Introduction

For thousands of years, natural plants have been seen as a valuable source of medicinal agents with great potential of treating infectious diseases and with lesser side effects compared with synthetic drug agents (Iwu et al, 1999). In most parts of the world and Africa in particular, vast populations of people in the rural areas still depend on medicinal plants for the treatment of infections. The global need for the use of plants as an alternative prevention and treatment option is prompted by a number of factors. These include the fact that phytomedicines are cheaper than conventional drugs and may not be associated with adverse side effects like hypersensitivity on the host (Saraf, 2010); they can possibly replace conventional antibiotics to which human pathogens have developed resistance (Ali et al., 2011).

Alchornea cordifolia, of the family Euphorbiaceae is a multi-stemmed shrub or small tree commonly called ‘Christmas bush’ or ‘dovewood’, ‘ubobo’ in Urhobo; and ‘evwa’ in Isoko languages of Delta State, Nigeria. It is pantropical and geographically distributed in secondary forests sometimes near water, moist or marshy places (Dalziel, 1956). The leaf extracts have been reportedly used for treating urinary tract infections, infected wound, dental caries, diarrhoea, cough, chest pain, venereal diseases, dermatoses,  conjunctivitis, stomach ulcers, bronchitis, toothache and anaemia (Kambu et al., 1990; Le Grand and Wondergem, 1987). Stem pith is chewed to treat tachycardia and rubbed on the chest to treat respiratory problems (Mavar-Manga et al., 2007) as well as enteric infections.

Tooth decay is a progressive destruction of enamel, dentine and cementum, mediated by microbial activity at the tooth surface (Steve, 1997). Though hundreds of bacteria are present, only twenty two predominant types have been identified to inhabit the oral cavity (Topezian et al., 2002). There is need for a cheap and alternative source of treating these oral infections. In this study, the inhibitory effects of A. cordifolia on four bacterial causative agents of dental caries and toothache were tested in vitro in order to validate its traditional use in the treatment of tooth decay.

Materials and Methods

Plant selection and preparation

Alchornea cordifolia whole plants were collected from the banks of River Ethiope, Abraka. They were identified at the herbarium of the University of Nigeria, Nsukka. The leaves were cleaned by washing with distilled water and sorted to eliminate any old, damaged leaves. The tender stems of A. cordifolia were broken and the pith neatly removed.

The leaves and stem pith of the plant were each ground using a porcelain pestle and mortar. Juice was extracted from each plant material and filtered with sterile filter papers to obtain clear extract.

For concentrated extracts, the crushed plant materials were weighed with a balance before the juice was extracted and filtered into a sterile bottle. The weight of each extract was noted before the concentration process was initiated. The volume of each concentrated extract was also noted and doubling dilutions of each was made.

Sources of Test Organisms

The test bacteria were isolated from the mouths of undergraduates in Novena University, Ogume, Delta State, Nigeria. Sterile swab sticks were used to swab the tongues, teeth, gums and palates of individuals and placed in peptone water. Each sample was taken to the laboratory in ice packs and inoculated into agar. Bacterial species were isolated, characterized and identified according to standard methods (Murray et al., 2007; Holt 1994).

Standardization of test organisms

A standard stock of the bacteria isolates were prepared by suspending a loop full of each microbial growth in about 10 ml of nutrient broth. After incubation at 37°C for 12 h, the turbidity was adjusted to be visually comparable with a 0.5 McFarland’s standard giving a bacterial load of about 1 – 2 × 10⁸ cfu/ml (Murray et al., 2007).

Susceptibility test

This was done in triplicates. The agar-well diffusion method described by NCCLS (2000) was employed in the susceptibility testing. Suspensions of the bacterial isolates were made in sterile normal saline and adjusted to the 0.5 McFarland’s standard. Each Mueller Hinton (MH) agar plate was uniformly seeded by means of sterile swab dipped in the suspension and streaked on the surface of the agar. The plates were left on the bench for excess fluid to be absorbed. Wells of 5 mm in diameter and 4 mm deep were punched in the MH agar with a sterile cork-borer. Approximately 100 μl of the extracts were dropped into each well to fill them. The setup was allowed to stabilize for 3 hours before being incubated at 37°C for 24 hours as described previously by (Shahidi, 2004). The mean zones of inhibition were then measured for all the individual isolates. A positive control well was equally filled with gentamycin (35mg/mL).

Determination of Minimum Inhibitory Concentration (MIC)

The MICs of the leaf and stem pith extracts of A. cordifolia were determined for each of the test organisms using the dilution susceptibility technique. A 0.5 MacFarland standard of each of the bacteria were inoculated into Mueller Hinton agar. Thereafter, serial dilutions of the extract were carried out to give the following extract concentration: 1:2, 1:4, 1:8, 1:16 and 1:32, respectively. Using the sterile cork borer, holes were perforated on the plates and filled with each dilution of the extract. Negative control plates with no bacterial inoculation, were simultaneously maintained. All plates were incubated at 37^oC for 24 h. Zones of inhibition were measured and recorded in millimetres (mm).

Statistical analysis

The data obtained were analyzed by descriptive statistics. The mean and standard deviation of the triplicate susceptibility tests were calculated. Results were expressed as Mean ± SD.

Results and Discussion

Four Gram positive bacterial species were found to be present in the oral samples investigated. These include Actinomyces viscosus, Staphylococcus aureus, Lactobacillus casei and Streptococcus mutans. This agrees with the observation that the oral cavity is a primary portal for Staphylococcus species as well as Actinomyces (Lampe et al., 2008). Lactobacillus species and Streptococcus mutans are microbial species that form part of the microbiota of the oral cavity. The link between the activities of these organisms and oral diseases is well established (Jenkinson and Lamont, 2005). The prevalence of S.mutans and Lactobacillus in carious teeth has been described (Nishikawara et al., 2006).

The pith juice of the plant significantly inhibited all four bacterial species with zones of inhibition ranging between 28.7 ± 1.7 mm and 43.0 ± 2.0 mm (Table 1). This validates its traditional use for the treatment of oral infections. However, A. viscosus, S. aureus and S. mutans gave inhibition zones that compared well with the gentamycin standard (Table 1). S. aureus was most susceptible with a zone of inhibition of 43.0 ± 2.0 mm which is higher than the 41.3±1.7mm given by the standard. The susceptibility shown by S. aureus over other bacterial isolates is of great significance since reports abound that this organism has developed resistance to many antibiotics, sometimes making its clinical management difficult (Willey et al., 2008). On the other hand, the leaf juice of the plant did not inhibit S. aureus and S. mutans. While L. casei gave an inhibition zone of 9.0 ± 2.0 mm and A. viscosus, 12.3 ± 1.7 mm.

The concentrated leaf extracts (Table 2) at 575.0 mg/ml inhibited all the isolates significantly with zones of inhibition which ranged from 14.0 ± 1.0 mm to 25.3 ± 0.5mm. S. aureus was most susceptible. At a concentration of 287.8 mg/ml, S. aureus and S. mutans were susceptible while A. viscosus and L. casei were resistant. At a concentration of 143.8 mg/ml, S. aureus was the only organism that was susceptible to the extract. On the other hand, the concentrated pith extract did not inhibit A. viscosus and S. mutans.  However, S. aureus was susceptible at a concentration of 536.0 mg/ml. A remarkable result was observed with L. casei which became susceptible at a concentration of 268.0 mg/ml with a mean zone of inhibition of 15.3±0.5 mm but the zone of inhibition reduced greatly (7.2±0.6 mm) at the concentration of 134.0 mg/ml.

The minimum inhibitory concentrations of the extracts are shown in Table 3. The highest dilution of 143.8 mg/ml of the leaf extract inhibited S. aureus while the lowest dilution of 575.0 mg/ml inhibited both A. viscosus and L. casei. On the other hand, the minimum inhibitory concentration of the pith extract on L. casei is 134.0 mg/ml and 535.0 mg/ml for S. aureus.

Conclusion

The in vitro potency of the leaf and pith extracts of A. cordifolia on oral infections has been demonstrated in this work. The fresh pith of Alchornea cordifolia is by this investigation, found to be a very effective antibacterial agent against these organisms and thus a good treatment for oral infections. The traditional use of the leaves for the management of dental caries and toothache caused by these bacteria is scientifically validated in this research. However, the leaves are more effective when concentrated and can be prepared for commercial use or incorporated into herbal toothpastes.

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