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In vitro evaluation of antimicrobial and antioxidant activities of some Tunisian vegetables
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South African Journal of Botany 78 (2012) 252 – 256 www.elsevier.com/locate/sajb
In vitro evaluation of antimicrobial and antioxidant activities of some Tunisian vegetables H. Edziri a , S. Ammar c , L. Souad c , M.A. Mahjoub c , M. Mastouri b , M. Aouni a , Z. Mighri c , L. Verschaeve d, e,⁎ a
c
Laboratoire des maladies transmissibles et des substances biologiquement actives, Faculté de Pharmacie-5000-Monastir, Tunisie b Laboratoire de Microbiologie C H U Fattouma Bouguiba-5000-Monastir, Tunisie Laboratoire de chimie des substances naturelles et de synthèse organique 99/UR/12-26, Faculté des sciences de Monastir, 5000 Monastir, Tunisie d Scientific Institute of Public Health, O.D. Epidemiology & Surveillance, Laboratory of Toxicology, Brussels, Belgium e Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium Received 10 May 2011; received in revised form 29 September 2011; accepted 30 September 2011
Abstract The aim of the present study was the investigation of antimicrobial and antioxidant activities of aqueous and methanolic extracts obtained from some Tunisian vegetables. The antimicrobial activity was evaluated by the microdilution method. Total phenolic contents were determined by the Folin–Ciocalteu colorimetric method. The antioxidant activity was evaluated using the DPPH assay. Among tested extracts, the methanolic extract of Apium graveolens had the best antifungal activity against Candida albicans, Candida kreussei and Candida parapsilosis with MIC values ranging between 0.08 and 0.31 mg/ml. Concerning the antioxidant activity we conclude that aqueous extracts of A. graveolens, Solanum melongena and Anethum graveolens showed the best antioxidant activity with IC50 of 20 μg/ml. These results may suggest that methanolic extracts of Solanum tuberosum and A. graveolens possess good antimicrobial activity, and therefore, they can be used in biotechnological fields as natural preservative ingredients in food and/or pharmaceutical industry. © 2011 SAAB. Published by Elsevier B.V. All rights reserved. Keywords: Antimicrobial activity; Antioxidant activity; Total phenolic content; Vegetables
1. Introduction Oxidative damage can result when the critical balance between free radical generation and antioxidant defenses is unfavorable. These oxidative damages could be retarded by endogenous defense systems such as catalase, superoxide dismutase, and the glutathione peroxidase system, but these systems are not completely efficient (Rock et al., 1996). Oxidative damage has been hypothesized to play a key role in cardiovascular disease, cancer initiation, cataract formation, the aging process, inflammatory diseases, and a variety of neurological disorders (Rock et al., ⁎ Corresponding author at: Scientific Institute of Public Health, O.D. Epidemiology & Surveillance, Laboratory of Toxicology, Brussels, Belgium. E-mail address: [email protected] (L. Verschaeve).
1996). The antioxidant defense system has both enzymatic and non-enzymatic components that prevent radical formation, remove radicals before damage can occur, repair oxidative damage, eliminate damaged molecules, and prevent mutations (Gordon, 1996). A diet rich in vegetables and fruit may provide protection against cardiovascular disease (Ness and Powles, 1997), several common cancers (Steinmetz and Potter, 1996). Importance of natural antioxidants for medical and food application has been underlined by numerous works as reported by Spigno and De Faveri (2007). Serdula et al. (1996) showed that the frequency of intake of fresh fruit and vegetables increased as the level of physical activity increased, and that consumption of fruit and vegetables was lower in those who reported that they were sedentary, heavy smokers, or heavy drinkers.
0254-6299/$ - see front matter © 2011 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2011.09.012
H. Edziri et al. / South African Journal of Botany 78 (2012) 252–256
The presence and growth of pathogenic microorganisms (bacteria, mold, viruses, fungi) in food may cause spoilage and result in a reduction in its quality and quantity (Ohshima and Bartsch, 1994). This microbial contamination still poses important public health and economic concerns for the human society. The exploration of naturally occurring antimicrobials for food preservation receives increasing attention due to consumer awareness of natural food products and a growing concern of microbial resistance toward conventional preservatives (Gould, 1995). The aim of the present study was the investigation of the antimicrobial and antioxidant activities of aqueous and methanolic extracts obtained from some Tunisian vegetables. 2. Materials and methods 2.1. Plant material
253
2.3. Determination of total phenolic content Total phenolic content in each extract was determined using Folin–Ciocalteu reagent according to the method of Singleton and Rosi (1965). Forty microliters of extract (1 mg/ml) was mixed with 200 μl Folin–Ciocalteu reagent (Sigma-Aldrich, Germany) and 1160 μl of distilled water, followed by 600 μl 20% sodium carbonate (Na2CO3) 3 min later. The mixture was shaken for 2 h at room temperature and absorbance was measured at 765 nm. All tests were performed in triplicate. Gallic acid (Sigma-Aldrich, Germany) was used as a standard. The concentration of total phenolic compounds (TPC) was determined as μg gallic acid equivalents (GAE)/mg extract using the following equation obtained from a standard gallic acid graph (R 2 = 0.9877): Absorbance ¼ 0:0012 gallic acid ðμgÞ−0:0034
All the vegetables were collected from different locations in Tunisia. They are presented in Table 1. 2.2. Extracts preparation 2.2.1. Aqueous extraction The vegetables were cut into small parts. Distilled water (200 ml) was added to 50 g of each sample in a conical flask. Then they were allowed to boil for 1 h. The extract was filtered using a Whatman no. 1 paper. The extract was lyophilized and it was stored at − 25 °C until use. 2.2.2. Methanolic extraction Each sample (50 g) was incubated in a glass flask with 200 ml of methanol (90%) for 3 days under magnetic stirrer. Solvent was evaporated under vacuum at 70 °C to get crude extracts and it was stored at − 25 °C until use.
2.4. Antibacterial and antifungal activities 2.4.1. Microrganisms The microorganism strains used in the biological assays are listed in Table 1. Different American-Type Cell Culture (ATCC) reference bacteria and fungi as well as clinical isolates including methicillin resistant Staphylococcus aureus (MRSA) strain were used. Their ATCC number is given in the legend of Table 1. 2.4.2. Antibacterial and antifungal screening The minimum inhibitory concentrations (MIC) were determined by the microwell dilution method (Zgoda and Porter, 2001) with some modifications as follows. The inoculum (10 6 cfu/ml) was prepared from 24 h broth cultures and suspensions were adjusted to 0.5 McFarland standard turbidity. All the extracts dissolved in 10% of DMSO were first diluted to
Table 1 Antibacterial activity of vegetable extracts using microdilution test MICa minimal inhibitory concentration; values given as mg/ml; MIC positive control: levofloxacin (E. coli 0.61 μg/ml, P. aeruginosa 0.3 μg/ml, S. aureus 0.3 μg/ml, E. faecalis 1.22 μg/ml). MBCb: minimal bactericidal concentration; values given as mg/ml; Mec methanolic extract, Aqd aqueous extract, E.c: Escherichia coli ATCC 25922; P.a: Pseudomonas aeruginosa ATCC 27950; S.a: Staphylococcus aureus ATCC 25923; E.f: Enterococcus faecalis ATCC 29212. Plant species
E.c Used parts
Raphanus sativus
Fruit
Petroselinum sativum
Leaves
Apium graveolens
Leaves
Beta vulgaris var cicla
Leaves
Solanum melongena
Fruit
Solanum tuberosum
Fruit
Anethum graveolens
Leaves
P.a
E.f
S.a
Extracts
MICa
MBCb
MIC
MBC
MIC
MBC
MIC
MBC
c
2.5 5 1.25 5 1.25 5 1.25 5 5 5 0.312 5 5 5
5 10 5 10 5 10 5 10 10 10 0.625 10 10 10
5 5 2.5 5 5 5 1.25 2.5 5 5 0.312 5 2.5 5
5 10 10 10 10 10 5 5 10 10 0.625 10 10 10
2.5 5 5 5 0.312 5 2.5 1.25 5 5 1.5 5 2.5 5
5 5 10 10 1.25 10 10 2.5 10 10 1.25 10 10 10
2.5 5 5 5 1.25 5 1.25 5 5 5 0.312 5 2.5 5
5 10 10 10 5 10 5 10 10 10 1.25 10 10 10
Me Aqd Me Aq Me Aq Me Aq Me Aq Me Aq Me Aq
H. Edziri et al. / South African Journal of Botany 78 (2012) 252–256
highest concentration (20 mg/ml) to be tested, and then serial two-fold dilution was made in a concentration range from 0.016 to 20 mg/ml in sterile water. The 96-well plates were prepared by dispensing into each well 95 μl of MH broth (bacteria) or Sabouraud dextrose broth (yeast) and 5 μl of the inoculum. A 100 μl from extracts initially prepared at the concentration of 20 mg/ml was added into the first wells. Then, 100 μl from their serial dilutions was transferred into six consecutive wells. The last well containing 195 μl of broth without compound and 5 μl of the inoculum on each strip, was used as negative control. The final volume in each well was 200 μl. Control tests with the solvent DMSO (10%) employed to dissolve the plant extracts were performed for all assays and showed no inhibition of microbial growth. The MIC of each extract was defined as the lowest concentration which inhibited either bacterial or candidal growth, after incubation at 37 °C between 18 and 24 h. The minimal bactericidal concentration (MBC) and the minimal fungicidal concentration (MFC) were determined by subculture on blood agar at 37 °C between 18 and 24 h. Levofloxacin was used as antibacterial positive control, and Amphotericin B for the anticandidal one. 2.5. Antioxidant activity by DPPH assay DPPH radical-scavenging activity was determined by the method of Mitsuda et al. (1966). Briefly, a 0.5 ml aliquot of DPPH methanol solution (25 mg/ml) was added to 0.5 ml sample solution at different concentrations. The mixture was shaken vigorously and allowed to stand at room temperature in the dark for 30 min. Then the absorbance was measured at 517 nm in a spectrophotometer. IC50 value (μg/ml) is the inhibitory concentration at which DPPH radicals were scavenged by 50% and was obtained by interpolation from linear regression analysis. Trolox was used for comparison as positive control. 2.6. Statistical analysis The analytical values of the antimicrobial activity measurement (MICs) represent means of two replicates, done in two different experiments. Data obtained were subject to one-way analysis of variance (ANOVA) and Student t-test analysis. Significance was assumed at p ≤ 0.05 at least. For bacterial counts, data from three independent replicate trials (for each treatment) were pooled and the mean values and standard deviations were determined. Differences between samples were determined by Student's t-test and were considered to be significant when p ≤ 0.05 at least. 3. Results and discussions 3.1. Total phenolic content
and 2. The total phenolic content of aqueous extracts varied from 152.14 to 405.75 mg of catechin equivalent (CE/g) extract. Solanum melongena was found to have the highest phenolic contents (405.75 mg CE/g). Apium graveolens, Anethum graveolens, Beta vulgaris var cicla, Raphanus sativus, Solanum tuberosum and Petroselinum sativum were relatively rich in phenolic compounds with concentrations of 244.65; 237.84; 199.35; 171.94; 163.15; 152.14 mg CE/g. Compared with aqueous extracts, methanolic extracts of A. graveolens, A. graveolens, S. melongena and P. sativum had the highest total phenolic content. 3.2. Antibacterial and antifungal activities The results of the antibacterial and antifungal activities of methanol and aqueous extracts of studied vegetables are summarized in Tables 1 and 2. Aqueous and methanolic extract of all plants were effective in inhibiting the growth of all the tested strains with MICs varied between 0.31 and 10 mg/ml. These two extracts showed also antifungal activity against Candida species. Among all tested extracts those of S. tuberosum had the highest antibacterial activity against all tested bacteria, with MICs values between 1.5 and 0.31 mg/ml. All vegetable extracts showed antifungal activity against all Candida species but methanolic extract of A. graveolens had the best antifungal activity against Candida albicans, Candida kreussei and Candida parapsilosis with MIC values ranging between 0.08 and 0.31 mg/ml (Table 2) and with MFC values ranging between 0.08 and 0.63 mg/ml (Table 2). With regards to the total phenolic content we conclude that these active extracts contained a higher amount of phenols. The observed antibacterial and antifungal activities of the extracts of vegetables might also be attributed to the high quantity of polyphenols, which are known to possess efficient antibacterial activity (Velioglu et al., 1998). Consequently phenolic compounds have been reported to be responsible for antimicrobial properties (Penna et al., 2001). 450
Total phenolic contents (mg CE/g extract)
254
S.m
400 350 300 A.p A.g
250 B.v
200 P.s
S.t
R.s
150 100 50 0
The antioxidant activity of plant extracts has been correlated to their total phenolic content due to their property of scavenging free radicals (Singleton and Rosi, 1965). The TPC was expressed in mg of catechin equivalents (mg CE/g of extract). The total phenolic content of the extracts is given in Figs. 1
Aqueous extracts
Fig. 1. Total phenolic content of aqueous extracts of vegetables; mg CE/g: milligram catechin equivalent per gram extract; P.s: Petroselinum sativum; S.t: Solanum tuberosum; R.s: Raphanus sativus; B.v: Beta vulgaris var cicla; A.g: Anethum graveolens; A.p: Apium graveolens; S.m: Solanum melongena.
Total Phenolic content (mg CE/g extract)
H. Edziri et al. / South African Journal of Botany 78 (2012) 252–256 500 P.s
450
S.m
255
Table 3 IC50 values obtained for the vegetable extracts in DPPH assay Me: methanolic extract, Aq: aqueous extract.
A.g
400 A.p
Plant species
Used parts
Extracts
IC50 (μg/ml)
Raphanus sativus
Fruit
Petroselinum sativum
Leaves
Apium graveolens
Leaves
Beta vulgaris var cicla
Leaves
Solanum melongena
Fruit
Solanum tuberosum
Fruit
Anethum graveolens
Leaves
Trolox
–
Me Aq Me Aq Me Aq Me Aq Me Aq Me Aq Me Aq –
640.2 ± 0.4 10.3 ± 0.2 230.5 ± 0.3 250.1 ± 0.1 30.1 ± 0.4 20.0 ± 0.2 600.1 ± 0.1 20.0 ± 0.3 20.0 ± 0.3 20.0 ± 0.4 800.1 ± 0.2 20.0 ± 0.1 90.5 ± 0.1 20.0 ± 0.2 23.1 ± 0.01
350 300 250 200
R.s
S.t
B.v
150 100 50 0
Methanolic extracts
Fig. 2. Total phenolic content of methanol extracts of vegetables; mg CE/g: milligram catechin equivalent per gram of extract; P.s: Petroselinum sativum; S.t: Solanum tuberosum; R.s: Raphanus sativus; B.v: Beta vulgaris var cicla; A.g: Anethum graveolens; A.p: Apium graveolens; S.m: Solanum melongena.
In addition the obtained results confirmed the earlier studies that methanol is the best solvent for extraction of antimicrobial substances compared with the other solvents (Penna et al., 2001). In previous work we showed that 4-(methylthio)-3-butenyl isothiocyanate (MTBITC) is a principal isothiocyanate (ITC) having pungency in R. sativus for a long period. However, its biological effect was first identified as antimicrobial to Escherichia coli, S. aureus, Saccharomyces cerevisiae, and Aspergillus oryzae in 1982 (Esaki and Onozaki, 1982; Hashem and Saleh, 1999). 3.3. Antioxidant activity by DPPH assay The radical DPPH has a violet color due to the unpaired nitrogen electron and, after reaction with the oxygen atom of a radical scavenger (antioxidant compound) reduced DPPH-H is formed which is yellow. Table 3 shows the IC50 values of the DPPH assay where the results from the various vegetables extract are compared with a Trolox with recognized antioxidant activity.
IC50 values denote the concentration of each sample which required to scavenge 50% free radicals or to prevent lipid peroxidation by 50%. All aqueous extracts except P. sativum showed higher DPPH radical scavenging activity with IC50 ranging from 10.3 to 20 μg/ml. Aqueous extracts of A. graveolens, B. vulgaris var cicla, S. melongena, S. tuberosum and A. graveolens showed the highest antioxidant activity with IC50 of 20 μg/ ml lower than the positive control Trolox. The antioxidant activity of vegetable extracts has been correlated to their content of phenolic components (Velioglu et al., 1998) due to their property of scavenging free radicals. Therefore, it is important to consider the effect of the total phenolic quantity in the antioxidant activity of the vegetable extracts. In previous work, L-tryptophan, and 7-[3-(3,4-dihydroxy4-hydroxymethyl-tetrahydro-furan-2-yloxy)-4,5-dihydroxy-6hydroxymethyl-tetrahydro-pyran-2-yloxy]-5-hydroxy-2-(4hydroxy-3-methoxy-phenyl)-chromen-4-one were isolated
Table 2 Antifungal activity of selected vegetable extracts using microdilution test. C.a: Candida albicans ATCC 90028; C.g: Candida glabrata ATCC 90030; C.k: Candida kreussei ATCC 6258; C.p: Candida parapsilosis ATCC 22019; Me: methanolic extract, Aq; aqueous extract; MICa minimal inhibitory concentration; values given as mg/ml; positive control with Amphotericin B (MFC 0.5 μg/ml). MBCb: minimal fungicidal concentration; values given as mg/ml. Plant species
C.g
C.a
C.k
C.p
Used parts
Extracts
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
Raphanus sativus
Fruit
Petroselinum sativum
Leaves
Apium graveolens
Leaves
Beta vulgaris var cicla
Leaves
Solanum melongena
Fruit
Solanum tuberosum
Fruit
Anethum graveolens
Leaves
Me Aq Me Aq Me Aq Me Aq Me Aq Me Aq Me Aq
5 5 1.25 5 5 5 1.25 2.5 5 5 1.25 5 5 5
5 10 5 10 10 10 5 5 10 10 5 10 5 10
2.5 2.5 1.25 5 0.156 5 2.5 1.25 5 5 1.25 5 5 5
10 2.5 5 10 0.625 10 10 2.5 10 10 5 10 5 10
1.25 2.5 1.25 5 0.078 5 5 2.5 5 5 1.25 1.25 2.5 5
1.25 2.5 5 10 0.078 10 10 1.25 10 10 5 1.25 2.5 10
2.5 5 2.5 5 0.312 5 2.5 1.25 5 5 2.5 5 2.5 5
10 10 10 10 0.625 10 10 2.5 10 10 10 10 5 10
256
H. Edziri et al. / South African Journal of Botany 78 (2012) 252–256
from A. graveolens and they exhibited good antioxidant activity at concentrations of 125 and 250 μg/ml. Tryptophan, a precursor for indole biosynthesis, is well documented for its effect on central nervous system and for its therapeutic use in depression, mania and aggression (Young et al., 1984). 4. Conclusion In conclusion, antioxidant and antimicrobial activities which could be derived from their compounds such as flavonoids, polyphenol compounds and vitamin C. By coupling the results of different assays for evaluating antioxidant activity of these vegetables from different solvents, we can conclude that aqueous extracts showed the best antiradical activity with DPPH assay. Among all tested extracts we conclude that methanolic extract of S. tuberosum had the highest antibacterial activity. This study shows that methanolic extract of A. graveolens had the best antifungal activity against C. albicans, C. kreussei and C. parapsilosis. Moreover development of extraction methods and fractionations of extracts should be carried out in order to further investigate their possible use as food preservatives. This potential could be adopted by the food industry. References Esaki, H., Onozaki, H., 1982. Antimicrobial action of pungent principles in radish root. Eiyo To Shokuryo 35, 207–211. Gordon, M.H., 1996. Dietary antioxidants in disease prevention. Natural Product Research 13, 265–273. Gould, G.W., 1995. Homeostatic mechanisms during food preservation by combined methods. In: Barbosa-Canovas, G., Welti-Chanes, J. (Eds.), Food Preservation by Moisture Control. Lancaster Techromic Publishing Co., Berlin, pp. 87–95. Hashem, F.A., Saleh, M.M., 1999. Antimicrobial components of some Cruciferae plants. Phytotherapy Research 13, 329–332.
Edited by L McGaw
Mitsuda, H., Yasumoto, K., Iwami, K., 1966. Antioxidative action of indole compounds during the autoxidation of linoleic acid. Eiyo To Shokuryo 19, 210–214. Ness, A.R., Powles, J.W., 1997. Dietary habits and mortality in vegetarians and health conscious people. Several uncertainties still exist. British Medical Journal 11, 48–149. Ohshima, H., Bartsch, H., 1994. Quantitative estimation of endogenous nitrosation in humans by monitoring N-nitrosoproline excreted in the urine. Cancer Research 41, 3658–3662. Penna, C., Marino, S., Vivot, E., Cruaňes, M.C., Muňoz, J.D., Cruaňes, J., Ferraro, G., Gutkind, G., Martino, V., 2001. Antimicrobial activity of Argentine plants used in the treatment of infectious diseases. Isolation of active compounds from Sebastiania brasiliensis. Journal of Ethnopharmacology 77, 37–40. Rock, C.L., Jacob, R.A., Bowen, P.E., 1996. Update on the biological characteristics of the antioxidant micronutrients: vitamin C, vitamin E, and the carotenoids. Journal of American Diet Association 96, 693–702. Serdula, M.K., Byers, T., Mokdad, A.H., Simoes, E., Mendlein, J.M., Coates, R.J., 1996. The association between fruit and vegetable intake and chronic disease risk factors. Epidemiology 7, 161–165. Singleton, V.L., Rosi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. American Journal of Enolitic Viticum 16, 144–158. Spigno, G., De Faveri, D.M., 2007. Antioxidants from grape stalks and marc: influence of extraction procedure on yield, purity and antioxidant power of the extracts. Journal of Food Engineering 78, 793–801. Steinmetz, K.A., Potter, J.D., 1996. Vegetables, fruit, and cancer prevention: a review. Journal of American Diet Association 96, 1027–1039. Velioglu, Y.S., Mazza, G., Gao, L., Omah, B.D., 1998. Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. Journal of Agriculture and Food Chemistry 46, 4113–4117. Young, S.N., Chouinard, G., Annable, L., Morand, C., Ervin, F.R., 1984. Progress in tryptophan and serotonin research. In: Schlossberger, H., Kochen, W., Linzen, H., Steinhart, H. (Eds.), The Therapeutic Action of Tryptophan in Depression, Mania and Aggression. Walter de Gruyter & Co., Berlin, pp. 182–186. Zgoda, J.R., Porter, J.R., 2001. A convenient microdilution method for screening natural products against bacteria and fungi. Pharmaceutical Biology 39, 221–225.
South African Journal of Botany 78 (2012) 252 – 256 www.elsevier.com/locate/sajb
In vitro evaluation of antimicrobial and antioxidant activities of some Tunisian vegetables H. Edziri a , S. Ammar c , L. Souad c , M.A. Mahjoub c , M. Mastouri b , M. Aouni a , Z. Mighri c , L. Verschaeve d, e,⁎ a
c
Laboratoire des maladies transmissibles et des substances biologiquement actives, Faculté de Pharmacie-5000-Monastir, Tunisie b Laboratoire de Microbiologie C H U Fattouma Bouguiba-5000-Monastir, Tunisie Laboratoire de chimie des substances naturelles et de synthèse organique 99/UR/12-26, Faculté des sciences de Monastir, 5000 Monastir, Tunisie d Scientific Institute of Public Health, O.D. Epidemiology & Surveillance, Laboratory of Toxicology, Brussels, Belgium e Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium Received 10 May 2011; received in revised form 29 September 2011; accepted 30 September 2011
Abstract The aim of the present study was the investigation of antimicrobial and antioxidant activities of aqueous and methanolic extracts obtained from some Tunisian vegetables. The antimicrobial activity was evaluated by the microdilution method. Total phenolic contents were determined by the Folin–Ciocalteu colorimetric method. The antioxidant activity was evaluated using the DPPH assay. Among tested extracts, the methanolic extract of Apium graveolens had the best antifungal activity against Candida albicans, Candida kreussei and Candida parapsilosis with MIC values ranging between 0.08 and 0.31 mg/ml. Concerning the antioxidant activity we conclude that aqueous extracts of A. graveolens, Solanum melongena and Anethum graveolens showed the best antioxidant activity with IC50 of 20 μg/ml. These results may suggest that methanolic extracts of Solanum tuberosum and A. graveolens possess good antimicrobial activity, and therefore, they can be used in biotechnological fields as natural preservative ingredients in food and/or pharmaceutical industry. © 2011 SAAB. Published by Elsevier B.V. All rights reserved. Keywords: Antimicrobial activity; Antioxidant activity; Total phenolic content; Vegetables
1. Introduction Oxidative damage can result when the critical balance between free radical generation and antioxidant defenses is unfavorable. These oxidative damages could be retarded by endogenous defense systems such as catalase, superoxide dismutase, and the glutathione peroxidase system, but these systems are not completely efficient (Rock et al., 1996). Oxidative damage has been hypothesized to play a key role in cardiovascular disease, cancer initiation, cataract formation, the aging process, inflammatory diseases, and a variety of neurological disorders (Rock et al., ⁎ Corresponding author at: Scientific Institute of Public Health, O.D. Epidemiology & Surveillance, Laboratory of Toxicology, Brussels, Belgium. E-mail address: [email protected] (L. Verschaeve).
1996). The antioxidant defense system has both enzymatic and non-enzymatic components that prevent radical formation, remove radicals before damage can occur, repair oxidative damage, eliminate damaged molecules, and prevent mutations (Gordon, 1996). A diet rich in vegetables and fruit may provide protection against cardiovascular disease (Ness and Powles, 1997), several common cancers (Steinmetz and Potter, 1996). Importance of natural antioxidants for medical and food application has been underlined by numerous works as reported by Spigno and De Faveri (2007). Serdula et al. (1996) showed that the frequency of intake of fresh fruit and vegetables increased as the level of physical activity increased, and that consumption of fruit and vegetables was lower in those who reported that they were sedentary, heavy smokers, or heavy drinkers.
0254-6299/$ - see front matter © 2011 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2011.09.012
H. Edziri et al. / South African Journal of Botany 78 (2012) 252–256
The presence and growth of pathogenic microorganisms (bacteria, mold, viruses, fungi) in food may cause spoilage and result in a reduction in its quality and quantity (Ohshima and Bartsch, 1994). This microbial contamination still poses important public health and economic concerns for the human society. The exploration of naturally occurring antimicrobials for food preservation receives increasing attention due to consumer awareness of natural food products and a growing concern of microbial resistance toward conventional preservatives (Gould, 1995). The aim of the present study was the investigation of the antimicrobial and antioxidant activities of aqueous and methanolic extracts obtained from some Tunisian vegetables. 2. Materials and methods 2.1. Plant material
253
2.3. Determination of total phenolic content Total phenolic content in each extract was determined using Folin–Ciocalteu reagent according to the method of Singleton and Rosi (1965). Forty microliters of extract (1 mg/ml) was mixed with 200 μl Folin–Ciocalteu reagent (Sigma-Aldrich, Germany) and 1160 μl of distilled water, followed by 600 μl 20% sodium carbonate (Na2CO3) 3 min later. The mixture was shaken for 2 h at room temperature and absorbance was measured at 765 nm. All tests were performed in triplicate. Gallic acid (Sigma-Aldrich, Germany) was used as a standard. The concentration of total phenolic compounds (TPC) was determined as μg gallic acid equivalents (GAE)/mg extract using the following equation obtained from a standard gallic acid graph (R 2 = 0.9877): Absorbance ¼ 0:0012 gallic acid ðμgÞ−0:0034
All the vegetables were collected from different locations in Tunisia. They are presented in Table 1. 2.2. Extracts preparation 2.2.1. Aqueous extraction The vegetables were cut into small parts. Distilled water (200 ml) was added to 50 g of each sample in a conical flask. Then they were allowed to boil for 1 h. The extract was filtered using a Whatman no. 1 paper. The extract was lyophilized and it was stored at − 25 °C until use. 2.2.2. Methanolic extraction Each sample (50 g) was incubated in a glass flask with 200 ml of methanol (90%) for 3 days under magnetic stirrer. Solvent was evaporated under vacuum at 70 °C to get crude extracts and it was stored at − 25 °C until use.
2.4. Antibacterial and antifungal activities 2.4.1. Microrganisms The microorganism strains used in the biological assays are listed in Table 1. Different American-Type Cell Culture (ATCC) reference bacteria and fungi as well as clinical isolates including methicillin resistant Staphylococcus aureus (MRSA) strain were used. Their ATCC number is given in the legend of Table 1. 2.4.2. Antibacterial and antifungal screening The minimum inhibitory concentrations (MIC) were determined by the microwell dilution method (Zgoda and Porter, 2001) with some modifications as follows. The inoculum (10 6 cfu/ml) was prepared from 24 h broth cultures and suspensions were adjusted to 0.5 McFarland standard turbidity. All the extracts dissolved in 10% of DMSO were first diluted to
Table 1 Antibacterial activity of vegetable extracts using microdilution test MICa minimal inhibitory concentration; values given as mg/ml; MIC positive control: levofloxacin (E. coli 0.61 μg/ml, P. aeruginosa 0.3 μg/ml, S. aureus 0.3 μg/ml, E. faecalis 1.22 μg/ml). MBCb: minimal bactericidal concentration; values given as mg/ml; Mec methanolic extract, Aqd aqueous extract, E.c: Escherichia coli ATCC 25922; P.a: Pseudomonas aeruginosa ATCC 27950; S.a: Staphylococcus aureus ATCC 25923; E.f: Enterococcus faecalis ATCC 29212. Plant species
E.c Used parts
Raphanus sativus
Fruit
Petroselinum sativum
Leaves
Apium graveolens
Leaves
Beta vulgaris var cicla
Leaves
Solanum melongena
Fruit
Solanum tuberosum
Fruit
Anethum graveolens
Leaves
P.a
E.f
S.a
Extracts
MICa
MBCb
MIC
MBC
MIC
MBC
MIC
MBC
c
2.5 5 1.25 5 1.25 5 1.25 5 5 5 0.312 5 5 5
5 10 5 10 5 10 5 10 10 10 0.625 10 10 10
5 5 2.5 5 5 5 1.25 2.5 5 5 0.312 5 2.5 5
5 10 10 10 10 10 5 5 10 10 0.625 10 10 10
2.5 5 5 5 0.312 5 2.5 1.25 5 5 1.5 5 2.5 5
5 5 10 10 1.25 10 10 2.5 10 10 1.25 10 10 10
2.5 5 5 5 1.25 5 1.25 5 5 5 0.312 5 2.5 5
5 10 10 10 5 10 5 10 10 10 1.25 10 10 10
Me Aqd Me Aq Me Aq Me Aq Me Aq Me Aq Me Aq
H. Edziri et al. / South African Journal of Botany 78 (2012) 252–256
highest concentration (20 mg/ml) to be tested, and then serial two-fold dilution was made in a concentration range from 0.016 to 20 mg/ml in sterile water. The 96-well plates were prepared by dispensing into each well 95 μl of MH broth (bacteria) or Sabouraud dextrose broth (yeast) and 5 μl of the inoculum. A 100 μl from extracts initially prepared at the concentration of 20 mg/ml was added into the first wells. Then, 100 μl from their serial dilutions was transferred into six consecutive wells. The last well containing 195 μl of broth without compound and 5 μl of the inoculum on each strip, was used as negative control. The final volume in each well was 200 μl. Control tests with the solvent DMSO (10%) employed to dissolve the plant extracts were performed for all assays and showed no inhibition of microbial growth. The MIC of each extract was defined as the lowest concentration which inhibited either bacterial or candidal growth, after incubation at 37 °C between 18 and 24 h. The minimal bactericidal concentration (MBC) and the minimal fungicidal concentration (MFC) were determined by subculture on blood agar at 37 °C between 18 and 24 h. Levofloxacin was used as antibacterial positive control, and Amphotericin B for the anticandidal one. 2.5. Antioxidant activity by DPPH assay DPPH radical-scavenging activity was determined by the method of Mitsuda et al. (1966). Briefly, a 0.5 ml aliquot of DPPH methanol solution (25 mg/ml) was added to 0.5 ml sample solution at different concentrations. The mixture was shaken vigorously and allowed to stand at room temperature in the dark for 30 min. Then the absorbance was measured at 517 nm in a spectrophotometer. IC50 value (μg/ml) is the inhibitory concentration at which DPPH radicals were scavenged by 50% and was obtained by interpolation from linear regression analysis. Trolox was used for comparison as positive control. 2.6. Statistical analysis The analytical values of the antimicrobial activity measurement (MICs) represent means of two replicates, done in two different experiments. Data obtained were subject to one-way analysis of variance (ANOVA) and Student t-test analysis. Significance was assumed at p ≤ 0.05 at least. For bacterial counts, data from three independent replicate trials (for each treatment) were pooled and the mean values and standard deviations were determined. Differences between samples were determined by Student's t-test and were considered to be significant when p ≤ 0.05 at least. 3. Results and discussions 3.1. Total phenolic content
and 2. The total phenolic content of aqueous extracts varied from 152.14 to 405.75 mg of catechin equivalent (CE/g) extract. Solanum melongena was found to have the highest phenolic contents (405.75 mg CE/g). Apium graveolens, Anethum graveolens, Beta vulgaris var cicla, Raphanus sativus, Solanum tuberosum and Petroselinum sativum were relatively rich in phenolic compounds with concentrations of 244.65; 237.84; 199.35; 171.94; 163.15; 152.14 mg CE/g. Compared with aqueous extracts, methanolic extracts of A. graveolens, A. graveolens, S. melongena and P. sativum had the highest total phenolic content. 3.2. Antibacterial and antifungal activities The results of the antibacterial and antifungal activities of methanol and aqueous extracts of studied vegetables are summarized in Tables 1 and 2. Aqueous and methanolic extract of all plants were effective in inhibiting the growth of all the tested strains with MICs varied between 0.31 and 10 mg/ml. These two extracts showed also antifungal activity against Candida species. Among all tested extracts those of S. tuberosum had the highest antibacterial activity against all tested bacteria, with MICs values between 1.5 and 0.31 mg/ml. All vegetable extracts showed antifungal activity against all Candida species but methanolic extract of A. graveolens had the best antifungal activity against Candida albicans, Candida kreussei and Candida parapsilosis with MIC values ranging between 0.08 and 0.31 mg/ml (Table 2) and with MFC values ranging between 0.08 and 0.63 mg/ml (Table 2). With regards to the total phenolic content we conclude that these active extracts contained a higher amount of phenols. The observed antibacterial and antifungal activities of the extracts of vegetables might also be attributed to the high quantity of polyphenols, which are known to possess efficient antibacterial activity (Velioglu et al., 1998). Consequently phenolic compounds have been reported to be responsible for antimicrobial properties (Penna et al., 2001). 450
Total phenolic contents (mg CE/g extract)
254
S.m
400 350 300 A.p A.g
250 B.v
200 P.s
S.t
R.s
150 100 50 0
The antioxidant activity of plant extracts has been correlated to their total phenolic content due to their property of scavenging free radicals (Singleton and Rosi, 1965). The TPC was expressed in mg of catechin equivalents (mg CE/g of extract). The total phenolic content of the extracts is given in Figs. 1
Aqueous extracts
Fig. 1. Total phenolic content of aqueous extracts of vegetables; mg CE/g: milligram catechin equivalent per gram extract; P.s: Petroselinum sativum; S.t: Solanum tuberosum; R.s: Raphanus sativus; B.v: Beta vulgaris var cicla; A.g: Anethum graveolens; A.p: Apium graveolens; S.m: Solanum melongena.
Total Phenolic content (mg CE/g extract)
H. Edziri et al. / South African Journal of Botany 78 (2012) 252–256 500 P.s
450
S.m
255
Table 3 IC50 values obtained for the vegetable extracts in DPPH assay Me: methanolic extract, Aq: aqueous extract.
A.g
400 A.p
Plant species
Used parts
Extracts
IC50 (μg/ml)
Raphanus sativus
Fruit
Petroselinum sativum
Leaves
Apium graveolens
Leaves
Beta vulgaris var cicla
Leaves
Solanum melongena
Fruit
Solanum tuberosum
Fruit
Anethum graveolens
Leaves
Trolox
–
Me Aq Me Aq Me Aq Me Aq Me Aq Me Aq Me Aq –
640.2 ± 0.4 10.3 ± 0.2 230.5 ± 0.3 250.1 ± 0.1 30.1 ± 0.4 20.0 ± 0.2 600.1 ± 0.1 20.0 ± 0.3 20.0 ± 0.3 20.0 ± 0.4 800.1 ± 0.2 20.0 ± 0.1 90.5 ± 0.1 20.0 ± 0.2 23.1 ± 0.01
350 300 250 200
R.s
S.t
B.v
150 100 50 0
Methanolic extracts
Fig. 2. Total phenolic content of methanol extracts of vegetables; mg CE/g: milligram catechin equivalent per gram of extract; P.s: Petroselinum sativum; S.t: Solanum tuberosum; R.s: Raphanus sativus; B.v: Beta vulgaris var cicla; A.g: Anethum graveolens; A.p: Apium graveolens; S.m: Solanum melongena.
In addition the obtained results confirmed the earlier studies that methanol is the best solvent for extraction of antimicrobial substances compared with the other solvents (Penna et al., 2001). In previous work we showed that 4-(methylthio)-3-butenyl isothiocyanate (MTBITC) is a principal isothiocyanate (ITC) having pungency in R. sativus for a long period. However, its biological effect was first identified as antimicrobial to Escherichia coli, S. aureus, Saccharomyces cerevisiae, and Aspergillus oryzae in 1982 (Esaki and Onozaki, 1982; Hashem and Saleh, 1999). 3.3. Antioxidant activity by DPPH assay The radical DPPH has a violet color due to the unpaired nitrogen electron and, after reaction with the oxygen atom of a radical scavenger (antioxidant compound) reduced DPPH-H is formed which is yellow. Table 3 shows the IC50 values of the DPPH assay where the results from the various vegetables extract are compared with a Trolox with recognized antioxidant activity.
IC50 values denote the concentration of each sample which required to scavenge 50% free radicals or to prevent lipid peroxidation by 50%. All aqueous extracts except P. sativum showed higher DPPH radical scavenging activity with IC50 ranging from 10.3 to 20 μg/ml. Aqueous extracts of A. graveolens, B. vulgaris var cicla, S. melongena, S. tuberosum and A. graveolens showed the highest antioxidant activity with IC50 of 20 μg/ ml lower than the positive control Trolox. The antioxidant activity of vegetable extracts has been correlated to their content of phenolic components (Velioglu et al., 1998) due to their property of scavenging free radicals. Therefore, it is important to consider the effect of the total phenolic quantity in the antioxidant activity of the vegetable extracts. In previous work, L-tryptophan, and 7-[3-(3,4-dihydroxy4-hydroxymethyl-tetrahydro-furan-2-yloxy)-4,5-dihydroxy-6hydroxymethyl-tetrahydro-pyran-2-yloxy]-5-hydroxy-2-(4hydroxy-3-methoxy-phenyl)-chromen-4-one were isolated
Table 2 Antifungal activity of selected vegetable extracts using microdilution test. C.a: Candida albicans ATCC 90028; C.g: Candida glabrata ATCC 90030; C.k: Candida kreussei ATCC 6258; C.p: Candida parapsilosis ATCC 22019; Me: methanolic extract, Aq; aqueous extract; MICa minimal inhibitory concentration; values given as mg/ml; positive control with Amphotericin B (MFC 0.5 μg/ml). MBCb: minimal fungicidal concentration; values given as mg/ml. Plant species
C.g
C.a
C.k
C.p
Used parts
Extracts
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
Raphanus sativus
Fruit
Petroselinum sativum
Leaves
Apium graveolens
Leaves
Beta vulgaris var cicla
Leaves
Solanum melongena
Fruit
Solanum tuberosum
Fruit
Anethum graveolens
Leaves
Me Aq Me Aq Me Aq Me Aq Me Aq Me Aq Me Aq
5 5 1.25 5 5 5 1.25 2.5 5 5 1.25 5 5 5
5 10 5 10 10 10 5 5 10 10 5 10 5 10
2.5 2.5 1.25 5 0.156 5 2.5 1.25 5 5 1.25 5 5 5
10 2.5 5 10 0.625 10 10 2.5 10 10 5 10 5 10
1.25 2.5 1.25 5 0.078 5 5 2.5 5 5 1.25 1.25 2.5 5
1.25 2.5 5 10 0.078 10 10 1.25 10 10 5 1.25 2.5 10
2.5 5 2.5 5 0.312 5 2.5 1.25 5 5 2.5 5 2.5 5
10 10 10 10 0.625 10 10 2.5 10 10 10 10 5 10
256
H. Edziri et al. / South African Journal of Botany 78 (2012) 252–256
from A. graveolens and they exhibited good antioxidant activity at concentrations of 125 and 250 μg/ml. Tryptophan, a precursor for indole biosynthesis, is well documented for its effect on central nervous system and for its therapeutic use in depression, mania and aggression (Young et al., 1984). 4. Conclusion In conclusion, antioxidant and antimicrobial activities which could be derived from their compounds such as flavonoids, polyphenol compounds and vitamin C. By coupling the results of different assays for evaluating antioxidant activity of these vegetables from different solvents, we can conclude that aqueous extracts showed the best antiradical activity with DPPH assay. Among all tested extracts we conclude that methanolic extract of S. tuberosum had the highest antibacterial activity. This study shows that methanolic extract of A. graveolens had the best antifungal activity against C. albicans, C. kreussei and C. parapsilosis. Moreover development of extraction methods and fractionations of extracts should be carried out in order to further investigate their possible use as food preservatives. This potential could be adopted by the food industry. References Esaki, H., Onozaki, H., 1982. Antimicrobial action of pungent principles in radish root. Eiyo To Shokuryo 35, 207–211. Gordon, M.H., 1996. Dietary antioxidants in disease prevention. Natural Product Research 13, 265–273. Gould, G.W., 1995. Homeostatic mechanisms during food preservation by combined methods. In: Barbosa-Canovas, G., Welti-Chanes, J. (Eds.), Food Preservation by Moisture Control. Lancaster Techromic Publishing Co., Berlin, pp. 87–95. Hashem, F.A., Saleh, M.M., 1999. Antimicrobial components of some Cruciferae plants. Phytotherapy Research 13, 329–332.
Edited by L McGaw
Mitsuda, H., Yasumoto, K., Iwami, K., 1966. Antioxidative action of indole compounds during the autoxidation of linoleic acid. Eiyo To Shokuryo 19, 210–214. Ness, A.R., Powles, J.W., 1997. Dietary habits and mortality in vegetarians and health conscious people. Several uncertainties still exist. British Medical Journal 11, 48–149. Ohshima, H., Bartsch, H., 1994. Quantitative estimation of endogenous nitrosation in humans by monitoring N-nitrosoproline excreted in the urine. Cancer Research 41, 3658–3662. Penna, C., Marino, S., Vivot, E., Cruaňes, M.C., Muňoz, J.D., Cruaňes, J., Ferraro, G., Gutkind, G., Martino, V., 2001. Antimicrobial activity of Argentine plants used in the treatment of infectious diseases. Isolation of active compounds from Sebastiania brasiliensis. Journal of Ethnopharmacology 77, 37–40. Rock, C.L., Jacob, R.A., Bowen, P.E., 1996. Update on the biological characteristics of the antioxidant micronutrients: vitamin C, vitamin E, and the carotenoids. Journal of American Diet Association 96, 693–702. Serdula, M.K., Byers, T., Mokdad, A.H., Simoes, E., Mendlein, J.M., Coates, R.J., 1996. The association between fruit and vegetable intake and chronic disease risk factors. Epidemiology 7, 161–165. Singleton, V.L., Rosi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. American Journal of Enolitic Viticum 16, 144–158. Spigno, G., De Faveri, D.M., 2007. Antioxidants from grape stalks and marc: influence of extraction procedure on yield, purity and antioxidant power of the extracts. Journal of Food Engineering 78, 793–801. Steinmetz, K.A., Potter, J.D., 1996. Vegetables, fruit, and cancer prevention: a review. Journal of American Diet Association 96, 1027–1039. Velioglu, Y.S., Mazza, G., Gao, L., Omah, B.D., 1998. Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. Journal of Agriculture and Food Chemistry 46, 4113–4117. Young, S.N., Chouinard, G., Annable, L., Morand, C., Ervin, F.R., 1984. Progress in tryptophan and serotonin research. In: Schlossberger, H., Kochen, W., Linzen, H., Steinhart, H. (Eds.), The Therapeutic Action of Tryptophan in Depression, Mania and Aggression. Walter de Gruyter & Co., Berlin, pp. 182–186. Zgoda, J.R., Porter, J.R., 2001. A convenient microdilution method for screening natural products against bacteria and fungi. Pharmaceutical Biology 39, 221–225.