Aqueous extract of Epimedium sagittatum (Sieb. et Zucc.) Maxim. induces liver injury in mice via pyroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Epimedium sagittatum (Sieb. et Zucc.) Maxim. has been used traditionally in Asia. It can dispel wind and cold, tonify the kidney, and strengthen bones and tendons. However, adverse effects of E. sagittatum have been reported, and the underlying mechanisms remain unclear. AIM OF THE STUDY: This study aimed to investigate liver injury caused by an aqueous extract of E. sagittatum in Institute of Cancer Research (ICR) mice and explore its potential mechanisms. MATERIALS AND METHODS: Dried E. sagittatum leaves were decocted in water to prepare aqueous extracts for ultra-high performance liquid chromatography analysis. Mice were administered an aqueous extract of E. sagittatum equivalent to either 3 g raw E. sagittatum/kg or 10 g raw E. sagittatum/kg once daily via intragastric injection for three months. The liver weights and levels of the serum biochemical parameters including alanine transaminase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), total bilirubin (TBIL), and alkaline phosphatase were measured. Hematoxylin-eosin staining was performed for histopathology. Apoptosis was detected using the TUNEL apoptosis assay kit. IL-1ß was detected using ELISA kits. Proteomics was used to identify the differentially expressed proteins. Western blot analysis was performed to determine the levels of proteins significantly affected by the aqueous extract of E. sagittatum. RESULTS: E. sagittatum treatment increased the liver weights and liver coefficients, and ALT and AST levels significantly increased (p < 0.05). A high dose of E. sagittatum significantly increased LDH and TBIL levels (p < 0.05). Ruptured cell membranes and multiple sites of inflammatory cell infiltration were also observed. No evidence of apoptosis was observed. IL-1ß levels were significantly increased (p < 0.05). The expressions of PIK3R1, p-MAP2K4, p-Jun N-terminal kinase (JNK)/JNK, p-c-Jun, VDAC2, Bax, and CYC were upregulated, whereas that of Bcl-2 was inhibited by E. sagittatum. The expression of cleaved caspase-1 was significantly increased; however, its effects on GSDMD and GSDMD-N were significantly decreased. The expression levels of cleaved caspase-3 and its effector proteins GSDME and GSDME-N significantly increased. CONCLUSIONS: Our results suggest that the aqueous extract of E. sagittatum induces liver injury in ICR mice after three months of intragastric injection via inflammatory pyroptosis.

In vitro simulated saliva, gastric, and intestinal digestion followed by faecal fermentation reveals a potential modulatory activity of Epimedium on human gut microbiota.

Herba Epimedii, known for its rich array of bioactive ingredients and widespread use in ethnopharmacological practices, still lacks a comprehensive understanding of its gastrointestinal biotransformation. In this study, we qualitatively explored the dynamic changes in Epimedium sagittatum components during in vitro simulated digestions, with a quantitative focus on its five major flavonoids. Notably, significant metabolism of E. sagittatum constituents occurred in the simulated small intestinal fluid and colonic fermentation stages, yielding various low molecular weight metabolites. Flavonoids like kaempferol glycosides were fully metabolized in the simulated intestinal fluid, while hyperoside digestion occurred during simulated colon digestion. Colonic fermentation led to the production of two known bioactive isoflavones, genistein, and daidzein. The content and bioaccessibility of the five major epimedium flavonoids-icariin, epimedin A, epimedin B, epimedin C, and baohuoside I-significantly increased after intestinal digestion. During colon fermentation, these components gradually decreased but remained incompletely metabolized after 72 h. Faecal samples after E. sagittatum fermentation exhibited shift towards dominance by Lactobacillus (Firmicutes), Bifidobacterium (Actinobacteria), Streptococcus (Firmicutes), and Dialister (Firmicutes). These findings enhance our comprehension of diverse stages of Herba Epimedii constituents in the gut, suggesting that the primary constituents become bioaccessible in the colon, where new bioactive compounds may emerge.

Comprehensive characterization of Epimedium-Rhizoma drynariae herb pair in rat plasma, urine, and feces metabolic profiles by UHPLC-Q-Orbitrap HRMS combined with diagnostic extraction strategy and multicomponent pharmacokinetic study by UHPLC-MS/MS.

Epimedium-Rhizoma drynariae (EP-RD) was a well-known herb commonly used to treat bone diseases in traditional Chinese medicine. Nevertheless, there was incomplete pharmacokinetic behavior, metabolic conversion and chemical characterization of EP-RD in vivo. Therefore, this study aimed to establish metabolic profiles combined with multicomponent pharmacokinetics to reveal the in vivo behavior of EP-RD. Firstly, the diagnostic product ions (DPIs) and neutral losses (NLs) filtering strategy combined with UHPLC-Q-Orbitrap HRMS for the in vitro chemical composition of EP-RD and metabolic profiles of plasma, urine, and feces after oral administration of EP-RD to rats were proposed to comprehensively characterize the 47 chemical compounds and the 97 exogenous in vivo (35 prototypes and 62 metabolites), and possible biotransformation pathways of EP-RD were proposed, which included phase I reactions such as hydrolysis, hydrogenation, dehydrogenation, hydroxylation, dehydroxylation, isomerization, and demethylation and phase II reactions such as glucuronidation, acetylation, methylation, and sulfation. Moreover, a UHPLC-MS/MS quantitative approach was established for the pharmacokinetic analysis of seven active components: magnoflorine, epimedin A, epimedin B, epimedin C, icariin, baohuoside II, and icariin II. Results indicated that the established method was reliably used for the quantitative study of plasma active ingredients after oral administration of EP-RD in rats. Compared to oral EP alone, the increase in area under curves and maximum plasma drug concentration (P < 0.05). This study increased the understanding of the material basis and biotransformation profiles of EP-RD in vivo, which was of great significance in exploring the pharmacological effects of EP-RD.

[Determination of 13 chemical components of Epimedii Folium in pharmacopoeia by UPLC method combined with quantitative analysis of multicomponents by single-marker].

The quantitative analysis of multicomponents by single-marker(QAMS) was established for 13 chemical components of Epimedii Folium, including neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuoside Ⅰ, so as to investigate the feasibility and accuracy of this method in evaluating the quality of Epimedii Folium materials from different origins and different varieties. Through the scientific and accurate investigation of the experimental method, the external standard method was used to determine the content of 13 chemical components in epimedium brevieornu. At the same time, icariin was used as the internal standard, and the relative correction factors of icariin with neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuoside Ⅰ were established, respectively. The contens of neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuosideⅠ in Epimedii Folium were calculated by QAMS. Finally, the difference between the measured value and the calculated value was compared to verify the accuracy and scientific nature of QAMS in the determination. The relative correction factor of each component had better repeatability, and there was no significant difference between the results of the external standard method and those of QAMS. With icariin as the internal standard, QAMS simultaneously determining neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuoside Ⅰ can be used for quantitative analysis of Epimedii Folium.

Icariin attenuates vascular endothelial dysfunction by inhibiting inflammation through GPER/Sirt1/HMGB1 signaling pathway in type 1 diabetic rats.

Icariin, a flavonoid glycoside, is extracted from Epimedium. This study aimed to investigate the vascular protective effects of icariin in type 1 diabetic rats by inhibiting high-mobility group box 1 (HMGB1)-related inflammation and exploring its potential mechanisms. The impact of icariin on vascular dysfunction was assessed in streptozotocin (STZ)-induced diabetic rats through vascular reactivity studies. Western blotting and immunofluorescence assays were performed to measure the expressions of target proteins. The release of HMGB1 and pro-inflammation cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The results revealed that icariin administration enhanced acetylcholine-induced vasodilation in the aortas of diabetic rats. It also notably reduced the release of pro-inflammatory cytokines, including interleukin-8 (IL-8), IL-6, IL-1ß, and tumor necrosis factor-alpha (TNF-α) in diabetic rats and high glucose (HG)-induced human umbilical vein endothelial cells (HUVECs). The results also unveiled that the pro-inflammatory cytokines in the culture medium of HUVECs could be increased by rHMGB1. The increased release of HMGB1 and upregulated expressions of HMGB1-related inflammatory factors, including advanced glycation end products (RAGE), Toll-like receptor 4 (TLR4), and phosphorylated p65 (p-p65) in diabetic rats and HG-induced HUVECs, were remarkably suppressed by icariin. Notably, HMGB1 translocation from the nucleus to the cytoplasm in HUVECs under HG was inhibited by icariin. Meanwhile, icariin could activate G protein-coupled estrogen receptor (GPER) and sirt1. To explore the role of GPER and Sirt1 in the inhibitory effect of icariin on HMGB1 release and HMGB-induced inflammation, GPER inhibitor and Sirt1 inhibitor were used in this study. These inhibitors diminished the effects of icariin on HMGB1 release and HMGB1-induced inflammation. Specifically, the GPER inhibitor also negated the activation of Sirt1 by icariin. These findings suggest that icariin activates GPER and increases the expression of Sirt1, which in turn reduces HMGB1 translocation and release, thereby improving vascular endothelial function in type 1 diabetic rats by inhibiting inflammation.

Flavonoid extracted from Epimedium attenuate cGAS-STING-mediated diseases by targeting the formation of functional STING signalosome.

Hyperactivation of the cyclic-GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signalling pathway has been shown to be associated with the development of a variety of inflammatory diseases, and the discovery of an inhibitor of the cGAS-STING signalling pathway holds great promise in the therapeutic interventions. Epimedium flavonoid (EF), a major active ingredient isolated from the medicinal plant Epimedium, has been reported to have good anti-inflammatory activity, but its exact mechanism of action remains unclear. In the present study, we found that EF in mouse bone marrow-derived macrophages (BMDMs), THP-1 (Tohoku Hospital Pediatrics-1) as well as in human peripheral blood mononuclear cells (hPBMC) inhibited the activation of the cGAS-STING signalling pathway, which subsequently led to a decrease in the expression of type I interferon (IFN-ß, CXCL10 and ISG15) and pro-inflammatory cytokines (IL-6 and TNF-α). Mechanistically, EF does not affect STING oligomerization, but inhibits the formation of functional STING signalosome by attenuating the interaction of interferon regulatory factor 3 (IRF3) with STING and TANK-binding kinase 1 (TBK1). Importantly, in vivo experiments, EF has shown promising therapeutic effects on inflammatory diseases mediated by the cGAS-STING pathway, which include the agonist model induced by DMXAA stimulation, the autoimmune inflammatory disease model induced by three prime repair exonuclease 1 (Trex1) deficiency, and the non-alcoholic steatohepatitis (NASH) model induced by a pathogenic amino acid and choline deficiency diet (MCD). To summarize, our study suggests that EF is a potent potential inhibitor component of the cGAS-STING signalling pathway for the treatment of inflammatory diseases mediated by the cGAS-STING signalling pathway.

Icariin mitigates anxiety-like behaviors induced by hemorrhagic shock and resuscitation via inhibiting of astrocytic activation.

BACKGROUND: Abnormal activation of astrocytes in the amygdala contributes to anxiety after hemorrhagic shock and resuscitation (HSR). Nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)-associated epigenetic reprogramming of astrocytic activation is crucial to anxiety. A bioactive monomer derived from Epimedium icariin (ICA) has been reported to modulate NF-κB signaling and astrocytic activation. PURPOSE: The present study aimed to investigate the effects of ICA on post-HSR anxiety disorders and its potential mechanism of action. METHODS: We first induced HSR in mice through a bleeding and re-transfusion model and selectively inhibited and activated astrocytes in the amygdala using chemogenetics. Then, ICA (40 mg/kg) was administered by oral gavage once daily for 21 days. Behavioral, electrophysiological, and pathological changes were assessed after HSR using the light-dark transition test, elevated plus maze, recording of local field potential (LFP), and immunofluorescence assays. RESULTS: Exposure to HSR reduced the duration of the light chamber and attenuated open-arm entries. Moreover, HSR exposure increased the theta oscillation power in the amygdala and upregulated NF-κB p65, H3K27ac, and H3K4me3 expression. Contrarily, chemogenetic inhibition of astrocytes significantly reversed these changes. Chemogenetic inhibition in astrocytes was simulated by ICA, but chemogenetic activation of astrocytes blocked the neuroprotective effects of ICA. CONCLUSION: ICA mitigated anxiety-like behaviors induced by HSR in mice via inhibiting astrocytic activation, which is possibly associated with NF-κB-induced epigenetic reprogramming.

Study on the intervention effect of Epimedium before and after suet-oil-processed on kidney yang deficiency rats based on intestinal flora and fecal metabolomics.

Epimedium is a Chinese herbal medicine commonly used in clinical practice to reinforce yang. Previous studies have shown that Epimedium fried with suet oil based has the best effect on warming kidney and promoting yang. Evidence suggests a relationship between kidney yang deficiency syndrome (KYDS) and metabolic disorders of the intestinal microflora. However, the specific interaction between KYDS and the intestinal microbiome, as well as the internal regulatory mechanism of the KYDS intestinal microbiome regulated by Epimedium fried with suet oil, remain unclear. The purpose of this study was to investigate the regulatory effects of different processed products of Epimedium on intestinal microflora and metabolites in rats with kidney yang deficiency, and to reveal the processing mechanism of Epimedium fried with suet oil warming kidney and helping yang. 16 S rRNA and LC-MS/MS technology were used to detect fecal samples. Combined with multivariate statistical analysis, differential intestinal flora and metabolites were screened. Then the content of differential bacteria was then quantified using quantitative real-time fluorescence PCR. Furthermore, the correlation between differential bacterial flora and metabolites was analyzed using Spearman's method. The study found that the composition of intestinal flora in rats with kidney yang deficiency changed compared to healthy rats. Epimedium fried with suet oil could increase the levels of beneficial bacteria, while significantly reducing the levels of harmful bacteria. Real-time quantitative PCR results were consistent with 16 S rRNA gene sequencing analysis. Fecal metabolomics revealed that KYDS was associated with 30 different metabolites, involving metabolic pathways steroid hormone biosynthesis etc. Moreover, differential bacteria were closely correlated with potential biomarkers. Epimedium could improve metabolic disorders associated with KYDS by acting on the intestinal flora, with Epimedium fried with suet oil demonstrating the most effective regulatory effect. Its potential mechanism may involve the regulation of abnormal metabolism and the impact on the diversity and structure of the intestinal flora.

[Improvement of solubility of epimedium flavonoid secondary glycoside components by traditional Chinese medicine polysaccharides and its mechanism].

The poor solubility of insoluble components of traditional Chinese medicine(TCM) is an important factor restricting the development of its preparations. Natural polysaccharides of TCM can be used as functional components to increase the solubility of insoluble components. Epimedium flavonoid secondary glycoside components(EFSGC) have been shown to have positive effects on the prevention and treatment of osteoporosis, but they exhibit poor solubility. Therefore, the strategy of solubilizing EFSGC with TCM polysaccharides was adopted, and its effect on the permeability and stability of EFSGC was evaluated in this study. Based on the equilibrium solubility experiment of EFSGC, it was found that Panax notoginseng crude polysaccharide(PNCP) had the best solubilization effect on EFSGC among the ten kinds of TCM polysaccharides, which increased the solubility of EFSGC from 0.8 mg·mL~(-1) to 13.3 mg·mL~(-1). It should be noted that after the solubilization of EFSGC by preparation technology, the effects on permeability and stability should be considered. Therefore, this study also investigated these two properties. The results showed that PNCP increased the effective transmittance of EFSGC from 50.5% to 71.1%, which could increase the permeability of EFSGC significantly. At the same time, it could improve the stability of EFSGC in the simulated gastric juice environment. In order to explain the solubilization mechanism of PNCP on EGSGC, critical micelle concentration, particle size, potential, differential scanning calorimetry, and infrared spectroscopy were analyzed. It was preliminarily inferred that the mechanism was as follows: PNCP and EFSGC could self-assemble into aggregates for solubilization by intermolecular hydrogen bonding interaction in water. In summary, PNCP can not only improve the solubility of EFSGC but also improve its permeability and stability. This study lays the foundation for the application of TCM polysaccharides as a functional component to solubilize insoluble components.

Structural identification of an polysaccharide isolated from Epimedium brevicornum and its beneficial effect on promoting osteogenesis in osteoblasts induced by high glucose.

AIM: Diabetes osteoporosis (DOP) is a chronic bone metabolic disease induced by diabetes, whose morbidity continues to increase. Epimedium brevicornum Maxim (EB), a popular Chinese traditional medicine, has been used to treat bone diseases in China for thousands of years. But its material basis and specific mechanism of action are not clear. METHODS: Epimedium brevicornum crude polysaccharide (EPE) is the main component, in this research the characterized the structure of EBPC1 purified from EPE was detected and its effects on cell proliferation, differentiation, and cytoskeletal in osteoblasts induced by high glucose. RESULTS: The molecular weight of EBPC1 was 10.5 kDa. It was mainly comprised of glucose and galactose, and the backbone of EBPC1 was→4)-α-D-Galp-(1→4)-α-D-Galp-(1→6)-ß-D-Galp-(1→6)-ß-D-Galp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→. The results from in vitro experiments revealed that EBPC1 significantly increased alkaline phosphatase (ALP) activity and mineralized nodule formation in primary osteoblasts, also significantly up-regulated expression of Alp mRNA and Runx2 mRNA in the presence of EBPC1 pretreatment. Moreover, EBPC1 modulated apoptosis via the regulation of Bax/Bcl2. CONCLUSION: These results indicate that EBPC1 treatment can promote osteogenesis during DOP, which can ameliorate apoptosis by regulating Bax/Bcl2 and accelerating osteogenesis in osteoblasts.

Advancements in the Biotransformation and Biosynthesis of the Primary Active Flavonoids Derived from Epimedium.

Epimedium is a classical Chinese herbal medicine, which has been used extensively to treat various diseases, such as sexual dysfunction, osteoporosis, cancer, rheumatoid arthritis, and brain diseases. Flavonoids, such as icariin, baohuoside I, icaritin, and epimedin C, are the main active ingredients with diverse pharmacological activities. Currently, most Epimedium flavonoids are extracted from Epimedium plants, but this method cannot meet the increasing market demand. Biotransformation strategies promised huge potential for increasing the contents of high-value Epimedium flavonoids, which would promote the full use of the Epimedium herb. Complete biosynthesis of major Epimedium flavonoids by microbial cell factories would enable industrial-scale production of Epimedium flavonoids. This review summarizes the structures, pharmacological activities, and biosynthesis pathways in the Epimedium plant, as well as the extraction methods of major Epimedium flavonoids, and advancements in the biotransformation and complete microbial synthesis of Epimedium flavonoids, which would provide valuable insights for future studies on Epimedium herb usage and the production of Epimedium flavonoids.

Flavonoids and prenylhydroquinones from the prepared folium of Epimedium sagittatum Maxim. and their inhibition against phosphodiesterase5A.

An effort to identify novel active substances of the prepared folium of Epimedium sagittatum Maxim. (PFES) that was an important herb for male erectile dysfunction (ED) was taken. At present, phosphodiesterase-5A (PDE5A) is the most important target of new drugs for the treatment of ED. Therefore, the inhibition ingredients in PFES were systematically screened for the first time in this study. Eleven compounds, including eight new flavonoids and three prenylhydroquinones were isolated: sagittatosides DN (1-11), and their structures were elucidated by spectra and chemical analyses. Among them, a novel prenylflavonoid with oxyethyl group (1) was obtained and three prenylhydroquinones (9-11) were firstly isolated from Epimedium. All compounds were analyzed for the inhibition against PDE5A by molecular docking, and they all showed significant binding affinity as same as sildenafil. Their inhibitory activities were verified, and the results showed compound 6 had significant inhibition against PDE5A1. The isolation of new flavonoids and prenylhydroquinones with inhibitory activities of PDE5A from PFES implied that this herb might be a good source for the treatment of ED agents finding.

Research progress on the extraction technology and activity study of Epimedium polysaccharides.

More pharmacological effects of polysaccharides from traditional Chinese medicines have been discovered in recent years. Epimedium has been used for thousands of years as a traditional Chinese medicine in China. Water-soluble Epimedium polysaccharides is one of the main ingredients of Epimedium, which is one of the main active ingredients of Epimedium, mainly composed of mannose, rhamnose, galacturonic acid, glucose, and galactose. The extraction methods of Epimedium polysaccharides including hot water extraction, cellulase extraction, ultrasonic extraction, microwave-assisted extraction, ultrasound compound enzyme and ultra-high pressure extraction, they affect the yield of Epimedium polysaccharides. The characteristics of deproteinization including enzyme deproteinization, macroporous resin deproteinization and Sevag methods are introduced respectively. Some chemical modification methods of Epimedium polysaccharides are also involved such as phosphorylation, sulfation, selenization, and lipids encapsulated. Epimedium polysaccharides have a variety of pharmacological activities, including immune promotion, reproduction promotion, anti-osteoporosis, anti-tumor, antioxidant, anti-fatigue and antivirus, also beneficial to nervous and hematopoietic systems. At present, the research of Epimedium polysaccharides has been in depth. In this paper, the research progress on extraction, purification, chemical modification methods and pharmacological activity of Epimedium polysaccharides summarized. The aim is to provide reference for further research and development of Epimedium polysaccharides.

Screening and characterization of a ß-xylosidase from Bifidobacterium breve K-110 and its application in the biotransformation of the total flavonoids of epimedium to icariin with α-l-rhamnosidase.

Among the flavonoids of epimedium, epimedin B, epimedin C, and icariin are considered to be representative components and their structures are quite similar. Besides sharing the same backbone, the main difference is the sugar groups attached at the positions of C-3 and C-7. Despite their structural similarities, their potencies differ significantly, and only icariin is currently included in the Chinese Pharmacopoeia as a quality marker (Q-marker) for epimedium flavonoids. Furthermore, icariin has the functions of anti-aging, anti-inflammation, antioxidation, anti-osteoporosis, and ameliorating fibrosis. We used bioinformatics to look for the GH43 family ß-xylosidase genes BbXyl from Bifidobacterium breve K-110, which has a length of 1347 bp and codes for 448 amino acids. This will allow us to convert epimedin B and epimedin C into icariin in a specific way. The expression level of recombinant BbXyl in TB medium containing 1 % inulin as carbon source, with an inducer concentration of 0.05 mmol/L and a temperature of 28 °C, was 86.4 U/mL. Previous studies found that the α-l-rhamnosidase BtRha could convert epoetin C to produce icariin, so we combined BbXyl and BtRha to catalyze the conversion of epimedium total flavonoids in vitro and in vivo to obtain the product icariin. Under optimal conditions, in vitro hydrolysis of 5 g/L of total flavonoids of epimedium eventually yielded a concentration of icariin of 678.1 µmol/L. To explore the conversion of total flavonoids of epimedium in vivo. Under the optimal conditions, the yield of icariin reached 97.27 µmol/L when the total flavonoid concentration of epimedium was 1 g/L. This study is the first to screen xylosidases for the targeted conversion of epimedin B to produce icariin, and the first to report that epimedin B and epimedin C in the raw epimedium flavonoids can convert efficiently to icariin by a collaborative of ß-xylosidase and α-l-rhamnosidase.

Sub-chronic toxicity of an aqueous extract of Epimedium sagittatum (Sieb. Et Zucc.) Maxim. in rats.

Epimedium sagittatum (Sieb. et Zucc.) Maxim., a traditional medicinal plant in Asia, is widely used in clinical settings but its safety in vivo is unclear. This study investigated the sub-chronic toxicity of E. sagittatum aqueous extract to rats with a 13-week daily intragastric administration of 7.5, 15, or 30 g/kg. Nine constituents of the aqueous extract were identified by ultra-performance liquid chromatography (UPLC). Organ weights, organ coefficients, serum biochemistry parameters, histopathology, and metabolomic analysis were performed. In female rats, treatment increased the liver, thymus, and adrenal gland coefficients (p < 0.05). Liver, pancreas, and adrenal gland injury were observed. The levels of six metabolites were altered by the treatment (p < 0.05). In male rats, treatment altered liver, heart, and thymus coefficients (p < 0.05) and liver, adrenal gland, and heart injury were observed. The levels of 11 metabolites were altered (p < 0.05). The no-observed-adverse-effect level was not determined but would be below 7.5 g/kg in rats treated for 13 weeks. In female rats, E. sagittatum may injure the liver and pancreas and dysregulate the biosynthesis of phenylalanine, tyrosine, tryptophan, valine, leucine, and isoleucine and the metabolism of phenylalanine. In male rats, the extract may injure the liver and adrenal gland and dysregulate the biosynthesis of valine, leucine, and isoleucine and the metabolism of pyruvate.

Improvement of solubility of epimedium flavonoid secondary glycoside components by traditional Chinese medicine polysaccharides and its mechanism / 中国中药杂志

The poor solubility of insoluble components of traditional Chinese medicine(TCM) is an important factor restricting the development of its preparations. Natural polysaccharides of TCM can be used as functional components to increase the solubility of insoluble components. Epimedium flavonoid secondary glycoside components(EFSGC) have been shown to have positive effects on the prevention and treatment of osteoporosis, but they exhibit poor solubility. Therefore, the strategy of solubilizing EFSGC with TCM polysaccharides was adopted, and its effect on the permeability and stability of EFSGC was evaluated in this study. Based on the equilibrium solubility experiment of EFSGC, it was found that Panax notoginseng crude polysaccharide(PNCP) had the best solubilization effect on EFSGC among the ten kinds of TCM polysaccharides, which increased the solubility of EFSGC from 0.8 mg·mL~(-1) to 13.3 mg·mL~(-1). It should be noted that after the solubilization of EFSGC by preparation technology, the effects on permeability and stability should be considered. Therefore, this study also investigated these two properties. The results showed that PNCP increased the effective transmittance of EFSGC from 50.5% to 71.1%, which could increase the permeability of EFSGC significantly. At the same time, it could improve the stability of EFSGC in the simulated gastric juice environment. In order to explain the solubilization mechanism of PNCP on EGSGC, critical micelle concentration, particle size, potential, differential scanning calorimetry, and infrared spectroscopy were analyzed. It was preliminarily inferred that the mechanism was as follows: PNCP and EFSGC could self-assemble into aggregates for solubilization by intermolecular hydrogen bonding interaction in water. In summary, PNCP can not only improve the solubility of EFSGC but also improve its permeability and stability. This study lays the foundation for the application of TCM polysaccharides as a functional component to solubilize insoluble components.

[Rapid prediction of flavonoid content in Epimedium sagittatum by infrared spectroscopy].

Epimedii Folium is a well-known Chinese herbal medicine with the effect of nourishing kidney and strengthening Yang. Its main active ingredients are flavonoids. In this study, 60 samples of Epimedium sagittatum were collected for the determination of total flavonoids(TF) including the total amount of epimedin A, epimedin B, epimedin C, and icariin(abbreviated as ABCI) specified in the Chinese Pharmacopoeia as well as rhamnosylicariside Ⅱ and icariside Ⅱ. The calibration parameters of "first derivativemultiva-riate scattering correction in 1 900-650 cm~(-1) band(4-point smoothing)" and "first derivativestandard normal variable correction in 4 000-650 cm~(-1) full band(4-point smoothing)" were confirmed respectively. The quantitative model was established via Fourier infrared spectroscopy plus attenuated total reflection(FTIR-ATR) accessory combined with partial least squares(PLS) method and then used to predict the flavonoid content of 11 validation sets. The average prediction accuracy for ABCI in calibration set and validation set was 98.985% and 96.087%, respectively. The average prediction accuracy for TF in calibration set and validation set was 98.998% and 94.771%, respectively. These results indicated that FTIR-ATR combined with PLS model could be used for rapid prediction of flavonoid content in E. sagittatum, with the prediction accuracy above 94.7%. The establishment of this method provides a new solution for the detection of a large number of E. sagittatum samples.

Molecular Mechanism of Epimedium Extract against Ischemic Stroke Based on Network Pharmacology and Experimental Validation.

Ischemic stroke exhibits high morbidity, disability, and mortality, and treatments for ischemic stroke are limited despite intensive research. The potent neuroprotective benefits of Epimedium against ischemic stroke have gained lots of interest. Nevertheless, systematic research on the direct role and mechanisms of Epimedium in ischemic stroke is still lacking. Network pharmacology analysis coupled with experimental verification was utilized to systematically evaluate the potential pharmacological mechanism of Epimedium against ischemic stroke. The TCMSP database was used to mine the bioactive ingredients and Epimedium's targets. The DrugBank, OMIM, and GeneCards databases were employed to identify potential targets of ischemic stroke. GO and KEGG pathway analyses were also carried out. The interaction between active components and hub targets was confirmed via molecular docking. An experimental ischemic stroke model was used to evaluate the possible therapeutic mechanism of Epimedium. As a result, 23 bioactive compounds of Epimedium were selected, and 30 hub targets of Epimedium in its function against ischemic stroke were identified, and molecular docking results demonstrated good binding. The IL-17 signaling pathway was revealed as a potentially significant pathway, with the NF-κB and MAPK/ERK signaling pathways being involved. Furthermore, in vivo experiments demonstrated that Epimedium treatment could improve neurological function and reduce infarct volume. Additionally, Epimedium reduced the activation of microglia and astrocytes in both the ischemic penumbra of the hippocampus and cerebral cortex following ischemic stroke. Western blot and RT-qPCR analyses demonstrated that Epimedium not only depressed the expression of IL-1ß, TNF-α, IL-6, and IL-4 but also inhibited the NF-κB and MAPK/ERK signaling pathways. This study applied network pharmacology and in vivo experiment to explore possible mechanism of Epimedium's role against ischemic stroke, which provides insight into the treatment of ischemic stroke.

New prenylated flavonoid glycosides derived from Epimedium wushanense by ß-glucosidase hydrolysis and their testosterone production-promoting effects.

Six new prenylated flavonoid glycosides, including four new furan-flavonoid glycosides wushepimedoside A-D (1-4) and two new prenyl flavonoid derivatives wushepimedoside E-F (5-6), and one know analog epimedkoreside B (7) were isolated from biotransformation products of the aerial parts of Epimedium wushanense. Their structures were elucidated according to comprehensive analysis of HR-MS and NMR spectroscopic data, and the absolute configurations were assigned using experimental and calculated electronic circular dichroism (ECD) data. The regulatory activity of compounds 1-7 on the production of testosterone in primary rat Leydig cells were investigated, and 4 and 5 exhibited testosterone production-promoting activities. Molecular docking analysis suggested that bioactive compounds 4 and 5 showed the stable binding with 3ß-HSD and 4 also had good affinity with Cyp17A1, which suggested that these compounds may regulate testosterone production through stimulating the expression of the above two key proteins.

Deciphering the molecular mechanism underlying the effects of epimedium on osteoporosis through system bioinformatic approach.

Epimedium has gained widespread clinical application in Traditional Chinese Medicine, with the functions of promoting bone reproduction, regulating cell cycle and inhibiting osteoclastic activity. However, its precise cellular pharmacological therapeutic mechanism on osteoporosis (OP) remains elusive. This study aims to elucidate the molecular mechanism of epimedium in the treatment of OP based on system bioinformatic approach. Predicted targets of epimedium were collected from TCMSP, BATMAN-TCM and ETCM databases. Differentially expressed mRNAs of OP patients were obtained from Gene Expression Omnibus database by performing Limma package of R software. Epimedium-OP common targets were obtained by Venn diagram package for further analysis. The protein-protein interaction network was constructed using Cytoscape software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were carried out by using clusterProfiler package. Molecular docking analysis was conducted by AutoDock 4.2 software to validate the binding affinity between epimedium and top 3 proteins based on the result of protein-protein interaction. A total of 241 unique identified epimedium targets were screened from databases, of which 62 overlapped with the targets of OP and were considered potential therapeutic targets. The results of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that these targets were positive regulation of cell cycle, cellular response to oxidative stress and positive regulation of cell cycle process as well as cellular senescence, FoxO, PI3K-Akt, and NF-kappa B signaling pathways. Molecular docking showed that epimedium have a good binding activity with key targets. Our study demonstrated the multitarget and multi-pathway characteristics of epimedium on OP, which elucidates the potential mechanisms of epimedium against OP and provides theoretical basis for further drug development.