Anti-Diabetic Effect And Mechanism Of Active Components Ilex Kudingcha And Its Chemical Composition

Ilex kudincha C. J. Tseng is belongs to the Aquifoliaceae family, whichmainly grown in Hainan, Hubei, Guangdong and Guangxi area of China.The leaves of Ilex kudingcha are used as an ethnomedicine in the treatment ofsymptoms related with diabetes mellitus and obesity throughout the centuriesin China. Nowadays it becomes the research topic in the study of medicinalplants for the treatment ofcardiovascular-related diseases, diabetes mellitusand obesity. The present study investigated the anti-diabetic activities of anactive components group obtained from Ilex kudingcha in alloxan-inducedtype II diabetic mice. The project was based on the anti-diabetic activitiestogether with the method of bioassay-guided fractionation. The chemicalcomposition of active components group of Ilex kudincha was analyzed andidentification with LC-MS/MS. Meanwhile, the mechanism of theanti-diabetic activities of lex kudincha was study by the method of real timeRT-PCR from the perspective of molecular biology.The project was first to study the hypoglycemic effect of Ilex kudincha ontype II diabetic mice. Type II diabetes were induced by a high-fat diet andalloxan. A high-fat diet contained basic diet (78.8%), egg yolk (10%), lard oil(10%), cholesterol (1%), and cholate (0.2%). The mice were fed this diet forone month, then combined with a once low-dose fresh alloxan (90mg·Kg1). The levels of serum total cholesterol (TC), serum glucose (GLU), triglyceride(TG), superoxide dismutase (SOD), malondialdehyde (MDA) andnonesterified fatty acid (NEFA) were investigated in type II diabetic mice afterone month of treatment of10g·kg-1·d-1Ilex kudincha. The study wasperformed to prove the hypoglycemic effect of Ilex kudincha.Second, the Ilex kudincha decoction was separated into three fractions (A,B and C) in order to investigate the effects of different fractions of Ilexkudincha decoction on the levels of serum TC, GLU, TG, SOD, MDA andNEFA in type I hyperglycemic mice. The hyperglycemic mice were inducedby a single high dose intraperitoeal injection of200mg·Kg1body weightfresh alloxan solution. Twenty-four hours after the alloxan administration,fasting blood was collected from caudal vein of all the animals to determinethe glucose concentration. The hypoglycemic effects were evaluated by thelevels of several diabetes-related serum parameters including GLU、TC、TG、SOD、MDA and NEFA after one month treatments of fractions A, B and C of15g·kg-1·d-1Ilex kudincha.Then the hypoglycemic effects of the active components group from Ilexkudincha on type II diabetic mice was investigated including the animalexperiments, the mechanism of its anti-diabetic activities and the chemicalcomposition of the active components group from Ilex kudingcha. Mouse wasfed a high-fat diet for4weeks to prepare the hyperlipidemia mouse. Thehyperlipidemia mouse was treated with a twice low-dose intraperitonealinjection of fresh alloxan (60mg·kg1×2of body weight) for establishment oftype II diabetic mouse. The active components group of low, medium and highdose groups were treated with1.27g,2.54g and3.81g powder·Kg1bodyweight. The1.27g,2.54g and3.81g active components group powder wereequivalent to5g,10g, and15g Ilex kudingcha. Food and water intake werechecked every4days. And other serum biochemical parameters including GLU、TC、TG、SOD、MDA and NEFA were investigated after4weeks oftreatments. For the chemical composition study, the active components group(fraction A) was thawed at room temperature, dissolved in80%aqueousmethanol (10mg·mL1of methanol), and used directly forLC-DAD/HR-ESI-TOF-MS analysis. The study of the mechanism ofanti-diabetic activities was based on the principles of molecular biology andusing the technology of quantitative Real-Time RT-PCR. Four genes wereexamined by real-time RT-PCR. The representative genes were selectedaccording to metabolic functions in terms of gluconeogenesis (G6pc),glycolysis (Gck), lipid metabolism (Fas), and cholesterol synthesis (Hmgcr).The results were:(1) Ilex kudincha decoction can significantly reduceserum GLU, NEFA, TC, TG and MDA levels. Also, the serum SOD activity issignificantly increased in type II diabetic mice treated with Ilex kudinchadecoction for one month. The therapeutic effect of Ilex kudincha was proved.(2) The fraction A of Ilex kudincha decoction can significantly decreaseserum GLU and TC levels, and increase serum SOD activity in type Ihyperglycemic mice. The fractions B and C do not possessed significantanti-diabetic activity.(3) The active components group (fraction A) of low, medium and highdose groups could significantly reduce the body weight, food and water intakeand serum GLU levels in type II diabetic mice. Especially, the high dose groupcould significantly decrease serum TC, TG and NEFA levels and increaseserum SOD activity in type II diabetic mice.(4) The3-Hydroxy-3-methylglutaryl coenzyme A reductase andglucokinase were upregulated significantly, while fatty acid synthetase,glucose-6-phosphatase catalytic enzyme was downregulated in diabetic miceafter treatment of the active components group from Ilex kudingcha. Themechanism of the hypoglycemic and hypolipidemic effects of the active components group from Ilex kudingcha including the increased excretion ofbile acid and cholesterol induced a deficiency in hepatic cholesterol and itsderivatives, leading to the potent induction of cholesterol synthesis andcholesterol uptake and the activation of glycolysis and inactivation ofgluconeogenesis in liver.(5) In the study, sixteen components including three chlorogenic acidsisomers (neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid),three dicaffeoylquinic acids isomers, four flavonoids, and six triterpenoidsaponins were identified or characterized by their MS/MS spectra and LCretention time.The project subject to the Traditional Chinese Medicine theory as a guideto investigate the anti-diabetic activity of Ilex kudingcha which was more inline with the experience of the clinical application of Ilex kudingcha. Thestudy successfully used the modern advanced technology and methods such asLC-MS/MS technology in basic research material of anti-diabetic activity ofIlex kudingcha. The active components was identified and the mechanism ofhypoglycemic and hypolipidemic effects of Ilex kudingcha were clarified bythe quantitative real-time RT-PCR technique which laid a solid foundation forthe development and utilization of Ilex kudingcha.