Thaweesak Juengwatanatrakul

Comparative analysis of the chemical constituents of two varieties of Pueraria candollei

A high-performance liquid chromatography (HPLC) method was developed to determine the contents of miroestrol and deoxymiroestrol in the tubers of Pueraria candollei var. mirifica and P. candollei var. candollei. The linear detection ranges were 0.78-25.00 μg/mL for miroestrol and 1.56-25.00 μg/mL for deoxymiroestrol. The limit of detection (LOD) and limit of quantification (LOQ) were 0.2 and 0.78 μg/mL, respectively, for miroestrol and 0.78 and 1.56 μg/mL, respectively, for deoxymiroestrol. Our results suggest that both varieties of P. candollei can produce miroestrol and deoxymiroestrol and that the developed HPLC method can be applied for quality control of plants and their products.

Preparation of a specific monoclonal antibody to asiaticoside for the development of an enzyme-linked immunosorbent assay

Asiaticoside (AS), the major active component of Centella asiatica (L.) Urban, is used as a memory enhancer and for wound healing. We have successfully prepared monoclonal antibodies (MAbs) against AS, and developed an enzyme-linked immunosorbent assay (ELISA) system for its determination. AS was conjugated to the carrier protein bovine serum albumin (BSA), which acted as an immunogen. In order to confirm its immunogenicity, the ratio of hapten in the AS-BSA conjugate was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). After immunization, hybridomas secreting MAbs against AS were produced by fusing splenocytes with the mouse myeloma cell line, SP2/0-Ag14. After the screening, anti-asiaticoside MAb 2B4 was obtained. Weak cross-reactivities occurred with madecassoside (7.08%), but no cross-reactivities were observed with other related triterpenoid glycosides (<0.01%). The assay was suitable for quantitating AS in the range of 0.78 to 50 µg mL(-1). A good correlation of AS concentrations in crude extracts of C. asiatica between ELISA and HPLC methods was obtained (r(2) = 0.999). The contents of AS in various cultivated C. asiatica samples were assayed by the newly established ELISA. The recovery rates of AS in the samples were in the range of 95-103% with coefficients of variation of <10%. The intra- and inter-assay variations were 3.9 and 4.5%, respectively. The ELISA method described should prove useful as an analytical tool for quality control and standardization of medicinal plants and pharmaceutical products containing AS.

Bimodal action of miroestrol and deoxymiroestrol, phytoestrogens from Pueraria candollei var. mirifica, on hepatic CYP2B9 and CYP1A2 expressions and antilipid peroxidation in mice.

Miroestrol and deoxymiroestrol are phytoestrogens isolated from Pueraria candollei var. mirifica. The influence of miroestrol and dexoymirosestrol on hepatic cytochrome P450 (P450) enzymes and antioxidative activity in brain was examined in C57BL/6 mice compared with that of a synthetic female sex hormone estradiol. We hypothesized that miroestrol and deoxymiroestrol would induce CYP2B9 expression, whereas CYP1A2 expression would be suppressed compared with estradiol. Miroestrol and deoxymiroestrol treatment significantly increased uterus weight and volume. In addition, both of these phytoestrogens induced the expression of CYP2B9 and suppressed the expression of CYP1A2, as expected. Hepatic P450 activities correspondingly showed that both compounds increased benzyloxyresorufin O-dealkylase activity, whereas methoxyresorufin O-dealkylase activity was reduced. These observations suggested that miroestrol and deoxymiroestrol might affect hepatic P450 enzymes, including the CYP2B9 and CYP1A2 P450 isoforms. Assessment of lipid peroxidation demonstrated that miroestrol and deoxymiroestrol markedly decreased levels of malondialdehyde formation in the mouse brain. This is the first report suggesting miroestrol and deoxymiroestrol as potential alternative medicines to estradiol because of their distinctive ability to regulate mouse hepatic P450 expression and their beneficial antioxidative activities.

High performance enzyme-linked immunosorbent assay for determination of miroestrol, a potent phytoestrogen from Pueraria candollei

Pueraria candollei associated preparation is widely applied in folk Thai medicine for rejuvenating purpose in aged people, which correlated with its pharmacological activities reported by pre-clinical and clinical trials. Therefore, standardized products of this plant are needed by consumers and health care personnel. Miroestrol, a potent and stable phytoestrogen in P. candollei, exhibited potential to be biomarker for quality control of P. candollei samples in research or industrial levels. Indirect competitive enzyme-linked immunosorbant assay (ELISA) for miroestrol determination was developed and validated by using polyclonal antibody from rabbit immunization. The polyclonal antibody recognized specifically to miroestrol, which exhibited cross-reactivity to deoxymiroestrol and isomiroestrol with 6.68% and 1.05%, respectively. The linearity range of measurement was 0.73-3000 ng mL(-1), which coefficient of variation (CV) of both intra- and inter-plate determination was less than 5%. With spiked samples of known amount miroestrol, the percentages of recovery were 98.80-104.37% and 98.31-106.69% in P. candollei and its involved product samples, respectively. Validated ELISA was comparable with published HPLC method (R(2)=0.9996) (Yusakul et al.) in samples with various miroestrol contents. For application, the P. candollei involved preparations contained miroestrol 0.695±0.037-12.108±0.285 μg g(-1) dry wt. The developed ELISA was high performance for miroestrol determination, which could be applied for P. candollei quality control in research fields and industrial productions.

Highly selective and sensitive determination of deoxymiroestrol using a polyclonal antibody-based enzyme-linked immunosorbent assay

Pueraria candollei-associated products are of interest to worldwide consumers for their rejuvenating and cosmetic purposes. In addition, clinical trials have supported the beneficial effects of P. candollei on the alleviation of menopausal symptoms. Deoxymiroestrol, which was reported as the most potent phytoestrogen in the tuberous root of P. candollei, exhibited potential as a biomarker of P. candollei-derived samples and products. A polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed for deoxymiroestrol determination. The raised antibody bound specifically to deoxymiroestrol with very low cross reactivities of 1.26 and 0.42% to structurally related miroestrol and isomiroestrol, respectively. The linear range was 0.73-3000.00 ng mL(-1), and the coefficients of variation for both the intra- and inter-plate determinations were less than 5%. In samples spiked with a known amount of deoxymiroestrol, the recoveries were 99.82-102.58% in P. candollei samples and 98.07-106.33% in its products samples. In comparison with other analytical methods, the validated ELISA was comparable to published HPLC-UV methods for samples with high deoxymiroestrol content (R(2)=0.9993). Furthermore, ELISA can be used for samples with deoxymiroestrol concentrations that are too small to detect by HPLC and for conditions when other chemicals cause interference with chromatographic analysis. For the P. candollei-derived products, the preparations contained 0.154-10.998 µg g(-1) dry wt. Our ELISA successfully measured deoxymiroestrol content with high sensitivity, selectivity, accuracy and rapidity. Therefore, this ELISA showed potential for dosage standardization of P. candollei-associated samples.

Down regulation of gene related sex hormone synthesis pathway in mouse testes by miroestrol and deoxymiroestrol.

Miroestrol and deoxymiroestrol are phytoestrogens isolated from tuberous root of Pueraria candollei var. mirifica. Modulatory effects of miroestrol and deoxymiroestrol on enzymes involved in sex-hormone synthesis pathway in male C57BL/6 mice were investigated using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Miroestrol and deoxymiroestrol suppressed the expressions of 3β-HSD, 17β-HSD1, and CYP17 while CYP19 mRNA expression was slightly decreased. In addition, the expression of 17β-HSD2 was induced in correlation with those did by estradiol. These observations supported that miroestrol and deoxymiroestrol could exhibit the same effect as estradiol regarding regulation of testicular gene related sex hormone synthesis pathway.

Preparation and application of a monoclonal antibody against the isoflavone glycoside daidzin using a mannich reaction-derived hapten conjugate.

INTRODUCTION Daidzin and its aglycone daidzein are major pharmacologically active compounds of soybean (Glycine max), kudzu (Pueraria lobata), and kwao kruea khao (P. mirifica). Pharmacological activities of daidzin are mediated by its more potent metabolites daidzein and equol; however, daidzin is the predominant compound found in these medicinal plants, and the efficacy and safety of equol depend on the amount of daidzin consumed. OBJECTIVE To develop a specific monoclonal antibody (MAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) for standardisation of daidzin content in herbal medicines or nutraceuticals. METHODOLOGY The Mannich reaction was used for the synthesis of a highly immunogenic conjugate between daidzin and a cationised carrier protein. Splenocytes of hyperimmunised mice were fused with myeloma cells to generate a hybridoma secreting antibody against daidzin. RESULTS The icELISA showed high selectivity and acceptable sensitivity for daidzin determination (1.56-100 ng/mL) with high reproducibility (coefficients of variation were < 5%). The icELISA was a reliable analytical method for daidzin in Glycine max, Pueraria lobata and P. mirifica, for which daidzin recoveries from spiked samples were 98.99-104.94%. Daidzin content of these plant-derived products determined using the icELISA were in close agreement with those determined by a HPLC-UV method. CONCLUSION The icELISA is useful for specific daidzin determination because of its reliability, low cost, speed and high throughput.

Enzyme-linked immunosorbent assay by enhanced chemiluminescence detection for the standardization of estrogenic miroestrol in Pueraria candollei Graham ex Benth.

Miroestrol (ME) is a potent phytoestrogen from the P. candollei tuberous root. It has been approved for use in clinical trials due to its beneficial effect on disorders associated with estrogen deficiency. To ensure medical efficacy and safety, high performance analytical methods for ME analysis are required to standardize products from the P. candollei root. An enhanced chemiluminescence enzyme-linked immunosorbent assay (ECL-ELISA) was developed and validated using a polyclonal antibody against ME and a chemiluminescent system of luminol-H2 O2 -horseradish peroxidase-4-(1-imidazolyl) phenol. The ECL-ELISA system exhibited linearity over a concentration range of 0.31-10.00 ng mL(-1) , for which the relative standard variation (%RSD) was less than 10% for both intra- and interplate determinations. The ECL-ELISA is reliable for the determination of ME as reflected by the high recovery percentage (101.22-103.06%). As a comparative analysis, the ME content in each sample determined by ECL-ELISA was correlated with high coefficients of determination with colorimetric ELISA (R(2)  = 0.998) and high performance liquid chromatography (HPLC) (R(2)  = 0.998) methods. The ECL-ELISA method could be applied to all of the commercial products containing P. candollei root, when the products contain between 0.706 ± 0.046 and 13.123 ± 0.794 µg g(-1) dry wt. of ME. This method is useful as a high performance analytical method for the quantity control of ME in raw materials and end products at both the research and industrial levels.

Preparation of a Single-Chain Variable Fragment and a Recombinant Antigen-Binding Fragment against the Anti-Malarial Drugs, Artemisinin and Artesunate, and Their Application in an ELISA

Two different recombinant antibodies, a single-chain variable fragment (scFv) and an antigen-binding fragment (Fab), were prepared against artemisinin (AM) and artesunate (AS) and were developed for use in an enzyme-linked immunosorbent assay (ELISA). The recombinant antibodies, which were derived from a single monoclonal antibody against AM and AS (mAb 1C1) prepared by us, were expressed by Escherichia coli cells and their reactivity and specificity were characterized. As a result, to obtain sufficient signal in indirect ELISA, a much greater amount of a first antibody was needed in the use of scFv due to the differences of the secondary antibody and conformational stability. Therefore, we focused on the development of the recombinant Fab antibodies and applied it to indirect competitive ELISA. The specificity of the Fab was similar to that of mAb 1C1 in that it showed specific reactivity toward AM and AS only. The sensitivity of the icELISA (0.16 μg/mL to 40 μg/mL for AM and 8.0 ng/mL to 60 ng/mL for AS) was sufficient for analysis of antimalarial drugs, and its utility for quality control of analysis of Artemisia spp. was validated. The Fab expression and refolding systems provided a good yield of high-quality antibodies. The recombinant antibody against AM and AS provides an essential component of an economically attractive immunoassay and will be useful in other immunochemical applications for the analysis and purification of antimalarial drugs.

Production of polyclonal antibody against madecassoside and development of immunoassay methods for analysis of triterpene glycosides in Centella asiatica.

INTRODUCTION Centella asiatica (L.) Urban consists of two major triterpene glycosides, asiaticoside (AS) and madecassoside (MA), as active components used for wound healing and enhancing memory. OBJECTIVE To produce a polyclonal antibody against madecassoside (MA-PAb) and develop enzyme-linked immunosorbent assay (ELISA) and Eastern blotting methods for quantitative analysis of triterpene glycosides in Centella asiatica. METHODS An ELISA method was developed using polyclonal antibody against MA. An Eastern blotting method on the PES membrane was established for determination of MA and AS. The immunoassays were validated for sensitivity, precision, specificity and accuracy. RESULTS The prepared MA-PAb shows specificity to MA and AS. The measuring range of triterpene glycosides was 0.39-50 µg/mL using the ELISA method. An Eastern blotting method was developed for determining individual MA and AS, which could be detected in the range of 62.5-500 ng. The limit of detection for MA and AS was 31.25 ng. The two methods developed showed good specificity, precision, and accuracy, and also correlated with high-performance liquid chromatography. CONCLUSION These immunoassays have several advantages that include high sensitivity as well as being rapid and facile for determination of the triterpene glycosides in C. asiatica.