10 melanoma cell were 145.98±7.49 µg/ml and 121.60±6.20µg/ml for 24h and 48h respectively. DAPI staining showed DNA fragmentation which was further confirmed by ladder-like appearance of DNA by gel electrophoresis and comet assay. PE extract has prevented the cell proliferation and migration of the treated cancer cell. GC-MS analysis shows the presence of three anticancer compound geranylgeraniol, squalene and beta-sitosterol.

Conclusion: The present study established that PE extract of Bixa orellana seed possesses potent anticancer and apoptotic inductive potential which can further be explored in-vivo model for possible cytotoxic activity.

Keywords: Bixa orellana, GC-MS, Geranylgeraniol, Comet assay, Annexin V/PI staining, AO/EB staining


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1. Smith-Warner. Increasing vegetable and fruit intake: randomized intervention and monitoring in an at-risk population. Cancer Epidemiol Biomarkers Prev 2000;9:307-17.
2. Cragg GM, DJ Newman. Antineoplastic agents from natural sources: achievements and future directions. Expert Opin Invest Drugs 2000;9:2783-97.
3. Hirano T, K Abe, Gotoh M, Oka k. Citrus flavone tangeretin inhibits leukaemic HL-60 cell growth partially through induction of apoptosis with less cytotoxicity on normal lymphocytes. Br J Cancer 1995;72:1380-8.
4. Chiao C, Carothers AM, Grunberger D, Solomon G, Preston GA, Barrett JC. Apoptosis and altered redox state induced by caffeic acid phenethyl ester (CAPE) in transformed rat fibroblast cells. Cancer Res 1995;55:3576-83.
5. Jiang MC, Yang-Yen HF, Yen JJ, Lin JK. Curcumin induces apoptosis in immortalized NIH 3T3 and malignant cancer cell lines. Nutr Cancer 1996;26:111-20.
6. Butler MS, Robertson AA, Cooper MA. Natural products to drugs: natural product-derived compounds in clinical trials. Nat Prod Rep 2008;25:475-516.
7. Yu Wanga, Kedi Xua, Lin Linb, Yuanjiang Panb, Xiaoxiang Zhenga. Geranyl flavonoids from the leaves of Artocarpus altilis. Phytochemistry 2007;68:1300-6.
8. Diepgen TL, Mahler V. The epidemiology of skin cancer. Br J Dermatol 2002;146:1-6.
9. Garbe C1, McLeod GR, Buettner PG. Time trends of cutaneous melanoma in queensland, australia and central europe. Cancer 2000;89:1269-78.
10. Daniela AV, Marina Suênia de AV, Túlio Flávio Accioly de Lima e Moura, Fernanda Nervo Raffin, Márcia Rosa de Oliveira, Camilo Flamarion de Oliveira Franco, et al. Traditional uses, chemical constituents, and biological activities of bixa orellana L.: a review. Sci World J 2014. doi.org/10.1155/2014/857292. [Article in Press]
11. Mu Y M YT, Nishi Y, Tanaka A, Saito M, Jin CH, Mukasa C, et al. Saturated FFAs, palmitic acid and stearic acid, induce apoptosis in human granulosa cells. Endocrinology 2001;142:3590-7.
12. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.
13. Yang X1, Luo P, Yang B, He Q. Antiangiogenesis response of endothelial cells to the antitumor drug 10-methoxy-9-nitrocamptothecin. Pharmacol Res 2006;54:334-40.
14. Machana S, Weerapreeyakul N, Barusrux S, Thumanu K, Tanthanuch W. Synergistic anticancer effect of the extracts from Polyalthia evecta caused apoptosis in human hepatoma (HepG2) cells. Asian Pac J Trop Biomed 2012;2:589-96.
15. Sheng Y, Pero RW, Amiri A, Bryngelsson C. Induction of apoptosis and inhibition of proliferation in human tumor cells treated with extracts of Uncaria tomentosa. Anticancer Res 1998;18:3363-8.
16. Lima CF, M Fernandes-Ferreira, C Pereira-Wilson. Phenolic compounds protect HepG2 cells from oxidative damage: relevance of glutathione levels. Life Sci 2006;79:2056-68.
17. Ohizumi H, Masuda Y, Nakajo S, Sakai I, Ohsawa S, Nakaya K. Geranylgeraniol is a potent inducer of apoptosis in tumor cells. J Biochem 1995;117:11-3.
18. Masuda Y NM, Nakajo S, Nakaya K. Geranylgeraniol potently induces caspase-3-like activity during apoptosis in human leukemia U937 cells. Biochem Biophys Res Commun 1997;234:641-5.
19. Reddy LH, Couvreur P. Squalene: a natural triterpene for use in disease management and therapy. Adv Drug Delivery Rev 2009;61:1412-26.
20. Gupta R, Sharma AK, Dobhal MP, Sharma MC, Gupta RS. Antidiabetic and antioxidant potential of beta-sitosterol in streptozotocin-induced experimental hyperglycemia. J Diabetes 2011;3:29-37.
21. Baskar AA, Ignacimuthu S, Paulraj GM, Al Numair KS. Chemopreventive potential of beta-sitosterol in experimental colon cancer model--an in vitro and In vivo study. BMC Complementary Altern Med 2010;10:10-24.
22. Ward TH, J Cummings, Dean E, Greystoke A, Hou JM, Backen A, et al. Biomarkers of apoptosis. Br J Cancer 2008;99:841–6.
23. Anand AS BS, Chandan BK, Handa SS, Jaggi BS, Prabhakar A. Synergistic composition of the bioactive fraction isolated from Barleria prionitis Linn and a method of treatment for hepatotoxicity, immuno-deficiency and fatigue and a process thereof. Patent No. 6664236; 2002.
24. Ma XH, Zheng CJ, Han LY, Xie B, Jia J, Cao ZW, et al. Synergistic therapeutic actions of herbal ingredients and their mechanisms from molecular interaction and network perspectives. Drug Discovery Today 2009;14:579-88.
25. Boulaaba M, Mkadmini K, Tsolmon S, Han J, Smaoui A, Kawada K, et al. In vitro antiproliferative effect of arthron mum indicum extracts on caco-2 cancer cells through cell cycle control and related phenol LC-TOF-MS identification. J Evidence-Based Complementary Altern Med 2013. doi.org/10.1155/ 2013/529375. [Article in Press].
26. Chen GY, G Nunez. Sterile inflammation: sensing and reacting to damage. Nat Rev Immunol 2010;10:826-37.
27. Oboh G, Akomolafe TL, Adefegha SA, Adetuyi AO. Inhibition of cyclophosphamide-induced oxidative stress in rat brain by polar and non-polar extracts of annatto (Bixa orellana) seeds. Exp Toxicol Pathol 2011;63:257-62.
28. Lopes MV, Desoti VC, Caleare AO, Nakamura T, Silva SO, Nakamura CV. Mitochondria superoxide anion production contributes to geranylgeraniol-induced death in leishmania amazonensis. J Evidence-Based Complementary Altern Med 2012. doi:10.1155/2012/298320. [Epub 04 Dec 2012].
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How to Cite
Kumar, Y., and L. Periyasamy. “GC-MS ANALYSIS AND IN-VITRO CYTOTOXIC STUDIES OF BIXA ORELLANA SEED EXTRACT AGAINST CANCER CELL LINE”. International Journal of Pharmacy and Pharmaceutical Sciences, Vol. 8, no. 1, Dec. 2015, pp. 408-13, https://innovareacademics.in/journals/index.php/ijpps/article/view/9631.
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