De Novo Assembly and Comparative Analysis of Mitochondrial Genomes of Two Pueraria montana Varieties.

Pueraria montana is a species with important medicinal value and a complex genetic background. In this study, we sequenced and assembled the mitochondrial (mt) genomes of two varieties of P. montana. The mt genome lengths of P. montana var. thomsonii and P. montana var. montana were 457,390 bp and 456,731 bp, respectively. Both P. montana mitogenomes showed a multi-branched structure consisting of two circular molecules, with 56 genes annotated, comprising 33 protein-coding genes, 18 tRNA genes (trnC-GCA and trnM-CAU are multi-copy genes), and 3 rRNA genes. Then, 207 pairs of long repeats and 96 simple sequence repeats (SSRs) were detected in the mt genomes of P. montana, and 484 potential RNA-editing sites were found across the 33 mitochondrial protein-coding genes of each variety. Additionally, a syntenic sequence analysis showed a high collinearity between the two mt genomes. This work is the first to analyze the mt genomes of P. montana. It can provide information that can be used to analyze the structure of mt genomes of higher plants and provide a foundation for future comparative genomic studies and evolutionary biology research in related species.

Identification of Pueraria spp. through DNA barcoding and comparative transcriptomics.

BACKGROUND: Kudzu is a term used generically to describe members of the genus Pueraria. Kudzu roots have been used for centuries in traditional Chinese medicine in view of their high levels of beneficial isoflavones including the unique 8-C-glycoside of daidzein, puerarin. In the US, kudzu is seen as a noxious weed causing ecological and economic damage. However, not all kudzu species make puerarin or are equally invasive. Kudzu remains difficult to identify due to its diverse morphology and inconsistent nomenclature. RESULTS: We have generated sequences for the internal transcribed spacer 2 (ITS2) and maturase K (matK) regions of Pueraria montana lobata, P. montana montana, and P. phaseoloides, and identified two accessions previously used for differential analysis of puerarin biosynthesis as P. lobata and P. phaseoloides. Additionally, we have generated root transcriptomes for the puerarin-producing P. m. lobata and the non-puerarin producing P. phaseoloides. Within the transcriptomes, microsatellites were identified to aid in species identification as well as population diversity. CONCLUSIONS: The barcode sequences generated will aid in fast and efficient identification of the three kudzu species. Additionally, the microsatellites identified from the transcriptomes will aid in genetic analysis. The root transcriptomes also provide a molecular toolkit for comparative gene expression analysis towards elucidation of the biosynthesis of kudzu phytochemicals.

Electrochemical Profile Recording for Pueraria Variety Identification.

The rapid identification of plant variety is valuable in both academic studies and crop production. However, rapid and accurate identification has been difficult because many varieties have very similar morphological characteristics and are susceptible to the effects of the growing environment. In this work, we established an electrochemical method for recording the electro-active profile of compounds in plant tissue. Because the chemical composition of different varieties is largely controlled by their genes, rather than a growing environment, this method has considerable potential for variety identification. Three varieties of Pueraria with sixteen locations were collected for confirming the feasibility of the proposed methodology. Principal component analysis and peak ratio analysis have been used for grouping the sample data. The results indicate the electrochemical profiles of three varieties can be distinguished using their voltammetric data.

Evaluation of white Kwao Krua (Pueraria candollei Grah. ex Benth.) products sold in Thailand by molecular, chemical, and microscopic analyses.

The tuberous roots of Pueraria candollei Grah. ex Benth. (Fabaceae), commonly known as white Kwao Krua, are used to relieve menopausal symptoms in Thai traditional medicine because they contain phytoestrogens. Black and red Kwao Krua crude drugs exist as well, but they have different botanical origins and pharmacological activities. There is a high demand for white Kwao Krua products, but because of the limited availability of the plant material, it is suspected that the adulteration and misidentification of white Kwao Krua crude drugs and products occur. In this study, we authenticated white Kwao Krua products collected from Thai herbal markets by molecular, chemical, and microscopic analyses. The nucleotide sequences in the internal transcribed spacer (ITS) and trnH-psbA regions of 23 samples of authentic P. candollei were analyzed, and both regions were found to have intraspecific DNA polymorphisms. Based on the single nucleotide polymorphisms in the ITS1 region, species-specific primer sets of P. candollei were designed to authenticate white Kwao Krua and differentiate it from red and black Kwao Krua. Only the PCR products of KWP02 were not amplified by the primer sets. Isoflavonoid contents and microscopic features were used to support the results of molecular analysis to clarify the botanical origin of white Kwao Krua. Molecular, chemical and microscopic methods confirmed that all the Thai Kwao Krua products examined in this study contained authentic "white Kwao Krua" as claimed on their labels.

Efficacy and safety of Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham for menopausal women: A systematic review of clinical trials and the way forward.

ETHNOPHARMACOLOGICAL RELEVANCE: Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham (commonly termed P. mirifica, PM) growing in upland Thailand has a long history as a postmenopausal rejuvenant therapy for indigenants. Its amelioration of menopause symptoms in clinical trials was assessed. MATERIALS AND METHODS: International and Thai databases were searched from inception to February 2017. Clinical trials investigating effects of PM menopausal or postmenopausal women were included. Outcomes were self-reported menopausal symptoms, serum reproductive hormones, urino-genital tract function, and bone surrogates. Methodological quality was assessed by Cochrane risk-of-bias v2.0, and a 22-parameter quality score based on the CONSORT checklist for herbal medicines. RESULTS: Eight studies (9 articles) used data from 309 menopausal patients. Five-studies demonstrated that PM was associated with climacteric scores reduced by ~50% compared to baseline. Other PM studies using limited numbers of placebo participants suggested improved vaginal and other urogenital tract symptoms. Bone alkaline phosphatase halved (suggesting lowered bone turnover). Variable serum reproductive hormone levels suggested menopausal status differed between studies. PM active ingredients and sources were not defined. Adverse event rates (mastodynia, vaginal spotting, dizziness) were similar in all groups (PM, conjugated equine estrogen, and placebos) but serum C-reactive protein doubled. These studies had design and reporting deficiencies, high risks of biases, and low quality scores. CONCLUSIONS: The efficacy of PM on menopausal symptoms remains inconclusive because of methodological short-comings especially placebo effects inherent in self-assessment/recall questionnaires and no PM standardization. PM efficacy and safety need a fundamental re-appraisal by: (i) cohort (retro- and prospective) studies on current users to define its traditional use for rejuvenation; (ii) tightly coupling long-term efficacy to safety of well-defined PM and multiple end-points; (iii) using study design related to current understanding of menopause progression and estrogen pharmacology (iv) robust pharmacovigilance.

Parsing polyphyletic Pueraria: Delimiting distinct evolutionary lineages through phylogeny.

Several taxonomic and phylogenetic studies have hypothesized polyphyly within Pueraria DC., a genus comprising 19 species (24 with varieties) including the highly invasive Pueraria montana var. lobata (Kudzu) introduced to the U.S.A. about 150years ago. Previous efforts to investigate monophyly of the genus have been hampered by limited taxon sampling or a lack of comprehensive evolutionary context that would enable definitive taxonomic associations. This work presents a comprehensive phylogenetic investigation of Pueraria within the context of tribe Phaseoleae (Leguminosae). Polyphyly was found to be more extensive than previously thought, with five distinct lineages spread across the tribe and spanning over 25mya of divergence strongly supported by two chloroplast and one nuclear marker, AS2, presented here as a phylogenetic marker for the first time. Our phylogenies support taxonomic revisions to rectify polyphyly within Pueraria, including the resurrection of Neustanthus, moving one species to Teyleria, and the creation of two new genera, Haymondia and Toxicopueraria (taxonomic revisions published elsewhere).

Heterogeneity of clonal patterns among patches of kudzu, Pueraria montana var. lobata, an invasive plant.

BACKGROUND AND AIMS: Viny species are among the most serious invasive plants, and better knowledge of how vines grow to dominate landscapes is needed. Patches may contain a single genotype (i.e. genet), a competitively dominant genet or many independent but interacting genets, yet the clonal structure of vining species is often not apparent. Molecular markers can discriminate among the genetic identities of entwined vines to reveal the number and spatial distribution of genets. This study investigated how genets are spatially distributed within and among discrete patches of the invasive vine kudzu, Pueraria montana var. lobata, in the United States. It was expected that ramets of genets would be spatially clustered within patches, and that an increase in the number of genets within a patch would be associated with a decrease in the average size of each genet. METHODS: Six discrete kudzu patches were sampled across 2 years, and 1257 samples were genotyped at 21 polymorphic allozyme loci. Variation in genotypic and genetic diversity among patches was quantified and patterns of genet interdigitation were analysed. KEY RESULTS: Substantial genotypic and genetic variation occurred within and among patches. As few as ten overlapping genets spanned up to 68 m(2) in one patch, while >90 % of samples were genetically unique in another patch. Genotypic diversity within patches increased as mean clone size decreased, although spatially widespread genets did not preclude interdigitation. Eight genets were shared across ≥2 patches, suggesting that vegetative dispersal can occur among patches. CONCLUSIONS: Genetically unique kudzu vines are highly interdigitated. Multiple vegetative propagules have become established in spatially discrete patches, probably through the movement of highway construction or maintenance machinery. The results suggest that common methods for controlling invasive vines (e.g. mowing) may inadvertently increase genotypic diversity. Thus, understanding vine architecture and growth has practical implications.

Differentiating Puerariae Lobatae Radix and Puerariae Thomsonii Radix using HPTLC coupled with multivariate classification analyses.

Puerariae Lobatae Radix (PLR), the root of Pueraria lobata, is a traditional Chinese medicine for treating diabetes and cardiovascular diseases. Puerariae Thomsonii Radix (PTR), the root of Pueraria thomsonii, is a closely related species to PLR and has been used as a PLR substitute in clinical practice. The aim of this study was to compare the classification accuracy of high performance thin-layer chromatography (HPTLC) with that of ultra-performance liquid chromatography (UPLC) in differentiating PLR from PTR. The Matlab functions were used to facilitate the digitalisation and pre-processing of the HPTLC plates. Seven multivariate classification methods were evaluated for the two chromatographic methods. The results demonstrated that the HPTLC classification models were comparable to the UPLC classification models. In particular, k-nearest neighbours, partial least square-discriminant analysis, principal component analysis-discriminant analysis and support vector machine-discriminant analysis showed the highest rate of correct species classification, whilst the lowest classification rate was obtained from soft independent modelling of class analogy. In conclusion, HPTLC combined with multivariate analysis is a promising technique for the quality control and differentiation of PLR and PTR.

The use of cycleave PCR for the differentiation of the rejuvenating herb species Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea), and Mucuna macrocarpa (Black Kwao Khruea), and the simultaneous detection of multiple DNA targets in a DNA admixture.

Kwao Khruea, the tuberous roots of Pueraria candollei Graham ex Benth. (White Kwao Khruea), Butea superba Roxb. (Red Kwao Khruea), and Mucuna macrocarpa Wall. (Black Kwao Khruea), are used as rejuvenating herbs in traditional medicine in many tropical countries. Although Kwao Khruea has attracted strong interest because of its rejuvenation properties, each species is used for specific purposes and effects. P. candollei shows estrogenic effects in females. In contrast, B. superba and M. macrocarpa show androgenic effects in males. The potential misidentification of dried tuberous roots of various Kwao Khruea species might cause problems in the drug market, especially when they are reduced into powders. A cycleave PCR, which is based on the sequence of chloroplast matK gene, was developed to differentiate P. candollei, B. superba, and M. macrocarpa. The results showed that cycleave PCR is able to identify specific Kwao Khruea species. A multiplex cycleave PCR was optimized for the simultaneous detection of two different DNA targets in a DNA admixture. The specificity of this technique was confirmed by its ability to distinguish M. macrocarpa from five related Mucuna species. Cycleave PCR can be a specific, sensitive, and rapid method for the identification of medicinal plants and crude plant samples.

Botanical origin of dietary supplements labeled as "Kwao Keur", a folk medicine from Thailand.

In the course of our study on the quality of dietary supplements in Japan, both the internal transcribed spacer (ITS) sequence of nrDNA and the rps16 intron sequence of cpDNA of products labeled as "Kwao Keur" were investigated. As a result, the DNA sequence of Pueraria candollei var. mirifica, which is the source plant of Kwao Keur, was observed in only about half of the products. Inferred from the determined sequences, source plants in the other products included Medicago sativa, Glycyrrhiza uralensis, Pachyrhizus erosus, and Ipomoea batatas, etc. These inferior products are estimated to lack the efficacy implied by their labeling. In order to guarantee the quality of dietary supplements, it is important to identify the source materials exactly; in addition, an infrastructure that can exclude these inferior products from the market is needed for the protection of consumers from potential damage to their health and finances. The DNA analysis performed in this study is useful for this purpose.

Differentiation of Pueraria lobata and Pueraria thomsonii using partial least square discriminant analysis (PLS-DA).

The aims of the study were to differentiate Pueraria lobata from its related species Pueraria thomsonii and to examine the raw herbal material used in manufacturing kudzu root granules using partial least square discriminant analysis (PLS-DA). Sixty-four raw materials of P. lobata and P. thomsonii and kudzu root-labelled granules were analysed by ultra performance liquid chromatography. To differentiate P. lobata from P. thomsonii, PLS-DA models using the variables selected from the entire chromatograms, genetic algorithm (GA), successive projection algorithm (SPA), puerarin alone and six selected peaks were employed. The models constructed by GA and SPA demonstrated superior classification ability and lower model's complexity as compared to the model based on the entire chromatographic matrix, whilst the model constructed by the six selected peaks was comparable to the entire chromatographic model. The model established by puerarin alone showed inferior classification ability. In addition, the PLS-DA models constructed by the entire chromatographic matrix, GA, SPA and the six selected peaks showed that four brands out of seventeen granules were mislabelled as P. lobata. In conclusion, PLS-DA is a promising procedure for differentiating Pueraria species and determining raw material used in commercial products.

Discrimination of the Thai rejuvenating herbs Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea), and Mucuna collettii (Black Kwao Khruea) using PCR-RFLP.

The tuberous roots of Pueraria candollei (White Kwao Khruea), Butea superba (Red Kwao Khruea) and Mucuna collettii (Black Kwao Khruea), which belong to the family Leguminosae, are used as rejuvenating herbs in traditional Thai medicine. Although all of these species have an indication for rejuvenation, each differs in its medicinal properties. Two varieties of P. candollei, var. mirifica and var. candollei, affect females, whereas B. superba and M. collettii exhibit effects on males. However, the identification of these roots according to the name "Kwao Khruea" is confusing due to the similarity in their features. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) was utilised to identify plant origin. The partial matK gene was amplified and subjected to restriction enzyme digestion with DdeI and TaqI. The restriction fragments generated differed in number and size. To test the reliability of the method, an admixture of the different Kwao Khruea species containing equal amounts of DNA was tested. The results showed combined restriction patterns, and each species could be detected in the background of the others. The method was also used to authenticate eight different crude drugs sold as various types of Kwao Khruea in Thai markets. The results showed that the misidentification of commercial drugs remains a problem in crude drug markets. The PCR-RFLP analysis developed here provides a simple and accurate discrimination of these rejuvenating "Kwao Khruea" species.

White Kwao Krua variety classification by botanical characteristics and ISSR-Touchdown PCR technique.

White Kwao Krua [Pueraria candollei Grah. var. mirifica (Airy Shaw et Suvatabandhu) Niyomdham] is a herb used as an ingredient in supplementary and cosmetic. The tuberous roots of White Kwao Krua (WKK) contain estrogen-like substances. Seeds of WKK, collected from Prachuab Khiri Khan, were planted and propagated in the farm of Suranaree University of Technology, and their genetic backgrounds were ambiguous. Thirty six plants of WKK in the same age were sampled for classification using 7 botanical characteristics and DNA fingerprint by ISSR-Touchdown PCR technique. The relationship of the 7 botanical characteristics, using principle component analysis (PCA), showed the WKK plants fell into 3 groups. In the first group was plant number 34, which was distinguished from the other plants by its small leaf size. The second group consisted of 23 plants with elliptic leaf shape, acute leaf base, and acuminate leaf apex. The third group consisted of 12 plants with ovate leaf shape, obtuse leaf base, and cuspidate leaf apex. The ISSR-Touchdown PCR technique with 41 primers detected 355 loci of DNA with an average of 8.6 loci per primer. The sizes of DNA ranged between 280 bp to 1550 bp. Two hundred ninety three loci exhibited polymorphisms (82.54%) and the rest 62 loci were monomorphic (17.46%). The polymorphism information content (PIC) was between 0.0315-0.9779 (average 0.4779) and number of effective alleles per locus (Ne) ranged between 1.1250-1.8541 (average 1.5544). Unweighted pair group method with arithmetic mean (UPGMA), Jaccard similarity coefficient and PCA were used to find the construction of genetic relationship of WKK. The genetic similarity (GS) of WKK ranged between 0.50-0.86 (average 0.77). At the GS of 0.56 from cluster analysis, the WKK varieties could be divided into 2 major groups. The first group comprised of plant number 34 and 7, and the second group could be further divided in 2 subgroups at GS of 0.69. None of the WKK plants was identical in genetic, and they were expected to be derived from 5 genetic sources. These results showed that applying of the ISSR-Touchdown PCR technique could be used to classify WKK efficiently.

[Analysis of genetic relationships of Pueraria thomsonii based on SRAP markers].

OBJECTIVE: To determine the interspecies relationships of 18 Pueraria thomsonii cultivars in molecular level. METHOD: Eighteen P. thomsonii cultivars were evaluated by using sequence-related amplified polymorphism (SRAP) markers, with P. lobata and P. peduncularis as contrast species. Systematic relationships were constructed based on the UPGMA method by TREECONW software. RESULT: The results showed that 22 primer pairs produced 338 loci, out of which 216 were polymorphic, the percentage of polymorphic loci was 63.9%. An average of 15.4 loci and 9.8 polymorphic loci were generated by each pair of primers. Genetic distance was analyzed by TREECONW software. Genetic distance of 18 P. thomsonii were changed from 0.004 7 to 0.265 8, with an average of 0.316. Using cluster analysis (UPGMA) based on those polymorphism bands amplified with SRAP primers, the 22 cultivars were classified into three groups, groups 1 with 18 P. thomsonii, group 2 with 3 P. lobata, and group 3 with 1 P. peduncularis. Most of the P. thomsonii from the same region were not in the same group. CONCLUSION: SRAP markers could be a good marker for genetic relationship research in the P. thomsonii.

Comparative study of Puerariae lobatae and Puerariae thomsonii by HPLC-diode array detection-flow injection-chemiluminescence coupled with HPLC-electrospray ionization-MS.

An on-line HPLC-diode array detection-flow injection chemiluminescence (HPLC-DAD-FICL) method was applied to estimate the difference of Puerariae lobatae and Puerariae thomsonii. Their chemical and active profiles could be obtained by HPLC-DAD-FICL in one run. Seventeen compounds in two species were tentatively identified by HPLC-electrospray ionization-MS (HPLC-ESI-MS) method. The main antioxidants were rapidly screened by active fingerprints coupled with MS data. Similarity and Hierarchical clustering analysis (HCA) were used to distinguish different samples. The results suggested that the chemical fingerprints of 16 batches of samples were similar by similarity evaluation, while HCA could discriminate the two species. The active fingerprints of Puerariae lobatae and Puerariae thomsonii were significantly different. More antioxidants were found in Puerariae lobatae than in Puerariae thomsonii. Main antioxidants, including 3'-hydroxypuerarin, genistein 8-C-glycoside-xyloside, puerarin, 6″-O-xylosylpuerarin, mirificin and daidzein in two species, may be reasonable markers for the discrimination of the two species. The integrated fingerprint based on the chemical and active characteristics may provide an objective quality evaluation for Puerariae lobatae and Puerariae thomsonii.

[Classification of Pueraria lobata in different geographical regions].

OBJECTIVE: To classify Pueraria lobata originated from different geographical regions based on ITS,psbK-psbI and trnH-psbA information. METHOD: Twenty-four samples of P. lobata were collected from northeast China, north China, central China and northwest China. DNA extraction, PCR, sequence and genotypes/haplotypes analysis were performed . RESULT: ITS1, 5.8S and ITS2 varied only 1 bp respectively, psbK-psbI 2 bps; trnH-psbA varied 1 bp and 10 bp deletion. CONCLUSION: Based on the variation of ITS,psbK-psbI and trnH-psbA, 4 genotypes and 2 haplotypes were identified, respectively.

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.

Correlation of antioxidant activity and major isoflavonoid contents of the phytoestrogen-rich Pueraria mirifica and Pueraria lobata tubers.

The antioxidant activity of wild Pueraria mirifica collected from 28 of the 76 provinces of Thailand and Pueraria lobata collected from China were assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. P. mirifica tuberous extracts showed weak antioxidant activity in comparison with alpha-tocopherol. Six plant samples exhibited stronger antioxidant activity than the mean value of the P. mirifica population. In addition, the mean value of the P. mirifica population indicated significantly lower antioxidant activity than P. lobata. The analysis of the antioxidant activity of isoflavonoids revealed that puerarin and daidzein exhibited the same level of antioxidant activity as alpha-tocopherol. The results showed convincingly that puerarin and daidzein in the plant tubers may play an important role in antioxidant activity. The correlation analysis between antioxidant activity and major isoflavonoid contents of plant tubers indicated a significant correlation only with puerarin and a significant lack of correlation with daidzin, daidzein and genistein.

[Herbalogical textual research On "Gegen"].

OBJECTIVE: To provide further research with literature evidence of the variety origin, traditional effect and dosage. METHOD: Textual research on medicinal works of past dynasties. RESULT: The origin plants are Pueraria lobata, P. thomosonii and P. eduli. Its present effect is the same as ancient literature. However the fresh juice of the plant used for the treatment of bitter by mad dog and aborticide as literature recorded should be proved by further researches. The dosage is different among past dynasties. CONCLUSION: As one of the often used Chinese materia medica, Gegen has a bright prospect.

Molecular authentication of Radix Puerariae Lobatae and Radix Puerariae Thomsonii by ITS and 5S rRNA spacer sequencing.

In the present study, we examined nuclear DNA sequences in an attempt to reveal the relationships between Pueraria lobata (Willd). Ohwi, P. thomsonii Benth., and P. montana (Lour.) Merr. We found that internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA are highly divergent in P. lobata and P. thomsonii, and four types of ITS with different length are found in the two species. On the other hand, DNA sequences of 5S rRNA gene spacer are highly conserved across multiple copies in P. lobata and P. thomsonii, they could be used to identify P. lobata, P. thomsonii, and P. montana of this complex, and may serve as a useful tool in medical authentication of Radix Puerariae Lobatae and Radix Puerariae Thomsonii.