Journal of Ginseng Research

The Korean Society of Ginseng (고려인삼학회)
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A novel protocol for batch-separating gintonin-enriched, polysaccharide-enriched, and crude ginsenoside-containing fractions from Panax ginseng

  • Rami Lee (Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University) ;
  • Han-Sung Cho (Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University) ;
  • Ji-Hun Kim (Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University) ;
  • Hee-Jung Cho (Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University) ;
  • Sun-Hye Choi (Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University) ;
  • Sung-Hee Hwang (Department of Pharmaceutical Engineering, College of Health Sciences, Sangji University) ;
  • Hyewon Rhim (Center for Neuroscience, Korea Institute of Science and Technology) ;
  • Ik-Hyun Cho (Department of Convergence Medical Science, Department of Science in Korean Medicine, Graduate School, Kyung Hee University) ;
  • Man-Hee Rhee (Laboratory of Physiology and Cell Signalling, Department of Veterinary Medicine, College of Veterinary Medicine, Kyungpook National University) ;
  • Do-Geun Kim (Dementia Research Group, Korea Brain Research Institute) ;
  • Hyoung-Chun Kim (Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University) ;
  • Seung-Yeol Nah (Ginsentology Research Laboratory and Department of Physiology, College of Veterinary Medicine, Konkuk University)
  • Received : 2022.07.26
  • Accepted : 2022.10.11
  • Published : 2023.05.01
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Abstract

Background: Ginseng contains three active components: ginsenosides, gintonin, and polysaccharides. After the separation of 1 of the 3 ingredient fractions, other fractions are usually discarded as waste. In this study, we developed a simple and effective method, called the ginpolin protocol, to separate gintonin-enriched fraction (GEF), ginseng polysaccharide fraction (GPF), and crude ginseng saponin fraction (cGSF). Methods: Dried ginseng (1 kg) was extracted using 70% ethanol (EtOH). The extract was water fractionated to obtain a water-insoluble precipitate (GEF). The upper layer after GEF separation was precipitated with 80% EtOH for GPF preparation, and the remaining upper layer was vacuum dried to obtain cGSF. Results: The yields of GEF, GPF, and cGSF were 14.8, 54.2, and 185.3 g, respectively, from 333 g EtOH extract. We quantified the active ingredients of 3 fractions: L-arginine, galacturonic acid, ginsenosides, glucuronic acid, lysophosphatidic acid (LPA), phosphatidic acid (PA), and polyphenols. The order of the LPA, PA, and polyphenol content was GEF > cGSF > GPF. The order of L-arginine and galacturonic acid was GPF >> GEF = cGSF. Interestingly, GEF contained a high amount of ginsenoside Rb1, whereas cGSF contained more ginsenoside Rg1. GEF and cGSF, but not GPF, induced intracellular [Ca2+]i transient with antiplatelet activity. The order of antioxidant activity was GPF > GEF = cGSF. Immunological activities (related to nitric oxide production, phagocytosis, and IL-6 and TNF-α release) were, in order, GPF > GEF = cGSF. The neuroprotective ability (against reactive oxygen species) order was GEF > cGSP > GPF. Conclusion: We developed a novel ginpolin protocol to isolate 3 fractions in batches and determined that each fraction has distinct biological effects.

Keywords

Acknowledgement

This study was supported by Konkuk University in 2022.

References

  1. Park HJ, Kim DH, Park SJ, Kim JM, Ryu JH. Ginseng in traditional herbal prescriptions. J Ginseng Res 2012;36(3):225-41. https://doi.org/10.5142/jgr.2012.36.3.225
  2. Jia L, Zhao YQ. Current evaluation of the millennium phytomedicine-ginseng (I): etymology, pharmacognosy, phytochemistry, market and regulations. Curr Med Chem 2009;16(19):2475-84. https://doi.org/10.2174/092986709788682146
  3. Tyler VE. What pharmacists should know about herbal remedies. Journal of the American Pharmaceutical Association 1996;NS36(1):29-37. https://doi.org/10.1016/S1086-5802(16)30004-3
  4. Shibata S, Fujita M, Itokawa H, Tanaka O, Ishii T. Studies on the constituents of Japanese and Chinese crude drugs. Xi. Panaxadiol, a sapogenin of ginseng roots. Chemical & Pharmaceutical Bulletin 1963;11:759-61. https://doi.org/10.1248/cpb.11.759
  5. Wagner-Jauregg T, Roth M. [On panaxol, a new constituent of "red" ginseng root]. Pharmaceutica Acta Helvetiae 1962;37:352-9.
  6. Ratan ZA, Youn SH, Kwak YS, Han CK, Haidere MF, Kim JK, Min H, Jung YJ, Hosseinzadeh H, Hyun SH. Adaptogenic effects of Panax ginseng on modulation of immune functions. J Ginseng Res 2021;45(1):32-40. https://doi.org/10.1016/j.jgr.2020.09.004
  7. Zhao B, Lv C, Lu J. Natural occurring polysaccharides from Panax ginseng C. A. Meyer: a review of isolation, structures, and bioactivities. Int J Biol Macromol 2019;133:324-36. https://doi.org/10.1016/j.ijbiomac.2019.03.229
  8. Lee SJ, In G, Han ST, Lee MH, Lee JW, Shin KS. Structural characteristics of a red ginseng acidic polysaccharide rhamnogalacturonan I with immunostimulating activity from red ginseng. J Ginseng Res 2020;44(4):570-9. https://doi.org/10.1016/j.jgr.2019.05.002
  9. Pyo MK, Choi SH, Shin TJ, Hwang SH, Lee BH, Kang J, Kim HJ, Lee SH, Nah SY. A simple method for the preparation of crude gintonin from ginseng root, stem, and leaf. J Ginseng Res 2011;35(2):209-18. https://doi.org/10.5142/jgr.2011.35.2.209
  10. Hwang SH, Lee BH, Choi SH, Kim HJ, Jung SW, Kim HS, Shin HC, Park HJ, Park KH, Lee Gintonin MK. A novel ginseng-derived lysophosphatidic acid receptor ligand, stimulates neurotransmitter release. Neurosci Lett 2015;584: 356-61. https://doi.org/10.1016/j.neulet.2014.11.007
  11. Choi SH, Jung SW, Lee BH, Kim HJ, Hwang SH, Kim HK, Nah SY. Ginseng pharmacology: a new paradigm based on gintonin-lysophosphatidic acid receptor interactions. Front Pharmacol 2015;6.
  12. Jeong SM, Lee JH, Kim S, Rhim H, Lee BH, Kim JH, Oh JW, Lee SM, Nah SY. Ginseng saponins induce store-operated calcium entry in Xenopus oocytes. Brit J Pharmacol 2004;142(3):585-93. https://doi.org/10.1038/sj.bjp.0705797
  13. Kanzaki T, Morisaki N, Shiina R, Saito Y. Role of transforming growth factorbeta pathway in the mechanism of wound healing by saponin from Ginseng Radix rubra. Br J Pharmacol 1998;125(2):255-62. https://doi.org/10.1038/sj.bjp.0702052
  14. Lee JH, Jeong SM, Lee BH, Kim JH, Ko SR, Kim SH, Lee SM, Nah SY. Effect of calmodulin on ginseng saponin-induced Ca2+-activated Cl- channel activation in Xenopus laevis oocytes. Archives of Pharmacal Research 2005;28(4):413-20. https://doi.org/10.1007/BF02977670
  15. Reay JL, Kennedy DO, Scholey AB. Single doses of Panax ginseng (G115) reduce blood glucose levels and improve cognitive performance during sustained mental activity. J Psychopharmacol 2005;19(4):357-65. https://doi.org/10.1177/0269881105053286
  16. Wei XY, Yang JY, Wang JH, Wu CF. Anxiolytic effect of saponins from Panax quinquefolium in mice. J Ethnopharmacol 2007;111(3):613-8. https://doi.org/10.1016/j.jep.2007.01.009
  17. Park IH, Kim NY, Han SB, Kim JM, Kwon SW, Kim HJ, Park MK, J H. Park Three new dammarane glycosides from heat processed ginseng. Archives of Pharmacal Research 2002;25(4):428-32. https://doi.org/10.1007/BF02976595
  18. Cho HJ, Choi SH, Kim HJ, Lee BH, Rhim H, Kim HC, Hwang SH, S Y. Nah Bioactive lipids in gintonin-enriched fraction from ginseng. J Ginseng Res 2019;43(2):209-17. https://doi.org/10.1016/j.jgr.2017.11.006
  19. Proctor JTA, Lee JC, Lee Ginseng SS. Production in Korea. Hortscience 1990;25(7):746-50. https://doi.org/10.21273/HORTSCI.25.7.746
  20. Jorgensen NR, Geist ST, Civitelli R, Steinberg TH. ATP- and gap junctiondependent intercellular calcium signaling in osteoblastic cells. The Journal of Cell Biology 1997;139(2):497-506. https://doi.org/10.1083/jcb.139.2.497
  21. Grynkiewicz G, Poenie M, Tsien A RY. New generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985;260(6):3440-50. https://doi.org/10.1016/S0021-9258(19)83641-4
  22. Arnao MB, Casas JL, Delrio JA, Acosta M, Garciacanovas F. An enzymatic colorimetric method for measuring naringin using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (abts) in the presence of peroxidase. Anal Biochem 1990;185(2):335-8. https://doi.org/10.1016/0003-2697(90)90304-R
  23. Prior RL, Hoang H, Gu L, Wu X, Bacchiocca M, Howard L, Hampsch-Woodill M, Huang D, Ou B, Jacob R. Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance capacity (ORAC(FL))) of plasma and other biological and food samples. J Agr Food Chem 2003;51(11):3273-9. https://doi.org/10.1021/jf0262256
  24. Endale M, Lee WM, Kamruzzaman SM, Kim SD, Park JY, Park MH, Park TY, Park HJ, Cho JY, Rhee MH. Ginsenoside-Rp1 inhibits platelet activation and thrombus formation via impaired glycoprotein VI signalling pathway, tyrosine phosphorylation and MAPK activation. Brit J Pharmacol 2012;167(1):109-27. https://doi.org/10.1111/j.1476-5381.2012.01967.x
  25. Wang J, Gao WY, Zhang J, Zuo BM, Zhang LM, Huang LQ. Production of ginsenoside and polysaccharide by two-stage cultivation of Panax quinquefolium L. cells. Vitro Cell Dev-Pl 2012;48(1):107-12. https://doi.org/10.1007/s11627-011-9396-x
  26. Slinkard K, Singleton VL. Total phenol analysis - automation and Comparison with manual methods. Am J Enol Viticult 1977;28(1):49-55. https://doi.org/10.5344/ajev.1977.28.1.49
  27. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem 1956;28(3):350-6. https://doi.org/10.1021/ac60111a017
  28. Bitter T, Muir HM. A modified uronic acid carbazole reaction. Anal Biochem 1962;4(4):330-&.
  29. Hyun SH, Kim SW, Seo HW, Youn SH, Kyung JS, Lee YY, In G, Park CK, C K. Han Physiological and pharmacological features of the non-saponin components in Korean Red Ginseng. J Ginseng Res 2020;44(4):527-37. https://doi.org/10.1016/j.jgr.2020.01.005
  30. Lee SM, Bae BS, Park HW, Ahn NG, Cho BG, Cho YL, Y S. Kwak characterization of Korean red ginseng (panax ginseng meyer): history, preparation method, and chemical composition. J Ginseng Res 2015;39(4):384-91. https://doi.org/10.1016/j.jgr.2015.04.009
  31. Cho YJ, Choi SH, Lee RM, Cho HS, Rhim H, Kim HC, Kim BJ, Kim JH, Nah SY. Protective effects of gintonin on reactive oxygen species-induced HT22 cell damages: involvement of LPA1 receptor-BDNF-AKT signaling pathway. Molecules 2021;26(14).
  32. Choi SH, Jung SW, Kim HS, Kim HJ, Lee BH, Kim JY, Kim JH, Hwang SH, Rhim H, Kim HC, et al. A brief method for preparation of gintonin-enriched fraction from ginseng. J Ginseng Res 2015;39(4):398-405. https://doi.org/10.1016/j.jgr.2015.05.002
  33. Kim HM, Song Y, Hyun GH, Long NP, Park JH, Hsieh YSY, S W. Kwon characterization and antioxidant activity determination of neutral and acidic polysaccharides from panax ginseng C. A. Meyer. Molecules 2020;25(4).
  34. Chen F, Huang GL. Antioxidant activity of polysaccharides from different sources of ginseng. Int J Biol Macromol 2019;125:906-8. https://doi.org/10.1016/j.ijbiomac.2018.12.134
  35. Irfan M, Jeong Dahye, Saba Evelyn, Kwon Hyuk-Woo, Shin Jung-Hae, Jeon BoRa, et al. Gintonin modulates platelet function and inhibits thrombus formation via impaired glycoprotein VI signaling. Platelets 2019;30(5):589-98. https://doi.org/10.1080/09537104.2018.1479033
  36. Jin YR, Yu JY, Lee JJ, You SH, Chung JH, Noh JY, Im JH, Han XH, Kim TJ, Shin KS, et al. Antithrombotic and antiplatelet activities of Korean red ginseng extract. Basic Clin Pharmacol 2007;100(3):170-5. https://doi.org/10.1111/j.1742-7843.2006.00033.x
  37. Lim DS, Bae KG, Jung IS, Kim CH, Yun YS, Song JY. Anti-septicaemic effect of polysaccharide from Panax ginseng by macrophage activation. The Journal of Infection 2002;45(1):32-8. https://doi.org/10.1053/jinf.2002.1007
  38. Shin JY, Song JY, Yun YS, Yang HO, Rhee DK, Pyo S. Immunostimulating effects of acidic polysaccharides extract of Panax ginseng on macrophage function. Immunopharmacology and Immunotoxicology 2002;24(3):469-82. https://doi.org/10.1081/IPH-120014730
  39. Hwang SH, Shin TJ, Choi SH, Cho HJ, Lee BH, Pyo MK, Lee JH, Kang J, Kim HJ, Park CW, et al. Gintonin, newly identified compounds from ginseng, is novel lysophosphatidic acids-protein complexes and activates G protein-coupled lysophosphatidic acid receptors with high affinity. Molecules and Cells 2012;33(2):151-62. https://doi.org/10.1007/s10059-012-2216-z
  40. Pyo MK, Choi SH, Shin TJ, Hwang SH, Lee BH, Kang J, Kim HJ, Lee SH, Nah SY. A simple method for the preparation of crude gintonin from ginseng root, stem, and leaf. J Ginseng Res 2011;35(2):209-18. https://doi.org/10.5142/jgr.2011.35.2.209
  41. Zuo X, Li Q, Y F, Ma LJ, Tian Z, Zhao M, Fan D, Zhao Y, Mao YH, Wan JB, et al. Ginsenosides Rb2 and Rd2 isolated from Panax notoginseng flowers attenuate platelet function through P2Y12-mediated cAMP/PKA and PI3K/Akt/Erk1/2 signaling. Food & Function 2021;12(13):5793-805. https://doi.org/10.1039/D1FO00531F
  42. Irfan M, Kim M, Rhee MH. Anti-platelet role of Korean ginseng and ginsenosides in cardiovascular diseases. J Ginseng Res 2020;44(1):24-32. https://doi.org/10.1016/j.jgr.2019.05.005
  43. Lee JG, Lee YY, Wu B, Kim SY, Lee YJ, Yun-Choi HS, Park JH. Inhibitory activity of ginsenosides isolated from processed ginseng on platelet aggregation. Die Pharmazie 2010;65(7):520-2.
  44. Seo JY, Lee CW, Choi DJ, Lee J, Lee JY, Y I. Park Ginseng marc-derived lowmolecular weight oligosaccharide inhibits the growth of skin melanoma cells via activation of RAW264.7 cells. Int Immunopharmacol 2015;29(2):344-53. https://doi.org/10.1016/j.intimp.2015.10.031