[Genetic and chemical discrimination of traditional Tibetan medicine seabuckthorn based on DNA barcode and ¹H-NMR metabolic method].

To differentiate three medicinal Hippopahe species of seabuckthorn, a combined genetic and chemical identification method was established in this study. ITS2 and psbA-trnH were tested for identification of 3 species of seabuckthorn. Detection of the kimura 2-parameter (K2P) distance, the neighbor-joining (NJ) tree and the barcoding gap were used to assess the identification efficiency. ¹H-NMR based metabolic method was applied to acquire the profile of metabolites. PCA was used to analysis the metabolite data. The results indicated that DNA barcode combined ¹H-NMR based metabolic method is a powerful tool for the identification of 3 medicinal Hippopahe species of seabuckthorn. The finding demonstrated that different genetic variation and chemical constituents existed among 3 medicinal Hippopahe species of seabuckthorn. The combined identification method will improve the reliability of species discrimination and could be applicable to much other ethnic medicine which has various origins in China.

[Stud on quality standard for Aconiti Tatsienensis Radix].

OBJECTIVE: To establish the quality standard for Aconiti Tatsiehensis Radix. METHODS: TLC was used to detect yunaconi- tine and talasamine in Aconiti Tatsiensis Radix. HPLC was adopted to determine the content of yunaconitine. Moisture,ash,acid insolu- ble ash and ethanol soluble extractive were determined according the procedures recorded in the Appendix of Chinese Pharmacopoeia (2010 edition). The microscopic identification was also carried out. RESULTS: Aconiti Tatsienensis Radix had obvious microscopic char- acteristics,and its TLC identification had a good resolution with clear spots. An average content of moisture was 11. 48%, ash was 4. 83% ,acid insoluble was 1. 81%, ethanol soluble extractive was 20. 46% and yunaconitine was 0. 23%. CONCLUSION: The established standard is acceptable for quality evaluation of Aconiti Tatsienensis Radix.

[Population characteristics and ecological suitability of Saussureae hieracioides].

OBJECTIVE: To analyze the population characteristics and the appropriate producing area of Saussureae hieracioides in China. METHODS: Chuanxibei plateau, one of the main producing areas of Saussureae hieracioides, was selected as the analytical basal place. Ecological methods were used to investigate the density and biomass of Saussureae hieracioides. Traditional Chinese Medicine Geographic Information System (TCMGIS-II) was used to analyze the appropriate producing area of Saussureae hieracioides. RESULTS: Saussureae hieracioides could form the dominant species in its distribution area. The proper region (with similarity of 90% - 100%) of Saussureae hieracioides accounted for 338 776.89 km2, including 5 provinces/municipalities and 226 counties/cities. The largest area among them was Tibet Autonomous Region with area of 148 175.55 km2, followed by Sichuan Province (110 216.46 km2), Qinghai Province (62 947.61 km2), Gansu Province (16 233.09 km2) and Yunnan Province (1 177.18 km2). CONCLUSION: TCMGIS is much valuable to the recognition of formation of producing area, the division of adaptive area, introduction and acclimatization of medicinal materials, it also provides a scientific reference for the introduction and cultivation of Saussureae hieracioides.

[Determination of skimmin, scopolin and umbelliferone in Tibetan medicine Saussurea hieracioides by HPLC].

This study is aimed to establish a high-performance liquid chromatography (HPLC) method for simultaneous determination of skimmin, scopolin and umbelliferone in Saussurea hieracioides. Samples were analyzed on a Wondasil C18-WR column (4.6 mm x 250 mm, 5 microm) with methanol (A) and water containing 0.1% phosphate (B) as mobile phases for gradient elution at a flow rate of 1.0 mL x min(-1). The detection wavelength and column temperature were set at 325 nm and 35 degrees C, respectively, and the sample size was 10 microL. The results showed that skimmin, scopolin and umbelliferone were simultaneously achieved within 40 min under the above conditions. A good linearity was observed in the range of 0.18-5.6 microg (r = 1.000 0), 0.060-1.8 microg (r = 0.999 9), 0.032-0.97 microg (r = 0.999 8) for skimmin, scopolin and umbelliferone, respectively, with the average recoveries of 99.16% (RSD = 0.41%), 100.3% (RSD = 0.79%), 102.2% (RSD = 0.87%). The method is simple, accurate and reproducible and can be used for the quality control of S. hieracioides.

[Study on three different species tibetan medicine sea buckthorn by 1H-NMR-based metabonomics].

The 1H-NMR fingerprints of three different species tibetan medicine sea buckthorn were established by 1H-HMR metabolomics to find out different motablism which could provide a new method for the quality evaluation of sea buckthorn. The obtained free induction decay (FID) signal will be imported into MestReNova software and into divide segments. The data will be normalized and processed by principal component analysis and.partial least squares discriminant analysis to perform pattern recognition. The results showed that 25 metabolites belonging to different chemical types were detected from sea buckthorn,including flavonoids, triterpenoids, amino acids, carbohydrates, fatty acids, etc. PCA and PLS-DA analysis showed three different varietiest of sea buckthorn that can be clearly separated by the content of L-quebrachitol, malic acid and some unidentified sugars, which can be used as the differences metabolites of three species of sea buckthorn. 1H-NMR-based metabonomies method had a holistic characteristic with sample preparation and handling. The results of this study can offer an important reference for the species identification and quality control of sea buckthorn.

Acetylation of histones associated with the p21WAF1/CIP1 gene by butyrate is not sufficient for p21WAF1/CIP1 gene transcription in human colorectal adenocarcinoma cells.

Butyric acid is well recognized as a histone deacetylase (HDAC) inhibitor, and changes in histone acetylation are thought to alter gene expression. The mechanism by which sodium butyrate (NaB) induces p21WAF1/CIP1, a critical gene involved in the antiproliferative effect of NaB, was studied at the chromatin level. Using chromatin immunoprecipitation (ChIP) assay, acetylation of histone H3 was observed at the proximal region of the promoter within 30 min of NaB exposure and this extended to the distal region within 2 hr. By contrast, histone H4 was acetylated both at the proximal and the distal regions of the promoter within 30 min. NaB did not influence other histone modifications. NaB stimulated recruitment of the transcription factors ZBP89 and Sp1 as well as GCN5, but did not influence recruitment of Sp3, HDAC1, p300, or CBP. As recruitment of HDAC1 to the promoter appeared not to account for NaB-induced changes in histone acetylation, we aimed to influence HDAC activity by altering its phosphorylation status. The kinase inhibitor, H7, suppressed p21WAF1/CIP1 mRNA in both the absence and the presence of NaB without influencing the butyrate-induced hyperacetylation of H3 and H4 associated with the p21WAF1/CIP1 promoter. These results suggest that acetylation of histones at the p21WAF1/CIP1 promoter is not sufficient for NaB to exert antiproliferative effects via transcription of the p21WAF1/CIP1 gene. Induction of p21WAF1/CIP1 transcription by the phosphatase inhibitor, okadaic acid, in the absence of changes in association of acetylated histones with the p21WAF1/CIP1 promoter provides further evidence of the importance of phosphorylation to p21WAF1/CIP1 transcription.

Sodium butyrate inhibits cell growth and stimulates p21WAF1/CIP1 protein in human colonic adenocarcinoma cells independently of p53 status.

Butyric acid, one of the short-chain fatty acids produced by microbial fermentation in the colon, exhibits antiproliferative activities in various cancer cell lines. The initial objective of the study was to assess whether the effect of sodium butyrate (NaB) on cell growth differed by p53 status of the cells. Four human colorectal adenocarcinoma cell lines were used: HT29 (p53 point mutation), Caco2 (p53 truncation), LS513 (p53 wild type), and Lovo (p53 wild type). NaB significantly inhibited cell growth in all four cell lines. NaB arrested HT29 and LS513 cells in G0/G1 and Caco2 and Lovo in G2-phase. A second objective was to determine whether NaB similarly affected the cyclin-dependent kinase inhibitor, p21WAF1/CIP1. In all cell lines, p21 mRNA levels were immediately elevated after NaB exposure, and p21 protein levels were increased within 6 h. NaB increased p21 promoter activity in both Caco2 and Lovo, suggesting p53 independence. NaB did not influence p21 mRNA stability. Although three DNase I hypersensitivity sites were identified in the region of the p21 gene, induction of p21 mRNA by NaB was not accompanied by relaxation of the chromatin in the region of the p21 gene.