| Phloretin(Pht)is a hydrophobic polyphenolic compound mainly found in apples,strawberries,camellia and other plants.Extensive research studies have reported that Pht has a wide range of pharmacological activities,such as antioxidative,inhibiting tyrosinase activity,antitumor,hypoglycemic activity.Because of its safe,non-toxic and rich physiological functions,it has a great application prospect in the fields of food,medicine,cosmetics and so on.However,due to the poor water solubility,poor stability and low bioaccessibility of Pht,the application of Pht in these fields is limited.Therefore,how to improve the water solubility and bioaccessibility of phloretin is particularly important.In order to solve the problems in the application of hydrophobic substances,a variety of nanocarriers with different properties have been developed.Among them,soybean lecithin(SL)and chitosan(CS)have been widely used in novel drug delivery systems,such as nanoparticles,because of their biocompatibility and biodegradation.Diabetic nephropathy(DN)is one of the main causes of death in diabetic patients.In this paper,Phloretin-loaded Soybean lecithin-chitosan Nanoparticles(Pht-SL-CS NPs)were prepared by electrostatic adsorption between SL and CS,and the structure and properties of Pht-SL-CS NPs were also explored,and then the protective effect and the possible mechanism on DN were also discussed.The main conclusions are as follows:(1)According to the single factor experiment and orthogonal experiments,the optimal preparation process of soybean lecithin-chitosan nanoparticles(SL-CS NPs)was as follows:chitosan concentration was 0.10 mg/mL,CS/SL mass ratio was 1:25,ethanol content was 8%,pH value was 4,the magnetic stirring time was 2 h.It was proved that the encapsulation efficiency(EE)of 3 mg Pht was 98.12%±0.10%,which was higher than that of any group of experiments in the orthogonal experiment.(2)The effect of the addition of Pht on the EE and Loading Efficiency(LE)of Pht-SL-CS NPs was explored.The results showed that when the concentration of Pht was 250 ?g/mL,EE was 92.71%±0.67%,LE was 6.76%±0.06%,after embedding,the solubility of Pht was increased by 9.97 times;the structure of Pht-SL-CS NPs was characterized:the results of Fourier Transform Infrared Spectroscopy(FTIR)indicated that Pht has been successfully coated in the lipid core of nanoparticles;X-ray Diffractometer(XRD)showed that Pht exists in Pht-SL-CS NPs in an amorphous state,which improves the water solubility of Pht;the appearance and reproducibility of the prepared Pht-SL-CS NPs indicated that the prepared Pht-SL-CS NPs had a spherical structure,small particle size,no large particles,uniform distribution and good reproducibility.(3)The effects of mannitol,bovine serum albumin and glycerol on the reconstructed particle size of Pht-SL-CS NPs were compared.The protective effect of glycerol was the best,when the amount of glycerol added was 4%(w/v).There was no significant difference between the particle size of Pht-SL-CS+4%GNPs reconstituted before freeze-drying(p>0.05);the effect of each solution on Pht under 25? natural light,25? opaque and 4? opaque conditions was explored,and the results showed that Pht NPs greatly improved light stability and storage stability;the bioaccessibility of Pht in each solution was investigated through in vitro simulated digestion experiment,and the results showed that after embedding,the bioaccessibility of Pht were increased significantly(p<0.05),and using glycerol as a protectant in lyophilization can significantly improve the decrease of biological accessibility after lyophilization resolution(p<0.05);the inhibitory effect of each solution on ?-glucosidase was explored,and the inhibitory activity was as follows:Pht 1%DMSO solution>Pht-SL-CS+4%G NPs>Pht-SL-CS+4%G NPs redissolution>Pht-SL-CS NPs>Pht-SL-CS NPs redissolution>Acarbose.(4)Intraperitoneal injection of streptozotocin(STZ)was used to establish a diabetic rat model.Pht-SL-CS+4%G NPs(Pht NPs for short)had no significant effect on the body weight of diabetic rats,but had a certain effect of lowering blood glucose;diabetes induced by STZ can cause extremely significant weight gain in kidney and liver tissue(p<0.001),and low-dose Pht NPs can significantly slow down the increase in kidney and liver(p<0.05),so it may have a certain kidney and liver protection in diabetic rats;the results of the effect on the kidney function of diabetic rats showed that Pht NPs can effectively alleviate renal lesion,and all indicators were not significantly different from CN group and Emg group(p>0.05);the renal histopathology and fibrosis observation results of diabetic rats showed that different doses of Pht NPs can alleviated kidney lesion and fibrosis,the effect was similar to that of the positive drug Emg.However,the Pht group still had kidney damage and a large number of blue-stained areas,which proved that low-dose Pht cannot reduce the degree of kidney damage and fibrosis.After coating,Pht NPs can more effectively play the physicological function in the body;the results of antioxidant effect on renal of diabetic rats showed Pht NPs had anti-oxidative stress effect,thereby preventing the renal damage;the results of the effect on the TGF-?1/Smad2 signaling pathway in the kidney of diabetic rats showed that the intervention of Pht NPs made expression of TGF-?1 and Smad2 was extremely significantly down-regulated(p<0.001),indicating that Pht NPs may alleviate renal fibrosis in diabetic rats by regulating the TGF-?1/Smad2 signaling pathway,thereby exerting its renal protection.In summary,Pht NPs can not only significantly improve the water solubility,storage stability,light stability and bioaccessibility of Pht,but also have strong ?-glucosidase inhibitory activity.The results of animal experiments in a diabetic rat model induced by STZ showed that Pht NPs can alleviate renal lesion and fibrosis.The specific mechanism involved improving the oxidative stress of kidney and inhibiting the TGF-?1/Smad2 signaling pathway.This article provides a theoretical basis for the development of Pht NPs in inhibiting ?-glucosidase and preventing diabetic nephropathy. |
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