| 1 BackgroundAs environmental pollution, people lifestyle changes, malignant tumor has become a threat to human life and health that can not be neglected. In recent years, research on antitumor therapy has been in continuous development, but there was no breakthrough so far, conquer malignant tumors is still one of the main scientific problems. Because of the characteristics which include integrity, multiple targets and the risk-benefit ratio, Chinese medicine treatment of the tumor has a good curative effect, so excavated traditional Chinese medicine or traditional Chinese medicine compound which has a significant anti-tumor effect from traditional medicine of China has become an important research approach of anti-tumor at domestic and overseas regions. Antike capsule was composed of toad skin(Corium bofonis) and angelica(Angelica sinensis), which has obvious curative effect to malignant tumor in clinical based a lot of practices. However, ambiguous of composition, mechanism as well as prescription principle have firmly restricted the widely application and international recognition of Antike capsule. Therefore, conduct a comprehensive composition analysis for Antike capsule, illustrate its pharmacodynamic material basis, functional mechanism and prescription principle, and then screen the molecular formula with clear composition, exact target and reasonable compatibility. It is not only significant for the secondary development of Antike capsule, but also meaningful adaptation for the internationalization and modernization of traditional Chinese medicine. For this purpose, this study will apply the method of traditional Chinese medicine fingerprint and serum pharmacochemistry, to identify the main components, explore the major antitumor active ingredients, on this basis, take advantages of pharmacokinetics research to clarify the scientific and reasonable of Antike capsule compatibility preliminarily. 2 Methods 2.1 Composition analysis and quality control of Antike capsule: Using high performance liquid chromatography(HPLC) method to establish the analysis method of fingerprint and multicomponents quantitative determination. On this basis, filtered the characteristic chromatographic peaks in the chromatograms, refered to the known reference substance for the chemical identification and content determination. Chromatographic conditions: Angilent Zorbax SB-C18 column(4.6 × 250 mm 5 μm), Angilent Zorbax SB-C18 4.6 × 12.5 mm guard column, column temperature: 30℃. Mobile phase was performed with acetonitrile(A) and 0.1% glacial acetic acid-0.5% potassium dihydrogen phosphate buffered solution(adjust p H=2.4 with phosphoric acid)(B), the method of gradient elution time application as follows: 8% acetonitrile(0 min), 30% acetonitrile(20 min), 40% acetonitrile(45 min), 50% acetonitrile(70 min), 40% acetonitrile(75 min), and 8% acetonitrile(80 min), flow rate: 0.8 m L/min. Sample quantity: 20 μL. Detection wavelength: 296 nm. 2.2 Constituents migrating to blood analysis of Antike capsule: Refluxed Antike capsule, toad skin and angelica powder with methanol at 70 ℃to prepare the extracting solution, after concentration, make use of vacuum freeze dryer to volatilize moisture, so as to prepare the lyophilized powder of Antike capsule, toad skin and angelica extractive. With the method of serum pharmacochemistry, analysised the chromatogram of rats’ serum after gavage administration of Antike lyophilized powder. Compared chromatograms of Antike lyophilized powder, drug serum, blank serum and reference substance to identify the constituents migrating to blood of Antike. 2.3 Pharmacokinetics study of toad skin, angelica and their prescription in rats: Established the HPLC method of simultaneously content determination of ferulic acid, senkyunolide I, telocinobufagin and bufalin in rat plasma. Chromatographic conditions: Angilent Zorbax SB-C18 column(4.6 × 250 mm 5 μm), Angilent Zorbax SB-C18 4.6 × 12.5 mm guard column, column temperature: 30℃. Mobile phase was performed with acetonitrile(A) and 0.1% glacial acetic acid-0.5% potassium dihydrogen phosphate buffered solution(adjust p H=2.4 with phosphoric acid)(B), the method of gradient elution time application as follows: 10% acetonitrile(0 min), 20% acetonitrile(6 min), 40% acetonitrile(40 min), 50% acetonitrile(50 min), 30% acetonitrile(55 min), and 10% acetonitrile(60 min), flow rate: 0.8 m L/min. Sample quantity: 20 μL. Detection wavelength: 296 nm. Draw the plasma concentration-time curve of 4 ingredients after single administration of toad skin, angelica and compatible administration of two compositions. Compared the pharmacokinetic parameters difference of 4 ingredients in 3 groups, discussed the influence of pharmacokinetic rule after compatibility. 2.4 Experimental studies of bufalin in combination with ferulic acid on antitumous effect in vitro: With MTT method, analyzed the cytotoxic effect of bufalin and ferulic acid to hepatoma cell lines which include Hep G2, HCCLM3, Hep-3B, Bel-7404 and QGY-7703, then calculated the half inhibitory concentration(IC50). Make use of Chou-Talalay method to analyze the combination index(CI) of bufalin and ferulic acid to five hepatoma cell lines. Take advantage of flow cytometry(FCM) to analyze the influence on the cell cycle distribution of bufalin, ferulic acid and their compatibility. 3 Results 3.1 Composition analysis and quality control of Antike capsule: There are 28 chromatographic peaks were screened as common characteristics peaks. After identification, in which 16 peaks are ferulic acid, gamabufotalin, senkyunolide I, senkyunolide H, arenobufagin, telocinobufagin, desacetylcinobufotalin, bufotalin, cinobufotalin, bufalin, coniferyl ferulate, cinobufagin, senkyunolide A, resibufogenin, ligustilide and n-butylidenephthalide. At the same time, it set up a method of simultaneously content determination of 16 gredients in Antike capsule, this method is stable and feasible, with good precision and high repeatability, each component recovery is at the range of 94.86~103.10%, which is suitable for the quality control of Antike capsule. 3.2 Constituents migrating to blood analysis of Antike capsule: After gavage administration of Antike lyophilized powder to rats, 9 constituents migrating to blood were distinguished in rat serum, after identification, 4 constituents were confirmed as ferulic acid, senkyunolide I which derived from angelica and telocinobufagin, bufalin which derived from toad skin. These 4 constituents may be the major antitumor active ingredients in Antike capsule. 3.3 Pharmacokinetics study of toad skin, angelica and their prescription in rats: Compared the pharmacokinetic parameters difference of 4 ingredients in 3 groups, it indicate that compatibility of toad skin and angelica can remarkably increase the peak plasma concentration and area under the curve(P﹤0.05), decrease the mean residence time, plasma clearance and apparent volume of distribution(P﹤0.05) for ferulic acid and senkyunolide I; at the same time, compatibility can prominently decrease the peak plasma concentration and area under the curve(P﹤0.05), increase the mean residence time, plasma clearance and apparent volume of distribution(P﹤0.05)for telocinobufagin, bufalin. It prompt that compatibility could dramatically accelerate absorption, reduce the drug plasma clearance, improve the bioavailability of ferulic acid and senkyunolide I as well as prolong the time of drug action, promote the drug distribution to the tissue. 3.4 Experimental studies of bufalin in combination with ferulic acid on antitumous effect in vitro: According to MTT results, bufalin was with high cytotoxicity and presented a dose-dependent manner, the IC50 of bufalin to hepatoma cell lines Hep G2, HCCLM3, Hep-3B, Bel-7404, QGY-7703 were 57.60, 83.13, 74.16, 27.47, 52.31 μM respectively, ferulic acid was with low cytotoxicity and the IC50 of ferulic acid to five hepatoma cell lines were 1.98, 1.89, 3.20, 0.97, 1.16 m M respectively. Chou-Talalay method analysis results show that when compatibility of bufalin and ferulic acid to five hepatoma cell lines, the CI is greater than 1.0 in most concentrations, which means these two drugs show mutual antagonistic function. According to the flow cytometry technique analysis, bufalin can block Hep G2 cells in G2/M phase(P﹤0.05), ferulic acid has no significantly affect on the G1/G0 phase and S phase of Hep G2 cells, when compatibility of the two drugs, it shows stronger block function of G2/M phase than bufalin alone(P﹤0.05). 4 ConclusionsThe present study established the HPLC fingerprint of Antike capsule for the first time, in which confirmed 28 common chromatographic peaks and the identified 16 ingredients. Took advantage of serum pharmacochemistry method, detected 9 constituents migrating to blood of Antike capsule in which attributed 4 constituents were ferulic acid and senkyunolide I derive from Angelica as well as bufalin and telocinobufagin derive from Toad skin. Meanwhile it revealed the pharmacokinetic characteristics of these 4 components in vivo. In addition to this, this research discussed the bufalin and ferulic acid compatibility combination effects to tumor cells and the cell cycle distribution. The results provided a reliable research approach to further illustrate the scientific rationality of Antike prescription, it also offered reference for original creation of Antike molecular medicine. |
PDF Full Text Request