Ecological niche modeling of Astragalus membranaceus var. mongholicus medicinal plants in Inner Mongolia, China.

Radix Astragali is commonly used in traditional Chinese medicine, and its quality is closely related to ecological factors, such as climate and soil, in the production area. To provide high-quality Radix Astragali to Chinese and foreign markets, we used maximum entropy model and statistical analysis method, combined with data on ecological factors, Astragalus membranaceus var. mongholicus geographical distribution, and index component content to predict the ecological suitability distribution of A. membranaceus var. mongholicus and establish the relationship between astragaloside IV and calycosin-7-glucoside in this species and ecological factors. Subsequently, we could determine the suitability regionalization of high-quality A. membranaceus var. mongholicus in Inner Mongolia, China. The results showed that the standard deviation of seasonal changes in temperature (40.6%), precipitation in October (15.7%), vegetation type (14.3%), soil type (9.2%), and mean sunshine duration in the growing season (9.1%) were the top five most influential factors out of the 17 main ecological factors affecting the distribution of A. membranaceus var. mongholicus. The standard deviation of seasonal changes in temperature, precipitation in October, precipitation in April, soil pH, and mean sunshine duration in the growing season were found to be the key ecological factors affecting the accumulation of astragaloside IV and calycosin-7-glucoside in A. membranaceus var. mongholicus. The regions with the highest-quality A. membranaceus var. mongholicus were distributed in Baotou (Guyang County), Hohhot (Wuchuan County), and central Wulanchabu (Chahar Right Middle Banner, Chahar Right Back Banner, and Shangdu County) and its surroundings in Inner Mongolia. Baotou, Hohhot, and their surrounding areas were the main traditional production areas of A. membranaceus var. mongholicus, and central Wulanchabu was a potentially suitable distribution area of this species. The main production areas were consistent with the actual production base of A. membranaceus var. mongholicus. This study therefore provides a scientific basis to guide the cultivation of A. membranaceus var. mongholicus.

Transcriptome sequencing and characterization of Astragalus membranaceus var. mongholicus root reveals key genes involved in flavonoids biosynthesis.

BACKGROUND: Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao is a traditional medicinal herb of Leguminosae since it contains bioactive compounds such as flavonoids, which have significant pharmacological effects on immunity and antioxidant. However, the scanty genomic and transcriptome resources of Astragalus membranaceus have hindered further exploration of its biosynthesis and accumulation mechanism. OBJECTIVE: This project aim to further improve our understanding of the relationship between transcriptional behavior and flavonoids content of A. mongholicus. METHODS: The accumulation of flavonoids and related gene expression in five different developmental stages (A: vegetative, B: florescence, C: fruiting, D: fruit ripening and E: defoliating stages) of A. mongholicus root were studied by combining UV spectrophotometry and transcriptomic techniques. The de novo assembly, annotation and functional evaluation of the contigs were performed with bioinformatics tools. RESULTS: After screening and assembling the raw data, there were a total of 158,123 unigenes with an average length of 644.89 bp were finally obtained, which has 8362 unigenes could be jointly annotated by NR, SwissProt, eggNOG, GO, KEGG and Pfam databases. KEGG enrichment analysis was performed on differentially expressed genes(DEGs)in the four groups (A vs. B, B vs. C, C vs. D, D vs. E). The results showed that many DEGs in each group were significantly enriched to flavonoids biosynthesis related pathways. Among them, a number of 86 were involved in the biosynthesis of isoflavonoid (12), flavonoid (5) and phenylpropanoid (69). Further analysis of these DEGs revealed that the expression levels of key genes such as PAL, 4CL, CCR, COMT, DFR, etc. were all down-regulated at the fruiting stage, and then raised at the fruit ripening stage. This expression pattern was similar to the accumulation trend of total flavonoids content. CONCLUSIONS: In summary, this comprehensive transcriptome dataset allowed the identification of genes associated with flavonoids metabolic pathways. The results laid a foundation for the biosynthesis and regulation of flavonoids. It also provided a scientific basis for the most suitable harvest time and resource utilization of A. mongholicus.

Discovery of chemical markers for identifying species, growth mode and production area of Astragali Radix by using ultra-high-performance liquid chromatography coupled to triple quadrupole mass spectrometry.

BACKGROUND: Astragali Radix (AR) is a well-known Chinese herbal medicine. The quality of AR can be affected by many factors such as species, growth mode and production area, but there are still no chemical markers to distinguish it. PURPOSE: To explore chemical markers for improving the quality assessment of AR and discover chemical markers for identifying species, growth mode and production area of AR. METHODS: A highly sensitive, efficient and accurate method based on ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) for simultaneous quantitative determination of 14 major chemical components (five flavonoids and nine triterpene saponins) in 94 batches of AR from China, Republic of Korea and Germany was developed for the first time. To explore chemical markers and assess changes in the contents of 14 compounds in the 94 batches of AR samples from different regions, hierarchical clustering analysis (HCA) and principal component analysis (PCA) were performed. RESULTS: Astragaloside III was not only an important chemical marker for distinguishing two species of AR, i.e.: Astragalus mongholicus and A. membranaceus, but also a potential chemical marker for the classification of cultivated and semi-wild AR. In addition, in the batches of cultivated AR, the content of isoastragaloside II and cyclocephaloside II were greater in batches from the region of Shaanxi Province than that of other Provinces in China, but the content of calycosin-7-O-ß-D-glucoside and astragaloside IV, which are the quality control markers of AR required by the Chinese Pharmacopoeia, were higher than that of other Provinces in China. In addition, the content of calycosin-7-O-ß-D-glucoside, ononin, calycosin and astragaloside I could be used to identify samples of AR collected from China, Republic of Korea and Germany. CONCLUSION: This UHPLC-QQQ-MS/MS method could be applied to the quantitative evaluation of AR and could be an important and meaningful reference to develop chemical markers for quality control of AR.

Enhanced Production of Two Bioactive Isoflavone Aglycones in Astragalus membranaceus Hairy Root Cultures by Combining Deglycosylation and Elicitation of Immobilized Edible Aspergillus niger.

A cocultivation system of Astragalus membranaceus hairy root cultures (AMHRCs) and immobilized food-grade fungi was established for the enhanced production of calycosin (CA) and formononetin (FO). The highest accumulations of CA (730.88 ± 63.72 µg/g DW) and FO (1119.42 ± 95.85 µg/g DW) were achieved in 34 day-old AMHRCs cocultured with immobilized A. niger (IAN) for 54 h, which were 7.72- and 18.78-fold higher than CA and FO in nontreated control, respectively. IAN deglycosylation could promote the formation of CA and FO by conversion of their glycoside precursors. IAN elicitation could intensify the generation of endogenous signal molecules involved in plant defense response, which contributed to the significantly up-regulated expression of genes in CA and FO biosynthetic pathway. Overall, the coupled culture of IAN and AMHRCs offered a promising and effective in vitro approach to enhance the production of two health-promoting isoflavone aglycones for possible nutraceutical and pharmaceutical uses.

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In order to explore the ecological factors affecting the growth of Astragalu smembranaceus var. mongholicus, we investigated the resource distribution, habitat characteristics and growth conditions of wild and cultivated A. membranaceus var. mongholicus by fixed-plot observation, survey method, and literature. These data were analyzed by traditional Chinese medicine GIS-2 (TCMGS-2) to obtain the most suitable areas of A. membranaceus var. mongholicus within Inner Mongolia. The results showed that the production areas of cultivated A. membranaceus var. mongholicus were mainly located in Wuchuan County, Guyang County and other 15 counties, which were cha-racterized by the altitude higher than 1000 m, with soil type of sand, gravel and calcareous clay. The wild A. membranaceus var. mongholicus was distributed mainly in the eastern Inner Mongolia and germinated in sunny place, which preferred to the cold dry climate and sandy loam soil or gra-vel but avoided damp heavy clay soils. There are 43 counties of 94460.30 km2 for wild A. membranaceus var. mongholicus, and 32 counties of 76013.93 km2 for cultivated one within Inner Mongolia, with a similarity coefficient of ecological factors greater than 95%.

[Investigation on Main Cultivation of Astragalus membranaceus var. mongholicus and Commercial Specifications Grades].

OBJECTIVE: To investigate the main cultivation areas of Astragalus membranaceus var. mongholicus, and to clarify its present cultivation status and commercial specification grades. METHODS: Astragalus membranaceus var. mongholicus cultivated in Hunyuan and Ying County of Shanxi, Longxi, Weiyuan and Min County of Gansu and Wuchuan, Guyang and Chifeng of Inner Mongolia were investigated, including their ecological environment, cultivation history and present situation, harvesting and processing as well as commercial specification grades. RESULTS: The cultivation methods of Astragalus membranaceus var. mongholicus changed greatly in the main cultivation areas. In Gansu and Inner Mongolia, the current major cultivation method was 2 - 3 years of seedling transplantation method, while the 3 - 5 years of seed-directly-sowed method in Inner Mongolia had been almost abandoned, and the 6 - 8 years of mountain direct seeding method was still in application in Hunyuan county of Shanxi province. On the other hand, the harvest method of Astragalus membranaceus var. mongholicus had been gradually changed from manual harvesting in ancient times to the current mechanical harvesting although the latter method did more damage to environment of cultivation areas and the resource of Astragalus membranaceus var. mongholicus in regions Hunyuan county of Shanxi. Through investigating the processing methods of Astragalus membranaceus var. mongholicus in different cultivation areas, it was found that processing method varied from area to area, and no unified specifications had been formulated on processing methods in market. In addition, the current processing method was too complicated in processing the slices of prepared Astragali Radix and most were self processing by farmers themselves, creating market disorders. Moreover, the Astragali Radix products were basically without grading classification but sold as bulk cargo. CONCLUSION: It is urgent to unify the specifications and grades of Astragali Radix product, and standardize its processing methods.

Efficient production of isoflavonoids by Astragalus membranaceus hairy root cultures and evaluation of antioxidant activities of extracts.

In this study, Astragalus membranaceus hairy root cultures (AMHRCs) were established as an attractive alternative source for the efficient production of isoflavonoids (IF). A. membranaceus hairy root line II was screened as the most efficient line and was confirmed by PCR amplification of rolB, rolC and aux1 genes. Culture parameters of AMHRCs were systematically optimized, and five main IF constituents were quali-quantitatively determined by LC-MS/MS. Under optimal conditions, the total IF accumulation of 34 day old AMHRCs was 234.77 µg/g dry weight (DW). This yield was significantly higher compared to that of 3 year old field grown roots (187.38 µg/g DW). Additionally, in vitro antioxidant assays demonstrated that AMHRC extracts exhibited antioxidant activities with lower IC50 values (1.40 and 1.73 mg/mL) as compared to those of field grown roots (1.96 and 2.17 mg/mL). Overall, AMHRCs may offer a promising and continuous product platform for naturally derived, high quality and valuable nutraceuticals.

[Study on method and its optimization of improving seed germination of Astragalus membranaceus as gansu traditional medicinal herb].

OBJECTIVE: To break the hard testa and improve seed germination situation of Astragalus membranaceus var. mongholicus, in order to solve the problems of low success rate of seed germination and seedling. METHODS: Longxi Astragalus membranaceus var. mongholicus seed was treated by soaking seed with 75% alcohol and concentrated sulfuric acid, warm-water incubating, grinding and comprehensive treating with warm-water incubating, grinding and sand culture. Its seed germination situation was evaluated by germination potential, germination rate and germination index. RESULTS: Different processing methods significantly improved seed germination with different effect. Comprehensive treatment with warm-water incubating, grinding and sand culture was the best one on Astragalus membranaceus var. mongholicus seed germination. Its germination potential, germination rate and germination index was 66.04%, 87.70% and 1.34,respectively. CONCLUSION: Comprehensive treatment with warm-water incubating, grinding and sand culture is an economic and effective processing method, which is suitable for actual production.

[Autotoxicity of aqueous extracts from plant of cultivated Astragalus membranaceus var. mongholicus].

OBJECTIVE: To exploring the relationship between continuous cropping obstacle and autotoxicity of Astragalus membranaceus var. mongholicus, autotoxic effect of plant aqueous extract were determined. METHODS: Distilled water (CK), aqueous extract of plant, including root, stem and leaf (12.5, 25, 50 and 100 mg/mL respectively)were applied to testing their effect on early growth of Astragalus membranaceus var. mongholicus. Specifically, seed germination rate, germination index, emergence rate, elongation of radical and embryo, and seedling vigor index were determined. RESULTS: The aqueous extract of root, stem, and leaf at 25 mg/mL significantly inhibited the seed germination and seedling growth of Astragalus membranaceus var. mongholicus, and this inhibitory effect generally increased with the increase of the concentration of aqueous extracts. To the comprehensive allelopathic effect, the extracts from Astragalus membranaceus var. mongholicus stem were more inhibitory than those from leaf and root. The germination index and seedling vigor index were more sensitive to extract than other determined parameters. CONCLUSION: Aqueous extracts from Astragalus membranaceus var. mongholicus plant gave inhibitory effects on Astragalus. membranaceus var. mongholicus germination and seedling growth, and this inhibitory effect generally increased with the increases of aqueous extract concentration at a certain ranges. In conclusion, there is an autotoxicity in continuous cropping of Astragalus membranaceus var. mongholicus.

[Status and analysis of Astragali Radix resource in China].

To explore the status of the resources of Astragali Radix, a survey on its germplasm resources was carried out. Some conclusions can be drawn for Astragali Radix: the major source is the cultivated Astragalus mongolicus. The new major cultivation areas for A. mongolicus and A. membranaceus are Shandong and Gansu province. The semi-wildly planting model in Shanxi province maintains the genuine trait of Astragali Radix, but its yield is limited, and now a combination model has been developed. The major problems for Astragali Radix are the selection of planting sites, the rot root and difficulty in collecting and processing. Several developmental proposals for Astragali Radix were put forward including rational distribution of planting areas, establishment of standard system, development and standardization of producing technologies.

[Regulation of Vitreoscilla hemoglobin on biosynthesis of astragaloside IV].

In the present study, the regulation of Vitreoscilla hemoglobin (VHb) on astragaloside IV biosynthesis was investigated. An intermediate expression vector consisting of the CaMV35S promoter fused to the vgb and nopaline synthase terminator was transferred into Astragalus membranaceus via Agrobacterium rhizogenes. The transgenic hairy roots were confirmed by PCR amplification and Southern blot hybridization. The expression of vgb in transgenic hairy roots was confirmed by RT-PCR. After 15 days cultivation, the dry weight and growth rate of transgenic hairy roots were higher than that of the non-transgenic hairy root. ELSD-HPLC analysis showed that astragaloside IV content of transgenic hairy roots was 5 to 6 times of non-transgenic hairy root control and 10 to 12 times of Radix Astragali from Shanxi Province. These results suggested that the expression of vgb promoted the growth of transgenic hairy roots, and increased the content of astragaloside IV.

[Effects of nitrogen, phosphorus and potassium application on growth and active ingredient of Astragalus membranaceus].

OBJECTIVE: To study the effects of fertilizers with the different proportional of nitrogen, phosphorus and potassium on growth and active ingredient of Astragalus membranaceus. METHOD: Field experiment was conducted based on the D-saturation optimal design with three factors of nitrogen, phosphorus and potassium. The effects on growth and active ingredient of A. membranaceus were analyzed. RESULT: Fertilization promoted the seedling growth and provided abundant supply of nutrition for growth of root, yield and accumulation of active ingredient at the later growth stage, and increased the accumulation of dry matter of stem-leaf and root system. The effect of nitrogen, phosphorus and potassium application on the total dry matter accumulation of A. membranaceus was as following: nitrogen > potassium > phosphorus; the effect on the stem-leaf dry matter accumulation was as following: nitrogen > phosphorus > potassium; the effect on the root dry matter accumulation was as following: nitrogen > potassium > phosphorus. Nitrogen, phosphorus and potassium fertilizer increased the root yield of A. membranaceus. Obviously, the effect on the root yield was as following: nitrogen > potassium > phosphorus. The application of different proportional with nitrogen, phosphorus and potassium increased the content of polysaccharide and astragaloside, but had no distinct effect on the content of total flavonoids. The effect on the content of polysaccharide was as following: potassium > phosphorus > nitrogen, but the effect on the conten,t of astragaloside was as following: nitrogen > potassium > phosphorus. CONCLUSION: Nitrogen and potassium fertilizer application had more important effect on growth, yield and the contents of polysaccharide and astragaloside in A. membranaceus. During medicinal plants cultivation process, it should pay attention to the application of nitrogen fertilizer and potassium fertilizer and make balance application of nitrogen, phosphorus and potassium fertilizer.

[Study on difference of biological characteristics and resistance to powdery mildew of different Astragalus populations].

OBJECTIVE: To study difference among populations which belong to Astragalus membranaceus and A. membranaceus var. mongholicus on morphology, habit, characteristics of physiology and resistance to powdery mildew, and classify them in order to provide theoretical basis for breeding and improving varieties. METHOD: Morphology, habits, isozyme and soluble protein electrophoretograms were compared among the populations. They were categorized by cluster analysis based on those electrophoretograms. Different ability of resistance to powdery mildew was also studied through comparing disease indices among six populations. RESULT: The results showed A. membranaceus var. mongholicus was distinctly different from A. membranaceus. There was a special type in colonies of A. membranaceus, which showed hairy upper epidermis of leaflets and later florescence. CONCLUSION: Astragalus for medicine could be categorized in three types A. membranaceus var. mongholicus, A. membranaceus early florescence type and A. membranaceus late florescence type. Among them A. membranaceus var. mongholicus is most resistant to powdery mildew, while A. membranaceus is easily infected, and the early florescence type is even more easily infected.

[Quantitative analysis of calycosin glycoside and formononetin in Radix astragali from different sources].

OBJECTIVE: To establish the quantitative methods for calycosin glycoside and formononetin in Radix Astragali, and the samples from different sources were analyzed, in order to supply the basis for the quality control of Radix Astragali. METHOD: The content of calycosin glycoside and formononetin in 59 samples of Radix Astragali from eight with different provinces was analyzed by HPLC-DAD. RESULT: The contents of calycosin glycoside and formononetin in Radix Astragali from different sources, with different cultivating method or in different ages differed markedly, and the results showed that the quality of samples from Shannxi, Innermongolia and Shanxi were better than other sources, and the semi-wild samples were better than other cultiving samples, moreover the shorter age, the better quality. CONCLUSION: This simple, accurate and reproducible method could use to determine the contents of isoflavanoids in Radix Astragali.

[Study on accumulation of dry matter and aborption of N, P and K of Astragalus membranaceus].

OBJECTIVE: To study Astragalus membranaceus absorption characteristic of nitrogen, phosphorus and potassium at different growth stages. METHOD: Through the field experiment and the sampling analysis, the absorbing capacity of nitrogen, phosphorus and potassium as well as the growth of plant at different growth stages in A. membranaceus were analyzed. RESULT: The dry matter accumulation reached 88.22% of the total accumulation in 100-163 days after seedling emergence, The content of N, P, K in the stem was higher than that in the root system. Nitrogen, phosphorus and potassium absorbing capacity in the stem, the leaf and the root systems was N > K > P. In the whole growth period, nitrogen accumulation reached the highest, and followed with the accumulation of potassium, and the accumulation of phosphorus was the lowest. In the last phase of the exuberant growth period, the accumulation intensity of nitrogen, phosphorus and potassium reduced. During the harvesting time, the accumulation intensity of nitrogen and potassium increased, and the accumulation intensity of phosphorus remained stable. CONCLUSION: The dry matter accumulation reached the maximum in 100-163 days after seedling emergence. The absorption strength of nitrogen, phosphorus and potassium in aerial part reached the maximum in 100-132 days after seeding emergence, the maximum absorption comes earlier than that of phosphorus and potassium, at that time the needs of nutrients reach the highest. For producing of 100 kg A. membranaceus 2.32 kg N, 0.323 kg P2O5, 1.62 kg K2O are needed to be absorbed from soil and fertilizer.

[Standard operating procedure for Astragalus membranaceus].

In order to ensure the superior quality and safety of the raw materials of the Chinese herbal medicine, a standard operating procedure for astragalus was established on the base of the GAP of the Chinese herbal medicine as well as practice investigation and experiments. This standard operating procedure provides the technical requirements for astragalus's growing, field management, controlling of the diseases and pests, harvesting, processing, packing, storing, transporting and quality monitoring.

Astragaloside IV and polysaccharide production by hairy roots of Astragalus membranaceus in bioreactors.

Hairy roots of Astragalus membranaceus were grown in bioreactors up to 30 l for 20 d. Cultures from a 30 l airlift bioreactor gave 11.5 g l dry wt with 1.4 mg g(-1) astragaloside IV, similar to cultures from 250 ml and 1 l flasks, but greater than yields from a 10 l bioreactor (dry wt 9.4 g l(-1), astragaloside IV 0.9 mg g(-1)). Polysaccharide yields were similar amongst the different bioreactors (range 25-32 mg g(-1)). The active constituent content of the cells approached that of plant extracts, indicating that large scale hairy root cultures of A. membranaceus has the potential to provide an alternative to plant crops without compromising yield or pharmacological potential.

[Induction and identification of polyploid of Astragalus membranaceus var. mongholicus].

In this article, improved agar painting method, with semi-solid of 0.2% colchicine and 0.1% agar, was adopted to treat apical buds of Astragalus membranaceus var. mongholicus (Bge.) Hsiao seedlings. Obtained plants were proved to be tetraploids by identification of biological characteristics and chromosome numbers.

[Presearch on preventing the medicinal plant diseases with Trichoderma harzianum preparation].

OBJECTIVE: To control the medicinal plant diseases with the preparation of Trichoderma harzianum. METHOD: Antagonistic action of the preparation to the pathogens of the medicinal plants in vitro, and controling effects of the preparation on these diseases in greenhouse and in the field were tested. RESULT: The test in vitro showed that Trichoderma harzianum, used as a biocontrol factor, had stronger antagonistic action to Fusarium equiseti, Sclerotinia sp. and Rhizoctonia solani which were the medicinal plant pathogens of Astragalus membranaceus, Glehnia littoralis and Panax quinquefolium respectively. Biological controling effects on sclerotium root rot of Glehnia littoralis were 83.6% and 72.5% respectively in greenhouse and in the field with the preparation of Trichoderma harzianum. And controling effects on root rot of Astragalus membranaceus and seedling damping-off of Panax quinquefolium were 80% and 60% respectively in the field. The dosage of the preparation used in the field was 10 g.m-2. CONCLUSION: The preparation of Trichoderma harzianum can be used as a substitute for such chemicals as Carbendazim. Using the preparation to control medicinal plant diseases provides a technical safeguard for the good agricultural practice of medicinal plants.