Analysis of Quality Markers Related to Expelling Phlegm and Arresting Coughing of Trichosanthis Fructus Based Pharmacokinetics and Multivariate Statistical Methods / 中国实验方剂学杂志

Objective:Based on pharmacokinetics， the antitussive and expectorant related quality markers （Q-marker） of Trichosanthis Fructus were screened from diosmetin-7-<italic>O</italic>-glucopyranoside， diosmetin， apigenin， vanillic acid and cinnamic acid， and the candidate Q-marker was evaluated by multivariate statistical method. Method:Six healthy rats were randomly selected and the 70% ethanol extract of Trichosanthis Fructus （dose of 20 g·kg^-1） was given by intragastric administration. Blood was collected from the orbital vein at different time points， and the plasma concentrations of 5 components （diosmetin-7-<italic>O</italic>-glucopyranoside， diosmetin， apigenin， vanillic acid and cinnamic acid） from Trichosanthis Fructus were detected simultaneously by high performance liquid chromatography-triple quadrupole tandem mass spectrometry （HPLC-QqQ-MS/MS）. The main detection conditions were as following：mobile phase of 0.2% formic acid aqueous solution （A）-acetonitrile （B） for gradient elution （0-4 min， 6%-23%B； 4-5 min， 23%-59.5%B； 5-10 min， 59.5%-60%B）， flow rate of 0.5 mL·min^-1， the detection wavelength at 254 nm， electrospray ionization （ESI）， positive ion mode detection， multiple reaction monitoring （MRM） mode scanning， scanning range of <italic>m</italic>/<italic>z</italic> 50-1 500. Diosmetin-7-<italic>O</italic>-glucopyranoside， diosmetin， apigenin and vanillic acid with clear pharmacokinetic behaviors were selected as candidate Q-marker about antitussive and expectorant of Trichosanthis Fructus. The contents of these components in 9 batches of medicinal materials were determined and the main detection conditions were the same as the pharmacokinetic study. SPSS 21.0 was used for cluster analysis and principal component analysis （PAC） based on the results of determination. Result:The pharmacokinetic results showed that the area under concentration-time curve （AUC_0-<italic>_t</italic>） of 4 components （diosmetin-7-<italic>O</italic>-glucopyranoside， diosmetin， apigenin and vanillic acid） were （111.28±9.94）， （27.08±2.76）， （1 376.12±101.86）， （631.32±64.72） μg·h·L^-1， respectively. The 9 batches of Trichosanthis Fructus samples were clustered into 3 groups by systematic cluster analysis. The clustering results were related to the variety of Trichosanthis Fructus and also affected by the origin. The PCA results showed that the comprehensive scores of Gaotang Trichosanthis Fructus， Shanxi Trichosanthis Fructus， Hebei Ben Trichosanthis Fructus were 1.919， 1.356 and 0.299， respectively， ranking in the top 3 among all samples. The comprehensive scores of Nongkeyuan No. 1， Hebei Trichosanthis Fructus and Nongkeyuan No. 2 were -0.804， -1.085， -1.120， respectively， which were in the last 3 positions among all samples. Conclusion:The pharmacokinetic characteristics and quality evaluation of diosmetin-7-<italic>O</italic>-glucopyranoside， diosmetin， apigenin and vanillic acid meet the requirements about antitussive and expectorant related Q-marker of Trichosanthis Fructus.

Effect of Chaenomelis Fructus Alcohol Extract on Arthritis Inflammation and Apoptosis of Synovial Cells in Rheumatoid Arthritis Model / 中国实验方剂学杂志

Objective:To study the protective effect and mechanism of Chaenomelis Fructus alcohol extract (CFE) on the synovium of rheumatoid arthritis (RA). Method:Sixty male SD rats were randomly divided into normal group and model group. RA model was made by injection of complete Freund's adjuvant， and then was randomly divided into model group， CFE low， medium and high dose group and Tripterygium glycoside group according to the inflammatory score. The CFE groups (0.15，0.30，0.60 g·kg-1·d-1) had intragastric administration once a day for 30 d after the model establishment. The blank control group and the model group were given the same volume saline water by gavage. After all the drugs were given， the blood， joint tissues and synovium tissue of rats were collected. The levels of interleukin-1β (IL-1β)， interleukin-6 (IL-6)， interleukin-10 (IL-10) and tumor necrosis factor (TNF)-α in serum were detected by enzyme linked immunosorbent assay (ELISA)， the pathological changes of synovium were observed by hematoxylin eosin (HE) staining， and the expressions of B-cell lymphoma-2 (Bcl-2)，Bcl-2 associated X protein (Bax) and death factor (Fas) in joints were detected by Western blot. Result:Compared with normal group， the swelling degree and inflammation index of rats' feet in model group increased significantly， the levels of inflammatory factors IL-1β， IL-6， TNF-α in serum increased (P<0.01)， anti-inflammatory factor IL-10 decreased (P<0.01)， the protein expression of Bax， Fas and Bcl-2 increased， and the statistical results of Bcl-2 showed significant difference (P<0.05). Compared with model group， the swelling degree and inflammatory index of the plantar of RA rats were improved in the middle and high dose groups of CFE (P<0.01)， the pathological changes such as synovial tissue hyperplasia and inflammatory cell infiltration were reduced in each dose group， and the levels of IL-1β， IL-6 and TNF-α in serum were reduced (P<0.05)， anti inflammatory factor IL-10 increased (P<0.05)， the disorder of inflammatory cytokine in the model was corrected， Bax， Fas expression increased， Bcl-2 protein expression decreased (P<0.01). Conclusion:CFE can reduce the degree of inflammation in RA joint and has obvious anti RA effects， which may be related to the apoptosis of synoviocytes induced by CFE.

Comparative Study on Content of Medicinal Materials from Three Varieties of Sambucus williamsii / 中国实验方剂学杂志

Objective:To determine the content of active ingredients in root bark,stems,leaves and fruits of three varieties of Sambucus williamsii (Saline-toleratedvariety,Natural Red No. 1 variety,Qingzhou No.15 variety),and identify the chemical patterns of the chemical components. Method:The content of polysaccharides,flavonoids,phenolic acids and triterpene were determined by UV spectrophotometry (UV) colorimetry. The content of anthocyanin was determined by pH differential method. Ursolic acid,oleanolic acid,quercetin,protocatechuic acid,morroniside content were determined by high performance liquid chromatography (HPLC). The root bark,stems,leaves and fruits of three varieties were analyzed by chemical pattern recognition. Result:The quality of different samples was further evaluated by F3 and F6 comprehensive scores in principal component analysis. It was found that the root bark and leaves were higher in the comprehensive ranking,and the results were basically consistent with the quantitative results. The contents of polysaccharide and morroniside in Natural Red No. 1 variety root bark were the highest,which were 161.09 mg·g-1 and 8.33 mg·g-1,respectively. Flavonoids,rutin and quercetin were the highest in the Natural Red No. 1 variety leaf,which were 71.93,4.09 and 3.57 mg·g-1,respectively. Triterpene was the highest in Natural Red No. 1 variety fruit, which was 47.84 mg·g-1. Phenolic acid and anthocyanin were the highest in Saline-tolerated variety fruits,which were 11.25 mg·g-1 and 94.32 mg·g-1,respectively. The Saline-tolerated variety stem had the highest oleanolic acid content, which was 1.41 mg·g-1. Saline-tolerated variety leaf had the highest contents of ursolic acid and gallic acid,which were 2.12 and 0.34 mg·g-1, respectively. The highest content of protocatechuic acid in Qingzhou No. 15 variety root bark, which was 0.12 mg·g-1. Conclusion:The content of Natural Red No. 1 variety is higher than that of the other two varieties,with a good quality.

Cloning and expression analysis of cinnamate 4-hydroxylase (C4H) reductase gene from Aquilaria sinensis / 中国中药杂志

The study aimed to clone the open reading frame of cinnamate 4-hydroxylase (C4H) from Aquilaria sinensis and analyze the bioinformatics and expression of the gene. One unique sequence containing C4H domain was discovered in our previous reported wound transcriptome dataset of A. sinensis. The open reading frame of C4H was cloned by RT-PCR strategy with the template of mixed RNA extracted from A. sinensis stem which treated by different wound time. The bioinformatic analysis of this gene and its corresponding protein was performed. C4H expression profiles in responds to MeJA (methyl jasmonate) application were analyzed by real-time PCR. The length of C4H open reading frame (ORF) was 1 515 bp, encoding 514 amino acids. The GenBank accession number is KF134783. Inducible-experiments showed that the genes were induced by mechanical wound as well as MeJA induction, and reached the highest expression level at 8 h and 20 h, respectively. The full-length cDNA of C4H and its expression patterns will provide a foundation for further research on its function in the molecular mechanisms of aromatic compounds and flavonoids biosynthesis.

Cloning, prokaryotic expression, and functional identification of a sesquiterpene synthase gene (AsSS4) from Aquilaria sinensis / 药学学报

A sesquiterpene synthase (AsSS4) full-length open reading frame (ORF) cDNA was cloned from wounded stems of Aquilaria sinensis by RT-PCR method. The result showed that the ORF of AsSS4 was 1,698 bp encoding 565 amino acids. Prokaryotic expression vector pET28a-AsSS4 was constructed and transformed into E. coli BL21 (DE3) pLysS. Recombinant AsSS4 protein was obtained after induction by IPTG and SDS-PAGE analysis with a MW of 64 kD. Enzymatic reactions using farnesyl pyrophosphate showed that recombinant AsSS4 protein purified by Ni-agarose gel yielded five sesquiterpene compounds, cyclohexane, 1-ethenyl-1-methyl-2, 4-bis(1-methylethenyl)-, β-elemene, α-guaiene, α-caryophyllene and δ-guaiene. This paper reported the first cloning and functional characterization of AsSS4 gene from A. sinensis, which will establish a foundation for future studies on the molecular mechanisms of wound-induce agarwood formation in A. sinensis

Salvianic acid A inhibits lipopolysaccharide-induced apoptosis through regulating glutathione peroxidase activity and malondialdehyde level in vascular endothelial cells / 中国天然药物

AIM@#To find out the role of salvianic acid A (SAA) in the protection of vascular endothelial cells (VEC) and its possible mechanism in vitro.@*METHODS@#The ingredient at various concentrations was added to human umbilical vein endothelial cells (HUVEC) treated with 0.5 μmol·L(-1) lipopolysaccharide (LPS) for 24 h. Apoptotic morphological changes of cells were observed under inverted phase contrast microscope; the cell viability was quantified using MTT assay. Nuclear fragmentation of cells was observed under laser scanning confocal microcope after being stained with acridinorange. Cell cycle distribution was detected by flow-cytometry after being stained with propidium iodide (PI). The activities of glutathione peroxidase (GPH-PX) as well as maleic dialdehyde (MDA) level in cells were measured by spectrophotometric methods as described in the assay kits.@*RESULTS@#Apoptotic morphological changes and the decrease of cell viability of these cells were obviously inhibited by SAA in a dose-dependent manner. Furthermore, the abnormal cell cycle distribution, the decrease of GSH-Px activity and the increase of MDA level induced by LPS were markedly reversed.@*CONCLUSION@#SAA exerts protective effect on VEC induced by LPS via an antioxidative mechanism.

Genetic diversity of germplasm resources of Lonicera japonica by AFLP analysis / 中国中药杂志

<p><b>OBJECTIVE</b>This study aimed to analyze the genetic diversity and genetic relationship of germplasm resources of Lonicera japonica in main producing areas of China and provide reference for developing new varieties of L. japonica.</p><p><b>METHOD</b>Using 6 primer combinations, 13 germplasm of L. japonica were analyzed by AFLP marker. The genetic distance was worked out by using DPS V3.01 software, and the cluster was conducted based on UPGMA.</p><p><b>RESULT</b>A total of 435 bands were obtained including 191 polymorphic ones. The average polymorphic frequency was 43.9%. Cluster analysis showed that the relationship of cultivated variety from the same genuine area was near, and the classification result based on AFLP marker of germplasm of L. japonica from Shandong province was basically consistent with those on their morphological character.</p><p><b>CONCLUSION</b>AFLP marker can indicate the abundant genetic diversity of L. japonica and provide theoretical evidence for reasonable utilization and breeding new cultivar of L. japonica in molecular level.</p>

Studies on chemical constituents in fruits of Eucalyptus globulus / 中国中药杂志

<p><b>OBJECTIVE</b>To study the chemical constituents in the fruits of Eucalyptus globulus Labill.</p><p><b>METHOD</b>The chemical constituents were isolated by various column chromatographic methods and structurally elucidated by IR, NMR and MS evidences.</p><p><b>RESULT</b>Fifteen compounds were obtained and identified as beta-sitosterol (1), betulinic acid (2), stigmasterol (3), euscaphic acid (4), 2a-Hydroxybetulinic acid (5), macrocarpal B (6), macrocarpal A (7), oleanolic acid (8), 3,4,3'-O-trimethylellagic acid (9), 3-O-methylellagic acid 4'-O-(2"-O-acetyl )-alpha-L-rhamnopyranoside (10), camaldulenside (cypellocarpin C, 11), 3-O-methylellagic acid 4'-O-alpha-L-rhamnopyranoside (12), 3-O-methylellagic acid (13), ellagic acid (14), and gallic acid (15).</p><p><b>CONCLUSION</b>Compounds 4 and 5 from genera Eucalyptus, 1, 3 and 11 from plant E. globulus, and 6, 7, 9 and 15 from the fruits of E. globulus were isolated for the first time.</p>

Studies on micromorphological characters of fruit coats in cultivated Fructus Trichosanthis in Shandong Province / 中国中药杂志

<p><b>OBJECTIVE</b>To explore the affinity relation among land race of Trichosanthes kirilowii and to provide evidence tor the classification and authentication.</p><p><b>METHOD</b>Using scanning electron microscope, characteristics of the pericarp surface were studied comparatively.</p><p><b>RESULT</b>The pericarp surface of the cultivated Fructus Trichosanthis in Shandong showed the characteristics of generality and diversity.</p><p><b>CONCLUSION</b>The results of the study could be used for the identification of the cultivated Fructus Trichosanthis in Shandong.</p>

The relationship of angiotensin I-converting enzyme gene polymorphism with diabetic retinopathy and diabetes myocardial infarction / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the relationship of angiotensin I-converting enzyme (ACE) gene polymorphism to diabetic retinopathy and diabetes myocardial infarction.</p><p><b>METHODS</b>ACE insertion/deletion(I/D) polymorphism was determined by PCR.</p><p><b>RESULTS</b>No evidence showed that ACE gene was associated with diabetic retinopathy. By comparison of the type 2 diabetes patients with myocardial infarction versus those without-myocardial infarction, it was found that the frequencies of homozygote DD (41.2% versus 33.2%) and of allele D (64.7% versus 55.0%) increased remarkably; the difference was statistically significant (P<0.05).</p><p><b>CONCLUSION</b>Allele D(RR=1.50) and genotype DD(RR=1.33) seemed to be a genetic risk factor for type 2 diabetes myocardial infarction.</p>