Phytochemical, antioxidant, antibacterial, and α-amylase inhibitory properties of different extracts from betel leaves

Industrial Crops and Products 62 (2014) 47–52

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Phytochemical, antioxidant, antibacterial, and ␣-amylase inhibitory properties of different extracts from betel leaves Leila Nouri a , Abdorreza Mohammadi Nafchi a,∗ , A.A. Karim b a b

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

a r t i c l e

i n f o

Article history: Received 3 May 2014 Received in revised form 8 August 2014 Accepted 11 August 2014 Keywords: Betel leaf Solvent extraction Phenolic compound Antioxidant Antimicrobial activity

a b s t r a c t Antimicrobial and antioxidant activities, phenol content, and ␣-amylase inhibitory effects of a local variety of betel leaves were evaluated. The effects of various solvents (methanol, ethanol, acetone, and ethyl acetate) on phenols and antioxidant activities were also studied. Methanol and ethanol (90%) extracts showed maximum phenolic contents (205.2 and 202.9 mg GAE/g, respectively). Maximum flavonoid contents were determined using 90% acetone (82.5 mg CE/g), and the highest inhibition percentage of 2,2-diphenyl-1-picrylhydrazyl radical was exhibited by 90% ethanol (percent inhibition, 94%). ␣-Amylase activity assay showed that ␣-amylase inhibitory activities were positively correlated with the total phenolic content of ethanol and methanol. Considering antimicrobial activities, we found that all of the Gram-positive bacteria and Gram-negative bacteria were inhibited by betel leaf extract except Pseudomonas aeruginosa. Our results could provide a basis of future studies on betel leaves used in food and pharmaceutical applications. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Studies have been conducted to identify antioxidants from natural sources, which are suitable alternatives to synthetic antioxidants (Gharibi et al., 2013; Suppakul et al., 2006). Natural antioxidants can be used to develop new nutraceutical products that can prevent oxidative damage in the body (Bazylko et al., 2013; Wong et al., 2006). Antioxidants exhibit functional potential against major components involved in age-related diseases, such as type 2 diabetes linked hyperglycemia, cardiovascular diseases, and cancer (Espín et al., 2007; Fraga, 2010; Ranilla et al., 2010; Rowland, 1999). Type 2 diabetes is a metabolic disorder characterized by chronic hyperglycemia with disorders in carbohydrate, protein, and fat metabolism because of a disturbance in insulin function (Ali et al., 2006). Polyphenolic compounds or phytochemicals present in plants are important components of the human diet; phytochemicals can be used to regulate oxidation and stress-related chronic diseases, such as diabetes and cardiovascular diseases (Kwon et al., 2008). ␣-Amylase is a key enzyme involved in starch hydrolysis. If ␣-amylase involved in the breakdown and absorption of

∗ Corresponding author. Tel.: +98 232 522 5045; fax: +98 232 522 5039. E-mail addresses: [email protected], [email protected] (A. Mohammadi Nafchi). http://dx.doi.org/10.1016/j.indcrop.2014.08.015 0926-6690/© 2014 Elsevier B.V. All rights reserved.

carbohydrates is inhibited, post-prandial blood glucose level can be reduced; this mechanism can be used as a strategy to control hyperglycemia related to type 2 diabetes (Kwon et al., 2008). However, currently used synthetic ␣-amylase inhibitors, such as acarbose, can cause several complications, including flatulence, meteorism, abdominal distention, and possibly diarrhea (Kwon et al., 2008). Microbial contamination is one of the main parameters that can reduce food quality and shelf-life and compromise product safety. The growth of microorganisms may cause food-borne diseases or spoilage in food products; oxidation in food products also cause proteins and lipids to deteriorate, thereby altering the color, flavor, and texture of products (Suppakul et al., 2006). Hence, the prevention of microbial contamination is directly relevant in food processing. Chemical composition, antioxidant, and antimicrobial activity of lots of medicinal plants recently have been investigated (Bhat et al., 2012; Li et al., 2013; Skotti et al., 2014). Nikolic´ et al. (2014) were reported chemical composition and in vitro biological activity of Thymus essential oils (EO). They showed that succeeds in creating comparable and quantitative data for usage of Thymus oils against oral microorganisms. Finding new antimicrobial agents with various chemical structures and neo-mechanisms of action is essential. The increasing morbidity of new infectious diseases and resistance to current antibiotics in clinical use (Nair and Chanda, 2008).

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Betel leaves is widely grown in Asian regions, such as India, Sri Lanka, Malaysia, and Philippines. Betel leaves exhibit wound healing, gastro-protective, and hepato-protective activities; these leaves also elicit anti-fertility and anti-motility effects in washed human spermatozoa (Arambewela et al., 2005). Furthermore, betel leaves show antioxidant activities (Santhakumari et al., 2003; Wang et al., 1999). Although a few studies on the antioxidant properties of betel leaves have been conducted, studies on the antidiabetic (Santhakumari et al., 2003) and antimicrobial (Nair and Chanda, 2008) activities of betel leaves and extract are limited. To the best of our knowledge different extraction and antimicrobial, antioxidant, and anti-diabetic properties of betel leave mutually has not been investigated. So our study aimed to evaluate the antioxidant, antimicrobial, and ␣-amylase inhibitory properties of the phenolics in betel leaves extracted with aqueous and organic solvents. 2. Materials and methods 2.1. Plant materials Fresh betel leaves with no apparent physical damage were purchased from a local wet market in Penang, Malaysia. Betel leaves were obtained from a local variety of Piper betel L. (Fig. 1), a creeper plant that grows to a large bush of vines and leaves. The leaves are used for chewing and medicinal purposes, as well as in ceremonies in traditional Malay weddings as part of a bridegroom’s offering. The taxonomic identity of P. betel was determined by taxonomists in the Department of Biology (Herbarium Center; Voucher Number: 11240), Universiti Sains Malaysis, Pulau Pinang, Malaysia. 2.2. Chemicals, microorganisms and reagents Folin-Ciocalteu’s (FC) phenol reagent, sodium carbonate, sodium acetate, sodium nitrite, sodium hydroxide, aluminum chloride, 2,2-diphenyl-1-picrylhydrazyl (DPPH◦ ), gallic acid, catechin, rutin, 3,5-dinitrosalicylic acid, potassium sodium tartrate tetrahydrate, soluble starch, and human salivary ␣-amylase enzyme (A1031 type XIII-A) were supplied by Sigma-Aldrich® (St. Louis, MO, USA). Ethanol, methanol, acetone, and ethyl acetate were purchased from R&M chemicals (Essex, UK). All of the reagents and chemicals used in the study were of analytical grade. Media and chemicals used in antibacterial experiments were nutrient agar and nutrient broth from Merck, Germany, M¨vellerHinton agar, plate count agar, ampicillin, and ringer tablets purchased from Oxoid, England. Following ten clinical oral isolates were tested: Staphylococcus aureus, Staphylococcus epidermis, Bacillus cereus, Bacillus subtilis, Listeria monocytogenes, Escherichia coli, Salmonella typhimurium, Salmonella enterididis, Klebsiella pneumonia, and Pseudomonas aeruginosa. The reference strains were obtained from the microbiology laboratory (School of Industrial Technology, USM, Malaysia). 2.3. Extraction of polyphenols Leaves were washed with clean water, cut into small pieces (approximately 1 cm × 1 cm), and placed in a freeze dryer (model 7754511, Labconco Corporation, Kansas City, Missouri, USA) for 48 h. The freeze–dried samples were ground to a powder by using a stainless-steel grinder and stored in a freezer until they were used. For each experiment, 1 g of freeze–dried sample was weighed and placed in a beaker containing 100 ml of solvent for 24 h. The beaker was then placed on a hotplate (Model FSMQ 143551, Fisher Scientific, Kuala Lumpur, Malaysia) and constantly stirred at 1200 rpm by using a magnetic stirrer at room temperature

(25 ± 1 ◦ C). The extract was filtered under suction with Whatman No. 1 filter paper. The remaining residue on the filter paper was placed back in the same flask and stirred for another 3 h and re-extracted twice according to the same procedure until the residue became colorless. Light exposure was avoided throughout the extraction process. Extraction was conducted in triplicate. Four different 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 evaluated by the FC assay, as described in a previous method ((Singleton et al., 1999) with slight modifications. In brief, 40 ␮l of the extract was mixed with 1.8 ml of the FC reagent. The FC reagent was prediluted 10 times using deionized water. After 5 min, 1.2 ml of (7.5% w/v) sodium carbonate solution was added. The solutions were then mixed and kept for 1 h at room temperature. Absorbance was determined using UV–visible spectrophotometer (Shimadzu UV-160APC, Japan) at 765 nm. The calibration curve was prepared using 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 mg gallic acid equivalents per 1 g of sample. 2.5. Determination of total flavonoids The total flavonoid (TF) content of the betel leaves were determined according to colorimetric assay (Zhishen et al., 1999). Approximately, 1 ml of betel leaves extract was mixed with 4 ml of deionized water and 0.3 ml of 5% (w/v) NaNO2 . The mixture was 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. Approximately, 2.4 ml of deionized water was immediately added to obtain a final volume of 10 ml. The mixture was shaken vigorously and the absorbance of the mixture was determined at 510 nm by using a UV–visible spectrophotometer (Shimadzu UV-160APC, Japan). Catechin was used to prepare a standard solution with different concentrations (20, 40, 60, 80, and 100 mg/l, r2 = 0.996). Results were indicated as mg catechin equivalents (CE) per 1 g on a dry weight basis of a specimen. 2.6. Determination of total flavonols Flavonol is one of the major classes of flavonoids. Flavonols exhibit antioxidant properties that can be determined using a paper-based method described by (Abdel-Hameed, 2009). Approximately, 1 ml of betel leaf extract (10 mg/ml) was mixed with 1 ml of aluminum trichloride solution (20 mg/ml) and 3 ml of sodium acetate solution (50 mg/ml). The mixture was kept for 2.5 h at room temperature and observed at 440 nm by using a UV–visible spectrophotometer (Shimadzu UV-160APC, Japan). Different concentrations 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 the determinations were performed in triplicate. 2.7. DPPH◦ free radical scavenging assay The antioxidant activity of betel leaves was analyzed by evaluating the effects of free radical scavenging against the radicals of DPPH◦ . The experiment was performed using a method described previously (de Ancos et al., 2002). In brief, 10 ␮l of betel leaf extract was thoroughly vortex-mixed with 90 ␮l of deionized water and 3.9 ml of 25 mM DPPH◦ methanolic solution. The mixture was kept in a dark place for 30 min. After 30 min, the absorbance was determined against methanol without DPPH◦ as blank at 515 nm by using

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Fig. 1. Betel leaves in a natural habitat in Pulau Pinang, Malaysia.

a UV-visible spectrophotometer (Shimadzu UV-160APC, Japan). DPPH◦ radical scavenging was expressed as percentage, which was calculated using the following equation: % inhibition of DPPH◦ =

(Abscontrol − Abssample ) (Abscontrol )

× 100

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

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

3. Results and discussion

2.8. ˛-Amylase inhibitory assay

3.1. Effect of different solvents on betel leaf extraction

The ␣-amylase inhibitory assay of betel leaf extracts was evaluated according to a previously described method (Ranilla et al., 2010). In brief, 0.5 ml of extract was mixed with 0.5 ml of ␣-amylase solution (0.5 mg/ml) with 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl). The mixture was incubated at room temperature for 10 min and 0.5 ml of starch solution (1%) in 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) was added. The resulting mixture was incubated at room temperature for 10 min, and the reaction was terminated using 1 ml of dinitrosalicylic acid color reagent. At this time, the test tubes were placed in a water bath (100 ◦ C and 5 min) and cooled until room temperature was reached. The mixture was then diluted with 10 ml of deionized water, and absorbance was determined at 540 nm. The absorbances of blank (buffer instead of extract and amylase solution) and control (buffer instead of extract) samples were also determined. The inhibition of ␣-amylase was calculated using the following equation: % inhibition of ␣-Amylase =

(Abscontrol − Abssample ) (Abscontrol )

× 100

where Abscontrol corresponds to the absorbances of the solution without extract (buffer instead of extract) and with ␣-amylase solution and Abssample corresponds to the solution with extract and ␣-amylase solution. 2.9. Antimicrobial assay The antimicrobial activity of betel leaf extracts was assessed using the disc diffusion method with slight modifications (Arias et al., 2004). Petri dishes (9 cm in diameter) with 25 ml of M¨vellerHinton agar were prepared. Disks (diameter = 6 mm) were soaked with 20 ␮l of the betel leaf extracts were placed on the agar. A disk containing ampicillin was used as a control treatment. The Petri dishes were placed in an incubator (35 ◦ C) for 16–20 h. All of the experiments were performed in triplicate, and results were expressed as diameters (mm) of inhibition.

Total phenolic (TP) contents of betel leaves measured using Folin-Ciocalteu’s colorimetric method are presented in Table 1. The TP contents of betel leaf extracts ranged from 59.38 to 202.92 mg GAE/g and 58.54 to 205.20 mg GAE/g using ethanol and methanol solvents, respectively. The TP values of acetone- and ethyl acetate-extracted samples ranged from 47.68 to 191.50 mg GAE/g and from 46.45 to 128.43 mg GAE/g, respectively. In waterextracted samples, the TP content was 71.30 mg GAE/g. The total flavonoid TF contents of betel leaves extracted from different solvents and different concentrations are presented in Table 1. The TF contents of the ethanol extract of betel leaves ranged from 12.13 to 78.8 mg CE/g. The TF contents of methanol-, acetone-, and ethyl acetate-extracted samples ranged from 12.25 to 80.59 mg CE/g, from 12.10 to 82.55 mg CE/g, and from 10.97 to 42.05 mg CE/g, respectively. In water-extracted samples, the TF content was 30.88 mg CE/g. The flavonol content ranged from 0.21 to 1.4 mg RE/g, from 0.32 to 2.5 mg RE/g, from 0.69 to 2.43 mg RE/g, and from 0.11 to 0.43 mg RE/g in ethanol-, methanol-, acetone-, and ethyl acetateextracted samples, respectively. In water-extracted samples, the flavonol content was 0.12 mg RE/g. The DPPH◦ radical scavenging activities of the extracts ranged from 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. In water-extracted samples, the % inhibition of DPPH◦ was 52.63%. In Table 1, 90% methanol and 90% ethanol extracts contained higher polyphenolic contents and antioxidant activities than other solvents in this study. Flavonoids are the most common group of phenolic compounds widely distributed in all parts of plants, particularly in photosynthesizing cells. Flavonoids contain different structures, degrees of hydroxylation and polymerization, substitutions and conjugations. Flavonoids also exhibit different chemical properties because of their varying characteristics. All of the flavonoids contain a simple

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Table 1 Total phenolic, flavonoids, flavonols, and DPPH◦ of betel leaves. Total flavonoids (mg CE/g dry weight)

Total flavonols (mg RE/g dry weight)

DPPH◦ (%)

71.30 ± 0.86j

30.88 ± 0.56h

0.12 ± 0.00lm

52.63 ± 0.52k

30 50 70 90 100

59.38 ± 0.90k 82.29 ± 0.48i 147.96 ± 0.81e 202.92 ± 0.71a 195.92 ± 0.92b

12.13 ± 0.12jk 14.30 ± 0.15ij 47.96 ± 0.36f 78.78 ± 0.51bc 75.22 ± 0.56d

0.21 ± 0.00kl 0.44 ± 0.01h 0.81 ± 0.00f 1.40 ± 0.01d 0.83 ± 0.02f

75.01 ± 0.61hi 79.20 ± 0.38fg 85.97 ± 0.49 cd 93.99 ± 0.40a 89.95 ± 0.67b

30 50 70 90 100

58.54 ± 0.32k 97.32 ± 0.66h 140.15 ± 0.63f 205.20 ± 0.44a 192.72 ± 0.78bc

12.25 ± 0.21jk 15.11 ± 0.48i 47.12 ± 0.33f 80.59 ± 0.91ab 76.52 ± 0.69 cd

0.32 ± 0.01ij 0.67 ± 0.01g 1.76 ± 0.02c 2.50 ± 0.05a 1.37 ± 0.01d

73.17 ± 0.52i 78.43 ± 0.45g 84.38 ± 0.36de 89.10 ± 0.46bc 87.18 ± 1.21 cd

30 50 70 90 100

47.68 ± 0.16l 93.39 ± 0.63h 159.81 ± 1.38d 191.50 ± 01.21c 131.11 ± 0.80g

12.10 ± 0.20jk 12.20 ± 0.22jk 56.38 ± 0.89e 82.55 ± 0.60a 76.67 ± 0.69cd

0.70 ± 0.01 g 0.69 ± 0.01 g 2.43 ± 0.04ab 2.35 ± 0.02c 1.25 ± 0.02e

69.44 ± 0.80j 73.20 ± 0.35i 77.46 ± 0.81gh 86.58 ± 0.74cd 81.77 ± 0.48ef

30 50 70 90 100

128.43 ± 1.27g 67.32 ± 0.71j 46.45 ± 0.82l 48.10 ± 0.21l 59.58 ± 0.43k

42.05 ± 0.40g 32.32 ± 0.32h 10.97 ± 0.18k 11.10 ± 0.08k 13.17 ± 0.20ijk

0.11 ± 0.00m 0.43 ± 0.01h 0.39 ± 0.00hi 0.11 ± 0.00m 0.23 ± 0.00jk

41.14 ± 0.32m 44.27 ± 0.38lm 46.11 ± 0.34l 45.89 ± 0.58l 55.51 ± 0.63k

Solvent

%

Water

100

Total phenolic (mg GAE/g dry weight)

Ethanol

Methanol

Acetone

Ethyl acetate

Values are mean ± SE (n = 3). Different letters in columns shows significant differences at 5% level of probability.

C6–C3–C6 carbon skeleton. Furthermore, flavonoids exhibit a wide range of pharmacological activities, such as antioxidative, antidiabetic, anti-inflammatory, antiallergic, antiviral, gastro-protective, and hepato-protective activities (Yao et al., 2004). Phenolic compounds and flavonoids are effective antioxidants (Vermerris and Nicholson, 2006). The protective effects of phenolic compounds and flavonoids are directly related to their ability to scavenge free radicals (Fraga, 2010). Free radicals can be harmful if they are present in an unprotected body (Dasgupta and De, 2004). Phenols undergo oxidation, producing toxic compounds, which elicit inhibitory effects on pathogenic microorganisms (Vermerris and Nicholson, 2006). Ethanolic and methanolic extracts (90%) with the highest phenolic and antioxidant activities were chosen for this study because of their anti-microbial and ␣-amylase inhibitory activity.

3.2. ˛-Amylase inhibitory assay Extracts with higher phenolic compounds and antioxidant activities exhibit a higher ␣-amylase inhibitory activity at different dilutions. In Fig. 2, the increased dilution of extracts correspondingly increased ␣-amylase inhibitory activity. This result indicated that high concentrations of phenolic compounds exhibited high ␣amylase inhibitory activity. Hence, if phenolic contents are reduced substantially, ␣-amylase inhibitory activity can be decreased significantly. Therefore, ethanol or methanol can be used to control glucose uptake related to hyperglycemia; this result is consistent with that in a previous study (Ranilla et al., 2010). Previous studies showed that in vitro analysis can be performed to provide additional insights into the possible inhibitory effects of key enzymes related to type 2 diabetes and hyperglycemia; these effects related

Table 2 Antibacterial assay of betel leaves extracted with ethanol and methanol 90%. Microorganism

Diameter of inhibition zone (mm)* Deionized water

Ethanol 90%

Methanol 90%

Ampicillin

Gram-positive bacteria Staphylococcus aureus Staphylococcus epidermis Bacillus cereus Bacillus subtilis Listeria monocytogenes

11.5 ± 0.29aD 10.3 ± 0.33abD 9.3 ± 0.44bC 9.2 ± 0.17bC 9.8 ± 0.60abC

14.5 ± 0.29aC 11.8 ± 0.17cdC 11.7 ± 0.17cdB 10.3 ± 0.44eB 11.0 ± 0.29deB

15.3 ± 0.60aB 12.9 ± 0.67bcB 11.8 ± 0.33cdB 10.0 ± 0.00deB 10.7 ± 0.44deB

20.7 ± 44aA 20.5 ± 29aA 18.7 ± 33cA 17.5 ± 76deA 17.8 ± 44dA

Gram-negative bacteria Escherichia coli Salmonella Typhimurium Salmonella enterididis Klebsiella pneumonia Pseudomonas aeruginosa

7.0 ± 0.15bD 10.3 ± 0.60aC 10.2 ± 0.33abC 9.5 ± 0.29bD NA

10.6 ± 0.29deB 12.7 ± 0.23bcB 13.2 ± 0.15abB 12.5 ± 0.32bcB NA

9.6 ± 0.20eC 12.7 ± 0.44bcB 13.8 ± 0.17abB 11.3 ± 0.33cdeC NA

17.5 ± 76deA 19.8 ± 44abA 19.8 ± 60abA 17.8 ± 33dA 17.2 ± 44eA

* Values are means ± SE (n = 3). Values with different small letter show significant difference between different microorganisms at same percentage of the extract at 5% level of probability. Values with different capital letter show significant difference between different extract concentrations for same microorganism.

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systems. Solvents, such as water, ethanol, methanol, petroleum ether, acetone, chloroform, and ethyl acetate, are routinely used for extraction of polyphenols and other bioactive compounds from plant sources. Previous researches describe acetone and methanol as yielding maximum extraction of phenolic compounds compared to other solvents (Ganesan et al., 2008) and therefore these two solvents were used in this research. Recovery of polyphenols and other antioxidant compounds from plant materials depends greatly on the compound solubility in a particular solvent, the polarity of the solvents, viscosity, and vapor pressure. Thus, solvents, such as methanol or acetone, can easily diffuse into the pores of the plant materials to leach out the active constituents. Among the different factors that contribute to varied results for polyphenols and antioxidant of a single plant source, the chemical nature of the compounds, the extraction solvents used, and the assay employed are the major ones (Bhat et al., 2012; Naczk and Shahidi, 2006). So, the variations observed in the results of antioxidant compounds in the Betel leave can be attributed directly to the polarity and type of used solvent. Fig. 2. Amylase inhibitory assay (%) of betel leaves extract with methanol and ethanol (90%) in comparison with catechin and gallic acid as standard. Values of the bars represent the mean ± SE (n = 3). Different letters shows significant differences at 5% level of probability.

to diabetes can be attributed to the phenolic content and antioxidant activity of medicinal plants and herbs (Ranilla et al., 2010). Post-prandial hyperglycemia should be decreased, and this technique is one of the therapeutic approaches to treat diabetes. This technique can also be performed by delaying glucose absorption because of the inhibitory effects on hydrolyzing enzymes (␣-amylase and ␣-glucosidase) in the digestive system. Enzyme inhibitors can retard carbohydrate digestion, increase carbohydrate digestion time, and decrease glucose absorption rate; therefore, this increase in post-prandial plasma glucose was prevented. Plants contribute to the treatment of type 2 diabetes, particularly when resources are limited and modern treatment is not available; plants are also used to treat diabetes when the side effects of inhibitors and antidiabetic agents, such as acarbose, voglibose, and miglitol, become unbearable (Ali et al., 2006).

4. Conclusion The type and percentage of solvents improved the extraction from betel leaves. Ethanol (90%) and methanol (90%) were the most effective solvents and showed the highest amount of TP contents and DPPH◦ (%) related to antioxidant activity. Other experiments in this study were performed using ethanol and methanol as solvents because the researchers assumed that the high amount of phenolic contents and the high percentage of antioxidant activities increased antimicrobial and ␣-amylase inhibitory activities. Ethanolic extraction also showed a higher ␣-amylase inhibitory activity than methanolic extraction. However, no significant difference in antimicrobial activities was observed between ethanol and methanol extractions when these extracts were used against Gram-negative bacteria or Gram-positive bacteria.

References 3.3. Antimicrobial assay The results of the disc diffusion assay (Table 2) indicated that betel leaf extract exhibited good antimicrobial activity against Gram-negative bacteria and Gram-positive bacteria. All of the extracts showed antimicrobial activity against the pathogens used in this study except Gram-negative P. aeruginosa. Our observations are consistent with those in a previous study (Suppakul et al., 2006). Different sensitivities between Gram-negative bacteria and Gram-positive bacteria have been attributed to the morphological differences of pathogens. For example, Gram-negative bacteria contain a strong phospholipid membrane in their outer layer with structural lipopolysaccharide constituents, and these constituents are accounted for the impermeability of the cell wall against lipophilic solutes; porins provide a selective barrier against hydrophilic solutes. Gram-positive bacteria are more susceptible to antimicrobials because they are surrounded by only one outer peptidoglycan layer providing an insufficient permeability barrier (Arias et al., 2004). Some of the polyphenolic compounds promote the permeability of the cell membrane to K+ and H+ , which can inhibit ATP synthesis by consuming a proton motive force (Suppakul et al., 2006). 3.4. Effects of extraction solvents Extraction of bioactive compounds is a vital step in accurately quantifying levels from a plant material using various solvent

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