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Effects of Hibiscus sabdariffa calyx anthocyanins and ascorbate on 2, 4- dinitrophenylhydrazine-induced changes in the activities of antioxidant enzymes in rabbits

Augustine O Olusola*

Department of Biochemistry, Faculty of Science,dekunlejasin University,kungbakoko, Ondo State, Nigeria

*Corresponding Author:
Augustine O Olusola
Department of Biochemistry, Faculty of Science,dekunlejasin University,kungbakoko, Ondo State, Nigeria
Tel: +2348053447560

Received date: 20/03/2014; Revised date: 25/05/2014; Accepted date: 27/05/2014

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Abstract

The effects of anthocyanin-rich extract of the calyces of Hibiscus sabdariffa (HS) Linn and ascorbate on the 2, 4- dinitrophenylhydrazine (DNPH)-induced changes in the levels of antioxidant enzymes of rabbits were evaluated in this study. The organs examined were the blood, brain and liver. Thirty male adult rabbits used for the study were divided into six groups. Group 1, the control took only water while animals in groups 2,3,5 and 6 received 100 mg/kg body weight of the extracts once daily for 28 days. After 22nd day of treatment, the rabbits in groups 4, 5 and 6 received 28 mg/kg body weight of DNPH for the remaining 5 days of treatment, after which the animals were sacrificed. Exposure of rabbits to DNPH (28 mg/kg body weight) caused significant (P<0.05) increase in catalase and superoxide dismutase activities relative to the DNPH-free group. The activity of glucose-6-phosphate dehydrogenase was also significantly (p<0.05) elevated in the serum following DNPH treatment when compared to control. However, pre-treatment with (100 mg/kg body weight) HS anthocyanins and ascorbate separately provided varying degrees of protection against DNPH-induced biochemical changes. Relative to the controls, the extract and ascorbate treatments significantly (P<0.05) decreased the activities of the antioxidant enzymes. Examined separately and compared, both treatments appeared to have offered effective protection against DNPH-induced oxidative damage, though the anthocyanin isolate appeared to be more effective in this capacity. Our findings show that Hibiscus sabdariffa anthocyanins are probably more potent antioxidants than ascorbate.

Keywords

Hibiscus sabdariffa,nthocyanin-rich extract,scorbate,ntioxidant enzymes, 2,4-dinitrophenylhydrazine.

Introduction

Hibiscus sabdariffa Linn (Roselle) belongs to the family of Malvaceae, which is native to old World tropics, probably in the East Indies; now cultivated throughout the tropics [5]. The vegetable is widely grownnd commonly useds port herb or soup in the northern part of Nigeria. In Nigeria especially in the northern part, the extract of the red calyces is consumeds beverage knowns zobo.

Ethnobotanical information regarding Hibiscus sabdariffa reveals the following medicinal uses: diuretic, diaphoretic,ntibacterialgent,ntifungalgent, mild laxative, sedative,ntihypertensive, gastrointestinal disorder treatment, hypercholesterolemia treatment, kidney stone treatment, liver damage treatment,gent for decreasing the viscosity of the blood,ndgent for treating thefter effects of drunkenness [1,7].mong the chemical constituents of the flowerre the flavonoids, gossypetine, hibiscetine,nthocyaninnd sabdaretine [20]. Certainmounts of delphinidin-3-monoglucosidend cyaniding-3-monoglucoside which constitute thenthocyaninsrelso present [9].

Some studies have reported that Hibiscus sabdariffa is effective for decreasing the levels of total lipids, cholesterolnd triacylglycerol, suggesting the possibility that Hibiscus sabdariffa functionss hypolipidemicgent [7,13]. Studies on the effect of Hibiscus sabdariffa calyx extract on thectivities ofntioxidant enzymesre however, scanty. The present study is thereforeimedt evaluating the effect of Hibiscus sabdariffanthocyaninss compared toscorbate, on 2,4-dinitrophenylhydrazine-induced changes in thectivity ofntioxidant enzymes in rabbits.

Phenylhydrazinend its derivatives 2, 4-dinitrophenylhydrazinere toxicgents. Their toxicction has beenttributed to theirbility to undergouto oxidation. This increased oxidant potential enables them to oxidize enzymes, membrane proteinnd hemoglobin. Phenylhydrazine isble to initiate lipid peroxidation in membrane phospholipids [8] while 2,4-dinitrophenylhydrazine has been shown to be capable of inducing lipid peroxidationnd other oxidative damage in rabbits [14,15,16] nd rats [10]. Thebility of 2,4-DNPH to induce lipid peroxidationnd other free radical damage makes itnppropriate model toxicant for testing the claim that the extract of Hibiscus sabdariffa Linn calyces can protect tissues from oxidative stress-induced changesnd otherttendant biochemical changes.

Materials and Methods

Plant material

Fresh calyces of H. sabdariffa were harvested from Botanical Gardens University of Ilorin, Kwara State, Nigeria. They were dried under continuousir-flow maintainedt 25 0C until constant weight. Identificationnd taxonomical classifications were donet herbarium of the Department of Plant Biologynd Biotechnology, University of Benin, Benin City, Edo-State, Nigeria.

Animals

Thirty (30) rabbits (Oryctolagus cuniculus) used for this research work were obtained from private breeder in Benin City. Thenimals weighed 800-1000 g on purchasend were in very good state of healths confirmed by veterinary physician. Thenimals were housed in twos (same sex) in improvised rabbit cages composed of wire mesh (100cmX40cmX30cm) under 14 hr/10 hr light/dark regimen. They were fed with growers mash (obtained from Bendel Floursnd Feed Mill, Ewu, Edo State, Nigeria)nd waterd libitum. Thenimals were protected from parasite infestation by proper veterinary management throughout the duration of the treatment.

Preparation ofnthocyanin-rich extract from plant materials

Anthocyanin-rich extract from Hibiscus sabdariffa calyces was preparedccording to the method described in our previous reports (Ologundudu etl., 2009a, b).

Experimental design

Thirty (30) rabbits weighing 800-1000 g were used for this research work. They were randomly selected into six (6) experimental groupss shown below. The experiment lasted for 28 days.

Group 1: Water treated control. Each rabbit was given distilled water, 2.5 ml/kg body weight

Group 2:nthocyanin-rich extract of H. sabdariffa wasdministeredt dose of 100 mg/kg body weight, to each rabbit in this group by gavage.

Group 3:scorbate wasdministeredt dose of 100 mg/kg body weight, to each rabbit in this group by gavage.

Group 4: 2, 4-DNPH wasdministeredt dose of 28 mg/kg body weight intraperitoneally to each rabbit in this group during the last 5 days of the 28-day study period before sacrifice.

Group 5:nthocyanin-rich extract of H. sabdariffa wasdministeredt dose of 100 mg/kg body weight for 28 days to each rabbit in this groupccompanied with 28 mg/kg body weight of 2, 4-dinitrophenylhydrazinedministered intraperitoneally daily from day 24 (5 days 2,4-DNPH treated) before sacrifice.

Group 6:scorbate wasdministeredt dose of 100 mg/kg body weight for 28 days to each rabbit in this groupccompanied with 28 mg/kg body weight of 2, 4-dinitrophenylhydrazinedministered intraperitoneally daily from day 24 (5 days 2,4-DNPH treated) before sacrifice.

Biochemical determinations

Catalasectivity was determined by the method of Sinha (1971) by following its decomposition of H2O2. The superoxide dismutasectivity was determined by the method of Misrand Fridovich (1972). Thectivity of glucose-6-phosphate dehydrogenase was determined usingssay kit obtained from Randox Laboratories, UK. The method iss described in the manual/leaflet.

Statisticalnalysis

The data obtained were subjected to standard statisticalnalysis of variance (ANOVA) using the SAS software (SAS Inst. Inc.1999). Treatment means were compared using the Duncan procedure of the same software. The significance level was sett P<0.05.

Results

The effects of DNPH, HSnthocyaninsndscorbate on thectivities of G6PD in the serumnd liver of rabbitsre presented in Table 1.dministration of DNPH (Group 4) significantly (p<0.05) increased the level of G6PD in the serum. Rabbits that received thenthocyanin-rich extractndscorbate separately before DNPHdministration (Groups 5nd 6) did not showltered level of the enzyme in both serumnd the liver when compared with control but were significantly reduced in the serum when compared with DNPH only group.

pharmacology-toxicological-studies-Effects-anthocyanins-serum

Table 1: Effects of DNPH, HS anthocyanins and ASB on the activities of G6PD in the serum and liver of rabbits

The effects of DNPH, HSnthocyaninsndscorbate on the specificctivities of catalase in the serum, livernd brain of rabbits is presented in Table 2.dministration of DNPH (Group 4) caused significant (p<0.05) depletion of serum catalasectivity but caused significant increase in the level of the enzyme in both the brainnd the liver when compared with control (Group 1).Relative to control, treatment withnthocyanin extractlone (Group 2) caused significant rise in the liver level of the enzyme. Rabbits that receivednthocyaninlone (Group 5)nd those that receivedscorbatelone (Group 6) before DNPHdministration showed significant increase in serum level of catalasend significant decrease in its levels in the livernd brain when compared with those exposed to DNPH only (Group 4).

pharmacology-toxicological-studies-Effects-anthocyanins-ascorbate

Table 2: Effects of DNPH, HS anthocyanins and ascorbate on serum, liver and brain catalase activities.

The effects of DNPH, HSnthocyaninsndscorbate on the specificctivities of SOD in the serum, livernd brain of rabbits is presented in Table 3. DNPH treatment significantly (p<0.05) reduced thectivity of SOD in the serum but caused significant increase in itsctivity in livernd brain relative to control (Group1). Groups treated withnthocyaninndscorbate separately before DNPHdministration (Groups 5nd 6) did not showny significant difference in thectivities of the enzyme in the tissues when compared with control.

pharmacology-toxicological-studies-anthocyanins-serum-liver

Table 3: Effects of DNPH, HS anthocyanins and ASB on the specific activities of SOD in the serum, liver and brain.

Discussion

Recently,ttention has been focused on the protective role of naturally occurringntioxidants in biological systems,nd on the mechanisms ofction. Phenolic compounds, whichre widely distributed in plants,re currently believed to bentioxidants capable of preventing oxidative damage in living systems (Wang etl, 2000; Stanner etl, 2004).nthocyaninsre phenolic compounds,nd theirntioxidant roles,s compared toscorbate were investigated in this study.

Glucose-6-phosphate dehydrogenase

Tissue toxicity was induced bydministration of DNPHnd the toxicity was established by the significantly increased serum specificctivity of glucose-6-phosphate dehydrogenase [6,18]. This property of DNPH has been well characterizednd it stems from its cellular disruptionnd oxidative damage resulting in hemolysis. The integrity of erythrocyte membrane is maintained by reduced glutathione whose level in turn depends on the cellular level of NADPH, metabolic product of the reaction of glucose-6-phosphate dehydrogenase in pentose phosphate pathway. Therefore the observed significant increase in the specificctivity of glucose-6-phosphate dehydrogenase under the condition of DNPHdministration was toxic response, which was necessary for the maintenance of erythrocyte membrane integritynd prevention of oxidative damage [18].

Treatment with each ofscorbatendnthocyanin isolate did not showny significant effect on the level of G6PD in livernd serum, but prophylacticdministration of each of them before DNPH treatment offered significant protections evidenced by the significant reduction in the serum levels of specificctivity of glucose-6-phosphate dehydrogenase of rabbits pretreated with each of the extractndscorbate prior to DNPH treatment, when compared with groupdministered with DNPHlone [18,22]. This indicated that thentioxidant preparations exhibit prophylactic-type of protectiongainst chemical damage in the blood cells.lso there was no significant difference between the two treatment groups (Groups 5nd 6) which indicate that the effectiveness of bothnthocyaninsndscorbate in counteracting the DNPH-induced changes in thectivity of G6PD is comparable. The liver is rich but notbsolute source of glucose-6-phosphate dehydrogenase, but itsctivity in the liver homogenate can providen insight into likely basal free radical generation during course of normal cellular metabolism in the livernd the response of the cells to the generated free radicals.lso since some rabbits were treated with DNPH, there is need to understand how liver cells manage the oxidative damagend its rippling effects.

The recorded increased specificctivity of glucose-6-phosphate dehydrogenase in the liver,s result ofnthocyaninsndscorbatedministrations compared to the water control may point to thebility of the preparations to individually induce the expressionnd the specificctivity of the enzyme in the liver cells [17,19].

Administration of DNPH resulted in decreasedctivity of glucose-6-phosphate dehydrogenase in liver homogenate. This property of DNPH has been well characterizednd it stems from its cellular disruptionnd oxidative damage [8,15,16]. These factors result in cell necrosisnd subsequent loss of intracellular glucose-6-phosphate dehydrogenasend the pH changess the enzyme leaks out of the hepatocytes into the blood, thus,ccounting for the reduced specificctivity of glucose-6-phosphate dehydrogenase under condition of DNPHdministration in the liver. This latter phenomenon couldlso be responsible for the elevated level of the enzyme observed in the serum ofnimals treated with DNPH [12,16]. Prophylacticdministration of each of thentioxidant preparationsnd subsequent treatment of rabbits with DNPH revealed similar pattern forscorbatendnthocyanin isolate indicating the higherbility ofnthocyanin to scavenge free radicals produced by DNPHdministrationnd reduce the expression of the genes for glucose-6-phosphate dehydrogenase compared withscorbate [4].

Catalase

DNPHdministration resulted in significant (P<0.05) increase in the specificctivity of catalase in livernd brain compared to the water control. This can beccounted for by the cellular need to detoxify the increased hydrogen peroxide produced during DNPH toxicity.ntioxidant enzymes suchs catalase, superoxide dismutase,nd glutathione-s-transferasend glutathione peroxidasere present in oxygen handling cells whichre the first line of cellular defensegainst oxidative injuries decomposing O2nd H2O2 before they interact to form more reactive radicals [2,8,14,16]. The increase in thectivity of catalase in the DNPH-treated models is necessary for effective protection. The significant reduction in serumctivity of catalase is result of hemotoxicction of DNPH which caused the depression of the enzyme productiont gene levelnd consequent depletion of the enzyme level in the serum.

Priordministration ofnthocyanin extractnd vitamin C, followed by treatment with DNPH resulted in significantly decreased specificctivity of catalase in livernd brain compared with those treated with DNPHlone while thectivity of the enzyme in the serum was significantly increased relative to DNPH- treated rabbits,nd maintainedt control levels.gain the results obtained for the brain under this treatment, showed thatnthocyanins may be more effective in counteracting the oxidant effect of DNPH thanscorbates shown by the significant difference in the catalasectivity of the livernd brain of Groups 5nd 6.

It islso noteworthy that comparing thectivities of catalase in the three tissues, the serum has the lowest. This lowctivity could possibly be due to othervailablegents for detoxifying hydrogen peroxide, these include the presence of dietaryntioxidantsnd bilirubin which is product of heme degradation [8]ptly present in the blood cellsnd circulation.

Superoxide dismutase (SOD)

Like glucose-6-phosphate dehydrogenase, superoxide dismutase islso widely distributed enzyme in tissuesnd its roles one of the early enzymes catalyzing detoxification of superoxide has been well documented [3]. It does not only represent the first major enzyme of superoxide metabolizing enzyme, its deficiency has been linked to life-threatening pathologic conditions [6]. Its determination in this research was significant because it represents the chief enzyme that prevent peroxidation initiated when superoxide radicalsttack the unsaturated fattycyl groups of phospholipids; reaction which heralds other downstream reactions like hemolysis in red blood cellsnd necrosis of tissues [22].

The significant reduction in the specificctivity of SOD in the serum of rabbits treated with DNPHlone is due to the fact that DNPH induces hemolysis in the red blood cells. Cell lyses is occasioned principally by free radical reactions on membrane lipids which ultimately depletend thus, lowering serum specificctivity of superoxide dismutase [16].

The livernd brain of groups treated withnthocyanin extract (Group 2)ndscorbate (Group 3) showed no significant difference in the specificctivity of superoxide dismutase when compared with each othernd the water control. However, Group 2 showed higher specificctivity of SOD than Group 3, even though the difference is insignificant. On the other hand, treatment with DNPH resulted in significantly increased specificctivity of superoxide dismutase compared to the water control (P<0.05). Theccumulation of DNPH in the liver must have significantly increased the rate of superoxide generation possibly higher than what the basal enzymectivity could cope with. Therefore, to salvage the cells from the peroxidativection of superoxidend tissue necrosis,n SOS response was induced to increase the cellular expression of superoxide dismutase thus,ccounting for the significant increase in the superoxide dismutasectivity of livernd brain under DNPHdministration. Prophylacticdministration of thenthocyanin extractnd vitamin C, followed by treatment with DNPH resulted in significantly decreased specificctivity of superoxide dismutase compared with those treated with DNPHlone.NOVA however showed no significant difference between the two groups. The results, therefore, show that thenthocyanin extractndscorbate showed comparable potencies probably due to their similar polarities.

References

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