Development of an Assay System for Saikosaponin a Using

munoplate (MaxiSorptmSurface, Nalge NUNC Roskilde, Denmark) then it was treated with 300ml PBS containing 5% skim milk (SPBS) for 1h to reduce non-spe...

0 downloads 36 Views 71KB Size
66

Biol. Pharm. Bull. 27(1) 66—71 (2004)

Vol. 27, No. 1

Development of an Assay System for Saikosaponin a Using Antisaikosaponin a Monoclonal Antibodies Shu-hang ZHU, Shin-ichi SHIMOKAWA, Hiroyuki TANAKA, and Yukihiro SHOYAMA* Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University; 3–1–1 Maidashi, Higashi-ku, Fukuoka 812–8582, Japan. Received June 27, 2003; accepted September 13, 2003 For immunization, saikosaponin a (SSa) was conjugated with bovine serum albumin (BSA). The hapten number in an antigen conjugate was determined to be eleven by matrix-assisted laser adsorption/ionization timeof-flight mass spectrometry (MALDI-TOF Mass). Hybridomas secreting monoclonal antibodies (MAb) against SSa were produced by fusing splenocytes immunized with SSa-BSA conjugate and a hypoxanthine-aminopterinthymidine-sensitive (HAT) mouse myeloma cell line, P3-X63-Ag8-653. A high specific MAb against SSa was selected from hybridomas using enzyme-linked immunosorbent assay (ELISA) analysis. Weak cross-reactivities occurred with saikosaponin c, b2 and d, which are stereochemical and/or functional isomers of SSa, but no crossreactivities were observed with other related steroidal glycosides. The full range of the assay extends 26 ng/ml to 1.5 m g/ml of SSa. Good correlation of SSa concentrations in a crude extract of Bupleuri radix between ELISA and HPLC methods was obtained after hydrolysis of acyl saikosaponins by treatment with a mild alkaline solution. The newly established ELISA has been applied for the quantitative assay of SSa in the Bupleuri radix and the Kampo medicines (TCM) prescribed with Bupleuri radix. Key words saikosaponin a; monoclonal antibody; ELISA; bovine serum albumin (BSA) conjugate; hybridoma; Kampo medicine

Bupleuri radix (Bupleurum species root) is one of the most important crude drugs used in many Kampo medicines, prescribed with other crude drugs for many diseases. It is believed that a part of these pharmaceutical properties are due to saikosaponins having a typical oleanan-type skeleton as an aglycon (Fig. 1). As a major saponin, saikosaponin a (SSa) has anti-cancer,1) anti-inflammation,2) corticosterone secreting3) and plasma-cholesterol decreasing activities.4) Although immunoassay systems using monoclonal antibody (MAb) are now indispensable in various investigations, not as many are available for naturally occurring bioactive compounds having smaller molecular weights, except against drugs like morphine. In our ongoing study of the formation of MAbs against naturally occurring bioactive compounds, we have established MAbs against forskolin,5) solamargine,6) opium alkaloid,7) marijuana compounds,8) glycyrrhizin,9) crocin,10) ginsenoside Rb111) and Rg1,12) and developed their applica-

Fig. 1.

Structure of Saikosaponins

∗ To whom correspondence should be addressed.

tions, such as a new eastern blotting13) and an immuno affinity column chromatography.14) No formation of MAb against SSa has been reported yet, although immunological approaches for assaying quantities of SSa using the anti-SSa polyclonal antibody have been investigated by Jung et al.15) Polyclonal antibodies cross-reacted with random proteins non-specifically and induced a problem for the quantitation of hapten in plasma. We herein report the formation of MAb against a major pharmacologically active compound, SSa in Bupleuri radix, and the development of enzyme-linked immunosorbent assay (ELISA) for quantitation of SSa using an anti-SSa MAb. MATERIALS AND METHODS Materials Saikosaponin a, b2, c and d were purchased from Wako Pure Chemical Ind., Ltd. (Osaka, Japan). Bovine serum albumin (BSA) and human serum albumin (HSA) were obtained from Pierce (Rockford, IL, U.S.A.). Peroxidase-labeled anti-mouse IgG was obtained from ICN Pharmaceuticals., Inc. (U.S.A.). All other chemicals were standard commercial products of analytical grade. The roots of Bupleurum species were purchased from Nakai Koshindo Co., Ltd. (Kobe, Japan). The commercial Kampo medicines provided by Tsumura & Co. (Tokyo, Japan) and Teikoku Seiyaku Co., Ltd. (Kagawa, Japan) were kindly provided by Mr. T. Somehara, Saga Medical School. Synthesis of SSa-Carrier Protein Conjugates SSa-carrier protein conjugates were synthesized by a modification of the procedure already used for solamargine,6) which is based on the method of Erlanger and Beiser.16) To the H2O solution (0.6 ml) containing NaIO4 (10 mg), MeOH solution (1 ml) of SSa (10 mg) was added dropwise and stirred at room temperature for 1 h. To the above reaction mixture, 50 mM carbonate buffer solution (pH 9.6, 1 ml) containing BSA (6.4 mg) was added. The reaction mixture was adjusted to pH 10 with 1 M Na2CO3 solution and stirred at room temperature for 5 h. The

e-mail: [email protected]

© 2004 Pharmaceutical Society of Japan

January 2004

reaction mixture was dialyzed against H2O 5 times, then lyophilized to give SSa-BSA conjugate (SSa-BSA) (5.3 mg). SSa-HSA was also synthesized in the same manner as that of SSa-BSA. Determination of Hapten Number of SSa-Carrier Protein Conjugate by Matrix-Assisted Laser Adsorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry The hapten number in the SSa-BSA conjugate was determined by MALDI-TOF mass spectrometry. A small amount (1—10 pmol) of antigen conjugate was mixed with a 103-fold molar excess of sinapinic acid in an aqueous solution containing 0.15% trifluoroacetic acid. The mixture was subjected to a JMS-ELITE TOF mass monitor and irradiated with a N2 laser (337 nm, 150 ns pulse). The ions formed by each pulse were accelerated by a 20 kV potential into a 2.0 m evacuated tube and detected using a compatible computer, as previously reported.17) Immunization and Hybridization BALB/c female mice were immunized with the SSa-BSA conjugate and MAbs were generated as described for previous protocols in this laboratory,5) with modification. The first immunization (50 m g protein) was executed by an intraperitoneal injection using a 1 : 1 emulsion in Freund’s complete adjuvant. The second and third immunizations (50 m g protein in each injection) were injected as a 1 : 1 emulsion in Freund’s incomplete adjuvant biweekly. On the third day after the final immunization (100 m g protein), splenocytes were isolated and fused with a hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line, P3-X63-Ag8-U1 (P3U1), by the polyethylene glycol (PEG) method.18) Hybridomas secreting MAb reactive to SSa were cloned by the limited dilution method.19) Established hybridomas were cultured in eRDF medium supplemented with 10 m g/ml insulin, 35 m g/ml of transferrin, 20 m M ethanolamine and 25 nM selenium (ITES).20) Purification of MAb Anti-SSa MAbs were purified using a Protein G FF column (0.46311 cm, Amersham Pharmacia Biotech, Uppsala, Sweden) as previously reported.5) The cultured medium (200 ml each) containing the IgG was adjusted to pH 7 with 1 M Tris solution and subjected to the column, then the column was washed with 10 mM phosphate buffer. Adsorbed IgG was eluted with 100 mM citrate buffer (pH 2.7). The eluted IgG was neutralized with 1 M Tris solution (pH 9), then dialyzed against phosphate buffered saline pH 7.4 (PBS) 3 times, and finally lyophilized. Direct ELISA Using SSa-HSA The reactivity of MAbs to SSa-HSA was determined by a direct ELISA method. SSa-HSA dissolved in 50 mM sodium carbonate buffer (1 m g/ml; 100 m l) was adsorbed to the wells of a 96 well-immunoplate (MaxiSorptm Surface, Nalge NUNC Roskilde, Denmark) then it was treated with 300 m l PBS containing 5% skim milk (SPBS) for 1 h to reduce non-specific adsorption. The plate was washed three times with PBS containing 0.05% Tween 20 (TPBS) and reacted with 100 m l of MAb for 1 h. The plate was washed three times with TPBS, then the MAb was reacted with 100 m l of 1000 times-diluted peroxidase-labeled anti-mouse IgG (Organon Teknika Cappel Products, U.S.A.) for 1 h. After washing the plate three times with TPBS, 100 m l of substrate solution, 0.1 M citrate buffer (pH 4.0) containing 0.003% H2O2 and 0.3 mg/ml 2,29-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (Wako, Osaka, Japan), was added to each well and

67

incubated for 10 min. Absorbance at 405 nm was measured with a micro plate reader (MODEL 450 Microplate Reader BIO-RAD Laboratories, U.S.A.). All reactions were carried out at 37 °C. Competitive ELISA A 96-well immunoplate, to which had been adsorbed 100 m l of 1 m g/ml SSa-HSA was treated with 300 m l SPBS for 1 h to reduce non-specific adsorption. Fifty microliters of various concentrations of SSa or samples dissolved in 10% of MeOH solution was separately incubated with 50 m l of anti-SSa MAb solution for 1 h. The plate was washed three times with TPBS, and then the anti-SSa MAb was combined with 100 m l of a 1 : 1000 dilution of peroxidase-labeled anti-mouse IgG for 1 h. After washing the plate three times with TPBS, 100 m l of substrate solution was added to each well and incubated for 10 min. The absorbance was measured by microplate reader at 405 nm. The cross-reactivities of SSa and related compounds were determined according to Weiler and Zenk’s equation.21) Quantitative Analysis of Saikosaponins by HPLC Samples of SSa (400, 200, 100, 50, 25 m g/ml) in MeOH were freshly prepared and assayed by HPLC against standards of SSa. HPLC analysis was performed on a Model LC-10AD (Shimadzu Co., Ltd.) Pump connected with TSK-gel ODS120A (4.6 I.D.3250 mm. Tosoh Co., Ltd.), equipped with a UV-8 Model II Spectrophotometer (Tosoh Co., Ltd.) detected at 203 nm. The mobile phase was 50 mM KH2PO4 buffer containing 44% of CH3CN. Sample Preparation Dried samples (20 mg) of various Bupleuri radix were powdered, extracted with MeOH (0.5 ml) under sonication 5 times, filtered, and then evaporated. For the elimination of an acyl group from acylated saikosaponins, the extracts were treated with 5% of KOH in MeOH at room temperature for 2 h, as previously reported,22) then neutralized with 1 M HCl in MeOH, and assayed by ELISA. Correlation between HPLC and ELISA Analysis The interpolated concentrations of the samples obtained by each method were then compared by linear regression analysis. RESULTS Direct Determination of Hapten-Carrier Protein Conjugate by MALDI-TOF Mass Spectrometry Hapten number was analyzed by the MALDI-TOF mass spectra. A broad peak coinciding with SSa-BSA conjugate appeared from m/z 69000 to 83000 centering at around m/z 75476, although a sharp molecular peak was obtained, as previously reported.8) Its broadness might depend on the random cleavage of a sugar moiety in SSa by NaIO4. Using experimental results and a molecular weight of BSA, 66433, the calculated values of the SSa component (MW 780) are from 3 to 22 (11 in average) molecules of SSa conjugated with BSA. The hapten number was estimated to be enough for immunization, compared with the previous result.6) The number of SSa contained in the SSa-HSA conjugate was also determined to be around 7 molecules by its spectrum (data not shown). Cross-Reactivities of Anti-SSa MAbs and Assay Sensitivity Cross-reactivities are the most important factor in phytochemical investigations in which there are many structurally related compounds (see Fig. 1). Assay specificities were examined by competitive ELISA with various related

68

Vol. 27, No. 1

Table 1.

Cross-Reactivities (%) of Anti-SSa-MAbs

Compound

1G6

3G10

2A8

4B9

Saikosaponin a Saikosaponin b2 Saikosaponin c Saikosaponin d Digitonin Solasonine Deoxychokic acid Glycyrrhizin Ergosterol Solamargine Ginsenoside Rb1 Cholesterol Sitosterol

100 ,0.25 2.65 3.76 ,0.25 ,0.25 0.45 ,0.25 ,0.25 ,0.25 ,0.25 ,0.25 ,0.25

100 63.77 28.52 15.62 1.42 ,0.25 ,0.25 ,0.25 ,0.25 ,0.25 ,0.25 ,0.25 ,0.25

100 2.92 36.31 3.69 ,0.25 ,0.25 ,0.25 ,0.25 ,0.25 ,0.25 ,0.25 ,0.25 ,0.25

100 10.58 49.23 20.89 ,0.25 ,0.25 ,0.25 ,0.25 ,0.25 ,0.25 ,0.25 ,0.25 ,0.25

The cross-reactivities of saikosaponins and related compounds were determined according to Weiler’s equation (E. W. Weiler et al., 1976).21)

Fig. 2.

compounds, then calculated following Weiler and Zenk’s equation.21) MAbs reacted with related compounds having various differences of cross-reactivity. Some characteristic MAbs are indicated in Table 1. MAb 1G6 reacted only with structurally related saikosaponin c (SSc) and saikosaponin d (SSd), weakly resulting in the high specificity of MAb. An undetectable cross-reaction with other steroidal compounds was shown. When comparing the cross-reactivities between SSa having a b -hydroxyl group at the C-16 position and SSd (a -hydroxyl group), all MAbs only had small cross-reactivities against SSd (see Fig. 1). From this, the hydroxyl group at the C-16 position is distinguished stereochemically. In our investigation, immunization for a sugar moiety may be loose because MAb 4B9 has 49% cross-reactivity against SSc, which has a different sugar constituent compared to SSa. This phenomenon might depend on the preparation method of the antigen by which the sugar moiety was randomly opened by NaIO4 and then conjugated with BSA. This indicates a good agreement with a broad peak in the MALDITOF mass spectrum. Wide cross-reactivities were found in 4B9, indicating that SSd was 21%, together with the 49% for SSc, as discussed above. MAb 3G10 cross-reacted with saikosaponin b2 (SSb2), SSc and SSd at a rate of 63.8, 28.5 and 15.6%, respectively, easily suggesting that the ether ring between the C-13 and C-28 position in SSa might be opened in a mouse body during the immunization period, and then immunized. This wide crossreaction is the major advantage of the antibody reagent used in this ELISA, because when total saikosaponin contents in body fluid and/or plant sample are needed, this MAb can be widely available, similar to the results of anti-solamargine MAb.6) From these results, we decided that the concentration of the major saikosaponin, SSa, in the crude drug of Bupleuri radix can be analyzed by ELISA using MAb 1G6. On the other hand, when it is necessary to determine the concentration of total saikosaponins in the extracts of Kampo medicines, MAb 3G10 functions well for quantitative analysis because the ether ring at the C-13 and C-28 positions opens during the extraction process, as previously reported.23) Quantitative Analysis of SSa by ELISA Using MAb 1G6 Figure 2 indicates the calibration curve of SSa using MAb 1G6, showing that the full measuring range of the

Calibration Curve of SSa by ELISA with MAb 1G6

Various concentrations of SSa were incubated with MAb 1G6 in the microtiter plate adsorbed with SSa-HSA (1 m g/ml). After washing with TPBS, the wells were again incubated with peroxidase-labeled anti-mouse IgG. Absorbance was measured at 405 nm.

Table 2. Variations among ELISA Runs for the Analysis of SSa Using MAb 1G6 SSa concentration (ng/ml) 200 300 400 500 600

CV (%) Intra-assay

Inter-assay

1.72 3.14 2.63 3.53 4.47

4.96 3.69 4.51 6.16 6.06

The measured values were the mean6S.D. for five plates and five replicate wells for each concentration within one plate from five consecutive days. The variations in replicates from well to well and plate to plate are defined as intra-assay and inter-assay variation, respectively.

assay covers from 26 ng/ml to 1.5 m g/ml. Since the available measuring range of the HPLC method performed in our laboratory was 25 to 400 m g/ml, it becomes evident that the ELISA is about 1000 times more sensitive than that of the HPLC method. Assay Variation Since reproducibility and precision are integral criteria for an immunoassay, the variations between replicates from well to well (intra-assay) and plate to plate (inter-assay) were measured (Table 2). It is known that the intra-assay variations are generally lower than the inter-assay’s. It became evident that the newly developed ELISA method didn’t exceed general ranges, not only of the intraassay variations but also the inter-assay variations. From these results, this ELISA system using MAb 1G6 is available as a new analytical method for SSa. Factors related to variations are suggested to include the quality of the hapten conjugate, coating, plate wells and multi-channel pipette, edge effects due to evaporation, uneven temperature during incubation, day-to-day variation in the preparation of the samples, and so on. Thus, a new standard curve should be created for each assay in order to reduce the variation. Recovery of SSa by ELISA Used MAb 1G6 For recovery experiments, the extract of Bupleuri radix was used. For each level, three samples were analyzed. The recovery was

January 2004 Table 3.

69

Recovery of SSa in Standard Spiked Bupleuri Radix Samples Determined by ELISA

Spiked level (m g)

Sample weight (mg)

Expected amounta) (m g/mg)

Measured amountb) (m g/mg)

Recoveryc) (%)

0d) 12.5 25 50

20.3 20.9 20.5 20.1

— 0.598 1.219 2.487

Control* 0.591 1.292 2.476

— 98.81 105.92 99.56

a) Expected amount5spiked level/sample weight. b) Measured amount5determined amount of ELISA2control* amount. c) Recovery5measured amount/expected amount3100%. d) Control: the sample without spiking.

calculated from the added SSa (12.5, 25.0, 50.0 m g, respectively) in the same concentration ranges, indicating 98.8, 105.9 and 99.6%, respectively (Table 3). From these results, it is evident that the ELISA using MAb 1G6 can be routinely used for phytochemical investigations involving crude plant extracts and/or Kampo medicines without any complicated pretreatment, as previously performed by HPLC,24) because of MAb’s high sensitivity and specificity. Correlation of SSa in Crude Extracts of Bupleuri Radix between HPLC and ELISA Using MAb 1G6 In order to compare the correlation between HPLC and ELISA, we also determined the SSa concentrations in various Bupleuri radix using HPLC. The correlation coefficient was calculated from fitting a straight line analyzed by ELISA and HPLC methods. Figure 3A shows the correlation of SSa concentration in the Bupleuri radix between HPLC and ELISA, indicating scattering (r50.831). Ebata et al.25) isolated acetyl and/or malonyl SSa from the Bupleuri radix. From this evidence, such acyl saikosaponins may affect the correlation between both assay systems, since it is easily suggested that acyl SSa’s have a similar cross-reactivity with SSa for the already discussed reason that the sugar moiety in saikosaponins is not related to its cross-reactivity. Therefore, the crude extract was treated with a mild alkaline solution, as previously reported, to give SSa without any change in aglycon skelton, similarly to saikogenin F.22) Figure 3B shows the correlation of SSa concentration in Bupleuri radix between both assay systems, improving the correlation ratio to be r50.999. From this result, the crude extract of Bupleuri radix should be treated with a mild alkaline solution before the ELISA assay, since it becomes evident that the reason for scattering of the correlation between HPLC and ELISA are the effect of acyl SSa. Concentrations of SSa in Various Bupleuri Radix Table 4 indicates the concentrations of SSa in various Bupleuri radix analyzed by ELISA and HPLC after the treatment with a mild alkaline condition. One milligram or less of Bupleuri radix powders is enough for the ELISA, although HPLC analysis needs much more,24) approximately 1 g, because the sensitivity of ELISA is about 1000 times that of HPLC, as already described. The concentration of SSa analyzed by ELISA and HPLC correlated well, suggesting that the mild alkaline treatment was necessary as a only pretreatment for its analysis. Quantitative Analysis of SSa in the Kampo Medicines by ELISA Since many Kampo medicines consist of the crude drug of the Bupleurum species, along with other crude drugs, SSa was analyzed by ELISA for the standardization and quality control of Kampo medicines, which present a big problem in clinical use due to the wide variation of Bupleuri radix quality caused by differences in the growth location

Fig. 3. Correlation between the Values of SSa Obtained by ELISA and HPLC before a Mild Alkaline Treatment (A) and after a Mild Alkaline Treatment (B) Table 4. SSa Concentration of Various Bupleuri Radix Samples Determined by ELISA and HPLC Origin (Growth locales) Japan (Mishima) Japan China (Kita) China (Tianjin) China (Hebei) China

Concentration of SSa (m g/mg dwt.) ELISA by 1G6

HPLC

4.3960.09 2.0760.10 8.9860.26 6.1460.20 3.6060.18 3.6560.18

4.3660.01 2.0560.02 8.8760.31 5.9360.24 3.6360.18 3.6160.20

and species.23) Table 5 shows the concentrations of SSa. Because the acyl and/or malonyl SSa will be decomposed during the processing, the concentrations of SSa were measured by the developed ELISA without a mild alkaline treatment. The concentrations of SSa in these Kampo medicines are proportional to

70

Vol. 27, No. 1

Table 5.

Determination of SSa in the Kampo Medicines by ELISA Using MAb 1G6 ELISA by 1G6 (m g/mg)

Percentage of Bupleuri radix

Sho-saiko-to

0.76460.011

25%

Bupleuri radix 6.0 g, Pinelliae tuber 6.0 g, Scutellariae radix 3.0 g, Ginseng radix 3.0 g, Zizyphi fructus 3.0 g, Zingiberis rhizoma 1.0 g, and Glycyrrhizae radix 2.0 g

Dai-saiko-to

0.61660.015

23%

Bupleuri radix 6.0 g, Pinelliae tuber 6.0 g, Scutellariae radix 3.0 g, Aurantii fructus immaturus 3.0 g, Paeoniae radix 3.0 g, Zizyphi fructus 3.0 g, Zingiberis rhizoma 1.5 g, Rhei rhizoma 0.5 g

Saiko-kyoyo-to

0.68260.002

23%

Bupleuri radix 5.0 g, Pinelliae tuber 4.0 g, Scutellariae radix 2.0 g, Cinnamomi cortex 2.0 g, Paeoniae radix 2.0 g, Zizyphi fructus 2.0 g, Ginseng radix 2.0 g, Glycyrrhizae radix 1.5 g, Zingiberis rhizoma 1.0 g

Sai-boku-to

0.73760.003

20%

Bupleuri radix 7.0 g, Pinelliae tuber 5.0 g, Hoelen 5.0 g, Scutellariae radix 3.0 g, Magnoliae cortex 3.0 g, Zizyphi fructus 3.0 g, Ginseng radix 3.0 g, Glycyrrhizae radix 2.0 g, Perillae herba 2.0 g, Zingiberis rhizoma 2.0 g

Saiko-ka-ryukotsuborei-to

0.89760.002

17%

Bupleuri radix 5.0 g, Pinelliae tuber 4.0 g, Cinnamomi cortex 3.0 g, Hoelen 3.0 g, Scutellariae radix 2.5 g, Zizyphi fructus 2.5 g, Ginseng radix 2.5 g, Ostreae testa 2.5 g, Fossilia ossis mastodi 2.5 g, Rhei rhizoma 1.0 g, Zingiberis rhizoma 0.5 g

Sai-rei-to

0.37860.020

14%

Bupleuri radix 5.0 g, Alismatis rhizoma 5.0 g, Pinelliae tuber 4.0 g, Scutellariae radix 3.0 g, Atractylodis lanceae rhizoma 3.0 g, Polyporus 3.0 g, Hoelen 3.0 g, Cinnamomi cortex 2.5 g, Zizyphi fructus 2.5 g, Ginseng radix 2.5 g, Glycyrrhizae radix 2.0 g, Zingiberis rhizoma 1.0 g

Kami-shoyo-san

0.30460.002

13%

Bupleuri radix 3.0 g, Paeoniae radix 3.0 g, Angelicae radix 3.0 g, Atractylodis rhizoma 3.0 g, Hoelen 3.0 g, Gardeniae fructus 2.0 g, Moutan cortex 2.0 g, Glycyrrhizae radix 1.5 g, Zingiberis rhizoma 1.0 g, Menthae herba 1.0 g

Jyumi-haidoku-to

0.23560.002

11%

Pruni cortex 3.0 g, Platycodi radix 3.0 g, Bupleuri radix 3.0 g, Cnidii rhizoma 3.0 g, Hoelen 3.0 g, Saposhnikoviae radix 3.0 g, Querci cortex 3.0 g, Glycyrrihzae radix 2.0 g, Schizonepetae spica 2.0 g, Angelicae radix 2.0 g, Zingiberis rhizoma 1.0 g

0%

Pinelliae tuber 6.0 g, Zingiberis rhizoma 3.0 g, Glycyrrhizae radix 3.0 g, Cinnamomi cortex 3.0 g, Schisandrae fructus 3.0 g, Asiasari radix 3.0 g, Paeoniae radix 3.0 g, Ephedrae herba 3.0 g

Kampo medicines

Sho-seiryu-to

ND

Composition of Kampo medicines

Although the occurrence of Bupleuri radix was 17% in Saiko-ka-ryukotsu-borei-to, the concentration of SSa was higher than in the others. This phenomenon depended upon neutralizing the acidic extract by CaCO3 contained in Ostreae testa, suggesting that the neutral condition inhibited the hydrolysis of SSa.23) ND: not detectable.

Table 6. Influence of Glycyrrhizae Radix and Ginseng Radix in Shosaiko-to on Determination of SSa by ELISA Using MAb 1G6 Measured amount (m g/ml)

Percentage of b) Bupleuri radix

Sho-saiko-toa)

0.8660.003

25.00%

Sho-saiko-to without Glycyrrhizae radix

0.8860.02

27.27%

Sho-saiko-to without Ginseng radix

0.8660.01

28.57%

Sho-saiko-to without Glycyrrhizae radix and Ginseng radix

0.9160.01

31.58%

Various Sho-saiko-to

Sho-saiko-to without Bupleuri radix

ND

0%

a) Sho-saiko-to contains Bupleuri radix 6.0 g, Pinelliae tuber 6.0 g, Scutellariae radix 3.0 g, Ginseng radix 3.0 g, Zizyphi fructus 3.0 g, Zingiberis rhizoma 1.0 g, and Glycyrrhizae radix 2.0 g, respectively. b) Measured amount5determined amount of ELISA/percentage of Bupleuri radix. Samples were extracted by reflux in 250 ml water for 2 h. The extracted solutions were lyophilized after filtration. ND: not detectable.

the occurrence of Bupleuri radix in an individual prescription. In order to confirm the non-specific detection of crude drugs which contain structurally related saponins compared to SSa, we prepared extracts of Sho-saiko-to, which is one of the most widely used Kampo medicines in Japan (Table 6). When we removed Panax ginseng and Glycyrrhiza uralensis, which contained ginsenosides and glycyrrhizin, respectively, from the Sho-saiko-to prescription, almost the same concentration of SSa was obtained, resulting in no non-specific

affinity with other crude drugs. Thus, the detection of SSa in the Sho-saiko-to prescription did not occur without Bupleuri radix. Therefore, the ELISA is suitable for the analysis of SSa in a complicated background without any pretreatment. DISCUSSION Compared with the anti-SSa polyclonal antibody,15) our MAb-1G6 has higher specificity against SSa. We can produce our MAb in the same quality at any time, which is important to the stability of an assay system, whereas a polyclonal antibody cannot be expected to have such a character. Also, no investigation of SSa involved in crude plant extracts and/or Kampo medicines using anti-SSa antibody has been reported before this study. Compared with the HPLC method, the newly established ELISA methodology may make possible the easy in vitro assay of biomedical samples; therefore, it will be suitable for large numbers of samples, or of small size samples in the clinical usage of SSa. Standardization of Bupleuri radix in the Kampo medicines is difficult because the concentration of saikosaponins depends on the growth locales, the time of harvest and the part of the root, as indicated in Table 4 where we analyzed various samples of Bupleuri radix, although its control is important for clinical use. However, since a high sensitive and rapid assay system has been established against a marker compound, SSa in this study, it may be possible to solve at least the quality control of the Kampo medicines prescribed with Bupleuri radix. Moreover, since an ELISA

January 2004

dose not need any organic solvent compared with HPLC, it may be possible to be called an ecological assay system. In conclusion, the ELISA could be used to survey for low concentrations of SSa in plants and/or in experimental animals and humans.26) Moreover, it is possible to study a large number of plantlet culture and a small sample size in vitro for the breeding of Bupleurum species yielding a high concentration of SSa in our ongoing study of medicinal plants.27—31) Acknowledgments The research in this paper was supported in part by a Grant-in-Aid (No. 08457586 for Y.S.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the research fund of Kyushu University Foundation, and the research fund of the Japan Kampo Medicine Manufacturers Association from Japan Kampo Medicine Manufacturers. REFERENCES 1) 2)

3) 4) 5) 6) 7) 8) 9) 10)

Motoo Y., Sawabu N., Cancer Lett., 86, 91—95 (1994). Bermejo Benito P., Abad Martinez M. J., Silvan Sen A. M., Sanz Gomez A., Fernandez Matellano L., Sanchez Contreras S., Diaz Lanza A. M., Life Sci., 63, 1147—1156 (1998). Nose M., Amagaya S., Ogihara Y., Chem. Pharm. Bull., 37, 2736— 2740 (1989). Hattori T., Ito M., Suzuki Y., Nippon Yakurigaku Zasshi, 97, 13—21 (1991). Sakata R., Shoyama Y., Murakami H., Cytotechnology, 16, 101—108 (1994). Ishiyama M., Shoyama Y., Murakami H., Shinohara H., Cytotechnology, 18, 153—158 (1996). Shoyama Y., Fukada T., Murakami H., Cytotechnology, 19, 55—61 (1996). Tanaka H., Goto Y., Shoyama Y., J. Immunoassay, 17, 321—342 (1996). Tanaka H., Shoyama Y., Biol. Pharm. Bull., 21, 1391—1393 (1998). Xuan L., Tanaka H., Xu Y., Shoyama Y., Cytotechnology, 29, 65—70 (1999).

71 11) 12) 13) 14) 15) 16) 17) 18) 19) 20) 21) 22) 23) 24) 25) 26) 27) 28) 29)

30)

31)

Tanaka H., Fukuda N., Shoyama Y., Cytotechnology, 29, 115—120 (1999). Fukuda N., Tanaka H., Shoyama Y., Cytotechnology, 34, 197—204 (2000). Fukuda N., Tanaka H., Shoyama Y., Biol. Pharm. Bull., 22, 219—220 (1999). Fukuda N., Tanaka H., Shoyama Y., J. Nat. Prod., 63, 283—285 (2000). Jung D. W., Shibuya M., Ebizuka Y., Yoshimatsu K., Shimomura K., Sung C. K., Chem. Pharm. Bull., 46, 1140—1143 (1998). Erlanger B. F., Beiser S. M., Proc. Natl. Acad. Sci. U.S.A., 52, 68—74 (1964). Goto Y., Shima Y., Morimoto., Shoyama Y., Murakami H., Kusai A., Nijima K., Org. Mass Spectrom., 29, 668—671 (1994). Galfre G., Milstein C., Methods Enzymol., 73, 3—46 (1981). Goding J. W., J. Immunol. Methods, 39, 285—308 (1980). Murakami H., Masui H., Sato G. H., Sueoka N., Chow T. P., KanoSueoka T., Proc. Natl. Acad. Sci. U.S.A., 79, 1158—1162 (1982). Weiler E. W., Zenk M. H., Phytochemistry, 15, 1537—1545 (1976). Kitagawa I., Taniyama T., Yoshikawa M., Ikenishi Y., Nakagawa Y., Chem. Pharm. Bull., 37, 2961—2970 (1989). Akahori A., Gendaitoyoigaku., 1, 45—50 (1980). Kanazawa H., Nagata Y., Matsushima Y., Tomoda M., Takai N., J. Chromatogr. A., 630, 408—414 (1993). Ebata N., Nakajima K., Taguchi H., Mitsuhashi H., Chem. Pharm. Bull., 38, 1432—1434 (1990). Shan S., Tanaka H., Hayashi J., Shoyama S., “The Pharmaceutical Society of Japan, Abstract II,” 2001, p. 112. Shoyama Y., Zhu X. X., Nakai R., Shiraishi S., Kohda H., Plant Cell Rep., 16, 450—453 (1997). Matsumoto M., Shoyama Y., Nishioka I., Iwai H., Wakimoto S., Plant Cell Rep., 7, 636—638 (1989). Shoyama Y., Chen S., Tanaka H., Sasaki Y., Sashida Y., “Biotechnology in Agriculture and Forestry, Medicinal and Aromatic Plants,” 10th ed., Vol. 41, ed. by Bajaj Y. P. S., Springer-Verlag, Berlin, 1998, pp. 464—479. Shoyama Y., Matsushita H., Shu X. X., Kishira H., “Biotechnology in Agriculture and Forestry Somatic Embryogenesis and Synthetic Seed,” Vol. 31, 2nd ed., ed. by Bajaj Y. P. S., Springer-Verlag, Berlin, 1994, pp. 343—356. Shoyama Y., Nishioka I., Hatano K., “Biotechnology in Agriculture and Forestry, Medicinal and Aromatic Plants,” 3rd ed., Vol. 15, ed. by Bajaj Y. P. S., Springer-Verlag, Berlin, 1991, pp. 1—13.