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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.
Subject(s)
Chromatography, High Pressure Liquid , Epimedium , Tandem Mass Spectrometry , Chromatography, Liquid , Multivariate AnalysisABSTRACT
OBJECTIVE：To establish HPLC fingerprint of different products of suet oil-baked Epimedium brevicornum ，and to screen the optimal baking technology. METHODS ：HPLC method was adopted. Using icariin as reference ，HPLC fingerprints of 22 batches of samples were drawn. The similarity was evaluated by using Similarity Evaluation System of TCM Chromatographic Fingerprint（2012 edition），and common peaks were confirmed. HCA ，PCA and OPLS-DA analysis were performed by SIMCA 14.1 statistical software. Taking variable importance in the project ＞1 as criteria ，biomarkers affecting the quality difference of suet oil-baked E. brevicornum were screened ；using mass marker as index ，the baking technology was screened by baking technology. RESULTS：There were 18 common peaks in 22 batches of samples. The similarities were between 0.831 and 0.991. Totally 7 common peaks were identified ，i.e. epimedin A ，epimedin B ，epimedin C ，icariin，sagittatoside A ，sagittatoside B ，baohuoside Ⅰ. The 22 batches of samples were clustered into two categories ，S19-S22 were clustered into category Ⅰ and S 1-S18 were clustered into category Ⅱ. The category Ⅱ was sub-clustered into category Ⅱa（S15-S18），category Ⅱb（S10-S14），category Ⅱc （S1-S9）；the result of PCA analysis was consistent with above results. OPLS-DA showed that the biomarkers affecting the quality difference were icariin ，sagittatoside B and baohuoside Ⅰ. The results of kinetic studies showed that the content of icariin when baked at 180 ℃ for 25 min or 190 ℃ for 20 min，that of baohuoside Ⅰ when baked at 180 ℃ for 30 min or 190 ℃ for 15 min and that of epimedin B when baked at 210 ℃ for 18 min were the highest ；according to above results ，the optimal baking technology was baking at 180 ℃ for 25-30 min or 190 ℃ for 15-20 min. CONCLUSIONS ：Established fingerprint is stable ， reliable and reproducible. The multivariate statistical analysis can be used for the changes of chemical components in E. brevicornum under different baking condition and preliminary selection of baking technology.
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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.
Subject(s)
Epimedium , Classification , Metabolism , Estrogens , Flavonoids , Signal TransductionABSTRACT
Objective To establish a method that can accurately and efficiently screen potential pharmaceutical agents targeting Nuclear Receptor Retinoid X receptor-α (RXRα) from Traditional Chinese Medicine .Methods Based on the principle of Receptor-Ligand Recognition ,the fusion protein of His-RXRαLBD and the active fractions were mixed to incubate for capturing and identif-ying potential ligand of RXRα,and then the identified ligand was evaluated for RXRα-dependent anti-inflammation effect .Results Baohuoside Ⅰ from Epimedium brevicornum Maxim .was identified as a RXRα binder ,and could induce RXRα-dependent anti-in-flammation effect .Conclusion The method was an efficient strategy to accurately identify active compounds from natural products , to expedite the discovery of natural RXRαregulator ,and also to share the thought with other receptors in identifying ligand .
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This study was aimed to optimize the extraction technology of Wei-Kang (WK) Capsule. Orthogonal test and comprehensive evaluation were used to optimize the extraction process of compound preparation. The icarrin, to-tal ginkgo flavone glycosides and the dried decocting rates were used as index components for optimizing the effect of ethanol concentration, ethanol volume, extraction duration and extraction frequency. The results showed that the opti-mal ethanol extraction technology was adding 12 times of 60% ethanol and extract for 3 times with 1 h for each ex-traction time. It was concluded that the optimized extraction technology was stable, practical, scientific and reason-able, which can be used in the large-scale industrial production.
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Objective To identify the constituents and metabolites of epimedium in rat plasma by rapid-resolution liquid chromatography-time of flight mass spectrometry (RRLC-TOF/MS) after intragastric administration, so as to explore the active ingredients and metabolites of epimedium in vivo. Methods The separation was performed on a SHISEIDO MG C18 reverse phase column (3. 0 mm×100 mm, 3 μm). The mobile phase consisted of water containing 0. 1% formic acid and acetonitrile was used as gradient elute. The flow ratewas 0. 6 mL/min, post-column split ratio was 3) 1, and the temperature of column was 25°C. Time-of-flight mass spectrometer(TOF/MS) and electrospray ion source (ESI) were applied for qualitative analysis under positive ion mode, with the mass scan range being m/z 100-1 000. Results Three prototype constituents and 4 metabolites of epimedium were identified in rat plasma. Conclusion A rapid and efficient RRLC-TOF/MS method has been established for studying the constituentsand metabolites of epimedium in vivo, which provides a reference for further pharmacodynamics experiments.
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Objective To investigate the effects of the traditional herb-epimedium on the expression of interleukin-6 (IL-6) mRNA in bone of ovariectomized rat. Methods Forty female rats were randomly allocated into 4 groups, 10 in each: ovariectomized (OVX) group, sham operation group, OVX followed by epimedium (group 3)or nilestriol (group 4) for 3 months respectively. All rats were then sacrificed,and total RNA were directly isolated from their right tibia. Interleukin-6 mRNA expression was detected by relative semiquantitative reverse transcription-polymerase chain reaction technique. Lumbar bone mineral density (BMD) was measured by dual-energy X ray absorptiometry before sacrifice. Results The BMD of epimedium group was significantly higher than that in the OVX group ( P
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Objective To study the chemical constituents of Epimedium brevicornum.Methods Compounds were isolated with various chromatographic techniques,their structures were elucidated by physicochemical methods and spectral analysis.Results Ten compounds were obtained and identified as p-nitroethylphenol(Ⅰ),salidroside(Ⅱ),5,7,4'-trihydroxy-8,3'-diprenylflavone(Ⅲ),kaempferol-3-O-?-L-dirhamnoside(Ⅳ),baohuoside-Ⅰ(Ⅴ),sagittatoside B(Ⅵ),2″-O-rhamnosylicariside Ⅱ(Ⅶ),7,3',4',5'-tetrahydroxyflavanol(Ⅷ),desmethylicaritin(Ⅸ),and 1-O-?-glucopyransosyl-1,4-dihydroxy-2-(3'-hydroxy-3'-methylbutyl)benzene(Ⅹ).Conclusion Compounds Ⅰ,Ⅱ,Ⅳ,and Ⅹ are isolated and identified from the plants of Epimedium L.for the first time and compound Ⅵ is isolated from the title plant for the first time.
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Objective To study antidepressant effects of Epimedium brevicornum extracts.Methods(Behavioral) despair models of male mice,tails suspension test(TST),and forced swimming test(FST) were used to evaluate the effects of E.brevicornum extracts on behavioral,monoamine oxidas(MAOA) and monoamine oxidase B(MAO-B) activities in brain and liver tissue,and MDA level in liver tissue of mouse.Reserpine antagonistic model was also used to investigate possible antidepressant mechanisms of E.brevicornum extracts.Results The extracts of E.brevicornum(25,50, 100,and 200 mg/kg) significantly reduced the duration of murine immobility in TST and FST,inhibited MAO-A and MAO-B activities in brain and liver of mice,and reversed the elevated liver MDA level in mice in TST.There was no significant amelioration in the decreases of body temperature in mice of Reserpine antagonistic model.Conclusion(E.brevicornum) extracts possesses the definite antidepressant properties.
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Objective To investigate the effect of total flavonoids of Herba Epimodii (HEF) on the expression of core binding factor?1 (Cbfal) in the bone of ovariectomized rats.Methods Fifty-four female SD rats were allocated into 6 groups (9 in each): sham operation group,ovariectomized (OVX) group,OVX followed by three kinds of HEF doses(40,80 and 160 mg?kg~(-1)?d~(-1))and nilestriol (0.1 mg?kg~(-1)?week~(-1))for 12 weeks respectively.Bone mineral density (BMD) of whole body was determined by DEXA.The levels of osteocalcin (BGP) and estradiol (E_2) in serum were measured by radioimmunologic method.All rats were then sacrificed,and total RNA were directly isolated from the skull.Relative quantification of Cbfal mRNA expression was detected by real time quantitative PCR.Results Serum E_2 and BMD of whole body in the OVX group were lower than those in sham group significantly after 12 weeks (both P0.05).Relative quantification of Cbfal mRNA expression in the OVX group was significantly lower than that in sham group after 12 weeks (P
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Objective To investigate the effects of total flavonoids of Herba Epimedii(HEF)on the metabolism of typeⅠcollagen and the expression of cathepsin K in the bone of ovariectomized(OVX)rats. Methods Fifty-four female SD rats were allocated into 6 groups;OVX group,sham operation group,OVX rats followed by three doses of HEF(40,80 and 160 mg?kg~(-1)?d~(-1))and nilestriol(0.1 mg?kg~(-1)?d~(-1))for 12 weeks respectively.Bone mineral density(BMD)of whole body was determined by dual-energy X-ray absoptiometry.The level of cross-linked N-telopeptide of typeⅠcollagen(NTx)in the urine were determined by ELISA.The amounts of typeⅠcollagen protein and cathepsin K protein in bone tissue were detected by immunohistochemical method and Western blotting.Results Compared with OVX group,the total BMD values in the HEF treated groups were increased(all P＜0.05),and the expression levels of typeⅠcollagen in three HEF treated groups rose significantly in a dose-dependent manner after 12 week,and simultaneously,both the expression of cathepsin K in bone and the level of NTx/Cr were reduced markedly(P＜0.05),being most significant(P＜0.01)in the group treated with the highest dose of HEF(160 mg?kg~(-1)?d~(-1)).Conclusion HEF seems to be able to elevate BMD and improve bone quality of rats via promoting synthesis and inhibiting proteolysis and absorption of typeⅠcollagen in the bone.