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.

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.

Effects of Pueraria candollei var mirifica (Airy Shaw and Suvat.) Niyomdham on Ovariectomy-Induced Cognitive Impairment and Oxidative Stress in the Mouse Brain

The effects of the phytoestrogen-enriched plant Pueraria mirifica (PM) extract on ovari-ectomy (OVX)-induced cognitive impairment and hippocampal oxidative stress in mice were investigated. Daily treatment with PM and 17β-estradiol (E2) significantly elevated cognitive behavior as evaluated by using the Y maze test, the novel object recognition test (NORT), and the Morris water maze test (MWM), attenuated atrophic changes in the uterus and decreased serum 17β-estradiol levels. The treatments significantly ameliorated ovariectomy-induced oxidative stress in the hippocampus and serum by a decrease in malondialdehyde (MDA), an enhancement of superoxide dismutase, and catalase activity, including significantly down-regulated expression of IL-1β, IL-6 and TNF-α proinflammatory cytokines, while up-regulating expression of PI3K. The present results suggest that PM extract suppresses oxidative brain damage and dysfunctions in the hippocampal antioxidant system, including the neuroinflammatory system in OVX animals, thereby preventing OVX-induced cognitive impairment. The present results indicate that PM exerts beneficial effects on cognitive deficits for which menopause/ovariectomy have been implicated as risk factors.

Detection of puerarin from Pueraria mirifica tuber and its formulated cosmetic products

Pueraria mirifica (PM) has traditionally been used to relief postmenopausal symptoms. Recently, its extract has been developed into various cosmetic products to promote skin rejuvenation and youthfulness. This study investigated the phytochemicals of PM tuber and compared between the tuber flesh and its outer peel. Puerarin which is one of the major isoflavones and being considered as the marker compound was used to determine the presence of PM extract in local cosmetic products. Puerarin could be ionized by a mass spectrometer at both negative and positive modes. The peak ionized at the negative mode showed to have a narrower peak width (0.2 min) and higher signal-to-noise ratio (30) for pueararin (1 mg/L). The results also found PM extract contained many C- and O-glycosylated isoflavones, especially from its peel extract. This explains the peel extract showed to have four times higher antiradical activity than those of flesh extract. Puerarin from the cosmetic products was recovered via successive methanolic sonication and followed by liquid-liquid extraction using ethyl acetate. Puerarin was successfully partitioned from the highly complex chemical mixture of cosmetic products with the recovery ranged from 89.1 % to 115 %. Hence, isoflavones was found to be higher at the outer peels than its tuber flesh. A simple and reliable method has been developed to analyse the presence of PM extract in cosmetic products based on the detection of puerarin after successive extraction via methanolic sonication and ethyl acetate partition.

Comparative Pharmacokinetics of Puerarin Alone and in Pueraria mirifica Extract in Female Cynomolgus Monkeys

Pueraria mirifica is an endemic Thai plant that has been used for rejuvenation and in the relief of various aging diseases. Puerarin is one of the major isoflavones found in this plant and shows several pharmacological activities in relation to the Thai traditional use of P. mirifica. Therefore, comparative pharmacokinetics of pure puerarin alone and that in a P. mirifica extract in cynomolgus monkeys were conducted in order to investigate the pharmacokinetic profiles of the 2 preparations. To this end, puerarin and P. mirifica extract, at an equivalent dose of 10 mg/kg of puerarin, were orally dosed to adult female monkeys for 7 consecutive days. A single intravenous injection of puerarin at a dose of 1 mg/kg was also peformed. Serial blood samples and excreta were collected from 0 – 24 h and 0 – 48 h after dosing. Determination of the puerarin levels and its metabolites in biological samples was conducted by liquid chromatography tandem mass spectrometry. Plasma levels of aspartate aminotransferase, alanine aminotransferase, and creatinine fluctuated in the normal range, with no abnormal physical signs in the animal. The absolute oral bioavailability of puerarin was approximately 1% in both preparations. Accumulation of puerarin was found after oral dosing for 7 consecutive days in both groups. Major metabolites of puerarin found in monkeys were hydroxylation and deglycosylation products. A negligible amount of unchanged puerarin was detected in urine and feces. Pharmacokinetic profiles obtained from this study could help to design the prescribed remedy of puerarin and P. mirifica extract phytopharmaceutical products for human use.