Daidzein Hydroxylation by CYP81E63 Is Involved in the Biosynthesis of Miroestrol in Pueraria mirifica.

White Kwao Krua (Pueraria candollei var. mirifica), a Thai medicinal plant, is a rich source of phytoestrogens, especially isoflavonoids and chromenes. These phytoestrogens are well known; however, their biosynthetic genes remain largely uncharacterized. Cytochrome P450 (P450) is a large protein family that plays a crucial role in the biosynthesis of various compounds in plants, including phytoestrogens. Thus, we focused on P450s involved in the isoflavone hydroxylation that potentially participates in the biosynthesis of miroestrol. Three candidate P450s were isolated from the transcriptome libraries by considering the phylogenetic and expression data of each tissue of P. mirifica. The candidate P450s were functionally characterized both in vitro and in planta. Accordingly, the yeast microsome harboring PmCYP81E63 regiospecifically exhibited either 2' or 3' daidzein hydroxylation and genistein hydroxylation. Based on in silico calculation, PmCYP81E63 had higher binding energy with daidzein than with genistein, which supported the in vitro result of the isoflavone specificity. To confirm in planta function, the candidate P450s were then transiently co-expressed with isoflavone-related genes in Nicotiana benthamiana. Despite no daidzein in the infiltrated N. benthamiana leaves, genistein and hydroxygenistein biosynthesis were detectable by liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Additionally, we demonstrated that PmCYP81E63 interacted with several enzymes related to isoflavone biosynthesis using bimolecular fluorescence complementation studies and a yeast two-hybrid analysis, suggesting a scheme of metabolon formation in the pathway. Our findings provide compelling evidence regarding the involvement of PmCYP81E63 in the early step of the proposed miroestrol biosynthesis in P. mirifica.

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.

Transcriptome analysis of Pueraria candollei var. mirifica for gene discovery in the biosyntheses of isoflavones and miroestrol.

BACKGROUND: Pueraria candollei var. mirifica, a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown. RESULTS: Miroestrol biosynthesis was reconsidered and the most plausible mechanism starting from the isoflavonoid daidzein was proposed. A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers. A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidates involved in these biosynthetic pathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid and miroestrol profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica. To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor (AtMYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins led to the production of the isoflavone genistein. CONCLUSIONS: Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for identifying biosynthetic genes and transcription factors possibly involved in the isoflavonoid and miroestrol biosyntheses in P. candollei var. mirifica.

Development of a simple and rapid method for the detection of isomiroestrol in Pueraria candollei by an immunochromatographic strip test.

Pueraria candollei (P. candollei) is a traditional Thai herb widely used for estrogen replacement therapy because it contains many unique chromenes that possess potent estrogenic activity, one of which is known as isomiroestrol. Since isomiroestrol is a promising compound that is solely present in P. candollei, it can be used as an identifying marker for standardization of P. candollei. Here, we developed a lateral-flow immunochromatographic strip (ICS) test using a colloidal gold nanoparticle-conjugated anti-isomiroestrol monoclonal antibody (12C1-mAb) for the detection of isomiroestrol in plant samples and products of P. candollei. The advantages of the developed ICS over an enzyme-linked immunosorbent assay are its simplicity and rapidity, as the ICS test can be completed 15 min after dipping the strip into the analyte solution. The detectable concentration of isomiroestrol was 7.0 µg/mL. Considering the demand for the standardization of P. candollei due to concerns regarding its quality, our ICS test using isomiroestrol as an identifying marker would be effective and useful to assess the presence of isomiroestrol.

A lateral flow colloidal gold-based immunoassay for rapid detection of miroestrol in samples of White Kwao Krua, a phytoestrogen-rich plant.

White Kwao Krua (WKK)-derived products have been used worldwide as dietary supplements to relieve climacteric symptoms in menopausal women. Miroestrol is a unique chromene found in WKK tuberous roots that corresponds to the estrogenic activity of WKK. However, miroestrol naturally accumulates at low levels in WKK samples, which are difficult to detect. The development of a rapid and sensitive assay to detect miroestrol in numerous products derived from this plant would be a practical and useful method to guarantee the quality of raw materials. To allow rapid and easy qualitative detection of miroestrol, a lateral flow immunoassay (LFIA) using a colloidal gold-labeled monoclonal antibody (mAb) against miroestrol was developed. The qualitative LFIA was based on the competition of free miroestrol in the sample and immobilized miroestrol-conjugated proteins on the strip for a limited number of antibodies in the detection reagent. Anti-miroestrol mAb was colored by colloidal gold labels and used as the detection reagent in LFIA. Anti-mouse immunoglobulin G was used to indicate the functioning of the LFIA system. The detection limit of the LFIA was 0.156 µg of miroestrol. The LFIA was applied to determine the miroestrol content in WKK samples and products. The result was compared with the validated enzyme-linked immunosorbent assay (ELISA) and demonstrated a correlative outcome. This study shows that the developed LFIA is practical and suitable for detecting small amounts of miroestrol in WKK samples. This qualitative assay is more rapid in screening miroestrol in WKK samples (within 10 min) than conventional methods (ELISA and HPLC).

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.