prophylactic role of averrhoa carambola (star fruit) extract against

Research ArticleProphylactic Role of Averrhoa carambola (Star Fruit) Extractagainst Chemically Induced Hepatocellular Carcinoma in SwissAlbino Mice

Ritu Singh Jyoti Sharma and P K Goyal

Radiation amp Cancer Biology Laboratory Department of Zoology University of Rajasthan Jaipur 302 004 India

Correspondence should be addressed to P K Goyal pkgoyal2002gmailcom

Received 4 January 2014 Accepted 10 January 2014 Published 19 February 2014

Academic Editor Mustafa F Lokhandwala

Copyright copy 2014 Ritu Singh et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Liver cancer remains one of the severe lethal malignancies worldwide and hepatocellular carcinoma (HCC) is the most commonformThe current study was designed to evaluate the prophylactic role of the fruit of Averrhoa carambola (star fruit or Kamrak) ondiethylnitrosamine- (DENA-) induced (15mgkg bwt single ip injection) and CCl

4-promoted (16 gkg bwt in corn oil thrice a

week for 24 weeks) liver cancer in Swiss albino mice Administration of ACE was made orally at a dose of 25mgkg bwtday for 5consecutive days and it was withdrawn 48 hrs before the first administration of DENA (preinitiational stage) CCl

4was given after 2

weeks of DENA administration A cent percent tumor incidence was noted in carcinogen treated animals while ACE administrationresulted in a considerable reduction in tumor incidence tumor yield and tumor burden Further ACE treatment brings out asignificant reduction in lipid peroxidation (119875 lt 0001) alongwith an elevation in the activities of enzymatic antioxidants (superoxidedismutase 119875 lt 0001 and catalase 119875 lt 0001) nonenzymatic antioxidant (reduced glutathione 119875 lt 0001) and total proteins(119875 lt 0001) when compared to the carcinogen treated control These results demonstrate that ACE prevents the DENACCl

4

induced adverse physical and biochemical alterations during hepatic carcinogenesis in mice This study suggests the prophylacticrole of Averrhoa carambola against hepatocellular carcinoma in mice therefore it could be employed for the further screening as agood chemopreventive natural supplement against cancer

1 Introduction

Cancer is a global challenge as this disease remains thesecond largest cause of death around the world with somepredictions that it will move into the top rank in coming timeCancer accounts for one out of every eight deaths annuallyIncrease in life expectancy and adoption of western diet andlifestyles owing to tobacco abuse andwidespread exposure tocarcinogens are some of the major key factors for increasingthe burden of cancer in the developing countries like India

Hepatocellular carcinoma (HCC) is one of the mostfrequent malignant tumors worldwide and a leading causeof cancer related death killing 5 lacs people annually [1]HCC has been linked to diverse etiologies including chronichepatitis B and C viral infection and alcohol exposure [2]Due to the high tolerance of liver HCC is seldom detected atthe early stage and treatment has a poor prognosis in most ofthe cases making it a significant global health problem [3ndash5]

The recurrence rates of HCC are also very high and long-term survival rate of the patients has not improved muchfrom the past few decades Surgery including transplanta-tion resection is currently the most effective treatment forHCC

Since the liver is the major site of metabolism of ingestedmaterials it is more susceptible to carcinogenic insult DENA(diethylnitrosamine) is a potent carcinogen entering the envi-ronment through the food chain [6] DENA is synthesizedendogenously and found in work place processed meatstobacco smoke whiskey and wide variety of foods and it isalso produced from metabolism of some drugs [7] In addi-tion DENA is extensively used as a solvent in fiber industrysoftener for copolymer additive for lubricants in condensersto increase the dielectric constant and for the synthesis of 11-diethylhydrazine [8] It is a potent hepatocarcinogen knownto cause perturbations in the nuclear enzymes involved inDNA repair or replication [9]

Hindawi Publishing CorporationAdvances in Pharmacological SciencesVolume 2014 Article ID 158936 8 pageshttpdxdoiorg1011552014158936

2 Advances in Pharmacological Sciences

Free radicals and other reactive oxygen species (ROS) areconstantly formed in the human body accumulation whichcauses oxidative damage Normal cells have evolved defensemechanisms for protection against this oxidative damageby developing multiple antioxidative defenses [10] If suchdelicate balance of free radical production and antioxidantdefenses goes out of control it results in the pathology ofseveral human diseases including cancer atherosclerosismalaria rheumatoid arthritis and neurodegenerative dis-eases [11 12]

Chemoprevention is a pharmacological way of interfer-ence in order to arrest or reverse the process of carcinogene-sis Chemopreventive substances are identified on the basisof their antioxidant antimutagenic and anti-inflammatoryactivities capable of arresting proliferation and enhancingapoptosis which are the major criteria for their anticar-cinogenic activity Progress in the area of chemopreventionduring the past two decades has been very impressiveAccumulating epidemiological and experimental evidenceshave revealed the chemopreventive influence of number ofnaturally occurring compounds and their role in preventionof the diseases [13ndash17]

Herbal products are gaining progressively attention thesedays for primary health care owing to less toxicity bettercompatibility with the body and high efficacy against freeradical mediated diseases Many studies have suggested thata healthy diet especially fruits and vegetables that are rich innatural antioxidants is efficacious to prevent oxidative stressand thus plays a vital role in cancer prevention [18]

Averrhoa carambola L (Oxalidaceae) is also known asthe star fruit tree Studies have shown that the fruit of Acarambola has several medicinal properties and it is richin antioxidants which act against reactive oxygen speciesThe ripe star fruit has digestive and biliousness propertiesIt is also a good source of vitamin C and used to treatheadache vomiting coughing hangovers and eczemas [1920] Furthermore it is used as an appetite stimulant diureticantidiarrheal and febrifugal agent In addition the extractobtained through the leaves of such planthas been used in thetreatment of diabetes [21]

Looking into the pharmacological and medicinal proper-ties of this plant the present study has been targeted to inves-tigate the possible anticancer potential of A carambola fruitextract against chemical induced hepatocellular carcinoma inmammals

2 Materials and Methods

21 Chemicals Diethylnitrosamine (DENA) and carbontetrachloride (CCl

4) were purchased from Sigma Chemical

Co (St Louis MO USA) DENA at a dose 15mgkg bwt(single ip injection in normal saline) was injected to initiatehepatic carcinogenesis while CCl

4(16 gkg bwt) in 1 1

dilution with corn oil was given orally to animals by gavageto stimulate liver cell proliferation and regeneration

22 Preparation of Plant Extract Carambola fruits werecleaned air dried and grinded into the form of fine powder

The powder was extracted with 90 ethyl alcohol usingSoxhlet apparatus and concentrated by evaporating its liquidcontents The required dose for further treatment was pre-pared by dissolving the extract in DDW

23 Animals Swiss albino mice (3-week old) were takenfor the experiment from an inbred colony and they wereprovided feed and water ad libitum All studies were carriedout in accordance with the guidelines of the InstitutionalAnimal Ethics Committee amp INSA New Delhi

24 Dose Selection of ACE For deciding the optimum doseexperiments were conducted inwhich Swiss albinomiceweredivided into different groups and were given orally Averrhoacarambola extract (ACE) at the dose of 05 15 25 50 and75mgkg bwtdaymganimalday Animals from each groupwere observed for 30 days for any sign of sickness morbiditymortality gait weight behavioral alterations and so forthand were necropsied on 16th and 31st day Various doses ofACE were selected (ie 15 25 and 50mgkgbwtanimal)from the above doses after evaluation of various biochemicalparameters in the liver of mice Out of these 25mg dose wasfound to be the optimum dose for this experiment

25 Chemopreventive Activity of ACE Animals for thisexperiment were divided into the following groups

Group I Negative Control (Vehicle Treated Normal Mice) Inthis group animals were given single ip injection of normalsaline and later administered with corn oil by oral gavagethree times in a week for the entire experimental period thatis for 24 weeks

Group II Positive Control (Carcinogen Treated) The animalsin this group were given DENA in normal saline After 2weeks of DENA administration CCl

4was given 3 times in

a week until the end of the experiment

Group III Drug Treated Control In this group the animalswere administered Averrhoa carambola extract (ACE) at adose of 25mgkgbwtanimalday for the entire experimentalperiod

Group IV ACE Treated Experimental The animals of thisgroup were provided ACE at a dose of 25mgkg bwtdayfor 5 consecutive days ACE source was withdrawn 48 hrsbefore the first administration of DENA CCl

4was given after

2 weeks as 3 times a week for 24 weeksThe following parameters were taken into account for the

study

(1) Morphological

(i) Body Weight The weights of the animals fromeach group were recorded at the beginning andat the termination of the experiments

(ii) Tumor Incidence It is the number of micecarrying at least 1 tumor expressed as percentageincidence

Advances in Pharmacological Sciences 3

Table 1 Variations in bodyweight liver weight andmorphometry of liver tumor afterDENACCl4 treatmentwith orwithoutACE treatment

Group Body weight (gm) Liver weight (gm) TumorInitial Final Incidence () Burden Yield

I 1310 plusmn 148 3160 plusmn 037 254 plusmn 092 0 0 0II 1260 plusmn 034 3500 plusmn 044 390 plusmn 054 100 24 24III 1540 plusmn 183 3383 plusmn 097 260 plusmn 028 0 0 0IV 1250 plusmn 119 3093 plusmn 022 305 plusmn 033 80 81 1012

(iii) Tumor Yield It refers to the total numberof tumors per group (number of tumorstotalnumber of mice)

(iv) Tumor Burden The average number of tumorsper tumor bearing mouse (total number oftumors in all micetotal number of tumor bear-ing mice)

(2) Biochemical All the animals were autopsied after theend of experiment that is 24 weeks and the wholeliver was taken out from each mice Biochemicalanalysis for the following parameters was performedin the liver

(i) Lipid Peroxidation (LPO) The level of LPOin liver was measured in terms of thiobarbi-turic acid reactive substances by the methodof Ohkhawa et al [22] Briefly thiobarbituricacid (08) sodium dodecyl sulfate (01) andacetic acid (20) were added to 100mL of thetissue homogenate for 60min It was cooledand extracted with N-butanol-pyridine and theoptical density was recorded at 532 nm Thecontent of TBAS was expressed in nmolmg

(ii) Glutathione (GSH) The level of reduced GSHwas estimated by the method of Moron et al[23]TheGSH content in the liver wasmeasuredspectrophotometrically using Ellmanrsquos reagentwith 551015840-dithiobis 2-nitrobenzoic acid (DTNB)as a coloring agent according to the method ofBeutler et al [24]The absorbance was recordedat 412 nm with levels expressed as nmolmg ofprotein

(iii) Catalase (CAT) The enzyme activity wasassayed in the liver by the method of Aebi[25] The content was estimated at 240 nm bymonitoring the disappearance of H2O2

(iv) SuperoxideDismutase (SOD)The activity of thisenzyme was measured by utilizing the methodof S Marklund and G Marklund [26]

(v) Total ProteinsTheprotein contents in liver weremeasured by the method of Lowry et al [27]The absorbance was recorded at 680 nm

3 Results and Discussion

There was no considerable change in the average body weightof DENA (Group II) ACE (Group III) and DENA + ACE

0

20

40

60

80

100

III

IIIIV

0

100

0

80

Tum

or in

cide

nce (

)

Groups

Figure 1 Variations in tumor incidence () after DENACCl4

induced hepatic carcinogenesis withwithout ACE administration

(Group IV) treated animals when compared to vehicle treatedcontrol (Group I) However the ACE treated mice (GroupIII) exhibited a spontaneous gain in body weight as similarto the control mice The mice receiving DENA and CCl

4

(carcinogen control) exhibited a slight increase in meanbody weight from that of the untreated (Group I) and ACEtreated mice (Group III) till the end of experiment On thecontrary liver weight was found to be significantly higher inDENA treated animals as compared to the ACE treated onceFurther no tumor appeared on the liver of the vehicle treatedand ACE treated control animals while DENA treated micehad the appearance of the tumor incidence as 100 On theother hand such tumor appearancewas reduced to 80whenDENA (Group III) treated mice were orally administeredACE (Group IV) Similarly treatment with the carambolafruit extract leads to reduction in tumor burden and tumoryield to 3375 and 4216 respectively (Table 1 Figures 1 2and 3)

Morphologically several small white-grayish foci weredetected on the liver of DENA treated mice at the end of theexperimentation (ie 24 weeks) However animals of vehicletreated control (Group I) as well as ACE treated control(Group III) did not show any such foci on the liver Ontreatment of ACE with DENA (Group IV) the number ofvisible foci was found to be radically decreased and the liversurface was much smoother

Induction of oxidative stress by DENACCl4was evi-

denced in the liver by the increase in LPO level and fall inthe activities of GSH SOD CAT and total proteins contentThe levels of LPO in liver were measured to be significantlyraised (119875 lt 0001) to 1291 plusmn 138 nmolemg tissue whilethe activities of GSH SOD and catalase as well as the levelof total proteins in the liver were obtained to be significantly


0

5

10

15

20

25

I II III IV

0

24

0

81

Tum

or b

urde

n

Groups

Figure 2 Variations in tumor burden in DENACCl4induced

hepatic carcinogenesis withwithout ACE administration

0

5

10

15

20

25

I II III IV

0

24

0

1012

Tum

or y

ield

Groups

Figure 3 Variations in tumor yield after DENACCl4induced


lower (119875 lt 0001) to 051 plusmn 002 120583molegm tissue 1259 plusmn208Umg tissue 206plusmn042Umg tissue 3331plusmn595mggmrespectively than the vehicle treated control values

Pretreatment of mice with the A carambola fruit extract(25mgkg bwt for 5 consecutive days and was withdrawn48 hrs before the first administration of DENA) significantly(119875 lt 0001) lowered down the activity of LPO to 492 plusmn089 nmolemg tissue GSH to 158 plusmn 045 120583molegm tissueSOD to 1305plusmn105Umg tissue catalase to 395plusmn050Umgtissue and the level of total proteins to 11411plusmn 1155mggm(Figures 4 5 6 7 and 8)

Cancer continues to be a great challenge to scientistsand practitioners interested in its biology prevention andtherapy Therefore the search for new chemopreventive andantitumor agents as more effective and less toxic than theexisting ones has kindled great interest in research forphytochemicals

HCC is a complex disease with multiple underlyingpathogenic mechanisms caused by a variety of risk factorsHepatic carcinogenesis has been intensively studied in exper-imental animals and numerous chemical compounds havebeen demonstrated to be carcinogenic to liver cells DENAis used as hepatocarcinogen in this study causing the tumorof the liver and not affecting any other organ It has beenused as an initiating agent in hepatocarcinogenic two-stageprotocols that is initiation and promotion model

0

5

10

15

20

I II III IV

LPO

(nm

olm

g)

Treatment groups

lowast

lowast

Figure 4 Variations in LPO levels after DENACCl4induced hep-

atic carcinogenesis withwithout ACE administration Significancelevelmdashnormal versus carcinogen treated control carcinogen treatedcontrol versus ACE treated experimental lowast119875 lt 0001

0

2

4

6

8

I II III IVTreatment groups

lowast

lowast

GSH

(120583m

ole

gm ti

ssue

)

Figure 5 Variations in GSH level after DENACCl4induced hep-


There is a significant increase in the liver weight of micereceiving DENA and CCl

4as compared to vehicle treated

control It may be because of the presence of tumors and theincreased size of liver in such animals The increase in thesize of liver might be because hypertrophy took place in theliver to compensate the damage induced by the carcinogensAfter 24 weeks of DENA treatment hyperplastic nodulesdeveloped as a consequence of the appearance of renewedhepatocytes degenerated hepatocytes oval cells and fibroticchanges

Marked elevations in biochemical parameters like LPOand the fall in GSH SOD catalase and total protein levelsin the liver reflect the degree of hepatocellular dysfunctionswhich indicates that reactive oxygen species induced byDENA play an important role in DENA-induced hepaticcarcinogenesis Therefore it is suggested that oxidative stressis one of the major causes of DENA-induced hepatic carcino-genesis

Oxidative stress is the state of imbalance between thelevel of antioxidant defense system and production of reactiveoxygen species (ROS) Increased generation of ROS anddecreased antioxidant enzymes in liver tissue has been


0

5

10

15

20

I II III IV

CAT

(Um

g tis

sue)

Treatment groups

lowast

lowast

Figure 6 Variations in CAT level after DENACCl4induced hep-


0

5

10

15

20

25

I II III IV

SOD

(Um

g tis

sue)

Treatment groups

lowast

lowast

Figure 7 Variations in SOD level after DENACCl4induced hep-


reported in many models of DENA-induced hepatocellularcarcinoma [28ndash31]

The implication of reactive oxygen species (ROS) incarcinogenic nitrosamines likeDENAandCCl

4toxic hepatic

injury is well documented [32] It has been reported that ROSplay a major role in tumor promotion through interactionwith critical macromolecules including lipids DNA DNArepair systems and other enzymes [33] Increased O

2con-

centration and production of ROS such as superoxide radical( lowastO2) hydroxyl radical (OHlowast) and hydrogen peroxide

cause oxidative stress in biological tissues It may also actas tumor initiator by directly activating oncogenes throughmutagenesis

Herbal drugs play a role in the management of variousliver disorders in addition to other natural healing processesof the liver [34]There are studies which show that medicinalplants with hepatoprotective properties mediate their protec-tion via antioxidant and free radical scavenging activities [35ndash37]

Further the incidences of hepatic tumors were found tobe significantly decreased in the experimental group (ACEtreated) than the carcinogen treated control The increase inthe activities of the antioxidant enzymes in the experimentalmice is attributed to the major antioxidative compounds

0

50

100

150

200

I II III IV

Tota

l pro

tein

s (m

ggm

)

Treatment groups

lowast

lowast

Figure 8 Variations in total proteins level after DENACCl4

induced hepatic carcinogenesis withwithout ACE administrationSignificance levelmdashnormal versus carcinogen treated control car-cinogen treated control versus ACE treated experimental lowast119875 lt0001

present in the Averrhoa carambola fruit These include cate-chin epicatechin proanthocyanidins and saponins [38 39]Polyphenols and flavonoids are known to have hepatoprotec-tive role [40 41]

In the present study fruit extract of Averrhoa carambolaprevented the progression ofDENA-induced hepatic carcino-genesis Data presented here demonstrated that administra-tion of ACE reversed the decrease in GSH CAT SOD andtotal proteins induced by DENA in liver tissues Significantreduction in the LPO levels elicited by carambola andenhanced GSH SOD catalase and proteins levels suggestthe protection of structural integrity of hepatocytes cellmembrane or stimulatory effects on hepatic regenerationalso reflecting the recovery of liver from the toxic effects ofDENA and CCl

4towards the normal liver cell functions

Lipid peroxidation plays an important role in the processof carcinogenesis [42] and may lead to the formation ofseveral toxic products such as malondialdehyde (MDE)and 4-hydroxynonenal These products can attack cellulartargets including DNA thereby inducing mutagenicity andcarcinogenicity [43] Increase in lipid peroxidation has beenreported during DENA-induced hepatocarcinogenesis [44]An elevated level of lipid peroxidation during liver carcino-genesis was also observed in DENA treated control miceduring the present study Administration of carambola fruitextract resulted in a significant decrease in the levels ofhepatic lipid peroxidation The phytosterols present in thefruit of carambola showed to mediate the decrease in lipidperoxidation [45 46]

Free radical scavenging enzymes such as superoxide dis-mutase (SOD) protect the biological systems from oxidativestress SOD and CAT provide the first defense against oxygentoxicity by catalyzing the dismutation of superoxide anion tohydrogen peroxide and decomposition of hydrogen peroxideto water and molecular oxygen Earlier reports showed thedecreased activities of SOD and CAT in hepatoma [47]The current study showed a significant decrease in SODand CAT activity in mice treated with DENA Decreasedactivities of SOD and CAT in DENA-treated mice could be


due to overutilization of these nonenzymatic and enzymaticantioxidants to scavenge the products of lipid peroxidationOn the other hand there was a significant increase in SODand CAT activities in group treated with plant extract It maybe due to presence of the ascorbic acid which is known forits quenching abilities of the free radicals as well as for theconjugation with cytotoxic genotoxic and lipid peroxidationproducts to ultimately lead their excretion [47 48]

Glutathione is required to maintain the normal reducedstate of cells and to counteract all the deleterious effectsof oxidative stress Thus GSH is involved in many cellularprocesses including the detoxification of endogenous andexogenous compounds The elevated level of GSH protectscellular proteins against oxidation through glutathione redoxcycle and also directly detoxifies reactive species [49] whilethe increased level of glutathione reductase helps in main-taining the basal level of cellular GSH [50] Administrationof DENA depleted the level of glutathione (GSH) in thisstudy Such depletion is also reported in many studies [51ndash53] It has been proposed that glutathione peroxidase isresponsible for the detoxification of hydrogen peroxide inlow concentration whereas catalase comes into play whenglutathione peroxidase is saturated with the substrate [54]GSH level was observed significantly higher in ACE treatedmice than the carcinogen alone treated ones

4 Conclusion

The exact mechanism of the chemopreventive action ofACE against DENA-induced hepatic tumor is not studied inthe present experiment but this investigation demonstratesthat the Averrhoa carambola fruit extract has a prophylacticrole against chemical induced hepatic carcinogenesis in themammals

Conflict of Interests

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

Acknowledgment

The authors are thankful to Council of Scientific amp IndustrialResearch New Delhi India for providing the financialassistance in the form of Senior Research Fellowship to MsRitu Singh under the supervision of Professor P K Goyal

References

[1] T Severi H van Malenstein C Verslype and J F van PeltldquoTumor initiation and progression in hepatocellular carcinomarisk factors classification and therapeutic targetsrdquo Acta Phar-macologica Sinica vol 31 no 11 pp 1409ndash1420 2010

[2] S Badvie ldquoHepatocellular carcinomardquo Postgraduate MedicalJournal vol 76 no 891 pp 4ndash11 2000

[3] K J Jeena K L Joy and R Kuttan ldquoEffect of Emblicaofficinalis Phyllanthus amarus and Picrorrhiza kurroa onN-nitrosodiethylamine induced hepatocarcinogenesisrdquo CancerLetters vol 136 no 1 pp 11ndash16 1999

[4] F X Bosch J Ribes R Cleries and M Dıaz ldquoEpidemiology ofHepatocellular carcinomardquo Clinics in Liver Disease vol 9 pp191ndash211 2005

[5] H B El-Serag and K L Rudolph ldquoHepatocellular carcinomaepidemiology and molecular carcinogenesisrdquo Gastroenterologyvol 132 no 7 pp 2557ndash2576 2007

[6] G Mittal A P Brar and G Soni ldquoImpact of hypercholes-terolemia on toxicity of N-nitrosodiethylamine biochemicaland histopathological effectsrdquo Pharmacological Reports vol 58no 3 pp 413ndash419 2006

[7] L Verna J Whysner and G M Williams ldquoN-Nitrosodi-ethylaminemechanistic data and risk assessment bioactivationDNA-adduct formation mutagenicity and tumor initiationrdquoPharmacology andTherapeutics vol 71 no 1-2 pp 57ndash81 1996

[8] D J Liano A Blanck P Eneroth J Gustafsson and IP Hallstrom ldquoDiethylnitrosamine causes pituitary damagedisturbs hormone levels and reduces sexual dimorphism ofcertain liver functions in the ratrdquoEnvironmental Health Perspec-tives vol 109 no 9 pp 943ndash947 2001

[9] K Pashupathy and R K Bhattacharya Schriftenr Forschun-gszentJuliech Bilateral Sem Int Bur vol 29 pp 159ndash161 1998

[10] B Demple and L Harrison ldquoRepair of oxidative damage toDNA enzymology and biologyrdquoAnnual Review of Biochemistryvol 63 pp 915ndash948 1994

[11] O Lux and D Naidoo ldquoBiological variability of superoxidedismutase and glutathione peroxidase in bloodrdquo Redox Reportvol 1 pp 331ndash335 1995

[12] U Bandyopadhyay O Das and R K Banerjee ldquoReactiveoxygen species oxidative damage and pathogenesisrdquo CurrentScience vol 77 no 5 pp 658ndash666 1999

[13] G Block B Patterson and A Subar ldquoFruit vegetables andcancer prevention a review of the epidemiological evidencerdquoNutrition and Cancer vol 18 no 1 pp 1ndash29 1992

[14] K A Steinmetz and J D Potter ldquoVegetables fruit and cancerprevention a reviewrdquo Journal of the American Dietetic Associa-tion vol 96 no 10 pp 1027ndash1039 1996

[15] M J Wargovich ldquoExperimental evidence for cancer preventiveelements in foodsrdquoCancer Letters vol 114 no 1-2 pp 11ndash17 1997

[16] M A Eastwood ldquoInteraction of dietary antioxidants in vivohow fruit and vegetables prevent diseaserdquo QJMed vol 92 no9 pp 527ndash530 1999

[17] E J Park and JM Pezzuto ldquoBotanicals in cancer chemopreven-tionrdquo Cancer and Metastasis Reviews vol 21 no 3-4 pp 231ndash255 2002

[18] P Vitaglione FMorisco N Caporaso andV Fogliano ldquoDietaryantioxidant compounds and liver healthrdquo Critical Reviews inFood Science and Nutrition vol 44 no 7-8 pp 575ndash586 2004

[19] A D Correa Plantas Medicinais Do Cultivo a TerapeuticaEditora Vozes Petropolis Brazil 1998

[20] R O G Carolino R O Beleboni A B Pizzo et al ldquoConvulsantactivity and neurochemical alterations induced by a fractionobtained from fruit Averrhoa carambola (Oxalidaceae Gera-niales)rdquoNeurochemistry International vol 46 no 7 pp 523ndash5312005

[21] M Provasi C E Oliveira M C Martino L G Pessini RB Bazotte and D A G Cortez ldquoAvaliacao da toxicidade edo potencial antihiperglicemiante da Averrhoa carambola LOxalidaceaerdquoActa Scientiarum vol 23 no 3 pp 665ndash669 2001

[22] H Ohkhawa N Ohishi and K Yogi ldquoAssay for lipid peroxida-tion in animal tissue by thiobarbituric acid reactionrdquoAnalyticalBiochemistry vol 95 pp 351ndash358 1979


[23] M A Moron J W Depierre and B Mannervik ldquoLevels of glu-tathione glutathione reductase and glutathione S-transferaseactivities in rat lung and liverrdquo Biochimica et Biophysica Actavol 582 no 1 pp 67ndash78 1979

[24] E Beutler O Duron B M Kelly and E Eutler ldquoImprovedmethod for the determination of blood glutathionerdquoThe Journalof laboratory and Clinical Medicine vol 61 pp 882ndash888 1963

[25] H Aebi ldquoCatalase in vitrordquo in Method in Enzymology SP Colowick and N O Kaplan Eds vol 105 pp 121ndash126Academic Press New York NY USA 1984

[26] S Marklund and G Marklund ldquoInvolvement of the superoxideanion radical in the autoxidation of pyrogallol and a convenientassay for superoxide dismutaserdquo European Journal of Biochem-istry vol 47 no 3 pp 469ndash474 1974

[27] O H Lowry N J Rosenbrough A L Farr and R J LandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[28] S Kweon K A Park and H Choi ldquoChemopreventive effectof garlic powder diet in diethylnitrosamine-induced rat hepa-tocarcinogenesisrdquo Life Sciences vol 73 no 19 pp 2515ndash25262003

[29] G Ramakrishnan H R Raghavendran R Vinodhkumar andT Devaki ldquoSuppression of N-nitrosodiethylamine inducedhepatocarcinogenesis by silymarin in ratsrdquo Chemico-BiologicalInteractions vol 161 no 2 pp 104ndash114 2006

[30] A S Yadav and D Bhatnagar ldquoChemo-preventive effect ofstar anise inN-nitrosodiethylamine initiated and phenobarbitalpromoted hepato-carcinogenesisrdquo Chemico-Biological Interac-tions vol 169 no 3 pp 207ndash214 2007

[31] V Sivaramakrishnan P N Shilpa V R Praveen and D SNiranjali ldquoAttenuation of N-nitrosodiethylamine-induced hep-atocellular carcinogenesis by a novel flavonol-MorinrdquoChemico-Biological Interactions vol 171 no 1 pp 79ndash88 2008

[32] A Ravid and R Koren ldquoThe role of reactive oxygen species inthe anticancer activity of vitamin Drdquo Anticancer Research vol164 pp 357ndash367 2003

[33] T W Kensler and M A Trush ldquoRole of oxygen radicals intumor promotionrdquo Environmental Mutagenesis vol 6 no 4 pp593ndash616 1984

[34] A Subramoniam D A Evans S Rajasekharan and P Push-pangadan ldquoHepatoprotective activity of Trichopus zeylanicusextract against paracetamolmdashinduced hepatic damage in ratsrdquoIndian Journal of Experimental Biology vol 36 no 4 pp 385ndash389 1998

[35] A A Adeneye and A S Benebo ldquoProtective effect of theaqueous leaf and seed extract of Phyllanthus amarus on gen-tamicin and acetaminophen-induced nephrotoxic ratsrdquo Journalof Ethnopharmacology vol 118 no 2 pp 318ndash323 2008

[36] S R Parmar P H Vashrambhai and K Kiran ldquoHepato-protective activity of some plants extract against paracetamolinduced hepatotoxicity in ratsrdquo Journal of Herbal Medicine andToxicology vol 4 no 2 pp 101ndash106 2010

[37] A V Rupal D M Savalia and A V R L NarasimhacharyaldquoPlant extracts as biotermiticidesrdquoElectronic Journal of Environ-mental Sciences vol 4 pp 73ndash77 2011

[38] G Shui and L P Leong ldquoAnalysis of polyphenolic antioxidantsin star fruit using liquid chromatography and mass spectrome-tryrdquo Journal of Chromatography A vol 1022 no 1-2 pp 67ndash752004

[39] J Ghosh J Das P Manna and P C Sil ldquoCytoprotective effectof arjunolic acid in response to sodium fluoride mediatedoxidative stress and cell death via necrotic pathwayrdquo Toxicologyin Vitro vol 22 no 8 pp 1918ndash1926 2008

[40] L H Yao Y M Jiang J Shi et al ldquoFlavonoids in food and theirhealth benefitsrdquo Plant Foods for Human Nutrition vol 59 no 3pp 113ndash122 2004

[41] M Meydani ldquoEffect of functional food ingredients vitamin Emodulation of cardiovascular diseases and immune status in theelderlyrdquo The American Journal of Clinical Nutrition vol 71 no6 pp 1665ndash1668 2000

[42] M C Banakar S K Paramasivan M B Chattopadhyayet al ldquo1120572 25-dihydroxyvitamin D3 prevents DNA damageand restores antioxidant enzymes in rat hepatocarcinogenesisinduced by diethylnitrosamine and promoted by phenobarbi-talrdquo World Journal of Gastroenterology vol 10 no 9 pp 1268ndash1275 2004

[43] L L de Zwart J H Meerman J N Commandeur and N PVermeulen ldquoBiomarkers of free radical damage applications inexperimental animals and in humansrdquo Free Radical Biology andMedicine vol 26 no 1-2 pp 202ndash226 1999

[44] P V Jeyabal M B Syed M Venkataraman J K Sambandhamand D Sakthisekaran ldquoApigenin inhibits oxidative stress-induced macromolecular damage in N-nitrosodiethylamine(NDEA)-induced hepatocellular carcinogenesis in Wistaralbino ratsrdquo Molecular Carcinogenesis vol 44 no 1 pp 11ndash202005

[45] Y Yoshida andEANiki ldquoAntioxidant effects of phytosterol andits componentsrdquo Journal of Nutritional Science and Vitaminol-ogy vol 49 no 4 pp 277ndash280 2003

[46] G Ferretti T Bacchetti S Masciangelo and V BicchiegaldquoEffect of phytosterols on copper lipid peroxidation of humanlow-density lipoproteinsrdquoNutrition vol 26 no 3 pp 296ndash3042010

[47] R Corrocher M Casaril G Bellisola et al ldquoSevere impairmentof antioxidant system in human hepatomardquo Cancer vol 58 no8 pp 1658ndash1662 1986

[48] J Sowell B Frei and J F Stevens ldquoVitamin C conjugatesof genotoxic lipid peroxidation products structural character-ization and detection in human plasmardquo Proceedings of theNational Academy of Sciences of the United States of Americavol 101 pp 17964ndash17969 2004

[49] B Ketterer ldquoGlutathione S-transferases and prevention of cel-lular free radical damagerdquo Free Radical Research vol 28 no 6pp 647ndash658 1998

[50] J Lopez-Baria J A Barcena and J A Bocanegra ldquoStructuremechanism functions and regulatory properties of glutathionereductaserdquo in Glutathione J V B Raton Ed pp 105ndash116 CRCPress Boca Raton Fla USA 1990

[51] A K Bansal M Bansal G Soni and D Bhatnagar ldquoProtectiverole of Vitamin E pre-treatment on N-nitrosodiethylamineinduced oxidative stress in rat liverrdquo Chemico-Biological Inter-actions vol 156 no 2-3 pp 101ndash111 2005

[52] V Sivaramakrishnan P N Shilpa V R Praveen Kumar andS Niranjali Devaraj ldquoAttenuation of N-nitrosodiethylamine-induced hepatocellular carcinogenesis by a novel flavonol-Morinrdquo Chemico-Biological Interactions vol 171 no 1 pp 79ndash88 2008


[53] K Pradeep C V Mohan K Gobianand and S KarthikeyanldquoSilymarin modulates the oxidant-antioxidant imbalance dur-ing diethylnitrosamine induced oxidative stress in ratsrdquo Euro-pean Journal of Pharmacology vol 560 no 2-3 pp 110ndash1162007

[54] G F Gaetani S Galiano L Canepa A M Ferraris and HN Kirkman ldquoCatalase and glutathione peroxidase are equallyactive in detoxification of hydrogen peroxide in human erythro-cytesrdquo Blood vol 73 no 1 pp 334ndash339 1989

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

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AntibioticsInternational Journal of

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Drug DeliveryJournal of



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Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


Free radicals and other reactive oxygen species (ROS) areconstantly formed in the human body accumulation whichcauses oxidative damage Normal cells have evolved defensemechanisms for protection against this oxidative damageby developing multiple antioxidative defenses [10] If suchdelicate balance of free radical production and antioxidantdefenses goes out of control it results in the pathology ofseveral human diseases including cancer atherosclerosismalaria rheumatoid arthritis and neurodegenerative dis-eases [11 12]

Chemoprevention is a pharmacological way of interfer-ence in order to arrest or reverse the process of carcinogene-sis Chemopreventive substances are identified on the basisof their antioxidant antimutagenic and anti-inflammatoryactivities capable of arresting proliferation and enhancingapoptosis which are the major criteria for their anticar-cinogenic activity Progress in the area of chemopreventionduring the past two decades has been very impressiveAccumulating epidemiological and experimental evidenceshave revealed the chemopreventive influence of number ofnaturally occurring compounds and their role in preventionof the diseases [13ndash17]

Herbal products are gaining progressively attention thesedays for primary health care owing to less toxicity bettercompatibility with the body and high efficacy against freeradical mediated diseases Many studies have suggested thata healthy diet especially fruits and vegetables that are rich innatural antioxidants is efficacious to prevent oxidative stressand thus plays a vital role in cancer prevention [18]

Averrhoa carambola L (Oxalidaceae) is also known asthe star fruit tree Studies have shown that the fruit of Acarambola has several medicinal properties and it is richin antioxidants which act against reactive oxygen speciesThe ripe star fruit has digestive and biliousness propertiesIt is also a good source of vitamin C and used to treatheadache vomiting coughing hangovers and eczemas [1920] Furthermore it is used as an appetite stimulant diureticantidiarrheal and febrifugal agent In addition the extractobtained through the leaves of such planthas been used in thetreatment of diabetes [21]

Looking into the pharmacological and medicinal proper-ties of this plant the present study has been targeted to inves-tigate the possible anticancer potential of A carambola fruitextract against chemical induced hepatocellular carcinoma inmammals

2 Materials and Methods

21 Chemicals Diethylnitrosamine (DENA) and carbontetrachloride (CCl

4) were purchased from Sigma Chemical

Co (St Louis MO USA) DENA at a dose 15mgkg bwt(single ip injection in normal saline) was injected to initiatehepatic carcinogenesis while CCl

4(16 gkg bwt) in 1 1

dilution with corn oil was given orally to animals by gavageto stimulate liver cell proliferation and regeneration

22 Preparation of Plant Extract Carambola fruits werecleaned air dried and grinded into the form of fine powder

The powder was extracted with 90 ethyl alcohol usingSoxhlet apparatus and concentrated by evaporating its liquidcontents The required dose for further treatment was pre-pared by dissolving the extract in DDW

23 Animals Swiss albino mice (3-week old) were takenfor the experiment from an inbred colony and they wereprovided feed and water ad libitum All studies were carriedout in accordance with the guidelines of the InstitutionalAnimal Ethics Committee amp INSA New Delhi

24 Dose Selection of ACE For deciding the optimum doseexperiments were conducted inwhich Swiss albinomiceweredivided into different groups and were given orally Averrhoacarambola extract (ACE) at the dose of 05 15 25 50 and75mgkg bwtdaymganimalday Animals from each groupwere observed for 30 days for any sign of sickness morbiditymortality gait weight behavioral alterations and so forthand were necropsied on 16th and 31st day Various doses ofACE were selected (ie 15 25 and 50mgkgbwtanimal)from the above doses after evaluation of various biochemicalparameters in the liver of mice Out of these 25mg dose wasfound to be the optimum dose for this experiment

25 Chemopreventive Activity of ACE Animals for thisexperiment were divided into the following groups

Group I Negative Control (Vehicle Treated Normal Mice) Inthis group animals were given single ip injection of normalsaline and later administered with corn oil by oral gavagethree times in a week for the entire experimental period thatis for 24 weeks

Group II Positive Control (Carcinogen Treated) The animalsin this group were given DENA in normal saline After 2weeks of DENA administration CCl

4was given 3 times in

a week until the end of the experiment

Group III Drug Treated Control In this group the animalswere administered Averrhoa carambola extract (ACE) at adose of 25mgkgbwtanimalday for the entire experimentalperiod

Group IV ACE Treated Experimental The animals of thisgroup were provided ACE at a dose of 25mgkg bwtdayfor 5 consecutive days ACE source was withdrawn 48 hrsbefore the first administration of DENA CCl

4was given after

2 weeks as 3 times a week for 24 weeksThe following parameters were taken into account for the

study

(1) Morphological

(i) Body Weight The weights of the animals fromeach group were recorded at the beginning andat the termination of the experiments

(ii) Tumor Incidence It is the number of micecarrying at least 1 tumor expressed as percentageincidence















0

20

40

60

80

100

III

IIIIV

0

100

0

80

Tum

or in

cide

nce (

)

Groups




4






0

5

10

15

20

25

I II III IV

0

24

0

81

Tum

or b

urde

n

Groups



0

5

10

15

20

25

I II III IV

0

24

0

1012

Tum

or y

ield

Groups







0

5

10

15

20

I II III IV

LPO

(nm

olm

g)

Treatment groups

lowast

lowast



0

2

4

6

8


lowast

lowast

GSH

(120583m

ole

gm ti

ssue

)









0

5

10

15

20

I II III IV

CAT

(Um

g tis

sue)

Treatment groups

lowast

lowast



0

5

10

15

20

25

I II III IV

SOD

(Um

g tis

sue)

Treatment groups

lowast

lowast





4toxic hepatic


2con-





0

50

100

150

200

I II III IV

Tota

l pro

tein

s (m

ggm

)

Treatment groups

lowast

lowast











4 Conclusion




Acknowledgment


References





























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of















0

20

40

60

80

100

III

IIIIV

0

100

0

80

Tum

or in

cide

nce (

)

Groups




4






0

5

10

15

20

25

I II III IV

0

24

0

81

Tum

or b

urde

n

Groups



0

5

10

15

20

25

I II III IV

0

24

0

1012

Tum

or y

ield

Groups







0

5

10

15

20

I II III IV

LPO

(nm

olm

g)

Treatment groups

lowast

lowast



0

2

4

6

8


lowast

lowast

GSH

(120583m

ole

gm ti

ssue

)









0

5

10

15

20

I II III IV

CAT

(Um

g tis

sue)

Treatment groups

lowast

lowast



0

5

10

15

20

25

I II III IV

SOD

(Um

g tis

sue)

Treatment groups

lowast

lowast





4toxic hepatic


2con-





0

50

100

150

200

I II III IV

Tota

l pro

tein

s (m

ggm

)

Treatment groups

lowast

lowast











4 Conclusion




Acknowledgment


References





























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

5

10

15

20

25

I II III IV

0

24

0

81

Tum

or b

urde

n

Groups



0

5

10

15

20

25

I II III IV

0

24

0

1012

Tum

or y

ield

Groups







0

5

10

15

20

I II III IV

LPO

(nm

olm

g)

Treatment groups

lowast

lowast



0

2

4

6

8


lowast

lowast

GSH

(120583m

ole

gm ti

ssue

)









0

5

10

15

20

I II III IV

CAT

(Um

g tis

sue)

Treatment groups

lowast

lowast



0

5

10

15

20

25

I II III IV

SOD

(Um

g tis

sue)

Treatment groups

lowast

lowast





4toxic hepatic


2con-





0

50

100

150

200

I II III IV

Tota

l pro

tein

s (m

ggm

)

Treatment groups

lowast

lowast











4 Conclusion




Acknowledgment


References





























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

5

10

15

20

I II III IV

CAT

(Um

g tis

sue)

Treatment groups

lowast

lowast



0

5

10

15

20

25

I II III IV

SOD

(Um

g tis

sue)

Treatment groups

lowast

lowast





4toxic hepatic


2con-





0

50

100

150

200

I II III IV

Tota

l pro

tein

s (m

ggm

)

Treatment groups

lowast

lowast











4 Conclusion




Acknowledgment


References





























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




4 Conclusion




Acknowledgment


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

prophylactic role of averrhoa carambola (star fruit) extract against

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