沙冬青种子总黄酮纯化工艺及免疫活性研究

发布时间：2018-08-26 11:45

【摘要】：沙冬青为豆科旱生植物,沙冬青种子总黄酮是以其种子为原料提取分离的总黄酮。已有研究表明,沙冬青种子总黄酮具有良好的抗病毒活性。但到目前为止,有关沙冬青种子总黄酮含量的测定方法及相关免疫调节活性的研究尚属空白。为深入探索沙冬青种子总黄酮的纯化工艺和其免疫调节活性,本试验从以下几个方面进行了研究。(1)沙冬青种子总黄酮测定方法的建立:采用4种显色方法对沙冬青种子总黄酮进行显色,通过紫外波长扫描分别确定对照品槲皮素及沙冬青种子总黄酮的最佳吸收波长,分别建立标准曲线,并对各测定方法进行方法学验证,利用新建立的测定方法对沙冬青种子总黄酮进行含量测定。(2)沙冬青种子总黄酮大孔树脂纯化工艺研究:选择AB-8、D101、S-8型大孔树脂通过静态吸附及解析试验,筛选出AB-8型大孔树脂进行动态试验,分别从上样液浓度、上样液体积、上样液流速、pH、洗脱液乙醇浓度、洗脱流速、洗脱液体积对吸附及洗脱效果的影响进行考察,从而确定AB-8型大孔树脂最佳纯化工艺条件;将纯化后的总黄酮进一步通过乙酸乙酯进行萃取,提高其总黄酮纯度。(3)沙冬青种子总黄酮免疫活性研究:采用环磷酰胺(CTX)建立小鼠免疫抑制模型,再分别给予免疫抑制小鼠不同剂量浓度的沙冬青种子总黄酮[(高剂量(250mg/mL)、中剂量组(150mg/mL)、低剂量组(50mg/mL)],通过免疫器官脏器指数、细胞因子(IL-2、IL-4)、碳粒廓清率的测定及器官组织结构观察,探索沙冬青种子总黄酮对小鼠体内免疫调节作用。同时,探索不同剂量浓度的沙冬青种子总黄酮对体外小鼠脾淋巴细胞增殖的影响。结果表明,(1)以槲皮素为对照品,采用AlCl3-CH4O法显色后,沙冬青种子总黄酮在303nm和335nm处均出现明显的最大吸收峰,通过方法学验证可知AlCl3-CH4O法在335nm处精密度(RSD=0.541%)、重现性(RSD=1.05%)、稳定性(RSD=0.62%)均在误差范围以内,其加样回收率达到102.57%,可准确测定沙冬青种子总黄酮的含量。(2)AB-8型大孔树脂在上样液浓度为6mg/mL、上样液pH=5.5、上样液体积5BV、上样液流速3.5BV/h的条件下吸附,以70%乙醇、洗脱液流速1.5BV/h、洗脱液体积6BV条件下洗脱后沙冬青种子总黄酮干物质中含量由纯化前5.69%提高到41.07%;用AB-8树脂纯化后的总黄酮配置成一定浓度溶液后采用乙酸乙酯双水相萃取,总黄酮纯度提升到76.15%。(3)与CTX模型组比较,纯化沙冬青种子总黄酮中剂量组小鼠脾脏指数4.431±0.574,胸腺指数为3.162±0.524极显著增大(P0.01)。(4)与CTX模型组比较,纯化沙冬青种子总黄酮中剂量组小鼠体内IL-2含量为541.3±33.41、IL-4含为44.819±2.257极显著升高(P0.01)。(5)与CTX模型组比较,纯化沙冬青种子总黄酮中剂量组小鼠碳粒廓清指数K为0.0203±0.0089,吞噬指数α为4.592±0.3285吞噬能力极显著增强(P0.01)。(6)高纯度沙冬青种子总黄酮中剂量组(150mg/kg)与CTX相比,小鼠脾脏中成熟淋巴细胞数量增加,白髓面积相对扩大,脾小体数量增多且生发中心相对扩大。胸腺中胸腺小叶体积相对增大,皮质区增厚,皮质胸腺细胞数量相对增多,密度增加。(7)体外脾淋巴细胞增殖试验表明,单纯给药组与阴性对照组比较,在一定浓度范围内(10?320μg/mL)细胞增殖与给药浓度呈正相关,且细胞增殖数量增加极显著(P0.01),随给药浓度增加,细胞增殖能力增强;低于10μg/mL后活细胞数量增加不显著(P0.05)。ConA诱导+给药组与阴性对照组比较,活细胞数量极显著增加(P0.01);当给药浓度低于5μg/mL后,ConA诱导+给药组活细胞数量较ConA阳性对照组差异不显著(P0.05)。本研究建立了沙冬青种子总黄酮检测方法,为准确检测其含量提供了依据。同时,建立了沙冬青种子总黄酮纯化工艺,有效提高了其干物质的纯度。通过体内免疫活性调节试验及体外脾淋巴细胞增殖试验,证明沙冬青种子总黄酮在一定浓度范围内,具有明显促进动物机体免疫系统活化和增强机体免疫能力的作用。
[Abstract]:Ammopiptanthus mongolicus is a legume xerophyte. The total flavonoids of Ammopiptanthus mongolicus seeds are extracted from its seeds. Studies have shown that the total flavonoids of Ammopiptanthus mongolicus seeds have good antiviral activity. In order to further explore the purification process and immunomodulatory activity of total flavonoids from the seeds of Ammopiptanthus mongolicus, the following aspects were studied in this experiment: (1) Establishment of a method for the determination of total flavonoids in the seeds of Ammopiptanthus mongolicus: Four coloration methods were used to develop the total flavonoids in the seeds of Ammopiptanthus mongolicus, and the control substances quercetin and Ammopiptanthus Mongolic The optimum absorption wavelength of total flavonoids in the seeds of Ammopiptanthus mongolicus was determined by a new method. (2) Purification process of total flavonoids in the seeds of Ammopiptanthus mongolicus by macroporous resin AB-8, D101, S-8 was studied. AB-8 macroporous resin was screened out for dynamic test. The effects of sample concentration, sample volume, sample flow rate, pH, eluent ethanol concentration, elution flow rate and eluent volume on adsorption and elution were investigated to determine the best purification process conditions of AB-8 macroporous resin. The purity of total flavonoids was improved by extracting with ethyl acetate in one step. (3) Study on the immune activity of total flavonoids from Ammopiptanthus mongolicus seeds: Immunosuppressive model of mice was established by using cyclophosphamide (CTX), and then total flavonoids from seeds of Ammopiptanthus mongolicus mongolicus were given to immunosuppressive mice at different doses [(250 mg/mL), middle dose group (150 mg/mL), low dose group (5 mg/mL). The effects of total flavonoids from the seeds of Ammopiptanthus mongolicus on the proliferation of spleen lymphocytes in mice in vitro were studied by means of organ index, cytokines (IL-2, IL-4), carbon clearance rate and organ structure observation. The maximum absorption peaks of total flavonoids in the seeds of Ammopiptanthus mongolicus were observed at 303 nm and 335 nm. The precision (RSD = 0.541%) and reproducibility (RSD = 1.05%) and stability (RSD = 0.62%) of AlCl3-CH4O method at 335 nm were determined by methodological verification. The content of total flavonoids in Ammopiptanthus mongolicus seeds can be accurately determined by 57%. (2) The content of total flavonoids in Ammopiptanthus mongolicus seeds can be determined by AB-8 macroporous resin under the conditions of sample concentration of 6mg/mL, sample pH=5.5, sample volume of 5BV, sample flow rate of 3.5BV/h, 70% ethanol, elution flow rate of 1.5BV/h and elution volume of 6BV. The purity of total flavonoids was increased to 76.15%. (3) Compared with the CTX model group, the spleen index of mice in the middle dose group of total flavonoids from the seeds of Ammopiptanthus mongolicus was 4.431 [0.574] and the thymus index was 3.162 [0.524] significantly increased (P 0.05). (4) Compared with the CTX model group, the content of IL-2 and IL-4 in the middle dose group were 541.3 (6) Compared with CTX, the number of mature lymphocytes, the area of white pulp, the number of splenic corpuscles and the germinal center of the mice spleen were increased. The thymic lobules in the thymus gland were relatively enlarged, the cortical areas were thickened, and the cortical thymocytes were enlarged. (7) Compared with the negative control group, the proliferation of splenic lymphocytes was positively correlated with the concentration of 10?320 ug/mL, and the proliferation of splenic lymphocytes was significantly increased (P 0.01). Compared with the negative control group, the number of living cells in the ConA-induced + administration group increased significantly (P 0.01). When the concentration was lower than 5 ug/ml, the number of living cells in the ConA-induced + administration group was not significantly different from that in the ConA-positive control group (P 0.05). At the same time, the purification process of total flavonoids from the seeds of Ammopiptanthus mongolicus was established, and the purity of dry matter was effectively improved. The results showed that the total flavonoids from the seeds of Ammopiptanthus mongolicus could obviously promote the immune system of animals in a certain range of concentration by regulating the immune activity in vivo and proliferating the spleen lymphocytes in vitro. And enhance the immune function of the body.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S853.7

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