research article kolaviron and l-ascorbic acid attenuate...

Research ArticleKolaviron and L-Ascorbic Acid Attenuate Chlorambucil-InducedTesticular Oxidative Stress in Rats

Ebenezer Tunde Olayinka and Ayokanmi Ore

Biochemistry Unit Department of Chemical Sciences Ajayi Crowther University PMB 1066 Oyo Oyo State Nigeria

Correspondence should be addressed to Ebenezer Tunde Olayinka etolayinkaacuedung

Received 28 May 2014 Revised 14 August 2014 Accepted 4 September 2014 Published 17 September 2014

Academic Editor Robert H Rice

Copyright copy 2014 E T Olayinka and A Ore This 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

Chlorambucil (4-[4-[bis(2-chloroethyl)amino]phenyl]butanoic acid) is an alkylating agent indicated in chronic lymphocyticleukaemia Kolaviron (KV) a biflavonoid complex from Garcinia kola and L-ascorbic acid (AA) are known to protect againstoxidative damage in vivo This study evaluates the protective capacity of KV and AA on chlorambucil-induced oxidative stressin the testes of rat Twenty male Wistar rats (180ndash200 g) were randomized into four groups I control II chlorambucil (02mgkgbw) III 02mgkg chlorambucil and 100mgkgKV and IV 02mgkg chlorambucil and 100mgkgAAAfter 14 days of treatmentsresults indicated that chlorambucil caused significant reduction (119875 lt 005) in testicular vitamin C and glutathione by 32 and 39respectively relative to control Similarly activities of testicular GST SOD and CAT reduced significantly by 48 47 and 49respectively in chlorambucil-treated rats relative to control Testicular MDA and activities of ALP LDH and ACP were increasedsignificantly by 53 51 64 and 70 respectively in the chlorambucil-treated rat However cotreatment with KV and AAoffered protection and restored the levels of vitamin C GSH and MDA as well as SOD CAT GST ACP ALP and LDH activitiesOverall kolaviron and L-ascorbic acid protected against chlorambucil-induced damage in the testes of the rat

1 Introduction

The upsurge in the synthesis and use of anticancer agenthas generated questions about the safety of these substanceson normal tissues Most drugs administered in cancerchemotherapy are well known to be associated with toxicside effects [1] Among the most commonly reported toxiceffects are those associated with organs such as the liverkidneys testes and other functional parameters of the bloodand immune system [2ndash6]

Chlorambucil (4-[4-[bis (2-chloroethyl) amino] phenyl]butanoic acid) Figure 1(a) is an orally available alkylatingagent indicated in the treatment of chronic leukaemia [3 7]Chlorambucil is rapidly absorbed from the gastrointestinaltract and distributed to the liver kidneys and other organsThemetabolism of chlorambucil occurs predominantly in theliver by the hepatic drug metabolising enzymes leading tothe formation of 3-(4-dehydrochlorambucil and phenylaceticacid mustard 2-(4-(bis(2-chloroethyl)amino)phenyl)acetic

acid) [8] Phenylacetic acidmustard is further metabolized toinactive products which are excreted in the urine and faeces[9]

Chlorambucil and its derivatives form covalent bondswith proteins and DNA of neoplastic cells resulting instructural and functional damage to DNA [10] While chlo-rambucil and its metabolites are cytotoxic to cancer cellsthey may also be toxic to normal body cells Toxic effectsof chlorambucil in the form of haematological toxicityhepatotoxicity nephrotoxicity neurotoxicity and so forthhave been reported [3 4 11ndash13] Recent study from ourlaboratory suggested that the mechanism of toxicities elicitedby chlorambucil may involve formation of free radicals ordepletion of cellular antioxidant reserve [14]

The testes have evolved a formidable antioxidant defenceto protect against the damaging effects of free radicals andoxidants which may have negative implication on spermato-genesis [15] These comprise the nonenzymatic antioxidantsincluding glutathione ascorbic acid and tocopherol and

Hindawi Publishing CorporationJournal of ToxicologyVolume 2014 Article ID 587015 9 pageshttpdxdoiorg1011552014587015

2 Journal of Toxicology

enzymatic antioxidants such as catalase superoxide dismu-tase glutathione peroxidase glutathione reductase and glu-tathione S-transferaseThe testes are rich in lipid componentsand therefore exposure to high level of prooxidants maydeplete the antioxidant reserve thereby exposing the cellto excessive level of lipid peroxidation and ultimately tissuedamage [16]

The natural antioxidant kolaviron is a fraction of thedefatted ethanol extract ofGarcinia kola containing Garciniabiflavonoids GB1 and GB2 and kolaflavanone (Figure 1(c))Kolaviron is of high safety profile and its antioxidant prop-erties have been extensively studied in vivo [17] Moreover itis known to significantly prevent drug and chemical-inducedorgan toxicity and oxidative damage in experimental animalmodels [14 18ndash20]

Many classes of antineoplastic agents are known togenerate high levels of oxidative stress in biological systems[21] leading to tissue injuries Injuries elicited by antineo-plastic agents on vital organs have been reported in mostcases but with few reports on reproductive organs particu-larly the testes Fewer reports are also available concerningchemotherapy-induced injuries due to generation of potentoxidants or disruptions of testicular antioxidant system bythese agents Besides proper functioning of the testicularredox homeostasis is essential to efficient spermatogenesisand overall function of the testes Chlorambucil has beenreported in our previous study to induce oxidative stress inthe liver of rats [14] It is assumed that the administrationof antioxidants along with chlorambucil may offer someprotection against generation of reactive oxygen species(ROS) and oxidative damage to the testes In addition if thereactive species and oxidative stress caused by chlorambucilare responsible only for its toxic effects antioxidants sup-plementation may reduce the severity of these toxic effectswithout interfering with the antineoplastic activity of thedrug Evidence suggests that administration of antioxidantsalong with chemotherapeutic agents has been found toeffectively relieve toxic side effects [22ndash24] In most casesantioxidant has improved the cytotoxicity of antineoplasticagents For instance vitamin C is reported to enhance theantineoplastic activity of doxorubicin cisplatin and paclitaxilin human breast carcinoma cells [25] Also combination ofvitamins A C and E improved the antitumor activity ofdoxorubicin in mice [26] Several studies have also reportedthe potential of the plant derived flavonoids to inhibit theproliferation of cancer cells [27ndash30] However the presentstudy was designed specifically to evaluate the protective roleof kolaviron a biflavonoid complex and L-ascorbic acid onchlorambucil-induced oxidative stress in the testes of rats

2 Materials and Methods

21 Chemicals and Assay Kits Chlorambucil (Leukeran) wasa product of Aspen Pharm Trading Limited Dublin 1Ireland 1-Chloro-24-dinitrobenzene (CDNB) 5101584051015840-di-thiobis-2-nitrobenzoic acid (DTNB) glutathione (GSH)epinephrine hydrogen peroxide (H

2O2) and thiobarbituric

acid (TBA) were all purchased from Sigma ChemicalCompany (London UK) Assay kits for alkaline phosphatase

(ALP) and acid phosphatase (ACP) were obtained fromRandox Laboratories Ltd (Antrim UK) Assay kit for lactatedehydrogenase (LDH) was a product of Cypress DiagnosticsBelgium All other reagents used were of analytical gradeand were obtained from British Drug House (BDH) PooleEngland

22 Extraction of Kolaviron Kolaviron was extracted fromthe fresh seeds of Garcinia kola (35 kg) and characterizedaccording to the method of Iwu et al [31] Briefly thepowdered seeds were extracted with light petroleumether (bp 40ndash60∘C) in a Soxhlet extractor for 24 hr Thedefatted dried marc was repacked and then extracted withmethanol The extract was concentrated and diluted to twiceits volume with distilled water and extracted with ethylacetate (625 L) The concentrated ethyl acetate fractionyielded a golden yellow solid termed kolaviron whichhas been shown to consist of Garcinia biflavonoid GB-1(31015840101584041015840410158401015840101584055101584010158407710158401015840-heptahydroxy-3810158401015840 biflavanone) GB-2(310158401015840410158404101584010158401015840551015840101584051015840101584010158407710158401015840-octahydroxy-3810158401015840-biflavanone) andkolaflavanone (310158401015840410158404101584010158401015840551015840101584051015840101584010158407710158401015840octahydroxy-4101584010158401015840-me-thoxy-3810158401015840-biflavanone) (Figure 1) The purity and identityof kolaviron were determined by subjecting it to thin-layerchromatography (TLC) using Silica gel GF 254-coated platesand solvent mixture of methanol and chloroform in a ratio1 4 vv The separation revealed the presence of three bandswhich were viewed under UV light at a wavelength of 254 nmwith RF values of 048 071 and 076 [32]

23 Animal Selection and Care Twenty inbred male rats(Wistar strain) weighing between 180 and 200 g wereobtained from the animal holding unit of the Department ofChemical Sciences Ajayi Crowther University Oyo NigeriaThe rats were acclimatised under laboratory conditions priorto the commencement of the studyThe animals were housedin wire meshed cages maintained at standard conditions oftemperature and humidity with an alternating light cycle(12 hr lightdark) They were fed with standard rat pellet(Ladokun feeds Ibadan Nigeria) and were supplied waterad libitum The experimental protocol relating to animalhandling conformed to the Guidelines of the National Insti-tute of HealthmdashGuide for the Care and Use of LaboratoryAnimals (NIH Publication number 85-23 revised 1985 USDepartment of Health Education and Welfare BethesdaMA)

24 Drug Treatments The rats were randomised into fourexperimental groups (IndashIV) of 5 animals each Group I(control) animals were administered distilled water Animalsin group II received chlorambucil at a dose of 02mgkg bodyweight (bw) Group III animals were coadministered chlo-rambucil (02mgkg bw) and kolaviron (100mgkg bw)Animals in group IV were coadministered chlorambucil(02mgkg bw) and L-ascorbic acid (100mgkg bw) Thedose of chlorambucil administered to the animals was basedon the recommended dose in the treatment of Hodgkinrsquoslymphoma (01-02mgkgday) [33] while doses of ascorbicacid and kolavironwere selected based on the results obtained

Journal of Toxicology 3

O

OHN

Cl

Cl

(a)

OH

O O

HO

HO

HO

(b)

O

O

O

O

OH

OH

OH

OHOH

OHOH OH

OHOH

HO

HO

GB1 H HGB2 HKolaflavanone H OMe

R1 R2 R3 R4

R1

R2

R3

R4

(c)

Figure 1 Chemical structure of (a) chlorambucil (4-[4-[bis(2-chloroethyl) amino] phenyl] butanoic acid) (b) L-ascorbic acid and (c)kolaviron

from previous studies in experimental animals [34 35] Eachof the drug doses was delivered orally in 1mL solution oncedaily using an oral cannulaThe vehicle for chlorambucil andascorbic acid was water while kolaviron was administered incorn oilThis has been included in the revisedmanuscript andhighlighted All the treatments lasted for a period of 14 days

25 Animal Sacrifice and Collection of Testes Animals weresacrificed 24 hours after the last treatments by cervical dislo-cation and the testes were carefully excised from each animalfor preparation of cytosolic fraction and histopathologicalanalysis Testicular samples for histopathological analysiswere immediately fixed in Bouinrsquos solution for 24 hours

26 Preparation of Cytosolic Fractions The testes excisedfrom rats were blotted of blood stains rinsed in ice-cold115 KCl and homogenized in 4 volumes of ice-cold 001Mphosphate buffer (pH74)Thehomogenateswere centrifugedat 12500 g for 15min at minus4∘C (Eppendorf UK) and the super-natants termed the postmitochondrial fractions (PMF) werealiquoted and used for biochemical assays

27 Determination of Testicular Protein Content The proteinconcentration in the testicular homogenate was determinedby theBiuretmethod ofGornall et al [36] using bovine serumalbumin as standard

28 Assay of Testicular SOD CAT and GST The methodof Misra and Fridovich [37] as described by Magwere et al[38] was used for the determination of testicular superoxidedismutase (SOD) activity by measuring the inhibition ofautooxidation of epinephrine at pH 102 and 30∘C Testicularcatalase activity was determined by the method of Asru[39] by measuring the reduction of dichromate in aceticacid to chromic acetate at 570 nm Glutathione S-transferase

(GST) activity was determined by the method described byHabig et al [40] using 1-chloro-24-dinitrobenzene (CDNB)as substrate

29 Assay of Testicular GSH Ascorbic Acid and Level ofLipid Peroxidation Testicular reduced glutathione (GSH)level was determined according to the method of Jollow et al[41] The chromophoric product resulting from the reactionof Ellmanrsquos reagent with the reduced glutathione 2-nitro-5-thiobenzoic acid possesses a molar absorption at 412 nmwhich was measured in a spectrophotometer Reduced GSHis proportional to the absorbance at 412 nm The ascorbicacid concentration was determined according to the methodof Jagota and Dani [42] Ascorbic acid in testicular samplesreacts with Folinrsquos reagent an oxidizing agent to give a bluecolor which has its maximum absorption at 760 nm Theextent of lipid peroxidation (LPO) was estimated by themethod of Varshney and Kale [43] The method involved thereaction between malondialdehyde (MDA) a product of lipidperoxidation and thiobarbituric acid to yield a stable pinkchromophore with maximum absorption at 532 nm

210 Assay of Testicular ACP ALP and LDH Testicularacid phosphatase (ACP) alkaline phosphatase (ALP) andlactate dehydrogenase (LDH) activities were determinedusing Randox diagnostic kits ACP and ALP activities weredetermined by the method of Tietz [44] The p-nitrophenolformed by the hydrolysis of p-Nitrophenyl phosphate confersyellowish colour on the reactionmixture and its intensity canbe monitored at 405 nm to give a measure of enzyme activityDetermination of testicular LDH activity was based on themethod of Cabaud and Wroblewski [45]

211 Testicular Histology Themethod of Baker and Silverton[46]was employed for the processing of the testicular samplesfor histopathological studies Fixed testicular samples were


dehydrated in graded ethanol and embedded in paraffin waxA thin section was made from each testicular tissue and wasstainedwith hematoxylin and eosin followed by examinationunder a light microscope

212 Statistical Analysis The results were expressed as meanof 5 replicates plusmn SD Data obtained were subjected to one-way analysis of variance (ANOVA) and complemented withDuncanrsquos multiple range test using SigmaPlot StatisticalSoftware A value of 119875 lt 005 was accepted as statisticallysignificant

3 Results

31 Protective Effect of Kolaviron and L-Ascorbic Acid onChlorambucil-Induced Changes in Testicular SOD CAT andGST Activities in Rats Following two-week exposure tochlorambucil the testicular activities of SOD CAT andGST were significantly decreased by 47 49 and 48respectively in chlorambucil-treated animals in comparisonwith the controls (Table 1) Kolaviron and ascorbic acidcosupplementation with chlorambucil significantly attenu-ated the activities of these testicular antioxidant enzymestoward the control levels

32 Protective Effect of Kolaviron and L-Ascorbic Acid onChlorambucil-Induced Changes in Testicular Level of GSHAscorbic Acid and MDA in Rats As presented in Figure 2a significant decrease by 39 and 32 in testicular reducedglutathione and vitamin C (nonenzymic antioxidants) wasobserved following chlorambucil exposure for two weekswith a corresponding significantly elevated testicular MDAcontent (a product of lipid peroxidation) However coad-ministration of chlorambucil with kolaviron and vitamin Cacid respectively significantly ameliorated the decrease intesticular GSH vitamin C and increase in MDA (Figure 2)

33 Protective Effect of Kolaviron and L-Ascorbic Acid onChlorambucil-Induced Changes in Activities of Testicular ACPALP and LDH The testicular acid phosphatase (ACP) activ-ity (Figure 3(a)) was significantly reduced by 70 while alka-line phosphatase (ALP) and lactate dehydrogenase (LDH)activities showed a significant increase by 59 and 64respectively in the chlorambucil-treated animals with respectto the control (Figures 3(a) 3(b) and 3(c)) Kolaviron orascorbic acid cosupplementation with chlorambucil signif-icantly restored the activities of these testicular enzymestoward control

34 Protective Role of Kolaviron and L-Ascorbic Acid againstChlorambucil-Induced Damage to Testicular Histology Rep-resentative photomicrographs of the testicular sections fromrats are shown in Figure 4 The histological study of thetesticular section of the control rats showed a typical normalcellular architecture with no lesion However chlorambucilexposure for two weeks resulted in formation of immaturegerminal cells in the lumen (Figure 4(b))However coadmin-istration of chlorambucil with kolaviron and ascorbic acid

restored the testicular histoarchitecture with distinct cellulararrangements as observed for control animals

4 Discussion

This study investigated the protective role of orally admin-istered kolaviron and ascorbic acid in chlorambucil-inducedtesticular toxicity in rat Chlorambucil is an anticancer agentindicated for chronic leukaemia andmay have the capacity toinduce oxidative stress or deplete tissue antioxidant reserveduring chemotherapy [14] The testis is an organ involved inspermatogenesis and is normally exposed to a low oxygenlevel Despite the low oxygen level the testes are known to bevulnerable to oxidative stress and peroxidative damage dueto the abundance of highly unsaturated fatty acids [15] Tes-ticular oxidative stress plays an important role in conditionsknown to be detrimental to male fertility These conditionsinclude exposure to certain drugs and environmental toxi-cants Series of studies on antineoplastic agents have shownthat they can cause oxidative stress in the testes with resultingdisturbance in testicular functions [6 47] However the testespossess formidable antioxidant defence systems comprisingboth enzymatic and nonenzymatic antioxidants The enzy-matic constituents of this defence system are made up ofcatalase (CAT) superoxide dismutase (SOD) glutathione S-transferase (GST) and glutathione peroxidase (GPx) Thenonenzymatic constituents include the ascorbic acid (AA)reduced glutathione (GSH) and vitamin E

In this study we observed a significant reduction intesticular activities of superoxide dismutase (SOD) catalase(CAT) and glutathione S-transferase as a result of CLBadministration SOD catalyzes the reaction involving a rapiddismutation of superoxide radical to hydrogen peroxide anddioxygen while CAT converts the hydrogen peroxide formedin this process and other cellular processes into water andmolecular oxygen Reduction in the activities of SOD andCAT by chlorambucil may predispose the testes to oxidativedamage [47] Glutathione S-transferase (GST) on the otherhand is an enzyme involved in the detoxification of ingestedxenobiotics in the liver GST activity is also present in othertissues including the testes where it functions as part of theantioxidant defense mechanism that scavenge and suppressthe formation of ROS [48] In addition to the formation ofglutathione S-conjugates of drugs GST also possesses antiox-idant capabilities It catalyses the reduction of peroxide-containing compounds in the cell and this peroxidase activityexhibited by GST is however dependent on availability ofGSH [48] Coadministration of kolaviron and L-ascorbic acidoffers protection against oxidative stress in the testes of theanimal by increasing the activities of antioxidant enzymes inthe experimental animals

AA is a vital antioxidant of the aqueous phase of the celland rapidly scavenges free radicals It also plays an importantrole in the regeneration of vitamin Emdashthe membrane boundantioxidant [49] The AA in the testes is maintained in areduced state byGSHdependent dehydroascorbate reductasein the testes Suppression of testicular AA and GSH followingadministration of antineoplastic drugs has been reported


Table 1 Protective effect of kolaviron and L-ascorbic acid on chlorambucil-induced changes in testicular SOD CAT and GST activities inrats

Treatment groups SOD (units) CAT (120583molesminmg protein) GST (nmoleminmg protein)Control 386 plusmn 19 814 plusmn 01 188 plusmn 12

CLB 204 plusmn 18lowast

412 plusmn 03lowast

974 plusmn 08lowast

CLB + KV 340 plusmn 16lowastdagger

714 plusmn 02lowastdagger


CLB + AA 328 plusmn 25lowastdagger



Results are expressed as mean plusmn SD (119899 = 5) SOD superoxide dismutase CAT catalase GST glutathione S-transferase CLB chlorambucil (02mgKg bw)KV kolaviron (100mgKg bw) and AA ascorbic acid (100mgKg bw) lowastSignificantly different from control daggerSignificantly different from CLB

0

5

10

15

20

25

30

Control CLB CLB + KV CLB + AA

GSH

(120583g

g te

stes)

lowast

dagger

dagger

(a)

0

5

10

15

20

25

30

35

lowast

dagger

dagger

Asc

orbi

c aci

d (120583

gm

L)


(b)

0

10

20

30

40

50

60

70

MD

A (n

mol

em

g pr

otei

n)

lowast

dagger

dagger


(c)

Figure 2 Protective effect of kolaviron and L-ascorbic acid on chlorambucil-induced changes in testicular level of GSH ascorbic acid andMDA in rats Results are expressed asmeanplusmn SD (119899 = 5) GSH reduced glutathioneMDAmalondialdehyde CLB chlorambucil (02mgKgbw) KV kolaviron (100mgKg bw) and AA ascorbic acid (100mgKg bw) lowastSignificantly different from CTRL daggerSignificantly differentfrom CLB

in several studies [50 51] The observed reduction may becaused by the presence of free radicals generated duringthe metabolism of the drug resulting in the consumptionof the available AA and GSH in the testes Reduction intesticular activities of enzymatic and levels of nonenzymaticantioxidants can predispose the testes to excessive oxidativestress Suppression of testicular antioxidant system has been

widely associated with oxidative stress Uncontrolled oxida-tive stress may result in membrane lipid peroxidation andultimately testicular damage and loss of testicular functionsThe decrease in the activities of SOD CAT and GST in CLB-exposed rat testicular cells may have increased the observedlevel of lipid peroxidation This observation is consistentwith previous reports [52ndash54] The observed reduction in


0

5

10

15

20

25

30

35

40AC

P (U

L)

lowast

daggerdagger


(a)

0

100

200

300

400

500

600

ALP

(UL

)

lowast

daggerdagger


(b)

0

5

10

15

20

25

30

35

40

LDH

(UL

)

lowast

dagger

dagger


(c)

Figure 3 Protective effect of kolaviron and L-ascorbic acid on chlorambucil-induced changes in testicular ACP ALP and LDH in rats Resultsare expressed as mean plusmn SD (119899 = 5) ACP acid phosphatase ALP alkaline phosphatase LDH lactate dehydrogenase CLB chlorambucil(02mgKg bw) KV kolaviron (100mgKg bw) and AA ascorbic acid (100mgKg bw) lowastSignificantly different fromCTRL daggerSignificantlydifferent from CLB

testicular lipid peroxidation may be related to the protectionof the antioxidant defense system in the rats by kolaviron andAA [55]

Activities of testicular marker enzymes such as acidphosphate (ACP) alkaline phosphate (ALP) and lactatedehydrogenase (LDH) are considered functional indicatorsof spermatogenesis The reduction in the activity of ACPby the administration of CLB was supported by Ananthanand Kumaran [56] where administration of Mancozeb (afungicide of ethylene-bis-dithiocarbamate group) signifi-cantly decreased the activity of ACP in the testis of ratSpermatogenic cells contain acid phosphatase and the specificactivity of this enzyme increases as the germ cells differ-entiate from spermatogonia into spermatocytes and sper-matids [57] Lysosomal acid phosphatases also participate inthe intracellular digestion of endogenous and phagocytosedexogenous compounds containing phosphate residues In the

acrosome they may be involved in the penetration of thespermatozoon through the egg [57] The increase in theactivity of ACP in the testes of animals administered KVand AA may be related to increased lysosomal activity in thetestes ALP and LDH play an important role in the processof spermatogenesis and have been shown to be vital forsperm survival and motility [58] The increased activity oftesticular ALP and LDH observed in this study may reflecttesticular degeneration [53] LDH is closely associated withspermatogenesis and testicular development Increase in theactivity of this enzyme in the chlorambucil-treated rats mayalso be resulting fromadaptation to improve spermatogenesisand testicular development from oxidative damage [59] KVand AA effectively attenuated the activities of ACP ALP andLDH

The degeneration of testicular histological structure byCLB may be a result of oxidative damage This can also result


(a) (b)

(c) (d)

Figure 4 Photomicrograph showing protective role of kolaviron and L-ascorbic acid on chlorambucil-induced damage on testicularmorphology (hematoxylin and eosin stain) (a) Control no visible lesions seen (b) Chlorambucil group there are immature germinal cellsin the lumen (c) Chlorambucil + kolaviron group no visible lesions seen (d) chlorambucil + ascorbic acid group no visible lesions seen

in the degeneration of germ cells and poor spermquality [60]KV and AA supplementation prevented the degeneration ofgerm cells as evidenced by histological evaluation Potentialevidences from the literature supporting the role of ROS-mediated oxidative stress may be responsible for the mech-anism of chlorambucil-induced testicular toxicity observed[14] The observed attenuation properties of KV and AA inthis study may be due to their antioxidant properties [23 61]which may be involved in the scavenging of radical speciesgenerated by CLB

5 Conclusion

From our present study it may be suggested that chlo-rambucil impairs testicular antioxidant system and causesdegenerative changes in the germ cells However kolavironand ascorbic acid positivelymodulate the effect of the drug onthe antioxidant status and effectively prevent chlorambucil-induced testicular toxicity

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] N Vardi H Parlakpinar B Ates A Cetin and A OtluldquoAntiapoptotic and antioxidant effects of 120573-carotene againstmethotrexate-induced testicular injuryrdquo Fertility and Sterilityvol 92 no 6 pp 2028ndash2033 2009

[2] P E Kintzel and R T Dorr ldquoAnticancer drug renal toxicityand elimination dosing guidelines for altered renal functionrdquoCancer Treatment Reviews vol 21 no 1 pp 33ndash64 1995

[3] J Tomenendalova JMayerMDoubekDHorky K Rehakovaand J Doubek ldquoChlorambucil and fludarabine as a new pre-transplant conditioning for patients with chronic lymphocyticleukemia results of in vivo experimentsrdquoVeterinarni Medicinavol 53 no 10 pp 564ndash571 2008


[4] G Pearse A Pietersma J Cunliffe et al ldquoTime-course study ofthe immunotoxic effects of the anticancer drug chlorambucil inthe ratrdquo Toxicologic Pathology vol 37 no 7 pp 887ndash901 2009

[5] G Ramadori and S Cameron ldquoEffects of systemic chemother-apy on the liverrdquo Annals of Hepatology vol 9 no 2 pp 133ndash1432010

[6] E Yulug S Turedi A Alver and C Kahraman ldquoEffects ofresveratrol on methotrexate-induced testicular damage in ratsrdquoThe Scientific World Journal vol 2013 Article ID 489659 6pages 2013

[7] G C Kundu J R Schullek and I B Wilson ldquoThe alkylatingproperties of chlorambucilrdquo Pharmacology Biochemistry andBehavior vol 49 no 3 pp 621ndash624 1994

[8] A McLean D Newell G Baker and T Connors ldquoThemetabolism of chlorambucilrdquo Biochemical Pharmacology vol29 no 14 pp 2039ndash2047 1980

[9] D Plumb Plumbrsquos Veterinary DrugHandbook Blackwell Stock-holm Wis USA 2005

[10] A Puckowska D Drozdowska M Rusak T Bielawski IBruzgo and K Midura-Nowaczek ldquoAmino and chlorambucilanalogues of pentamidine synthesis and biological examina-tionsrdquo Acta Poloniae Pharmaceutica Drug Research vol 69 no1 pp 63ndash73 2012

[11] N Pichon M Debette-Gratien F Cessot et al ldquoAcutecholestatic hepatitis due to chlorambucilrdquo GastroenterologieClinique et Biologique vol 25 no 2 pp 202ndash203 2001

[12] A Nicolle S J Proctor and G P Summerfield ldquoHigh dosechlorambucil in the treatment of lymphoid malignanciesrdquoLeukemia and Lymphoma vol 45 no 2 pp 271ndash275 2004

[13] N Lameire V Kruse and S Rottey ldquoNephrotoxicity of anti-cancer drugsmdashan underestimated problemrdquo Acta Clinica Bel-gica vol 66 no 5 pp 337ndash345 2011

[14] E T Olayinka A Ore and K A Fashiku ldquoKolaviron and L-ascorbic acid ameliorates chlorambucilmdashinduced hepatic andrenal toxicity in ratrdquo International Journal of Toxicology andApplied Pharmacology vol 4 no 1 pp 23ndash32 2014

[15] R J Aitken and S D Roman ldquoAntioxidant systems andoxidative stress in the testesrdquo in Molecular Mechanism in Sper-matogenesis C Y Cheng Ed pp 154ndash171 Landes Bioscienceand Springer Science 2008

[16] C Mylonas and D Kouretas ldquoLipid peroxidation and tissuedamagerdquo In Vivo vol 13 no 3 pp 295ndash309 1999

[17] E O Farombi I A Adedara A B Oyenihi E Ekakitie andS Kehinde ldquoHepatic testicular and spermatozoa antioxidantstatus in rats chronically treated with Garcinia kolaseedrdquoJournal of Ethnopharmacology vol 146 no 2 pp 536ndash542 2013

[18] E O Farombi I A Adedara S A Akinrinde O O Ojo andA S Eboh ldquoProtective effects of kolaviron and quercetin oncadmium-induced testicular damage and endocrine pathologyin ratsrdquo Andrologia vol 44 no 4 pp 273ndash284 2012

[19] I A Adedara P P Mathur and E O Farombi ldquoKolavironprevents ethylene glycol monoethyl ether-induced testicularapoptosis via down-regulation of stress proteins FasFas-Land caspases expressions in ratsrdquo Toxicology Mechanisms andMethods vol 23 no 9 pp 689ndash696 2013

[20] OAAdaramoyeOOAkanni and EO Farombi ldquoNevirapineinduces testicular toxicity in Wistar rats reversal effect ofkolaviron (biflavonoid from Garcinia kola seeds)rdquo Journal ofBasic and Clinical Physiology and Pharmacology vol 24 no 4pp 313ndash320 2013

[21] K A Conklin ldquoChemotherapy-associated oxidative stressimpact on chemotherapeutic effectivenessrdquo Integrative CancerTherapies vol 3 no 4 pp 294ndash300 2004

[22] J A Drisko J Chapman and V J Hunter ldquoThe use of antiox-idant therapies during chemotherapyrdquo Gynecologic Oncologyvol 88 no 3 pp 434ndash439 2003

[23] S M Sagar ldquoShould patients take or avoid antioxidant supple-ments during anticancer therapy An evidence-based reviewrdquoCurrent Oncology vol 12 no 2 pp 44ndash54 2005

[24] A M Seufi S S Ibrahim T K Elmaghraby and E EHafez ldquoPreventive effect of the flavonoidquercetin on hepaticcancer in rats via oxidantantioxidant activity molecular andhistological evidencesrdquo Journal of Experimental and ClinicalCancer Research vol 28 no 1 article 80 2009

[25] C M Kurbacher U Wagner B Kolster P E AndreottiD Krebs and H W Bruckner ldquoAscorbic acid (vitamin C)improves the antineoplastic activity of doxorubicin cisplatinand paclitaxel in human breast carcinoma cells in vitrordquo CancerLetters vol 103 no 2 pp 183ndash189 1996

[26] V S Mosienko L T Khasanova V N Sukolinskii T SMorozkina and V N Sukolinskiı ldquoAbstract Effectiveness ofcombined action of vitamins A E and C and Cyclophosphaneor Adriamycin on growth of transpslanted tumors in micerdquoEksperimentalaia Oncologiia vol 12 pp 55ndash57 1990

[27] J A Choi J Y Kim J Y Lee et al ldquoInduction of cell cyclearrest and apoptosis in human breast cancer cells by quercetinrdquoInternational Journal of Oncology vol 19 no 4 pp 837ndash8442001

[28] C S Ong E Tran T T T Nguyen et al ldquoQuercetin-inducedgrowth inhibition and cell death in nasopharyngeal carcinomacells are associated with increase in Bad and hypophosphory-lated retinoblastoma expressionsrdquo Oncology Reports vol 11 no3 pp 727ndash733 2004

[29] R G Beniston and M S Campo ldquoQuercetin elevates p27Kip1and arrests both primary and HPV16 E6E7 transformedhuman keratinocytes in G1rdquoOncogene vol 22 no 35 pp 5504ndash5514 2003

[30] GDiCarloNMascolo AA Izzo and F Capasso ldquoFlavonoidsold and new aspects of a class of natural therapeutic drugsrdquo LifeSciences vol 65 no 4 pp 337ndash353 1999

[31] M M Iwu O A Igboko C O Okunji and M S TempestaldquoAntidiabetic and aldose reductase activities of biflavanones ofGarcinia kolardquo Journal of Pharmacy and Pharmacology vol 42no 4 pp 290ndash292 1990

[32] O A Adaramoye E O Farombi E O Adeyemi and G OEmerole ldquoComparative study on the antioxidant properties offlavonoids of Garcinia kola seedsrdquo Pakistan Journal of MedicalSciences vol 21 no 3 pp 331ndash339 2005

[33] J Tomenendalova J Mayer M Doubek et al ldquoToxicity of high-dose chlorambucil inWistar ratsrdquoActa Veterinaria Brno vol 77no 4 pp 595ndash602 2008

[34] E O Farombi and I A Nwaokeafor ldquoAnti-oxidant mechanismsof kolaviron Studies on serum lipoprotein oxidation metalchelation and oxidative membrane damage in ratsrdquoClinical andExperimental Pharmacology and Physiology vol 32 no 8 pp667ndash674 2005

[35] S O Malomo A Ore and M T Yakubu ldquoIn vitro and in vivoantioxidant activities of the aqueous extract of Celosia argentealeavesrdquo Indian Journal of Pharmacology vol 43 no 3 pp 278ndash285 2011


[36] A G Gornall C J Bardwawill and M M David ldquoDetermi-nation of serum proteins by means of the biuret reactionrdquo TheJournal of biological chemistry vol 177 no 2 pp 751ndash766 1949

[37] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquoThe Journal of Biological Chemistry vol 247 no 10pp 3170ndash3175 1972

[38] T Magwere Y S Naik and J A Hasler ldquoEffects of chloroquinetreatment on antioxidant enzymes in rat liver and kidneyrdquo FreeRadical Biology and Medicine vol 22 no 1-2 pp 321ndash327 1996

[39] A K Sinha ldquoColorimetric assay of catalaserdquo Analytical Bio-chemistry vol 47 no 2 pp 389ndash394 1972

[40] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases The first enzymatic step in mercapturic acidformationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974

[41] D J Jollow J R Mitchell N Zampaglione and J R GilletteldquoBromobenzene induced liver necrosis Protective role of glu-tathione and evidence for 34 bromobenzene oxide as thehepatotoxic metaboliterdquo Pharmacology vol 11 no 3 pp 151ndash169 1974

[42] S K Jagota and H M Dani ldquoA new colorimetric techniquefor the estimation of vitamin C using folin phenol reagentrdquoAnalytical Biochemistry vol 127 no 1 pp 178ndash182 1982

[43] R Varshney and R K Kale ldquoEffects of calmodulin antagonistson radiation-induced lipid peroxidation in microsomesrdquo Inter-national Journal of Radiation Biology vol 58 no 5 pp 733ndash7431990

[44] N M Tietz Clinical Guide to Laboratory Tests SaundersPhiladelphia Pa USA 3rd edition 1995

[45] P G Cabaud and F Wroblewski ldquoColorimetric measurementof lactic dehydrogenase activity of body fluidsrdquo The AmericanJournal of Clinical Pathology vol 30 no 3 pp 234ndash236 1958

[46] F J Baker and R E Silverton Introduction to Medical Labora-tory Technology Butter worth London UK 6th edition 1985

[47] J Yeh B S Kim and J Peresie ldquoReproductive toxic effectsof cisplatin and its modulation by the antioxidant sodium 2-mercaptoethanesulfonate (Mesna) in female ratsrdquo ReproductiveBiology Insights vol 4 no 1 pp 17ndash27 2011

[48] P J Sherratt and J D Hayes ldquoGlutathione S-transferasesrdquo inEnzyme Systems thatMetabolise Drugs andOther Xenobiotics CIoannides Ed pp 219ndash252 John Wiley amp Sons London UK2002

[49] K M Robert K G Daryl A M Peter and W R Victor ldquoVita-mins and Mineralsrdquo in Harperrsquos Biochemistry R K Murray DK Granner P A Mayes and V W Rodwell Eds vol 25 pp169ndash649 Appleton Lange New York NY USA 2000

[50] Y O Ilbey E Ozbek A Simsek A Otunctemur M Cekmenand A Somay ldquoPotential chemoprotective effect of melatoninin cyclophosphamide- and cisplatin-induced testicular damagein ratsrdquo Fertility and Sterility vol 92 no 3 pp 1124ndash1132 2009

[51] L Patil and R Balaraman ldquoEffect of melatonin on doxorubicininduced testicular damage in ratsrdquo International Journal ofPharmTech Research vol 1 no 3 pp 879ndash884 2009

[52] E T Olayinka A Ore and O O Akinnawo ldquoProtective roleof ethanolic extract of Sorghum bicolor leaf sheath againstcadmium-induced oxidative stress in ratsrdquo International Journalof Pharmacology Biomedical Research vol 2 no 4 pp 254ndash2602011

[53] P Seema S S Swathy and M Indira ldquoProtective effect of sele-nium on nicotine-induced testicular toxicity in ratsrdquo BiologicalTrace Element Research vol 120 no 1ndash3 pp 212ndash218 2007

[54] K Jana P K Samanta and D Kumar De ldquoNicotine diminishestesticular gametogenesis steroidogenesis and steroidogenicacute regulatory protein expression in adult albino rats possibleinfluence on pituitary gonadotropins and alteration of testicularantioxidant statusrdquo Toxicological Sciences vol 116 no 2 pp647ndash659 2010

[55] O A Adaramoye and M Arisekola ldquoKolaviron a biflavonoidcomplex fromGarcinia kola seeds ameliorates ethanol-inducedreproductive toxicity in male wistar ratsrdquo Nigerian Journal ofPhysiological Sciences vol 28 no 1 pp 9ndash15 2012

[56] G Ananthan and B Kumaran ldquoEffect of mancozeb on thespecific activities of testicular phosphatases and Protectiverole of vitamin C in Albino ratsrdquo Bullettin of EnvironmentalPharmacology and Life Sciences vol 2 no 7 pp 56ndash61 2013

[57] T Vanha-Perttula J P Mather C W Bardin S B Moss and AR Bellve ldquoAcid phosphatases in germinal and somatic cells ofthe testesrdquo Biology of reproduction vol 35 no 1 pp 1ndash9 1986

[58] P Kaur and M P Bansal ldquoInfluence of selenium inducedoxidative stress on spermatogenesis and lactate dehydrogenase-X in mice testisrdquo Asian Journal of Andrology vol 6 no 3 pp227ndash232 2004

[59] S O Abarikwu E O Farombi and A B Pant ldquoKolavironbiflavanoids of garcinia kola seeds protect atrazine-inducedcytotoxicity in primary cultures of rat leydig cellsrdquo InternationalJournal of Toxicology vol 31 no 4 pp 407ndash415 2012

[60] S Kim I Lee H Baek C Moon S Kim and J KimldquoProtective effect of diallyl disulfide on cyclophosphamide-induced testicular toxicity in ratsrdquo Laboratory Animal Researchvol 29 no 4 pp 204ndash211 2013

[61] K Iqbal A A Khan and K K Khattak ldquoBiological significanceof ascorbic acid (vitaminC) in humanhealth a reviewrdquoPakistanJournal of Nutrition vol 3 no 1 pp 5ndash13 2004

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


enzymatic antioxidants such as catalase superoxide dismu-tase glutathione peroxidase glutathione reductase and glu-tathione S-transferaseThe testes are rich in lipid componentsand therefore exposure to high level of prooxidants maydeplete the antioxidant reserve thereby exposing the cellto excessive level of lipid peroxidation and ultimately tissuedamage [16]

The natural antioxidant kolaviron is a fraction of thedefatted ethanol extract ofGarcinia kola containing Garciniabiflavonoids GB1 and GB2 and kolaflavanone (Figure 1(c))Kolaviron is of high safety profile and its antioxidant prop-erties have been extensively studied in vivo [17] Moreover itis known to significantly prevent drug and chemical-inducedorgan toxicity and oxidative damage in experimental animalmodels [14 18ndash20]

Many classes of antineoplastic agents are known togenerate high levels of oxidative stress in biological systems[21] leading to tissue injuries Injuries elicited by antineo-plastic agents on vital organs have been reported in mostcases but with few reports on reproductive organs particu-larly the testes Fewer reports are also available concerningchemotherapy-induced injuries due to generation of potentoxidants or disruptions of testicular antioxidant system bythese agents Besides proper functioning of the testicularredox homeostasis is essential to efficient spermatogenesisand overall function of the testes Chlorambucil has beenreported in our previous study to induce oxidative stress inthe liver of rats [14] It is assumed that the administrationof antioxidants along with chlorambucil may offer someprotection against generation of reactive oxygen species(ROS) and oxidative damage to the testes In addition if thereactive species and oxidative stress caused by chlorambucilare responsible only for its toxic effects antioxidants sup-plementation may reduce the severity of these toxic effectswithout interfering with the antineoplastic activity of thedrug Evidence suggests that administration of antioxidantsalong with chemotherapeutic agents has been found toeffectively relieve toxic side effects [22ndash24] In most casesantioxidant has improved the cytotoxicity of antineoplasticagents For instance vitamin C is reported to enhance theantineoplastic activity of doxorubicin cisplatin and paclitaxilin human breast carcinoma cells [25] Also combination ofvitamins A C and E improved the antitumor activity ofdoxorubicin in mice [26] Several studies have also reportedthe potential of the plant derived flavonoids to inhibit theproliferation of cancer cells [27ndash30] However the presentstudy was designed specifically to evaluate the protective roleof kolaviron a biflavonoid complex and L-ascorbic acid onchlorambucil-induced oxidative stress in the testes of rats

2 Materials and Methods

21 Chemicals and Assay Kits Chlorambucil (Leukeran) wasa product of Aspen Pharm Trading Limited Dublin 1Ireland 1-Chloro-24-dinitrobenzene (CDNB) 5101584051015840-di-thiobis-2-nitrobenzoic acid (DTNB) glutathione (GSH)epinephrine hydrogen peroxide (H

2O2) and thiobarbituric

acid (TBA) were all purchased from Sigma ChemicalCompany (London UK) Assay kits for alkaline phosphatase

(ALP) and acid phosphatase (ACP) were obtained fromRandox Laboratories Ltd (Antrim UK) Assay kit for lactatedehydrogenase (LDH) was a product of Cypress DiagnosticsBelgium All other reagents used were of analytical gradeand were obtained from British Drug House (BDH) PooleEngland

22 Extraction of Kolaviron Kolaviron was extracted fromthe fresh seeds of Garcinia kola (35 kg) and characterizedaccording to the method of Iwu et al [31] Briefly thepowdered seeds were extracted with light petroleumether (bp 40ndash60∘C) in a Soxhlet extractor for 24 hr Thedefatted dried marc was repacked and then extracted withmethanol The extract was concentrated and diluted to twiceits volume with distilled water and extracted with ethylacetate (625 L) The concentrated ethyl acetate fractionyielded a golden yellow solid termed kolaviron whichhas been shown to consist of Garcinia biflavonoid GB-1(31015840101584041015840410158401015840101584055101584010158407710158401015840-heptahydroxy-3810158401015840 biflavanone) GB-2(310158401015840410158404101584010158401015840551015840101584051015840101584010158407710158401015840-octahydroxy-3810158401015840-biflavanone) andkolaflavanone (310158401015840410158404101584010158401015840551015840101584051015840101584010158407710158401015840octahydroxy-4101584010158401015840-me-thoxy-3810158401015840-biflavanone) (Figure 1) The purity and identityof kolaviron were determined by subjecting it to thin-layerchromatography (TLC) using Silica gel GF 254-coated platesand solvent mixture of methanol and chloroform in a ratio1 4 vv The separation revealed the presence of three bandswhich were viewed under UV light at a wavelength of 254 nmwith RF values of 048 071 and 076 [32]

23 Animal Selection and Care Twenty inbred male rats(Wistar strain) weighing between 180 and 200 g wereobtained from the animal holding unit of the Department ofChemical Sciences Ajayi Crowther University Oyo NigeriaThe rats were acclimatised under laboratory conditions priorto the commencement of the studyThe animals were housedin wire meshed cages maintained at standard conditions oftemperature and humidity with an alternating light cycle(12 hr lightdark) They were fed with standard rat pellet(Ladokun feeds Ibadan Nigeria) and were supplied waterad libitum The experimental protocol relating to animalhandling conformed to the Guidelines of the National Insti-tute of HealthmdashGuide for the Care and Use of LaboratoryAnimals (NIH Publication number 85-23 revised 1985 USDepartment of Health Education and Welfare BethesdaMA)

24 Drug Treatments The rats were randomised into fourexperimental groups (IndashIV) of 5 animals each Group I(control) animals were administered distilled water Animalsin group II received chlorambucil at a dose of 02mgkg bodyweight (bw) Group III animals were coadministered chlo-rambucil (02mgkg bw) and kolaviron (100mgkg bw)Animals in group IV were coadministered chlorambucil(02mgkg bw) and L-ascorbic acid (100mgkg bw) Thedose of chlorambucil administered to the animals was basedon the recommended dose in the treatment of Hodgkinrsquoslymphoma (01-02mgkgday) [33] while doses of ascorbicacid and kolavironwere selected based on the results obtained


O

OHN

Cl

Cl

(a)

OH

O O

HO

HO

HO

(b)

O

O

O

O

OH

OH

OH

OHOH

OHOH OH

OHOH

HO

HO


R1 R2 R3 R4

R1

R2

R3

R4

(c)














3 Results






4 Discussion








412 plusmn 03lowast

974 plusmn 08lowast








0

5

10

15

20

25

30


GSH

(120583g

g te

stes)

lowast

dagger

dagger

(a)

0

5

10

15

20

25

30

35

lowast

dagger

dagger

Asc

orbi

c aci

d (120583

gm

L)


(b)

0

10

20

30

40

50

60

70

MD

A (n

mol

em

g pr

otei

n)

lowast

dagger

dagger


(c)





0

5

10

15

20

25

30

35

40AC

P (U

L)

lowast

daggerdagger


(a)

0

100

200

300

400

500

600

ALP

(UL

)

lowast

daggerdagger


(b)

0

5

10

15

20

25

30

35

40

LDH

(UL

)

lowast

dagger

dagger


(c)







(a) (b)

(c) (d)



5 Conclusion




References




































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


O

OHN

Cl

Cl

(a)

OH

O O

HO

HO

HO

(b)

O

O

O

O

OH

OH

OH

OHOH

OHOH OH

OHOH

HO

HO


R1 R2 R3 R4

R1

R2

R3

R4

(c)














3 Results






4 Discussion








412 plusmn 03lowast

974 plusmn 08lowast








0

5

10

15

20

25

30


GSH

(120583g

g te

stes)

lowast

dagger

dagger

(a)

0

5

10

15

20

25

30

35

lowast

dagger

dagger

Asc

orbi

c aci

d (120583

gm

L)


(b)

0

10

20

30

40

50

60

70

MD

A (n

mol

em

g pr

otei

n)

lowast

dagger

dagger


(c)





0

5

10

15

20

25

30

35

40AC

P (U

L)

lowast

daggerdagger


(a)

0

100

200

300

400

500

600

ALP

(UL

)

lowast

daggerdagger


(b)

0

5

10

15

20

25

30

35

40

LDH

(UL

)

lowast

dagger

dagger


(c)







(a) (b)

(c) (d)



5 Conclusion




References




































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




3 Results






4 Discussion








412 plusmn 03lowast

974 plusmn 08lowast








0

5

10

15

20

25

30


GSH

(120583g

g te

stes)

lowast

dagger

dagger

(a)

0

5

10

15

20

25

30

35

lowast

dagger

dagger

Asc

orbi

c aci

d (120583

gm

L)


(b)

0

10

20

30

40

50

60

70

MD

A (n

mol

em

g pr

otei

n)

lowast

dagger

dagger


(c)





0

5

10

15

20

25

30

35

40AC

P (U

L)

lowast

daggerdagger


(a)

0

100

200

300

400

500

600

ALP

(UL

)

lowast

daggerdagger


(b)

0

5

10

15

20

25

30

35

40

LDH

(UL

)

lowast

dagger

dagger


(c)







(a) (b)

(c) (d)



5 Conclusion




References




































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





412 plusmn 03lowast

974 plusmn 08lowast








0

5

10

15

20

25

30


GSH

(120583g

g te

stes)

lowast

dagger

dagger

(a)

0

5

10

15

20

25

30

35

lowast

dagger

dagger

Asc

orbi

c aci

d (120583

gm

L)


(b)

0

10

20

30

40

50

60

70

MD

A (n

mol

em

g pr

otei

n)

lowast

dagger

dagger


(c)





0

5

10

15

20

25

30

35

40AC

P (U

L)

lowast

daggerdagger


(a)

0

100

200

300

400

500

600

ALP

(UL

)

lowast

daggerdagger


(b)

0

5

10

15

20

25

30

35

40

LDH

(UL

)

lowast

dagger

dagger


(c)







(a) (b)

(c) (d)



5 Conclusion




References




































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

5

10

15

20

25

30

35

40AC

P (U

L)

lowast

daggerdagger


(a)

0

100

200

300

400

500

600

ALP

(UL

)

lowast

daggerdagger


(b)

0

5

10

15

20

25

30

35

40

LDH

(UL

)

lowast

dagger

dagger


(c)







(a) (b)

(c) (d)



5 Conclusion




References




































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(a) (b)

(c) (d)



5 Conclusion




References




































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

research article kolaviron and l-ascorbic acid attenuate...

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