| Objective:China has become the country with the highest incidence of diabetes in the world,and the CCMR-3B study showed that 42% of patients with type 2 diabetes mellitus(T2DM)had dyslipidemia,and medium-chain fatty acids have the effects of regulating blood lipids,improving glucose tolerance,regulating glucolipid metabolism and balancing intestinal flora.In this experiment,octanoic acid and decanoic acid were prepared as nano-emulsion formulations to reduce the irritation of octanoic acid and mask its undesirable odor.The octanoic acid-decanoic acid nano-emulsion(OA-DA-NE)with few excipients and high stability was prepared by the combined method of high-energy emulsification technology through single-factor test and response surface optimization method;the quality of OA-DA-NE was evaluated;to explore the effects of OA-DA-NE on glucolipid metabolism,insulin resistance and intestinal homeostasis in T2 DM rats.Methods:1.Preparation of OA-DA-NE by high-energy emulsification method,with the mean particle size and polydispersity coefficient as evaluation indexes,and determination of optimal formulation and optimal preparation process conditions of nano-emulsion by single-factor and response surface methods;2.The type of OA-DA-NE was identified by staining method,the appearance of the emulsion was observed by naked eye,the particle size,polydispersity coefficient and Zeta potential of OA-DA-NE were determined by Malvern laser particle size meter,the methylation conditions of acid-catalyzed method were optimized by single-factor and orthogonal tests,and the content of octanoic acid and decanoic acid in OA-DA-NE was detected by gas chromatography after methylation by this method,the encapsulation rate of octanoic acid and decanoic acid in OA-DA-NE was measured by ultrafiltration centrifugation,and the stability of OA-DA-NE was investigated by centrifugation test,Tyndall effect,bright light test,acceleration test and long-term test.3.A total of 80 male SD rats were randomly divided into a normal group,10 of which were fed the basal diet,and the other 70 were fed the high-fat diet for 5 weeks,and then intraperitoneally injected STZ at the dose of 35 mg/kg to establish T2 DM rat model.FBG≥ 11.1 mmol/L within one week,and symptoms such as polydipsia,polydipsia,polyuria and weight loss were considered as the model.The model rats were randomly divided into model group,metformin positive group(200 mg/kg),OA-DA-NE high-dose group(0.8 g/kg),OA-DA-NE medium-dose group(0.4 g/kg)and OA-DA-NE low-dose group(0.2 g/kg)according to blood glucose values.The rats were administrated intragastric once a day for 4 weeks.The body weight and intake of food and water of the rats were monitored every day,fasting blood glucose was measured every week.OGTT test was performed after administration,fresh sterile feces were collected from rats,and then sacrificed for blood sampling.Automatic biochemical analyzer was used to detect lipid(TC,TG,LDL-C,HDL-C),liver function(AST,ALT)and renal function(UREA,CREA)indexes of rats,and ELISA was used to detect the contents of FINS,GHb,IL-6 and TNF-α in serum.HOMR-IR,liver index and kidney index were calculated,pathological tissue sections of pancreas,liver and kidney were observed,and intestinal flora in stool of rats was detected by 16 S r RNA method.Results:1.The optimal prescription for the OA-DA-NE was 10.0% oil phase(7.5% octanoic acid + 2.5% decanoic acid),6.0% Ethoxylated hydrogenated castor oil,and 84.0%ultrapure water.The optimal process conditions for OA-DA-NE were: shear speed 13000 rpm,shear time 1 min,homogenization pressure 350 bar,and homogenization 3 times.2.The OA-DA-NE was O/W type;the appearance was homogeneous milky white,with good butyral phenomenon,and no delamination and emulsion breakage after standing,and good flowability;the average particle size was(79.12 ± 0.48)nm,PDI was(0.117 ± 0.003),and zeta potential was(-11.7 ± 0.37)m V.The drug loading amounts of octanoic acid and decanoic acid in the nano-emulsion were(73.91 ± 0.62)mg/g and(23.87 ± 0.32)mg/g,and the Entrapment efficiency of octanoic acid and decanoic acid were(98.20 ± 0.06)% and(97.17 ± 0.14)%.The stability tests showed that the OA-DA-NE had better centrifugal stability;better storage advantages at 4℃ and 25℃;poor stability under high temperature and strong light conditions;good stability in long-term test and accelerated test.The methyl esterification method of octanoic acid-decanoic acid is acidcatalyzed,and its optimal methyl esterification conditions were: reflux temperature of65℃,reflux time of 50 min,catalyst concentration of 2% sulfuric acid-methanol solution,and catalyst addition amount of 2m L.3.After modeling,the FBG of each group was ≥ 11.1 mmol/L,which was significantly higher than that of the normal group(P<0.01),the body weight was significantly lower than that of the normal group(P<0.01),and the symptoms of polydipsia,polydipsia and polyuria appeared,indicating that the modeling was successful.After medication intervention,FBG value in medication groups was significantly decreased compared with model group(P<0.01),body weight increased,food intake and water intake also improved.The area under OGTT curve in model group was significantly higher than that in normal group(P<0.01),and the area under OGTT curve in drug administration groups was significantly lower than that in model group(P<0.01),indicating that the OA-DA-NE could control blood sugar and improve the control ability of blood sugar in rats.FINS and HOMR-IR in the model group were significantly higher than those in the normal group(P<0.01 or P<0.05),while those in the administration groups were lower than those in the model group(P<0.01),indicating that OA-DA-NE could improve insulin resistance in type 2 diabetic rats.The levels of TC,TG and LDL-C in model group were significantly higher than those in normal group(P<0.01),and the level of HDL-C in model group was significantly lower than that in normal group(P<0.01).After administration,the levels of TC,TG and LDL-C in each group were decreased compared with that in model group(P<0.01 or P<0.05),while the level of HDL-C was increased(P>0.05),indicating that OA-DA-NE could improve lipid metabolism disorder in type 2 diabetic rats.AST,ALT,liver index,UREA,CREA and kidney index in model group were significantly increased compared with model group(P<0.01).The levels of AST and CREA in all administration groups were significantly lower than those in model group(P<0.01 or P<0.05),there were significant differences in ALT levels in other administration groups except low-dose group(P<0.01 or P<0.05).There were significant differences in UREA level of other drug administration groups except medium and low dose groups(P<0.01 or P<0.05).These results indicate that the OA-DA-NE could alleviate the damage of liver and kidney caused by type 2 diabetes,and had a certain protective effect on liver and kidney.The contents of IL-6 and TNF-α in the model group were significantly higher than those in the normal group(P<0.01),and the contents of IL-6 and TNF-α in each administration group were significantly lower than those in the model group(P<0.01),indicating that the contents of IL-6 and TNF-α in serum could be reduced by the OA-DANE.HE staining results showed that there were tissue lesions in pancreas,liver and kidney of model group rats,and the lesions of pancreas,liver and kidney were improved after administration,while no obvious pathological changes were observed in liver and kidney.Intestinal flora sequencing results showed that the richness and diversity of intestinal flora in model group were significantly decreased compared with that in normal group(P<0.01 or P<0.05),and the abundance of Firmicutes and Allobaculum increased,the abundance of Bacteroidota,unclassified＿Muribaculaceae and Ligilactobacillus decreased,the F/B ratio was significantly higher than that in normal group,indicating that intestinal flora was disordered and unbalanced.After administration,the richness and diversity of intestinal flora in each treatment groups were increased(P<0.01),the abundance of Firmicutes and Allobaculum decreased,the abundance of Bacteroidota,unclassified＿Muribaculaceae,Ligilactobacillus and Blautia increased,the F/B ratio was lower than that in model group,and the species abundance of intestinal flora in each treatment groups tended to be in normal group,indicating that OA-DA-NE could balance intestinal flora and maintain intestinal homeostasis in T2 DM rats.Conclusion:1.The OA-DA-NE with high drug loading capacity,low excipients and high stability was prepared by high-speed shear combined with high pressure homogenization method,and its particle size,polydispersity coefficient and Zeta potential meet the requirements of nano-emulsion.2.The OA-DA-NE can relieve the symptoms of polydipsia,polyphagia,polyuria and weight loss in type 2 diabetic rats,reduce fasting blood glucose value,improve pancreatic resistance and glucose tolerance,regulate lipid metabolism,inhibit the release of pro-inflammatory factors,reduce the damage of pancreas,liver and kidney tissue,improve the richness and diversity of intestinal flora,and improve the balance of intestinal flora.The effect of 0.4 mg/kg OA-DA-NE on T2 DM rats was the best after continuous intragastric administration for 4 weeks. |
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