| Deinagkistrodon acutus is one of the ten most venomous snakes in China.After the toxin enters the body of the injured person,there will be edema,pain,tissue infection,necrosis and areas where snake wounds are common at home and abroad in Asia,Africa and South America,herbs are commonly used to rescue snake wound patients,Saxifraga Stolonifera Meerb.,as a traditional national medicine in China,is commonly in the folk to treat snake wounds,and the effect is obvious.Therefore,this article uses the Miao medicine S.stolonifera to explore the activity and mechanism of the D.acutus venom.The results of the study are as follows:(1)Optimization of response surface extraction technology of aqueous extract of S.stolonifera and correlation between total phenol(TP)content and inhibiting phospholipase(PLA2)activity of D.acutus venom.The results showed that when the particle is 60 mesh,the material to liquid ratio is 1:50,the ultrasonic power is 250W,and the ultrasonic time is 1.8h,the aqueous extract of S.stolonifera and its inhibitory rate(PIR)of the PLA2 activity in D.acutus venom can reach the best ultrasonic-assisted extraction process conditions.Under the optimized conditions,the content of TP of S.stolonifera(expressed by the mass of phenolic acid compounds equivalent to gallic acid per gram of dried samples)was(8.69±0.046)g GA/100 g,and the inhibition rate of PLA2 activity(PIR)was(40.91±0.21)%;the correlation analysis showed that there was a correlation between the content of TP and the experimental results of PIR(P<0.01),it is speculated that the TP of S.stolonifera is the material basis for inhibiting D.acutus venom.(2)Study on the inhibition of main enzymes in Deinagkistrodon acutus venom in vitro by aqueous extracts of S.stolonifera.First,The PLA2 enzyme,proteolytic enzyme and hyaluronidase in D.acutus venom were selected for in vitro experiments.Experiments on inhibiting the activities of PLA2 enzyme and proteolytic enzyme were carried out by agarose plate method.With the increase of the concentration of the aqueous extract of S.stolonifera,the inhibitory activity gradually increases,the EC50 values of the inhibitory activity of aqueous extract of S.stolonifera on the activities of PLA2 enzyme and proteolytic enzymes in D.acutus venom were 0.115 mg/mL and 0.026 mg/mL,the smaller EC50 value is,the better the inhibition effect is.The UV spectrophotometry was used to inhibit the activity of hyaluronidase,the results showed that the inhibitory activity increased with the increase of the concentration of aqueous extract of S.stolonifera,the EC50 value of aqueous extract of S.stolonifera for inhibiting the hyaluronidase activity in D.acutus venom was 0.238 mg/mL.Ji Desheng snake tablets as a positive control experiment,to explore the inhibitory effects of aqueous extract of S.stolonifera and Ji Desheng snake tablets on the activities of the PLA2 enzyme,proteolytic enzyme and hyaluronidase of venom of D.acutus venom at the same concentration.The experimental results showed that the inhibitory rates of the aqueous extract of S.stolonifera and Ji Desheng snake tablets on the PLA2 enzyme of D.acutus venom were(41.2±0.27)%and(41.7±0.2)%;the inhibitory rates of the aqueous extract of S.stolonifera and Ji Desheng snake tablets on the proteolytic enzyme of D.acutus venom were(29.4±0.17)%and(31.8±0.15)%;the inhibitory rate of the aqueous extract of S.stolonifera and Ji Desheng snake tablets on the hyaluronic acid activity of D.acutus venom were(42.1±0.17)%and(40±0.25)%.According to the significant difference analysis,there is no significant difference in the inhibitory rate of the aqueous extract of S.stolonifera and Ji Desheng snake tablets on the PLA2 enzyme,proteolytic enzyme and hyaluronic acid activities of D.acutus venom.The results indicated that the anti-venom effect of aqueous extract of S.stolonifera was similar to that of Ji Desheng snake tablets.(3)Study on the inhibitory effect of aqueous extract of S.stolonifera on the activity in vivo induced by D.acutus venom.Bleeding activity experiment and edema activity experiment caused by D.acutus venom were selected for in vivo experimental design.Accroding to intraperitoneal injection for the experiment of inhibiting bleeding activity in mice,3 hours after injection,they were sacrificed,after dissection,it was founded that the skin at the injection site produced blood halo,measurement showed that,with the increase of the concentration of the aqueous extract of S.stolonifera,the diameter of the blood halo gradually decreased,when the mass ratio of D.acutus snake venom to aqueous extract of S.stolonifera is 1:500,the effect of inhibiting bleeding activity is the best,the inhibition rate was(64.2±0.17)%;by injecting the right hind foot of mice,the activity of inhibiting edema in mice was tested.After 0,1,2 and 3 hours,the degree of edema of the right posterior foot was measured,with the increase of the concentration of the aqueous extract of S.stolonifera,the inhibition effect was gradually enhanced;while the inhibition effect decreases with the increase of time,when the mass ratio of D.acutus snake venom to aqueous extract of S.stolonifera is 1:500,the inhibition effect wasmost obvious after 1 h of injection,the inhibition rate was(63.5±0.22)%,therefore,it is better to treat the bite of D.acutus venom as soon as possible.In summary,in this paper,Optimization of response surface extraction technology of aqueous extract of S.stolonifera and correlation between total phenol(TP)content and inhibiting phospholipase(PLA2)activity of D.acutus venom were determined.The results showed that the TP of S.stolonifera was the material basis for inhibiting D.acutus venom,and through in vitro and in vivo inhibitory activity experiments showed that the aqueous extract of S.stolonifera had an inhibitory effect on the activity of D.acutus venom,preliminarily explored the mechanism of S.stolonifera inhibiting D.acutus,it provides a theoretical basis for further development and research of Anti-D.acutus venom drugs. |
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