Extraction and Isolation of capsaicin from Capsicum annum L (CO-1) variety by High-performance liquid chromatography and its Bioactivities

H. Deepa¹ , A. Subhashini²

Section:Research Paper, Product Type: Journal-Paper
Vol.7 , Issue.2 , pp.82-91, Apr-2020

Online published on Apr 30, 2020

Copyright © H. Deepa , A. Subhashini . This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
 

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Abstract :
Capsaicin is a nutritional factor, the active component of chilli, which is responsible for the pungent component of chilli. Chilli has diverse uses as a spice, condiment, culinary supplement, Medicine, vegetable and ornamental plant. Aim of the study: Stimulation and enhancement of capsaicin content by polyploidy induction, quantification of enhanced capsaicin, its cytotoxicity and Bioactivity studies. Materials and methods: Healthy 3 varieties (CO-1, K2, Jwala) of Capsicum annum L. (chilli) was selected for enhancement of capsaicin content by polyploidy induction. The variety showing high capsaicin content in the fifth generation was chosen for further study. The enhanced capsaicin was isolated by silica gel column chromatography and purified using HPLC. Cytotoxicity was studied in onion root tips by root tip squash method and Vero cell lines. Anti-inflammatory activity by inhibition of Albumin denaturation method and Anti-cancer activity by MTT assay was studied using the enhanced capsaicin. Results: Of the 3 chilli varieties selected, CO-1 showed high and enhanced capsaicin content in the fifth generation. By HPLC, with a retention time of 1.368 min, 72.93% purity was obtained against control having a retention time of 1.337min of 41.4% purity recorded. Cytotoxicity of onion root tips showed no cell abnormalities and hence was found to be non-toxic to the cells. In the Vero cell line assay, the IC50 value and the viability of the cell line was 60.39% in treated and 58.79% in control of 100 µg/ml concentration. Anti-inflammatory activity of enhanced Capsaicin also showed maximum inhibition rate of 81.23% against 76.26% in control using 100 µg/ml. In Anti-cancer activity, IC50 dose was 6.25μg/ml in treated whereas untreated capsaicin showed 25μg/ml. Thus, enhanced Capsaicin shows a significant anti-inflammatory and anti-cancer activity.

Key-Words / Index Term :
chilli, capsaicin, HPLC, inhibition, anti-inflammatory, anti-cancer

References :
[1]. Perucka, M. Materska, “Phenylalanine ammonia-lyase and antioxidant activities of a lipophilic fraction of fresh pepper fruits Capsicum annuum L”, Innovat. Food Sci. Emerg. Tech 2, pp.189-192, 2001.
[2]. Deal, C.L. Schnitzer, T.J. Lipstein, E. Seibold, J.R. Stevens, R.M.; Levy, M.D. Albert, D.s Renold, F, “Treatment of arthritis with topical capsaicin: A double-blind trial”. Clin. Ther. 13, pp.383-395, 1999.
[3]. Y. Zewdie, P.W. Bosland, “Evaluation of genotype, environment and genotype-by-environment interaction for capsaicinoids in Capsicum annuum L”. Euphytica, 111 pp.185-190, 2000.
[4]. D.J. Moore, D.M. Moore, “Synergistic Capsicum-tea mixtures with anticancer activity”, J. Pharm. Pharmacol. 55, pp.987-994, 2003.
[5]. J. Szolcsanyi, “Forty years in capsaicin research for sensory pharmacology and physiology”. Neuropeptides, 38, pp.377-384, 2004.
[6]. Bali, P.N. and Tandon, S.L, “Colchicine induced tetraploidy in Linaria vulgaris”. Curr. Sci., 26, pp.256-258, 1956.
[7]. Raghuvanshi, S. and Joshi, S, “Cytomorphological studies on the colchiploids of Capsicum frutescens L”. Cytologia, 225, pp.61-78, 1964.
[8]. Swanson, C.P, wilian Co., N.Y. and Kihlman, B.A. “The chromosomal aberration by ionizing radiations and chemical mutagens”. pp. 239-252, 1957.
[9]. Bhattacharya A, Chattopadhyay A, Mazumdar D, Chakravarty A and Pal S. “Antioxidant constituents and enzyme activities in chilli peppers. International Journal of Vegetable Science” 16, pp.201–211, 2010.
[10]. Luo XJ, Peng J and Li YJ. “Recent advances in the study on capsaicinoids and capsinoids”. European Journal of Pharmacology; 650, pp.1–7, 2011.
[11]. Adams, D. O., & Hamilton, T. A. “The cell biology of macrophage activation”. Annual Review of Immunology, 2, pp.283–318, 1984.
[12]. Tsai, T. H., Tsai, P. J., & Ho, S. C, “Antioxidant and anti- inflammatory activities of several commonly used spices”. Journal of Food Science, 70, pp.93–97, 2005.
[13]. Park, E., Lee, S. M., Lee, J., & Kim, J. H. “Anti-inflammatory activity of mulberry leaf extract through inhibition of NF-kB”. Journal of Functional Foods, 5, pp.178–186, 2013.
[14]. Skidgel, R. A., Gao, X. P., Brovkovych, V., Rahman, A., Jho, D., Predescu, S., Standiford, T. J., & Malik, A. B, “Nitric oxide stimulates macrophage inflammatory protein-expression in sepsis”. Journal of Immunology, 169, pp.2093–2101, 2002.
[15]. Morre, D.J., Chueh, P.J. and Morre, D.M, “Capsaicin inhibits preferentially the NADH oxidase and growth of transformed cells in culture”. Proceedings of the National Academy of Sciences. 92: pp.1831-1835, 1995.
[16]. . Morre, D.J., Sun, E., Geilen, C., Wu, L.Y., de-Cabo, R., Krasagakis, K., Orfanos, C.E. and Morre, D.M. “Capsaicin inhibits plasma membrane NADH oxidase and growth of human and mouse melanoma lines”. European Journal of Cancer. 32A, pp.1995-2003, 1996.
[17]. Macho, A., Blazquez, M.V., Navas, P. and Munoz, E, “Induction of apoptosis by vanilloid compounds does not require de novo gene transcription and activator protein 1 activity”. Cell Growth and Differentiation. 9, pp.277-286, 1998
[18]. . Macho, A., Calzado, M.A., Munoz-Blanco, J., Gomez-Diaz, C., Gajate, C., Mollinedo, F., Navas, P. and Munoz, E, “Selective induction of apoptosis by capsaicin in transformed cells: the role of reactive oxygen species and calcium”. Cell Growth and Differentiation. 6, pp.155-165, 1999.
[19]. Macho, A., Lucena, C., Calzado, M.A., Blanco, M., Donnay, I., Appendino, G. and Munoz, E, “Phorboid 20-homovanillates induce apoptosis through a VR1-independent mechanism”. Chemistry and Biology. 7, pp.483-492, 2000.
[20]. Wolvetang, E.J., Larm, J.A., Moutsoulas, P. and Lawen, A, “Apoptosis induced by inhibitors of the plasma membrane NADH-oxidase involves Bcl-2 and calcineurin”. Cell Growth and Differentiation. 7, pp.1315-1325, 1996.
[21]. Matsufuji, H., Nakamuro, H., Chino, M. and Mitsuharo, T, “Antioxidant activity of capsanthin and the fatty acid esters in paprika (Capsicum annuum)”. Journal of Agricultural and Food Chemistry. 46: pp.3462-3472, 1998.
[22]. J. Zheng, Y.Zhou, Y, Li, D.-P. Xu, S. Li, H.-B.Li, “Spices for prevention and treatment of cancers”, Nutrients 8 (8), pp.495, 2016.
[23]. M.D. Collins, L. Mayer-Wasmund, P.W. Bosland, “Improved method for quantifying capsaicinoids in Capsicum using high-performance liquid chromatography”, Hort. Sci., 30, pp.137-139, 1995.
[24]. H.W. Lewis, C.J. Moody, “Experimental Organic Chemistry: Principles and Practice (Illustrated ed.)”, pp.159–173, 1989.
[25]. T. Mosmann, “Rapid colourimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays”. J. Immun. Method., 65, pp.55–63, 1983.
[26]. Y. Mizushima, M. Kobayashi M. “Interaction of anti‐inflammatory drugs with serum proteins, especially with some biologically active proteins”. J. Pharm. Pharmacol. 20, 169‐ 173, 1968.
[27]. S. Sakat, A.R. Juvekar, M.N. Gambhire, “In vitro antioxidant and anti-inflammatory activity of methanol extract of Oxalis corniculata Linn”. Int. J. Pharm. Pharmacol. Sci. 2, pp.146-155, 2010.
[28]. Yacine Badjah Hadj Ahmed, Zeid Abdullah Al Othman, Mohamed Abdelaty Habila and Ayman Abdel Ghafar. “Determination of Capsaicin and Dihydrocapsaicin in Capsicum Fruit Samples using High-Performance Liquid Chromatography”. Molecules, 16, pp.8919-8929, 2011.
[29]. Bindu Thapa, Natasa Skalko-Basnet, Akihito Takano, Kazuo Masuda, and Purusotam Basnet, “High-Performance Liquid Chromatography Analysis of Capsaicin Content in 16 Capsicum Fruits from Nepal”. J Med Food 12 (4), pp.908–913, 2009.
[30]. Saksit chanthai, Jureerat Juangsamoot, Chalerm Ruangviriyachai and Suchila Techawongstien 2012. “Determination of Capsaicin and Dihydrocapsaicin in Some Chilli Varieties using Accelerated Solvent Extraction Associated with Solid-Phase Extraction Methods and RP-HPLC-Fluorescence”. E-Journal of Chemistry, 9(3), pp.1550-1561, 2012.
[31]. Mônica Rosa Bertão, Milena Cristina Moraes, Darío Abel Palmieri, Luciana Pereira Silva and Regildo Márcio Gonçalves da Silva, “Cytotoxicity, genotoxicity and antioxidant activity of extracts from Capsicum spp. Res. J. Med. Plants, 10, pp.265-275, 2016.
[32]. Liljana Koleva Gudeva, Sasa Mitrev, Viktorija Maksimova, Dusan Spasov, “Content of capsaicin extracted from hot pepper (Capsicum annuum ssp. microcarpum L.) and its use as an ecopesticide”. Hem. Ind. 67 (4) pp.671–675, 2013.
[33]. Adnan Jassim Mohammed, Al–Fartosy Sameerah, Ahmed Zearah, “Antioxidant, Antibacterial and Cytotoxicity Activities of Flavonoid Extract from Capsicum annum L”. Seeds Iraqi national journal of chemistry, volume 49, pp.100-112, 2013.
[34]. Reshma, K.P. Arun P. Brindha, “In Vitro Anti-Inflammatory, Antioxidant and Nephroprotective Studies on Leaves of Aegle marmelos and Ocimum sanctum”s, Asian J Pharm Clin Res, 7, 2014.
[35]. Manjunatha H. thriveni Vasanth Kumar, Rajesh KP, “Anti-inflammatory activity of curcumin and capsaicin augmented in combination”. Int J Pharm Pharm Sci, Vol 9, Issue 6, pp.145-149, 2017.
[36]. Jolayemi AT, Ojewole JAO, “Comparative anti-inflammatory properties of Capsaicin and ethyl- acetate extract of Capsicum frutescens Linn [Solanaceae] in rats”. African Health Sciences; 13(2): pp.357 – 361, 2013.
[37]. Muraleedharan G. Nair and Yunbao Liu, “Capsaicinoids in the Hottest Pepper Bhut Jolokia and its Antioxidant and Anti-inflammatory Activities”. Natural Product Communications Vol. 5 (1) pp.91-95, 2010.
[38]. N.J. Amruthraj, J.P. Preetam Raj, S. Saravanan, L. Antoine Lebel, “In Vitro Studies on Anticancer Activity of Capsaicinoids from Capsicum Chinense Against Human Hepatocellular Carcinoma Cells”, Int. J. Pharmacy Pharm. Sci. 6, pp.254-558, 2014.
[39]. Raj, John, “In Vitro Studies on Anticancer Activity Of Capsaicinoids From Capsicum Chinense Against Human Hepatocellular Carcinoma Cells. Int J Pharm Pharm Sci. 6. pp. 254-558, 2014.
[40]. Rosa Raybaudi-Massiliaa, Al´ırica I. Su´arezb, Francisco Arveloc,d, Alexandra Zambrano, Felipe Sojoc,d, Marıa I. Calderon-Gabaldon, and Jonathan Mosqueda-Melgara, “Cytotoxic, Antioxidant and Antimicrobial Properties of Red Sweet Pepper (Capsicum Annuum L. var. Llanero´n) Extracts: In Vitro Study”. International Journal of Food Studies, Volume 6, pp.222–231, 2017.
[41]. Mehmet Berkoza, Metin Yildirim, Gulhan Arvasc, Omer Turkmen, Oruc Allahverdiyeve, “Effect of capsaicin on transcription factors in 3T3-L1 cell line”. Eastern Journal of Medicine 20, pp.34-45, 2015.