Effect of drying processing methods on different specifications of Sophorae Flos based on comprehensive statistical analysis / 中国中药杂志

To investigate the effects of six common drying methods on the quality of different specifications of Sophorae Flos, in order to select their suitable drying methods. According to appearance and morphology, Sophorae Flos was divided into the following three specifications: flower bud type(HL), half-open type(BK) and blooming type(SK). All specifications of samples were treated with shade-drying method(25 ℃, natural temperature), sun-drying method, hot-air-drying method(60, 105 ℃), and drying method(60 ℃) after steaming. The contents of total flavonoids, rutin, narcissus, quercetin, isorhamnetin, and Fe~(3+) reducing ability, DPPH free radical scavenging ability, ABTS free radical scavenging ability and fluorescence recovery after photobleaching(FRAP) were detected by UV, HPLC and colorimetry, respectively. Principal component analysis(PCA), cluster analysis(CA) and correlation analysis were used to comprehensively evaluate the quality of samples. According to the results, there were significant differences in the effect of drying methods on different specifications of samples. The drying method(60 ℃) after steaming was suitable for HL and BK, while the hot-air-drying method(60 ℃) was suitable for SK. When the fresh medicinal materials could not be treated in time, they should be spread out in a cool and ventilated place. Under high and low temperature conditions, the quality of three specifications of Sophorae Flos would be reduced. The hot-air-drying method(105 ℃) and shade-drying method(25 ℃) were not suitable for the treatment of fresh flowers and flower buds of Sophora japonicus. There were obviously differences of chemical compositions and antioxidant activities among the three specifications of samples. Therefore, the specifications of medicinal materials should be controlled to ensure the uniform quality. The study provided the abundant data reference for the selection of appropriate drying methods for the three specifications of Sophorae Flos, and useful exploration for the classification and processing of medicinal materials of flowers.

Identification of Coptis chinensis root rot disease pathogenic Fusarium spp. fungi / 中国中药杂志

Root rot disease is vital disease of Coptis chinensis, it has bursted in most producing area in recent years, and has caused severe damage. To identify the pathogenic fungi, Fusarium spp. fungi were isolated from rot root, of which the pathogenic fungi were screened with inoculation on C. chinensis root and plant, and identified with molecular and morphological method. The 20 Fusarium spp. fungi were obtained, of which 5 displayed high pathogenicity. It was deduced that F. oxysporum, F. solani and F. tricinctum were the pathogen, possibly pioneer pathogen of C. chinensis root rot disease. Among which F. oxysporum was dominant and deserved to pay more attention. High temperature and high humidity can increase pathogenicity of Fusarium spp. So the global climate warming may lead to temperature rising of C. chinensis producing area and favor the pathogen fungi, which may be one of the main factors leading to bursting of C. chinensis root rot disease. To control the root rot, beside developing and using pesticide, producing base should be moved to a high altitude area.

Quality grading standard of Citrus aurantium seedlings / 中国中药杂志

Forty-three annual Citrus aurantium grafted seedlings from Chongqing, Sichuan, Hunan, Jiangxi and other main producing areas were collected, and the plant height, rootstock diameter, scion diameter, root length, root diameter, lateral root number, root breadth, branch number, branch length, green leaf number, leaf length, leaf width, thorns and other indicators were measured. Through the K-cluster analysis of SPSS 19.0 software, the classification standards were obtained. Combined with the production practice, plant height, scion diameter and branch number were taken as the quality classification indexes of C. aurantium seedlings(annual grafted seedlings), and three classification standards were established. If it does not meet the three-level standard, it is unqualified seedling and cannot be used as seedling. It is suggested to use the first and second level seedlings in production.

Diversity of culturable fungi isolated from Coptis chinensis rot root in Shizhu city of Chongqing / 中国中药杂志

Root rot disease restricts the Coptis chinensis industry in Shizhu of Sichuan province. To disclose fungi composition and pathogen in rot root,so as to prevent and treat the root rot disease,the C. chinensis rot root of 5 years from 4 areas in Shizhu were collected in 3 seasons respectively. The fungi were isolated and molecularly and morphological identified,followed with population statistics. 437 fungi were isolated,belonging to 5 subphylum,11 classes,16 orders,22 families and 28 genus respectively. There are great difference among the fungi compositions of different area,year and sampling season,while there was no obvious variation rule. Ilyonectria sp.,Pythium sp.,Phoma sp,Trichoderma sp.are dominant genus,while Pythium sp.,Ilyonectria sp.,Phoma sp.,Fusarium sp. may contain root rot pathogen. Antagonistic bacteria may be screened from the strains of Trichoderma sp. isolated.

Research for dynamic changes of growth and alkaloids of Coptis chinensis / 中国中药杂志

With Coptis chinensis in high-yielding soil as the object,the growth regularity of plant and dynamic change of alkaloid content was studied. The plant growth model of C. chinensis was constructed. The plant height equation was =3.030 9+0.732 6-0.009 6²,the number of leaves equation was =111.882 6-2 234.881 7/+15 218.960 8/²-31 740.960 8/³,the leaf area equation was =-217.136 1+30.552 2-0.359 0²,the roots talk biomass equation was =-2.748 8+0.210 3+0.006 4²,the number of rootstalk equation was =-1.246 0+0.192 6+0.000 8²,the fibrous root biomass equation was =-4.973 5+0.589 4 -0.002 6². The results indicated that the number of leaves and leaf area were increasing continuously after seedling transplanting,the leaf area of 3-year-old C. chinensis reached a maximum value of 425.83 cm²/plant,after declining.The number of leave of 5-year-old C. chinensis reached a maximum value of 70.91. With the increasing of years of growth, the number of rootstalk and rootstalk biomass of C. chinensis was increasing continuously. The biomass growth of 3-year-old and 4-year-old rootstalk was the fastest in the whole development stage of C. chinensis,the annual increase of more than 300%. The change curve of rootstalk number, rootstalk biomass and fibrous root biomass in the whole growth stage was a s-type.The dry matter partition of leafwas the highest in 1-year-old C. chinensis, and then gradually decreased,the change trend of dry matter partition of rootstalk was just the opposite, the dry matter partition of fibrous root increases with the increase of the growing year, reaching the maximum value in 3-year-old, then gradually lower trend. The root-shootratio of 1-year-old C. chinensis was the smallest, then gradually increases, the growth center gradually shifted to the roots from stems and leaves, The weight of underground part of 3-year-old C. chinensis exceeded the aboveground part, the 5-year-old C. chinensis root-shoot ratio reached the maximum value of 1.91:1.With the increasing of years of growth, the contents of coptisine, berberine, epiberberine and palmatine in rootstalk was increasing continuously. The jatrorrhizine content in 2-year-old C. chinensis was significantly lower than that in other years, the content was no significant change after that. The columbamine content reached a maximum value in 3-year-old C. chinensis,then the decreased gradually. The content of magnoflorine gradually increased and reached maximum value in 5-year-old C. chinensis.

Transcriptome analysis reveals candidate genes involved in flavonols biosyhthesis in Sophora japonica / 中国中药杂志

In order to study the pathways of biosynthesis of flavonoids in Sophora japonica, 113 797 unigenes were obtained by Trinity software, with an average length of 803 bp, of which 72 752 unigenes were obtained from the database by high-throughput sequencing, and a total of 38 891 SSR loci were searched. Through the metabolic pathway analysis, we found that there were 135 unigenes involved in the biosynthesis of flavonoids and 959 unigene involved in other secondary metabolic pathways. Further analysis of genes involved in rutin biosynthesis revealed that 24 were associated with CHS, 52 were associated with FLS, and 11 were associated with UFGT. The obtained data of S. japonica transcriptome lays the foundation for studying the pathways of biosynthesis of flavonoids in S. japonica and provides theoretical basis for the formation of the quality of S. japonica.

Transcriptome characterization for Scrophularia ningpoensis based on high-throughput sequencing technology and related genes for synthesis of terpenoid compounds / 中国中药杂志

To investigate the profile of gene function and search for SSR, a new technology of high-throughput Solexa/Illumina sequencing was used to generate the root transcriptome of Scrophularia ningpoensis, and 65 602 036 raw reads were obtained. Based on the bioinformatics analysis and Trinity, 73 983 unigenes were obtained with an average length of 823 bp. The comparison of sequence homology in database showed that 56 389 unigenes had different degrees of homology. A total of 520 metabolic pathways related genes and 191 relDODO transcription factors were identified by the Swiss-Prot, GO, KEGG and COG.The 11 659 SSRs were found by MISA and the highest frequency was AG/CT. In this study, we obtained numerous SSRs to provide references for the study of functional gene cloning and genetic diversity of S. ningpoensis. The key genes involved in the secondary metabolism are the basis for the study of biosynthesis and regulatory mechanism of the secondary metabolites.

Research on bacteria microecology in root rot rhizosphere soil of Coptis chinensis produced in Shizhu city / 中国中药杂志

Illumina Hiseq 2500 high-throughput sequencing platform was used to study the bacteria richness and diversity, the soil enzyme activities, nutrients in unplanted soil, root-rot and healthy rhizophere soil of Coptis chinensis for deeply discussing the mechanism of the root-rot of C. chinensis. The high-throughput sequencing result showed that the artificial cultivation effected the bacteria community richness and diversity. The bacteria community richness in healthy and diseased rhizosphere soil showed significant lower than that of in unplanted soil (P<0.05) and declined bacteria diversity. The bacteria community richness in root-rot rhizosphere soil increased significantly than that of health and unplanted soil and the diversity was lower significant than that of unplanted soil (P<0.05). The results of soil nutrients and enzyme activities detected that the pH value, available phosphorus and urease activity decreased and the sucrase activity increased significantly (P<0.05). The content of organic carbon and alkaline hydrolysis nitrogen the catalase and urease activity in root rot soil samples was significantly lower than that of healthy soil samples (P<0.05). However, the contents of available phosphorus and available potassium were significantly in root-rot sample higher than that of healthy soil samples (P<0.05). Comprehensive analysis showed that the artificial cultivation declined the bacteria community richness and diversity. The bacteria community richness decreased significantly and the decreased diversity may be the cause of the root-rot. Meanwhile, the decrease of carbon and the catalase activity may be another cause of the root-rot in C. chinensis produced in Shizhu city, Chongqing province.

Effect of nitrogen, phosphorus and potassium fertilizers on Flos Sophorae Immaturus yield and quality in hilly area of Chongqing / 中国中药杂志

With Sophora japonica at the flowering stage as the object, the effect of nitrogen, phosphorus and potassium fertilizers on the yield composition factors, yield and quality of Flos Sophorae Immaturus (FSI) was studied. The results indicated that in early spring, nitrogen, phosphorus and potassium fertilizer on the amplification rate of S. japonica, FSI yield composition, yield and quality were different significantly, middle to high nitrogen (1.5-2.0 kg/plant) significantly increased the level of panicled clusters, raceme and flower bud number and yield. Phosphorus (1.5-2.0 kg/plant) could significantly increase the total buds of flower number and yield, potassium showed no significant increase in yield and yield components. Comprehensively considering yield and quality of FSI, nitrogen 1.5-2.0 kg/plant, phosphorus 1.5-2.0 kg/plant and potassium 0.6-0.9 kg/plant are appropriate.

Analysis on genetic diversity and genetic relationship of medicinal species in Coptis by SCoT / 中国中药杂志

The genetic diversity and genetic relationship among four medicinal species of Coptis were detected by the approach of sequence-related amplified polymorphism (SCoT). The associated genetic parameters were calculated by POPGENE1.31. The systematic diagram of genetic relationship were clustered by TREECONW. The results showed that a total of 434 bands were produced by using 28 primers, of which 430 were polymorphic loci. There was a high level of genetic diversity among species (PPB=99.1%,Na=1.990 6,Ne=1.329 3,H=0.212 2,I=0.337 8). However, genetic diversity was lower within species, the average of genetic parameters wasPPB=16.8%,Na=1.168 2, Ne=1.073 0,H=0.043 7,I=0.067 7. The Nei's genetic differentiation coefficient was 0.794 0, that indicated that most of the genetic variation existed among species. By clustering analysis, different individuals gathered in the same group. The results confirmed that SCoT marker can be used as one of the effective methods to reveal the genetic diversity and relationship among medicinal species of Coptis.

Identification and phylogenetic analysis of endophytic fungi isolated from Scrophularia ningpoensis / 中国中药杂志

The endophytic fungi from root, main stem, branch and leaf of Scrophularia ningpoensis were isolated and identified from Wulong and Chongqing, and the population diversity analysis and phylogenetic analysis were followed. The result indicated that, as to population diversity index, S. ningpoensis from Wulong： leaf>main stem=branch>root, branch from Chongqing>branch from Wulong. Fifty-eight endophytic fungi were obtained, most of which were the pathogens of the plant. Colletotrichum was the prevailing genus, of which C. gloeosporioides and C. boninense were the prevailing strains. Leaf and seedlings might be the main path of infection. Endophytic fungi and pathogen might convert to each other, influenced by such factors as environment, genotype et al.

Study on associated plants and spatial distribution pattern of Dipsacus asperoides populations / 中国中药杂志

In order to understand the associated species and the population distribution pattern, the investigation of 32 sample plotsfrom the main natural distribution area in Dipsacus asperoides community was carried by quadrat method .The results showed that there were 156 species, which belong to 131 genera and 60 families. There were more species in the two dominant families, Asteraceae and Rosaceae. There were many types of associated, but most appeared at a low frequency. The vegetation type were mostly herbaceous and shrub species, which accounted for 77.6% of the total species. The value of t was greater than t0.05 by methods of variance/mean, showed the difference was significant and the distribution pattern of D. asperoides were cluster distribution. The determination results of seven aggregation intensity index also showed that D. asperoides population accorded with the characteristics of cluster distribution（C>1，K>0，Ca>0，m*>1，m*/m>1，I>0，GI>0）.

Investigate optimum conditions and determinate changes of β-glucosidase activity in Scrophularia root under different drying conditions / 中国中药杂志

To explore the optimum conditions of β-glucosidase activity in Scrophularia root by using pNPG method. The extraction conditions and reaction conditions (such as extraction liquid type, reaction system, reaction time, temperature, and substrate concentration) were screened by using monofactorial experiment and homogeneous design. Then the changes of β-glucosidase activity in Scrophularia root were detected at the drying temperature of 40-100 ℃. The results showed that citric acid phosphate buffer had better extraction effect, and the maximum absorbance produced by enzymatic reaction was present at 50 ℃ environment after reaction for 30 min. Homogeneous design experiment determined that the optimal conditions were as follows: optimal extraction liquid pH 7.0; enzymatic reaction system pH 6.0; substrate concentration 20 mmol•L⁻¹. The change of enzyme activity was affected by drying temperature and water loss rate. In the drying temperature of 60-100 ℃, the enzyme activity was reduced rapidly with the increase in water loss rate, while the activity was seen even with 0% of water at 40 and 50 ℃. This study has laid the theoretical foundation for research of hydrolysis mechanism of iridoid glycosides and optimum drying process.

Study on seed quality test and quality standard of Lonicera macranthoides / 中国中药杂志

Referring to the rules for agricultural seed testing (GB/T 3543-1995) issued by China, the test of sampling, purity, thousand seed weight, moisture, viability, relative conductivity and germination rate had been studied for seed quality test methods of Lonicera macranthoides. The seed quality from 38 different collection areas was measured to establish quality classification standard by K-means clustering. The results showed that at least 7.5 g seeds should be sampled, and passed 20-mesh sieve for purity analysis.The 500-seed method used to measure thousand seed weight. The moisture was determined by crushed seeds dried in high temperature (130±2) ℃ for 3 h.The viability determined by 25 ℃ 0.1% TTC stained 5h in dark. 1.0 g seeds soaked in 50 ml ultra pure water in 25 ℃ for 12 hours to determine the relative conductivity. The seed by 4 ℃stratification for 80 days were cultured on paper at 15 ℃. Quality of the seeds from different areas was divided into three grades. The primary seed quality classification standard was established.The I grade and II grade were recommend use in production.

Study on influence factors of seed germination and seeding growth of Lonicera macranthoides / 中国中药杂志

In order to improve reproductive efficiency and quality standard, the influence factors of seed germination and seeding growth of Lonicera macranthoides werew studied. The fruit and seed morphological characteristics of L. macranthoides were observed, the seed water absorbing capacity was determined, and different wet sand stratification time, temperature and germination bed treatment were set up. The effects of the parameters on seed germination and seedling growth were analysed. There was no obstacles of water absorption on L. macranthoides seed, quantity for 22 h water absorption was close to saturation. In the first 80 d, with the increase of the stratification time, seed initial germination time was shortened, germination rate and germination potential was improved. Stratification for 100 d, germination rate decreased. At 15 ℃, seed germination and seedling growth indicators were the best. The seedling cotyledon width in light was significantly higher than that in dark. Seeds on the top of paper and top of sand germination rate, germination potential, and germination index was significantly higher than that of other germination bed and mildew rate is low. The optimal conditions of seeds germination test was stratified in 4 ℃ wet sand for 80 d, 15 ℃ illuminate culture on the top of paper or top of sand. The first seeding counting time was the 4th day after beginning the test, the final time was the 23th day. The germination potential statistical time was the 13th day after beginning the test.

Detection of Echinoderm Microtubule Associated Protein Like 4-Anaplastic Lymphoma Kinase Fusion Genes in Non-small Cell Lung Cancer Clinical Samples by a Real-time Quantitative Reverse Transcription Polymerase Chain Reaction Method / 中国医学科学院学报

Objective To establish a real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) for the rapid, sensitive, and specific detection of echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion genes in non-small cell lung cancer. Methods The specific primers for the four variants of EML4-ALK fusion genes (V1, V2, V3a, and V3b) and Taqman fluorescence probes for the detection of the target sequences were carefully designed by the Primer Premier 5.0 software. Then, using pseudovirus containing EML4-ALK fusion genes variants (V1, V2, V3a, and V3b) as the study objects, we further analyzed the lower limit, sensitivity, and specificity of this method. Finally, 50 clinical samples, including 3 ALK-fluorescence in situ hybridization (FISH) positive specimens, were collected and used to detect EML4-ALK fusion genes using this method. Results The lower limit of this method for the detection of EML4-ALK fusion genes was 10 copies/μl if no interference of background RNA existed. Regarding the method's sensitivity, the detection resolution was as high as 1% and 0.5% in the background of 500 and 5000 copies/μl wild-type ALK gene, respectively. Regarding the method's specificity, no non-specific amplification was found when it was used to detect EML4-ALK fusion genes in leukocyte and plasma RNA samples from healthy volunteers. Among the 50 clinical samples, 47 ALK-FISH negative samples were also negative. Among 3 ALK-FISH positive samples, 2 cases were detected positive using this method, but another was not detected because of the failure of RNA extraction. Conclusion The proposed qRT-PCR assay for the detection of EML4-ALK fusion genes is rapid, simple, sensitive, and specific, which is deserved to be validated and widely used in clinical settings.

Analysis on genetic diversity among wild Dipsacus asperoides by SCoT / 中国中药杂志

To investigate the genetic diversity among wild Dipsacus asperoides in China, 66 germplasmic resources of D. asperoides were analyzed by Start Codon Targeted Polymorphism (SCoT) molecular markers. Genetic distance was calculated by TREECONW software and the systematic diagram of genetic relationship was clustered by UPGMA method. The results showed that the totals of 181 bands were detected using 20 primers , among which 109 were polymorphic bands. The average percentage of polymorphic bands was 60.13%. Genetic distance changed from 0.030 6 to 0.181 4. The clustering results showed that there was no significant correlation between the classification of the wild D. asperoides and their geographical origin. The relatively high genetic diversity of D. asperoides provides the basis for breeding new varieties.

Quality classification standard of Scrophularia ningpoensis seedlings / 中国中药杂志

The morphological indexes of the Scuophularia ningpoensis seedlings including the longth, diameter and weight were measured, clustering analysis was used to set up the standard quality grading of seedlings of S. Ningpoensis by SPSS. Field experiment was carried out to measure the indicators of plants growth and development, the yield and the quality. The results showed that the growth and yield of class I seedlings were better than those of class II and III. The content of main active ingredients was affected barely by seedlings classification. To ensure the quality, class II seedlings or above should be used for plantation. The established quality classification standard of S. ningpoensis seedlings was scientific and feasible, and provides the basis for the standardized cultivation of S. ningpoensis.

Analysis on genetic diversity and genetic relationship of medicinal species in Dipsacus from China by SRAP / 中国中药杂志

The author detected the genetic diversity and genetic relationship within and among eight medicinal species of Dipsacus by the approach of sequence-related amplified polymorphism (SRAP). The associated genetic parameters were calculated by POPGENE 1.31. The Genetic distance was calculated by TREECONW and the systematic diagrams of genetic relationship were clustered by UPG-MA. The results showed that, using 26 primers, a total of 558 bands were produced, of which 539 were polymorphic loci. There was a high level of genetic diversity among species (PPB = 96.59%, Na = 1.9659, Na = 1.3375, H = 0.2143, I = 0.3423). However, genetic diversity was lower within species, the average of genetic parameters was PPB = 6.97%, Na = 1.0697, Na = 1.0311, H = 0.0187, I = 0.0291. The Nei's genetic differentiation coefficient was 0.9126, indicated that most of the genetic variation existed among species. By clustering analysis, different individuals gathered in the same group and the classified result of SRAP marker between traditional modal characters was almost same. The results confirmed that SRAP marker can be used as one of the effective methods to reveal the genetic diversity and relationship among medicinal species of Dipsacus.

Impact of storage conditions and time on herb of Lonicera macranthoides / 中国中药杂志

To study the effect of different storage conditions and storage time on herb quality of Lonicera macranthoides, different packaging materials including vacuum plastic bags, plastic bags, woven bags, sealed with endometrial bags, paper bags, sack bags were selected for the study under different storage conditions including room temperature, 5 degrees C refrigerator, low temperature of - 20 degrees C refrigerator and desiccator. Twenty-four batches of samples were used for the study, and active ingredients were determined. The experimental results showed that the ingredients in each storage group changed with the storage time, storage conditions (storage environment, packaging). Under the same storage time, the storage environment (temperature, humidity) had effect on the stability of herb quality. Low temperature had less effect on herb quality. The effect of packaging on herb quality was as following: plastic vacuum packaging > woven with endometrial sealed packaging > plastic bag > woven bag > sack bags > paper bags. Under the same storage conditions, with the increase of storage time, caffeic acid content increased slowly, and other five ingredients content decreased gradually. Storage time affected significantly on the intrinsic quality (chemical composition) and appearance of herb. It is suggested that low temperature (5 degrees C), dark and sealed storage are suitable for storage of L. macranthoides herb, the storage time should be not more than 24 months.