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Journal of Stress Physiology & Biochemistry, Vol. 10 No. 2 2014, pp. 190-199 ISSN 1997-0838 Original Text Copyright © 2014 by Al Maqtari and Nagi
ORIGINAL ARTICLE
Screening of Salt-stress, Antioxidant Enzyme, and Antimicrobial Activity of Leave extracts of Mangroves Avicennia marina L. from Hodaidah, Yemen
Maher A. Al Maqtari*1, Hisham M. Nagi2
1 Department of Chemistry, Faculty of Science, Sana'a University
2 Department of Earth and Environmental Sciences, Faculty of Science, Sana'a University *E-Mail: [email protected]
Received January 31, 2014
In the present study the salinity stress, antioxidant enzyme and antimicrobial activities of leaf extract of Avicennia marinawere investigated. As visualized from SDS-PAGE, no differences was found in number of protein band, but the intensities of several protein bands having apparent molecular mass by reduced severely in salt treated samples with enhanced activities of CAT, POX and GPX. Escherichia coli (ATCC8739), Staphylococus aureus (ATCC 6538), and Bacillus subtilis (ATCC6633) and fungus (Candida albicans ATCC 2091, and Aspergillus niger ATCC 16404) were used as the test pathogenic bacteria and fungi, respectively in this study. The Avicennia marina extract possessed antibacterial activity against E. coli, S. aureus, and B. subtilis (12, 6, and 7 mm respectively), with antifungal activity against C. albicans and A. niger (9 and 10 mm).
Key words: Salinity, Antioxidant enzymes, Antimicrobial effect, Mangrove, Avicennia marina
ORIGINAL ARTICLE
Screening of Salt-stress, Antioxidant Enzyme, and Antimicrobial Activity of Leave extracts of Mangroves Avicennia marina L. from Hodaidah, Yemen
Maher A. Al Maqtari*1, Hisham M. Nagi2
1 Department of Chemistry, Faculty of Science, Sana'a University
2 Department of Earth and Environmental Sciences, Faculty of Science, Sana'a University *E-Mail: [email protected]
Received January 31, 2014
In the present study the salinity stress, antioxidant enzyme and antimicrobial activities of leaf extract of Avicennia marinawere investigated. As visualized from SDS-PAGE, no differences was found in number of protein band, but the intensities of several protein bands having apparent molecular mass by reduced severely in salt treated samples with enhanced activities of CAT, POX and GPX. Escherichia coli (ATCC8739), Staphylococus aureus (ATCC 6538), and Bacillus subtilis (ATCC6633) and fungus (Candida albicans ATCC 2091, and Aspergillus niger ATCC 16404) were used as the test pathogenic bacteria and fungi, respectively in this study. The Avicennia marina extract possessed antibacterial activity against E. coli, S. aureus, and B. subtilis (12, 6, and 7 mm respectively), with antifungal activity against C. albicans and A. niger (9 and 10 mm).
Key words: Salinity, Antioxidant enzymes, Antimicrobial effect, Mangrove, Avicennia marina
Mangroves are evergreen woody plants that grow at the interface between land and sea, and form the dominant intertidal vegetation in tropical and subtrobical regions (Blasco, 1984). These plants, and the associated microbes, fungi, plants, and animals, constitute the mangrove forest community or mangal (Tomlinson, 1994). Mangroves trees are characterized with broad leaves, aerial roots, like pneumatophores or stilt roots, and viviparous germinated seedlings (Naskarand Mandal, 1999).
Area of mangrove habitats in Yemen was estimated to be approximately 22.55 km2 (Nagi et al., 2012). Yemen's coastal and marine ecosystems which include extensive mangroves such as Avicennia marina and Rhizophora mucronata that is only growing in two locations (Nagi and Abubakr, 2013), coral reefs, and sea grass areas are of major economic importance for fisheries and tourism. A. marina is a mangrove tree species that is extraordinarily adaptable with a wide latitudinal range closely associated with its flexible growth
pattern (Vioux-Chagnoleau et al., 2006).
Mangrove habitats along with their associated biota are of extreme importance, as a large coastal population depends on these resources for their livelihood. They have been used as traditional medicine in South Asian countries including Yemen (EPA, 2009). Recently, it has been strongly recommended that mangroves should be considered as a valuable source for chemical constituents with potential medicinal and agricultural values (Bandarnayake, 2002). Avicennia marina (Forssk.) Vierh. (Avicenniaceae) has received some attention in determining its important chemical constituents. Phenolic compounds are secondary plant metabolites and are involved in a wide range of specialized physiological functions. They are very important for the normal growth, development and defense mechanisms of plants (Miles et al., 1998). These compounds are capable of inhibiting free radicals, and hence can retard the aging process (Maisuthisakul et al., 2007).
Extracts and chemicals from mangroves are used mainly in folkloric medicine (e.g. bush medicine), as insecticides and pesticides and these practices continue to this day (Natarajan et al., 2003). Methanol extract of Excoecaria agallocha leaves and shoots (Ravikumar et al., 2010) and antifungal activity of methanol extract of E. agallocha and Bruguiera gymnorrhiza trunks are some other examples of pharmaceutical potential of mangrove plants.
There are no reports on salinity stress, antioxidant and antimicrobial activities of A. marina in Yemen, the present study was designed and carried out based on the reasons mentioned above. In this study, A. marina leaf extract was examined to evaluate the antioxidant potential and the same crude extracts were also used to indicate the
antimicrobial activities of the samples.
MATERIALS AND METHODS
Preparation of plant extract
Mangrove leaves of A. marina were collected from Al Rage'ah Island (14° 54' 53" N and 42° 55' 39" E) which is located in front of Al Gabbanah village north of Al Hodaidah city. The collected leaf samples are air dried and then ground with sterilized distilled water. After grinding, filtration process conducted and the filtrate collected with Whatman No.1 filter paper and evaporated at 40°C for 48 hours. The residue was preserved for experimental analysis.
Plant materials and culture conditions
The preliminary experiments were carried out on A. marina at different concentrations of NaCl (50, 120, 190, 260, 330, 400, and 500 mM) for 0, 6, 12, 18, 23, 35, and 50 days in order to determine the viable range of salinities. Since, 500 mM NaCl was found lethal for this plant and at 330 mM NaCl, the plant can survive for more than 25 d, therefore, 330 mM NaCl was chosen as suitable concentration for investigating long-term effect of salinity in A. marina. The cultures were aerated continuously with an air bubbler. Leafs of plant from different days of treatment were collected to measure the experimental parameters and the experiments were repeated twice with three replicas in each treatment.
Extraction and estimation of total leaf protein
Total leaf protein was extracted by the acetone-TCA precipitation method as described earlier by (Damerval et al., 1986) and estimated following the method of (Lowry et al., 1951). Bovine serum albumin was used as standard protein.
Analysis of protein profile of leaf by SDS-PAGE
Leaf samples were harvested from control and
NaCl treated plants after 7, 14, 30 and 45 days of treatment for analysis of soluble protein by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). Samples (0.5 g) were homogenized with 2 ml of a buffer containing 50 mm Tris(hydroxymethyl) aminomethane (Tris)-Glycine (pH 8.3), 0.5 m sucrose, 50 mm EDTA, 0.1 m KCl, 2 mm PMSF and 0.1% (v/v) 2-
mercaptoethanolin a chilled pestle and mortar at 4 0C. The homogenate was centrifuged in a refrigerated centrifuge at 14,000r.p.m for 10 min. Protein concentration in the supernatant samples was estimated according to the method of (Laemmli, 1970). The supernatants were stored in small aliquots at 85 0C for SDS-PAGE. Supernatant samples (40 ^g protein) were mixed with equal volumes of solubilizing buffer [62.5 mm Tris-HCl, pH 6.8, 20% (w/v) glycerol, 2%(w/v) SDS, 5% (v/v) 2-mercaptoethanol and 0.01% bromophenol blue] and heated for 4 min at 95 0C, cooled on ice before loading on 12.5% polyacrylamide slab gels. Gels were made according to (Laemmli, 1970).
A 10 % separating gel containing 375 mm Tris-HCl, pH 8.8, 0.1% (w/v) SDS, 0.05% (w/v) ammonium persulfate and 0.4 ^l-TEMED was used for resolving the polypeptides whereas a 4% stacking gel containing 125 mm Tris-HCl, pH 6.8. Extraction of antioxidative enzymes and their assays
Extraction of enzymes and assays:
Two grams of young leaf buds were macerated to powder with liquid nitrogen with a mortar-pestle; then 0.1 g PVP and 5 ml of extraction buffer (consisting of 1 M Sucrose, 0.2 M Tris-HCL and 0.056 M P-Marcaptoethanol; pH adjusted at 8.5) was added and homogenized. The extractants were centrifuged at 10,000 rpm for 20 min at 480C. The
supernatant were collected and used for the assay of catalase (CAT), peroxidase (POX) and guaicol peroxidase (GPX).
Estimation of Catalase (CAT)
Catalase was measured according to (Sadasivam and Manickam, 1991) by change in absorbance at 240 nm. An assay mixture contained 3 ml H2O2 phosphate buffer (0.64 ml of H2O2 diluted to 100 ml with 0.1M phosphate buffer pH 7.0) and 0.2 ml enzyme extract. The amount of enzyme required to change the absorption (A OD) by 0.01 min-1 mg-1 protein was taken as unit enzyme activity. Estimation of Peroxidase (POX)
Peroxidase activity was measured according to (Maehly and Chance, 1954) by following the change in absorbance at 470 nm due to guiacol oxidation in the presence of H2O2 and enzyme. The assay mixture 2 ml 0.1M phosphate buffer (pH 7.0), 1 ml 20 mM guaiacol, 0.05 ml H2O2 (20 mM) and 0.5 ml enzyme extract. The amount of enzyme required to change the absorption (A OD) by 0.01 min-1 mg-1 protein was taken as unit enzyme activity. Estimation of guaiacol peroxidase (GPX)
GPX activity was measured
spectrophotometrically at 25°C by following the method of (Tatiana et al., 1999). The reaction mixture (2ml) consisted of 50 mM potassium phosphate (pH 7.0), 2 mM H2O2, and 2.7 mM guaiacol. The reaction was started by the addition of an enzyme extract equivalent to 5 ^g protein. The formation of tetra guaiacol was measured at 470nm (£=26.6 mM-1cm-1).
Screening for antimicrobial activity of Avicennia marina Extracts
The inhibition activity on bacteria (Escherichia coli ATCC8739, Staphylococus aureus ATCC 6538, and Bacillus subtilis ATCC6633) and fungus
(Candida albicans ATCC 2091, and Aspergillus niger ATCC 16404) Were tested using Agar well diffusion method (He and Zhou, 2007). The 1 mL of bacterial and fungus cultured at equal turbidity of McFarland No.0.5 was swab and placed into the surface of Mueller-Hinton Agar. The agar media was punctured into 6 holes per each culture plates of 0.5 cm diameter. 5, 10, 15, 20, 25, and 30^l of A. marina extracts were poured into 5 holes of agar and another hole was used as control (without the
A. marina extract). The culture plates were incubated Finally, and the diameters of inhibition zone (DIZ) were measured in millimeter (mm). The experiment was repeated three times and the mean values were presented. Data were expressed as means ± standard deviations (SD) of triplicate experiments.
RESULTS
Changes in total leaf soluble protein content
Total soluble protein content in A. marina decreased upon exposure to 500mM NaCl for a short period of 7 days (Figure 1). Total soluble leaf protein content in A. marina decreased 3.4 fold after 7 days of salt treatment as compared to control.
Changes in leaf protein SDS-PAGE profiling
The leaf protein was extracted from control and salt treated plant leaves samples and analyzed by SDS-PAGE (Figure 2). SDS-PAGE analysis of total protein profiling indicated that no differences were found in number of protein band upon exposure to salt treatment. But the intensities of several protein bands having apparent molecular mass by reduced severely in salt treated samples compared to control in A. marina.
Effect of salinity on antioxidant enzymes
The effect of NaCl on the catalase, guaiacol peroxidase, and peroxidase activities in the leaves at 500 mM NaCl concentration in different days of treatment is presented in Table 1. There was a steady increase in all the enzyme activities in A. marina.
Antimicrobial activity assay
The result of antimicrobial activity of A. marina extracts was presented in Table 2 and Figure 3. Extract of leaves showed antimicrobial activity against E. coli, S. aureus, and B. subtiliss, and antifungal activity against the growth of C. albicans and A. niger.
Figure 1: Effect of NaCl on total soluble proteins in leaves of Avicennia marina in different days for 500mM NaCl treatment.
7d 5d 3d 1d C Figure 2: Effect of NaCl on total soluble proteins in
leaves of Avicennia marina in different days for 500mM NaCl treatment.
A B c
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Figure 3: Anti-microbial activity of different levels from leave extract of Avicennia marina (A) with Escherichia coli. (B) with Staphylococus aureus. (C) with Bacillus subtilis. (Antifungal activity not shown)
Table 1. Effects of 500 mMNaCl treatment on enzyme GPX, POX, CAT and Protein content in leaves of
Avicennia marina in different days of treatment
Control 500mM NaCl
1d 3d 5d 7d
Protein (mg) 45.41 30.20 15.13 15.13 13.22
CAT U/mg of protein 0.173±0.113 0.186±0.013 0.224±0.012 0.276±0.011 0.332±0. 113
POX U/mg of protein 1.034±0.023 1.265±0.043 1.454±0.054 1.856±0.013 2.234±0.003
GPX U/mg of protein 2.122±0.003 2.321±0.003 2.445±0.002 3.765±0.001 4.683±0.002
Table 2: Anti-microbial activity of different levels from leave extract of Avicennia marina.
Microorganisms levels of Mangroves extract
30 ці 25 ці 20 ці 15 ці 10 ці 5 ці
Inhibition zone, mm
Escherichia coli ATCC8739 12 11 10 8 7 4
Staphylococus aureus ATCC 6538 6 0 0 0 0 0
Bacillus subtilis ATCC6633 7 6 5 0 0 0
Candida albicans ATCC 2091 9 8 6 0 0 0
Aspergillus niger ATCC 16404 10 9 8 0 0 0
DISCUSSION
The total protein content of leaf gradually decreased with increasing concentration of NaCl. This decrease in protein content might be due to stimulation of protein hydrolysis (Uprety and Sari, 1976), or the increasing activity of acid and alkaline proteases. As reported earlier, levels of free amino acid increase as a result of salt stress in B. parviflora (Parida et al., 2002).
In this study, three antioxidant enzymes were estimated, i.e. CAT, POX, and GPX. A quantitative study of these enzymes from saline and fresh water grown plants revealed that enzymes activity were higher in salt-stressed plant.
Increase in CAT activity is supposed to be an adaptive trait possibly helping to overcome the damage to the tissue metabolism by reducing toxic levels of H2O2 produced during cell metabolism and protection against oxidative (Bor et al., 2003). (Takemura et al., 2000) reported an inductive response in CAT activity in the mangrove B. gymnorrhiza under salt stress. Similarly in the present study, the salt induced enhancement of CAT activity in A. marina may suggest its effective scavenging mechanism to remove H2O2 and imparting tolerance against salinity induced oxidative stress.
POX activity of A. marina was increased with increasing salt concentrations. This result agrees with Aegiceras corniculatum (Manikandan and Venkatesan, 2004). The enzyme POX involves in the decomposition of cosubstrates such as phenolic compounds and/or antioxidants. GPX activity also increases when exposed to salt stress. Exposure to high NaCl imposes oxidative stress due to changes in the osmotic and ionic environment in plant (Allakhverdiev et al., 2000).
It is important to study scientifically plants that have been used in traditional medicines to determine potential sources of novel antimicrobial compounds (Hammer et al., 2001). Plants are employed as important source for traditional medications (Neves et al., 2009).
Mangrove plant A. marina extracts showed antimicrobial activity against E. coli, S. aureus, and
B. subtilis. The A. marina specie studied, was effective against E. coli, S. aureus and B. subtilisat (5, 10, 15, 20, 25, 30nl), (30 ^l) and (20, 25, 30 ^l), respectively. The inhibition zone was found to be better in E. coli. The result for antibacterial activity agrees with many leaves extract of mangrove, (Imdadul et al., 2011a; Natarajan et al., 2011; Imdadul et al., 2011b).
Antifungal metabolites mangrove plant leaves include alkaloids, flavonoids and related
compounds, fatty acids, oxygen heterocyclics, proanthocyanidins, quinones, stilbenes, terpenoids and triterpenoid, saponins. These compounds have toxicological characteristics, such as anti-fungal activity (Bandaranayake, 2002). The antifungal activity was effective against fungus at (20, 25, 30^l) for both C. albicans and A. niger. Different levels of mangrove extract have been used to consider its antimicrobial effect. Fatty acids are widely occurring compounds in natural fats and dietary oils, and they are known to have antibacterial and antifungal properties (Agoramoorthy et al., 2007; Shelar et al., 2012).
ACKNOWLEDGEMENTS
The authors are very grateful to Yemen Standardization, Meteorology and Quality Control for help in analytical analysis and to the faculty of sciences', Sana'a University for providing infrastructure facilities. Authors are also grateful to Mr. Aref Abdullah Hamoud, and Mr. Yaser Al Ghubair DG of EPA Hodaidah Branch for helping in collecting the samples
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