Acanthopanax koreanum which is a shrub belonging to the Araliaceae family and is distributed in Northeast Asia, has been used as a tonic and to treat rheumatism, allergies, hepatitis, and diabetes [1,2]. Phytochemical profile of A. koreanum have been documented with several lignans and diterpenes [3,4] along with, especially, various lupane-type triterpene saponins, which are considered as major constituents [5,6].

In the case of qualitative and/or quantitative analytical approaches of ginsenosides, thin layer chromatography (TLC) [7], high performance liquid chromatography (HPLC) [8,9], and liquid chromatographymass spectrometry (LC-MS) [10] are used frequently. Recently, it becomes evident that an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody (MAb) against small molecular has been developed for natural products as a highly sensitive, specific, and simple methodology. In this regard, we have succeed in the preparation of MAb against ginsenoside Rb₁ (G-Rb₁) [11]. ginsenoside Rg₁ [12], and G-Re [13], and established a new immunostaining method, eastern blotting for G-Rb₁ and -Rg₁[14]. Additionally, in our previous study, the combination of ELISA and eastern blotting methods using anti-GRb ₁ MAb was applied for the identification of G-Rb₁ in Panax species and traditional Chinese medicines containing lower concentrations of G-Rb₁ [15]. This paper herein deals with the finding and determination of G-Rb₁ in A. koreanum of Araliaceae family by using ELISA and eastern blotting monitoring.

Materials

Polyethersulfone (PES) membranes (Immobilon-N) and glass microfiber filter sheets were from Millipore Corporation (Bedford, MA, USA), and bovine (BSA) and human (HSA) serum albumins were from Pierce (Rockford, IL, USA). Peroxidase-labeled anti-mouse IgG was from Organon Teknika Cappel Products (West Chester, PA, USA). Standard G-Rb₁, -Rc, -Rd, -Re and -Rg₁ were from Wako Pure Chemical Industries, Ltd (Osaka, Japan). All chemicals and solvents used were of analytical grade and water was filtered by a Millipore ultrapure water system (Milli-Q Direct, Merk Millipore, Germany).

Sample preparation

A. koreanum samples were colleted by Dr. Susumu Isoda from Herbal Garden in Faculty of Pharmaceutical Science, Showa University, and authenticated by one of the authors (YS). Dried samples of the plant (roots, stems, and leaves) (50 mg individually) were powdered then extracted with methanol (5 mL) under sonication five times. They were then filtered and the combined extract was diluted with 20% methanol for ELISA and eastern blotting.

Eastern blotting and double staining

After adaptation with procedures from Tanaka et al. [15], A. koreanum extracts were loaded onto two TLC plates and developed with n-BuOH–EtOAc–H₂O (15:1:4). Of which, one developed TLC plate was dried and stained with H₂SO₄ and another plate was dried followed by spraying with a blotting solution mixture of isopropanolmethanol- H₂O (1:4:16, v/v/v). Next, the treated TLC plate was placed on a stainless steel plate then covered with a PES membrane sheet. After covering with a glass microfiber filter sheet, it was pressed evenly for 70 s with a 120°C hot. The PES membrane was separated from the plate and dried then dipped in water containing NaIO₄ (10 mg/mL) while stirring at room temperature for 1 h. After washing with water, 50 mM carbonate buffer solution containing BSA was added followed by stirring for 5 h. The PES membrane was then washed twice with PBS containing 0.05% Tween-20 (TPBS) for 5 min then washed with water. It was then immersed in anti-G-Rb₁ MAb and shaken at 4°C overnight. In the next day, the membrane was washed twice with TPBS-water and 1000 times diluted of peroxidase-labeled goat anti-mouse IgG in PBS containing 0.2% gelatin (GPBS) was then added followed by stirring at room temperature for 1 h. The obtained membrane was then rinsed twice with TPBS and water, and treated with 1 mg/mL 4-chloro-1- naphthol-0.03% H₂O₂ in PBS solution freshly prepared before use for 10 min at room temperature.

For the staining by anti-G-Rg₁ MAb, the blotted PES membrane was treated in the same procedure as anti-G-Rb1 MAb except that it was exposed to 2 mg/10 mL 3-amino-9-ethylcarbazole-0.03% H₂O₂ in acetate buffer (0.05 M, pH 5.0) containing 0.5 mL of N,Ndimethylformamide.

Competitive ELISA

G-Rb₁ concentrations in A. koreanum were analyzed by ELISA [14]. G-Rb₁-HSA (100 μL of 1 μg/mL) was adsorbed onto the wells of a 96-well immunoplate and treated with 300 μL PBS containing 5% skimmed milk (S-PBS) for 1 h to reduce non-specific adsorption. Different concentrations of G-Rb₁ (50 μL) or samples diluted in 20% MeOH were incubated with 50 μL G-Rb₁ MAb solution for 1 h. The sample plates were washed three times with T-PBS and incubated with 100 μL of a 1000-fold dilution of peroxidase-labeled goat antimouse IgG for 1 h. Next, the plates were washed again plates with T-PBS and then 100 μL substrate solution (100 mM citrate buffer, pH 4.0, containing 0.003% H₂O₂ and 0.3 mg/mL of 2,2-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS; Wako Pure Chemical, Osaka, Japan) was added to each well followed by incubation for 15 min. The absorbance was recorded on micro-plate reader (Immuno Mini NJ-2300, Nalge Nunc, Roskilde, Denmark) at 405 nm. All reactions were carried out at 37°C.

While PES membranes treated with NaIO₄ solution and reactive BSA protein enhanced the fixation of ginsenoside-BSA conjugation, small cross-reactivities against anti-G-Rb1 MAb like G-Rc (0.024%) and G-Rd (0.02%) were also observed [11]. It is noticeable that only G-Rb₁, G-Rc and G-Rd having protopanaxadiol as an aglycone could be stained and this became evident that the specific reactivity of the sugar part in the ginsenoside molecule against MAb might be modified by NaIO₄ treatment of the ginseng saponin on the membrane. This phenomenon is important for the surveys of saponins having the same aglycone like ginsenosides. Furthermore, we used two MAbs, anti-G-Rb₁ MAb and anti-G-Rg₁ MAb for eastern blotting as previously reported [16]. In this case, two types of ginsenoside having protopanaxadiol and protopanaxatriol as an aglycone can be stained separately. Purple color and blue color indicate ginsenosides having protopanaxatriol and protopanaxadiol as aglycone, respectively as indicated in previous paper [16].

As shown in Figure 1, although H₂SO₄ staining detected clearly all standard ginsenosides without changing color, the TLC profile of A. koreanum crude extract revealed complicated spot patterns indicating that ginsenosides are ambiguously determined. However, the evidence of double eastern blotting unambiguously indicated that no ginsenoside having protopanaxatriol as an aglycone was detected in the A. koreanum crude extract because of no purple spot appeared. On the other hand, blue spots were observed meaning that protopanaxadiol type ginsenosides are occured in A. koreanum. In addition, it is clear that R_f value on TLC reflects the sugar number in general. Taken together, based on these evidences, it is suggested that A. koreanum contains small amount of G-Rb₁ and a more polar ginsenoside which cannot be longer analyzed due to its trace amount (Figure 1).

Figure 1: (A) Eastern blotting profile for the standard ginsenosides and MeOH extract of A. koreanum (leaf) (B) TLC plate treated with 10% H₂SO₄.

Next, we analyzed A. koreanum leaves crude extract by competitive ELISA using anti-G-Rb1 MAb (Figure 2) in order to confirm the existence and concentration of G-Rb₁ resulting in 0.000016% dry wt. of G-Rb₁. The roots and stems were also analyzed separately by the same manner finding concentrations of 0.000039% and 0.000014% dry wt., respectively. In the view point of naturally occurring compounds, the concentrations of G-Rb₁ in A. koreanum is extremely low, therefore, it has become evident that chromatographic purification and analyses of G-Rb₁ have been unaffordable to date. To the best of our knowledge, occurrence of G-Rb₁ and dammarane-type saponins in the Acanthopanax genus had not been reported previously.

Figure 2: Calibration curve for G-Rb1 various concentrations of G-Rb₁ were incubated with MAb in wells pre-coated with G-Rb1 HSA (1 µg/mL). After washing with PBS containing 0.05% of Tween 20, the wells were incubated again with peroxidase-labeled anti-mouse IgG. Absorbance was measured at 405 nm.

In conclusion, it is found that A. koreanum might be a new resource of G-Rb₁ and more analytical studies are suggested to accumulate G-Rb₁ from the medicinal materials. This study further support potential and promising application of MAb such as eastern blotting and ELISA for surveying ginsenoside sources.

This work was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS). One of the authors (N.H.T) is grateful to the JSPS for a Postdoctoral Research Fellowship at Nagasaki International University.