Keywords

1 Introduction

With the rapid development of the molecular biosciences and their biotechnological applications, immunoassays using monoclonal antibodies (MAbs) against drugs and low-molecular-weight bioactive compounds have become an important tool due to their specificity for receptor binding analyses, enzyme assays, and quantitative and qualitative analytical techniques in both animals and plants. The immunoblotting method is based on a western blotting technique that utilizes antigen–antibody binding properties and has provided a specific and sensitive detection method for higher molecule analytes, such as peptides and proteins. Previously, we prepared many MAbs against naturally occurring bioactive compounds such as forskolin (Sakata et al. 1994), crocin (Xuan et al. 1999) , solamargine (Ishiyama et al. 1996), opium alkaloids (Shoyama et al. 1996), marijuana compounds (Tanaka et al. 1996), ginsenosides (Tanaka et al. 1996; Fukuda et al. 2000a) , saikosaponin A (Zhu et al. 2004), paeoniflorin (Lu et al. 2003), sennosides (Morinaga et al. 2000; Morinaga et al. 2001) , ginkgolic acid (Loungratana et al. 2004), glycyrrhizin (Shan et al. 2001), and berberine (Kim et al. 2004) and established individual competitive enzyme-linked immunosorbent assays (ELISAs) as highly sensitive, specific, and simple methodologies. As an extension of this approach, an immunostaining method using anti-solamargine MAb was established by us (Tanaka et al. 1997).

Ginseng, the crude drug of the Panax ginseng root, is one of the most important components of traditional Chinese medicine (TCM) . It has been used to enhance stamina and the capacity to cope with fatigue and physical stress; it has been used as a tonic against cancers, disturbances of the central nervous system, and hypothermia; and it has been used to boost carbohydrate and lipid metabolism, immune function, the cardiovascular system, and radioprotections. Its major active components are the ginsenosides, which consist of protopanaxatriol and/or protopanaxadiol that has a dammarane skeleton in their molecules. It is well-known that the concentrations of ginsenosides vary in the ginseng root or the root extracts depending on the method of extraction, subsequent treatment (Kitagawa et al. 1989), or even the season of its collection (Tanaka 1989). Therefore, standardization of quality is required in TCM. Moreover, Panax japonicus is morphologically different from P. ginseng. It is evident that major components of ginsenosides are oleanane-type saponins such as chikusetsusaponins. Therefore, the content of ginsenosides might be low.

To control the quality of ginseng prescribed in TCM, we previously prepared anti-G-Rb1, anti-G-Rg1, and anti-G-Re MAbs (Tanaka et al. 1999; Fukuda et al. 2000a) , We also prepared an ELISA, a new staining method for ginsenosides (eastern blot) , and an immunoaffinity concentration method for quantitative analysis of samples containing low concentrations of G-Rb1 (Fukuda et al. 2000b) .

As part of our ongoing studies on MAbs against naturally occurring bioactive compounds, we review here a new eastern blotting method for ginsenosides in TCM-prescribed ginseng and the double staining for ginsenosides in the crude drug of the Panax species using anti-G-Rb1 and anti-G-Rg1 MAbs. The immunoaffinity concentration of G-Rb1 using an immunoaffinity column is also discussed.

2 Eastern Blotting (Tanaka et al. 1997)

Ginsenosides were applied to a silica gel TLC plate and developed with n-BuOH/EtOAc/H2O (15:1:4). The developed TLC plate was dried and sprayed with a blotting solution mixture of i-PrOH/MeOH/H2O (1:4:20 by volume). It was placed on a stainless steel plate and covered with a PVDF membrane. Next, a glass microfiber filter sheet was placed over the plate, and the whole assembly was pressed evenly for 50 s with a 120 °C hot plate as previously described (Tanaka et al. 1997) with some modifications. The PVDF membrane was separated from the TLC plate and dried.

The blotted PVDF membrane was dipped in water containing NaIO4 and stirred at room temperature for 1 h. After washing with water, 50 mM carbonate buffer solution (pH 9.6) containing BSA was added and stirred at room temperature for 3 h. After the PVDF membrane was washed with PBS, it was treated with PBS containing 5% skim milk for 3 h to reduce nonspecific adsorption. The PVDF membrane was immersed in anti-G-Rb1 MAb and stirred at room temperature for 1 h. After the PVDF membrane was washed twice with PBS containing 0.05% Tween 20 and water, a 1:1000 dilution of peroxidase-labeled goat anti-mouse IgG in PBS containing 0.2% gelatin was added and stirred at room temperature for 1 h. The PVDF membrane was washed twice with TPBS and water and then exposed to freshly prepared 1 mg/mL 4-chloro-1-naphthol/0.03% H2O2 in PBS for 10 min at room temperature. The reaction was stopped by washing with water, and the immunostained PVDF membrane was allowed to dry.

For staining by anti-G-Rg1 MAb, the blotted PVDF membrane was treated in the same way as was the anti-G-Rb1 MAb except it was exposed to 0.2 mg/mL of 3-amino-9-ethylcarbazole/0.03% H2O2 in acetate buffer (0.05 M, pH 5.0) containing 5% N,N-dimethylformamide.

3 Preparation of Immunoaffinity Column Using Anti-G-Rb1 MAb and Immunoaffinity Concentration of G-Rb1 (Fukuda et al. 2000b)

Purified anti-G-Rb1 MAb in diluted Bio-Rad Affi-Gel Hz coupling buffer was dialyzed against the coupling buffer two times. NaIO4 solution was added to the MAb solution and stirred gently at room temperature. After the reaction, glycerol was added to the reaction mixture and stirred to inactivate NaIO4 before being dialyzed. Affi-Gel Hz hydrazide gel was added to the above reaction mixture, resulting in a hydrazone gel and an immunoaffinity gel, which were packed into a plastic minicolumn.

The ginseng root extracts were redissolved in PBS and then filtered with a 0.45-μm MILEX-HV filter (Millipore) to remove insoluble portions. The filtrate was loaded on the immunoaffinity column and allowed to stand overnight at 4 °C. The column was washed with the washing buffer solution (40 mL) and then eluted with 100 mM AcOH buffer containing 0.5 M KSCN and 20% MeOH (pH 4.0). The fraction containing G-Rb1 was used for ELISA to determine its concentration and then subjected to TLC with CHCl3/MeOH/H2O (7:4:1) and n-BuOH/AcOH/H2O (15:1:4), used as developing solvents, followed by eastern blotting.

Concentration of ginsenoside from the crude extracts of the P. japonicus root by immunoaffinity column was carried out in the same way as was for P. ginseng as described above. Individual fractions containing ginsenosides were performed for eastern blotting as indicated above.

4 Eastern Blotting of Ginsenosides

Although western blotting is a common assay for separating substances of high molecular weight, it has not been used for small molecules, as direct immunostaining of such compounds on a TLC plate has not been performed to date. Therefore, a new method for small molecular compounds is needed. Moreover, fixing them onto the membrane also requires a new methodology. Previously, we successfully separated the functional groups of small molecular compounds such as solasodine glycosides into a part of an epitope and fixed them onto a membrane as follows (Tanaka et al. 1997). The blotted PVDF membrane was treated with NaIO4 solution. This reaction enhanced the fixing of solasodine glycoside via solasodine glycoside–BSA conjugates on the PVDF membrane. The PVDF membrane incubated in the absence of NaIO4 was essentially free of staining for solasodine glycoside. We have applied this new methodology to various glycosides such as glycyrrhizin (Shan et al. 2001) and saikosaponins (Morinaga et al. 2006a) . In this paper, we investigate the eastern blotting of ginsenosides.

The H2SO4 staining and eastern blotting of ginsenoside standards and the TCM using anti-G-Rb1 MAb are shown in Fig. 21.1. It is impossible to determine the ginsenosides by TLC stained with H2SO4 because of the complicated profile, shown in Fig. 21.1. However, clear staining of G-Rb1 was obtained using eastern blotting. Although H2SO4 staining detected all standard compounds, eastern blotting showed only limited staining of G-Rb1 and two other ginsenosides, G-Rc and G-Rd, cross-reactivities of which were 0.02% (Fig. 21.1). The eastern blotting method was considerably more sensitive than H2SO4 staining. Furthermore, Kikyoto and Daiokanzoto prescriptions, which do not contain ginseng, showed no spot of G-Rb1. This finding suggests that aglycon, protopanaxadiol, and a part of the sugars may be of importance to immunization and may function as an epitope in the structure of ginsenosides. In addition, the specific reactivity of the sugar moiety in the ginsenoside molecule against anti-G-Rb1 MAb may be modified by NaIO4 treatment of ginsenosides on the PVDF membrane, causing G-Rc and G-Rd to become detectable by eastern blotting.

Fig. 21.1
figure 1

Eastern blotting of ginsenosides in traditional Chinese medicine (TCM) prescriptions by anti-ginsenoside Rb1 MAb. Samples: 1, Kikyoto; 2, Daiokanzoto; 3, Ninjin’yoeito; 4, Shikunshito; 5, Ninjinto; 6, Hangeshashinto; 7, Sho-saiko-to; 8, crude extract of ginseng. Samples 1 and 2 do not contain ginseng. Standard of ginsenosides indicated G-Rg1, G-Re, G-Rd, G-Rc, and G-Rb1 from upper

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We report here a new methodology to separate the G-Rb1 molecule into two functional parts. The sugar parts are oxidized to give dialdehydes, which react with lysine and/or arginine amino groups of proteins that can bind strongly to the adsorbent membrane, PVDF. The aglycon part of the G-Rb1 molecule is bound and can be visualized by using the anti-G-Rb1 MAb. The method is shown diagrammatically in Fig. 21.2.

Fig. 21.2
figure 2

Mechanism of eastern blotting

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Although purple staining of G-Rg1 was expected because of the 3-amino-9-ethylcarbazole substrate, all ginsenosides in the mixture of anti-G-Rg1 and anti-G-Rb1 MAbs and the pair of substrates, including G-Rg1, G-Re, G-Rd, G-Rc, and G-Rb1, were stained blue (data not shown). This finding suggested that the sensitivities of the 3-amino-9-ethylcarbazole and 4-chloro-1-naphthol substrates might be different. Therefore, we performed successive staining of the membrane using anti-G-Rg1 and anti-G-Rb1. Finally, we succeeded in the double staining of the ginsenosides, in which G-Rg1 and G-Re were stained purple and the others were stained blue, as shown in Fig. 21.3. These results indicate that both antibodies can distinguish among individual aglycons, protopanaxatriol, and protopanaxadiol. For this application, the crude extracts of various Panax species were analyzed using the double-staining system (Fukuda et al. 2001) , allowing all ginsenosides to be determined (Fig. 21.3).

Fig. 21.3
figure 3

Double staining of eastern blotting for ginsenosides contained in various ginseng samples using anti-G-Rb1 and anti-G-Rg1 monoclonal antibodies: (a) TLC profile stained by sulfuric acid; (b) eastern blotting by anti-G-Rb1 and anti-G-Rg1 monoclonal antibodies I, II, III, IV, V, and VI indicated white ginseng, red ginseng, fibrous ginseng (Panax ginseng) , Panax notoginseng, Panax quinquefolius, and Panax japonicus, respectively. Upper purple color spots and lower blue color spots were stained by anti-G-Rg1 and anti-G-Rb1 monoclonal antibodies, respectively

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Interestingly, the staining color may indicate pharmacological activity. For example, the purple spots show ginsenosides that have the stimulation activity as the central nervous system. The blue color shows ginsenosides that have the depression effect for the central nervous system. Moreover, the Rf value of the ginsenosides is roughly the same as the number of sugars attached to the aglycon. Therefore, both analyses indicate that it is possible that the aglycon and the number of sugars elucidate the structure of ginsenosides.

We also investigated the Araliaceae species by eastern blotting using anti-G-Rb1 MAb (Fig. 21.4). ELISA analysis and the eastern blotting profile of Kalopanax pictus Nakai (Fig. 21.4b, line 13, as indicated by an arrow) suggest that this species may contain G-Rb1. Using this information, we successfully isolated G-Rb1 from the bark of K. pictus even though the concentration is 0.0009% dry weight as reported previously (Tanaka et al. 2005).

Fig. 21.4
figure 4

Ginsenosides in Araliaceae plants analyzed by eastern blotting. Arrow indicates Kalopanax pictus

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5 Immunoaffinity Concentration by Immunoaffinity Column Conjugated with MAb for the Determination of Ginsenosides (Fukuda et al. 2000b)

To confirm the concentration for G-Rb1 by immunoaffinity column conjugated with anti-G-Rb1 MAb, a crude extract of P. ginseng roots was loaded onto the immunoaffinity column and washed with the washing solvent. The fractions 1–8 (0–16 mL) containing overcharged G-Rb1 were determined by ELISA. G-Rc, G-Rd, G-Re, and G-Rg1 were also detected in these fractions by the eastern blotting procedure (Fig. 21.5). A sharp peak appeared around fractions 19–25 (40–52 mL), which contained G-Rb1. However, G-Rb1 purified by the immunoaffinity column was still contaminated by a small amount of malonyl-G-Rb1 as detected by eastern blotting. This compound has almost the same cross-reactivity as G-Rb1 (data not shown). Therefore, the mixture was treated with a mild alkaline solution at room temperature for 1 h to give pure G-Rb1. Overcharged G-Rb1, eluted with washing solution, was repeatedly loaded and finally isolated in pure form. These results confirmed that the immunoaffinity column can concentrate G-Rb1 from the ginsenoside mixture.

Fig. 21.5
figure 5

Elution profile of Panax ginseng crude extract used immunoaffinity column monitoring by ELISA using anti-G-Rb1 MAb

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After washing, fractions were deionized, and the solvent was lyophilized. Figure 21.6 shows the TLC profile of the purification step. Lines 1, 2, and 3 were the crude extract, the washing fraction, and the eluted fraction, respectively. Interestingly, the washing fraction contained all of the compounds in the ginseng crude extract except G-Rb1. It is evident that the G-Rb1 molecule can be eliminated using an immunoaffinity column conjugated with anti-G-Rb1 MAb and that the washing fraction is knocked out only by the G-Rb1antigen. Therefore, we named this washing fraction a knockout extract. This knockout extract may be useful for the determination of real pharmacologically active principles in TCM. The antibody was stable when exposed to the eluent and the immunoaffinity column, indicating almost no decrease in capacity (20 μg/mL gel) after repeated use of more than 10 cycles under the same conditions, as reported for a single-step separation of forskolin from a crude extract of Coleus forskohlii root (Yanagihara et al. 1996). Furthermore, because we succeeded in the preparation of MAbs having a wide cross-reactivity with molecules such as anti-solamargine MAb (Ishiyama et al. 1996), anti-saikosaponin A (Zhu et al. 2006) , and G-Re (Morinaga et al. 2006b) , related total saponins can be concentrated using an immunoaffinity column conjugated with Mab, as reported previously in the case of solasodine glycosides (Putalun et al. 1999).

Fig. 21.6
figure 6

Preparation of knockout extract eliminated G-Rb1 from Panax ginseng crude extract using immunoaffinity column conjugated with anti-G-Rb1 MAb. Lines 1, 2, and 3 indicate crude extract, knockout extract, and purified G-Rb1, respectively. Red spot shows G-Rb1

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Panax japonicus is distributed in Japan and China and is morphologically different from the other Panax species. Yahara et al. reported that no G-Rb1 was found in P. japonicus (Yahara et al. 1977) but instead isolated oleanane-type saponins called chikusetsusaponins and determined their structure. Morita et al. examined the varieties of P. japonicus by chemical analysis of saponins (Morita et al. 1985) and found that the concentration of G-Rb1 may exist at trace levels. However, we found higher concentrations of G-Rb1 using ELISA compared to previous reports (Fukuda et al. 2000c), although approximately half the concentration of G-Rb1 was found using HPLC analysis as was found using ELISA. To clarify these differences, we used an immunoaffinity column for concentration of G-Rb1. The crude root extract of P. japonicus was loaded onto the immunoaffinity column and washed with the washing solvent and an elution solvent, as previously discussed. Figures showed the H2SO4 staining (21.7a) and the eastern blotting (21.7b) profiles of the two fractions separated by the immunoaffinity column. Fraction 1 eluted with the washing solvent showed many spots, including chikusetsusaponins, similar to the original extract of P. japonicus. However, fraction 2 contained a higher concentration of compound 1, although two other bands were still detected with eastern blotting. Compound 1 clearly indicated a dammarane saponin having a protopanaxadiol framework and three sugars, a number similar to the Rf value of G-Rd, suggesting that compound 1 is chikusetsusaponin III. We were ultimately able to identify compound 1 as chikusetsusaponin III in a direct comparison with an authentic sample.

Fig. 21.7
figure 7

Purification and determination of ginsenosides of P. japonicus by immunoaffinity column and eastern blotting

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A clear unknown band of compound 2 appeared in fraction 5 eluted with the elution solvent. G-Rb1 was not detected by eastern blotting (Fig. 21.7b), although it was detected by TLC (Fig. 21.7a). This finding suggests that compound 2 has a molecular structure and cross-reactivity similar to those of G-Rb1 and seems to be related to the ginseng saponin that has a protopanaxadiol aglycon. Moreover, compound 2 might have the same sugar fragments but possesses five sugar moieties, as indicated by their Rf values. This evidence suggests that compound 2 might be chikusetsusaponin III-20-O-gentiobiose, chikusetsusaponin IV, which was identified by direct comparison with an authentic sample (Fukuda et al. 2000c). Therefore, we concluded that P. japonicus did not contain G-Rb1 but did contain chikusetsusaponin IV and that it has the same aglycon and the same sugar component as chikusetsusaponin.