摘要
目的设计优化3-OH根皮苷酶法制备工艺,探究3-OH根皮苷体内抗白癜风活性,为实现3-OH根皮苷的量产及白癜风防治提供科学依据。方法以湖北海棠嫩叶水提液中根皮苷为底物,在含抗坏血酸、多酚氧化酶(PPO)粗酶液的体系中制备3-OH根皮苷。以3-OH根皮苷的摩尔产率为指标,选取根皮苷浓度、pH、温度、反应时间、抗坏血酸浓度、酶浓度、搅拌速度等因素,通过单因素试验、普拉克特-伯曼(PB)试验、最陡爬坡试验、中心综合设计(CCD)-效应面法等方法,优化3-OH根皮苷制备工艺条件,反应体系放大至250 L验证制备工艺可行性。外用氢醌乳膏涂抹C57BL/6小鼠背部皮肤构建白癜风模型,连续造模75 d,造模第20天将小鼠随机分为空白组、模型组、阳性药8-甲氧补骨脂素组(8-mop,4.25 mg·kg^(-1))、3-OH根皮苷低、中、高剂量组(10、20、40 mg·kg^(-1)),灌胃给药55 d,各组小鼠每5d脱毛1次,同时对造模区域进行摄像,毛发脱色评分。给药结束后摘眼球取血,使用试剂盒检测小鼠血清酪氨酸酶(TYR)、胆碱酯酶(CHE)活性、丙二醛(MDA)含量。取小鼠背部皮损处皮肤,切片经苏木精-伊红(HE)染色,在光学显微镜下观察50个毛囊,对含有黑色素的毛囊进行计数。结果3-OH根皮苷的最佳制备工艺为:根皮苷1.70 mmol·L^(-1)、反应时间6.0 h、PPO浓度25.0 U·mL^(-1)。放大实验,反应液经大孔树脂除杂,浓缩,干燥得纯度为70.32%的3-OH根皮苷粗品334.80 g,提取率为8.37%,进一步分离纯化可得到纯度为98.10%的3-OH根皮苷纯品,得率为68.50%。体内活性实验表明,与模型组相比,3-OH根皮苷低、中、高剂量组小鼠毛发脱色情况显著改善,毛发脱色评分显著降低(P<0.05)、血清MDA含量、CHE活性显著降低(P<0.05),含黑色素毛囊个数、血清TYR活性显著上升(P<0.05)。结论可通过湖北海棠嫩叶水提液酶法高效提取制备3-OH根皮苷,且具有量化生产的可行性。3-OH根
OBJECTIVE To design and optimize the enzymatic preparation process of 3-hydroxyphloridzin and investigate the in vivo anti-vitiligo activity of 3-hydroxyphloridzin,thus to provide scientific basis for achieving the mass production of 3-hydroxyphloridzin and vitiligo prevention and treatment.METHODS 3-hydroxyphloridzin was prepared in a system containing L-ascorbic acid and crude polyphenol oxidase(PPO)solution,with phloridzin from the aqueous extract of young leaves of Malus hupehensis being the substrate.The molar yield of 3-hydroxyphloridzin was taken as the index,and factors including phloridzin concentration,pH,temperature,reaction time,L-ascorbic acid concentration,enzyme concentration,and stirring speed were selected to optimize the preparation process conditions of 3-hydroxyphloridzin by a complete randomized design,Placket-Burman(PB)test,path of steepest ascent method,and central composite design(CCD)-effect surface method.To verify the feasibility of the preparation process,the reaction system was scaled up to 250 L.Besides,Vitiligo model was constructed by applying hydroquinone cream to the back skin of C57BL/6 mice for 75 consecutive days,and the mice were randomly divided into blank group,model group,positive group(8-methoxypsoralen(8-mop),4.25 mg·kg^(-1)),and 3-hydroxyphloridzin low,medium,and high dosage group(10,20,and 40 mg·kg^(-1))on the 20th day of modeling,and were administered with 55 days of gavage.The mice in each group were depilated every 5 days,while the modeling area was videotaped and the hair decoloration was scored.At the end of the administration,blood was taken by removing the mice eyeballs,and the serum tyrosinase(TYR),cholinesterase(CHE)activity,and malondialdehyde(MDA)content of mice were detected using the kit.In addition,skin was taken from the dorsal lesions of mice and sections were stained with hematoxylin-eosin(HE),then under a light microscope,50 hair follicles were observed to count those containing melanin RESULTS The optimal preparation process of 3-hydroxyphloridzin
作者
雷晶鹏
汪鋆植
余海立
谢阳
邓张双
邓改改
杨梦瑶
许培勇
税先樊
LEI Jingpeng;WANG Junzhi;YU Haili;XIE Yang;DENG Zhangshuang;DENG Gaigai;YANG Mengyao;Xu Peiyong;SHUI Xianfan(Hubei Provincial Key Laboratory of Natural Products Research and Development,Three Gorges University,Yichang 443002,China;Angel Yeast Co.,Ltd,Yichang 443003,China;Hubei Province Badong County Guandukou Town Agricultural Service Center,Yichang 444399,China)
出处
《中国药学杂志》
CAS
CSCD
北大核心
2024年第11期1011-1022,共12页
Chinese Pharmaceutical Journal
基金
湖北农业土著品种基因资源多样性保护与可持续利用项目资助(HBGC-2021-167C)。
