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Objective:To develop an HPLC method for the simultaneous determination of four active ingredients ( psoralen, ber-gapten, desmethylwedelolactone and wedelolactone) in Xichuan pills. Methods: A Hypersil C18 column was used for the chromato-graphic separation of the four analytes. The mobile phase consisting of methanol-acetonitrile (2∶ 1) and 0. 5% glacial acetic acid solu-tion was used for gradient elution. The flow rate was 1. 1 ml·min-1 . The column temperature was 25℃. Psoralen and bergapten were detected at 222 nm, and desmethylwedelolactone and wedelolactone were detected at 351 nm. Results:There was good linear relation-ship between the concentration of psoralen, bergapten, desmethylwedelolactone and wedelolactone and peak area within the concentra-tion range of 5.380-107.600 μg·ml-1(r =0.999 5),6.870-137.400 μg·ml-1(r =0.999 7),4.580-91.600 μg·ml-1(r =0. 999 2) and 7. 300-146. 000 μg·ml-1(r=0. 999 9),respectively. The average recovery(RSD) was 96. 82%(0. 77%), 98. 81%(1. 40%),99. 06%(1. 20%) and 98. 39% (0. 80%), respectively (n=6). Conclusion:The developed method is accurate, highly sensitive and well reproducible, which can be used for the determination of the four active ingredients in Xichuan pills.
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OBJECTIVE:To establish a method for simultaneous determination of the contents of ligustroflavone,specnuezhe-nide,demethylwedelolactone and wedelolactone in Zibu ganshen pill. METHODS:Dual-wavelength HPLC was performed on the column of Elite C18 with mobile phase of acetonitrile-0.5%acetic acid(gradient elution)at flow rate of 0.9 ml/min,column temper-ature was 25 ℃,detection wavelengths were 224 nm(0-30 min) and 351 nm (30-50 min),and the injection volume was 20 μl. RESULTS:The linear range was 6.75-135.00μg/ml(r=0.999 5)for ligustroflavone,6.54-130.80μg/ml(r=0.999 8)for specnuezhe-nide,4.90-98.00μg/ml(r=0.999 4)for demethylwedelolactone and 6.42-128.40μg/ml(r=0.999 6)for wedelolactone;RSDs of pre-cision,stability and reproducibility tests were no more than 1.25%;average recoveries were 96.15%-99.96%(RSD<2%,n=6). CONCLUSIONS:The method is rapid,sensitive and accurate,and can be used for the contents determination of ligustroflavone, specnuezhenide,demethylwedelolactone and wedelolactone in Zibu ganshen pill.
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Objective To study the effects of wedelolactone on the expression of P-glycoprotein (P-gp) and to explore the multi-drug resistance reversing mechanism of wedelolactone in K562/A02 cells in vitro.Methods The half maximal inhibitory concentration of ADM in K562/A02 was determined by MTT method.The protein expression level of P-gp was determined by Western blot after wedelolactone pretreatment.Results Wedelolactone remarkably enhanced chemo-sensitivity to ADM of K562/A02 cells.After 0.2, 2, 20 μmol/L different concentration of wedelolactone treatment in 24 h, the relative reversal efficiency of K562/A02 to ADM was 23.5％, 47.1％ and 67.7％, respectively.according to the results of Western blot, wedelolactone was shown to efficiently inhibit the expression of P-gp (P＜0.05).The relative efficiency of K562/A02 to ADM was 25.4％,46％ and 55.6％, respectively.Conclusion Wedelolactone could modulate P-gp expression, and P-gp expression down regulation may be one of the MDR reversal mechanisms in K562/A02 cells by wedelolactone.
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Following optimization of extraction, separation and analytical conditions, a rapid, sensitive and simple reverse-phase high performance liquid chromatography-photo diode array (HPLC-PDA) method has been developed for the identification and quantification of wedelolactone in different extracts of Eclipta alba. The separation of wedelolactone was achieved on a C18 column using the solvent system consisting of a mixture of methanol: water: acetic acid (95: 5: 0.04) as a mobile phase in isocratic elution mode followed by photo diode array detection at 352 nm. The developed method was validated as per the guidelines of the International Conference on Harmonization (ICH). Calibration curve presented good linear regression (r²>0.998) within the test range and the maximum relative standard deviation (RSD, %) values for intra-day assay were found to be 0.15, 1.30 and 1.1 for low (5 µg/mL), medium (20 µg/mL) and high (80 µg/mL) concentrations of wedelolactone. For inter-day assay the maximum RSD (%) values were found to be 2.83, 1.51 and 2.06 for low, medium and high concentrations, respectively. Limit of detection (LOD) and limit of quantification (LOQ) were calculated to be 2 and 5 µg/mL respectively. Analytical recovery of wedelolactone was greater than 95%. Wedelolactone in different extracts of Eclipta alba was identified and quantified using the developed HPLC method. The validated HPLC method allowed precise quantitative analysis of wedelolactone in Eclipta. alba extracts.
Desenvolveu-se método rápido, sensível e simples de Cromatografia Líquida de Alta Eficiência em fase reversa, utilizando-se arranjo de fotodiodo (HPLC-PDA), visando à separação, extração e às condições analíticas para a identificação e quantificação de wedelolactona em diferentes extratos de Eclipta alba. A separação de wedelolactona foi efetuada por meio de uma coluna C18, utilizando mistura de metanol:água:ácido acético (95:5:0.04) como fase móvel, em sistema de eluição isocrática, seguida de detecção por arranjo de fotodiodo a 352 nm. O método desenvolvido foi validado de acordo com as diretrizes da Conferência Internacional de Harmonização (ICH). As curvas de calibração apresentaram boa regressão linear (r²>0,998), dentro dos intervalos de teste, e os valores máximos de desvio padrão relativo (RSD,%) dos ensaios intra-dia foram 0,15, 1,30 e 1,1 para concentrações de wedelolactona baixa (5 µg/mL), média (20 µg/mL) e elevada (80 µg/mL) Para o ensaio inter-dia,os máximos de RSD (%) foram 2,83, 1,51 e 2,06 para as concentrações baixa, média e alta, respectivamente. O Limite de Detecção (LD) e o Limite de Quantificação (LOQ) foram de 2 e 5 µg/mL, respectivamente. A recuperação analítica de wedelolactona foi maior do que 95%. A wedelolactona em diferentes extratos de Eclipta alba foi identificada e quantificada pelo método de HPLC desenvolvido. O método de HPLC validado permitiu a análise quantitativa precisa de wedelolactona em extratos de Eclipta alba.
Subject(s)
Chromatography, High Pressure Liquid/methods , Validation Study , Eclipta/classification , Wedelia/classification , Chromatography, Reverse-Phase/instrumentationABSTRACT
Wedelolactone possesses a wide range of biological activities and is used for the treatment of hepatitis, cirrhosis. A simple HPTLC method has been developed for the quantification of wedelolactone. The samples were dissolved in methanol and linear ascending development was carried out in twin trough glass chamber saturated with mobile phase consisting of toluene: ethyl acetate: acetone: formic acid (6:2:1:1v/v/v/v). Spectrodensitometric scanning was performed by TLC scanner III (CAMAG) in absorbance mode at the wavelength of 351nm.The system was found to give compact spots for wedelolactone (Rf value of 0.39 ± 0.03). The linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.9963 in the concentration range 400-800 ng/spot with respect to peak area. According to the ICH guidelines the method was validated for accuracy, precision, recovery, and robustness. The wedelolactone content quantified from herbal formulations was found well within limits. Statistical analysis of the data showed that the method is reproducible and selective for the estimation of wedelolactone.
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Objecave To explore the protective effect of active component of Eclipta on hepatic injury induced by Concanavalin A(ConA)in mice. Methods Mouse hepatic injury models were established by injection of ConA(15 mg/kg)via tail vein.The effect of Eclipta extracts on serum alanine aminotransferase (ALT) and liver histology in both normal mice and hepatic injury mice was examined.A pure compound was obtained by means of bioactivity-guided isolation,and IR,~(13)C-NMR, ~1H-NMR,~1H-~(13)C HMBC and ~1H-~(13)C HMQC were used for structural identiffcation. Results Eclipta extracts decreased serum ALT in hepatic injury mice induced by ConA.and demonstrated anti-apoptosis effect induced by ConA in hepatoeytes.Wedelolaetone,a eoumarin,was isolated from Eclipta,and its structure was identified on the basis of spectroscopic analysis.Wedelolactone inhibited ConA-induced T cell proliferation selectively,and completely antagonized the effect of ConA at the concentration of 10μg/mL. Conclusion Eclipta herb has hepatoprotective effect.Wedelolactone,the anti-inflammatory and anti-apoptosis active component of Eclipta,might serve as a lead compound for developing new antiinflammatory drugs.
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Objective To study their chemical components of Eclipta prostrata and identify their chemical structures. Methods The compounds were isolated by chromatography, purified by Sephadex LH-20 gel, identified by spectral analyses, and compared with the published data. Results Ten compounds were isolated and identified as 2, 2', 5″, 2″-terthiophene-5-carboxylic acid (Ⅰ), ?-sitosterol (Ⅱ), apigenin (Ⅲ), quercetin (Ⅳ), luteolin (Ⅴ), wedelolactone (Ⅵ), demethyl wedelolactone (Ⅶ), ecliptasaponin C (Ⅷ), luteoloside (Ⅸ), and linarin (Ⅹ). Conclusion Compound Ⅰ is a new compound from nature and compound Ⅹ is obtained from E. prostrata for the first time.
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Small molecular weight compounds from Mandevilla velutina and from Eclipta prostata were found to be active against snakebite.