应用成骨细胞生物色谱法研究牡丹皮促进骨折愈合的效应物质

发布时间：2018-06-07 02:15

本文选题：细胞固相色谱法 + 牡丹皮水提液　；参考：《南京中医药大学》2017年硕士论文

【摘要】：目的:建立MC3T3-E1成骨细胞固相色谱方法,研究骨折合剂君药牡丹皮促进骨折愈合的效应物质;并应用MC3T3-E1成骨细胞增殖和分化效应验证所得结合物质的活性,为牡丹皮在临床治疗骨折提供实验依据,也为骨折合剂的进一步开发提供基础。方法:采用Agilent5TC-C18色谱柱,0.4%的甲酸水溶液-乙腈为流动相梯度洗脱,检测波长为254nm,柱温为30℃,进样量为5μL,流速为1mL·min-1,建立牡丹皮水提液的指纹图谱,并对其三个主要成分没食子酸、芍药苷和丹皮酚进行定量研究。建立MC3T3-E1成骨细胞固相色谱法:采用高温灭菌法处理牡丹皮水提液,药液与MC3T3-E1成骨细胞共同孵育4 h,洗脱4次后将细胞失活,使结合效应成分解离下来,再将细胞进行裂解,使进入细胞内的效应成分释放出来。应用HPLC和HPLC/MS对解离液和裂解液中的物质成分进行分析。采用MTT法和ALP活性测定法,研究筛选出的准活性成分对MC3T3-E1成骨细胞活性的影响。结果:得到10批牡丹皮的对照指纹图谱和共有峰30个,其中4号色谱峰为没食子酸、7号色谱峰为没食子酸甲酯、14号色谱峰为芍药内酯苷、16号色谱峰为芍药苷、21号色谱峰为1,2,3,4,6-O-五没食子酰葡萄糖(PGG)、30号色谱峰为丹皮酚。各批次牡丹皮水提液的指纹图谱及对照图谱相似度均大于0.9。三个主成分方法学考察的结果显示:没食子酸、芍药苷和丹皮酚的线性范围分别在1.691～270 μg·mL-1(R2=0.9997)、1.847～250 μg·mL-1(R2=0.9999)、0.181～29.0μg·mL-1(R2=0.9999)与峰面积呈良好的线性关系;平均回收率(n=3)在94.12%～100.01%;含量测定结果显示,牡丹皮中没食子酸、芍药苷和丹皮酚的平均含量分别为 0.34%、0.54%和 0.0091%,RSD 分别为 5.01%、5.40%和 2.41%。应用MC3T3-E1成骨细胞固相色谱法,筛选分析得到4个主要的准活性成分,其中与细胞膜结合的成分为没食子酸和1,2,3,4,6-五没食子酰葡萄糖(PGG),进入细胞内部的成分为没食子酸甲酯和芍药内酯苷。药理学验证结果显示:0.6～1.6 mg/mL的没食子酸对MC3T3-E1成骨细胞的增殖与分化均具有明显的促进作用(P0.05,0.001);0.5～1.0 mg/mL的没食子酸甲酯对MC3T3-E1成骨细胞的增殖与分化均具有明显的促进作用(P0.05,0.01,0.001);4.0 mg/mL的芍药内酯苷对MC3T3-E1成骨细胞的增殖具有明显的促进作用(P0.05),但0.5～4.0 mg/mL的芍药内酯苷对MC3T3-E1成骨细胞的分化具有抑制作用;1.2mg/mL的1,2,3,4,6-0-五没食子酰葡萄糖对MC3T3-E1成骨细胞的增殖与分化均具有明显的促进作用(P0.05,0.01)。结论:1)本课题建立的牡丹皮水提液的HPLC指纹图谱,方法简便、重复性好;没食子酸、芍药苷和丹皮酚多成分检测的方法学考察结果表明,该分析方法稳定可靠,为本课题牡丹皮的提取工艺提供质控标准。2)本课题建立的MC3T3-E1成骨细胞固相色谱法通过验证,说明方法可行性,可用于牡丹皮促进骨折愈合成分的筛选。3)通过课题研究,初步确定牡丹皮中促进骨折愈合的效应物质为没食子酸、没食子酸甲酯与1,2,3,4,6-五没食子酰葡萄糖(PGG)。
[Abstract]:Objective: to establish a solid phase chromatography of MC3T3-E1 osteoblast to study the effect substance of fracture healing agent peony skin to promote fracture healing, and to verify the activity of the combined substance using the proliferation and differentiation effect of MC3T3-E1 osteoblast, and provide the basis for the clinical treatment of fracture of peony skin, and also provide the further development of fracture mixture. Methods: using Agilent5TC-C18 column, 0.4% formic acid solution - acetonitrile for mobile phase gradient elution, detection wavelength of 254nm, column temperature of 30 C, 5 mu L and 1mL. Min-1, the fingerprint of water extract of peony skin was established, and the quantitative study on three main ingredients gallic acid, paeoniflorin and paeonol was established. MC 3T3-E1 osteoblast solid phase chromatography: the aqueous extract of peony skin was treated with high temperature sterilization. The liquid was incubated with MC3T3-E1 osteoblasts for 4 h. The cells were deactivated after 4 times of elution, and the combined effect components were dissociated and the cells were cracked to release the effect into the cells. HPLC and HPLC/MS were applied to the dissociation fluid and crack. Analysis of the substances in the solution. The effects of the selected quasi active components on the activity of MC3T3-E1 osteoblasts were studied by MTT and ALP activity. Results: the control fingerprint and the total peak of 10 batches of peony were obtained. 30 of the peaks were gallic acid, the peak of No. 7 was methyl gallate, and the peak of No. 14 was peony. The peak of No. 16 chromatographic peak is paeoniflorin, the peak of No. 21 is 1,2,3,4,6-O- five galloyl glucose (PGG), and the peak of No. 30 is paeonol. The similarity of the fingerprint and the control map of the water extracts of each batches of peony peel are greater than that of the three principal component methods of 0.9.. 1.691 to 270 mu g / mL-1 (R2=0.9997), 1.847 to 250 mu g mL-1 (R2=0.9999), 0.181 ~ 29 G. ML-1 (R2=0.9999) had a good linear relationship with peak area. The average recovery rate (n=3) was 94.12% to 100.01%. The content determination showed that the average content of paeoniflorin in Peony peel was 0.34%, 0.54% and 0, respectively. .0091%, RSD are 5.01%, 5.40% and 2.41%. using MC3T3-E1 osteoblast solid phase chromatography, and 4 major quasi active components are screened and analyzed. The components of the cell membrane are gallic acid and 1,2,3,4,6- five galloyl glucose (PGG), and the components into the cells are methyl gallate and paeoniflorin. The results showed that 0.6 ~ 1.6 mg/mL gallic acid had an obvious promoting effect on the proliferation and differentiation of MC3T3-E1 osteoblasts (P0.05,0.001); 0.5 ~ 1 mg/mL gallate methyl gallate had an obvious promoting effect on the proliferation and differentiation of MC3T3-E1 osteoblasts (P0.05,0.01,0.001); 4 mg/mL of paeoniflorin to MC3T3-E1 The proliferation of osteoblasts had an obvious promoting effect (P0.05), but 0.5 ~ 4 mg/mL of paeoniflorin had inhibitory effect on the differentiation of MC3T3-E1 osteoblasts; 1,2,3,4,6-0- five galloyl glucose of 1.2mg/mL had an obvious promoting effect on the proliferation and differentiation of MC3T3-E1 osteoblasts (P0.05,0.01). Conclusion: 1) the establishment of this subject was established. The HPLC fingerprint of the water extract of peony skin is simple and reproducible. The method of detecting the multicomponent analysis of gallic acid, paeoniflorin and paeonol shows that the method is stable and reliable, and provides quality control standard.2 for the extraction process of peony skin in this subject. The solid phase chromatography of MC3T3-E1 osteoblasts established by this project has been verified by the method of solid phase chromatography. To illustrate the feasibility of the method, it can be used to screen.3 for healing of fracture healing in Peony skin). Through the study of the subject, the effects of gallic acid, methyl gallate and 1,2,3,4,6- five galloyl glucose (PGG) are preliminarily identified in the skin of peony skin.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284;R285

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