baohuoside-i and icariin

Cancer is one of the leading causes of deaths worldwide. Compounds derived from traditional Chinese medicines have been an important source of anticancer drugs and adjuvant agents to potentiate the efficacy of chemotherapeutic drugs and improve the side effects of chemotherapy. Herba Epimedii is one of most popular herbs used in China traditionally for the treatment of multiple diseases, including osteoporosis, sexual dysfunction, hypertension and common inflammatory diseases. Studies show Herba Epimedii also possesses anticancer activity. Flavonol glycosides icariin and icariside II are the main bioactive components of Herba Epimedii. They have been found to possess anticancer activities against various human cancer cell lines in vitro and mouse tumor models in vivo via their effects on multiple biological pathways, including cell cycle regulation, apoptosis, angiogenesis, and metastasis, and a variety of signaling pathways including JAK2-STAT3, MAPK-ERK, and PI3k-Akt-mTOR. The review is aimed to provide an overview of the current research results supporting their therapeutic effects and to highlight the molecular targets and action mechanisms.

Topics: Drugs, Chinese Herbal; Flavonoids; Humans; Neoplasms; Phytotherapy

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.

Topics: Epimedium; Flavonoids; Polysaccharides; Solubility

Topics: Flavonoids; Glycoside Hydrolases; Hot Temperature; Hydrolysis

Topics: Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Multidrug Resistance-Associated Proteins; Osteosarcoma; Phosphorylation; Rhodamine 123; STAT3 Transcription Factor; Triterpenes

Topics: Animals; Cell Differentiation; Drug Evaluation, Preclinical; Flavonoids; Humans; Least-Squares Analysis; Osteoclasts; Plant Extracts; Plants, Medicinal; Quantitative Structure-Activity Relationship

A crude β-glucosidase has been produced from Trichoderma viride and used to explore a simple method to prepare icariside II from icariin. The crude enzyme has been studied by zymography method and used for hydrolysis of ICA. To achieve a high conversion rate of ICA, various factors have been studied including pH, reaction time, temperature, initial concentration of enzyme, and initial concentration of ICA through central composite design experiments. In the condition of the optimum hydrolysis parameters with pH 4.0, 41°C, 1.0 mg/mL ICA, and 9.8 U/mL crude β-glucosidase, the conversion rate of ICA reached 95.03% at 1 h. Moreover, the cytotoxicity test showed that ICA II performed inhibition effects on proliferation of A549 cell, while ICA has no cytotoxicity. It indicated that the hydrolysis transformation study of ICA is valuable for exploration of active new drugs.

Topics: beta-Glucosidase; Biotransformation; Cell Line, Tumor; Cell Proliferation; Cytotoxins; Flavonoids; Humans; Temperature; Trichoderma

Biotransformations of icariin (1) by cell suspension cultures of Glycyrrhiza uralensis and Morus alba yielded two new metabolites, icaruralins A and B (2 and 3), and one known metabolite, baohuoside I (4). Their structures were determined on the basis of extensive spectroscopic analysis. This is the first report that the cell suspension cultures of G. uralensis and M. alba possess deglycosylation functionality.

Topics: Biotransformation; Flavonoids; Glycyrrhiza uralensis; Molecular Structure; Morus; Nuclear Magnetic Resonance, Biomolecular; Plant Roots

Epimedium L. is well known medicinal genus of Chinese pharmacopoeia. Various species are ethno-botanically used against diseases of eye and kidney, impotence, asthma, arthritis and hypertension; besides being used as analeptic, expectorant, antibacterial, hypoglycemic, vasodilator and refrigerant. Recent studies have attributed most of these medicinal properties to its flavonoid glycosides, especially Icariin which is the major pharmacologically active constituent. Icariin has been found to possess effective aphrodisiac, antioxidant, immunomodulatory, hepatoprotective, cardioprotective, vasodilatory, antidepressant and anti-osteoporosis activities. Icariside-II, another active constituent, has cytotoxic and cytostatic effects on 6 cancer cell-lines, and immunosuppressive effects on allograft rejection. In this present study, Epimedium elatum Morr. and Decne., the only species of this genus growing in Indian subcontinent, has been investigated for its medicinal value by determining the content of pharmacologically active constituents, Icariin and Icariside-II, by HPLC method. HPLC analysis of alcohol extract of its shade dried parts was performed with reverse phase C-18 column. The mobile phase for Icariin was acetonitrile-water in gradient mode; while for Icariside-II, it was methanol-water. The effluent was monitored at 270 nm. The results have revealed an appreciable content of Icariin and Icariside-II in its aerial and underground parts; the content being higher in populations growing at higher altitudes. The substantial presence of pharmacologically active constituents, Icariin and Icariside-II, in this species of Epimedium, signifies its value as a medicinal plant.

Topics: Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Epimedium; Flavonoids

Total flavonoids in Herba epimedii (HEP) have been demonstrated to protect against bone loss and bone deterioration associated with estrogen deficiency without exerting any uterotrophic effects. However, it is unclear how flavonoids in HEP exert their protective effects on bone and if different flavonoids exert estrogenic actions in bone cells via similar mechanism of actions. The present study aims to investigate the bone anabolic effects of four major flavonoids isolated from HEP, namely icariin, baohuoside-I, epimedin B and sagittatoside A as well as the mechanism involved in mediating their estrogenic actions in rat osteoblastic-like UMR-106 cells. All tested compounds significantly stimulated the cell proliferation rate, alkaline phosphate (ALP) activity and osteoprotegerin (OPG)/receptor activator of nuclear factor κ-B ligand (RANKL) mRNA expression in UMR-106 cells and their effects could be abolished by co-incubation with 10(-6)M ICI 182,780. None of the flavonoids exhibited binding affinities toward ERα and ERβ. However, sagittatoside A selectively activated estrogen response element (ERE)-luciferase activity via ERα. In addition, icariin and sagittatoside A induced ERα phosphorylation at serine 118 residue. Taken together, our results indicated that all four flavonoids from HEP stimulated ER-dependent osteoblastic functions in UMR-106 cells, but only two of them appeared to exert their actions by ligand-independent activation of ERα. Our study provides evidence to support the hypothesis that the estrogen-like protective effects on bone by flavonoids are mediated via mechanisms that are distinct from the classical actions of estrogen.

Topics: Animals; Blotting, Western; Cell Differentiation; Cells, Cultured; Diterpenes; Drugs, Chinese Herbal; Estrogen Receptor alpha; Estrogens; Flavonoids; Glucosides; Immunoenzyme Techniques; Osteoblasts; Rats; Real-Time Polymerase Chain Reaction; Response Elements; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

To optimize the process of Icraiin be hydrolyzed to Baohuoside I by cellulase by Plackett-Burman design combined with Central Composite Design (CCD) response surface methodology.. To select the main influencing factors by Plackett-Burman design, using CCD response surface methodology to optimize the process of Icraiin be hydrolyzed to Baohuoside I by cellulase. Taking substrate concentration, the pH of buffer and reaction time as independent variables, with conversion rate of icariin as dependent variable,using regression fitting of completely quadratic response surface between independent variable and dependent variable,the optimum process of Icraiin be hydrolyzed to Baohuoside I by cellulase was intuitively analyzed by 3D surface chart, and taking verification tests and predictive analysis.. The best enzymatic hydrolytic process was as following: substrate concentration 8. 23 mg/mL, pH 5. 12 of buffer,reaction time 35. 34 h.. The optimum process of Icraiin be hydrolyzed to Baohuoside I by cellulase is determined by Plackett-Burman design combined with CCD response surface methodology. The optimized enzymatic hydrolytic process is simple, convenient, accurate, reproducible and predictable.

Topics: Cellulase; Flavonoids; Hydrolysis

This study aims to observe different factors which affected the bionic enzymatic hydrolysis of icariin into baohuoside I and to optimize the reaction conditions in order to provide research foundation for building a novel bionic enzymolysis drug delivery system. To simulate the environment in vivo, 37 degrees C was set as the temperature and artificial intestinal juice and gastric juice were selected as the buffer solutions. Taking the conversion of baohuoside I as index, the effects of the kinds of enzyme, enzyme activity, substrate concentration, reaction time, pancreatin in artificial intestinal juice and surfactant on the conversion of baohuoside I were investigated. The results showed that cellulase, beta-glucosidase and snailase were all inactive in artificial gastric juice and no baohuoside I generated. Pancreatin in artificial intestinal juice couldn't significantly influence the activity of beta-glucosidase or snailase (P > 0.05), but noticeably decrease the activity of cellulase (P < 0.05). In artificial intestinal juice, the conversion of baohuoside I was highest by using beta-glucosidase, and the optimum reaction conditions were determined as follows: enzyme activity 10 U x mL(-1), substrate concentration 1 mg x mL(-1), 3 g x L(-1) rhamnolipid and reaction time 3 h. Under this condition, the conversion of baohuoside I was 99.8%.

Topics: Animals; beta-Glucosidase; Cellulase; Flavonoids; Hydrolases; Hydrolysis; Pancreatin; Snails; Surface-Active Agents

Accumulating evidences suggest that Herba epimedii has the potential benefits against osteoporosis. However, previous studies were focused on the crude extract, total flavonoids (TF) and icariin (ICA), and the detailed molecular mechanisms of action and structure-activity relationship (SAR) remain unclear. Herein we aimed to systematically investigate the effects of Herba epimedii flavonoids (HEF) on the activity of osteoclasts, and explore the potential SAR. Both ICA and baohuoside-1 (BS) significantly inhibited the proliferation of RAW 264.7 cells (IC(50) 25 μM and 67 μM, respectively). Treatment of ICA resulted in G2/M arrest and apoptosis in RAW 264.7 cells as early as 12 h. Besides, HEF remarkably suppressed vitamin D-induced differentiation of osteoclasts in rabbit bone marrow cells and the bone resorption of rabbit mature osteoclasts in vitro. It is notable that the inhibitory effect of 100 μM ICA and BS on osteoclast formation is almost 90%; and the inhibition rate on bone resorption is 50% and 80%, respectively. Besides, RANKL-induced osteoclast formation from RAW 264.7 cells and the expression of TRAP, CA II, CTSK and MMP-9 was significantly reduced by the treatment of 25 μM HEF and 17β-estradiol (ES), and the inhibitory strength increases in the order TF < ES < ICA < BS, which was blocked by ICI182780 suggesting that the regulation of osteoclast activity might be ER dependent. Furthermore, the free hydroxyl group at C-7 of BS played an important role in the SAR for anti-osteoclast action. To conclude, HEF could regulate the formation and activity of osteoclasts by inhibiting the proliferation and differentiation, inducing apoptosis and cell cycle arrest and suppressing bone resorption of osteoclasts. Changes in osteoclast activity are probably mediated predominantly by interaction with nuclear estrogen receptors and via mitochondrial pathway. HEF, especially BS, has great potential for the prevention and treatment of osteoporosis.

Topics: Acid Phosphatase; Animals; Animals, Newborn; Apoptosis; Bone Resorption; Cell Cycle Checkpoints; Cell Differentiation; Cell Division; Cell Line; Cell Proliferation; Drugs, Chinese Herbal; Epimedium; Estradiol; Flavonoids; Fulvestrant; G2 Phase; Inhibitory Concentration 50; Isoenzymes; Matrix Metalloproteinase 9; Molecular Structure; Monocytes; Osteoclasts; Rabbits; RANK Ligand; Reverse Transcriptase Polymerase Chain Reaction; Tartrate-Resistant Acid Phosphatase

This study was to comprehensively evaluate the chemical quality of main species of epimedium planted in China. The contents of 5 marker compounds, epimedin A, epimedin B, epimedin C, icariin and baohuoside I, as well as total flavonoids of 22 samples of 8 officinal species of Epimedium were determined by HPLC and UV, separately. Some physical and chemical tests (H2O, total ash, acid-insoluble ash and EtOH extract) were also carried out to investigate their chemical qualities. There were significant differences in types and contents of prenyl-flavonoid glycosides such as epimedin A, epimedin B, epimedin C, icariin and baohuoside I in different species, meanwhile, the physical and chemical parameters results also showed that there were obvious differences in chemical quality among different species of epimedium herb. The results provide theoretical and experimental basis for the establishment of comprehensive quality assessment system of epimedium in China.

Topics: Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Epimedium; Flavonoids; Plants, Medicinal; Quality Control; Species Specificity; Spectrophotometry, Ultraviolet

Epimedium was obtained from different habitats, and their bioactive components for inhibiting RAW264.7 were screened by MTT assay. Results indicate that epimedium from different habitats displayed significant different activities. By means of model population analysis (MPA), a latent bioactive component, baohuoside-I was got. Activity of baohuoside-I wasvalidated and prior to icariin. MPA can be used for bioactive components screening.

Topics: Cell Proliferation; Cytological Techniques; Drugs, Chinese Herbal; Epimedium; Flavonoids; Humans; Osteoclasts; Plants, Medicinal

To investigate the effects of icariin and icariside II on eNOS expression and NOS activity in endothelial cells and possible mechanisms using EGFR over-expressed porcine aorta endothelial (PAE) cell line.. The EGFR gene was transfected into PAE cells and genetic stable cell line (PAE-EGFR) was selected. 12.5 μmol/L of icariin and of icariside II were used to treat the PAE and PAE-EGFR cells respectively for 48 h, the eNOS expression in each group was observed. EGF was also used to treat the cells to observe the regulatory effects of icariin and icariside II on NOS activity. The regulatory effects of icariin and icariside II on NOS activity were also observed, and sildenafil was used as a control.. Western blot showed that the basic value of eNOS expression was higher in PAE-EGFR group compared with that in PAE group, both of icariin and icariside II increased the eNOS expression in PAE and PAE-EGFR group (P<0.01), and the value of eNOS expression was higher in PAE-EGFR group than that in PAE group. In the PAE-EGFR cell line, the NOS activity reached (15.37 ± 1.49) u/mg when the concentration of icariside II was 10(-8) mol/L, which was 4.66 u/mg more than that in the PAE cell line. When the concentration reached 10(-7), 10(-6) or 10(-5) mol/L, the change of NOS activity in PAE-EGFR group was greater than that in PAE group (P<0.01). icariin also increased the NOS activity in PAE and PAE-EGFR cells, but the activity was 20% lower compared with icariside II group, however, Sildenafil showed no influence on NOS activity.. Icariin and icariside II may increase the eNOS expression through activating EGF-EGFR pathway in PAE cell, by which endothelial cells function could be regulated and the better effect was noted in icariside II compared to icariin.

Topics: Animals; Aorta; Cells, Cultured; Endothelial Cells; Epidermal Growth Factor; ErbB Receptors; Flavonoids; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Swine

Compounds that delay aging might also postpone age-related diseases and extend healthspan in humans. Icariin is a flavonol extracted from several plant species of the Epimedium family. The icariin and its metabolic derivatives have been shown to exert wide protective effects in age-related diseases. However, whether icariin and its derivatives have the potency of delaying aging remains unclear. Here, we report that icariin and its derivative icariside II extend C. elegans lifespan. Using HPLC, we found high level of icariside II in the animals treated with icariin, suggesting icariside II is the bioactive form in vivo of icariin. Icariside II also increased the thermo and oxidative stress tolerance, slowed locomotion decline in late adulthood and delayed the onset of paralysis mediated by polyQ and Aβ(1-42) proteotoxicity. The lifespan extension effect of icariside II is dependent on the insulin/IGF-1 signaling (IIS) since the daf-16(mu86) and daf-2(e1370) failed to show any lifespan extension upon icariside II treatment. Consistently, icariside II treatment upregulates the expression of DAF-16 targets in the wild-type. Moreover, our data suggests that the heat shock transcription factor HSF-1 has a role in icariside II-dependent lifespan extension further implicating the IIS pathway. In conclusion, we demonstrate a novel natural compound, icariside II as the bioactive form of icariin, extends the healthspan via IIS pathway in C. elegans.

Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Flavonoids; Insulin; Insulin-Like Growth Factor I; Longevity

It has been reported that icariin and icariside II, two flavonoid glycosides coming from herba epimedii, which have a closely structural relationship, show some pharmacological effects such as preventing osteoporosis, cancer and depression. The content of natural icariside II is very low in herba epimedii, but it is the main component in vivo after the administration of herba epimedii. More icariside II can be obtained from icariin by enzymatic hydrolysis method than by traditional isolation method. This study focuses on finding a simple and feasible method to prepare icariside II from icariin by enzymatic hydrolysis, so as to meet the request for further pharmacologic actions study. Icariin was obtained successively with 90% ethanol extraction, isolation on macroporous resin and purification on silica gel chromatography. Enzymatic hydrolysis conditions were tested for the bioconversion of icariin into icariside II by orthogonal array design. The structures of isolated icariin and produced icariside II were identified by UV, IR, ESIMS, (1)H NMR, (13)C NMR, and DEPT spectroscope. Enzymatic hydrolysis experiment showed that icariin could be transformed into icariside II with the action of beta-glucosidase and the optimum reaction conditions were determined as follows: 50 degrees C, 0.2 M disodium hydrogen phosphate and citric acid buffer system (pH6.0), the ratio of icariin/enzyme is 1:1 and reaction time 5 h. By using this enzymatic condition, 95.5 mg icariside II (with the purity of 99.1%) was obtained eventually by transforming 200 mg icariin.

Topics: Berberidaceae; beta-Glucosidase; Drug Compounding; Flavonoids; Hydrolysis; Molecular Structure

To investigate the effects of icariin and it's main metabolites-icariside II on the osteogenic differentiation of rat bone marrow stromal cells (rBMSCs).. rBMSCs were cultured by adherence screening method, icariin and icariside II were supplemented into the culture at 5 x 10(-5) mol/L respectively. The osteogenic differentiation markers including alkaline phosphatase (ALP) activity, CFU-F(ALp), osteocalcin secretion, calcium deposition and mineralized bone modulus were compared among the icariin-supplemented group, icariside II and the control. The gene expressions of bFGF, IGF-1, Osterix and Runx-2 were examined by RT-Real Time PCR.. Both icariside II and icariin significantly improved ALP activity, CFU-F(ALP) amount, osteocalcin secretion, calcium deposition and mineralized modulus. Besides, they enhanced the gene expressions of bFGF, IGF-1, Osterix and Runx-2. Icariside II was obviously stronger than icariin at the above activities.. Icariside II is stronger than icariin at enhancing the osteogenic differentiation of rBMSCs, suggesting that icariin can be administered via oral and it's metabolites are the effective constitutes for antiosteoporosis activity.

Topics: Alkaline Phosphatase; Animals; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Drugs, Chinese Herbal; Female; Fibroblast Growth Factor 2; Flavonoids; Insulin-Like Growth Factor I; Osteocalcin; Osteogenesis; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stromal Cells

A series of icariin derivatives were synthesized. Their multidrug resistance (MDR) reversal activities were evaluated by MTT assay and the results indicated that the derivatives were the potent modulators of MDR. It was showed that the derivatives significantly increased the intracellular accumulation of ADR in MCF-7/ADR cells compared with drug sensitive MCF-7 cells. The results of bi-directional assay and reverse transcription polymerase chain reaction (RT-PCR) assay showed that the derivatives had high inhibitory activity against P-gp efflux function and significantly down-regulated on the expression of P-gp.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Caco-2 Cells; Cell Line, Tumor; Chemistry, Pharmaceutical; Drug Design; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Flavonoids; Humans; Inhibitory Concentration 50; Models, Chemical; Reverse Transcriptase Polymerase Chain Reaction; Tetrazolium Salts; Thiazoles; Time Factors

Three kinds of prenylated flavonols, icariside I, icariside II, and icaritin, were isolated from an icariin hydrolysate and their effects on melanogenesis evaluated based on mushroom tyrosinase inhibition and quantifying the melanin contents in melanocytes. Although none of the compounds had an effect on tyrosinase activity, icariside II and icaritin both effectively inhibited the melanin contents with an IC50 of 10.53 and 11.13 MM, respectively. Whereas icariside II was obtained from a reaction with beta-glucosidase and cellulase, the icariin was not completely converted into icariside II. Thus, for the high-purity production of icariside II, the reaction was optimized using the response surface methodology, where an enzyme concentration of 5.0 mg/ml, pH 7, 37.5 degrees C;, and 8 h reaction time were selected as the central conditions for the central composite design (CCD) for the enzymatic hydrolysis of icariin into icariside II using cellulase. Empirical models were developed to describe the relationships between the operating factors and the response (icariside II yield). A statistical analysis indicated that all four factors had a significant effect (p<0.01) on the icariside II production. The coefficient of determination (R2) was good for the model (0.9853), and the optimum production conditions for icariside II was an enzyme concentration of 7.5 mg/ml, pH 5, 50 degrees C, and 12 h reaction time. A good agreement between the predicted and experimental data under the designed optimal conditions confirmed the usefulness of the model. A laboratory pilot scale was also successful.

Topics: Animals; Biotechnology; Cell Line, Tumor; Chromatography, High Pressure Liquid; Epimedium; Flavones; Flavonoids; Hydrogen-Ion Concentration; Hydrolysis; Mass Spectrometry; Melanins; Melanocytes; Mice; Mice, Inbred C57BL; Monophenol Monooxygenase; Umbelliferones

The purpose is to determine absorption mechanism of five bioactive prenylated flavonoids (baohuoside I, icariin, epimedine A, B, and C) present in heat-processed Epimedium koreanum Nakai (Yin Yanghuo).. Transport of five prenylated flavonoids present in heat-processed herbs were studied in the human intestinal Caco-2 model and the perfused rat intestinal model.. In the perfused rat intestinal model, prenylated flavonoids with a monoglucosidic bond (e.g., icariin) was rapidly hydrolyzed into corresponding metabolites (e.g., baohuoside I). In the Caco-2 model, apical to basolateral permeability of a monoglycoside baohuoside I (1.46 x 10(-6) cm/sec) was more than 2 folds greater than four prenylated flavonoids with 2 or more sugar moieties (<0.6 x 10(-6) cm/sec). The slow apical to basolateral transport of baohuoside I was the result of efflux. This efflux was carrier-mediated and active since its transport was vectorial, concentration- and temperature-dependent with activation energies greater than 15 kcal/mol. Efflux of baohuoside I was significantly suppressed by inhibitors of BCRP and MRP2, whereas efflux of icariin was significantly inhibited only by p-glycoprotein inhibitor verapamil. Because YHH is often heat-processed for better efficacy, we determined and found the optimal condition for increasing contents of more bioavailable flavonoids (i.e., baohuoside I) to be 160-170 degrees C for 5-7 min.. Poor bioavailability of prenylated flavonoids results from their poor intrinsic permeation and transporter-mediated efflux. Heat processing parameters may be optimized to preserve the herb's bioavailable flavonoids, which help retain and improve its efficacy during processing.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biological Availability; Caco-2 Cells; Epimedium; Flavonoids; Hot Temperature; Humans; Hydrolysis; Intestinal Absorption; Intestine, Small; Male; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Perfusion; Permeability; Prenylation; Rats; Rats, Sprague-Dawley; Time Factors

Herba Epimedii (family Berberidaceae), Yinyanghuo in Chinese, is one of the commonly used Chinese medicines. Flavonoids are considered as its active components. In this study, a reliable pressurized liquid extraction (PLE) and HPLC method was developed for simultaneous determination of 15 flavonoids, namely hexandraside E, kaempferol-3-O-rhamnoside, hexandraside F, epimedin A, epimedin B, epimedin C, icariin, epimedoside C, baohuoside II, caohuoside C, baohuoside VII, sagittatoside A, sagittatoside B, 2''-O-rhamnosyl icariside II and baohuoside I in different species of Epimedium. The analysis was performed by using a Zorbax SB-C18 analytical column (250 mm x 4.6 mm I.D., 5 microm) at gradient elution of water and acetonitrile with diode-array detection (270 nm). All calibration curves showed good linearity (r(2)>0.9997) within test ranges. The LOD and LOQ were lower than 1.31 ng and 2.62 ng on column, respectively. The RSD for intra- and inter-day of 15 analytes was less than 3.8% at three levels, and the recoveries were 90.5-106.8%. The validated method was successfully applied for the analysis of 15 flavonoids in different species of Epimedium which had great variation on the contents of investigated flavonoids. Hierarchical clustering analysis based on the characteristics of 15 investigated compound peaks in HPLC profiles showed that 26 samples were divided into three main clusters, which were in accordance with their flavonoid contents. Four flavonoids including epimedin A, B, C and icariin were optimized as markers for quality control of the species of Epimedium used as Yinyanghuo.

Topics: Chromatography, High Pressure Liquid; Epimedium; Flavonoids; Reproducibility of Results

A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was developed and validated for the simultaneous quantitative determination of icariin and its two major metabolites, icariside I and icariside II in rat plasma. The analytes were extracted by liquid-liquid extraction with ethyl acetate after internal standard (daidzein) spiked. The separation was performed by a ZORBAX SB-C(18) column (3.5 microm, 2.1 mm x 100 mm) and a C(18) guard column (5 microm, 4.0 mm x 2.0mm) with an isocratic mobile phase consisting of acetonitrile-water-formic acid (50:50:0.05, v/v/v) at a flow rate of 0.25 mL/min. The Agilent G6410A triple quadrupole LC-MS system was operated under the multiple reaction monitoring (MRM) mode using the electrospray ionization technique in positive mode. The nominal retention times for icariin, icariside I, icariside II and daidzein were 1.21, 1.88, 2.34 and 1.35 min, respectively. The lower limits of quantification (LLOQ) of icariin, icariside I and icariside II of the method were 1.0, 0.5 and 0.5 ng/mL, respectively. The method was linear for icariin and its metabolites with correlation coefficients >0.995 for all analytes. The intra-day and inter-day accuracy and precision of the assay were less than 12.5%. This method has been applied successfully to a pharmacokinetic study involving the intragastric administration of icariin to rats.

Topics: Administration, Oral; Animals; Chromatography, Liquid; Epimedium; Flavones; Flavonoids; Isoflavones; Male; Medicine, Chinese Traditional; Rats; Rats, Wistar; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Tandem Mass Spectrometry; Umbelliferones

A sensitive, specific and accurate LC-MS/MS method was developed for simultaneous determination of icariin, icariside II and osthole in rat plasma. With carbamazepine as the internal standard, plasma samples were prepared by protein precipitation with acetonitrile. Analysis was carried out on an ACQUITY UPLC BEH C(18) column with a linear gradient and 0.1% aqueous acetic acid and acetonitrile were used as mobile phase. Detection was performed by means of electrospray ionization mass spectrometry in positive ion mode with multiple reaction monitoring. Linear calibration curves of icariin, icariside II and osthole were obtained over the concentration ranges of 2.00-200, 2.00-200 and 2.00-500 ng/ml, respectively. The intra- and inter-day precisions were within 8.0% and 14%, and the accuracy was from -6.0% to 9.0%. The method was successfully applied to pharmacokinetic studies of icariin, icariside II and osthole in rats after oral administration of Gushudan extract.

Topics: Animals; Chromatography, Liquid; Coumarins; Drug Stability; Drugs, Chinese Herbal; Female; Flavonoids; Male; Rats; Rats, Wistar; Reproducibility of Results; Tandem Mass Spectrometry