Protective effects of pomegranate (Punica granatum) juice on testes against carbon tetrachloride intoxication in rats

Carbon tetrachloride (CCl4; CAS Number 56-23-5) and Tris–HCl buffer were purchased from Sigma (St. Louis, MO, USA). Perchloric acid, thiobarbituric acid (TBA) and trichloroacetic acid (TCA) were purchased from Merck. All other chemicals and reagents used in this study were of analytical grade. Double-distilled water was used as the solvent.

All experimental procedures involving animals were conducted in accordance with the guidelines of the National Program for Science and Technology of Faculty of Science, King Saud University. The study protocol was approved (No. 1/3/12337) by Ethical Committee of King Saud University (KSU), Riyadh, of the joined work between College of Science (KSU) and Zoology Department (Helwan University).

Ten kg of pomegranates were washed and manually peeled, without separating the seeds. Juice was obtained using a commercial blender (Braun, Germany), filtrated with a buchner funnel and immediately diluted with distilled water to volume of 1:3 and stored at -20°C for no longer than 2 months [13].

Pomegranate juice stability was assessed by measuring initial total phenolic content and evaluating the alterations after 2 and 3 days of exposure to the same conditions as the juice supplied to the animals. The total polyphenol content of the pomegranate juice was 74.8 μg gallic acid equivalent/ml juice, determined following the standard Folin-Ciocalteu method. The content of polyphenol was not markedly affected after long time storage.

Three replicates from juice were centrifuged in an eppendorf tube (2 min at 1400 rpm) and filtered through a 0.45 μm filter. A liquid chromatography apparatus 1290 series from Agilent Technologies, including a degasser, a binary pump delivery system, and an automatic liquid sampler, was used and coupled to Agilent Triple Quad Model 6460 mass spectrometer detectors. The HPLC column was a ZORBAX Eclipse plus C18 (4.6 × 150 mm, 5 μm) from Agilent Technologies (Agilent Technologies, Palo Alto, CA, USA). Separation was carried out by acetic acid (2%; A) and acetonitrile (B). The following multistep linear gradient was applied: 0 min, 5% B; 2 min, 7% B; 4 min, 9% B; 6 min, 12% B; 8 min, 15% B; 9 min, 16% B; 10 min, 17% B; 11 min, 17.5% B; 12 min, 18% B; 14 min, 20% B; 16 min, 28% B; 18 min, 100% B; 22 min, 100% B; 23 min, 5% B. The initial conditions were maintained for 5 min. The flow rate was set at 0.80 ml/min throughout the gradient. The injection volume in the HPLC system was 2.5 μl. The chromatograms were registered at 280 and 360 nm. Separation was carried out at 30°C. MS analysis was carried out using electrospray ionization (ESI) interface in negative ionization mode.

To study the protective effects of pomegranate on carbon tetrachloride mediated reproductive toxicity, twenty eight adult male rats were randomly allocated to four groups of seven rats of each. Group I (Con) served as control and received 300 μl of saline by intraperitoneal (i.p.) injection route each week. Group II (CCl₄) received weekly i.p. injection of 2 ml CCl₄/kg body weight (bwt) for 10 weeks as described by sohn et al. [14]. Group III (Pom) received juice supplied on dark water bottles and renewed every 2–3 days [13] and the animals of group IV (Pom + CCl₄) received pomegranate juice as group III for 2 weeks before and concurrent with CCl₄ treatment that injected intraperitoneally for 10 weeks at a dose of 2 ml of CCl₄ per kg bwt. After one week of the last i.p. injection of CCl₄, blood samples were collected from all animals by cardiac puncture (under anaesthesia with chloroform). Right testes was promptly excised, weighed and homogenized immediately to give 50% (w/v) homogenate in ice-cold medium containing 50 mM Tris–HCl, pH, 7.4. The homogenate was centrifuged at 3000 rpm for 10 min at 4°C. The supernatant (10%) was used for the various biochemical determinations.

Relative weight of testes was calculated as left testes weight/body weight × 100.

Homogenates of testes were used in determination of superoxide dismutase (SOD) [18], catalase (CAT) [19], glutathione peroxidase (GPx) [20], glutathione-S-transferase (GST) [21] and glutathione reductase (GR) [22].

Quantitative measurement of serum testosterone, follicle stimulating hormone (FSH) and luteinizing hormone (LH) were carried out adopting ELISA technique using kits specific for rats purchased from BioVendor (Gunma, Japan) according to the protocol provided with each kit.

The testes tissues were collected and immediately fixed with 10% buffered formalin, and embedded in paraffin. Sections (4–5 μm) were prepared and then stained with hematoxylin and eosin dye for photomicroscopic observations.

Results were expressed as the mean ± standard error of the mean (SEM). Data for multiple variable comparisons were analyzed by one-way analysis of variance (ANOVA). For the comparison of significance between groups, Duncan’s test was used as a post hoc test according to the statistical package program (SPSS version 17.0) and figures were drawn with Origin (version 8). All p values are two-tailed and p < 0.05 was considered as significant for all statistical analysis in this study.

The phytochemical fingerprint of pomegranate juice was determined using a electrospray ionization mass spectrometry (ESI-MS). HPLC-MS technique is an important method used for identifying complex mixtures, especially the phenolics or its fraction found in the plant, either by using standard compounds (target identification) or by comparing mass spectrum obtained with literatures (tentative identification). This method is useful to avoid replication, safe time, and money used in isolation and identification of known compounds. In the current study, pomegranate juice was subjected to HPLC-ESI-MS analysis. HPLC-ESI-MS experiment allowed the identification of a total of 41 compounds. Hydrolyzable tannins were the main class of (poly)phenolics identified in pomegranate juice. A broad number of anthocyanins, non-coloured flavonoids and phenolic acids were also found. Other phytochemicals, such as lignans, were also observed. Likewise, several organic acids were detected (Figure 1).

The 41 compounds were identified by the interpretation of their fragmentation patterns obtained from the mass spectrum. Data provided by literature information was employed for the comprehensive evaluation of the juice. The retention times and mass spectrum data along with peak assignments for compounds identified using negative ionization are described in Additional file 1: Table S1. As shown in Additional file 1: Table S1, m/z 301 (-) ions from MS analysis are evidence for the presence of ellagic acid precursor and HHDP in the molecules. Ellagitannins were also detected in the pomegranate juice assessed. They were distinguished by their characteristic fragment ion spectra yielding sequential losses of galloyl (m/z 152), gallate (m/z 170). Gallotannins, composed by monomeric and dimeric galloyl moieties linked to a hexose sugar were also detected. Six compounds matching the molecular ion m/z 331 were observed and considered as gallotannins. The different flavonoids belonging to four subclasses of non-coloured flavonoids (flavan-3-ols, flavonols, dihydrochalcones and flavanones) were detected. The flavan-3-ols detected was (+)-gallocatechin. Kaempferol, phlorizin and quercetin displayed flavonols, dihydrochalcones and flavanones subclasses, respectively. A total of three phenolic acid derivatives were found in pomegranate juice in this study, vanillic acid, its hexoside and syringetin-hexoside. In chromatographic analysis, organic acids were separated and identified by comparison with published data. Citric acid and its derivative have been pointed out as the main organic acids in pomegranate juices. As summarized in Additional file 1: Table S1, six anthocyanins were detected. Anthocyanins are the phenolics responsible for the red colour of pomegranate juice. The anthocyanin profile comprised cyanidin, pelargonidin, and delphinidin.

Additionally, gas chromatography–mass spectrometry (GC-MS) analysis experiment was performed. GC-MS chromatogram of the pomegranate juice showed 33 peaks indicating the presence of 33 phytochemical constituents (Additional file 2: Figure S1 and Table S2).

The toxicity of CCl₄ on testes weight and the relative testes weight were represented in Figure 2. The injection of rats with CCl₄ caused a significant increase in testes weight (1.4 g; p < 0.05) and relative testes weight by 74.63% comparing to the control group. Treatment with pomegranate juice erased the CCl₄ toxicity and significantly improved testes weight and relative testes weight when compared with the CCl₄ group which helps in reducing edema in the testes due to fluid accumulation, however, pomegranate juice failed to return the testes weight and relative testes weight (1 g and 23.88%, respectively) to the control values. Supplementation of pomegranate juice on its own did not change the testes weight (0.78 g) and relative testes weight as compared with that in the control group.

The LPO level is widely used as a marker of free-radical mediated lipid peroxidation. The results of the LPO assays in the testes homogenates are shown in Figure 3. LPO level in the CCl₄-treated group (65.9%) was significantly higher than in the vehicle-control group. Supplementation of pomegranate juice significantly decreased CCl₄-induced testisticular lipid peroxidation. NO reacts with O₂ ^•- and leads to the formation of ONOO^- (peroxynitrite), which contributes to reproductive toxicity due to its cytotoxicity properties. CCl₄ injection caused a significant increase (58.4%; p < 0.05) in NO content in testes homogenates compared to the control group. This oxidant molecule was significantly reduced (20.8%) when animals were supplemented with pomegranate juice showing the ameliorative effects of pomegranate juice (Figure 3).

Antioxidant enzymes such as CAT, SOD, GPx, GR and GST, as well as, glutathione as a non-enzymatic antioxidant substance were estimated in the present study. There was a significant decrease in GSH in the testes homogenates of CCl₄ group as compared to the control group (-22.90% at p < 0.05). The supplementation of rats with pomegranate juice pre- and concurrent with CCl₄ injection caused a significant increase in GSH not only when compared with CCl₄ group but also with the control group (Table 1). GPx, GR and GST activities were also significantly decreased in the testisticular tissues of rats inoculated with CCl₄ (Table 1), but pomegranate juice was able to significantly elevate these parameters after 10 weeks of CCl₄ injection.

Carbon tetrachloride decreased the activities of SOD and CAT to approximately -55.81% and -29.87%, respectively, compared to the control group (Figure 4). Rats supplemented with pomegranate together with CCl₄ for 12 weeks experienced a significant increase in SOD and CAT compared to the CCl₄ group (Figure 4).

The mean values of the serum hormones; testosterone, luteinizing hormone and follicle stimulating hormone are shown in Figure 5. After treatment of rats with CCl₄ for 10 weeks, the mean values of testosterone, LH and FSH were decreased as compared to the control group (-72.1%, -65.4% and -19.0%, respectively). In the (Pom + CCl₄) treated group, testosterone levels were restored to control values. Serum level of LH in this group was increased significantly as compared with the CCl₄-treated group (p < 0.05), but it still decreased significantly compared to the control group. Mean values of FSH in the (Pom + CCl₄) treated group did not differ from the CCl₄-treated group. Statistically significant increase in the levels of testosterone, LH and FSH were observed in rats treated with pomegranate juice on its own as compared to the control group.

Among rats given daily pomegranate juice supplements alone, there were no marked changes in testicular histology relative to controls (Figure 6C). Thus normal spermatogenesis, well preserved Sertoli cells and well delineated tubular basement membrane were observed. The interstitium between tubules and Leydig cells was also intact. However, in the CCl₄-treated group, differences were observed in the histology of testes, where complete swallowing of seminiferous tubules was exhibited while in other areas of the section the tubular basement membranes of seminiferous tubules were identified, but most of the germ cells were degenerated, especially the ones involving highly differentiated germ cells along with deformed sperm. The ground substance within the interstitium also partially disappeared and was replaced by fibroblast and inflammatory cells (Figure 6B). In the (Pom + CCl₄)-treated group, those toxic effects were ameliorated (Figure 6D).