Journal of Nutrition and Health

The Korean Nutrition Society (한국영양학회)
권호 표지
DOI QR코드

DOI QR Code

Antioxidant activity of ethanol extract of Lycium barbarum's leaf with removal of chlorophyll

클로로필을 제거한 영하구기엽 에탄올 추출물의 항산화 활성

  • Kim, Ji Eun (Department of Food and Nutrition, Chungam National University) ;
  • Bae, Su Mi (Department of Food and Nutrition, Chungam National University) ;
  • Nam, You Ree (Department of Food and Nutrition, Chungam National University) ;
  • Bae, Eun Young (Department of Food and Nutrition, Chungam National University) ;
  • Ly, Sun Yung (Department of Food and Nutrition, Chungam National University)
  • 김지은 (충남대학교 식품영양학과) ;
  • 배수미 (충남대학교 식품영양학과) ;
  • 남유리 (충남대학교 식품영양학과) ;
  • 배은영 (충남대학교 식품영양학과) ;
  • 이선영 (충남대학교 식품영양학과)
  • Received : 2018.11.21
  • Accepted : 2018.12.28
  • Published : 2019.02.28
Download PDF
⟨ Previous Next ⟩

Abstract

Purpose: The aim of this study was to estimate the antioxidant activities of 50%, 70%, and 100% ethanol extracts of Lycium barbarum leaf and chlorophyll removal extract. Methods: The antioxidant activities were estimated by measuring total polyphenol content and by assays of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfate) (ABTS) radical scavenging activities and ferric reducing antioxidant power (FRAP). In addition, reactive oxygen species (ROS) production, DNA fragmentation, and antioxidant enzyme (superoxide dismutase and catalase) activities of the extracts were measured in hydrogen peroxide ($H_2O_2$)-stressed HepG2 cells. Results: The total polyphenol content, DPPH and ABTS radical scavenging activities, and FRAP value of the extracts increased in an ethanol concentration-dependent manner. The antioxidant activities of the chlorophyll-removal extracts were much higher than those of the chlorophyll-containing extracts. Cytotoxicity was not observed in HepG2 cells with extracts up to $1,000{\mu}g/mL$. All extracts inhibited ROS production in a concentration-dependent manner from $31.3{\mu}g/mL$ and inhibited DNA damage at $250{\mu}g/mL$. The SOD and catalase activities of cell lines treated with the extracts and $H_2O_2$ were similar to those of normal cells, indicating a strong protective effect. Conclusion: Lycium barbarum leaf extracts had high antioxidant activities and protected $H_2O_2$-stressed HepG2 cells. Since the chlorophyll-removal extract exhibited higher antioxidant activities than the chlorophyll-containing ones and the cytoprotective effect was similar, chlorophyll removal extract of Lycium barbarum leaf could be developed as ingredients of functional food and cosmetics.

본 연구에서는 구기엽의 항산화 활성을 확인하고자 50%, 70%, 100% 에탄올 추출물과 100% 에탄올 추출물에서 클로로필을 제거한 시료를 포함하여 총 4종 시료의 항산화 활성을 비교하였다. 항산화능을 측정하기 위하여 총 폴리페놀 함량, DPPH와 ABTS radicals 소거능, FRAP assay를 통해 시료의 항산화능을 평가하였다. 또한 $H_2O_2$처리로 산화적 스트레스를 유발한 HepG2세포주에서 추출물 들이 세포보호 효과를 나타낼 수 있는지 알아보고자 ROS 생성, DNA fragmention, 세포의 항산화효소활성 (SOD와 catalase)을 측정하였다. 연구결과, 총 폴리페놀 함량, DPPH와 ABTS radicals 소거능, 그리고 FRAP value에서 모두 추출용매의 알코올 농도가 높을수록 증가하였고 클로로필 제거 시료가 제거 전 시료에 비하여 월등히 높았다(p < 0.05). HepG2세포에서 세포독성을 확인한 결과 모든 시료에서 $1,000{\mu}g/mL$까지 세포독성을 유발하지 않았다. 모든 추출물들은 $31.3{\mu}g/mL$ 농도부터 $H_2O_2$에 의해 증가한 ROS생성량을 농도 의존적으로 감소시키는 효과를 보였다 (p < 0.05). 모든 추출물은 $250{\mu}g/mL$의 농도로 배양액에 처리하였을 때 세포의 DNA 손상을 억제하는 효과를 보여주었다 (p < 0.05). 또한 $H_2O_2$처리한 세포에 비하여 추출물들을 함께 처리한 세포군의 SOD와 catalase 활성은 정상세포과 유사한 수준으로 강력한 보호효과를 보여주었다. 결론적으로 본 연구에서 사용한 구기엽 에탄올 추출물들은 항산화능이 높으며 고농도까지 세포독성을 보이지 않고 $H_2O_2$에 의해 손상된 간세포를 보호할 수 있는 효능을 가지고 있음을 확인할 수 있었다. 특히 클로로필 제거구기엽 에탄올 추출물은 항산화능이 높고 세포독성과 세포보호 효과가 클로로필 제거전 구기엽 에탄올 추출물과 유사하게 나타나 건강기능식품이나 화장품의 소재로 활용하기에 적절한 소재로 생각된다.

Keywords

References

  1. Lee BC, Park JS, Kwak TS, Moon CS. Variation of chemical properties in collected boxthorn varieties. Korean J Breed 1998; 30(3): 267-272.
  2. Kwon KD, Park WJ, Kim SA. Buy decision making factors and marketing strategies of Lycium chinense: focused on Cheongyang region. Korean J Agric Manage Policy 2007; 34(2): 422-443
  3. Park YJ, Kim M, Bae SJ. Enhancement of anticarcinogenic effect by combination of Lycii fructus with vitamin C. J Korean Soc Food Sci Nutr 2002; 31(1): 143-148. https://doi.org/10.3746/jkfn.2002.31.1.143
  4. Park JS, Park JD, Lee BC, Choi KJ, Ra SW, Chang KW. Effects of extracts from various parts of Lycium chinense Mill. on proliferation of mouse spleen cells. Korean J Med Crop Sci 2000; 8(4): 291-296.
  5. Kang K, Jung J, Koh KH, Lee CH. Hepatoprotective effects of Lycium chinense mill fruit extracts and fresh fruit juice. Korean J Food Sci Technol 2006; 38(1): 99-103.
  6. Sung SH, Park SH. Effect of Lycii Fructus powder on lipid metabolism in 1% cholesterol fed rats. Korean J Food Cult 2008; 23(4): 521-528.
  7. Kim TS, Park WJ, Ko SB, Kang MH. Development of extracts of Lycii folium having high antioxidant activity. J Korean Soc Food Sci Nutr 2008; 37(10): 1318-1322. https://doi.org/10.3746/jkfn.2008.37.10.1318
  8. Chernomorsky SA, Segelman AB. Biological activities of chlorophyll derivatives. N J Med 1988; 85(8): 669-673.
  9. Solymosi K, Mysliwa-Kurdziel B. Chlorophylls and their derivatives used in food industry and medicine. Mini Rev Med Chem 2017; 17(13): 1194-1222.
  10. Skovsen E, Snyder JW, Lambert JD, Ogilby PR. Lifetime and diffusion of singlet oxygen in a cell. J Phys Chem B 2005; 109(18): 8570-8573. https://doi.org/10.1021/jp051163i
  11. Nurhayati N, Suendo V. Isolation of chlorophyll a from spinach leaves and modification of center ion with Zn2+: study on its optical stability. Matematika Sains, 2011; 16(2): 65-70.
  12. Ozkan G, Ersus Bilek S. Enzyme-assisted extraction of stabilized chlorophyll from spinach. Food Chem 2015; 176: 152-157. https://doi.org/10.1016/j.foodchem.2014.12.059
  13. Tanielian C, Wolff C. Mechanism of physical quenching of singlet molecular oxygen by chlorophylls and related compounds of biological interest. Photochem Photobiol 1988; 48(3): 277-280. https://doi.org/10.1111/j.1751-1097.1988.tb02821.x
  14. Sanchez-Valle V, Chavez-Tapia NC, Uribe M, Mendez-Sanchez N. Role of oxidative stress and molecular changes in liver fibrosis: a review. Curr Med Chem 2012; 19(28): 4850-4860. https://doi.org/10.2174/092986712803341520
  15. Feng Y, Wang N, Ye X, Li H, Feng Y, Cheung F, et al. Hepatoprotective effect and its possible mechanism of Coptidis rhizoma aqueous extract on carbon tetrachloride- induced chronic liver hepatotoxicity in rats. J Ethnopharmacol 2011; 138(3): 683-690. https://doi.org/10.1016/j.jep.2011.09.032
  16. Singal AK, Jampana SC, Weinman SA. Antioxidants as therapeutic agents for liver disease. Liver Int 2011; 31(10): 1432-1448. https://doi.org/10.1111/j.1478-3231.2011.02604.x
  17. Palma HE, Wolkmer P, Gallio M, Correa MM, Schmatz R, Thome GR, et al. Oxidative stress parameters in blood, liver and kidney of diabetic rats treated with curcumin and/or insulin. Mol Cell Biochem 2014; 386(1-2): 199-210. https://doi.org/10.1007/s11010-013-1858-5
  18. Lee CK, Kim NY, Han YN, Choi JW. Effects of pretreated Korean red ginseng on carbon tetrachloride and galactosamine-induced hepatotoxicity in rats. J Ginseng Res 2003; 27(1): 1-10 https://doi.org/10.5142/JGR.2003.27.1.001
  19. Folin O, Denis W. On phosphotungstic-phosphomolybdic compounds as color reagents. J Biol Chem 1912; 12: 239-243. https://doi.org/10.1016/S0021-9258(18)88697-5
  20. Blois MS. Antioxidant determinations by the use of a stable free radical. Nature 1958; 181(4617): 1199-1200. https://doi.org/10.1038/1811199a0
  21. Fellegrini N, Ke R, Yang M, Rice-Evans C. Screening of dietary carotenoids and carotenoid-rich fruit extracts for antioxidant activities applying 2,2′-azinobis (3-ethylenebenzothiazoline-6-sulfonic acid radical cation decolorization assay. Methods Enzymol 1999; 299: 379-389. https://doi.org/10.1016/S0076-6879(99)99037-7
  22. Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay. Anal Biochem 1996; 239(1): 70-76. https://doi.org/10.1006/abio.1996.0292
  23. Skotti E, Anastasaki E, Kanellou G, Polissiou M, Tarantilis PA. Total phenolic content, antioxidant activity and toxicity of aqueous extracts from selected Greek medicinal and aromatic plants. Ind Crops Prod 2014; 53: 46-54. https://doi.org/10.1016/j.indcrop.2013.12.013
  24. Liu SC, Lin JT, Hu CC, Shen BY, Chen TY, Chang YL, et al. Phenolic compositions and antioxidant attributes of leaves and stems from three inbred varieties of Lycium chinense Miller harvested at various times. Food Chem 2017; 215: 284-291. https://doi.org/10.1016/j.foodchem.2016.06.072
  25. Nirmal NP, Benjakul S. Use of tea extracts for inhibition of polyphenoloxidase and retardation of quality loss of Pacific white shrimp during iced storage. Lebenson Wiss Technol 2011; 44(4): 924-932. https://doi.org/10.1016/j.lwt.2010.12.007
  26. Rattaya S, Benjakul S, Prodpran T. Extraction, antioxidative, and antimicrobial activities of brown seaweed extracts, Turbinaria ornata and Sargassum polycystum, grown in Thailand. Int Aquat Res 2015; 7(1): 71-16.
  27. Olatunde OO, Benjakul S, Vongkamjan K. Antioxidant and antibacterial properties of guava leaf extracts as affected by solvents used for prior dechlorophyllization. J Food Biochem 2018; 42(5): e12600. https://doi.org/10.1111/jfbc.12600
  28. Benjakul S, Kittiphattanabawon P, Shahidi F, Maqsood S. Antioxidant activity and inhibitory effects of lead(Leucaena leucocephala) seed extracts against lipid oxidation in model systems. Food Sci Technol Int 2013; 19(4): 365-376. https://doi.org/10.1177/1082013212455186
  29. Khalaf NA, Shakya AK, Al-Othman A, El-Agbar Z, Farah H. Antioxidant activity of some common plants. Turk J Biol 2008; 32: 51-55.
  30. Swargiary A, Daimari A, Daimari M, Basumatary N, Narzary E. Phytochemicals, antioxidant, and anthelmintic activity of selected traditional wild edible plants of lower Assam. Indian J Pharmacol 2016; 48(4): 418-423. https://doi.org/10.4103/0253-7613.186212
  31. Benjakul S, Kittiphattanabawon P, Sumpavapol P, Maqsood S. Antioxidant activities of lead (Leucaena leucocephala) seed as affected by extraction solvent, prior dechlorophyllisation and drying methods. J Food Sci Technol 2014; 51(11): 3026-3037. https://doi.org/10.1007/s13197-012-0846-1
  32. Lanfer-Marquez UM, Barros RM, Sinnecker P. Antioxidant activity of chlorophylls and their derivatives. Food 2005; 38(8-9): 885-891. https://doi.org/10.1016/j.foodres.2005.02.012
  33. Sanchez-Gonzalez I, Jimenez-Escrig A, Saura-Calixto F. In vitro antioxidant activity of coffees brewed using different procedures (Italian, espresso and filter). Food Chem 2005; 90(1-2): 133-139. https://doi.org/10.1016/j.foodchem.2004.03.037
  34. Albishi T, John IA, Al-Khalifa AS, Shahidi F. Antioxidative phenolic constituents of skins of onion varieties and their activities. J Funct Foods 2013; 5(3): 1191-1203. https://doi.org/10.1016/j.jff.2013.04.002
  35. Yoo HJ, Ahn C, Narantuya L. Extractions of chlorophyll from spinach and mate powders and their dyeability on fabrics. J Korean Soc Clothing Text 2013; 37(3): 413-423. https://doi.org/10.5850/JKSCT.2013.37.3.413
  36. Conforti F, Ioele G, Statti GA, Marrelli M, Ragno G, Menichini F. Antiproliferative activity against human tumor cell lines and toxicity test on Mediterranean dietary plants. Food Chem Toxicol 2008; 46(10): 3325-3332. https://doi.org/10.1016/j.fct.2008.08.004
  37. Qi B, Ji Q, Wen Y, Liu L, Guo X, Hou G, et al. Lycium barbarum polysaccharides protect human lens epithelial cells against oxidative stress-induced apoptosis and senescence. PLoS One 2014; 9(10): e110275. https://doi.org/10.1371/journal.pone.0110275
  38. Ceccarini MR, Vannini S, Cataldi S, Moretti M, Villarini M, Fioretti B, et al. Codini M. In vitro protective effects of Lycium barbarum berries cultivated in Umbria(Italy) on human hepatocellular carcinoma cells. BioMed Res Int 2016; 2016: 7529521.
  39. Bobek P. Dietary tomato and grape pomace in rats: effect on lipids in serum and liver, and on antioxidant status. Br J Biomed Sci 1999; 56(2): 109-113.
  40. Erzurum SC, Lemarchand P, Rosenfeld MA, Yoo JH, Crystal RG. Protection of human endothelial cells from oxidant injury by adenovirus-mediated transfer of the human catalase cDNA. Nucleic Acids Res 1993; 21(7): 1607-1612. https://doi.org/10.1093/nar/21.7.1607
  41. Cohen G, Dembiec D, Marcus J. Measurement of catalase activity in tissue extracts. Anal Biochem 1970; 34(1): 30-38. https://doi.org/10.1016/0003-2697(70)90083-7

Cited by

  1. 염증유도 RAW264.7 세포와 동물모델에서 구기자와 구기엽의 항염 효능 vol.52, pp.2, 2019, https://doi.org/10.4163/jnh.2019.52.2.129
  2. 와송이 고지방 식이로 유도된 비만 쥐의 간내 지질 및 항산화 대사에 미치는 영향 vol.47, pp.4, 2019, https://doi.org/10.4014/mbl.1908.08008
  3. 국내 수집 야생버섯류 추출물의 생리활성 비교 vol.19, pp.1, 2021, https://doi.org/10.14480/jm.2021.19.1.41