Natural Products Chemistry & Research

Ginseng saponins, commonly called ginsenosides, are the principal components in Panax species and have been occurred in several other Araliaceae plants. According to different aglycones, ginsenosides can be classified into three types: the 20 (S)-protopanaxadiol type such as ginsenosides Rb₁, Rc, Rb₂, and Rd, the 20 (S)-protopanaxatriol type such as ginsenosides Rg₁ and Re, and the oleanolic acid type including ginsenoside Ro and polyacetyleneginsenoside Ro, respectively [1]. These compounds have been widely investigated for their effects on disturbances of the central nervous system, hypothermia and tumor metastasis, and for their antioxidant, antidiabetes, antiaging and radioprotective activities [2]. Especially, ginsenoside Rb₁, one of major ginsenosides, exhibited remarkable effects on the central nervous system [2,3] and drug-induced memory impairment [4]. In addition, the regulation of ChAT, NGF and trkA mRNA expression by ginsenoside Rb₁ in the rat brain was observed [3]. Therefore, ginsenosides including ginsenoside Rb₁ have played as important resources in the development of new drugs [5].

For qualitative and quantitative analyses of ginsenosides, thin layer chromatography (TLC) [6], high performance liquid chromatography (HPLC) [7,8], and liquid chromatography-mass spectrometry (LCMS) [9] have been routinely used. Recently, an enzyme-linked immunosorbent assay (ELISA) system has been opened for natural product analysis as the most promising methodology. We have developed the preparation of monoclonal antibodies (MAbs) against ginsenosides Rb₁ [10], -Rg₁ [11], and -Re [12]. Furthermore, we set up the ELISA using individual MAbs and applied for the quantitative analysis of ginsenosides [13,14]. On the other hand, regarding immunostaining we succeeded to immunostain the steroidal alkaloid glycosides using anti-solamargine MAb [15] in the first stage, then established the immunostaining methods for G-Rb₁ and -Rg₁ [13]. According to development of new staining method for glycosides, we named this methodology as eastern blotting for immunostaining of glycyrrhizin [16].

Eastern blotting fingerprint for ginsenosides

The ginsenoside Rb₁, Rc, Rd, Rg₁ mixture was applied to TLC plates and developed with n-BuOH-EtOAc-H₂O (15:1:4, v/v/v). One TLC plate developed was sprayed and stained with H₂SO₄. Another TLC plate developed was blotted on the PVDF membrane by heating at around 120°C for short period, the PVDF membrane was treated with NaIO₄ solution to release aldehyde groups in sugar moieties, then reacted with proteins such as bovine serum albumin (BSA) resulting in ginsenoside-BSA conjugates which can fix on the PVDF membrane. On the other hand, an aglycone and a part of sugar moiety as an antigen can be stained by MAb like western blotting. Therefore, it became evident that ginsenosides having small cross-reactivities for anti-ginsenosides Rb₁ and anti-ginsenoside Rg₁ MAbs could be stained in the case of eastern blotting for ginsenosides, suggesting that the specific reactivity of the sugar moiety in the ginsenoside molecule against MAb might be modified by NaIO₄ treatment of the ginsenoside on the membrane, resulting that other ginsenosides having protopanaxatriol as an aglycone such as ginsenosides Re, R_f and Rg₃ can be detectable by eastern blotting using anti-ginsenoside Rg₁ MAb although 3.3% of cross-reactivity for ginsenoside Re [13] (Figure 1). Likewise, ginsenosides possessing protopaxadiol as an aglycone like ginsenoside Rc, Rd and Rb₂ can be stained using anti-ginsenoside Rb₁ MAb [17] (Figure 2). This finding is important for the surveys of saponins having two types of aglycone like ginsenosides.

Lanes 1, 2, 3, 4, 5 and 6 indicate ginsenoside Rb, Rc, Rd, Re, Rg and chikusetsusaponin IV, respectively. Lanes I, II, III, IV, V and VI are white ginseng, red ginseng, fibrous ginseng, P. notoginseseng, P. quinquefolium and P. japonicus, respectively.
Figure 1: Fingerprinting of ginsenoside standards and ginsenosides in various ginseng by H₂SO₄ (left) and eastern blotting (right) using anti-ginsenoside Rg1 monoclonal antibody.

Lanes 1, chikusesusaponin IV; II, III, IV, V and VI indicate white ginseng, red ginseng, fibrous ginseng, P. notoginseseng, P. quinquefolium and P. japonicus, respectively.
Figure 2: Fingerprinting of ginsenosides in various ginseng by H₂SO₄ (left) and eastern blotting (right) using anti-ginsenoside Rb1 monoclonal antibody.

Analysis of ginsenoside by eastern blotting fingerprinting

Determination of ginsenoside Rb₁ from Acanthopanax koreanum Nakai: Acanthopanax koreanum (Araliaceae), which is a perennial shrub and distributed in Northeast Asia, has been used as a tonic and for treatment of rheumatism, allergies, hepatitis, and diabetes [18,19]. Bioactive constituents of A. koreanum have been reported including several lignans, diterpenes [20,21], and lupane-type triterpene glycosides, which are considered as major constituents [22,23]. Since we have established the combination of ELISA and eastern blotting methods using anti-ginsenoside Rb₁ MAb for the identification of ginsenoside Rb₁ in Panax species and traditional Chinese medicines [14] as a high sensitive and rapid method, the finding and determination of ginsenoside Rb₁ in A. koreanum of Araliaceae family by using ELISA and eastern blotting monitoring will be reviewed in this section.

Figure 3 showed the double eastern blotting staining (A) and the H₂SO₄ staining (B) profiles of ginsenoside standards (right) and crude extracts of A. koreanum (left) using anti-ginsenoside Rb₁ and antiginsenoside Rg₁ MAbs. Although H₂SO₄ staining detected clearly all standard ginsenosides without color differences, the TLC profile of A. koreanum crude extract revealed complicated fingerprinting patterns indicating that ginsenosides are ambiguously determined. From double eastern blotting we confirmed that no ginsenoside having protopanaxatriol as an aglycone was contained in the A. koreanum crude extract because no purple spot appeared. On the other hand, blue spots were detected meaning that protopanaxadiol type ginsenosides are contained in A. koreanum. It is clear that R_f value on TLC reflects the sugar number in general. From these evidences it is easily suggested that A. koreanum contains small amount of ginsenoside Rb₁ and a more polar ginsenoside which cannot be longer analyzed due to its trace amount [24].

Purple spots are protopanaxtriol saponins and blue spots are protopanaxdiol saponins.
Figure 3: Fingerprinting of ginsenoside standards and crude extract of Acanthopanax koreanum by H₂SO₄ (right) and eastern blotting (left) using antiginsenoside Rb1 and Rg1 monoclonal antibodies.

We analyzed A. koreanum leaves crude extract by competitive ELISA using anti-ginsenoside Rb₁ MAb in order to confirm the existence and concentration of ginsenoside Rb₁ resulting in 0.000016% dry wt. of ginsenoside Rb₁. The roots and stems were also analyzed separately by the same manner finding concentrations of 0.000039% and 0.000014% dry wt., respectively. The concentrations of ginsenoside Rb₁ in the sample is extremely low, therefore, it has been suggested that chromatographic purification and analyses of ginsenoside Rb₁ have been unaffordable to date. To our knowledge, this is the first evidence of ginsenoside Rb₁ in Acanthopanax species [24]. The results further support potential and promising application of MAb such as eastern blotting and ELISA for surveying ginsenoside sources.

Determination of ginsenosides from Panax japonicas: P. japonicas, which is wildly distributed in Japan and China, is morphologically different from the other Panax species. Regarding its constituents, Yahara et al. reported that no ginsenoside Rb₁ was found and isolated several dammarane-type saponins structurally related to ginsenoside like chikusetusaponin I~VI and oleanane-type saponins named as chikusetsusaponins as major components [25]. In addition, Morita et al. examined the varieties of P. japonicus by chemical analysis of saponins [26]. From these results, the concentration of ginsenoside Rb₁ might be at trace levels. However, higher concentration compared with previous reports was determined by ELISA [13], although relatively half the concentration of ginsenoside Rb₁ was detected by HPLC analysis compared with ELISA. In order to confirm these differences, immunoaffinity column chromatography was employed for immunoaffinity concentration of ginsenoside Rb₁. The crude root extract of P. japonicus was subjected to the immunoaffinity column and first washed with the washing solvent (PBS; fraction 1) and then with elution solvent (AcOH buffer and 20% MeOH (fraction 2) [14].

Figure 4 shows the H₂SO₄ staining (A) and the eastern blotting (B) profiles of the two fractions separated by the immunoaffinity column. Fraction 1 eluted with the washing solvent showed two spots in the eastern blotting profile. A clear unknown spot appeared in fraction 2 eluted with the elution solvent. However, ginsenoside Rb₁ was not detected by eastern blotting although it was determined by TLC as indicated in (Figure 4). The unknown compound is proposed to resemble molecular structure and a similar cross-reaction with ginsenoside Rb₁, and likely a ginseng saponin having an aglycone of panaxadiol. Moreover, this compound might have the same sugar fragments in the molecule, but possess an additional sugar moiety compared with ginsenodise Rb₁, as indicated by their R_f values. These evidences revealed that an unknown compound contained in fraction 2 is chikusetsusaponin VI comparing with the authentic sample directly (Figure 5).

Figure 4: Purification and determination of ginsenosides of P. japonicas by immunoaffinity column and eastern blotting.

The eastern blotting fingerprinting of fraction 1 indicated that an unknown ginsenoside has protopanaxadiol as an aglycone and three sugar components compared to the R_f value of ginsenoside Rc. Therefore, we determined that the unknown compound contained in fraction 1 is chikusetsusaponin III compared with the authentic sample (Figure 5). From this investigation we confirmed that the saponins having protopanaxdiol as an aglycone can be separated by the immunoaffinity column conjugated with anti-ginsenoside Rb₁ MAb. Moreover, we concluded that the combination of immunoaffinity concentration and ELISA is suitable for samples containing lower concentrations of ginsenoside Rb₁ since the immunoaffinity column can concentrate the ginsenoside Rb₁ concentration [14].

Figure 5: Identified compound 1 and 2 by using immunoaffinity column and eastern blotting and their structures together with ginsenosides Rb1, Rc and Rd in Panax japonicas.

Isolation of ginsenosides by using eastern blotting fingerprinting

Isolation of ginsenoside Rb₁ from Kalopanax pictus Nakai: A number of Araliaceae species, including Panax species, have been used as tonics in Asian folk medicine. On the basis of phytochemical study, Araliaceous plants have been documented to contain similar constituents such as ginsenoside, depending on their chemotaxonomical classification. Various analytical methods have been used to analyze ginsenosides, and of them, ELISA appears to be the most promising methodology, considerably. This section reviewed the distribution of ginsenosides in Araliaceous species and isolation of ginsenoside Rb₁ from the bark of Kalopanax pictus using ELISA and eastern blotting monitoring. Since the bark of K. pictus (Figure 6 line 13) indicated a positive band, the crude extract was analyzed by competitive ELISA using anti-ginsenoside Rb₁ MAb, resulting in 0.0009% dry wt. of ginsenoside Rb₁. The crude extract was subjected to repeated silica gel column chromatography guided with eastern blotting using anti-ginsenoside Rb₁ MAb to afford compound 1. Compound 1 was consistent with that of ginsenoside Rb₁ by eastern blotting, and the physical and spectroscopic data (¹H- and ¹³C-NMR) of compound 1 resembled to those of authentic ginsenoside Rb₁. Further analyses of the stem bark and leaves by competitive ELISA revealed that a higher concentration of ginsenoside Rb₁ was occurred in the leaves (0.0037% dr. wt.) than in the bark (0.0009% dr. wt.) [27]. To our knowledge, this is the first isolation of ginsenoside Rb₁ from K. pictus although the isolation of various oleanane saponins was reported [28,29]. These findings suggested that K. pictus might be a new resource of ginsenoside Rb₁, and moreover the immunoaffinity column conjugated with antiginsenoside Rb₁ MAb [13] is readily applicable for purification of ginsenoside Rb₁ in the final stage of the separation process.

Figure 6: Detection of Ginsenosides in Alariaceae plants detected by H₂SO₄ (A) and eastern blotting (B).

Isolation of new ginsenosides from Panax quenquefolium: American ginseng (Panax quinquefolium L.), which is mainly cultivated in the USA, Canada and China, has been widely used as a tonic and functional foods in various forms such as decoction, powder, tea, capsule, etc. like Asian ginseng (P. ginseng, CA. Meyer). These conventional ginseng products have been reported to have a wide array of pharmacological and physiological actions including antiaging, antidiabetic, anticarcinogenic, analgesic, antipyretic, antistress, and antifatigue, respectively [30-33]. The dammarane-type saponins (ginsenosides) are the major active constituents in American ginseng and with the development of modern chromatography, there are more than 30 ginsenosides such as ginsenosides Rb₁, Rb₂, Rg₁, Rc, Rd, and Re reported [34,35]. However, in comparison with a number of researches on Asian ginseng, the study on American ginseng and its constituents is much less extensive.

Figure 7 showed the double eastern blotting staining (A) and H₂SO₄ staining (B) profiles of crude extracts of Panax species including American ginseng (line 5) using anti-ginsenoside Rb₁ and anti-ginsenoside Rg₁ MAbs. In general, since H₂SO₄ staining detected all compounds, the TLC profile stained by this reagent showed complicated fingerprinting patterns so that ginsenosides are ambiguously indicated. On the contrary, eastern blotting obviously and selectively revealed positive spots regarding ginsenosides. The crude extracts of American ginseng clearly indicated ginsenosides Rg₁, Re, Rd, Rc and Rb₁ as major ginsenosides. Besides, there have minor spots with the similar color indicating lower R_f value than ginsenoside Rc and ginsenoside Rb₁ ((Figure 7-A-line 5) in respect to ginsenoside Rb₁ (blue) or ginsenoside Rg₁ (purple), suggesting more polar ginsenosides. Accordingly, two minor dammarane-type saponins, namely quinquenosides Ja (1) and Jb (2), were isolated from the American ginseng extract for the first time by immunoassayguided fractionation and chromatography separation ((Figure 8). Their structures were identified as 6-O-[α-L-rhamnopyranosyl(1→2)- β-D-glucopyranosyl]-20-O-[β-D-glucopyranosyl(1→4)-β-Dglucopyranosyl]- 3β,6α,12β,20β-tetrahydroxydammar-24-ene (1) and 3-O-[β-D- glucopyranosyl(1→2)-β-D-glucopyranosyl]-20- O-{[α-L-arabinofuranosyl(1→6)-β-D-glucopyranosyl](1→6)-β-D glucopyranosyl}-3β,12β,20β-trihydroxydammar-24-ene (2) on the basis of chemical and spectroscopic methods [36]. The results further supported potential and promising application

Figure 7: Staining of ginsenosides in Panax species by eastern blotting (A) and H₂SO₄ (B).

Figure 8: Structures of the two new ginsenosides from P. quinquefolium.

It is well known that ginsenosides possesses wide pharmacological activities, and one of them, ginsenoside Rg₃ is now an anti-cancer drug in China suggesting that the other ginsenosides may have the possibility of drug development. However, since such ginsenosides can be supplied from ginsengs resulting in high cost performance of ginsenosides, a new supplement system of ginsenosides is needed. From these aims in this review the eastern blotting using anti-ginsenoside Rb₁ and/or anti-ginsenoside Rg₁ MAbs was applied to analyze the distribution of ginsenosides in Araliaceous plants, like A. koreanum and K. pictus. From A. koreanum we determined ginsenoside Rb₁ by ELISA and eastern blotting using anti-ginsenoside Rb₁ MAb. P. quinquefolium were tested, and the structure elucidations of two new ginsenosides from P. quinquefolium were succeeded. The combination of eastern blotting and immunoaffinity column conjugated with anti-ginsenoside Rb₁ MAb makes it evident to separate ginsenosides having small crossreactivity with anti-ginsenoside Rb₁ MAb. In the case of P. japonicus we used the immunoaffinity column conjugated with anti-ginsenoside Rb₁ and separated ginsenosides having the same aglycone with ginsenoside Rb₁, and finally determined two ginsenosides. This biotechnological system has a great possibility to widely apply for quick separation of ginsenosides. In fact we succeeded to separate all solasodine glycosides in Solanum khasianum fruits by once immunoaffinity column conjugated with anti-solamargine MAb [37]. The results further supported potential and promising application of MAb such as eastern blotting for surveying pharmacologically active resources.

One of the authors (N.H.T) is grateful to the Japan Society for the Promotion of Science (JSPS) for a Postdoctoral Research Fellowship (P10423) at Nagasaki International University.