Specific PCR for Identification of Astragalus membranaceus var. mongholicus Seeds， A. membranaceus Seeds， and Adulterants / 中国实验方剂学杂志

ObjectiveTo establish a method based on specific polymerase chain reaction （PCR） that can accurately and rapidly identify Astragalus membranaceus var. mongholicus （AMM） seeds and A. membranaceus （AM） seeds. MethodThe Chloroplast Genome Information Resource （CGIR） and IdenDSS were used to obtain the characteristic DNA fragments of AMM and AM， and the specific single nucleotide polymorphism （SNP） sites of AMM and AM were screened out， on the basis of which the specific primers MG-F/MG-R of AMM and MJ-F/MJ-R of AM were designed. The specific PCR method for identifying AMM and AM was established and optimized， and the specificity and applicability of the method were investigated. ResultThe specific PCR conditions for the identification of AMM were primers MG-F/MG-R， annealing at 62 ℃， and 28 cycles. After PCR amplification and gel electrophoresis， the specific band appeared at about 220 bp， with no band for the seeds of AM or adulterants. The specific PCR conditions for identifying the AM were primers MJ-F/MJ-R， annealing at 58 ℃， and 28 cycles. After PCR amplification and gel electrophoresis， the band appeared at about 150 bp， with no band of AMM or adulterants. ConclusionThe specific PCR method established in this study can accurately and quickly identify the seeds of AMM and AM， which provides a basis for the classification and accurate identification of Astragalus seeds and adulterants.

Analysis of Fusarium oxysporum infection on changes of Astragalus metabolism by metabolomic approach / 药学学报

italic>Astragalus is a commonly used Chinese medicinal material in traditional Chinese medicine (TCM), and with the increase of planting area in recent years, the damage of Astragalus root rot has worsened year by year, which seriously affecting its quality and yield. Fusarium oxysporum is one of the main pathogens causing root rot in astragalus. In this study, UPLC-Q-TOF-MS based metabolomic approach combined with multivariate statistical analysis were used to analyze the metabolite changes of Astragalus in response to F. oxysporum infection. The results showed that 62 metabolites in the Astragalus had significant changes after inoculation of F. oxysporum. Polar metabolites included 40 flavonoids, 8 saponins, 2 nucleosides, 1 vitamin, 1 organic acid, 1 amino acid; while lipid metabolites included 3 fatty acids, 1 diradylglycerols, 2 lysophosphatidylcholine, 1 lysophosphatidylglycerol, 1 phosphatidylinositol, 1 sterol lipid. Among these differential metabolites, the relative content of flavonoids, vitamin B2, tryptophan and salicylic acid were increased, while the relative content of saponins were decreased. Correlation analysis showed that the flavonoids were positively correlated with each other, and positively correlated with most lipids, but negatively correlated with most saponins. In addition, studies have shown that F. oxysporum infection is not an influencing factor for the generation of malonyl substitution of flavonoid. This study elucidates the effect of F. oxysporum infection on Astragalus from the perspective of plant metabolism, which provides a basis for exploring the interaction mechanism between the Astragalus and F. oxysporum and further promoting molecular breeding.

Targeted metabolomic analysis of phenylpropanoid metabolites in Astragalus membranaceus var. mongholicus in response to Fusarium solani infection / 药学学报

Root rot severely restricts the sustainable development of Astragalus membranaceus var. mongholicus (AMM) industry. Resistance breeding is an economical and environmentally safe way to manage the disease and its key lies in the obtaining of resistance indicators. This study aimed to quickly and accurately screen the resistance-related (RR) metabolites so as to provide reference for the screening of indicators of AMM breeding for resistance. LC-MS-based targeted metabolomics and real-time quantitative PCR technology were employed, in combination with multivariate statistical analysis, in analyzing the dynamic changes of phenylpropanoid metabolites in AMM in response to root rot pathogen Fusarium solani (FS) infection and identifying the differential metabolites. The LC-MS method established showed high sensitivity; each metabolite had a good linear relationship (R2 ≥ 0.968 9) in the corresponding linear range of the respective standard curve; the recoveries and the relative standard deviations (RSDs) (n = 6) ranged from 70% to 107% and from 1.2% to 9.9%, respectively. Obvious disturbances were observed in the changes of the targeted metabolites in AMM infected by FS. These metabolites, compared with the mock-inoculated (CK) group, showed different up or down regulation with time series. Calycosin-7-O-β-D-glucoside, ononin, calycosin and formononetin were identified as differential metabolites, and they all belong to flavonoids. The first three compounds were significantly negatively correlated (r ≤ -0.97, P < 0.05) with the content of FS in the root of AMM. As potential RR metabolites, they are helpful in obtaining promising resistance indicators for AMM against FS infection.

Effect of EBR on Seed Germination， Seedling Growth and Key Enzyme Activities for Flavonoid Biosynthesis in Astragalus membranaceus var. mongholicus Under PEG-induced Drought Stress / 中国实验方剂学杂志

Objective:To study the effects of 24-epibrassinolide （EBR） at different concentrations on seed germination， seedling growth and key enzyme activities for flavonoid biosynthesis in <italic>Astragalus membranaceus </italic>var.<italic> mongholicus</italic> under polyethylene glycol （PEG）-induced drought stress， in order to provide theoretical reference for standardizing the drought-resistant cultivation techniques of <italic>A. membranaceus </italic>var.<italic> mongholicus</italic>. Method:The seeds of <italic>A. membranaceus </italic>var.<italic> mongholicus </italic>were soaked in EBR solution at different concentrations（0.001， 0.01， 0.1， 1， 10 μmol·L^-1） followed by foliar spraying to explore their effects on seed germination， seedling growth， photosynthesis， chlorophyll and malondialdehyde （MDA） contents， and key enzyme activities for flavonoid biosynthesis under drought stress induced by 20% polyethylene glycol （PEG）-6000. Result:Compared with the control（CK）group， PEG-induced drought stress led to an obvious decrease in germination potential， germination rate， germination index， vigor index， relative germination rate， plant height， root length， aboveground and root dry weight， net photosynthetic rate （<italic>Pn</italic>）， stomata conductance （<italic>Gs</italic>）<italic>， </italic>transpiration rate （<italic>Tr</italic>）， chlorophyll contents and chlorophyll a/b ratio， while a significant increase in intercellular carbon dioxide concentration （<italic>Gi</italic>）， MDA， flavonoids contents， and key enzyme activities for flavonoid biosynthesis such as phenylalanine ammonia lyase （PAL）. The treatment with exogenous EBR solution at the suitable concentration significantly enhanced the adaptation of <italic>A. membranaceus </italic>var.<italic> mongholicus </italic>seeds and seedlings to PEG-induced drought stress， manifested as significantly elevated germination potential， germination rate， germination index， vigor index， relative germination rate， plant height， root length， aboveground and root dry weight， <italic>Pn</italic>， <italic>Gs， Tr</italic>， chlorophyll a/b ratio， chlorophyll and flavonoids contents and key enzyme activities for flavonoid biosynthesis like PAL while lowered <italic>Ci</italic> and MDA contents. The optimal concentration of EBR solution was 0.1 μmol·L^-1. Conclusion:Exogenous EBR solution at the suitable concentration ameliorates the inhibitory effect of 20% PEG stress against seed germination and seedling growth of <italic>A. membranaceus </italic>var.<italic> mongholicus</italic>， reduces the oxidative damage in leaves， and improves the stress resistance to a certain extent by up-regulating the key enzyme activities and promoting flavonoid synthesis.

Analysis of quality difference based on Astragalus membranaceus var. mongholicus in genuine region / 中国中药杂志

This work is to establish the fingerprint of Astragalus membranaceus var. mongholicus by HPLC-ELSD method, and to analyze the simulated wildness degree of A. membranaceus var. mongholicus in the genuine region of Inner Mongolia, Ningxia and Gansu. Compared with wild A. membranaceus var. mongholicus, the quality differences of A. membranaceus var. mongholicus in the genuine region were analyzed by identification of chromatographic peaks and similarity evaluation, cluster analysis(CA), principal components analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA). HPLC fingerprints of A. membranaceus var. mongholicus in different genuine regions are established. The qualitative analysis of mass spectrometry identified 18 components. The similarity evaluation shows that the similarity of 32 batches of A. membranaceus var. mongholicus samples was 0.688-0.993. Among them, the similarity of samples in Shanxi, Inner Mongolia, Ningxia is 0.688-0.993, 0.835-0.989, 0.934-0.988, respectively and the similarity of samples in Gansu is 0.729-0.876 except No. 25 sample. The results of CA show that the samples of A. membranaceus var. mongholicus can be grouped into four categories according to the production area except the No. 11 and No. 25 samples. The results of PCA indicate that 32 batches of A. membranaceus var. mongholicus samples can be clustered according to quality and origin, and the quality of A. membranaceus var. mongholicus in Inner Mongolia is the closest to the wild breed. The results of OPLS-DA indicate that there are six components that can distinguish the wild and domestic A. membranaceus var. mongholicus, which are malonylastragaloside Ⅰ, astragaloside Ⅰ, calycosin-7-O-β-D-glycoside-6″-O-malonate, calycosin-7-O-β-D-glycoside, formononetin-7-O-β-D-glycoside-6″-O-malonate, and astrapterocarpan-3-O-β-D-glycoside-6″-O-malonate. The established method can be used to analyze differences between A. membranaceus var. mongholicus origin and planting environment, and can provide references for the protection and replacement of wild A. membranaceus var. mongholicus resources, and the cultivation, processing and production of A. membranaceus var. mongholicus.

Study on Suitability Zoning of Astragalus membranaceus var. mongholicus in Dingxi City Based on MaxEnt and ArcGIS / 中国药房

OBJECTIVE：To study the ecological suitability zoning of Astragalus membranaceus var. mongholicus in Dingxi city. METHODS ：Taking 1 001 batches of A. membranaceus var. mongholicus in Dingxi city as the investigation samples （each natural village with A. membranaceus var. mongholicus cultivation as the collection unit ），the longitude and latitude information of them were obtained from Dingxi economic crop technology promotion station ，and 55 environmental ecological factors （including climate，terrain，soil，etc.）were obtained from the Grid Database of Spatial Information of TCM Resources . Combined with the information of longitude ，latitude and environmental ecological factors ，the maximum entropy model （MaxEnt model ） was established with 75％ samples as the training set ，and the main ecological factors were screened out ，and 25％ samples were set as the validation set for model validation. Then the suitable growing areas of A. membranaceus var. mongholicus were divided by using ArcGIS. RESULTS ：Established MaxEnt model had good prediction （the area under the working characteristic curve of subjects in training set and verification set was 0.970 and 0.968）. Eight main ecological factors ，such as altitude ，precipitation and temperature ， were selected （the total contribution rate was 98.90％）. The comprehensive analysis found that the altitude was 1 800 to 2 650 m， the average precipitation in April was 25 to 50 mm，the lowest temperature of the coldest month was －16 to －8 ℃，the wettest monthly precipitation was 95 to 110 mm，and the seasonal variation of temperature was 70 to 80，the average temperature in December was －6 to －3 ℃ ，the average precipitation in October was 30 to 50 mm，and the average precipitation in December was 0 to 10 mm，which was the suitable environmental parameter for the growth of A. membranaceus var. mongholicus in Dingxi city. Based the results of ArcGIS analysis ，in Dingxi city ，A. membranaceus var. mongholicus was generally suitable for growth ；in the northwest of Lintao county ，the north of Anding district ，the south of Tongwei county ，a small area in the south of Longxi county and the south of Minxian county ，the border between Weiyuan county ，Zhangxian county and Minxian county ，A membranaceus var. mongholicus was not suitable for growth. CONCLUSIONS： Ther results of established model is in E-mail：961308817@qq.com line with the actual investigation ， can provide reference for the planting regional planning of A. membranaceus var. mongholicus in Dingxi city.

Ater-soluble Protein Profiles of Astragalus membranaceus var. mongholicus and A. membranaceus / 中国实验方剂学杂志

Objective::Astragalus membranaceus var. mongholicus and A. membranaceus are medicinal Astragalus, which are closely related and similar in composition, but with unclear medicinal value. Water-soluble protein profiles for A. membranaceus var. mongholicus and A. membranaceus were established to explore the differences between the two kinds of Astragalus Radix. Method::The water-soluble protein components were obtained through water ultrasonic extraction and acetone precipitation. After digested with trypsin, the obtained peptides were analyzed by nano ESI-LC-MS/MS method. Proteome Discovery 1.4 software was used to identify the proteins by comparing with the legume protein database, and the different expression water-soluble proteins were analyzed by the label-free quantitative software SIEVE. Finally, relevant information for common expression proteins, including classification, molecular function, involved biological process and signaling pathway, were analyzed by bioinformatics. Result::There were 920 and 717 specific proteins identified for A. membranaceus var. mongholicus and A. membranaceus, respectively. Totally 472 proteins were found to be co-expressed, in which 21 were differentially expressed, such as PR-10 protein, NDK-1 protein, glutelin A2, and phospholipase D. There were 14 highly expressed proteins in A. membranaceus var. mongholicus and 7 highly expressed proteins in A. membranaceus. Conclusion::There are significant differences in water-soluble protein profiles for two kinds of Astragalus Radix. Specific proteins, differentially expressed proteins and common expressed proteins can provide references for the identification of A. membranaceus var. mongholicus and A. membranaceus. It also can be used to define pharmacological mechanisms and search for drug action targets.

Effect of Transplanting Densities on Growth, Development, Yield and Quality of Astragalus membranaceus var. mongholicus / 中国实验方剂学杂志

Objective::Astragali Radix is an important medicinal and edible herb. To achieve standardized cultivation of Astragali Radix and improve cultivation results, Astragalus membranaceus var. mongholicus was cultivated with a row spacing of 30 cm and different plant spacing (8, 10, 12, 14, 16 cm) in a test base of Longxi County, Gansu Province. Method::The growth and development dynamics of green strains and the yield and quality of medicinal materials were measured, and the comprehensive evaluation of membership function was used to determine the rational transplanting density and explore the standard production technology of A. membranaceus var. mongholicus. Result::The transplanting topping had a significant effect on the growth and development of A. membranaceus var. mongholicus under the condition of the row spacing of 30 cm. With the increase of topping, the biomass of astragalus on the ground decreased, and the ratio of root to shoot increased, but the yield of medicinal material per unit area decreased. The appearance traits were improved as the transplant density decreased. Under the row spacing of 14 cm, the content of astragaloside and calycosin-7-glucoside were the highest. Under the row spacing of 8-16 cm, the content of root extract of Astragali Radix was better than the Chinese Pharmacopoeia standard. The comprehensive evaluation index of each plant spacing treatment was 14 cm>16 cm>10 cm>8 cm>12 cm in turn. Conclusion::Combined with the comprehensive evaluation of yield and economic benefit, the optimal transplanting plant spacing and row spacing were 30 cm×14 cm (with the density of 238 100 strains/hm2). Under this density, A. membranaceus var. mongholicus grows vigorously, with thick stems, big root crowns, a high yield and high quality.

Comparative analysis of twenty-five compounds in different parts of Astragalus membranaceus var. mongholicus and Astragalus membranaceus by UPLC-MS/MS / 药物分析学报

As a traditional Chinese medicine, the root of Astragalus membranaceus var. mongholicus (AMM) or A. membranaceus (AM) has been widely used in China and other Asian countries for thousands of years. Till now, the flavonoids, phenolic acids and saponins are considered as the main active components contributing to their therapeutic effect in these plants. In order to clarify the distribution and contents of these compounds in different organs of these plants, a rapid and sensitive analytical method for simultaneous determination of 25 active compounds including seven types (i.e. dihydroflavones, iso-flavane, isoflavones, flavones, pterocarpans, phenolic acid and saponins) within 10 min was established using ultra-pressure liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Then, the established method was fully validated and successfully applied to the determination of the contents of these analytes in different parts (root, rhizome, stem, leaf and flower) of AMM and AM. The results indicated that the contents of the same type of compounds in two different species plants were significantly different. Moreover, the obvious differences were also found for the distribution and con-tents of different type of compounds in five organs of the same species. The present study could provide necessary information for the rational development and utilization of AMM and AM resource.

Analysis on SSR information in transcriptome of Astragalus membranaceus var. mongholicus and its polymorphism / 中国中药杂志

In order to enrich the library of SSR and provide more powerful tools for molecular marker-assisted breeding in Astragalus membranaceus var. mongholicus, simple sequence repeats (SSR) loci in its transcriptome were searched in 18 040 unigenes (>=1 kb) by using MISA. SSR loci information was analyzed and SSR primers were designed by Primer 3. Furthermore, 110 pairs of primers were randomly selected for the polymorphic analysis on 20 plants collected from different habitats. A total of 5 640 SSRs were found in the transcriptome of A. membranaceus var. mongholicus, distributed in 4 462 unigenes with the distribution frequency of 31.26%. SSR loci occurred every 6 514 bp. Mono-nucleotide repeat was the main type, accounted for as much as 36.72% of all SSRs, followed by tri-nucleotide(32.57%) and di-nucleotide(27.73%) repeat motif. Among all 75 repeat types, A/T(2 026) was the predominant one followed by AG/CT(1 179), AAG/CTT(477). For validating the availability of the SSR primers designed using Primer 3, 110 pairs of primers were randomly selected for PCR amplification. Among them, 97 pairs of primers (88.18%) produced clear and reproductive bands. Using 19 pairs of primers showed polymorphism, 20 plants were divded into two groups by UPGMA. There are numerous SSRs in A. membranaceus var. mongholicus transcriptome with high frequency and various types, this will provide the abundant candidate molecular markers for genetic diversity, molecular identification, and marker-assisted breeding study for this plant.

Evolution and characteristics of system,assessing quality by distinguishing features of traditional Chinese medicinal materials, of Dao-di herbs of Astragali Radix / 中国中药杂志

"Assessing the quality by distinguishing features of traditional Chinese medicinal materials" is a characteristic quality evaluation system of traditional Chinese medicine, and it is also the basis of "Rating according to characters and setting the price by the grade" on the market. Astragali Radix was regarded as a famous traditional Chinese medicine (TCM), and this paper has carried out herbal textual research on the development and formation of the concept, "assessing the quality by distinguishing features of traditional Chinese medicinal materials", of Astragali Radix. The authentic medicine producing areas of Astragalus in China have experienced a great change, Gansu , Sichuan and adjacent areas before the Tang Dynasty; Shanxi during the Tang and Song Dynasty. The concept, "assessing the quality by distinguishing features of traditional Chinese medicinal materials", of Astragali Radix was formed in the Song and Ming Dynasty and still used today, which described as that the shape is "straight as an arrow"; the texture is "soft as cotton"; the section looks like" gold well and jade hurdle"; it was sweet in taste and has beany flavor. The system, "assessing the quality by distinguishing features of traditional Chinese medicinal materials", of Astragali Radix has undergone the adjustments from "true or false" to "good or bad", advance with the times, pick out the advantages from others and absorb the experience of traditional identification actively. Besides, it always returns to laconism from erudition and was summarized highly. Assessing the quality by distinguishing features of traditional Chinese medicinal materials and commodity specifications have the same root, so the former has reference meaning to revise the latter.

Review of Astragali Radix / 中草药·英文版

Astragali Radix (AR), known as Huangqi in China, is one of the most popular herbal medicines learnt worldwide to reinforce Qi (the vital energy). AR is traditionally prepared from the dried roots of Astragalus membranaceus or A. membranaceus var. mongholicus. It has been reported to have cardiotonic, hepatoprotective, hypotensive, immunostimulant, anti-aging, anti-oxidative, antidiabetic, and anti-inflammatory activities. The bioactive compounds were found to be flavonoids, saponins, polysaccharides, amino acids, and some trace elements. The present paper reviews the studies on AR including history, phytochemistry studies, pharmacological functions, and clinical application in recent years.