白鲜皮多糖硫酸酯化修饰及抗银屑病作用研究
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篇名: 白鲜皮多糖硫酸酯化修饰及抗银屑病作用研究
TITLE:
摘要: 目的:对白鲜皮精制多糖(DDP-Ⅲ)进行硫酸酯化修饰,并比较其酯化修饰前后的结构特征及抗银屑病作用。方法:采用DEAE-52阴离子交换纤维素柱、Sephadex G-100葡聚糖凝胶柱等对白鲜皮多糖(DDP)进行分离纯化,得DDP-Ⅲ;用1-苯基-3-甲基-5-吡唑啉酮衍生化后,采用高效液相色谱法测定其单糖组成。用酯化试剂(无水吡啶+氯磺酸)对DDP-Ⅲ进行硫酸酯化修饰,得SDDP-Ⅲ;采用氯化钡-明胶浊度法测定其硫酸根取代度,并通过红外光谱、拉曼光谱、扫描电镜比较修饰前后的结构特征。将雄性ICR小鼠随机分为正常组、模型组、阳性组(雷公藤多苷,20 mg/kg)和DDP-Ⅲ/SDDP-Ⅲ低、中、高剂量组(均分别为56、112、224 mg/kg)。除正常组小鼠外敷凡士林外,其余各组小鼠均外敷咪喹莫特乳膏复制银屑病模型。各给药组小鼠灌胃相应药物溶液0.4 mL,正常组与模型组小鼠灌胃等体积水,每天1次,连续14 d。末次给药2 h后,采用酶联免疫吸附测定法检测各组小鼠血清白细胞介素17(IL-17)、IL-23含量,采用苏木精-伊红染色法观察其近尾部皮肤鳞片,并记录有颗粒层鳞片数。结果:DDP-Ⅲ由甘露糖、鼠李糖、葡萄糖醛酸、半乳糖醛酸、葡萄糖组成。SDDP-Ⅲ的硫酸根取代度为0.65。红外光谱、拉曼光谱分析结果显示,除与DDP-Ⅲ有相同的特征吸收峰外,SDDP-Ⅲ分别在1 255、823 cm-1和1 240、815 cm-1处有硫酸根基团的特征吸收峰。扫描电镜分析结果显示,DDP-Ⅲ呈片状,表面光滑、平整,排列紧密;SDDP-Ⅲ呈块状或颗粒状,有孔洞结构,排列疏松。动物实验结果显示,与正常组比较,模型组小鼠皮损表皮明显增厚,颗粒层明显减少,其血清IL-17、IL-23含量均显著升高,有颗粒层鳞片数显著减少(P<0.05或P<0.01);与模型组比较,各给药组小鼠上述症状均有不同程度的改善,阳性组、DDP-Ⅲ高剂量组以及SDDP-Ⅲ中、高剂量组小鼠血清IL-17、IL-23含量均显著下降,有颗粒层鳞片数均显著升高,且SDDP-Ⅲ中、高剂量组上述指标均显著优于DDP-Ⅲ同剂量组(P<0.05或P<0.01)。结论:DDP-Ⅲ含有甘露糖等5种单糖成分。DDP-Ⅲ、SDDP-Ⅲ均具有一定的抗银屑病作用,且经硫酸酯化修饰的SDDP-Ⅲ的作用更强;这种作用可能与抑制IL-23/IL-17信号通路有关。
ABSTRACT: OBJECTIVE: To conduct sulfated modification of polysaccharide from Dictamnus dasycarpus (DDP-Ⅲ), and to compare structure characteristics and anti-psoriasis activity of DDP-Ⅲ before and after sulfated modification. METHODS: DDP-Ⅲ was separated and purified with DEAE-52 anion exchange cellulose column and Sephadex G-100 column. After derived with 1-phenyl-3-methyl-5-pyrazolone, HPLC was used to determine the composition of its monosaccharide. SDDP-Ⅲ was synthesized using esterification reagent (anhydrous pyridine+chlorosulfonic acid) to modify DDP-Ⅲ. The degree of sulfate substitution was determined by barium chloride-gelatin turbidimetric method. The structures were compared by IR, Raman spectrum and SEM before and after modification. The male ICR mice were randomly divided into normal group, model group, positive group (tripterygium glycosides, 20 mg/kg) and DDP-Ⅲ/SDDP-Ⅲ low-dose, medium-dose and high-dose groups (56, 112, 224 mg/kg). Except that normal group was given vaseline for external use, and other groups were given Imiquimod cream for external use to induce psoriasis model. At the same time, administration groups were given relevant medicine intragastrically 0.4 mL, and both normal group and model group were given constant volume of water intragastrically, once a day, for consecutive 14 d. Two hours after last medication, the serum contents of IL-17 and IL-23 were determined by ELISA. The skin scales near the tail were observed by HE staining, and the number of scales with granular layer was recorded. RESULTS: DDP-Ⅲ was composed of mannose, rhamnose, glucuronic acid, galacturonic acid and glucose. The degree of sulfate substitution was 0.65 for SDDP-Ⅲ. IR and Raman spectrum showed that the characteristic absorption peaks of sulfate radical group appeared near 1 255 cm-1 and 823 cm-1, 1 240 cm-1 and 815 cm-1 for SDDP-Ⅲ, except for same characteristic absorption peak as DDP-Ⅲ. SEM analysis showed that DDP-Ⅲ was flaky, smooth and tightly arranged; SDDP-Ⅲ was massive or granular with porous structure and loose arrangement. Animal experiment showed that compared with normal group, the epidermis of skin lesion was significantly thickened and the granular layer was significantly reduced; serum contents of IL-17 and IL-23 were increased significantly, while the number of scales with granular layer was decreased significantly (P<0.05 or P<0.01). Compared with model group, above symptoms of administration groups were improved to different extent, and serum contents of IL-17 and IL-23 in positive group, DDP-Ⅲ high-dose groups, SDDP-Ⅲ medium-dose and high-dose groups were decreased significantly; the number of scales with granular layer was increased significantly, and above indexes of SDDP-Ⅲ medium-dose and high-dose groups were significantly better than corresponding DDP-Ⅲ group (P<0.05 or P<0.01). CONCLUSIONS: DDP-Ⅲ contains five monosaccharide components such as mannose, etc. Both DDP-Ⅲ and SDDP-Ⅲ possess anti- psoriasis effects, and SDDP-Ⅲ exhibits stronger anti-psoriasis effect than DDP-Ⅲ. Its mechanism may be associated with inhibiting IL-23/IL-17 signaling pathway.
期刊: 2019年第30卷第8期
作者: 张凯,张宇,王丽红,张义秀,王宇亮,赵统超,赵宏,王瑞瑞
AUTHORS: ZHANG Kai,ZHANG Yu,WANG Lihong,ZHANG Yixiu,WANG Yuliang,ZHAO Tongchao,ZHAO Hong,WANG Ruirui
关键字: 白鲜皮;多糖;硫酸酯化修饰;银屑病;炎症因子;信号通路
KEYWORDS: Dictamnus dasycarpus; Polysaccharides; Sulfated modification; Psoriasis; Inflammatory factor; Signaling pathway
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