山银花茎叶与花化学成分和抗炎活性比较研究

Comparative study of chemical constituents and anti-inflammatory activities of stems/leaves and flowers of Flos Lonicerae

目的比较山银花茎叶和花提取物的主要成分及抗炎活性差异。方法通过D101大孔树脂制备富含酚酸与三萜类成分的山银花茎叶及花提取部位,采用HPLC测定样品中6种酚酸和3种三萜含量。利用脂多糖(lipopolysaccharides,LPS)诱导RAW264.7细胞炎症模型,将细胞分为空白对照组、模型组、地塞米松组(32μg·mL^(-1))、山银花茎叶组(25、50、100、200μg·mL^(-1))、山银花组(29.5、59、118、236μg·mL^(-1)),共5组。空白对照组和模型组给予等量的无血清培养基,其余组以含各药物的无血清培养基预处理2 h后,加入浓度为1μg·mL^(-1)的LPS,继续培养24 h。采用Griess法检测一氧化氮(nitric oxide,NO)释放量。将小鼠随机分为空白对照组、模型组、地塞米松组(5 mg·kg^(-1))、山银花茎叶组(1.25、2.5、5 g·kg^(-1))和山银花组(1.25、2.5、5 g·kg^(-1)),连续灌胃给药7 d后,除空白对照组外,其余各组小鼠于右耳内外侧涂抹巴豆油(40μL)建立耳郭肿胀模型和左侧足趾皮下注射1%角叉菜胶(30μL)建立足趾肿胀模型。4 h后,测定各组小鼠耳郭和足趾肿胀度,计算肿胀抑制率,采用ELISA法测定小鼠足肿胀组织中白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis facor-α,TNF-α)和前列腺素E2(prostaglandin E2,PGE2)的含量。结果山银花茎叶中总酚酸(P<0.01)和隐绿原酸(P<0.05)含量高于山银花,总三萜(P<0.01)、灰毡毛忍冬皂苷乙(P<0.01)、灰毡毛忍冬皂苷甲(P<0.05)含量低于山银花。与空白对照组比较,模型组RAW264.7细胞NO水平、小鼠耳郭肿胀度和足趾肿胀度以及足肿胀组织中炎症因子(IL-1β、IL-6、TNF-α、PGE2)水平均显著升高(P<0.01)。与模型组比较,山银花茎叶组(50、100、200μg·mL^(-1))和山银花组(59、118、236μg·mL^(-1))RAW264.7细胞NO水平降低(P<0.01);山银花茎叶组(1.25、2.5、5 g·kg^(-1))和山银花组(1.25、2.5、5 g·kg^(-1))耳郭肿胀度明显降低(P<0.01),耳肿胀抑制率升高(P<0.01);山银花茎叶组(2.5、5 g·kg^(-1))和山银花组(2.5、5 g·kg^(-1))足趾肿胀度降低(P<0.01),足肿胀抑制率升高(P<0.01);山银花茎叶组(1.25、2.5、5 g·kg^(-1))和山银花组(1.25、2.5、5 g·kg^(-1))足肿胀组织中IL-1β、IL-6、TNF-α和PGE2的含量降低(P<0.01)。结论山银花茎叶和花具有相似的酚酸和三萜成分,但其含量存在显著差异。山银花茎叶和花提取物对急性炎症均有明显抑制作用,其机制可能与抑制外周TNF-α、IL-6、IL-1β、PGE2和NO炎症因子的表达有关。

Objective To compare the differences of the chemical constituents and anti-inflammatory activities of the stem/leaf and flower of Flos Lonicerae.Methods The stem/leaf and flower extracts of Flos Lonicerae rich in phenolic acid and triterpene constituents were prepared using D101 macroporous resin.HPLC method was applied to determine the content of six phenolic acids and three triterpenes in the samples.Lipopolysaccharides(LPS)was used to induce RAW264.7 inflammatory cell model.Cells were divided into five groups:blank control group,model group,dexamethasone group(32μg·mL^(-1)),Flos Lonicerae stem/leaf group(25,50,100,200μg·mL^(-1)),and Flos Lonicerae flower group(29.5,59,118,236μg·mL^(-1)).The blank control and model groups were given equal amounts of serum-free medium,while the remaining groups were pretreated with serum-free medium containing each drug for two hours.LPS at a concentration of 1 g·mL^(-1)was added after 2 h of pretreatment,and continued to culture for another 24 h.Griess assay was used to examine the nitric oxide(NO)concentration.Mice were randomly divided into blank control group,model group,dexamethasone group(5 mg·kg^(-1)),Flos Lonicerae stem/leaf group(1.25,2.5,5 g·kg^(-1)),and Flos Lonicerae flower group(1.25,2.5,5 g·kg^(-1)).After continuous intragastric administration for 7 d,except for the blank control group,the remaining groups of mice were coated with croton oil(40μL)on the inner and outer sides of the right ear to establish an ear swelling model,and subcutaneously injected with 1%carrageenan(30μL)on the left toe to establish a foot swelling model.The ear and foot swelling degrees of each group of mice were measured and the swelling inhibition rate was calculated four hours later.ELISA method was used to measure the interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),and prostaglandin E2(PGE2)content in foot swelling tissue.Results The content of the total phenolic acid(P<0.01)and cryptochlorogenic acid(P<0.05)in Flos Lonicerae stem/leaf was higher than that in Flos Lonicerae flower;while the content of total triterpenoids(P<0.01),macranthoidin B(P<0.01),and macranthoidin A(P<0.05)in Flos Lonicerae stem/leaf was lower than that in Flos Lonicerae flowers.Compared with the blank control group,the release level of NO in RAW264.7 cells,the swelling degree of mice ear and foot,and the levels of inflammatory factors(IL-1β,IL-6,TNF-α,and PGE2)in swelling tissues increased significantly in the model group(P<0.01).Compared with the model group,the release level of NO in RAW264.7 cells decreased in Flos Lonicerae stem/leaf group(50,100,200μg·mL^(-1))and Flos Lonicerae flower group(59,118,236μg·mL^(-1))(P<0.01);the Flos Lonicerae stem/leaf group(1.25,2.5,5 g·kg^(-1))and Flos Lonicerae flower group(1.25,2.5,5 g·kg^(-1))(P<0.01)showed a significant decrease in the ear swelling degree and an increase in inhibition rate;the Flos Lonicerae stem/leaf group(2.5,5 g·kg^(-1))and Flos Lonicerae flower group(2.5,5 g·kg^(-1))(P<0.01)showed a decrease in foot swelling degree and an increase in the inhibition rate;the content of IL-1β,IL-6,TNF-α,and PGE2 in foot swelling tissues decreased in Flos Lonicerae stem/leaf group(2.5,5 g·kg^(-1))and Flos Lonicerae flower group(2.5,5 g·kg^(-1))(P<0.05,P<0.01).Conclusion The stems/leaves and flowers of Flos Lonicerae have similar phenolic acid and triterpenoid constituents,but there are significant differences in their content.Extracts from Flos Lonicerae stems/leaves and flowers have significant inhibitory effects on acute inflammation,and its mechanism may be related to the inhibition of the expressions of peripheral inflammatory factors of TNF-α,IL-6,IL-1β,PGE2 and NO.