| Stephania cepharantha Hayata, wildly grow in Zhejiang, Jiangsu, Anhui, Fujian, Hubei and other provinces belongs to the family of Menispermaceae. Its root has long been used as a traditional Chinese medicine. It is well known that Menispermaceae plants are rich in alkaloids, especially bisbenzylisoquinoline(BBI) such as cepharanthine, isotetrandrine, berbamine, cycleanine, L- isociorydine, cepharanoline, cepharamine and so on. Many pharmaceutical activities of BBI including anti- inflammatory, immunomodulatory, anti- leukopenia, bronchial asthma, anti-cancer, hypotensive, anti-arrhythmia, myocardial protection and reversal of multi drug resistance have been reported. However, information about the preparation, biological activity and phytochemical composition of Stephania cepharantha Hayata are limited.In the present stuy, alkaloid rich extract was prepared from the dry root of Stephania cepharantha Hayata and its main alkaloids were identified. Then, the leukogenic effect of this extract was evaluated by oral and intravenous injection administration, finally the mechanism of leukogenic effects were discussed.(1)Preparation of S.cepharantha extractPowdered root of S.cepharmantha was extracted with 0.5 % sulfuric acid and the extract solution was divided into two equal parts. O ne part of the solution was used for pH gradient precipitation and the other part was used for cation resin absorption to obtain alkaloids. The pH values of the solution were successively adjusted to 5, 7, 9, 11 with 10 % aqueous ammonia and precipitations at each p H value were obtained by centrifugation, and the precipitation was washed with distilled water until no NH4+ detected. The collected precipitation was suspended in water and extracted with ethyl acetate. The alkaloid extract was obtained by evaporation the solvent. Another part of solution was used for cation resin absorption. Sodium styrene sulfonate cationic resin(H+) was used to absorb the alkaloids. Brifely, acid extract solution was subjected into a column packed with cationic resin at a suitable speed. After the satura tion absorption was reached, the column was clearned with 5 column volumes of distill water. Then, the resin was taken out and let it drip dried. The drip dried resin was put into a reflux device and the pH value was adjusted to pH9 using 10 % aqueous ammo nia. Twenty minutes after the p H adjustment, dichloromethane was added and refluxed for 30 mins. The dichloromethane solution was washed to neutral using distill water followed by anhydrous sodium sulfate drying and filtration. Finally, the extract was obtained by evaporating the solvent.As a result, the extract weights obtained by pH gradient precipitation at p H5, pH7, pH9 and pH11 were 0.396 g, 0.504 g, 1.654 g and 0.087 g, respectively. The weight obtained by cation resin absorption was 0.450 g. And preliminary analsis by HPLC showed that alkaloids in the extract obtained by cation resin absorption were more complicated than that in the extracts obtained by p H gradient precipitation. Furthermore, pH gradient precipitation was more convenient to operate than cation resin absorption.(2) Identification the extract componentsHPLC chromatogram showed that there were, at least, 3 components in the extract counted more than 50 % in peak area. The 3 major peaks were collected using a preparative HPLC and identified as berbamine, isotetrandrine, and cepharanthine using NMR and MS.The major alkaloids in the extracts were quantified by HPLC using isolated berbamine, isoletrandrine and cepharanthine as reference. The result showed that the content of berbamine, isotetrandrine and cepharanthine in the extracts obtained by pH gradient precipitation at different p H value and cation resin absorption were varied. The total contents of these 3 major alkaloids in the extracts obtained by pH gradient precipitation at pH5, p H7, pH9, pH11 and by cation resin absorption were 149.69%, 241.3, 739.6, 13.89 and 502.1 mg/g, respectively. The data indicated that extract with highest alkaloid content was prepared by pH gradient precipitation at p H9, where the contents of berbamine, isotetrandrine, and cepharanthine were 196.8 mg/g, 429.6 mg/g and 113.2 mg/g, respectively. So the preferable extraction method for the extraction of bisbenzylisoquinoline alkaloids from S.cepharantha was deduced as: The powder of the plant material is extracted with 0.5 % sulfuric acid aqueous solution. After that, adjust the p H value of the sulfuric acid aqueous solution to p H5 using 10 % aqueous ammonia, discard the precipitate(if any) and the supernatant was collected by centrifugation. And then, the pH value of the collected supernatant was adjusted to pH9 using 10 % aqueous ammonia and collect the precipitate by centrifugation. The collected precipitate is washed and suspended in distilled water. Finally, the alkaloids are extracted from the suspending using ethyl acetate and the BBI rich extract is obtained by evaporation the solvent.As the extract prepared by p H gradient precipitation at p H9 had the highest content of bisbenzylisoquinoline alkaloid(BBI), it was used for the further study.(3) Effects of the extract on leucopenia by oral administrationLeucopenia in mice was induced by cyclophosphamide(CY) at 80 mg/kg by intraperitoneal injection for 3 days, by which the white blood cell count(WBC) was significantly lower than normal mice. Specifically, mice were divided into normal control group(received saline only), model group(received C Y only), positive control(orally received C Y + DY tablet 100 mg/kg) and treatment groups(orally received CY + EX(extract of S.cepharantha) at 5, 10, 20 mg/kg, respectively). The treatment was performed for 6 days. O n the 3rd day and 6t h day of the treatment, the whole blood was collected from ophthalmic venous plexus of mice and peripheral blood WBC counts and leukocytes counts were determined. Mice were sacrificed after the blood collection on 6t h day of the reatment and spleen and thymus were weighed and calculated the spleen index and thymus index.The injectiong of CY caused remarkably reduction in white blood cell(WBC),neutrophile granulocyte in peripheral blood when compared with control. Treatments with DY tablets, EX, however, WBC and neutrophile granulocytes were significantly recovered when compared with C Y injection, and at the end of the treatment, they returned to the normal level which were s ignificantly different from the C Y group. The dose-response relationships of EX were also observed at the end of 3 and 6 days treatment.Compared with the control group, C Y injection reduced the body weight, indices of spleen(IS) and thymus(IT) s ignificantly. Treatments with DY tablet, EX were restored the altered body wight, IS and IT by C Y injection. The dose response of these restorations was also observed.(4) The effects of the extract on leucopenia by intravenous injectionSimilarly, the leucopenia was induced by cyclophosphamide at 80 mg/kg for 3 days. The EX, however, was administered by intravenous injection at the doses of 1.25, 2.5, 5 mg/kg for 7 days. WBC count, red blood cells, platelets, hemoglobin count, spleen and thymus weight, body weight, bone marrow cells, bone marrow DNA content were determined. The results indicated that intravenous injection of the extract effectively mitigated the leucopenia induced by cyclophosphamide. Bone marrow nucleated cells and DN A content were recovered by intravenous injection of the extracts. Conclusion: Administration by intravenous can reverse the leucopenia induced by cyclophosphamide, it can also compete the bone marrow supression induced by cyclophosphamide. |
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