Research Article Chemopreventive Properties and Toxicity...

Research ArticleChemopreventive Properties and Toxicity of Kelulut Honey inSprague Dawley Rats Induced with Azoxymethane

Latifah Saiful Yazan12 Muhamad Firdaus Shyfiq Muhamad Zali1 Razana Mohd Ali3

Nurul Amira Zainal1 Nurulaidah Esa2 Sarah Sapuan2 Yong Sze Ong2 Yin Sim Tor2

Banulata Gopalsamy1 Fui Ling Voon1 and Sharifah Sakinah Syed Alwi1

1Department of Biomedical Science Faculty of Medicine and Health Sciences Universiti Putra Malaysia (UPM)43400 Serdang Selangor Malaysia2Laboratory of Molecular Biomedicine Institute of Bioscience Universiti Putra Malaysia (UPM) 43400 Serdang Selangor Malaysia3Department of Pathology Faculty of Medicine and Health Sciences Universiti Putra Malaysia (UPM)43400 Serdang Selangor Malaysia

Correspondence should be addressed to Latifah Saiful Yazan latifahsyupmedumy

Received 24 February 2016 Revised 10 May 2016 Accepted 22 June 2016

Academic Editor Koichiro Wada

Copyright copy 2016 Latifah Saiful Yazan et al 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

Ethnopharmacological Relevance Colon cancer has been a major problem worldwide Kelulut honey (KH) is produced by thestingless bees from Trigona species and has strong antioxidant activities that could be one of the potential chemopreventive agentsfrom natural resources Aim of This Study This study investigated the chemopreventive properties and toxicity of KH in SpragueDawley rats induced with azoxymethane (AOM)Material and Method Twenty-four male Sprague Dawley rats aged 5 weeks weredivided into 4 groups (G1) untreated group not induced with AOM (G2) untreated group induced with AOM (G3) treated groupinduced with AOM and (G4) treated group not induced with AOM Injection of AOM (15mgkg) was via intraperitoneal routeonce a week for two subsequent weeksThe treatment groups were given oral administration of KH (1183mgkg body weight) twicedaily for 8 weeks Results Treatment with KH significantly reduced the total number of aberrant crypt foci (ACF) and aberrantcrypts (AC) and crypt multiplicity KH was not toxic to the animals since the level of blood profile parameters liver enzymes andkidney functions was in normal range Conclusions The current finding shows that KH has chemopreventive properties in ratsinduced with colorectal cancer and also was found not toxic towards the animals

1 Introduction

Cancer has been a major problem worldwide Althoughcolon cancer has been ranked the fourth the incidence isrising dramatically Colon cancer ranks the first amongmalesand third among females [1] There are several treatmentmodalities for colon cancer such as chemotherapy surgeryand radiotherapy Nevertheless chemotherapy has a fewadverse effects such as neutropenia (reduction of leucocytes)diarrhea and thromboembolic events (blockage of bloodvessel) [2]Hence numerous researches have been carried outin finding new alternatives for management of colon cancer

One of the alternative managements is via chemopre-vention Chemoprevention is defined as the use of natural

or synthetic substances to reduce the risk of developingcancer or to reduce the chance of cancer to recur Thus itis important to inhibit the development of epigenetic andgenetic alterations that are part of the process by which clonalproliferation of cells with abnormal genetic content cascadesinto dysplastic and later malignant cells [3] In colon cancerit is an alternative approach to reduce the mortality whichinvolves long-term use of a variety of chemopreventive agentsthat can inhibit development of neoplasms in the large bowel[4]

Kelulut honey (KH) is produced by the stingless beesfrom Trigona species The honey produced by this stinglessbee is stored in clusters of small resin pots near theirnest Meanwhile honey that is produced by normally found

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 4036926 6 pageshttpdxdoiorg10115520164036926

2 BioMed Research International

bee is stored in hexagonal-shaped combs [5] KH is morediluted compared to other types of honey and it has distincttaste (sour like taste) as well as aroma [6] TraditionallyKH is used for antiageing enhancing libido and immunesystem killing bacteria treatment of bronchial phlegm andrelieving sore throat cough and cold [7] KH has beenproven to possess various pharmacological properties suchas anti-inflammatory [8] antioxidant [5] antiageing [9] andantibacterial properties [10] Since KH has strong antioxidantactivities it could be one of the potential chemopreventiveagents from natural resources

2 Materials and Methods

21 ChemicalsTest Materials Azoxymethane was purchasedfrom Aldrich Sigma (St Louis United States) hematoxylinand eosin stain was purchased fromCellPath (Mochdre UK)10 formalin was purchased from Fisher Scientific (Lough-borough UK) and RCL2 was purchased from Alphaleys(Plaisir France) Kelulut honey (KH) was provided by Mar-bawi Food Processing and Trading Kuala Kangsar PerakMalaysia

22 Experimental Animals A total of 24 male Sprague Daw-ley rats with body weight of 180 to 220 g at the age of 5weeks were used Prior to the experiment the rats wereacclimatized a week with free access to food and water adlibitum The animals were housed in cages (119899 = 3) understandard laboratory conditions with 12-hour darklight cycleat 20ndash24∘C with 40ndash50 relative humidity Ethic approvalwas obtained from the Institutional Animal Care and UseCommittee (IACUC) ofUniversiti PutraMalaysia prior to thestudy (UPMIACUCFYP2015FPSK003)

23 Experimental Procedures The rats were divided into 4groups (119899 = 6) which were (G1) negative control group(not induced with AOM without treatment) (G2) positivecontrol group (induced with AOM without treatment)(G3) treatment group (induced with AOM treated withKH (1183mgkg)) and (G4) toxicity group (not inducedwith AOM treated with KH) After acclimatization AOM(15mgkg) was injected intraperitoneally into the rats for twosubsequent weeks once per week on the first day of eachweek Next KH was administered orally twice daily in themorning and at night for eight weeks The body weight ofthe rats was recorded every three days The dosage of KHwas calculated based on human intake Upon terminationthe rats were fasted for 8 hours before blood collection Priorto blood sampling the rats were anesthetized with ketamine-xylazine (75mgkg 5mgkg) by intraperitoneal injectionThe blood sample (7mL) was collected by cardiac punctureusing a 26 G 1210158401015840 needle (Terumo Belgium Europe) intononheparinized (5mL) and EDTA-containing tube (2mL)for biochemical (Roche Cobas USA) and haematological(ABX Pentra 60 Japan) analyses respectively The bloodcollected in nonheparinized tube was then centrifuged at10000 rpm for 10 minutes to separate the serum The organs

(colon liver and kidney) were removed and weighed beforebeing fixed in 10 formalin

24 Determination of Incidence of Aberrant Crypt Foci andCrypt Multiplicity The colon was processed by first dividingit into three parts which are proximal colon (near the cae-cum) middle colon and distal colon (towards the rectum)Each part was cut into strip and placed in a cassette Forliver it was grossed from a lobe of the liver and placed intoa cassette For kidney the organ was cut into half and placedin a cassette Next the organs that had been grossed wereprocessed using an automated tissue processor (LEICA TP1020 USA) where the machine runs dehydration clearingand impregnating process The organs were being embeddedin molten paraffin with appropriate molds to support thetissue during sectioning processThe blocks were trimmed at16 120583m thickness and sectioned at thickness of 04 120583m usinga microtome The thin sections of the tissue samples wereplaced in a water bath and fished onto glass slides The slidesprepared will be dewaxed first before being stained withhematoxylin and eosin (HampE) followed by mounting with P-xylene-bis-pyridinium bromide (DPX) and observed undera light microscope The total number of ACF per colon totalnumber of aberrant crypts (AC) and crypt multiplicity ofeach rat in all groups were calculated

25 Statistical Analysis The data were analysed by StatisticalPackage for Social Sciences (SPSS) version 21 One-wayANOVA analysis was performed followed by Duncanrsquos testThe data was presented as mean plusmn SEM Value of 119901 lt 005was considered as significant

3 Results

31 Effects of KH on Body Weight Figure 1 shows the bodyweight of all groups throughout the experiment There was agradual increase in the body weight throughout the 8 weeksof studyThehighest bodyweightwas noted inG2 followed byG1 G3 and G4 However there was no significant differencein the body weight among all groups (119901 gt 005)

32 Effects of KH on Blood Profile The red blood cell(RBC) haemoglobin (Hb) packed cell volume (PCV) meancorpuscular volume (MCV) mean corpuscular haemoglobinconcentration (MCHC) white blood cell (WBC) and throm-bocytes level of rats in 4 different groups are summarizedin Table 1 Data show no significant difference in the bloodprofile among all groups (119901 gt 005)

33 Effects of KH on Liver Enzymes and Kidney FunctionsTable 2 shows the liver enzymes and kidney function tests ofrats in 4 different groups show no significant difference in allthe parameters tested among all groups (119901 gt 005)

34 Effects of KH on Colon Length Table 3 shows the effectsof KH on the length of the colon in all groups throughoutthe experiment The highest colon length was noted inG1 followed by G3 G4 and G2 However there was no

BioMed Research International 3

Table 1 Effects of KH on the blood profile of rats after 8 weeks of study The data were analysed by using one-way ANOVA and the valuesare expressed as mean plusmn SEM Value of 119901 lt 005 was considered significant

Parameter GroupNormal 1 2 3 4

RBC (times1012L) 630ndash942 805 plusmn 007 831 plusmn 014 815 plusmn 012 840 plusmn 020Hb (gL) 100ndash170 15116 plusmn 122 15016 plusmn 149 15316 plusmn 132 15340 plusmn 294PCV (LL) 034ndash053 045 plusmn 00054 046 plusmn 00094 044 plusmn 00042 046 plusmn 00098MCV (fL) 500ndash778 5683 plusmn 040 5550 plusmn 092 5416 plusmn 047 5525 plusmn 062MCHC (gL) 2820ndash3410 33000 plusmn 272 32583 plusmn 479 34533 plusmn 204 32960 plusmn 128WBC (times109L) 29ndash209 745 plusmn 088 876 plusmn 036 932 plusmn 125 1012 plusmn 095Thrombocyte (times109L) 6850ndash14360 64200 plusmn 2799 71200 plusmn 3143 66350 plusmn 2654 64840 plusmn 5200

Table 2 Effects of KH on the liver enzymes and kidney functions of rats after 8 weeks of study The data were analysed by using one-wayANOVA and the values are expressed as mean plusmn SEM Value of 119901 lt 005 was considered significant


ALT (UL) 1750ndash3230 3936 plusmn 097 3793 plusmn 160 4108 plusmn 186 3588 plusmn 216ALP (UL) 5680ndash12800 9966 plusmn 507 8916 plusmn 344 8983 plusmn 261 9620 plusmn 251AST (UL) 4570ndash8080 136517 plusmn 970 14585 plusmn 1767 16486 plusmn 1642 15078 plusmn 1634Creatinine (120583molL) 5300ndash10600 6216 plusmn 060 6633 plusmn 160 6350 plusmn 138 6560 plusmn 180Urea (mmolL) 180ndash710 483 plusmn 0061 563 plusmn 019 551 plusmn 022 5740 plusmn 020

Table 3 Effects of KH on the colon length of the rats after 8 weeksof study The data were analysed by using one-way ANOVA andthe values are expressed as mean plusmn SEM Value of 119901 lt 005 wasconsidered significant

Group Length (cm)1 198667 plusmn 0806912 176833 plusmn 1309053 185167 plusmn 0671034 177400 plusmn 073865

significant difference in colon length among all groups (119901 gt005)

35 Effects of KH on Aberrant Crypt Foci Table 4 shows thetotal ACF and total aberrant crypts (AC)The total number ofACF and AC and crypt multiplicity were significantly lowerin G3 compared to G2 (119901 lt 005)

36 Histological Classification of Aberrant Crypt Foci ACFcan be classified according to the number of crypts per focifor example 1 crypt 2 crypts 3 crypts andge4 crypts per focusas being shown in Figure 2

4 Discussion

This study was carried out to investigate the chemopreventiveproperties of KH towards Sprague Dawley rats induced withAOM In this study AOM was proven to induce ACF forma-tion as reported by Bird [11] In this model ACF formation isused as biomarker to identify the progression of developmentof colon cancer KH has chemopreventive properties against

Wee

k 0

Wee

k 1

Wee

k 2

Wee

k 3

Wee

k 4

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k 5

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k 6

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k 7

Wee

k 8

G1G2

G3G4

200

250

300

350

400

450

500

Body

wei

ght (

g)

Figure 1 Effects of KH on the body weight of rats after 8 weeks ofstudyThe data were analysed using one-wayANOVA and the valueswere expressed as mean plusmn SEM Value of 119901 lt 005 was consideredsignificant

azoxymethane-induced colon cancer based on the reductionof total number of ACF total number of AC and cryptmultiplicity

Gribel and Pashinskiı [12] reported that honey possessesmoderate anticolon cancer activity Even though they usednormal honey Souza et al [13] stated that there is only aslight difference in properties between honey produced byApis bees and honey produced by stingless bees It is believed


Table 4 Effects of KH on the incidence of azoxymethane-induced aberrant crypt foci of rats after 8 weeks of study The data were analysedby using one-way ANOVA and the values are expressed as mean plusmn SEM Value of 119901 lt 005 was considered significant

Group Total number of ACF Number of crypts Total aberrant crypts(AC)1 2 3 ge4

Normal 367 plusmn 0333 200 plusmn 0577 167 plusmn 0882 0 0 533 plusmn 1202Negative 2933 plusmn 4667 1633 plusmn 2963 633 plusmn 0667 433 plusmn 0882 267 plusmn 0332 5200 plusmn 7371KH-treated 1067 plusmn 2028 733 plusmn 1333 300 plusmn 0577 100 plusmn 0577 0 1500 plusmn 3000

(a) (b)

(c) (d)

(e)

Figure 2 Photomicrograph of (a) normal crypt (b) 1 crypt (c) 2 crypts (d) 3 crypts and (e) ge4 crypts stained with hematoxylin and eosinNormal crypt is characterized by constant internal and diameter between the crypts and higher proportion of goblet cells (G) while ACFexhibit darker stain with reduced number of goblet cells (400x magnification)


that one of the components of KH that is responsible for itsanticolon cancer properties is caffeic acid ester [14] Accord-ing to Xiang et al [15] caffeic acid esters suppressed thetranscriptional activity of 120573-catenin and hence can becomean agent against colon cancer Caffeic acid phenethyl ester(CAPE) is a phenolic antioxidant which has biological andpharmacological functions that include immunoregulationanti-inflammatory antiviral antibacterial and antitumoractivities [16] CAPE has antiproliferative and apoptosis-inducing effect in various tumor cells in vitro and in vivo[17 18] According to Borrelli et al [19] CAPE inhibited thedevelopment of AOM-induced ACF in the colon of rats

In traditional usage honey including KH is taken twicedaily usually in the morning and at night Based on thedensity the daily intake of KH for human is 18933mgkgIn this study we were trying to mimic the way KH is beingconsumed by human The dose of KH for rat consumption iscalculated according to FDA dose translation formula Thusa rat received 1183mgkg bodyweight of KH

Surprisingly in this study ACF were also found in thenormal groupThis could be due to the fact that normal bodymutation also occurs Mutation can occur in body because ofseveral factors such as food surroundings and exposure tocarcinogenesis agents [20]

KH was proven to be not toxic to the animals There wasno reduction in bodyweight According to Peterson et al [21]when there is a presence of toxicity the body weight of thestudied organism should be decreased In addition the levelsof parameters for blood profile liver enzymes and kidneyfunctions were in normal range According to Wang et al[22] abnormal values of blood profile liver enzymes andkidney function are an indication of toxicity

In summary KH has chemopreventive properties in ratsinduced with colon cancer by reducing the development ofACF AC and crypt multiplicity KH was also to be found nottoxic towards the animals

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This research was fully supported by the ResearchGrant fromthe Universiti Putra Malaysia

References

[1] National Cancer Patient RegistrymdashColorectal Cancer 2010httpwwwcrcgovmyreports

[2] T Andre C Boni L Mounedji-Boudiaf et al ldquoOxaliplatinfluorouracil and leucovorin as adjuvant treatment for coloncancerrdquo The New England Journal of Medicine vol 350 no 23pp 2343ndash2351 2004

[3] D Das N Arber and J A Jankowski ldquoChemoprevention ofcolorectal cancerrdquo Digestion vol 76 no 1 pp 51ndash67 2007

[4] P A Janne and R J Mayer ldquoChemoprevention of colorectalcancerrdquo The New England Journal of Medicine vol 342 no 26pp 1960ndash1968 2000

[5] S P KekN L Chin YA Yusof SWTan andL S Chua ldquoTotalphenolic contents and colour intensity of Malaysian honeysfrom the Apis spp and Trigona spp beesrdquo Agriculture andAgricultural Science Procedia vol 2 pp 150ndash155 2014

[6] F C Biluca F Della Betta G P de Oliveira et al ldquo5-HMF andcarbohydrates content in stingless bee honey by CE before andafter thermal treatmentrdquo Food Chemistry vol 159 pp 244ndash2492014

[7] S A S A Barakhbah ldquoHoney in the Malay traditionrdquoMalaysian Journal of Medical Sciences vol 14 no 1 pp 101ndash1272007

[8] A Sabir C R Tabbu P Agustiono and W Sosroseno ldquoHisto-logical analysis of rat dental pulp tissue capped with propolisrdquoJournal of Oral Science vol 47 no 3 pp 135ndash138 2005

[9] H Afrouzan V Bankova G Tahmasebi M Bigdeli and MPopova ldquoComparison of gymnosperms and angiosperms plantson quality and quantity of propolisrdquo Pharmacognosy Magazinevol 3 no 9 pp 21ndash25 2007

[10] M I Zainol KMohdYusoff andMYMohdYusof ldquoAntibacte-rial activity of selected Malaysian honeyrdquo BMC Complementaryand Alternative Medicine vol 13 article no 129 2013

[11] R P Bird ldquoObservation and quantification of aberrant crypts inthe murine colon treated with a colon carcinogen preliminaryfindingsrdquo Cancer Letters vol 37 no 2 pp 147ndash151 1987

[12] N V Gribel and V G Pashinskiı ldquoThe antitumor properties ofhoneyrdquo Voprosy Onkologii vol 36 no 6 pp 704ndash709 1990

[13] B Souza D Roubik O Barth et al ldquoComposition of stinglessbee honey setting quality standardsrdquo Interciencia vol 31 no 12pp 867ndash875 2006

[14] C V Rao D Desai B Simi N Kulkarni S Amin and B SReddy ldquoInhibitory effect of caffeic acid esters on azoxymethane-induced biochemical changes and aberrant crypt foci formationin rat colonrdquo Cancer Research vol 53 no 18 pp 4182ndash41881993

[15] D Xiang D Wang Y He et al ldquoCaffeic acid phenethyl esterinduces growth arrest and apoptosis of colon cancer cells viathe 120573-cateninT-cell factor signalingrdquo Anti-Cancer Drugs vol17 no 7 pp 753ndash762 2006

[16] Y-J He B-H Liu D-B Xiang Z-Y Qiao T Fu and Y-HHe ldquoInhibitory effect of caffeic acid phenethyl ester on thegrowth of SW480 colorectal tumor cells involves 120573-cateninassociated signaling pathway down-regulationrdquo World Journalof Gastroenterology vol 12 no 31 pp 4981ndash4985 2006

[17] H-C Kuo W-H Kuo Y-J Lee W-L Lin F-P Chou and T-H Tseng ldquoInhibitory effect of caffeic acid phenethyl ester on thegrowth of C6 glioma cells in vitro and in vivordquo Cancer Lettersvol 234 no 2 pp 199ndash208 2006

[18] N Orsolic S Terzic Z Mihaljevic L Sver and I BasicldquoEffects of local administration of propolis and its polyphenoliccompounds on tumor formation and growthrdquo Biological ampPharmaceutical Bulletin vol 28 no 10 pp 1928ndash1933 2005

[19] F Borrelli A A Izzo G Di Carlo et al ldquoEffect of a propolisextract and caffeic acid phenethyl ester on formation of aberrantcrypt foci and tumors in the rat colonrdquo Fitoterapia vol 73 no1 pp S38ndashS43 2002

[20] R W Taylor M J Barron G M Borthwick et al ldquoMito-chondrial DNA mutations in human colonic crypt stem cellsrdquoJournal of Clinical Investigation vol 112 no 9 pp 1351ndash13602003

[21] R E Peterson M D Seefeld B J Christian C L Potter C KKelling and R E Keesey ldquoThe wasting syndrome in 2 3 7 8-tetrachlorodibenzo-p-dioxin toxicity basic features and their


interpretationrdquo in Banbury Report Biological Mechanisms ofDioxin Action A Poland and R Kimbrough Eds vol 18 pp291ndash308 Cold Spring Harbor Laboratory New York NY USA1984

[22] T C Wang Y P Su T Y Hsu C C Yang and C C Lin ldquo28-Day oral toxicity study of the aqueous extract from spider brake(Pteris multifida Poiret) in ratsrdquo Food and Chemical Toxicologyvol 45 no 9 pp 1757ndash1763 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

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Oxidative Medicine and Cellular Longevity


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


bee is stored in hexagonal-shaped combs [5] KH is morediluted compared to other types of honey and it has distincttaste (sour like taste) as well as aroma [6] TraditionallyKH is used for antiageing enhancing libido and immunesystem killing bacteria treatment of bronchial phlegm andrelieving sore throat cough and cold [7] KH has beenproven to possess various pharmacological properties suchas anti-inflammatory [8] antioxidant [5] antiageing [9] andantibacterial properties [10] Since KH has strong antioxidantactivities it could be one of the potential chemopreventiveagents from natural resources

2 Materials and Methods

21 ChemicalsTest Materials Azoxymethane was purchasedfrom Aldrich Sigma (St Louis United States) hematoxylinand eosin stain was purchased fromCellPath (Mochdre UK)10 formalin was purchased from Fisher Scientific (Lough-borough UK) and RCL2 was purchased from Alphaleys(Plaisir France) Kelulut honey (KH) was provided by Mar-bawi Food Processing and Trading Kuala Kangsar PerakMalaysia

22 Experimental Animals A total of 24 male Sprague Daw-ley rats with body weight of 180 to 220 g at the age of 5weeks were used Prior to the experiment the rats wereacclimatized a week with free access to food and water adlibitum The animals were housed in cages (119899 = 3) understandard laboratory conditions with 12-hour darklight cycleat 20ndash24∘C with 40ndash50 relative humidity Ethic approvalwas obtained from the Institutional Animal Care and UseCommittee (IACUC) ofUniversiti PutraMalaysia prior to thestudy (UPMIACUCFYP2015FPSK003)

23 Experimental Procedures The rats were divided into 4groups (119899 = 6) which were (G1) negative control group(not induced with AOM without treatment) (G2) positivecontrol group (induced with AOM without treatment)(G3) treatment group (induced with AOM treated withKH (1183mgkg)) and (G4) toxicity group (not inducedwith AOM treated with KH) After acclimatization AOM(15mgkg) was injected intraperitoneally into the rats for twosubsequent weeks once per week on the first day of eachweek Next KH was administered orally twice daily in themorning and at night for eight weeks The body weight ofthe rats was recorded every three days The dosage of KHwas calculated based on human intake Upon terminationthe rats were fasted for 8 hours before blood collection Priorto blood sampling the rats were anesthetized with ketamine-xylazine (75mgkg 5mgkg) by intraperitoneal injectionThe blood sample (7mL) was collected by cardiac punctureusing a 26 G 1210158401015840 needle (Terumo Belgium Europe) intononheparinized (5mL) and EDTA-containing tube (2mL)for biochemical (Roche Cobas USA) and haematological(ABX Pentra 60 Japan) analyses respectively The bloodcollected in nonheparinized tube was then centrifuged at10000 rpm for 10 minutes to separate the serum The organs

(colon liver and kidney) were removed and weighed beforebeing fixed in 10 formalin

24 Determination of Incidence of Aberrant Crypt Foci andCrypt Multiplicity The colon was processed by first dividingit into three parts which are proximal colon (near the cae-cum) middle colon and distal colon (towards the rectum)Each part was cut into strip and placed in a cassette Forliver it was grossed from a lobe of the liver and placed intoa cassette For kidney the organ was cut into half and placedin a cassette Next the organs that had been grossed wereprocessed using an automated tissue processor (LEICA TP1020 USA) where the machine runs dehydration clearingand impregnating process The organs were being embeddedin molten paraffin with appropriate molds to support thetissue during sectioning processThe blocks were trimmed at16 120583m thickness and sectioned at thickness of 04 120583m usinga microtome The thin sections of the tissue samples wereplaced in a water bath and fished onto glass slides The slidesprepared will be dewaxed first before being stained withhematoxylin and eosin (HampE) followed by mounting with P-xylene-bis-pyridinium bromide (DPX) and observed undera light microscope The total number of ACF per colon totalnumber of aberrant crypts (AC) and crypt multiplicity ofeach rat in all groups were calculated

25 Statistical Analysis The data were analysed by StatisticalPackage for Social Sciences (SPSS) version 21 One-wayANOVA analysis was performed followed by Duncanrsquos testThe data was presented as mean plusmn SEM Value of 119901 lt 005was considered as significant

3 Results

31 Effects of KH on Body Weight Figure 1 shows the bodyweight of all groups throughout the experiment There was agradual increase in the body weight throughout the 8 weeksof studyThehighest bodyweightwas noted inG2 followed byG1 G3 and G4 However there was no significant differencein the body weight among all groups (119901 gt 005)

32 Effects of KH on Blood Profile The red blood cell(RBC) haemoglobin (Hb) packed cell volume (PCV) meancorpuscular volume (MCV) mean corpuscular haemoglobinconcentration (MCHC) white blood cell (WBC) and throm-bocytes level of rats in 4 different groups are summarizedin Table 1 Data show no significant difference in the bloodprofile among all groups (119901 gt 005)

33 Effects of KH on Liver Enzymes and Kidney FunctionsTable 2 shows the liver enzymes and kidney function tests ofrats in 4 different groups show no significant difference in allthe parameters tested among all groups (119901 gt 005)

34 Effects of KH on Colon Length Table 3 shows the effectsof KH on the length of the colon in all groups throughoutthe experiment The highest colon length was noted inG1 followed by G3 G4 and G2 However there was no













4 Discussion


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G1G2

G3G4

200

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Body

wei

ght (

g)








(a) (b)

(c) (d)

(e)








Competing Interests


Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























4 Discussion


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k 0

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k 1

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k 5

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k 6

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k 7

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k 8

G1G2

G3G4

200

250

300

350

400

450

500

Body

wei

ght (

g)








(a) (b)

(c) (d)

(e)








Competing Interests


Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















(a) (b)

(c) (d)

(e)








Competing Interests


Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Competing Interests


Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Chemopreventive Properties and Toxicity...

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Transcript of Research Article Chemopreventive Properties and Toxicity...