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Industrial Crops and Products 62 (2014) 47–52

Contents lists available at ScienceDirect

Industrial Crops and Products

jo ur nal home p age: www.elsev ier .com/ locate / indcrop

hytochemical, antioxidant, antibacterial, and �-amylase inhibitoryroperties of different extracts from betel leaves

eila Nouria, Abdorreza Mohammadi Nafchia,∗, A.A. Karimb

Food Biopolymer Research Group, Food Science and Technology Department, Damghan Branch, Islamic Azad University, Damghan, Semnan, IranFood Biopolymer Research Group, Food Technology Division, School of Industrial Technology, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia

r t i c l e i n f o

rticle history:eceived 3 May 2014eceived in revised form 8 August 2014ccepted 11 August 2014

eywords:etel leaf

a b s t r a c t

Antimicrobial and antioxidant activities, phenol content, and �-amylase inhibitory effects of a local vari-ety of betel leaves were evaluated. The effects of various solvents (methanol, ethanol, acetone, and ethylacetate) on phenols and antioxidant activities were also studied. Methanol and ethanol (90%) extractsshowed maximum phenolic contents (205.2 and 202.9 mg GAE/g, respectively). Maximum flavonoidcontents were determined using 90% acetone (82.5 mg CE/g), and the highest inhibition percentage of2,2-diphenyl-1-picrylhydrazyl radical was exhibited by 90% ethanol (percent inhibition, 94%). �-Amylase

olvent extractionhenolic compoundntioxidantntimicrobial activity

activity assay showed that �-amylase inhibitory activities were positively correlated with the total phe-nolic content of ethanol and methanol. Considering antimicrobial activities, we found that all of theGram-positive bacteria and Gram-negative bacteria were inhibited by betel leaf extract except Pseu-domonas aeruginosa. Our results could provide a basis of future studies on betel leaves used in food andpharmaceutical applications.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Studies have been conducted to identify antioxidants fromatural sources, which are suitable alternatives to synthetic antiox-

dants (Gharibi et al., 2013; Suppakul et al., 2006). Naturalntioxidants can be used to develop new nutraceutical productshat can prevent oxidative damage in the body (Bazylko et al., 2013;

ong et al., 2006). Antioxidants exhibit functional potential againstajor components involved in age-related diseases, such as type 2

iabetes linked hyperglycemia, cardiovascular diseases, and cancerEspín et al., 2007; Fraga, 2010; Ranilla et al., 2010; Rowland, 1999).

Type 2 diabetes is a metabolic disorder characterized by chronicyperglycemia with disorders in carbohydrate, protein, and fatetabolism because of a disturbance in insulin function (Ali et al.,

006).Polyphenolic compounds or phytochemicals present in plants

re important components of the human diet; phytochemicals

an be used to regulate oxidation and stress-related chronic dis-ases, such as diabetes and cardiovascular diseases (Kwon et al.,008). �-Amylase is a key enzyme involved in starch hydroly-is. If �-amylase involved in the breakdown and absorption of

∗ Corresponding author. Tel.: +98 232 522 5045; fax: +98 232 522 5039.E-mail addresses: fstiau@damghaniau.ac.ir, fstiau@gmail.com

A. Mohammadi Nafchi).

ttp://dx.doi.org/10.1016/j.indcrop.2014.08.015926-6690/© 2014 Elsevier B.V. All rights reserved.

carbohydrates is inhibited, post-prandial blood glucose level canbe reduced; this mechanism can be used as a strategy to con-trol hyperglycemia related to type 2 diabetes (Kwon et al., 2008).However, currently used synthetic �-amylase inhibitors, such asacarbose, can cause several complications, including flatulence,meteorism, abdominal distention, and possibly diarrhea (Kwonet al., 2008).

Microbial contamination is one of the main parameters that canreduce food quality and shelf-life and compromise product safety.The growth of microorganisms may cause food-borne diseases orspoilage in food products; oxidation in food products also causeproteins and lipids to deteriorate, thereby altering the color, fla-vor, and texture of products (Suppakul et al., 2006). Hence, theprevention of microbial contamination is directly relevant in foodprocessing.

Chemical composition, antioxidant, and antimicrobial activityof lots of medicinal plants recently have been investigated (Bhatet al., 2012; Li et al., 2013; Skotti et al., 2014). Nikolic et al. (2014)were reported chemical composition and in vitro biological activityof Thymus essential oils (EO). They showed that succeeds in creatingcomparable and quantitative data for usage of Thymus oils againstoral microorganisms.

Finding new antimicrobial agents with various chemical struc-tures and neo-mechanisms of action is essential. The increasingmorbidity of new infectious diseases and resistance to currentantibiotics in clinical use (Nair and Chanda, 2008).

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Betel leaves is widely grown in Asian regions, such as India,ri Lanka, Malaysia, and Philippines. Betel leaves exhibit woundealing, gastro-protective, and hepato-protective activities; these

eaves also elicit anti-fertility and anti-motility effects in washeduman spermatozoa (Arambewela et al., 2005). Furthermore, betel

eaves show antioxidant activities (Santhakumari et al., 2003; Wangt al., 1999). Although a few studies on the antioxidant propertiesf betel leaves have been conducted, studies on the antidiabeticSanthakumari et al., 2003) and antimicrobial (Nair and Chanda,008) activities of betel leaves and extract are limited. To the bestf our knowledge different extraction and antimicrobial, antioxi-ant, and anti-diabetic properties of betel leave mutually has noteen investigated. So our study aimed to evaluate the antioxidant,ntimicrobial, and �-amylase inhibitory properties of the phenolicsn betel leaves extracted with aqueous and organic solvents.

. Materials and methods

.1. Plant materials

Fresh betel leaves with no apparent physical damage were pur-hased from a local wet market in Penang, Malaysia. Betel leavesere obtained from a local variety of Piper betel L. (Fig. 1), a creeperlant that grows to a large bush of vines and leaves. The leaves aresed for chewing and medicinal purposes, as well as in ceremonies

n traditional Malay weddings as part of a bridegroom’s offering.he taxonomic identity of P. betel was determined by taxonomistsn the Department of Biology (Herbarium Center; Voucher Number:1240), Universiti Sains Malaysis, Pulau Pinang, Malaysia.

.2. Chemicals, microorganisms and reagents

Folin-Ciocalteu’s (FC) phenol reagent, sodium carbonate,odium acetate, sodium nitrite, sodium hydroxide, aluminum chlo-ide, 2,2-diphenyl-1-picrylhydrazyl (DPPH◦), gallic acid, catechin,utin, 3,5-dinitrosalicylic acid, potassium sodium tartrate tetrahy-rate, soluble starch, and human salivary �-amylase enzymeA1031 type XIII-A) were supplied by Sigma-Aldrich® (St. Louis,

O, USA).Ethanol, methanol, acetone, and ethyl acetate were purchased

rom R&M chemicals (Essex, UK). All of the reagents and chemicalssed in the study were of analytical grade.

Media and chemicals used in antibacterial experiments wereutrient agar and nutrient broth from Merck, Germany, Mveller-inton agar, plate count agar, ampicillin, and ringer tabletsurchased from Oxoid, England.

Following ten clinical oral isolates were tested: Staphylococ-us aureus, Staphylococcus epidermis, Bacillus cereus, Bacillus subtilis,isteria monocytogenes, Escherichia coli, Salmonella typhimurium,almonella enterididis, Klebsiella pneumonia, and Pseudomonaseruginosa. The reference strains were obtained from the microbi-logy laboratory (School of Industrial Technology, USM, Malaysia).

.3. Extraction of polyphenols

Leaves were washed with clean water, cut into small piecesapproximately 1 cm × 1 cm), and placed in a freeze dryer (model754511, Labconco Corporation, Kansas City, Missouri, USA) for8 h. The freeze–dried samples were ground to a powder by using atainless-steel grinder and stored in a freezer until they were used.

For each experiment, 1 g of freeze–dried sample was weighed

nd placed in a beaker containing 100 ml of solvent for 24 h.he beaker was then placed on a hotplate (Model FSMQ 143551,isher Scientific, Kuala Lumpur, Malaysia) and constantly stirredt 1200 rpm by using a magnetic stirrer at room temperature

d Products 62 (2014) 47–52

(25 ± 1 ◦C). The extract was filtered under suction with What-man No. 1 filter paper. The remaining residue on the filter paperwas placed back in the same flask and stirred for another 3 hand re-extracted twice according to the same procedure until theresidue became colorless. Light exposure was avoided throughoutthe extraction process. Extraction was conducted in triplicate. Fourdifferent solvents (methanol, ethanol, acetone, and ethyl acetate)with five different concentrations in deionized water (30%, 50%,70%, 90%, and 100%, v/v) and 100% deionized water were used.

2.4. Determination of total phenolic content

The total phenolic (TP) content of betel leaves were evaluatedby the FC assay, as described in a previous method ((Singletonet al., 1999) with slight modifications. In brief, 40 �l of the extractwas mixed with 1.8 ml of the FC reagent. The FC reagent was pre-diluted 10 times using deionized water. After 5 min, 1.2 ml of (7.5%w/v) sodium carbonate solution was added. The solutions werethen mixed and kept for 1 h at room temperature. Absorbancewas determined using UV–visible spectrophotometer (ShimadzuUV-160APC, Japan) at 765 nm. The calibration curve was preparedusing a standard Gallic acid solution (20, 40, 60, 80, and 100 mg/l,r2 = 0.997). Results were expressed on a dry weight basis as mggallic acid equivalents per 1 g of sample.

2.5. Determination of total flavonoids

The total flavonoid (TF) content of the betel leaves were deter-mined according to colorimetric assay (Zhishen et al., 1999).Approximately, 1 ml of betel leaves extract was mixed with 4 mlof deionized water and 0.3 ml of 5% (w/v) NaNO2. The mixturewas stored for 5 min and 0.3 ml of (10% w/v) AlCl3 was added.After 6 min, NaOH (2 ml of 1 M solution) was added. Approxi-mately, 2.4 ml of deionized water was immediately added to obtaina final volume of 10 ml. The mixture was shaken vigorously andthe absorbance of the mixture was determined at 510 nm by usinga UV–visible spectrophotometer (Shimadzu UV-160APC, Japan).Catechin was used to prepare a standard solution with differentconcentrations (20, 40, 60, 80, and 100 mg/l, r2 = 0.996). Resultswere indicated as mg catechin equivalents (CE) per 1 g on a dryweight basis of a specimen.

2.6. Determination of total flavonols

Flavonol is one of the major classes of flavonoids. Flavonolsexhibit antioxidant properties that can be determined using apaper-based method described by (Abdel-Hameed, 2009). Approx-imately, 1 ml of betel leaf extract (10 mg/ml) was mixed with 1 mlof aluminum trichloride solution (20 mg/ml) and 3 ml of sodiumacetate solution (50 mg/ml). The mixture was kept for 2.5 h atroom temperature and observed at 440 nm by using a UV–visiblespectrophotometer (Shimadzu UV-160APC, Japan). Different con-centrations of rutin was used in a standard solution (20, 40, 60, 80,and 100 mg/l, r2 = 0.996) to prepare a calibration curve. All of thedeterminations were performed in triplicate.

2.7. DPPH◦ free radical scavenging assay

The antioxidant activity of betel leaves was analyzed by evalu-ating the effects of free radical scavenging against the radicals ofDPPH◦. The experiment was performed using a method describedpreviously (de Ancos et al., 2002). In brief, 10 �l of betel leaf extract

was thoroughly vortex-mixed with 90 �l of deionized water and3.9 ml of 25 mM DPPH◦ methanolic solution. The mixture was keptin a dark place for 30 min. After 30 min, the absorbance was deter-mined against methanol without DPPH◦ as blank at 515 nm by using

L. Nouri et al. / Industrial Crops and Products 62 (2014) 47–52 49

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UV-visible spectrophotometer (Shimadzu UV-160APC, Japan).PPH◦ radical scavenging was expressed as percentage, which wasalculated using the following equation:

inhibition of DPPH◦ = (Abscontrol − Abssample)(Abscontrol)

× 100

here the absorbance of the solution without extract (DPPH◦ only)s expressed as Abscontrol and the solution combined with thextract is expressed by Abssample.

.8. ˛-Amylase inhibitory assay

The �-amylase inhibitory assay of betel leaf extracts was eval-ated according to a previously described method (Ranilla et al.,010). In brief, 0.5 ml of extract was mixed with 0.5 ml of �-amylaseolution (0.5 mg/ml) with 0.02 M sodium phosphate buffer (pH 6.9ith 0.006 M NaCl). The mixture was incubated at room tempera-

ure for 10 min and 0.5 ml of starch solution (1%) in 0.02 M sodiumhosphate buffer (pH 6.9 with 0.006 M NaCl) was added. The result-

ng mixture was incubated at room temperature for 10 min, and theeaction was terminated using 1 ml of dinitrosalicylic acid coloreagent. At this time, the test tubes were placed in a water bath100 ◦C and 5 min) and cooled until room temperature was reached.he mixture was then diluted with 10 ml of deionized water, andbsorbance was determined at 540 nm. The absorbances of blankbuffer instead of extract and amylase solution) and control (buffernstead of extract) samples were also determined. The inhibition of-amylase was calculated using the following equation:

inhibition of �-Amylase = (Abscontrol − Abssample)(Abscontrol)

× 100

here Abscontrol corresponds to the absorbances of the solutionithout extract (buffer instead of extract) and with �-amylase solu-

ion and Abssample corresponds to the solution with extract and-amylase solution.

.9. Antimicrobial assay

The antimicrobial activity of betel leaf extracts was assessedsing the disc diffusion method with slight modifications (Ariast al., 2004). Petri dishes (9 cm in diameter) with 25 ml of Mveller-inton agar were prepared. Disks (diameter = 6 mm) were soakedith 20 �l of the betel leaf extracts were placed on the agar. A

isk containing ampicillin was used as a control treatment. Theetri dishes were placed in an incubator (35 ◦C) for 16–20 h. Allf the experiments were performed in triplicate, and results werexpressed as diameters (mm) of inhibition.

tat in Pulau Pinang, Malaysia.

2.10. Statistical analysis

Data were analyzed using SPSS software (SPSS Inc. IL, Chicago,USA). ANOVA and Tukey tests were performed to compare dif-ferences between samples at 5% significance level. Values werepresented as means ± standard errors. All of the experiments wereperformed in triplicate.

3. Results and discussion

3.1. Effect of different solvents on betel leaf extraction

Total phenolic (TP) contents of betel leaves measured usingFolin-Ciocalteu’s colorimetric method are presented in Table 1.The TP contents of betel leaf extracts ranged from 59.38 to202.92 mg GAE/g and 58.54 to 205.20 mg GAE/g using ethanol andmethanol solvents, respectively. The TP values of acetone- and ethylacetate-extracted samples ranged from 47.68 to 191.50 mg GAE/gand from 46.45 to 128.43 mg GAE/g, respectively. In water-extracted samples, the TP content was 71.30 mg GAE/g.

The total flavonoid TF contents of betel leaves extracted fromdifferent solvents and different concentrations are presented inTable 1. The TF contents of the ethanol extract of betel leavesranged from 12.13 to 78.8 mg CE/g. The TF contents of methanol-,acetone-, and ethyl acetate-extracted samples ranged from 12.25to 80.59 mg CE/g, from 12.10 to 82.55 mg CE/g, and from 10.97to 42.05 mg CE/g, respectively. In water-extracted samples, the TFcontent was 30.88 mg CE/g.

The flavonol content ranged from 0.21 to 1.4 mg RE/g, from0.32 to 2.5 mg RE/g, from 0.69 to 2.43 mg RE/g, and from 0.11 to0.43 mg RE/g in ethanol-, methanol-, acetone-, and ethyl acetate-extracted samples, respectively. In water-extracted samples, theflavonol content was 0.12 mg RE/g.

The DPPH◦ radical scavenging activities of the extracts rangedfrom 75.01% to 93.99%, from 73.11% to 89.01%, from 69.44%to 86.58%, and from 41.14% to 55.1% in ethanol-, methanol-,acetone-, and ethyl acetate-extracted samples, respectively. Inwater-extracted samples, the % inhibition of DPPH◦ was 52.63%.

In Table 1, 90% methanol and 90% ethanol extracts containedhigher polyphenolic contents and antioxidant activities than othersolvents in this study.

Flavonoids are the most common group of phenolic compoundswidely distributed in all parts of plants, particularly in photosyn-

thesizing cells. Flavonoids contain different structures, degrees ofhydroxylation and polymerization, substitutions and conjugations.Flavonoids also exhibit different chemical properties because oftheir varying characteristics. All of the flavonoids contain a simple

50 L. Nouri et al. / Industrial Crops and Products 62 (2014) 47–52

Table 1Total phenolic, flavonoids, flavonols, and DPPH◦ of betel leaves.

Solvent % Total phenolic(mg GAE/g dry weight)

Total flavonoids(mg CE/g dry weight)

Total flavonols(mg RE/g dry weight)

DPPH◦ (%)

Water 100 71.30 ± 0.86j 30.88 ± 0.56h 0.12 ± 0.00lm 52.63 ± 0.52k

Ethanol30 59.38 ± 0.90k 12.13 ± 0.12jk 0.21 ± 0.00kl 75.01 ± 0.61hi50 82.29 ± 0.48i 14.30 ± 0.15ij 0.44 ± 0.01h 79.20 ± 0.38fg70 147.96 ± 0.81e 47.96 ± 0.36f 0.81 ± 0.00f 85.97 ± 0.49 cd90 202.92 ± 0.71a 78.78 ± 0.51bc 1.40 ± 0.01d 93.99 ± 0.40a

100 195.92 ± 0.92b 75.22 ± 0.56d 0.83 ± 0.02f 89.95 ± 0.67b

Methanol30 58.54 ± 0.32k 12.25 ± 0.21jk 0.32 ± 0.01ij 73.17 ± 0.52i50 97.32 ± 0.66h 15.11 ± 0.48i 0.67 ± 0.01g 78.43 ± 0.45g70 140.15 ± 0.63f 47.12 ± 0.33f 1.76 ± 0.02c 84.38 ± 0.36de90 205.20 ± 0.44a 80.59 ± 0.91ab 2.50 ± 0.05a 89.10 ± 0.46bc

100 192.72 ± 0.78bc 76.52 ± 0.69 cd 1.37 ± 0.01d 87.18 ± 1.21 cd

Acetone30 47.68 ± 0.16l 12.10 ± 0.20jk 0.70 ± 0.01 g 69.44 ± 0.80j50 93.39 ± 0.63h 12.20 ± 0.22jk 0.69 ± 0.01 g 73.20 ± 0.35i70 159.81 ± 1.38d 56.38 ± 0.89e 2.43 ± 0.04ab 77.46 ± 0.81gh90 191.50 ± 01.21c 82.55 ± 0.60a 2.35 ± 0.02c 86.58 ± 0.74cd

100 131.11 ± 0.80g 76.67 ± 0.69cd 1.25 ± 0.02e 81.77 ± 0.48ef

Ethyl acetate30 128.43 ± 1.27g 42.05 ± 0.40g 0.11 ± 0.00m 41.14 ± 0.32m50 67.32 ± 0.71j 32.32 ± 0.32h 0.43 ± 0.01h 44.27 ± 0.38lm70 46.45 ± 0.82l 10.97 ± 0.18k 0.39 ± 0.00hi 46.11 ± 0.34l90 48.10 ± 0.21l 11.10 ± 0.08k 0.11 ± 0.00m 45.89 ± 0.58l

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100 59.58 ± 0.43k 13

alues are mean ± SE (n = 3). Different letters in columns shows significant differen

6–C3–C6 carbon skeleton. Furthermore, flavonoids exhibit a wideange of pharmacological activities, such as antioxidative, antidi-betic, anti-inflammatory, antiallergic, antiviral, gastro-protective,nd hepato-protective activities (Yao et al., 2004).

Phenolic compounds and flavonoids are effective antioxidantsVermerris and Nicholson, 2006). The protective effects of phenolicompounds and flavonoids are directly related to their ability tocavenge free radicals (Fraga, 2010). Free radicals can be harmful ifhey are present in an unprotected body (Dasgupta and De, 2004).henols undergo oxidation, producing toxic compounds, whichlicit inhibitory effects on pathogenic microorganisms (Vermerrisnd Nicholson, 2006).

Ethanolic and methanolic extracts (90%) with the highest phe-olic and antioxidant activities were chosen for this study becausef their anti-microbial and �-amylase inhibitory activity.

able 2ntibacterial assay of betel leaves extracted with ethanol and methanol 90%.

Microorganism Diameter of inhibition zone (mm)*

Deionized water Et

Gram-positive bacteriaStaphylococcus aureus 11.5 ± 0.29aD 14Staphylococcus epidermis 10.3 ± 0.33abD 11Bacillus cereus 9.3 ± 0.44bC 11Bacillus subtilis 9.2 ± 0.17bC 10Listeria monocytogenes 9.8 ± 0.60abC 11

Gram-negative bacteriaEscherichia coli 7.0 ± 0.15bD 10Salmonella Typhimurium 10.3 ± 0.60aC 12Salmonella enterididis 10.2 ± 0.33abC 13Klebsiella pneumonia 9.5 ± 0.29bD 12Pseudomonas aeruginosa NA NA

* Values are means ± SE (n = 3). Values with different small letter show significant diffevel of probability. Values with different capital letter show significant difference betwe

.20ijk 0.23 ± 0.00jk 55.51 ± 0.63k

5% level of probability.

3.2. ˛-Amylase inhibitory assay

Extracts with higher phenolic compounds and antioxidant activ-ities exhibit a higher �-amylase inhibitory activity at differentdilutions. In Fig. 2, the increased dilution of extracts correspond-ingly increased �-amylase inhibitory activity. This result indicatedthat high concentrations of phenolic compounds exhibited high �-amylase inhibitory activity. Hence, if phenolic contents are reducedsubstantially, �-amylase inhibitory activity can be decreased sig-nificantly. Therefore, ethanol or methanol can be used to controlglucose uptake related to hyperglycemia; this result is consistentwith that in a previous study (Ranilla et al., 2010). Previous studies

showed that in vitro analysis can be performed to provide addi-tional insights into the possible inhibitory effects of key enzymesrelated to type 2 diabetes and hyperglycemia; these effects related

hanol 90% Methanol 90% Ampicillin

.5 ± 0.29aC 15.3 ± 0.60aB 20.7 ± 44aA

.8 ± 0.17cdC 12.9 ± 0.67bcB 20.5 ± 29aA

.7 ± 0.17cdB 11.8 ± 0.33cdB 18.7 ± 33cA

.3 ± 0.44eB 10.0 ± 0.00deB 17.5 ± 76deA

.0 ± 0.29deB 10.7 ± 0.44deB 17.8 ± 44dA

.6 ± 0.29deB 9.6 ± 0.20eC 17.5 ± 76deA

.7 ± 0.23bcB 12.7 ± 0.44bcB 19.8 ± 44abA

.2 ± 0.15abB 13.8 ± 0.17abB 19.8 ± 60abA

.5 ± 0.32bcB 11.3 ± 0.33cdeC 17.8 ± 33dA NA 17.2 ± 44eA

erence between different microorganisms at same percentage of the extract at 5%en different extract concentrations for same microorganism.

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Fraga, C.G., 2010. Plant Phenolics and Human Health: Biochemistry, Nutrition, andPharmacology. Wiley, Hoboken, NJ.

Ganesan, P., Kumar, C.S., Bhaskar, N., 2008. Antioxidant properties of methanol

ig. 2. Amylase inhibitory assay (%) of betel leaves extract with methanol andthanol (90%) in comparison with catechin and gallic acid as standard. Values of thears represent the mean ± SE (n = 3). Different letters shows significant differencest 5% level of probability.

o diabetes can be attributed to the phenolic content and antioxi-ant activity of medicinal plants and herbs (Ranilla et al., 2010).

Post-prandial hyperglycemia should be decreased, and thisechnique is one of the therapeutic approaches to treat diabetes.his technique can also be performed by delaying glucose absorp-ion because of the inhibitory effects on hydrolyzing enzymes�-amylase and �-glucosidase) in the digestive system. Enzymenhibitors can retard carbohydrate digestion, increase carbohydrateigestion time, and decrease glucose absorption rate; therefore,his increase in post-prandial plasma glucose was prevented. Plantsontribute to the treatment of type 2 diabetes, particularly whenesources are limited and modern treatment is not available; plantsre also used to treat diabetes when the side effects of inhibitorsnd antidiabetic agents, such as acarbose, voglibose, and miglitol,ecome unbearable (Ali et al., 2006).

.3. Antimicrobial assay

The results of the disc diffusion assay (Table 2) indicated thatetel leaf extract exhibited good antimicrobial activity againstram-negative bacteria and Gram-positive bacteria. All of thextracts showed antimicrobial activity against the pathogens usedn this study except Gram-negative P. aeruginosa. Our observationsre consistent with those in a previous study (Suppakul et al., 2006).

Different sensitivities between Gram-negative bacteria andram-positive bacteria have been attributed to the morphologi-al differences of pathogens. For example, Gram-negative bacteriaontain a strong phospholipid membrane in their outer layerith structural lipopolysaccharide constituents, and these con-

tituents are accounted for the impermeability of the cell wallgainst lipophilic solutes; porins provide a selective barrier againstydrophilic solutes. Gram-positive bacteria are more susceptibleo antimicrobials because they are surrounded by only one outereptidoglycan layer providing an insufficient permeability barrierArias et al., 2004). Some of the polyphenolic compounds pro-

ote the permeability of the cell membrane to K+ and H+, whichan inhibit ATP synthesis by consuming a proton motive forceSuppakul et al., 2006).

.4. Effects of extraction solvents

Extraction of bioactive compounds is a vital step in accuratelyuantifying levels from a plant material using various solvent

d Products 62 (2014) 47–52 51

systems. Solvents, such as water, ethanol, methanol, petroleumether, acetone, chloroform, and ethyl acetate, are routinely usedfor extraction of polyphenols and other bioactive compounds fromplant sources. Previous researches describe acetone and methanolas yielding maximum extraction of phenolic compounds comparedto other solvents (Ganesan et al., 2008) and therefore these twosolvents were used in this research. Recovery of polyphenols andother antioxidant compounds from plant materials depends greatlyon the compound solubility in a particular solvent, the polarity ofthe solvents, viscosity, and vapor pressure. Thus, solvents, such asmethanol or acetone, can easily diffuse into the pores of the plantmaterials to leach out the active constituents. Among the differ-ent factors that contribute to varied results for polyphenols andantioxidant of a single plant source, the chemical nature of the com-pounds, the extraction solvents used, and the assay employed arethe major ones (Bhat et al., 2012; Naczk and Shahidi, 2006). So, thevariations observed in the results of antioxidant compounds in theBetel leave can be attributed directly to the polarity and type ofused solvent.

4. Conclusion

The type and percentage of solvents improved the extractionfrom betel leaves. Ethanol (90%) and methanol (90%) were the mosteffective solvents and showed the highest amount of TP contentsand DPPH◦ (%) related to antioxidant activity. Other experimentsin this study were performed using ethanol and methanol as sol-vents because the researchers assumed that the high amount ofphenolic contents and the high percentage of antioxidant activ-ities increased antimicrobial and �-amylase inhibitory activities.Ethanolic extraction also showed a higher �-amylase inhibitoryactivity than methanolic extraction. However, no significant dif-ference in antimicrobial activities was observed between ethanoland methanol extractions when these extracts were used againstGram-negative bacteria or Gram-positive bacteria.

References

Abdel-Hameed, E.-S.S., 2009. Total phenolic contents and free radical scaveng-ing activity of certain Egyptian Ficus species leaf samples. Food Chem. 114,1271–1277.

Ali, H., Houghton, P.J., Soumyanath, A., 2006. [alpha]-Amylase inhibitory activityof some Malaysian plants used to treat diabetes; with particular reference toPhyllanthus amarus. J. Ethnopharmacol. 107, 449–455.

Arambewela, L., Arawwawala, M., Ratnasooriya, W., 2005. Antidiabetic activities ofaqueous and ethanolic extracts of Piper betle leaves in rats. J. Ethnopharmacol.102, 239–245.

Arias, M.E., Gomez, J.D., Cudmani, N.M., Vattuone, M.A., Isla, M.I., 2004. Antibacterialactivity of ethanolic and aqueous extracts of Acacia aroma Gill. ex Hook et Arn.Life Sci. 75, 191–202.

Bazylko, A., Granica, S., Filipek, A., Piwowarski, J., Stefanska, J., Osinska, E., Kiss, A.K.,2013. Comparison of antioxidant, anti-inflammatory, antimicrobial activity andchemical composition of aqueous and hydroethanolic extracts of the herb ofTropaeolum majus L. Ind. Crops Prod. 50, 88–94.

Bhat, R., Liong, M.-T., Abdorreza, M.N., Karim, A.A., 2012. Evaluation of free radi-cal scavenging activity and antioxidant potential of a few popular green leafyvegetables of Malaysia. Int. J. Food Prop. 16, 1371–1379.

Dasgupta, N., De, B., 2004. Antioxidant activity of Piper betle L. leaf extract in vitro.Food Chem. 88, 219–224.

de Ancos, B., Sgroppo, S., Plaza, L., Cano, M.P., 2002. Possible nutritional and health-related value promotion in orange juice preserved by high-pressure treatment.J. Sci. Food Agric. 82, 790–796.

Espín, J.C., García-Conesa, M.T., Tomás-Barberán, F.A., 2007. Nutraceuticals: facts andfiction. Phytochemistry 68, 2986–3008.

extract and its solvent fractions obtained from selected Indian red seaweeds.Bioresour. Technol. 99, 2717–2723.

Gharibi, S., Tabatabaei, B.E.S., Saeidi, G., Goli, S.A.H., Talebi, M., 2013. Total phenoliccontent and antioxidant activity of three Iranian endemic Achillea species. Ind.Crops Prod. 50, 154–158.

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K

L

N

N

N

R

R

2 L. Nouri et al. / Industrial Cro

won, Y., Apostolidis, E., Shetty, K., 2008. In vitro studies of eggplant (Solanum mel-ongena) phenolics as inhibitors of key enzymes relevant for type 2 diabetes andhypertension. Bioresour. Technol. 99, 2981–2988.

i, S., Li, S.-K., Gan, R.-Y., Song, F.-L., Kuang, L., Li, H.-B., 2013. Antioxidant capacitiesand total phenolic contents of infusions from 223 medicinal plants. Ind. CropsProd. 51, 289–298.

aczk, M., Shahidi, F., 2006. Phenolics in cereals, fruits and vegetables: occurrence,extraction and analysis. J. Pharm. Biomed. Anal. 41, 1523–1542.

air, R., Chanda, S., 2008. Antimicrobial activity of Terminalia catappa, Manilkarazapota and Piper betel leaf extract. Ind. J. Pharm. Sci. 70, 390–393.

ikolic, M., Glamoclija, J., Ferreira, I.C.F.R., Calhelha, R.C., Fernandes, Â., Markovic, T.,Markovic, D., Giweli, A., Sokovic, M., 2014. Chemical composition, antimicrobial,antioxidant and antitumor activity of Thymus serpyllum L., Thymus algerien-sis Boiss. and Reut and Thymus vulgaris L. essential oils. Ind. Crops Prod. 52,183–190.

anilla, L.G., Kwon, Y.-I., Apostolidis, E., Shetty, K., 2010. Phenolic compounds,antioxidant activity and in vitro inhibitory potential against key enzymes

relevant for hyperglycemia and hypertension of commonly used medic-inal plants, herbs and spices in Latin America. Bioresour. Technol. 101,4676–4689.

owland, I., 1999. Optimal nutrition: fibre and phytochemicals. Proceedings of theNutrition Society 58, 415–419.

d Products 62 (2014) 47–52

Santhakumari, P., Prakasam, A., Pugalendi, K., 2003. Modulation of oxidative stressparameters by treatment with Piper Betle leaf in streptozotocin induced diabeticrats. Ind. J. Pharmacol. 35, 373–378.

Singleton, V.L., Orthofer, R., Lamuela-Raventos, R.M., 1999. Analysis of total phenolsand other oxidation substrates and antioxidants by means of Folin-Ciocalteureagent. Method Enzymol. 299, 152–178.

Skotti, E., Anastasaki, E., Kanellou, G., Polissiou, M., Tarantilis, P.A., 2014. Total phe-nolic content, antioxidant activity and toxicity of aqueous extracts from selectedGreek medicinal and aromatic plants. Ind. Crops Prod. 53, 46–54.

Suppakul, P., Sanla-Ead, N., Phoopuritham, P., 2006. Antimicrobial and antioxidantactivities of betel oil. Kasetsart J. Nat. Sci. 40, 91–100.

Vermerris, W., Nicholson, R., 2006. Phenolic Compound Biochemistry. Springer.Wang, C.K., Su, H.Y., Lii, C.K., 1999. Chemical composition and toxicity of taiwanese

betel quid extract. Food Chem. Toxicol. 37, 135–144.Wong, S.P., Leong, L.P., William Koh, J.H., 2006. Antioxidant activities of aqueous

extracts of selected plants. Food Chem. 99, 775–783.Yao, L.H., Jiang, Y.M., Shi, J., TomÁS-BarberÁN, F.A., Datta, N., Singanusong, R., Chen,

S.S., 2004. Flavonoids in food and their health benefits. Plant Foods Hum. Nutr.(Formerly Qualitas Plantarum) 59, 113–122.

Zhishen, J., Mengcheng, T., Jianming, W., 1999. The determination of flavonoidcontents in mulberry and their scavenging effects on superoxide radicals. FoodChem. 64, 555–559.