Establishment of specific chromatogram，chemical pattern recognition analysis and identification with different origins and counterfeit products of Uncariae Ramulus Cum Uncis / 中国药房

OBJECTIVE To establish the ultra-high liquid chromatography （UPLC） characteristic spectrum of Uncariae Ramulus Cum Uncis from different producing areas， to conduct chemical pattern recognition analysis， and to identify the medicinal materials of their different origins and counterfeit products. METHODS UPLC method was adopted to establish the characteristic spectra of 43 batches of Uncariae Ramulus Cum Uncis from different origins； cluster analysis combined with principal component analysis were used to analyze their quality； Uncariae Ramulus Cum Uncis from different origins and counterfeit products were identified. RESULTS UPLC specific spectrum of Uncariae Ramulus Cum Uncis was established， and 13 common peaks were calibrated； peak 2 was identified as catechin， peak 3 as chlorogenic acid， peak 4 as cryptochlorogenic acid， peak 7 as isochlorogenic acid B， peak 8 as isodehydroguotenine， peak 9 as isooguotenine， peak 10 as dehydroguotenine， peak 11 as isochlorogenic acid C， peak 12 as goutenine， and peak 13 as camptothecin. Through cluster analysis， the medicinal materials of 43 batches of Uncariae Ramulus Cum Uncis could be divided into 5 categories according to their different origins. Further principal component analysis revealed that the principal component comprehensive scores of Uncariae Ramulus Cum Uncis produced in Jiangxi and Hunan were relatively high， ranging from 0.264 to 2.904. The specific chromatogram could effectively distinguish among the different origins and their counterfeit products of Uncariae Ramulus Cum Uncis. CONCLUSIONS The established UPLC specific chromatogram can be used for quality control of Uncariae Ramulus Cum Uncis， and the study found that the quality of Uncariae Ramulus Cum Uncis from Jiangxi and Hunan provinces is relatively good.

Identification of Gastrodia elata and its hybrid by polymerase chain reaction / 中国中药杂志

Gastrodia elata is a kind of traditional Chinese medicinal materials and has good medicinal value. G. elata is divided into five varieties, which includes G. elata f. elata(proto variant), G. elata f. glauca, G. elata f. viridis, G. elata f. flavid and G. elata f. alba. Among them, G. elata f. elata and G. elata f. glauca have excellent characteristics and higher contents of gastrodin and polysaccharides. The hybrid of G. elata f. elata and G. elata f. glauca is present in markets, but the characteristics between hybrid and parent are not obvious and distinguished quickly and accurately. The aim of this study is to establish a PCR specific PCR identification method, which can identify G. elata f. elata, G. elata f. glauca and their hybrid. Based on the re-sequencing results of G. elata, we screened for the single nucleotide polymorphism(SNP) variation sites, and designed two pairs of specific primers(W291-F/W291-R and H255-F/H255-R). We further collected G. elata f. elata, G. elata f. glauca and their hybrid samples from different regions, established and optimized PCR method, and investigated and verified their tolerance and applicability. The results showed that when the annealing temperature was 48 ℃ and the number of cycles was 33, 255 bp specific band were obtained from G. elata f. glauca and hybrid by using specific primers W291-F/W291-R. When the annealing temperature was 51 ℃ and the number of cycles was 33, 291 bp specific band were obtained from G. elata f. elata and hybrid by using specific primers H255-F/H255-R. Our method could be used as a promising method to identify G. elata f. elata, G. elata f. glauca and their hybrid.

Quantitative taxonomic study on agronomic traits of cultivated Gastrodia elata / 中国中药杂志

To better understand the formation mechanism of Gastrodia elata traits, the agronomic traits of aboveground tissues and tubers were measured and analyzed in this study. It has shown that the color and thickness of the stems of the 39 samples of the G. elata collected are affected by the germplasm and variation. Clustering analysis of 39 agronomic traits of G. elata was conducted with Ward's method and Euclidean distance. The threshold of 11.0 was divided into three groups, namely hybrid G. elata, G. elata f. elata and G. elata f. glauca. Simultaneously, the correlation analysis, coefficient of variation analysis, factor analysis and cluster analysis of 13 agronomic traits of 105 G. elata tuber samples were carried out. The results showed that the weight of G. elata was significantly positively correlated with tuber length and width. The agronomic traits of tuber were highly variable, and the depth of variability of the scar was the largest and 13 agronomic traits could be divided into 6 types of factors and the contribution up to 89.348%, furthermore, tuber length factor, width and weight factor contributed more than 20%, indicating that it is of great significance for distinguishing G. elata germplasm. Cluster analysis was performed by Ward's method and Euclidean distance, with 8.0 as the threshold can be divided into three categories in the light of the origin of the source, 33 samples from Shanxi and Hubei are clustered into one category, and 19 samples from Yunnan and Guizhou are clustered into one group, and the remaining samples are grouped into one category. This study will provide a basis for the identification and purification of G. elata germplasm and germplasm resources.

Survey on Woods Species and Its Relation with Yield and Quality of Gastrodia elata / 中国实验方剂学杂志

Objective:As a source of energy for Armillaria mellea and Gastrodia elata，the woods species as fungus material of G.elata are diverse and play an important role in the development of G.elata industry. In order to explore the impact of different woods species on the quality of G. elata，the plant origins and lignocellulose content of the woods，the yield and quality of G. elata per unit area were systemically analyzed through literature research and investigation on production bases. Method:G. elata and its cultivated woods were collected from four main producing areas （Guizhou, Yunnan, Hubei, and Shaanxi），and the the plant origins of the woods were identified by DNA fragments. The content of lignocellulose in the woods was determined by high-performance liquid chromatography and loss-on-ignition method. The content of polysaccharides of G. elata from these 4 areas was determined by Phenol-sulfuric acid method. The yields and polysaccharide content of G. elata cultivated with different woods species were compared and their correlation with the woods was analyzed. Result:The woods as fungus material of G. elata were diverse in species, and betulaceae was the most widely used species in cultivation of G. elata. There were differences in the composition ratio of lignocellulose in the woods. Nyssaceae had the highest cellulose content，Moraceae had the highest hemicellulose content and Rosaceae had the highest lignin content. Different woods species had certain effects on the yield and polysaccharide content of G. elata. The maximum yield of G. elata was 1 285.51 g and the lowest yield was 379.30 g. The average mass fraction of polysaccharide content was 241.1 mg·g-1，with a range of 87.95-411.2 mg·g-1. The yield and polysaccharide content of G. elata were highly positively correlated with the cellulose content of the woods, and highly negatively correlated with the lignin content. Conclusion:Different woods have a significant impact on the yield and quality of G. elata. Choosing the appropriate woods species will be beneficial to the absorption of nutrients for A. mellea and the yield increase of G. elata. This study can provide a scientific basis for the selection of woods species during the cultivation of G. elata.

Identification of Cervi Cornu Pantotrichum and Cervi Cornu by Allele-specific PCR / 中国实验方剂学杂志

Objective: To obtain a rapid,efficiency and convenient polymerase Chain reaction(PCR) identification method for medicinal Cervi Cornu Pantotrichum,Cervi Cornu and its common adulterates. Method: Based on three single nucleotide polymorphisms (SNP) of Cytb gene DNA sequences among Cervus nippon,C. elaphus and its adulterants,a pair of species-specific primers (LR-238.F and LR-238.R) was designed,the reaction conditions were optimized,and the PCR method for identification was explored and verified in terms of tolerance and feasibility. Result: Through the established allele-specific PCR method,under the annealing temperature of 56℃ and cycle number of 35,250 bp of fragments were amplified from DNA templates of Cervi Cornu Pantotrichum,Cervi Cornu and its subspecies in origin animal samples as well as herbal medicines. All of the adulterants species of Przewalskium albirostris,Cervus eldi,Odocoileus hemionus,Dama dama,Alces alces,Elaphurus davidianus,Capreolus pygargus,Rusa unicolor and Rangifer tarandus were negative by the PCR assay. Conclusion: The identification primer is highly specific,and the allele-specific PCR identification method established in this paper can accurately identify the medicinal Cervi Cornu Pantotrichum and Cervi Cornu.

Optimization of an Activity Assay of Coprinus Cinereus Peroxidase

ABSTRACT To seek a simple, rapid and sensitive Coprinus cinereus Peroxidase (CIP) activity assay, a convenient one-factor-at-a-time (OFAT) method and a response surface methodology (RSM) were used. The recombinant CIP expressed in Pichia pastoris was purified with the Ni-NTA spin column. Based on the results of catalytic efficiency (kcat/Km) analysis, 2,2'-azinobis (ethylbenzthiazoline -6-sulfonate) (ABTS) was selected as the optimal enzyme substrate. Results of the OFAT method showed that enzymatic reaction performed in 0.1 mol/L sodium acetate (pH 5.0) buffer in a 200-µl reaction mixture containing 0.5 mmol/L ABTS, 10 mmol/L hydrogen peroxide (H2O2), 49.7 ng CIP at 25°C gave an average CIP activity of 88 U/mL. The ABTS and H2O2 concentrations were then further optimized to improve the sensitivity of the assay. To do that, RSM was conducted through central composite design, and a reduced quadratic model with good fit regression equation was generated. ANOVA analysis of this model indicated that the concentrations of ABTS and H2O2 and their interaction had significant impact on the assay sensitivity. The optimal reaction mixture was determined to include an initial ABTS concentration of 0.82 mmol/L 49.7 ng CIP and 16.36 mmol/L H2O2, and the activity under this condition was determined to be 138.89 U/mL.

Effect of Chuanhuang No. 1 recipe on renal function and micro-inflammation in phase 3 chronic kidney disease patients / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the effect of Chuanhuang No.1 Recipe (CHR) on renal function and micro-inflammation in phase 3 chronic kidney disease (CKD) patients.</p><p><b>METHODS</b>Totally 60 phase 3 CKD patients were randomly assigned to the treatment group (treated by CHR) and the control group (treated by Losartan Potassium), 30 in each group. All patients received basic treatment. Patients in the treatment group took CHR decoction, 400 mL each time, one dose per day, while those in the control group took Losartan Potassium, 50-100 mg per day. All medication lasted for 24 weeks. Changes of serum creatinine (SCr), blood urea nitrogen (BUN), estimated glomerular filtration rate (eGFR), serum uric acid (UA), 24 h urinary protein excretion (24 h U-pro), urinary microalbumin (U-Alb), high-sensitivity C-reactive protein (hs-CRP), serum tumor necrosis factor (TNF)-alpha, and serum IL-6 were detected and compared before and after treatment. Efficacy was also compared.</p><p><b>RESULTS</b>Compared with before treatment, SCr and BUN significantly decreased in the treatment group (P<0.05, P<0.01); eGFR in- creased (P<0.05). Only UA obviously decreased in the control group (P<0.05), but with no obvious change in SCr, BUN, or eGFR. Compared with before treatment, 24 h U-pro decreased after treatment in the treatment group (P<0.05), but with less decreased level when compared with the control group. U- Alb was also significantly decreased in the control group (P<0.01). There was statistical difference in 24 h U-pro and U-Alb between the two groups after treatment (P<0.05). Compared with before treatment, hs-CRP obviously decreased after treatment in the two groups, but serum levels of TNF-alpha and IL-6 obviously decreased only in the treatment group (P<0.05). The total effective rate was obviously higher in the treatment group than in the control group (70.00% vs. 43.33%, P<0.01).</p><p><b>CONCLUSION</b>CHR could efficiently improve the renal function of phase 3 CKD patients and alleviate the micro-inflammation.</p>