Metabolite profiling of four major flavonoids of Herba Epimedii in zebrafish

The zebrafish model organism was applied first in a metabolic study of icariin, baohuoside I, epimedin A and epimedin C, which are flavonoids in Herba Epimedii. Metabolites of these compounds in zebrafish after exposure for 24 h were identified by HPLC-ESI-MS, whereby the separation was performed with a Zorbax C-18 column using a gradient elution of 0.05% formic acid acetonitrile-0.05% formic acid water. The quasi-molecular ions of compounds were detected in simultaneous negative and positive ionization modes. Metabolic products of icariin and epimedin C via cleavage of glucose residue instead of rhamnose residues were found, which coincided with the results using regular metabolic analysis methods. In addition, the zebrafish model was used to predict the metabolism of the trace component epimedin A, whose metabolic mechanisms haven't been clearly elucidated with the current metabolism model. The metabolic pathway of epimedin A in zebrafish was similar to those of its homologue icariin and epimedin C. Our study demonstrated that the zebrafish model can successfully imitate the current models in elucidating metabolic pathways of model flavonoids, which has advantages of lower cost, far less amount of compound needed, easy set up and high performance. This novel model can also be applied in quickly predicting the metabolism of Chinese herb components, especially trace compounds.     

Simultaneous determination of 15 flavonoids in Epimedium using pressurized liquid extraction and high-performance liquid chromatography

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.     

An MC3T3-E1 Cell Line Biomembrane Extraction and HPLC–ESI-MS n Method for Simultaneous Analysis of Potential Anti-Osteoporosis Components of Epimedium koreanum

Aerial parts of Epimedium koreanum Nakai have been used as Herba Epimedii in China. Its anti-osteoporosis effect has attracted much attention in recent years. In this study, a method involving osteoblastic cell (MC3T3-E1 cell line) extraction and high-performance liquid chromatography–electrospray ionisation–mass spectrometry (HPLC–ESI-MS ⁿ ) was developed for screening of potential anti-osteoporosis agents in E.?koreanum. Four compounds identified as epimedin?A, epimedin?B, epimedin?C and icariin were found to interact with MC3T3-E1 cells and possessed potent osteoblast-stimulating activity as evaluated by cell proliferation [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay] and differentiation [alkaline phosphatase (ALP) activity and Ca content] in?vitro. The results suggest that these four flavonoids are the anti-osteoporosis constituents of Herba Epimedii and that the method combining MC3T3-E1 cell extraction with HPLC–ESI-MS ⁿ is rapid and effective in screening anti-osteoporosis agents from traditional Chinese medicines.     

Phenolic compounds from the aerial parts of Clematis viticella L. and their in vitro anti-inflammatory activities*

Phytochemical investigations on the EtOH extract of Clematis viticella led to the isolation of six flavonoid glycosides, isoorientin (1), isoorientin 3′-O-methyl ether (2), quercetin 7-O-α-L-rhamnopyranoside (3), quercetin 3,7-di-O-α-L-rhamnopyranoside (4), manghaslin (5) and chrysoeriol 7-O-β-D-glucopyranoside (6), one phenylethanol derivative, hydroxytyrosol (7), along with three phenolic acids, caffeic acid (8), (E)-p-coumaric acid (9) and p-hydroxybenzoic acid (10). The structures of the isolates were elucidated on the basis of NMR and HR-MS data. All compounds were isolated from C. viticella for the first time. Compounds 7 and 8 showed significant anti-inflammatory activity at 100 μM by reducing the release of NO in LPS-stimulated macrophages comparable to positive control indomethacin. Compounds 3 and 7 exhibited anti-inflammatory activity through lowering the levels of TNF-α while 1, 3 and 5 decreased the levels of neopterin better than the positive controls.     

A new strategy for the discovery of epimedium metabolites using high-performance liquid chromatography with high resolution mass spectrometry

In this paper, a new strategy of drug metabolite discovery and identification was established using high-performance liquid chromatography with high resolution mass spectrometry (HPLC–HRMS) and a mass spectral trees similarity filter (MTSF) technique. The MTSF technique was developed as a means to rapidly discover comprehensive metabolites from multiple active components in a complicated biological matrix. Using full-scan mass spectra as the stem and data-dependent subsequent stage mass spectra to form branches, the HRMS and multiple-stage mass spectrometric data from detected compounds were converted to mass spectral trees data. Potential metabolites were discovered based on the similarity between their mass spectral trees and that known compounds or metabolites in a mass spectra trees library. The threshold value for match similarity scores was set at above 200, allowing approximately 80% of interference to be filtered out. A total of 115 metabolites of five flavonoid monomers (epimedin A, epimedin B, epimedin C, icariin, and baohuoside I) and herbal extract of epimedium were discovered and identified in rats via this new strategy. As a result, a metabolic profile for epimedium was obtained and a metabolic pathway was proposed. In addition, comparing to the widely used neutral loss filter (NLF), product ion filter (PIF), and mass defect filter (MDF) techniques, the MTSF technique was shown superior efficiency and selectivity for discovering and identifying metabolites in traditional Chinese medicine (TCM).     

Phenolic constituents from Wissadula periplocifolia (L.) C. Presl. and anti-inflammatory activity of 7,4′-di-O-methylisoscutellarein

This study reports the first phenolics from Wissadula genus (Malvaceae) and the anti-inflammatory activity of 7,4′-di-O-methylisoscutellarein. Using chromatographic methods, five phenolic compounds were isolated from aerial parts of Wissadula periplocifolia (L.) C. Presl. The compounds were identified as 4-hydroxybenzoic acid, 3-hydroxybenzoic acid, trans-cinnamic acid, tamgermanetin and 7,4′-di-O-methylisoscutellarein using spectroscopic methods. The flavone 7,4′-di-O-methylisoscutellarein showed anti-inflammatory activity by inhibiting neutrophils recruitment in a mice model of pleurisy and by decreasing significantly the production of cytokines IL-1β and TNF-α.     

Effect of stability of internal standard on quantification of 15 flavonoids in Epimedium using CZE

A CZE method was developed for the simultaneous determination of 15 flavonoids, including epimedin B, epimedin A, hexandraside F, epimedin C, icariin, sagittatoside B, sagittatoside A, hexandraside E, 2′′‐O‐rhamnosyl icariside II, baohuoside VII, baohuoside I, caohuoside C, epimedoside C, baohuoside II, and kaempferol‐3‐O‐rhamnoside, in different species of Epimedium, and the effect of stability of internal standard (IS) on quantification was also investigated. As a result, rutin was not available for use as an IS because of its unstable property in sample solution, which suggested that the stability of IS both in standards and sample solution should be considered for the analysis. Using stable daidzein as IS, the analysis was performed within 35 min by using 50 mM borax buffer containing 20% ACN as a modifier (pH 10.0), while separation voltage was 25 kV and temperature was at 30°C. The method was validated to be accurate, simple, and repeatable, and was successfully applied to the analysis of 36 samples from 17 species of Epimedium.     

Secoiridoid Constituents from the Fruits of Ligustrum lucidum

Four new secoiridiod glucosides, p‐hydroxyphenethyl 7‐β‐D ‐glucosideelenolic acid ester ( 1 ), 6′‐elenolylnicotiflorine ( 2 ), 6′′′‐acetylnicotiflorine ( 3 ), and oleoside 7‐ethyl 11‐methyl ester ( 4 ), as well as six known glucosides, nuezhenide ( 5 ), Gl‐3 ( 6 ), nicotiflorine ( 7 ), isonuezhenide ( 8 ), neonuezhenide ( 9 ), and oleoside 11‐methyl ester ( 10 ) were isolated from the fruits of Ligustrum lucidum. Their structures were elucidated by spectroscopic methods. Compound 4 was an artifact produced during extraction.     

Activity-guided investigation of Carissa carandas (L.) roots for anti-inflammatory constituents

The present study was structured to investigate the anti-inflammatory potential of the extracts, fractions and compounds isolated from Carissa carandas (L.) roots. Bioassay guided fractionation of methanol extract based on inhibitory potential towards proinflammatory mediators (TNF-α, IL-1β and nitric oxide (NO)) led to the identification of stigmasterol (1), lupeol (2), oleanolic acid (3), carissone (4) and scopoletin (5) as potential anti-inflammatory agents. Carissone (4) (IC₅₀ = 20.1 ± 2.69 μg/mL) and scopoletin (5) (IC₅₀ = 24.6 ± 1.36 μg/mL) exhibited significant inhibition of NO production comparable to specific NO inhibitor (L-NAME; IC₅₀ = 19.82 ± 1.64 μg/mL) without affecting the cell viability. Also, 4 and 5 at a concentration of 30 μM were found to inhibit 41.88–53.44% of TNF-α and IL-1β. To the best of our knowledge, this is the first report displaying the anti-inflammatory effects of C. carandas (L.) roots, partially mediated by inhibition of TNF-α, IL-1β and NO.     

Synthesis, Reactions, and Anti-inflammatory Activity of Heterocyclic Systems Fused to a Thiophene Moiety Using Citrazinic Acid As Synthon

Summary. A series of pyridines, pyrimidinones, and oxazinones were synthesized as anti-inflammatory agents using citrazinic acid (2,6-dihydroxyisonicotinic acid) as a starting material. Acryloyl pyridine was treated with cyanothioacetamide to give cyano pyridine-thione, which was reacted with ethyl chloroacetate to yield the corresponding amino ester. The ester was hydrolysed to the sodium salt, which was treated with acetic anhydride to afford 2-methyloxazinone, which was treated with ammonium acetate to afford 2-methylpyrimidinone followed by methylation with methyl iodide to yield 2,3-dimethylpyrimidinone. In addition, the oxazinone derivative was reacted with aniline or hydrazine hydrate to give 3-phenyl- or 3-aminopyrimidinones. The latter reacted with thiophene-2-carboxaldehyde or phenylisothiocyanate to afford Schiff’s bases or thiosemicarbazides. 3-Aminopyrimidinone was treated with phthalic anhydride or 1,2,4,5-benzenetetracarboxylic acid dianhydride or toluene-3,5-diisocyanate to afford the corresponding imide, bis-imide, and bis-semicarbazide derivatives. The pharmacological screening showed that many of these compounds have good anti-inflammatory activity comparable to Prednisolone^? as reference drug.     

Studies on target tissue distribution of ginsenosides and epimedium flavonoids in rats after intravenous administration of Jiweiling freeze‐dried powder

A simple and rapid liquid chromatography–mass spectrometry (LC‐MS) method was developed and validated for analysis of ginsenoside Rb₁, Rb₂, Rc, Rd, Re, Rf, Rg₁, icariin and epimedin A, B, C in rat target tissues (spinal cord, brain, muscle and sciatic nerve) after intravenous administration of Jiweiling freeze‐dried powder using genistein as an internal standard (IS). The tissue samples were treated by protein precipitation with methanol prior to HPLC and chromatographic separation was performed on a C₁₈ column utilizing a gradient elution program with acetonitrile and 0.1% formic acid aqueous. Electrospray ionization (ESI) source was employed and the 11 analytes and IS were detected by multiple reaction monitoring (MRM) scanning under the negative ionization mode. Higher sensitivity was achieved and the optimized mass transition ion‐pairs (m/z) for quantitation were selected. The calibration curves were linear over the investigated concentration ranges with correlation coefficients higher than 0.995. The intra‐ and inter‐day RSDs were all less than 10% with the relative error (RE) within ±9.3%. The mean extraction recoveries for all compounds were between 93.3 and 106%. The proposed method was successfully applied to investigate the target tissue distribution of the 11 compounds in rat after intravenous administration of Jiweiling freeze‐dried powder. Copyright © 2011 John Wiley & Sons, Ltd.     

工业制备高效液相色谱法制备淫羊藿中的黄酮系列对照品

淫羊藿甙和朝藿定A、B、C是淫羊藿中重要的活性成分,本研究应用工业制备高效液相色谱从淫羊藿粗提物中分离制备了这4个成分。淫羊藿粗提物经大孔吸附树脂粗分离获得相应的组分后,利用工业制备高效液相色谱完成精制纯化。采用自装填Chromatorex C18制备柱(220 mm×77 mm, 10 μm),乙腈-水(26:74或30:70, v/v)为流动相进行洗脱,在35 min内,实现了这4种成分的基线分离及规模制备。从300 g粗提物(总黄酮含量约20%)中获得淫羊藿甙33 g、朝藿定C 4.6 g、朝藿定B 3.7 g和朝藿定A 0.6 g,产品纯度均达到98%以上。此方法通过两步分离即可实现这4种成分的完全分离,具有快速高效、产品纯度高的特点,适于淫羊藿中淫羊藿甙、朝藿定A、B、C系列对照品的规模制备。     

New chemical constituents from the fruits of Zanthoxylum armatum and its in vitro anti-inflammatory profile

Chemical investigations on the fruits of Zanthoxylum armatum Roxb. (Rutaceae) led to the isolation of two new constituents characterised as 2α-methyl-2β-ethylene-3β-isopropyl-cyclohexan-1β, 3α-diol (1) and phenol-O-β-D-arabinopyranosyl-4′-(3″, 7″, 11″, 15″-tetramethyl)-hexadecan-1″-oate (2) along with known compounds m-methoxy palmityloxy benzene (3), acetyl phenyl acetate (4), linoleiyl-O-α-D-xylopyranoside (5), m-hydroxyphenoxy benzene (6) and palmitic acid (7). The chemical structures were established with the help of physical, chemical and spectroscopic methods. The anti-inflammatory potential of isolated compounds 1 and 2 was evaluated using in vitro target-based anti-inflammatory activity in LPS-stimulated primary peritoneal macrophages isolated from mice. Production of pro-inflammatory cytokines (TNF-α and IL-6) was significantly inhibited by the treatment of isolated compounds 1 and 2 in a dose-dependent manner.     

Rapid characterization and determination of multiple components in Bu‐Shen‐Yi‐Qi‐Fang by high‐performance liquid chromatography coupled to electrospray ionization and quadrupole time‐of‐flight mass spectrometry

In this study, a qualitative and quantitative analysis using high‐performance liquid chromatography coupled to electrospray ionization and quadrupole time‐of‐flight mass spectrometry was performed for the quality control of Bu‐Shen‐Yi‐Qi‐Fang, a traditional Chinese formula used for asthma. Thirty‐four compounds, including flavonoids, isoflavonoids, triterpenoid saponins, and iridoid glycosides were identified or tentatively characterized by comparing their retention times and mass spectra with those of authentic standards or literature data. Sixteen components were considered as the main bioactive constituents of Bu‐Shen‐Yi‐Qi‐Fang and they were chosen as the chemical markers in quantitative analysis, including catalpol, leonuride, calycosin‐7‐O‐β‐d ‐glucoside, hyperoside, acteoside, formononetin‐7‐O‐β‐d ‐glucoside, epimedin A, calycosin, icariin, epimedin B, epimedin C, formononetin, astragaloside IV, astragaloside II, baohuoside‐I, and astragaloside I. The total run time was 20 min. It was found that the calibration curves for all analytes showed good linearity (R² > 0.99) within the test ranges. The relative standard deviations for intra‐ and inter‐day precisions were below 3.9 and 11.7%, respectively. The accuracy was evaluated by the recovery test within the range of 89.20–110.71% with the relative standard deviation < 4.8%. The sample was stable for at least 48 h at 4°C. The results showed that the new approach was effective for the quality control of Bu‐Shen‐Yi‐Qi‐Fang.     

Simultaneous determination of seven flavonoids in Epimedium by liquid chromatography-tandem mass spectrometry method

In this paper, a sensitive and specific liquid chromatography-electrospray ionisation-mass spectrometry （LC-ESI-MS） method has been developed and validated for the identification and determination of seven flavonoids, namely epimedin A, epimedin B, epimedin C, icariin, sagittatoside B, 2＂-O-rhamnosyl icariside II, and baohuoside I in Epimedium from different sources.     

Anti-inflammatory,antioxidant and antiangiogenic activities of diosgenin isolated from traditional medicinal plant,Costus speciosus (Koen ex.Retz.) Sm

Costus speciosus is an important medicinal plant widely used in several indigenous medicinal formulations. The present study was conducted to evaluate the in vitro anti-inflammatory, antioxidant and antiangiogenic activities of diosgenin isolated from C. speciosus. The diosgenin was isolated from C. speciosus by HPTLC and its biological activities were studied by different protocols. The results demonstrated that LPS stimulated TNF-α generation in RAW 264.7 macrophage culture supernatant up to 3.7-fold of the control and that sample treatment (50 μg/mL) resulted in a highly significant inhibitory effect on LPS-stimulated TNF-α (p < 0.01) in a similar manner to methotrexate inhibitory effect. The tested sample possessed an effective antioxidant scavenging affinity against DPPH radicals as compared with the standard antioxidant activity of vitamin C. The results presented here may suggest that diosgenin isolated from C. speciosus possess anticancer, apoptotic and inhibitory effects on cell proliferation.     

Comparative pharmacokinetic study of nine bioactive components in osteoarthritis rat plasma using ultra-performance liquid chromatography–tandem mass spectrometry after single and combined oral administration of Epimedii Folium and Chuanxiong Rhizoma extracts

The herb pair Epimedii Folium–Chuanxiong Rhizoma (EF–CR), derived from the classical traditional Chinese medicine ‘Xian Ling Pi San’, has a distinctive compatibility therapeutic profile and is clinically safe and effective. This study aimed to investigate and compare the pharmacokinetic characteristics of nine analytes in osteoarthritis (OA) rat plasma after the oral administration of EF, CR or a combination of these two herbs. We developed an ultra-performance liquid chromatography method coupled with quadrupole linear ion-trap mass spectrometry to simultaneously quantify and assess the pharmacokinetics of icariin, epimedin A, epimedin B, epimedin C, icariside I, icariside II, ferulic acid, ligustilide and senkyunolide A of the EF–CR pair in the plasma of osteoarthritic rats. The pharmacokinetic parameters showed that the absorption of multiple components was significantly enhanced and residence time was prolonged in the EF–CR group (P < 0.05) compared to the single-herb group. These parameters revealed that the combination of EF and CR exhibited synergistic effects of the nine bioactive components, suggesting the potential application of the EF–CR combination for the treatment of OA.     

Inhibitory effect of silibinin on tumour necrosis factor-alpha and hydrogen peroxide production by human monocytes

Silibinin is a chemically defined flavonoid and the main active component of silymarin, a polyphenolic complex from Silybum marianum, which has anti-inflammatory, hepatoprotective and anticarcinogenic properties. Monocytes obtained from healthy individuals were incubated with silibinin to evaluate cell viability, hydrogen peroxide (H(2)O(2)) release and tumour necrosis factor-alpha (TNF-α) production by these cells. The duration of treatment and different silibinin concentrations had no significant effect on cell viability. Monocytes showed a dose-dependent inhibitory effect on H(2)O(2) release by phorbol myristate acetate-stimulated monocytes in silibinin concentrations ranging from 6.25 to 50 μg mL(-1). Significant inhibition of TNF-α production by lipopolysaccharide-stimulated monocytes was observed at concentrations of 12.5, 50 and 100 μg mL(-1) of silibinin. These results suggest that silibinin exerts antioxidant and anti-inflammatory properties on human monocytes through an inhibitory effect on H(2)O(2) release and on TNF-α production, respectively.     

Synthesis of unsymmetric bile pigments: Mesobilirubin-VIIIα, 17-desvinyl-17-ethyl-bilirubin-VIIIα and 12-despropionic acid-12-ethyl-mesobilirubin-XIIIα

The unnatural bile pigments 17-desvinyl-17-ethylbilirubin-VIIIα, mesobilirubin-VIIIα and 12-despropionic acid-12-ethyl mesobilirubin-XIIIα were synthesized via (1) “reverse scrambling” of bilirubin-XIIIα or mesobilirubin-IXα with mesobilirubin-IVα or etiobilirubin-IV-γ or (2) following coupling of xanthobilirubic acid methyl ester with ψ-xanthobilirubic acid methyl ester, or coupling of xanthobilirubic acid methyl ester with kryptopyrromethenone.     

Synthesis and anti-inflammatory and antimicrobial activities of some novel 2-methylquinazolin-4(3H)-one derivatives bearing urea,thiourea and sulphonamide functionalities

A variety of novel 2-methylquinazolin-4(3H)-one derivatives (1–29) bearing urea, thiourea and sulphonamide functionalities at position 3 of biological interest have been synthesized and screened for their anti-inflammatory (TNF-α and IL-6) and antimicrobial activities (antibacterial and antifungal). “Biological evaluation study revealed that the compounds 3, 4, 6, 9, 16 and 18 were found to have promising anti-inflammatory activity (up to 62–84% TNF-α and 73–92% IL-6 inhibitory activity) at concentration of 10 μM with reference to standard dexamethasone (75% TNF-α and 84% IL-6 inhibitory activities at 1 μM) and 7, 10, 12, 23, 25 and 28 have antimicrobial activity at MIC of 10–30 μg/mL against selected pathogenic bacteria and fungi.”