Preparation,Modification,and Regulation Mechanism Of Glucose Metabolism Of 4,4-dimethylsterols From Rice Bran Oil

Phytosterols are the main components of the unsaponifiables of vegetable oils,including4-desmethylsterols,4-monomethylsterols and 4,4-dimethylsterols.4-Desmethylsterols are known to reduce total cholesterol（TC）and low-density lipoprotein cholesterol（LDL-C）,whereas 4,4-dimethylsterols have received little attention.In recent years,4,4-dimethylsterols have been found to enhance insulin release fromβ-cells in rats with type 2 diabetes mellitus,suggesting that 4,4-dimethylsterols may regulate glucose metabolism and have the potential to ameliorate the development of type 2 diabetes mellitus.Meanwhile,4,4-dimethylsterols are byproducts of ferulic acid production fromγ-oryzanol from rice bran oil,which have not been properly used.Therefore,the separation,purification,structural modification of 4,4-dimethylsterols from rice bran oil and its molecular mechanism of regulating glucose metabolism were studied in this paper.The main research results are as follows:Firstly,a flash chromatography method was established for the separation of 4,4-dimethylsterols and 4-desmethylsterols as byproducts ofγ-oryzanol hydrolysis.The purity and molecular structure of the products were identified.The results showed that the R_S of flash chromatography was 2.25.The yield of 4,4-dimethylsterols and 4-desmethylsterols were41.76±2.78%and 36.78±1.70%with the total yield of 78.53±1.07%,and the purity was93.37±0.38%and 98.24±0.79%,respectively.At the same concentration,the absorbance value of 4,4-dimethylsterols was higher than that of 4-desmethylsterols at the maximum absorption wavelength of 210 nm.FT-IR could distinguish them at 881cm^-1.GC-MS results showed that4,4-dimethylsterols were composed of cycloartenol（35.21%）and 24-methylenecycloartanol（61.79%）with an average molecular weight was 421.87.The 4-desmethylsterols were composed of campesterol（20.60%）,campestanol（12.95%）,stigmasterol（7.03%）,stigmastanol（8.08%）,sitosterol（30.47%）and sitostanol（19.11%）with an average molecular weight of402.52.Secondly,the inhibitory effects of 4,4-dimethylsterols and 4-desmethylsterols onα-glucosidase were compared,and the mechanism of interaction was elucidated by enzyme inhibition kinetics,multispectral and molecular docking techniques.The results showed that the inhibitory effect of 4,4-dimethylsterols onα-glucosidase with an IC₅₀ value of 0.71±0.12mg/m L was about 5 times that of acarbose,and higher than that of 4-desmethylsterols.The inhibition kinetics showed thatα-glucosidase inhibition by 4-desmethylsterols was non-competitive with the inhibition constant K_i value of 4.23×10-4 mol/L.Fluorescence quenching experiments showed that quenching belonged to static quenching accompanied by fluorescence resonance energy transfer,and there was only one or one kind of potential binding sites in the interaction.The results of thermodynamic analysis showed that theΔS°（-154.61 J/mol.K）,ΔH°（-65.83 k J/mol）andΔG°（-19.75～-17.90 k J/mol）of the enzyme binding process were all negative values,which was a spontaneous exothermic process driven by hydrogen bond and van der Waals force.Molecular docking suggested the presence of van der Waals forces between4,4-dimethylsterols andα-glucosidase residues Tyr158,Glu411,Asp352,Arg442,Phe303,Phe178,Gln279,Arg315,His280,Phe314 and Lys156.The hydrogen bond is formed between theα-glucosidase residue Arg-442 and the OH at the C-3 position of 4,4-dimethylsterols.Therefore,it was speculated that 4,4-dimethylsterols could be used as the potential hypoglycemic active component during the digestion phase of carbohydrate hydrolysis to glucose.Thirdly,4,4-dimethylsterol oleates were synthesized by acyl chloride esterification in order to improve the bioavailability.The reaction products were purified by flash chromatography,and then characterized.The results showed that the molar ratio of oleyl chloride to 4,4-dimethylsterold was 1.1:1,and the reaction was carried out at 313 K for 60 min,the conversion of 4,4-dimethylsterols reached 99.27%.The reaction followed the second-order kinetic model（R²=0.9628-0.9977）with activation energy and pre-exponential factor of 15.54k J/mol and 1.78×10³ L/（mol.min）,respectively.FT-IR found that the-OH stretching vibration at 3404 cm^-1 disappeared,and a-C=O strong absorption peak signal appeared at 1732 cm^-1,indicating the existence of ester bond.¹H NMR and ¹³C NMR results showed that the chemical shifts of 3-H and 3-C had 1.28 and 1.48 ppm downfield shift,respectively,which further confirmed the existence of ester bond.UPLC-Q-TOF-MS results showed that 4,4-dimethylsterol oleates were composed of cycloartenol oleate and 24-methylenecycloartanol oleate.Finally,the hypoglycemic activities of 4,4-dimethylsterols and their oleates were compared,and the molecular mechanism of their regulation of liver glucose metabolism was elucidated by db/db mouse model,insulin-resistant Hep G2 cell model and q PCR technology.The results showed that compared with the model group,the blood glucose levels of db/db mice in 4,4-dimethylsterol high-dose group and 4,4-dimethylsterol oleate group were significantly decreased by 26.9%and 32.9%,respectively（p<0.05）,insulin level decreased by 17.35%and30.44%,HOMA-IR decreased by 46.7%and 60.4%,respectively,indicating that 4,4-dimethylsterols and their oleates could improve hyperglycemia and insulin-resistance in db/db mice.4,4-dimethylsterols and their oleates（300μg/m L）significantly increased by 28.18%and57.30%glucose consumption,and by 22.85%and 51.35%glycogen content in insulin-resistant Hep G2 cells（p<0.05）,which is beneficial for glucose uptake and glycogen storage in cells.Meanwhile,4,4-dimethylsterols and their oleates significantly inhibited the activities of gluconeogenic key enzymes PEPCK and G6P in insulin-resistant Hep G2 cells（p<0.05）,and PEPCK activity was decreased by 16.25%and 21.98%,and G6P activity was decreased by10.61%and 18.89%,respectively.4,4-dimethylsterols and their oleates promoted glycogen synthesis in insulin-resistant Hep G2 cells mainly by down-regulating GSK3βgene expression and up-regulating GYS2 gene expression,and inhibited gluconeogenesis by down-regulating PEPCK and G6P gene expression by inhibiting FOXO1 transcriptional activity.In conclusion,high purity 4,4-dimethylsterols and their oleates were prepared from byproducts ofγ-oryzanol hydrolysis.We elucidated the molecular mechanism of 4,4-dimethylsterols and their oleates in regulating hepatic glucose metabolism by promoting the expression of key genes during glycogen synthesis and inhibiting the expression of key genes during gluconeogenesis.It was of great significance to improve the added value of rice bran oil deep processing products and explore the application of 4,4-dimethylsterols.