Antimicrobial and antioxidant activities of the fruits of bemavo , a variety of Ravenala madagascariensis Sonn . ( Strelitziaceae )

This work was designed to evaluate the antibacterial and antioxidant activities of the fruit extracts from bemavo, a variety of Ravenala madagascariensis. Methanolic extracts from pericarp (PME), seed (SME) and aril (AME) were tested against 5 Gram positive and 6 Gram negative of pathogenic bacteria using disk diffusion and microdilution methods. At 1000 μg/ml, all extracts prevented selectively the growth of at least 5 of the tested bacteria with Inhibition Zone Diameter (IZD) ranking from 8.5 mm to 19 mm. The best activity was recorded with PME which was active against 81.8 % of the studied bacteria with 63.6 % of IZD higher than 14 mm: 15 mm on Yersinia enterocolitica, Salmonella enterica, Shigella flexneri, and Listeria monocytogenes, 16 mm on Staphylococcus aureus, 18 mm on Enteroacter aerogenes, and 19 mm on Vibrio fischeri. Clostridium perfringens and Bacillus cereus were resistant to the three extracts. The great majority (97 %) of MCI recorded were ≤1000 μg/ml, 84.8 % were ≤500 μg/ml, 12.1 % between 500 μg/ml and 1000 μg/ml. Enterobacter aerogenes was the most sensitive to the three extracts. The three extracts were bactericidal against Enterobacter aerogenes and Clostridium perfringens, bacteriostatic against Salmonella enterica and Bacillus cereus and bactericidal or bacteriostatic against the remaining strains. The antioxidant activity of extracts was determined by the method using free radical scavenging against DPPH. Compared with ascorbic acid (IC50=7.320 μg/ml), SME (IC50=0.568 μg/ml) and AME (IC50=3.792 μg/ml) were 12.9 and 6.5 times more active respectively. Phenolic compounds present in three extracts could be responsible for antimicrobial and antioxidant activities of the bemavo fruit methanol extracts.


Introduction
Infectious diseases remain the main cause of the high mortality rates recorded in the developing nations. Resistance of bacterial pathogens to antibacterial drugs is a global concern. The number of multi drug resistant microbial strains and the appearance of the strains with reduced susceptibility to antibiotics are continuously increasing.
Many diseases such as development of cell injury, aging, cardiovascular and neurodegenerative diseases, autoimmune disorders, rheumatoid arthritis and cancer are related to overproduction of free radicals and reactive oxygen species (ROS) [1,2,3].
World Health Organization (WHO) reported that about 80 % of the globe population rely on plants or its derivative products for the treatment of various sicknesses [4]. Search for new drugs with low cost, more potential, without adverse effects is being pursued in several laboratories all around the world. Bioactive secondary metabolites produced by plants have proven to possess a wide range of therapeutic effects for human diseases [5]. Considerable attention has been devoted to medicinal plants with antimicrobial and antioxidant properties [5,6].
With the richness of its biodiversity and the high level of its endemism (80 %), Madagascar flora is a potential source of medicinal plants. Ravenala madagascariensis (Strelitziaceae) is one of those endemic medicinal plants. Also known as the traveler's palm or traveler's tree, this plant has been considered as an iconic symbol of Madagascar [7]. It is naturalized in the Mascarene Islands and widely planted as an ornamental tree throughout the tropics [8,9]. In Madagascar, Ravenala madagascariensis is a multi-purpose plant, used primarily for construction (house, roof, fence etc.) but also as food, tools and medicines [10,11]. It is traditionally used to treat some diseases like, diabetes, inflammations, arterial hypertension, tooth decay [12,13] and wounds [14]. Investigations on Ravenala madagascariensis from other countries showed that aerial parts exhibited antimicrobial activity) [15,16], leaf extract has antioxidant activity [17]. However, it is noteworthy to emphasis that, in Madagascar, there are 4 varieties of the Ravenala genus (Malama, Hiranirana, Bemavo and Horonorona) which are different by their macromorphological characters, growth habits and habitat preferences [12,18,19]. However, in these works hereabove cited, the name of the variety was not mentioned.
The objective of this study was to evaluate the antibacterial and the antioxidant potential of the fruits of bemavo, a variety of Ravenala madagascariensis.

Plant material
Bemavo is a variety of Ravenala madagascariensis growing in open areas between 200 and 600 m of altitude [8]. It is single-stemmed, without offshoots at the base and with yellow leaf sheaths that was at the origin of its vernacular name (figure 1). Fruits were dried in the shade until their dehiscence. The fruits parts (pericarp, aril and seed) were separately ground into fine powders and then stored at -20 °C.

Bacterial strains
Eleven pathogenic strains supplied by the National Center for Application of Pharmaceutical Research (CNARP) including 5 Gram (+) and 6 Gram (-) were used (Table 1).

Preparation of the methanolic extracts
In a Soxhlet apparatus, exhaustive extractions were successively carried out with hexane, ethyl acetate and methanol from 100 g of pericarp, aril and seed powder. All solvent extracts were evaporated to dryness using a rotary evaporator at 40 °C under reduced pressure. Methanolic extracts of pericarp, (PME), aril (AME) and seeds (SME) were used for all tests.

Phytochemical screening
The qualitative chemical analysis of the different fruit extracts was carried out according to the methods used by Firdouse et al. [20].

Antibacterial assays
Antibacterial activities were assessed by measuring the Inhibition Zone Diameter (IZD) using the disk diffusion method [21,22] and determining the Minimum inhibitory (MIC) and the Minimum bactericidal concentration (MBC) using the microdilution method [23]. PME, AME and SME extracts dissolved in Dimethyl sulfoxide (DMSO) 10 % were used for all antibacterial assays.

Determination of IZD
Two (2) ml of inoculum, equivalent to 0.5 Mc Farland (10 8 CFU/ml), were spread on the surface of a solid medium of Mueller-Hinton Agar. Sterilized filter paper disks (6 mm diameter) (BioMérieux, REF54991) were impregnated with 10 µl of each extract. The concentration of each extract was 1 mg/ml /disk which is a concentration generally used to assess the antibacterial potential of plant extracts [24]. Impregnated disks were placed on the agar surface and incubated at 37 °C. After 24 h of incubation, IZDs were measured and results were interpreted according to the IZD scale of Ponce et al. [25] and Celikel and Kavas [26]: bacteria are not sensitive for IZD ≤ 8 mm; sensitive for 9 mm ≤ IZD ≤ 14 mm; very sensitive for 15 mm ≤ IZD ≤ 19 mm and extremely sensitive for IZD ≥ 20 mm. Imepenem was used as reference antibiotic and the experiments were carried out in triplicate.

Determination of antioxidant activity
The antioxidant capacity of PME, SME and AME, was evaluated by the method using free radical scavenging against DPPH (2, 2-Diphenyl-1-Pycryl Hydrazyl) [29]. One (1) ml of 0.004 % DPPH in methanol was added to 1 ml of methanol extract at different concentrations ranking from 0 to 120 µg/ml depending on the extract. In the case of free scavenging activity, the purple DPPH was reduced to a yellow compound. After 30 min of incubation at room temperature and in the dark, the optical density was measured at 517 nm with a spectrophotometer using methanol sample as blank. A mixture of 1ml of DPPH and 1 ml of methanol was used as negative control and ascorbic acid as positive reference. Assays were made in triplicate.
The decrease of the optical density was then converted into percentage of inhibition (I %), according to the equation hereafter: Free scavenging activity (I %) = 100 X Abs DPPH − Abs assay Abs DPPH With, Abs DPPH: Absorbance of DPPH at 517 nm.
Abs assay: Absorbance of extract solution in methanol at 517 nm The IC50 (in µg/ml) which is the extract concentration inducing the 50 % loss of DPPH antioxidant activity was determined by linear regression of I % values. The lower the IC50 value the higher the extract antioxidant activity [30].

Statistical analysis of data
The results obtained by the disk diffusion method were expressed as mean values ± standard deviations of three separate replicates.
One way analysis of variance (ANOVA) followed by Newman Keuls comparison test with statitcf software MS-DOS 6.21 was used for statistical analysis. Statistical estimates were done with a confidence interval of 95 %.

Phytochemical results
The chemical families present in the PME, SME and AME are shown in Table 2. Leucoanthocyanins, tannins, polyphenols and steroids were detected in the three extracts. Flavonoids, triterpenes and desoxyoses were present in SME and AME but not in PME. Quinones, saponins, irridoids and alkaloids were not found in the three extracts.

Antimicrobial activity
As shown in Table 3, active IZD ranked from 8.5 to 19 mm. Values are averages of three separate replicates ± standard deviations PME, SME and AME displayed antibacterial activity with selective effects. They were active against both Gram (+) and Gram (-) germs. Clostridium perfringens and Bacillus cereus were the only resistant strains to the three extracts.
PME was proved to be the most efficient extract: it was active against 9 of the 11 bacteria tested (81.8 %) with IZD ranking from 11 mm (Escherichia coli) to 19 mm (Vibrio fischeri) and the majority of IZD values (63.6 %) was higher than 14 mm. AME showed a moderate activity against 5 of the 11 bacteria tested (45.6 %) with IZD ranking from 10 mm (Yersinia enterocolitica and Shigella flexneri) to 12.5 mm (Salmonella enterica). SME was the least effective extract: against the 5 sensitive bacteria, IZD values ranked from 8.5 mm (Shigella flexneri) to 10 mm (Salmonella enterica and Vibrio fischeri).The three extracts were less efficient than reference antibiotic Imipenem at 10 µg/ml.
The values of MIC, MBC and the MBC/MIC ratios are presented in Table 4.
All the three extracts prevented the growth of the 11 bacteria tested. Enterobacter aerogenes was the most sensitive to the three extracts. The great majority of MIC values ranked from 100 µg/ml to 1000 µg/ml: 84.8 % were ≤ 500 µg/ml, 12.1 % between 500 µg/ml and 1000 µg/ml and 3.1 % higher than 1000 µg/ml.
The effect of PME, SME and AME was bactericidal (MBC/MCI ≤ 4) against Salmonella enterica and Bacillus cereus, bacteriostatic (MBC/MCI > 4) against Enterobacter aerogenes and Clostridium perfringens whereas variable according to the extract against the remaining strains.

Antioxidant activity
As ascorbic acid, PME, SME and AME had concentration dependent effects on the reduction of DPPH (Figure 2) and showed a highly significant (p= 0.0000) antioxidant activity.

Figure 2
Inhibition percentage (I %) of DPPH in the presence of different concentrations of PME, SME, AME and ascorbic acid (AC ASC), p= 0.0000.
The IC50 values, determined from the linear curve of free scavenging activity I % in terms of extract concentration in µg/ml, are presented in Table 5. Table 5 IC50 of Ascorbic acid, AME, SME and PME

Extracts
Ascorbic acid PME SME AME IC50 % in µg/ml 7.320 8.925 0.568 3.792 In comparison with ascorbic acid which is a pure product, AME and SME were more active and PME was somewhat less active. SME had the highest antioxidant capacity with an IC50 12.9 times less than ascorbic acid 6.7 and 15.7 times than AME and PME respectively.

Discussion
Preliminary phytochemical screening revealed that all fruit parts from the bemavo variety of R. madagascariensis contained the same chemical families (phenolic compounds and triterpenes). However, it did not provide information about the number, the nature and the respective amounts of each chemical group in each fruit part. These phytochemicals were also present in other parts (leaves and roots) of R. madagascariensis the variety name of which is unknown [31,3,16].
All the fruit extracts showed selective inhibitory activity against the tested bacterial strains. However in some cases, the disk diffusion and the microdilution assays gave different results. Bioactive compounds probably diffused little or not at all in solid medium. With disk diffusion method, PME was by far the most efficient extract. It clearly differed from SME and AME by the number of susceptible strains (9 of 11) and the inhibition level. However, with microdilution method there was no much difference between the 3 extracts. That might be due to the fact that PME contained active molecule(s) absent or in low amounts in AME and SME. In liquid medium, the activity of these molecules might be prevented by antagonist interactions which were low or non-existent in solid medium.
With disk diffusion Vibrio fischeri (IZD = 19 mm) was the most susceptible to PME, Salmonella enterica (IZD = 12.5 mm) to AME and Salmonella enterica and Vibrio fischeri (IZD = 10 mm) for SME. Enterobacter aerogenes (MIC = 125 µg/ml) was the most sensitive to the three extracts with microdilution assay. According to Fabry et al. [32], with MIC values below 8 mg/ ml, PME, SME and AME were considered having noteworthy antibacterial activity against all the tested bacterial strains. In reference to the more restrictive scale of Dalmarco et al. [33], 85 % of the bemavo fruit extracts had moderate (CMI ≤ 500 µg/ml) and 12 % low (500 µg/ml < CMI ≥ 2000 µg/ml ) antibacterial activity.
Comparison of our results with those of other authors was not easy because the extracts, the antibacterial assays and the bacterial strains used were different. PME, SME and AME (great majority of CMI ≤ 1 mg/ml) were by far more efficient than leaf extract from R. madagascariensis of Nigeria (75 mg/ml ≤ CMI ≥ 150 mg/ml) [16]. They also were more active (9 mm ≥ IZD ≤ 29 mm) than leaf extract from Bangladeshi R. madagascariensis (IZD < 9 mm) [15].
The effect nature of PME, SME and AME depended upon the bacteria but bactericidal activity (MBC/MCI >4) was the most recorded.
All extracts exhibited a highly significant (p = 0.0000) scavenging activity against DPPH. In comparison with ascorbic acid (IC50 = 7.320 µg/ml) used as reference antioxidant, the bemavo fruit extracts displayed high antioxidant activities: PME (IC50 = 8.925 µg/ml); SME (IC50 = 0.568 µg/ml) and AME (IC50 = 3.792 µg/ml), the highest scavenging activity against the 1,1-dipheny1-2-picrylhydrazyl radical, Comparison of the IC50 of PME, SME and AME with those of other plant crude extracts determined by DPPH assay is shown in Table 6.  [30] According to the literature, the antioxidant and antimicrobial activities were correlated with phenolic compounds [36,6]. The antibacterial and antioxidant activities of the bemavo fruit could therefore be induced by those secondary metabolites present in its pericarp, aril and seeds.
Further chemical and biological investigations will still be needed to determine the number and the identities of the active principles responsible for the antibacterial and the antioxidant properties.

Conclusion
In conclusion, all fruit parts of R. madagascariensis possessed an antibacterial activity which confirmed its traditional uses as antiseptics and to treat diarrhea and tooth decay. They could constitute sources of natural antibacterial agents and antioxidants to treat diseases related to oxidation stress.
Investigations on the other Ravenala varieties, Malama, Hiranirana and Horonorona are ongoing.