Isoflavonoids, including isoflavones, isoflavans, and pterocarpans, the principal components in Astragalus membranaceus, have a great deal of versatile health-promoting benefits. In this work, as a continuation of our search for bioactive constituents from A. membranaceus, a fast high-performance liquid chromatography-diode array detection-multiple-stage mass spectrometry method was first used to analyze the isoflavonoid profile of A. membranaceus roots extract. Twelve diverse isoflavonoids in subclasses of isoflavones, isoflavans, and pterocarpans present in glycoside/aglycone pair forms were tentatively characterized; of those 12, eight major isoflavonoids were finally isolated and simultaneously quantified by the established fast UHPLC method. Furthermore, the results confirmed for the first time that Astragalus isoflavonoid aglycones could attenuate mesangial cell proliferation and extracellular matrix (ECM) accumulation triggered by high glucose levels, and the primary mechanism might be via protecting intracellular antioxidant enzymes activities and enhancing endogenous antioxidant function to lower levels of cellular oxidative damage induced by high glucose levels. Collectively, diverse Astragalus isoflavonoid antioxidants have the potential to ameliorate high-glucose-induced mesangial cell dysfunction through the regulation of cellular antioxidant defense.

OBJECTIVE

To observe the TH cell subset function in children with recurrent tonsillitis (RT) at the remission stage and to study the effects of astragalus membranacus (AM) on TH cell subset function.

METHODS

The peripheral blood mononuclear cells (PBMC) from 27 children with RT at the remission stage were stimulated with either phytohemagalutinin (PHA) (RT-PHA group) or PHA together with AM (RT-AM group) and were then cultured in vitro for 48 hrs. The samples from 21 healthy children stimulated with PHA were used as the Control group. The levels of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in the supernatants of PBMC were detected using ELISA.

RESULTS

The IFN-gamma level and the ratio of IFN-gamma/IL-4 in the RT-PHA group were statistically lower than those in the Control group (P < 0.01). The level of IFN-gamma and the ratio of IFN-gamma/IL-4 in the RT-AM group were markedly higher than those in the RT-PHA group (P < 0.01), but were significantly lower than those in the Control group (P < 0.05). There were no differences in the IL-4 level among the three groups.

CONCLUSIONS

TH1 cell subset dysfunction may exit in RT children at the remission stage, suggesting that TH1 cell subset dysfunction plays an important role in the pathogenesis of RT. AM can improve TH1 cell subset function and therefore shows an important significance in treating RT.

Astragalus Polysaccharide (APS), as the main active ingredient of Astragalus membranaceus, has extensive biological activities related to immune, metabolic, and anti-oxidative regulatory processes. Previous studies have proven that piRNAs could play important roles in genital gland. This study aimed to identify the differentially expressed piRNAs in chicken testes in response to dietary APS supplements and further evaluate the roles of these piRNAs related to the effect of dietary APS supplements on testicular changes. We generated piRNA expression profiles of testes from breeding cocks fed without or with extra APS. As results, there were 42 up-regulated and 86 down-regulated piRNAs in APS group, compared with the control group meeting the criteria of P<0.05 and fold change <0.67 or fold change >1.5. The potential targets were subsequently annotated against the Kyoto Encyclopedia of Genes and Genomes databases. The results revealed that dietary APS supplements could regulate tight junction pathways by regulating the piRNA expression profiles, which were related to the regulation of a better testicular condition for spermatogenesis. Our results provided a novel insight into the effect of dietary APS supplements on testicular piRNA expression profiles and its potential roles in testicular condition regulation.

The interaction between immune cells and hepatic stellate cells (HSCs) can modulate the development of hepatic fibrosis. It can also regulate hepatic fibrosis and liver cirrhosis caused by excessive deposition of extracellular matrix (ECM). This article reviews the action mechanism of immune cells on liver fifibrosis and the effect of Astragalus membranaeus and its active components on immune cells. In-depth study of interaction between immune cells and HSCs on the pathogenesis of liver fifibrosis, and the regulatory effect of Astragalus membranaeus and its active components on immune mechanism will provide new insights in the treatment of liver fifibrosis.

OBJECTIVE

To study separation and purification of flavonids with ethanol/phosphate aqueous two-phase system.

METHODS

The diversity of phase separation ability and the distribution of target products in various systems were taken as indicators to analyze aqueous two-phase extraction systems and phase diagrams formed by ethanol and some common salts, screen out EtOH/ K2HPO4 system as the optimla system for extracting total flavonids, and study the impact of proportion of components in EtOH/K2 HPO4 system on the partition coefficient and phase ratio of flavonids.

CONCLUSIONS

The EtOH/K2 HPO4 system with omegaEtOH 36.05% and omegaKHPO4 18.20% has been proved as the optimal conditions for separating and purifying total flavonoids of Astragalus (TFA). Under this optimal condition, the partition coefficient and the extraction yield of TFA reached 10.33 and 96.6%, respectively. After extraction, the contents of A. membranaceus saponins and A. membranaceus polysaccharides in top and bottom phases were determined at the same time, showing that A. membranaceus saponins in the removal rate reached 92.01%, and A. membranaceus polysaccharides were totally concentrated in bottom water phase, indicating a removal rate of 100%. Therefore, this is beneficial to separate and purify total flavonids from A. membranaceus crude extracts.

BACKGROUND

Astragaloside, which is one of the main components of Astragalus membranaceus, has been widely used in the treatment of congestive heart failure in China, and it can protect cardiomyocytes. Its mechanism of action remains unclear. Therefore, the present study was carried out to investigate the influence of astragaloside on rat cardiomyocytes stimulated with endothelin-1 (ET-1), and explored the underlying mechanism.

METHODS

ET-1 was used to stimulate primary rat cardiomyocytes and establish a cardiomyocyte hypertrophy model. Different astragaloside doses were administered in combination with ET-1. Cardiomyocyte hypertrophy and apoptosis were examined using transmission electron microscopy (TEM) and flow cytometry, respectively. The molecular mechanism was explored by analyzing the mRNA of the vitamin D receptor (VDR), cytochrome P450 family 27 subfamily B member 1(CYP27B), cytochrome P450 family 24 subfamily A member 1(CYP24A) and renin mRNA levels by quantificational real-time polymerase chain reaction(qRT-PCR).

RESULTS

Rat cardiomyocyte hypertrophy model was established successfully. Astragaloside administration significantly affected cell apoptosis and significantly inhibited ET-1-induced cardiomyocyte hypertrophy in a dose-dependent manner. Astragaloside treatment affected the expression of signaling molecules in the vitamin D axis.

CONCLUSIONS

Astragaloside inhibits ET-1-induced cardiomyocyte hypertrophy. This effect can be reversed by regulating the levels of the relevant factors in the vitamin D axis.

The root of Astragalus membranaceus also known as Huang Qi in China is a common traditional Chinese herb with extensive pharmacological activities. Huangqi injection (HI), a common preparation of Huang Qi, was wildly co-administered with gliquidone to treat diabetes mellitus and diabetic nephropathy in clinic of China. The aim of the study was to investigate the effect of HI on the pharmacokinetics and hepatic uptake of gliquidone and related mechanism to ensure the safety and efficacy of their usage. Normal rats (n = 5) and streptozotocin (STZ)-induced diabetic rats (n = 5) were administered orally with 120 mg/kg gliquidone alone or in combination with 8 ml/kg HI (i.v.), HI was given intravenously 5 min before that of gliquidone. The plasma concentration of gliquidone was determined by HPLC-fluorescence. In addition, hepatic uptake of gliquidone with or without HI was evaluated in fresh primary rat hepatocytes. Co-administration of HI with gliquidone could significantly increased the bioavailability of gliquidone in normal and STZ-induced diabetic rats (n = 5); the concentration of gliquidone in fresh primary rat hepatocytes was greatly decreased by HI. This study suggested that when HI and gliquidone co-administering to normal and diabetic rats, the pharmacokinetics of gliquidone was greatly changed, and these changes connect with HI inhibiting hepatic uptake of gliquidone, and transportation of gliquidone across liver mucosal membrane inhibited by HI would be the main mechanisms.

A generic strategy based on chemical fingerprinting is proposed for the differentiation of polysaccharides from Astragalus membranaceus (AEP) and A. mongholicus (AOP), using multiple chromatographic and mass spectrometric techniques. Complete and mild acid hydrolysis and Smith degradation were employed for the depolymerization of polysaccharides. The corresponding digested products were efficiently separated and detected using GC-MS, HILIC-ELSD and HR-ESI^--MS. The resulting bottom-up fingerprinting reflected the variations in native polysaccharides, which may be attributed to the three structural levels, which are monosaccharide compositions, glycosidic linkages, and skeletal structure. Similarity analysis from GC-MS and HILIC-ELSD fingerprinting showed that the correlation coefficients from homologous species were more than 0.914, whereas those from heterologous species were less than 0.796. It also noted that characteristic peak area ratio of Man/Gal in Smith fingerprinting could be used a feasible parameter for direct discrimination of AEP and AOP. Principal component analysis from m/z fingerprinting data resulted in clear clustering of AEP and AOP based on changes of oligosaccharides in mild acid hydrolyzates. By combining the accurate m/z, ESI^--MS/MS and methylation assays, the structures of a series of hexose glycopolymers in mild acid hydrolyzates were characterized by predominant 1,6 glycosidic linkages along with minor 1,4 glycosidic linkages. The established chemical fingerprinting is not only an interconnected structure mapping but also a powerful approach for the evaluation of polysaccharides from traditional Chinese medicines.

Standard Huangqi (Astragalus membranaceus (Fisch) Bge. Var. monghlicus (Bge.) Hsiao) and its counterfeit Ciguogancao (Glychrrhiza pallidiflora Maxim.) can be discriminated and identified by using multi-steps infrared maro--fingerprint method. In the 1D-IR spectra, the peak intensity at 1 737 cm(-1) for Ciguogancao, which is the stretching vibration peak of C==O, is much stronger than that of Huangqi. It's proved that the organic ester compounds in Ciguogancao are much more than Huangqi. In the secondary derivative spectra, it's easy to find the fingerprint characteristic peaks of CaC2O4 in the infrared spectra of Ciguogancao, but not in that of Huangqi. Besides, both secondary derivative spectra also have main characteristic peaks, which are the skeletal stretching of aromatic, round 1463, 1511 and 1596 cm(-1), but Ciguogancao aslo has one shoulder peak at 1 453 cm(-1). In the 2D-IR spectra, both have three auto-peaks at 1070, 1095 and 1140 cm(-1), which are the autopeaks of glucoside, but the strongest auto-peak of Huangqi is at 1140 cm(-1) and that of Ciguogancao's is at 1 090 cm(-1). The spectra testified that the organic ester compounds, aromatic compounds and glucoside compounds in Huangqi and its counterfeit Ciguogancao were different. The method not only can identify standard Huangqi and its counterfeit Ciguogancao rapidly, but also provides useful information about the differences in organic ester compounds, aromatic compounds and glucoside between Huangqi and its counterfeit Ciguogancao. It's proved that multi-steps infrared maro-fingerprint method can be used to analyze and distinguish Huangqi and its counterfeit Ciguogancao.

OBJECTIVE

To study the effects of Astragalus polysaccharide (APS), the primary effective component of the Chinese herb medicine Astragalus membranaceus (frequently used for its anti-hepatic fibrosis effects), on nanoscale mechanical properties of liver sinusoidal endothelial cells (SECs).

METHODS

Using endothelial cell medium as the control, 5 experimental groups were established utilizing different concentrations of APS, i.e. 12.5, 25, 50, 100, and 200 μg/mL. By using atomic force microscopy along with a microcantilever modified with a silicon dioxide microsphere as powerful tools, the value of Young's modulus in each group was calculated. SAS 9.1 software was applied to analyze the values of Young's modulus at the pressed depth of 300 nm. Environmental scanning electron microscopy was performed to observe the surface microtopography of the SECs.

RESULTS

The value of Young's modulus in each APS experimental group was significantly greater than that of the control group: as APS concentration increased, the value of Young's modulus presented as an increasing trend. The difference between the low-concentration (12.5 and 25 μg/mL) and high-concentration (200 μg/mL) groups was statistically significant (P<0.05), but no significant differences were observed between moderateconcentration (50 and 100 μg/mL) groups versus low- or high-concentration groups (P>0.05). Surface topography demonstrated that APS was capable of increasing the total area of fenestrae.

CONCLUSIONS

The values of Young's modulus increased along with increasing concentrations of APS, suggesting that the stiffness of SECs increases gradually as a function of APS concentration. The observed changes in SEC mechanical properties may provide a new avenue for mechanistic research of anti-hepatic fibrosis treatments in Chinese medicine.

This study aimed to determine the effect of crude drugs on the dynamic viscoelasticity and angiogenic property of soft polymer materials, in vitro. Two kinds of polyethyl methacrylates, and crude drugs (Astragalus membranaceus Bunge [HQ] and Salvia miltiorrhiza Bunge [DS]) were used in their powdered forms. And, acetyl tributyl citrate and ethyl alcohol were used in the liquid form. The dynamic viscoelasticity of each specimen was measured after 0, 1, 3, 7, 14 and 28 days of immersion in distilled water. The CellPlayer angiogenesis PrimeKit assay was used to test angiogenesis. Significant differences in dynamic viscoelasticity were observed among the materials. Specimens containing 1 wt% HQ showed higher angiogenic activity than those containing 5 wt% and 10 wt% HQ, and DS. Our results suggest that the addition of low amounts of crude drugs to soft polymer materials may promote angiogenesis in human tissues.

Astragalus membranaceus is a famous herb found among medicinal and food plants in East and Southeastern Asia. The Nrf2-ARE assay-guided separation of an extract from Jing liqueur led to the identification of a nontoxic Nrf2 activator, methylnissolin-3-O-β-d-glucopyranoside (MNG, a component of A. membranaceus). Nrf2 activation by MNG has not been reported before. Using Western Blot, RT-qPCR and imaging, we investigated the cytoprotective effect of MNG against hydrogen peroxide-induced oxidative stress. MNG induced the expression of Nrf2, HO-1 and NQO1, accelerated the translocation of Nrf2 into nuclei, and enhanced the phosphorylation of AKT. The MNG-induced expression of Nrf2, HO-1, and NQO1 were abolished by Nrf2 siRNA, while the MNG-induced expression of Nrf2 and HO-1 was abated and the AKT phosphorylation was blocked by LY294002 (a PI3K inhibitor). MNG reduced intracellular ROS generation. However, the protection of MNG against the H₂O₂ insult was reversed by Nrf2 siRNA with decreased cell viability. The enhancement of Nrf2 and HO-1 by MNG upon H₂O₂ injury was reduced by LY294002. These data showed that MNG protected EA.hy926 cells against oxidative damage through the Nrf2/HO-1 and at least partially the PI3K/Akt pathways.

Keywords: Astragalus membranaceus; EA.hy926 cells; MNG; Nrf2; PI3K.

Although astragaloside IV protects from acute myocardial infarction (AMI)-induced chronic heart failure (CHF), the underlying mechanism of action is unclear. We determined the potential therapeutic effect of astragaloside IV using molecular docking approaches and validated the findings by the ligation of the left anterior descending (LAD) coronary artery-induced AMI rat model. The interaction between astragaloside IV and myeloid differentiation factor 88 (MyD88) was evaluated by SwissDock. To explore the mechanisms underlying the beneficial effects of astragaloside IV in the LAD coronary artery ligation-induced AMI model, we administered the rats with astragaloside IV for 4 weeks. Hemodynamic indexes were used to evaluate the degree of myocardial injury in model rats. The histopathological changes in myocardium were detected by hematoxylin & eosin (H&E) staining and Masson's staining. Myocardium homogenate contents of collagen I and collagen III were evaluated by ELISA. The level of myocardial hydroxyproline (HYP) was determined by alkaline hydrolysis. Immunohistochemistry was used to examine collagen I. Western blotting was used to examine relevant proteins. As per the molecular docking study results, astragaloside IV may act on MyD88. Furthermore, astragaloside IV improved hemodynamic disorders, alleviated pathological changes, and reduced abnormal collagen deposition and myocardial HYP in vivo. Astragaloside IV significantly reduced the overexpression of TLR4, MyD88, NF-Κb, and TGF-β, which further validated the molecular docking findings. Hence, astragaloside IV ameliorates AMI by reducing inflammation and blocking TLR4/MyD88/NF-κB signaling. These results indicate that astragaloside IV may alleviate AMI. PRACTICAL APPLICATIONS: Astragaloside IV, a small active substance extracted from Astragalus membranaceus, has demonstrated potent protective effects against cardiovascular ischemia/reperfusion, diabetic nephropathy, and other diseases. Molecular docking experiments showed that astragaloside IV might act on the myeloid differentiation factor 88 (MyD88). Astragaloside IV can effectively reduce the overexpression of TLR4, MyD88, and NF-κB p65, indicating that astragaloside IV inhibits inflammation via TLR4/MyD88/NF-κB signaling pathway. These results indicate that astragaloside IV may alleviate acute myocardial infarction.

Keywords: TLR4/MyD88/NF-κB signaling pathway; acute myocardial infarction; astragaloside IV; molecular docking.

Astragalus membranaceus (Fisch.) Bge. var. mongholicus, which is used in traditional Chinese medicine, contains several bioactive ingredients. The root-associated microbial communities play a crucial role in the production of secondary metabolites in plants. However, the correlation of root-associated bacteria and fungi with the bioactive ingredients production in A. mongholicus has not been elucidated. This study aimed to examine the changes in soil properties, root bioactive ingredients, and microbial communities in different cultivation years. The root-associated bacterial and fungal composition was analyzed using high-throughput sequencing. The correlation between root-associated bacteria and fungi, soil properties, and six major bioactive ingredients were examined using multivariate correlation analysis. Results showed that soil properties and bioactive ingredients were distinct across different cultivation years. The composition of the rhizosphere microbiome was different from that of the root endosphere microbiome. The bacterial community structure was affected by the cultivation year and exhibited a time-decay pattern. Soil properties affected the fungal community composition. It was found that 18 root-associated bacterial operational taxonomic units (OTUs) and four fungal OTUs were positively and negatively correlated with bioactive ingredient content, respectively. The abundance of Stenotrophomonas in the rhizosphere was positively correlated with astragaloside content. Phyllobacterium and Inquilinus in the endosphere were positively correlated with the calycosin content. In summary, this study provided a new opportunity and theoretical reference for improving the production and quality of in A. mongholicus, which thus increase the pharmacological value of A. mongholicus.

Keywords: Phyllobacterium; Stenotrophomonas; bioactive ingredients; rhizosphere; root endosphere.

Ethnopharmacological relevance: Cardiovascular and cerebrovascular diseases have become a severe threat for human health worldwide, however, optimal therapeutic options are still developed. Zhilong Huoxue Tongyu capsule (ZL capsule) is mainly composed of Astragalus membranaceus, Leech, Earthworm, Cinnamomum cassia and Sargentodoxa cuneata, having functions of replenishing qi and activating blood, dispelling wind and reducing phlegm. It is an expanded application on the basis of traditional uses of above TCMs, acquiring a satisfactory curative effect on cardiovascular and cerebrovascular diseases over twenty years.

Aim of the study: To comprehensively summarize the main components of ZL capsule, understand the mechanisms of ZL capsule, and conclude clinical regimens of ZL capsule for cardiovascular and cerebrovascular diseases.

Materials and methods: We selected network pharmacology technology to analyze main active compounds and predict underlying mechanism of ZL capsule against atherosclerosis. Molecular docking was performed to simulate the interaction pattern between the active components of ZL capsule and putative targets. Further, PubMed, Web of Science, China National Knowledge Infrastructure and Google Scholar were used to search literatures, with the key words of "Zhilong Huoxue Tongyu capsule", "cardiovascular and cerebrovascular diseases", "atherosclerosis", "clinical study" and their combinations, mainly from 2000 to 2020.

Results: Both network pharmacology analysis, molecular docking and animal experiments studies confirmed that mechanisms of ZL capsule plays the role of anti-inflammatory, anti-apoptosis and promoting angiogenesis in treating cardiovascular and cerebrovascular diseases by multi-components acting on multi-targets via multi-pathways. Over 1000 clinical cases were benefited from the treatment of ZL capsule, suggesting a holistic concept of "the same therapy for different myocardial and cerebral diseases".

Conclusions: For the first time, this systematic review may supply meaningful information for further studies to explore material basis and pharmacodynamics of ZL capsule and also provide a basis for sharing the "Chinese patent medicine" for cardiovascular and cerebrovascular diseases.

Keywords: Atherosclerosis; Cardiovascular and cerebrovascular diseases; Mechanism; Network pharmacology; Zhilong Huoxue Tongyu capsule.

Background: This paper aimed to verify how a supplementation of rower's diet with Astragalus Membranaceus Root (AMR) modulated their immune system response to maximal physical exertion.

Methods: The double-blind study included 18 members of the Polish Rowing Team assigned to the supplemented group (n = 10), and the placebo group (n = 8). The participants performed a 2000 m test on a rowing ergometer at the beginning and at the end of the six-week of intensive training camp during which the supplemented group received 500 mg of AMR. Blood samples were obtained prior to, 1 min after completing, and 24 h after the exertion test. The levels of interleukin 2 (IL2), interleukin 4 (IL4), interleukin 10 (IL10), interferon ɤ (IFN-ɣ), and lactic acid were determined. Subpopulations of T regulatory lymphocytes [CD4+/CD25+/CD127-] (Treg), cytotoxic lymphocytes [CD8+/TCRαβ+] (CTL), natural killer cells [CD3-/CD16+/CD56+] (NK), and TCRδγ-positive cells (Tδγ) were determined with flow cytometry.

Results: After the camp, the initial NK and Treg levels sustained at the baseline, while Tδγ counts increased relative to the levels in the placebo group. In the supplemented subgroup, a decrease in IL2 level in reaction to maximal exertion clearly deepened while the change in IL-2/IL-10 level induced by the recovery after this exertion clearly increased, relative to the changes in the placebo group.

Conclusions: AMR restored the immunological balance in strenuously trained athlets through a stabilization of NK and Treg cells with a positive trend in Tδγ towards Th1 response during restitution by cytokine IL2 modulation.

Keywords: Astragalus Membranaceus Root; Immunosuppression; Strenuous exercise; Supplementation.

Astragalus membranaceus is a functional food with multiple bioactivities. It presents differentiated health benefits due to origins. Polysaccharides (APS) are the leading bioactive macromolecules of A. membranaceus, which are highly related to its health benefits. However, the effect of origin on the structural characteristics of APSs remains unclear. In this work, polysaccharides from four origins were isolated and identified by NMR. The results showed APSs of four origins had identical monosaccharide composition and glycosidic linkage. Rhamnogalacturonan II pectins and α-(1→4)-glucan were the dominant polysaccharides. However, the level of methyl ester in pectins varied to a large extent. The molecular weight profiles of APSs were also different. Inner Mongolia APS had the largest percentage of 20-40 kDa polysaccharides. Molecular weight and methyl ester level were two important parameters determining the difference of APSs from four origins. These results were helpful to recognize the origin-related quality of A. membranaceus.

Keywords: NMR; glycosidic linkage; monosaccharide composition; pectin.

Background: Cisplatin is a frequently used and effective chemotherapy drug in clinical practice, but severe side effects limit its use, among which nephrotoxicity is considered the most serious and prolonged damage to the body. Astragalus membranaceus (AM) is a well-known herbal medicine, and modern pharmacological studies have confirmed its antioxidant, immunomodulatory, and antiapoptotic effects. Clinical studies have shown that AM and its active components can attenuate cisplatin-induced kidney damage, but the molecular mechanism has not been fully expounded.

Materials and methods: First, the components and targets information of AM were collected from the TCMSP, and the relevant targets of cisplatin-induced kidney damage were accessed from the GeneCards and OMIM databases. Then, the core targets were selected by the Venn diagram and network topology analysis, which was followed by GO and KEGG pathway enrichment analysis. Finally, we construct a component-target-pathway network. Furthermore, molecular docking was carried out to identify the binding activity between active components and key targets.

Results: A total of 20 active components and 200 targets of AM and 646 targets related to cisplatin-induced kidney damage were obtained. 91 intersection targets were found between AM and cisplatin-induced kidney damage. Then, 16 core targets were identified, such as MAPK1, TNF-α, and p53. Furthermore, GO and KEGG pathway enrichment analysis suggested that MAPK, Toll-like receptor, and PI3K-Akt signaling pathways may be of significance in the treatment of cisplatin-induced kidney damage by AM. Molecular docking indicated that quercetin and kaempferol had high binding affinities with many core targets.

Conclusion: In summary, the active components, key targets, and signaling pathways of AM in the treatment of cisplatin-induced kidney damage were predicted in this study, which contributed to the development and application of AM.

Doxorubicin (DOX) is widely used against cancer but carries the risk of a progressive cardiomyopathy. Astragalus polysaccharides (ASP) is the main active ingredient of Astragalus membranaceus Bunge. It has been proved to be effective against DOX-induced cardiomyopathy. However, its therapeutic mechanism is not yet well explored. In this study, a metabolomics approach based on hydrophilic interaction liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HILIC-Q-TOFMS) was developed to characterize the metabolic fluctuations associated with DOX cardiomyopathy and elucidate the underlying mechanisms of the protective effects of ASP. By the combination of HILIC-Q-TOFMS and multivariate and univariate data analysis, we identified 22 polar serum metabolites associated with DOX cardiomyopathy, 12 of which were significantly reversed when the animals were co-treated with ASP through two main metabolic pathways, i.e., sphingolipid and glycerophospholipid metabolism. Furthermore, it was found that ASP could alleviate DOX-induced cardiomyopathy by decreasing the levels of acid sphingomyelinase, acid ceramidase and phospholipase A2 and increasing the levels of sphingomyelin synthase to regulate the sphingolipid and glycerophospholipid metabolic disorder. These results revealed that sphingolipid and glycerophospholipid metabolism may be significantly responsible for DOX cardiomyopathy, which is also a major mechanism for the action of ASP against DOX cardiomyopathy.

Keywords: Astragalus polysaccharides; Cardiotoxicity; Doxorubicin; HILIC-Q-TOFMS; Metabolomics.

Formononetin is one of the main active compounds of traditional Chinese herbal medicine Astragalus membranaceus. However, disposition of formononetin via sulfonation pathway remains undefined. Here, expression-activity correlation was performed to identify the contributing of SULT1A3 to formononetin metabolism. Then the sulfonation of formononetin and excretion of its sulfate were investigated in SULT1A3 overexpressing human embryonic kidney 293 cells (or HKE-SULT1A3 cells) with significant expression of breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 4 (MRP4). As a result, formononetin sulfonation was significantly correlated with SULT1A3 protein levels (r = 0.728; p < 0.05) in a bank of individual human intestine S9 fractions (n = 9). HEK-SULT1A3 cells catalyzed formononetin formation of a monosulfate metabolite. Sulfate formation of formononetin in HEK-SULT1A3 cell lysate followed the Michaelis-Menten kinetics (V_max = 13.94 pmol/min/mg and K_m = 6.17 μM). Reduced activity of MRP4 by MK-571 caused significant decrease in the excretion rate (79.1%-94.6%) and efflux clearance (85.3%-98.0%) of formononetin sulfate, whereas the BCRP specific inhibitor Ko143 had no effect. Furthermore, silencing of MRP4 led to obvious decrease in sulfate excretion rates (>32.8%) and efflux clearance (>50.6%). It was worth noting that the fraction of dose metabolized (f_met), an indicator of the extent of drug sulfonation, was also decreased (maximal 26.7%) with the knockdown of MRP4. In conclusion, SULT1A3 was of great significance in determining sulfonation of formononetin. HEK-SULT1A3 cells catalyzed formononetin formation of a monosulfate. MRP4 mainly contributed to cellular excretion of formononetin sulfate and further mediated the intracellular sulfonation of formononetin.

Keywords: HEK293 cells; MRP4; efflux transporter; formononetin; sulfonation.

This study explored the effects of Astragalus membranaceus polysaccharide (APS) on intestinal inflammatory damage of goslings infected with parvovirus ('gosling plague'). A total of 90 healthy goslings were randomly divided into three groups; control, infected or APS treated, respectively. Goslings in the infection and APS treatment groups were inoculated with 0.3 ml allantoic fluid containing goose parvovirus (ELD₅₀ = 1×10³/0.3 ml) by intramuscular injection and the control group were injected with saline (0.3 ml) twice a day for 15 days. Blood serum and the jejunum from each gosling were collected at 5, 10 and 15 days after the start of the experiment to detect the activities of SOD and GSH-Px, levels of MDA, sIgA, IL-1β, IL-6 and TNF-α, the mRNA expression of IL-1β, IL-6, LITAF, NF-κB, COX-2 and PGE2, pathological damage in the jejunum and serum IgG, IgM, C3, C4, IFN-γ levels. After APS treatment, SOD and GSH-Px activities increased, MDA content decreased; sIgA, IL-1β, IL-6 and TNF-α protein content, and IL-1β, IL-6, LITAF, NF-κB, COX-2 and PGE2 mRNA expression decreased in the jejunal tissue, serum IgG, IgM, C3, C4, IFN-γ significantly increased and pathological damage of jejunum significantly improved. In conclusion, APS reduced intestinal inflammatory damage in goslings infected with parvovirus by improving the immune and antioxidant functions of goslings.

Keywords: Astragalus membranaceus polysaccharide; Parvivirus; gosling; infectious diseases; inflammatory injury; intestine.