Antioxidant, Anti-inflammatory and Hypolipidemic Properties of Apple Flavonols

144 Pages·2012·1.82 MB·English

Antioxidant, Anti-inflammatory and Hypolipidemic Properties of
Apple Flavonols
by
SatvirSekhon-Loodu
Submittedinpartialfulfilmentoftherequirements
forthedegreeofMasterofScience
at
DalhousieUniversity
Halifax,NovaScotia
inco-operationwith
NovaScotiaAgriculturalCollege
Truro,NovaScotia
August2012
©CopyrightbySatvirSekhon-Loodu,2012
DALHOUSIEUNIVERSITY
NOVA SCOTIAAGRICULTURALCOLLEGE
The undersigned hereby certify that they have read and recommend to the Faculty of
Graduate Studies for acceptance a thesis entitled “Antioxidant, Anti-inflammatory and
Hypolipidemic Properties of Apple Flavonols” by Satvir Sekhon-Loodu in partial
fulfilmentoftherequirementsfor thedegreeofMasterofScience.
Dated: August23,2012
Co-Supervisors: _________________________________
_________________________________
Readers: _________________________________
_________________________________
ii
DALHOUSIEUNIVERSITY
AND
NOVA SCOTIAAGRICULTURALCOLLEGE
DATE: 23.08.2012
AUTHOR: Satvir Sekhon-Loodu
TITLE: Antioxidant,Anti-inflammatoryandHypolipidemicPropertiesofApple
Flavonols.
DEPARTMENTOR SCHOOL: DepartmentofEnvironmentalSciences
DEGREE: MSc CONVOCATION: May YEAR: 2013
Permission is herewith granted to DalhousieUniversityto circulate and to have copied for
non-commercial purposes, at its discretion, the above title upon the request of individuals
orinstitutions.Iunderstandthatmythesis willbe electronicallyavailabletothepublic.
The author reserves other publication rights, and neither the thesis nor extensive extracts
fromitmaybeprintedor otherwisereproducedwithouttheauthor’s written permission.
The author attests that permission has been obtained for the use of any copyrighted
material appearing in the thesis (other than the brief excerpts requiring only proper
acknowledgementinscholarlywriting), andthatallsuchuseisclearlyacknowledged.
_______________________________
SignatureofAuthor
iii
Dedication
Idedicatethisdocumenttomyhusbandforhissupport,trust,motivation&beingwith
meateverystepofthisjourney.
iv
Table of Contents
LISTOFTABLES...........................................................................................................viii
LISTOFFIGURES...........................................................................................................ix
ABSTRACT........................................................................................................................x
LISTOFABBREVATIONS.............................................................................................xi
ACKNOWLEDGEMENTS.............................................................................................xiii
CHAPTER1.0 INTRODUCTION.....................................................................1
CHAPTER2.0 OBJECTIVES............................................................................5
CHAPTER3.0 LITERATUREREVIEW.........................................................6
3.1INFLAMMATIONDISORDERS............................................................................6
3.1.1InflammatoryCytokines InvolvedinEndothelialDysfunction..........................8
3.1.2Hyperlipidemiaand Inflammation......................................................................9
3.1.3Non-SteroidalAnti-inflammatoryDrugs..........................................................11
3.1.4ReactiveOxygen Speciesand Inflammation....................................................12
3.2POLYPHENOLICS-ANTIOXIDANTSAND ANTI-INFLAMMATORY
PROPERTIES...............................................................................................................14
3.2.1BioactiveBenefits ofPolyphenolics.................................................................15
3.2.2 Flavonoids ........................................................................................................15
3.2.3Biochemical Facts of Flavonoids.....................................................................16
3.2.4RelationshipofFlavonolsandMetabolicDiseases..........................................16
3.2.5AppleasaRich Sourceof Flavonols................................................................18
3.3 FAT INTAKEANDMETABOLIC DISEASE......................................................18
3.3.1DietaryFattyAcid AbsorptionandDistribution..............................................19
3.3.2 FattyAcidsandtheirRoleinInflammation.....................................................20
3.4ISOLATION ANDPURIFICATIONOFFLAVONOIDS....................................24
3.4.1ExtractionofPolyphenolics..............................................................................24
3.4.2DifferentTechniquesforPurificationof Flavonols..........................................25
3.5ESTERIFICATIONOFFLAVONOLS..................................................................28
3.5.1 Factors InfluencingAcylationReaction...........................................................30
3.6RESEARCHANIMALMODEL............................................................................33
v
CHAPTER4.0 ANTIOXIDANTABILITYOFFRACTIONATEDAPPLEPEEL
PHENOLICSTO INHIBIT FISHOILOXIDATION......................................................35
4.1ABSTRACT............................................................................................................35
4.2INTRODUCTION...................................................................................................36
4.3MATERIALSANDMETHODS............................................................................38
4.3.1Chemicals andReagents...................................................................................38
4.3.2Materials...........................................................................................................38
4.3.3ExtractionofApplePeel...................................................................................39
4.3.4 FractionationofApplePeelPolyphenolics......................................................39
4.3.5UPLC-MS/MSAnalysisofPhenolicFractions................................................40
4.3.6TheBulk Fish OilModel System.....................................................................41
4.3.7TotalPhenolicContentbyFolin-Ciocalteu(FC)ReagentAssay.....................42
4.3.8ThiobarbituricAcid ReactiveSubstances (TBARS)Assay.............................42
4.3.9 FerricReducingAntioxidantPower(FRAP)Assay.........................................43
4.3.10DPPH· ScavengingActivity...........................................................................43
4.3.11StatisticalAnalysis.........................................................................................44
4.4RESULTS................................................................................................................44
4.4.1DistributionofPhenolicCompounds...............................................................44
4.4.2TotalPhenolicsand AntioxidantCapacityof ApplePeelExtractand
Fractions ……………………………………………………………………………48
4.4.3InhibitionofFishOilOxidationbyApplePolyphenolics................................51
4.4.4CorrelationAnalysisofTotalPhenolicContentandAntioxidantActivity......54
4.5DISCUSSION.........................................................................................................57
CHAPTER5.0 MODULATIONOFLIPID PROFILE AND INFLAMMATORY
(cid:38)(cid:60)(cid:55)(cid:50)(cid:46)(cid:44)(cid:49)(cid:40)(cid:54)(cid:3)(cid:37)(cid:60)(cid:3)(cid:36)(cid:51)(cid:51)(cid:47)(cid:40)(cid:3)(cid:41)(cid:47)(cid:36)(cid:57)(cid:50)(cid:49)(cid:50)(cid:47)(cid:54)(cid:15)(cid:3)(cid:550)(cid:22)(cid:3)(cid:51)(cid:56)(cid:41)(cid:36)(cid:15)(cid:3)(cid:55)(cid:43)(cid:40)(cid:44)(cid:53)(cid:3)(cid:38)(cid:50)(cid:48)(cid:37)(cid:44)(cid:49)(cid:36)(cid:55)(cid:44)(cid:50)(cid:49)(cid:3)(cid:36)(cid:49)(cid:39)(cid:3)
ISQUERCITRIN-EICOSAPENTAENOIC ACIDESTER INHYPERLIDEMIC RATS...
……………………………………………………………………………………………62
5.1ABSTRACT............................................................................................................62
5.2INTRODUCTION...................................................................................................63
5.3MATERIALSANDMETHODS............................................................................65
5.3.1Chemicals andApparatus.................................................................................65
5.3.2Animal Modeland ExperimentalDesign.........................................................65
vi
5.3.3CollectionandStorageofBloodandTissueSamples......................................67
5.3.4 LipidProfileAnalysisinRatSerum.................................................................68
5.3.5QuantificationofLiverCholesterolandCholesterylEster..............................69
5.3.6QuantificationofLiverTriacylglycerols..........................................................69
5.3.7Cytokines Analysis usingCytometricBead Array...........................................70
5.3.8DeterminationofCRPinSerum.......................................................................71
5.3.9DeterminationofSerum IL-6...........................................................................72
5.3.10Determinationof SerumAdiponectin.............................................................72
5.3.11UPLC-MS/MSanalysisof applepeelphenoliccompounds..........................73
5.3.12StatisticalAnalysis.........................................................................................73
5.4RESULTS................................................................................................................74
5.4.1Compositionof Flavonolrich-Apple PeelConcentrate....................................74
5.4.2.Synthesis ofEPAEstersofIsoquercitrin(Quercetin-3-O-glucoside).............75
5.4.3EffectofDifferent DietarySupplements on Food Intake, Weightof Body,
Liverand Spleen........................................................................................................78
5.4.4Serum and Liver LipidProfiles........................................................................79
5.4.5Serum InflammatoryCytokines........................................................................82
5.5DISCUSSION.........................................................................................................86
CHAPTER6.0 CONCLUSIONS.................................................................................95
6.1ANTIOXIDANT, ANTI-INFLAMMATORYANDHYPOLIPIDEMIC
ACTIVITIESOFAPPLEFLAVONOLS…………..……………………..…………. 95
6.2RECOMMENDATIONS FOR FUTURERESEARCH.........................................97
REFERENCES.................................................................................................................99
APPENDIX:Supplementarydata………………………………………………...……130
vii
LIST OF TABLES
Table 4.1. Concentration of major polyphenolics (mg/L) recovered after
fractionation of crude extract from frozen apple peels using UPLC-
MS/MS……………………………………………………………............ 46
Table 4.2. Concentration of major polyphenolics (mg/L) recovered after
fractionation of crude extract prepared from dried apple peels using UPLC-
MS/MS……………………………………………………........................ 47
Table 4.3. Total phenolic content (FC) and antioxidant capacity (FRAP and DPPH)
of crude extracts and corresponding fractions prepared from dried (DP),
frozen applepeel(FP)andcorrespondingfractions……………………….. 50
Table 4.4. Pearson correlation coefficients to exhibit linear relationship among the
antioxidant capacity measures (FRAP, DPPH and TBARS values) and the
phenolic compounds analyzed by FC and UPLC-MS/MS in the different
fractions offrozenapplepeels…………………………………. 55
Table 4.5. Pearson correlation coefficients to exhibit linear relationship among the
antioxidant capacity measures (FRAP, DPPH and TBARS values) and the
phenolic compounds analyzed by FC and UPLC-MS/MS in the different
fractions ofdriedapplepeels...................................................................... 56
Table5.1.Compositionofthehigh-fatdiet……………..………………................ 67
Table 5.2. Concentrations of phenolic compounds in Northern Spy apple peel
concentrate(AF)preparedusingreverse-phasechromatography………… 74
Table 5.3. Food intake, body weight gain, liver weight and spleen weight of the
treatment groupsofhyperlipidemicrats….................................................. 78
viii
LIST OF FIGURES
Figure2.1.Obesityinducedinflammatoryevents……………………………………. 7
Figure2.2. Linkbetween reactiveoxygenspecies andinflammation………………… 13
Figure.2.3. Fatdigestion andabsorption………………………………………………
20
(cid:41)(cid:76)(cid:74)(cid:88)(cid:85)(cid:72)(cid:3)(cid:21)(cid:17)(cid:23)(cid:17)(cid:3)(cid:54)(cid:87)(cid:85)(cid:88)(cid:70)(cid:87)(cid:88)(cid:85)(cid:68)(cid:79)(cid:3)(cid:68)(cid:81)(cid:71)(cid:3)(cid:80)(cid:72)(cid:87)(cid:68)(cid:69)(cid:82)(cid:79)(cid:76)(cid:70)(cid:3)(cid:86)(cid:92)(cid:81)(cid:87)(cid:75)(cid:72)(cid:86)(cid:76)(cid:86)(cid:3)(cid:82)(cid:73)(cid:3)(cid:83)(cid:85)(cid:76)(cid:80)(cid:68)(cid:85)(cid:92)(cid:3)(cid:550)-(cid:25)(cid:3)(cid:68)(cid:81)(cid:71)(cid:3)(cid:550)-3 PUFA by
22
elongasesanddesaturases…………………………………………………..
Figure 2.(cid:24)(cid:17)(cid:3)(cid:39)(cid:72)(cid:83)(cid:76)(cid:70)(cid:87)(cid:76)(cid:82)(cid:81)(cid:3)(cid:82)(cid:73)(cid:3)(cid:87)(cid:75)(cid:72)(cid:3)(cid:70)(cid:72)(cid:79)(cid:79)(cid:88)(cid:79)(cid:68)(cid:85)(cid:3)(cid:68)(cid:85)(cid:68)(cid:70)(cid:75)(cid:76)(cid:71)(cid:82)(cid:81)(cid:76)(cid:70)(cid:3)(cid:68)(cid:70)(cid:76)(cid:71)(cid:3)(cid:68)(cid:81)(cid:71)(cid:3)(cid:550)-3 PUFA metabolism
involvedininflammation…………………………………………………... 23
Figure2.6. Structureofflavonoidwiththreerings…..………….……………………..
28
Figure 4.1. Percent inhibition of fish oil oxidation by polyphenolics in dried and
frozen apple peel fractions, BHT and (cid:302)-tocopherol at concentration of 200
μg total phenolics/mL in fish oil using TBARS
assay…………………........………………………………………………..
52
Figure 4.2. Concentration dependent inhibition of fish oil oxidation by major phenolic
compoundsfoundin applepeelusingTBARSassay………..………...... 53
Figure 5.1. Structure of the synthesized isoquercitrin-eicosapentaenoic acid ester
compound;(6-{3,5-dihydroxy-2-[3-(4-ydroxyphenyl)propanoyl]phenoxy}-
3,4,5-trihydroxy tetrahydro-2H-pyran-2-yl)methy(5Z,8Z,11Z,14Z,17Z)-
76
5,8,11,14,17-eicosapentaenoate………………………………………….
Figure 5.2. NMR spectrum of (6-{3,5-dihydroxy-2-[3-(4-hydroxyphenyl) propanoyl]
phenoxy}-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl (5Z,8Z,11Z,
14Z,17Z)-5,8,11,14,17-eicosapentaenoate…………………...................... 77
Figure 5.3. Serum triacylglycerols (TG), HDL-cholesterol (HDL-C) and non-HDL-
cholesterol levels in six groups of high-fat diet fed
rats………………..……………………………………………………….. 80
Figure 5.4. Liver total cholesterol (TC), free cholesterol (FC), triacylglycerols (TG)
andcholesteryl ester(CE)levelsinsix groups ofhigh-fat diet fed rat…… 81
Figure5.5. Serum concentrations of adiponectin, C-reactiveprotein,TNF-(cid:302) and IFN-(cid:534)(cid:3)
insix groupsofhigh-fat dietfedrat.……………………………………… 83
Figure 5.6. Serum concentrations of IL-6, IL-10, IL-1(cid:302), IL-2 and IL-4 in six groups of
85
high-fatdiet fed rats……………………………………………………….
ix
ABSTRACT
Obesity is considered an underlying risk factor for metabolic disease including
cardiovascular disease (CVD) and diabetes. The fractions containing flavonols from
apple peel were evaluated for their antioxidant, anti-inflammatory, and hypolipidemic
properties using in vitro and in vivo experimental model systems. The fractionated
polyphenolics from apple peels showed a strong antioxidant property protecting against
heat-induced oxidation of polyunsaturated fatty acids present in fish oil. Apple flavonols
(AF), eicosapentaenoic acid (EPA) and the isoquercitrin-EPA ester (QE) significantly
reduced serum triacylglycerols and elevated the high density lipoprotein (HDL)-
cholesterol compared to the high fat control group. C-reactive protein and interleukin-6
were also reduced compared to the high fat control group and inflammation induced by
lipopolysaccharides. Serum adiponectin and interferon-(cid:534)(cid:3) (cid:70)(cid:82)(cid:81)(cid:70)(cid:72)(cid:81)(cid:87)(cid:85)(cid:68)(cid:87)(cid:76)(cid:82)(cid:81)(cid:86)(cid:3) (cid:90)(cid:72)(cid:85)(cid:72)(cid:3)
significantly altered by QE treatment. Overall, AF and QE exhibited anti-inflammatory
and hypolipidemic effects under in vivo conditions. These beneficial physiological
propertiesand modeofactionofAFand QEneed tobefurtherinvestigated.
x