Punicalagin Improved the Oxidant-antioxidant Status in Male New Zealand White Rabbits

Punicalagin is one of the largest known polyphenols. It has a molecular weight of 1084.722 g/Mol and has strong antioxidant properties since it has a total of 16 separable -OH groups (Sohrab et al., 2014). This phenolic compound contains ellagitannins such as gallic acid and glucose-bound ellagic acid. Once consumed, all of these ellagitannins are capable of being hydrolyzed to ellagic acid. Consequently, ellagic acid is released into the blood and stay at high concentrations for a long time (Cerda et al., 2003).

Pomegranate (Punica granatum), Terminalia catappa, Terminalia myriocarpa and Combretum molle plants can be mentioned as the most important sources of punicalagin (Martel et al., 2016). Pomegranate fruit is pressed as a whole during fruit juice processing, resulting in 2 g/L of ellagitannin passes to pomegranate juice (Gil et al., 2000). Among other fruit shells such as bananas, mangoes and coconuts, pomegranate peel has the highest antioxidant capacity (Okonogi et al., 2007).

Due to the high antioxidant properties of pressed pomegranate juice and tropical almond leaf, it is possible to come across in vivo and in vitro studies with these products. However, the number of in vivo studies performed with the active substance, punicalagin, is limited. Although antimicrobial (Machado et al., 2002; Silva et al., 2015), hepatoprotective (Lin et al., 2001), neuroprotective (Yaidikar et al., 2014), anti-inflammatory (Lin et al., 1999; Jean-Gilles et al., 2013), antidiabetic (Nagappa et al., 2003) and spermatological (Yildiz -Gulay and Gulay, 2019) effects of punicalagin were reported, the literature lacks direct studies on the possible antioxidant properties of punicalagin in rabbits. Therefore, the current study was aimed to evaluate the tolerable dose, possible health benefits and protective effects of punicalagin in male rabbits.

This study was approved by the Ethics Committee of Burdur Mehmet Akif Ersoy University (2015.25.11/159) and supported by TUBITAK (project no: 116O027). Prior to the experiment, 24 male New Zealand White rabbits (7 to 10 months old and 2.4 to 3.7 kg body weight) were individually kept in a galvanised cage and acclimated to laboratory conditions (50-55% humidity, 22 ± 2^oC temperature and 14:10 hour light: dark cycle) and at the Experimental Animals Units of Burdur Mehmet Akif Ersoy University, Faculty of Veterinary Medicine, Burdur, Tukey in 2017.
 
Experimental design
 
Following the acclimation period, rabbits were randomly divided into 4 groups (n=6). The control group (C) received distilled water by oral gavages for 9 weeks. P1, P2 and P10 groups received daily oral gavages of 1, 2 and 10  mg/kg body weights of punicalagin in distilled water for 9 weeks, respectively. Oral gavage applications were performed between 08:00 and 09:30 before morning feedings.

The study protocol in the current study for punicalagin was as follows: C= daily oral distilled water (1 ml distilled water for 1 kg body weight); P1= daily oral 1 mg/kg of punicalagin in distilled water (1 ml of distilled water contained 1 mg of punicalagin); P2= daily oral 2 mg/kg of punicalagin in distilled water (1 ml of distilled water contained 2 mg of punicalagin); P10= daily oral 10 mg/kg of punicalagin in distilled water (1 ml of distilled water contained 10 mg of punicalagin). The rabbits were weighted ones a week and the dose of punicalagin was adjusted weekly according to the individual live weights for each rabbit.  The appropriate dose calculated for each rabbit was administered directly into the stomach by gavage fixed to the syringe.

Water and feed were given as ad libitum. Rabbits were fed with standard commercial rabbit diet (Korkuteli Yem Gýda Sanayi-Antalya/Turkey; 6.93% crude ash, 17.0% crude protein, 12.68% crude cellulose, 3.67% crude oil, 0.49% calcium and 0.46% phosphorus) and daily feed intake was measured ones a week.
 
Blood collection
 
At the end of the experiment, rabbits were fasted for 12 hr prior to the blood collection. Blood samples from each rabbit were collected from the ear artery. Blood samples in anticoagulant free tubes were centrifuged at 1457 x g for 30 min. The serum from each sample was stored at -20^oC until assayed for biochemical parameters. Tubes containing EDTA were used immediately for red blood cell (RBC), white blood cell (WBC) and platelet (PLT) analysis.
 
Hematological and biochemical parameters
 
Hematological parameters (RBC, WBC, PLT-Abacus Junior Vet SN-100702) and biochemical parameters [total cholesterol, LDL, HDL, amylase, lipase, CRP (C-reactive protein) and GGT (Gamma-glutamyl transpeptidase)-Achitech C8000 auto-analyzer; bilirubin, albumin, glucose, AST (Aspartate aminotransferase), ALT (Alanine amino transferase), ALP (Alkaline phosphatase)- Gesan Chem 200 auto-analyzer) were analyzed by using auto analyzers.
 
Euthanasia and collection of wet organs
 
Following blood collection, euthanasia was performed under isoflurane anesthesia. Immediately after the euthanasia, the cavum abdominis and thorax were opened. The liver, kidneys,  spleen, heart and lungs were washed with PBS chilled to 5^oC. Immediately after noting the individual organ weights, the caudal lobe of the liver and right kidney of each rabbit were stored at -80^oC.

Oxidant-antioxidant parameters
 
Liver and kidney tissues for oxidant and antioxidant parameters were prepared according to the manufacturer’s instructions. Malondialdehyde – MDA (Cat No: SG-50252; SinoGeneClon Biotech Co., Ltd.; the detection limits of 6-350 ng/mL, inter- and intra assay precision of <10%), superoxide-dismutase – SOD (Cat No: SG-0061Rb; the detection limits of 30-1000 pg/mL, inter- and intra assay precision of <10%), catalase – CAT (Cat No: SG-50185; the detection limits of 1-36 pg/mL, inter- and intra assay precision of <10%) and glutathione peroxidase – GPx (Cat No: SG-0120Rb; the detection limits of 33-2000 pg/mL, inter- and intra assay precision of <10%) levels of the liver and kidney samples were assayed using ELISA kits (SinoGeneClon Biotech Co., Ltd., China). The results were read at 450 nm.
 
Statistical analysis
 
Results are presented as mean ± SD. The data were analyzed by PROC GLM procedure using the SAS statistical package. The minimum level of significance was set at p<0.05.

In general, no clinical side effects were apparent due to any of the punicalagin treatments. Moreover, there was no significant difference between the control and any of the treatment groups in terms of live body weight and feed intake at the end of the study. Correspondingly, no significant changes were observed in any of the organ weights at the end of the experiment (Table 1). Similar results were observed in rats with pomegranate extract at a daily dose of 180 mg/kg (Patel et al., 2008). In another study, pomegranate extract containing 4800 mg/kg/day negatively affected feed intake and body weight after 37 days (Cerda et al., 2003). However, the authors directly mixed pomegranate peel product with the rat diet. It was emphasized that adding the peel mixture to the diet may affect the palatability and the flavor.



Data in hematological parameters are shownin Table 2. No statistical difference was found among the study groups in any hematological parameters examined. RBC, WBC and PLT numbers were not affected by punicalagin treatment.In a similar study in rats, an oral dose of 4800 mg/kg/day punicalagin for 37 days had no effect on hematological parameters (Cerda et al., 2003). In another study, an oral pomegranate extract (60, 200 and 600 mg/kg/day) had no negative effect on RBC and hemoglobin in rats (Patel et al., 2008).  Although there was an increase in hematocrit and MCV at the two highest doses, it was concluded that the reason was not treatment-related (Patel et al., 2008).



The biochemical parameters are given in Table 3. At the end of the study, no notable changes in biochemical parameters were observed in the control or punicalagin treated rabbits. In addition, no statistical difference was observed in any of the biochemical parameters tested among the groups.The biochemical parameters, such as amylase and lipase are considered as an important indicator in determining pancreatic damage and inflammation and therefore pancreatic diseases are evaluated clinically by serum amylase and lipase levels (Lott, 1982). GGT is an enzyme found in the kidney, liver, spleen, gallbladder and pancreas. Thus, GGT is one of the most important indicators used in the diagnosis and monitoring of the liver and biliary tract (Goldberg, 1980). CRP is the most important protein secreted in our body as an inflammatory response (Erlinger et al., 2004) and its levels are elevated in patients with cardiac tissue injuries, chronic renal failure and severe peripheral vascular disease (Naji, 2017). Moreover, serum ALT, AST and ALP levels provide information about liver functions (Henderson and Moss, 2005).



The oral punicalagin administration, including the highest dose of 10 mg/kg/day,  showed no adverse effect on any of these biochemical parameters used to predict the tissue damage of liver, pancreas, heart, spleen and kidney. The level of these enzymes was not altered when compared to the rabbits in the control group. Similarly, in a study conducted in humans, it has been reported that administering ellagitannin-enriched polyphenols orally at a dose of 1420 mg/day for 28 days did not have a negative effect on serum ALT, AST, ALP levels (Haber et al., 2007). Administration of 600 mg/kg/day dose for 90 days in rats also did not cause an increase in the serum levels of these enzymes (Patel et al., 2008).

The treatment effect on oxidant and antioxidant parameters is given in Table 4. At the end of the experiment, none of the punicalagin treatments caused significant changes in liver or kidney GPx levels. However, the liver and kidney MDA levels decreased in P2 and P10 groups compared to the controls (P<0.05). Furthermore, the liver and kidney SOD levels increased significantly in the two highest doses of punicalagin groups compared to the C group (P<0.05). Consequently, the levels of liver and kidney CAT were significantly higher for rabbits in P2 and P10 groups (P<0.04) and P1, P2 and P10 groups (P<0.05), respectively.



MDA affects ion exchange from cell membranes, leading to cross-linking of compounds in the membrane and has negative consequences, such as changes in ion permeability and enzyme activity. SOD, CAT and GPx are intracellular enzymatic antioxidants. SOD catalyzes the superoxide radical to  H₂O₂ and O₂ (Sen et al.  2010). With this feature, SOD forms the first line of defense against reactive oxygen species (ROS). In the current study, punicalagin decreased MDA levels in liver and kidney tissues and increased SOD values. In an in vitro study, punicalagin showed an important antioxidant activity in goat liver tissues exposed to oxidative stress (Yaidikar and Thakur, 2001). Similarly, punicalagin increased SOD levels in the tissue levels (Sun et al., 2017). The protective effects of punicalagin on liver tissue were also observed in rats treated with carbon tetrachloride (Vora et al., 2015). In addition, there are indications that pomegranate juice protects liver tissue in rats with experimental hepatitis (Amal et al., 2012). The pharmacological effects of tannins and flavonoids are associated with antioxidant activities that may result from their ability to cleanse free radicals, chelate metal ions and apply synergistic effects with other antioxidant metabolites (Niki et al., 2005; Manna et al., 2006; Raja et al., 2007). The results of the current study suggested that punicalagin may reduce oxidative damage directly or indirectly by protecting the antioxidant defense system, removing ROS from the system, or suppressing lipid peroxidation.

Punicalagin had no apparent negative effect on the parameters measured. Moreover, the effects of 2 and 10 mg/kg/day of punicalagin on oxidant and antioxidant enzymes in the liver and kidney tissues were promising. Overall, the oxidant and antioxidant parameters were better than the control rabbits. Therefore, our study indicated punicalagin up to 10 mg/kg/day dose can be used in male New Zealand White rabbits and have positive effects as an antioxidant. In addition, 2 mg/kg/day punicalagin was considered to be sufficient for positive effects. Due to the low toxicity of punicalagin, it may be a suitable antioxidant candidate for clinical applications.

All authors declared that there is no conflict of interest.