[Multiplex PCR method for simultaneous identification of Xanthii Fructus and its adulterants].

The purpose of this study is to establish a molecular method to identify Xanthii Fructus and two adulterants, the fruits of Xanthium mongolicum and X. italicum. Xanthii Fructus is the fruit of X. sibiricum, which is a Chinese herbal medicine used clinically to treat allergic rhinitis. The fruits of X. mongolicum and X. italicum have strong morphological similarities with Xanthii Fructus, while their safety of medication cannot be guaranteed. The genomes of X. sibiricum, X. mongolicum, and X. italicum were sequenced, which generated sequences of 2.21, 2.24, and 2.54 Gb, respectively. Based on the 76 specific contigs screened out by BLASTN and Bowtie 2, the corresponding primers were designed by Primer 5.0. Three pairs of primers with stable amplification efficiency and good reproducibility were screened out to establish a multiplex PCR method based on the PCR amplification results. Further, the annealing temperature, the amount of DNA template, the number of cycles, different DNA polymerases, and different PCR thermal cyclers were optimized. Fragments of 262 bp and 458 bp from X. sibiricum, 260, 454, and 927 bp from X. mongolicum, and 260 bp and 926 bp from X. italicum were amplified under the following conditions: the annealing temperature of 52 ℃, 35 cycles, 30 ng template DNA. Then, the established method was used to detect 18 samples of X. sibiricum, 17 samples of X. mongolicum, and 12 samples of X. italicum. The results showed that all the samples had positive results, which were consistent with the morphological identification results, thus proving the stability and reliability of the established method. Combining genome sequencing technology and multiplex PCR method to identify Xanthii Fructus and its adulterants can not only obtain the difference in genetic background but also facilitate the design of reliable primers. The multiplex PCR have high specificity and repeatability, providing a new method for the molecular identification of Xanthii Fructus.

[Extracts of Poria cocos polysaccharides improves alcoholic liver disease in mice via CYP2E1 and NF-κB inflammatory pathways].

The present study investigated the effect of extract of Poria cocos polysaccharides(PCP) on cytochrome P450 2 E1(CYP2 E1) and nuclear factor κB(NF-κB) inflammatory signaling pathways in alcoholic liver disease(ALD) mice and explored its protective effect and mechanism. Sixty male C57 BL/6 N mice of SPF grade were randomly divided into a control group, a model group, a positive drug group(bifendate, 200 mg·kg~(-1)), and high-(200 mg·kg~(-1)) and low-dose(50 mg·kg~(-1)) PCP groups. Gao-binge mo-del was induced and the mice in each group were treated correspondingly. Liver morphological and pathological changes were observed and organ index was calculated. Serum levels of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were detected. Malondialdehyde(MDA) and superoxide dismutase(SOD) in liver tissues were detected by assay kits. The levels of interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) were detected by ELISA. The activation of macrophages was observed by immunofluorescence staining and protein expression of CYP2 E1, Toll-like receptor 4(TLR4), NF-κB p65, and phosphorylated NF-κB p65(p-NF-κB p65) were analyzed by Western blot. The ALD model was properly induced. Compared with the model group, the PCP groups significantly improved the pathological injury of liver tissues. Immunofluorescence staining revealed that compared with the model group, the groups with drug intervention showed decreased macrophages in liver tissues. Additionally, the PCP groups showed reduced ALT, AST, MDA, IL-6, and TNF-α(P<0.05), and potentiated activity of SOD(P<0.01). PCP extract has the protective effect against alcoholic liver injury in mice, and the underlying mechanism may be related to the regulation of the expression of CYP2 E1 and inhibition of TLR4/NF-κB inflammatory signaling pathway to reduce oxidative stress and inflammatory injury, thereby inhibiting the development of ALD.

[Research progress on chemical constituents and pharmacological effects of Glycyrrhizae Radix et Rhizoma and discussion of Q-markers].

Glycyrrhizae Radix et Rhizoma, a traditional Chinese herbal medicine, mainly contains triterpenoids, flavonoids, polysaccharides, coumarins and volatile oils with many pharmacological activities such as anti-tumor, anti-bacterial, anti-viral, anti-inflammatory, immune regulatory and anti-fibrotic effects. The widespread applications of Glycyrrhizae Radix et Rhizoma in food, medicine and chemical industries make its demand increase gradually. Therefore, the quality guarantee of the medicinal is of great value. Starting from the elaboration of chemical components and pharmacological effects of Glycyrrhizae Radix et Rhizoma and the introduction to the concept of quality marker(Q-marker), this study analyzed the Q-markers of Glycyrrhizae Radix et Rhizoma from the aspects of plant phylogene-tics, chemical component specificity, traditional efficacy, traditional medicinal properties, absorbed components, different processing methods and so on, which provides reference for quality evaluation, development and utilization of Glycyrrhizae Radix et Rhizoma.

[Review of chemical composition, pharmacological effects, and clinical application of Salviae Miltiorrhizae Radix et Rhizoma and prediction of its Q-markers].

Salviae Miltiorrhizae Radix et Rhizoma is a Chinese herbal medicine that promotes blood circulation to remove blood stasis, nourishes blood to tranquilize the mind, and cools blood to disperse carbuncles. Salviae Miltiorrhizae Radix et Rhizoma has microcirculation-improving, blood vessel-dilating, atherosclerosis-preventing, anti-inflammatory, anti-tumor, and blood pressure-and blood lipid-lowering activities. As research progresses, the chemical composition, pharmacological effect, and clinical application of Salviae Miltiorrhizae Radix et Rhizoma have attracted much attention. We reviewed the research progress in this field. Based on the concept of quality marker(Q-marker) in traditional Chinese medicine, the Q-markers of Salviae Miltiorrhizae Radix et Rhizoma were predicted and analyzed from the aspects of quality transfer, traceability, ingredient specificity, association between ingredients and pharmacological effects, ingredient predictability, and compounding environment. This review provides a scientific basis for the quality control of Salviae Miltiorrhizae Radix et Rhizoma and its preparations.

[Biosynthetic pathways of Polygonatum cyrtonema polysaccharide and diosgenin based on its transcriptomic data].

Polygonatum cyrtonema belongs to the plant family Liliaceae, and its dried rhizome is one of the sources of Chinese traditional medicine of Polygonati Rhizoma. It possesses the dual function as both medicine and food. Its main chemical components are polysaccharides and saponins. In order to understand the biosynthesis pathway of polysaccharides and diosgenin in P. cyrtonema, the corresponding transcriptomic data were obtained by extracting and sequencing the RNA of four parts of P. cyrtonema, namely, leaves, stems, rhizomes and roots. By adopting BGISEQ-500 sequencing platform, 42.03 Gb data were retrieved. Subsequently, the de novo assembly was carried out by Trinity software to obtain 137 233 transcripts, of which 68.13% of unigenes were annotated in seven databases including KEGG, GO, NR, NT, SwissProt, Pfam and KOG. Transcripts that may be involved in the biosynthesis of polysaccharides and diosgenin were analyzed by data mining. With help of qPCR, we validated expression data of four genes that were possibly involved in the biosynthesis of target metabolites. This experiment provides data for the study of biosynthetic pathways of P. cyrtonema secondary metabolites and the clarification of related structural gene functions.

[Simultaneous determination and analysis of amino acids and nucleosides in Dendrobium by UHPLC-QTRAP-MS/MS].

By using multivariate statistical analysis to evaluate essential quality, and provide scientific basis for their comprehensive utilization, we established an UHPLC-QTRAP-MS/MS method for the fast, precise, efficient determination of 21 kinds of amino acids and 10 kinds of nucleosides in different species of Dendrobium. The analysis was performed on a Waters XBridge Amide column(2.1 mm×100 mm,3.5 µm) with elution by mobile phase of 0.2% formic acid in water-0.2% formic acid in acetonitrile at a flow rate of 0.2 mL·min~(-1) with the column temperature at 30 ℃. The target compounds were analyzed by the positive ion multiple reaction monitoring(MRM) mode. The comprehensive evaluation of different species of Dendrobium was carried out by PCA and TOPSIS analysis. All 21 kinds of amino acids and 10 nucleosides showed good linearity among certain concentration range(r>0.999), the RSDs of the stability, precision, and repeatability tests were less than 3.0%. The recovery rate was in the range from 93.31% to 107.5%, and RSD was in the range of 1.1%-3.7%. The comprehensive evaluation index obtained with PCA showed that D. huoshanense was significantly higher than others regarding amino acids and D. officinale has higher nucleosides than other species. The biggest C_i difference of TOPSIS was 68.7%, and comprehensive evaluation showed that D. huoshanense produced the highest comprehensive quality. The method is precise, fast and efficient and can provide reliable basis for further researches and intrinsic quality control of Dendrobium.

[Cloning and quantitative expression analysis of GMPP gene from Dendrobium huoshanense].

In order to understand the function of GDP-mannose pyrophosphorylase(GMPP) function and its regulation in polysaccharide biosynthesis mechanism in Dendrobium. D. huoshanense was used to clone GMPP gene. GMPP gene expression in D. huoshanense,D. officinale and D. moniliforme was also determined by qPCR. The results showed that the length of D. huoshanense GMPP gene c DNA sequence is 1 867 bp,containing 1 245 bp open reading frame(ORF),encoding 415 amino acids. Phylogenetic tree analysis showed that D. huoshanense,D. officinale and D. moniliforme are closely related with GMPP taken into consideration. Bioinformatics analysis demonstrated that GMPP sequence similarity among the three species reached as high as 99%. qPCR results indicated that GMPP genes was highly expressed in stem of D. huoshanense compared with its leaf,flower and root. According to GMPP gene expression profile in D. huoshanense,D. officinale and D. moniliforme grown in Huoshan area,it was clear that GMPP in D. huoshanense showed the highest expression level. Furthermore,our findings of GMPP gene expression profile will facilitate future researches into its polysaccharide biosynthetic mechanism.

[Therapeutic effect and mechanism of three kinds of Dendrobium on constipation in rats with spleen Yin deficiency].

Books on Chinese herbal medicines have shown that Dendrobium has the effect of nourishing Yin and reinforcing Yin,usually used for constipation induced by spleen Yin deficiency in clinical application. D. huoshanense,as an independent species among many species of Dendrobium,has no experimental studies about its effects on spleen Yin deficiency-type constipation. The purpose of this experiment was to illustrate the therapeutic effect of D. huoshanense on the constipation of spleen Yin deficiency type in rats,investigate its preliminary mechanism,and compare it with the D. officinale and D. nobile contained in the Chinese Pharmacopoeia to clarify its characteristics. The spleen Yin deficiency model was replicated in 70 rats by the composite factor method,and then the model rats were randomly divided into 7 groups: model group,Liuwei Dihuang Pills group( LWDHP),D. huoshanense high( DHS-H),medium( DHS-M),low( DHS-L) dose groups,D. nobile group( DNS),and D. officinale group( DOS),and another 10 rats were used as normal group( Normal). After 7 continuous days of administration,the fecal water content and intestine propulsion rate of each group were detected. HE staining was used to observe the pathological damage of ileum and colon in each group. Immunohistochemistry and Western blot were used to detect aquaporin 3( AQP3) expressions,while the expression levels of the somatostatin( SS) and motilin( MTL) in the ileum of each group were detected by enzyme-linked immunosorbent assay. The results showed that as compared with the model group,the rats in each drug-administered group had increased number of fecal pellets,increased fecal water content,and the increased intestinal propulsion rate( P<0. 01),while the pathological damage of the ileum and colon was significantly reduced; the expression of AQP3 protein was significantly decreased( P<0. 01); the level of MTL was significantly increased and the level of SS was decreased( P<0. 01). All DHS groups showed a good dose-effect relationship,and the same dose treatment effect was equivalent to that of DOS,but it was superior to DNS. Therefore,DHS has a significant therapeutic effect on constipation of spleen Yin deficiency type,and its mechanism may be related to intestinal motility and water-liquid metabolism,with a good therapeutic effect.

[Cluster analysis of Dendrobium officinale from different populations through agronomy characterization].

Through resources investigation and sample collection,a total number of 392 Dendrobium officinale from 38 different populations,9 provinces were processed for measuring and observing.Fourteen agronomy characterizations like stem height,stem diameter,number of node in stem were selected for further classification.The cluster analysis was performed using Ward and Euclidean method.The results showed that the threshold of genetic distance was 2.77.Thirtyeight populations were divided into 6 groups.The factor analysis showed that,the leaf shape,stem shape,pitch length and leaf color were very important factors for classification.This study establish the foundation for analyzing the genetic relationship of D.officinale from different populations.

[Establishment of DNA identity card and analysis of genetic similarity among 58 varieties in Lonicera japonica].

A total of 58 varieties in Lonicera japonica from 20 producing areas were amplified by 22 pairs of SSR primers. Seven pairs of polymorphic primers were screened and their primers were used to establish DNA identity card and analyze genetic similarity.All the 58 varieties could be distinguished each other by the DNA identity card constituted by 7 pairs of core SSR primers.The genetic similarity coefficients of 58 varieties ranged from 0.366 7 to 0.916 7 by using PopGene32（vesion1.32）. Furthermore, all the varieties consistency were classified into 4 groups and constructed an evaluation table according to cluster analysis by an un-weighted pair-group average method with arithmetic mean. As expected, the results of cluster and evaluation table reflected 58 varieties relatives, which provide reference information for the selection of fine germplasm of L. japonica and the theoretical basis for the study of Dao-di herbs.

[Simultaneous determination of five saponins in Bupleuri Radix by HPLC-DAD dual wavelength method].

Epoxy ether type and isophthalene type saponin are the main saponins of Bupleurum chinense. However,due to the difference of their UV spectrum,there is no quantitative method for simultaneous determination of these two kinds of saponins. In this paper,a dual-wavelength high performance liquid chromatography(HPLC) was developed for simultaneous determination of five saponins in epoxidized ether(saikosaponin a,c,d) and isosorbide type(saikosaponin b1,b2). The mobile phase was eluted with acetonitrile-water(0.1% phosphoric acid) gradient at a column temperature of 30 °C and a flow rate of 1.0 mL·min⁻¹. The detection wavelengths were 208 nm for saikosaponins a,c, and d, and 254 nm for saikosaponins b1 and b2. The results showed that the separation of five kinds of saikosaponin was good, with the linear range of 9.70-1 935.00(r=0.999 4),8.20-1 380.00(r=0.999 3),6.90-1 640.00(r=0.999 0),5.25-630.00(r=0.999 4), and 5.15-618.00 mg·L⁻¹(r=0.999 5), respectively. The average recoveries were 97.70%-100.2% and the RSD was less than 3%(n=6). The method is simple,rapid and reproducible. It can be used for the determination of five kinds of saikosaponins in B. chinense.

[Next generation sequencing and transcriptome analysis of root bark from Paeonia suffruticosa cv. Feng Dan].

Moutan Cortex is an important traditional Chinese medicine, "Fengdan Pi" was known as Dao-di herbs from the root bark of Paeonia suffruticosa cv. Feng Dan for its extracted various active components. However, the genetic basis for their activity is virtually unknown. The transcriptome of the root bark from "Fengdan" was sequenced using the Illumina HiSeq 4000 sequencing platform. The clean reads were then de novo assembled into 72 997 unigenes. Among them, the number of unigenes which could been annotated by dataset Nr and GO was 41 139 and 34 592. The 20 016 unigenes could been annotated by KEGG dataset, which were involved in 5 major categories, 34 middle categories, and 352 metabolism pathways. The number of unigenes which were mapped to the phenylpropanoid biosynthesis pathway, terpenoid backbone biosynthesis pathway, terpenoid biosynthesis pathway, alkaloid biosynthesis pathway, and flavonoid biosynthesis pathway was 214, 104, 152, 55 and 36 respectively, suggesting that they are involves in these pathways of pharmaceutically important. Furthermore, there also showed remarkable differences in groups which enrichment ratio of the different expressed gene compared. In addition, a total of 9 939 SSRs were identified from the sequence of 72 997 unigenes. This study not only provides many valuable basal data which was important gene in the synthesis pathway of secondary metabolites with gene searching, but also has important significance to find molecular marker in germplasm for breeding and improvement.

[Optimization of method for determination of Salviae Miltiorrhizae Radix et Rhizoma].

OBJECTIVE: To optimize the method in the Chinese Pharmacopoeia for determining Salviae Miltiorrhizae Radix et Rhizoma. METHOD: Tanshinone II(A) and salvianolic acid B were selected as the index in optimization of the sample preparation method of Salviae Miltiorrhizae Radix et Rhizoma in Chinese Pharmacopoeia. Orthogonal test was used to optimize the extraction process of Salviae Miltiorrhizae Radix et Rhizoma, and concentration of contents were detected by high performance liquid chromatography method. A detection of using methanol-water (85: 15) at wavelength of 270 nm was employed for tanshinone II(A) and a detection of using methanol-acetonitrile-formic acid-water (30:10:1: 59) at wavelength of 286 nm was employed for salvianolic acid B. RESULT: The optimized extraction process of tanshinone II(A) and salvianolic acid B was: extracted by 90% methanol and reflux twice (0.5 h each time) at 75 degrees C, extracted by 70% methanol and reflux twice (1.5 h each time) at 75 degrees C, respectively. CONCLUSION: Optimized extraction and determination methods could be used to reflect the content of tanshinone II(A) and salvianolic acid B in Salviae Miltiorrhizae Radix et Rhizoma more accurately and efficiently.

[HPLC Characteristic Fingerprint of "Boju" Chrysanthemum morifolium].

OBJECTIVE: To provide a scientific basis for quality control of "Boju" Chrysanthemum morifolium by establishing a HPLC characteristic fingerprint. METHODS: The HPLC analysis was performed on a Spursil C18 chromatographic column (250 mm x 4. 6 mm, 5 µm), and the mobile phase was acetonitrile-0. 1% phosphoric acid in a gradient mode with the flow rate of 1. 0 mL/min. The detection wavelength was 325 nm and the temperature of column was 30 °C. RESULTS: The common pattern of HPLC characteristic chromatographic profile was established. There were 16 common peaks, five of which were identified in the pattern. The similarities of 10 batches of "Boju" Chrysanthemum monrfolium were evaluated, and all of them were greater than 0. 900. CONCLUSION: This analysis method of HPLC characteristic chromatographic fingerprint is simple and reproducible, and it can provide a scientific basis for identification and quality evaluation of "Boju" Chrysanthemum monfolium.

[Determination of Trace Elements in Different Parts of Boju and Root Soil by ICP-MS].

OBJECTIVE: To explore the contents and enrichment status of the trace elements in Boju by using ICP-MS. METHODS: Trace elements of the different parts of Boju and the soil near the roots were determined by microwave digestion-ICP-MS and the correlation of the data obtained was tested by using the statistical software. RESULTS: There were differences among the contents of the eight elements in the different parts of Boju and the soil; there were obvious differences in the enrichment factor of eight elements in the different parts of Boju, which ranged from 0.0054 to 0.9205; there was a significant positive correlation between Fe and Mg in the inflorescence and the contents of Fe was positively correlated with the soil; for Al, Ba and Mg, there were significant positive correlations between its soil contents and leaves contents. CONCLUSION: Boju absorbs the inorganic elements selectively,the yield and quality of Chinese medicinal materials can be improved by improving the cultivation.

Untargeted metabonomics and TLR4/ NF-κB signaling pathway analysis reveals potential mechanism of action of Dendrobium huoshanense polysaccharide in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is marked by hepatic steatosis accompanied by an inflammatory response. At present, there are no approved therapeutic agents for NAFLD. Dendrobium Huoshanense polysaccharide (DHP), an active ingredient extracted from the stems of Dendrobium Huoshanense, and exerts a protective effect against liver injury. However, the therapeutic effects and mechanisms of action DHP against NAFLD remain unclear. DHP was extracted, characterized, and administered to mice in which NAFLD had been induced with a high-fat and high-fructose drinking (HFHF) diet. Our results showed that DHP used in this research exhibits the characteristic polysaccharide peak with a molecular weight of 179.935 kDa and is composed primarily of Man and Glc in a molar ratio of 68.97:31.03. DHP treatment greatly ameliorated NAFLD by significantly reducing lipid accumulation and the levels of liver function markers in HFHF-induced NAFLD mice, as evidenced by decreased serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC) and total triglyceride (TG). Furthermore, DHP administration reduced hepatic steatosis, as shown by H&E and Oil red O staining. DHP also inhibited the Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway expression, thereby reducing levels of hepatic proinflammatory cytokines. Besides, untargeted metabolomics further indicated that 49 metabolites were affected by DHP. These metabolites are strongly associated the metabolism of glycine, serine, threonine, nicotinate and nicotinamide, and arachidonic acid. In conclusion, DHP has a therapeutic effect against NAFLD, whose underlying mechanism may involve the modulation of TLR4/NF-κB, reduction of inflammation, and regulation of the metabolism of glycine, serine, threonine, nicotinate and nicotinamide metabolism, and arachidonic acid metabolism.

[Analysis of trace elements in chrysanthemum from different habitats with ICP-MS].

OBJECTIVE: To establish a method for determination of trace elements in chrysanthemum and analyze trace elements in chrysanthemum from different habitats. METHODS: The preprocessing for determination of trace elements in chrysanthemum was carried out by microwave digestion,the content of trace elements in chrysanthemum was determined by ICP-MS. RESULTS: The content of trace elements of chryscmthemum varied with the habitats. CONCLUSION: The contents of trace elements as well as heavy metals of Chrysanthemum relate to its species and habitats to a certain degree.

[Dynamic accumulation of dry substance and active components in root of Peucedanum praeruptorum].

OBJECTIVE: To study the dynamic accumulation regulation of dry substance and four coumarin constituents in root of Peucedanum praeruptorum, so to provide the basis for optimal harvest time. METHOD: Roots of P. praeruptorum were collected periodically. The dry weight of the roots was measured and the content of four coumarins was determined by HPLC method. RESULT: The dry substance accumulation of the root reached the maximum amount and the coumarin contents maintain stability at the end of the vegetative growth period. The coumarins of roots decline significantly in the reproductive growth period. CONCLUSION: The most optimum collecting time of root of P. praeruptorum cultivated in Ningguo county in Anhui province should be from December to next February.

[The influence of different processing on the content of flavones of Dendranthema morifolium].

OBJECTIVE: To investigate the effect of different processing methods on the content of luteolin-7-O-beta-D-glucoside, acacetin-7-O-beta-D-glucoside, luteolin, and acacetin of Dendranthema morifolium. METHODS: The optimal conditions were achieved on phenomenex, synergi Fusion-RP (5 microm, 4.6 mm x 250 mm)analytical column with a gradient mobile phase consisting of methanol and 0.1% phosphoric acid and detection wavelength set at 350 nm. RESULTS: The significant effect of different processing methods on the content of luteolin-7-O-beta-D-glucoside, acacetin-7-O-beta-D-glucoside, luteolin and acacetin was proved. CONCLUSION: Different processing methods have significant effect on flavonoids of Dendranthema morifolium, it should be paid attention to drying temperature while sulphur fumigation.

Poria cocos polysaccharide prevents alcohol-induced hepatic injury and inflammation by repressing oxidative stress and gut leakiness.

Alcoholic liver disease (ALD) is a major worldwide chronic liver disease accompanied by hepatic inflammation, gut leakiness, and abnormal oxidative stress. Our previous study demonstrated substantial hepatoprotective activity of the active Poria cocos polysaccharide (PCP-1C). The present study explored whether PCP-1C protects against ALD among hepatic inflammation, gut leakiness, and abnormal oxidative stress. The results showed that PCP-1C significantly improved alcohol-induced liver injury by decreasing serum biochemical parameters, alleviating hepatic steatosis, and reducing lipid accumulation caused by ALD. Moreover, PCP-1C treatment reduced hepatic inflammation by inhibiting the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway and also improved hepatocyte apoptosis by inhibiting the cytochrome P450 2E1 (CYP2E1)/reactive oxygen species (ROS)/mitogen-activated protein kinases (MAPKs) signaling pathway. Regarding intestinal protection, PCP-1C could repair the intestinal barrier and reduce lipopolysaccharide (LPS) leakage. Generally, PCP-1C exerts a positive therapeutic effect on ALD, which may play a pivotal of decreasing inflammatory factor release, inhibiting oxidative stress and apoptosis, and improving intestinal barrier injury.