Metabolomics study of renal fibrosis and intervention effects of total aglycone extracts of Scutellaria baicalensis in unilateral ureteral obstruction rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Scutellariae Radix (Scutellaria baicalensis Georgi) is a well-known Traditional Chinese Medicine (TCM) which mainly contains flavonoids. Our previous studies have demonstrated that total aglycone extracts of Scutellaria baicalensis (TAES) can improve kidney disease in rats. AIM OF THE STUDY: To investigate the renal fibrosis (RF) pathogenesis and TAES treatment mechanism in unilateral ureteral obstruction (UUO) rats, using a metabolomics approach based on gas chromatography-mass spectrometry (GC/MS). METHODS: Rats with RF were divided into 6 groups with rats subjected to sham operation as normal control. The effects of TAES on some RF closely related parameters in UUO rats were investigated. A metabolomics method, based on GC/MS, was developed to monitor metabolic alterations in urine. Multivariate data analysis was utilized to identify biomarkers potentially associated with RF and the anti-RF activity of TAES. Ontology-based enrichment analysis by BiNChE and pathway analysis by MetPA aid in the interpretation of difference metabolites. RESULTS: After 10 days of treatment, the parameters of renal function begin returning to normal, and the abnormal high expressions of genes associated with extracellular matrix (ECM) were relived. In the metabolomics study, metabolic perturbations induced by UUO were reversed after treatment and TAES showed a dose-dependent therapy effect on RF, meanwhile, 18 potential biomarkers associated with RF were identified. Enrichment analysis of metabolites shows an over representation of mostly alkane-alpha, omega-diamine and alpha, omega-dicarboxylic acid, and these biomarkers are primarily involved in Glycine, serine and threonine metabolism, Retinol metabolism, Arginine and proline metabolism and Fructose and mannose metabolism. CONCLUSIONS: Our findings indicate that TAES have positive effects on UUO-induced RF in rats, meanwhile, metabolomics method coupled with metabolites enrichment analysis is a useful tool for revealing the pathogenesis of diseases and action mechanism of TCM on the whole body.

Development and characterization of simple sequence repeat (SSR) markers based on a full-length cDNA library of Scutellaria baicalensis.

Scutellaria baicalensis Georgi is an herbaceous perennial plant used as one of the staple Chinese herbal medicines in China with a long officinal history. However, research on S. baicalensis is currently limited due to the lack of genome and gene expression information. A full-length cDNA library from leaves and roots of S. baicalensis subjected to water deficit and heat, conditions that have been shown to affect baicalein accumulation, was constructed. There were 6491 expressed sequence tags (ESTs) obtained. UniGenes were assembled by BLAST similarity searches and annotated with Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). A total of 78 simple sequence repeats (SSRs) were identified and SSR markers associated with the active ingredients of S. baicalensis were selected. EST-SSR transferability was determined from 5 populations from different areas. This study is the first to produce a large volume of gene expression data from S. baicalensis to facilitate gene discovery in S. baicalensis and provide an important resource for molecular genetic and functional genomic studies in this species.

Cloning and characterization of a flavonol synthase gene from Scutellaria baicalensis.

Flavonols are the most abundant of all the flavonoids and play pivotal roles in a variety of plants. We isolated a cDNA clone encoding flavonol synthase from Scutellaria baicalensis (SbFLS). The SbFLS cDNA is 1011 bp long, encodes 336 amino acid residues, and belongs to a family of 2-oxoglutarate-dependent dioxygenases. The overall structure of SbFLS is very similar to that of Arabidopsis thaliana anthocyanidin synthase (AtANS), with a ß jelly-roll fold surrounded by tens of short and long α-helices. SbFLS was constitutively expressed in the roots, stems, leaves, and flowers, with particularly high expression in the roots and flowers. SbFLS transcript levels in the roots were 376-, 70-, and 2.5-fold higher than in the leaves, stems, and flowers. The myricetin content was significantly higher than that of kaempferol and quercetin. Therefore, we suggest that SbFLS mediates flavonol formation in the different organs of S. baicalensis. Our study may contribute to the knowledge of the role of FLS in S. baicalensis.

[Locality identification of Chinese medicinal plant Scutellaria baicalensis (Lamiaceae) population-level DNA barcoding].

Scutellaria baicalensis is an important traditional Chinese medicine and Scutellaria flavonoids have received worldwide attention in recent years. It is the basis of controlling quality of S. baicalensis to develop a reliable genetic marker system used to identify locality of origin. Because of the characteristics of maternal inherited and high-rate of evolution, the cpDNA intergenic spacer can effectively elucidate the degree of genetic variation in different areas of the same species (populations), which can be used as the population-level DNA barcoding to locality identify. In this study, we have used the molecular phylogeography analysis for the three cpDNA intergenic spacers atpB-rbcL, trnL-trnF and psbA-trnH of 17 wild populations from different localities, which reveals the 20 haplotypes, including 13 polymorphic sites and constitutes a shallow gene tree. The authers have divided the haplotypes of S. baicalensis into three grades of population-level DNA barcoding according to the frequence and geographic distribution: 3 highest-frequency haplotypes as area-population-level DNA barcoding, 3 haplotypes were mainly shared by 2-3 adjacent populations as region-population-level DNA barcoding, and there are also 8 unique-population haplotypes as unique-population-level DNA barcoding. The result of this study reveals that population-level DNA barcoding is a reliable genetic marker used to locality identify of S. baicalensis.

Comparison of the major flavonoid content of S. baicalensis, S. lateriflora, and their commercial products.

According to the notification for definition of pharmaceuticals from the Director-General of the Pharmaceutical and Food Safety Bureau, Ministry of Health Labour and Welfare of Japan, the roots of Scutellaria baicalensis (Chinese skullcap) and S. lateriflora (skullcap) are classified as "the raw materials exclusively used as pharmaceuticals", but their aerial parts are classified as "non-pharmaceuticals" so, in principle, there are no health claims for these materials and no descriptions of drug-like dosages or administration directions. Dried root of S. baicalensis is also registered in Japanese Pharmacopoeia XV as scutellaria root. Scutellaria root is considered to have the adverse drug reactions of interstitial pneumonia and drug-induced hepatopathy in kampo medicines (Japanese traditional herbal formulations), and baicalin, its major constituent, is considered to be the cause of the adverse reaction. This study was conducted to evaluate the validity of this borderline between pharmaceuticals and non-pharmaceuticals by analyzing the amounts of four flavonoids, including baicalin, in the roots, stems, and leaves of S. baicalensis and S. lateriflora, and in the commercial products herbal tea and dietary supplements prepared from S. lateriflora. These flavonoids were found in the root of S. baicalensis; its aerial parts, however, did not contain them. On the other hand, the amounts of those flavonoids in the aerial parts of S. lateriflora were larger than in the root. Herbal tea and dietary supplements of S. lateriflora obtained commercially also contained those flavonoids, and the dietary supplements contained amounts of them comparable with that in kampo medicine. These results suggest that classification that the aerial parts of S. lateriflora as non-pharmaceuticals in Japan needs reconsideration.

[Quality evaluation of seed of Scutellaria baicalensis from different habitats].

OBJECTIVE: To study the seed quality of Scutellaria baicalensis Georgi. METHOD: Collecting seeds from different habitats and studying the thousand-seeds weight, plumpness rate and germination percentage. RESULT: The seeds from Ningxia and Juxian (Shandong province) are better than those from other habitats. Its plumpness rate is above 95%, thousand-seeds weight is 2.2 g and germination percentage is above 90%. CONCLUSION: There are obvious differences in quality of seeds of Scutellaria baicalensis from different habitats.

[Recognition of three classes of skullcaps by FTIR spectroscopy combined with artificial neural networks].

In order to recognition of three classes of skullcaps (cultivated, wild Scutellaria baicalensis Georgi and Scutellaria viscidula Bge) three kinds of models of artificial neural networks (ANN), nonlinear-linear, linear-linear and nonlinear-nonlinear model, were used combined with their infrared spectra. Skullcaps samples were collected by Fourier Transform Infrared (FTIR) spectra. 42 samples were gathered as a train set, and 34 samples as a test set, then their supervision trains were performed using three models each. When the summation of error square of train target was selected as 0.01, the correct rate for recognition of three classes of skullcaps using each ANN was 100% for the train set, but was different for the test set, which depended on the number of node in hidden layer, S1. It was found that with the increase of S1, the correct rate would decrease oppositely. This may be caused by the high degree of the non-linearity of the networks, so that the models of networks were not fit for the train of this kind of sample set. When using linear-linear model of ANN varied with S1 in some extent, the correct rate was generally about 85%. Recognizability obtained using nonlinear-linear model of ANN was the best. Its correct rate of recognition was > 97% when S1 = 3, and so this method can be used to recognize three of skullcaps simply, rapidly, and accurately.