Green tea extract protects endothelial progenitor cells from oxidative insult through reduction of intracellular reactive oxygen species activity

Document Type: Original Article


1 Medical Research Center, Faculty of Medicine, Maranatha Christian University, Jl. Prof. Drg. Suria Sumantri 65, Bandung 40164, West Java, Indonesia

2 Biomolecular and Biomedical Research Center, Aretha Medika Utama, Jl. Babakan Jeruk 2 no 9, Bandung, 40163, West Java, Indonesia

3 Faculty of Pharmacy, Pancasila University, Jl Jagakarsa, Jakarta, Indonesia

4 Department of Medical Chemistry and Biochemistry, Faculty of Medicine, Lambung Mangkurat University, Banjarmasin, South Kalimantan, Indonesia

5 Department of Pharmacology, Faculty of Medicine, Brawijaya University, Jl. Veteran, Malang, 65145, East Java, Indonesia

6 Stem Cell and Cancer Institute, JL. A. Yani no.2 Pulo Mas, Jakarta, 13210, Indonesia


Objective(s):Many studies have reported that tea consumption decreases cardiovascular risk, but the mechanisms remain unclear. Green tea is known to have potent antioxidant and free radical scavengingactivities. This study aimed to investigate whether green tea extract (GTE) can protect endothelial progenitors cells (EPCs) against oxidative stress through antioxidant mechanisms.
Materials and Methods: Mononuclear cells (MNCs) were isolated from peripheral blood by density gradient centrifugation with Ficoll. The cells were then plated on fibronectin-coated culture dishes. After 7 days of culture, EPCs were characterized as adherent cells double positive for DiI-ac-LDL uptake and lectin binding. EPCs were further identified by assessing the expression of CD34/45, CD133, and KDR. EPCs were then treated with hydrogen peroxide (H2O2) at doses of 50, 100, 200 µM and incubated with or without GTE (25 µg/ml). The intracellular reactive oxygen species (ROS) levels were detected by flow cytometry using a 2’,7’-dichlorofluorescein diacetate (DCF-DA) fluorescentprobe.
Results: GTE ameliorated the cell viability of EPCs induced by H2O2 at doses of 50, 100, 200 µM for about 25.47, 22.52, and 11.96% higher than controls, respectively. GTE also decreased the intracellular ROS levels of EPCs induced by H2O2 at doses of 50, 100, 200 µM for about 84.24, 92.27, and 93.72% compared to controls, respectively.
Conclusion: GTE improves cell viability by reducing the intracellular ROS accumulation in H2O2-induced EPCs.


1. Montorsi P, Ravagnani PM, Galli S, Ali SG, Briganti A, Salonia A, et al. The triad of endothelial dysfunction, cardiovascular disease, and erectile dysfunction: clinical implications. European Urology Supplements 2009; 8:58-66.

2. Versari D, Daghini E, Virdis A, Ghiadoni L, taddei S. Endothelial dysfunction as a target for prevention of cardiovascular disease. Diabetes Care 2009; 32:S314-S321.

3. Walter DH, Rittig K, Bahlmann FH, Kirchmair R, Silver M, Murayama T, et al. Statin therapy accelerates reendothelialization: A novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells. Circulation 2002; 105:3017–3024.

4. Sata M. Molecular strategies to treat vascular diseases: Circulating vascular progenitor cell as a potential target for prophylactic treatment of atherosclerosis. Circ J 2003; 67:983–991.

5. Siddique A, Shantsila E, Lip GYH, Varma C. Endothelial progenitor cells: what use for the Cardiologist. J  Angiogenesis Res 2010; 2:6.

6. Lenk K, Uhlemann M, Schuler G, Adams V. Role of endothelial progenitor cells in the beneficial effects of physical exercise on atherosclerosis and coronary artery disease. J Appl Physiol 2011; 111:321–328.

7. Mavromatis K, Aznaouridis K, Mheid IA, Veledar E, Dhawan S, Murrow JR, et al. Circulating proangiogenic cell activity is associated with cardiovascular disease risk. J Biomol Screening 2012; 17:1163-1170.

8. Hodgson JM, Croft KD. Tea flavonoids and cardiovascular health. Mol Aspects Med 2010; 31:495-502.

9. Hodgson JM. Tea flavonoids and cardiovascular disease. Asia Pac J Clin Nutr 2008; 17:288-290.

10. Amic D, Davidovic-Amic D, Beslo D, Rastija V, Lucic B, Trinajstic N. SAR and QSAR of the antioxidant activity of flavonoids.Curr Med Chem 2007; 14:827-845.

11. Saeed N, Khan MR, Shabbir M. Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Complementary and Altern Med 2012;12:1-12.

12. Widowati W, Ratnawati H, Mozefis T, Pujimulyani D, Yelliantty Y. Hypolipidemic and antioxidant effects of black tea extract and quercetin in atherosclerotic rats. Int J Med Pharm Sci  Eng 2013; 7:373-380.

13. Costa LM, Gouveia ST, Nobrega JA. Comparison of heating extraction procedures for Al, Ca, Mg and Mn in tea samples. Ann Sci 2002; 18:313–318.

14. Macfarlane A, Macfarlane I. The empire of tea: the remarkable history of the plant that took over the world. Newark: Audible Studios; 2010.

15. Rietveld A, Wiseman S. Antioxidant effects of tea: Evidence from human clinical trials. J Nutr 2003; 133:3285–3292.

16. Arab L, Khan F, Lam H. Tea consumption and cardiovascular disease risk1,2,3. Am J Clin 2013; 98:1651S-1659S.

17. Kokubo Y, Iso H, Saito I, Yamagishi K, Yatsuya H, Ishihara J,  et al. The impact of green tea and coffee consumption on the reduced risk of stroke incidence in japanese population. The Japan Public Health Center-Based Study Cohort. Stroke 2013; 44:1369-1373.

18. McCullough ML, Peterson JJ, Patel R, Jacques PF, Shah R, Dwyer JT. Flavonoid intake and cardiovascular disease mortality in a prospective cohort of US adults 1,2,3,4. Am J Clin Nutr 2012; 95:454–464.

19. Stangl V, Lorenz M, Stangl K. The role of tea and tea flavonoids in cardiovascular health. Mol Nutr Food Res 2006; 50:218-228.

20. Heiss C, Keen CL, Kelm M. Flavanols and cardiovascular disease prevention. European Heart J 2010; 31:2583–2592.

21. Mink PJ, Scrafford CG, Barraj LM, Harnack L, Hong CP, Nettleton JA, et al. Flavonoid intake and cardiovascular disease mortality : a prospective study in postmenopausal women 1,2,3,4.  Am J Clin Nutr 2007; 85:895–909.

22. Boyer J, Liu RH. Apple phytochemicals and their health benefits. Nutrition J 2004; 3:5.

23. Terao J, Kawai Y, Murota K. Vegetable flavonoids and cardiovascular disease. Asia Pac J Clin Nutr 2008; 17:291-293.

24. Manning J, Roberts JC. Analysis of catechin content of commercial green tea products. J Herbal Pharmacother 2003; 3:19-32.

25. Doss MX, Potta SP, Hescheler J, Sachinidis A. Trapping of growth factors by catechins: a possible therapeutical target for prevention of proliferative diseases. J Nutr Biochem 2005; 16:259-266.

26. Tiwari P, Kumar B, Kaur M, Kaur G, Kaur H. Phytochemical screening and extraction : A Review. Internationale Pharmaceutica Sciencia 2011; 1:98-106.

27. Widowati W, Wijaya L, Wargasetia TL, Bachtiar I, Yellianty, Laksmitawati DR. Antioxidant, anticancer, and apoptosis-inducing effects of Piper extracts in HeLa cells. J Exp Integr Med 2013; 3:225-230.

28. Widowati W, Herlina T, Ratnawati H, Mozef T, Risdian C. Antioxidant and platelet aggregation inhibitor activities of black tea (Camellia sinensis L.) extract and fractions. Med Plants 2011; 3:21-26.

29. Jang A, Liu X-D, Shin M-H, Lee B-D, Lee S-K, Lee J-H, et al. Antioxidative potential of raw breast meat from broiler chicks fed a dietary medicinal herb extract mix.  Poultry Sci 2008; 87:2382–2389.

30. Thring TSA, Hili P, Naughton DP. Anti-collagenase, anti-elastase and antioxidant activities of extracts from 21 plants. BMC Complement Altern Med 2009; 9:27. 

31. Etoundi CB, Kuaté D, Ngondi JL, Oben J. Anti-amylase, anti-lipase and antioxidant effects of aqueous extracts of some Cameroonian spices. J Nat Products 2010; 3:165-171.

32. Mishra A, Bapat MM, Tilak JC, Devasagayam TPA. Antioxidant activity of Garcinia indica (kokam) and its syrup. Curr Sci 2006; 91:90-93.

33. Adedapo AA, Jimoh FO, Afolayan AJ, Masika PJ. Antioxidant activities and phenolic contents of the methanol extracts of the stems of Acokanthera oppositifolia and Adenia gummifera. BMC Complement and Altern Med 2008; 8:54.

34. Chen J, Wang X, Zhu J, Shang Y, Guo X, Sun J. Effects of Ginkgo biloba extract on number and activity of endothelial progenitor cells from peripheral blood. J Cardiovasc Pharmacol 2004; 43:347–352.

35. Vasa M, Fichtlscherer S, Adler K, Aicher A, Martin H, Zeiher AM, et al. Increase in circulating endothelial progenitor cells by statin therapy in patients with stable coronary artery disease. Circulation 2001; 103:2885-2890.

36. Bickford PC, Tan J, Shytle RD, Sanberg CD, Elbadri N, Sanberg R. Nutraceuticals synergistically promote proliferation on human stem cells. Stem Cells and Dev 2006; 15:118-123.

37. Gu J, Wang CQ, Fan HH, Ding HY, Xie XL, Xu YM, et al. Effect of resveratrol on endothelial progenitor cells and their contributions to reendothelialization in intima-injured rats. J Cardiovasc Pharmacol 2006; 47:711-721.

38. Jie G,  Lin Z, Zhang L, Lv H,  He P,  Zhao B. Free radical scavenging effect of Pu-erh tea extracts and their protective effect on oxidative damage in human fibroblast cells. J Agric Food Chem 2006; 54:8058-8064.

39. Stolzing A, Scutt A. Effect of reduced culture temperature on antioxidant defences of mesenchymal stem cells. Free Rad Bio & Med 2006; 41:326-338.

40. Hristov M, Weber C. Endothelial progenitor cells: Characterization, pathophysiology, and possible clinical relevance. J Cell Mol Med 2004; 8:498-508.

41. Hirschi KK, Ingram DA, Yoder MC. Assessing identity,
phenotype, and fate of endothelial progenitor cells. Arterioscler Thromb Vasc Biol 2008; 28:1584-1595.

42. Suzuki K, Sakata N, Kitani A, Hara M, Hirose T, Hirose W, et al. Characterization of human monocytic cell line, U937, in taking up acetylated low-density lipoprotein and cholesteryl ester accumulation A flow cytometric and HPLC study. Biochim Biophys Acta 1990; 1042:210–216.

43. Malich G, Markovic B, Winder C. The sensitivity and specificity of the MTS tetrazolium assay for detecting the in vitro cytotoxicity of 20 chemicals using human cell lines. Toxicology 1997; 124:179-192.

44. Huang WY, Lin YR, Ho RF, Liu HY, Lin YS. Effects of water solutions on extracting green tea leaves. 2013; 2013:368350. 

45. Miguel F, Augusto AC, Gurgueira SA. Effect of acute vs chronic H2O2-induced oxidative stress on antioxidant enzyme activities. Free Radic Res 2009; 43:340-347.

46. Stone JR, Yang S. Hydrogen peroxide: a signaling messenger. Antioxid Redox Signal 2006; 8:243-270.

47. Gülden M, Jess A, Kammann J, Maser E, Seibert H. Cytotoxic potency of H2O2 in cell cultures: Impact of cell concentration and exposure time. Free Rad Biol Med 2010; 49:1298–1305.

48. Temple MD, Perrone GG, Dawes IW. Complex cellular responses to reactive oxygen species. Trends Cell Biol 2005; 15:319–326.

49. Finkel T, Holbrook NJ. Oxidants, oxidative stress and biology of ageing. Nature 2000; 408:239–247.

50. Lee SH, Kim JY, Yoo SY, Kwon SM. Cytoprotective effect of dieckol on human endothelial progenitor cells (hEPCs) from oxidative stress-induced apoptosis. Free Radic Res 2013; 47:526-534.

51. Dernbach E, Urbich C, Brandes RP, Hofmann WK, Zeiher AM, Dimmeler S. Antioxidative stress-associated genes in circulating progenitor cells: evidence for enhanced resistance against oxidative stress. Blood 2004; 104:3591–3597.

52. He T, Peterson TE, Holmuhamedov EL, Terzic A, Caplice NM, Oberley LW, Katusic ZS. Human endothelial progenitor cells tolerate oxidative stress due to intrinsically high expression of manganese superoxide dismutase. Arterioscler Thromb Vasc Biol 2004; 24:2021–2027.

53. Cao Y, Xu ZC, He XJ, Dang XQ, Yi ZW, Zeng XQ. Protective effects of cathecin on apoptosis of endothelial progenitor cells induced by H2O2 in rats. Zhongguo Dang Dai Er Ke Za Zhi 2009; 11:61-64.