Can Fig and Olive Ameliorate the toxicity Induced by 2-nitropropane in some organs of mice? role of inflammatory versus anti-inflammatory genes

Elham A.Abd-Allah,Nouf S.Al-Abbas,Mona M.Atia,Fawzia Alzahrani,El-Mokhtar M.Ahmed,Soad S.Ali,Soad K.Al Jaouni

Elham A.Abd-Allah,Department of Zoology,Faculty of Science,the New Valley University,El-Kharga 72511,Egypt

Nouf S.Al-Abbas,Department of chemistry,Al Leith University College,Umm Al-Qura University,Mecca 21955,Saudi Arabia

Mona M.Atia,Department of Zoology,Faculty of Science,Assiut University,Assiut 71684,Egypt

Fawzia Alzahrani,Anatomy Department(Cytology and Histology),Faculty of Medicine(FM),King Abdulaziz University,Jeddah 21589,Saudi Arabia(KAU)

El-Mokhtar M.Ahmed,Department of Microbiology and Immunology,Faculty of Medicine,Assiut University,Assiut 71684,Egypt

Soad S.Ali,Department of Histology and Yousef Abdulatif Jameel Scientific Chair of Prophetic Medicine,Faculty of Medicine,King Abdul Aziz University,Jeddah 21589,Saudi Arabia

Soad K.Al Jaouni,Department of Hematology/Pediatric Oncology and Head of Yousef Abdulatif Jameel Scientific Chair of Prophetic Medicine,Faculty of Medicine,King Abdul Aziz University,Jeddah 21589,Saudi Arabia

Abstract OBJECTIVE:To investigate the efficacy of Fig fruit powder and olive on hepatic,renal and splenic injury induced by 2-nitropropane (2-NP) in mice,especially if they were used in combination.METHODS:A total of 40 adult BALB/c male mice weighting 25-30 g/each.Mice were categorized into five groups (8 each).Group 1 as negative control.Group 2 as positive control group intraperitoneally injected with 2-NP (100 mg/kg b.w.) 3 times/weekly for eight weeks.Group 3 injected with 2-NP and were orally supplemented with Fig(300 mg/kg).Group 4 injected with 2-NP and were orally supplemented with olive (100 mg/kg).Group 5 injected with 2-NP and were orally supplemented with mixture of Fig and olive (3∶1 respectively).RESULTS:Histopathological observation of liver in mice treated with 2-NP showed cellular degeneration,pyknosis,and congestion of the portal vein.In kidney there were disorganization of the cortical tissues,cellular necrosis and plenty of inflammatory lymphocytic aggregation.Significant elevations in liver function parameters(alanine aminotransferase and aspartate aminotransferase),mRNA expression levels of tumor necrosis factor-α,nicotinamide adenine dinucleotide phosphate oxidase and cyclooxygenase were detected as anti-inflammatory markers and 5-lipoxygenase,interleukin-1β and interleukin-6 as inflammatory biomarkers for liver and spleen,also significant elevations was detected in lipid peroxidation levels.The levels of antioxidants,glutathione,glutathione peroxidase,catalase and superoxide dismutase were significantly decreased.CONCLUSION:our findings indicated that Fig fruit powder and olive protected against hepatic,renal and splenic injury induced with 2-NP in mice,especially if they were used in combination.

Keywords:Ficus;olea;antioxidants;toxicity tests;2-nitropropane;inflammatory genes

INTRODUCTION

2-nitropropane (2-NP) is a nitro alkane,used as a fundamental of paints and inks,in the manufacture of chemicals as developed solvent and can be found in cigarette smoke.12-NP has been observed to cause hepatotoxicity in occupationally exposed humans,2and in rats and rabbits.3The mechanism of hepatotoxicity which exerts by 2-NP is not obviously understood,but many authors suggested that the metabolism of 2-NP may increase reactive oxygen species ROSs levels and cause cellular damage.4,5Intense liver damage,as well as some kidney damage,has been observed in workers who are poisoned from acute and chronic (short-term) inhalation exposure to 2-NP.Recent studies suggested that if those factory workers exposed to prolonged inhalation of 2-NP they will be suffer from severe headaches,nausea,pulmonary irritation,vomiting and diarrhea,in the workers.Also in animals frequently exposed to 2-NP by inhalation have damaged liver.6

Figs (Ficus carica L.) family Moraceae is a famous plant,grows in Egypt and many other countries.Fig possesses a very nutritious value and therapeutic properties.7Fig leaves,phloem,fruits,seeds and latex are anti-inflammatory mediators used traditionally in the treatment of jaundice,diarrhea,nutritional anemia.8Extraction of Fig leaves are used as traditional medication for diabetic treatment.9Hepatoprotective,hypoglycemic,antifungal,antispasmodic,antipyretic (reduce fever),anthelmintic,antioxidant and antimutagenic activities have been reported.10

The leaf decoction is taken as a medication for diabetes and kidneys calcifications and liver.11Ficus carica (Fig tree)has been widely investigated for its proteolytic enzymes,12minerals and sugars,13triterpenoids and amino acids,14and organic acids.15

The olive plant(Olea europaea L.)from family Oleaceae,and in specific,its leaves since earliest times have been used for the treatment of injuries,fever,diabetes,hypertension,atherosclerosis and gout.16The olive trees grow mainly in moderate and equatorial regions such as south west Asia.17Olive oil consists of important chemical compounds,including glycerol compounds and small quantities of free fatty acids,which together give glycerides.18It also contains unknown resinous components and phenols (mainly oleuropein and hydroxytyrosol),tocopherols and other compounds exhibit a significant role on the health and a group of related natural products with potent antioxidant properties.19,20In Olea europaea,oleuropein,demethyl-oleuropein,ligstroside,and oleoside are the major phenolic oleosides,21while verbascoside is the main hydroxycinnamic derivative of the olive fruit.22Oleuropein is usually the most prominent phenolic compound in olive types and can reach concentrations of up to 140 mg/g on a dry matter basis in young olives23and 60-90 mg/g of dry matter in the leaves.24

It is well known that oleuropein elicits anti-inflammatory effects by preventing lypoxygenase activity and the production of leukotriene.25So,if the diet rich in olive,this will be useful for diseases associated with oxidative damage such as coronary heart disease,cancer,and also for aging.Consumption of olive oil lowed coronary risk and reduced breast-cancer risk.26

Recent studies proved that the crude extracts,at small concentrations have antiproliferative activity and can prevent cell spread of human breast carcinoma and human urinary bladder carcinoma.27Further pharmacological activity of oleuropein includes various wound healing properties due to its vasodilatory,28anti-platelet aggregation.29Hypotensive,30anti-rheumatic,31diuretic32and hypothermic33effects.

Taking these in consideration,the aim of the present study was to investigate the effect of Fig powder,olive crude extract and the combination of the two fruits on toxicological parameters and estimate the role of some antioxidant defenses (enzymatic and non-enzymatic) to inhibit the damaging effects of reactive oxygen species(ROS) generated after administration of 2-NP on experimental liver,kidney and spleen in mice.

METHODS

Preparation of extracts

Extraction of crude extracts of olive fruits:olive fruits(the fruits of the olives are purchased in full maturity,where the fruits are colored in full black) washed,cut into small pieces,dried in oven at 40 ℃(the olive fragments were dried at this temperature in order to preserve the active materials in olive),and minced.Then,we mixed the dough with five folds of distilled water.Then,the mixture was put in the reflex at 100 ℃temperature for 3 h.After that,the extract was filtered and placed in the rotary evaporator to allow to the fluid to be concentrated.Then,in oven we dried the crude extract at 45 ℃.34After the crude extract has been dried,it was collected and put in safekeeping at－20 ℃until used.35

Extraction of crude extracts of Fig:Fig fruits were shade dried and grounded.Methanol (80%) was used as hydroalcoholic solution for socking in a glass beaker,two hundred grams of grounded Fig fruit was socked in 80% methanol for 3 d.Beaker was closed with aluminum foil and saved in laboratory.Each day the Beaker was shaken for 10 min.For rough filtration,soaked material was filtered through several layers of muslin cloth.Then,this rough filtrate was filtered through a Whatman # 01 filter paper.By using a rotary vacuum evaporator,the above filtrate was vaporized at 40 ℃under reduced pressure until the concentrate was condensed to 1/3rd of initial volume.The extract will be dark brown in color with soma residues.The extract,which obtained was then again filtered through a Whatman # 01 filter paper to remove residues.Finally,we get dark extract which will be stored in freezer.36

Animals,experimental design and doses

We bought for this experiment a total of 40 adult BALB/c male mice weighting 25-30 g/each from the Theodor Bilharz Institute (Cairo,Egypt).All animals well-preserved in a specific location (pathogen-free).The experimental animal protocols were performed according to guidelines agreed by the Institutional Animal Care and Use Committee,which were approved by Assiut University.Also,all experimental procedures were done according to the Declaration of Helsinki and the recommendations for the care and use of experimental animals established by the National Institutes of Health(NIH).All animals were acclimatized in plastic cages (8 animals per cage) inside a well-ventilated room (animal room,Zoology Department,Faculty of Science,Assiut University),one-week prior the experiment.The animals were kept under typical laboratory conditions (temperature of 23 ℃,relative humidity of 60%-0%,and a 12 h light/dark cycle) and were fed a diet of standard commercial pellets (contain 20%crude protein and 11%crude fiber,rich in protein and energy) and water containing libitum.After 1 week of acclimatization,mice were randomly categorized into five main groups (8 mice each);the powder of Fig and the dough of olive were mixed with distilled water to prepare the doses of the two extracts.

Animal treatments

Mice of group 1 served as negative control group intraperitoneally injected with normal saline (0.9 % NaCl),3 times/weekly for eight weeks.

Mice of group 2 served as positive control group intraperitoneally injected with 2-NP (100 mg/kg body weight(3 times/weekly for eight weeks.37

Mice of group 3 intraperitoneally injected with 2-NP(100 mg/kg body) and were orally supplemented with Fig (300 mg/kg) via drinking water,3 times/weekly for eight weeks.38

Mice of group 4 intraperitoneally injected with 2-NP(100 mg/kg body) and were orally supplemented with olive (100 mg/kg) via drinking water,3 times/weekly for eight weeks.39

Mice of group 5 intraperitoneally injected with 2-NP(100 mg/kg body) and were orally supplemented with mixture of Fig and Olive (3∶1 respectively)viadrinking water.

Liver,kidney and spleen toxicity were induced in mice in the latter four groups(n=32).

Chemicals and reagent kits

2-NP,thiobarbituric acid,glutathione(GSH),superoxide dismutase,epinephrine,and 5,5'-dithio-bis-(2-nitrobenzoic acid;DTNB) were purchased from Sigma-Aldrich (St.Louis,MO,USA).Activities of GSH peroxidase (GSH-Px),glutathione S-transferase(GSH-ST),glucose-6-phosphte dehydrogenase(G6PD) and Catalase (CAT) were determined using GSH-Px,GSH-ST,G6PD and CAT commercial kits(Bio-Diagnostic Company,Cairo,Egypt).All other chemicals were obtained from local sources with highest analytical grade.

Sample collection

All animals were sacrificed at day 57 post-2-NP injection and blood were collected from the heart to obtain plasma for biochemical studies.We stored the plasma at－80 ℃until use.Removed liver,kidney and spleen,washed in saline (－ 4 ℃),cut into small pieces.Part was immersed in 10% neutral buffered formalin for further paraffin processing and sectioning for light microscope examination.Other samples were homogenized in 0.1 M cold phosphate buffered (PBS)(pH 7.4) using IKA Yellow line DI homogenizer (18 Disperser,Germany)at 6000 rpm for 1 h at 4 ℃.Supernatant was frozen at－20 ℃for biochemical assays.For RNA extraction and gene expression analysis another samples were suspended in Trizol solution.

Assessment of parameters

Using the commercial available kits,alanine aminotransferase (ALT) and aspartate aminotransferase(AST) were analyzed according to the method described by Reitman and Frankel.40Serum and tissue total proteins were determined calorimetrically using the method of Lowryet al.41Hepatic,renal and splenic TBARS were assayed according to the method described by Mihara and Uchiyama.42GSH levels were measured deepening on the method of Beutleret al.43The Super oxide dismutase(SOD)activity was assessed by the method of Misra and Fridovich.44CAT,GSH-Px,GSH-ST and G6PD were determined according to the methods described by Aebi,45Paglia and Valentine,46Habiget al,47andKornberget al,48respectively.

Gene expression analyses

The effect of Fig,olive and co-administration of both on the expression of a panel of selected genes involved in the inflammation and toxicity of liver,kidney and spleen were investigated using real-time quantitative polymerase chain reaction(RT-qPCR).

The primers used for RT-qPCR analysis were designed using the Primer Express 1.5 software (Applied Biosystems).The mouse primers were designed in Table 1.

Table 1 Mouse primers used for RT-qPCR analysis

After performing the indicated treatments,RNA was extracted and then the corresponding cDNA was prepared and then real time PCR was applied for gene expression analysis.49All cDNA samples were managed in a 96-well plate using the following cycling conditions:10 min at 95 ℃,and 40 cycles at 95 ℃for 15 s ended by one min.at 60 ℃for annealing and extension.The data were analyzed according to Livak and Schmittgen.50

Histopathological analysis

After blood withdrawal anesthetized animals were euthanized by cervical dislocation then dissected for extraction of liver,kidney and spleen pieces of each organs 2 mm×2 mm were fixed in 10% buffered for 24 h and routinely processed for paraffin embedding.Sections 5 μ were cut from prepared blocks and stained with hematoxylin and eosin (HE).51The stained slices were examined by the light microscope (Olympus CX31,Shinjuku,Japan)and photographed using a digital camera (Olympus,Camedia C-5060,Shinjuku,Japan).

Statistical analysis

The results were analyzed statistically using one-way analysis of variance followed by Newman-keuls Multiple Comparison Test as a post-test.These analyses were executed using computer prism program for windows,version 7.0 (Graph pad software,Inc,San Diego,CA,USA).The level of significance between groups was accepted atP<0.05,0.01,0.0001or 0.0001,and the data were expressed as mean ± standard error of mean.

RESULTS

In the present study,it was observed that G6PD and lipid peroxidation(LPO)(supplementary Figures 1,2),ALT,AST (supplementary Figures 3,4),activities were significantly increased in 2-NP treated mice when compared to control group.However,activities of CAT,GSH-Px,GSH-ST,SOD and GSH were significantly decreased (supplementary Figures 5-9).In animal group given Fig,olive or the combination of Fig and olive along with 2-NP these changes were modified where ALT,AST,LPO were found to be decreased while G6PD and CAT,GPx,GST,SOD and GSH were elevated by different grads as compared with 2-NP treated group.

TNF-α,IL-6,IL-1β,NOX2,COX-2 and 5LO levels of liver and spleen were significantly increased in mice administrated with 2-NP compared to control group.Significant reduction in all raised parameters were observed when Fig,olive or their combination were given along with 2-NP compared with 2-NP treated group(supplementary Figures 10,11).

Histopathological examination of the hepatic and renal tissues cleared that 2-NP caused severe change confined as degenerative changes in some areas,inflammatory leucocytic infiltration,and hydropic degeneration,vacuolation of the cytoplasm and congestion of the hepatic portal veins compared with control rats.Co-treatment of mice with Fig,olive or the combination of Fig and olive revealed a noticeable repair,restoration and improvement of the general structure and arrangement of hepatocytes (Figure 1).In renal tissues,there were disorganization of the cortical kidney tissues,degeneration,extravasated erythrocytes and necrosis of their epithelial cells,much lymphocytic aggregation is appeared in many areas through the damaged kidney sections compared with 2-NP treated group.In Co-treatment of mice with Fig there was a repair of the general structure with some degeneration of the epithelial cells lining of the proximal and distal tubules.In olive co-treated mice there was less of cellular degeneration in tubules narrowing of the urinary spaces.Less of Malpighian corpuscles underwent some symptoms of injury.While,Co-treatment of mice with Fig and,showed that,the renal cortex tubules and renal corpuscles have almost restored their normal structure which showed normality in their epithelial cells.It was found that Olive and Fig co-treatments exerted more protective effect than each one alone (Figure 2).

Figure 1 Effect of Fig and olive on liver histopathology induced by 2-NP stained with hematoxylin and eosin

Figure 2 Effect of Fig and olive on liver histopathology induced by 2-NP stained with hematoxylin and eosin

DISCUSSION

Imbalance between oxidative stress and antioxidant defense capabilities is the most common underling cause of cancer development or enhancement.In the biological systems the major defense systems against free radicals occurred via enzymatic and non-enzymatic antioxidants.52

Liver is a main site for metabolism of exogenous substances(pesticides,drugs,metals),which led to the formation of metabolites,and this may be more or less toxic than the parental compound.53Regarding kidney,the high volume of blood runs through it where large amounts of toxins were filtered and later may concentrate in the kidney tubules,with subsequent marked tissue damage leading to kidney failure.Similar concentration of ROS can also occur in kidney tissue resulting in oxidative stress damage to renal tubular cells and derangement of kidney functions.54

2-NP was used in experimental work to induce oxidative damage,lipid peroxidation in hepatic tissue.55Oxidative stress,DNA damage,and induced hepatocyte apoptosis are the main factors behind hepatic damage induced by 2-NP as reported by Lodoviciet al.56

The liver has been implicated as the principal site of 2-NP metabolism,57and the most important indicators for hepatocellular damages are the elevation of serum ALT and AST.58,59In this study,administration of 2-NP was found to alter both kidney and liver functions evidenced by a significant increase in serum levels of ALT and AST as compared to control group.It is generally thought that aminotransferase (ALT and AST) elevations consequent to cell membrane disruption by generated free radicles and release of enzymes into circulation.60

In case of chemical hepatotoxicity,it was found that during the later phase of injury,serum ALT activity was dramatically increases,either as a result of direct hepatocyte damage and necrosis or associated inflammation.61Sallieet al62demonstrated also that hepatocyte cellular necrosis and damaged cellular membrane by oxidative stress result in release of cytoplasmic enzymes represented by the rise of serum AST and ALT levels.The present work demonstrated that the Co-treatment of Fig,olive extracts and their combination in mice showed reduction of ALT and AST levels compared to 2-NP treated animals.

Literature review showed that living cells possess potent antioxidative enzymes and non-enzymatic mechanisms (Vitamin C,reduced glutathione) to discourage the damaging effects of reactive oxygen species created after administration of 2-NP.63Lu64reported that in cases of liver damage,GSH,a well-known ROS scavenger,plays a chief protective role.GSH is considered a very important powerful reducing agent.65SOD,GST,GSH-Px and CAT are also well-known to be effective for elimination the hazardous effect of ROS.66

COX-2 was considered the most common inflammatory mediator that has a significant role in inflammatory processes progression as well as cancer development.67Also,similar action in mediation of both acute and chronic inflation was attributed to TNF.68From the mediators which are responsible for the inflammation,there is the main one,the pathway of cyclooxygenase(COX).69Inhibition of COX-2,the inducible COX isoform was found to play a major role in treating inflammatory conditions.70Alternatively,recent information has indicated that arachidonic acid,5-lipoxygenase(5-LO) and leukotrienes (LTs);the potent chemotactic agent for leukocytes,are strong mediators for inflammatory as well as of allergic reaction.71

In 2007 Horrilloet al72supported the concept in cell proliferation,neo-angiogenesis,COX-2 and 5-LO pathways are implicated,73and in the development of liver inflammation and fibrosis.

Cangemiet al74and Del Benet al,75detected an elevation of serum sNOX2 levels in a number of chronic inflammatory and metabolic diseases.Consequently,the current study showed an increase of LPO levels and G6PD in studied tissues(liver,spleen and kidney)with a significant decrease of the enzymatic antioxidant as CAT,GSH-Px,GSH-ST,SOD and non-enzymatic antioxidant GSH activities after 2-NP-treatment.Additionally,2-NP caused significant increase in TNF-α,NOX-2,IL-1β,IL-6,COX-2 and 5LO inflammatory mediators.The present findings are much similar to those reported by Borgeset al76and Hasegawaet al.77The authors reported an increase in hepatic lipid peroxidation levels with elevation of ALT and AST of the plasma activities in addition decrease in CAT activity following 2-NP intoxication.

Also,the present data showed that Pro-inflammatory cytokines involved in up-regulation of inflammatory reactions are mainly produced by activated macrophages.Several studies confirm that the process of pathological pain was attributed to many pro-inflammatory cytokines such as TNF-α,IL-1β and IL-6,from these cytokines,there are TNF-α which recognized as the essential one to prompt systemic responses to sepsis and injury.78

The nicotinamide adenine dinucleotide phosphate oxidases (NOXs) are from the most famous sources caused the development of reactive oxygen species(ROS),from those there are NOX1,NOX2,and NOX4 are the most predominant counterparts which are expressed in the liver,the role of phagocytic NOX2 was documented in Kupffer cell and in inflammation and the activity neutrophil phagocytes.79In agreement with this theory,our present work reported elevation of expression of NOX2 mRNA in liver and spleen upon treatment of mice with 2-NP 3 times/weekly for eight weeks.

Borgeset al76observed lymphocytic infiltration and confluent necrosis in centro-lobular zone of 2-NP treated mice liver.In the present study,the liver tissues of 2-NP-treated mice showed leucocytic infiltration,hydropic degeneration,vacuolation of the cytoplasm and congestion of portal vein,in renal sections,there were degeneration of the epithelial cells in major parts of cortical region with lymphocytic aggregations in many areas through the sections from damaged kidney.Administration Fig,olive or combination of them to 2-NP treated mice showed that most of the hepatocytes restored the normal feature and appearance kidney parenchyma including renal corpuscles and tubules showed evident preservation with few foci of degeneration of the epithelial cells lining of the proximal and distal tubules.

Accordingly,the present study demonstrated that Co-treatment of Fig,olive extracts and their combination led to enhanced activities of SOD,CAT.GPx,GST and GSH while G6PDH and LPO levels were significantly decreased compared to 2-NP treated mice.Furthermore,the histopathological notes mostly boost the results that obtained from enzyme assays,Hepatic tissue showed an improvement of the damaged hepatocytes which looked normal compared to 2-NP treated mice.In renal sections the renal cortex tubules and renal corpuscles have almost restored their normal structure which showed normality in their epithelial cells.

Visioliet al80showed that administration of catecholic phenolic compounds from olive oil (oleuropein) to intoxicated volunteers showed dose dependent decrease in lipid peroxidation and urinary of 8-iso-PGF2α excretion.Oleuropein was found to exert anti-inflamtory effect via inhibiting production of leukotriene B4 and lipoxygenase activity.

Oleuropein from olive fruit may inhibit free radical formation throughout its capability for Chelation metal ions,such as Fe and Cu,which were well known to act as catalysts for free radical generation reactions.81From the present research,we can have concluded that oleuropein,the main glycoside present in olives,has been associated with reduction of the inflammatory and necrotic effect caused by 2-NP.

In earlier studies,extracts of other species of Ficus were examined as hepatoprotective agent.The oral dose of 400,500 mg/kg of Ficus hispida methanolic leaf extract was found to have a significant amolerating effect against hepatotoxicity induced by paracetamol in rats.38In agreement with this study,Aghelet al82reported that,the histological construction of liver sections of mice treated with different Ficus carica leaf extracts showed more or less normal histological features with less necrotic changes,minimum degree of fatty infiltration and decreased infiltration of lymphocytes comparable to the negative control group.

The present study showed that Figs contain some effective components which can ameliorate oxidative stress and antioxidant parameters in 2-NP treated mice.These compounds are phenolic in nature and act as antioxidant.The effective antioxidant activity of phenolic compounds may be related to its action as scavenger and inhibitors of lipid peroxidation.83On the other hand,the phenolic compound Ferulic acid (FA),a most abundant natural compound in F.C.L.and its leaves can be categorized as natural antioxidant scavenger for free radicals.In this respect,it was reported that FA applies its protective effect for tissues by modulating lipid peroxidation and improving antioxidant defense system,and this may be attributed to the properties of phenolic compounds as antioxidants which existing in Figs and its leaves.84Finally,the answer of the question why co-treatment with Fig and olive extracts has the better effect on ameliorating the histological and bioassay results of 2-NP treated mice is because the high total polyphenols content in the mixture of olive and Fig extracts which can increase the antioxidant activity,these outcomes are in agreement with those described by Kiralanet al85who demonstrated a linear correlation between phenolic contents and antioxidant activity.

In conclusion,findings detailed here from the present study reveal significant role of ROS in pathogenesis of the 2-NP induced liver,kidney and spleen toxicity.We can also have concluded that,the extracts from olive and Fig fruits contain considerable number and amount of healthy compounds namely polyphenols and flavonoids,which act as antioxidant defenses,that can be react as free radical scavengers which delayed the oxidation and damage of tissues.After this study we can say that the combined dose of Figs and olives was found to have strong protective effect in the prevention of 2-NP induced toxicity and malfunctions in the studied organs than using them singly.In addition,it is safe to use the natural antioxidants rather than the artificial ones.

ACKNOWLEDGEMENTS

The authors thank Yousef Abdullatif Jameel,Chair of Prophetic Medical Applications (YAJCPMA),Faculty of Medicine,King Abdulaziz University,Jeddah,Saudi Arabia,for his support to this study.