| Xianglian pills is composed of Coptis Chinensis (Huanglian) and Radix Aucklandiae (Muxiang), can clear heat and remove dampness and so on. It is used to treate heat dysentery, tenesmus, abdominal pain, bacillary dysentery, enteritis. Huanglian count for 80% of preparation, is principal agents of Xianglian pills, and Muxiang play an important supporting role, Evodia Rutaecarpa inhibit the bitter cold nature of Huanglian. In recent years, studies on quality control of Xianglian pills were based on determinating berberine. With the increasingly modernizating of Chinese medicine, the quality control of Chinese medicine preparations can not meet the needs of the development of modern medicine, whose only the content of an active component is determeted. For enhancing the quality control level of Xianglian pills, three alkaloids and two sesquiterpene lactones were quantitatively determinated, and the fingerprints of three alkaloids in Xianglian pills were established.As we all know, depending on its various composition, Chinese medicine possesses a wealth of activities. The studies about dynamic changing of active components in vivo can provide a theoretical basis for explaining the mechanism of their activities, and provide the scientific basis for clinical treatment. Now, the researchs on dynamic changes in vivo of Xianglian pills have not seen. In view of this, two sesquiterpene lactones, costunolide and dehydrocostuslactone, which come from Xianglian pills, whose pharmacokinetics in mice were studied. The distribution of various tissues in mice of two active components mentioned above were researched in detaile. In addition, in order to provide the material basis for studies in vivo, we have established RP-HPLC semi-preparative method of costunolide and dehydrocostuslactone. The study includes following three aspects:1 Determination of five active components in Xianglian Pills1.1 This study established method of determinating palmatine, jatrorrhizine and berberine in Xianglian Pills, and the micellar liquid chromatography method which utilized photodiode array detection (PAD) was developed for this study. The contents of palmatine, jatrorrhizine and berberine are assaied with chromatographic conditions as follows:Kromasil ODS(5μm,4.6 mm×150 mm) with 30℃, isocratic mobile phase consist 0.2 mol.L-1NaH2PO4-7.00 mmol.L-1 aqueous solution of sodium dodecyl sulfate-acetonitrile (35:35:30, V:V:V) at a flow rate of 1.0mL·min-1, and detective wavelength was 350 nm. The calibration curves of palmatine, jatrorrhizine and berberine in Xianglian Pills were:A= 3.37×104X-2.65x 104 (r= 0.9999), A= 4.58×103X-2.88×103 (r = 0.9997), A= 5.62 x 103 X-9.46×103 (r= 0.9994), the mean recovery of them was found to be 87.67% (RSD=0.17),102.37% (RSD=0.98),96.81% (RSD=3.13), respectively. In accordance with above method, we determined contents of palmatine, jatrorrhizine and berberine in self-made and commercially available Xianglian Pills, the results show their average contents were 45.77 mg.g-1,3.89 mg.g-1,9.88 mg.g-1 (Feci Pharmaceuticals); 86.53 mg.g-1,16.66 mg.g-1,18.59 mg.g-1 (self-made), respectively.1.2 This study established method of assaying costunolide and dehydrocostuslacton in Xianglian Pills, and the RP-HPLC method which utilized photodiode array detection (PAD) was developed for this study. The contents of costunolide and dehydrocostuslacton were assaied with chromatographic conditions as follows:Hypersil ODS (5μm,4.6 mm x 250 mm) with 30℃, mobile phase consist acetonitrile-water (70:30, V:V) at a flow rate of 1.0 mL·min-1, and detective wavelength was 210 nm. The calibration curves of costunolide and dehydrocostuslacton in Xianglian Pills were:A= 1.30 x 105 X-3.38 x 104 (r= 0.9998),0.0223-0.178 mg. ml-1; A= 1.68×105 X-3.34×104 (r= 0.9993), 0.023-0.182 mg. ml-1, the mean recovery of them was found to be 98.50% (RSD=0.53),95.30% (RSD=0.32), respectively. In accordance with above method, we determined contents of costunolide and dehydrocostuslacton in self-made and commercially available Xianglian Pills, the results showed their average contents were 1.23 mg.g-1,1.17 mg.g-1 (Foci Pharmaceuticals); 2.41 mg.g-1,2.13 mg.g-1 (self-made).The determination of costunolide and dehydrocostuslacton contained in Muxiang comprised of Xianglian Pills was carried out, the average content of costunolide and dehydrocostuslacton were 14.93 mg.g-1,13.16 mg.g-1 (Huirentang Church); 15.67 mg.g-1, 11.19 mg.g-1 (Huanghe City); but costunolide and dehydrocostuslacton have not detected in Qingmuxiang.1.3 Under the same chromatography condition with determination of palmatine, jatrorrhizine and berberine, the fingerprint determination of Alkaloids was established. Palmatine, jatrorrhizine and berberine were choosed as the reference substance.10 common peaks were achieved after analyzing the fingerprint of 6 batches of commercial available and 9 batches of self-made Xianglian Pills, and we developed the common pattern of Xianglian Pills. The similarities analysis showed that, the similarity of 15 batches of Xianglian Pills was 99.80%,99.81% and 99.81% (self-made),99.80%, 99.82% and 99.79% (self-made),99.76%,99.80% and 99.81% (self-made),98.15%, 98.12% and 98.29% (Foci Pharmaceuticals),98.22%,98.24% and 98.27% (Hubei Pharmaceutical Company), the average similarity was 99.17%. The results also showed both the self-made and commercially Xianglian Pills, and the preparation itself had a good correlation. Under the same chromatographic conditions, the fingerprinting of Huanglian was carried out, and there are good correaltions among the Huanglian and Xianglian Pills, the similarity was rang 99.51% to 99.78%, the average similarity was 99.59%, RSD was 0.076%. There was no significant different between Huanglian purchased different drugstore, between pre-and post processed and preparation. However, after processing, the similarity between Huanglian and preparation was closer to 1, which demonstrated the species, origin, quality and treatment process of Huanglian was similar to Xianglian Pills in a certain extent.The results showed that the determination method established of five kinds of components was simple, sensitive, accurate and reproducible. It not only was used to determinated five kinds of components in Xianglian Pills and medical materials comprised of this preparation, but also could provide reference with quality control for the preparation which contained Muxiang and Huanglian. Evaluating the similarity, matching peak of fingerprint of medical materials and preparations, combining with determination of active components mentioned above, such multi-index evaluation could applied to control quality of Chinese medicine preparation, and the method is simple, reproducible and had a certain operability.2 Development of HPLC method for costunolide and dehydrocostuslactone in mice plasma and tissues:Application to pharmacokinetic study 2.1 An RP-HPLC method was developed in this paper for the determination of costunolide and dehydrocostuslactone in mice plasma. Under the same chromatography condition with determination of costunolide and dehydrocostuslactone, and results showed that good resolution between two components, no endogenous interfering peaks were observed, the intra-day and inter-day RSD of the assay method for the two components were less than 5% and mean recovery was within the 86.5 to 101.8% range, the lowest detection limit and lower limit of quantitation was 21.27 ng.mL-1 and 785 ng.mL-1; 71 ng.mL-1 and 2.62μg.mL-1, respectively. And this method was used for pharmacokinetic of ostunolide and dehydrocostuslactone after single oral ethanol extraction of Muxiang in mice. We choosed 14 groups of Kunming mice (n= 6), blood samples (0.5 mL) were collected into a heparinized Eppendoff tube and mixed gently and immediately at 0,0.25,0.5,0.75,1.0,1.5,2.0,2.5,3.0,5.0,8.0,12.0,24.0,36.0 h after administration. Following protein precipitatted by acetonitrile, all organic layer was drawn into respective centrifuge tube and evaporated to dryness. The residue was reconstituted with methanol for analysis. The peak concentrations of costunolide and dehydrocostuslactone were observed 30 minutes in the plasma with values of 0.0133 and 0.0308 mg-mL"1. Using a computer program DAS Version 1.0 software meant for calculation of model independent parameters, blood drug concentration versus time profiles of costunolide and dehydrocostuslactone fitted a two-compartment model, the t1/2a and ti/2p of them were 0.690 and 9.908,0.292 and 11.094 h, respectively. The corresponding coefficients (R2 values) for the both of the components were 0.997 and 0.999 respectively, and the pharmacokinetic results for them were reasonable. The absorption of costunolide was faster than dehydrocostuslactone, but eliminate was slow.2.2 The mice were subsequently euthanized by cervical vertebrae dislocation, and afterward whole stomach, lung, intestine, kidney and brain, liver, spleen, were collected (stomach and intestine were cleaned by water). The appropriate amount tissues containing costunolide and dehydrocostuslacton were homogenized with 2 times amount of normal saline, then 4 times amount of acetonitrile was added and vortexed to mix for 5 min, and centrifuged at 4,000 rpm for 5 min. The supernatant (about 5 mL) was separated and allowed to evaporate in vacuum oven at 50℃. The evaporated residue was reconstituted with 500μL of methanol and 20μL of the reconstituted sample was injected into the HPLC system for analysis.The peak concentrations of costunolide and dehydrocostuslactone in tissues were all in the order of C stomach>C lung>C intestine>C kidney > C brain, there were 0.108 and 0.159 mg-g-1; 0.069 and 0.078 mg-g-1; 0.010 and 0.048 mg·g-1; 0.0059 and 0.0069 mg-g-1; 0.0027 and 0.0037 mg-g-1, and corresponding concentration ratio of stomach/plasma, lung/plasma, intestine/plasma, kidney/plasma and brain/plasma were 8.08 and 5.16; 5.17 and 2.52; 0.75 and 1.56; 0.45 and 0.22; 0.20 and 0.12, respectively. We have not detected contunolide and dehydrocostuslactone in liver and spleen, possible reasons as follow:1. Contunolide and dehydrocostuslactone were small molecular liposoluble compounds without liver metabolism, and were directly absorbed through the gastric or intestinal mucosa; 2. The uptake efficiency of liver and spleen for costunolide and dehydrocostuslactone was lower; 3. Due to the size of the spleen is small, the sample pretreatment may lead to its content below the detection limit.The results showed that both the active component could be rapidly distributed in the tissues, and the order of the maximum concentration in the organs was exactly same, the order was stomach, lung, intestine, kidney and brain. The concentration of two components could be seen in the gastrointestinal concentrations were high, which fully consistent with their treatment.These studies not only presented basic theory for interpretation of the mechanism of costunolide and dehydrocostuslactone, but also provided for its clinical application with more scientific basis.3 Separation and preparation of costunolide and dehydrocostuslactone from Muxiang by reversed phase preparative liquid chromatographyThe classical column chromatography and semi-preparative reversed phase liquid chromatography were used to isolate costunolide and dehydrocostuslactone. Their chemical structures were identified as costunolide and dehydrocostuslactone by spectroscopic methods including UV, TLC, NMR. The preparation of sesquiterpene compounds had high purity and reliable, simple, rapid, and could be used for separation sesquiterpene from food and medicine. |
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