Protective effect of Samultang and its four herbal plants against reactive oxygen species in vitro and cellular system

  • Nam, Mi Na (Department of Food Science and Nutrition, Pusan National University) ;
  • Lee, Ah Young (Department of Food Science, Gyeongnam National University of Science and Technology) ;
  • Sin, Seung Mi (Gyeongnam Oriental Anti-aging Institute) ;
  • Goo, Young-Min (Gyeongnam Oriental Anti-aging Institute) ;
  • Cho, Eun Ju (Department of Food Science and Nutrition, Pusan National University)
  • Received : 2019.01.18
  • Accepted : 2019.05.17
  • Published : 2019.09.01


Oxidative stress and overproduction of free radicals have been reported to be a major pathological hallmark of neurodegenerative diseases. Samultang has been known as a beneficial agent to treat liver disease and cardiovascular diseases. However, the anti-oxidant activities and neuro-protective effects of Samultang against oxidative stress still have not been evaluated yet. The aim of the present study was to investigate the anti-oxidant and protective effects of Samultang and its four herbal plants, Paeonia lactiflora (PL), Ligusticum striatum (LS), Rehmannia glutinosa (RG), and Angelica gigas (AG), in vitro system and in SH-SY5Y neuronal cells. The extracts of Samultang strongly increased the radical scavenging activities of 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical (${\cdot}OH$), and nitric oxide (NO) in a concentration-dependent manner. Furthermore, we investigated the protective effects of Samultang on cellular damage against oxidative stress induced by hydrogen peroxide ($H_2O_2$) in SH-SY5Y cells. Treatment with Samultang alleviated the oxidative stress from $H_2O_2$ by increasing the cell viability and decreasing the intracellular reactive oxygen species levels. Based on these results, we further investigated the radical scavenging effects of PL, LS, RG, and AG. In our results, PL had the highest DPPH, ${\cdot}OH$, and NO radical scavenging activities. Thus, PL has a crucial role in Samultang, which has anti-oxidative and neuro-protective effects. The present research suggests that Samultang and PL have protective roles against oxidative stress from $H_2O_2$-induced neuronal cell death.


  1. Agholome L, Lindstrom T, Kagedal K, Marcusson J, Hallbeck M. 2010. An in vitro model for neuroscience: Differentiation of SH-SY5Y cells into cells with morphological and biochemical characteristics of mature neurons. Journal of Alzheimer's Disease 20:1069-1082.
  2. Althaus JS, Oien TT, Fici GJ, Scherch HM, Sethy VH, VonVoigtlander PF. 1994. Structure activity relationships of peroxynitrite scavengers an approach to nitric oxide neurotoxicity. Research Communications in Chemical Pathology and Pharmacology 83:243-254.
  3. Aruoma OI. 1998. Free radicals, oxidative stress, and antioxidants in human health and disease. Journal of the American Oil Chemists' Society 75:199-212.
  4. Ashok BT, Ali R. 1999. The aging paradox: Free radical theory of aging. Experimental Gerontology 34:293-303.
  5. Butterfield DA, Sultana R. 2011. Methionine-35 of $A{\beta}(1-42)$: Importance for oxidative stress in Alzheimer disease. Journal of Amino Acids 2011:Article ID 198430.
  6. Cabiscol E, Tamarit J, Ros J. 2000. Oxidative stress in bacteria and protein damage by reactive oxygen species. International Microbiology 3:3-8.
  7. Cathcart R, Schwiers E, Ames BN. 1983. Detection of picomole levels of hydroperoxides using a fluorescent dichlorofluorescein assay. Analytical Biochemistry 134:111-116.
  8. Chen X, Guo C, Kong J. 2012. Oxidative stress in neurodegenerative diseases. Neural Regeneration Research 7:376-385.
  9. Cheung YT, Lau WK, Yu MS, Lai CS, Yeung SC, So KF, Chang RC. 2009. Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research. Neurotoxicology 30:127-135.
  10. Choi IY, Kim SJ, Kang TH, Lee BH, Lee JH, Lee JY, Kim HM, Hong SH, Um JY. 2006. Anti-inflammatory effect of Samultang in human mast cell line HMC-1. Oriental Pharmacy and Experimental Medicine 6:237-244.
  11. Choi MA, Kim ML, Park CS. 2008. The antibacterial and antioxidative activities of Samultang ingredient extracts. Korean Journal of Food and Cookery Science 24:52-58. [in Korean]
  12. Chung SK, Osawa T, Kawakishi S. 1997. Hydroxyl radical-scavenging effects of spices and scavenger from brown mustard (Brassica nigra). Bioscience, Biotechnology, and Biochemistry 61:118-123.
  13. Dawson TM, Dawson VL, Snyder SH. 1992. A novel neuronal messenger molecule in brain: the free radical, nitric oxide. Annals of Neurology 32:297-311.
  14. Floyd RA, Carney JM. 1992. Free radical damage to protein and DNA: Mechanisms involved and relevant observations on brain undergoing oxidative stress. Annals of Neurology 32:S22-S27.
  15. Fordel E, Thijs L, Martinet W, Lenjou M, Laufs T, Van Bockstaele D, Moens L, Dewilde S. 2006. Neuroglobin and cytoglobin overexpression protects human SH-SY5Y neuroblastoma cells against oxidative stress-induced cell death. Neuroscience Letters 410:146-151.
  16. Fulda S, Gorman AM, Hori O, Samali A. 2010. Cellular stress responses: cell survival and cell death. International Journal of Cell Biology 2010:1-23.
  17. Hatano T, Edamatsu R, Hiramatsu M, Mori A, Fujita Y, Yasuhara T, Yoshida T, Okuda T. 1989. Effects of the interaction of tannins with co-existing substances. VI: effects of tannins and related polyphenols on superoxide anion radical, and on 1,1-diphenyl-2-picrylhydrazyl radical. Chemical and Pharmaceutical Bulletin 37:2016-2021.
  18. Heo JI, Kim JH, Lee JM, Kim SC, Park JB, Kim J, Lee JY. 2013. Antioxidant activity and its mechanisms of Paeonia lactiflora Pall extract. Natural Product Sciences 19:49-53.
  19. Kang TH, Baek HY, Kim YC. 2000. Protective effect of jakyak-gamcho-tang extract and its consituents against tBHP-induced oxidative stress damage in HT22 cells. The American Journal of Chinese Medicine 33:181-189.
  20. Kim DH, Kim SM, Cho HG, Cha YS, Heo Y, Moon BS, Cho KH. 2000. Effects of Samul-tang on nitric oxide induced-cytotoxicity in C6 glial cell. The Journal of Korean Oriental Internal Medicine 21:535-542. [in Korean]
  21. Kim JH, Lee JK, Ha HK, Seo CS, Lee HY, Jung DA, Lee NH, Lee JA, Huang DS, Shin HK. 2009. Analysis of studies on Samul-tang for fundamental establishment of evidence based medicine. Journal of Physiology & Pathology in Korean Medicine 23:779-788. [in Korean]
  22. Kim YC, Lee JH, Woo HJ. 2003. Effect of Samul-tang (Siwu-tang) on procollagen synthesis in cultured murine hepatic non-parenchymal cells. Korean Journal of Oriental Medicine 24:120-126. [in Korean]
  23. Klepac N, Relja M, Klepac R, Heimovi S, Babi T, Trkulja V. 2007. Oxidative stress parameters in plasma of Huntington's disease patients, asymptomatic Huntington's disease gene carriers and healthy subjects. Journal of Neurology 254:1676-1683.
  24. Labbadia J, Morimoto RI. 2013. Huntington's disease: Underlying molecular mechanisms and emerging concepts. Trends in Biochemical Sciences 38:378-385.
  25. Lee G, Joo JC, Choi BY, Lindroth AM, Park SJ, Park YJ. 2016. Neuroprotective effects of Paeonia Lactiflora extract against cell death of doparminergic SH-SY5Y cells is mediated by epigenetic modulation. BMC Complementary and Alternative Medicine 16:208.
  26. Lee GW, Kim MS. 2009. Water extract of Samultang reduces apoptotic cell death by $H_{2}O_{2}$-indcued oxidative injury in SK-N-MC cells. Korean Journal of Physiology & Pharmacology 13:139-145.
  27. Lee HW, Kim H, Ryuk JA, Kil KJ, Ko BS. 2014. Hemopoietic effect of extracts from constituent herbal medicines of Samul-tang on phenylhydrazine-induced hemolytic anemia in rats. International Journal of Clinical and Experimental Pathology 7:6179-6185.
  28. Lee IC, Kim BH, Kim MK. 2010. Biological activity and single oral dose toxicity of Dansam-samultang. Laboratory Animal Research 26:385-391. [in Korean]
  29. Lehtinen MK, Bonni A. 2006. Modeling oxidative stress in the central nervous system. Current Molecular Medicine 6:871-881.
  30. Lopes FM, Schroder R, da Frota ML Jr, Zanotto-Filho A, Muller CB, Pires AS, Meurer RT, Colpo GD, Gelain DP, Kapczinski F, Moreira JC, Fernandes Mda C, Klamt F. 2010. Comparison between proliferative and neuron-like SH-SY5Y cells as an in vitro model for Parkinson disease studies. Brain Research 1337:85-94.
  31. Marcocci L, Maguire JJ, Droylefaix MT, Packer L. 1994. The nitric oxide-scavenging properties of Ginkgo biloba extract EGb761. Biochemical and Biophysical Research Communications 201:748-755.
  32. Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assay. Journal of Immunological Methods 65:55-63.
  33. Ray PD, Huang BW, Tsuji Y. 2012. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cellular Signalling 24:981-990.
  34. Ryu JH, Kim MS, Hwang YS, Yook CS. 2001. Anxiolytic effects of the three kinds of traditional Chinese medicine, Shin-Ki-Hwan, Bo-Jung-Ik-Ki-Tang, and Sa-Mul-Tang, using the elevated plus-maze test. Biomolecules & Therapeutics 9:125-130. [in Korean]
  35. Sainani GS, Manika JS, Sainani RG. 1997. Oxidative stress: A key factor in pathogenesis of chronic diseases. Med Update 1:1-5.
  36. Shin MK. 1996. New clinical therapies of oriental traditional medicine. pp. 389-390. Younglim Press, Seoul, Korea. [in Korean]
  37. Stanely Mainzen Prince P, Menon VP. 2001. Antioxidant action of Tinospora cordifolia root extract in alloxan diabetic rats. Phytotherapy Research 15:213-218.
  38. Szuster-Ciesielska A, Daniluk J, Kandefer-Szerszen M. 2001. Alcohol-related cirrhosis with pancreatitis. The role of oxidative stress in the progression of the disease. Archivum Immunologiae et Therapiae Experimentalis 49:139-146.
  39. Tagami K, Niwa K, Lian Z, Gao J, Mori H, Tamaya T. 2004. Preventive effect of Juzen-taiho-to on endometrial carcinogenesis in mice is based on Shimotsu-to constituent. Biological and Pharmaceutical Bulletin 27:156-161.
  40. Um JN, Min JW, Joo KS, Kang HC. 2017. Antioxidant, anti-wrinkle activity and whitening effect of fermented mixture extracts of Angelica gigas, Paeonia lactiflora, Rehmannia chinensis and Cnidum officinale. Korean Journal of Medicinal Crop Science 25:152-159. [in Korean]
  41. Wang KH, Lin RD, Hsu FL, Huang YH, Chang HC, Huang CY, Lee MH. 2006. Cosmetic applications of selected traditional Chinese herbal medicines. Journal of Ethnopharmacology 106:353-359.
  42. Watanabe H, Ni JW, Ohta H, Ni XH, Matsumoto K. 1991. A kampo prescription, shimotsu-to, improves scopolamine-induced spatial cognitive deficits in rats. Japanese Journal of Psychopharmacology 11:215-222.