Research Article Dietary Supplementation of Calendula...

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Hindawi Publishing Corporation ISRN Nutrition Volume 2013, Article ID 538427, 9 pages http://dx.doi.org/10.5402/2013/538427 Research Article Dietary Supplementation of Calendula officinalis Counteracts the Oxidative Stress and Liver Damage Resulted from Aflatoxin Mohamed A. Hamzawy, 1 Ezzeldein S. M. El-Denshary, 2 Nabila S. Hassan, 3 Fathia A. Mannaa, 4 and Mosaad A. Abdel-Wahhab 5 1 Pharmacology and Toxicology Department, College of Pharmacy, Misr University for Science and Technology, Al-Motamayez District, P.O. Box 77, 6th October City, Egypt 2 Pharmacology and Toxicology Department, Faculty of Pharmacy, Cairo University, Cairo 11787, Egypt 3 Pathology Department, National Research Center, Dokki, Cairo 12311, Egypt 4 Medical Physiology Department, National Research Center, Dokki, Cairo 12311, Egypt 5 Food Toxicology and Contaminants Department, National Research Center, Dokki, Cairo 12311, Egypt Correspondence should be addressed to Mosaad A. Abdel-Wahhab; mosaad [email protected] Received 25 November 2012; Accepted 10 January 2013 Academic Editors: M. Holecek, M. G. Nikolaidis, H. Schr¨ oder, and C. Shing Copyright © 2013 Mohamed A. Hamzawy et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. is study was conducted to evaluate the total phenolic compounds, the antioxidant properties, and the hepatorenoprotective potential of Calendula officinalis extract against aflatoxins (AFs-) induced liver damage. Six groups of male Sprague-Dawley rats were treated for 6 weeks included the control; the group fed AFs-contaminated diet (2.5 mg/kg diet); the groups treated orally with Calendula extract at low (CA1) and high (CA2) doses (500 and 1000 mg/kg b.w); the groups treated orally with CA1 and CA2 one week before and during AFs treatment for other five weeks. e results showed that the ethanol extract contained higher phenolic compounds and posses higher 1,1-diphenyl 1-2-picryl hydrazyl (DPPH) radical scavenging activity than the aqueous extract. Animals fed AFs-contaminated diet showed significant disturbances in serum biochemical parameters, inflammatory cytokines, and the histological and histochemical pictures of the liver accompanied by a significant increase in malondialdehyde (MDA) and a significant decrease in superoxide dismutase (SOD) and glutathione peroxidase (GPx) in liver. Calendula extract succeeded to improve the biochemical parameters, inflammatory cytokines, decreased the oxidative stress, and improved the histological pictures in the liver of rats fed AFs-contaminated diet in a dose-dependent manner. It could be concluded that Calendula extract has potential hepatoprotective effects against AFs due to its antioxidant properties and radical scavenging activity. 1. Introduction Mycotoxins are fungal metabolites toxic to humans and animals, commonly found as contaminants of food or feed [1]. Aflatoxins (AFs) are principally produced by Aspergillus flavus and Aspergillus parasiticus. Among these, aflatoxin B 1 (AFB 1 ) is the predominant form as cereal and oilseed contaminants and presents the highest toxic potential [1], being hepatotoxic and carcinogenic in human and animals [2]. e biotransformation of AFB 1 by liver microsomal enzymes resulted in toxic metabolites AFB 1 -8, 9-epoxide. e toxic effects of AFs mostly arise from the binding of this particular epoxide derivative to cellular macromolecules such as DNA and proteins [3]. AFB 1 is classified by the International Agency of Research on Cancer (IARC) as Group 1 carcinogen [4]. is mycotoxin is also mutagenic, teratogenic, and immunosuppressive in farm and laboratory animals [5] and mainly affects the cell-mediated immunity [6]. Reactive oxygen species such as the hydroxyl radical, superoxide anion, and hydrogen peroxide, which are gener- ated as a result of this metabolism, are also involved in the toxic mechanism of AFs [710]. Calendula officinalis L., a member of the Asteraceae family, is an annual plant with yellow to orange flowers, mostly seen in the Mediterranean region and has been cultivated as a food and medicinal plant since the Middle

Transcript of Research Article Dietary Supplementation of Calendula...

Hindawi Publishing CorporationISRN NutritionVolume 2013 Article ID 538427 9 pageshttpdxdoiorg1054022013538427

Research ArticleDietary Supplementation of Calendula officinalis Counteractsthe Oxidative Stress and Liver Damage Resulted from Aflatoxin

Mohamed A Hamzawy1 Ezzeldein S M El-Denshary2 Nabila S Hassan3

Fathia A Mannaa4 and Mosaad A Abdel-Wahhab5

1 Pharmacology and Toxicology Department College of PharmacyMisr University for Science and Technology Al-Motamayez DistrictPO Box 77 6th October City Egypt

2 Pharmacology and Toxicology Department Faculty of Pharmacy Cairo University Cairo 11787 Egypt3 Pathology Department National Research Center Dokki Cairo 12311 Egypt4Medical Physiology Department National Research Center Dokki Cairo 12311 Egypt5 Food Toxicology and Contaminants Department National Research Center Dokki Cairo 12311 Egypt

Correspondence should be addressed to Mosaad A Abdel-Wahhab mosaad abdelwahhabyahoocom

Received 25 November 2012 Accepted 10 January 2013

Academic Editors M Holecek M G Nikolaidis H Schroder and C Shing

Copyright copy 2013 Mohamed A Hamzawy et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

This study was conducted to evaluate the total phenolic compounds the antioxidant properties and the hepatorenoprotectivepotential of Calendula officinalis extract against aflatoxins (AFs-) induced liver damage Six groups of male Sprague-Dawley ratswere treated for 6 weeks included the control the group fed AFs-contaminated diet (25mgkg diet) the groups treated orally withCalendula extract at low (CA1) and high (CA2) doses (500 and 1000mgkg bw) the groups treated orally with CA1 and CA2 oneweek before and during AFs treatment for other five weeks The results showed that the ethanol extract contained higher phenoliccompounds and posses higher 11-diphenyl 1-2-picryl hydrazyl (DPPH) radical scavenging activity than the aqueous extractAnimals fed AFs-contaminated diet showed significant disturbances in serum biochemical parameters inflammatory cytokinesand the histological and histochemical pictures of the liver accompanied by a significant increase in malondialdehyde (MDA)and a significant decrease in superoxide dismutase (SOD) and glutathione peroxidase (GPx) in liver Calendula extract succeededto improve the biochemical parameters inflammatory cytokines decreased the oxidative stress and improved the histologicalpictures in the liver of rats fed AFs-contaminated diet in a dose-dependent manner It could be concluded that Calendula extracthas potential hepatoprotective effects against AFs due to its antioxidant properties and radical scavenging activity

1 Introduction

Mycotoxins are fungal metabolites toxic to humans andanimals commonly found as contaminants of food or feed[1] Aflatoxins (AFs) are principally produced by Aspergillusflavus and Aspergillus parasiticus Among these aflatoxinB1(AFB1) is the predominant form as cereal and oilseed

contaminants and presents the highest toxic potential [1]being hepatotoxic and carcinogenic in human and animals[2] The biotransformation of AFB

1by liver microsomal

enzymes resulted in toxic metabolites AFB1-8 9-epoxide

The toxic effects of AFs mostly arise from the binding ofthis particular epoxide derivative to cellular macromolecules

such as DNA and proteins [3] AFB1is classified by the

International Agency of Research on Cancer (IARC) asGroup 1 carcinogen [4] This mycotoxin is also mutagenicteratogenic and immunosuppressive in farm and laboratoryanimals [5] and mainly affects the cell-mediated immunity[6] Reactive oxygen species such as the hydroxyl radicalsuperoxide anion and hydrogen peroxide which are gener-ated as a result of this metabolism are also involved in thetoxic mechanism of AFs [7ndash10]

Calendula officinalis L a member of the Asteraceaefamily is an annual plant with yellow to orange flowersmostly seen in the Mediterranean region and has beencultivated as a food and medicinal plant since the Middle

2 ISRN Nutrition

Ages [11] It has been used in the treatment of inflammationand skin wounds [12] In the early Indian andArabic culturesas well as in ancient Greece and Rome C officinalis was usedas colourant for fabrics foods and cosmetics [13] NowadaysC officinalis is approved for food use in USA and appears inthe Food and Drug Administrationrsquos list of GRAS (GenerallyRecognized as Safe) substances It has a high economic valueas an herbal medicine and is widely used in cosmeticsperfumes pharmaceutical preparations and in food [11]C officinalis contains a high number of carotenoids suchas flavoxanthin lutein rubixanthin 120573-carotene g-caroteneand lycopene [14] It has been cited that 120573-carotene maywork synergistically with vitamin E [15] Lycopene anothercarotenoid has been found to have antioxidant antimi-crobial and antiproliferative properties Research suggeststhat it can be very protective against prostate cancer [16]C officinalis has been studied extensively for its beneficialeffects on humans Literature has shown that an herbal teamade from C officinalis could improve the symptoms ofcolitis duodenal ulcers and gastroduodenitis [16] Althoughconsiderable work has been done on C officinalis extractsno report is available on its role against the oxidative stressgenerated by natural toxicants Therefore the aims of thepresent study was to determine the total phenolic contentthe radical scavenging activity of the ethanolic and aqueousextract of Calendula in vitro and to evaluate the possiblehepatoprotective effects of the extract against oxidative stressinduced by aflatoxins in rats

2 Material and Methods

21 Chemicals and Kits Aflatoxins standards were purchasedfrom Sigma Chemical Co (St Louis MO USA) Alanineaminotransferase (ALT) aspartate aminotransferase (AST)glutathione peroxidase (GPx) and superoxide dismutase(SOD) were purchased from Randox (Antrim UK) Alkalinephosphatase (ALP) total protein (TP) albumin and creati-nine were purchased from QCA (AMPOSTA Spain) Ureawas purchased from Prodia (Korbach Germany) Lipid per-oxide formation was evaluated as malondialdehyde (MDA)and was purchased from Oxis Research Co (USA) Alphafetoprotein (AFP) was purchased fromMonobind Inc (LakeForest USA) Interleukin-1120573 (IL-1120573) and tumor necrosisfactor-alpha (TNF-alpha) were purchased from Orgenium(Helsinki Finland) All other chemicals were of the highestanalytical grade available

22 Aflatoxin Preparation Aflatoxins were produced viafermentation of rice by Aspergillus parasiticus NRRL 2999The fermented rice was autoclaved dried and ground to apowder and AFs content was measured by HPLC [17] Therice powder was incorporated into the basal diet to providethe desired level of 25mgkg diet The diet containing AFswas analyzed and the presence of parent AFs was confirmedand determined as mentioned above

23 PlantMaterial Calendula officinaliswas purchased fromthe local market at Cairo The plant was identified by the

Department of Medicinal Plants National Research Center(NRC) and the voucher was kept in the herbarium of NRC

24 Preparation of Calendula Extracts Dried and groundflowers and leaves of Calendula officinalis (50 g) were sub-jected to extraction with 400mL of ethanol (95) or distilledwater for 48 hrs The extracts were filtered and the ethanolextract was concentrated under the reduced pressure ofnitrogen and completely evaporated in a vacuum oven at atemperature not exceeding 40∘C until constant weights wereobtained The aqueous extract was dried using Freeze Dryersystem (Dura-Dry Freeze Dryer Model PAC-TC-V4 FTSsystem Inc Stone Ridge NY USA)

25 Determination of Total Phenolic Contents The concen-tration of phenolics in the extracts was determined usingthe method of Jayaprakasha and Rao [18] In brief 5mg ofeach extracts was dissolved in a 10mL mixture of acetoneand water (6 4 vv) Samples (02mL) were mixed with 1mLof 10-folds diluted Folin-Ciocalteu reagent and 08mL ofsodium carbonate solution (75) The absorbance was mea-sured at 765 nm using UV-160 IPC UV-visible spectropho-tometer (Shimadzu Japan) after 30min at room temperatureEstimation of phenolic compounds as catechin equivalents(CE) was carried out using standard curve of catechin [19]

26 Evaluation of Radical Scavenging Activity (RSA) by 11-Diphenyl 1-2-Picryl Hydrazyl (DPPH) Assay Crude extractswere dissolved in methanol to obtain a concentration of200 ppm and 02mL of this solution was completed to4mL by MeOH then 1mL of DPPH (609 times 10minus5molL)solution in the same solvent was then added The absorptionwas monitored after 10min at 516 nm The reference sample(Blank) was 1mL of DPPH solution and 4mL MeOH Thecapacity of antioxidants to quench DPPH radical was deter-mined according to Nogala-Kalucka et al [20] and calculatedaccording to the following equation

RSA = (Absorbance of control sample

minus absorbance of extract sample)

times (absorbance of control sample)minus1 times 100

(1)

27 Experimental Animals Three-month-old male SpragueDawley rats (100ndash150 g) were purchased from Animal HouseColony National Research Centre Dokki Cairo Egypt Ani-mals were maintained on standard lab diet (protein 1604fat 363 fiber 41 gkg of metabolizable energy 1208MJ)housed in filter-top polycarbonate cages in a room free fromany source of chemical contamination artificially illuminated(12 h darklight cycle) and thermally controlled (25 plusmn 1∘C) atthe Animal House Lab National Research Centre After anacclimatization period of 1 week the animals were dividedinto six groups (10 ratsgroup) and housed in filter-toppolycarbonate cages All animals received humane care incompliance with the guidelines of the Animal Care and UseCommittee of the National Research Center

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Table 1 Yield total phenolic compounds and DPPH radicalscavenging activity of the ethanol of Calendula

Parameter Ethanolic extractYield (g100 g plant) 799 plusmn 049Total phenolic compounds (120583gg) 13600 plusmn 2188DPPH radical scavenging activity () 9630

28 Experimental Design Animals within different treat-ment groups were treated daily for 6 weeks and includedthe control the group fed AFs-contaminated diet (25mgkgdiet) the groups treated orally with the low (CE1) and high(CE2) doses of Calendula extract (500 and 1000mgkg bw)and the groups pretreated orally withCalendula extract at thetwo tested doses one week before and during AFs treatmentfor another five weeks At the end of the treatment periodall animals were fasted for 12 hr and blood samples werecollected from the retro-orbital venous plexus from eachanimal under ether anesthesia Blood samples were left toclot and the sera were separated using cooling centrifugationat 3000 rpm for 15min and stored at minus20∘C until analysisThe sera were used for the determination of ALT AST ALPtotal protein albumin urea creatinine AFP TNF-120572 and IL-1120573 according to the kits instructions

After the collection of blood samples all animals werekilled by cervical dislocation and sample of the liver wasweighed (approximately 005ndash01 g) and homogenized inphosphate buffer (pH 74) to give 20 wv homogenate Thishomogenate was centrifuged at 1700 rpm at 4∘C for 10minand the supernatant was stored at minus70∘C until analysis Thissupernatant was used for the assessment of GPx MDA andSOD according to the kits instructions Another sample ofeach liver was removed and placed in 10 of natural formalinfor histological and histochemical examinations [21]

29 Statistical Analysis All data were statistically analyzedby analysis of variance (ANOVA) using the General LinearModel Procedure of the Statistical Analysis System [22] Thesignificance of the differences among treatment groups wasdetermined byWaller-Duncan k-ratio [23] All statements ofsignificance were based on probability of 119875 le 005

3 Results

31 Total Phenolic Content and DPPH Scavenging ActivityThe results of total phenolic contents and DPPH scavengingactivity of the aqueous and ethanolic extracts of Calendulaextract are presented in Table 1 The results revealed that thetotal phenolic content of the ethanolic extract of Calendulawas higher than that in the aqueous extract Moreover theDPPH scavenging activity of the ethanolic extract showedhigher radical scavenging activity than the aqueous extractTherefore the ethanolic extract was used for the biologicalassay

32 The Biological Assay Along the study period no mortal-ity was observed in the groups treated with Calendula at the

0

5

10

15

20

25

30

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6

ControlAFsCE1

CE2

Food

inta

ke (g

)

AFs + CE1AFs + CE2

Figure 1 Effect of the treatment with ethanol extract of Calendulaon food intake of rats fed AFs-contaminated diet during the exper-imental period (AFs aflatoxins CE1 low dose of Calendula extract(500mgkg bw) CE2 high dose of Calendula extract (1000mgkgbw))

0

50

100

150

200

250

300

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6

Body

wei

ght (

g)

ControlAFsCE1

CE2AFs + CE1AFs + CE2

Figure 2 Effect of the treatment with ethanol extract of Calendulaon body weight of rats fed AFs-contaminated diet during theexperimental period (AFs aflatoxins CE1 low dose of Calendulaextract (500mgkg bw) CE2 high dose of Calendula extract(1000mgkg bw))

two tested doses either alone or one week before and duringfeeding AFs-contaminated diet Effect of different treatmentson the body weight changes is illustrated at Figure 1 Animalsfed AFs-contaminated diet showed a significant reductionof the body weight whereas animals treated with CE1 orCE2 were comparable to the control CE1 or CE2 succeededto normalize body weight when treated with animals fedAFs-contaminated diet The results indicate that animals fedAFs-contaminated diet demonstrated a significant decreaseof feed intake but animals treated with either CE1 or CE2were comparable to control Groups fed AFs-contaminateddiet and treated with CE1 or CE2 showed a significantimprovement of feed intake This result was pronounced inthe group treated with the high dose (Figure 2)

The effects of different treatments on serum biochemicalparameters are depicted in Table 2 These results indicatedthat animals fed AFs-contaminated diet showed a significantincrease in serum ALT AST ALP urea creatinine and AFPaccompanied with a significant decrease in total proteinand albumin Animals treated with the CE1 or CE2 were

4 ISRN Nutrition

Table 2 Effect of ethanol extract of Calendula on serum biochemical parameters in rats fed aflatoxins-contaminated diet

Parameter GroupsControl AFs CE1 CE2 CE1 + AFs CE2 + AFs

AST (UmL) 24832 plusmn 314a 33034 plusmn 422d 2394 plusmn 896b 24060 plusmn 642a 2830 plusmn 42c 25764 plusmn 324a

ALT (UmL) 7536 plusmn 137a 13251 plusmn 405c 732 plusmn 202a 7412 plusmn 269a 1092 plusmn 177b 7752 plusmn 133a

ALP (UL) 10675 plusmn 623a 23451 plusmn 532b 9367 plusmn 324a 10436 plusmn 333a 15671 plusmn 343c 11137 plusmn 261d

TP (mgdL) 999 plusmn 141a 435 plusmn 031b 923 plusmn 046a 1044 plusmn 161a 895 plusmn 027c 927 plusmn 023c

Albumin (mgdL) 344 plusmn 020a 188 plusmn 012b 311 plusmn 007a 337 plusmn 012a 307c plusmn 014 316 plusmn 022a

Urea (mgdL) 151 plusmn 014a 344 plusmn 031b 172 plusmn 013a 161 plusmn 013a 234 plusmn 012a 184 plusmn 032a

Creatinine (mgdL) 077 plusmn 021a 333 plusmn 014b 075 plusmn 013a 076 plusmn 014a 203 plusmn 002c 087 plusmn 013d

AFP (ngmL) 152 plusmn 078a 464 plusmn 022b 153 plusmn 017a 144 plusmn 016a 277 plusmn 031c 159 plusmn 023a

Within each row means superscript with different letters are significantly different at 119875 le 005AFs aflatoxinsCE1 low dose of calendula extract (500mgkg bw)CE2 high dose of calendula extract (1000mgkg bw)

Table 3 Effect of ethanol extract of calendula on liver antioxidants and lipid peroxidation in rats fed aflatoxins-contaminated diet

Parameter GroupControl AFs CE1 CE2 CE1 + AFs CE2 + AFs

SOD (Umg liver protein) 29937 plusmn 1033a 17232 plusmn 711b 30549 plusmn 2167a 31021 plusmn 832a 28157 plusmn 1504c 28532 plusmn 932a

GPx (Umg liver protein) 3366 plusmn 254a 1461 plusmn 120b 5403 plusmn 145c 5649 plusmn 212a 3561 plusmn 143a 3743 plusmn 156a

MDA (nmolmg liver protein) 3732 plusmn 212a 8431 plusmn 244b 2271 plusmn 228c 2031 plusmn 144a 4526 plusmn 129d 4232 plusmn 213a

Within each row means superscript with different letters are significantly different at 119875 le 005AFs aflatoxinsCE1 low dose of calendula extract (500mgkg bw)CE2 high dose of calendula extract (1000mgkg bw)

comparable to the controls in all biochemical parametersTreatment with the extract of the two tested dose one weekand during feeding AFs-contaminated diet succeeded toinduce a significant improvement of all biochemical parame-ters towards the control values

The existing results proved that AFs-contaminated dietresulted in a significant decrease in GPx and SOD activ-ities accompanied with a significant increase of MDAlevel (Table 3) However no significant effect was observedbetween those treated with CE1 or CE2 alone and the controlgroup Furthermore animals fed AFs-contaminated diet andtreated with CE1 or CE2 demonstrated a significant increaseof GPx and SOD activities with a significant decrease ofMDAlevel compared to the AFs-treated group The pronouncedimprovement has been showed at the high dose of extract(CA2) especially in SOD which was comparable to control

The effects of different treatments on inflammatorycytokine TNF-120572 and IL-1120573were illustrated in (Table 4) AFs-treated rats showed a significant increase of the inflammatorycytokines however animals treated with either CE1 or CE2were comparable to the controls Treatment with CE1 or CE2one week before and during AFs treatment succeeded toinduce a significant improvement in TNF-120572 and IL-1120573 in adose-dependent manner The higher dose demonstrated asignificant improvement of IL-1120573 toward the control level

The biochemical results obtained in the current studywere confirmed by the histopathological examination of theliver tissue Microscopic examination of the liver sectionof a control rat showed branching and anatomizing cords

radiating from the central vein with vesicular nuclei andsome binucleated cells and separated by sinusoids whichlined by flat endothelial cell and kupfer cells (Figure 3(a))The liver section of a rat fed AFs-contaminated diet showedthe disorganization and damage in hepatocytes architec-ture accompanied with hepatocytes necrosis apoptosis andfibrosis around the blood vessels (Figure 3(b)) The liversection of a rat treated with CE1 or CE2 showed hepatocyteswith normal architecture in the central and the portal veinsaccompanied with few aggregations of inflammatory cells in-between the hepatocytes (Figure 3(c))

The liver section of rats treated with CE1 one week beforeand during AFs-treatment showed marked improvement inhepatocytes architecture no vacuoles or fatty droplets weredetected Some cells have eosinophilic cytoplasm and deeplystained nuclei around the central and portal vein areas andmononuclear cellular infiltration are scattered around theportal tracts (Figure 3(d)) Moreover the liver section of ratsfed AFs-contaminated diet and treated with CE2 showednormal histological picture with few fatty degeneration andinterstitial hemorrhage around the central vein (Figure 3(e))

The microscopic examination of liver sections stainedwith Periodic-Sheif reagent stain (PAS) for glycogen demon-stration revealed that the control liver showed a strongreaction in the liver section (Figure 4(a))The liver of animalsfed AFs-contaminated diet showed a weak reaction in thedamaged cell while a strong reaction was noticed in intactcells (Figure 4(b)) The liver of the animals treated with CE1or CE2 showed different types of reaction strong reaction

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Table 4 Effect of ethanol extract of calendula on serum inflammatory cytokine in rats fed aflatoxins-contaminated diet

Parameter GroupControl AFs CE1 CE2 CE1 + AFs CE2 + AFs

TNF-120572 (pgmL) 5834 plusmn 312a 13112 plusmn 332b 5837 plusmn 207a 6072 plusmn 192a 8647 plusmn 231c 6324 plusmn 166d

IL-1120573 (pgmL) 066 plusmn 007a 234 plusmn 012b 065 plusmn 004a 084 plusmn 022a 158 plusmn 004c 077 plusmn 021a

Within each row means superscript with different letters are significantly different at 119875 le 005AFs aflatoxinsCE1 low dose of calendula extract (500mgkg bw)CE2 high dose of calendula extract (1000mgkg bw)

CV

Hepatocytes

(HX and E times400)

(a)

FN

(HX and E times400)

(b)

Hepatocytes

Inf

CV

(HX and E times400)

(c)

CVPV

PV

(HX and E times200)

(d)

Fatty degeneration

(HX and E times400)

(e)

Figure 3 A photomicrograph in liver section from (a) a control rat branching and anatomizing cords radiating from the central vein CVThehepatocytes that are having vesicular nuclei and some binucleated cells are separated by sinusoids lined by flat endothelial cell and kupfer cells(b) a rat fed AFs-contaminated diet showing hepatocytes necrosis (N) and apoptosis (yellow arrows) and fibrosis around the blood vessels(F) (c) A rat treated with CE1 or CE2 showing the normal hepatocytes with vesicular nuclei and evident mononuclear cellular infiltrationaround the central vein (d) a rat fed AFs-contaminated diet and treated with CE1 showing marked improvement in hepatocytes architectureno vacuoles or fatty droplets were detected Some cells have eosinophilic cytoplasm and deeply stained nuclei around the central and portalvein areas (arrows) and mononuclear cellular infiltration are scattered around the portal tracts (e) A rat fed AFs-contaminated diet andtreated with CE2 showing normal histological picture with few fatty degeneration and interstitial hemorrhage around the central vein

around the central vein andweak reaction around portal veinof hepatocytes (Figure 4(c))The liver of the animals fed AFs-contaminated diet and treated with CE1 showed that weakreaction in some damaged cells which scattered in all thesection (Figure 4(d)) However the liver section of animalstreatedwithCE2 showedmarked improvement although lowreaction in hepatocytes was still demonstrated (Figure 4(e))

4 Discussion

Previous reports indicated that the type of extraction ofactive ingredient compounds from plant material dependsmainly on the type of solvent used [24] In the currentstudy the total phenolic compounds of the ethanol extractof Calendula were higher than that in the aqueous extract

However the ethanolic extract showed a higher DPPH (11-diphenyl 1-2-picryl hydrazyl) free radical scavenging activitycompared to the aqueous extract Cetkovic et al [25] showedsimilar scavenging potential against DPPH hydroxyl andperoxyl radicals and suggested that all of the extracts obtainedscavenged all radicals in concentration-dependant mannerMoreover Preethi et al [26] tested the antioxidant potentialof various leaves extracts of Calendula against DPPH ABTS(221015840-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) nitricoxide and superoxide radicals and reported lower scavengingproperty towards DPPH whereas they exerted higher radicalscavenging effects against the other radicals tested

In a similar study Danila et al [27] screened the waterand alcoholic extracts of Calendula grown in Romania for itsantioxidant activity and reported that Calendula contained

6 ISRN Nutrition

(a) (b)

(c) (d)

(e)

Figure 4 Photomicrographs in liver sections stained with Periodic-Sheif reagent-stain (PAS) for glycogen demonstration from (a)control rats showing a strong reaction of PAS (b) rats fed AFs-contaminated diet showing a weak reaction in the damaged cellwhile a strong reaction in intact cells (c) rats treated with CE1 orCE2 showing different types of reaction as well as strong reactionaround the central vein and weak reaction around portal vein ofhepatocytes (d) rats fed with AFs contaminated diet treated withCE1 showing that weak reaction in some damaged cells whichscattered in all the section and (e) rats fed with AFs contaminateddiet and treated with CE2 showing marked improvement althoughlow reaction in hepatocytes were still demonstrated (PAS reactiontimes100)

a high total phenol amount and scavenging activity againstDPPH and ABTS radicals DallrsquoAcqua et al [28] investigatedantioxidant activity of Calendula arvensis used in Sardinianfolk medicine using DPPH radical scavenging assay andreported that the extract showed a high radical scavengingeffect towards DPPH The radical scavenging propertieswere attributed to the presence of flavonoid and triterpenederivatives in the extract [29]

In the in vivo study the selective dose of AFs andCalendula was literature-based [30 31] respectively Theresults revealed a significant reduction of body weight andfood intake in animals fed AFs-contaminated diet Theseresults were in agreement with those reported in the literatureof aflatoxicosis [32ndash38] The reduction in body weight inthe animals fed AFs-contaminated diet alone may be due tothe effects of AFs on the balance between orexigenic andanorexigenic circuits that regulate the homeostatic loop ofbody weight regulation leading to cachexia [34] Moreover

AFs exposure may lead to significant reduction of leptin[35] accompanied with high levels of cortisol IL-6 andinsulin resistance which together act to influence the feedingresponse causing weight loss in patients with pancreaticcancer [36] Osborne et al [37] suggested that AFs ingestionaffect various digestive enzymatic activities that give riseto a malabsorption syndrome characterized by steatorrheahypocarotenoidemy and to lowering of bile pancreatic lipasetrypsin and amylase Furthermore Lesson et al [38] reportedthat the 89 epoxide metabolite of AFB

1may covalently bind

to DNA and proteins which then alters enzymatic processessuch as gluconeogenesis Krebs cycle or fatty acid synthesis

Animals fed AFs-contaminated diet showed a significantincrease of ALT AST ALP urea and createnine whichindicate changes in the hepatic tissues and biliary system [39]structural damaging of liver integrity since these enzymes arecytoplasmic in location and released into plasma as a result ofcellular damage [8 40] It is also likely that alteration in ALPactivity affects membrane permeability and the transport ofmetabolites Moreover increase in urea and createnine levelobserved in the current study in AFs-treated group clearlyindicated the harmful and stressful effect on renal tissue [30]Taken together the increased level of urea and the decreasedlevel of albumin and total protein (TP) indicate the inhibitionof protein synthesis and increase of protein catabolism andorrenal dysfunction [41 42]These results clearly indicated thatAFs had stressful effects on the hepatic and renal tissuesconsistent with those reported in the literature of aflatoxicosis[43 44]

Animals treated with AFs showed a significant increasein AFP which is considered specific biomarkers for livercancer This increase in AFP may be due to induction ofexpression of mRNAs of liver alpha-fetoprotein [45 46] Onthe other hand aflatoxin B

1-induced hepatocarcinogenesis

associated with defective DNA-damage response by passingp53 activation [47] and modulation of insulin-like growthfactor 2 dependent signal axis (IGF-2) [48] Similar to theseobservations Sell et al [49] and Yang et al [45] reported thatAFB1administration resulted in the elevation of serum AFP

level in both duck and ratsThe current study showed that animals fed AFs-con-

taminated diet suffer from oxidative stress as indicated bythe significant increment of lipid peroxidation (MDA) andthe significant reduction of enzymatic antioxidant such assuperoxide dismutase (SOD) and glutathione peroxidase(GPx) These results are in agreement with previous studiessuggested that oxidative stress may be due to direct effectof AFs or by the metabolites formed and the free radicalsgenerated during the formation of these metabolites [3446 50] Moreover the reduction of protein synthesis inAFs-treated animals may affect certain metal ions (ie ironand copper) which play an important role in free radicalproduction and liberation [10] In normal state metal ionsare bonded to transfer proteins such as ceruloplasmin andtransferrin [51] and play a crucial role of SOD CAT and GPxactivities which constitute the enzymatic antioxidant defensesystem of the cell displayed bidirectional alterations either inthe form of increase or decrease depending on the particulartissue Previous studies demonstrated that the mechanism of

ISRN Nutrition 7

AFs-induced liver injury may be due to those reactive andtoxic metabolites of AFs and the liver necrosis begins whenthe glutathione stores are almost exhausted [34 35 52]

Tumor necrosis factor-alpha (TNF-120572) and interleukin-1 alpha (IL-1120572) are produced by macrophages and theyplay an important role in tumor conditions [35] TNF-120572is an essential factor in tumor promotion [53] and IL-1polymorphisms are important mediators in the inflamma-tory process [54] In the current study ingestion of AFs-contaminated diet significantly increased TNF-120572 and IL-1120573 suggesting that AFs preferentially affects macrophagefunctions In particular it decouples the close correlationsusually observed between transcriptional and translationalcontrols of IL-1120572 and TNF-120572 production by these cells [55]In this concern Barton et al [56] stated that TNF-120572 plays acausal role in the development of liver injury and it plays amajor role in modulating mycotoxin-induced hepatotoxicityMoreover TNF-120572 has been proven to play an importantrole in inflammation in mediating the proliferation anddifferentiation of immune cells and development of immuneresponse [57] Hopkins [55] and Abdel-Wahhab et al [35]reported that directly proportional relationship has beenobserved between transcriptional and translational controlsof IL-1 and TNF-120572 production by hepatocytes Consequentlythe current results indicated that aflatoxicosis may induceTNF-120572 and IL-1 due to activation of caspase-3 which is oneof the apoptotic mediator [58] and the significant elevationof inflammatory cytokinemay affect themRNA expression ofthese mediators [46]

The current results indicated that the ethanolic extract ofCalendula showed a potential protective effect against AFstoxicity Animals treated withCalendula extract did not showany sign of toxicity In this concern the safety of the ethanolextract of Calendula was suggested and the oral LD

50in rats

was estimated by 4640mgkg bw [59] The results reportedherein indicated that animals treated with Calendula extractshowed insignificant increase in food intake and body weightgain Moreover Calendula extract succeeded to induce asignificant improvement in the biochemical parameters andthe histological and histochemical pictures Taken togetherthe results of the in vitro study and the in vivo evaluationsuggested that there are an antioxidant and free radicalscavenging properties of this extract and this improvementmore pronounced with the high dose (CA2) [29] In thisconcern previous study suggested that Calendula plays animportant role in hepaticcytolysis inhibition and improvesliver integrity in CCl

4intoxicated rats [60] In referring to

previous literature hepatoprotective activity of Calendulamay be due to its modulatory effect on cytochrome P450which may interfere on aflatoxin active metabolite produc-tion [61]MoreoverCalendula extract showed renoprotectiveaction against cisplatin-induced renal toxicity through itsmodulatory effect against myelosuppression of the renalsystem

The existing data revealed anticarcinogenicity of Cal-endula extract indicated by significant reduction of AFPand this effect was pronounced in the animals treatedwith the high dose (CA2) Several reports suggested thatthe anticancer properties of Calendula may be due to its

chemoprotector properties against hepatocarcinogenesis inrats beside antitumoural activity in vivo in the Ehrlichmouse carcinoma model due to its ability to stimulate T-lymphocytes B-lymphocytes and cluster of differentiation(CD4+) [62] Moreover Calendula plays a crucial role inprevention of DNA damaging [63] These results were inagreement with those reported previously [26 31 62]

The results also suggested that Calendula extract pos-sesses anti-inflammatory activity which may be due to itshigher content of carvacol Carvacol has been described asa promoter for liver regeneration and inhibitor of TNF-120572and IL-6 level in rats undergoing partial hepatectomy [64]Furthermore Tsai et al [65] reported that caravacl couldreduce TNF-120572 and IL-1120573 levels in intoxicated rats through theinhibition of cycloxygensae (COX) enzymes activity mRNAexpression and its protein in lipopoly saccharides-inducedinflammation

The histopathological and histochemical changes in liverconfirmed the biochemical results and revealed that ani-mals fed AFs-contaminated diet showed severe histologicalchanges typical to those reported in the literature [10 39 66]However animals treated with the extract in combinationwith AFs resulted in a significant improvement in liver tissuessimilar to that reported previously [61]

5 Conclusion

It can be concluded that the ethanolic and aqueous extracts ofCalendula officinalis have a valuable quantity of total phenoliccompounds and have DPPH radical scavenging activityMoreover the ethanolic extract showed higher phenoliccontent and radical scavenging property compared to theaqueous extract The ethanolic extract exhibited hepatoreno-protective properties against aflatoxin-induced liver injury ina dose-dependent manner due to its antioxidant free radicalscavenging activities and anti-inflammatory properties

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

This work was full supported by College of Pharmacy MisrUniversity for Science and Technology 6th October CityEgypt Faculty of Pharmacy Cairo University Cairo EgyptNational Research Center Dokki Cairo Egypt

References

[1] CAST ldquoMycotoxins risks in plant animal and humanrdquo inPotential Economic Costs ofMycotoxins inUnited States pp 136ndash142 CAST Task Force Reports 2003

[2] J A Nogueira S K Ono-Nita M E Nita et al ldquo249 TP53mutation has high prevalence and is correlated with larger andpoorly differentiated HCC in Brazilian patientsrdquo BMC Cancervol 9 article 204 2009

8 ISRN Nutrition

[3] S Rawal J E Kim and R Coulombe Jr ldquoAflatoxin B1in

poultry toxicology metabolism and preventionrdquo Research inVeterinary Science vol 89 no 3 pp 325ndash331 2010

[4] International Agency for Research in Carcinogenesis ldquoAfla-toxinsrdquo Monograph on the Evaluation of Carcinogenic Risks toHumans vol 56 p 245 1993

[5] IPCS-WHO ldquoAflatoxinsrdquo in International Programme onChem-ical Safety WHO Food Additives Series 40 pp 1ndash73 WorldHealth Organization Geneva Switzerland 1998

[6] J H Williams T D Phillips P E Jolly J K Stiles C M JollyandDAggarwal ldquoHuman aflatoxicosis in developing countriesareview of toxicology exposure potential health consequencesand interventionsrdquo American Journal of Clinical Nutrition vol80 no 5 pp 1106ndash1122 2004

[7] K C Choi W T Chung J K Kwon et al ldquoInhibitory effectsof quercetin on aflatoxin B1-induced hepatic damage in micerdquoFood and Chemical Toxicology vol 48 no 10 pp 2747ndash27532010

[8] D S El-Agamy ldquoComparative effects of curcumin and resver-atrol on aflatoxin B 1-induced liver injury in ratsrdquo Archives ofToxicology vol 84 no 5 pp 389ndash396 2010

[9] M A Abdel-Wahhab A A Ibrahim A A El-Nekeety NS Hassan and A A Mohamed ldquoPanax ginseng CA Meyerextract counteracts the oxidative stress in rats fed multi-mycotoxins-contaminated dietrdquo Comunicata Scientiae vol 3no 3 pp 143ndash153 2012

[10] M A Hamzawy E S El-Denshary N S Hassan F A Mannaaand M A Abdel-Wahhab ldquoAntioxidant and hepatorenoprotec-tive effect of thyme vulgaris extract in rats during aflatoxicosisrdquoGlobal Journal Pharmacology vol 6 no 2 pp 106ndash117 2012

[11] Z C Gazim C M Rezende S R Fraga T I E Svidzinskiand D A G Cortez ldquoAntifungal activity of the essential oilfrom Calendula officinalis L (Asteraceae) growing in BrazilrdquoBrazilian Journal of Microbiology vol 39 no 1 pp 61ndash63 2008

[12] L R Della A Tubaro S Sosa H Becker S Saar and D IsaacldquoThe role of triperpenoids in the topical antiinflamatory activityofCalendula officinalisflowersrdquoPlantaMedica vol 60 no 6 pp516ndash520 1994

[13] Chrysolite 2010 httphealthwikinutcomCalendula-officin-alis-Pot-Marigold11v4g8pw

[14] A Pintea C Bele S Andrei and C Socaciu ldquoHPLC analysis ofcarotenoids in four varieties of Calendula officinalis L flowersrdquoActa Biologica Szegediensis vol 247 pp 37ndash40 2003

[15] R A Jacob ldquoThe integrated antioxidant systemrdquo NutritionResearch vol 15 no 5 pp 755ndash766 1995

[16] K Dahan M Fennal and N B Kumar ldquoLycopene in the pre-vention of prostate cancerrdquo Journal of the Society for IntegrativeOncology vol 6 no 1 pp 29ndash36 2008

[17] J E Hutchins andWMHagler ldquoRapid liquid chromatographicdetermination of aflatoxins in heavily contaminated cornrdquoJournal of the Association of Official Analytical Chemists vol 66no 6 pp 1458ndash1465 1983

[18] G K Jayaprakasha and L J Rao ldquoPhenolic constituents fromthe lichen parmotrema stuppeum (Nyl) Hale and their antioxi-dant activityrdquo Zeitschrift fur Naturforschung C vol 55 no 11-12pp 1018ndash1022 2000

[19] G K Jayaprakasha T Selvi and K K Sakariah ldquoAntibacterialand antioxidant activities of grape (Vitis vinifera) seed extractsrdquoFood Research International vol 36 no 2 pp 117ndash122 2003

[20] M Nogala-Kalucka J Korczak M Dratwia E Lampart-Szczapa A Siger and M Buchowski ldquoChanges in antioxidant

activity and free radical scavenging potential of rosemaryextract and tocopherols in isolated rapeseed oil triacylglycerolsduring accelerated testsrdquo Food Chemistry vol 93 no 2 pp 227ndash235 2005

[21] R A Drury E A Wallington and R Cancerson CarltonrsquosHistopathological Techniques Oxford University Press OxfordUK 4th edition 1980

[22] SAS IInstitute SAS Userrsquos Guide Statistics SAS Institute CaryNC USA

[23] R A Waller and D B Duncan ldquoA Bayes rule for the symmetricmultiple comparisons problemsrdquo Journal of American StatisticalAssociation vol 64 no 328 pp 1484ndash1503 1969

[24] L Majhenic M Skerget and Z Knez ldquoAntioxidant and antimi-crobial activity of guarana seed extractsrdquo Food Chemistry vol104 no 3 pp 1258ndash1268 2007

[25] G S Cetkovic S M Djilas J M Canadanovic-Brunet and VT Tumbas ldquoAntioxidant properties of marigold extractsrdquo FoodResearch International vol 37 no 7 pp 643ndash650 2004

[26] K C Preethi G Kuttan and R Kuttan ldquoAntioxidant potentialof an extract ofCalendula officinalis flowers in vitro and in vivordquoPharmaceutical Biology vol 44 no 9 pp 691ndash697 2006

[27] AODanila F Gatea andG L Radu ldquoPolyphenol compositionand antioxidant activity of selected medicinal herbsrdquoChemistryof Natural Compounds vol 47 no 1 pp 22ndash26 2011

[28] S DallrsquoAcqua R Cervellati M C Loi and G InnocentildquoEvaluation of in vitro antioxidant properties of some tradi-tional Sardinian medicinal plants investigation of the highantioxidant capacity of Rubus ulmifoliusrdquo Food Chemistry vol106 no 2 pp 745ndash749 2008

[29] T Ercetin F S Senol I Erdogan Orhan and G TokerldquoComparative assessment of antioxidant and cholinesteraseinhibitory properties of the marigold extracts from Calendulaarvensis L and Calendula officinalis Lrdquo Industrial Crops andProducts vol 36 no 1 pp 203ndash208 2012

[30] M A Abdel-Wahhab and S E Aly ldquoAntioxidants and radicalscavenging properties of vegetable extracts in rats fed aflatoxin-contaminated dietrdquo Journal of Agricultural and Food Chemistryvol 51 no 8 pp 2409ndash2414 2003

[31] C Gladine C Morand E Rock D Gruffat D Bauchart andD Durand ldquoThe antioxidative effect of plant extracts rich inpolyphenols differs between liver and muscle tissues in rats fedn-3 PUFA rich dietsrdquo Animal Feed Science and Technology vol139 no 3-4 pp 257ndash272 2007

[32] R H Rauber P Dilkin L Z Giacomini C A Araujo deAlmeida and C A Mallmann ldquoPerformance of Turkey poultsfed different doses of aflatoxins in the dietrdquo Poultry Science vol86 no 8 pp 1620ndash1624 2007

[33] SM RustemeyerW R Lamberson D R Ledoux et al ldquoEffectsof dietary aflatoxin on the health and performance of growingbarrowsrdquo Journal of Animal Science vol 88 no 11 pp 3624ndash3630 2010

[34] M A Abdel-Wahhab N S Hassan A A El-Kady et al ldquoRedginseng extract protects against aflatoxin B

1and fumonisins-

induced hepatic pre-cancerous lesions in ratsrdquo Food and Chem-ical Toxicology vol 48 no 2 pp 733ndash742 2010

[35] M A Abdel-Wahhab H H Ahmed and M M HagazildquoPrevention of aflatoxin B1-initiated hepatotoxicity in rat bymarine algae extractsrdquo Journal of Applied Toxicology vol 26 no3 pp 229ndash238 2006

[36] M D Barber D C McMillan A M Wallace J A RossT Preston and K C H Fearon ldquoThe response of leptin

ISRN Nutrition 9

interleukin-6 and fat oxidation to feeding in weight-losingpatients with pancreatic cancerrdquo British Journal of Cancer vol90 no 6 pp 1129ndash1132 2004

[37] D J Osborne W E Huff P B Hamilton and H R BurmeisterldquoComparison of ochratoxin aflatoxin and T-2 toxin for theireffects on selected parameters related to digestion and evidencefor specific metabolism of carotenoids in chickensrdquo PoultryScience vol 61 no 8 pp 1646ndash1652 1982

[38] S Lesson G Diaz and J Summers Poultry Metabolic Disordersand Mycotoxins University Books Montreal Canada 1995

[39] M A Abdel-Wahhab S A Nada and F A Khalil ldquoPhys-iological and toxicological responses in rats fed aflatoxin-contaminated diet with or without sorbent materialsrdquo AnimalFeed Science and Technology vol 97 no 3-4 pp 209ndash219 2002

[40] A A El-Nekeety S R Mohamed A S Hathout N S HassanS E Aly and M A Abdel-Wahhab ldquoAntioxidant properties ofThymus vulgaris oil against aflatoxin-induce oxidative stress inmale ratsrdquo Toxicon vol 57 no 7-8 pp 984ndash991 2011

[41] N Jindal S K Mahipal and N K Mahajan ldquoToxicity ofaflatoxin B1 in broiler chicks and its reduction by activatedcharcoalrdquo Research in Veterinary Science vol 56 no 1 pp 37ndash40 1994

[42] M A Abdel-Wahhab and S E Aly ldquoAntioxidant property ofNigella sativa (black cumin) and Syzygium aromaticum (clove)in rats during aflatoxicosisrdquo Journal of Applied Toxicology vol25 no 3 pp 218ndash223 2005

[43] D M Miller and D M Wilson Veterinary Diseases Relatedto Aflatoxins the Toxicology of Aflatoxins Academic Press SanDiego Calif USA 1994

[44] M A Abdel-Wahhab and A M Kholif ldquoMycotoxins in animalfeeds and prevention strategies a reviewrdquo Asian Journal ofAnimal Sciences vol 2 no 1 pp 7ndash25 2008

[45] C F Yang J Liu S Wasser H M Shen C E L Tan andC N Ong ldquoInhibition of ebselen on aflatoxin B1-inducedhepatocarcinogenesis in Fischer 344 ratsrdquo Carcinogenesis vol21 no 12 pp 2237ndash2243 2000

[46] S H Abdel-Aziem A M Hassan and M A Abdel-WahhabldquoDietary supplementation with whey protein and ginsengextract counteracts oxidative stress and DNA damage in ratsfed an aflatoxin-contaminated dietrdquoMutationResearch vol 723no 1 pp 65ndash71 2011

[47] O Gursoy-Yuzugullu H YuzugulluM Yilmaz andMOzturkldquoAflatoxin genotoxicity is associated with a defective DNAdamage response bypassing p53 activationrdquo Liver Internationalvol 31 no 4 pp 561ndash571 2011

[48] T Ubagai T Kikuchi T Fukusato and Y Ono ldquoAflatoxin B1

modulates the insulin-like growth factor-2 dependent signalingaxisrdquo Toxicology in Vitro vol 24 no 3 pp 783ndash789 2010

[49] S Sell K L Xu W E Huff L F Kabena R B Harveryand H A Dunsford ldquoAflatoxin exposure produces serumalphafetoprotein elevations and marked oval cell proliferationin young male Pekin ducklingsrdquo Pathology vol 30 no 1 pp34ndash39 1998

[50] M Kanbur G Eraslan Z S Sarica andO Aslan ldquoThe effects ofevening primrose oil on lipid peroxidation induced by subacuteaflatoxin exposure in micerdquo Food and Chemical Toxicology vol49 no 9 pp 1960ndash1964 2011

[51] E Gitto S Pellegrino P Gitto I Barberi and R J ReiterldquoOxidative stress of the newborn in the pre- and postnatalperiod and the clinical utility of melatoninrdquo Journal of PinealResearch vol 46 no 2 pp 128ndash139 2009

[52] M A Abdel-Wahhab A Said and A Huefner ldquoNMR andradical scavenging activities of patuletin from Urtica urensagainst aflatoxin B

1rdquo Pharmaceutical Biology vol 43 no 6 pp

515ndash525 2005[53] M Suganuma E Sueoka N Sueoka S Okabe and H Fujiki

ldquoMechanisms of cancer prevention by tea polyphenols based oninhibition of TNF-120572 expressionrdquo BioFactors vol 13 no 1-4 pp67ndash72 2000

[54] S Rollinson A P Levene F K Mensah et al ldquoGastric marginalzone lymphoma is associated with polymorphisms in genesinvolved in inflammatory response and antioxidative capacityrdquoBlood vol 102 no 3 pp 1007ndash1011 2003

[55] S J Hopkins ldquoThe pathophysiological role of cytokinesrdquo LegalMedicine vol 5 no 1 pp S45ndashS57 2003

[56] C C Barton E X Barton P E Ganey S L Kunkel and RA Roth ldquoBacterial lipopolysaccharide enhances aflatoxin B

1

hepatotoxicity in rats by a mechanism that depends on tumornecrosis factor 120572rdquo Hepatology vol 33 no 1 pp 66ndash73 2001

[57] J Cao L Jiang X Zhang et al ldquoBoric acid inhibits LPS-inducedTNF-120572 formation through a thiol-dependent mechanism inTHP-1 cellsrdquo Journal of Trace Elements in Medicine and Biologyvol 22 no 3 pp 189ndash195 2008

[58] A R M A Meik S K Abdel-Ghaffar and I El-GibalyldquoAflatoxin B1 induces apoptosis in rat liver protective effect ofmelatoninrdquo Neuroendocrinology Letters vol 22 no 6 pp 417ndash426 2001

[59] CIREP ldquoFinal report on the safety assessment ofCalendula offic-inalis Extract and Calendula officinalisrdquo International JournalToxicology vol 20 pp 13ndash20 2001

[60] M A Rusu M Tamas C Puica I Roman and M SabadasldquoThe hepatoprotective action of ten herbal extracts in CCl

4

intoxicated liverrdquo Phytotherapy Research vol 19 no 9 pp 744ndash749 2005

[61] K C Preethi and R Kuttan ldquoHepato and reno protective actionof Calendula officinalis L Flower extractrdquo Indian Journal ofExperimental Biology vol 47 no 3 pp 163ndash168 2009

[62] B P Muley S S Khadabadi and N B Banarase ldquoPhytochem-ical constituents and pharmacological activities of Calendulaofficinalis Linn (Asteraceae) a reviewrdquo Tropical Journal ofPharmaceutical Research vol 8 no 5 pp 455ndash465 2009

[63] T Frankic K Salobir and J Salobir ldquoThe comparison of invivo antigenotoxic and antioxidative capacity of two propyleneglycol extracts of Calendula officinalis (marigold) and vitaminE in young growing pigsrdquo Journal of Animal Physiology andAnimal Nutrition vol 93 no 6 pp 688ndash694 2009

[64] M Uyanoglu M Canbek E Aral and K Husnu Can BaserldquoEffects of carvacrol upon the liver of rats undergoing partialhepatectomyrdquo Phytomedicine vol 15 no 3 pp 226ndash229 2008

[65] M L Tsai C C Lin W C Lin and C H Yang ldquoAntimicrobialantioxidant and anti-inflammatory activities of essential oilsfromfive selected herbsrdquoBioscience Biotechnology Biochemistryvol 75 no 10 pp 1977ndash1983 2011

[66] K Mayura M A Abdel-Mahhab K S McKenzie et alldquoPrevention of maternal and developmental toxicity in rats viadietary inclusion of common aflatoxin sorbents potential forhidden risksrdquo Toxicological Sciences vol 41 no 2 pp 175ndash1821998

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

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Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 ISRN Nutrition

Ages [11] It has been used in the treatment of inflammationand skin wounds [12] In the early Indian andArabic culturesas well as in ancient Greece and Rome C officinalis was usedas colourant for fabrics foods and cosmetics [13] NowadaysC officinalis is approved for food use in USA and appears inthe Food and Drug Administrationrsquos list of GRAS (GenerallyRecognized as Safe) substances It has a high economic valueas an herbal medicine and is widely used in cosmeticsperfumes pharmaceutical preparations and in food [11]C officinalis contains a high number of carotenoids suchas flavoxanthin lutein rubixanthin 120573-carotene g-caroteneand lycopene [14] It has been cited that 120573-carotene maywork synergistically with vitamin E [15] Lycopene anothercarotenoid has been found to have antioxidant antimi-crobial and antiproliferative properties Research suggeststhat it can be very protective against prostate cancer [16]C officinalis has been studied extensively for its beneficialeffects on humans Literature has shown that an herbal teamade from C officinalis could improve the symptoms ofcolitis duodenal ulcers and gastroduodenitis [16] Althoughconsiderable work has been done on C officinalis extractsno report is available on its role against the oxidative stressgenerated by natural toxicants Therefore the aims of thepresent study was to determine the total phenolic contentthe radical scavenging activity of the ethanolic and aqueousextract of Calendula in vitro and to evaluate the possiblehepatoprotective effects of the extract against oxidative stressinduced by aflatoxins in rats

2 Material and Methods

21 Chemicals and Kits Aflatoxins standards were purchasedfrom Sigma Chemical Co (St Louis MO USA) Alanineaminotransferase (ALT) aspartate aminotransferase (AST)glutathione peroxidase (GPx) and superoxide dismutase(SOD) were purchased from Randox (Antrim UK) Alkalinephosphatase (ALP) total protein (TP) albumin and creati-nine were purchased from QCA (AMPOSTA Spain) Ureawas purchased from Prodia (Korbach Germany) Lipid per-oxide formation was evaluated as malondialdehyde (MDA)and was purchased from Oxis Research Co (USA) Alphafetoprotein (AFP) was purchased fromMonobind Inc (LakeForest USA) Interleukin-1120573 (IL-1120573) and tumor necrosisfactor-alpha (TNF-alpha) were purchased from Orgenium(Helsinki Finland) All other chemicals were of the highestanalytical grade available

22 Aflatoxin Preparation Aflatoxins were produced viafermentation of rice by Aspergillus parasiticus NRRL 2999The fermented rice was autoclaved dried and ground to apowder and AFs content was measured by HPLC [17] Therice powder was incorporated into the basal diet to providethe desired level of 25mgkg diet The diet containing AFswas analyzed and the presence of parent AFs was confirmedand determined as mentioned above

23 PlantMaterial Calendula officinaliswas purchased fromthe local market at Cairo The plant was identified by the

Department of Medicinal Plants National Research Center(NRC) and the voucher was kept in the herbarium of NRC

24 Preparation of Calendula Extracts Dried and groundflowers and leaves of Calendula officinalis (50 g) were sub-jected to extraction with 400mL of ethanol (95) or distilledwater for 48 hrs The extracts were filtered and the ethanolextract was concentrated under the reduced pressure ofnitrogen and completely evaporated in a vacuum oven at atemperature not exceeding 40∘C until constant weights wereobtained The aqueous extract was dried using Freeze Dryersystem (Dura-Dry Freeze Dryer Model PAC-TC-V4 FTSsystem Inc Stone Ridge NY USA)

25 Determination of Total Phenolic Contents The concen-tration of phenolics in the extracts was determined usingthe method of Jayaprakasha and Rao [18] In brief 5mg ofeach extracts was dissolved in a 10mL mixture of acetoneand water (6 4 vv) Samples (02mL) were mixed with 1mLof 10-folds diluted Folin-Ciocalteu reagent and 08mL ofsodium carbonate solution (75) The absorbance was mea-sured at 765 nm using UV-160 IPC UV-visible spectropho-tometer (Shimadzu Japan) after 30min at room temperatureEstimation of phenolic compounds as catechin equivalents(CE) was carried out using standard curve of catechin [19]

26 Evaluation of Radical Scavenging Activity (RSA) by 11-Diphenyl 1-2-Picryl Hydrazyl (DPPH) Assay Crude extractswere dissolved in methanol to obtain a concentration of200 ppm and 02mL of this solution was completed to4mL by MeOH then 1mL of DPPH (609 times 10minus5molL)solution in the same solvent was then added The absorptionwas monitored after 10min at 516 nm The reference sample(Blank) was 1mL of DPPH solution and 4mL MeOH Thecapacity of antioxidants to quench DPPH radical was deter-mined according to Nogala-Kalucka et al [20] and calculatedaccording to the following equation

RSA = (Absorbance of control sample

minus absorbance of extract sample)

times (absorbance of control sample)minus1 times 100

(1)

27 Experimental Animals Three-month-old male SpragueDawley rats (100ndash150 g) were purchased from Animal HouseColony National Research Centre Dokki Cairo Egypt Ani-mals were maintained on standard lab diet (protein 1604fat 363 fiber 41 gkg of metabolizable energy 1208MJ)housed in filter-top polycarbonate cages in a room free fromany source of chemical contamination artificially illuminated(12 h darklight cycle) and thermally controlled (25 plusmn 1∘C) atthe Animal House Lab National Research Centre After anacclimatization period of 1 week the animals were dividedinto six groups (10 ratsgroup) and housed in filter-toppolycarbonate cages All animals received humane care incompliance with the guidelines of the Animal Care and UseCommittee of the National Research Center

ISRN Nutrition 3

Table 1 Yield total phenolic compounds and DPPH radicalscavenging activity of the ethanol of Calendula

Parameter Ethanolic extractYield (g100 g plant) 799 plusmn 049Total phenolic compounds (120583gg) 13600 plusmn 2188DPPH radical scavenging activity () 9630

28 Experimental Design Animals within different treat-ment groups were treated daily for 6 weeks and includedthe control the group fed AFs-contaminated diet (25mgkgdiet) the groups treated orally with the low (CE1) and high(CE2) doses of Calendula extract (500 and 1000mgkg bw)and the groups pretreated orally withCalendula extract at thetwo tested doses one week before and during AFs treatmentfor another five weeks At the end of the treatment periodall animals were fasted for 12 hr and blood samples werecollected from the retro-orbital venous plexus from eachanimal under ether anesthesia Blood samples were left toclot and the sera were separated using cooling centrifugationat 3000 rpm for 15min and stored at minus20∘C until analysisThe sera were used for the determination of ALT AST ALPtotal protein albumin urea creatinine AFP TNF-120572 and IL-1120573 according to the kits instructions

After the collection of blood samples all animals werekilled by cervical dislocation and sample of the liver wasweighed (approximately 005ndash01 g) and homogenized inphosphate buffer (pH 74) to give 20 wv homogenate Thishomogenate was centrifuged at 1700 rpm at 4∘C for 10minand the supernatant was stored at minus70∘C until analysis Thissupernatant was used for the assessment of GPx MDA andSOD according to the kits instructions Another sample ofeach liver was removed and placed in 10 of natural formalinfor histological and histochemical examinations [21]

29 Statistical Analysis All data were statistically analyzedby analysis of variance (ANOVA) using the General LinearModel Procedure of the Statistical Analysis System [22] Thesignificance of the differences among treatment groups wasdetermined byWaller-Duncan k-ratio [23] All statements ofsignificance were based on probability of 119875 le 005

3 Results

31 Total Phenolic Content and DPPH Scavenging ActivityThe results of total phenolic contents and DPPH scavengingactivity of the aqueous and ethanolic extracts of Calendulaextract are presented in Table 1 The results revealed that thetotal phenolic content of the ethanolic extract of Calendulawas higher than that in the aqueous extract Moreover theDPPH scavenging activity of the ethanolic extract showedhigher radical scavenging activity than the aqueous extractTherefore the ethanolic extract was used for the biologicalassay

32 The Biological Assay Along the study period no mortal-ity was observed in the groups treated with Calendula at the

0

5

10

15

20

25

30

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6

ControlAFsCE1

CE2

Food

inta

ke (g

)

AFs + CE1AFs + CE2

Figure 1 Effect of the treatment with ethanol extract of Calendulaon food intake of rats fed AFs-contaminated diet during the exper-imental period (AFs aflatoxins CE1 low dose of Calendula extract(500mgkg bw) CE2 high dose of Calendula extract (1000mgkgbw))

0

50

100

150

200

250

300

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6

Body

wei

ght (

g)

ControlAFsCE1

CE2AFs + CE1AFs + CE2

Figure 2 Effect of the treatment with ethanol extract of Calendulaon body weight of rats fed AFs-contaminated diet during theexperimental period (AFs aflatoxins CE1 low dose of Calendulaextract (500mgkg bw) CE2 high dose of Calendula extract(1000mgkg bw))

two tested doses either alone or one week before and duringfeeding AFs-contaminated diet Effect of different treatmentson the body weight changes is illustrated at Figure 1 Animalsfed AFs-contaminated diet showed a significant reductionof the body weight whereas animals treated with CE1 orCE2 were comparable to the control CE1 or CE2 succeededto normalize body weight when treated with animals fedAFs-contaminated diet The results indicate that animals fedAFs-contaminated diet demonstrated a significant decreaseof feed intake but animals treated with either CE1 or CE2were comparable to control Groups fed AFs-contaminateddiet and treated with CE1 or CE2 showed a significantimprovement of feed intake This result was pronounced inthe group treated with the high dose (Figure 2)

The effects of different treatments on serum biochemicalparameters are depicted in Table 2 These results indicatedthat animals fed AFs-contaminated diet showed a significantincrease in serum ALT AST ALP urea creatinine and AFPaccompanied with a significant decrease in total proteinand albumin Animals treated with the CE1 or CE2 were

4 ISRN Nutrition

Table 2 Effect of ethanol extract of Calendula on serum biochemical parameters in rats fed aflatoxins-contaminated diet

Parameter GroupsControl AFs CE1 CE2 CE1 + AFs CE2 + AFs

AST (UmL) 24832 plusmn 314a 33034 plusmn 422d 2394 plusmn 896b 24060 plusmn 642a 2830 plusmn 42c 25764 plusmn 324a

ALT (UmL) 7536 plusmn 137a 13251 plusmn 405c 732 plusmn 202a 7412 plusmn 269a 1092 plusmn 177b 7752 plusmn 133a

ALP (UL) 10675 plusmn 623a 23451 plusmn 532b 9367 plusmn 324a 10436 plusmn 333a 15671 plusmn 343c 11137 plusmn 261d

TP (mgdL) 999 plusmn 141a 435 plusmn 031b 923 plusmn 046a 1044 plusmn 161a 895 plusmn 027c 927 plusmn 023c

Albumin (mgdL) 344 plusmn 020a 188 plusmn 012b 311 plusmn 007a 337 plusmn 012a 307c plusmn 014 316 plusmn 022a

Urea (mgdL) 151 plusmn 014a 344 plusmn 031b 172 plusmn 013a 161 plusmn 013a 234 plusmn 012a 184 plusmn 032a

Creatinine (mgdL) 077 plusmn 021a 333 plusmn 014b 075 plusmn 013a 076 plusmn 014a 203 plusmn 002c 087 plusmn 013d

AFP (ngmL) 152 plusmn 078a 464 plusmn 022b 153 plusmn 017a 144 plusmn 016a 277 plusmn 031c 159 plusmn 023a

Within each row means superscript with different letters are significantly different at 119875 le 005AFs aflatoxinsCE1 low dose of calendula extract (500mgkg bw)CE2 high dose of calendula extract (1000mgkg bw)

Table 3 Effect of ethanol extract of calendula on liver antioxidants and lipid peroxidation in rats fed aflatoxins-contaminated diet

Parameter GroupControl AFs CE1 CE2 CE1 + AFs CE2 + AFs

SOD (Umg liver protein) 29937 plusmn 1033a 17232 plusmn 711b 30549 plusmn 2167a 31021 plusmn 832a 28157 plusmn 1504c 28532 plusmn 932a

GPx (Umg liver protein) 3366 plusmn 254a 1461 plusmn 120b 5403 plusmn 145c 5649 plusmn 212a 3561 plusmn 143a 3743 plusmn 156a

MDA (nmolmg liver protein) 3732 plusmn 212a 8431 plusmn 244b 2271 plusmn 228c 2031 plusmn 144a 4526 plusmn 129d 4232 plusmn 213a

Within each row means superscript with different letters are significantly different at 119875 le 005AFs aflatoxinsCE1 low dose of calendula extract (500mgkg bw)CE2 high dose of calendula extract (1000mgkg bw)

comparable to the controls in all biochemical parametersTreatment with the extract of the two tested dose one weekand during feeding AFs-contaminated diet succeeded toinduce a significant improvement of all biochemical parame-ters towards the control values

The existing results proved that AFs-contaminated dietresulted in a significant decrease in GPx and SOD activ-ities accompanied with a significant increase of MDAlevel (Table 3) However no significant effect was observedbetween those treated with CE1 or CE2 alone and the controlgroup Furthermore animals fed AFs-contaminated diet andtreated with CE1 or CE2 demonstrated a significant increaseof GPx and SOD activities with a significant decrease ofMDAlevel compared to the AFs-treated group The pronouncedimprovement has been showed at the high dose of extract(CA2) especially in SOD which was comparable to control

The effects of different treatments on inflammatorycytokine TNF-120572 and IL-1120573were illustrated in (Table 4) AFs-treated rats showed a significant increase of the inflammatorycytokines however animals treated with either CE1 or CE2were comparable to the controls Treatment with CE1 or CE2one week before and during AFs treatment succeeded toinduce a significant improvement in TNF-120572 and IL-1120573 in adose-dependent manner The higher dose demonstrated asignificant improvement of IL-1120573 toward the control level

The biochemical results obtained in the current studywere confirmed by the histopathological examination of theliver tissue Microscopic examination of the liver sectionof a control rat showed branching and anatomizing cords

radiating from the central vein with vesicular nuclei andsome binucleated cells and separated by sinusoids whichlined by flat endothelial cell and kupfer cells (Figure 3(a))The liver section of a rat fed AFs-contaminated diet showedthe disorganization and damage in hepatocytes architec-ture accompanied with hepatocytes necrosis apoptosis andfibrosis around the blood vessels (Figure 3(b)) The liversection of a rat treated with CE1 or CE2 showed hepatocyteswith normal architecture in the central and the portal veinsaccompanied with few aggregations of inflammatory cells in-between the hepatocytes (Figure 3(c))

The liver section of rats treated with CE1 one week beforeand during AFs-treatment showed marked improvement inhepatocytes architecture no vacuoles or fatty droplets weredetected Some cells have eosinophilic cytoplasm and deeplystained nuclei around the central and portal vein areas andmononuclear cellular infiltration are scattered around theportal tracts (Figure 3(d)) Moreover the liver section of ratsfed AFs-contaminated diet and treated with CE2 showednormal histological picture with few fatty degeneration andinterstitial hemorrhage around the central vein (Figure 3(e))

The microscopic examination of liver sections stainedwith Periodic-Sheif reagent stain (PAS) for glycogen demon-stration revealed that the control liver showed a strongreaction in the liver section (Figure 4(a))The liver of animalsfed AFs-contaminated diet showed a weak reaction in thedamaged cell while a strong reaction was noticed in intactcells (Figure 4(b)) The liver of the animals treated with CE1or CE2 showed different types of reaction strong reaction

ISRN Nutrition 5

Table 4 Effect of ethanol extract of calendula on serum inflammatory cytokine in rats fed aflatoxins-contaminated diet

Parameter GroupControl AFs CE1 CE2 CE1 + AFs CE2 + AFs

TNF-120572 (pgmL) 5834 plusmn 312a 13112 plusmn 332b 5837 plusmn 207a 6072 plusmn 192a 8647 plusmn 231c 6324 plusmn 166d

IL-1120573 (pgmL) 066 plusmn 007a 234 plusmn 012b 065 plusmn 004a 084 plusmn 022a 158 plusmn 004c 077 plusmn 021a

Within each row means superscript with different letters are significantly different at 119875 le 005AFs aflatoxinsCE1 low dose of calendula extract (500mgkg bw)CE2 high dose of calendula extract (1000mgkg bw)

CV

Hepatocytes

(HX and E times400)

(a)

FN

(HX and E times400)

(b)

Hepatocytes

Inf

CV

(HX and E times400)

(c)

CVPV

PV

(HX and E times200)

(d)

Fatty degeneration

(HX and E times400)

(e)

Figure 3 A photomicrograph in liver section from (a) a control rat branching and anatomizing cords radiating from the central vein CVThehepatocytes that are having vesicular nuclei and some binucleated cells are separated by sinusoids lined by flat endothelial cell and kupfer cells(b) a rat fed AFs-contaminated diet showing hepatocytes necrosis (N) and apoptosis (yellow arrows) and fibrosis around the blood vessels(F) (c) A rat treated with CE1 or CE2 showing the normal hepatocytes with vesicular nuclei and evident mononuclear cellular infiltrationaround the central vein (d) a rat fed AFs-contaminated diet and treated with CE1 showing marked improvement in hepatocytes architectureno vacuoles or fatty droplets were detected Some cells have eosinophilic cytoplasm and deeply stained nuclei around the central and portalvein areas (arrows) and mononuclear cellular infiltration are scattered around the portal tracts (e) A rat fed AFs-contaminated diet andtreated with CE2 showing normal histological picture with few fatty degeneration and interstitial hemorrhage around the central vein

around the central vein andweak reaction around portal veinof hepatocytes (Figure 4(c))The liver of the animals fed AFs-contaminated diet and treated with CE1 showed that weakreaction in some damaged cells which scattered in all thesection (Figure 4(d)) However the liver section of animalstreatedwithCE2 showedmarked improvement although lowreaction in hepatocytes was still demonstrated (Figure 4(e))

4 Discussion

Previous reports indicated that the type of extraction ofactive ingredient compounds from plant material dependsmainly on the type of solvent used [24] In the currentstudy the total phenolic compounds of the ethanol extractof Calendula were higher than that in the aqueous extract

However the ethanolic extract showed a higher DPPH (11-diphenyl 1-2-picryl hydrazyl) free radical scavenging activitycompared to the aqueous extract Cetkovic et al [25] showedsimilar scavenging potential against DPPH hydroxyl andperoxyl radicals and suggested that all of the extracts obtainedscavenged all radicals in concentration-dependant mannerMoreover Preethi et al [26] tested the antioxidant potentialof various leaves extracts of Calendula against DPPH ABTS(221015840-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) nitricoxide and superoxide radicals and reported lower scavengingproperty towards DPPH whereas they exerted higher radicalscavenging effects against the other radicals tested

In a similar study Danila et al [27] screened the waterand alcoholic extracts of Calendula grown in Romania for itsantioxidant activity and reported that Calendula contained

6 ISRN Nutrition

(a) (b)

(c) (d)

(e)

Figure 4 Photomicrographs in liver sections stained with Periodic-Sheif reagent-stain (PAS) for glycogen demonstration from (a)control rats showing a strong reaction of PAS (b) rats fed AFs-contaminated diet showing a weak reaction in the damaged cellwhile a strong reaction in intact cells (c) rats treated with CE1 orCE2 showing different types of reaction as well as strong reactionaround the central vein and weak reaction around portal vein ofhepatocytes (d) rats fed with AFs contaminated diet treated withCE1 showing that weak reaction in some damaged cells whichscattered in all the section and (e) rats fed with AFs contaminateddiet and treated with CE2 showing marked improvement althoughlow reaction in hepatocytes were still demonstrated (PAS reactiontimes100)

a high total phenol amount and scavenging activity againstDPPH and ABTS radicals DallrsquoAcqua et al [28] investigatedantioxidant activity of Calendula arvensis used in Sardinianfolk medicine using DPPH radical scavenging assay andreported that the extract showed a high radical scavengingeffect towards DPPH The radical scavenging propertieswere attributed to the presence of flavonoid and triterpenederivatives in the extract [29]

In the in vivo study the selective dose of AFs andCalendula was literature-based [30 31] respectively Theresults revealed a significant reduction of body weight andfood intake in animals fed AFs-contaminated diet Theseresults were in agreement with those reported in the literatureof aflatoxicosis [32ndash38] The reduction in body weight inthe animals fed AFs-contaminated diet alone may be due tothe effects of AFs on the balance between orexigenic andanorexigenic circuits that regulate the homeostatic loop ofbody weight regulation leading to cachexia [34] Moreover

AFs exposure may lead to significant reduction of leptin[35] accompanied with high levels of cortisol IL-6 andinsulin resistance which together act to influence the feedingresponse causing weight loss in patients with pancreaticcancer [36] Osborne et al [37] suggested that AFs ingestionaffect various digestive enzymatic activities that give riseto a malabsorption syndrome characterized by steatorrheahypocarotenoidemy and to lowering of bile pancreatic lipasetrypsin and amylase Furthermore Lesson et al [38] reportedthat the 89 epoxide metabolite of AFB

1may covalently bind

to DNA and proteins which then alters enzymatic processessuch as gluconeogenesis Krebs cycle or fatty acid synthesis

Animals fed AFs-contaminated diet showed a significantincrease of ALT AST ALP urea and createnine whichindicate changes in the hepatic tissues and biliary system [39]structural damaging of liver integrity since these enzymes arecytoplasmic in location and released into plasma as a result ofcellular damage [8 40] It is also likely that alteration in ALPactivity affects membrane permeability and the transport ofmetabolites Moreover increase in urea and createnine levelobserved in the current study in AFs-treated group clearlyindicated the harmful and stressful effect on renal tissue [30]Taken together the increased level of urea and the decreasedlevel of albumin and total protein (TP) indicate the inhibitionof protein synthesis and increase of protein catabolism andorrenal dysfunction [41 42]These results clearly indicated thatAFs had stressful effects on the hepatic and renal tissuesconsistent with those reported in the literature of aflatoxicosis[43 44]

Animals treated with AFs showed a significant increasein AFP which is considered specific biomarkers for livercancer This increase in AFP may be due to induction ofexpression of mRNAs of liver alpha-fetoprotein [45 46] Onthe other hand aflatoxin B

1-induced hepatocarcinogenesis

associated with defective DNA-damage response by passingp53 activation [47] and modulation of insulin-like growthfactor 2 dependent signal axis (IGF-2) [48] Similar to theseobservations Sell et al [49] and Yang et al [45] reported thatAFB1administration resulted in the elevation of serum AFP

level in both duck and ratsThe current study showed that animals fed AFs-con-

taminated diet suffer from oxidative stress as indicated bythe significant increment of lipid peroxidation (MDA) andthe significant reduction of enzymatic antioxidant such assuperoxide dismutase (SOD) and glutathione peroxidase(GPx) These results are in agreement with previous studiessuggested that oxidative stress may be due to direct effectof AFs or by the metabolites formed and the free radicalsgenerated during the formation of these metabolites [3446 50] Moreover the reduction of protein synthesis inAFs-treated animals may affect certain metal ions (ie ironand copper) which play an important role in free radicalproduction and liberation [10] In normal state metal ionsare bonded to transfer proteins such as ceruloplasmin andtransferrin [51] and play a crucial role of SOD CAT and GPxactivities which constitute the enzymatic antioxidant defensesystem of the cell displayed bidirectional alterations either inthe form of increase or decrease depending on the particulartissue Previous studies demonstrated that the mechanism of

ISRN Nutrition 7

AFs-induced liver injury may be due to those reactive andtoxic metabolites of AFs and the liver necrosis begins whenthe glutathione stores are almost exhausted [34 35 52]

Tumor necrosis factor-alpha (TNF-120572) and interleukin-1 alpha (IL-1120572) are produced by macrophages and theyplay an important role in tumor conditions [35] TNF-120572is an essential factor in tumor promotion [53] and IL-1polymorphisms are important mediators in the inflamma-tory process [54] In the current study ingestion of AFs-contaminated diet significantly increased TNF-120572 and IL-1120573 suggesting that AFs preferentially affects macrophagefunctions In particular it decouples the close correlationsusually observed between transcriptional and translationalcontrols of IL-1120572 and TNF-120572 production by these cells [55]In this concern Barton et al [56] stated that TNF-120572 plays acausal role in the development of liver injury and it plays amajor role in modulating mycotoxin-induced hepatotoxicityMoreover TNF-120572 has been proven to play an importantrole in inflammation in mediating the proliferation anddifferentiation of immune cells and development of immuneresponse [57] Hopkins [55] and Abdel-Wahhab et al [35]reported that directly proportional relationship has beenobserved between transcriptional and translational controlsof IL-1 and TNF-120572 production by hepatocytes Consequentlythe current results indicated that aflatoxicosis may induceTNF-120572 and IL-1 due to activation of caspase-3 which is oneof the apoptotic mediator [58] and the significant elevationof inflammatory cytokinemay affect themRNA expression ofthese mediators [46]

The current results indicated that the ethanolic extract ofCalendula showed a potential protective effect against AFstoxicity Animals treated withCalendula extract did not showany sign of toxicity In this concern the safety of the ethanolextract of Calendula was suggested and the oral LD

50in rats

was estimated by 4640mgkg bw [59] The results reportedherein indicated that animals treated with Calendula extractshowed insignificant increase in food intake and body weightgain Moreover Calendula extract succeeded to induce asignificant improvement in the biochemical parameters andthe histological and histochemical pictures Taken togetherthe results of the in vitro study and the in vivo evaluationsuggested that there are an antioxidant and free radicalscavenging properties of this extract and this improvementmore pronounced with the high dose (CA2) [29] In thisconcern previous study suggested that Calendula plays animportant role in hepaticcytolysis inhibition and improvesliver integrity in CCl

4intoxicated rats [60] In referring to

previous literature hepatoprotective activity of Calendulamay be due to its modulatory effect on cytochrome P450which may interfere on aflatoxin active metabolite produc-tion [61]MoreoverCalendula extract showed renoprotectiveaction against cisplatin-induced renal toxicity through itsmodulatory effect against myelosuppression of the renalsystem

The existing data revealed anticarcinogenicity of Cal-endula extract indicated by significant reduction of AFPand this effect was pronounced in the animals treatedwith the high dose (CA2) Several reports suggested thatthe anticancer properties of Calendula may be due to its

chemoprotector properties against hepatocarcinogenesis inrats beside antitumoural activity in vivo in the Ehrlichmouse carcinoma model due to its ability to stimulate T-lymphocytes B-lymphocytes and cluster of differentiation(CD4+) [62] Moreover Calendula plays a crucial role inprevention of DNA damaging [63] These results were inagreement with those reported previously [26 31 62]

The results also suggested that Calendula extract pos-sesses anti-inflammatory activity which may be due to itshigher content of carvacol Carvacol has been described asa promoter for liver regeneration and inhibitor of TNF-120572and IL-6 level in rats undergoing partial hepatectomy [64]Furthermore Tsai et al [65] reported that caravacl couldreduce TNF-120572 and IL-1120573 levels in intoxicated rats through theinhibition of cycloxygensae (COX) enzymes activity mRNAexpression and its protein in lipopoly saccharides-inducedinflammation

The histopathological and histochemical changes in liverconfirmed the biochemical results and revealed that ani-mals fed AFs-contaminated diet showed severe histologicalchanges typical to those reported in the literature [10 39 66]However animals treated with the extract in combinationwith AFs resulted in a significant improvement in liver tissuessimilar to that reported previously [61]

5 Conclusion

It can be concluded that the ethanolic and aqueous extracts ofCalendula officinalis have a valuable quantity of total phenoliccompounds and have DPPH radical scavenging activityMoreover the ethanolic extract showed higher phenoliccontent and radical scavenging property compared to theaqueous extract The ethanolic extract exhibited hepatoreno-protective properties against aflatoxin-induced liver injury ina dose-dependent manner due to its antioxidant free radicalscavenging activities and anti-inflammatory properties

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

This work was full supported by College of Pharmacy MisrUniversity for Science and Technology 6th October CityEgypt Faculty of Pharmacy Cairo University Cairo EgyptNational Research Center Dokki Cairo Egypt

References

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[2] J A Nogueira S K Ono-Nita M E Nita et al ldquo249 TP53mutation has high prevalence and is correlated with larger andpoorly differentiated HCC in Brazilian patientsrdquo BMC Cancervol 9 article 204 2009

8 ISRN Nutrition

[3] S Rawal J E Kim and R Coulombe Jr ldquoAflatoxin B1in

poultry toxicology metabolism and preventionrdquo Research inVeterinary Science vol 89 no 3 pp 325ndash331 2010

[4] International Agency for Research in Carcinogenesis ldquoAfla-toxinsrdquo Monograph on the Evaluation of Carcinogenic Risks toHumans vol 56 p 245 1993

[5] IPCS-WHO ldquoAflatoxinsrdquo in International Programme onChem-ical Safety WHO Food Additives Series 40 pp 1ndash73 WorldHealth Organization Geneva Switzerland 1998

[6] J H Williams T D Phillips P E Jolly J K Stiles C M JollyandDAggarwal ldquoHuman aflatoxicosis in developing countriesareview of toxicology exposure potential health consequencesand interventionsrdquo American Journal of Clinical Nutrition vol80 no 5 pp 1106ndash1122 2004

[7] K C Choi W T Chung J K Kwon et al ldquoInhibitory effectsof quercetin on aflatoxin B1-induced hepatic damage in micerdquoFood and Chemical Toxicology vol 48 no 10 pp 2747ndash27532010

[8] D S El-Agamy ldquoComparative effects of curcumin and resver-atrol on aflatoxin B 1-induced liver injury in ratsrdquo Archives ofToxicology vol 84 no 5 pp 389ndash396 2010

[9] M A Abdel-Wahhab A A Ibrahim A A El-Nekeety NS Hassan and A A Mohamed ldquoPanax ginseng CA Meyerextract counteracts the oxidative stress in rats fed multi-mycotoxins-contaminated dietrdquo Comunicata Scientiae vol 3no 3 pp 143ndash153 2012

[10] M A Hamzawy E S El-Denshary N S Hassan F A Mannaaand M A Abdel-Wahhab ldquoAntioxidant and hepatorenoprotec-tive effect of thyme vulgaris extract in rats during aflatoxicosisrdquoGlobal Journal Pharmacology vol 6 no 2 pp 106ndash117 2012

[11] Z C Gazim C M Rezende S R Fraga T I E Svidzinskiand D A G Cortez ldquoAntifungal activity of the essential oilfrom Calendula officinalis L (Asteraceae) growing in BrazilrdquoBrazilian Journal of Microbiology vol 39 no 1 pp 61ndash63 2008

[12] L R Della A Tubaro S Sosa H Becker S Saar and D IsaacldquoThe role of triperpenoids in the topical antiinflamatory activityofCalendula officinalisflowersrdquoPlantaMedica vol 60 no 6 pp516ndash520 1994

[13] Chrysolite 2010 httphealthwikinutcomCalendula-officin-alis-Pot-Marigold11v4g8pw

[14] A Pintea C Bele S Andrei and C Socaciu ldquoHPLC analysis ofcarotenoids in four varieties of Calendula officinalis L flowersrdquoActa Biologica Szegediensis vol 247 pp 37ndash40 2003

[15] R A Jacob ldquoThe integrated antioxidant systemrdquo NutritionResearch vol 15 no 5 pp 755ndash766 1995

[16] K Dahan M Fennal and N B Kumar ldquoLycopene in the pre-vention of prostate cancerrdquo Journal of the Society for IntegrativeOncology vol 6 no 1 pp 29ndash36 2008

[17] J E Hutchins andWMHagler ldquoRapid liquid chromatographicdetermination of aflatoxins in heavily contaminated cornrdquoJournal of the Association of Official Analytical Chemists vol 66no 6 pp 1458ndash1465 1983

[18] G K Jayaprakasha and L J Rao ldquoPhenolic constituents fromthe lichen parmotrema stuppeum (Nyl) Hale and their antioxi-dant activityrdquo Zeitschrift fur Naturforschung C vol 55 no 11-12pp 1018ndash1022 2000

[19] G K Jayaprakasha T Selvi and K K Sakariah ldquoAntibacterialand antioxidant activities of grape (Vitis vinifera) seed extractsrdquoFood Research International vol 36 no 2 pp 117ndash122 2003

[20] M Nogala-Kalucka J Korczak M Dratwia E Lampart-Szczapa A Siger and M Buchowski ldquoChanges in antioxidant

activity and free radical scavenging potential of rosemaryextract and tocopherols in isolated rapeseed oil triacylglycerolsduring accelerated testsrdquo Food Chemistry vol 93 no 2 pp 227ndash235 2005

[21] R A Drury E A Wallington and R Cancerson CarltonrsquosHistopathological Techniques Oxford University Press OxfordUK 4th edition 1980

[22] SAS IInstitute SAS Userrsquos Guide Statistics SAS Institute CaryNC USA

[23] R A Waller and D B Duncan ldquoA Bayes rule for the symmetricmultiple comparisons problemsrdquo Journal of American StatisticalAssociation vol 64 no 328 pp 1484ndash1503 1969

[24] L Majhenic M Skerget and Z Knez ldquoAntioxidant and antimi-crobial activity of guarana seed extractsrdquo Food Chemistry vol104 no 3 pp 1258ndash1268 2007

[25] G S Cetkovic S M Djilas J M Canadanovic-Brunet and VT Tumbas ldquoAntioxidant properties of marigold extractsrdquo FoodResearch International vol 37 no 7 pp 643ndash650 2004

[26] K C Preethi G Kuttan and R Kuttan ldquoAntioxidant potentialof an extract ofCalendula officinalis flowers in vitro and in vivordquoPharmaceutical Biology vol 44 no 9 pp 691ndash697 2006

[27] AODanila F Gatea andG L Radu ldquoPolyphenol compositionand antioxidant activity of selected medicinal herbsrdquoChemistryof Natural Compounds vol 47 no 1 pp 22ndash26 2011

[28] S DallrsquoAcqua R Cervellati M C Loi and G InnocentildquoEvaluation of in vitro antioxidant properties of some tradi-tional Sardinian medicinal plants investigation of the highantioxidant capacity of Rubus ulmifoliusrdquo Food Chemistry vol106 no 2 pp 745ndash749 2008

[29] T Ercetin F S Senol I Erdogan Orhan and G TokerldquoComparative assessment of antioxidant and cholinesteraseinhibitory properties of the marigold extracts from Calendulaarvensis L and Calendula officinalis Lrdquo Industrial Crops andProducts vol 36 no 1 pp 203ndash208 2012

[30] M A Abdel-Wahhab and S E Aly ldquoAntioxidants and radicalscavenging properties of vegetable extracts in rats fed aflatoxin-contaminated dietrdquo Journal of Agricultural and Food Chemistryvol 51 no 8 pp 2409ndash2414 2003

[31] C Gladine C Morand E Rock D Gruffat D Bauchart andD Durand ldquoThe antioxidative effect of plant extracts rich inpolyphenols differs between liver and muscle tissues in rats fedn-3 PUFA rich dietsrdquo Animal Feed Science and Technology vol139 no 3-4 pp 257ndash272 2007

[32] R H Rauber P Dilkin L Z Giacomini C A Araujo deAlmeida and C A Mallmann ldquoPerformance of Turkey poultsfed different doses of aflatoxins in the dietrdquo Poultry Science vol86 no 8 pp 1620ndash1624 2007

[33] SM RustemeyerW R Lamberson D R Ledoux et al ldquoEffectsof dietary aflatoxin on the health and performance of growingbarrowsrdquo Journal of Animal Science vol 88 no 11 pp 3624ndash3630 2010

[34] M A Abdel-Wahhab N S Hassan A A El-Kady et al ldquoRedginseng extract protects against aflatoxin B

1and fumonisins-

induced hepatic pre-cancerous lesions in ratsrdquo Food and Chem-ical Toxicology vol 48 no 2 pp 733ndash742 2010

[35] M A Abdel-Wahhab H H Ahmed and M M HagazildquoPrevention of aflatoxin B1-initiated hepatotoxicity in rat bymarine algae extractsrdquo Journal of Applied Toxicology vol 26 no3 pp 229ndash238 2006

[36] M D Barber D C McMillan A M Wallace J A RossT Preston and K C H Fearon ldquoThe response of leptin

ISRN Nutrition 9

interleukin-6 and fat oxidation to feeding in weight-losingpatients with pancreatic cancerrdquo British Journal of Cancer vol90 no 6 pp 1129ndash1132 2004

[37] D J Osborne W E Huff P B Hamilton and H R BurmeisterldquoComparison of ochratoxin aflatoxin and T-2 toxin for theireffects on selected parameters related to digestion and evidencefor specific metabolism of carotenoids in chickensrdquo PoultryScience vol 61 no 8 pp 1646ndash1652 1982

[38] S Lesson G Diaz and J Summers Poultry Metabolic Disordersand Mycotoxins University Books Montreal Canada 1995

[39] M A Abdel-Wahhab S A Nada and F A Khalil ldquoPhys-iological and toxicological responses in rats fed aflatoxin-contaminated diet with or without sorbent materialsrdquo AnimalFeed Science and Technology vol 97 no 3-4 pp 209ndash219 2002

[40] A A El-Nekeety S R Mohamed A S Hathout N S HassanS E Aly and M A Abdel-Wahhab ldquoAntioxidant properties ofThymus vulgaris oil against aflatoxin-induce oxidative stress inmale ratsrdquo Toxicon vol 57 no 7-8 pp 984ndash991 2011

[41] N Jindal S K Mahipal and N K Mahajan ldquoToxicity ofaflatoxin B1 in broiler chicks and its reduction by activatedcharcoalrdquo Research in Veterinary Science vol 56 no 1 pp 37ndash40 1994

[42] M A Abdel-Wahhab and S E Aly ldquoAntioxidant property ofNigella sativa (black cumin) and Syzygium aromaticum (clove)in rats during aflatoxicosisrdquo Journal of Applied Toxicology vol25 no 3 pp 218ndash223 2005

[43] D M Miller and D M Wilson Veterinary Diseases Relatedto Aflatoxins the Toxicology of Aflatoxins Academic Press SanDiego Calif USA 1994

[44] M A Abdel-Wahhab and A M Kholif ldquoMycotoxins in animalfeeds and prevention strategies a reviewrdquo Asian Journal ofAnimal Sciences vol 2 no 1 pp 7ndash25 2008

[45] C F Yang J Liu S Wasser H M Shen C E L Tan andC N Ong ldquoInhibition of ebselen on aflatoxin B1-inducedhepatocarcinogenesis in Fischer 344 ratsrdquo Carcinogenesis vol21 no 12 pp 2237ndash2243 2000

[46] S H Abdel-Aziem A M Hassan and M A Abdel-WahhabldquoDietary supplementation with whey protein and ginsengextract counteracts oxidative stress and DNA damage in ratsfed an aflatoxin-contaminated dietrdquoMutationResearch vol 723no 1 pp 65ndash71 2011

[47] O Gursoy-Yuzugullu H YuzugulluM Yilmaz andMOzturkldquoAflatoxin genotoxicity is associated with a defective DNAdamage response bypassing p53 activationrdquo Liver Internationalvol 31 no 4 pp 561ndash571 2011

[48] T Ubagai T Kikuchi T Fukusato and Y Ono ldquoAflatoxin B1

modulates the insulin-like growth factor-2 dependent signalingaxisrdquo Toxicology in Vitro vol 24 no 3 pp 783ndash789 2010

[49] S Sell K L Xu W E Huff L F Kabena R B Harveryand H A Dunsford ldquoAflatoxin exposure produces serumalphafetoprotein elevations and marked oval cell proliferationin young male Pekin ducklingsrdquo Pathology vol 30 no 1 pp34ndash39 1998

[50] M Kanbur G Eraslan Z S Sarica andO Aslan ldquoThe effects ofevening primrose oil on lipid peroxidation induced by subacuteaflatoxin exposure in micerdquo Food and Chemical Toxicology vol49 no 9 pp 1960ndash1964 2011

[51] E Gitto S Pellegrino P Gitto I Barberi and R J ReiterldquoOxidative stress of the newborn in the pre- and postnatalperiod and the clinical utility of melatoninrdquo Journal of PinealResearch vol 46 no 2 pp 128ndash139 2009

[52] M A Abdel-Wahhab A Said and A Huefner ldquoNMR andradical scavenging activities of patuletin from Urtica urensagainst aflatoxin B

1rdquo Pharmaceutical Biology vol 43 no 6 pp

515ndash525 2005[53] M Suganuma E Sueoka N Sueoka S Okabe and H Fujiki

ldquoMechanisms of cancer prevention by tea polyphenols based oninhibition of TNF-120572 expressionrdquo BioFactors vol 13 no 1-4 pp67ndash72 2000

[54] S Rollinson A P Levene F K Mensah et al ldquoGastric marginalzone lymphoma is associated with polymorphisms in genesinvolved in inflammatory response and antioxidative capacityrdquoBlood vol 102 no 3 pp 1007ndash1011 2003

[55] S J Hopkins ldquoThe pathophysiological role of cytokinesrdquo LegalMedicine vol 5 no 1 pp S45ndashS57 2003

[56] C C Barton E X Barton P E Ganey S L Kunkel and RA Roth ldquoBacterial lipopolysaccharide enhances aflatoxin B

1

hepatotoxicity in rats by a mechanism that depends on tumornecrosis factor 120572rdquo Hepatology vol 33 no 1 pp 66ndash73 2001

[57] J Cao L Jiang X Zhang et al ldquoBoric acid inhibits LPS-inducedTNF-120572 formation through a thiol-dependent mechanism inTHP-1 cellsrdquo Journal of Trace Elements in Medicine and Biologyvol 22 no 3 pp 189ndash195 2008

[58] A R M A Meik S K Abdel-Ghaffar and I El-GibalyldquoAflatoxin B1 induces apoptosis in rat liver protective effect ofmelatoninrdquo Neuroendocrinology Letters vol 22 no 6 pp 417ndash426 2001

[59] CIREP ldquoFinal report on the safety assessment ofCalendula offic-inalis Extract and Calendula officinalisrdquo International JournalToxicology vol 20 pp 13ndash20 2001

[60] M A Rusu M Tamas C Puica I Roman and M SabadasldquoThe hepatoprotective action of ten herbal extracts in CCl

4

intoxicated liverrdquo Phytotherapy Research vol 19 no 9 pp 744ndash749 2005

[61] K C Preethi and R Kuttan ldquoHepato and reno protective actionof Calendula officinalis L Flower extractrdquo Indian Journal ofExperimental Biology vol 47 no 3 pp 163ndash168 2009

[62] B P Muley S S Khadabadi and N B Banarase ldquoPhytochem-ical constituents and pharmacological activities of Calendulaofficinalis Linn (Asteraceae) a reviewrdquo Tropical Journal ofPharmaceutical Research vol 8 no 5 pp 455ndash465 2009

[63] T Frankic K Salobir and J Salobir ldquoThe comparison of invivo antigenotoxic and antioxidative capacity of two propyleneglycol extracts of Calendula officinalis (marigold) and vitaminE in young growing pigsrdquo Journal of Animal Physiology andAnimal Nutrition vol 93 no 6 pp 688ndash694 2009

[64] M Uyanoglu M Canbek E Aral and K Husnu Can BaserldquoEffects of carvacrol upon the liver of rats undergoing partialhepatectomyrdquo Phytomedicine vol 15 no 3 pp 226ndash229 2008

[65] M L Tsai C C Lin W C Lin and C H Yang ldquoAntimicrobialantioxidant and anti-inflammatory activities of essential oilsfromfive selected herbsrdquoBioscience Biotechnology Biochemistryvol 75 no 10 pp 1977ndash1983 2011

[66] K Mayura M A Abdel-Mahhab K S McKenzie et alldquoPrevention of maternal and developmental toxicity in rats viadietary inclusion of common aflatoxin sorbents potential forhidden risksrdquo Toxicological Sciences vol 41 no 2 pp 175ndash1821998

Submit your manuscripts athttpwwwhindawicom

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ISRN Nutrition 3

Table 1 Yield total phenolic compounds and DPPH radicalscavenging activity of the ethanol of Calendula

Parameter Ethanolic extractYield (g100 g plant) 799 plusmn 049Total phenolic compounds (120583gg) 13600 plusmn 2188DPPH radical scavenging activity () 9630

28 Experimental Design Animals within different treat-ment groups were treated daily for 6 weeks and includedthe control the group fed AFs-contaminated diet (25mgkgdiet) the groups treated orally with the low (CE1) and high(CE2) doses of Calendula extract (500 and 1000mgkg bw)and the groups pretreated orally withCalendula extract at thetwo tested doses one week before and during AFs treatmentfor another five weeks At the end of the treatment periodall animals were fasted for 12 hr and blood samples werecollected from the retro-orbital venous plexus from eachanimal under ether anesthesia Blood samples were left toclot and the sera were separated using cooling centrifugationat 3000 rpm for 15min and stored at minus20∘C until analysisThe sera were used for the determination of ALT AST ALPtotal protein albumin urea creatinine AFP TNF-120572 and IL-1120573 according to the kits instructions

After the collection of blood samples all animals werekilled by cervical dislocation and sample of the liver wasweighed (approximately 005ndash01 g) and homogenized inphosphate buffer (pH 74) to give 20 wv homogenate Thishomogenate was centrifuged at 1700 rpm at 4∘C for 10minand the supernatant was stored at minus70∘C until analysis Thissupernatant was used for the assessment of GPx MDA andSOD according to the kits instructions Another sample ofeach liver was removed and placed in 10 of natural formalinfor histological and histochemical examinations [21]

29 Statistical Analysis All data were statistically analyzedby analysis of variance (ANOVA) using the General LinearModel Procedure of the Statistical Analysis System [22] Thesignificance of the differences among treatment groups wasdetermined byWaller-Duncan k-ratio [23] All statements ofsignificance were based on probability of 119875 le 005

3 Results

31 Total Phenolic Content and DPPH Scavenging ActivityThe results of total phenolic contents and DPPH scavengingactivity of the aqueous and ethanolic extracts of Calendulaextract are presented in Table 1 The results revealed that thetotal phenolic content of the ethanolic extract of Calendulawas higher than that in the aqueous extract Moreover theDPPH scavenging activity of the ethanolic extract showedhigher radical scavenging activity than the aqueous extractTherefore the ethanolic extract was used for the biologicalassay

32 The Biological Assay Along the study period no mortal-ity was observed in the groups treated with Calendula at the

0

5

10

15

20

25

30

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6

ControlAFsCE1

CE2

Food

inta

ke (g

)

AFs + CE1AFs + CE2

Figure 1 Effect of the treatment with ethanol extract of Calendulaon food intake of rats fed AFs-contaminated diet during the exper-imental period (AFs aflatoxins CE1 low dose of Calendula extract(500mgkg bw) CE2 high dose of Calendula extract (1000mgkgbw))

0

50

100

150

200

250

300

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6

Body

wei

ght (

g)

ControlAFsCE1

CE2AFs + CE1AFs + CE2

Figure 2 Effect of the treatment with ethanol extract of Calendulaon body weight of rats fed AFs-contaminated diet during theexperimental period (AFs aflatoxins CE1 low dose of Calendulaextract (500mgkg bw) CE2 high dose of Calendula extract(1000mgkg bw))

two tested doses either alone or one week before and duringfeeding AFs-contaminated diet Effect of different treatmentson the body weight changes is illustrated at Figure 1 Animalsfed AFs-contaminated diet showed a significant reductionof the body weight whereas animals treated with CE1 orCE2 were comparable to the control CE1 or CE2 succeededto normalize body weight when treated with animals fedAFs-contaminated diet The results indicate that animals fedAFs-contaminated diet demonstrated a significant decreaseof feed intake but animals treated with either CE1 or CE2were comparable to control Groups fed AFs-contaminateddiet and treated with CE1 or CE2 showed a significantimprovement of feed intake This result was pronounced inthe group treated with the high dose (Figure 2)

The effects of different treatments on serum biochemicalparameters are depicted in Table 2 These results indicatedthat animals fed AFs-contaminated diet showed a significantincrease in serum ALT AST ALP urea creatinine and AFPaccompanied with a significant decrease in total proteinand albumin Animals treated with the CE1 or CE2 were

4 ISRN Nutrition

Table 2 Effect of ethanol extract of Calendula on serum biochemical parameters in rats fed aflatoxins-contaminated diet

Parameter GroupsControl AFs CE1 CE2 CE1 + AFs CE2 + AFs

AST (UmL) 24832 plusmn 314a 33034 plusmn 422d 2394 plusmn 896b 24060 plusmn 642a 2830 plusmn 42c 25764 plusmn 324a

ALT (UmL) 7536 plusmn 137a 13251 plusmn 405c 732 plusmn 202a 7412 plusmn 269a 1092 plusmn 177b 7752 plusmn 133a

ALP (UL) 10675 plusmn 623a 23451 plusmn 532b 9367 plusmn 324a 10436 plusmn 333a 15671 plusmn 343c 11137 plusmn 261d

TP (mgdL) 999 plusmn 141a 435 plusmn 031b 923 plusmn 046a 1044 plusmn 161a 895 plusmn 027c 927 plusmn 023c

Albumin (mgdL) 344 plusmn 020a 188 plusmn 012b 311 plusmn 007a 337 plusmn 012a 307c plusmn 014 316 plusmn 022a

Urea (mgdL) 151 plusmn 014a 344 plusmn 031b 172 plusmn 013a 161 plusmn 013a 234 plusmn 012a 184 plusmn 032a

Creatinine (mgdL) 077 plusmn 021a 333 plusmn 014b 075 plusmn 013a 076 plusmn 014a 203 plusmn 002c 087 plusmn 013d

AFP (ngmL) 152 plusmn 078a 464 plusmn 022b 153 plusmn 017a 144 plusmn 016a 277 plusmn 031c 159 plusmn 023a

Within each row means superscript with different letters are significantly different at 119875 le 005AFs aflatoxinsCE1 low dose of calendula extract (500mgkg bw)CE2 high dose of calendula extract (1000mgkg bw)

Table 3 Effect of ethanol extract of calendula on liver antioxidants and lipid peroxidation in rats fed aflatoxins-contaminated diet

Parameter GroupControl AFs CE1 CE2 CE1 + AFs CE2 + AFs

SOD (Umg liver protein) 29937 plusmn 1033a 17232 plusmn 711b 30549 plusmn 2167a 31021 plusmn 832a 28157 plusmn 1504c 28532 plusmn 932a

GPx (Umg liver protein) 3366 plusmn 254a 1461 plusmn 120b 5403 plusmn 145c 5649 plusmn 212a 3561 plusmn 143a 3743 plusmn 156a

MDA (nmolmg liver protein) 3732 plusmn 212a 8431 plusmn 244b 2271 plusmn 228c 2031 plusmn 144a 4526 plusmn 129d 4232 plusmn 213a

Within each row means superscript with different letters are significantly different at 119875 le 005AFs aflatoxinsCE1 low dose of calendula extract (500mgkg bw)CE2 high dose of calendula extract (1000mgkg bw)

comparable to the controls in all biochemical parametersTreatment with the extract of the two tested dose one weekand during feeding AFs-contaminated diet succeeded toinduce a significant improvement of all biochemical parame-ters towards the control values

The existing results proved that AFs-contaminated dietresulted in a significant decrease in GPx and SOD activ-ities accompanied with a significant increase of MDAlevel (Table 3) However no significant effect was observedbetween those treated with CE1 or CE2 alone and the controlgroup Furthermore animals fed AFs-contaminated diet andtreated with CE1 or CE2 demonstrated a significant increaseof GPx and SOD activities with a significant decrease ofMDAlevel compared to the AFs-treated group The pronouncedimprovement has been showed at the high dose of extract(CA2) especially in SOD which was comparable to control

The effects of different treatments on inflammatorycytokine TNF-120572 and IL-1120573were illustrated in (Table 4) AFs-treated rats showed a significant increase of the inflammatorycytokines however animals treated with either CE1 or CE2were comparable to the controls Treatment with CE1 or CE2one week before and during AFs treatment succeeded toinduce a significant improvement in TNF-120572 and IL-1120573 in adose-dependent manner The higher dose demonstrated asignificant improvement of IL-1120573 toward the control level

The biochemical results obtained in the current studywere confirmed by the histopathological examination of theliver tissue Microscopic examination of the liver sectionof a control rat showed branching and anatomizing cords

radiating from the central vein with vesicular nuclei andsome binucleated cells and separated by sinusoids whichlined by flat endothelial cell and kupfer cells (Figure 3(a))The liver section of a rat fed AFs-contaminated diet showedthe disorganization and damage in hepatocytes architec-ture accompanied with hepatocytes necrosis apoptosis andfibrosis around the blood vessels (Figure 3(b)) The liversection of a rat treated with CE1 or CE2 showed hepatocyteswith normal architecture in the central and the portal veinsaccompanied with few aggregations of inflammatory cells in-between the hepatocytes (Figure 3(c))

The liver section of rats treated with CE1 one week beforeand during AFs-treatment showed marked improvement inhepatocytes architecture no vacuoles or fatty droplets weredetected Some cells have eosinophilic cytoplasm and deeplystained nuclei around the central and portal vein areas andmononuclear cellular infiltration are scattered around theportal tracts (Figure 3(d)) Moreover the liver section of ratsfed AFs-contaminated diet and treated with CE2 showednormal histological picture with few fatty degeneration andinterstitial hemorrhage around the central vein (Figure 3(e))

The microscopic examination of liver sections stainedwith Periodic-Sheif reagent stain (PAS) for glycogen demon-stration revealed that the control liver showed a strongreaction in the liver section (Figure 4(a))The liver of animalsfed AFs-contaminated diet showed a weak reaction in thedamaged cell while a strong reaction was noticed in intactcells (Figure 4(b)) The liver of the animals treated with CE1or CE2 showed different types of reaction strong reaction

ISRN Nutrition 5

Table 4 Effect of ethanol extract of calendula on serum inflammatory cytokine in rats fed aflatoxins-contaminated diet

Parameter GroupControl AFs CE1 CE2 CE1 + AFs CE2 + AFs

TNF-120572 (pgmL) 5834 plusmn 312a 13112 plusmn 332b 5837 plusmn 207a 6072 plusmn 192a 8647 plusmn 231c 6324 plusmn 166d

IL-1120573 (pgmL) 066 plusmn 007a 234 plusmn 012b 065 plusmn 004a 084 plusmn 022a 158 plusmn 004c 077 plusmn 021a

Within each row means superscript with different letters are significantly different at 119875 le 005AFs aflatoxinsCE1 low dose of calendula extract (500mgkg bw)CE2 high dose of calendula extract (1000mgkg bw)

CV

Hepatocytes

(HX and E times400)

(a)

FN

(HX and E times400)

(b)

Hepatocytes

Inf

CV

(HX and E times400)

(c)

CVPV

PV

(HX and E times200)

(d)

Fatty degeneration

(HX and E times400)

(e)

Figure 3 A photomicrograph in liver section from (a) a control rat branching and anatomizing cords radiating from the central vein CVThehepatocytes that are having vesicular nuclei and some binucleated cells are separated by sinusoids lined by flat endothelial cell and kupfer cells(b) a rat fed AFs-contaminated diet showing hepatocytes necrosis (N) and apoptosis (yellow arrows) and fibrosis around the blood vessels(F) (c) A rat treated with CE1 or CE2 showing the normal hepatocytes with vesicular nuclei and evident mononuclear cellular infiltrationaround the central vein (d) a rat fed AFs-contaminated diet and treated with CE1 showing marked improvement in hepatocytes architectureno vacuoles or fatty droplets were detected Some cells have eosinophilic cytoplasm and deeply stained nuclei around the central and portalvein areas (arrows) and mononuclear cellular infiltration are scattered around the portal tracts (e) A rat fed AFs-contaminated diet andtreated with CE2 showing normal histological picture with few fatty degeneration and interstitial hemorrhage around the central vein

around the central vein andweak reaction around portal veinof hepatocytes (Figure 4(c))The liver of the animals fed AFs-contaminated diet and treated with CE1 showed that weakreaction in some damaged cells which scattered in all thesection (Figure 4(d)) However the liver section of animalstreatedwithCE2 showedmarked improvement although lowreaction in hepatocytes was still demonstrated (Figure 4(e))

4 Discussion

Previous reports indicated that the type of extraction ofactive ingredient compounds from plant material dependsmainly on the type of solvent used [24] In the currentstudy the total phenolic compounds of the ethanol extractof Calendula were higher than that in the aqueous extract

However the ethanolic extract showed a higher DPPH (11-diphenyl 1-2-picryl hydrazyl) free radical scavenging activitycompared to the aqueous extract Cetkovic et al [25] showedsimilar scavenging potential against DPPH hydroxyl andperoxyl radicals and suggested that all of the extracts obtainedscavenged all radicals in concentration-dependant mannerMoreover Preethi et al [26] tested the antioxidant potentialof various leaves extracts of Calendula against DPPH ABTS(221015840-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) nitricoxide and superoxide radicals and reported lower scavengingproperty towards DPPH whereas they exerted higher radicalscavenging effects against the other radicals tested

In a similar study Danila et al [27] screened the waterand alcoholic extracts of Calendula grown in Romania for itsantioxidant activity and reported that Calendula contained

6 ISRN Nutrition

(a) (b)

(c) (d)

(e)

Figure 4 Photomicrographs in liver sections stained with Periodic-Sheif reagent-stain (PAS) for glycogen demonstration from (a)control rats showing a strong reaction of PAS (b) rats fed AFs-contaminated diet showing a weak reaction in the damaged cellwhile a strong reaction in intact cells (c) rats treated with CE1 orCE2 showing different types of reaction as well as strong reactionaround the central vein and weak reaction around portal vein ofhepatocytes (d) rats fed with AFs contaminated diet treated withCE1 showing that weak reaction in some damaged cells whichscattered in all the section and (e) rats fed with AFs contaminateddiet and treated with CE2 showing marked improvement althoughlow reaction in hepatocytes were still demonstrated (PAS reactiontimes100)

a high total phenol amount and scavenging activity againstDPPH and ABTS radicals DallrsquoAcqua et al [28] investigatedantioxidant activity of Calendula arvensis used in Sardinianfolk medicine using DPPH radical scavenging assay andreported that the extract showed a high radical scavengingeffect towards DPPH The radical scavenging propertieswere attributed to the presence of flavonoid and triterpenederivatives in the extract [29]

In the in vivo study the selective dose of AFs andCalendula was literature-based [30 31] respectively Theresults revealed a significant reduction of body weight andfood intake in animals fed AFs-contaminated diet Theseresults were in agreement with those reported in the literatureof aflatoxicosis [32ndash38] The reduction in body weight inthe animals fed AFs-contaminated diet alone may be due tothe effects of AFs on the balance between orexigenic andanorexigenic circuits that regulate the homeostatic loop ofbody weight regulation leading to cachexia [34] Moreover

AFs exposure may lead to significant reduction of leptin[35] accompanied with high levels of cortisol IL-6 andinsulin resistance which together act to influence the feedingresponse causing weight loss in patients with pancreaticcancer [36] Osborne et al [37] suggested that AFs ingestionaffect various digestive enzymatic activities that give riseto a malabsorption syndrome characterized by steatorrheahypocarotenoidemy and to lowering of bile pancreatic lipasetrypsin and amylase Furthermore Lesson et al [38] reportedthat the 89 epoxide metabolite of AFB

1may covalently bind

to DNA and proteins which then alters enzymatic processessuch as gluconeogenesis Krebs cycle or fatty acid synthesis

Animals fed AFs-contaminated diet showed a significantincrease of ALT AST ALP urea and createnine whichindicate changes in the hepatic tissues and biliary system [39]structural damaging of liver integrity since these enzymes arecytoplasmic in location and released into plasma as a result ofcellular damage [8 40] It is also likely that alteration in ALPactivity affects membrane permeability and the transport ofmetabolites Moreover increase in urea and createnine levelobserved in the current study in AFs-treated group clearlyindicated the harmful and stressful effect on renal tissue [30]Taken together the increased level of urea and the decreasedlevel of albumin and total protein (TP) indicate the inhibitionof protein synthesis and increase of protein catabolism andorrenal dysfunction [41 42]These results clearly indicated thatAFs had stressful effects on the hepatic and renal tissuesconsistent with those reported in the literature of aflatoxicosis[43 44]

Animals treated with AFs showed a significant increasein AFP which is considered specific biomarkers for livercancer This increase in AFP may be due to induction ofexpression of mRNAs of liver alpha-fetoprotein [45 46] Onthe other hand aflatoxin B

1-induced hepatocarcinogenesis

associated with defective DNA-damage response by passingp53 activation [47] and modulation of insulin-like growthfactor 2 dependent signal axis (IGF-2) [48] Similar to theseobservations Sell et al [49] and Yang et al [45] reported thatAFB1administration resulted in the elevation of serum AFP

level in both duck and ratsThe current study showed that animals fed AFs-con-

taminated diet suffer from oxidative stress as indicated bythe significant increment of lipid peroxidation (MDA) andthe significant reduction of enzymatic antioxidant such assuperoxide dismutase (SOD) and glutathione peroxidase(GPx) These results are in agreement with previous studiessuggested that oxidative stress may be due to direct effectof AFs or by the metabolites formed and the free radicalsgenerated during the formation of these metabolites [3446 50] Moreover the reduction of protein synthesis inAFs-treated animals may affect certain metal ions (ie ironand copper) which play an important role in free radicalproduction and liberation [10] In normal state metal ionsare bonded to transfer proteins such as ceruloplasmin andtransferrin [51] and play a crucial role of SOD CAT and GPxactivities which constitute the enzymatic antioxidant defensesystem of the cell displayed bidirectional alterations either inthe form of increase or decrease depending on the particulartissue Previous studies demonstrated that the mechanism of

ISRN Nutrition 7

AFs-induced liver injury may be due to those reactive andtoxic metabolites of AFs and the liver necrosis begins whenthe glutathione stores are almost exhausted [34 35 52]

Tumor necrosis factor-alpha (TNF-120572) and interleukin-1 alpha (IL-1120572) are produced by macrophages and theyplay an important role in tumor conditions [35] TNF-120572is an essential factor in tumor promotion [53] and IL-1polymorphisms are important mediators in the inflamma-tory process [54] In the current study ingestion of AFs-contaminated diet significantly increased TNF-120572 and IL-1120573 suggesting that AFs preferentially affects macrophagefunctions In particular it decouples the close correlationsusually observed between transcriptional and translationalcontrols of IL-1120572 and TNF-120572 production by these cells [55]In this concern Barton et al [56] stated that TNF-120572 plays acausal role in the development of liver injury and it plays amajor role in modulating mycotoxin-induced hepatotoxicityMoreover TNF-120572 has been proven to play an importantrole in inflammation in mediating the proliferation anddifferentiation of immune cells and development of immuneresponse [57] Hopkins [55] and Abdel-Wahhab et al [35]reported that directly proportional relationship has beenobserved between transcriptional and translational controlsof IL-1 and TNF-120572 production by hepatocytes Consequentlythe current results indicated that aflatoxicosis may induceTNF-120572 and IL-1 due to activation of caspase-3 which is oneof the apoptotic mediator [58] and the significant elevationof inflammatory cytokinemay affect themRNA expression ofthese mediators [46]

The current results indicated that the ethanolic extract ofCalendula showed a potential protective effect against AFstoxicity Animals treated withCalendula extract did not showany sign of toxicity In this concern the safety of the ethanolextract of Calendula was suggested and the oral LD

50in rats

was estimated by 4640mgkg bw [59] The results reportedherein indicated that animals treated with Calendula extractshowed insignificant increase in food intake and body weightgain Moreover Calendula extract succeeded to induce asignificant improvement in the biochemical parameters andthe histological and histochemical pictures Taken togetherthe results of the in vitro study and the in vivo evaluationsuggested that there are an antioxidant and free radicalscavenging properties of this extract and this improvementmore pronounced with the high dose (CA2) [29] In thisconcern previous study suggested that Calendula plays animportant role in hepaticcytolysis inhibition and improvesliver integrity in CCl

4intoxicated rats [60] In referring to

previous literature hepatoprotective activity of Calendulamay be due to its modulatory effect on cytochrome P450which may interfere on aflatoxin active metabolite produc-tion [61]MoreoverCalendula extract showed renoprotectiveaction against cisplatin-induced renal toxicity through itsmodulatory effect against myelosuppression of the renalsystem

The existing data revealed anticarcinogenicity of Cal-endula extract indicated by significant reduction of AFPand this effect was pronounced in the animals treatedwith the high dose (CA2) Several reports suggested thatthe anticancer properties of Calendula may be due to its

chemoprotector properties against hepatocarcinogenesis inrats beside antitumoural activity in vivo in the Ehrlichmouse carcinoma model due to its ability to stimulate T-lymphocytes B-lymphocytes and cluster of differentiation(CD4+) [62] Moreover Calendula plays a crucial role inprevention of DNA damaging [63] These results were inagreement with those reported previously [26 31 62]

The results also suggested that Calendula extract pos-sesses anti-inflammatory activity which may be due to itshigher content of carvacol Carvacol has been described asa promoter for liver regeneration and inhibitor of TNF-120572and IL-6 level in rats undergoing partial hepatectomy [64]Furthermore Tsai et al [65] reported that caravacl couldreduce TNF-120572 and IL-1120573 levels in intoxicated rats through theinhibition of cycloxygensae (COX) enzymes activity mRNAexpression and its protein in lipopoly saccharides-inducedinflammation

The histopathological and histochemical changes in liverconfirmed the biochemical results and revealed that ani-mals fed AFs-contaminated diet showed severe histologicalchanges typical to those reported in the literature [10 39 66]However animals treated with the extract in combinationwith AFs resulted in a significant improvement in liver tissuessimilar to that reported previously [61]

5 Conclusion

It can be concluded that the ethanolic and aqueous extracts ofCalendula officinalis have a valuable quantity of total phenoliccompounds and have DPPH radical scavenging activityMoreover the ethanolic extract showed higher phenoliccontent and radical scavenging property compared to theaqueous extract The ethanolic extract exhibited hepatoreno-protective properties against aflatoxin-induced liver injury ina dose-dependent manner due to its antioxidant free radicalscavenging activities and anti-inflammatory properties

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

This work was full supported by College of Pharmacy MisrUniversity for Science and Technology 6th October CityEgypt Faculty of Pharmacy Cairo University Cairo EgyptNational Research Center Dokki Cairo Egypt

References

[1] CAST ldquoMycotoxins risks in plant animal and humanrdquo inPotential Economic Costs ofMycotoxins inUnited States pp 136ndash142 CAST Task Force Reports 2003

[2] J A Nogueira S K Ono-Nita M E Nita et al ldquo249 TP53mutation has high prevalence and is correlated with larger andpoorly differentiated HCC in Brazilian patientsrdquo BMC Cancervol 9 article 204 2009

8 ISRN Nutrition

[3] S Rawal J E Kim and R Coulombe Jr ldquoAflatoxin B1in

poultry toxicology metabolism and preventionrdquo Research inVeterinary Science vol 89 no 3 pp 325ndash331 2010

[4] International Agency for Research in Carcinogenesis ldquoAfla-toxinsrdquo Monograph on the Evaluation of Carcinogenic Risks toHumans vol 56 p 245 1993

[5] IPCS-WHO ldquoAflatoxinsrdquo in International Programme onChem-ical Safety WHO Food Additives Series 40 pp 1ndash73 WorldHealth Organization Geneva Switzerland 1998

[6] J H Williams T D Phillips P E Jolly J K Stiles C M JollyandDAggarwal ldquoHuman aflatoxicosis in developing countriesareview of toxicology exposure potential health consequencesand interventionsrdquo American Journal of Clinical Nutrition vol80 no 5 pp 1106ndash1122 2004

[7] K C Choi W T Chung J K Kwon et al ldquoInhibitory effectsof quercetin on aflatoxin B1-induced hepatic damage in micerdquoFood and Chemical Toxicology vol 48 no 10 pp 2747ndash27532010

[8] D S El-Agamy ldquoComparative effects of curcumin and resver-atrol on aflatoxin B 1-induced liver injury in ratsrdquo Archives ofToxicology vol 84 no 5 pp 389ndash396 2010

[9] M A Abdel-Wahhab A A Ibrahim A A El-Nekeety NS Hassan and A A Mohamed ldquoPanax ginseng CA Meyerextract counteracts the oxidative stress in rats fed multi-mycotoxins-contaminated dietrdquo Comunicata Scientiae vol 3no 3 pp 143ndash153 2012

[10] M A Hamzawy E S El-Denshary N S Hassan F A Mannaaand M A Abdel-Wahhab ldquoAntioxidant and hepatorenoprotec-tive effect of thyme vulgaris extract in rats during aflatoxicosisrdquoGlobal Journal Pharmacology vol 6 no 2 pp 106ndash117 2012

[11] Z C Gazim C M Rezende S R Fraga T I E Svidzinskiand D A G Cortez ldquoAntifungal activity of the essential oilfrom Calendula officinalis L (Asteraceae) growing in BrazilrdquoBrazilian Journal of Microbiology vol 39 no 1 pp 61ndash63 2008

[12] L R Della A Tubaro S Sosa H Becker S Saar and D IsaacldquoThe role of triperpenoids in the topical antiinflamatory activityofCalendula officinalisflowersrdquoPlantaMedica vol 60 no 6 pp516ndash520 1994

[13] Chrysolite 2010 httphealthwikinutcomCalendula-officin-alis-Pot-Marigold11v4g8pw

[14] A Pintea C Bele S Andrei and C Socaciu ldquoHPLC analysis ofcarotenoids in four varieties of Calendula officinalis L flowersrdquoActa Biologica Szegediensis vol 247 pp 37ndash40 2003

[15] R A Jacob ldquoThe integrated antioxidant systemrdquo NutritionResearch vol 15 no 5 pp 755ndash766 1995

[16] K Dahan M Fennal and N B Kumar ldquoLycopene in the pre-vention of prostate cancerrdquo Journal of the Society for IntegrativeOncology vol 6 no 1 pp 29ndash36 2008

[17] J E Hutchins andWMHagler ldquoRapid liquid chromatographicdetermination of aflatoxins in heavily contaminated cornrdquoJournal of the Association of Official Analytical Chemists vol 66no 6 pp 1458ndash1465 1983

[18] G K Jayaprakasha and L J Rao ldquoPhenolic constituents fromthe lichen parmotrema stuppeum (Nyl) Hale and their antioxi-dant activityrdquo Zeitschrift fur Naturforschung C vol 55 no 11-12pp 1018ndash1022 2000

[19] G K Jayaprakasha T Selvi and K K Sakariah ldquoAntibacterialand antioxidant activities of grape (Vitis vinifera) seed extractsrdquoFood Research International vol 36 no 2 pp 117ndash122 2003

[20] M Nogala-Kalucka J Korczak M Dratwia E Lampart-Szczapa A Siger and M Buchowski ldquoChanges in antioxidant

activity and free radical scavenging potential of rosemaryextract and tocopherols in isolated rapeseed oil triacylglycerolsduring accelerated testsrdquo Food Chemistry vol 93 no 2 pp 227ndash235 2005

[21] R A Drury E A Wallington and R Cancerson CarltonrsquosHistopathological Techniques Oxford University Press OxfordUK 4th edition 1980

[22] SAS IInstitute SAS Userrsquos Guide Statistics SAS Institute CaryNC USA

[23] R A Waller and D B Duncan ldquoA Bayes rule for the symmetricmultiple comparisons problemsrdquo Journal of American StatisticalAssociation vol 64 no 328 pp 1484ndash1503 1969

[24] L Majhenic M Skerget and Z Knez ldquoAntioxidant and antimi-crobial activity of guarana seed extractsrdquo Food Chemistry vol104 no 3 pp 1258ndash1268 2007

[25] G S Cetkovic S M Djilas J M Canadanovic-Brunet and VT Tumbas ldquoAntioxidant properties of marigold extractsrdquo FoodResearch International vol 37 no 7 pp 643ndash650 2004

[26] K C Preethi G Kuttan and R Kuttan ldquoAntioxidant potentialof an extract ofCalendula officinalis flowers in vitro and in vivordquoPharmaceutical Biology vol 44 no 9 pp 691ndash697 2006

[27] AODanila F Gatea andG L Radu ldquoPolyphenol compositionand antioxidant activity of selected medicinal herbsrdquoChemistryof Natural Compounds vol 47 no 1 pp 22ndash26 2011

[28] S DallrsquoAcqua R Cervellati M C Loi and G InnocentildquoEvaluation of in vitro antioxidant properties of some tradi-tional Sardinian medicinal plants investigation of the highantioxidant capacity of Rubus ulmifoliusrdquo Food Chemistry vol106 no 2 pp 745ndash749 2008

[29] T Ercetin F S Senol I Erdogan Orhan and G TokerldquoComparative assessment of antioxidant and cholinesteraseinhibitory properties of the marigold extracts from Calendulaarvensis L and Calendula officinalis Lrdquo Industrial Crops andProducts vol 36 no 1 pp 203ndash208 2012

[30] M A Abdel-Wahhab and S E Aly ldquoAntioxidants and radicalscavenging properties of vegetable extracts in rats fed aflatoxin-contaminated dietrdquo Journal of Agricultural and Food Chemistryvol 51 no 8 pp 2409ndash2414 2003

[31] C Gladine C Morand E Rock D Gruffat D Bauchart andD Durand ldquoThe antioxidative effect of plant extracts rich inpolyphenols differs between liver and muscle tissues in rats fedn-3 PUFA rich dietsrdquo Animal Feed Science and Technology vol139 no 3-4 pp 257ndash272 2007

[32] R H Rauber P Dilkin L Z Giacomini C A Araujo deAlmeida and C A Mallmann ldquoPerformance of Turkey poultsfed different doses of aflatoxins in the dietrdquo Poultry Science vol86 no 8 pp 1620ndash1624 2007

[33] SM RustemeyerW R Lamberson D R Ledoux et al ldquoEffectsof dietary aflatoxin on the health and performance of growingbarrowsrdquo Journal of Animal Science vol 88 no 11 pp 3624ndash3630 2010

[34] M A Abdel-Wahhab N S Hassan A A El-Kady et al ldquoRedginseng extract protects against aflatoxin B

1and fumonisins-

induced hepatic pre-cancerous lesions in ratsrdquo Food and Chem-ical Toxicology vol 48 no 2 pp 733ndash742 2010

[35] M A Abdel-Wahhab H H Ahmed and M M HagazildquoPrevention of aflatoxin B1-initiated hepatotoxicity in rat bymarine algae extractsrdquo Journal of Applied Toxicology vol 26 no3 pp 229ndash238 2006

[36] M D Barber D C McMillan A M Wallace J A RossT Preston and K C H Fearon ldquoThe response of leptin

ISRN Nutrition 9

interleukin-6 and fat oxidation to feeding in weight-losingpatients with pancreatic cancerrdquo British Journal of Cancer vol90 no 6 pp 1129ndash1132 2004

[37] D J Osborne W E Huff P B Hamilton and H R BurmeisterldquoComparison of ochratoxin aflatoxin and T-2 toxin for theireffects on selected parameters related to digestion and evidencefor specific metabolism of carotenoids in chickensrdquo PoultryScience vol 61 no 8 pp 1646ndash1652 1982

[38] S Lesson G Diaz and J Summers Poultry Metabolic Disordersand Mycotoxins University Books Montreal Canada 1995

[39] M A Abdel-Wahhab S A Nada and F A Khalil ldquoPhys-iological and toxicological responses in rats fed aflatoxin-contaminated diet with or without sorbent materialsrdquo AnimalFeed Science and Technology vol 97 no 3-4 pp 209ndash219 2002

[40] A A El-Nekeety S R Mohamed A S Hathout N S HassanS E Aly and M A Abdel-Wahhab ldquoAntioxidant properties ofThymus vulgaris oil against aflatoxin-induce oxidative stress inmale ratsrdquo Toxicon vol 57 no 7-8 pp 984ndash991 2011

[41] N Jindal S K Mahipal and N K Mahajan ldquoToxicity ofaflatoxin B1 in broiler chicks and its reduction by activatedcharcoalrdquo Research in Veterinary Science vol 56 no 1 pp 37ndash40 1994

[42] M A Abdel-Wahhab and S E Aly ldquoAntioxidant property ofNigella sativa (black cumin) and Syzygium aromaticum (clove)in rats during aflatoxicosisrdquo Journal of Applied Toxicology vol25 no 3 pp 218ndash223 2005

[43] D M Miller and D M Wilson Veterinary Diseases Relatedto Aflatoxins the Toxicology of Aflatoxins Academic Press SanDiego Calif USA 1994

[44] M A Abdel-Wahhab and A M Kholif ldquoMycotoxins in animalfeeds and prevention strategies a reviewrdquo Asian Journal ofAnimal Sciences vol 2 no 1 pp 7ndash25 2008

[45] C F Yang J Liu S Wasser H M Shen C E L Tan andC N Ong ldquoInhibition of ebselen on aflatoxin B1-inducedhepatocarcinogenesis in Fischer 344 ratsrdquo Carcinogenesis vol21 no 12 pp 2237ndash2243 2000

[46] S H Abdel-Aziem A M Hassan and M A Abdel-WahhabldquoDietary supplementation with whey protein and ginsengextract counteracts oxidative stress and DNA damage in ratsfed an aflatoxin-contaminated dietrdquoMutationResearch vol 723no 1 pp 65ndash71 2011

[47] O Gursoy-Yuzugullu H YuzugulluM Yilmaz andMOzturkldquoAflatoxin genotoxicity is associated with a defective DNAdamage response bypassing p53 activationrdquo Liver Internationalvol 31 no 4 pp 561ndash571 2011

[48] T Ubagai T Kikuchi T Fukusato and Y Ono ldquoAflatoxin B1

modulates the insulin-like growth factor-2 dependent signalingaxisrdquo Toxicology in Vitro vol 24 no 3 pp 783ndash789 2010

[49] S Sell K L Xu W E Huff L F Kabena R B Harveryand H A Dunsford ldquoAflatoxin exposure produces serumalphafetoprotein elevations and marked oval cell proliferationin young male Pekin ducklingsrdquo Pathology vol 30 no 1 pp34ndash39 1998

[50] M Kanbur G Eraslan Z S Sarica andO Aslan ldquoThe effects ofevening primrose oil on lipid peroxidation induced by subacuteaflatoxin exposure in micerdquo Food and Chemical Toxicology vol49 no 9 pp 1960ndash1964 2011

[51] E Gitto S Pellegrino P Gitto I Barberi and R J ReiterldquoOxidative stress of the newborn in the pre- and postnatalperiod and the clinical utility of melatoninrdquo Journal of PinealResearch vol 46 no 2 pp 128ndash139 2009

[52] M A Abdel-Wahhab A Said and A Huefner ldquoNMR andradical scavenging activities of patuletin from Urtica urensagainst aflatoxin B

1rdquo Pharmaceutical Biology vol 43 no 6 pp

515ndash525 2005[53] M Suganuma E Sueoka N Sueoka S Okabe and H Fujiki

ldquoMechanisms of cancer prevention by tea polyphenols based oninhibition of TNF-120572 expressionrdquo BioFactors vol 13 no 1-4 pp67ndash72 2000

[54] S Rollinson A P Levene F K Mensah et al ldquoGastric marginalzone lymphoma is associated with polymorphisms in genesinvolved in inflammatory response and antioxidative capacityrdquoBlood vol 102 no 3 pp 1007ndash1011 2003

[55] S J Hopkins ldquoThe pathophysiological role of cytokinesrdquo LegalMedicine vol 5 no 1 pp S45ndashS57 2003

[56] C C Barton E X Barton P E Ganey S L Kunkel and RA Roth ldquoBacterial lipopolysaccharide enhances aflatoxin B

1

hepatotoxicity in rats by a mechanism that depends on tumornecrosis factor 120572rdquo Hepatology vol 33 no 1 pp 66ndash73 2001

[57] J Cao L Jiang X Zhang et al ldquoBoric acid inhibits LPS-inducedTNF-120572 formation through a thiol-dependent mechanism inTHP-1 cellsrdquo Journal of Trace Elements in Medicine and Biologyvol 22 no 3 pp 189ndash195 2008

[58] A R M A Meik S K Abdel-Ghaffar and I El-GibalyldquoAflatoxin B1 induces apoptosis in rat liver protective effect ofmelatoninrdquo Neuroendocrinology Letters vol 22 no 6 pp 417ndash426 2001

[59] CIREP ldquoFinal report on the safety assessment ofCalendula offic-inalis Extract and Calendula officinalisrdquo International JournalToxicology vol 20 pp 13ndash20 2001

[60] M A Rusu M Tamas C Puica I Roman and M SabadasldquoThe hepatoprotective action of ten herbal extracts in CCl

4

intoxicated liverrdquo Phytotherapy Research vol 19 no 9 pp 744ndash749 2005

[61] K C Preethi and R Kuttan ldquoHepato and reno protective actionof Calendula officinalis L Flower extractrdquo Indian Journal ofExperimental Biology vol 47 no 3 pp 163ndash168 2009

[62] B P Muley S S Khadabadi and N B Banarase ldquoPhytochem-ical constituents and pharmacological activities of Calendulaofficinalis Linn (Asteraceae) a reviewrdquo Tropical Journal ofPharmaceutical Research vol 8 no 5 pp 455ndash465 2009

[63] T Frankic K Salobir and J Salobir ldquoThe comparison of invivo antigenotoxic and antioxidative capacity of two propyleneglycol extracts of Calendula officinalis (marigold) and vitaminE in young growing pigsrdquo Journal of Animal Physiology andAnimal Nutrition vol 93 no 6 pp 688ndash694 2009

[64] M Uyanoglu M Canbek E Aral and K Husnu Can BaserldquoEffects of carvacrol upon the liver of rats undergoing partialhepatectomyrdquo Phytomedicine vol 15 no 3 pp 226ndash229 2008

[65] M L Tsai C C Lin W C Lin and C H Yang ldquoAntimicrobialantioxidant and anti-inflammatory activities of essential oilsfromfive selected herbsrdquoBioscience Biotechnology Biochemistryvol 75 no 10 pp 1977ndash1983 2011

[66] K Mayura M A Abdel-Mahhab K S McKenzie et alldquoPrevention of maternal and developmental toxicity in rats viadietary inclusion of common aflatoxin sorbents potential forhidden risksrdquo Toxicological Sciences vol 41 no 2 pp 175ndash1821998

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Research and TreatmentAIDS

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Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 ISRN Nutrition

Table 2 Effect of ethanol extract of Calendula on serum biochemical parameters in rats fed aflatoxins-contaminated diet

Parameter GroupsControl AFs CE1 CE2 CE1 + AFs CE2 + AFs

AST (UmL) 24832 plusmn 314a 33034 plusmn 422d 2394 plusmn 896b 24060 plusmn 642a 2830 plusmn 42c 25764 plusmn 324a

ALT (UmL) 7536 plusmn 137a 13251 plusmn 405c 732 plusmn 202a 7412 plusmn 269a 1092 plusmn 177b 7752 plusmn 133a

ALP (UL) 10675 plusmn 623a 23451 plusmn 532b 9367 plusmn 324a 10436 plusmn 333a 15671 plusmn 343c 11137 plusmn 261d

TP (mgdL) 999 plusmn 141a 435 plusmn 031b 923 plusmn 046a 1044 plusmn 161a 895 plusmn 027c 927 plusmn 023c

Albumin (mgdL) 344 plusmn 020a 188 plusmn 012b 311 plusmn 007a 337 plusmn 012a 307c plusmn 014 316 plusmn 022a

Urea (mgdL) 151 plusmn 014a 344 plusmn 031b 172 plusmn 013a 161 plusmn 013a 234 plusmn 012a 184 plusmn 032a

Creatinine (mgdL) 077 plusmn 021a 333 plusmn 014b 075 plusmn 013a 076 plusmn 014a 203 plusmn 002c 087 plusmn 013d

AFP (ngmL) 152 plusmn 078a 464 plusmn 022b 153 plusmn 017a 144 plusmn 016a 277 plusmn 031c 159 plusmn 023a

Within each row means superscript with different letters are significantly different at 119875 le 005AFs aflatoxinsCE1 low dose of calendula extract (500mgkg bw)CE2 high dose of calendula extract (1000mgkg bw)

Table 3 Effect of ethanol extract of calendula on liver antioxidants and lipid peroxidation in rats fed aflatoxins-contaminated diet

Parameter GroupControl AFs CE1 CE2 CE1 + AFs CE2 + AFs

SOD (Umg liver protein) 29937 plusmn 1033a 17232 plusmn 711b 30549 plusmn 2167a 31021 plusmn 832a 28157 plusmn 1504c 28532 plusmn 932a

GPx (Umg liver protein) 3366 plusmn 254a 1461 plusmn 120b 5403 plusmn 145c 5649 plusmn 212a 3561 plusmn 143a 3743 plusmn 156a

MDA (nmolmg liver protein) 3732 plusmn 212a 8431 plusmn 244b 2271 plusmn 228c 2031 plusmn 144a 4526 plusmn 129d 4232 plusmn 213a

Within each row means superscript with different letters are significantly different at 119875 le 005AFs aflatoxinsCE1 low dose of calendula extract (500mgkg bw)CE2 high dose of calendula extract (1000mgkg bw)

comparable to the controls in all biochemical parametersTreatment with the extract of the two tested dose one weekand during feeding AFs-contaminated diet succeeded toinduce a significant improvement of all biochemical parame-ters towards the control values

The existing results proved that AFs-contaminated dietresulted in a significant decrease in GPx and SOD activ-ities accompanied with a significant increase of MDAlevel (Table 3) However no significant effect was observedbetween those treated with CE1 or CE2 alone and the controlgroup Furthermore animals fed AFs-contaminated diet andtreated with CE1 or CE2 demonstrated a significant increaseof GPx and SOD activities with a significant decrease ofMDAlevel compared to the AFs-treated group The pronouncedimprovement has been showed at the high dose of extract(CA2) especially in SOD which was comparable to control

The effects of different treatments on inflammatorycytokine TNF-120572 and IL-1120573were illustrated in (Table 4) AFs-treated rats showed a significant increase of the inflammatorycytokines however animals treated with either CE1 or CE2were comparable to the controls Treatment with CE1 or CE2one week before and during AFs treatment succeeded toinduce a significant improvement in TNF-120572 and IL-1120573 in adose-dependent manner The higher dose demonstrated asignificant improvement of IL-1120573 toward the control level

The biochemical results obtained in the current studywere confirmed by the histopathological examination of theliver tissue Microscopic examination of the liver sectionof a control rat showed branching and anatomizing cords

radiating from the central vein with vesicular nuclei andsome binucleated cells and separated by sinusoids whichlined by flat endothelial cell and kupfer cells (Figure 3(a))The liver section of a rat fed AFs-contaminated diet showedthe disorganization and damage in hepatocytes architec-ture accompanied with hepatocytes necrosis apoptosis andfibrosis around the blood vessels (Figure 3(b)) The liversection of a rat treated with CE1 or CE2 showed hepatocyteswith normal architecture in the central and the portal veinsaccompanied with few aggregations of inflammatory cells in-between the hepatocytes (Figure 3(c))

The liver section of rats treated with CE1 one week beforeand during AFs-treatment showed marked improvement inhepatocytes architecture no vacuoles or fatty droplets weredetected Some cells have eosinophilic cytoplasm and deeplystained nuclei around the central and portal vein areas andmononuclear cellular infiltration are scattered around theportal tracts (Figure 3(d)) Moreover the liver section of ratsfed AFs-contaminated diet and treated with CE2 showednormal histological picture with few fatty degeneration andinterstitial hemorrhage around the central vein (Figure 3(e))

The microscopic examination of liver sections stainedwith Periodic-Sheif reagent stain (PAS) for glycogen demon-stration revealed that the control liver showed a strongreaction in the liver section (Figure 4(a))The liver of animalsfed AFs-contaminated diet showed a weak reaction in thedamaged cell while a strong reaction was noticed in intactcells (Figure 4(b)) The liver of the animals treated with CE1or CE2 showed different types of reaction strong reaction

ISRN Nutrition 5

Table 4 Effect of ethanol extract of calendula on serum inflammatory cytokine in rats fed aflatoxins-contaminated diet

Parameter GroupControl AFs CE1 CE2 CE1 + AFs CE2 + AFs

TNF-120572 (pgmL) 5834 plusmn 312a 13112 plusmn 332b 5837 plusmn 207a 6072 plusmn 192a 8647 plusmn 231c 6324 plusmn 166d

IL-1120573 (pgmL) 066 plusmn 007a 234 plusmn 012b 065 plusmn 004a 084 plusmn 022a 158 plusmn 004c 077 plusmn 021a

Within each row means superscript with different letters are significantly different at 119875 le 005AFs aflatoxinsCE1 low dose of calendula extract (500mgkg bw)CE2 high dose of calendula extract (1000mgkg bw)

CV

Hepatocytes

(HX and E times400)

(a)

FN

(HX and E times400)

(b)

Hepatocytes

Inf

CV

(HX and E times400)

(c)

CVPV

PV

(HX and E times200)

(d)

Fatty degeneration

(HX and E times400)

(e)

Figure 3 A photomicrograph in liver section from (a) a control rat branching and anatomizing cords radiating from the central vein CVThehepatocytes that are having vesicular nuclei and some binucleated cells are separated by sinusoids lined by flat endothelial cell and kupfer cells(b) a rat fed AFs-contaminated diet showing hepatocytes necrosis (N) and apoptosis (yellow arrows) and fibrosis around the blood vessels(F) (c) A rat treated with CE1 or CE2 showing the normal hepatocytes with vesicular nuclei and evident mononuclear cellular infiltrationaround the central vein (d) a rat fed AFs-contaminated diet and treated with CE1 showing marked improvement in hepatocytes architectureno vacuoles or fatty droplets were detected Some cells have eosinophilic cytoplasm and deeply stained nuclei around the central and portalvein areas (arrows) and mononuclear cellular infiltration are scattered around the portal tracts (e) A rat fed AFs-contaminated diet andtreated with CE2 showing normal histological picture with few fatty degeneration and interstitial hemorrhage around the central vein

around the central vein andweak reaction around portal veinof hepatocytes (Figure 4(c))The liver of the animals fed AFs-contaminated diet and treated with CE1 showed that weakreaction in some damaged cells which scattered in all thesection (Figure 4(d)) However the liver section of animalstreatedwithCE2 showedmarked improvement although lowreaction in hepatocytes was still demonstrated (Figure 4(e))

4 Discussion

Previous reports indicated that the type of extraction ofactive ingredient compounds from plant material dependsmainly on the type of solvent used [24] In the currentstudy the total phenolic compounds of the ethanol extractof Calendula were higher than that in the aqueous extract

However the ethanolic extract showed a higher DPPH (11-diphenyl 1-2-picryl hydrazyl) free radical scavenging activitycompared to the aqueous extract Cetkovic et al [25] showedsimilar scavenging potential against DPPH hydroxyl andperoxyl radicals and suggested that all of the extracts obtainedscavenged all radicals in concentration-dependant mannerMoreover Preethi et al [26] tested the antioxidant potentialof various leaves extracts of Calendula against DPPH ABTS(221015840-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) nitricoxide and superoxide radicals and reported lower scavengingproperty towards DPPH whereas they exerted higher radicalscavenging effects against the other radicals tested

In a similar study Danila et al [27] screened the waterand alcoholic extracts of Calendula grown in Romania for itsantioxidant activity and reported that Calendula contained

6 ISRN Nutrition

(a) (b)

(c) (d)

(e)

Figure 4 Photomicrographs in liver sections stained with Periodic-Sheif reagent-stain (PAS) for glycogen demonstration from (a)control rats showing a strong reaction of PAS (b) rats fed AFs-contaminated diet showing a weak reaction in the damaged cellwhile a strong reaction in intact cells (c) rats treated with CE1 orCE2 showing different types of reaction as well as strong reactionaround the central vein and weak reaction around portal vein ofhepatocytes (d) rats fed with AFs contaminated diet treated withCE1 showing that weak reaction in some damaged cells whichscattered in all the section and (e) rats fed with AFs contaminateddiet and treated with CE2 showing marked improvement althoughlow reaction in hepatocytes were still demonstrated (PAS reactiontimes100)

a high total phenol amount and scavenging activity againstDPPH and ABTS radicals DallrsquoAcqua et al [28] investigatedantioxidant activity of Calendula arvensis used in Sardinianfolk medicine using DPPH radical scavenging assay andreported that the extract showed a high radical scavengingeffect towards DPPH The radical scavenging propertieswere attributed to the presence of flavonoid and triterpenederivatives in the extract [29]

In the in vivo study the selective dose of AFs andCalendula was literature-based [30 31] respectively Theresults revealed a significant reduction of body weight andfood intake in animals fed AFs-contaminated diet Theseresults were in agreement with those reported in the literatureof aflatoxicosis [32ndash38] The reduction in body weight inthe animals fed AFs-contaminated diet alone may be due tothe effects of AFs on the balance between orexigenic andanorexigenic circuits that regulate the homeostatic loop ofbody weight regulation leading to cachexia [34] Moreover

AFs exposure may lead to significant reduction of leptin[35] accompanied with high levels of cortisol IL-6 andinsulin resistance which together act to influence the feedingresponse causing weight loss in patients with pancreaticcancer [36] Osborne et al [37] suggested that AFs ingestionaffect various digestive enzymatic activities that give riseto a malabsorption syndrome characterized by steatorrheahypocarotenoidemy and to lowering of bile pancreatic lipasetrypsin and amylase Furthermore Lesson et al [38] reportedthat the 89 epoxide metabolite of AFB

1may covalently bind

to DNA and proteins which then alters enzymatic processessuch as gluconeogenesis Krebs cycle or fatty acid synthesis

Animals fed AFs-contaminated diet showed a significantincrease of ALT AST ALP urea and createnine whichindicate changes in the hepatic tissues and biliary system [39]structural damaging of liver integrity since these enzymes arecytoplasmic in location and released into plasma as a result ofcellular damage [8 40] It is also likely that alteration in ALPactivity affects membrane permeability and the transport ofmetabolites Moreover increase in urea and createnine levelobserved in the current study in AFs-treated group clearlyindicated the harmful and stressful effect on renal tissue [30]Taken together the increased level of urea and the decreasedlevel of albumin and total protein (TP) indicate the inhibitionof protein synthesis and increase of protein catabolism andorrenal dysfunction [41 42]These results clearly indicated thatAFs had stressful effects on the hepatic and renal tissuesconsistent with those reported in the literature of aflatoxicosis[43 44]

Animals treated with AFs showed a significant increasein AFP which is considered specific biomarkers for livercancer This increase in AFP may be due to induction ofexpression of mRNAs of liver alpha-fetoprotein [45 46] Onthe other hand aflatoxin B

1-induced hepatocarcinogenesis

associated with defective DNA-damage response by passingp53 activation [47] and modulation of insulin-like growthfactor 2 dependent signal axis (IGF-2) [48] Similar to theseobservations Sell et al [49] and Yang et al [45] reported thatAFB1administration resulted in the elevation of serum AFP

level in both duck and ratsThe current study showed that animals fed AFs-con-

taminated diet suffer from oxidative stress as indicated bythe significant increment of lipid peroxidation (MDA) andthe significant reduction of enzymatic antioxidant such assuperoxide dismutase (SOD) and glutathione peroxidase(GPx) These results are in agreement with previous studiessuggested that oxidative stress may be due to direct effectof AFs or by the metabolites formed and the free radicalsgenerated during the formation of these metabolites [3446 50] Moreover the reduction of protein synthesis inAFs-treated animals may affect certain metal ions (ie ironand copper) which play an important role in free radicalproduction and liberation [10] In normal state metal ionsare bonded to transfer proteins such as ceruloplasmin andtransferrin [51] and play a crucial role of SOD CAT and GPxactivities which constitute the enzymatic antioxidant defensesystem of the cell displayed bidirectional alterations either inthe form of increase or decrease depending on the particulartissue Previous studies demonstrated that the mechanism of

ISRN Nutrition 7

AFs-induced liver injury may be due to those reactive andtoxic metabolites of AFs and the liver necrosis begins whenthe glutathione stores are almost exhausted [34 35 52]

Tumor necrosis factor-alpha (TNF-120572) and interleukin-1 alpha (IL-1120572) are produced by macrophages and theyplay an important role in tumor conditions [35] TNF-120572is an essential factor in tumor promotion [53] and IL-1polymorphisms are important mediators in the inflamma-tory process [54] In the current study ingestion of AFs-contaminated diet significantly increased TNF-120572 and IL-1120573 suggesting that AFs preferentially affects macrophagefunctions In particular it decouples the close correlationsusually observed between transcriptional and translationalcontrols of IL-1120572 and TNF-120572 production by these cells [55]In this concern Barton et al [56] stated that TNF-120572 plays acausal role in the development of liver injury and it plays amajor role in modulating mycotoxin-induced hepatotoxicityMoreover TNF-120572 has been proven to play an importantrole in inflammation in mediating the proliferation anddifferentiation of immune cells and development of immuneresponse [57] Hopkins [55] and Abdel-Wahhab et al [35]reported that directly proportional relationship has beenobserved between transcriptional and translational controlsof IL-1 and TNF-120572 production by hepatocytes Consequentlythe current results indicated that aflatoxicosis may induceTNF-120572 and IL-1 due to activation of caspase-3 which is oneof the apoptotic mediator [58] and the significant elevationof inflammatory cytokinemay affect themRNA expression ofthese mediators [46]

The current results indicated that the ethanolic extract ofCalendula showed a potential protective effect against AFstoxicity Animals treated withCalendula extract did not showany sign of toxicity In this concern the safety of the ethanolextract of Calendula was suggested and the oral LD

50in rats

was estimated by 4640mgkg bw [59] The results reportedherein indicated that animals treated with Calendula extractshowed insignificant increase in food intake and body weightgain Moreover Calendula extract succeeded to induce asignificant improvement in the biochemical parameters andthe histological and histochemical pictures Taken togetherthe results of the in vitro study and the in vivo evaluationsuggested that there are an antioxidant and free radicalscavenging properties of this extract and this improvementmore pronounced with the high dose (CA2) [29] In thisconcern previous study suggested that Calendula plays animportant role in hepaticcytolysis inhibition and improvesliver integrity in CCl

4intoxicated rats [60] In referring to

previous literature hepatoprotective activity of Calendulamay be due to its modulatory effect on cytochrome P450which may interfere on aflatoxin active metabolite produc-tion [61]MoreoverCalendula extract showed renoprotectiveaction against cisplatin-induced renal toxicity through itsmodulatory effect against myelosuppression of the renalsystem

The existing data revealed anticarcinogenicity of Cal-endula extract indicated by significant reduction of AFPand this effect was pronounced in the animals treatedwith the high dose (CA2) Several reports suggested thatthe anticancer properties of Calendula may be due to its

chemoprotector properties against hepatocarcinogenesis inrats beside antitumoural activity in vivo in the Ehrlichmouse carcinoma model due to its ability to stimulate T-lymphocytes B-lymphocytes and cluster of differentiation(CD4+) [62] Moreover Calendula plays a crucial role inprevention of DNA damaging [63] These results were inagreement with those reported previously [26 31 62]

The results also suggested that Calendula extract pos-sesses anti-inflammatory activity which may be due to itshigher content of carvacol Carvacol has been described asa promoter for liver regeneration and inhibitor of TNF-120572and IL-6 level in rats undergoing partial hepatectomy [64]Furthermore Tsai et al [65] reported that caravacl couldreduce TNF-120572 and IL-1120573 levels in intoxicated rats through theinhibition of cycloxygensae (COX) enzymes activity mRNAexpression and its protein in lipopoly saccharides-inducedinflammation

The histopathological and histochemical changes in liverconfirmed the biochemical results and revealed that ani-mals fed AFs-contaminated diet showed severe histologicalchanges typical to those reported in the literature [10 39 66]However animals treated with the extract in combinationwith AFs resulted in a significant improvement in liver tissuessimilar to that reported previously [61]

5 Conclusion

It can be concluded that the ethanolic and aqueous extracts ofCalendula officinalis have a valuable quantity of total phenoliccompounds and have DPPH radical scavenging activityMoreover the ethanolic extract showed higher phenoliccontent and radical scavenging property compared to theaqueous extract The ethanolic extract exhibited hepatoreno-protective properties against aflatoxin-induced liver injury ina dose-dependent manner due to its antioxidant free radicalscavenging activities and anti-inflammatory properties

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

This work was full supported by College of Pharmacy MisrUniversity for Science and Technology 6th October CityEgypt Faculty of Pharmacy Cairo University Cairo EgyptNational Research Center Dokki Cairo Egypt

References

[1] CAST ldquoMycotoxins risks in plant animal and humanrdquo inPotential Economic Costs ofMycotoxins inUnited States pp 136ndash142 CAST Task Force Reports 2003

[2] J A Nogueira S K Ono-Nita M E Nita et al ldquo249 TP53mutation has high prevalence and is correlated with larger andpoorly differentiated HCC in Brazilian patientsrdquo BMC Cancervol 9 article 204 2009

8 ISRN Nutrition

[3] S Rawal J E Kim and R Coulombe Jr ldquoAflatoxin B1in

poultry toxicology metabolism and preventionrdquo Research inVeterinary Science vol 89 no 3 pp 325ndash331 2010

[4] International Agency for Research in Carcinogenesis ldquoAfla-toxinsrdquo Monograph on the Evaluation of Carcinogenic Risks toHumans vol 56 p 245 1993

[5] IPCS-WHO ldquoAflatoxinsrdquo in International Programme onChem-ical Safety WHO Food Additives Series 40 pp 1ndash73 WorldHealth Organization Geneva Switzerland 1998

[6] J H Williams T D Phillips P E Jolly J K Stiles C M JollyandDAggarwal ldquoHuman aflatoxicosis in developing countriesareview of toxicology exposure potential health consequencesand interventionsrdquo American Journal of Clinical Nutrition vol80 no 5 pp 1106ndash1122 2004

[7] K C Choi W T Chung J K Kwon et al ldquoInhibitory effectsof quercetin on aflatoxin B1-induced hepatic damage in micerdquoFood and Chemical Toxicology vol 48 no 10 pp 2747ndash27532010

[8] D S El-Agamy ldquoComparative effects of curcumin and resver-atrol on aflatoxin B 1-induced liver injury in ratsrdquo Archives ofToxicology vol 84 no 5 pp 389ndash396 2010

[9] M A Abdel-Wahhab A A Ibrahim A A El-Nekeety NS Hassan and A A Mohamed ldquoPanax ginseng CA Meyerextract counteracts the oxidative stress in rats fed multi-mycotoxins-contaminated dietrdquo Comunicata Scientiae vol 3no 3 pp 143ndash153 2012

[10] M A Hamzawy E S El-Denshary N S Hassan F A Mannaaand M A Abdel-Wahhab ldquoAntioxidant and hepatorenoprotec-tive effect of thyme vulgaris extract in rats during aflatoxicosisrdquoGlobal Journal Pharmacology vol 6 no 2 pp 106ndash117 2012

[11] Z C Gazim C M Rezende S R Fraga T I E Svidzinskiand D A G Cortez ldquoAntifungal activity of the essential oilfrom Calendula officinalis L (Asteraceae) growing in BrazilrdquoBrazilian Journal of Microbiology vol 39 no 1 pp 61ndash63 2008

[12] L R Della A Tubaro S Sosa H Becker S Saar and D IsaacldquoThe role of triperpenoids in the topical antiinflamatory activityofCalendula officinalisflowersrdquoPlantaMedica vol 60 no 6 pp516ndash520 1994

[13] Chrysolite 2010 httphealthwikinutcomCalendula-officin-alis-Pot-Marigold11v4g8pw

[14] A Pintea C Bele S Andrei and C Socaciu ldquoHPLC analysis ofcarotenoids in four varieties of Calendula officinalis L flowersrdquoActa Biologica Szegediensis vol 247 pp 37ndash40 2003

[15] R A Jacob ldquoThe integrated antioxidant systemrdquo NutritionResearch vol 15 no 5 pp 755ndash766 1995

[16] K Dahan M Fennal and N B Kumar ldquoLycopene in the pre-vention of prostate cancerrdquo Journal of the Society for IntegrativeOncology vol 6 no 1 pp 29ndash36 2008

[17] J E Hutchins andWMHagler ldquoRapid liquid chromatographicdetermination of aflatoxins in heavily contaminated cornrdquoJournal of the Association of Official Analytical Chemists vol 66no 6 pp 1458ndash1465 1983

[18] G K Jayaprakasha and L J Rao ldquoPhenolic constituents fromthe lichen parmotrema stuppeum (Nyl) Hale and their antioxi-dant activityrdquo Zeitschrift fur Naturforschung C vol 55 no 11-12pp 1018ndash1022 2000

[19] G K Jayaprakasha T Selvi and K K Sakariah ldquoAntibacterialand antioxidant activities of grape (Vitis vinifera) seed extractsrdquoFood Research International vol 36 no 2 pp 117ndash122 2003

[20] M Nogala-Kalucka J Korczak M Dratwia E Lampart-Szczapa A Siger and M Buchowski ldquoChanges in antioxidant

activity and free radical scavenging potential of rosemaryextract and tocopherols in isolated rapeseed oil triacylglycerolsduring accelerated testsrdquo Food Chemistry vol 93 no 2 pp 227ndash235 2005

[21] R A Drury E A Wallington and R Cancerson CarltonrsquosHistopathological Techniques Oxford University Press OxfordUK 4th edition 1980

[22] SAS IInstitute SAS Userrsquos Guide Statistics SAS Institute CaryNC USA

[23] R A Waller and D B Duncan ldquoA Bayes rule for the symmetricmultiple comparisons problemsrdquo Journal of American StatisticalAssociation vol 64 no 328 pp 1484ndash1503 1969

[24] L Majhenic M Skerget and Z Knez ldquoAntioxidant and antimi-crobial activity of guarana seed extractsrdquo Food Chemistry vol104 no 3 pp 1258ndash1268 2007

[25] G S Cetkovic S M Djilas J M Canadanovic-Brunet and VT Tumbas ldquoAntioxidant properties of marigold extractsrdquo FoodResearch International vol 37 no 7 pp 643ndash650 2004

[26] K C Preethi G Kuttan and R Kuttan ldquoAntioxidant potentialof an extract ofCalendula officinalis flowers in vitro and in vivordquoPharmaceutical Biology vol 44 no 9 pp 691ndash697 2006

[27] AODanila F Gatea andG L Radu ldquoPolyphenol compositionand antioxidant activity of selected medicinal herbsrdquoChemistryof Natural Compounds vol 47 no 1 pp 22ndash26 2011

[28] S DallrsquoAcqua R Cervellati M C Loi and G InnocentildquoEvaluation of in vitro antioxidant properties of some tradi-tional Sardinian medicinal plants investigation of the highantioxidant capacity of Rubus ulmifoliusrdquo Food Chemistry vol106 no 2 pp 745ndash749 2008

[29] T Ercetin F S Senol I Erdogan Orhan and G TokerldquoComparative assessment of antioxidant and cholinesteraseinhibitory properties of the marigold extracts from Calendulaarvensis L and Calendula officinalis Lrdquo Industrial Crops andProducts vol 36 no 1 pp 203ndash208 2012

[30] M A Abdel-Wahhab and S E Aly ldquoAntioxidants and radicalscavenging properties of vegetable extracts in rats fed aflatoxin-contaminated dietrdquo Journal of Agricultural and Food Chemistryvol 51 no 8 pp 2409ndash2414 2003

[31] C Gladine C Morand E Rock D Gruffat D Bauchart andD Durand ldquoThe antioxidative effect of plant extracts rich inpolyphenols differs between liver and muscle tissues in rats fedn-3 PUFA rich dietsrdquo Animal Feed Science and Technology vol139 no 3-4 pp 257ndash272 2007

[32] R H Rauber P Dilkin L Z Giacomini C A Araujo deAlmeida and C A Mallmann ldquoPerformance of Turkey poultsfed different doses of aflatoxins in the dietrdquo Poultry Science vol86 no 8 pp 1620ndash1624 2007

[33] SM RustemeyerW R Lamberson D R Ledoux et al ldquoEffectsof dietary aflatoxin on the health and performance of growingbarrowsrdquo Journal of Animal Science vol 88 no 11 pp 3624ndash3630 2010

[34] M A Abdel-Wahhab N S Hassan A A El-Kady et al ldquoRedginseng extract protects against aflatoxin B

1and fumonisins-

induced hepatic pre-cancerous lesions in ratsrdquo Food and Chem-ical Toxicology vol 48 no 2 pp 733ndash742 2010

[35] M A Abdel-Wahhab H H Ahmed and M M HagazildquoPrevention of aflatoxin B1-initiated hepatotoxicity in rat bymarine algae extractsrdquo Journal of Applied Toxicology vol 26 no3 pp 229ndash238 2006

[36] M D Barber D C McMillan A M Wallace J A RossT Preston and K C H Fearon ldquoThe response of leptin

ISRN Nutrition 9

interleukin-6 and fat oxidation to feeding in weight-losingpatients with pancreatic cancerrdquo British Journal of Cancer vol90 no 6 pp 1129ndash1132 2004

[37] D J Osborne W E Huff P B Hamilton and H R BurmeisterldquoComparison of ochratoxin aflatoxin and T-2 toxin for theireffects on selected parameters related to digestion and evidencefor specific metabolism of carotenoids in chickensrdquo PoultryScience vol 61 no 8 pp 1646ndash1652 1982

[38] S Lesson G Diaz and J Summers Poultry Metabolic Disordersand Mycotoxins University Books Montreal Canada 1995

[39] M A Abdel-Wahhab S A Nada and F A Khalil ldquoPhys-iological and toxicological responses in rats fed aflatoxin-contaminated diet with or without sorbent materialsrdquo AnimalFeed Science and Technology vol 97 no 3-4 pp 209ndash219 2002

[40] A A El-Nekeety S R Mohamed A S Hathout N S HassanS E Aly and M A Abdel-Wahhab ldquoAntioxidant properties ofThymus vulgaris oil against aflatoxin-induce oxidative stress inmale ratsrdquo Toxicon vol 57 no 7-8 pp 984ndash991 2011

[41] N Jindal S K Mahipal and N K Mahajan ldquoToxicity ofaflatoxin B1 in broiler chicks and its reduction by activatedcharcoalrdquo Research in Veterinary Science vol 56 no 1 pp 37ndash40 1994

[42] M A Abdel-Wahhab and S E Aly ldquoAntioxidant property ofNigella sativa (black cumin) and Syzygium aromaticum (clove)in rats during aflatoxicosisrdquo Journal of Applied Toxicology vol25 no 3 pp 218ndash223 2005

[43] D M Miller and D M Wilson Veterinary Diseases Relatedto Aflatoxins the Toxicology of Aflatoxins Academic Press SanDiego Calif USA 1994

[44] M A Abdel-Wahhab and A M Kholif ldquoMycotoxins in animalfeeds and prevention strategies a reviewrdquo Asian Journal ofAnimal Sciences vol 2 no 1 pp 7ndash25 2008

[45] C F Yang J Liu S Wasser H M Shen C E L Tan andC N Ong ldquoInhibition of ebselen on aflatoxin B1-inducedhepatocarcinogenesis in Fischer 344 ratsrdquo Carcinogenesis vol21 no 12 pp 2237ndash2243 2000

[46] S H Abdel-Aziem A M Hassan and M A Abdel-WahhabldquoDietary supplementation with whey protein and ginsengextract counteracts oxidative stress and DNA damage in ratsfed an aflatoxin-contaminated dietrdquoMutationResearch vol 723no 1 pp 65ndash71 2011

[47] O Gursoy-Yuzugullu H YuzugulluM Yilmaz andMOzturkldquoAflatoxin genotoxicity is associated with a defective DNAdamage response bypassing p53 activationrdquo Liver Internationalvol 31 no 4 pp 561ndash571 2011

[48] T Ubagai T Kikuchi T Fukusato and Y Ono ldquoAflatoxin B1

modulates the insulin-like growth factor-2 dependent signalingaxisrdquo Toxicology in Vitro vol 24 no 3 pp 783ndash789 2010

[49] S Sell K L Xu W E Huff L F Kabena R B Harveryand H A Dunsford ldquoAflatoxin exposure produces serumalphafetoprotein elevations and marked oval cell proliferationin young male Pekin ducklingsrdquo Pathology vol 30 no 1 pp34ndash39 1998

[50] M Kanbur G Eraslan Z S Sarica andO Aslan ldquoThe effects ofevening primrose oil on lipid peroxidation induced by subacuteaflatoxin exposure in micerdquo Food and Chemical Toxicology vol49 no 9 pp 1960ndash1964 2011

[51] E Gitto S Pellegrino P Gitto I Barberi and R J ReiterldquoOxidative stress of the newborn in the pre- and postnatalperiod and the clinical utility of melatoninrdquo Journal of PinealResearch vol 46 no 2 pp 128ndash139 2009

[52] M A Abdel-Wahhab A Said and A Huefner ldquoNMR andradical scavenging activities of patuletin from Urtica urensagainst aflatoxin B

1rdquo Pharmaceutical Biology vol 43 no 6 pp

515ndash525 2005[53] M Suganuma E Sueoka N Sueoka S Okabe and H Fujiki

ldquoMechanisms of cancer prevention by tea polyphenols based oninhibition of TNF-120572 expressionrdquo BioFactors vol 13 no 1-4 pp67ndash72 2000

[54] S Rollinson A P Levene F K Mensah et al ldquoGastric marginalzone lymphoma is associated with polymorphisms in genesinvolved in inflammatory response and antioxidative capacityrdquoBlood vol 102 no 3 pp 1007ndash1011 2003

[55] S J Hopkins ldquoThe pathophysiological role of cytokinesrdquo LegalMedicine vol 5 no 1 pp S45ndashS57 2003

[56] C C Barton E X Barton P E Ganey S L Kunkel and RA Roth ldquoBacterial lipopolysaccharide enhances aflatoxin B

1

hepatotoxicity in rats by a mechanism that depends on tumornecrosis factor 120572rdquo Hepatology vol 33 no 1 pp 66ndash73 2001

[57] J Cao L Jiang X Zhang et al ldquoBoric acid inhibits LPS-inducedTNF-120572 formation through a thiol-dependent mechanism inTHP-1 cellsrdquo Journal of Trace Elements in Medicine and Biologyvol 22 no 3 pp 189ndash195 2008

[58] A R M A Meik S K Abdel-Ghaffar and I El-GibalyldquoAflatoxin B1 induces apoptosis in rat liver protective effect ofmelatoninrdquo Neuroendocrinology Letters vol 22 no 6 pp 417ndash426 2001

[59] CIREP ldquoFinal report on the safety assessment ofCalendula offic-inalis Extract and Calendula officinalisrdquo International JournalToxicology vol 20 pp 13ndash20 2001

[60] M A Rusu M Tamas C Puica I Roman and M SabadasldquoThe hepatoprotective action of ten herbal extracts in CCl

4

intoxicated liverrdquo Phytotherapy Research vol 19 no 9 pp 744ndash749 2005

[61] K C Preethi and R Kuttan ldquoHepato and reno protective actionof Calendula officinalis L Flower extractrdquo Indian Journal ofExperimental Biology vol 47 no 3 pp 163ndash168 2009

[62] B P Muley S S Khadabadi and N B Banarase ldquoPhytochem-ical constituents and pharmacological activities of Calendulaofficinalis Linn (Asteraceae) a reviewrdquo Tropical Journal ofPharmaceutical Research vol 8 no 5 pp 455ndash465 2009

[63] T Frankic K Salobir and J Salobir ldquoThe comparison of invivo antigenotoxic and antioxidative capacity of two propyleneglycol extracts of Calendula officinalis (marigold) and vitaminE in young growing pigsrdquo Journal of Animal Physiology andAnimal Nutrition vol 93 no 6 pp 688ndash694 2009

[64] M Uyanoglu M Canbek E Aral and K Husnu Can BaserldquoEffects of carvacrol upon the liver of rats undergoing partialhepatectomyrdquo Phytomedicine vol 15 no 3 pp 226ndash229 2008

[65] M L Tsai C C Lin W C Lin and C H Yang ldquoAntimicrobialantioxidant and anti-inflammatory activities of essential oilsfromfive selected herbsrdquoBioscience Biotechnology Biochemistryvol 75 no 10 pp 1977ndash1983 2011

[66] K Mayura M A Abdel-Mahhab K S McKenzie et alldquoPrevention of maternal and developmental toxicity in rats viadietary inclusion of common aflatoxin sorbents potential forhidden risksrdquo Toxicological Sciences vol 41 no 2 pp 175ndash1821998

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

ISRN Nutrition 5

Table 4 Effect of ethanol extract of calendula on serum inflammatory cytokine in rats fed aflatoxins-contaminated diet

Parameter GroupControl AFs CE1 CE2 CE1 + AFs CE2 + AFs

TNF-120572 (pgmL) 5834 plusmn 312a 13112 plusmn 332b 5837 plusmn 207a 6072 plusmn 192a 8647 plusmn 231c 6324 plusmn 166d

IL-1120573 (pgmL) 066 plusmn 007a 234 plusmn 012b 065 plusmn 004a 084 plusmn 022a 158 plusmn 004c 077 plusmn 021a

Within each row means superscript with different letters are significantly different at 119875 le 005AFs aflatoxinsCE1 low dose of calendula extract (500mgkg bw)CE2 high dose of calendula extract (1000mgkg bw)

CV

Hepatocytes

(HX and E times400)

(a)

FN

(HX and E times400)

(b)

Hepatocytes

Inf

CV

(HX and E times400)

(c)

CVPV

PV

(HX and E times200)

(d)

Fatty degeneration

(HX and E times400)

(e)

Figure 3 A photomicrograph in liver section from (a) a control rat branching and anatomizing cords radiating from the central vein CVThehepatocytes that are having vesicular nuclei and some binucleated cells are separated by sinusoids lined by flat endothelial cell and kupfer cells(b) a rat fed AFs-contaminated diet showing hepatocytes necrosis (N) and apoptosis (yellow arrows) and fibrosis around the blood vessels(F) (c) A rat treated with CE1 or CE2 showing the normal hepatocytes with vesicular nuclei and evident mononuclear cellular infiltrationaround the central vein (d) a rat fed AFs-contaminated diet and treated with CE1 showing marked improvement in hepatocytes architectureno vacuoles or fatty droplets were detected Some cells have eosinophilic cytoplasm and deeply stained nuclei around the central and portalvein areas (arrows) and mononuclear cellular infiltration are scattered around the portal tracts (e) A rat fed AFs-contaminated diet andtreated with CE2 showing normal histological picture with few fatty degeneration and interstitial hemorrhage around the central vein

around the central vein andweak reaction around portal veinof hepatocytes (Figure 4(c))The liver of the animals fed AFs-contaminated diet and treated with CE1 showed that weakreaction in some damaged cells which scattered in all thesection (Figure 4(d)) However the liver section of animalstreatedwithCE2 showedmarked improvement although lowreaction in hepatocytes was still demonstrated (Figure 4(e))

4 Discussion

Previous reports indicated that the type of extraction ofactive ingredient compounds from plant material dependsmainly on the type of solvent used [24] In the currentstudy the total phenolic compounds of the ethanol extractof Calendula were higher than that in the aqueous extract

However the ethanolic extract showed a higher DPPH (11-diphenyl 1-2-picryl hydrazyl) free radical scavenging activitycompared to the aqueous extract Cetkovic et al [25] showedsimilar scavenging potential against DPPH hydroxyl andperoxyl radicals and suggested that all of the extracts obtainedscavenged all radicals in concentration-dependant mannerMoreover Preethi et al [26] tested the antioxidant potentialof various leaves extracts of Calendula against DPPH ABTS(221015840-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) nitricoxide and superoxide radicals and reported lower scavengingproperty towards DPPH whereas they exerted higher radicalscavenging effects against the other radicals tested

In a similar study Danila et al [27] screened the waterand alcoholic extracts of Calendula grown in Romania for itsantioxidant activity and reported that Calendula contained

6 ISRN Nutrition

(a) (b)

(c) (d)

(e)

Figure 4 Photomicrographs in liver sections stained with Periodic-Sheif reagent-stain (PAS) for glycogen demonstration from (a)control rats showing a strong reaction of PAS (b) rats fed AFs-contaminated diet showing a weak reaction in the damaged cellwhile a strong reaction in intact cells (c) rats treated with CE1 orCE2 showing different types of reaction as well as strong reactionaround the central vein and weak reaction around portal vein ofhepatocytes (d) rats fed with AFs contaminated diet treated withCE1 showing that weak reaction in some damaged cells whichscattered in all the section and (e) rats fed with AFs contaminateddiet and treated with CE2 showing marked improvement althoughlow reaction in hepatocytes were still demonstrated (PAS reactiontimes100)

a high total phenol amount and scavenging activity againstDPPH and ABTS radicals DallrsquoAcqua et al [28] investigatedantioxidant activity of Calendula arvensis used in Sardinianfolk medicine using DPPH radical scavenging assay andreported that the extract showed a high radical scavengingeffect towards DPPH The radical scavenging propertieswere attributed to the presence of flavonoid and triterpenederivatives in the extract [29]

In the in vivo study the selective dose of AFs andCalendula was literature-based [30 31] respectively Theresults revealed a significant reduction of body weight andfood intake in animals fed AFs-contaminated diet Theseresults were in agreement with those reported in the literatureof aflatoxicosis [32ndash38] The reduction in body weight inthe animals fed AFs-contaminated diet alone may be due tothe effects of AFs on the balance between orexigenic andanorexigenic circuits that regulate the homeostatic loop ofbody weight regulation leading to cachexia [34] Moreover

AFs exposure may lead to significant reduction of leptin[35] accompanied with high levels of cortisol IL-6 andinsulin resistance which together act to influence the feedingresponse causing weight loss in patients with pancreaticcancer [36] Osborne et al [37] suggested that AFs ingestionaffect various digestive enzymatic activities that give riseto a malabsorption syndrome characterized by steatorrheahypocarotenoidemy and to lowering of bile pancreatic lipasetrypsin and amylase Furthermore Lesson et al [38] reportedthat the 89 epoxide metabolite of AFB

1may covalently bind

to DNA and proteins which then alters enzymatic processessuch as gluconeogenesis Krebs cycle or fatty acid synthesis

Animals fed AFs-contaminated diet showed a significantincrease of ALT AST ALP urea and createnine whichindicate changes in the hepatic tissues and biliary system [39]structural damaging of liver integrity since these enzymes arecytoplasmic in location and released into plasma as a result ofcellular damage [8 40] It is also likely that alteration in ALPactivity affects membrane permeability and the transport ofmetabolites Moreover increase in urea and createnine levelobserved in the current study in AFs-treated group clearlyindicated the harmful and stressful effect on renal tissue [30]Taken together the increased level of urea and the decreasedlevel of albumin and total protein (TP) indicate the inhibitionof protein synthesis and increase of protein catabolism andorrenal dysfunction [41 42]These results clearly indicated thatAFs had stressful effects on the hepatic and renal tissuesconsistent with those reported in the literature of aflatoxicosis[43 44]

Animals treated with AFs showed a significant increasein AFP which is considered specific biomarkers for livercancer This increase in AFP may be due to induction ofexpression of mRNAs of liver alpha-fetoprotein [45 46] Onthe other hand aflatoxin B

1-induced hepatocarcinogenesis

associated with defective DNA-damage response by passingp53 activation [47] and modulation of insulin-like growthfactor 2 dependent signal axis (IGF-2) [48] Similar to theseobservations Sell et al [49] and Yang et al [45] reported thatAFB1administration resulted in the elevation of serum AFP

level in both duck and ratsThe current study showed that animals fed AFs-con-

taminated diet suffer from oxidative stress as indicated bythe significant increment of lipid peroxidation (MDA) andthe significant reduction of enzymatic antioxidant such assuperoxide dismutase (SOD) and glutathione peroxidase(GPx) These results are in agreement with previous studiessuggested that oxidative stress may be due to direct effectof AFs or by the metabolites formed and the free radicalsgenerated during the formation of these metabolites [3446 50] Moreover the reduction of protein synthesis inAFs-treated animals may affect certain metal ions (ie ironand copper) which play an important role in free radicalproduction and liberation [10] In normal state metal ionsare bonded to transfer proteins such as ceruloplasmin andtransferrin [51] and play a crucial role of SOD CAT and GPxactivities which constitute the enzymatic antioxidant defensesystem of the cell displayed bidirectional alterations either inthe form of increase or decrease depending on the particulartissue Previous studies demonstrated that the mechanism of

ISRN Nutrition 7

AFs-induced liver injury may be due to those reactive andtoxic metabolites of AFs and the liver necrosis begins whenthe glutathione stores are almost exhausted [34 35 52]

Tumor necrosis factor-alpha (TNF-120572) and interleukin-1 alpha (IL-1120572) are produced by macrophages and theyplay an important role in tumor conditions [35] TNF-120572is an essential factor in tumor promotion [53] and IL-1polymorphisms are important mediators in the inflamma-tory process [54] In the current study ingestion of AFs-contaminated diet significantly increased TNF-120572 and IL-1120573 suggesting that AFs preferentially affects macrophagefunctions In particular it decouples the close correlationsusually observed between transcriptional and translationalcontrols of IL-1120572 and TNF-120572 production by these cells [55]In this concern Barton et al [56] stated that TNF-120572 plays acausal role in the development of liver injury and it plays amajor role in modulating mycotoxin-induced hepatotoxicityMoreover TNF-120572 has been proven to play an importantrole in inflammation in mediating the proliferation anddifferentiation of immune cells and development of immuneresponse [57] Hopkins [55] and Abdel-Wahhab et al [35]reported that directly proportional relationship has beenobserved between transcriptional and translational controlsof IL-1 and TNF-120572 production by hepatocytes Consequentlythe current results indicated that aflatoxicosis may induceTNF-120572 and IL-1 due to activation of caspase-3 which is oneof the apoptotic mediator [58] and the significant elevationof inflammatory cytokinemay affect themRNA expression ofthese mediators [46]

The current results indicated that the ethanolic extract ofCalendula showed a potential protective effect against AFstoxicity Animals treated withCalendula extract did not showany sign of toxicity In this concern the safety of the ethanolextract of Calendula was suggested and the oral LD

50in rats

was estimated by 4640mgkg bw [59] The results reportedherein indicated that animals treated with Calendula extractshowed insignificant increase in food intake and body weightgain Moreover Calendula extract succeeded to induce asignificant improvement in the biochemical parameters andthe histological and histochemical pictures Taken togetherthe results of the in vitro study and the in vivo evaluationsuggested that there are an antioxidant and free radicalscavenging properties of this extract and this improvementmore pronounced with the high dose (CA2) [29] In thisconcern previous study suggested that Calendula plays animportant role in hepaticcytolysis inhibition and improvesliver integrity in CCl

4intoxicated rats [60] In referring to

previous literature hepatoprotective activity of Calendulamay be due to its modulatory effect on cytochrome P450which may interfere on aflatoxin active metabolite produc-tion [61]MoreoverCalendula extract showed renoprotectiveaction against cisplatin-induced renal toxicity through itsmodulatory effect against myelosuppression of the renalsystem

The existing data revealed anticarcinogenicity of Cal-endula extract indicated by significant reduction of AFPand this effect was pronounced in the animals treatedwith the high dose (CA2) Several reports suggested thatthe anticancer properties of Calendula may be due to its

chemoprotector properties against hepatocarcinogenesis inrats beside antitumoural activity in vivo in the Ehrlichmouse carcinoma model due to its ability to stimulate T-lymphocytes B-lymphocytes and cluster of differentiation(CD4+) [62] Moreover Calendula plays a crucial role inprevention of DNA damaging [63] These results were inagreement with those reported previously [26 31 62]

The results also suggested that Calendula extract pos-sesses anti-inflammatory activity which may be due to itshigher content of carvacol Carvacol has been described asa promoter for liver regeneration and inhibitor of TNF-120572and IL-6 level in rats undergoing partial hepatectomy [64]Furthermore Tsai et al [65] reported that caravacl couldreduce TNF-120572 and IL-1120573 levels in intoxicated rats through theinhibition of cycloxygensae (COX) enzymes activity mRNAexpression and its protein in lipopoly saccharides-inducedinflammation

The histopathological and histochemical changes in liverconfirmed the biochemical results and revealed that ani-mals fed AFs-contaminated diet showed severe histologicalchanges typical to those reported in the literature [10 39 66]However animals treated with the extract in combinationwith AFs resulted in a significant improvement in liver tissuessimilar to that reported previously [61]

5 Conclusion

It can be concluded that the ethanolic and aqueous extracts ofCalendula officinalis have a valuable quantity of total phenoliccompounds and have DPPH radical scavenging activityMoreover the ethanolic extract showed higher phenoliccontent and radical scavenging property compared to theaqueous extract The ethanolic extract exhibited hepatoreno-protective properties against aflatoxin-induced liver injury ina dose-dependent manner due to its antioxidant free radicalscavenging activities and anti-inflammatory properties

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

This work was full supported by College of Pharmacy MisrUniversity for Science and Technology 6th October CityEgypt Faculty of Pharmacy Cairo University Cairo EgyptNational Research Center Dokki Cairo Egypt

References

[1] CAST ldquoMycotoxins risks in plant animal and humanrdquo inPotential Economic Costs ofMycotoxins inUnited States pp 136ndash142 CAST Task Force Reports 2003

[2] J A Nogueira S K Ono-Nita M E Nita et al ldquo249 TP53mutation has high prevalence and is correlated with larger andpoorly differentiated HCC in Brazilian patientsrdquo BMC Cancervol 9 article 204 2009

8 ISRN Nutrition

[3] S Rawal J E Kim and R Coulombe Jr ldquoAflatoxin B1in

poultry toxicology metabolism and preventionrdquo Research inVeterinary Science vol 89 no 3 pp 325ndash331 2010

[4] International Agency for Research in Carcinogenesis ldquoAfla-toxinsrdquo Monograph on the Evaluation of Carcinogenic Risks toHumans vol 56 p 245 1993

[5] IPCS-WHO ldquoAflatoxinsrdquo in International Programme onChem-ical Safety WHO Food Additives Series 40 pp 1ndash73 WorldHealth Organization Geneva Switzerland 1998

[6] J H Williams T D Phillips P E Jolly J K Stiles C M JollyandDAggarwal ldquoHuman aflatoxicosis in developing countriesareview of toxicology exposure potential health consequencesand interventionsrdquo American Journal of Clinical Nutrition vol80 no 5 pp 1106ndash1122 2004

[7] K C Choi W T Chung J K Kwon et al ldquoInhibitory effectsof quercetin on aflatoxin B1-induced hepatic damage in micerdquoFood and Chemical Toxicology vol 48 no 10 pp 2747ndash27532010

[8] D S El-Agamy ldquoComparative effects of curcumin and resver-atrol on aflatoxin B 1-induced liver injury in ratsrdquo Archives ofToxicology vol 84 no 5 pp 389ndash396 2010

[9] M A Abdel-Wahhab A A Ibrahim A A El-Nekeety NS Hassan and A A Mohamed ldquoPanax ginseng CA Meyerextract counteracts the oxidative stress in rats fed multi-mycotoxins-contaminated dietrdquo Comunicata Scientiae vol 3no 3 pp 143ndash153 2012

[10] M A Hamzawy E S El-Denshary N S Hassan F A Mannaaand M A Abdel-Wahhab ldquoAntioxidant and hepatorenoprotec-tive effect of thyme vulgaris extract in rats during aflatoxicosisrdquoGlobal Journal Pharmacology vol 6 no 2 pp 106ndash117 2012

[11] Z C Gazim C M Rezende S R Fraga T I E Svidzinskiand D A G Cortez ldquoAntifungal activity of the essential oilfrom Calendula officinalis L (Asteraceae) growing in BrazilrdquoBrazilian Journal of Microbiology vol 39 no 1 pp 61ndash63 2008

[12] L R Della A Tubaro S Sosa H Becker S Saar and D IsaacldquoThe role of triperpenoids in the topical antiinflamatory activityofCalendula officinalisflowersrdquoPlantaMedica vol 60 no 6 pp516ndash520 1994

[13] Chrysolite 2010 httphealthwikinutcomCalendula-officin-alis-Pot-Marigold11v4g8pw

[14] A Pintea C Bele S Andrei and C Socaciu ldquoHPLC analysis ofcarotenoids in four varieties of Calendula officinalis L flowersrdquoActa Biologica Szegediensis vol 247 pp 37ndash40 2003

[15] R A Jacob ldquoThe integrated antioxidant systemrdquo NutritionResearch vol 15 no 5 pp 755ndash766 1995

[16] K Dahan M Fennal and N B Kumar ldquoLycopene in the pre-vention of prostate cancerrdquo Journal of the Society for IntegrativeOncology vol 6 no 1 pp 29ndash36 2008

[17] J E Hutchins andWMHagler ldquoRapid liquid chromatographicdetermination of aflatoxins in heavily contaminated cornrdquoJournal of the Association of Official Analytical Chemists vol 66no 6 pp 1458ndash1465 1983

[18] G K Jayaprakasha and L J Rao ldquoPhenolic constituents fromthe lichen parmotrema stuppeum (Nyl) Hale and their antioxi-dant activityrdquo Zeitschrift fur Naturforschung C vol 55 no 11-12pp 1018ndash1022 2000

[19] G K Jayaprakasha T Selvi and K K Sakariah ldquoAntibacterialand antioxidant activities of grape (Vitis vinifera) seed extractsrdquoFood Research International vol 36 no 2 pp 117ndash122 2003

[20] M Nogala-Kalucka J Korczak M Dratwia E Lampart-Szczapa A Siger and M Buchowski ldquoChanges in antioxidant

activity and free radical scavenging potential of rosemaryextract and tocopherols in isolated rapeseed oil triacylglycerolsduring accelerated testsrdquo Food Chemistry vol 93 no 2 pp 227ndash235 2005

[21] R A Drury E A Wallington and R Cancerson CarltonrsquosHistopathological Techniques Oxford University Press OxfordUK 4th edition 1980

[22] SAS IInstitute SAS Userrsquos Guide Statistics SAS Institute CaryNC USA

[23] R A Waller and D B Duncan ldquoA Bayes rule for the symmetricmultiple comparisons problemsrdquo Journal of American StatisticalAssociation vol 64 no 328 pp 1484ndash1503 1969

[24] L Majhenic M Skerget and Z Knez ldquoAntioxidant and antimi-crobial activity of guarana seed extractsrdquo Food Chemistry vol104 no 3 pp 1258ndash1268 2007

[25] G S Cetkovic S M Djilas J M Canadanovic-Brunet and VT Tumbas ldquoAntioxidant properties of marigold extractsrdquo FoodResearch International vol 37 no 7 pp 643ndash650 2004

[26] K C Preethi G Kuttan and R Kuttan ldquoAntioxidant potentialof an extract ofCalendula officinalis flowers in vitro and in vivordquoPharmaceutical Biology vol 44 no 9 pp 691ndash697 2006

[27] AODanila F Gatea andG L Radu ldquoPolyphenol compositionand antioxidant activity of selected medicinal herbsrdquoChemistryof Natural Compounds vol 47 no 1 pp 22ndash26 2011

[28] S DallrsquoAcqua R Cervellati M C Loi and G InnocentildquoEvaluation of in vitro antioxidant properties of some tradi-tional Sardinian medicinal plants investigation of the highantioxidant capacity of Rubus ulmifoliusrdquo Food Chemistry vol106 no 2 pp 745ndash749 2008

[29] T Ercetin F S Senol I Erdogan Orhan and G TokerldquoComparative assessment of antioxidant and cholinesteraseinhibitory properties of the marigold extracts from Calendulaarvensis L and Calendula officinalis Lrdquo Industrial Crops andProducts vol 36 no 1 pp 203ndash208 2012

[30] M A Abdel-Wahhab and S E Aly ldquoAntioxidants and radicalscavenging properties of vegetable extracts in rats fed aflatoxin-contaminated dietrdquo Journal of Agricultural and Food Chemistryvol 51 no 8 pp 2409ndash2414 2003

[31] C Gladine C Morand E Rock D Gruffat D Bauchart andD Durand ldquoThe antioxidative effect of plant extracts rich inpolyphenols differs between liver and muscle tissues in rats fedn-3 PUFA rich dietsrdquo Animal Feed Science and Technology vol139 no 3-4 pp 257ndash272 2007

[32] R H Rauber P Dilkin L Z Giacomini C A Araujo deAlmeida and C A Mallmann ldquoPerformance of Turkey poultsfed different doses of aflatoxins in the dietrdquo Poultry Science vol86 no 8 pp 1620ndash1624 2007

[33] SM RustemeyerW R Lamberson D R Ledoux et al ldquoEffectsof dietary aflatoxin on the health and performance of growingbarrowsrdquo Journal of Animal Science vol 88 no 11 pp 3624ndash3630 2010

[34] M A Abdel-Wahhab N S Hassan A A El-Kady et al ldquoRedginseng extract protects against aflatoxin B

1and fumonisins-

induced hepatic pre-cancerous lesions in ratsrdquo Food and Chem-ical Toxicology vol 48 no 2 pp 733ndash742 2010

[35] M A Abdel-Wahhab H H Ahmed and M M HagazildquoPrevention of aflatoxin B1-initiated hepatotoxicity in rat bymarine algae extractsrdquo Journal of Applied Toxicology vol 26 no3 pp 229ndash238 2006

[36] M D Barber D C McMillan A M Wallace J A RossT Preston and K C H Fearon ldquoThe response of leptin

ISRN Nutrition 9

interleukin-6 and fat oxidation to feeding in weight-losingpatients with pancreatic cancerrdquo British Journal of Cancer vol90 no 6 pp 1129ndash1132 2004

[37] D J Osborne W E Huff P B Hamilton and H R BurmeisterldquoComparison of ochratoxin aflatoxin and T-2 toxin for theireffects on selected parameters related to digestion and evidencefor specific metabolism of carotenoids in chickensrdquo PoultryScience vol 61 no 8 pp 1646ndash1652 1982

[38] S Lesson G Diaz and J Summers Poultry Metabolic Disordersand Mycotoxins University Books Montreal Canada 1995

[39] M A Abdel-Wahhab S A Nada and F A Khalil ldquoPhys-iological and toxicological responses in rats fed aflatoxin-contaminated diet with or without sorbent materialsrdquo AnimalFeed Science and Technology vol 97 no 3-4 pp 209ndash219 2002

[40] A A El-Nekeety S R Mohamed A S Hathout N S HassanS E Aly and M A Abdel-Wahhab ldquoAntioxidant properties ofThymus vulgaris oil against aflatoxin-induce oxidative stress inmale ratsrdquo Toxicon vol 57 no 7-8 pp 984ndash991 2011

[41] N Jindal S K Mahipal and N K Mahajan ldquoToxicity ofaflatoxin B1 in broiler chicks and its reduction by activatedcharcoalrdquo Research in Veterinary Science vol 56 no 1 pp 37ndash40 1994

[42] M A Abdel-Wahhab and S E Aly ldquoAntioxidant property ofNigella sativa (black cumin) and Syzygium aromaticum (clove)in rats during aflatoxicosisrdquo Journal of Applied Toxicology vol25 no 3 pp 218ndash223 2005

[43] D M Miller and D M Wilson Veterinary Diseases Relatedto Aflatoxins the Toxicology of Aflatoxins Academic Press SanDiego Calif USA 1994

[44] M A Abdel-Wahhab and A M Kholif ldquoMycotoxins in animalfeeds and prevention strategies a reviewrdquo Asian Journal ofAnimal Sciences vol 2 no 1 pp 7ndash25 2008

[45] C F Yang J Liu S Wasser H M Shen C E L Tan andC N Ong ldquoInhibition of ebselen on aflatoxin B1-inducedhepatocarcinogenesis in Fischer 344 ratsrdquo Carcinogenesis vol21 no 12 pp 2237ndash2243 2000

[46] S H Abdel-Aziem A M Hassan and M A Abdel-WahhabldquoDietary supplementation with whey protein and ginsengextract counteracts oxidative stress and DNA damage in ratsfed an aflatoxin-contaminated dietrdquoMutationResearch vol 723no 1 pp 65ndash71 2011

[47] O Gursoy-Yuzugullu H YuzugulluM Yilmaz andMOzturkldquoAflatoxin genotoxicity is associated with a defective DNAdamage response bypassing p53 activationrdquo Liver Internationalvol 31 no 4 pp 561ndash571 2011

[48] T Ubagai T Kikuchi T Fukusato and Y Ono ldquoAflatoxin B1

modulates the insulin-like growth factor-2 dependent signalingaxisrdquo Toxicology in Vitro vol 24 no 3 pp 783ndash789 2010

[49] S Sell K L Xu W E Huff L F Kabena R B Harveryand H A Dunsford ldquoAflatoxin exposure produces serumalphafetoprotein elevations and marked oval cell proliferationin young male Pekin ducklingsrdquo Pathology vol 30 no 1 pp34ndash39 1998

[50] M Kanbur G Eraslan Z S Sarica andO Aslan ldquoThe effects ofevening primrose oil on lipid peroxidation induced by subacuteaflatoxin exposure in micerdquo Food and Chemical Toxicology vol49 no 9 pp 1960ndash1964 2011

[51] E Gitto S Pellegrino P Gitto I Barberi and R J ReiterldquoOxidative stress of the newborn in the pre- and postnatalperiod and the clinical utility of melatoninrdquo Journal of PinealResearch vol 46 no 2 pp 128ndash139 2009

[52] M A Abdel-Wahhab A Said and A Huefner ldquoNMR andradical scavenging activities of patuletin from Urtica urensagainst aflatoxin B

1rdquo Pharmaceutical Biology vol 43 no 6 pp

515ndash525 2005[53] M Suganuma E Sueoka N Sueoka S Okabe and H Fujiki

ldquoMechanisms of cancer prevention by tea polyphenols based oninhibition of TNF-120572 expressionrdquo BioFactors vol 13 no 1-4 pp67ndash72 2000

[54] S Rollinson A P Levene F K Mensah et al ldquoGastric marginalzone lymphoma is associated with polymorphisms in genesinvolved in inflammatory response and antioxidative capacityrdquoBlood vol 102 no 3 pp 1007ndash1011 2003

[55] S J Hopkins ldquoThe pathophysiological role of cytokinesrdquo LegalMedicine vol 5 no 1 pp S45ndashS57 2003

[56] C C Barton E X Barton P E Ganey S L Kunkel and RA Roth ldquoBacterial lipopolysaccharide enhances aflatoxin B

1

hepatotoxicity in rats by a mechanism that depends on tumornecrosis factor 120572rdquo Hepatology vol 33 no 1 pp 66ndash73 2001

[57] J Cao L Jiang X Zhang et al ldquoBoric acid inhibits LPS-inducedTNF-120572 formation through a thiol-dependent mechanism inTHP-1 cellsrdquo Journal of Trace Elements in Medicine and Biologyvol 22 no 3 pp 189ndash195 2008

[58] A R M A Meik S K Abdel-Ghaffar and I El-GibalyldquoAflatoxin B1 induces apoptosis in rat liver protective effect ofmelatoninrdquo Neuroendocrinology Letters vol 22 no 6 pp 417ndash426 2001

[59] CIREP ldquoFinal report on the safety assessment ofCalendula offic-inalis Extract and Calendula officinalisrdquo International JournalToxicology vol 20 pp 13ndash20 2001

[60] M A Rusu M Tamas C Puica I Roman and M SabadasldquoThe hepatoprotective action of ten herbal extracts in CCl

4

intoxicated liverrdquo Phytotherapy Research vol 19 no 9 pp 744ndash749 2005

[61] K C Preethi and R Kuttan ldquoHepato and reno protective actionof Calendula officinalis L Flower extractrdquo Indian Journal ofExperimental Biology vol 47 no 3 pp 163ndash168 2009

[62] B P Muley S S Khadabadi and N B Banarase ldquoPhytochem-ical constituents and pharmacological activities of Calendulaofficinalis Linn (Asteraceae) a reviewrdquo Tropical Journal ofPharmaceutical Research vol 8 no 5 pp 455ndash465 2009

[63] T Frankic K Salobir and J Salobir ldquoThe comparison of invivo antigenotoxic and antioxidative capacity of two propyleneglycol extracts of Calendula officinalis (marigold) and vitaminE in young growing pigsrdquo Journal of Animal Physiology andAnimal Nutrition vol 93 no 6 pp 688ndash694 2009

[64] M Uyanoglu M Canbek E Aral and K Husnu Can BaserldquoEffects of carvacrol upon the liver of rats undergoing partialhepatectomyrdquo Phytomedicine vol 15 no 3 pp 226ndash229 2008

[65] M L Tsai C C Lin W C Lin and C H Yang ldquoAntimicrobialantioxidant and anti-inflammatory activities of essential oilsfromfive selected herbsrdquoBioscience Biotechnology Biochemistryvol 75 no 10 pp 1977ndash1983 2011

[66] K Mayura M A Abdel-Mahhab K S McKenzie et alldquoPrevention of maternal and developmental toxicity in rats viadietary inclusion of common aflatoxin sorbents potential forhidden risksrdquo Toxicological Sciences vol 41 no 2 pp 175ndash1821998

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 ISRN Nutrition

(a) (b)

(c) (d)

(e)

Figure 4 Photomicrographs in liver sections stained with Periodic-Sheif reagent-stain (PAS) for glycogen demonstration from (a)control rats showing a strong reaction of PAS (b) rats fed AFs-contaminated diet showing a weak reaction in the damaged cellwhile a strong reaction in intact cells (c) rats treated with CE1 orCE2 showing different types of reaction as well as strong reactionaround the central vein and weak reaction around portal vein ofhepatocytes (d) rats fed with AFs contaminated diet treated withCE1 showing that weak reaction in some damaged cells whichscattered in all the section and (e) rats fed with AFs contaminateddiet and treated with CE2 showing marked improvement althoughlow reaction in hepatocytes were still demonstrated (PAS reactiontimes100)

a high total phenol amount and scavenging activity againstDPPH and ABTS radicals DallrsquoAcqua et al [28] investigatedantioxidant activity of Calendula arvensis used in Sardinianfolk medicine using DPPH radical scavenging assay andreported that the extract showed a high radical scavengingeffect towards DPPH The radical scavenging propertieswere attributed to the presence of flavonoid and triterpenederivatives in the extract [29]

In the in vivo study the selective dose of AFs andCalendula was literature-based [30 31] respectively Theresults revealed a significant reduction of body weight andfood intake in animals fed AFs-contaminated diet Theseresults were in agreement with those reported in the literatureof aflatoxicosis [32ndash38] The reduction in body weight inthe animals fed AFs-contaminated diet alone may be due tothe effects of AFs on the balance between orexigenic andanorexigenic circuits that regulate the homeostatic loop ofbody weight regulation leading to cachexia [34] Moreover

AFs exposure may lead to significant reduction of leptin[35] accompanied with high levels of cortisol IL-6 andinsulin resistance which together act to influence the feedingresponse causing weight loss in patients with pancreaticcancer [36] Osborne et al [37] suggested that AFs ingestionaffect various digestive enzymatic activities that give riseto a malabsorption syndrome characterized by steatorrheahypocarotenoidemy and to lowering of bile pancreatic lipasetrypsin and amylase Furthermore Lesson et al [38] reportedthat the 89 epoxide metabolite of AFB

1may covalently bind

to DNA and proteins which then alters enzymatic processessuch as gluconeogenesis Krebs cycle or fatty acid synthesis

Animals fed AFs-contaminated diet showed a significantincrease of ALT AST ALP urea and createnine whichindicate changes in the hepatic tissues and biliary system [39]structural damaging of liver integrity since these enzymes arecytoplasmic in location and released into plasma as a result ofcellular damage [8 40] It is also likely that alteration in ALPactivity affects membrane permeability and the transport ofmetabolites Moreover increase in urea and createnine levelobserved in the current study in AFs-treated group clearlyindicated the harmful and stressful effect on renal tissue [30]Taken together the increased level of urea and the decreasedlevel of albumin and total protein (TP) indicate the inhibitionof protein synthesis and increase of protein catabolism andorrenal dysfunction [41 42]These results clearly indicated thatAFs had stressful effects on the hepatic and renal tissuesconsistent with those reported in the literature of aflatoxicosis[43 44]

Animals treated with AFs showed a significant increasein AFP which is considered specific biomarkers for livercancer This increase in AFP may be due to induction ofexpression of mRNAs of liver alpha-fetoprotein [45 46] Onthe other hand aflatoxin B

1-induced hepatocarcinogenesis

associated with defective DNA-damage response by passingp53 activation [47] and modulation of insulin-like growthfactor 2 dependent signal axis (IGF-2) [48] Similar to theseobservations Sell et al [49] and Yang et al [45] reported thatAFB1administration resulted in the elevation of serum AFP

level in both duck and ratsThe current study showed that animals fed AFs-con-

taminated diet suffer from oxidative stress as indicated bythe significant increment of lipid peroxidation (MDA) andthe significant reduction of enzymatic antioxidant such assuperoxide dismutase (SOD) and glutathione peroxidase(GPx) These results are in agreement with previous studiessuggested that oxidative stress may be due to direct effectof AFs or by the metabolites formed and the free radicalsgenerated during the formation of these metabolites [3446 50] Moreover the reduction of protein synthesis inAFs-treated animals may affect certain metal ions (ie ironand copper) which play an important role in free radicalproduction and liberation [10] In normal state metal ionsare bonded to transfer proteins such as ceruloplasmin andtransferrin [51] and play a crucial role of SOD CAT and GPxactivities which constitute the enzymatic antioxidant defensesystem of the cell displayed bidirectional alterations either inthe form of increase or decrease depending on the particulartissue Previous studies demonstrated that the mechanism of

ISRN Nutrition 7

AFs-induced liver injury may be due to those reactive andtoxic metabolites of AFs and the liver necrosis begins whenthe glutathione stores are almost exhausted [34 35 52]

Tumor necrosis factor-alpha (TNF-120572) and interleukin-1 alpha (IL-1120572) are produced by macrophages and theyplay an important role in tumor conditions [35] TNF-120572is an essential factor in tumor promotion [53] and IL-1polymorphisms are important mediators in the inflamma-tory process [54] In the current study ingestion of AFs-contaminated diet significantly increased TNF-120572 and IL-1120573 suggesting that AFs preferentially affects macrophagefunctions In particular it decouples the close correlationsusually observed between transcriptional and translationalcontrols of IL-1120572 and TNF-120572 production by these cells [55]In this concern Barton et al [56] stated that TNF-120572 plays acausal role in the development of liver injury and it plays amajor role in modulating mycotoxin-induced hepatotoxicityMoreover TNF-120572 has been proven to play an importantrole in inflammation in mediating the proliferation anddifferentiation of immune cells and development of immuneresponse [57] Hopkins [55] and Abdel-Wahhab et al [35]reported that directly proportional relationship has beenobserved between transcriptional and translational controlsof IL-1 and TNF-120572 production by hepatocytes Consequentlythe current results indicated that aflatoxicosis may induceTNF-120572 and IL-1 due to activation of caspase-3 which is oneof the apoptotic mediator [58] and the significant elevationof inflammatory cytokinemay affect themRNA expression ofthese mediators [46]

The current results indicated that the ethanolic extract ofCalendula showed a potential protective effect against AFstoxicity Animals treated withCalendula extract did not showany sign of toxicity In this concern the safety of the ethanolextract of Calendula was suggested and the oral LD

50in rats

was estimated by 4640mgkg bw [59] The results reportedherein indicated that animals treated with Calendula extractshowed insignificant increase in food intake and body weightgain Moreover Calendula extract succeeded to induce asignificant improvement in the biochemical parameters andthe histological and histochemical pictures Taken togetherthe results of the in vitro study and the in vivo evaluationsuggested that there are an antioxidant and free radicalscavenging properties of this extract and this improvementmore pronounced with the high dose (CA2) [29] In thisconcern previous study suggested that Calendula plays animportant role in hepaticcytolysis inhibition and improvesliver integrity in CCl

4intoxicated rats [60] In referring to

previous literature hepatoprotective activity of Calendulamay be due to its modulatory effect on cytochrome P450which may interfere on aflatoxin active metabolite produc-tion [61]MoreoverCalendula extract showed renoprotectiveaction against cisplatin-induced renal toxicity through itsmodulatory effect against myelosuppression of the renalsystem

The existing data revealed anticarcinogenicity of Cal-endula extract indicated by significant reduction of AFPand this effect was pronounced in the animals treatedwith the high dose (CA2) Several reports suggested thatthe anticancer properties of Calendula may be due to its

chemoprotector properties against hepatocarcinogenesis inrats beside antitumoural activity in vivo in the Ehrlichmouse carcinoma model due to its ability to stimulate T-lymphocytes B-lymphocytes and cluster of differentiation(CD4+) [62] Moreover Calendula plays a crucial role inprevention of DNA damaging [63] These results were inagreement with those reported previously [26 31 62]

The results also suggested that Calendula extract pos-sesses anti-inflammatory activity which may be due to itshigher content of carvacol Carvacol has been described asa promoter for liver regeneration and inhibitor of TNF-120572and IL-6 level in rats undergoing partial hepatectomy [64]Furthermore Tsai et al [65] reported that caravacl couldreduce TNF-120572 and IL-1120573 levels in intoxicated rats through theinhibition of cycloxygensae (COX) enzymes activity mRNAexpression and its protein in lipopoly saccharides-inducedinflammation

The histopathological and histochemical changes in liverconfirmed the biochemical results and revealed that ani-mals fed AFs-contaminated diet showed severe histologicalchanges typical to those reported in the literature [10 39 66]However animals treated with the extract in combinationwith AFs resulted in a significant improvement in liver tissuessimilar to that reported previously [61]

5 Conclusion

It can be concluded that the ethanolic and aqueous extracts ofCalendula officinalis have a valuable quantity of total phenoliccompounds and have DPPH radical scavenging activityMoreover the ethanolic extract showed higher phenoliccontent and radical scavenging property compared to theaqueous extract The ethanolic extract exhibited hepatoreno-protective properties against aflatoxin-induced liver injury ina dose-dependent manner due to its antioxidant free radicalscavenging activities and anti-inflammatory properties

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

This work was full supported by College of Pharmacy MisrUniversity for Science and Technology 6th October CityEgypt Faculty of Pharmacy Cairo University Cairo EgyptNational Research Center Dokki Cairo Egypt

References

[1] CAST ldquoMycotoxins risks in plant animal and humanrdquo inPotential Economic Costs ofMycotoxins inUnited States pp 136ndash142 CAST Task Force Reports 2003

[2] J A Nogueira S K Ono-Nita M E Nita et al ldquo249 TP53mutation has high prevalence and is correlated with larger andpoorly differentiated HCC in Brazilian patientsrdquo BMC Cancervol 9 article 204 2009

8 ISRN Nutrition

[3] S Rawal J E Kim and R Coulombe Jr ldquoAflatoxin B1in

poultry toxicology metabolism and preventionrdquo Research inVeterinary Science vol 89 no 3 pp 325ndash331 2010

[4] International Agency for Research in Carcinogenesis ldquoAfla-toxinsrdquo Monograph on the Evaluation of Carcinogenic Risks toHumans vol 56 p 245 1993

[5] IPCS-WHO ldquoAflatoxinsrdquo in International Programme onChem-ical Safety WHO Food Additives Series 40 pp 1ndash73 WorldHealth Organization Geneva Switzerland 1998

[6] J H Williams T D Phillips P E Jolly J K Stiles C M JollyandDAggarwal ldquoHuman aflatoxicosis in developing countriesareview of toxicology exposure potential health consequencesand interventionsrdquo American Journal of Clinical Nutrition vol80 no 5 pp 1106ndash1122 2004

[7] K C Choi W T Chung J K Kwon et al ldquoInhibitory effectsof quercetin on aflatoxin B1-induced hepatic damage in micerdquoFood and Chemical Toxicology vol 48 no 10 pp 2747ndash27532010

[8] D S El-Agamy ldquoComparative effects of curcumin and resver-atrol on aflatoxin B 1-induced liver injury in ratsrdquo Archives ofToxicology vol 84 no 5 pp 389ndash396 2010

[9] M A Abdel-Wahhab A A Ibrahim A A El-Nekeety NS Hassan and A A Mohamed ldquoPanax ginseng CA Meyerextract counteracts the oxidative stress in rats fed multi-mycotoxins-contaminated dietrdquo Comunicata Scientiae vol 3no 3 pp 143ndash153 2012

[10] M A Hamzawy E S El-Denshary N S Hassan F A Mannaaand M A Abdel-Wahhab ldquoAntioxidant and hepatorenoprotec-tive effect of thyme vulgaris extract in rats during aflatoxicosisrdquoGlobal Journal Pharmacology vol 6 no 2 pp 106ndash117 2012

[11] Z C Gazim C M Rezende S R Fraga T I E Svidzinskiand D A G Cortez ldquoAntifungal activity of the essential oilfrom Calendula officinalis L (Asteraceae) growing in BrazilrdquoBrazilian Journal of Microbiology vol 39 no 1 pp 61ndash63 2008

[12] L R Della A Tubaro S Sosa H Becker S Saar and D IsaacldquoThe role of triperpenoids in the topical antiinflamatory activityofCalendula officinalisflowersrdquoPlantaMedica vol 60 no 6 pp516ndash520 1994

[13] Chrysolite 2010 httphealthwikinutcomCalendula-officin-alis-Pot-Marigold11v4g8pw

[14] A Pintea C Bele S Andrei and C Socaciu ldquoHPLC analysis ofcarotenoids in four varieties of Calendula officinalis L flowersrdquoActa Biologica Szegediensis vol 247 pp 37ndash40 2003

[15] R A Jacob ldquoThe integrated antioxidant systemrdquo NutritionResearch vol 15 no 5 pp 755ndash766 1995

[16] K Dahan M Fennal and N B Kumar ldquoLycopene in the pre-vention of prostate cancerrdquo Journal of the Society for IntegrativeOncology vol 6 no 1 pp 29ndash36 2008

[17] J E Hutchins andWMHagler ldquoRapid liquid chromatographicdetermination of aflatoxins in heavily contaminated cornrdquoJournal of the Association of Official Analytical Chemists vol 66no 6 pp 1458ndash1465 1983

[18] G K Jayaprakasha and L J Rao ldquoPhenolic constituents fromthe lichen parmotrema stuppeum (Nyl) Hale and their antioxi-dant activityrdquo Zeitschrift fur Naturforschung C vol 55 no 11-12pp 1018ndash1022 2000

[19] G K Jayaprakasha T Selvi and K K Sakariah ldquoAntibacterialand antioxidant activities of grape (Vitis vinifera) seed extractsrdquoFood Research International vol 36 no 2 pp 117ndash122 2003

[20] M Nogala-Kalucka J Korczak M Dratwia E Lampart-Szczapa A Siger and M Buchowski ldquoChanges in antioxidant

activity and free radical scavenging potential of rosemaryextract and tocopherols in isolated rapeseed oil triacylglycerolsduring accelerated testsrdquo Food Chemistry vol 93 no 2 pp 227ndash235 2005

[21] R A Drury E A Wallington and R Cancerson CarltonrsquosHistopathological Techniques Oxford University Press OxfordUK 4th edition 1980

[22] SAS IInstitute SAS Userrsquos Guide Statistics SAS Institute CaryNC USA

[23] R A Waller and D B Duncan ldquoA Bayes rule for the symmetricmultiple comparisons problemsrdquo Journal of American StatisticalAssociation vol 64 no 328 pp 1484ndash1503 1969

[24] L Majhenic M Skerget and Z Knez ldquoAntioxidant and antimi-crobial activity of guarana seed extractsrdquo Food Chemistry vol104 no 3 pp 1258ndash1268 2007

[25] G S Cetkovic S M Djilas J M Canadanovic-Brunet and VT Tumbas ldquoAntioxidant properties of marigold extractsrdquo FoodResearch International vol 37 no 7 pp 643ndash650 2004

[26] K C Preethi G Kuttan and R Kuttan ldquoAntioxidant potentialof an extract ofCalendula officinalis flowers in vitro and in vivordquoPharmaceutical Biology vol 44 no 9 pp 691ndash697 2006

[27] AODanila F Gatea andG L Radu ldquoPolyphenol compositionand antioxidant activity of selected medicinal herbsrdquoChemistryof Natural Compounds vol 47 no 1 pp 22ndash26 2011

[28] S DallrsquoAcqua R Cervellati M C Loi and G InnocentildquoEvaluation of in vitro antioxidant properties of some tradi-tional Sardinian medicinal plants investigation of the highantioxidant capacity of Rubus ulmifoliusrdquo Food Chemistry vol106 no 2 pp 745ndash749 2008

[29] T Ercetin F S Senol I Erdogan Orhan and G TokerldquoComparative assessment of antioxidant and cholinesteraseinhibitory properties of the marigold extracts from Calendulaarvensis L and Calendula officinalis Lrdquo Industrial Crops andProducts vol 36 no 1 pp 203ndash208 2012

[30] M A Abdel-Wahhab and S E Aly ldquoAntioxidants and radicalscavenging properties of vegetable extracts in rats fed aflatoxin-contaminated dietrdquo Journal of Agricultural and Food Chemistryvol 51 no 8 pp 2409ndash2414 2003

[31] C Gladine C Morand E Rock D Gruffat D Bauchart andD Durand ldquoThe antioxidative effect of plant extracts rich inpolyphenols differs between liver and muscle tissues in rats fedn-3 PUFA rich dietsrdquo Animal Feed Science and Technology vol139 no 3-4 pp 257ndash272 2007

[32] R H Rauber P Dilkin L Z Giacomini C A Araujo deAlmeida and C A Mallmann ldquoPerformance of Turkey poultsfed different doses of aflatoxins in the dietrdquo Poultry Science vol86 no 8 pp 1620ndash1624 2007

[33] SM RustemeyerW R Lamberson D R Ledoux et al ldquoEffectsof dietary aflatoxin on the health and performance of growingbarrowsrdquo Journal of Animal Science vol 88 no 11 pp 3624ndash3630 2010

[34] M A Abdel-Wahhab N S Hassan A A El-Kady et al ldquoRedginseng extract protects against aflatoxin B

1and fumonisins-

induced hepatic pre-cancerous lesions in ratsrdquo Food and Chem-ical Toxicology vol 48 no 2 pp 733ndash742 2010

[35] M A Abdel-Wahhab H H Ahmed and M M HagazildquoPrevention of aflatoxin B1-initiated hepatotoxicity in rat bymarine algae extractsrdquo Journal of Applied Toxicology vol 26 no3 pp 229ndash238 2006

[36] M D Barber D C McMillan A M Wallace J A RossT Preston and K C H Fearon ldquoThe response of leptin

ISRN Nutrition 9

interleukin-6 and fat oxidation to feeding in weight-losingpatients with pancreatic cancerrdquo British Journal of Cancer vol90 no 6 pp 1129ndash1132 2004

[37] D J Osborne W E Huff P B Hamilton and H R BurmeisterldquoComparison of ochratoxin aflatoxin and T-2 toxin for theireffects on selected parameters related to digestion and evidencefor specific metabolism of carotenoids in chickensrdquo PoultryScience vol 61 no 8 pp 1646ndash1652 1982

[38] S Lesson G Diaz and J Summers Poultry Metabolic Disordersand Mycotoxins University Books Montreal Canada 1995

[39] M A Abdel-Wahhab S A Nada and F A Khalil ldquoPhys-iological and toxicological responses in rats fed aflatoxin-contaminated diet with or without sorbent materialsrdquo AnimalFeed Science and Technology vol 97 no 3-4 pp 209ndash219 2002

[40] A A El-Nekeety S R Mohamed A S Hathout N S HassanS E Aly and M A Abdel-Wahhab ldquoAntioxidant properties ofThymus vulgaris oil against aflatoxin-induce oxidative stress inmale ratsrdquo Toxicon vol 57 no 7-8 pp 984ndash991 2011

[41] N Jindal S K Mahipal and N K Mahajan ldquoToxicity ofaflatoxin B1 in broiler chicks and its reduction by activatedcharcoalrdquo Research in Veterinary Science vol 56 no 1 pp 37ndash40 1994

[42] M A Abdel-Wahhab and S E Aly ldquoAntioxidant property ofNigella sativa (black cumin) and Syzygium aromaticum (clove)in rats during aflatoxicosisrdquo Journal of Applied Toxicology vol25 no 3 pp 218ndash223 2005

[43] D M Miller and D M Wilson Veterinary Diseases Relatedto Aflatoxins the Toxicology of Aflatoxins Academic Press SanDiego Calif USA 1994

[44] M A Abdel-Wahhab and A M Kholif ldquoMycotoxins in animalfeeds and prevention strategies a reviewrdquo Asian Journal ofAnimal Sciences vol 2 no 1 pp 7ndash25 2008

[45] C F Yang J Liu S Wasser H M Shen C E L Tan andC N Ong ldquoInhibition of ebselen on aflatoxin B1-inducedhepatocarcinogenesis in Fischer 344 ratsrdquo Carcinogenesis vol21 no 12 pp 2237ndash2243 2000

[46] S H Abdel-Aziem A M Hassan and M A Abdel-WahhabldquoDietary supplementation with whey protein and ginsengextract counteracts oxidative stress and DNA damage in ratsfed an aflatoxin-contaminated dietrdquoMutationResearch vol 723no 1 pp 65ndash71 2011

[47] O Gursoy-Yuzugullu H YuzugulluM Yilmaz andMOzturkldquoAflatoxin genotoxicity is associated with a defective DNAdamage response bypassing p53 activationrdquo Liver Internationalvol 31 no 4 pp 561ndash571 2011

[48] T Ubagai T Kikuchi T Fukusato and Y Ono ldquoAflatoxin B1

modulates the insulin-like growth factor-2 dependent signalingaxisrdquo Toxicology in Vitro vol 24 no 3 pp 783ndash789 2010

[49] S Sell K L Xu W E Huff L F Kabena R B Harveryand H A Dunsford ldquoAflatoxin exposure produces serumalphafetoprotein elevations and marked oval cell proliferationin young male Pekin ducklingsrdquo Pathology vol 30 no 1 pp34ndash39 1998

[50] M Kanbur G Eraslan Z S Sarica andO Aslan ldquoThe effects ofevening primrose oil on lipid peroxidation induced by subacuteaflatoxin exposure in micerdquo Food and Chemical Toxicology vol49 no 9 pp 1960ndash1964 2011

[51] E Gitto S Pellegrino P Gitto I Barberi and R J ReiterldquoOxidative stress of the newborn in the pre- and postnatalperiod and the clinical utility of melatoninrdquo Journal of PinealResearch vol 46 no 2 pp 128ndash139 2009

[52] M A Abdel-Wahhab A Said and A Huefner ldquoNMR andradical scavenging activities of patuletin from Urtica urensagainst aflatoxin B

1rdquo Pharmaceutical Biology vol 43 no 6 pp

515ndash525 2005[53] M Suganuma E Sueoka N Sueoka S Okabe and H Fujiki

ldquoMechanisms of cancer prevention by tea polyphenols based oninhibition of TNF-120572 expressionrdquo BioFactors vol 13 no 1-4 pp67ndash72 2000

[54] S Rollinson A P Levene F K Mensah et al ldquoGastric marginalzone lymphoma is associated with polymorphisms in genesinvolved in inflammatory response and antioxidative capacityrdquoBlood vol 102 no 3 pp 1007ndash1011 2003

[55] S J Hopkins ldquoThe pathophysiological role of cytokinesrdquo LegalMedicine vol 5 no 1 pp S45ndashS57 2003

[56] C C Barton E X Barton P E Ganey S L Kunkel and RA Roth ldquoBacterial lipopolysaccharide enhances aflatoxin B

1

hepatotoxicity in rats by a mechanism that depends on tumornecrosis factor 120572rdquo Hepatology vol 33 no 1 pp 66ndash73 2001

[57] J Cao L Jiang X Zhang et al ldquoBoric acid inhibits LPS-inducedTNF-120572 formation through a thiol-dependent mechanism inTHP-1 cellsrdquo Journal of Trace Elements in Medicine and Biologyvol 22 no 3 pp 189ndash195 2008

[58] A R M A Meik S K Abdel-Ghaffar and I El-GibalyldquoAflatoxin B1 induces apoptosis in rat liver protective effect ofmelatoninrdquo Neuroendocrinology Letters vol 22 no 6 pp 417ndash426 2001

[59] CIREP ldquoFinal report on the safety assessment ofCalendula offic-inalis Extract and Calendula officinalisrdquo International JournalToxicology vol 20 pp 13ndash20 2001

[60] M A Rusu M Tamas C Puica I Roman and M SabadasldquoThe hepatoprotective action of ten herbal extracts in CCl

4

intoxicated liverrdquo Phytotherapy Research vol 19 no 9 pp 744ndash749 2005

[61] K C Preethi and R Kuttan ldquoHepato and reno protective actionof Calendula officinalis L Flower extractrdquo Indian Journal ofExperimental Biology vol 47 no 3 pp 163ndash168 2009

[62] B P Muley S S Khadabadi and N B Banarase ldquoPhytochem-ical constituents and pharmacological activities of Calendulaofficinalis Linn (Asteraceae) a reviewrdquo Tropical Journal ofPharmaceutical Research vol 8 no 5 pp 455ndash465 2009

[63] T Frankic K Salobir and J Salobir ldquoThe comparison of invivo antigenotoxic and antioxidative capacity of two propyleneglycol extracts of Calendula officinalis (marigold) and vitaminE in young growing pigsrdquo Journal of Animal Physiology andAnimal Nutrition vol 93 no 6 pp 688ndash694 2009

[64] M Uyanoglu M Canbek E Aral and K Husnu Can BaserldquoEffects of carvacrol upon the liver of rats undergoing partialhepatectomyrdquo Phytomedicine vol 15 no 3 pp 226ndash229 2008

[65] M L Tsai C C Lin W C Lin and C H Yang ldquoAntimicrobialantioxidant and anti-inflammatory activities of essential oilsfromfive selected herbsrdquoBioscience Biotechnology Biochemistryvol 75 no 10 pp 1977ndash1983 2011

[66] K Mayura M A Abdel-Mahhab K S McKenzie et alldquoPrevention of maternal and developmental toxicity in rats viadietary inclusion of common aflatoxin sorbents potential forhidden risksrdquo Toxicological Sciences vol 41 no 2 pp 175ndash1821998

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

ISRN Nutrition 7

AFs-induced liver injury may be due to those reactive andtoxic metabolites of AFs and the liver necrosis begins whenthe glutathione stores are almost exhausted [34 35 52]

Tumor necrosis factor-alpha (TNF-120572) and interleukin-1 alpha (IL-1120572) are produced by macrophages and theyplay an important role in tumor conditions [35] TNF-120572is an essential factor in tumor promotion [53] and IL-1polymorphisms are important mediators in the inflamma-tory process [54] In the current study ingestion of AFs-contaminated diet significantly increased TNF-120572 and IL-1120573 suggesting that AFs preferentially affects macrophagefunctions In particular it decouples the close correlationsusually observed between transcriptional and translationalcontrols of IL-1120572 and TNF-120572 production by these cells [55]In this concern Barton et al [56] stated that TNF-120572 plays acausal role in the development of liver injury and it plays amajor role in modulating mycotoxin-induced hepatotoxicityMoreover TNF-120572 has been proven to play an importantrole in inflammation in mediating the proliferation anddifferentiation of immune cells and development of immuneresponse [57] Hopkins [55] and Abdel-Wahhab et al [35]reported that directly proportional relationship has beenobserved between transcriptional and translational controlsof IL-1 and TNF-120572 production by hepatocytes Consequentlythe current results indicated that aflatoxicosis may induceTNF-120572 and IL-1 due to activation of caspase-3 which is oneof the apoptotic mediator [58] and the significant elevationof inflammatory cytokinemay affect themRNA expression ofthese mediators [46]

The current results indicated that the ethanolic extract ofCalendula showed a potential protective effect against AFstoxicity Animals treated withCalendula extract did not showany sign of toxicity In this concern the safety of the ethanolextract of Calendula was suggested and the oral LD

50in rats

was estimated by 4640mgkg bw [59] The results reportedherein indicated that animals treated with Calendula extractshowed insignificant increase in food intake and body weightgain Moreover Calendula extract succeeded to induce asignificant improvement in the biochemical parameters andthe histological and histochemical pictures Taken togetherthe results of the in vitro study and the in vivo evaluationsuggested that there are an antioxidant and free radicalscavenging properties of this extract and this improvementmore pronounced with the high dose (CA2) [29] In thisconcern previous study suggested that Calendula plays animportant role in hepaticcytolysis inhibition and improvesliver integrity in CCl

4intoxicated rats [60] In referring to

previous literature hepatoprotective activity of Calendulamay be due to its modulatory effect on cytochrome P450which may interfere on aflatoxin active metabolite produc-tion [61]MoreoverCalendula extract showed renoprotectiveaction against cisplatin-induced renal toxicity through itsmodulatory effect against myelosuppression of the renalsystem

The existing data revealed anticarcinogenicity of Cal-endula extract indicated by significant reduction of AFPand this effect was pronounced in the animals treatedwith the high dose (CA2) Several reports suggested thatthe anticancer properties of Calendula may be due to its

chemoprotector properties against hepatocarcinogenesis inrats beside antitumoural activity in vivo in the Ehrlichmouse carcinoma model due to its ability to stimulate T-lymphocytes B-lymphocytes and cluster of differentiation(CD4+) [62] Moreover Calendula plays a crucial role inprevention of DNA damaging [63] These results were inagreement with those reported previously [26 31 62]

The results also suggested that Calendula extract pos-sesses anti-inflammatory activity which may be due to itshigher content of carvacol Carvacol has been described asa promoter for liver regeneration and inhibitor of TNF-120572and IL-6 level in rats undergoing partial hepatectomy [64]Furthermore Tsai et al [65] reported that caravacl couldreduce TNF-120572 and IL-1120573 levels in intoxicated rats through theinhibition of cycloxygensae (COX) enzymes activity mRNAexpression and its protein in lipopoly saccharides-inducedinflammation

The histopathological and histochemical changes in liverconfirmed the biochemical results and revealed that ani-mals fed AFs-contaminated diet showed severe histologicalchanges typical to those reported in the literature [10 39 66]However animals treated with the extract in combinationwith AFs resulted in a significant improvement in liver tissuessimilar to that reported previously [61]

5 Conclusion

It can be concluded that the ethanolic and aqueous extracts ofCalendula officinalis have a valuable quantity of total phenoliccompounds and have DPPH radical scavenging activityMoreover the ethanolic extract showed higher phenoliccontent and radical scavenging property compared to theaqueous extract The ethanolic extract exhibited hepatoreno-protective properties against aflatoxin-induced liver injury ina dose-dependent manner due to its antioxidant free radicalscavenging activities and anti-inflammatory properties

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

This work was full supported by College of Pharmacy MisrUniversity for Science and Technology 6th October CityEgypt Faculty of Pharmacy Cairo University Cairo EgyptNational Research Center Dokki Cairo Egypt

References

[1] CAST ldquoMycotoxins risks in plant animal and humanrdquo inPotential Economic Costs ofMycotoxins inUnited States pp 136ndash142 CAST Task Force Reports 2003

[2] J A Nogueira S K Ono-Nita M E Nita et al ldquo249 TP53mutation has high prevalence and is correlated with larger andpoorly differentiated HCC in Brazilian patientsrdquo BMC Cancervol 9 article 204 2009

8 ISRN Nutrition

[3] S Rawal J E Kim and R Coulombe Jr ldquoAflatoxin B1in

poultry toxicology metabolism and preventionrdquo Research inVeterinary Science vol 89 no 3 pp 325ndash331 2010

[4] International Agency for Research in Carcinogenesis ldquoAfla-toxinsrdquo Monograph on the Evaluation of Carcinogenic Risks toHumans vol 56 p 245 1993

[5] IPCS-WHO ldquoAflatoxinsrdquo in International Programme onChem-ical Safety WHO Food Additives Series 40 pp 1ndash73 WorldHealth Organization Geneva Switzerland 1998

[6] J H Williams T D Phillips P E Jolly J K Stiles C M JollyandDAggarwal ldquoHuman aflatoxicosis in developing countriesareview of toxicology exposure potential health consequencesand interventionsrdquo American Journal of Clinical Nutrition vol80 no 5 pp 1106ndash1122 2004

[7] K C Choi W T Chung J K Kwon et al ldquoInhibitory effectsof quercetin on aflatoxin B1-induced hepatic damage in micerdquoFood and Chemical Toxicology vol 48 no 10 pp 2747ndash27532010

[8] D S El-Agamy ldquoComparative effects of curcumin and resver-atrol on aflatoxin B 1-induced liver injury in ratsrdquo Archives ofToxicology vol 84 no 5 pp 389ndash396 2010

[9] M A Abdel-Wahhab A A Ibrahim A A El-Nekeety NS Hassan and A A Mohamed ldquoPanax ginseng CA Meyerextract counteracts the oxidative stress in rats fed multi-mycotoxins-contaminated dietrdquo Comunicata Scientiae vol 3no 3 pp 143ndash153 2012

[10] M A Hamzawy E S El-Denshary N S Hassan F A Mannaaand M A Abdel-Wahhab ldquoAntioxidant and hepatorenoprotec-tive effect of thyme vulgaris extract in rats during aflatoxicosisrdquoGlobal Journal Pharmacology vol 6 no 2 pp 106ndash117 2012

[11] Z C Gazim C M Rezende S R Fraga T I E Svidzinskiand D A G Cortez ldquoAntifungal activity of the essential oilfrom Calendula officinalis L (Asteraceae) growing in BrazilrdquoBrazilian Journal of Microbiology vol 39 no 1 pp 61ndash63 2008

[12] L R Della A Tubaro S Sosa H Becker S Saar and D IsaacldquoThe role of triperpenoids in the topical antiinflamatory activityofCalendula officinalisflowersrdquoPlantaMedica vol 60 no 6 pp516ndash520 1994

[13] Chrysolite 2010 httphealthwikinutcomCalendula-officin-alis-Pot-Marigold11v4g8pw

[14] A Pintea C Bele S Andrei and C Socaciu ldquoHPLC analysis ofcarotenoids in four varieties of Calendula officinalis L flowersrdquoActa Biologica Szegediensis vol 247 pp 37ndash40 2003

[15] R A Jacob ldquoThe integrated antioxidant systemrdquo NutritionResearch vol 15 no 5 pp 755ndash766 1995

[16] K Dahan M Fennal and N B Kumar ldquoLycopene in the pre-vention of prostate cancerrdquo Journal of the Society for IntegrativeOncology vol 6 no 1 pp 29ndash36 2008

[17] J E Hutchins andWMHagler ldquoRapid liquid chromatographicdetermination of aflatoxins in heavily contaminated cornrdquoJournal of the Association of Official Analytical Chemists vol 66no 6 pp 1458ndash1465 1983

[18] G K Jayaprakasha and L J Rao ldquoPhenolic constituents fromthe lichen parmotrema stuppeum (Nyl) Hale and their antioxi-dant activityrdquo Zeitschrift fur Naturforschung C vol 55 no 11-12pp 1018ndash1022 2000

[19] G K Jayaprakasha T Selvi and K K Sakariah ldquoAntibacterialand antioxidant activities of grape (Vitis vinifera) seed extractsrdquoFood Research International vol 36 no 2 pp 117ndash122 2003

[20] M Nogala-Kalucka J Korczak M Dratwia E Lampart-Szczapa A Siger and M Buchowski ldquoChanges in antioxidant

activity and free radical scavenging potential of rosemaryextract and tocopherols in isolated rapeseed oil triacylglycerolsduring accelerated testsrdquo Food Chemistry vol 93 no 2 pp 227ndash235 2005

[21] R A Drury E A Wallington and R Cancerson CarltonrsquosHistopathological Techniques Oxford University Press OxfordUK 4th edition 1980

[22] SAS IInstitute SAS Userrsquos Guide Statistics SAS Institute CaryNC USA

[23] R A Waller and D B Duncan ldquoA Bayes rule for the symmetricmultiple comparisons problemsrdquo Journal of American StatisticalAssociation vol 64 no 328 pp 1484ndash1503 1969

[24] L Majhenic M Skerget and Z Knez ldquoAntioxidant and antimi-crobial activity of guarana seed extractsrdquo Food Chemistry vol104 no 3 pp 1258ndash1268 2007

[25] G S Cetkovic S M Djilas J M Canadanovic-Brunet and VT Tumbas ldquoAntioxidant properties of marigold extractsrdquo FoodResearch International vol 37 no 7 pp 643ndash650 2004

[26] K C Preethi G Kuttan and R Kuttan ldquoAntioxidant potentialof an extract ofCalendula officinalis flowers in vitro and in vivordquoPharmaceutical Biology vol 44 no 9 pp 691ndash697 2006

[27] AODanila F Gatea andG L Radu ldquoPolyphenol compositionand antioxidant activity of selected medicinal herbsrdquoChemistryof Natural Compounds vol 47 no 1 pp 22ndash26 2011

[28] S DallrsquoAcqua R Cervellati M C Loi and G InnocentildquoEvaluation of in vitro antioxidant properties of some tradi-tional Sardinian medicinal plants investigation of the highantioxidant capacity of Rubus ulmifoliusrdquo Food Chemistry vol106 no 2 pp 745ndash749 2008

[29] T Ercetin F S Senol I Erdogan Orhan and G TokerldquoComparative assessment of antioxidant and cholinesteraseinhibitory properties of the marigold extracts from Calendulaarvensis L and Calendula officinalis Lrdquo Industrial Crops andProducts vol 36 no 1 pp 203ndash208 2012

[30] M A Abdel-Wahhab and S E Aly ldquoAntioxidants and radicalscavenging properties of vegetable extracts in rats fed aflatoxin-contaminated dietrdquo Journal of Agricultural and Food Chemistryvol 51 no 8 pp 2409ndash2414 2003

[31] C Gladine C Morand E Rock D Gruffat D Bauchart andD Durand ldquoThe antioxidative effect of plant extracts rich inpolyphenols differs between liver and muscle tissues in rats fedn-3 PUFA rich dietsrdquo Animal Feed Science and Technology vol139 no 3-4 pp 257ndash272 2007

[32] R H Rauber P Dilkin L Z Giacomini C A Araujo deAlmeida and C A Mallmann ldquoPerformance of Turkey poultsfed different doses of aflatoxins in the dietrdquo Poultry Science vol86 no 8 pp 1620ndash1624 2007

[33] SM RustemeyerW R Lamberson D R Ledoux et al ldquoEffectsof dietary aflatoxin on the health and performance of growingbarrowsrdquo Journal of Animal Science vol 88 no 11 pp 3624ndash3630 2010

[34] M A Abdel-Wahhab N S Hassan A A El-Kady et al ldquoRedginseng extract protects against aflatoxin B

1and fumonisins-

induced hepatic pre-cancerous lesions in ratsrdquo Food and Chem-ical Toxicology vol 48 no 2 pp 733ndash742 2010

[35] M A Abdel-Wahhab H H Ahmed and M M HagazildquoPrevention of aflatoxin B1-initiated hepatotoxicity in rat bymarine algae extractsrdquo Journal of Applied Toxicology vol 26 no3 pp 229ndash238 2006

[36] M D Barber D C McMillan A M Wallace J A RossT Preston and K C H Fearon ldquoThe response of leptin

ISRN Nutrition 9

interleukin-6 and fat oxidation to feeding in weight-losingpatients with pancreatic cancerrdquo British Journal of Cancer vol90 no 6 pp 1129ndash1132 2004

[37] D J Osborne W E Huff P B Hamilton and H R BurmeisterldquoComparison of ochratoxin aflatoxin and T-2 toxin for theireffects on selected parameters related to digestion and evidencefor specific metabolism of carotenoids in chickensrdquo PoultryScience vol 61 no 8 pp 1646ndash1652 1982

[38] S Lesson G Diaz and J Summers Poultry Metabolic Disordersand Mycotoxins University Books Montreal Canada 1995

[39] M A Abdel-Wahhab S A Nada and F A Khalil ldquoPhys-iological and toxicological responses in rats fed aflatoxin-contaminated diet with or without sorbent materialsrdquo AnimalFeed Science and Technology vol 97 no 3-4 pp 209ndash219 2002

[40] A A El-Nekeety S R Mohamed A S Hathout N S HassanS E Aly and M A Abdel-Wahhab ldquoAntioxidant properties ofThymus vulgaris oil against aflatoxin-induce oxidative stress inmale ratsrdquo Toxicon vol 57 no 7-8 pp 984ndash991 2011

[41] N Jindal S K Mahipal and N K Mahajan ldquoToxicity ofaflatoxin B1 in broiler chicks and its reduction by activatedcharcoalrdquo Research in Veterinary Science vol 56 no 1 pp 37ndash40 1994

[42] M A Abdel-Wahhab and S E Aly ldquoAntioxidant property ofNigella sativa (black cumin) and Syzygium aromaticum (clove)in rats during aflatoxicosisrdquo Journal of Applied Toxicology vol25 no 3 pp 218ndash223 2005

[43] D M Miller and D M Wilson Veterinary Diseases Relatedto Aflatoxins the Toxicology of Aflatoxins Academic Press SanDiego Calif USA 1994

[44] M A Abdel-Wahhab and A M Kholif ldquoMycotoxins in animalfeeds and prevention strategies a reviewrdquo Asian Journal ofAnimal Sciences vol 2 no 1 pp 7ndash25 2008

[45] C F Yang J Liu S Wasser H M Shen C E L Tan andC N Ong ldquoInhibition of ebselen on aflatoxin B1-inducedhepatocarcinogenesis in Fischer 344 ratsrdquo Carcinogenesis vol21 no 12 pp 2237ndash2243 2000

[46] S H Abdel-Aziem A M Hassan and M A Abdel-WahhabldquoDietary supplementation with whey protein and ginsengextract counteracts oxidative stress and DNA damage in ratsfed an aflatoxin-contaminated dietrdquoMutationResearch vol 723no 1 pp 65ndash71 2011

[47] O Gursoy-Yuzugullu H YuzugulluM Yilmaz andMOzturkldquoAflatoxin genotoxicity is associated with a defective DNAdamage response bypassing p53 activationrdquo Liver Internationalvol 31 no 4 pp 561ndash571 2011

[48] T Ubagai T Kikuchi T Fukusato and Y Ono ldquoAflatoxin B1

modulates the insulin-like growth factor-2 dependent signalingaxisrdquo Toxicology in Vitro vol 24 no 3 pp 783ndash789 2010

[49] S Sell K L Xu W E Huff L F Kabena R B Harveryand H A Dunsford ldquoAflatoxin exposure produces serumalphafetoprotein elevations and marked oval cell proliferationin young male Pekin ducklingsrdquo Pathology vol 30 no 1 pp34ndash39 1998

[50] M Kanbur G Eraslan Z S Sarica andO Aslan ldquoThe effects ofevening primrose oil on lipid peroxidation induced by subacuteaflatoxin exposure in micerdquo Food and Chemical Toxicology vol49 no 9 pp 1960ndash1964 2011

[51] E Gitto S Pellegrino P Gitto I Barberi and R J ReiterldquoOxidative stress of the newborn in the pre- and postnatalperiod and the clinical utility of melatoninrdquo Journal of PinealResearch vol 46 no 2 pp 128ndash139 2009

[52] M A Abdel-Wahhab A Said and A Huefner ldquoNMR andradical scavenging activities of patuletin from Urtica urensagainst aflatoxin B

1rdquo Pharmaceutical Biology vol 43 no 6 pp

515ndash525 2005[53] M Suganuma E Sueoka N Sueoka S Okabe and H Fujiki

ldquoMechanisms of cancer prevention by tea polyphenols based oninhibition of TNF-120572 expressionrdquo BioFactors vol 13 no 1-4 pp67ndash72 2000

[54] S Rollinson A P Levene F K Mensah et al ldquoGastric marginalzone lymphoma is associated with polymorphisms in genesinvolved in inflammatory response and antioxidative capacityrdquoBlood vol 102 no 3 pp 1007ndash1011 2003

[55] S J Hopkins ldquoThe pathophysiological role of cytokinesrdquo LegalMedicine vol 5 no 1 pp S45ndashS57 2003

[56] C C Barton E X Barton P E Ganey S L Kunkel and RA Roth ldquoBacterial lipopolysaccharide enhances aflatoxin B

1

hepatotoxicity in rats by a mechanism that depends on tumornecrosis factor 120572rdquo Hepatology vol 33 no 1 pp 66ndash73 2001

[57] J Cao L Jiang X Zhang et al ldquoBoric acid inhibits LPS-inducedTNF-120572 formation through a thiol-dependent mechanism inTHP-1 cellsrdquo Journal of Trace Elements in Medicine and Biologyvol 22 no 3 pp 189ndash195 2008

[58] A R M A Meik S K Abdel-Ghaffar and I El-GibalyldquoAflatoxin B1 induces apoptosis in rat liver protective effect ofmelatoninrdquo Neuroendocrinology Letters vol 22 no 6 pp 417ndash426 2001

[59] CIREP ldquoFinal report on the safety assessment ofCalendula offic-inalis Extract and Calendula officinalisrdquo International JournalToxicology vol 20 pp 13ndash20 2001

[60] M A Rusu M Tamas C Puica I Roman and M SabadasldquoThe hepatoprotective action of ten herbal extracts in CCl

4

intoxicated liverrdquo Phytotherapy Research vol 19 no 9 pp 744ndash749 2005

[61] K C Preethi and R Kuttan ldquoHepato and reno protective actionof Calendula officinalis L Flower extractrdquo Indian Journal ofExperimental Biology vol 47 no 3 pp 163ndash168 2009

[62] B P Muley S S Khadabadi and N B Banarase ldquoPhytochem-ical constituents and pharmacological activities of Calendulaofficinalis Linn (Asteraceae) a reviewrdquo Tropical Journal ofPharmaceutical Research vol 8 no 5 pp 455ndash465 2009

[63] T Frankic K Salobir and J Salobir ldquoThe comparison of invivo antigenotoxic and antioxidative capacity of two propyleneglycol extracts of Calendula officinalis (marigold) and vitaminE in young growing pigsrdquo Journal of Animal Physiology andAnimal Nutrition vol 93 no 6 pp 688ndash694 2009

[64] M Uyanoglu M Canbek E Aral and K Husnu Can BaserldquoEffects of carvacrol upon the liver of rats undergoing partialhepatectomyrdquo Phytomedicine vol 15 no 3 pp 226ndash229 2008

[65] M L Tsai C C Lin W C Lin and C H Yang ldquoAntimicrobialantioxidant and anti-inflammatory activities of essential oilsfromfive selected herbsrdquoBioscience Biotechnology Biochemistryvol 75 no 10 pp 1977ndash1983 2011

[66] K Mayura M A Abdel-Mahhab K S McKenzie et alldquoPrevention of maternal and developmental toxicity in rats viadietary inclusion of common aflatoxin sorbents potential forhidden risksrdquo Toxicological Sciences vol 41 no 2 pp 175ndash1821998

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 ISRN Nutrition

[3] S Rawal J E Kim and R Coulombe Jr ldquoAflatoxin B1in

poultry toxicology metabolism and preventionrdquo Research inVeterinary Science vol 89 no 3 pp 325ndash331 2010

[4] International Agency for Research in Carcinogenesis ldquoAfla-toxinsrdquo Monograph on the Evaluation of Carcinogenic Risks toHumans vol 56 p 245 1993

[5] IPCS-WHO ldquoAflatoxinsrdquo in International Programme onChem-ical Safety WHO Food Additives Series 40 pp 1ndash73 WorldHealth Organization Geneva Switzerland 1998

[6] J H Williams T D Phillips P E Jolly J K Stiles C M JollyandDAggarwal ldquoHuman aflatoxicosis in developing countriesareview of toxicology exposure potential health consequencesand interventionsrdquo American Journal of Clinical Nutrition vol80 no 5 pp 1106ndash1122 2004

[7] K C Choi W T Chung J K Kwon et al ldquoInhibitory effectsof quercetin on aflatoxin B1-induced hepatic damage in micerdquoFood and Chemical Toxicology vol 48 no 10 pp 2747ndash27532010

[8] D S El-Agamy ldquoComparative effects of curcumin and resver-atrol on aflatoxin B 1-induced liver injury in ratsrdquo Archives ofToxicology vol 84 no 5 pp 389ndash396 2010

[9] M A Abdel-Wahhab A A Ibrahim A A El-Nekeety NS Hassan and A A Mohamed ldquoPanax ginseng CA Meyerextract counteracts the oxidative stress in rats fed multi-mycotoxins-contaminated dietrdquo Comunicata Scientiae vol 3no 3 pp 143ndash153 2012

[10] M A Hamzawy E S El-Denshary N S Hassan F A Mannaaand M A Abdel-Wahhab ldquoAntioxidant and hepatorenoprotec-tive effect of thyme vulgaris extract in rats during aflatoxicosisrdquoGlobal Journal Pharmacology vol 6 no 2 pp 106ndash117 2012

[11] Z C Gazim C M Rezende S R Fraga T I E Svidzinskiand D A G Cortez ldquoAntifungal activity of the essential oilfrom Calendula officinalis L (Asteraceae) growing in BrazilrdquoBrazilian Journal of Microbiology vol 39 no 1 pp 61ndash63 2008

[12] L R Della A Tubaro S Sosa H Becker S Saar and D IsaacldquoThe role of triperpenoids in the topical antiinflamatory activityofCalendula officinalisflowersrdquoPlantaMedica vol 60 no 6 pp516ndash520 1994

[13] Chrysolite 2010 httphealthwikinutcomCalendula-officin-alis-Pot-Marigold11v4g8pw

[14] A Pintea C Bele S Andrei and C Socaciu ldquoHPLC analysis ofcarotenoids in four varieties of Calendula officinalis L flowersrdquoActa Biologica Szegediensis vol 247 pp 37ndash40 2003

[15] R A Jacob ldquoThe integrated antioxidant systemrdquo NutritionResearch vol 15 no 5 pp 755ndash766 1995

[16] K Dahan M Fennal and N B Kumar ldquoLycopene in the pre-vention of prostate cancerrdquo Journal of the Society for IntegrativeOncology vol 6 no 1 pp 29ndash36 2008

[17] J E Hutchins andWMHagler ldquoRapid liquid chromatographicdetermination of aflatoxins in heavily contaminated cornrdquoJournal of the Association of Official Analytical Chemists vol 66no 6 pp 1458ndash1465 1983

[18] G K Jayaprakasha and L J Rao ldquoPhenolic constituents fromthe lichen parmotrema stuppeum (Nyl) Hale and their antioxi-dant activityrdquo Zeitschrift fur Naturforschung C vol 55 no 11-12pp 1018ndash1022 2000

[19] G K Jayaprakasha T Selvi and K K Sakariah ldquoAntibacterialand antioxidant activities of grape (Vitis vinifera) seed extractsrdquoFood Research International vol 36 no 2 pp 117ndash122 2003

[20] M Nogala-Kalucka J Korczak M Dratwia E Lampart-Szczapa A Siger and M Buchowski ldquoChanges in antioxidant

activity and free radical scavenging potential of rosemaryextract and tocopherols in isolated rapeseed oil triacylglycerolsduring accelerated testsrdquo Food Chemistry vol 93 no 2 pp 227ndash235 2005

[21] R A Drury E A Wallington and R Cancerson CarltonrsquosHistopathological Techniques Oxford University Press OxfordUK 4th edition 1980

[22] SAS IInstitute SAS Userrsquos Guide Statistics SAS Institute CaryNC USA

[23] R A Waller and D B Duncan ldquoA Bayes rule for the symmetricmultiple comparisons problemsrdquo Journal of American StatisticalAssociation vol 64 no 328 pp 1484ndash1503 1969

[24] L Majhenic M Skerget and Z Knez ldquoAntioxidant and antimi-crobial activity of guarana seed extractsrdquo Food Chemistry vol104 no 3 pp 1258ndash1268 2007

[25] G S Cetkovic S M Djilas J M Canadanovic-Brunet and VT Tumbas ldquoAntioxidant properties of marigold extractsrdquo FoodResearch International vol 37 no 7 pp 643ndash650 2004

[26] K C Preethi G Kuttan and R Kuttan ldquoAntioxidant potentialof an extract ofCalendula officinalis flowers in vitro and in vivordquoPharmaceutical Biology vol 44 no 9 pp 691ndash697 2006

[27] AODanila F Gatea andG L Radu ldquoPolyphenol compositionand antioxidant activity of selected medicinal herbsrdquoChemistryof Natural Compounds vol 47 no 1 pp 22ndash26 2011

[28] S DallrsquoAcqua R Cervellati M C Loi and G InnocentildquoEvaluation of in vitro antioxidant properties of some tradi-tional Sardinian medicinal plants investigation of the highantioxidant capacity of Rubus ulmifoliusrdquo Food Chemistry vol106 no 2 pp 745ndash749 2008

[29] T Ercetin F S Senol I Erdogan Orhan and G TokerldquoComparative assessment of antioxidant and cholinesteraseinhibitory properties of the marigold extracts from Calendulaarvensis L and Calendula officinalis Lrdquo Industrial Crops andProducts vol 36 no 1 pp 203ndash208 2012

[30] M A Abdel-Wahhab and S E Aly ldquoAntioxidants and radicalscavenging properties of vegetable extracts in rats fed aflatoxin-contaminated dietrdquo Journal of Agricultural and Food Chemistryvol 51 no 8 pp 2409ndash2414 2003

[31] C Gladine C Morand E Rock D Gruffat D Bauchart andD Durand ldquoThe antioxidative effect of plant extracts rich inpolyphenols differs between liver and muscle tissues in rats fedn-3 PUFA rich dietsrdquo Animal Feed Science and Technology vol139 no 3-4 pp 257ndash272 2007

[32] R H Rauber P Dilkin L Z Giacomini C A Araujo deAlmeida and C A Mallmann ldquoPerformance of Turkey poultsfed different doses of aflatoxins in the dietrdquo Poultry Science vol86 no 8 pp 1620ndash1624 2007

[33] SM RustemeyerW R Lamberson D R Ledoux et al ldquoEffectsof dietary aflatoxin on the health and performance of growingbarrowsrdquo Journal of Animal Science vol 88 no 11 pp 3624ndash3630 2010

[34] M A Abdel-Wahhab N S Hassan A A El-Kady et al ldquoRedginseng extract protects against aflatoxin B

1and fumonisins-

induced hepatic pre-cancerous lesions in ratsrdquo Food and Chem-ical Toxicology vol 48 no 2 pp 733ndash742 2010

[35] M A Abdel-Wahhab H H Ahmed and M M HagazildquoPrevention of aflatoxin B1-initiated hepatotoxicity in rat bymarine algae extractsrdquo Journal of Applied Toxicology vol 26 no3 pp 229ndash238 2006

[36] M D Barber D C McMillan A M Wallace J A RossT Preston and K C H Fearon ldquoThe response of leptin

ISRN Nutrition 9

interleukin-6 and fat oxidation to feeding in weight-losingpatients with pancreatic cancerrdquo British Journal of Cancer vol90 no 6 pp 1129ndash1132 2004

[37] D J Osborne W E Huff P B Hamilton and H R BurmeisterldquoComparison of ochratoxin aflatoxin and T-2 toxin for theireffects on selected parameters related to digestion and evidencefor specific metabolism of carotenoids in chickensrdquo PoultryScience vol 61 no 8 pp 1646ndash1652 1982

[38] S Lesson G Diaz and J Summers Poultry Metabolic Disordersand Mycotoxins University Books Montreal Canada 1995

[39] M A Abdel-Wahhab S A Nada and F A Khalil ldquoPhys-iological and toxicological responses in rats fed aflatoxin-contaminated diet with or without sorbent materialsrdquo AnimalFeed Science and Technology vol 97 no 3-4 pp 209ndash219 2002

[40] A A El-Nekeety S R Mohamed A S Hathout N S HassanS E Aly and M A Abdel-Wahhab ldquoAntioxidant properties ofThymus vulgaris oil against aflatoxin-induce oxidative stress inmale ratsrdquo Toxicon vol 57 no 7-8 pp 984ndash991 2011

[41] N Jindal S K Mahipal and N K Mahajan ldquoToxicity ofaflatoxin B1 in broiler chicks and its reduction by activatedcharcoalrdquo Research in Veterinary Science vol 56 no 1 pp 37ndash40 1994

[42] M A Abdel-Wahhab and S E Aly ldquoAntioxidant property ofNigella sativa (black cumin) and Syzygium aromaticum (clove)in rats during aflatoxicosisrdquo Journal of Applied Toxicology vol25 no 3 pp 218ndash223 2005

[43] D M Miller and D M Wilson Veterinary Diseases Relatedto Aflatoxins the Toxicology of Aflatoxins Academic Press SanDiego Calif USA 1994

[44] M A Abdel-Wahhab and A M Kholif ldquoMycotoxins in animalfeeds and prevention strategies a reviewrdquo Asian Journal ofAnimal Sciences vol 2 no 1 pp 7ndash25 2008

[45] C F Yang J Liu S Wasser H M Shen C E L Tan andC N Ong ldquoInhibition of ebselen on aflatoxin B1-inducedhepatocarcinogenesis in Fischer 344 ratsrdquo Carcinogenesis vol21 no 12 pp 2237ndash2243 2000

[46] S H Abdel-Aziem A M Hassan and M A Abdel-WahhabldquoDietary supplementation with whey protein and ginsengextract counteracts oxidative stress and DNA damage in ratsfed an aflatoxin-contaminated dietrdquoMutationResearch vol 723no 1 pp 65ndash71 2011

[47] O Gursoy-Yuzugullu H YuzugulluM Yilmaz andMOzturkldquoAflatoxin genotoxicity is associated with a defective DNAdamage response bypassing p53 activationrdquo Liver Internationalvol 31 no 4 pp 561ndash571 2011

[48] T Ubagai T Kikuchi T Fukusato and Y Ono ldquoAflatoxin B1

modulates the insulin-like growth factor-2 dependent signalingaxisrdquo Toxicology in Vitro vol 24 no 3 pp 783ndash789 2010

[49] S Sell K L Xu W E Huff L F Kabena R B Harveryand H A Dunsford ldquoAflatoxin exposure produces serumalphafetoprotein elevations and marked oval cell proliferationin young male Pekin ducklingsrdquo Pathology vol 30 no 1 pp34ndash39 1998

[50] M Kanbur G Eraslan Z S Sarica andO Aslan ldquoThe effects ofevening primrose oil on lipid peroxidation induced by subacuteaflatoxin exposure in micerdquo Food and Chemical Toxicology vol49 no 9 pp 1960ndash1964 2011

[51] E Gitto S Pellegrino P Gitto I Barberi and R J ReiterldquoOxidative stress of the newborn in the pre- and postnatalperiod and the clinical utility of melatoninrdquo Journal of PinealResearch vol 46 no 2 pp 128ndash139 2009

[52] M A Abdel-Wahhab A Said and A Huefner ldquoNMR andradical scavenging activities of patuletin from Urtica urensagainst aflatoxin B

1rdquo Pharmaceutical Biology vol 43 no 6 pp

515ndash525 2005[53] M Suganuma E Sueoka N Sueoka S Okabe and H Fujiki

ldquoMechanisms of cancer prevention by tea polyphenols based oninhibition of TNF-120572 expressionrdquo BioFactors vol 13 no 1-4 pp67ndash72 2000

[54] S Rollinson A P Levene F K Mensah et al ldquoGastric marginalzone lymphoma is associated with polymorphisms in genesinvolved in inflammatory response and antioxidative capacityrdquoBlood vol 102 no 3 pp 1007ndash1011 2003

[55] S J Hopkins ldquoThe pathophysiological role of cytokinesrdquo LegalMedicine vol 5 no 1 pp S45ndashS57 2003

[56] C C Barton E X Barton P E Ganey S L Kunkel and RA Roth ldquoBacterial lipopolysaccharide enhances aflatoxin B

1

hepatotoxicity in rats by a mechanism that depends on tumornecrosis factor 120572rdquo Hepatology vol 33 no 1 pp 66ndash73 2001

[57] J Cao L Jiang X Zhang et al ldquoBoric acid inhibits LPS-inducedTNF-120572 formation through a thiol-dependent mechanism inTHP-1 cellsrdquo Journal of Trace Elements in Medicine and Biologyvol 22 no 3 pp 189ndash195 2008

[58] A R M A Meik S K Abdel-Ghaffar and I El-GibalyldquoAflatoxin B1 induces apoptosis in rat liver protective effect ofmelatoninrdquo Neuroendocrinology Letters vol 22 no 6 pp 417ndash426 2001

[59] CIREP ldquoFinal report on the safety assessment ofCalendula offic-inalis Extract and Calendula officinalisrdquo International JournalToxicology vol 20 pp 13ndash20 2001

[60] M A Rusu M Tamas C Puica I Roman and M SabadasldquoThe hepatoprotective action of ten herbal extracts in CCl

4

intoxicated liverrdquo Phytotherapy Research vol 19 no 9 pp 744ndash749 2005

[61] K C Preethi and R Kuttan ldquoHepato and reno protective actionof Calendula officinalis L Flower extractrdquo Indian Journal ofExperimental Biology vol 47 no 3 pp 163ndash168 2009

[62] B P Muley S S Khadabadi and N B Banarase ldquoPhytochem-ical constituents and pharmacological activities of Calendulaofficinalis Linn (Asteraceae) a reviewrdquo Tropical Journal ofPharmaceutical Research vol 8 no 5 pp 455ndash465 2009

[63] T Frankic K Salobir and J Salobir ldquoThe comparison of invivo antigenotoxic and antioxidative capacity of two propyleneglycol extracts of Calendula officinalis (marigold) and vitaminE in young growing pigsrdquo Journal of Animal Physiology andAnimal Nutrition vol 93 no 6 pp 688ndash694 2009

[64] M Uyanoglu M Canbek E Aral and K Husnu Can BaserldquoEffects of carvacrol upon the liver of rats undergoing partialhepatectomyrdquo Phytomedicine vol 15 no 3 pp 226ndash229 2008

[65] M L Tsai C C Lin W C Lin and C H Yang ldquoAntimicrobialantioxidant and anti-inflammatory activities of essential oilsfromfive selected herbsrdquoBioscience Biotechnology Biochemistryvol 75 no 10 pp 1977ndash1983 2011

[66] K Mayura M A Abdel-Mahhab K S McKenzie et alldquoPrevention of maternal and developmental toxicity in rats viadietary inclusion of common aflatoxin sorbents potential forhidden risksrdquo Toxicological Sciences vol 41 no 2 pp 175ndash1821998

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

ISRN Nutrition 9

interleukin-6 and fat oxidation to feeding in weight-losingpatients with pancreatic cancerrdquo British Journal of Cancer vol90 no 6 pp 1129ndash1132 2004

[37] D J Osborne W E Huff P B Hamilton and H R BurmeisterldquoComparison of ochratoxin aflatoxin and T-2 toxin for theireffects on selected parameters related to digestion and evidencefor specific metabolism of carotenoids in chickensrdquo PoultryScience vol 61 no 8 pp 1646ndash1652 1982

[38] S Lesson G Diaz and J Summers Poultry Metabolic Disordersand Mycotoxins University Books Montreal Canada 1995

[39] M A Abdel-Wahhab S A Nada and F A Khalil ldquoPhys-iological and toxicological responses in rats fed aflatoxin-contaminated diet with or without sorbent materialsrdquo AnimalFeed Science and Technology vol 97 no 3-4 pp 209ndash219 2002

[40] A A El-Nekeety S R Mohamed A S Hathout N S HassanS E Aly and M A Abdel-Wahhab ldquoAntioxidant properties ofThymus vulgaris oil against aflatoxin-induce oxidative stress inmale ratsrdquo Toxicon vol 57 no 7-8 pp 984ndash991 2011

[41] N Jindal S K Mahipal and N K Mahajan ldquoToxicity ofaflatoxin B1 in broiler chicks and its reduction by activatedcharcoalrdquo Research in Veterinary Science vol 56 no 1 pp 37ndash40 1994

[42] M A Abdel-Wahhab and S E Aly ldquoAntioxidant property ofNigella sativa (black cumin) and Syzygium aromaticum (clove)in rats during aflatoxicosisrdquo Journal of Applied Toxicology vol25 no 3 pp 218ndash223 2005

[43] D M Miller and D M Wilson Veterinary Diseases Relatedto Aflatoxins the Toxicology of Aflatoxins Academic Press SanDiego Calif USA 1994

[44] M A Abdel-Wahhab and A M Kholif ldquoMycotoxins in animalfeeds and prevention strategies a reviewrdquo Asian Journal ofAnimal Sciences vol 2 no 1 pp 7ndash25 2008

[45] C F Yang J Liu S Wasser H M Shen C E L Tan andC N Ong ldquoInhibition of ebselen on aflatoxin B1-inducedhepatocarcinogenesis in Fischer 344 ratsrdquo Carcinogenesis vol21 no 12 pp 2237ndash2243 2000

[46] S H Abdel-Aziem A M Hassan and M A Abdel-WahhabldquoDietary supplementation with whey protein and ginsengextract counteracts oxidative stress and DNA damage in ratsfed an aflatoxin-contaminated dietrdquoMutationResearch vol 723no 1 pp 65ndash71 2011

[47] O Gursoy-Yuzugullu H YuzugulluM Yilmaz andMOzturkldquoAflatoxin genotoxicity is associated with a defective DNAdamage response bypassing p53 activationrdquo Liver Internationalvol 31 no 4 pp 561ndash571 2011

[48] T Ubagai T Kikuchi T Fukusato and Y Ono ldquoAflatoxin B1

modulates the insulin-like growth factor-2 dependent signalingaxisrdquo Toxicology in Vitro vol 24 no 3 pp 783ndash789 2010

[49] S Sell K L Xu W E Huff L F Kabena R B Harveryand H A Dunsford ldquoAflatoxin exposure produces serumalphafetoprotein elevations and marked oval cell proliferationin young male Pekin ducklingsrdquo Pathology vol 30 no 1 pp34ndash39 1998

[50] M Kanbur G Eraslan Z S Sarica andO Aslan ldquoThe effects ofevening primrose oil on lipid peroxidation induced by subacuteaflatoxin exposure in micerdquo Food and Chemical Toxicology vol49 no 9 pp 1960ndash1964 2011

[51] E Gitto S Pellegrino P Gitto I Barberi and R J ReiterldquoOxidative stress of the newborn in the pre- and postnatalperiod and the clinical utility of melatoninrdquo Journal of PinealResearch vol 46 no 2 pp 128ndash139 2009

[52] M A Abdel-Wahhab A Said and A Huefner ldquoNMR andradical scavenging activities of patuletin from Urtica urensagainst aflatoxin B

1rdquo Pharmaceutical Biology vol 43 no 6 pp

515ndash525 2005[53] M Suganuma E Sueoka N Sueoka S Okabe and H Fujiki

ldquoMechanisms of cancer prevention by tea polyphenols based oninhibition of TNF-120572 expressionrdquo BioFactors vol 13 no 1-4 pp67ndash72 2000

[54] S Rollinson A P Levene F K Mensah et al ldquoGastric marginalzone lymphoma is associated with polymorphisms in genesinvolved in inflammatory response and antioxidative capacityrdquoBlood vol 102 no 3 pp 1007ndash1011 2003

[55] S J Hopkins ldquoThe pathophysiological role of cytokinesrdquo LegalMedicine vol 5 no 1 pp S45ndashS57 2003

[56] C C Barton E X Barton P E Ganey S L Kunkel and RA Roth ldquoBacterial lipopolysaccharide enhances aflatoxin B

1

hepatotoxicity in rats by a mechanism that depends on tumornecrosis factor 120572rdquo Hepatology vol 33 no 1 pp 66ndash73 2001

[57] J Cao L Jiang X Zhang et al ldquoBoric acid inhibits LPS-inducedTNF-120572 formation through a thiol-dependent mechanism inTHP-1 cellsrdquo Journal of Trace Elements in Medicine and Biologyvol 22 no 3 pp 189ndash195 2008

[58] A R M A Meik S K Abdel-Ghaffar and I El-GibalyldquoAflatoxin B1 induces apoptosis in rat liver protective effect ofmelatoninrdquo Neuroendocrinology Letters vol 22 no 6 pp 417ndash426 2001

[59] CIREP ldquoFinal report on the safety assessment ofCalendula offic-inalis Extract and Calendula officinalisrdquo International JournalToxicology vol 20 pp 13ndash20 2001

[60] M A Rusu M Tamas C Puica I Roman and M SabadasldquoThe hepatoprotective action of ten herbal extracts in CCl

4

intoxicated liverrdquo Phytotherapy Research vol 19 no 9 pp 744ndash749 2005

[61] K C Preethi and R Kuttan ldquoHepato and reno protective actionof Calendula officinalis L Flower extractrdquo Indian Journal ofExperimental Biology vol 47 no 3 pp 163ndash168 2009

[62] B P Muley S S Khadabadi and N B Banarase ldquoPhytochem-ical constituents and pharmacological activities of Calendulaofficinalis Linn (Asteraceae) a reviewrdquo Tropical Journal ofPharmaceutical Research vol 8 no 5 pp 455ndash465 2009

[63] T Frankic K Salobir and J Salobir ldquoThe comparison of invivo antigenotoxic and antioxidative capacity of two propyleneglycol extracts of Calendula officinalis (marigold) and vitaminE in young growing pigsrdquo Journal of Animal Physiology andAnimal Nutrition vol 93 no 6 pp 688ndash694 2009

[64] M Uyanoglu M Canbek E Aral and K Husnu Can BaserldquoEffects of carvacrol upon the liver of rats undergoing partialhepatectomyrdquo Phytomedicine vol 15 no 3 pp 226ndash229 2008

[65] M L Tsai C C Lin W C Lin and C H Yang ldquoAntimicrobialantioxidant and anti-inflammatory activities of essential oilsfromfive selected herbsrdquoBioscience Biotechnology Biochemistryvol 75 no 10 pp 1977ndash1983 2011

[66] K Mayura M A Abdel-Mahhab K S McKenzie et alldquoPrevention of maternal and developmental toxicity in rats viadietary inclusion of common aflatoxin sorbents potential forhidden risksrdquo Toxicological Sciences vol 41 no 2 pp 175ndash1821998

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom