沙棘果不同极性部位体外及体内抗氧化作用研究

In Vitro and in Vivo Antioxidant Effect of Different Polar Parts of Seabuckthorn

目的 探究沙棘果不同极性部位体外抗氧化能力及体内抗氧化作用,明确有效部位,为拓展沙棘果研发利用、优化制备工艺提供依据。方法 以沙棘果为原料,95%乙醇提取后依次用石油醚、乙酸乙酯、正丁醇进行萃取,得到不同极性部位提取物,采用清除1,1-二苯基-2-三硝基苯肼自由基(DPPH·)法、超氧阴离子自由基(O_(2)·^(-))法及羟自由基(·OH)法,比较不同极性部位体外抗氧化作用。将110只小鼠随机分为空白组(Control),模型组(Model),沙棘果乙酸乙酯层低剂量组(YD,0.015 g/kg)、沙棘果乙酸乙酯层中剂量组(YZ,0.045 g/kg)、沙棘果乙酸乙酯层高剂量组(YG,0.135 g/kg),沙棘果正丁醇层低剂量组(ZD,0.04 g/kg)、沙棘果正丁醇层中剂量组(ZZ,0.13 g/kg)、沙棘果正丁醇层高剂量组(ZG,0.39 g/kg)及沙棘果醇提取物低剂量组(SD,0.16 g/kg)、沙棘果醇提取物中剂量组(SZ,0.5 g/kg)、沙棘果醇提取物高剂量组(SG,1.5 g/kg),每组10只。除Control外,其余各组每日腹腔注射浓度为0.05 g/mL D-半乳糖(D-gal)溶液,剂量为10 g/0.1 mL,Control注入等量生理盐水,连续60 d。造模第11天开始灌胃给药,给药组分别灌胃沙棘果不同极性部位提取物低、中、高剂量,Control与Model灌胃蒸馏水。末次给药后,考察小鼠体质量变化、脏器指数,对比各组小鼠的血清抗氧化能力,包括血清总抗氧化能力(total antioxidant capacity,T-AOC)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-PX)、超氧化物歧化酶(superoxide dismutase,SOD)以及丙二醛(malondialdehyde,MDA)水平,观察心脏、肺脏、肾脏的病理变化。结果 沙棘果不同极性部位均可清除DPPH·、O_(2)·~-、·OH,具有体外抗氧化活性,其中沙棘果醇提取部位对DPPH·清除率高达76.09%,清除效果最强;体内实验结果显示,YG、ZZ、ZG、SZ、SG血清GSH-PX、SOD、T-AOC水平增高(P<0.05),且均可抑制MDA的积累(P<0.01)。其中SG血清GSH-PX抗氧化水平为(872.73±165.39)U/mL、SOD为(180.02±12.33)U/mL、T-AOC为(9.25±1.45)U/mL,高于Model(P<0.01),MDA水平为(4.93±2.07)nmol/mL,相比于Model有所下降(P<0.05)。HE染色结果表明,沙棘果不同极性部位对心脏、肺脏、肾脏组织细胞及结构损伤具有较好的修复作用。结论 沙棘果不同极性部位均具有一定的抗氧化作用,其中沙棘果醇提取物抗氧化能力最强,其余各部位抗氧化能力与极性呈正相关。

Objective To investigate the antioxidant capacity in vitro and antioxidant effect in vivo of different polar parts of seabuckthorn,to clarify the effective parts,and to provide bases for the development-utilization expansion and manufacture technique optimization of seabuckthorn.Methods Using seabuckthorn as the raw material,95%ethanol extraction was followed by petroleum ether,ethyl acetate and n-butanol extraction in turns to obtain extracts from different polar parts.The antioxidant effect in vitro of different polar parts were compared through the methods of clearing 1,1-diphenyl-2-trinitrophenylhydrazine radicals(DPPH·),superoxide anion radicals(O_(2)·^(-)),and hydroxyl radicals(·OH).110 mice were randomly divided into blank group(CON),model group(MOD),low-dose group of sea buckthorn ethyl acetate layer(YD,0.015 g/kg),medium-dose group of sea buckthorn ethyl acetate layer(YZ,0.045 g/kg),high-dose group of sea buckthorn ethyl acetate layer(YG,0.135 g/kg),low-dose group of sea buckthorn n-butanol layer(ZD,0.04 g/kg),medium-dose group of sea buckthorn n-butanol layer(ZZ,0.13 g/kg),high-dose group of sea buckthorn n-butanol layer(ZG,0.39 g/kg),and low-dose group of sea buckthorn alcohol extract(SD,0.16 g/kg),medium-dose group of sea buckthorn alcohol extract(SZ,0.5 g/kg),and high-dose group of sea buckthorn alcohol extract(SG,1.5 g/kg),with 10 mice in each group.Excepting for CON,the other groups were injected intraperitoneally with a concentration of 0.05 g/mL D-galactose(D-gal)solution at a dose of 10 g/0.1 mL.CON was injected with an equal amount of physiological saline for 60 consecutive days.Starting from the 11th day of modeling,the drug was administered by gavage,with low,medium,and high doses of extracts from different polar parts of sea buckthorn,as well as distilled water for CON and MOD.After the last administration,the body weight and organ index of mice were examined,and the serum antioxidant capacity was compared in mice from each group,including total antioxidant capacity(T-AOC),glutathione peroxidase(GSH-PX),superoxide dismutase(SOD),and malondialdehyde(MDA)levels.The pathological changes of heart,lung and kidney were observed.Results All different polar parts of seabuckthorn could remove DPPH·,O_(2)·-and·OH,and also were antioxidant active in vitro.Among them,the alcoholic extract part of seabuckthorn had the strongest effect with DPPH·clearance rate up to 76.09%.The results of the experiments in vivo showed that the activity of serum GSH-PX,SOD and T-AOC were significantly increased in YG,ZZ,ZG,SZ,SG(P<0.05),and all of which were effective in inhibiting the accumulation of MDA(P<0.01).Among them,the antioxidant levels of serum GSH-PX,serum SOD and T-AOC from seabuckthorn alcohol extract were respectively(872.73±165.39)U/mL,(180.02±12.33)U/mL and(9.25±1.45)U/mL in SG,which were higher than MOD(P<0.01).The MDA level was(4.93±2.07)nmol/mL,which was lower than MOD(P<0.05).The results of HE stains showed that every polar part of seabuckthorn had significant repairing effects on histiocytes and structural damage of heart,lung and kidney.Conclusion All different polar parts of seabuckthorn have certain antioxidant effects,among which,the alcoholic extract of seabuckthorn has the strongest antioxidant capacity,and the activity of each extract part is positively correlated with polarity.