Preparation Conditions Of Garcinia Kola Heckel Ethanol Extract And Its Effect On Renal Damage In Type 2 Diabetes

Diabetic nephropathy（DN）is the most important microvascular complication of diabetes,and also the main cause of end-stage renal disease.At present,the trend of using natural products to treat diabetes is more and more obvious.Garcinia kola Heckel is rich in flavonoids,cardiac glycosides,steroids,saponins and other active ingredients,so it is often used to treat hepatitis,cough,bacterial infections and other diseases in African folk.At the same time,studies have shown that G.kola has antioxidant,hypoglycemic,hypolipidemic and other pharmacological effects,however,there is a shortage of pharmacological research on diabetic nephropathy.Therefore,the total flavonoids extraction rate was used as an indicator for the optimized process of ethanol extracts of G.kola（GK）extracted by ultrasound-assisted enzymatic extraction in this study.And the purpose of this research is to determine the influence and mechanism of GK on diabetic nephropathy.This study investigated the effects of seven factors on the extraction rate of GK,including cellulase concentration,liquid-solid ratio,ethanol concentration,p H,enzymolysis time,ultrasound time,and enzymdrolysis temperature.And on this basis,the four most influential factors were selected.And the process conditions of total flavonoids from GK for ultrasound-assisted enzymatic extraction were optimized by Design-Expert software.After that,high-glucose and high-fat diets were paired with low-dose streptozotocin（HGHFD/STZ）-induced type 2 diabetic nephropathy rat model was used to explore the effect of GK on diabetes-induced kidney damage.After 8 weeks of administration,the levels of urine protein,UA,Cr,and BUN in urine and serum were measured by kits.Then the kidney tissues were weighed and the levels of IL-1β,IL-6,TNF-α,MCP-1,SOD,MDA,CAT,and GSH-Px were tested by the appropriate kits.The pathological damage in renal tissue were observed by H&E,Masson and PAS staining.The number of apoptotic cells in kidney tissues was detected by IHC and the amount of cleaved-caspase 3/9 was measured by western blot.In vitro experiments,H₂O₂-induced HBZY-1 cells was used to investigate the cellular protections of GK.The contents of ROS,SOD,MDA and ATP were tested.The mitochondrial membrane potential and the number of apoptotic positive cells were detected by kits.And the expression levels of caspase 3/9,mitochondrial fission and mitochondrial damage were measured by western blot,TEM and Mito Tracker staining.Results have shown that four most influential factors,namely,cellulase concentration,liquid-solid ratio,enzymatic hydrolysis time and enzymatic hydrolysis temperature,were found based on the results of single-factor experiments.Then the optimal extraction conditions of GK by response surface methodology was as follows:cellulase concentration for 0.52 mg/m L,liquid-solid ratio for 50:1,enzymolysis time for2 h,enzymolysis temperature for 50℃.Under these conditions,the extraction yield of total flavonoids is 88.95%.Subsequently,animal experiments have shown that GK significantly reduced urine volume and renal index in T2DM rats,and GK improved the glomerular filtration function,inhibited oxidative stress and inflammation in renal tissues.The section results showed that GK inhibited the accumulation of glycogen and reduced the thickness of glomerular basement membrane in renal tissues.And the apoptosis in renal tissue was attenuated by reducing the expression levels of cleaved-caspase 3/9.Cell experiments have shown that GK has a protective effect on H₂O₂-induced HBZY-1 cells.The antioxidant capacity was improved after GK administration.Besides,GK restored mitochondrial function by modifying mitochondrial membrane potential and diminishing mitochondrial fission.In addition,GK inhibited apoptosis by reducing the expression levels of cleaved-caspase 3/9.To sum up,the process conditions of GK were adjusted,which providing an experimental basis for the future development and utilization.At the same time,in vivo study has shown that GK improved the levels of oxidative stress and inflammation in the renal tissues on T2DM rats by inhibiting apoptosis of tissues and cells.In vitro study has shown that the mechanism of GK in protecting H₂O₂-induced HBZY-1 cells may be related to the regulation of mitochondrial fission and caspases-mediated apoptotic signaling pathways.