Preparation Of Epicatechin Gallate And Digoxin-loaded Solid Lipid Nanoemulsion And Their Effects On Myocardial Contractility In Isolated Rat Hearts

Catechins are one of the most important bioactive components in tea.Our previous studies showed that epicatechin gallate?ECG?was capable of increasing myocardial contraction in isolated rat hearts through the activation of membrane translocation of protein kinase C?,which is Ca²⁺-independent.It suggested that ECG may be a novel candidate drug with inotropy effects.However,this effect is limited due to its poor hydrophobicity and bioavailability.Digoxin is commonly used as inotropic drugs through inhibiton of Na⁺/K⁺-ATPase to increase intracelluar calcium contration for the treatment of patients with heart failure in Clinic,but its effective contration is very narrow and eaisly led to side effects.In view of this,Solid lipid nanoemulsion?SLNe?loading ECG or Digoxin with the physiological concentration was prepared in this thesis to test whether the nano-emulsions could improve the ECG bioavailability and stability in gastrointestinal tract and further to test whether there is a synergistic effect when combination usage.Firstly,a response surface method was applied to optimize the preparing of nanoemulsions using glycerol monostearate?GM?as wrapping material and poloxamer188 as the emulsifier,by film-ultrasonic technology.The particle size?diameter?,Zeta potential,Encapsulation efficiency?EE%?were selected as the responding values.The results of ECG-loaded SLNe have shown that the nanoparticle is spherical with the diameter of 233.10±14.65 nm when the mass ratio of GM and ECG is 19.23:1 with the0.27%addition of poloxamer 188 under the film-ultrasonication at 518.4 watt for 315seconds.Its Zeta potential,polydispersity index?PDI?,Encapsulation efficiency?EE%?and drug loading content?DLC%?are at-32.38±4.05 mV,0.25±0.06,79.61±6.01%and3.94±0.23%,respectively.Moreover,when the mass ratio of GM and Digoxin is 15.02:1with the 0.20%addition of poloxamer 188 under the film-ultrasonication at 489.6 watt for310 seconds,the prepared Digoxin-loaded SLNe also has a spherical nanoparticle with the diameter of 210.50±4.61 nm.Its Zeta potential,PDI,EE%and DLC%are at-30.33±5.81mV,0.31±0.05,71.93±2.17%and 4.49±0.30%,respectively.Those data suggest that the film-ultrasonication is applicable to prepare ECG-loaded SLNe and Digoxin-loaded SLNe with the Encapsulation of GM.Secondly,ECG-loaded SLNe and Digoxin-loaded SLNe were simulatorily digested in vitro.ECG-loaded SLNe and Digoxin-loaded SLNe have no changesinmouth digestion for 10 min.The digested SLNe was further digested in simulatory gastric digestion for 120 min.It found that the cumulative release rates of ECG and Digoxin are12.77±3.44%,0%respectively.After the 120 min in vitro small intestine digestion,the cumulative release of ECG and Digoxin are 21.77±1.08%?19.43±3.15%respectively.The results suggest that both of ECG-loaded SLNe and Digoxin-loaded SLNe are relative stable during in vitro mouth and gastrointestinal digestion.Finally,Classic Langendorff perfusion system with online Power Lab monitoring software was used to test the inotropic effects of ECG,Digoxin and their nanoemulsion at physiological concentration and further to test their synergistic effects in isolated hearts of male Sprague-Dawley rats at 10 to 11 weeks old.The results of Left Ventricular myocardial contractility indicators as LVSP,LVDP and±dp/dt have been shown that:?1?ECG at the range of 0.01 to 0.05?g/mL has a dose-dependent effect.Compared to Control group,LVSP at ECG 0.05?g/mL group significantly increased by 5.80±0.03%?P<0.05?.Digoxin aLso has a dose-dependent effect.When its concentration at 0.10 or0.40?M,the LVSP significantly increased by 5.90±0.09%?P<0.05?and 17.90±0.28%?P<0.05?respectively compared to the control group.?2?The combination of ECG 0.05?g/mL and Digoxin 0.10?M aLso has synergistic effects on the myocardial contractility,in which the LVSP increased by 17.90±0.20%,compared to those of the ECG alone and Digoxin alone at 5.80±0.03%,5.90±0.09%respectively?P<0.05?.?3?Compared to the control group,ECG-loaded SLNe 0.05?g/mL significantly increased LVSP by 14.60±0.39%?P<0.05?while Digoxin-loaded SLNe 0.10?M increased by 14.00±0.38%?P<0.05?.?4?The combination of ECG-loaded SLNe 0.05?g/mL and Digoxin-loaded SLNe0.10?M has synergistic effects on the myocardial contractility in isolated rat hearts,in which the LVSP increased by 38.40±0.89%,compared to those of the ECG-loaded SLNe alone and Digoxin-loaded SLNe alone at 14.60±0.39%,14.00±0.38%,respectively.The total results have suggested that there was a synergistic effect of the combination of ECG and Digoxin.Moreover,this synergistic effect increaseed remarkally after nanoemulsion.Our findings would facilitate the exploration of a novel inotropic agent or dietary supplement with the combination of ECG and Digoxin.