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.

[Aqueous extract of Epimedium sagittatum mitigates pulmonary fibrosis in mice].

This study aims to investigate the intervention effect of the aqueous extract of Epimedium sagittatum Maxim on the mouse model of bleomycin(BLM)-induced pulmonary fibrosis, so as to provide data support for the clinical treatment of pulmonary fibrosis. Ninety male C57BL/6N mice were randomized into normal(n=10), model(BLM, n=20), pirfenidone(PFD, 270 mg·kg~(-1), n=15), and low-, medium-, and high-dose E. sagittatum extract(1.67 g·kg~(-1), n=15; 3.33 g·kg~(-1), n=15; 6.67 g·kg~(-1), n=15) groups. The model of pulmonary fibrosis was established by intratracheal instillation of BLM(5 mg·kg~(-1)) in the other five groups except the normal group, which was treated with an equal amount of normal saline. On the day following the modeling, each group was treated with the corresponding drug by gavage for 21 days. During this period, the survival rate of the mice was counted. After gavage, the lung index was calculated, and the morphology and collagen deposition of the lung tissue were observed by hematoxylin-eosin(HE) and Masson staining, respectively. The levels of reactive oxygen species(ROS) in lung cell suspensions were measured by flow cytometry. The levels of glutathione peroxidase(GSH-Px), total superoxide dismutase(T-SOD), and malondialdehyde(MDA) the in lung tissue were measured. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling(TUNEL) was employed to examine the apoptosis of lung tissue cells. The content of interleukin-6(IL-6), chemokine C-C motif ligand 2(CCL-2), matrix metalloproteinase-8(MMP-8), transforming growth factor-beta 1(TGF-ß1), alpha-smooth muscle actin(α-SMA), E-cadherin, collagen Ⅰ, and fibronectin in the lung tissue was measured by enzyme-linked immunosorbent assay(ELISA). The expression levels of F4/80, Ly-6G, TGF-ß1, and collagen Ⅰ in the lung tissue were determined by immunohistochemistry. The mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue were determined by qRT-PCR. The content of hydroxyproline(HYP) in the lung tissue was determined by alkaline hydrolysation. The expression of α-SMA and E-cadherin was detected by immunofluorescence, and the protein levels of α-SMA, vimentin, E-cadherin in the lung tissue were determined by Western blot. The results showed the aqueous extract of E. sagittatum increased the survival rate, decreased the lung index, alleviated the pathological injury, collagen deposition, and oxidative stress in the lung tissue, and reduced the apoptotic cells. Furthermore, the aqueous extract of E. sagittatum down-regulated the protein levels of F4/80 and Ly-6G and the mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue, reduced the content of IL-6, CCL-2, and MMP-8 in the alveolar lavage fluid. In addition, it lowered the levels of HYP, TGF-ß1, α-SMA, collagen Ⅰ, fibronectin, and vimentin, and elevated the levels of E-cadherin in the lung tissue. The aqueous extract of E. sagittatum can inhibit collagen deposition, alleviate oxidative stress, and reduce inflammatory response by regulating the expression of the molecules associated with epithelial-mesenchymal transition, thus alleviating the symptoms of bleomycin-induced pulmonary fibrosis in mice.

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.

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.

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.

Direct Flavonoid-Focused Chemical Comparison among Three Epimedium Plants by Online Liquid Extraction-High Performance Liquid Chromatography-Tandem Mass Spectrometry.

It is usually a tedious task to profile the chemical composition of a given herbal medicine (HM) using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) due to the time-consuming sample preparation and laborious post-acquisition data processing procedures. Even worse, some labile compounds may face degradation risks when exposed to organic solvents for a relatively long period. As one of the most popular HMs, the promising therapeutic benefits of Epimedii Herba (Chinese name: Yinyanghuo) are well defined; however, the chemical profile, and in particular those flavonoids that have been claimed to be responsible for the efficacy, remains largely unknown. Attempts are devoted here to achieve direct LC-MS measurement and efficient post-acquisition data processing, and chemome comparison among three original sources of Epimedii Herba, such as Epimedium sagittatum (Esa), E. pubescens (Epu), and E. koreanum (Eko) was employed to illustrate the strategy utility. A home-made online liquid extraction (OLE) module was introduced at the front of the analytical column to comprehensively transfer the compounds from raw materials onto the LC-MS instrument. A mass defect filtering approach was programmed to efficiently mine the massive LC-MS dataset after which a miniature database was built involving all chemical information of flavonoids from the genus Epimedium to draw a pentagonal frame to rapidly capture potential quasi-molecular ions (mainly [M-H]-). A total of 99 flavonoids (66 in Esa, 84 in Eko, and 66 in Epu) were captured, and structurally annotated by summarizing the mass fragmentation pathways from the mass spectrometric data of authentic compounds and an in-house data library as well. Noteworthily, neutral loss of 144 Da was firstly assigned to the neutral cleavage of rhamnosyl residues. Significant species-differences didn't occur among their chemical patterns. The current study proposed a robust strategy enabling rapid chemical profiling of, but not limited to, HMs.

Yellow light promotes the growth and accumulation of bioactive flavonoids in Epimedium pseudowushanense.

The plant species of the genus Epimedium L. are well-known traditional Chinese medicinal herbs with special therapeutic effects on human beings and animals in invigorating sexuality and strengthening muscles and bones. In large-scale cultivating Epimedium that is a typical shade plant species, they are arbitrarily covered with black colored shade nets. However, their optimal growth conditions, especially light, are still less understood. During the investigation of different light qualities on the growth of Epimedium pseudowushanense, it was found that, all the values of plant growth characteristics (except shoot number) and photosynthetic characteristics were lower under red, yellow, or blue light treatment than under white light treatment. However, yellow light treatment had beneficial effects on shoot number, dry biomass (per plant) as well as net photosynthesis rate (Pn) and maximal apparent quantum efficiency (AQY) in E. pseudowushanense when compared with red or blue light treatment. More importantly, we found that E. pseudowushanense accumulated higher levels of bioactive flavonoids under yellow light treatment than under white, red, or blue light treatment. Furthermore, both RNAseq and qPCR analyses revealed that yellow light could highly up-regulate the expression levels of flavonoid biosynthetic genes, in particular CHS1, F3H1, PT_5, and raGT_5 that possibly contributed to the enhanced accumulation of bioactive flavonoids in E. pseudowushanense. Taken together, our study revealed that yellow light is the optimal light for the growth of E. pseudowushanense. Our results provided key information on how to improve the cultivation condition and concurrently enhance the accumulation of bioactive flavonoids in E. pseudowushanense.

[Effective components and signaling pathways of Epimedium brevicornumbased on network pharmacology].

The aim of this paper was to find out the active components of Epimedium brevicornum using network pharmacology, and find the potential targets and mechanisms. The TCMSP database was used to screen the active ingredients, and TTD and DrugBank databases were used to predict the potential targets with the literature mining. The pathway annotation was used to enrich and analyze the active ingredients and potential targets of E. brevicornum. The results showed that E. brevicornum had34 potential target active ingredients, including 21 flavones components, such as icariin, epimedin A, epimedin B, epimedin C, Yinyanghuo A, Yinyanghuo C and so on, 2 lignans involved in (+)-cycloolivil and olivil, 3 sterols consisting of sitosterol, 24-epicampesterol and poriferast-5-en-3beta-ol. The main predicted targets included Ptgs2, NCOA6, RANK, OPG, WNT9B, PTH1R, BMPs, SMAD4A and so on. There were 88 signaling pathways involved in 10 signaling pathways which was related to inflammation, such as NF-kappa B signaling pathway, T cell receptor signaling pathway, IL-17 signaling pathway and 10 pathways which was related to cancer included breast cancer, bladder cancer, pancreatic cancer and so on, and estrogen related signaling pathways included estrogen signaling pathway. This laid the foundation for the discovery of the active components of Epimedium and the study on its mechanism of action.

Understanding the molecular mechanisms underlying the effects of light intensity on flavonoid production by RNA-seq analysis in Epimedium pseudowushanense B.L.Guo.

Epimedium pseudowushanense B.L.Guo, a light-demanding shade herb, is used in traditional medicine to increase libido and strengthen muscles and bones. The recognition of the health benefits of Epimedium has increased its market demand. However, its resource recycling rate is low and environmentally dependent. Furthermore, its natural sources are endangered, further increasing prices. Commercial culture can address resource constraints of it.Understanding the effects of environmental factors on the production of its active components would improve the technology for cultivation and germplasm conservation. Here, we studied the effects of light intensities on the flavonoid production and revealed the molecular mechanism using RNA-seq analysis. Plants were exposed to five levels of light intensity through the periods of germination to flowering, the flavonoid contents were measured using HPLC. Quantification of epimedin A, epimedin B, epimedin C, and icariin showed that the flavonoid contents varied with different light intensity levels. And the largest amount of epimedin C was produced at light intensity level 4 (I4). Next, the leaves under the treatment of three light intensity levels ("L", "M" and "H") with the largest differences in the flavonoid content, were subjected to RNA-seq analysis. Transcriptome reconstruction identified 43,657 unigenes. All unigene sequences were annotated by searching against the Nr, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. In total, 4008, 5260, and 3591 significant differentially expressed genes (DEGs) were identified between the groups L vs. M, M vs. H and L vs. H. Particularly, twenty-one full-length genes involved in flavonoid biosynthesis were identified. The expression levels of the flavonol synthase, chalcone synthase genes were strongly associated with light-induced flavonoid abundance with the highest expression levels found in the H group. Furthermore, 65 transcription factors, including 31 FAR1, 17 MYB-related, 12 bHLH, and 5 WRKY, were differentially expressed after light induction. Finally, a model was proposed to explain the light-induced flavonoid production. This study provided valuable information to improve cultivation practices and produced the first comprehensive resource for E. pseudowushanense transcriptomes.

Isolation and functional characterization of a R2R3-MYB regulator of the anthocyanin biosynthetic pathway from Epimedium sagittatum.

KEY MESSAGE: A R2R3-MYB transcription factor EsAN2 was isolated from Epimedium sagittatum and functionally characterized to regulate the anthocyanin biosynthetic pathway. Epimedium plants are used widely both as traditional Chinese medicinal herbs and ornamental perennials. Anthocyanins, acting as major contributors to plant color diversity, their biosynthesis are regulated by a series of transcription factors, including MYB, bHLH and WD40 protein. Previously, a MYB transcription factor involved in regulation of the anthocyanin pathway from Epimedium sagittatum, EsMYBA1 has been isolated, but was found to be expressed mostly in leaves. In this research, another MYB transcription factor, designated as EsAN2, was isolated from flowers by the screening of E. sagittatum EST database. Preferential expression of EsAN2 in flowers and flower buds was found. Ectopic expression of EsAN2 in tobacco significantly enhanced the anthocyanin biosynthesis and accumulation, both in leaves and flowers. Most structural genes of the anthocyanin biosynthetic pathway were strongly upregulated, as well as two bHLH regulators (NtAn1a and NtAn1b) in old leaves of tobacco overexpressing EsAN2, compared to the control plants. While only three structural genes, chalcone synthase (CHS), chalcone isomerase (CHI) and anthocyanidin synthase (ANS), were upregulated by EsAN2 ectopic expression in tobacco flowers. Yeast two-hybrid assay showed that EsAN2 was capable of interacting with four bHLH regulators of the anthocyanin biosynthetic pathway. These results suggest that EsAN2 is involved in regulation of the anthocyanin biosynthesis in Epimedium flowers. Identification and characterization of EsAN2 provide insight into the coloration of Epimedium flowers and a potential candidate gene for metabolic engineering of flavonoids in the future.

Intestinal Absorption and Metabolism of Epimedium Flavonoids in Osteoporosis Rats.

Herba Epimdii is a traditional Chinese medicine used to treat osteoporosis. Its main pharmacological ingredients are flavonoids. In previous studies conducted in healthy animals, we showed that epimedium flavonoids could be hydrolyzed into secondary glycosides or aglycon by intestinal flora or enzymes, thereby enhancing their absorption and antiosteoporosis activity. To study the medicine in the pathologic state, epimedium flavonoids were incubated with intestinal mucosa and feces in vitro and intestinal perfusion in situ to explore the differences in absorption and metabolism between sham and osteoporosis rats. For osteoporosis rats, the hydrolysis rates of icariin, epimedin A, epimedin B, and epimedin C incubated with intestinal flora for 1 hour were reduced by 0.19, 0.26, 0.19, and 0.14, respectively, compared with that in sham rats. Hydrolysis rates were reduced by 0.21, 0.24, 0.08, and 0.31 for icariin, epimedin A, epimedin B, and epimedin C incubated with duodenal enzymes for 1 hour and by 0.13, 0.09, 0.07, and 0.47 for icariin, epimedin A, epimedin B, and epimedin C incubated with jejunum enzymes, respectively, compared with the sham group. In addition, the apparent permeability coefficient and elimination percentage of the four epimedium flavonoids in the duodenum, jejunum, ileum, and colon decreased by 29%-44%, 32%-50%, 40%-56%, and 27%-53% compared with that in sham rats, respectively. The main metabolites of the four epimedium flavonoids were the same for the two groups after intestinal perfusion, or flora and enzyme incubation. In conclusion, the amount and activity of intestinal flora and enzymes changed in ovariectomized rats, which affected the intestinal absorption and hydrolysis of epimedium flavonoids whose structures contain 7-glucose.

The growth and medicinal quality of Epimedium wushanense are improved by an isolate of dark septate fungus.

CONTEXT: Seven dark-septate endophytic (DSE) fungi have been isolated from the roots of Epimedium wushanense T. S. Ying (Berberidaceae), an important medicinal plant with various pharmacological activities. OBJECTIVE: The current study explores the effects of seven DSE fungi on the growth and accumulation of bioactive compounds in E. wushanense. MATERIALS AND METHODS: Each 1-month-old E. wushanense seedling was inoculated with one of the seven DSE fungi and was grown under greenhouse conditions for 90 d. The molecular identification of the fungi was based on the ITS1-5.8S-ITS2 nuclear ribosomal gene cluster. RESULTS: The results showed that the influence of DSE fungi inoculation varied between strains. Inoculation with DSE8 not only significantly enhanced plant height, root length, leaf area, leaf number, and shoot and root biomass but also improved the total flavonoid and icariin content, with an increase ranging from 20.24% to 237.97%. Three of the seven DSE fungi caused the inoculated plants to die, and the remaining three DSE strains showed neutral or negative effects on plant growth and the accumulation of bioactive compounds. According to the ITS sequence, DSE8 is congeneric to the genus Leptodontidium. DISCUSSION AND CONCLUSION: The findings indicate that application of DSE8 may be valuable to facilitate the cultivation of E. wushanense with a higher biomass and improved medicinal quality.

Icariin attenuates hypoxia-induced oxidative stress and apoptosis in osteoblasts and preserves their osteogenic differentiation potential in vitro.

OBJECTIVES: Icariin, a prenylated flavonol glycoside isolated from traditional Chinese medicinal herb of the genus Epimedium, has been demonstrated to be a potential alternative therapy for osteoporosis, and its action mechanism so far has been mainly attributed to its phytoestrogenic property. As blood supply to bone is considerably reduced with ageing and by the menopause, we hypothesized that icariin treatment would reduce bone loss by preventing ischaemia-induced hypoxic damages to bone. MATERIALS AND METHODS: To investigate effects of icariin treatment on cultured rat calvarial osteoblasts exposed to hypoxic conditions (2% oxygen). RESULTS: Compared to normoxic control, cell viability decreased with time to 50% by 48 h in the hypoxic group, and icariin attenuated the reduction, dose dependently, with 10(-6) and 10(-5)  m concentrations showing significant protective effects. Icariin also inhibited increase of lactate dehydrogenase activity in culture media. Measurements on oxidative stress, cell cycling and cell survival indicated that icariin protected osteoblasts by reducing production of reactive oxygen species and malondialdehyde, increasing superoxide dismutase activity, arresting the cell cycle and inhibiting apoptosis. Icariin also preserved osteogenic differentiation potential of the hypoxic cells in a dose-dependent manner, compared to the hypoxia alone group, as revealed by increased levels of RUNX-2, OSX and BMP-2 gene expression, alkaline phosphatase activity, and formation of mineralized nodules. CONCLUSIONS: Our results demonstrated that icariin attenuated oxidative stress and apoptosis and preserved viability and osteogenic potential of osteoblasts exposed to hypoxia in vitro, and suggested that its anti-osteoporotic effect may be attributed to its anti-hypoxic activity and phytoestrogenic properties.

[Morphological character of growth cycle for Epimedium acuminatim and icariin content analysis].

OBJECTIVE: To study the relationship between morphological characteristics of growth cycle for Epimedium acuminatium and accumulation level of secondary metabolites. METHOD: After making habitat clear, methods of morphology observation, specimen preparation, organism charting were applied to record morphological characteristics of E. acuminatium at different stages. HPLC was employed to detect icariin content of different parts of the plant at the stage of vegetative growth and sexual reproduction. RESULT: The growth cycle of E. acuminatium was divided into vegetative growth stage and sexual and asexual reproduction coexistence stage. Seven to eight years were needed for the plants flowering and seeding of E. acuminatimcan in open land of forest edge, but within good nutrition conditions, growth cycle is shortened into three to four years. The difference of icariin content in two growth stages was not significant. CONCLUSION: Morphological change of lower and foliar organ can be utilized to estimate growth age. But after applying sexual propagation for both root and stem of grown plants, it's been difficult to determine their growth year. If individual biomass or yield is economical, plants can be harvested in both vegetative growth and sexual reproduction stages.

Effect of Chinese traditional herb Epimedium grandiflorum C. Morren and its extract Icariin on osteoarthritis via suppressing NF-kappaB pathway.

Osteoarthritis (OA), which is also called degenerative arthritis, is the leading cause of disabilities in the old people. The Chinese traditional herb Epimedium grandiflorum had long been found to attenuate osteoarthritis process, but the detailed mechanism was not clear. To study the mechanisms of E. grandiflorum in the treatment of osteoarthritis, rabbit osteoarthritis model combined with D-galactose was used. After different treatments for 10 weeks, cartilage sections were analyzed by immunohistochemistry for uPA, uPAR and PAI expression level. E. grandiflorum could significantly attenuate OA condition and decrease uPA, uPAR and PAI expression. The extract of E. grandiflorum, icariin also had a similar effect when compared with E. grandiflorum treatment alone. Rabbit chondrocytes were further isolated to be stimulated by TNFalpha combined with different reagents treatment. Here, icariin treatment significantly reduced nuclear factor kappa B NF-kappaB (P65) activity, decreased uPA expression level and increased Ikappabetaalpha protein level. The results indicated that E. grandiflorum and its extract icariin could attenuate OA condition, reduce the expression of uPA and uPAR and increase PAI in experimental rabbit model and this effect may be conducted by suppressing NF-kappaB activity by increasing IkappaBalpha level.

A R2R3-MYB transcription factor from Epimedium sagittatum regulates the flavonoid biosynthetic pathway.

Herba epimedii (Epimedium), a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. The bioactive components in herba epimedii are mainly prenylated flavonol glycosides, end-products of the flavonoid pathway. Epimedium species are also used as garden plants due to the colorful flowers and leaves. Many R2R3-MYB transcription factors (TFs) have been identified to regulate the flavonoid and anthocyanin biosynthetic pathways. However, little is known about the R2R3-MYB TFs involved in regulation of the flavonoid pathway in Epimedium. Here, we reported the isolation and functional characterization of the first R2R3-MYB TF (EsMYBA1) from Epimedium sagittatum (Sieb. Et Zucc.) Maxim. Conserved domains and phylogenetic analysis showed that EsMYBA1 belonged to the subgroup 6 clade (anthocyanin-related MYB clade) of R2R3-MYB family, which includes Arabidopsis AtPAP1, apple MdMYB10 and legume MtLAP1. EsMYBA1 was preferentially expressed in leaves, especially in red leaves that contain higher content of anthocyanin. Alternative splicing of EsMYBA1 resulted in three transcripts and two of them encoded a MYB-related protein. Yeast two-hybrid and transient luciferase expression assay showed that EsMYBA1 can interact with several bHLH regulators of the flavonoid pathway and activate the promoters of dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS). In both transgenic tobacco and Arabidopsis, overexpression of EsMYBA1 induced strong anthocyanin accumulation in reproductive and/or vegetative tissues via up-regulation of the main flavonoid-related genes. Furthermore, transient expression of EsMYBA1 in E. sagittatum leaves by Agrobacterium infiltration also induced anthocyanin accumulation in the wounded area. This first functional characterization of R2R3-MYB TFs in Epimedium species will promote further studies of the flavonoid biosynthesis and regulation in medicinal plants.

Variation of medicinal components in a unique geographical accession of horny goat weed Epimedium sagittatum Maxim. (Berberidaceae).

Herbal Epimedium species have been widely in Traditional Chinese Medicine for sexual enhancement, immunity improvement, anticancer and anti-aging treatment, with flavonoids and polysaccharides being the major active components. However, exhaustive depletion of wild sources warrants germplasm evaluation and quality resource exploration. A preliminarily analysis had previously indicated that a specific local geographic accession of Epimedium sagittatum found in Luotian (LT) county of Hubei Province (China) had a much higher content of total flavonoids and polysaccharides. In this study, we further investigated the medicinal component variation in the LT type under different light intensities and in different regions by the common-garden experiment. The results indicated a light intensity range of 40-160 µmol/m²/s was the most suitable for the synthesis and accumulation of total flavonoids, while polysaccharide accumulation was negatively correlated with the light intensity. Icariin was the component displaying the highest content among flavonoids, and the content of major flavonoid bioactive components was relatively stable in the third year after cultivation. There was significant correlation between the major flavonol glycoside constituents and the geographic location, and Central China followed by Northern China were the highly suitable regions for cultivation of LT type E. sagittatum. The results revealed that there was a functional balance between flavonoids and polysaccharides at different developmental stages, and the best harvesting stage should consider the primary contents of interest. This study provides important information on the exploration of quality resources, further breeding approaches and cultivation practices of E. sagittatum, and thus the important insights to enhance our understanding of quality control of traditional medicinal plants.

[Variation of icariin and total flavonoid of Epimedium acuminatum in different parts and habitats].

OBJECTIVE: Through comparative study on contents of icariin and total flavonoid of Epimedium acuminatum in different habitats and parts, the distribution and correlation of the two components were observed. METHOD: Twenty-four sample spots in four habitats were set up, and the whole plant of these samples was divided as following: leaf, root, stem and rhizome. Total flavonoid and icariin of samples were determined by using the UV method and the HPLC, respectively. The data was analyzed by SPSS 17.0. RESULT: The distribution of icariin in different parts had the pattern: leaf > root > stem > rhizome, the total flavonoid content was higher in leaf, but it showed no difference in other parts. Habitat had certain effect on icariin accumulation in E. acuminatum. The significant differences of total flavonoid content in E. acuminatum from different parts were not observed. CONCLUSION: The distribution of icariin from different parts and habitats has high selectivity. Metabolism and accumulation of flavonoid content in each part have no difference. Flavonoid content is less affected by environment. Considering the distribution of icariin and flavonoid content in every part and the growth strategy in different habitats comprehensively, it is reasonable to harvest the aerial part for the sustainable utilization of E. acuminatum.

[Distribution and influencing factors of magnoflorine in Epimedium].

Eighty samples of Epimedium from 29 species and were determined in this study. The content of magnoflorine in leaves range between 0. 003% and 2. 603%. The results showed that the content of magnoflorine was quite stable within species except E. wushanense, E. acuminatum, E. hunanense. Genetic factors might be the main influencing ones. The contents of different parts and different collecting time of the medicinal materials were variable.

[Simultaneous determination of ginsenosides and epimedium flavonoids in rat urine by HPLC-UV-ELSD].

OBJECTIVE: To develop and validate a HPLC-UV-ELSD method for the simultaneous determination of ginsenosides and epimedium flavonoids in rat urine after intravenous administration of Jiweiling freeze-dried powder. METHOD: Chromatographic separation was performed on a C18 HPLC column, with gradient elution of acetonitrile and water as mobile phase. An UV detector was used at detection wavelength of 220 nm. An evaporative light scattering detector (ELSD) was used at drift tube temperature of 80 degrees C and gas pressure of 172.4 kPa. RESULT: The calibration curves were linear over the investigated concentration ranges with all correlation coefficients higher than 0.998. The a intra- and inter-day RSD were less than 9.1% and the relative errors were verage extraction recoveries for all compounds were between 88.67% and 101.2%. The within the range of -11.58% to 10.89%. CONCLUSION: The proposed method showed appropriate accuracy and selectivity and was successfully applied to the rat urine samples analysis of saponins and flavonoids after intravenous administration of Jiweiling freeze-dried powder, which may provide some references to the apprehension of the action mechanism and clinical application.