Introduction

Diabetes mellitus is the most popular endocrine disorder that affects more than 285 million people worldwide. This number is expected to grow to 438 million by 2030, corresponding to 7.8% of the adult population. In addition to the primary complications of diabetes, this disease is associated by increased risk of hypertension, dyslipidemia, decreased fibrinolytic activity, severe atherosclerosis, increased platelet aggregation, and also poor wound healing (Ahmed et al. 2014).

Kidney is one of the organs that is affected in diabetic patients. However, the exact pathogenesis of poor nephropathy in diabetic patients is not clearly understood; the decrease of proximal and distal cell capacity and also the oxidative and inflammatory changes are suggested to be the main causes (Musabayane 2012).

Renal hypertrophy and glomerular hyperfiltration are two known complications that occur in the initial stages of diabetes mellitus (Hostetter et al. 1981). Further, renal disorders following diabetes mellitus may have a specific pattern compared to the other renal diseases (McCrary et al. 1981). Some studies had revealed that in early diabetes, glomerular hyperfiltration and renal hypertrophy could be reversed by insulin treatment (Mogensen and Anderson 1975; Christiansen et al. 1982). Whereas, in chronic diabetes, glomerular hyperfiltration could be improved by severe control of blood glucose level, but renal hypertrophy is irreversible (Wiseman et al. 1985). Although renal hypertrophy and glomerular hyperfiltration play a pivotal role in the progression of diabetic nephropathy, the relationship between them is still unclear (Hostetter 2003).

The enormous costs of modern medicines demonstrate that alternative strategies are needed for better management of diabetes and its related problems (Rahimi et al. 2005). Some plants have the high contents of tannins, saponins, flavonoids, naphthaquinone, triterpenes, and alkaloids. So, they can increase the quality and rate of healing of wounds (Abdel-Barry et al. 1997; Pushparaj et al. 2000). The World Health Organization (WHO) suggested that there should be further studies on antidiabetic properties of medicinal plants (WHO 1981).

In Iranian traditional medicine, herbal medicines have been the basis of treatment and cure for various diseases and physiological conditions. Allium saralicum R.M. Fritsch known as “Valak Soori” in local regions is one of the most important herbal medicines, which is widely consumed in the west of Iran. This plant belongs to the family Amaryllidaceae and Allioideae subfamily. Allium saralicum has a long history of use in Kurdish traditional medicine for treatment of digestive, nervous, and infectious diseases. But there is no evidence of its antidiabetic and/or its nephroprotective effects (Block 2010; Davies 1992; Foroughi et al. 2016).

Therefore, this study was designed to evaluate antihyperglycemic effect of Allium saralicum and its protective effects on renal structural changes in STZ-induced diabetic mice using design-based and unbiased stereological methods.

Materials and methods

Plant collection

Allium saralicum (AS) at maturity was harvested from the Kermanshah suburb during April 2015. The plant was identified for the first time, and a voucher specimen (no. 2738RUH) has been deposited at the Herbarium of Research Center of Faculty of Agriculture, Razi University, Kermanshah, Iran.

Plant extraction

Leaves of the plant were shade dried for 1 week. Dried aerial part of the plants was ground, and about 150 g of the obtained powder was extracted with 450 mL of 100% ethanol for 2 h at 40 °C with continuous shaking. The extract was left for 24 h at room temperature; then, it was filtered through Whatman paper no. 2. The extract was concentrated in rotary evaporator (Panchun Scientific Co., Kaohsiung, Taiwan) then lyophilized.

Animals

Thirty-five healthy male Balb/c mice weighing 36 ± 3 g were provided by the Center of Laboratory Animal Breeding, Kermanshah University of Medical Sciences. The animals were housed under standard environmental (25 ± 3 °C temperature and 12:12-h light and dark) and nutritional (standard pellet diet and water ad libitum) conditions during the experiment.

Institutional Ethics Committee approval was obtained, and all procedures performed in the study were in compliance with the desired ethical rules.

Diabetes was induced by a single intraperitoneal (IP) administration of 60 mg/kg bw of streptozotocin (Sigma, St. Louis, MO, USA). Fasting blood glucose (FBG) level was assessed everyday by glucometer strips. A blood glucose level upper than 250 mg/dL was considered diabetic.

Experimental design

The mice were divided into five following groups (n = 7):

  1. (I)

    Control group (C) which received 200 μL normal saline orally.

  2. (II)

    Untreated-diabetic group (UTD).

  3. (III)

    Treated diabetic mice which received 0.5 mg/kg glibenclamide for 15 days (TDG).

  4. (IV)

    Treated diabetic mice which received 200 μg/kg of the ethanolic extract of AS for 15 days.

  5. (V)

    Treated diabetic mice which received 400 μg/kg of the ethanolic extract of AS for 15 days.

Blood sampling

Blood samples were collected daily via tail vein to assess the blood glucose level by glucometer strip. At the end of the experiment, all animals were weighed and euthanized with deep chloroform inhalation. Immediately blood samples were drawn from the animals’ heart. The samples were centrifuged at 10,000 rpm for 15 min and serum separated.

Stereological study

Volume density

After dissection, the left kidneys were removed and then weighed. They were placed in 10% neutral buffered formalin solution for 5 days. Immersion method was then used to determine the primary volume of the kidney. For estimation of final volume of the organs, the amount of tissue shrinkage must be specified (Gundersen et al. 1992; Braendgaard and Gundersen 1986). Isotropic uniform random (IUR) sections must be obtained for estimating tissue shrinkage and tubular length (Gundersen et al. 1992; Nyengaard 1999). Theses sections were obtained using orientator method. Totally, 7–10 slab were obtained from each kidney through orientator method. A circular piece was sampled from a kidney slab, and the area of this piece was calculated. The slabs and circular piece were processed, sectioned (5-μm thicknesses), and stained by Periodic Acid Schiff (PAS) method. The area of the circular piece was calculated again, and tissue shrinkage was estimated as (Mandarim-de-Lacerda 2003)

$$ \mathrm{Volumeshrinkage}=1-{\left(\frac{\mathrm{AA}}{\mathrm{AB}}\right)}^{1.5} $$

where AA and AB are the area of the circular piece after and before tissue processing. The total volume of the organ was then estimated using

$$ {V}_{\mathrm{final}}={V}_{\mathrm{primary}}\times \left(1-\mathrm{volume}\ \mathrm{shrinkage}\right) $$

Tissue sections were examined using a microscope (Olympus CX2, Japan) connected to a video camera (Dinocapture ver.5, dino-lit.com 30.5 mm) and a P4 PC, and the stereological parameters were estimated. The fractional volume of the renal structures was estimated using a point probe (with an area of 100 cm2 and containing 25 points) and following the formula:

$$ {V}_v=\frac{P_{\mathrm{structure}}}{P_{\mathrm{reference}}} $$

“Pstructure” = sum of points dealt with the interested structures.

“Preference” = sum of points dealt with the reference space.

Length density

The length density of the tubules and vessels was estimated using a counting probe (740 × 740 μm) and the following formula:

$$ {L}_v=2\times \frac{\sum Q}{a\left(\mathrm{frame}\right)\times \sum \mathrm{frame}} $$

∑Q = sum of the tubules counted, a (frame) = probe area, 547,600 μm2, and ∑frame = total number of the counted frames.

Numerical density

Physical disector procedure (Sterio 1984) was applied for estimating the numerical density of glomeruli. Two parallel sections with 20-μm distance (first and fifth sections) were prepared. The first section as reference plane and the fifth section as look-up plane. Two counting probes with an area of 547,600 μm2 were attached on the monitor at the final magnification ×135, and the numerical density was estimated using

$$ {N}_V=\frac{\sum {Q}^{-}}{a\left(\mathrm{frame}\right)\times h\times \sum P} $$

∑Q− = sum of the counted glomeruli, a (frame) = probe area, ∑P = total number of the examined fields, and h = disector height. The absolute value of each parameter was estimated by multiplying its density by the reference space (Mandarim-de-Lacerda 2003).

Statistical analysis

The data were analyzed by SPSS software, version 22.0 (SPSS Inc., Chicago, IL, USA). The one-way ANOVA and Tukey’s post hoc test were used to compare the mean groups, and p < 0.05 was accepted as statistically significant.

Results

Effect of AS on blood glucose level

The blood glucose levels of untreated diabetic mice increased to approximately 170% (p ≤ 0.05) of the control mice in a time-dependent manner (Table 1). However, treatment of STZ diabetic mice with the ethanolic extract of As at high dose (400 μg/kg) could significantly (p ≤ 0.05) reduce the blood glucose levels similar to the glibenclamide—treated at the end of the experiment. Also, the difference between low and high dose of AS was significant (p ≤ 0.05) at 10–13 and 14–16 days. The ethanolic extract of AS has most effect on days 10–13 of the experiment (Table 1).

Table 1 Blood glucose level on different days in the control and experimental groups treated with AS (mean ± SD)
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Effect of AS on body weight

The body weight decreased significantly (p ≤ 0.05) in untreated diabetic animals compared to the control ones. High dose of AS could improve the body weight as well as the glibenclamide. The difference between AS 200 group and untreated diabetic mice was not significant (p > 0.05) (Table 2).

Table 2 Animal weight (g), kidney weight (mg), absolute volume of the kidney (mm3), and absolute volume (mm3) of cortex and medulla of the control and experimental groups treated with AS (mean ± SD)
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Effect of AS on stereological parameters

The data of the kidney weight, mean absolute volume of the kidney, and its subcomponents in treated and untreated diabetic groups are presented in Tables 2 and 3. The results showed that the kidney weight and volume were increased 42 and 56% (p ≤ 0.05), respectively, in the untreated diabetic mice when compared to the control ones. Cortical volume increased 80% (p ≤ 0.05) in this group, but the increase of medullary volume was not significant (p ≥ 0.05) compared to the control one. High dose (400 μg/kg) of AS could significantly (p ≤ 0.05) improve the kidney weight and consequently renal volume compared to the low dose (200 μg/kg).

Table 3 Absolute volume (mm3) of proximal and distal convoluted tubules (PCT, DCT), collecting ducts (CD), loop of Henle (LH), interstitial tissues (IT), and vessels (VES) in the control and experimental groups treated with AS (mean ± SD)
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The volume of PCT (proximal convoluted tubule), DCT (distal convoluted tubule), vessels (VES), and interstitial tissue (IT) was increased significantly (p ≤ 0.05) in untreated diabetic mice in comparison with the control ones (Table 3). Administration of AS at high dose to the diabetic mice could significantly (p ≤ 0.05) decrease the volume of the above structures in comparison with the untreated diabetic group. Low dose of As has no significant (p > 0.05) effect on DCT and collecting duct (CD) volume compared to the untreated diabetic and TDG groups.

The length of the PCT, DCT, LH, and vessels was significantly (p ≤ 0.05) increased in untreated diabetic mice compared to the control ones (Table 4). High dose of AS could significantly (p ≤ 0.05) decrease the length of the PCT and vessels compared to the untreated diabetic group.

Table 4 Absolute length (m) of the proximal and distal convoluted tubules (PCT, DCT), collecting ducts (CD), loop of Henle (LH), and vessels (VES) in the control and experimental groups treated with AS (mean ± SD)
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The glomerular volume was increased significantly (p ≤ 0.05) following diabetes induction, and treatment with high dose of AS could significantly (p ≤ 0.05) improve glomerular hypertrophy as compared to the untreated group.

The glomerular number in the untreated diabetic mice was reduced significantly (p ≤ 0.05) as compared to the control ones. The glomerular number loss was prevented significantly (p ≤ 0.05) with high dose (400 μg/kg) of AS as compared to the untreated ones (Table 5).

Table 5 Absolute volume (mm3) and number of the glomeruli (GLOM) in the control and experimental groups treated with treated with AS (mean ± SD)
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Discussion

This study was the first attempt to evaluate the ethanolic extract of Allium saralicum as antidiabetic and nephroprotective agent using stereological methods. Diabetes mellitus is the most common endocrine disease that affects many populations in the world. In this disease, the purpose of oral treatment is reducing blood glucose to impede later complications (Salvatore and Giugliano 1996).

In the present study, 60 mg/kg of STZ was used for diabetes induction in all mice. This dose can partially damage the beta cells of islets of Langerhans, nephron, and hepatocytes resulting in inexpressive insulin secretion resulting in type 2 diabetes, nephrotoxicity, hepatotoxicity, and hypercholesterolemia (Weir et al. 1981; Heidland et al. 1996; Rabkin et al. 1996).

During the short-term study, the administration of AS extract produced significant antihyperglycemic activity and improved the renal morphological changes at a dose of 400 μg/kg in diabetic mice. In accordance with the obtained results, numerous studies have shown the antidiabetic effects of other plants from genus Allium and family Amaryllidaceae (Eidi et al. 2006; Thomson et al. 2007). These features can be attributed to the sulfur compounds which are found in these plants.

There is some evidence that antidiabetic effects of garlic can be attributed to the antioxidant properties of S-allyl cysteine sulfoxide (Mathew and Augusti 1973; Augusti et al. 1996). Stimulation of pancreatic beta cells for secretion of more insulin or release of bound insulin is another mechanism which can lead to hypoglycemic activity (Jain and Vyas 1975). Accordingly, it is reported that administration of garlic oil or diallyl sulfide to STZ-induced diabetic rats resulted in increased serum insulin levels (Liu et al. 2005).

In the present study, diabetic mice showed some degree of renal hypertrophy which was mainly due to the enlargement of the cortex and its subcomponents. These changes was improved significantly with high dose of AS. It was previously shown that other plants including garlic, ginger (Qattan et al. 2008), and Ginkgo biloba (Welt et al. 2007) can also prevent renal hypertrophy in experimentally induced diabetes. It is well established that renal hypertrophy can be treated at the beginning of the diabetes. However, belated treatment is not successful (Stackhouse et al. 1990; Gondwe et al. 2008). The pathogenesis of the renal hypertrophy can be attributed to the overproduction of oxygen-free radicals following hyperglycemia and inducible nitric oxide synthase (iNOS) which is expressed in response to cytokines (Chaiyasut et al. 2011; Sharma and Ziyadeh 1995; DeRubertis and Craven 1994). Therefore, compounds with antioxidant properties can ameliorate these changes and inhibit the progression of diabetic nephropathy. AS is mainly composed of linolenic acid, gamma tocopherol, and vit E that might be contributed to its antiradical/antioxidant efficacy (Sherkatolabbasieh et al. 2016).

The results of serum glucose levels showed that AS at high dose could restore the blood glucose level toward the normal level. Although there was no significant difference between the experimental doses of AS and classic antidiabetic drug, glibenclamide until day 9 of the experiment, high dose of AS showed its maximum activity between days 10 and 13 with a significant difference as compared to the glibenclamide. But the difference between these was not significant at the end of the experiment (14–16). This indicates that ethanolic extract of AS at high dose only is as effective and potent as glibenclamide and also can acts faster than it.

Conclusion

In conclusion, the present results show that Allium saralicum at high dose may be useful for controlling blood glucose level and alleviation of diabetic complications such as nephropathy generally observed in diabetic patients.