[Effect of origin, tree age, and harvesting time on content of flavonoids and terpene lactones in Ginkgo Folium].

The content of total flavonol glycosides in Ginkgo Folium in the planting bases was determined by high performance liquid chromatography(HPLC).The samples were extracted by reflux with methanol-25% hydrochloric acid.The HPLC conditions were as follows: Agilent ZORBAX SB-C_(18) column(4.6 mm×250 mm, 5 µm), isocratic elution with mobile phase of 0.4% phosphoric acid solution-methanol(45∶55), flow rate of 1 mL·min~(-1), column temperature of 30 ℃, detection wavelength of 360 nm, and injection vo-lume of 10 µL.A method for the determination of terpene lactones in Ginkgo Folium was established based on ultra-high performance liquid chromatograph-triple-quadrupole/linear ion-trap tandem mass spectrometry(UPLC-QTRAP-MS/MS).The UPLC conditions were as below: gradient elution with acetonitrile-0.1% formic acid, flow rate of 0.2 mL·min~(-1), column temperature of 30 ℃, sample chamber temperature of 10 ℃, and injection volume of 10 µL.The ESI~+and multiple reaction monitoring(MRM) were adopted for the MS.The above methods were used to determine the content of total flavonol glycosides and terpene lactones in 99 batches of Ginkgo Folium from 6 planting bases, and the results were statistically analyzed.The content of flavonoids and terpene lactones in Ginkgo Folium from different origins, from trees of different ages, harvested at different time, from trees of different genders, and processed with different methods was compared.The results showed that the content of total flavonol glucosides in 99 Ginkgo Folium samples ranged from 0.38% to 2.08%, and the total content of the four terpene lactones was in the range of 0.03%-0.87%.The method established in this study is simple and reliable, which can be used for the quantitative analysis of Ginkgo Folium.The research results lay a basis for the quality control of Ginkgo Folium.

[Effect of heavy metal lead on anticoagulant activity of leech based on label-free quantitative proteomics].

Whitmania pigra is the most widely distributed species of leeches in the market. In this study, the effect of heavy metal lead pollution on the anticoagulant activity of Wh. pigra was studied and the potential mechanism was explored. Pb(NO_3)_2 was used to contaminate the breeding soil which was then used to rear Wh. pigra for 50 days(lead-contaminated group, LC group), and meanwhile the blank control group(CG group) was set. Proteins were extracted from the obtained leech samples, and the differentially expressed proteins between LC and CG groups were analyzed by label-free proteomics technology. In this study, a total of 152 differentially expressed proteins were screened out, of which 93 proteins were up-regulated and 59 proteins were down-regulated in LC group. Bioinformatics analysis showed that the biological processes enriched with the differentially expressed proteins were mainly vesicle-mediated transport and transport positive regulation; the enriched cell components were mainly endocytosis vesicles and apical plasma membrane; the enriched molecular functions mainly included carbohydrate binding. The differentially expressed proteins were enriched in 76 KEGG pathways, which mainly involved metabolic pathways, biosynthesis of secondary metabolites, and bacterial invasion of epithelial cells. In this study, two differentially expressed proteins with Antistasin domain were presumed, which provides reference for further exploring the regulatory mechanism and signal transduction underlying the effect of lead pollution on the anticoagulant activity of leech.

[Determination of caffeine content in Ginkgo Folium by ultra high performance liquid chromatography-triple quadrupole mass spectrometry].

In this experiment, an ultra-high performance liquid chromatographytandem triple quadrupole mass spectrometry was established for the determination of caffeine in commercially available Ginkgo Folium. The samples were extracted by ultrasonic method with methanol, and separated on Waters CORTECS T3 column(2.1 mm×100 mm, 2.7 µm), with mobile phase of 0.1% formic acid solution-0.1% formic acid acetonitrile solution for gradient elution, at flow rate of 0.3 mL·min~(-1); column temperature of 30 ℃, and injection volume of 2 µL. Mass spectrometry was conducted at ESI~+ multiple reaction monitoring(MRM) mode; quantitative analysis was conducted with external standard method. The results showed that in the range of 0.099 6-9.96 ng·mL~(-1), there was a good linear relationship between the mass concentration of caffeine and the peak area, R~2=0.999; the average recovery was 84.51%, with RSD of 6.2%. The results of precision, repeatability and stability showed that the RSD was 5.1%, 5.9%, 7.2%, respectively. The content range of caffeine in 10 batches of Ginkgo Folium was 1.52-60.86 µg·kg~(-1). In conclusion, this method is accurate, reliable and reproducible, which provides a reference for the safety study of Ginkgo Folium.

[Diversity of fungi on surface of Pheretima].

Traditional Chinese medicines( TCMs) are easily contaminated by fungi during planting,harvesting,processing,transportation and storage. The 2015 version of Chinese Pharmacopoeia stipulates the detection of aflatoxin in Dilong. After reviewing the literature,it has been found that there are no domestic and foreign scholars who have studied the surface fungi of Dilong. Pheretima,known as Dilong in China,is a commonly used TCMs in animal. In this experiment,8 batches of Dilong were collected from retail pharmacies in Beijing. The fungi on the surface of Dilong were cultured by traditional plate method and the single strain was obtained by the top purification method. The fungal colony morphology,microstructure characteristics and DNA barcode were used to isolate and identify the fungi. At the same time,based on Illumina Hi Seq 2500 high-throughput sequencing platform,the diversity of fungi on the surface of Dilong was analyzed. The results showed that 287 strains of 9 species of fungi were isolated and identified by plate method. Combined with 3 kinds of identification method,eight of nine fungi could be identified,respectively,Aspergillus niger,Penicillium,Alternaria nees,A. flavus,and Penicillium oxalicum,Humicola sp.,Talaromyces purpurogenus and A. insuetus,1 kind of fungi was not identified yet. Among them,Penicillium and Aspergillus were the dominant genus. The results of high-throughput sequencing belonged to 2 boundaries,6 gates,19 classes,44 orders,98 families,127 genus and 121 species in different classification levels. Wallemia,Aspergillus and Cordyceps were the dominant genus,and the relative abundances are 63. 33%,15. 28%,and 10. 28%,respectively. Through the diversity study on the surface fungi of Dilong in Beijing retail pharmacies,it can provide a reference for its safe storage and clinical use.

Genetic differentiation of Pseudoregma bambucicola population based on mtDNA COII gene.

mtDNA COII gene sequences were identified and analyzed using different types of software, namely, MEGA5.0, DNAMAN, and DnaSP5.0 in four Chinese provinces, namely, Sichuan, Zhejiang, Guizhou and Shanghai. Analysis of molecular genetic variation and its genetic structure and differentiation, combined with NJ tree, MP tree analysis and analysis of molecular variance (AMOVA), at Fst = 0.0582 conclude that the genetic differentiation is low, gene flow is Nm = 8.0911, and gene exchange is sufficient. However, for the geographic populations of Pseudoregma bambucicola in the four provinces, their gene exchange is relatively weak at Nm = 0.8284, whereas the genetic differentiation is high at Fst = 0.3764. Based on the data, total nucleotide diversity between the populations is 0.00158 ±â€¯0.00021. The results showed that the total population of Tajima's D and Fu's Fs results are D = -0.885 and Fs = 0.226, respectively. The experimental numerical results showed that this total population is not significant (P > 0.10), indicating that nine different geographic populations are short-term. No expansion occurred in the internal population. This study provided a theoretical and practical basis for the comprehensive prevention and control of P. bambucicola.

[Effect of lead pollution on anticoagulant effect of Whitmania pigra based on in vitro anticoagulation experiment].

Leech has a good anticoagulant activity and is one of the raw materials for treatment of many cardiovascular and cerebrovascular diseases. This study was based on in vitro anticoagulant experiments( APTT and PT) to investigate the effects of lead contamination on the anticoagulant effect of leech. At present,the Hirudo circulating in the market are dominated by Whitmania pigra,therefore Wh. pigra were cultivated under a different lead pollution for 50 days. Then,the effects of Wh. pigra extract,extracting from different cultivating environment,on activated partial thrombin time( APTT) and prothrombin time( PT) were determined by automatic coagulation instrument. The results showed that the Wh. pigra extract significantly prolonged the APTT compared with the saline group.The APTT of the lead-high residual Wh. pigra was shorter than that of the blank Wh. pigra. The Wh. pigra extracts from different treatment groups had little effect on PT. The results showed that the lead residue in the Wh. pigra increased with the increase of lead in the cultured soil,the lead residual of the Pb-H group was( 10. 66±2. 79) mg·kg~(-1),which exceeded the lead limit specified in the 2015 edition of the Chinese Pharmacopoeia. The results indicated that growth environment pollution is one of the important factors causing excessive lead in Wh. pigra. Lead pollution will reduce the anticoagulant effect of Wh. pigra and affect its clinical efficacy.

Aphicidal Activity of an Ageraphorone Extract From Eupatorium adenophorum Against Pseudoregma bambucicola (Homoptera: Aphididae, Takahashi).

The bamboo aphid, Pseudoregma bambucicola, is an important insect pest of bamboo that affects normal bamboo growth and induces sooty molds. The control of P. bambucicola involves the application of chemicals, such as imidacloprid, to which many species are resistant. In this study, we isolate a novel botanical pesticide (9-oxo-10,11-dehydro-ageraphorone) from an Eupatorium adenophorum(Asteraceae: Compositae) petroleum ether extract and test the aphicidal activity of this compound against P. bambucicola in laboratory bioassay and field-based experiments. This ageraphorone compound at a concentration of 2 mg/ml caused 73.33% mortality (corrected mortality [Subtracted the mortality of the negative control]: 70%) of P. bambucicola by laboratory bioassay within 6 h. Even at lower concentrations, this compound caused greater 33% mortality (corrected mortality: 30%) of aphids. Field experiments with naturally infested bamboo plants showed that two applications of 2 mg/ml ageraphorone to infested plants completely cleared infestations within 30 d. These effects were similar to those of the positive control (imidacloprid). These results reveal that 9-oxo-10,11-dehydro-ageraphorone exhibits significant aphicidal activity against bamboo aphids. We suggest that future research be directed at developing this ageraphorone compound from E. adenophorum as an aphicidal agent for biocontrol.

[Research of DNA barcode of Teseudinis Carapax et Planstrum and its adulterants based on COI gene sequence].

OBJECTIVE: To use COI gene on the Mauremys reevesii and its adulterants by molecular identification. Search a rapid, accurate method of identification of Teseudinis Carapax et Planstrum and its adulterants. METHOD: We collected 8 species of the authentic and adulterants of teseudinis carapax et planstrum in a nationwide then, extracted DNA, got the COI sequences. Use ContigExpress, Dnaman, Edit Sequence and Mega 5 to analyze the variable site and construct the N-J tree. RESULT: Compare with the authentic Teseudinis Carapax et Planstrum, the adulterant exist lots of variable site. The N-J tree Indicates that the same genus belong together and each species belong to relatively independent branch. CONCLUSION: Based on the COI gene, the technology of DNA bar code can be a excellent identification of Teseudinis Carapax et Planstrum and its adulterants.

[DNA identification of Zijingpi's adulterant species Schisandra sphenanthera based on NCBI nucleotide database analysis].

OBJECTIVE: To provide basis for quality control of Zijingpi, DNA identification was used based on NCBI nucleotide database analysis. METHOD: Firstly, total DNA of Zijingpi was extracted. Secondly, the ITS sequence was amplified by PCR with universal primer of ITS and PCR products was directly sequenced after purification. Finally, ITS sequence similarity and phylogenetic tree were used for identification. RESULT: The ITS sequence information of the mainstream commercial drugs of Zijingpi was obtained. CONCLUSION: It is firstly reported that the mainstream commercial drugs of Zijingpi was the bark of Schisandra sphenanthera.

Rapid discrimination of Chinese red ginseng and Korean ginseng using an electronic nose coupled with chemometrics.

Red ginseng is a precious and widely used traditional Chinese medicine. At present, Chinese red ginseng and Korean ginseng are both commonly found on the market. To rapidly and nondestructively discriminate between Chinese red ginseng and Korean ginseng, an electronic nose coupled with chemometrics was developed. Different red ginseng samples, including Chinese red ginseng (n=30) and Korean ginseng (South Korean red ginseng and North Korean red ginseng n=26), were collected. The metal oxide sensors on an electronic nose were used to measure the red ginseng samples. Multivariate statistical analyses, including principal component analysis (PCA), discriminant factorial analysis (DFA) and soft independent modeling of class analogy (SIMCA), were employed. All of the samples were analyzed by PCA. Most of the samples were used to set up DFA and SIMCA models, and then the remaining samples (Nos. 9, 10, 17, 18, 29, 30, 34, 43, 44, 50, and 51) were projected onto the DFA and SIMCA models in the form of black dots to validate the models. The results indicated that Chinese red ginseng and Korean ginseng were successfully discriminated using the electronic nose coupled with PCA, DFA and SIMCA. The checking scores of the DFA and SIMCA models were 100. The samples projected onto the DFA and SIMCA models were all correctly discriminated. The DFA and SIMCA models were robust. Electronic nose technology is a rapid, accurate, sensitive and nondestructive method to discriminate between Chinese red ginseng and Korean ginseng.

[Research and investigation in medicine of Baima Tibetan in Pingwu Sichuan].

OBJECTIVE: To investigate and collect medicinal resources and methods of prevention and treatment of diseases in Baima Tibetan of Pingwu Sichuan in order to rescue and protect the ethenological medicine. METHOD: Through visiting, field survey in four ethnic townships of Baima Tibetan in Pingwu Sichuan as well as referring literatures to collect information and data analysis of them the investigation was carried out. RESULT: The investigation area showed rich medical resources. Rheumatism, stomach disease, pharyngitis and trauma are local common diseases which caused by local climate, diet and life style and so on. The Baima Tibetan are good at using local herb singly and simply to treat disease, using cold water medicine and powder snuffing are their own characters. CONCLUSION: It is an extremely urgent issue to rescue Baima Tibetan medicine which is disappearing by the influence of the Han nationality culture.

[Relationships among Cyrtotrachelus buqueti larval density and wormhole number and bamboo shoot damage degree].

In August of 2007 and 2008, a field investigation was made to study the relationships among Cyrtotrachelus buqueti larval density and wormhole number and bamboo shoot damage degree in Sichuan Province. The three pairs of variables, i. e., C. buqueti larval density and wormhole number, C. buqueti larval density and bamboo shoot damage degree, and C. buqueti wormhole number and bamboo shoot damage degree, fitted cubic equations well, with the correlation coefficients at P = 0.001. Based on these mathematical models, the forecast tables for C. buqueti larval density and bamboo shoot damage degree were established, and the thresholds of C. buqueti larval density and wormhole number were 0.13 and 0.40 individual per bamboo, respectively.

The discovery,genetic analysis and gene mapping of earliness rice (Oryza sativa L.) D64B.

D64B, an achromatic early mutation, was discovered from indica rice preserver line D63B. D64B were crossed with inhomogeneous sterile lines, preserver lines and restorer lines, most of their F1 shared the same heading dates as D64B. The results indicated that D64B possessed the characteristic of dominant earliness. To analyze the earliness trait, positive and negative crosses F2 and BC1 from the cross of D64B and Shuhui 527 were cultivated. Their heading dates showed double apices distributing with same apex values. According to apex vale value -103 d, plants of the F2 and BC1 were separated into early plants and late plants, which were tested with Chi2 value. The results suggested that the segregation ratio of number of early to late plant fitted to 3:1 and 1:1 for positive and negative cross F2 and BC1, respectively, and that the earliness of D64B was controlled by a single dominant major gene. To map the gene, the polymorphisms between D64B and Shuhui 527 was analyzed with reported 356 SSR primers and 59 primers showed polymorphisms. The earliness-lateness near isogenic pools, early plants and late plants from F2 of Shuhui 527 x D64B were further amplified with the 59 primers. The results indicated that there were polymorphisms on both RM279 and RM71. Their results being analyzed with MAPMAKER/EXP3.0 software, the dominant major gene was located on the top arm of rice chromosome 2 and between the two SSR markers, RM279 and RM71, with genetic distance of 12.6 cM and 13.3 cM, respectively. According to reported data, the gene was first discovered and tentatively named as Ef-3(t). In breeding practice, sterile line D64A had been bred with D64B.