【摘要】：沙冬青為豆科旱生植物,沙冬青種子總黃酮是以其種子為原料提取分離的總黃酮。已有研究表明,沙冬青種子總黃酮具有良好的抗病毒活性。但到目前為止,有關(guān)沙冬青種子總黃酮含量的測(cè)定方法及相關(guān)免疫調(diào)節(jié)活性的研究尚屬空白。為深入探索沙冬青種子總黃酮的純化工藝和其免疫調(diào)節(jié)活性,本試驗(yàn)從以下幾個(gè)方面進(jìn)行了研究。(1)沙冬青種子總黃酮測(cè)定方法的建立:采用4種顯色方法對(duì)沙冬青種子總黃酮進(jìn)行顯色,通過(guò)紫外波長(zhǎng)掃描分別確定對(duì)照品槲皮素及沙冬青種子總黃酮的最佳吸收波長(zhǎng),分別建立標(biāo)準(zhǔn)曲線,并對(duì)各測(cè)定方法進(jìn)行方法學(xué)驗(yàn)證,利用新建立的測(cè)定方法對(duì)沙冬青種子總黃酮進(jìn)行含量測(cè)定。(2)沙冬青種子總黃酮大孔樹脂純化工藝研究:選擇AB-8、D101、S-8型大孔樹脂通過(guò)靜態(tài)吸附及解析試驗(yàn),篩選出AB-8型大孔樹脂進(jìn)行動(dòng)態(tài)試驗(yàn),分別從上樣液濃度、上樣液體積、上樣液流速、pH、洗脫液乙醇濃度、洗脫流速、洗脫液體積對(duì)吸附及洗脫效果的影響進(jìn)行考察,從而確定AB-8型大孔樹脂最佳純化工藝條件;將純化后的總黃酮進(jìn)一步通過(guò)乙酸乙酯進(jìn)行萃取,提高其總黃酮純度。(3)沙冬青種子總黃酮免疫活性研究:采用環(huán)磷酰胺(CTX)建立小鼠免疫抑制模型,再分別給予免疫抑制小鼠不同劑量濃度的沙冬青種子總黃酮[(高劑量(250mg/mL)、中劑量組(150mg/mL)、低劑量組(50mg/mL)],通過(guò)免疫器官臟器指數(shù)、細(xì)胞因子(IL-2、IL-4)、碳粒廓清率的測(cè)定及器官組織結(jié)構(gòu)觀察,探索沙冬青種子總黃酮對(duì)小鼠體內(nèi)免疫調(diào)節(jié)作用。同時(shí),探索不同劑量濃度的沙冬青種子總黃酮對(duì)體外小鼠脾淋巴細(xì)胞增殖的影響。結(jié)果表明,(1)以槲皮素為對(duì)照品,采用AlCl3-CH4O法顯色后,沙冬青種子總黃酮在303nm和335nm處均出現(xiàn)明顯的最大吸收峰,通過(guò)方法學(xué)驗(yàn)證可知AlCl3-CH4O法在335nm處精密度(RSD=0.541%)、重現(xiàn)性(RSD=1.05%)、穩(wěn)定性(RSD=0.62%)均在誤差范圍以內(nèi),其加樣回收率達(dá)到102.57%,可準(zhǔn)確測(cè)定沙冬青種子總黃酮的含量。(2)AB-8型大孔樹脂在上樣液濃度為6mg/mL、上樣液pH=5.5、上樣液體積5BV、上樣液流速3.5BV/h的條件下吸附,以70%乙醇、洗脫液流速1.5BV/h、洗脫液體積6BV條件下洗脫后沙冬青種子總黃酮干物質(zhì)中含量由純化前5.69%提高到41.07%;用AB-8樹脂純化后的總黃酮配置成一定濃度溶液后采用乙酸乙酯雙水相萃取,總黃酮純度提升到76.15%。(3)與CTX模型組比較,純化沙冬青種子總黃酮中劑量組小鼠脾臟指數(shù)4.431±0.574,胸腺指數(shù)為3.162±0.524極顯著增大(P0.01)。(4)與CTX模型組比較,純化沙冬青種子總黃酮中劑量組小鼠體內(nèi)IL-2含量為541.3±33.41、IL-4含為44.819±2.257極顯著升高(P0.01)。(5)與CTX模型組比較,純化沙冬青種子總黃酮中劑量組小鼠碳粒廓清指數(shù)K為0.0203±0.0089,吞噬指數(shù)α為4.592±0.3285吞噬能力極顯著增強(qiáng)(P0.01)。(6)高純度沙冬青種子總黃酮中劑量組(150mg/kg)與CTX相比,小鼠脾臟中成熟淋巴細(xì)胞數(shù)量增加,白髓面積相對(duì)擴(kuò)大,脾小體數(shù)量增多且生發(fā)中心相對(duì)擴(kuò)大。胸腺中胸腺小葉體積相對(duì)增大,皮質(zhì)區(qū)增厚,皮質(zhì)胸腺細(xì)胞數(shù)量相對(duì)增多,密度增加。(7)體外脾淋巴細(xì)胞增殖試驗(yàn)表明,單純給藥組與陰性對(duì)照組比較,在一定濃度范圍內(nèi)(10?320μg/mL)細(xì)胞增殖與給藥濃度呈正相關(guān),且細(xì)胞增殖數(shù)量增加極顯著(P0.01),隨給藥濃度增加,細(xì)胞增殖能力增強(qiáng);低于10μg/mL后活細(xì)胞數(shù)量增加不顯著(P0.05)。ConA誘導(dǎo)+給藥組與陰性對(duì)照組比較,活細(xì)胞數(shù)量極顯著增加(P0.01);當(dāng)給藥濃度低于5μg/mL后,ConA誘導(dǎo)+給藥組活細(xì)胞數(shù)量較ConA陽(yáng)性對(duì)照組差異不顯著(P0.05)。本研究建立了沙冬青種子總黃酮檢測(cè)方法,為準(zhǔn)確檢測(cè)其含量提供了依據(jù)。同時(shí),建立了沙冬青種子總黃酮純化工藝,有效提高了其干物質(zhì)的純度。通過(guò)體內(nèi)免疫活性調(diào)節(jié)試驗(yàn)及體外脾淋巴細(xì)胞增殖試驗(yàn),證明沙冬青種子總黃酮在一定濃度范圍內(nèi),具有明顯促進(jìn)動(dòng)物機(jī)體免疫系統(tǒng)活化和增強(qiáng)機(jī)體免疫能力的作用。
[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.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S853.7

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