桑黄黄酮类成分及制剂研究
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摘要
桑黄属担子菌亚门、多孔菌科,是一种珍贵的药用真菌。在我国传统中药中用于治疗痢疾、盗汗、血崩等。日本则作为利尿剂使用。研究表明,桑黄具有抗肿瘤、增强免疫以及抗纤维化作用。该真菌中含有三萜、多糖和黄酮等活性成分。桑黄是少有的含有黄酮化合物的药用真菌,黄酮类化合物具有抗氧化、扩张血管、降血脂和抗癌作用。
本文研究总黄酮的提取纯化、体内外药效以及制剂工艺,为桑黄菌开发利用提供实验依据。主要的研究内容如下：
1.以总黄酮提取率为考察指标,比较五个品种中桑黄总黄酮的含量,结果：桑桑黄>杨桑黄>白桦桑黄>黑桦桑黄>松桑黄。
2.采用超声辅助提取,设计正交试验,对桑黄中黄酮的提取工艺进行研究。结果：以总黄酮提取率为指标,超声法提取桑黄总黄酮的最佳工艺为40-60目细粉,乙醇浓度70%,提取温度45℃,料液比1：30,提取时间20 min,超声功率50W,
3.将桑黄总黄酮粗提物分别经大孔吸附树脂、聚酰胺、正丁醇等方法预处理后,通过Fe3+还原实验、还原力测定、Fe2+络合实验、DPPH清除率实验等方法来进行抗氧化活性研究,与同等质量的桑黄总黄酮粗提物和桑黄多糖的抗氧化活性进行对比,研究桑黄总黄酮各种纯化方法对抗氧化活性的影响。结果显示粗品抗氧化活性较强,纯化后提高了黄酮的含量,抗氧化活性并没有提高,其中大孔吸附树脂纯化的精制黄酮的纯度较高,抗氧化活性也高,桑黄多糖的抗氧化活性最低。
4.建立D-半乳糖衰老小鼠模型后,各给药组小鼠用桑黄醇提物和水提物分别灌胃一段时间后,检测小鼠脑组织中一氧化氮合酶(NOS)活力、一氧化氮(NO)含量。与衰老模型对照组相比,给药组的NOS活力和NO含量均明显降低,水提部位和醇提部位给药效果没有显著差异；通过灌胃高脂饲料建立小鼠高血脂症模型后,按照高、中、低剂量灌胃桑黄总黄酮提取物,测定体重、脏器系数、血清SOD以及脑NO等指标。结果表明桑黄总黄酮具有明显的降血脂作用。
5.以崩解时限为指标,采用单因素考察和正交设计实验,对桑黄总黄酮分散片处方及制剂工艺进行筛选、优化。最优处方和工艺是MCC:乳糖(6:2)为填充剂,5%PVPK17乙醇溶为粘合剂,崩解剂为立崩,含量7%,压力3kg。按优选处方和工艺制备的桑黄总黄酮分散片符合《中国药典》中有关分散片的要求。
6.选用氯化铝为受电子试剂,采用荷移分光光度法进行总黄酮含量测定,并进行方法学考察。结果A=0.028C-0.001,R2=0.9994,线性范围为1-40μg·ml-1,平均回收率为99.59%。
7.建立RP-HPLC测定桑黄黄酮分散片中柚皮素含量的方法：YWG-C18(4.6mm×150mm,10μm)色谱柱,检测波长288 nm,柱温为40℃,流动相为水-甲醇-冰醋酸(59:40:1),流速为0.6ml·min-1。标准曲线为A=9.499×105C+698.8,回归系数R2=0.9994,浓度范围1-100μg·ml-1,方法精密度RSD为1.1%(n=6);平均回收率为103.3%,RSD为1.5%(n=9)；重复性实验RSD为0.3%(n=6)：样品在10h内测量结果稳定；含量为0.0343%,RSD为0.7%(n=6)。
Phellinus Igniarius (Polyporaceae, Basidiomycotin) is a rare leechdom fungus. It was one kind of Chinese Tradition Medicines to treat many diseases like dysenteria, night-sweat, uterine bleeding and as emictoria in Japan. Researchs respected that it had a good effect on cancer, fibrosis and can enhance i-immunity. It contains active components of triterpene, polysaccharide, flavone, and it is a rare fungus which contains flavone compounds had the effect of anti-OX, broaden blood vessel and antitumous.
The articles mainly studys the extraction, depuration, pharmacodynamic action and praeparatum technology for exploitation and utilization, the main reaserach contents have been listed as followings:
1. The extraction rate of total flavonoids as the indictor, compare the five different species of Phellinus Igniarius, the contents of total flavonoids listed from higher to lower was Phellinus yucatanensis, Phellinus vaninii with Betula platyphylla Suk., Phellinus igniarius with Betula dahurica Pall., Phellinus igniarius, Phellinus pini.
2. Optimum conditions for extracting flavone from Phellinus vaninii sporophore by ultrasound-assisted method were studied using monofactorial and orthogonal testing. It was concluded that the optimal conditions were:particle size 40-60 mesh, alcohol ratio 70%, extraction temperature 45℃, ratio of liquid to solid by 30:1, extraction time 20 minutes, power 50W.
3. For studying on Antioxidation effects of total flavonoids from Phellinus vaninii by different purification methods, experiments on Fe3+ disoxidation, reducing power, Fe2+ complexation, DPPH clearance were adopt, with comparising with crude flavonoids and polysaccharide from Phellinus vaninii. Result displays that the effect of crude flavonoids is the best but the content of flavonoids had raised that purified through macroreticular resins chromatography, polyamide chromatography, n-butanol extraction.
4. Senium mouse models administered D-GAL by hypodermic injection was raised with alcoh extract and aqueous extract from Phellinus vaninii by intragastric administration respectively, after 15 days detected NOS and NO in brain. Result displays that the both contents are cut down and the effects of dosage parts have no difference. Hyperlipemia mouse models were administered higher, middle, lower dose total flavonoids from Phellinus vaninii by lavage, they were killed after 15 days and were and detected organ quotient, MDA、SOD of blood-serum and No in brain, result displays that total flavonoids from Phellinus vaninii have the role in lowering blood-fat.
5. The disintigrate as the index, monofactorial and orthogonal experimental tests were used to optimizate dispersible tablet preparation technology of total flavonoids from Phellinus vaninii, the best was MCC:LYP(6:2)as loading agent,5%PVP K17 in alcoh as binder,7% super-CMS as disintegrating agent, pressure 3kg.
6. To establish a method of ultraviolet spectrophotometry after charge-transfer, aluminum chloride was selected as the acceptor; the contents of total flavonoids in extract of Phellinus vaninii were assayed. The result:A=0.028C-0.001, R2=0.9994, linear within range 1～40μg·ml-1, average recovery 99.59%.
7. RP-HPLC method for determination of naringenin in flavone dispersible tablet of Phellinus vaninii was set up. YWG-C18 column (4.6mm×250mm,5μm) was used. The detection wavelength was set at 288nm. The mobile phase consisted of methanol-water-aceticacid (40:59:1) with the flow rate 0.6 ml·min-1 and column temperature at 40℃. The standard curve A=9.499×105C+698.8 (R2=0.9994), linear within range 1～100μg·ml-1. The method RSD was RSD 1.1%(n=6), the average recovery of assay was 103.3%(RSD=1.5%, n=9), the RSD of reproducibility experimentation was 0.3%(n=6) and the test was stable in 10 hours. The results:the content of naringenin in flavone dispersible tablet of Phellinus vaninii was 0.0343% (RSD=0.7%,n=6)
本文研究总黄酮的提取纯化、体内外药效以及制剂工艺,为桑黄菌开发利用提供实验依据。主要的研究内容如下：
1.以总黄酮提取率为考察指标,比较五个品种中桑黄总黄酮的含量,结果：桑桑黄>杨桑黄>白桦桑黄>黑桦桑黄>松桑黄。
2.采用超声辅助提取,设计正交试验,对桑黄中黄酮的提取工艺进行研究。结果：以总黄酮提取率为指标,超声法提取桑黄总黄酮的最佳工艺为40-60目细粉,乙醇浓度70%,提取温度45℃,料液比1：30,提取时间20 min,超声功率50W,
3.将桑黄总黄酮粗提物分别经大孔吸附树脂、聚酰胺、正丁醇等方法预处理后,通过Fe3+还原实验、还原力测定、Fe2+络合实验、DPPH清除率实验等方法来进行抗氧化活性研究,与同等质量的桑黄总黄酮粗提物和桑黄多糖的抗氧化活性进行对比,研究桑黄总黄酮各种纯化方法对抗氧化活性的影响。结果显示粗品抗氧化活性较强,纯化后提高了黄酮的含量,抗氧化活性并没有提高,其中大孔吸附树脂纯化的精制黄酮的纯度较高,抗氧化活性也高,桑黄多糖的抗氧化活性最低。
4.建立D-半乳糖衰老小鼠模型后,各给药组小鼠用桑黄醇提物和水提物分别灌胃一段时间后,检测小鼠脑组织中一氧化氮合酶(NOS)活力、一氧化氮(NO)含量。与衰老模型对照组相比,给药组的NOS活力和NO含量均明显降低,水提部位和醇提部位给药效果没有显著差异；通过灌胃高脂饲料建立小鼠高血脂症模型后,按照高、中、低剂量灌胃桑黄总黄酮提取物,测定体重、脏器系数、血清SOD以及脑NO等指标。结果表明桑黄总黄酮具有明显的降血脂作用。
5.以崩解时限为指标,采用单因素考察和正交设计实验,对桑黄总黄酮分散片处方及制剂工艺进行筛选、优化。最优处方和工艺是MCC:乳糖(6:2)为填充剂,5%PVPK17乙醇溶为粘合剂,崩解剂为立崩,含量7%,压力3kg。按优选处方和工艺制备的桑黄总黄酮分散片符合《中国药典》中有关分散片的要求。
6.选用氯化铝为受电子试剂,采用荷移分光光度法进行总黄酮含量测定,并进行方法学考察。结果A=0.028C-0.001,R2=0.9994,线性范围为1-40μg·ml-1,平均回收率为99.59%。
7.建立RP-HPLC测定桑黄黄酮分散片中柚皮素含量的方法：YWG-C18(4.6mm×150mm,10μm)色谱柱,检测波长288 nm,柱温为40℃,流动相为水-甲醇-冰醋酸(59:40:1),流速为0.6ml·min-1。标准曲线为A=9.499×105C+698.8,回归系数R2=0.9994,浓度范围1-100μg·ml-1,方法精密度RSD为1.1%(n=6);平均回收率为103.3%,RSD为1.5%(n=9)；重复性实验RSD为0.3%(n=6)：样品在10h内测量结果稳定；含量为0.0343%,RSD为0.7%(n=6)。
Phellinus Igniarius (Polyporaceae, Basidiomycotin) is a rare leechdom fungus. It was one kind of Chinese Tradition Medicines to treat many diseases like dysenteria, night-sweat, uterine bleeding and as emictoria in Japan. Researchs respected that it had a good effect on cancer, fibrosis and can enhance i-immunity. It contains active components of triterpene, polysaccharide, flavone, and it is a rare fungus which contains flavone compounds had the effect of anti-OX, broaden blood vessel and antitumous.
The articles mainly studys the extraction, depuration, pharmacodynamic action and praeparatum technology for exploitation and utilization, the main reaserach contents have been listed as followings:
1. The extraction rate of total flavonoids as the indictor, compare the five different species of Phellinus Igniarius, the contents of total flavonoids listed from higher to lower was Phellinus yucatanensis, Phellinus vaninii with Betula platyphylla Suk., Phellinus igniarius with Betula dahurica Pall., Phellinus igniarius, Phellinus pini.
2. Optimum conditions for extracting flavone from Phellinus vaninii sporophore by ultrasound-assisted method were studied using monofactorial and orthogonal testing. It was concluded that the optimal conditions were:particle size 40-60 mesh, alcohol ratio 70%, extraction temperature 45℃, ratio of liquid to solid by 30:1, extraction time 20 minutes, power 50W.
3. For studying on Antioxidation effects of total flavonoids from Phellinus vaninii by different purification methods, experiments on Fe3+ disoxidation, reducing power, Fe2+ complexation, DPPH clearance were adopt, with comparising with crude flavonoids and polysaccharide from Phellinus vaninii. Result displays that the effect of crude flavonoids is the best but the content of flavonoids had raised that purified through macroreticular resins chromatography, polyamide chromatography, n-butanol extraction.
4. Senium mouse models administered D-GAL by hypodermic injection was raised with alcoh extract and aqueous extract from Phellinus vaninii by intragastric administration respectively, after 15 days detected NOS and NO in brain. Result displays that the both contents are cut down and the effects of dosage parts have no difference. Hyperlipemia mouse models were administered higher, middle, lower dose total flavonoids from Phellinus vaninii by lavage, they were killed after 15 days and were and detected organ quotient, MDA、SOD of blood-serum and No in brain, result displays that total flavonoids from Phellinus vaninii have the role in lowering blood-fat.
5. The disintigrate as the index, monofactorial and orthogonal experimental tests were used to optimizate dispersible tablet preparation technology of total flavonoids from Phellinus vaninii, the best was MCC:LYP(6:2)as loading agent,5%PVP K17 in alcoh as binder,7% super-CMS as disintegrating agent, pressure 3kg.
6. To establish a method of ultraviolet spectrophotometry after charge-transfer, aluminum chloride was selected as the acceptor; the contents of total flavonoids in extract of Phellinus vaninii were assayed. The result:A=0.028C-0.001, R2=0.9994, linear within range 1～40μg·ml-1, average recovery 99.59%.
7. RP-HPLC method for determination of naringenin in flavone dispersible tablet of Phellinus vaninii was set up. YWG-C18 column (4.6mm×250mm,5μm) was used. The detection wavelength was set at 288nm. The mobile phase consisted of methanol-water-aceticacid (40:59:1) with the flow rate 0.6 ml·min-1 and column temperature at 40℃. The standard curve A=9.499×105C+698.8 (R2=0.9994), linear within range 1～100μg·ml-1. The method RSD was RSD 1.1%(n=6), the average recovery of assay was 103.3%(RSD=1.5%, n=9), the RSD of reproducibility experimentation was 0.3%(n=6) and the test was stable in 10 hours. The results:the content of naringenin in flavone dispersible tablet of Phellinus vaninii was 0.0343% (RSD=0.7%,n=6)
引文
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[1]崔德福主编.药剂学(第五版).人民卫生出版社.2006.
[2]雷同康.分散片的处方和工艺[J].中国医药工业杂志.1999,30(2)：87-96.
[3]史宁,吴久鸿.口腔崩解片的研究进展[J].解放军药学学报.2005,21(1)：56-68.
[4]Mulliken,R.S.J.Am. Chem [J]. Soc.1952,74:811
[5]林珊,王世铭一,王琼生.新型高分子一多酸电荷转移配合物的合成与表征[J].功能高分子学报,2005,18(2)：239-242.
[6]Justin M. Notestein, Enrique Iglesia, Alexander Katz. Photoluminescence and Charge-Transfer Complexes of Calixarenes Grafted on TiO2 Nanoparticles[J]. Chem. Mater,2007,19:4998-5005.
[7]V.N, Shevchuk. Photo-stimulate charge transfer and luminescence in rare earth gallium garnet crystals [J]. Radiation Measurements,2007,42:704-707.
[8]徐婉珍,李春香,陆晓青等.亚甲基蓝-红霉素电荷转移体系与光度法联用测定红霉素的研究[J].化学世界,2007：152-154.
[9]李方,张进,张新申等.胶束及反胶束微环境中的槲皮素金属配合物[J].无机化学学报,2005,21(4)：565-570.
[10]Galati, E.M.Monforte, M.T.Kirijavanein, S. Forestieri, A. M.Trovato, A.Tripodo, M. M. Ⅱ Farmaco.1994,49:709-714.
[11]项炯华.柚皮素的药理研究及其在临床上的应用[J].中华医学研究杂志,2007,(05)：428-430.
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