柿鞣质水提取液对白色念珠菌生物膜的体外抑制作用
发布时间：2018-07-29 12:11
【摘要】：口腔念珠菌病是口腔粘膜真菌性感染的常见疾病,主要为白色念珠菌感染。白色念珠菌是一种广泛寄居在宿主体内的条件致病真菌,在机体正常免疫的情况下,一般不发病,但当宿主免疫力降低,机体平衡系统被打破时,白色念珠菌则会引起机会性感染。目前由于免疫抑制剂、激素和抗生素在临床上的广泛应用,机体菌群失调随之越来越普遍,白色念珠菌机会性感染率日益上升,发病率逐年升高。由于传统抗真菌药物的使用以及白色念珠菌生物膜的形成,导致其耐药性逐步增强,耐药机制越来越复杂,从而增加了生物膜相关性感染的治疗难度。目前能有效治疗且不引起相关脏器损伤的抗真菌药物极为有限,因而越来越多的研究者试图从中草药中寻找新型抗真菌药物应用于临床,或与传统抗真菌药物联合应用,开辟除了传统抗真菌药物治疗的另一片治疗天地。鞣质于植物界中广泛存在,大部分生药中均含有鞣质类化合物,其具有抗病毒、抗菌、抗肿瘤和癌变等作用,且毒副作用小,易于提取。柿子中鞣质含量较高,在我国被广泛种植,产量丰富。因此本研究使用经乙醇超声提取而出的柿鞣质水提取液,采用XTT还原比色法,观察柿鞣质水提取液对白色念珠菌生物膜的体外抑制作用,为临床治疗白色念珠菌病提供实验依据。目的:在体外构建白色念珠菌生物膜的模型,对其进行抗真菌药物敏感性实验时发现,以生物膜形式存在的白色念珠菌菌株对传统用药均表现出高度耐药性,所以临床治疗白色念珠菌感染有很大难度。鞣质能凝固微生物体内的原生质并作用于多种酶,能抑制多种病菌生长。因此本实验研究了柿鞣质水提取液对白色念珠菌生物膜的体外抑制作用,进一步了解柿鞣质水提取液对白色念珠菌生物膜的影响。方法:1柿鞣质的提取对盖柿全柿果实打碎研磨,乙醇浸泡后进行超声提取,浓缩液经蒸馏水稀释,AB-8大孔树脂吸附除糖,旋转蒸发,使用聚砜中空纤维超滤膜超滤,获得的柿鞣质溶液的分子量为10000,利用Folin-Denis法测定其鞣质的含量。取全柿鞣质水提取液,使用0.22μm滤菌器过滤除菌,4℃避光保存备用。2白色念珠菌生物膜的制备将白色念珠菌接种于YPD培养基中,30℃摇床过夜培养,离心采集菌体,PBS洗涤菌体2~3次,以RPMI-1640培养基配成浓度为1x106cfu·m L-1的标准菌液。将100μL标准菌液接种于96孔培养板中,另加RPMI-1640培养基补足体积至200μL,37℃静置培养24h,形成生物膜结构。倒置显微镜下观察白色念珠菌生物膜的形成。3柿鞣质水提取液对白色念珠菌生物膜的影响采用XTT还原比色法观察柿鞣质水提取液对白色念珠菌成膜的影响,评估不同浓度的柿鞣质水提取液对不同时间白色念珠菌的药敏性,倒置显微镜观察药物作用后白色念珠菌生物膜的形态学变化。结果:1柿鞣质水提取液的鞣质含量为7.42 mg/m L。2柿鞣质水提取液对白色念珠菌生物膜形成以及菌丝态细胞的生长具有明显抑制作用(P0.05),其作用与药物浓度呈正相关,其中3.7100mg/m L和1.8550 mg/m L柿鞣质水提取液对白色念珠菌生物膜形成的抑制率超过90%。3柿鞣质水提取液对不同时间白色念珠菌的药物敏感性不完全一致,高浓度柿鞣质水提取液(3.7100 mg/m L和1.8550 mg/m L)对不同时间白色念珠菌生物膜的抑制率均大于90%,中低浓度柿鞣质水提取液(0.9275 mg/m L,0.4638 mg/m L,0.2319 mg/m L,0.1159 mg/m L)同一浓度的条件下,随着白色念珠菌生物膜的成熟,柿鞣质水提取液对其的抑制率有下降趋势(P0.05)。结论:1柿鞣质水提取液对白色念珠菌生物膜形成以及菌丝态细胞的生长具有明显抑制作用(P0.05),其作用与药物浓度呈正相关。2柿鞣质水提取液对不同时间白色念珠菌的药物敏感性不完全一致,低浓度柿鞣质水提取液同一浓度的条件下,随着白色念珠菌生物膜的成熟,柿鞣质水提取液对其的抑制率有下降趋势。
[Abstract]:Oral candidiasis is a common disease of fungal infection in oral mucous membrane, mainly Candida albicans infection. Candida albicans is a pathogenic fungus which is widely distributed in the host. In the normal immunity of the body, it usually does not occur. But when the host's immunity is reduced and the body's balance system is broken, Candida albicans will be Due to the widespread application of immunosuppressants, hormones and antibiotics in the clinic, the disorder of organism flora is becoming more and more common. The opportunistic infection rate of Candida albicans is increasing and the incidence is increasing year by year. Because of the use of traditional antifungal drugs and the formation of Candida albicans biofilm, the drug resistance of Candida albicans is caused. More and more researchers are trying to find new antifungal drugs from Chinese herbal medicine for clinical use, or with traditional antifungal drugs, and therefore more and more researchers are trying to find new antifungal drugs from Chinese herbal medicine, or to traditional antifungal drugs. Combined application, opening up another field of treatment in addition to traditional antifungal drugs. Tannins exist widely in the plant community. Most of the raw drugs contain tannin compounds, which have antiviral, antibacterial, anti-tumor and cancerous effects, and have small toxic and side effects and are easy to extract. In this study, the extraction of tannin water from persimmon extracted by ethanol was used in this study. The XTT reduction colorimetric method was used to observe the inhibitory effect of persimmon tannin water extract on Candida albicans biofilm in vitro, and to provide experimental basis for clinical treatment of Candida albicans. It is found that Candida albicans in the form of biofilm are highly resistant to traditional drugs, so it is very difficult to treat Candida albicans in clinical. Tannin can solidify the protoplasm of microbes and act on many kinds of enzymes and inhibit the growth of many kinds of bacteria. The effect of persimmon tannin water extract on Candida albicans biofilm in vitro was studied, and the effect of tannin extract of persimmon on Candida albicans biofilm was further understood. Method: the extraction of 1 persimmon tannin was broken and lapping to the fruit of persimmon persimmon fruit. After ethanol immersion, ultrasonic extraction was carried out after ethanol immersion. The concentration solution was diluted by distilled water, and AB-8 macroporous resin was adsorbed. Sugar, rotary evaporation and ultrafiltration of polysulfone hollow fiber ultrafiltration membrane, the molecular weight of the tannin solution of persimmon was 10000. The content of tannin was determined by Folin-Denis method. The whole persimmon tannin water extract was extracted, the 0.22 mu m filter was used to filter the bacteria, and the preparation of the.2 Candida albicans biofilm at 4 degrees centigrade was inoculated to YPD In the medium, 30 degrees centigrade rocking bed for overnight culture, centrifugation to collect bacteria, PBS scrubbing bacteria 2~3 times, the standard bacteria solution with a concentration of 1x106cfu. M L-1 with RPMI-1640 medium. The standard bacteria solution of 100 mu L was inoculated into 96 hole culture plate, the volume of RPMI-1640 medium was added to 200 micron, and 24h was incubated at 37 DEG C, and the biofilm structure was formed. Inverted microscope was used to form a biofilm structure. The effect of.3 persimmon water extract on Candida albicans biofilm by observing the formation of Candida albicans biofilm, the effect of tannin extract of persimmon on the membrane of Candida albicans was observed by XTT reduction colorimetry, and the drug sensitivity of different concentration of persimmon tannin extract to albicans at different time was evaluated by inverted microscope to observe the drug The morphological changes of the biofilm of Candida albicans after action. Results: the tannin content of 1 persimmon tannin extract is 7.42 mg/m L.2 persimmon water extract, which has obvious inhibitory effect on the formation of Candida albicans biofilm and the growth of mycelial cells (P0.05), and its effect is positively related to the drug concentration, of which 3.7100mg/m L and 1.8550 mg/m L The inhibition rate of persimmon tannin water extraction on the formation of Candida albicans biofilm exceeded 90%.3 persimmon tannin extract to different drug sensitivity of Candida albicans at different time. The inhibition rate of high concentration persimmon tannin water extract (3.7100 mg/m L and 1.8550 mg/m L) was greater than 90%, low medium concentration at different time. Under the same concentration of 0.9275 mg/m L, 0.4638 mg/m L, 0.2319 mg/m L and 0.1159 mg/m L in persimmon, the inhibition rate of persimmon tannin water extract decreased (P0.05). Conclusion: 1 persimmon tannin extraction solution to Candida albicans biofilm formation and mycelial cell growth The effect was obviously inhibited (P0.05), and its effect was positively related to the drug concentration..2 persimmon water extract was not identical to the drug sensitivity of Candida albicans at different time. Under the same concentration of low concentration persimmon tannin water extract, with the maturation of the Candida albicans biofilm, the inhibition rate of the tannin water extract of persimmon was decreased. Trend.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.5
[Abstract]:Oral candidiasis is a common disease of fungal infection in oral mucous membrane, mainly Candida albicans infection. Candida albicans is a pathogenic fungus which is widely distributed in the host. In the normal immunity of the body, it usually does not occur. But when the host's immunity is reduced and the body's balance system is broken, Candida albicans will be Due to the widespread application of immunosuppressants, hormones and antibiotics in the clinic, the disorder of organism flora is becoming more and more common. The opportunistic infection rate of Candida albicans is increasing and the incidence is increasing year by year. Because of the use of traditional antifungal drugs and the formation of Candida albicans biofilm, the drug resistance of Candida albicans is caused. More and more researchers are trying to find new antifungal drugs from Chinese herbal medicine for clinical use, or with traditional antifungal drugs, and therefore more and more researchers are trying to find new antifungal drugs from Chinese herbal medicine, or to traditional antifungal drugs. Combined application, opening up another field of treatment in addition to traditional antifungal drugs. Tannins exist widely in the plant community. Most of the raw drugs contain tannin compounds, which have antiviral, antibacterial, anti-tumor and cancerous effects, and have small toxic and side effects and are easy to extract. In this study, the extraction of tannin water from persimmon extracted by ethanol was used in this study. The XTT reduction colorimetric method was used to observe the inhibitory effect of persimmon tannin water extract on Candida albicans biofilm in vitro, and to provide experimental basis for clinical treatment of Candida albicans. It is found that Candida albicans in the form of biofilm are highly resistant to traditional drugs, so it is very difficult to treat Candida albicans in clinical. Tannin can solidify the protoplasm of microbes and act on many kinds of enzymes and inhibit the growth of many kinds of bacteria. The effect of persimmon tannin water extract on Candida albicans biofilm in vitro was studied, and the effect of tannin extract of persimmon on Candida albicans biofilm was further understood. Method: the extraction of 1 persimmon tannin was broken and lapping to the fruit of persimmon persimmon fruit. After ethanol immersion, ultrasonic extraction was carried out after ethanol immersion. The concentration solution was diluted by distilled water, and AB-8 macroporous resin was adsorbed. Sugar, rotary evaporation and ultrafiltration of polysulfone hollow fiber ultrafiltration membrane, the molecular weight of the tannin solution of persimmon was 10000. The content of tannin was determined by Folin-Denis method. The whole persimmon tannin water extract was extracted, the 0.22 mu m filter was used to filter the bacteria, and the preparation of the.2 Candida albicans biofilm at 4 degrees centigrade was inoculated to YPD In the medium, 30 degrees centigrade rocking bed for overnight culture, centrifugation to collect bacteria, PBS scrubbing bacteria 2~3 times, the standard bacteria solution with a concentration of 1x106cfu. M L-1 with RPMI-1640 medium. The standard bacteria solution of 100 mu L was inoculated into 96 hole culture plate, the volume of RPMI-1640 medium was added to 200 micron, and 24h was incubated at 37 DEG C, and the biofilm structure was formed. Inverted microscope was used to form a biofilm structure. The effect of.3 persimmon water extract on Candida albicans biofilm by observing the formation of Candida albicans biofilm, the effect of tannin extract of persimmon on the membrane of Candida albicans was observed by XTT reduction colorimetry, and the drug sensitivity of different concentration of persimmon tannin extract to albicans at different time was evaluated by inverted microscope to observe the drug The morphological changes of the biofilm of Candida albicans after action. Results: the tannin content of 1 persimmon tannin extract is 7.42 mg/m L.2 persimmon water extract, which has obvious inhibitory effect on the formation of Candida albicans biofilm and the growth of mycelial cells (P0.05), and its effect is positively related to the drug concentration, of which 3.7100mg/m L and 1.8550 mg/m L The inhibition rate of persimmon tannin water extraction on the formation of Candida albicans biofilm exceeded 90%.3 persimmon tannin extract to different drug sensitivity of Candida albicans at different time. The inhibition rate of high concentration persimmon tannin water extract (3.7100 mg/m L and 1.8550 mg/m L) was greater than 90%, low medium concentration at different time. Under the same concentration of 0.9275 mg/m L, 0.4638 mg/m L, 0.2319 mg/m L and 0.1159 mg/m L in persimmon, the inhibition rate of persimmon tannin water extract decreased (P0.05). Conclusion: 1 persimmon tannin extraction solution to Candida albicans biofilm formation and mycelial cell growth The effect was obviously inhibited (P0.05), and its effect was positively related to the drug concentration..2 persimmon water extract was not identical to the drug sensitivity of Candida albicans at different time. Under the same concentration of low concentration persimmon tannin water extract, with the maturation of the Candida albicans biofilm, the inhibition rate of the tannin water extract of persimmon was decreased. Trend.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.5
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