Insights from the first chromosome-level genome assembly of the alpine gentian Gentiana straminea Maxim.

Gentiana straminea Maxim. is a perennial herb and mainly distributed in the Qinghai-Tibetan Plateau. To adapt to the extreme environment, it has developed particular morphological, physiological, and genetic structures. Also, rich in iridoids, it is one of the original plants of traditional Chinese herb 'Qinjiao'. Herein, we present its first chromosome-level genome sequence assembly and compare it with the genomes of other Gentiana species to facilitate the analysis of genomic characteristics. The assembled genome size of G. straminea was 1.25 Gb, with a contig N50 of 7.5 Mb. A total of 96.08% of the genome sequences was anchored on 13 pseudochromosomes, with a scaffold N50 of 92.70 Mb. A total of 54,310 protein-coding genes were predicted, 80.25% of which were functionally annotated. Comparative genomic analyses indicated that G. straminea experienced two whole-genome duplication events after the γ whole-genome triplication with other eudicots, and it diverged from other Gentiana species at ~3.2 Mya. A total of 142 enzyme-coding genes related to iridoid biosynthesis were identified in its genome. Additionally, we identified differences in the number and expression patterns of iridoid biosynthetic pathway genes in G. straminea compared with two other Gentiana species by integrating whole-genome sequence and transcriptomic analyses.

Effective Parts of Gentiana straminea Maxim Attenuates Hypoxia-Induced Oxidative Stress and Apoptosis.

Hypoxia occurs in physiological situations and several pathological situations, inducing oxidative stress. G straminea Maxim (G.s Maxim) is a traditional Tibetan medicine that exerts several biological effects. This study focused on the protective effects of G.s Maxim in hypoxia-induced oxidative stress and apoptosis. We found that G.s Maxim significantly increased survival and reduced oxidative stress in hypoxic mice. Various extraction parts of G.s Maxim showed antioxidant activity and significantly improved survival in hypoxia-injured PC12 cells. G.s Maxim reduced hypoxia-induced cell apoptosis and leakage of lactate dehydrogenase. Hypoxic cells had increased malondialdehyde levels but reduced superoxide dismutase activity and G.s Maxim reversed these effects. Moreover, G.s Maxim suppressed hypoxia-induced apoptosis by inducing protein expression of B cell leukemia/lymphoma-2 and reducing the expression of hypoxia-inducible factor-1α, Bcl-2-associated X, and nuclear factor-k-gene binding. These findings suggest that G.s Maxim attenuates hypoxia-induced injury associated with oxidative stress and apoptosis.

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.

Gentiana straminea supplementation improves feed intake, nitrogen and energy utilization, and methane emission of Simmental calves in northwest China.

OBJECTIVE: Native plants can be used as additives to replace antibiotics to improve ruminant feed utilization and animal health. An experiment was conducted to evaluate the effects of Gentiana straminea (GS) on nutrient digestibility, methane emissions, and energy metabolism of Simmental calves. METHODS: Thirty-two (5-week-old) male Simmental clves, with initial body weight (BW) of 155±12 kg were fed the same basal diet of concentrates (26%), alfalfa hay (37%), and oat hay (37%) and were randomly separated into four treatment groups according to the amount of GS that was added to their basal diet. The four different groups received different amounts of GS as a supplement to their basal diet during whole experiment: (0 GS) 0 mg/kg BW, the control; (100 GS) 100 mg/kg BW; (200 GS) 200 mg/kg BW; and (300 GS) 300 mg/kg BW. RESULTS: For calves in the 200 GS and 300 GS treatment groups, there was a significant increase in dry matter (DM) intake (p<0.01), average daily gain (ADG) (p<0.05), organic matter intake (p<0.05), DM digestibility (p<0.05), neutral detergent fibre (NDF) digestibility (p<0.05), and acid detergent fibre (ADF) digestibility (p<0.05). Dietary GS supplementation result in quadratic increases of DM intake (p<0.01), ADG (p<0.05), NDF intake (p<0.05), and ADF intake (p<0.05). Supplementing the basal diet with GS significantly increased nitrogen (N) retention (p<0.001) and the ratio of retention N to N intake (p<0.001). Supplementing the basal diet with GS significantly decreased methane (CH4) emissions (p<0.01), CH4/BW0.75 (p<0.05) and CH4 energy (CH4-E) (p<0.05). Dietary GS supplementation result in quadratic increases of CH4 (p<0.01) and CH4/DM intake (p<0.01). Compared with 0 GS, GS-supplemented diets significantly improved their gross energy intake (p<0.05). The metabolizable energy and digestive energy intake were significantly greater for calves in the 100 GS and 200 GS calves than for 0 GS calves (p<0.05). CONCLUSION: From this study, we conclude that supplementing calf diets with GS could improve utilization of feed, energy, and N, and may reduce CH4 emissions without having any negative effects on animal health.

Gentiana straminea Maxim. polysaccharide decolored via high-throughput graphene-based column and its anti-inflammatory activity.

Gentiana straminea Maxim. exhibits various biological activities. However, the purification and functions of polysaccharides in Gentiana straminea Maxim. have never been reported. Herein, by proposing a flexible 3D graphene-based decoloration column (3DD column), Gentiana straminea Maxim. polysaccharide (GMP) was high-throughput obtained and its anti-inflammatory activity was investigated. Benefiting from the large macroporous network of 3D NH2-graphene oxide hydrogel with selective adsorption towards pigments, the 3DD column exhibits high decoloration ratio (96.41%). In addition, the 3DD column provides superior practical functionality as compared to the traditional approaches, which are time-consuming and need toxic solvents, and exhibiting widespread-application for the purification of polysaccharide from other common plant species. More importantly, the decolored GMP as a natural product has promising anti-inflammatory activity on RAW264.7 cells without negative impact on cell viability. Overall, this work reveals a new functional polysaccharides and provides a flexible approach for polysaccharide decoloration, exhibiting a promising prospect for natural polysaccharides in practical application of pharmaceutical.

Quantitative analysis of the profiles of twelve major compounds in Gentiana straminea Maxim. Roots by LC-MS/MS in an extensive germplasm survey in the Qinghai-Tibetan plateau.

ETHNOBOTANICAL RELEVANCE: Gentiana straminea Maxim. is a well-known Tibetan traditional herb, which has been used to treat rheumatic arthritis, iceteric hepatitis, and other diseases for thousands years. However, there is still lack of comprehensive active constituents profiling of this species throughout the Qinghai-Tibet Plateau (QTP). AIM OF STUDY: This study was designed to provide a comprehensive quality map of G.straminea germplasm based on twelve active constituents (loganic acid, gentiopicroside, swertiamarin, sweroside, 6-O-ß-D-glucosylgentiopicroside, oleanic acid, morroniside, trilobatin, isoorientin, isovite, Shanzhisidemethylester and quercetin) on the QTP. MATERIALS AND METHODS: G.straminea root samples collected throughout QTP in the flowering period were analyzed by the LC-MS/MS. Statistics analysis methods PCA, clustering and ecological regions analysis for G.straminea constituents differentiation was demonstrated. RESULTS: The active constituents varied greatly across the QTP; the majority of constituents were secoiridoid derivatives, with gentiopicroside being the most abundant compound. Most constituents were significantly affected by the latitudes and altitudes other than longitudes. PCA and hierarchical clustering analysis showed that all samples could be separated into six distinct groups, and 15 populations showed the highest constituent abundances. Further, geographical region analysis showed that the highest quality populations mainly located near the source region of Yellow River, especially in the Qinghai and Sichuan areas. Additionally, correlation analysis showed that there were relationships among genetiopicroside, loganic acid, and other compounds, which might be related to the enzymatic pathways involved in the metabolism of these constituents. CONCLUSION: LC-MS/MS method allowed separation of quality profiling of G.straminea on the QTP, 15 populations showed the highest constituent abundances. In six geographical groups, the highest quality populations mainly located near the source region of Yellow River, especially in the Qinghai and Sichuan areas, which may be due to the climate caused by the westerlies and Indian Ocean monsoons in the QTP.

Traceability of Geographical Origin in Gentiana straminea by UPLC-Q Exactive Mass and Multivariate Analyses.

The root of Gentiana straminea Maxim. (Gentianaceae), is officially listed as "Qin-Jiao" in the Chinese Pharmacopoeia for the treatment of rheumatic arthritis, icteric hepatitis, constipation, pain, and hypertension. To establish the geographical origin traceability in G. straminea, its chemical profiles were determined by a UPLC-Q exactive mass spectrometer, from which 43 compounds were identified by comparing retention times and mass spectrometry. Meanwhile, a pair of isomers (loganin and secologanol) was identified by mass spectrometry based on their fragmentation pathway. A total of 42 samples from difference habitats were determined by an UPLC-Q exactive mass spectrometer and the data were assayed with multivariate statistical analysis. Eight characteristic compounds were identified to determine the geographical origin of the herb. To estimate the key characteristic markers associated with pharmacological function, the inhibiting activities of nitric oxide (NO) production in lipopolysaccharide (LPS)-induced macrophages were examined. This finding is crucial in realizing the determination of botanical origin and evaluating the quality of G. straminea.

Assessment of genetic diversity on Gentiana straminea based on ISSR markers / 中草药

Objective: To assess the genetic diversity of Gentiana straminea (Gentianaceae). Methods: Intersimple sequence repeat (ISSR) markers were used. Twenty-eight populations (83 individuals) of G. straminea were sampled from Sichuan, Qinghai, Gansu provinces, and Autonomous Region of Tibet. ISSR data were analyzed with the program POPGEN, and a UPGMA dendrogram was constructed. Results: PCR products corresponding to 183 alleles at 95 loci, amplified by the seven primers were scored. Eighty-eight loci were polymorphic. Expected heterozygosity (He) and Shannon's information index (Im) were 0.288 2 and 0.437 1, respectively. Nei's coefficient of genetic differentiation (Gst) and gene flow (Nm) were 0.678 3 and 0.237 1 at the population level, respectively. The genetic distance varied from 0.074 3 to 0.490 0. G. straminea populations were mainly divided into two braches by UPGMA tree. Conclusion: Genetic diversity level of G. straminea is high, and genetic diversity level among populations of G. straminea is higher than that in one population; Its autogenous variation mainly exist among populations; Hereditary capacity has certain correlation with geographical distribution. This work contributes basic information for variety identification, species in situ conservation, and enviroment investigation on the influence of heredity differentiated and Chinese Materia Medica genuine study. The congruence between genetic distance and geographic distances is high.

Authentication of Gentiana straminea Maxim. and its substitutes based on chemical profiling of iridoids using liquid chromatography with mass spectrometry.

A liquid chromatography-electrospray ionization-ion-trap mass spectrometry (MSn ) method was established and applied for authentication of Gentiana straminea from the four substitutes, G. tibetica, G. lhassica, G. waltonii and G. robusta, based on chemical profiling of the principal iridoid glucosides aided by a quadrupole time-of-flight mass spectrometry. The fragmentation pathways of the three representative iridoid glucosides, loganic acid, gentiopicroside and sweroside, were investigated by MSn analysis in negative ion mode, which assisted the characterization of analogs detected in the chromatographic profiling of the tested Gentiana species. In total, 25 iridoids were identified or tentatively characterized from G. straminea and four substitutes, in which 7-O-(4''-O-glucosyl)coumaroyl-loganic acid and 7-O-coumaroyl-loganic acid are diagnostic in G. straminea and can serve as the proposed chemical markers to discriminate it from morphologically similar substitutes.

[Ecology suitability study of Chinese materia medica Gentianae Macrophyllae Radix].

This paper is aimed to predict ecology suitability distribution of Gentianae Macrophyllae Radix and search the main ecological factors affecting the suitability distribution. The 313 distribution information about G. macrophylla, 186 distribution information about G. straminea, 343 distribution information about G. dauricaand 131 distribution information about G. crasicaulis were collected though investigation and network sharing platform data . The ecology suitable distribution factors for production Gentianae Macrophyllae Radix was analyzed respectively by the software of ArcGIS and MaxEnt with 55 environmental factors. The result of MaxEnt prediction was very well (AUC was above 0.9). The results of predominant factors analysis showed that precipitation and altitude were all the major factors impacting the ecology suitable of Getiana Macrophylla Radix production. G. macrophylla ecology suitable region was mainly concentrated in south of Gansu, Shanxi, central of Shaanxi and east of Qinghai provinces. G. straminea ecology suitable region was mainly concentrated in southwest of Gansu, east of Qinghai, north and northwest of Sichuan, east of Xizang province. G. daurica ecology suitable region was mainly concentrated in south and southwest of Gansu, east of Qinghai, Shanxi and north of Shaanxi province. G. crasicaulis ecology suitable region was mainly concentrated in Sichuan and north of Yunnan, east of Xizang, south of Gansu and east of Qinghai province. The ecological suitability distribution result of Gentianae Macrophyllae Radix was consistent with each species actual distribution. The study could provide reference for the collection and protection of wild resources, meanwhile, provide the basis for the selection of cultivation area of Gentiana Macrophylla Radix.

The complete chloroplast genome of Gentiana straminea (Gentianaceae), an endemic species to the Sino-Himalayan subregion.

Endemic to the Sino-Himalayan subregion, the medicinal alpine plant Gentiana straminea is a threatened species. The genetic and molecular data about it is deficient. Here we report the complete chloroplast (cp) genome sequence of G. straminea, as the first sequenced member of the family Gentianaceae. The cp genome is 148,991bp in length, including a large single copy (LSC) region of 81,240bp, a small single copy (SSC) region of 17,085bp and a pair of inverted repeats (IRs) of 25,333bp. It contains 112 unique genes, including 78 protein-coding genes, 30 tRNAs and 4 rRNAs. The rps16 gene lacks exon2 between trnK-UUU and trnQ-UUG, which is the first rps16 pseudogene found in the nonparasitic plants of Asterids clade. Sequence analysis revealed the presence of 13 forward repeats, 13 palindrome repeats and 39 simple sequence repeats (SSRs). An entire cp genome comparison study of G. straminea and four other species in Gentianales was carried out. Phylogenetic analyses using maximum likelihood (ML) and maximum parsimony (MP) were performed based on 69 protein-coding genes from 36 species of Asterids. The results strongly supported the position of Gentianaceae as one member of the order Gentianales. The complete chloroplast genome sequence will provide intragenic information for its conservation and contribute to research on the genetic and phylogenetic analyses of Gentianales and Asterids.

Ecology suitability study of Chinese materia medica Gentianae Macrophyllae Radix / 中国中药杂志

This paper is aimed to predict ecology suitability distribution of Gentianae Macrophyllae Radix and search the main ecological factors affecting the suitability distribution. The 313 distribution information about G. macrophylla, 186 distribution information about G. straminea, 343 distribution information about G. dauricaand 131 distribution information about G. crasicaulis were collected though investigation and network sharing platform data . The ecology suitable distribution factors for production Gentianae Macrophyllae Radix was analyzed respectively by the software of ArcGIS and MaxEnt with 55 environmental factors. The result of MaxEnt prediction was very well (AUC was above 0.9). The results of predominant factors analysis showed that precipitation and altitude were all the major factors impacting the ecology suitable of Getiana Macrophylla Radix production. G. macrophylla ecology suitable region was mainly concentrated in south of Gansu, Shanxi, central of Shaanxi and east of Qinghai provinces. G. straminea ecology suitable region was mainly concentrated in southwest of Gansu, east of Qinghai, north and northwest of Sichuan, east of Xizang province. G. daurica ecology suitable region was mainly concentrated in south and southwest of Gansu, east of Qinghai, Shanxi and north of Shaanxi province. G. crasicaulis ecology suitable region was mainly concentrated in Sichuan and north of Yunnan, east of Xizang, south of Gansu and east of Qinghai province. The ecological suitability distribution result of Gentianae Macrophyllae Radix was consistent with each species actual distribution. The study could provide reference for the collection and protection of wild resources, meanwhile, provide the basis for the selection of cultivation area of Gentiana Macrophylla Radix.

Genetic diversity of germplasm resources of Gentiana straminea from Tibet of China / 中草药

Objective: To study the genetic diversity of 17 samples of Gentiana straminea from different habitats of Tibet. Methods: Sampling in populations was carried out, and the related species G. crassicaulis from the same area was as the out group; nr DNA ITS sequences were amplified and analyzed; UPGMA systematic tree was constructed. Results: One polymorphic site of G. straminea from Tibet was found; the six samples of out group have the identical DNA ITS sequences. Conclusion: The difference between G. straminea and the out group is obvious; seventeen samples of G. straminea from different habitats are divided into two groups; genetic diversity is more abundant; the result could provide the basic reference for the quality evaluation and the construction of core germplasm of G. straminea in Tibet.

Non-iridoid constituents from roots of Gentiana straminea / 中草药

Objective To study the non-iridoid constituents from the roots of Gentiana straminea.Methods The compounds were repeatedly separated and purified on column chromatography of silica gel,Sephadex LH-20,and ODS,and their structures were identified on the basis of spectral and chemical methods.Results Twelve compounds were isolated from 70% ethanol extract of G.straminea and were identified as(-)-syringaresinol-4,4′-bis-?-O-D-glucopyranoside(1),gentiaphyllide-D(2),erythrecentaurin(3),macrophyiioside D(4),gentiaphyllide-E(5),roburic acid(6),uvaol(7),?-sitosterol(8),daucosterol(9),gentianose(10),?-glucose(11),and ?-glucose(12),respectively.Conclusion Compounds 1-6 and 10 are isolated from this plant for the first time.

HPLC-Fingerprint of Qinghai genuine medicinal herb——Gentiana straminea / 中草药

Objective To establish the fingerprint of Gentiana straminea from Qinghai Province by HPLC. Methods By orthogonal design and related methods based on information theories and chemometrics to optimize the proper HPLC conditions so that the fingerprint spectra of ten groups of different samples have been formed. Results The experimental results are the fact that the common fingerprint peaks, precision, and repeatability in the fingerprint totally meet the relative regulations required in Technical Requirements of the Fingerprint Research in Injection of Chinese Materia Medica (Tentatine Standard). Conclusion The chromatographic fingerprint can be used to identify and evaluate the quality of G. straminea.

HPLC fingerprint of Gentiana straminea Maxim / 中成药

AIM: To establish the HPLC fingerprint of Genti ana straminea Maxim. METHODS: It was adopted gradient with Methanol and 0.4% phospho ric acid, the detection wavelength was at 230nm and recording time. And it w as compared with a software published by Zhejiang University. RESULTS: The constituents of Gentiana straminea Maxim. were well se parated by HPLC, and the similarity was more than 0.90 . CONCLUSION: The method can be used as the fingerprint of G entiana straminea Maxim.