Chloroplast DNA reveals genetic population structure in Sinomenium acutum in subtropical China / 中草药·英文版

OBJECTIVE@#The population density and diversity of Sinomenium acutum (Menispermaceae) have been greatly reduced recently by overharvesting for medicinal purposes in China. Therefore, it is urgent that the remaining populations are investigated, and that strategies for the utilization and conservation of this species are developed. This study aimed to find the possible glacial refugia and define the genetic diversity of S. acutum for its proper utilization and conservation.@*METHODS@#A total of 77 S. acutum samples were collected from four locations, Qinling Mountains, Daba Mountains, Dalou Mountains, and Xuefeng Mountains, in subtropical China. Genetic diversity among and between these populations were phylogenetically analyzed using four chloroplast DNA molecular markers (atpI-atpH, trnQ-5'rps16, trnH-psbA and trnL-trnF).@*RESULTS@#A total of 14 haplotypes (C1 to C14) were found in collected samples. Haplotypes C1 and C3 were shared among all populations, with C3 as the ancestral haplotype. Haplotypes C11 and C12 diverged the most from C3 and other haplotypes. No obvious phylogeographic structure was found in four locations using the GST/NST test. There is no evidence of rapid demographic expansion in S. acutum based on the mismatch distribution, and the results of Tajima's D test, and Fu's FS test. Our analyses of molecular variance revealed a high level of genetic variation within populations. In contrast, the genetic differentiation among S. acutum populations was low, indicating frequent gene flow.@*CONCLUSION@#Xuefeng, Dalou, and Daba Mountains were possible glacial refugia for the populations of S. acutum. C1, C3, C11 and C12 haplotypes of S. acutum should be carefully preserved and managed for their genetic value.

Discovery, isolation and structural identification of alkaloid glycosides in six traditional Chinese medicine such as Coptis chinensis / 中国中药杂志

Alkaloids are important active ingredients occurring in many traditional Chinese medicines, and alkaloid glycosides are one of their existence forms. The introduction of saccharide units improves the water solubility of alkaloid glycosides thus presenting better biological activity.Because of the low content in plants, alkaloid glycosides have been not comprehensively studied. In this study, ultrahigh performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry(UPLC-QTOF-MS/MS) was employed to identify and analyze the alkaloid glycosides in Coptis chinensis, Phellodendron chinense, Menispermum dauricum, Sinomenium acutum, Tinospora sagittata and Stephania tetrandra. The results showed that except Tinospora sagittata, the other five herbal medicines contained alkaloid glycosides. Furthermore, the alkaloid glycosides in each herbal medicine were identified based on UV absorption spectra, quasimolecular ion peaks in MS, fragment ions information in the MS/MS, and previous literature reports. A total of 42 alkaloid glycosides were identified. More alkaloid glycosides were identified in C. chinensis and Menispermum dauricum, and eleven in C. chinensis were potential new compounds. Furthermore, the alkaloid glycosides in the water extract of C. chinensis were coarsely se-parated by macroporous adsorption resin, purified by column chromatography with D151 cation exchange resin, ODS and MCI, combined with semi-preparative high performance liquid chromatography. Two new alkaloid glycosides were obtained, and their structures were identified by mass spectrometry and NMR data as(S)-7-hydroxy-1-(p-hydroxybenzyl)-2,2-N,N-dimethyl-1,2,3,4-tetrahydroisoquinoline-6-O-β-D-glucopyranoside and(S)-N-methyltetrahydropalmatubine-9-O-β-D-glucopyranoside, respectively. This study is of great significance for enriching the information about the chemical composition and the in-depth development of C. chinensis. Meanwhile, it can provide a reference for rapid identification and isolation of alkaloid glycosides from other Chinese herbal medicines.

Analysis of sinomenine synthesis pathway based on transcriptome sequencing in Sinomenii Caulis / 中草药

Objective: In order to explore the expression of sinomenine content control genes, synthetic control sites and expression pathway. Methods: In this study, high performance liquid chromatography (HPLC) was used to determine the content of sinomenine in the roots and stems of 49 Sinomenii Caulis in six populations. Two populations with large multiple differences in sinomenine content were selected, namely Shanxi Baoji and Guizhou Zunyi. The most representative of them were selected, and their roots and stems were taken for transcriptome sequencing and named as HR/LR and HS/LS. Results: Sequencing results showed that 355 201 transcripts were obtained by splicing clean reads, including 275 491 Unigene transcripts. There were 23 562 and 37 143 differentially expressed genes in HR/LR and HS/LS, respectively. GO database analysis showed that the functions of these differentially expressed genes were significantly enriched in aspartic-type endopeptidase activity and aspartic-type peptidase activity, it is speculated that these two enzymes might be encoded. The results of KEGG enrichment explained that the differentially expressed genes were involved in carbohydrate metabolism, protein binding to cell membrane and vitamin C synthesis. The results of qRT-PCR verified the expression of upstream key genes of the isoquinoline alkaloid synthesis pathway and found that it was positively correlated with the accumulation of sinomenine. Conclusion: This study provided a preliminary understanding of the molecular mechanism that caused the difference in sinomenine content, and provided a reference for further understanding of the accumulation rules and synthesis pathways of sinomenine.

Bioinformatics Based Therapeutic Effects of Sinomenium Acutum / 中国结合医学杂志

OBJECTIVE@#To decipher the possible mechanisms of Sinomenium Acutum (SA) in treating diseases by a bioinformatics method.@*METHODS@#SA ingredients were searched according to Chinese Pharmacopoeia, Chinese Medicine Dictionary and Traditional Chinese Medicines Database (TCMD). Active compounds and target proteins of SA were acquired through the Pubchem platform. Pathway, network and function analyses of SA were performed with ingenuity pathway analysis (IPA), a bioinformatics analysis platform. Disease, biofunction-target networks were established with Cytoscape.@*RESULTS@#Eighteen ingredients from SA were obtained. Seven active ingredients with 31 active target proteins were acquired according to PubChem Bioassay test. By IPA analysis, 277 canonical pathways belonging to 17 function categories were collected, 23 kinds of diseases, 21 categories bio-functions were obtained. Based on P value, calculated by IPA, the top 5 significant pathway of SA targets include phosphatidylinositol 3 kinase/Akt (PI3K/Akt) signaling, prostate cancer signaling, macrophage migration inhibitory factor (MIF) regulation of innate immunity, Guanosine-binding protein coupled receptor (GPCR) signaling, and ataxia telangiectasia mutated protein (ATM) signaling. Disease and bio-function network analysis indicated that mitogen activated protein kinase 1 (MAPK1), MAPK3, p65 nuclear factor κB (RELA), nuclear factor of κB inhibitor alpha (NFκBIA), interleukin 1β(IL-1β), prostaglandin G/H synthase 2 (PTGS2) and tumor protein 53 (TP53) were the critical targets in various diseases treated by SA.@*CONCLUSION@#In the different view of target, pathway, disease and bio-function, inflammation was found to be a central theme in many chronic conditions. SA could be used not only as an anti-inflammatory agent, but also for the treatment of cancers, neurological diseases, psychological disorders and metabolic diseases.

Sinomenine, an Alkaloid Derived from Sinomenium acutum Potentiates Pentobarbital-Induced Sleep Behaviors and Non-Rapid Eye Movement (NREM) Sleep in Rodents

Sinomenium acutum has been long used in the preparations of traditional medicine in Japan, China and Korea for the treatment of various disorders including rheumatism, fever, pulmonary diseases and mood disorders. Recently, it was reported that Sinomenium acutum, has sedative and anxiolytic effects mediated by GABA-ergic systems. These experiments were performed to investigate whether sinomenine (SIN), an alkaloid derived from Sinomenium acutum enhances pentobarbital-induced sleep via γ-aminobutyric acid (GABA)-ergic systems, and modulates sleep architecture in mice. Oral administration of SIN (40 mg/kg) markedly reduced spontaneous locomotor activity, similar to diazepam (a benzodiazepine agonist) in mice. SIN shortened sleep latency, and increased total sleep time in a dose-dependent manner when co-administrated with pentobarbital (42 mg/kg, i.p.). SIN also increased the number of sleeping mice and total sleep time by concomitant administration with the sub-hypnotic dosage of pentobarbital (28 mg/kg, i.p.). SIN reduced the number of sleep-wake cycles, and increased total sleep time and non-rapid eye movement (NREM) sleep. In addition, SIN also increased chloride influx in the primary cultured hypothalamic neuronal cells. Furthermore, protein overexpression of glutamic acid decarboxylase (GAD(65/67)) and GABA(A) receptor subunits by western blot were found, being activated by SIN. In conclusion, SIN augments pentobarbital-induced sleeping behaviors through GABA(A)-ergic systems, and increased NREM sleep. It could be a candidate for the treatment of insomnia.

Effects of Sinomenium acutum Polysaccharide on Oxidative Stress and Hepatocyte Apoptosis in Mice with Acute Alcoholic Liver Injury / 中国药房

OBJECTIVE:To explore the protective effect and its mechanisms of Sinomenium acutum polysaccharide on mice with acute alcoholic liver injury. METHODS:60 mice were randomly divided into blank control group (normal saline),model group (normal saline),bifendate group (positive control,150 mg/kg) and S. acutum polysaccharide low-dose,medium-dose, high-dose groups(100,200,400 mg/kg),10 in each group,intragastrically administrated,once a day,for continual 7 d. 1 h af-ter last administration,mice received 50% ethanol (0.1 mL/10 g) intragastrically to induce acute alcoholic liver injury model ex-cept for those in blank control group. After 12 h,alanine aminotransferase(ALT),aspartate aminotransferase(AST)levels in se-rum,malondialdehyde(MDA),superoxide dismutase(SOD),glutathione(GSH),glutathione peroxidase(GSH-Px)levels in liv-er tissue of mice were determined;hematoxylin-eosin staining was conducted to observe the pathological changes in liver tissue;flow cytometry was used to detect the cell apoptosis rate. RESULTS:Compared with blank control group,mice in model group showed pathological changes in edema,disordered cell arrangement and local necrosis;ALT and AST levels in serum,MDA level in liver tissue and hepatocyte apoptotic rate were significantly increased,while the SOD,GSH and GSH-Px levels in liver tissue were significantly decreased,with statistical significances (P<0.01). Compared with model group,cell degeneration and necrosis degree of mice were improved in S. acutum polysaccharide medium-dose and high-dose groups;except for cell apoptosis rate of liver in S. acutum polysaccharide low-dose group was not decreased significantly,the above-mentioned indicators in other treatment groups were significantly improved(P<0.05 or P<0.01). CONCLUSIONS:S. acutum polysaccharide shows obvious protective effect on mice with acute alcoholic liver injury,its mechanism might re-late to anti-oxidation stress and inhibiting hepatocyte apoptosis.

Reversed-phase ion-pair chromatography in determination of sinomenine contents in Sinomenium acutum / 第二军医大学学报

Objective: To establish a reversed-phase ion-pair chromatography (RP-IPC) method for determination of sinomenine content in Sinomenium acutum. Methods: The IPC method was established using a column of Eclipse Plus C18(4. 6 mmX250 mm, 5 μm) with a mobile phase of acetonitrile and buffer phosphate (0. 03 mol/L potassium dihydrogen phosphate and 0. 03 mol/L sodium heptane sulfonic acid, adjusted to pH 4. 0 by phosphoric acid) 18 : 82. The flow rate was 1. 0 ml/min; the detection wavelength was set at 262 nm, and the temperature was at 40°C. Results: The linearity was obtained over a concentration range of 0. 025-0. 5 mg/ml(r=1. 000). The low limit of detection was 2. 5 μg/ml. The intra-day precisions of low, medium, and high concentrations of sinomenine were 1.54%, 0. 70%, and 0. 45%, and the inter-day precisions were 1. 54%, 0. 70%, and 0. 45%, respectively. The average recovery was 99. 9%(RSD=2. 9%, n=6), which met the requirement for analysis. Conclusion: The present method is simple, fast, and accurate; it is suitable for determination of sinomenine content in Sinomenium acutum.