Annals of Agricultural Science (2013) 58(1), 27–32

Faculty of Agriculture, Ain Shams University

Annals of Agricultural Science

www.elsevier.com/locate/aoas

ORIGINAL ARTICLE

Antioxidant activity and hepatoprotective effect

of pomegranate peel and whey powders in rats

I.S. Ashoush a,*, O.I. El-Batawy a, Gehan A. El-Shourbagy b

a Food Science Department, Faculty of Agriculture, Ain Shams University, Shoubra El-Kheima, Cairo, Egyptb Food Science Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt

Received 5 November 2012; accepted 19 November 2012Available online 27 February 2013

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KEYWORDS

Pomegranate peel;

Whey powder;

Antioxidant;

Hepatoprotective effect;

Rats

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niversity.

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Abstract The antioxidant activity of pomegranate peel powder (PPP) and whey powder (WP) was

evaluated, their hepatoprotective effect of each alone or in combination (PPWP) at equal levels was

also evaluated in Wistar rats against carbon tetrachloride (CCL4) induced liver injury. The hepato-

protective activity was assessed using various biochemical parameters and histopathological studies.

The results indicated that both PPP and WP exhibited antioxidant activity. Also, rats fed on diets

supplemented with 10% PPP, 10% WP or 10% of their mixture (PPWP) for 28 days showed a

potential hepatoprotective effects compared to liver injury control group (IC). They succeeded to

restore the biochemical parameters and improved the histological alteration of the liver. This

improvement was pronounced in the group received PPWP. It could be concluded that whey pow-

der should be incorporated with pomegranate peel powder when used as ingredients in functional

foods for people suffering from liver diseases.ª 2013 Faculty of Agriculture, Ain Shams University. Production and hosting by Elsevier B.V.

Open access under CC BY-NC-ND license.

Introduction

The liver is an amazingly complex organ which virtually affects

every physiological process of the body. Our body is protectedfrom various injurious substances and toxic metabolic byprod-ucts by the liver, which has been absorbed from intestinal

01001843122.

om (I.S. Ashoush).

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tract. Xenobiotics are often reported to cause potential hepaticdamage.

Liver diseases constitute a major problem of worldwide

proportions. Carbon tetrachloride (CCl4) is a well knownhepatotoxin that is widely used to induce acute-toxic liver in-jury in a large range of laboratory animals (Sahu, 2007). The

toxic effects of CCl4 are the consequences of production of freeradicals. These reactive free radicals initiate cell damagethrough two major mechanisms of covalent binding and lipid

peroxidation. Lipid peroxidative process has been shown toaugment collagen synthesis and fibrosis. Hence, antioxidantsmay play a role in inhibiting the liver injury induced during celldamage (Slater, 1984).

Foods rich in natural antioxidants have been proposed as atool to prevent and cure liver damage (Morisco et al., 2008).The pomegranate is one of the important dietary sources of

on and hosting by Elsevier B.V. Open access under CC BY-NC-ND license.

28 I.S. Ashoush et al.

antioxidant phenolics Murthy et al. (2002) and Ozgen et al.(2008). Pomegranate peel is recognized for its many health-promoting qualities and apparent wound-healing properties

(Chidambara et al., 2004), anticancer property (Jeune et al.,2005), antiatherosclerotic and antioxidative capacities (Tzulkeret al., 2007).

Whey is a general term that typically denotes the translu-cent liquid part of milk that remains following the process(coagulation and curd removal) of cheese manufacturing.

Whey also is one of two major protein groups of bovine milk,accounting for 20% of the milk while casein accounts for theremainder (Haffman and Falvo, 2004). Whey proteins areincreasingly being used for nutritional purposes because they

consistently received high score in traditional tests of proteinquality. The content of total essential amino acids andbranched-chain amino acids is superior in whey protein than

in most dietary proteins (Helaine et al., 2001).Because milk whey is an abundant by-product in cheese

manufacture (9 l of whey are produced from 10 l of milk dur-

ing cheese making (Manso and Lopez-Fandino, 2004) it isinexpensive source of high nutritional quality protein for usesas a physiologically functional food ingredient. So, the use of

whey protein in formulating products is increasing due to thenutritional and health benefits attributed to these proteins(Onwulata et al., 2004). These proteins exhibit potent antioxi-dant activities by inducing cellular biosynthesis of glutathione

(GSH), which can boost the immune system and detoxifypotential carcinogens and its role in cancer treatment wasreported by (Counous, 2000; Haug et al., 2007 and Bayram

et al., 2008). In an earlier work, Eason et al. (2004) showed thatwhey powder reduced mammary tumor.

The objectives of the present study were to investigate the

potential hepatoprotective of pomegranate peel powder(PPP) as a natural source for antioxidant and whey powder(WP) which is using in formulating products as well as their

combination (PPWP) against CCL4 induced liver injury inWister rats.

Table 1 Compositions of the experimental diets (%).

Component % NC IC PPP WP PPWP

Corn starch 65.2 65.2 55.2 55.2 55.2

Casein 18 18 18 18 18

Corn oil 7 7 7 7 7

Cellulose 5 5 5 5 5

Salt mixture 3.5 3.5 3.5 3.5 3.5

Vitamin mixture 1 1 1 1 1

Choline bitartrate 0.3 0.3 0.3 0.3 0.3

Pomegranate peel powder – – 10 – 5

Whey powder – – – 10 5

Materials and methods

Materials

The full ripe pomegranate fruits (Punica granatum L.) wonder-ful variety was obtained from the local market. Whey powdercontaining 11% protein, was purchased from Green Land for

Food Industries 10Th of Ramadan City, Egypt. While, Com-mercial kits used for determining alanine aminotransferase(ALT), aspartate aminotransferase (AST), malondialdehyde

(MDA) and reduced glutathione (GSH) were obtained fromBiodiagnostic Co. Dokki, Egypt. Carbon tetrachloride, so-dium carbonate and methanol were obtained from El-Gomho-

reya Co., Cairo, Egypt. 1,1-diphenyl-2-picrylhydrazyl radical(DPPH) and Folin–Ciocalteus phenol reagent were purchasedfrom Sigma–Aldrich Inc. (St. Louis, MO, USA).

Animals

Wistar rats with an average weight of 140 g were obtainedfrom the Organization of Biological Products and Vaccines

(Helwan Farm, Cairo, Egypt).

Preparation of pomegranate peels powder (PPP)

Pomegranate fruits were washed by distilled water then peeledand their edible portions were carefully separated. The peelswere air dried in a ventilated oven at 40 �C for 48 h and ground

to a fine powder.

Methods

Total phenolic content

The total phenolic content of the PPP was methanolic ex-tracted and determined according to Singleton et al. (1999).

The reaction mixture contained 0.5 ml of Folin–Ciocalteu re-agent, 0.5 ml of 7.5% Na2CO3 and 0.5 ml of the PPP extract.The absorbance was measured at 765 nm after the mixture was

stirred and allowed to stand for 30 min. The results were ex-pressed as gallic acid equivalents (GAE) per gram PPP (mgGAE/g powder) by reference to the gallic acid standard curve.

DPPH radical scavenge activity %

The ability of the PPP methanolic extract to scavenge 1,1-di-phenyl-2-picrylhydrazyl radical (DPPH) free radicals were

determined by the method described by Singh et al. (2002).While, the free radical scavenging activity of the whey powderaqueous extract was determined according to the method ofKennedy et al. (1995)

Biological experiment

The experiment was conducted on forty male Wistar rats, they

were housed in screen-bottomed aluminum cages in roomsmaintained at 25 ± 1 �C with alternating cycles of light anddark of 12 h duration. The animals were fed on basal dietaccording to AIN-93 guidelines (Reeves et al., 1993) and were

provided with water ad libitum during the experimentalperiod.

The animals were randomly divided into five groups with

eight rats in each group. Group one was reserved as normalcontrol (NC), groups two–five animals were administratedinterperitoneal (IP) injection with single dose of 0.5 ml/kg

body weight (2:5 v/v CCl4/paraffin). Group two kept as injurycontrol (IC), group three received basal diet with 10% pome-granate peel powder (PPP), group four received basal diet with

10% whey powder (WP) and group five received basal dietwith a combination of 5% pomegranate peel and 5% wheypowder (PPWP) for 28 days. The composition of the experi-mental diets is shown in Table 1, the changes in body weight

Antioxidant activity and hepatoprotective effect of pomegranate peel and whey powders in rats 29

were recorded weekly, blood samples were also taken from theretro-orbital plexus of the eyes from all animals of each groupat the end of the experiment; the organ (liver) was excised

immediately after bleeding and weighted. Plasma was obtainedfrom blood samples by centrifugation at 1500 rpm for 15 minat an ambient temperature for analysis.

Liver function

The activities of plasma alanine aminotransferase (ALT) andaspartate aminotransferase (AST) were determined colorimet-

rically at 505 nm according to the method adopted by Reitmanand Frankel (1957).

Oxidative stress parameter

The extent of lipid peroxidation in plasma was determined bymeasurement of malondialdehyde (MDA) formation at534 nm using the thiobarbituric acid reactive substances

(TBARS) method as described by Ohkawa et al. (1979). Thereduced glutathione (GSH) in the plasma was estimated byits reaction with dithio-bis-2-nitrobenzoic acid (DTNB)

according to the method described by Beutler et al., 1963.

Tissue sampling

Autopsy samples were taken from the liver of the different

groups of rats and fixed in 10% formol saline for 24 h. Wash-ing was done with tap water then serial dilutions of alcohol(methyl, ethyl and absolute ethyl) for dehydration. Specimens

were cleared in xylene embedded in paraffin at 56� in hot airoven for 24 h. Paraffin bees wax tissue blocks were preparedfor sectioning at 4 lm thickness by slidge microtome. The ob-tained tissue sections were collected on glass slides, deparaffi-

nized and stained by hematoxylin and eosin stains forhistopathological examinations through the light microscope(Banchroft et al., 1996).

Statistical analysis

Data were expressed as mean values ± SD of eight rats in eachgroup. Statistical analysis was performed using one way anal-

ysis of variance (ANOVA) followed by Duncan’s MultipleRange Test with P < 0.05 being considered statistically signif-icant. Statistical analysis was conducted with SAS program

(SAS, 1996).

Results and discussion

Total phenolics content and antioxidant activity

From the data presented in Table 2, it could be noticed that thepomegranate peel powder (PPP) is a good source of total phen-olics and had a great free radical scavenging activity. While,

whey powder (WP) recorded the least scavenging activity com-

Table 2 Total phenolics content and antioxidant activity of

pomegranate peel powder (PPP) and whey powder (WP).

Scavenging activity % Total phenolic (mg/g)

PPP 96.24 10.96

WP 72.15 –

pared to PPP. These results are in agreement with those ofIbrahium (2010) and El-Kady et al. (2010).

Body weight gain and relative liver weight in rats

The initial body weights of all rats groups were not signifi-cantly different, however, after 28 days of feeding; body weight

gain were significantly lower in liver injury control group (IC)treated with CCl4 as compared to other groups (Table 3). Onthe other hand, rats fed on diets supplemented with pomegran-

ate peel (PPP), whey powder (WP) and their mixture (PPWP)occurred higher increased in body weight gain compared to IC.These results hypothesized that the applied treatments may im-

prove appetite and enhance weight gain. From the same table,it could be noticed that the relative weight of liver was signif-icantly higher in the IC group than in the other groups (Table3). These results are in agreement with the findings of Osman

et al. (2011) showed that CCl4-treated rats increased the rela-tive organ weights of liver.

Liver function parameters

The liver is a major target organ for toxicity of xenobiotics anddrugs, because most of the orally ingested. The present study

was focused on investigating the role of pomegranate peel,whey powder and their combination against CCl4 induced hep-atotoxicity and to find its possible mode of action inhepatoprotection.

Rats subjected to CCl4 developed significant hepatocellulardamage as evident from the plasma activities of ALT and ASTcompared to normal values, and which have been used as reli-

able markers of hepatotoxicity (Table 4). Supplemented dietwith pomegranate peel powder (PPP), whey powder (WP)and their combination (PPWP) exhibited a significant reduc-

tion in the levels of ALT and AST as compared with liver in-jury control rats group. This was also reported by Manal andAbd El-Megeid (2006), Ibrahium (2010) and Hamad et al.

(2011).

Oxidative stress parameters

Increased lipid peroxidation is generally believed to be an

important underlying cause of the initiation of oxidative stressrelated to various tissue injury, cell death, and further progres-sion of many acute and chronic diseases (Halliwell and Gutter-

idge, 1999).Table 5 shows the changes in the levels of plasma thiobar-

bituric acid reactive substances (TBARS) as an indication of

lipid oxidation in plasma of rats groups. Supplementation withpomegranate peel powder (PPP), whey powder (WP) and theircombination (PPWP) decreased the levels of plasma TBARS

comparable to liver injury control group. The significantreduction in the levels of TBARS confirms that PPP, WPand PPWP could effectively protect against free radicals in-duced by CCl4.

Glutathione acts as an antioxidant both intracellularly andextracellularly in conjuction with various enzymatic processesthat reduce hydrogen peroxide and hydroperoxides (Kadiska

et al., 2000). Carbon tetrachloride-induced hepatotoxicity isassociated with reduced antioxidant levels (Basu, 2003).

Table 3 Effects of pomegranate peel powder, whey powder and their mixture on body weight gain and relative liver weight in rats.

Parameters NCA ICA PPP WP PPWP

Body weight (g)

Initial 158.4 ± 2.4a 160.3 ± 5.1a 158.2 ± 4.9a 161.9 ± 5.1a 159.8 ± 3.9a

Final 207.3 ± 3.2c 189.6 ± 2.7d 216.4 ± 4.9b 210.1 ± 5.7c 221.6 ± 2.5a

Gain 48.9 ± 5.0b 29.4 ± 3.9c 58.3 ± 5.8a 48.3 ± 4.8b 61.8 ± 3.5a

Relative liver weight (g/100 g BW)

7.34 ± 0.13a 9.03 ± 0.26b 7.44 ± 0.22a 7.75 ± 0.94a 7.69 ± 0.85a

Means having different superscripts in the same row are significantly different (P 6 0.05).A NC, normal control; IC, injury control.

Table 4 Effect of pomegranate peel powder, whey powder

and their mixture on the activities of ALT and AST in normal

and CCl4-treated rats.

Groups ALT (U/ml) AST (U/ml)

Normal control (NC) 45.89 ± 4.8c 48.92 ± 3.2d

CCl4 control (IC) 113.23 ± 4.8a 106.29 ± 2.8a

CCl4 + PPP 48.14 ± 3.1bc 59.71 ± 1.27c

CCl4 +WP 51.22 ± 5.8b 67.16 ± 1.29b

CCl4 + PPWP 44.28 ± 3.2c 58.13 ± 0.8c

Means having different superscripts in the same column are sig-

nificantly different (P 6 0.05).

Table 5 Effect of pomegranate peel powder, whey powder

and their mixture on plasma TBARS and GSH in normal and

CCl4-treated rats.

Groups MDA (nmol/ml) GSH (mg/dl)

Normal control (NC) 0.17 ± 0.03d 23.34 ± 1.5a

CCl4 control (IC) 0.51 ± 0.03a 9.94 ± 0.84d

CCl4 + PPP 0.35 ± 0.03b 17.94 ± 1.3c

CCl4 +WP 0.38 ± 0.01b 16.74 ± 1.2c

CCl4 + PPWP 0.23 ± 0.04c 20.77 ± 1.04b

Means having different superscripts in the same column are sig-

nificantly different (P 6 0.05).

Micrograph 1 Liver of normal control rat (H & E, 40·).

Micrograph 2 Liver of CCl4-treated rat (H & E, 64·).

30 I.S. Ashoush et al.

Changes in GSH content of different groups has been

shown in (Table 5), rats treated with CCl4 alone significantlylowered the plasma glutathione (GSH) level. While, supple-mentation with PPP, WP and their combination (PPWP) im-proved the level of glutathione in the plasma. This result is

in agreement with that of Abdou et al. (2012), who revealedthat pomegranate possesses protective effects against CCl4genotoxicity and hepatotoixicity in animal models. In this re-

spect Basu (2003) indicated that Carbon tetrachloride-inducedhepatotoxicity is associated with reduced antioxidant levels.

Histopathological examination

Hepatic injury through carbon tetrachloride (CCl4) induced li-pid peroxidation is well known and has been extensively used

in the experimental models to understand the cellular mecha-nisms behind oxidative damage and further to evaluate thetherapeutic potential of dietary antioxidants.

Liver injury was evaluated by histopathological approachin Micrograph 1–5 and (Table 6). The normal control group(NC) showed a normal histological structure of the portal

area, central veins (cv) and surrounding hepatocytes (h)(Micrograph 1).

Focal necrosis with inflammatory cells infiltration was de-

tected in the hepatic parenchyma is associated with dilatationin the portal vein (PV) and inflammatory cells infiltration inthe portal area (m) (Micrograph 2).

The liver sections obtained from animals treated with CCl4plus pomegranate peel powder (PPP) showed dilatation in cen-tral veins (cv) (Micrograph 3).

The liver sections obtained from animals treated with CCl4plus whey powder (WP) showed oedema (o) with few inflam-

Micrograph 4 Liver of whey powder and CCl4-treated rat (H &

E, 40·).

Micrograph 5 Liver of mixture of pomegranate peel and whey

powders and CCl4-treated rat (H & E, 64·).

Table 6 Effect of pomegranate peel powder, whey powder

powders and their mixture on CCl4 induced histopathological

changes in rat liver.

Treatment Necrosis Portal

inflammation

Congestion Total

Normal control (NC) – – – –

CCl4 control (IC) +++ +++ +++ ++++

CCl4 + PPP – – ++ ++

CCl4 + WP – ++ + +++

CCl4 + PPWP – + – +

–: Nil; +: Mild; ++: Moderate; +++: Severe; ++++ Very

severe effect.

Micrograph 3 Liver of pomegranate peel powder and CCl4-

treated rat (H & E, 40·).

Antioxidant activity and hepatoprotective effect of pomegranate peel and whey powders in rats 31

matory cells infiltration (arrow) and dilatation in the portalvein (pv) (Micrograph 4).

The liver sections obtained from animals treated with CCl4plus mixture of pomegranate peel and whey powder (PPWP)showed few inflammatory cells infiltration in the portal area(arrow) with newly formed bile ductules (d) associated with

diffuse kupffer cells proliferation in between the hepatocytes.These results suggest that pomegranate peel and whey powders

showed better protection towards CCl4 induced liver damage

(Micrograph 5).The histopathological changes in pretreated groups with

pomegranate peel, whey powders and their mixture as com-

pared to that induced with CCl4 alone indicated marked pro-tective effects of these substances. Also, the histopathologicalobservation in the pretreated groups indicated that the changeswere less in pomegranate peel and its mixture with whey trea-

ted groups as compared with whey treated group (Table 6).

Conclusion

Based on the aforementioned results, it could be concludedthat pomegranate peel powder and whey powder exhibited apotential antioxidant. They were able singly or in combination

to protect liver against the oxidative stress of CCl4. Pomegran-ate peel powder was effective than whey powder however, thecombined treatment was found to be more effective than the

single treatment. So, the mixture of pomegranate peel powderand whey powder could be used as natural antioxidants to en-hance the antioxidant properties of functional food.

Acknowledgment

The authors would like to thank Prof. Dr. Adel Bakeer Kho-loussy, Professor of Pathology, Faculty of Veterinary Medi-cine, Cairo University, for interpretation the slides of

histopathology.

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