Original Papers
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A Sensitive and Specific Indirect Competitive Enzyme-Linked Immunosorbent Assay for Detection of Paeoniflorin and Its Application in Pharmacokinetic Interactions between Paeoniflorin and Glycyrrhizinic Acid Authors
Yan Zhao 1, Huihua Qu 2, Xueqian Wang 1, Yue Zhang 1, Wenchao Shan 3, Yan Zhao 1*, Qingguo Wang 1
Affiliations
1
3
Key words " paeoniflorin l " monoclonal antibody l " enzyme‑linked immunol sorbent assay (ELISA) " pharmacokinetics l " glycyrrhizic acid l
received revised accepted
October 29, 2014 January 6, 2015 March 8, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1545913 Published online April 9, 2015 Planta Med 2015; 81: 765–770 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence *Yan Zhao School of Basic Medical Sciences Beijing University of Chinese Medicine 11 Beisanhuandong Road, Chaoyang District Beijing 100029 China Phone: + 64 28 67 05 Fax: + 64 28 67 05 [email protected] Correspondence Qingguo Wang School of Basic Medical Sciences Beijing University of Chinese Medicine 11 Beisanhuandong Road, Chaoyang District Beijing 100029 China Phone: + 64 28 68 21 Fax: + 64 28 68 21 [email protected]
School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing, China Scientific Research Experiment Center, Beijing University of Chinese Medicine, Beijing, China College of Traditional Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing, China
Abstract
Abbreviations
!
!
This study aimed to develop an indirect competitive enzyme-linked immunosorbent assay based on monoclonal antibodies against paeoniflorin to study the effects of different doses of glycyrrhizinic acid on the pharmacokinetics of paeoniflorin in mice. An anti-paeoniflorin monoclonal antibody was produced from a hybridoma created through a fusion of splenocytes immunized with paeoniflorin-bovine serum albumin and conjugated with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma cell line SP2/0. The resultant antibody was used to develop and validate a rapid, specific and sensitive, indirect competitive enzyme-linked immunosorbent assay for the measurement of paeoniflorin (linear range 4.8–312.5 ng · mL−1). The intraday and interday precision values of the indirect competitive ELISA method were well within the recommended range (≤ 10 %), and the recovery rate was, on average, 101.13 %. Pharmacokinetic parameters obtained from mouse blood samples at various intervals following the oral administration of paeoniflorin and glycyrrhizic acid at three doses (1 : 0.3, 1 : 1, 1 : 3, respectively) demonstrated that the highest dose of glycyrrhizic acid inhibits the absorption of paeoniflorin.
AUC0-t: BSA: Cmax: CR: GA: GR: HAT: icELISA: MAb: MRT: OVA: PBST: PEG: PES: PF: PF‑BSA: PF‑OVA: PR: RSD: SGD: SPBS: Tmax: TCM: TMB:
Introduction ! " Fig. 1) is the main active compoPaeoniflorin (l nent in PR, a monoterpene glycoside with anti-inflammatory, antirheumatic, immunomodulatory [1], analgesic [2], and neuroprotective properties [3]. Several methods are used to determine the
area under the curve bovine serum albumin mean maximum concentration in plasma cross-reactivity glycyrrhizic acid Glycyrrhizae Radix et Rhizoma hypoxanthine-aminopterinthymidine indirect competitive enzyme-linked immunosorbent assay monoclonal antibody mean residence time ovalbumin phosphate-buffered saline containing 0.05 % Tween 20 polyethylene glycol polyethersulfone paeoniflorin paeoniflorin conjugated with bovine serum albumin paeoniflorin conjugated with ovalbumin Paeoniae Radix Alba relative standard deviation Shaoyao-Gancao decoction phosphate-buffered saline containing 5 % skim milk time of maximum concentration traditional Chinese medicine 3,3′,5,5′-tetramethylbenzidine
concentration of paeoniflorin in medicines and biological samples [4–6], with HPLC/MS/MS being the most commonly used method and displaying high sensitivity in pharmacokinetic studies [7–9]. The enzyme-linked immunosorbent assay (ELISA) using MAbs is another important method for the qualitative or quantitative detection of these
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Fig. 1 Structure of paeoniflorin.
products, with benefits including small scale, high reproducibility under various circumstances and minimal pretreatment [10, 11]. The ELISA method is very suitable for biological samples due to the need for only a very small amount of sample, which allows for simultaneous use across a large number of samples [11]. While Zhaohua Lu [12] and colleagues have developed an ELISA for the detection of paeoniflorin and albiflorin, it remains difficult to evaluate the contents of PF and profile the pharmacokinetics of paeoniflorin in animals or humans. Therefore, it is necessary to establish an ELISA with enough specificity and sensitivity to accurately monitor small amounts of paeoniflorin in various samples. The increasing use of botanical herb extracts and their prescription as therapeutic agents has led to an increase in investigations of more and more bioactive compound interactions between different herbs [13–15], most of which are associated with pharmacokinetics [16]. Under TCM guidance, the combination of PR and GR is typically used to treat spasmodic abdominal pain accompanying acute gastroenteritis [17], muscle cramps [18], asthma [19], and dysmenorrhea [20]. This combination has been used for many years with favorable results, though the dosage of the herb can affect such beneficial effects. Several studies have shown that the greatest efficacy against spasmolysis is obtained with a dosage proportion of 1 : 1 PR to GR, and that this effect improved as the dosage increased [21, 22]. On the other hand, a better curative effect against intractable hiccups was found when the dosage proportion of PR and GR was 3 : 1 [23]. In addition, PR and GR act as analgesics when PR is more abundant than GR (3 : 2 or 2 : 1) [24]. It is apparent from these studies that the various biological effects of the herbs depend heavily on the different compounds and their dosage proportions, and that these effects can be studied by pharmacokinetics. The pharmacokinetics and assessment of PF and GA have been widely reported [25–27], but the pharmacokinetics of PF in the presence of GA at different doses has not yet been investigated. Therefore, studying the interaction of these efficacious components through pharmacokinetics could provide a theoretical basis and clinical guidance for the rational and effective use of traditional Chinese medicines. In the present study, we first generated a new anti-PF monoclonal antibody and developed a sensitive and specific ELISA to efficiently measure the concentration of PF. Furthermore, this method was applied to explore the pharmacokinetics of PF in the presence of different doses of GA.
Results and Discussion !
A competitive binding assay in which the anti-PF MAb binds either free PF or a PF‑OVA conjugate adsorbed onto a polystyrene microtiter plate was established. Under these conditions, the linear range of the assay extended from 4.8 to 312.5 ng · mL−1, as in-
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Fig. 2 Standard curve of inhibition by PF using MAbs in an icELISA. Various concentrations of PF were incubated with anti-PF MAb in a 96-well ELISA plate precoated with PF‑OVA (1 µg · mL−1). Error bars: ± 1 SD; the data represent the mean of five replicates.
" Fig. 2. The sensitivity of the antibody was satisfacdicated in l tory, with an IC50 value of 42.72 ng · mL−1 and a limit of detection (LOD) of 4.75 ng · mL−1 in buffer. Specificity is the most important factor in determining the value of an antibody and the accuracy of an immunoassay, which was evaluated by cross reactivity. Because each compound has multiple antigenic determinants, it has the ability to stimulate the immune system to produce specific antibodies when conjugated with a carrier protein. In 2003, Zhaohua Lu et al. screened four hybridomas secreting monoclonal antibodies against PF; these antibodies all had a high CR with albiflorin (143.7 %, 89.56 %, 68.96 %, and 46.27 %, respectively) [12], indicating that they are four distinct monoclonal antibodies against different epitopes of PF. In this study, the anti-PF MAb had little CR with albiflorin (< 0.27%) and no CR with various other compounds (< 0.01 %), as " Table 1, suggesting that our new monoclonal antishown in l body against PF differs from the previously reported antibodies. In addition, the minor CR seen with albiflorin confirms the accuracy of the PF assay without the interference of albiflorin, which is advantageous for the establishment of a quality control method and pharmacokinetic study using this anti-PF MAb. The concentrations of PF in four traditional Chinese medicine decoctions (Sini San, Shaoyao Gancao Tang, Guizhi Tang, and Zhen" Table 2) were measured and compared to wu Tang, as shown in l the results of those acquired by performing high-performance liquid chromatography (HPLC), allowing us to verify the reliability of our method. The correlation was then calculated from a plot developed by regression analysis based on the values obtained " Fig. 3. The concentrations from ELISA and HPLC, as indicated in l of PF in the four traditional Chinese medicine decoctions, as measured by our developed ELISA, were highly consistent with those obtained by HPLC from the same samples (R2 = 0.9918). From these results, it is apparent that this ELISA method can be applied to determine PF in medicine, which will be very useful and more convenient in product quality control and quantification in the future. The accuracy and variation of the assay were evaluated by the RSDs of intra- (well to well) and inter- (plate to plate and day to day) assay data of the icELISA using our anti-PF MAb. A standard
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Table 2 The four Chinese medicines.
Classification
Compound
CRs (%)
Traditional
Terpene
paeoniflorin albiflorin ginsenoside Rb1 ginsenoside Re ginsenoside Rg1 notoginsenoside glycyrrhizic acid glycyrrhetic acid saikosaponin a saikosaponin d gentiopicrin geniposide deoxycholic acid cholic acid puerarin baicalin baicalein hyperoside rutin hesperidin naringin chlorogenic acid ferulic acid rheum emodin rheinic acid berberine salvianolic acid curcumin gastrodin amygdalin
100 < 0.27 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01
Chinese
Steroid Flavonoid
Phenylpropanoids Anthraquinones Alkaloid Other
curve for the ELISA was established with various concentrations of PF in plasma, which allowed for the calculation of RSDs in the sextuplicate wells (intra-assay) and multiple plates (inter-assay) on " Table 3 shows that the intra-assay RSDs six consecutive days. l were less than 3.1 % and the inter-assay RSDs were lower than 6.3 %, which indicates that this assay is very accurate and stable. A recovery experiment was performed to evaluate the reliability of the assay. Briefly, mouse plasma was spiked with purified PF at doses of 50, 100, and 200 ng. Recovery was calculated using the equation: recovery (%) = measured amount/added amount " Table 4, the average recovery rate was × 100%. As shown in l 101.13 % from 50 to 200 ng of spiked PF, suggesting that this assay is sufficiently reliable for the determination of PF in plasma. There are many documented instances of positive interactions between drugs [28], indicating the potential significance of using herbal combinations that are based on TCM theory. PR and GR formed SGD, which was reported originally in the Treatise on Febrile and Miscellaneous Diseases both written by Zhang Zhongjing in the last years of the Han dynasty in China (approximately 206 B. C. to 200 A. D.). In the clinical practice of TCM science, SGD has been used to treat systremma, gastrospasm, intercostal neuralgia, stomachache, abdominal pain, and dysmenorrhea over a very long period of time and has produced quite a favorable effect. A pharmacokinetic study on SGD has shown that the absorption degrees of PF and GA have enhanced after compatibility, which gave evidence that the compatibility of PR and GR was reasonable [29]. As the major effective constituents of PR and GR, PF and GA are used as the phytochemical markers for their quality control, respectively, in the Chinese Pharmacopoeia [30]. The pharmaco-
Composition of Kampo medicine
Percentage of Paeoniae
medicines
Radix Alba
Si-ni-San
Shao-yao-gancao-tang Gui-zhi-tang
Zhen-wu-tang
Paeoniae Radix Alba, Auranth Fructus Immaturus, Bupleuri Radix, Glycyrrhizae Radix Et Rhizoma Paeoniae Radix Alba, Glycyrrhizae Radix Et Rhizoma Cinnamomi Ramulus, Paeoniae Radix Alba, Glycyrrhizae Radix Et Rhizoma, Jujubae Fructus, Zingiberis Rhizoma Recens Poria, Paeoniae Radix Alba, Atractylodis Macrocephalae Rhizoma, Aconiti Lateralis Radix Praeparata, Zingiberis Rhizoma Recens
1/3
1/2 3/13
3/14
Fig. 3 Correlation between the content of PF determined by ELISA and HPLC.
Table 3 Intra- and interday PF precision in mice plasma. PF (ng · mL−1)
RSD (%) Intra-assay
Inter-assay
2.5 5 10 20 40
1.8 2.3 2.6 3.1 2.8
2.8 4.1 3.3 5.5 6.3
Table 4 Recovery of PF from mice plasma. Added amount (ng)
Measured amount (ng)
Recovery (%)
50 100 200
51.24 ± 0.36 101.27 ± 2.15 199.25 ± 3.18
102.48 101.27 99.63 Average 101.13
kinetic determinations of PF [31, 32] and GA [33, 34] in PR and GR based on blood samples have been widely reported, and there are interactions between PR and GR [25, 35, 36]. However, there are no reports regarding the effect of different doses and dose ratios on the pharmacokinetics of PF and GA in mice, so far. In this study, we measured and plotted the mean concentration of PF in
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Table 1 CRs (%) of anti-PF MAb against various compounds using icELISA.
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high-throughput detection in four hours. These show that the developed icELISA method is simpler, more efficient, and more sensitive than HPLC. However, it cannot be denied that the developed icELISA cannot monitor the PF metabolizing process in vivo, and the cross-reaction of the monoclonal antibody to the structure-related compound is hard to avoid, while there is no such limit to HPLC.
Materials and Methods !
Materials and reagents
Fig. 4 Mean concentration-time profiles of PF in mouse blood after oral administration for group 1 (GA: PF = 0.3 : 1), group 2 (GA: PF = 1 : 1), and group 3 (GA: PF = 3 : 1) (n = 6). (Color figure available online).
mouse plasma as a function of time in three groups, as shown in " Fig. 4. The pharmacokinetic parameters of mouse plasma saml ples following oral administration of different rations of PF and " Table 5. GA are shown in l An increased glycyrrhizic acid content significantly (p < 0.05) reduced the absorption of paeoniflorin when comparing groups 1 and 3, which was reflected by the AUC0–6 (decreased from 849.08 µg · min · mL−1 to 561.11 µg · min · mL−1) and Cmax (decreased from 10.62 µg · mL−1 to 5.28 µg · mL−1) values, whereas the MRT increased 1.5-fold when the dosage of GA was increased 10fold. The Cmax and AUC0–6 values exhibited a linear relationship (R2 = 0.9808), demonstrating that the AUC of PF is dose-related. With the content of GA increasing, the Cmax and AUC0–6 values of PF decreased gradually, while the MRT was extended, which implies that the absorption of PF was inhibited when the content of GA increased to a certain amount, but the residence time of PF was prolonged, which is beneficial for PF to work in vivo. A further study will focus on the pharmacodynamics of the different combinations of PF and GA to explore the mechanism of the result of this study. The results of the present study demonstrate that our newly developed ELISA based on a new MAb against PF provides a reliable and sensitive approach for the determination of PF content in medicines and plasma. Due to its sensitivity, the detection of PF in plasma needs small amounts of blood, which allows for the blood to continuously get to several time points from the same mouse without killing it. Besides, using the icELISA method, we need minimal pretreatment steps before detection and achieve
Paeoniflorin (purity 98%) and various other compounds were purchased from Dingguo changsheng Biotechnology. BSA, OVA, and Freundʼs complete and incomplete reagents were obtained from Sigma-Aldrich. All other chemicals and reagents were of analytical grade and were purchased from Sinopharm Chemical Reagents.
Synthesis of antigen conjugates PF-carrier protein conjugates, namely PF‑BSA and PF‑OVA, were synthesized by a periodate oxidation procedure according to a previous methodology with some modifications [12], and detected by ultraviolet visible spectrophotometry (UV) [37]. The result was shown in a previous study [38].
Preparation of the paeoniflorin antibody Immunization: Female BALB/C mice (7 weeks of age) were purchased from Vital River Laboratories. The mice were fed a standard rodent diet 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 the Beijing University of Chinese Medicine, China (2013BZHYLL00 106). The mice were immunized as described by previous reports [39]. Briefly, the mice were injected subcutaneously with 100 µg of PF‑BSA conjugates in 0.15 mL phosphate buffer emulsified with the same volume of Freundʼs complete adjuvant. Injections were repeated at 2-week intervals (weeks 2, 4, and 6) with the same volume of Freundʼs incomplete adjuvant. The final immunization lacked adjuvant and was administered as a booster for three days prior to cell fusion. Mouse blood was obtained from the tail vein one day after the booster, and sera were isolated for the titer test by direct ELISA.
Cell fusion and preparation of anti-paeoniflorin monoclonal antibody On the third day after the final immunization, splenocytes were isolated and fused with the SP2/0 HAT-sensitive mouse myeloma cell line using the PEG method [40, 41]. Briefly, 1 mL of PEG was
Table 5 Pharmacokinetic parameters of PF in three mouse treatment groups (mean ± SD, n = 6). Parameter
Group 1
Group 2
Group 3
Cmax (µg · mL−1) Tmax (min) AUC0–6 h (µg · min · mL−1) MRT (min) T1/2 (min) Vd (L/Kg) Clearance (mL/min · Kg)
10.62 ± 4.78 25.83 ± 12.81 849.08 ± 249.13 193.51 ± 48.16 134.10 ± 33.37 12.41 ± 3.43# 72.87 ± 13.22
7.16 ± 2.30 30.83 ± 10.21 625.78 ± 149.55 226.31 ± 142.18 156.83 ± 98.53 18.30 ± 3.97# 112.65 ± 43.74
5.28 ± 1.92* 23.33 ± 4.08 561.11 ± 107.94* 301.24 ± 73.00* 208.76 ± 50.59* 33.39 ± 7.04 116.10 ± 8.44
* p < 0.05 (compared with group 1); # p < 0.05 (compared with group 3)
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Purification of anti-paeoniflorin monoclonal antibody The ascites obtained from the abdominal cavity of Balb/c mice implanted with the established hybridoma were purified by the caprylic acid-ammonium sulfate method [44].
Analytical procedure Direct ELISA: The reactivity of anti-PF MAb to PF‑OVA was determined by direct ELISA. PF‑OVA (100 µl/well) was diluted in 50 mM carbonate buffer (pH 9.6), added into the wells of a 96well immunoplate in different columns and incubated for 1 h. This was then treated with 200 µl of blocking buffer (SPBS) for 1 h to eliminate nonspecific adsorption. The plate was washed three times with washing buffer (PBST) and filled with 100 µL of MAb solution at various dilutions in different rows. After 1 h, the plate was washed three times with PBST and incubated with 100 µl of a 10 000-fold diluted peroxidase-labeled goat antimouse IgG solution for 1 h, followed by another wash with PBST for three times. Then, 100 µl of the substrate solution (0.1 mM citrate buffer (pH 4) containing 0.75 % H2O2 and 2 mg/mL TMB) was added to each well, and the plate was incubated for 15 min. The reaction was then terminated by adding 50 µl of 2 M sulfuric acid to each well. All reactions were carried out at 37 °C. The absorbance was determined at 450 nm using a Biotek ELx 800 microplate reader.
Indirect competitive enzyme-linked immunosorbent assay The reactivity of anti-PF MAb to PF was determined by icELISA. PF‑OVA (1 : 10 000, 100 µL/well) was adsorbed in the wells of a 96-well immunoplate, which was then treated with 200 µl of 5 % SPBS for 1 h to reduce nonspecific adsorption. The plate was washed three times with PBST, and 50 µL of various concentrations of PF solution were incubated with 50 µL of MAb solution for 1 h. The subsequent process was the same as that described for direct ELISA.
tions of PF in the four TCM decoctions were compared using HPLC and icELISA methods in parallel. Chromatographic conditions: An Agilent series 1260 HPLC instrument equipped with a quaternary pump, a diode-array detector, an autosampler, and a column compartment was used for analyses. The sample was separated on a Zorbax SB‑C18 column (5 µm, ϕ 4.6 × 150 mm, Agilent), with the mobile phase consisting of CH3CN (A) and water containing 0.1 % acetic acid (B). A gradient program was used as follows: 10% A in the first 5 min, linearly increased to 25% A at 15 min, then linearly decreased to 10 % A at 16 min using a mobile phase flow rate of 1.0 mL/min. The chromatogram was recorded at 230 nm and the spectral data for all peaks were obtained in the range of 190–400 nm. The column temperature was kept constant at 30 °C.
Determination of paeoniflorin in plasma Animals and drug administration: Kunming mice from Vital River Laboratory Animal Technology were maintained on a 12-h lightdark cycle (light from 7 : 00 a. m. to 7 : 00 p. m.) in a temperatureand humidity-controlled room (22 ± 2 °C and 50 ± 10%, respectively). Mice were treated after a 1-week acclimation period and were fasted for 12 h before the experiment with free access to water. All procedures were approved by the Committee on Ethics of Animal Experiments, Beijing University of Chinese Medicine, China. The mice were divided into three groups: group 1 [GA: PF, 0.3 : 1 (w/w)], group 2 [GA: PF, 1 : 1 (w/w)], and group 3 [GA: PF, 3 : 1 (w/w)]. The amount of the orally administered drugs was equivalent to 80 mg/kg body weight of PF. Blood collection and preparation: At the selected time points (5, 10, 15, 25, 35, 45, 60, 90, 120, 150, 180, 210, 240, and 360 min), 10 µl of tail vein blood was extracted and placed into 0.5 ml EP tubes treated with heparin sodium (1 mg · mL−1). Blood was centrifuged at 1000 g for 10 min at 4 °C, and the supernatant was taken and diluted 20 times for analyses. Determination of PF in plasma and data analyses: After sample processing, the concentrations of PF in the plasma samples were measured by the established enzyme immunoassay. The icELISA procedures were performed as described above, with the exception that the mouse plasma was tested. The standards and the blank control were prepared in the control mouse plasma. Cmax, Tmax, terminal elimination half-life, AUC0-t, and MRT were calculated using a non-compartment model in Kinetica version 4.4 (Thermo Fisher Scientific). Variance analysis (ANOVA) was used for the statistical analyses.
Specificity of anti-paeoniflorin monoclonal antibody The specificity of the antibody was determined by assessing CR with PF analogs that are known to compete with the binding of the antibody for the coating antigen in icELISA. CR was determined by comparing the IC50 of the competitors with that of PF and was calculated according to the equation developed by Weiler and Zenk [45].
Correlations between the indirect competitive enzyme-linked immunosorbent assay and high-performance liquid chromatography for the determination of paeoniflorin Sample preparation: The four TCM decoctions were produced and diluted with 90% ethanol, followed by filtration through a 0.45µm PES membrane prior to assaying. PF was dried to a constant weight and accurately weighed before being dissolved in methanol. It was then further diluted in carbonate buffer prior to filtration through a 0.45-µm microporous membrane. The concentra-
Acknowledgements !
The authors would like to thank Yifei Li and Xin Su for their assistance in providing the synthesis of haptens for conjugates and immunization. This research was supported by the National Natural Science Foundation of China (81 274 043, 81 373 542), the National Key Basic Research Development Program (973 program) (2011CB505 101), and the Classical Prescription Basic Research Team of Beijing University of Chinese Medicine.
Conflict of Interest !
The authors declare no conflicts of interest.
References 1 Li PP, Liu DD, Liu YJ, Song SS, Wang QT, Chang Y, Wu YJ, Chen JY, Zhao WD, Zhang LL, Wei W. BAFF/BAFF‑R involved in antibodies production of
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added after the blended splenocytes, and myeloma cells (at the ratio of 5 : 1, respectively) were centrifuged and incubated for 1 min at 37 °C. HAT medium was added, and the hybridoma was then transferred to 96-well plates for cell culture. Cells producing MAbs reactive to PF were identified by competitive ELISA and cloned by the limiting dilution method [42, 43]. The established hybridoma was cultured in HT medium.
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2
3
4
5
6
7
8
9
10
11
12
13
14 15 16
17 18
19
20
21
22
rats with collagen-induced arthritis via PI3K‑Akt-mTOR signaling and the regulation of paeoniflorin. J Ethnopharmacol 2012; 141: 290–300 Yu HY, Mu DG, Chen J, Yin W. Suppressive effects of intrathecal paeoniflorin on bee venom-induced pain-related behaviors and spinal neuronal activation. Pharmacol 2011; 88: 159–166 Hu ZX, Xu L, Yan R, Huang Y, Liu G, Zhou WX, Zhang YX. Advance in studies on effect of paeoniflorin on nervous system. Zhongguo Zhong Yao Za Zhi 2013; 38: 297–301 Xie Y, Jiang ZH, Zhou H, Ma WZ, Wong YF, Liu ZQ, Liu L. The pharmacokinetic study of sinomenine, paeoniflorin and paeonol in rats after oral administration of a herbal product Qingfu Guanjiesu capsule by HPLC. Biomed Chromatogr 2014; 28: 1294–1302 Hwang YH, Kim T, Cho WK, Jang D, Ha JH, Ma JY. Food-and gender-dependent pharmacokinetics of paeoniflorin after oral administration with Samul-tang in rats. J Ethnopharmacol 2012; 142: 161–167 Fiang J, Zhao Y, Wang J, Wei S, Wei Z, Li R, Zhu Y, Sun Z, Xiao X. Comparative pharmacokinetic study of paeoniflorin and albiflorin after oral administration of Paeoniae Radix Alba Rubra in normal rats and the acute cholestasis hepatitis rats. Fitoterapia 2012; 83: 415–421 Ma M, Feng F, Sheng Y, Cui S, Liu H. Development and evaluation of an efficient HPLC/MS/MS method for the simultaneous determination of pseudoephedrine and cetirizine in human plasma: application to phase-I pharmacokinetic study. J Chromatogr B 2007; 846: 105–111 Nair SN, Menon S, Shailajan S. A liquid chromatography/electrospray ionization tandem mass spectrometric method for quantification of asiatic acid from plasma: application to pharmacokinetic study in rats. J Rapid Commun Mass Spectrom 2012; 26: 1899–1908 Cao S, Zhou G. Pharmacokinetic interactions between ilaprazole and clarithromycin following ilaprazole, clarithromycin and amoxicillin triple therapy. Acta Pharmacol Sin 2012; 33: 1095–1100 Shan S, Tanaka H, Shoyama Y. Enzyme-linked immunosorbent assay forglycyrrhizin using anti-glycyrrhizin monoclonal antibody and an eastern blotting technique for glucuronides of glycyrrhetic acid. Anal Chem 2001; 73: 5784–5790 Qu HH, Zhang GL, Li YF, Sun H, Sun Y, Zhao Y, Wang QG. Development of an enzyme-linked immunosorbent assay based on anti-puerarin monoclonal antibody and its applications. J Chromatogr B 2014; 953: 120–125 Lu ZH, Morinaga O, Tanaka H, Shoyama Y. A quantitative ELISA using monoclonal antibody to survey paeoniflorin and albiflorin in crude drugs and traditional Chinese herbal medicines. Biol Pharm Bull 2003; 26: 862–866 Aruna D, Naidu MUR. Pharmacodynamic interaction studies of Ginkgo biloba with cilostazol and clopidogrel in healthy human subjects. Br J Clin Pharmacol 2007; 63: 333–338 Meijerman I, Beijnen JH, Schellens JHM. Herb-drug interactions in oncology: focus on mechanisms of induction. Oncologist 2006; 11: 742–752 Flanagan D. Understanding the grapefruit-drug interaction. Gen Dent 2004; 53: 282–285 Brazier NC, Levine MA. Drug-herb interaction among commonly used conventional medicines: a compendium for health care professionals. Am J Ther 2003; 10: 163–169 Zhai YJ. Clinical applications of Shaoyao-Gancao-Tang. Lishizhen Med Mater Med Res 2003; 14: 302–303 Hinoshita F, Ogura Y, Suzuki Y, Hara S, Yamada A, Tanaka N, Marumo F. Effect of orally administered shao-yao-gan-cao-tang (Shakuyaku-kanzo-to) on muscle cramps in maintenance hemodialysis patients: a preliminary study. Am J Chin Med 2003; 31: 445–453 Cai WR, Qian H, Zhu YH, Song K, Zhang H, Wang Z, Tang J. Experimental study on the anti-allergic and anti-asthmatic effects of Peony-Licorice decoction. Chin J Integr Tradit Western Med Intens Crit Care 2000; 7: 341–342 Tanaka T. A novel anti-dysmenorrhea therapy with cyclic administration of two Japanese herbal medicines. Clin Exp Obstet Gynecol 2003; 30: 95–98 Zhong ZY, Gong AS, Han J, Jing L, Rong XL, Wu QH. Studies on the optimal proportion of Shaoyaogancao decoction by orthogonal design and linear regression. Pharmacol Clin Chin Mater Med 2005; 21: 7–10 Chen LH, Feng Y, Xu DS, Li JS, Zhang L. Effect of Shaoyao-Gancao effective components on contractile activity of rat stomach muscle strips. Pharmacol Clin Chin Mater Med 2007; 23: 1–4
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23 Li H. 52 cases of intractable hiccups cured with Shaoyao Gancao decoction. Nei Mongol J Trad Chin Med 2011; 14: 9–10 24 Liu HQ. Experience of Liu Zhijian in application Shaoyao Gancao decoction. J Emerg Tradit Chin Med 2006; 15: 279–280 25 Xu CH, Wang P, Wang Y, Yang Y, Li DH, Li HF, Wu XZ. Pharmacokinetic comparisons of two different combinations of Shaoyao-Gancao Decoction in rats: competing mechanisms between paeoniflorin and glycyrrhetinic acid. J Ethnopharmacol 2013; 149: 443–452 26 Sun YM, Yang Y, Li D, Pu TT, Xie XH, Wang Q. Effect of paeoniflorin on the pharmacokinetics of glycyrrhizic acid and glycyrrhetinic acid in rats. Med Plant 2012; 47: 599–603 27 Liu H, Shan JJ, Kang A, Hui F, Chen LT, Zhang W, Di LQ. Effects of glycyrrhizic acid and glycyrrhetinic acid on in vivo pharmacokinetic parameters of paeoniflorin in rats. Chin Trad Herbal Drugs 2013; 44: 1610–1614 28 Mc Elnay JC, Wang ZS. The part of the drug interaction and mechanism: in vitro, intestines and metabolic parts of the interaction. Foreign Med Sci Pharm 1982; 1: 42–46 29 Xiang Q, Cheng G, Chen J. Pharmacokinetics and bioavailability of Paeonia and Glycyrrhizae Rad. decoction in rats. Chin Pharm J 2000; 35: 615–618 30 State Pharmacopoeia Commission. Pharmacopoeia of the Peopleʼs Republic of China, Part 1. Beijing: China Medical Science Press; 2010: 80, 96–97 31 Feng C, Liu M, Shi X, Yang W, Kong D, Duan K, Wang Q. Pharmacokinetic properties of paeoniflorin, albiflorin and oxypaeoniflorin after oral gavage of extracts of Radix Paeoniae Rubra and Radix Paeoniae Alba in rats. J Ethnopharmacol 2010; 130: 407–413 32 Liu J, Wang JS, Kong LY. Comparative pharmacokinetics of paeoniflorin in plasma of vascular dementia and normal rats orally administrated with Danggui-Shaoyao-San or pure paeoniflorin. Fitoterapia 2011; 82: 466–473 33 He P, Jia SW, Wu MC, Li LF, Guo YJ. Pharmacokinetics of glycyrrhizic acid in mice and human plasma protein binding. Chin Pharmacol Bull 1998; 14: 89 34 Gao YQ, Wu J, Jiang RW, Zhao GP. Determination of cinnamic acid and glycyrrhizic acid in rat serum and its pharmacokinetics after oral administration of Dangguisini decoction. Chin Med Mat 2011; 34: 408–411 35 Shen L, Cong WJ, Lin X, Hong YL, Hu RW, Feng Y, Xu DS, Ruan KF. Characterization using LC/MS of the absorption compounds and metabolites in rat plasma after oral administration of a single or mixed decoction of Shaoyao and Gancao. Chem Pharm Bull 2012; 60: 712–721 36 Chen Y, Wang J, Wang L, Chen L, Wu Q. Absorption and interaction of the main constituents from the traditional Chinese drug pair ShaoyaoGancao via a Caco-2 cell monolayer model. Molecules 2012; 17: 14 908–14 917 37 Wen HY, Zhang HY, Wang YR, Zhang XJ, Hu P. Investigation of the interaction between bovine serum album in with paeoniflorin and loganin. Chin J Pharm Anal 2010; 30: 6–11 38 Qu HH, Zhao Y, Su X, He NN, Sun Y, Kong H, Zhao Y, Wang QG. Synthesis and identification of artificial antigens of paeoniflorin. China J Chin Mater Med 2014; 39: 2043–2046 39 Zhang Y, Qu HH, Wu TT, Xue J, Sun Y, Sun H, Li YF, Zhao Y, Wang QG. Preparation and identification of anti-glycyrrhizic acid monoclonal antibody. Chin J Pharm Anal 2013; 33: 770–774 40 Galfrè G, Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol 1981; 73: 3–46 41 North SM, Styles JM, Hobbs SM, Dean CJ. Monoclonal antibodies to rat sarcomata. I. Immunization procedures and source of lymphoid cells for hybridoma production. Immunology 1982; 47: 397–405 42 Goding JW. Antibody production by hybridomas. J Immunol Methods 1980; 39: 285–308 43 Johnson DR. Murine monoclonal antibody development. In: Paul S, editor. Antibody engineering protocols. New York: Humana Press Inc.; 1995: 123–137 44 Xiao ZH, Huang ZL, Lin YX, Dong B, Tian SJ. Research of purification methods of ascites monoclonal antibody. Chin Med Biotechnol 2013; 8: 425–428 45 Weiler EW, Zenk MH. Radioimmunoassay for the determination of digoxin and related compounds in Digitalis lanata. Phytochemistry 1976; 15: 1537–1545
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A Sensitive and Specific Indirect Competitive Enzyme-Linked Immunosorbent Assay for Detection of Paeoniflorin and Its Application in Pharmacokinetic Interactions between Paeoniflorin and Glycyrrhizinic Acid Authors
Yan Zhao 1, Huihua Qu 2, Xueqian Wang 1, Yue Zhang 1, Wenchao Shan 3, Yan Zhao 1*, Qingguo Wang 1
Affiliations
1
3
Key words " paeoniflorin l " monoclonal antibody l " enzyme‑linked immunol sorbent assay (ELISA) " pharmacokinetics l " glycyrrhizic acid l
received revised accepted
October 29, 2014 January 6, 2015 March 8, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1545913 Published online April 9, 2015 Planta Med 2015; 81: 765–770 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence *Yan Zhao School of Basic Medical Sciences Beijing University of Chinese Medicine 11 Beisanhuandong Road, Chaoyang District Beijing 100029 China Phone: + 64 28 67 05 Fax: + 64 28 67 05 [email protected] Correspondence Qingguo Wang School of Basic Medical Sciences Beijing University of Chinese Medicine 11 Beisanhuandong Road, Chaoyang District Beijing 100029 China Phone: + 64 28 68 21 Fax: + 64 28 68 21 [email protected]
School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing, China Scientific Research Experiment Center, Beijing University of Chinese Medicine, Beijing, China College of Traditional Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing, China
Abstract
Abbreviations
!
!
This study aimed to develop an indirect competitive enzyme-linked immunosorbent assay based on monoclonal antibodies against paeoniflorin to study the effects of different doses of glycyrrhizinic acid on the pharmacokinetics of paeoniflorin in mice. An anti-paeoniflorin monoclonal antibody was produced from a hybridoma created through a fusion of splenocytes immunized with paeoniflorin-bovine serum albumin and conjugated with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma cell line SP2/0. The resultant antibody was used to develop and validate a rapid, specific and sensitive, indirect competitive enzyme-linked immunosorbent assay for the measurement of paeoniflorin (linear range 4.8–312.5 ng · mL−1). The intraday and interday precision values of the indirect competitive ELISA method were well within the recommended range (≤ 10 %), and the recovery rate was, on average, 101.13 %. Pharmacokinetic parameters obtained from mouse blood samples at various intervals following the oral administration of paeoniflorin and glycyrrhizic acid at three doses (1 : 0.3, 1 : 1, 1 : 3, respectively) demonstrated that the highest dose of glycyrrhizic acid inhibits the absorption of paeoniflorin.
AUC0-t: BSA: Cmax: CR: GA: GR: HAT: icELISA: MAb: MRT: OVA: PBST: PEG: PES: PF: PF‑BSA: PF‑OVA: PR: RSD: SGD: SPBS: Tmax: TCM: TMB:
Introduction ! " Fig. 1) is the main active compoPaeoniflorin (l nent in PR, a monoterpene glycoside with anti-inflammatory, antirheumatic, immunomodulatory [1], analgesic [2], and neuroprotective properties [3]. Several methods are used to determine the
area under the curve bovine serum albumin mean maximum concentration in plasma cross-reactivity glycyrrhizic acid Glycyrrhizae Radix et Rhizoma hypoxanthine-aminopterinthymidine indirect competitive enzyme-linked immunosorbent assay monoclonal antibody mean residence time ovalbumin phosphate-buffered saline containing 0.05 % Tween 20 polyethylene glycol polyethersulfone paeoniflorin paeoniflorin conjugated with bovine serum albumin paeoniflorin conjugated with ovalbumin Paeoniae Radix Alba relative standard deviation Shaoyao-Gancao decoction phosphate-buffered saline containing 5 % skim milk time of maximum concentration traditional Chinese medicine 3,3′,5,5′-tetramethylbenzidine
concentration of paeoniflorin in medicines and biological samples [4–6], with HPLC/MS/MS being the most commonly used method and displaying high sensitivity in pharmacokinetic studies [7–9]. The enzyme-linked immunosorbent assay (ELISA) using MAbs is another important method for the qualitative or quantitative detection of these
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Fig. 1 Structure of paeoniflorin.
products, with benefits including small scale, high reproducibility under various circumstances and minimal pretreatment [10, 11]. The ELISA method is very suitable for biological samples due to the need for only a very small amount of sample, which allows for simultaneous use across a large number of samples [11]. While Zhaohua Lu [12] and colleagues have developed an ELISA for the detection of paeoniflorin and albiflorin, it remains difficult to evaluate the contents of PF and profile the pharmacokinetics of paeoniflorin in animals or humans. Therefore, it is necessary to establish an ELISA with enough specificity and sensitivity to accurately monitor small amounts of paeoniflorin in various samples. The increasing use of botanical herb extracts and their prescription as therapeutic agents has led to an increase in investigations of more and more bioactive compound interactions between different herbs [13–15], most of which are associated with pharmacokinetics [16]. Under TCM guidance, the combination of PR and GR is typically used to treat spasmodic abdominal pain accompanying acute gastroenteritis [17], muscle cramps [18], asthma [19], and dysmenorrhea [20]. This combination has been used for many years with favorable results, though the dosage of the herb can affect such beneficial effects. Several studies have shown that the greatest efficacy against spasmolysis is obtained with a dosage proportion of 1 : 1 PR to GR, and that this effect improved as the dosage increased [21, 22]. On the other hand, a better curative effect against intractable hiccups was found when the dosage proportion of PR and GR was 3 : 1 [23]. In addition, PR and GR act as analgesics when PR is more abundant than GR (3 : 2 or 2 : 1) [24]. It is apparent from these studies that the various biological effects of the herbs depend heavily on the different compounds and their dosage proportions, and that these effects can be studied by pharmacokinetics. The pharmacokinetics and assessment of PF and GA have been widely reported [25–27], but the pharmacokinetics of PF in the presence of GA at different doses has not yet been investigated. Therefore, studying the interaction of these efficacious components through pharmacokinetics could provide a theoretical basis and clinical guidance for the rational and effective use of traditional Chinese medicines. In the present study, we first generated a new anti-PF monoclonal antibody and developed a sensitive and specific ELISA to efficiently measure the concentration of PF. Furthermore, this method was applied to explore the pharmacokinetics of PF in the presence of different doses of GA.
Results and Discussion !
A competitive binding assay in which the anti-PF MAb binds either free PF or a PF‑OVA conjugate adsorbed onto a polystyrene microtiter plate was established. Under these conditions, the linear range of the assay extended from 4.8 to 312.5 ng · mL−1, as in-
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Fig. 2 Standard curve of inhibition by PF using MAbs in an icELISA. Various concentrations of PF were incubated with anti-PF MAb in a 96-well ELISA plate precoated with PF‑OVA (1 µg · mL−1). Error bars: ± 1 SD; the data represent the mean of five replicates.
" Fig. 2. The sensitivity of the antibody was satisfacdicated in l tory, with an IC50 value of 42.72 ng · mL−1 and a limit of detection (LOD) of 4.75 ng · mL−1 in buffer. Specificity is the most important factor in determining the value of an antibody and the accuracy of an immunoassay, which was evaluated by cross reactivity. Because each compound has multiple antigenic determinants, it has the ability to stimulate the immune system to produce specific antibodies when conjugated with a carrier protein. In 2003, Zhaohua Lu et al. screened four hybridomas secreting monoclonal antibodies against PF; these antibodies all had a high CR with albiflorin (143.7 %, 89.56 %, 68.96 %, and 46.27 %, respectively) [12], indicating that they are four distinct monoclonal antibodies against different epitopes of PF. In this study, the anti-PF MAb had little CR with albiflorin (< 0.27%) and no CR with various other compounds (< 0.01 %), as " Table 1, suggesting that our new monoclonal antishown in l body against PF differs from the previously reported antibodies. In addition, the minor CR seen with albiflorin confirms the accuracy of the PF assay without the interference of albiflorin, which is advantageous for the establishment of a quality control method and pharmacokinetic study using this anti-PF MAb. The concentrations of PF in four traditional Chinese medicine decoctions (Sini San, Shaoyao Gancao Tang, Guizhi Tang, and Zhen" Table 2) were measured and compared to wu Tang, as shown in l the results of those acquired by performing high-performance liquid chromatography (HPLC), allowing us to verify the reliability of our method. The correlation was then calculated from a plot developed by regression analysis based on the values obtained " Fig. 3. The concentrations from ELISA and HPLC, as indicated in l of PF in the four traditional Chinese medicine decoctions, as measured by our developed ELISA, were highly consistent with those obtained by HPLC from the same samples (R2 = 0.9918). From these results, it is apparent that this ELISA method can be applied to determine PF in medicine, which will be very useful and more convenient in product quality control and quantification in the future. The accuracy and variation of the assay were evaluated by the RSDs of intra- (well to well) and inter- (plate to plate and day to day) assay data of the icELISA using our anti-PF MAb. A standard
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Table 2 The four Chinese medicines.
Classification
Compound
CRs (%)
Traditional
Terpene
paeoniflorin albiflorin ginsenoside Rb1 ginsenoside Re ginsenoside Rg1 notoginsenoside glycyrrhizic acid glycyrrhetic acid saikosaponin a saikosaponin d gentiopicrin geniposide deoxycholic acid cholic acid puerarin baicalin baicalein hyperoside rutin hesperidin naringin chlorogenic acid ferulic acid rheum emodin rheinic acid berberine salvianolic acid curcumin gastrodin amygdalin
100 < 0.27 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01
Chinese
Steroid Flavonoid
Phenylpropanoids Anthraquinones Alkaloid Other
curve for the ELISA was established with various concentrations of PF in plasma, which allowed for the calculation of RSDs in the sextuplicate wells (intra-assay) and multiple plates (inter-assay) on " Table 3 shows that the intra-assay RSDs six consecutive days. l were less than 3.1 % and the inter-assay RSDs were lower than 6.3 %, which indicates that this assay is very accurate and stable. A recovery experiment was performed to evaluate the reliability of the assay. Briefly, mouse plasma was spiked with purified PF at doses of 50, 100, and 200 ng. Recovery was calculated using the equation: recovery (%) = measured amount/added amount " Table 4, the average recovery rate was × 100%. As shown in l 101.13 % from 50 to 200 ng of spiked PF, suggesting that this assay is sufficiently reliable for the determination of PF in plasma. There are many documented instances of positive interactions between drugs [28], indicating the potential significance of using herbal combinations that are based on TCM theory. PR and GR formed SGD, which was reported originally in the Treatise on Febrile and Miscellaneous Diseases both written by Zhang Zhongjing in the last years of the Han dynasty in China (approximately 206 B. C. to 200 A. D.). In the clinical practice of TCM science, SGD has been used to treat systremma, gastrospasm, intercostal neuralgia, stomachache, abdominal pain, and dysmenorrhea over a very long period of time and has produced quite a favorable effect. A pharmacokinetic study on SGD has shown that the absorption degrees of PF and GA have enhanced after compatibility, which gave evidence that the compatibility of PR and GR was reasonable [29]. As the major effective constituents of PR and GR, PF and GA are used as the phytochemical markers for their quality control, respectively, in the Chinese Pharmacopoeia [30]. The pharmaco-
Composition of Kampo medicine
Percentage of Paeoniae
medicines
Radix Alba
Si-ni-San
Shao-yao-gancao-tang Gui-zhi-tang
Zhen-wu-tang
Paeoniae Radix Alba, Auranth Fructus Immaturus, Bupleuri Radix, Glycyrrhizae Radix Et Rhizoma Paeoniae Radix Alba, Glycyrrhizae Radix Et Rhizoma Cinnamomi Ramulus, Paeoniae Radix Alba, Glycyrrhizae Radix Et Rhizoma, Jujubae Fructus, Zingiberis Rhizoma Recens Poria, Paeoniae Radix Alba, Atractylodis Macrocephalae Rhizoma, Aconiti Lateralis Radix Praeparata, Zingiberis Rhizoma Recens
1/3
1/2 3/13
3/14
Fig. 3 Correlation between the content of PF determined by ELISA and HPLC.
Table 3 Intra- and interday PF precision in mice plasma. PF (ng · mL−1)
RSD (%) Intra-assay
Inter-assay
2.5 5 10 20 40
1.8 2.3 2.6 3.1 2.8
2.8 4.1 3.3 5.5 6.3
Table 4 Recovery of PF from mice plasma. Added amount (ng)
Measured amount (ng)
Recovery (%)
50 100 200
51.24 ± 0.36 101.27 ± 2.15 199.25 ± 3.18
102.48 101.27 99.63 Average 101.13
kinetic determinations of PF [31, 32] and GA [33, 34] in PR and GR based on blood samples have been widely reported, and there are interactions between PR and GR [25, 35, 36]. However, there are no reports regarding the effect of different doses and dose ratios on the pharmacokinetics of PF and GA in mice, so far. In this study, we measured and plotted the mean concentration of PF in
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Table 1 CRs (%) of anti-PF MAb against various compounds using icELISA.
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high-throughput detection in four hours. These show that the developed icELISA method is simpler, more efficient, and more sensitive than HPLC. However, it cannot be denied that the developed icELISA cannot monitor the PF metabolizing process in vivo, and the cross-reaction of the monoclonal antibody to the structure-related compound is hard to avoid, while there is no such limit to HPLC.
Materials and Methods !
Materials and reagents
Fig. 4 Mean concentration-time profiles of PF in mouse blood after oral administration for group 1 (GA: PF = 0.3 : 1), group 2 (GA: PF = 1 : 1), and group 3 (GA: PF = 3 : 1) (n = 6). (Color figure available online).
mouse plasma as a function of time in three groups, as shown in " Fig. 4. The pharmacokinetic parameters of mouse plasma saml ples following oral administration of different rations of PF and " Table 5. GA are shown in l An increased glycyrrhizic acid content significantly (p < 0.05) reduced the absorption of paeoniflorin when comparing groups 1 and 3, which was reflected by the AUC0–6 (decreased from 849.08 µg · min · mL−1 to 561.11 µg · min · mL−1) and Cmax (decreased from 10.62 µg · mL−1 to 5.28 µg · mL−1) values, whereas the MRT increased 1.5-fold when the dosage of GA was increased 10fold. The Cmax and AUC0–6 values exhibited a linear relationship (R2 = 0.9808), demonstrating that the AUC of PF is dose-related. With the content of GA increasing, the Cmax and AUC0–6 values of PF decreased gradually, while the MRT was extended, which implies that the absorption of PF was inhibited when the content of GA increased to a certain amount, but the residence time of PF was prolonged, which is beneficial for PF to work in vivo. A further study will focus on the pharmacodynamics of the different combinations of PF and GA to explore the mechanism of the result of this study. The results of the present study demonstrate that our newly developed ELISA based on a new MAb against PF provides a reliable and sensitive approach for the determination of PF content in medicines and plasma. Due to its sensitivity, the detection of PF in plasma needs small amounts of blood, which allows for the blood to continuously get to several time points from the same mouse without killing it. Besides, using the icELISA method, we need minimal pretreatment steps before detection and achieve
Paeoniflorin (purity 98%) and various other compounds were purchased from Dingguo changsheng Biotechnology. BSA, OVA, and Freundʼs complete and incomplete reagents were obtained from Sigma-Aldrich. All other chemicals and reagents were of analytical grade and were purchased from Sinopharm Chemical Reagents.
Synthesis of antigen conjugates PF-carrier protein conjugates, namely PF‑BSA and PF‑OVA, were synthesized by a periodate oxidation procedure according to a previous methodology with some modifications [12], and detected by ultraviolet visible spectrophotometry (UV) [37]. The result was shown in a previous study [38].
Preparation of the paeoniflorin antibody Immunization: Female BALB/C mice (7 weeks of age) were purchased from Vital River Laboratories. The mice were fed a standard rodent diet 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 the Beijing University of Chinese Medicine, China (2013BZHYLL00 106). The mice were immunized as described by previous reports [39]. Briefly, the mice were injected subcutaneously with 100 µg of PF‑BSA conjugates in 0.15 mL phosphate buffer emulsified with the same volume of Freundʼs complete adjuvant. Injections were repeated at 2-week intervals (weeks 2, 4, and 6) with the same volume of Freundʼs incomplete adjuvant. The final immunization lacked adjuvant and was administered as a booster for three days prior to cell fusion. Mouse blood was obtained from the tail vein one day after the booster, and sera were isolated for the titer test by direct ELISA.
Cell fusion and preparation of anti-paeoniflorin monoclonal antibody On the third day after the final immunization, splenocytes were isolated and fused with the SP2/0 HAT-sensitive mouse myeloma cell line using the PEG method [40, 41]. Briefly, 1 mL of PEG was
Table 5 Pharmacokinetic parameters of PF in three mouse treatment groups (mean ± SD, n = 6). Parameter
Group 1
Group 2
Group 3
Cmax (µg · mL−1) Tmax (min) AUC0–6 h (µg · min · mL−1) MRT (min) T1/2 (min) Vd (L/Kg) Clearance (mL/min · Kg)
10.62 ± 4.78 25.83 ± 12.81 849.08 ± 249.13 193.51 ± 48.16 134.10 ± 33.37 12.41 ± 3.43# 72.87 ± 13.22
7.16 ± 2.30 30.83 ± 10.21 625.78 ± 149.55 226.31 ± 142.18 156.83 ± 98.53 18.30 ± 3.97# 112.65 ± 43.74
5.28 ± 1.92* 23.33 ± 4.08 561.11 ± 107.94* 301.24 ± 73.00* 208.76 ± 50.59* 33.39 ± 7.04 116.10 ± 8.44
* p < 0.05 (compared with group 1); # p < 0.05 (compared with group 3)
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Purification of anti-paeoniflorin monoclonal antibody The ascites obtained from the abdominal cavity of Balb/c mice implanted with the established hybridoma were purified by the caprylic acid-ammonium sulfate method [44].
Analytical procedure Direct ELISA: The reactivity of anti-PF MAb to PF‑OVA was determined by direct ELISA. PF‑OVA (100 µl/well) was diluted in 50 mM carbonate buffer (pH 9.6), added into the wells of a 96well immunoplate in different columns and incubated for 1 h. This was then treated with 200 µl of blocking buffer (SPBS) for 1 h to eliminate nonspecific adsorption. The plate was washed three times with washing buffer (PBST) and filled with 100 µL of MAb solution at various dilutions in different rows. After 1 h, the plate was washed three times with PBST and incubated with 100 µl of a 10 000-fold diluted peroxidase-labeled goat antimouse IgG solution for 1 h, followed by another wash with PBST for three times. Then, 100 µl of the substrate solution (0.1 mM citrate buffer (pH 4) containing 0.75 % H2O2 and 2 mg/mL TMB) was added to each well, and the plate was incubated for 15 min. The reaction was then terminated by adding 50 µl of 2 M sulfuric acid to each well. All reactions were carried out at 37 °C. The absorbance was determined at 450 nm using a Biotek ELx 800 microplate reader.
Indirect competitive enzyme-linked immunosorbent assay The reactivity of anti-PF MAb to PF was determined by icELISA. PF‑OVA (1 : 10 000, 100 µL/well) was adsorbed in the wells of a 96-well immunoplate, which was then treated with 200 µl of 5 % SPBS for 1 h to reduce nonspecific adsorption. The plate was washed three times with PBST, and 50 µL of various concentrations of PF solution were incubated with 50 µL of MAb solution for 1 h. The subsequent process was the same as that described for direct ELISA.
tions of PF in the four TCM decoctions were compared using HPLC and icELISA methods in parallel. Chromatographic conditions: An Agilent series 1260 HPLC instrument equipped with a quaternary pump, a diode-array detector, an autosampler, and a column compartment was used for analyses. The sample was separated on a Zorbax SB‑C18 column (5 µm, ϕ 4.6 × 150 mm, Agilent), with the mobile phase consisting of CH3CN (A) and water containing 0.1 % acetic acid (B). A gradient program was used as follows: 10% A in the first 5 min, linearly increased to 25% A at 15 min, then linearly decreased to 10 % A at 16 min using a mobile phase flow rate of 1.0 mL/min. The chromatogram was recorded at 230 nm and the spectral data for all peaks were obtained in the range of 190–400 nm. The column temperature was kept constant at 30 °C.
Determination of paeoniflorin in plasma Animals and drug administration: Kunming mice from Vital River Laboratory Animal Technology were maintained on a 12-h lightdark cycle (light from 7 : 00 a. m. to 7 : 00 p. m.) in a temperatureand humidity-controlled room (22 ± 2 °C and 50 ± 10%, respectively). Mice were treated after a 1-week acclimation period and were fasted for 12 h before the experiment with free access to water. All procedures were approved by the Committee on Ethics of Animal Experiments, Beijing University of Chinese Medicine, China. The mice were divided into three groups: group 1 [GA: PF, 0.3 : 1 (w/w)], group 2 [GA: PF, 1 : 1 (w/w)], and group 3 [GA: PF, 3 : 1 (w/w)]. The amount of the orally administered drugs was equivalent to 80 mg/kg body weight of PF. Blood collection and preparation: At the selected time points (5, 10, 15, 25, 35, 45, 60, 90, 120, 150, 180, 210, 240, and 360 min), 10 µl of tail vein blood was extracted and placed into 0.5 ml EP tubes treated with heparin sodium (1 mg · mL−1). Blood was centrifuged at 1000 g for 10 min at 4 °C, and the supernatant was taken and diluted 20 times for analyses. Determination of PF in plasma and data analyses: After sample processing, the concentrations of PF in the plasma samples were measured by the established enzyme immunoassay. The icELISA procedures were performed as described above, with the exception that the mouse plasma was tested. The standards and the blank control were prepared in the control mouse plasma. Cmax, Tmax, terminal elimination half-life, AUC0-t, and MRT were calculated using a non-compartment model in Kinetica version 4.4 (Thermo Fisher Scientific). Variance analysis (ANOVA) was used for the statistical analyses.
Specificity of anti-paeoniflorin monoclonal antibody The specificity of the antibody was determined by assessing CR with PF analogs that are known to compete with the binding of the antibody for the coating antigen in icELISA. CR was determined by comparing the IC50 of the competitors with that of PF and was calculated according to the equation developed by Weiler and Zenk [45].
Correlations between the indirect competitive enzyme-linked immunosorbent assay and high-performance liquid chromatography for the determination of paeoniflorin Sample preparation: The four TCM decoctions were produced and diluted with 90% ethanol, followed by filtration through a 0.45µm PES membrane prior to assaying. PF was dried to a constant weight and accurately weighed before being dissolved in methanol. It was then further diluted in carbonate buffer prior to filtration through a 0.45-µm microporous membrane. The concentra-
Acknowledgements !
The authors would like to thank Yifei Li and Xin Su for their assistance in providing the synthesis of haptens for conjugates and immunization. This research was supported by the National Natural Science Foundation of China (81 274 043, 81 373 542), the National Key Basic Research Development Program (973 program) (2011CB505 101), and the Classical Prescription Basic Research Team of Beijing University of Chinese Medicine.
Conflict of Interest !
The authors declare no conflicts of interest.
References 1 Li PP, Liu DD, Liu YJ, Song SS, Wang QT, Chang Y, Wu YJ, Chen JY, Zhao WD, Zhang LL, Wei W. BAFF/BAFF‑R involved in antibodies production of
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added after the blended splenocytes, and myeloma cells (at the ratio of 5 : 1, respectively) were centrifuged and incubated for 1 min at 37 °C. HAT medium was added, and the hybridoma was then transferred to 96-well plates for cell culture. Cells producing MAbs reactive to PF were identified by competitive ELISA and cloned by the limiting dilution method [42, 43]. The established hybridoma was cultured in HT medium.
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rats with collagen-induced arthritis via PI3K‑Akt-mTOR signaling and the regulation of paeoniflorin. J Ethnopharmacol 2012; 141: 290–300 Yu HY, Mu DG, Chen J, Yin W. Suppressive effects of intrathecal paeoniflorin on bee venom-induced pain-related behaviors and spinal neuronal activation. Pharmacol 2011; 88: 159–166 Hu ZX, Xu L, Yan R, Huang Y, Liu G, Zhou WX, Zhang YX. Advance in studies on effect of paeoniflorin on nervous system. Zhongguo Zhong Yao Za Zhi 2013; 38: 297–301 Xie Y, Jiang ZH, Zhou H, Ma WZ, Wong YF, Liu ZQ, Liu L. The pharmacokinetic study of sinomenine, paeoniflorin and paeonol in rats after oral administration of a herbal product Qingfu Guanjiesu capsule by HPLC. Biomed Chromatogr 2014; 28: 1294–1302 Hwang YH, Kim T, Cho WK, Jang D, Ha JH, Ma JY. Food-and gender-dependent pharmacokinetics of paeoniflorin after oral administration with Samul-tang in rats. J Ethnopharmacol 2012; 142: 161–167 Fiang J, Zhao Y, Wang J, Wei S, Wei Z, Li R, Zhu Y, Sun Z, Xiao X. Comparative pharmacokinetic study of paeoniflorin and albiflorin after oral administration of Paeoniae Radix Alba Rubra in normal rats and the acute cholestasis hepatitis rats. Fitoterapia 2012; 83: 415–421 Ma M, Feng F, Sheng Y, Cui S, Liu H. Development and evaluation of an efficient HPLC/MS/MS method for the simultaneous determination of pseudoephedrine and cetirizine in human plasma: application to phase-I pharmacokinetic study. J Chromatogr B 2007; 846: 105–111 Nair SN, Menon S, Shailajan S. A liquid chromatography/electrospray ionization tandem mass spectrometric method for quantification of asiatic acid from plasma: application to pharmacokinetic study in rats. J Rapid Commun Mass Spectrom 2012; 26: 1899–1908 Cao S, Zhou G. Pharmacokinetic interactions between ilaprazole and clarithromycin following ilaprazole, clarithromycin and amoxicillin triple therapy. Acta Pharmacol Sin 2012; 33: 1095–1100 Shan S, Tanaka H, Shoyama Y. Enzyme-linked immunosorbent assay forglycyrrhizin using anti-glycyrrhizin monoclonal antibody and an eastern blotting technique for glucuronides of glycyrrhetic acid. Anal Chem 2001; 73: 5784–5790 Qu HH, Zhang GL, Li YF, Sun H, Sun Y, Zhao Y, Wang QG. Development of an enzyme-linked immunosorbent assay based on anti-puerarin monoclonal antibody and its applications. J Chromatogr B 2014; 953: 120–125 Lu ZH, Morinaga O, Tanaka H, Shoyama Y. A quantitative ELISA using monoclonal antibody to survey paeoniflorin and albiflorin in crude drugs and traditional Chinese herbal medicines. Biol Pharm Bull 2003; 26: 862–866 Aruna D, Naidu MUR. Pharmacodynamic interaction studies of Ginkgo biloba with cilostazol and clopidogrel in healthy human subjects. Br J Clin Pharmacol 2007; 63: 333–338 Meijerman I, Beijnen JH, Schellens JHM. Herb-drug interactions in oncology: focus on mechanisms of induction. Oncologist 2006; 11: 742–752 Flanagan D. Understanding the grapefruit-drug interaction. Gen Dent 2004; 53: 282–285 Brazier NC, Levine MA. Drug-herb interaction among commonly used conventional medicines: a compendium for health care professionals. Am J Ther 2003; 10: 163–169 Zhai YJ. Clinical applications of Shaoyao-Gancao-Tang. Lishizhen Med Mater Med Res 2003; 14: 302–303 Hinoshita F, Ogura Y, Suzuki Y, Hara S, Yamada A, Tanaka N, Marumo F. Effect of orally administered shao-yao-gan-cao-tang (Shakuyaku-kanzo-to) on muscle cramps in maintenance hemodialysis patients: a preliminary study. Am J Chin Med 2003; 31: 445–453 Cai WR, Qian H, Zhu YH, Song K, Zhang H, Wang Z, Tang J. Experimental study on the anti-allergic and anti-asthmatic effects of Peony-Licorice decoction. Chin J Integr Tradit Western Med Intens Crit Care 2000; 7: 341–342 Tanaka T. A novel anti-dysmenorrhea therapy with cyclic administration of two Japanese herbal medicines. Clin Exp Obstet Gynecol 2003; 30: 95–98 Zhong ZY, Gong AS, Han J, Jing L, Rong XL, Wu QH. Studies on the optimal proportion of Shaoyaogancao decoction by orthogonal design and linear regression. Pharmacol Clin Chin Mater Med 2005; 21: 7–10 Chen LH, Feng Y, Xu DS, Li JS, Zhang L. Effect of Shaoyao-Gancao effective components on contractile activity of rat stomach muscle strips. Pharmacol Clin Chin Mater Med 2007; 23: 1–4
Zhao Y et al. A Sensitive and …
Planta Med 2015; 81: 765–770
23 Li H. 52 cases of intractable hiccups cured with Shaoyao Gancao decoction. Nei Mongol J Trad Chin Med 2011; 14: 9–10 24 Liu HQ. Experience of Liu Zhijian in application Shaoyao Gancao decoction. J Emerg Tradit Chin Med 2006; 15: 279–280 25 Xu CH, Wang P, Wang Y, Yang Y, Li DH, Li HF, Wu XZ. Pharmacokinetic comparisons of two different combinations of Shaoyao-Gancao Decoction in rats: competing mechanisms between paeoniflorin and glycyrrhetinic acid. J Ethnopharmacol 2013; 149: 443–452 26 Sun YM, Yang Y, Li D, Pu TT, Xie XH, Wang Q. Effect of paeoniflorin on the pharmacokinetics of glycyrrhizic acid and glycyrrhetinic acid in rats. Med Plant 2012; 47: 599–603 27 Liu H, Shan JJ, Kang A, Hui F, Chen LT, Zhang W, Di LQ. Effects of glycyrrhizic acid and glycyrrhetinic acid on in vivo pharmacokinetic parameters of paeoniflorin in rats. Chin Trad Herbal Drugs 2013; 44: 1610–1614 28 Mc Elnay JC, Wang ZS. The part of the drug interaction and mechanism: in vitro, intestines and metabolic parts of the interaction. Foreign Med Sci Pharm 1982; 1: 42–46 29 Xiang Q, Cheng G, Chen J. Pharmacokinetics and bioavailability of Paeonia and Glycyrrhizae Rad. decoction in rats. Chin Pharm J 2000; 35: 615–618 30 State Pharmacopoeia Commission. Pharmacopoeia of the Peopleʼs Republic of China, Part 1. Beijing: China Medical Science Press; 2010: 80, 96–97 31 Feng C, Liu M, Shi X, Yang W, Kong D, Duan K, Wang Q. Pharmacokinetic properties of paeoniflorin, albiflorin and oxypaeoniflorin after oral gavage of extracts of Radix Paeoniae Rubra and Radix Paeoniae Alba in rats. J Ethnopharmacol 2010; 130: 407–413 32 Liu J, Wang JS, Kong LY. Comparative pharmacokinetics of paeoniflorin in plasma of vascular dementia and normal rats orally administrated with Danggui-Shaoyao-San or pure paeoniflorin. Fitoterapia 2011; 82: 466–473 33 He P, Jia SW, Wu MC, Li LF, Guo YJ. Pharmacokinetics of glycyrrhizic acid in mice and human plasma protein binding. Chin Pharmacol Bull 1998; 14: 89 34 Gao YQ, Wu J, Jiang RW, Zhao GP. Determination of cinnamic acid and glycyrrhizic acid in rat serum and its pharmacokinetics after oral administration of Dangguisini decoction. Chin Med Mat 2011; 34: 408–411 35 Shen L, Cong WJ, Lin X, Hong YL, Hu RW, Feng Y, Xu DS, Ruan KF. Characterization using LC/MS of the absorption compounds and metabolites in rat plasma after oral administration of a single or mixed decoction of Shaoyao and Gancao. Chem Pharm Bull 2012; 60: 712–721 36 Chen Y, Wang J, Wang L, Chen L, Wu Q. Absorption and interaction of the main constituents from the traditional Chinese drug pair ShaoyaoGancao via a Caco-2 cell monolayer model. Molecules 2012; 17: 14 908–14 917 37 Wen HY, Zhang HY, Wang YR, Zhang XJ, Hu P. Investigation of the interaction between bovine serum album in with paeoniflorin and loganin. Chin J Pharm Anal 2010; 30: 6–11 38 Qu HH, Zhao Y, Su X, He NN, Sun Y, Kong H, Zhao Y, Wang QG. Synthesis and identification of artificial antigens of paeoniflorin. China J Chin Mater Med 2014; 39: 2043–2046 39 Zhang Y, Qu HH, Wu TT, Xue J, Sun Y, Sun H, Li YF, Zhao Y, Wang QG. Preparation and identification of anti-glycyrrhizic acid monoclonal antibody. Chin J Pharm Anal 2013; 33: 770–774 40 Galfrè G, Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol 1981; 73: 3–46 41 North SM, Styles JM, Hobbs SM, Dean CJ. Monoclonal antibodies to rat sarcomata. I. Immunization procedures and source of lymphoid cells for hybridoma production. Immunology 1982; 47: 397–405 42 Goding JW. Antibody production by hybridomas. J Immunol Methods 1980; 39: 285–308 43 Johnson DR. Murine monoclonal antibody development. In: Paul S, editor. Antibody engineering protocols. New York: Humana Press Inc.; 1995: 123–137 44 Xiao ZH, Huang ZL, Lin YX, Dong B, Tian SJ. Research of purification methods of ascites monoclonal antibody. Chin Med Biotechnol 2013; 8: 425–428 45 Weiler EW, Zenk MH. Radioimmunoassay for the determination of digoxin and related compounds in Digitalis lanata. Phytochemistry 1976; 15: 1537–1545
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