ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ENDEMICTREE ABIES NUMIDICA GROWING IN BABOR MOUNTAINS FROM ALGERIA

Abstract

The aim of present study was to determine the antimicrobial and antioxidant activity of essential oil and different extracts from Abies numidica leaves. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was used to detect in vitro antioxidant activity. Antibacterial activity was tested against nine bacterial species, representing both Gram positive and Gram negative bacteria. Antifungal activity was evaluated using two Candida species. The results indicate that the crude methanolic extracts and the essential oil resulted in the highest antioxidant activities compared to other solvents. All extracts of A. numidica showed antimicrobial activity against both Gram positive and Gram negative bacteria, as well as fungi, especially methanolic extracts and essential oil. A. numidica could be exploited for the isolation of bioactive compounds which could be a potential source for antioxidant and antimicrobial activities.

Headnote

ABSTRACT

The aim of present study was to determine the antimicrobial and antioxidant activity of essential oil and different extracts from Abies numidica leaves. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was used to detect in vitro antioxidant activity.Antibacterial activity was tested against nine bacterial species, representing both Gram positive and Gram negative bacteria. Antifungal activity was evaluated using two Candida species. The results indicate that the crude methanolic extracts and the essential oil resulted in the highest antioxidant activities compared to other solvents.All extracts of A. numidica showed antimicrobial activity against both Gram positive and Gram negative bacteria, as well as fungi, especially methanolic extracts and essential oil. A. numidica could be exploited for the isolation of bioactive compounds which could be a potential source for antioxidant and antimicrobial activities.

KEY WORDS: Antifungal activity, DPPH, essential oil, bioactive, methanolic extracts.

INTRODUCTION

The beneficial effect of antioxidants and antibiotics on the maintenance of health in human has become an important subject that has engaged many scientists across the world over the last decade.

Antioxidants have significant inhibition roles, not only on undesirable changes in the flavor and nutritional quality of food, but also on tissue damage in various human diseases such as inflammation, cancer, and atherosclerosis (Rajendran et al., 2014). To avoid or delay this autoxidation process, conventional artificial antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), propyl gallate (PG) and tertiary butyl hydroquinone (TBHQ) have been used for more than five decades. However, these synthetic antioxidants have been suspected to cause serious negative health effects. Recently synthetic products are being restricted in the industries, because of harmful effects observed such as human toxicity and environmental pollution. For this reason, there is a growing interest in studies of natural additives as potential antioxidants (Roby et al., 2013). The substitution of synthetic antioxidants by plant materials has caused great interest in nutrition research.

The development of bacterial resistance to presently available antibiotics necessitated the search for new antimicrobial agents. The antimicrobial activity of plant oils and extracts has formed the basis of many applications, including raw and possessed potential as natural agents for food preservation, pharmaceuticals, alternative medicine and natural therapies (Dung et al., 2008)

During the last few decades, the global interest in the study of various medicinal plants has increased rapidly due to their antibacterial and antioxidant activities, low toxicity and the potential to be a cheaper alternative to costly synthetic drugs (Benderradji et al., 2014; Farjana et al., 2014).

The family Pinaceae, one of the major conifer taxa, is divided into several genera of which Abies, Picea and Pinus are among the largest. Conifers of the genus Abies Mill. play an important ecological role in forest ecosystems which cover large parts of the northern hemisphere. Currently 51 species have been described in the genus Abies; of these, ten species and subspecies are distributed around the Mediterranean Sea (Liepelt et al., 2010). The fir of Numidia (Abies numidica De Lannoy ex Carrière) is an endemic plant growing in Algeria, and is often used as folk medicines. A. numidica appears only on the tops of mountains of Babor and Tababort from Algeria, where these Forests cover only a few hundred hectares (Quezel, 1985, 1998). However, to the best of our knowledge, there are so far no published reports on bioactivity of this plant.

The aim of this work was to evaluate the antioxidant and antimicrobial activities of the different extracts of Abies numidica.

MATERIALS AND METHODS

Plant material: Abies numidica is a tree growing up to 20 m tall. Whorled branches, spread horizontally, the last erect. The leaves are needle-like, moderately flattened, glossy dark green with a patch of greenish-white stomata near the tip above, and with two greenish-white bands of stomata below. The cones are glaucous green with a pink or violet tinge, maturing brown, 13-20 cm long (Quezel and Santa, 1962; Schutt and Lang, 1991).

Aerial parts of A. numidica were collected during Marsh 2015 from mountains of Babor in Algeria (Figure 1). Plant materials were identified by Dr. Bounar Rabah according to "Now Flora of Algeria" (Quezel and Santa, 1962) (Figure 2) and voucher specimens deposited in the Department of Natural and Life Sciences, Faculty of Sciences, Mohamed Boudiaf University, M'sila, Algeria.

Preparation of extracts: Leaves of A. numidica were air-dried at room temperature and then ground at approximately 0.2 to 0.4 mm. The dried powder (100 g) was extracted by soaking with 1 L of 70% (v/v) methanol for 24 h at room temperature. After filtration, the residue was processed similarly with the same amount of solvent. The crude methanol extract (MeOH extract) was concentrated to dryness under reduced pressure at 40°C with a rotary evaporator, and stored at 4°C until further use. Five grams from the methanol extract was further fractionated by successive solvent extraction with ethyl acetate (EtOAc fraction), and then with n-butanol saturated water (nBuOH fraction).The crude extract using purified water was subjected to successive appropriate solvent with increased polarity (ethyl acetate and n-butanol) under a continuous reflux setup in a Soxhlet extractor.

Essential oil: Air-dried plant materials (leaves) were subjected to hydrodistillation using a Clevenger type apparatus (Clevenger, 1928), according to the method recommended in the European Pharmacopoeia 4 (C E, 2002). The essential oil was dried over anhydrous sodium sulphate and then stored in sealed glass vials at 4°C prior to analysis. The yield of essential oil was determined in triplicate.

DPPH free radical scavenging assay: The evaluation of the free radical-scavenging activity of essential oil, crude ethanol extract, ethyl acetate fraction and n-butanol fraction was based on the measurement of the reducing ability of antioxidants toward the 1,1-diphenyl2-picrylhydrazyl (DPPH) 2,2-diphenyl-1picrylhydrazyl DPPH radical.Scavenging of DPPH radical was assayed following the method of Dung et al. (2008), with some modification. Solutions with different extract concentrations were prepared and 25 µL of each solution was added to 150 µL of DPPH solution (63.4µM) and 125 µL of 96 % ethanol. The mixture was left to stand for 1h in the dark at room temperature. Reduction of the amount of the DPPH radical present was measured using a decrease in the absorption value at 517 nm. The extract concentration providing 50% inhibition (IC50) was calculated using a graph of the scavenging effect percentage against the extract concentration. The scavenging effect percentage was calculated from the formula:

(ProQuest: ... denotes formula omitted.)

where ADPPH is the absorbance of a negative control (blank sample containing the same amount of solvent and DPPH solution) and AS is the absorbance of the sample. IC50 values denote the concentration of sample, which is required to scavenge 50% of DPPH free radicals. The IC50 values were calculated from linear regression analysis. Butylated hydroxyl toluene (BHT) was used as a positive control.

Determination of antimicrobial activity

Test microorganisms: Antimicrobial activity was tested against nine strains of bacteria (Bacillus subtilis ATCC 10876, Klebsiella pneumoniae ATCC532, Staphylococcus aureus ATCC 6538, S. epidermidis ATCC 12228, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC27853, Pseudomonas syringaepv. Tomato 1086, Escherichia coli ATCC25922, Micrococcus luteus ATCC 533) and two strains of fungi (Candida albicans ATCC 24433 and Candida tropicalis R2 CIP 203). For experiments, the bacterial strains were grown on Müeller-Hinton agar at 37°C for 18 hours whereas Sabouraud dextrose agar at 35°C for 24 hours was used for yeasts.

Disc diffusion assay: Antimicrobial activity of A. numidica essential oil, crude methanol and solvent extracts was evaluated by the disc diffusion test, according to the standard M2-A8 from Clinical Laboratory Standards Institute ( CLSI, 2003) for bacteria and according to NCCLS (2004), document M44-A for yeasts. Briefly, the essential oil and the different test solvent extracts (methanol, ethyl acetate and nbutanol) were loaded to the sterilized sterile 6 mm discs, and then the impregnated discs with 50 μl at a concentration of 100 mg/ml was prepared with dimethyl sulphoxide (DMSO). DMSO was used as control. The inoculum was prepared in a sterile solution of 0.85% sodium chloride and the optical density of the suspension was adjusted to 0.5 McFarland (1 × 108 colony forming units (CFU)/mL). Then the Müeller-Hinton Agar plates for bacteria and Mueller-Hinton Agar + 2% Glucose and 0.5 µg/mL Methylene Blue Dye (GMB) medium for yeasts were inoculated, allowed to dry and the discs previously prepared were placed over the agar. The plates were incubated at 37°C for 48 h under microaerobic conditions for bacteria and at 35°C for 20 to 24 hours of incubation for yeasts and after incubation the diameter of the inhibition zone was measured. All the assays were done in triplicates and the results were given in mean ± SD. Standard antibiotics such as gentamicin for pathogenic bacteria and fluconazole for pathogenic fungi served as positive controls.

Statistical analysis: All experiments were repeated 3 times. The data are expressed as the mean ± SD (standard deviation). Analysis of variance (ANOVA) was carried out to determine significant differences by the SAS statistical software version 9.0 (SAS Institute, Inc., Cary, NC, USA). The significance of the difference was checked by the Duncan test, and differences were considered significant at p < 0.05.

RESULTS AND DISCUSSION

Total yield: The yields of the different solvent extracts and the essential oil obtained are presented in Table1. The results demonstrated a significant (P < 0.05) difference in the extract yields among the solvent systems used.

From this table (1), we noted that the extraction yields with methanol resulted in the highest amount of total extractable compounds whereas the lowest yields were obtained in essential oil. In fact, this variation in the yields can be attributed to the polarities of the different compounds present in the leaves of A. numidica. The extraction yield of the essential oil was 0.260 ± 0.036 % in the dry basis, which was less than that reported before (Ramdani et al., 2014). Differences in the extraction yields could be derived from several factors such as genetic factors, environmental conditions, sample pretreatment, extraction methods and extraction conditions (Cho et al., 2011; Khoudja et al., 2014; Dong et al., 2015).

DPPH free radical scavenging activity: The antioxidant potential of essential oil, methanol, ethyl acetate, and n-butanol extracts of A. numidica was evaluated on the basis of their ability to scavenge stable free DPPH radicals. The concentration required to attain 50% radical-scavenging effect (IC50) was determined from the results of a series of concentrations tested. A lower IC50 value corresponds to a larger scavenging activity.

Scavenging activity increased with the increase in the concentration of essential oil and different extracts of A. numidica leaves. Crude methanolic extract and essential oil showed significantly higher activity than the other extracts, as the amount of sample required to decrease the DPPH concentration by 50 % (IC50) was found to be 0.282 and 0.288 mg/mL for crude methanolic extract and essential oil, respectively (Table 1). The experimental data reveals that these extracts and essential oil are likely to have the effect of scavenging free radical. However, the IC50 value for extracts and essential oil were much lower than BHT used in the present study.

The difference in preparation of the extracts might affect DPPH radical activity of them. Phenolic compounds such as flavonoids are known to be potential antioxidants due to their ability to scavenge free radicals and active oxygen species such as singlet oxygen, superoxide anion radical and hydroxyl radicals (Orak et al., 2011; Djouossi et al., 2015). Therefore, the presence of such compounds could be responsible for the antioxidant activity found in the crude methanolic extract and the essential oil. To our best knowledge this is the first report on characterization of antioxidant properties of A. numidica.

Antimicrobial activity: The results from the antimicrobial activity of the essential oil and the three extracts tested by disc diffusion method are summarized in Table 2. The essential oil and all extracts of A. numidica showed an inhibitory effect on the growth of all tested microbial strains. The rate of inhibition was determined by measuring the diameter of the clear zone around the soaked filter paper (Table 2 and Figure 3).

The diameters of inhibition zones ranged from 52.67 ± 2.52 to 8.67 ± 1.15 mm. The best result was obtained for methanolic extract and the essential oil (Table 2). The variation in antimicrobial activity between different tested bacterial and fungal strains using essential oil extracts, fluconazole and gentamicin (P < 0.05) was statistically significant. Essential oil and different extracts of A. numidica showed significant antibacterial activity, especially against M. luteus, S. aureus and E. faecalis, a recognized pathogenic species. The oil and different extracts also efficiently inhibited the growth of C. albicans and C. tropicalis. Antimicrobial activity could be due to the presence of a higher concentration of the alcohol-soluble active molecules in A. numidica. The effectiveness of essential oil and extracts against a wide range of microorganisms is related to their hydrophobicity, which enables them to integrate into the lipids of the cell membrane and mitochondria, rendering them permeable and leading to leakage of cell contents (Horvathova et al., 2014). The antimicrobial results of current study are in accordance with literature, where extracts of many Abies species were effective against pathogenic bacteria and fungi (Bagci and Digrak, 1996; Vishnoi et al., 2007; Xia et al., 2012; Ramdani et al., 2014; Poaty et al., 2015). Previous studies clearly revealed that the essential oil and different extracts have strong activity against bacteria and yeast, because these phenols and flavonoids have a strong antimicrobial potential (Hossain et al., 2014; Al-Jadidi and Hossain, 2015; Stefanovic et al., 2015). Therefore, it is necessary to conduct a more profound research on the relationship between antimicrobial activity and chemical structure of each phenol and flavonoid compound in the A. numidica extract.

CONCLUSION

The present study has shown that the crude methanol extract and essential oil of A. numidica gave the highest antioxidant activity, followed by the n-butanol fraction and the ethyl acetate fraction was the least active. The crude methanol extract and essential oil, proven to have very good potential as a source of antimicrobial activity.

In summary, the current study proved in vitro antioxidant and antimicrobial activity of A. numidica essential oil and different extracts. The identification of the bioactive compounds and study of mechanisms of actions are necessary prior to application. Further research should be done in order to determine the relation-ship between bioactivities and chemical structure of each compound in the A. numidica extracts.

ACKNOWLEDGEMENTS:

This work was funded by the Algerian Ministry of Higher Education and Scientific Research. We greatly acknowledge Department of Natural and Life Sciences, Faculty of Sciences, University of Mohamed Boudiaf-M'sila and Laboratory of Applied Microbiology, Department of Microbiology, Faculty of Natural and Life Sciences, University Ferhat Abbas-Setif, Algeria for their constant encouragement.

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Cite this article:

Ghadbane Mouloud, Bounar Rabah, Khellaf Rebbas, Medjekal Samir, Belhadj Hani, Benderradji Laid, Smaili Tahar, Harzallah Daoud (2016), ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ENDEMICTREE ABIES NUMIDICA GROWING IN BABOR MOUNTAINS FROM ALGERIA, Global J Res. Med. Plants & Indigen. Med., Volume 5(1): 19-28

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Source of Support: Algerian Ministry of Higher Edu-cation and Scientific Research

Conflict of Interest: None Declared