Supplementation of Citrus maxima fruits peel powder improves glucose intolerance and prevents oxidative stress in liver of alloxan-induced diabetic rats

Abstract

The present investigation was conducted to evaluate the effect of Citrus maxima fruit peel supplementation in alloxan-induced diabetic rats. Diabetes was induced in male Long Evans rats by intraperitoneal injection of alloxan monohydrate (90 mg/kg body weight). Blood glucose level, oral glucose tolerance, and liver enzyme markers were evaluated. Moreover, histopathological examinations were also conducted using in liver sections to examine inflammation and fibrosis in the liver. Alloxan administered animals showed significant body weight loss and poor glucose tolerance. Alloxan administration also increased the liver marker enzymes’ activities and increased oxidative stress parameters compared to control rats. Citrus maxima fruit peel supplementation for 21 days significantly (p < 0.05) reverted the glucose intolerance and liver enzymes activities to near normal levels. Moreover, Citrus maxima fruit peel supplementation prevented oxidative stress in liver of alloxan-induced diabetic rats. Our investigation also showed that alloxan administration in rats causes inflammatory cells’ infiltration and fibrosis in the liver which is ameliorated by Citrus maxima fruit peel supplementation. Our investigation suggests that Citrus maxima fruit peel supplementation can ameliorate alloxan-induced diabetes and its complications. The antioxidant properties of the fruit probably play a major role in the observed effects.

Keywords

Alloxan inflammation fibrosis oxidative stress

Abbreviation

AGEs

advanced glycation end-products

AID

alloxan-induced diabetic

ALT

alanine aminotransferase

ALP

alkaline phosphatase

ANOVA

Anlysis of varience

APOP

advanced protein oxidation product

AST

aspartate aminotransferase

AUC

Area under the Curve

CMPP

Citrus maxima fruit peel powder

Diabetes mellitus

GLUT2

Glucose Uptake Transporter 2

GPx

glutathione peroxidase

GSH

reduced glutathione

MDA

malondialdehyde

NAFLD

non-alcoholic fatty liver disease

nitric oxide

OGTT

Oral Glucose Tolerance Test

PPG

Peak plasma glucose

ROS

reactive oxygen species

SOD

superoxide dismutase

1 Introduction

Diabetes mellitus (DM) is a chronic metabolic disease that has complicated the lives of millions of people worldwide. Beside other manifestations, the main hallmark of this disease is a deficiency of secretion and reduced activity of endogenous insulin, leading to a hyperglycemic condition [1]. Over a long time, diabetes leads to a lot of debilitating complications such as retinopathy, nephropathy, peripheral neuropathy, etc. [1, 2]. For some time now, it has been recognized that DM could also lead to hepatic damage, although the exact mechanism of hepatic dysfunction in diabetes has not been completely elucidated [3]. Markers of hepatic enzyme activity such as alanine aminotransferase (ALT), and alkaline phosphatase (ALP) and aspartate aminotransferase (AST) have been found to increase in plasma of diabetic animals, suggestive of damage to the liver [4]. Indeed, examination of liver tissue has demonstrated the occurrence of lesions, steatohepatitis and periportal fibrosis in diabetic animals [5].

DM is a condition that often needs to be controlled through lifestyle modifications on the part of the patient. These include the adoption of regimens of regular exercise or modifications of diet such as intake of food with low sugar content etc. [1]. Recently, interest has revived in the scientific and medical community to use natural product supplementation in the diet to treat chronic diseases for which few or no satisfactory therapeutic agents exist. Apart from a chronic hyperglycemic condition, DM is associated with other pathological irregularities such as lipoprotein abnormalities and cellular oxidative stress [2, 6]. Natural plant products that are rich in hypoglycemic and antioxidant properties would, therefore, be of benefit in DM.

Citrus fruits are good examples of plant products that are highly rich in antioxidant chemicals. The plants which bear these fruits, i.e., Citrus plants, belong to the family Rutaceae [7, 8]. These plants are largely found in certain South Asian and Mediterranean countries [7]. Citrus fruits largely owe their antioxidant properties to the presence of high concentrations of polyphenolic compounds known as flavonoids [8]. A particular category of flavonoids, the flavanones, are relatively abundant in Citrus fruits. Citrus flavanones are often present in both the aglycone and glycoside forms. Important flavanones include the aglycones naringenin and hesperetin, and their glycoside forms, naringin, neohesperidin, narirutin, and hesperidin. Naringin and neohesperidin are neohesperidosides whereas narirutin and hesperidin are rutinosides [9]. Flavanones are potent scavengers of free radicals [10]. For example, supplementation with naringin in the diet increased cellular levels of antioxidant enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) in diabetic animals [11, 12]. In addition to their antioxidant capabilities, several of these flavonoids have marked anti-inflammatory, anti-diabetic, and hepatoprotective activities [9 , 13]. Hesperidin and naringin have been shown to improve hyperglycemia through regulation of activities of hepatic glucose metabolizing enzymes that are involved in glycolysis and gluconeogenesis [14]. Use of naringin in particular in high-carbohydrate, high-fat diet fed obese rats led to attenuation of hepatic lipid accumulation and fibrosis relative to controls [15].

Citrus fruits, being rich in these flavonoids, are therefore a potential source of naturally derived chemicals that would be of benefit in ameliorating diabetic conditions and associated long-term hepatic complications. Citrus maxima is a citrus plant that is indigenous to Bangladesh, India and East Asia [7]. The major flavonoids it contains are neohesperidin and naringin [16]. Compared to other citrus species, its role in different disease conditions has been largely unexplored. The fruit of this species is referred to as pomelo in English and is widely consumed by the indigenous population. It is the fruit pulp that is consumed, and the fruit peel is mostly thrown away. However, the peel contains the highest concentration of flavanones as compared to the pulp and seeds [13, 17]. Therefore, a rich source of beneficial flavonoids largely goes to waste. The peel also contains high amounts of other phenolic derivatives such as caffeic acid, (-)-epicatechin, gallic acid, etc. which are also quite powerful antioxidants [7]. Alloxan, a selectively cytotoxic drug, was chosen to produce diabetic animal models. Alloxan induces diabetes through the destruction of pancreatic beta cells, halting the production of insulin. In this study, using alloxan-induced diabetic rat models; the potential beneficial effect of CMPP on diabetes and diabetes-associated hepatic complications were investigated.

2 Materials and methods

2.1 Plant sample collection and identification

Citrus maxima fruits were collected from the local market, and the peels were removed and sundried to prepare coarse powder. Citrus maxima fruits were also identified by the expert Mr. Sarker Nasir Uddin, Senior Scientific Officer, National Herbarium, Mirpur, Dhaka, Bangladesh. A voucher specimen (Acc. number 40844) was deposited in National Herbarium, Dhaka, Bangladesh, for future reference.

2.2 Ethical approval number

The study protocol was approved by Ethical Committee of Department of Pharmaceutical Sciences, North South University for animal care and experimentation. The ethical approval number is AEC 001-2015 which was approved on 05-08-2015.

2.3 Induction of diabetes in rats

The Animal House of Department of Pharmaceutical Sciences, North South University supplied us Long Evans male rats (190–270 g, 12–14 weeks of age). These animals were kept in individual cages. The room temperature was maintained at 25±3°C with a 12 dark/light cycles. All the animals had free access to standard laboratory feed and water ad libitum. Rats were treated with alloxan at a dose of 90 mg/kg subcutaneously and given glucose solution orally to prevent immediate hypoglycemia due to alloxan administration. Several days later, blood glucose levels were checked for hyperglycemia e.g. fasting blood glucose > 9 mmol/L which is considered as diabetes. Hyperglycemic rats were sorted out from the normoglycemic rats and used in further studies.

2.4 Animals and treatment

To study the effects of Citrus maxima, rats were equally divided into four groups (six rats in each group)-

Group I (Control)- Rats were supplemented with normal food and water every day for three weeks.

Group II (Diabetic)- Rats were supplemented with normal food and water every day for three weeks.

Group III- Control rats were supplemented with Citrus maxima (0.5% supplement with 100 g crushed food) every day for three weeks.

Group IV- Diabetic rats were supplemented with Citrus maxima (0.5% supplement with 100 g crushed food) every day for three weeks.

Animals were checked for variations in body weight on a daily basis. After 21 days, all animals in all groups were weighed and kept in food deprivation state (but normal water was provided in this stage) for 14 hours to perform the oral glucose tolerance test (OGTT). A glucose solution of 2 gm/kg was administered to each rat through gastric gavage (orally) and blood glucose level was monitored in tail blood by a glucometer at every 30 minutes up to 120 minutes. The next day, all rats were sacrificed using a high dose of ketamine anesthesia. Blood was collected from the abdominal aorta immediately after deep anesthesia from each rat in tubes containing citrate buffer solution, and all internal organs such as pancreas, heart, kidney, spleen, and liver were also harvested. These organs were then weighed immediately after collection, and stored in neutral buffered formalin (pH 7.4) for histological analysis and in the freezer at –20°C for further studies. The collected blood was centrifuged at 8000 rpm, the plasma separated and then stored in a refrigerator at –20°C for further analysis.

2.5 Assessment of hepatotoxicity

Liver function marker enzymes such as- alanine aminotransferase (ALT), and alkaline phosphatase (ALP) and aspartate aminotransferase (AST), were estimated in plasma by using commercial kits (Diatec diagnostic kits, Hungary) according to the manufacturer’s protocol.

2.6 Preparation of tissue sample for the assessment of oxidative stress markers

For determination of oxidative stress markers, heart, liver and kidney tissue were homogenized in 10 volumes of phosphate buffer containing (pH 7.4) and centrifuged at 12,000×g for 30 min at 4°C. The supernatant was collected and used for the determination of protein and enzymatic studies as described below.

Lipid peroxidation in plasma and liver were estimated by measuring thiobarbituric acid reactive substances (TBARS) following the previously described method [18]. Nitric oxide (NO) was determined according to the method described by Tracy et al. as nitrate and nitrite [19]. NO level was measured by using the standard curve and expressed as nmol/gm of tissue. Determination of AOPP levels was performed by the method previously described by Witko-Sarsat [20] and Tiwari [21]. CAT activities were determined using a previously described method by Chance and Maehly [22]. Reduced glutathione was estimated by the method of Jollow et al. [23].

2.7 Histopathological determination

For microscopic evaluation, liver tissue was fixed in neutral buffered formalin and embedded in paraffin, sectioned at five μm and subsequently stained with hematoxylin/eosin to observe the architecture of liver tissue and inflammatory cell infiltration. Sirius red staining was also performed in each group for the determination of fibrosis. Stained sections were then studied and photographed under a light microscope (Zeiss) at 40X magnification.

2.8 Statistical analysis

All values are expressed as mean±standard error of the mean (SEM). The results were evaluated using the One-way ANOVA followed by Bonferroni or Newman- Keul’s test using Graph Pad Prism Software. Statistical significance was considered at p < 0.05 in all cases.

3 Results

3.1 Effect on organ wet weight

Table 1 shows the effect of various treatments on the rats’ organ wet weight. Both the liver and spleen wet weight were higher in the AID group of rats relative to the control groups. Treatment with CMPP prevented the increase in liver and spleen wet weight of AID rats.

Table 1
Effect of Citrus maxima on body weight, and organ weight of alloxan-treated rats

Parameters Control Alloxan Control+ Citrus maxima Alloxan+ Citrus maxima

Initial Body weight (g) 184.27±0.91ns 183.42±0.59ns 197.67±1.84ns 207.15±4.62ns

Final Body weight (g) 240.70±1.63a 203.62±7.57b 242.42±3.14a 264.38±3.34a

Food Intake (g) 19.60±0.27ns 19.18±0.22ns 22.21±1.89ns 20.60±1.66ns

Water Intake (g) 20.00±0.25a 21.03±0.11a 23.57±2.36a 21.73±1.84a

Liver wet weight (g/100 g of body weight) 3.47±0.17ns 3.93±0.23ns 3.53±0.15ns 3.54±0.04ns

Spleen wet weight (g/100 g of body weight) 0.30±0.02a 0.44±0.04b 0.33±0.01a 0.34±0.01a

Parameters	Control	Alloxan	Control+ Citrus maxima	Alloxan+ Citrus maxima
Initial Body weight (g)	184.27±0.91ns	183.42±0.59ns	197.67±1.84ns	207.15±4.62ns
Final Body weight (g)	240.70±1.63a	203.62±7.57b	242.42±3.14a	264.38±3.34a
Food Intake (g)	19.60±0.27ns	19.18±0.22ns	22.21±1.89ns	20.60±1.66ns
Water Intake (g)	20.00±0.25a	21.03±0.11a	23.57±2.36a	21.73±1.84a
Liver wet weight (g/100 g of body weight)	3.47±0.17ns	3.93±0.23ns	3.53±0.15ns	3.54±0.04ns
Spleen wet weight (g/100 g of body weight)	0.30±0.02a	0.44±0.04b	0.33±0.01a	0.34±0.01a

Data are presented as mean±SEM, n = 6. Statistical analysis was done as One way ANOVA with Bonferroni post hoc test, which was conducted using Prism software (USA). Statistical significance was considered as p < 0.05 in all cases. b vs a, signifies alloxan vs. control or other treatment groups which are significantly different at p < 0.05. ns- not significant.

3.2 Effect on plasma glucose concentration in the oral glucose tolerance test (OGTT)

Plasma glucose levels of AID rats were higher than that of the other groups of rats throughout 120 minutes of the Oral Glucose Tolerance Test (OGTT). The peak plasma glucose (PPG) concentration was reached at 30 minutes in all groups of rats. The PPG concentration in AID rats was 12 mmol/L compared to about 8 mmol/L in the other groups. Figure 1 shows that the Area under the Curve (AUC) of plasma glucose concentration over time was significantly higher for the AID rats group compared to both the control groups. Supplementation of CMPP in AID rats significantly brought down the AUC in comparison to the control groups.

Fig.1

Effect of Citrus maxima on oral glucose tolerance test in alloxan-induced diabetic rats. Data are presented as mean±SEM, n = 6. Statistical analysis was done as One way ANOVA with Bonferroni post hoc test, which was conducted using Prism software (USA). Statistical significance was considered as p < 0.05 in all cases.

3.3 Effect on biochemical parameters of liver functions

Biochemical analyses of plasma and liver functions revealed that AID rats exhibited a significantly higher level of plasma AST, ALT, and ALP concentrations in comparison to the control group (Table 2). CMPP supplementation in AID rats precluded the rise in the plasma levels of these liver activity biomarkers. Control group of rats treated with CMPP alone exhibited plasma levels of liver enzymes comparable to that of the control group.

Table 2
Effect of Citrus maxima on biochemical parameters of alloxan-treated rats

Parameters Control Alloxan Control + Citrus maxima Alloxan + Citrus maxima

Plasma

AST(U/L) 27.18±2.65a 57.42±4.26b 30.15±1.93a 37.32±6.55a

ALT(U/L) 28.71±1.82a 55.99±6.58b 25.84±3.15a 34.45±3.15a

ALP(U/L) 53.26±3.62a 81.07±5.76b 40.81±2.85a 47.49±4.35a

MDA(nmol/mL) 16.94±1.70a 41.05±2.18b 26.07±2.23a 21.97±1.42a

NO (nmol/mL) 7.63±0.51a 10.93±0.93b 4.49±0.19a 5.93±0.38a

APOP 384.44±27.78a 575.71±36.01b 325.71±16.89a 378.89±17.97a

GSH (nmol/mL) 11.43±0.73a 7.16±0.24b 11.72±1.0a 9.04±0.37a

Catalase (U/min) 6.50±0.50ns 4.0±0.37ns 6.00±0.68ns 7.17±1.11ns

Liver

NO (nmol/ g tissue) 14.55±1.74a 21.57±0.86b 11.92±0.75a 14.80±0.43a

MDA (nmol/ g tissue) 43.77±1.20a 69.67±3.79b 48.38±2.67a 56.72±3.17a

APOP (nmol/ g tissue equivalent of Chloramine T) 681.59±55.13a 1000.63 + 33.75b 640.32±32.68a 637.14±48.69a

GSH (ng/g tissue) 18.23±1.81a 11.35±0.78b 19.38±2.29a 15.42±0.57a

Parameters	Control	Alloxan	Control + Citrus maxima	Alloxan + Citrus maxima
Plasma
AST(U/L)	27.18±2.65a	57.42±4.26b	30.15±1.93a	37.32±6.55a
ALT(U/L)	28.71±1.82a	55.99±6.58b	25.84±3.15a	34.45±3.15a
ALP(U/L)	53.26±3.62a	81.07±5.76b	40.81±2.85a	47.49±4.35a
MDA(nmol/mL)	16.94±1.70a	41.05±2.18b	26.07±2.23a	21.97±1.42a
NO (nmol/mL)	7.63±0.51a	10.93±0.93b	4.49±0.19a	5.93±0.38a
APOP	384.44±27.78a	575.71±36.01b	325.71±16.89a	378.89±17.97a
GSH (nmol/mL)	11.43±0.73a	7.16±0.24b	11.72±1.0a	9.04±0.37a
Catalase (U/min)	6.50±0.50ns	4.0±0.37ns	6.00±0.68ns	7.17±1.11ns
Liver
NO (nmol/ g tissue)	14.55±1.74a	21.57±0.86b	11.92±0.75a	14.80±0.43a
MDA (nmol/ g tissue)	43.77±1.20a	69.67±3.79b	48.38±2.67a	56.72±3.17a
APOP (nmol/ g tissue equivalent of Chloramine T)	681.59±55.13a	1000.63 + 33.75b	640.32±32.68a	637.14±48.69a
GSH (ng/g tissue)	18.23±1.81a	11.35±0.78b	19.38±2.29a	15.42±0.57a

Data are presented as mean±SEM, n = 6. Statistical analysis was carraied out as One way ANOVA with Bonferroni post hoc test, using Prism software (USA). Statistical significance was considered as p < 0.05 in all cases. b vs a, signifies alloxan vs. control or other treatment groups which are significantly different at p < 0.05. ns- not significant.

3.4 Effect on oxidative stress markers and antioxidant enzymes

To assess the oxidative stress of alloxan-induced diabetes and effect of CMPP supplementation, the malondialdehyde (MDA), nitric oxide (NO), advanced protein oxidation product (APOP), and reduced glutathione (GSH) levels in the plasma and liver were measured (Table 2). Relative to the control group, the concentration of lipid peroxidation product malondialdehyde (MDA) was significantly higher in both the liver homogenate (69.67±3.79 nmol/mL vs. 43.77±1.20 nmol/mL) and the plasma (41.05±2.18 nmol/mL vs. 16.94±1.70 nmol/mL) of AID rats. AID rats supplemented with CMPP had a plasma and liver MDA levels much lower than that of AID rats without CMPP supplementation (liver: 56.72±3.17 vs. 69.67±3.79 nmol/mL; plasma: 21.97±1.42 vs. 41.05±2.18 nmol/mL). However, the CMPP supplemented AID rats had MDA levels still higher than that of the control group of rats.

On a similar note, the plasma and liver levels of nitric oxide (measured as nitrate) and advanced peroxidation products (APOP) were found to be significantly elevated in AID rats compared to the control group. The concentration of NO in liver homogenates and plasma of AID rats were 21.57±0.86 nmol/mL and 10.93±0.93 nmol/mL respectively compared to 14.55±1.74 nmol/mL and 7.63±0.51 nmol/mL in control rats. Concentration of APOP in liver homogenates and plasma of AID rats were 1000.63±33.75 nmol/mL and 575.71±36.01 nmol/mL respectively compared to 681.59±55.13 nmol/mL and 384.44±27.78 nmol/mL in control rats. Treatment with CMPP in AID rats was able to significantly prevent a rise in plasma and liver NO levels that were seen in AID rats without CMPP supplementation. It was noted that CMPP supplementation, when given to a control group of rats, produced a decrease in plasma and liver NO and APOP levels below that of the levels found in the control group without CMPP supplementation.

The level of antioxidant enzyme GSH was also affected by alloxan-induced diabetes. AID rats showed a significant decrease in liver and plasma concentration of GSH compared to the controls (liver: 11.35±0.78 nmol/mL vs 18.23±1.81 nmol/mL; plasma: 7.16±0.24 nmol/mL vs 11.43±0.73 nmol/mL). CMPP supplementation prevented the attenuation of GSH levels in both liver and plasma, although the GSH levels were lower than that of the control group. It was also observed that GSH levels in liver and plasma of control rats given CMPP supplementation were higher than the control rats without CMPP supplementation. However, no significant changes were observed in catalase activity in plasma among the groups tested.

3.5 Liver inflammation and fibrosis markers

Inflammation in the liver was seen in rats treated with Alloxan. A massive surge of inflammatory cells was found in the centrilobular part of a liver section stained with H & E staining in alloxan-treated rats group (Fig. 2). Necrotized tissue scar and ballooning of the hepatocytes were also seen in the liver of alloxan-treated rats (Fig. 2). CMPP supplementation prevented the inflammatory cell infiltration in the liver of alloxan-induced diabetic rats (Fig. 2). Liver fibrosis was evaluated histologically by visualizing the red color of collagen fibers using Sirius red stain. In sections stained with Sirius red, the connective tissue (CT) septa, containing collagen fibers, were not demarcated around the classical hepatic lobules in control rats (Fig. 3). In contrast, the collagen fibers were heavily deposited around portal tracts and central veins in the Alloxan-intoxicated group and extended from central vein to portal tract (Fig. 3). CMPP supplementation prevented the collagen deposition and fibrosis in the liver of alloxan-induced diabetic rats (Fig. 3).

Fig.2

Effect of Citrus maxima peel powder supplementation on hepatic inflammation in alloxan-induced diabetic rats. A, Control; B, Control+ Citrus maxima; C, Alloxan; D, Alloxan+ Citrus maxima. Magnification 40 X.

Fig.3

Effect of Citrus maxima peel powder supplementation on hepatic fibrosis in alloxan induced diabetic rats. A, Control; B, Control+ Citrus maxima; C, Alloxan; D, Alloxan+ Citrus maxima. Magnification 40 X.

4 Discussion

Diabetes is a pathological condition that may induce hepatic damage over time. The current study demonstrates that hyperglycemia, as well as diabetes associated liver damage, is alleviated in alloxan-induced diabetic rats that are given supplements of citrus maxima peel powder (CMPP). Plasma glucose levels were found to be significantly lower in CMPP fed rats compared to controls. This might have been brought about by the action of certain citrus flavonoids on the glucose-regulating enzymes in the liver as evident from some studies. Naringenin has been shown to reduce plasma glucose, triglycerides, and cholesterol in diabetic cells [24]. In a study carried out in db-/db- mice, the flavonoid naringin induced a significant decrease in activities of hepatic glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, enzymes that play a major role in hepatic gluconeogenesis [25]. Caffeic acid, which is present in very high amounts in CMPP [7], has been reported to lower blood glucose through enhancement of enzyme glucokinase and concurrent down regulation of the enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in C57BL/KsJ-db/db mice [26]. Hepatic gluconeogenesis accounts for 50–60% of the endogenous production of glucose [24], and therefore inhibition of key rate-limiting enzymes of this process holds great promise in effective control of blood glucose levels. Another way via which citrus flavonoids in Citrus maxima induce lowering of plasma glucose could be through inhibitory effects on carbohydrate digestion. Amylase-catalysed starch digestion was markedly inhibited by citrus flavonoids hesperidin and naringin [27, 28], and therefore regulation of absorption of carbohydrates in the diet by flavonoids remains a viable option.

Apart from lowering of blood glucose, CMPP also lowered serum activities of hepatic enzymes such as AST, ALT, ALP, etc. in AID rats relative to controls. This was indicative of attenuation of liver damage brought about by alloxan-induced diabetes. This seems to be in line with previous studies where the citrus flavonoid naringin was able to significantly lower raised plasma transaminase activities in rats with heavy metal-induced liver toxicity [29, 30]. Moreover, in hyperglycemic and hypoinsulinemic conditions such as that in diabetes, there is an increase in lipid deposition in the liver [31]. High levels of lipid accumulation in the liver have been linked to increased levels of inflammation, oxidative damage, and hepatic fibrosis [5]. In our study, feeding of CMPP to the alloxan induced rats led to an overall lowering of hepatic lipid deposition. It was also found that hepatic fibrosis in CMPP fed alloxan induced rats was lower than that of alloxan induced rats, suggesting possible beneficial effects of citrus flavonoids on the prognosis of diabetes-induced liver damage. CMPP also contains high levels of (-)-epicatechin [7], a powerful antioxidant, which is known to exert powerful anti-inflammatory effects, thereby, reducing progression of hepatic fibrosis [32, 33]. It is therefore highly likely that the compound above had a significant role to play in attenuating liver damage in our AID rats.

Diabetic conditions lead to an up-regulation of glucose transporter 2 (GLUT2) in the liver, resulting in a higher uptake of carbohydrates into the liver [34]. A certain portion of the carbohydrates is converted into fatty acids in the liver [35], and therefore an increase in hepatic carbohydrate uptake would mean an increase in hepatic fatty acid production. Enhanced conversion of fatty acids to triglycerides and their subsequent storage in parenchymal tissues of the liver is a predisposing factor for the development of non-alcoholic fatty liver disease (NAFLD) and steatohepatitis [36]. Indeed, clinically, the prevalence of NAFLD is significantly higher in diabetic patients than in non-diabetic patients [37]. The fact that CMPP has an inhibitory effect on hepatic lipid deposition is probably due to the presence of certain citrus flavonoids in Citrus maxima, which have been known to alter expression of specific GLUT proteins in the body. Studies have reported significant attenuation of hepatic GLUT2 expression in diabetic mice by naringin and hesperidin [14]. Therefore, it is quite likely that citrus flavonoids in CMPP might have exerted the glucose-lowering action in the liver through down-regulation of hepatic GLUT2 expression.

Presence of high levels of lipids in the liver also increases the chances of oxidative stress as increased oxidation of free fatty acids in liver result in the generation of higher levels of reactive oxygen species (ROS) [38]. A significant increase in ROS and a marked reduction in defensive antioxidants have been reported in the liver tissues of diabetic animals [39]. High ROS levels and increased lipid peroxidation products in the liver are instrumental in bringing about structural and functional change in hepatocytes that ultimately lead to their apoptosis [40]. This is accompanied by secretion of pro-fibrogenic and pro-inflammatory cytokines in the vicinity of those cells, leading to stimulation of hepatic stellate (HSCs) cells [41]. The HSCs once activated start proliferation and produce collagen, deposition of which in the liver is manifested in the form of liver fibrosis [42]. Diabetes has also been known to cause collagen deposition and hepatic fibrosis through another process. Long-term diabetic conditions lead to the production of advanced glycation end-products (AGEs) [43]. AGE formation is a non-enzymatic process where sugar molecules react with proteins and lipids in a series of non-oxidative and oxidative reactions [43]. The receptor for AGEs (RAGE) acts as a transcriptional factor to induce a variety of gene expression [44]. RAGE-activated pathways are known to induce inflammation through pro-inflammatory cytokine production, enhance oxidative stress, and stimulate HS cell activation [44].

In this study, tissue markers of oxidative stress were found to be lower, and the levels of antioxidant enzymes were found to be higher in CMPP fed alloxan induced rats relative to alloxan induced rats. This happens to illustrate another key pathway through which citrus flavonoids could be protective in diabetes-induced liver damage. Earlier, it was pointed out that attenuation of GLUT2 expression was a viable mechanism through which citrus flavonoids could prevent excessive lipid accumulation and thus reduce the formation of possibly damaging lipid peroxidation products. The fact that many citrus flavonoids possess powerful antioxidant capabilities suggest that, in addition to playing a key protective role in the pathway above, flavonoids in CMPP are also able to directly prevent the formation of high levels of ROS and thereby prevent hepatic fibrosis in yet another supplementary fashion. Flavonoids owe their exceptionally powerful scavenging ability of free radicals to their ability to donate hydrogen to radicals and thereby stabilizing them [45]. Among other flavonoids, naringin and naringenin which are found in significant amounts in CMPP, are strong scavengers of superoxide ions and hydroxyl radicals [46]. They also significantly inhibit the enzyme xanthine oxidase which is a source of superoxide ions [46]. Also, citrus flavonoids have anti-inflammatory actions. For example, hesperidin can inhibit kinases and phosphodiesterase enzymes involved in cellular signaling pathways that mediate inflammation [47]. Therefore, CMPP might also protect the liver in diabetic conditions by limiting the recruitment of inflammatory mediators in hepatic tissues.

5 Conclusion

In summary, our investigation showed that CMPP supplementation prevents the glucose intolerance and reduces oxidative stress in alloxan-induced diabetic rats. Moreover, CMPP supplementation prevented the inflammatory cell infiltration and fibrosis in the liver of diabetic rats. This agrees with our previous research where we demonstrated that CMPP supplementation prevented oxidative stress, fibrosis and hepatic damage in rats treated with carbon tetrachloride [7]. The beneficial effect showed by CMPP supplementation in diabetic rats could be attributed to the presence of high amount of polyphenolic antioxidants present in the peel powder. Further studies are required to establish the efficacy of CMPP supplementation in a clinical setup.

Author contribution

Anayt Ulla and Md Ashraful Alam equally contributed in this research, drafted the manuscript and analyzed the data. Anayt Ulla, Mosfiqur Rahman, and D M Isha Olive Khan housekeeping the animals and given all treatment required for the study. Biswajit Sikder, Monirul Islam, Tarikur Rahman and Nilima Rahman participated in tissue sample collection and biochemical analysis. Hasan Mahmud Reza, Preeti Jain and Nusrat Subhan supervised the project and drafted the final version of the manuscript.

Conflict of interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Footnotes

Acknowledgments

The authors gratefully acknowledge the logistic support provided by the Department of Pharmaceutical Sciences, North South University Bangladesh.

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