Gorawit Yusakul

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.

Simultaneous determination of soy isoflavone glycosides, daidzin and genistin by monoclonal antibody-based highly sensitive indirect competitive enzyme-linked immunosorbent assay

Soy isoflavones are known as major bioactive compounds in soybean (Glycine max), which is an indispensable food. Despite their utility, the consumption of isoflavones has recently been limited because they exhibit oestrogenic and topoisomerase II inhibitory effects. To assess their intake limitation, accurate, sensitive, and effective quantitative analyses are necessary. In this study, we produced the monoclonal antibody (MAb) against daidzin (DZ) and applied it to an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the simultaneous determination of DZ and genistin (GEN), which are known as two major soy isoflavone glycosides in soy products. Using the DZ-MAb, we developed a sensitive icELISA method, where the limit of detection for DZ and GEN was 1.95ng/ml. Several validation analyses revealed that the icELISA is sufficiently accurate and sensitive to be used to assess the overconsumption of soy isoflavones, which would lead to the safe dietary intake of soy products.

Colloidal gold-based indirect competitive immunochromatographic assay for rapid detection of bioactive isoflavone glycosides daidzin and genistin in soy products

Daidzin (DZ) and genistin (GEN) are two major soy isoflavone glycosides isolated from soybeans. Soy products containing isoflavones have recently been widely accepted for commercial use. However, the Japanese Government has suggested that soy isoflavone intake should be limited because of their estrogenic effects due to their interactions with estrogen receptors. In this study, we established a one-step indirect competitive immunochromatographic assay (ICA) for rapid and sensitive detection of total isoflavone glycosides (DZ and GEN) using gold nanoparticles conjugated with a monoclonal antibody against DZ. This assay was able to be completed in 15min following the immersion of a test strip in an analyte solution. Furthermore, the limit of detection for the total amount of isoflavone glycosides was ∼125ngmL(-1). Considering that the major soy isoflavone glycosides found in soy products are DZ and GEN, this study demonstrates the potential use of ICA for the assessments of over consumption of isoflavones in soy supplements and foods, which would increase the safe dietary intake of soy products.

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.

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.

Anti-miroestrol polyclonal antibodies: A comparison of immunogen preparations used to obtain desired antibody properties

Immunogen quality is one important factor that contributes to desirable antibody characteristics. Highly specific antibodies against miroestrol can be used to develop a quality control immunoassay for Pueraria candollei products. In this study, we investigated how various immunogen preparations affect antibody properties. The results show that immunogen prepared using the Mannich reaction provides antibodies with higher specificity and sensitivity against miroestrol than immunogen prepared with the periodate reaction. The results suggest the Mannich reaction maintains the original structure of miroestrol and generates useful antibodies for developing immunoassays.

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.

Determination of iriflophenone 3-c-β-d-glucoside from aquilaria spp. by an indirect competitive enzyme-linked immunosorbent assay using a specific polyclonal antibody

Polyclonal antibody against iriflophenone 3-C-β-d-glucoside (IP3G), a major compound from the leaves of Aquilaria spp., was produced for the development of an enzyme-linked immunosorbent assay (ELISA). The results showed that the antibodies were specific for IP3G. The produced antibody has low cross reactivity with iriflophenone 3,5-C-β-d-diglucopyranoside (13%), genkwanin 5-O-β-primeveroside (3.55%) and no cross reactivity found in other compounds. The range of ELISA assay extends from 100 to 1560 ng/mL with coefficient of variation (CV) 1.19% to 2.07% for intra-assay and 3.76% to 7.15% for inter-assay precision levels. The recovery rates of IP3G in the leaves of Aquilaria spp. were in the range of 96.0% to 99.0% with CV 4.50% to 5.32%. A correlation between ELISA and high-performance liquid chromatography methods was obtained when analysis of IP3G in the plant samples (R(2) = 0.9321). These results suggest that the developed ELISA method can be applied to determine IP3G content with high specificity, rapidity, and simplicity. The developed immunosorbent assay in this study provides a useful tool for the analysis of IP3G in plant samples and products.