紫花地丁总黄酮的提取工艺及抗炎作用研究  

Study on Extraction Process and Anti-inflammatory Effect of Total Flavonoids from Viola yedoensis

作　　者：陈志良 刘聪 廖自文 武玉娇 邢建康 龙小青 赵雯雪 王振[1] 赵鑫[1] 邵永斌[1] 罗燕[1] CHEN Zhiliang;LIU Cong;LIAO Ziwen;WU Yujiao;XING Jiankang;LONG Xiaoqing;ZHAO Wenxue;WANG Zhen;ZHAO Xin;SHAO Yongbin;LUO Yan(Shihezi University,Shihezi 832003,China)

机构地区：[1]石河子大学,石河子832003

出　　处：《中国畜牧兽医》2024年第11期5051-5063,共13页China Animal Husbandry & Veterinary Medicine

基　　金：石河子大学创新发展专项(CXFZ202209);石河子大学2023年国家级大学生创新创业训练计划项目(202310759010)。

摘　　要：【目的】利用响应面法优化紫花地丁总黄酮的提取工艺,采用液相色谱-串联质谱法(LC-MS/MS)鉴定化合物组成,并研究其抗炎活性。【方法】用芦丁标准品测定510 nm处的吸光度,以吸光度(Y)为纵坐标,以样品浓度(X)为横坐标,建立黄酮标准曲线。紫花地丁打成粉末过100目筛网、乙醇浸泡12 h、利用超声波仪超声提取,以紫花地丁粉末与乙醇的比例(料液比)、超声提取的时间(超声时间)、所用乙醇的浓度(乙醇浓度)为考察因素,以总黄酮提取量综合评分为响应值,结合Box-Behnken响应面法优化紫花地丁总黄酮提取工艺。利用D101型大孔树脂对提取的紫花地丁总黄酮进行纯化,通过LC-MS/MS法鉴定其化合物组成。60只6周龄健康昆明种小鼠随机均分成空白组、模型组、低剂量组(150 mg/kg)、中剂量组(300 mg/kg)、高剂量组(600 mg/kg)、左氧氟沙星组(5 mg/kg),除空白组外其余各组小鼠鼻腔滴注LPS(1 g/L,50μL/只)建立急性肺损伤(ALI)模型,造模成功后用药干预1周(1次/d,连续7 d);末次给药1 h后采集血液,采血后进行麻醉、处死,采集肺脏;HE染色观察肺损伤程度,ELISA法检测小鼠血清白细胞介素-1β(IL-1β)、IL-6、IL-10、肿瘤坏死因子-α(TNF-α)水平。【结果】料液比1∶20、提取时间22 min、乙醇浓度73%时紫花地丁总黄酮提取量最高,为25.134 mg/g。经鉴定,提取物中含37种黄酮(槲皮素、木犀草素、芹菜素等),少量异黄酮类、糖类、香豆素类、木脂素、单萜类、倍半萜类物质。病理切片显示,各给药组与模型组相比,肺组织出血点逐渐减少、面积缩小,有不同程度恢复:高剂量组与左氧氟沙星组的肺组织在颜色与形态上见轻微损伤:低、中剂量组小鼠肺组织相比模型组损伤程度减轻,但仍有一定程度水肿和部分出血点。ELISA结果表明,经给药后,低、中、高剂量组的TNF-α、IL-β、IL-6、IL-10水平相比模型组均有不同程度【Objective】The aim of this study was to optimize the extraction process of total flavonoids from Viola yedoensis by response surface methodology,identify the composition of compounds by liquid chromatography-tandem mass spectrometry(LC-MS/MS),and study its anti-inflammatory activity.【Method】The absorbance at 510 nm was determined by rutin standard,and the standard curve of flavonoids was established with absorbance(Y)as ordinate and sample concentration(X)as abscissa.The powder of Viola yedoensi was sieved through 100 mesh,soaked in ethanol for 12 h,and ultrasonically extracted by ultrasonic instrument.The ratio of Viola yedoensis powder to ethanol(solid-liquid ratio),ultrasonic extraction time(ultrasonic time),and ethanol concentration(ethanol concentration)were used as investigation factors.The extraction process of total flavonoids from Viola yedoensis was optimized by Box-Behnken response surface methodology with comprehensive score of total flavonoids extraction.The total flavonoids extracted from Viola yedoensis were purified by D101 macroporous resin,and the composition of the compounds was identified by LC-MS/MS method.Sixty 6-week-old healthy Kunming mice were randomly divided into blank,model,low dose(150 mg/kg),medium dose(300 mg/kg),high dose(600 mg/kg)and levofloxacin(5 mg/kg)groups.Except for blank group,LPS(1 g/L,50μL/mouse)was instilled into the nasal cavity to establish an acute lung injury(ALI)model.After successful modeling,the drug intervention was performed for 1 week(1 time/d,continuous 7 days).Blood was collected 1 h after the last administration,followed by anesthesia,execution,and lung collection.The degree of lung injury was observed by HE staining.The levels of interleukin-1β(IL-1β),IL-6,IL-10 and tumor necrosis factor-α(TNF-α)in serum of mice were detected by ELISA.【Result】When the solid-liquid ratio was 1∶20,the extraction time was 22 min,and the ethanol concentration was 73%,the extraction amount of total flavonoids was the highest,which was 25.134 mg/g.It was iden

关 键 词：紫花地丁 黄酮 响应面法 鉴定 急性肺损伤 抗炎 

分 类 号：S854.5[农业科学—临床兽医学]