'Ajwa' dates (Phoenix dactylifera L.) extract ameliorates isoproterenol-induced cardiomyopathy through downregulation of oxidative, inflammatory and apoptotic molecules in rodent model.
Background/Purpose: Ajwa, a special variety of Saudi Arabian dates (Phoenix dactylifera L) is a rich source of nutrients, fibers and bioactive molecules. While previous studies have shown the therapeutic value of dates phytoconstituents in liver and kidney diseases etc., its cardioprotective potential remains elusive. We therefore, investigated the cardioprotective effect of lyophilized Ajwa extract (AJLE) ex vivo as well as in vivo.
Methods: Ex vivo cardioprotective effect of AJLE was evaluated on DCFH-toxicated cardiomyoblast cells (H9C2). In vivo hemodynamics, cardiac function, serum cardiac enzymes, myocardial antioxidant, inflammatory and apoptotic biomarkers as well as histopathological parameters were studied in IPS-injured Wistar rat heart tissues.
Results: AJLE (250 [micro]g/ml) attenuated the cytotoxicity and enhanced the H9C2 proliferation by up to 40%. Oral administration of AJLE (250 and 500 mg/kg.bw) prevented the depletion of endogenous antioxidants (CAT, SOD, NP-SH and NO) and myocyte injury marker enzymes, and inhibited lipid peroxidation (MDA, MPO). Moreover, AJLE downregulated the expressions of proinflammatory cytokines (IL-6, IL-10 and TNF-[alpha]) and apoptotic markers (caspase-3 and Bax), and upregulated the anti-apototic protein Bd2. Histological data showed that AJLE pretreatment reduced myonecrosis, edema, and infiltration of inflammatory cells and restored the cardiomyocytes architecture.
Conclusion: Taken together, our data revealed that AJLE had strong antioxidant, hypolipidimic, cardioprotective, anti- inflammatory and anti-apoptotic potential against myocardial damage. This further endorses the use of Ajwa in Arabian traditional medicine against cardiovascular diseases.
Dates are considered as one of the oldest cultivated fruits by desert civilizations. In the Middle East, the Saudi Arabian city of Medina exclusively cultivates a special variety of soft-dry date (Phoenix dactylifera L.), locally known as "Ajwa". Dates are a rich source of nutrients such as iron, potassium, calcium, vitamin A, vitamin B3 and folic acid (Mallhiet al., 2014). Dates contains abundant levels of fatty acids such as oleic and linoleic acids as well as proteinogenic and non-proteinogenic amino acids (Ali et al., 2014; Mallhi et al., 2014). Bioactive molecules that foster antioxidant properties of dates include polyphenols, tannins, carotenoids triterpenoids and flavonoids (Al-Farsi and Lee, 2008; Martin-Sanchez et al., 2014).
In traditional or folk medicine, dates are shown to be beneficial in the treatment of a variety of health conditions. In experimental animal models, date fruits have been demonstrated to exhibit hepatoprotective (El Arem et al., 2014a), nephroprotective (El Arem et al., 2014b), antimicrobial (Mahdhi et al., 2013), anti-genotoxic (Diab and Aboul-Ela, 2012), anti-allergic (Karasawa and Otani, 2012), neuroprotective (Pujari et al., 2010), antiviral (Jassim and Naji, 2010) and antifungal (Boulenouar et al., 2011) properties. The dates pollen extract was previously shown to mitigate cadmium-induced testicular toxicity by activating endocrine and antioxidant systems (El-Neweshy et al., 2013). Oral administration of date extracts also modulated immune responses by influencing cytokine levels in vivo (Boghdadi et al., 2012). The polyphenols and polysaccharides present in date fruits were previously shown to stimulate cellular immunity in mice by enhancing mRNA levels of interferon-[gamma] (Karasawa et al., 2011; Ragab et al., 2013).
Despite extensive studies on the pharmacological properties of dates and its constituents, the effects of date consumption on cardiovascular functions have not been reported so far. In the present study, we therefore, examined the biological effects of lyophilized extract of Ajwa dates (AJLE) on cultured human H9C2 cells as well as experimentally-induced myocardial injury in rats. Using a previously described isoproterenol (ISP) rat model of cardiac injury (Ojha et al., 2008) and histological analysis, we show that antioxidant effects conferred by AJLE administration is a decisive molecular mechanism in amelioration of ISP-induced cardiac injury.
2. Materials and methods
2.1. Plant material collection and authentication
The fresh ripen fruits of Ajwa (P. dactylifera L.) were collected from a village of Medina region of Saudi Arabia. The date fruit was authenticated by a plant taxonomist at the College of Pharmacy, King Saud University and a voucher specimen (No. 15387) was deposited at the college herbarium.
2.2. Extraction of plant materials
The pulp of the dried Ajwa fruits was manually separated from the pits and pulverized into powder. About 650 g of the powder was soaked in 2 1 of cold distilled water. After 24 h, the solution was filtered and lyophilized to dryness (Christ Alpha 1-4 LSC Freeze Dryer).
2.3. Ex vivo cardioprotective activity assay
Human cardiomyoblast cells (H9C2) were grown in DMEM medium, supplemented with 10% heat-inactivated bovine serum (Gibco) and lx penicillin-streptomycin (Gibco) at 37[degrees]C in a humified chamber with 5% C[O.sub.2] supply. H9C2 cells were seeded ([10.sup.4] cells/well, in triplicate) in a 96-well flat-bottom plate (Becton-Dickinson Labware) and grown over night. Ex vivo cardioprotection of AJLE ([IC.sub.50]: 628.66 [micro]g/ml) was determined against 2,7-dichlorofluorescein (DCFH; Sigma) cytotoxicity ([IC.sub.50]: 125 [micro]g/ml), using MTT assay (MTT-Cell proliferation Assay Kit, Tervigen). Four doses of AJLE (31.25, 62.5, 125, and 250 [micro]g/ml) were prepared in DMSO (<0.1%, final), followed by dilution in DMEM media. The culture monolayer were replenished with DMEM containing 125 [micro]g/ml DCFH plus a dose of AJLE, including untreated as well as DCFH only-treated controls. The treated cells were incubated for 48 h at 37 [degrees]C followed by MTT assay as per the manufacturer's instruction. The optical density (OD) was recorded at 570 nm in a microplate reader (BioTek, ELx800) and cell survival fraction was determined.
A direct visual investigation was made under an inverted microscope (Optica, 40x and 100x) to observe any morphological changes in the cells cultured with different concentrations of AJLE and/or DCFH at 24 and 48 h.
2.5. Animals and ethical compliances
Albino male Wistar rats weighing approximately (200-250 g) were obtained from the Experimental Animal Care Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. The animals were kept under controlled environmental conditions (23 [degrees]C and 12 h light/dark cycle). The animals had free access to commercially available standard rodent diet and water and were fed ad libitum. The study was approved by Research Ethics Committee of College of Pharmacy, King Saud University (Riyadh, Saudi Arabia). The animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals by the Animal Care Center.
2.6. Acute toxicity test
Wistar rats were divided into different test groups comprising of six animals, each. The test was performed using increasing oral dose of AJLE from 50-3000 mg/kg body weight (b.w.). The rats were observed continuously for 1 h and then at 30 min intervals for 4 h for any gross behavioral change and general motor activities like writhing, convulsion, response to tail pinching, gnawing, pupil size, fecal output, feeding behavior, etc., followed by another 72 h for healthy survival. Because the Ajwa dates are commonly consumed as nutritional food in large quantities, randomly selected doses of 250 and 500 mg/kg b.w. were used in experimental rats.
2.7. Induction of experimental myocardial infarction (MI) in rats
Isoproterenol (ISP), a synthetic nonselective [beta]2-adrenergic agonist (a synthetic catecholamine), is a well-accepted noninvasive drug to induce MI in rat model (Rona et al., 1959; Ojha et al., 2012).The physiopathological and morphological changes of ISP-induced myocardial necrosis are similar to those observed in humans (Assmann, 1979). Therefore, to induce MI, ISP was dissolved in saline and injected subcutaneously (85 mg/kg b.w.) (Rona et al., 1959) for two consecutive days at the interval of 24 h.
2.8. Experimental groups and study design
The animals were randomly divided into four experimental groups, each containing eight rats. Group I (normal group; control) animals received normal saline using intragastric tube for 21 days, and saline was administered (500 [micro]l/rat, s.c.) on day 20 and 21 (24 h interval). Group II (ISP control; ISP) animals received normal saline for 21 days, and received IPS (85 mg/kg, s.c.) on day 20 and 21 (24 h interval). Group III (AJLE+ISP) animals received AJLE (250 mg/kg/day) orally for 21 days along with concurrent administration of ISP (85 mg/kg, s.c. at 24 h interval) on day 20 and 21. Group IV (AJLE+ISP) animals received AJLE (500 mg/kg/day) and ISP (85 mg/kg, s.c. at 24 h interval) on day 20 and 21. The optimized doses of AJLE (250 and 500 mg/kg) and the duration of pretreatment (21 days) were based on a pilot study in our laboratory (data not shown). During the experimental period, the b.w. of the rats was monitored at regular intervals.
2.9. Blood collection, tissue preparations and protein estimation
On day 21, i.e., 24 h after the second administration or 48 h after the first administration of ISP, the rats were euthanized with diethyl ether and blood samples were collected through retro-orbital plexus. Serum samples were separated by centrifugation at 4000 g for 15 min and stored at -80 [degrees]C for biochemical analyses. Subsequently, the animals were sacrificed and heart tissues were dissected. The tissues were homogenized in ice cold phosphate buffer saline (PBS; 50 mM; pH 7.4) to obtain a 10% (w/v) tissue homogenate. Heart tissue homogenates of different experimental groups were centrifuged at 12000 g for 45 min at 4 [degrees]C and the supernatants were used for estimation of various biochemical parameters. Total protein concentration in tissue samples was measured using Lowry reagent (Sigma-Aldrich) against bovine serum albumin (BSA) as standard.
2.10. Evaluation of serum myocardial injury markers
Levels of serum AST (aspartate transaminase), ALT (alanine transaminase), [gamma]-glutamyl transferase (GGT), ALP (alkaline phosphatase), LDH (lactate dehydrogenase) and CK-MB (creatine kinase-MB isoenzyme) were estimated using respective commercial kits and employing auto blood analyzer (Siemens, Dimension Xpandplus, USA).The relative weight of the heart for each experimental animal group was also recorded as indices of cardiac hypertrophy.
2.11. Estimation of serum lipids and lipoproteins
Serum levels of total cholesterol, triglycerides and HDL-C were estimated by Assmann method (Ohkawa et al., 1979) using commercially available enzymatic kits (Siemens, India) and employing auto blood analyzer (Siemens, Dimension Xpand plus, USA, USA). Serum LDL-cholesterol was calculated by the Friedewald formula: LDL-C= Total cholesterol - [HDL-C + (Triglycerides/5)]. Similarly, VLDL-C was calculated as follows: VLDL-C = Triglycerides/5.
2.12. Evaluation of oxidative stress markers
Malondialdehyde (MDA), indicative of lipid peroxidation (LPO) formation, was assessed according to its absorbance by spectrophotometry (Zhouet al., 2008). Myeloperoxidase (MPO) activity, an indirect index of heart injury was assessed by measuring the [H.sub.2][O.sub.2]-dependent oxidation of o-dianizidine. One unit (U) of enzyme activity was defined as the amount of the MPO present per gram of tissue weight that caused a change in absorbance of 1.0/min at 460 nm at 37 [degrees]C (Hillegass et al., 1990), Contents of cardiac NP-SH (Sedlak and Lindsay, 1968), protein (Lowry et al., 1951), superoxide dismutase (SOD) (Peskin and Winterbourn, 2000), and catalase (CAT) (Aebi, 1974) were determined by established methods. Production of nitric oxide (NO) was assessed by quantification of its related end products, nitrite/nitrate. In the assay, nitrate is converted to nitrite by nitrate reductase and total nitrite is measured using the Griess reaction (Green and Reed, 1998). The levels of cardiac tissue TP, TBARS, MPO, NP-SH, SOD, CAT and NO were determined using commercially available kits.
2.13. Determination of proinflammatory cytokines
The concentrations of proinflammatory cytokines: TNF-[alpha], 1L-6 and IL- 10 in the heart tissue samples were estimated using commercially available ELISA kits (R&D Systems, USA). Absorbance was recorded at 450 nm by using Multiskan[R]96-well Plate Reader (MTX Lab Systems Inc., Vienna, VA, USA). The protein level of tissue supernatant was estimated and concentrations of TNF-[alpha], IL-6 and 1L-10 were expressed as ng/mg of protein.
2.14. Preparation of cardiac tissue total protein and nuclear extracts and estimation of apoptotic protein expression
Frozen myocardial tissues from different experimental groups were homogenized in ice-cold RIPA buffer containing 1% protease inhibitor cocktail (Sigma-Aldrich) to get total protein extracts. After centrifugation at 12,000 g for 20 min at 4 [degrees]C, the supernatants were collected and analyzed for caspase-3, Bax, Bcl2 and [beta]-actin expressions using western blot (Towbin et al., 1979). Briefly, 25 [micro]g of protein was separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The membrane was incubated with the indicated primary antibody, followed by secondary antibody conjugated with horseradish peroxidase (HRP). Protein bands were detected using an enhanced chemiluminescence and then exposed to X-ray film. Levels of Caspase-3, Bax and Bcl2 were normalized by comparison to [beta]-actin (internal control). Similarly, nuclear extracts were prepared by using NE-PER nuclear extraction kit (Pierce Biotechnology, Rockford, IL, USA) containing 1% protease inhibitor cocktail (Sigma-Aldrich) according to the manufacturer's protocol and used for NF-[kappa]B-DNA binding assay. The protein contents were measured by using Lowery method.
2.15. NF-[kappa]B (p65)-DNA binding assay
NF-[kappa]B-DNA binding activity was analyzed using the NF-[kappa]B (p65) transcription factor ELISA kit (Cayman Chemical Company, Ann Arbor, MI). Briefly, nuclear extracts were incubated in the oligonucleotide-coated wells where the oligonucleotide sequence contains the NF-[kappa] response element consensus-binding site. After washing, samples were incubated by addition of specific primary antibody directed against NF-[kappa]B. A secondary antibody conjugated to HRP was added to provide a sensitive colorimetric readout at 450 nm.
2.16. Histopathological studies
Heart tissues were sliced in small pieces and immersed in neutral buffered 10% formalin for 24 h. The fixed tissues were processed routinely, embedded in paraffin (to get paraffin sections; 4-5 [micro]), sectioned, deparaffinized and rehydrated using the standard techniques (Bancroft and Gamble). The sections were then stained with Haematoxylin-Eosin dye and studied for histopathological changes under the light microscope (Olympus, Germany).
2.17. In vitro antioxidant/free-radical scavenging assay
The free-radical scavenging ability of AJLE against DPPH was evaluated as previously described (Brand et al., 1995). In the presence of an antioxidant which can donate an electron to DPPH, the purple color, typical for free DPPH radical decays, and the change in absorbency ([lambda] = 517 nm) was measured. The test provided information on the ability of a compound to donate a hydrogen atom, on the number of electrons a given molecule can donate, and on the mechanism of antioxidant action. While AJLE was re-dissolved in methanol at various concentrations (10,50,100,500 and 1000 [micro]g/ml), 125 [micro]l of DPPH was prepared in 375 [micro]l of methanol (1 mM final). After 30 min incubation at 25 [degrees]C, the decrease in absorbance (Abs) was measured optically. The free-radical scavenging activity was calculated using the equation:
%Free - radical scavenging activity = [Abs.sub.control] - [Abs.sub.sample] / [Abs.sub.control] x 100
2.18. Statistical analysis
Results were expressed as mean [+ or -] S.E.M. Total variation present in a set of data was estimated by one-way analysis of variance (ANOVA) followed by Dunnet's-test. P < 0.01 was considered significant.
3.1. Ex vivo cardioprotective potential of AJLE
Cardioprotective effect of AJLE against DCF-induced cardiotoxicity was investigated on cultured C9H2 cells. While DCFH (125 [micro]g/ml)-toxicated cardiomyocytes were recovered to about 25% with 62.5 [micro]g/ml of AJLE supplementation with 125 and 250 [micro]g/ml of AJLE further enhanced the cardiomyocytes proliferation by up to 40% (Fig. 1). Therefore, AJLE at the best minimal dose of 125 [micro]g/ml showed the cardioprotective effect where there was no further significant enhancement with 250 [micro]g/ml dose.
3.2. Effect of AJLE on animal's heart to body weight ratio
ISP treatment led to a significant increase in animal heart/body weight ratio in comparison with the control group (Fig. 2). However, AJLE treatment caused a significant decrease in the heart/body weight ratio as compared to ISP control group.
3.3. In vivo effect of AJLE on biochemical markers
Firstly, we explored the cardiovascular effect of AJLE treatment in ISP-treated rats by examining the levels of biomarkers in serum. To this end, ISP (85 mg/kg) significantly enhanced serum levels of AST, ALT, GGT, ALP, LDH and CK, an effect significantly and dosedependently abated by administration of AJLE (250 and 500 mg/kg), suggesting that AJLE ameliorated ISP-triggered tissue damage in vivo (Table 1). Secondly, we examined the effect of AJLE on lipid metabolism by determining the levels of serum lipoproteins. ISP treatment elicited increase in serum levels of cholesterol, triglycerides, LDL-C and VLDL-C and decrease in HDL-C. These effects were significantly abated by AJLE in a dose-dependent manner (Table 3), indicating that AJLE had a robust effect on ISP-induced modification of serum lipid profile in rats. To elucidate the mechanisms involved in the cardioprotective effects of AJLE, we analyzed the redox-sensitivity of AJLE in ISP-induced cardiac injury. The antioxidative enzyme activities of SOD, CAT NP-SH and NO were significantly decreased in the cardiac tissues of ISP-treated rats, as compared to normal control rats (Table 2). AJLE pretreatment (250 and 500 mg/kg) along with ISP treatment exhibited significant increases in SOD, CAT, NP-SH and NO levels in dose dependent manners (p < 0.01 and p < 0.01 for SOD, p < 0.01 and p < 0.01 for CAT, p < 0.01 and p < 0.01 for NP-SH and p < 0.01 and p < 0.01 for NO) whereas, ISP-treated rats demonstrated significant increases in levels of MDA and MPO, the marker of cardiac oxidative stress and inflammation, respectively (Table 2). AJLE pretreatment (250 and 500 mg/kg) along with ISP treatment showed significant decrease in MDA and MPO levels in dose-dependent manners (Table 2).
3.4. Downregulation of proinflammatory cytokines levels by AJLE
To elucidate the mechanisms involved in the cardioprotective effects of AJLE, we analyzed the inflammatory cytokines levels of AJLE treatment in ISP-induced cardiac injury. While the levels of IL-6, IL-10 and TNF-[alpha] were significantly increased in the cardiac tissues of ISPtreated rats as compared to control group, AJLE (250 and 500 mg/kg) along with ISP treatment exhibited significant decreases in the cytokines levels in dose-dependent manner (p < 0.01 and p < 0.01 for TNF-[alpha], p < 0.01 and p < 0.01 for IL-6, p < 0.01 and p<0.01 for IL-10) (Fig. 3). Moreover, ISP significantly increased NF-/cB-DNA binding activity (Fig. 4, upper panel) compared to normal control rats. AJLE treatment at 250 and 500 mg/kg significantly reduced the DNAbinding activity when compared to ISP alone treated rats.
3.5. Suppression of myocardial proapoptotic protein by AJLE
ISP administration significantly increased the myocardial expression of Bax, and caspases-3 proteins and decreased the Bcl2 expression when compared to control rats. Gel electrophoresis analysis of DNA extracted from heart tissues exhibited marked DNA smearing with fragmentation in the ISP treated rats. AJLE pretreatment (250 and 500 mg/kg) along with ISP effectively reduced the myocardial expression of Bax and caspase-3, and increased the Bcl2 level (Fig. 4, lower panel).
3.6. Free-radical scavenging activity of AJLE
AJLE showed moderate free-radical scavenging activity in vitro (Table 4.). It was able to reduce the stable DPPH to the yellow-colored DPPH at high concentrations (500 and 1000 |ig/ml) only.
3.7. Histological improvement of injured myocardiac tissues by AJLE
Histopathological analysis of rat cardiac tissues revealed that ISP elicited inflammatory lesions led to various myocardial structure disorders of muscle fibers as well as infiltration of acute inflammatory cells, including extravasation of red blood cells (Fig. 5A and B). Other structural changes observed were interstitial edema and the appearance of vacuoles in ISP alone treated group. AJLE treatment mitigated ISP-induced microscopic inflammatory changes in cardiac tissues, indicating that AJLE potently improved the tissue lesions in dose-dependent manner (Fig. 5C and D).
The present study validates the therapeutic potential of Saudi Arabian Ajwa dates (AJLE) in myocardiac infarction under ex vivo as well as in vivo experimental conditions. The ex vivo cardioprotective activity of AJLE against DCFH-induced cytotoxicity was studied on cultured H9C2 cells. Notably, DCFH is generally used to measure ex vivo or in vitro oxidative stress generated by free radicals through the principle of oxidation of DCFH to the fluorescent DCF (Rota et al., 1999). In this study, we used DCFH because of its high cytotoxicity on a variety of human cell lines (personal observation). AJLE treatment not only protected the cardiomyocytes against DCFH-induced toxicity, but also promoted cell recovery and proliferation. These findings were in line with our microscopic examination of the cell morphology under the same experimental conditions. To further validate the ex vivo results, the in vivo studies were carried out in Wistar rats.
AJLE administration resulted in improvement of hemodynamic biomarkers and histological indices in ISP-triggered myocardial injury. ISP, an [alpha]-adrenergic agonist (a synthetic catecholamine) induces myocardial damage in rats and mimics human myocardial infarction (Colucci, 1990). This is therefore, a well-established experimental model to investigate possible pharmacological effects of new drugs on the myocardium. It has been reported that catecholamines exert toxic effects on heart muscle by altering its function, biochemical and structural integrity (Stanton and Schwartz, 1967). In the present study, subsequent ISP pretreatment significantly enhanced heart weight while body weight remained unchanged, resulting in an increase in heart weight to body weight ratio. The increase in heart weight might be ascribed to increased water content and edematous conditions in intramuscular space (Upaganlawar et al., 2009; Upaganlawar et al., 2011). The 1% increase in myocardial water content may lead to 10% decrease in myocardial function (Laine and Allen, 1991). These results proposed that AJLE improved ISP-induced cardiac injury and ameliorated abnormal heart function. ISP may further compromise endogenous antioxidant defense mechanisms in the cardiac muscle (Gunjal et al., 2010). It is evident from the present study that ISP treatment caused a pronounced elevation of serum cardiac enzymes including AST, LDH and CK besides other enzymes such as ALT, ALP and GGT. The heart muscle contains high concentrations of AST, LDH, CK that are released in the circulation under metabolic disturbances (Ragab et al., 2013). Treatment of rats with ISP fosters hypoxia (Subashini and Sumathi 2012) and causes infarction and/or necrosis of the cardiac muscle of the rats concomitant with oxidative damage (Arya et al., 2010). Mechanisms underlying ISP-triggered cardiac injury are however, largely not understood. Nevertheless, mounting evidence suggests that production of reactive oxygen species (ROS) in the injured myocardium mediates ISPelicited effects (Ojha et al., 2012). A similar protective effect on various marker enzymes by AjLE was observed in lead-induced oxidative stress in rabbits (Sunmonu and Afolayan 2010). Thus, a robust reduction in CK and LDH enzymes points to cardioprotective effects of AJLE. The increases in ROS or depletion of antioxidants may induce increased oxidative stress that may cause myocardial ischemia (Rona, 1985; Sawyer et al., 2002). Enzymes such as SOD, CAT, and GSH (Sawyer et al., 2002) are the main defense against oxidative stress through eliminating oxygen radicals, such as superoxide and hydrogen peroxide, and by preventing the production of hydroxyl radicals. In this respect, our in vivo data revealed that the decreased activities of SOD, CAT, and NP-SH in the ISP alone group were significantly replenished by AJLE. Also, our in vitro assay confirmed the free-radical scavenging activity AJLE (500 [micro]g/ml). These results suggested that AJLE could strengthen the myocardial antioxidative defense system against oxidative stress. These observations were in line with our ex vivo data on cardioprotection by AJLE against DCFH-induced cardiotoxicty.
LPO plays a pivotal role in triggering myocardial necrosis (Habib and Ibrahim, 2011; Vayalil, 2012). Along those lines, ISP challenge to rats causes an increase in LPO in myocytes which is an important marker of oxidative injury to cardiac tissue (Nirmala and Puvanakrishnan, 1996). MDA, an end product of LPO was significantly elevated in the ISP-intoxicated group, which indicated pathological degeneration and necrosis of the myocardium as a result of oxidative stress (Zhou et al., 2006; Zhou et al., 2008). Our results showed that AJLE antagonized the enhancement of MDA level, thus, supporting the view that antioxidant properties of AJLE orchestrate its cardioprotective effects. Remarkably, several studies have shown that dates contain various antioxidant bioactive components (Ondrejickova et al., 1990; Devi and Vijayaraghavan, 2007; Al-Yahya et al., 2013) which, at least in theory, can block lipid peroxidation generated by ISP (Zhang et al., 2013). Moreover, our study showed that ISP administration caused a significant reduction in the content of cardiac NPS, an important defense mechanism against free radicals (Zhang et al., 2013). It has been suggested that MPO could also serve as a sensitive predictor for myocardial infarction (Brennan et al., 2003). Accordingly, ISP treatment induced a significant increase in MPO levels, indicative of necrosis induced cardiac inflammation. In our studies, AJLE treatment significantly decreased the MPO level, indicating that it inhibited neutrophil infiltration to cardiac tissues. Furthermore, we observed that AJLE administration significantly enhanced serum level of HDL while reducing those of cholesterol and triglyceride. Elevated serum concentrations of cholesterol, triglycerides and LDL foster the development of atherosclerosis. ISP was previously shown to increase the levels of circulatory lipids which, in turn, contribute to the pathophysiology of ISP-induced cardiovascular toxicity (Chilton, 2004). Compelling evidence suggests that decreased LDL level can significantly reduce the risk of major coronary diseases (Al-Shahib and Marshall, 2003; Hasan et al., 2010). From our data, it is intriguing to speculate that administration of Ajwa is beneficial in reducing the risk of cardiovascular diseases in humans.
Ajwa dates are known to contain large amount of natural fibers, apart from various micronutrients and minerals. It has been recommended that increased dietary fiber could be a safe and practical strategy in reducing cholesterol (Bazzano et al., 2003) because increased blood cholesterol is an important risk factor for the development of coronary artery disease (Brown et al., 1999). Epidemiological studies indicate that dietary fiber intake protects against cardiovascular disease (Kris-Etherton et al., 1988; Glore et al., 1994) including lowering of total and LDL cholesterol (Pereira et al., 2004). The hypolipidemic potential of AJLE could therefore, possibly reverse the ISP-induced myocardial infarction in the present study due to its high fiber contents (Ithayarasi and Devi, 1997). This observed effect could be attributed to the hyperactivity of extrahepatic lipoprotein lipase (Yagyu et al., 1999) which mediate lipolysis (Milei et al., 1978) and may, thus, contribute to hypocholesterolemia and hypotriglyceridemia in AJLE-treated rats. Previous reports have shown that ISP treatment induces expressions of myocardial proinflammatory cytokines, TNF-[alpha] and 1L-6 (Prabhu et al., 2009; Yang et al., 2013). The [beta]-adrenergic blockade treatment could exert beneficial effects on myocardial injury which is accompanied by selective reductions in myocardial expression of proinflammatory cytokines, IL-6, IL-10 and TNF-[alpha] (Deten et al., 2003; Ramani et al., 2004). Therefore, inhibition of proinflammatory cytokines is an important approach to protect myocardial damage. In our study, the increased levels of cardiac TNF-[alpha], IL-6 and IL-10 in ISP-treated rats were significantly normalized in AJLE treated (250 and 500 mg/kg) rats in a dose-dependent manners. We also observed AJLE mediated significant decrease in pro-apoptotic molecules, caspase-3 and Bax, and increase in pro-cell survival molecule, Bcl2 against IPS. We further analyzed the NF-[kappa]B-DNA binding activity by ELISA and found dose-dependent down regulation of NF-[kappa]B-DNA binding activity in AJLE pretreated rats. Recently, it was found that bioactive compounds from Ajwa dates possess antioxidant and anti-inflammatory activity (Jenkins et al., 2009). The antioxidant activity of AJLE fruit is attributed to phytochemical compounds such as phenolic acids, flavonoids, anthocyanins, etc., as well as the mineral selenium (Al-Farsi and Lee, 2008; Allaith, 2008).
In view of the modulatory effects of AJLE treatment on ISP-induced changes in serum biomarkers, lipids and redox status of cardiac tissue, we microscopically examined changes in cardiac tissue. Histopathological analysis revealed that ISP elicited myocardial structure disorders of muscle fibers as well as infiltration of acute inflammatory cells, along with extravasation of red blood cells. Other structural changes included interstitial edema and the appearance of vacuoles in ISP-treated rats. AJLE treatment mitigated ISP-induced microscopic inflammatory changes in cardiac tissues, indicating that AJLE potentially and dose-dependently cured the morphological disorders. Taken together, our in vitro and in vivo data confirmed the promising cardioprotective potential of Saudi Ajwa dates by using two different cytotoxins under two different physiological conditions.
Our present study revealed that AJLE pretreatment had strong antioxidant, hypolipidimic, cardioprotective, anti-apoptotic, and antiinflammatory potential against experimental myocardial damage. This further endorses the use of Ajwa dates in Arabian traditional medicine in the treatment
and prevention of cardiovascular diseases.
Received 10 August 2015
Revised 29 October 2015
Accepted 30 October 2015
Conflict of interest
The authors declare that there is no conflict of interest.
The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group Project No. RG-1435-053.
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Mohammed Al-Yahya (a,c), Mohammad Raish (b), Mansour S. AlSaid (a,c), Ajaz Ahmad (d), Ramzi A. Mothana (a,c), Mohammed Al-Sohaibani (e), Mohammed S. Al-Dosari (a), Mohammad K. Parvez (a), *, Syed Rafatullah (c)
(a) Department of Pharmacognosy, College of Pharmacy, KingSaud University, Riyadh 11451, Saudi Arabia
(b) Department of Pharmaceutics, College of Pharmacy, KingSaud University. Riyadh 11451, Saudi Arabia
(c) Center for Medicinal, Aromatic and Poisonous Plants Research, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
(d) Department of Clinical Pharmacy. College of Pharmacy, KingSaud University, Riyadh 11451, Saudi Arabia
(e) Department of Pathology, King Khalid University Hospital, King Saud University, Riyadh 11451, Saudi Arabia
Abbreviations: AST, aspartate transaminase; ALP, alkaline phosphatase; ALT, alanine transaminase; AJLE, lyophilized extract of Ajwa dates; BSA, bovine serum albumin; b.w, body weight; CAT, catalase; CK-MB, creatine kinase-MB isoenzyme; DCFH. 2,7-dichlorofluorescein; DMEM, Dulbecco's modified Eagle medium; DPPH, 2,2-diphenyl-1-picrylhydrazyl; CGT, [gamma]-glutamyl transferase; HRP, horseradish peroxidase; 1L, interleukin; ISP, Isoproterenol; LDH, lactate dehydrogenase; LPO, lipid peroxidation; MDA, Malondialdehyde; MI, myocardial infarction; MPO, Myeloperoxidase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; NF-Kb, nuclear factor kappa B; NO, nitric oxide; NP-SH, nonprotein sulphydydryl; OD, optical density; s.c, subcutaneous; SOD, superoxide dismutase; TBARS, thiobarbituric acid reacting substance; TNF, Tumor necrosis factor; w/v, weight by volume.
* Corresponding author. Tel.: +96 61 4675132; fax: +9661 4677245.
E-mail address: firstname.lastname@example.org (M.K. Parvez).
Table 1 Effect of AJLE on serum marker enzymes of control and experimental rats. Group Normal (control) ISP (85 mg/kg) (a) SGOT (U/L) 75.81 [+ or -] 154.50 [+ or -] 3.29 4.46 *** SGPT (U/L) 27.80 [+ or -] 100.38 [+ or -] 1.38 4.20 *** GGT (U/L) 5.16 [+ or -] 11.60 [+ or -] 0.26 0.28 *** ALP (U/L) 275.50 [+ or -] 434.83 [+ or -] 7.79 12.06 *** LDH (U/L) 88.91 [+ or -] 185.66 [+ or -] 3.27 6.25 *** CK (U/L) 140 [+ or -] 199.16 [+ or -] 3.50 7.84 *** Group AJLE (250 mg/kg) + ISP (b) AJLE (500 mg/kg) + ISP (b) SGOT (U/L) 143.83 [+ or -] 96.98 [+ or -] 3.37 3.60" SGPT (U/L) 87.30 [+ or -] 48.63 [+ or -] 2.08 * 3.99 *** GGT (U/L) 10.50 [+ or -] 7.26 [+ or -] 0.44 0.39 *** ALP (U/L) 369.00 [+ or -] 325.16 [+ or -] 18.84 * 7.63 *" LDH (U/L) 153.16 [+ or -] 131.33 [+ or -] 5.36" 4.70 *** CK (U/L) 181.16 [+ or -] 161.50 [+ or -] 6.83 4.12" All values represent mean [+ or -] SEM. * p < 0.05. ** p < 0.01. *** p < 0.001. ANOVA, followed by Dunnett's multiple comparison test. (a) As compared with normal group. (b) As compared with only ISP only group. Table 2 Effect of AJLE on oxidative stress markers of control and experimental rats. Group Normal (control) ISP (85 mg/kg) (a) CAT (U/mg) 46.615 [+ or -] 0.620 16.886 [+ or -] 0.375 SOD (U/mg) 8.21 [+ or -] 0.402 3.724 [+ or -] 0.337 NP-SH (nmol/g) 6.703 [+ or -] 0.491 3.191 [+ or -] 0.266 NO ([[micro]M/L) 35.849 [+ or -] 0.969 14.27 [+ or -] 0.476 MDA (nmol/g) 0.663 [+ or -] 0.1132 9.668 [+ or -] 0.956 MPO (U/g) 4.144 [+ or -] 0.038 18.719 [+ or -] 0.2431 Group AJLE (250 mg/kg) + ISP (b) CAT (U/mg) 26.824 [+ or -] 0.370 ** (b) SOD (U/mg) 5.702 [+ or -] 0.138 ** (b) NP-SH (nmol/g) 3.728 [+ or -] 0.344 ** (a) NO ([[micro]M/L) 19.47 [+ or -] 0.435 ** (b) MDA (nmol/g) 4.745 [+ or -] 0.487 ** (b) MPO (U/g) 10.628 [+ or -] 0.215 ** (b) Group AJLE (500 mg/kg)+ ISP (b) CAT (U/mg) 36.264 [+ or -] 0.556 ** (b) SOD (U/mg) 7.106 [+ or -] 0.222 * (b) NP-SH (nmol/g) 5.01 [+ or -] 0.330 * (b) NO ([[micro]M/L) 25.17 [+ or -] 0.610 ** (b) MDA (nmol/g) 2.02 [+ or -] 0.259 * (b) MPO (U/g) 7.236 [+ or -] 0.174 ** (b) All values represent mean [+ or -] SEM. * p < 0.05. ** p < 0.01. *** p < 0.001. ANOVA, followed by Dunnett's multiple comparison test. (a) As compared with normal group. (b) As compared with only ISP only group. Table 3 Effect of AJLE on serum lipid metabolism and serum lipoproteins of control and experimental rats. Group Normal (control) ISP (85 mg/kg) (a) Cholesterol 92.93 [+ or -] 201.66 [+ or -] (mg/dl) 4.09 4.83 Triglycerides 74.01 [+ or -] 154.33 [+ or -] (mg/dl) 3.81 3.20 HDL-C 53.58 [+ or -] 27.96 [+ or -] (mg/dl) 2.89 1.49 LDL-C 14.80 [+ or -] 30.86 [+ or -] (mg/dl) 0.76 0.64 VLDL-C 24.54 [+ or -] 142.83 [+ or -] (mg/dl) 8.77 6.20 Group AJLE (250 mg/kg)+ ISP (b) AJLE (500 mg/kg)+ ISP (b) Cholesterol 164.33 [+ or -] 128.66 [+ or -] (mg/dl) 5.31 **, (b) 3.83 ***, (b) Triglycerides 130.00 [+ or -] 103.51 [+ or -] (mg/dl) 2.86 ***, (b) 3.46 ***, (b) HDL-C 38.16 [+ or -] 41.10 [+ or -] (mg/dl) 3.29 *, (b) 3.18 **, (b) LDL-C 26.00 [+ or -] 20.70 [+ or -] (mg/dl) 0.57 ***, (b) 0.69 ***, (b) VLDL-C 100.16 [+ or -] 67.86 [+ or -] (mg/dl) 8.48 **, (b) 5.96 ***, (b) All values represent mean [+ or -] SEM. * p < 0.05. ** p < 0.01. *** p < 0.001. ANOVA, followed by Dunnett's multiple comparison test. (a) As compared with normal group. (b) As compared with only ISP only group. Table 4 In vitro free-radical scavenging activity of the AJLE. ([micro]g/ml) Radical scavenging activity (%) 10 50 100 500 1000 AJLE 10.4 19.1 32.6 57.5 73.5 Ascorbic acid 21.0 68.4 81.9 93.5 93.8
Please note: Some tables or figures were omitted from this article.