Quality Analysis of Different Specification Grades of Astragalus membranaceus var. mongholicus (Huangqi) from Hunyuan, Shanxi.

Background: Huangqi is a famous Chinese medicinal material whose Dao-di producing area is Hunyuan, Shanxi. Huangqi produced in Hunyuan, Shanxi, were divided into several different specifications and grades according to the diameters and different positions of root system. Objective: This article investigates the quantitative characteristics of chemical compositions in different specifications and grades of Astragalus membranaceus var. mongholicus roots, aiming to elucidate the correlation between specifications and/or grades and chemical compositions in Huangqi. Methods: Based on the field investigation, samples of Huangqi collected from Hunyuan, Shanxi, were divided into different specifications and grades. The content of seven flavonoids and five saponins in Astragalus membranaceus var. mongholicus roots of different specifications and grades were determined simultaneously by HPLC-diode-array detection-evaporative light-scattering detection (HPLC-DAD-ELSD). Results: Huangqi was processed by traditional methods, and its commercial specification was classified by different parts of the root system, such as ge-da-tou, hong-lan-qi, zheng-bai-qi, fu-bai-qi, mao-wei-zi, and qi-jian. The total content of seven flavonoids and five saponins in ge-da-tou, qi-jian were lower. The total content of seven flavonoids in hong-lan-qi was much higher, while that of five saponins was much lower. The total content of seven flavonoids in lateral roots or fibrous roots were higher, and that of five saponins was lower, such as zheng-bai-qi, fu-bai-qi, and mao-wei-zi. According to the root diameters, Huangqi was classified to special grade, grade I, grade II, grade III, grade IV, or grade V. Among six grades of Huangqi, the total content of seven flavonoids in grade III, grade IV, and grade V were lower, while the total content of five saponins in them were much higher. Conclusions: There is an obvious difference on the distribution pattern of contents of seven flavonoids and five saponins in Huangqi of different specifications and grades, which provide a certain scientific basis for the quality evaluation of Huangqi. Highlights: The content of seven flavonoids and five saponins in Huangqi were determined by HPLC-DAD-ELSD. The relationship between the commercial specification grades and chemical components of Dao-di herbs Astragalus membranaceus var. mongholicus (Huangqi ) from Hunyuan, Shanxi were revealed, which provided a chemical basis for the classification of commercial specification grades of dao-herbs Astragalus membranaceus var. mongholicus (Huangqi ) from Hunyuan, Shanxi.

Nuclear magnetic resonance based metabolomic differentiation of different Astragali Radix.

Astragali Radix (AR) is one of the most popular herbal medicines in traditional Chinese medicine (TCM). Wild AR is believed to be of high quality, and substitution with cultivated AR is frequently encountered in the market. In the present study, two types of ARs (wild and cultivated) from Astragalus membranaceus (Fisch.) Bge. and A. membranaceus var. mongholicus (Bge.) Hsiao, growing in different regions of China, were analyzed by NMR profiling coupled with multivariate analysis. Results showed that both could be differentiated successfully and cultivation patterns or growing years might have greater impact on the metabolite compositions than the variety; the metabolites responsible for the separation were identified. In addition, three extraction methods were compared and the method (M1) was used for further analysis. In M1, the extraction solvent composed of water, methanol, and chloroform in the ratio of 1 : 1 : 2 was used to obtain the aqueous methanol (upper layer) and chloroform (lower layer) fractions, respectively, showing the best separation. The differential metabolites among different methods were also revealed. Moreover, the sucrose/glucose ratio could be used as a simple index to differentiate wild and cultivated AR. Meanwhile, the changes of correlation pattern among the differential metabolites of the two varieties were found. The work demonstrated that NMR-based non-targeted profiling approach, combined with multivariate statistical analysis, can be used as a powerful tool for differentiating AR of different cultivation types or growing years.

Distinguishing Astragalus mongholicus and Its Planting Soil Samples from Different Regions by ICP-AES.

"Daodi herb" enjoys a good reputation for its quality and clinical effects. As one of the most popular daodi herbs, Astragalus membranaceus (Fisch.) Bge var. mongholicus (Bge.) Hsiao (A. membranaceus) is popularly used for its anti-oxidant, anti-inflammatory and immune-enhancing properties. In this study, we used inductively coupled plasma atomic emission spectrometry (ICP-AES) technique to investigate the inorganic elements contents in A. mongholicu and its soil samples from daodi area (Shanxi) and non-daodi areas (Inner Mongolia and Gansu). A total of 21 inorganic elements (Pb, Cd, As, Hg, Cu, P, K, Zn, Mn, Ca, Mg, Fe, Se, B, Al, Na, Cr, Ni, Ba, Ti and Sr) were simultaneously determined. Principal component analysis (PCA) was performed to differentiate A. mongholicu and soil samples from the three main producing areas. It was found that the inorganic element characteristics as well as the uptake and accumulation behavior of the three kinds of samples were significantly different. The high contents of Fe, B, Al, Na, Cr and Ni could be used as a standard in the elements fingerprint to identify daodi and non-daodi A. Mongholicus. As the main effective compounds were closely related to the pharmacodynamics activities, the inter-relationships between selected elements and components could reflect that the quality of A. Mongholicus from Shanxi were superior to others to a certain degree. This finding highlighted the usefulness of ICP-AES elemental analysis and evidenced that the inorganic element profile can be employed to evaluate the genuineness of A. mongholicus.

Intraspecific and heteroplasmic variations, gene losses and inversions in the chloroplast genome of Astragalus membranaceus.

Astragalus membranaceus is an important medicinal plant in Asia. Several of its varieties have been used interchangeably as raw materials for commercial production. High resolution genetic markers are in urgent need to distinguish these varieties. Here, we sequenced and analyzed the chloroplast genome of A. membranaceus (Fisch.) Bunge var. mongholicus (Bunge) P.K. Hsiao using the next generation DNA sequencing technology. The genome was assembled using Abyss and then subjected to gene prediction using CPGAVAS and repeat analysis using MISA, Tandem Repeats Finder, and REPuter. Finally, the genome was subjected phylogenetic and comparative genomic analyses. The complete genome is 123,582 bp long, containing only one copy of the inverted repeat. Gene prediction revealed 110 genes encoding 76 proteins, 30 tRNAs, and four rRNAs. Five intra-specific hypermutation loci were identified, three of which are heteroplasmic. Furthermore, three gene losses and two large inversions were identified. Comparative genomic analyses demonstrated the dynamic nature of the Papilionoideae chloroplast genomes, which showed occurrence of numerous hypermutation loci, frequent gene losses, and fragment inversions. Results obtained herein elucidate the complex evolutionary history of chloroplast genomes and have laid the foundation for the identification of genetic markers to distinguish A. membranaceus varieties.

Microscopic research on a multi-source traditional Chinese medicine, Astragali Radix.

Astragali Radix is a widely and commonly used Chinese herbal medicine, which is derived from roots of Astragalus membranaceus var. mongholicus and Astragalus membranaceus. To find a quick and reliable method of distinguishing these two species of Astragali Radix and of determining the age of a sample, microscopic characteristics of the two species were compared using light microscopy. The results showed that the microscopic characteristics, such as number of layers of phellem, continuing lignified xylem bundles within spring wood and lignified parenchyma cells in the central part of the xylem could be used for the differentiation of the root of A. membranaceus from the root of A. membranaceus var. mongholicus. Growth rings (annual rings) were found for the first time in the roots of both species, and could determine the age of a sample. For the first time, radial fibers in both species of Astragali Radix and pipette-shaped fibers in A. membranaceus var. mongholicus were found. The structure of "rotten heart" cork tissue (decayed central xylem) and tubular cork tissue was carefully studied, and the arranged order of tissues in both "rotten heart" and tubular cork tissues is phelloderm and phellem from outside to inside, which is contrary to that in the periderm.

[Between Hengshanhuangqi and Chuanhuangqi based on metabolomics and ITS2 sequences].

To compare the differences between Hengshanhuangqi (HH) and Chuanhuangqi (CH) at molecular level, 1H NMR based plant metabolomics approach was used to reveal the chemical difference between HH and CH. Then, the contents of astragaloside IV and calycosin-7-O-beta-D-glucoside, the marker compounds specified in China Pharmacopoeia, were determined. In addition, the ITS2 fragments of HH and CH were sequenced. Twenty-three metabolites were identified in the 1H NMR spectrum, and the principal component analysis showed CH and HH could be separated clearly. HH contained more aspartic acid, GABA, citric acid, astragaloside IV and calycosin-7-O-beta-D-glucoside, while CH contained more threonine, alanine, acetic acid, choline, arginine, fructose and sucrose. And the astragaloside IV is almost undetectable in CH. In addition, the ITS2 fragment sequences of HH and CH were different at eight bases. Thus, the HH and CH showed significant differences chemically and genetically.

[Molecular identification of astragali radix and its adulterants by ITS sequences].

OBJECTIVE: To explore a new method for identification Astragali Radix from its adulterants by using ITS sequence. METHOD: Thirteen samples of the different Astragali Radix materials and 6 samples of the adulterants of the roots of Hedysarum polybotrys, Medicago sativa and Althaea rosea were collected. ITS sequence was amplified by PCR and sequenced unidirectionally. The interspecific K-2-P distances of Astragali Radix and its adulterants were calculated, and NJ tree and UPGMA tree were constructed by MEGA 4. RESULT: ITS sequences were obtained from 19 samples respectively, there were Astragali Radix 646-650 bp, H. polybotrys 664 bp, Medicago sativa 659 bp, Althaea rosea 728 bp, which were registered in the GenBank. Phylogeny trees reconstruction using NJ and UPGMA analysis based on ITS nucleotide sequences can effectively distinguish Astragali Radix from adulterants. CONCLUSION: ITS sequence can be used to identify Astragali Radix from its adulterants successfully and is an efficient molecular marker for authentication of Astragali Radix and its adulterants.

[A taxonomic study on the original plant of radix astragali].

There are dispute about the status of taxonomy among Astragalus membranaceus (Fisch.) Bge, A. membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao. and A. pallidipurpureus stat. nov. The varieties and taxa of the complex are still in need of revision. With molecular biology study used trnH-psbA intergenic region, the taxonomic revision of Radix Astragali has been made. A. pallidipurpureus stat. nov is suggested as a new species.

Molecular identification of Astragalus membranaceus at the species and locality levels.

Astragalus membranaceus (Fisch.) Bunge, a commonly used Chinese medicinal material, from certain localities contains more favourable trace elements and fewer harmful trace elements than those from other localities. Therefore, there is a need to distinguish Astragalus membranaceus from different localities. Internal transcribed spacer 1 (ITS1) of the nuclear ribosomal RNA gene of 23 Astragalus membranaceus samples were sequenced to confirm the species of the samples. Arbitrarily primed polymerase chain reaction (APPCR) was then used to obtain unique fingerprints for each sample using several primers. The presence and absence of bands were used for calculating mean similarity indexes among the samples. It was found that the Heilongjiang samples were markedly distinguishable from samples of other localities. In addition, bands common for samples from the same locality were also identified and used to distinguish samples from Neimengu and Shanxi. Therefore, Astragalus membranaceus from these provinces, the major cultivation places in China, can be differentiated.

[Identification of the habitats of Radix Astragali using the correlative characteristic peaks selected by rough sets].

OBJECTIVE: Correlative characteristic peaks of HPLC selected by rough sets were employed to identify the habitats of Radix Astragali. METHOD: Using HPLC fingerprints of Radix Astragali sampled from different sources as examples, the reduct of attributes was achieved via rough sets, and therefore characteristic peaks correlated to identification of habitats were selected. Hierarchical cluster analysis was served for identifying the habitats of Radix Astragali. RESULTS: The ability of identification only using correlative characteristic peaks is more powerful than the one of whole peaks collected from HPLC fingerprints, and satisfied prediction for unknown samples is achieved. CONCLUSION: Correlative characteristic peaks obtained from rough sets can be regarded as a distinctive foundation for identifying the habitats of Radix Astragali.

Application of 3' untranslated region (UTR) sequence-based amplified polymorphism analysis in the rapid authentication of Radix astragali.

Radix astragali (root of Astragalus membranaceus) is an important traditional Chinese medicine. It has been used as a tonic herb for thousands of years in China. The water extract of the roots has a wide range of immunopotentiating effects and has been proven to be efficacious as an adjunct cancer therapy. Authentication of the herbal plant is routinely required for general practice in the field of herbal medicine. To facilitate rapid identification of numerous varieties of Radix astragali that are circulating on the herb markets, a rapid molecular genetic method, named 3' untranslated region (3' UTR) sequence-based amplified polymorphism (UAP), has been developed. A cDNA library was first built from transcripts of an authentic A. membranaceus species. Several cDNA clones specific to A. membranaceus were identified through subtractive hybridization of the A. membranaceus cDNA library with Arabidopsis total cellular RNA. On the basis of these cDNA sequences of the 3' untranslated region (3' UTR) of selected cDNA clones, a Polymerase Chain Reaction (PCR) was performed on genomic DNAs of the dry roots of several putative A. membranaceus. PCR fragment length polymorphism was found between A. membranaceus and its relatives. By using this method, it was possible to differentiate the authentic A. membranaceus root from those putative ones obtained from herbal medicine markets. To the authors' knowledge, this is the first paper applying UAP in the authentication of traditional Chinese medicine plants.

[Determination of calycosin-7-O-beta-D-glucopyranoside in radix astragali by HPLC].

OBJECTIVE: TO establish a RP-HPLC method for the determination of calycosin-7-O-beta-D-glucopyranoside in Radix Astragali, and to analyse the calycosin-7-O-beta-D-glucopyranoside content of ten samples of Radix Astragali, collected from different regions. METHOD: A Polaris C18(250 mm x 4.6 mm, 5 microns) column was used and a mixture of methanol-water (30:70) was used as the mobile phase at a flow rate of 1.0 mL.min-1. The column temperature was 25 degrees C and the UV detection wavelength was 254 nm. RESULT: The calibration curve was in good linearity over the range of 0.0106-2.12 micrograms with the regression equation Y = 3035. 97 X - 14.85(r = 0.9999). The average recovery was 95.8% (n = 5, RSD = 1.3%). CONCLUSION: The method is simple, quick, sensitive and reproducible. In all of the samples, the calycosin-7-O-beta-D-glucopyranoside contents differ markedly.