Research ArticleInhibitory Potential of Turbinaria ornata againstKey Metabolic Enzymes Linked to Diabetes

P S Unnikrishnan K Suthindhiran and M A Jayasri

Marine Biotechnology and Biomedicine Laboratory School of Biosciences and Technology VIT University Vellore 632007 India

Correspondence should be addressed to M A Jayasri jayasrimavitacin

Received 28 February 2014 Revised 4 June 2014 Accepted 5 June 2014 Published 24 June 2014

Academic Editor Yoshifumi Saisho

Copyright copy 2014 P S Unnikrishnan et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

One of the therapeutic approaches in treating diabetes is to reduce postprandial hyperglycemia by inhibiting major carbohydratehydrolyzing enzymes In the present study crude extracts of marine seaweed Turbinaria ornata were tested for their antidiabeticpotential using enzyme inhibitory assays (120572-amylase 120572-glucosidase and dipeptidyl peptidase-IV) Among the tested extractsmethanol and acetone extracts showed significant inhibitory effects on 120572-amylase (IC

502509 120583gmL)120572-glucosidase (5356 120583gmL)

and dipeptidyl peptidase-4 (552 120583gmL) respectively Free radical scavenging activity of these extracts was analyzed using DPPHassay (65) Extracts were tested for in vitro toxicity using DNA fragmentation assay haemolytic assay and MTT assay Noneof the extracts showed toxicity in tested models Furthermore GC-MS analysis of lead extracts showed the presence of majorcompounds hentriacontane z z-6 28-heptatriactontadien-2-one 8-heptadecene and 1-heptacosanol Our findings suggest thatTurbinaria ornata can be used as a potential source for further in vivo studies in controlling hyperglycemia

1 Introduction

Diabetes mellitus is a metabolic disorder which affects theendocrine system of the body characterized by defects incarbohydrate lipid and protein metabolism [1] Globally thefrequency of disorder is rising gradually patients sufferingfrom this disorder are unable to turn out or respond properlyto insulin produced in the body [2] Control of postprandialhyperglycemia prevents chronic vascular complications andit is a widely accepted goal for the management of type-2diabetes [3] 120572-Amylase and 120572-glucosidase are the two majorenzymes which play a key role in the digestion of carbo-hydrates by hydrolysis of inner 120572-1 4-glucosidic linkages instarch and several other polysaccharides and the uptake ofglucose by intestinal 120572-glucosidases [4] One of the effectivestrategies for the management of diabetes is by retardingthe starch digestion rate by inhibiting these enzymes leadingto the reduction of postprandial hyperglycemia in diabeticsubjects [5] A recent approach for the development of anantidiabetic drug is based on the development of incretinanalogues and dipeptidyl peptidase-IV (DPP-IV) inhibitorswhich play a crucial role in the glucose homeostasis by

promoting 120572 and 120573 cell function [6] It also downregulatesthe gastric emptying and gastric acid secretion to reducethe postprandial glucose level [7 8] Currently there areseveral commercially availableDPP-IV inhibitors (sitagliptinsaxagliptin and vildagliptin) to block DPP-IV action therebyprolonging the half-life and biological activity of incretinhormones [9] On the other hand some pharmacokineticsstudies reveal that DPP-IV inhibitors mainly vildagliptinare not safe for the patients with severe liver problems [1011] Similarly other synthetic hypoglycemic agents (acarboseand voglibose) that inhibit 120572-amylase and 120572-glucosidase cancause hepatic and gastrointestinal disorders [12] Experimen-tal and clinical evidences show that hyperglycemia increasesthe production of free radicals and induces oxidative stressleading to various diseases like diabetes mellitus cardiac dis-orders inflammatory and neurodegenerative diseases [13ndash15] Hence there is a need for new safe and efficaciousantioxidant and antidiabetic agents

Natural sources such as marine flora and fauna bacteriafungi and higher plants are of new interest in the develop-ment of antidiabetic agents Among them marine seaweedsrepresent one of the richest sources of bioactive compounds

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 783895 10 pageshttpdxdoiorg1011552014783895

2 BioMed Research International

which are widely used in pharmaceutical research [16ndash18] Polyphenols and sulfated polysaccharides present inseaweeds have been proven for antiviral antitumoral anti-inflammatory and anticoagulant activity [17ndash19] Seaweedscontain prolific source of bioactive compounds which canbe exploited for the treatment of major chronic diseases likediabetes through the inhibition of starch digesting enzymesand the regulation of glucose induced oxidative stress [20 21]

In the present study antidiabetic and antioxidant effect offive different solvent extracts of marine seaweed Turbinariaornata has been evaluated T ornata a wide spread speciesbelonging to Phaeophyceae is rich in fucoids and sulphatedpolysaccharides [22 23] It has been previously reported forits antioxidant anti-inflammatory and cytotoxicity activityon murine melanoma and colon cancer cells [23ndash25] Tothe best of our knowledge this is the first report on Tornata elucidating three different antidiabetic mechanisms(120572-amylase 120572-glucosidase and DPP-IV) in vitro and theextracts were also tested for their in vitro toxicity

2 Materials and Methods

21 Chemicals Porcine pancreatic 120572-amylase dinitrosali-cylic acid (DNSA) acarbose p-nitrophenyl 120572-D-gluco-pyranoside diprotin A (Ile-Pro-Ile) DPP-IV from porcinekidney and Gly-pro-p-nitroanilide (GPPN) were purchasedfrom Sigma Bangalore India DPPH butylated hydroxytoluene (BHT) was obtained from Himedia Mumbai 120572-Glucosidase anddimethyl sulphoxide (DMSO)were obtainedfrom SRL Mumbai

22 Collection and Processing of Seaweed Fresh brown sea-weeds were collected from Mandapam coastal region (Lat-itude 9∘1710158401015840N Longitude 79∘1110158401015840E) Gulf of Mannar Tamil-nadu South India on low tide during October 2012 Thevoucher specimen was deposited in the Marine Biotech-nology and Biomedicine Laboratory VIT University Theseaweeds were cleaned and holdfasts were removed andstored at minus20∘C immediately Before extraction they wereshade-dried powdered and stored in air-tight containers atminus20∘C

23 Preparation of Seaweed Extracts The dried seaweedsamples (25 g) were milled and extracted using 250mL ofvarious solvents such as petroleum ether benzene ethylacetate acetone and methanol for 24 h by using Soxhletapparatus Each filtrate was concentrated to dryness underreduced pressure using rotary evaporator (Super Fit Rotavapmodel PBU-6 India)The samples were lyophilized by usingfreeze dryer (Lark Penguin Classic Plus India) and storedin a refrigerator at 2ndash8∘C for use in subsequent experimentsPrior to experiments various concentrations were preparedfrom 250 to 1000 120583gmL

24 Preliminary Phytochemical Screening Preliminary phy-tochemical screenings were carried out as per the standardprotocols of Harborne [26]

25 22-Diphenyl-1-picrylhydrazyl (DPPH)Radical ScavengingActivity Free radical scavenging activity was determinedaccording to the method of Mensor et al [27] Briefly 500120583Lof 03mM alcoholic solution of DPPH (Himedia India) wasadded to 25mL of test samples at varying concentrations(250ndash1000120583gmL) After incubation of samples in the darkfor 30 minutes the optical density was measured at 518 nmin the UV-visible spectrophotometer (Systronics AU-2700India) using methanol as blank and synthetic antioxidantbutylated hydroxy toluene (BHT) as positive control Theexperiments were performed in triplicates and scavengingactivity was expressed as percentage inhibition using thefollowing formula

Inhibition = [(Abscontrol minus Abssamples)

Abscontrol] times 100 (1)

26 In Vitro 120572-Amylase Inhibition Assay The 120572-amylaseinhibitory activity was determined as described in Jayasriet al [28] with minor modifications Briefly 250 120583L of algalextracts with varying concentrations (250ndash1000120583gmL) and250 120583L of 002M sodium phosphate buffer (pH 69 with0006M sodium chloride) containing 120572-amylase (porcinepancreatic 120572-amylase Sigma St Louis USA) solution(05mgmL) were incubated for 10min at 25∘C After prein-cubation 250120583L of 1 starch solution in 002M sodiumphosphate buffer (pH 69 with 0006M sodium chloride) wasadded to each tube at 5 sec intervals The reaction mixtureswere then incubated at 25∘C for 10min The reaction wasstopped with 500 120583L dinitrosalicylic acid (Sigma St LouisUSA) colour reagent The tubes were then incubated in aboiling water bath for 5min and cooled to room temperatureThe reaction mixture was then diluted by adding 5mL ofdistilled water and absorbance was measured at 540 nmin the UV-visible spectrophotometer (Systronics AU-2700India) The experiments were performed in triplicate and the120572-amylase inhibitory activity was calculated as percentageinhibition using the formula

Inhibition = [(Abscontrol minus Abssamples)

Abscontrol] times 100 (2)

27 In Vitro 120572-Glucosidase Inhibition Assay The 120572-gluco-sidase inhibitory activity was determined as described byJayasri et al [28] with slight modifications Briefly 50120583L ofalgal extracts with varying concentrations (250ndash1000 120583gmL)and 100 120583L of 01M phosphate buffer (pH-69) containing 120572-glucosidase (SRL India) solution (10UmL) were incubatedin 96-well plates at 25∘C for 10 minutes After preincubation50 120583L of 5mM p-nitrophenyl 120572-D-glucopyranoside (SigmaSt Louis USA) in 01M phosphate buffer (pH-69) wasadded to each well at 5 sec intervals The reaction mixturewas then incubated at 25∘C for 5min After incubationabsorbance readings were recorded at 405 nm using a platereader (Bio-TEK USA) and compared to the control whichcontained 50 120583L of buffer solution in place of algal extractThe experiments were performed in triplicate and the 120572-glucosidase inhibitory activity was calculated as percentage

BioMed Research International 3

inhibition The percentage of enzyme inhibition was calcu-lated as follows

Inhibition = [(Abscontrol minus Abssamples)

Abscontrol] times 100 (3)

28 In Vitro Dipeptidyl Peptidase-IV (DPP-IV) InhibitionAssay DPP-IV inhibitory activity was determined accordingto the method of Al-Masri et al [29] Standard diprotin A(Sigma St Louis USA) was diluted to various concentrations(02 04 08 16 32 and 64 120583gmL) using Tris-HCl buffer(50mM pH 75) and the final volume was made to 35 120583Land DPP-IV enzyme (15 120583L) (Sigma St Louis USA) wasadded to the above mixture One unit of enzyme activity wasdefined as the amount of enzyme that catalyzes the releaseof 1 120583mol para-nitroaniline (pNA) from the substrateminunder assay conditions After adding the enzyme themixturewas preincubated for 10min at 37∘C to enhance the bindingcapacity of the inhibitorThis was followed by the addition of50120583L of (02mM) Gly-pro-p-nitroanilide (Sigma St LouisUSA) in Tris-HCl as a substrate Final incubation was done at37∘C for 30min and the reaction was terminated by additionof 25 120583L of 25 glacial acetic acid The absorbance wasmeasured at 405 nm using microtiter plate reader (Bio-TEKUSA) Experiments were done in triplicate and the resultsobtained were compared with negative control

29 DPP-IV Inhibition Assay of Seaweed Extract Seaweedextracts were dissolved in dimethyl sulphoxide to makea stock concentration of 1000 120583gmL From the stock thefollowing concentrations were prepared (25 10 40 and80 120583gmL) in Tris-HCl buffer (50mM pH 75) in a totalvolume of 100 120583Lwell The assay was performed in triplicateaccording to standardized procedure of diprotin A Thepercentage of DPP-IV inhibition was calculated as follows

Inhibition = [(Abscontrol minus Abssamples)

Abscontrol] times 100 (4)

210 Maintenance of Cell Line J774 cell line (mouse macro-phage) was obtained from National Centre for Cell SciencePune India The cells were grown in Dulbeccorsquos ModifiedEaglersquos Medium (DMEM) supplemented with 10 (vv) heat-inactivated foetal bovine serum (FBS) 2mM l-glutamineand 100UmL of penicillinstreptomycin with 5 CO

2at

37∘C in a humidified incubator Exponentially growing cellswere used for the experiment

211 Cytotoxicity Assay Briefly cells were seeded at a densityof 5 times 105 cellsmL and allowed to attach for 24 h in 300120583Lof medium incubated at 37∘C and 5 CO

2 After 24 h

seaweed extracts were added to the medium at variousconcentrations (1000ndash250120583gmL) and incubated for 24 hAt the end of treatment cells were incubated with MTT[3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bro-mide] (5mgmL) for 4 h The formazan crystals formedwere dissolved inDMSO (dimethyl sulfoxide) after aspirating

the medium The extent of cytotoxicity was measured spec-trophotometrically at 630 nm with a microplate reader (Bio-TEK USA)

212 DNA Fragmentation Assay Acridine orange-ethidiumbromide staining was used to characterize the morphologicalchanges and apoptotic body formation assessed by fluores-cent microscopy Briefly J774 cells (5 times 105) were seeded onsterile glass cover slips in six-well tissue culture plates andtreated with test extracts (1000 120583gmL) for 24 h The mediumwas removed and washed twice with PBS (phosphate-buffered saline) Cells grown on cover slips were loaded withacridine orange-ethidium bromide (4 120583gmL) of ratio (1 1)and incubated for 15min at 37∘C in the dark Cells wereviewed under Weswox LED fluorescent microscope (FM-3000) attached with a camera and photographs were takenunder 40 and 100x magnification at fluorescent conditions

213 Haemolytic Assay The seaweed extracts were evaluatedfor their haemolytic activity as described by Malagoli [30]The blood sample was collected from a healthy volunteer(A+ve) using ethylene diamine tetra acetic acid (EDTA) as ananticoagulant Whole blood was washed thrice in 9 volumesof sterile 09 NaCl saline solution after each washing thecells were centrifuged (150timesg for 5min) and the supernatantwas discardedThe final pellet obtained was diluted 19 (vv)in sterile 09 NaCl saline solution and then 124 (vv) insterile Dulbeccorsquos phosphate buffer saline (D-PBS) pH 70containing 05mM boric acid and 1mM calcium chlorideThe haemolytic activity of seaweed extracts was tested underin vitro conditions in 96-well plates Briefly eachwell received100 120583L of 085 NaCl solution containing 10mM CaCl

2

100 120583L of normal saline served as negative control 01Triton X-100 as a positive control and test extracts of variousconcentrations (250ndash1000120583gmL) were added to each wellThen eachwell received 100 120583L of human erythrocytes dilutedin D-PBS After 30min incubation at room temperaturethe samples were centrifuged and supernatant was used tomeasure the absorbance of liberated haemoglobin at 630 nm[31]

214 GCMSAnalysis GCMS (Perkin Elmer Clarus 680GCcoupled to a Clarus 600 MS) analysis was performed for thedetection of major compounds present in various extracts ofT ornata Column used in GCMS was Elite-5MS (300m025mmID and 250 120583mdf) Carrier gas used was helium ata constant flow rate of 1mLmin EI mode with electronenergy set at 70 eV was used 1 120583L of extract diluted withmethanol was injected into GCMS and the compounds wereidentified based on the molecular structure molecular massand calculated fragment ratio of resolved spectra with thatof mass spectra available from the library Spectral data wereinterpreted using the database of National Institute StandardAnd Technology (NIST)

215 Statistical Analysis All the data reportedwere expressedas mean plusmn SEM Statistical analysis was performed using

4 BioMed Research International

(a) (b)

Figure 1 (a)Macroscopic view of Turbinaria ornata collected fromMandapam coastal region in Gulf ofMannar Tamilnadu India Stiff erectstalks hard thick leaves (blades) characterized by lateral ridges and outermarginal blade with stiff row of spines are the unique characteristicsof this species (b) Microscopic image of Turbinaria ornata (magnification 40x)

Table 1 Qualitative phytochemical screening of various extracts of Turbinaria ornata (+ present minus absent)

Phytochemicals Pet ether Benzene Ethyl acetate Methanol AcetoneAlkaloids + + + + +Phenols + + + + +Flavanoids + + + + +Proteins and amino acid + + + + +Lipids + + + + +Carbohydrates + + + + +Saponins minus minus + minus minus

Glycosides minus minus + + minus

Tannins + + + minus minus

Oils and fats + minus minus minus minus

two-way ANOVA The values were considered to be signif-icantly different when the 119875 value was lt00001 compared tothe baseline values Software employed for statistical analysiswas Graph-Pad Prism Version 5

3 Results

31 Identification of Collected Seaweeds The samples wereidentified based on the morphological characteristics inresponse to environmental conditions stiff erect stalks hardthick leaves (blades) characterized by lateral ridges and outermarginal bladewith stiff rowof spines (Figure 1) Based on theabove characters seaweed species was identified as T ornataand authenticated byDr P Kaladharan principle scientist andscientist in charge Calicut Regional Centre of CentralMarineFisheries Research Institute

32 Phytochemical Analysis of T ornata Qualitative anal-ysis of the crude extracts of T ornata showed the pres-ence of major phytochemicals like alkaloids phenols fla-vanoids proteins lipids carbohydrates glycosides tanninsand saponins in all the studied extracts (Table 1)

33 22-Diphenyl-1-picrylhydrazyl (DPPH)Radical ScavengingActivity The antioxidant potential of T ornata was studiedthrough its scavenging ability of the stable radical DPPH

Acetone extract showed significant scavenging ability onDPPH (65) at a concentration of 1000120583gmL when com-pared with that of a standard BHT (97) However none ofthe extracts exhibited higher activity than BHT at the sameconcentration (Figure 2)

34 In Vitro 120572-Amylase Inhibition Assay Inhibitory effect ofT ornata on 120572-amylase is shown in Figure 3(a) Among thefive extracts studied (petroleum ether benzene ethyl acetatemethanol and acetone) three extracts showed (petroleumether methanol and acetone) significant 120572-amylase inhi-bition at all the tested concentrations (250 500 750 and1000 120583gmL) Methanol extract of T ornata showed max-imum inhibition of 965 with an IC

50of 2509 120583gmL

followed by acetone extract (876) at a concentration of1000 120583gmL

35 In Vitro 120572-Glucosidase Inhibition Assay The 120572-gluco-sidase inhibitory activity of T Ornata extracts was deter-mined using p-nitrophenyl 120572-D-glucopyranoside as a sub-strate and these were compared with a standard acarbose(88) (Figure 3(b)) At a concentration of 1000 120583gmL ace-tone extract showed maximum inhibition (876) with anIC50value of 5356 120583gmL The inhibitory activity of acetone

extracts was found to be more potent and similar whencompared to the positive control acarbose

BioMed Research International 5

0

50

250 500 750 1000

100

150

Petroleum etherBenzeneEthyl acetate

MethanolAcetoneBHT

Concentration (120583gmL)

Inhi

bitio

n (

)

a

a

a

aa

a

a a a a

a

aaa a a

a

a

a

aa

a

a

a

Figure 2 DPPH radical scavenging activity of petroleum etherbenzene ethyl acetatemethanol and benzene extracts ofTurbinariaornata BHT is used as positive control and absorbance wasmeasured at 517 nm Values are means plusmn SD (119899 = 3) (a119875 lt 00001considered as significant)

36 In Vitro Dipeptidyl Peptidase-IV (DPP-IV) InhibitionAssay T ornata extracts were evaluated for their mode ofaction to stimulate insulin secretion through inhibition ofDPP-IV enzyme The effectiveness of various extracts wasevaluated on the basis of percentage inhibition and IC

50

values obtained Among the five different solvent extracts(petroleum ether benzene ethyl acetate methanol andacetone) methanol extracts showed maximum percentageinhibition of 554 with an IC

50value of 552120583gmL at a

maximum concentration of 80 120583gmL All the results werecompared with a standard diprotin A (Figure 4)

37 Cytotoxic Effect of T ornata Extracts on J774 Cell Line Toevaluate the cytotoxic effect of the extracts J774 cell line wasincubated for 24 h with various concentrations of extracts Asshown in Figure 5(a) the decrease in viability correlates withthe increase in concentration MTT assay indicates that J774cells treated with various extracts of T ornata were safe anddid not show any toxic effect against the cell treated at a lowerconcentration of 250120583gmL The observed cytotoxicity wasfound to be 257 when treated with methanol extract of Tornata 50 of cells were found to be viable when treatedwitha higher concentration of 1000120583gmL

38 DNA Fragmentation Assay Staining cells with acridineorange-ethidium bromide is used to measure the proportionof viable apoptotic and necrotic cells J774 cells treated witha maximum concentration of 1000120583gmL of algal extractswere found to be viable and there was no change in cellularmorphology including chromatin condensation membraneblebbing and fragmented nuclei (Figure 6)

39 Haemolytic Assay The lead extracts methanol (278)and acetone (461) were found to be nontoxic at a dosageof 1000120583gmL (Figure 5(b)) Also the extracts did not showany erythrocyte membrane damage at all the tested concen-trations

310 GC-MS Analysis of the Acetone Extract of T ornataThe crude extracts were analyzed by GC-MS and comparedwith the retention time (RT) and mass spectra availablein the data library of NIST The GC-MS analysis revealsthe presence of major bioactive compounds like hentriacon-tane z z-6 28-heptatriactontadien-2-one 8-heptadeceneand 1-heptacosanol Chromatograms obtained were shown inFigure 7

4 Discussion

Marine seaweeds are good source of dietary fibres polysac-charides polyphenols polyunsaturated fatty acids mineralsand vitamins which possess various biological activities likeantidiabetic anti-inflammatory and antioxidant activity [32]Although this evidence seems to be valid detailed researchshould be carried out to ensure the presence of various phy-tochemicals present in the seaweed extracts In the currentstudy marine seaweed T ornata was extracted and screenedfor both nonpolar and polar compounds using varioussolvents based on polarity Qualitative analysis showed thepresence of major bioactive compounds like alkaloids phe-nols flavanoids glycosides lipids carbohydrates proteinstannins oils and fats Presence of these bioactive moleculesin various extracts of this seaweed may be responsible forvarious biological activities (antiviral anti-inflammatory andanticoagulant) [17ndash19]

The objective of this study was to investigate the antidia-betic and antioxidant potential of marine seaweed T ornataand to enumerate major phytocompounds present in theextracts One of the major causes for diabetes is due tothe increased production of reactive oxygen species orimpaired antioxidant defense systemwhich leads to oxidativedamage of beta cells [33] In this study acetone extract ofT ornata showed significant scavenging capability againstDPPH (65) at a maximum concentration of 1000 120583gmLcompared with a positive control BHT (97) As previouslyreported by Ananthi et al and Chattopadhyay et al [2325] the fucoidan fraction of Turbinaria conoides (61) ata concentration of 1000 120583gmL and aqueous extract of Tornata (80) at a concentration of 500 120583gmL showed thesimilar scavenging capability on DPPH radicals Our resultswere similar to these reports which confirm the antioxidantactivity of T ornata

In the present study the edible seaweed T ornata wasevaluated for in vitro 120572-amylase 120572-glucosidase and DPP-IV inhibitory property Among the five extracts studiedpetroleum ether acetone and methanol extracts showedsignificant percentage inhibition Methanol extract of Tornata showed maximum percentage inhibition on 120572-amylase (965)with an IC

50value of 2509120583gmLwithmore

inhibition than positive control acarbose (88) Acetone

6 BioMed Research International

0

50

100

150In

hibi

tion

()

Petroleum etherMethanol

AcetoneAcarbose

a

aa

a

a

a

a

a a

a

aa a

a a a

250 500 750 1000Concentration (120583gmL)

IC50 value of methanol extract 2509 120583gmL

(a)

0

20

40

60

80

100

Petroleum etherMethanol

AcetoneAcarbose

Inhi

bitio

n (

)

a a

a

a

aa

a

aa

a

a

aa

a

a

a

250 500 750 1000Concentration (120583gmL)

IC50 value of acetone extract 5356 120583gmL

(b)

Figure 3 (a) In vitro 120572-amylase inhibitory activity of petroleum ether methanol and acetone extracts of Turbinaria ornata Absorbance wasmeasured at 540 nm (b) In vitro 120572-glucosidase inhibitory activity of petroleum ether methanol and acetone extracts of Turbinaria ornataacarbose is used as positive control and absorbance was measured at 405 nm Values are means plusmn SD (119899 = 3) (a119875 lt 00001 consideredsignificant)

3002 04 08 16 32 64

40

50

60

70

Inhi

bitio

n (

)

aa

a

a

a

a

Concentration (120583gmL)

(a)

025 10 40 80

20

40

60

Inhi

bitio

n (

) a a a a aa

a

a

a aa

a

aa

a a

aa

a a

Concentration (120583gmL)

Petroleum etherBenzeneAcetone

Ethyl acetateMethanol

IC50 value of methanol extract 552 120583gmL

(b)

Figure 4 (a) DPP-IV inhibitory activity of diprotin A as positive control (b) DPP-IV inhibitory activity of petroleum ether benzene ethylacetate methanol and acetone extracts of Turbinaria ornata Absorbance was measured at 405 nm Values are means plusmn SD (119899 = 3) (a119875 lt00001 considered significant)

extract of T ornata showed more potent inhibition on120572-glucosidase (843) with an IC

50value of 5356 120583gmL

at a concentration of 1000120583gmL similar to the positivecontrol acarbose (88) The DPP-IV inhibitory potential ofmethanol extract of T ornata showed (554) inhibitionwith an IC

50value of 552120583gmL which is comparable with

that of positive control diprotin A (65) Similar findingswere shown by two brown seaweeds Ecklonia stoloniferaOkumura and Eisenia bicyclis [34] Several studies reportedthat the 120572-amylase and 120572-glucosidase inhibitory action of theclass Phaeophyceae [35 36] supports that these algal speciesare excellent source for antidiabetic agents Apart from 120572-amylase and 120572-glucosidase T ornata extracts also showedsignificant DPP-IV inhibition Based on these results this

seaweed can be used as alternative therapy for diabetes byraising the levels of endogenous incretin hormones (GLP-1and GIP)

In this study we also investigated the effects of crudeextracts (methanol acetone) of T ornata in macrophagecell line (J774) The cytotoxic effects of the extracts weredetermined by using anMTT assay After 24 h exposure 250ndash1000 120583gmL of methanol and acetone extracts significantlydecreased cell viability by increasing concentration As pre-viously reported seaweed extracts which have higher phenolcontent may explain the level of cytotoxicity at higher con-centrations (1000 120583gmL) [36 37] whereby up to 500120583gmLof seaweed extracts did not show any effect on J774 cellviability andmore than 90of cells were found to be viable at

BioMed Research International 7

0

50

100

150C

ell v

iabi

lity

() a a

aa

aa a

a

250 750 1000500

MethanolAcetone

Concentration (120583gmL)

(a)

0250 750 1000500

2

4

6

MethanolAcetone

Cell

lysis

()

a a a

a

a

a

a

a

Concentration (120583gmL)

(b)

Figure 5 (a) Cell viability determined byMTT assay J774 cells treated with methanol and acetone extracts of Turbinaria ornata Absorbancewasmeasured at 630 nm (b)Haemolytic activity ofmethanol and acetone extracts ofTurbinaria ornata Absorbance wasmeasured at 630 nmValues are means plusmn SD (119899 = 3) (a119875 lt 00001 considered significant)

Control

(a)

TO M

(b)

Control

(c)

Treated

(d)

Control

(e)

Treated

(f)

Figure 6 (a) Fluorescent photomicrographs (100x magnification) of J774 cells treated without seaweed extracts (b) J774 cells treated withTurbinaria ornatamethanol extract (TOM) (c) Phase contrast microscopic image of J774 cells treated with media alone (control) (d) Phasecontrast microscopic image of J774 cells treated with test extracts ((e)-(f)) Microscopic image (40x magnification) of in vitro haemolyticactivity

8 BioMed Research International

A

R3A-(13IS-1200)

B

500

100(

)

01000 1500 2000 2500 3000

Time

515e8TIC

Scan El+

OH

317

0308

130

28

294

528

36

277

627

17

264

3257

525

05

243

924

33

236

423

59

228

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017

64

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015

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141

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3

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A

B

5111

31

33333337030

333

8130

3333333333

2829

45

28

22222222236

277

627

22222

1726

4325

75

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524

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71764

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516

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16

110

151

214

18

164

0

18

188181818181881881881888888818881881888811111143444444444444

C

Figure 7 Gas chromatogram of acetone extract of Turbinaria ornata (A) 8-heptadecene (B) 1-heptacosanol and (C) hentriacontane

a concentration of 250120583gmL Staining cells with fluorescentdyes is used to differentiate the nuclear morphology of liveand apoptotic cells J774 cells were analyzed in the presenceof acridine orange and ethidium bromide to determinethe nuclear morphology of viable and necrotic cells Cellsstained green represent viable cells and ethidium bromideselectively stains the cells that have lost membrane integrityand stains DNA orange In this study acridine orange-ethidium bromide staining revealed that there was no changein cellular morphology including chromatin condensationmembrane blebbing and fragmented nuclei occurring dueto the treatment of extracts even at higher concentration(1000 120583gmL) Our results indicate that the tested extracts didnot induce haemolysis at a concentration of (1000120583gmL)Based on the above results the methanol and acetone extractsof T ornata were found to be nontoxic under in vitroconditions

GC-MS analysis revealed the presence of major bioac-tive compounds like hentriacontane z z-6 28-heptatriact-ontadien-2-one 8-heptadecene and 1-heptacosanol Hentri-acontane is the major component present in Oldenlandiadiffusa a natural source for the treatment of cancer in Asia[38] and also has reported activities like anti-inflammatoryand antioxidant properties [38 39] Currently there is noinformation regarding the antidiabetic effects of hentria-contane Further purification and characterization of activecompounds along with mechanism studies will only giveconclusive data on the antidiabetic property of tested algae

5 Conclusions

The present study indicates that crude extracts of T ornatapossess in vitro 120572-amylase 120572-glucosidase and DPP-IV

inhibitory activity Due to their strong enzyme inhibitoryactivity they are expected to suppress the glycemic responsein diabetic conditions Additionally the extracts are capableof scavenging free radicals in vitro toxicological parametersindicate that the IC

50of methanol and acetone extracts of T

ornata for 120572-amylase 120572-glucosidase and DPP-IV inhibitionare greatly below cytotoxic levels Future studies on in vivoantidiabetic activity of selected extracts and identification ofmajor compounds responsible for antidiabetic action are inprogress These studies will reveal the possible antidiabeticmechanisms of T ornata

Conflict of Interests

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

Acknowledgments

The authors wish to thank Department of BiotechnologyGovernment of India for their financial support (Grantno BTBio-CARe033472010-11) and VIT University forproviding all necessary facilities

References

[1] K G Alberti and P Z Zimmet ldquoDefinition diagnosis andclassification of diabetes mellitus and its complications Part1 diagnosis and classification of diabetes mellitus provisionalreport of a who consultationrdquo Diabetic Medicine vol 15 no 7pp 539ndash553 1998

[2] O P Gupta and S Phatak ldquoPandemic trends in prevalenceof diabetes mellitus and associated coronary heart disease in

BioMed Research International 9

India-their causes and preventionrdquo International Journal ofDiabetes in Developing Countries vol 23 pp 37ndash50 2003

[3] R R Ortiz-Andrade S Garcıa-Jimenez P Castillo-EspanaG Ramırez-Avila R Villalobos-Molina and S Estrada-Sotoldquo120572-Glucosidase inhibitory activity of the methanolic extractfrom Tournefortia hartwegiana an anti-hyperglycemic agentrdquoJournal of Ethnopharmacology vol 109 no 1 pp 48ndash53 2007

[4] G M Gray ldquoCarbohydrate digestion and absorption Role ofthe small intestinerdquo The New England Journal of Medicine vol292 no 23 pp 1225ndash1230 1975

[5] C A Tarling K Woods R Zhang et al ldquoThe search fornovel human pancreatic 120572-amylase inhibitors high-throughputscreening of terrestrial and marine natural product extractsrdquoChemBioChem vol 9 no 3 pp 433ndash438 2008

[6] R Mentlein B Gallwitz and W E Schmidt ldquoDipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide gluca-gon-like peptide-1(7-36)amide peptide histidine methionineand is responsible for their degradation in human serumrdquoEuropean Journal of Biochemistry vol 214 no 3 pp 829ndash8351993

[7] T Vilsboslashll T Krarup C F Deacon S Madsbad and J J HolstldquoReduced postprandial concentrations of intact biologicallyactive glucagon-like peptide 1 in type 2 diabetic patientsrdquoDiabetes vol 50 no 3 pp 609ndash613 2001

[8] M A Nauck U Niedereichholz R Ettler et al ldquoGlucagon-like peptide 1 inhibition of gastric emptying outweighs itsinsulinotropic effects in healthy humansrdquo American Journal ofPhysiologymdashEndocrinology and Metabolism vol 273 no 5 ppE981ndashE988 1997

[9] A-M Lambeir S Scharpe and I DeMeester ldquoDPP4 inhibitorsfor diabetesmdashwhat nextrdquo Biochemical Pharmacology vol 76no 12 pp 1637ndash1643 2008

[10] [EMEA] EuropeanMedicines Agency ldquoEuropean public assess-ment report (EPAR)mdashJanuviardquo 2007 httpwwwemaeuropaeuema

[11] [EMEA] EuropeanMedicines Agency ldquoEuropean public assess-ment report (EPAR)mdashGalvusrdquo 2007 httpwwwemaeuropaeuema

[12] A Murai K Iwamura M Takada K Ogawa T Usui andJ-I Okumura ldquoControl of postprandial hyperglycaemia bygalactosyl maltobionolactone and its novel anti-amylase effectin micerdquo Life Sciences vol 71 no 12 pp 1405ndash1415 2002

[13] B Halliwell and O I Aruoma ldquoDNA damage by oxygen-derived species Its mechanism and measurement in mam-malian systemsrdquo FEBS Letters vol 281 no 1-2 pp 9ndash19 1991

[14] J Zhou N Hu Y-L Wu Y-J Pan and C-R Sun ldquoPreliminarystudies on the chemical characterization and antioxidant prop-erties of acidic polysaccharides from Sargassum fusiformerdquoJournal of Zhejiang University Science B vol 9 no 9 pp 721ndash727 2008

[15] D A Butterfield A Castenga C B Pocernich J DrakeG Scapagnini and V Calabrese ldquoNutritional approaches tocombat oxidative stress in Alzheimerrsquos diseasesrdquo Journal ofNutritional Biochemistry vol 13 pp 444ndash461 2002

[16] A M S Mayer and V K B Lehmann ldquoMarine pharmacologyin 1998 marine compounds with antibacterial anticoagu-lant antifungal anti-inflammatory anthelmintic antiplateletantiprotozoal and antiviral activities with actions on the cardio-vascular endocrine immune and nervous systems and othermiscellaneous mechanisms of actionrdquo Pharmacologist vol 42pp 62ndash69 2000

[17] A Cumashi N A Ushakova M E Preobrazhenskaya et alldquoA comparative study of the anti-inflammatory anticoagulantantiangiogenic and antiadhesive activities of nine differentfucoidans from brown seaweedsrdquoGlycobiology vol 17 no 5 pp541ndash552 2007

[18] T Ghosh K Chattopadhyay M Marschall P Karmakar PMandal and B Ray ldquoFocus on antivirally active sulfatedpolysaccharides from structure-activity analysis to clinicalevaluationrdquo Glycobiology vol 19 no 1 pp 2ndash15 2009

[19] V H Pomin and P A S Mourao ldquoStructure biology evolutionand medical importance of sulfated fucans and galactansrdquoGlycobiology vol 18 no 12 pp 1016ndash1027 2008

[20] S-H Lee J-S Han S-J Heo J-Y Hwang and Y-J JeonldquoProtective effects of dieckol isolated fromEcklonia cava againsthigh glucose-induced oxidative stress in human umbilical veinendothelial cellsrdquo Toxicology in Vitro vol 24 no 2 pp 375ndash3812010

[21] S-H Lee F Karadeniz M-M Kim and S-K Kim ldquo120572-Glucosidase and 120572-amylase inhibitory activities of phlorogluci-nal derivatives from edible marine brown alga Ecklonia cavardquoJournal of the Science of Food and Agriculture vol 89 no 9 pp1552ndash1558 2009

[22] A Rohfritsch C Payri V Stiger and F Bonhomme ldquoMolec-ular and morphological relationships between two closelyrelated speciesTurbinaria ornata andT conoides (SargassaceaePhaeophyceae)rdquo Biochemical Systematics and Ecology vol 35no 2 pp 91ndash98 2007

[23] S Ananthi H R B Raghavendran A G Sunil V Gayathri GRamakrishnan and H R Vasanthi ldquoIn vitro antioxidant and invivo anti-inflammatory potential of crude polysaccharide fromTurbinaria ornata (Marine Brown Alga)rdquo Food and ChemicalToxicology vol 48 no 1 pp 187ndash192 2010

[24] F Asari T Kusumi and H Kakisawa ldquoTurbinaric acid acytotoxic secosqualene carboxylic acid from the brown algaTurbinaria ornatardquo Journal of Natural Products vol 52 no 5pp 1167ndash1169 1989

[25] N Chattopadhyay T Ghosh S Sinha K ChattopadhyayP Karmakar and B Ray ldquoPolysaccharides from Turbinariaconoides structural features and antioxidant capacityrdquo FoodChemistry vol 118 no 3 pp 823ndash829 2010

[26] J B Harborne ldquoMethods of extraction and isolationrdquo inPhytochemical Methods vol 3 pp 60ndash66 Chapman amp Hall1998

[27] L L Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plant extracts for antioxidant activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[28] M A Jayasri A Radha and T L Mathew ldquo120572-amylase and120572-glucosidase inhibitory activity of Costus pictus D Don inthe management of diabetesrdquo Journal of Herbal Medicine andToxicology vol 3 pp 91ndash94 2009

[29] IM Al-MasriM KMohammad andMO Tahaa ldquoInhibitionof dipeptidyl peptidase IV (DPP IV) is one of the mechanismsexplaining the hypoglycemic effect of berberinerdquo Journal ofEnzyme Inhibition and Medicinal Chemistry vol 24 no 5 pp1061ndash1066 2009

[30] D Malagoli ldquoA full-length protocol to test hemolytic activityof palytoxin on human erythrocytesrdquo Invertebrate SurvivalJournal vol 4 pp 92ndash94 2007

[31] V R Muzykantov G P Samokhin M D Smirnov andS P Domogatsky ldquoHemolytic complement activity assay in

10 BioMed Research International

microtitration platesrdquo Journal of Applied Biochemistry vol 7 no3 pp 223ndash227 1985

[32] A-R Kim T-S Shin M-S Lee et al ldquoIsolation and identifi-cation of phlorotannins from ecklonia stolonifera with antioxi-dant and anti-inflammatory propertiesrdquo Journal of Agriculturaland Food Chemistry vol 57 no 9 pp 3483ndash3489 2009

[33] J W Baynes ldquoChemical modification of proteins by lipids indiabetesrdquo Clinical Chemistry and Laboratory Medicine vol 41no 9 pp 1159ndash1165 2003

[34] H E Moon M N Islam B R Ahn et al ldquoProtein tyrosinephosphatase 1B and 120572-glucosidase inhibitory phlorotanninsfrom edible brown algae Ecklonia stolonifera and Eiseniabicyclisrdquo Bioscience Biotechnology and Biochemistry vol 75 no8 pp 1472ndash1480 2011

[35] E Apostolidis P D Karayannakidis Y-I Kwon C M Leeand N P Seeram ldquoSeasonal variation of phenolic antioxidant-mediated a-glucosidase inhibition of Ascophyllum nodosumrdquoPlant Foods forHumanNutrition vol 66 no 4 pp 313ndash319 2011

[36] F Nwosu J Morris V A Lund D Stewart H A Ross andG J McDougall ldquoAnti-proliferative and potential anti-diabeticeffects of phenolic-rich extracts from edible marine algaerdquo FoodChemistry vol 126 no 3 pp 1006ndash1012 2011

[37] G Galati and P J OBrien ldquoPotential toxicity of flavonoids andother dietary phenolics significance for their chemopreventiveand anticancer propertiesrdquo Free Radical Biology and Medicinevol 37 no 3 pp 287ndash303 2004

[38] S-J KimW-S Chung S-S Kim S-G Ko and J-Y Um ldquoAnti-inflammatory effect of Oldenlandia diffusa and its constituenthentriacontane through suppression of caspase-1 activation inmouse peritoneal macrophagesrdquo Phytotherapy Research vol 25no 10 pp 1537ndash1546 2011

[39] G Agoramoorthy M Chandrasekaran V Venkatesalu andM J Hsu ldquoAntibacterial and antifungal activities of fatty acidmethyl esters of the blind-your-eye mangrove from IndiardquoBrazilian Journal of Microbiology vol 38 no 4 pp 739ndash7422007

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

2 BioMed Research International

which are widely used in pharmaceutical research [16ndash18] Polyphenols and sulfated polysaccharides present inseaweeds have been proven for antiviral antitumoral anti-inflammatory and anticoagulant activity [17ndash19] Seaweedscontain prolific source of bioactive compounds which canbe exploited for the treatment of major chronic diseases likediabetes through the inhibition of starch digesting enzymesand the regulation of glucose induced oxidative stress [20 21]

In the present study antidiabetic and antioxidant effect offive different solvent extracts of marine seaweed Turbinariaornata has been evaluated T ornata a wide spread speciesbelonging to Phaeophyceae is rich in fucoids and sulphatedpolysaccharides [22 23] It has been previously reported forits antioxidant anti-inflammatory and cytotoxicity activityon murine melanoma and colon cancer cells [23ndash25] Tothe best of our knowledge this is the first report on Tornata elucidating three different antidiabetic mechanisms(120572-amylase 120572-glucosidase and DPP-IV) in vitro and theextracts were also tested for their in vitro toxicity

2 Materials and Methods

21 Chemicals Porcine pancreatic 120572-amylase dinitrosali-cylic acid (DNSA) acarbose p-nitrophenyl 120572-D-gluco-pyranoside diprotin A (Ile-Pro-Ile) DPP-IV from porcinekidney and Gly-pro-p-nitroanilide (GPPN) were purchasedfrom Sigma Bangalore India DPPH butylated hydroxytoluene (BHT) was obtained from Himedia Mumbai 120572-Glucosidase anddimethyl sulphoxide (DMSO)were obtainedfrom SRL Mumbai

22 Collection and Processing of Seaweed Fresh brown sea-weeds were collected from Mandapam coastal region (Lat-itude 9∘1710158401015840N Longitude 79∘1110158401015840E) Gulf of Mannar Tamil-nadu South India on low tide during October 2012 Thevoucher specimen was deposited in the Marine Biotech-nology and Biomedicine Laboratory VIT University Theseaweeds were cleaned and holdfasts were removed andstored at minus20∘C immediately Before extraction they wereshade-dried powdered and stored in air-tight containers atminus20∘C

23 Preparation of Seaweed Extracts The dried seaweedsamples (25 g) were milled and extracted using 250mL ofvarious solvents such as petroleum ether benzene ethylacetate acetone and methanol for 24 h by using Soxhletapparatus Each filtrate was concentrated to dryness underreduced pressure using rotary evaporator (Super Fit Rotavapmodel PBU-6 India)The samples were lyophilized by usingfreeze dryer (Lark Penguin Classic Plus India) and storedin a refrigerator at 2ndash8∘C for use in subsequent experimentsPrior to experiments various concentrations were preparedfrom 250 to 1000 120583gmL

24 Preliminary Phytochemical Screening Preliminary phy-tochemical screenings were carried out as per the standardprotocols of Harborne [26]

25 22-Diphenyl-1-picrylhydrazyl (DPPH)Radical ScavengingActivity Free radical scavenging activity was determinedaccording to the method of Mensor et al [27] Briefly 500120583Lof 03mM alcoholic solution of DPPH (Himedia India) wasadded to 25mL of test samples at varying concentrations(250ndash1000120583gmL) After incubation of samples in the darkfor 30 minutes the optical density was measured at 518 nmin the UV-visible spectrophotometer (Systronics AU-2700India) using methanol as blank and synthetic antioxidantbutylated hydroxy toluene (BHT) as positive control Theexperiments were performed in triplicates and scavengingactivity was expressed as percentage inhibition using thefollowing formula

Inhibition = [(Abscontrol minus Abssamples)

Abscontrol] times 100 (1)

26 In Vitro 120572-Amylase Inhibition Assay The 120572-amylaseinhibitory activity was determined as described in Jayasriet al [28] with minor modifications Briefly 250 120583L of algalextracts with varying concentrations (250ndash1000120583gmL) and250 120583L of 002M sodium phosphate buffer (pH 69 with0006M sodium chloride) containing 120572-amylase (porcinepancreatic 120572-amylase Sigma St Louis USA) solution(05mgmL) were incubated for 10min at 25∘C After prein-cubation 250120583L of 1 starch solution in 002M sodiumphosphate buffer (pH 69 with 0006M sodium chloride) wasadded to each tube at 5 sec intervals The reaction mixtureswere then incubated at 25∘C for 10min The reaction wasstopped with 500 120583L dinitrosalicylic acid (Sigma St LouisUSA) colour reagent The tubes were then incubated in aboiling water bath for 5min and cooled to room temperatureThe reaction mixture was then diluted by adding 5mL ofdistilled water and absorbance was measured at 540 nmin the UV-visible spectrophotometer (Systronics AU-2700India) The experiments were performed in triplicate and the120572-amylase inhibitory activity was calculated as percentageinhibition using the formula

Inhibition = [(Abscontrol minus Abssamples)

Abscontrol] times 100 (2)

27 In Vitro 120572-Glucosidase Inhibition Assay The 120572-gluco-sidase inhibitory activity was determined as described byJayasri et al [28] with slight modifications Briefly 50120583L ofalgal extracts with varying concentrations (250ndash1000 120583gmL)and 100 120583L of 01M phosphate buffer (pH-69) containing 120572-glucosidase (SRL India) solution (10UmL) were incubatedin 96-well plates at 25∘C for 10 minutes After preincubation50 120583L of 5mM p-nitrophenyl 120572-D-glucopyranoside (SigmaSt Louis USA) in 01M phosphate buffer (pH-69) wasadded to each well at 5 sec intervals The reaction mixturewas then incubated at 25∘C for 5min After incubationabsorbance readings were recorded at 405 nm using a platereader (Bio-TEK USA) and compared to the control whichcontained 50 120583L of buffer solution in place of algal extractThe experiments were performed in triplicate and the 120572-glucosidase inhibitory activity was calculated as percentage

BioMed Research International 3

inhibition The percentage of enzyme inhibition was calcu-lated as follows

Inhibition = [(Abscontrol minus Abssamples)

Abscontrol] times 100 (3)

28 In Vitro Dipeptidyl Peptidase-IV (DPP-IV) InhibitionAssay DPP-IV inhibitory activity was determined accordingto the method of Al-Masri et al [29] Standard diprotin A(Sigma St Louis USA) was diluted to various concentrations(02 04 08 16 32 and 64 120583gmL) using Tris-HCl buffer(50mM pH 75) and the final volume was made to 35 120583Land DPP-IV enzyme (15 120583L) (Sigma St Louis USA) wasadded to the above mixture One unit of enzyme activity wasdefined as the amount of enzyme that catalyzes the releaseof 1 120583mol para-nitroaniline (pNA) from the substrateminunder assay conditions After adding the enzyme themixturewas preincubated for 10min at 37∘C to enhance the bindingcapacity of the inhibitorThis was followed by the addition of50120583L of (02mM) Gly-pro-p-nitroanilide (Sigma St LouisUSA) in Tris-HCl as a substrate Final incubation was done at37∘C for 30min and the reaction was terminated by additionof 25 120583L of 25 glacial acetic acid The absorbance wasmeasured at 405 nm using microtiter plate reader (Bio-TEKUSA) Experiments were done in triplicate and the resultsobtained were compared with negative control

29 DPP-IV Inhibition Assay of Seaweed Extract Seaweedextracts were dissolved in dimethyl sulphoxide to makea stock concentration of 1000 120583gmL From the stock thefollowing concentrations were prepared (25 10 40 and80 120583gmL) in Tris-HCl buffer (50mM pH 75) in a totalvolume of 100 120583Lwell The assay was performed in triplicateaccording to standardized procedure of diprotin A Thepercentage of DPP-IV inhibition was calculated as follows

Inhibition = [(Abscontrol minus Abssamples)

Abscontrol] times 100 (4)

210 Maintenance of Cell Line J774 cell line (mouse macro-phage) was obtained from National Centre for Cell SciencePune India The cells were grown in Dulbeccorsquos ModifiedEaglersquos Medium (DMEM) supplemented with 10 (vv) heat-inactivated foetal bovine serum (FBS) 2mM l-glutamineand 100UmL of penicillinstreptomycin with 5 CO

2at

37∘C in a humidified incubator Exponentially growing cellswere used for the experiment

211 Cytotoxicity Assay Briefly cells were seeded at a densityof 5 times 105 cellsmL and allowed to attach for 24 h in 300120583Lof medium incubated at 37∘C and 5 CO

2 After 24 h

seaweed extracts were added to the medium at variousconcentrations (1000ndash250120583gmL) and incubated for 24 hAt the end of treatment cells were incubated with MTT[3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bro-mide] (5mgmL) for 4 h The formazan crystals formedwere dissolved inDMSO (dimethyl sulfoxide) after aspirating

the medium The extent of cytotoxicity was measured spec-trophotometrically at 630 nm with a microplate reader (Bio-TEK USA)

212 DNA Fragmentation Assay Acridine orange-ethidiumbromide staining was used to characterize the morphologicalchanges and apoptotic body formation assessed by fluores-cent microscopy Briefly J774 cells (5 times 105) were seeded onsterile glass cover slips in six-well tissue culture plates andtreated with test extracts (1000 120583gmL) for 24 h The mediumwas removed and washed twice with PBS (phosphate-buffered saline) Cells grown on cover slips were loaded withacridine orange-ethidium bromide (4 120583gmL) of ratio (1 1)and incubated for 15min at 37∘C in the dark Cells wereviewed under Weswox LED fluorescent microscope (FM-3000) attached with a camera and photographs were takenunder 40 and 100x magnification at fluorescent conditions

213 Haemolytic Assay The seaweed extracts were evaluatedfor their haemolytic activity as described by Malagoli [30]The blood sample was collected from a healthy volunteer(A+ve) using ethylene diamine tetra acetic acid (EDTA) as ananticoagulant Whole blood was washed thrice in 9 volumesof sterile 09 NaCl saline solution after each washing thecells were centrifuged (150timesg for 5min) and the supernatantwas discardedThe final pellet obtained was diluted 19 (vv)in sterile 09 NaCl saline solution and then 124 (vv) insterile Dulbeccorsquos phosphate buffer saline (D-PBS) pH 70containing 05mM boric acid and 1mM calcium chlorideThe haemolytic activity of seaweed extracts was tested underin vitro conditions in 96-well plates Briefly eachwell received100 120583L of 085 NaCl solution containing 10mM CaCl

2

100 120583L of normal saline served as negative control 01Triton X-100 as a positive control and test extracts of variousconcentrations (250ndash1000120583gmL) were added to each wellThen eachwell received 100 120583L of human erythrocytes dilutedin D-PBS After 30min incubation at room temperaturethe samples were centrifuged and supernatant was used tomeasure the absorbance of liberated haemoglobin at 630 nm[31]

214 GCMSAnalysis GCMS (Perkin Elmer Clarus 680GCcoupled to a Clarus 600 MS) analysis was performed for thedetection of major compounds present in various extracts ofT ornata Column used in GCMS was Elite-5MS (300m025mmID and 250 120583mdf) Carrier gas used was helium ata constant flow rate of 1mLmin EI mode with electronenergy set at 70 eV was used 1 120583L of extract diluted withmethanol was injected into GCMS and the compounds wereidentified based on the molecular structure molecular massand calculated fragment ratio of resolved spectra with thatof mass spectra available from the library Spectral data wereinterpreted using the database of National Institute StandardAnd Technology (NIST)

215 Statistical Analysis All the data reportedwere expressedas mean plusmn SEM Statistical analysis was performed using

4 BioMed Research International

(a) (b)

Figure 1 (a)Macroscopic view of Turbinaria ornata collected fromMandapam coastal region in Gulf ofMannar Tamilnadu India Stiff erectstalks hard thick leaves (blades) characterized by lateral ridges and outermarginal blade with stiff row of spines are the unique characteristicsof this species (b) Microscopic image of Turbinaria ornata (magnification 40x)

Table 1 Qualitative phytochemical screening of various extracts of Turbinaria ornata (+ present minus absent)

Phytochemicals Pet ether Benzene Ethyl acetate Methanol AcetoneAlkaloids + + + + +Phenols + + + + +Flavanoids + + + + +Proteins and amino acid + + + + +Lipids + + + + +Carbohydrates + + + + +Saponins minus minus + minus minus

Glycosides minus minus + + minus

Tannins + + + minus minus

Oils and fats + minus minus minus minus

two-way ANOVA The values were considered to be signif-icantly different when the 119875 value was lt00001 compared tothe baseline values Software employed for statistical analysiswas Graph-Pad Prism Version 5

3 Results

31 Identification of Collected Seaweeds The samples wereidentified based on the morphological characteristics inresponse to environmental conditions stiff erect stalks hardthick leaves (blades) characterized by lateral ridges and outermarginal bladewith stiff rowof spines (Figure 1) Based on theabove characters seaweed species was identified as T ornataand authenticated byDr P Kaladharan principle scientist andscientist in charge Calicut Regional Centre of CentralMarineFisheries Research Institute

32 Phytochemical Analysis of T ornata Qualitative anal-ysis of the crude extracts of T ornata showed the pres-ence of major phytochemicals like alkaloids phenols fla-vanoids proteins lipids carbohydrates glycosides tanninsand saponins in all the studied extracts (Table 1)

33 22-Diphenyl-1-picrylhydrazyl (DPPH)Radical ScavengingActivity The antioxidant potential of T ornata was studiedthrough its scavenging ability of the stable radical DPPH

Acetone extract showed significant scavenging ability onDPPH (65) at a concentration of 1000120583gmL when com-pared with that of a standard BHT (97) However none ofthe extracts exhibited higher activity than BHT at the sameconcentration (Figure 2)

34 In Vitro 120572-Amylase Inhibition Assay Inhibitory effect ofT ornata on 120572-amylase is shown in Figure 3(a) Among thefive extracts studied (petroleum ether benzene ethyl acetatemethanol and acetone) three extracts showed (petroleumether methanol and acetone) significant 120572-amylase inhi-bition at all the tested concentrations (250 500 750 and1000 120583gmL) Methanol extract of T ornata showed max-imum inhibition of 965 with an IC

50of 2509 120583gmL

followed by acetone extract (876) at a concentration of1000 120583gmL

35 In Vitro 120572-Glucosidase Inhibition Assay The 120572-gluco-sidase inhibitory activity of T Ornata extracts was deter-mined using p-nitrophenyl 120572-D-glucopyranoside as a sub-strate and these were compared with a standard acarbose(88) (Figure 3(b)) At a concentration of 1000 120583gmL ace-tone extract showed maximum inhibition (876) with anIC50value of 5356 120583gmL The inhibitory activity of acetone

extracts was found to be more potent and similar whencompared to the positive control acarbose

BioMed Research International 5

0

50

250 500 750 1000

100

150

Petroleum etherBenzeneEthyl acetate

MethanolAcetoneBHT

Concentration (120583gmL)

Inhi

bitio

n (

)

a

a

a

aa

a

a a a a

a

aaa a a

a

a

a

aa

a

a

a

Figure 2 DPPH radical scavenging activity of petroleum etherbenzene ethyl acetatemethanol and benzene extracts ofTurbinariaornata BHT is used as positive control and absorbance wasmeasured at 517 nm Values are means plusmn SD (119899 = 3) (a119875 lt 00001considered as significant)

36 In Vitro Dipeptidyl Peptidase-IV (DPP-IV) InhibitionAssay T ornata extracts were evaluated for their mode ofaction to stimulate insulin secretion through inhibition ofDPP-IV enzyme The effectiveness of various extracts wasevaluated on the basis of percentage inhibition and IC

50

values obtained Among the five different solvent extracts(petroleum ether benzene ethyl acetate methanol andacetone) methanol extracts showed maximum percentageinhibition of 554 with an IC

50value of 552120583gmL at a

maximum concentration of 80 120583gmL All the results werecompared with a standard diprotin A (Figure 4)

37 Cytotoxic Effect of T ornata Extracts on J774 Cell Line Toevaluate the cytotoxic effect of the extracts J774 cell line wasincubated for 24 h with various concentrations of extracts Asshown in Figure 5(a) the decrease in viability correlates withthe increase in concentration MTT assay indicates that J774cells treated with various extracts of T ornata were safe anddid not show any toxic effect against the cell treated at a lowerconcentration of 250120583gmL The observed cytotoxicity wasfound to be 257 when treated with methanol extract of Tornata 50 of cells were found to be viable when treatedwitha higher concentration of 1000120583gmL

38 DNA Fragmentation Assay Staining cells with acridineorange-ethidium bromide is used to measure the proportionof viable apoptotic and necrotic cells J774 cells treated witha maximum concentration of 1000120583gmL of algal extractswere found to be viable and there was no change in cellularmorphology including chromatin condensation membraneblebbing and fragmented nuclei (Figure 6)

39 Haemolytic Assay The lead extracts methanol (278)and acetone (461) were found to be nontoxic at a dosageof 1000120583gmL (Figure 5(b)) Also the extracts did not showany erythrocyte membrane damage at all the tested concen-trations

310 GC-MS Analysis of the Acetone Extract of T ornataThe crude extracts were analyzed by GC-MS and comparedwith the retention time (RT) and mass spectra availablein the data library of NIST The GC-MS analysis revealsthe presence of major bioactive compounds like hentriacon-tane z z-6 28-heptatriactontadien-2-one 8-heptadeceneand 1-heptacosanol Chromatograms obtained were shown inFigure 7

4 Discussion

Marine seaweeds are good source of dietary fibres polysac-charides polyphenols polyunsaturated fatty acids mineralsand vitamins which possess various biological activities likeantidiabetic anti-inflammatory and antioxidant activity [32]Although this evidence seems to be valid detailed researchshould be carried out to ensure the presence of various phy-tochemicals present in the seaweed extracts In the currentstudy marine seaweed T ornata was extracted and screenedfor both nonpolar and polar compounds using varioussolvents based on polarity Qualitative analysis showed thepresence of major bioactive compounds like alkaloids phe-nols flavanoids glycosides lipids carbohydrates proteinstannins oils and fats Presence of these bioactive moleculesin various extracts of this seaweed may be responsible forvarious biological activities (antiviral anti-inflammatory andanticoagulant) [17ndash19]

The objective of this study was to investigate the antidia-betic and antioxidant potential of marine seaweed T ornataand to enumerate major phytocompounds present in theextracts One of the major causes for diabetes is due tothe increased production of reactive oxygen species orimpaired antioxidant defense systemwhich leads to oxidativedamage of beta cells [33] In this study acetone extract ofT ornata showed significant scavenging capability againstDPPH (65) at a maximum concentration of 1000 120583gmLcompared with a positive control BHT (97) As previouslyreported by Ananthi et al and Chattopadhyay et al [2325] the fucoidan fraction of Turbinaria conoides (61) ata concentration of 1000 120583gmL and aqueous extract of Tornata (80) at a concentration of 500 120583gmL showed thesimilar scavenging capability on DPPH radicals Our resultswere similar to these reports which confirm the antioxidantactivity of T ornata

In the present study the edible seaweed T ornata wasevaluated for in vitro 120572-amylase 120572-glucosidase and DPP-IV inhibitory property Among the five extracts studiedpetroleum ether acetone and methanol extracts showedsignificant percentage inhibition Methanol extract of Tornata showed maximum percentage inhibition on 120572-amylase (965)with an IC

50value of 2509120583gmLwithmore

inhibition than positive control acarbose (88) Acetone

6 BioMed Research International

0

50

100

150In

hibi

tion

()

Petroleum etherMethanol

AcetoneAcarbose

a

aa

a

a

a

a

a a

a

aa a

a a a

250 500 750 1000Concentration (120583gmL)

IC50 value of methanol extract 2509 120583gmL

(a)

0

20

40

60

80

100

Petroleum etherMethanol

AcetoneAcarbose

Inhi

bitio

n (

)

a a

a

a

aa

a

aa

a

a

aa

a

a

a

250 500 750 1000Concentration (120583gmL)

IC50 value of acetone extract 5356 120583gmL

(b)

Figure 3 (a) In vitro 120572-amylase inhibitory activity of petroleum ether methanol and acetone extracts of Turbinaria ornata Absorbance wasmeasured at 540 nm (b) In vitro 120572-glucosidase inhibitory activity of petroleum ether methanol and acetone extracts of Turbinaria ornataacarbose is used as positive control and absorbance was measured at 405 nm Values are means plusmn SD (119899 = 3) (a119875 lt 00001 consideredsignificant)

3002 04 08 16 32 64

40

50

60

70

Inhi

bitio

n (

)

aa

a

a

a

a

Concentration (120583gmL)

(a)

025 10 40 80

20

40

60

Inhi

bitio

n (

) a a a a aa

a

a

a aa

a

aa

a a

aa

a a

Concentration (120583gmL)

Petroleum etherBenzeneAcetone

Ethyl acetateMethanol

IC50 value of methanol extract 552 120583gmL

(b)

Figure 4 (a) DPP-IV inhibitory activity of diprotin A as positive control (b) DPP-IV inhibitory activity of petroleum ether benzene ethylacetate methanol and acetone extracts of Turbinaria ornata Absorbance was measured at 405 nm Values are means plusmn SD (119899 = 3) (a119875 lt00001 considered significant)

extract of T ornata showed more potent inhibition on120572-glucosidase (843) with an IC

50value of 5356 120583gmL

at a concentration of 1000120583gmL similar to the positivecontrol acarbose (88) The DPP-IV inhibitory potential ofmethanol extract of T ornata showed (554) inhibitionwith an IC

50value of 552120583gmL which is comparable with

that of positive control diprotin A (65) Similar findingswere shown by two brown seaweeds Ecklonia stoloniferaOkumura and Eisenia bicyclis [34] Several studies reportedthat the 120572-amylase and 120572-glucosidase inhibitory action of theclass Phaeophyceae [35 36] supports that these algal speciesare excellent source for antidiabetic agents Apart from 120572-amylase and 120572-glucosidase T ornata extracts also showedsignificant DPP-IV inhibition Based on these results this

seaweed can be used as alternative therapy for diabetes byraising the levels of endogenous incretin hormones (GLP-1and GIP)

In this study we also investigated the effects of crudeextracts (methanol acetone) of T ornata in macrophagecell line (J774) The cytotoxic effects of the extracts weredetermined by using anMTT assay After 24 h exposure 250ndash1000 120583gmL of methanol and acetone extracts significantlydecreased cell viability by increasing concentration As pre-viously reported seaweed extracts which have higher phenolcontent may explain the level of cytotoxicity at higher con-centrations (1000 120583gmL) [36 37] whereby up to 500120583gmLof seaweed extracts did not show any effect on J774 cellviability andmore than 90of cells were found to be viable at

BioMed Research International 7

0

50

100

150C

ell v

iabi

lity

() a a

aa

aa a

a

250 750 1000500

MethanolAcetone

Concentration (120583gmL)

(a)

0250 750 1000500

2

4

6

MethanolAcetone

Cell

lysis

()

a a a

a

a

a

a

a

Concentration (120583gmL)

(b)

Figure 5 (a) Cell viability determined byMTT assay J774 cells treated with methanol and acetone extracts of Turbinaria ornata Absorbancewasmeasured at 630 nm (b)Haemolytic activity ofmethanol and acetone extracts ofTurbinaria ornata Absorbance wasmeasured at 630 nmValues are means plusmn SD (119899 = 3) (a119875 lt 00001 considered significant)

Control

(a)

TO M

(b)

Control

(c)

Treated

(d)

Control

(e)

Treated

(f)

Figure 6 (a) Fluorescent photomicrographs (100x magnification) of J774 cells treated without seaweed extracts (b) J774 cells treated withTurbinaria ornatamethanol extract (TOM) (c) Phase contrast microscopic image of J774 cells treated with media alone (control) (d) Phasecontrast microscopic image of J774 cells treated with test extracts ((e)-(f)) Microscopic image (40x magnification) of in vitro haemolyticactivity

8 BioMed Research International

A

R3A-(13IS-1200)

B

500

100(

)

01000 1500 2000 2500 3000

Time

515e8TIC

Scan El+

OH

317

0308

130

28

294

528

36

277

627

17

264

3257

525

05

243

924

33

236

423

59

228

222

03

220

721

24

212

020

37

203

320

28

192

218

49

181

017

64

172

516

47

161

015

12

141

8

164

0

184

3

C

A

B

5111

31

33333337030

333

8130

3333333333

2829

45

28

22222222236

277

627

22222

1726

4325

75

250

524

39

243

323

222

6423

22

5922

82

220

322

07

21

2224

212

020

37

203

320

28

192

218

49

181

017

71764

172

516

47

16

110

151

214

18

164

0

18

188181818181881881881888888818881881888811111143444444444444

C

Figure 7 Gas chromatogram of acetone extract of Turbinaria ornata (A) 8-heptadecene (B) 1-heptacosanol and (C) hentriacontane

a concentration of 250120583gmL Staining cells with fluorescentdyes is used to differentiate the nuclear morphology of liveand apoptotic cells J774 cells were analyzed in the presenceof acridine orange and ethidium bromide to determinethe nuclear morphology of viable and necrotic cells Cellsstained green represent viable cells and ethidium bromideselectively stains the cells that have lost membrane integrityand stains DNA orange In this study acridine orange-ethidium bromide staining revealed that there was no changein cellular morphology including chromatin condensationmembrane blebbing and fragmented nuclei occurring dueto the treatment of extracts even at higher concentration(1000 120583gmL) Our results indicate that the tested extracts didnot induce haemolysis at a concentration of (1000120583gmL)Based on the above results the methanol and acetone extractsof T ornata were found to be nontoxic under in vitroconditions

GC-MS analysis revealed the presence of major bioac-tive compounds like hentriacontane z z-6 28-heptatriact-ontadien-2-one 8-heptadecene and 1-heptacosanol Hentri-acontane is the major component present in Oldenlandiadiffusa a natural source for the treatment of cancer in Asia[38] and also has reported activities like anti-inflammatoryand antioxidant properties [38 39] Currently there is noinformation regarding the antidiabetic effects of hentria-contane Further purification and characterization of activecompounds along with mechanism studies will only giveconclusive data on the antidiabetic property of tested algae

5 Conclusions

The present study indicates that crude extracts of T ornatapossess in vitro 120572-amylase 120572-glucosidase and DPP-IV

inhibitory activity Due to their strong enzyme inhibitoryactivity they are expected to suppress the glycemic responsein diabetic conditions Additionally the extracts are capableof scavenging free radicals in vitro toxicological parametersindicate that the IC

50of methanol and acetone extracts of T

ornata for 120572-amylase 120572-glucosidase and DPP-IV inhibitionare greatly below cytotoxic levels Future studies on in vivoantidiabetic activity of selected extracts and identification ofmajor compounds responsible for antidiabetic action are inprogress These studies will reveal the possible antidiabeticmechanisms of T ornata

Conflict of Interests

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

Acknowledgments

The authors wish to thank Department of BiotechnologyGovernment of India for their financial support (Grantno BTBio-CARe033472010-11) and VIT University forproviding all necessary facilities

References

[1] K G Alberti and P Z Zimmet ldquoDefinition diagnosis andclassification of diabetes mellitus and its complications Part1 diagnosis and classification of diabetes mellitus provisionalreport of a who consultationrdquo Diabetic Medicine vol 15 no 7pp 539ndash553 1998

[2] O P Gupta and S Phatak ldquoPandemic trends in prevalenceof diabetes mellitus and associated coronary heart disease in

BioMed Research International 9

India-their causes and preventionrdquo International Journal ofDiabetes in Developing Countries vol 23 pp 37ndash50 2003

[3] R R Ortiz-Andrade S Garcıa-Jimenez P Castillo-EspanaG Ramırez-Avila R Villalobos-Molina and S Estrada-Sotoldquo120572-Glucosidase inhibitory activity of the methanolic extractfrom Tournefortia hartwegiana an anti-hyperglycemic agentrdquoJournal of Ethnopharmacology vol 109 no 1 pp 48ndash53 2007

[4] G M Gray ldquoCarbohydrate digestion and absorption Role ofthe small intestinerdquo The New England Journal of Medicine vol292 no 23 pp 1225ndash1230 1975

[5] C A Tarling K Woods R Zhang et al ldquoThe search fornovel human pancreatic 120572-amylase inhibitors high-throughputscreening of terrestrial and marine natural product extractsrdquoChemBioChem vol 9 no 3 pp 433ndash438 2008

[6] R Mentlein B Gallwitz and W E Schmidt ldquoDipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide gluca-gon-like peptide-1(7-36)amide peptide histidine methionineand is responsible for their degradation in human serumrdquoEuropean Journal of Biochemistry vol 214 no 3 pp 829ndash8351993

[7] T Vilsboslashll T Krarup C F Deacon S Madsbad and J J HolstldquoReduced postprandial concentrations of intact biologicallyactive glucagon-like peptide 1 in type 2 diabetic patientsrdquoDiabetes vol 50 no 3 pp 609ndash613 2001

[8] M A Nauck U Niedereichholz R Ettler et al ldquoGlucagon-like peptide 1 inhibition of gastric emptying outweighs itsinsulinotropic effects in healthy humansrdquo American Journal ofPhysiologymdashEndocrinology and Metabolism vol 273 no 5 ppE981ndashE988 1997

[9] A-M Lambeir S Scharpe and I DeMeester ldquoDPP4 inhibitorsfor diabetesmdashwhat nextrdquo Biochemical Pharmacology vol 76no 12 pp 1637ndash1643 2008

[10] [EMEA] EuropeanMedicines Agency ldquoEuropean public assess-ment report (EPAR)mdashJanuviardquo 2007 httpwwwemaeuropaeuema

[11] [EMEA] EuropeanMedicines Agency ldquoEuropean public assess-ment report (EPAR)mdashGalvusrdquo 2007 httpwwwemaeuropaeuema

[12] A Murai K Iwamura M Takada K Ogawa T Usui andJ-I Okumura ldquoControl of postprandial hyperglycaemia bygalactosyl maltobionolactone and its novel anti-amylase effectin micerdquo Life Sciences vol 71 no 12 pp 1405ndash1415 2002

[13] B Halliwell and O I Aruoma ldquoDNA damage by oxygen-derived species Its mechanism and measurement in mam-malian systemsrdquo FEBS Letters vol 281 no 1-2 pp 9ndash19 1991

[14] J Zhou N Hu Y-L Wu Y-J Pan and C-R Sun ldquoPreliminarystudies on the chemical characterization and antioxidant prop-erties of acidic polysaccharides from Sargassum fusiformerdquoJournal of Zhejiang University Science B vol 9 no 9 pp 721ndash727 2008

[15] D A Butterfield A Castenga C B Pocernich J DrakeG Scapagnini and V Calabrese ldquoNutritional approaches tocombat oxidative stress in Alzheimerrsquos diseasesrdquo Journal ofNutritional Biochemistry vol 13 pp 444ndash461 2002

[16] A M S Mayer and V K B Lehmann ldquoMarine pharmacologyin 1998 marine compounds with antibacterial anticoagu-lant antifungal anti-inflammatory anthelmintic antiplateletantiprotozoal and antiviral activities with actions on the cardio-vascular endocrine immune and nervous systems and othermiscellaneous mechanisms of actionrdquo Pharmacologist vol 42pp 62ndash69 2000

[17] A Cumashi N A Ushakova M E Preobrazhenskaya et alldquoA comparative study of the anti-inflammatory anticoagulantantiangiogenic and antiadhesive activities of nine differentfucoidans from brown seaweedsrdquoGlycobiology vol 17 no 5 pp541ndash552 2007

[18] T Ghosh K Chattopadhyay M Marschall P Karmakar PMandal and B Ray ldquoFocus on antivirally active sulfatedpolysaccharides from structure-activity analysis to clinicalevaluationrdquo Glycobiology vol 19 no 1 pp 2ndash15 2009

[19] V H Pomin and P A S Mourao ldquoStructure biology evolutionand medical importance of sulfated fucans and galactansrdquoGlycobiology vol 18 no 12 pp 1016ndash1027 2008

[20] S-H Lee J-S Han S-J Heo J-Y Hwang and Y-J JeonldquoProtective effects of dieckol isolated fromEcklonia cava againsthigh glucose-induced oxidative stress in human umbilical veinendothelial cellsrdquo Toxicology in Vitro vol 24 no 2 pp 375ndash3812010

[21] S-H Lee F Karadeniz M-M Kim and S-K Kim ldquo120572-Glucosidase and 120572-amylase inhibitory activities of phlorogluci-nal derivatives from edible marine brown alga Ecklonia cavardquoJournal of the Science of Food and Agriculture vol 89 no 9 pp1552ndash1558 2009

[22] A Rohfritsch C Payri V Stiger and F Bonhomme ldquoMolec-ular and morphological relationships between two closelyrelated speciesTurbinaria ornata andT conoides (SargassaceaePhaeophyceae)rdquo Biochemical Systematics and Ecology vol 35no 2 pp 91ndash98 2007

[23] S Ananthi H R B Raghavendran A G Sunil V Gayathri GRamakrishnan and H R Vasanthi ldquoIn vitro antioxidant and invivo anti-inflammatory potential of crude polysaccharide fromTurbinaria ornata (Marine Brown Alga)rdquo Food and ChemicalToxicology vol 48 no 1 pp 187ndash192 2010

[24] F Asari T Kusumi and H Kakisawa ldquoTurbinaric acid acytotoxic secosqualene carboxylic acid from the brown algaTurbinaria ornatardquo Journal of Natural Products vol 52 no 5pp 1167ndash1169 1989

[25] N Chattopadhyay T Ghosh S Sinha K ChattopadhyayP Karmakar and B Ray ldquoPolysaccharides from Turbinariaconoides structural features and antioxidant capacityrdquo FoodChemistry vol 118 no 3 pp 823ndash829 2010

[26] J B Harborne ldquoMethods of extraction and isolationrdquo inPhytochemical Methods vol 3 pp 60ndash66 Chapman amp Hall1998

[27] L L Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plant extracts for antioxidant activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[28] M A Jayasri A Radha and T L Mathew ldquo120572-amylase and120572-glucosidase inhibitory activity of Costus pictus D Don inthe management of diabetesrdquo Journal of Herbal Medicine andToxicology vol 3 pp 91ndash94 2009

[29] IM Al-MasriM KMohammad andMO Tahaa ldquoInhibitionof dipeptidyl peptidase IV (DPP IV) is one of the mechanismsexplaining the hypoglycemic effect of berberinerdquo Journal ofEnzyme Inhibition and Medicinal Chemistry vol 24 no 5 pp1061ndash1066 2009

[30] D Malagoli ldquoA full-length protocol to test hemolytic activityof palytoxin on human erythrocytesrdquo Invertebrate SurvivalJournal vol 4 pp 92ndash94 2007

[31] V R Muzykantov G P Samokhin M D Smirnov andS P Domogatsky ldquoHemolytic complement activity assay in

10 BioMed Research International

microtitration platesrdquo Journal of Applied Biochemistry vol 7 no3 pp 223ndash227 1985

[32] A-R Kim T-S Shin M-S Lee et al ldquoIsolation and identifi-cation of phlorotannins from ecklonia stolonifera with antioxi-dant and anti-inflammatory propertiesrdquo Journal of Agriculturaland Food Chemistry vol 57 no 9 pp 3483ndash3489 2009

[33] J W Baynes ldquoChemical modification of proteins by lipids indiabetesrdquo Clinical Chemistry and Laboratory Medicine vol 41no 9 pp 1159ndash1165 2003

[34] H E Moon M N Islam B R Ahn et al ldquoProtein tyrosinephosphatase 1B and 120572-glucosidase inhibitory phlorotanninsfrom edible brown algae Ecklonia stolonifera and Eiseniabicyclisrdquo Bioscience Biotechnology and Biochemistry vol 75 no8 pp 1472ndash1480 2011

[35] E Apostolidis P D Karayannakidis Y-I Kwon C M Leeand N P Seeram ldquoSeasonal variation of phenolic antioxidant-mediated a-glucosidase inhibition of Ascophyllum nodosumrdquoPlant Foods forHumanNutrition vol 66 no 4 pp 313ndash319 2011

[36] F Nwosu J Morris V A Lund D Stewart H A Ross andG J McDougall ldquoAnti-proliferative and potential anti-diabeticeffects of phenolic-rich extracts from edible marine algaerdquo FoodChemistry vol 126 no 3 pp 1006ndash1012 2011

[37] G Galati and P J OBrien ldquoPotential toxicity of flavonoids andother dietary phenolics significance for their chemopreventiveand anticancer propertiesrdquo Free Radical Biology and Medicinevol 37 no 3 pp 287ndash303 2004

[38] S-J KimW-S Chung S-S Kim S-G Ko and J-Y Um ldquoAnti-inflammatory effect of Oldenlandia diffusa and its constituenthentriacontane through suppression of caspase-1 activation inmouse peritoneal macrophagesrdquo Phytotherapy Research vol 25no 10 pp 1537ndash1546 2011

[39] G Agoramoorthy M Chandrasekaran V Venkatesalu andM J Hsu ldquoAntibacterial and antifungal activities of fatty acidmethyl esters of the blind-your-eye mangrove from IndiardquoBrazilian Journal of Microbiology vol 38 no 4 pp 739ndash7422007

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 3

inhibition The percentage of enzyme inhibition was calcu-lated as follows

Inhibition = [(Abscontrol minus Abssamples)

Abscontrol] times 100 (3)

28 In Vitro Dipeptidyl Peptidase-IV (DPP-IV) InhibitionAssay DPP-IV inhibitory activity was determined accordingto the method of Al-Masri et al [29] Standard diprotin A(Sigma St Louis USA) was diluted to various concentrations(02 04 08 16 32 and 64 120583gmL) using Tris-HCl buffer(50mM pH 75) and the final volume was made to 35 120583Land DPP-IV enzyme (15 120583L) (Sigma St Louis USA) wasadded to the above mixture One unit of enzyme activity wasdefined as the amount of enzyme that catalyzes the releaseof 1 120583mol para-nitroaniline (pNA) from the substrateminunder assay conditions After adding the enzyme themixturewas preincubated for 10min at 37∘C to enhance the bindingcapacity of the inhibitorThis was followed by the addition of50120583L of (02mM) Gly-pro-p-nitroanilide (Sigma St LouisUSA) in Tris-HCl as a substrate Final incubation was done at37∘C for 30min and the reaction was terminated by additionof 25 120583L of 25 glacial acetic acid The absorbance wasmeasured at 405 nm using microtiter plate reader (Bio-TEKUSA) Experiments were done in triplicate and the resultsobtained were compared with negative control

29 DPP-IV Inhibition Assay of Seaweed Extract Seaweedextracts were dissolved in dimethyl sulphoxide to makea stock concentration of 1000 120583gmL From the stock thefollowing concentrations were prepared (25 10 40 and80 120583gmL) in Tris-HCl buffer (50mM pH 75) in a totalvolume of 100 120583Lwell The assay was performed in triplicateaccording to standardized procedure of diprotin A Thepercentage of DPP-IV inhibition was calculated as follows

Inhibition = [(Abscontrol minus Abssamples)

Abscontrol] times 100 (4)

210 Maintenance of Cell Line J774 cell line (mouse macro-phage) was obtained from National Centre for Cell SciencePune India The cells were grown in Dulbeccorsquos ModifiedEaglersquos Medium (DMEM) supplemented with 10 (vv) heat-inactivated foetal bovine serum (FBS) 2mM l-glutamineand 100UmL of penicillinstreptomycin with 5 CO

2at

37∘C in a humidified incubator Exponentially growing cellswere used for the experiment

211 Cytotoxicity Assay Briefly cells were seeded at a densityof 5 times 105 cellsmL and allowed to attach for 24 h in 300120583Lof medium incubated at 37∘C and 5 CO

2 After 24 h

seaweed extracts were added to the medium at variousconcentrations (1000ndash250120583gmL) and incubated for 24 hAt the end of treatment cells were incubated with MTT[3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bro-mide] (5mgmL) for 4 h The formazan crystals formedwere dissolved inDMSO (dimethyl sulfoxide) after aspirating

the medium The extent of cytotoxicity was measured spec-trophotometrically at 630 nm with a microplate reader (Bio-TEK USA)

212 DNA Fragmentation Assay Acridine orange-ethidiumbromide staining was used to characterize the morphologicalchanges and apoptotic body formation assessed by fluores-cent microscopy Briefly J774 cells (5 times 105) were seeded onsterile glass cover slips in six-well tissue culture plates andtreated with test extracts (1000 120583gmL) for 24 h The mediumwas removed and washed twice with PBS (phosphate-buffered saline) Cells grown on cover slips were loaded withacridine orange-ethidium bromide (4 120583gmL) of ratio (1 1)and incubated for 15min at 37∘C in the dark Cells wereviewed under Weswox LED fluorescent microscope (FM-3000) attached with a camera and photographs were takenunder 40 and 100x magnification at fluorescent conditions

213 Haemolytic Assay The seaweed extracts were evaluatedfor their haemolytic activity as described by Malagoli [30]The blood sample was collected from a healthy volunteer(A+ve) using ethylene diamine tetra acetic acid (EDTA) as ananticoagulant Whole blood was washed thrice in 9 volumesof sterile 09 NaCl saline solution after each washing thecells were centrifuged (150timesg for 5min) and the supernatantwas discardedThe final pellet obtained was diluted 19 (vv)in sterile 09 NaCl saline solution and then 124 (vv) insterile Dulbeccorsquos phosphate buffer saline (D-PBS) pH 70containing 05mM boric acid and 1mM calcium chlorideThe haemolytic activity of seaweed extracts was tested underin vitro conditions in 96-well plates Briefly eachwell received100 120583L of 085 NaCl solution containing 10mM CaCl

2

100 120583L of normal saline served as negative control 01Triton X-100 as a positive control and test extracts of variousconcentrations (250ndash1000120583gmL) were added to each wellThen eachwell received 100 120583L of human erythrocytes dilutedin D-PBS After 30min incubation at room temperaturethe samples were centrifuged and supernatant was used tomeasure the absorbance of liberated haemoglobin at 630 nm[31]

214 GCMSAnalysis GCMS (Perkin Elmer Clarus 680GCcoupled to a Clarus 600 MS) analysis was performed for thedetection of major compounds present in various extracts ofT ornata Column used in GCMS was Elite-5MS (300m025mmID and 250 120583mdf) Carrier gas used was helium ata constant flow rate of 1mLmin EI mode with electronenergy set at 70 eV was used 1 120583L of extract diluted withmethanol was injected into GCMS and the compounds wereidentified based on the molecular structure molecular massand calculated fragment ratio of resolved spectra with thatof mass spectra available from the library Spectral data wereinterpreted using the database of National Institute StandardAnd Technology (NIST)

215 Statistical Analysis All the data reportedwere expressedas mean plusmn SEM Statistical analysis was performed using

4 BioMed Research International

(a) (b)

Figure 1 (a)Macroscopic view of Turbinaria ornata collected fromMandapam coastal region in Gulf ofMannar Tamilnadu India Stiff erectstalks hard thick leaves (blades) characterized by lateral ridges and outermarginal blade with stiff row of spines are the unique characteristicsof this species (b) Microscopic image of Turbinaria ornata (magnification 40x)

Table 1 Qualitative phytochemical screening of various extracts of Turbinaria ornata (+ present minus absent)

Phytochemicals Pet ether Benzene Ethyl acetate Methanol AcetoneAlkaloids + + + + +Phenols + + + + +Flavanoids + + + + +Proteins and amino acid + + + + +Lipids + + + + +Carbohydrates + + + + +Saponins minus minus + minus minus

Glycosides minus minus + + minus

Tannins + + + minus minus

Oils and fats + minus minus minus minus

two-way ANOVA The values were considered to be signif-icantly different when the 119875 value was lt00001 compared tothe baseline values Software employed for statistical analysiswas Graph-Pad Prism Version 5

3 Results

31 Identification of Collected Seaweeds The samples wereidentified based on the morphological characteristics inresponse to environmental conditions stiff erect stalks hardthick leaves (blades) characterized by lateral ridges and outermarginal bladewith stiff rowof spines (Figure 1) Based on theabove characters seaweed species was identified as T ornataand authenticated byDr P Kaladharan principle scientist andscientist in charge Calicut Regional Centre of CentralMarineFisheries Research Institute

32 Phytochemical Analysis of T ornata Qualitative anal-ysis of the crude extracts of T ornata showed the pres-ence of major phytochemicals like alkaloids phenols fla-vanoids proteins lipids carbohydrates glycosides tanninsand saponins in all the studied extracts (Table 1)

33 22-Diphenyl-1-picrylhydrazyl (DPPH)Radical ScavengingActivity The antioxidant potential of T ornata was studiedthrough its scavenging ability of the stable radical DPPH

Acetone extract showed significant scavenging ability onDPPH (65) at a concentration of 1000120583gmL when com-pared with that of a standard BHT (97) However none ofthe extracts exhibited higher activity than BHT at the sameconcentration (Figure 2)

34 In Vitro 120572-Amylase Inhibition Assay Inhibitory effect ofT ornata on 120572-amylase is shown in Figure 3(a) Among thefive extracts studied (petroleum ether benzene ethyl acetatemethanol and acetone) three extracts showed (petroleumether methanol and acetone) significant 120572-amylase inhi-bition at all the tested concentrations (250 500 750 and1000 120583gmL) Methanol extract of T ornata showed max-imum inhibition of 965 with an IC

50of 2509 120583gmL

followed by acetone extract (876) at a concentration of1000 120583gmL

35 In Vitro 120572-Glucosidase Inhibition Assay The 120572-gluco-sidase inhibitory activity of T Ornata extracts was deter-mined using p-nitrophenyl 120572-D-glucopyranoside as a sub-strate and these were compared with a standard acarbose(88) (Figure 3(b)) At a concentration of 1000 120583gmL ace-tone extract showed maximum inhibition (876) with anIC50value of 5356 120583gmL The inhibitory activity of acetone

extracts was found to be more potent and similar whencompared to the positive control acarbose

BioMed Research International 5

0

50

250 500 750 1000

100

150

Petroleum etherBenzeneEthyl acetate

MethanolAcetoneBHT

Concentration (120583gmL)

Inhi

bitio

n (

)

a

a

a

aa

a

a a a a

a

aaa a a

a

a

a

aa

a

a

a

Figure 2 DPPH radical scavenging activity of petroleum etherbenzene ethyl acetatemethanol and benzene extracts ofTurbinariaornata BHT is used as positive control and absorbance wasmeasured at 517 nm Values are means plusmn SD (119899 = 3) (a119875 lt 00001considered as significant)

36 In Vitro Dipeptidyl Peptidase-IV (DPP-IV) InhibitionAssay T ornata extracts were evaluated for their mode ofaction to stimulate insulin secretion through inhibition ofDPP-IV enzyme The effectiveness of various extracts wasevaluated on the basis of percentage inhibition and IC

50

values obtained Among the five different solvent extracts(petroleum ether benzene ethyl acetate methanol andacetone) methanol extracts showed maximum percentageinhibition of 554 with an IC

50value of 552120583gmL at a

maximum concentration of 80 120583gmL All the results werecompared with a standard diprotin A (Figure 4)

37 Cytotoxic Effect of T ornata Extracts on J774 Cell Line Toevaluate the cytotoxic effect of the extracts J774 cell line wasincubated for 24 h with various concentrations of extracts Asshown in Figure 5(a) the decrease in viability correlates withthe increase in concentration MTT assay indicates that J774cells treated with various extracts of T ornata were safe anddid not show any toxic effect against the cell treated at a lowerconcentration of 250120583gmL The observed cytotoxicity wasfound to be 257 when treated with methanol extract of Tornata 50 of cells were found to be viable when treatedwitha higher concentration of 1000120583gmL

38 DNA Fragmentation Assay Staining cells with acridineorange-ethidium bromide is used to measure the proportionof viable apoptotic and necrotic cells J774 cells treated witha maximum concentration of 1000120583gmL of algal extractswere found to be viable and there was no change in cellularmorphology including chromatin condensation membraneblebbing and fragmented nuclei (Figure 6)

39 Haemolytic Assay The lead extracts methanol (278)and acetone (461) were found to be nontoxic at a dosageof 1000120583gmL (Figure 5(b)) Also the extracts did not showany erythrocyte membrane damage at all the tested concen-trations

310 GC-MS Analysis of the Acetone Extract of T ornataThe crude extracts were analyzed by GC-MS and comparedwith the retention time (RT) and mass spectra availablein the data library of NIST The GC-MS analysis revealsthe presence of major bioactive compounds like hentriacon-tane z z-6 28-heptatriactontadien-2-one 8-heptadeceneand 1-heptacosanol Chromatograms obtained were shown inFigure 7

4 Discussion

Marine seaweeds are good source of dietary fibres polysac-charides polyphenols polyunsaturated fatty acids mineralsand vitamins which possess various biological activities likeantidiabetic anti-inflammatory and antioxidant activity [32]Although this evidence seems to be valid detailed researchshould be carried out to ensure the presence of various phy-tochemicals present in the seaweed extracts In the currentstudy marine seaweed T ornata was extracted and screenedfor both nonpolar and polar compounds using varioussolvents based on polarity Qualitative analysis showed thepresence of major bioactive compounds like alkaloids phe-nols flavanoids glycosides lipids carbohydrates proteinstannins oils and fats Presence of these bioactive moleculesin various extracts of this seaweed may be responsible forvarious biological activities (antiviral anti-inflammatory andanticoagulant) [17ndash19]

The objective of this study was to investigate the antidia-betic and antioxidant potential of marine seaweed T ornataand to enumerate major phytocompounds present in theextracts One of the major causes for diabetes is due tothe increased production of reactive oxygen species orimpaired antioxidant defense systemwhich leads to oxidativedamage of beta cells [33] In this study acetone extract ofT ornata showed significant scavenging capability againstDPPH (65) at a maximum concentration of 1000 120583gmLcompared with a positive control BHT (97) As previouslyreported by Ananthi et al and Chattopadhyay et al [2325] the fucoidan fraction of Turbinaria conoides (61) ata concentration of 1000 120583gmL and aqueous extract of Tornata (80) at a concentration of 500 120583gmL showed thesimilar scavenging capability on DPPH radicals Our resultswere similar to these reports which confirm the antioxidantactivity of T ornata

In the present study the edible seaweed T ornata wasevaluated for in vitro 120572-amylase 120572-glucosidase and DPP-IV inhibitory property Among the five extracts studiedpetroleum ether acetone and methanol extracts showedsignificant percentage inhibition Methanol extract of Tornata showed maximum percentage inhibition on 120572-amylase (965)with an IC

50value of 2509120583gmLwithmore

inhibition than positive control acarbose (88) Acetone

6 BioMed Research International

0

50

100

150In

hibi

tion

()

Petroleum etherMethanol

AcetoneAcarbose

a

aa

a

a

a

a

a a

a

aa a

a a a

250 500 750 1000Concentration (120583gmL)

IC50 value of methanol extract 2509 120583gmL

(a)

0

20

40

60

80

100

Petroleum etherMethanol

AcetoneAcarbose

Inhi

bitio

n (

)

a a

a

a

aa

a

aa

a

a

aa

a

a

a

250 500 750 1000Concentration (120583gmL)

IC50 value of acetone extract 5356 120583gmL

(b)

Figure 3 (a) In vitro 120572-amylase inhibitory activity of petroleum ether methanol and acetone extracts of Turbinaria ornata Absorbance wasmeasured at 540 nm (b) In vitro 120572-glucosidase inhibitory activity of petroleum ether methanol and acetone extracts of Turbinaria ornataacarbose is used as positive control and absorbance was measured at 405 nm Values are means plusmn SD (119899 = 3) (a119875 lt 00001 consideredsignificant)

3002 04 08 16 32 64

40

50

60

70

Inhi

bitio

n (

)

aa

a

a

a

a

Concentration (120583gmL)

(a)

025 10 40 80

20

40

60

Inhi

bitio

n (

) a a a a aa

a

a

a aa

a

aa

a a

aa

a a

Concentration (120583gmL)

Petroleum etherBenzeneAcetone

Ethyl acetateMethanol

IC50 value of methanol extract 552 120583gmL

(b)

Figure 4 (a) DPP-IV inhibitory activity of diprotin A as positive control (b) DPP-IV inhibitory activity of petroleum ether benzene ethylacetate methanol and acetone extracts of Turbinaria ornata Absorbance was measured at 405 nm Values are means plusmn SD (119899 = 3) (a119875 lt00001 considered significant)

extract of T ornata showed more potent inhibition on120572-glucosidase (843) with an IC

50value of 5356 120583gmL

at a concentration of 1000120583gmL similar to the positivecontrol acarbose (88) The DPP-IV inhibitory potential ofmethanol extract of T ornata showed (554) inhibitionwith an IC

50value of 552120583gmL which is comparable with

that of positive control diprotin A (65) Similar findingswere shown by two brown seaweeds Ecklonia stoloniferaOkumura and Eisenia bicyclis [34] Several studies reportedthat the 120572-amylase and 120572-glucosidase inhibitory action of theclass Phaeophyceae [35 36] supports that these algal speciesare excellent source for antidiabetic agents Apart from 120572-amylase and 120572-glucosidase T ornata extracts also showedsignificant DPP-IV inhibition Based on these results this

seaweed can be used as alternative therapy for diabetes byraising the levels of endogenous incretin hormones (GLP-1and GIP)

In this study we also investigated the effects of crudeextracts (methanol acetone) of T ornata in macrophagecell line (J774) The cytotoxic effects of the extracts weredetermined by using anMTT assay After 24 h exposure 250ndash1000 120583gmL of methanol and acetone extracts significantlydecreased cell viability by increasing concentration As pre-viously reported seaweed extracts which have higher phenolcontent may explain the level of cytotoxicity at higher con-centrations (1000 120583gmL) [36 37] whereby up to 500120583gmLof seaweed extracts did not show any effect on J774 cellviability andmore than 90of cells were found to be viable at

BioMed Research International 7

0

50

100

150C

ell v

iabi

lity

() a a

aa

aa a

a

250 750 1000500

MethanolAcetone

Concentration (120583gmL)

(a)

0250 750 1000500

2

4

6

MethanolAcetone

Cell

lysis

()

a a a

a

a

a

a

a

Concentration (120583gmL)

(b)

Figure 5 (a) Cell viability determined byMTT assay J774 cells treated with methanol and acetone extracts of Turbinaria ornata Absorbancewasmeasured at 630 nm (b)Haemolytic activity ofmethanol and acetone extracts ofTurbinaria ornata Absorbance wasmeasured at 630 nmValues are means plusmn SD (119899 = 3) (a119875 lt 00001 considered significant)

Control

(a)

TO M

(b)

Control

(c)

Treated

(d)

Control

(e)

Treated

(f)

Figure 6 (a) Fluorescent photomicrographs (100x magnification) of J774 cells treated without seaweed extracts (b) J774 cells treated withTurbinaria ornatamethanol extract (TOM) (c) Phase contrast microscopic image of J774 cells treated with media alone (control) (d) Phasecontrast microscopic image of J774 cells treated with test extracts ((e)-(f)) Microscopic image (40x magnification) of in vitro haemolyticactivity

8 BioMed Research International

A

R3A-(13IS-1200)

B

500

100(

)

01000 1500 2000 2500 3000

Time

515e8TIC

Scan El+

OH

317

0308

130

28

294

528

36

277

627

17

264

3257

525

05

243

924

33

236

423

59

228

222

03

220

721

24

212

020

37

203

320

28

192

218

49

181

017

64

172

516

47

161

015

12

141

8

164

0

184

3

C

A

B

5111

31

33333337030

333

8130

3333333333

2829

45

28

22222222236

277

627

22222

1726

4325

75

250

524

39

243

323

222

6423

22

5922

82

220

322

07

21

2224

212

020

37

203

320

28

192

218

49

181

017

71764

172

516

47

16

110

151

214

18

164

0

18

188181818181881881881888888818881881888811111143444444444444

C

Figure 7 Gas chromatogram of acetone extract of Turbinaria ornata (A) 8-heptadecene (B) 1-heptacosanol and (C) hentriacontane

a concentration of 250120583gmL Staining cells with fluorescentdyes is used to differentiate the nuclear morphology of liveand apoptotic cells J774 cells were analyzed in the presenceof acridine orange and ethidium bromide to determinethe nuclear morphology of viable and necrotic cells Cellsstained green represent viable cells and ethidium bromideselectively stains the cells that have lost membrane integrityand stains DNA orange In this study acridine orange-ethidium bromide staining revealed that there was no changein cellular morphology including chromatin condensationmembrane blebbing and fragmented nuclei occurring dueto the treatment of extracts even at higher concentration(1000 120583gmL) Our results indicate that the tested extracts didnot induce haemolysis at a concentration of (1000120583gmL)Based on the above results the methanol and acetone extractsof T ornata were found to be nontoxic under in vitroconditions

GC-MS analysis revealed the presence of major bioac-tive compounds like hentriacontane z z-6 28-heptatriact-ontadien-2-one 8-heptadecene and 1-heptacosanol Hentri-acontane is the major component present in Oldenlandiadiffusa a natural source for the treatment of cancer in Asia[38] and also has reported activities like anti-inflammatoryand antioxidant properties [38 39] Currently there is noinformation regarding the antidiabetic effects of hentria-contane Further purification and characterization of activecompounds along with mechanism studies will only giveconclusive data on the antidiabetic property of tested algae

5 Conclusions

The present study indicates that crude extracts of T ornatapossess in vitro 120572-amylase 120572-glucosidase and DPP-IV

inhibitory activity Due to their strong enzyme inhibitoryactivity they are expected to suppress the glycemic responsein diabetic conditions Additionally the extracts are capableof scavenging free radicals in vitro toxicological parametersindicate that the IC

50of methanol and acetone extracts of T

ornata for 120572-amylase 120572-glucosidase and DPP-IV inhibitionare greatly below cytotoxic levels Future studies on in vivoantidiabetic activity of selected extracts and identification ofmajor compounds responsible for antidiabetic action are inprogress These studies will reveal the possible antidiabeticmechanisms of T ornata

Conflict of Interests

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

Acknowledgments

The authors wish to thank Department of BiotechnologyGovernment of India for their financial support (Grantno BTBio-CARe033472010-11) and VIT University forproviding all necessary facilities

References

[1] K G Alberti and P Z Zimmet ldquoDefinition diagnosis andclassification of diabetes mellitus and its complications Part1 diagnosis and classification of diabetes mellitus provisionalreport of a who consultationrdquo Diabetic Medicine vol 15 no 7pp 539ndash553 1998

[2] O P Gupta and S Phatak ldquoPandemic trends in prevalenceof diabetes mellitus and associated coronary heart disease in

BioMed Research International 9

India-their causes and preventionrdquo International Journal ofDiabetes in Developing Countries vol 23 pp 37ndash50 2003

[3] R R Ortiz-Andrade S Garcıa-Jimenez P Castillo-EspanaG Ramırez-Avila R Villalobos-Molina and S Estrada-Sotoldquo120572-Glucosidase inhibitory activity of the methanolic extractfrom Tournefortia hartwegiana an anti-hyperglycemic agentrdquoJournal of Ethnopharmacology vol 109 no 1 pp 48ndash53 2007

[4] G M Gray ldquoCarbohydrate digestion and absorption Role ofthe small intestinerdquo The New England Journal of Medicine vol292 no 23 pp 1225ndash1230 1975

[5] C A Tarling K Woods R Zhang et al ldquoThe search fornovel human pancreatic 120572-amylase inhibitors high-throughputscreening of terrestrial and marine natural product extractsrdquoChemBioChem vol 9 no 3 pp 433ndash438 2008

[6] R Mentlein B Gallwitz and W E Schmidt ldquoDipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide gluca-gon-like peptide-1(7-36)amide peptide histidine methionineand is responsible for their degradation in human serumrdquoEuropean Journal of Biochemistry vol 214 no 3 pp 829ndash8351993

[7] T Vilsboslashll T Krarup C F Deacon S Madsbad and J J HolstldquoReduced postprandial concentrations of intact biologicallyactive glucagon-like peptide 1 in type 2 diabetic patientsrdquoDiabetes vol 50 no 3 pp 609ndash613 2001

[8] M A Nauck U Niedereichholz R Ettler et al ldquoGlucagon-like peptide 1 inhibition of gastric emptying outweighs itsinsulinotropic effects in healthy humansrdquo American Journal ofPhysiologymdashEndocrinology and Metabolism vol 273 no 5 ppE981ndashE988 1997

[9] A-M Lambeir S Scharpe and I DeMeester ldquoDPP4 inhibitorsfor diabetesmdashwhat nextrdquo Biochemical Pharmacology vol 76no 12 pp 1637ndash1643 2008

[10] [EMEA] EuropeanMedicines Agency ldquoEuropean public assess-ment report (EPAR)mdashJanuviardquo 2007 httpwwwemaeuropaeuema

[11] [EMEA] EuropeanMedicines Agency ldquoEuropean public assess-ment report (EPAR)mdashGalvusrdquo 2007 httpwwwemaeuropaeuema

[12] A Murai K Iwamura M Takada K Ogawa T Usui andJ-I Okumura ldquoControl of postprandial hyperglycaemia bygalactosyl maltobionolactone and its novel anti-amylase effectin micerdquo Life Sciences vol 71 no 12 pp 1405ndash1415 2002

[13] B Halliwell and O I Aruoma ldquoDNA damage by oxygen-derived species Its mechanism and measurement in mam-malian systemsrdquo FEBS Letters vol 281 no 1-2 pp 9ndash19 1991

[14] J Zhou N Hu Y-L Wu Y-J Pan and C-R Sun ldquoPreliminarystudies on the chemical characterization and antioxidant prop-erties of acidic polysaccharides from Sargassum fusiformerdquoJournal of Zhejiang University Science B vol 9 no 9 pp 721ndash727 2008

[15] D A Butterfield A Castenga C B Pocernich J DrakeG Scapagnini and V Calabrese ldquoNutritional approaches tocombat oxidative stress in Alzheimerrsquos diseasesrdquo Journal ofNutritional Biochemistry vol 13 pp 444ndash461 2002

[16] A M S Mayer and V K B Lehmann ldquoMarine pharmacologyin 1998 marine compounds with antibacterial anticoagu-lant antifungal anti-inflammatory anthelmintic antiplateletantiprotozoal and antiviral activities with actions on the cardio-vascular endocrine immune and nervous systems and othermiscellaneous mechanisms of actionrdquo Pharmacologist vol 42pp 62ndash69 2000

[17] A Cumashi N A Ushakova M E Preobrazhenskaya et alldquoA comparative study of the anti-inflammatory anticoagulantantiangiogenic and antiadhesive activities of nine differentfucoidans from brown seaweedsrdquoGlycobiology vol 17 no 5 pp541ndash552 2007

[18] T Ghosh K Chattopadhyay M Marschall P Karmakar PMandal and B Ray ldquoFocus on antivirally active sulfatedpolysaccharides from structure-activity analysis to clinicalevaluationrdquo Glycobiology vol 19 no 1 pp 2ndash15 2009

[19] V H Pomin and P A S Mourao ldquoStructure biology evolutionand medical importance of sulfated fucans and galactansrdquoGlycobiology vol 18 no 12 pp 1016ndash1027 2008

[20] S-H Lee J-S Han S-J Heo J-Y Hwang and Y-J JeonldquoProtective effects of dieckol isolated fromEcklonia cava againsthigh glucose-induced oxidative stress in human umbilical veinendothelial cellsrdquo Toxicology in Vitro vol 24 no 2 pp 375ndash3812010

[21] S-H Lee F Karadeniz M-M Kim and S-K Kim ldquo120572-Glucosidase and 120572-amylase inhibitory activities of phlorogluci-nal derivatives from edible marine brown alga Ecklonia cavardquoJournal of the Science of Food and Agriculture vol 89 no 9 pp1552ndash1558 2009

[22] A Rohfritsch C Payri V Stiger and F Bonhomme ldquoMolec-ular and morphological relationships between two closelyrelated speciesTurbinaria ornata andT conoides (SargassaceaePhaeophyceae)rdquo Biochemical Systematics and Ecology vol 35no 2 pp 91ndash98 2007

[23] S Ananthi H R B Raghavendran A G Sunil V Gayathri GRamakrishnan and H R Vasanthi ldquoIn vitro antioxidant and invivo anti-inflammatory potential of crude polysaccharide fromTurbinaria ornata (Marine Brown Alga)rdquo Food and ChemicalToxicology vol 48 no 1 pp 187ndash192 2010

[24] F Asari T Kusumi and H Kakisawa ldquoTurbinaric acid acytotoxic secosqualene carboxylic acid from the brown algaTurbinaria ornatardquo Journal of Natural Products vol 52 no 5pp 1167ndash1169 1989

[25] N Chattopadhyay T Ghosh S Sinha K ChattopadhyayP Karmakar and B Ray ldquoPolysaccharides from Turbinariaconoides structural features and antioxidant capacityrdquo FoodChemistry vol 118 no 3 pp 823ndash829 2010

[26] J B Harborne ldquoMethods of extraction and isolationrdquo inPhytochemical Methods vol 3 pp 60ndash66 Chapman amp Hall1998

[27] L L Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plant extracts for antioxidant activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[28] M A Jayasri A Radha and T L Mathew ldquo120572-amylase and120572-glucosidase inhibitory activity of Costus pictus D Don inthe management of diabetesrdquo Journal of Herbal Medicine andToxicology vol 3 pp 91ndash94 2009

[29] IM Al-MasriM KMohammad andMO Tahaa ldquoInhibitionof dipeptidyl peptidase IV (DPP IV) is one of the mechanismsexplaining the hypoglycemic effect of berberinerdquo Journal ofEnzyme Inhibition and Medicinal Chemistry vol 24 no 5 pp1061ndash1066 2009

[30] D Malagoli ldquoA full-length protocol to test hemolytic activityof palytoxin on human erythrocytesrdquo Invertebrate SurvivalJournal vol 4 pp 92ndash94 2007

[31] V R Muzykantov G P Samokhin M D Smirnov andS P Domogatsky ldquoHemolytic complement activity assay in

10 BioMed Research International

microtitration platesrdquo Journal of Applied Biochemistry vol 7 no3 pp 223ndash227 1985

[32] A-R Kim T-S Shin M-S Lee et al ldquoIsolation and identifi-cation of phlorotannins from ecklonia stolonifera with antioxi-dant and anti-inflammatory propertiesrdquo Journal of Agriculturaland Food Chemistry vol 57 no 9 pp 3483ndash3489 2009

[33] J W Baynes ldquoChemical modification of proteins by lipids indiabetesrdquo Clinical Chemistry and Laboratory Medicine vol 41no 9 pp 1159ndash1165 2003

[34] H E Moon M N Islam B R Ahn et al ldquoProtein tyrosinephosphatase 1B and 120572-glucosidase inhibitory phlorotanninsfrom edible brown algae Ecklonia stolonifera and Eiseniabicyclisrdquo Bioscience Biotechnology and Biochemistry vol 75 no8 pp 1472ndash1480 2011

[35] E Apostolidis P D Karayannakidis Y-I Kwon C M Leeand N P Seeram ldquoSeasonal variation of phenolic antioxidant-mediated a-glucosidase inhibition of Ascophyllum nodosumrdquoPlant Foods forHumanNutrition vol 66 no 4 pp 313ndash319 2011

[36] F Nwosu J Morris V A Lund D Stewart H A Ross andG J McDougall ldquoAnti-proliferative and potential anti-diabeticeffects of phenolic-rich extracts from edible marine algaerdquo FoodChemistry vol 126 no 3 pp 1006ndash1012 2011

[37] G Galati and P J OBrien ldquoPotential toxicity of flavonoids andother dietary phenolics significance for their chemopreventiveand anticancer propertiesrdquo Free Radical Biology and Medicinevol 37 no 3 pp 287ndash303 2004

[38] S-J KimW-S Chung S-S Kim S-G Ko and J-Y Um ldquoAnti-inflammatory effect of Oldenlandia diffusa and its constituenthentriacontane through suppression of caspase-1 activation inmouse peritoneal macrophagesrdquo Phytotherapy Research vol 25no 10 pp 1537ndash1546 2011

[39] G Agoramoorthy M Chandrasekaran V Venkatesalu andM J Hsu ldquoAntibacterial and antifungal activities of fatty acidmethyl esters of the blind-your-eye mangrove from IndiardquoBrazilian Journal of Microbiology vol 38 no 4 pp 739ndash7422007

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

4 BioMed Research International

(a) (b)

Figure 1 (a)Macroscopic view of Turbinaria ornata collected fromMandapam coastal region in Gulf ofMannar Tamilnadu India Stiff erectstalks hard thick leaves (blades) characterized by lateral ridges and outermarginal blade with stiff row of spines are the unique characteristicsof this species (b) Microscopic image of Turbinaria ornata (magnification 40x)

Table 1 Qualitative phytochemical screening of various extracts of Turbinaria ornata (+ present minus absent)

Phytochemicals Pet ether Benzene Ethyl acetate Methanol AcetoneAlkaloids + + + + +Phenols + + + + +Flavanoids + + + + +Proteins and amino acid + + + + +Lipids + + + + +Carbohydrates + + + + +Saponins minus minus + minus minus

Glycosides minus minus + + minus

Tannins + + + minus minus

Oils and fats + minus minus minus minus

two-way ANOVA The values were considered to be signif-icantly different when the 119875 value was lt00001 compared tothe baseline values Software employed for statistical analysiswas Graph-Pad Prism Version 5

3 Results

31 Identification of Collected Seaweeds The samples wereidentified based on the morphological characteristics inresponse to environmental conditions stiff erect stalks hardthick leaves (blades) characterized by lateral ridges and outermarginal bladewith stiff rowof spines (Figure 1) Based on theabove characters seaweed species was identified as T ornataand authenticated byDr P Kaladharan principle scientist andscientist in charge Calicut Regional Centre of CentralMarineFisheries Research Institute

32 Phytochemical Analysis of T ornata Qualitative anal-ysis of the crude extracts of T ornata showed the pres-ence of major phytochemicals like alkaloids phenols fla-vanoids proteins lipids carbohydrates glycosides tanninsand saponins in all the studied extracts (Table 1)

33 22-Diphenyl-1-picrylhydrazyl (DPPH)Radical ScavengingActivity The antioxidant potential of T ornata was studiedthrough its scavenging ability of the stable radical DPPH

Acetone extract showed significant scavenging ability onDPPH (65) at a concentration of 1000120583gmL when com-pared with that of a standard BHT (97) However none ofthe extracts exhibited higher activity than BHT at the sameconcentration (Figure 2)

34 In Vitro 120572-Amylase Inhibition Assay Inhibitory effect ofT ornata on 120572-amylase is shown in Figure 3(a) Among thefive extracts studied (petroleum ether benzene ethyl acetatemethanol and acetone) three extracts showed (petroleumether methanol and acetone) significant 120572-amylase inhi-bition at all the tested concentrations (250 500 750 and1000 120583gmL) Methanol extract of T ornata showed max-imum inhibition of 965 with an IC

50of 2509 120583gmL

followed by acetone extract (876) at a concentration of1000 120583gmL

35 In Vitro 120572-Glucosidase Inhibition Assay The 120572-gluco-sidase inhibitory activity of T Ornata extracts was deter-mined using p-nitrophenyl 120572-D-glucopyranoside as a sub-strate and these were compared with a standard acarbose(88) (Figure 3(b)) At a concentration of 1000 120583gmL ace-tone extract showed maximum inhibition (876) with anIC50value of 5356 120583gmL The inhibitory activity of acetone

extracts was found to be more potent and similar whencompared to the positive control acarbose

BioMed Research International 5

0

50

250 500 750 1000

100

150

Petroleum etherBenzeneEthyl acetate

MethanolAcetoneBHT

Concentration (120583gmL)

Inhi

bitio

n (

)

a

a

a

aa

a

a a a a

a

aaa a a

a

a

a

aa

a

a

a

Figure 2 DPPH radical scavenging activity of petroleum etherbenzene ethyl acetatemethanol and benzene extracts ofTurbinariaornata BHT is used as positive control and absorbance wasmeasured at 517 nm Values are means plusmn SD (119899 = 3) (a119875 lt 00001considered as significant)

36 In Vitro Dipeptidyl Peptidase-IV (DPP-IV) InhibitionAssay T ornata extracts were evaluated for their mode ofaction to stimulate insulin secretion through inhibition ofDPP-IV enzyme The effectiveness of various extracts wasevaluated on the basis of percentage inhibition and IC

50

values obtained Among the five different solvent extracts(petroleum ether benzene ethyl acetate methanol andacetone) methanol extracts showed maximum percentageinhibition of 554 with an IC

50value of 552120583gmL at a

maximum concentration of 80 120583gmL All the results werecompared with a standard diprotin A (Figure 4)

37 Cytotoxic Effect of T ornata Extracts on J774 Cell Line Toevaluate the cytotoxic effect of the extracts J774 cell line wasincubated for 24 h with various concentrations of extracts Asshown in Figure 5(a) the decrease in viability correlates withthe increase in concentration MTT assay indicates that J774cells treated with various extracts of T ornata were safe anddid not show any toxic effect against the cell treated at a lowerconcentration of 250120583gmL The observed cytotoxicity wasfound to be 257 when treated with methanol extract of Tornata 50 of cells were found to be viable when treatedwitha higher concentration of 1000120583gmL

38 DNA Fragmentation Assay Staining cells with acridineorange-ethidium bromide is used to measure the proportionof viable apoptotic and necrotic cells J774 cells treated witha maximum concentration of 1000120583gmL of algal extractswere found to be viable and there was no change in cellularmorphology including chromatin condensation membraneblebbing and fragmented nuclei (Figure 6)

39 Haemolytic Assay The lead extracts methanol (278)and acetone (461) were found to be nontoxic at a dosageof 1000120583gmL (Figure 5(b)) Also the extracts did not showany erythrocyte membrane damage at all the tested concen-trations

310 GC-MS Analysis of the Acetone Extract of T ornataThe crude extracts were analyzed by GC-MS and comparedwith the retention time (RT) and mass spectra availablein the data library of NIST The GC-MS analysis revealsthe presence of major bioactive compounds like hentriacon-tane z z-6 28-heptatriactontadien-2-one 8-heptadeceneand 1-heptacosanol Chromatograms obtained were shown inFigure 7

4 Discussion

Marine seaweeds are good source of dietary fibres polysac-charides polyphenols polyunsaturated fatty acids mineralsand vitamins which possess various biological activities likeantidiabetic anti-inflammatory and antioxidant activity [32]Although this evidence seems to be valid detailed researchshould be carried out to ensure the presence of various phy-tochemicals present in the seaweed extracts In the currentstudy marine seaweed T ornata was extracted and screenedfor both nonpolar and polar compounds using varioussolvents based on polarity Qualitative analysis showed thepresence of major bioactive compounds like alkaloids phe-nols flavanoids glycosides lipids carbohydrates proteinstannins oils and fats Presence of these bioactive moleculesin various extracts of this seaweed may be responsible forvarious biological activities (antiviral anti-inflammatory andanticoagulant) [17ndash19]

The objective of this study was to investigate the antidia-betic and antioxidant potential of marine seaweed T ornataand to enumerate major phytocompounds present in theextracts One of the major causes for diabetes is due tothe increased production of reactive oxygen species orimpaired antioxidant defense systemwhich leads to oxidativedamage of beta cells [33] In this study acetone extract ofT ornata showed significant scavenging capability againstDPPH (65) at a maximum concentration of 1000 120583gmLcompared with a positive control BHT (97) As previouslyreported by Ananthi et al and Chattopadhyay et al [2325] the fucoidan fraction of Turbinaria conoides (61) ata concentration of 1000 120583gmL and aqueous extract of Tornata (80) at a concentration of 500 120583gmL showed thesimilar scavenging capability on DPPH radicals Our resultswere similar to these reports which confirm the antioxidantactivity of T ornata

In the present study the edible seaweed T ornata wasevaluated for in vitro 120572-amylase 120572-glucosidase and DPP-IV inhibitory property Among the five extracts studiedpetroleum ether acetone and methanol extracts showedsignificant percentage inhibition Methanol extract of Tornata showed maximum percentage inhibition on 120572-amylase (965)with an IC

50value of 2509120583gmLwithmore

inhibition than positive control acarbose (88) Acetone

6 BioMed Research International

0

50

100

150In

hibi

tion

()

Petroleum etherMethanol

AcetoneAcarbose

a

aa

a

a

a

a

a a

a

aa a

a a a

250 500 750 1000Concentration (120583gmL)

IC50 value of methanol extract 2509 120583gmL

(a)

0

20

40

60

80

100

Petroleum etherMethanol

AcetoneAcarbose

Inhi

bitio

n (

)

a a

a

a

aa

a

aa

a

a

aa

a

a

a

250 500 750 1000Concentration (120583gmL)

IC50 value of acetone extract 5356 120583gmL

(b)

Figure 3 (a) In vitro 120572-amylase inhibitory activity of petroleum ether methanol and acetone extracts of Turbinaria ornata Absorbance wasmeasured at 540 nm (b) In vitro 120572-glucosidase inhibitory activity of petroleum ether methanol and acetone extracts of Turbinaria ornataacarbose is used as positive control and absorbance was measured at 405 nm Values are means plusmn SD (119899 = 3) (a119875 lt 00001 consideredsignificant)

3002 04 08 16 32 64

40

50

60

70

Inhi

bitio

n (

)

aa

a

a

a

a

Concentration (120583gmL)

(a)

025 10 40 80

20

40

60

Inhi

bitio

n (

) a a a a aa

a

a

a aa

a

aa

a a

aa

a a

Concentration (120583gmL)

Petroleum etherBenzeneAcetone

Ethyl acetateMethanol

IC50 value of methanol extract 552 120583gmL

(b)

Figure 4 (a) DPP-IV inhibitory activity of diprotin A as positive control (b) DPP-IV inhibitory activity of petroleum ether benzene ethylacetate methanol and acetone extracts of Turbinaria ornata Absorbance was measured at 405 nm Values are means plusmn SD (119899 = 3) (a119875 lt00001 considered significant)

extract of T ornata showed more potent inhibition on120572-glucosidase (843) with an IC

50value of 5356 120583gmL

at a concentration of 1000120583gmL similar to the positivecontrol acarbose (88) The DPP-IV inhibitory potential ofmethanol extract of T ornata showed (554) inhibitionwith an IC

50value of 552120583gmL which is comparable with

that of positive control diprotin A (65) Similar findingswere shown by two brown seaweeds Ecklonia stoloniferaOkumura and Eisenia bicyclis [34] Several studies reportedthat the 120572-amylase and 120572-glucosidase inhibitory action of theclass Phaeophyceae [35 36] supports that these algal speciesare excellent source for antidiabetic agents Apart from 120572-amylase and 120572-glucosidase T ornata extracts also showedsignificant DPP-IV inhibition Based on these results this

seaweed can be used as alternative therapy for diabetes byraising the levels of endogenous incretin hormones (GLP-1and GIP)

In this study we also investigated the effects of crudeextracts (methanol acetone) of T ornata in macrophagecell line (J774) The cytotoxic effects of the extracts weredetermined by using anMTT assay After 24 h exposure 250ndash1000 120583gmL of methanol and acetone extracts significantlydecreased cell viability by increasing concentration As pre-viously reported seaweed extracts which have higher phenolcontent may explain the level of cytotoxicity at higher con-centrations (1000 120583gmL) [36 37] whereby up to 500120583gmLof seaweed extracts did not show any effect on J774 cellviability andmore than 90of cells were found to be viable at

BioMed Research International 7

0

50

100

150C

ell v

iabi

lity

() a a

aa

aa a

a

250 750 1000500

MethanolAcetone

Concentration (120583gmL)

(a)

0250 750 1000500

2

4

6

MethanolAcetone

Cell

lysis

()

a a a

a

a

a

a

a

Concentration (120583gmL)

(b)

Figure 5 (a) Cell viability determined byMTT assay J774 cells treated with methanol and acetone extracts of Turbinaria ornata Absorbancewasmeasured at 630 nm (b)Haemolytic activity ofmethanol and acetone extracts ofTurbinaria ornata Absorbance wasmeasured at 630 nmValues are means plusmn SD (119899 = 3) (a119875 lt 00001 considered significant)

Control

(a)

TO M

(b)

Control

(c)

Treated

(d)

Control

(e)

Treated

(f)

Figure 6 (a) Fluorescent photomicrographs (100x magnification) of J774 cells treated without seaweed extracts (b) J774 cells treated withTurbinaria ornatamethanol extract (TOM) (c) Phase contrast microscopic image of J774 cells treated with media alone (control) (d) Phasecontrast microscopic image of J774 cells treated with test extracts ((e)-(f)) Microscopic image (40x magnification) of in vitro haemolyticactivity

8 BioMed Research International

A

R3A-(13IS-1200)

B

500

100(

)

01000 1500 2000 2500 3000

Time

515e8TIC

Scan El+

OH

317

0308

130

28

294

528

36

277

627

17

264

3257

525

05

243

924

33

236

423

59

228

222

03

220

721

24

212

020

37

203

320

28

192

218

49

181

017

64

172

516

47

161

015

12

141

8

164

0

184

3

C

A

B

5111

31

33333337030

333

8130

3333333333

2829

45

28

22222222236

277

627

22222

1726

4325

75

250

524

39

243

323

222

6423

22

5922

82

220

322

07

21

2224

212

020

37

203

320

28

192

218

49

181

017

71764

172

516

47

16

110

151

214

18

164

0

18

188181818181881881881888888818881881888811111143444444444444

C

Figure 7 Gas chromatogram of acetone extract of Turbinaria ornata (A) 8-heptadecene (B) 1-heptacosanol and (C) hentriacontane

a concentration of 250120583gmL Staining cells with fluorescentdyes is used to differentiate the nuclear morphology of liveand apoptotic cells J774 cells were analyzed in the presenceof acridine orange and ethidium bromide to determinethe nuclear morphology of viable and necrotic cells Cellsstained green represent viable cells and ethidium bromideselectively stains the cells that have lost membrane integrityand stains DNA orange In this study acridine orange-ethidium bromide staining revealed that there was no changein cellular morphology including chromatin condensationmembrane blebbing and fragmented nuclei occurring dueto the treatment of extracts even at higher concentration(1000 120583gmL) Our results indicate that the tested extracts didnot induce haemolysis at a concentration of (1000120583gmL)Based on the above results the methanol and acetone extractsof T ornata were found to be nontoxic under in vitroconditions

GC-MS analysis revealed the presence of major bioac-tive compounds like hentriacontane z z-6 28-heptatriact-ontadien-2-one 8-heptadecene and 1-heptacosanol Hentri-acontane is the major component present in Oldenlandiadiffusa a natural source for the treatment of cancer in Asia[38] and also has reported activities like anti-inflammatoryand antioxidant properties [38 39] Currently there is noinformation regarding the antidiabetic effects of hentria-contane Further purification and characterization of activecompounds along with mechanism studies will only giveconclusive data on the antidiabetic property of tested algae

5 Conclusions

The present study indicates that crude extracts of T ornatapossess in vitro 120572-amylase 120572-glucosidase and DPP-IV

inhibitory activity Due to their strong enzyme inhibitoryactivity they are expected to suppress the glycemic responsein diabetic conditions Additionally the extracts are capableof scavenging free radicals in vitro toxicological parametersindicate that the IC

50of methanol and acetone extracts of T

ornata for 120572-amylase 120572-glucosidase and DPP-IV inhibitionare greatly below cytotoxic levels Future studies on in vivoantidiabetic activity of selected extracts and identification ofmajor compounds responsible for antidiabetic action are inprogress These studies will reveal the possible antidiabeticmechanisms of T ornata

Conflict of Interests

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

Acknowledgments

The authors wish to thank Department of BiotechnologyGovernment of India for their financial support (Grantno BTBio-CARe033472010-11) and VIT University forproviding all necessary facilities

References

[1] K G Alberti and P Z Zimmet ldquoDefinition diagnosis andclassification of diabetes mellitus and its complications Part1 diagnosis and classification of diabetes mellitus provisionalreport of a who consultationrdquo Diabetic Medicine vol 15 no 7pp 539ndash553 1998

[2] O P Gupta and S Phatak ldquoPandemic trends in prevalenceof diabetes mellitus and associated coronary heart disease in

BioMed Research International 9

India-their causes and preventionrdquo International Journal ofDiabetes in Developing Countries vol 23 pp 37ndash50 2003

[3] R R Ortiz-Andrade S Garcıa-Jimenez P Castillo-EspanaG Ramırez-Avila R Villalobos-Molina and S Estrada-Sotoldquo120572-Glucosidase inhibitory activity of the methanolic extractfrom Tournefortia hartwegiana an anti-hyperglycemic agentrdquoJournal of Ethnopharmacology vol 109 no 1 pp 48ndash53 2007

[4] G M Gray ldquoCarbohydrate digestion and absorption Role ofthe small intestinerdquo The New England Journal of Medicine vol292 no 23 pp 1225ndash1230 1975

[5] C A Tarling K Woods R Zhang et al ldquoThe search fornovel human pancreatic 120572-amylase inhibitors high-throughputscreening of terrestrial and marine natural product extractsrdquoChemBioChem vol 9 no 3 pp 433ndash438 2008

[6] R Mentlein B Gallwitz and W E Schmidt ldquoDipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide gluca-gon-like peptide-1(7-36)amide peptide histidine methionineand is responsible for their degradation in human serumrdquoEuropean Journal of Biochemistry vol 214 no 3 pp 829ndash8351993

[7] T Vilsboslashll T Krarup C F Deacon S Madsbad and J J HolstldquoReduced postprandial concentrations of intact biologicallyactive glucagon-like peptide 1 in type 2 diabetic patientsrdquoDiabetes vol 50 no 3 pp 609ndash613 2001

[8] M A Nauck U Niedereichholz R Ettler et al ldquoGlucagon-like peptide 1 inhibition of gastric emptying outweighs itsinsulinotropic effects in healthy humansrdquo American Journal ofPhysiologymdashEndocrinology and Metabolism vol 273 no 5 ppE981ndashE988 1997

[9] A-M Lambeir S Scharpe and I DeMeester ldquoDPP4 inhibitorsfor diabetesmdashwhat nextrdquo Biochemical Pharmacology vol 76no 12 pp 1637ndash1643 2008

[10] [EMEA] EuropeanMedicines Agency ldquoEuropean public assess-ment report (EPAR)mdashJanuviardquo 2007 httpwwwemaeuropaeuema

[11] [EMEA] EuropeanMedicines Agency ldquoEuropean public assess-ment report (EPAR)mdashGalvusrdquo 2007 httpwwwemaeuropaeuema

[12] A Murai K Iwamura M Takada K Ogawa T Usui andJ-I Okumura ldquoControl of postprandial hyperglycaemia bygalactosyl maltobionolactone and its novel anti-amylase effectin micerdquo Life Sciences vol 71 no 12 pp 1405ndash1415 2002

[13] B Halliwell and O I Aruoma ldquoDNA damage by oxygen-derived species Its mechanism and measurement in mam-malian systemsrdquo FEBS Letters vol 281 no 1-2 pp 9ndash19 1991

[14] J Zhou N Hu Y-L Wu Y-J Pan and C-R Sun ldquoPreliminarystudies on the chemical characterization and antioxidant prop-erties of acidic polysaccharides from Sargassum fusiformerdquoJournal of Zhejiang University Science B vol 9 no 9 pp 721ndash727 2008

[15] D A Butterfield A Castenga C B Pocernich J DrakeG Scapagnini and V Calabrese ldquoNutritional approaches tocombat oxidative stress in Alzheimerrsquos diseasesrdquo Journal ofNutritional Biochemistry vol 13 pp 444ndash461 2002

[16] A M S Mayer and V K B Lehmann ldquoMarine pharmacologyin 1998 marine compounds with antibacterial anticoagu-lant antifungal anti-inflammatory anthelmintic antiplateletantiprotozoal and antiviral activities with actions on the cardio-vascular endocrine immune and nervous systems and othermiscellaneous mechanisms of actionrdquo Pharmacologist vol 42pp 62ndash69 2000

[17] A Cumashi N A Ushakova M E Preobrazhenskaya et alldquoA comparative study of the anti-inflammatory anticoagulantantiangiogenic and antiadhesive activities of nine differentfucoidans from brown seaweedsrdquoGlycobiology vol 17 no 5 pp541ndash552 2007

[18] T Ghosh K Chattopadhyay M Marschall P Karmakar PMandal and B Ray ldquoFocus on antivirally active sulfatedpolysaccharides from structure-activity analysis to clinicalevaluationrdquo Glycobiology vol 19 no 1 pp 2ndash15 2009

[19] V H Pomin and P A S Mourao ldquoStructure biology evolutionand medical importance of sulfated fucans and galactansrdquoGlycobiology vol 18 no 12 pp 1016ndash1027 2008

[20] S-H Lee J-S Han S-J Heo J-Y Hwang and Y-J JeonldquoProtective effects of dieckol isolated fromEcklonia cava againsthigh glucose-induced oxidative stress in human umbilical veinendothelial cellsrdquo Toxicology in Vitro vol 24 no 2 pp 375ndash3812010

[21] S-H Lee F Karadeniz M-M Kim and S-K Kim ldquo120572-Glucosidase and 120572-amylase inhibitory activities of phlorogluci-nal derivatives from edible marine brown alga Ecklonia cavardquoJournal of the Science of Food and Agriculture vol 89 no 9 pp1552ndash1558 2009

[22] A Rohfritsch C Payri V Stiger and F Bonhomme ldquoMolec-ular and morphological relationships between two closelyrelated speciesTurbinaria ornata andT conoides (SargassaceaePhaeophyceae)rdquo Biochemical Systematics and Ecology vol 35no 2 pp 91ndash98 2007

[23] S Ananthi H R B Raghavendran A G Sunil V Gayathri GRamakrishnan and H R Vasanthi ldquoIn vitro antioxidant and invivo anti-inflammatory potential of crude polysaccharide fromTurbinaria ornata (Marine Brown Alga)rdquo Food and ChemicalToxicology vol 48 no 1 pp 187ndash192 2010

[24] F Asari T Kusumi and H Kakisawa ldquoTurbinaric acid acytotoxic secosqualene carboxylic acid from the brown algaTurbinaria ornatardquo Journal of Natural Products vol 52 no 5pp 1167ndash1169 1989

[25] N Chattopadhyay T Ghosh S Sinha K ChattopadhyayP Karmakar and B Ray ldquoPolysaccharides from Turbinariaconoides structural features and antioxidant capacityrdquo FoodChemistry vol 118 no 3 pp 823ndash829 2010

[26] J B Harborne ldquoMethods of extraction and isolationrdquo inPhytochemical Methods vol 3 pp 60ndash66 Chapman amp Hall1998

[27] L L Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plant extracts for antioxidant activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[28] M A Jayasri A Radha and T L Mathew ldquo120572-amylase and120572-glucosidase inhibitory activity of Costus pictus D Don inthe management of diabetesrdquo Journal of Herbal Medicine andToxicology vol 3 pp 91ndash94 2009

[29] IM Al-MasriM KMohammad andMO Tahaa ldquoInhibitionof dipeptidyl peptidase IV (DPP IV) is one of the mechanismsexplaining the hypoglycemic effect of berberinerdquo Journal ofEnzyme Inhibition and Medicinal Chemistry vol 24 no 5 pp1061ndash1066 2009

[30] D Malagoli ldquoA full-length protocol to test hemolytic activityof palytoxin on human erythrocytesrdquo Invertebrate SurvivalJournal vol 4 pp 92ndash94 2007

[31] V R Muzykantov G P Samokhin M D Smirnov andS P Domogatsky ldquoHemolytic complement activity assay in

10 BioMed Research International

microtitration platesrdquo Journal of Applied Biochemistry vol 7 no3 pp 223ndash227 1985

[32] A-R Kim T-S Shin M-S Lee et al ldquoIsolation and identifi-cation of phlorotannins from ecklonia stolonifera with antioxi-dant and anti-inflammatory propertiesrdquo Journal of Agriculturaland Food Chemistry vol 57 no 9 pp 3483ndash3489 2009

[33] J W Baynes ldquoChemical modification of proteins by lipids indiabetesrdquo Clinical Chemistry and Laboratory Medicine vol 41no 9 pp 1159ndash1165 2003

[34] H E Moon M N Islam B R Ahn et al ldquoProtein tyrosinephosphatase 1B and 120572-glucosidase inhibitory phlorotanninsfrom edible brown algae Ecklonia stolonifera and Eiseniabicyclisrdquo Bioscience Biotechnology and Biochemistry vol 75 no8 pp 1472ndash1480 2011

[35] E Apostolidis P D Karayannakidis Y-I Kwon C M Leeand N P Seeram ldquoSeasonal variation of phenolic antioxidant-mediated a-glucosidase inhibition of Ascophyllum nodosumrdquoPlant Foods forHumanNutrition vol 66 no 4 pp 313ndash319 2011

[36] F Nwosu J Morris V A Lund D Stewart H A Ross andG J McDougall ldquoAnti-proliferative and potential anti-diabeticeffects of phenolic-rich extracts from edible marine algaerdquo FoodChemistry vol 126 no 3 pp 1006ndash1012 2011

[37] G Galati and P J OBrien ldquoPotential toxicity of flavonoids andother dietary phenolics significance for their chemopreventiveand anticancer propertiesrdquo Free Radical Biology and Medicinevol 37 no 3 pp 287ndash303 2004

[38] S-J KimW-S Chung S-S Kim S-G Ko and J-Y Um ldquoAnti-inflammatory effect of Oldenlandia diffusa and its constituenthentriacontane through suppression of caspase-1 activation inmouse peritoneal macrophagesrdquo Phytotherapy Research vol 25no 10 pp 1537ndash1546 2011

[39] G Agoramoorthy M Chandrasekaran V Venkatesalu andM J Hsu ldquoAntibacterial and antifungal activities of fatty acidmethyl esters of the blind-your-eye mangrove from IndiardquoBrazilian Journal of Microbiology vol 38 no 4 pp 739ndash7422007

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 5

0

50

250 500 750 1000

100

150

Petroleum etherBenzeneEthyl acetate

MethanolAcetoneBHT

Concentration (120583gmL)

Inhi

bitio

n (

)

a

a

a

aa

a

a a a a

a

aaa a a

a

a

a

aa

a

a

a

Figure 2 DPPH radical scavenging activity of petroleum etherbenzene ethyl acetatemethanol and benzene extracts ofTurbinariaornata BHT is used as positive control and absorbance wasmeasured at 517 nm Values are means plusmn SD (119899 = 3) (a119875 lt 00001considered as significant)

36 In Vitro Dipeptidyl Peptidase-IV (DPP-IV) InhibitionAssay T ornata extracts were evaluated for their mode ofaction to stimulate insulin secretion through inhibition ofDPP-IV enzyme The effectiveness of various extracts wasevaluated on the basis of percentage inhibition and IC

50

values obtained Among the five different solvent extracts(petroleum ether benzene ethyl acetate methanol andacetone) methanol extracts showed maximum percentageinhibition of 554 with an IC

50value of 552120583gmL at a

maximum concentration of 80 120583gmL All the results werecompared with a standard diprotin A (Figure 4)

37 Cytotoxic Effect of T ornata Extracts on J774 Cell Line Toevaluate the cytotoxic effect of the extracts J774 cell line wasincubated for 24 h with various concentrations of extracts Asshown in Figure 5(a) the decrease in viability correlates withthe increase in concentration MTT assay indicates that J774cells treated with various extracts of T ornata were safe anddid not show any toxic effect against the cell treated at a lowerconcentration of 250120583gmL The observed cytotoxicity wasfound to be 257 when treated with methanol extract of Tornata 50 of cells were found to be viable when treatedwitha higher concentration of 1000120583gmL

38 DNA Fragmentation Assay Staining cells with acridineorange-ethidium bromide is used to measure the proportionof viable apoptotic and necrotic cells J774 cells treated witha maximum concentration of 1000120583gmL of algal extractswere found to be viable and there was no change in cellularmorphology including chromatin condensation membraneblebbing and fragmented nuclei (Figure 6)

39 Haemolytic Assay The lead extracts methanol (278)and acetone (461) were found to be nontoxic at a dosageof 1000120583gmL (Figure 5(b)) Also the extracts did not showany erythrocyte membrane damage at all the tested concen-trations

310 GC-MS Analysis of the Acetone Extract of T ornataThe crude extracts were analyzed by GC-MS and comparedwith the retention time (RT) and mass spectra availablein the data library of NIST The GC-MS analysis revealsthe presence of major bioactive compounds like hentriacon-tane z z-6 28-heptatriactontadien-2-one 8-heptadeceneand 1-heptacosanol Chromatograms obtained were shown inFigure 7

4 Discussion

Marine seaweeds are good source of dietary fibres polysac-charides polyphenols polyunsaturated fatty acids mineralsand vitamins which possess various biological activities likeantidiabetic anti-inflammatory and antioxidant activity [32]Although this evidence seems to be valid detailed researchshould be carried out to ensure the presence of various phy-tochemicals present in the seaweed extracts In the currentstudy marine seaweed T ornata was extracted and screenedfor both nonpolar and polar compounds using varioussolvents based on polarity Qualitative analysis showed thepresence of major bioactive compounds like alkaloids phe-nols flavanoids glycosides lipids carbohydrates proteinstannins oils and fats Presence of these bioactive moleculesin various extracts of this seaweed may be responsible forvarious biological activities (antiviral anti-inflammatory andanticoagulant) [17ndash19]

The objective of this study was to investigate the antidia-betic and antioxidant potential of marine seaweed T ornataand to enumerate major phytocompounds present in theextracts One of the major causes for diabetes is due tothe increased production of reactive oxygen species orimpaired antioxidant defense systemwhich leads to oxidativedamage of beta cells [33] In this study acetone extract ofT ornata showed significant scavenging capability againstDPPH (65) at a maximum concentration of 1000 120583gmLcompared with a positive control BHT (97) As previouslyreported by Ananthi et al and Chattopadhyay et al [2325] the fucoidan fraction of Turbinaria conoides (61) ata concentration of 1000 120583gmL and aqueous extract of Tornata (80) at a concentration of 500 120583gmL showed thesimilar scavenging capability on DPPH radicals Our resultswere similar to these reports which confirm the antioxidantactivity of T ornata

In the present study the edible seaweed T ornata wasevaluated for in vitro 120572-amylase 120572-glucosidase and DPP-IV inhibitory property Among the five extracts studiedpetroleum ether acetone and methanol extracts showedsignificant percentage inhibition Methanol extract of Tornata showed maximum percentage inhibition on 120572-amylase (965)with an IC

50value of 2509120583gmLwithmore

inhibition than positive control acarbose (88) Acetone

6 BioMed Research International

0

50

100

150In

hibi

tion

()

Petroleum etherMethanol

AcetoneAcarbose

a

aa

a

a

a

a

a a

a

aa a

a a a

250 500 750 1000Concentration (120583gmL)

IC50 value of methanol extract 2509 120583gmL

(a)

0

20

40

60

80

100

Petroleum etherMethanol

AcetoneAcarbose

Inhi

bitio

n (

)

a a

a

a

aa

a

aa

a

a

aa

a

a

a

250 500 750 1000Concentration (120583gmL)

IC50 value of acetone extract 5356 120583gmL

(b)

Figure 3 (a) In vitro 120572-amylase inhibitory activity of petroleum ether methanol and acetone extracts of Turbinaria ornata Absorbance wasmeasured at 540 nm (b) In vitro 120572-glucosidase inhibitory activity of petroleum ether methanol and acetone extracts of Turbinaria ornataacarbose is used as positive control and absorbance was measured at 405 nm Values are means plusmn SD (119899 = 3) (a119875 lt 00001 consideredsignificant)

3002 04 08 16 32 64

40

50

60

70

Inhi

bitio

n (

)

aa

a

a

a

a

Concentration (120583gmL)

(a)

025 10 40 80

20

40

60

Inhi

bitio

n (

) a a a a aa

a

a

a aa

a

aa

a a

aa

a a

Concentration (120583gmL)

Petroleum etherBenzeneAcetone

Ethyl acetateMethanol

IC50 value of methanol extract 552 120583gmL

(b)

Figure 4 (a) DPP-IV inhibitory activity of diprotin A as positive control (b) DPP-IV inhibitory activity of petroleum ether benzene ethylacetate methanol and acetone extracts of Turbinaria ornata Absorbance was measured at 405 nm Values are means plusmn SD (119899 = 3) (a119875 lt00001 considered significant)

extract of T ornata showed more potent inhibition on120572-glucosidase (843) with an IC

50value of 5356 120583gmL

at a concentration of 1000120583gmL similar to the positivecontrol acarbose (88) The DPP-IV inhibitory potential ofmethanol extract of T ornata showed (554) inhibitionwith an IC

50value of 552120583gmL which is comparable with

that of positive control diprotin A (65) Similar findingswere shown by two brown seaweeds Ecklonia stoloniferaOkumura and Eisenia bicyclis [34] Several studies reportedthat the 120572-amylase and 120572-glucosidase inhibitory action of theclass Phaeophyceae [35 36] supports that these algal speciesare excellent source for antidiabetic agents Apart from 120572-amylase and 120572-glucosidase T ornata extracts also showedsignificant DPP-IV inhibition Based on these results this

seaweed can be used as alternative therapy for diabetes byraising the levels of endogenous incretin hormones (GLP-1and GIP)

In this study we also investigated the effects of crudeextracts (methanol acetone) of T ornata in macrophagecell line (J774) The cytotoxic effects of the extracts weredetermined by using anMTT assay After 24 h exposure 250ndash1000 120583gmL of methanol and acetone extracts significantlydecreased cell viability by increasing concentration As pre-viously reported seaweed extracts which have higher phenolcontent may explain the level of cytotoxicity at higher con-centrations (1000 120583gmL) [36 37] whereby up to 500120583gmLof seaweed extracts did not show any effect on J774 cellviability andmore than 90of cells were found to be viable at

BioMed Research International 7

0

50

100

150C

ell v

iabi

lity

() a a

aa

aa a

a

250 750 1000500

MethanolAcetone

Concentration (120583gmL)

(a)

0250 750 1000500

2

4

6

MethanolAcetone

Cell

lysis

()

a a a

a

a

a

a

a

Concentration (120583gmL)

(b)

Figure 5 (a) Cell viability determined byMTT assay J774 cells treated with methanol and acetone extracts of Turbinaria ornata Absorbancewasmeasured at 630 nm (b)Haemolytic activity ofmethanol and acetone extracts ofTurbinaria ornata Absorbance wasmeasured at 630 nmValues are means plusmn SD (119899 = 3) (a119875 lt 00001 considered significant)

Control

(a)

TO M

(b)

Control

(c)

Treated

(d)

Control

(e)

Treated

(f)

Figure 6 (a) Fluorescent photomicrographs (100x magnification) of J774 cells treated without seaweed extracts (b) J774 cells treated withTurbinaria ornatamethanol extract (TOM) (c) Phase contrast microscopic image of J774 cells treated with media alone (control) (d) Phasecontrast microscopic image of J774 cells treated with test extracts ((e)-(f)) Microscopic image (40x magnification) of in vitro haemolyticactivity

8 BioMed Research International

A

R3A-(13IS-1200)

B

500

100(

)

01000 1500 2000 2500 3000

Time

515e8TIC

Scan El+

OH

317

0308

130

28

294

528

36

277

627

17

264

3257

525

05

243

924

33

236

423

59

228

222

03

220

721

24

212

020

37

203

320

28

192

218

49

181

017

64

172

516

47

161

015

12

141

8

164

0

184

3

C

A

B

5111

31

33333337030

333

8130

3333333333

2829

45

28

22222222236

277

627

22222

1726

4325

75

250

524

39

243

323

222

6423

22

5922

82

220

322

07

21

2224

212

020

37

203

320

28

192

218

49

181

017

71764

172

516

47

16

110

151

214

18

164

0

18

188181818181881881881888888818881881888811111143444444444444

C

Figure 7 Gas chromatogram of acetone extract of Turbinaria ornata (A) 8-heptadecene (B) 1-heptacosanol and (C) hentriacontane

a concentration of 250120583gmL Staining cells with fluorescentdyes is used to differentiate the nuclear morphology of liveand apoptotic cells J774 cells were analyzed in the presenceof acridine orange and ethidium bromide to determinethe nuclear morphology of viable and necrotic cells Cellsstained green represent viable cells and ethidium bromideselectively stains the cells that have lost membrane integrityand stains DNA orange In this study acridine orange-ethidium bromide staining revealed that there was no changein cellular morphology including chromatin condensationmembrane blebbing and fragmented nuclei occurring dueto the treatment of extracts even at higher concentration(1000 120583gmL) Our results indicate that the tested extracts didnot induce haemolysis at a concentration of (1000120583gmL)Based on the above results the methanol and acetone extractsof T ornata were found to be nontoxic under in vitroconditions

GC-MS analysis revealed the presence of major bioac-tive compounds like hentriacontane z z-6 28-heptatriact-ontadien-2-one 8-heptadecene and 1-heptacosanol Hentri-acontane is the major component present in Oldenlandiadiffusa a natural source for the treatment of cancer in Asia[38] and also has reported activities like anti-inflammatoryand antioxidant properties [38 39] Currently there is noinformation regarding the antidiabetic effects of hentria-contane Further purification and characterization of activecompounds along with mechanism studies will only giveconclusive data on the antidiabetic property of tested algae

5 Conclusions

The present study indicates that crude extracts of T ornatapossess in vitro 120572-amylase 120572-glucosidase and DPP-IV

inhibitory activity Due to their strong enzyme inhibitoryactivity they are expected to suppress the glycemic responsein diabetic conditions Additionally the extracts are capableof scavenging free radicals in vitro toxicological parametersindicate that the IC

50of methanol and acetone extracts of T

ornata for 120572-amylase 120572-glucosidase and DPP-IV inhibitionare greatly below cytotoxic levels Future studies on in vivoantidiabetic activity of selected extracts and identification ofmajor compounds responsible for antidiabetic action are inprogress These studies will reveal the possible antidiabeticmechanisms of T ornata

Conflict of Interests

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

Acknowledgments

The authors wish to thank Department of BiotechnologyGovernment of India for their financial support (Grantno BTBio-CARe033472010-11) and VIT University forproviding all necessary facilities

References

[1] K G Alberti and P Z Zimmet ldquoDefinition diagnosis andclassification of diabetes mellitus and its complications Part1 diagnosis and classification of diabetes mellitus provisionalreport of a who consultationrdquo Diabetic Medicine vol 15 no 7pp 539ndash553 1998

[2] O P Gupta and S Phatak ldquoPandemic trends in prevalenceof diabetes mellitus and associated coronary heart disease in

BioMed Research International 9

India-their causes and preventionrdquo International Journal ofDiabetes in Developing Countries vol 23 pp 37ndash50 2003

[3] R R Ortiz-Andrade S Garcıa-Jimenez P Castillo-EspanaG Ramırez-Avila R Villalobos-Molina and S Estrada-Sotoldquo120572-Glucosidase inhibitory activity of the methanolic extractfrom Tournefortia hartwegiana an anti-hyperglycemic agentrdquoJournal of Ethnopharmacology vol 109 no 1 pp 48ndash53 2007

[4] G M Gray ldquoCarbohydrate digestion and absorption Role ofthe small intestinerdquo The New England Journal of Medicine vol292 no 23 pp 1225ndash1230 1975

[5] C A Tarling K Woods R Zhang et al ldquoThe search fornovel human pancreatic 120572-amylase inhibitors high-throughputscreening of terrestrial and marine natural product extractsrdquoChemBioChem vol 9 no 3 pp 433ndash438 2008

[6] R Mentlein B Gallwitz and W E Schmidt ldquoDipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide gluca-gon-like peptide-1(7-36)amide peptide histidine methionineand is responsible for their degradation in human serumrdquoEuropean Journal of Biochemistry vol 214 no 3 pp 829ndash8351993

[7] T Vilsboslashll T Krarup C F Deacon S Madsbad and J J HolstldquoReduced postprandial concentrations of intact biologicallyactive glucagon-like peptide 1 in type 2 diabetic patientsrdquoDiabetes vol 50 no 3 pp 609ndash613 2001

[8] M A Nauck U Niedereichholz R Ettler et al ldquoGlucagon-like peptide 1 inhibition of gastric emptying outweighs itsinsulinotropic effects in healthy humansrdquo American Journal ofPhysiologymdashEndocrinology and Metabolism vol 273 no 5 ppE981ndashE988 1997

[9] A-M Lambeir S Scharpe and I DeMeester ldquoDPP4 inhibitorsfor diabetesmdashwhat nextrdquo Biochemical Pharmacology vol 76no 12 pp 1637ndash1643 2008

[10] [EMEA] EuropeanMedicines Agency ldquoEuropean public assess-ment report (EPAR)mdashJanuviardquo 2007 httpwwwemaeuropaeuema

[11] [EMEA] EuropeanMedicines Agency ldquoEuropean public assess-ment report (EPAR)mdashGalvusrdquo 2007 httpwwwemaeuropaeuema

[12] A Murai K Iwamura M Takada K Ogawa T Usui andJ-I Okumura ldquoControl of postprandial hyperglycaemia bygalactosyl maltobionolactone and its novel anti-amylase effectin micerdquo Life Sciences vol 71 no 12 pp 1405ndash1415 2002

[13] B Halliwell and O I Aruoma ldquoDNA damage by oxygen-derived species Its mechanism and measurement in mam-malian systemsrdquo FEBS Letters vol 281 no 1-2 pp 9ndash19 1991

[14] J Zhou N Hu Y-L Wu Y-J Pan and C-R Sun ldquoPreliminarystudies on the chemical characterization and antioxidant prop-erties of acidic polysaccharides from Sargassum fusiformerdquoJournal of Zhejiang University Science B vol 9 no 9 pp 721ndash727 2008

[15] D A Butterfield A Castenga C B Pocernich J DrakeG Scapagnini and V Calabrese ldquoNutritional approaches tocombat oxidative stress in Alzheimerrsquos diseasesrdquo Journal ofNutritional Biochemistry vol 13 pp 444ndash461 2002

[16] A M S Mayer and V K B Lehmann ldquoMarine pharmacologyin 1998 marine compounds with antibacterial anticoagu-lant antifungal anti-inflammatory anthelmintic antiplateletantiprotozoal and antiviral activities with actions on the cardio-vascular endocrine immune and nervous systems and othermiscellaneous mechanisms of actionrdquo Pharmacologist vol 42pp 62ndash69 2000

[17] A Cumashi N A Ushakova M E Preobrazhenskaya et alldquoA comparative study of the anti-inflammatory anticoagulantantiangiogenic and antiadhesive activities of nine differentfucoidans from brown seaweedsrdquoGlycobiology vol 17 no 5 pp541ndash552 2007

[18] T Ghosh K Chattopadhyay M Marschall P Karmakar PMandal and B Ray ldquoFocus on antivirally active sulfatedpolysaccharides from structure-activity analysis to clinicalevaluationrdquo Glycobiology vol 19 no 1 pp 2ndash15 2009

[19] V H Pomin and P A S Mourao ldquoStructure biology evolutionand medical importance of sulfated fucans and galactansrdquoGlycobiology vol 18 no 12 pp 1016ndash1027 2008

[20] S-H Lee J-S Han S-J Heo J-Y Hwang and Y-J JeonldquoProtective effects of dieckol isolated fromEcklonia cava againsthigh glucose-induced oxidative stress in human umbilical veinendothelial cellsrdquo Toxicology in Vitro vol 24 no 2 pp 375ndash3812010

[21] S-H Lee F Karadeniz M-M Kim and S-K Kim ldquo120572-Glucosidase and 120572-amylase inhibitory activities of phlorogluci-nal derivatives from edible marine brown alga Ecklonia cavardquoJournal of the Science of Food and Agriculture vol 89 no 9 pp1552ndash1558 2009

[22] A Rohfritsch C Payri V Stiger and F Bonhomme ldquoMolec-ular and morphological relationships between two closelyrelated speciesTurbinaria ornata andT conoides (SargassaceaePhaeophyceae)rdquo Biochemical Systematics and Ecology vol 35no 2 pp 91ndash98 2007

[23] S Ananthi H R B Raghavendran A G Sunil V Gayathri GRamakrishnan and H R Vasanthi ldquoIn vitro antioxidant and invivo anti-inflammatory potential of crude polysaccharide fromTurbinaria ornata (Marine Brown Alga)rdquo Food and ChemicalToxicology vol 48 no 1 pp 187ndash192 2010

[24] F Asari T Kusumi and H Kakisawa ldquoTurbinaric acid acytotoxic secosqualene carboxylic acid from the brown algaTurbinaria ornatardquo Journal of Natural Products vol 52 no 5pp 1167ndash1169 1989

[25] N Chattopadhyay T Ghosh S Sinha K ChattopadhyayP Karmakar and B Ray ldquoPolysaccharides from Turbinariaconoides structural features and antioxidant capacityrdquo FoodChemistry vol 118 no 3 pp 823ndash829 2010

[26] J B Harborne ldquoMethods of extraction and isolationrdquo inPhytochemical Methods vol 3 pp 60ndash66 Chapman amp Hall1998

[27] L L Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plant extracts for antioxidant activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[28] M A Jayasri A Radha and T L Mathew ldquo120572-amylase and120572-glucosidase inhibitory activity of Costus pictus D Don inthe management of diabetesrdquo Journal of Herbal Medicine andToxicology vol 3 pp 91ndash94 2009

[29] IM Al-MasriM KMohammad andMO Tahaa ldquoInhibitionof dipeptidyl peptidase IV (DPP IV) is one of the mechanismsexplaining the hypoglycemic effect of berberinerdquo Journal ofEnzyme Inhibition and Medicinal Chemistry vol 24 no 5 pp1061ndash1066 2009

[30] D Malagoli ldquoA full-length protocol to test hemolytic activityof palytoxin on human erythrocytesrdquo Invertebrate SurvivalJournal vol 4 pp 92ndash94 2007

[31] V R Muzykantov G P Samokhin M D Smirnov andS P Domogatsky ldquoHemolytic complement activity assay in

10 BioMed Research International

microtitration platesrdquo Journal of Applied Biochemistry vol 7 no3 pp 223ndash227 1985

[32] A-R Kim T-S Shin M-S Lee et al ldquoIsolation and identifi-cation of phlorotannins from ecklonia stolonifera with antioxi-dant and anti-inflammatory propertiesrdquo Journal of Agriculturaland Food Chemistry vol 57 no 9 pp 3483ndash3489 2009

[33] J W Baynes ldquoChemical modification of proteins by lipids indiabetesrdquo Clinical Chemistry and Laboratory Medicine vol 41no 9 pp 1159ndash1165 2003

[34] H E Moon M N Islam B R Ahn et al ldquoProtein tyrosinephosphatase 1B and 120572-glucosidase inhibitory phlorotanninsfrom edible brown algae Ecklonia stolonifera and Eiseniabicyclisrdquo Bioscience Biotechnology and Biochemistry vol 75 no8 pp 1472ndash1480 2011

[35] E Apostolidis P D Karayannakidis Y-I Kwon C M Leeand N P Seeram ldquoSeasonal variation of phenolic antioxidant-mediated a-glucosidase inhibition of Ascophyllum nodosumrdquoPlant Foods forHumanNutrition vol 66 no 4 pp 313ndash319 2011

[36] F Nwosu J Morris V A Lund D Stewart H A Ross andG J McDougall ldquoAnti-proliferative and potential anti-diabeticeffects of phenolic-rich extracts from edible marine algaerdquo FoodChemistry vol 126 no 3 pp 1006ndash1012 2011

[37] G Galati and P J OBrien ldquoPotential toxicity of flavonoids andother dietary phenolics significance for their chemopreventiveand anticancer propertiesrdquo Free Radical Biology and Medicinevol 37 no 3 pp 287ndash303 2004

[38] S-J KimW-S Chung S-S Kim S-G Ko and J-Y Um ldquoAnti-inflammatory effect of Oldenlandia diffusa and its constituenthentriacontane through suppression of caspase-1 activation inmouse peritoneal macrophagesrdquo Phytotherapy Research vol 25no 10 pp 1537ndash1546 2011

[39] G Agoramoorthy M Chandrasekaran V Venkatesalu andM J Hsu ldquoAntibacterial and antifungal activities of fatty acidmethyl esters of the blind-your-eye mangrove from IndiardquoBrazilian Journal of Microbiology vol 38 no 4 pp 739ndash7422007

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

6 BioMed Research International

0

50

100

150In

hibi

tion

()

Petroleum etherMethanol

AcetoneAcarbose

a

aa

a

a

a

a

a a

a

aa a

a a a

250 500 750 1000Concentration (120583gmL)

IC50 value of methanol extract 2509 120583gmL

(a)

0

20

40

60

80

100

Petroleum etherMethanol

AcetoneAcarbose

Inhi

bitio

n (

)

a a

a

a

aa

a

aa

a

a

aa

a

a

a

250 500 750 1000Concentration (120583gmL)

IC50 value of acetone extract 5356 120583gmL

(b)

Figure 3 (a) In vitro 120572-amylase inhibitory activity of petroleum ether methanol and acetone extracts of Turbinaria ornata Absorbance wasmeasured at 540 nm (b) In vitro 120572-glucosidase inhibitory activity of petroleum ether methanol and acetone extracts of Turbinaria ornataacarbose is used as positive control and absorbance was measured at 405 nm Values are means plusmn SD (119899 = 3) (a119875 lt 00001 consideredsignificant)

3002 04 08 16 32 64

40

50

60

70

Inhi

bitio

n (

)

aa

a

a

a

a

Concentration (120583gmL)

(a)

025 10 40 80

20

40

60

Inhi

bitio

n (

) a a a a aa

a

a

a aa

a

aa

a a

aa

a a

Concentration (120583gmL)

Petroleum etherBenzeneAcetone

Ethyl acetateMethanol

IC50 value of methanol extract 552 120583gmL

(b)

Figure 4 (a) DPP-IV inhibitory activity of diprotin A as positive control (b) DPP-IV inhibitory activity of petroleum ether benzene ethylacetate methanol and acetone extracts of Turbinaria ornata Absorbance was measured at 405 nm Values are means plusmn SD (119899 = 3) (a119875 lt00001 considered significant)

extract of T ornata showed more potent inhibition on120572-glucosidase (843) with an IC

50value of 5356 120583gmL

at a concentration of 1000120583gmL similar to the positivecontrol acarbose (88) The DPP-IV inhibitory potential ofmethanol extract of T ornata showed (554) inhibitionwith an IC

50value of 552120583gmL which is comparable with

that of positive control diprotin A (65) Similar findingswere shown by two brown seaweeds Ecklonia stoloniferaOkumura and Eisenia bicyclis [34] Several studies reportedthat the 120572-amylase and 120572-glucosidase inhibitory action of theclass Phaeophyceae [35 36] supports that these algal speciesare excellent source for antidiabetic agents Apart from 120572-amylase and 120572-glucosidase T ornata extracts also showedsignificant DPP-IV inhibition Based on these results this

seaweed can be used as alternative therapy for diabetes byraising the levels of endogenous incretin hormones (GLP-1and GIP)

In this study we also investigated the effects of crudeextracts (methanol acetone) of T ornata in macrophagecell line (J774) The cytotoxic effects of the extracts weredetermined by using anMTT assay After 24 h exposure 250ndash1000 120583gmL of methanol and acetone extracts significantlydecreased cell viability by increasing concentration As pre-viously reported seaweed extracts which have higher phenolcontent may explain the level of cytotoxicity at higher con-centrations (1000 120583gmL) [36 37] whereby up to 500120583gmLof seaweed extracts did not show any effect on J774 cellviability andmore than 90of cells were found to be viable at

BioMed Research International 7

0

50

100

150C

ell v

iabi

lity

() a a

aa

aa a

a

250 750 1000500

MethanolAcetone

Concentration (120583gmL)

(a)

0250 750 1000500

2

4

6

MethanolAcetone

Cell

lysis

()

a a a

a

a

a

a

a

Concentration (120583gmL)

(b)

Figure 5 (a) Cell viability determined byMTT assay J774 cells treated with methanol and acetone extracts of Turbinaria ornata Absorbancewasmeasured at 630 nm (b)Haemolytic activity ofmethanol and acetone extracts ofTurbinaria ornata Absorbance wasmeasured at 630 nmValues are means plusmn SD (119899 = 3) (a119875 lt 00001 considered significant)

Control

(a)

TO M

(b)

Control

(c)

Treated

(d)

Control

(e)

Treated

(f)

Figure 6 (a) Fluorescent photomicrographs (100x magnification) of J774 cells treated without seaweed extracts (b) J774 cells treated withTurbinaria ornatamethanol extract (TOM) (c) Phase contrast microscopic image of J774 cells treated with media alone (control) (d) Phasecontrast microscopic image of J774 cells treated with test extracts ((e)-(f)) Microscopic image (40x magnification) of in vitro haemolyticactivity

8 BioMed Research International

A

R3A-(13IS-1200)

B

500

100(

)

01000 1500 2000 2500 3000

Time

515e8TIC

Scan El+

OH

317

0308

130

28

294

528

36

277

627

17

264

3257

525

05

243

924

33

236

423

59

228

222

03

220

721

24

212

020

37

203

320

28

192

218

49

181

017

64

172

516

47

161

015

12

141

8

164

0

184

3

C

A

B

5111

31

33333337030

333

8130

3333333333

2829

45

28

22222222236

277

627

22222

1726

4325

75

250

524

39

243

323

222

6423

22

5922

82

220

322

07

21

2224

212

020

37

203

320

28

192

218

49

181

017

71764

172

516

47

16

110

151

214

18

164

0

18

188181818181881881881888888818881881888811111143444444444444

C

Figure 7 Gas chromatogram of acetone extract of Turbinaria ornata (A) 8-heptadecene (B) 1-heptacosanol and (C) hentriacontane

a concentration of 250120583gmL Staining cells with fluorescentdyes is used to differentiate the nuclear morphology of liveand apoptotic cells J774 cells were analyzed in the presenceof acridine orange and ethidium bromide to determinethe nuclear morphology of viable and necrotic cells Cellsstained green represent viable cells and ethidium bromideselectively stains the cells that have lost membrane integrityand stains DNA orange In this study acridine orange-ethidium bromide staining revealed that there was no changein cellular morphology including chromatin condensationmembrane blebbing and fragmented nuclei occurring dueto the treatment of extracts even at higher concentration(1000 120583gmL) Our results indicate that the tested extracts didnot induce haemolysis at a concentration of (1000120583gmL)Based on the above results the methanol and acetone extractsof T ornata were found to be nontoxic under in vitroconditions

GC-MS analysis revealed the presence of major bioac-tive compounds like hentriacontane z z-6 28-heptatriact-ontadien-2-one 8-heptadecene and 1-heptacosanol Hentri-acontane is the major component present in Oldenlandiadiffusa a natural source for the treatment of cancer in Asia[38] and also has reported activities like anti-inflammatoryand antioxidant properties [38 39] Currently there is noinformation regarding the antidiabetic effects of hentria-contane Further purification and characterization of activecompounds along with mechanism studies will only giveconclusive data on the antidiabetic property of tested algae

5 Conclusions

The present study indicates that crude extracts of T ornatapossess in vitro 120572-amylase 120572-glucosidase and DPP-IV

inhibitory activity Due to their strong enzyme inhibitoryactivity they are expected to suppress the glycemic responsein diabetic conditions Additionally the extracts are capableof scavenging free radicals in vitro toxicological parametersindicate that the IC

50of methanol and acetone extracts of T

ornata for 120572-amylase 120572-glucosidase and DPP-IV inhibitionare greatly below cytotoxic levels Future studies on in vivoantidiabetic activity of selected extracts and identification ofmajor compounds responsible for antidiabetic action are inprogress These studies will reveal the possible antidiabeticmechanisms of T ornata

Conflict of Interests

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

Acknowledgments

The authors wish to thank Department of BiotechnologyGovernment of India for their financial support (Grantno BTBio-CARe033472010-11) and VIT University forproviding all necessary facilities

References

[1] K G Alberti and P Z Zimmet ldquoDefinition diagnosis andclassification of diabetes mellitus and its complications Part1 diagnosis and classification of diabetes mellitus provisionalreport of a who consultationrdquo Diabetic Medicine vol 15 no 7pp 539ndash553 1998

[2] O P Gupta and S Phatak ldquoPandemic trends in prevalenceof diabetes mellitus and associated coronary heart disease in

BioMed Research International 9

India-their causes and preventionrdquo International Journal ofDiabetes in Developing Countries vol 23 pp 37ndash50 2003

[3] R R Ortiz-Andrade S Garcıa-Jimenez P Castillo-EspanaG Ramırez-Avila R Villalobos-Molina and S Estrada-Sotoldquo120572-Glucosidase inhibitory activity of the methanolic extractfrom Tournefortia hartwegiana an anti-hyperglycemic agentrdquoJournal of Ethnopharmacology vol 109 no 1 pp 48ndash53 2007

[4] G M Gray ldquoCarbohydrate digestion and absorption Role ofthe small intestinerdquo The New England Journal of Medicine vol292 no 23 pp 1225ndash1230 1975

[5] C A Tarling K Woods R Zhang et al ldquoThe search fornovel human pancreatic 120572-amylase inhibitors high-throughputscreening of terrestrial and marine natural product extractsrdquoChemBioChem vol 9 no 3 pp 433ndash438 2008

[6] R Mentlein B Gallwitz and W E Schmidt ldquoDipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide gluca-gon-like peptide-1(7-36)amide peptide histidine methionineand is responsible for their degradation in human serumrdquoEuropean Journal of Biochemistry vol 214 no 3 pp 829ndash8351993

[7] T Vilsboslashll T Krarup C F Deacon S Madsbad and J J HolstldquoReduced postprandial concentrations of intact biologicallyactive glucagon-like peptide 1 in type 2 diabetic patientsrdquoDiabetes vol 50 no 3 pp 609ndash613 2001

[8] M A Nauck U Niedereichholz R Ettler et al ldquoGlucagon-like peptide 1 inhibition of gastric emptying outweighs itsinsulinotropic effects in healthy humansrdquo American Journal ofPhysiologymdashEndocrinology and Metabolism vol 273 no 5 ppE981ndashE988 1997

[9] A-M Lambeir S Scharpe and I DeMeester ldquoDPP4 inhibitorsfor diabetesmdashwhat nextrdquo Biochemical Pharmacology vol 76no 12 pp 1637ndash1643 2008

[10] [EMEA] EuropeanMedicines Agency ldquoEuropean public assess-ment report (EPAR)mdashJanuviardquo 2007 httpwwwemaeuropaeuema

[11] [EMEA] EuropeanMedicines Agency ldquoEuropean public assess-ment report (EPAR)mdashGalvusrdquo 2007 httpwwwemaeuropaeuema

[12] A Murai K Iwamura M Takada K Ogawa T Usui andJ-I Okumura ldquoControl of postprandial hyperglycaemia bygalactosyl maltobionolactone and its novel anti-amylase effectin micerdquo Life Sciences vol 71 no 12 pp 1405ndash1415 2002

[13] B Halliwell and O I Aruoma ldquoDNA damage by oxygen-derived species Its mechanism and measurement in mam-malian systemsrdquo FEBS Letters vol 281 no 1-2 pp 9ndash19 1991

[14] J Zhou N Hu Y-L Wu Y-J Pan and C-R Sun ldquoPreliminarystudies on the chemical characterization and antioxidant prop-erties of acidic polysaccharides from Sargassum fusiformerdquoJournal of Zhejiang University Science B vol 9 no 9 pp 721ndash727 2008

[15] D A Butterfield A Castenga C B Pocernich J DrakeG Scapagnini and V Calabrese ldquoNutritional approaches tocombat oxidative stress in Alzheimerrsquos diseasesrdquo Journal ofNutritional Biochemistry vol 13 pp 444ndash461 2002

[16] A M S Mayer and V K B Lehmann ldquoMarine pharmacologyin 1998 marine compounds with antibacterial anticoagu-lant antifungal anti-inflammatory anthelmintic antiplateletantiprotozoal and antiviral activities with actions on the cardio-vascular endocrine immune and nervous systems and othermiscellaneous mechanisms of actionrdquo Pharmacologist vol 42pp 62ndash69 2000

[17] A Cumashi N A Ushakova M E Preobrazhenskaya et alldquoA comparative study of the anti-inflammatory anticoagulantantiangiogenic and antiadhesive activities of nine differentfucoidans from brown seaweedsrdquoGlycobiology vol 17 no 5 pp541ndash552 2007

[18] T Ghosh K Chattopadhyay M Marschall P Karmakar PMandal and B Ray ldquoFocus on antivirally active sulfatedpolysaccharides from structure-activity analysis to clinicalevaluationrdquo Glycobiology vol 19 no 1 pp 2ndash15 2009

[19] V H Pomin and P A S Mourao ldquoStructure biology evolutionand medical importance of sulfated fucans and galactansrdquoGlycobiology vol 18 no 12 pp 1016ndash1027 2008

[20] S-H Lee J-S Han S-J Heo J-Y Hwang and Y-J JeonldquoProtective effects of dieckol isolated fromEcklonia cava againsthigh glucose-induced oxidative stress in human umbilical veinendothelial cellsrdquo Toxicology in Vitro vol 24 no 2 pp 375ndash3812010

[21] S-H Lee F Karadeniz M-M Kim and S-K Kim ldquo120572-Glucosidase and 120572-amylase inhibitory activities of phlorogluci-nal derivatives from edible marine brown alga Ecklonia cavardquoJournal of the Science of Food and Agriculture vol 89 no 9 pp1552ndash1558 2009

[22] A Rohfritsch C Payri V Stiger and F Bonhomme ldquoMolec-ular and morphological relationships between two closelyrelated speciesTurbinaria ornata andT conoides (SargassaceaePhaeophyceae)rdquo Biochemical Systematics and Ecology vol 35no 2 pp 91ndash98 2007

[23] S Ananthi H R B Raghavendran A G Sunil V Gayathri GRamakrishnan and H R Vasanthi ldquoIn vitro antioxidant and invivo anti-inflammatory potential of crude polysaccharide fromTurbinaria ornata (Marine Brown Alga)rdquo Food and ChemicalToxicology vol 48 no 1 pp 187ndash192 2010

[24] F Asari T Kusumi and H Kakisawa ldquoTurbinaric acid acytotoxic secosqualene carboxylic acid from the brown algaTurbinaria ornatardquo Journal of Natural Products vol 52 no 5pp 1167ndash1169 1989

[25] N Chattopadhyay T Ghosh S Sinha K ChattopadhyayP Karmakar and B Ray ldquoPolysaccharides from Turbinariaconoides structural features and antioxidant capacityrdquo FoodChemistry vol 118 no 3 pp 823ndash829 2010

[26] J B Harborne ldquoMethods of extraction and isolationrdquo inPhytochemical Methods vol 3 pp 60ndash66 Chapman amp Hall1998

[27] L L Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plant extracts for antioxidant activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[28] M A Jayasri A Radha and T L Mathew ldquo120572-amylase and120572-glucosidase inhibitory activity of Costus pictus D Don inthe management of diabetesrdquo Journal of Herbal Medicine andToxicology vol 3 pp 91ndash94 2009

[29] IM Al-MasriM KMohammad andMO Tahaa ldquoInhibitionof dipeptidyl peptidase IV (DPP IV) is one of the mechanismsexplaining the hypoglycemic effect of berberinerdquo Journal ofEnzyme Inhibition and Medicinal Chemistry vol 24 no 5 pp1061ndash1066 2009

[30] D Malagoli ldquoA full-length protocol to test hemolytic activityof palytoxin on human erythrocytesrdquo Invertebrate SurvivalJournal vol 4 pp 92ndash94 2007

[31] V R Muzykantov G P Samokhin M D Smirnov andS P Domogatsky ldquoHemolytic complement activity assay in

10 BioMed Research International

microtitration platesrdquo Journal of Applied Biochemistry vol 7 no3 pp 223ndash227 1985

[32] A-R Kim T-S Shin M-S Lee et al ldquoIsolation and identifi-cation of phlorotannins from ecklonia stolonifera with antioxi-dant and anti-inflammatory propertiesrdquo Journal of Agriculturaland Food Chemistry vol 57 no 9 pp 3483ndash3489 2009

[33] J W Baynes ldquoChemical modification of proteins by lipids indiabetesrdquo Clinical Chemistry and Laboratory Medicine vol 41no 9 pp 1159ndash1165 2003

[34] H E Moon M N Islam B R Ahn et al ldquoProtein tyrosinephosphatase 1B and 120572-glucosidase inhibitory phlorotanninsfrom edible brown algae Ecklonia stolonifera and Eiseniabicyclisrdquo Bioscience Biotechnology and Biochemistry vol 75 no8 pp 1472ndash1480 2011

[35] E Apostolidis P D Karayannakidis Y-I Kwon C M Leeand N P Seeram ldquoSeasonal variation of phenolic antioxidant-mediated a-glucosidase inhibition of Ascophyllum nodosumrdquoPlant Foods forHumanNutrition vol 66 no 4 pp 313ndash319 2011

[36] F Nwosu J Morris V A Lund D Stewart H A Ross andG J McDougall ldquoAnti-proliferative and potential anti-diabeticeffects of phenolic-rich extracts from edible marine algaerdquo FoodChemistry vol 126 no 3 pp 1006ndash1012 2011

[37] G Galati and P J OBrien ldquoPotential toxicity of flavonoids andother dietary phenolics significance for their chemopreventiveand anticancer propertiesrdquo Free Radical Biology and Medicinevol 37 no 3 pp 287ndash303 2004

[38] S-J KimW-S Chung S-S Kim S-G Ko and J-Y Um ldquoAnti-inflammatory effect of Oldenlandia diffusa and its constituenthentriacontane through suppression of caspase-1 activation inmouse peritoneal macrophagesrdquo Phytotherapy Research vol 25no 10 pp 1537ndash1546 2011

[39] G Agoramoorthy M Chandrasekaran V Venkatesalu andM J Hsu ldquoAntibacterial and antifungal activities of fatty acidmethyl esters of the blind-your-eye mangrove from IndiardquoBrazilian Journal of Microbiology vol 38 no 4 pp 739ndash7422007

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 7

0

50

100

150C

ell v

iabi

lity

() a a

aa

aa a

a

250 750 1000500

MethanolAcetone

Concentration (120583gmL)

(a)

0250 750 1000500

2

4

6

MethanolAcetone

Cell

lysis

()

a a a

a

a

a

a

a

Concentration (120583gmL)

(b)

Figure 5 (a) Cell viability determined byMTT assay J774 cells treated with methanol and acetone extracts of Turbinaria ornata Absorbancewasmeasured at 630 nm (b)Haemolytic activity ofmethanol and acetone extracts ofTurbinaria ornata Absorbance wasmeasured at 630 nmValues are means plusmn SD (119899 = 3) (a119875 lt 00001 considered significant)

Control

(a)

TO M

(b)

Control

(c)

Treated

(d)

Control

(e)

Treated

(f)

Figure 6 (a) Fluorescent photomicrographs (100x magnification) of J774 cells treated without seaweed extracts (b) J774 cells treated withTurbinaria ornatamethanol extract (TOM) (c) Phase contrast microscopic image of J774 cells treated with media alone (control) (d) Phasecontrast microscopic image of J774 cells treated with test extracts ((e)-(f)) Microscopic image (40x magnification) of in vitro haemolyticactivity

8 BioMed Research International

A

R3A-(13IS-1200)

B

500

100(

)

01000 1500 2000 2500 3000

Time

515e8TIC

Scan El+

OH

317

0308

130

28

294

528

36

277

627

17

264

3257

525

05

243

924

33

236

423

59

228

222

03

220

721

24

212

020

37

203

320

28

192

218

49

181

017

64

172

516

47

161

015

12

141

8

164

0

184

3

C

A

B

5111

31

33333337030

333

8130

3333333333

2829

45

28

22222222236

277

627

22222

1726

4325

75

250

524

39

243

323

222

6423

22

5922

82

220

322

07

21

2224

212

020

37

203

320

28

192

218

49

181

017

71764

172

516

47

16

110

151

214

18

164

0

18

188181818181881881881888888818881881888811111143444444444444

C

Figure 7 Gas chromatogram of acetone extract of Turbinaria ornata (A) 8-heptadecene (B) 1-heptacosanol and (C) hentriacontane

a concentration of 250120583gmL Staining cells with fluorescentdyes is used to differentiate the nuclear morphology of liveand apoptotic cells J774 cells were analyzed in the presenceof acridine orange and ethidium bromide to determinethe nuclear morphology of viable and necrotic cells Cellsstained green represent viable cells and ethidium bromideselectively stains the cells that have lost membrane integrityand stains DNA orange In this study acridine orange-ethidium bromide staining revealed that there was no changein cellular morphology including chromatin condensationmembrane blebbing and fragmented nuclei occurring dueto the treatment of extracts even at higher concentration(1000 120583gmL) Our results indicate that the tested extracts didnot induce haemolysis at a concentration of (1000120583gmL)Based on the above results the methanol and acetone extractsof T ornata were found to be nontoxic under in vitroconditions

GC-MS analysis revealed the presence of major bioac-tive compounds like hentriacontane z z-6 28-heptatriact-ontadien-2-one 8-heptadecene and 1-heptacosanol Hentri-acontane is the major component present in Oldenlandiadiffusa a natural source for the treatment of cancer in Asia[38] and also has reported activities like anti-inflammatoryand antioxidant properties [38 39] Currently there is noinformation regarding the antidiabetic effects of hentria-contane Further purification and characterization of activecompounds along with mechanism studies will only giveconclusive data on the antidiabetic property of tested algae

5 Conclusions

The present study indicates that crude extracts of T ornatapossess in vitro 120572-amylase 120572-glucosidase and DPP-IV

inhibitory activity Due to their strong enzyme inhibitoryactivity they are expected to suppress the glycemic responsein diabetic conditions Additionally the extracts are capableof scavenging free radicals in vitro toxicological parametersindicate that the IC

50of methanol and acetone extracts of T

ornata for 120572-amylase 120572-glucosidase and DPP-IV inhibitionare greatly below cytotoxic levels Future studies on in vivoantidiabetic activity of selected extracts and identification ofmajor compounds responsible for antidiabetic action are inprogress These studies will reveal the possible antidiabeticmechanisms of T ornata

Conflict of Interests

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

Acknowledgments

The authors wish to thank Department of BiotechnologyGovernment of India for their financial support (Grantno BTBio-CARe033472010-11) and VIT University forproviding all necessary facilities

References

[1] K G Alberti and P Z Zimmet ldquoDefinition diagnosis andclassification of diabetes mellitus and its complications Part1 diagnosis and classification of diabetes mellitus provisionalreport of a who consultationrdquo Diabetic Medicine vol 15 no 7pp 539ndash553 1998

[2] O P Gupta and S Phatak ldquoPandemic trends in prevalenceof diabetes mellitus and associated coronary heart disease in

BioMed Research International 9

India-their causes and preventionrdquo International Journal ofDiabetes in Developing Countries vol 23 pp 37ndash50 2003

[3] R R Ortiz-Andrade S Garcıa-Jimenez P Castillo-EspanaG Ramırez-Avila R Villalobos-Molina and S Estrada-Sotoldquo120572-Glucosidase inhibitory activity of the methanolic extractfrom Tournefortia hartwegiana an anti-hyperglycemic agentrdquoJournal of Ethnopharmacology vol 109 no 1 pp 48ndash53 2007

[4] G M Gray ldquoCarbohydrate digestion and absorption Role ofthe small intestinerdquo The New England Journal of Medicine vol292 no 23 pp 1225ndash1230 1975

[5] C A Tarling K Woods R Zhang et al ldquoThe search fornovel human pancreatic 120572-amylase inhibitors high-throughputscreening of terrestrial and marine natural product extractsrdquoChemBioChem vol 9 no 3 pp 433ndash438 2008

[6] R Mentlein B Gallwitz and W E Schmidt ldquoDipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide gluca-gon-like peptide-1(7-36)amide peptide histidine methionineand is responsible for their degradation in human serumrdquoEuropean Journal of Biochemistry vol 214 no 3 pp 829ndash8351993

[7] T Vilsboslashll T Krarup C F Deacon S Madsbad and J J HolstldquoReduced postprandial concentrations of intact biologicallyactive glucagon-like peptide 1 in type 2 diabetic patientsrdquoDiabetes vol 50 no 3 pp 609ndash613 2001

[8] M A Nauck U Niedereichholz R Ettler et al ldquoGlucagon-like peptide 1 inhibition of gastric emptying outweighs itsinsulinotropic effects in healthy humansrdquo American Journal ofPhysiologymdashEndocrinology and Metabolism vol 273 no 5 ppE981ndashE988 1997

[9] A-M Lambeir S Scharpe and I DeMeester ldquoDPP4 inhibitorsfor diabetesmdashwhat nextrdquo Biochemical Pharmacology vol 76no 12 pp 1637ndash1643 2008

[10] [EMEA] EuropeanMedicines Agency ldquoEuropean public assess-ment report (EPAR)mdashJanuviardquo 2007 httpwwwemaeuropaeuema

[11] [EMEA] EuropeanMedicines Agency ldquoEuropean public assess-ment report (EPAR)mdashGalvusrdquo 2007 httpwwwemaeuropaeuema

[12] A Murai K Iwamura M Takada K Ogawa T Usui andJ-I Okumura ldquoControl of postprandial hyperglycaemia bygalactosyl maltobionolactone and its novel anti-amylase effectin micerdquo Life Sciences vol 71 no 12 pp 1405ndash1415 2002

[13] B Halliwell and O I Aruoma ldquoDNA damage by oxygen-derived species Its mechanism and measurement in mam-malian systemsrdquo FEBS Letters vol 281 no 1-2 pp 9ndash19 1991

[14] J Zhou N Hu Y-L Wu Y-J Pan and C-R Sun ldquoPreliminarystudies on the chemical characterization and antioxidant prop-erties of acidic polysaccharides from Sargassum fusiformerdquoJournal of Zhejiang University Science B vol 9 no 9 pp 721ndash727 2008

[15] D A Butterfield A Castenga C B Pocernich J DrakeG Scapagnini and V Calabrese ldquoNutritional approaches tocombat oxidative stress in Alzheimerrsquos diseasesrdquo Journal ofNutritional Biochemistry vol 13 pp 444ndash461 2002

[16] A M S Mayer and V K B Lehmann ldquoMarine pharmacologyin 1998 marine compounds with antibacterial anticoagu-lant antifungal anti-inflammatory anthelmintic antiplateletantiprotozoal and antiviral activities with actions on the cardio-vascular endocrine immune and nervous systems and othermiscellaneous mechanisms of actionrdquo Pharmacologist vol 42pp 62ndash69 2000

[17] A Cumashi N A Ushakova M E Preobrazhenskaya et alldquoA comparative study of the anti-inflammatory anticoagulantantiangiogenic and antiadhesive activities of nine differentfucoidans from brown seaweedsrdquoGlycobiology vol 17 no 5 pp541ndash552 2007

[18] T Ghosh K Chattopadhyay M Marschall P Karmakar PMandal and B Ray ldquoFocus on antivirally active sulfatedpolysaccharides from structure-activity analysis to clinicalevaluationrdquo Glycobiology vol 19 no 1 pp 2ndash15 2009

[19] V H Pomin and P A S Mourao ldquoStructure biology evolutionand medical importance of sulfated fucans and galactansrdquoGlycobiology vol 18 no 12 pp 1016ndash1027 2008

[20] S-H Lee J-S Han S-J Heo J-Y Hwang and Y-J JeonldquoProtective effects of dieckol isolated fromEcklonia cava againsthigh glucose-induced oxidative stress in human umbilical veinendothelial cellsrdquo Toxicology in Vitro vol 24 no 2 pp 375ndash3812010

[21] S-H Lee F Karadeniz M-M Kim and S-K Kim ldquo120572-Glucosidase and 120572-amylase inhibitory activities of phlorogluci-nal derivatives from edible marine brown alga Ecklonia cavardquoJournal of the Science of Food and Agriculture vol 89 no 9 pp1552ndash1558 2009

[22] A Rohfritsch C Payri V Stiger and F Bonhomme ldquoMolec-ular and morphological relationships between two closelyrelated speciesTurbinaria ornata andT conoides (SargassaceaePhaeophyceae)rdquo Biochemical Systematics and Ecology vol 35no 2 pp 91ndash98 2007

[23] S Ananthi H R B Raghavendran A G Sunil V Gayathri GRamakrishnan and H R Vasanthi ldquoIn vitro antioxidant and invivo anti-inflammatory potential of crude polysaccharide fromTurbinaria ornata (Marine Brown Alga)rdquo Food and ChemicalToxicology vol 48 no 1 pp 187ndash192 2010

[24] F Asari T Kusumi and H Kakisawa ldquoTurbinaric acid acytotoxic secosqualene carboxylic acid from the brown algaTurbinaria ornatardquo Journal of Natural Products vol 52 no 5pp 1167ndash1169 1989

[25] N Chattopadhyay T Ghosh S Sinha K ChattopadhyayP Karmakar and B Ray ldquoPolysaccharides from Turbinariaconoides structural features and antioxidant capacityrdquo FoodChemistry vol 118 no 3 pp 823ndash829 2010

[26] J B Harborne ldquoMethods of extraction and isolationrdquo inPhytochemical Methods vol 3 pp 60ndash66 Chapman amp Hall1998

[27] L L Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plant extracts for antioxidant activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[28] M A Jayasri A Radha and T L Mathew ldquo120572-amylase and120572-glucosidase inhibitory activity of Costus pictus D Don inthe management of diabetesrdquo Journal of Herbal Medicine andToxicology vol 3 pp 91ndash94 2009

[29] IM Al-MasriM KMohammad andMO Tahaa ldquoInhibitionof dipeptidyl peptidase IV (DPP IV) is one of the mechanismsexplaining the hypoglycemic effect of berberinerdquo Journal ofEnzyme Inhibition and Medicinal Chemistry vol 24 no 5 pp1061ndash1066 2009

[30] D Malagoli ldquoA full-length protocol to test hemolytic activityof palytoxin on human erythrocytesrdquo Invertebrate SurvivalJournal vol 4 pp 92ndash94 2007

[31] V R Muzykantov G P Samokhin M D Smirnov andS P Domogatsky ldquoHemolytic complement activity assay in

10 BioMed Research International

microtitration platesrdquo Journal of Applied Biochemistry vol 7 no3 pp 223ndash227 1985

[32] A-R Kim T-S Shin M-S Lee et al ldquoIsolation and identifi-cation of phlorotannins from ecklonia stolonifera with antioxi-dant and anti-inflammatory propertiesrdquo Journal of Agriculturaland Food Chemistry vol 57 no 9 pp 3483ndash3489 2009

[33] J W Baynes ldquoChemical modification of proteins by lipids indiabetesrdquo Clinical Chemistry and Laboratory Medicine vol 41no 9 pp 1159ndash1165 2003

[34] H E Moon M N Islam B R Ahn et al ldquoProtein tyrosinephosphatase 1B and 120572-glucosidase inhibitory phlorotanninsfrom edible brown algae Ecklonia stolonifera and Eiseniabicyclisrdquo Bioscience Biotechnology and Biochemistry vol 75 no8 pp 1472ndash1480 2011

[35] E Apostolidis P D Karayannakidis Y-I Kwon C M Leeand N P Seeram ldquoSeasonal variation of phenolic antioxidant-mediated a-glucosidase inhibition of Ascophyllum nodosumrdquoPlant Foods forHumanNutrition vol 66 no 4 pp 313ndash319 2011

[36] F Nwosu J Morris V A Lund D Stewart H A Ross andG J McDougall ldquoAnti-proliferative and potential anti-diabeticeffects of phenolic-rich extracts from edible marine algaerdquo FoodChemistry vol 126 no 3 pp 1006ndash1012 2011

[37] G Galati and P J OBrien ldquoPotential toxicity of flavonoids andother dietary phenolics significance for their chemopreventiveand anticancer propertiesrdquo Free Radical Biology and Medicinevol 37 no 3 pp 287ndash303 2004

[38] S-J KimW-S Chung S-S Kim S-G Ko and J-Y Um ldquoAnti-inflammatory effect of Oldenlandia diffusa and its constituenthentriacontane through suppression of caspase-1 activation inmouse peritoneal macrophagesrdquo Phytotherapy Research vol 25no 10 pp 1537ndash1546 2011

[39] G Agoramoorthy M Chandrasekaran V Venkatesalu andM J Hsu ldquoAntibacterial and antifungal activities of fatty acidmethyl esters of the blind-your-eye mangrove from IndiardquoBrazilian Journal of Microbiology vol 38 no 4 pp 739ndash7422007

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

8 BioMed Research International

A

R3A-(13IS-1200)

B

500

100(

)

01000 1500 2000 2500 3000

Time

515e8TIC

Scan El+

OH

317

0308

130

28

294

528

36

277

627

17

264

3257

525

05

243

924

33

236

423

59

228

222

03

220

721

24

212

020

37

203

320

28

192

218

49

181

017

64

172

516

47

161

015

12

141

8

164

0

184

3

C

A

B

5111

31

33333337030

333

8130

3333333333

2829

45

28

22222222236

277

627

22222

1726

4325

75

250

524

39

243

323

222

6423

22

5922

82

220

322

07

21

2224

212

020

37

203

320

28

192

218

49

181

017

71764

172

516

47

16

110

151

214

18

164

0

18

188181818181881881881888888818881881888811111143444444444444

C

Figure 7 Gas chromatogram of acetone extract of Turbinaria ornata (A) 8-heptadecene (B) 1-heptacosanol and (C) hentriacontane

a concentration of 250120583gmL Staining cells with fluorescentdyes is used to differentiate the nuclear morphology of liveand apoptotic cells J774 cells were analyzed in the presenceof acridine orange and ethidium bromide to determinethe nuclear morphology of viable and necrotic cells Cellsstained green represent viable cells and ethidium bromideselectively stains the cells that have lost membrane integrityand stains DNA orange In this study acridine orange-ethidium bromide staining revealed that there was no changein cellular morphology including chromatin condensationmembrane blebbing and fragmented nuclei occurring dueto the treatment of extracts even at higher concentration(1000 120583gmL) Our results indicate that the tested extracts didnot induce haemolysis at a concentration of (1000120583gmL)Based on the above results the methanol and acetone extractsof T ornata were found to be nontoxic under in vitroconditions

GC-MS analysis revealed the presence of major bioac-tive compounds like hentriacontane z z-6 28-heptatriact-ontadien-2-one 8-heptadecene and 1-heptacosanol Hentri-acontane is the major component present in Oldenlandiadiffusa a natural source for the treatment of cancer in Asia[38] and also has reported activities like anti-inflammatoryand antioxidant properties [38 39] Currently there is noinformation regarding the antidiabetic effects of hentria-contane Further purification and characterization of activecompounds along with mechanism studies will only giveconclusive data on the antidiabetic property of tested algae

5 Conclusions

The present study indicates that crude extracts of T ornatapossess in vitro 120572-amylase 120572-glucosidase and DPP-IV

inhibitory activity Due to their strong enzyme inhibitoryactivity they are expected to suppress the glycemic responsein diabetic conditions Additionally the extracts are capableof scavenging free radicals in vitro toxicological parametersindicate that the IC

50of methanol and acetone extracts of T

ornata for 120572-amylase 120572-glucosidase and DPP-IV inhibitionare greatly below cytotoxic levels Future studies on in vivoantidiabetic activity of selected extracts and identification ofmajor compounds responsible for antidiabetic action are inprogress These studies will reveal the possible antidiabeticmechanisms of T ornata

Conflict of Interests

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

Acknowledgments

The authors wish to thank Department of BiotechnologyGovernment of India for their financial support (Grantno BTBio-CARe033472010-11) and VIT University forproviding all necessary facilities

References

[1] K G Alberti and P Z Zimmet ldquoDefinition diagnosis andclassification of diabetes mellitus and its complications Part1 diagnosis and classification of diabetes mellitus provisionalreport of a who consultationrdquo Diabetic Medicine vol 15 no 7pp 539ndash553 1998

[2] O P Gupta and S Phatak ldquoPandemic trends in prevalenceof diabetes mellitus and associated coronary heart disease in

BioMed Research International 9

India-their causes and preventionrdquo International Journal ofDiabetes in Developing Countries vol 23 pp 37ndash50 2003

[3] R R Ortiz-Andrade S Garcıa-Jimenez P Castillo-EspanaG Ramırez-Avila R Villalobos-Molina and S Estrada-Sotoldquo120572-Glucosidase inhibitory activity of the methanolic extractfrom Tournefortia hartwegiana an anti-hyperglycemic agentrdquoJournal of Ethnopharmacology vol 109 no 1 pp 48ndash53 2007

[4] G M Gray ldquoCarbohydrate digestion and absorption Role ofthe small intestinerdquo The New England Journal of Medicine vol292 no 23 pp 1225ndash1230 1975

[5] C A Tarling K Woods R Zhang et al ldquoThe search fornovel human pancreatic 120572-amylase inhibitors high-throughputscreening of terrestrial and marine natural product extractsrdquoChemBioChem vol 9 no 3 pp 433ndash438 2008

[6] R Mentlein B Gallwitz and W E Schmidt ldquoDipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide gluca-gon-like peptide-1(7-36)amide peptide histidine methionineand is responsible for their degradation in human serumrdquoEuropean Journal of Biochemistry vol 214 no 3 pp 829ndash8351993

[7] T Vilsboslashll T Krarup C F Deacon S Madsbad and J J HolstldquoReduced postprandial concentrations of intact biologicallyactive glucagon-like peptide 1 in type 2 diabetic patientsrdquoDiabetes vol 50 no 3 pp 609ndash613 2001

[8] M A Nauck U Niedereichholz R Ettler et al ldquoGlucagon-like peptide 1 inhibition of gastric emptying outweighs itsinsulinotropic effects in healthy humansrdquo American Journal ofPhysiologymdashEndocrinology and Metabolism vol 273 no 5 ppE981ndashE988 1997

[9] A-M Lambeir S Scharpe and I DeMeester ldquoDPP4 inhibitorsfor diabetesmdashwhat nextrdquo Biochemical Pharmacology vol 76no 12 pp 1637ndash1643 2008

[10] [EMEA] EuropeanMedicines Agency ldquoEuropean public assess-ment report (EPAR)mdashJanuviardquo 2007 httpwwwemaeuropaeuema

[11] [EMEA] EuropeanMedicines Agency ldquoEuropean public assess-ment report (EPAR)mdashGalvusrdquo 2007 httpwwwemaeuropaeuema

[12] A Murai K Iwamura M Takada K Ogawa T Usui andJ-I Okumura ldquoControl of postprandial hyperglycaemia bygalactosyl maltobionolactone and its novel anti-amylase effectin micerdquo Life Sciences vol 71 no 12 pp 1405ndash1415 2002

[13] B Halliwell and O I Aruoma ldquoDNA damage by oxygen-derived species Its mechanism and measurement in mam-malian systemsrdquo FEBS Letters vol 281 no 1-2 pp 9ndash19 1991

[14] J Zhou N Hu Y-L Wu Y-J Pan and C-R Sun ldquoPreliminarystudies on the chemical characterization and antioxidant prop-erties of acidic polysaccharides from Sargassum fusiformerdquoJournal of Zhejiang University Science B vol 9 no 9 pp 721ndash727 2008

[15] D A Butterfield A Castenga C B Pocernich J DrakeG Scapagnini and V Calabrese ldquoNutritional approaches tocombat oxidative stress in Alzheimerrsquos diseasesrdquo Journal ofNutritional Biochemistry vol 13 pp 444ndash461 2002

[16] A M S Mayer and V K B Lehmann ldquoMarine pharmacologyin 1998 marine compounds with antibacterial anticoagu-lant antifungal anti-inflammatory anthelmintic antiplateletantiprotozoal and antiviral activities with actions on the cardio-vascular endocrine immune and nervous systems and othermiscellaneous mechanisms of actionrdquo Pharmacologist vol 42pp 62ndash69 2000

[17] A Cumashi N A Ushakova M E Preobrazhenskaya et alldquoA comparative study of the anti-inflammatory anticoagulantantiangiogenic and antiadhesive activities of nine differentfucoidans from brown seaweedsrdquoGlycobiology vol 17 no 5 pp541ndash552 2007

[18] T Ghosh K Chattopadhyay M Marschall P Karmakar PMandal and B Ray ldquoFocus on antivirally active sulfatedpolysaccharides from structure-activity analysis to clinicalevaluationrdquo Glycobiology vol 19 no 1 pp 2ndash15 2009

[19] V H Pomin and P A S Mourao ldquoStructure biology evolutionand medical importance of sulfated fucans and galactansrdquoGlycobiology vol 18 no 12 pp 1016ndash1027 2008

[20] S-H Lee J-S Han S-J Heo J-Y Hwang and Y-J JeonldquoProtective effects of dieckol isolated fromEcklonia cava againsthigh glucose-induced oxidative stress in human umbilical veinendothelial cellsrdquo Toxicology in Vitro vol 24 no 2 pp 375ndash3812010

[21] S-H Lee F Karadeniz M-M Kim and S-K Kim ldquo120572-Glucosidase and 120572-amylase inhibitory activities of phlorogluci-nal derivatives from edible marine brown alga Ecklonia cavardquoJournal of the Science of Food and Agriculture vol 89 no 9 pp1552ndash1558 2009

[22] A Rohfritsch C Payri V Stiger and F Bonhomme ldquoMolec-ular and morphological relationships between two closelyrelated speciesTurbinaria ornata andT conoides (SargassaceaePhaeophyceae)rdquo Biochemical Systematics and Ecology vol 35no 2 pp 91ndash98 2007

[23] S Ananthi H R B Raghavendran A G Sunil V Gayathri GRamakrishnan and H R Vasanthi ldquoIn vitro antioxidant and invivo anti-inflammatory potential of crude polysaccharide fromTurbinaria ornata (Marine Brown Alga)rdquo Food and ChemicalToxicology vol 48 no 1 pp 187ndash192 2010

[24] F Asari T Kusumi and H Kakisawa ldquoTurbinaric acid acytotoxic secosqualene carboxylic acid from the brown algaTurbinaria ornatardquo Journal of Natural Products vol 52 no 5pp 1167ndash1169 1989

[25] N Chattopadhyay T Ghosh S Sinha K ChattopadhyayP Karmakar and B Ray ldquoPolysaccharides from Turbinariaconoides structural features and antioxidant capacityrdquo FoodChemistry vol 118 no 3 pp 823ndash829 2010

[26] J B Harborne ldquoMethods of extraction and isolationrdquo inPhytochemical Methods vol 3 pp 60ndash66 Chapman amp Hall1998

[27] L L Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plant extracts for antioxidant activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[28] M A Jayasri A Radha and T L Mathew ldquo120572-amylase and120572-glucosidase inhibitory activity of Costus pictus D Don inthe management of diabetesrdquo Journal of Herbal Medicine andToxicology vol 3 pp 91ndash94 2009

[29] IM Al-MasriM KMohammad andMO Tahaa ldquoInhibitionof dipeptidyl peptidase IV (DPP IV) is one of the mechanismsexplaining the hypoglycemic effect of berberinerdquo Journal ofEnzyme Inhibition and Medicinal Chemistry vol 24 no 5 pp1061ndash1066 2009

[30] D Malagoli ldquoA full-length protocol to test hemolytic activityof palytoxin on human erythrocytesrdquo Invertebrate SurvivalJournal vol 4 pp 92ndash94 2007

[31] V R Muzykantov G P Samokhin M D Smirnov andS P Domogatsky ldquoHemolytic complement activity assay in

10 BioMed Research International

microtitration platesrdquo Journal of Applied Biochemistry vol 7 no3 pp 223ndash227 1985

[32] A-R Kim T-S Shin M-S Lee et al ldquoIsolation and identifi-cation of phlorotannins from ecklonia stolonifera with antioxi-dant and anti-inflammatory propertiesrdquo Journal of Agriculturaland Food Chemistry vol 57 no 9 pp 3483ndash3489 2009

[33] J W Baynes ldquoChemical modification of proteins by lipids indiabetesrdquo Clinical Chemistry and Laboratory Medicine vol 41no 9 pp 1159ndash1165 2003

[34] H E Moon M N Islam B R Ahn et al ldquoProtein tyrosinephosphatase 1B and 120572-glucosidase inhibitory phlorotanninsfrom edible brown algae Ecklonia stolonifera and Eiseniabicyclisrdquo Bioscience Biotechnology and Biochemistry vol 75 no8 pp 1472ndash1480 2011

[35] E Apostolidis P D Karayannakidis Y-I Kwon C M Leeand N P Seeram ldquoSeasonal variation of phenolic antioxidant-mediated a-glucosidase inhibition of Ascophyllum nodosumrdquoPlant Foods forHumanNutrition vol 66 no 4 pp 313ndash319 2011

[36] F Nwosu J Morris V A Lund D Stewart H A Ross andG J McDougall ldquoAnti-proliferative and potential anti-diabeticeffects of phenolic-rich extracts from edible marine algaerdquo FoodChemistry vol 126 no 3 pp 1006ndash1012 2011

[37] G Galati and P J OBrien ldquoPotential toxicity of flavonoids andother dietary phenolics significance for their chemopreventiveand anticancer propertiesrdquo Free Radical Biology and Medicinevol 37 no 3 pp 287ndash303 2004

[38] S-J KimW-S Chung S-S Kim S-G Ko and J-Y Um ldquoAnti-inflammatory effect of Oldenlandia diffusa and its constituenthentriacontane through suppression of caspase-1 activation inmouse peritoneal macrophagesrdquo Phytotherapy Research vol 25no 10 pp 1537ndash1546 2011

[39] G Agoramoorthy M Chandrasekaran V Venkatesalu andM J Hsu ldquoAntibacterial and antifungal activities of fatty acidmethyl esters of the blind-your-eye mangrove from IndiardquoBrazilian Journal of Microbiology vol 38 no 4 pp 739ndash7422007

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 9

India-their causes and preventionrdquo International Journal ofDiabetes in Developing Countries vol 23 pp 37ndash50 2003

[3] R R Ortiz-Andrade S Garcıa-Jimenez P Castillo-EspanaG Ramırez-Avila R Villalobos-Molina and S Estrada-Sotoldquo120572-Glucosidase inhibitory activity of the methanolic extractfrom Tournefortia hartwegiana an anti-hyperglycemic agentrdquoJournal of Ethnopharmacology vol 109 no 1 pp 48ndash53 2007

[4] G M Gray ldquoCarbohydrate digestion and absorption Role ofthe small intestinerdquo The New England Journal of Medicine vol292 no 23 pp 1225ndash1230 1975

[5] C A Tarling K Woods R Zhang et al ldquoThe search fornovel human pancreatic 120572-amylase inhibitors high-throughputscreening of terrestrial and marine natural product extractsrdquoChemBioChem vol 9 no 3 pp 433ndash438 2008

[6] R Mentlein B Gallwitz and W E Schmidt ldquoDipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide gluca-gon-like peptide-1(7-36)amide peptide histidine methionineand is responsible for their degradation in human serumrdquoEuropean Journal of Biochemistry vol 214 no 3 pp 829ndash8351993

[7] T Vilsboslashll T Krarup C F Deacon S Madsbad and J J HolstldquoReduced postprandial concentrations of intact biologicallyactive glucagon-like peptide 1 in type 2 diabetic patientsrdquoDiabetes vol 50 no 3 pp 609ndash613 2001

[8] M A Nauck U Niedereichholz R Ettler et al ldquoGlucagon-like peptide 1 inhibition of gastric emptying outweighs itsinsulinotropic effects in healthy humansrdquo American Journal ofPhysiologymdashEndocrinology and Metabolism vol 273 no 5 ppE981ndashE988 1997

[9] A-M Lambeir S Scharpe and I DeMeester ldquoDPP4 inhibitorsfor diabetesmdashwhat nextrdquo Biochemical Pharmacology vol 76no 12 pp 1637ndash1643 2008

[10] [EMEA] EuropeanMedicines Agency ldquoEuropean public assess-ment report (EPAR)mdashJanuviardquo 2007 httpwwwemaeuropaeuema

[11] [EMEA] EuropeanMedicines Agency ldquoEuropean public assess-ment report (EPAR)mdashGalvusrdquo 2007 httpwwwemaeuropaeuema

[12] A Murai K Iwamura M Takada K Ogawa T Usui andJ-I Okumura ldquoControl of postprandial hyperglycaemia bygalactosyl maltobionolactone and its novel anti-amylase effectin micerdquo Life Sciences vol 71 no 12 pp 1405ndash1415 2002

[13] B Halliwell and O I Aruoma ldquoDNA damage by oxygen-derived species Its mechanism and measurement in mam-malian systemsrdquo FEBS Letters vol 281 no 1-2 pp 9ndash19 1991

[14] J Zhou N Hu Y-L Wu Y-J Pan and C-R Sun ldquoPreliminarystudies on the chemical characterization and antioxidant prop-erties of acidic polysaccharides from Sargassum fusiformerdquoJournal of Zhejiang University Science B vol 9 no 9 pp 721ndash727 2008

[15] D A Butterfield A Castenga C B Pocernich J DrakeG Scapagnini and V Calabrese ldquoNutritional approaches tocombat oxidative stress in Alzheimerrsquos diseasesrdquo Journal ofNutritional Biochemistry vol 13 pp 444ndash461 2002

[16] A M S Mayer and V K B Lehmann ldquoMarine pharmacologyin 1998 marine compounds with antibacterial anticoagu-lant antifungal anti-inflammatory anthelmintic antiplateletantiprotozoal and antiviral activities with actions on the cardio-vascular endocrine immune and nervous systems and othermiscellaneous mechanisms of actionrdquo Pharmacologist vol 42pp 62ndash69 2000

[17] A Cumashi N A Ushakova M E Preobrazhenskaya et alldquoA comparative study of the anti-inflammatory anticoagulantantiangiogenic and antiadhesive activities of nine differentfucoidans from brown seaweedsrdquoGlycobiology vol 17 no 5 pp541ndash552 2007

[18] T Ghosh K Chattopadhyay M Marschall P Karmakar PMandal and B Ray ldquoFocus on antivirally active sulfatedpolysaccharides from structure-activity analysis to clinicalevaluationrdquo Glycobiology vol 19 no 1 pp 2ndash15 2009

[19] V H Pomin and P A S Mourao ldquoStructure biology evolutionand medical importance of sulfated fucans and galactansrdquoGlycobiology vol 18 no 12 pp 1016ndash1027 2008

[20] S-H Lee J-S Han S-J Heo J-Y Hwang and Y-J JeonldquoProtective effects of dieckol isolated fromEcklonia cava againsthigh glucose-induced oxidative stress in human umbilical veinendothelial cellsrdquo Toxicology in Vitro vol 24 no 2 pp 375ndash3812010

[21] S-H Lee F Karadeniz M-M Kim and S-K Kim ldquo120572-Glucosidase and 120572-amylase inhibitory activities of phlorogluci-nal derivatives from edible marine brown alga Ecklonia cavardquoJournal of the Science of Food and Agriculture vol 89 no 9 pp1552ndash1558 2009

[22] A Rohfritsch C Payri V Stiger and F Bonhomme ldquoMolec-ular and morphological relationships between two closelyrelated speciesTurbinaria ornata andT conoides (SargassaceaePhaeophyceae)rdquo Biochemical Systematics and Ecology vol 35no 2 pp 91ndash98 2007

[23] S Ananthi H R B Raghavendran A G Sunil V Gayathri GRamakrishnan and H R Vasanthi ldquoIn vitro antioxidant and invivo anti-inflammatory potential of crude polysaccharide fromTurbinaria ornata (Marine Brown Alga)rdquo Food and ChemicalToxicology vol 48 no 1 pp 187ndash192 2010

[24] F Asari T Kusumi and H Kakisawa ldquoTurbinaric acid acytotoxic secosqualene carboxylic acid from the brown algaTurbinaria ornatardquo Journal of Natural Products vol 52 no 5pp 1167ndash1169 1989

[25] N Chattopadhyay T Ghosh S Sinha K ChattopadhyayP Karmakar and B Ray ldquoPolysaccharides from Turbinariaconoides structural features and antioxidant capacityrdquo FoodChemistry vol 118 no 3 pp 823ndash829 2010

[26] J B Harborne ldquoMethods of extraction and isolationrdquo inPhytochemical Methods vol 3 pp 60ndash66 Chapman amp Hall1998

[27] L L Mensor F S Menezes G G Leitao et al ldquoScreening ofBrazilian plant extracts for antioxidant activity by the use ofDPPH free radical methodrdquo Phytotherapy Research vol 15 pp127ndash130 2001

[28] M A Jayasri A Radha and T L Mathew ldquo120572-amylase and120572-glucosidase inhibitory activity of Costus pictus D Don inthe management of diabetesrdquo Journal of Herbal Medicine andToxicology vol 3 pp 91ndash94 2009

[29] IM Al-MasriM KMohammad andMO Tahaa ldquoInhibitionof dipeptidyl peptidase IV (DPP IV) is one of the mechanismsexplaining the hypoglycemic effect of berberinerdquo Journal ofEnzyme Inhibition and Medicinal Chemistry vol 24 no 5 pp1061ndash1066 2009

[30] D Malagoli ldquoA full-length protocol to test hemolytic activityof palytoxin on human erythrocytesrdquo Invertebrate SurvivalJournal vol 4 pp 92ndash94 2007

[31] V R Muzykantov G P Samokhin M D Smirnov andS P Domogatsky ldquoHemolytic complement activity assay in

10 BioMed Research International

microtitration platesrdquo Journal of Applied Biochemistry vol 7 no3 pp 223ndash227 1985

[32] A-R Kim T-S Shin M-S Lee et al ldquoIsolation and identifi-cation of phlorotannins from ecklonia stolonifera with antioxi-dant and anti-inflammatory propertiesrdquo Journal of Agriculturaland Food Chemistry vol 57 no 9 pp 3483ndash3489 2009

[33] J W Baynes ldquoChemical modification of proteins by lipids indiabetesrdquo Clinical Chemistry and Laboratory Medicine vol 41no 9 pp 1159ndash1165 2003

[34] H E Moon M N Islam B R Ahn et al ldquoProtein tyrosinephosphatase 1B and 120572-glucosidase inhibitory phlorotanninsfrom edible brown algae Ecklonia stolonifera and Eiseniabicyclisrdquo Bioscience Biotechnology and Biochemistry vol 75 no8 pp 1472ndash1480 2011

[35] E Apostolidis P D Karayannakidis Y-I Kwon C M Leeand N P Seeram ldquoSeasonal variation of phenolic antioxidant-mediated a-glucosidase inhibition of Ascophyllum nodosumrdquoPlant Foods forHumanNutrition vol 66 no 4 pp 313ndash319 2011

[36] F Nwosu J Morris V A Lund D Stewart H A Ross andG J McDougall ldquoAnti-proliferative and potential anti-diabeticeffects of phenolic-rich extracts from edible marine algaerdquo FoodChemistry vol 126 no 3 pp 1006ndash1012 2011

[37] G Galati and P J OBrien ldquoPotential toxicity of flavonoids andother dietary phenolics significance for their chemopreventiveand anticancer propertiesrdquo Free Radical Biology and Medicinevol 37 no 3 pp 287ndash303 2004

[38] S-J KimW-S Chung S-S Kim S-G Ko and J-Y Um ldquoAnti-inflammatory effect of Oldenlandia diffusa and its constituenthentriacontane through suppression of caspase-1 activation inmouse peritoneal macrophagesrdquo Phytotherapy Research vol 25no 10 pp 1537ndash1546 2011

[39] G Agoramoorthy M Chandrasekaran V Venkatesalu andM J Hsu ldquoAntibacterial and antifungal activities of fatty acidmethyl esters of the blind-your-eye mangrove from IndiardquoBrazilian Journal of Microbiology vol 38 no 4 pp 739ndash7422007

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

10 BioMed Research International

microtitration platesrdquo Journal of Applied Biochemistry vol 7 no3 pp 223ndash227 1985

[32] A-R Kim T-S Shin M-S Lee et al ldquoIsolation and identifi-cation of phlorotannins from ecklonia stolonifera with antioxi-dant and anti-inflammatory propertiesrdquo Journal of Agriculturaland Food Chemistry vol 57 no 9 pp 3483ndash3489 2009

[33] J W Baynes ldquoChemical modification of proteins by lipids indiabetesrdquo Clinical Chemistry and Laboratory Medicine vol 41no 9 pp 1159ndash1165 2003

[34] H E Moon M N Islam B R Ahn et al ldquoProtein tyrosinephosphatase 1B and 120572-glucosidase inhibitory phlorotanninsfrom edible brown algae Ecklonia stolonifera and Eiseniabicyclisrdquo Bioscience Biotechnology and Biochemistry vol 75 no8 pp 1472ndash1480 2011

[35] E Apostolidis P D Karayannakidis Y-I Kwon C M Leeand N P Seeram ldquoSeasonal variation of phenolic antioxidant-mediated a-glucosidase inhibition of Ascophyllum nodosumrdquoPlant Foods forHumanNutrition vol 66 no 4 pp 313ndash319 2011

[36] F Nwosu J Morris V A Lund D Stewart H A Ross andG J McDougall ldquoAnti-proliferative and potential anti-diabeticeffects of phenolic-rich extracts from edible marine algaerdquo FoodChemistry vol 126 no 3 pp 1006ndash1012 2011

[37] G Galati and P J OBrien ldquoPotential toxicity of flavonoids andother dietary phenolics significance for their chemopreventiveand anticancer propertiesrdquo Free Radical Biology and Medicinevol 37 no 3 pp 287ndash303 2004

[38] S-J KimW-S Chung S-S Kim S-G Ko and J-Y Um ldquoAnti-inflammatory effect of Oldenlandia diffusa and its constituenthentriacontane through suppression of caspase-1 activation inmouse peritoneal macrophagesrdquo Phytotherapy Research vol 25no 10 pp 1537ndash1546 2011

[39] G Agoramoorthy M Chandrasekaran V Venkatesalu andM J Hsu ldquoAntibacterial and antifungal activities of fatty acidmethyl esters of the blind-your-eye mangrove from IndiardquoBrazilian Journal of Microbiology vol 38 no 4 pp 739ndash7422007

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of