Journal List > J Nutr Health > v.52(1) > 1117286

J Nutr Health. 2019 Feb;52(1):26-35. Korean.
Published online Feb 28, 2019.
© 2019 The Korean Nutrition Society
Antioxidant activity of ethanol extract of Lycium barbarum's leaf with removal of chlorophyll
Ji Eun Kim,1 Su Mi Bae,1 You Ree Nam,1 Eun Young Bae,1,2 and Sun Yung Ly1,2
1Department of Food and Nutrition, Chungam National University, Daejeon 34134, Korea.
2Convergence Research Center for Natural Products, Chungnam National University, Daejeon 34134, Korea.

To whom correspondence should be addressed. tel: +82-42-821-6838, Email:
Received Nov 21, 2018; Revised Dec 17, 2018; Accepted Dec 28, 2018.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.



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.


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 (H2O2)-stressed HepG2 cells.


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 µg/mL. All extracts inhibited ROS production in a concentration-dependent manner from 31.3 µg/mL and inhibited DNA damage at 250 µg/mL. The SOD and catalase activities of cell lines treated with the extracts and H2O2 were similar to those of normal cells, indicating a strong protective effect.


Lycium barbarum leaf extracts had high antioxidant activities and protected H2O2-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.

Keywords: antioxidant; Lycium barbarum; HepG2 cell; chlorophyll removal


Fig. 1
Effect of the Lycium barbarum leave's ethanol extracts (LL50, LL70, LL100, LL100 Ch-) on HepG2 cell viability. LL 50, 70 and 100: Lycium barbarum's leaf extracted with 50, 70 and 100% ethanol, respectively; Group LL100 Ch-: chlorophyll removal ethanol extract. Values are mean ± standard deviation of three replicate determinations (n = 3). Different letters above the bars indicate statistically significant differences (p < 0.05).
Click for larger image

Fig. 2
Effects of (A) 50% ethanol extracs, (B) 70% ethanol extract, (C) 100% ethanol extract of Lycium barbarum's leaf and (D) chlorophyll removal extract with 100% ethanol from Lycium barbarum's leaf on the production of intracellular ROS level in H2O2-treated HepG2 cells. Values are mean ± SD of three replicate determinations (n = 3). Different superscripts (a–c) in a column indicate significant differences at p < 0.05 by Duncan's multiple range test (*: p < 0.05, compared to treated only H2O2).
Click for larger image


Table 1
Chlorophyll a and total polyphenol contents of Lycium barbarum's leaf extracts
Click for larger image

Table 2
Antioxidant capacities of Lycium barbarum's leaf extracts
Click for larger image

Table 3
Levels of DNA damage expressed as tail DNA, tail length, and tail moment in HepG2 cells with or without Lycium barbarum's leaf extracts
Click for larger image

Table 4
Effects of Lycium barbarum's leaf extracts on the antioxidant enzyme activities in HepG2 cells
Click for larger image


This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1D1A3B03028628).

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.
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.
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.
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. Özkan G, Ersus Bilek S. Enzyme-assisted extraction of stabilized chlorophyll from spinach. Food Chem 2015;176:152–157.
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.
14. Sánchez-Valle V, Chávez-Tapia NC, Uribe M, Méndez-Sánchez N. Role of oxidative stress and molecular changes in liver fibrosis: a review. Curr Med Chem 2012;19(28):4850–4860.
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.
16. Singal AK, Jampana SC, Weinman SA. Antioxidants as therapeutic agents for liver disease. Liver Int 2011;31(10):1432–1448.
17. Palma HE, Wolkmer P, Gallio M, Corrêa MM, Schmatz R, Thomé 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.
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.
19. Folin O, Denis W. On phosphotungstic-phosphomolybdic compounds as color reagents. J Biol Chem 1912;12:239–243.
20. Blois MS. Antioxidant determinations by the use of a stable free radical. Nature 1958;181(4617):1199–1200.
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.
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.
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.
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.
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.
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):1–16.
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
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.
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.
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.
32. Lanfer-Marquez UM, Barros RM, Sinnecker P. Antioxidant activity of chlorophylls and their derivatives. Food 2005;38(8-9):885–891.
33. Sánchez-González I, Jiménez-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.
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.
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.
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.
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
38. Ceccarini MR, Vannini S, Cataldi S, Moretti M, Villarini M, Fioretti B, Codini M, et al. 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.
41. Cohen G, Dembiec D, Marcus J. Measurement of catalase activity in tissue extracts. Anal Biochem 1970;34(1):30–38.