绞股蓝籽油食品安全毒理学评价及抗衰老研究

Food safety toxicology evaluation and anti-aging analysis of Gynostemma pentaphyllum seed oil

【目的】对绞股蓝籽油(GPSO)进行毒理学分析与评价,为绞股蓝籽油的生物学功能研究及其新资源的开发提供科学依据。【方法】通过小鼠急性经口毒性试验、小鼠骨髓嗜多染红细胞微核试验和小鼠精子畸形试验,对GPSO进行食品安全毒理学评估。在小鼠急性经口毒性试验中,于小鼠禁食16h后,各试验组分别经口一次性灌胃0,4.6,10.0及21.5g/kg GPSO,连续观察14d,记录小鼠的饮食、运动、排泄、中毒表现及死亡情况;在小鼠骨髓嗜多染红细胞微核试验中,试验组小鼠以2.5,5.0,10.0g/kg的剂量分别灌胃GPSO,空白对照组以环磷酰胺为阳性药物腹腔注射给药,每天1次,持续5d,处死后取骨髓制作骨髓细胞涂片,镜下观察并计算微核率;在小鼠精子畸形试验中,以性成熟雄性小鼠为试验动物,其中试验组小鼠以2.5,5.0,10.0g/kg的剂量分别灌胃GPSO,空白对照组以环磷酰胺为阳性药物腹腔注射给药,每天1次,持续5d,在最后1次灌胃7,14,21,28和35d后分别处死小鼠,取附睾制作精子涂片,镜检并计算精子畸形率。通过注射D-半乳糖建立衰老模型,同时以绞股蓝籽油作为抗衰老试验药物,按2.0,3.0,4.0mL/kg的剂量进行灌胃给药,60d后检测小鼠体质量、各脏器指数及血清中总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)、高密度脂蛋白(HDL)浓度,以及小鼠肝脏、脑组织中超氧化物歧化酶(SOD)活性、总抗氧化能力(T-AOC)、过氧化氢酶(CAT)活性及丙二醛(MDA)含量,并观察小鼠肝脏组织的病理变化。【结果】绞股蓝籽油对雌雄小鼠急性经口半数致死剂量LD50值大于10.0g/kg,依据急性毒性剂量分级标准属于实际无毒物质;小鼠骨髓嗜多染红细胞微核试验表明,绞股蓝籽油3个剂量组中雌雄小鼠骨髓嗜多染红细胞微核率为2.0‰~3.8‰,与空白对照组相比无显著差异(P>0.05),证明绞股蓝籽油不会对小鼠骨髓嗜多染红细胞微核数造成显著影响;小鼠精子畸形试验表明,绞股蓝籽油低、中、高剂量组与空白对照组之间无显著差异(P>0.05),表明绞股蓝籽油对小鼠精子无致畸变作用。抗衰老试验表明,与空白对照组相比,衰老模型组小鼠TC、TG、LDL浓度均显著升高(P<0.05),HDL浓度显著降低(P<0.05),肝、脑组织中SOD活性和T-AOC能力降低(P<0.05),MDA含量升高(P<0.05),证实衰老小鼠建模成功;绞股蓝籽油低、中、高剂量组均可缓解因注射D-半乳糖导致的小鼠饮食量下降、毛皮光泽度变差、行动缓慢、体质量增加缓慢甚至下降等症状;与衰老模型组相比,绞股蓝籽油可以显著降低小鼠血清中的TC、TG、LDL浓度(P<0.05),显著提高血清中的HDL浓度(P<0.05);小鼠肝、脑组织生化指标测定发现,与衰老模型组相比,GPSO中、低剂量组能极显著增强SOD活性(P<0.05),显著或极显著降低MDA含量(P<0.05),极显著提高T-AOC能力(P<0.05),表明绞股蓝籽油可能通过增强机体的总抗氧化能力从而有效清除多余的自由基,降低机体细胞损伤程度,间接保护正常细胞,从而起到抗衰老作用。【结论】初步判定绞股蓝籽油无明显毒副作用,且有一定的抗衰老活性。

【Objective】This study evaluated the toxicology of Gynostemma pentaphyllum seeds oil（GPSO）to provide basis for studying biological function of GPSO and developing new resources.【Method】Mice acute oral toxicity test,mouse bone marrow polychromatic erythrocyte micronucleus test,and mouse sperm deformation test were conducted to evaluate the food safety toxicology.For mice acute oral toxicity test,after fasting for 16 h,each group of mice was orally administered with 0,4.6,10.0 and 21.5 g/kg of GPSO.Then the mice were continuously observed for 14 d,and the diet,exercise,excretion,poisoning performance and death were recorded.For mouse bone marrow polychromatic erythrocyte micronucleus test,the mice in experimental groups were treated with 2.5,5.0 and 10.0 g/kg of GPSO while the blank control group was injected intraperitoneally with cyclophosphamide as a positive drug once a day for 5 days.Bone marrow cells were sacrificed,the incidence of micronuclei was observed and calculated under microscope.For mouse sperm deformation test,male mice were selected and treated with 2.5,5.0 and 10.0 g/kg of GPSO while the blank control group was injected intraperitoneally with cyclophosphamide as a positive drug once per day for 5 days.Mice were sacrificed at the last time after 7,14,21,28 and 35 days,epididymis were taken to produce sperm smears for microscopy observation and calculating sperm deformity rate.The food safety toxicological evaluation of GPSO was carried out by above three tests.D-galactose was injected to establish an aging model while the GPSO was administered as an anti-aging test drug at doses of 2.0,3.0,and4.0 mL/kg.After 60 days,mice organ index and levels of total cholesterol（TC）,triglyceride（TG）,low density lipoprotein（LDL）and high density lipoprotein（HDL）in serum were determined.The activities superoxide dismutase（SOD）,total antioxidant capacity（T-AOC）,catalase（CAT）activity and malondialdehyde（MDA）in liver and brain tissues of mice were observed and the pathological changes of liver tissues were observed.【Result】The acute oral lethal dose LD50 value of GPSO on male and female mice was greater than 10.0 g/kg,and GPSO was actual non-toxic substance according to the acute toxicity dose classification standard.The micronucleus test of mouse bone marrow polychromatic erythrocytes showed that the micronucleus rate of bone marrow polychromatic erythrocytes in male and female mice was between2.0‰-3.8‰,without significant difference compared with control group（P〉0.05）.It was proved that GPSO did not significantly affect the micronuclei of bone marrow polychromatic erythrocytes in mice.Mice sperm abnormalities test showed that GPSO-L,M,and H dose groups had no significant difference compared with blank control group.Anti-aging study showed that the contents of TC,TG and LDL in aging group were significantly higher（P〈0.05）and HDL was significantly lower（P〈0.05）compared with the control.Liver and brain biochemical markers were detected in mice,GPSO could significantly enhance SOD activity（P〈0.05）,reduce MDA content（P〈0.05）,and improve T-AOC capacity（P〈0.05）.It was indicated that GPSO may effectively enhance the total antioxidant capacity to effectively remove excess free radicals,reduce the degree of cell damage,indirectly protect normal cells,and play a role in anti-aging.【Conclusion】It is preliminarily confirmed that GPSO showed no significant toxic effect while had certain anti-aging activity.