The mechanism of "Trichosanthis Fructus-Allii Macrostemonis Bulbus" on phlegm and blood stasis syndrome-related cardiovascular diseases based on network pharmacology and experimental verification / 药学学报

This study aimed to investigate the mechanism of "Trichosanthis Fructus-Allii Macrostemonis Bulbus" (GX) on phlegm and blood stasis syndrome (PBSS) rats combining the methods of network pharmacology and experimental verification. Animal experiment ethical requirements were approved by the Ethical Committee Experimental Animal Center of Anhui University of Chinese Medicine (grant number: AHUCM-rats-2021070). Based on the HPLC-Q-TOF-MS analysis and database, 69 chemical constituents of GX and 163 targets of GX for the treatment of phlegm and blood stasis-related cardiovascular diseases were obtained. Then, key targets such as serine/threonine kinase 1 (Akt1), tumor necrosis factor (TNF), interleukin 6 (IL6), vascular endothelial growth factor A (VEGFA), cellular tumor antigen p53 (Tp53) were screened. Pathway analysis showed that the targets of GX in the treatment of phlegm and blood stasis-relate cardiovascular diseases were mainly involved in PI3K/Akt signaling pathway, sphingolipid metabolism, platelet activation, hypoxia inducible factor-1 (HIF-1), ras-proximate-1 (rap1) and other signaling pathways. In addition, molecular docking analysis showed that apigenin, cucurbitacin D, linolenic acid and kaempferol and other key components had potential binding ability with Akt1, TNF, IL6, VEGFA and Tp53. In the animal experiments, compared to the phlegm and blood stasis syndrome group, GX could significantly improve the traditional Chinese medicine syndrome score, blood lipid, vascular endothelial structure disorders and reduce serum endothelin-1 (ET-1) level, increase serum nitric oxide (NO) and endothelial nitric oxide synthase (eNOS) levels, which could restore aortic endothelial function. In addition, the expression of intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in aorta could be significantly reduced, which could improve the vascular endothelial injury of aorta. Western blot revealed that GX could significantly decrease the phosphorylation levels of phosphoinositide 3-kinase (PI3K) and Akt in aorta. This study revealed the mechanism of GX in treatment of phlegm and blood stasis-relate cardiovascular diseases is consistent with the characteristics of multiple ingredients, multiple targets and multiple pathways. In addition, this study also clarified that the reversal of pathological of phlegm and blood stasis syndrome rats may be related to GX inhibiting PI3K/Akt signaling pathway, which could improve vascular inflammation and vascular endothelial function injury.

Five new spirosterol saponins from Allii Macrostemonis Bulbus / 中国天然药物

Five new spirostanol saponins (1-5) and seven known compounds (6-12) were isolated from the n-butanol fraction of 75% ethanol extract of Allii Macrostemonis Bulbus. The identification and structural elucidation of all the isolates were performed through extensive 1D and 2D NMR experiments, HR-ESI-MS data analysis and comparisons with literature values. Antioxidant evaluation showed that compounds 6-11 exhibited certain scavenging effects on ABTS radical, where compounds 6, 7 and 11 had IC50 values of 0.208, 0.057 and 0.014 mg·mL-1, respectively.

Study on effect of "Trichosanthis Fructus-Allii Macrostemonis Bulbus" on atherosclerosis in ApoE~(-/-) mice based on liver metabonomics / 中国中药杂志

In this study, ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS)-based liver metabolomics approach was used to explore the mechanism of "Trichosanthis Fructus-Allii Macrostemonis Bulbus" in improving atherosclerosis(AS) of mice with apolipoprotein E gene knockout(ApoE~(-/-)). AS mouse model was induced by high-fat diet. The pathological and biochemical indexes such as the histopathological changes, body weight, liver weight, blood lipid level and inflammatory factors in the liver of mice were determined. The metabolic profiling of mice liver samples was performed with UPLC-Q-TOF-MS. Multiple statistical analysis methods including partial least squares discriminant analysis(PLS-DA) and orthogonal partial least squares discriminant analysis(OPLS-DA) were employed to screen and identify biomarkers. The levels of related enzymes including LCAT, sPLA2, EPT1 and ACER1 were detected. The results showed that "Trichosanthis Fructus-Allii Macrostemonis Bulbus" significantly reduced the areas of aortic plaque and fat vacuoles of liver in AS mice and decreased the accumulation of lipid droplets and liver coefficient. "Trichosanthis Fructus-Allii Macrostemonis Bulbus" also regulated the levels of blood lipid and inflammatory injury in the liver. The metabolites of the control group, the model group and the "Trichosanthis Fructus-Allii Macrostemonis Bulbus" group could be distinguished significantly. Fifteen potential biomarkers related to AS were discovered and preliminarily identified, seven of which could be regulated by "Trichosanthis Fructus-Allii Macrostemonis Bulbus" in a trend of returning to normal. Metabolic pathway analysis screened out two major metabolic pathways. "Trichosanthis Fructus-Allii Macrostemonis Bulbus" obviously regulated the levels of LCAT, sPLA2, EPT1 and ACER1. It was inferred that "Trichosanthis Fructus-Allii Macrostemonis Bulbus" could play a major role in AS treatment by regulating glycerophospholipid and sphingolipid metabolism disorders in the liver, with the mechanism probably relating to the intervention of the expression of LCAT, sPLA2, EPT1 and ACER1.

Effect of Trichosanthis Fructus-Allii Macrostemonis Bulbus on Formation of Atherosclerotic Plaque in ApoE-/- Mice Based on Autophagy of Vascular Smooth Muscle / 中国实验方剂学杂志

Objective:To explore the effect of Trichosanthis Fructus-Allii Macrostemonis Bulbus medicine on the proliferation and autophagy levels of aortic plaque vascular smooth muscle cells （VSMCs） in ApoE^-/- mice with atherosclerosis （AS）. Method:A total of 40 ApoE^-/- mice were fed with high-fat diet to replicate AS animal models. They were randomly divided into model group， Trichosanthis Fructus-Allii Macrostemonis Bulbus group， rapamycin group and atorvastatin group， and 10 mice with normal diet C57BL/6J mice were the blank group. The blank group and the model groups were given normal saline by gavage， while Trichosanthis Fructus-Allii Macrostemonis Bulbus group， rapamycin group and atorvastatin group were given corresponding drugs by gavage for 8 weeks. After the experiment， the mice were sacrificed. Total cholesterol （TC）， triglyceride （TG）， high-density lipoprotein cholesterol （HDL-C） and low-density lipoprotein cholesterol （LDL-C） levels were detected by the Microplate reader， the ratio of the aortic plaque area to the total area was observed and measured by staining with aortic gross oil red O. Western blot method was used to detect the proliferation-related protein proliferating cell nuclear antigen （PCNA） and <italic>α</italic>-smooth muscle actin （<italic>α</italic>-SMA） levels of VSMCs in the aortic media. Transmission electron microscopy was used to observe the autophagosomes of VSMCs and detect the expressions of VSMCs autophagy-related proteins Beclin-1， light chain proteinⅡ （LC3Ⅱ） and p62. Result:Compared with the model group， the Trichosanthis Fructus-Allii Macrostemonis Bulbus group showed significant reduction in the aortic lipid accumulation and plaque area of AS mice and the levels of TC， TG， LDL-C （<italic>P</italic><0.01） and increase of HDL-C （<italic>P</italic><0.05）. Trichosanthis Fructus-Allii Macrostemonis Bulbus significantly reduced the levels of proliferation-related antigens PCNA and <italic>α</italic>-SMA in aortic VSMCs （<italic>P</italic><0.01）， and inhibited the excessive proliferation of VSMCs. Trichosanthis Fructus-Allii Macrostemonis Bulbus significantly up-regulated Beclin-1 and LC3Ⅱ in aortic VSMCs protein expression， decreased p62 accumulation （<italic>P</italic><0.01）， increased the expressions of VSMCs autophagosomes， and increased the autophagy level of VSMCs. Conclusion:Trichosanthis Fructus-Allii Macrostemonis Bulbus regulates blood lipid levels in AS mice， and inhibits the excessive proliferation of aortic VSMCs and plaque formation in the aorta of AS mice. The mechanism may be related to the up-regulation of the autophagy activity of VSMCs.

Mechanism of Action of Trichosanthis Fructus-Allii Macrostemonis Bulbus Herb Pairs Against Hyperlipidemia Based on Network Pharmacology / 中国实验方剂学杂志

Objective:Based on the hyperlipidemia rat model and network pharmacology technology, the mechanism of action of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs against hyperlipidemia was analyzed. Method:The levels of blood lipids and inflammatory factors were measured through prophylactic administration of low, medium and high-dose Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs in hyperlipidemia rats. The active ingredients of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs were screened out through Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP) and text mining. The targets of active ingredients screened through the Swiss Target Prediction, Similarity ensemble approach (SEA), DrugBank database. The disease targets were collected through Therapeutic Target Database (TTD), Online Mendelian Inheritance in Man (OMIM), DrugBank, DisGeNET database. The targets of active ingredients and disease target were integrated, and screened through topological parameters to gain the main candidate targets of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs against hyperlipidemia. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and the gene ontology (GO) functional enrichment analysis were conducted through ClueGO and Database for Annotation, Visualization and Integrated Discovery (DAVID), respectively. The traditional Chinese medicine-chemical ingredient-target network model, and the target-pathway network model were constructed through Cytoscape, and their crosstalk target and signal pathway were analyzed. Result:Animal experiments showed that the prophylactic administration of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs significantly reduced the levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) in serum of rats with hyperlipidemia, increased high-density lipoprotein (HDL-C) levels, and inhibited the expressions of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). According to the findings, 27 active ingredients, such as mandenol, diosmetin and α-spinasterol, might be the main active ingredients of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs, 16 crosstalk targets and 10 signal pathways might be the main therapeutic targets and pathways, main targeting apolipoprotein A1 (APOA1), apolipoprotein A2 (APOA2), apolipoprotein C3 (APOC3), lipoprotein lipase (LPL), low-density lipoprotein receptor (LDLR) and other crosstalk targets affected cholesterol metabolism, bile secretion, peroxisome proliferator activated receptor (PPAR) signaling pathway in regulating the lipid level, targeting tumor necrosis factor (TNF), IL-6, interleukin-1β (IL-1β), mitogen-activated protein kinase 1 (MAPK1), C-C motif chemokine 2 (CCL2) and other crosstalk targets affected tumor necrosis factor (TNF) signaling pathway, Toll-like receptor signaling pathway, interleukin-17 (IL-17) signaling pathway, and hypoxia inducible factor (HIF) signaling pathway in regulating the inflammatory factor level. The DAVID database for GO enrichment analysis showed that the hyperlipidemia was treated mainly through biological processes, such as inflammation, lipid localization, storage and lipid metabolism. Conclusion:These findings can predict the mechanism of action of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs against hyperlipidemia, and provide a theoretical basis for the material basis and clinical application of Trichosanthis Fructus-Allii Macrostemonis Bulbus herb pairs.

Study on the Effect and Mechanism of Allii Macrostemonis Bulbus on Blood Lipid Levels in Hyperlipi-demia Model Rats / 中国药房

OBJECTIVE:To investigate the effect and mechanism of Allii macrostemonis bulbus on blood lipid levels in hyperlipidemia model rats,and to provide reference for clinical use of Allii macrostemonis bulbus to reduce blood lipid. METHODS:A total of 10 normal rats were included in normal control group and given common diet. Other 50 rats were given hyperlipid diet to induce hyperlipidemia rat model. 40 model rats were randomly divided into model group(hyperlipid diet),Allii macrostemonis bulbus low-dose,medium-dose and high-dose groups(0.83,1.67,2.50 g/kg,fed by hyperlipid diet which containing 10%Allii macrostemonis bulbus 8.3,16.7,25.0 g/kg,fill with hyperlipid feed in patients with insufficient food intake). After fed for 45 d,the contents of TC,TG,LDL-C and HDL-C in serum of rats were detected. Liver,spleen,renal and cardiac indexes of rats were calculated. mRNA expression of low density lipoprotein receptor(LDLR)and liver X-receptor α(LXRα)were detected in liver tissue of rats. RESULTS:Compared with normal control group,the contents of TC and LDL-C in serum and liver index of rats were increased significantly in model group,while the content of HDL-C in serum and mRNA expressions of LDLR and LXR α in liver tissue were decreased significantly,with statistical significance(P<0.01). Compared with model group,the contents of TC and LDL-C in serum were decreased significantly in Allii macrostemonis bulbus groups,while the content of HDL-C was increased significantly. mRNA expressions of LDLR and LXR α in liver tissue were increased significantly in Allii macrostemonis bulbus medium-dose and high-dose groups,while liver and spleen indexes were decreased significantly,with statistical significance(P<0.05 or P<0.01). CONCLUSIONS:Allii macrostemonis bulbus shows good blood-lipid lowering effect,the mechanism of which may be associated with up-regulating mRNA expressions of LDLR and LXRα in liver tissue.

HPLC fingerprint analysis on Qibai Pingfei Granules and multi-components determination / 中草药

Objective: To establish an HPLC fingerprint of the compounds in Qibai Pingfei Granules (QPG), and to make a quantitative analysis. Methods: Sample was extracted by 50% methanol. Phenomenex Luna C18 column (250 mm × 4.6 mm, 5 μm) was used with a mobile phase of methanol-0.2% formic acid gradient elution. The flow rate was 1.0 mL/min, the detection wavelength was 250 nm, and the column temperature was 30℃. The chemical component fingerprint similarity of 10 batches of QPG was calculated with Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012) published by National Pharmacopoeia Committee and the common peaks were identified by reference compounds. Results: Fingerprints of 10 batches of QPG were established and the similarities to the common mode were above 0.96. Totally 25 common peaks were found. Among them, peak 1 belonged to Pheretima, peaks 2 and 3 belonged to Pheretima and Ginseng Radix et Rhizoma (GRR), peak 4 belonged to GRR, Schisandrae Chinensis Fructus (SCF), and Chuanxiong Rhizoma (CR), peak 5 belonged to GRR, Allii Macrostemonis Bulbus (AMB), CR, and Pheretima, peaks 6, 8, 22, 23, 24, and 25 belonged to SCF, peak 7 belonged to Astragali Radix (AR) and SCF, peak 9 belonged to GRR and AMB, peak 10 belonged to Descurainiae Semen Lepidii Semen (DSLS) and AMB, peaks 11, 12, 13, 15, and 16 belonged to CR, peak 14 belonged to AR and DSLS, peaks 17, 18, 19, 20, and 21 belonged to AR. Based on the retention time, and UV absorption spectra of reference compounds, six constituents including caffeic acid (peak 12), ferulic acid (peak 13), schizandrol A (peak 22), schisantherin A (peak 23), deoxyschizandrin (peak 24), and schisandrin (peak 25) were identified. The linear ranges of caffeic acid, ferulic acid, schizandrol A, schisantherin A, deoxyschizandrin, and schisandrin were 3.38-108.02, 3.60-115.33, 2.99- 95.61, 2.81-89.77, 3.26-104.17, and 2.89-92.45 μg/mL, respectively. In 10 batches of QPG samples, the contents were as follows: caffeic acid of 0.412-0.429 mg/g, ferulic acid of 0.302-0.317 mg/g, schizandrol of A 0.182-0.195 mg/g, schisantherin A of 0.179-0.195 mg/g, deoxyschizandrin of 0.203-0.215 mg/g, and schisandrin of 0.131-0.144 mg/g, the amount of each indicator composition among different batches changed a litte, and the sample quality is stable. Conclusion: The method has good precision, reproducibile, stability, separation, and can be used for the quality control of QPG.

Phytochemistry and pharmacology of Allii Macrostemonis Bulbus, a traditional Chinese medicine / 中国天然药物

Allii Macrostemonis Bulbus (Xiebai in Chinese), as a famous traditional Chinese medicine, has great medicinal and dietary values since ancient times. In China, the dry bulbs of Allium macrostemon and Allium chinense are both used as its original plants. Pharmacological studies have revealed that both of them could increase plasminogen activator activity and prolong the effect of coagulation to achieve antiplatelet aggregation which validates their traditional uses for the treatment of thoracic obstruction and cardialgia in clinics. Besides, several other significant activities, including lipid-lowering, anti-atherosclerosis, antitumor, antispasmodic, antibacterial, antioxidant, and insecticidal activities, have already been reported. The volatile oils, nitrogenous compounds, and steroidal saponins are the major beneficial compounds. Among them, steroidal saponins are considered as the characteristic constituents. In this review, the current information concerning the phytochemistry and pharmacology of Allii Macrostemonis Bulbus is summarized comprehensively. In addition, several research future perspectives are presented, especially the mechanism of bioactive components and fraction from the bulbs of Allium macrostemon and Allium chinense.

Steroidal saponins from Allii Macrostemonis Bulbus / 中草药

Objective: To study the steroidal saponins from Allii Macrostemonis Bulbus. Methods The silica gel and ODS column chromatographies were used for the isolation and purification of compounds from Allii Macrostemonis Bulbus. Their structures were identified through comparing with references and spectral analyses. Results: Three steroidal saponins were isolated from the 70% ethanol extract from Allii Macrostemonis Bulbus and identified as 5β-spirostane-25(27)-en-3β, 12β-diol-3-O-β-D-glucopyranosyl- (1→2)-β-D-galactopyranoside (1), (25R)-5β-spirostane-3β, 12β-diol-3-O-β-D-glucopyranosyl- (1→2)-β-D-galactopyranoside (2), and 5β-spirostane-25(27)-en-2β, 3β-diol-3-O-β-D-glucopyranosyl- (1→2)-β-D-galactopyranoside (3). Conclusion: Compound 1 is a new compound named macrostemonoside S. Compound 2 is isolated from Allii Macrostemonis Bulbus for the first time.