antiamylase, anticholinesterases, antiglycation, and...

Research ArticleAntiamylase Anticholinesterases Antiglycation and GlycationReversing Potential of Bark and Leaf ofCeylon Cinnamon (Cinnamomum zeylanicum Blume) In Vitro

Sirimal Premakumara Galbada Arachchige1

Walimuni Prabhashini Kaushalya Mendis Abeysekera1

andWanigasekera Daya Ratnasooriya23

1Herbal Technology Section (HTS) Modern Research amp Development Complex (MRDC) Industrial Technology Institute (ITI)503A Halbarawa Gardens Malabe Sri Lanka2Department of Zoology Faculty of Science University of Colombo Colombo Sri Lanka3Faculty of Allied Health Sciences General Sir John Kotelawala Defence University Ratmalana Sri Lanka

Correspondence should be addressed to Sirimal Premakumara Galbada Arachchige gaspitilk

Received 3 June 2017 Revised 25 July 2017 Accepted 3 August 2017 Published 30 August 2017

Academic Editor Luigi Milella

Copyright copy 2017 Sirimal Premakumara Galbada Arachchige et al This is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided theoriginal work is properly cited

Ethanol (95) and dichloromethane methanol (DCM M 1 1 vv) bark extracts (BEs) and leaf extracts (LEs) of authenticatedCeylon cinnamon (CC)were studied for antiamylase antiglucosidase anticholinesterases and antiglycation and glycation reversingpotential in bovine serum albumin- (BSA-) glucose and BSA-methylglyoxal models in vitro Further total proanthocyanidins(TP) were quantified Results showed significant differences (119901 lt 005) between bark and leaf extracts for the studied biologicalactivities (except antiglucosidase) and TP BEs showed significantly high (119901 lt 005) activities for antiamylase (IC50 214 plusmn 2ndash215 plusmn10 120583gmL) antibutyrylcholinesterase (IC50 2662 plusmn 166ndash3609 plusmn 083 120583gmL) and glycation reversing in BSA-glucose model(EC50 9433 plusmn 181ndash10716 plusmn 395 120583gmL) compared to LEs In contrast glycation reversing in BSA-methylglyoxal (EC50 ethanol12215 plusmn 601 120583gmL) and antiglycation in both BSA-glucose (IC50 ethanol 1522 plusmn 047 120583gmL) and BSA-methylglyoxal models(IC50 DCM M 27829 plusmn 855 120583gmL) were significantly high (119901 lt 005) in leaf Compared to the reference drugs used some of thebiological activities were significantly (119901 lt 005) high (BEs BChE inhibition and ethanol leaf BSA-glucosemediated antiglycation)some were comparable (BEs BSA-glucose mediated antiglycation) and some were moderate (BEs and LEs antiamylase AChEinhibition and BSA-MGOmediated antiglycation DCM M leaf BSA-glucose mediated antiglycation) TP were significantly high(119901 lt 005) in BEs compared to LEs (BEs and LEs 109790 plusmn 7301ndash138153 plusmn 4593 and 30952 plusmn 281ndash43424 plusmn 1412mg cyanidinequivalentsg extract resp) In conclusion both bark and leaf of CC possess antidiabetic properties and thus may be useful inmanaging diabetes and its complications

1 Introduction

Diabetes mellitus is one of the most prevalent chronicmetabolic diseases worldwide [1 2] It affected about 387million peopleworldwide in 2014 and the number is projectedto increase by another 205 million people by 2035 [1] Themajor categories of diabetes include type 1 and type 2 which ischaracterized by chronic hyperglycemia resulting from abso-lute or relative deficiencies in insulin secretion andactivity

[2] Prolonged hyperglycemic condition in diabetes patientsinduces nonenzymatic glycation reaction and leads to pro-duction of multitude of heterogeneous end products whichare known as advanced glycation end products (AGEs) [3ndash7]Low molecular weight carbonyl compounds such as glyoxaland methylglyoxal (MGO) behave as precursors of AGEs[5ndash8] They form adducts on proteins inducing cellulardysfunctions leading to long-term diabetes complicationssuch as retinopathy neuropathy and nephropathy [3ndash7] and

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 5076029 13 pageshttpsdoiorg10115520175076029

2 Evidence-Based Complementary and Alternative Medicine

several age related diseases such as Alzheimerrsquos diseaseatherosclerosis arthritis pulmonary fibrosis renal failureand cancer [3ndash7] Accumulation of AGEs in the brain isinvolved in extensive protein cross linking oxidative stressandneuronal cell death leading to neurodegenerative diseasesand most commonly Alzheimerrsquos disease [6 9] CurrentlyAlzheimerrsquos disease is even referred to as type 3 diabetesas it can be explained through AGEs and oxidation [9]and insulin and the cholinergic hypothesis [10 11] Naturalproducts reported to have antidiabetic activity all over theworld for centuries [12] Antidiabetic drugs nutraceuticalsand functional foods derived from plant sources have highdemand as they are natural and safe alternatives tomany syn-thetic drugs [13 14] Cinnamon one of the oldest and mostfrequently consumed spices worldwide belongs to the genusCinnamomum and there are different species of cinnamonworldwide [15 16] Among several species of cinnamon in theworld Ceylon cinnamon is the ldquotrue cinnamonrdquo the worldover based on its unique taste aroma and phytochemicalcomposition [15 16] Currently Sri Lanka is the leadingexporter of true cinnamon with 85 of world market shareand 145 market share for all types of cinnamon worldwideAccording to the recent statistics nearly 50 of exportearnings of minor agricultural crops in the country comefrom Ceylon cinnamon [17]

Cinnamon is reported to have several pharmacologi-cal activities including some antidiabetic related propertiesworldwide [15 16 18 26 27] However main problem inmany of these publications that there is no proper authen-tication for the experimental cinnamon sample [15] Hencethere is no strong evidence to confirm that these reportedbiological activities are from authenticated Ceylon cinnamon(true cinnamon) since the genus contains four economicallyimportant cinnamon species such as Cinnamomum zeylan-icum or Cinnamomum verum (Ceylon cinnamon or truecinnamon)Cinnamomumaromaticum (Cinnamomumcassiaor Chinese cinnamon) Cinnamomum burmannii (KorintjeJava or Indonesian cinnamon) and Cinnamomum loureiroi(Vietnamese or Saigon cinnamon) [28] On the other handwithin the country there are no in-depth studies on antidi-abetic activity of authenticated Ceylon cinnamon (exceptRanasinghe et al [29]) even though it is the most economicalminor agricultural crop in Sri Lanka Further the studiesconductedworldwide so far on antidiabetic activity of Ceyloncinnamon (true cinnamon) mainly focused on bark extractsand only 3 studies [20 25 30] are available on antidiabeticactivity of leaf extracts to date Further as yet there are nopublish studies on antiamylase antiglycation and glycationreversing activities of bark and antiglucosidase antiglycationand glycation reversing potential of leaf of authenticated Cey-lon cinnamon (true cinnamon) worldwide Previous investi-gations on antiamylase antiglucosidase anticholinesterasesantiglycation and glycation reversing activities of bark andleaf of Cinnamomum species are given in Table 1 The aimof this study was to evaluate antiamylase antiglucosidaseanticholinesterases antiglycation and glycation reversingpotential of both bark and leaf of authenticated Ceyloncinnamon viawidely used well established sensitive specificreliable and reproducible in vitro bioassays

2 Materials and Methods

21 Chemicals and Reagents Soluble starch bovine serumalbumin (BSA) D-glucose 120572-glucosidase (type V from rice)p-nitrophenyl 120572-D-glucopyranoside acarbose trichloro-acetic acid (TCA) acetylcholinesterase (AChE) from electriceel (Type-VI-S) butyrylcholinesterase (BChE) from horseserum acetylthiocholine butyrylthiocholine 551015840-dithio-bis-(2-nitrobenzoic) acid (DTNB) methylglyoxal (MGO) 35-dinitrosalicylic acid (DNS) dimethyl sulfoxide (DMSO)galantamine rutin cyanidin chloride and ammoniumiron(III) sulfate dodecahydrate were purchased from Sigma-Aldrich USA 120572-Amylase (Bacillus amyloliquefaciens) waspurchased from Roche Diagnostics USA All the otherchemicals and reagents were of analytical grade

22 Collection and Preparation of Ceylon Cinnamon AlbaGrade Bark and Leaf Samples Fresh cinnamon leaves werecollected from cinnamon cultivations of LB spices (Pvt)Ltd Aluthwala Galle Sri Lanka Alba grade cinnamon barksamples (alba grade cinnamon has the lowest quill thicknessmaximum 6mm according to the grading of cinnamonquills based on the quill thickness) [31] were collected fromcinnamon factories of LB spices (Pvt) Ltd AluthwalaGalle Sri Lanka and G P De Silva and Sons Spice (Pvt)Ltd Ambalangoda Sri Lanka The alba grade bark sampleswere authenticated by Dr Chandima Wijesiriwardena Prin-ciple Research Scientist Industrial Technology Institute SriLanka and leaf samples (voucher number CZB-KA) wereauthenticated byMr NPT Gunawardena Officer In-ChargeNational Herbarium Department of National Botanic Gar-dens Peradeniya Sri Lanka The specimens of each bark andleaf samples (HTS-CIN-1) and photographic evidence weredeposited at the Pharmacognosy Laboratory Herbal Tech-nology Section Industrial Technology Institute Sri LankaFresh leaves were air-dried at room temperature (30 plusmn 2∘C)for 7 days The air-dried leaves and bark were groundpowdered and stored at minus20∘C until used for the extraction

23 Preparation of Extracts

231 Preparation of Ethanolic Extracts Powdered bark andleaf samples (20 g) were extracted in 200mL of 95 ethanolfor 4-5 h in a Soxhlet extractor (4ndash6 cycles) until the solventbecame colorless The extracts were filtered evaporatedand freeze-dried (Christ-Alpha 1ndash4 Freeze dryer BiotechInternational Germany) Freeze-dried extracts were stored atminus20∘C until used for analysis

232 Preparation of Dichloromethane Methanol (DCM M)Extracts Powdered bark and leaf (20 g) samples wereextracted in 200mL of dichloromethane methanol(DCM M) at a ratio of (1 1 vv) at room temperature(30 plusmn 2∘C) for 7 days with occasional shaking The extractswere filtered evaporated freeze-dried and stored at minus20∘Cuntil used for analysis

24 Antiamylase Activity The antiamylase activity of barkand leaf extracts of Ceylon cinnamon were carried out

Evidence-Based Complementary and Alternative Medicine 3

Table1Antidiabetic

activ

ityof

Cinn

amom

umspeciesinvitro

Cinn

amom

umspecies

Partusedextract

Activ

ityRe

ferences

Antia

mylasea

ctivity

Czeylanicu

mlowast

Bark

aqueou

sextract

IC50123plusmn0

02m

gmL

Adisa

kwattana

etal2011[18]

Carom

aticu

m(cassia

)lowastBa

rkaqueou

sextract

IC50177plusmn

005

mgmL

Adisa

kwattana

etal2011[18]

Cloureir

oi(Saigon

cinn

amon

)lowastBa

rkaqueou

sextract

IC50gt4

00m

gmL

Adisa

kwattana

etal2011[18]

Czeylanicu

m(C

verum)lowast

Bark

hydroalcoho

licextract

(50

50vvw

ater

ethano

l)IC5025120583g

mL

Beejmoh

unetal2014

[19]

Cverum

Isop

ropano

lleafextract

IC501120583g

mL

Ponn

usam

yetal2011[20]

Czeylanicu

mlowast

Bark

aqueou

sextract

77inhibitio

nat25

mgmL72inhibitio

nat125mgmLand

51inhibitio

nat5m

gmL

Ranilla

etal2010

[21]

Antiglucosid

asea

ctivitym

altaseandsucraseinh

ibition

Czeylanicu

mlowast

Bark

aqueou

sextract

100

and95inhibitio

nat25and05m

gmLrespectiv

elyRa

nilla

etal2010

[21]

Czeylanicu

mlowast

Bark

aqueou

sextract

IC50120583g

mL077plusmn0

04maltase042plusmn0

02sucrase

Adisa

kwattana

etal2011[18]

Carom

aticu

m(cassia

)lowastBa

rkaqueou

sextract

IC50120583g

mL085plusmn0

04maltase088plusmn0

33sucrase

Adisa

kwattana

etal2011[18]

Cloureir

oi(Saigon

cinn

amon

)lowastBa

rkaqueou

sextract

IC50120583g

mL096plusmn0

03maltasegt4

00sucrase

Adisa

kwattana

etal2011[18]

Antiglyc

ationactiv

ity

Cinn

amon

(Cinna

mom

umspeciesu

sedno

tmentio

ned)lowast

Ethylacetateandbu

tano

lsolub

lefractio

nsof

bark

water

extract

dilutedwith

ethano

l

BSA-

glucosea

ntiglycatio

nethylacetates

olub

lefractio

nsgt4

0tolt90

inhibitio

nat200p

pmbutanolsolublefractionslt1

0togt80

inhibitio

nat200p

pmBS

A-MGOantig

lycatio

nethylacetatesolublefractionslt1

0tolt80

inhibitio

nat25m

gmLbu

tano

lsolub

lefractio

ns

lt10to

nearly40inhibitio

nat25m

gmL

Peng

etal2008

[22]

Cverumlowast

Bark

methano

lextract

BSA-

glucosea

ntiglycatio

nIC5026120583g

mL

HoandCh

ang2012

[23]

AChE

andBC

hEinhibitory

activ

ity

Czeylanicu

mBa

rkethano

lextract

AChE

4083plusmn0

005

inhibitio

nat100120583g

mLBC

hE515

3plusmn

0005inhibitio

nat100120583g

mL

Kumar

etal2012

[24]

Czeylanicu

mMethano

licleafextract

IC50A

ChE7778plusmn0

03120583g

mLBC

hE8862plusmn1

72120583g

mL

Dalaietal2014

[25]

lowastEx

perim

entalcinnamon

sampleh

asno

authentic

ation

AChE

acetylch

olinesteraseB

ChE

butyrylcho

linesterase


according to the method of Bernfeld [32] with some modifi-cations Briefly a reaction volume of 1mL containing 50 120583L ofethanolic and DCM M bark and leaf extracts (bark extracts625 125 250 500 and 1000 120583gmL 119899 = 4 leaf extracts 937518750 375 750 and 1500120583gmL 119899 = 4) 40 120583L of starch(1 wv) and 50 120583L of enzyme (5120583gmL) in 100mM sodiumacetate buffer (pH 60) were incubated at 40∘C for 15minAfter the incubation period 05mL of DNS reagent wasadded and placed in a boiling water bath for 5min Thenreaction mixtures were cooled in a water bath containing iceand absorbance readings were recorded at 540 nm using a96-well microplate reader (SpectraMax PLUS 384 MolecularDevices Inc USA) Control of the experiment contains allthe reagents except extracts whereas sample blanks werewithout the enzyme Acarbose was used as the positive con-trol (625ndash100 120583gmL) Antiamylase activity ( inhibition)was given as IC50 values (concentration of bark and leafextracts and positive control that inhibited the hydrolysisof starch by 50) Inhibition was calculated using thefollowing

Inhibition () = [119860119888 minus (119860 119904 minus 119860119887)119860119888 ] lowast 100 (1)

where 119860119888 is the absorbance of the control 119860119887 is theabsorbance of sample blanks and119860 119904 is the absorbance in thepresence of bark and leaf extracts

25 Antiglucosidase Activity Antiglucosidase activity ofbark and leaf extracts of Ceylon cinnamon was carriedout according to the method of Matsui et al [33] withminor modifications in 96-well microplates A reactionvolume of 01mL containing 4mM p-nitrophenyl-120572-D-glucopyranoside 50mUmL of 120572-glucosidase and 40 120583L ofethanolic and DCM M bark and leaf extracts (25 50 100200 and 400120583gmL 119899 = 4) in 50mM sodium acetate buffer(pH 58) were incubated at 37∘C for 30min After theincubation period reaction was stopped by adding 50 120583Lof 01M Na2CO3 Then absorbance readings were taken at405 nm using a 96-well microplate reader Reaction mixturewithout extract was used as the control and reaction mixturewith the extract and without enzyme was used as the sampleblank Acarbose a clinical 120572-glucosidase inhibitor was usedas the positive control Antiglucosidase activity ( inhibition)was calculated by using the following


where 119860119888 is the absorbance of the control (100 enzymeactivity) 119860119887 is the absorbance produced by cinnamonextracts (sample blank) and 119860 119904 is the absorbance of thesample in the presence of cinnamon bark or leaf extracts oracarbose

26 Anticholinesterase Activity AChE and BChE inhibitoryactivities of bark and leaf extracts of Ceylon cinnamon wereperformed according to the method of Ellman et al [34] with

some modifications in 96-well microplates A reaction vol-ume of 200120583L containing 01M sodiumphosphate buffer (pH80) 15003mU of AChEBChE (10 120583L) enzyme and 50 120583Lof different concentrations of bark and leaf extracts (bothbark and leaf for AChE 50 100 200 400 and 800 120583gmLbark BChE 625 125 25 50 and 100 120583gmL leaf BChE25 50 100 200 and 400 120583gmL) and the positive controlswere preincubated for 15min at 25∘C The reaction wasthen initiated by the addition of 1020 120583L of 2mM acetylth-iocholinebutyrylthiocholine and 20 120583L of 05mM DNTBThe hydrolysis of acetylthiocholinebutyrylthiocholine wasmonitored by the formation of yellow colored 5-thio-2-nitrobenzoate anion for a period of 10min for BChE and20min for AChE at 412 nm using 96-well microplate reader(SpectraMax Plus384 Molecular Devices USA) Galantaminewas used as the positive control (AChE 039ndash25 120583gmLBChE 125ndash200 120583gmL) Control incubations were carriedout in the same way while replacing extracts with bufferThe kinetic parameter 119881max was used to calculate the inhibition and anticholinesterase activity was given as IC50values (the concentrations of bark and leaf extracts andthe positive control that inhibited the hydrolysis of acetyl-cholinebutyrylthiocholine by 50)

The percentage inhibition was calculated as

Inhibition () = (119860119862 minus 119860119878)119860119862 times 100 (3)

where 119860119862 is 119881max of the control and 119860119878 is 119881max of the sampleor galantamine

27 Antiglycation Activity

271 BSA-Glucose Glycation Inhibitory Activity This assaywas carried out according to the method of Matsuura etal [35] with some modifications A reaction mixture of1mL containing 800120583g BSA 400mM glucose and 50120583L ofethanolic and DCM M bark and leaf extracts (625 125 2550 75 and 100 120583gmL 119899 = 4) in 50mM phosphate buffer(pH 74) containing 002 sodium azide were incubatedfor 40 h at 60∘C The 600 120583L of each reaction mixture wastransferred to 15mL Eppendorf tubes and 60120583L of 100(wv) TCA was added mixed well and allowed to standat room temperature (25 plusmn 2∘C) for 30min Then samplemixtures were centrifuged at 15000 rpm at 4∘C for 4min andsupernatants were discarded The AGEs-BSA precipitate wasthen dissolved in 1mL of phosphate buffer saline (pH 10)and the fluorescence intensity was measured at an excitationand emission wave lengths of 370 nm and 440 nm using a96-well florescence microplate reader (SpectraMax GeminiEM Molecular Devices Inc USA) Rutin was used as thepositive control (625ndash100 120583gmL) Antiglycation activity (inhibition) was calculated using the following equation IC50values (concentration of bark and leaf extracts and rutinthat inhibited the formation of AGEs by 50) were alsocalculated

Inhibition () = [(119865119888 minus 119865119887) minus (119865119904 minus 119865119904119887)(119865119888 minus 119865119887) ] lowast 100 (4)


where 119865119888 is the florescence of incubated BSA glucose andDMSO (control)119865119887 is the florescence of incubated BSA alone(blank) 119865119904 is the florescence of the incubated BSA glucoseand cinnamon leaf or bark extracts or the positive controland 119865119904119887 is the florescence of incubated BSA with the leaf orbark extracts or the positive control

272 BSA-MGO Glycation Inhibitory Activity This assay wascarried out according to the method reported by Lunce-ford and Gugliucci [36] with some modifications Reactionvolume of 1mL containing 1mg BSA 5mM MGO anddifferent concentrations of ethanolic and DCM M bark andleaf extracts (25 50 100 200 and 400 120583gmL 119899 = 6) in01M phosphate buffer containing 02 gL sodium azide wereincubated at 37∘C for 6 days After the incubation periodflorescence was measured at an excitation and emissionwavelengths of 370 and 440 nm using 96-well florescencemicroplate reader Control experiments were conducted in anidentical way while replacing extracts with 01M phosphatebuffer For sample blanks MGO solution was replaced with01M phosphate buffer Rutin was used as the positive control(625ndash200120583gmL) Antiglycation activity (inhibition ) wascalculated as described in BSAglucose model by replacingglucose with MGO

28 Glycation Reversing Activity

281 BSA-Glucose Glycation Reversing Activity This assaywas carried out according to the method of Premakumaraet al [37] with some modifications A reaction mixturecontaining 800120583g BSA and 400mM glucose in 1mL of50mM phosphate buffer (pH 74) containing 002 sodiumaside (wv) was incubated at 60∘C for 40 h Then 600 120583L ofeach reaction mixtures was transferred to 15mL Eppendorftubes and 60120583L of 100 (wv) TCA was added stirred welland allowed to stand at room temperature for 30min Thensample mixtures were centrifuged at 15000 rpm at 4∘C for4min and supernatants were discarded The resulting AGEs-BSA precipitates were dissolved in 50mM phosphate buffer(pH 74) added with 125 25 50 100 150 and 200 120583gmL barkand leaf extracts (119899 = 6) in a final reaction volume of 1mLand were incubated at 60∘C for 40 h After cooling 60 120583Lof 100 (wv) TCA was added stirred and centrifuged at15000 rpm at 4∘C for 4min The resulting precipitates werethen dissolved in 1mL of phosphate buffer saline (pH 10) andfluorescence intensity was measured at an excitation wavelength of 370 nm and emission wave length of 440 nm usinga 96-well florescence microplate reader Percentage glycationreversing was calculated using the following equation andresults were given as EC50 values (concentration of bark andleaf extracts that reversed the AGEs by 50)

Glycation reversing ()= [(119865119888 minus 119865119887) minus (119865119904 minus 119865119904119887)(119865119888 minus 119865119887) ] lowast 100 (5)

where 119865119888 is the florescence of incubated BSA glucose andDMSO (control)119865119887 is the florescence of incubated BSA alone

(blank) 119865119904 is the florescence of the incubated BSA glucoseand barkleaf extracts and 119865119904119887 is the florescence of incubatedBSA with the barkleaf extracts

282 BSA-MGO Glycation Reversing Activity This assaywas performed according to the method of Lunceford andGugliucci [36] and Premakumara et al [37] with minormodifications Reaction mixture containing 1mg BSA and5mM MGO in 1mL of 01M phosphate buffer pH 74 wasincubated at 37∘C for 6 days The test solution also contained02 gL NaN3 to assure an aseptic conditionThen aliquots of600 120583L were transferred to 15mL Eppendorf tubes and 60 120583Lof 100 (wv) TCA was added stirred and centrifuged at15000 rpm at 4∘C for 4min and supernatants were removedThe resulting precipitates were dissolved in 01M phosphatebuffer (pH 74) and added with 375 75 150 300 and600 120583gmL (119899 = 4) bark and leaf extracts to a final reactionvolume of 1mL for incubation at 37∘C for 6 days Afterthe incubation florescence was measured at an excitationwave length of 370 nm and emission wave length of 440 nmusing a 96-well florescence microplate reader Percentageglycation reversing was calculated as described in BSAglucose reversing model via replacing glucose with MGO

29 Total Proanthocyanidin Content The total proantho-cyanidin content of bark and leaf extracts of Ceylon cinna-mon was quantified by butanol-HCl assay method describedby Porter et al [38] with minor modifications Reactionvolumes of 36mL containing 05mL of extracts in methanol(assay concentration ethanolic and DCM M bark and leafextracts 025mgmL 119899 = 6 each) 3mL of butanol-HClreagent (95 5 vv) and 100 120583L of 2 ammonium iron(III)sulfate dodecahydrate in 2M HCl were added to 10mLscrew capped test tubes mixed well and incubated at 95∘Cin a water bath for 40min Sample blanks were carriedout in the same way without heating After the incubationperiod samples were allowed to cool to room temperatureand absorbance was recorded at 550 nm Cyanidin chloride(0016 0031 0063 0125 and 025mgmL 119899 = 3) wasused as the standard Results were expressed as mg cyanidinequivalents per g of extract of cinnamon barkleaf

210 Statistical Analysis Data of each experiment were sta-tistically analyzed using SAS version 612 One way analysisof variance (ANOVA) and the Duncanrsquos Multiple RangeTest (DMRT) were used to determine the differences amongtreatment means 119901 lt 005 was regarded as significant

3 Results

31 Antiamylase Activity of Bark and Leaf Extracts of CeylonCinnamon Both bark and leaf extracts demonstrated anti-amylase activity in a dose-dependent manner (ethanol barkDCM M bark ethanol leaf DCM M leaf 1199032 = 099 100100 and 095 resp) However bark extracts showed signifi-cantly higher activity (119901 lt 005) compared to leaf extractsAntiamylase activity between ethanol and DCM M barkextracts were statistically non-significant (119901 gt 005)The IC50


Table 2 Antiamylase activity

Extract inhibition 120583gmLConcentration (120583gmL)

Bark 6250 125 250 500 1000 IC50Ethanol 2442 plusmn 222 3235 plusmn 110 5691 plusmn 207 7413 plusmn 053 9228 plusmn 123 215 plusmn 10bDCM M 1851 plusmn 059 3020 plusmn 060 5785 plusmn 047 7384 plusmn 254 7619 plusmn 511 214 plusmn 2bLeaf 9375 18750 375 750 1500 IC50Ethanol 434 plusmn 237 1084 plusmn 190 2078 plusmn 251 4314 plusmn 246 7749 plusmn 203 943 plusmn 28aDCM M minus478 plusmn 206 070 plusmn 286 181 plusmn 506 983 plusmn 291 1759 plusmn 124 mdashData represented as mean plusmn SEM (119899 = 4 each) Mean IC50 values in the column superscripted by different letters are significantly different at 119901 lt 005Ethanol bark DCM M bark ethanol leaf and DCM M leaf 1199032 = 099 100 100 and 095 respectively IC50 acarbose 13388 plusmn 44120583gmL DCM Mdichloromethane methanol

Table 3 Antiglucosidase activity

Extract inhibitionEthanol bark minus811 plusmn 220DCM M bark minus572 plusmn 489Ethanol leaf minus867 plusmn 319DCM M leaf minus705 plusmn 086Data represented as mean plusmn SEM (119899 = 4 each) inhibition at 400120583gmLIC50 acarbose 047 plusmn 001 120583gmL

values of ethanolic bark and DCM M bark were 215 plusmn 10and 214 plusmn 2 120583gmL respectively Among the studied leafextracts ethanolic leaf extract had high antiamylase activity(IC50943 plusmn 28 120583gmL) than DCM M leaf extract (1759 plusmn124 inhibition at 15mgmL) Further both bark and leafextracts showed moderate antiamylase activity comparedto the standard drug acarbose (IC5013388 plusmn 254 120583gmL)The dose-response relationship of bark and leaf extracts forantiamylase activity is given in Table 2

32 Antiglucosidase Activity of Bark and Leaf Extracts ofCeylon Cinnamon Both ethanolic and DCM M bark andleaf extracts did not show antiglucosidase activity even atthe highest studied concentration of 400120583gmL Results ofantiglucosidase activity of bark and leaf extracts were given inTable 3 Acarbose a clinical 120572-glucosidase inhibitor had anti-glucosidase activity as IC50 = 047 plusmn 001 120583gmL

33 Anticholinesterase Activity of Bark and Leaf Extracts ofCeylon Cinnamon Both ethanolic and DCM M bark andleaf extracts of Ceylon cinnamon showed both AChE andBChE inhibitory activities However inhibition of BChEwas more prominent compared to AChE inhibition in bothbark and leaf extracts Bark extracts showed dose-dependent(ethanol bark and DCM M bark 1199032 = 097 each) and signifi-cantly high (ethanol bark andDCM Mbark IC50 3609plusmn083and 2662 plusmn 166 120583gmL resp) (119901 lt 005) BChE inhibitioncompared to the standard drug galantamine (IC50 7480 plusmn353 120583gmL) On the other hand BChE inhibition of leafextracts although dose-dependent (ethanol leaf andDCM Mleaf 1199032 = 094 and 098 resp) was moderate (ethanol leaf andDCM M leaf IC50 34060plusmn1823 and 26196plusmn1956 120583gmL

resp) Further DCM M extracts showed significantly high(119901 lt 005) activity than ethanol extracts in both bark andleaf In complete contrast AChE inhibition of bark and leafextracts showed dose-dependent (ethanol bark DCM Mbark ethanol leaf and DCM M leaf 1199032 = 092 094 095and 099 resp) but significantly low (119901 lt 005) activitywith respect to standard drug galantamineThe IC50 values ofethanol bark DCM M bark ethanol leaf DCM M leaf andgalantamine were 80488 plusmn 4869 96668 plusmn 6318 81096 plusmn7998 87935 plusmn 6800 and 252 plusmn 017 120583gmL respectivelyThe dose-response relationships of ethanol and DCM Mbark and leaf extracts for acetyl and butyrylcholinesteraseinhibitory activities are given in Table 4

34 Antiglycation Potential of Bark and Leaf Extracts ofCeylon Cinnamon

341 BSA-Glucose Glycation Inhibitory Activity Bothethanolic and DCM M bark and leaf extracts showed dose-dependent antiglycation activity (ethanol bark DCM Mbark ethanol leaf and DCM M leaf 1199032 = 089 099 100 and096 resp) IC50 values of bark and leaf extracts ranged from1942plusmn126ndash2080plusmn268 to 1522plusmn047ndash4262plusmn167 120583gmLrespectively Ethanol leaf had the highest BSA-glucoseglycation inhibitory activity (IC50 1522 plusmn 047 120583gmL)Further both bark extracts showed similar (ethanoland DCM M bark extracts IC50 1942 plusmn 126 and2080 plusmn 268 120583gmL resp) and DCM M leaf showed lowestantiglycation activity (IC50 4262 plusmn 167 120583gmL) Antigly-cation activity of ethanolic leaf and bark extracts wassignificantly higher (119901 lt 005) and comparable compared tothe positive control rutin (IC50 2188 plusmn 282 120583gmL) Dose-response relationships of ethanolic and DCM M bark andleaf extracts of Ceylon cinnamon are given in Figure 1

342 BSA-MGO Glycation Inhibitory Activity Both barkand leaf extracts of Ceylon cinnamon showed BSA-MGOglycation inhibitory activity The inhibitory activity of BSA-MGOglycationwas dose-dependent (ethanol bark DCM Mbark ethanol leaf and DCM M leaf 1199032 = 095 099 098and 095 resp) andmoderate compared to the standard drugrutin (IC50 6335 plusmn 067 120583gmL) The IC50 values of bark andleaf extracts ranged from 35738 plusmn 308ndash39259 plusmn 2088 to


Table4Anticho

linesterasesa

ctivity

Extract

inhibitio

n120583g

mL

Con

centratio

n(120583g

mL)

50100

200

400

800

IC50

Acetylcholinesterase

inhibitory

activ

ity

Ethano

lbark

1046plusmn2

132868plusmn1

973011plusmn2

373690plusmn2

20

5213plusmn0

48

80488plusmn4

869

b

DCM

Mbark

1977plusmn2

163234plusmn0

61

3788plusmn0

1140

26plusmn0

99

4769plusmn1

0596668plusmn6

318

a

Ethano

lleaf

684plusmn1

441344plusmn2

523008plusmn0

08

3504plusmn1

5946

33plusmn3

75

81096plusmn7

998a

DCM

Mleaf

minus1066plusmn3

65

403plusmn1

371493plusmn2

313434plusmn1

584913plusmn0

63

8793

5plusmn6

800

a

Bark

625

125

2550

100

IC50

Butyrylch

olinesterase

inhibitory

activ

ityEthano

l850plusmn2

94

1717plusmn3

69

4214plusmn3

01

6606plusmn1

197510plusmn0

75

3609plusmn0

83c

DCM

M90

6plusmn2

98

3679plusmn2

69

5060plusmn4

42

6716plusmn0

92

7897plusmn1

682662plusmn1

66d

Leaf

2550

100

200

400

IC50

Butyrylch

olinesterase

inhibitory

activ

ityEthano

l542plusmn4

07

2584plusmn2

20

3528plusmn1

144262plusmn2

66

4922plusmn1

8134060plusmn18

23a

DCM

M657plusmn2

122295plusmn2

03

3745plusmn0

38

4228plusmn0

63

5748plusmn1

812619

6plusmn19

56b

Datar

epresented

asmeanplusmnSE

M(119899=4each)MeanIC50values

inthec

olum

nsuperscriptedby

different

lette

rsaresignificantly

different

at119901lt005Statistic

alanalysiswas

carriedou

tseparately

fora

cetylch

oline

esteraseandbu

tyrylch

olinee

steraseinhibitory

assaysIC 50galantam

ineacetylcholinee

steraseinhibitory

activ

ity252plusmn017120583gmLIC50galantam

inebu

tyrylch

olinee

steraseinhibitory

activ

ity7480plusmn353120583gmL

Ethano

lbarkDCM

Mbarkethanolleafand

DCM

Mleaf1199032

=097097094and

098respectively

for

butyrylch

olinee

sterase

inhibitory

activ

ityE

thanolbarkD

CMM

barkethanolleafand

DCM

Mleaf

1199032=092094095and

099respectively

for

acetylcholinee

sterase

inhibitory

activ

ityD

CMM

dichlorom

ethane

methano

l


Ethanol bark Ethanol leaf

20 40 60 80 100 1200Concentration (gmL)

minus5

5

15

25

35

45

55

65

75

85

95

in

hibi

tion

DCM M bark DCM M leaf

Figure 1 Antiglycation activity via BSA-glucose model Datarepresented as mean plusmn SEM (119899 = 4 each) IC50 values ethanolleaf ethanol bark DCM M bark and DCM M leaf 1522 plusmn 047c1942plusmn126b 2080plusmn268b and 4262plusmn167a gmL respectively IC50values superscripted by different letters are significantly different at119901 lt 005 Ethanol leaf ethanol bark DCM M bark and DCM Mleaf 1199032 = 100 089 099 and 096 respectively IC50 rutin 2188 plusmn282 120583gmL DCM M dichloromethane methanol

27829 plusmn 855ndash34928 plusmn 821 120583gmL respectively DCM Mextracts of both bark and leaf showed significantly (119901 lt005) high activity compared to ethanol extracts The orderof potency of BSA-MGO glycation inhibitory activity wasDCM M leaf gt ethanol leaf = DCM M bark gt ethanol barkDose-response relationships of ethanolic and DCM M barkand leaf extracts of Ceylon cinnamon are given in Figure 2


351 BSA-Glucose Glycation Reversing Activity Both barkand leaf extracts showed significant and dose-dependent(ethanol bark DCM M bark ethanol leaf and DCM M leaf1199032 = 097 096 099 and 099 resp) BSA-glucose glycationreversing activity IC50 values of bark and leaf extracts rangedfrom 9433 plusmn 181ndash10716 plusmn 395 to 12120 plusmn 201ndash19942 plusmn902 120583gmL respectively Bark extracts showed significantlyhigh activity than leaf extracts (119901 lt 005) The orderof potency of BSA-glucose glycation reversing activity wasDCM M bark gt ethanol bark gt ethanol leaf gtDCM M leafDose-response relationships of ethanolic and DCM M barkand leaf extracts of Ceylon cinnamon are given in Figure 3

352 BSA-MGO Glycation Reversing Activity Both ethano-lic and DCM M bark and leaf extracts of Ceylon cinna-mon showed dose-dependent (ethanol bark DCM M barkethanol leaf and DCM M leaf 1199032 = 094 096 099 and090 resp) BSA-MGO glycation reversing activity However


100 200 300 400 5000Concentration (gmL)

0

10

20

30

40

50

60

70

in

hibi

tion


Figure 2 Antiglycation activity via BSA-MGO model Data rep-resented as mean plusmn SEM (119899 = 4 each) IC50 values DCM Mleaf ethanol leaf DCM M bark and ethanol bark 27829 plusmn 855c34928 plusmn 821b 35738 plusmn 308b and 39259 plusmn 2088a 120583gmL respec-tively IC50 values superscripted by different letters are significantlydifferent at 119901 lt 005 DCM M leaf ethanol leaf DCM M barkand ethanol bark 1199032 = 095 098 099 and 095 respectively IC50rutin 6335 plusmn 067 120583gmL DCM M dichloromethane methanolMethylgloxal MGO



0

10

20

30

40

50

60

70

80

90

in

hibi

tion


Figure 3 Glycation reversing activity via BSA-glucose model Datarepresented as mean plusmn SEM (119899 = 6 each) EC50 values DCM Mbark ethanol bark ethanol leaf and DCM M leaf 9433 plusmn 181d10716 plusmn 395c 12120 plusmn 201b and 19942 plusmn 902a 120583gmL respec-tively EC50 values superscripted by different letters are significantlydifferent at 119901 lt 005 DCM M bark ethanol bark ethanol leaf andDCM M leaf 1199032 = 096 097 099 and 099 respectively DCM Mdichloromethane methanol


Ethanol barkEthanol leaf

200 400 600 8000Concentration (gmL)

minus5

5

15

25

35

45

55

65

75

85

in

hibi

tion

DCM M leafDCM M bark

Figure 4 Glycation reversing activity via BSA-MGO model Datarepresented as mean plusmn SEM (119899 = 4 each) EC50 values ethanol leafDCM M bark and ethanol bark 12215 plusmn 601c 28780 plusmn 320band 32283 plusmn 176a 120583gmL respectively EC50 values superscriptedby different letters are significantly different at 119901 lt 005 Ethanolleaf ethanol bark and DCM M bark 1199032 = 099 094 and 096respectively DCM M dichloromethane methanol MethylgloxalMGO

Table 5 Total proanthocyanidin content of bark and leaf extracts

Extract mg cyanidinequivalentsg of extract

DCM M bark 138153 plusmn 4593aEthanol bark 109790 plusmn 7301bEthanol leaf 43424 plusmn 1412cDCM M leaf 30952 plusmn 281dData represented as mean plusmn SEM (119899 = 6 each) Mean values in the columnsuperscripted by different letters are significantly different at 119901 lt 005

ethanolic leaf extract showed the highest reversing abilitywhile DCM M extract of leaf showed the lowest reversingactivity The order of potency of BSA-MGO glycation revers-ing activity was ethanol leafgtDCM Mbarkgt ethanol barkgtDCM M leaf Dose-response relationships of ethanolic andDCM Mbark and leaf extracts of Ceylon cinnamon for BSA-MGO glycation reversing are given in Figure 4

36 Total ProanthocyanidinContent Total proanthocyanidincontent of ethanolic and DCM M bark and leaf extracts ofCeylon cinnamon is given in Table 5 Mean total proan-thocyanidin content of bark and leaf extracts of cinna-mon ranged from 30952 plusmn 281 to 138153 plusmn 4593mgcyanidin equivalentsg extract Both bark extracts had sig-nificantly high total proanthocyanidin content (109790 plusmn7301ndash138153 plusmn 4593mg cyanidin equivalentsg extract)than both leaf extracts (30952 plusmn 281ndash43424 plusmn 1412mg

cyanidin equivalentsg extract) (119901 lt 005)The order ofmeantotal proanthocyanidin content was DCM M bark gt ethanolbark gt ethanol leaf gt DCM M leaf

4 Discussion

A range of selected antidiabetic properties [antiamylaseantiglucosidase anticholinesterases antiglycation and gly-cation reversing activities] of alba grade bark and leaf ofCeylon cinnamon were evaluated using well establishedwidely used sensitive specific validated and internationallyaccepted antidiabetic bioassays in vitro [32ndash37] Alba gradebark of Ceylon cinnamon was used since which is the mosthighly priced cinnamon grade in the international trade(due to its finest quill thickness unique aroma and taste)Leaf extracts were also evaluated for antidiabetic relatedproperties as leaf is claimed to have antidiabetic activity in SriLankan traditional knowledge [39] and folklore Ethanol andDCM M bark and leaf extracts were used as these extractshave been previously used in the investigation of antioxidantsand antioxidant activity [40] and antilipidemic activity invitro [16]120572-Amylase and 120572-glucosidases are the key enzymesinvolved in starch digestion process [18] Thus inhibitors ofthese enzymes can play a key role in the management ofdiabetes Both bark (IC50 214 plusmn 2ndash215 plusmn 10 120583gmL) and leaf(IC50 943 plusmn 28 120583gmL) of Ceylon cinnamon showed anti-amylase activity Antiamylase activity of both bark extractswas significantly high compared to both leaf extracts whileit was moderate compared to the reference drug acarbose(IC50 13388 plusmn 44 120583gmL) Previous investigation on 120572-amylase inhibitory activity of bark of some economicallyimportant Cinnamomum species such as C zeylanicum Caromaticum and C loureiroi showed that it had antiamylaseactivity and activity as IC50 values 123 plusmn 002 177 plusmn 005and gt400mgmL respectively [18] According to the aboveresearch bark of C zeylanicum had the highest antiamylaseactivity among the studied economically important Cin-namomum species Further Beejmohun et al [19] reportedantiamylase activity of bark of C zeylanicum as IC50 value25 120583gmL Compared to the above studies antiamylase activ-ity of bark of Ceylon cinnamon was ranging from 6 timeshigher to 25 times lower in the present study The presentstudy was conducted using ethanolic and DCM M extractsof authenticated bark of Ceylon cinnamon and Bacillusamyloliquefaciens 120572-amylase as the source of amylase On theother hand the studies conducted by Adisakwattana et al [18]andBeejmohun et al [19]were usedwater and hydroalcoholicextracts of bark and porcine pancreatic 120572-amylase as thesource of amylase Further the cinnamon samples used inboth studies were not authenticated Therefore discrepancyobserved between present study and previous investigationson antiamylase activity may be due to the use of differentsolvents extraction procedures source of 120572-amylase anduse of cinnamon samples without proper authenticationCompared to bark leaf ofC zeylanicum (C verum)was rarelyinvestigated for antiamylase activity to date Research carriedout by Ponnusamy et al [20] reported that antiamylaseactivity of leaf extract of C verum as IC50 value 1 120583gmL


Compared to above study 120572-amylase inhibitory activityof leaf of Ceylon cinnamon was nearly 950 times lowerin the present study Isopropanol leaf extract and humanpancreatic 120572-amylase were used by Ponnusamy et al [20]to evaluate the antiamylase activity of leaf of C verumTherefore discrepancy observed between present study andstudy conducted by Ponnusamy et al [20] on 120572-amylaseinhibitory activity of leaf may be due to the use of differentsolvents extraction procedures source of 120572-amylase anduse of cinnamon samples without proper authentication Tothe best of our knowledge except our research this is theonly available report on antiamylase activity of leaf of anyCinnamomum species worldwide

A recent study has shown that C zeylanicum barkpossesses 120572-glucosidase inhibitory activity [21] Howeverboth bark and leaf extracts of Ceylon cinnamon did notshow 120572-glucosidase inhibition in the present study at studiedconcentrations The inhibitory activity observed by Ranillaet al [21] was at high concentrations (100 inhibition at25mgmL and 95 inhibition at 05mgmL) and it isbeyond the maximum concentration (400 120583gmL) used inour experiments Nevertheless some recent studies haveshown that Ceylon cinnamon bark has intestinal maltaseand sucrase inhibitory activities [15 18] Besides the barkexcept our research on antiglucosidase activity of leaf ofCeylon cinnamon none of the leaf extracts of Cinnamomumspecies reported to have antiglucosidase activity to dateHowever ability to impair postprandial intestinal glucoseabsorption by inhibiting the activity of enzymes involved incarbohydrate metabolism (120572ndashamylase and 120572ndashglucosidase) byboth bark [15 18 21] and leaf of Ceylon cinnamon in thepresent and previous studies indicates its potential use asfood supplements nutraceuticals and functional foods in themanagement of diabetes and related complications

Prolonged hyperglycemic condition in diabetes patientsinduces formation of AGEs which are positively correlatedwith development and progression of several diabetes com-plications and age related diseases [3ndash7] The process ofAGEs formation through protein glycation is not a singlestep reaction [4 6] This reaction can be broadly dividedinto three stages as early middle and late stage glycation[41] In this study inhibitory activity of Ceylon cinnamon onprotein glycation in BSA-glucose and BSA-MGOmodels rep-resents early and middle stages of protein glycation processrespectively [36] Reversing of already formed AGEs andorcross link braking is another vital approach for attenuationof AGEs related complications [6 42] To date very fewcompounds are known to have AGE cross links breakingcapacity 13-Thiazolium derivatives such as N-phenyl-13-thiazolium bromide (PTB) and N-phenacyl-45-dimethyl-13-thiazolium chloride (alagebrium chloride) are impor-tant protein crosslink breakers However these compoundsreported to have limited efficacy in in vivo studies [6 42]Therefore discovery of inhibitors which can inhibit all stagesof glycation process and reversing of already formed AGEswould offer a potential therapeutic approach for the preven-tion of diabetes complications and AGEs related pathologies

The results demonstrated for the first time that bothbark and leaf of authenticated true Ceylon cinnamon can

inhibit both early and middle stages of protein glycationprocess Inhibition of early stage protein glycation was morepotent (ethanolic leaf extract IC50 1522 plusmn 047 120583gmL)or comparable (bark extracts IC50 1942 plusmn 126ndash2080 plusmn268 120583gmL) to the reference standard rutin (IC50 2188 plusmn282 120583gmL) while it was moderate in middle stage proteinglycation in both bark and leaf extracts Previous investi-gation on antiglycation activity of cinnamon by Peng et al[22] reported that different fractions including ethyl acetateand 1-butanol fractions of water extract of bark of cinnamonhad BSA-glucose glycation inhibition with 662 and 595respectively at a concentration of 200 ppm Further Ho andChang [23] reported that IC50 value of BSA-glucose glycationinhibitory activity of cinnamon as 26 120583gmL Comparedto the above studies the BSA-glucose glycation inhibitoryactivity of bark of authenticated Ceylon cinnamon in thepresent study is similar to IC50 value reported by Ho andChang [23] However the cinnamon samples used in boththe studies were not authenticated and hence contradictoryabout the cinnamon species used in those experiments Onthe other hand antiglycation activity of leaf of C zeylanicumis not reported to date Therefore this is the first study toreport that both bark and leaf extracts of authenticated trueCeylon cinnamon can inhibit both early and middle stages ofprotein glycation process Further both bark and leaf extractsexhibited the glycation reversing ability in both BSA-glucoseand BSA-MGO glycated products and this is the first reportof glycated products reversing ability of any Cinnamomumspecies worldwide The presence of antiglycation potentialof bark and leaf of Ceylon cinnamon indicates its ability toameliorate various diabetes and age related complications

Alzheimerrsquos disease is characterized by inadequate pro-duction of acetylcholine in the brain and recently it isreferred as type 3 diabetes as insulin plays a significant rolein the expression of choline acetyltransferase the enzymeresponsible for the synthesis of acetylcholine [10 11] Bothbark and leaf of Ceylon cinnamon showed moderate AChE(IC50 80488 plusmn 4869ndash96668 plusmn 631 120583gmL) and moderate tohigh BChE inhibitory activities (IC50 2662 plusmn 166ndash34060 plusmn1823 120583gmL) compared to the reference drug galantamine(IC50 AChE 252 plusmn 017 120583gmL BChE 7480 plusmn 353 120583gmL)Both AChE and BChE play an important role in cholinergicsignaling A recent research has shown that reduction ofAChE activity can be compensated by increasing BChEactivity since BChE can even hydrolyze acetylcholine whenAChE levels are depleted in Alzheimerrsquos patients [43 44]Therefore currently BChE inhibitors such as cymserineanalogues and the dual inhibitor of both AChE and BChEsuch as rivastigmine are used therapeutically for treatingAlzheimerrsquos disease and other related dementias [43] AsCeylon cinnamon bark and leaf demonstrated both AChEand BChE inhibitory activities consumption in daily lifecould increase the acetylcholine level and would be beneficialformanagement of Alzheimerrsquos diseaseThis is the first reportof AChE and BChE inhibitory activities of authenticated leafof Ceylon cinnamon worldwide

Several research studies have clearly shown that oxidativestress plays a key role in pathological processes observed indiabetes mellitus [3ndash7] The use of antioxidant therapy has


shown beneficial effects for management of pathologies asso-ciated with oxidative stress in diabetes patients [45] Furtherseveral researches have shown that antioxidants includingphenolic compounds play an important role in mediatingantiglycation activity and inhibitory activity towards amylaseand cholinesterase enzymes [37 46] In our previous studyCeylon cinnamon bark and leaf extracts were shown to havehigh antioxidant activity and phenolic contents [40] Furtherin the present study both bark extracts showed high andboth leaf extracts showed moderate total proanthocyani-dins content Therefore the observed antidiabetic relatedproperties of Ceylon cinnamon may be attributed at leastpartly to phenolic compounds including proanthocyanidinsand other antioxidants present in both bark and leaf Thedifferences observed in studied biological activities in barkand leaf may be ascribed to the differences in compositionand concentration of bioactive compounds present in barkand leaf extracts [20 40]

The present study includes some interesting and impor-tant novel findings such as antiamylase antiglycation andglycation reversing activity of bark and antiglucosidaseantiglycation and glycation reversing ability of leaf of authen-ticated true cinnamon worldwide Further this is the firstcomparative research on bark and leaf of authenticatedCeylon cinnamon for antidiabetic activity (antiamylase andanticholinesterases activities) and its effect onmanagement ofdiabetic complications (antiglycation and glycation reversingactivities) Therefore important findings on antidiabeticrelated properties ofCeylon cinnamonwould help to enhancethe usage among consumers of local and international andit might create a positive financial impact to Sri Lanka ascurrently Ceylon cinnamon is the true cinnamon the worldover and the main contributor of the export earnings fromspices in the country

5 Conclusions

It is concluded that both bark and leaf of Ceylon cinna-mon ldquotrue cinnamonrdquo exhibit antidiabetic related properties(mediated via antiamylase and anticholinesterases) and abil-ity to impair development of diabetic complications due toantiglycation and glycation reversing activities In generalbark showed high antiamylase and antibutyrylcholinesteraseactivities compared to leaf whereas leaf showed high antigly-cation and glycation reversing activities compared to barkThus consumption of Ceylon cinnamon bark and leaf as adietary supplement may play a vital role in the managementof diabetes and its related complications Further mostimportantly findings of this study added value to leaf ofCeylon cinnamon and indicate its potential in developingpromising novel antidiabetic food supplements nutraceu-ticals and functional foods and use in adjuvant therapyin the management of diabetes and related complicationsworldwide

Conflicts of Interest

There are no conflicts of interest in any form between theauthors

Acknowledgments

This work was supported by the Sri Lankan treasury (Grantno TG 1160)

References

[1] International Diabetes FederationDiabetes Atlas InternationalDiabetes Federation Brussels Belgium 6th edition 2014

[2] American Diabetes Association ldquoClassification and diagnosisof diabetesrdquo Diabetes Care vol 38 supplement 1 pp S8ndashS162015

[3] S Brings T Fleming M Freichel M Muckenthaler S Herzigand P Nawroth ldquoDicarbonyls and advanced glycation end-products in the development of diabetic complications andtargets for interventionrdquo International Journal of MolecularSciences vol 18 no 5 p 984 2017

[4] C Henning and M A Glomb ldquoPathways of the Maillard reac-tion under physiological conditionsrdquo Glycoconjugate Journalvol 33 no 4 pp 499ndash512 2016

[5] K Nowotny T Jung A Hohn D Weber and T GruneldquoAdvanced glycation end products and oxidative stress in type 2diabetes mellitusrdquo Biomolecules vol 5 no 1 pp 194ndash222 2015

[6] I Sadowska-Bartosz and G Bartosz ldquoPrevention of proteinglycation by natural compoundsrdquo Molecules vol 20 no 2 pp3309ndash3334 2015

[7] S Dornadula B Elango P Balashanmugam R Palanisamy andR Kunka Mohanram ldquoPathophysiological insights of methyl-glyoxal induced type-2 diabetesrdquo Chemical Research in Toxicol-ogy vol 28 no 9 pp 1666ndash1674 2015

[8] N Rabbani M Xue and P J Thornalley ldquoDicarbonyls andglyoxalase in disease mechanisms and clinical therapeuticsrdquoGlycoconjugate Journal vol 33 no 4 pp 513ndash525 2016

[9] S Y Ko H A Ko and K H Chu ldquoThe possible mechanismof Advanced Glycation End Products (AGEs) for Alzheimerrsquosdiseaserdquo PLoS ONE vol 10 no 11 Article ID e0143345 2015

[10] J W Ashford ldquoTreatment of Alzheimerrsquos disease the legacyof the cholinergic hypothesis neuroplasticity and future direc-tionsrdquo Journal of Alzheimerrsquos Disease vol 47 no 1 pp 149ndash1562015

[11] F G De Felice M V Lourenco and S T Ferreira ldquoHowdoes brain insulin resistance develop in Alzheimerrsquos diseaserdquoAlzheimerrsquos amp Dementia vol 10 no 1 pp S26ndashS32 2014

[12] R Chawla PThakur andA Chowdhry ldquoEvidence based herbaldrug standardization approach in coping with challenges ofholistic management of diabetes a dreadful lifestyle disorder of21st centuryrdquo Journal of DiabetesMetabolic Disorders vol 12 no35 2013

[13] B Bigliardi and F Galati ldquoInnovation trends in the foodindustry the case of functional foodsrdquo Trends in Food Scienceand Technology vol 31 no 2 pp 118ndash129 2013

[14] M Padmavathi ldquoChronic diseasemanagementwith nutraceuti-calsrdquo International Journal of Pharmaceutical Science Inventionvol 2 no 4 pp 1ndash11 2013

[15] P Ranasinghe R Jayawardana P Galappaththy G R Con-stantine N de Vas Gunawardana and P Katulanda ldquoEfficacyand safety of lsquotruersquo cinnamon (Cinnamomum zeylanicum) as apharmaceutical agent in diabetes a systematic review andmeta-analysisrdquo Diabetic Medicine vol 29 no 12 pp 1480ndash1492 2012


[16] W P K M Abeysekera S P G Arachchige and W D Rat-nasooriya ldquoBark extracts of ceylon cinnamon possess antilipi-demic activities and bind bile acids in vitrordquo Evidence-BasedComplementary and Alternative Medicine vol 2017 Article ID7347219 pp 1ndash10 2017

[17] Anonymous AgStat Pocket Book of Agricultural Statistics vol10 Socio Economic and Planning Centre Department ofAgriculture Peradeniya Sri Lanka 2013

[18] S Adisakwattana O Lerdsuwankij U Poputtachai AMinipun and C Suparpprom ldquoInhibitory activity of cinnamonbark species and their combination effect with acarbose againstintestinal 120572-glucosidase and pancreatic 120572-amylaserdquo Plant Foodsfor Human Nutrition vol 66 no 2 pp 143ndash148 2011

[19] V Beejmohun M Peytavy-Izard C Mignon et al ldquoAcuteeffect of Ceylon cinnamon extract on postprandial glycemiaalpha-amylase inhibition starch tolerance test in rats andrandomized crossover clinical trial in healthy volunteersrdquo BMCComplementary and Alternative Medicine vol 14 no 1 article351 2014

[20] S Ponnusamy R Ravindran S Zinjarde S Bhargava and A RKumar ldquoEvaluation of traditional Indian antidiabeticmedicinalplants for human pancreatic amylase inhibitory effect in vitrordquoEvidence-Based Complementary and Alternative Medicine vol2011 Article ID 515647 pp 1ndash10 2011

[21] L G Ranilla Y-I Kwon E Apostolidis and K Shetty ldquoPhe-nolic compounds antioxidant activity and in vitro inhibitorypotential against key enzymes relevant for hyperglycemia andhypertension of commonly used medicinal plants herbs andspices in Latin Americardquo Bioresource Technology vol 101 no12 pp 4676ndash4689 2010

[22] X Peng K-W Cheng J Ma et al ldquoCinnamon bark proantho-cyanidins as reactive carbonyl scavengers to prevent the forma-tion of advanced glycation endproductsrdquo Journal of Agriculturaland Food Chemistry vol 56 no 6 pp 1907ndash1911 2008

[23] S Ho and P Chang ldquoInhibitory effects of several spiceson inflammation caused by advanced glycation endproductsrdquoAmerican Journal of Plant Sciences vol 3 no 7A Article ID21047 pp 995ndash1002 2012

[24] S Kumar Brijeshlata and S Dixit ldquoScreening of traditionalindian spices for inhibitory activity of acetylcholinesteraseand butyrylcholinesterase enzymesrdquo International Journal ofPharma and Bio Sciences vol 3 no 1 pp P59ndashP65 2012

[25] M K Dalai S Bhadra S K Chaudhary J Chanda A Bandy-opadhyay and P K Mukherjee ldquoAnticholinesterase activityof Cinnamomum zeylanicum L leaf extractrdquo Tang [HumanitasMedicine] vol 4 no 2 pp 111-116 2014

[26] R A Anderson and A M Roussel ldquoCinnamon glucose andinsulin sensitivityrdquo in Nutraceuticals Glycemic Health and Type2 Diabetes V Pasupuleti and J W Anderson Eds pp 127ndash140IFT press series Wiley-Blackwell Publishing 2008

[27] K N Prasad B Yang X Dong et al ldquoFlavonoid contents andantioxidant activities from Cinnamomum speciesrdquo InnovativeFood Science and Emerging Technologies vol 10 no 4 pp 627ndash632 2009

[28] P Chen J Sun and P Ford ldquoDifferentiation of the four majorspecies of cinnamons (C burmannii C verum C cassia andC loureiroi) using a flow injection mass spectrometric (FIMS)fingerprintingmethodrdquo Journal of Agricultural and Food Chem-istry vol 62 no 12 pp 2516ndash2521 2014

[29] P Ranasinghe S Perera M Gunatilake et al ldquoEffects of Cin-namomum zeylanicum (Ceylon cinnamon) on blood glucose

and lipids in a diabetic and healthy rat modelrdquo PharmacognosyResearch vol 4 no 2 pp 73ndash79 2012

[30] M Tailang B K Gupta andA Sharma ldquoAntidiabetic activity ofalcoholic extract ofCinnamomum zeylanicum Leaves in alloxoninduced diabetic ratsrdquo Peoples Journal of Scientific Research vol1 pp 9ndash11 2008

[31] SLSI Specification for Ceylon Cinnamon Sri Lanka Standardsvol 29 Sri Lanka Standards Institution (SLSI) Colombo SriLanka 4th edition 2010

[32] P Bernfeld ldquoAmylases alpha and betardquo inMethods in Enzymol-ogy S P Colowick and N O Kaplan Eds pp 149ndash158 NewYork Academic Press New York NY USA 1955

[33] T Matsui T Ueda T Oki K Sugita N Terahara and K J Mat-sumoto ldquoAlpha-glucosidas inhibition isolated anthocyaninsrdquoJournal of Agricultural and Food Chemistry vol 49 pp 1948ndash1951 2001

[34] G L Ellman K D Courtney V Andres Jr and R M Feather-stone ldquoA new and rapid colorimetric determination of acetyl-cholinesterase activityrdquo Biochemical Pharmacology vol 7 no2 pp 88ndash95 1961

[35] N Matsuura T Aradate C Sasaki et al ldquoScreening system forthe Maillard reaction inhibitor from natural product extractsrdquoJournal of Health Science vol 48 no 6 pp 520ndash526 2002

[36] N Lunceford and A Gugliucci ldquoIlex paraguariensis extractsinhibit AGE formation more efficiently than green teardquo Fitoter-apia vol 76 no 5 pp 419ndash427 2005

[37] G A S Premakumara W K S M Abeysekera W D Ratna-sooriya N V Chandrasekharan and A P Bentota ldquoAntioxi-dant anti-amylase and anti-glycation potential of brans of someSri Lankan traditional and improved rice (Oryza sativa L)varietiesrdquo Journal of Cereal Science vol 58 no 3 pp 451ndash4562013

[38] L J Porter L N Hrstich and B G Chan ldquoThe conversionof procyanidins and prodelphinidins to cyanidin and delphini-dinrdquo Phytochemistry vol 25 no 1 pp 223ndash230 1985

[39] Anonymous Ayurveda Pharmacopoeia vol 1 Part III Depart-ment of Ayurveda Colombo Sri Lanka 1976

[40] W P K M Abeysekera G A S Premakumara and W DRatnasooriya ldquoIn vitro antioxidant properties of bark andleaf extracts of Ceylon Cinnamon (Cinnamomum zeylanicumBlume)rdquo Tropical Agricultural Research vol 24 no 2 pp 128ndash138 2013

[41] V P Singh A Bali N Singh and A S Jaggi ldquoAdvancedglycation end products and diabetic complicationsrdquoTheKoreanJournal of Physiology and Pharmacology vol 18 no 1 pp 1ndash142014

[42] R Nagai D B Murray T O Metz and J W Baynes ldquoChela-tion A fundamental mechanism of action of AGE inhibitorsAGE breakers and other inhibitors of diabetes complicationsrdquoDiabetes vol 61 no 3 pp 549ndash559 2012

[43] M Bajda A Wieckowska M Hebda N Guzior C A Sotrifferand B Malawska ldquoStructure-based search for new inhibitors ofcholinesterasesrdquo International Journal ofMolecular Sciences vol14 no 3 pp 5608ndash5632 2013

[44] G F Makhaeva S V Lushchekina N P Boltneva et alldquoConjugates of 120574-Carbolines and phenothiazine as new selectiveinhibitors of butyrylcholinesterase and blockers of NMDAreceptors for Alzheimer diseaserdquo Scientific Reports vol 5Article ID 13164 2015


[45] S R Zatalia and H Sanusi ldquoThe role of antioxidants in thepathophysiology complications and management of diabetesmellitusrdquo Acta medica Indonesiana vol 45 no 2 pp 141ndash1472013

[46] D Szwajgier ldquoAnticholinesterase activity of selected phenolicacids and flavonoidsmdashinteraction testing in model solutionsrdquoAnnals of Agricultural and Environmental Medicine vol 22 no4 pp 690ndash694 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


several age related diseases such as Alzheimerrsquos diseaseatherosclerosis arthritis pulmonary fibrosis renal failureand cancer [3ndash7] Accumulation of AGEs in the brain isinvolved in extensive protein cross linking oxidative stressandneuronal cell death leading to neurodegenerative diseasesand most commonly Alzheimerrsquos disease [6 9] CurrentlyAlzheimerrsquos disease is even referred to as type 3 diabetesas it can be explained through AGEs and oxidation [9]and insulin and the cholinergic hypothesis [10 11] Naturalproducts reported to have antidiabetic activity all over theworld for centuries [12] Antidiabetic drugs nutraceuticalsand functional foods derived from plant sources have highdemand as they are natural and safe alternatives tomany syn-thetic drugs [13 14] Cinnamon one of the oldest and mostfrequently consumed spices worldwide belongs to the genusCinnamomum and there are different species of cinnamonworldwide [15 16] Among several species of cinnamon in theworld Ceylon cinnamon is the ldquotrue cinnamonrdquo the worldover based on its unique taste aroma and phytochemicalcomposition [15 16] Currently Sri Lanka is the leadingexporter of true cinnamon with 85 of world market shareand 145 market share for all types of cinnamon worldwideAccording to the recent statistics nearly 50 of exportearnings of minor agricultural crops in the country comefrom Ceylon cinnamon [17]

Cinnamon is reported to have several pharmacologi-cal activities including some antidiabetic related propertiesworldwide [15 16 18 26 27] However main problem inmany of these publications that there is no proper authen-tication for the experimental cinnamon sample [15] Hencethere is no strong evidence to confirm that these reportedbiological activities are from authenticated Ceylon cinnamon(true cinnamon) since the genus contains four economicallyimportant cinnamon species such as Cinnamomum zeylan-icum or Cinnamomum verum (Ceylon cinnamon or truecinnamon)Cinnamomumaromaticum (Cinnamomumcassiaor Chinese cinnamon) Cinnamomum burmannii (KorintjeJava or Indonesian cinnamon) and Cinnamomum loureiroi(Vietnamese or Saigon cinnamon) [28] On the other handwithin the country there are no in-depth studies on antidi-abetic activity of authenticated Ceylon cinnamon (exceptRanasinghe et al [29]) even though it is the most economicalminor agricultural crop in Sri Lanka Further the studiesconductedworldwide so far on antidiabetic activity of Ceyloncinnamon (true cinnamon) mainly focused on bark extractsand only 3 studies [20 25 30] are available on antidiabeticactivity of leaf extracts to date Further as yet there are nopublish studies on antiamylase antiglycation and glycationreversing activities of bark and antiglucosidase antiglycationand glycation reversing potential of leaf of authenticated Cey-lon cinnamon (true cinnamon) worldwide Previous investi-gations on antiamylase antiglucosidase anticholinesterasesantiglycation and glycation reversing activities of bark andleaf of Cinnamomum species are given in Table 1 The aimof this study was to evaluate antiamylase antiglucosidaseanticholinesterases antiglycation and glycation reversingpotential of both bark and leaf of authenticated Ceyloncinnamon viawidely used well established sensitive specificreliable and reproducible in vitro bioassays

2 Materials and Methods

21 Chemicals and Reagents Soluble starch bovine serumalbumin (BSA) D-glucose 120572-glucosidase (type V from rice)p-nitrophenyl 120572-D-glucopyranoside acarbose trichloro-acetic acid (TCA) acetylcholinesterase (AChE) from electriceel (Type-VI-S) butyrylcholinesterase (BChE) from horseserum acetylthiocholine butyrylthiocholine 551015840-dithio-bis-(2-nitrobenzoic) acid (DTNB) methylglyoxal (MGO) 35-dinitrosalicylic acid (DNS) dimethyl sulfoxide (DMSO)galantamine rutin cyanidin chloride and ammoniumiron(III) sulfate dodecahydrate were purchased from Sigma-Aldrich USA 120572-Amylase (Bacillus amyloliquefaciens) waspurchased from Roche Diagnostics USA All the otherchemicals and reagents were of analytical grade

22 Collection and Preparation of Ceylon Cinnamon AlbaGrade Bark and Leaf Samples Fresh cinnamon leaves werecollected from cinnamon cultivations of LB spices (Pvt)Ltd Aluthwala Galle Sri Lanka Alba grade cinnamon barksamples (alba grade cinnamon has the lowest quill thicknessmaximum 6mm according to the grading of cinnamonquills based on the quill thickness) [31] were collected fromcinnamon factories of LB spices (Pvt) Ltd AluthwalaGalle Sri Lanka and G P De Silva and Sons Spice (Pvt)Ltd Ambalangoda Sri Lanka The alba grade bark sampleswere authenticated by Dr Chandima Wijesiriwardena Prin-ciple Research Scientist Industrial Technology Institute SriLanka and leaf samples (voucher number CZB-KA) wereauthenticated byMr NPT Gunawardena Officer In-ChargeNational Herbarium Department of National Botanic Gar-dens Peradeniya Sri Lanka The specimens of each bark andleaf samples (HTS-CIN-1) and photographic evidence weredeposited at the Pharmacognosy Laboratory Herbal Tech-nology Section Industrial Technology Institute Sri LankaFresh leaves were air-dried at room temperature (30 plusmn 2∘C)for 7 days The air-dried leaves and bark were groundpowdered and stored at minus20∘C until used for the extraction

23 Preparation of Extracts

231 Preparation of Ethanolic Extracts Powdered bark andleaf samples (20 g) were extracted in 200mL of 95 ethanolfor 4-5 h in a Soxhlet extractor (4ndash6 cycles) until the solventbecame colorless The extracts were filtered evaporatedand freeze-dried (Christ-Alpha 1ndash4 Freeze dryer BiotechInternational Germany) Freeze-dried extracts were stored atminus20∘C until used for analysis

232 Preparation of Dichloromethane Methanol (DCM M)Extracts Powdered bark and leaf (20 g) samples wereextracted in 200mL of dichloromethane methanol(DCM M) at a ratio of (1 1 vv) at room temperature(30 plusmn 2∘C) for 7 days with occasional shaking The extractswere filtered evaporated freeze-dried and stored at minus20∘Cuntil used for analysis

24 Antiamylase Activity The antiamylase activity of barkand leaf extracts of Ceylon cinnamon were carried out


Table1Antidiabetic

activ

ityof

Cinn

amom

umspeciesinvitro

Cinn

amom

umspecies

Partusedextract

Activ

ityRe

ferences

Antia

mylasea

ctivity

Czeylanicu

mlowast

Bark

aqueou

sextract

IC50123plusmn0

02m

gmL

Adisa

kwattana

etal2011[18]

Carom

aticu

m(cassia

)lowastBa

rkaqueou

sextract

IC50177plusmn

005

mgmL

Adisa

kwattana

etal2011[18]

Cloureir

oi(Saigon

cinn

amon

)lowastBa

rkaqueou

sextract

IC50gt4

00m

gmL

Adisa

kwattana

etal2011[18]

Czeylanicu

m(C

verum)lowast

Bark

hydroalcoho

licextract

(50

50vvw

ater

ethano

l)IC5025120583g

mL

Beejmoh

unetal2014

[19]

Cverum

Isop

ropano

lleafextract

IC501120583g

mL

Ponn

usam

yetal2011[20]

Czeylanicu

mlowast

Bark

aqueou

sextract

77inhibitio

nat25

mgmL72inhibitio

nat125mgmLand

51inhibitio

nat5m

gmL

Ranilla

etal2010

[21]

Antiglucosid

asea

ctivitym

altaseandsucraseinh

ibition

Czeylanicu

mlowast

Bark

aqueou

sextract

100

and95inhibitio

nat25and05m

gmLrespectiv

elyRa

nilla

etal2010

[21]

Czeylanicu

mlowast

Bark

aqueou

sextract

IC50120583g

mL077plusmn0

04maltase042plusmn0

02sucrase

Adisa

kwattana

etal2011[18]

Carom

aticu

m(cassia

)lowastBa

rkaqueou

sextract

IC50120583g

mL085plusmn0

04maltase088plusmn0

33sucrase

Adisa

kwattana

etal2011[18]

Cloureir

oi(Saigon

cinn

amon

)lowastBa

rkaqueou

sextract

IC50120583g

mL096plusmn0

03maltasegt4

00sucrase

Adisa

kwattana

etal2011[18]

Antiglyc

ationactiv

ity

Cinn

amon

(Cinna

mom

umspeciesu

sedno

tmentio

ned)lowast

Ethylacetateandbu

tano

lsolub

lefractio

nsof

bark

water

extract

dilutedwith

ethano

l

BSA-

glucosea

ntiglycatio

nethylacetates

olub

lefractio

nsgt4

0tolt90

inhibitio

nat200p


0togt80

inhibitio

nat200p

pmBS

A-MGOantig

lycatio


0tolt80

inhibitio

nat25m

gmLbu

tano

lsolub

lefractio

ns

lt10to

nearly40inhibitio

nat25m

gmL

Peng

etal2008

[22]

Cverumlowast

Bark

methano

lextract

BSA-

glucosea

ntiglycatio

nIC5026120583g

mL

HoandCh

ang2012

[23]

AChE

andBC

hEinhibitory

activ

ity

Czeylanicu

mBa

rkethano

lextract

AChE

4083plusmn0

005

inhibitio

nat100120583g

mLBC

hE515

3plusmn

0005inhibitio

nat100120583g

mL

Kumar

etal2012

[24]

Czeylanicu

mMethano

licleafextract

IC50A

ChE7778plusmn0

03120583g

mLBC

hE8862plusmn1

72120583g

mL

Dalaietal2014

[25]

lowastEx

perim

entalcinnamon

sampleh

asno

authentic

ation

AChE

acetylch

olinesteraseB

ChE

butyrylcho

linesterase



























3 Results
















Table4Anticho

linesterasesa

ctivity

Extract

inhibitio

n120583g

mL

Con

centratio

n(120583g

mL)

50100

200

400

800

IC50


inhibitory

activ

ity

Ethano

lbark

1046plusmn2

132868plusmn1

973011plusmn2

373690plusmn2

20

5213plusmn0

48

80488plusmn4

869

b

DCM

Mbark

1977plusmn2

163234plusmn0

61

3788plusmn0

1140

26plusmn0

99

4769plusmn1

0596668plusmn6

318

a

Ethano

lleaf

684plusmn1

441344plusmn2

523008plusmn0

08

3504plusmn1

5946

33plusmn3

75

81096plusmn7

998a

DCM

Mleaf

minus1066plusmn3

65

403plusmn1

371493plusmn2

313434plusmn1

584913plusmn0

63

8793

5plusmn6

800

a

Bark

625

125

2550

100

IC50

Butyrylch

olinesterase

inhibitory

activ

ityEthano

l850plusmn2

94

1717plusmn3

69

4214plusmn3

01

6606plusmn1

197510plusmn0

75

3609plusmn0

83c

DCM

M90

6plusmn2

98

3679plusmn2

69

5060plusmn4

42

6716plusmn0

92

7897plusmn1

682662plusmn1

66d

Leaf

2550

100

200

400

IC50

Butyrylch

olinesterase

inhibitory

activ

ityEthano

l542plusmn4

07

2584plusmn2

20

3528plusmn1

144262plusmn2

66

4922plusmn1

8134060plusmn18

23a

DCM

M657plusmn2

122295plusmn2

03

3745plusmn0

38

4228plusmn0

63

5748plusmn1

812619

6plusmn19

56b

Datar

epresented

asmeanplusmnSE


inthec

olum

nsuperscriptedby

different

lette

rsaresignificantly

different


alanalysiswas

carriedou

tseparately

fora

cetylch

oline

esteraseandbu

tyrylch

olinee

steraseinhibitory

assaysIC 50galantam

ineacetylcholinee

steraseinhibitory

activ


inebu

tyrylch

olinee

steraseinhibitory

activ


Ethano

lbarkDCM

Mbarkethanolleafand

DCM

Mleaf1199032

=097097094and

098respectively

for

butyrylch

olinee

sterase

inhibitory

activ

ityE

thanolbarkD

CMM

barkethanolleafand

DCM

Mleaf

1199032=092094095and

099respectively

for

acetylcholinee

sterase

inhibitory

activ

ityD

CMM

dichlorom

ethane

methano

l




minus5

5

15

25

35

45

55

65

75

85

95

in

hibi

tion









0

10

20

30

40

50

60

70

in

hibi

tion





0

10

20

30

40

50

60

70

80

90

in

hibi

tion






minus5

5

15

25

35

45

55

65

75

85

in

hibi

tion









4 Discussion













5 Conclusions




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table1Antidiabetic

activ

ityof

Cinn

amom

umspeciesinvitro

Cinn

amom

umspecies

Partusedextract

Activ

ityRe

ferences

Antia

mylasea

ctivity

Czeylanicu

mlowast

Bark

aqueou

sextract

IC50123plusmn0

02m

gmL

Adisa

kwattana

etal2011[18]

Carom

aticu

m(cassia

)lowastBa

rkaqueou

sextract

IC50177plusmn

005

mgmL

Adisa

kwattana

etal2011[18]

Cloureir

oi(Saigon

cinn

amon

)lowastBa

rkaqueou

sextract

IC50gt4

00m

gmL

Adisa

kwattana

etal2011[18]

Czeylanicu

m(C

verum)lowast

Bark

hydroalcoho

licextract

(50

50vvw

ater

ethano

l)IC5025120583g

mL

Beejmoh

unetal2014

[19]

Cverum

Isop

ropano

lleafextract

IC501120583g

mL

Ponn

usam

yetal2011[20]

Czeylanicu

mlowast

Bark

aqueou

sextract

77inhibitio

nat25

mgmL72inhibitio

nat125mgmLand

51inhibitio

nat5m

gmL

Ranilla

etal2010

[21]

Antiglucosid

asea

ctivitym

altaseandsucraseinh

ibition

Czeylanicu

mlowast

Bark

aqueou

sextract

100

and95inhibitio

nat25and05m

gmLrespectiv

elyRa

nilla

etal2010

[21]

Czeylanicu

mlowast

Bark

aqueou

sextract

IC50120583g

mL077plusmn0

04maltase042plusmn0

02sucrase

Adisa

kwattana

etal2011[18]

Carom

aticu

m(cassia

)lowastBa

rkaqueou

sextract

IC50120583g

mL085plusmn0

04maltase088plusmn0

33sucrase

Adisa

kwattana

etal2011[18]

Cloureir

oi(Saigon

cinn

amon

)lowastBa

rkaqueou

sextract

IC50120583g

mL096plusmn0

03maltasegt4

00sucrase

Adisa

kwattana

etal2011[18]

Antiglyc

ationactiv

ity

Cinn

amon

(Cinna

mom

umspeciesu

sedno

tmentio

ned)lowast

Ethylacetateandbu

tano

lsolub

lefractio

nsof

bark

water

extract

dilutedwith

ethano

l

BSA-

glucosea

ntiglycatio

nethylacetates

olub

lefractio

nsgt4

0tolt90

inhibitio

nat200p


0togt80

inhibitio

nat200p

pmBS

A-MGOantig

lycatio


0tolt80

inhibitio

nat25m

gmLbu

tano

lsolub

lefractio

ns

lt10to

nearly40inhibitio

nat25m

gmL

Peng

etal2008

[22]

Cverumlowast

Bark

methano

lextract

BSA-

glucosea

ntiglycatio

nIC5026120583g

mL

HoandCh

ang2012

[23]

AChE

andBC

hEinhibitory

activ

ity

Czeylanicu

mBa

rkethano

lextract

AChE

4083plusmn0

005

inhibitio

nat100120583g

mLBC

hE515

3plusmn

0005inhibitio

nat100120583g

mL

Kumar

etal2012

[24]

Czeylanicu

mMethano

licleafextract

IC50A

ChE7778plusmn0

03120583g

mLBC

hE8862plusmn1

72120583g

mL

Dalaietal2014

[25]

lowastEx

perim

entalcinnamon

sampleh

asno

authentic

ation

AChE

acetylch

olinesteraseB

ChE

butyrylcho

linesterase



























3 Results
















Table4Anticho

linesterasesa

ctivity

Extract

inhibitio

n120583g

mL

Con

centratio

n(120583g

mL)

50100

200

400

800

IC50


inhibitory

activ

ity

Ethano

lbark

1046plusmn2

132868plusmn1

973011plusmn2

373690plusmn2

20

5213plusmn0

48

80488plusmn4

869

b

DCM

Mbark

1977plusmn2

163234plusmn0

61

3788plusmn0

1140

26plusmn0

99

4769plusmn1

0596668plusmn6

318

a

Ethano

lleaf

684plusmn1

441344plusmn2

523008plusmn0

08

3504plusmn1

5946

33plusmn3

75

81096plusmn7

998a

DCM

Mleaf

minus1066plusmn3

65

403plusmn1

371493plusmn2

313434plusmn1

584913plusmn0

63

8793

5plusmn6

800

a

Bark

625

125

2550

100

IC50

Butyrylch

olinesterase

inhibitory

activ

ityEthano

l850plusmn2

94

1717plusmn3

69

4214plusmn3

01

6606plusmn1

197510plusmn0

75

3609plusmn0

83c

DCM

M90

6plusmn2

98

3679plusmn2

69

5060plusmn4

42

6716plusmn0

92

7897plusmn1

682662plusmn1

66d

Leaf

2550

100

200

400

IC50

Butyrylch

olinesterase

inhibitory

activ

ityEthano

l542plusmn4

07

2584plusmn2

20

3528plusmn1

144262plusmn2

66

4922plusmn1

8134060plusmn18

23a

DCM

M657plusmn2

122295plusmn2

03

3745plusmn0

38

4228plusmn0

63

5748plusmn1

812619

6plusmn19

56b

Datar

epresented

asmeanplusmnSE


inthec

olum

nsuperscriptedby

different

lette

rsaresignificantly

different


alanalysiswas

carriedou

tseparately

fora

cetylch

oline

esteraseandbu

tyrylch

olinee

steraseinhibitory

assaysIC 50galantam

ineacetylcholinee

steraseinhibitory

activ


inebu

tyrylch

olinee

steraseinhibitory

activ


Ethano

lbarkDCM

Mbarkethanolleafand

DCM

Mleaf1199032

=097097094and

098respectively

for

butyrylch

olinee

sterase

inhibitory

activ

ityE

thanolbarkD

CMM

barkethanolleafand

DCM

Mleaf

1199032=092094095and

099respectively

for

acetylcholinee

sterase

inhibitory

activ

ityD

CMM

dichlorom

ethane

methano

l




minus5

5

15

25

35

45

55

65

75

85

95

in

hibi

tion









0

10

20

30

40

50

60

70

in

hibi

tion





0

10

20

30

40

50

60

70

80

90

in

hibi

tion






minus5

5

15

25

35

45

55

65

75

85

in

hibi

tion









4 Discussion













5 Conclusions




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of










































3 Results
















Table4Anticho

linesterasesa

ctivity

Extract

inhibitio

n120583g

mL

Con

centratio

n(120583g

mL)

50100

200

400

800

IC50


inhibitory

activ

ity

Ethano

lbark

1046plusmn2

132868plusmn1

973011plusmn2

373690plusmn2

20

5213plusmn0

48

80488plusmn4

869

b

DCM

Mbark

1977plusmn2

163234plusmn0

61

3788plusmn0

1140

26plusmn0

99

4769plusmn1

0596668plusmn6

318

a

Ethano

lleaf

684plusmn1

441344plusmn2

523008plusmn0

08

3504plusmn1

5946

33plusmn3

75

81096plusmn7

998a

DCM

Mleaf

minus1066plusmn3

65

403plusmn1

371493plusmn2

313434plusmn1

584913plusmn0

63

8793

5plusmn6

800

a

Bark

625

125

2550

100

IC50

Butyrylch

olinesterase

inhibitory

activ

ityEthano

l850plusmn2

94

1717plusmn3

69

4214plusmn3

01

6606plusmn1

197510plusmn0

75

3609plusmn0

83c

DCM

M90

6plusmn2

98

3679plusmn2

69

5060plusmn4

42

6716plusmn0

92

7897plusmn1

682662plusmn1

66d

Leaf

2550

100

200

400

IC50

Butyrylch

olinesterase

inhibitory

activ

ityEthano

l542plusmn4

07

2584plusmn2

20

3528plusmn1

144262plusmn2

66

4922plusmn1

8134060plusmn18

23a

DCM

M657plusmn2

122295plusmn2

03

3745plusmn0

38

4228plusmn0

63

5748plusmn1

812619

6plusmn19

56b

Datar

epresented

asmeanplusmnSE


inthec

olum

nsuperscriptedby

different

lette

rsaresignificantly

different


alanalysiswas

carriedou

tseparately

fora

cetylch

oline

esteraseandbu

tyrylch

olinee

steraseinhibitory

assaysIC 50galantam

ineacetylcholinee

steraseinhibitory

activ


inebu

tyrylch

olinee

steraseinhibitory

activ


Ethano

lbarkDCM

Mbarkethanolleafand

DCM

Mleaf1199032

=097097094and

098respectively

for

butyrylch

olinee

sterase

inhibitory

activ

ityE

thanolbarkD

CMM

barkethanolleafand

DCM

Mleaf

1199032=092094095and

099respectively

for

acetylcholinee

sterase

inhibitory

activ

ityD

CMM

dichlorom

ethane

methano

l




minus5

5

15

25

35

45

55

65

75

85

95

in

hibi

tion









0

10

20

30

40

50

60

70

in

hibi

tion





0

10

20

30

40

50

60

70

80

90

in

hibi

tion






minus5

5

15

25

35

45

55

65

75

85

in

hibi

tion









4 Discussion













5 Conclusions




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























Table4Anticho

linesterasesa

ctivity

Extract

inhibitio

n120583g

mL

Con

centratio

n(120583g

mL)

50100

200

400

800

IC50


inhibitory

activ

ity

Ethano

lbark

1046plusmn2

132868plusmn1

973011plusmn2

373690plusmn2

20

5213plusmn0

48

80488plusmn4

869

b

DCM

Mbark

1977plusmn2

163234plusmn0

61

3788plusmn0

1140

26plusmn0

99

4769plusmn1

0596668plusmn6

318

a

Ethano

lleaf

684plusmn1

441344plusmn2

523008plusmn0

08

3504plusmn1

5946

33plusmn3

75

81096plusmn7

998a

DCM

Mleaf

minus1066plusmn3

65

403plusmn1

371493plusmn2

313434plusmn1

584913plusmn0

63

8793

5plusmn6

800

a

Bark

625

125

2550

100

IC50

Butyrylch

olinesterase

inhibitory

activ

ityEthano

l850plusmn2

94

1717plusmn3

69

4214plusmn3

01

6606plusmn1

197510plusmn0

75

3609plusmn0

83c

DCM

M90

6plusmn2

98

3679plusmn2

69

5060plusmn4

42

6716plusmn0

92

7897plusmn1

682662plusmn1

66d

Leaf

2550

100

200

400

IC50

Butyrylch

olinesterase

inhibitory

activ

ityEthano

l542plusmn4

07

2584plusmn2

20

3528plusmn1

144262plusmn2

66

4922plusmn1

8134060plusmn18

23a

DCM

M657plusmn2

122295plusmn2

03

3745plusmn0

38

4228plusmn0

63

5748plusmn1

812619

6plusmn19

56b

Datar

epresented

asmeanplusmnSE


inthec

olum

nsuperscriptedby

different

lette

rsaresignificantly

different


alanalysiswas

carriedou

tseparately

fora

cetylch

oline

esteraseandbu

tyrylch

olinee

steraseinhibitory

assaysIC 50galantam

ineacetylcholinee

steraseinhibitory

activ


inebu

tyrylch

olinee

steraseinhibitory

activ


Ethano

lbarkDCM

Mbarkethanolleafand

DCM

Mleaf1199032

=097097094and

098respectively

for

butyrylch

olinee

sterase

inhibitory

activ

ityE

thanolbarkD

CMM

barkethanolleafand

DCM

Mleaf

1199032=092094095and

099respectively

for

acetylcholinee

sterase

inhibitory

activ

ityD

CMM

dichlorom

ethane

methano

l




minus5

5

15

25

35

45

55

65

75

85

95

in

hibi

tion









0

10

20

30

40

50

60

70

in

hibi

tion





0

10

20

30

40

50

60

70

80

90

in

hibi

tion






minus5

5

15

25

35

45

55

65

75

85

in

hibi

tion









4 Discussion













5 Conclusions




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















minus5

5

15

25

35

45

55

65

75

85

95

in

hibi

tion









0

10

20

30

40

50

60

70

in

hibi

tion





0

10

20

30

40

50

60

70

80

90

in

hibi

tion






minus5

5

15

25

35

45

55

65

75

85

in

hibi

tion









4 Discussion













5 Conclusions




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















minus5

5

15

25

35

45

55

65

75

85

in

hibi

tion









4 Discussion













5 Conclusions




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























5 Conclusions




Acknowledgments


References























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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