柿鞣質(zhì)水提取液對白色念珠菌生物膜的體外抑制作用
發(fā)布時間：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見疾病,主要為白色念珠菌感染。白色念珠菌是一種廣泛寄居在宿主體內(nèi)的條件致病真菌,在機體正常免疫的情況下,一般不發(fā)病,但當(dāng)宿主免疫力降低,機體平衡系統(tǒng)被打破時,白色念珠菌則會引起機會性感染。目前由于免疫抑制劑、激素和抗生素在臨床上的廣泛應(yīng)用,機體菌群失調(diào)隨之越來越普遍,白色念珠菌機會性感染率日益上升,發(fā)病率逐年升高。由于傳統(tǒng)抗真菌藥物的使用以及白色念珠菌生物膜的形成,導(dǎo)致其耐藥性逐步增強,耐藥機制越來越復(fù)雜,從而增加了生物膜相關(guān)性感染的治療難度。目前能有效治療且不引起相關(guān)臟器損傷的抗真菌藥物極為有限,因而越來越多的研究者試圖從中草藥中尋找新型抗真菌藥物應(yīng)用于臨床,或與傳統(tǒng)抗真菌藥物聯(lián)合應(yīng)用,開辟除了傳統(tǒng)抗真菌藥物治療的另一片治療天地。鞣質(zhì)于植物界中廣泛存在,大部分生藥中均含有鞣質(zhì)類化合物,其具有抗病毒、抗菌、抗腫瘤和癌變等作用,且毒副作用小,易于提取。柿子中鞣質(zhì)含量較高,在我國被廣泛種植,產(chǎn)量豐富。因此本研究使用經(jīng)乙醇超聲提取而出的柿鞣質(zhì)水提取液,采用XTT還原比色法,觀察柿鞣質(zhì)水提取液對白色念珠菌生物膜的體外抑制作用,為臨床治療白色念珠菌病提供實驗依據(jù)。目的:在體外構(gòu)建白色念珠菌生物膜的模型,對其進(jìn)行抗真菌藥物敏感性實驗時發(fā)現(xiàn),以生物膜形式存在的白色念珠菌菌株對傳統(tǒng)用藥均表現(xiàn)出高度耐藥性,所以臨床治療白色念珠菌感染有很大難度。鞣質(zhì)能凝固微生物體內(nèi)的原生質(zhì)并作用于多種酶,能抑制多種病菌生長。因此本實驗研究了柿鞣質(zhì)水提取液對白色念珠菌生物膜的體外抑制作用,進(jìn)一步了解柿鞣質(zhì)水提取液對白色念珠菌生物膜的影響。方法:1柿鞣質(zhì)的提取對蓋柿全柿果實打碎研磨,乙醇浸泡后進(jìn)行超聲提取,濃縮液經(jīng)蒸餾水稀釋,AB-8大孔樹脂吸附除糖,旋轉(zhuǎn)蒸發(fā),使用聚砜中空纖維超濾膜超濾,獲得的柿鞣質(zhì)溶液的分子量為10000,利用Folin-Denis法測定其鞣質(zhì)的含量。取全柿鞣質(zhì)水提取液,使用0.22μm濾菌器過濾除菌,4℃避光保存?zhèn)溆谩?白色念珠菌生物膜的制備將白色念珠菌接種于YPD培養(yǎng)基中,30℃搖床過夜培養(yǎng),離心采集菌體,PBS洗滌菌體2~3次,以RPMI-1640培養(yǎng)基配成濃度為1x106cfu·m L-1的標(biāo)準(zhǔn)菌液。將100μL標(biāo)準(zhǔn)菌液接種于96孔培養(yǎng)板中,另加RPMI-1640培養(yǎng)基補足體積至200μL,37℃靜置培養(yǎng)24h,形成生物膜結(jié)構(gòu)。倒置顯微鏡下觀察白色念珠菌生物膜的形成。3柿鞣質(zhì)水提取液對白色念珠菌生物膜的影響采用XTT還原比色法觀察柿鞣質(zhì)水提取液對白色念珠菌成膜的影響,評估不同濃度的柿鞣質(zhì)水提取液對不同時間白色念珠菌的藥敏性,倒置顯微鏡觀察藥物作用后白色念珠菌生物膜的形態(tài)學(xué)變化。結(jié)果:1柿鞣質(zhì)水提取液的鞣質(zhì)含量為7.42 mg/m L。2柿鞣質(zhì)水提取液對白色念珠菌生物膜形成以及菌絲態(tài)細(xì)胞的生長具有明顯抑制作用(P0.05),其作用與藥物濃度呈正相關(guān),其中3.7100mg/m L和1.8550 mg/m L柿鞣質(zhì)水提取液對白色念珠菌生物膜形成的抑制率超過90%。3柿鞣質(zhì)水提取液對不同時間白色念珠菌的藥物敏感性不完全一致,高濃度柿鞣質(zhì)水提取液(3.7100 mg/m L和1.8550 mg/m L)對不同時間白色念珠菌生物膜的抑制率均大于90%,中低濃度柿鞣質(zhì)水提取液(0.9275 mg/m L,0.4638 mg/m L,0.2319 mg/m L,0.1159 mg/m L)同一濃度的條件下,隨著白色念珠菌生物膜的成熟,柿鞣質(zhì)水提取液對其的抑制率有下降趨勢(P0.05)。結(jié)論:1柿鞣質(zhì)水提取液對白色念珠菌生物膜形成以及菌絲態(tài)細(xì)胞的生長具有明顯抑制作用(P0.05),其作用與藥物濃度呈正相關(guān)。2柿鞣質(zhì)水提取液對不同時間白色念珠菌的藥物敏感性不完全一致,低濃度柿鞣質(zhì)水提取液同一濃度的條件下,隨著白色念珠菌生物膜的成熟,柿鞣質(zhì)水提取液對其的抑制率有下降趨勢。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R781.5
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