Ameliorative effect of Citrus aurantifolia and Cinnamomum burmannii extracts on diabetic complications in a hyperglycemic rat model

Purpose: To evaluate the effects of Citrus aurantifolia and Cinnamomum burmannii extracts (Jermanis) on the number of pancreatic β-cells and levels of superoxide dismutase (SOD), low-density lipoprotein (LDL), and transforming growth factor-beta (TGF-β) in β-cells. Methods: Various doses of a combination of extracts of C. aurantifolia and C. burmannii were given orally to 25 male Wistar rats (Rattus norvegicus) every 2 h for 30 days. Doses of 100, 300 and 500 mg/kg/day C. aurantifolia extract were considered low, medium and high doses, respectively, while, 200, 400 and 800 mg/kg/day C. burmannii extract were considered low, medium and high doses, respectively. LDL and SOD levels in blood serum were analyzed spectrophotometrically. TGF-β expression was evaluated by immunohistochemistry. Pancreatic tissue sections (diameter of a β-cell) were evaluated by light microscopy after hematoxylin and eosin staining. Results: The combination of C. aurantifolia and C. burmannii extracts increased SOD levels, TGF-β expression and the number of β-cells and decreased LDL levels in hyperglycemic rats. The results indicate that the highest doses of C. aurantifolia and C. burmannii increased the number of β-cells in the islets of Langerhans. The combined extracts of C. aurantifolia and C. burmannii significantly affected pancreatic cell regeneration. Conclusion: The combination of C. aurantifolia and C. burmannii extracts may be a promising alternative preventative medicine for management of diabetic complications in patients with hyperglycemia.


INTRODUCTION
Diabetes mellitus (DM) is a chronic disease that is increasing globally, including Indonesia.DM is related to economic status and lifestyle changes [1].The prevalence of DM in urban regions is approximately 5.7 % [2,3].In the United Kingdom, DM (all types) is estimated to affect approximately 2.4 million people (prevalence of 4 %), of which 1.4 million (prevalence of 2.5 %) are diagnosed, while 1 million go undiagnosed [4].
If DM is not well controlled, it leads to NAD(P)H oxidation, which induces the production of free radicals [5].Excessive production of free radicals can trigger oxidative stress, which can damage lipids and thereby increase the level of lowdensity lipoprotein (LDL) in the blood [6].Free radicals can damage the smooth muscle cells of blood vessels, which are a source of superoxide dismutase (SOD) [7].
As the effects of free radicals in DM can lead to complications, additional antioxidants are required.Citrus aurantifolia and Cinnamomum burmannii contain flavonoids with antioxidant properties and have a strong protective effect against radiation exposure [8,9].One of the growth factors involved in pancreatic organogenesis is transforming growth factor-beta (TGF-β) [10][11][12].
In this study, hyperglycemic rats were fed a highcholesterol diet and treated with a combination of extracts from C. aurantifolia and C. burmannii, which have antioxidant activity.The effect of the extract treatment on the complications caused by hyperglycemia and the high-cholesterol diet were assessed.A previous study showed that flavonoid antioxidants derived from green tea decreased cholesterol, glucose and triglyceride levels in rats on a high-fat diet [13].Therefore, the aim of this study was to evaluate the effect of a combination C. aurantifolia and C. burmannii extracts on SOD and LDL levels, TGF-β expression and the number of β-cells in the islets of Langerhans in a hyperglycemic rat model.Our results showed that a combination of C. aurantifolia and C. burmannii extracts is a promising therapeutic medicine to prevent diabetic complications.

EXPERIMENTAL Preparation of rats
Twenty-five male rats (Rattus novergicus) were acclimatized under laboratory conditions for 1 week with adequate feeding.On the last day, fasting blood glucose levels were measured, and the rats with normal blood sugar levels (90 -110 mg/dl) were selected.The rats were intraperitoneally injected with streptozotocin at 30 mg/kg body weight (BW).A week after streptozotocin induction, blood glucose levels were measured after fasting for 6 hours.Rats with a sugar level > 250 mg/dl (using a blood glucose meter) were considered hyperglycemic.

Preparation of Citrus aurantifolia and
Cinnamomum burmannii extracts C. aurantifolia juice was filtered using flannelette and then dried using a freeze dryer.C. burmannii was powdered using size 40 mesh.Then, 1 kg C. burmannii powder was dissolved in 7 l 95% ethanol, with occasional stirring for 24 hours, and then filtered.The filtrate was collected, and the precipitate was macerated for 24 hours, and remaceration was conducted for 2-5 days.The collected filtrate was evaporated at 60˚C until it turned into a thick liquid.This thick liquid was considered the extract.
Prior to treatment, rats were adapted to laboratory conditions for 7 days to adjust to the environment (e.g., cage changes and feeding times) and were fed a standard feed diet during this adaptation period.
In groups 3, 4 and 5, a few days after alloxan induction, the rat's blood glucose levels were examined immediately because the extract was given when the blood glucose level was above 200 mg/dl (5-6 days).The treatment was carried out over the course 30 days, then the glucose level, LDL, and SOD were examined.

Examination of fasting blood serum LDL (lowdensity lipoprotein) level
The blood (~ 3 mL) was collected from rats' heart and then centrifuged at 3000 rpm for 20 minutes.Then serum was collected and analyzed.LDL measurements were conducted using specific colorimetric tests (Horiba ABX Diagnostics, Montpellier, France) and an automated analysis system (COBAS MIRA, Roche, Basel, Switzerland).LDL levels were then measured using ABX PENTRA CP (ABX Pentra, Montpellier, France).

Measurement of superoxide dismutase content
The blood (~ 3 mL) was centrifuged at 3500 rpm for 20 min.The supernatant was collected and centrifuged at 6000 rpm for 10 minutes at 4˚C and then transferred to Eppendorf tubes.The supernatant was diluted with xanthine, xanthine oxidase, nitroblue tetrazolium (NBT), and phosphate-buffered saline (PBS).The sample was diluted with PBS to a total volume of 3500 μL and incubated at 30°C for 30 minutes (until the sample turned purple).The SOD level was measured by UV/Vis spectrophotometry (500 -600 nm).

Preparation of pancreatic sections for histological examination
The pancreas was solidified in a paraffin block and then cut into 5 μm sections using a microtome.Sections were dried using a hot plate at 38 -40 °C and then stored in an incubator at 38 -40 °C for 24 h.

Evaluation of transforming growth factor-beta expression
TGF-β expression was determined using immunohistochemical methods.Prepared slides were washed with PBS (pH 7.4) and then 3 % Hydrogen Peroxide (H 2 O 2 ) for 20 min.The slides were washed again with PBS (pH 7.4) for 5 minutes (three times).The slides were blocked with 5 % fetal bovine serum for 1 hour and then washed with PBS (pH 7.4) for 5 min (three times).The prepared slides were incubated with primary anti-rat TGF-β antibody [antibodiesonline GmbH, Schloss-Rahe-Str.15, 52072 Aachen, Germany] overnight at 4 °C and then washed with PBS (pH 7.4) for 5 min (three times).
The slides were incubated with a secondary antibiotin antibody (Santa Cruz Biotechnology, USA) for 1 h at room temperature, washed with PBS (pH 7.4) for 5 min (three times) and incubated with streptavidin-horseradish peroxidase for 40 min.The slides were washed with PBS (pH 7.4) for 5 minutes (three washes) and then incubated for 10 minutes with diaminobenzidine (DAB).
The slides were washed with PBS (pH 7.4) for 5 min (three washes).Counterstaining was performed using Mayer's hematoxylin for 10 min.The slides were washed and dried in Entellan mounting medium and covered with a cover glass.Positive TGF-β expression was indicated by a brown staining color.The percentage of cells positive for TGF-β expression was calculated using Axiovision software.

Pancreatic tissue repair evaluation using hematoxylin and eosin staining
For hematoxylin and eosin staining, tissues were first deparaffinized and then placed in storied xylol for 5 minutes.During the rehydration stage, the prepared tissues were placed in a graded ethanol series (95, 90, 80 and 70 %) for 5 min and then soaked in distilled water for 5 min.Hematoxylin was added for 10 minutes until the sections showed strong staining.The tissues were washed with water for 30 min and then rinsed with distilled water before staining with eosin for 5 min.Then, dehydration was performed by subjecting the tissues to a series of ethanol washes consisting of 80, 90, 95 % and absolute ethanol.The tissues were cleared by placing in 1,2 xylol and were then dried and mounted with Entellan.

Statistical analysis
Data were analyzed using the Statistical Package for the Social Sciences (SPSS) version 1.9.The data were assessed for normal distribution and variance, and differences between groups were determined by one-way ANOVA with Tukey's test.Differences between groups were considered significant at p <0.05.

RESULTS
The results showed that the extracts of C. aurantifolia and C. burmannii had a significant effect on blood glucose levels (p = 0.035) (Figure 1).There was no difference between the negative control group (G1) and the G4 group that consumed C. aurantifolia (300 mg/kg/day) and C. burmannii (400 mg/kg/day).The C. aurantifolia and C. burmannii extracts lowered the glucose level by 53.2 %.The decrease in glucose levels in the other groups were not significant.
The C. aurantifolia and C. burmannii extracts also had a significant effect on SOD levels in the blood (p = 0.00).There was a significant difference between the negative control group (K1) and the groups K3, K4 and K5.However, there was no significant difference between the groups that were treated with the extracts of C. aurantifolia and C. burmannii.The results showed that the highest increase in SOD level occurred when the dose of C. aurantifolia was 300 mg/kg/day and the dose of C. burmannii was 400 mg/kg/day.The extracts of C. aurantifolia and C. burmannii was able to increase the SOD level by 75.86 % in group K4, 73.4 % in group K3 and 66.9 % in group K5 (Figure 2).The LDL levels (Figure 3) showed that the extracts of C. aurantifolia and C. burmannii had a significant effect on cholesterol levels (p = 0.00).There were significant differences among the negative control (G1), G2, G3, G4 and G5 groups, with the lowest LDL levels found in the G1 and G5 groups (treated with 500 and 800 mg/kg/day C. aurantifolia and C. burmannii extracts, respectively).

DISCUSSION
The results showed that extracts of C. aurantifolia and C. burmannii had a significant effect on blood glucose and SOD levels.The extracts lowered the glucose level by 53.2%, and the 300 mg/kg BW/day C. aurantifolia and 400 mg/kg BW/day C. burmannii doses increased the SOD level.The antioxidant defense system stimulates the cellular repair response in the presence of oxidative stress [16].SOD plays an important role in protecting cells from oxidative stress.Oxidative stress is initiated by an increase in the production of free radicals.Auto-oxidation of glucose can decrease the concentration of low-molecular-weight antioxidants in tissue and disrupt the enzymatic activity of antioxidant defenses [17].In DM patients, a decrease in the SOD level is related to production of various markers of oxidative stress, such as lipid hydroperoxide, conjugated dienes and protein carbonyl [18].C. aurantifolia contains high levels of antioxidants.Evaluation of SOD activity in 140 types of plants showed very high SOD activity in C. aurantifolia and C. burmannii [19].
Another benefit of antioxidants is that they minimize the formation of advanced glycation end products (AGEs) in the polyol pathway (sorbitol-aldose reductase pathway).A reduction in tissue accumulation of sorbitol would suppress fructose synthesis; thus, the process of nonenzymatic glycosylation would also be suppressed.Ascorbic acid works extracellularly in blood vessels and intracellularly in endothelial cells.Extracellularly, this antioxidant suppresses formation of superoxide radicals, which are formed during the glucose auto-oxidation process [21].Some citrus species such as C. aurantifolia, Citrus aurantium, Citrus sinensis and Citrus grandis have been confirmed to have antidiabetic properties [22].The flavonoids in C. aurantifolia juice were found to reduce oxidative stress in diabetic rats [23].
Hyperglycemic conditions increase the oxidation of NAD(P)H, which induces the production of free radicals.Excessive free radical production triggers oxidative stress and damages blood vessels [6].Under these conditions, the smooth muscle cells of blood vessel walls become damaged, leading to decreased SOD production [7].C. burmannii is also capable of decreasing blood glucose levels [24].C. burmannii contains methylhydroxychalcone polymer (MHCP) and other substances that increase the uptake of glucose and insulin receptor phosphorylation [23].
The analysis of LDL levels showed that the extracts of C. aurantifolia and C. burmannii had a significant effect on cholesterol levels.Lime extract contains citric acid, which can lower the pH in the digestive tract.Acidic conditions in the gastrointestinal tract stimulate the formation of bile salts to neutralize the acid.Bile salts are the end product of cholesterol metabolism.Under acidic conditions in the digestive system, more cholesterol is metabolized, and the blood cholesterol level decreases [25].Khan et al [24] also found that C. aurantifolia lowered LDL levels in blood.In the treatment group K5, the acidic conditions in the digestive tract stimulated the pancreas to increase the production of liquid sodium bicarbonate formed from cholesterol, leading to a decrease in the blood cholesterol level.The blood cholesterol showed a direct relationship with the LDL level (the higher the blood cholesterol level, the higher the level of LDL and vice versa).
Compared with the control group (K1), higher LDL cholesterol levels were observed in rats with DM that did not receive extract treatment (K2 group).This was due to the high-fat diet given to K2 rats to induce hyperglycemia (DM), which also increased the LDL level.Furthermore, DM patients also suffer from dyslipidemia [23], because DM is associated with altered HDL cholesterol levels due to HDL catabolism.Increased HDL catabolism occurs due to an increase in hepatic lipase activity in liver cells.Glucose levels can be used as an indicator of increased hepatic lipase activity.An increase in glucose levels can occur as a result of insulin resistance [22].
C. aurantifolia contains flavonoid compounds such as naringin and hesperidin, which can increase the extent of hyperlipidemia in animals with type 2 DM by regulating fatty acid and cholesterol metabolism.C. aurantifolia extract also affects the expression of genes encoding enzymes involved in glucose metabolism [20].
C. burmannii contains MHCP, an active compound that acts as an insulin mimic.The mechanisms affected by MHCP include insulin receptor phosphorylation, glucose uptake and glycogen synthesis [22].In DM patients, MHCP may decrease insulin resistance, which in turn can help control the release of non-esterified fatty acids from adipose tissue and increase the activation of lipoprotein lipase in adipose tissue [24].

CONCLUSION
The combination of C. aurantifolia and C. burmannii extracts may be a promising alternative preventative medicine for management of diabetic complications in patients with hyperglycemia.

Figure 1 :
Figure 1: Effect of the extract of C. aurantifolia and C. burmannii on blood glucose levels in fasting Wistar rats after treatment.

Figure 2
Figure 2 Effect of the combined extract of C. aurantifolia and C. burmannii on SOD levels (mg/mL) in fasting Wistar rats after treatment

Figure 3 :
Figure 3: Low-density lipoprotein blood levels (mg/dl) in fasting Wistar rats after treatment

Figure 4 :
Effect of the extract of C. aurantifolia and C. burmannii on the number of β-cells (a) and TGF-β expression (b) The Ethics Committee of Polytechnic of Health, The Ministry of Health in Malang approved this The experimental procedures were performed according to the principles of the Institute for Laboratory Animal Research in the Guide for the Care and Use of Laboratory Animals [14].