Simultaneous Determination of Calycosin-7-O-β-d-Glucoside,Ononin, Calycosin,Formononetin, Astragaloside IV,and Astragaloside II in Rat Plasma After Oral Administration of Radix Astragali Extraction for Their Pharmacokinetic Studies by Ultra-Pressure Liquid Chromatography with Tandem Mass Spectrometry

A sensitive and reliable ultra-pressure liquid chromatography with tandem mass spectrometry (UPLC–MS) was developed and validated for simultaneous quantification of six main bioactive components, i.e., calycosin-7-O-β-d-glucoside, ononin, calycosin, formononetin, astragaloside IV, and astragaloside II in rat plasma after oral administration of the 95 % ethanol extraction from Radix Astragali. Plasma samples were extracted with Waters Oasis^TM HLB 1 cc (30 mg) Extraction Cartridges (SPE) separated on an UPLC? BEH C18 column and detected by MS with electro spray ionization interface in positive selective ion monitoring mode. Calibration curves offered linear ranges of two orders of magnitude with r ² > 0.99. The method had the lower limit quantification of 1.30, 0.73, 1.17, 2.33, 0.63, and 0.83 ng/mL for ononin, calycosin, calycosin-7-O-β-d-glucoside, formononetin, astragaloside IV, and astragaloside II, respectively, with precision less than 10 %. The RSD of intra- and inter-day variations ranged from 1.66 to 6.46 and 3.39 to 6.58 %. This developed method was applied subsequently to pharmacokinetic studies of the six compounds in rats successfully. The proposed method was for the first time to compare the pharmacokinetic difference between calycosin-7-O-β-d-glucoside and calycosin in rat plasma, so as between ononin and formononetin, and studied to the astragaloside II pharmacokinetics in rat plasma.     

Simultaneous determination of calycosin-7-O-beta-D-glucoside, ononin, astragaloside IV, astragaloside I and ferulic acid in rat plasma after oral administration of Danggui Buxue Tang extract for their pharmacokinetic studies by liquid chromatography-mass spectrometry

A sensitive and reliable high-performance liquid chromatography-mass spectrometry (HPLC-MS) was developed and validated for simultaneous quantification of five main bioactive components, i.e., calycosin-7-O-beta-D-glucoside, ononin, astragaloside IV, astragaloside I and ferulic acid in rat plasma after oral administration of Danggui Buxue Tang (DBT) extract. Plasma samples were extracted with solid-phase extraction (SPE) separated on an Inertsil ZORBAX C(18) column and detected by MS with electrospray ionization (ESI) interface in negative selective ion monitoring (SIM) mode. Calibration curves offered linear ranges of two orders of magnitude with r(2)>0.99. The method had the lower limit quantification of 0.55, 0.46, 1.07, 1.12 and 4.6 ng/mL for calycosin-7-O-beta-D-glucoside, ononin, astragaloside IV, astragaloside I and ferulic acid, respectively, with precision less than 10%. The RSD of intra- and inter-day variations ranged from 2.10% to 6.19% and 2.37% to 6.72%. This developed method was subsequently applied to pharmacokinetic studies of the five compounds in rats successfully.     

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

BackgroundAstragali 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.PurposeTo explore chemical markers for improving the quality assessment of AR and discover chemical markers for identifying species, growth mode and production area of AR.MethodsA 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.ResultsAstragaloside 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-β-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-β-D-glucoside, ononin, calycosin and astragaloside I could be used to identify samples of AR collected from China, Republic of Korea and Germany.ConclusionThis 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.     

Isolation and purification of flavonoid glucosides from Radix Astragali by high-speed counter-current chromatography

High-speed counter-current chromatography methods, combined with resin chromatography were applied to the separation and purification of flavonoid glycosides from the Chinese medicinal herb, Radix Astragali. Five flavonoid glycosides, namely calycosin-7-O-β-d-glucoside, ononin, (6aR, 11aR)-9,10-dimethoxypterocarpan-3-O-β-d-glucoside, (3R)-2′-hydroxy-3′,4′-dimethoxyisoflavan-7-O-β-d-glucoside and calycosin-7-O-β-d-glucoside-6′′-O-acetate, were obtained. Among them, calycosin-7-O-β-d-glucoside-6′′-O-acetate was preparatively separated from Radix Astragali for the first time. Their structures were identified by ESI–MS, ¹H NMR, ¹³C NMR, and 2D NMR.     

Metabolomics-based optimal koji fermentation for tyrosinase inhibition supplemented with Astragalus radix

The present study was focused on improving the quality of rice koji by fermentation with a selected Aspergillus oryzae strain and a plant Astragalus radix. A. oryzae KCCM 60345 was used as main inoculant and the Astragalus radix was added as supplement in rice koji preparation. LC-MS based metabolite analysis and tyrosinase inhibitory activities were studied for different time periods. A. oryzae KCCM 60345 fermented rice koji supplemented with Astragalus showed higher tyrosinase inhibition activity at 4 d of fermentation and metabolite analysis with PCA and PLS-DA indicated differences in kojic acid, calycosin-7-O-β-D-glucoside, ononin, calycosin, and formononetin as compared with other forms of rice koji fermentation. By correlation analysis between metabolites and tyrosinase inhibitory activity, calycosin and kojic acid were identified as major tyrosinase inhibitors. Based on these results, we concluded that A. oryzae KCCM 60345 supplemented with Astragalus radix is useful for whitening effects, and we identified optimal conditions for rice koji preparation.     

Ultraviolet irradiation induces accumulation of isoflavonoids and transcription of genes of enzymes involved in the calycosin-7-O-β-d-glucoside pathway in Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao

Isoflavonoids are a group of phenolic secondary metabolites found almost exclusively in leguminous plants. Formononetin, calycosin and calycosin-7-O-β-d-glucoside (CG) are isoflavonoid products in the CG pathway. Accumulation of the three isoflavonoids plus daidzein and expression of six genes of enzymes involved in the CG pathway were studied in Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao with ultraviolet (UV) irradiation. Our results showed that (1) main isoflavonoids in roots, stems and leaves were CG, daidzein and calycosin, respectively; they accumulated significantly under the induction of UV irradiation during 8 days but their content declined later; (2) expression of six genes of enzymes involved in the CG pathway was inhibited slightly at early stage but the expression was increased greatly afterward; (3) chalcone synthase, chalcone reductase and chalcone isomerase were expressed to their individual maximum level within shorter hours than were cinnamate 4-hydroxylase, isoflavone synthase (IFS) and isoflavone 3'-hydroxylase and (4) more calycosin but less daidzein accumulated in leaves. IFS was highly expressed in leaves, which might lead to high accumulation of the common precursor of daidzein and 2,7-dihydroxy-4'-O-methoxy-isoflavanone, the latter of which would be converted to formononetin, calycosin and CG via a series of reactions. Little daidzein accumulated in leaves, which suggested that rather than be converted to daidzein, the 2,7,4'-trihydroxyisoflavanone was probably more easily caught by 2-hydroxyisoflavanone 4'-O-methyltransferase and hence provided more precursors for formononetin. The findings were discussed in terms of the influence of UV irradiation in the accumulation of isoflavonoids.     

Effects of progressive drought stress on the physiology,antioxidative enzymes and secondary metabolites of Radix Astragali

The dried roots of Radix Astragali are widely used in traditional Chinese medicine, and flavonoids present in the root of this herb have been implicated in its bioactivity. We subjected 2-year-old Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao to a progressive drought stress over 14 days of water withholding and studied the physiological and biochemical responses and secondary metabolite accumulation. Results indicated that drought stress reduced relative water content, reduced yield, but increased electrolyte leakage, malondialdehyde, proline and soluble sugar content. Mild and moderate drought stress enhanced some antioxidative enzyme activity to protect plants from damaging, but antioxidative enzyme activity was limited by severe stress. The calycosin-7-O-β-d-glycoside and ononin content of plant roots was enhanced with degree of drought stress, whereas calycosin and formononetin levels did not differ significantly until 14 days. These results demonstrate that Radix Astragali can adapt to water stress by up-regulating antioxidant enzymes and accumulation of osmotic agents, and a certain degree of drought stress can enhance accumulation of some flavonoids, potentially facilitating higher yields of pharmacological activity of calycosin-7-O-β-d-glycoside production.     

Pharmacokinetics-based comprehensive strategy to identify multiple effective components in Huangqi decoction against liver fibrosis

BackgroundHuangqi decoction (HQD) has been used to treat chronic liver diseases since the 11th century, but the effective components in HQD against liver fibrosis have not been definitively clarified.PurposeTo investigate and identify multiple effective components in HQD against liver fibrosis using a pharmacokinetics-based comprehensive strategy.MethodsThe absorbed representative components in HQD and their metabolites were detected in human plasma and urine using high-resolution mass spectrometry combined with a database-directed method, and then pharmacokinetics in multiple HQD components in human plasma was analyzed by ultra-performance liquid chromatography coupled with triple-quadruple mass spectrometry. Furthermore, the anti-fibrotic effect of potential effective HQD components was studied in LX-2 cells and that of a multi-component combination of HQD (MCHD) was verified in a mouse CCl₄-induced hepatic fibrosis model.ResultsTwenty-four prototype components in HQD and 17 metabolites were identified in humans, and the pharmacokinetic characteristics of 14 components were elucidated. Among these components, astragaloside IV, cycloastragenol, glycyrrhizic acid, glycyrrhetinic acid, liquiritigenin, and isoliquiritigenin downregulated the mRNA expression of α-SMA; cycloastragenol, calycosin-7-O-β-D-glucoside, formononetin, glycyrrhetinic acid, liquiritin, and isoliquiritin downregulated the mRNA expression of Col I; and calycosin, liquiritigenin, isoliquiritigenin, cycloastragenol, and glycyrrhetinic accelerated the apoptosis of LX-2 cells. MCHD reduced serum aminotransferase activity and hepatic collagen fibril deposition in mice with CCl₄-induced hepatic fibrosis.ConclusionUsing the pharmacokinetics-based comprehensive strategy, we revealed that multiple effective HQD components act together against liver fibrosis.     

Interactions between thrombin and natural products of Millettia nitita var. hirsutissima using capillary zone electrophoresis

A sensitive and selective high-performance analytical method based on capillary zone electrophoresis (CZE) was developed for investigating interactions between natural products isolated from Millettia nitita var. hirsutissima and thrombin qualitatively and quantitatively for the first time. The results showed that, compared with positive and negative control, the compounds ZYY-5 (genistein-8-C-β-d-apiofuranosyl-(1→6)-O-β-d-glucopyranoside), ZYY-6 (calycosin), ZYY-8 (isoliquiritigenin), ZYY-9 (formononetin), ZYY-12 (gliricidin), ZYY-13 (8-O-methylretusin), FJ-2 (dihydrokaempferol), FJ-3 (biochanin), FJ-5 (afromosin) and XC-2 (hirsutissimiside F) interacted with thrombin, while ZYY-1 (sphaerobioside), ZYY-2 (formononetin-7-O-β-d-apiofuranosyl-(1→6)-O-β-d-glucopyranoside), ZYY-3 (genistein-5-methylether-7-O-α-l-rhamnopyranosyl-(1→6)-O-β-d-glucopyranoside), ZYY-4 (retusin-7,8-O-β-d-diglucopyranoside), ZYY-7 (symplocoside), ZYY-10 (ononin), ZYY-11 (genistin), ZYY-14 (afromosin-7-O-β-d-glucopyranoside), ZYY-15 (lanceolarin), FJ-1 (liquiritigenin), FJ-4 (7,2-dihydroxy,4-methoxyisoflavan) and XC-1 (sphaerobioside) had no binding to thrombin. This indicated that the reported CZE method for the determination of compound–thrombin interactions is powerful, sensitive and fast, and requires less amounts of reagents, and further, it can be employed as a reliable alternative to other methods.     

2种黄芪幼苗对缺硫胁迫的生理响应机制

以药用植物膜荚黄芪（Astragalus membranaceus（Fisch.）Bge.）及其变种蒙古黄芪（Astragalus membranaceus Bge.var.mongholicus Hsiao）幼苗为材料，在水培条件下研究了两种黄芪幼苗在不同程度缺硫条件下的生物量积累、光合参数、次生代谢产物积累等的变化规律。结果显示：与供应Hoagland全营养液相比，低硫和无硫供应处理均显著抑制了两种黄芪幼苗根系和地上部的伸长生长以及生物量的累积，并且引起了植株叶片失绿变黄、根系褐变等一系列缺硫症状。此外，缺硫还导致了两种黄芪幼苗叶片叶绿素含量降低以及光合参数下降、全株各部位细胞膜脂过氧化程度增加。相对膜荚黄芪而言，不同程度缺硫对蒙古黄芪生长发育的影响较小。进一步研究发现，在低硫或无硫条件下，膜荚黄芪植株各部位总酚含量出现了明显下降，而蒙古黄芪根系和叶片总酚含量却因缺硫处理而显著升高。同时我们还发现，硫营养缺乏诱导了蒙古黄芪叶片非光化学能量耗散的升高以及植株各部位主要异黄酮物质的大量积累。我们推断，营养液中硫元素供应的减少引起了黄芪幼苗硫营养不良，影响了植株叶绿素合成，降低了植株的光合能力，并同时引起了全株的氧化胁迫，最终使得黄芪幼苗生长发育受到抑制；蒙古黄芪在缺硫胁迫下提高了光保护能力，合成了较多的异黄酮类物质，有效缓解了缺硫胁迫对其生长的影响，从而在面对缺硫胁迫时比膜荚黄芪表现出了更强的耐性。     

Substance P receptor blocker,aprepitant, inhibited cutaneous and other neurogenic inflammation side effects of the EGFR1-TKI,erlotinib

Molecular and Cellular Biochemistry - Calycosin-7-O-β-d-glucoside (CG) is the component of Astragali Radix, and the aim of the present study is to investigate whether CG protects myocardium...     

Chemical and biological assessment of Angelica herbal decoction: Comparison of different preparations during historical applications

The commonly used Angelica herbal decoction today is Danggui Buxue Tang (DBT), which is a dietary supplement in treating menopausal irregularity in women, i.e. to nourish "Qi" and to enrich "Blood". According to historical record, many herbal decoctions were also named DBT, but the most popular formulation of DBT was written in Jin dynasty (1247 AD) of China, which contained Astragali Radix (AR) and Angelicae Sinensis Radix (ASR) with a weight ratio of 5:1. However, at least two other Angelica herbal decoctions recorded as DBT were prescribed in Song (1155 AD) and Qing dynasties (1687 AD). Although AR and ASR are still the major components in the DBT herbal decoctions, they are slightly varied in the herb composition. In order to reveal the efficiency of different Angelica herbal decoctions, the chemical and biological properties of three DBT herbal extracts were compared. Significantly, the highest amounts of AR-derived astragaloside III, astragaloside IV, calycosin and formononetin and ASR-derived ferulic acid were found in DBT described in 1247 AD: this preparation showed stronger activities in osteogenic, estrogenic and erythropoetic effects than the other two DBT. The current results supported the difference of three DBT in chemical and biological properties, which could be a result of different herbal combinations. For the first time, this study supports the popularity of DBT described in 1247 AD.     

HPLC-DAD-ELSD Combined Pharmacodynamics and Serum Medicinal Chemistry for Quality Assessment of Huangqi Granule

Objective

To more scientifically and reasonably control the quality of Huangqi Granules, preliminary studies on the pharmacodynamics and serum pharmacochemistry of this medicine were performed. DPPH and MTT experiments showed that water extracts of Huangqi Granules had good antioxidant activity and increased immunity. Timed blood samples collected 5 min, 15 min, and 30 min after oral administration of a set amount of Huangqi Granules were collected and tested using UPLC-ESI-MS/MS. As a result, calycosin-7-O-β-D-glucoside, ononin, calycosin, astragaloside IV, and formononetin were found to exist in rat blood after dosing, indicating that the five chemical compounds might have pharmacological activity, and based on this result, they were designated biomarkers for quality control of Huangqi Granules. Consequently, a simple, rapid and efficient method was developed in the present study for the simultaneous determination of the five characteristic compounds in Huangqi Granules using HPLC-DAD-ELSD.

Materials and Methods

The separation was performed using an Agilent Hypersil ODS column (4.6 × 250 mm, 5 μm) at 30 ℃. The mobile phase was composed of water (solvent A) and acetonitrile (solvent B) with a flow rate of 1 mL/min. The drift tube temperature of the ELSD system was set to 85 ℃, and the nitrogen pressure was 3.5 bar.

Results

All five characteristic compounds had good linear behavior with r2 values greater than 0.9972. The recoveries varied from 96.31% to 101.22%. Subsequently, the developed method was applied to evaluate the quality of Huangqi Granules from different batches, and hierarchical clustering analysis (HCA) was used to analyze the classification of the samples based on the values of the five compounds.

Conclusion

The established HPLC method combined with HCA proved to be effective to evaluate the quality of Huangqi Granules.     

膜荚黄芪中的异黄酮化合物

从上海崇明产膜荚黄芪(Astragalus membranaceus(Fisch.)Bunge)根的乙醇提取物中分离鉴定了6个异黄酮化合物:8,3＇-二羟基-7,4＇-二甲氧基异黄酮、奥刀拉亭-7-O-β-D-葡萄吡喃糖甙、芒柄花素、7,3＇-二羟基-8,4＇-二甲氧基异黄酮、毛蕊异黄酮和毛蕊异黄酮-7-O-β-D葡萄吡喃糖甙。其中,前两个为新化合物。     

黄芪化学成分与生态因子的相关性

以主成分分析(PCA)和相关性分析(CA)法研究黄芪中主要有效成分与生态因子的相关性,探究影响黄芪主要成分积累的生态因子.结果表明: 山西产地黄芪中的黄芪甲苷、毛蕊异黄酮苷、芒柄花苷、山奈酚和黄芪多糖的含量显著高于内蒙古和甘肃两地.影响黄芪化学成分含量的气候因子主要为年均相对湿度、年日照时数及7月均温.影响黄芪化学成分含量的土壤元素主要为钙,且钙在一定范围内与毛蕊异黄酮苷、山柰酚、芒柄花苷、槲皮素、黄芪多糖的含量呈负相关.     

Flavonoids from Glycyrrhiza pallidiflora hairy root cultures

Three flavonoids named licoagrosides D, E and F together with four known flavonoids, medicarpin 3-O-glucoside, calycosin 7-O-glucoside, formononetin 7-O-(6"-malonylglucoside) and 2'-hydroxyformononetin 7-O-glucoside were isolated from Glycyrrhiza pallidiflora hairy root cultures. Their structures were determined on the basis of spectroscopic evidence. Licoagrosides E and F are the first examples of a 6a-hydroxypterocarpan glycoside and an alpha-O-glycosidic alpha-hydroxydihydrochalcone, respectively.     

A novel biotransformation of astragalosides to astragaloside IV with the deacetylation of fungal endophyte Penicillium canescens

Under the deacetylation of fungal endophyte Penicillium canescens, which was isolated from pigeon pea, a novel and highly efficient biotransformation method of astragalosides to astragaloside IV in Radix Astragali was investigated. After single factor tests of the biotransformation procedure, the optimum biotransformation conditions were confirmed as the liquid solid ratio 20:1, the biotransformation temperature 30 °C, time 36 h and pH 7, respectively. Final content of astragaloside IV in Radix Astragali reached 7.66 ± 0.44 mg/g, which was 5.51-fold to that of untreated one and contents of astragaloside I and astragaloside II significantly decreased. The immobilized Ca-alginate gel beads with P. canescens could be reused at least for 13 runs. This is the first report that fungal endophyte was applied for the biotransformation of astragalosides to astragaloside IV in Radix Astragali and this novel high-efficiency biotransformation method will be an alternative to enhance the content of astragaloside IV in Radix Astragali in commercial process.     

Solid-phase extraction-liquid chromatographic method for the determination and pharmacokinetic studies of albiflorin and paeoniflorin in rat serum after oral administration of Si-Wu decoction

A sensitive and rapid high-performance liquid chromatography (HPLC) method with solid-phase extraction (SPE) to simultaneously determine albiflorin and paeoniflorin in rat serum was described. Serum samples were pretreated with solid-phase extraction using Extract-Clean cartridges, and the extracts were analyzed by HPLC on a reversed-phase C(18) column and a mobile phase of acetonitrile-0.03% formic acid (17:83 (v/v)) with ultraviolet detection at 230 nm. Pentoxifylline was used as the internal standard (IS). The linear ranges of the calibration curves were 29-1450 ng/ml for albiflorin and 10-2000 ng/ml for paeoniflorin. The intra- and inter-day precisions (R.S.D.) were 相似文献   

引种栽培黄芪药材中主要黄酮类成分含量比较研究

种质资源的筛选和评价对于黄芪的规范化栽培与种植具有重要意义。本研究即分别采用分光光度法、高效液相色谱法测定引种至山西浑源黄芪道地产区不同来源黄芪药材中总黄酮和毛蕊异黄酮、芒柄花素的含量,并进行相关性分析。结果表明:上述成分不仅仅与黄芪栽培方式、产地有关,还与种质来源密切相关;山西、陕西地区黄芪种质适宜在当地栽培和野生种植,而甘肃、内蒙古黄芪引种到山西浑源地区黄酮类物质含量较原产地为高,适宜大面积引种;黄芪药材中毛蕊异黄酮和芒柄花素的含量呈显著的正相关。该研究结果将为黄芪的规范化种植、优良品种选育及质量评价体系的优化提供新的科学依据。     

Investigation of Antioxidant Interactions between Radix Astragali and Cimicifuga foetida and Identification of Synergistic Antioxidant Compounds

The medicinal plants of Huang-qi (Radix Astragali) and Sheng-ma (Cimicifuga foetida) demonstrate significantly better antioxidant effects when used in combination than when used alone. However, the bioactive components and interactional mechanism underlying this synergistic action are still not well understood. In the present study, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay was employed to investigate the antioxidant capacity of single herbs and their combination with the purpose of screening synergistic antioxidant compounds from them. Chromatographic isolation was performed on silica gel, Sephadex LH-20 columns and HPLC, and consequently to yield formononetin, calycosin, ferulic acid and isoferulic acid, which were identified by their retention time, UV λ_max, MS and MS/MS data. The combination of isoferulic acid and calycosin at a dose ratio of 1∶1 resulted in significant synergy in scavenging DPPH radicals and ferric reducing antioxidant power (FRAP) assay. Furthermore, the protective effects of these four potential synergistic compounds were examined using H₂O₂-induced HepG2 Cells bioassay. Results revealed that the similar synergy was observed in the combination of isoferulic acid and calycosin. These findings might provide some theoretical basis for the purported synergistic efficiency of Huang-qi and Sheng-ma as functional foods, dietary supplements and medicinal drugs.