G-Rb1 concentration in various ginseng samples
1. Introduction
Worldwide demand of herbal medicines has increased in recent years owing to rising interest in the health benefits. Among with this, the quality control of plant extracts and plant-derived medicines is growing in importance to ensure their efficacy and safety. Effective quality control of the traditional Chinese medicines (TCM) and plant crude extracts requires the rapid and sensitive methods for separation and quantification of bioactive compounds. Various methods have been employed for the separation and quantification of certain constituents in medicinal plants or herbal medicines. However, the current methods in use are not necessarily optimal approaches. For example, separation and quantification of glycyrrhizin (GC), the main active constituent in licorice (
Immunoassay systems using monoclonal antibody (MAb) against drugs and small molecular weight bioactive compounds have become an important tool for studies on receptor binding assays, enzyme assays, and quantitative and qualitative analytical techniques both
In this chapter, we focus on the immunoaffinity purification to separate and concentrate the target bioactive compounds from the crude extract. Our approaches effectively succeeded one-step purification of target compounds by MAb-conjugated immunoaffinity column, which leads to high-sensitivity detection and isolation of target compounds. In addition, the immunoaffinity column can prepare the knockout (KO) extract which contains all components except an antigen molecule, and KO extract will be useful for the pharmacological investigation to reveal the real effects of bioactive compound in the crude extract.The information in this chapter may provide new insight into quality control of plant-derived medicines.
2. Preparation of anti-ginsenoside Rb1 immunoaffinity column and its application
Ginseng, the root of
Ginsenoside Rb1 (G-Rb1) is one of the main ginsenosides responsible for many pharmaceutical actions of ginseng [27]. G-Rb1 has various biological activities, including facilitating acquisition and retrieval of memory [28], scavenging free radicals [29], inhibition of calcium over-influx into neurons [30], and preserving the structural integrity of the neurons [31]. In order to develop efficient quality control of ginseng, we have prepared anti-G-Rb1 MAb, set up of enzyme-linked immunosorbent assay (ELISA), and a new immunostaining method named Eastern blotting [8,32]. Furthermore, we established an immunoaffinity column against G-Rb1 and its application for one-step isolation from crude extract of ginseng root [19,32]. Herein we describe the preparation of anti-G-Rb1 immunoaffinity column and it applications for identification and concentration of G-Rb1.
2.1. Preparation of MAb and immunoaffinity column against G-Rb1
2.1.1. Analytical methodology for determination of hapten number in antigen, hapten-carrier protein conjugate
The first step for the MAb production is the synthesis of a hapten-carrier protein conjugate. Bovine serum albumin (BSA) conjugated with G-Rb1 was produced for the preparation of specific MAb in mouse [8]. There had been no direct and appropriate methods for the determination of haptens conjugated carrier proteins without differential UV analysis, radiochemical or chemical methods. Therefore, immunization by the injection of hapten-carrier protein conjugate was unreliable. Wengatez
2.1.2. Preparation of anti-G-Rb1 MAb and ELISA as an assay system
A hybridoma-producing MAb reactive to G-Rb1 was obtained by general procedure and classified into IgG2b which had κ light chains [8]. The reactivity of IgG type MAb, 9G7 was tested by varying antibody concentration and by performing a dilution curve. The antibody concentration was selected for competitive ELISA. The free MAb following competition is bound to polystyrene microtiter plates precoated with G-Rb1-human serum albumin (HSA). Under these conditions, the full measurement range of the assay extends from 20 to 400 ng/mL. The cross-reactivity against G-Rc and G-Rd, which possess a diglucose moiety attached to the C-3 hydroxy group, were weak compared with G-Rb1 (0.024 and 0.020 %, respectively). G-Re and G-Rg1 showed no cross-reactivity (less than 0.005 %). It is evident that the MAb reacted only with a small number of structurally related G-Rb1 molecules, and very weakly and did not react with other steroidal compounds.
2.1.3. Preparation of anti-G-Rb1 immunoaffinity column and appropriate buffer systems for separation of G-Rb1
The purified IgG (10 mg) was treated by NaIO4 to give dialdehyde group in sugar moiety which was coupled to Affi-Gel Hz hydrazide gel resulting in a hydrozone-type immunoaffinity gel [32]. The immunoaffinity gel was packed into plastic mini-column (Figure 2). Due to examine the optimal conditions of adsorption and elution, 400 µg of G-Rb1 was dissolved in phosphate buffered saline (PBS) and loaded on anti-G-Rb1 affinity column. After washing with washing buffer (20 mM PB containing 0.5 M NaCl), various buffer solutions for elution were loaded on the column, and then the recovery efficiency was determined by ELISA. The G-Rb1 concentration was somewhat increased by eluting with a 20 mM phosphate buffer containing 0.5 M KSCN and 10 % MeOH. When the 20 mM phosphate buffer was changed to 100 mM AcOH buffer (pH 4), the elution ability reached the optimal level. Although 20 % MeOH could enhance the elution of G-Rb1, higher MeOH concentration of over 20 % was ineffective. Thus, 100 mM AcOH buffer containing 0.5 M KSCN and 20 % MeOH could be used as an elution buffer in the immunoaffinity chromatography.
2.2. Purification of G-Rb1 by immunoaffinity column
2.2.1. One-step purification of G-Rb1 from crude extract of P. ginseng roots by anti-G-Rb1 immunoaffinity column
A crude extract (3.8 mg) of
This methodology is effective for the rapid and simple purification of G-Rb1 and may open up a wide field of comparable studies with other families of saponins for which an acceptable method for one-step separation has not yet been developed. Furthermore, to separate the total ginseng saponins, a wide cross-reactive MAb against ginsenoside, like anti G-Re MAb which showed wide cross-reactivity, could be designed [34]. A combination of immunoaffinity column, Eastern blotting and ELISA could be used to survey low concentrations of ginsenoside Rb1 of plant origin and/or in experimental animals and human. In fact we have succeeded in the isolation of G-Rb1 from a different plant,
2.2.2. Isolation and determination of unknown compounds related to G-Rb1 by anti-G-Rb1 immunoaffinity column
Several species of ginseng are known to exist and contain different amount and kinds of ginsenosides.
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To clarify the unknown compounds bound to anti-G-Rb1 MAb, the crude extract of
Compound 1 has three sugar moieties in a molecule because that the
These data suggested that the anti-G-Rb1 immunoaffinity column could isolate some unknown structurally resemble compounds having cross-reactivity against anti-G-Rb1MAb. Therefore, this purification system will be applied to survey new compounds related to target compound of MAb. In our previous studies, we demonstrated the immunoaffinity purification against all solasodine glycosides from crude extract by one-step purification. In this case, all solasodine glycoside have almost same cross-reactivity against anti-solamargine MAb [12].
2.2.3. Preparation of G-Rb1 knockout extract by anti-G-Rb1 immunoaffinity column
The capacity of this anti-G-Rb1immunoaffinity column is 20 µg of G-Rb1/ml gel [32]. By loading the samples not to exceed the binding capacity against G-Rb1, this immunoaffinity column becomes possible to remove all G-Rb1 from crude ginseng extract. Figure 6 showed H2SO4 staining of TLC of the purification steps by the immunoaffinity column. Lane 1 and 2 were spotted the standard of ginsenosides (G-Rd, G-Rc, G-Rb1, G-Rg1, and G-Re). Lane A, B, and C were the crude extract, the washing fraction, and the eluted fraction, respectively. In the crude extract (lane A), all spots of ginsenosides were clearly detected. On the other hand, the washing fraction (lane B) contained all of the ginsenosides in the crude extract except G-Rb1. Furthermore, the spot of G-Rb1 was detected in the eluted fractions (lane C). These data strongly indicated that G-Rb1 molecule in the ginseng extract can be eliminated by an anti-G-Rb1 immunoaffinity column and the washing fractions was knockout only by the antigen molecule, G-Rb1. Thus, we named the washing fractions a knockout (KO) extract [39,40]. This KO extract may be useful for the determination of real pharmacologically active principle in the TCMs.
3. Glycyrrhizin-knockout extract and its application for in vitro assay
Licorice (
3.1. Preparation of GC-KO extract by anti-GC immunoaffinity column and the characterization of GC-KO extract
Our previous study demonstrated the preparation of anti-GC MAb [11]. The cross-reactivities of the anti-GC MAb against glycyrrhetic acid-3-
The immunoaffinity column against GC was prepared by coupling the purified 60 mg of the anti-GC MAb to 25 ml of an Affi-Gel Hz gel [11]. To eliminate GC from licorice extract, 12 mg of licorice crude extract (GC content: 1275.0 µg) in loading buffer (5 % MeOH) was applied on the anti-GC MAb immunoaffinity column, and then the loading buffer was continuously circulated through the column to enhance the binding efficiency. After overnight circulation at 4 °C, the unbound fraction was separated. The column was washed with washing buffer (5 % MeOH) and then eluted with elution buffer (20 mM phosphate buffer containing 30 % MeOH). After separation, each fraction was deionized and the solvent was lyophilized. Figure 7 showed the recovery ratio of GC checked by ELISA. In the unbound fraction, 3.50 µg of GC (0.27% of the applied GC) was detected. On the other hand, 1269.26 µg of GC (99.55% of the applied GC) was obtained in the bound fraction. These data indicate that the anti-GC column could eliminate 99.55 % of the loading GC. Thus, we named this unbound fraction “GC-knockout (GC-KO) extract” [50].
To further characterize GC-KO extract, the TLC analysis and Eastern blotting were performed [50]. As shown in Figure 8A, several spots including GC were detected in licorice extract (Lane B). However, the spot of GC was completely disappeared in GC-KO extract, although all other spots were clearly detected (lane C). Eastern blotting by anti-GC MAb indicated that GC was detected in licorice extract (Figure 8B, lane B), but the spot of GC was disappeared in GC-KO extract (Figure 8B, lane C). Therefore, these data suggest that GC was specifically eliminated from licorice extract by anti-GC MAb immunoaffinity column.
3.2. in vitro Assay by GC-KO extract prepared by anti-GC immunoaffinity column
Nitric oxide (NO), synthesized by NO synthase (NOS) from L-arginine, is an important regulatory/modulatory mediator for several physiological processes [51]. However, during inflammatory process, a large amount of NO is produced by inducible NOS (iNOS) stimulated by bacterial lipopolysaccharide (LPS) and inflammatory cytokines participates in the pathogenesis of inflammatory diseases [52]. Overproduced NO synthesized by iNOS triggers a variety of inflammatory diseases including sepsis, psoriasis, arthritis, multiple sclerosis, and systemic lupus
In LPS-treated mouse RAW264 macrophages, licorice extract inhibited NO production and iNOS expression. At 100 µg/mL of licorice extract, iNOS protein and mRNA were completely suppressed [50].
We next examined the inhibitory effect of GC alone, GC-KO extract and the combined treatment with GC and GC-KO extract on NO production [50]. Since 100 µg of licorice extract contains 10.6 ±0.618 µg of GC,the cells were pre-treated with licorice extract (100 µg/ml), GC-KO extract (89.4 µg/ml), or the combination of GC-KO extract (89.4 µg/ml) and GC (10.6 µg/ml). Figure9A indicated that the treatment of licorice extract led to a marked suppression of NO production as compared to LPS treatment [inhibition ratio (IR) 57.7% ]. Interestingly, the inhibitory effect of GC-KO extract was lower (IR 17.8% ) compared with licorice extract although GC alone could not block NO production as indicated above. On the other hand, the combined treatment with GC-KO extract and GC significantly improved the inhibitory ability (IR 33.5% ). To determine whether the combinational effect of GC-KO extract and GC was related to iNOS expression, we performed Western blotting. As shown in Figure 9B, the treatment of GC-KO extract diminished the inhibitory ability of LE on iNOS expression, and addition of GC to GC-KO extract could improve it. These data suggest that GC alone cannot suppress iNOS expression, but combinational inhibition of iNOS expression may occur when GC coexists with the other constituents contained in licorice extract. The
4. Conclusion
In this chapter, we introduce the unique strategy of one-step purification of target compounds from crude extract by anti-natural compound specific MAb-conjugated immunoaffinity column. The immunoaffinity column conjugated with anti-G-Rb1 MAb could purify the G-Rb1 from
Acknowledgments
This work was funded by the Asahi Beer Science Promoting Foundation and Takeda Science Foundation. The research in this paper was also supported in part by Sasakawa Scientific Research Grant from Japan Science Society and “Science and Technology Research Partnership for Sustainable Development (SATREPS)” supported by the Japan Science and Technology Agency (JST) and the Japan International Cooperation Agency (JICA).
References
- 1.
Ong E. S. 2002 Chemical assay of glycyrrhizin in medicinal plants by pressurized liquid extraction (PLE) with capillary zone electrophoresis (CZE). 25 13 825 831 - 2.
Tan T. W. Huo Q. Ling Q. 2002 Purification of glycyrrhizin from glycyrrhiza uralensis fisch with ethanol/phosphate aqueous two phase system. 24 17 1417 1420 - 3.
Sakata R. Shoyama Y. Murakami H. 1994 Production of monoclonal antibodies and enzyme immunoassay for typical adenylate cyclase activator, Forskolin. 16 2 101 108 - 4.
Xuan L. Tanaka H. Xu Y. Shoyama Y. 1999 Preparation of monoclonal antibody against crocin and its characterization. 29 1 65 70 - 5.
Lu Z. Morinaga O. Tanaka H. Shoyama Y. 2003 A quantitative ELISA using monoclonal antibody to survey paeoniflorin and albiflorin in crude drugs and traditional Chinese herbal medicines. 26 6 862 866 - 6.
Shoyama Y. Fukada T. Murakami H. 1996 Production of monoclonal antibodies and ELISA for thebaine and codeine. 19 1 55 61 - 7.
Kim J. S. Tanaka H. Shoyama Y. 2004 Immunoquantitative analysis for berberine and its related compounds using monoclonal antibodies in herbal medicines. 129 1 87 91 - 8.
Tanaka H. Fukuda N. Shoyama Y. 1999 Formation of monoclonal antibody against a major ginseng component, ginsenoside Rb1 and its characterization. ;29 1 115 120 - 9.
Fukuda N. Tanaka H. Shoyama Y. 2000 Formation of monoclonal antibody against a major ginseng component, ginsenoside Rg1 and its characterization Monoclonal antibody for a ginseng saponin. 34 3 197 204 - 10.
Zhu S. Shimokawa S. Tanaka H. Shoyama Y. 2004 Development of an assay system for saikosaponin a using anti-saikosaponin a monoclonal antibodies. 27 1 66 71 - 11.
Shan S. J. Tanaka H. Shoyama Y. 2001 Enzyme-linked immunosorbent assay for glycyrrhizin using anti-glycyrrhizin monoclonal antibody and an eastern blotting technique for glucuronides of glycyrrhetic acid. 73 24 5784 5790 - 12.
Ishiyama M. Shoyama Y. Murakami H. Shinohara H. 1996 Production of monoclonal antibodies and development of an ELISA for solamargine. 18 3 153 158 - 13.
Morinaga O. Tanaka H. Shoyama Y. 2000 Production of monoclonal antibody against a major purgative component, sennoside A, its characterization and ELISA. 125 8 1109 1113 - 14.
Morinaga O. Nakajima S. Tanaka H. Shoyama Y. 2001 Production of monoclonal antibodies against a major purgative component, sennoside B, their characterization and use in ELISA. 126 8 1372 1376 - 15.
Tanaka H. Goto Y. Shoyama Y. 1996 Monoclonal antibody based enzyme immunoassay for marihuana (cannabinoid) compounds. 17 4 321 342 - 16.
Loungratana P. Tanaka H. Shoyama Y. 2004 Production of monoclonal antibody against ginkgolic acids in Ginkgo biloba Linn. 32 2 33 48 - 17.
Yanagihara H. Sakata R. Minami H. Shoyama Y. Murakami H. 1996 Immunoaffinity column chromatography against forskolin using an anti-forskolin monoclonal antibody and its application. 335 1-2 63 70 - 18.
Putalun W. Tanaka H. Yukihira S. 1999 Rapid separation of solasodine glycosides by an immunoaffinity column using anti-solamargine monoclonal antibody. 31 1-2 151 156 - 19.
Fukuda N. Tanaka H. Shoyama H. 2000 Isolation of the pharmacologically active saponin ginsenoside Rb1 from ginseng by immunoaffinity column chromatography. 63 2 283 285 - 20.
Xu J. Tanaka H. Shoyama Y. 2007 One-step immunochromatographic separation and ELISA quantification of glycyrrhizin from traditional Chinese medicines. 850 1-2 53 58 - 21.
Gillis C. N. 1997 Panax ginseng pharmacology: a nitric oxide link? 54 1 1 8 - 22.
Liu C. X. Xiao P. G. 1992 Recent advances on ginseng research in China. 36 1 27 38 - 23.
Yu H. Zhang C. Lu M. Sun F. Fu Y. Jin F. 2007 Purification and characterization of new special ginsenosidase hydrolyzing multi-glycisides of protopanaxadiolginsenosides, ginsenosidase type I. 55 2 231 235 - 24.
Kitagawa I. Taniyama T. Yoshikawa M. Ikenishi Y. Nakagawa Y. 1989 Chemical studies on crude drug processing. IV. Chemical structures of malonyl-ginsenosides Rb1, Rb2, Re and Rd isolated from the root of Panax Ginseng C.A. Meyer. 37 11 2961 2970 - 25.
Tanaka O. 1989 Saponin-composition of Panax species. In: Shibata S, Ohtsuka Y, Saito H. (eds.) Recentadvances in ginseng studies. Tokyo Hirokawa Publishing 43 47 - 26.
Fuzzati N. 2004 Analysis methods of ginsenosides. 812 1-2 119 33 - 27.
Washida D. Kitanaka S. 2003 Determination of polyacetylenes and ginsenosides in Panax species using high performance liquid chromatography. 51 11 1314 1317 - 28.
Mook-Jung I. Hong H. S. Boo J. H. Lee K. H. Yun S. H. Cheong M. Y. Joo I. Huh K. Jung M. W. 2001 Ginsenoside Rb1 and Rg1 improve spatial learning and increase hippocampal synaptophysin level in mice. 63 6 509 515 - 29.
Lim J. H. Wen T. C. Matsuda S. Tanaka J. Maeda N. Peng H. Aburaya J. Ishihara K. Sakanaka M. 1997 Protection of ischemic hippocampal neurons by ginsenoside Rb1, a main ingredient of ginseng root. 28 3 191 200 - 30.
Liu M. Zhang J. 1995 Effects of ginsenoside Rb1 and Rg1 on synaptosomal free calcium level, ATPase and calmodulin in rat hippocampus. 108 7 544 547 - 31.
Jiang K. Y. Qian Z. N. 1995 Effects of Panax notoginseng saponins on posthypoxic cell damage of neurons in vitro. 16 5 399 402 - 32.
Fukuda N. Tanaka H. Shoyama Y. 2000 Applications of ELISA, western blotting and immunoaffinity concentration for survey of ginsenosides in crude drugs of Panax species and traditional Chinese herbal medicines. 125 8 1425 1429 - 33.
Wengatz I. Schmid R. D. Kreißig S. Wittmann C. Hock B. Ingendoh A. Hillenkamp F. 1992 Determination of the hapten density of immuno-conjugates by matrix-assisted UV laser desorption/ionization mass spectrometry. 25 11 1983 1997 - 34.
Morinaga O. Tanaka H. Shoyama Y. 2006 Detection and quantification of ginsenoside Re in ginseng samples by a chromatographic immunostaining method using monoclonal antibody against ginsenoside Re. 830 1 100 104 - 35.
Tanaka H. Fukuda N. Yahara S. Isoda S. Yuan C. S. Shoyama Y. 2005 Isolation of ginsenoside Rb1 from Kalopanax pictus by eastern blotting using anti-ginsenoside Rb1 monoclonal antibody. 19 3 255 258 - 36.
Yahara S. Kasai R. Tanaka O. 1977 New dammarane type saponins of leaves of Panax japonicus C.A. Meyer. (1). Chikusetsusaponins L5, L9a and L10. 25 8 2041 2047 - 37.
Morita T. Tanaka O. Kohda H. 1985 Saponin composition of rhizomes of Panax japonicus collected in South Kyushu, Japan, and its significance in oriental traditional medicine. 33 9 3852 3858 - 38.
Kohda H. Tanaka S. Yamaoka Y. Ohhara Y. 1991 Saponins from Amaranthus hypochondriacus. 39 10 2609 2612 - 39.
Tanaka H. Fukuda N. Shoyama Y. 2007 Eastern blotting and immunoaffinity concentration using monoclonal antibody for ginseng saponins in the field of traditional chinese medicines. 55 10 3783 3787 - 40.
Wang C. A. Shoyama Y. 2006 Herbal medicine: identification, analysis, and evaluationstrategies. In: Yuan CS, Bieber EJ, Bauer BA (eds)second edition.United Kingdom Informa Healthcare 51 70 - 41.
Kim S. C. Byun S. H. Yang C. H. Kim C. Y. Kim J. W. Kim S. G. 2004 Cytoprotective effects of Glycyrrhizae radix extract and its active component liquiritigenin against cadmium-induced toxicity (effects on bad translocation and cytochrome c-mediated PARP cleavage). 197 3 239 251 - 42.
Fuchikami J. Isohama Y. Sakaguchi M. Matsuda M. Kucota T. Akie Y,k. Fujino A. Miyata T. 2004 Effect of glycyrrhizin on late asthmatic responses induced by antigen inhalation in guinea pigs. 94 Suppl.1 251 - 43.
Asl M. N. Hosseinzadeh H. 2008 Review of pharmacological effects of Glycyrrhiza sp. and its bioactive compounds. 22 6 709 724 - 44.
Jakkula M. Boucher T. A. Beyendorff U. Conn S. M. Johnson J. E. Nolan C. J. Peine C. J. Albrecht J. H. 2004 A randomized trial of Chinese herbal medicines for the treatment of symptomatic hepatitis C. 164 12 1341 1346 - 45.
Yanagawa Y. Ogura M. Fujimoto E. 2004 Effects and cost of glycyrrhizin in the treatment of upper respiratory tract infections in members of the Japanese maritime self-defense force: Preliminary report of a prospective, randomized, double-blind, controlled, parallel-group, alternate-day treatment assignment clinical trial. 65 1 26 33 - 46.
Schalm S. W. Brouwer J. T. Bekkering F. C. van Rossum T. G. 1999 New treatment strategies in non-responder patients with chronic hepatitis C. 31 Suppl.1 1184 1188 - 47.
Coon J. T. Ernst E. 2004 Complementary and alternative therapies in the treatment of chronic hepatitis C: a systematic review. 40 3 491 500 - 48.
Chin Y. W. Jung H. A. Liu Y. Su B. N. Castoro J. A. Keller W. J. Pereira M. A. Kinghorn A. D. 2007 Anti-oxidant constituents of the roots and stolons of licorice (Glycyrrhiza glabra). 55 12 4691 4697 - 49.
Morinaga O. Fujino A. Tanaka H. Shoyama Y. 2005 An on-membrane quantitative analysis system for glycyrrhizin in licorice roots and traditional Chinese medicines. 383 4 668 672 - 50.
Uto T. Morinaga O. Tanaka H. Shoyama Y. 2012 Analysis of the synergistic effect of glycyrrhizin and other constituents in licorice extract onlipopolysaccharide-induced nitric oxide production using knock-out extract. 417 1 473 478 - 51.
Moncada S. Palmer R. M. Higgs E. A. 1991 Nitric oxide: physiology, pathophysiology, and pharmacology. 43 2 109 142 - 52.
Blantz R. C. Munger K. 2002 Role of nitric oxide in inflammatory conditions. 90 4 373 378 - 53.
Kröncke K. D. Fehsel K. Kolb-Bachofen V. 1998 Inducible nitric oxide synthase in human diseases. 113 2 147 156