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ObjectiveTo develop a quality control method for the simultaneous determination of multiple active components in Nymphaeae Flos aiming at the problems of the single index for quality control and the relatively low overall quality control level. MethodUltra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）was used to identify and select the index components for quality control with the mobile phase of 0.1% formic acid aqueous solution（A）-acetonitrile（B）for gradient elution （0-2 min， 3%-8%B； 2-4 min， 8%-10%B； 4-13 min， 10%-15%B； 13-19 min， 15%-20%B； 19-26 min， 20%-45%B） at a flow rate of 0.4 mL·min-1， detection wavelength of 350 nm， electrospray ionization（ESI）， negative ion scanning mode， ion source temperature of 120 ℃， scanning range of m/z 100-1 200， transmit collision energy of 6 eV for low-energy scanning and 25-50 eV for high-energy scanning. High performance liquid chromatography（HPLC）was used to establish the quality control method for the simultaneous determination of multi-index components with the mobile phase of 0.2% phosphoric acid aqueous solution（A）-acetonitrile（B） for gradient elution（0-30 min， 12%-15%B； 30-60 min， 15%-22%B； 60-90 min， 22%-40%B）and detection wavelength of 350 nm. The preparation method of the test solution for content determination was refluxing extraction for 60 min with 80 times the amount of 70% methanol. ResultBy comparing the retention time， ultraviolet absorption characteristics， MS and MS/MS spectrometric signals in the samples with the reference substances， 8 active components with high contents， including brevifolincarboxylic acid， ellagic acid， rutin， nicotiflorin， astragalin， quercetin， quercetin-3-methylether and kaempferol， were identified qualitatively from Nymphaeae Flos， which were selected as the index components for quality control. Under the established HPLC conditions， the above 8 components could be well separated（resolution>1.5）， and showed good linearity（r=0.999 9）between the concentration ranges of 1.99-99.6， 1.76-176， 1.52-75.8， 3.60-180， 0.964-96.4， 1.18-118， 1.94-96.8， 1.04-104 mg·L-1 and the peak areas， respectively. The detection limits of them were 10-49 μg·L-1， and the limits of quantitation were 34-164 μg·L-1. The average recoveries were 97.12%-103.1% with the relative standard deviations （RSDs） were 1.1%-2.2%. ConclusionA quality control method for simultaneous determination of the multiple active components in Nymphaeae Flos have been developed， which is simple， accurate and reproducible， and it can provide a scientific basis for the formulation of quality standard of this herb and lay a research foundation for the transformation of Uygur hospital preparations containing Nymphaeae Flos into new drugs.
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OBJECTIVE： To optimize the extraction technology of verbascoside from Cistanche tubulosa， and to provide reference for further development and comprehensive utilization of C. tubulosa. METHODS： The content of verbascoside in C. tubulosa was determined by HPLC. The determination was performed on Inertsil-ODS-3V column with mobile phase consisted of methanol-0.2％ formic acid aqueous solution （40 ∶ 60， V/V） at the flow rate of 1 mL/min. The column temperature was 30 ℃， the detection wavelength was 330 nm， and the sample size was 10 μL. Using extraction rate of verbascoside as index， soaking time， ethanol concentration， liquid-solid ratio， extraction time and extraction times were investigated by single factor tests. According to the results of above tests， ethanol concentration， liquid-solid ratio and extraction time were optimized by Box-Behnken response surface methodology. The verification tests were carried out on the optimized extraction technology. RESULTS： The linear range of verbascoside was 18.65-932.4 μg/mL. The optimal extraction technology included that ethanol concentration 63％， liquid-solid ratio 8 ∶ 1 （mL/g）， soaking for 2 h， extraction time 1.5 h， extracting for 2 times. The extraction rates of verbascoside in the three parallel verification tests were 78.21％， 76.95％， 79.34％， respectively. The relative errors of those to predicted value 76.76％ were 1.89％， 0.25％， 3.36％. CONCLUSIONS： The optimized extraction technology of verbascoside from C. tubulosa is stable and feasible， and is suitable for the extraction of verbascoside.
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Objective To establish the chromatographic conditions of isochlorogenic acid A and isochlorogenic acid C in vernonia anthelmintica. Methods By changing the mobile phase,flow rate,column temperature and other chromatographic conditions,the best chromatographic conditions was we pursued to established. Results The linear relationship between the concentration of isochlorogenic acid A and the peak area was between 5. 825-69. 9 μg·mL-1, and the concentration of isochlorogenic acid C,was between 5.15-61.80 μg·mL-1and the peak area was good . The sample recovery rates of the two groups were 98.70%-101.92%(RSD＝1.04%,n＝9)、95.99%-102.52%(RSD＝1.90%,n＝9). Conclusion The method is simple,rapid, accurate and reliable for the determination of isochlorogenic acid A and isochlorogenic acid C in Vernonia anthelmintica and also for the quality control of the raw material.
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OBJECTIVE:To investigate the in vitro transdermal absorption properties of galangin and effects of different pene-tration enhancers on its transdermal behaviors,and provide reference for developing skin preparations using galangin as APIs in the treatment of vitiligo. METHODS:HPLC was used to determine the galangin content. Using cumulative permeation rate (Q) and the transdermal rate(J)of galangin as indexes,the effect of absorption of receiving solution [20%,40% polyethylene glycol 400 (PEG400)solution and 30% ethanol solution] and rotating rate(200,300,400 r/min)on galangin in complete skin of mice were investigated,as well as the azone(1%,3%,5%)and propylene glycol(10%,20%,40%)alone or combination on its penetra-tion promotion. And the transdermal properties of galangin in complete skin,exfoliating skin,dermis skin of rats and mice were de-tected. RESULTS:The best permeability of complete skin of mice showed in 40% PEG400 solution at rotating speed of 300 r/min with 5% azone alone,J was 3.2570 μg/(cm2·h). Js of complete skin,exfoliating skin,dermis skin of mice were 2.7199,34.016, 33.874 μg/(cm2·h),respectively;and those of rats were 0.4996,9.5124,17.406 μg/(cm2·h). CONCLUSIONS:Galangin can penetrate the complete skin of mice and rats,however,the penetration quantity is far lower than exfoliating skin and dermis skin.
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OBJECTIVE:To study the extraction rate and speciation of antivitiligo-related elements in Opercalina turperthum by water extraction and semi-bionic extraction. METHODS:Water extraction and semi-bionic extraction were respectively used. Wa-ter-soluble state and suspension state in extract of O. turperthum were separated by microporous filtering film;organic and inorgan-ic trace elements in water-soluble state were separated by macroporous resin. The contents of Cu,Zn,Fe,Ca,Mg,Mn and Sr were detected by flame atomic adsorption spectrophotometry,and speciation analysis was conducted. RESULTS:After water extrac-tion,extraction rates of 7 trace elements were 40.47%-72.49%;ratio of suspended particles was 3.69%-8.78%;ratio of organic state/inorganic state was 104.36% in water-soluble state of Sr and 3.94%-48.39% in water-soluble state of Cu,Zn,Fe,Ca,Mg, Mn. After semi-bionic extraction,except for Mn,extraction rates of trace elements were higher than water extraction,extraction rates of Cu,Zn,Fe,Ca,Mg,Sr were 77.69%-90.19%;ratio of suspended particles was 0.39%-8.57%;the ratio of organic state/inorganic state was 72.74%-180.79% in water-soluble state of elements. CONCLUSIONS:Cu,Zn,Fe,Ca,Mg,Mn and Sr in O. turperthum are dissolved easily,mainly existing in the form of inorganic state. After semi-bionic extraction,except for Mn,the dis-solution rate of other elements and the proportion of organic trace elements after dissolution increase significantly. Both dissolution and effective utilization rate of trace elements by semi-bionic extraction are higher than water extraction.
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OBJECTIVE:To study the effects of butin in Vernohia anthelmintica(VW)on proliferation of human immortal ke-ratinocyte cell strain HaCaT and cell secretory factors,and explore the mechanism of butin in VW in the treatment of vitiligo. METHODS:MTT method was used to determine the survival rate of HaCaT cells cultured by 0 (blank control),0.1,0.5,1.0, 5.0,10.0 μg/mL of butin for 48 h. Enzyme-linked immunosorbent assay was used to determine the contents of cell secretory factors as endothelin 1 (ET-1),ET-3,melanocyte stimulating hormone (MSH),stem cell factor (SCF),basic fibroblast growth factor (bFGF)in culture medium after HaCaT cells were cultured by 0.5,1.0,5.0 μg/mL of butin for 48 h. RESULTS:Compared with blank control,cell survival rate was increased to varying degrees after cultured by 0.1-5.0μg/mL of butin for 48 h,while decreased after cultured by 10.0 μg/mL of butin. Contents of ET-1,SCF,bFGF in culture medium were significantly increased after cultured by 0.5,1.0,5.0μg/mL of butin for 48 h(P<0.01);and contents of ET-3,MSH in culture medium were significantly increased af-ter cultured by 1.0,5.0 μg/mL of butin for 48 h(P<0.01). CONCLUSIONS:Butin can promote the proliferation of HaCaT cells, the mechanism may be associated with promoting the secretion of cell secretory factors of ET-1,ET-3,MSH,SCF,bFGF.
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OBJECTIVE:To study the effects of butin in Vernohia anthelmintica(VW)on proliferation of human immortal ke-ratinocyte cell strain HaCaT and cell secretory factors,and explore the mechanism of butin in VW in the treatment of vitiligo. METHODS:MTT method was used to determine the survival rate of HaCaT cells cultured by 0 (blank control),0.1,0.5,1.0, 5.0,10.0 μg/mL of butin for 48 h. Enzyme-linked immunosorbent assay was used to determine the contents of cell secretory factors as endothelin 1 (ET-1),ET-3,melanocyte stimulating hormone (MSH),stem cell factor (SCF),basic fibroblast growth factor (bFGF)in culture medium after HaCaT cells were cultured by 0.5,1.0,5.0 μg/mL of butin for 48 h. RESULTS:Compared with blank control,cell survival rate was increased to varying degrees after cultured by 0.1-5.0μg/mL of butin for 48 h,while decreased after cultured by 10.0 μg/mL of butin. Contents of ET-1,SCF,bFGF in culture medium were significantly increased after cultured by 0.5,1.0,5.0μg/mL of butin for 48 h(P<0.01);and contents of ET-3,MSH in culture medium were significantly increased af-ter cultured by 1.0,5.0 μg/mL of butin for 48 h(P<0.01). CONCLUSIONS:Butin can promote the proliferation of HaCaT cells, the mechanism may be associated with promoting the secretion of cell secretory factors of ET-1,ET-3,MSH,SCF,bFGF.
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ObjectiveTo investigate the effects of extracts ofLiaoxuan Kaxifu Powders (LE) on proliferation and transcription of IL-8 and ICAM-1 in HaCaT induced by TNF-α; To discuss its mechanism of action.Methods Cultured HaCaT was assigned into normal group, TNF-α group and low-, medium- and high-dose of LE group. Every group was induced by 40 ng/mL TNF-α except for normal group, and then LE groups were treated by different concentrations (8, 40, 200μg/mL) of LE for 48 h. The proliferation of HaCaT was evaluated by MTT and the apoptosis was detected by inverted fluorescence microscope. Levels of IL-8 and ICAM-1 in HaCaT were assessed by ELISA, and their mRNA expressions was detected by semi-quantitative RT-PCR.Results Compared with normal group, the absorbency of HaCaT and the contents and mRNA expressions of IL-8 and ICAM-1 increased in TNF-α group (P<0.05,P<0.01); compared with TNF-α group, LE of all dose groups could significantly inhibit the absorbency, decrease the contents and mRNA expressions of IL-8 and ICAM-1 (P<0.05,P<0.01).Conclusion LE is able to inhibit the proliferation of HaCaT induced by TNF-α, and the mechanism is probably related to promoting apoptosis and down-regulating the gene expressions of IL-8 and ICAM-1, and then maintaining the normal level of HaCaT.
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Objective To investigate the effect of acteoside on injury PC12 cells induced by glutamate. Methods PC12 cells were assigned into normal control group, model control group, positive drug group and acteoside(AS) treated group. Every group was treated by 1. 5 mmol·L-1 glutamate for 24 hours except for the control group, and the injury was antagonized by 10 μmol·L-1 Vit E and acteoside at different concentration(15. 625, 31. 25, 62. 5, 125 and 250 μmol·L-1 ). Cell morphology was observed by inverted microscope, cell survival was determined with MTT, LDH activity was measured by enzyme label kit, the MDA content and SOD activity were measured by TBA kit and WST kit, and the ROS was measured by Elisa kit. Results Compared with the model control group, all doses of acteoside could significantly improve the PC12 cell morphology and survival (P<0. 05), inhibit LDH activity and production of MDA and ROS (P<0. 05), increase the activity of SOD (P<0. 05), except for the lowest dose group. Conclusion Acteoside has protective effects on PC12 cells injured by glutamate.
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Objective To investigate the effects of galangin on gene expressions of Nrf2 andγ-GCS in oxidative damage of A375 cell;To discuss its protective mechanism for anti-oxidative damage. Methods A375 melanoma cells were induced oxidative stress to establish oxidative damage model by 700μmol/L H2O2. The study was divided into normal group, model group, positive medicine group and high-, medium-, and low-dose galangin groups. All administration groups were given relevant medicine for cultivation. Cell viability was detected by MTT;ROS content was detected by ELISA;the gene expressions of Nrf2 andγ-GCS were detected by RT-PCR.Results Compared with normal group, cell viability decreased significantly;ROS content increased significantly;the gene expressions of Nrf2 andγ-GCS decreased significantly in the model group (P<0.05,P<0.01). Compared with model group, cell viability increased, ROS content decreased, the gene expressions of Nrf2 andγ-GCS increased significantly in all administration groups (P<0.05,P<0.01). Conclusion Galangin may activate Nrf2 signal path to realize the protective effect on A375 cellular oxidation damage through upregulating the expressions of Nrf2 andγ-GCS to promote the integration of Nrf2 and antioxidant response element and relevant regulatory enzymes.
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Aim To investigate the effect of acteoside (AS)on the expression of caspase-3 in cerebral cortex of mouse models of Alzheimer’s disease(AD).Meth-ods Kunming (KM)strain mice were assigned into control group,model group,positive control group (VitE)and acteoside group.Every group was induced by a combination of D-galactose(i.p.60mg·kg -1 · d -1 )and AlCl3 (i.g.5mg·kg -1 ·d -1 )for 60ds ex-cept for control group,then mice were treated by dif-ferent concentrations(30,60,1 20 mg·kg -1 ·d -1 )of acteoside for 30ds.During the time,mice were in-duced continuously by a combination of D-galactose and AlCl3 .The learning and memory of mice were de-tected by step-down test,the activity of AChE in serum and brain of mice was measured by chemical colorime-try,the structure changes in cerebral cortex were ob-served by HE staining,and the expression of caspase-3 in cerebral cortex was analyzed through the immunohis-tochemical staining.Results Compared with model group,acteoside could improve the learning and mem-ory abilities(P <0.05 or P <0.01 ),decrease the ac-tivity of AChE in serum and brain(P <0.05 or P <0.01 ),and improve the morphology and number of neuron in cerebral cortex(P <0.01 ).Moreover,acte-oside could significantly inhibit the expression of caspase-3 in cerebral cortex (P <0.05,P <0.01 ). Conclusion Acteoside has significantly protective effects on brain damage of mice induced by a combina-tion of D-galactose and AlCl3 , and it′s protective mechanism probably relate to inhibiting the expression of caspase-3 and maintainings the normal morphology and number of neuron in cerebral cortex.
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Objective To study the effects of the gingerol on the melanogenesis in melanoma B16 cells. Methods Melanoma cells were cultured with gingerol at 12. 5, 25. 0, 50. 0, 100. 0, 200. 0 μmol · L-1 and positive control drug hydroquinone,respectively,using Dulbecco's modified eagle's medium(DMEM) as the blank control group. The cell proliferation was measured by methyl thiazolyltet tetrazolium ( MTT) colorimetric assay. The tyrosinase activity and melanin content were measured by colorimetry assay. Results Gingerol at different concentrations had inhibitory effect on B16 cell proliferation compared with the blank control group ( P < 0. 05 or P < 0. 01), the inhibition rate being more than 48% at the dosage of 200. 0 μmol·L-1 . Tyrosinase activity was inhibited significantly compared with blank control group(P<0. 05 or P<0. 01),the inhibition rate being up to 50% at the dosage of 200 μmol·L-1 . Melanin content was also decreased at all levels of gingerol compared with blank control group(P<0. 05 or P<0. 01),but not in a dose-dependent manner. The inhibition rate of melanin content reached the plateau at gingerol levels greater than 25. 0 μmol · L-1 . Conclusion Gingerol can inhibit the cell proliferation,tyrosinase activity and decrease melanin synthesis in certain range of concentrations.
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Objective To investigate the effect of Galangin of different purity on melanocyte proliferation and melanin synthesis of A375-HaCaT co-culture system. Methods The A375-HaCaT co-culture system was established with Transwell technology, and 0.1, 0.5, 1.0, 5.0, 10 μg/mL Galangin of the purity of 70%and 90%was used to act on the co-culture system. Cell proliferation, melanin content and tyrosinase of A375 were measured by MTT assay, NaOH lysis assay and L-DOPA oxidation assay respectively. The cytokines such as ET-1 and SCF in HaCaT were detected by ELISA. Results A375 cells in co-culture system grew well, and 0.1, 0.5, 1.0, 5.0, 10 μg/mL Galangin of the purity of 70% and 90% had up-regulation effect on cell proliferation, melanin content and tyrosinase of A375. Moreover, Galangin could increase the expression level of ET-1 and SCF of HaCaT at more than 0.5 μg/mL, and the effect of Galangin of 70% purity was better than 90% purity. Conclusion Galangin could promote the cell proliferation, melanin content and tyrosinase of A375, and mechanism of the pathways is probably related to the up-regulation on the expression of ET-1 and SCF of HaCaT.
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<p><b>OBJECTIVE</b>To establish the fingerprint of Vernonia anthelmintica extracts of different ethanol concentrations to study the effect of common peak components on the proliferation of A375 human melanoma cells and the correlation between fingerprint and pharmacodynamics of V. anthelmintica extracts, in order to provide both theoretical basis and data support for establishing a traditional Chinese medicine quality control mode with the combination of fingerprint and pharmacology.</p><p><b>METHOD</b>HPLC was applied to establish fingerprint of V. anthelmintica extracts of different ethanol concentrations and the similarity evaluation was made. MTT was used to study the proliferation of A375 human melanoma cells. Grey relation analysis was adopted to analyze the correlation between fingerprint and pharmacology of V. anthelmintica.</p><p><b>RESULT</b>The HPLC fingerprint of different V. anthelmintica ethanol extracts showed 13 common peaks with the proliferation effect on A375 human melanoma cells. The 4th peak had the largest contribution and highest correlation to cell proliferation.</p><p><b>CONCLUSION</b>Different V. anthelmintica ethanol extracts have different fingerprints, which show a certain correlation between their common peaks and effect on cell proliferation.</p>
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Humanos , Linhagem Celular Tumoral , Proliferação de Células , Melanoma , Tratamento Farmacológico , Patologia , Extratos Vegetais , Farmacologia , Vernonia , QuímicaRESUMO
Objective To sieve the bioactive constituents of Plumbago zeylanica, serum pharmacochemistry research was performed. Method Based on the establishment of HPLC fingerprints of Plumbago zeylanica, the constituents absorbed into blood were determined by comparing the HPLC fingerprints of the methanol extracts, medicated serum samples and blank serum sample. Results One compound absorbed into blood was detected, the other might be metabolites of the original constituent. Conclusion The one onstituent absorbed into blood was possible bioactive component of Plumbago zeylanica.
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Objective:Vernonia anthelmintica,with complicated components,is a common medicine for the treatment of vitiligo.The purpose of this study was to establish and optimize the separation conditions of high performance liquid chromatography fingerprints(HPLC-FPs) for Vernonia anthelmintica(L.) Willd.Methods: We investigated the HPLC-FPs of different extraction samples by the retention times and relative areas of common peaks in the fingerprints.Results: We established the optimum separation conditions of HPLC-FPs for Vernonia anthelmintica(L.) Willd,and obtained 19 common peaks in the fingerprints.The HPLC-FPs of the 85% ethanol extract were quite similar to those of the semi-bionic extract(SBE) but very different from those of the water extract of Vernonia anthelmintica(L.) Willd.The No.1,4,6,11,12,14 and 16-19 peaks were the endemic ones of the HPLC-FPs of the 85% ethanol extract and SBE of Vernonia anthelmintica(L.) Willd,while No.20-30 were the endemic peaks of the HPLC-FPs of the water extract.Conclusion: The results of our study has provided experimental evidence for further investigation into the constituents absorbed into blood,pharmaceutical technology and pharmacodynamics of Vernonia anthelmintica(L.) Willd.
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OBJECTIVE:To establish a determination method for trace elements of Plumbago zeylanica. METHODS:Induct- ively coupled plasma emission spectrum (ICP-AES) was used to determine seven trace elements of P. zeylanica, i.e. Cu, Zn,Fe,Ca,Mg,Mn and Sr simultaneously. Se was determined by atomic fluorescence (AFS) method. RESULTS: The RSD ranged from 0.78% to 3.01%(n=5),while the recovery was within 91.9%～105.8%. The result obtained was satisfactory. Se took a large proportion in P. zeylanica, followed by Cu,Zn,Fe,Ca,Mg,Mn,Sr.CONCLUSION: The method is brief, rapid and accurate for the determination of P. zeylanica. The study provide reference for future pharmacological study of P. zeylanica.
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Objective To optimize the best condition of semi-bionic extraction for Vernonia anthelmintica.Methods The best condition of the semi-bionic extraction for V.anthelmintica by uniform design was optimized with total HPLC integrate area of common peaks in fingerprints,total flavonoids and dried extract weight as the indexes.Results According to industry production condition,the best technical condition:pH values of 80% ethanol for the 1st,2nd,and 3rd decoctions were in order of 6.0,6.5,and 8.0,and the extraction time were 1,0.5,and 0.5 h,respectively.Conclusion It is better for V.anthelmintica extracted by semi-bionic extraction,and the parametrs are optimized by uniform design.