A comprehensive analytical method based on ultra-fast liquid chromatography coupled with triple quadrupole/linear ion trap tandem mass spectrometry(UFLC-QTRAP-MS/MS) was established for simultaneous determination of the content of 45 bioactive constituents including flavonoids, alkaloids, amino acids, phenolic acids, and nucleosides in Epimedium brevicornum. The multiple bioactive constituents in leaves, petioles, stems and rhizomes of E. brevicornum were analyzed. The gradient elution was performed at 30 ℃ in an XBridge~® C_(18) column(4.6 mm×100 mm, 3.5 μm) with 0.4% formic acid aqueous solution-acetonitrile as the mobile phase at a flow rate of 0.8 mL·min~(-1). Single factor experiment and response surface methodology were employed to optimize the extraction conditions. Multivariate statistical analyses including systematic cluster analysis(SCA), principal component analysis(PCA), partial least squares discriminant analysis(PLS-DA), and one-way analysis of variance(One-way ANOVA) were carried out to classify the samples from different parts and identify different constituents. Grey relation analysis(GRA) and entropy weight-TOPSIS analysis were performed to build a multi-index comprehensive evaluation model for different parts of E. brevicornum. The results showed that there was a good relationship between the mass concentrations of 45 constituents and the corresponding peak areas, with the correlation coefficients(r) not less than 0.999 0. The precision, repeatability, and stability of the established method were good for all the target constituents in this study, with the relative standard deviations(RSDs) less than 5.0%(0.62%-4.9%) and the average recovery of 94.51%-105.7%. The above results indicated that the bioactive constituents varied in different parts of E. brevicornum, and the overall quality followed the trend of leaves > petioles > rhizomes > stems. This study verified the rationality of the Chinese Pharmacopoeia(2020 edition) stipulating that the medicinal part of E. brevicornum is the leaf. Moreover, our study indicated that the rhizome had the potential for medicinal development. The established method was accurate and reliable, which can be used to comprehensive evaluate and control the quality of E. brevicornum. This study provides data reference for clarifying the medicinal parts and rationally utilizing the resources of E. brevicornum.
Chromatography, High Pressure Liquid ; Epimedium ; Tandem Mass Spectrometry ; Chromatography, Liquid ; Multivariate Analysis

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-β1), 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-β1, 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-β1, 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.
Mice ; Male ; Animals ; Pulmonary Fibrosis/metabolism* ; Transforming Growth Factor beta1/metabolism* ; Epimedium/metabolism* ; Fibronectins/metabolism* ; Matrix Metalloproteinase 7/therapeutic use* ; Matrix Metalloproteinase 8/therapeutic use* ; Vimentin/metabolism* ; Interleukin-6/metabolism* ; Mice, Inbred C57BL ; Lung ; Collagen/metabolism* ; Bleomycin/toxicity* ; RNA, Messenger/metabolism* ; Cadherins/metabolism*

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.
Medicine, Chinese Traditional ; Flavonoids/chemistry* ; Glycosides ; Epimedium/chemistry* ; Solubility ; Cardiac Glycosides ; Polysaccharides/chemistry*

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.
Epimedium/chemistry* ; Flavonoids/chemistry* ; Plant Leaves ; Least-Squares Analysis ; Spectrophotometry, Infrared

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 3β-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.
Animals ; Epimedium/chemistry* ; Flavonoids/chemistry* ; Furans ; Glycosides/chemistry* ; Hydrolysis ; Male ; Molecular Docking Simulation ; Molecular Structure ; Rats ; Testosterone ; beta-Glucosidase/metabolism*

Four new prenylflavonoid glycosides, namely koreanoside H-K (1-4), together with eleven known ones (5-15) were isolated from the leaves of Epimedium koreanum Nakai. Their structures were elucidated by 1D NMR, 2D NMR, HR-ESI-MS, IR and UV. The identification of the sugar moieties was carried out by means of acid hydrolysis and HPLC analysis of their derivatives. It is worth noting that compound 3 and compound 4 were elucidated to contain fucose and quinovose moieties, which were two extremely rare sugar units from the genus Epimedium. The anti-pulmonary fibrosis activity of the new compounds was evaluated using A549 cell line. Compounds 1, 2 and 4 showed significant anti-pulmonary fibrosis activities.
Chromatography, High Pressure Liquid ; Epimedium/chemistry* ; Glycosides/pharmacology* ; Plant Extracts/pharmacology* ; Plant Leaves/chemistry*

Coix ; Epimedium ; Fatigue ; Humans ; Hypoxia ; Plant Leaves

This study evaluated the effects of epimedium polysaccharide(EPS) on the solubility of icariin and baohuoside Ⅰ so as to preliminary explore its solubilization function and the underlying mechanism. The solubility of these two insoluble flavonoids in water and polysaccharide solutions was compared by high performance liquid chromatography, and the mechanism was investigated by diffe-rential scanning calorimetry(DSC) and critical micelle concentration determination. The results indicated that their solubilization in crude EPS solutions was concentration-dependent. The solubility of icariin and baohuoside Ⅰ in 20 mg·mL~(-1) EPS-1-1 was 9.05 times and 5.76 times that in water, respectively; while their solubility in 20 mg·mL~(-1) EPS-2-1 was 10.55 and 8.39 times that in water, respectively. The change of the DSC thermograms suggested the formation of new complexes from icariin and baohuoside Ⅰ with polysaccharides. The critical micelle concentrations proved the micellar properties of both EPS-1-1 and EPS-2-1. In short, EPS can significantly increase the solubility of icariin and baohuoside Ⅰ, the mechanism of which may be related to the formation of micellar complexes between EPS and insoluble flavonoids.
Epimedium ; Flavonoids ; Polysaccharides ; Solubility

OBJECTIVE: To observe the effects of Epimedium polysaccharides (EPS) on bone marrow hematopoietic function and Th17/Treg balance in aplastic anemia (AA) mice, and preliminarily explore its therapeutic mechanism. METHODS: Forty BALB/C mice were randomly divided into control (control), model (model), stanozolol (stanozolol) and epimedium polysaccharide (EPS) group, with 10 mice in each group. Except for the control group, Acetophenazine, Gy irradiation and cyclophosphamide triple application were used to establish AA models for the other groups. After the model was established, the stanozolol group was intragastrically administered with 4 mg/kg stanozolol suspension, the EPS group was intragastrically administered with 100 mg/kg epimedium polysaccharide, while the control group and the model group were given an equal volume of 0.9% sodium chloride solution by gavage once a day, for 14 consecutive days. The automatic animal blood analyzer was used to detect the changes in peripheral blood hemoglobin (Hb), red blood cells (RBC), white blood cells (WBC) and platelets (PLT), flow cytometry was used to detect the proportion of Treg and Th17 cells, the levels of interleukin 2 (IL-2), interleukin 11 (IL-11) and tumor necrosis factor α (TNF-α) were detected by ELISA, the number of nucleated bone marrow cells was counted, HE staining and immunohistochemical staining were used to detect the number, the proliferation and apoptosis of bone marrow cells, Western blot was used to detect the expression of signal transducer and activator of transcription 3 (STAT3), retinoic acid receptor-related orphan receptor γ (RORγt), transducer and activator of transcription 5 (STAT5) and fork head transcription factor 3 (Foxp3). RESULTS: Compared with the model group, the levels of Hb, RBC, WBC and PLT in the peripheral blood of mice in stanozolol and EPS group significantly increased, the proportion of Th17 cells was significantly reduced, and the proportion of Treg cells significantly increased. The levels of IL-2 and TNF-α in serum were significantly reduced (P<0.05), the level of IL-11 significantly increased (P<0.05), the number of bone marrow nucleated cells significantly increased (P<0.05), the positive rate of Ki-67 significantly increased (P<0.05) and the positive rate of Caspase-3 was significantly reduced (P<0.05). At the same time, the protein expression of STAT3 and RORγt significantly decreased, and the protein expression of STAT5 and Foxp3 increased, the difference showed statistically significant (P<0.05). CONCLUSION EPS can promote the recovery of bone marrow hematopoietic function in AA mice and improve Th17/Treg imbalance, the mechanism may be related to the inhibition of STAT3/RORγt expression and promotion of STAT5/Foxp3 expression.
Anemia, Aplastic ; Animals ; Bone Marrow ; Epimedium ; Mice ; Mice, Inbred BALB C ; Polysaccharides ; T-Lymphocytes, Regulatory ; Th17 Cells

In this study, the growth index including plant height, compound leaf area, specific leaf area, leaf water content, number of branches, and leaf biomass per plant and the icariin flavonoids such as epimedin A, epimedin B, epimedin C and icariin of Epimedium pseudowushanense were determined on 30 d and 60 d under light intensity(18.2±2.5) μmol·m~(-2)·s~(-1)(L1) and(90.9 ±2.5) μmol·m~(-2)·s~(-1)(L2), and white light as control, red light, blue light and yellow light were used as three light quality treatments, to study the effect of light quality on the growth and flavonoids accumulation of E. pseudowushanense. The E. pseudowushanense was sui-table for growth under L1 light intensity, the blue light treatment significantly reduced the leaf area, but had little effect on the stem height, the red light treatment and the yellow light treatment had no obvious effect on the stem height and leaf area, but the yellow light treatment significantly increased the germination of new branches, and had a sustained promoting effect, and the biomass was significantly higher than the white light treatment at 60 d. The content of icariin flavonoids in red light, blue light and yellow light treatment was higher than that in white light treatment at 30 d and 60 d under L1 light intensity, while yellow light treatment promoted the synthesis of icariin flavonoids to the largest extent, which was 1.8 and 1.9 times of white light treatment(30 d and 60 d).Under L2 light intensity, the effect of strong light on promoting stem germination became the main factor, while the yellow light treatment showed no significant effect on promoting stem germination, and the red light treatment exhibited a significant effect on reducing leaf area. Icariin flavonoids under red light, blue light and yellow light treatment were all lower than that under white light treatment, that is, the effect of white light treatment on the synthesis of icariin flavonoids is better than red light, blue light and yellow light treatment. When the time of strong light treatment was longer, the degradation range of icariin flavonoids in other light treatment appeared, while red light treatment promotes the synthesis of icariin flavonoids. Therefore, the influence of light quality on E. pseudowushanense is quite different under different light intensity, no matter from growth index or flavonoid content index. The results support that the biomass and icariin flavonoid content can be increased by providing appropriate red and yellow light.
Drugs, Chinese Herbal ; Epimedium ; Flavonoids ; Plant Leaves