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Objective:To investigate and compare the effects of water extracts of Whitmania pigra Whitman and Hirudinaria ma-nillensis Lesson on the angiogenesis of Tg (kdrl:mCherry) zebrafish. Methods:The zebrafish embryos 6-8 hours after fertilization (6-8hpf) were transferred to the culture medium containing Whitmania pigra Whitman or Hirudinaria manillensis Lesson extract at different concentrations, and the culture medium containing the drugs was replaced every 24 h. And then, at 72 hpf, the larvalmorphology and intersegmental vessels were observed under a microscope. The hatchability of 48-and 72-hpf embryos, and the number of intersegmen-tal vessels and the heart rate of 72-hpf juveniles were measured. Results:Compared with the control group, when the concentration of Whitmania pigra Whitmanis was higher than 30μg· ml-1 , and the concentration of Hirudinaria manillensis Lesson was higher than 20μg· ml-1, the number of intersegmental vessels was significantly reduced (P<0. 01). Compared with the control group, at 48 hpf, when the concentration of the drug groups was higher than 40 μg· ml-1 , the hatchability of the two groups significantly decreased ( P<0. 01);at 72 hpf, the hatchability of Whitmania pigra Whitman decreased significantly at the concentration of 100 μg·ml-1 (P<0.01), while the hatchability of Hirudinaria manillensis Lesson decreased significantly at the concentration of 80 μg· ml-1(P <0. 01). There was no obvious yolk sac edema, pericardial edema and spine curvature in the two groups. The heart rate decreased sig-nificantly (P<0. 01), while was still within the normal range. Conclusion:Both Whitmania pigra Whitman and Hirudinaria manillen-sis Lesson have notable anti-angiogenic activity, and the anti-angiogenesis activity of Hirudinaria manillensis Lesson is stronger. They both have effects on the development of zebrafish embryos, while the toxicity is not obvious.
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Objective To establish a pre-column derivation HPLC-UV method for the content determination of hyodeoxycholic acid in artificial calculus bovis. Methods The hyodeoxycholic acid was derived by 2-bromo-2’-acetonaphthoneat using triethylamine as the catalyst in 60 ℃ water bath. After that, a HPLC-UV method was established to determine the content of hyodeoxycholic acid in artificial calculus bovis. Results When the derivatising time at 60 ℃ water bath was 50 min, the radio of the molar amount of derived reagents and hyodeoxycholic was over 20:1 and the radio of catalyst and hyodeoxycholic was over 15:1; the reaction was completed. The calibration curve was linear within the range of 0.1–2 μg for hyodeoxycholic acid (r=0.999 7), and the average recovery was 97.85% (RSD=1.6%). In this sample, the content of hyodeoxycholic is 4.12%. Conclusion The method is with high sensitivity, highly reproducible, reliable and accurate for the content determination of hyodeoxycholic acid in artificial calculus bovis.
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Objective:To investigate the enantiomer separation for atropine by capillary electrophoresis .Methods:Capillary elec-trophoresis was used with an elastic quartz capillary column (60 cm ×75 mm, effective length of 40 cm).The concentration of phos-phate buffer was 30 mmol· L-1 .The high and time of injection was 10 cm and 5 s, respectively.The detection wavelength was 225 nm.The best separation conditions were selected including the type and concentration of chiral resolving agent , pH of the buffer solu-tion, operating voltage and organic solvent.Results:The optimum conditions of separation were as follows:the pH of buffer solution was 7.0, the concentration of S-β-CDP was 10 mg· ml-1 , and the operating voltage was 12 kV.Conclusion: The method is simple and fast, which can be used to se parate the optical isomers of atrpo ine.
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Objective:To compare the anti-thrombin activity and the effects on the coagulation pathway between Whitmania pigra Whitman and Hirudinaria manillensis to provide scientific reference for the anticoagulation mechanism revelation for Hirudo. Methods:Anti-thrombin titration and chromogenic substrate assay-extraction-HPLC were applied to study the anti-thrombin activity of Whitmania pigra Whitman and Hirudinaria manillensis. APTT, PT and TT were determined by a clotting assay to compare the effects on the path-way of blood clotting. Results:The anticoagulation activity order measured by anti-thrombin titration was living Hirudinaria manillensis> dried Hirudinaria manillensis > > dried Whitmania pigra Whitman. The results of chromogenic substrate assay-extraction-HPLC in-dicated that the low dose of aqueous extract promoted the thrombin activity, while the high dose inhibited the thrombin activity. Hirudi-naria manillensis significantly inhibited the activity of thrombin, while Whitmania pigra Whitman showed weak anti-thrombin activity only at the higher dose. All leeches could prolong APTT, PT and TT. However, living Hirudinaria manillensis mainly affected TT, and dried Hirudinaria manillensis mainly affected APTT. Dried Whitmania pigra Whitman dramatically influenced APTT and TT. All the results indicated that the anticoagulant activity of Whitmania pigra Whitmanis was significantly higher than that of Hirudinaria manillen-sis. Conclusion:There are notable differences in the anti-thrombin activity and the effect on the pathway of blood clotting between Whitmania pigra Whitman and Hirudinaria manillensis.
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Objective To establish a capillary electrophoresis method to separate ephedrine and psedudoephedrine. Methods RM-β-CD and HP-β-CD were set as additives. A capillary electrophoresis method was set up. The effects of types and concentrations of additives, the concentrations and pH values of buffered solution, running voltage and organic solvent on the separation of ephedrine and psedudoephedrine were investigated.Results Ephedrine and pseudoephedrine could be successfully separated by using either RM-β-CD or HP-β-CD as additives. When RM-β-CD was used as additive, the best separation conditions were as follows: separation voltage 10 kV, 25 mmol/L Tris-H3PO4 (pH 2.42), 20 mg/mL of RM-β-CD. Under the conditions, the resolution of ephedrine and pseudoephedrine was 1.56 and they were separated successfully within 13 min. When HP-β-CD was used as additive, the best separation conditions were as follows: separation voltage 10 kV, 25 mmol/L Tris-H3PO4 (pH 3.00), 50 mg/mL of HP-β-CD. Under the conditions, the resolution of ephedrine and pseudoephedrine was 2.73 and they were separated successfully within 15 min.ConclusionThis method is reliable, rapid and repeatable. It can be used as separation determination method for ephedrine and pseudoephedrine.
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Objective To investigate the factors affecting the stability of the volatile oil extracted fromYinqiaosan Decoction.Methods The main chemical compositions and the extraction repetitiveness of the compound volatile oil were determined by GC-MS, and the stability of multiple extracted volatile oil was studied. Absorbance of the compound volatile oil was used as the evaluation index, and the factors affecting the stability of the of the compound volatile oil were investigated, such as illumination, temperatures and pH values of volatile oil solution and metal ions.Results The results of the GC-MS chromatograph indicated that the main chemical compositions of the compound volatile oil extracted fromYinqiaosan Decoction twice were the same. The results of the stability of the volatile oil showed that the preservation temperature and illumination affected the stability of the volatile oil to a certain extent. The absorbance values of the compound volatile oil changed slowly when it was stored at a relatively low temperature (4℃) and shielded from light, and it was less stable when stored in normal temperature and under illumination. Meanwhile, the absorbance of the compound volatile oil changed quickly in acid or alkaline solutions and was in instability. The metal ions, such as Cu2+ and Fe3+, have chemical reactions with the compositions of the compound volatile oil and there was a big change in the UV-Vis spectrum of the compound volatile oils.Conclusion The compound volatile oil should be stored at a relatively low temperature (4℃) and shielded from light. At the same time, it should be stored avoiding acids, alkaline and the metal ions, such as Cu2+ and Fe3+, to guarantee its stability. This study provides a reference for the preservation conditions and the preparation conditions of the compound volatile oil extracted fromYinqiaosan Decoction.
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The poor chemical stability and solubility, easy volatile and other shortcomings of TCM volatile oils can be improved after they are included by cyclodextrin. Therefore, cyclodextrin inclusion technology is widely used in the preparation process of volatile oil. This article reviewed the domain of cyclodextrin inclusion of volatile oils, the types of cyclodextrin, inclusion technologies, characterization for inclusion, components changes before and after inclusion, and dissolution characteristics of cyclodextrin inclusion.