Salazinic acid attenuates male sexual dysfunction and testicular oxidative damage in streptozotocin-induced diabetic albino rats†

Acute toxicity study

The acute toxicity study on male albino rats has exposed the non-toxic aptitude of Sa up to 100 mg kg⁻¹ body weight (b.w) dosage and the median lethal dose (LD₅₀) value of Sa could not be estimated due to no mortality. Thus, 1/10 and 1/20 of determined tolerated dose of Sa were selected as high dose (10 mg kg⁻¹) and low dose (5 mg kg⁻¹) for the current study.

Body weight and relative reproductive organs weight

At the beginning of the study, there was no significant change in the bodyweight of albino rats was observed between normal and all treated groups. At the end of 5^th and 9^th week, the body weight of diabetic control group rats was significantly lowered (P < 0.0001) than the normal control group (Table 1). However, Sa (5 and 10 mg kg⁻¹ b.w) and sildenafil citrate (a clinically unapproved anti-diabetic drug) administration had significantly improved the body weight of animals compared to the diabetic control group at the end of 5^th and 9^th week (P < 0.0001) (Table 1).

In association to normal control rats, a significant decrease in the relative weight of reproductive organs, namely testis (P < 0.001), penile (P < 0.0001), seminal vesicles (P < 0.0001), prostate gland (P < 0.0001), and cauda epididymis (P < 0.0001), were detected in the diabetic control rats (Table 1). The oral administration of Sa and sildenafil citrate prevented a significant reduction in the relative weight of reproductive organs compared to the diabetic control rats (Table 1). Overall, sildenafil citrate-treated group showed almost equivalent recovery of body weight and relative reproductive organ weights as that of the Sa-treated group at 10 mg kg⁻¹ b.w (Table 1).

Biochemical parameters

From week 0^th to 9^th, a gradual rise of fasting plasma glucose (FPG) levels (P < 0.0001) were noticed in the diabetic control group compared to the normal control group. Contrarily, Sa-treated groups significantly (P < 0.0001) reduced FPG levels compared to the diabetic rats in a dose-dependent way. In contrast to normal control, the serum level of insulin from week 0^th to 9^th was sharply decreased (P < 0.0001) in diabetic control rats. Sa administration steadily improved (P < 0.0001) the serum insulin levels compared to diabetic control rats in a dose-dependent way. However, the high dose of Sa treatment showed more potency in controlling serum FPG and insulin levels than sildenafil citrate (5 mg kg⁻¹ b.w) (Table 2). This finding confirms the ability of Sa to increase insulin concentration to lower plasma glucose, proving its antidiabetic activity.

At the end of 0 week, significant (P < 0.0001) variations of the serum concentrations of malondialdehyde (MDA), glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and reactive oxygen species (ROS) were observed in STZ-induced diabetic rats compared to the normal control group (Fig. 2). Simultaneously, treatment with Sa for 9 weeks significantly reduced MDA (P < 0.0001) and ROS (P < 0.001) concentrations with significant improvement in GSH (P < 0.0001), CAT (P < 0.0001), SOD (P < 0.0001) and GPx (P < 0.001–0.0001) concentrations in serum compared with those of the diabetic control rats in a dose-dependent way. At the end of week 9^th, all the serum oxidative stress parameters were almost similar in normal control, Sa-treated (10 mg kg⁻¹ b.w), and sildenafil citrate-treated groups (Fig. 2).

On the end of week 9^th, a significant reduction of serum hormones such as testosterone (T) (P < 0.0001), follicle-stimulating hormone (FSH) (P < 0.0001) and luteinizing hormone (LH) (P < 0.05) levels and significantly higher levels of tumor necrosis factor (TNF)-α (P < 0.0001) was observed in diabetic control group compared to normal control group (Fig. 3). In a dose-dependent manner, the oral treatment of Sa for 9 weeks significantly raised the above specified hormonal levels and significantly lowered TNF-α levels compared to the diabetic control group. The higher concentration of Sa-treated group was more potent than sildenafil citrate-treated group in elevating the serum hormonal levels (T, FSH and LH). As the serum T concentration is inversely proportional with the plasma glucose levels, the elevated T levels could be associated with androgen releasing activity, as well as antidiabetic effects of Sa. Moreover, the serum TNF-α levels were almost similar in both Sa-treated (10 mg kg⁻¹ b.w) and sildenafil citrate-treated groups (Fig. 3), suggest that Sa could inhibit inflammation to alleviate diabetic male reproduction dysfunction.

Similarly, an abnormal regulation (P < 0.0001) of lipid parameters including total cholesterol (TC), triglyceride (TG), and very-low-, low- and high-density lipoprotein (VLDL, LDL and HDL) content were observed in the diabetic control group compared to normal control (Fig. 4). In contrast to diabetic control group, Sa-treated groups showed a significant (P < 0.0001) reduction of TC, TG, VLDL and LDL contents with a significant (P < 0.0001) rise in HDL content in a dose-dependent way. At the end of week 9^th, the content of lipid parameters was almost similar in normal control, Sa-treated (10 mg kg⁻¹ b.w), and sildenafil citrate-treated groups (Fig. 4).

As compared to the normal control group, significantly (P < 0.0001) elevated levels of liver enzymes namely alanine transferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), were observed in the diabetic control group (Fig. 5A–C). Both, low and high doses of Sa-treated groups showed a significant (P < 0.0001) reduction of ALT, AST and ALP activities compared to the diabetic control group, in a dose-dependent way. In contrast to sildenafil citrate-treated group, a highest reduction in the ALT and ALP activities were noticed in high dose of Sa (Fig. 5A–C).

Also, the levels of the liver biomarkers, including creatinine (Cr) (P < 0.0001), urea (U) and total bilirubin (TBIL) (P < 0.0001), were significantly increased in the diabetic control group, and a remarkable reduction was observed in Sa-treated (P < 0.05–0.0001) and sildenafil citrate-treated (P < 0.0001) groups (Fig. 5D–F). The highest reduction in the U and TBIL levels were observed in the high dose of Sa-treated group, compared to the sildenafil citrate-treated group (Fig. 5D–F). On the other hand, a significant reduction of total protein (TP) levels was noticed in diabetic control group (P < 0.0001) and a significant recovery was observed in Sa-treated (P < 0.05–0.0001) and sildenafil citrate-treated (P < 0.0001) groups. However, a highest decrease in the TP level was observed in the sildenafil citrate-treated group, compared to the sildenafil citrate-treated group (Fig. 5G).

Testicular oxidative stress markers

The diabetic control group rats showed a significant (P < 0.0001) reduction of testicular concentrations of SOD, GPx, CAT and GSH with elevated MDA concentrations in testicular homogenate than the normal control group (Fig. 6). The oral supplementation of Sa significantly restored the levels of SOD (P < 0.0001), GPx (P < 0.0001), CAT (P < 0.001–0.0001), GSH and MDA (P < 0.0001) in the testicular tissues compared to the diabetic control group. However, treatment with Sa at 10 mg kg⁻¹ b.w showed more potent effects in controlling the antioxidant profile in the testicular tissues of diabetic rats compared to the sildenafil citrate-treated group (Fig. 6). This finding reflects that the elevated antioxidant enzymes convert free radicals into neutralized substances via scavenging ability in diabetic male rats and ultimately reduce oxidative damages. Therefore, as an effective antioxidant agent, Sa can defend the integrity of sperm membranes against oxidative stress conditions.

Sexual behavior of Sa-treated diabetic albino rats

The chronic diabetic conditions in the disease control group had significantly (P < 0.001–0.0001) altered the sexual function in male diabetic rats compared to the normal control group (Table 3). Our findings indicate that the oral supplementation of Sa and sildenafil citrate had significantly decreased mount latency (ML) (P < 0.001–0.0001), ejaculation latency (EL) (P < 0.05), intromission latency (IL) (P < 0.05–0.001), and post-ejaculatory interval (PEI), and significant improvement in mount frequency (MF) (P < 0.001–0.0001) and intromission frequency (IF) (P < 0.001–0.0001) compared to diabetic control group. The 10 mg kg⁻¹ b.w of Sa-treated group had shown more prominent results in improving male sexual activities compared to the sildenafil citrate group (Table 3), which specifies the restoration of T levels in rats.

The observations of the mating test revealed that the diabetic control group animals had remarkably reduced fertilizing capabilities such as number of females mated (4/12), mating success (33.34%), number of females pregnant (3/12), number of foetuses (5.0 ± 1.0; P < 0.0001) and corpora lutea (11.67 ± 0.58) formed, and male fertility (42.68 ± 6.87%; P < 0.0001) compared to the normal control group (Table 4). In a dose-dependent manner, Sa (low and high doses) treatment had improved the mating success (58.34 and 83.34%) by the number of females mated (7/12 and 10/12) and the number of females pregnant (6/12 and 10/12) compared to the diabetic control group. Also, the number of foetuses (8.00 ± 0.89 and 10.50 ± 0.71 (P < 0.0001)) and corpora lutea (12.33 ± 0.82 and 12.30 ± 0.48) formed per female rat were improved with Sa at 5 and 10 mg kg⁻¹ b.w doses, respectively, compared to the diabetic control group (Table 4). The male fertility percentage of Sa-treated groups (low and high doses) significantly increased to 64.99 ± 7.20 and 85.38 ± 5.17% (P < 0.0001), respectively, compared to the diabetic control group. On the other hand, sildenafil citrate-treated group (5 mg kg⁻¹ b.w) showed almost equivalent fertilizing capability as that of a high dose of Sa in STZ-induced diabetic male albino rats (Table 4).

Epididymal sperm characteristics

A significant (P < 0.0001) drop in sperm count (36.08 ± 2.51 million cells per mL), motility (48.33 ± 2.07%) and viability (45.67 ± 2.80%) with significantly (P < 0.0001) more abnormalities (18.17 ± 2.14%) and detached head from sperm (41.00 ± 3.22%) in sperm morphology were detected in the diabetic control rats (Video S1^†) compared to normal control rats (Table 5). In contrast to the diabetic control group, Sa-treated groups counteracted the reduction in all the above sperm parameters in a dose-dependent way. However, animals that received high dose of Sa showed the best reduction effects in sperm parameters than the sildenafil citrate-treated group (Table 5; Videos S2 and S3^†), which clearly justifies its ability to improve the quality of sperm, gonadotropin levels and spermatogenesis.

Histopathological examination

The slides of testicular tissues in the normal control group visualized an intact basal membrane (no interstitial space) lined with Leydig cells and healthy matured seminiferous tubules containing various stages of spermatogonial cells in the germinal epithelium zone (Fig. 7A). In the diseased control group, huge interstitial spaces were noticed between the seminiferous tubules with a great irregularity and/or degeneration in the order of spermatogenic cells, and no Leydig cells (Fig. 7B). On the other hand, oral treatment of Sa or sildenafil citrate exhibited marked development in the testicular structure and restoration in the series of spermatogenic cells (Fig. 7C–E). However, the recovery in Sa-treated groups was proportional to their dosages. Furthermore, a significant (P < 0.0001) reduction of testicular lesion score (TLS), mean Johnson’s score (MJS), mean seminiferous tubule diameter (MSTD) and mean seminiferous tubule diameter (MSTD) were detected in the diabetic control group compared to normal control rats (Table 6). Over 9 weeks of treatment with Sa at 5 (P < 0.05) and 10 (P < 0.0001) mg kg⁻¹ b.w significantly improved the early-mentioned factors compared with the diabetic control group in a dose-dependent manner. Besides, Sa at 10 mg kg⁻¹ b.w showed almost equivalent improvement in the TLS, MJS, MSTD and HGE as that of a sildenafil citrate-treated group (Table 6).

In silico studies

Drug likeliness

The physicochemical properties of the Sa were studied using DruLiTo software. A new chemical entity, that is intended to be used as a drug should follow Lipinski’s rule. The rule states that a drug should have molecular weight ≤500, hydrogen bond donor (HBD) ≤5, hydrogen bond acceptor (HBA) ≤10, and calculated logarithm of the partition coefficient (C log P) ≤5. DruLiTo also provides a convenient method of predicting other molecular properties that a drug should possess. Based on the Lipinski’s rule, the Sa follows and has no violations. The details of drug likeliness of Sa were furnished in Table 8.

Absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies

The ADMET profile of a molecule should be evaluated during the early stages of drug discovery and design to avoid drug withdrawal from the market.²⁷ Using these descriptors, it is possible to determine whether a compound is absorbed, distributed, metabolized, excreted, as well as if it is toxic. Although there are different in vitro methods for establishing ADMET profiles, in silico determination is a faster, cheaper, and life-saving method of determining ADMET profiles.²⁸

In addition to being nontoxic, ideal drug candidates should exhibit acceptable ADME characteristics. Based on SwissADME, ProTox-II and admetSAR we examined the ADME profiles, including drug-likeness, partition coefficients, solubility, human gastrointestinal absorption (HIA), blood–brain barrier (BBB), and cytochrome P450 inhibition, of the identified molecules (Table 8).²⁹

One of the most important properties of ADMET is its ability to absorb drugs from the human gut. HIA plays a pivotal role in drug transport to the target cells.³⁰ A higher HIA resulted in improved intestinal absorption of the compound. In this context, Sa showed HIA values greater than 0.9, indicating good membrane permeation. A Pan-Assay Interference Structural (PAINS) alert was used to determine the toxicity of compounds with desirable physicochemical properties. This assay is also referred to as a toxicophore test because of the presence of group elements that affect biological processes by interfering with DNA or proteins, which can cause fatal conditions such as cancer and hepatotoxicity.³¹ PAINS analysis provides information on the potential toxicity of a molecule. However, the Sa had 0 PAINS structural alerts, indicating their non-toxic nature (Table 8). The results from ProTox server indicated Sa (with LD₅₀ of 962 mg kg⁻¹) have toxicity class 4 (1 is for the most and 6 is for the least toxicity level). It exhibits inactive towards hepatotoxicity, carcinogenicity, immunotoxicity and cytotoxicity as mentioned in the Table 8.

These obtained predictions reveal acceptable properties od Sa, in terms of intestinal adsorption, distribution, permeability and toxicity across the blood–brain barrier, and therefore an interesting candidate for further studies.

In vitro antioxidant activity

Sa was respectively exposed to ABTS⁺·,³² DPPH·,³² superoxide radical,³³ Fe³⁺ ion reducing power³³ assays previously described. Each experiment was performed in triplicate and results were reported as IC₅₀ values. Particularly, to each known concentration (25–100 μg mL⁻¹) of Sa and ascorbic acid (standard drug) added either 1 mL of ABTS⁺· solution, 0.004% DPPH solution, nicotinamide adenine dinucleotide (73 μM) and nitroblue tetrazolium (50 μM), or 1% potassium ferricyanide and 0.1% ferric chloride. The mixture was incubated at appropriate temperature and time duration and absorbance at the specific wavelength (ABTS⁺·: 37 °C, 6 min, 750 nm; DPPH·: 37 °C, 30 min, 517 nm; superoxide: 37 °C, 30 min, 562 nm; Fe³⁺ ion: 37 °C, 30 min, 700 nm) was assessed against the blank.

In vitro antidiabetic activity

Sa was respectively exposed to each of the seven inhibitory assays, namely AR,³³ DPP-IV,³⁴ LIP,³⁵ protein glycation,³⁶ α-amylase and α-glucosidase³³ as previously described. Each experiment was performed in triplicate and results were reported as IC₅₀ values. To each known concentration (25–100 μg mL⁻¹) of Sa and acarbose (standard drug), added specific volume/amount of the corresponding enzyme and the substrate. The mixture was then incubated at a certain temperature and time duration, followed by measuring absorbance at particular wavelength against the blank.

Animals

Forty male and 120 female albino rats weighing 190–200 g were employed for the present experiment. Seven days before the experiment, all animals were fed under standard diet and adapted to the laboratory environment. The present experimental protocol was designed and executed as per OECD regulations and approved by the Institutional Animal Ethics Committee (No. 516/PO/c/01/IAEC) of the University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, India.

Acute toxicity study

The standardized normal male albino rats were orally dosed with 100 mg kg⁻¹ body weight (b.w) of Sa and carefully observed for 14 days. During this time, the behavioral parameters and mortality rate were recorded to determine the LD₅₀.³²

Induction of diabetes

Overnight fasted male albino rats were intraperitoneally (ip) induced with 55 mg kg⁻¹ b.w of STZ and allowed them to have free access to drink glucose solution (5% v/w). After 72 h, the animals having FPG levels of above 250 mg dL⁻¹ were considered as diabetic and used for the subsequent experiment.³²

Experimental design

The diabetic male albino rats were randomly categorized into groups (n = 6) and six other normal male albino rats were also included as the normal control group. The normal control and diabetic control groups were assigned only 0.5% sodium carboxymethyl cellulose. The standard group was administered with sildenafil citrate (5 mg kg⁻¹ b.w), while sample groups treated with Sa at 5 and 10 mg kg⁻¹ b.w for 9 successive weeks, respectively. The FPG levels and body weights of all groups were measured at the end of the 0^th (first day), 5^th and 9^th week.

Mating and copulatory behavior tests

On the 8^th week of the experiment, each treated diabetic male albino rat was paired with two normal virgin female albino rats in a separate cage for 5 days. Every morning, vaginal smear analysis was conducted on each female rat to identify the spermatozoa, which is used as a successful mating indicator. The positive (with the presence of spermatozoa) female rats were separated for 10 days, and their pregnancy was confirmed by euthanizing with an overdose of diethyl ether (ip), and the total number of foetuses and corpora lutea were ascertained.⁶

On day 63rd, the mating behaviour test was conducted on all animals after being treated with the test compound at certain doses. Initially, female albino rats were ovariectomized and subcutaneously injected 0.1 mL of 3-benzoyloxy-17b-estrol (48 h prior copulatory behaviour experiment) and 500 μg progesterone (4 h prior test). Each treated male albino rat was permitted 10 min of adaptation time to experimental conditions (cages in dim red light) and later paired with sexually receptive rats on a one-to-one basis. Finally, male mating behaviours such as ML, MF, EL, IL, IF, and PEI, were assessed. The copulatory behaviour test was terminated only in the cases where the male or female rats were unsuccessful to commence sexual behaviour.^6,13

Biochemical estimation

By piercing of retro-orbital plexus under diethyl ether, fasting blood samples were collected from treated male albino rats at the end of the 0^th, 5^th and 9^th week, and serum was separated by centrifugation of these blood samples for 15 min at 3500 rpm. The FPG levels were determined by using commercially available kits (Accu-Chek Glucometer, Roche Diabetes Care, India), and serum insulin levels, T, gonadotropins (FSH and LH) and TNF-α were measured by using ELISA kits (Novatec, India) according to the manufacturer’s guidelines. The levels of MDA and GSH and antioxidant enzyme activities such as CAT, SOD, GPx and ROS in serum were assessed using the corresponding assay kits purchased from Biochrome Scientific (India) according to the manufacturer’s information. In addition, ALT, AST, ALP, Cr, U, TP, TBIL, serum levels of TC, TG, VLDL, LDL and HDL, were assessed by using the corresponding commercially available kits (Biochrome Scientific, India) as per the manufacturer’s guidelines. Lastly, male albino rats were sacrificed with an overdose of diethyl ether (ip), and reproductive organs were detached and stored in 10% formalin.

Weights of reproductive organs

The dissected reproductive organs (testis, penile, cauda epididymis, seminal vesicle and prostate) were weighed (Shimadzu ATX224 Analytical Balance, Japan; capacity: 0.1 mg to 220 g) and the relative weight of each organ [(individual organ weight/body weight of the particular animal) × 100] was calculated.

Assessment of epididymal sperm characteristics

From the cauda epididymis, semen was gently sucked by using a red blood pipette (0.5 mark) and diluted up to 101 mark with normal saline and used for below sperm characteristics.¹³

Assessment of testicular oxidative stress markers

In ice-cold phosphate buffer solution (4 °C), the testis specimens were homogenized (Heidolph homogenizer Diax 900, Germany), centrifuged at 10 000 rpm for 15 min (4 °C), and the obtained supernatants were stored at −80 °C. The antioxidant enzyme activities such as SOD, GPx and CAT, and levels of GSH and MDA in testicular homogenates were assessed using the corresponding assay kits purchased from Biochrome Scientific (India) according to the manufacturer’s information.

Histopathological examination

The testes stored in 10% formalin were embedded in paraffin and subjected to microtome (5 μm). The obtained slices were stained with hematoxylin-eosin and examined microscopically (x400, Optscopes Classic IS 500, Germany). The MSTD and the HGE were calculated from 20 cross-sections of seminiferous tubules (mostly or nearly circular in shape) from three best slides from the lower, middle and upper parts of testes. Similarly, the MJS (range: 1–10) was evaluated to identify the spermatogenesis (testicular injury) in seminiferous tubules (50 in number).

In silico study

Prediction of drug likeliness

To predict drug likeliness, DruLiTo was used. Sa was subjected to DruLiTo that runs the algorithm to predict whether the molecules possess drug likeliness or not. DruLiTo is a java enabled application that is available at http://www.niper.gov.in/pi_dev_tools/DruLiToWeb/DruLiTo_index. html, which aids in easy prediction about the drug likeliness of molecule.