| Objective: To investigate the toxic and side effects of Panax notoginseng(PN)with different particle sizes on Caenorhabditis elegans in large doses,and to determine the safe dosage of PN.To study the longevity effect of PN on C.elegans and its possible molecular mechanism under safe dose,and to determine the best particle size.To study the dissolution and stability of saponins from PN with different particle sizes in vitro,and to provide certain theoretical basis for clinical application of PN and further development of new dosage forms and preparations.Methods: Using C.elegans as a model,acute exposure of 10,20,40mg/m L ethanol extracts of PN(EPN)to nematodes for 48 h and chronic exposure of 1,2,4 mg/m L EPN to nematodes for 8 d.The worms were used to detect survival rate,reproduction,body length,exercise behavior,apoptosis number,ROS level of each group of nematodes to determine the safe dosage of PN.The C.elegans was cultured with0.8,1.6 mg/m L EPN for 4 d,and the life span and reproduction of each group of C.elegans were measured to determine the optimal particle size of PN.The survival rate of nematodes under oxidative stress and heat stress were measured by cultured of ethanol extract of PN fine powder(FPE),ROS level in nematodes after 1 h of oxidative stress was measured,lipid content,SOD activity level and CAT activity level in nematodes were measured,and expression level of age-related genes in nematodes were measured.The effects of EPN on the expression of HSP-16.2∷GFP in transgenic nematodes CL2070 and SOD-3∷GFP in transgenic nematodes CF1553 were measured,and the effects of PN on the life span of transgenic nematode CF1038 and CB1370 under oxidative stress were measured,so as to observed the anti-aging effect of PN on C.elegans and its effect on the regulation of IIS signal pathway.The dissolution of PN with different particle sizes in different p H solutions were studied,and the accelerated stability of saponins from PN with different particle sizes were determined to provide reference for further development of PN.Results:(1)Acute exposure of 10,20,40 mg/m L EPN to nematodes for 48 h reduced survival rate,reproductive ability and locomotor ability of nematodes,increased the number of apoptosis cells and ROS level in nematodes,and the 40 mg/m L FPE showed the most significant effect.(2)Chronic exposure of 1,2,4 mg/m L EPN to nematodes for 8 days,only 4 mg/m L group reduced survival rate,reproductive ability and exercise ability of nematodes,increased apoptosis number and ROS level in nematodes,and the FPE group showed the most significant effect,while the 1,2 mg/m L groups showed no obvious toxic and side effects.(3)0.8 and 1.6 mg/m L EPN prolongs the life of C.elegans without affecting its reproductive ability.Compared with the control group,the FPE increases the survival rate of nematodes under acute oxidative stress and acute heat stress,reduced the lipid content and ROS level in nematodes after oxidative stress,and increased SOD activity and CAT activity in nematodes.(4)Compared with the control group,the expression of daf-2 m RNA was significantly decreased,and the expression of daf-16,sod-3,hsp-16.2 m RNA were significantly increased in the administration group of FPE,and the expression of HSP-16.2∷GFP in the transgenic nematode CL2070 and SOD-3∷GFP in the transgenic nematode CF1553 were significantly increased,while the life span of daf-2 and daf-16 transgenic nematodes were not significantly affected.(5)In hydrochloric acid solution with p H=1,the three saponins mainly contained in PN were rapidly degraded,and their dissolution curves could not be measured.In pure water,buffer solution with p H=4.5 and p H=6.8,the dissolution rates of MP and FP were higher than that of CP,but the dissolution rate of MP and FP were not different.In the three dissolution media,the higher the p H,the higher the cumulative dissolution rate of PNS.In the accelerated test,the stabilities of PN powder with different particle sizes were basically the same.Conclusion: Large dose(10,20,40 mg/m L)of EPN acute exposed to nematodes for 48 h,the results show that large dose of PN had certain toxic and side effects,and the fine powder has the most significant effect.Chronic exposure of 1,2,4 mg/m L EPN to the nematodes for 8 d shows that the safe dose administered to nematodes is below 2 mg/m L.The administration of 0.8 and 1.6 mg/m L EPN to nematodes showed that EPN can prolong the life of nematodes and has certain anti-aging effect,and FPE has the strongest anti-aging effect,which may be realized by adjusting IIS signal pathway.The dissolution and stability of medium powder and fine powder are basically the same.Combined with pharmacodynamics research,it is suggested to use fine powder orally,providing reference for further development of PN powder. |
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