Pueraria Candollei Research Articles

Enhancement of isoflavonoid production and release in Pueraria candollei cell suspension culture using elicitors for improving pharmacological activities

Enhancement of isoflavonoid production and release in Pueraria candollei cell suspension culture using elicitors for improving pharmacological activities

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

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. This study aims to develop, characterise, and validate ICA with a monoclonal antibody exhibiting similar reactivity against Mi and Dmi (MD-mAb). 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). 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. 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.

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.

Bioimprinting as a Receptor for Detection of Kwakhurin

Bioimprinting was performed against ovalbumin (OVA) to confer its binding cavities for kwakhurin (Kwa), an isoflavonoid, produced solely by Pueraria candollei var. mirifica (P. candollei). The characterization of bioimprinted-OVA (biOVA), evaluated by an enzyme-linked immunosorbent assay (ELISA), revealed that it functioned as a specific receptor for Kwa. Using biOVA, two systems, i.e., an indirect competitive ELISA (icELISA) and the even simpler and more rapid competitive enzyme-linked bioimprinted-protein assay (cELBIA), were developed as novel techniques for the quantitative analysis of Kwa in P. candollei and its related products. The two analysis methods were found to have limits of detection (LOD) of 4.0 and 2.5 µg/mL, respectively. The high reliability of the developed icELISA and cELBIA using biOVA was also demonstrated by various validation analyses. Subsequently, bioimprinting was performed using eight other proteins to investigate them as candidate scaffolds for the generation of binding cavities for Kwa. Interestingly, two bioimprinted-IgG monoclonal antibodies (biMAbs) recognized Kwa, but their original binding affinity to hapten was lost. That is, the MAbs obtained a new binding ability to Kwa in exchange for their original binding affinity, raising the possibility that biMAb could be alternatively used as a probe for the quantitative analysis of Kwa as well as biOVA. This is the first report of small molecules recognition by MAbs used as proteins for bioimprinting.

Development of a DNA barcode library of plants in the Thai Herbal Pharmacopoeia and Monographs for authentication of herbal products

Traditional herbal medicine has long been practiced as a method of health care in many countries worldwide. The usage of herbal products has been increasing and is expected to continue to do so in the future. However, admixture and adulteration are concerns regarding the quality of herbal medicine, including its safety and efficacy. We aimed to develop a reference DNA barcode library of plants listed in the Thai Herbal Pharmacopoeia (THP) and Monographs of Selected Thai Materia Medica (TMM) (n = 101 plant species) using four core barcode regions, namely, the ITS2, matK, rbcL and trnH-psbA intergenic spacer regions, for authentication of the plant origin of raw materials and herbal products. Checking sequences from samples obtained from local markets and the Thai Food and Drug Administration (Thai FDA) against our digital reference DNA barcode system revealed the authenticity of eighteen out of twenty tested samples as claimed on their labels. Two samples, no. 3 and 13, were not Cyanthillium cinereum (L.) H.Rob. and Pueraria candollei Wall. ex Benth. as claimed, respectively. They were recognized as Emilia sonchifolia (L.) DC. and Butea superba (Roxb.), respectively. Hence, it is important for the Thai FDA or regulatory agencies to immediately initiate strict enforcement for the development of pharmacopoeial standards as well as revisions or modifications of available regulatory guidelines and to implement close monitoring for the quality control of herbal products in terms of authentication before they enter the herbal market. The centralized digital reference DNA barcode database developed here could play a very important role in monitoring or checking the authenticity of medicinal plants.

Magnetized water: A way to enhance isoflavonoids in cultured Pueraria candollei var. mirifica cells

Magnetic fields can alter plant growth and development. In this study, the effect of magnetized water on the growth and secondary metabolite production of Pueraria candollei var. mirifica (PM) suspension cells was evaluated. Magnetized water was generated by passing water through a magnetic field (400 mT) at a 6 L/min flow rate. The suspension cells exposed to magnetized water for 10 (M2, 1.37 g) and 5 min (M1, 1.23 g) showed 21% and 9% higher biomass accumulation, respectively, compared with the control cells after 5 days. Compared with that in the control cells, the biomass was increased by approximately 43% when the cells were exposed to M2 (3.01 g) after 28 days. In addition, the use of magnetized water altered the accumulation pattern of isoflavonoids after 5 days of treatment. Isoflavonoid accumulation was the highest after treatment with M2 for 5 days and yielded 4.20 mg/g dry weight (DW) daidzein, 0.58 mg/g DW genistein, and 3.98 mg/g DW kwakhurin. When the cells were maintained in the treatment for a relatively long duration (28 days), a short span of magnetization (M1) resulted in the highest accumulation of isoflavonoids with the amounts of 3.51, 0.55, and 2.58 mg/g DW for daidzein, genistein, and kwakhurin, respectively. After 28 days of exposure to magnetized water, the expression of 2-hydroxyisoflavanone synthase and 2-hydroxyisoflavanone dehydratase increased, and this phenomenon coincided with the increased amount of isoflavonoids in the suspension cells. The magnetized water-treated PM cells also exhibited higher antioxidant activities than the control cells. The lowest half-maximal inhibitory concentration (37.93 mg/mL) was observed in the cells treated with magnetized water after 15 min (M3). The enhanced total antioxidant activity of the cells treated with M3 may be attributed to the presence of other compounds in addition to isoflavonoids. This study represents an innovative attempt at a low-cost and environmentally friendly approach that can enhance the growth, secondary metabolite production, and antioxidant activity of PM suspension cells.

Assessment of 28-day oral exposure to Pueraria candollei var. mirifica (Fabaceae) roots on pituitary-ovarian axis function and selected metabolic parameters in ovary-intact rats

Pueraria candollei var. mirifica (Fabaceae) root (PMR) has recently been developed as a potential selective estrogen receptor modulator (SERM) in menopausal women. Nowadays, many premenopausal women also take dietary PMR supplements, however, the exact biological effects of PMR have not been evaluated. This study included the application of the OECD guideline 407 for the assessment of 28-day oral exposure to PMR on pituitary-ovarian (PO) axis function and metabolic parameters in the premenopausal rat model. Ovary-intact adult rats were orally administrated with 10, 100, 750, 1000, and 1500 mg/kg body weight (BW)/day of PMR powder. The positive estrogenic group was given 2 mg 17β-estradiol (E2)/kg BW/day. Serum levels of reproductive hormones, lipid and thyroid parameters, estrous cycle determination, and histomorphometric and histopathological evaluations of the anterior pituitary, ovary, uterus, vagina, mammary gland, and liver were investigated. PMR displayed neutral effects on uterine, vaginal, and body weights, and circulating E2 and prolactin levels. PMR exerted E2-like effects by i) reducing ovarian and increasing hepatic weights, ii) decreasing serum gonadotropins, iii) lowering serum lipids without altering thyroid parameters, iv) increasing the prevalence of abnormal estrous cycles with prolonged estrus, v) increasing nuclear diameter of anterior pituitary cells, vi) decreasing ovarian size and follicular numbers and increasing follicular degeneration, vii) thickening of uterine myometrium and luminal epithelium, and vaginal epithelium, and viii) induction of mammary alveolar hyperplasia and ductal secretion. Unlike E2, the appearance of very small numbers of focal microvesicular steatosis in hepatocytes demonstrated mild toxicity at high PMR doses. This is the first report that high-dose PMR exerted actions exactly like E2 on gonadotrope-ovarian axis function and histology, lipid, and thyroid parameters without affecting uterine and vaginal growth in ovary-intact rats according to OECD guidelines.

Study on characterization of chalcone synthase gene from \(\textit{Pueraria lobata}\) and \(\textit{Pueraria mirifica}\) in Vietnam

Two species of genus Pueraria ((Pueraria lobata (synonym: Pueraria montana var. lobata) and Pueraria mirifica (synonym: Pueraria candollei var. mirifica)) are traditional plants used in medicine since ancient times. These plants have been used and became commercially crucial indigenous medicinal plants. Currently, both roots and flowers of P. mirifica are used as a dietary supplement and functional food for women because of their rich source of phytoestrogen and nutrition. However, little information of genes on both species of Pueraria genus (P. lobata and P. mirifica) are known in Vietnam. The purpose of this research is to support more understanding about Chalcone synthase (CHS) genes by determining and sequence analyzing an encoding region of CHS genes that were isolated from P. lobata and P. mirifica. The full-length open reading frame (ORF) sequence CHS was identified with 1170 bp which encodes 389 amino acids by Sanger sequencing. The isolated CHS gene of P. lobata has no difference in sequence with CHS reported on GenBank (D10223.1), whereas a difference of 26 nucleotide positions in CHS sequence of P. mirifica compared with the published gene sequence (JQ409456.1) as consequential having 97.78% genetic similarity. The CHS genes sequence of P. lobata and P. mirifica are homologous with 98.4% because of having 19 nucleotide differences. Chalcone-stilbene synthase N-C terminal, PLN03173, CHS-like, BH0617, fabH are some important domains predicting the CHS genes. Especially, the family signature ‘GVLFGFGPGLTI’ motif of CHS gene as a part of the active-site scaffold contributes to decide the product of cyclization reactions performing the stereochemistry of cyclization which was also observed in P. lobata and P. mirifica, but it was not included for all members in Fabaceae family. With in sillico analysis, the P. lobata and P. mirifica CHS sequences have highly conserved regions to maintain their structure and function, so that it needs further studies to clarify these points.

Fragment antigen-binding (Fab) antibody-based lateral flow immunoassay for rapid and sensitive detection of potent phytoestrogen, deoxymiroestrol.

Pueraria candollei is an ingredient of Thai herbal medicine, dietary supplements, and cosmetics. The in vitro and in vivo studies of this plant supported anti-osteoporotic activity and used for hormone replacement therapy. Deoxymiroestrol shows the most potent phytoconstituent in tuberous root of P. candollei with estrogenic activity. The quality controls are important for good agricultural practice (GAP) and good manufacturing practice (GMP) of plant-derived raw materials. The rapid detection of lateral flow immunoassay (LFIA) using colloidal gold is simply method, easy visualize detection and produce less waste than conventional chromatographic detection. In this study, LFIA for qualitative detection of deoxymiroestrol using antigen-binding fragment antibody (Fab) was developed. The result showed that the developed LFIA displays specific detection of deoxymiroestrol. Cross reactivity of this method was analyzed with miroestrol, isomiroestrol and methylisomiroestrol which showed 39.97%, 7.71% and 5.72%, respectively. After optimal condition, limit of detection (LOD) for deoxymiroestrol is 250ng/ml. Plant samples were applied to strip test compare with indirect competitive ELISA using polyclonal antibody to confirm the application of LFIA. The results of LFIA method were comparable with those from ELISA. This developed lateral flow immunoassay can apply to detect deoxymiroestrol for the rapid testing. The developed method can use for quality control in plant samples as deoxymiroestrol is biomarker compound in P. candollei.

Simple preparation and analysis of a phytoestrogen-rich extract of Pueraria candollei var. mirifica and its in vitro estrogenic activity

As a source of phytoestrogens, Pueraria candollei var. mirifica (PM) is popular in phytopharmaceutical product development for estrogen replacement therapy. It is a challenging process to extract a large amount of phytoestrogens from a complex plant mixture and to quantify these compounds for standardization. In this study, the 95 % (v/v) crude ethanol extract of the PM root cortex was reextracted with 0–20 % (v/v) aqueous ethanol. The results from the quantitative analysis showed that 10 % (v/v) ethanol is the lowest solvent ratio that provided a high phytoestrogen content. More than 90 % of the potent phytoestrogens (i.e., deoxymiroestrol and miroestrol) and 80 % of the isoflavonoids (i.e., puerarin, daidzin, daidzein, genistein, and kwakhurin) were distributed into the derived soluble extract. In contrast, the insoluble extract contained a low level of phytoestrogens and was concentrated with less polar unidentified compounds. The extraction efficiency was confirmed by the estrogenic activity of the soluble extract on the proliferation of MCF-7 cells. The results also indicated that deoxymiroestrol mainly contributed to the total proliferative effects of PM, while the antiproliferative or cytotoxic effects occurred from treatment with the insoluble compounds, which were removed by the extraction method. Therefore, this extraction method is helpful to enhance the known phytoestrogen content and simplify the extraction of chemical constituents from PM as an ingredient in healthcare products.

Enzyme-linked immunosorbent assay using fragment antigen-binding (Fab) antibody for determination of deoxymiroestrol, a potent phytoestrogen from Pueraria candollei

ABSTRACTDeoxymiroestrol is the most potent phytoestrogen in the chromene group found in Pueraria candollei. The estrogenic activity of P. candollei, functions as hormone replacement therapy in postmenopausal women. This study established an enzyme-linked immunosorbent assay (ELISA) for quantifying deoxymiroestrol using fragment antigen-binding (Fab) antibody to improve analytical performance. Our ELISA specifically detected deoxymiroestrol in the range of 31.25–1,000 ng/mL. Intra- and inter-assay precision was 1.48%–7.11% and 0.58%–9.31%, respectively, the recovery of authentic deoxymiroestrol spiked into samples was 99.77%–101.61%, and the limit of detection was 30.80 ng/mL. The findings of P. candollei root bark determined using our developed ELISA with Fab antibody and an ELISA with anti-deoxymiroestrol polyclonal antibody were consistent (R2 = 0.9807). The developed ELISA can be applied to the quality control of deoxymiroestrol derived from P. candollei.

Transformation of Pueraria candollei var. mirifica phytoestrogens using immobilized and free β-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 β-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 β-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 β-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 β-glucosidase was the best fit by quadratic models, which had adjusted R2 values between 0.8625 and 0.9318. Immobilized β-glucosidase can be reused up to nine times and maintained efficacy of greater than 90%. Treatment of the PM extract with β-glucosidase increased the estrogenic activity of the PM extract by 8.71- to 23.2-fold compared to that of the untreated extract. Thus, β-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.

Quantification of methylisomiroestrol, a phytoestrogen of Pueraria candollei, by enzyme-linked immunosorbent assay in comparison with high-performance liquid chromatography

Pueraria candollei is a phytoestrogen-rich herb used to treat estrogen deficiency disorders; however, quality control of P. candollei-related health products is required for consistency of clinical outcomes. Estrogenically active (+)-7-O-methylisomiroestrol could be a potential chemical marker that facilitates the prediction of the overall estrogenic activity of P. candollei. The analytical performance of ELISA using newly produced monoclonal antibodies against methylisomiroestrol was compared with HPLC analysis. The developed indirect competitive ELISA (icELISA) was highly sensitive to methylisomiroestrol for detection, with an LOQ of 2.9 ng/mL, whereas the LOQ was 1.15 μg/mL by HPLC. The results from method validation indicated acceptable precision (1.71–6.37 % and 0.13–2.40 %) and accuracy (99.23–102.54 % and 96.84–101.88 %) of the methylisomiroestrol analysis using icELISA and HPLC. These methods were effectively applied for the determination of the methylisomiroestrol content in P. candollei samples. Apart from the plant tubers, the stem was observed as a source of methylisomiroestrol. The developed ELISA was more effective than HPLC in detecting a small quantity of methylisomiroestrol in the plant samples [0.23 × 10−3% (w/w) to 0.628 × 10−3% (w/w) dry weight]. Therefore, the ELISA could be a useful tool for the standardization of P. candollei, which is the crucial step to improve the quality of plant-derived products.

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

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. This research aims to develop a simple analytical method for identification and to differentiate PM from red Kwao Krua and other Pueraria species. 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. 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. 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.

Kwakhurin-magnetic particles conjugates enable fast enzyme immunoassay for the detection of kwakhurin in Pueraria candollei.

Kwakhurin (Kwa) is one of the unique isoflavonoids produced in Pueraria candollei var. mirifica (P. candollei), which has long been used as folk medicine for rejuvenation in Thailand. Recently, the use of P. candollei-derived products has widely spread among Japanese women for cosmetic purposes. Correspondingly, there has been an increase in the number of reports regarding possible health hazards caused by estrogenic activity inherent to the plant; thus, the need for a detailed evaluation of the phytoestrogen content of P. candollei-derived products has gained a sense of urgency in recent years. This study aims to develop a rapid enzyme immunoassay that can be applied to the quantitative analysis of Kwa in P. candollei and its derived products. A rapid and sensitive immunoassay was developed with a combination of Kwa-specific monoclonal antibody (MAb 11F) and Kwa-magnetic particles (MPs) conjugates, which increased the surface area of the solid phase, resulting in a decrease in the immunoreaction time. This novel MPs-based enzyme immunoassay (MPs-EIA) was used to determine Kwa concentration in the range from 2.44 to 78.1 ng/mL with a limit of detection of 1.90 ng/mL. Validation analyses revealed that the proposed MPs-EIA protocol was sufficiently precise and accurate for effective quantitative analysis of Kwa in P. candollei and its derived products.

Comparative analysis of the chemical constituents from the tuberous root and stem of Pueraria candollei var. mirifica and evaluation of their estrogenic activity

Background: Pueraria candollei var.mirifica (PC) is a tuberous plant enriched with bioactive phytoestrogens. PC is currently high demanded in the global markets as dietary supplement products, which resulted in a shortage of tuberous root from the natural resource and the field cultivation. Materials and Methods: We compare phytoestrogens contents from the tuberous root and stem of PC and their estrogenic activities on MCF-7 cells proliferation. Results: The root and stem of PC accumulated different contents of phytoestrogens. The highest amount of total isoflavonoids and total chromenes was found in the root bark of PC. However, the stem bark (10 μg/mL crude extract) which contains a high level of chromenes and low amount of isoflavonoids exhibited higher growth stimulation of MCF-7 cells (127.25% ± 4.43% relative proliferative effect [RPE] as compared to estradiol [10−10 M]) than root bark at the same concentration with 116.32% ± 3.59% RPE. After the solubilization of the ethanol extract with 10% (v/v) aqueous ethanol, the soluble fraction of crude extract of stem stimulated proliferation of MCF-7 cells without cell growth suppression effect at high concentration (100 μg/mL) as observed in the insoluble fraction of the extract. Conclusion: We suggested that the soluble part of crude extract might be less toxic than the ethanolic crude extract and its non-polar components. Our study opens the possibility of using the stem of PC as a new alternative source of bioactive phytoestrogens for dietary supplement products.

(+)-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.

Correction to: Optimizing Pueraria candollei var. mirifica cell suspension culture for prolonged maintenance and decreased variation of isoflavonoid from single cell lines

The Acknowledgements section in the original article was incomplete. The complete section is shown below. Acknowledgements This research was supported by Rachadapisek Sompote Fund for Postdoctoral Fellowship and for New Faculty Member Chulalongkorn University, Bangkok and Thailand Research Fund Project No. MRG5680090. TM was a recipient of the Research assistant scholarship from the Graduate School, Chulalongkorn University.

Pueraria candollei var. mirifica-Induced CYP1A1 and CYP1A2 expression in human choriocarcinoma bewo cells

Background: The human placenta metabolizes many endogenous substances, drugs, and xenobiotics. Cytochrome P450 family 1 (CYP1) is expressed in both early- and full-term placenta. The Thai medicinal plant Pueraria candollei var. mirifica (PM) is traditionally consumed for rejuvenation and has neuroprotective, anti-osteoporotic, and antioxidant activities. Objectives: The objective of this study was to compare the effects of PM and the CYP1A inducer β-naphthoflavone (BNF) on the expression of CYP1, aryl hydrocarbon receptor (AHR), AHR nuclear translocator (ARNT), and the transporter ABCG2. Materials and Methods: Human choriocarcinoma BeWo cells were treated with BNF (10 μM), ethanolic extract of PM (PM-EtOH), or column chromatographic fractions of PM-EtOH (F2, F4, and F6) at 1, 10, and 100 μg/mL for 24 h. The mRNA expression of target genes was determined using real-time quantitative polymerase chain reaction. The activity of ethoxyresorufin-O-deethylase (EROD), a marker for CYP1, was measured at the RNA harvesting time point. Results and Discussion: PM-EtOH, F2, and F4 significantly induced EROD activity and expression of CYP1A1 and CYP1A2 while CYP1B1 and AHR were slightly suppressed and ARNT was unchanged. ABCG2 was slightly induced by F2. Therefore, the expression of CYP1 in BeWo cells appears to be independent of the AHR/ARNT regulatory pathway. Conclusion: The use of PM-containing products at high quantities or for long periods during pregnancy is of concern due to likely herb–drug interactions and toxicological risks through activation of CYP1A1 and CYP1A2 transcription.

Investigation of the estrogenic activity of Pueraria candollei variety mirifica extract on rats

Background: Pueraria candollei variety mirifica (PM) has been widely used as ingredient in many rejuvenating products. In this study, we aimed to assess the estrogenic activity of PM extract grown in Vietnam.Methods: Estrogenic activity of PM extract was estimated on immature female rats by using uterotrophic method to measure the weight of the reproductive organs. Estrogenic activity of PM extract also was investigated in mature female ovariectomized rats by evaluating the vaginal cells growth, reproductive organs weight, serum estradiol concentration.Results: Our results showed that PM extract at doses of 100 mg/kg, 200 mg/kg had increased the reproductive organs weight in immature rats and female ovariectomized rats. In addition, PM extract had increased the serum estradiol concentration and the vaginal cells growth by increasing the percentage of keratinocytes in female ovariectomized rats.Conclusions: Our results showed that PM extract has strong estrogenic activity in rats.