Studies on the Antidiabetic Activities of Cordyceps militaris Extract in Diet-Streptozotocin-Induced Diabetic Sprague-Dawley Rats

Cordyceps

Abstract

Due to substantial morbidity and high complications, diabetes mellitus is considered as the third “killer” in the world. A search for alternative antidiabetic drugs from herbs or fungi is highly demanded. Our present study aims to investigate the antidiabetic activities of Cordyceps militaris on diet-streptozotocin-induced type 2 diabetes mellitus in rats. Diabetic rats were orally administered with water extract or alcohol extract at 0.05 g/kg and 2 g/kg for 3 weeks, and then, the factors levels related to blood glucose, lipid, free radicals, and even nephropathy were determined. Pathological alterations on liver and kidney were examined. Data showed that, similar to metformin, Cordyceps militaris extracts displayed a significant reduction in blood glucose levels by promoting glucose metabolism and strongly suppressed total cholesterol and triglycerides concentration in serum. Cordyceps militaris extracts exhibit antioxidative effects indicated by normalized superoxide dismutase and glutathione peroxidase levels. The inhibitory effects on blood urea nitrogen, creatinine, uric acid, and protein revealed the protection of Cordyceps militaris extracts against diabetic nephropathy, which was confirmed by pathological morphology reversion. Collectively, Cordyceps militaris extract, a safe pharmaceutical agent, presents excellent antidiabetic and antinephropathic activities and thus has great potential as a new source for diabetes treatment.

1. Introduction

Diabetes mellitus (DM) is characterized by chronic hyperglycaemia which is resulted by the defects of insulin secretion or action. Diabetes patients suffer with a series of metabolic disorders in carbohydrate, fat, and proteins [1]. Noninsulin-dependent diabetes mellitus (NIDDM), caused by insulin resistance, is known as the most common form of diabetes (type 2 diabetes) [2]. According to statistics, till 2025, 8 billion people in the whole world will suffer with type 2 diabetes. Additionally, various complications including cardiovascular disease, nephropathy, neuropathy, retinopathy, and hyperlipemia are observed in most diabetes patients [3].

As a cosmopolitism tough problem, no satisfactory therapeutic regimen can cure diabetes although most of them normalize blood glucose and fat levels, possess hypotensive activity, and improve microcirculation [4]. Traditional therapy only focuses on pancreatic islet function recovery and blood glucose regulation, which fails to control the diabetic complications [5]. As reported previously, insulin injection and some oral antihyperglycemic agents, such as metformin and pioglitazone, display undesirable adverse effects [6]. Pioglitazone induces hepatocellular-cholestatic liver injury and metformin causes diarrhea and nausea or vomiting [7]. Additionally, weight gain, hypoglycemia, edema, gastrointestinal disturbances, and insulin resistance are observed in diabetes patients who receive long-term insulin treatment [8]. Meanwhile, diabetes mellitus requires lifelong medication, and the economic burden of patients should receive attention [9]. Due to the limitation of existing antidiabetic agents, a search for alternative treatment is highly demanded.

Herbal medicine turns out to be a valuable reservoir for novel drugs due to its few side effects [10]. Amount of research demonstrated that natural products possess antidiabetic activity with less adverse effects and show great auxiliary therapeutic effect on complications [1112]. Cordyceps militaris, an anamorph of Cordyceps sinensis, is advertised as a Chinese herb with antioxidant [13], immunomodulatory [14], anticancer, and anti-inflammatory pharmacological [15] effects. Cordyceps polysaccharides, the richest and most important activity component, display a hypoglycemic activity [16]. Additionally, several studies have shown that water extracts of Cordyceps militaris possess notable activity via increment of insulin secretion and cholinergic activation in normal Wistar rats [1718]. Excitingly, separated research finds that Cordyceps militaris can be used for kidney protection [19]. However, the regulatory effects of polysaccharide-enriched fraction of Cordyceps militaris on Sprague-Dawley rats with diabetes have not been reported yet.

We therefore hypothesized that Cordyceps militaris extracts may show antidiabetic, hypolipidemic, and even antinephritic effects. To test this hypothesis, the present study aims to investigate the related biological activities of Cordyceps militaris extracts via in vivo experiments. After treatment with polysaccharide-enriched fractions of Cordyceps militaris, the changes of serum fasting glucose levels, pyruvate kinase activity, triglyceride (TG), and total cholesterol in experimental diabetic Sprague-Dawley rats were detected. Several indexes associated with oxidation resistance and hypolipidemic activity were also determined. Furthermore, the therapeutic effects of Cordyceps militaris extracts on diabetic nephropathy were detected through histopathologic morphology observation and four indexes analysis including blood urea nitrogen (BUN), uric acid (UA), creatinine, and urine protein.

2. Methods

2.1. Submerged Fermentation of Cordyceps militaris

Cordyceps militaris (NBRC9787; obtained from National Biological Resource Center, Japan) was cultured in a rotary shaker incubator (10 L, Biostat B; Germany) at 150 rpm for 5 days and the cultured temperature was 26°C. The cultured medium was as follows: glucose, 20 g/L; peptone, 10 g/L; yeast extract powder, 18 g/L; KH2PO4, 3 g/L; MgSO4·7H2O, 3 g/L; (NH4)2SO4, 10 g/L; ZnCl2, 0.01 g/L; Vitamin B1, 0.24 g/L. The mycelium pellets were harvested and lyophilized for further using.

2.2. Cordyceps militaris Extract Preparation

As reported previously [20], the water and the alcohol extract from Cordyceps militaris were prepared as follows: 100 g mycelial powder was extracted two times in double distilled water at 80°C for 3 h. After centrifuging at 5000 rpm for 10 min, using Sevag reagent [V (n-butanol) : V (chloroform) = 1 : 4, 50 mL], the proteins that existed in the extracts were removed [21]. After concentration, the water extract (WE) was freeze-dried and stored in vacuum environment. Similarly, the alcohol extract (AE) was prepared using alcohol distillation at 60°C for 3 h followed by proteins removing and freeze-drying. The content of the total polysaccharides was  mg/g in WE and  mg/g in AE.

2.3. In Vivo Experiment in Animal Model of Diabetes

Experimental protocol was approved by the Lab Animal Centre of Jilin University (licence number SCXK-(JI) 2006-0001). Sprague-Dawley male rats weighing 120–140 g were housed in groups of two in clear plastic cages and maintained on a 12 h light/dark cycle (lights on 07:00–19:00 h) at °C with water and food available ad libitum.

The experimental protocol for diabetic rat model establishment and drug administration was shown in Figure 1. To produce experimental model of diabetes, 42 male Sprague-Dawley rats were administrated with a modified high fat high sucrose diet (HFHSD; 68.8% standard chow, 20% sucrose, 10% lard, 0.2% cholesterol, and 1% salt mixture; purchased from the Lab Animal Centre of Jilin University, Jilin, China) [22] for 8 weeks followed by the injection of 30 mg/kg streptozotocin (STZ) for 3 days (i.p., once per day) [23]. During the experiment, 5% glucose solution was fed to rats 4 h after STZ injection to prevent hypoglycaemia. Rats with fasting serum glucose levels between 11 mmol/L and 26 mmol/L were identified as severe diabetic groups for further study [24]. Another 7 male Sprague-Dawley rats feeding with normal diet for 8 weeks and injected with citrate buffer for 3 days served as control group (CT) which were treated with normal saline orally for another 3 weeks. All diet-STZ-induced diabetic rats were separated for 6 groups randomly as follows and received drug administration for 3 weeks (once a day):diabetic model group (DM; ): treatment with normal saline orally;metformin (DH) group (): treatment with 120 mg/kg metformin orally;low dose AE treated group (): treatment with 0.05 g/kg AE orally;high dose AE treated group (): treatment with 2 g/kg AE orally;low dose WE treated group (): treatment with 0.05 g/kg WE orally;high dose WE treated group (): treatment with 2 g/kg WE orally.

After 3-week treatment, food intake, water intake, and urine excretion in all rats were monitored within 16 h. Blood and urine samples were collected, and the fasting serum glucose, pyruvate kinase (PK), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), triglycerides (TG), total cholesterol, blood urea nitrogen (BUN), uric acid (UA), creatinine, and urine protein levels were determined. All the assay kits were obtained from Nanjing Biotechnology Co. Ltd. (Nanjing, China). After oral glucose tolerance test, animals were sacrificed by administration of 200 mg/kg pentobarbital; meanwhile, liver and kidney were collected and fixed in 4% paraformaldehyde.

2.4. Oral Glucose Tolerance Test (OGTT)

As shown in Figure 1, after 3-week treatment in diet-STZ-induced diabetic rats, an oral glucose tolerance test (OGTT) was performed. After a 12 h fast, all the experimental rats were received physiological saline, metformin, AE, or WE, respectively, as described above; 30 min later, 2 g/kg of glucose was orally given to all the rats. Blood samples were collected at 0, 30, 60, and 120 min to detect the blood glucose levels using Glucose Assay Kit (Nanjing Biotechnology Co. Ltd., Jiangsu, China). Calculation of the area under the blood glucose curve (AUC) was made according to  [25]:

2.5. Histopathological Examination

Collected tissues were immerged in 4% paraformaldehyde for 48 h and then dehydrated in gradient ethanol (50%, 70%, 80%, 90%, 95%, and 100%) step by step. Samples were immerged in xylene for 30 min and incubated with first paraffin at 65°C overnight. After embedding in wax, tissues were cut into serial sections at 5 μm thickness using microtome (Leica, Germany) and spread over microscopy slides. Sections were deparaffinized with fresh xylene for 10 min, hydrated with gradient ethanol (100%, 90%, 80%, and 70%), and then washed with double distilled water for three times. The sections were analyzed via haematoxylin and eosin staining (H&E staining) [26] and examined by a light microscope digital camera (Nikon Instruments, Tokyo, Japan).

 

2.6. Statistical Analysis

All values were expressed as mean ± SD. One-way analysis of variance (ANOVA) was used to detect statistical significance followed by post hoc multiple comparisons (Dunn’s test). A value of  was considered to be significant.

3. Results

3.1. Bodyweight, Food and Water Intakes, and the Urine Excretion Monitoring

Compared with CT group, DM rats consumed more food and water (; Table 1); meanwhile, more urine excretion in DM rats was noted (; Table 1). Similar to DH-administrated rats, both AE and WE treatment at 0.05 g/kg and 2 g/kg strikingly decreased urine excretion and water intakes in diet-STZ-induced diabetic rats compared with DM rats; however, no significant changes in food intakes were observed (Table 1; ). Compared with CT, the growth of diet-STZ-induced diabetic rats was inhibited strongly (; Figure 2); however, after 3-week 2 g/kg AE and WE and 120 mg/kg DH treatment, the bodyweight was increased significantly compared with DM group (; Figure 2, Table 1).