Cloning and expression of SmDXS2 gene in Swertia mussotii / 中国中药杂志

1-deoxy-D-xylulose-5-phosphate synthase2(DXS2) is the first key enzyme of the MEP pathway,which plays an important role in terpene biosynthesis of plants. According to the data of Swertia mussotii transcriptome, DXS2 gene(Gen Bank number MH535905) was cloned and named as Sm DXS2. The bioinformatics results showed that Sm DXS2 has no intron,with a 2 145 bp open reading frame encoding a polypeptide of 714 amino acids. They are belonging to 20 kinds of amino acids,and the most abundant amino acids include Ala,Gly and Trp. The predicted protein molecular weight was 76. 91 k Da and its theoretical isoelectric point(p I) was6. 5,which belonging to a hydrophilic protein. α-Helix and loop were the major motifs of predicted secondary structure of DXS2. The three function domains are TPP＿superfamily,Transket＿pyr＿ superfamily and Transketolase＿C superfamily,respectively. The Sm DXS2 protein shared high identity with other DXS2 proteins of plants. Phylogenetic analysis showed that Sm DXS2 protein is grouped with the gentian DXS2 protein. The recombinant protein of Sm DXS2 gene in Escherichia coli was approximately 92. 00 k Da(containing sumo-His tag protein 13 k Da),which was consistent with the anticipated size.This work will provide a foundation for further functional research of Sm DXS2 protein and increasing the product of iridoid compound by genetic engineering in S. mussotii.

Correlation Between Chemical Constituents and Ecological Factors of Tibetan Medicine Swertia mussotii / 中国实验方剂学杂志

Objective:The HPLC fingerprinting of Swertia mussotii was established to study the correlation between chemical components and ecological factors in different areas. Method:The fingerprint of S. mussotii was established by high performance liquid chromatography (HPLC),and the evaluation and analysis were made based on the " Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A Edition" promulgated by the National Pharmacopoeia Commission. The analysis was carried out on a Wondasil C18 column(4.6 mm×250 mm,5 μm), with methanol-0.2%phosphoric acid as the mobile phase for gradient elution,and the column temperature was set at 30℃. The flow rate was 1.0 mL·min-1, and the detection wavelength was set at 245 nm. And data on ecological factors in each sample habitat, such as climate and soil, were collected. The gray correlation and bivariate analysis were carried out on the chemical constituents and ecological factors of medicinal materials in different areas using DPS data processing system and SPSS 21.0 statistical software. Result:The HPLC fingerprint of S. mussotii was established,a total of 12 common fingerprint peaks were marked, and the chemical constituent of the seven peaks were determined. The chemical constituents, such as swertiamain and mangiferin of S. mussotii, were significantly correlated with ecological factors. Moreover,the chemical constituents were obviously affected by the monthly average temperature range,annual precipitation,precipitation seasonality in the climatic factors,the soil organic carbon ratio and soil pH in the soil factors. Conclusion:The chemical constituents of S. mussotii have a correlation with the external ecological factors,the findings could provide a basis for the artificial planting of the medicinal material and the scientific connotation of the " environment-based" theory for Tibetan medicines.

Cloning and bioinformatics analysis of SmMK gene in Swertia mussotii / 中草药

Objective: In order to identify the function of the mevalonate kinase (MK) which is a key enzyme of the mevalonate pathway (MVA) in Swertia mussotii, and to improve the study of MVA in S. mussotii. Methods: According to the SmMK gene sequence of transcriptome of S. mussotii, the specific primers were designed, the cDNA complete sequences was obtained by RT-PCR and the sequence was analyzed using bioinformatics. Prokaryotic expression vector MBP-SmMK was constructed and transformed into Escherichia coli Rosetta (DE3) for expression. Results: The results showed that SmMK cDNA complete sequences had a length of 1 164 bp encoding 387 amino acid residues. The SmMK protein shared high identity with other MK proteins of plants. And the protein signal peptide, transmembrane region, location, secondary, and tertiary structures were analyzed and forecasted. The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size, which was 40 970. Conclusion: This work will provide a foundation for research the SmMK protein functional and study MCA in S. mussotii. At the same time, it will supply the basis to improve the production of the isoprenoids.

Cloning and expression of geranyl pyrophosphate synthase gene in Swertia mussotii / 中草药

Objective: To clone the geranyl pyrophosphate synthase gene from Swertia mussotii (SmGPPS), analyze the bioinformation of SmGPPS, and perform the gene expression. Methods: According to the SmGPPS gene sequence of transcriptome of S. mussotii, the specific primers were designed, the cDNA complete sequences was obtained by RT-PCR and the sequence was analyzed using bioinformatics. Prokaryotic expression vector pET-28a-SmGPPS was constructed and transformed into Escherichia coli BL-21 (DE3) for expression under 37℃ and induced by 1 mmol/L IPTG. The relative expression of gene SmGPPS in the leaf, stem, and flower of S. mussotii was also studied. Results: The results showed that SmGPPS cDNA complete sequences had a length of 1 119 bp encoding 372 amino acid residues. And the protein secondary and tertiary structures were analyzed and forecasted. The SmGPPS protein shared high identity with other GPPS proteins of plants. The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size. Relative RT-PCR analysis indicated that SmGPPS showed the highest transcript abundance in the leaf. Conclusion: This work will provide a foundation for further functional research of SmGPPS protein and increasing the product of iridoid compound by genetic engineering in S. mussotii.

Application of remote sensing and GIS in study of suitability distribution of Swertia mussotii, a Tibetan medicine in Sichuan province / 中国中药杂志

Swertia mussotii is a kind of rare medicinal materials, the relevant researches are mainly concentrated on its medicinal efficacy and medicinal value till now, researches of adaptive distribution by applying remote sensing and GIS are relatively less. This study is to analyze the adaptive distribution of S.mussotii in Sichuan province by applying remote sensing and GIS technology, and provide scientific basis for the protection and development of wild resources, artificial cultivation and adjustment of Chinese medicine industrial distribution in Sichuan province. Based on literature review and ecological factors such as altitude, annual precipitation and annual average temperature, this study extracted ecological factors, overlay analysis in GIS, as well as combining GPS field validation data by means of remote sensing and GIS, discusses the adaptive distribution of SMF sin Sichuan province. ①The area of adaptive distribution of S. mussotii in Sichuan province is 1 543.749 km², mainly in Dege county, Ganzi county, Daofu county, Kangding county, Barkam, Jinchuan county, Xiaojin county, Danba county, Daocheng county, Xiangcheng county, Xinlong county, Aba county, Muli county and other counties and cities, accounts for about 7.25% in total area. ② Combining statistical information and field validation, this study found that S. mussotii adaptive distribution gained by remote sensing and GIS is in conformity with its actual distribution. The study shows that remote sensing and GIS technology are feasible to obtain the S. mussotii adaptive distribution, they can further be applied to studies on adaptive distributions of other rare Chinese medicinal herb.

Resources Survey on Rare and Endangered Medicinal Plant Swertia mussotii Franch / 世界科学技术-中医药现代化

The resource of rare and endangered medicinal plant Swertia mussotii Franch. in Tibet, Qinghai and Sichuan province were surveyed by ways of documents, interview, quadrat and market investigation. The results indi-cated that Swertia mussotii Franch. mainly distributed in Zuogong and Mangkang of Tibet, Yushu of Qinghai province, Shiqu, Daofu, Kangding, Maerkang, Jinchuan and Xiaojin of Sichuan province. According to the height above sea level, the distribution altitude was from 2 300 m (Kangding of Sichuan province) to 3 900 m (Mangkang of Tibet). There are distributions of Swertia mussotii Franch. within the scope of 2 600 m. The illumination, water, soil, temperature and altitude had significant influence on the distribution, growth and reserve of Swertia mussotii Franch. from different angles. In recent years, there was huge increase of market requirement in Swertia mussotii Franch. which were used in Tibetan medicine Zangyinc he n. Excess collection was the primary cause of rapid decreasing in resource of Swertia mussotii Franch.. It was suggested to strengthen the management of rational collection, as well as to accelerate the development of cultivation and production.

Chemical constituents from Swertia mussotii / 中草药

Objective: To study the chemical constituents of Swertia mussotii. Methods: Various chromatographic methods were employed to isolate the compounds and their structures were established by spectroscopic analysis. Results: Nineteen compounds were isolated from 75% ethanol extract of S. mussotii and identified as 1-hydroxyl-3, 4, 7, 8-tetramethoxyxanthone (1), 1, 7- dihydroxyl-3-methoxyxanthone (2), 1, 3, 7-trihydroxyxanthone (3), 1, 3, 7, 8-tetrahydroxyxanthone (4), 1, 3, 8-trihydroxyl-7- methoxyxanthone (5), 1, 3-dihyfroxyl-7, 8-dimethoxyxanthone (6), 1, 5, 8-trihydroxyl-3, 4-dimethoxyxanthone (7), 1-hydroxyl-3, 4, 5, 8-tetramethoxyxanthone (8), 1-hydroxyl-3, 5, 8-trimethoxyxanthone (9), (S)-(+)-gentiolactone (10a), (R)-(-)-gentiolactone (10b), 1, 8-dihydroxyl-3, 7-dimethoxyxanthone (11), 1-hydroxyl-3, 7, 8-trimethoxyxanthone (12), 1, 7-dihydroxyl-3, 8-dimethoxyxanthone (13), 1, 7, 8-trihydroxyl-3-methoxyxanthone (14), 1, 3, 5, 8-tetrahydroxyxanthone (15), 1, 7-dihydroxyl-3, 4, 8-trimethoxyxanthone (16), mangiferin (17), and oleanolic acid (18), respectively. Conclusion: Compounds 1-9 are firstly isolated from S. mussotii. Compounds 10a and 10b are firstly isolated from the plants in Swertia L.

Study on Swertia mussotii Franch. by HPLC fingerprinting method combined with chemical pattern recognition approaches / 中国药学杂志

OBJECTIVE: To study the quality control method of Swertia mussotii Franch. METHODS: A high performance liquid chromatographic method was developed to establish the fingerprint of Swertia mussotii Franch. Nineteen samples from various batches were analyzed, furthermore, PCA were used to differentiate and evaluate the whole fingerprints. Some characteristic peaks were i-dentified preliminarily with HPLC-MS method. RESULTS: Eleven peaks were identified by MS. Swertiamarin and swertianolin were the key components for quality control. CONCLUSION: The method provides an academic reference for controlling the quality of Swertia mussotii Franch.

Therapeutic effect of Swertia Mussotii Franch on immunological liver injury in mice / 西安交通大学学报(医学版)

Objective To observe the therapeutic effect of Swertia mussotii Franch is extracted by alcohol on immunological liver injury in mice,and further evaluate its immun-regulating effect in decreasing aminotransferase and protecting heptocytes. Methods BCG vaccine and lipopolysaccharide were intravenously injected to establish liver injury model.The effects of Swertia mussotii Franch on liver injury were contrasted. Results The high-,medium-and low-dose Swertia mussotii Franch groups which had been administered for 10 consecutive days,had significantly inhibited serum ALT and AST acticities of immunological liver injury;Swertia mussotii Franch could effectively prevent immunological liver injury by BCG+LPS. Conclusion Swertia mussotii Franch can protect mice from immunological liver injury and protect the function of liver.

Purification technology of iridoid glycosides and xanthones from Swertia mussotii by macroporous resin / 中草药

Objective To study the separation and purification technology of iridoid glycosides and xanthones from Swertia mussotii by macroporous resin.Methods The dynamic adsorption and desorption characteristics of macroporous adsorption resins HPD-300,HPD-400,HPD-600,AB-8,DM-301,and D-101-Ⅰ for swertiamarin and swertianolin were investigated.On the base of the investigation,the better macroporous adsorption resin was chosen to finish the experiments.Results The adsorption and elution characteristics of HPD-300 used to separate and purify iridoid glycosides and xanthones were very good.In the course of adsorption,the amount of used adsorption is 0.9 g dried medicinal herb/mL resin,with about 8 pH value of adsorption solution,and the volumetric rate is 2 BV/h; In the course of elution,the resin column chromatography was eluted gradiently with 7 BV 20% and 5 BV 70% EtOH after removing impurities with water.The elution rates were both more than 90%,and the contents of iridoid glycosides and xanthones were both up to 50% in the two dried fractions of 20% and 70% ethanolic elutions.Conclusion HPD-300 is an ideal resin with the best enrichment for separating and purifying both iridoid glucosioles and xanthanoes simultanously.