total phenol content, antioxidant activities and α-glucosidase inhibition of sesame cake extracts

TOTAL PHENOL CONTENT, ANTIOXIDANT ACTIVITIES ANDa-GLUCOSIDASE INHIBITION OF SESAME CAKE EXTRACTSM.V. RESHMA1, L.K. NAMITHA, A. SUNDARESAN and CHALLA RAVI KIRAN

National Institute for Interdisciplinary Science and Technology (CSIR), Agro Processing and Natural Products Division, Thiruvathapuram, India

1Corresponding author. M.V. Reshma,National Institute for Interdisciplinary Scienceand Technology (formerly RRL), Council ofScientific and Industrial Research, Trivandrum695019, India. TEL: +91-949-6100937; FAX:+91-471-491712; EMAIL:[email protected]

Manuscript Region of Origin: India

Received for Publication November 17, 2011Accepted for Publication May 29, 2012

doi:10.1111/j.1745-4514.2012.00671.x

ABSTRACTSesame cake, the byproduct obtained after the removal of oil is presently used ascattle field. Present study evaluates the a-glucosidase, a-amylase inhibition andantioxidant properties of black sesame cake extracts. For that purpose, defattedseeds were sequentially extracted with ethyl acetate, methanol, methanol-water70:30 (v/v) and water. Among the extracts tested, methanol extract demonstratedbetter antioxidant activities (2,2-diphenyl-1-picrylhydrazyl, hydroxyl and super-oxide radical) and total phenol content. But the total flavonoid content and thetotal reducing power was high for methanol-water. Most active methanol extractwas further screened for a-glucosidase and a-amylase inhibition. The extractshowed strong a-glucosidase inhibitory potential and mild a-amylase inhibition.The study indicated that the extraction yield and the antioxidant activities werestrongly dependent on the solvent, antioxidant assays and extract concentration.These results demonstrated that sesame meal can be exploited as source of proteinand bioactive for the development of functional food.

PRACTICAL APPLICATIONSesame seed is one of the most important oil seed crops in the world. Defattedsesame meal, the byproduct obtained after the removal of oil is mainly used as acattle feed aside from being a good source of protein and antioxidants. This studyconducted using sesame meal extracts has revealed the inhibitory potential ofthe extracts against two carbohydrate digestive enzymes: a-glucosidase anda-amylase. The meal extract showed the presence of phenolics and flavonoids andalso showed antioxidant activities. This study thus provides the biochemical ratio-nale for further in vivo studies and utilization of this byproduct for developmentof functional food for the management of diabetes.

INTRODUCTION

Sesame (Sesamum indicum Linn.) seed is one of the mostimportant oil seed crops in the world. Besides providinghighly stable oil, sesame seed is also a source of protein-richmeals and is used in sweetmeats and confectionery foods,and has varieties of medicinal properties. Sesame oil plays aprominent role in Indian Ayurvedic medicine and Tibetanmedicine. In the Chinese system of medicine, dried sesameflowers are used in curing alopecia, frostbite and constipa-tion. Several studies have reported the health-promotingeffects of sesame, and this can be attributed to the presence

of bioactive compounds including lignans and phenols(Miyahara et al. 2001; Sankar et al. 2005).

Recent studies have shown that phenolic phytochemicalshave high antioxidant activity and certain therapeutic prop-erties (Vattem et al. 2005) including antidiabetic and anti-hypertension activity (Kwon et al. 2006). Diabetes is asignificant health problem affecting an estimated 180million people worldwide. During onset and developmentof type 2 diabetes, cellular balance of carbohydrate andlipid metabolism is affected by improper glucose metabo-lism. A sudden rise in blood glucose levels, causing hyperg-lycemia in type 2 diabetes patients happens due to

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Journal of Food Biochemistry ISSN 1745-4514

1Journal of Food Biochemistry •• (2012) ••–•• © 2012 Wiley Periodicals, Inc.

hydrolysis of starch by pancreatic a-amylase and uptakeof glucose by intestinal a-glucosidase (Apostolidis et al.2007). An effective strategy for type 2 diabetes managementis the strong inhibition of intestinal a-glucosidase and mildinhibition of pancreatic a-amylase (Krentz and Bailey2005).

a-Glucosidase and a-amylase are the key enzymesinvolved in the metabolism of carbohydrates. a-Amylasedegrades complex carbohydrates to oligosaccharides anddisaccharides, which are ultimately converted intomonosaccharides by a-glucosidases. Liberated glucose isthen absorbed by the gut and results in postprandial hyper-glycemia. Inhibition of intestinal a-glucosidases limitspostprandial glucose levels by delaying the process of carbo-hydrate hydrolysis and absorption, making such inhibitorsuseful in the management of type 2 diabetes (Shinde et al.2008).

Many researchers have attempted separating bioactivecompounds from sesame for in vitro/in vivo studies, nutra-ceutical applications and have reported the presence of lowmolecular weight phenolic compounds such as p-coumaricacid, o-coumaric acid, p-hydroxybenzoic acid, vanillic acidand isoferulic acid, also lignan compounds (sesamin andsesamolin) and tetranotriterpenoids. Some researchershave also reported antioxidant activities of sesame cakeand hull (Chang et al. 2002; Suja et al. 2004; Lee et al.2005; Shahidi et al. 2006; Reshma et al. 2010). Most of thestudies reported so far are on antioxidant activitiesof sesame seed and very few on a-glucosidase anda-amylase inhibition of sesame seed. In this study, wereport the a-glucosidase, a-amylase inhibition, totalphenol, total flavonoid content (TFC), antioxidant activitystudies and proximate composition of defatted blacksesame cake.

MATERIALS AND METHODS

Chemicals

2,2-Diphenyl-1-picrylhydrazyl (DPPH), nitro blue tetrazo-lium (NBT), phenazinemethosulphate (PMS), catechin,a-glucosidase type 1 from bakers yeast, p-nitro phenyla–dglucopyranoside (pNPG), a-amylase from Aspergillusoryzae and dinitrosalicylic acid (DNSA) were purchasedfrom Sigma Chemical Co. (St. Louis, MO). Thiobarbituricacid (TBA) was purchased from CDH (New Delhi, India),ethylenediamine tetraacetic acid (EDTA), NaH2PO4, Na2

HPO4 and Na2CO3 were from E-Merck (New Delhi, India)Ltd. Deoxyribose, trichloroacetic acid (TCA), ascorbic acid,gallic acid and Folin–Ciocalteau reagent from SiscoResearch Lab (Maharashtra, India). All the other chemicalsand solvents used were of standard analytical grade.

Sample Preparation

Black sesame seeds purchased from local market (Trivan-drum, India) were used for this study. The seeds used werecarefully cleaned to remove foreign particles.

Extraction Procedure Sequential Extractionof Sesame

Crushed black sesame seeds (500 g) were defatted withhexane for 12 h in a soxhlet apparatus and oil was separatedfrom the solvent using a rotary vacuum evaporator. Themeal thus obtained was sequentially extracted for 10–12 h atambient temperature (30C) with ethyl acetate, methanol,methanol-water 70:30 (v/v) and water, in the increasingorder of solvent polarity, as shown in Fig. 1. The extractswere filtered and solvent stripped off using a rotary vacuumevaporator, weighed and used for further analysis.

yield of extract weight of extractweight of sample

%( ) =×100

SCREENING OF ANTIOXIDANTACTIVITY

Total Phenolic Content (TPC)

The TPC of defatted sesame meal extracts (DSME) wasdetermined using Folin–Ciocalteu reagent (Singleton andRossi 1965). For every 100–500 mL of samples, distilledwater, 0.5 mL of Folin–Ciocalteu reagent and 1 mL of 20%sodium Carbonate were added. The contents were mixedand allowed to stand for 90 min. Absorbance of the solutionwas recorded at 760 nm using Shimadzu ultraviolet (UV)-visible spectrophotometer (Kyoto, Japan). The TPC wasexpressed as gallic acid equivalence in mg/100 g of thesample, using a standard curve plotted by taking 0–1,000 mggallic acid/mL.

TFC

The amount of total flavonoids was estimated by the alumi-num chloride method suggested by Chiang et al. (2002).Standard solution of catechin (20–100 mg) were separatelymixed with 0.3 mL of 10% aluminum chloride, 0.3 mL of5% sodium nitrite and 2 mL of NaOH and the volume wasmade to 10 mL with water. The absorbance of the reactionmixture was measured at 510 nm with UV-visible spectro-photometer. The amount of 10% aluminum chloride wassubstituted by the same amount of distilled water in blank.

a-GLUCOSIDASE INHIBITION OF SESAME CAKE M.V. RESHMA ET AL.

2 Journal of Food Biochemistry •• (2012) ••–•• © 2012 Wiley Periodicals, Inc.

Similarly, 100–500 mL of DSME was reacted with aluminumchloride for determination of flavonoid content asdescribed earlier.

Determination of DPPH RadicalScavenging Activity

The method described by Brand-Williams et al. (1995) wasused to assess antioxidant activity of DSME. The assaymixture contained 2.5 mL of 130 mM DPPH (final concen-tration 83.3 mM) dissolved in absolute methanol. Sample atdifferent concentrations was added to DPPH and the final

volume was adjusted to 3 mL. The mixture was shaken vig-orously on a vortex mixer and then incubated for 90 min atambient temperature in dark. A control run was also per-formed by taking 2.5 mL of DPPH and 0.5 mL methanolunder the same reaction condition. Absorbance was mea-sured spectrophotometrically at 517 nm. DPPH radicalscavenging activity of the DSME was calculated as followsand expressed as IC50 (mg/mL).

DPPH radical scavenging activityabsorbance of sample

%( )= −1 aabsorbance of control( ) ×100

Gallic acid was used as reference standard.

Black sesame seed

Crushed and extracted with n- Hexane

Oil + solvent Defatted sesame meal

Solvent stripped off Extracted with ethyl acetate

Sesame oil

Meal Extract 1

Extracted with Methanol

Meal Extract 2

Extracted with Methanol-Water70:30(v/v)

Meal Extract 3

Extracted with Water

Meal Extract 4 FIG. 1. SCHEME FOR SEQUENTIALEXTRACTION OF UNROASTED BLACK SESAMESEED

M.V. RESHMA ET AL. a-GLUCOSIDASE INHIBITION OF SESAME CAKE


Hydroxyl Radical Scavenging Activity2-Deoxyribose is oxidized by ·OH that is produced by theFenton reaction and degraded to malondialdehyde. Thereaction mixture contained 0.45 mL of 0.2 M sodium phos-phate buffer (pH 7.0), 0.15 mL of 10 mM 2-deoxyribose,0.15 mL of 10 mM FeSO4–EDTA, 0.15 mL of 10 mM H2O2,0.525 mL of water and 0.1 mL of sample solution in anEppendorf tube. The reaction was started by the addition ofH2O2. After incubation at 37C for 4 h, the reaction wasstopped by adding 0.75 mL of 2.8% (w/v) TCA and 0.75 mLof 1.0% (w/v) of TBA in 50 mM NaOH, the solution wasboiled for 10 min, and then cooled in water. The absorbanceof the solution was measured spectrophotometrically at520 nm. Hydroxyl radical scavenging ability of DSME wasevaluated as the inhibition rate of 2-deoxyribose oxidationby hydroxyl radicals (Halliwell et al. 1987) and comparedwith reference standard catechin.

Superoxide Anion Scavenging Activity

Superoxide anion scavenging activity was assayed accordingto the method of Kakkar et al. (1984). Superoxide radicalswere generated by the PMS/nicotinamide adeninedinucle-otide (PMS/NADH) system by oxidation of NADH andassayed by the reduction of NBT. The reaction mixture con-tained 100 mM Tris HCl buffer of pH 7.4 (1.58 g tris HClbuffer in 100 mL distilled water), 300 mM NBT (4.9 mgNBT/20 mL distilled water), 120 mM PMS (0.74 mg PMS/20 mL tris HCl buffer), 936 mM NADH (13.28 mg NADH/20 mL tris HCl buffer). Various concentration of DSME wastaken, made up to 1 mL using methanol. To the testsamples, 250 mL NBT, 250 mL NADH and 250 mL PMS wereadded. The reaction was started by the addition of 100 mL ofPMS (120 mM) to the mixture. After 5 min of incubation at25C, absorbance was measured at 560 nm. The percentageinhibition of superoxide anion generation was calculatedusing the following formula:

% inhibition absorbance of sampleabsorbance of control

= −() ×

11100

IC50 values of DSME were compared with catechin.

Determination of Reducing Power

The reducing properties are generally associated with thepresence of reductones, which have been shown to exertantioxidant action by breaking the free radical chain bydonating a hydrogen atom. The reductive potential of theextract was determined according to the method of Oyaizu(1986). Different concentrations of DSME were mixed withbuffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide(K3Fe[CN]6) (2.5 mL, 1% w/v). The mixture was incubated

at 50C for 20 min. A portion (2.5 mL) of TCA (10%w/v)was added to the mixture, which was then centrifuged for10 min. The upper layer of solution (2.5 mL) was mixedwith distilled water (2.5 mL) and FeCl3 (0.5 mL, 0.1% w/v),and the absorbance was measured at 700 nm in a spectro-photometer. The results are represented as 1 mg gallic acidequivalent, i.e., those extracts with low concentrationhaving absorbance equivalent to the absorbance of 1 mggallic acid are more powerful reducing agents.

a-Glucosidase Assay

The assay is modification of the procedure of Pistia-Brueggeman and Hollingsworth (2001). a-glucosidase(20 mL, 1.25 U/mL) was premixed with 200 mL of variousconcentrations of methanol extracts, made up in a 50-mMphosphate buffer at pH 6.8 and incubated for 5 min at 37C.In 50 mM of phosphate buffer, 1 mM pNPG (200 mL) wasadded to initiate the reaction, and the mixture was furtherincubated at 37C for 20 min. The reaction was terminatedby the addition of 500 mL of 1 M Na2CO3 and the finalvolume was made up to 1.50 mL. a-Glucosidase activity wasdetermined spectrophotometrically at 405 nm on a Bioradmultimode microplate reader (BioTek Instruments Inc.,Winooski, VT) by measuring the quantity of p-nitrophenolreleased from pNPG. The assay was performed in triplicate.The concentration of the extract that causes an inhibition inthe a-glucosidase activity by 50% under the assay condi-tions was the IC50 values.

a-Amylase Inhibition Assay

This method was adopted from Apostolidis et al. (2007).500 mL of the most active methanol extract and 500 mL of0.02 M sodium phosphate buffer (pH 6.9 with 0.006 Msodium chloride) containing a-amylase solution (0.5 mg/mL) were incubated at 25C for 10 min. After pre incuba-tion, 500 mL of 1% starch solution in 0.02 M sodiumphosphate buffer (pH 6.9 with 0.006 M sodium chloride)was added to each tube at timed intervals. The reaction wasincubated at 25C for 10 min. The reaction was stopped with1 mL of DNSA color reagent. The test tubes were then incu-bated in boiling water bath for 5 min and cooled to roomtemperature. The reaction mixture was then diluted afteradding 10 mL of distilled water and absorbance was read at540 nm.

% inhibition control extract control= −( )[ ] ( )A A A540 540 540

Proximate Analysis of DSM

The meal obtained after sequential extraction were analyzedfor the contents of moisture, crude protein, crude fat andash according to the AOAC method (AOAC 1984).



Statistical Analysis

All results were expressed as mean � standard deviation(n = 3).

RESULTS AND DISCUSSION

Yield, TPC and TFC

Yield, TPC and TFC of black sesame seed are shown inTables 1 and 2. Dried and crushed black sesame seeds weresequentially extracted using solvents (hexane, ethyl acetate,methanol, methanol-water 70:30 [v/v] and water) in theincreasing order of polarity. The oil content of sesame seeds(47.90 � 0.32%) obtained through hexane extraction wassimilar to the earlier reports of earlier workers (Kamal-Eldin and Appelqvist 1994) where a yield of 30% is reportedin the wild and 50% in the cultivated seeds. Because of itshigh oil content (about 50%), black sesame seeds are con-sidered a good source of edible oil.

Phenolic acids and their derivatives are widely distributedin plants and are reported to show antioxidant properties.In our study, we found that most of the polyphenol in DSMwere extracted into methanol (2.62 � 0.14%) followed

by water (1.76 � 0.09%), methanol-water 70:30 (v/v)(1.27 � 0.09%), ethyl acetate (0.69 � 0.05%) and hexane(0.07 � 0.03%). TPC in all the extracts combined was6.41 � 0.08%. TPC in DSM was then calculated, by consid-ering the yield obtained for each extract and % of TPC ineach extract. TPC of DSM (by combining the values of TPCobtained for all extracts) was 0.41 � 0.01%. Earlier workershave reported that 75.3% of phenolic compounds in DSMwere present as soluble esters and 24.7% as insolubleresidue (Dabrowski and Sosulski 1984).

The flavonoids are the largest single group of phenoliccompounds, which includes catechins and anthocyanidins,and have been proven to display a wide range of pharmaco-logical and biochemical properties. On investigating theTFC of DSM, we noticed that the extractability of fla-vonoids was high with methanol-water 70:30 (v/v)(1.74 � 0.11%) unlike TPC. The TFC in all the extractscombined was 3.89 � 0.05%. Total content of flavonoid inDSM was 0.22 � 0.02% and was calculated as mentionedfor TPC. TFC in DSM was also found lower than TPC. Theresults of TPC and TFC indicated the presence of polarcompounds in the extracts. In various studies, the antioxi-dant of plant extracts was correlated with the concentrationof flavonoids (Cakir et al. 2003).

DPPH Radical Scavenging Activity

The DPPH assay measures the ability of the extract todonate hydrogen to the DPPH radical resulting in bleachingof the DPPH solution. The greater the bleaching action,the higher the antioxidant activity, and is reflected bylower IC50 value. The free-radical scavenging activity ofthe extracts of sesame evaluated using the DPPH methodis shown in Table 3. The ability to scavenge DPPH radical(IC50) by sesame extracts was in the order of methanol

TABLE 1. YIELD OF EXTRACTS USING DIFFERENT SOLVENTS

Extracts †Yield (%) ‡Yield (%)

Hexane extract (oil) 47.90 � 0.32 –Ethyl acetate 1.81 � 0.05 3.78 � 0.20Methanol 4.41 � 0.01 9.46 � 0.12Methanol-water 70:30 (v/v) 2.70 � 0.16 5.96 � 0.35Water 1.89 � 0.05 4.98 � 0.48

† 100 g seed base.‡ based on defatted sesame meal (DSM).

TABLE 2. TPC AND TFC CONTENT OF DSMEExtracts †TPC (%) ‡TPC (%) †TFC (%) ‡TFC (%)

Hexane extract (oil) 0.07 � 0.03 0.03 � 0.00 NS NSEthyl acetate 0.69 � 0.05 0.03 � 0.00 0.84 � 0.02 0.03 � 0.01Methanol 2.62 � 0.14 0.24 � 0.01 0.89 � 0.04 0.08 � 0.02Methanol-water 70:30 (v/v) 1.27 � 0.09 0.07 � 0.00 1.74 � 0.11 0.09 � 0.02Water 1.76 � 0.09 0.07 � 0.01 0.42 � 0.04 0.02 � 0.01TPC content in all extracts

combined6.41 � 0.08 – – –

TFC content in all Extractscombined

– – 3.89 � 0.05 –

TPC content in DSM – 0.41 � 0.01 – –TFC content in DSM – – – 0.22 � 0.02

† % calculated based on extract.‡ % calculated based on DSM.TPC, total phenol content; TFC, total flavonoid content; DSME, defatted sesame meal extracts;NS, not significant.Values were the means of three replicates � standard deviation.



(61.79 � 1.63 mg/mL) > water (348.44 � 9.08 mg/mL) >methanol-water 70:30 (v/v) (480.29 � 6.57 mg/mL) > ethylacetate (636.36 � 12.49 mg/mL). IC50 value of gallic acid was1.95 � 0.52 mg/mL. The varied radical scavenging activity ofthe extracts depended on the amount of total phenolic ineach fraction. DPPH radical scavenging activity of sesameextracts may primarily be related to their hydrogen dona-tion ability. Some researchers has reported that ethanolextract of white sesame coat showed 94.9% scavengingeffect at a concentration of 10 mg (Chang et al. 2002).

Hydroxyl Radical Scavenging Activity

The scavenging activity of sesame extracts against hydroxylradical inhibition was reviewed using Fenton reaction(Table 3) and compared with reference standard Catechin.The IC50 values were observed in the following order:methanol (5.97 � 0.50 mg/mL) > catechin (6.40 � 0.67 mg/mL) > ethyl acetate (137.17 � 3.98 mg/mL) > water(1,206.33 � 15.44 mg/mL). Very low concentration ofmethanol and ethyl acetate extracts were effective againstsuppression of hydroxyl radical generated by the Fentonreaction than scavenging DPPH radical. The extractscompete with deoxyribose for the availability of hydroxylradical, thus reduce the formation of TBA reactive sub-stances (TBARS) resulting in better activity. Hydroxylradical is the most reactive oxygen species and it bears theshortest half-life compared with other reactive oxygenspecies. The ability of a substance to inhibit deoxyribosedegradation under the reaction condition is a measure of itsability to interfere with site specific Fenton chemistry(Aruoma 2003).

Superoxide Anion Scavenging Activity

The extracts (DSME) showed the ability to quench the O2–

radicals generated from the PMS/NADH reaction and

the results are shown in Fig. 2. Among the extractstested methanol extract was found to be most active(48.73 � 1.98 mg/mL) followed by water (974.85 �

12.45 mg/mL), ethyl acetate (1,055.22 � 14.56 mg/mL) andmethanol-water 70:30 (v/v) (1,322.33 � 19.65 mg/mL).Methanol extract was also found to be more active than thereference standard catechin (74.79 � 1.53 mg/mL). A similarstudy conducted by Visavadiya et al. (2009) on the aqueousextract and ethanol extract of sesame has shown that theethanol extract had a pronounced influence on their super-oxide radical scavenging activity as compared with theiraqueous extract. The activity shown here can be relatedto TPC, which have been believed as potent superoxidescavenger.

Determination of Reducing Power

The reducing power observed in the present study was doneaccording to the method of Oyaizu (1986), and the results

TABLE 3. DPPH, HYDROXYL RADICALSCAVENGING ACTIVITY AND TRP OFDEFATTED SESAME MEAL EXTRACTS

Sample DPPH (IC50 mg/mL)Hydroxyl radical scavengingactivity (IC50 mg/mL)

TRP (1 mg gallic acidequivalent)

Ethyl acetate 636.36 137.17 146.30�12.49 �3.98 �4.28

Methanol 61.79 5.97 161.04�1.63 �0.50 �3.36

Methanol/water70:30 (v/v)

480.29 NS 98.20�6.57 �9.00

Water 348.44 1206.33 409.25�9.08 �15.44 �11.00

Gallic acid 1.95 – –�0.52

Catechin – 6.40 –�0.67

DPPH, 2,2-diphenyl-1-picrylhydrazyl; TRP, total reducing power; NS, not significant.

Catechin Ethylacetate Methanol Methanol-Water Water0

200

400

600

800

1000

1200

1400

Con

cent

rati

on(μ

g/m

l)

FIG. 2. SUPEROXIDE RADICAL SCAVENGING ACTIVITIES (IC50) OFDEFATTED SESAME MEAL EXTRACTS (DSME) AND CATECHIN



are expressed as 1 mg gallic acid equivalent (Table 3).Among the extracts tested methanol-water 70:30 (v/v) wasfound more active (98.2 � 9.0 mg/mL) followed by ethylacetate (146.30 � 4.28 mg/mL), methanol (161.04 �

3.36 mg/mL) and water (409.25 � 11.0 mg/mL). The extractswith a low concentration having absorbance equivalentto 1 mg gallic acid are more powerful reducing agents. Inour study, we noticed that the extracts having high TFChas showed more activity than methanol extractshaving high TPC. Duh (1998) reported that the reducingproperties of antioxidants are generally associated withthe presence of reductones. In reducing power assay,polyphenols reduced ferricyanide ions i.e., (Fe[CN]6)3–

to ferrocyanide ions i.e (Fe[CN]6)4–, which further on, react-ing with Fe3+, gives rise to prussian blue-colored complex(ferric ferrocyanide) i.e., Fe4[Fe{CN}6]3). Phenolic acids andphenols having more number of hydrolysable OH groupsattached to rings act as more powerful reducing agents asthey have more electron- or H.-donating ability resulting inthe termination of free-radical chain reactions (Andersenet al. 2003).

a-Glucosidase Inhibition anda-amylase Inhibition

a-Amylases are endoglucanases, which hydrolyse the inter-nal alpha 1,4 glucosidic linkages in starch. a-Glucosidase isone of the glucosidases located in the brush border surfacemembrane of intestinal cells and is a key enzyme for carbo-hydrate digestion. These enzymes have been recognized astherapeutic targets for modulation of postprandial hyperg-lycemia. Amylase and a-glucosidase inhibitors are known toreduce postprandial hyperglycemia by partially inhibitingthe enzymatic hydrolysis of complex carbohydrates andhence may delay the absorption of glucose. Acarbose, vogli-bose and miglitol are synthetic inhibitors used either aloneor in combination with insulin secretogogues for patientswith type II diabetes. However, theses inhibitors arereported to cause several side effects. Several other safernatural inhibitors are reported from plant resources(Shobana et al. 2009).

In this study, we checked the a-glucosidase inhibitoryactivities of methanol extract of DSM and the valueobtained were compared with standard acarbose (Fig. 3).The enzyme inhibition of methanol extract was in thedose-dependent manner and the IC50 value was 375 �

5 mg/mL and of standard acarbose was 444.30 � 2 mg/mL.There are recent studies showing the association ofTPC, antioxidant activity and a-glucosidase inhibition.Plant phenolic compounds are reported to modulate theenzymatic breakdown of carbohydrate by inhibiting amy-lases and glucosidases (McDougall et al. 2005). Thus, the

inhibition of enzyme in this study can be related to thepresence of phenolic compounds in the methanol extract ofDSM.

On checking the a-amylase inhibitory activities ofmethanol extract of DSM, we found that the extract showedonly low inhibitory activity against a-amylase. Themaximum inhibition shown was only 16.95% and theconcentration was 750 � 15 mg/mL (Fig. 4). On furtherincreasing the concentration the extract showed a decreasedinhibitory activity. Mild inhibition of pancreatic a amylaseand strong inhibition of intestinal a- glucosidase is consid-ered as an effective strategy for type 2 diabetic management(Krentz and Bailey 2005).

Proximate Analysis

Besides the presence of phenolics and flavonoids, DSM isalso a source of protein (31.02 � 2.57%), carbohydrates(56.47 � 5.61%) and ash (11.87 � 1.39%) (Table 4). Pres-

0 100 200 300 400 50020

30

40

50

60

Inh

ibit

ion(

%)

Concentration(mg/ml)

Concentration(mg/ml)

b

100 200 300 400 50010

20

30

40

50

60

70

%In

hib

itio

n

a

FIG. 3. (a) a-GLUCOSIDASE INHIBITION ACTIVITY OF METHANOLEXTRACT (b) a-GLUCOSIDASE INHIBITION ACTIVITY OF ACARBOSE



ence of these compounds point out the possibility of utiliz-ing DSM for fortification and functional food development.

The goal of this study was to provide in vitro evidence fora-glucosidase and a-amylase inhibition, which is lessreported in sesame. The results of this study also show thatthe antioxidant activity of the extracts depends on thesolvent, the concentration of the extract and the antioxidantactivity assay. The extract concentration was a determinantfactor of the prooxidant and antioxidant action. It wasobserved that the extracts obtained using polar solventswere considerably more effective radical scavengers thanthose obtained using less polar solvents. Change in thepolarity of the solvents alters its ability to dissolve a selectedgroup of antioxidant compounds and influences activityestimation. As observed, extracts with higher antioxidantcapacity also had higher polyphenolic content. Radical scav-enging studies observed in this investigation can also beattributed to the presence of lignan glycosides, flavonoid,tannins and polysaccharides. Thus, this study also pointsout the possibility of exploring the utilization of sesamecake not only as a potential source of protein, but also asa source of health-protective bioactive for developingfunctional food.

CONFLICT OF INTEREST STATEMENT

The authors declare that there are no conflicts of interest.

ACKNOWLEDGMENT

This work is part of Supra Institutional Project funded byCSIR, India.

REFERENCES

ANDERSEN, M.L., LAURIDSEN, R.K. and SKIBSTED, L.H.2003. Optimising the use of phenolic compounds in foods.In Phytochemical Functional Foods, (I. Johnson and G.Williamson, eds.) pp. 315–346, Woodhead Publishing Ltd,Cambridge.

AOAC. 1984. Official Method of Analysis, 14th Ed., Association ofOfficial Analytical Chemists, VA, USA, Washington, DC.

APOSTOLIDIS, E., KWON, Y.-I. and SHETTY, K. 2007.Inhibitory potential of herb, fruit and fungal enrichedcheese against key enzymes linked to type2 diabetes andhypertension. Innov. Food Sci. Emerg. Technol. 8, 46–54.

ARUOMA, O.I. 2003. Methodological considerations forcharacterizing potential antioxidant actions of bioactivecomponents in plant foods. Mutat. Res. 523, 9–20.

BRAND-WILLIAMS, W., CUVELIER, M.E. and BERSET, C.1995. Use of a free radical method to evaluate antioxidantactivity. LWT – Food Sci. Technol. 28, 25–30.

CAKIR, A., MAVI, A., YILDIRIM, A., DURU, M.E.,HARMANDAR, H. and KAZAZ, C. 2003. Isolation andcharacterization of antioxidant phenolic compounds from theaerial parts of Hypericum hyssopifolium L. by activity-guidedfractionation. J. Ethnopharmacol. 87, 73–83.

CHANG, L.W., YEN, W.J., HUANG, S.C. and DUH, P.D. 2002.Antioxidant activity of sesame coat. Food Chem. 78, 347–354.

CHIANG, C.C., YANG, M.H., WEN, H.M. and CHERN, J.C.2002. Estimation of total flavonoid content in propolis by twocomplementary colorimetric methods. Yaowu. Shipin. Fenxi.10, 178–182.

DABROWSKI, K.J. and SOSULSKI, F.W. 1984. Composition offree and hydrolyzable phenolic acids in defatted flours of tenoilseeds. J. Agric. Food Chem. 32, 128–130.

DUH, P.D. 1998. Antioxidant activity of budrock (Arctium lappaL.): It’s scavenging effect on free radical and active oxygen. J.Am. Oil Chem. Soc. 75, 455–461.

HALLIWELL, B., GUTTERIDGE, J.M.C. and ARUOMA, O.I.1987. The deoxyribose method: A simple “test-tube” assay fordetermination of rate constants for reactions of hydroxylradicals. Anal. Biochem. 165, 215–219.

KAKKAR, P., DAS, B. and VISWANATHAN, P.N. 1984. Amodified spectrophotometric assay of superoxide dismutase.Indian J. Biochem. Biophys. 21, 130–132.

KAMAL-ELDIN, A. and APPELQVIST, L.A. 1994. Variation infatty acid composition of the different acyl lipids in seed oilsfrom four Sesamum species. J. Am. Oil Chem. Soc. 71,135–139.

KRENTZ, A.J. and BAILEY, C.J. 2005. Oral antidiabetic agents –current role in type 2 diabetes mellitus. Drugs 65, 385–411.

400 600 800 1000 1200

3

6

9

12

15

18 I

nhib

itio

n(%

)

Concentration (μg/mL)

FIG. 4. a- AMYLASE ACTIVITY OF METHANOL EXTRACT

TABLE 4. PROXIMATE COMPOSITION OF DEFATTED SESAME MEAL

Moisture (%) 0.25 � 0.04

Fat (%) 0.40 � 0.06Protein (%) 31.02 � 2.57†Carbohydrate (%) 56.47 � 5.61Ash (%) 11.87 � 1.39

† By difference.



KWON, Y.-I., VATTEM, D.A. and SHETTY, K. 2006. Evaluationof clonal herbs of Lamiaceae species for management ofdiabetes and hypertension. Asia Pac. J. Clin. Nutr. 15,107–118.

LEE, S.C., JEONG, S.M., KIM, S.Y., NAM, K.C. and AHN, D.U.2005. Effect of far-infrared irradiation on the antioxidantactivity of defatted sesame meal extracts. J. Agric. Food Chem.53, 1495–1498.

MCDOUGALL, G.J., SHPIRO, F., DOBSON, P., SMITH, P.,BLAKE, A. and STEWART, D. 2005. Different polyphenoliccomponents of soft fruit inhibit a-amylase and a-glucosidase.J.Agric. Food. Chem. 53, 2760–2766.

MIYAHARA, Y., HIBASAMI, H., KATSUZAKI, H., IMAI, K. andKOMIYA, T. 2001. Sesamolin from sesame seed inhibitsproliferation by inducing apoptosis in human lymphoidleukemia Molt 4B cells. Int. J. Mol. Med. 7, 369–371.

OYAIZU, M. 1986. Studies on products of browningreaction-antioxidative activities of products of browningreaction prepared from glucosamine. Jpn. J. Nutr. 44,307–315.

PISTIA-BRUEGGEMAN, G. and HOLLINGSWORTH, R.I.2001. A preparation and screening strategy for glycosidaseinhibitors. Tetrahedron 57, 8773–8778.

RESHMA, M.V., BALACHANDRAN, C., ARUMUGHAN, C.,SUNDERASAN, A., SUKUMARAN, D., THOMAS, S. andSARITHA, S.S. 2010. Extraction, separation andcharacterisation of sesame oil lignan for nutraceuticalapplications. Food Chem. 120, 1041–1046.

SANKAR, D., SAMBANDAM, G., RAO, M.R. and PUGALENDI,K.V. 2005. Modulation of blood pressure, lipid profiles and

redox status in hypertensive patients taking different edibleoils. Clin. Chim. Acta 355(1–2), 97–104.

SHAHIDI, F., LIYANA-PATHIRANA, C.M. and WALL, D.S.2006. Antioxidant activity of white and black sesame seedsand their hull fractions. Food Chem. 99, 478–483.

SHINDE, J., TALDONE, T., BARLETTA, M., KUNAPARAJU, N.,HU, B., KUMAR, S., PLACIDO, J. and ZITO, S.W. 2008.Alpha-glucosidase inhibitory activity of Syzygium cumini(Linn.) Skeels seed kernel in vitro and in Goto-Kakizaki (GK)rats. Carbohydr. Res. 343, 1278–1281.

SHOBANA, S., SREERAMA, Y.N. and MALLESHI, N.G. 2009.Composition and enzyme inhibitory properties of fingermillet (Eleusine coracana L.) seed coat phenolics: Mode ofinhibition of alpha-glucosidase and pancreatic amylase. FoodChem. 115, 1268–1273.

SINGLETON, V.L. and ROSSI, J.A. 1965. Colorimetry of totalphenolics with phosphomolybdic-phosphotungstic acidreagents. Am. J. Enol. Vitic. 16, 144–158.

SUJA, K.P., ABRAHAM, J.T., THAMIZH, S.N., JAYALEKSHMY,A. and ARUMUGHAN, C. 2004. Antioxidant efficacy ofsesame cake extract in vegetable oil protection. Food Chem.84, 393–400.

VATTEM, D.A., GHAEDIAN, R. and SHETTY, K. 2005.Enhancing health benefits of berries through phenolicantioxidant enrichment: Focus on cranberry. Asia Pac. J. Clin.Nutr. 14, 120–130.

VISAVADIYA, N.P., SONI, B. and DALWADI, N. 2009. Freeradical scavenging and antiatherogenic activities of Sesamumindicum seed extracts in chemical and biological modelsystems. Food Chem. Toxicol. 47, 2507–2515.



total phenol content, antioxidant activities and α-glucosidase inhibition of sesame cake extracts

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