there is a global increase in the prevalence of diabetes mellitus ( dm ) which is predominantly related to changing lifestyle and the resulting surge in obesity . dm is a metabolic disease characterized by chronic hyperglycemia resulting from defects in insulin metabolism and impaired function of carbohydrate , lipid , and protein metabolism that leads to long - term complications . the liver plays a central role in whole body lipid and carbohydrate metabolism . the liver dysfunction observed in type 1 and 2 diabetes is described as steatohepatitis , and many authors assign it to a wide group of liver pathologies called nonalcoholic fatty liver disease ( nafld ) [ 13 ] . chronic oxidative stress mediated by glucolipotoxicity is involved in the onset and progression of diabetic liver dysfunction in both animal models and humans [ 46 ] . hence , regulating liver cell homeostasis and protecting the cells against oxidative surrounding are the most important , which calls for an effective antioxidant therapy as alternative in the management of nafld [ 710 ] . recently , it has been reported that increased reactive oxygen species ( ros ) generation activates stress - sensitive intracellular signaling pathways such as nuclear factor - kappa b ( nf-b ) , extracellular signal - regulated kinase ( erk ) , c - jun n - terminal kinase ( jnk ) , and p38 mitogen - activated protein ( map ) kinase during hepatic dysfunction [ 1113 ] . several pathological responses have been suggested to induce diabetic complications and these include hyperglycemia , oxidative stress - induced nf-b transcription , and apoptosis , which all take place synergistically and reciprocally [ 1416 ] . mapks participate in apoptotic signal transduction as a response to various factors , including ros , inflammatory cytokines , ultraviolet lights , radiation , heat , and osmotic shock . therefore , novel approaches are necessary to identify therapeutic agents which can act with pleiotropic effects including antioxidant properties to prevent and/or treat dm and its secondary complications . the young leaves of t. sinensis are used as a vegetable , and mature leaves are used in chinese traditional medicine for the treatment of enteritis , dysentery , and eye infections . previous studies revealed that t. sinensis leaves are rich in flavonoids with good radical scavenging abilities [ 1921 ] . our previous studies and others have shown that quercetin is the major flavonoid of t. sinensis leaves [ 22 , 23 ] . pharmacological investigations have demonstrated that quercetin has diverse biological effects such as antioxidant , anticancer , anti - inflammatory , and cardioprotective activities [ 27 , 28 ] . quercetin also plays a crucial role in aldose reductase inhibition [ 29 , 30 ] . recently , it was found that quercetin has a strong effect on blood glucose levels in alloxan induced hyperglycemia which is mediated by the blunting of free radical induced toxicity [ 31 , 32 ] . therefore , quercetin may be one of the main hyperglycemia and dyslipidemia counteracting constituents of t. sinensis leaves . however , relatively little attention has been paid to the antihyperglycemic activity of quercetin from t. sinensis leaves ( qtl ) , and studies on the effects of qtl in mouse models of diabetes induced by a high - carbohydrate / high - fat diet ( hfd ) and alloxan have to our best knowledge not been reported to date . our current study was carried out to determine whether qtl could suppress the hyperglycemia and liver damage induced by hfd - alloxan treatment in diabetic mice . the results reveal qtl as a promising dietary agent able to reduce the secondary complications of diabetes mellitus by ameliorating oxidative stress in the liver . leaves of t. sinensis were collected in shaanxi province , china , in august 2015 and identified by experts in the college of forestry , northwest a&f university , china . blood glucose ( bg ) was measured using kits from shanghai rongsheng biotechnology co. , china . total cholesterol ( tc ) , triglyceride ( tg ) , low density lipoprotein - cholesterol ( ldl - c ) , high density lipoprotein cholesterol ( hdl - c ) , nitric oxide ( no ) , plasma alanine aminotransferase ( alt ) , and aspartate aminotransferase ( ast ) activities were measured using kits from the nanjing jiancheng bioengineering research institute , china . insulin levels were determined using a radioimmunoassay kit from beijing biosino biotechnology co. , china . polyclonal rabbit antibodies against p65 , p38 , erk , caspase-9 , and caspase-3 were purchased from cell signaling technologies ( beverly , ma , usa ) . antibodies against  - actin were purchased from santa cruz biotechnology ( santa cruz , ca , usa ) . t. sinensis leaves ( 1  :  10 , w / v ) were soaked in a 70% ethanol solvent for 2.5  h and sonicated in an ultrasonic bath at 200  khz at 55c for 45  min . the residue was diluted with h2o and extracted using ethyl acetate ( etoac ) in triplicate . subsequently etoac fractions were chromatographed over silica gel ( n - hexane / etoac / meoh ) to obtain 13 fractions . fraction 8 , eluted with etoac / meoh ( 8  :  1 ) , was further separated and purified by capillary electrophoresis using silica gel column chromatography to yield quercetin . the structure of isolate was determined by reverse phase high performance liquid chromatography in comparison with authentic quercetin ( the national institute for the control of pharmaceutical and biological products , beijing , china ) . male chinese kunming mice weighing 20  2  g ( n = 60 ) were obtained from the experimental animal center of xi'an jiaotong university ( xi'an , china ) and were acclimated for 1 week before being randomly assigned to different experimental groups . the mice were maintained on a 12  h light / dark cycle on a standard chow diet until experimental analysis . the experimental animal protocol was approved by the experimental animal ethics committee of xi'an jiaotong university . after adaptation for 1 week , the mice were randomly divided into four groups ( n = 15 per group , 5 mice per cage ) : ( 1 ) normal ; ( 2 ) normal + 200  mg / kg  b.w./d qtl ; ( 3 ) dm groups : high - carbohydrate / high - fat diet- ( hfd- ) alloxan treatment ( hfd , 52.6% standard laboratory chow , 10% lard , 15% sucrose , 15% yolk powder , 5% casein , 1.2% cholesterol , 0.2% bile salt , 0.6% calcium bicarbonate , and 4.73  kcal / gram ) ; ( 4 ) dm + 200  mg / kg  b.w./d qtl . after 4 weeks of dietary manipulation , mice fed with hfd were injected intraperitoneally with 0.04% alloxan dissolved in sterile normal saline in a dose of 100  mg / kg  b.w . the mice were allowed to continue to feed on their respective diets until the termination of the experiment . three weeks after alloxan injection , the animals were sacrificed by euthanization with isofluorane after fasting for 8  h ; plasma and liver were collected , weighed , shock frozen in liquid nitrogen , and stored at 80c for further analysis . blood samples were collected by cardiac puncture , and plasma was obtained by centrifuging the blood at 8000  g for 15  min at 4c . fasting blood glucose levels were monitored periodically with the tail prick method using kits based on glucose oxidase method . blood glucose levels were expressed as mmol / l . for the oral glucose tolerance test , mice were fasted for 8 hours but given water freely and either left untreated or treated with qtl 90 minutes before the test was performed using d - glucose at a dose of 2  g / kg  b.w . blood was drawn from the tail vein before treatment and at 0 , 30 , and 120 minutes after the glycemic load . the levels of insulin , tc , tg , hdl - c , and ldl - c , as well as the activities of alt and ast in sera , were measured according to the commercial kits instructions . livers were thawed , weighed , and homogenized in buffer ( 5  mm tris - hcl , 2  mm edta , ph 7.4 ) on ice . homogenates were centrifuged ( 150  g , 10  min , 4c ) and the supernatants were used immediately for the assays . whole protein lysates of liver tissue were extracted using ripa buffer ( 25  mm tris - hcl ( ph 7.6 ) , 150  mm nacl , 1% np-40 , 1% sodium deoxycholate , and 0.1% sds , thermo fisher scientific ) supplemented with 1% protease inhibitor cocktail and 1% phenylmethylsulfonyl fluoride . aliquots containing 50  g of protein were loaded onto an 8% sds - page gel , transblotted onto a polyvinylidene difluoride ( pvdf ) membrane ( bio - rad laboratories , usa ) , blocked for 1  h at room temperature with 5% bovine serum albumin in tbs buffer , and subsequently incubated overnight with respective primary antibodies against p65 , p38 , erk , caspase-9 , caspase-3 , and  - actin ( internal control ) . the membrane was then incubated with secondary hrp conjugated antibodies ( zb 2301 , zhongshan golden bridge biotechnology co. ltd . , the bound complexes were detected with a chemiluminescence solution ( ecl ) purchased from millipore ( # wbkls0050 , merck millipore , usa ) according to the manufacturer 's instructions . the immunoblot bands were quantified using densitometric analysis , and the ratio of each protein to  - actin was calculated and normalized to the values of the mice fed the normal chow ( set as 1 ) . slices of liver tissue were fixed in 10% formalin for 1 week at room temperature . the specimens were subsequently dehydrated in a graded series of ethanol solutions , cleared in xylene , and embedded in paraffin wax . tissue blocks were sectioned into sections of 5  m thickness using a rotary microtome and stained with hematoxylin and eosin ( h&e ) . histological changes in the stained sections were inspected under a light microscope by a pathologist without knowledge of the experimental groups the samples belonged to . portion of liver ( about 1  mm ) from the control and experimental groups of mice was fixed in 3% glutaraldehyde in sodium phosphate buffer ( 200  mm , ph 7.4 ) for 3  h at 4c . tissue samples were washed with the same buffer and additionally fixed in 1% osmium tetroxide and in sodium phosphate buffer ( 200  mm , ph 7.4 ) for 1  h at 4c . the samples were washed again with the same buffer for 3  h at 4c , dehydrated with a graded series of ethanol solutions , and embedded in araldite . thin sections were cut using a lkbum4 ultramicrotome equipped with a diamond knife ( diatome , aldermaston , berkshire , england ) , mounted on a copper grid , and stained with 2% uranyl acetate and reynolds lead citrate . the grids were examined under a hitachi h-7650 transmission electron microscope ( hitachi high - technologies corporation , japan ) . means and standard deviations were used for statistical analysis . significance of differences between means was evaluated using one - way analysis of variance ( anova ) ; frequency data were compared using ridit scoring . table 1 presents the changes in body weight , blood glucose levels , and plasma insulin levels in normal and diabetic mice treated with qtl . qtl treatment did not affect weight gain , blood glucose levels , and insulin levels of normal mice ; however it significantly lowered the weight gain ( p < 0.01 ) , blood glucose levels ( p < 0.01 ) , and insulin levels ( p < 0.01 ) within the diabetic groups ( table 1 ) . as shown in figures 1(a ) and 1(b ) , the glucose tolerance and auc ( area under curve ) of the diabetic control mice were significantly worse than those of normal controls ( p < 0.05 , p < 0.01 , resp . ) , and both the diabetic and control groups treated with qtl showed a significant improvement ( p < 0.05 , p < 0.01 , resp . ) in their tolerance performance . 
 table 2 shows the effect of qtl on lipid metabolic parameters . at termination , tg , tc , and ldl - c levels in the diabetic groups were significantly ( p < 0.01 ) higher than those from normal controls , indicating a disorder of lipid metabolism in the untreated diabetic groups ( table 2 ) . qtl treatment in the diabetic groups significantly ( p < 0.01 ) decreased tg and tc levels . additionally , hdl - c levels were significantly ( p < 0.01 ) lower in diabetic groups compared to those of the normal diet and normal diet supplemented with qtl groups . even although qtl treatment did not result in significant downregulation of ldl - c levels ( p > 0.05 ) in the diabetic mice ( table 2 ) , it significantly ( p < 0.01 ) improved hdl - c levels in this group . as shown in figures 2(a ) and 2(b ) , qtl had no notable effect on the serum no levels and inos activity of normal mice . however , hfd - alloxan induced diabetic mice showed significantly ( p < 0.01 ) enhanced hepatic no levels and inos activity , and qtl treatment led to a significant restoration of no levels and inos activity towards normal values ( p < 0.01 , p < 0.01 , resp . ) when compared with nontreated diabetic mice ( figures 2(a ) and 2(b ) ) . alt and ast activity in diabetic mice was significantly higher ( p < 0.01 , p < 0.01 , resp . ) than that in normal mice and normal mice treated with qtl ( figures 2(c ) and 2(d ) ) . after 42  d of oral qtl administration , the alt and ast activity in the treated diabetic mice was lower than that in the untreated diabetic mice , and no significant difference ( p > 0.05 ) was found for alt activity between normal mice and qtl treated diabetic mice . the higher levels of alt observed in the diabetic mice could indicate a certain degree of hepatic damage , and these results consequently indicated that qtl protects against hepatic injury induced in type 2 diabetic mice . in the control animals , a normal liver architecture was observed with a normal central vein and dense cytoplasm with no granulocytes . in the diabetes model group , the liver showed marked alterations as evidenced by extensive cell necrosis , congestion , karyolysis , infiltration , and inflammation ( figure 3 ) . administration of qtl to diabetic mice ameliorated these deleterious effects in a dose - dependent manner . the ultrastructural changes observed in the hepatocytes of normal and diabetic mice are shown in figures 4(a)4(d ) . figure 4(a ) is representative of the electron micrographs of hepatocytes of normal mice showing normal cellular organelles , mitochondria , and rough endoplasmic reticulum , as well as a nucleus with intact nuclear membrane and normal chromatin . similar architecture is observed in the electron micrographs of normal mice treated with qtl ( figure 4(b ) ) . the electron micrographs of hepatocyte of diabetic mice ( figure 4(c ) ) revealed the decrease of organelle regeneration , swelling in the cisternae of the rough endoplasmic reticulum and mitochondrial cristae , fusion or disappearance of mitochondrial crests , degranulation of rough endoplasmic reticulum , increased smooth endoplasmic reticulum and lipid accumulation , and cells with dark and light cytoplasm . the electron micrograph of cells from treated diabetic animals ( figure 4(d ) ) shows the protective effect of qtl on hepatocyte in diabetic mice , as illustrated by either absent or significantly reduced swelling of the rough endoplasmic reticulum cisternae and mitochondrial cristae , reduction of smooth endoplasmic reticulum , and accumulation of parallel rough endoplasmic reticulum cisternae dotted with ribosomes . the expression levels of p65 , p38 , erk , caspase-9 , and caspase-3 proteins in the liver were analyzed ( figure 5 ) . a clear increase in nf-b / p65 protein levels was produced in the diabetic mice ( p < 0.01 ) , induced by treatment with hfd - alloxan . western blotting and the subsequent measurement of band intensity relative to -actin indicated that treatment with qtl markedly ( p < 0.01 ) inhibited hfd - alloxan induced nf-b the expression of p38 and erk was significantly increased ( p < 0.01 and p < 0.01 , resp . ) in diabetic mice compared to normal mice , whereas only erk enhanced levels were significantly decreased by qtl administration ( p < 0.01 ) . caspase-9 and caspase-3 protein expressions were also higher in diabetic mice than normal mice , and again the expression levels of markers were significantly decreased in the qtl treated group ( p < 0.01 and p < 0.01 , resp . ) over the years , various medicinal plants and their extracts rich in polyphenolic / flavonoid compounds have been reported to be effective against reactive oxygen species mediated damage by enhancing antioxidants defenses and reducing hyperglycemia in alloxan or streptozotocin - induced diabetes . in the present study , we investigated the possible usefulness of the potent antioxidant quercetin isolated from t. sinensis leaves in a mouse diabetes model induced by hfd - alloxan treatment . previous studies showed that 0.12  g / kg  b.w and 0.06  g / kg  b.w . of total flavonoid from t. sinensis could significantly decrease the blood glucose levels in mice with alloxan induced diabetes . a study by cao et al . demonstrated that 100  mg / kg  b.w quercetin ameliorated intracellular stress , led to better regulated gene expression , and reduced embryonic malformations in a diabetic pregnancy model . jeong et al . fed a diet containing 0.04% quercetin ( equivalent to 60~120  mg / kg  b.w . ) or 0.08% of the diet ( equivalent to 120~240  mg / kg  b.w . ) to c57bl / ksj - db / db mice for 6 weeks . the results showed quercetin to be effective in improving hyperglycemia , dyslipidemia , and antioxidant status in type 2 diabetes . evaluated the effects of quercetin intake on cardiovascular risk factors and inflammatory biomarkers in 72 women with type 2 diabetes in a double - blind randomized clinical trial . the results found that quercetin supplementation reduced systolic blood pressure significantly but had no effect on other cardiovascular risk factors and inflammatory biomarkers . considering the biological effects of quercetin , we chose 200  mg / kg  b.w . daily oral treatment of normal mice with qtl ( 200  mg / kg  b.w . ) for 49  d led only to nonsignificant differences in body weight gain , blood glucose , insulin , tc , hdl - c levels , alt activity , no levels , and inos activity . qtl significantly reduced the tg , ldl - c levels , and ast activity in the sera of normal mice . furthermore , qtl induced a significant decline in the protein levels of bax , caspase-9 , and caspase-3 in livers of normal mice . quercetin was reported to possess -glucosidase inhibitory activity in vitro   as well as a hyperglycemia ameliorating activity in vivo which most likely exerts by blunting free radical induced toxicity in type 2 diabetes mellitus . in the present study , administration of qtl to diabetic mice led to a significant decrease in body weight gain and fasting blood glucose , as well as an increase in serum insulin levels . metabolic disturbances and their consequences in diabetes mellitus are well known , and hyperlipidemia is one of a set of the disorders characteristics of diabetes . some studies also showed that quercetin can significantly decrease blood cholesterol levels in rats fed with a high cholesterol diet that induces lipemic - oxidative injury , leading the authors to consider it as an effective hypocholesterolemic agent . we have also assessed the serum levels of tc , tg , ldl - c , and hdl - c , and qtl treatment that resulted in a significant recovery in the levels of these lipid profile biosensors . this shows that qtl has the ability to lower the level of ldl cholesterol while at the same time boosting the hdl cholesterol level . it has been reported that diabetic mice had significantly increased levels of inos expression and activity in the serum [ 37 , 38 ] . excess no produced by inos has been reported to induce deleterious effects in the livers of diabetic mice . our data showed that treatment with qtl caused a marked reduction in no production and inos activity in the serum . these parameters showed that qtl suppressed the oxidative stress caused by hfd - alloxan induced diabetes . epidemiological studies show that diabetic patients are at higher risk of developing chronic liver diseases and hepatocellular carcinoma . in experimental models of diabetes , both a high - carbohydrate / high - fat diet and alloxan exert their toxic effects on the liver and other organs in addition to pancreatic -cells . the insulin insufficiency and hyperglycemia that result from -cell necrosis further augment liver damage through reactive free radical mediated lipid peroxidation of hepatocellular membrane [ 39 , 40 ] . in this study , the diabetic hyperglycemia induced by hfd - alloxan treatment produced elevated levels of ast and alt in the serum , which are considered highly indicative of liver dysfunction . these results , together with the observed histological changes , indicated that qtl protects against hepatic injury in diabetic mice . accumulating evidence indicates that hyperglycemia induced oxidative stress leads to peripheral tissue injury in diabetic patients [ 4143 ] . oxidative stress is associated with the modulation of nf-b , mapk , and caspase dependent pathways in experimental model of diabetic liver injury [ 4446 ] . nf-b is one of the most ubiquitous transcription factors and regulates the expression of genes required for cellular proliferation , the inflammatory response , and cell adhesion . the expression of inos is also regulated by nf-b , which is ubiquitous in the cytoplasm as a p50 and p65 heterodimer [ 47 , 48 ] . jnk and p38 mapk are identified as  stress activated kinases  that are generally related to inflammation and apoptosis . erk supports cellular proliferation by responding to epidermal growth factors ( egfs ) and other extrinsic mitogenic signals . in this study , hfd - alloxan treatment was able to activate the nf-b and mapk pathways in mouse hepatocytes of mice , whereas the expressions of p65 and erk proteins were markedly reduced by qtl administration . the caspase cascade is the most prevalent pathway for apoptotic signal transduction in liver cells . activation of caspase-3 is a key step in both the extrinsic and intrinsic apoptotic pathways and is often considered to be the point of no return in the apoptotic signaling cascade . interestingly , the present study revealed that qtl treatment of diabetic mice significantly suppressed hepatic caspase-9 and caspase-3 protein expression . the results presented here suggest that qtl could prevent apoptosis - induced hepatic damage , at least in part , through the amelioration of oxidative stress - induced diabetic liver injury . the present study shows that quercetin from toona sinensis leaves exhibits significant antihyperglycemic and liver cell protective effects in a high - carbohydrate / high - fat diet and alloxan induced mouse model of diabetes . the observed liver cell protection by quercetin may involve inhibition of pathways related to oxidative stress . these results will be crucial to understand the role t. sinensis leaves may play as alternative or supplementary therapies for clinical treatment of diabetes .
the development of diabetes mellitus is related to oxidant stress induced by a high carbohydrate / high - fat diet ( hfd ) . 
 quercetin , as a major bioactive component in toona sinensis leaves ( qtl ) , is a natural antioxidant . 
 however , the exact mechanism by which qtl ameliorate diabetes mellitus is still unknown . in this study 
 , we investigated the hypoglycemic effects and hepatocytes protection of qtl on hfd and alloxan induced diabetic mice . 
 intragastric administration of qtl significantly reduced body weight gain , serum glucose , insulin , total cholesterol , triglyceride , low density lipoprotein - cholesterol , alanine aminotransferase , and aspartate aminotransferase serum levels compared to those of diabetic mice . 
 furthermore , it significantly attenuated oxidative stress , as determined by lipid peroxidation , nitric oxide content , and inducible nitric oxide synthase activity and as a result attenuated liver injury . 
 qtl also significantly suppressed the diabetes - induced activation of the p65/nf-b and erk1/2/mapk pathways , as well as caspase-9 and caspase-3 levels in liver tissues of diabetic mice . 
 finally , micrograph analysis of liver samples showed decreased cellular organelle injury in hepatocytes of qtl treated mice . 
 taken together , qtl can be viewed as a promising dietary agent that can be used to reduce the risk of diabetes mellitus and its secondary complications by ameliorating oxidative stress in the liver .