Journal of Chromatography B, 985 (2015) 197–205

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Development of ELISA for detection of Rh1 and Rg2 and potential method of immunoaffinity chromatography for separation of epimers Huihua Qu a , Yan Wang b , Wenchao Shan c , Yue Zhang b , Huibin Feng c , Jiayang Sai d , Qingguo Wang b , Yan Zhao b,∗ a

Center of Scientific Experiment, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China School of Basic Medical Sciences, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China c School of Chinese Materia Medica, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China d The third affiliated hospital, Beijing University of Chinese Medicine, 11 Beisanhuandong Road, Chaoyang District, Beijing 100029, China b

a r t i c l e

i n f o

Article history: Received 29 August 2014 Accepted 27 January 2015 Available online 3 February 2015 Keywords: Ginsenoside-Rh1 20(R,S)-ginsenoside-Rg2 Monoclonal antibody Indirect competitive enzyme-linked immunosorbent assay Immunoaffinity chromatography

a b s t r a c t In this work, hybridomas producing anti-ginsenoside-Rh1 monoclonal antibodies (MAbs) were generated. These MAbs were subsequently used to create indirect competitive enzyme-linked immunosorbent assays (icELISAs). A linear correlation was obtained for G-Rh1 concentrations in the range from 26 to 512 ng/mL. The regression equation was y = 1.979 − 0.201 Log2 (X) with a regression coefficient of 0.9898. Precision and accuracy of the icELISA method were evaluated by the variations between replicates from well to well (intra-assay) and plate to plate (inter-assay). The recovery rates ranged from 93.16% to 108.43%. Testing with the icELISA demonstrated that the MAbs were specific for 20(S)-Rh1 and 20(S)Rg2 with no cross-reactivity against 20(R)-Rh1 and 20(R)-Rg2. The immunoaffinity chromatography column (IAC) was constructed by covalently coupling monoclonal antibody (MAb) against G-Rh1 to CNBractivated Sepharose 4B. When 20(R)-type-Rg2 passed through the IAC column, it was adsorbed, but the amount adsorbed was lower than that when 20(S)-type-Rg2 ran through the column. The differences in adsorption between the 20(S) and 20(R) type ginsenosides bring a new approach or method to separate 20(S)-Rg2 and 20(R)-Rg2 by IAC. Our results indicate that the icELISA is a sensitive and efficient approach for the identification of epimers, and the application of IAC using MAbs against small molecules provides a totally new thought and potential method for resolving epimers. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Ginseng, (Panax ginseng C.A. Meyer), is an alternative and conventional medicine used worldwide to combat cancer [1], regulate the central nervous system [2], reduce blood pressure [3] and protect against the effects of UVB radiation [4]. Ginsenosides, the active ingredients in ginseng, have a broad range of therapeutic applications. The basic structure of a ginsenoside consists of a tetracyclic triterpenoid and many chiral carbons. There are two stereoisomers present for each ginsenoside, which is due to chirality of carbon20 (C-20). These stereoisomers are called epimers. It is probable

Abbreviations: MAb, monoclonal antibody; IAC, immunoaffinity chromatography column; BSA, bovine serum albumin; OVA, ovalbumin; HAT, hypoxanthine– aminopterin–thymidine; HT, hypoxanthine–thymidine; PEG, polyethylene glycol; TMB, 3,3,5,5-tetramethylbenzidine; PBS, phosphate buffered saline; CBS, carbonate buffer solution; DW, deionized water; icELISA, indirect competitive ELISA. ∗ Corresponding author. Tel.: +86 1 6428 6705; fax: +86 1 6428 6821. E-mail address: [email protected] (Y. Zhao). http://dx.doi.org/10.1016/j.jchromb.2015.01.037 1570-0232/© 2015 Elsevier B.V. All rights reserved.

that the two different epimers of each ginsenoside have distinct biological characteristics. For example, it was found that 20(S)ginsenoside Rg3, but not 20(R)-ginsenoside Rg3, could inhibit Ca2+ , K+ and Na+ channel currents in a dose- and voltage-dependent manner, as well as inhibit androgen-dependent and independent prostate cancer cell proliferation [5–8]. Therefore, separation of 20S/R-epimers is interesting, important and would assist academic, industrial and pharmaceutical research. The ginsenoside Rh1 and Rg2 are protopanaxatriol-type compounds and are two of the major active components in the roots, stems and leaves of P. ginseng. G-Rh1 has extensive pharmacological activity in the nervous, cardiovascular, and circulatory systems. Furthermore, it has antitumor, immunoregulatory, memory improving, anti-allergy and anti-inflammatory functions [9–13]. This suggests that G-Rh1 has strong potential as a new therapeutic agent. G-Rh1 constitutes about 0.001% of ginseng [14]. The current analytical techniques used to assay Rh1 levels include high performance liquid chromatography (HPLC) [15] and liquid chromatography–mass spectrometry (LC–MS) [16]. However, these methods are far from satisfactory for analytical purposes

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in terms of sensitivity and reproducibility. Therefore, a rapid and sensitive method for monitoring the Rh1 concentration in Chinese medicines and/or Chinese herbal compounds, as well as for use in pharmacological research, is needed. The ginsenoside Rg2 has blood pressure increasing, anti-shock and heart therapy capabilities [17,18]. 20(S)-Rg2 and 20(R)-Rg2 are a pair of epimers of Rg2 that potentially have different biochemical and pharmacological properties. However, this has not been confirmed and the pharmacokinetic profiles of the Rg2 epimers remain unknown [19,20]. Thus, the resolution between 20(S)- and 20(R)epimers is important for further studies. However, the 20(S)- and 20(R)-epimers are particularly challenging to separate using conventional chromatographic approaches due to the overlap between most of their chemical and physical properties. This is due to the fact that there is only one stereochemical difference, which occurs at C20 (Fig. 1). Therefore, a preparative technique that is both simple and effective is necessary to achieve better isolation/purification between these two stereoisomers. Immunoaffinity methods utilize specific and reversible interactions between antibodies and their cognate antigens, and are highly effective at separating and purifying target analytes from complex matrices. Numerous reports have been published documenting the many uses of IAC, including studies on toxins, veterinary drugs and pesticide residues. Landsteiner was the first to demonstrate that antibodies can distinguish between stereoisomers and enantiomers. [21] Since then, highly sensitive immunological methods have been described using antibodies with stereospecificity to d- or l-a-amino acids in order to determine enantiomeric purity [22]. These antibodies were successfully applied in enzyme-linked immunosorbent assays (ELISAs) for detection of various amino acids in the presence of 100,000-fold excess of their respective enantiomers [23]. Also, indirect competitive and non-competitive ELISAs were used to test the stereospecificity of anti-L-T3 antibodies [22]. Using these assays, the IAC method was proved to be both simpler and more successful than other column chromatography at distinguishing between and separating enantiomers. However, there are currently no published reports on the resolution of 20(S)and 20(R)-ginsenosides using IAC. Previously, characteristic detection using MAbs specific for GRb1 [24], G-Rg1 [25], G-Rf [26], G-Re [27] and G-Rg3 [28], and polyclonal antibodies specific for G-Rg1 [29], G-Rf and G-Rg2 [30] have been reported. In this study, we first created and characterized MAbs specific for G-Rh1. Then, we developed an ELISA-based method to determine the concentration of G-Rh1 and G-Rg2 in traditional Chinese medicines (TCM). Furthermore, IACs were generated and could be employed as a promising method for resolution of 20(S)-Rg2 and 20(R)-Rg2.

obtained from Sigma-Aldrich (USA). 96 well-immunoplates were purchased from Corning (NY, USA). 3,3,5,5-Tetramethylbenzidine (TMB) was supplied by Sigma (St. Louis, MO, USA). Peroxidaselabeled anti-mouse IgG was provided by Organon Teknika Cappel Products (West Chester, PA, USA). Chinese medicines were obtained from the Beijing Tong Ren Tang Group (Beijing, China). All other chemicals and reagents of analytical grade were obtained from Sinopharm Chemical Reagents (Beijing, China). CNBr-activated Sepharose 4B was from GE Healthcare (USA).

2.1.2. Instruments The electronic balance (BS124-S) was from Sartorius (Gottingen, Germany). The spectrophotometric microtiter plate reader used for absorbance measurements was from Thermo Fisher Scientific (Multiskan MK3, Thermo Company, USA). Deionized water was prepared using an ultraclass UV plus water purification system (SG Company, Goettingen, Germany). Immunoreactions were carried out in an electro-heated standing-temperature cultivator (DRP9082) from Samsung Experiment Instrument Co., Ltd. (Shanghai, China). The refrigerated high-speed centrifuge was from USTC Zonkia Scientific Instruments Co., Ltd. (Anhui, China).

2.1.3. Buffers and solutions The composition of buffers and solutions used in this study is as follows. Phosphate buffered saline (PBS), pH 7.4: NaCl (137 mmol/L), Na2 HPO4 ·12H2 O (10 mmol/L), KCl (2.68 mmol/L) and KH2 PO4 (1.47 mmol/L). Carbonate buffer solution (CBS), pH 9.6: Na2 CO3 (15 mmol/L) and NaHCO3 (35 mmol/L). Washing buffer: PBS with 0.05% Tween-20 (PBS-T). Blocking buffer: 10 mg/mL gelatin in PBS (GPBS). TMB substrate solution: combination of Part A (0.5 mL added. Stock made of 24.3 mL of 0.1 mol/mL citric acid, 25.7 mL of 0.2 mol/mL Na2 HPO4 and 50 mL deionized water), Part B (10 mL added. Stock made of 2 mg of TMB dissolved in 1 mL of methanol) and Part C (32 ␮L of 0.75% H2 O2 added). Stopping solution: 2 mol/mL H2 SO4 , 1 mmol HCl (pH 4.0, 20 mL/g) and coupling buffer (0.1 M NaHCO3 containing 0.5 M NaCl, pH 8.3). 2.1.4. Sample preparation 15 TCM decoctions were prepared in accordance with the traditional method [31]. Radix Ginseng extract and 43 herbs extracts were prepared as follows: powdered herbs (30 g) were extracted with boiling water (60 mL) for 30 min and filtered through gauze. After adding ethanol to 70%, the solution was stored at 4 ◦ C overnight and filtered. Next, the solvent were vaporized to obtain dry powder and diluted in DW (10 mL).

2. Experimental 2.1. Materials and methods

2.2. Synthesis of the G-Rh1-BSA conjugate and G-Rh1-OVA conjugate

2.1.1. Chemicals and reagents G-Re (purity: 99.50%, HPLC; STA-25142009), G-Rg1 (purity: 99.00%, HPLC; STA-25442011), G-Rb1 (purity: 98.00%, HPLC; STA24842006), 20(S)-G-Rh1 (purity: 98.00%, HPLC; STA-46500000), 20(R)-G-Rh1 (purity: 98.00%, HPLC; STA-56600000), 20(S)-G-Rh2 (purity: 98.000%, HPLC; STA-09842004), 20(S)-G-Rg2 (purity: 98.00%, HPLC; STA-46800000), 20(S)-G-Rg3 (purity: 97.00%, HPLC; STA-38513006), G-Rc (purity: 98.00%, HPLC; STA-75200000) and G-Rd (purity: 98.00%, HPLC; STA-59700000) were purchased from Shanghai Standard Biotech Co. Ltd., (Shanghai, China). Bovine serum albumin (BSA), ovalbumin (OVA), complete and incomplete Freund’s adjuvants, hypoxanthine–aminopterin–thymidine (HAT), hypoxanthine–thymidine (HT) and polyethylene glycol (PEG) were

Conjugates were synthesized using a periodate oxidation procedure modified from a previously described method [32,33]. First, 2 mL of G-Rh1 (10.0 mg in 50% MeOH) were added drop-wise to 1 mL of an aqueous solution containing NaIO4 (3.5 mg), then this solution was stirred at room temperature for 1 h. Subsequently, 1 mL of 50 mmol CBS (pH 9.6) containing BSA (10.0 mg) was added to the reaction. The pH was adjusted to 9.6 using a 1 M Na2 CO3 solution and the mixture was stirred for an additional 6 h period at room temperature. The reaction was then dialyzed six times against deionized water (DW). Following this, the G-Rh1-BSA conjugate was aliquotted and stored at −4 ◦ C until used for detection and immunization procedures. G-Rh1-OVA was synthesized according to a similar protocol.

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Fig. 1. Chemical structures of 20(S)- and 20(R)-ginsenoside-Rg2.

2.3. Immunization 6 week old, female Balb/c mice were purchased from Vital River Laboratories (Beijing, China). Mice were fed a standard rodent diet (Keaoxieli animal feed Co. Ltd., Beijing, China) ad libitum and housed in an environmentally controlled (23 ± 2 ◦ C; 12 h light/dark cycle) animal facility. All experimental protocols were approved by the Committee on Ethics of Animal Experiments at Beijing University of Chinese Medicine, China. G-Rh1-BSA conjugates (50 ␮g) were injected into the back of the mice by multipoint subcutaneous injection every other week. The first immunization was consisted of G-Rh1-BSA conjugates emulsified with an equal volume of Freund’s complete adjuvant, while the second and third immunizations consisted of G-Rh1-BSA conjugates with Freund’s incomplete adjuvant. The final immunization consisted of 100 ␮g of immunogen without adjuvant was injected 3 days prior to cell fusion. Blood samples were obtained from the tail vein 1 day after the final immunization and the serum was collected for titration of G-Rh1-specific antibodies using icELISA with G-Rh1-OVA as a coating antigen.

dissolved in CBS, then 100 ␮L was added to each well of a 96-well maxisorp immunoplate and incubated for 1 h. Each well was then treated with 200 ␮L GPBS for 1 h to block non-specific absorption. The plate was washed three times with PBS-T prior to the addition of 100 ␮L MAb solutions at a range of dilutions. After a 1 h incubation, the plate was washed three times and incubated with 100 ␮L peroxidase-labeled goat anti-mouse IgG solution (1:10,000) for 30 min. The plate was then washed three times, and 100 ␮L TMB substrate solution was added to each well for a 15 min incubation. The reaction was terminated by adding 50 ␮L 2 M H2 SO4 , and the absorbance was measured at 450 nm using a Biotek ELx 800 microplate reader. All reactions were carried out at 37 ◦ C. The reactivity of the anti-G-Rh1 MAbs was determined using an icELISA. The protocol for this assay was identical to that used for the iELISA, except that the primary antibodies used were a mixture of 50 ␮L G-Rh1 and 50 ␮L anti-G-Rh1 MAbs and the subsequent incubation was 1 h. 2.7. Characteristics of anti-G-Rh1 MAbs and recovery rate of the assay

2.4. Cell fusion and preparation of the anti-G-Rh1 MAbs Three days following the final immunization, splenocytes were isolated and fused according to the PEG method with a HAT sensitive mouse myeloma cell line, SP2/0, which was obtained from Sciencell Research Laboratory (Carlsbad, CA, USA) [34,35]. Resulting hybridomas producing monoclonal antibodies reactive to G-Rh1 were identified using icELISA and were cloned by the limiting dilution method [36]. The established hybridomas were cultured in HT medium. 2.5. Production and purification of anti-G-Rh1 MAbs Hybridomas were transplanted into the abdominal cavity of 10 week old, male Balb/c mice that had been injected with Freund’s incomplete adjuvant the day before. After 5–7 days, fluid was drained from the resulting ascites. This fluid was then purified by centrifugation at 4 ◦ C at 8000 r/min for 10 min, followed by caprylic acid precipitation and protein quantification. 2.6. The establishment of iELISA and icELISA The reactivity of the anti-G-Rh1 MAbs with G-Rh1-OVA was determined using an indirect ELISA (iELISA). G-Rh1-OVA was

A calibration curve and standard curve of inhibition were established using icELISA. The sensitivity of the assay corresponds to the concentration at which a 50% inhibition is reached. The linear range of the calibration curve represents the test range of the assay. The cross-reactivity of G-Rh1 and structurally related compounds were calculated according to Weiler’s equation [37]. The recovery rate was determined using a previously described method [38]. 2.8. Determination of the amount of G-Rh1 and G-Rg2 in 15 TCMs The concentration of G-Rh1 and G-Rg2 were then determined by icELISAs. 2.9. Preparation of IACs The protective additive in the CNBr-activated Sepharose 4B (15 g) was washed away using 1 mmol HCl in the Z chromatography column (26 mm × 200 mm, Shanghai Jingke instrument Co. Ltd., Shanghai, China). The purified anti-G-Rh1 MAbs were coupled to a CNBr-activated Sepharose 4B slurry in coupling buffer. This was subsequently used to prepare the immunoaffinity column. Excess anti-G-Rh1 MAbs were washed away with five volumes of coupling solution at a flow rate of 1.0 mL/min, followed by two alternative pH

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buffers. (Alternative pH buffer A: 0.1 M Tris–HCl, (pH 8.0, containing 0.5 M NaCl). Alternative pH buffer B: 0.1 M acetic acid/sodium acetate buffer (pH 4.0, containing 0.5 M NaCl)). The medium was washed with five medium volumes of each alternative pH buffer for three cycles. Following experimental use, the IACs were washed and equilibrated with DW and stored at 4 ◦ C until reuse. The entire procedure was carried out at room temperature except for specified incubations. All of the procedures mentioned above were carried out according to the GE Healthcare Instructions. 2.10. Purification of G-Rh1 and G-Rg2 using IACs from Radix Ginseng Appropriate amounts (2 mL) of water extract (EXT) of Radix Ginseng were loaded on top of the medium, followed by washing with DW at a flow rate of 1.0 mL/min. The filtrate fractions were collected as knockout extracts (KO-EXT). The column was then washed with 0.1 M glycine-HCL buffer (pH 2.7) containing 0.5 M NaCl and the eluent fractions were collected. All procedures were carried out at room temperature. The concentrations were determined by icELISA. 2.11. Standard solutions preparation and analysis by HPLC 20(S)- and 20(R)-ginsenoside Rg2 were weighed and dissolved in a 50:50 v/v methanol–water solution in order to prepare standard solutions at a concentration of 1 mg/mL. Then, a series of sample solutions was prepared by mixing the two standard solutions at ratios of 1:1, 4:1, and 10:1 (S type:R type). Puerarin was added to the solutions as an internal standard. The concentration level of puerarin in Sample 1, 2, 3, 4, 5 is 20, 44, 12, 16, 15 ␮g/mL respectively. The HPLC was performed using an Agilent 1260 series liquid chromatographer (Agilent Technologies, Palo Alto, CA, USA); Phenomenex Luna C18(2) 100A column (5 ␮m, 250 mm × 4.60 mm, Phenomenex, USA). The components were separated via a gradient elution using water (solvent A) and acetonitrile (solvent B) with a constant flow rate of 1.0 mL/min. The gradient profile was: 0–5 min went from 10% to 30% solvent B, 5–20 min went from 30% to 50% solvent B. Each sample was injected and monitored at 203 nm with puerarin as an internal standard. The column was held at 30 ◦ C. The solutions were filtered through a 0.22 ␮m polyethersulfone membrane before injecting of the final solution into the HPLC instrument (10 ␮L) for analysis. This analysis was repeated six times. 2.12. Resolution of 20(S)- and 20(R)-Rg2 Samples were diluted and loaded onto the prepared IACs, and then the columns were washed with 5 medium volumes of ultrapure water. Then, elution of samples was initiated with 0.1 M glycine-HCl buffer (pH 2.7) containing 0.5 M NaCl. 3. Results and discussion 3.1. Generation of anti-G-Rh1 MAbs Six-week old female Balb/c mice were immunized with 50 ␮g G-Rh1-BSA conjugate. Following three immunizations with conjugate, the serum titer reached 32,000 and obvious competition was observed. Following a booster immunization of 100 ␮g, splenocytes were harvested from the immunized mice and prepared within 72 h for fusion with the HAT sensitive SP2/0 myeloma cell line according to our laboratory protocols. Hybridomas producing anti-G-Rh1 MAbs were cloned using limiting dilutions after confirmation of anti-G-Rh1 antibody production by screening with icELISA. Ascites

Fig. 2. Calibration curve of inhibition by 20(S)-G-Rh1 using icELISA. A range of concentrations of G-Rh1 were incubated with anti-G-Rh1 (1/10,000) in wells precoated with G-Rh1-OVA (1:4000).

induced by transplant of these hybridomas contained 43.02 mg/mL of anti-G-Rh1 antibody before purification and 4.66 mg/mL after purification, where the antibody protein represented 10.83% of the total protein content. Comparison of the anti-G-Rh1 antibody titers before and after purification revealed only minor differences. The anti-G-Rh1 MAbs generated were named 1G8C8. 3.2. Assay sensitivity and specificity The optimal concentrations of coating antigen (G-Rh1-OVA) and anti-G-Rh1 MAbs were determined by icELISA. The icELISA was optimized and performed with G-Rh1-OVA (1:4000) and anti-GRh1 MAbs (1:10,000), and a calibration curve was generated. A range of 20(S)-G-Rh1 amounts were added in order to compete with the coated antigen and create a standard curve of inhibition. Fig. 2 displays the competitive inhibition between anti-G-Rh1 MAbs and G-Rh1-OVA resulting from increasing concentrations of G-Rh1. This provides a calibration curve of G-Rh1 in the icELISA. Under these conditions, a linear relationship between optical density and concentrations in the range of 26–512 ng/mL of G-Rh1 (R2 = 0.9898) was observed. The affinity of the monoclonal antibody was obtained by the method of ELISA according to the equation reported by Friguet [39], and it is intimately related with the sensitivity of the antibody. The sensitivity of the antibody was satisfactory with an IC50 of 125 ng/mL, same with that before purification and a limit of detection (LOD) of 26 ng/mL in buffer. The affinity constant of monoclonal antibody was 9.16 × 106 L/mol. The anti-G-Rh1 MAbs cross-reacted with 20(S)-type-hydroxylG-Rg2 (470.65%), G-Rg3 (13.88%) and G-Rh2 (12.25%), which are structurally related to G-Rh1, but did not cross-react with 20(R)G-Rh1, 20(R)-G-Rg2 or 20(S)-type-glucosyl group ginsenosides (0.09%) (G-Rb1, G-Re, G-Rg1, G-Rc, G-Rd) (Table 1). These results suggest that MAbs (1G8C8) may recognize the chemical structures containing the hydroxyl group at C-20(S), because the configuration of the hydroxyl group at C-20(S) of panaxadiol and/or panaxtrol saponin molecules is critical for the cross-reaction. Calibration curves of inhibition by 20(S)-G-Rh2, 20(S)-G-Rg2 and 20(S)-G-Rg3 are shown in Fig. 3. In addition, the icELISA using these MAbs was also specific to 20(S)-G-Rg2, 20(S)-G-Rg3 and 20(S)-G-Rh2 compared with other ginsenosides, and can be widely used in measuring the total amount of 20(S)-type-hydroxyl-ginsenosides. Overall, this assay provides a tool with increased sensitivity for G-Rh2, G-Rg3, G-Rh1 and G-Rg2.

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Fig. 3. Calibration curve of inhibition by 20(S)-G-Rg2, 20(S)-G-Rg3 and 20(S)-G-Rh2 using icELISA. A range of concentrations of 20(S)-G-Rg2, 20(S)-G-Rg3 and 20(S)-G-Rh2 were incubated with anti-G-Rh1 (1/10,000) in wells precoated with ginsenoside G-Rh1-OVA (1:4000).

TCMs have been widely used for treating disease in China for thousands of years. In TCM theory, compatibility refers to the combination of two or more herbs based on clinical settings and the properties of the herbs. About 500 herbal drugs that make up more than 100,000 formulas have been commonly used during the past 2000 years. For example, ginseng is frequently prescribed in conjunction with Astragalus, Paeonia, Angelica and Rhizoma atractylodis. Formulas containing ginseng, such as Yu-ping-feng-san, Si-jun-zi-tang and Xiao-chaihu-tang, are used to treat deficiency syndromes and also have anti-inflammatory, antioxidant, antihepatic fibrosis, antitumor, antithrombotic and antiplatelet activities. Based on the cross reaction of 43 herb extracts (Table 2), it was found that the anti-G-Rh1 MAbs can also be widely used in the field of pharmacokinetics and for the quantitative and qualitative analysis of TCMs.

3.4. Assay recovery rate The recovery rate was measured to evaluate the reliability of the assay. Several concentrations of G-Rh1 were incubated with 50 ␮L of anti-G-Rh1 MAbs. For each level, four samples were analyzed and the amount of G-Rh1 was determined by icELISA. Recovery of G-Rh1 ranged from 93.16% to 108.43% (Table 4). The accuracy and consistency of the data indicate that this assay is sufficiently reliable for determination of G-Rh1 in a variety samples.

3.3. Accuracy and variation of the icELISA The accuracy and variation of the anti-G-Rh1 MAb icELISA were evaluated in terms of the relative standard deviations (RSDs) of intra-(well-to-well and triplicates) and inter-(plate-to-plate and day-to-day for 3 consecutive days)assays. The RSDs were less than 7.06% and 6.93% for intra-assay and inter-assay variation, respectively (Table 3). These results indicate that this assay is highly accurate and consistent. However, the accuracy of determination could be affected by several factors, such as coating concentration, plate wells and uneven temperature. Therefore, a new standard curve should be created each time the assay is performed to reduce variation.

Fig. 4. IAC elution profile of Radix ginseng EXT monitored by icELISA using anti-GRh1 MAbs. From 0 to 24th tube contains the KO-EXT fraction, and from 25th to 46th tube contains the eluent fraction with a peak around 31st tube.

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Table 1 Cross-reactivity (%) of MAb 1G8C8 to various compounds was determined by icELISA according to Weiler’s equation.

Table 2 Cross-reactivity (%) of anti-G-Rh1 MAbs to various crude extracts was determined by icELISA according to Weiler’s equation.

Classification

Compound

Cross-reactivity (%)

Compound

Cross-reactivity (%)

Terpene

Ginsenoside Rg2 (S) Ginsenoside Rh1 (S) Ginsenoside Rg3 (S) Ginsenoside Rh2 (S) Ginsenoside Rh1 (R) Ginsenoside Rg2 (R) Ginsenoside Rb1 Ginsenoside Re Ginsenoside Rg1 Ginsenoside Rc Ginsenoside Rd Ginsenoside Ro Notoginsenoside Glycyrrhizic acid Glycyrrhetic acid Saikosaponin Paeoniflorin Gentiopicrin Gardenia jasminoides Ellis

470.65% 100.00% 13.88% 12.25% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09%

Flavonoid

Puerarin Baicalin Baicalein Hyperoside Rutin Hesperidin Naringin

0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09%

Phenylpropanoids

Chlorogenic acid Ferulic acid

0.09% 0.09%

Steride

Cholic acid Deoxycholic acid

0.09% 0.09%

Anthraquinones

Rheumemodin Rheinic acid

0.09% 0.09%

Alkaloid

Berberine

0.09%

Other

Salvianolic acid Curcumin Gastrodin Amygdalin

0.09% 0.09% 0.09% 0.09%

Radix Ginseng Radix Puerariae Radix Knoxiae Fructus Evodiae Cortex Magnolia Officindis Cortex Phellodendri Cortex Fraxini Flos Genkwa Fructus Gardeniae Fructus Aurantii Immaturus Fructus Trichosanthis Fructus Schisandrae Chinensis Fructus Crotonis Herba Artemisiae Scopariae Herba Andrographis Medulla Tetrapanacis Polyporus Radix Paeoniae Alba FlosInulae Radix Scutellariae Radix Bupleuri Radix Angelicae Sinensis Radix Kansui Radix Platycodonis Radix Ophiopogonis Radix Phytolaccae Radix Asparagi Radix Pulsatillae Radix Astragali Radix Arctii Radix Aconite Lateralis Preparata Radix Glycyrrhizae Radix Et Rhizoma Rhei Radix Vladimiriae Ramulus Cinnamomi Rhizome Pinelliae Rhizome Coptidis Herba Asari Rhizome Alismatis Rhizome Anemarrhenae Rhizome Cimicifugae Rhizome Curcuma Longae Semen Lepidii, Semen Descurainiae Semen Armeniacae Amarum

100.00% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09% 0.09%

3.5. G-Rh1 and G-Rg2 measurement in Radix Ginseng and TCMs We measured the concentration of G-Rh1 and G-Rg2 in TCMs. The results are listed in Table 5. The Du Shen Decoction contained only Radix Ginseng and G-Rg2 + G-Rh1 content was calculated as 0.011%, which is consistent with previous report. [40] Based on these data, we are confident that this ELISA method can be applied to determine G-Rh1 and G-Rg2 levels in herbs, and, therefore, can serve as a useful and convenient method for measurement of specific substances in pharmacokinetic and target studies.

3.6. Purification of G-Rg2 by IAC Fig. 4 shows that high purity G-Rh1 and G-Rg2 can be obtained using IACs. The water extract of Radix Ginseng was loaded onto the IACs and then the columns were washed with DW and eluted by 0.1 M glycine-HCl buffer (pH 2.7) containing 0.5 M NaCl. The amount of loading sample is over maximum capacity. The G-Rh1 and G-Rg2 concentration in the collected fractions was determined by the competitive ELISA. A peak of G-Rh1 and G-Rg2 is observed in the elution profile indicating the successful separation of G-Rh1 and G-Rg2 from water extract of Radix Ginseng. The elution concentration was 358.66 ␮g/mL, the knockout (overload) concentration was 1643.51 ␮g/mL and the loading concentration of the sample was 2 mg/mL. Based on this, the recovery was 100.61%.

Table 3 Assay accuracy and variation of the icELISA. Accuracy was evaluated by measuring four concentrations of G-Rh1 in six replicate wells of each. Data are presented as the mean ± SD. Intra-assay was tested by the six replicate wells and the inter-assay was carried out in 3 days. And they were described by the coefficient of variation (CV%) based on the mean ± SD of the measurements from the six replicate wells. G-Rh1(ng/mL)

0 16 128 1024

CV% Intra-assay

Inter-assay

2.09 1.34 6.36 7.06

3.94 6.93 6.25 3.64

Table 4 Assay recovery rate of G-Rh1 by icELISA. Data represents the mean ± SD from triplicate wells of each sample. The concentration (recovered level) was calculated using the equation of calibration curve as presented in Fig. 2. The recovery rate was calculated as follows: recovery (%) = (measured amount − control)/spiked amount × 100%. Spiked level (ng/mL)

Recovered level (ng/mL)

30 50 90 120

27.95 54.22 92.01 123.32

± ± ± ±

2.06 3.09 3.51 8.00

Recovery (%) 93.16 108.43 102.23 102.76

H. Qu et al. / J. Chromatogr. B 985 (2015) 197–205

203

Table 5 Total concentration of G-Rg2 + G-Rh1 in TCMs as measured by ELISA. TCM

Mean ± S.D. (␮g/mL)

Percentage of Ginseng radix

Composition of Kampo medicine

Du Shen Decoction Shen Fu Decoction Sheng Mai Yin Si Jun Zi Decoction

2.42 ± 0.32 2.67 ± 0.66 4.12 ± 0.14 1.75 ± 0.31

100% 43% 38% 27%

Li Zhong Decoction

0.53 ± 0.06

25%

Xiao Chaihu Decoction

0.95 ± 0.11

11%

Guizhi Xinjia Decoction

1.04 ± 0.18

14%

Guizhi jia Renshen Decoction

0.83 ± 0.21

19%

Banxia Xiexin Decoction

1.15 ± 0.06

12%

Zhigancao Decoction

0.14 ± 0.01

Sini jia Renshen Decoction

1.32 ± 0.43

Wuzhuyu Decoction

0.32 ± 0.05

6.7%

Bu Zhong Yi Qi Decoction

1.09 ± 0.05

9.5%

Fuzi Decoction

1.01 ± 0.15

Gegen huangqin huang lian Decoction

NDa

Radix Ginseng 9 g Radix Ginseng 9 g, Radix Aconiti Praeparata 12 g Radix Ginseng 9 g, Radix Ophipogonis 9 g, Fructus Schisandrae 6 g Radix Ginseng 9 g, Rhizome Atractylodis Macrocephalae 9 g, Paria 9 g, Radix Glycyrrhizae Praeparata 6 g Radix Ginseng 9 g, Rhizoma Zingiberis 9 g, Rhizoma Atractylodis Macrocephalae 9 g, Radix Glycyrrhizae Praeparata 9 g Radix Bupleuri 16 g, Radix Scutellariae 6 g, Rhizoma Zingiberis Recens 6 g, Radix Ginseng 6 g, Rhizoma Pinelliae 6 g, Radix Glycyrrhizae Praeparata 6 g, Fructus Ziziphi Jujubae 2 Pcs Radix Paeoniae Alba 12 g, Radix Ginseng 9 g, Ramulus Cinnamomi 9 g, Radix Glycyrrhizae Praeparata 6 g, Rhizoma Zingiberis 12 g, Fructus Ziziphi Jujubae 3 Pcs Ramulus Cinnamomi 12 g, Radix Glycyrrhizae Praeparata 9 g, Rhizima Atractylodis Macrocephalae 9 g, Radix Ginseng 9 g, Rhizoma Zingiberis 9 g Rhizoma Pinelliae 9 g, Radix Scutellariae 6 g, Rhizoma Coptidis 3 g, Rhizoma Zingiberis 6 g, Radix Ginseng 6 g, Radix Glycyrrhizae Praeparata 6 g, Fructus Ziziphi Jujubae 3 Pcs Radix Glycyrrhizae Praeparata 12 g, Rhizoma Zingiberis 9 g, Ramulus Cinnamomi 9 g, Radix Ginseng 6 g, Radix Rehmanniae51 g, Colla Corii Asini 6 g, Radix Ophiopogonis 9 g, Fructus Cannabis 9 g, Fructus Ziziphi Jujubae 9 Pcs Crude Radix Aconiti 10 g, Rhizoma Zingiberis 6 g, Radix Glycyrrhizae Praeparata 4 g, Radix Ginseng 4 g Fructus Evodiae 6 g, Radix Ginseng 2 g, Fructus ZiziphiJujubae 2 Pcs, Rhizoma Zingiberis Recens 12 g Radix AstragaliSeuHedysari12 g, Radix GlycyrrhizaePraeparata6 g, Radix Ginseng 4 g, Radix AngelicaeSinensis 2 g, Pericarpium CitriReticulatae 4 g, Rhizoma Cimicifugae 4 g, Radix Bupleuri 4 g, Rhizima Atractylodis Macrocephalae 6 g Radix Aconiti Praeparata 15 g, Paria 9 g, Radix Ginseng 6 g, Rhizima Atractylodis Macrocephalae 12 g, Radix Paeoniae Alba 9 g Radix Puerariae 6 g, Radix Scutellariae 4 g,Rhizoma Coptidis 1 g, Radix Glycyrrhizae Praeparata 1 g

3.8%

17%

12%

All data are presented as the mean ± SD (1 extraction/6 measurements). NDa = not detectable.

3.7. Potential resolution of 20(S) and (R)-type-ginsenosides by IACs 20 (R,S)-Ginsenoside-Rg2 is an anti-shock agent that is prescribed as a racemate. Previously, several methods for isolation and characterization of (20 S)- and (20R)-ginsenoside have been published, for example HPLC, silica gel chromatography and recrystallization for ginsenoside Rg2 and Rg3 [41,19]. The HPLC method was developed to simultaneously analyze the enantiomers of 20(R)-ginsenoside-Rg2 and 20(S)-ginsenoside-Rg2, where enantiomeric separation and identification were successfully achieved using a Diamonsil ODS C18 reversed-phase column [42]. In this study, we provided a new approach and method for resolving 20(S)-G-Rg2 and 20(R)-G-Rg2 using IACs with anti-Rh1 antibody. Mixture of 20(S)-G-Rg2 and 20(R)-G-Rg2 was created at ratios of 1:1, 4:1, and 10:1, respectively. These samples were loaded onto a prepared IAC and washed with 5 medium volumes of the washing solvent (deionized water), followed by the elution solvent (0.1 M glycine-HCl buffer (pH 2.7) containing 0.5 M NaCl). The

washing fractions (WFs) and elution fractions (ELs) were collected. The WFs were evaporated and the dry residue was redissolved to a 50:50 v/v methanol–water solution. Then, the loading sample and WFs were injected into the HPLC instrument for analysis (see Fig. 5). The peaks were identified by comparing the retention time and the UV spectra of the samples to the chemical standards. The results were shown in Table 6. Amount of 20(S)-G-Rg2 and 20(R)-G-Rg2 in WF fractions was calculated by internal standard method [43]. Cross-reaction analyses revealed that the anti-G-Rh1 MAbs has no cross-reactivity with 20(R)-type-hydroxyl-ginsenosides and 20glucosyl group ginsenosides. When the sample 2 passed through the IACs, 20(R)-type-Rg2 could also be adsorbed by the MAbs, thus causing the amounts to be lower than 20(S)-type-Rg2. This result was surprising and the reason behind it is unclear. The difference in adsorption between the 20(S) and 20(R) type ginsenosides suggests that IACs has the potential for resolution of both epimers. The relative quantities of S-type-Rg2 in the ELs obtained from samples 3 and 4 following IAC significantly increased, reaching more than 2:1 and 10:1. Furthermore, the ELs obtained from sample 5 only contained

Table 6 Amount of 20(S)-G-Rg2 and 20(R)-G-Rg2 using IAC measured by HPLC. WF fractions means washing fractions, EL fractions means elution fractions. Amount of 20(S)-G-Rg2 and 20(R)-G-Rg2 in WF fractions was calculated by internal standard method. Amount of 20(S)-G-Rg2 and 20(R)-G-Rg2 in EL fractions was calculated as follows: amount of 20(S/R)-G-Rg2 in EL fraction = amount of 20(S/R)-G-Rg2 in sample – amount of 20(S/R)-G-Rg2 in WF fraction. No.

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

Sample

WF fractions

EL fractions

S/mg

R/mg

S:R

S/mg

R/mg

S:R

S/mg

R/mg

S:R

2.00 0 0.80 2.40 3.00

0 1.00 0.80 0.60 0.30

– – 1:1 4:1 10:1

0.09 0 0.27 0.25 0.37

0 0.47 0.54 0.34 0.3

– – 1:2 1:1.4 12:1

1.91 0 0.53 2.15 2.63

0 0.53 0.26 0.26 0

– – 2:1 8:1 –

204

H. Qu et al. / J. Chromatogr. B 985 (2015) 197–205

Fig. 5. HPLC analysis of loading samples and WFs. Sample 1 and sample 2 were prepared with pure 20(S)-G-Rg2 and 20(R)-G-Rg2 respectively. The ratio of S:R type G-Rg2 is increasing in sample-3,4,5. In sample 5 (S:R = 10:1), the mass of 20(R)-G-Rg2 in washing fraction is equal to the loading sample, which indicates there is pure 20(S)-G-Rg2 in the elution fraction.

S-type-Rg2, but no R-type-Rg2. These results indicate that the differences in adsorption between 20(S)- and 20(R)-ginsenoside-Rg2 have potential for resolution using the IAC method. Although this study demonstrates the potential of IAC for separation of 20(S)- and (R)-type-ginsenosides, the columns used are only capable of separating a limited amount of the epimers. Therefore, further research is needed to increase the amounts of the epimers that can be separated on a single column. 4. Conclusions In the present study, anti-G-Rh1 MAbs were generated for the first time, and an icELISA was established using these MAbs. This icELISA was proved to be a sensitive and efficient approach with great potential for measuring 20(S)-G-Rh1 and 20(S)-G-Rg2 in TCMs and herbs. Furthermore, the knockout or purification of 20(S)-G-Rh1 and 20(S)-G-Rg2 by IAC is an efficient potential method that could be used to interpret the efficacy of TCM material foundations. These methods allow for comparative studies on the effects of extracts and knockout-target-compound extracts, which is a promising new strategy to learn about the unique characteristics of Chinese herbs or compound recipes. Furthermore,

the icELISA and IAC established above also provide a simple and effective potential methodology to identify and resolve chiral compounds, although, according to our work, this needs to be further validated.

Acknowledgements The authors wish to thank Boston Professional Group (BPG) Editing for providing language help and proof reading the article. This research was supported by the National Natural Science Foundation of China (81373542, 81473338, 81430102), Beijing University of Chinese Medicine (The Team of Applied Basic Research of Classical Prescription).

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