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LWT - Food Science and Technology 59 (2014) 707e712

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LWT - Food Science and Technology

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Antioxidant and antiglycemic potentials of a standardized extract ofSyzygium malaccense

Bavani Arumugam a, Thamilvaani Manaharan b, Chua Kek Heng a, Umah R. Kuppusamy a,Uma D. Palanisamy c, *

a Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysiab Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysiac School of Medicine and Health Sciences, Monash University, Sunway Campus, 46150, Bandar Sunway, Malaysia

a r t i c l e i n f o

Article history:Received 3 March 2014Received in revised form12 June 2014Accepted 17 June 2014Available online 28 June 2014

Keywords:AntiglycemicAntioxidantMyricitrinSyzygium malaccense

* Corresponding author. Tel.: þ60 3 55145840; fax:E-mail addresses: umadevi.palanisamy@monash.e

(U.D. Palanisamy).

http://dx.doi.org/10.1016/j.lwt.2014.06.0410023-6438/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

The present study was designed to identify the ability of the leaf extract from Syzygium malaccense (L.)Merr. & L.M. Perry to scavenge DPPH, ABTS and NO radicals; to inhibit the carbohydrate-hydrolyzingenzymes a-glucosidase and a-amylase; and, eventually, to identify and quantify its bioactive com-pound(s). The S. malaccense leaf extract was a far better scavenger of DPPH and ABTS than of nitric oxide.It also inhibited a-glucosidase more significantly than the positive control, acarbose, but was a poor a-amylase inhibitor. Myricitrin was identified by using high-performance liquid chromatography (HPLC)and liquid chromatography-mass spectrometry (LCMS) techniques as the major bioactive compoundpresent in the extract. The percent yield of myricetin derivatives in the extract was determined to be3.3 ± 0.05%. The presence of the potent antioxidant and antihyperglycemic agent myricitrin in theS. malaccense leaf extract indicates the potential use of the extract in the management of diabetesmellitus and its related complications.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Increased blood glucose levels are believed to accelerate thegeneration of free radicals (Maritim, Sanders, & Watkins, 2003) viavarious mechanisms (Baynes& Thorpe,1999). Imbalance in radical-generating and endogenous free-radical-scavenging defense sys-tems leads to oxidative stress and results in oxidative damage andtissue injury, the hallmark of diabetes and its related complications.The interconnection between free radicals and oxidative stress inthe pathogenesis of diabetes and its co-morbidities is well estab-lished (Baynes & Thorpe, 1999; Maritim et al., 2003). Therefore,natural antioxidants originating from various plants and their de-rivatives became a wise option for the management of oxidativestress-induced diabetes (Bajaj & Khan, 2012; Pereira, Valentao,Pereira, & Andrade, 2009).

þ60 3 55146323.du, upalanisamy@yahoo.com

Syzygium malaccense (L.) Merr. & L.M. Perry falls under thefamily of Myrtaceae. It is locally known as the ‘Malay apple’ andwasoriginally found in Malaysia and India. Various parts of the planthave been applied in traditional medicine. Interestingly, its barkextract has been shown to effectively serve as a hypoglycemic agentthat improved the fasting blood-sugar level and the liver-glycogendepletion and reduced diabetes-induced hyperlipidemia in diabeticrats (Bairy, Sharma, & Shalini, 2005). The plant extract ofS. malaccense is believed to be able to prevent the development ofdiabetes-induced cataractogenesis based on its strong inhibitoryeffect towards aldose reductase (Guzman & Guerrero, 2005). Theabove findings clearly suggest the potential use of S. malaccense inthe management of diabetes mellitus.

The present study is designed to evaluate the antioxidant andantiglycemic characteristics of the leaf extract of S. malaccense. Inaddition, the bioactive compounds potentially responsible for itsantioxidant and anti-hyperglycemic activities will be identifiedthrough a bioassay-guided fractionation technique. The findingsfrom this study are expected to provide the probable underlyingprinciples of the potential use of S. malaccense as an anti-hyperglycemic agent and its ability to manage oxidative stress-induced diabetes mellitus.

B. Arumugam et al. / LWT - Food Science and Technology 59 (2014) 707e712708

2. Materials and methods

2.1. Analytical reagents and chemicals

Quercetin dihydrate was the product of Calbiochem (Darmstadt,Germany). Acetonitrile (HPLC grade), formic acid and dimethylsulfoxide (DMSO) were obtained from Friendemann Schmidt(Germany). Acarbose and myricitrin (purity > 99.0%) were pur-chased from Sigma Chemical Co., Ltd. (St. Louis, MO). All other re-agents used were of analytical grade. All of the experiments wereperformed at 28 �C unless otherwise stated.

2.2. Collection of plant and preparation of S. malaccense leaf extract

The leaves of S. malaccense were collected from a plantation inJohor Bahru, Malaysia and authenticated (sample number:PID220712-15) by the Herbarium at the Forest Research Institute ofMalaysia (FRIM). The leaves were washed with distilled water, left todry in a circulating oven at 40 �C and powderized. Ethanol was addedto the powderized leaves in a 1:20 ratio (w/v), extraction was per-formed at 37 �C for 24 h on an orbital shaker at 200 rpm and the finalsuspension mixture was filtered using Whatman® grade 114 filterpapers. The ethanol filtrate obtained was concentrated using a rotaryevaporator. The dried residue was recovered, weighed and recon-stituted with absolute ethanol to a concentration of 50 mg/ml andstored at 4 �C for not more than twoweeks, prior to the evaluation ofits total phenolic content, antioxidant and antiglycemic activities.

2.3. Antioxidant assays

2.3.1. Determination of total phenolic content (TPC)The TPC was analyzed according to the method reported by Oki

et al. (2002) with slight modifications, using 96-well microtiterplates.TheS.malaccense leafextract (50ml)was incubatedwith50ml of10% (v/v) Folin-Ciocalteu phenol reagent for threeminutes in the darkat room temperature. This was followed by the addition of 100 ml of a10% (w/v) sodium carbonate (Na2CO3) solution and one hour of in-cubation before the absorbance was measured spectrophotometri-callyat 750nmonaBenchmark™Plusmicroplate spectrophotometer(Bio-Rad, USA). The mean TPC value was expressed as the amount ofthe phenolic content (mg gallic acid equivalents, GAE) in one gram ofextract.

2.3.2. DPPH radical-scavenging assayADPPH assaywas performed according to themethod described

by Gerhauser et al. (2003) with some modifications, using 96-wellmicrotiter plates. The extract (5 ml) and quercetin (positive control)of various concentrations were mixed with 195 ml of a 100 mMethanolic solution of the DPPH reagent. The absorbance was readafter incubation in the dark for 20 min at 515 nm.

2.3.3. ABTS radical-scavenging assayAnABTS assaywasperformedaccording to themethoddescribed

by Kanagasabapathy,Malek, Kuppusamy, and Vikineswary (2011) in96-well microtiter plates with slight modifications. The samples(10 ml) were mixed with 90 ml of ABTS. þ reagent. The absorbancewas measured after four minutes at 734 nm.

2.3.4. Nitric oxide (NO) radical-scavenging assayAnNOassaywasperformedaccording to themethoddescribedby

Ebrahimzadeh, Nabavi, Nabavi, Bahramian, and Bekhradnia (2010)with some modifications, using 96-well microtiter plates. Ten mi-croliters of the samples were mixed with 90 ml of the SNP reagent(final concentration of SNP: 10mM), prepared freshly in a phosphatebuffered solution (pH 7.4). Themixturewas incubated for 2.5 h in the

presence of light. Then, 100 ml of pre-mixed Griess-Ilosvay's nitritereagent was added to the wells, which were incubated for 30 min inthe dark, and the absorbance was measured at 540 nm.

2.3.5. Determination of IC50 values for free-radical-scavengingassays

The free radical (DPPH, ABTS and NO)-scavenging activities ofthe extract and quercetin were calculated according to thefollowing formula:

Percentage of radical quenchedð%Þ ¼ Acontrol � Asample

Acontrol� 100;

where A ¼ absorbance.The control was prepared by replacing the sample volume with

the respective diluents (ethanol for the extract; DMSO for quer-cetin). The absorbances of the samples at various concentrationswithout a reagent (substituted with water) were excluded toeliminate the sample color and background interferences.

The scavenging activity of the extract was expressed in IC50 value,the concentration of the sample required to scavenge 50% of theradicals.

2.4. a-amylase and a-glucosidase enzyme-inhibition assays

The diluted extracts and acarbose (positive control) at variousconcentrations in the reaction buffer were subjected to a-amylaseand a-glucosidase enzyme-inhibition assays as described byManaharan, Appleton, Ming, and Palanisamy (2012).

The a-amylase enzyme inhibition assay: Eighty microliters ofsamples were incubated with 40 ml of the amylase enzyme for10 min at room temperature. This was followed by the addition of40 ml of starch solution and incubation at 37 �C for 10 min. Finally,80 ml of 3,5,-dinitrosalicylic acid solution was added and themixture was incubated for 10 min at 95 �C to detect the presence ofreducing sugar. Absorbance was read at 540 nm.

The a-glucosidase inhibition assay: Twenty microliters of freshDL-dithiothreitol solution (1mM)was added to 20ml of samples. Thiswas followed by the addition of 20 ml of 6 mM 4-nitrophenyl-a-D-glucopyranoside solution and 20 ml of a-glucosidase enzyme (0.4 U/ml in0.1Msodiumphosphate buffer, pH6.8 supplementedwith0.2%BSA) to themixture. The reactionmixture was incubated at 37 �C for15 min. Finally, the reactionwas stopped with 80 ml of 0.2 M sodiumcarbonate solution and absorbance was read at 400 nm.

The percentage of inhibition of the samples was calculated ac-cording to the following formula:

Percentage of inhibitionð%Þ ¼ Acontrol � Asample

Acontrol� 100;

where A ¼ absorbance.The control was prepared without the sample by replacing the

sample volume with buffer. The absorbance of the samples andtheir contributions in the reaction at various concentrationswithout the enzyme (substituted with buffer) were excluded toeliminate the sample color and background interferences.

The inhibitory activity of the extract was computed from theplot of the percentage of inhibition against the concentration of thesamples. The IC50 values were expressed as the concentration of thesamples required to inhibit 50% of the enzymatic activity.

2.5. Bioassay-guided fractionation of the S. malaccense leaf extractby HPLC

The leaf extract of S. malaccense (10 mg/ml in ethanol) filteredusing syringe filter (0.20 mm) was injected into an Agilent 1200

Fig. 1. Chromatogram of (A) the standardized ethanolic extract of the Syzygium mal-accense leaf and (B) myricitrin (1 mg/ml) on an analytical HPLC with a detectionwavelength of 350 nm. The fractions were eluted using a water:acetonitrile gradientsystem. The solvent gradient consisted of 0% acetonitrile for 2 min, 0e100% acetonitrilefor 30 min and 100% acetonitrile for 3 min with a flow rate of 2 ml/min (injectionvolume: 10 ml). The inset indicates the retention times of the major peak of the extractand of myricitrin.

B. Arumugam et al. / LWT - Food Science and Technology 59 (2014) 707e712 709

series analytical high performance-liquid chromatography (HPLC)system (Germany). The extract was fractionated using a Chromo-lith® Performance RP-18 endcapped column (4.6 � 100 mm; 5 mm)(Merck, Darmstadt, Germany) with an injection volume of 10 ml at40 �C. The mobile phase consisted of solvent A, water with 0.1%formic acid, and solvent B, acetonitrile with 0.1% formic acid. Thefractions were eluted through a gradient profile at a flow rate of2 ml/min as follows: 0% of solvent B for 2 min; 0e100% of solvent Bfor 30 min; 100% of solvent B for 3 min and finally 100%e10% ofsolvent B for 2 min to recondition the column. Fifty-four fractionswere detected by a UV detector (210 nm) and were collected in 96-well microtiter plates. The solvents were evaporated using aScanSpeed 40 centrifugal evaporator (ScanVac®, Labogene,Denmark), and the resulting residues were tested for their anti-oxidant capacities (DPPH, ABTS and NO radical-scavengingactivities).

The most active fractions were further analyzed on an Agilent1290 Infinity liquid chromatography (LC) system (Gemany) coupledto an Agilent 6520 Accurate-Mass Q-TOF mass spectrometer with adual ESI source (USA). The active compounds were separated usingan Agilent Zorbax SB-C18 Narrow-Bore (2.1 � 150 mm, 3.5 mm)column with an injection volume of 4 ml. The mobile phase con-sisted of 0.1% formic acid in water (A) and 90% acetonitrile in waterwith 0.1% formic acid (B). A gradient elution was performed at aflow rate of 0.6 ml/min as follows: initially, 10% B; 0e13 min,10e100% B; 5 min, 100% B and the final 4 min, 100e10% B torecondition the column. Nitrogen was used as the sheath gas. Thecapillary temperature and the voltage were set to 300 �C and 4 kV,respectively. The mass ranges of 100e2000 m/z and 115e2000 m/zwere scanned in the positive and the negative full-ion monitoringmodes, respectively. The data were analyzed using the AgilentMassHunter Qualitative Analysis B.05.00 software and the METLINdatabase.

2.6. Quantitative analysis of myricetin derivatives

The analysis was performed on the Agilent 1200 series analyt-ical HPLC system using a Chromolith® Performance RP-18 end-capped column (4.6 � 100 mm; 5 mm) with an injection volume of10 ml at 40 �C. The same gradient elution method used for theS. malaccense leaf extract was also used for commercial myricitrin(dissolved in ethanol) at a flow rate of 2 ml/min. Standard curveswere constructed using various concentrations of myricitrin(0.1e1.0 mg/ml) against the peak area under the curve at 350 nm.The myricetin derivatives in the leaf extract were quantified usingthe equation derived from the standard curve.

2.7. Statistical analysis

All experiments were performed in triplicate. The values wereexpressed as the mean ± standard deviation (unless otherwisestated) and were calculated from triplicate measurements within asingle run using Microsoft Excel 2007 or GraphPad Prism version5.00.

3. Results and discussion

3.1. Extraction and preparation of the S. malaccense leaf extract

The ethanolic extraction of the powderized S. malaccenseconsistently resulted in a percent yield of 8.87 ± 0.12. The percentyield was found to be comparable to the results obtained earlierby Noreen, Sarrano, Perera, and Bohlin (1998), who reported a13.6% yield using 70% (v/v) ethanol as the extraction solvent.A standardized HPLC profile of the extract was established based

on the conditions described previously (Section 2.5) to ensurethe uniformity of the S. malaccense leaf extract (Fig. 1; Chro-matogram A).

3.2. Antioxidant property and TPC of S. malaccense leaf extract

The ethanolic extract was tested for its antioxidant propertyusing DPPH, ABTS and NO free-radical-scavenging assays. DPPHscavenging is a commonly assessed parameter in identification ofplants with potential free radical scavenging activity(Ebrahimzadeh, Pourmorad, & Hafezi, 2008) whereas ABTS scav-enging assay is known for its sensitivity (Ling et al., 2009). Recog-nition of plants with good nitric oxide scavenging property wouldbe of interest, as nitric oxides are commonly related to manypathological and inflammation conditions (Moncada, Palmer, &Higgs, 1991). Table 1 compares the IC50 (mg/ml) values ofS. malaccense in these three assays using quercetin as the positivecontrol (Ebrahimzadeh et al., 2010; Zaporozhets, Krushynska,Lipkovska, & Barvinchenko, 2004). Generally, the IC50 values ofthe extract were higher than the IC50 values of quercetin in all of theassays, which is expected because the latter is a pure compound.Among the three different antioxidant assays, the extract was ableto scavenge DPPH and ABTS radicals far better than the nitric oxideradicals. The total phenolic content in the ethanolic S. malaccenseleaf extract was determined to be 125.81 ± 0.003 mg gallic acidequivalent g�1 extract. (One gram of the extract corresponds to11.27 g of dry weight of powderized leaves of S. malaccense ascalculated from the percent yield of 8.87 ± 0.12).

3.3. Antiglycemic potential of S. malaccense leaf extract

The enzymes, a-amylase and a-glucosidase are responsible forcarbohydrate digestion and have been identified to be the thera-peutic targets for the management of postprandial hyperglycemia(Shobana, Sreerama, & Malleshi, 2009). Acarbose is a drug thatserves as an inhibitor of the enzymes a-glucosidase and a-amylaseand is commonly used in the management of early-stage diabetes

Table 1Antioxidant activity of leaf extract of Syzygium malaccense.

Scavenging assays IC50 values (mg/ml)

Quercetin Syzygium malaccense

DPPH 5.73 ± 1.05 16.65 ± 1.17ABTS 4.95 ± 1.08 47.27 ± 1.048NO 36.20 ± 1.12 333.00 ± 1.187

Values ¼ mg/ml ± standard error; final concentration.

Table 2Antiglycemic property of ethanolic leaf extract of Syzygium malaccense.

Enzyme inhibition assays IC50 values (mg/ml)

Acarbose Syzygium malaccense

a-glucosidase 388.50 ± 1.03 207.60 ± 1.07a-amylase 2.25 ± 1.03 >1000

Values ¼ mg/ml ± standard error.

Table 3Antioxidant activities of fractions following bioassay-guided fractionation.

Scavenging assays % Inhibition

F1 F2

DPPH 30.31 47.24ABTS 74.62 97.82NO 44.00 51.52

Concentration used: 0.5 mg/ml.

B. Arumugam et al. / LWT - Food Science and Technology 59 (2014) 707e712710

mellitus (Cheplick, Kwon, Bhowmik, & Shetty, 2010). The IC50

values determined for acarbose in this study for both a-glucosidaseand a-amylase (Table 2) were comparable to those reported byManaharan et al. (2011). The ethanolic extract of the S. malaccenseleaves displayed a far better a-glucosidase inhibitory activity thanthat of acarbose (Table 2), reflecting its potential antiglycemicproperties. A similar effect was not observed in its ability to inhibita-amylase. Similar studies investigating the anti-diabetic proper-ties of an Syzygium aqueum extract, conducted by Manaharan et al.(2012), showed that the extract was a far better a-glucosidase in-hibitor. Interestingly, Jung et al. (2006) have reported that the activecompound from the methanolic bark extract of S. malaccense, cas-uarine 6-O-a-glucoside, was found to effectively inhibit a-glucosidase.

The relationships among the TPC and the antioxidant andantiglycemic properties have been well described in previousstudies (Cheplick et al., 2010; Manaharan et al., 2011, 2012). In thepresent study, the ethanolic leaf extract of S. malaccense displayed amoderate total phenolic content with reasonable free-radical-scavenging properties and, importantly, served as a good inhibi-tor of the enzyme a-glucosidase.

Fig. 2. Bioassay-guided fractionation of the ethanolic extract of the Syzygium malaccense leconsisted of 0% acetonitrile for 2 min, 0e100% acetonitrile for 30 min and 100% acetonitrile fcollected with the major peaks F1 and F2.

3.4. Bioassay-guided fractionation and the identification andquantification of bioactive components

The ethanolic leaf extract of S. malaccense was subjected to ananalytical HPLC system. The fractions collected at retention times of0.90 (F1) and 8.08min (F2) (Fig. 2) were found to be themost activefractions, with F2 being more active than F1 when tested for radical(DPPH, ABTS and NO)-scavenging ability (Table 3).

Interestingly, the active compounds present in the major F2peak (Fig. 2) were predominantly derivatives of myricetin. Thepresence of myricetin 3-O-L-rhamnoside (myricitrin), which pro-duced a significant fragment peak at m/z 463.0891 [M�H]- (Fig. 3aand d), was found to be notably more prominent than myricetin 3-alpha-L-arabinofuranoside, which produced a significant fragmentpeak at m/z 449.0735 [M�H]- (Fig. 3a and c), and myricetin 30-glucoside, which produced a significant fragment peak at m/z479.0836 [M�H]- (Fig. 3a and b) when analyzed in the negativefull-ion-monitoring mode (Fig. 3). In addition, the retention time ofcommercially available myricitrin coincided with that of the majorpeak of F2 (Fig. 1; detection wavelength: 350 nm) when a similargradient method was performed on an analytical HPLC system. Theamount of myricetin derivatives in the S. malaccense leaf extractwas quantified to be 33 mg/g extract (One gram of the extractcorresponds to 11.27 g of dry weight of powderized leaves ofS. malaccense) using calibration curves established on the analyticalHPLC system at 350 nm (data not shown). On the other hand, F1was identified to contain mostly gallic acid, which may havecontributed to its antioxidant activities (Lu, Nie, Belton, Tang, &Zhao, 2006).

Myricitrin has been considered as a chemosystemic indicator ofthe genus Syzygium (Tian et al., 2011) and identified as a vital activecomponent in Syzygium cumini (L.) Skeels, Syzygium forrestii andSyzygium samarangense. These plants have been shown to possess

af (10 mg/ml) on HPLC with a detection wavelength of 210 nm. The solvent gradientor 3 min with a flow rate of 2 ml/min (injection volume: 10 ml). Fifty-four fractions were

Fig. 3. The separation and identification of active compounds in F2 of Syzygium malaccense on a liquid chromatography system coupled to a Q-TOF mass spectrometer with a dualESI source. The mobile phase consisted of 0.1% formic acid in water, A, and 90% acetonitrile in water with 0.1% formic acid, B. The solvent gradient consisted of 10% B initially,10e100% B for 13 min and 100% B for 5 min with a flow rate of 0.6 ml/min (injection volume: 4 ml). The capillary temperature and the voltage were 300 �C and 4 kV, respectively. Themass ranges of 115e2000 m/z were scanned in the negative full-ion-monitoring mode. The data were analyzed using the Agilent MassHunter Qualitative Analysis B.05.00 softwareand the METLIN database. (a) Chromatogram (count vs retention time) of F2. The inset shows the abundance of myricetin derivatives in F2. (bed) Mass spectra (count vs m/z) andchemical structures of: (b) myricetin 30-glucoside, (c) myricetin 3-alpha-L-arabinofuranoside and (d) myricitrin.

B. Arumugam et al. / LWT - Food Science and Technology 59 (2014) 707e712 711

significant antioxidant and anti-diabetic properties (Ayyanar &Subash-Babu, 2012; Simirgiotis et al., 2008; Tian et al., 2011). Theroles of the flavanoid rhamnoside, myricitrin, as a potent antioxi-dant and antiglycemic agent have been well documented (Chen,Zhuang, Li, Shen, & Zheng, 2013; Manaharan et al., 2012;Manaharan, Ming, & Palanisamy, 2013; Sun et al., 2013). The abil-ity of the leaf extract of S. malaccense to scavenge free radicals andinhibit the enzyme alpha-glucosidase in this study can be attrib-uted to the presence of myricitrin as the major active compound.

4. Conclusions

In conclusion, the leaf extract of S. malaccense displayed goodfree-radical-scavenging properties, as determined by DPPH and

ABTS radical-scavenging assays but it was a weak scavenger of NO.The extract showed significant antiglycemic properties, as evi-denced by its ability to inhibit the enzyme a-glucosidase. Thefunctional molecules present in the extract were identified to bemyricetin derivatives as the major active components besides gallicacid. The present study is the first report to describe a standardizedpreparation and the antiglycemic potential of the ethanolic leafextract of S. malaccense and to establish the presence of a3.3 ± 0.05% yield of myricetin derivatives in the crude extract.

Acknowledgements

The technical assistance of Ms. Adillah Akhasan andMs. Tee TingYee is greatly appreciated. B. Arumugam is grateful to the University

B. Arumugam et al. / LWT - Food Science and Technology 59 (2014) 707e712712

of Malaya (UM) and the Ministry of Education (MoE) for financialassistance. This study was supported by the High Impact ResearchMoE Grant UM.C/HIR/MoE/MED/11 (E000042-20001) from theMinsitry of Education Malaysia and a Postgraduate Research Grant(PG018/2012B) awarded to B. Arumugam by UM, Malaysia.

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