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Drug-receptor interaction plays an important role in a series of biological effects, such as cell pro-liferation, immune response, tumor metastasis, and drug delivery. Therefore, the research on drug-re-ceptor interaction is growing rapidly. The equilibrium dissociation constant (KD) is the basic parameter to evaluate the binding property of the drug-receptor. Thus, a variety of analytical methods have been established to determine the KD values, including radioligand binding assay, surface plasmon resonance method, fluorescence energy resonance transfer method, affinity chromatography, and isothermal ti-tration calorimetry. With the invention and innovation of new technology and analysis method, there is a deep exploration and comprehension about drug-receptor interaction. This review discusses the differ-ent methods of determining the KD values, and analyzes the applicability and the characteristic of each analytical method. Conclusively, the aim is to provide the guidance for researchers to utilize the most appropriate analytical tool to determine the KD values.
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It is very difficult to comprehensively achieve the quality control of traditional Chinese medicine (TCM), because some problems such as indicator simplification and lack of correlation between efficacy and indicator are ex-isted in the common quality control of TCM. Combined with the practical experience of our research work, this paper outlined the characteristics and applications of cell membrane chromatography (CMC) and discussed that CMC method can be used for quality control of TCM.
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In China.traditional Chinese medicines (TCMs) have been used in clinical applications for thousands of years.The successful hyphenation of high-performance liquid chromatography (HPLC) and mass spectrometry (MS) has been applied widely in TCMs and biological samples analysis.Undoubtedly.HPLC/MS technique has facilitated the understanding of the treatment mechanism of TCMs.We reviewed more than 350 published papers within the last 5 years on HPLC/MS in the analysis of TCMs.The present review focused on the applications of HPLC/MS in the component analysis,metabolites analysis,and pharmacokinetics of TCMs etc.50% of the literature is related to the component analysis of TCMs,which show that this field is the most popular type of research.In the metabolites analysis,HPLC coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry has been demonstrated to be the powerful tool for the characterization of structural features and fragmentation behavior patterns.This paper presented a brief overview of the applications of HPLC/MS in the analysis of TCMs.HPLC/MS in the fingerprint analysis is reviewed elsewhere.
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A rapid method for the simultaneous determination of berberine (BBR), matrine (MT) and oxymatrine (OMT) by nonaqueous capillary electrophoresis (NACE) was developed. Optimum separation of the analytes was obtained on a 50cm×50μm i.d. fused-silica capillary using a non-aqueous buffer system of 70mM ammonium acetate, 7.0% acetic acid and 10% acetonitrile at 25kV and 20℃. The relative standard deviations (R.S.D.) of the migration times and peak areas of the three active components were 0.06%-0.20% and 0.12%-3.41% for berberine, 0.11%-0.60% and 0.74%-1.63% for matrine, 0.15% and 0.45% for oxymatrine, respectively. Detection limits of berberine, matrine and oxymtrine were 0.18μg/mL, 4.08μg/mL and 4.16μg/mL, respectively. In the tested concentration range, good linear relationships (0.9992 for berberine, 0.9988 for matrine and 0.9988 for oxymatrine) were observed. The linear calibration ranges were 0.45-360.0μg/mL for berberine, 8.16-408.0μg/mL for matrine and 20.8-416.0μg/mL for oxymatrine. This method has been successfully applied to the phytochemical analysis of alkaloids extracts from two commonly used traditional Chinese herbal drugs: Sophora flavescens Ait. (Kushen) and Cortex phellodendri chinensis (Huangbai) and their medicinal preparations.
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<p><b>OBJECTIVE</b>To investigate the absorption kinetics of atractylenolide I in intestines of rats and the influence of P-glycoprotein (P-gp) on the absorption.</p><p><b>METHOD</b>The absorption kinetics was investigated using the method of in situ intestine absorption in rats and the samples were determined by HPLC.</p><p><b>RESULT</b>Atractylenolide I is absorbed quite well at all segments of intestine in rats and no specific absorption was founded in different segment. When the concentration of perfusion solution was increased contrarily the absorption rate constant (Ka) kept at the same level. Compared Ka of three different concentration of perfusion solution with variance analysis method, Ka of atractylenolide I had no significant differences. But the Ka values were significently increased in the presence of P-gp inbibitor, verapamil or digoxin.</p><p><b>CONCLUSION</b>Atractylenolide I can be classified into high penetrating drug. Passive diffusion dominates the absorptive transport behivior of atractylenolide I. Atractylenolide I can be absorbed in the whole intestinal segments and there is not a preferntial absorption zone in the intestine. The absorption and secretion of atractylenolide I are mediated by the efflux transport system, P-gp.</p>
Subject(s)
Animals , Male , Rats , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Metabolism , Intestinal Absorption , Intestines , Chemistry , Metabolism , Kinetics , Lactones , Pharmacokinetics , Rats, Sprague-Dawley , Sesquiterpenes , PharmacokineticsABSTRACT
Objective To investigate the molecular mechanisms that are responsible for anti-inflammatory effect of usnic acid (UA), the effects of UA from usnea longissm on tumor necrosis factor-α(TNF-α) and nitric oxide (NO) production in peritoneal macrophages has been examined. Methods The different concentrations of UA were added to peritoneal macrophages. The TNF-α and NO production in peritoneal macrophages were examined with mouse TNF-α ELISA kit and NO content by measuring the amount of nitrite (NO-2μmol/L) formed in the medium using Griess reaction. The activity of inducible nitric oxide synthase (i-NOS) was determined using i-NOS detection kit and the TNF-α mRNA expression was tested by reverse transcriptase polymerase chain reaction (RT-PCR). Results UA decreased the TNF-α and NO level in LPS-stimulated peritoneal macrophages in dose-dependent manner, the IC50 values were 12.8μmol/L and 5.7μmol/L respectively. RT-PCR analysis indicated that UA could inhibit TNF-α mRNA expression; the activity analysis of i-NOS indicated that UA could inhibit the activity of i-NOS. Conclusion UA could inhibit the TNF-α and NO production in peritoneal macrophages, it may be associated with the anti-inflammatory activity of UA.
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Objective To discover the effective component of Rhizoma Tupistrae chinensis with inhibiting angiogenesis. Methods Several parts of Rhizoma Tupistrae chinensis were obtained by using different extraction solvents. They were screened by the model of ECV304 cell membrane chromatography with anti-VEGF antibody as a control drug. The inhibition of effective component (ZGQ-C) on ECV304 proliferation in vitro was examined by MTT assay. Results The inhibition rate (40.51%,51.11%,63.54%,74.32%,81.26%) was correlated with the ZGQ-C concentration. The ECV304 cell had significantly changed in morphology. Conclusion The ZGQ-C is an effective component for inhibiting angiogenesis. The screening results on the model have a good correlation with that of MTT assay.
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Aim To investigate the bio-affinities of ligustilide and butylidenephthalide to rat aortic smooth muscle cells and the inhibitory effects of them on bFGF-stimulated proliferation of rat vascular smooth muscle cell (VSMC). Methods VSMCs were cultured from rat aorta pectoralis and identified by an immunohistochemical method. The bio-affinities between solute (ligustilide or butylidenephthalide) and cell membrane were measured by rat aortic cell membrane chromatography (CMC). The inhibitory effects of ligustilide and butylidenephthalide on bFGF-stimulated VSMC proliferation were evaluated by MTT colorimetric method. Results Both ligustilide and butylidenephthalide had selective affinities to rat aortic smooth muscle cell as the same as verapamil, one of the calcium ion antagonists. They could potently separately (P ＜ 0.05 ), but had no effects on the normal VSMC growth. Conclusion Both ligustilide and butylidenephthalide can inhibit the abnormal proliferation of VSMC induced by bFGF.
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Objective To study the effect and mechanism of taspine on wound healing and fibroblast proliferation. Methods The effect of taspine on skin wound was observed in vivo. The different concentration of taspine hydrochloride was added to L929 fibroblast cultivated in vitro, and lactate dehydrogenase was detected and MTT method was applied to observe effect of taspine on fibroblast proliferation. Results The local application of taspine 3 mg/Ml and 1.5 mg/mL accelerated the healing of skin wounded. In vitro, 0.01～0.5 μg/mL of taspine hydrochloride showed no effect on the change of lactate dehydrogenase activity and fibroblast proliferation. Conclusion Taspine is a kind of active alkaloid from leontice robustum which can enhance wound healing, its mechanism on wound healing is not by means of accelerating the proliferation of fibroblast, other mechanisms are necessary for being further studied.
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OBJECTIVE:To prepare loperamide hydrochloride oral solution,to establish its quality control method,and to study its stability.METHODS:Loperamide hydrochloride oral solution was prepared by using macrogol-400as the anxiliary solvent,water as the solvent,a reversed phase HPLC method was established to determination the content of principal agent loperamide hydrochloride and the method described in Chinese Pharmacopeia was applied to study its stability.RESULTS:A good linear relationship was obtained in the loperamide hydrochloride at concentration of0.05～0.5mg/ml(r=0.9995);The average recoveries of loperamide hydrochloride at the high,medium and low concentrations were100.6%、101.5%and99.1%respectively,with RSD being1.03%、0.49%and0.56%respectively;The stability showed no evident change as compared with before.CONCLUSION:The loperamide hydrochloride oral solution shows the advantages of simple preparation procedure,satisfactory stability and controllable quality.
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OBJECTIVE: To study the effect of taspine hydrochloride (TA/HCl) on skin wound healing in rats and its mechanism. METHODS: Bilateral round wounds were made on the backs of SD rats. The effect of TA/HCl on the skin wound was evaluated through determining closure time and contracting ability of the skin wound, observing histopathological characteristics and measuring contents of hydroxyproline (Hyp) and protein in the wound tissue. RESULTS: The closure time of the skin wounds was significantly shorter in the TA/HCl-treated groups than that in the model group. The percentages of wound contraction were higher in the TA/HCl-treated groups than that in the dimethyl sulfoxide (DMSO) control group of the same group (P<0.05 or P<0.01) on the 3rd to 14th days after wounding. The content of the protein in the wound tissue in the TA/HCl-treated group (2 mg/ml) was higher than that in the model group (P<0.05) on the 3rd to 7th days after wounding, and it arrived at the peak on the 7th day and gradually decreased to the normal level in skin tissue on the 14th to 21st days after wounding. The contents of Hyp in the wound tissues in the TA/HCl-treated groups were higher than that in the model group (P<0.05 or P<0.015) on the 3rd to 21st days after wounding, and they arrived at the peak on the 14th day and at the normal level in skin tissue on the 21st day. Histopathological test results showed that TA/HCl could promote the formation of newly born capillaries in the early period of the wound healing. CONCLUSION: TA/HCl has the ability of promoting skin wound healing in rats, and it can also accelerate the growth of newly born capillaries and raise the production of protein and collagen in wound tissue.
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Objective To establish a qualitative and quanti ta tive method with RP-HPLC for controlling the quality of Cortex Moutan. Methods The experimental conditions of the RP-HPLC method wer e as follows: planetsil C 18 column (150 mm?4.6 mm, 5 ?m), mobile phase of methanol-water-acetic acid ( 44∶56∶0.1), flow rate at 1.0 mL?min -1, detection wavelength at 240 nm and room temperature. The qualitative fingerprint of Cortex Moutan and the quantitative measurement of paeonol wer e carried out under the chromatographic conditions mentioned above. Res ults Eleven comparatively stable peaks were detected among 13 differ ent samples, from which 10 common peaks used as index peaks for qualitative iden tification were conformed by correlating the peak areas. At the same time, the c ontents of paeonol in samples range from 1.32% to 2.78%. Conclusion The analytical method with qualitative fingerprint of Cortex Moutan and quantitative measurement of paeonol can effectively be used to control the quality of Cortex Moutan.
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Purpose The aim is to prepare ouabain polyclonal antibody F(ab)2 fragment and to estimate its molecular weight.Methods[ KG*2 [ WTBZ]Ouabain polyclonal antibody was obtained from immunized rabbits.The antibod y was digested with pepsin.The resulting products were analyzed and the molecular weig ht of F(ab)2 fragment was estimated by HPSEC.The immune activity was detec ted by ELISA.Results 100 mg of ouabain polyclonal antibody wa s dige sted by 2 mg of pepsin for 18 hours at pH 3.0 and active ouabain polyclonal anti body F(ab)2 framgment was obtained.Its molecular weight was 107 kD.Concl usion The active ouabain polyclonal antibody F(ab)2 fragment coul d be prepared by digesting its antibody with pepsin.
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AIM To study the differences of the pharmacokinetics and tissue distribution of two enantiomers of nicardipine in the rabbit. METHODS Biological samples were diluted by 1 mol*L-1 NaOH solution and extracted with n-hexane - ethyl acetate (1∶1). The concentrations of S-nicardipine and R-nicardipine in samples were determined by coupled achiral C18 column (150 mm×4.6 mm, 5 μm) and chiral OJ column (250 mm×4.6 mm, 10 μm) chromatography. RESULTS The racemic nicardipine and the enantiomers in sample were separated well by the coupled system. The linear range was 55-550 ng*mL-1 for both enantiomers. The within-day and between-days RSD (n=5) were 5.25% and 8.97%, and the relative recoveries were 99.99% and 97.10% for R- and S- enantiomer, respectively. The mean Tmax, Cmax and AUC values were (2.49±0.03) h, (134±2) ng*mL-1 and (1082±32) ng*mL-1*h for S-nicardipine and (1.24±0.05) h, (109±2) ng*mL-1 and (778±22) ng*mL-1*h for R-nicardipine. The concentration of S-nicardipine were generally higher than that of R-nicardipine in main target tissues and cells. CONCLUSION There were significant differences between the two enantiomers of nicardipine in rabbit in pharmacokinetics and tissue distribution.
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Objective: To build an extraction method of ferulic acid from Ligusticum Chuanxiong Hort.Methods: The solvent varieties, polarities, and extraction methods were systematically studied by HPLC with ferulic acid as marker.Results: The more effective way to extract ferulic acid from Ligusticum Chuanxiong Hort was effluent extraction with 8 times amount of 40% alcohol under reflux for three hours.Conclusion: The solvent varieties and extraction method showed greater effects than others on the extraction efficiency of ferulic acid.
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Objective: To build an extraction method of coumarins from Radix Angelicae Dahuricae. Methods: The solvent varieties, polarities, and extraction methods were systematically studied by TLC with imperatorin as marker.Results: The more effective way to extract coumarins from Radix Angelicae Dahurica was refluent extraction with 4 times amount of 95% alcohol twice (one hour each time).Conclusion: The Solvent varieties and extraction method showed greater effects than others on the extraction efficiency of imperatorin.
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AIM: To prepare the solid dispersions of angelica dahurica coumarins and measure their dissolution characteristics. METHODS: The solid dispersions were obtained by using the melted and dissolved methods with PEG6000, PVP and poloxamer188 as carriers respectively.The existing state of the coumarins in the solid dispersions were identificated by the differential scanning calorimetery. The dissolution characteristics of the solid dispersions in vitro were analyzed by HPLC method under the chromatographic conditions with ThermoC_(18)(150 mm?4.6 mm,5 ?m) as analytial column and methanol/water(70∶30)as mobile phase,using imperatorin as testing index. RESULTS: The melted and dissolved methods can be used to prepare the solid dispersions.The coumarins were completely dispersed in carrier and formed an euctics mixture with the carrier.The dissolution rates of all solid dispersion were increased obviously. CONCLUSION: The solid dispersion prepared with PVP can increase the solubility and dissolution of the coumarins.
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AIM: To study the pharmacokinetics of ligustilide in the volatile oil from Angelica Sinensis(Oliv.) Diels in the rabbit. METHODS: HPLC method for ligustilide determination in the blood was developed.The HPLC system consisted of C_(18) column using MeOH-H_2O(65∶35,v/v) as mobile phase at a flow rate of 1.0 mL/min and UV detection at 236 nm. RESULTS: Linear calibration curves were obtained over the concentration range of 0.40 ?g?mL~(-1)～10.00 ?g?mL~(-1) for ligustilide.The minimum limit detection was 0.40 ?g?mL~(-1).The recovery of ligusitilide in blood was 90.90% with RSD 2.74%. CONCLUSION: After oral administration of volatile oil,intracorporal process of ligustilide in rabbit accords with 2-compartment model with 1 st order absorption,(2.6638) h and 108.88 h are obtained as t_(1/2?) and t_(1/2?) respectively.
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Objective To compare the specificities of the cell membrane stationary phases(CMSP) with cell membrane chromatography(CMC).Methods Cell chromatographic columns were constructed for both rat aorta tissue cells and cultured rat aorta smooth muscle cells.Then the chromatographic affinities of ten ligands of ?-adrenergic receptor(?-AR) with both said chromatographic columns were investigated.Capacity factors(k'),as a chromatographic parameter,were calculated.Results The correlation analysis showed a positive correlation between the rat aorta tissue CMSP and the cultured rat aorta smooth muscle cell CMSP,with correlation factor of r=0.923,P
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Objective To establish a qualitative and quantitative method for controlling the quality of Radix Puerariae and its preparation. Methods The qualitative fingerprint of effective part (extract part of ethyl acetate) was obtained by using RP-HPLC with methanol-water (42∶58) as mobile phase and UV detection at 250 nm . The quantitative measurement of effective components (puerarin and daidzein) was finished under the above chromatographic conditions. And the 11 kinds of different samples of Radix Puerariae were analyzed by the method. Results Under the qualitative conditions, the 5 common peaks in RP-HPLC fingerprint of the effective part could be used as index peaks for qualitative identification. In RP-HPLC quantitative analysis, the recoveries of the method were 98.0% with RSD of 1.30% for puerarin and 95.4% with RSD of 1.61% for daidzein, respectively. The amount of puerarin and daidzein in Radix Puerariae was 0.213%～3.626% and 0.012%～ 0.049% , respectively. Conclusion The analytical method with qualitative fingerprint of effective part and quantitative measurement of effective components of Radix Puerariae can be accurately used for controlling its quality.