Ameliorating effects of Astragalus maximus methanolic extract on inflammation and oxidative stress in streptozotocin-induced diabetic rats

: Our results revealed that the oral administration of A. maximus methanolic extract might be considered a natural product for controlling and treating diabetes mellitus; however, more investigations are desired to determine the action mechanisms of its extract


Introduction
Diabetes mellitus is a metabolic disorder in the body in which the ability to produce insulin is lost or the body becomes resistant to insulin and the produced insulin cannot perform its normal function, thus, many tissues such as the pancreas, kidney, liver, brain, and heart are affected (1,2).It has been proven that insulin resistance and decreased insulin discharge are the principal causes of type 2 diabetes, which result in impaired glucose oxidation and hyperglycemia (3,4).Along with many chemical drugs that are used to control diabetes, the use of therapeutic supplements such as herbal supplements can be effective in reducing diabetes and its complications (5,6).It has been customary to use plants in the treatment of diseases in almost all countries of the world (7)(8)(9).Having minimal side effects, low cost, and availability of these plants have caused the gradual replacement of chemical drugs by plants (8,9).
Herbs belonging to the Astragalus genus from the Fabaceae family have nearly 3000 species, with two main distribution centers in America and Eurasia (10).Astragalus spp, due to the presence of compounds such as flavonoids, saponins, and polysaccharides, has various activities such as strengthening the body's immune system, regulating blood cholesterol, preventing diabetes, treating digestive disorders, treating allergies, and strengthening memory (11,12).Recent studies have reported that Astragalus spp.displayed various biological effects in modern pharmacology, e.g., anticancer, antioxidant, antimicrobial, neuroprotective, and hepatoprotective activities (12,13).This work was designed to study the effect of Astragalus maximus methanolic extract (AMME) in streptozotocin-diabetic rats.

Plant
The plant materials (aerial parts) were collected from the rural regions of Noorabad city in the western regions of Iran, Lorestan province, and were identified by a botanist from the Department of Botany, Lorestan University, Khorramabad, Iran.A voucher specimen was stored at Lorestan University of Medical Sciences (No. 2827).

Preparing the methanolic extract
The dried aerial parts were extracted by maceration technique with 70% methanol.By a rotary evaporator, the alcoholic part was thrown out and the extract was reserved at minus 20°C for other tests (14).

Phytochemical study
The phytochemical examination of the AMME was accomplished to discover the presence of tannins, saponins, alkaloids, flavonoids, and glycosides, etc based on a previous investigation by means of some reagents, e.g., Mayer and Dragendorff 's reagents for alkaloids, Mg and HCl for flavonoids, 1% gelatin for tannins, FeCl 2 and H 2 SO 4 for glycosides, and suds production for saponins (15).

Total phenolic and flavonoids compounds
The total phenolic and flavonoid compounds of AMME were determined based on the Folin-Ciocalteu's method and the aluminum chloride (AlCl 3 ) colorimetric assay based on the previous studies (16).The total phenolic compounds were expressed in terms of milligrams of gallic acid per gram (GAE/g) of extract, while the total flavonoid compounds were expressed in terms of milligrams of quercetin per gram (mg QE/g) of extract.

In vitro antidiabetic effects by evaluating α-amylase inhibition activity
The level of α-amylase inhibition by AMME was determined based on a previous study with some modifications (17).Briefly, sodium phosphate buffer (200 μL) was mixed with alpha-amylase (20 μL) and AMME at concentrations of 25-200 μg/ml.The combination was kept for 10 minutes at 37°C and then, 1% starch solution was added to the test tube, and kept again at 37°C.By adding the dinitrosalicylic acid reagent and stopping the reaction, the optical density of the combination was determined at 540 nm.The control tube was without AMME.The % inhibition was considered based on the following formula:

% of inhibition= Absorbance of control -Absorbance of extract/ Absorbance of control
All tests were completed in triplicate and the IC 50 was calculated by Probit test.

Animals
Forty-eight adult male Wistar rats (200-250 g) were acquired from the care and reproduction of laboratory animals of Lorestan University of Medical Sciences (Khorramabad, Iran).Mice were stored in a room with a light/dark cycle of 12:12 hours at a temperature of 21±2 o C.

Establishment of animal model of diabetes
To induce diabetes, streptozotocin (65 mg/kg/bw) was intraperitoneally injected to the tested mice.Three days after injection, the rats were fasted for 12 hours and blood samples were collected from the mice's tail.Then, the fasting blood sugar was studied by enzyme method.If the fasting blood sugar was equal to or >250 mg/dL, it was measured as a diabetic rat (18).

Study design
Mice were accidentally distributed into six groups containing 8 mice per each, including non-diabetic mice, diabetic mice that were orally treated with AMME at 75, 150, and 300 mg/kg/d for 28 days, and diabetic animals that were orally treated with glibenclamide (0.6 mg/kg/d).

Determination of the blood glucose and insulin
The blood glucose was measured via the commercial kits (Pars Azmon, Iran).Serum insulin level was also measured via a rat ELISA kit (Pars Azmon, Iran).

Measurement of the biochemical factors
On the 29 th day after the induction of diabetes, blood sampling was collected from the hearts of the mice and centrifuged for 10 minutes at 15 000 rpm and the serum specimens were collected.The serum levels of total proteins, triglyceride, cholesterol, as well as kidney and liver enzymes were examined by Pars Azmon diagnostic kits.

Measurement of oxidant/antioxidant enzymes
The tissue levels of oxidant/antioxidant enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), and lipid peroxidation (LPO) were determined in the pancreatic homogenates by Pars Azmon diagnostic kits.

Assessment of the pro-inflammatory cytokines
The pancreatic levels of some pro-inflammatory cytokines, e.g., interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the tested rats were assessed according to the commercial ELISA kits (Carmania Parsgen Co, Iran) based on the producer instructions.

Statistical analysis
Data were analyzed by SPSS software version 26.0.The differences between groups were assessed by one-way analysis of variance and the results were reported at a significance level of 5%.

Phytochemical analysis
The phytochemical examination exhibited the appearance of flavonoids, saponins, terpenoids, and polysaccharides in AMME.The total phenolic and flavonoid contents were 3.41 (mg GAE/g DW) and 1.46 (mg QE/g DW) respectively.
Evaluating α-amylase inhibition activity AMME inhibited the α-amylase as a dose-dependent response.The IC 50 values for AMME and acarbose as the control drug were 18.1 and 4.2 μg/mL, respectively (Figure 1).

Effects of AMME on the biochemical factors
In diabetic rats, the levels of glucose, cholesterol, and triglyceride were considerably (P < 0.001) elevated compared to the non-diabetic rats.However, the level of insulin was considerably (P < 0.001) declined in diabetic rats compared to the non-diabetic rats.In the diabetic rats receiving the AMME at doses of 150 and 300 mg/kg a significant reduction (P < 0.05) in the levels of glucose, cholesterol, and triglyceride and an elevation in the level of insulin were observed (Table 1).In diabetic rats, the levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bilirubin were elevated when compared with the non-diabetic rats; nevertheless, diabetic rats treated with AMME at doses of 150 and 300 mg/kg for 28 days displayed improved levels of AST, ALT, ALP, and bilirubin compared with diabetic rats treated with normal saline (P < 0.001) (Table 2).
In diabetic rats, the levels of kidney function parameters of creatinine (Cr) and urea (Ur) were significantly raised when compared with the non-diabetic rats; however, diabetic rats treated with AMME at doses of 150 and 300 mg/kg for 28 days displayed improved levels of Cr, Ur, uric acid, total protein, and albumin compared with diabetic rats treated with normal saline (P < 0.001) (Table 3).
Effects of AMME on the oxidant/antioxidant enzymes Diabetes resulted in a significant reduction in the tissue activity of antioxidant enzymes (P < 0.01).However, diabetic rats treated with AMME at doses of 150 and 300 mg/kg for 28 days exhibited noticeable enhancement  in the tissue activity levels of SOD, CAT, and GST (P < 0.001).Diabetes also caused an elevation in tissue malondialdehyde (MDA) level in the tested mice; however, after treatment of diabetic rats with AMME at doses of 150 and 300 mg/kg for 28 days, the tissue level of MDA was declined significantly (P < 0.001) when compared with diabetic rats (Table 4).Following the treatment of diabetic rats with either 150 or 300 mg/kg AMME for 28 days, a significant reduction in the pancreas levels of TNF-α and IL-1β were observed when compared with diabetic rats (Figure 2).

Discussion
This work was designed to study the effect of A. maximus methanolic extract in streptozotocin-diabetic rats.The phytochemical examination exhibited the appearance of flavonoids, saponins, terpenoids, and polysaccharides in AMME.The total phenolic and flavonoid contents were 3.41 (mg GEA/g DW) and 1.46 (mg QE/g DW), respectively.In line with our results, in previous studies these compounds have been the main compounds of Astragalus spp.(19)(20)(21).Flavonoids and phenols from herbal medicines are valuable alternative medications for diabetes mellitus.These compounds are promising molecules that might produce novel drug discoveries (22).Phenolic compounds have exhibited their anti-diabetic effects through various mechanisms such as improving the insulin sensitivity and glucose uptake, inhibition of α-glucosidase/α-amylase enzymes, activation of AMPK and PPAR pathways, and modulating the oxidative stress and inflammation (23).Previous investigations reported the antidiabetic effects of A. membranaceus and its phytochemical ingredients, e.g., phenols, flavonoids, saponins, and polysaccharides for treating the types 1 and 2 diabetes mellitus through the modulation of inflammatory and apoptotic cytokines, as well as increasing the antioxidant activities and insulin sensitivity (24).AMME inhibited α-amylase dose-dependently.In the diabetic rats receiving the AMME at doses of 150 and 300 mg/kg a significant reduction (P < 0.05) in the levels of glucose, cholesterol, and triglyceride was observed, while they displayed an elevated level of insulin.Consequently, AMME may be displayed an insulin-like activity through releasing insulin from the pancreas and improving the serum glucose level of diabetic rats.It has been previously proven that herbal extracts most likely increase the consumption of glucose by the adjacent tissues, prevent glucose absorption in the kidneys, and increase the release of insulin from the islets cells of the pancreas (25).We reported diabetic rats treated with AMME at doses of 150 and 300 mg/kg for 28 days displayed improved levels of AST, ALT, ALP, bilirubin, Cr, Ur, uric acid, total protein, and albumin compared with diabetic rats treated with normal saline.These promising effects on liver and kidney function are probably due to the positive effects of herbal extract in ameliorating the liver and kidney injuries induced by streptozotocin (26).
Our results revealed that diabetes results in a significant reduction in the tissue activity of antioxidant enzymes.However, diabetic rats treated with AMME at doses of 150 and 300 mg/kg for 28 days exhibited that the tissue activity levels of SOD, CAT, and GST were noticeably enhanced.Diabetes also caused an elevation in tissue MDA level in the tested mice; however, after treatment of diabetic rats with AMME at doses of 150 and 300 mg/kg for 28 days, the tissue level of MDA was declined when compared with diabetic rats.Diabetes results in an elevation in oxidative stress and lipid peroxidation; where there is a direct association between diabetes symptoms and lipid peroxidation.In addition, it has been proven that elevated blood sugar results in a significant reduction in the activity of antioxidant enzymes and subsequently a rise in free radicals (27)(28)(29).Therefore, we can suggest that AMME, through increasing the antioxidant enzymes and reducing the oxidative stress, is able to control diabetes in rats.Inflammatory responses are well-known as key factors in the development of diabetes and are thus linked to elevated insulin resistance and declined reaction in insulin target organs (30).The treatment of diabetic rats with either 150 or 300 mg/kg AMME for 28 days resulted in significant reduction of TNF-α and IL-1β levels in the pancreas, when compared with diabetic, indicating that AMME through its anti-inflammatory effects may control diabetes in rats.

Conclusion
This study showed that the oral administration of A. maximus methanolic extract in diabetic rats with streptozotocin displayed a potent anti-diabetic activity through increasing insulin release and ameliorating inflammation and oxidative stress; however, more investigations are desired to determine the mechanism effects of the extract.
Authors' contribution HS designed, HS, NS, and HRM performed experiments and collected data, RR wrote the draft, HRM and JGY discussed the results and strategy, and MNM supervised, directed, and managed the study.All authors approved the final version to be published.

Conflict of interests
The authors declare no conflict of interest.

Ethical considerations
This study was approved by the ethics committee of Lorestan University of Medical Sciences, Khorramabad, Iran, with the ethics number of IR.LUMS.REC.1401.219.

Funding/Support
The authors declare that they have not received any financial support from any organization.

Table 1 .
Antidiabetic effects of Astragalus maximus methanolic extract and glibenclamide on the serum levels of some biochemical parameters in the diabetic rats AMME, Astragalus maximus methanolic extract; GLB, glibenclamide; Diab, diabetic.*** P < 0.001 in comparison with the diabetic rats treated with normal saline.

Table 2 .
The effects of Astragalus maximus methanolic extract and glibenclamide on the serum levels of liver enzymes in healthy and diabetic rats

Table 3 .
The effect of Astragalus maximus methanolic extract and glibenclamide on the serum level of kidney parameters in non-diabetic and diabetic rats