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LWT - Food Science and Technology xxx (2014) 1e7

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LWT - Food Science and Technology

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Evaluation of antioxidative, antibacterial and probiotic growthstimulatory activities of Sesamum indicum honey containing phenoliccompounds and lignans

Angira Das a, Sanjukta Datta b, Sayani Mukherjee a, Sreedipa Bose a, Santinath Ghosh b,Pubali Dhar a, *

a Laboratory of Food Science and Technology, Food and Nutrition Division, University of Calcutta, 20B Judges Court Road, Kolkata 700027, Indiab Department of Chemical Technology, University College of Science and Technology, University of Calcutta, 92, A.P.C. Road, Kolkata 700009, India

a r t i c l e i n f o

Article history:Received 13 September 2012Received in revised form11 November 2014Accepted 22 November 2014Available online xxx

Keywords:Sesamum indicum (sesame) honeyFlavonoidSesame lignanProbioticAgarose gel electrophoresis

* Corresponding author. Tel.: þ91 9433122560; faxE-mail address: pubalighoshdhar@yahoo.co.in (P. D

http://dx.doi.org/10.1016/j.lwt.2014.11.0440023-6438/© 2014 Elsevier Ltd. All rights reserved.

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a b s t r a c t

Seven Sesamum indicum (sesame) honey samples were collected from Hooghly district of West Bengal,India and analyzed for polyphenol and flavonoid content along with their in vitro free radical scavengingactivities. Antibacterial activity and stimulatory effect on multiplication of probiotic bacteria wereevaluated. Antioxidant markers like IC50 value for DPPH (1, 1-diphenyl-2-picrylhydrazyl) and FRAP (ferricreducing antioxidant power) of sesame honey were positively correlated to its polyphenolic content(28.9 ± 0.6 mg GAE/100 g) and color intensity (r ranges between 0.872 and 0.931). Four flavonoids viz.,apigenin, quercetin, myricetin, rutin have been identified along with one cinnamic acid derivative (ferulicacid) and two sesame lignans (sesamin and episesamin) by High performance liquid chromatographywhich can be used as a tool for authentication of sesame honey. Antibacterial activity of sesame honeyagainst some enteropathogenic bacteria Escherichia coli, Vibrio cholerae, S. Typhi, S. Typhimurium werestudied. Minimum inhibitory concentration was found to be lowest against S. Typhi (12.5% w/v) and S.Typhimurium (12.5% w/v). Plasmid DNA degradation by sesame honey administration evinced its mo-lecular level of action. Interestingly, it was also found that sesame honey exhibited significant growthpromoting property of probiotic strains such as Lactobacillus acidophilus and Bifidobacterium bifidum.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Honey is a supersaturated solution of sugars, of which fructose(38% w/w) and glucose (31% w/w) are the main contributors, con-taining a wide range of minor constituents like phenolic acids,flavonoids, certain enzymes, carotenoid like substances, aminoacids, organic acids, Maillard reaction products, vitamins andminerals. Its composition is rather variable and primarily dependson the botanical and geographical origin of the floral source,although certain external factors also play a role, such as seasonaland environmental factors and its processing.

Phenolic compounds in honey are mainly flavonoid, aglyconesand phenolic acid derivatives like benzoic acids, cinnamics acidsand their respective esters (Tom�as Barber�an, Martos, Ferreres,Radovic, & Anklam, 2001).

: þ91 33 23519755.har).

l., Evaluation of antioxidativepounds and lignans, LWT

Hitherto, phenolic compounds have been in use as potentialchemical markers for authenticating the geographical and botanicalorigin of honey. Flavonoids, the most common phenolics in floralhoneys are characteristic profiles of the plants which are used bythe bees to collect nectar or honeydew. These bioactive compo-nents present in honey varies corresponding to the different plantsources (Martos, Ferreres, & Tom�as Barber�an, 2000). A strong cor-relation exists between the antioxidant activity of honeys and theirphenolic composition and especially the total phenolic content(Gheldof & Engeseth, 2002). The antibacterial activity of honeyagainst several pathogens and its dependence on the floral originand their phenolics has been widely reported (Al et al., 2009;Finola, Lasagno, & Marioli, 2007). Thus, characterization of phe-nolics and other components in honey that might have antioxidantand antibacterial properties is essential to improve our knowledgeabout honey as a source of nutraceutical and would also be animportant tool to contribute to their authentication.

Several studies on the flavonoid composition of honeys havebeen carried out in different parts of theworld (Martos et al., 2000).

, antibacterial and probiotic growth stimulatory activities of Sesamum- Food Science and Technology (2014), http://dx.doi.org/10.1016/

A. Das et al. / LWT - Food Science and Technology xxx (2014) 1e72

However, Indian honeys lack documentation in the literature abouttheir phenolic profile and therapeutic potential. Although Sesamumindicum (sesame) honey has been widely used as a domestic ay-urvedic remedy in India, yet its antioxidative activity has not beenreported so far. The selective activity of sesame honey againstgastrointestinal pathogenic bacteria, while its augmentative effecton multiplication of probiotic bacteria is a novel aspect in terms ofits therapeutic implication and has been well elucidated in ourstudy. The phenolic and lignan profile of sesame honey and theirassociation with in vitro antioxidative, antibacterial and growthstimulatory activity of probiotics has been assessed. The presentstudy thus opens up vistas of opportunities in use of Indian honeyfor therapeutic applications inter alia other benefits.

2. Materials and methods

2.1. Chemicals

DPPH (1, 1-diphenyl-2-picrylhydrazyl) and TPTZ (2, 4, 6-tri (2-pyridyl)-s-triazine) were purchased from E. Merck India Pvt. Ltd.,Kolkata, India. FolineCiocalteu's phenol reagent, ferulic acid,quercetin, apigenin, myricetin and rutin were obtained from SigmaAldrich Chemical Co., Milwaukee, Wis., U.S.A.

2.2. Bacterial strains and media used

The test organisms Escherichia coli (ATCC 25922), Vibrio cholera569B, S. Typhi D641 and S. Typhimurium SGT340 were suppliedfrom National Institute of Cholera and other Enteric Disease(NICED), Kolkata, India. Lactobacillus acidophilus (MTCC 447) andBifidobacterium bifidum (ATCC 700541) were bought fromMicrobialType Culture collection (MTCC) Chandigarh, India and AmericanType Culture Collection (ATCC), U.S.A. respectively.

Trypticase Soy Broth (TSB) and nutrient agar medium werepurchased from SISCO Research Laboratories Pvt Ltd. Mumbai, In-dia. De Man, Rogosa, Sharpe (MRS) Broth and MRS agar mediumwere procured from HIMEDIA, Hi Media laboratories Pvt Ltd,Vadhani Ind. Est, LBS Marg, Mumbai, India.

2.3. Honey samples

Seven sesame honey samples were obtained from differentbeekeeper's association fromHooghly district ofWest Bengal, India.The honey samples were collected in 2011e2012 flowering seasons.All the samples were stored at 0e4 �C and analyzed at the earliestin such a way that none of the samples exceeded the storage periodbeyond 3 months. The honey samples were kept at room temper-ature overnight before the analyses were performed.

2.4. Total polyphenol and flavonoid content and in vitroantioxidative assays

2.4.1. Total polyphenol contentThe FolineCiocalteumethod (Dhar, Chaudhury, Mallik,&Ghosh,

2011) was used to determine total polyphenol content. Gallic acid(20e100 mg) was used as standard.

2.4.2. Flavonoid contentThe flavonoid content was determined using the Dowd method

as adapted by Arvouet, Vennat, Pourrat, and Legret (1994). The totalflavonoid content was determined using a standard curve withquercetin (20e100 mg) standard and was expressed as mg ofquercetin equivalents (QE)/100 g of honey.

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2.4.3. Color intensity ABS450Sesame honey samples were diluted to 50% (w/v) with warm

(45e50 �C) double distilled water and the solution was filteredthrough a Whatman No. 1 paper. Color intensity was thenmeasured using a spectrophotometer (PerkineElmer Lambda 25) at450 and 720 nm and the difference in absorbance was expressed asmAU (Beretta, Granata, Ferrero, Orioli, & Facino, 2005).

2.4.4. DPPH radical scavenging activity2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity of

honey samples was measured as described by Beretta et al. (2005)with some modifications. 0.75 ml of honey samples (25e100 mg/ml) was mixed with 1.5 ml of DPPH in methanol (0.02 mg/ml), withmethanol serving as the blank. After 15 min of incubation at roomtemperature, the absorbance was measured at 517 nm. Themean ofthree IC50 (concentration causing 50% inhibition of DPPH) values ofthe honey samples were determined graphically.

2.4.5. FRAP assayThe ferric reducing ability of honey was determined by FRAP

assay (Benzie & Strain, 1996) with some modifications. WorkingFRAP reagent was prepared by mixing 10 volumes of 300 mMol/Lacetate buffer, pH 3.6 with 1 volume of 10mMol/L 2, 4, 6-tripyridyl-s-triazine in 40 mMol/L hydrochloric acid and with 1 volume of20mMol/L ferric chloride. 400 ml of honey solution (containing 0.1 gof honey/ml) was added to 3 ml of freshly prepared FRAP reagent.The absorbance was measured at 593 nm after 4 min incubation at37 �C. The difference between this absorbance and the sampleblank (honey solution with distilled water), was calculated to getthe final absorbance. Aqueous solutions of known Fe2þ concen-tration, in the range of 100e500 mMol/L (FeSO4.7H2O)were used forcalibration. The reducing ability of honey was expressed as mMol ofFe2þ equivalent/g of honey.

2.5. Determination of phenolic compounds and sesame lignan

2.5.1. Sample extraction for HPLC analysis (columnchromatography)

Sesame honey samples were extracted for HPLC analysis bycolumn chromatography according to the method of Tom�asBarber�an et al. (2001). 100 g of each sample were mixed with fiveparts of water (pH 2 with HCl) until completely fluid and filteredthrough cotton to remove solid particles. The filtrate was thenpassed through a column (25 � 2 cm) of Amberlite XAD-2 (FlukaChemie; pore size ¼ 9 nm, particle size ¼ 0.3e1.2 mm). Thephenolic compounds remain on the column, while sugars and otherpolar compounds elute with the aqueous solvent. The column waswashed with acid water (pH 2 with HCl, 100 ml) and subsequentlywith distilled water (300ml). Thewhole phenolic fractionwas theneluted withmethanol (300ml) and taken to dryness under reducedpressure (40 �C). The residue was dissolved in 5 ml of water andextracted with diethyl ether (5 ml � 3). The ether extracts werecombined, concentrated under reduced pressure, and dissolved in0.5 ml of methanol for HPLC analysis.

2.5.2. HPLC analysis for flavonoid identificationAccording to China et al. (2011), a gradient elution was

employed for flavonoids with a mobile phase consisting of50 mMol/L H3PO4, pH 2.5 (solution A), and acetonitrile (solution B)which is as follows: isocratic elution 95% A/5% B (v/v), 0e5 min;linear gradient from 95% A/5% B to 50% A/50% B, 5e55min; isocraticelution 50% A/50% B, 55e65 min; linear gradient from 50% A/50% Bto 95% A/5% B, 65e67 min; post-time 6 min before the next injec-tion. The system, WATERS 2487 is equipped with a C-18 column(Nova-Pak C18, 3.9 � 150 mm). 340 nm and 370 nm wavelengths

, antibacterial and probiotic growth stimulatory activities of Sesamum- Food Science and Technology (2014), http://dx.doi.org/10.1016/

Table 1Polyphenol and flavonoid content and in vitro antioxidative assays of sesame honeysamples. Data are expressed as Mean ± SD.

Parameters Sesame honey (n ¼ 7)

Total polyphenol (mg GAE/100 g) 28.9 ± 0.6Total flavonoid (mg QE/100 g) 13.2 ± 1.8Color intensity ABS450 (in mAU) 1180 ± 1.2DPPH radical scavenging activity (IC50 in mg/ml) 39.5 ± 0.4FRAP value (mMol Fe (II) equivalence/L) 2.75 � 106 ± 4.8

A. Das et al. / LWT - Food Science and Technology xxx (2014) 1e7 3

were selected for the detection. The flow rate of the mobile phasewas 0.8 ml/min, and the injection volume was 20 ml. The peakswere identified in comparison to authentic standards.

2.5.3. HPLC analysis of sesame lignan identificationLipid was extracted from 50 g of sesame honey using 100 ml of

Chloroform (Kapoulas, Mastronicolis, & Galanos, 1977). Sesamelignans were then purified by the method of Fukuda, Nagada,Osawa and Namiki (1986) and analyzed by an HPLC method. Theinstrument was provided with binary HPLC pump 1525 andWatersdual absorbance UV detector 2487. The column was Nova-Pakbonded C-18 (size: 4.6 � 150 mm) having microparticulate silicaof particle size of 5 mm. A total of 20 ml of the sample solution wasinjected. The mobile phase was methanol/water (65:35, v/v) at aflow rate of 0.8 ml/min 290 nm wavelengths was selected for thedetection. The peaks were identified in comparison to authenticstandards.

2.6. Evaluation of antibacterial effect

2.6.1. Agar well diffusion assayAntibacterial activity of sesame honey was determined by agar

well-diffusion method against four enteropathogenic microorgan-isms, viz. E. coli (ATCC 25922), V. cholera 569B, S. Typhi D641 and S.Typhimurium SGT340. An aliquot of 100 ml of an overnight culturewas diluted in saline solution to about 106 CFU/ml and was spreadon nutrient agar plates. Wells (7 mm in diameter) were made inagar using a sterilized stainless steel borer. Eachwell was filled with100 ml of 50% (w/v) honey solution. The plates were left at roomtemperature for 30 min to allow diffusion of materials in media.Ampicillin, streptomycin, chloramphenicol, ciprofloxacin, azi-thromycin, doxycycline, nalidixic acid, ofloxacin, tetracycline wereused as controls. Plates were incubated at 37 �C for 24 h, untilvisible growth of testmicroorganismswas evident in control plates.Inhibition zones in mm (including well diameter) around wellswere measured. Zone of inhibition formed by commercial antibi-otics were compared with the zone size interpretative chart sup-plied by HIMEDIA (CLSI, 2008).

2.6.2. Determination of minimum inhibitory concentration (MIC)The minimum inhibitory concentration (MIC) of sesame honey

was determined using the broth dilution method in screw cappedtubes (10mm� 100mm) (Tan et al. 2009). Fifty percent (w/v) stocksolution of each type of honey was prepared using TSB. Furtherdilutions were done to obtain honey concentrations of 6.25%, 7.5%,8.75%, 10%, 11.25%, 12.5%, 15%, 17.5%, 20%, 22.5% and 25% (w/v).Experimental screw tube was filled with 0.1 ml of inoculums(106 CFU/ml), 0.4 ml of diluted honey and 1 ml of TSB. The tubeswere then incubated at optimal temperature of 37 �C for 18 h.Positive control and negative control tubes contain(TSB þ Inoculums) and (TSB þ Inoculums þ Antibiotics) respec-tively. All honey solutions were freshly prepared before each assayand were filtered through Millipore membrane filter (poresize ¼ 0.22 mm).

2.6.3. DNA degradation assays by agarose gel electrophoresisThe extent of DNA damage induced in plasmid DNA was fol-

lowed by the difference in migration pattern on agarose gel elec-trophoresis. The reaction mixture was conducted in a total volumeof 20 ml containing 2 ml of pUC 18 DNA,10 ml of 50% (w/v) honey and8 ml of double distilled water andwas incubated at 37 �C for 5 h. Thereaction mixture was then mixed with 2 ml of 6� gel loading dye,loaded into 1% (w/v) agarose gel and run at 50 V for 30 min fol-lowed by 15 min run time at 100 V in a submarine gel

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electrophoresis apparatus. The DNA was visualized and photo-graphed using gel doc system (Brudzynski, Abubaker, & Miotto,2012).

2.7. Effect of sesame honey on the growth of probiotics

The effect of sesame honey on the growth of two probioticstrains L. acidophilus (MTCC 447) and B. bifidum (ATCC 700541)were evaluated by two different methods. For determination ofgrowth curve pattern of probiotic strains, sesame honey supple-mented at 5% (w/v) level to carbohydrate freeMRS broth (modified)were filter sterilized with 0.22 mmmembrane filter (Millipore, USA)and inoculated with L. acidophilus (106 CFU/ml) and B. bifidum(106 CFU/ml) served as experimental culture. MRS broth inoculatedwith probiotic strains served as control. All the tubes were thenincubated anaerobically at 37 �C for 24 h. The growth rate ofL. acidophilus and B. bifidumweremeasured spectrophotometricallyat 600 nm at 2 h interval using sterilized broth as blank.

For plate count method, the experimental and control cultureswere diluted in MRS broth and were plated on MRS agar medium.The effect of sesame honey on L. acidophilus and B. bifidum weredetermined comparing the number of CFU in the presence of ses-ame honey against those obtained from controls.

2.8. Statistical analysis

All analyses were carried out in triplicate and the data wereexpressed as means ± standard deviations (SD). Differences be-tween means and correlation at the 95% (p < 0.05) confidence levelwere considered statistically significant. Correlations were ob-tained by Pearson's correlation coefficient (r) in bivariate linearcorrelations using SPSS software (version 19.0).

3. Results and discussions

3.1. In vitro antioxidative potential

The total polyphenolic content and flavonoid content of sesamehoney were found to be 28.9 ± 0.6 mg GAE and 13.2 ± 1.8 QE per100 g of honey respectively (Table 1). Its flavonoid content isconsiderably higher than that of European honeys e.g., Eucalyptushoney (2e2.5 mg QE/100 g), sunflower and rape honey (1.5e2 mgQE/100 g) while that of polyphenol content is comparable to someSlovenian and Romanian honeys ranging from 4.48 to 24.14 and23.0 to 125.0 mg GAE/100 g, respectively (Al et al., 2009; Bertoncelj,Dobersek, Jamnik,& Golob, 2007; Martos et al., 2000). In this study,we used a spectrophotometric quantification of flavonoids withaluminum chloride, which has previously been described for thequantification of flavonoids in propolis extracts (Arvouet et al.,1994).

The color of sesame honey was found to be dark brown withABS450 value of 1180 ± 1.2 mAU (Table 1). The color intensity(ABS450) is related to the presence of pigments (carotenoids, fla-vonoids, Maillard reaction products) which are known to have

, antibacterial and probiotic growth stimulatory activities of Sesamum- Food Science and Technology (2014), http://dx.doi.org/10.1016/

Table 2Correlation matrix (Pearson correlation coefficient).

Polyphenol Flavonoid ABS450 DPPH(1/IC50)

Flavonoid 0.726ABS450 0.942 0.681DPPH(IC50) 0.872 0.513 0.878FRAP 0.923 0.578 0.931 0.902

A. Das et al. / LWT - Food Science and Technology xxx (2014) 1e74

antioxidant activity. The reported ABS450 values for some lightcolored Acacia and Lime honeys are in the range of 70e123 mAUwhile that of dark brown Strawberry and Buckwheat honeys are inthe range of 2245e3413 mAU (Beretta et al., 2005; Bertoncelj et al.,2007). A high positive correlation observed between color intensityand polyphenol content (r ¼ 0.942), IC50 value of DPPH (r ¼ 0.878),and FRAP (r ¼ 0.931) which supported the fact that darker honeyshave good radical scavenging properties which may be due to thepresence of polyphenols (Table 2).

The DPPH assay measures hydrogen atom (or one electron)donating activity and hence provides a measure of antioxidantactivity. Table 1 showed the DPPH radical scavenging abilityexpressed as IC50 on the DPPH radical, the amount of antioxidantnecessary to decrease the initial concentration of DPPH by 50%.Mean IC50 value of sesame honey samples was found to be39.5 ± 0.4 mg/ml. A high correlation existed between 1/IC50 andpolyphenol content (r¼ 0.872) of sesame honey lead us to concludethat phenolic compound also has an antiradical potency (Table 2).

To gain further insight on the antioxidant activity of honey, FRAPvalue of sesame honey samples were analysed. FRAP assay is asimple direct test that is widely used for antioxidant activity and isbased on the ability of the analyte to reduce the Fe3þ/Fe2þ couple.Mean FRAP value of sesame honey found to be2.75�106 ± 4.8 mMol Fe(II)/(Table 1) was quite high as compared tothat of Slovenian fir and forest honey, 4.785 � 105 and

Fig. 1. (A). HPLC chromatogram of sesame honey at 340 nm for flavonoid detection. The pea370 nm for flavonoid detection. The peaks identified are 1 e rutin, 2 e myricetin, 3 e que

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4.264 � 105 mMol Fe(II)/L respectively (Bertoncelj et al., 2007). Asdepicted in Table 2, strong positive correlation existed betweenFRAP and total polyphenol content (r ¼ 0.923) was indicative of thefact that polyphenols are one of the major component responsiblefor reduction of Fe3þ to Fe2þ (Table 2).

3.2. HPLC analysis of flavonoids and sesame lignan

Themain problem in the analysis of flavonoids fromhoney is thevery high sugar content, which makes the extraction of flavonoidsand sample preparation for HPLC analysis difficult. Liquideliquidpartitions produce inconvenient interphases which do not permitthe complete recovery of flavonoids. However, this problem hasbeen solved by using XAD-2 resin (Tom�as Barber�an et al., 2001). Allsesame honey samples analysed contained variable amounts of fivephenolic compounds (one phenolic acid and four flavonoids). Thefour flavonoids identified in sesame honey include rutin, quercetin,apigenin and myricetin while that of phenolic acid is ferulic acid(Fig. 1A and B).

Two important sesame lignans viz. sesamin and episesamin,were identified (Fig. 2) in the lipid fraction of the sesame honey.Sesame lignans were 97.5% (w/w) pure as observed in the HPLCchromatogram (65.5:34.5 sesamin/episesamin, w/w). These uniquesesame lignans can be used for the authentication of sesame honey.

3.3. Antibacterial activity

With the rise in prevalence of antibiotic resistant bacteria, honeyis increasingly valued for its antibacterial activity. The results ofantibacterial activity of sesame honey against test organisms areillustrated in Tables 3 and 4. In our study, the growth of bacterialspecies that cause gastric infections, such as S. Typhi, S. Typhimu-rium and E. coli were inhibited by sesame honey with inhibitionzone respectively of 30 mm, 33 mm and 29 mm at the

ks identified are 1-ferulic acid, 2-apigenin. (B). HPLC chromatogram of sesame honey atrcetin.

, antibacterial and probiotic growth stimulatory activities of Sesamum- Food Science and Technology (2014), http://dx.doi.org/10.1016/

Fig. 2. HPLC chromatogram of sesame lignans detected from lipid fraction of sesame honey. Detector UV 290 nm. Peak 1 e sesamin, Peak 2 e episesamin.

Table 4Minimum inhibitory concentration (MIC) of sesame honey.

Microorganisms MIC [in % (w/v)] Tetracycline [mg/ml]

E. coli 15 ± 0.001 0.02 ± 0.001S. Typhi 12.5 ± 0.001 0.005 ± 0.001S. Typhimurium 12.5 ± 0.002 0.025 ± 0.001

Values are expressed in mean ± SEM (n ¼ 7).

A. Das et al. / LWT - Food Science and Technology xxx (2014) 1e7 5

concentration of 50% (w/v). No clear zone of inhibition wasobserved against V. cholera. MIC values suggested that all the ses-ame honey samples exhibited good antibacterial activity againstthe tested enteropathogens. S. Typhi and S. Typhimuriumwere themost susceptible species, yielding the lowest MICs of 12.5% (w/v) ascompared to E. coli with MIC of 15% (w/v).

Antibacterial activity of honey is primarily due to its combinedaction of acidity, osmolarity, hydrogen peroxide and phenoliccompounds (Molan, 1992). Agarose gel electrophoresis was con-ducted as an attempt to explain the antibacterial activity of sesamehoney (Fig. 3). Lane 1 shows DNA ladder in the range of 0.5e12 kb.Lane 2 exhibits pUC 18 control plasmid DNA and lane 3 denotes apositive control lane wherein pUC 18 DNA is digested with Dnase(0.1 U Dnase I for 5 min). Incubation of sesame honey with pUC 18plasmid DNA led to partial degradation of plasmid DNA convertingsupercoiled DNA to open or linear form of DNA (lane 4). No DNAfragmentation was observed when pUC 18 plasmid DNA wasincubated with H2O2 alone (lane 5). DNA degradation caused bysesame honey did not require H2O2. Therefore, sesame honey ap-pears to contain necessary components to induce DNA degradation,which in turn explain its antibacterial activity.

Table 3Antibacterial activity of sesame honey using agar well diffusion assay.

Microorganism Diameter of inhibition zone (mm)a

Sesame honey Ab Sc Cd C

E. coli 29S ± 0.5 15I ± 0.5 17S ± 0.5 21S ± 1 2S. Typhi 30S ± 1 23S ± 0 14I ± 0 26S ± 0.5 2S. Typhimurium 33S ± 1 0R ± 0.5 19 ± 0 25S ± 0 2V. cholera 0R ± 0 10R ± 0 10S ± 0.5 12R ± 0 2

S-Sensitive; R-Resistant; I-Intermediate.a Values are expressed in mean ± SD (n ¼ 7).b Ampicillin.c Streptomycin.d Chloramphenicol.e Ciprofloxacin.f Azithromycin.g Doxycyclin.h Nalidixic acid.i Ofloxacin.j Tetracyclin.

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Molan (1992) observed that non peroxide dependent antibac-terial activity of honey is due to phytochemical componentsderived from the floral source of nectar used and so not all honeyspossess similar activity. HPLC analysis of sesame honey phenolicsidentified the presence of four flavonoids viz., quercetin, apigenin,rutin and myricetin (Fig. 1A and B). Antibacterial activity of quer-cetin and apigenin is partially attributed to the inhibition of E. coliDNA gyrase (Ohemeng, Schwender, Fu, & Barrett, 1993). Mirzoeva,Grishanin, and Calder (1997) had reported that quercetin increasesmembrane permeability and dissipation of membrane potential,transport and motility. Myricetin, however, inhibits E. coli Dna Bhelicase, required for its DNA replication (Griep, Blood, Larson,Koepsell, & Hinrichs, 2007). Bernard et al. (1997) had exhibited

fe Af Dog Nah Ofi Tj

5S ± 0 22S ± 0 18S ± 0 20S ± 0 27S ± 0.5 20S ± 0.58S ± 0 22S ± 0 25S ± 0.5 10R ± 0 26S ± 1 24S ± 0.58S ± 0.5 29S ± 0 14I ± 0.5 19I ± 0.5 24S ± 1 13I ± 12S ± 0.5 20S ± 0.5 0R ± 0 10R ± 0 0R ± 0 25I ± 0.5

, antibacterial and probiotic growth stimulatory activities of Sesamum- Food Science and Technology (2014), http://dx.doi.org/10.1016/

Fig. 3. Agarose gel electrophoresis showing extent of plasmid DNA degradation bysesame honey. Lane 1 DNA Ladder (0.5 kbe12 kb); Lane 2 pUC 18 control plasmid DNA;Lane 3 plasmid DNA treated with Dnase (0.1 U Dnase I for 5 min); Lane 4 plasmid DNAtreated with sesame honey; Lane 5 plasmid DNA treated with H2O2 (2.5 mMol/L).

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Fig. 5. Bifidobacterium bifidum grown in MRS broth with or without sesame honey ascarbohydrate source. Values were expressed as mean ± SD (n ¼ 7). ▪ representsgrowth of B. bifidum without sesame honey and C represents growth of B. bifidumwith sesame honey.

A. Das et al. / LWT - Food Science and Technology xxx (2014) 1e76

that rutin induces topoisomerase IV mediated DNA cleavage lead-ing to SOS response and growth inhibition of E. coli. Sesaminwhichis known to have antibacterial activity (Jianxin, 2006) had also beenidentified in the lipid fraction of sesame honey samples. It could besuggested that the flavonoids and the sesamin present in sesame

0 5 10 15 20 25-0.05

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Fig. 4. Lactobacillus acidophilus grown in MRS broth with or without sesame honey ascarbohydrate source. Values were expressed as mean ± SD (n ¼ 7). : representsgrowth of L. acidophilus without sesame honey and C represents growth ofL. acidophilus with sesame honey.

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honey might be contributory to the antibacterial potential of ses-ame honey exhibited in our study.

3.4. Growth stimulatory effect on probiotic strains

In this study, 5 ml of honey solution was added for preparationof 1 L of modified MRS containing 20 g of honey carbohydrates.Honey carbohydrates substituted the dextrose (20 g/L) present inMRS broth. Following 24 h of incubation, L. acidophilus (Fig. 4) andB. bifidum (Fig. 5) bacteria attained higher populations when grownin carbohydrate free MRS broth supplemented with sesame honeythan unsupplemented MRS broth. In the sample set the bacterialcultures were grown in selective broth in presence of the sesamehoney. The sets containing the sesame honey showed significantlyhigher (p < 0.05) rate of growth compared to the control asmeasured by spectrophotometric analysis of the growth at 600 nm.In the case of bifidobacteria, statistically significant higher cellnumbers were obtained when this organism was grown in thepresence of 5% (w/v) of sesame honey. A 5% (w/v) sesame honeylevel was selected because previous studies determined this opti-mum level in stimulating growth and activity of commercially usedbifidobacteria as well as human intestinal bifidobacteria (Chick,Shin, & Ustunol, 2001; Kajiwara, Gandhi, & Ustunol, 2002).

For determining the quantitative growth promoting activity ofthe sesame honey, plate count method was used. It was observedthat after 24 h incubation, sesame honey supplemented culturemedia induced a statistically significant increase (p < 0.05) ofL. acidophilus and B. bifidum growth, in comparison with the cellcount obtained from control culture (probiotic strains grown onlyin MRS broth). The CFU count/ml of two sets in triplicate is shownin Table 5.

Table 5Growth stimulatory effect of sesame honey on L. acidophilus and B. bifidum.

L. acidophilus B. bifidum

Initial CFU/ml 1 � 105 ± 0.001 1 � 105 ± 0.001Sample CFU/ml 1.2 � 109 ± 0.004 2.3 � 109 ± 0.003Control CFU/ml 3.93 � 108 ± 0.002 4.8 � 108 ± 0.003

Values are expressed in mean ± SEM (n ¼ 7).

, antibacterial and probiotic growth stimulatory activities of Sesamum- Food Science and Technology (2014), http://dx.doi.org/10.1016/

A. Das et al. / LWT - Food Science and Technology xxx (2014) 1e7 7

The GI microflora is in a dynamic equilibrium that may bealtered by diet, medication, stress, aging and various other envi-ronmental factors. The balance of the GI microflora by stimulatingthe growth and/or activity of beneficial microorganisms such asbifidobacteria and lactobacilli and suppressing the potentiallydeleterious bacteria (Kolida, Tuohy, & Gibson, 2002; Sanders, 1993)is a potential area for research by food microbiologist. The non-digestible carbohydrates, a variety of oligosaccharides that occurnaturally in foods such as fruits, vegetables, milk and honey(Tannock, 1999) serves as prebiotic (Crittenden, 1999). Our hy-pothesis is that sesame honey selectively supports growth ofbeneficial intestinal microflora such as lactobacilli andbifidobacteria.

4. Conclusion

This study showed that the seven sesame honey samples fromWest Bengal, India exhibited potential antioxidant/radical scav-enging activity. This could be attributed to its color intensity andpolyphenol content manifested by the positive correlation coeffi-cient observed. Moreover, good quality flavonoids and sesamelignans have been identified in sesame honey which may alsocontribute to its biomedical potential. India is home to diverse andunique floral resources that are exploited by the apiculture basedindustries. The present work contributes conspicuously to the preexisting limited reports on antimicrobial effect of honey. At thesame time, the galvanizing effects of sesame honey in promotingprobiotic growth has also been elucidated at in vitro level, hithertonot reported anywhere. Thus the outcome of the present studymaybenefit both the Indian apiary industry and the health care sector.These findings indicate that there is an opportunity for Indianapiarists to share in the lucrative medicinal honey market.

Acknowledgments

Authors thank Dr. Mahua Ghosh, Department of ChemicalTechnology, University of Calcutta for her technical help. Wethankfully acknowledge the support of Mr. Debjyoti Paul forrendering his support in the final preparation of the manuscript.This research was supported financially by the University GrantsCommission, Government of India.

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