Antidiabetic effect of hydro-methanol extract of Prunus cerasus L fruits and identification of its bioactive compounds

Purpose: To investigate the antidiabetic effect of hydro-methanol extract of Prunus cerasus fruit extract. Methods: The antidiabetic activity was assessed in alloxan-induced diabetic rats. The effect of P. cerasus fruit extract on plasma fasting blood glucose (FBG), insulin, C-peptide, total protein, glycosylated hemoglobin (HbA1c), total hemoglobin, reduced glutathione (GSH), vitamins E and C, ceruloplasmin, lipid profile, histology of the pancreas, and expression of glucose transporter type 4 (GLUT-4) were determined using standard procedures. Liquid chromatography-mass spectrometry was used for phytochemical analysis. Results: Alloxan-induced diabetes significantly reduced plasma levels of insulin, C-peptide, total hemoglobin and total protein, and significantly increased FBG and HbA1c levels (p < 0.05). However, after treatment with the extract, changes in the levels of these parameters were significantly and dosedependently reversed (p < 0.05). The extract also increased the levels of GSH, vitamins E and C, and. Alloxan-induced DM significantly increased the levels of triacylglycerols (TGs), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C), and significantly reduced the level of total cholesterol (TC) at different time points (p < 0.05). However, after treatment, the levels of TG, LDL-C and HDL-C declined but TC level was significantly elevated timeand dose-dependently by the extract (p< 0.05). The extract upregulated the expression of GLUT-4 mRNA in soleus muscle and adipose tissue. LC-MS analysis revealed that the extract contained chlorogenic acid, rutin, diadzin, amygdalin, quercetin and naringenin. Conclusion: The results obtained in this study have shown that hydromethanol extract of P. cerasus fruits exhibits remarkable antidiabetic effects.


INTRODUCTION
Prunus cerasus belongs to the Rosaceae family and it is locally known as sour cherry. Its fruits are rich in nutrients and a number of bioactive compounds such as polyphenols and flavonoids [1]. Studies have shown that plant materials rich in natural antioxidants such as flavonoids and phenolics lower the risk of cardiovascular disease (CVD), neurodegenerative disorders, oxidative stress, cancer, and Diabetes mellitus (DM) [2].The antioxidant and anti-inflammatory properties of extracts of P. cerasus have been attributed to the presence of polyphenols in the plant [3].Metabolic disorders and oxidative stress play key roles in the pathogenesis of DM [4].Type 1DM (TIDM) is due to insulin deficiency, while type 2 DM (T2DM) is caused by insulin resistance.
Plant extracts rich in antioxidant molecules have been shown to be beneficial in the treatment of DM [5,6]. At present, studies on the antidiabetic properties of extracts of P. cerasus are scanty. The aim of this study was to investigate the antidiabetic effect of hydro-methanol extract of P. cerasus fruits and its bioactive components.

EXPERIMENTAL Collection and extraction of P. cerasus fruits
Fresh fruits of P. cerasus were collected from the wild and identified at the Department of Botany, Central South University, Changsha, Hunan, China. A voucher specimen was prepared and kept at the herbarium of the department. The fruits were dried in an incubator for 2 days at 40°C, crushed separately in an electric grinder, and then pulverized.
A portion of the powder (50 g) was dissolved in a 2:3 volume ratio of water and methanol (100 ml water and 150 ml methanol) and kept in the incubator at 37°C for 36 h. The slurry was stirred intermittently at intervals of 2 h, and left overnight. The mixture was thereafter filtered and the filtrate concentrated using a vacuum rotatory evaporator. The resultant concentrate was freeze-dried by lyophilization.

Liquid chromatography-mass spectrometry (LC-MS)
A portion of the dried hydro-methanol extract (9g) was subjected to LC-MS for characterization as described previously [7].

Acute toxicity study of hydro-methanol extract of P. cerasus fruits
This was performed according to Lorke's method.

Induction
of DM and grouping of experimental rats Twenty five adult male Wistar rats weighing 170.0 to 190.0 g (mean weight = 180.0 ± 5.80 g) were randomly assigned to five groups of five rats each: normal control group, extract control group, diabetic control group and two treatment groups. With the exception of normal control and extract control groups, DM was experimentally induced in the rats by intravenous injection of alloxan (150 mg/kg bwt). After 72 h, rats with plasma glucose levels >200 mg/dL were classified as diabetic. The diabetic control group was not treated, while the treatment groups received 100 or 200 mg/kg bwt extract/day orally for 60 days.
Diabetes was not induced in the extract control group, but it received 200 mg/kg bwt of extract. The animal experimental protocols were approved by the ethical committee for animal welfare of Weifang People's hospital, Weifang, People's Republic of China (approval no. YXT134539. All the procedures were performed as per the international ethical guidelines for animal studies [8].

Determination of blood glucose level
The levels of FBG in all the groups were estimated at zero time (day 0), day 30 and day 60 during the study.

Determination of serum levels of GSH, vitamins E and C, and ceruloplasmin
The serum levels of GSH, vitamins E and C, and ceruloplasmin were determined in normal and diabetic rats after 60 days of treatment.

Determination of levels of insulin and other plasma proteins
The levels of insulin, C-peptide, total hemoglobin, HbA1c, and total protein were determined in the serum of normal and diabetic rats.

Lipid profiles, tissue histology and expression of GLUT-4
The expressions of GLUT-4in soleus muscle and adipose tissue were determined using real-time quantitative polymerase chain reaction (qRT-PCR), while histological examination of the pancreas was performed using hematoxylin and eosin (H & E) staining. The levels of plasma lipids were also determined.

Statistical analysis
Data are expressed as mean ± SD, and statistical analysis was performed using SPSS (20.0). Groups were compared using Student ttest. Values of p < 0.05 were considered statistically significant.

Oral LD 50 of hydro-methanol extract of P. cerasus fruits
Doses of the extract (≤ 5000 mg/kg bwt) did not produce mortality and significant changes in rat behavior. Macroscopic pathological examination of rat organs revealed no visible damage to pancreatic tissue. The LD 50 of hydro-methanol extract of P. cerasus fruits in the rats was therefore taken to be> 5000 mg/kg (Table 1).

Levels of FBG
Alloxan-induced DM significantly increased FBG level in the rats. However, after treatment with extract, FBG was significantly reduced in the treatment groups, relative to diabetic control group at different time points (p < 0.05).There was no significant difference in FBG between the two treatment groups at day 0 (p > 0.05). However, at day 30 and day 60, FBG levels were significantly reduced in 200 mg/kg bwt group, relative to the 100 mg/kg group (p < 0.05). These results are shown in Table 2.

Levels of GSH, vitamins E and C, and ceruloplasmin
Alloxan-induced DM significantly reduced the plasma levels of GSH, vitamins E and C, and ceruloplasmin (p < 0.05). However, after treatment with extract, the plasma levels of these parameters were significantly and dosedependently increased in the treatment groups, when compared with diabetic control group (p < 0.05; Table 3).

Insulin and other plasma proteins
As shown in Table 4, alloxan-induced DM significantly reduced plasma levels of insulin, Cpeptide, total hemoglobin and total protein, and significantly increased HbA1c level (p< 0.05). However, after treatment, changes in the levels of these parameters were significantly and dosedependently reversed (p< 0.05).

Lipid profiles
Alloxan-induced DM significantly increased serum TG, LDL-C and HDL-C, and significantly reduced serum TC at the different time points (p < 0.05). However, after extract treatment, the levels of TG, LDL-C and HDL-C were significantly reduced, and TC level was significantly elevated time-and dosedependently (p < 0.05; Figure 1).

Outcome of histological examination
The results of histological examination showed that the extract alone did not elicit any significant changes in histology of rat pancreas. However, alloxan-induced diabetic rats exhibited pancreatic injury which was characterized by significant reductions in the numbers of islets cells and diameters of pancreatic islets. Islets of Langerhans were severely reduced in diabetic control pancreas, relative to the normal control pancreas. However, the pancreatic injury was markedly mitigated on treatment of the diabetic rats with hydro-methanol extract of P. cerasus fruits. Treatment with 100 mg/kg bwt of the extract induced moderate expansion of islets of Langerhans but the degree of injury was significantly reduced (Figure 2).

GLUT-4 mRNAs levels in soleus muscle and adipose tissue
There were significant reductions in GLUT-4 mRNA levels in adipose tissue and soleus muscle of diabetic control rats, relative to normal control group (p < 0.05). However, after treatment of diabetic rats with extract, the level of expression of GLUT-4 mRNA in soleus muscle and adipose tissue was significantly upregulated (p < 0.05; Figure 3).

Identification of bioactive compounds in hydromethanol extract of P. cerasus fruits
As shown in Figures 4 and 5, chlorogenic acid, rutin, diadzin, amygdalin, quercetin, naringenin and gallic acid were identified in hydro-methanol extract P. cerasus fruits.

DISCUSSION
The present study investigated the antidiabetic effect of hydro-methanol extract of P. cerasus fruits, and identified its bioactive compounds. The results of acute toxicity study showed that the LD 50 of the extract in rats was greater than 5000 mg/kg bwt, an indication that the extract is non-toxic. It has been reported that alloxan causes DM by generating reactive oxygen species (ROS) which in turn destroy βcells [20].Oxidative stress has been reported to contribute to the pathogenesis of DM [4]. Increased incidence of diabetic complications is due to increased free radical generation or reduced antioxidant defense responses [9].
In this study, alloxan-induced DM significantly reduced plasma levels of insulin, C-peptide, total hemoglobin and total protein, and significantly increased FBG and HbA1c levels. However, after treatment with hydro-methanol extract of P. cerasus fruits, changes in the levels of these parameters were significantly dosedependently reversed. The extract also potentiated the non-enzyme antioxidants. It is likely that the extract inhibited lipid peroxidation and alloxan-induced oxidative stress in the diabetic rats. The increase in the level of insulin after treatment with extract may also be attributed to the stimulatory effect of the extract on the pancreatic β-cells which survived after induction of DM. Extract-promoted restoration of pancreatic β-cells may also be responsible for the enhanced insulin secretion.
The generation of free radicals in situations of severe oxidative stress leads to modification of cellular proteins [10]. Studies have shown that the level of plasma total protein is reduced in DM [11].
In this study, alloxan-induced DM significantly increased the levels of TG, LDL-C and HDL-C, and significantly reduced the level of TC at the different time points. However, after treatment with extract, the levels of TG, LDL-C and HDL-C were significantly reduced, while TC level was significantly elevated time-and dosedependently. Increased level of TG in the diabetic rats suggests a condition of hypertriglyceridemia which may be due to overproduction of very low-density lipoprotein (VLDL) cholesterol by the liver. These results suggest that hydro-methanol extract of P. cerasus fruits may normalized altered lipid profiles in diabetic rats.
In this study, the islets of Langerhans in the alloxan-induced rat pancreas were necrotic and damaged. However, treatment with the extract remarkably restored these histopathological changes and induced a distinct granulated and protective effect on pancreatic β-cells.
Studies have shown that DM leads to marked reduction in GLUT-4 expression [5,6]. In this study, there were significant reductions in GLUT-4 mRNA levels in adipose tissue and soleus muscle of diabetic control rats, relative to normal control group. However, after treatment of diabetic rats with extract, the level of expression of GLUT-4 mRNA in soleus muscle and adipose tissue was significantly upregulated.
Results from LC-MS revealed the presence of chlorogenic acid, rutin, diadzin, amygdalin, quercetin, naringenin and gallic acid in the extract of P. cerasus fruits, an indication that this extract may be a rich source of important flavonoids and phenolic compounds. Studies have shown that phenolic compounds function as potent antioxidants, and phytochemicals such as rutin, naringenin, and quercetin exert beneficial effect on DM [7,12,13]. Thus, the antidiabetic effect of this extract could be attributed to its active phytochemical constituents such as flavonoids and phenolic compounds.

CONCLUSION
The results obtained in this study show that the hydro-methanol extract of P. cerasus fruits is a rich source of important flavonoids and phenolic compounds which synergistically contribute to its antidiabetic effect.