本文選題：組合提取物 + 抗敏止癢　； 參考：《東北農(nóng)業(yè)大學》2015年博士論文

【摘要】：本課題組前期對具有抗敏止癢功效的植物進行過大量的篩選工作,在此基礎上本論文從抗敏、止癢、修復途徑出發(fā),通過多種功效評價方法,從10種植物提取物中篩選出功效最佳的仙人掌、苦參和麥冬提取物,并進行組合提取,得到三種植物組合提取物。運用分析化學、藥理學和細胞生物學等手段闡明了組合提取物的抗敏活性物質(zhì)基礎、抗敏功效、安全性及其作用途徑。旨在開發(fā)出可應用于護膚品中的天然抗敏植物原料,也為其應用提供理論依據(jù)和數(shù)據(jù)支撐。主要研究內(nèi)容如下:將前期篩選的10種具有一定抗敏止癢功效的植物分別進行提取,得到10種提取物凍干粉。通過透明質(zhì)酸酶體外抑制試驗快速判定10種植物提取物抗敏效果;通過磷酸組胺致豚鼠皮膚瘙癢試驗判定10種植物提取物的止癢效果;通過皮膚刺激修復試驗判定10種植物提取物對皮膚的修復效果。從中篩選出透明質(zhì)酸酶活性抑制率最高的米邦塔仙人掌提取物,對磷酸組胺所致豚鼠皮膚瘙癢的抑制作用最強的苦參提取物,對皮膚刺激修復作用最佳的麥冬提取物。從抗敏、止癢、修復三個途徑入手解決皮膚過敏問題,將三種提取物組合提取,通過均勻設計試驗,優(yōu)選出最佳配比,即仙人掌:苦參:麥冬為7:1:3,為使工藝相對簡單和操作方便,利于實現(xiàn)工業(yè)化生產(chǎn),采用活性炭脫色法和樹脂吸附離子的方法進行工藝優(yōu)化研究,制備工藝如下:苦參和麥冬分別粗粉碎,過20目篩,取5g苦參和15g麥冬溶解于1100mL去離子水中,加熱至90℃攪拌提取1h,降溫至50℃,再加入35g仙人掌粉,保溫攪拌提取1h,冷卻至室溫。將提取液100目濾網(wǎng)粗濾后,5000 r?min-1離心10min,取上清液,上清液過0.8μm孔徑紙板精濾,得到澄清透亮精濾液。精濾液經(jīng)過活性炭脫色后(活性炭添加量為1.0%、脫色溫度為80℃、脫色時間為0.5h),過活化好的離子交換樹脂(陽離子001×7,陰離子樹脂D309),將去離子后的料液收集,濃縮,制備成凍干粉。測定凍干粉中的活性成分含量,其中多糖含量為634.6mg?g-1,蛋白含量為38.4mg?g-1,多酚含量為56.8mg?g-1,氧化苦參堿含量為4.98mg?g-1,苦參堿含量為3.12mg?g-1,多糖為組合提取物的主要成分。通過透明質(zhì)酸酶活性抑制試驗,驗證了同濃度下的組合提取物比單一的各組分提取物抗敏效果更佳。采用豚鼠磷酸組胺致癢反應試驗、低分子右旋糖酐誘發(fā)小鼠皮膚瘙癢試驗和小鼠腹腔毛細血管通透性試驗,綜合評價了組合提取物的抗敏功效。組合提取物高劑量與氟輕松組比較差異無統(tǒng)計學意義(p0.05),這表明組合提取物能減輕磷酸組胺對皮膚造成的瘙癢癥狀,具有一定的止癢功效;組合提取物3個劑量組都能減少低分子右旋糖酐致小鼠瘙癢發(fā)作次數(shù)、延長潛伏時間,高劑量組與模型組相比,差異有統(tǒng)計學意義(**p0.01);小鼠腹腔毛細血管通透性的影響實驗同樣表明,與模型組相比較,氟輕松軟膏、組合提取物高劑量組對降低毛細血管的通透性有顯著作用(*P0.05),能大大降低皮膚紅腫癥狀的表現(xiàn)程度,具有一定的抗敏功效。采用紅細胞溶血試驗、BCOP試驗和人體斑貼試驗對組合提取物進行安全性評價。紅細胞溶血試驗結果表明,組合提取物在一般化妝品配方中添加的濃度范圍(1%)下,溶血率均小于5%,說明組合提取物溫和無刺激,安全性高,低濃度和高濃度下都可在護膚品中安全添加;BCOP試驗和人體斑貼試驗可以看出,組合提取物對牛角膜和人體皮膚無任何刺激性,涂抹含有組合提取物的乳液后,還能減弱刺激源對皮膚所造成的損傷,在一定程度上保護表皮細胞膜結構完整性及屏障功能,提高皮膚的耐受性,真正具有修復受損肌膚減輕過敏癥狀的功效。同時也對組合提取物化妝品配方劑型的應用進行了研究,得到了不同劑型的護膚品的配方及制備工藝。在具有抗敏止癢功效及安全性保障前提之下,對組合提取物的抗敏途徑及作用機理進行了探究。通過DPPH自由基清除試驗,發(fā)現(xiàn)組合提取物能夠清除自由基,減弱自由基對人體嗜堿細胞和肥大細胞細胞膜的破壞,從而間接降低組胺的釋放量,起到抗敏止癢的功效。TEWL試驗結果表明,組合提取物能提升皮膚屏障功能,降低皮膚中水分的散失量,從而在一定程度上能提升皮膚的自我保護能力,降低敏感程度。經(jīng)成分分析,組合提取物中多糖成分主要起到減少水分散失,增強屏障功能的效果。建立了PMA和A23187聯(lián)合誘導KU812細胞組胺釋放模型,用來評價提取物對組胺釋放的影響,結果表明,經(jīng)PMA和A23187聯(lián)合誘導后,模型對照組組胺含量明顯升高,與空白對照組比較有顯著性差異(**p0.01)。KU812細胞經(jīng)組合提取物處理后,組胺含量明顯降低,高、中、低劑量組抑制率分別為45.23%、39.91%和33.45%,這表明組合提取物能通過抑制組胺的釋放量,從而減輕過敏癥狀,起到抗敏的功效。采用PMA聯(lián)合鈣離子載體誘導方法建立KU812細胞脫顆粒體外模型,結果顯示,經(jīng)PMA和A23187誘導后,KU812細胞釋放TNF-α、IL-1β、IL-6、IL-8促炎細胞因子,使用組合提取物干預后,高、中、低劑量的組合提取物均可顯著下調(diào)TNF-α、IL-1β、IL-6、IL-8的表達水平,且呈劑量依賴性。說明組合提取物可以通過抑制組胺和一系列促炎因子的釋放,從而減輕過敏反應中出現(xiàn)的皮膚病理損傷等癥狀。
[Abstract]:On the basis of this, we screened out the best efficacy of cactus, Radix Sophorae and ophiopogon extract from 10 kinds of plant extracts, and obtained three plants. Using analytical chemistry, pharmacology and cell biology, the anti sensitive active material basis, anti sensitization effect, safety and the way of action of the combined extracts were elucidated by means of analytical chemistry, pharmacology and cell biology. The purpose was to develop natural anti sensitive plant materials used in skin care products and provide theoretical basis and data support for their use. The following: 10 kinds of plants with certain anti sensitive antipruritic effect were extracted and 10 kinds of freeze-dried powder were obtained. Through in vitro inhibition test of hyaluronidase, the antipruritic effect of 10 kinds of plant extracts was determined, and the antipruritic effect of 10 plant extracts was determined through the test of the skin pruritus of guinea pigs induced by histamine phosphate; The skin irritation repair test was used to determine the effect of 10 plant extracts on the skin. The highest inhibitory rate of hyaluronidase activity was screened from the extract of the mica cactus, the best extract of the Sophora flavescens on the skin pruritus of guinea pigs caused by histamine phosphate, the best extract of Ophiopogon japonicus on the skin irritation and repair. To stop the itching and repair three ways to solve the skin allergy problem, three kinds of extracts were extracted, and the optimum ratio was selected through the uniform design test, namely, cactus: Radix Sophorae japonicus was 7:1:3, in order to make the process relatively simple and convenient to operate, it was beneficial to the industrialization of production, the method of activated carbon decolorization and resin adsorption ion were used. Process optimization research, preparation process as follows: Sophora Flavescentis and ophiopogon pulverized respectively, 20 mesh sieves, 5g Sophora Flavescentis and 15g Ophiopogon dissolved in 1100mL deionized water, heated to 90 centigrade to extract 1H, cooling to 50 centigrade, and then adding 35g cactus powder, insulating and stirring to extract 1H, cooling to room temperature. After extracting 100 mesh filter net, 5000 r? Min-1 centrifuged 10min, With the supernatant, the supernatant is filtered with 0.8 m pore size, and the clear and transparent filtrate is clarified. After the filtrate is decolorized by activated carbon (the amount of activated carbon is 1%, the decolorization temperature is 80, and the decolorization time is 0.5h), the activated ion exchange resin (cation 001 * 7, negative resin D309) is used to collect, concentrate and prepare the deionized liquid. The content of active ingredients in freeze-dried powder was determined. The content of the active ingredients in the freeze-dried powder was 634.6mg? G-1, the content of the protein was 38.4mg? G-1, the content of polyphenols was 56.8mg? G-1, the content of oxymatrine was 4.98mg? G-1, the content of matrine was 3.12mg? G-1, and the polysaccharide was the main component of the combination extract. The same concentration was verified through the inhibition test of hyaluronidase activity. The combined extract was better than the single component extract. The itching test of guinea pig histamine phosphate, the skin pruritus test of mice and the peritoneal capillary permeability test were induced by low molecular dextran, and the anti sensitive work effect of the combined extract was evaluated. The high dose of combined extract was compared with that of the fluororelaxed group. The difference was not statistically significant (P0.05), which showed that the combined extract could reduce the pruritus caused by histamine phosphate and had a certain antipruritic effect. The 3 dose groups of combined extract could reduce the number of itching attacks and prolong the latency time of mice with low molecular dextran, and the high dose group had a significant difference compared with the model group. (**p0.01) the effect of the capillary permeability of the mice abdominal cavity also showed that compared with the model group, the fluororelaxed ointment and the high dose group of the combined extract had significant effect on reducing the permeability of capillaries (*P0.05), which could greatly reduce the performance of red and swelling symptoms of the skin, and had certain anti sensitive efficacy. The erythrocyte hemolysis test, B COP test and human patch test were used to evaluate the safety of the combined extracts. The results of red blood cell hemolysis test showed that the hemolysis rate of the combined extract in the general cosmetic formula (1%) was less than 5%, indicating that the combined extract was mild and non irritating and safe, and it could be safe in the skin care products under low concentration and high concentration. BCOP test and human patch test show that the combination extract has no irritation to the bovine cornea and human skin. After smearing the emulsion containing combined extracts, it can also weaken the damage caused by the source of the stimulus, to a certain extent, protect the integrality and barrier function of the epidermal cell membrane and improve the tolerance of the skin. It has the effect of restoring the damaged skin and alleviated the allergy symptoms. At the same time, the application of the combination formulation of the combined extract cosmetics was also studied, and the formula and preparation process of the skin care products with different dosage forms were obtained. Under the premise of anti sensitive antipruritic effect and safety guarantee, the anti sensitive ways and mechanism of the combination extract were carried out. Through the DPPH free radical scavenging test, it is found that the combination extracts can remove free radicals and weaken the destruction of free radicals to the membrane of human basophilic cells and mast cells, thereby indirectly reducing the release of histamine and acting as anti sensitive antipruritic. The results of.TEWL test show that the combined extract can improve the skin barrier function and reduce the skin. The loss of moisture in the medium can improve the self protection ability and reduce the sensitivity of the skin to a certain extent. By the analysis of composition, the composition of polysaccharide in the combined extract plays a major role in reducing the loss of water and enhancing the barrier function. A combined PMA and A23187 induced KU812 cell histamine release model is established to evaluate the extract pairs. The effect of amine release showed that after the combined induction of PMA and A23187, the content of histamine in the model control group was significantly higher than that in the blank control group (**p0.01), the content of histamine decreased significantly, high, middle and low dosage group inhibition rates were 45.23%, 39.91% and 33.45% respectively after the combination of the blank control group. By inhibiting the release of histamine, it alleviated the allergy symptoms and played an antiallergic effect. The PMA combined with calcium ion carrier induction method was used to establish a KU812 cell degranulation model. The results showed that KU812 cells released TNF- alpha, IL-1 beta, IL-6, IL-8 proinflammatory cytokines after PMA and A23187 induction. The combination extract of low dose can significantly reduce the expression level of TNF- alpha, IL-1 beta, IL-6, IL-8, and is dose-dependent. It shows that the combined extract can inhibit the release of histamine and a series of pro-inflammatory factors, thus alleviated the symptoms of skin pathological injury in the allergic reaction.
【學位授予單位】：東北農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R283.6;R285.5

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相關期刊論文 前1條

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本文編號：1926617