Determination of six short-chain fatty acids in rat feces using headspace solid-phase dynamic extraction coupled with GC-MS.

Short-chain fatty acids (SCFAs) are organic acids with carbon atoms less than six, released through fermentation products by intestinal microbiome, having multiple physiological activities. Considering weak acidity and high volatility, derivatization or liquid-liquid extraction is essential, which is time consuming. Headspace-solid-phase dynamic extraction (HS-SPDE) coupled with gas chromatography-mass spectrometry is automated and effortless to determine SCFAs in rat feces. The extraction procedure is performed by aspirating and discharging the headspace cyclically through a steel needle, coated with an inner polyethylene glycol sorbent. The key parameters of SPDE were optimized including coating type, incubation time and temperature, and number of extraction strokes. Besides, salting-out was conducted. Then, a method by HS-SPDE-GC-MS was established and validated. It only took 3-min incubation time, 4.5 min extraction time, and 13 min chromatographic separation in a run. The recovery, linearity, limit of quantification, and stability were evaluated. Then, the proposed method was applied to analyze rat feces including 18 rats with liver injury and 23 normal controls. Mann-Whitney U test indicated that the concentrations of six SCFAs in normal rat feces were higher than those with liver injury. This method provides a choice for fast, solvent-free, automated, and high-throughput analysis of SCFAs.

Untargeted Metabolomics Based on UPLC-Q-Exactive-Orbitrap-MS/MS Revealed the Differences and Correlations between Different Parts of the Root of Paeonia lactiflora Pall.

BACKGROUND: Paeonia lactiflora Pall. (PLP) is a plant with excellent ornamental and therapeutic value that can be utilized in traditional Chinese medicine as Paeoniae Radix Alba (PRA) and Paeoniae Radix Rubra (PRR). PRA must undergo the "peeling" process, which involves removing the cork and a portion of the phloem. PLP's biological function is strongly linked to its secondary metabolites, and the distribution of metabolites in different regions of the PLP rhizome causes changes in efficacy when PLP is processed into various therapeutic compounds. METHODS: The metabolites of the cork (cor), phloem (phl), and xylem (xyl) were examined in the roots of PLP using a metabolomics approach based on UPLC-Q-Exactive-Orbitrap-MS/MS (UPLC-MS/MS), and the differential metabolites were evaluated using multivariate analysis. RESULTS: Significant changes were observed among the cor, phl, and xyl samples. In both positive and negative ion modes, a total of 15,429 peaks were detected and 7366 metabolites were identified. A total of 525 cor-phl differential metabolites, 452 cor-xyl differential metabolites, and 328 phl-xyl differential metabolites were evaluated. Flavonoids, monoterpene glycosides, fatty acids, sugar derivatives, and carbohydrates were among the top 50 dissimilar chemicals. The key divergent metabolic pathways include linoleic acid metabolism, galactose metabolism, ABC transporters, arginine biosynthesis, and flavonoid biosynthesis. CONCLUSION: The cor, phl, and xyl of PLP roots exhibit significantly different metabolite types and metabolic pathways; therefore, "peeling" may impact the pharmaceutical effect of PLP. This study represents the first metabolomics analysis of the PLP rhizome, laying the groundwork for the isolation and identification of PLP pharmacological activity, as well as the quality evaluation and efficacy exploration of PLP.

Efficient Separation of Proteins and Polysaccharides from Dendrobium huoshanense Using Aqueous Two-Phase System with Ionic Liquids.

By applying the hydrophilic ionic liquid, 1-butyl-3-methylimidazolium chloride ([C4mim]Cl), and inorganic salts (K3PO4), an ionic liquid aqueous two-phase system (ILATPS) was established for the separation of Dendrobium huoshanense polysaccharides (DhPs) and proteins. The effects of inorganic salt concentration, IL quantity, crude DhPs concentration, pH value and temperature were studied to achieve the optimal condition. With the best combination of ILATPS (1.75 g K3PO4, 1.25 g [C4mim]Cl, 10 mg crude DhPs and 5.0 mL ddH2O at pH 7.0 under 25 °C), the extraction efficiency rates for DhPs and proteins were 93.4% and 90.2%, respectively. The processed DhPs retrieved from the lower salt-rich phase comprised mannose, glucose, galactose, arabinose, and galacturonic acid with a molar ratio of 185:71:1.5:1:1 and the molecular weight was 2.14 × 105 Da. This approach is fast, simple and environmentally friendly. It provides a new insight into purifying functional polysaccharides of plant origin.

Comparison of the Antioxidant Activities and Polysaccharide Characterization of Fresh and Dry Dendrobium officinale Kimura et Migo.

It is generally believed that fresh Dendrobium officinale (FDO) has more significant pharmacological activity than dried Dendrobium officinale (DDO); however, the difference has not been clearly shown. Our study compared their antioxidant properties both in vitro and in vivo, and the molecular weight arrangement and monosaccharide composition of the fresh Dendrobium officinale polysaccharides (FDOPs) and the dried Dendrobium officinale polysaccharides (DDOPs) were analyzed by HPLC-GPC and GC-MS. The results showed that the FDO and its polysaccharides had more significant effects on scavenging DPPH, ABTS, and hydroxyl radicals than the DDO. In addition, both the FDO and DDO significantly reduced lipid peroxidation levels and increased the SOD, T-AOC, CAT, and GSH levels in mice with acute liver damage caused by CCl4, while the FDO and its polysaccharides were more effective. Histopathological analysis further verified the protective effect of the Dendrobium polysaccharides on CCl4-induced liver injury. The determination of the polysaccharides revealed that the polysaccharide and mannose contents of the FDO were significantly higher than their dried counterparts, and the homogeneous arrangement of the polysaccharides in the FDO was degraded into three polysaccharide fragments of different molecular weights in the DDO. Overall, our data identified differences in the antioxidant activities of the FDO and DDO, as well as the reasons for these differences.

[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.

Putative genes in alkaloid biosynthesis identified in Dendrobium officinale by correlating the contents of major bioactive metabolites with genes expression between Protocorm-like bodies and leaves.

BACKGROUND: Dendrobium officinale, an endangered Chinese herb, possesses extensive therapeutic effects and contains bioactive ingredients such as major polysaccharides, alkaloids, and minimal flavonoids. We first obtained the protocorm-like bodies (PLBs) of this plant through tissue culture in order to determine the distribution of the main secondary metabolites in each organelle and the PLBs. We then analyzed the correlation between gene expression level from comparative transcriptome sequencing and metabolite content in different organs to identify putative genes encoding enzymes involved in the biosynthesis of polysaccharides, alkaloids, and flavonoids. RESULTS: We used seeds as explants for protocorm induction and PLB propagation of D. officinale. The optimal medium formula for PLB propagation was 1/2 MS + α-NAA 0.5 mg·L- 1 + 6-BA 1.0 mg·L- 1 + 2, 4-D 1.5-2.0 mg·L- 1 + potato juice 100 g·L- 1. Stems, PLBs and leaves of D. officinale had the highest content of polysaccharides, alkaloids and flavonoids, respectively. Naringenin was only produced in stem; however, PLBs with high alkaloid content can replace other organs producing alkaloids. The hot water extraction method outperformed the ultrasound-assisted extraction method for extracting polysaccharides from D. officinale. A comparative transcriptome analysis of PLBs and leaves of D. officinale revealed differential expression of genes encoding enzymes involved in polysaccharide, alkaloid and flavonoid biosynthetic pathways. Putative genes encoding enzymes involved in these biosynthetic pathways were identified. Notably, we identified genes encoding the alkaloid biosynthesis enzymes strictosidine ß-D-Glucosidase, geissoschizine synthase and vinorine synthase in D. officinale. CONCLUSIONS: The identification of candidate genes encoding enzymes involved in metabolite biosynthesis will help to explore and protect this endangered species and facilitate further analysis of the molecular mechanism of secondary metabolite biosynthesis in D. officinale.

Determination and analysis of monosaccharides in Polygonatum cyrtonema Hua polysaccharides from different areas by ultra-high-performance liquid chromatography quadrupole trap tandem mass spectrometry.

We wanted to explore a new method for the determination of monosaccharides in Polygonatum cyrtonema Hua polysaccharide using the ultra-high-performance liquid chromatography quadrupole trap tandem mass spectrometry. In this study, hydrochloric acid was used instead of trifluoroacetic acid to hydrolyze polysaccharides, and hydrolysis time was greatly reduced from 5-9 h to 1 h. The 1-phenyl-3-methyl-5-pyrazolone was used for pre-column derivatization of monosaccharides. The parameters of linearity (R2  > 0.999), stability (1.63-2.52%), intra-day and inter-day precision (0.69-0.95%, 1.81-2.77%), repeatability (1.89-2.65%), and recovery (97.63-102.24%) of the method were verified. Satisfactory validation results showed this method could be used to determine the target components. The results indicated the polysaccharide contained glucose, mannose, rhamnose, galactose, ribose, and arabinose. Technique for order preference by similarity to an ideal solution and principal component analysis were used to build an evaluation model based on the monosaccharide composition. The evaluation results showed that the samples from the Qingyang County of Anhui Province were the best when the monosaccharides were used as the evaluation index. Therefore, a new method was established to detect the monosaccharide content of polysaccharides from Polygonatum cyrtonema Hua and comprehensively evaluate the quality of Chinese medicines with polysaccharides as the main active ingredient.

Determination of monosaccharides in Lycium barbarum fruit polysaccharide by an efficient UHPLC-QTRAP-MS/MS method.

INTRODUCTION: Pre-column derivatisation using 1-phenyl-3-methyl-5-pyrazolone (PMP) is a common method for monosaccharide determination. Herein, a specific ultra-high-performance liquid chromatography quadrupole trap tandem mass spectrometry (UHPLC-QTRAP-MS/MS) method was developed and used for the determination of monosaccharide composition of Lycium barbarum polysaccharide (LBP). OBJECTIVE: Exploration of a new efficient method for monosaccharide determination. METHODS: In our study, hydrochloric acid was used to hydrolyse the polysaccharide obtained from L. barbarum fruits. Principal component analysis (PCA) and technique for order of preference by similarity to ideal solution (TOPSIS) analysis were used for comprehensive evaluation of the samples. The results showed that LBP was composed of seven monosaccharides: galactose, arabinose, mannose, rhamnose, xylose, ribose, and glucose. The linear relationship of the seven monosaccharides was optimum within a certain concentration range. Quantitative recoveries of the seven monosaccharides from the samples ranged from 94.76% to 102.11%. RESULTS: A rapid quantitative detection method was established, in which the hydrolysis time was reduced from 12 h to 2 h. By using LBP as one of the indexes to evaluate the quality of L. barbarum, L. barbarum from Zhongning County, Ningxia Province, was identified as having the best quality among the varieties tested. CONCLUSION: The UHPLC-QTRAP-MS/MS pre-column derivatisation method used in this study was simple, accurate, and sensitive, with good repeatability, and can be used for quality evaluation and origin distinction of L. barbarum and other medicinal plants.

[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.

Comparison of the chromatographic fingerprint, multicomponent quantitation and antioxidant activity of Salvia miltiorrhiza Bge. between sweating and nonsweating.

Salvia miltiorrhiza Bge. is a traditional Chinese medicine applied in the treatment of various diseases in clinical practice. In the course of its processing, S. miltiorrhiza Bge. is usually processed by sweating. This study employed 10-component contents determination coupled with high-performance liquid chromatography (HPLC) fingerprint and antioxidant activity to investigate the effect of sweating on S. miltiorrhiza Bge. so as to evaluate the quality of S. miltiorrhiza Bge. The HPLC method was performed using C18 and 0.05% phosphoric acid aqueous solution-acetonitrile with a gradient elution system. It was validated for linearity, precision, repeatability, stability and recovery. Similarity analysis, principal components analysis and antioxidant activity assays were used to compare sweated S. miltiorrhiza Bge. (SSM) and nonsweated S. miltiorrhiza Bge. (NSSM). SSM and NSSM showed good similarities in HPLC fingerprint (>0.9), but principal components analysis could classify the HPLC fingerprint and 10-component quantitation analysis. Meanwhile, the antioxidant activity of SSM was significantly higher than that of NSSM (p < 0.01). The results of this study indicated that sweating could alter the content of chemical constituents in S. miltiorrhiza Bge., and could also improve its antioxidant activity. In addition, the method not only affords a viable strategy for comparing SSM and NSSM and assessing the quality of S. miltiorrhiza Bge., but also provides a reference for other herbal medicine that suffers from sweating.

[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.