- Email: [email protected]
Antioxidant and antibacterial activities of Nephelium lappaceum L. extracts
Available online at www.sciencedirect.com
LWT - Food Science and Technology 41 (2008) 2029e2035 www.elsevier.com/locate/lwt
Antioxidant and antibacterial activities of Nephelium lappaceum L. extracts Nont Thitilertdecha a, Aphiwat Teerawutgulrag b, Nuansri Rakariyatham b,* a b
Division of Biotechnology, Graduate School, Chiang Mai University, Chiang Mai 50200, Thailand Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand Received 27 June 2007; received in revised form 25 January 2008; accepted 30 January 2008
Abstract Ether, methanolic and aqueous extracts of lyophilized rambutan (Nephelium lappaceum L.) peels and seeds were evaluated for phenolic contents, antioxidant and antibacterial activities. High amounts of phenolic compounds were found in the peel extracts and the highest content was in the methanolic fraction (542.2 mg/g dry extract). Several potential antioxidant activities, including reducing power, b-carotene bleaching, linoleic peroxidation and free radical scavenging activity, were evaluated. The peel extracts exhibited higher antioxidant activity than the seed extracts in all methods determined (P < 0.05). The methanolic fraction was found to be the most active antioxidant as shown by their 50% DPPH inhibition concentration, 4.94 mg/mL. The results indicated this fraction exhibited greater DPPH radical scavenging activity than BHT and ascorbic acid (0.32 g dry extract/g BHT or ascorbic acid). Antibacterial activity against eight bacterial strains was assessed by disc diffusion and broth macrodilution methods. All peel extracts exhibited antibacterial activity against five pathogenic bacteria. The most sensitive strain, Staphylococcus epidermidis, was inhibited by the methanolic extract (MIC 2.0 mg/mL). Ó 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
Keywords: Nephelium lappaceum; Antioxidant; Antibacterial; Phenolics
1. Introduction Reactive oxygen species (ROS) generated from both living organisms and exogenous sources (Halliwell & Gutteridge, 1999), initiate reactions which damage biological molecules and also play an important causative role in disease initiation (Croft, 1999; Halliwell, 1996). Lipid oxidation, caused by free radicals, is one of the major factors for the deterioration of food products during processing and storage. Effective synthetic antioxidants such as butylated hydroxytoluene (BHT) have been used for industrial processing but these are suspected of being responsible for liver damage and carcinogenesis (Barlow, 1990). Recently, there is an increasing interest in finding natural antioxidants from plant materials to replace synthetic ones.
* Corresponding author. Tel.: þ66 53 943341; fax: þ66 53 892277. E-mail address: [email protected] (N. Rakariyatham).
Plants contain a large variety of substances possessing antioxidant activity, such as vitamin C, vitamin E, carotenes, xanthophylls, tannins and phenolics (Chanwitheesuk, Teerawutgulrag, & Rakariyatham, 2005). Sources of natural antioxidants are primarily plant phenolics that can be found in all parts of the plant (Pratt, 1992). The plant phenolic compounds, such as flavonoids exhibit antioxidant properties due to their high redox potential (Cook & Samman, 1996). They also exhibit a wide range of biological activity, antimicrobial activity, anticarcinogenicity and antiproliferation, and many biological activities can be attributed to their antioxidant properties (Ren, Qiao, Wang, Zhu, & Zhang, 2003; Tapiero, Tew, Ba, & Mathe´, 2002). Interestingly, recent research has revealed that fruit peels and seeds, such as grape seeds and peels (Jayaprakasha, Selvi, & Sakariah, 2003; Jayaprakasha, Singh, & Sakariah, 2001; Negro, Tommasi, & Miceli, 2003), pomegranate peel (Singh, Murthy, & Jayaprakasha, 2002), sweet orange peel (Anagnostopoulou, Kefalas, Papageorgiou, Assimopoulou, & Boskou,
0023-6438/$34.00 Ó 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2008.01.017
2030
N. Thitilertdecha et al. / LWT - Food Science and Technology 41 (2008) 2029e2035
2006) and mango seed kernel (Kabuki et al., 2000) may potentially possess antioxidant and/or antimicrobial properties. The current study focuses on the possibility of using rambutan peel and seed waste as source of low-cost natural antioxidant and antimicrobial. 2. Materials and methods 2.1. Preparation of extracts Lyophilized peels and seeds of Nephelium lappaceum L. were powdered and then extracted with ether (three times). The residue was then extracted with methanol (three times) and finally with water (three times). After filtration, the combined ether and methanolic extracts were evaporated under vacuum to absolute dryness and the aqueous extract was lyophilized. 2.2. Determination of the total phenolic contents The amounts of phenolic compounds in the extracts were determined according to the method of Waterman and Mole (1994) using FolineCiocalteu method and catechin was used as the standard phenolic compound. The extract solution in appropriate solvent (0.1 mL) was transferred to a volumetric flask containing 7.9 mL of distilled water. After that, 0.5 mL of FolineCiocalteu reagent was added. Three minutes later, 1.5 mL of 200 g/L sodium carbonate solution was added. Subsequently, the shaken mixture was allowed to stand for 2 h at room temperature and then measured at 760 nm. The experiment was carried out in triplicate and the total phenolic compounds were expressed as catechin equivalents. 2.3. Determination of antioxidative activity 2.3.1. Reducing power The reducing power was based on the method described previously by Yildirim, Mavi, and Kara (2001). Different concentrations of extracts and BHT (SigmaeAldrich GmbdH, Germany) (50e200 mg/mL) in 1 mL of methanol were mixed with 2.5 mL of phosphate buffer (0.2 mol/L, pH 6.6) and 2.5 mL of 10 g/L potassium ferricyanide. The mixture was incubated at 50 C for 30 min. An aliquot (2.5 mL) of 100 g/L trichloroacetic acid was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. Finally, 2.5 mL of the upper layer solution was mixed with 2.5 mL of distilled water and 0.5 mL of 1 g/L FeCl3, and the absorbance of the resulting solution was measured at 700 nm. 2.3.2. b-Carotene linoleate model system The antioxidant activity of rambutan peel and seed extracts was determined according to the method of Jayaprakasha et al. (2001). First, 0.2 mg of b-carotene in 1 mL of chloroform, 20 mg of linoleic acid and 200 mg of Tween-40 were mixed. Chloroform was removed using nitrogen gas and the resulting mixture was topped up to 50 mL using oxygenated water and mixed well. Aliquots (5 mL) of the emulsion were pipetted
into different test tubes containing 0.2 mL of extracts in methanol at different concentrations. BHT was used for comparative standard. A control containing 0.2 mL of methanol and 5 mL of the above emulsion was prepared. The tubes were placed at 50 C in the water bath. Absorbance was taken at zero time (t ¼ 0) at 470 nm. Measurement of absorbance was continued until the color of b-carotene disappeared in the control (t ¼ 180 min) at 15 min intervals. A mixture prepared as above without b-carotene served as blank. The experiment was carried out in triplicate. The antioxidant activity of the extracts was evaluated in terms of the bleaching of b-carotene using the following equation: Antioxidant activity ¼ 1 ðA0 At Þ= A00 A0t 100 Where A0 and A00 were the absorbance measured at zero time of the incubation for test sample and control, respectively, and At and A0t were the absorbance measured for the test sample and the control, respectively, after incubation for 180 min. Extract concentration providing 50% inhibition (IC50) was calculated from the graph of inhibition percentage against extract concentration. 2.3.3. Linoleic peroxidation method The antioxidant activity of rambutan peel and seed extracts was based on the thiocyanate method described previously by Jayaprakasha et al. (2001) with slight modifications. The extracts were dissolved in methanol for stock solutions. BHT was used as comparative standard. The solution containing stock extract solution or BHT at various concentrations in 2.5 mL of potassium phosphate buffer (0.04 mol/L, pH 7.0) was transferred to 2.5 mL of linoleic acid emulsion containing 0.28 g Tween-40, 0.28 g linoleic acid dissolved in potassium phosphate buffer (0.04 mol/L, pH 7.0). A control, containing 2.5 mL linoleic acid emulsion and 2.5 mL potassium phosphate buffer (0.04 mol/L, pH 7.0) was prepared. The mixture was incubated at 37 C for 64 h. Aliquots (0.1 mL) were drawn from the incubation mixture at 8-h intervals and then mixed with 5.0 mL of 75% (v/v) ethanol, 0.1 mL of 300 g/L ammonium thiocyanate and 0.1 mL of 20 mmol/L ferrous chloride in 0.96 mol/L HCl and allowed to stand at room temperature for 3 min. The absorbance was measured at 500 nm. The experiments on antioxidant activity were run in triplicate and the percent inhibition of lipid peroxidation was calculated using the following formula: Percent inhibition ¼ 100 ½A1 =A0 100 Where A0 was the absorbance of the control reaction and A1 was the absorbance in the presence of the extracts. The antioxidant activities were illustrated as IC50 values.
2.3.4. DPPH radical scavenging activity The stable free radical scavenging activity was determined by the 1,1-diphenyl-2-picryl-hydracyl (DPPH ) (Sigmae Aldrich GmbdH, Germany) method of Gu¨lc¸ın, Oktay, Kirec¸ci, and Ku¨frevıo˘glu (2003). A 0.1 mmol/L DPPH solution in methanol was prepared, and then 1 mL of this solution was
N. Thitilertdecha et al. / LWT - Food Science and Technology 41 (2008) 2029e2035
mixed with 3 mL of extract at different concentrations (0.78e 400 mg/mL). A control, containing 1 mL of DPPH solution and 3 mL of methanol was prepared. The mixture was incubated at room temperature for 30 min and then the absorbance was measured at 517 nm. The ability to scavenge the DPPH radical was calculated as percent DPPH scavenging using the following equation:
2031
determined as the lowest concentration of the extract that demonstrated no visible growth. 2.6. Statistical analysis
%DPPH scavenging ¼ ½ðA0 A1 Þ=A0 100 Where A0 was the absorbance of the control and A1 was the absorbance of the mixture containing extracts. IC50 of reference antioxidant compounds, BHT, quercetin and ascorbic acid (SigmaeAldrich GmbdH, Germany), were used for comparison to IC50 of the extracts. 2.4. Bacterial cultures The microorganisms used in this study consisted of eight strains of pathogenic bacteria, viz. Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Vibrio cholerae, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis. All bacterial strains were obtained from the Department of Clinical Microbiology, Faculty of Associated Medical Sciences, Chiang Mai University. The bacteria were grown and maintained on nutrient agar slants. The inoculated agar slants were incubated at 37 C. 2.5. Antibacterial assays 2.5.1. Disc diffusion assay The antibacterial activity was based on disc diffusion method (Bauer, Kirby, Sherris, & Truck, 1966) using bacterial cell suspension whose concentration was equilibrated to a 0.5 McFarland standard. A 100 mL of each bacterial suspension was spread on a MuellereHinton agar plate. Sterile paper discs (6 mm diameter) were impregnated with 20 mL of each extract dissolved in the solvent used for extraction at 125 mg/mL. The discs were allowed to dry and then placed on the inoculated agar. Discs with the solvent used for dissolution were used as negative control and 10 mg streptomycin discs were used as positive controls. The plates were incubated at 37 C for 24 h. After incubation time, zone of inhibition was measured. The experiment was performed in triplicate. 2.5.2. Minimum inhibition concentrations (MICs) Minimum inhibition concentrations of the extracts were evaluated for the bacterial strains which were determined as being sensitive to the extracts in the disc diffusion assay. A broth macrodilution method was used, as previously described by Nakamura et al. (1999) with a slight modification. Serial twofold dilutions of each extract were prepared in 10% (v/v) dimethylsulfoxide (DMSO), and 30 mL of each dilution was added to 3 mL of MuellereHinton broth. These were inoculated with 30 mL of culture of the test bacterial strains. After incubation of the cultures at 37 C, the MIC value was
All experimental results were expressed as means S.D. Analysis of variance was performed by ANOVA procedures. Correlation coefficient (R) was used to determine two variables. SPSS software was used for statistical calculations. The results with P < 0.05 were regarded to be statistically significant. 3. Results and discussion In this study, phenolic content, antioxidant activity and antibacterial activity of various N. lappaceum peel and seed extracts were determined. The extraction yields (g/100 g lyophilized rambutan) from various extractants, i.e. ether, methanol and water are presented in Table 1. The total phenolic contents of the extracts were determined by FolineCiocalteu method. The high amounts of phenolic compounds were found in the peel extracts in the following order: methanolic fraction (542.2 mg/g), aqueous fraction (393.2 mg/g) and ether fraction (293.3 mg/g) (Table 1). Although low amounts of phenolic compounds were observed in the seed extracts, the methanolic fraction of seed extract contained the highest phenolic content (58.5 mg/g). The extracts obtained by various solvent extractions were determined for their antioxidant activities. The extracts were investigated for the reductive capabilities by using the potassium ferricyanide reduction method. The reducing ability may serve as a significant indicator of potential antioxidant activity (Meir, Kanner, Akiri, & Hadas, 1995). The peel and seed extracts increased in reducing powers with increasing concentration (Fig. 1) and the peel extracts exhibited higher reducing ability than the seed extracts. The methanolic extract showed the highest activity, followed by the aqueous and ether extracts. When compared to the BHT, the methanolic fraction showed significantly (P < 0.05) higher activity at all concentrations. Yen and Chen (1995) reported that the extract which Table 1 Extraction yields and total phenolic content of Nephelium lappaceum L. extracts Extraction yield (g/100 g)
Total phenolicsf
Peel extracts Ether Methanol Aqueous
2.84 25.1 2.06
293.3 0.5c 542.2 0.0a 393.2 7.4b
Seed extracts Ether Methanol Aqueous
21.2 4.80 7.29
7.4 1.8e 58.5 0.4d 3.3 0.2e
Extracts
Values are expressed as means S.D. aee Means the column followed by different letters are significantly different (P < 0.05). f Express as mg catechin/1 g dry extract.
N. Thitilertdecha et al. / LWT - Food Science and Technology 41 (2008) 2029e2035
2032
300
1.2
d
250
1
IC50 ( g/mL)
absorbance at 700 nm
1.4
0.8 0.6 0.4 0.2
200 150 c 100 50
b
0 0
50
100
150
200
0
concentration ( g/mL) Fig. 1. Reducing powers of Nephelium lappaceum L. extracts. (,), (-), (:) are the ether, methanolic and aqueous extracts of the peel; (), (6), (C) are the ether, methanolic and aqueous extracts of the seed and (B) is BHT, butylated hydroxytoluene.
showed a reducing power could function as an electron donor and also could reduce the oxidized intermediates generated from the lipid peroxidation reaction. For their antioxidant activity, the extracts were determined for their capability to delay b-carotene bleaching in the coupled oxidation of b-carotene/linoleic acid model system. Fig. 2 presents the antioxidant activity of the extracts and BHT. Like the reducing power, high activity was found in the peel extracts and lower activity was found in the seed extracts. The methanolic and aqueous fractions of the peel showed potent antioxidative activity with no statistical difference (P > 0.05) when compared to the antioxidant standard, BHT. The total antioxidant activity by lipid peroxidation based on ferric thiocyanate method was also evaluated. The peel extracts still showed statistically higher activity than that of the seed extracts (P < 0.05). The antioxidant activity (IC50) of the peel extracts is ranked as follows: methanolic fraction (0.46 mg/mL) > aqueous fraction (0.55 mg/mL) > ether fraction (0.81 mg/mL) (Fig. 3).
a
a
a
PE
PM
PA
a SE
SM
SA
BHT
Fig. 3. Antioxidant activity of Nephelium lappaceum L. extracts in the lipid peroxidation method. PE, PM, PA are the ether, methanolic and aqueous extracts of the peel; SE, SM, SA are the ether, methanolic and aqueous extracts of the seed; BHT, butylated hydroxytoluene. aed means the column followed by different letters are significantly different (P < 0.05).
The free radical scavenging capacity of the extracts against common free radicals (DPPH ) in vitro were further determined. Fig. 4 shows the dose-response curve of DPPH scavenging activities of the extracts. The results indicated that the peel extracts exhibited a potential free radical scavenging activity. The half inhibition concentration (IC50) of the radical scavenging activity of the peel extracts were calculated and are illustrated in Table 2. The results revealed that the extract with the highest effective radical scavenging activity was the methanolic fraction (4.94 mg/mL), followed by the aqueous extract (9.67 mg/mL) and the ether extract (17.3 mg/mL), while lower activities were found in the seed extracts (>400 mg/mL, data not shown). BHT and ascorbic acid revealed less effective activities (P < 0.05) than the methanolic extract (0.32 mg extract/mg reference compounds) and the aqueous extract (0.64 mg extract/mg reference compounds), while quercetin showed stronger radical scavenging activity than these two extracts (>1 mg extract/mg quercetin). The ether fraction
1400 100
scavenging activity (%)
d
1200
IC50 ( g/mL)
1000 800 600 400 c b
200 a
a
a
PE
PM
PA
a
0 SE
SM
SA
BHT
80 60 40 20 0 0
10
20
30
40
50
60
concentration ( g/mL) Fig. 2. Antioxidant activity of Nephelium lappaceum L. extracts in the bcarotene/linoleic acid system. PE, PM, PA are the ether, methanolic and aqueous extracts of the peel; SE, SM, SA are the ether, methanolic and aqueous extracts of the seed; BHT, butylated hydroxytoluene. aed means the column followed by different letters are significantly different (P < 0.05).
Fig. 4. Percent free radical scavenging activity of Nephelium lappaceum L. extracts. (,), (-), (:) are the ether, methanolic and aqueous extracts of the peel; (), (6), (C) are the ether, methanolic and aqueous extracts of the seed and (B) is BHT, butylated hydroxytoluene.
N. Thitilertdecha et al. / LWT - Food Science and Technology 41 (2008) 2029e2035 Table 2 The 50% inhibition concentrations (IC50) for DPPH radical scavenging activity of Nephelium lappaceum L. peel extracts and comparison with the four reference antioxidant compounds: BHT, quercetin, a-tocopherol, and ascorbic acid
Extracts
Ether Methanol Aqueous
IC50 (mg/mL) 17.3 1.03 4.94 0.26 9.67 0.87
Table 4 The Minimum inhibition concentration (MIC) values of the extracts of Nephelium lappaceum L. peel extract Microorganism
Quercetin
Ascorbic acid
1.14 0.10 0.32 0.01 0.64 0.07
22.0 0.87 6.31 0.91 12.3 1.07
1.14 0.09 0.32 0.05 0.64 0.08
Values are expressed as means S.D. BHT; butylated hydroxytoluene.
showed the lowest activity among peel extracts and antioxidant standards. According to these results, the antioxidant activity increased proportionally with the phenolic content. Correlations between phenolic content and antioxidant activity were investigated. There was a substantial correlation between the phenolic content versus reducing power (R2 ¼ 0.96), antioxidant activity as inhibition of b-carotene bleaching (R2 ¼ 0.61), antioxidant activity as thiocyanate method (R2 ¼ 0.68) and free radical scavenging activity (R2 ¼ 0.96). Thus, it can be noted that the strong antioxidant properties may be attributed to the phenolic components in the extracts. The significant correlation between the phenolic content and the antioxidant activity of various vegetable extracts has been previously observed (Velioglu, Mazza, Gao, & Oomah, 1998). There are many types of compounds possessing antioxidant activity in higher plants (Larson, 1988) and the phenolic compounds were highlighted to be the potential antioxidants (Yu et al., 2005). The fact that phenolic compounds possess a high potential to scavenge radicals can be explained by their ability to donate a hydrogen atom from their phenolic hydroxyl groups (Sawa, Nakao, Akaike, Ono, & Maeda, 1999). The inhibitory effects on lipid peroxidation and autoxidation of linoleic acid have been attributed to the radical scavenging activity (Hatano et al., 2002). The antibacterial activity of the peel and seed extracts of N. lappaceum L. were determined against eight strains of
MIC value Ether extract (mg/mL)
Methanol extract (mg/mL)
Aqueous extract (mg/mL)
Gram negative bacteria Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Salmonella typhi Vibrio cholerae
ND ND ND ND 62.5
ND ND 62.5 ND 15.6
ND ND 125 ND 15.6
Gram positive bacteria Enterococcus faecalis Staphylococcus aureus Staphylococcus epidermidis
62.5 31.2 31.2
15.6 31.2 2.0
62.5 31.2 15.6
mg Dry extract/mg reference compounds BHT
2033
ND; not determined.
pathogenic bacteria as shown in Table 3. The solvents used for control and all rambutan seed extracts did not show any activity (the results not shown). As shown in Table 3, all rambutan peel extracts exhibited a potent activity against the tested bacteria except E. coli, K. pneumoniae and S. typhi, and only the ether extract did not inhibit the growth of P. aeruginosa. To compare the sensitivity of the bacterial strains to the extracts, the extracts that exhibited antibacterial activity in disc diffusion assay were submitted to the minimum inhibition concentration (MIC) test. The MIC values are illustrated in Table 4. There were substantial differences between the MICs of various extracts. The methanolic fraction had the most effective antibacterial activity. The bacterium S. epidermidis was the most sensitive strain to the methanolic extracts of N. lappaceum peel (MIC 2.0 mg/mL). These results verify the earlier studies that methanol is the better solvent for more consistent extraction of antimicrobial substances compared to the other solvents (Ahmad, Mehmood, & Mohammad, 1998; Natarajan et al., 2005). Similar to antioxidant activity, only fractions containing a high phenolic content of the peel extracts exhibited the antibacterial activity. Phenolic compounds have also been reported
Table 3 Antimicrobial activity of the extracts of Nephelium lappaceum L. peel (2.5 mg/disc) against the tested microorganisms based on disc diffusion method Microorganism
Inhibition zone in diameter (mm) Ether
Methanol
Aqueous
Streptomycin
Gram negative bacteria Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Salmonella typhi Vibrio cholerae
e e e e 12.25 1.31
e e 7.50 0.00 e 16.75 0.62
e e 7.75 0.85 e 17.25 0.62
16.00 0.47 15.00 1.08 13.00 0.71 16.25 0.24 17.75 0.47
Gram positive bacteria Enterococcus faecalis Staphylococcus aureus Staphylococcus epidermidis
7.50 0.41 7.00 0.29 10.00 0.20
11.75 0.47 8.50 0.82 16.50 0.83
7.25 0.62 7.50 0.53 13.25 0.34
7.25 0.47 18.00 0.82 18.25 1.25
Values are means S.D (mm) of three separate experiments; ‘‘e’’, no inhibition zone.
2034
N. Thitilertdecha et al. / LWT - Food Science and Technology 41 (2008) 2029e2035
to be responsible for antimicrobial properties. Penna et al. (2001) isolated two antibacterial phenolic compounds, methyl gallate (MIC 128 mg/mL) and protocatechuic acid (MIC 128 mg/mL) from Sebastiania brasiliensis. Seven antibacterial phenolic compounds, viz. ethyl gallate, caffeic acid, dihydrokaempferol, eriodictyol, quercetin, 3,30 ,50 ,5,7-pentahydroflavanone and ()-epicatechin were isolated from Gleditsia sinensis Lam. (Zhou, Li, Wang, Liu, & Wu, 2007). Thus, it may be concluded that the phenolic compounds in the N. lappaceum extracts could be the main components which possess the antioxidant and antibacterial properties. 4. Conclusion This study has demonstrated the antioxidant and antibacterial activities of various extracts from N. lappaceum L. peel and seed parts. More potential activities were found in the peel extracts than the seed extracts. Methanol was a better solvent for extraction of antioxidant and antibacterial substances compared to the other solvents by providing high extraction yields and also strong antioxidant and antibacterial activities. The activities determined in the extracts could be attributed to the phenolic components. Thus, the N. lappaceum L. peel can be considered as an easily accessible source of natural antioxidants and antibacterial agents. We are continuing our efforts to identify the antioxidative and antibacterial phenolic compounds in the methanolic fraction of N. lappaceum L. peel by further fractionation and analysis. Acknowledgement The authors thank Mr. James F. Maxwell, Department of Biology, Faculty of Science, Chiang Mai University, for the plant identification. This research was financially supported by the Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph. D. Program, and from the Department of Chemistry, Faculty of Science and Graduate School, Chiang Mai University, Thailand. References Ahmad, I., Mehmood, Z., & Mohammad, E. (1998). Screening of some Indian medicinal plants for their antimicrobial properties. Journal of Ethnopharmacology, 62, 183e193. Anagnostopoulou, M. A., Kefalas, P., Papageorgiou, V. P., Assimopoulou, A. N., & Boskou, D. (2006). Radical scavenging activity of various extracts and fractions of sweet orange peel (Citrus sinensis). Food Chemistry, 94, 19e25. Barlow, S. M. (1990). Toxicological aspects of antioxidants used as food additives. In B. J. F. Hudson (Ed.), Food antioxidants. London, New York: Elsevier Applied Science. Bauer, A. W., Kirby, W. M. M., Sherris, J. C., & Truck, M. (1966). Antibiotic susceptibility testing by a standardized single disc method. American Journal of Clinical Pathology, 45, 493e496. Chanwitheesuk, A., Teerawutgulrag, A., & Rakariyatham, N. (2005). Screening of antioxidant activity and antioxidant compounds of some edible plants of Thailand. Food Chemistry, 92, 491e497. Cook, N. C., & Samman, S. (1996). Flavonoids-chemistry, metabolism, cardioprotective effects, and dietary sources. Journal of Nutritional Biochemistry, 7, 66e76.
Croft, K. D. (1999). Antioxidant effects of plant phenolic compounds. In T. K. Basu, N. J. Temple, & M. L. Garg (Eds.), Antioxidants in human health and disease (pp. 109e112). New York: CABI Publishing. _ Oktay, M., Kırec¸cı, E., & Ku¨frevıo˘glu, O ¨ . I_ (2003). Screening of Gu¨lc¸ın, I., antioxidant and antimicrobial activities of anise (Pimpinella anisum L.) seed extracts. Food Chemistry, 83, 371e382. Halliwell, B. (1996). Antioxidants in human health and disease. Annual Review of Nutrition, 16, 33e50. Halliwell, B., & Gutteridge, J. M. C. (1999). Free radicals in biology and medicine. Oxford: Oxford University Press. pp. 27e56. Hatano, T., Miyatake, H., Natsume, M., Osakabe, N., Takizawa, T., & Ito, H., et al. (2002). Proanthocyanidin glycosides and related polyphenols from cacao liquor and their antioxidant effects. Phytochemistry, 59, 749e758. Jayaprakasha, G. K., Selvi, T., & Sakariah, K. K. (2003). Antibacterial and antioxidant activities of grape (Vitis vinisfera) seed extracts. Food Research International, 36, 117e122. Jayaprakasha, G. K., Singh, R. P., & Sakariah, K. K. (2001). Antioxidant activity of grape seed (Vitis vinifera) extracts on peroxidation models in vitro. Food Chemistry, 73, 285e290. Kabuki, T., Nakajima, H., Arai, M., Ueda, S., Kuwabara, Y., & Dosako, S. (2000). Characterization of novel antimicrobial compounds from mango (Mangifera indica L.) kernel seeds. Food Chemistry, 71, 61e66. Larson, R. A. (1988). The antioxidant of higher plants. Phytochemistry, 27, 969e978. Meir, S., Kanner, J., Akiri, B., & Hadas, S. P. (1995). Determination and involvement of aqueous reducing compounds in oxidative defense systems of various senescing leaves. Journal of Agricultural and Food Chemistry, 43, 1813e1819. Nakamura, C. V., Ueda-Nakamura, T., Bando, E., Melo, A. F. N., Cortez, D. A. G., & Filho, B. P. D. (1999). Antibacterial activity of Ocimum gratissimun L. essential oil. Memo´rias do Instituto Oswaldo Cruz, 94(5), 675e678. Natarajan, D., Britto, S. J., Srinivasan, K., Nagamurugan, N., Mohanasundari, C., & Perumal, G. (2005). Anti-bacterial activity of Euphorbia fusiformis e a rare medicinal herb. Journal of Ethnopharmacology, 102, 123e126. Negro, C., Tommasi, L., & Miceli, A. (2003). Phenolic compounds and antioxidant activity from red grape marc extracts. Bioresource Technology, 87, 41e44. Penna, C., Marino, S., Vivot, E., Cruanˇes, M. C., Munˇoz, J.de. D., Cruanˇes, J., & Ferraro, G., et al. (2001). Antimicrobial activity of Argentine plants used in the treatment of infectious diseases: isolation of active compounds from Sebastiania brasiliensis. Journal of Ethnopharmacology, 77, 37e40. Pratt, D. E. (1992). Natural antioxidants from plant material. In I. M. T. Huang, C. T. Ho, & C. Y. Lee (Eds.), Phenolic compounds in food and their effects on health (pp. 54e72). New York: American Chemical Society. Ren, W., Qiao, Z., Wang, H., Zhu, L., & Zhang, L. (2003). Flavonoids: promising anticancer agents. Medicinal Research Reviews, 23, 519e534. Sawa, T., Nakao, M., Akaike, T., Ono, K., & Maeda, H. (1999). Alkylperoxyl radical-scavenging activity of various flavonoids and other phenolic compounds: implications for the anti-tumor-promotor effect of vegetables. Journal of Agricultural and Food Chemistry, 47, 397e402. Singh, R. P., Murthy, K. N. C., & Jayaprakasha, G. K. (2002). Studies on the antioxidant activity of pomegranate (Punica granatum) peel and seed extracts using in vitro models. Journal of Agricultural and Food Chemistry, 50, 81e86. Tapiero, H., Tew, K. D., Ba, G. N., & Mathe´, G. (2002). Polyphenol: do they play a role in the prevention of human pathologies? Biomedicine & Pharmacotherapy, 56, 200e207. Velioglu, Y. S., Mazza, G., Gao, L., & Oomah, B. D. (1998). Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. Journal of Agricultural and Food Chemistry, 46, 4113e4117. Waterman, P. G., & Mole, S. (1994). Analysis of phenolic plant metabolites. Oxford: Blackwell Scientific Publications. p. 84.
N. Thitilertdecha et al. / LWT - Food Science and Technology 41 (2008) 2029e2035 Yen, G.-C., & Chen, H.-Y. (1995). Antioxidant activity of various tea extracts in relation to their antimutagenicity. Journal of Agricultural and Food Chemistry, 43, 27e32. Yildirim, A., Mavi, A., & Kara, A. A. (2001). Determination of antioxidant and antimicrobial activities of Rumex crispus L. extracts. Journal of Agricultural and Food Chemistry, 49, 4083e4089.
2035
Yu, J., Wang, L., Walzem, R. L., Miller, E. G., Pike, L. M., & Patil, B. S. (2005). Antioxidant activity of citrus limonoids, flavonoids, and coumarins. Journal of Agricultural and Food Chemistry, 53, 2009e2014. Zhou, L., Li, D., Wang, J., Liu, Y., & Wu, J. (2007). Antibacterial phenolic compounds from the spines of Gleditsia sinensis Lam. Natural Product Research, 21, 283e291.
LWT - Food Science and Technology 41 (2008) 2029e2035 www.elsevier.com/locate/lwt
Antioxidant and antibacterial activities of Nephelium lappaceum L. extracts Nont Thitilertdecha a, Aphiwat Teerawutgulrag b, Nuansri Rakariyatham b,* a b
Division of Biotechnology, Graduate School, Chiang Mai University, Chiang Mai 50200, Thailand Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand Received 27 June 2007; received in revised form 25 January 2008; accepted 30 January 2008
Abstract Ether, methanolic and aqueous extracts of lyophilized rambutan (Nephelium lappaceum L.) peels and seeds were evaluated for phenolic contents, antioxidant and antibacterial activities. High amounts of phenolic compounds were found in the peel extracts and the highest content was in the methanolic fraction (542.2 mg/g dry extract). Several potential antioxidant activities, including reducing power, b-carotene bleaching, linoleic peroxidation and free radical scavenging activity, were evaluated. The peel extracts exhibited higher antioxidant activity than the seed extracts in all methods determined (P < 0.05). The methanolic fraction was found to be the most active antioxidant as shown by their 50% DPPH inhibition concentration, 4.94 mg/mL. The results indicated this fraction exhibited greater DPPH radical scavenging activity than BHT and ascorbic acid (0.32 g dry extract/g BHT or ascorbic acid). Antibacterial activity against eight bacterial strains was assessed by disc diffusion and broth macrodilution methods. All peel extracts exhibited antibacterial activity against five pathogenic bacteria. The most sensitive strain, Staphylococcus epidermidis, was inhibited by the methanolic extract (MIC 2.0 mg/mL). Ó 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
Keywords: Nephelium lappaceum; Antioxidant; Antibacterial; Phenolics
1. Introduction Reactive oxygen species (ROS) generated from both living organisms and exogenous sources (Halliwell & Gutteridge, 1999), initiate reactions which damage biological molecules and also play an important causative role in disease initiation (Croft, 1999; Halliwell, 1996). Lipid oxidation, caused by free radicals, is one of the major factors for the deterioration of food products during processing and storage. Effective synthetic antioxidants such as butylated hydroxytoluene (BHT) have been used for industrial processing but these are suspected of being responsible for liver damage and carcinogenesis (Barlow, 1990). Recently, there is an increasing interest in finding natural antioxidants from plant materials to replace synthetic ones.
* Corresponding author. Tel.: þ66 53 943341; fax: þ66 53 892277. E-mail address: [email protected] (N. Rakariyatham).
Plants contain a large variety of substances possessing antioxidant activity, such as vitamin C, vitamin E, carotenes, xanthophylls, tannins and phenolics (Chanwitheesuk, Teerawutgulrag, & Rakariyatham, 2005). Sources of natural antioxidants are primarily plant phenolics that can be found in all parts of the plant (Pratt, 1992). The plant phenolic compounds, such as flavonoids exhibit antioxidant properties due to their high redox potential (Cook & Samman, 1996). They also exhibit a wide range of biological activity, antimicrobial activity, anticarcinogenicity and antiproliferation, and many biological activities can be attributed to their antioxidant properties (Ren, Qiao, Wang, Zhu, & Zhang, 2003; Tapiero, Tew, Ba, & Mathe´, 2002). Interestingly, recent research has revealed that fruit peels and seeds, such as grape seeds and peels (Jayaprakasha, Selvi, & Sakariah, 2003; Jayaprakasha, Singh, & Sakariah, 2001; Negro, Tommasi, & Miceli, 2003), pomegranate peel (Singh, Murthy, & Jayaprakasha, 2002), sweet orange peel (Anagnostopoulou, Kefalas, Papageorgiou, Assimopoulou, & Boskou,
0023-6438/$34.00 Ó 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2008.01.017
2030
N. Thitilertdecha et al. / LWT - Food Science and Technology 41 (2008) 2029e2035
2006) and mango seed kernel (Kabuki et al., 2000) may potentially possess antioxidant and/or antimicrobial properties. The current study focuses on the possibility of using rambutan peel and seed waste as source of low-cost natural antioxidant and antimicrobial. 2. Materials and methods 2.1. Preparation of extracts Lyophilized peels and seeds of Nephelium lappaceum L. were powdered and then extracted with ether (three times). The residue was then extracted with methanol (three times) and finally with water (three times). After filtration, the combined ether and methanolic extracts were evaporated under vacuum to absolute dryness and the aqueous extract was lyophilized. 2.2. Determination of the total phenolic contents The amounts of phenolic compounds in the extracts were determined according to the method of Waterman and Mole (1994) using FolineCiocalteu method and catechin was used as the standard phenolic compound. The extract solution in appropriate solvent (0.1 mL) was transferred to a volumetric flask containing 7.9 mL of distilled water. After that, 0.5 mL of FolineCiocalteu reagent was added. Three minutes later, 1.5 mL of 200 g/L sodium carbonate solution was added. Subsequently, the shaken mixture was allowed to stand for 2 h at room temperature and then measured at 760 nm. The experiment was carried out in triplicate and the total phenolic compounds were expressed as catechin equivalents. 2.3. Determination of antioxidative activity 2.3.1. Reducing power The reducing power was based on the method described previously by Yildirim, Mavi, and Kara (2001). Different concentrations of extracts and BHT (SigmaeAldrich GmbdH, Germany) (50e200 mg/mL) in 1 mL of methanol were mixed with 2.5 mL of phosphate buffer (0.2 mol/L, pH 6.6) and 2.5 mL of 10 g/L potassium ferricyanide. The mixture was incubated at 50 C for 30 min. An aliquot (2.5 mL) of 100 g/L trichloroacetic acid was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. Finally, 2.5 mL of the upper layer solution was mixed with 2.5 mL of distilled water and 0.5 mL of 1 g/L FeCl3, and the absorbance of the resulting solution was measured at 700 nm. 2.3.2. b-Carotene linoleate model system The antioxidant activity of rambutan peel and seed extracts was determined according to the method of Jayaprakasha et al. (2001). First, 0.2 mg of b-carotene in 1 mL of chloroform, 20 mg of linoleic acid and 200 mg of Tween-40 were mixed. Chloroform was removed using nitrogen gas and the resulting mixture was topped up to 50 mL using oxygenated water and mixed well. Aliquots (5 mL) of the emulsion were pipetted
into different test tubes containing 0.2 mL of extracts in methanol at different concentrations. BHT was used for comparative standard. A control containing 0.2 mL of methanol and 5 mL of the above emulsion was prepared. The tubes were placed at 50 C in the water bath. Absorbance was taken at zero time (t ¼ 0) at 470 nm. Measurement of absorbance was continued until the color of b-carotene disappeared in the control (t ¼ 180 min) at 15 min intervals. A mixture prepared as above without b-carotene served as blank. The experiment was carried out in triplicate. The antioxidant activity of the extracts was evaluated in terms of the bleaching of b-carotene using the following equation: Antioxidant activity ¼ 1 ðA0 At Þ= A00 A0t 100 Where A0 and A00 were the absorbance measured at zero time of the incubation for test sample and control, respectively, and At and A0t were the absorbance measured for the test sample and the control, respectively, after incubation for 180 min. Extract concentration providing 50% inhibition (IC50) was calculated from the graph of inhibition percentage against extract concentration. 2.3.3. Linoleic peroxidation method The antioxidant activity of rambutan peel and seed extracts was based on the thiocyanate method described previously by Jayaprakasha et al. (2001) with slight modifications. The extracts were dissolved in methanol for stock solutions. BHT was used as comparative standard. The solution containing stock extract solution or BHT at various concentrations in 2.5 mL of potassium phosphate buffer (0.04 mol/L, pH 7.0) was transferred to 2.5 mL of linoleic acid emulsion containing 0.28 g Tween-40, 0.28 g linoleic acid dissolved in potassium phosphate buffer (0.04 mol/L, pH 7.0). A control, containing 2.5 mL linoleic acid emulsion and 2.5 mL potassium phosphate buffer (0.04 mol/L, pH 7.0) was prepared. The mixture was incubated at 37 C for 64 h. Aliquots (0.1 mL) were drawn from the incubation mixture at 8-h intervals and then mixed with 5.0 mL of 75% (v/v) ethanol, 0.1 mL of 300 g/L ammonium thiocyanate and 0.1 mL of 20 mmol/L ferrous chloride in 0.96 mol/L HCl and allowed to stand at room temperature for 3 min. The absorbance was measured at 500 nm. The experiments on antioxidant activity were run in triplicate and the percent inhibition of lipid peroxidation was calculated using the following formula: Percent inhibition ¼ 100 ½A1 =A0 100 Where A0 was the absorbance of the control reaction and A1 was the absorbance in the presence of the extracts. The antioxidant activities were illustrated as IC50 values.
2.3.4. DPPH radical scavenging activity The stable free radical scavenging activity was determined by the 1,1-diphenyl-2-picryl-hydracyl (DPPH ) (Sigmae Aldrich GmbdH, Germany) method of Gu¨lc¸ın, Oktay, Kirec¸ci, and Ku¨frevıo˘glu (2003). A 0.1 mmol/L DPPH solution in methanol was prepared, and then 1 mL of this solution was
N. Thitilertdecha et al. / LWT - Food Science and Technology 41 (2008) 2029e2035
mixed with 3 mL of extract at different concentrations (0.78e 400 mg/mL). A control, containing 1 mL of DPPH solution and 3 mL of methanol was prepared. The mixture was incubated at room temperature for 30 min and then the absorbance was measured at 517 nm. The ability to scavenge the DPPH radical was calculated as percent DPPH scavenging using the following equation:
2031
determined as the lowest concentration of the extract that demonstrated no visible growth. 2.6. Statistical analysis
%DPPH scavenging ¼ ½ðA0 A1 Þ=A0 100 Where A0 was the absorbance of the control and A1 was the absorbance of the mixture containing extracts. IC50 of reference antioxidant compounds, BHT, quercetin and ascorbic acid (SigmaeAldrich GmbdH, Germany), were used for comparison to IC50 of the extracts. 2.4. Bacterial cultures The microorganisms used in this study consisted of eight strains of pathogenic bacteria, viz. Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Vibrio cholerae, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis. All bacterial strains were obtained from the Department of Clinical Microbiology, Faculty of Associated Medical Sciences, Chiang Mai University. The bacteria were grown and maintained on nutrient agar slants. The inoculated agar slants were incubated at 37 C. 2.5. Antibacterial assays 2.5.1. Disc diffusion assay The antibacterial activity was based on disc diffusion method (Bauer, Kirby, Sherris, & Truck, 1966) using bacterial cell suspension whose concentration was equilibrated to a 0.5 McFarland standard. A 100 mL of each bacterial suspension was spread on a MuellereHinton agar plate. Sterile paper discs (6 mm diameter) were impregnated with 20 mL of each extract dissolved in the solvent used for extraction at 125 mg/mL. The discs were allowed to dry and then placed on the inoculated agar. Discs with the solvent used for dissolution were used as negative control and 10 mg streptomycin discs were used as positive controls. The plates were incubated at 37 C for 24 h. After incubation time, zone of inhibition was measured. The experiment was performed in triplicate. 2.5.2. Minimum inhibition concentrations (MICs) Minimum inhibition concentrations of the extracts were evaluated for the bacterial strains which were determined as being sensitive to the extracts in the disc diffusion assay. A broth macrodilution method was used, as previously described by Nakamura et al. (1999) with a slight modification. Serial twofold dilutions of each extract were prepared in 10% (v/v) dimethylsulfoxide (DMSO), and 30 mL of each dilution was added to 3 mL of MuellereHinton broth. These were inoculated with 30 mL of culture of the test bacterial strains. After incubation of the cultures at 37 C, the MIC value was
All experimental results were expressed as means S.D. Analysis of variance was performed by ANOVA procedures. Correlation coefficient (R) was used to determine two variables. SPSS software was used for statistical calculations. The results with P < 0.05 were regarded to be statistically significant. 3. Results and discussion In this study, phenolic content, antioxidant activity and antibacterial activity of various N. lappaceum peel and seed extracts were determined. The extraction yields (g/100 g lyophilized rambutan) from various extractants, i.e. ether, methanol and water are presented in Table 1. The total phenolic contents of the extracts were determined by FolineCiocalteu method. The high amounts of phenolic compounds were found in the peel extracts in the following order: methanolic fraction (542.2 mg/g), aqueous fraction (393.2 mg/g) and ether fraction (293.3 mg/g) (Table 1). Although low amounts of phenolic compounds were observed in the seed extracts, the methanolic fraction of seed extract contained the highest phenolic content (58.5 mg/g). The extracts obtained by various solvent extractions were determined for their antioxidant activities. The extracts were investigated for the reductive capabilities by using the potassium ferricyanide reduction method. The reducing ability may serve as a significant indicator of potential antioxidant activity (Meir, Kanner, Akiri, & Hadas, 1995). The peel and seed extracts increased in reducing powers with increasing concentration (Fig. 1) and the peel extracts exhibited higher reducing ability than the seed extracts. The methanolic extract showed the highest activity, followed by the aqueous and ether extracts. When compared to the BHT, the methanolic fraction showed significantly (P < 0.05) higher activity at all concentrations. Yen and Chen (1995) reported that the extract which Table 1 Extraction yields and total phenolic content of Nephelium lappaceum L. extracts Extraction yield (g/100 g)
Total phenolicsf
Peel extracts Ether Methanol Aqueous
2.84 25.1 2.06
293.3 0.5c 542.2 0.0a 393.2 7.4b
Seed extracts Ether Methanol Aqueous
21.2 4.80 7.29
7.4 1.8e 58.5 0.4d 3.3 0.2e
Extracts
Values are expressed as means S.D. aee Means the column followed by different letters are significantly different (P < 0.05). f Express as mg catechin/1 g dry extract.
N. Thitilertdecha et al. / LWT - Food Science and Technology 41 (2008) 2029e2035
2032
300
1.2
d
250
1
IC50 ( g/mL)
absorbance at 700 nm
1.4
0.8 0.6 0.4 0.2
200 150 c 100 50
b
0 0
50
100
150
200
0
concentration ( g/mL) Fig. 1. Reducing powers of Nephelium lappaceum L. extracts. (,), (-), (:) are the ether, methanolic and aqueous extracts of the peel; (), (6), (C) are the ether, methanolic and aqueous extracts of the seed and (B) is BHT, butylated hydroxytoluene.
showed a reducing power could function as an electron donor and also could reduce the oxidized intermediates generated from the lipid peroxidation reaction. For their antioxidant activity, the extracts were determined for their capability to delay b-carotene bleaching in the coupled oxidation of b-carotene/linoleic acid model system. Fig. 2 presents the antioxidant activity of the extracts and BHT. Like the reducing power, high activity was found in the peel extracts and lower activity was found in the seed extracts. The methanolic and aqueous fractions of the peel showed potent antioxidative activity with no statistical difference (P > 0.05) when compared to the antioxidant standard, BHT. The total antioxidant activity by lipid peroxidation based on ferric thiocyanate method was also evaluated. The peel extracts still showed statistically higher activity than that of the seed extracts (P < 0.05). The antioxidant activity (IC50) of the peel extracts is ranked as follows: methanolic fraction (0.46 mg/mL) > aqueous fraction (0.55 mg/mL) > ether fraction (0.81 mg/mL) (Fig. 3).
a
a
a
PE
PM
PA
a SE
SM
SA
BHT
Fig. 3. Antioxidant activity of Nephelium lappaceum L. extracts in the lipid peroxidation method. PE, PM, PA are the ether, methanolic and aqueous extracts of the peel; SE, SM, SA are the ether, methanolic and aqueous extracts of the seed; BHT, butylated hydroxytoluene. aed means the column followed by different letters are significantly different (P < 0.05).
The free radical scavenging capacity of the extracts against common free radicals (DPPH ) in vitro were further determined. Fig. 4 shows the dose-response curve of DPPH scavenging activities of the extracts. The results indicated that the peel extracts exhibited a potential free radical scavenging activity. The half inhibition concentration (IC50) of the radical scavenging activity of the peel extracts were calculated and are illustrated in Table 2. The results revealed that the extract with the highest effective radical scavenging activity was the methanolic fraction (4.94 mg/mL), followed by the aqueous extract (9.67 mg/mL) and the ether extract (17.3 mg/mL), while lower activities were found in the seed extracts (>400 mg/mL, data not shown). BHT and ascorbic acid revealed less effective activities (P < 0.05) than the methanolic extract (0.32 mg extract/mg reference compounds) and the aqueous extract (0.64 mg extract/mg reference compounds), while quercetin showed stronger radical scavenging activity than these two extracts (>1 mg extract/mg quercetin). The ether fraction
1400 100
scavenging activity (%)
d
1200
IC50 ( g/mL)
1000 800 600 400 c b
200 a
a
a
PE
PM
PA
a
0 SE
SM
SA
BHT
80 60 40 20 0 0
10
20
30
40
50
60
concentration ( g/mL) Fig. 2. Antioxidant activity of Nephelium lappaceum L. extracts in the bcarotene/linoleic acid system. PE, PM, PA are the ether, methanolic and aqueous extracts of the peel; SE, SM, SA are the ether, methanolic and aqueous extracts of the seed; BHT, butylated hydroxytoluene. aed means the column followed by different letters are significantly different (P < 0.05).
Fig. 4. Percent free radical scavenging activity of Nephelium lappaceum L. extracts. (,), (-), (:) are the ether, methanolic and aqueous extracts of the peel; (), (6), (C) are the ether, methanolic and aqueous extracts of the seed and (B) is BHT, butylated hydroxytoluene.
N. Thitilertdecha et al. / LWT - Food Science and Technology 41 (2008) 2029e2035 Table 2 The 50% inhibition concentrations (IC50) for DPPH radical scavenging activity of Nephelium lappaceum L. peel extracts and comparison with the four reference antioxidant compounds: BHT, quercetin, a-tocopherol, and ascorbic acid
Extracts
Ether Methanol Aqueous
IC50 (mg/mL) 17.3 1.03 4.94 0.26 9.67 0.87
Table 4 The Minimum inhibition concentration (MIC) values of the extracts of Nephelium lappaceum L. peel extract Microorganism
Quercetin
Ascorbic acid
1.14 0.10 0.32 0.01 0.64 0.07
22.0 0.87 6.31 0.91 12.3 1.07
1.14 0.09 0.32 0.05 0.64 0.08
Values are expressed as means S.D. BHT; butylated hydroxytoluene.
showed the lowest activity among peel extracts and antioxidant standards. According to these results, the antioxidant activity increased proportionally with the phenolic content. Correlations between phenolic content and antioxidant activity were investigated. There was a substantial correlation between the phenolic content versus reducing power (R2 ¼ 0.96), antioxidant activity as inhibition of b-carotene bleaching (R2 ¼ 0.61), antioxidant activity as thiocyanate method (R2 ¼ 0.68) and free radical scavenging activity (R2 ¼ 0.96). Thus, it can be noted that the strong antioxidant properties may be attributed to the phenolic components in the extracts. The significant correlation between the phenolic content and the antioxidant activity of various vegetable extracts has been previously observed (Velioglu, Mazza, Gao, & Oomah, 1998). There are many types of compounds possessing antioxidant activity in higher plants (Larson, 1988) and the phenolic compounds were highlighted to be the potential antioxidants (Yu et al., 2005). The fact that phenolic compounds possess a high potential to scavenge radicals can be explained by their ability to donate a hydrogen atom from their phenolic hydroxyl groups (Sawa, Nakao, Akaike, Ono, & Maeda, 1999). The inhibitory effects on lipid peroxidation and autoxidation of linoleic acid have been attributed to the radical scavenging activity (Hatano et al., 2002). The antibacterial activity of the peel and seed extracts of N. lappaceum L. were determined against eight strains of
MIC value Ether extract (mg/mL)
Methanol extract (mg/mL)
Aqueous extract (mg/mL)
Gram negative bacteria Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Salmonella typhi Vibrio cholerae
ND ND ND ND 62.5
ND ND 62.5 ND 15.6
ND ND 125 ND 15.6
Gram positive bacteria Enterococcus faecalis Staphylococcus aureus Staphylococcus epidermidis
62.5 31.2 31.2
15.6 31.2 2.0
62.5 31.2 15.6
mg Dry extract/mg reference compounds BHT
2033
ND; not determined.
pathogenic bacteria as shown in Table 3. The solvents used for control and all rambutan seed extracts did not show any activity (the results not shown). As shown in Table 3, all rambutan peel extracts exhibited a potent activity against the tested bacteria except E. coli, K. pneumoniae and S. typhi, and only the ether extract did not inhibit the growth of P. aeruginosa. To compare the sensitivity of the bacterial strains to the extracts, the extracts that exhibited antibacterial activity in disc diffusion assay were submitted to the minimum inhibition concentration (MIC) test. The MIC values are illustrated in Table 4. There were substantial differences between the MICs of various extracts. The methanolic fraction had the most effective antibacterial activity. The bacterium S. epidermidis was the most sensitive strain to the methanolic extracts of N. lappaceum peel (MIC 2.0 mg/mL). These results verify the earlier studies that methanol is the better solvent for more consistent extraction of antimicrobial substances compared to the other solvents (Ahmad, Mehmood, & Mohammad, 1998; Natarajan et al., 2005). Similar to antioxidant activity, only fractions containing a high phenolic content of the peel extracts exhibited the antibacterial activity. Phenolic compounds have also been reported
Table 3 Antimicrobial activity of the extracts of Nephelium lappaceum L. peel (2.5 mg/disc) against the tested microorganisms based on disc diffusion method Microorganism
Inhibition zone in diameter (mm) Ether
Methanol
Aqueous
Streptomycin
Gram negative bacteria Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Salmonella typhi Vibrio cholerae
e e e e 12.25 1.31
e e 7.50 0.00 e 16.75 0.62
e e 7.75 0.85 e 17.25 0.62
16.00 0.47 15.00 1.08 13.00 0.71 16.25 0.24 17.75 0.47
Gram positive bacteria Enterococcus faecalis Staphylococcus aureus Staphylococcus epidermidis
7.50 0.41 7.00 0.29 10.00 0.20
11.75 0.47 8.50 0.82 16.50 0.83
7.25 0.62 7.50 0.53 13.25 0.34
7.25 0.47 18.00 0.82 18.25 1.25
Values are means S.D (mm) of three separate experiments; ‘‘e’’, no inhibition zone.
2034
N. Thitilertdecha et al. / LWT - Food Science and Technology 41 (2008) 2029e2035
to be responsible for antimicrobial properties. Penna et al. (2001) isolated two antibacterial phenolic compounds, methyl gallate (MIC 128 mg/mL) and protocatechuic acid (MIC 128 mg/mL) from Sebastiania brasiliensis. Seven antibacterial phenolic compounds, viz. ethyl gallate, caffeic acid, dihydrokaempferol, eriodictyol, quercetin, 3,30 ,50 ,5,7-pentahydroflavanone and ()-epicatechin were isolated from Gleditsia sinensis Lam. (Zhou, Li, Wang, Liu, & Wu, 2007). Thus, it may be concluded that the phenolic compounds in the N. lappaceum extracts could be the main components which possess the antioxidant and antibacterial properties. 4. Conclusion This study has demonstrated the antioxidant and antibacterial activities of various extracts from N. lappaceum L. peel and seed parts. More potential activities were found in the peel extracts than the seed extracts. Methanol was a better solvent for extraction of antioxidant and antibacterial substances compared to the other solvents by providing high extraction yields and also strong antioxidant and antibacterial activities. The activities determined in the extracts could be attributed to the phenolic components. Thus, the N. lappaceum L. peel can be considered as an easily accessible source of natural antioxidants and antibacterial agents. We are continuing our efforts to identify the antioxidative and antibacterial phenolic compounds in the methanolic fraction of N. lappaceum L. peel by further fractionation and analysis. Acknowledgement The authors thank Mr. James F. Maxwell, Department of Biology, Faculty of Science, Chiang Mai University, for the plant identification. This research was financially supported by the Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph. D. Program, and from the Department of Chemistry, Faculty of Science and Graduate School, Chiang Mai University, Thailand. References Ahmad, I., Mehmood, Z., & Mohammad, E. (1998). Screening of some Indian medicinal plants for their antimicrobial properties. Journal of Ethnopharmacology, 62, 183e193. Anagnostopoulou, M. A., Kefalas, P., Papageorgiou, V. P., Assimopoulou, A. N., & Boskou, D. (2006). Radical scavenging activity of various extracts and fractions of sweet orange peel (Citrus sinensis). Food Chemistry, 94, 19e25. Barlow, S. M. (1990). Toxicological aspects of antioxidants used as food additives. In B. J. F. Hudson (Ed.), Food antioxidants. London, New York: Elsevier Applied Science. Bauer, A. W., Kirby, W. M. M., Sherris, J. C., & Truck, M. (1966). Antibiotic susceptibility testing by a standardized single disc method. American Journal of Clinical Pathology, 45, 493e496. Chanwitheesuk, A., Teerawutgulrag, A., & Rakariyatham, N. (2005). Screening of antioxidant activity and antioxidant compounds of some edible plants of Thailand. Food Chemistry, 92, 491e497. Cook, N. C., & Samman, S. (1996). Flavonoids-chemistry, metabolism, cardioprotective effects, and dietary sources. Journal of Nutritional Biochemistry, 7, 66e76.
Croft, K. D. (1999). Antioxidant effects of plant phenolic compounds. In T. K. Basu, N. J. Temple, & M. L. Garg (Eds.), Antioxidants in human health and disease (pp. 109e112). New York: CABI Publishing. _ Oktay, M., Kırec¸cı, E., & Ku¨frevıo˘glu, O ¨ . I_ (2003). Screening of Gu¨lc¸ın, I., antioxidant and antimicrobial activities of anise (Pimpinella anisum L.) seed extracts. Food Chemistry, 83, 371e382. Halliwell, B. (1996). Antioxidants in human health and disease. Annual Review of Nutrition, 16, 33e50. Halliwell, B., & Gutteridge, J. M. C. (1999). Free radicals in biology and medicine. Oxford: Oxford University Press. pp. 27e56. Hatano, T., Miyatake, H., Natsume, M., Osakabe, N., Takizawa, T., & Ito, H., et al. (2002). Proanthocyanidin glycosides and related polyphenols from cacao liquor and their antioxidant effects. Phytochemistry, 59, 749e758. Jayaprakasha, G. K., Selvi, T., & Sakariah, K. K. (2003). Antibacterial and antioxidant activities of grape (Vitis vinisfera) seed extracts. Food Research International, 36, 117e122. Jayaprakasha, G. K., Singh, R. P., & Sakariah, K. K. (2001). Antioxidant activity of grape seed (Vitis vinifera) extracts on peroxidation models in vitro. Food Chemistry, 73, 285e290. Kabuki, T., Nakajima, H., Arai, M., Ueda, S., Kuwabara, Y., & Dosako, S. (2000). Characterization of novel antimicrobial compounds from mango (Mangifera indica L.) kernel seeds. Food Chemistry, 71, 61e66. Larson, R. A. (1988). The antioxidant of higher plants. Phytochemistry, 27, 969e978. Meir, S., Kanner, J., Akiri, B., & Hadas, S. P. (1995). Determination and involvement of aqueous reducing compounds in oxidative defense systems of various senescing leaves. Journal of Agricultural and Food Chemistry, 43, 1813e1819. Nakamura, C. V., Ueda-Nakamura, T., Bando, E., Melo, A. F. N., Cortez, D. A. G., & Filho, B. P. D. (1999). Antibacterial activity of Ocimum gratissimun L. essential oil. Memo´rias do Instituto Oswaldo Cruz, 94(5), 675e678. Natarajan, D., Britto, S. J., Srinivasan, K., Nagamurugan, N., Mohanasundari, C., & Perumal, G. (2005). Anti-bacterial activity of Euphorbia fusiformis e a rare medicinal herb. Journal of Ethnopharmacology, 102, 123e126. Negro, C., Tommasi, L., & Miceli, A. (2003). Phenolic compounds and antioxidant activity from red grape marc extracts. Bioresource Technology, 87, 41e44. Penna, C., Marino, S., Vivot, E., Cruanˇes, M. C., Munˇoz, J.de. D., Cruanˇes, J., & Ferraro, G., et al. (2001). Antimicrobial activity of Argentine plants used in the treatment of infectious diseases: isolation of active compounds from Sebastiania brasiliensis. Journal of Ethnopharmacology, 77, 37e40. Pratt, D. E. (1992). Natural antioxidants from plant material. In I. M. T. Huang, C. T. Ho, & C. Y. Lee (Eds.), Phenolic compounds in food and their effects on health (pp. 54e72). New York: American Chemical Society. Ren, W., Qiao, Z., Wang, H., Zhu, L., & Zhang, L. (2003). Flavonoids: promising anticancer agents. Medicinal Research Reviews, 23, 519e534. Sawa, T., Nakao, M., Akaike, T., Ono, K., & Maeda, H. (1999). Alkylperoxyl radical-scavenging activity of various flavonoids and other phenolic compounds: implications for the anti-tumor-promotor effect of vegetables. Journal of Agricultural and Food Chemistry, 47, 397e402. Singh, R. P., Murthy, K. N. C., & Jayaprakasha, G. K. (2002). Studies on the antioxidant activity of pomegranate (Punica granatum) peel and seed extracts using in vitro models. Journal of Agricultural and Food Chemistry, 50, 81e86. Tapiero, H., Tew, K. D., Ba, G. N., & Mathe´, G. (2002). Polyphenol: do they play a role in the prevention of human pathologies? Biomedicine & Pharmacotherapy, 56, 200e207. Velioglu, Y. S., Mazza, G., Gao, L., & Oomah, B. D. (1998). Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. Journal of Agricultural and Food Chemistry, 46, 4113e4117. Waterman, P. G., & Mole, S. (1994). Analysis of phenolic plant metabolites. Oxford: Blackwell Scientific Publications. p. 84.
N. Thitilertdecha et al. / LWT - Food Science and Technology 41 (2008) 2029e2035 Yen, G.-C., & Chen, H.-Y. (1995). Antioxidant activity of various tea extracts in relation to their antimutagenicity. Journal of Agricultural and Food Chemistry, 43, 27e32. Yildirim, A., Mavi, A., & Kara, A. A. (2001). Determination of antioxidant and antimicrobial activities of Rumex crispus L. extracts. Journal of Agricultural and Food Chemistry, 49, 4083e4089.
2035
Yu, J., Wang, L., Walzem, R. L., Miller, E. G., Pike, L. M., & Patil, B. S. (2005). Antioxidant activity of citrus limonoids, flavonoids, and coumarins. Journal of Agricultural and Food Chemistry, 53, 2009e2014. Zhou, L., Li, D., Wang, J., Liu, Y., & Wu, J. (2007). Antibacterial phenolic compounds from the spines of Gleditsia sinensis Lam. Natural Product Research, 21, 283e291.