The Deoxymiroestrol and Isoflavonoid Production and Their Elicitation of Cell Suspension Cultures of Pueraria candollei var. mirifica: from Shake Flask to Bioreactor.

To address the high demand for Pueraria candollei var. mirifica (PM) used as the active ingredient in health products and its difficulty to cultivate in the field, the growth and production of deoxymiroestrol (DME) and isoflavonoid (ISF) phytoestrogens in PM cell suspensions were studied. In a 125-mL shake flask, the cell suspension produced DME [78.7 ± 8.79-116 ± 18.2 µg/g dry weight (DW)] and ISF (140 ± 6.83-548 ± 18.5 µg/g DW), which are the predominant ISF glycosides. While ISF aglycones accumulated in the PM cell suspension cultured in the airlift bioreactor. The DME content was increased to 976 ± 79.6 µg/g DW when the PM cell suspension was cultured in the 5-L scale bioreactor. The production of DME and ISF was enhanced by elicitors including methyl jasmonate (MJ), yeast extract (YE), and chitosan (CHI). MJ produced the highest induction of DME accumulation, while ISF accumulation was the highest with YE treatment. Analysis of catalase activity implied that the elicitors enhanced ROS production, which resulted in the enhancement of DME and ISF production and accumulation in PM cell suspension cultures. PM cell suspension culture is a promising source of beneficial PM phytoestrogens that exhibit bioactivity that may useful for the treatment of menopausal symptoms.

Development of a colloidal gold nanoparticle-based immunochromatographic strip for the one-step detection of miroestrol and puerarin.

Pueraria candollei or White Kwao Krua (Leguminosae) is an indigenous plant in Thailand which has long been used in Thai traditional medicine. The tuberous root of this plant is widely used for rejuvenation, particularly in elder women. Among the bioactive compounds in P. candollei, miroestrol and puerarin exhibit estrogenic activity. This study aims to develop an immunochromatographic strip (ICS) with a colloidal gold-based detection system for the simultaneous detection of miroestrol and puerarin in a one-step analysis. The developed method is sensitive and specific for the detection of miroestrol and puerarin in raw materials and marketed products. The detection limits of miroestrol and puerarin were 0.15 and 4.5 µg, respectively. In addition, the results from the developed ICS were confirmed with an enzyme-linked immunosorbent assay and presented a good correlation between these two methods. This is the first report on the development of an ICS that can detect miroestrol and puerarin in one step. The developed ICS provides a simplified method for the detection of miroestrol and puerarin in P. candollei and Pueraria spp.

A New Highly Selective and Specific Anti-puerarin polyclonal Antibody for Determination of Puerarin Using a Mannich Reaction Hapten Conjugate.

BACKGROUND: Puerarin (PUE) is a phytoestrogen found in Pueraria candollei and Pueraria lobata. These plants are substantial for traditional medicine in various Asian countries. PUE is a key marker that can be found only in the Pueraria species. OBJECTIVE: To establish the method for determination of PUE content which is required for quality control of pharmaceutical products. MATERIALS AND METHODS: PUE-cationized bovine serum albumin conjugate was created via Mannich reaction. After the rabbit immunization, the obtain anti-PUE polyclonal antibody (PAb) was used to develop an enzyme-linked immunosorbent assay (ELISA). RESULTS: An anti-PUE PAb possess a great sensitivity and specificity. The cross-reactivity analysis shows no cross-reaction of an established antibody against other substances. In addition, we successfully developed an indirect competitive ELISA (icELISA) for the quantitative analysis of PUE. The result of method validation conforms to acceptance criteria and correlates with high-performance liquid chromatography, the reference method. The icELISA was applied to determine PUE content in Pueraria spp. plant samples and its derived pharmaceutical products. CONCLUSION: This highly specific immunogen was created from the Mannich reaction. An icELISA can also be applied to other research propose in the further studies. SUMMARY: The new immunogen conjugated (puerarin-cBSA) via Mannich reaction was successfully in rising of antibody against puerarin (PUE)The obtained anti-PUE polyclonal antibody (PAb) was high sensitivity and specificity to PUEAn indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed and validated using anti-PUE PAbThe established icELISA was applied to determine PUE content in various tuberous root of Pueraria sppMoreover, icELISA method can be applicable in Pueraria spp. derived products. Abbreviations used: PUE: Puerarin; PAb: Polyclonal antibody; ELISA: Enzyme-linked immunosorbent assay; icELISA: Indirect competitive ELISA; cBSA: Cationized bovine serum albumin.

A pilot pharmacokinetic study of miroestrol and deoxymiroestrol on rabbit sera using polyclonal antibody-based icELISA analysis.

Miroestrol (ME) and deoxymiroestrol (DME) are the most potent phytoestrogens and bioactive markers in Pueraria candollei var. mirifica tuberous roots. To understand their pharmacokinetic profiles, a pharmacokinetic study of ME and DME, at 0.43 and 0.21 mg per kg body weight, respectively, in three rabbits was performed after orally administering a single dose of P. candollei var. mirifica enriched fraction extract. Two established polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assays were validated to determine ME and DME in rabbit sera. In rabbits, the area under the 0- to 48-hr concentration-time curve of ME and DME were 854.92 and 1,692.84 ng·h/ml, respectively. The maximum concentration of ME was measured 1 hr after administration as 69.62 ± 8.28 ng/ml, and the maximum concentration of DME was measured at 3 hr as 81.8 ± 5.43 ng/ml. These results provide an initial approach for designing and studying the relationship between the ME and DME levels and their therapeutic effects based on their pharmacokinetic profiles.

Development of an enzyme-linked immunosorbent assay for the detection of isomiroestrol, an identical marker, in White Kwao Krua using a monoclonal antibody.

Pueraria candollei var. mirifica or White Kwao Krua (WKK) is a phytoestrogen-rich plant widely used among women to improve climacteric symptoms. Additionally, the tuberous roots of this plant have been added as an active ingredient for skin rejuvenation and breast enlargement effects in various functional foods. However, most of the products on the market containing WKK have not been sufficiently standardized with respect to the active compound or identical marker. To control the quality of these plant materials, an enzyme-linked immunosorbent assay (ELISA) using anti-isomiroestrol antibodies was established for the determination of isomiroestrol, an identical marker in WKK. Polyclonal and monoclonal antibodies against isomiroestrol were generated and their specificity characterized in this study. Monoclonal antibody 12C1 showed higher specificity to isomiroestrol and was thus selected to develop the ELISA. Based on the validation analysis and the tested performance of the developed ELISA in variably sourced WKK samples, the assay can provide an alternative approach that is reliable and highly sensitive for the quantitative analysis of isomiroestrol in plant.

Development of an Enzyme-Linked Immunosorbent Assay for Determination Of Miroestrol Using an Anti-miroestrol Monoclonal Antibody.

Miroestrol is a chromene with potent estrogenic activity present in Pueraria candollei, commonly known as White Kwao Krua. Although this compound is only present in low amounts in the plant, it plays an important role in the estrogenic action of P. candollei products. As a tool for further studies about the efficacy and safety of P. candollei as a phytoestrogenic supplement, we generated a novel monoclonal antibody against miroestrol. This anti-miroestrol monoclonal antibody was used to develop an immunoassay for the determination of miroestrol content, which can be used for quality control purposes of P. candollei. The developed ELISA against miroestrol has a calibration range of 10-780 ng/mL miroestrol, a limit of detection of 3.5 ng/mL, and a limit of quantitation of 12.2 ng/mL. According to the validation analysis, the established ELISA is precise, accurate, specific, and sensitive for miroestrol detection in plants. Furthermore, the anti-miroestrol monoclonal antibody was used to prepare an immunoaffinity column for the isolation of miroestrol from the tuberous root of P. candollei. The column provides a simple procedure for miroestrol isolation, with a capacity of 3.91 µg of miroestrol per 1 mL of immunogel.

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.

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.

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.

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.

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.

Enhanced plumbagin production from in vitro cultures of Drosera burmanii using elicitation.

Methyl jasmonate, 50 microM, 0.5 mg yeast extract/l and 100 mg chitosan/l stimulated plumbagin production in Drosera burmanii whole plant cultures after 6 days of elicitation. Yeast extract (0.5 mg/l) was the most efficient enhancing plumbagin production in roots of D. burmanii to 8.8 +/- 0.5 mg/g dry wt that was 3.5-fold higher than control plants.