A neutral heteropolysaccharide from Halenia elliptica D. Don: Extraction, structural characterization, antioxidant and antiaging activities.

Halenia elliptica D. Don (H. elliptica), which is also known as "heijicao" and "luanehuamao" in China, is recognised as a valuable Tibetan medicinal plant with polysaccharides as the main active ingredient. However, studies on the polysaccharides isolated from H. elliptica are few. A polysaccharide (HEPN-1) with a molecular weight of 10.80 kDa was mainly composed of Gal, Ara, Man, Glc, Rha and Fuc in a molar ratio of 25.56:24.52:4.58:3.37:2.62:1.00. Structural analysis showed that HEPN-1 had a backbone mainly consisting of 4-ß-Galp, 3,6-ß-Galp and 3,4,6-ß-Galp and branched chains that contained two arabinan (R1 and R2) and two heteropolysaccharide (R3 and R4) side chains. The branching degree of HEPN-1 was 0.52. Within the range of doses (75-300 µg/mL), HEPN-1 increased the enzyme activity of SOD, CAT and GSH-Px and decreased the MDA level in H2O2-induced RAW 264.7 cells in a dose-dependent manner. After 6 weeks of intragastric administration, 300 mg/kg HEPN-1 considerably improved the learning and memory deficits in mice and the antioxidant enzyme system. Moreover, the MDA formation in D-gal-induced aging mice was inhibited, possibly partly via the activation of the PI3K/Akt and Nrf2/HO-1 signalling pathways. Therefore, HEPN-1 could serve as a potential natural antioxidant to prevent aging.

Phenotypic Selection in Halenia elliptica D. Don (Gentianaceae), an Alpine Biennial with Mixed Mating System.

The transition from outcrossing to selfing is a common evolutionary trend in flowering plants, and floral traits change significantly with the evolution of selfing. Whether or not plant traits are subjected to selection remains an open question in species with mixed mating systems. We examined phenotypic selection in two populations of Halenia elliptica with different selfing rates. We found that the pollen-ovule ratio, seed size, plant height, spur length, and pollinator visitation rate in the population with the higher selfing rate were lower than those in the population with the lower selfing rate. Selfing provides reproductive assurance for populations when pollinator service is low, and the floral traits that are associated with selfing syndrome are evident in populations with a higher selfing rate but are subjected to weak selection in each of the two populations with different selfing rates. Directional selection for an early flowering time indicated that late blooming flowers could experience a risk of seed development in alpine environments, and for large plants, selection indicated that seed production could be limited by the available resources. The floral traits that are associated with pollinator attraction and specialization could be subjected to weak selection at the plant level as selfing evolves, and the selective pressures that are independent of pollinators might not change significantly; highlighting the selective biotic and abiotic pressures that shape the morphological traits of plant species and their independence from the mating system.

Multiple lines of evidence supports the two varieties of Halenia elliptica (Gentianaceae) as two species.

Delimiting species requires multiple sources of evidence. Here, we delimited two varieties of Halenia elliptica (Gentianaceae) using several lines of evidence, including morphological traits and mating system in a sympatric population, phylogenetic relationships based on nrITS and cpDNA (rpl16) data, and complete chloroplast genome sequences. Comparative analysis of 21 morphological traits clearly separates the two varieties of H. elliptica. Examination of the flowering process and pollination treatments indicate that H. elliptica var. grandiflora produces seeds via outcrossing, whereas H. elliptica var. elliptica produces seeds via mixed mating. Furthermore, hand-pollinated hybridization of the two varieties produced no seeds. Observations of pollinators showed that when bees began a pollination bout on H. elliptica var. grandiflora they preferred to continue pollinating this variety; however, when they began a pollination bout on H. elliptica var. elliptica, they showed no preference for either variety. Phylogenetic analysis confirmed the monophyly of H. elliptica, which was further divided into two monophyletic clades corresponding to the two varieties. A large number of variants from the chloroplast genomes reflected remarkable genetic dissimilarities between the two varieties of H. elliptica. We recommend that the two varieties of H. elliptica should be revised as two species (H. elliptica and H. grandiflora). Our findings indicate that H. elliptica varieties may have split into two separate species due to a shift in mating system, changes in flowering phenology and/or post-pollination reproductive isolation.

[Research on autophagy induced by two xanthone compounds in HepG2 cells].

To investigate the inhibitory effects of two xanthone compounds, 1-hydroxy-2,3,4,8-4 methoxy xanthone(here in after referred to as Fr15) and 1-hydroxy-2,3,4,6-4 methoxy xanthone(here in after referred to as Fr17), on the proliferation of hepatocellular carcinoma cells HepG2, and to further investigate their mechanism in combination with transcriptomics. Cell counting was used to detect the effects of two kinds of xanthone compounds Fr15 and Fr17(0, 0.03, 0.15, 0.3 mmoL·L~(-1)) on the proliferation of HepG2 cells; the effects of the two compounds Fr15 and Fr17 on HepG2 cell cycle were detected by flow cytometry; the changes of autophagosomes count in cells were observed under fluorescence microscope; the expression of autophagy marker proteins autophagy marker proteins SQSTM 1(p62) and microtubule associated protein 1 light chain 3 Ⅰ/Ⅱ(LC3 Ⅰ/Ⅱ) in the cells was detected by Western blot; the differentially expressed genes between the control group and the experimental group were analyzed by RNA-seq transcriptome sequencing; qRT-PCR was used to verify the differentially expressed genes in sequencing. The results showed that compounds Fr15 and Fr17 inhibited the proliferation of HepG2 cells with the increase of drug concentration and time. Flow cytometry showed that compounds Fr15 and Fr17 had little effect on HepG2 cell cycle. Fluorescence microscopy results showed that the number of autophagosomes in cells increased with the increase of drug concentration. Western blot showed that the expression of p62 protein was decreased and the expression of LC3-Ⅱ protein was significantly increased after drug addition. The results of RNA sequencing showed that 26 102 and 52 351 differentially expressed genes were obtained in Fr15 and Fr17 respectively. Analysis of KEGG showed that drug treatment had a great effect on autophagy pathway. qRT-PCR verified that 6 up-regulated genes were related to autophagy, and their trend was consis-tent with sequencing results, where all 6 genes showed an up-regulated trend. Two xanthone compounds Fr15 and Fr17 may inhibit proliferation of HepG2 cells by inducing autophagy.

Research on autophagy induced by two xanthone compounds in HepG2 cells / 中国中药杂志

To investigate the inhibitory effects of two xanthone compounds, 1-hydroxy-2,3,4,8-4 methoxy xanthone(here in after referred to as Fr15) and 1-hydroxy-2,3,4,6-4 methoxy xanthone(here in after referred to as Fr17), on the proliferation of hepatocellular carcinoma cells HepG2, and to further investigate their mechanism in combination with transcriptomics. Cell counting was used to detect the effects of two kinds of xanthone compounds Fr15 and Fr17(0, 0.03, 0.15, 0.3 mmoL·L~(-1)) on the proliferation of HepG2 cells; the effects of the two compounds Fr15 and Fr17 on HepG2 cell cycle were detected by flow cytometry; the changes of autophagosomes count in cells were observed under fluorescence microscope; the expression of autophagy marker proteins autophagy marker proteins SQSTM 1(p62) and microtubule associated protein 1 light chain 3 Ⅰ/Ⅱ(LC3 Ⅰ/Ⅱ) in the cells was detected by Western blot; the differentially expressed genes between the control group and the experimental group were analyzed by RNA-seq transcriptome sequencing; qRT-PCR was used to verify the differentially expressed genes in sequencing. The results showed that compounds Fr15 and Fr17 inhibited the proliferation of HepG2 cells with the increase of drug concentration and time. Flow cytometry showed that compounds Fr15 and Fr17 had little effect on HepG2 cell cycle. Fluorescence microscopy results showed that the number of autophagosomes in cells increased with the increase of drug concentration. Western blot showed that the expression of p62 protein was decreased and the expression of LC3-Ⅱ protein was significantly increased after drug addition. The results of RNA sequencing showed that 26 102 and 52 351 differentially expressed genes were obtained in Fr15 and Fr17 respectively. Analysis of KEGG showed that drug treatment had a great effect on autophagy pathway. qRT-PCR verified that 6 up-regulated genes were related to autophagy, and their trend was consis-tent with sequencing results, where all 6 genes showed an up-regulated trend. Two xanthone compounds Fr15 and Fr17 may inhibit proliferation of HepG2 cells by inducing autophagy.

The complete chloroplast genome of a medical herb, Halenia elliptica D.Don (Gentianaceae), from Qinghai-Tibet Plateau in China.

Halenia elliptica D.Don (Gentianaceae) is one of the genuine medicinal species in Qinghai-Tibet Plateau, China. Here we report the first chloroplast (cp) genome of H. elliptica using Illumina HiSeq X Ten platform. The length of its complete cp genome is 153,341 bp, containing four sub-regions; a large single-copy region (LSC) of 82,811 bp and a small single-copy region (SSC) of 18,278 bp, which are separated by a pair of inverted repeat regions (IRs) of 26,126bp each. The complete cp genome of H. elliptica contains 129 genes, including 84 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The overall GC content of the cp genome is 38.1%. The phylogenetic analysis, based on 15 cp genomes, suggested that H. elliptica is closely related to Halenia corniculata (L.) Cornaz and Swertia species.

Analysis of the Phylogenetic Relationships of 3 Basic Plants of Tibetan Medicine “Dida”Based on ISSR Technology / 中国药房

OBJECTIVE： To study the phylogenetic relationships of 3 basic plants of Tibetan medicine “Dida”， such as Swertia puricea， Wertia mileensis， Halenia elliptica. METHODS： ISSR technology was used for PCR amplification of 9 samples of S. puricea （ZT-1 to ZT-5 from Gantongsi in Dali Cangshan， ZC-1 to ZC-4 from Binchuan county of Dali）， 2 samples of W. mileensis （QYD-1 to QYD-2） and 2 samples of H. elliptica （HM-1 to HM-2）. Using DNA genome of S. puricea as template， 8 primers were screened and used for PCR reaction. The PCR amplification products were read by hand， the original data matrix was established， and the polymorphic band ratio was calculated. At the same time， genetic similarity coefficient was calculated by using NTSYS 2.1 software， and UPGMA method was used to draw cluster diagram. RESULTS： A total of 113 clear and identifiable amplification product bands were obtained by 8 ISSR primers. The rate of polymorphic site was 100％. The genetic similarity coefficients for totally 13 samples of S. puricea， W. mileensis and H. elliptica ranged 0.301-0.500. Intraspecific genetic similarity coefficients for 9 samples of S. puricea ranged from 0.752 to 0.929. The cluster analysis showed， when the range line was 0.410， 13 samples could be divided into three groups， i.e. S. puricea， W. mileensis， H. elliptica； when the range line was 0.780， 9 samples of S. purpurea could be divided into 2 subgroups， one of which was only sample ZT-1 collected from Gantongsi in Cangshan， and the other contained the remaining 8 samples. CONCLUSIONS： ISSR technology can be used to identify S. punicea， S. glabra and H. elliptica at the molecular level. S. punicea has some genetic relationship with S. glabra and H. elliptica， but the genetic relationship is relatively distant and the genetic difference is large. S. punicea from two different locations in Dali area has little genetic difference and close relationship， but it shows abundant genetic diversity.

Transcriptome Analysis and Microsatellite Markers Development of a Traditional Chinese Medicinal Herb Halenia elliptica D. Don (Gentianaceae).

Halenia elliptica is a popular Chinese medicinal herb that is used to treat jaundice disease and virus hepatitis, and its wild populations have been reduced significantly due to overharvesting recently. However, effective conservation could not be implemented because of the lack of genomic information and genetic markers. In this study, a de novo transcriptome of H elliptica was sequenced using the NGS Illumina, and 132 695 unigenes with the length >200 bp (base pairs) were obtained. Among them, a total of 32 109 unigenes were scanned to develop simple sequence repeats (SSRs). Based on NCBI (National Center for Biotechnology Information) nonredundant database (Nr), these SSR sequences were annotated and assigned into gene ontology categories. In addition, we designed 126 pairs of SSR primers for polymerase chain reaction amplification, of which 12 pairs were identified to be polymorphic among 40 individuals from 8 populations. We then used the 12 polymorphic SSRs to construct a UPGMA dendrogram of the 40 individuals. In addition, a significant correlation between the genetic relationship and the geographic distance was found, suggesting a phylogeographic structure in H elliptica. Moreover, 2 of these SSRs were also successfully amplified in a related species Veratrilla baillonii, suggesting their cross-species transferability. Generally, the SSR markers with high polymorphisms identified in this study provide valuable genetic resources and represent an initial step for exploring the genetic diversity and population histories of H elliptica and its related species.

Equipped for Migrations Across High Latitude Regions? Reduced Spur Length and Outcrossing Rate in a Biennial Halenia elliptica (Gentianaceae) With Mixed Mating System Along a Latitude Gradient.

Halenia (Gentianaceae) originated from the mountain regions of East Asia, and diversified in America following long migrations via Beringia. While Halenia elliptica, one species of the genus in China, migrated toward high latitudes in China. Spur length of H. elliptica is highly variable. We examined the relationship between spur length and mating pattern along a latitude gradient. Field experiments were performed in two populations of H. elliptica, and we found that this species could produce seeds via both autonomous selfing and the aid of pollinators, suggesting a mixed mating system. In seven populations of H. elliptica along a latitudinal gradient, we found a trend of decrease in spur length with the increase of latitude. Based on molecular data from 11 microsatellite loci, we found that multilocus outcrossing rate decreased with the increase of latitude while the estimated inbreeding depression increased significantly, indicating that a high degree of inbreeding depression might have prevented evolution toward complete selfing in the high latitude populations with short spur length, and thus maintained mixed mating system of H. elliptica. Our results suggest that the mixed mating system of this species might be helpful in overcoming pollinator scarcity in newly colonized populations toward high latitudes after its origination in the mountain regions of China, and the decrease of spur length in the high latitude populations could result from reduced resource allocation to pollinator associated traits.

Interaction effects on cytochrome P450 both in vitro and in vivo studies by two major bioactive xanthones from Halenia elliptica D. Don.

The major components, 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1) and 1,5-dihydroxy-2,3-dimethoxy-xanthone (HM-5) isolated from Halenia elliptica D. Don (Gentianaceae), could cause vasodilatation in rat coronary artery with different mechanisms. In this work, high-performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LCMS-IT-TOF) was used to clarify the metabolic pathways, and CYP450 isoform involvement of HM-1 and HM-5 were also studied in rat. At the same time, in vivo inhibition effects of HM-1 and ethyl acetate extracts from origin herb were studied. Three metabolites of HM-5 were found in rat liver microsomes (RLMs); demethylation and hydroxylation were the major phase I metabolic reactions for HM-5. Multiple CYP450s were involved in metabolism of HM-1 and HM-5. The inhibition study showed that HM-5 inhibited Cyp1a2, 2c6 and 2d2 in RLMs. HM-1 inhibited activities of Cyp1a2, Cyp2c6 and Cyp3a2. In vivo experiment demonstrated that both HM-1 and ethyl acetate extracts could inhibit Cyp3a2 in rats. In conclusion, the metabolism of xanthones from the origin herb involved multiple CYP450 isoforms; in vitro, metabolism of HM-5 was similar to that of its parent drug HM-1, but their inhibition effects upon CYP450s were different; in vivo, Cyp3a2 could be inhibited by HM-1 and ethyl acetate extracts.

Ethnopharmacological, chemical, and pharmacological aspects of Halenia elliptica: A comprehensive review.

Traditional Tibetan medicine (TTM) is an old traditional medical system, which is an effective and natural method of improving physical and mental health, and has been widely spread in the western part of China for centuries. Halenia elliptica (H. elliptica) D. Don, known as "Jiadiranguo" (Tibetan medicine name) is one of the most important herbal medicine in TTM that is from the genus Halenia (family: Gentianaceae). The whole herb can be used as a medicine to treat hepatobiliary diseases and xeransis, and possesses many biological and pharmacological activities including heat clearing, bile benefiting, liver soothing, digestion promoting, blood nursing, detoxification activities, and so on. In modern research, H. elliptica can be used to treat acute or chronic hepatitis, especially hepatitis B. In addition, the chemical compounds of the herb have potent antihepatitis B virus (anti-HBV) activity in vitro. As an important TTM, further studies on H. elliptica can lead to the development of new drugs and therapeutics for various diseases, and more attention should be paid on the aspects of how to utilize it better.

In vitro identification of cytochrome P450 isoforms responsible for the metabolism of 1-hydroxyl-2,3,5-trimethoxy-xanthone purified from Halenia elliptica D. Don.

1-Hydroxyl-2,3,5-trimethoxyxanthone (HM-1) is one of the main constituents extracted from Halenia elliptica D. Don, which is a traditionally used Tibetan medicinal plant. The aim of this study was to illustrate the proposed metabolic pathways of HM-1 and identify which cytochrome P450 (CYP450) isoforms involved in its metabolism by using pooled human liver microsomes (HLMs) and recombinant CYP450 isoforms with selective chemical inhibitors. Metabolites were identified by high performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LCMS(n)-ESI-IT-TOF) and nuclear magnetic resonance spectroscopy (hydrogen-1 NMR and carbon-13 NMR). Three metabolites (M1-M3) were identified, which demonstrated that demethylation and hydroxylation were the major Phase I metabolic reactions for HM-1 in HLMs. The structure of another metabolite (M4) was still unclear. The enzymatic kinetics of M1 (K(m)=23.19±14.20 µM) and M2 (Km=32.06±17.09 µM) exhibited substrate inhibition; whereas, the formation of M3 (K(m)=5.73±0.70 µM) and M4 (K(m)=16.43±5.12 µM) displayed Michaelis-Menten kinetics. The intrinsic clearance (V(max)/K(m)) of M3 was highest among these metabolites, suggesting that M3 was the major metabolite of HM-1. Moreover, CYP3A4 and CYP2C8 were the primary CYP450 isoform responsible for the metabolism of HM-1. CYP1A2, CYP2A6, CYP2B6, CYP2C9 and CYP2C19 were also involved in HM-1 metabolism, especially in the formation of M3. This study finally provides evidence of substrate inhibition and metabolism-based drug-drug interaction for the medicinal preparations containing HM-1 used in clinic.

Protective Effects of Ethanol-extract of Halenia elliptica on Chemical Hepatic Injury in Mice / 中药新药与临床药理

Objective To investigate the protective effects of ethanol-extract of Halenia elliptica D. Don on chemical hepatic injury in mice. Methods Mouse models of chemical hepatic injury were induced by intraperitoneal injection of 0.12 %CCL4. Extract of Halenia elliptica D. Don by alcohol was administered,and serum ALT,AST activities and liver glycogen level were measured in mice. Results Compared with the models,the enzyme activities of ALT and AST were significantly reduced and the content of liver glycogen was significantly increased in the ethanol-ex tract of Halenia elliptica groups. Conclusion It is indicated that ethanol-extract of Halenia elliptica D. Don has an effect in protecting the liver.

Identification of new chemical constituents of Tibetan medicinal herb Halenia elliptica / 中草药

Object To separate and characterize the chemical constituents of a plateau plant Halenia elliptica D Don Methods Elemental analysis (EA), 1HNMR and 13 CNMR, MS, FTIR and UV spectrometry, as well as DSC were employed Results Two needle shaped crystal chemical constituents obtained from H elliptica were confirmed to be 1 hydroxy 3, 7, 8 trimethoxyxanthone and 1, 7 dihydroxy 3, 8 dimethoxyxanthone, respectively Conclusion This is the first time for these two chemical constituents to be separated from this Tibetan medicinal plant