Key Words

Acacia hockii, antifungal, antibacterial, antioxidant, phytochemical

Introduction

Many strains of bacteria and fungi pose a very serious public health problem all over the world especially in resource poor African countries [1]. Currently, the prevalence of microorganism showing resistance to antibiotics increases dramatically. Infectious diseases are indeed the source of more than 17 million deaths a year worldwide, including more than half in African continent [2]. Thus, there is an urgency to develop novel antimicrobial compounds. Traditional Medicine using medicinal plants constituted a good alternative for the discovery of new antimicrobial compounds. The use of medicinal plants has long been an interaction between human and his environment. Particularly in developing countries, traditional medicine using plants to provide health care cover more than 80% of the population [3]. These medicinal plants are rich sources of antimicrobial agents. Medicinal plants have become in recent years an area of interest because of their effectiveness and accessibility.It is important to note that efficacy of this plants is due to the presence of secondary metabolite known as phytochemicals [4]. The objective of the present work was to investigate the antimicrobial and antioxidant activities of aqueous and ethanolic extracts of leafy stems of Acacia hockii collected from Tanguiéta (North-Benin) against a range of microorganisms including Gram-positive, Gram-negative bacteria and fungi.

Materials and Methods

Plant material

The leafy stems of Acacia hockii were harvested in April 2015 from Tanguiéta, department of Atacora in North Benin. They were authenticated by Professor YEDOMONHAN Hounnankpon, botanist from University of Abomey-Calavi. Specimen was deposited in the National Herbarium of University of Abomey-Calavi in Benin under the identification code (AA6615/HNB). The samples were dried in the laboratory conditions (22 ± 3°C) and then reduced to powder using an electric mill (MARLEX)

Preparation of plant extracts

Acacia hockii powder (200 g) was extracted with 1 L of distilled water using a heating mantle with temperature controller (Electromantle MA Solid State Stirrer, 60°C). After 30 min of extraction, the resulting mixture was filtered through Whatman paper. The residue obtained was again extracted two (02) times using same procedure. The resulting filtrate was concentrated using a rotary evaporator and the extract was stored at 4°C. For solvent extraction, the air-dried powder of Acacia hockii (200 g) was extracted with ethanol (850 ml) by mechanical stirring for 24 hours at laboratory temperature (22 ± 3°C). The mixture obtained after maceration was filtered through Whatman paper (Qualitative Circles 150 mm Cat No. 1001 150). The same extraction was repeated twice (02) during 1 hour. The filtrates were concentrated under reduced pressure using a rotary evaporator (Buchi Rotavapor R II).

Preliminary Phytochemical Screening

Aqueous and ethanolic extracts of Acacia hockii were subjected to preliminary phytochemical screening to verify the presence of chemical constituents. Tannins, alkaloids, flavonoids, steroids, coumarins, saponins, naphthoquinones, triterpenes, lignans, pigments, anthracene derivatives have been investigated as described previously [5, 6].

Antibacterial activity

Tested microorganisms

Each of both extracts of Acacia hockii was tested against a microorganism panel, including Gram positive bacteria: Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis CIP8039 and Staphylococcus aureus methicillin resistant (SARM) and Gram negative bacteria: Escherichia coli CIP 53126, Pseudomonas aeruginosa CIP 82118,) obtained from the laboratory of Biophotonics and Pharmacology, University of Strasbourg in France. The microorganisms were maintained on agar media at 4°C and subcultured for 24 h before use.

Growth inhibition effect of extracts at 10 mg/ml

Before evaluating the antibacterial properties of Acacia hockii aqueous and ethanolic extracts, a first assay was carried out at a concentration of 10 mg/ml. This assay is used generally to eliminate the extracts which are not active at this concentration [7]. The growth inhibitory effect of extracts against bacteria was evaluated by the microplate method using p-iodonitrotetrazolium (INT) as viability indicator [8]. The inhibition of growth is evaluated by the mixtures color change in the wells. Active extract do not change color.

Minimum Inhibitory Concentration (MIC) and total activity

The minimum inhibitory concentration (MIC) of plant extracts was determined according to the microdilution method using p-iodonitrotetrazolium (INT) as an indicator of bacteria viability [9]. Briefly, bacteria were incubated for 18 h with extracts at various concentrations ranging from 5 to 0.078 mg/ml. INT was used as indicator of bacteria viability. After 1 hour of incubation with INT, the MICs, which are the lowest concentration of extract that inhibits bacterial growth, were determined. The total activity of each extract was calculated by dividing the MICs with the amount of extract obtained from 1 g of plant material [10]. The total activity indicates the volume in which the active extract can be diluted without losing its inhibitory effect on bacteria growth. [11].

Antifungal assay

Test organisms

The antifungal activity of Acacia hockii extracts was investigated against three fungal strains of the genus Aspergillus: Aspergillus parasiticus CMBB20, Aspergillus ochraceus CMBB91 and Aspergillus clavatus NCPT24, obtained from the laboratory of biochemistry and molecular biology at the University of Abomey-Calavi.

Antifungal assay

The antifungal activity of extracts was assessed against mycelia development and sporulation of fungi as described previously [12]. Briefly, the extracts were reconstituted to a concentration of 1 mg/ml in the culture medium (Potato Dextrose Agar). 10 ml of the mixture were poured into sterile petri dishes. After 15 minutes of polymerization, 100 spores prepared in the tween (5%) were deposited in the center of the petri dishes which were then incubated at 25 °C for 5 days. All assays were performed in triplicate and fluconazol (100 µg/ml) was used as positive control. The activity of the extracts was evaluated by determining the percentage of inhibition (PI) against mycelial growth and sporulation of fungi. The percentage of inhibition was calculated for each extract according to the formula below:

In which Av control and represent the average diameter of the mycelia or estimated number of spores of control and Av tested extract the average diameter of the mycelia or estimated number of spores in petri dishes contained extracts.

DPPH Radical-Scavenging Activity

The antioxidant assay of extracts was evaluated as described previously [13]. A range of eight concentrations of extracts obtained by two-fold dilution (100 to 0.78 µg/ml ) was tested in this assay. The reaction mixture consists of 0.75 ml of extract and 1.5 ml of the 2% solution of DPPH in methanol. The blank sample consists of 0.75 ml of methanol and 1.5 mL of DPPH (2%). Ascorbic acid was used as positive control. All tests were performed in triplicate. The mixture was incubated in the dark at room temperature. After 15 min of incubation, absorbencies were read at 517 nm with a spectrophotometer (VWR UV- 1600PC). The inhibition percentage (IP) of DPPH radical was determined according to the formula below: IP (%) = [(Ab – As)/ Ab] x 100, were As is the sample absorbance (tested extract solution) and Ab is the blank absorbance.

Statistical analysis

Data were presented as mean ± SD. The graphical representation of the data was performed using the Graph Pad Prism 5.0 software (Microsoft, USA).

Results and Discussion

Phytochemical results

Biological activity of plant extracts depends strongly on the type and amount of active compounds. Phytochemical analysis of aqueous and ethanolic extracts of Acacia hockii leafy stems are presented in Table 1. The phytochemical analysis revealed the presence of all the searched groups of secondary metabolites in the ethanol extract except naphthoquinones. The aqueous extract also contains all secondary metabolites except coumarins and naphthoquinones. Our results are consistent with previous study in which flavonoids, tannins, saponins and triterpenes were revealed in the ethanol extract of the leaves of Acacia hockii [14]. As far as we know, few chemical data on Acacia hockii was available in the literature. However, previous studies of related species revealed the presence of saponin and tannins in Acacia sieberiana [15], alkaloids, carbohydrates, saponins, Tannins, Flavonoids, cardiacglycosides and anthraquinone in Acacia nilotica [16]. Other related species have also confirmed the presence of secondary metabolites similar to those found in Acacia hockii [17]. However, some differences were observed. This could be attributed to the species and phenology of the study sites [6].

Antibacterial activity

Growth inhibition effect of extracts at 10 mg/mL

The antibacterial activity of aqueous extracts and ethanol (10 mg/ml) of A. hokii against tested bacteria was reported in Table 2. Both extracts showed strong antibacterial activity by inhibiting growth of Gram positive (Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus aureus methicillin resistant) and Gram negative (Escherichia coli and Pseudomonas aeruginosa). These results could be justified by the presence in the extracts of some groups of compounds such as tannins, saponins, terpenoids. The antimicrobial activity of these secondary metabolites was reported [18].

Minimum Inhibitory Concentrations (MIC) and Total Activity

The Minimum Inhibitory Concentrations (MIC) and Total Activity of extracts are recorded in table 3. The results of antibacterial activity showed that the MICs ranged from 0.62 mg/ml to 5 mg/ml. Ethanolic extract was the most active extract with a MIC of 0.62 mg/ml against P. aeruginosa followed by the aqueous extract with a MIC of 1.25 mg/ml of against S. epidermidis. No antibacterial activity of A. hockii is found in the literature. However, the anti-inflammatory and Antipyretic Activities have been assessed [19, 20]. In the present study, the antimicrobial activity of A. hockii extracts could be attributed to the presence in these extracts of actives compounds such as flavonoids, terpenoids, alkaloids, coumarins and tannins [21-24]. Flavonoids are known as antimicrobial agents through various mechanisms like inhibition of nucleic acid synthesis, inhibition of cytoplasmic membrane function and energy metabolism [25]. Antimicrobial activity of terpenoids is associated with the rupture of the bacterial cell membrane [26]. Forthermore, coumarins represent a large group of compounds that have been reported to possess a wide range of biological activities including antimicrobial [27, 28]. Tannins are known antimicrobial agents that could inhibit the growth of microorganisms by precipitating the microbial protein [29]. The presence of these active principles could explain the antibacterial activity of Acacia hockii extracts. The antibacterial activity of actives extracts were also quantified by dividing the mass of extract from 1 g of the plant material by the MIC value. Our results showed that the total activity is depending on the extraction solvent and to the tested bacterial. The highest total activity was obtained with ethanolic extract against P. aeruginosa (TA = 201.76 ml) while lower activity was obtained with aqueous extract against Staphylococcus aureus methicillin resistant with a total activity of 29.45 ml.

Antifungal assay

The antifungal activity of Acacia hockii was investigated on mycelial growth and sporulation of three Aspergillus strains. The inhibitory effects of extracts are shown in Tables 4 and 5. The Percentage of Inhibition (PI) of extracts on mycelia growth ranging from 36.62% to 78.80% (Table 4). Ethanolic extract was the most active with a PI values of 78.80% against A. ochraceus, 44.56% against A. parasiticus and 36.62% against A. clavatus. The aqueous extract was moderately active against A.ochraceus (IP = 65.84%), A. parasiticus (IP = 54.05%) and A. clavatus (IP = 45.50%). At the same concentration, antifungal activity of A. hockii extracts was more pronounced on sporulation than the mycelial growth. The Inhibitory Percentage of extracts against sporulation of fungi ranged from 61.36 to 95.88% (Table 5). Both extracts (aqueous and ethanolic) showed interesting activity against A parasiticus (IP = 95.88%) and A. ochraceus (IP = 94.57%) respectively. The antifungal activity of A. hockii extracts could be justified by the presence of terpenoids, flavonoids, coumarins and tannins known for their antifungal properties [25, 30-32].

Antioxidant activity

In the present study, in vitro antioxidant activity of aqueous and ethanolic extracts of Acacia hockii was investigated using diphenylpicrylhydrazyl (DPPH) scavenging assay. DPPH is a stable free radical which accepts an electron or hydrogen radical to become a stable diamagnetic molecule [33]. Antioxidants induce a reduction of DPPH radicals causing a decrease of the absorbance as a result of a visual discoloration from purple to yellow. Interestingly, results of Figure 1 shows that both extracts of Acacia hockii exhibited an inhibition of DPPH at dose dependent. Both extract, ethanolic and aqueous, showed higher scavenging activity of 97.47% and 95.66% respectively at 100 µg/ml. In comparison, the ethanolic extract showed the highest radical scavenging effect than the aqueous extract. This observation could be attributed to the difference in the degree of solubility of the bioactive compounds in the two solvents which guaranteed the extraction of more phytochemicals compounds in the ethanolic than in the aqueous extract. These phytochemicals would be probably the flavonoids, coumarins and tannins. The antioxidant activity of these secondary metabolites was demonstrated [34-36]

Table 1: Phytochemical constituents of leafy stem of A. hockii

Plant extracts
Phytochemical components EtOH H2O
Alkaloid + +
Coumarin +
Naphtoquinone
Lignan + +
Flavonoid + +
Pigment + +
Saponin + +
Tannin + +
Triterpene + +

EtOH: ethanol, H2O: aqueous; (+) = present and (-) = indicates the absence of the compound tested.

Table 2: Antibacterial activity of A. hockii extracts at 10 mg/ml

Growth inhibition effect of extract at 10 mg/ml
Gram (+) Gram (-)
Extracts S. a S.a.m.r S. ep E. f P. a E. coli
Ethanol + + + + + +
Aqueous + + + + + +

+: sensitive ; S.a.m.r :Staphylococus aureus meticillin resitant ; S.ep : Staphylococus epidermidis ; P. a : Pseudomonas aeruginosa ; S. a : Staphylococcus aureus

Table 3: Minimum Inhibitory Concentration and total activity of A. hockii extracts

Minimum Inhibitory Concentration (mg/ml)
Gram (+) bacteria Gram (-) bacteria
Extracts S.aureus S.a.m.r S. epidermidis E.faecalis P.aeruginosa E. coli
Ethanol 1.25 1.25 1.25 1.25 0.62 1.25
Aqueous 5 >5 1.25 5 2.5 2.5
Gentamicin 0.31 0.15 0.15 0.31 0.31 0.31
Total activity (ml/g)
Ethanol 100.88 100.88 100.88 100.88 201.76 100.88
Aqueous 29.45 ?29.45 117.82 29.45 58.91 58.91

S.a.m.r :Staphylococus aureus meticillin resitant

Table 4: Inhibitory effect of A. hockii extracts against mycelia growth of fungi.

Inhibition percentage of mycelia growth (%)
Fungi Extracts A. parasiticus A.clavatus A. ochraceus
EtOH 44.56 ± 1.94 36.62 ± 1.69 78.80 ± 0.00
Aqueous 54.05 ± 1.46 45.50 ± 1.69 65.84 ± 1.57
Fluconazol 65.72 ± 0.97 70.84 ± 1.69 84.68 ± 1.57

Table 5: Inhibitory effect of A. hockii’s extracts against sporulation of fungi.

Inhibition percentage of sporulation (%)
Fungi Extract A. parasiticus A.clavatus A. ochraceus
EtOH 72.64 ± 0.55 71.47 ± 1.42 94.57 ± 1.00
Aqueous 95.88 ± 0.59 88.51 ± 0.60 61.36 ± 0.63
Fluconazol 96.14 ± 0.29 92.35 ± 0.58 96.51 ± 0.23

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Figure 1: Radical scavanging activity of Acacia hockii extract

Conclusion

The present study revealed that aqueous and ethanolic extracts of Acacia hockii seeds possess antimicrobial property, which could be explored in the treatment of bacterial diseases and in control of some fungal infections. This study also showed that A. hockii extracts have antioxidant activity at dose dependent manner. Aqueous and ethanolic extracts of A. hockii could be useful in pharmaceutical and phytotherapeutic applications. However, Additional studies such as safety, active compounds isolation and structure elucidation should be carried out for more extensive investigations on Acacia hockii specie