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Macedonian Journal of Medical Sciences. 2009 Dec 15; 2(4):311-318.doi:10.3889/MJMS.1857-5773.2009.0073Basic Science

Potential Effects of Bee Honey and Propolis Against theToxicity of Ochratoxin A in Rats

Zakaria El-Khayat 1, Ahmed R. Ezzat 3, Mahmoud S. Arbid2, Wafaa I. Rasheed1 and Tahany R. Elias1

1Medical Biochemistry Department, National Research Center (NRC), Tahreer Street Doki, Cairo 002, Egypt; 2PharmacologyDepartment NRC, Cairo 002, Egypt; 3Zoology Department Ain Shams University, Cairo 002, Egypt

Key words:Bee honey, propolis, ochratoxin A,antioxidant defense system,peroxidation, liver, kidney.

Correspondence:Wafaa Ibrahim Rasheed, AssistantProfessor,National Research CENTER, MedicalBiochemistry, Tahreer Street Doki,Cairo 002, Egypte-mail: wafaa.rasheed@yahoo.com

Received: 31-Aug-2009Revised: 14-Oct-2009Accepted: 02-Nov-2009Online first: 17-Nov-2009

Abstract

Background. In the recent years, extensive research work has been focused on the useof natural materials as antioxidants against the toxic oxidative materials to ameliorate their toxicand cell damaging effects.

Aim. To evaluate the antioxidant effects of bee honey and propolis against OA-inducedoxidative stress in liver and kidney in rats.

Materials and Methods. 64 albino rats divided into 8 groups, group 1 as control, groups 2- 4 received an oral dose of OA, honey and propolis respectively for four weeks, groups 5and 6 were treated with a weekly dose of OA concomitant with a daily dose of bee honeyin group 5 and propolis in group 6, groups 7and 8 were treated with a daily dose of bee honeyin group 7 and propolis in group 8 and single weekly dose of OA then adminstrered startingthe second week of treatment. After 4 weeks, blood samples, liver and kidney tissues werecollected for the subsequent determinations.

Results. The study showed that OA exerted toxic effects on both liver and kidney tissuesmanifested as elevated serum alanine aminotransferase (ALT), gamma glutamyl transferase(γ GT), creatinine and cholestrol. OA also caused perturbation in liver and kidney antioxidantsystem reflected as diminished reduced glutathione (GSH), oxidized glutathione (GSSG)content and also decrease in glutathione peroxidase (GPX) and superoxide dismutase (SOD)activity. The level of malondialdehyde (MDA) which is a lipid peroxidation product waselevated. Bee honey (BH) and propolis (PR) ameliorated the toxic effects of OA on liver andkidney tissues with significant reduction of mean serum levels of ALT, γ GT, cholesterol andcreatinine. Also BH and PR improved the reduction in the antioxidant parameters of the liverand kidney (GSH, GSSG content and GPX , SOD activity) caused by OA administration. Thelevel of MDA was also significantly decreased.

Conclusion. Bee honey and propolis ameliorated OA-induced oxidative stress in the liver andkidney through their role in scavenging free radicals and preventing lipid peroxidation.

IntroductionIn the recent years, extensive research work

has been focused on the use of natural antioxidantproducts against the toxic oxidative materials toameliorate their toxic and cell damaging effects.

Ochratoxin A (OA), which is a toxic metabolite

OPENACCESS

produced by Aspergillus Ochraceus was reported tocause anaemia and to increase the levels of ALT, AST,alkaline phosphatase, triglycerides, cholesterol, uricacid and creatinine (1, 2). Khan et al., 1989 (3) havealso shown that OA enhanced lipid peroxidation.

Propolis (bee glue) is a resinous hive product

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collected by honey bees from various plant sources. Itis a popular folk medicine possessing a broad spectrumof biological activities (4). Hepatoprotective, renalprotective and therapeutic effects of propolis ethanolextract were observed (5, 6). The pharmacologicaleffects of bee propolis include reduction of the bloodpressure, protection of the liver tissue against carbontetrachloride, protection against stomach ulcerformation and maintenance of serum glucose (7).

Bee honey is a functional food which has aunique composition and antimicrobial properties (8). Itis a natural product with very complex chemicalcomposition, composed primarily of fructose andglucose but also contains 4 to 5% fructooligo-saccharides, which serve as prebiotic agents (9). Itcontains more than 180 substances, including aminoacids, vitamins, minerals and enzymes (10). Honey isused in medicine in the treatment of chronic skinulcers, gastrointestinal diseases, chest diseases aswell as in surgery (11). It is also used as chemicalantiseptic, antibacterial and antifungal agent (12).

The aim of this study was to evaluate theantioxidant effects of bee honey and propolis againstOA-induced oxidative stress in liver and kidney in rats.

Materials and Methods

Experimental animals

Male albino (Sprague Dawely) strain rats withan average weight of (100–200 g), obtained from theanimal house of the National Research Center, Dokki,Egypt. They were maintained on OA-free standardlaboratory diet (protein: 16.04%, fat: 3.6%, fiber: 4.1%and metabolic energy: 2887 kcal/kg) and water adlibitum. The animals were kept in ideal laboratoryconditions. The experiment was carried-out inaccordance with the national regulations of animalwelfare and Institutional Animal Ethical Committee(IAEC), National Research Center.

Preparation of ochratoxin (OA)

Ochratoxin A (OA), a toxic fungal metaboliteproduced by Aspergillus Ochraceus was preparedaccording to the method of Frohlich et al.,1988 (13).

Preparation of honey samples

The honey was collected form Saint Katherinearea in Sinai Peninsula. Samples were prepared witha modification of the method proposed by Schade etal.,1958 (14). A sample of 25 g of honey was weighed

into a sterile jar and then 25 ml of sterile water wasadded. The honey was then dissolved rapidly byshaking at room temperature.

Preparation of propolis samples

The propolis was collected form SaintKatherine area. Samples were prepared with amodification of the method proposed by Hegazi, 1997(15). A sample of 5 g of propolis was dissolved in 50 mlof distilled water, heated gently and shaken at roomtemperature.

Treatment protocol

64 male rats were divided into 8 experimentalgroups each of 8 animals, (treatment duration was 4weeks). The first group represented a control group,treated daily with 10% NaHCO

3 solution as a vehicle.

The second group was given a weekly oral dose of OAin NaHCO

3 (1 mg/kg b.w equivalent to 1/5 LD

50), the rest

of the days, the NaHCO3 vehicle was administered

daily. The third group was treated with a daily oral doseof (1 mg/kg b.w) honey in NaHCO

3. The fourth group

was treated orally with a daily dose of (1 mg/kg b.w)propolis in NaHCO

3.

The fifth group, each rat was given a weekly

dose of OA (1/5 LD50

) in NaHCO3 concomitant with a

daily dose of bee honey (1 mg/kg b.w) throughout theduration of the experiment. The sixth group, eachanimal was administered a weekly dose of OA (1/5LD

50) in NaHCO

3 concomitant with a daily dose of

propolis (1 mg/kg b.w) throughout the duration of theexperiment. In the seventh group, each rat was treatedwith a daily dose of bee honey (1 mg/kg b.w) for 4weeks, and a single weekly dose of OA wasadministered, starting the second week from the initiationof the honey treatment. The eighth group, each rat wastreated in a way similar to that of group seven exceptthat honey was replaced by propolis.

Blood and tissue sampling

At the end of the experiment, blood sampleswere obtained from the retro-orbital plexus (16) usingheparinized capillary tubes. Samples were left to clot,then centrifuged at 3000 r.p.m. for 15 minutes toseparate blood serum. Serum was stored at -20oC untilused for the determination of creatinine, totalcholesterol, alanine aminotransferase and γ-glutamyl-transferase. Animals were rapidly sacrificed for excisionof the liver and kidney. They were removed quickly andplaced in iced normal saline, perfused with the samesolution to remove blood cells, blotted on filter paperand frozen at –80oC.

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Maced J Med Sci. 2009 Dec 15; 2(4):311-318.

Homogenate preparation

The liver and kidney tissues were cut intosmall pieces and homogenized in 5 ml cold buffer (0.5g of Na

2HPO

4 and 0.7 g of NaH

2PO

4 per

500 ml

deionized water, pH 7.4) per gram tissue, thencentrifuged at 4000 rpm for 20 minutes at 5oC, thesupernatant was used for the assay of the content of(GSH) and (GSSG), the activity of (GPX) and (SOD)and the level of (MDA).

Biochemical analysis

Serum creatinine was assayed according tothe method of Bartles, et al.,1972 (17) using BioMerieux kit, France. Serum total cholesterol wasperformed according to the method of Richmond,1973(18) using commercial kit supplied by Randox, U.S.A.Serum alanine aminotransferase was determinedaccording to the method described by Reitman andFrankel, 1957 (19) using a Bio Merieux kit, France.Serum gamma-glutamyl transferase (γ-GT) wasdetermined by a kinetic colourimetric method accordingto Nielsen et al., 1978 (20) using a kit supplied bySclavo Diagnostics, Italy. Reduced glutathione (GSH)content was determined in liver and kidney accordingto the method described by Ellman, 1959 (21).Estimation of oxidized glutathione (GSSG) content inliver and kidney was made according to the method ofRall and Lehninger 1952 (22). Glutathione peroxidase(GPX) activity in liver and kidney was determined usingthe Ransel kit according to Kraus and Ganthen 1980(23). Estimation of superoxide dismutase (SOD) activityin liver and kidney was made according to the methodof Suttle, 1986 (24) using Randox Kit. Tissuemalondialdhyde (MDA) was performed according tothe method of Draper and Hadley, 1990 (25).

Statistical Analysis

Statistical analysis was performed using SPSSprogram, version 9.05 and Microsoft Excel 2003. Thedata was expressed as mean ± standard deviation(SD). Independent samples T-test was performed todetermine the specific differences between means.The results are considered to be significant when pvalue is less than 0.05 and highly significant.

ResultsThe results of this study are presented in

Table 1 and Figures 1 - 5. Table 1 shows the effect of

Table 1: Effect of oral treatment with ocratoxin A (OA), propolis (PR), bee honey (BH) and their combinations on serumalanine aminotransferase (ALT), serum γγγγγ-glutamyl transferase (γγγγγ-GT), serum total cholestrol and serum creatinine.

Values are expressed as means ±SE. Numbers between parentheses indicate the percentage of change in comparison with the corresponding control value. Means within a row with a commonsuperscript are not significantly different (P > 0.05).

Figure 1: Effect of oral treatment with ochratoxin A (OA), beehoney (BH) , propolis (PR) and their combination on liver andkidney reduced glutathione (GSH) content (micromol /g tissue)in rats. Means with a common superscript are not significantlydifferent(p>0.05).

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ochratoxin A (OA), bee honey (BH), propolis (PR), andtheir combinations on serum alanine aminotransferase(ALT), gamma-glutamyl transferase (γ-GT), cholesteroland creatinine in rats after 28 days.

There were no significant differences betweenthe groups of rats treated with either honey or propolisand the control group regarding these serumparameters.

OA administration caused significant elevationin serum ALT, γ-GT, cholesterol and creatinine.Significant decrease in their mean serum levels wasobserved in the groups treated with honey or propoliseither before or after OA compared to OA-treatedgroup, but the ameliorating effect of BH or PRadministration before OA was better than that after OA.Nonetheless, the mean levels of all these serumparameters did not return back to the control value.

Figures 1- 3 show the effect of oral treatmentwith OA, BH, PR and their combination on liver andkidney reduced glutathione (GSH), oxidized glutathione(GSSG), and glutathione peroxidase (GPX). It isapparent that there was a significant decrease in thecontent of GSH, GSSG and activity of GPX in the rats

Figure 2: Effect of oral treatment with ochratoxin A (OA), beehoney (BH) , propolis (PR) and their combination on liver andkidney Oxidized glutathione (GSSG) content (micromol/g tissue)in rats. Means with a common superscript are not significantlydifferent(p>0.05).

Figure 3: Effect of oral combination with ochratoxin A (OA), beehoney (BH), propolis (PR) and their combination on liver andkidney glutathione peroxidase (GPX) activity (U/g tissue) ) inrats. Means with a common superscript are not significantlydifferent (p>0.05).

Figure 4: Effect of oral treatment with ochratoxin A (OA), beehoney (BH) ), propolis (PR) and their combination on liver andkidney superoxide dismutase (SOD) activity (unit/g tissue) israts.

treated with OA compared to the control group.Improvement was observed in the groups treated withBH or PR either before or after OA and the change wasstatistically significant, yet their levels did not reachthose of the control.

Figure 4 shows similar results as regard theeffect on liver and kidney superoxide dismutase enzyme(SOD) but the improvement in case of treatment withbee honey and propolis before OA was better than thatafter OA.

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Maced J Med Sci. 2009 Dec 15; 2(4):311-318.

a a a aa

c

c

d

dd d

d

d

b

b

Figure 5 shows the effect of OA, BH, PR andtheir combination on liver and kidney lipid peroxidationproduct, malondialdhyde (MDA). OA significantlyincreased its mean level above that of the control.Treatment with BH or PR either before or after OAinhibited the OA induced increase in the MDA, althoughits mean level is still higher than that of the control. Alsofigures 1- 5 show that treatment with either BH or PRalone has no significant effect on either of the studiedparameters as compared to the control group.

DiscussionOur results revealed that ochratoxin A has

toxic effects on the liver as shown by the elevated meanserum levels of ALT and γ-GT. These results are inagreement with those of Nada et al.,1996 and Richard,2007 (1, 2).

The liver is the most sensitive organ topreoxidative damage because it is rich in oxidizablesubstances. The increment of the oxidative stress onthe cells of the liver and the consequent decrease in theantioxidant ability of the cells result in the occurrenceof aggressive cellular damage to the liver cells withdestruction of their membranes and the release of the

enzymes into the blood stream (26, 27).

The more severe the liver damage the higherthe release of the liver enzymes (28). In our experimenta pronounced decrease in the activities of both serumALT and γ-G T was detected after oral treatment withbee honey or propolis either before or after OAadministration. These results indicate a markedhepatoprotection induced by both agents. Thisprotective effect may be due to the antioxidant effect ofboth honey and propolis which was previously confirmed(4, 29, 30).

In the current study marked increase in themean serum cholesterol level was found in the OAsupplemented group which may reflect the impairmentof liver function and particularly lipid metabolism (1).Significant improvement in the serum cholesterol levelin the groups of animals treated with propolis or beehoney either before or after OA supplementation wasobserved. Langseth, 2000 (31) obtained similar resultsand he explained that flavonoids in the bee honey wereresponsible for such effect.

In this study nephrotoxicity was manifestedby inhibition of kidney function as indicated by increasedmean serum creatinine level.OA acts essentially in theproximal renal tubules, inhibiting the enzymephosphoenol-pyruvate carboxylase, which is a lipidperoxidant, and it alters the structural and functionalrenal ability to metabolize calcium (32). Morphologically,the kidney shows atrophy and sclerosis of the proximaltubules. Functionally, tubular functions are reduced(33). Our results coincides with others by Kuiper-Goodman and Scott 1989 (34) and Marquardt et al.,1990 (35).

Effective reduction of serum creatinine levelwas noticed after administration of bee honey orpropolis. These results are probably due to theantioxidant protective effect of propolis and honeywhich could have accumulated in the cells of theproximal convoluted tubule of the kidney where propoliswas reported to be collected and secreted (36).

Reduced glutathione (GSH) is an importantnaturally occurring antioxidant and its level in a tissueis considered a critical determinant of the threshold fortissue injury. Our results proved OA toxic effectssimilar to those of cadmium resulting in a significantdepletion of GSH in both liver and kidney cells whichmight have led to their damage due to enhancing of lipidperoxidation (37).

OA combines with iron, facilitating itsreduction. The iron–OA complex produces the

Figure 5: Effect of oral treatment with ochratoxin A (OA), beehoney (BH), propolis (PR) and their combination on liver andkidney lipid peroxidation product malondialdehyde (MDA) content(m mol/g tissue) in rats. Means with a common superscript arenot significantly different (p>0.05).

Live

r &

kid

ney

MD

A co

nten

t (μ

mol

/g t

issu

e) 2.5

2.0

1.5

1.0

0.5

3.0

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extremely damaging hydroxyl radical in the presenceof NADPH cytochrome p-450 reductase system, thisradical species may be partly responsible for OAtoxicity (38, 39). Similar results were obtained for liverand kidney oxidized glutathione (GSSG) with OAtreatment. Since GSH represents the substrate ofglutathione peroxidase which catalyzes the formationof GSSG, so if the reduced glutathione decreases, thisis likely to be followed by a decrease of oxidizedglutathione (40).

Propolis or honey treatment ameliorated thiseffect in both kidney and liver but to a some extent asthe values were still lower than that of the control group.This protective effect is probably a result of theirantioxidant and free- radical scavenging propertieswhich in turn help to maintain the intracellular level ofboth reduced glutathione and oxidized glutathione(41).

The present results proved significant increaseof lipid peroxidation secondary to OA administrationwhich is manifested by high malondialdehyde level.MDA is an end product of lipid peroxidation and it isconsidered a late biomarker of oxidative stress andcellular damage (42). Pervious studies suggested thatthe toxicity of OA may be the result of three majoreffects: inhibition of ATP synthesis, inhibition of proteinsynthesis and enhanced lipid peroxidation (35). Similarresults were obtained by others (39, 43) whodemonstrated that OA enhanced lipid peroxidationwhen added to liver and kidney microsomes in vitro orwhen administered to rats in vivo. They ascribed thiseffect to an OA- stimulated NADPH-dependent andascorbate -dependent lipid peroxidation with iron beingan essential cofactor.

The efficiency of several ochtratoxins(ochratoxins A, B, C, alpha and O- methyl ochratoxinC) to enhance lipid peroxidation was also related to thepresence of a reactive phenolic hydroxyl group in thesecompounds capable of complexing with transitionalmetals. Furthermore, substantial lipid peroxidationoccurs only when cellular defense mechanisms havebeen weakened or overcomed by prolonged oxidativestress (43, 44). Lipid peroxidation was greatly reducedin the groups of rats treated with propolis or bee honeybefore or after OA treatment, as manifested bydecreased MDA level.

The antioxidant effects of bee honey wasattributed to its constituents of the most importantantioxidant trace elements and to the antioxidantactivity of its flavonoid compounds. Therefore beehoney has been suggested to be able to decrease the

nitric oxide and lipid peroxidation . Similarly propoliswas reported to decrease lipid perodxidation (45-48)

Concerning the effect of OA on the activity ofliver and kidney glutathione peroxidase (GPX) andsuperoxide dismutase (SOD), significant decrease intheir activity was observed in the OA treated groupcompared to the control group. The reduction in theiractivity is probably due to the increase inmalondialdehyde level caused by OA (49).

The study showed significant improvement inGPX and SOD activity after treatment with bee honeyor propolis. This improvement plays an important rolein the cellular defense against the oxidative stress ofOA (50). In case of SOD the improvement was betterwhen they were administered before OA. Machlin andBendich 1987 (51) explained that bee honeyconstituents include the antioxidant trace elementsiron, zinc and selenium which are essential cofactorsfor the enzymatic antioxidant defense systemrepresented by catalase, superoxide dismutase andglutathione peroxidase.

ConclusionOA is a concern of public health by the

consumption of contaminated food. The kidney andliver are its main target tissues. This study provides aneasy and relatively cheap natural products (bee honeyand propolis) as natural antioxidants for protectingagainst oxidative toxic effects of OA. The resultsconfirmed counteracting most of OA –induced hepato-and nephrotoxicity by these bee products and showedthat their administration improved the tissue specificantioxidant enzymes, reduced lipid peroxides thusenhance tissue antioxidant defense capacity.

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