Establishment of an Enzyme-Linked Immunosorbent Assay and Application on Determination of Ginsenoside Re in Human Saliva

Abstract

This work describes an immunochemical approach for the quality control of Panax ginseng and a pharmacological study of ginsenoside Re, a major bioactive constituent in P. ginseng, using an enzyme-linked immunosorbent assay. A hybridoma secreting monoclonal antibody against ginsenoside Re was produced by fusing splenocytes immunized with a ginsenoside Re-bovine serum albumin conjugate with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma SP2/0 cell line. The method, at an effective measuring range of 7.8–500 ng · mL⁻¹ of ginsenoside Re, successfully detected ginsenoside Re in Chinese traditional herb prescriptions. The results demonstrate that we generated a novel and reliable assay system for measuring ginsenoside Re in Chinese medicines more efficiently. Futhermore, we determined the ginsenoside Re concentrations in the saliva of six healthy adults after the oral administration of a ginseng capsule to study the pharmacokinetics of ginsenoside Re in human saliva.

Key words

ginsenoside Re - ginsenoside Rg1 - monoclonal antibody - enzyme-linked immunosorbent assay - human saliva

^* These two authors contributed equally to this work.

• References

• 1 Kim EH, Kim IH, Ha JA, Choi KT, Pyo S, Rhee DK. Antistress effect of red ginseng in brain cells is mediated by TACE repression via PADI4. J Ginseng Res 2013; 37: 315-323
• 2 Yu CH, Wang CZ, Yuan CS. Progress in anti-cancer research of American ginseng: with an example of colorectal cancer. Yao Xue Xue Bao 2013; 48: 986-992
• 3 Mucalo I, Jovanovski E, Rahelic D, Bozikov V, Romic Z, Vuksan V. Effect of American ginseng (Panax quinquefolius L.) on arterial stiffness in subjects with type-2 diabetes and concomitant hypertension. J Ethnopharmacol 2013; 150: 148-153
• 4 Kim HJ, Kim P, Shin CY. A comprehensive review of the therapeutic and pharmacological effects of ginseng and ginsenosides in central nervous system. J Ginseng Res 2013; 37: 8-29
• 5 Lee H, Lee JY, Song KC, Kim J, Park JH, Chun KH, Hwang GS. Protective Effect of Processed Panax ginseng, Sun Ginseng on UVB-irradiated Human Skin Keratinocyte and Human Dermal Fibroblast. J Ginseng Res 2012; 36: 68-77
• 6 Lee MY, Seo CS, Shin IS, Kim YB, Kim JH, Shin HK. Evaluation of oral subchronic toxicity of soshiho-tang water extract: the traditional herbal formula in rats. Evid Based Complement Alternat Med 2013; 10: 1155
• 7 Lee JJ, Kim T, Cho WK, Ma JY. Antithrombotic and antiplatelet activities of soshiho-tang extract. BMC Complement Altern Med 2013; 13: 137
• 8 Tanaka H, Fukuda N, Shoyama Y. Formation of monoclonal antibody against a major ginseng component, ginsenoside Rb1 and its characterization. Cytotechnology 1999; 29: 115-120
• 9 Peng L, Sun S, Xie LH, Wicks SM, Xie JT. Ginsenoside Re: pharmacological effects on cardiovascular system. Cardiovasc Ther 2012; 30: 183-188
• 10 Chen LM, Zhou XM, Cao YL, Hu WX. Neuroprotection of ginsenoside Re in cerebral ischemia-reperfusion injury in rats. J Asian Nat Prods Res 2007; 10: 439-445
• 11 Xie JT, Mehendale SR, Li X, Quigg R, Wang X, Wang CZ, Wu JA, Aung HH, Rue PA, Bell GI, Yuan CS. Antidiabetic effect of ginsenoside Re in ob/ob mice. Biochim Biophys Acta 2005; 1740: 319-325
• 12 Song X, Chen J, Sakwiwatkul K, Li R, Hu S. Enhancement of immune responses to influenza vaccine (H3N2) by ginsenoside Re. Int Immunopharmacol 2010; 10: 351-356
• 13 Lee BB, Shim IS, Lee HJ, Hahm DH. Effect of ginsenoside Re on depression- and anxiety-like behaviors and cognition memory deficit induced by repeated immobilization in rats. J Microbiol Biotechnol 2012; 22: 708-720
• 14 Putalun W, Fukuda N, Tanaka H, Shoyama Y. A one-step immunochromatographic assay for detecting ginsenosides Rb1 and Rg1. Anal Bioanal Chem 2004; 378: 1338-1341
• 15 Morinaga O, Tanaka H, Shoyama Y. Detection and quantification of ginsenoside Re in ginseng samples by a chromatographic immunostaining method using monoclonal antibody against ginsenoside Re. J Chromatogr B Analyt Technol Biomed Life Sci 2006; 830: 100-104
• 16 Nan TG, Wu SQ, Zhao HW, Tan WM, Li ZH, Zhang QC, Wang BM. Development of a secondary antibody thio-functionalized microcantilever immunosensor and an ELISA for measuring ginsenoside Re content in the herb ginseng. Anal Chem 2012; 84: 4327-4333
• 17 Sritularak B, Morinaga O, Yuan CS, Shoyama Y, Tanaka H. Quantitative analysis of ginsenosides Rb1, Rg1, and Re in American ginseng berry and flower samples by ELISA using monoclonal antibodies. J Nat Med 2009; 63: 360-363
• 18 Yuan CS, Tanaka H. Bioactivity of American ginseng by knockout extract preparation using monoclonal antibody. Curr Drug Discov Technol 2011; 8: 32-41
• 19 Nokes DJ, Enquselassie F, Nigatu W, Vyse AJ, Cohen BJ, Brown DWG, Cutts FT. Has oral fluid the potential to replace serum for the evaluation of population immunity levels? A study of measles, rubella and hepatitis B in rural Ethiopia. Bull World Health Organ 2001; 79: 588-595
• 20 Raju KS, Taneja I, Singh SP. Wahajuddin. Utility of noninvasive biomatrices in pharmacokinetic studies. Biomed Chromatogr 2013; 27: 1354-1366
• 21 Teeninga N, Guan Z, Freijer J, Ruiter AF, Ackermans MT, Kist-van Holthe JE, van Gelder T, Nauta J. Monitoring prednisolone and prednisone in saliva: a population pharmacokinetic approach in healthy volunteers. Ther Drug Monit 2013; 35: 485-492
• 22 Kawaguchi M, Matsuo R. Problems in clinical tests using saliva and the future prospects for alternative methods for pharmacokinetic blood tests. Nihon Yakurigaku Zasshi 2013; 141: 295
• 23 Yuki D, Kikuchi A, Miura N, Kakehi A, Onozawa M. Good relationship between saliva cotinine kinetics and plasma cotinine kinetics after smoking one cigarette. Regul Toxicol Pharmacol 2013; 67: 240-245
• 24 Gorodischer R, Burtin P, Verjee Z, Hwang P, Koren G. Is saliva suitable for therapeutic monitoring of anticonvulsants in children: an evaluation in the routine clinical setting. Ther Drug Monit 1997; 19: 637-642
• 25 Drummer OH. Introduction and review of collection techniques and applications of drug testing of oral fluid. Ther Drug Monit 2008; 30: 203-206
• 26 Bosker WM, Huestis MA. Oral fluid testing for drugs of abuse. Clin Chem 2009; 55: 1910-1931
• 27 Weiler EW, Zenk MH. Radioimmunoassay for the determination of digoxin and related compounds in Digitalis lanata . Phytochemistry 1976; 15: 1537-1545
• 28 Wang SP, Hu YY, Tan W, Wu X, Chen R, Cao JL, Chen MW, Wang YT. Compatibility art of traditional Chinese medicine: from the perspective of herb pairs. J Ethnopharmacol 2012; 2: 412-423
• 29 Liu X, Wu WY, Jiang BH, Yang M, Guo DA. Pharmacological tools for the development of traditional Chinese medicine. Trends Pharmacol Sci 2013; 34: 620-628
• 30 Li X, Tang MH, Zhang F, Yuan Z, Chen LJ, Wang XH. Quantitative determination of ginsenoside Re and Rg1 in human plasma by ultra performance liquid chromatography-tandem mass spectrometry. Sichuan Da Xue Xue Bao Yi Xue Ban 2009; 40: 912-917
• 31 Lv C, Li O, Zhang X, He B, Xu H, Yin Y, Liu R, Liu J, Chen X, Bi K. Simultaneous quantitation of polygalaxanthone III and four ginsenosides by ultra-fast liquid chromatography with tandem mass spectrometry in rat and beagle dog plasma after oral administration of Kai-Xin-San: application to a comparative pharmacokinetic study. J Sep Sci 2014; 37: 1103-1110
• 32 Morinaga O, Uto T, Yuan CS, Tanaka H, Shoyama Y. Evaluation of a new eastern blotting technique for the analysis of ginsenoside Re in American ginseng berry pulp extracts. Fitoterapia 2010; 81: 284-288
• 33 Liu J, Ding JY, Zhou QY, He L, Wang ZT. Studies on influence of fungal elicitor on hairy root of Panax ginseng biosynthesis ginseng saponin and biomass. Zhongguo Zhong Yao Za Zhi 2004; 29: 302-305
• 34 Ishiyama M, Shoyama Y, Murakami H, Shinohara H. Production of monoclonal antibodies and development of an ELISA for solamargine. Cytotechnology 1996; 18: 153-158
• 35 Schreck RR, Warburton D, Miller OJ, Beiser SM, Erlanger BF. Chromosome structure as revealed by a combined chemical and immunochemical procedure. Proc Natl Acad Sci U S A 1973; 70: 804-807
• 36 Galfre G, Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol 1981; 73: 3-46
• 37 North SM, Styles JM, Hobbs SM, Dean CJ. Monoclonal antibodies to rat sarcomata. I. Immunization procedures and source of lymphoid cells for hybridoma production. Immunology 1982; 47: 397-405
• 38 Johnson DR. Murine monoclonal antibody development. Methods Mol Biol 1995; 51: 123-137
• 39 Liu HF, Ma J, Winter C, Bayer R. Recovery and purification process development for monoclonal antibody production. Landes Bioscience 2010; 10: 480-499