)joebxj1vcmjtijoh$psqpsbujpo ...downloads.hindawi.com/archive/2014/549287.pdfdecapitated under mild...

Research ArticleIn Vitro Studies on the Antioxidant Property andInhibition of 120572-Amylase 120572-Glucosidase and AngiotensinI-Converting Enzyme by Polyphenol-Rich Extracts fromCocoa (Theobroma cacao) Bean

Ganiyu Oboh Ayokunle O Ademosun Adedayo O Ademiluyi Olasunkanmi S OmojokunEsther E Nwanna and Kuburat O Longe

Functional Foods and Nutraceuticals Unit Biochemistry Department Federal University of TechnologyPMB 704 Akure 340252 Nigeria

Correspondence should be addressed to Olasunkanmi S Omojokun sunjokslivecom

Received 19 May 2014 Revised 9 July 2014 Accepted 2 September 2014 Published 8 September 2014

Academic Editor Shahid Pervez

Copyright copy 2014 Ganiyu Oboh 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

Background This study sought to investigate the antidiabetic and antihypertensive mechanisms of cocoa (Theobroma cacao) beanthrough inhibition of 120572-amylase 120572-glucosidase angiotensin-1 converting enzyme and oxidative stress Methodology The totalphenol and flavonoid contents of the water extractable phytochemicals from the powdered cocoa bean were determined andthe effects of the extract on 120572-amylase 120572-glucosidase and angiotensin-1 converting enzyme activities were investigated in vitroFurthermore the radicals [11-diphenyl-2 picrylhydrazyl (DPPH) 22-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)hydroxyl (OH) and nitric oxide (NO)] scavenging ability and ferric reducing antioxidant property of the extract were assessedResults The results revealed that the extract inhibited 120572-amylase (181 plusmn 022mgmL) 120572-glucosidase (184 plusmn 017mgmL) andangiotensin-1 converting enzyme (0674 plusmn 006mgmL [lungs] 1006 plusmn 008mgmL [heart]) activities in a dose-dependent mannerand also showed dose-dependent radicals [DPPH (1694 plusmn 134mgmL) NO (698 plusmn 0886mgmL) OH (372 plusmn 026mgmL) andABTS (157 plusmn 106mmolTEACsdotg] scavenging ability Conclusion The inhibition of 120572-amylase 120572-glucosidase and angiotensin-1converting enzyme activities by the cocoa bean extract could be part of the possible mechanism by which the extract could manageandor prevent type-2 diabetes and hypertension

1 Introduction

The studies of cocoa and their related products have becomean area of interest owing to their health-promoting prop-erties In recent years cocoa and cocoa products namelycocoa powder dark chocolate and cocoa liquor have beenshown to suppress atherosclerosis and reduce the risk of heartdisease [1] increase dermal blood circulation and decreaseplatelet activation adhesion and function as well as functionas cancer protective agent by inhibiting the proliferation ofhuman cancer cells and also exerted hypoglycemic properties[2] owing to the presence of the phenolic compounds init Polyphenols have been researched for decades mostlybecause of their antioxidant properties [3]

Dietary antioxidants sourced from the diet such as fruitsand vegetables are capable of counteracting the damagingbut normal effects of the physiological process of oxidationreactions that occur in animal tissue [4] These antioxidantsare considered beneficial because of their protective roleagainst oxidative stress which is involved in the pathogenesisof multiple diseases such as cardiovascular and cerebrovas-cular diseases [5] Diabetes mellitus is a complex diseasethat is characterized by chronic hyperglycemia The peculiarclinical features like excessive urination thirst weight lossand secondary complications observed in diabetes mellitusare primarily an indication of the hyperglycemic state [6]Type 1 diabetes results from inadequate synthesis of insulin by120573-cells of the pancreas while type II diabetes is characterized

Hindawi Publishing CorporationPathology Research InternationalVolume 2014 Article ID 549287 6 pageshttpdxdoiorg1011552014549287

2 Pathology Research International

primarily by insulin resistance (a condition in which periph-eral cells do not respond normally to insulin) or 120573-celldysfunction [7]

Alpha-amylase is a prominent enzyme found in thepancreatic juice and saliva which breaks down large insolublestarch molecules into absorbable molecules [8] On the otherhand mammalian 120572-glucosidase is an enzyme found in themucosal brush border of the small intestine which catalyzesthe end step of digestion of disaccharides that are abun-dant in human diet to its corresponding monosaccharide[9] Effective means of lowering the levels of postpran-dial hyperglycemia have been offered by 120572-amylase and 120572-glucosidase inhibitors by delaying the breakdown of ingestedcarbohydrates in the small intestine thereby reducing thepostprandial blood glucose excursion [10] Several inhibitorsof 120572-amylase and 120572-glucosidase have been isolated frommedicinal plants to serve as an alternative drugwith increasedpotency and less adverse effects than existing synthetic drugs[11]

Angiotensin-I converting enzyme (ACE) activity hasbeen linked to hypertension Inhibition of ACE is considereda useful therapeutic approach in the treatment of high bloodpressure and dietary phenolic phytochemicals have shownpromising potential while previous in vitro and in vivo animaland clinical studies have also indicated the potential of spe-cific phenolic phytochemicals in hypertension managementwith absorption into the blood [12] However despite thedocumented antidiabetic [13] and antihypertensive potentialofTheobroma cacao [14] no previous report has been given onthe mechanism by which it exerts this effect

2 Materials and Methods

21 Chemicals All chemicals used were sourced from SigmaCorporation (St Louis MO) Except stated otherwise all thechemicals and reagents used are of analytical grade while thewater used was glass distilled

22 Plant Material Collection and Preparation Cocoa beanswere bought at a local market in Ibule-soro (a suburb ofAkure) near Federal University of Technology Akure OndoState Nigeria The authentication of the bean was done atthe Department of Crop Soil and Pest Management FederalUniversity of Technology Akure Nigeria Subsequently thedried beans were then milled into fine powder The aqueousextracts of the bean were prepared by soaking 5 g of thegrinded samples in 100mLof distilledwater for 24 hrs at 37∘CThe mixture was later filtered through Whatmann number2 filter paper and centrifuged at 4000 rpm to obtain a clearsupernatant which was then stored in the refrigerator forsubsequent analysis [15]

23 Total Phenol Determination The total phenol contentwas determined by mixing 02mL of the sample extract with25mL 10 Folin-Cioalteau reagent (vv) and 20mL of 75sodiumcarbonatewas subsequently addedThe reactionmix-ture was incubated at 45∘C for 40min and the absorbancewas measured at 765 nm using a spectrophotometer Gallic

acid was used as standard phenol the total phenol contentwas subsequently calculated as gallic acid equivalent [16]

24 Total Flavonoid Determination The total flavonoid con-tent was determined by mixing 05mL of appropriatelydiluted sample with 05mLmethanol 50 120583L 10AlC1

3 50120583L

1M Potassium acetate and 14mL distilled water and allowedto incubate at room temperature for 30min The absorbanceof the reactionmixturewas subsequentlymeasured at 415 nmquercetin is used as standard flavonoid The total flavonoidcontent was subsequently calculated as quercetin equivalentThe nonflavonoid polyphenols were taken as the differencebetween the total phenol and total flavonoid content [17]

25 Enzyme Inhibition Assay

251 Angiotensin I Converting Enzyme (ACE) InhibitionAssay Appropriate dilution of the aqueous extract (0ndash500120583L) and ACE solution (50 120583L 4mU) was incubated at37∘C for 15min The enzymatic reaction was initiated byadding 150120583L of 833mM of the substrate Bz - Gly - His -Leu in 125mMTris-HCl buffer (pH 83) to the mixture Afterincubation for 30min at 37∘C the reaction was arrested byadding 250120583Lof 1MHClTheGly-His bondwas then cleavedand the Bz-Gly produced by the reaction was extracted with15mL ethyl acetateThereafter themixturewas centrifuged toseparate the ethyl acetate layer then 1mL of the ethyl acetatelayer was transferred to a clean test tube and evaporated Theresidue was redissolved in distilled water and its absorbancewasmeasured at 228 nm [18]TheACE inhibitory activity wasexpressed as percentage () inhibition

252 120572-Amylase Inhibition Assay This was measured usingthe dinitrosalicylic acidmethod as described byWorthingtonBiochemical Corporation 1978 [19] Appropriate dilution ofthe pastes (500120583L) and 500120583L of 002M sodium phosphatebuffer (pH 69 with 0006M NaCl) containing pancreatic 120572-amylase (EC 3211) (05mgmL) were incubated at 25∘C for10min Then 500120583L of 1 starch solution in 002M sodiumphosphate buffer (pH 69 with 0006M NaCl) was addedto each tube The reaction mixtures was incubated at 25∘Cfor 10min and stopped with 10mL of dinitrosalicylic acidcolour reagent Thereafter the mixture was incubated in aboiling water bath for 5min and cooled to room temperatureThe reaction mixture was then diluted by adding 10mL ofdistilled water and absorbance measured at 540 nm TheEC50

(the extract concentration inhibiting 50 of the 120572-amylase activity) of the pastes was calculated

253 120572-Glucosidase Inhibition Assay The extract (50 120583L)and 100 120583L of 120572-glucosidase solution (10UmL) in 01Mphosphate buffer (pH 69) was incubated at 25∘C for 10minThen 50120583L of 5mM p-nitrophenyl-120572-D-glucopyranosidesolution in 01M phosphate buffer (pH 69) was added Themixtures were incubated at 25∘C for 5min before readingthe absorbance at 405 nm in the spectrophotometer The 120572-glucosidase inhibitory activity was expressed as percentageinhibition The EC

50of the pastes was calculated [20]

Pathology Research International 3

26 In Vitro Antioxidant Studies

261 Determination of Reducing Property The reducingproperty was determined by assessing the ability of thesample extract to reduce FeCl

3solution as described by

Pulido et al 2002 [21] Briefly appropriate dilutions (0ndash10mL) were mixed with 25mL 200mm sodium phosphatebuffer (ph 66) and 25mL of 1 potassium ferricyanideThe mixtures were incubated at 50∘C for 20min Thereafter25mL 10 trichloroacetic acid was added and subsequentlycentrifuged at 650 rpm for 10minThen 5mL of the resultingsupernatant was mixed with equal volume of water and 1mLof 01 ferric chloride The absorbance was taken at 700 nmagainst a reagent blank

262 DPPH Free Radical Scavenging Assay The free radicalscavenging ability of the extract against DPPH (11-diphenyl-2picrylhdrazyl) free radical was evaluated as described byGyamfi et al 1999 [22] Briefly appropriate dilution of theextracts (0ndash500120583L)wasmixedwith 1mL 04mMmethanolicsolution containing DPPH radicals the mixture was left inthe dark for 30min and the absorbance was taken at 516 nmThe DPPH free radical scavenging ability was subsequentlycalculated

263 ABTSsdot+ Radical Scavenging Ability Assay The ABTSsdot+(221015840-azino-bis(3-ethylbenzthiazoline-6-sulphoic acid) scav-enging ability of the extract was determined according tothe method described by Re et al 1999 [23] The ABTSsdot+was generated by reacting 7mMABTS aqueous solution withK2S2O8(245mmolL final concentration) in the dark for

16 hrs and adjusting the Abs 734 nm to 0700 with ethanolThereafter 200120583L of appropriate dilution of the extractwas added to 20mL ABTSsdot+ solution and the absorbancewas measured at 734 nm after 15min The trolox equivalentantioxidant capacity was subsequently calculated using troloxas the standard

264 Inhibition of the Fenton Reaction (Degradation ofDeoxyribose) The method of Halliwell and Gutteridge 1981[24] was used to determine the ability of the extract toprevent Fe2+H

2O2induced decomposition of deoxyribose

The extract (0ndash100 120583L) was added to a reaction mixturecontaining 120 120583L of 20mM deoxyribose 400 120583L of 01Mphosphate buffer 40120583L of 500120583M FeSO

4 and the volume

were made up to 800120583L with distilled water The reactionmixture was incubated at 37∘C for 30min and the reac-tion was then stopped by the addition of 05mL of 28trichloroacetic acid (TCA) This was followed by additionof 04mL of 06 thiobarbituric acid (TBA) solution Thetubes were subsequently incubated in boiling water for20min and the absorbance was measured at 532 nm in aspectrophotometer

265 Nitric Oxide Radical Scavenging Assay The scavengingeffect of the extract on nitric oxide (NO) radical was mea-sured according to the method of Marcocci et al 1994 [25]Samples of 100ndash400 120583L of the oil extract were added in the

test tubes to 1mL of Sodium nitroprusside solution (25mM)and tubes incubated at 37∘C for 2 hours An aliquot (05mL)of the incubation was removed and diluted with 03mLGriess reagent (1 sulphanilamide in 5 H

3PO4and 01

naphthlethylenediaminedihy drochloride) The absorbanceof the chromophore formed was immediately read at 570 nmagainst distilled water as blank with catechin (50 120583g) usedas standard Results were expressed as percentage radicalscavenging activity (RSA)

27 Lipid Peroxidation Assay

271 Preparation of Tissue Homogenates The rats weredecapitated under mild diethyl ether anaesthesia the pan-creas was rapidly isolated placed on ice and weighed Thistissue was subsequently homogenized in cold saline (110wv) with about 10-up-and down strokes at approximately1200 revmin in a Teflon glass homogenizerThe homogenatewas centrifuged for 10min at 3000timesg to yield a pellet that wasdiscarded and a low-speed supernatant (S1) that was kept forlipid peroxidation assay

272 Lipid Peroxidation and Thiobarbituric Acid ReactionsThe lipid peroxidation assay was carried out using themodifiedmethod of Ohkawa et al 1979 [26] Briefly 100 120583L Slfraction was mixed with a reaction mixture containing 30120583Lof 01M pH 74 Tris-HCl buffer extract (0ndash100120583L) and 30 120583Lof 250120583M freshly prepared FeSO

4(the procedure was also

carried out using 7 120583M sodium nitroprusside) The volumewas made up to 300 120583L by water before incubation at 37∘Cfor 1 hr The reaction was developed by adding 300 120583L 81Sodium doudecylsulphate (SDS) to the reaction mixture andthis was subsequently followed by the addition of 600 120583L ofacetic acidHCl (pH 34) and 600 120583L 08 thiobarbituric acid(TBA) This mixture was incubated at 100∘C for 1 hr and thethiobarbituric acid reactive species (TBARS) produced weremeasured at 532 nm Subsequently the lipid peroxidationwascalculated as MDA produced (percentage of control)

28 Data Analysis The results of three replicates were pooledand expressed as mean plusmn standard deviation (SD) One-way analysis of variance (ANOVA) and least significancedifference (LSD) were carried out [27] Significance wasaccepted at 119875 le 005 EC

50was determined using linear

regression analysis

3 Results

The total phenolic content reported as gallic acid equiva-lent total flavonoid content reported as quercetin equiv-alent ABTSlowast scavenging ability reported as trolox equiv-alent and ferric reducing antioxidant property reportedas ascorbic acid equivalent as presented in Table 1 were179mgsdotGAE100 g 684mgsdotQUE100 g 157mmolTEACsdotgand 1492mgAAEsdotg respectively

As shown in Table 2 the cocoa bean aqueous extractscavenged DPPH OH and NO radicals in concentration-dependent manners with EC

50values of 1694mgmL

372mgmL and 698mgmL respectively


Table 1 The total phenol content reported as gallic acid equivalenttotal flavonoid content reported as quercetin equivalent ABTSlowastscavenging ability reported as trolox equivalent antioxidant capacityand ferric reducing antioxidant property reported as ascorbic acidequivalent of aqueous extract of cocoa bean

Parameter Value (unit)Total phenol 179 plusmn 096 (mgsdotGAE100 g)Total flavonoid 684 plusmn 010 (mgsdotQUE100 g)ABTSlowast scavenging ability 157 plusmn 106 (mmolTEACsdotg)Ferric reducing antioxidantproperty 1492 plusmn 082 (mgAAEsdotg)

Values represent means plusmn standard deviation of triplicate readings

Table 2 EC50 values of DPPH OH and NO radical scavengingability of aqueous extract of cocoa bean

Parameter EC50 value (mgmL)DPPH radical scavenging ability 1694 plusmn 134OH radical scavenging ability 372 plusmn 026NO radical scavenging ability 698 plusmn 088Values represent means plusmn standard deviation of triplicate readings

The antioxidant effect of the cocoa powder by inter-acting the aqueous extract with isolated rat pancreas inthe presence of sodium nitroprusside (SNP) and Fe2+ asprooxidants was investigated As shown in Table 3 the EC

50

results show that the extract inhibits SNP (1147mgmL)and Fe2+ (212mgmL) induced lipid peroxidation in ratrsquospancreas In assessing the antihypertensive and antidia-betic potentials of the extract the results revealed that thebean extracts inhibited angiotensin-I converting enzyme120572-amylase and 120572-glucosidase activities The EC

50values

as presented in Table 3 were found to be 181mgmL (120572-amylase) 184mgmL (120572-glucosidase) 0674mgmL (ACE inthe lungs) and 101mgmL (ACE in the heart)

4 Discussion

Previous studies on the health benefits of cocoa have pri-marily focused on its effects on the risk of cardiovasculardiseases and reduction of blood glucose levels [28] with adearth of information on the possible mechanisms of actionThe inhibition of 120572-amylase slows down the breakdown ofstarch to disaccharide while the inhibition of 120572-glucosidaseslows down the breakdown of the disaccharides to the simplemonosaccharide glucose thereby reducing the amount ofglucose absorbed into the blood stream [10] Angiotensin-1converting enzyme (ACE) cleaves angiotensin I which is adecapeptide to produce angiotensin II an octapeptide andpotent vasoconstrictor that has been identified as amajor fac-tor in hypertensive conditions [29]Therefore ACE inhibitorshave been widely considered to prevent angiotensin II pro-duction in cardiovascular diseases and utilized in clinicalapplications since the discovery of ACE inhibitors in snakevenom [29] The inhibition of 120572-amylase and 120572-glucosidaseactivities by the cocoa powder aqueous extracts could have

Table 3 EC50 values of SNP and Fe2+ induced lipid peroxidation inratrsquos pancreas angiotensin-I converting enzyme inhibitory activityin the lungs and heart and 120572-amylase and 120572-glucosidase inhibitoryactivity of aqueous extract of cocoa bean

Parameter EC50 value (mgmL)SNP induced lipid peroxidation 1147 plusmn 107Fe2+ induced lipid peroxidation 212 plusmn 010ACE (lungs) 0674 plusmn 006ACE (heart) 1006 plusmn 008120572-amylase 181 plusmn 022120572-glucosidase 184 plusmn 017Values represent means plusmn standard deviation of triplicate readings

contributed to its use in the management of diabetes therebyslowing down the breakdown of starch to disaccharide Theextract inhibited both 120572-glucosidase and 120572-amylase in adose-dependent manner pointing to its potential of beingof great pharmaceutical importance in addressing some ofthe side effects (like flatulence and abdominal distention)associated with the drugs (AcarboseMiglitol andVoglibose)presently used for the management of diabetes Furthermorethe dose-dependent ACE inhibitory activity of the cocoapowder aqueous extract reveals that this could explain thelikely mechanism for its use in the treatment of hypertensionand possibly address the side effects of some class of antihy-pertensive drugs (diuretics beta blockers and synthetic ACEinhibitors) used in managing hypertension

Polyphenols have received wide attention because oftheir antioxidant properties which refers to their abilityto prevent damage from reactive oxygen species throughfree radical scavenging or prevent the generation of thesespecies by iron chelation as well as bind and inhibit theenzymes 120572-amylase and 120572-glucosidase [30] The antioxidantactivity of fruits and vegetables significantly increases withthe increase in polyphenol content in it [31] The total phenoland flavonoid content of this cocoa bean extract was found tobe 179mgsdotGAE100 g and 684mgsdotQUE100 g respectivelyThis finding was in agreement with previous study whichdemonstrated that high flavonoid (a class of polyphenol)content of cocoa extract correlated with its strong antioxidantcapacity [28]

The free radical scavenging ability of this extract was alsostudied making use of moderately stable nitrogen-centredradical species-ABTS radical [32] The result obtained hereagrees with the phenolic content in earlier research articleswhere correlations were reported between phenolic contentand antioxidant capacity of some plant foods [33]The extractwas found to also demonstrate strong free radical scavengingabilities as exemplified by their scavenging activity of moder-ately stable ABTSsdot+ NO OHminus and DPPH radicals in vitroHowever there is an agreement between the ABTSsdot+ NOOHminus and DPPH free radical scavenging ability which agreeswith earlier findings where plant antioxidant properties (freeradical scavenging ability) correlates with their phenoliccontent [34] A study showed that decrease in antioxidantcapacity in cocoa beverages stored at different temperatureand time was due to loss of its phenolic content [35]


Antioxidants neutralize the electrical charges on freeradicals and prevent them from taking electrons from othermolecules [36]They are needed to prevent the formation andoppose the actions of reactive oxygen and nitrogen specieswhich are generated in vivo and cause damage to DNA lipidsproteins and other biomolecules Antioxidants are naturallypowerful substances that help slow the aging process fightdiseases such as diabetes mellitus and hypertension andprevent some deadly and incurable diseases like cancer [37]The protective ability of the extract against Sodium nitro-prusside and Fe2+ induced lipid peroxidation in cultured ratsrsquopancreas assessed revealed that incubation of the pancreastissues in the presence of Fe2+ caused a significant (119875 lt 005)increase in the MDA content (181) Likewise incubation ofthe pancreas tissue in the presence of sodium nitroprussidecaused a significant (119875 lt 005) increase in the MDA content(249) These findings agree with earlier reports wherephenolics had been reported to be potent inhibitors of lipidperoxidation in several animal tissues [38] Sodium nitro-prusside a component of antihypertensive drugs is knownto cause cytotoxicity through the release of cyanide andornitric oxide (NO) NO is a universal neuronal messengerin the central nervous system and acts independently itmay also cause neuronal damage in cooperation with otherreactive oxygen species (ROS) [39] It could be suggestedthat the higher ACE inhibitory ability of the aqueous extractsof the cocoa powder aqueous extract is due to its higherNOlowast scavenging ability However we advise that further workshould be done to isolate the phytoconstituents in the cocoabean which may be responsible for eliciting the biologicalactivities Also in vitro works do not automatically translateto human body response hence we suggest that further invivo work should be carried out

5 Conclusion

Conclusively cocoa bean is rich in phenolic compoundsand exhibits a high antioxidant activity Its ingestion as afunctional food lowers plasma glucose level by inhibiting120572-amylase and 120572-glucosidase and could be of importancein the management of diabetes mellitus as it also addressessome side effects associatedwith synthetic antidiabetic drugsIn addition cocoa seed extract inhibition of ACE alsoexplains the likely mechanism for its use in the treatment ofhypertension Hence cocoa bean ingestion will improve thequality of life of hypertensive patients and the diabetics

Conflict of Interests

There is no conflict of interests regarding the publication ofthis paper

References

[1] J W Erdman Jr L Carson C Kwik-Uribe E M Evansand R R Allen ldquoEffects of cocoa flavanols on risk factorsfor cardiovascular diseaserdquo Asia Pacific Journal of ClinicalNutrition vol 17 no 1 pp 284ndash287 2008

[2] D Ramljak L J Romanczyk L J Metheny-Barlow et alldquoPentameric procyanidin from Theobroma cacao selectivelyinhibits growth of human breast cancer cellsrdquoMolecular CancerTherapeutics vol 4 no 4 pp 537ndash546 2005

[3] M Netzel G Strass I Bitsch R Konitz M Christmann andR Bitsch ldquoEffect of grape processing on selected antioxidantphenolics in red winerdquo Journal of Food Engineering vol 56 no2-3 pp 223ndash228 2003

[4] G Oboh ldquoAntioxidant properties of some commonly con-sumed and underutilized tropical legumesrdquo European FoodResearch and Technology vol 224 no 1 pp 61ndash65 2006

[5] M Alıa C Horcajo L Bravo and L Goya ldquoEffect of grapeantioxidant dietary fiber on the total antioxidant capacity andthe activity of liver antioxidant enzymes in ratsrdquo NutritionResearch vol 23 no 9 pp 1251ndash1267 2003

[6] OKlein J Lynge L Endahl BDamholt LNosek andTHeiseldquoAlbumin-bound basal insulin analogues (insulin detemir andNN344) comparable time-action profiles but less variabilitythan insulin glargine in type 2 diabetesrdquo Diabetes Obesity andMetabolism vol 9 no 3 pp 290ndash299 2007

[7] T Heise L Nosek B B Rooslashnn et al ldquoLower within-subjectvariability of insulin detemir in comparison toNPH insulin andinsulin glargine in people with type 1 diabetesrdquoDiabetes vol 53no 6 pp 1614ndash1620 2004

[8] A F Afifi A E Kamel A A Khalil M A Foaad E MFawziand and M Houseny ldquoPurification and characterizationof 120572-amylase from penicillium olsonii under the effect ofsome antioxidant vitaminsrdquoGlobal Journal of Biotechnology andBiochemistry vol 3 no 1 pp 14ndash12 2008

[9] V Manohar N A Talpur B W Echard S Lieberman andH G Preuss ldquoEffects of a water-soluble extract of maitakemushroom on circulating glucoseinsulin concentrations in KKmicerdquoDiabetes Obesity andMetabolism vol 4 no 1 pp 43ndash482002

[10] Y-I Kwon E Apostolidis and K Shetty ldquoEvaluation of pepper(Capsicum annuum) formanagement of diabetes and hyperten-sionrdquo Journal of Food Biochemistry vol 31 no 3 pp 370ndash3852007

[11] T Matsui I A Ogunwande K J M Abesundara and K Mat-sumoto ldquoAnti-hyperglycemic potential of natural productsrdquoMini-Reviews inMedicinal Chemistry vol 6 no 3 pp 349ndash3562006

[12] Y-I I Kwon D A Vattem and K Shetty ldquoEvaluation of clonalherbs of Lamiaceae species for management of diabetes andhypertensionrdquo Asia Pacific Journal of Clinical Nutrition vol 15no 1 pp 107ndash118 2006

[13] I Amin H A Faizul and R Azli ldquoEffect of cocoa powderextract on plasma glucose levels in hyperglycaemic ratsrdquo Nutri-tion and Food Science vol 34 pp 116ndash121 2004

[14] B Buijsse E J M Feskens F J Kok and D KromhoutldquoCocoa intake blood pressure and cardiovascular mortalitythe Zutphen Elderly Studyrdquo Archives of Internal Medicine vol166 no 4 pp 411ndash417 2006

[15] G Oboh R L Puntel and J B T Rocha ldquoHot pepper(Capsicum annuum Tepin and Capsicum chinese Habanero)prevents Fe2+-induced lipid peroxidation in brainmdashin vitrordquoFood Chemistry vol 102 no 1 pp 178ndash185 2007

[16] V L Singleton R Orthofer and R M Lamuela-RaventosldquoAnalysis of total phenols and other oxidation substrates andantioxidants by means of folin-ciocalteu reagentrdquo Methods inEnzymology vol 299 pp 152ndash178 1998


[17] A Meda C E Lamien M Romito J Millogo and O GNacoulma ldquoDetermination of the total phenolic flavonoid andproline contents in Burkina Fasan honey as well as their radicalscavenging activityrdquo Food Chemistry vol 91 no 3 pp 571ndash5772005

[18] D W Cushman and H S Cheung ldquoSpectrophotometric assayand properties of the angiotensin-converting enzyme of rabbitlungrdquo Biochemical Pharmacology vol 20 no 7 pp 1637ndash16481971

[19] Worthington Biochemical Corporation Enzyme and RelatedBiochemicals Worthington Biochemical Corporation Free-hold NJ USA 1978

[20] E Apostolidis Y-I Kwon and K Shetty ldquoInhibitory potentialof herb fruit and fungal-enriched cheese against key enzymeslinked to type 2 diabetes and hypertensionrdquo Innovative FoodScience and Emerging Technologies vol 8 no 1 pp 46ndash54 2007

[21] R Pulido L Bravo and F Saura-Calixto ldquoAntioxidant activityof dietary polyphenols as determined by a modified ferricreducingantioxidant power assayrdquo Journal of Agricultural andFood Chemistry vol 48 no 8 pp 3396ndash3402 2000

[22] M A Gyamfi M Yonamine and Y Aniya ldquoFree-radicalscavenging action of medicinal herbs from GhanaThonningiasanguinea on experimentally-induced liver injuriesrdquo GeneralPharmacology vol 32 no 6 pp 661ndash667 1999

[23] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[24] B Halliwell and J M C Gutteridge ldquoFormation of a thiobarbi-turic-acid-reactive substance from deoxyribose in the presenceof iron salts the role of superoxide and hydroxyl radicalsrdquo FEBSLetters vol 128 no 2 pp 347ndash352 1981

[25] LMarcocci J JMaguireM TDroy-Lefaix and L Packer ldquoThenitric oxide-scavenging properties of Ginkgo biloba extractEGb 761rdquo Biochemical and Biophysical Research Communica-tions vol 201 no 2 pp 748ndash755 1994

[26] H Ohkawa N Ohishi and K Yagi ldquoAssay for lipid peroxidesin animal tissues by thiobarbituric acid reactionrdquo AnalyticalBiochemistry vol 95 no 2 pp 351ndash358 1979

[27] J H Zar Biostatistical Analysis Prentice-Hall 1984[28] K A Cooper J L Donovan A I Waterhouse and G

Williamson ldquoCocoa and health a decade of researchrdquo BritishJournal of Nutrition vol 99 no 1 pp 1ndash11 2008

[29] A Villar M Paya and M C Terencio ldquoPlants with antihyper-tensive actionrdquo Fitoterap vol 57 pp 131ndash145 1986

[30] N R Perron and J L Brumaghim ldquoA review of the antioxidantmechanisms of polyphenol compounds related to iron bindingrdquoCell Biochemistry and Biophysics vol 53 no 2 pp 75ndash100 2009

[31] K Ghasemi Y Ghasemi and M A Ebrahimzadeh ldquoAntioxi-dant activity phenol and flavonoid contents of 13 citrus speciespeels and tissuesrdquo Pakistan Journal of Pharmaceutical Sciencesvol 22 no 3 pp 277ndash281 2009

[32] M Benderitter V Maupoil C Vergely F Dalloz F Briot and LRochette ldquoStudies by electron paramagnetic resonance of theimportance of iron in the hydroxyl scavenging properties ofascorbic acid in plasma effects of iron chelatorsrdquo Fundamentalamp Clinical Pharmacology vol 12 no 5 pp 510ndash516 1998

[33] J Sun Y-F Chu X Wu and R H Liu ldquoAntioxidant andantiproliferative activities of common fruitsrdquo Journal of Agricul-tural and Food Chemistry vol 50 no 25 pp 7449ndash7454 2002

[34] A A Oluwaseun and O Ganiyu ldquoAntioxidant properties ofmethanolic extracts of mistletoes (Viscum album) from cocoaand cashew trees in Nigeriardquo African Journal of Biotechnologyvol 7 no 17 pp 3138ndash3142 2008

[35] S Sang M-J Lee Z Hou C-T Ho and C S Yang ldquoStability oftea polyphenol (-)-epigallocatechin-3-gallate and formation ofdimers and epimers under common experimental conditionsrdquoJournal of Agricultural and Food Chemistry vol 53 no 24 pp9478ndash9484 2005

[36] G Oboh and J B T Rocha ldquoAntioxidant in foods a newchallenge for food processorsrdquo in Leading Edge AntioxidantsResearch H V Panglossi Ed pp 35ndash64 Nova Science Publish-ers New York NY USA 2007

[37] R H Liu ldquoHealth benefits of fruit and vegetables are fromadditive and synergistic combinations of phytochemicalsrdquo TheAmerican Journal of Clinical Nutrition vol 78 no 3 pp 517Sndash520S 2003

[38] I Klimczak M Małecka M Szlachta and A Gliszczynska-Swigło ldquoEffect of storage on the content of polyphenols vitaminC and the antioxidant activity of orange juicesrdquo Journal of FoodComposition and Analysis vol 20 no 3-4 pp 313ndash322 2007

[39] G Oboh and J B T Rocha ldquoAntioxidant and neuroprotectiveproperties of sour tea (Hibiscus sabdariffa calyx) and greentea (Camellia sinensis) on some pro-oxidant-induced lipidperoxidation in brain in vitrordquo Food Biophysics vol 3 no 4 pp382ndash389 2008

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


primarily by insulin resistance (a condition in which periph-eral cells do not respond normally to insulin) or 120573-celldysfunction [7]

Alpha-amylase is a prominent enzyme found in thepancreatic juice and saliva which breaks down large insolublestarch molecules into absorbable molecules [8] On the otherhand mammalian 120572-glucosidase is an enzyme found in themucosal brush border of the small intestine which catalyzesthe end step of digestion of disaccharides that are abun-dant in human diet to its corresponding monosaccharide[9] Effective means of lowering the levels of postpran-dial hyperglycemia have been offered by 120572-amylase and 120572-glucosidase inhibitors by delaying the breakdown of ingestedcarbohydrates in the small intestine thereby reducing thepostprandial blood glucose excursion [10] Several inhibitorsof 120572-amylase and 120572-glucosidase have been isolated frommedicinal plants to serve as an alternative drugwith increasedpotency and less adverse effects than existing synthetic drugs[11]

Angiotensin-I converting enzyme (ACE) activity hasbeen linked to hypertension Inhibition of ACE is considereda useful therapeutic approach in the treatment of high bloodpressure and dietary phenolic phytochemicals have shownpromising potential while previous in vitro and in vivo animaland clinical studies have also indicated the potential of spe-cific phenolic phytochemicals in hypertension managementwith absorption into the blood [12] However despite thedocumented antidiabetic [13] and antihypertensive potentialofTheobroma cacao [14] no previous report has been given onthe mechanism by which it exerts this effect

2 Materials and Methods

21 Chemicals All chemicals used were sourced from SigmaCorporation (St Louis MO) Except stated otherwise all thechemicals and reagents used are of analytical grade while thewater used was glass distilled

22 Plant Material Collection and Preparation Cocoa beanswere bought at a local market in Ibule-soro (a suburb ofAkure) near Federal University of Technology Akure OndoState Nigeria The authentication of the bean was done atthe Department of Crop Soil and Pest Management FederalUniversity of Technology Akure Nigeria Subsequently thedried beans were then milled into fine powder The aqueousextracts of the bean were prepared by soaking 5 g of thegrinded samples in 100mLof distilledwater for 24 hrs at 37∘CThe mixture was later filtered through Whatmann number2 filter paper and centrifuged at 4000 rpm to obtain a clearsupernatant which was then stored in the refrigerator forsubsequent analysis [15]

23 Total Phenol Determination The total phenol contentwas determined by mixing 02mL of the sample extract with25mL 10 Folin-Cioalteau reagent (vv) and 20mL of 75sodiumcarbonatewas subsequently addedThe reactionmix-ture was incubated at 45∘C for 40min and the absorbancewas measured at 765 nm using a spectrophotometer Gallic

acid was used as standard phenol the total phenol contentwas subsequently calculated as gallic acid equivalent [16]

24 Total Flavonoid Determination The total flavonoid con-tent was determined by mixing 05mL of appropriatelydiluted sample with 05mLmethanol 50 120583L 10AlC1

3 50120583L

1M Potassium acetate and 14mL distilled water and allowedto incubate at room temperature for 30min The absorbanceof the reactionmixturewas subsequentlymeasured at 415 nmquercetin is used as standard flavonoid The total flavonoidcontent was subsequently calculated as quercetin equivalentThe nonflavonoid polyphenols were taken as the differencebetween the total phenol and total flavonoid content [17]

25 Enzyme Inhibition Assay

251 Angiotensin I Converting Enzyme (ACE) InhibitionAssay Appropriate dilution of the aqueous extract (0ndash500120583L) and ACE solution (50 120583L 4mU) was incubated at37∘C for 15min The enzymatic reaction was initiated byadding 150120583L of 833mM of the substrate Bz - Gly - His -Leu in 125mMTris-HCl buffer (pH 83) to the mixture Afterincubation for 30min at 37∘C the reaction was arrested byadding 250120583Lof 1MHClTheGly-His bondwas then cleavedand the Bz-Gly produced by the reaction was extracted with15mL ethyl acetateThereafter themixturewas centrifuged toseparate the ethyl acetate layer then 1mL of the ethyl acetatelayer was transferred to a clean test tube and evaporated Theresidue was redissolved in distilled water and its absorbancewasmeasured at 228 nm [18]TheACE inhibitory activity wasexpressed as percentage () inhibition

252 120572-Amylase Inhibition Assay This was measured usingthe dinitrosalicylic acidmethod as described byWorthingtonBiochemical Corporation 1978 [19] Appropriate dilution ofthe pastes (500120583L) and 500120583L of 002M sodium phosphatebuffer (pH 69 with 0006M NaCl) containing pancreatic 120572-amylase (EC 3211) (05mgmL) were incubated at 25∘C for10min Then 500120583L of 1 starch solution in 002M sodiumphosphate buffer (pH 69 with 0006M NaCl) was addedto each tube The reaction mixtures was incubated at 25∘Cfor 10min and stopped with 10mL of dinitrosalicylic acidcolour reagent Thereafter the mixture was incubated in aboiling water bath for 5min and cooled to room temperatureThe reaction mixture was then diluted by adding 10mL ofdistilled water and absorbance measured at 540 nm TheEC50

(the extract concentration inhibiting 50 of the 120572-amylase activity) of the pastes was calculated

253 120572-Glucosidase Inhibition Assay The extract (50 120583L)and 100 120583L of 120572-glucosidase solution (10UmL) in 01Mphosphate buffer (pH 69) was incubated at 25∘C for 10minThen 50120583L of 5mM p-nitrophenyl-120572-D-glucopyranosidesolution in 01M phosphate buffer (pH 69) was added Themixtures were incubated at 25∘C for 5min before readingthe absorbance at 405 nm in the spectrophotometer The 120572-glucosidase inhibitory activity was expressed as percentageinhibition The EC

50of the pastes was calculated [20]












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