Anhydroicaritin suppresses tumor progression via the PI3K/AKT signaling pathway in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most malignant tumors. The in vitro experiments on the application of Anhydroicaritin (AHI), the active ingredient of Bushen Huayu Decoction, in HCC treatment remain limited, particularly regarding its molecular mechanism. METHODS: The TCMSP platform was used for drug ingredient screening. The GeneCards database and DisGeNET database are used to collect liver cancer targets. PPI network construction of active component-target intersection target was completed with string database. The GO and KEGG pathway analyses were performed via bioinformatics analysis. The molecular docking was used to confirm AHI's target proteins. The in vitro experiments were performed to validate the effect of AHI on HCC cell and explore the molecular mechanism by western blotting analysis. RESULTS: Through the intersection, 155 intersection targets are finally obtained. The top 15 active ingredients were quercetin, kaempferol, beta-sitosterol, luteolin, beta-carotene, Stigmasterol, naringenin, formononetin, baicalein, Anhydroicaritin, isorhamnetin, licochalcone, 7-O-methylisomucronulatol, aloe-emodin and 8-O-Methylreyusi. The molecular mocking analysis showed that the four active components (quercetin, kaempferol, luteolin and AHI) and targets had a good binding activity (affinity ≤ 5 kcal/mol). In vitro experiments reveled that AHI could suppress tumor proliferation, invasion and metastasis of HCC cells. Further analysis showed that AHI inhibited tumor growth by PI3K/AKT signal pathway in HCC. CONCLUSIONS: The Bushen Huayu Decoction and its active ingredient AHI could fight HCC. The potential mechanism may be associated with inhibiting the activation of PI3K/AKT signal pathway, which may serve as a potential treatment for HCC therapy.

Nanoengineered hydrogels as 3D biomimetic extracellular matrix with injectable and sustained delivery capability for cartilage regeneration.

The regeneration of articular cartilage remains a great challenge due to the difficulty in effectively enhancing spontaneous healing. Recently, the combination of implanted stem cells, suitable biomaterials and bioactive molecules has attracted attention for tissue regeneration. In this study, a novel injectable nanocomposite was rationally designed as a sustained release platform for enhanced cartilage regeneration through integration of a chitosan-based hydrogel, articular cartilage stem cells (ACSCs) and mesoporous SiO2 nanoparticles loaded with anhydroicaritin (AHI). The biocompatible engineered nanocomposite acting as a novel 3D biomimetic extracellular matrix exhibited a remarkable sustained release effect due to the synergistic regulation of the organic hydrogel framework and mesopore channels of inorganic mSiO2 nanoparticles (mSiO2 NPs). Histological assessment and biomechanical tests showed that the nanocomposites exhibited superior performance in inducing ACSCs proliferation and differentiation in vitro and promoting extracellular matrix (ECM) production and cartilage regeneration in vivo. Such a novel multifunctional biocompatible platform was demonstrated to significantly enhance cartilage regeneration based on the sustained release of AHI, an efficient bioactive natural small molecule for ACSCs chondrogenesis, within the hybrid matrix of hydrogel and mSiO2 NPs. Hence, the injectable nanocomposite holds great promise for use as a 3D biomimetic extracellular matrix for tissue regeneration in clinical diagnostics.

Enzymatic synthesis of anhydroicaritin, baohuoside and icariin.

Anhydroicaritin (1a), baohuoside (1b) and icariin (1c) were recognized as major pharmacologically active ingredients of Epimedium plants. Their primary means of acquisition were chemical isolation from plants. However, it suffers from low yield, environmental pollution and shortage of plants. Herein, to remedy these problems, biosynthesis was explored to obtain the three active ingredients. Fortunately, with SfFPT as 8-prenyltransferase, EpPF3RT and Ep7GT as glycosyltransferases, kaempferide (1) was transferred to 1a, 1b and 1c enzymatically. Thus, we report the details of this method. This approach represents a promising environmental friendly alternative for the production of these compounds from an abundant analogue.

Identifying the anti-metastasis effect of Anhydroicaritin on breast cancer: Coupling network pharmacology with experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Epimedium brevicornu Maxim. and Cullen corylifolium (L.) Medik. are part of a traditional Chinese medicine (TCM) drug pair (ECDP) widely used in the clinical treatment of breast cancer (BC). Both drugs have been proven to have anti-tumor effect. However, the active ingredients and molecular mechanism of ECDP remain to be explored. AIM OF THE STUDY: To explore the efficacy and potential mechanisms of actions of herb pair through network pharmacology and in vitro and in vivo experiments. MATERIALS AND METHODS: The active ingredients of ECDP were identified using high-performance liquid chromatography. The corresponding potential target genes for ECDP components and BC were extracted from established databases, and the protein-protein interaction network of shared genes was constructed using STRING database. The effective ingredients and targets of ECDP for BC were obtained through the TCMSP database and GeneCards database. The potential targets and pathways were selected through the protein interaction network and enrichment analysis. Proliferation and migration experiments in vitro and tumor growth in vivo were performed to evaluate the effects of Anhydroicaritin (AHI) on BC. RESULTS: AHI is the potential candidate active ingredient of ECDP through TCMSP. Molecular docking revealed that AHI has excellent binding ability with TP53, VEGFA, MMP2, and Met. In vitro experiment results showed that AHI inhibits the growth of MDA-MB-231, 4T1, MCF-7, and SK-BR-3 BC cells. The inhibitory effect of AHI on triple-negative BC cells is more obvious. With the increase of AHI concentration, the colony-forming, migration, and metastasis abilities of the MDA-MB-231 and 4T1 cells gradually decreases. In addition, Western blot and reverse transcription polymerase chain reaction analyses results indicated that AHI downregulates HIF-1α/VEGFA signaling in triple-negative BC cells. AHI inhibits tumor growth and lung metastasis while downregulating the expression of HIF-1α and VEGFA. CONCLUSION: AHI may play an anti-BC effect by inhibiting cancer cell proliferation, invasion, and metastasis. The results of this study may provide a theoretical basis for AHI research and the clinical application of ECDP in BC.

Anhydroicaritin Inhibits EMT in Breast Cancer by Enhancing GPX1 Expression: A Research Based on Sequencing Technologies and Bioinformatics Analysis.

Background: Breast cancer (BC) is the leading cause of cancer-related deaths among women worldwide. The application of advanced technology has promoted accurate diagnosis and treatment of cancer. Anhydroicaritin (AHI) is a flavonoid with therapeutic potential in BC treatment. The current study aimed to determine AHI's mechanism in BC treatment via RNA sequencing, comprehensive bioinformatics analysis, and experimental verification. Methods: Network pharmacology and MTT (3-(4,5)-dimethylthiazolyl-3,5- diphenyltetrazolium bromide) experiments were conducted to first confirm AHI's anti-BC effect. RNA sequencing was performed to identify the genes affected by AHI. Differential expression analysis, survival analysis, gene set enrichment analysis, and immune infiltration analysis were performed via bioinformatics analysis. Western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR) experiment, molecular docking, and drug affinity responsive target stability (DARTS) experiments were also performed to confirm AHI's direct effect on glutathione peroxidase 1 (GPX1) expression. Confocal immunofluorescence analysis was conducted to verify AHI's effect on the occurrence and development of epithelial-mesenchymal transition (EMT). Finally, BC nude mouse xenografts were established, and AHI's molecular mechanism on BC was explored. Results: Network pharmacology results demonstrated that AHI's therapeutic targets on BC were related to the proliferation, invasion, and metastasis of BC cells. AHI significantly inhibited the proliferation of 4T1 and MDA-MB-231 BC cells in the MTT experiments. RNA sequencing results showed that AHI upregulated the GPX1 expression in the 4T1 and MDA-MB-231 BC cells. Next, bioinformatics analysis revealed that GPX1 is less expressed in BC than in normal breast tissues. Patients with high GPX1 expression levels tended to have prolonged overall survival and disease-free survival than patients with low GPX1 expression levels in BC. Western blot and RT-PCR experiments revealed that AHI increased the protein and mRNA levels of GPX1. Molecular docking and DARTS experiments confirmed the direct binding combination between AHI and GPX1. After the evaluation of the EMT scores of 1,078 patients with BC, we found a potential anti-BC role of GPX1 possibly via suppression of the malignant EMT. The confocal immunofluorescence analysis showed that AHI increased E-cadherin expression levels and reduced vimentin expression levels in BC cells. Animal experiments showed that AHI significantly inhibited tumor growth. AHI also inhibited EMT by enhancing GPX1 and caspase3 cleavage, hence inhibiting EMT markers (i.e., N-cadherin and vimentin) and Ki-67. Conclusion: GPX1 plays a critical role in BC, which may be a biomarker for the prognosis. In addition, AHI suppressed EMT by increasing GPX1 expression, which may serve as a potential therapy for BC treatment.

Anti-HIV/SIV activity of icariin and its metabolite anhydroicaritin mainly involve reverse transcriptase.

AIDS, a serious fatal disease caused by the human immunodeficiency virus (HIV), is an epidemic disease for which no effective vaccine has been established. The current therapeutic interventions for AIDS have limited efficacy because they are unable to clear HIV infections and the continuous occurrence of resistant HIV strains. Therefore, the exploitation of new drugs to prevent the spread of AIDS remains a high priority. In this study, the effects of icariin and its metabolite anhydroicaritin on SIV/HIV replication were investigated. In CEM × 174 cells and PBMC cells, both icariin and anhydroicaritin can significantly inhibit HIV-1 or SIVmac251 replication. Furthermore, molecular docking studies revealed that icariin and anhydroicaritin can act on both HIV reverse transcriptase and protease but could not bind to integrase. Reverse transcriptase and protease inhibition biological assays showed that both icariin and anhydroicaritin could significantly inhibit only HIV reverse transcriptase. In summary, the two compounds can significantly inhibit HIV/SIV in vitro and their targets may be mainly involved with HIV reverse transcriptase.

Exploring active compounds of Jinhua Qinggan Granules for prevention of COVID-19 based on network pharmacology and molecular docking / 中草药

Objective: To explore the effective chemical constituents of Jinhua Qinggan Granules for treatment of coronavirus disease 2019 (COVID-19). Methods: The compounds and action targets of eleven herbal medicines in Jinhua Qinggan Granules were collected via TCMSP. The genes corresponding to the targets were queried by the UniProt database, then the “herbal medicine-compound-target” network was established by Cytoscape software. The gene ontology (GO) function enrichment analysis and KEGG pathway enrichment analysis were performed by DAVID to predict their mechanism. Molecular docking was used to analyze the binding force of the core effective compounds in the “herbal medicine-compound-target” network with SARS-CoV-2 3CL hydrolase and angiotensin converting enzyme II (ACE2). Results: The “herbal medicine-compound-target” network contained 154 compounds and 276 targets, and the key targets involved PTGS2, HSP90AB1, HSP90AA1, PTGS1, NCOA2, etc. GO function enrichment analysis revealed 278 items, including ATP binding, transcription factor activation and regulation of apoptosis process, etc. KEGG pathway enrichment screened 127 signaling pathways, including TNF, PI3K/Akt and HIF-1 signaling pathways related to lung injury protection. The results of molecular docking showed that formononetin, stigmasterol, beta-sitosterol, anhydroicaritin and other key compounds have a certain degree of affinity with SARS-CoV-2 3CL hydrolase and ACE2. Conclusion: The effective compounds in Jinhua Qinggan Granules regulate multiple signaling pathways via binding ACE2 and acting on targets such as PTGS2, HSP90AB1, HSP90AA1, PTGS1, NCOA2 for the prevention of COVID-19.

Mechanism of anti-fatigue of Epimedii Folium based on network pharmacology / 中草药

Objective: To study the anti-fatigue mechanism of Epimedii Folium by network pharmacology. Methods: The main active ingredients of Epimedii Folium and the targets of active ingredients were obtained by TCMSP. The GeneCards was used to predict and screen the anti-fatigue targets. The Cytoscape 3.6.1 software was used to construct the active ingredient-disease-target network. The protein interactions network was constructed using the String database. The GO enrichment and KEGG pathways of the targets were analyzed by using DAVID database. Results: Nine active ingredients were screened from Epimedii Folium, including chrysoeriol, kaempferol, anhydroicaritin, C-homoerythrinan,1,6-didehydro-3,15,16-trimethoxy-,(3.beta.)-, 8-(3-methylbut-2-enyl)-2-phenyl- chromone, luteolin, magnograndiolide, quercetin, 8-isopentenyl-kaempferol, which acted on 31 fatigue targets such as PPARG, GABRA1, CASP3, ICAM1, etc. Biological function analysis showed that the targets of Epimedii Folium included cellular response to hypoxia, regulation of apoptotic, positive regulation of nitric oxide biosynthetic, cellular response to hydrogen peroxide, cellular response to hyperoxia, and negative regulation of lipid storage. Signaling pathway analysis showed that Epimedii Folium exerted the anti-fatigue effect by regulating PI3K-Akt, P53, HIF-1, TNF, FoxO, ErbB, MAPK, and other pathways. Conclusion: This study reflects the characteristics of multi-component, multi-target, and multi-pathway of Epimedii Folium, which provides reference for further research on the mechanism of anti-fatigue effects of Epimedii Folium.

Epimedium koreanum Extract and Its Flavonoids Reduced Atherosclerotic Risk via Suppressing Modification of Human HDL.

Atherosclerosis is the key factor responsible for cardiovascular events, which is a major cause of morbidities and mortalities worldwide. It is well known that high-density lipoprotein (HDL) oxidation and glycation increases the risk for atherosclerosis. Epimedium koreanum has been used as a traditional oriental medicine for treating erectile dysfunction, kidney diseases, osteoporosis, and breast cancer. However, no reports on the effects of E. koreanum on HDL modification exist. In this study, we investigated the inhibitory effects of E. koreanum extract and its eight flavonoids, which are: (1) anhydroicaritin 3-O-rhamnoside, (2) ß-anhydroicaritin, (3-5) epimedins A-C, (6) epimedoside A, (7) icariin, and (8) des-O-methyl-ß-anhydroicaritin, against HDL modification. HDLs obtained from pooled human plasma samples were incubated in vitro with E. koreanum extract or each compound in the presence of copper sulfate or fructose. The HDL modifications were evaluated by measuring generation of conjugated dienes, production of thiobarbituric acid reactive substances, change in electrophoretic mobility of apoA-I, advanced glycation end products formation, and apoA-I aggregation. Consequently, E. koreanum extract and compound 8 suppressed HDL modification through inhibition of lipid peroxidation, apoA-I aggregation, negative charge increase, and AGEs formation. In particular, compound 8 showed more potent inhibitory effect on HDL modification than the extracts, suggesting its protective role against atherosclerosis via inhibition of HDL oxidation and glycation.

Anhydroicaritin, a SREBPs inhibitor, inhibits RANKL-induced osteoclastic differentiation and improves diabetic osteoporosis in STZ-induced mice.

Nowadays, more and more attention has been paid to osteoporosis caused by diabetes mellitus. Elevated levels of pro-inflammatory cytokines in diabetic patients activate the activity of osteoclasts through the RANKL/OPG pathway. The nuclear transcription factor SREBP2, a master regulator of cholesterol metabolism, has been found involved in osteoclastogenesis. In our previous study, we have identified anhydroicaritin as a potent inhibitor of transcription factor SREBPs, which improves dyslipidemia and insulin resistance. In this study, we demonstrated that anhydroicaritin could also decrease the level of SREBP2 and its target genes in osteoclasts induced by RANKL without significant cytotoxicity. Moreover, anhydroicaritin suppressed RANKL-induced osteoclasts differentiation. In STZ-induced diabetic mice model, we found that the osteoclasts were largely increased accompanied with deterioration of bone structure. Anhydroicaritin decreased the level of blood glucose and alleviated insulin resistance. More importantly, anhydroicaritin inhibited osteoclast differentiation and rescued diabetes-induced bone loss in vivo. In conclusion, anhydroicaritin, a potent SREBP2 inhibitor, inhibits the osteoclasts formation and improves diabetes-induced bone loss.

Analysis on chemical constituents of Epimedii Folium by UPLC-Q-TOF-MS / 中草药

Objective: To establish a quick method of ultra-performance liquid chromatography-quadruple time-of-fight mass spectrometry (UPLC-Q-TOF-MS) for the identification of chemical constituents in Epimedii Folium. Methods: The separation was performed on the chromatographic column of Waters Acquity UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm). The mobile phase consisted of 0.1% formic acid-water (A) and 0.1% formic acid-acetonitrile (B) was used as gradient elute. The flow rate was 0.4 mL/min gradient elution and column temperature was 35 ℃. The injection volume was 5 μL. The negative ion mode was used for TOF-MS scanning. The compounds were identified by retention time, accurate relative molecular mass, and fragment ions in mass spectrometry. Results: Based on the MS/MS of standards and compared with reference results, 29 compounds were discovered. Then 10 batches of Epimedii Folium sample were analyzed, and compounds in them were also identified. Finally, 16 constituents exiting in all samples were found. Conclusion: The results demonstrate that UPLC-Q-TOF-MS method is quick, accurate, and efficient for the identification of the compounds in Epimedii Folium. The identification of chemical compositions and the definition of compositions generally existed in Epimedii Folium which provides some experiment foundation for study of its efficiency substances and quality control.

Anhydroicaritin improves diet-induced obesity and hyperlipidemia and alleviates insulin resistance by suppressing SREBPs activation.

SREBPs play important roles in the regulation of lipid metabolism, and are closely related to the occurrence and development of many metabolic diseases. Small molecular inhibitors of SERBPs are important tools in developing efficient treatment of metabolic diseases. However, there are no listing drug targeting SREBPs. Therefore, there is an urgent need to develop highly specific small molecules that inhibit SREBPs. In this study, using a hepatocyte-based high-throughput screening, we identified anhydroicaritin (AHI) as a novel inhibitor of SREBPs. HepG2, HL-7702, and human primary hepatocytes were used to verify the effects of AHI. We explored the mechanism by which AHI blocks the binding of SCAP/SREBPs complex with Sec23α/24D via regulating LKB1/AMPK/mTOR pathway. AHI reduced liver cell lipid level by preventing de novo lipogenesis. In diet induced obese mice, AHI ameliorated obesity, insulin resistance, fatty accumulation in liver and hyperlipemia. In conclusion, AHI improves diet-induced obesity and alleviates insulin resistance by suppressing SREBPs maturation which is dependent on LKB1/AMPK/mTOR pathway. Thus, AHI can serve as a leading compound for pharmacological control of metabolic diseases.