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.

Study on UPLC fingerprint and content determination of mangiferin of Gentiana rhodantha / 中国药房

OBJECTIVE To provide reference for quality control of Gentiana rhodantha. METHODS Taking 52 batches of G. rhodantha as subject， ultra-high performance liquid chromatography （UPLC） fingerprint was adopted. The similarity of 52 batches of medicinal materials samples was evaluated by the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine （2004A edition）； the content of mangiferin was determined； chemometric analyses [cluster analysis， principal component analysis （PCA） and orthogonal partial least squares-discriminant analysis （OPLS-DA）] were performed. RESULTS UPLC fingerprints of 52 batches of G. rhodantha were established， 17 common peaks were identified， and 6 of them were identified， which were loganic acid （peak 1）， neomangiferin （peak 3）， swertiamarin （peak 5）， dangyin （peak 6）， mangiferin （peak 7） and isoorientin （peak 9）. The similarities of 52 batches of medicinal materials samples were all greater than 0.9； cluster analysis showed that S1-S46， S48-S52 clustered into one class， and S47 alone； PCA results showed that the cumulative variance contribution rate of the first six principal components was 82.928%； OPLS-DA results showed that the corresponding components of swertiamarin， mangiferin and chemical composition represented by peak 4， 14， 15， 16 were the main iconic components affecting the quality differences of G. rhodantha medicinal materials. The contents of mangiferin in 52 batches of medicinal material samples ranged from 18.2 to 101.0 mg/g， mostly in accordance with 2020 edition of Chinese Pharmacopoeia. CONCLUSIONS The established UPLC fingerprint and chemometric analysis methods combined with content determination method of mangiferin can comprehensively evaluate the quality of G. rhodantha.

Effect of Different Cultivation Modes on Microbial Diversity and Secondary Metabolites of Rhizosphere Soil of Gentiana crassicaulis / 中国实验方剂学杂志

ObjectiveTo investigate the effects of different cultivation modes on the yield of Gentiana crassicaulis and its microbial diversity and secondary metabolite content in the rhizosphere soil. MethodWith G. crassicaulis of different cultivation modes and its rhizosphere soil as the research objects， the composition of bacterial and fungal communities， dominant bacteria， and differential microorganisms in the rhizosphere soil were analyzed by high-throughput sequencing technology. HPLC was used to determine the content of iridoids in G. crassicaulis with different cultivation modes. ResultCompared with plastic film mulching， planting without mulch and intercropping of peony， white kidney bean， potato， and corn increased the yield of fresh products by 16.11%-17.68%， 22.48%-26.34%， 29.37%-32.19%， 34.82%-36.57%， and 35.34%-39.71%， respectively， and increased the yield of dry products by 19.75%-23.17%， 25.86%-29.32%， 30.18%-34.94%， 35.22%-39.87%， and 39.72%-43.73%. The total content of four iridoids， including gentiopicrin， loganic acid， sweroside， and swertiamarin， increased by 10.17%-37.83%， 5.93%-47.44%， 9.09%-28.84%， and 10.71%-28.57%， respectively. The diversity of bacterial and fungal communities in the rhizosphere soil increased significantly （P<0.05）. The relative abundance of pathogenic bacteria such as Sordariomycetes， Leotiomycetes， Tremellomycetes， Eurotiomycetes， Fusarium， and Cladophialophora decreased， and the proportions of beneficial bacteria such as Proteobacteria， Acidobacteria， and Actinobacteriota increased and they gradually became the dominant bacteria. ConclusionDifferent cultivation modes can affect the yield of G. crassicaulis and its microbial diversity and iridoid content in the rhizosphere soil. Cultivation without mulch and intercropping patterns have certain advantages， which can provide theoretical references for the planting of G. crassicaulis.

Analysis of Chemical Constituents in Different Parts of Gentiana straminea Based on UPLC-Q-TOF-MS/MS / 中国实验方剂学杂志

ObjectiveUltra-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry （UPLC-Q-TOF-MS/MS） was used to analyze the chemical constituents in the aerial part and roots of Gentiana straminea from different areas of Qinghai province， and the main chromatographic peaks and differential components of different parts were identified. MethodThe chromatographic separation was performed on a Waters ACQUITY UPLC HSS T3 column （2.1 mm×100 mm， 1.8 μm） with 0.1% formic acid aqueous solution （A）-acetonitrile （B） as the mobile phase for gradient elution （0-1 min， 1%-13%B； 1-5 min， 13%-18%B； 5-7 min， 18%-50%B； 7-9.5 min， 50%-60%B； 9.5-11 min， 60%-99%B； 11-14 min； 99%B； 14-15 min， 99%-1%B； 15-16 min， 1%B）， the column temperature at 40 ℃， and the flow rate of 0.3 mL·min-1. Electrospray ionization （ESI） and negative ion full scan mode were selected for the mass spectrometric conditions to analyze the samples， and the detection range was m/z 50-1 200. Chemical constituents of the aerial part were qualitatively analyzed with the reference substances， literature information and ChemSpider. Principal component analysis （PCA） and orthogonal partial least squares discriminant analysis （OPLS-DA） were used to analyze the classification trend， correlation and differential chemical components between aerial part and roots of G. straminea. ResultA total of 68 components， including 24 iridoids， 13 flavonoids， 8 triterpenoids， 6 xanthones， 5 fatty acids， 4 saccharides， 3 phenolic glycosides， 2 alkaloids， 2 sterols and 1 lignan， were preliminarily identified from the aerial part of G. straminea. Among them， 42 components were firstly reported in 4 Gentiana species included in the 2020 edition of Chinese Pharmacopoeia. Eight differential components were screened out， namely sucrose， maltotriose， loganic acid， shanzhiside methyl ester， 6′-O-β-D-glucosylgentiopicroside， swertiamarin， gentiopicrin and isovitexin. ConclusionThe aerial part of G. straminea is rich in chemical constituents and has good medicinal potential. There were significant differences in the chemical components between the aerial part and roots of G. straminea， and the main differential components were iridoids， which could provide a basis for exploring efficacy differences in different parts of G. straminea.

Herbal Textual Research on Gentianae Macrophyllae Radix in Famous Classical Formulas / 中国实验方剂学杂志

In this paper， through consulting relevant records in materia medica， medical and prescription books， and combining with modern literature， the name， origin， producing area， collection and processing of Gentianae Macrophyllae Radix in famous classical formulas from The Catalogue of Ancient Famous Classical Formulas （The First Batch） was systematically sorted out and textual research was carried out， in order to provide a basis for the development of the famous classical formulas containing Gentianae macrophyllae Radix. After textual research， it was found that Gentianae Macrophyllae Radix was the rectification of name in the past dynasties. In addition， there were other names such as Qinjiao， Qingua and Qinzhua. Gentiana macrophylla， G. straminea， G. dahurica and G. siphonantha were the main origin of this herb in ancient literature. Among them， G. macrophylla is the mainstream. In the Southern and Northern dynasties， G. straminea and G. macrophylla produced in northern Sichuan were recommended as the best. In the early Tang dynasty， G. macrophylla from the Liupan Mountain area at the border of Shanxi and Gansu provinces was the mainstream. During the Northern Song dynasty， G. siphonantha from Linxia and Qilian Mountain of Gansu province and G. macrophylla from eastern Shaanxi province were two new producing areas. In the Ming and Qing dynasties， the abundant base and production areas of Gentianae Macrophyllae Radix were gradually formed. In the past dynasties， harvesting was carried out in spring and autumn， and stored mainly by aeration drying or shade drying treatment. The processing methods are mainly the raw products after the net selection， cutting and drying， in addition to the frying， processing with wine and milk. G. macrophylla is recommended as the first choice for the herbal medicine involved in the famous classical formulas. Among them， wild products produced in Gansu and Shaanxi are the best， and raw products are recommended to be used. At the same time， it is suggested that G. siphonantha should be added to the subsequent edition of Chinese Pharmacopoeia as one of origins of Gentianae Macrophyllae Radix.

SNP double peak methods through chloroplast genome profiling for identifying Gentiana crassicaulis and two closely related species / 药学学报

italic>Gentiana crassicaulis Duthie ex Burk. is one of the plant sources of Gentianae Macrophyllae Radix (QinJiao). Gentiana tibetica King ex Hook. f. and Gentiana robusta King ex Hook. f. are relative species of G. crassicaulis. Due to the large intraspecific morphological variation, G. crassicaulis showed high morphological similarity with G. tibetica and G. robusta. And the distribution area of the three species overlaps to some extent, which makes it difficult to identify them. On the basis of morphological identification, the method of molecular identification of the three species was constructed in this study based on chloroplast genomes. The chloroplast genome of Gentiana tibetica is 148 765bp long, with LSC, SSC and IR 81 163 bp, 17 070 bp and 25 266 bp, respectively. The structure of the three is consistent. The chloroplast genome sequences of G. tibetica and G. crassicaulis are highly similar, and the number of variable sites is 9 (149 267 bp in total). Diagnostic SNP that could effectively identify the three species was screened and verified, and a dual-peak SNP detection method was established for the effective identification of each species and mixed samples. Our study provides basic data for the molecular identification of G. crassicaulis and its related species, and the arrangement of related Tibetan medicine.

DNA barcoding of Gentiana crassicaulis in Lijiang and evaluation of root processing methods / 药学学报

The key factors for producing the best quality Chinese herbal medicines are high-quality germplasm, suitable cultivation area and the proper processing methods for herbal raw materials. Gentiana crassicaulis in Gentiana (Sect. Cruciata), Gentianaceae is one of the original plants of the Chinese herb Qinjiao (Gentianae Macrophyllae Radix), and its type specimen was collected in Lijiang, Yunnan. There is a long planting history of the herb in this area. In this study a sampling plot was designated in these traditional planting areas. G. crassicaulis was planted and herbal raw materials were harvested from the plot. The raw materials were prepared locally and at a pharmaceutical factory in Shanghai using processing methods such as "sweating" or "no sweating", "slicing" or "no slicing" (whole root), and "stoving" or "no stoving" (air drying). The quality of all processed samples was evaluated. In addition, molecular markers were determined for identifying cultivated and wild samples from Lijiang, Yunnan. The results are as follows: ① Samples from the sampling plot and the field are taxonomically identified as Gentiana crassicaulis. ② A total of 270 sequences of trnC-GCA-petN, atpB-rbcL, psbN, ndhB-rps7 and ycf1 were obtained, and three genotypes were determined from the cultivated samples; the type III was shared by both cultivated and wild plants. Based on the molecular markers, a DNA barcoding method to identify cultivated and wild samples of G. crassicaulis from Lijiang, Yunnan was established. ③ Total content of loganic acid and gentiopicroside in all samples was ≥ 2.5%, and above the Chinese Pharmacopoeia (2020) limit. ④ In HPLC fingerprinting, 9 common peaks were assigned and similarity between all samples was > 0.999; and ⑤ In a PCA score plot all slice samples were clustered, while whole root samples were scattered. Therefore, our studies could provide basic data for optimizing the processing method, producing best quality Gentianae Macrophyllae Radix, and evaluating the quality of different ecotype varieties and the multiple origin of herbal medicines.

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.

Transcriptome analysis and exploration of genes involved in the biosynthesis of iridoids in Gentiana crassicaulis (Gentianaceae) / 药学学报

italic>Gentiana crassicaulis Duthie ex Burk. in Gentiana (Sect. Cruciata), Gentianaceae, is one of the original plants of both Gentianae Macrophyllae Radix and Tibetan herb Jie-Ji Na-Bao, which contain such bioactive iridoids as gentiopicroside, loganic acid and others. In this study, based on previous work, the transcriptome of G. crassicaulis was sequenced and analyzed to construct transcriptome databases of roots, stems, leaves and flowers. qRT-PCR verification was conducted for parts of unigenes that may be key enzymes in the pathway of iridoid biosynthesis. The results are as follows: ① a total of 159 534 unigenes were obtained, with an average length of 679 bp. According to the functional classification of GO, unigenes can be divided into 3 categories with 67 branches. The unigenes were aligned in the KOG database and were classified into 25 categories according to function. ② In the KEGG database, 215 unigenes were implicated in 20 standard secondary metabolism pathways. The analysis shows that 305 unigenes encoded 28 key enzymes in the pathway of iridoid biosynthesis, and their expression in different organs is different; and ③ qRT-PCR was approximately consistent with RNA-Seq results. The 7 annotated unigenes identified in this study, HMGS, DXS, MCS, GPPS, G10H, 7-DLNGT and STR, all had higher relative expression levels in the above-ground parts (stem, leaf and flower) than in the underground part (root). Iridoids are common active and index components of such traditional Chinese medicines as Qinjiao, Longdan, Dangyao, and Qingyedan, among others. Therefore, this work provides basic scientific data for further development including obtaining active components or intermediates through biotechnology, exploring the accumulation of effective components, evaluating the quality of different ecotype varieties, and identifying authentic biosynthesis pathways of medicinal materials.

Molecular markers based upon whole chloroplast genomes and identifying alpine Gentiana waltonii and G. lhassica (Gentianaceae) / 药学学报

As two original plants of Tibetan herb Jieji, Gentiana waltonii Burk. and Gentiana lhassica Burk. belong to Section Cruciata of Gentiana, Gentianaceae. Here, we report on whole chloroplast genome sequences in the alpine species, respectively, and the features of plastomes were investigated. The plastome of G. waltonii is 148 705 bp long (148 652 bp in G. lhassica) and encodes 112 genes, including 78 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes. Two pseudogenes, namely ψrps16 and ψinfA, were found in plastomes. In addition, two novel loci were detected, and a species-specific polymerase chain reaction assay was developed for differentiating G. waltonii and G. lhassica from 10 alpine species in Section Cruciata. Gentiana. Our study provides basic data for identifying Tibetan herbs, alpine species conservation and molecular phylogenetic studies of Gentiana and Gentianaceae.

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.

Correlation between quality and climatic factors for Gentiana officinalis from different producing area in Gansu province / 国际药学研究杂志

Objective: To investigate the correlation between the quality and climatic factors for Gentiana officinalis H. Smith from different producing area in Gansu province, China by grey correlation degree analysis combined with CRITIC-AHP weight ed evaluation. Methods: Contents of the total secoiridoid glycosides, total alcohol extracts and the metallic elements Ca, Fe and Zn were determined for the medicinal herb G. officinalis samples from 10 producing areas in Gansu, and their quality was estimated by the CRITIC-AHP weighted evaluation. The relationship between the quality and related climatic factors was then investigated by the grey correlation degree analysis. The climatic factors included the altitude, annual mean temperature, monthly mean temperature of Janu ary and July, annual mean relative humidity and the annual mean rainfall precipitation. Results : The content of the determined com ponents showed variation in the G. officinalis samples from the 10 different producing area. The altitude, annual mean temperature, monthly mean temperature of January and July, annual mean relative humidity and the annual mean rainfall all showed certain influ ence on the G. officinalis quality, and the biggest influence was the monthly mean temperature of July. Conclusion: The present re sults of the grey correlation degree analysis identifies the main climatic factors influencing the G. officinalis quality, which provides an experimental basis for the proper area selection and planting standardization for the G. officinalis production.

Comparative analysis of complete chloroplast genome sequences within Gentianaceae and significance of identifying species / 中草药

The family Gentianaceae are found mostly on the Qinghai-Tibet Plateau in China, which have important medicinal properties. Based on 27 published complete chloroplast genome sequences from Gentiana, Swertia, Halenia, Menyanthes, and Nymphoides of Gentianaceae, the chloroplast genome structure was analyzed. The cp genome sizes of 27 taxa range from 137 to 154 kb, and they contain 101-114 unique genes, including 67-80 protein-coding genes, 30 tRNA genes and four rRNA genes. Also, a Bayesian phylogenetic tree was constructed based on the 27 cp genome sequences with Pentalinon luteum (Apocynaceae) as the outgroup. The tree was topologically consistent with the treatments of traditional taxonomy, and the cp genome sequences have genus- or section-level resolution. In addition, we reviewed the significance of species identification within the family. These cp genome sequences could provide basic data for the endangered species conservation, the genetic analysis and pharmacognostic researches of herbs from Gentianaceae.

Study on transdermal permeation of ethanol extracts from roots of Gentiana crassicaulis in vitro and in vivo / 中草药

Objective: To establish a determination method for kinds of chemical components of iridoids in the roots of Gentiana crassicaulis in transdermal absorption liquid, and research its transdermal permeability, so as to provide scientific basis for transdermal delivery system, clinical medication and reform of the traditional forms of G. crassicaulis. Methods: Based on the results of the previous investigation, in this paper, using the roots of G. crassicaulis as the research subject, a certain concentration solution of G. crassicaulis extract was prepared by the alcohol extraction method. Three kinds of common penetration enhancers, azone, borneol and propylene glycol were used. The effects of single penetration enhancer and dual compound penetration enhancers on the transdermal penetration of five kinds of chemical components of loganic acid, shanzhiside methyl ester, swertimarin, gentiopicroside and sweroside in vitro and the three kinds of chemical components of gentiopicroside, loganic acid and swertimarin in vivo were quantitatively studied by the method of HPLC to investigate the transdermal permeability of G. crassicaulis extract in mice skin model. Results: According to the experimental results, compared to the control group, penetration enhancers significantly increased the absorption of five chemical components of G. crassicaulis in vitro. Transdermal absorption rates (J) of loganic acid, shanzhiside methyl ester, swertimarin, gentiopicroside and sweroside were 12.306 0, 1.248 8, 4.187 5, 153.030 0 and 5.012 6 μg/(cm2∙h), respectively. The transdermal enhancer effects of A (5% azone), B (5% borneol), C (5% propylene glycol), A + B (2.5% azone and 2.5% borneol), A + C (2.5% azone and 2.5% propylene glycol), A + C (2.5% borneol and 2.5% propylene glycol) were 9.73, 2.57, 13.94, 15.92 and 8.08 times faster than the control group, respectively. Among these, the group of A + C had a marked osmotic enhancer effect in vitro. In comparison with the control group, the in vivo percutaneous penetration test indicated that the dual compound penetration enhancers of 2.5% azone and 2.5% propylene glycol had a marked effect for the permeability enhancement. Conclusion: This study showed azone and propylene glycol significantly promoted the percutaneous penetration effect of loganic acid, shanzhiside methyl ester, swertimarin, gentiopicroside and sweroside of G. crassicaulis, and this study laid a foundation for the quality control of percutaneous drug delivery preparation of G. crassicaulis.

DNA fingerprinting and genetic diversity in a threatened alpine medicinal plant, Gentiana crassicaulis (Gentianaceae) using AFLP / 药学学报

Jieji Nabao is a common Tibetan herb. According to our ethnobotanical studies, one of its original plants is identified as Gentiana crassicaulis Duthie ex Burk. (Gentianaceae). Endemic to the Qinghai-Tibet Plateau, this medicinal alpine plant is a threatened species. In this study, 163 individuals from 20 populations of G. crassicaulis were collected throughout its geographical range and amplified fragment length polymorphism (AFLP) was used to investigate genetic variation in this species. A cluster analysis was performed on the AFLP data with Halenia elliptica and Gentiana straminea as the outgroups. From 64 pairs of AFLP primer combinations, 12 pairs were selected for amplification and a total of 315 bands were amplified, of which 254 bands were polymorphic, accounting for 80.63%. High genetic differentiation was detected between populations (87%), and low within populations (13%). The UPGMA (unweighted pair-group method with arithmetic means) tree was topologically consistent with the traditional taxonomic treatments at the species level, and the populations of G. crassicaulis were divided into two branches: one from Yunnan and Guizhou, the other from Tibet, Qinghai, Sichuan and Gansu. PCA analysis and the Mantel test showed that there was a positive correlation between genetic distance and geographical distance. In addition, combined with SSR and SNP markers within cpDNA, the genetic differentiation within the Sichuan population S1 was validated.

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.

A novel cost-effective medium for in vitro cultivation of Gentiana kurroo Royle using sugarcane bagasse

Objective: To develop a simple, cost-effective, and efficient medium by using sugarcane bagasse (SB) as abase material to replace the conventional Murashige and Skoog (MS) medium.Materials and Methods: Water extracts of SB along with some macronutrients and plant growth regulators weregelled with 0.7% agar-agar powder. Nodal segments of Gentiana kurroo were used as explants and inoculatedin the medium and placed in a growth chamber under standard conditions of light and temperature. Out of thetested combinations of plant growth regulators, 0.5 mg/l each of kinetin (KN) and 6-Benzylaminopurine (BAP)showed the excellent shoot multiplication and proliferation rate on the bagasse medium with the same potentialas on the MS medium with an average of 5–6 shoots/explant. In vitro rooting was obtained on half strength MSmedium supplemented with IBA (0.5 mg/l) with an average length of 7–8 cm and 20–25 roots/explant. Theplants were hardened in a mixture of clay loam and farmyard manure in 1:1(w/w) with 70%–80% survival ratewithout any phenotypic aberrations.Conclusion: The results from the present investigation indicate that SB can be used as a cost-effective substituteof MS medium for in vitro propagation of G. kurroo.

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.