Antibacterial and Antioxidant Properties of the Methanolic Extract of the Stem Bark of Pteleopsis hylodendron (Combretaceae)

Pteleopsis hylodendron (Combretaceae) is used in Cameroon and West Africa folk medicine for the treatment of various microbial infections (measles, chickenpox, and sexually transmitted diseases). The antibacterial properties of the methanolic extract and fractions from stem bark of Pteleopsis hylodendron were tested against three Gram-positive bacteria and eight Gram-negative bacteria using Agar-well diffusion and Broth microdilution methods. Antioxidant activities of the crude extract and fractions were investigated by DPPH radical scavenging activity and β-carotene-linoleic acid assays. The methanolic extract and some fractions exhibited antibacterial activities that varied between the bacterial species (ID = 0.00–25.00 mm; MIC = 781–12500 μg/mL and 0.24–1000 μg/mL). The activity of the crude extract is, however, very weak compared to the reference antibiotics (MIC = 0.125–128 μg/mL). Two fractions (FE and FF) showed significant activity (MIC = 0.97 μg/mL) while S. aureus ATCC 25922 was almost resistant to all the tested fractions. In addition, the crude extract and some fractions showed good antioxidant potential with inhibition values ranging from 17.53 to 98.79%. These results provide promising baseline information for the potential use of this plant as well as some of the fractions in the treatment of infectious diseases and oxidative stress.


Introduction
Since the successive introduction of various antibiotics into therapeutics, the sensitivity of pathogenic microorganisms changed a lot so that the proportion of antibiotically resistant strains is currently important [1], what involves an increase in seriousness of infectious diseases as gastroenteritis (GE) which are a problem of public health on a worldwide scale but especially in Africa [2]. Diarrhea, its main characteristic is a major cause of morbidity and mortality among children in developing countries. According to the World Health Organization (WHO), there are more than 2 million deaths per year [3]. Moreover, therapy with synthetic antibiotics is not always possible because of their high cost as well as toxicity due to their extended use. To overcome this problem, people in developing countries use preparations obtained from plants following folk tradition for their primary health care because of low cost with little or no undesirable side effects [4]. The plants represent a potential and almost inexhaustible source of new anti-infective compounds [5] and many of them are used to treat GE effectively [6].
Pteleopsis hylodendron Mildbr. belongs to the family Combretaceae commonly found in the forest regions of West and Central Africa. The genus Pteleopsis is represented in Africa by ten species but only P. hylodendron is found in Cameroon [7]. The aqueous decoction of the stem bark of P. hylodendron is used to treat measles, chickenpox, sexually transmitted diseases, GE, female sterility, liver and kidney disorders, as well as dropsy [8]. Phytochemical, antimicrobial, toxicity and antioxidant works have been previously reported of this plant [9,10]. In the same logic, we have analysed the stem bark of Pteleopsis hylodendron and report here the antibacterial activity on pathogenic bacteria of the gastro-intestinal tract and antioxidant activity of the crude extract and fractions.  [11]. The bacterial suspension (100 μL) was homogeneously seeded onto Petri dishes containing sterile molten MHA (20 mL). The sterile 6 mm diameter wells were impregnated (50 μL) with different concentrations of plant extract (10, 5, and 2.5 mg-200, 100, and 50 μg/mL). The dishes were kept for 1 h at room temperature for the diffusion of the extract. Subsequently, dishes were incubated at 35 • C for 24 h. Antibiotics (Amoxicillin, Ciprofloxacin and Gentamicin) were used as positive control (10 μg-200 μg/mL) and 10% aqueous DMSO was used as negative control. Results were evaluated by measuring the inhibition zones around each well. The assay was done in triplicate and the mean diameters recorded as inhibition zones. We considered that an extract is active when ID was up to 20 mm, and then the strain is known as sensitive; moderately active when ID was between 10 and 20 mm, and then the strain is known as moderate; little or not active when ID was between 0 and 10 mm, and then the strain is known as little sensitive or resistant [12,13].

Broth Microdilution Method.
Minimum inhibitory concentrations (MICs) were determined using Mueller Hinton Broth (MHB) by microdilution method [14]. A twofold serial dilution of the crude extract (12.50-0.024 mg/mL) and fractions (1000-1.953 μg/mL and 500-0.242 μg/mL). A negative control (10%, v/v aqueous DMSO, medium and inoculum) and positive control (10%, v/v aqueous DMSO, medium, inoculum and water-soluble antibiotics) were included. Each well of 96-well sterile microtitre plate received 100 μL of MHB, 100 μL of test substances and 100 μL of the bacterial inoculum (1.5 × 10 8 CFU/mL). The plates were covered and incubated at 35 • C for 24 h. As an indicator of bacterial growth, 50 μL p-iodonitrotetrazolium violet (INT) dissolved in water was added to the wells and incubated at 35 • C for 30 min. MIC values are recorded as the lowest concentration of the substance that completely inhibited bacterial growth that is, the solution in the well remained clear after incubation with INT. Minimum bactericidal concentrations (MBCs) were determined by plating 10 μL from each negative well and from the positive growth control on Mueller Hinton Agar. MBCs were defined as the lowest concentration yielding negative subcultures. The experiments were performed in triplicate. Amoxicillin, ciprofloxacin and gentamicin at the concentration ranging between 128 and 0.062 μg/mL served as positive control.

Antioxidant Assay
The free radical scavenging activity of the extract and fractions on the stable radical DPPH were estimated by the method of Mensor et al. [15]. 1.5 mL of a methanol solution of sample test at different concentrations (10, 50, 100, 500, and 1000 μg/mL) was mixed with a 0.3 mM DPPH methanol solution and kept for 30 min at room temperature. The decrease in the solution absorbance, due to proton donating of substances was measured at 517 nm. L-Ascorbic acid was used as positive control. The percentage of DPPH radical scavenging activity was calculated using the following formula: DPPH radical scavenging activity (%) (1)

β-Carotene-Linoleic Acid Assay.
In this assay antioxidant capacity is determined by measuring the inhibition of the volatile organic compounds and the conjugated diene hydroperoxides arising from linoleic acid oxidation [16]. A stock solution of β-carotene-linoleic acid mixture was prepared as follows: 1.5 mg β-carotene was dissolved in 3 mL of chloroform, and 75 μL linoleic acid and 600 mg tween 40 were added. Chloroform was completely evaporated using a vacuum evaporator. Then 150 mL distilled water saturated with oxygen was added with a vigorous shaking. 1340 μL of this reaction mixture was dispersed to test tubes and 160 μL extract (20 mg/mL) were added, and emulsion system was incubated at 55 • C for 105 min. Same procedure was repeated with L-Ascorbic acid used as a standard control and a blank. After this incubation period absorbance of the mixtures were measured at 492 nm. Antioxidative capacity of the extract was compared with those of ascorbic acid and blank.

Statistical
Analysis. Data were expressed as mean ± standard deviation. Statistical analysis was carried out using the Waller-Duncan's test. The 12.0 SPSS Windows software was used for this analysis. Differences were considered significant at P < .05.

Extraction and Fractionation.
The % yield of methanolic extract of P. hylodendron was 15.96%. F I (43.08%) and F J (24.54%) were the most abundant.

Phytochemical Screening.
Phytochemical screening revealed the presence of medicinally active constituents. The differences in the composition between crude extract and fractions and between fractions were noted. Except F A , all other substances contained at least one chemical group. Alkaloids, anthocyanins, anthraquinones, flavonoids, glycosides, phenols, polyphenols, saponins, and tannins were present in crude extract while coumarins, sterols, and triterpenes were absent. F B , F C , and F D had a similar chemical composition (alkaloids). It is the same for F G and F H (alkaloids, anthocyanins, anthraquinones, flavonoids, phenols, polyphenols, and tannins); F I and F J (flavonoids, glycosides, phenols, polyphenols, saponins, and tannins).  Tables 2 and 3.

Antibacterial
All the bacteria tested were inhibited by the methanolic extract (Table 2) with MIC ranging from 781-12500 μg/mL for all the bacteria, isolates, and Gram − ; 781-3125 μg/mL for the strains and Gram + . S. paratyphi A was the least sensitive (MIC = 12500 μg/mL). P. aeruginosa ATCC 27853, P. mirabilis and S. aureus were the most sensitive (MIC = 781 μg/mL). The important activity on S. aureus confirms the best activity obtained in solid medium; which revealed this germ as one of the most susceptible. Antibiotics exerted a higher inhibitory effect on bacterial (MIC = 0.125-128 μg/mL) than the methanolic extract.
The fractionation of the methanolic extract showed an inactivity of F A , F B , F C , and F D on all the bacteria tested (Table 3). On the contrary, F E and F F saw their activity increasing significantly. Indeed, on five of the eleven bacteria (E. coli, P. mirabilis, S. paratyphi B, E. faecalis ATCC 10541, S. aureus), MIC was 0.97 μg/mL, making the substances more active than reference antibiotics. F G had a fairly good activity (MIC = 0.24 μg/mL) on some bacteria (P. aeruginosa ATCC 27853, E. faecalis ATCC 10541, S. aureus) whereas F H , F I and F J were slightly active (MIC = 125-1000 μg/mL). S. aureus ATCC 25922 was almost resistant to all the fractions (MIC > 1000 μg/mL). The MBC/MIC ratio activity for all the bacteria tested varied between one (1) and eight (8) for the crude extract and between one (1) and twenty (20) for the fractions. According to Marmonier [18], plant extract and fractions exerted two types of activities: a bacteriostatic (MBC/MIC ≥ 4) and bactericidal activity (MBC/MIC ≤ 4). Methanolic extract and fractions of P. hylodendron were bactericidal on at least 63% and 27% of the bacteria respectively.

Antioxidant Activity.
The antioxidant activity of the methanolic extract and fractions was assessed by the DPPH and β-carotene-linoleic acid assays. The results are presented in Tables 4 and 5. Activity increased in a concentrationdependant manner compared to L-ascorbic acid (positive antioxidant control). At the concentrations of 500 and 1000 μg/mL the methanolic extract, F E , F F , F G , F H , F I , and F J showed a similar activity to that one of L-ascorbic acid. F H was the most antioxidant fraction (94.05-98.79%) while F A was the least antioxidant (1.86-21.26%).

Discussion and Conclusion
Phytochemical screening of the methanolic extract and fractions of P. hylodendron revealed the presence alkaloids, anthocyanins, anthraquinones, flavonoids, glycosides, phenols, polyphenols, saponins and tannins. Other investigators [19,20] have reported the presence of these components in  the Combretaceae family to which belongs the studied plant. However, Ngounou et al. [21] and Atta-Ur-Rahman et al. [22] working on the stem bark of P. hylodendron collected from East region of Cameroon revealed the presence of triterpenes which were absent in our sample. This difference can be attributed to the difference in the geographical region, soil composition, and age of the plant [17].
The antimicrobial activities of Pteleopsis species were reported [19,23]. Generally, the methanolic extract and some fractions of the stem bark of P. hylodendron showed variable antibacterial activities dose-dependant on the eleven bacterial strains and isolates tested. These broad spectra of action could be related to their chemical components [24]. Among these compounds, tannins induce an important antimicrobial activity because they have an ability to inactivate microbial adhesions, enzymes, cell envelope transport proteins, and so forth, [25]. Due to their ability to bind to proteins and metals, tannins also inhibit the growth of microorganisms through substrate and metal ion deprivation [26]. However, differences in chemical composition recorded between the crude extract and some fractions may explain their different degree of antimicrobial properties. Also, the amount of the active components in the crude extract may be diluted and fractionation may have increased their concentrations, thus the activities in the fractions [27]. Moreover, the differences in susceptibility may be explained by the differences in cell wall composition and/or genetic content of plasmids that can be easily transferred among Table 3: MIC and MBC (μg/mL) of the fractions from chromatography separation of P. hylodendron.

Bacteria
Parameters Fractions Reference antibiotics strains [28]. MIC values obtained from the extract by microdilution method revealed that S. aureus is the most sensitive. It was reported [29] that S. aureus is one of the most susceptible bacteria to the plant extracts. These values also showed that the Gram − and Gram + bacteria had a comparable susceptibility. This may suggest that the mode of action of the extract was not related to the cell wall composition. S. aureus ATCC 25922 which was inhibited completely by the methanolic extract at 3125 μg/mL, was almost resistant to all the fractions. This may suggest that this microbe required high concentrations of the substance tested and synergic effect of chemical compounds as extract. F A , F B , F C , and F D containing only alkaloids did not show any inhibitory effect on the bacteria tested. This may suggest these compounds which also present in the methanolic extract do not have a detectable antibacterial activity. However, alkaloids were reported to possess antibacterial activities [30]. F E and F F , most active had a comparable chemical composition that F G . Differences in activity between these fractions could be related to the absence of anthocyanins in F F and  anthraquinones in F E . Generally, it is difficult at the sight of results of the phytochemical screening to attribute the activities recorded to a chemical compounds group. As L-ascorbic acid, the methanolic extract and some fractions showed great antioxidant potentials. This particularly high activity could be attributed to the presence of phenolic compounds [31]. The antioxidant activity of phenolic compounds is mainly due to their redox properties, which can play an important role in neutralizing free radicals, quenching singlet and triplet oxygen species, or decomposing peroxides [32]. Numerous studies have suggested flavonoids, anthraquinones, anthocyanins and tannins [33,34] for antioxidant activity. Previous phytochemical investigations on this plant have reported the presence of ellagic acid derivatives as antioxidant source [22].
These results provide promising baseline information for the potential use of this plant as well as some of the fractions in the treatment of GE and oxidative stress. F E and F F by their high antibacterial activity could be the base of development of new antibacterial agents with broad spectra. Their purification and pharmacological and toxicity studies are essential.