黄花倒水莲3种成分含量测定及抗炎、调血脂作用研究

Experimental study on content determination of three components and antiinflammatory and hypolipidemic effects of Polygala fallax

目的 建立黄花倒水莲醇Polygala fallax Hemsl.提取物中3种成分的含量测定方法;探讨黄花倒水莲醇提取物抗炎、调血脂作用。方法 采用HPLC法测定黄花倒水莲醇提取物中3种成分——细叶远志皂苷、獐牙菜苦苷、芦丁的含量;建立二甲苯致小鼠耳肿胀、冰醋酸致小鼠腹腔毛细血管通透性增加、角叉菜胶致小鼠足跖肿胀炎症模型,评价黄花倒水莲醇提取物高、中、低剂量(40、20、10 g·kg^(-1))改善急、慢性炎症的药理作用;建立二甲苯致摘除双侧肾上腺小鼠耳肿胀、角叉菜胶致摘除双侧肾上腺小鼠足跖肿胀模型,判断其抗炎作用是否依赖下丘脑-垂体-肾上腺轴(HPAA)系统,试剂盒法测定足跖炎症组织丙二醛(MDA)、一氧化氮(NO)水平和超氧化物歧化酶(SOD)活性;采用高脂饲料喂食SD大鼠建立高脂大鼠模型,考察黄花倒水莲醇提取物高、中、低剂量(40、20、10 g·kg^(-1))对高脂大鼠血清总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)水平的影响。结果 HPLC法方法学考察各项指标符合规定,细叶远志皂苷、獐牙菜苦苷、芦丁的平均加样回收率均在97.13%~101.82%,RSD值均小于3.0%;黄花倒水莲醇提取物中细叶远志皂苷、獐牙菜苦苷、芦丁的质量分数分别为121.71、84.02、52.53μg·g^(-1)。与模型组比较,黄花倒水莲醇提取物高、中剂量对小鼠耳肿胀有明显的抑制作用(P<0.05、0.01),高、中、低剂量对小鼠腹腔毛细血管通透增加均有显著抑制作用(P<0.05、0.01),高、中、低剂量对角叉菜胶造成的小鼠足跖肿胀都有明显的抑制作用(P<0.05、0.01)。与模型组比较,在摘除双侧肾上腺小鼠中,黄花倒水莲醇提取物高、中剂量能显著抑制二甲苯致该模型小鼠耳肿胀(P<0.05、0.01),显著抑制角叉菜胶致该模型小鼠足跖肿胀(P<0.05、0.01);黄花倒水莲醇提取物高、中、低剂量均能显著降低足跖中MDA、NO水平(P<0.05、0.01),均能显著提高足跖中SOD活性(P<0.05、0.01)。ig高脂饲料28 d后,与模型组比较,黄花倒水莲醇提取物高、中、低剂量能显著降低大鼠血清的TC、TG和LDL-C水平(P<0.05)。结论 建立的HPLC含量测定方法专属性强、重复性好、准确度高,可用于黄花倒水莲醇提取物中细叶远志皂苷、獐牙菜苦苷、芦丁的含量测定;黄花倒水莲醇提取物具有抗炎活性,其抗炎作用的发挥不依赖HPAA系统,与减少MDA、NO水平,提高SOD活性有关;对高脂大鼠发挥明显的调血脂作用。

Objective To establish a method for the determination of three components in Polygala fallax alcohol extract(PFAE)to investigate the anti-inflammatory and hypolipidemic effects of PFAE.Methods The contents of three components(tenuifolin,swertiamarin and rutin)in PFAE were determined by HPLC.The inflammatory models such as xylene induced ear swelling in mice,acetic acid induced increased permeability of abdominal capillaries in mice,and carrageenan induced paw swelling inflammation in mice models were established to evaluate the pharmacological effects of high,medium and low doses(40,20,and 10 g·kg^(−1))PFAE on acute and chronic inflammation.The ear swelling model of mice with bilateral adrenalectomy induced by xylene and the paw swelling model of mice with bilateral adrenalectomy induced by carrageenan were established to determine whether its antiinflammatory effect depends on the hypothalamic-pituitary-adrenal axis(HPAA)system.The contents of malondialdehyde(MDA)and nitric oxide(NO)and the activity of superoxide dismutase(SOD)in the inflammatory tissue of the paw were determined by kits.A high-fat rat model was established by feeding SD rats with high-fat feed,and the effects of high,medium,and low doses(40,20,and 10 g·kg^(−1))of PFAE on serum total cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-C),and highdensity lipoprotein cholesterol(HDL-C)levels in hyperlipidemic rats were investigated.Results The indexes of methodological investigation were in accordance with the regulations.The average recovery rates of tenuifolin,swertiamarin and rutin were 97.13%—101.82%,and the RSD values were less than 3.0%.The mass fractions of tenuifolin,swertiamarin and rutin in the extract were 121.71,84.02 and 52.53μg·g^(−1),respectively.Compared with model group,except for the low dose group was not statistically significant,the high and middle dose groups of PFAE had significant inhibition on ear swelling in mice(P<0.05,0.01).The high,medium and low dose groups of PFAE had obvious inhibition on the permeability of abdominal capillary in mice(P<0.05,0.01),and the swelling of foot caused by carrageenan had significant inhibition(P<0.05,0.01).Compared with model group,in the mouse model of bilateral adrenalectomy,the high and medium dose groups of PFAE could significantly inhibit the ear swelling of the model mice induced by xylene(P<0.05,0.01),and can significantly inhibit carrageenan-induced paw swelling in the model mice(P<0.05,0.01),the high,medium and low dose groups of PFAE could significantly reduce the levels of MDA and NO in the toes,and significantly increase the content of SOD in the toes(P<0.05,0.01).After 28 days of high-fat diet,compared with the model group,high,medium,and low doses of PFAE significantly reduced the levels of TC,TG,and LDL-C in rat serum(P<0.05).Conclusion The content determination method established in this study has strong specificity,good repeatability and high accuracy,which can be used for the content determination of tenuifolin,swertiamarin and rutin in PFAE.PFAE has anti-inflammatory activity,and its anti-inflammatory effect may not depend on the hypothalamic pituitary adrenal axis(HPAA)system.At the same time,the anti-inflammatory mechanism of PFAE is related to reducing MDA,NO and increasing SOD activity.PFAE has obvious hypolipidemic effect on experimental hyperlipidemia.