Immunochromatographic assay for miroestrol and deoxymiroestrol, its cross-reactivity, and application in Pueraria mirifica (white Kwao Krua) analysis.

INTRODUCTION: Miroestrol (Mi) and deoxymiroestrol (Dmi) are trace, yet potent, phytooestrogens found in white Kwao Krua [Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham, PM]. However, the analysis of these substances is difficult because of complex matrix effects and their various analogues. In addition, alteration in the cross-reactivity of a gold nanoparticle (AuNP)-based immunochromatographic assay (ICA) resulting from the electrostatic adsorption between antibodies and AuNPs has not yet been evaluated. OBJECTIVES: This study aims to develop, characterise, and validate ICA with a monoclonal antibody exhibiting similar reactivity against Mi and Dmi (MD-mAb). MATERIALS AND METHODS: The ICA performance was validated for cross-reactivity and performance in comparison with those of indirect competitive enzyme-linked immunosorbent assays (icELISAs) with MD-mAb and mAb exhibiting specificity against Mi (Mi-mAb). RESULTS: The ICA showed a limit of detection (LOD) at 1 and 16 µg/mL for Mi and Dmi, respectively. The cross-reactivity of the ICA with Dmi was lower (6.25%) than that observed with the icELISA (120%). Cross-reactivity of ICA against other compounds of the PM was also correlated with those of icELISA; no false-positive/negative results were observed. The repeatability and reproducibility of the ICA were confirmed. The results obtained using ICA in samples of PM are correlated with the concentrations determined through icELISAs. CONCLUSION: An ICA with MD-mAb was constructed and validated. However, direct conjugation via the electrostatic adsorption of mAb-AuNPs was expected to alter the cross-reactivity of ICA, especially that of the analyte analogue Dmi.

Immunochromatographic assay for the detection of kwakhurin and its application for the identification of Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham.

INTRODUCTION: The plant Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham (PM), known by its common Thai name as white Kwao Krua, is sometimes misidentified because it presents similar botanical characteristics to those of Butea superba (red Kwao Krua). The phytochemicals in PM are phytoestrogens in the class of isoflavonoids, but Butea superba contains flavonoids that exhibit androgenic and antiestrogen effects. OBJECTIVES: This research aims to develop a simple analytical method for identification and to differentiate PM from red Kwao Krua and other Pueraria species. METHODS: A gold nanoparticle-based immunochromatographic assay (ICA) was developed for the detection of kwakhurin (Kwa), a unique compound found in PM. The parameters, including sensitivity, accuracy, precision, and specificity, were validated. All samples were analyzed using ICA and high-performance liquid chromatography with UV detector (HPLC-UV). The results of the two methods were compared for consistency checking. RESULTS: The cutoff limit of Kwa detection was 160 ng/mL, which was lower than in the HPLC-UV method. The repeatability and reproducibility of the ICA preparation and assembly showed high precision. The cross-reactivity to related isoflavonoids was less than 0.32%, which implied high specificity of the ICA for Kwa. Moreover, false-positive and false-negative results from other plant extracts were not observed. CONCLUSION: The developed ICA is applicable for distinguishing PM from red Kwao Krua and other Pueraria species. This simple analytical method can be applied for the identification of raw PM materials in the industrial and agricultural sectors.

Development of a colorless Centella asiatica (L.) Urb. extract using a natural deep eutectic solvent (NADES) and microwave-assisted extraction (MAE) optimized by response surface methodology.

This study outlines a green process for Centella asiatica (L.) Urb. (CA) extraction. Natural deep eutectic solvents (NADESs) and microwave-assisted extraction (MAE) were combined to provide a high bioactive compound yield and high antioxidant activity. Among the NADESs evaluated, the combination of acetylcholine chloride : malic acid : water (1 : 2 : 2): water (40 : 60) was the best for extraction. These conditions provide high madecassoside (MS) (21.7 mg g-1 dry weight) and asiaticoside (AS) (12.7 mg g-1 dry weight) yields, with greater than 80% (v/v) EtOH (13.3 mg g-1 MS and 7.80 mg g-1 AS). In addition, the extracts from this process showed higher antioxidant activity (IC50 = 0.26 mg mL-1) than the CA aqueous EtOH and water extracts. Moreover, the color of the extract products was less green than that of the extracts prepared using EtOH and aqueous EtOH as solvents, which are suitable for cosmeceutical products. Response surface methodology (RSM) was used for MAE optimization. The ANOVA data from the central composition design (CCD) of RSM were fitted with quadratic models yielding acceptable R 2 (>0.93), adjusted R 2 (>0.87), predicted R 2 (>0.81), and nonsignificant lack of fit (p > 0.05) values. The quadratic model was validated using optimal conditions (30 s, power 300 W, and a liquid to solid ratio 20 mL g-1), and the model validation showed more than 80% accuracy in both MS and AS yields. This research presented an effective green process for CA extraction, which resulted in an environmentally friendly CA extract requiring little energy consumption and no organic solvents.

(+)-7-O-Methylisomiroestrol, a new chromene phytoestrogen from the Pueraria candollei var. mirifica root.

(+)-7-O-Methylisomiroestrol (MeI), a novel chromene, was discovered as a phytoestrogen in the Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham (PM) root having been used as an active agent against oestrogen depletion disorders. The identification of PM phytochemicals is crucial for the development of standardised botanical drugs of PM. MeI was purified from the root cortex of PM, and its structure was elucidated using NMR and mass spectrometry. The content of MeI in the root bark of the PM root was 2.1-6.5 × 10-3% (w/w). The oestrogenic potency of MeI was stronger than that of isomiroestrol but less than that of deoxymiroestrol and miroestrol. Therefore, MeI is a new oestrogenic biomarker for the effective chemical standardisation of the PM extract for health product development.

Transformation of Pueraria candollei var. mirifica phytoestrogens using immobilized and free ß-glucosidase, a technique for enhancing estrogenic activity.

Pueraria candollei var. mirifica (PM) has a significant beneficial effect on postmenopausal symptoms associated with estrogen deficiency. However, the estrogenic activity and intestinal absorption of isoflavonoid glycosides derived from PM, such as daidzin and genistin, are significantly lower than those of their aglycones. To enhance the estrogenic activity of the PM extract, we developed ß-glucosidase and its immobilized form to increase the PM aglycone content (daidzein and genistein). The enzyme immobilization was done by alginate beads, and the resulting ß-glucosidase alginate beads have a diameter of about 0.20 cm. Response surface methodology (RSM) was used to optimize certain parameters, such as the pH, temperature, and ethanol concentration. The optimal conditions of ß-glucosidase for daidzein and genistein production were pH of 4.8-4.9, a temperature in the range 46.3-49.1 °C, and ethanol concentration of 10.0-11.0%. The ANOVA results indicated that the design experiment involving free and immobilized ß-glucosidase was the best fit by quadratic models, which had adjusted R 2 values between 0.8625 and 0.9318. Immobilized ß-glucosidase can be reused up to nine times and maintained efficacy of greater than 90%. Treatment of the PM extract with ß-glucosidase increased the estrogenic activity of the PM extract by 8.71- to 23.2-fold compared to that of the untreated extract. Thus, ß-glucosidase has a high potential for enhancing the estrogenic activity of PM constituents, and it can be applied on an industrial scale to increase the utility of these natural products.

Biocompatible natural deep eutectic solvent-based extraction and cellulolytic enzyme-mediated transformation of Pueraria mirifica isoflavones: a sustainable approach for increasing health-bioactive constituents.

The presence of specific gut microflora limits the biotransformation of Pueraria mirifica isoflavone (PMI) glycosides into absorbable aglycones, thus limiting their health benefits. Cellulolytic enzyme-assisted extraction (CAE) potentially solves this issue; however, solvent extraction requires recovery of the hydrophobic products. Here, we established the simultaneous transformation and extraction of PMIs using cellulolytic enzymes and natural deep eutectic solvents (NADESs). The NADES compositions were optimized to allow the use of NADESs as CAE media, and the extraction parameters were optimized using response surface methodology (RSM). The optimal conditions were 14.7% (v/v) choline chloride:propylene glycol (1:2 mol ratio, ChCl:PG) at 56.1 °C for the cellulolytic enzyme (262 mU/mL) reaction in which daidzin and genistin were extracted and wholly transformed to their aglycones daidzein and genistein. The extraction of PMIs using ChCl:PG is more efficient than that using conventional solvents; additionally, biocompatible ChCl:PG enhances cellulolytic enzyme activity, catalyzing the transformation of PMIs into compounds with higher estrogenicity and absorbability.