[Comprehensive evaluation of Pinellia ternata germplasm resources based on phenotypic trait classification].

The evaluation of germplasm resources is the prerequisite for the development, utilization, and conservation of Chinese medicinal resources. The selection of excellent germplasm is the key to the breeding and orderly production of Pinellia ternata. In this study, 21 germplasm materials of P. ternata from major production areas in China were collected and analyzed for population diversity after phenotypic preliminary screening. The results have revealed that the P. ternata population has abundant phenotypic variation, and the phenotypic changes could be divided into five phenotypes in terms of organ trait variation. Further analysis of variation in 20 quantitative traits of the population revealed that the coefficient of variation for adenosine content(339.05%) was the largest, while the coefficient of variation for the underground plant height(16.35%) was the smallest. Correlation analysis showed that there was a strong correlation among various traits, with 52 pairs of traits showing highly significant correlation(P<0.01) and 19 pairs of traits showing a significant correlation(P<0.05). The 21 germplasms in the test could be classified into three major clusters by cluster analysis, with Cluster Ⅱ having the highest number and content of nucleosides, making it suitable for the selection and breeding of P. ternata varieties with high content of nucleosides. The yield in Cluster Ⅲ was higher than that in other groups, making it suitable for the selection and breeding of P. ternata varieties with a high yield. All trait indicators could be simplified into five principal component factors through principal component analysis, and the cumulative contribution rate was up to 86.04%. Further, comprehensive analysis using membership function and stepwise regression analysis identified nine traits, such as plant height, main leaf length, and underground plant height as characteristic indicators for the comprehensive evaluation of germplasm resources of P. ternata. BX007, BX008, and BX005 were identified as germplasms with both high yield and high uridine content, with BX007 having the highest uridine content of 479.51 µg·g~(-1). It belonged to the germplasm of P. ternata with double bulbils and could be cultivated as a potential good variety. Based on the phenotypic classification of P. ternata, systematic resource evaluation was carried out in this study, which could lay a foundation for the excavation of genetic resources and the breeding of new varieties of P. ternata.

[Molecular identification and alkaloids content of endangered Chinese medicinal plant Coptis chinensis var. brevisepala].

According to the records of Chinese materia medica,Coptis chinensis var. brevisepala is an authentic Chinese medicinal plant highly recommended by ancient physicians since its rhizome is like a string of beads and has a good medicinal value. However,its medicinal components and values remain to be studied as it is endangered because of overexploitation. Therefore,this study aims to quantitatively determine its effective components based on UPLC-QTOF-MS,and to compare the contents of isoquinoline alkaloids in C.chinensis var. brevisepala with those in other Coptis species. Meanwhile,molecular methods accurately identified 12 batches of C. chinensis var. brevisepala,9 batches of C. chinensis,4 batches of C. deltoidea,and 1 batch of C. teeta. Gradient elution was performed with Waters CORTECS C18 column( 4. 6 mm× 150 mm,2. 7 µm) and the mobile phase acetonitrile-water with 0. 4% formic acid. Mass spectrometry was conducted in ESI positive mode. The quantitative results showed that 8 main alkaloids had a good linear relationship within the concentration range( R~2>0. 996),with the recovery rate of 95. 18%-105. 0% and the RSD of 0. 28%-3. 7%. Compared with that of other Coptis species,the rhizome of C. chinensis var. brevisepala had the highest contents of berberine and columbamine. The total content of the 8 alkaloids in C. chinensis var. brevisepala was similar to that in C. chinensis but higher than that of the other two species. PCA was performed to compare the alkaloids among the 4 species. Besides,the 8 alkaloids were evaluated in different parts of C. chinensis var. brevisepala. The results indicate that this method is reliable and efficient and can provide a reference for the quality research.

Population pharmacokinetics of oxcarbazepine active metabolite in Chinese paediatric epilepsy patients and its application in individualised dosage regimens.

PURPOSE: Oxcarbazepine (OXC) is an antiepileptic drug metabolised to active 10-monohydroxy derivative (MHD) following oral administration. There are no MHD population pharmacokinetic (PPK) models that describe the influence of genetic factors on MHD pharmacokinetics (PK). We developed a PPK model of MHD to investigate gene polymorphism of enzymes associated with MHD PK in Chinese paediatric epilepsy patients and evaluated its utility for dose individualisation. METHODS: Data were prospectively collected from 141 paediatric epilepsy patients (aged ≤ 14 years) who received OXC therapy at the First Affiliated Hospital of Fujian Medical University. The trough concentrations at steady state were determined by enzyme-multiplied immunoassay. Patients were genotyped for four single nucleotide polymorphisms (UGT2B7 802T>C, UGT1A9 I399C>T, ABCB1 3435C>T, and ABCB2 1249G>A). Patient gender, age, body weight (BW), hepatorenal function, and co-administrations were recorded. The PPK model was developed using nonlinear mixed-effects modelling software. The clinical performance of the final model was evaluated by including additional paediatric patients (n = 20) in the validation group. RESULTS: Oral clearance of MHD was significantly influenced by BW. The MHD PK was unrelated to the other covariates, such as the four single nucleotide polymorphisms and co-administration with new-generation antiepileptic drugs. The final BW-dependent exponent model showed the best fit with our data and predicted the trough concentrations in the validation group more accurately than the basic model. A new dosing strategy combining the dosage guideline and Bayesian method is proposed to individualise OXC regimens. CONCLUSION: A PPK model was established to estimate individual MHD clearance in paediatric patients taking OXC to develop individualised OXC dosing regimens for Chinese paediatric epilepsy patients.

[Comparative study on standard decoctions of Ophiopogonis Radix and Liriopes Radix].

Standard decoction is the core of the pharmacodynamics for water-soluble substance of Chinese materia medica. Its research is of great significance to the research and development of some single ingredients and the classical prescriptions,and it is the only way to transform traditional medication experience into industrial products. In this article,standard decoction research strategies were used for the comparison analysis of Ophiopogonis Radix from Zhejiang province(ZMD),Ophiopogonis Radix from Sichuan province(CMD),and Liriopes Radix(SMD). Regularities were present among different grades of CMD; potential quality markers and pH differences associated with SO2 residues were also found. Finally,the extract powder of Ophiopogonis Radix prepared by mass production process was analyzed and validated,and the results showed that the standard system could be used for the quality control of intermediates and final products. In conclusion,this study can provide reference for the clinical application of Ophiopogonis Radix medicines and provide testing method for higher quality with higher price.

[Correlative analysis of agronomic traits and quality of Panax ginseng saponins cultivation in farmland].

Cultivated ginseng in the farmland would become the mainly planting mode of Panax ginseng. However,there are relatively few cultivation ginseng varieties for farmland in China. Correlative analysis of qualitity and agronomic traits of P. ginseng cultivation in the farmland could provide a reference for the selection of excellent germplasm and new variety breeding of P. ginseng. In this study,the main index of saponin and agronomic traits of 4-6 years' samples were analyzed by UPLC and measured. The results show that there was significant difference in agronomic indexes of Damaya. The coefficient of variation of the root length( CV = 41. 97%) and fresh weight( CV = 31. 81%) were maximum,and the coefficient of variation of the stems thickness( 16. 72%) and root thickness were minimum. There was a significant correlation between yield and root thickness( P<0. 05). There was significant difference in drug yield of different harvest years( P<0. 05),and the yield of 6-years was 31. 52%-39. 69% higher than 4-years. However,there wasn't significant difference in total ginsenosides between 4 and 6 years old P. ginseng,but there was significant difference in ginseng Rg2,Rc and Rb2( P<0. 05),and the ginsenoside contents of different harvesting years were accorded with the criterion standards of 2015 Chinese Pharmacopoeia. There was no significant correlation between the saponin and the agronomic trait,while there was positive correlation with root thickness( P < 0. 05). Therefore,the stem diameter was positive correlation with yield of P. ginseng. Selection of the stem thickness of seedlings is beneficial to the increase of the yield and breeding of P. ginseng.

[Key techniques for precision cultivation of nitrogenous fertilizer of pollution-free ginseng].

Planting pollution-free farmland is the main mode of industrialization of ginseng cultivation, fine management of nitrogen fertilizer ginseng pollution-free farmland cultivation technology system is one of the key factors. In order to investigate the effect of nitrogenous fertilizer on the accumulation of ginseng biomass and saponins synthesis in vegetative growth stage, two-years-old ginsengs were used as test materials in this study. The test materials were cultivated by Hoagland medium with different nitrogen concentration (0，10，20，40 mg·L⁻¹) for 40 days. During the cultivation, photosynthetic rate was measured four times. After 40 days cultivation, chlorophyll content, stem diameter and the spatiotemporal expression of saponin synthesis related genes PgHMGR and PgSQE were tested. The results showed that there were significant differences in the photosynthetic rate and chlorophyll content among different nitrogen concentrations. The relative expression level of PgHMGR gene and PgSQE gene in root, stem and leaves of ginseng were different. Ginseng seedlings cultivated by 20 mg·L⁻¹ nitrogen possess the highest photosynthetic rate and chlorophyll content, while PgHMGR and PgSE showed the highest gene expression level. The optimal nitrogen concentration for the growth of 2-years-old ginseng might be 20 mg·L⁻¹ with 57.14 g ammonium nitrate each plant or pure 20.00 mg nitrogen each plant. It is concluded that this concentration is the most suitable concentration for the ginsenoside synthesis. Pollution-free ginseng with fine nitrogen fertilizer cultivation is conducive to the production of high quality and efficient ginseng medicinal materials. It lays a theoretical foundation for the rational fertilization and environment-friendly sustainable ecological ginseng planting industry.

[Research of pollution-free and precision cultivation system of Chinese herbal medicines].

The issues of disordering production and non-standard pesticide application are common in the production of Chinese herbal medicines. Aimed to above problems, research groups built the pollution-free and precision cultivation system of medicinal plants. This system mainly included the precise site selection of medicinal plants based on the GIS technology, modern omics-assisted breeding, metagenomics guiding the soil complex improvement, and the precise field management based on rational application of fertilizer and comprehensive control of disease. At present, the production and distribution of medicinal plants were performed in the many poor counties of the whole nation. The breeding platform of resistant varieties was built, and certificates of new and well-bred varieties were received, in the base of genetic backgrounds of the original species of medicinal plants. The disease incidences were declined after application of these resistant varieties. Additionally, chemical pesticide consumption of medicinal plants (such as Panax ginseng, P. notoginseng, Salvia miltiorrhiza, P. quinquefolium, Schisandra chinensis, Platycodon grandiflorum and P. grandiflorus etc.) reduced by 20%-80% based on the genetic testing technologies of plant diseases and insect pests and safety evaluation of pollution-free pesticides. The application of pollution-free and precision cultivation system of Chinese herbal medicines achieve significantly social, economic and ecological benefits.

Comprehensive Characterization for Ginsenosides Biosynthesis in Ginseng Root by Integration Analysis of Chemical and Transcriptome.

Herbgenomics provides a global platform to explore the genetics and biology of herbs on the genome level. Panax ginseng C.A. Meyer is an important medicinal plant with numerous pharmaceutical effects. Previous reports mainly discussed the transcriptome of ginseng at the organ level. However, based on mass spectrometry imaging analyses, the ginsenosides varied among different tissues. In this work, ginseng root was separated into three tissues-periderm, cortex and stele-each for five duplicates. The chemical analysis and transcriptome analysis were conducted simultaneously. Gene-encoding enzymes involved in ginsenosides biosynthesis and modification were studied based on gene and molecule data. Eight widely-used ginsenosides were distributed unevenly in ginseng roots. A total of 182,881 unigenes were assembled with an N50 contig size of 1374 bp. About 21,000 of these unigenes were positively correlated with the content of ginsenosides. Additionally, we identified 192 transcripts encoding enzymes involved in two triterpenoid biosynthesis pathways and 290 transcripts encoding UDP-glycosyltransferases (UGTs). Of these UGTs, 195 UGTs (67.2%) were more highly expressed in the periderm, and that seven UGTs and one UGT were specifically expressed in the periderm and stele, respectively. This genetic resource will help to improve the interpretation on complex mechanisms of ginsenosides biosynthesis, accumulation, and transportation.

[DNA barcoding identification of endangered medicinal plants of Orchidaceae].

In this study, DNA barcoding was used to validate the traditional morphological classification of medicinal plants of Orchidaceae. The 163 samples of 135 species belong to 49 genera which have been confirmed by morphological identification were collected. Candidate sequences, including matK, psbA-trnH and ITS2 sequences, were amplified, bidirectionally sequenced, and assembled. All the sequences were blasted to GenBank database at NCBI, then analyzed using Neighbor-joining tree method by MEGA 7.0. The results showed that the DNAs of 163 samples were successfully extracted. The amplification efficiency of matK, psbA-trnH and ITS2 sequences were 100%, 100% and 98.77%, respectively. The 487 sequences were obtained, 345 sequences of which have matched corresponding sequences in the GenBank database and 142 sequences were new sequences. The topology of NJ tree which were constructed with the matK sequences was better than the trees of psbA-trnH and ITS2 sequences. In conclusion, the matK, psbA-trnH and ITS2 sequences were complementary and suitable for identification of medicinal plants of Orchidaceae. DNA barcoding can be used as an auxiliary means for identification of medicinal plants of Orchidaceae.

[Research on technology regulation of non-polluted cultivation and strategies of Panax ginseng cultivation in farmland].

As the limit of the usage of available forest land, cultivated ginseng in the farmland would become the mainly Panax ginseng planting mode, meanwhile the non-polluted production technology would be the mainly development direction in the future. In this study, the non-polluted cultivation technology system of P. ginseng was established based on the research results of field investigation in the cultivated regions. The system includes suitable planting regions selecting, planting method, field management, pest control, harvesting & processing, and quality control. Aimed at the serious issues in the cultivation, research strategies have been provided to guarantee the sustainable development of the ginseng industry. The patterns of soil restoration after P. ginseng cultivation, establishment the platform of comprehensive disease & pest control, breeding new varieties for high stress tolerance and resistance, and a traceability system for P. ginseng cultivation. In all, these strategies was considered to largely developing of the ginseng industry in the green and sustainable way.

[Analysis of varieties and standards of Composite medicinal plants used in Dai medicine].

The study aims to analyze the varieties and standards of compositae medicinal plants used in Dai medicine. The results showed that there were 78 species (including varieties) compositae plants recorded in literatures, which belongs to 63 medicinal materials varieties. And 47 original plants (60.25%) were recorded in Chinese medicinal material standards. In those standards and literatures of Dai medicine, there are great differences in translated Chinese names, original plants, medicinal parts, and efficacy of medicinal plants. Therefore, the variety systematization and the quality standards of Dai medicine should be strengthened.

[Soil improvement promoted micro-ecology of farmlands for ginseng cultivation].

This study has revealed the change of the soil micro-ecology of farmlands, which used for ginseng cultivation, brought by comprehensive soil improvement. The process of soil improvement was described as follows： soil was sterilized using trichloronitromethane, and then perilla seeds were planted. After growing up, the perillas were turned over into the field and fermented, then organic fertilizer was added. Rotary tillages were carried out during the intervals. Physical and chemical properties of treated soil were measured, as well as microbial diversity, which was illustrated using 16s high through-put sequencing. The survival rate and growth data of ginseng seedlings were recorded. The analysis showed that after improvement, the soil organic matter content was increased and soil bulk density was decreased, compare to the controls, and the fertility in 0-20 cm of soil layer was increased in the treatment. Additionally, the soil microbial diversity was changed greatly. In detail, alpha diversity of the soil decreased after soil improvement while the beta diversity increased. In order to verify the achievement of soil improvement, ginseng seedlings were planted. Compared to the untreated land blocks, the survival rate of ginseng on improved blocks was increased up to 21.4%, and the ginseng physiological index were all better than the controls. Results showed that comprehensive soil improvements including soil sterilization, green manure planting and organic fertilization application effectively improved the soil micro-ecology in farmlands. This study will pave the way for the future standardization of ginseng cultivation on farmlands.

[DNA marker-assisted selection of medicinal plants (â ) .Breeding research of disease-resistant cultivars of Panax notoginseng].

DNA marker-assisted selection of medicinal plants is based on the DNA polymorphism, selects the DNA sequences related to the phenotypes such as high yields, superior quality, stress-resistance and so on according to the technologies of molecular hybridization, polymerase chain reaction and high-throughput sequencing, and assists the breeding of new cultivars. This study bred the first disease-resistant cultivar of notoginseng "Miaoxiang Kangqi 1" using the technology of DNA marker-assisted selection of medicinal plants and systematic breeding. The disease-resistant cultivar of notoginseng contained 12 special SNPs based on the analysis of Restriction-site Associated DNA Sequencing (RAD-Seq). Among the SNP (record_519688) was related to the root rot-resistant characteristics, which indicated this SNP could serve as genetic markers of disease-resistant cultivars and assist the systematic breeding. Compared to the conventional cultivated cultivars, the incidence rate of root-rot and rust-rot in notoginseng seedlings decreased by 83.6% and 71.8%, respectively. The incidence rate of root-rot respectively declined by 43.6% and 62.9% in notoginseng cultivation for 2 and 3 years compared with those of the conventional cultivated cultivars. Additionally, the potential disease-resistant groups were screened based on the relative SNP, and this model enlarged the target groups and advanced the breeding efficiency. DNA marker-assisted selection of medicinal plants accelerated the breeding and promotion of new cultivars, and guaranteed the healthy development of Chinese medicinal materials industry.

[Analysis of global ecology of Panax notoginseng in suitability and quality].

In this study, the Geographic Information System for Global Medicinal Plants(GMPGIS) was used to assess the global production and ecological adaptation of Panax notoginseng. Based on climate factors and soil types of P. notoginseng from 326 sampling sites, which cover both traditional and current major producing regions, as well as on the results of the ecological similarity computing analysis, we obtained the maximum ecological similarity areas for P. notoginseng worldwide. The results indicated that China was the most suitable ecological and cultivated area globally for P. notoginseng, accounting for more than 70% of the total cultivated area in the world. The United States, Brazil, Portugal, and other 22 countries also had a small amount of potential suitable producing area. China has eight potential suitable producing provinces, including Yunnan, Fujian, Guangxi, Guizhou, etc. The prediction is consistent with the new district of P. notoginseng reported in recent years, which verifies the accuracy of the prediction of GMPGIS. We conducted a literature analysis on resource regeneration and quality ecology on P. notoginseng, and summarized the cultivation, wild tending models, and effects of environmental factors on the quality of P. notoginseng. The results provide scientific basis for selection of P. notoginseng, as well as the introduction, cultivation, and production of P. notoginseng worldwide.

Clinical outcomes of toe amputation in patients with type 2 diabetes in Tianjin, China.

The aim of this study is to determine the predictors for reulceration, reamputation and mortality in patients with diabetes following toe amputation, and the impact of activities of daily living on clinical outcomes. This prospective cohort study included 245 patients who had undergone toe amputation (202 healing and 43 non-healing) and was followed for a 5-year period. Data regarding new foot ulceration, reamputation and mortality were recorded, and the patients' activities of daily living were evaluated. The rate of wound healing was 82·4%. The rate of follow-up in the healed group was 91·6%. In years 1, 3 and 5, the cumulative incidence of patients who developed a new foot ulcer was 27·3%, 57·2% and 76·4%, respectively, leading to reamputation in 12·5%, 22·3% and 47·1%, respectively. The cumulative mortality was 5·8%, 15·1% and 32·7% at 1, 3 and 5 years, respectively. Multivariate analysis showed that GHbA1c > 9% (75 mmol/mol) was identified as an independent predictor of impaired wound healing, reulceration and reamputation. An age of >70 years was identified as an independent predictor of reamputation, mortality and impairment of activities of daily living. Despite a satisfactory initial healing rate after the first toe amputation, with the extension course after the toe amputation, the long-term outcomes are not optimistic. In developing countries like China, taking measures to prevent reulceration and reamputation is very important for patients with diabetic foot minor amputations, especially following toe amputation.

[Study on botanical pesticides and its application in production of traditional Chinese medicine].

The issues including excessive pesticide residues and heavy metal contamination have become the bottle-neck in the development of Chinese herbal medicines. Compared with traditional chemical pesticides, biological pesticides, especially botanical pesticides, are more safe and environment-friendly, which were beneficial to the quality improvement Chinese medicinal materials. Though there exists a weak basic research and it is hard for promotion and regulation, the policy of good and the desire for botanical pesticides will accelerate its development, and replace traditional chemical pesticides gradually. This paper reviews the current situation of botanical pesticides, and gives some pertinence suggestions according to the existing problems and challenges. Research on botanical pesticides will become the key point to solve the problem of excessive pesticides residues and heavy metal contamination, and promote the healthy development of Chinese materia medica.

[Identification of Seeds and Seedlings of Chinese Medicinal Materials Using DNA Barcoding Technology:A Case Study in Paris polyphylla var. yunnanensis].

Objective: To establish a novel approach based on DNA barcode sequence, so as to guarantee the quality stability of Chinese medicinal materials. Methods: Eight species of Paris plants were collected, and a standard DNA barcode library was developed by ITS loci. Furthermore, the barcodes also used to identify the seed and seedling products that purchased from the markets. Results: ITS loci can stably and accurately distinguish Paris polyphylla var. yunnanensis and its adulterants. Conclusion: The seeds and seedlings of Chinese medicinal materials need to be properly authenticated before planting,and DNA barcoding has been found to be effective for this purpose.

[Disease of root-knot nematode and control strategy in medicinal plants].

Root-knot nematode (Meloidogyne spp.) is one of the important disease of medicinal plant cultivation and seriously hinders the sustainable development of traditional Chinese medicine industry. We introduced the main species, identification methods and control strategies of root-knot nematode diseases in the medicinal plants in this study. Identifications of morphology and molecular were the main tools for the distinction of root-knot nematodes at present. This study stated that integrated system was established for root-knot nematode control, including that integrated control technique was the first step, disease-resistant varieties with high yield were the basis, and normalized patterns of cultivation and management were the measure. These strategies would improve the sustainable development of medicinal plants and environmental protection.

[Progress in improvement of continuous monoculture cropping problem in Panax ginseng by controlling soil-borne disease management].

The continuous monoculture cropping problem severely has hindered the land resource of Panax ginseng cultivation and threatened the sustainable development of ginseng industry. There are comprehensive factors causing the continuous monoculture cropping problem, such as deterioration of soil physical and chemical properties, accumulation of allelochemical, increase of pesticide residue and heavy metal, imbalance of rhizospheric micro-ecosystem, and increase of soil-borne diseases. Among soil-borne disease was one of the key factors. More than 40 soil-borne diseases have been reported in the ginseng cultivation, especially, the diseases were more serious in the ginseng replanting land. Here main soil-borne diseases and their prevention way have been summarized, and we try to provide the effective improvement strategy of continuous monoculture cropping problem focusing on the disease control and offer reference for overcoming the ginseng continuous monoculture cropping problem.

[Quality Traceability System of TCM Based on Two-Dimensional Code].

Objective: To explore the quality traceability of traditional Chinese medicine based on quality two-dimensional code. Methods: Through reviewing and evaluating the current major tracing technologies and traceability system of traditional Chinese medicine, it was proposed that the concept of traditional Chinese medicine quality two-dimensional code, and on this basis, to build a quality traceability system and proposed concept model of online cloud platform of TCM quality traceability system, while the pros and cons were discussed. Results: This system was expected to break through the limitations of most of the original traceability system, not only to have information on the origin, circulation and processing steps, but also to track the intrinsic quality of the medicine; meanwhile the system expected to be used not only for Chinese herbal medicines, but also expected to be used to pieces, soups, Chinese patent drugs, etc. Conclusion: This study provides a theoretical basis for the establishment of quality traceability system of traditional Chinese medicine.