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Quick analysis of baicalin in Scutellariae Radix by enzyme-linked immunosorbent assay using a monoclonal antibody
Talanta 77 (2008) 346–350
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Talanta journal homepage: www.elsevier.com/locate/talanta
Quick analysis of baicalin in Scutellariae Radix by enzyme-linked immunosorbent assay using a monoclonal antibody Katsumi Kido a , Osamu Morinaga b , Yukihiro Shoyama b , Hiroyuki Tanaka a,∗ a b
Department of Phamacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan Faculty of Pharmaceutical Sciences, Nagasaki International University, Huis Ten Bosch 2825-7, Sasebo, Nagasaki 859-3298, Japan
a r t i c l e
i n f o
Article history: Received 22 May 2008 Received in revised form 19 June 2008 Accepted 19 June 2008 Available online 1 July 2008 Keywords: Baicalin Monoclonal antibody ELISA Scutellariae Radix
a b s t r a c t To establish an immunoassay for baicalin (BA), a hybridoma cell line (9D6) secreting a monoclonal antibody (MAb) against BA was prepared by cell fusion with splenocytes derived from a mouse immunized with BA–bovine serum albumin (BSA) conjugate and a myeloma cell line, SP2/0-Ag14. MAb 9D6 shows specific reactivity against BA and its aglycone, baicalein, but not against other natural products. We developed an enzyme-linked immunosorbent assay (ELISA) using MAb 9D6 in a competitive manner, ranging from 200 ng/mL to 2 g/mL. After validating the developed ELISA on the basis of intra- and inter-assays and a recovery experiment, it was found that the ELISA was not only simple, but also sufficiently reliable and accurate for quality control of Scutellariae Radix. It allowed determination of BA in complex and mixed materials, such as Kampo medicines. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Baicalin (BA) is the most abundant compound among more than 30 kinds of flavonoids, such as baicalein, wogonin, and wogonin 7-O--d-glucuronide, in Scutellaria baicalensis Georgi (Fig. 1 Labiatae). BA has been reported to have anti-allergic [1], antiinflammatory [2], anti-HIV [3,4], anti-cancer [5,6], anti-oxidant, and free-radical scavenging effects [7,8]. Recently, we found that BA must be the most important component in the defense mechanism including oxidative burst against plant disease and insect attack [9,10]. Scutellariae Radix (S. Radix), which is the dried root of S. baicalensis, is one of the most important crude drugs used widely in Kampo medicines in Japan. Because BA shows these characteristics, the concentration of BA in S. Radix used clinically should be ≥10% (w/w), as defined by the 15th edition of Japanese Pharmacopoeia. Kampo medicines containing S. Radix, which are clinically important, occasionally cause side effects [11]. For example, interstitial pneumonia has been aggravated if Shosaikoto containing S. Radix and interferon (IFN)-␣ are used for treatment of chronic liver diseases. In this case, baicalein is believed to be a candidate in this side effect [12]. Therefore, concentrations of baicalein and BA which is hydrolyzed to give baicalein in body fluids should be monitored precisely in Kampo medicines.
∗ Corresponding author. Tel.: +81 92 642 6668; fax: +81 92 642 6668. E-mail address: [email protected] (H. Tanaka). 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.06.034
Various analytical approaches have been investigated for the determination of BA in S. Radix, Kampo medicines, and biogenic materials. Thin-layer chromatography and gas chromatography–mass spectrometry were applied to qualitative analysis of BA [13]. High-performance liquid chromatography (HPLC) with ultraviolet detection has been adopted as a quality control for S. Radix in Japanese Pharmacopoeia. HPLC with electrochemical detection showed a high sensitivity in the pharmacokinetic study of BA [14–16]. To establish the fingerprints of S. Radix, micellar electrokinetic capillary electrophoretic methods were also reported to separate and determine BA and flavonoids [17–19]. We have been focusing on an immunochemical approach for determination of phytochemicals [20–24] because it is reproducible, rapid, and highly sensitive for simultaneous analysis. However, since immunoassays for BA have not yet been reported, a monoclonal antibody (MAb) against BA was prepared successfully, and then applied to an enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of BA in this study. 2. Experimental 2.1. Regents and materials BA and baicalein were purchased from Wako Pure Chemicals (Osaka, Japan). Bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma (Steinheim, Germany). Freund’s complete and incomplete adjuvants were obtained from Difco (Detroit, MI, USA). Peroxidase-labeled anti-mouse IgG was
K. Kido et al. / Talanta 77 (2008) 346–350
Fig. 1. Structure of baicalin (BA) and its related compounds.
purchased from MP Biomedicals (Solon, OH, USA). All other chemicals were standard commercial products of analytical-reagent grade. Samples of various S. Radixes were purchased from Tochimototenkaido Corporation (Osaka, Japan), Takasago Yakugyo Corporation (Osaka, Japan), Daido Corporation (Osaka, Japan), and Yamada Corporation (Osaka, Japan). Kampo medicines were procured from Tsumura & Co. (Tokyo, Japan), Sanwa Shoyaku Co., Ltd. (Tochigi, Japan), and Kotaro Kampo Seiyaku Co., Ltd. (Osaka, Japan). 2.2. Synthesis of immunogen 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC; 5 mg) was added to a mixture of BA (5 mg) and BSA (5 mg) in 50 mM carbonate buffer (pH 9.6). The mixture was stirred at room temperature for 6 h. After the solution was dialyzed five times against H2 O, the dialysate was lyophilized to yield the BA–BSA conjugate (8.6 mg). BA–HSA conjugate was also synthesized in the same manner.
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for antibodies recognizing BA by indirect and indirect competitive ELISAs using BA–HSA as a solid-phase antigen. On the fourth day after the final immunization (100 g protein), splenocytes were isolated and fused with an aminopterin-sensitive mouse myeloma cell line, SP2/0-Ag14, by the polyethylene glycol (PEG) method [27]. SP2/0-Ag14 used for the cell fusion was obtained from RIKEN BioResource Center (Ibaragi, Japan). Hybridomas were obtained selectively by cultivation in a medium containing hypoxanthine–aminopterin–thymidine (HAT); those producing MAb reactive to BA were cloned by the limiting dilution method [28]. An established hybridoma was cultured in eRDF medium (Toyko, Japan) supplemented with 10 g/mL insulin, 35 g/mL transferrin, 20 M ethanolamine, and 25 nM selenium [29]. 2.5. Indirect ELISA The reactivity of MAb to BA–HSA was determined by an indirect ELISA. BA–HSA (5 molecules of BA per molecule of HSA) (100 L, 1 g/mL) dissolved in 50 mM carbonate buffer at pH 9.6 was adsorbed to the wells of a 96-well immunoplate. It was then treated with 300 L of 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% of Tween 20 (TPBS) and reacted with 100 L of MAb solution to be tested for 1 h. The plate was washed three times with PBS containing 0.05% Tween 20 (TPBS) and incubated with 100 L of a 1000-fold diluted peroxidase-labeled anti-mouse IgG solution for 1 h. After washing the plate three times with TPBS, 100 L of substrate solution [0.1 M citrate buffer (pH 4) containing 0.003% H2 O2 and 0.3 mg/mL of 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)] was added to each well and incubated for 20 min. Absorbance at 405 nm was measured using a microplate reader. All reactions were carried out at 37 ◦ C.
2.3. Determination of hapten number in BA–carrier protein conjugates by matrix-assisted laser desorption/ionization (MALDI)-TOF mass spectrometry
2.6. Indirect competitive ELISA
The hapten number of the BA–BSA and BA–HSA conjugates was determined by MALDI-TOF-MS as previously described [25,26]. A small amount (1–10 pmol) of the antigen conjugate was mixed with a 1000-fold molar excess of sinapinic acid in an aqueous solution containing 0.15% trifluoroacetic acid. The mixture was analyzed with a JEOL Mass Spectrometer (JMS) time-of-flight (TOF) mass monitor and irradiated with an 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 long evacuated tube. The data were analyzed using the GRAMS/386 software (Galactic Industries, Salem, NH, USA).
BA–HSA (100 L, 1 g/mL) dissolved in 50 mM carbonate buffer (pH 9.6) was adsorbed to the wells of a 96-well immunoplate. It was then treated with 300 L SPBS for 1 h to reduce non-specific adsorption. Fifty microliters of various concentrations of BA or samples dissolved in 10% methanol were incubated with 50 L of MAb solution (1.7 ng/mL) for 1 h. The plate was washed three times with TPBS, and then incubated with 100 L of a 1000-fold diluted peroxidase-labeled anti-mouse IgG solution for 1 h. After washing the plate three times with TPBS, 100 L of ABTS solution was added to each well and incubated for 20 min. The absorbance at 405 nm was measured using a microplate reader. All reactions were carried out at 37 ◦ C.
2.4. Preparation of anti-BA MAb Male BALB/c mice (5 weeks old) were purchased from KBT Oriental Co. (Saga, Japan). Food (MF; Oriental Yeast Co., Tokyo, Japan) and water were provided ad libitum. Procedures and animal care were approved by the Committee on Ethics of Animal Experiments, Graduate School of Pharmaceutical Sciences, Kyushu University, Japan, and were conducted according to the Guidelines for Animal Experiments of the Graduate School of Pharmaceutical Sciences, Kyushu University. The mice were injected with BA–BSA four times. Immunization was initiated by intraperitoneal injection of the conjugate (50 g) in phosphate-buffered saline (PBS) emulsified with an equal volume of Freund’s complete adjuvant. As a second immunization, 50 g of the conjugate in Freund’s incomplete adjuvant was injected intraperitoneally 2 weeks after the initial injection. Mice were bled 4 days after the boosts, and the sera were monitored
2.7. Specificity and sensitivity of the assay The cross-reactivities (CRs, %) of BA and related compounds were determined according to Weiler’s equation [30]: CR (%) =
Concentration of BA at A/A0 = 50% Concentration of cross-reacting substance at A/A0 = 50% × 100
A and A0 are the absorbance in the presence and absence of the test compound, respectively. 2.8. HPLC instruments and conditions The chromatography system used in this study consisted of a LC10AD (Shimadzu, Kyoto, Japan), a UV-8 model II detector (Tosoh,
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Tokyo, Japan), and a Chromatopac C-R5A data analyzer system (Shimadzu). The analytical column was Cosmosil 5C18 -AR column (4.6 mm i.d. × 150 mm, Nacalai Tesque, Kyoto, Japan) maintained at room temperature. The mobile phase comprised CH3 CN–H2 O (3:7) containing 60 mM H3 PO4 and a flow rate of 1.0 mL/min. A 10-L aliquot of each sample was injected and monitored at 254 nm. 2.9. Sample preparation Dried samples (10 mg) of various S. Radixes and Kampo medicines were powdered, and extracted five times with MeOH (1 mL) under sonication. The extracted solution was filtered, and the combined extracted solution was adjusted to a volume of 10 mL with MeOH. The adjusted solution was diluted with H2 O to prepare a sample solution suitable for the indirect competitive ELISA. 2.10. Recovery experiment Various amounts of BA were added to a dried powder of S. Radix (10 mg). The amount of BA in the unspiked sample was determined to be 1.09 mg. The spiked sample was extracted with MeOH by sonication and filtered. The combined extract was adjusted to 10 mL with MeOH, diluted to prepare a 10% MeOH solution, and assayed by the indirect competitive ELISA. The recovery rate of BA was calculated from the spiked and recovered amounts in the same concentration ranges. Recovery (%) =
Measured amount − 1.09 × 100 Spiked amount
Fig. 2. Direct determination of BA–BSA conjugates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). [M+H]+ and [M+2H]2+ are single- and double-protonated molecules of BA–BSA, respectively.
production of antibodies by the immunized mice, their spleen was extirpated to prepare splenocytes including antibody-secreting cells for cell fusion. Cell fusion was performed with the splenocytes and SP2/0 myeloma cells following the procedure established in this laboratory [31]. A hybridoma producing MAb reactive to BA was obtained through a series of screenings with the indirect and indirect competitive ELISAs and cloning by the limiting dilution method. The MAb, designated 9D6, was classified as IgG2a having light chains.
3. Results and discussion
3.3. Assay sensitivity and assay specificity
3.1. Synthesis of the BA–BSA conjugate and direct determination of the hapten–carrier protein conjugates by MALDI-TOF mass spectrometry
An optimal concentration of anti-BA MAb applied to the indirect competitive ELISA was measured by the indirect ELISA with various concentrations of MAb 9D6. This optimal concentration was 1.7 ng/mL, which was selected for the indirect competitive ELISA to define sensitivity and specificity of the MAb because it was the minimum concentration of the MAb providing a sufficient signal to detect the interaction between the MAb and BA–HSA as a solidphase antigen. The indirect competitive ELISA was developed using this concentration of MAb and polystyrene microtiter plates precoated with 1 g/mL of BA–HSA. In addition, a 10% MeOH was used as a diluted solution for standard BA and samples, because addition of MeOH improved solubility of BA in the solutions at circumneutral condition. Fig. 3 shows inhibition of the interaction between the MAb and BSA-HSA using various concentrations of BA, providing a standard curve of BA in the indirect competitive ELISA. Under these conditions, a calibration curve for BA was generated in the linear range from 200 ng/mL to 2.0 g/mL (R2 = 0.994), which revealed that the sensitivity of the assay with a detection limit of 100 ng/mL was improved at least 5-fold compared with the HPLC method [32]. Specificity of MAb 9D6 was analyzed by comparing its crossreactivities to compounds structurally related to BA and other natural products. Table 1 lists the CRs of the MAb examined by the indirect competitive ELISA, and calculated using the concentration of BA yielding midrange values on the basis of the method reported by Weiler et al. [30]. MAb 9D6 recognized not only BA, but also baicalein (CR: 51.4%) indicating a slight reactivity to wogonin (0.73%), although no detection against other flavonoids such as rutin, hesperidin, and daidzein (Table 1). It is easily suggested that all of function in ring A of BA might be immunized because MAb 9D6 strongly reacts BA structurally related compounds. Com-
Since a low molecular weight compound (hapten) like BA is poorly immunogenic, a hapten–protein conjugate should be synthesized and used as an immunogen for developing antibodies against such compounds. BA possessing a carboxylic acid group in a molecule, can be conjugated with a protein by carbodiimide system. Therefore, BSA was selected as a carrier protein and readily coupled with BA using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) as a coupling reagent in this study. Fig. 2 shows the MALDI-TOF mass spectrum of the BA–BSA conjugate to validate its hapten number. A broad peak coinciding with the conjugate of BA and BSA appeared from m/z 66,900 to 74,900, centering at ∼m/z 70,200. Using the experimental result and a molecular weight of 66,433 for BSA, the calculated value of the BA component in the conjugate was 3767, suggesting 9 molecules of BA (on average) conjugated with a BSA molecule. This result revealed that adequate numbers of BA molecules were coupled with BSA, which meant that the BA–BSA conjugate could be used as an immunogen to evoke anti-BA antibodies in mice. BA–HSA was prepared as a solid-phase antigen for ELISA, and the hapten number of BA–HSA conjugate was also determined reliably from its spectrum to be at ∼5 molecules. 3.2. Production and characteristics of MAb against BA BALB/c mice were immunized with BA–BSA until the absorbance of more than 0.5 in the indirect ELISA was observed using 1600-fold diluted sera after 10 min of color development. After confirming the
K. Kido et al. / Talanta 77 (2008) 346–350
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Table 2 Variations among the indirect competitive ELISA runs using anti-BA MAb 9D6 R.S.D.a (%)
BA concentration (ng/mL)
0 156 313 625 1250 2500
Intra-assay
Inter-assay
3.01 1.29 1.14 2.01 7.50 4.05
1.82 2.38 5.92 5.29 5.06 4.90
The measured values were mean ± S.D. for 3 plates and triplicate wells for each concentration within one plate from 3 consecutive days. The variation on replicates from well to well and plate to plate are defined as intra- and inter-assay variation, respectively. a Relative standard deviation.
Fig. 3. Standard curve of inhibition by BA using MAb 9D6 in indirect competitive ELISA. Various concentrations of BA were incubated with MAb 9D6 (1.7 ng/mL) in a 96-well immunoplate precoated with BA–HSA (1 g/mL).
pared to baicalein and wogonin, two hydroxyl groups at C5 and C7 positions are same, but two functions at C6 and C8 are completely different. Since the MAb recognizes BA and baicalein more than wogonin, it is supposed that a hydroxyl group at C6 position of BA, which is uncommon among flavonoids, evoked specificity of MAb 9D6 to BA and baicalein. 3.4. Variation and accuracy of the indirect competitive ELISA using anti-BA MAb 9D6 Intra- and inter-assays were performed to evaluate the indirect competitive ELISA using anti-BA MAb 9D6. In the standard curve for the ELISA obtained using various concentrations of BA from 0 to 2.5 g/mL, variations were calculated using relative standard deviations (R.S.D.s) of measurements obtained from triplicate wells
Table 1 Cross-reactivities of anti-BA MAb (9D6) against flavonoids contained in Sculellariae Radix and other natural products Compound
Cross-reactivity (%)
Flavonoids Flavone Baicalin Baicalein Wogonin
100 51.41 0.73
Flavonol Rutin
<0.21
Flavanone Hesperidin
<0.21
Isoflavone Daidzein
(intra-assay) and from plate-to-plate (inter-assay) on 3 consecutive days. Among the ELISA runs, the R.S.D.s were <8% in intra-assay, and <6% in inter-assay, which is one of the verifications that this assay is sufficiently reliable (Table 2). Recovery rates of each spiked BA in S. Radix were calculated by the spiked and recovered amounts of BA in the same concentration ranges (Table 3). Table 3 indicates good recovery rates (av = 98%) with <7% of R.S.D.s (n = 3) from 0.25 to 1.0 mg of spiked BA. Taken together, these results suggest that the ELISA is sufficiently accurate to be applicable for quantitative determination of BA in products including S. Radixes. 3.5. BA determination in S. Radixes and Kampo medicines by the indirect competitive ELISA After the validation and optimization of the assay, concentrations of BA in various S. Radixes and Kampo medicines were determined by the indirect competitive ELISA using the antiBA MAb, and were compared with those determined by HPLC method. As shown in Table 4, the ELISA showed good linearity (R2 = 0.943) with HPLC method in case of the determination of BA in all the dried S. Radixes. However, the data by the ELISA are overall higher than those by HPLC method. From this result, it is speculated that other BA structurally related compounds in S. Radix like baicalein and wogonin may react with MAb 9D6 and influence on those quantitative data by the ELISA. In addition, these concentrations of BA by HPLC method were relatively lower than those in the S. Radixes defined by Japanese Pharmacopoeia (≥10%, w/w). This might depend on the not complete extraction by MeOH from S. Radixes. We realize that sample preparation should be improved to extract BA from samples as complete as possible. In the analytical trial of several Kampo medicines, the concentrations of BA in each sample except Kakkonto which does not contain S. Radix were Table 3 Recovery of BA in spiked samples determined by the ELISA Spiked amount (mg)
<0.21
Flavan-3-ol (+)-Catechin
<0.21
Glycyrrhizic acid Geniposide Saikosaponin a Magnolol Sennoside B Ginsenoside Rb1 Berberine
<0.21 <0.21 <0.21 <0.21 <0.21 <0.21 <0.21
0 0.25 0.5 1
Measured amount (mg)a 1.09 1.30 1.62 2.13
± ± ± ±
0.11 0.07 0.03 0.15
R.S.D.b
Recovery (%)c
[10.0] [5.38] [1.85] [7.04]
84 106 104 av = 98.0
a
Baicalin in spiked samples was extracted by methanol with sample preparation. Data were mean ± S.D. from triplicate samples for each level. b Relative standard deviation. c The zero spiked level was used as control when the percentage of recovery was calculated as follows: recovery (%) = (measured amount − 1.09)/spiked amount × 100.
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Table 4 BA concentrations in various Scutellariae Radixes and Kampo medicines determined by the ELISA and HPLC method Sample
ELISA Concentration (mean ± S.D., w/w%) [R.S.D.]a
HPLC Concentration (mean ± S.D., w/w%) [R.S.D.]
S. Radix 1 S. Radix 2 S. Radix 3 S. Radix 4 Daisaikoto Shosaikoto Ogonto Saikokeishito Saikokaryukotsuboreito Saikokaryukotsuboreito 2 Kakkonto
10.0 ± 0.5 [5.0] 13.8 ± 0.7 [5.1] 10.1 ± 0.3 [3.0] 11.9 ± 1.0 [8.4] 2.63 ± 0.06 [2.3] 2.87 ± 0.11 [3.8] 2.62 ± 0.20 [7.6] 2.74 ± 0.29 [11] 2.75 ± 0.04 [1.5] 2.41 ± 0.15 [6.2] N.D.b
7.5 10.9 6.7 8.5
± ± ± ±
0.2 [2.7] 0.4 [3.7] 0.5 [7.5] 0.6 [7.1]
Data were mean ± S.D. from triplicate analyses for each sample. a Relative standard deviation. b N.D.: not detected.
determined correctly without complicated sample pretreatments (Table 4). 4. Conclusion This is the first time to prepare MAb against BA and apply an indirect competitive ELISA for determination of BA in S. Radixes and Kampo medicines. It was evaluated that the ELISA was simple, rapid, and as reliable as compared with other instrumental analyses. The ELISA can be used as the next-generation quality control method of crude drugs and products containing BA, particularly Kampo medicines. Our data confirmed that the newly developed ELISA had sufficient sensitivity and accuracy to detect the total amount of BA and baicalein in Kampo medicine samples, resulting circumvention of side effects. Acknowledgments This study was supported in part by a Grant-in-Aid from JSPS’s Asian CORE Program, the Ministry of Education, Culture, Sports, Science and Technology of Japan, the research fund of Kyushu University Foundation.
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Quick analysis of baicalin in Scutellariae Radix by enzyme-linked immunosorbent assay using a monoclonal antibody Katsumi Kido a , Osamu Morinaga b , Yukihiro Shoyama b , Hiroyuki Tanaka a,∗ a b
Department of Phamacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan Faculty of Pharmaceutical Sciences, Nagasaki International University, Huis Ten Bosch 2825-7, Sasebo, Nagasaki 859-3298, Japan
a r t i c l e
i n f o
Article history: Received 22 May 2008 Received in revised form 19 June 2008 Accepted 19 June 2008 Available online 1 July 2008 Keywords: Baicalin Monoclonal antibody ELISA Scutellariae Radix
a b s t r a c t To establish an immunoassay for baicalin (BA), a hybridoma cell line (9D6) secreting a monoclonal antibody (MAb) against BA was prepared by cell fusion with splenocytes derived from a mouse immunized with BA–bovine serum albumin (BSA) conjugate and a myeloma cell line, SP2/0-Ag14. MAb 9D6 shows specific reactivity against BA and its aglycone, baicalein, but not against other natural products. We developed an enzyme-linked immunosorbent assay (ELISA) using MAb 9D6 in a competitive manner, ranging from 200 ng/mL to 2 g/mL. After validating the developed ELISA on the basis of intra- and inter-assays and a recovery experiment, it was found that the ELISA was not only simple, but also sufficiently reliable and accurate for quality control of Scutellariae Radix. It allowed determination of BA in complex and mixed materials, such as Kampo medicines. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Baicalin (BA) is the most abundant compound among more than 30 kinds of flavonoids, such as baicalein, wogonin, and wogonin 7-O--d-glucuronide, in Scutellaria baicalensis Georgi (Fig. 1 Labiatae). BA has been reported to have anti-allergic [1], antiinflammatory [2], anti-HIV [3,4], anti-cancer [5,6], anti-oxidant, and free-radical scavenging effects [7,8]. Recently, we found that BA must be the most important component in the defense mechanism including oxidative burst against plant disease and insect attack [9,10]. Scutellariae Radix (S. Radix), which is the dried root of S. baicalensis, is one of the most important crude drugs used widely in Kampo medicines in Japan. Because BA shows these characteristics, the concentration of BA in S. Radix used clinically should be ≥10% (w/w), as defined by the 15th edition of Japanese Pharmacopoeia. Kampo medicines containing S. Radix, which are clinically important, occasionally cause side effects [11]. For example, interstitial pneumonia has been aggravated if Shosaikoto containing S. Radix and interferon (IFN)-␣ are used for treatment of chronic liver diseases. In this case, baicalein is believed to be a candidate in this side effect [12]. Therefore, concentrations of baicalein and BA which is hydrolyzed to give baicalein in body fluids should be monitored precisely in Kampo medicines.
∗ Corresponding author. Tel.: +81 92 642 6668; fax: +81 92 642 6668. E-mail address: [email protected] (H. Tanaka). 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.06.034
Various analytical approaches have been investigated for the determination of BA in S. Radix, Kampo medicines, and biogenic materials. Thin-layer chromatography and gas chromatography–mass spectrometry were applied to qualitative analysis of BA [13]. High-performance liquid chromatography (HPLC) with ultraviolet detection has been adopted as a quality control for S. Radix in Japanese Pharmacopoeia. HPLC with electrochemical detection showed a high sensitivity in the pharmacokinetic study of BA [14–16]. To establish the fingerprints of S. Radix, micellar electrokinetic capillary electrophoretic methods were also reported to separate and determine BA and flavonoids [17–19]. We have been focusing on an immunochemical approach for determination of phytochemicals [20–24] because it is reproducible, rapid, and highly sensitive for simultaneous analysis. However, since immunoassays for BA have not yet been reported, a monoclonal antibody (MAb) against BA was prepared successfully, and then applied to an enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of BA in this study. 2. Experimental 2.1. Regents and materials BA and baicalein were purchased from Wako Pure Chemicals (Osaka, Japan). Bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma (Steinheim, Germany). Freund’s complete and incomplete adjuvants were obtained from Difco (Detroit, MI, USA). Peroxidase-labeled anti-mouse IgG was
K. Kido et al. / Talanta 77 (2008) 346–350
Fig. 1. Structure of baicalin (BA) and its related compounds.
purchased from MP Biomedicals (Solon, OH, USA). All other chemicals were standard commercial products of analytical-reagent grade. Samples of various S. Radixes were purchased from Tochimototenkaido Corporation (Osaka, Japan), Takasago Yakugyo Corporation (Osaka, Japan), Daido Corporation (Osaka, Japan), and Yamada Corporation (Osaka, Japan). Kampo medicines were procured from Tsumura & Co. (Tokyo, Japan), Sanwa Shoyaku Co., Ltd. (Tochigi, Japan), and Kotaro Kampo Seiyaku Co., Ltd. (Osaka, Japan). 2.2. Synthesis of immunogen 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC; 5 mg) was added to a mixture of BA (5 mg) and BSA (5 mg) in 50 mM carbonate buffer (pH 9.6). The mixture was stirred at room temperature for 6 h. After the solution was dialyzed five times against H2 O, the dialysate was lyophilized to yield the BA–BSA conjugate (8.6 mg). BA–HSA conjugate was also synthesized in the same manner.
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for antibodies recognizing BA by indirect and indirect competitive ELISAs using BA–HSA as a solid-phase antigen. On the fourth day after the final immunization (100 g protein), splenocytes were isolated and fused with an aminopterin-sensitive mouse myeloma cell line, SP2/0-Ag14, by the polyethylene glycol (PEG) method [27]. SP2/0-Ag14 used for the cell fusion was obtained from RIKEN BioResource Center (Ibaragi, Japan). Hybridomas were obtained selectively by cultivation in a medium containing hypoxanthine–aminopterin–thymidine (HAT); those producing MAb reactive to BA were cloned by the limiting dilution method [28]. An established hybridoma was cultured in eRDF medium (Toyko, Japan) supplemented with 10 g/mL insulin, 35 g/mL transferrin, 20 M ethanolamine, and 25 nM selenium [29]. 2.5. Indirect ELISA The reactivity of MAb to BA–HSA was determined by an indirect ELISA. BA–HSA (5 molecules of BA per molecule of HSA) (100 L, 1 g/mL) dissolved in 50 mM carbonate buffer at pH 9.6 was adsorbed to the wells of a 96-well immunoplate. It was then treated with 300 L of 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% of Tween 20 (TPBS) and reacted with 100 L of MAb solution to be tested for 1 h. The plate was washed three times with PBS containing 0.05% Tween 20 (TPBS) and incubated with 100 L of a 1000-fold diluted peroxidase-labeled anti-mouse IgG solution for 1 h. After washing the plate three times with TPBS, 100 L of substrate solution [0.1 M citrate buffer (pH 4) containing 0.003% H2 O2 and 0.3 mg/mL of 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)] was added to each well and incubated for 20 min. Absorbance at 405 nm was measured using a microplate reader. All reactions were carried out at 37 ◦ C.
2.3. Determination of hapten number in BA–carrier protein conjugates by matrix-assisted laser desorption/ionization (MALDI)-TOF mass spectrometry
2.6. Indirect competitive ELISA
The hapten number of the BA–BSA and BA–HSA conjugates was determined by MALDI-TOF-MS as previously described [25,26]. A small amount (1–10 pmol) of the antigen conjugate was mixed with a 1000-fold molar excess of sinapinic acid in an aqueous solution containing 0.15% trifluoroacetic acid. The mixture was analyzed with a JEOL Mass Spectrometer (JMS) time-of-flight (TOF) mass monitor and irradiated with an 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 long evacuated tube. The data were analyzed using the GRAMS/386 software (Galactic Industries, Salem, NH, USA).
BA–HSA (100 L, 1 g/mL) dissolved in 50 mM carbonate buffer (pH 9.6) was adsorbed to the wells of a 96-well immunoplate. It was then treated with 300 L SPBS for 1 h to reduce non-specific adsorption. Fifty microliters of various concentrations of BA or samples dissolved in 10% methanol were incubated with 50 L of MAb solution (1.7 ng/mL) for 1 h. The plate was washed three times with TPBS, and then incubated with 100 L of a 1000-fold diluted peroxidase-labeled anti-mouse IgG solution for 1 h. After washing the plate three times with TPBS, 100 L of ABTS solution was added to each well and incubated for 20 min. The absorbance at 405 nm was measured using a microplate reader. All reactions were carried out at 37 ◦ C.
2.4. Preparation of anti-BA MAb Male BALB/c mice (5 weeks old) were purchased from KBT Oriental Co. (Saga, Japan). Food (MF; Oriental Yeast Co., Tokyo, Japan) and water were provided ad libitum. Procedures and animal care were approved by the Committee on Ethics of Animal Experiments, Graduate School of Pharmaceutical Sciences, Kyushu University, Japan, and were conducted according to the Guidelines for Animal Experiments of the Graduate School of Pharmaceutical Sciences, Kyushu University. The mice were injected with BA–BSA four times. Immunization was initiated by intraperitoneal injection of the conjugate (50 g) in phosphate-buffered saline (PBS) emulsified with an equal volume of Freund’s complete adjuvant. As a second immunization, 50 g of the conjugate in Freund’s incomplete adjuvant was injected intraperitoneally 2 weeks after the initial injection. Mice were bled 4 days after the boosts, and the sera were monitored
2.7. Specificity and sensitivity of the assay The cross-reactivities (CRs, %) of BA and related compounds were determined according to Weiler’s equation [30]: CR (%) =
Concentration of BA at A/A0 = 50% Concentration of cross-reacting substance at A/A0 = 50% × 100
A and A0 are the absorbance in the presence and absence of the test compound, respectively. 2.8. HPLC instruments and conditions The chromatography system used in this study consisted of a LC10AD (Shimadzu, Kyoto, Japan), a UV-8 model II detector (Tosoh,
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Tokyo, Japan), and a Chromatopac C-R5A data analyzer system (Shimadzu). The analytical column was Cosmosil 5C18 -AR column (4.6 mm i.d. × 150 mm, Nacalai Tesque, Kyoto, Japan) maintained at room temperature. The mobile phase comprised CH3 CN–H2 O (3:7) containing 60 mM H3 PO4 and a flow rate of 1.0 mL/min. A 10-L aliquot of each sample was injected and monitored at 254 nm. 2.9. Sample preparation Dried samples (10 mg) of various S. Radixes and Kampo medicines were powdered, and extracted five times with MeOH (1 mL) under sonication. The extracted solution was filtered, and the combined extracted solution was adjusted to a volume of 10 mL with MeOH. The adjusted solution was diluted with H2 O to prepare a sample solution suitable for the indirect competitive ELISA. 2.10. Recovery experiment Various amounts of BA were added to a dried powder of S. Radix (10 mg). The amount of BA in the unspiked sample was determined to be 1.09 mg. The spiked sample was extracted with MeOH by sonication and filtered. The combined extract was adjusted to 10 mL with MeOH, diluted to prepare a 10% MeOH solution, and assayed by the indirect competitive ELISA. The recovery rate of BA was calculated from the spiked and recovered amounts in the same concentration ranges. Recovery (%) =
Measured amount − 1.09 × 100 Spiked amount
Fig. 2. Direct determination of BA–BSA conjugates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). [M+H]+ and [M+2H]2+ are single- and double-protonated molecules of BA–BSA, respectively.
production of antibodies by the immunized mice, their spleen was extirpated to prepare splenocytes including antibody-secreting cells for cell fusion. Cell fusion was performed with the splenocytes and SP2/0 myeloma cells following the procedure established in this laboratory [31]. A hybridoma producing MAb reactive to BA was obtained through a series of screenings with the indirect and indirect competitive ELISAs and cloning by the limiting dilution method. The MAb, designated 9D6, was classified as IgG2a having light chains.
3. Results and discussion
3.3. Assay sensitivity and assay specificity
3.1. Synthesis of the BA–BSA conjugate and direct determination of the hapten–carrier protein conjugates by MALDI-TOF mass spectrometry
An optimal concentration of anti-BA MAb applied to the indirect competitive ELISA was measured by the indirect ELISA with various concentrations of MAb 9D6. This optimal concentration was 1.7 ng/mL, which was selected for the indirect competitive ELISA to define sensitivity and specificity of the MAb because it was the minimum concentration of the MAb providing a sufficient signal to detect the interaction between the MAb and BA–HSA as a solidphase antigen. The indirect competitive ELISA was developed using this concentration of MAb and polystyrene microtiter plates precoated with 1 g/mL of BA–HSA. In addition, a 10% MeOH was used as a diluted solution for standard BA and samples, because addition of MeOH improved solubility of BA in the solutions at circumneutral condition. Fig. 3 shows inhibition of the interaction between the MAb and BSA-HSA using various concentrations of BA, providing a standard curve of BA in the indirect competitive ELISA. Under these conditions, a calibration curve for BA was generated in the linear range from 200 ng/mL to 2.0 g/mL (R2 = 0.994), which revealed that the sensitivity of the assay with a detection limit of 100 ng/mL was improved at least 5-fold compared with the HPLC method [32]. Specificity of MAb 9D6 was analyzed by comparing its crossreactivities to compounds structurally related to BA and other natural products. Table 1 lists the CRs of the MAb examined by the indirect competitive ELISA, and calculated using the concentration of BA yielding midrange values on the basis of the method reported by Weiler et al. [30]. MAb 9D6 recognized not only BA, but also baicalein (CR: 51.4%) indicating a slight reactivity to wogonin (0.73%), although no detection against other flavonoids such as rutin, hesperidin, and daidzein (Table 1). It is easily suggested that all of function in ring A of BA might be immunized because MAb 9D6 strongly reacts BA structurally related compounds. Com-
Since a low molecular weight compound (hapten) like BA is poorly immunogenic, a hapten–protein conjugate should be synthesized and used as an immunogen for developing antibodies against such compounds. BA possessing a carboxylic acid group in a molecule, can be conjugated with a protein by carbodiimide system. Therefore, BSA was selected as a carrier protein and readily coupled with BA using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) as a coupling reagent in this study. Fig. 2 shows the MALDI-TOF mass spectrum of the BA–BSA conjugate to validate its hapten number. A broad peak coinciding with the conjugate of BA and BSA appeared from m/z 66,900 to 74,900, centering at ∼m/z 70,200. Using the experimental result and a molecular weight of 66,433 for BSA, the calculated value of the BA component in the conjugate was 3767, suggesting 9 molecules of BA (on average) conjugated with a BSA molecule. This result revealed that adequate numbers of BA molecules were coupled with BSA, which meant that the BA–BSA conjugate could be used as an immunogen to evoke anti-BA antibodies in mice. BA–HSA was prepared as a solid-phase antigen for ELISA, and the hapten number of BA–HSA conjugate was also determined reliably from its spectrum to be at ∼5 molecules. 3.2. Production and characteristics of MAb against BA BALB/c mice were immunized with BA–BSA until the absorbance of more than 0.5 in the indirect ELISA was observed using 1600-fold diluted sera after 10 min of color development. After confirming the
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Table 2 Variations among the indirect competitive ELISA runs using anti-BA MAb 9D6 R.S.D.a (%)
BA concentration (ng/mL)
0 156 313 625 1250 2500
Intra-assay
Inter-assay
3.01 1.29 1.14 2.01 7.50 4.05
1.82 2.38 5.92 5.29 5.06 4.90
The measured values were mean ± S.D. for 3 plates and triplicate wells for each concentration within one plate from 3 consecutive days. The variation on replicates from well to well and plate to plate are defined as intra- and inter-assay variation, respectively. a Relative standard deviation.
Fig. 3. Standard curve of inhibition by BA using MAb 9D6 in indirect competitive ELISA. Various concentrations of BA were incubated with MAb 9D6 (1.7 ng/mL) in a 96-well immunoplate precoated with BA–HSA (1 g/mL).
pared to baicalein and wogonin, two hydroxyl groups at C5 and C7 positions are same, but two functions at C6 and C8 are completely different. Since the MAb recognizes BA and baicalein more than wogonin, it is supposed that a hydroxyl group at C6 position of BA, which is uncommon among flavonoids, evoked specificity of MAb 9D6 to BA and baicalein. 3.4. Variation and accuracy of the indirect competitive ELISA using anti-BA MAb 9D6 Intra- and inter-assays were performed to evaluate the indirect competitive ELISA using anti-BA MAb 9D6. In the standard curve for the ELISA obtained using various concentrations of BA from 0 to 2.5 g/mL, variations were calculated using relative standard deviations (R.S.D.s) of measurements obtained from triplicate wells
Table 1 Cross-reactivities of anti-BA MAb (9D6) against flavonoids contained in Sculellariae Radix and other natural products Compound
Cross-reactivity (%)
Flavonoids Flavone Baicalin Baicalein Wogonin
100 51.41 0.73
Flavonol Rutin
<0.21
Flavanone Hesperidin
<0.21
Isoflavone Daidzein
(intra-assay) and from plate-to-plate (inter-assay) on 3 consecutive days. Among the ELISA runs, the R.S.D.s were <8% in intra-assay, and <6% in inter-assay, which is one of the verifications that this assay is sufficiently reliable (Table 2). Recovery rates of each spiked BA in S. Radix were calculated by the spiked and recovered amounts of BA in the same concentration ranges (Table 3). Table 3 indicates good recovery rates (av = 98%) with <7% of R.S.D.s (n = 3) from 0.25 to 1.0 mg of spiked BA. Taken together, these results suggest that the ELISA is sufficiently accurate to be applicable for quantitative determination of BA in products including S. Radixes. 3.5. BA determination in S. Radixes and Kampo medicines by the indirect competitive ELISA After the validation and optimization of the assay, concentrations of BA in various S. Radixes and Kampo medicines were determined by the indirect competitive ELISA using the antiBA MAb, and were compared with those determined by HPLC method. As shown in Table 4, the ELISA showed good linearity (R2 = 0.943) with HPLC method in case of the determination of BA in all the dried S. Radixes. However, the data by the ELISA are overall higher than those by HPLC method. From this result, it is speculated that other BA structurally related compounds in S. Radix like baicalein and wogonin may react with MAb 9D6 and influence on those quantitative data by the ELISA. In addition, these concentrations of BA by HPLC method were relatively lower than those in the S. Radixes defined by Japanese Pharmacopoeia (≥10%, w/w). This might depend on the not complete extraction by MeOH from S. Radixes. We realize that sample preparation should be improved to extract BA from samples as complete as possible. In the analytical trial of several Kampo medicines, the concentrations of BA in each sample except Kakkonto which does not contain S. Radix were Table 3 Recovery of BA in spiked samples determined by the ELISA Spiked amount (mg)
<0.21
Flavan-3-ol (+)-Catechin
<0.21
Glycyrrhizic acid Geniposide Saikosaponin a Magnolol Sennoside B Ginsenoside Rb1 Berberine
<0.21 <0.21 <0.21 <0.21 <0.21 <0.21 <0.21
0 0.25 0.5 1
Measured amount (mg)a 1.09 1.30 1.62 2.13
± ± ± ±
0.11 0.07 0.03 0.15
R.S.D.b
Recovery (%)c
[10.0] [5.38] [1.85] [7.04]
84 106 104 av = 98.0
a
Baicalin in spiked samples was extracted by methanol with sample preparation. Data were mean ± S.D. from triplicate samples for each level. b Relative standard deviation. c The zero spiked level was used as control when the percentage of recovery was calculated as follows: recovery (%) = (measured amount − 1.09)/spiked amount × 100.
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Table 4 BA concentrations in various Scutellariae Radixes and Kampo medicines determined by the ELISA and HPLC method Sample
ELISA Concentration (mean ± S.D., w/w%) [R.S.D.]a
HPLC Concentration (mean ± S.D., w/w%) [R.S.D.]
S. Radix 1 S. Radix 2 S. Radix 3 S. Radix 4 Daisaikoto Shosaikoto Ogonto Saikokeishito Saikokaryukotsuboreito Saikokaryukotsuboreito 2 Kakkonto
10.0 ± 0.5 [5.0] 13.8 ± 0.7 [5.1] 10.1 ± 0.3 [3.0] 11.9 ± 1.0 [8.4] 2.63 ± 0.06 [2.3] 2.87 ± 0.11 [3.8] 2.62 ± 0.20 [7.6] 2.74 ± 0.29 [11] 2.75 ± 0.04 [1.5] 2.41 ± 0.15 [6.2] N.D.b
7.5 10.9 6.7 8.5
± ± ± ±
0.2 [2.7] 0.4 [3.7] 0.5 [7.5] 0.6 [7.1]
Data were mean ± S.D. from triplicate analyses for each sample. a Relative standard deviation. b N.D.: not detected.
determined correctly without complicated sample pretreatments (Table 4). 4. Conclusion This is the first time to prepare MAb against BA and apply an indirect competitive ELISA for determination of BA in S. Radixes and Kampo medicines. It was evaluated that the ELISA was simple, rapid, and as reliable as compared with other instrumental analyses. The ELISA can be used as the next-generation quality control method of crude drugs and products containing BA, particularly Kampo medicines. Our data confirmed that the newly developed ELISA had sufficient sensitivity and accuracy to detect the total amount of BA and baicalein in Kampo medicine samples, resulting circumvention of side effects. Acknowledgments This study was supported in part by a Grant-in-Aid from JSPS’s Asian CORE Program, the Ministry of Education, Culture, Sports, Science and Technology of Japan, the research fund of Kyushu University Foundation.
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