Transcriptome analysis and validation of key genes involved in biosynthesis of iridoids in Gentiana lhassica / 中国中药杂志

As the main chemical constituents, iridoids are widely distributed within Gentiana, Gentianaceae, with promising bioactivities. Based on the previous work, the transcriptome of G. lhassica, an original plant of Tibetan herb "Jieji Nabao", was sequenced and analyzed in this study, and the transcriptome databases of roots, stems, leaves, and flowers were constructed so as to explore unigenes that may encode the key enzymes in the biosynthetic pathway of iridoids. Then, qRT-PCR was used to validate the relative expression levels of 11 genes named AACT, DXS, MCS, HDS, IDI, GPPS, GES, G10H, 7-DLNGT, 7-DLGT, and SLS in roots, stems, leaves, and flowers. Also, the total contents of gentiopicroside and loganic acid were determined by HPLC, respectively. The results are as follows:(1)a total of 76 486 unigenes with an average length of 852 bp were obtained;(2)335 unigenes were involved in 19 stan-dard secondary metabolism pathways in KEGG database, with phenylpropanoid biosynthesis having the maximum number(75 unigenes), and no isoflavone biosynthetic pathway was annotated;(3)171 unigenes participatedin 27 key enzymes encoding in the biosynthetic pathway of iridoids, and 1-deoxy-D-xylulose-5-phosphate reductoisomerase(DXR) gene was highly expressed;(4)qRT-PCR results were approximately consistent with RNA-Seq data and the relative expression levels of the 11 genes were higher in the aboveground parts(stem, leaf, and flower) than in the underground part(root);(5)the total contents of gentiopicroside and loganic acid were higher in the aboveground parts(stem, leaf, and flower) than in the underground part(root), and the difference was significant. This study provides basic scientific data for accurate species identification, evaluation of germplasm resources, research on secondary pro-duct accumulation of medicinal plants within Gentianaceae, and protection of endangered alpine species.

DNA super-barcoding of several medicinal species in Gentiana from Yunnan province / 中国中药杂志

Gentiana is an important but complicated group in Gentianaceae. The genus covers numerous medicinal plants which are difficult to be identified. In the present study, several medicinal species in Gentiana from Yunnan province, including G. rigescens, G.rhodantha, and G. delavayi, were sequenced using the Illumina HiSeq 2500 system. Three complete chloroplast genome sequences were obtained after assembly and annotation. According to several published genome sequences of G. crassicaulis, the DNA super-barcoding of species in Gentiana was preliminarily carried out. The results revealed that chloroplast genomes of the three species were conservative with short lengths(146 944, 148 992, and 148 796 bp, respectively). The genomes encoded 114 genes, including 78 protein-coding genes, 30 tRNA genes, 4 rRNA genes, and 2 pseudogenes. Furthermore, these medicinal species in Yunnan province were identified using DNA super-barcoding based on chloroplast genomes. The results showed that the Gentiana species could be gathered into monophyletic branches with a high support value(100%). It indicated that DNA super-barcoding possessed obvious advantages in discriminating species in complicated genera. This study is expected to provide a scientific basis for the identification, utilization, and conservation of Gentiana species.

Rhodanthpyrone A and B play an anti-inflammatory role by suppressing the nuclear factor-κB pathway in macrophages

Macrophage-associated inflammation is crucial for the pathogenesis of diverse diseases including metabolic disorders. Rhodanthpyrone (Rho) is an active component of Gentiana rhodantha, which has been used in traditional Chinese medicine to treat inflammation. Although synthesis procedures of RhoA and RhoB were reported, the biological effects of the specific compounds have never been explored. In this study, the anti-inflammatory activity and mechanisms of action of RhoA and RhoB were studied in lipopolysaccharide (LPS)-stimulated macrophages. Pretreatment with RhoA and RhoB decreased inducible nitric oxide synthase and cyclooxygenase-2 expressions in RAW 264.7 cells and in thioglycollate-elicited mouse peritoneal macrophages. In addition, it downregulated transcript levels of several inflammatory genes in LPS-stimulated RAW 264.7 cells, including inflammatory cytokines/chemokines (Tnfa, Il6, and Ccl2) and inflammatory mediators (Nos2 and Ptgs2). Macrophage chemotaxis was also inhibited by treatment with the compounds. Mechanistic studies revealed that RhoA and RhoB suppressed the nuclear factor (NF)-κB pathway, but not the canonical mitogen activated protein kinase pathway, in LPS-stimulated condition. Moreover, the inhibitory effect of RhoA and RhoB on inflammatory gene expressions was attenuated by treatment with an NF-κB inhibitor. Our findings suggest that RhoA and RhoB play an anti-inflammatory role at least in part by suppressing the NF-κB pathway during macrophage-mediated inflammation.

An Innovative Scalp-Dyeing Technique with Gentian Violet Solution During Follicular Unit Extraction for White-Haired Follicular Units

There exist some restrictions and difficulties in performing follicular unit extraction (FUE) in white-haired patients, for several reasons. In this paper, we introduce a novel technique for visualizing white hair during the punching procedure and graft preparation in FUE for white-haired patients. In white-haired older male patients, we dyed the surrounding scalp skin purple with a gentian violet solution-stained toothpick. Our method has several advantages: surgeons can easily focus on the center of the follicular unit and rapidly perform punching, they can recognize the condition of the harvested follicular units during FUE, and the hair transplant team can secure a clear view for trimming and loading into the implanter. We suggest that scalp dyeing in difficult FUE procedures, especially in patients with white hair, may be a simple method that provides a good visualization for donor site harvesting and for microdissection.

Quality standard study on Tibetan medicine Gentianae Szechenyii Flos / 中国中药杂志

In order to efficiently control the quality of the Tibetan medicine Gentianae Szechenyii Flos, the quality standard was established in this study. The tests of water content, total ash and ethanol-soluble extractives of the crude drugs were carried out based on the methods recorded in appendix of Chinese Pharmacopeia (2010 edition, volume 1). The TLC method was established by using reference drug and gentiournoside A as reference substance, and a mixture of ethyl acetate-methanol-water-formic acid (7: 1.5: 1: 0.2) as the developing solvent system on silica gel G TLC plate. The content of gentiournoside A was assayed by HPLC on a Ultimate XB-C18 (4.6 mm x 250 mm, 5 μm) column, using methanol-water (0.02% phosphoric acid) (52:48) as the mobile phase at a flow rate of 1.0 mL x min(-1). The column temperature is 25 degrees C and the detection wavelength is at 240 nm. As a result, gentiournoside A and the other constituents were separated and presented the same fluorescence light comparing with the reference substance on TLC detected under the UV light(366 nm). The methodology validation for the assay of gentiournoside A showed that it was in a good linear correlation in the range of 10.01-400.32 mg x L(-1) with the regression equation of Y = 1 539.5X - 33.339 (r = 0.999 7), and the average recovery was 99.68% (RSD 1.92%). The mass fractions of gentiournoside A, water content, ethanol-soluble extractives of 19 batches samples were varied in the ranges of 14.48-31.51 mg x g(-1), 11.25% -12.74% and 24.21% - 31.60%, respectively, and total ash was 4.64% - 6.12% detected from 10 batches samples. The recommended standards of quantitative indexes are that the mass fractions of gentiournoside A and extractives are not less than 15.0 mg x g(-1) (1.5%) and 21.0%, respectively; the water and total ash are not more than 13.0% and 6.0%, respectively.

Studies on genetic diversity of three Tibetan herbs / 中国中药杂志

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.

DNA-based identification of Gentiana robusta and related species / 中国中药杂志

The alpine plant Gentiana robusta is an endemic species to the Sino-Himalayan subregion. Also, it is one of the original plants used as traditional Tibetan medicine Jie-Ji. We sequence the nuclear ribosomal internal transcribed spacer (ITS) regions, matK, rbcL, rpoC1, trnL (UAA), psbA-trnH, atpB-rbcL, trnS( GCU)-trnG(UCC), rpl20-rps12, trnL(UAA)-trnF( GAA) fragments of cp DNA in both G. robusta and such relative species as G. straminea, G. crassicaulis and G. waltonii. With Halenia elliptica as the outgroup, molecular systematic analysis reveals that G. robusta is a natural hybrid. G. straminea is the mother of hybrids, but the father is not very clear. In addition, the molecular markers for distinguishing G. robusta from the parental species or closely related species are identified, respectively. Our studies may provide valuable reference for the species identifications of medicinal plants with complex genetic backgrounds.

Hepatoprotective activity of Gentiana veitchiorum Hemsl. against carbon tetrachloride-induced hepatotoxicity in mice / 中国天然药物

AIM@#To study the hepatoprotective effect of methanol extract of Gentiana veitchiorum (MGV) against CCl4-induced oxidative stress and liver injury in mice.@*METHOD@#The acute hepatic model was developed by injection of 20% CCl4 in mice. ICR mice were divided into six groups, including control, CCl4, CCl4(+) silymarin, and CCl4(+) MGV (100, 200, and 400 mg·kg(-1)) groups. Hepatic enzymes including AST, ALT and ALP levels in serum, and antioxidant enzymes, including SOD, CAT and GPX activity in liver tissue, were determined. Histopathological examination and Western blot analysis were performed.@*RESULTS@#Oral administration of MGV at 200 and 400 mg·kg(-1) for 15 days dose-dependently inhibited the serum elevations of AST, ALT, and ALP, and recovered the reduction of SOD, CAT, and GPX in liver tissue. Hematoxylin and eosin staining examination performed in liver tissues suggested that MGV treatment ameliorated histopathological changes in CCl4-induced mice. Western blotting analysis implied that MGV increased HO-1 expression and recovered TNF-α alternation.@*CONCLUSION@#G. veitchiorum can protect the liver against CCl4-induced damage in mice, and this hepatoprotective effect was due at least in part to its ability through scavenging CCl4-associated free radical activities. The study provided in vivo evidence that G. veitchiorum can be used as a safe, cheap, and effective agent to reduce acute liver damage, supporting its folk medicine use.

Optimum harvest study of Gentiana crassicaulis in Ludian / 中国中药杂志

The paper is aimed to study the difference in yield and quality at different harvest time and determine the optimum harvest of planting Gentiana in Ludian traditional harvest period. The authors analyzed the variation in fresh weight, dry weight, dry discount rate, length, diameter, volume and the content of gentiopicroside, loganin acid, alcohol-soluble extract and total ash and made a comprehensive appraisal of yield, appearance quality and intrinsic quality by gray relational distance ideal Comprehensive Evaluation method. The results showed that there is a big difference in yield and quality both 2-year-old and 3-year-old Gentiana harvested in traditional harvest period and the comprehensive evaluation more better when harvested more later. It can be seen, Gentiana harvested the later had a better yield and quality in Ludian traditional harvest period. The harvest of Gentiana can be appropriate delayed depending on the particular circumstances of production.

Determination of polysaccharides content of Gentiana farreri from different producing areas based on anthrone-sulfuric acid method / 中国中药杂志

Study a method for the detemination of the content of polysaccharides in Gentiana farreri, and analysis of the content of polysaccharides from different producing areas. The results showed that using the anthrone-sulfuric acid method, simple operation, accurate result. Sample was measured at 620 nm absorbance after anthrone-sulfuric acid color, at this wavelength, solution absorption and glucose showed a good linear relationship; The linearity was in the range of 0.01-0.07 g x L(-1) (r = 0.996 7). The recovery rate was 99.41%, with RSD of 2.0%. Considering the experimental conditions, to determine the solid-liquid ratio 1:60, extracting time 50 min, concentration of ethanol 80%. The mass fraction of polysaccharides was the highest to reached 0.743% in G. farreri from Gansu Xiahe. This experiment has laid a good foundation for further study on G. farreri.

Varieties textual research and usage investigation of white flos gentianae, a Tibetan medicine / 中国中药杂志

<p><b>OBJECTIVE</b>To clarity the original plants and the main application varieties of White Flos Gentianae.</p><p><b>METHOD</b>Herbal textual research, wild specimen collection, investigation and collection of the samples from Tibetan hospital, Tibetan pharmaceutical factory and medical material market were carried out simultaneously to identify the original plants of White Flos Gentianae.</p><p><b>RESULT</b>The results of varieties textual research and specimen identification showed that Gentiana szechenyii, G. purdomii and G. algida were in accord with the record of Tibetan herbal textual The three species above were the original plants of White Flos Gentianae. The identification of 20 batches samples showed that G. szechenyii was the main application variety. The other varieties were only used in Tibetan hospitals. All the samples above were flowering branches.</p><p><b>CONCLUSION</b>It was necessary to strengthen the research on variety systematization of White Flos Gentianae make a further discussion on the taxonomy position of G. purdomii, G. algida and the white flos population. Its was also nessary to establish and improve the quality standard of different variety based on the principle of "one species, one name". The quality specification of White Flos Gentianae should be established and improved to standard clinical utilization and produce feeding. More study of resources investigation and cultivation of G. szechenyii should be carried on to meet the demand of produce and clinic.</p>

Performance Evaluation of Gentian Cystatin C on the Hitachi 7600 Automatic Analyzer / 임상검사와정도관리

BACKGROUND: Cystatin C, a 13-kDa protein synthesized in all nucleated cells, has been proposed as a replacement for serum creatinine in assessments of renal function. The Gentian Cystatin C immunoassay (Gentian, Norway) was recently developed using particle enhanced turbidimetric immunoassay. In this study, we evaluated the analytical performance of the Gentian Cystatin C immunoassay on the Hitachi 7600 Automatic Analyzer (Hitachi Ltd., Japan). METHODS: We performed precision and linearity studies using Hitachi Clinical Analyzer 7600 with Gentian reagent and compared the results to those obtained with the N Latex Cystatin C (Siemens, Germany) using a particle enhanced nephelometric immunoassay method performed on the Behring Nephelometer II (Siemens, Germany). We also analyzed the traceability of Gentian reagent and Siemens reagent to Cystatin C standard reference material, ERM-DA471/IFCC. RESULTS: The coefficient of variations (CVs) for within-run imprecision at low and high levels were 1.58% and 1.06% and the CVs for total imprecision at low and high levels were 2.53% and 2.09%, respectively. In the linearity test, the coefficient of determination (R2) was 0.9997 (range, 0.23 to 7.50 mg/L), and comparison with the results obtained by Siemens reagent showed an excellent correlation coefficient of 0.9982. In the traceability test, Gentian reagent is more accurate than Siemens reagent and the total accuracy was 96.0%. CONCLUSIONS: Gentian reagent provides good analytic performance on the Hitachi 7600 Automatic Analyzer and can be used for the diagnosis, treatment, monitoring, and risk assessment of renal function.

Quickly investigating the absorption ingredients of Gentianae Radix et Rhizoma by SEMAC / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a method for quickly investigating the absorption ingredients which could be used as the index of quality control of Gentianae Radix et Rhizoma.</p><p><b>METHOD</b>The absorption ingredients of Gentianae Radix et Rhizoma were investigated by using the model of in vitro everted intestinal sac (VEIS). The intestinal sac liquors of jejunum and ileum were collected at 6 intervals (15, 30, 45, 60, 90, 120 min) and gentiopicroside, loganin acid, swertiamarin and sweroside were detected by HPLC as the representative marker. The accumulative absorption quantity of gentiopicroside, loganin acid, swertiamarin and sweroside were calculated, respectively.</p><p><b>RESULT</b>Six components could be detected in intestinal sac. In different concentrations of Gentianae Radix et Rhizoma, gentiopicroside and swertiamarin in various intestinal sections were the linear absorption (R2 > 0.9), conformed to zero order absorption rate. In jejunum the constant of absorption rate (Ka) of gentiopicroside and swertiamarin increased with the raised dosage of Gentianae Radix et Rhizoma (P < 0.05), which indicated a passive absorption manner, and the value of Ka of high and middle dosage of those in ileum were higher than that of low dosage, and the difference of Ka between high and middle dosage were not significant, which indicated a positive absorption manner. The Ka of high and middle dosage of sweroside in ileum and jejunum were higher than that of low dosage (P < 0.05), and the difference of Ka between high and middle dosage were not significant, which indicated a positive absorption manner. The Ka of loganin acid in jejunum and ileum increased along with the raised dosage of Gentianae Radix et Rhizoma (P < 0.05), which indicated a passive absorption manner.</p><p><b>CONCLUSION</b>SEMAC could be used as a tool to investigate the absorption ingredients of Gentianae Radix et Rhizoma. Drug in intestine sac was selective, and the absorption part of intestine was also different.</p>

Identification of gentianae macrophyllae radix using the ITS2 barcodes / 药学学报

DNA barcoding is a rapidly developing frontier technology in the world and will be useful in promoting the quality control and standardization of traditional Chinese medicine. Until now, many studies concerning DNA barcoding have focused on leaf samples but rarely on Chinese herbal medicine. There are three issues involved in DNA barcoding for traditional Chinese medicinal materials: (1) the extraction methods for total DNA of the rhizomes of the medicinal materials; (2) intra-specific variation among samples from different places of origin; (3) accuracy and stability of this method. In this study, Gentianae Macrophyllae Radix was used to verify the stability and accuracy of DNA barcoding technology. Five regions (ITS2, psbA-trnH, matK, rbcL, and ITS) were tested for their ability to identify 86 samples of Gentianae Macrophyllae Radix and their adulterants. After improving the DNA extraction method, genomic DNA from all samples was successfully obtained. To evaluate each barcode's utility for species authentication, PCR amplification efficiency, genetic divergence, and species authentication were assessed. Among all tested regions only ITS2 locus showed 100% of PCR amplification and identification efficiencies. Based on the established method, we successfully identified two samples of Gentianae Macrophyllae Radix bought in pharmacy to the original species.

Correlation and path analysis on agronomic traits of Gentiana rigescens / 中国中药杂志

<p><b>OBJECTIVE</b>The interrelation of yield and agronomic traits of Gentiana rigescens was studied for the germplasm and breeding variety of this species.</p><p><b>METHOD</b>Twelve agronomic traits, root diameter, root length, root number, root biomass, stem diameter, plant height, the first branch number, leaf length, leaf width, leaf length/leaf width ratio, calyx length, and calyx number of G. rigescens from 26 wild populations in Yunnan were determined for correlation analysis, multiple stepwise regression analysis and path analysis.</p><p><b>RESULT</b>Correlation analysis showed that there were significantly positive correlation between the traits of aboveground part and the length, diameter, number, and biomass of the root. Multiple stepwise regression analysis showed that length, width, and number of root, plant height, the first branch number, and the calyx number were the main factors that affected the root biomass. Path analysis showed that the diameter, length, and number of the root, the stem height, and the first branch number had a direct positive effect on the root biomass.</p><p><b>CONCLUSION</b>The traits, such as high and strong stem, high number of first branch number and shrubby shape could be selected for the breeding and high yielding of G. rigescens.</p>

Study on seed biological characteristics of Gentiana crassicaulis / 中国中药杂志

<p><b>OBJECTIVE</b>To study seed biological characteristics of Gentiana crassicaulis.</p><p><b>METHOD</b>The samples were collected from five localities, and the morphological observation, germination test and viability TTC test were carried out.</p><p><b>RESULT</b>The seed morphological characters of all samples were similar to each other. The viability of all samples was similar to each other, but the germination rate of wild seed was the highest in all samples after stored for 8 months. Also, the seed energy of wild seed and sample 2008XR02 was higher than other three samples. There was no obvious correlation between thousand seed weight and germination rate.</p><p><b>CONCLUSION</b>The quality of wild seed and sample 2008XR02 was superior than that of the other three samples. The results can be used for basic data of in situ conservation and germplasm breeding of G. crassicaulis.</p>

Seed germination characteristics of Gentiana rigescens / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the influences of temperature, lightness, storage method, storage time, and gibberellin on seed germination of Gentiana rigescens.</p><p><b>METHOD</b>The germination rates of G. rigescens in different treatments were observed.</p><p><b>RESULT AND CONCLUSION</b>The most suitable temperature for the seed germination was 25 degrees C, at which the germination rate was 76.33%. The effect of lightness on the seeds was significantly; the germination rate of the seed was very low. Under the natural condition, the best storage method was dry storage (within 6 months), which could promote the after-ripening of the seed. 100-1 000 mg x L(-1) gibberellic acid could significantly reduce the seed germination time, and 500 mg x L(-1) gibberellic acid increased the germination rate of the seed to 95.00%.</p>

Transdermal delivery of Gentiana macrophylla complex components system under micro-needle conditions / 药学学报

The purpose of this study is to investigate the transdermal delivery characteristics of Gentiana macrophylla complex components system through different parts of the skin under micro-needles conditions. Two-chamber diffusion cells were used, different parts of isolated skin and micro-needle pretreated isolated mouse skin were applied separately, high performance liquid chromatography (HPLC) similarity evaluation methods were used to evaluate transdermal delivery characteristics of Gentiana macrophylla complex components system on receiving pool and the permeation rate and penetration amount of Gentiopicroside at different parts of mouse skin. In the 24 h, the similarity between receiving fluid which was on passive transdermal delivery and micro-needle transdermal delivery conditions and original fluid were ranged from 83.0% to 98.9%; By the micro-needle pretreatment with different parts of the mouse skin, the time that Gentiana macrophylla complex components system though abdominal skin to the receiving fluid which reached 90% similarity compared with that of original fluid was 4 h, which was 18 h at back skin and 12 h at neck skin separately. Micro-needles can be used as the ideal ingredients for traditional Chinese medicine complex transdermal delivery; transdermal absorption time delay could be greatly reduced and its bioavailability was improved. The permeation rate and similarity to original liquid of Chinese medicine complex components increased significantly in the abdominal skin relative to the neck and back skin under micro-needle conditions.

Quality standard of Gentiana rhodantha / 中国中药杂志

Based on the approaches of TLC identification, HPLC for assaying managiferin, and of the determination of water, total ash and acid-insoluble ash in 12 samples, collected from southwest of China, the quality standard of Gentiana rhodantha has been established. The results show the reference materia medica and mangiferin can be both well used as reference substances for TLC identification; the mass fractions of mangiferin is 0.7%-4.4% (average 2.8%), water 6.1%-8.6% (average 7.2%), total ash 3.7%-10.8% (average 6.6%) and acid-insoluble ash 0.2%-2.7% (average 1.3%). The recommended standards of quantitative indexes are that the mass fractions of mangiferin is not less than 2.0%, and the water, total ash and acid-insoluble ash are not more than 9.0%, 8.0% and 3.0% respectively.

Determination of five active constituents in aerial part of Tibetan medicine Gentiana straminea by HPLC / 中国中药杂志

A new RP-HPLC method was developed for the simultaneous determination of 5 active constituents, including loganic acid, swertiamarin, gentiopicroside, sweroside, isoorientin in aerial part of Gentiana straminea. Analysis was achieved on a Hypersil ODS analytical column (4.6 mm x 250 mm, 5 microm) eluted with methanol and water containing 0.02% phosphoric acid in gradient elution. The flow rate was 1 mL x min(-1), the column temperature was set at 35 degrees C and detection wavelength was set at 254 nm. The results showed that 5 active components were separated well and showed good linearity. The corelation coefficints and concentration ranges of the calibration curves were as follow: r = 0.999 9, 0.364-2.18 microg for loganic acid. r = 0.999 9, 0.275-1.65 (microg for swertiamarin. r = 0.999 9, 0.614-3.68 microg for gentiopicroside. r = 0.999 9, 0.065 6-0.394 microg for sweroside. r = 0.999 9, 0.089 9-0.539 microg for isoorientin. The contents of loganic acid and gentiopicroside far exceed that of the other 3 active components, and made up 60% in the total contents. The developed method was proved to be rapid, sensitive, accurate, credible and repeatable. It can be applied to quality control of aerial part of Tibetan medicine Gentiana straminea.