Uncovering the effects and molecular mechanism of Astragalus membranaceus (Fisch.) Bunge and its bioactive ingredients formononetin and calycosin against colon cancer: An integrated approach based on network pharmacology analysis coupled with experimental validation and molecular docking.

Colon cancer is a highly malignant cancer with poor prognosis. Astragalus membranaceus (Fisch.) Bunge (Huang Qi in Chinese, HQ), a well-known Chinese herbal medicine and a popular food additive, possesses various biological functions and has been frequently used for clinical treatment of colon cancer. However, the underlying mechanism is not fully understood. Isoflavonoids, including formononetin (FMNT) and calycosin (CS), are the main bioactive ingredients isolated from HQ. Thus, this study aimed to explore the inhibitory effects and mechanism of HQ, FMNT and CS against colon cancer by using network pharmacology coupled with experimental validation and molecular docking. The network pharmacology analysis revealed that FMNT and CS exerted their anticarcinogenic actions against colon cancer by regulating multiple signaling molecules and pathways, including MAPK and PI3K-Akt signaling pathways. The experimental validation data showed that HQ, FMNT and CS significantly suppressed the viability and proliferation, and promoted the apoptosis in colon cancer Caco2 and HT-29 cells. HQ, FMNT and CS also markedly inhibited the migration of Caco2 and HT-29 cells, accompanied by a marked increase in E-cadherin expression, and a notable decrease in N-cadherin and Vimentin expression. In addition, HQ, FMNT and CS strikingly decreased the expression of ERK1/2 phosphorylation (p-ERK1/2) without marked change in total ERK1/2 expression. They also slightly downregulated the p-Akt expression without significant alteration in total Akt expression. Pearson correlation analysis showed a significant positive correlation between the inactivation of ERK1/2 signaling pathway and the HQ, FMNT and CS-induced suppression of colon cancer. The molecular docking results indicated that FMNT and CS had a strong binding affinity for the key molecules of ERK1/2 signaling pathway. Conclusively, HQ, FMNT and CS exerted good therapeutic effects against colon cancer by mainly inhibiting the ERK1/2 signaling pathway, suggesting that HQ, FMNT and CS could be useful supplements that may enhance chemotherapeutic outcomes and benefit colon cancer patients.

Efficacy of Huangqi Injection in the Treatment of Hypertensive Nephropathy: A Systematic Review and Meta-Analysis.

Background: Huangqi injection (HQI) is the extract of Astragalus membranaceus (Fisch.) Bunge, which is widely used in the treatment of a variety of diseases in China. It is supposed to be an important adjuvant therapy for hypertensive nephropathy. Objective: To evaluate the efficacy of HQI combined with antihypertensive drugs in the treatment of hypertensive nephropathy. Materials and Methods: We systematically searched China National Knowledge Infrastructure (CNKI), Chinese Scientific Journals Database (VIP), Wanfang Knowledge Service Platform (WanfangData), Chinese Biomedical Database (CBM), EMBASE, PubMed and Cochrane Library from their inception to April 23st, 2021. All studies were independently screened by two auditors according to the inclusion and exclusion criteria. Randomized controlled trials comparing HQI in combination with antihypertensive drugs vs. antihypertensive drugs alone were extracted. Results: The meta-analysis included 15 studies involving 1,483 participants.The effect of HQI combined with antihypertensive drugs is better than that of antihypertensive drugs alone in regulating hypertensive nephropathy for reducing 24-h urinary total protein (24 h UTP) [WMD=-0.29, 95% CI (-0.40, -0.18), P = 0.000], microalbuminuria (mALB) [WMD = -17.04, 95% CI (-23.14, -10.94), P = 0.000], serum creatinine (SCr) [WMD = -40.39, 95% CI (-70.39, -10.39), P = 0.008], systolic blood pressure (SBP) [WMD = -9.50, 95% CI (-14.64, -4.37), P = 0.000], diastolic blood pressure (DBP) [WMD = -4.588, 95% CI (-6.036, -3.140), P = 0.000], cystatin-C (Cys-c) [WMD = -0.854, 95% CI (-0.99, -0.72), P = 0.000], blood urea nitrogen (BUN) [WMD = -4.155, 95% CI (-6.152, -2.157), P = 0.000]. Conclusion: The combination of HQI and antihypertensive drugs is more efficient in improving the related indexes of patients with hypertensive nephropathy than using antihypertensive drugs alone, and a moderate dose of HQI (no more than 30 mL) may benefit more. However, the quality of the methodology is low and the number of samples is small, the results need to be confirmed by more stringent randomized controlled trials.

Integrating Network Pharmacology and Experimental Validation to Investigate the Effects and Mechanism of Astragalus Flavonoids Against Hepatic Fibrosis.

Hepatic fibrosis (HF) represents the excessive wound healing where an excess amount of connective tissues is formed within the liver, finally resulting in cirrhosis or even hepatocellular carcinoma (HCC). Therefore, it is significant to discover the efficient agents and components to treat HF, thus restraining the further progression of hepatopathy. Astragalus membranaceus (Fisch.) Bunge [also called Astragali Radix (AR)] is a famous herb in traditional Chinese medicine (TCM), which possesses a variety of biological activities and exerts good therapeutic effects in the treatment of HF. Flavonoids account for the major active ingredients related to the AR pharmacological effects. Total AR flavonoids have been proved to exert inhibitory effects on hepatic fibrosis. This study aimed to further undertake network pharmacology analysis coupled with experimental validation and molecular docking to investigate the effects and mechanism of multiple flavonoid components from AR against liver fibrosis. The results of the network pharmacology analysis showed that the flavonoids from AR exerted their pharmacological effects against liver fibrosis by modulating multiple targets and pathways. The experimental validation data showed that the flavonoids from AR were able to suppress transforming growth factor beta 1 (TGF-ß1)-mediated activation of hepatic stellate cells (HSCs) and reduce extracellular matrix deposition in HSC-T6 cells via regulating the nuclear factor kappa B (NF-κB) signal transduction pathway. The results of the molecular docking study further showed that the flavonoids had a strong binding affinity for IκB kinase (IKKß) after docking into the crystal structure. The above results indicated that, flavonoids possibly exerted the anti-inflammatory effect on treating HF by mediating inflammatory signaling pathways. The potential mechanism of these flavonoids against liver fibrosis may be related to suppression of the NF-κB pathway through effective inhibition of IKKß. This study not only provides a scientific basis for clarifying the effects and mechanism of AR flavonoids against liver fibrosis but also suggests a novel promising therapeutic strategy for the treatment of liver fibrosis.

Estimation of genome sizes of Astragalus membranaceus based on flow cytometry and K-mer analysis / 中草药

Objective: To determinate the genome size and complexity of Astragalus membranaceus by using flow cytometry (FCM) and K-mer analysis, which can lay the foundation for the screening of functional genes of A. membranaceus. Methods Lycopersicon esculentum was served as an internal reference in this study. The mixed sample of A. membranaceus cell nucleus and L. esculentum cell nucleus was stained using propidium iodide (PI). The PI fluorescence intensities of the sample were measured by FCM. The genome size of A. membranaceus was calculated by comparing the multiple relationship between the peak of DNA content in the cells of A. membranaceus and L. esculentum. The genome of A. membranaceus was sequenced by using high-throughput sequencing technologies. The genome size of A. membranaceus was calculated by K-mer analysis. The hybridity percentage, repetitive sequence, and GC of A. membranaceus were estimated by bioinformatics analysis. Results The genome size of A. membranaceus was about 1 426 Mb. For K-mer analysis, more than 95 Gb high quality data from the genome was generated. The average genome size and sequencing coverage depth of A. membranaceus was about 1 456 Mb and 39 times respectively. The genome of A. membranaceus had obvious hybridity peak by K-mer method, and the hybridity percentage as high as 2.1%. Conclusion The genome size of A. membranaceus was about 1.45 Gb and the heterozygosity is high. These data would provide a reference for the genomic research in A. membranaceus.

Optimization of extraction process of Shenqi Qiangxin Tablets based on pharmacodynamic test and analytic hierarchy process / 中草药

Objective: To optimize the extraction technology of Shenqi Qiangxin Tablets (SQT). Methods: With the improvement of heart lesion of pharmacological model of isoproterenol induced heart failure in rats as the index, pharmacological efficacy test was used to screen extracting conditions of the technology. The extraction technology was optimized by analytic hierarchy process combined with principal component analysis, single factor and orthogonal tests using each content of solid matter, ginsenosides Rg1, Re as indexes. And the verification test was carried out by using solid mass and icariin content as indexes. Results: Pharmacological efficacy test showed that technology 4 was superior. The optimal extraction condition of technology 4 was as follow: five medicinal materials including red ginseng and astragalus were reflux extracted three times with 50% ethanol, 11 fold for the first time, 10 fold for the second and three times, 2.5 h for each extraction; Epimedium and the other two medicinal materials were decocted three times with water, 19 fold for the first time, 16 fold for the second and third times, 1.5 h for each decction. The verification test showed that the average yield of ethanol extracted solids was 19.78%, and the average extraction rate of ginsenoside Rg1 and Re was 77.52%; The average value of water extracted solids was 16.58%, and the average extraction rate of epimedium was 90.98% (RSD < 2.0%, n = 3). Conclusion: The optimized extraction technology was stable and feasible.

Cycloastragenol: An exciting novel candidate for age-associated diseases.

Cycloastragenol (CAG) is a triterpenoid saponin compound and a hydrolysis product of the main active ingredient in Astragalus membranaceus (Fisch.) Bunge. An increasing body of evidence has indicated that CAG has a wide spectrum of pharmacological functions, which are attracting attention in the research community. The aim of the present review paper was to review and elucidate the advanced study of CAG. The focus was on advanced studies of CAG in English and Chinese databases; the literature was collected and reviewed to summarize the latest efficacy, pharmacokinetics and adverse reactions of CAG. Extensive pharmacological effects have been attributed to CAG, including telomerase activation, telomere elongation, anti-inflammatory and anti-oxidative properties; CAG has also been reported to improve lipid metabolism. Clinical research has demonstrated that CAG activates telomerase in humans and ameliorates various biomarkers. CAG is absorbed through the intestinal epithelium via passive diffusion and undergoes first-pass hepatic metabolism. Within a certain dose range, oral CAG is relatively safe; however, underlying mechanisms associated with CAG are not clear, and thus, we should be aware of potential adverse reactions associated with CAG. According to existing studies and clinical trials, CAG is safe and has broad application prospects. However, further studies are required to fully understand its efficacy and potential adverse reactions, and to ensure the proper use of CAG is applied to treat diseases clinically.

Astragalus membranaceus (Fisch.) Bunge repairs intestinal mucosal injury induced by LPS in mice.

BACKGROUND: Astragalus membranaceus (Fisch.) Bunge is one of the most widely used traditional Chinese herbal medicines. It is used as immune stimulant, tonic, antioxidant, hepatoprotectant, diuretic, antidiabetic, anticancer, and expectorant. The purpose of the study was to investigate the curative effects of the decoction obtained from Astragalus membranaceus root in intestinal mucosal injury induced by LPS in mice. An LPS-induced intestinal mucosal injury mice model was applied in the study. METHODS: The mice were post-treated with Astragalus membranaceus decoction (AMD) for 4 days after 3 days LPS induction. ELISA kit was used to detect the content of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-4,IL-6 and IL-8 in the serum of each group mice. The morphological changes in intestinal mucosa at the end of the experiments were observed. Both VH (villus height) and CD (crypt depth) were measured using H&E-stained sections. RESULTS: There were significant differences in IL-1ß, IL-4,IL-6, IL-8 and TNF-α levels in AMD-treated group on the 7th day compared to the controls group. The VH was lower in duodenum, jejunum and the ileum in LPS-treated mice compared to the control animals. Similarly, there was also decrease in V/C. Compared to the control mice, for AMD-treated mice, VH and CD had no significantly differences. CONCLUSIONS: Astragalus membranaceus reduced intestinal mucosal damage and promoted tissue repair by inhibiting the expression of inflammatory cytokine.

Prediction of microRNAs and drought expression pattern analysis in Astragalus membranaceus / 中草药

Objective: To provide a good start for the study of miRNA in Astragalus membranaceus, the miRNAs and their targets were predicted using bioinformatics approach, then the drought-induced expression pattern was analyzed using quantitative RT-PCR. Methods: The deep sequencing data downloaded from public database were assembled using Trinity to establish the transcriptome database. Bioinformatics method was employed to predict miRNAs and their targets, functional classification analyses of targets were conducted. Stem-loop fluorescence quantitative PCR method was used to validate miRNAs, and the drought-induced expression patterns of miRNA were also determined. Results: A transcriptome database with 88 263 sequences was obtained. Based on the transcriptomic sequences, 17 miRNA, classified into 17 families, generated from 17 stem-loop precursors, were identified. A total of 145 genes were predicted to be regulated by these miRNAs, and these genes were involved into diversified biological processes including gene transcription regulation, substance metabolism, signal transduction, stress response, and post translational modification of protein. Eight miRNAs were randomly selected for expression analysis and the results indicated that miR2118 and miR166 might be involved in drought stress response. Conclusion: The miRNAs, the corresponding targets and the drought response miRNAs identified in this study will provide a solid basis for understanding the biological functions of miRNAs in A. membranaceus.

Integrative traditional Chinese medicine therapy reduces the risk of diabetic ketoacidosis in patients with type 1 diabetes mellitus.

ETHNOPHARMACOLOGICAL RELEVANCE: Life-long insulin is the standard treatment for type 1 diabetes mellitus (T1DM). The role of traditional Chinese medicine (TCM) in T1DM is still not clear. The aim of this study is to explore the prescription pattern of TCM and its impact on the risk of diabetic ketoacidosis (DKA) in patients with T1DM. MATERIALS AND METHODS: We retrieved samples from the registry for catastrophic illness patients from the National Health Insurance Research Database (NHIRD). Based on a frequency (1:4) matched case-control design, patients with T1DM in 2000-2011 were designated as cases (TCM users) and controls (non-TCM users). TCM treatment for patients with T1DM was analyzed. The incidence of DKA and the annual costs of emergency visits and hospitalizations were evaluated for all causes. RESULTS: Overall, 416 subjects were TCM users, whereas a total of 1608 matched subjects were classified as non-TCM users. The most common Chinese herbal formula and single herb is Liu-wei-di-huang-wan (Six-ingredient pill of Rehmannia) and Huang-qi (Radix Astragali; Astragalus membranaceus (Fisch.) Bunge, Astragalus membranaceus var. mongholicus (Bunge) P.K.Hsiao), respectively. Compared with non-TCM users, we found a 33% reduction in DKA incidence for all TCM users (aHR 0.67, 95% CI 0.56-0.81, p <0.000) and a 40% reduction for users receiving TCM treatment for more than 180 days (aHR 0.58, 95% CI 0.41-0.82, p <0.01). There were no significant differences between TCM users and non-users in the frequency and medical costs of emergency visits and hospitalizations. CONCLUSIONS: Integrative TCM use may reduce the risk of DKA in patients with T1DM. Our results suggest that TCM may have a substantial positive impact on the management of TIDM.

Analysis on codon bias of farnesyl pyrophosphate synthase gene in Astragalus membranaceus / 中草药

Objective: Farnesyl pyrophosphate synthase (FPS) is the key enzyme in biosynthesis pathway of astragaloside IV. The purpose of the experiment was to provide the theory basis for selecting appropriate expression systems and regulating the content of astragaloside IV. Method: FPS gene coding sequence was cloned based on Astragalus membranaceus from Changbai Mountain. Synonymous codons usage of FPS gene was analyzed by EMBOSS and Codon W programs and compared with the genome of other seven plants, such as Zea mays and Artemisia apiacea, and E. coli. Results: FPS gene of A. membranaceus was bias toward the codon with A and T at the third codon position and there are 22 codons showing the significant differences between FPS gene of A. membranaceus and E. coli genome. Conclution: The codons need to be optimized to improve the expression level of FPS gene in E. coli.

Isolation and purification of a lectin from roots of Astragalus membranaceus / 中草药

Objective To isolate and purify a lectin from the roots of Astragalus membranaceus.Methods The protein was purified using a combination of 20%—60% ammonium sulfate fraction and ConA-Sepharose 4B affinity chromatography.Results The purified protein appeared as a single band with molecular mass of 3.15?104 on SDS-PAGE and the relative molecular mass was estimated by gel filtration on a calibrated Superdex 75 column with apparent molecular weight of 3.35?104.This lectin was a glycoprotein with a neutral carbohydrate content of 10.7%.Conclusion A lectin is isolated and purified from the roots of A.membranaceus for the first time.It is a monomer glycoprotein and its specific activity is 391.9 U/mg.

Cloning and sequence analysis of a novel phenylalanine ammonia-lyase gene from Astragalus membranaceus / 中草药

Objective To clone and sequence the cDNA encoding phenylalanine ammonia-lyase(PAL)gene from Astragalus membranaceus.Methods RT-PCR and RACE Techniques were used to clone a phenylalanine ammonia-lyase gene from A.membranaceus roots with the total RNA as the template.Results The cloned gene named as AmPAL and the Genbank registry number is EF567076.Squence analysis showed that the full-length of AmPAL cDNA was 2 650 bp,including a 2 154 bp open reading frame(ORF).AmPAL was a new number of PAL family that consisted of 718 amino acids with prediated mole-cular weight of 7.805?104 and isoelectric point(PI)of 5.96.At the same time,AmPAL had the homo-logy with PAL of known leguminous plants and shared above 80% identity of amino acid sequences.Conclusion It is the first report that a novel PAL gene is cloned from A.membranaceus.This work lays a foundation for regulating phenylpropanoid pathway of medical plant with AmPAL.

Flavonoid constituents of Astragalus membranaceus var. mongholicus / 中草药

Objective To study the chemical constituents of Astragalus membranaceus var. mongholicus. Methods The constituents were isolated and purified by several chromatographic techniques and identified by chemicophysical properties and spectral analyses. Results Nine flavonoid compounds had been obtained from A. membranaceus var. mongholicus. They were determined as formononetin (Ⅰ), (3R)-8, 2′-dihydroxy-7, 4′-dimethoxy-isoflavane (Ⅱ), calycosin (Ⅲ), (6aR, 11aR)9, 10-dimethoxypterocarpan-3-O-?-D-glucoside (Ⅳ), 7, 2′-dihydroxy-3′, 4′-dimethoxy-isoflavane-7-O-?-D-glucoside (Ⅴ), formononetin-7-O-?-D-glucoside (Ⅵ), calycosin-7-O-?-D-glucoside (Ⅶ), pratensein-7-O-?-D-glucoside (Ⅷ), and genistin (Ⅸ), respectively. Conclusion Compound Ⅷ is obtained from the plants of Astragalus Linn. for the first time and compound Ⅱ is obtained from this plant for the first time. Compounds Ⅰ-Ⅶ show cell multiplication activity.

Morphologic characteristics and identification methods of seeds in Astragalus membranaceus var.mongholicus and A.membranaceus / 中草药

Objective To provide a method for identifying the seeds of Astragalus membranaceus var.mongholicus(AMM) and A.membranaceus(AM) and to provide basic research data for establishing relevant Standard Operating Practice(SOP) in accordance with GAP of traditional Chinese medicinal materials.Methods The seed morphologic characteristics and microcosmic structures were observed by eyes,light microscopes,and electron microscopes;the seed germination rates of AMM and AM were also compared.Results There was no obvious discrimination of the seed morphology between AMM and AM.There was obvious discrimination on the characteristic of germination-hole,the microcosmic structures of seed-umbilici and seed-coats of seeds between AMM and AM.The hard seed percentage for AMM was higher than that of AM,and its sprouting was not even and sprouting peak appeared later than that of AM.(Conclusion) The seeds of AMM and AM can be identified accuratly with electron microscopes.The patterns of germination-hole,microcosmic structures of seed-umbilici and seed-coats can be used as indices to identify the seeds of AMM and AM.The hard seed percentage and germination characteristics can be used to(identify) the seeds of AMM and AM subsidiarily.