The Therapeutic Effect Of Gynura Divaricata On Type 2 Diabetic Mice And Its Mechanism

Gynura divaricata(GD),an edible medicinal plant,has been shown to have good hypoglycaemic effects.In this study,fresh aerial parts of GD were collected,washed and lyophilised into powders for further research.Then,the species and contents of amino acids,Pigments and other active compounds(chlorogenic acid and its derivatives)contained in the plant were explored using HPLC-DAD and other analytical techniques.On this basis,we further studied the therapeutic effect of GD lyophilized powder on type 2 diabetic mice induced by the combination of streptozotocin(STZ)and a high-fat diet(HFD).Furthermore,the related mechanism was also studied.The experimental results of the basic composition analysis showed that GD has a variety of amino acids.The contents of valine,glutamic acid and aspartic acid were the highest,18.98%,11.29%and 10.61%of the total amino acids,respectively.It also contains 8 kinds of essential amino acids,which demonstrates the plant's high nutritional value as a vegetable.In addition,we also made a detailed analysis of the types and levels of pigments in GD.We found GD contains lutein,chlorophyll b,chlorophyll a and ?-carotene.Among the 4 kinds of pigments,the content of chlorophyll a was the highest,1.52 mg/g;the content of ?-carotene was the least,0.26 mg/g.Therefore,chlorophyll a is the main pigment in GD.Moreover,we also isolated and identified four compounds from the 70%methanol extract of GD,which were chlorogenic acid,3,4-dicafleoylquinic acid,3,5-dicaffeoylquinic acid,and 4,5-dicaffeoylquinic acid.The total amount of these four compounds reached 3.11%of lyophilized powder of GD aerial parts,which shows that GD contains a high content of chlorogenic acid and its three derivatives.In order to investigate the therapeutic effect of GD diet on diabetic mice and its mechanism,male ICR mice(3 weeks of age)were divided into two groups randomly:a normal group and a type 2 diabetic group.After 3 days of acclimation,mice in the normal group were continually fed with normal food.Mice in type 2 diabetic group were fed with a HFD for 4 weeks.After 4 weeks of HFD feeding,mice in the type 2 diabetic group were fasted for 12 hours,and each mouse was injected once with low-dose STZ(100 mg/kg.BW).Seven days after the injection,mice in the type 2 diabetic group were fasted for 10 hours and fasting blood glucose(FBG)was determined;mice with more than 11.1 mmol/L of FBG were considered to be diabetic and included in the study.The diabetic mice were randomly divided into 4 groups:the diabetic model group and three GD-treated diabetic groups with doses of 1.0%,5.0%,and 10.0%.Finally,the normal mice and the diabetic model mice received a normal diet.The three groups of GD-treated diabetic mice were given different doses of GD diets daily(the diets with 1.0%,5.0%,and 10.0%GD,respectively).The administration was given daily for 4 weeks and the weight and FBG were measured weekly.At last,blood,livers and pancreas were used for further investigation.Our results showed that the FBG level of mice in diabetic model group was consistently high,which was between 19.0-23.0 mmol/L.Moreover,serum insulin levels of this group were abnormally elevated.GD treatment significantly reduced FBG and fasting serum insulin levels in diabetic mice in a dose-dependent manner,which obviously alleviated hyperglycaemia and insulin resistance in diabetic mice.Oral administration of GD also significantly reduced the level of glycated serum protein and the activities of hepatic functional enzymes in the serum.What's more,GD played a good role in regulating blood lipids in diabetic mice.Our results indicated that GD could effectively protect the body from oxidative stress by increasing the antioxidant ability of liver and pancreas in GD-tareated diabetic mice,especially in mice of high-dose group.The histopathological examination of liver and pancreas showed that GD effectively prompted the repairation of the damaged liver and pancreatic tissue.In the present study,the effect of GD on the recovery of damaged pancreatic tissue in diabetic mice was further confirmed by immunohistochemistry.In order to further study the mechanism of hypoglycemic effect of GD lyophilized powder,we investigated the effects of GD diet on insulin signalling pathway,glucose metabolism,lipid metabolism and inflammatory response in the liver of diabetic mice by Western blot in detail.Results showed that GD further alleviated the liver insulin resistance in type 2 diabetic mice by activating the damaged PI3K/p-AKT pathway,improving glucose and lipid metabolism and reducing the inflammatory response.We also studied the effects of GD on the expression of some key proteins involved in the regulation of pancreatic function and islet cell apoptosis in the pancreas of diabetic mice by Western blot,The results showed that GD protected pancreatic structure and maintained pancreatic function in diabetic mice not only by improving the expression of proteins related to insulin secretion and maintenance of pancreatic function significantly,but also by down-regulating the expression of apoptosis-related factors to inhibit the apoptosis of pancreatic cells.This study is novel because the leaves and stems of GD were freeze dried to retain the plant's physicochemical properties and physiological activity to maintain as much of the plant's active chemicals as possible.However,the traditional methods of water or ethanol extraction reported in previous studies may lead to the loss or inactivation of active ingredients.All in all,GD lyophilized powder has a good therapeutic effect on type 2 diabetes and it significantly improved hepatic and pancreatic dysfunction in type 2 diabetes,which fully demonstrates that GD might be a promising food and medicine for the treatment of diabetes.