Difference analysis of chemical components in roots and rhizomes of 3 medicinal plants of Gentiana from the same origin / 中国药房

OBJECTIVE To provide reference for exploring alternative resources of Gentiana rigescens from the plants of Gentiana. METHODS The contents of four components （gentiopicroside， swertiamarin， swertioside and amarogentin） in the roots and rhizomes from 3 plants of Gentiana （G. rigescens， G. cephalantha， G. delavayi） were determined by high-performance liquid chromatography （HPLC）. The chemical compositions in the above roots and rhizomes were identified by ultra-performance liquid chromatography electrospray ionization quarter-time of flight mass spectrometry （UPLC-ESI-Q-TOF-MS）， and the differences were analyzed by principal component analysis （PCA）. RESULTS Four active components such as gentiopicroside， swertiamarin， swertioside and amarogentin were detected in the roots and rhizomes of G. rigescens and G. cephalantha， and the contents of the four components were similar in both. The contents of gentiopicroside in the root and rhizome of G. cephalantha and G.rigescens were more than four times of the limit standard of the Chinese Pharmacopoeia （Part Ⅰ） in 2020； However， only swertiamarin， swertioside and amarogentin were detected in the roots and rhizomes of G.delavayi， and the contents of swertioside and amarogentin were 34.12 and 8.81 times of those of G. rigescens， respectively. In addition， a total of 33 compounds 术。E-mail：515227235@qq.com were identified from the roots and rhizomes of 3 plants of Gentiana by UPLC-ESI-Q-TOF-MS， mainly iridoids. Additionally， G. rigescens and G. cephalantha contained xantones， G. delavayi contained flavonoids. PCA showed that there was a small difference between G. rigescens and G. cephalantha； however， there was a big difference between G. delavayi and G. rigescens. CONCLUSIONS The difference between the roots and rhizomes of G. cephalantha and G. rigescens from the same origin is small and there is substitutability； while the difference in the chemical components from roots and rhizomes between G. delavayi and G. rigescens is great and G. delavayi cannot be used as medicine instead of G. rigescens.

Q-marker prediction analysis of Gentiana rigescens Franch. based on HPLC and network pharmacology / 药学学报

Aa a characteristic medicinal plant in China, Gentiana rigescens Franch. has the function of protecting the liver and invigorating the spleen. At present, there are a few studies on the content determination method of characteristic components of G. rigescens, so it is necessary to establish a scientific and effective quality control method; In this study, The high performance liquid chromatography (HPLC) fingerprint of G. rigescens was established, based on literature reviewed and characteristic spectrum identified, the source range of G. rigescens quality marker (Q-marker) was screened. The effectiveness of the ingredients and the corresponding targets and pathways was analyzed through network pharmacology, and drew the diagram of ''component-target-pathway''. Qualitative and quantitative analysis of G. rigescens was performed by HPLC, and screen the main marker components leading to the differences between groups which were determined the Q-marker of G. rigescens; The literature and HPLC had determined that five iridoids were the main source of G. rigescens Q-marker. The network pharmacology (effectiveness) and qualitative and quantitative (detectability) analysis of G. rigescens from different producing areas confirmed that gentiopicroside, swertiamarin, and sweroside can be used as the main landmark components, and there were significant differences in their contents among different producing areas; The analysis of G. rigescens from different producing areas was carried out by network pharmacology and chemical fingerprints, it is confirmed can be used as potential Q-marker to provide sufficient theoretical basis for the quality control of G. rigescens in the later period.

Optimization of Primary Processing Technology of Gentiana rigescens by Box-Behnken Design-response Surface Method / 中国药房

OBJECTIVE：To optimize the primary processing technology of Gentiana rigesce ns. METHODS ：HPLC method was adopted for content determination of loganic acid ，swertiamarin and gentiopicroside in G. rigescens ，and overall desirability value （OD value ） of the contents of above 3 components was taken as index to carry out single factor test on blanching temperature，blanching time and drying temperature. Based on that ，Box-Behnken design-response surface methodology was used to optimize primary processing technology of G. rigescens . Validation test was also performed. The samples prepared by optimized technology were compared with those dried in the shade. RESULTS ：The optimal primary processing technology of G. rigescens included blanching time of 5 min，blanching temperature of 40 ℃ and drying temperature of 60 ℃. Validation test showed that the average OD value of the 3 components was 0.565 2，with a deviation of 0.94％ from the predicted value （0.570 6）. Compared with samples dried in the shade ，OD value of 3 components in samples prepared by optimized technology were increased significantly ， indicating the quality of the samples prepared by the optimized technology was better. CONCLUSIONS ：The optimal technology is stable and feasible ，and can be used for the primary processing of G. rigescens .

Distribution and diversity of endophytic fungi in Gentiana rigescens and cytotoxic activities / 中草药·英文版

Objective: In the present study, Gentiana rigescens was screened for fungi communities to clarify their diversity and community assemblage in hosts. Meanwhile, the identification and activity assays of the strains were also conducted. Methods: By culture-dependent (endophytic fungi isolations from plant sections) and culture-independent (metagenomic library and cloning from plant sections) techniques, fungi communities were studied. The metagenomic library was generated using direct DNA isolation of whole plants, plant radixes, plant stems, plant leaves, plant flowers and soils around the plant. Meanwhile, endophytes were isolated from all parts of G. rigescens plants. After fermentation of the fungi isolations, all the isolates were evaluated for their cytotoxicity against four kinds of human cancer cell lines (HCT116, BEL7404, A549, MDA-MB-231). Results: Eventually, 200 strains were isolated and 103 strains were further identified through the internal transcribed spacer (ITS, ITS1 and ITS2 regions) sequence by using the universal primers ITS5 and ITS4. A total of 59,106 fungal sequences corresponding to 374 putative operational taxonomic units (OTU) were identified by 454 pyrosequencing. Through 454 pyrosequencing, the main fungal genera were Sebacina, Botrytis, Mycosphaerella, Boletus and Gibberella, and the major fungal genera which were directly isolated were Aspergillus, Fusarium, Penicillium and Alternaria. Activity assays showed strains 5–26 (Aspergillus sp.) and 6–2 (Fusarium avenaceum) had the outstanding cytotoxicity to all the tested cell lines with IC50 values <5 μg/mL. Conclusion: This study revealed the abundance of endogenetic fungal resources and a variety of genetic information in G. rigescens by high-throughput 454 sequencing technology and fungi isolation methods. Activity assays indicated that endophytes were a promising natural source of potential anticancer agents.

Effects of Methyl Jasmonate on the Contents of 4 Kinds of Secoiridoid Substance in the Leaves of Gentiana rigescens / 中国药房

OBJECTIVE： To study the effects of exogenous methyl jasmonate on the contents of 4 kinds of secoiridoid substance （gentiopicrin， swertiamain， sweroside and amarogentin） and biochemical indexes in the leaves of Gentiana rigescens， and to provide scientific evidence for the application of methyl jasmonate in standardized planting of G. rigescens. METHODS： 2， 4， 6， 8， 10 d after spraying 10， 50， 100， 200 mg/L methyl jasmonate （spraying amount was 500 mL） for G. rigescens strain， the leaves of medicinal material were collected as sample， and the other leaves without spraying methyl jasmonate were collected as control. HPLC method was used to determine the contents of 4 kinds of secoiridoid substance in the leaves of G. rigescens after spraying 4 kinds of concentrations of methyl jasmonate for 10 d. The concentration of methyl jasmonate was optimized （the content of effective component was the highest）. HPLC method was adopted to determine the contents of 4 kinds of secoiridoid substance in the leaves of G. rigescens after spraying the optimal concentration of methyl jasmonate for different time. The levels of relevant biochemical indexes （SOD， POD， CAT， MDA） were determined. RESULTS： 10 d after spraying 10， 50， 100， 200 mg/L methyl jasmonate for G. rigescens strains， the contents of 4 kinds of secoiridoid substance in the leaves were increased to different extent. Compared with untreated leaves， 100 mg/L methyl jasmonate had the best effect after spraying， and the contents of gentiopicrin， swertiamain and sweroside in treated leaves were 1.88， 2.36 and 1.87 times of those in untreated leaves， respectively （P＜0.05）. 2， 4， 6， 8， 10 d after spraying 100 mg/L methyl jasmonate， the contents of 4 kinds of secoiridoid substance in treated leaves were higher than untreated leaves at corresponding stage； the content of secoiridoid had significant difference after spraying for 4 d （P＜0.05）. The contents of active components in general were relatively high after spraying for 6 d， and the contents of gentiopicrin， swertiamain， sweroside and amarogentin in treated leaves were 1.88， 1.88， 1.47， 1.82 times of those in untreated leaves， respectively （P＜0.05）. The levels of relevant biochemical indexes in treated leaves were increased significantly since 4 d of spraying， compared with untreated leaves （P＜0.05）. CONCLUSIONS： After spraying 100 mg/L methyl jasmonate for 6 d， the contents increase of 4 kinds of secoiridoid substance in the leaves of G. rigescens are most obvious， which may be associated with improving the levels of related biochemical indexes.

Study on the Contents of Secoiridoid Substances in the Leaves of Gentiana rigescens from Different Populations in Dali Bai Autonomous Prefecture / 中国药房

OBJECTIVE: To explore the contents of secoiridoid substances (gentiopicroside, swertiamarin, sweroside) in the leaves of Gentiana rigescens from different populations in Dali Bai Autonomous Prefecture (Shorted for Dali prefecture), and to provide reference for the selection of fine varieties, the development and utilization of G.rigescens. METHODS: The contents of secoiridoid substances in the leaves of G. rigescens from different populations in Dali prefecture were determined by HPLC, and the HPLC fingerprint was established. The contents of secoiridoid substances in the leaves of G. rigescens from different populations was studied by using single factor variance analysis, clustering analysis and principal component analysis. RESULTS: There were statistical significance in the contents of gentiamarin, swertiamarin and sweroside and the total contents in the leaves of G. rigescens from different populations in Dali prefecture (P<0. 05 or P<0. 01). Twelve common peaks were found in HPLC fingerprint, and HPLC fingerprint similarities of common peaks except Gantong population were all over 0. 972. Three peaks of them were identified as the characteristic peaks of gentiamarin, swertiamarin and sweroside which were correlated to the activities. The total contents of gentiamarin, swertiamarin and sweroside from different populations obtained by clustering analysis were consistent with that by HPLC fingerprint principal component analysis. CONCLUSIONS: There are great differences in the content of gentiamarin, swertiamarin and sweroside in the leaves of G. rigescens from different populations in Dali prefecture. The similarity of secoiridoid substances in the leaves of G. rigescens from different populations except Gantong population is higher.

Data fusion and multi-components quantitative analysis for identification and quality evaluation of Gentiana rigescens from different geographical origins / 中国中药杂志

The accumulation of secondary metabolites of traditional Chinese medicine (TCM) is closely related to its origins. The identification of origins and multi-components quantitative evaluation are of great significance to ensure the quality of medicinal materials. In this study, the identification of Gentiana rigescens from different geographical origins was conducted by data fusion of Fourier transform infrared (FTIR) spectroscopy and high performance liquid chromatography (HPLC) in combination of partial least squares discriminant analysis; meanwhile quantitative analysis of index components was conducted to provide an accurate and comprehensive identification and quality evaluation strategy for selecting the best production areas of G. rigescens. In this study, the FTIR and HPLC information of 169 G. rigescens samples from Yunnan, Sichuan, Guangxi and Guizhou Provinces were collected. The raw infrared spectra were pre-treated by multiplicative scatter correction, standard normal variate (SNV) and Savitzky-Golay (SG) derivative. Then the performances of FTIR, HPLC, and low-level data fusion and mid-level data fusion for identification were compared, and the contents of gentiopicroside, swertiamarin, loganic acid and sweroside were determined by HPLC. The results showed that the FTIR spectra of G. rigescens from different geographical origins were different, and the best pre-treatment method was SNV+SG-derivative (second derivative, 15 as the window parameter, and 2 as the polynomial order). The results showed that the accuracy rate of low- and mid-level data fusion (96.43%) in prediction set was higher than that of FTIR and HPLC (94.64%) in prediction set. In addition, the accuracy of low-level data fusion (100%) in the training set was higher than that of mid-level data fusion (99.12%) in training set. The contents of the iridoid glycosides in Yunnan were the highest among different provinces. The average content of gentiopicroside, as a bioactive marker in Chinese pharmacopoeia, was 47.40 mg·g⁻¹, and the maximum was 79.83 mg·g⁻¹. The contents of loganic acid, sweroside and gentiopicroside in Yunnan were significantly different from other provinces (<0.05). In comparison of total content of iridoid glycosides in G. rigescens with different geographical origins in Yunnan, it was found that the amount of iridoid glycosides was higher in Eryuan Dali (68.59 mg·g⁻¹) and Yulong Lijiang (66.68 mg·g⁻¹), significantly higher than that in Wuding Chuxiong (52.99 mg·g⁻¹), Chengjiang Yuxi (52.29 mg·g⁻¹) and Xundian Kunming (46.71 mg·g⁻¹) (<0.05), so these two places can be used as a reference region for screening cultivation and excellent germplasm resources of G. rigescens. A comprehensive and accurate method was established by data fusion of HPLC-FTIR and quantitative analysis of HPLC for identification and quality evaluation of G. rigescens, which could provide a support for the development and utilization of G. rigescens.

Efficient induction system of adventitious shoot and plantlet regeneration in Gentiana rigescens / 中草药

Objective: An efficient and stable regeneration system was developed to select the optimal protocol for plant regeneration with various explants of Gentiana rigescens. Methods: The study was designed as a L9(34) orthogonal experiment to explore the effects of different types and concentration of plant growth regulator combinations on adventitious shoot formation and plant regeneration, using different explants maintained on Murashige and Skoog (MS) medium after a single factor pre-experiment had been conducted. Results: Stem with buds was the optimal explant for indirect organogenesis with a frequency of callus induction of up to 97.73% obtained on MS + 6-BA 1.5 mg/L + NAA 1.0 mg/L + KT 0.05 mg/L after 10 dwith the axillary shoots starting to germinate along with the emergence of callus with a strong capacity of differentiation from the base; The callus started to generate green multiple shoots with a 100% rate after 15 d and a coefficient of differentiation in the clump shoot of 18.65. The multiplication coefficient was up to 63.58. However, the stem tip was the best explant for direct organogenesis with a propagation coefficient of up to 44.36 cultured on MS + 6-BA 0.5 mg/L + NAA 1.0 mg/L + KT 0.5 mg/L 30 days later. The plantlet derived from two explants was too weak for rooting and the weak multiplication plantlet was maintained on MS medium supplemented with 6-BA 1.5 mg/L, NAA 1.0 mg/L, KT 0.05 mg/L, and PP333 10 mg/L to rejuvenation for 60 d. The regeneration plant with 100% rooting rate was obtained by culturing on 1/2 MS + 6-BA 0.1 mg/L + NAA 2.0 mg/L for 45 d and more than 95% of plantlets survived after transplanting. Conclusion: The current study established a rapid propagation system which is helpful to protect the wild resources and high quality seedling propagation of G. rigescens, meanwhile providing scientific evidence for the research on genetic transformation.

Cloning and bioinformatics analysis of iridoid oxidase gene in Gentiana rigescens / 中草药

Objective To obtain the key enzyme gene involving in the monoterpenoid biosynthesis pathway, an iridoid oxidase gene (GrIDO) and its promoter were cloned from the leaves of Gentiana rigescens, and its bioinformatics analysis were also performed. Methods The gene specific primers were designed according to the GrIDO gene of transcriptome in G. rigescens. The open reading frame (ORF) of GrIDO gene was cloned by RT-PCR method. The bioinformation of GrIDO gene was analyzed by online softwares. Meanwhile, the gene specific primers were designed according to the cloned GrIDO gene, and the promoter of GrIDO gene was amplified by PCR method. Its sequence analysis was also performed. Results The length of GrIDO gene ORF (GenBank accession number KP722034) was 1 557 bp, which encoded a protein with 518 amino acids. Its relative molecular weight was 58 920 with the theoretical isoelectric point of 8.40. GrIDO was the member of cytochrome P450 superfamily and may localize in chloroplast. GrIDO was a hydrophilic stable protein without signal peptide and composed of mainly α-helix (51.07%) and loops (42.69%). GrIDO protein had a high similarity with CrIDO of Catharanthus roseus (85.83%) and their genetic relationship was close. The cloned GrIDO promoter had a length of 720 bp (GenBank accession number KT428570), which possessed cis-regulatory elements TATA-box and CAAT-box, and had cis-acting elements involved in the abscisic acid and MeJA responsiveness, part of a light responsive element and MYB transcription factor binding site. Conclusion The expression of GrIDO gene were regulated by multifactors. The GrIDO gene and its promoter were cloned from G. rigescens. This study will lay foundations for the functional research of GrIDO gene in monoterpenoid biosynthesis pathway.

Molecular cloning, differential expression, and bioinformatics analysis of G10H gene in Gentiana rigescens / 中草药

Objective: To clone the geraniol-10-hydroxylase (G10H) gene from Gentiana rigescens and analyze the gene expression. Methods: The cDNA sequence of G10H was obtained from G. rigescens using PCR technique. The physical and chemical properties, secondary structure and tertiary structure of G10H protein were forecasted and analyzed using related software. The expression of G10H gene was detected using real-time PCR in roots, stems, and leaves of G. rigescens. Results: The cloned G10H gene was 1 400 bp including 1 248 bp open reading frame and encoding a predicted protein of 415 amino acids. Bioinformatics predicted that the gene encoding protein molecular formula was C2131H3390N586O615S17, the isoelectric point was 7.62, the instability coefficient was 44.20, and the hydrophobic coefficient was -0.245. RT-PCR showed that G10H gene expressed in all organs. And the highest expression level was found in roots, while the lowest in the stems. Conclusion: This G10H gene is cloned from G. rigescens for the first time. The results provide a foundation for exploring the mechanism of gentiopicroside biosynthesis in G. rigescens.

Liver-protective and anti-inflammatory effects of extracts from different parts of Gentiana rigescens / 中成药

AIM To compare liver-protective and anti-inflammatory effects of extracts from root,stem,leaf,and flower of Gentiana rigescens.METHODS The mouse model for the immunological liver injury induced by Concanavalin A,and mouse ear swelling model for inflammation caused by dimethylbenzene were used for the comparison of the liver-protective or anti-inflammatory effects of four parts individually.RESULTS Four aqueous extracts of Gentiana rigescens showed the dose-dependent decrease in the activity of ALT and AST in serum and liver index,and alleviation of hepatic tissue injury induced by Concanavalin A in mice.The effects of the extracts from the leaf and root were better than those from the stem and flower.These extracts presented dose-dependent inhibition against the ear swelling caused by dimethylbenzene in mice.The effects of the extracts from the leaf and stem were better than those from the flower and root.CONCLUSION Extracts from the root and leaf of G.rigescens have liver-protective effect,and parts from the stem and leaf have anti-inflammatory effect.

HPLC characteristic fingerprints of Gentiana scabra Bge. and Gentiana rigescens Franch. based on the polarity orientation technology / 中国药学杂志

OBJECTIVE: To establish the HPLC characteristic fingerprints of Gentiana scabra Bge. and G. rigescens Franch. METHODS: The characteristic fingerprints were established based on polarity orientation technology. Similarity analysis, cluster analysis, and principal component analysis were used to evaluate the fingerprints. RESULTS: The HPLC fingerprints of ethyl acetate fraction from the water extract of G. scabra and G. rigescens showed obvious characteristics. The overall similarity of 21 batches of samples was 0.29-0.95. The overall similarity of G. scabra was 0.82-0.99 except S4. And that of G. rigescens was 0.75-0.99 among which 10 batches were greater than 0.90. In the cluster analysis, the samples were divided into three clusters: S4, G. scabra, and G. rigescens. In the principal component analysis, 21 batches of G. scabra. and G. rigescens could be clearly divided into two categories, while S4 was far away from the other samples of G. scabra. CONCLUSION: G scabra and G. rigescens can be identified effectively by the characteristic fingerprints. The method can be used for the quality control of Radix Gentianae.

Chemoetrics analysis of HPLC fingerprint of Gentiana rigescens from different multiple cropping systems and application in quality evaluation / 中国药学杂志

OBJECTIVE: To establish the HPLC fingerprint of Gentiana rigescens Franch. from different cultivation systems for the quality evaluation of the medicinal materials in combination with chemometrics analysis. METHODS: Four bioactive compounds, ie loganic acid, swertiamarin, gentiopicroside, and sweroside, were qualitatively and quantitatively analyzed. The chromatographic analysis was performed on a Shim-pack VP-ODS C18 column(4.6 mm×150 mm, 5 μm) at a temperature of 30℃. Linear gradient elution was used with mobile phase consisting of water with 0.1% formic acid(A) and acetonitrile(B). The flow rate was 1.00 mL·min-1. The injection volum was 10 μL. The detection wavelength was set at 241 nm. Chemometrics methods including hierarchical cluster, partial least squares discriminant analysis, and variable importance were used for this study. RESULTS: The calibration curves of the four compounds were linear in the range of 10-4000 μg·mL-1. The coefficients ranged from 0.9992 to 0.9999. The precision RSDs of the four compounds were 0.87%, 2.06%, 0.90% and 2.2%, respectively. The repeatability RSDs were 0.97%, 1.9%, 2.0% and 2.2%, respectively. The stability RSDs were lower than 2.2%. The recoveries of the four compounds varied from 97.12% to 103.81% with RSDs from 0.71% to 2.0%. The similarity analysis showed similarity values over 0.970 for most samples. The constituents of 70 samples collected from different cultivation systems were similar. However, the contents of those compounds varied significantly in the following order; gentiopicroside>swertiamarin>loganic acid>sweroside. The chemometrics characteristics of gentiopicroside was significantly different from the other compounds. The total content of the four compounds was the highest in the samples cultivated under Alnus nepalensis D. Don. The samples cultivated under Juglans regia Linn, had the highest content of loganic acid. The samples cultivated under Eucalyptus robusta Smith, had the highest content of swertiamarin. The samples cultivated under Cunninghamia lanceolata (Lamb.) Hook, had the highest content of sweroside. The results of partial least squares discriminant analysis and variable importance for the study showed that gentiopicroside (VIP=1.513) and swertiamarin (VIP=1.208) were important variables for discrimination of cultivation system. The compounds with retension time of more than 10 min were important for discrimination of cultivation condition too. CONCLUSION: HPLC fingerprint combined with contents of main bioactive compounds could provide method and theoretical basis for the quality evaluation of G. rigescens Franch. from different multiple cropping systems.

Quality evaluation and quality equivalence of Gentiana rigescens Franch. from different populations / 中国药学杂志

OBJECTIVE: To establish evaluation methods of the quality and quality equivalence of Gentiana rigescens Franch. from different populations. METHODS: The contents of the active ingredients, gentiopicroside, swertiamarin and sweroside in the roots of Gentiana rigescens Franch., were determined by HPLC, and the HPLC fingerprint was established. The quality and quality e-quivalence were evaluated by One-way ANOVA, cluster analysis and principal component analysis (PCA). RESULTS: The overall quality of Gentiana rigescens Franch. from different populations in Dali Yunnan was better. The content of gentiopicroside in the sample from Weishan population was lower than those in the other populations, while it was more than twice the national standard. There was significant difference (P<0.01) in the contents of gentiopicroside, swertiamarin, and sweroside in the roots of Gentiana rigescens Franch. from different populations. The quality equivalence of Gentiana rigescens Franch. from different populations was analyzed, and 11 common peaks in HPLC fingerprint were selected to evaluate the similarities of the crude drugs. The similarities of the characteristic peaks were all above 0.988. The three characteristic peaks of gentiopicroside, swertiamarin and sweroside which were related to the activity of Gentiana rigescens Franch were identified, and the qualities of different populations were basically equivalent. The total contents of gentiopicroside, swertiamarin and sweroside of Gentiana rigescens Franch. from different populations were similar as shown by cluster analysis and PCA of HPLC fingerprint. CONCLUSION: Jianchuan and Heqing polulations are quality germplasm populations according to the contents of effective components and HPLC fingerprint. The evaluation methods of quality and quality equivalence of Gentiana rigescens Franch. from different populations are established, which would provide the theoretical basis for further filtering of quality germplasm resources of Gentiana rigescens Franch. from wild populations.

Cloning, sequence analysis, and prokaryotic expression of 1-deoxy-D-xylulose-5- phosphate reductoisomerase GrDXR gene in Gentiana rigescens / 中草药

Objective: To clone the 1-deoxy-D-xylulose 5-phosphate reductoisomerase gene GrDXR which is the key enzyme involving in the terpenoid biosynthesis from young leaves of Gentiana rigescens, and to perform its sequence analysis and prokaryotic expression. Methods: According to the GrDXR gene sequence of G. rigescens transcriptome, a pair of primers were designed, and the ORF of cDNA sequence was obtained by RT-PCR. Then TA cloning, sequencing, and sequence analysis were performed. Prokaryotic expression vector pGEX-4T-1-GrDXR was constructed and transformed into Escherichia coli Rosetta (DE3) for expression under the induction of IPTG. Results: The ORF of GrDXR had a length of 1 425 bp coding for 474 amino acids. Sequence analysis showed that GrDXR was the member of DXR family. Results of phylogenic analysis showed that GrDXR was close to RvDXR, HbDXR, and CrDXR. The pGEX-4T-1-GrDXR recombinant plasmid was constructed and the stable prokaryotic expression system was obtained. The SDS-PAGE results displayed that the expressed proteins were consistent with the anticipated size. Conclusion: The GrDXR gene is successfully cloned, and the stable prokaryotic expression system is established. This study will provide a foundation for further purification, structural and functional research of GrDXR protein.

Cloning, sequence analysis, and prokaryotic expression of GrCYP450-17 gene in Gentiana rigescens / 中草药

Objective: To obtain the key enzyme gene GrCYP450 involving in the terpenoid biosynthesis, a CYP450-17 gene was cloned from young leaves of Gentiana rigescens, and its sequence analysis and prokaryotic expression were performed. Methods: According to the GrCYP450-17 gene sequence of transcriptome of triennial G. rigescens, a pair of primers were designed, and the ORF (Open reading frame)of cDNA sequences was obtained by RT-PCR. Then TA cloning, sequencing, and sequence analysis were performed. Prokaryotic expression vector pGEX-4T-1-GrCYP450-17 was constructed and transformed into E. coli Rosetta (DE3) for the expression under the induction of Isopropyl β-D-1-Thiogalactopyranoside (IPTG.). Results: The ORF of GrCYP450-17 has a length of 1 545 bp coding for 514 amino acids. Sequence analysis showed that GrCYP450-17 was the member of CYP714 family. Results of phylogenic analysis showed that GrCYP450-17 was close to SlCYP450 of tomato. The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size. Conclusion: The GrCYP450-17 gene is successfully cloned, and the stable prokaryotic expression system is established. This study will provide a foundation for the further purification, structural and functional researches of GrCYP450-17 protein.

Cloning, sequence analysis, and prokaryotic expression of GrGPPS gene in Gentiana rigescens / 中草药

Objective: To obtain the indispensable key enzyme involved in the monoterpene biosynthesis, the geranyl diphosphate synthase gene GrGPPS was cloned from Gentiana rigescens, and its sequence analysis and prokaryotic expression were performed. Methods: According to the GrGPPS gene sequence of transcriptome of triennial G. rigescens, a pair of specific primers was designed, and the full length of cDNA sequences was obtained by RT-PCR. Then TA cloning, sequencing and sequence analysing were performed. Prokaryotic expression vector pGEX-T-1-GrGPPS was constructed and transformed into Escherichia coli Rosetta for expression under 37°C and induced by 1 mmol/L IPTG. Results: The GrGPPS cDNA had a length of 1107 bp coding for 369 amino acids. Sequence analysis showed that GrGPPS was the member of "short-chain prenyltransferases" super family. Results of phylogenic analysis showed that GrGPPS was at the same evolutionary branch with AmGPPS. The SDS-PAGE results displayed that the expressed proteins were consistent with the anticipated size. Conclusion: The GrGPPS gene is cloned from G. rigescens, and the stable prokaryotic expression system of pGEX-T-1-GrGPPS is constructed. This work will provide a foundation for further purification, structure, and functional research of GrGPPS protein.

Comparison on HPLC fingerprints of pre-and post processed Radix et Rhizoma Gentiana rigescen with wine-fried method / 中成药

AIM: To establish HPLC fingerprint to make the difference between pre-and post processed Radix et Rhizoma Gentiana rigescen with wine-fried method. METHODS: Chromatographic fingerprint between pre-and post processed Radix et Rhizoma Gentiana rigescen with wine-fried was investigated by RP-HPLC and the gradient elution with acetonitrile and 0.1% phosphoric acid was applied to chromatographic separation(Alltima C_18(4.6 mm ?250 mm,5 ?m) column) for 65 min,and detection wavelength of 225 nm.Data were analysed by fingerprint similarity evaluation software to compare the similarity among the samples. RESULTS: The similitude degree of the10 batches of pre-and post processed Radix et Rhizoma Gentiana rigescen with wine-fried method were between 0.935-0.997 and 0.964-0.999,respectively.Comparing with the common processing of the HPLC fingerprint of Radix et Rhizoma Gentiana rigescen,the similarity of Radix et Rhizoma Gentiana rigescen processed with wine-fried was greater than 0.900. CONCLUSION: There have no significant deviation between semipolarity components of the pre-and post processed Radix et Rhizoma Gentiana rigescen with wine-fried method. The proportion of the main relative peak area has changed.