Isolation of ginsenoside Rb1 from Kalopanax pictus by eastern blotting using anti-ginsenoside Rb1...

GINSENOSIDES IN ARALIACEOUS SPECIES 255

Copyright © 2005 John Wiley & Sons, Ltd. Phytother. Res. 19, 255–258 (2005)

Copyright © 2005 John Wiley & Sons, Ltd.

PHYTOTHERAPY RESEARCHPhytother. Res. 19, 255–258 (2005)Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1675

SHORT COMMUNICATIONIsolation of Ginsenoside Rb1 from Kalopanaxpictus by Eastern Blotting Using Anti-ginsenoside Rb1 Monoclonal Antibody

Hiroyuki Tanaka1*, Noriko Fukuda1, Shoji Yahara2, Susumu Isoda3, Chun-Su Yuan4 andYukihiro Shoyama1

1Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku,Fukuoka 812-8582, Japan2Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Kumamoto University, Oe-honmachi 5-1, Kumamoto 860-0973, Japan3Department of Pharmacognosy, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo142-8555, Japan4Tang Center for Herbal Medicine Research, The Pritzker School of Medicine, The University of Chicago, Chicago, IL, USA

Araliaceous species containing ginsenosides, the major active component in Panax species, were surveyedusing ELISA and eastern blotting with anti-ginsenoside Rb1 monoclonal antibody. Immunoassay-guidedfractionation of the methanol-soluble extracts of Araliaceous species led to the isolation of a known com-pound in the bark of Kalopanax pictus Nakai. The known compound was identified as ginsenoside Rb1 byspectroscopic methods and by comparison with an authentic sample. The result provided evidence that acombination of two immunoassays using monoclonal antibodies against ginsenosides is a powerful means ofsurveying new ginsenoside resources. Copyright © 2005 John Wiley & Sons, Ltd.

Keywords: Kalopanax pictus; Araliaceous species; ginsenoside Rb1; monoclonal antibody; ELISA; eastern blotting.

Received 3 July 2004Accepted 14 December 2004

* Correspondence to: Dr H. Tanaka, Department of Pharmacognosy,Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.E-mail: [email protected]/grant Sponsor: Ministry of Education, Culture, Sports, Scienceand Technology, Japan.Contract /grant sponsor: Shorai Foundation for Science and Technology.Contract/grant sponsor: Sasakawa Scientific Research Grant.Contract/grant sponsor: Kyushu University Foundation.Contract/grant sponsor: Japan Kampo Medicine ManufacturersAssociation.

INTRODUCTION

Ginsenosides (ginseng saponins) have a uniqueaglycone, dammarane framework, and are the majoractive components in Panax species. According todifferent aglycones, ginsenosides can be classifiedinto three types: the 20(S)-protopanaxadiol type [e.g.ginsenosides Rb1 (G-Rb1), Rc (G-Rc), Rb2 (G-Rb2),Rd (G-Rd); and malonyl ginsenosides Rb1 (MG-Rb1), Rb2 (MG-Rb2) and Rc (MG-Rc)], the 20(S)-protopanaxatriol type [e.g. ginsenosides Rg1 (G-Rg1),Rf (G-Rf) and Re (G-Re)], and the oleanolic acid type[e.g. ginsenoside Ro (G-Ro)] (Shoji, 1990). These phar-macological activities have been widely investigatedfor their effects on disturbances of the central nervoussystem, hypothermia and tumor metastasis, and for theirantioxidant, antidiabetes (Chang et al., 1998), antiagingand radioprotective effects. One major ginsenoside,ginsenoside Rb1 (G-Rb1), has been investigated for itseffects on the central nervous system (Saito, 1989) and

more recently, Chang et al. (1998) reported the effectof G-Rb1 on drug-induced memory impairment. Xieet al. also found that American ginseng berry extractreduced blood glucose levels in ob/ob mice and con-firmed that ginsenoside Re is the active factor in thisprocess (Xie et al., 2002). Therefore, ginsenosides havebeen found to be important resources in the develop-ment of new drugs.

Almost all Araliaceous species, including Panaxspecies, have been used as tonics in Asian folk medi-cine. Therefore, it is highly possible that they mightcontain common compounds such as ginsenoside,depending on their chemotaxonomical classification.Many analytical approaches have been used to identifyginsenosides, and of these, enzyme-linked immuno-sorbent assay (ELISA) appears to be the most promis-ing. Due to the importance of dammarane saponins,Sankawa et al. (1982) investigated immunologicalapproaches for assaying quantities of ginsenoside usingpolyclonal antibodies. In our ongoing preparationof monoclonal antibodies (MAb), an ELISA wasestablished for G-Rb1 (Tanaka et al., 1999) and G-Rg1(Fukuda et al., 2000a). Moreover, a new immunostainingmethod named eastern blotting has been developedfor ginsenosides using anti-G-Rb1 and -G-Rg1 MAbto standardize the quality of ginsengs (Fukuda et al.,2000b). Therefore, a combination of ELISA and easternblotting methods might serve as a new system for sur-veying ginsenoside resources. This study investigatedthe distribution of ginsenosides in Araliaceous speciesand isolated G-Rb1 from the bark of Kalopanax pictusNakai using ELISA and eastern blotting monitoring.

256 H. TANAKA ET AL.


MATERIALS AND METHODS

Materials. Polyvinylidene difluoride (PVDF) mem-branes (Immobilon-N) and glass microfiber filter sheets(GF/A) were purchased from Millipore Corporation(Bedford, MA, USA), and bovine (BSA) and human(HSA) serum albumins were provided by Pierce(Rockford, IL, USA). Peroxidase-labeled anti-mouseIgG was purchased from Organon Teknika CappelProducts (West Chester, PA, USA). G-Rb1, -Rc, -Rd,-Re and -Rg1 were purchased from Wako Pure Chem-ical Industries, Ltd (Osaka, Japan).

Sample preparation. Araliaceous plants were collectedfrom the herbal garden of the Faculty of Pharmaceut-ical Sciences, Kyushu University, Fukuoka, Japanin 1998. Dried stem bark samples (50 mg) of variousAraliaceous plants were powdered then extractedwith methanol (5 mL) under sonication five times.They were then filtered and the combined extractwas diluted with 20% methanol for ELISA and easternblotting.

Eastern blotting. Eastern blotting was conducted aspreviously reported (Fukuda et al., 2000b) with slightmodification. Samples were applied to two TLC platesand developed with n-BuOH–EtOAc–H2O (15:1:4).One developed TLC plate was dried and stained withH2SO4, another was dried then sprayed with a blottingsolution mixture of isopropanol–methanol–H2O (1:4:16,by volume). The latter was placed on a stainless steelplate then covered with a PVDF membrane sheet.After covering with a glass microfiber filter sheet, itwas pressed evenly for 50 s with a 120 °C hot plate aspreviously described with slight modification. The PVDFmembrane was separated from the plate and dried thendipped in water containing NaIO4 while stirring at roomtemperature for 1 h. After washing with water, 50 mmcarbonate buffer solution containing BSA was addedfollowed by stirring for 3 h. The PVDF membrane wasthen washed twice with PBS containing 0.05% Tween20 (TPBS) for 5 min then washed with water. It wasthen immersed in anti-G-Rb1 MAb and stirred at roomtemperature for 1 h. After washing twice with TPBSand water, 1000 times dilution of peroxidase-labeledgoat anti-mouse IgG in PBS containing 0.2% gelatin(GPBS) was added followed by stirring at roomtemperature for 1 h. The PVDF membrane was thenwashed twice with TPBS and water, and exposedto 1 mg/mL 4-chloro-1-naphthol-0.03% H2O2 in PBSsolution freshly prepared before use for 10 min at roomtemperature.

K. pictus Nakai was collected from the herbal gardenof the Faculty of Pharmaceutical Sciences, KyushuUniversity, Fukuoka, Japan in February 1998. The speci-men was then brought to the Faculty of PharmaceuticalSciences. The air-dried and powdered bark of K. pictus(480 g) was extracted with MeOH then the extract (23 g)was dissolved in H2O. The filtrate was subjected tocolumn chromatography over MCI gel-CHP-20P elutingwith H2O, 50% MeOH and 100% MeOH, successivelyafforded the crude saponin fraction (MeOH eluent,5.7 g), which was treated with 4% NaOH at 70 °C for2 h. After cooling, the reaction mixture was passedthrough MCI gel-CHP-20P, washed with H2O until

neutral, and eluted with MeOH. The MeOH eluentwas then passed through Chromatorex NH (-NH2 form)and eluted with MeOH. It was evaporated under re-duced pressure to give neutral glycosides (2.0 g). Thisportion was chromatographed over silica gel using aCHCl3–MeOH–H2O mixture (7:3:0.5), and finally overChromatorex ODS using 78% MeOH to give compound1 (1.5 mg). 1H-NMR (pyridine d5) d: 0.82, 1.12, 1.29,1.62 (3H, s), 0.97, 1.67 (6H, s), 4.92 (1H, d, J = 7.9 Hz),5.11 (1H, d, J = 7.9 Hz), 5.14 (1H, d, J = 7.9 Hz), 5.38(1H, d, J = 7.3 Hz), 5.53 (1H, br s). 13C-NMR (pyridined5) d: 39.2, 26.8, 89.0, 39.7, 56.4, 18.5, 35.2, 40.1,50.3, 37.0, 30.9, 70.2, 49.5, 51.4, 30.9, 26.7, 51.7, 16.3,16.1, 83.5, 22.5, 36.2, 23.2, 126.0, 131.1, 25.8, 18.0,28.1, 16.6, 17.4 (C1–30), 105.4, 83.5, 77.2*, 71.7, 78.4**,62.9*** (glc C1–6), 106.1, 77.1*, 79.3, 71.7, 78.4**,62.9*** (glc′ C1–6), 98.1, 75.3****, 78.4**, 71.7,78.0**, 70.3 (glc″ C1–6), 105.1, 74.9****, 78.3**, 71.7,78.1**, 62.8*** (glc′″ C1–6). (*, **, ***, ****: assign-ments are interchangeable).

Figure 1. (A) Eastern blotting profiles for a standardizedginsenosides mixture and MeOH extracts of variousAraliaceous species. (B) A TLC plate treated with 10% H2SO4.1: Acanthopanax japonicus; 2: Acanthopanax spinosus; 3:Acanthopanax sieboldianus; 4: Acanthopanax divaricatus;5: Acanthopanax hypoleucus; 6: Eleutherococcus senticosus;7: Acanthopanax sieboldianus; 8: Aralia elata; 9: Acanthopanaxsciadophylloides; 10: Dendropanax trifidus; 11: Aralia cordata;12: Fatsia japonica; 13: Kalopanax pictus.

GINSENOSIDES IN ARALIACEOUS SPECIES 257


Figure 2. (A) Eastern blotting profiles for a standardized ginsenosides mixture and each fractionseparated by immunoaffinity column chromatography using anti-ginsenoside Rb1 MAb. (B) ATLC plate treated with 10% H2SO4.

RESULTS AND DISCUSSION

The eastern blotting method was previously developedfor ginsenosides (Fukuda et al., 2000b). In easternblotting, ginsenosides blotted to the PVDF membraneswere treated with NaIO4 solution and reacted with pro-teins such as BSA. This reaction enhanced the fixingof ginsenosides via ginsenoside-BSA conjugates on thePVDF membrane. On the other hand, aglycone andpart of the sugar components can be stained by MAbas with western blotting. In the case of eastern blottingfor ginsenosides, it was found that ginsenosides withsmall cross-reactivities for anti-G-Rb1 MAb could stillbe stained, suggesting that the specific reactivity ofthe sugar moiety in the ginsenoside molecule againstMAb might be modified by NaIO4 treatment of theginsenoside on the membrane, allowing G-Rd, G-Rc,G-Re and G-Rg1 detection by eastern blotting. Thisphenomenon is important for surveys of saponins withthe same aglycone as ginsenosides.

The crude extracts of Araliaceous plant samples wereanalysed as indicated in Fig.1. The TLC profile stainedby H2SO4 revealed complicated spot patterns indicat-ing that ginsenosides could not be determined (Fig. 1A),but suggesting oleanane saponins as previously reported.On the other hand, eastern blotting clearly showedsome positive spots in lines 7, 8, 11, 12 and 13. Sincethe bark of Kalopanax pictus Nakai (line 13) indicateda positive band, the crude extract was analysed by com-petitive ELISA using anti-G-Rb1 MAb, resulting

in 0.0009% dry wt. of G-Rb1. Since K. pictus Nakai(line 13) seemed to contain higher concentrations ofginsenoside, which might cross-react with anti-G-Rb1MAb, the crude extract was purified by silica gelcolumn chromatography using eastern blotting with anti-G-Rb1 MAb as indicated in Fig.2.

The band of compound 1 was identified with thatof G-Rb1 by eastern blotting and the physical andspectroscopic data (1H- and 13C-NMR) of compound 1were identical to that of authentic G-Rb1. The stembark and leaves were analysed separately by competi-tive ELISA and a higher concentration of G-Rb1 wasfound in the leaves (0.0037% dr. wt.) than in the bark(0.0009% dr. wt.). To our knowledge, this is the firstisolation of G-Rb1 from K. pictus although Porzel et al.(1992) and Shao et al. (1989) reported the isolationof oleanane saponins. It is suggested that the leavesof K. pictus might be a new resource of G-Rb1, andmoreover the immunoaffinity column conjugated withanti-G-Rb1 MAb (Fukuda et al., 2000b) might besuitable for purification of G-Rb1 in the final stage ofpurification.

Acknowledgements

This paper was supported in part by a Grant-in-Aid from the Ministryof Education, Culture, Sports, Science and Technology, Japan, theShorai Foundation for Science and Technology, the Sasakawa Scien-tific Research Grant from the Japan Science Society, the researchfund of Kyushu University Foundation, and the research fund ofJapan Kampo Medicine Manufacturers Association.

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Fukuda N, Tanaka H, Shoyama Y. 2000b. Applications of ELISA,Western blotting and immunoaffinity concentration forsurvey of ginsenosides in crude drugs of Panax speciesand traditional Chinese herbal medicines. Analyst 125: 1425–1429.

Porzel A, Van Song T, Schmidt J, Lischewski M, Adam G.1992. Studies on the chemical-constituents of Kalopanax-septemlobus. Planta Med 58: 481–482.

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