抗敏止痒植物组合提取物制备工艺、功效及作用途径研究

发布时间：2018-05-23 22:26

本文选题：组合提取物 + 抗敏止痒　；参考：《东北农业大学》2015年博士论文

【摘要】：本课题组前期对具有抗敏止痒功效的植物进行过大量的筛选工作,在此基础上本论文从抗敏、止痒、修复途径出发,通过多种功效评价方法,从10种植物提取物中筛选出功效最佳的仙人掌、苦参和麦冬提取物,并进行组合提取,得到三种植物组合提取物。运用分析化学、药理学和细胞生物学等手段阐明了组合提取物的抗敏活性物质基础、抗敏功效、安全性及其作用途径。旨在开发出可应用于护肤品中的天然抗敏植物原料,也为其应用提供理论依据和数据支撑。主要研究内容如下:将前期筛选的10种具有一定抗敏止痒功效的植物分别进行提取,得到10种提取物冻干粉。通过透明质酸酶体外抑制试验快速判定10种植物提取物抗敏效果;通过磷酸组胺致豚鼠皮肤瘙痒试验判定10种植物提取物的止痒效果;通过皮肤刺激修复试验判定10种植物提取物对皮肤的修复效果。从中筛选出透明质酸酶活性抑制率最高的米邦塔仙人掌提取物,对磷酸组胺所致豚鼠皮肤瘙痒的抑制作用最强的苦参提取物,对皮肤刺激修复作用最佳的麦冬提取物。从抗敏、止痒、修复三个途径入手解决皮肤过敏问题,将三种提取物组合提取,通过均匀设计试验,优选出最佳配比,即仙人掌:苦参:麦冬为7:1:3,为使工艺相对简单和操作方便,利于实现工业化生产,采用活性炭脱色法和树脂吸附离子的方法进行工艺优化研究,制备工艺如下:苦参和麦冬分别粗粉碎,过20目筛,取5g苦参和15g麦冬溶解于1100mL去离子水中,加热至90℃搅拌提取1h,降温至50℃,再加入35g仙人掌粉,保温搅拌提取1h,冷却至室温。将提取液100目滤网粗滤后,5000 r?min-1离心10min,取上清液,上清液过0.8μm孔径纸板精滤,得到澄清透亮精滤液。精滤液经过活性炭脱色后(活性炭添加量为1.0%、脱色温度为80℃、脱色时间为0.5h),过活化好的离子交换树脂(阳离子001×7,阴离子树脂D309),将去离子后的料液收集,浓缩,制备成冻干粉。测定冻干粉中的活性成分含量,其中多糖含量为634.6mg?g-1,蛋白含量为38.4mg?g-1,多酚含量为56.8mg?g-1,氧化苦参碱含量为4.98mg?g-1,苦参碱含量为3.12mg?g-1,多糖为组合提取物的主要成分。通过透明质酸酶活性抑制试验,验证了同浓度下的组合提取物比单一的各组分提取物抗敏效果更佳。采用豚鼠磷酸组胺致痒反应试验、低分子右旋糖酐诱发小鼠皮肤瘙痒试验和小鼠腹腔毛细血管通透性试验,综合评价了组合提取物的抗敏功效。组合提取物高剂量与氟轻松组比较差异无统计学意义(p0.05),这表明组合提取物能减轻磷酸组胺对皮肤造成的瘙痒症状,具有一定的止痒功效;组合提取物3个剂量组都能减少低分子右旋糖酐致小鼠瘙痒发作次数、延长潜伏时间,高剂量组与模型组相比,差异有统计学意义(**p0.01);小鼠腹腔毛细血管通透性的影响实验同样表明,与模型组相比较,氟轻松软膏、组合提取物高剂量组对降低毛细血管的通透性有显著作用(*P0.05),能大大降低皮肤红肿症状的表现程度,具有一定的抗敏功效。采用红细胞溶血试验、BCOP试验和人体斑贴试验对组合提取物进行安全性评价。红细胞溶血试验结果表明,组合提取物在一般化妆品配方中添加的浓度范围(1%)下,溶血率均小于5%,说明组合提取物温和无刺激,安全性高,低浓度和高浓度下都可在护肤品中安全添加;BCOP试验和人体斑贴试验可以看出,组合提取物对牛角膜和人体皮肤无任何刺激性,涂抹含有组合提取物的乳液后,还能减弱刺激源对皮肤所造成的损伤,在一定程度上保护表皮细胞膜结构完整性及屏障功能,提高皮肤的耐受性,真正具有修复受损肌肤减轻过敏症状的功效。同时也对组合提取物化妆品配方剂型的应用进行了研究,得到了不同剂型的护肤品的配方及制备工艺。在具有抗敏止痒功效及安全性保障前提之下,对组合提取物的抗敏途径及作用机理进行了探究。通过DPPH自由基清除试验,发现组合提取物能够清除自由基,减弱自由基对人体嗜碱细胞和肥大细胞细胞膜的破坏,从而间接降低组胺的释放量,起到抗敏止痒的功效。TEWL试验结果表明,组合提取物能提升皮肤屏障功能,降低皮肤中水分的散失量,从而在一定程度上能提升皮肤的自我保护能力,降低敏感程度。经成分分析,组合提取物中多糖成分主要起到减少水分散失,增强屏障功能的效果。建立了PMA和A23187联合诱导KU812细胞组胺释放模型,用来评价提取物对组胺释放的影响,结果表明,经PMA和A23187联合诱导后,模型对照组组胺含量明显升高,与空白对照组比较有显著性差异(**p0.01)。KU812细胞经组合提取物处理后,组胺含量明显降低,高、中、低剂量组抑制率分别为45.23%、39.91%和33.45%,这表明组合提取物能通过抑制组胺的释放量,从而减轻过敏症状,起到抗敏的功效。采用PMA联合钙离子载体诱导方法建立KU812细胞脱颗粒体外模型,结果显示,经PMA和A23187诱导后,KU812细胞释放TNF-α、IL-1β、IL-6、IL-8促炎细胞因子,使用组合提取物干预后,高、中、低剂量的组合提取物均可显著下调TNF-α、IL-1β、IL-6、IL-8的表达水平,且呈剂量依赖性。说明组合提取物可以通过抑制组胺和一系列促炎因子的释放,从而减轻过敏反应中出现的皮肤病理损伤等症状。
[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.
【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R283.6;R285.5

【参考文献】