Inhibitory effects of Mezoneuron benthamianum root extracts on oral cariogenic microorganisms and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging radical

Chewing sticks are commonly used as oral hygiene tool in Africa and reactive oxygen species have been linked to the cause of degenerative diseases because of their ability to induce oxidative damage to biological molecules. Plants have been a long source of dietary antioxidants as most plants have been found to exhibit excellent antioxidant potentials. Mezoneuron benthamianum is a plant that is used locally as chewing sticks in southwest, Nigeria, but its use as a therapeutic agent in dental caries, a disease caused by bacteria especially Streptococcus mutans is poorly investigated. This study was therefore designed to investigate the anticaries activities of the crude extracts of M. benthamianum root against four clinical oral pathogens (Staphylococccus aureus, Escherichia coli, Pseudomonas aeruginosa and Streptococcus mutans) and the antioxidant activities using 2,2-diphenyl-1-picrylhydrazyl (DPPH). The results of this study showed that M. benthamianum had a consistent activity against all the bacterial organisms tested, with the ethyl acetate extract having the highest anticaries activity with minimum inhibitory concentration values of 78 and 156 μg/mL, while the hexane extract had the least anticaries activity with MIC values of 2500 μg/ml against S. mutans and E. coli respectively. The results also revealed that the ethyl acetate and aqueous methanol extract exhibited a higher antioxidant activity (IC50 = 23.70 and 21.30 μg/mL) than standard ascorbic acid (IC50 = 38.20 μg/mL). This study demonstrated the anticaries and antioxidant potentials of M.benthamianum and therefore justifies the folkloric use of M. benthamianum in oral hygiene.


Background
Dental caries is a condition affecting half of the entire human population [1] and it is caused by bacteria found in the mouth especially Streptococcus mutans. It is a bacterial initiated demineralization of the hard tissue structures of the tooth [2]. Natural products have been used from the past for their antibacterial effect, but recently there have been investigations about promising agents for the prevention of oral diseases such as dental caries [3]. In addition, research has shown the potential of natural products in remineralising tooth surfaces affected by dental caries [4]. Hence, medicinal plant based extracts and their derivatives have been explored for the anti-cariogenic and anticancer effects [5,6]. Furthermore, the biological system undergoes metabolic processes involving redox reactions which generate reactive oxygen species (ROS). Reactive oxygen species have been linked to the causes of degenerative diseases such as cardiovascular disease, cancer, diabetes, hypertension and neurodegenerative disorders because of their ability to induce oxidative damage to biological molecules [7]. Normally, the ROS generated during metabolic process are detoxified by the antioxidant enzymes eg glutathione reductase by producing antioxidant compounds like glutathione and creatine, in order to achieve equilibrium between the ROS generation and its detoxification within the system. But when the generation of ROS overtakes the antioxidant defence of the cell, a phenomenon that leads to potential damage occurs, [8]. Plants have long been source of dietary antioxidants. It is believed that two-thirds of the world's plant species have medicinal importance, and almost all of these have excellent antioxidant potential [9]. In Africa, the use of chewing sticks is the most common means of maintaining oral hygiene and preventing dental caries and Mezoneuron benthamianum is plant that is used locally as a chewing stick in southwestern Nigeria [10] and It has been traditionally used in the management of several diseases including dysentery, urethral discharges, skin diseases and wounds [11]. However, its use as a therapeutic agent in preventing dental caries has not been extensively investigated. This study was therefore designed to investigate its antimicrobial activities against four oral clinical pathogens (Staphylococccus aureus, Escherichia coli, Pseudomonas aeruginosa and Streptococcus mutans) and antioxidant activities using 2,2diphenyl-1-picrylhydrazyl (DPPH).

Plant preparation
The plant samples of M. benthamianum Baill. were obtained in Ibadan and authenticated at the Department of Botany, University of Ibadan, Nigeria, where voucher specimen is being deposited in the herbarium. (No: UIH-22401).

Plant extraction
The dried, ground root of M. benthamianum (2.74 kg) was extracted by maceration with methanol (10 L × 2, for 72 h each) and the extracts were concentrated using rotary evaporator under reduced pressure to give dark brownish extract (200 g). This was then dissolved in aqueous methanol and was further partitioned with the aid of a separating funnel into hexane, ethyl acetate and aqueous methanol fractions to yield 5 g, 70 g and 100 g respectively.

Antimicrobial assay Microbial strains
The bacterial test strains used in this study were Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Streptococcus mutans isolated from carious teeth of patients at the dental clinic of the University College Hospital, University of Ibadan, Ibadan, Oyo State. In addition, three typed strains obtained from the American Type Culture Collection (ATCC) were also used namely: Staphylococcus aureus (ATCC 29213), Pseudomonas aeruginosa (ATCC 27855), E. coli (ATCC 35218) and the bacterial strains were maintained on agar slant at 4°C prior to being used.

Inoculation preparation
At least four well isolated colonies of the same type from a culture agar plate were selected and touched the top of colony with a loop and transferred to a tube containing 4 ml of a suitable broth such as tryptic soy broth (TSB). The suspension was incubated at 37°C and the size was adjusted to the 0.5 McFarland standard turbidity [12], approximately 1.5 × 10 6 CFU/ml.

Preparation of antimicrobial plates
The diluted methanol extracts were added to the melted and cooled medium in a ratio of 1 part extract sample agent to 9 parts medium (2 ml of plant extract to 18 ml of Mueller Hinton agar for each Petri dishes plate) with most susceptibility test. Gentamycin (0.62-5 μg/ml) was used as control for the microorganisms assay.

Inoculation of test organisms
Full each well of multiple-inoculator with inoculums test organisms and dip the tip of multiple-inoculator on Mueller Hinton Agar plates and incubate at 37°C for 24 h.
The antibacterial activity of the extracts was determined using the agar well diffusion technique with slight modification [13,14] and Gentamycin (Sigma) was used as a positive control. Nutrient agar plates were seeded with 100 μL of an overnight culture of each bacterial strain (equivalent to 10 7 -10 8 CFU/ml). The seeded plates were allowed to set and a standard cork borer of 8.00 mm diameter was used to cut uniform wells on the surface of the agar. Each plant extract was prepared into a concentration of 200 mg/mL from the dried extract by dissolving 2 g of the individual plant extract in 10 ml of methanol, then 2 ml of the mixture was diluted serially four times with 2 ml of the methanol to give 100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml and 6.25 mg/ml. The wells of the agar medium were then filled with 100 μL of each extract at the different concentrations of 6.25, 12.5, 25, 50 and 100 mg/ml using calibrated Pasteur pipette. Gentamicin at 10 μg/ml was used as the positive control while methanol as the negative control. The plates were left for 45 min to allow the diffusion of the extracts and controls through the agar medium after which they were incubated at 37°C for 24 h. Each test was carried out in duplicates. Zones of growth inhibition of the various concentrations of the extracts and the controls were measured. The mean zone of inhibition less than 8 mm is considered as no activity.

Determination of minimum inhibitory concentration and minimum bactericidal concentration
Minimum inhibitory concentration (MIC) was determined using the agar dilution method of [15] with slight modifications. The extract of 20 mg/ml was serially diluted in test tubes to give a final concentration in the range of 10, 5.0, 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078 mg/ mL. Two millilitre of each dilution of the extract was mixed with 18 ml of Mueller Hinton agar, poured into 10 cm diameter Petri dishes and allowed to set. After allowing the agar to dry for about 30 min, each plate was inoculated with 1:100 dilution of overnight broth cultures of each test organisms containing 1.0 × 108 CFU/ ml (according to 0.5 McFarland standard) and incubated for 24 h at 37°C. Nutrient agar plates with extract but without an organism and one containing only organism served as positive and negative control respectively. Each test was carried out in duplicate. The plates were then examined for the presence of growth after the incubation period. The least concentration that gave no visible colonies of the test organism was taken as the minimum inhibitory concentration of the extract.
The miminum bactericidal concentration (MBC) of the extracts was determined by taking samples from plates with no visible growth in the MIC assay and subcultured on to freshly prepared nutrient broth and later incubated at 37°C for 48 h. The MBC was taken as the lowest concentration of the extract that did not allow any bacterial growth on the surface of the agar plates or technically as the lowest concentration of extract which produced 99.9% killing of the inoculums.

DPPH antioxidant assay
The free radical scavenging activity of the compounds was measured by the 2,2-diphenyl-1\-picrylhydrazyl (DPPH) method described by, Brand-Williams et al., (1995) with slight modifications. Briefly, a 0.1 mM solution of DPPH in methanol was prepared and 1.0 ml of this solution was added to 0.5 ml of the samples dissolved in methanol and using a range of 12.5-200 μg/ml. After 20 min, the absorbance was measured at 517 nm. The DPPH radical scavenging activity was calculated according to the following equation: Where A o is the absorbance of the control and A I is the absorbance in the presence of the test substance. DPPH solution alone served as control (A o ). A graph of %

Results
The results of the antimicrobial activities of Mezoneuron benthamianum extracts (Tables 1 and 2 Table 2). The polar extracts of M. benthamianum are rich in polyphenols, which have been established to have antimicrobial and anticariogenic properties [5,16]. In addition, the results of the antioxidant activities of the crude extracts (Figs. 2 and 3) show that MBM, MBE and MBAM exhibited strong antioxidant activity as shown by their percentage scavenging activities as well as their IC 50 of 63.79, 23.7 and 21.3 μg/mL, while the

Discussion
Dental caries is the most prevalent disease affection the oral cavity. It cuts across different segment of the society. Although different microorganisms are implicated in dental caries, Streptococcus mutans is considered as the primary aetiological agent. Natural products are used routinely for oral hygiene and treatment of dental caries [3]. In Africa, especially Nigeria chewing sticks are commonly used in maintaining oral hygiene and preventing dental caries. While this is the first report of the antimicrobial activities of extracts of M. benthaminaum against cariogenic organisms, the antimicrobial potential of M. benthamianum can be attested to in previous reports. The report of [17], which reported the antimicrobial activities of the petroleum ether, chloroform and ethyl

Conclusion
In this study, the anticaries assay of the extracts of M. benthamianum revealed that the extracts possess inhibitory properties against the clinical isolates of the different cariogenic microorganisms including S. mutans. They also demonstrated a strong antioxidant activity against DPPH scavenging radical. This is the first report of the anticaries activities of Mezoneuron benthamianum extracts and its further corroborate the folkloric use of this plant in maintaining dental hygiene and preventing dental caries.