The standard sample of natural products is an essential standard reference to determine the quality of the product in the quality control of natural products. To develop a certified reference material(CRM) of swertioside according to the Work Guideline for Reference Materials(3): Reference Material-General Principles and Statistical Method for Certification(GB/T 15000.3-2008), swertioside was purified from whole plant of Swertia mussotii by extraction, isolation and Prep-HPLC to obtain certified reference material of swertioside. The structure of swertioside was identified by IR, UV, high-resolution MS, NMR. Thin layer chromatography, optical rotation, elemental analysis and melting point was carried out for the identification. The purity of the prepared sample was tested from different chromatographic elution conditions, thin layer chromatography and HPLC-MS. Swertioside was divided into 140 bottles, with 10 mg per bottle after homogeneity test, stability test and quantitative analysis. This CRM is 7-O-[α-L-rhamnopyranosyl-(1→2)-β-D-xylopyranosyl]; the homogeneity of the 95% confidence interval was good; the certified purity value was 98.66%, with a relative expanded uncertainty of 0.38%; the storage period was 36 months at 0-8 ℃. Therefore, the CRM of sakuranetin reached the technical requirements of CRM, and was accepted by SAC. Swertioside is successfully developed and can be used for determining content, evaluating test methods, detecting relevant products and controlling quality.
Certification ; Chromatography, High Pressure Liquid ; Mass Spectrometry ; Phytochemicals/standards* ; Reference Standards ; Swertia/chemistry*

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.
Amino Acid Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; Genes, Plant ; Iridoids ; Phylogeny ; Plant Proteins ; genetics ; Swertia ; enzymology ; genetics ; Transcriptome ; Transferases ; genetics

Swertiamarin (STM) is an iridoid compound that is present in the Gentianaceae swertia genus. Here we investigated antiapoptotic effects of STM on carbon tetrachloride (CCl₄)-induced liver injury and its possible mechanisms. Adult male Sprague Dawley rats were randomly divided into a control group, an STM 200 mg/kg group, a CCl₄ group, a CCl₄+STM 100 mg/kg group, and a CCl₄+STM 200 mg/kg group. Rats in experimental groups were subcutaneously injected with 40% CCl₄ twice weekly for 8 weeks. STM (100 and 200 mg/kg per day) was orally given to experimental rats by gavage for 8 consecutive weeks. Hepatocyte apoptosis was determined by TUNEL assay and the expression levels of Bcl-2, Bax, and cleaved caspase-3 proteins were evaluated by western blot analysis. The expression of TGF-β1, collagen I, collagen III, CTGF and fibronectin mRNA were estimated by qRT-PCR. The results showed that STM significantly reduced the number of TUNEL-positive cells compared with the CCl₄ group. The levels of Bax and cleaved caspase-3 proteins, and TGF-β1, collagen I, collagen III, CTGF, and fibronectin mRNA were significantly reduced by STM compared with the CCl₄ group. In addition, STM markedly abrogated the repression of Bcl-2 by CCl₄. STM also attenuated the activation of the PI3K/Akt pathway in the liver. These results suggested that STM ameliorated CCl₄-induced hepatocyte apoptosis in rats.
Adult ; Animals ; Apoptosis* ; Blotting, Western ; Carbon Tetrachloride ; Carbon* ; Caspase 3 ; Collagen ; Fibronectins ; Gentianaceae ; Hepatocytes ; Humans ; In Situ Nick-End Labeling ; Liver ; Male ; Rats* ; Rats, Sprague-Dawley ; Repression, Psychology ; RNA, Messenger ; Swertia

MISA (MicroSAtelite) software was employed to screen SSRs in 68 787 contigs of Swertia mussotii transcriptome sequences. 5 610 SSRs were distributed in 5 099 contigs which accounted for 7.41% of 68 787 contigs. There are 220 kinds of SSR motifs existing in S. mussotii transcriptome. On average, SSRs occurred every 12.60 kb in length. In the SSRs, the tri-nucleotide repeat motif was the most abundant (45.99%), followed by the di-nucleotide (41.62%). AT/TA and AAT/TTA were the main types of motif in di-, tri-nucleotide repeats. The repeat numbers of SSRs which from S. mussotii transcriptome SSRs were mainly from 5 to 10 and motif length of them mostly ranged from 12 bp to 30 bp. A total of 30 651 contigs were annotated, and only 1 447 SSRs were occurred in protein-coding regions. In the six repeat motifs, tri-nucleotide repeats were the most abundant in coding regions (928). There are abundant SSRs in S. mussotii transcriptome with high frequency and various types, indicating their usefulness in theory. This research may lay the foundation for designing the targeted SSR primers and developing SSR molecular markers by mining the information of SSRs loci in S. mussotii transcriptome sequences data.
Data Mining ; Medicine, Tibetan Traditional ; Microsatellite Repeats ; Plants, Medicinal ; genetics ; Swertia ; genetics ; Transcriptome

Fifteen known compounds were isolated from Swertia delavayi by silica gel, Sephadex LH-20 and Rp-18 column chromatographies. Based on extensive spectroscopic analysis (MS, 1H, 13C-NMR), their structures were identified aserythrocentaurin (1), erythrocentaurindimethylacetal (2), sweroside (3), swertiamarin (4), gentiopicroside (5), swertiakoside A (6), 2'-O-acetylswertiamarin (7), 4'-O-[(Z) -coumaroyl] swertiamarin (8), 1,5,8-trihydroxy-3-methoxyxanthone (9), 8-O-β-D-glucopyranosyl-1-hydroxy-2,3, 5-trimethoxyxanthone (10), 8-O-[β-D-xyl- opyranosyl-(1 --> 6)-β-D-glucopyranosyl]-7,8-dihydroxy-3-methoxyxanthone (11), isovitexin (12), β-sitosterol (13), daucosterol (14), and oleanolic acid (15). Among them, ten ones (14, 7-11, 13) were obtained from S. delavayi for the first time. The isolates were evaluated for their anti-HBV activities in HepG 2. 2. 15 cell line in vitro. The results showed that compound 1, 2, 6, 7, 9 and 12 exhibited significant inhibitory activity on HBV DNA replication with IC50 values from 0.05 to 1.46 mmol x L(-1).
Antiviral Agents ; chemistry ; isolation & purification ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Hepatitis B virus ; drug effects ; genetics ; Magnetic Resonance Imaging ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization ; Swertia ; chemistry

Chemical constituents of Swertia patens. The whole plant of air-dried Swertia patens was extracted with 90% EtOH. The water extract was suspended in H₂O and extracted with petroleum ether, EtOAc and n-BuOH, successively. The compounds were isola- ted and purified by column chromatography from the EtOAc fraction, and identified based on spectral analyses (MS, ¹H-NMR, ¹³C- NMR). Eighteen compounds were isolated and elucidated as 3, 4-dihydro-1H,6H,8H-naptho [1,2-c:4,5-c', d'dipyrano-1, 8-dione (1), angelone (2), gentiogenal (3), erythricin (4), erythrocentaurin (5), gentianine (6), swertiakoside B (7), swertiamarin (8), 2'-O-actylswertiamarin (9), amarogentin (10), 1, 3, 5-trihydroxyxanthone (11), 1, 3-dihydroxy-5-methoxyxanthone (12), 1-hydroxy- 2, 3, 5-trimethoxyxanthone (13), gentiocrucine (14), 3-hydroxyphenylketone (15), n-hexacosyl ester 4-hydroxy-trans-cinnamate (16), n-hexacosyl ester 4-hydroxy-cis-cinnamate (17), and cholest-4-en-3-one (18). Compounds 1-7, 9-18 were obtained from S. patens for the first time.
Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization ; Swertia ; chemistry

This present work is to study the chemical constituents of Swertia angustifolia. The whole plants of air-dried Swertia angustifolia was extracted with 90% EtOH. The water extract was suspended in H2O and extracted with petroleum ether, EtOAc and nBuOH, successively. The compounds were isolated and purified by column chromatography from the EtOAc fraction, and identified based on spectral analyses (MS, 1H-NMR, 13C-NMR). Fourteen compounds were isolated and characterized as 1, 8-dihydroxy-3, 7-dimethoxyxanthone (1), 1, 8-dihydroxy-3, 5, 7-trimethoxyxanthone (2), 7-hydroxy-3, 8-dimethoxyxanthone-1-O-β-D-glucopyranoside (3), 8-0-[β-D-xylopyranosyl-(1-6) -β-D-glucopyranosyl] -1, 7-dihydroxy-3-methoxyxanthone (4), (+) -syringaresinol (5), ferulic acid (6), trans-coniferyl aldehyde (7), sinapaldehyde (8), trans-coniferyl alcohol (9), 3, 4-dihydroxybenzoic acid (10), 2-hydroxybenzoic acid (11), isophthalic acid (12), 2-furoic acid (13), and 2-methyl-4(3H)-quinazolinone(14). Compounds 2-14 were obtained from this plant for the first time.
Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Mass Spectrometry ; Molecular Structure ; Swertia ; chemistry

The genetic diversity of three Tibetan herbs, i. e., Sang-Di, E-Dewa and Ye-Xingba (Tibetan names), was studied based on the field collection, specimen identification and DNA sequence analysis. Swertia hispidicalyx, Gentiana lhassica and Scrophularia dentata, as the original plants of the three Tibetan herbs, were collected and identified. The regions of ITS, matK, rbcL, rpoC1, trnL(UAA), psbA-trnH, atpB-rbcL, trnS (GCU)-trnG(UCC), rpl20-rps12, trnL(UAA)-trnF(GAA) and nadl 2nd intron were amplified and sequenced. The ITS regions of S. hispidicalyx and S. dentata were cloned and sequenced, and the sequences were classified into different genotypes. All the sequences were analyzed and compared with those of closely related species. Our studies may provide reference for the genetic diversity analysis and molecular identification of the three Tibetan herbs.
Genetic Variation ; Gentiana ; classification ; genetics ; Phylogeny ; Plant Proteins ; genetics ; Plants, Medicinal ; classification ; genetics ; Scrophularia ; classification ; genetics ; Swertia ; classification ; genetics ; Tibet

This study is to investigate the chemical constituents of Swertia kouitchensis. The whole plants of air-dried Swertia kouitchensis was extracted with 90% EtOH. The water extract was suspended in H2O and extracted with petroleum ether, EtOAc and n-BuOH, successively. The compounds were isolated and purified by column chromatography from the EtOAc fraction, and their structures were identified based on spectral analyses (MS, 1H-NMR, 13C-NMR). Twenty-eight compounds were obtained, and characterized as erythrocentaurin (1), erythrocentaurin dimethylacetal (2), swertiamarin (3), vogeloside (4), 2'-O- actylswertiamarin (5), swertianoside D (6), gentiocrucines A-B (7-8), gentiocrucine (9), 1-hydroxy-3, 7, 8-trimethoxyxanthone (10), 1-hydroxy-3, 5, 6-trimethoxyxanthone (11), 3-epitaraxerol (12), erythrodiol 3-O-palmitate (13), (+) -syringaresinol (14), caffeic acid (15), trans-coniferyl aldehyde (16), trans-coniferyl alcohol (17), 3, 4-dihydroxybenzoic acid (18), 4-hydroxy-3-methoxybenzoic acid (19), 3, 4-dihydroxybenzoic aldehyde (20), 2, 3-dihydroxybenzoic acid (21), 4-hydroxybenzoic acid (22), 3-acetoxybenzoic acid (23), 3-hydroxybenzoic acid (24), 3-hydroxybenzoic alcohol (25), nicotinic acid (26), 2-furoic acid (27), and uracil (28). Compounds 1-4, 6-28 were obtained from S. kouitchensis for the first time.
Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Magnetic Resonance Spectroscopy ; Mass Spectrometry ; Molecular Structure ; Swertia ; chemistry

OBJECTIVETo develop an HPLC method for simultaneous determination of swertiamarin, gentiopicroside, sweroside, mangiferin, erythrocentaurin, and to detect these five constituents in eight Qingyedans derived from Swertia mileensis, S. cincta, S. patens, S. punicea, S. delavayi, S. nervosa, S. macrosperma and S. yunnanensis.

METHODThe separation was carried out on a Thermo BDS Hypersil C18 (4. 6 mm x 250 mm, 5 microm) column eluted with mobile phase of water containing 0. 1% phosphoric acid and methanol (B) in gradient program (0-10 min, 18%-20% B; 10-30 min, 20%-35% B; 30-35 min, 35%-60% B). The column temperature was 32 degrees C , and the detection wavelength was set at 250, 260, 225 nm. The flow rate was 0. 7 mL . min-1 from 0 to 30 min, and be increased to 1. 0 mL . min-1 in 35 min.

RESULTThe five compounds were well separated. The linear response ranges of swertiamarin, gentiopicroside, sweroside, mangiferin, erythrocentaurin were 0. 072-13. 39, 0. 1204. 518, 0. 060-5. 050, 0. 025-1. 518, and 0. 031-0. 210 microg, respectively. The mean recoveries of five compounds were 97.03% -102. 7% (RSD 1. 8% -6.2% ). There are swertiamarin, gentiopicroside and sweroside in most samples, and mangiferin in half samples. But erythrocentaurin was only detected in a few samples. The contents of five compounds were different in different samples. The contents of swertiamarin in S. mileensis, S. patens, S. yunnanensis and S. delavayi are up to 34. 47-118.05 mg . g-1, the contents of gentiopicroside are up to 25. 91 mg . g-1 in S. cincta. In S. puncea all contents of swertiamarin, gentiopicroside, sweroside and mangiferin are higher, especially the content of sweroside. There are Xiao-Qingyedans and Da-Qingyedans called in markets, and they can be identified by the contents of swertiamarin, gentiopicroside and sweroside. S. punicea can be identified by the content of sweroside, and the ratio gentiopicroside/total content can be used for identification of S. cincta from other seven Qingyedan species.

CONCLUSIONThe method was certified to be accurate and reliable and can be used for identification and quality evaluation of traditional Chinese medicine Qingyedan derived from Swertia species.