ANTIPROLIFERATIVE AND APOPTOTIC EFFECTS OF THE ESSENTIAL OIL OF ORIGANUM ONITES AND CARVACROL ON HEP-G2 CELLS

Year 2011, Volume: 1 Issue: 2, 171 - 180, 17.06.2011

Hülya Sivas Özlem Tomsuk

Abstract

The essential oil Origanum onites L. and its phenolic constituent carvacrol were examined for their cytotoxic and apoptotic effects in a human hepatocellular carcinoma cells Hep-G2. WST-1 and neutral red uptake assays were performed to determine the inhibitory effects of the oil and carvacrol on the growth of the cells. Possible induction of apoptosis by Origanum oil and carvacrol was further investigated by acridine orange/ethidium bromide (AO/EB) staining. Results showed that the Origanum oil and carvacrol was significantly cytotoxic and induced apoptosis in Hep-G2 cells. IC50 value of essential oil and carvacrol was found about 0,009% (v/v) and 500 μM, respectively. After incubation of the cells with Origanum oil and carvacrol, characteristics of apoptotic morphology such as chromatin condensation, shrinkage of the cells and cytoplasmic blebbing was observed. In conclusion, both essential oil and its major constituent carvacrol significantly exhibited cytotoxic and apoptotic
activities in hepatocellular carcinoma cells, indicating its potential for use as an anticancer agent.

Keywords

-

Yaşam Bilimleri ve Biyoteknoloji

Abstract

Origanum onites L. uçucu yağı ve onun fenolik bileşeni olan karvakrolün insan kanserli karaciğer hücreleri Hep-G2 üzerindeki sitotoksik ve apoptotik etkileri çalışılmıştır. Hücrelerin çoğalması üzerine origanum uçucu yağının ve karvakrolün engelleyici etkilerini belirlemek için WST-1 ve nötral kırmızısı alım yöntemleri uygulanmıştır. Origanum yağı ve karvakrolün olası apoptotik etkisi akridinoranj/etidyumbromür (AO/EB) boyama yöntemiyle araştırılmıştır. Sonuçlar değerlendirildiğinde, Origanum yağı ve karvakrolün Hep-G2 hücrelerinde anlamlı derecede sitotoksik etkiye sahip olduğu ve bu hücrelerde apoptozu uyardığı gözlenmiştir. Uçucu yağın IC50 değeri % 0.009, karvakrolün 500 mM olarak belirlenmiştir. Origanum uçucu yağı ve karvakrol ile inkübasyonun ardından hücrelerin kromatin yoğunlaşması, hücre büzülmesi, sitoplazmik keselerin oluşumu gibi karakteristik apoptoz morfolojisi gösterdiği belirlenmiştir. Sonuç olarak, hem Origanum uçucu yağı hem de onun temel bileşeni olan karvakrol, insan kanserli karaciğer hücrelerinde anlamlı bir sitotoksik etki ve apoptotik aktivite sergilemiştir ve bu özelliklerinden dolayı, her ikisi de antikanser ajan olarak kullanılabilme potansiyeline sahiptir

Keywords

Origanum yağı, Karvakrol, Apoptoz, Sitotoksisite, Hep-G2

References

• Aligiannis, N., Kalpoutzakis, E., Mitaku, S. and Chinou, I.B. (2001). Composition and an- timicrobial activity of the essential oils of two Origanum species. J. Agric. Food Chem 49, 4168-4170.
• Al-Kalaldeh, J.Z., Abu-Dahab, R. and Afifi, F.U. (2010). Volatile oil composition and antiproliferative activity of Laurusnobilis, Origanum syriacum, Origanum vulgare, and Salvia triloba against human breast adenocarcinoma cells. Nutrition Research 30, 271-278.
• Arunasree, K.M. (2010). Anti-proliferative ef- fects of carvacrol on a human metastatic breast cancer cell line, MDA-MB 231. Phytomedicine 17(8-9), 581-588.
• Aslim, B. and Yucel, N. (2008). In vitro antim- icrobial activity of essential oil from en- demic Origanum minutiflorum on cipro- floxacin-resistant Campylobacter spp. Food Chemistry 107 602–606.
• Bakkali, F., Averbeck, S., Averbeck, D. and Idaomar, M. (2008). Biological effects of essential oils – A review. Food and Chemical Toxicology 46, 446–475.
• Başer, K.H.C., Özek, T., Tümen, G. and Sezik, E. (1993).Composition of the essential oils of Turkish Origanum species with commercial importance. Journal of Es- sential Oil Research 5, 619-623.
• Baser, K.H.C. (2008). Biological and pharma- cological activities of carvacrol and car- vacrol bearing essential oils. Current Pharmaceutical Design 14(29), 3106- 3119.
• Baydar, H., Osman Sağdiç, O., Özkan, G. and Karadoğan, T. (2004). Antibacterial activ- ity and composition of essential oils from Origanum, Thymbra and Satureja species with commercial importance in Turkey. Food Control 15 169–172.
• Busatta, C., Mossi, A.J., Rodrigues, M.R.A., Rogério Luis Cansian, R.L. and Oliveira, J.V. (2007). Evaluation of Origanum vul- gare essential oil as antimicrobial agent in. Brazilian Journal of Microbiology 38, 610–616
• Chinou, I., Liolios, C., Moreau, D. and Roussakis, C. (2007). Cytotoxic activity of Origanum dictamnus. Fitoterapia. 78(5), 342-4
• Choriannopoulos, N., Kalpoutzakis, E., Aligian- nis, N., Mitaku, N., Nychas, G.J. and Haroutounian, S.A. (2004). Essential oils of Satureja, Origanum and Thymusspe- cies: Chemical composition and antibacte- rial activities against foodborne patho- gens. J. Agric. Food Chem 52, 8261-8267 8261.
• Crowell, P.L., Ren, Z., Lin, S., Vedejs, E. and Gould, M.N. (1994). Structure-activity re- lationships among monoterpene inhibitors of protein isoprenylation and cell proliIH- UDtion. Biochem. Pharmacol. 47, 1405-15.
• Crowell, P.L. (1999). Prevention and therapy of cancer by dietary monoterpens. J. Nutr. 775S-778S.
• Ding, H.Y., Chou, T.H. and Liang, C.H. (2010). Antioxidant and antimelanogenic proper- ties of rosmarinic acid methyl ester from Origanum vulgare. Food Chemistry 123 254–262.
• Duke, R.C. (2004). Methods in Molecular Bio- logy, Vol. 282, Ed.: Brady, HJM. Humana Press.
• Fuentes, A.O., Gutiérrez, E.A., Señorans, F.J. and Reglero, G. (2010). International Journal of Food Microbiology 137 175– 180.
• Fotakis, G. and Timbrell, J.A. (2006). In vitro cytotoxicity assays: Comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Tox. Letters 160, 171- 177.
• Fuentes, A.O., Gutiérrez, E.A., Señorans, F.J. and Reglero, G. (2010). Supercritical fluid extraction of oregano (Origanum vulgare) essentials oils: Anti-inflammatory proper- ties based on cytokine response on THP-1 macrophages. Food and Chemical Toxi- cology 48 1568–1575.
• Horvathova, E., Turcaniova, V. and Slamenova, D. (2007). Comparative study of DNA- damaging and DNA-protective effects of selected components of essential plant oils in human leukemic cells K562. Neo- plasma 54(6), 478–483.
• He, L., Mo, H., Hadisusilu, S., Quresni, A.A. and Elson, C.E. (1997). Isoprenoids sup- press the growth of murine B16 melano- mas in vitro and in vivo. Biochemical and Molecular J. Nutr. 127(5), 668-674, 1997.
• Govaris, A., Solomakos, N., Pexara, A. and Chatzopoulou, P.S. (2010). The antim- icrobial effect of oregano essential oil, nisin and their combination against Sal- monella enteritidis in minced sheep meat during refrigerated storage. International Journal of Food Microbiology 137 175– 180.
• Goze, I., Cetin, A. and Goze, A. (2010). Investi- gation of effects of essential oils of Ori- ganum minutiflorum O Schwarz PH Davis and plants on angiogenesis in shell-lesschick embryo culture. African Journal of Bio- technology 9(14), 2156–2160.
• Ipek, E., Zeytinoglu, H., Okay, S., Tuylu, B.A., Kurkcuoglu, M.K. and Baser, K.H.C. (2005). Genotoxicity and antigenotoxicity of Origanum oil and carvacrol evaluated by Ames Salmonella/microsomal test. Food Chemistry 93 551–556.
• Jelnar, Z. Al-Kalaldeh, Rana Abu-Dahab and Fatma, U. Afifi. (2010). Volatile oil com- position and antiproliferative activity of Laurusnobilis, Origanum syriacum, Ori- ganum vulgare, and Salvia triloba against human breast adenocarcinoma cells. Nutrition Research 30, 4, 271-278
• Karkabounas, S., Kostoula, O.K., Daskalou, T., Veltsistas, P., Karamouzis, M., Zelovitis, I., Metsios, A., Lekkas, P., Evangelou, A.M., Kotsis, N. and Skoufos, I. (2006). Anticarcinogenic and antiplatelet effects of carvacrol. Experimental Oncology 28, 121–125.
• Karpouhtsis, I., Pardali, E., Feggou, E., Kokkini, S., Scouras, Z.G. and Tsipidou, P.M. (1998). Insecticidal and Genotoxic Activi- ties of Oregano Essential Oils. J. Agric. Food Chem 46, 1111-1115.
• Kim, D.K., Lillehoj, H.S., Lee, S.H., Jang, S.I and Bravo, D. (2010). High-throughput gene expression analysis of intestinal in- traepithelial lymphocytes after oral feed- ing of carvacrol, cinnamaldehyde, or Capsicumoleoresin. 89(1), 68-81. Poultry Science
• Kordali, S., Cakir, A., Ozer, H., Cakmakci, R., Kesdek, M. and Mete, E. (2008). Anti- fungal, phytotoxic and insecticidal prop- erties of essential oil isolated from Turk- ish Origanum acutidens and its three components, carvacrol, thymol and p- cymene. Bioresource Technology 99 8788–8795.
• Lampronti, I., Saab, A.M. and Gambari, R. (2006). Antiproliferative activity of essen- tial oils derived from plants belonging to the Magnoliophyta division. International Journal of Oncology 29, 989–995.
• Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: applica- tion to proliferation and cytotoxicity as- says. J. Immunol. Methods 65,55-63.
• Sivropoulou, A., Papanikolaou, E., Nikolaou, C., Kokkini, S., Lanaras, T. and Ar- senakis, M. (1996). Antimicrobial and cy- totoxic activities of Origanum essential oils. J. Agric. Food Chem 44, 1202-1205.
• Rao, A., Zhang, Y.Q., Muend, S. and Rao, R. (2010). Mechanism of antifungal activity of terpenoid phenols resembles calcium stress and inhibition of the TOR pathway. Antimicrob. Agents Chemother. 54, 5062- 69.
• Roula, A.M.M., Rida, F., Samer, B., Nisrine, E.C. and Elias, B. (2010). The apoptotic and anti-proliferative activity of Ori- ganummajorana extracts on human leu- kemic cell line. Leukemia Research 34(8), 1052-6.
• Souza, E.L., Stamford, T.L.M., Lima, E.O. and Trajano, V.N. (2007).Effectiveness of Origanum vulgare L. essential oil to in- hibit the growth of food spoiling yeasts. Food Control 18 409–413.
• Stammati, A., Bonsi, P., Zucco, F., Moezelaar, R., Alakomi, H.L. and von Wright, A. (1999). Toxicity of selected plant volatiles in microbial and mammalian short-term assay. Food and Chemical Toxicology 37(8), 813-823.
• Storia, A.L., Ercolini, D., Marinello, F., Pasqua, R.D., Villani, F. and Mauriello, G. (2011). Atomic force microscopy analysis shows surface structure changes in carvacrol- treated bacterial cells. Research in Micro- biology 162, 164-172.62, 164-172.
• Süntar, I., Akkol, E.K., Keleş, H., Oktem, A. and Başer, K.H.C. (2011). A novel wound healing ointment: A formulation of Hy- pericumperforatum oil and sage and oreg- ano essential oils based on traditional Turkish knowledge. Journal of Ethno- pharmacology 134, 89-96.
• Tabanca, N., Demirci, F., Ozek, T., Tumen, G. and Baser, K.H.C. (2001). Composition and antimicrobial activity of the essential oil of Origanum dolichosiphon P.H. Davis. Chemistry of Natural Products 37, 238-241.
• Ultee, A., Kets, E.P.W. and Smid, E.J. (1999). Mechanisms of action of carvacrol on the food-born pathogen Bacillus cereus. Appl. Environ. Microbiol. 65, 4606-10.
• Unlu, M., Ergene, E., Unlu, G.V. Zeytinoglu, H.S. and Vural, N. (2010). Composition, antimicrobial activity and in vitro cyto- toxicity of essential oil from Cinnamo- mum zeylanicum Blume (Lauraceae). Food and Chemical Toxicology 48 3274–3280.
• Vincenzi, M.D., Stammati, A., Vincenzi, A.D. and Silano, M. (2004). Constituents of aromaric plants: carvacrol. Fitoterapia 75, 801-804.
• Zeytinoglu, H., Incesu, Z. and Baser, K.H.C. (2003). Inhibition of DNA synthesis by carvacrol in Mouse myoblast cells bearing a human N-ras oncogene. Phytomedicine 10(4), 292-299.
• Xiao, J.X., Huang, G.Q., Zhu, C.P., Ren, D.D. and Zhang, S.H. (2007). Morphological study on apoptosis Hela cells induced by soyasaponins. Toxicol.In Vitro 21, 820– 826.