Antioxidant and Antibacterial Activities of Combretum nioroense Aubrév. Ex Keay (Combretaceae)

In this study, the antioxidant and antibacterial activities of acetone extract, ethyl acetate, n-butanol and n-hexane fractions of acetone extract from leaves of Combretum nioroense Aubrév. ex Keay were investigated. The total phenolics and total flavonoids contents in the fractions and acetone extract were determined by spectrophotometric methods using Folin-Ciocalteu and AlCl₃, respectively. Two methods were adopted to assess the antioxidant activities: the Ferric Reducing Antioxidant Power (FRAP) and the radical scavenging activity of 2, 2’-azinobis (3-ethylbenzothiozoline-6-sulfonate) radical cation (ABTS). The Minimum Inhibitory Concentrations (MICs) of the extract and fractions against pathogenic bacteria (4) and serotyped bacteria (4) from American Type Culture Collection (ATCC) were also determined using the agar-well diffusion method. The results showed that the butanol fraction, with the highest phenolic content, exhibited the best antioxidant and antibacterial activities as compared to the ethyl acetate fraction which contains more flavonoids.

INTRODUCTION

Infectious diseases caused by bacteria, viruses, fungi and other parasites are major causes of death in humans in spite of the enormous progress recorded by the modern medicine. The World Health Organization (2002) and UNAIDS (2007) reported that between 14 and 17 million people die each year due to infectious diseases. In developing countries, infectious diseases cause 43% mortality as against 1% in developed countries. In traditional medicine, Combretum species are used for many medicinal purposes such as diarrhea, pneumonia, gonorrhea, syphilis, malaria, hypertension and even cancer (Nacoulma, 1996; Fyhrquist et al., 2002).

Combretaceae is a large family with at least 475 species in the world, 244 species in Tropical Africa and 20 species in Burkina Faso (Thiombiano, 2005). Some species of the Combretaceae family have been investigated and have revealed to possess a large number of bioactive compounds and to exert strong antioxidant and antimicrobial activities. Among those plants are Combretum micranthum, Combretum erytrophyllum, Terminalia catappa (Martini and Eloff, 1998; Karou et al., 2005; Kloucek et al., 2005). Combretum nioroense, also a specy of this family, is less known; literature relates very few about its chemical structures and biological activities. In Burkina Faso, Combretum nioroense is localized in the eastern part of the country. It is used to fight against many diseases associated with microorganisms and the decoction of leaflets is used as drink for new born babies (Thiombiano, 2005).

The screening of plant extracts and plant products for antimicrobial activity has shown that higher plants represent a potential source of new anti-infective agents (Salvat et al., 2001; Ordoñez et al., 2003; Arias et al., 2004). Among the biological potential of medicinal plants, the antioxidant activity has gained an increase interest these last years because of the role they play in the prevention of chronic ailments such as heart disease, cancer, diabetes, hypertension, stroke and Alzheimer’s disease by combating oxidative stress (Liu, 2003; Riboli and Norat, 2003; Cole et al., 2005).

The aim of the present study was to evaluate the antioxidant and the antibacterial activities of acetone extract, n-hexane, ethyl acetate and n-butanol fractions of acetone extract of the leaves of Combretum nioroense.

MATERIALS AND METHODS

Chemicals: All reagents were of analytical grade: Folin Ciocalteu-reagent, NaH₂PO₄, Na₂HPO₄, sodium carbonate, aluminium trichloride (AlCl₃), gallic acid and quercetin were purchased from Sigma-Aldrich Chemie (Steinheim, Germany). 2, 2’-azinobis (3-ethylbenzothiazoline-6-sulphonate) ABTS; trichloroacetic acid, potassium persulfate, acetone, methanol, n-hexane, ethyl acetate and n-butanol were supplied by Fluka Chemie (Buchs, Switzerland). Potassium hexacyanoferrate [K₃Fe(CN)₆] was from Prolabo (Paris, France); ascorbic acid and iron trichloride were supplied by Labosi (Paris, France).

Plant material: The leaves of Combretum nioroense aubrev ex keay were collected in the Namounou region of Burkina Faso in June 2008 and identified by Prof. Millogo-Rasolodimby, a botanist from the University of Ouagadougou. Voucher specimen (CRV 01) was deposited in the Herbarium of Laboratoire de Biologie et d’Ecologie végétales UFR/SVT, University of Ouagadougou.

Preparation of plant extracts: Air-dried and powdered leaves (50 g) of Combretum nioroense were extracted by maceration at room temperature with 500 mL of acetone, during 48 h. The extract was subsequently filtered and concentrated to dryness at 40C under vacuum. Extract yield with respect to the dried plant was 6.5%. The dry residue (270 mg) was subjected to successive liquid-liquid fractionation (Eloff, 1998). Three fractions were obtained: the n-hexane fraction, the ethyl acetate fraction, the butanol fraction. Solvents have been eliminated and the different residues obtained were used for different biological activities.

Phytochemical screening: The phytochemical screening was conducted with the acetone extract for alkaloids, tannins, flavonoids, saponins, triterpenoids, steroids and coumarins using the method described by Ciulei (1982).

Determination of total phenolics and total flavonoids contents: The total phenolics of plant extract were determined by the Folin-Ciocalteu method (Lamien-Meda et al., 2008). The diluted aqueous solution of each extract (0.5 mL) was mixed with Folin Ciocalteu reagent (0.2 N, 2.5 mL). This mixture was allowed to stand at room temperature for 5 min and then sodium carbonate solution (75 g L^-1 in water, 2 mL) was added. After 2 h incubation, the absorbencies were measured at 760 nm against water blank. A standard calibration curve was plotted using gallic acid (0-200 mg L^-1). The results were expressed as mg of gallic acid equivalents GAE g^-1 of extract or fractions.

The total flavonoids were estimated according to the Dowd method as adapted by Lamien-Meda et al. (2008). A diluted methanolic solution (2 mL) of each extract was mixed with 2 mL of aluminium trichloride (AlCl₃) in methanol (2 %). The absorbance was read at 415 nm after 10 min against a blank consisting of 2 mL of methanol and 2 mL of plant extract (without AlCl₃). Quercetin was used as reference to produce the standard curve and the results were expressed as mg of quercetin equivalents (QE) g^-1 of extract or fractions.

Antioxidant activity
Iron (III) to iron (II) reduction activity (FRAP): The total antioxidant capacity of the plant extract was determined using the iron (III) reduction method (Hinneburg et al., 2006). One milliliter of the diluted aqueous solution of plant extract, at a concentration of 100 μg mL^-1, was mixed with phosphate buffer (0.2 M, pH 6.6, 2.5 mL) and 1% aqueous potassium hexacyanoferrate [K₃Fe(CN)₆] solution (2.5 mL). After 30 min incubation at 50C, 2.5 mL of a trichloroacetic acid 10 %, was added and the mixture was centrifuged at 3000 rpm for 10 min. Then, the upper layer solution (2.5 mL) was mixed with water (2.5 mL) and aqueous FeCl₃ (0.1 %) solution (0.5 mL). The absorbance was read at 700 nm and ascorbic acid was used to produce the calibration curve. The iron (III) reducing activity determination was expressed in mmol ascorbic acid equivalents per gram of extract or fractions.

ABTS radical cation decolorization assay: The radical scavenging capacity of antioxidants for the ABTS (2, 2’-azinobis-3-ethyl-benzothiazoline-6-sulphonate) radical cation was determined as described by Lamien-Meda et al. (2008). ABTS^•+ was generated by mixing a 7 mM aqueous solution of ABTS with 2.5 mM potassium persulfate (final concentration) followed by storage in the dark at room temperature for 12 h before use. The mixture was diluted with ethanol to give an absorbance of 0.700.02 units at 734 nm using spectrophotometer.

The diluted methanol solution of the extract (10 μL) was allowed to react with fresh ABTS^•+ solution (990 μL) and then the absorbance was measured 6 min after initial mixing. Ascorbic acid was used as a standard and the capacity of free radical scavenging was expressed as mmol ascorbic acid equivalents g^-1 of extract or fractions. Quercetin and gallic acid were used as positive controls.

Antibacterial study
Microorganisms: The microorganisms used in this study consisted of clinical isolates and collection/serotyped strains. The clinical isolates were obtained from biomedical laboratories. They were: Escherichia coli, Salmonella sp., Klebsiella pneumoniae, Staphylococcus aureus. The following serotyped strains used in this study are: Escherichia coli ATCC 25922; Salmonella typhimurium ATCC 13311; Staphylococcus aureus ATCC 6538 and Proteus mirabilis ATCC 35659. Before testing, pure cultures were realized with all the strains in Mueller Hinton Agar and Tryptic Soy Broth. The inocula were prepared by adjusting the turbidity of the suspension to match the 0.5 Mc Ferland standard.

Antibacterial tests: The agar-well diffusion method (Ojala et al., 2000) was used to evaluate the antibacterial activity. Minimum Inhibitory Concentrations (MICs) of the extract and fractions of Combretum nioroense were determined using the agar-well diffusion method. All the extract and fractions were diluted in Dimethyl Sulfoxide (DMSO) 25% to obtain series of concentrations of 20, 10, 5, 2.5, 1.25 and 0.625 mg mL^-1. The MIC was taken as the lowest concentration of extract or fractions that caused a clear to semi-clear inhibition zone around the hole after 24 h incubation at 37C.

Sterile petri dishes (d = 10 cm, Bibby Sterilin, UK) were prepared with a base layer of Müller-Hinton agar (Difco). Bacteria at density of 10⁶-10⁸ cfu were inoculated on solid agar. Holes (6 mm) were made in the agar with a sterile cork borer and filled with 50 μL of different dilutions of the extract and fractions. Petri dishes were incubated at 37C for 24 h. The diameters of the circular inhibition zones obtained were measured. Commercial antibiotic discs of Gentamicin and Amoxicillin were used as positive controls. DMSO 25% was used as a negative control.

Statistical analysis: Data are expressed as XSE (n = 5). Significant differences are determined by the Newman- Keuls Test Pharmacological Calculation System.

RESULTS AND DISCUSSION

The phytochemical screening revealed the presence of flavonoids, sterols/triterpens and tannins in the acetone extract of the leaves. The presence of these metabolites in some Combretum species has already been reported by Nacoulma (1996), Baba-Moussa et al. (1999), Simon et al. (2003) and Batawila et al. (2005).

Combretum nioroense leaves extracted with acetone yielded 6.5%. Three solvents of different polarities: n-hexane, ethyl acetate and n-butanol were used to extract phenolic compounds from the acetone extract. Yields of fractions are shown in Table 1.

Ethanol, methanol and acetone are the commonly used solvents to extract chemical compounds of the plant. Sun and Ho (2005) reported that acetone extracts more phenolic compounds than methanol and ethanol.

The total phenolics and total flavonoids contents in the acetone extract and the different fractions are consigned in Table 1. Table 1 shown that n-butanol extracted more phenolic compounds (375 mg GAE g^-1) than any other solvent. Table 1 also showed that the highest amount of flavonoids is found in the ethyl acetate fraction (239 mg QE g^-1). n-hexane, a much less polar solvent, contains the lowest quantity of phenolic and flavonoid compounds. The results showed that the acetone extract of leaves of Combretum nioroense contained 204.216.1 mg GAE g^-1 of extract (i.e. , 20.42% of the total extract) of phenolic compounds as against 37.801.04% in the leaves of Combretum micranthum (Karou et al., 2005). These results showed that Combretum micranthum contains more phenolic compounds than Combretum nioroense. However many research works reported that the contents in metabolites, phenolic and flavonoid compounds as well, are related to the nature of the soil and the microclimate (Millogo-Kone, 2008). The leaves of Combretum nioroense and those of Combretum micranthum were harvested almost at the same period of the year (June-August) but on different areas. Kana et al. (2005) demonstrated that flavonoid contents increased in October to December, with the tendency to decrease in May to June. Chaves et al. (1997) demonstrated that UV irradiation was the major inducer of the enhanced flavonoid secretion in the exudate. These results underline the ecophysiological role of the flavonoids in the protection of the plant against the damaging effects of UV irradiation.

Table 1:	Total phenolics and total flavonoids in extract/fractions of Combretum nioroense
AE: Acetone extract, EF: Ethyl acetate fraction, BF: Butanol fraction, HF: Hexane fraction. Different letters in the same column indicate significant difference (p<0.01)

Antioxidant activities of acetone extract and the various fractions from acetone extract were determined by using two test systems: the iron (III) to iron (II) reduction method (FRAP) and the ABTS method which consists in measuring the relative antioxidant ability of extract and fractions to scavenge the radical cation ABTS^.+ produced by the oxidation of 2, 2’-azinobis (3-ethylbenzothiazoline-6-sulphonate).

Statistical studies revealed that there is no significant difference between the results obtained with butanol fraction following the two methods (Table 2). Also, one can notice that with both methods (FRAP and ABTS), the highest antioxidant activity was obtained with the butanol fraction (1.860.13 mmol g^-1 through FRAP method and 1.800.41 mmol g^-1 for ABTS method) followed by the acetone extract (1.580.02 and 0.960.28 mmol g^-1, respectively for FRAP and ABTS methods), the ethyl acetate fraction (1.040.02 and 0.710.11 mmol g^-1, respectively for FRAP and ABTS methods) and the n-hexane fraction (0.430.02 and 0.240.08 mmol g^-1, respectively for FRAP and ABTS methods). Recent investigations have shown differences between the test systems for the determination of antioxidant activity (Schlesier et al., 2002; Nsimba et al., 2008). It was therefore recommended to use at least two methods, what we did.

Table 2:	Antioxidant activity of extract or fractions obtained from Combretum nioroense
AE: Acetone extract, EF: Ethyl acetate fraction, BF: Butanol fraction, HF: Hexane fraction. ABTS: 2, 2’-azinobis-3-ethyl-benzothiazoline-6-sulphonate, FRAP: Iron (III) to iron (II) reduction activity. Different letters in the same column indicate significant difference (p<0.01)

Several reports revealed relationship between antioxidant activity and the total phenolics and total flavonoids contents (Anna et al., 2003; Negro et al., 2003; Ramandeep and Geoffrey, 2005). The results of the present study confirmed these reports; butanol fraction contained the highest amount of phenolics and it exerted the highest antioxidant activity. It was also reported that antioxidant activity could be related to the nature of phenolics (Singh et al., 2007; Yu et al., 2007).

The results consigned in Table 3 are related to the diameters of the zones of inhibition. Table 3 shown that gentamicin and amoxicillin, two broad spectrum antibiotics, exerted strong inhibitions against the tested strains E. coli, S. aureus ATCC 6538 and S. typhimurium. But clinical isolate S. aureus showed resistance to the two antibiotics while it displayed susceptibility to all the extract and fractions. Indeed, with clinical isolate S. aureus the acetone extract, the ethyl acetate fraction and the butanol fraction gave, respectively 12, 12.5 and 12 mm. Those values showed no significant difference. Though efficient against all the tested strains, the butanol fraction exhibited a low antibacterial activity against E. coli. The butanol fraction was more effective than all the tested extracts while the hexane fraction had revealed to be the less active fraction. There was no significant difference (p<0.05) between the acetone extract and the ethyl acetate fraction against the clinical isolates and even for E. coli ATCC 25922.

The butanol fraction gave the lowest Minimal Inhibitory Concentrations (MIC). A very low MIC corresponds to a very strong antibacterial activity. The highest MICs (ranging from 10-20 mg mL^-1 and even more) were obtained with E. coli which means that this strain is the less sensitive to the tested extract and fractions. S. aureus 6538 and P. mirabilis ATCC 35659 showed very sensitive to all the tested extracts (Table 4). The resistance of clinical isolate S. aureus to many antibiotics had been demonstrated by Zéba (2006). Many research works had been conducted on methicillin resistant S. aureus and showed that medicinal plants exerted inhibition against methicillin resistant Staphylococcus strains.

Table 3:	Antibacterial effects of Combretum nioroense extract or fractions at 20 mg mL-1
AE: Acetone extract, EF: Ethyl acetate fraction, BF: Butanol fraction and HF: Hexane fraction, R: Resistant. -: Non determined. The values of Table 3 represent the diameters of the zones of inhibition in mm. The diameter of the hole is 6 mm

Table 4:	Minimum inhibitory concentrations (MIC) in mg mL-1
AE: Acetone extract, EF: Ethyl acetate fraction, BF: Butanol fraction and HF: Hexane fraction

Indeed, Voravuthikunchai and Kitpipit (2005) tested 10 medicinal plants against 35 clinical isolates of methicillin S. aureus and a serotyped S. aureus ATCC 25923. Nine out of the 10 medicinal plants showed effectiveness with MICs ranging from 0.05 to 3.2 mg mL^-1. In the literature, no antimicrobial study had been reported on Combretum nioroense but it was reported that many Combretum species have medical applications against various bacterial infections such as gonorrhea, syphilis and diarrhoea (Fyhrquist et al., 2002). Martini et al. (2004) showed that Combretum erythrophyllum exerted antimicrobial activity against Gram-positive and Gram-negative bacteria. They isolated, by bioassay-guided fractionation, seven flavonoids (apigenin, genkwanin, 5-hydroxy-7,4 dimethoxyflavone, rhamnocitrin, kaempferol, quercetin-5,3’-dimethylether, rhamnocitrin, rhamnazin) and demonstrated that all of them had good activity against Vibrio cholerae and Enterococcus faecalis.

CONCLUSION

The results obtained through this study clearly demonstrated that all the tested extract/fractions of Acetone extract and various fractions from acetone extract of Combretum niororense Aubrév. ex keay exerted antioxidant and antibacterial activities at different levels. The butanol fraction obtained from the acetone extract possessed the highest phenolic contents and demonstrated the best antioxidant and antibacterial activities. Those extracts had shown effectiveness against microorganisms responsible of infectious diseases thus justifying the successful use of Combretum nioroense for the treatment of diarrhea, dysenteries in traditional medicine.

Further investigations will be performed by:

ACKNOWLEDGMENTS

We are grateful to the International Foundation for Science (IFS) and also to the International Atomic Energy Agency (IAEA) for providing the facilities.