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Research ArticleReceived: 12 June 2009 Revised: 12 July 2009 Accepted: 13 July 2009 Published online in Wiley Interscience: 9 September 2009

(www.interscience.wiley.com) DOI 10.1002/jsfa.3748

Decalepis hamiltonii roots boost antioxidantstatus of rat liver and brainAnup Srivastavaa∗ and Thimmappa Shivanandappab

Abstract

BACKGROUND: Roots of Decalepis hamiltonii are traditionally consumed as pickles and juice for their health benefits. We haveearlier demonstrated the antioxidant property of the root extract and identified the constituent antioxidant molecules.

RESULTS: This paper reports the effect of multiple-dose (7, 15, and 30 days) treatment of Decalepis hamiltonii aqueousroot extract (DHA) (50 and 100 mg kg−1 body weight) on the antioxidant profile of rat liver and brain. Activities of theantioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR),and glutathione-S-transferase (GST) were increased and glutathione content was elevated in both liver and brain, apart fromreduction in the basal level of lipid peroxidation. DHA induced stronger antioxidant boost in brain by increasing the activitiesof SOD, CAT, and GPx compared to liver.

CONCLUSION: As failure to grapple with oxidative stress is an important factor in the etiology of several diseases, DHA’s effectson improvement of antioxidant status could provide a scientific justification for the health-promoting properties attributedto it.c© 2009 Society of Chemical Industry

Keywords: Decalepis hamiltonii; antioxidant enzymes; lipid peroxidation; glutathione

INTRODUCTIONThe oxidative damage of biological molecules is an importantevent in the development of a variety of human diseases thatresult from overwhelming of the biological defense systemagainst oxidative stress. The brain is considered highly sensitiveto oxidative damage as it is rich in easily peroxidizable fattyacids, consumes an inordinate fraction (20%) of total oxygenfor its relatively small weight (2%), and is relatively deficientin antioxidant defenses.1 There is substantial evidence thatoxidative stress is a causative or at least an ancillary factor inthe pathogenesis of major neurodegenerative diseases, includingParkinson’s disease, Alzheimer’s disease and amyotrophic lateralsclerosis, as well as in cases of stroke, trauma and seizures.2

Similarly, oxidative stress plays a central role in liver pathologiesand their progression.3

Antioxidants, especially those derived from natural sources,have potential applications in prevention and/or cure of humandisease.4,5 Natural products have been used for this purposesince ancient times and a trend is emerging recently for theirincreased use. A number of plants have been reported to pos-sess antioxidant properties like Bacopa monnieri, Salvia officinalis,and Acanthopanax senticosus. It has been demonstrated that an-tioxidants prevent hepatotoxicity by inhibiting lipid peroxidation,suppressing hepatocellular damage and enhancing antioxidantenzyme activity.6 Natural antioxidants have proven ability to sub-stantially reduce oxidative stress in neurons and thus are potentialcandidates for use as therapeutics in neurodegenerative diseasessuch as Parkinson’s and Alzhimer’s.7

Decalepishamiltonii (family: Asclepiadaceae) grows as a climbingshrub in the forests of peninsular India. Its tubers are traditionally

consumed as pickles and juice for its health-promoting properties.The roots are used in folk medicine and in ayurvedic preparationsof ancient Indian medicine.8 We have shown that the rootsof D. hamiltonii possess potent antioxidant properties andhypothesized that it may be associated with their health benefits.9

Our recent work has shown that the aqueous and methanolicextract of the roots of D. hamiltonii is a cocktail of potentantioxidants.10,11 In this paper, we describe the effect of multiple-dose treatment of aqueous extract of the roots of D. hamiltonii(with known antioxidant constituents) on the antioxidant profileof rat liver and brain which could scientifically validate its health-promoting effects.

MATERIALS AND METHODSChemicalsNicotinamide adenine dinucleotide phosphate reduced (NADPH),1-chloro-2,4-dinitrobenzene (CDNB), thiobarbituric acid (TBA),glutathione (GSH), oxidized glutathione (GSSG), glutathionereductase (GR), cumene hydroperoxide (CHP), pryogallol, bovine

∗ Correspondence to: Anup Srivastava, Department of Pathology, Center for FreeRadical Biology, University of Alabama at Birmingham, Birmingham, AL 35294,USA. E-mail: anup@uab.edu

a DepartmentofPathology,Center for FreeRadical Biology,UniversityofAlabamaat Birmingham, Birmingham, AL 35294, USA

b Department of Food Protectants and Infestation Control, Central FoodTechnological Research Institute, Mysore-570020, Karnataka, India

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serum albumin (BSA) and tetraethoxypropane were purchasedfrom Sigma Chemical Co. (St Louis, MO, USA). Trichloroaceticacid (TCA), hydrogen peroxide (H2O2), 5,5′dithiobis(2-nitrobenzoicacid) (DTNB) and other chemicals were purchased from SiscoResearch Laboratories (Mumbai, India). All the chemicals usedwere of highest purity grade available.

Preparation of the root powder and extractionTuberous roots of D. hamiltonii were washed with water, followedby crushing with a roller to separate the inner woody core from theouter fleshy layer. The fleshy portion was collected, dried at 40 ◦Cin a hot-air oven until they lost complete moisture (4–6 h) andthen finely powdered. The powder was used for extraction. Wereported earlier that aqueous extract of D. hamiltonii shows highantioxidant activity among the different solvent extracts.9 Theaqueous extract was prepared by homogenizing the root powderin warm water (50 ◦C) and allowing to stand for 24 h; it was thenfiltered with Whatman paper No. 1 and the filtrate was lyophilizedand weighed (170 g kg−1 root powder).

Animals and treatmentsSixty-day-old adult male Wistar rats (180–200 g) were dividedinto different groups, eight in each. The local ethics committeeapproved the design of the experiment and animal ethics werefollowed for animal handling. In a 90-day dietary toxicity study onrats it was established that the root extract of D. hamiltonii wassafe to the mammalian system. Aqueous extract of the roots ofD. hamiltonii was orally administered for 7, 15 and 30 consecutivedays at 50 and 100 mg kg−1 body weight (b.w.). Animals weresacrificed by anesthesia 24 h after the last administration; the liverand brain were perfused with saline, dissected out and processedimmediately for biochemical assays.

Thiobarbituric acid reactive substance (TBARS) estimationTBARS in the tissue homogenate was measured by the method ofOhkawa et al.12 Tissue homogenate (100 mg mL−1 in 50 mmol L−1

phosphate buffer, pH 7.4) was boiled in TCA (10%) and TBA(0.34%) for 15 min, cooled and centrifuged. Absorbance of thesupernatant was read at 535 nm. TBARS was calculated usingtetraethoxypropane as the standard.

Antioxidant enzymesTissue was homogenized (100 mg mL−1) in ice-cold 50 mmol L−1

phosphate buffer (pH 7.4), centrifuged at 10 000 × g for 20 min at4 ◦C and the supernatant was used to assay the antioxidant enzymeactivities. Superoxide dismutase (SOD) activity was measuredusing pyrogallol (2 mmol L−1) autoxidation in Tris buffer.13

Catalase (CAT) activity was measured using H2O2 as the substratein phosphate buffer.14 Glutathione peroxidase (GPx) activitywas measured by the indirect assay method using glutathionereductase. Cumene hydroperoxide (1 mmol L−1) and glutathione(0.25 mmol L−1) were used as substrates and oxidation of NADPHby glutathione reductase (0.25 U) in Tris buffer was monitored at340 nm.15 Glutathione reductase (GR) activity was estimated usingoxidized glutathione (20 mmol L−1) and NADPH (2 mmol L−1)in potassium phosphate buffer.16 Glutathione transferase (GST)activity was assayed by the method of Warholm et al.17 usingglutathione (20 mmol L−1) and CDNB (30 mmol L−1) as thesubstrates in phosphate buffer, and change in absorbance at344 nm was monitored.

GlutathioneTissue homogenate (100 mg mL−1) was prepared in trichloroaceticacid (50 mg mL−1 double-distilled water), centrifuged at 2000 × gfor 10 min and glutathione (GSH) in the deproteinized supernatantwas estimated by Ellman’s reagent with a standard curve.18

Protein content was estimated by the method of Lowry et al.19

with bovine serum albumin as the standard.

StatisticsAll the data are expressed as means ± SE of eight observations(n = 8) and significant difference between each of the groups wasstatistically analyzed by Duncan’s multiple range test (StatisticaSoftware, 1999). A difference was considered significant atP < 0.05.

RESULTSLipid peroxidationDHA treatment was effective in significantly lowering the basalLPO level in both liver and brain of the rat. The higher dose of DHA(100 mg kg−1 b.w.) used decreased basal LPO by 17% in brain andby 31% in liver over control after 30 days of DHA treatment (Fig. 1).

Antioxidant enzymesRat liver and brain showed increases in antioxidant enzymeactivities with DHA treatment. Rat brain showed a significantincrease in SOD activity after 5 days (25% increases at higherdose) of DHA treatment and was elevated further with theduration of treatment (52% increase after 30 days at higher dose).Liver also showed increase in SOD activity after 15 days of DHAtreatment which increased with the duration of treatment for

Figure 1. Multiple-dose treatment of D. hamiltonii aqueous extractdecreases the basal lipid peroxidation in rat: (A) brain; (B) liver. Control(no shading); DHA 50mg kg−1 b.w. (light shading); DHA 100 mg kg−1 b.w.(dark shading); ∗indicates significance P < 0.05.

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the lower dose (50 mg kg−1 b.w.) but not for the higher dose(46% increase at higher dose after 30 days) possibly because itwas the maximum responsive dose (Fig. 2). In brain, CAT activitysignificantly increased after 5 days of DHA treatment at lowerdose and was enhanced by 52% after 30 days at higher dose.Liver showed an increase of 28% in CAT activity after 15 days ofhigher-dose DHA treatment, which was raised further to 41% after30 days (Fig. 2). DHA was effective only after 15 days treatment inincreasing GPx activity in both rat liver and brain. DHA treatmentelevated GPx activity in rat brain by 35% and by 18% in liverover control at higher dose after 30 days (Fig. 3). DHA treatmentsignificantly increased GR activity in both rat liver and brain but itwas more pronounced in liver. DHA treatment increased GR activityeven at lower dose after 5 days and elicited the highest increaseof 65% and 34% in liver and brain, respectively, after 30 days athigher dose (Fig. 3). Rat liver showed significant increase in GSTactivity after 15 days with both doses of DHA treatment and waselevated further with the duration of treatment (33% increase after30 days at higher dose). Brain also showed significant increase inGST activity after 15 days of DHA treatment at higher dose only,which did not increase substantially with the duration of treatment(19% increase at higher dose after 30 days) (Fig. 4).

GlutathioneDHA treatment enhanced GSH level in both rat liver and brain after15 days. Liver and brain showed the highest increases of 44% and31%, respectively, after 30 days’ treatment with DHA higher dose(Fig. 4).

DISCUSSIONRoots of D. hamiltonii are traditionally consumed for their healthbenefits. However, there are no studies on the health-promoting

potential of D. hamiltonii. We have shown earlier that the roots ofD. hamiltonii possess antioxidant properties.9 Recently, we haveisolated and identified the active principles (4-hydroxyisophthalicacid, 14-aminotetradecanoic acid, 4-(1-hydroxy-1-methylethyl)-1-methyl-1,2-cyclohexane diol, 2-hydroxymethyl-3-methoxybenzal-dehyde, 2,4,8 trihydroxybicyclo[3.2.1]octan-3-one) and ellagicacid, which are responsible for the antioxidant activity of theaqueous extract of the roots of D. hamiltonii (Fig. 5).10,20 Theeffect of DHA on antioxidant status of the rat liver and brain wasinvestigated to test its efficacy in in vivo conditions.

Coordinated actions of various cellular antioxidants (like SOD,CAT, GPx, GR, GST and GSH) in mammalian cells are critical foreffectively detoxifying reactive oxygen species (ROS). Our studyshows that treatment of DHA boosted the antioxidant status ofthe liver and brain. Thirty-day treatment with DHA was mosteffective in reducing the basal (endogenous) LPO, enhancing GSHcontent, and increasing antioxidant enzyme activities in the brainand liver. There are very few studies showing neuroprotectiveaction of plant extracts. The antioxidant molecules isolated byactivity guided purification from DHA have demonstrated highfree radical scavenging activity (for superoxide, hydroxyl, lipidperoxide, nitric oxide, and DPPH radicals) as well as metal-chelating activity. These properties are potentially favorabletowards reducing the basal level of oxidative stress. Further,the mechanism by which plant extracts enhance antioxidantenzyme levels is not clearly understood, but positive effectsof plant-derived polyphenols on antioxidant enzyme activitiesin vivo have previously been reported.21,22 Some studies suggestthat the enhancement of phase II enzymes by phytochemicalantioxidants such as polyphenols present in aqueous plantextracts is achieved by upregulating the corresponding genesby interaction with antioxidant response elements (AREs) thattranscriptionally regulate these genes.23 It has been shown that

Figure 2. Multiple-dose treatment of D. hamiltonii aqueous extract increases SOD and CAT activities in rat: (A) brain; (B) liver. Control (no shading); DHA50mg kg−1 b.w. (light shading); DHA 100 mg kg−1 b.w. (dark shading); ∗indicates significance P < 0.05.

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Figure 3. Multiple-dose treatment of D. hamiltonii aqueous extract elevates GPx and GR activities in rat: (A) brain; (B) liver. Control (no shading); DHA50mg kg−1 b.w. (light shading); DHA 100 mg kg−1 b.w. (dark shading); ∗indicates significance P < 0.05.

Figure 4. Multiple-dose treatment of D. hamiltonii aqueous extract induces GST activity and GSH level in rat: (A) brain; (B) liver. Control (no shading); DHA50mg kg−1 b.w. (light shading); DHA 100 mg kg−1 b.w. (dark shading); ∗indicates significance P < 0.05.

the γ -glutamylcysteine synthetase (γ -GCS), a key enzyme inglutathione synthesis, is also transcriptionally regulated by AREs.24

It is known that treatments that induce expression of phase IIdetoxifying enzymes also result in elevated γ -GCS activity as wellas increased intracellular GSH levels.25 It is possible that, in thiscase too, the interaction of some compounds present in the DHA

with AREs in vivo would result in a higher antioxidant status apartfrom its free radical scavenging property.

Antioxidant intervention in therapeutic strategy for treat-ment of hepatological and neurological disorders is gainingsignificance.26,27 Natural plant products are being used in an-tioxidant therapy for degenerative disorders as they have minimal

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Figure 5. Antioxidant compounds present in the aqueous extract of the roots of Decalepis hamiltonii.

pathological and toxic side effects in contrast to those of a numberof synthetic drugs. It is known that dietary antioxidants and herbalextracts can significantly contribute to the modulation of com-plex mechanisms of degenerative diseases. Given their potentialcontribution to immune modulation, use of traditional medicineand food plant extracts in the management of biological disordersis of great interest. Understanding the molecular mechanisms ofprevention, oxidative stress and immune function will facilitatefuture therapeutic use of antioxidants.28 Research is now proceed-ing in parallel with efforts to demonstrate clinical efficacy of thesecondary metabolites of traditional medicine and food plants.

The emerging view is that antioxidant compounds could exertbeneficial effects on cells not only through their antioxidantpotential but also through the modulation of different pathwayssuch as signaling cascades, anti-apoptotic processes or geneexpression.29 With all these findings it can be concluded thatthe aqueous extract of roots of D. hamiltonii exhibits antioxidantactivity by inhibition of lipid peroxidation and enhancement ofantioxidant enzymes and GSH, and thus can be used in theprevention/treatment of various disorders where reactive oxygenspecies are involved. These results provide a scientific basis for thehealth-promoting attributes of D. hamiltonii.

ACKNOWLEDGEMENTSThis work was done at the Central Food Technological ResearchInstitute, Mysore, India. The authors wish to thank the Director ofthe Institute for his keen interest in this study. The first authoracknowledges the Council for Scientific and Industrial Research,New Delhi, for awarding a research fellowship.

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