Antidiabetic effects of the ethanolic extract of Allium saralicum R.M. Fritsch on streptozotocin‐induced diabetes in a mice model

Abstract Medicinal plants can protect different organs against diabetes‐induced oxidative stress due to their antioxidant compounds. The present study was designed to investigate the potential of Allium saralicum R.M. Fritsch (A. saralicum) ethanolic extract to alleviate the adverse effects of streptozotocin (STZ)‐induced diabetes in male BALB/c mice. Seventy male mice were randomly divided into seven groups (n = 10). Diabetes was experimentally induced by STZ (60 mg/kg bw). A. saralicum ethanolic extract with doses 5, 20, 80, and 320 mg/kg was administrated for 20 consecutive days in diabetic animals. Based on the obtained results, the untreated diabetic mice showed high blood glucose level, cholesterol, low‐density lipoprotein (LDL), white blood cells count (WBC), and platelets, as well as liver enzymes, urea, and creatinine. Administration of different doses of A. saralicum extract significantly reduced blood glucose level similar to glibenclamide. Also, the levels of catalase and superoxide dismutase enzymes restored toward normal level. All hepatic and renal function parameters as well as hematological parameters were improved following treatment with A. saralicum extract particularly at high doses. Histopathological studies showed a decrease in hepatic, renal, and pancreatic damage after treatment with A. saralicum extract. The results of the present work indicate that A. saralicum ethanolic extract can attenuate diabetic hepato‐renal, pancreatic, and hematological damages.


Streptozotocin (STZ), which is used for inducing diabetes,
is a very toxic agent for pancreas cells especially α and β cells (Brosius et al., 2009;Michalak et al., 2020). STZ causes DNA inconvenience and apoptosis in α and β cells as a nitrosourea class alkylating agent (Lenzen, 2008;Tesch & Allen, 2007). In addition to α and β cells, the liver and kidney are also sensitive to the toxicity of STZ (Rerup, 1970;Weiss, 1982), making it arduous to differentiate between diabetic hepatopathy and nephropathy (Tay et al., 2005).
Previous studies have indicated that STZ causes diabetes by changing the situation of antioxidant enzymes (Weiss, 1982). The results of many reports have revealed that ethnomedicinal plants by increasing the antioxidant enzymes levels have significant potentials for protecting of the pancreas, kidney, and liver against several toxins such as STZ Najafi et al., 2017).
Therefore, in the present experiment, we aimed to survey antidiabetic potentials of A. saralicum ethanolic extract in a mice model with focusing on biochemical, hematological, and histopathological approaches.

| Extract preparation
A. saralicum leaves were collected in summer, then milled after drying. 220 g of leaf powder was dissolved in 100% alcohol solution for 2 days. Then, the solution was filtered through paper (Whatman filter paper no.42, Millipore, USA. Cat No. 1442125) and dried in room temperature. Finally, 30 g of A. saralicum ethanolic extract was stored at 4℃ (Sherkatolabbasieh et al., 2017). The obtained extract was used for LC-Mass analysis.

| Experimental design
One day after induction of diabetic, the animals were classified into the several groups (n = 10) and treated through gavage for 20 days: 1. C: Healthy group treated by 0.5 ml normal saline.
The dose selection was performed according to the previous studies (Sherkatolabbasieh et al., 2017).

| Blood sampling
For measuring the fasting blood glucose, the blood was taken seven times (1-20 days) from the tail vein and asses by a glucometer. On day 20 of the experiment, 6 mg/kg of xylazine and 38 mg/kg of ketamine HCl were injected into the tail vein for euthanizing the animals.
Then, the bloods were extracted from the hearts for biochemical and hematological experiments.

| Determination of biochemical parameters
The collected samples were centrifuged for 16 min at 12,000 rpm and serum separated. In serum, the levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, total protein, conjugated bilirubin, total bilirubin, creatinine, urea, cholesterol, low-density lipoprotein (LDL), and highdensity lipoprotein (HDL) were analyzed by using diagnostic kits in Mehr laboratory, Iran.

| Determination of hematological parameters
In the hematological section, the blood samples were examined by a hematology analyzer. The parameters including white blood cell (WBC), red blood cell (RBC), hemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were assessed.

| Evaluation of the endogenous antioxidant enzymes activities
In this research, the levels of liver and kidney antioxidant enzymes, that is, catalase (CAT) and superoxide dismutase (SOD) were measured according to the Mohammadi et al., (2020) and Hemmati et al., (2020) methods, respectively.

| Histopathological assessment
In the histopathological section of the recent study, the pancreas, liver, and kidney samples were collected and investigated after preparing tissue sections. The volume density of the islets and B cells, percentage of B cells, number of islets, and average area of islets were measured.
In the liver sections, the enlargement and congestion in sinusoids, central veins, portal veins, and hepatic arteries, sinusoids hyperemia, fibrin and mononuclear cells leakage in pericentral veins and periportal zones, bile ducts proliferation, hepatocytes cellular and nuclear pleomorphism, eosinophilic cytoplasmic bodies and inclusion bodies in hepatocytes, hepatocytes necrosis, and liver fibrosis and cirrhosis were evaluated.
In the kidney, the enlargement and congestion in glomeruli, renal veins, and renal arteries, fibrin leakage in periglomerular zone, perirenal veins, and perirenal arteries, proximal convoluted tubules and distal convoluted tubules, cells necrosis, glomerular and tubular atrophy, and renal fibrosis were assessed.

| Statistical analysis
The normality of data was determined by Kolmogorov-Smirnov test and followed by one-way ANOVA test and post hoc Duncan test.
All of the statistical analyses were conducted using SPSS 22.0 (IBM SPSS Statistics for Windows, version XX) (IBM Corp.) and a p ≤ .05 was considered significant. The values are presented as mean ± SD.

| Effect of ASEE on fasting blood glucose concentration
The effect of ASEE on fasting blood glucose level in the diabetic mice is presented in Figure 1. There was no significant change in the blood glucose level of the control mice throughout the study.
The blood glucose levels of the untreated diabetic mice increased to approximately 520% (p ≤ .05) in a time-dependent manner.
However, treatment of the diabetic mice with ASEE at all doses significantly (p ≤ .05) decreased the blood glucose levels similar to the glibenclamide-treated mice at day 20 of the experiment. ASEE exerted its maximum effect on day 20 of the experiment.

| Histopathological findings
The histological sections of the liver in the untreated diabetic mice showed degenerative changes in the hepatocytes represented by  The dominant compounds are indicated in bold.

TA B L E 1
The components of ASEE analyzed by GC/MS F I G U R E 1 Fasting blood glucose levels on different days in the controls and ASEE-treated groups necrosis in the livers of the ASEE320-treated diabetic mice. The liver of the control group had normal structure (Table 2, Figure 2).
The kidneys of the control and ASEE-treated mice had normal structure. The proximal and distal convoluted tubules, renal corpuscles, glomerulus, and glomerular capsule had normal architecture, and in the untreated diabetic group, structural defects were seen in all of the above parameters. Microscopic examination of the kidneys of the diabetic mice treated with ASEE320 and ASEE80 did not show tubular necrosis or necrotic changes in the glomerular epithelium or glomerular and vascular hemorrhages (Table 3, Figure 3).
The effect of ASEE on histomorphometric findings of the pancreatic tissue in the diabetic mice is presented in Figure 4.
The number of pancreatic islets, volume density of the beta cells as well as the percentage of the beta cells showed a significant decrease (p ≤ .05) in the untreated diabetic mice compared to the normal control group. The volume density of pancreatic islets also showed a significant decline (p ≤ .05) following induction of diabetes. The pancreas of the diabetic mice treated with ASEE showed a slight increase in the size of pancreatic islets, having a few cells with hyperchromatic nucleus and regeneration of the beta cells in the center of islets. Also a regeneration process of pancreatic islets was more evident in ASEE-treated groups.
Although the number per square millimeter of the pancreatic islets, the volume density of the islets, and the volume density of the beta cells in the pancreas improved following administration of ASEE320, however, the percentage of beta cells, and the volume density of the beta cells in the pancreatic islets in the ASEEtreated mice were still significantly (p ≤ .05) lower than those of the control group.

TA B L E 2 Histopathological analysis of the liver in controls and ASEE-treated groups
No Liver Changes C UTD G20 ASEE5 ASEE20 ASEE80 ASEE320

| Effect of ASEE on liver biochemical parameters
The estimated values of the liver enzymes are presented in Figures 5-7.
STZ-induced hepatotoxicity increased ALP, AST, ALT, cholesterol, LDL, total, and conjugated bilirubin and decreased HDL, SOD, CAT, total protein, and albumin significantly (p ≤ .05) as compared to the control group.

Several doses of ASEE and glibenclamide could significantly (p ≤ .05)
decrease the raised levels of ALP, AST, ALT, cholesterol, LDL, total and conjugated bilirubin and increased HDL, SOD, CAT, total protein, and albumin significantly (p ≤ .05) as compared to the untreated group.

| Effect of ASEE on kidney biochemical parameters
The estimated values of the kidney biochemical parameters are depicted in Figure 8. STZ-induced diabetes increased urea and creatinine levels and decreased CAT and SOD levels significantly (p ≤ .05) compared to the control group. Different doses of ASEE could significantly (p ≤ .05) ameliorate the above parameters. There was no significant difference in the above-mentioned parameters (p ≤ .05) between ASEE320 and control groups.

| Effect of ASEE on hematological parameters
The differences (p ≤ .05) in the above factors (except for platelet and Hb levels) between ASEE320 and control groups (Figures 9-11).

| D ISCUSS I ON
The present study investigates the efficacy of ethanolic extract of Allium saralicum (A. saralicum) on streptozotocin-induced diabetes in male mice from various histopathological, hematological, and biochemical aspects.
The obtained results showed that the ethanolic extract of A. saralicum could significantly reduce the blood glucose level in STZ-induced diabetes. Such hypoglycemic effects of medicinal plants can be attributed to decrease in the rate and amount of intestinal absorption or increase in glucose uptake by peripheral tissues (Gupta et al., 2012;Hamden et al., 2001;Porchezhian et al., 2000). In addition, herbal extracts may have stimulatory effects on the remaining beta cells and more insulin production. Various studies have shown that the administration of plant extracts in laboratory diabetic animals can be effective in the reconstruction and repairment of the beta cells and Langerhans islands. Beta cells also have a remarkable potential for self-renewing in the early stages of diabetes (Cumaoğlu et al., 2011;Pepato et al., 2004). Therefore, by default, it can be assumed that the blood glucose levels in these groups. In addition, many researchers have suggested that the antidiabetic effects of some of the natural extracts can be attributed to their insulin-like effects, which enable them to decrease the blood glucose levels and serum lipids by controlling insulin (Shen et al., 2000;Zangeneh et al., 2018).
Dyslipidemia is one of the complications of hyperglycemia (Adeneye et al., 2010). This work showed that cholesterol and LDL levels decreased and HDL levels enhanced in the diabetic mice treated with ethanolic extract of A. saralicum. These results suggest that the ethanolic extract of A. saralicum has been able to improve the defect metabolism of the fatty acids in the streptozotocin-induced diabetic mice. Increased lipid decomposition and the release of free fatty acids from peripheral tissues are other mechanisms for increasing the lipids profile in diabetes (Chaiyasut et al., 2011). Previous studies have shown that some compounds, especially saponins and steroids, exert antihyperlipidemic effects by preventing intestinal absorption of lipids and also by preventing the activity of lipase enzymes (Hamden et al., 2001). The increase in the liver enzymes in diabetic mice may be due to diabetes-induced hepatic injuries (Rodrigues et al., 2010).

F I G U R E 7
The serum levels of liver SOD and CAT in the controls and ASEEtreated groups

F I G U R E 6 (a) Total protein and albumin levels and (b) Total bilirubin and conjugated bilirubin in the controls and ASEE-treated groups
The findings of the current work confirmed the signs of liver damage, such as dilation and congestion of the sinusoids, hepatic arteries, and veins. Treatment with different doses of A. saralicum ethanolic extract reduced the serum levels of transaminases and also improved the histopathologic alterations in the liver of the streptozotocin-induced diabetic animals. Also, diabetes can increase the bilirubin levels directly by damaging the bile ducts or by releasing from the muscles (Gaamoussi et al., 2010). The obtained results showed that the conjugated and total bilirubin levels restored toward the normal levels after treating with high doses of ethanolic extract of A. saralicum.
It has been well established that stress oxidative plays a pivotal role in the pathogenesis of diabetes and vascular complications.
Streptozotocin can increases reactive oxygen species (ROS) production and damages to the pancreas, leading to increased blood glucose level. These molecules are exacerbating factors in cellular injury, inflammation, cardiovascular diseases, and aging process. Therefore, antioxidants play a significant role in reducing diabetes complications and myocardial infarction. This fatty acid has an excellent inhibitory effect on NO and iNOS production. Also, the antioxidant effects of linolenic acid can be attributed to its ability to regulate the expression of TNFα as well as inflammatory interleukins (Sherkatolabbasieh et al., 2017). Moreover, other compounds in A. saralicum extract, such as phytol, neofitadine, and vitamin E, are potent antioxidant and antiinflammatory agents (Ren & Chung, 2007). Hematological indices were another parameter studied in this study. In general, the relationship between diabetes and anemia has been fully documented in previous studies (Mehdi & Toto, 2009;Weiss & Goodnough, 2005).
Several mechanisms can be considered for anemia associated with diabetes. Ferraro et al., (2011) in a study showed that diabetes affects the bone marrow cells and changes the microanatomy and physiology F I G U R E 8 (a) The serum levels of urea, creatinine, and (b) kidney SOD and CAT levels in the controls and ASEE-treated groups F I G U R E 9 The number of (a) WBC, (b) platelet, and (c) RBC in the controls and ASEE-treated groups of the bone marrow stem cells. In addition, it seems that one of the causes of diabetes mellitus-induced anemia is the glycosylation of the plasma membrane of the red blood cells. So that, hyperglycemia and protein oxidation lead to increased lipid peroxidation and ultimately diminish the fluidity and flexibility of the cell membrane and hemolysis of the red blood cells can be occurred (Kumar, 2012;Turk et al., 2002;Watala & Winocour, 1992). In this study, the number of white blood cells and platelets increased in the untreated diabetic animals, and the number of red blood cells, hemoglobin, MCV, MCH, MCHC, and PCV decreased significantly. Stookey et al., (2007) have shown that streptozotocin reduces the synthesis of MCH and MCHC, which indicates a defect in hemoglobin synthesis and a defect in osmotic pressure control and osmolality of the plasma. Treatment with A. saralicum extract, especially at high dose (320 mg/kg), improved the above-mentioned parameters. Peelman et al., (2004) suggested that leptin and its receptor are responsible for hemopoiesis. Ohshita et al., (2004) showed that white blood cell count is associated with some diseases, including