Effects of Cultured Cordycep militaris on Sexual Performance and Erectile Function in Streptozotocin-Induced Diabetic Male Rats



Cordyceps militaris (CM), a valuable edible and medicinal fungus, has been used as traditional medicine to treat health conditions, as well as hyposexuality in Asian societies for over a century. Due to the high demand, several artificial cultivation methods have been developed for their biological activities. In this study, CM was cultured on medium that contained white rice and silkworm pupae, and the levels of cordycepin and adenosine, as well as its aphrodisiac effects in diabetes-induced erectile dysfunction (DIED), were evaluated. Diabetic rats were induced by streptozotocin (STZ) injection and administered orally with CM (0.1, 0.5, and 1.0 g/kg BW/day) for 3 weeks. Diabetic rats in negative and positive control groups received vehicle and sildenafil citrate (5 mg/kg), respectively. Results showed the changes in mating behaviour in which mount latency and intromission latency were significantly increased in diabetic rats, compared with the normal control group. Diabetic rats also showed a significant reduction in intracavernosal pressure (ICP) response to cavernous nerve stimulation, sperm count, testosterone level, penile nitric oxide synthase (NOS), and testicular superoxide dismutase (SOD) activities, when compared to the normal control group. Administration of CM (0.1, 0.5, and 1.0 g/kg BW/day) reversed the effects of diabetes on the mating behaviour, and the ICP responses to electrical stimulation. Moreover, the levels of penile NOS, testicular SOD activities, testosterone, and sperm count were significantly increased, and testicular malondialdehyde (MDA) levels were significantly decreased in these treated diabetic rats. Diabetic rats treated with sildenafil showed a significant induction in intromission frequency and NOS and SOD activities, as well as a marked increase in ICP responses. These results suggest that CCM exerts its aphrodisiac effect, possibly through activating testosterone production and suppressing oxidative stress to enhance erectile function in diabetic rats.

1. Introduction

Erectile dysfunction or ED has been signified as an inability of the male to achieve a penile erection, as part of the overall multifaceted process of male sexual function [1]. ED status can arise in adult men of all ages, as its prevalence and incidence are associated with aging. The prevalence of ED in young men has been estimated to be as high as 30%, in which diabetes mellitus (DM) either type 1 or type 2 has a well-established and strong association with ED [23]. The pathophysiology of diabetes-induced erectile dysfunction (DIED) is multifactorial and several mechanisms of ED have been proposed in diabetic patients, including increased oxygen free radicals and impaired nitric oxide (NO) synthesis [4]. The chronic hyperglycemia can lead to endothelial dysfunction, which is manifested as the decreased bioavailability of NO, resulting in insufficient relaxation of vascular smooth muscle of the corpora cavernosal [5]. The current first-line therapy for diabetic ED is phosphodiesterase type 5 inhibitors (PDE5Is), such as sildenafil (Viagra®), tadalafil (Cialis®), and vardenafil (Levitra®). However, PDE5-Is has been shown to have some adverse effects, i.e., headache, abnormal vision, dyspepsia, flushing, nasal congestion, and back pain, which may impact negatively on patient’s lifestyle [6].

Alternative approaches, such as herbal medicine, have been adopted for sexual improvement for centuries. To date, many plants have been reported to possess aphrodisiac potential, and their effects on sexual behaviour have been validated [7]. In Asian countries, herbal supplements derived from Cordyceps species have been traditionally used as prosexual agent, one of which is Cordyceps militaris (CM), a valuable medicinal mushroom in the family Clavicipitaceae [8]. Nowadays, instead of harvesting from natural resources, CM has been cultivated using the artificial culture medium and similar bioactive contents and medicinal potential as wild Cordyceps has been reported [9]. Due to the growing demand, a number of culture techniques and the media formula to support growth of CM have been developed, and many bioactive ingredients have been isolated, such as adenosine, cordycepin, D-mannitol, polysaccharides, nucleosides, amino acid, essential oils, ergosterol peroxides, and xanthophylls [911]. Several scientific evidences related to the mechanisms and efficacy of this fungus, such as anticancer, antihypertensive, antioxidant, antiapoptotic, and hypoglycemic effects, have been reported [9101214]. Its positive effects in sexual function and testicular function have also been elucidated in young male rats [15], middle-aged rats [16], and aged male rats [17]. However, research on its bioactivity as prosexual agent in DIED is still scarce. The objective of the present study was to ascertain if CCM had aphrodisiac activity in STZ-induced diabetic rats and to elucidate the underlying mechanisms.

2. Materials and Methods

2.1. Preparation of CCM

Cordyceps militaris was obtained from the Department of Agriculture (DOA) in Thailand. CCM was prepared at the Department of Agricultural Science, Faculty of Agriculture, Natural Resources and Environment, Naresuan University. In brief, the mycelia were cultivated with modified of potato dextrose agar (MPDA) medium under stable conditions at 22°C for 2 weeks. The resultant culture was transferred to potato dextrose broth (PDB) medium, which was then incubated on rotary shaker at 22°C for 2 weeks before transferring the mycelium to sterilized rice cultured medium that contained white rice and silkworm pupae. The fruiting bodies with the inoculums were kept in 12 : 12 h light-dark at 18°C, 60-70% humidity until the mycelium had transformed into the fruiting bodies primordia. Then, the flasks were maintained at 22°C, 80-90% humidity for 64 days before the fruiting bodies had been reaped and immediately frozen at -20°C until used.

2.2. Detection of Cordycepin and Adenosine in CCM with High-Performance Liquid Chromatography (HPLC) Fingerprint Analysis

Cordycepin and adenosine in CCM were determined according to Huang et al. (2009) with some modifications. In brief, the fruiting bodies were dried in hot-air oven (55°C, 48 h). The dried samples were ground using a homogenizer, and 1.0 g of powder was added into 10 ml of methanol : water (50/50, ) and sonicated for 30 min, followed by centrifugation at 9,900 g for 15 min 2 times. The obtained supernatant was then filtered through a 0.45 μm filter membrane before injecting into the HPLC system (Shimadzu, Japan) with a column (Restek, Ultra IBD; , 5 μm particle size) set at 35°C. The mobile phase was a mixture of water and methanol (90 : 10; ) with the flow rate at 1 ml/min and a UV-vis detector at 254 nm. Five standard solutions of cordycepin and adenosine (Sigma Chemical, MO, USA) (20 μl) were prepared and injected into the HPLC to create standard calibration curves.

2.3. Animals

Eight-week-old Sprague-Dawley rats used in this study were specific-pathogen-free (SPF) grade and were purchased from M-CLEA Bioresource Co., Ltd. (Samut Prakan, Thailand). Procedures involving animal subjects were approved by the Naresuan University Animal Care and Use Committee (NUACUC). All animals were handled in accordance with the Guidelines for the Care and Use of Laboratory Animals (National Research Council of Thailand) with an effort to minimize animal suffering. Rats were maintained under controlled temperature (22 ± 1°C) and relative humidity () with 12 : 12 hours of reverse light and dark cycle at Naresuan University Centre of Animal Research (NUCAR), which has been accredited by AAALACi. All rats were fed ad libitum a standard diet (CP No. 082; C.P. Company, Bangkok, Thailand) and allowed free access to reverse osmosis (RO) water.

In order to induce diabetes, fifty male rats received a single intraperitoneal injection of STZ (60 mg/kg BW, i.p.) (Sigma-Aldrich, USA), which was dissolved in citrate acid buffer (pH 4.5). Ten male rats in the normal control group received only citrate buffer. After 72 h, fasting blood glucose (FBG) levels were checked using glucometer (Accu-Chek Performa, Roche). Rats with FBG levels higher than 200 mg/dl were used and divided into five groups: (I) DM control, (II) DM+CCM 0.1 g/kg, (III) DM+CCM 0.5 g/kg, (IV) DM+CCM 1.0 g/kg, and (V) DM+sildenafil 5 mg/kg. CCM were weighted and blended thoroughly with a blender and were given by daily oral gavage for 3 weeks. Sildenafil citrate was given only one time 30 min before mating behaviour test.

2.4. Surgical Procedure: Ovariectomy

For prevention of pregnancy, female rats were subjected to bilateral oophorectomy surgery before beginning the sexual function assessment. Each adult female rat was anaesthetized by 1.5-2.0% isoflurane (Piramal Critical Cares, Inc., USA) combined with oxygen. The lower abdominal skin and muscle were opened vertically 1 cm, and the uterine horn was pulled out and ligated before removal of the ovary, one at a time. The uterine horn was returned to the peritoneal cavity, and the wound was closed in two layers (abdominal muscle and skin) using sterile sutures. The skin was then disinfected with povidone iodine and covered with Fixomull Stretch®. Each rat received an intramuscular tramadol (5 mg/kg) to ameliorate postoperative pain and allowed at least two weeks for full recovery. The ovariectomized female rats were artificially brought into oestrus phase by the administration of estradiol (0.025 mg/kg) and progesterone (1 mg/kg) at 48 and 4 hours before mating, respectively.

2.5. Mating Behaviour Assessment

Mating tests were conducted in a custom made clear glass chamber . Each male rat was allowed to habituate in the chamber for 5 min before introducing a sexually receptive female rat into the chamber. The following male sexual behaviour parameters were calculated after monitoring for 30 min:(i)Mount latency (ML). The time interval between the introduction of the female and the first mount by the male(ii)Intromission latency (IL). The time interval between the introduction of the female and the intromission by the male(iii)Ejaculatory latency (EL). The time interval between the first intromission and ejaculation(iv)Mount frequency (MF). The number of mounts from the time of introduction of the female until ejaculation(v)Intromission frequency (IF). The number of intromissions from the time of introduction of the female until ejaculation(vi)Ejaculation frequency (EF). The number of ejaculations in a sexual cycle.

Their behaviour was recorded with the digital VDO camera (LYD-808C, China) for offline analysis by two observers to ensure accuracy.

2.6. In Vivo Assessment of Erectile Function

After completion of mating test, each male rat was anaesthetized with 2-2.5% isoflurane combination with oxygen. The ventilation rate, pulse rate, temperature, and heart rate were monitored via PhysioSuite® (Kent Scientific, USA). The carotid artery of rat was cannulated to measure mean arterial blood pressure (MAP) by using PowerLab® (AD Instruments, Australia). The penile skin was removed, and a polyethylene tube was inserted with heparinized saline via a 22gauge needle for measuring intracavernosal pressure (ICP). The lower abdomen was opened exposing the cavernous nerve, which was then stimulated via a copper bipolar electrode connected to the PowerLab®. The cavernous nerve was stimulated by electrostimulation, starting from 0.25, 0.50, 0.75, 1, 2, 3, 4, and 5 to 10 volts at a frequency of 20 Hz for 60 sec for each voltage. The results were recorded by LabChart (version 7.3.7; AD Instruments, Australia) connected to a computer. Since electrostimulation slightly lowered MAP, ICP was normalized by MAP as ICP/MAP. Upon completion of experiment, rats were euthanized, and their penis and left testis were immediately collected and stored at -80°C for further analysis. The relative weights of penis and testis were calculated by the following formula: .

2.7. Determination of Serum Testosterone and Sperm Concentration and Motility

Blood was collected from abdominal aorta and put into the nonanticoagulated tube and stored at 4°C before sending to the Biolab Medical Clinic, Phitsanulok, Thailand, for testosterone analysis. Semen was collected from the caudal epididymis and vas deferens and diluted in 1 M phosphate buffer saline (PBS) at 37°C. Then, 10 μl of sample was transferred into the Makler chamber and analysed under a light microscope. Sperm motility was recorded as video files for offline analysis. The number of sperm was counted and expressed as × 106 per milliliter according to the WHO manual.

2.8. Measurement of Penile Nitric Oxide Synthase (NOS) Activity

NOS activity in the penis was determined using the nitric oxide synthase assay kit (Cat. No 482702; Calbiochem®, Germany). The penile tissue was weighted and homogenized with ice-cold 1 M PBS (pH 7.4) by homogenizer (Ultra-turexT8, Germany). The sample was centrifuged at 10,000 g for 20 minutes and filtered through 0.45 μm membrane filter, and then the supernatant was obtained by centrifuging the sample at 100,000 g, 4°C for 15 min. The penile NOS activity was measured, following the manufacturer’s instructions. The absorbance at 540 nm was determined using a microplate reader (1401, LabSystem, Finland). Blank wells were used to normalize the yield.

2.9. Measurement of Superoxide Dismutase (SOD) Activity

Testicular SOD was measured using the superoxide dismutase assay kit (Calbiochem®, cat #574601, Merck Millipore). Testis tissue was homogenized with buffer (10% ), pH 7.2 (containing 1 mM EGTA, 70 mM sucrose, 20 mM HEPES, and 210 mM mannitol). The homogenate was centrifuged at 1,500 g for 5 min. The supernatant was collected and centrifuged at 10,000 g, 4°C for 15 min. The SOD activity in the sample was then processed according to the manufacturer’s instructions and determined by a microplate reader with the absorbance at 450 nm.

2.10. Measurement of Thiobarbituric Acid Reactive Substances (TBARS)

Concentrations of TBARS in testes tissues were measured according to the method of Ohkawa et al. [18] with some modifications. Testicular tissue was homogenized (10% ), in ice-cold 1 M phosphate buffer (pH 7.4). The homogenate was centrifuged at 4,000 g, 4°C for 15 min. Sample supernatant (100 μl) was added into the vial containing 1,500 μl of 20% acetic acid (pH 3.5), 200 μl of 8.1% sodium dodecyl sulphate (SDS), and 1,500 μl of 0.8% of thiobarbituric acid (TBA). The mixture was incubated for 60 min at 95°C and immediately cooled on ice, followed by centrifugation at 10,000 g for 3 min. The resulting supernatant was then used as the enzyme source for the determination of the malondialdehyde (MDA) level by the optical density (OD) measurement of the pink complex at 532 nm. MDA values were calculated using tetramethylpiperidine (TMP) as a standard curve and expressed as nmol/mg of protein that determined absorbance at a wavelength of 562 nm. Protein level was measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA).

2.11. Histological Examination of Testicular Tissues

The right testis was fixed in 10% neutral buffered formalin over 24 hours. The tissues were then processed with 70-100% ethanol and xylene, respectively. The infiltrated tissues were embedded in paraffin blocks and cut at 2 μm by using semiautomatic microtome (Leica, Germany). All slides were stained with hematoxylin and eosin (H&E), and histological changes were observed by light microscopy.

2.12. Statistical Analysis

The value of mating parameters, sperm concentration, NOS and SOD activities, and MDA level were presented as . ICP, MAP, and testosterone levels were presented as . Data were analysed using one-way analysis of variance (ANOVA) followed by Dunn’s post-hoc Multiple Comparison Test (Graph Pad Prism 7.4, GraphPad Software, San Diego, USA).  values of less than 0.05 were regarded as statistically significant.

3. Results and Discussion

3.1. Cordycepin and Adenosine Contents in CCM

Many species of Cordyceps are being cultivated in artificial medium for their medicinal and pharmaceutical properties. Cordycepin, a derivative of the nucleoside adenosine, has been shown to be the first and is the main active constituent isolated from Cordyceps sp. [9]. In this study, we performed an analysis of bioactive compounds and found that the adenosine and cordycepin contents in CCM were  and , respectively (Figure 1). This amount of cordycepin is relatively similar to that cultured in silk worm pupae medium () and higher than that obtained in brown rice medium (), as reported by Kang et al. [19]. However, cordycepin production could yield with a maximum of about 445 mg/l, when CM was cultured in submerged conditions [20].