The research aimed at carrying out a comparative study on the effect of Massularia acuminata fractions against bacterial and fungi isolates from the oral cavity. The Antimicrobial assay was carried on the following test organism; Staphylococcus aureus, Klebsiella pnuemoniae, Bacillus subtilis and Escherichia coli and Enterobacter aerogenes. From antifungal result, it was observed that the highest inhibition was observed in the ethanolic extract of Massularia acuminata on Rhyzopus stolonifer, Candida albican and Fusarium solani. The ethanolic and methanolic extracts were also observed from the results to have an intermediate sensitivity to Aspergillus favus, Candida tropicalis, Can dida albican, Rhyzopus stolonifer, Fusarium solani, while it was observed to be resistant to Fusarium solani, Rhyzopus stolonifer, Candida tropicalis, Aspergillus favus for all the partitions of the extracts. From the antifungal effect of the M. acuminata fractions, it could be inferred that the ethanolic and methanolic extracts were quite effective in the inhibition of the fungi infections and the methanolic extract proved very infective on Candida albican, Rhyzopus stolonifer and Fusarium solani. From the result of the antibacterial effect of M. acuminata fractions, the highest inhibition was observed by the methanolic extract of Massularia acuminata against all the Gram negative bacteria and Staphylococcus aureus. Also the aqueous extract also had a less sensitivity against all the test microbes, as all proved resistant. It could be inferred that the fractions of the extracts were unable to inhibit the growth of the strain of gram positive bacteria and probably, the potency could be increased, if the concentrations of the methanolic, ethanolic extracts of Massularia acuminata and the standard drug, tetracycline are increased. Results obtained showed that ethanol was a better and more powerful solvent than water. Whereas, the methanolic extract and the standard drug were quite effective in the inhibition of some gram negative bacteria, Klebsiella pnuemoniae and Enterobacter aerogenes. From the results obtained, it can be deduced that the ethanolic and methanolic extracts of Massularia acuminta possess antifungal and antibacterial potentials, compared with the aqueous extract and its potency as an inhibitor of bacterial and fungi organism found in the oral cavity.
Both the upper and lower parts of the human digestive tract harbor a complex ecosystem of bacteria, fungi, protozoa, and viruses, referred to as the microbiome. It begins to form even before birth, in the uterus, developing for another 2 - 3 years after birth to become a stable, fully functioning microbiome, until the physiological changes associated with senescence lead again to substantial shifts in its composition [
The plant parts: stem barks of Massularia acuminata were collected in Ute, in Owo Local Government area of Ondo State. The authentication were done in Department of Biotechnology, Adekunle Ajasin University, Akungba Akoko and re-authenticated at IFE Herbarium at Obafemi Awolowo University, Ile Ife. The stem bark were air-dried and crushed into fine powder. The powdered samples were extracted separately with methanol, ethanol and butanol with continuous agitation; the extracts were concentrated at 35˚C with rotary evaporator and water bath to dryness. A known amount of pulverized sample of Massularia acuminata (500 g) were percolated in 1 litre of distilled water and respective alcoholic solvents; with ratio of powder and solvent of 1:2, and with intermittent shaking and kept in the refrigerator for 48 hours. The solution were thereafter filtered using Whatman No. 1 filter paper and the filterate concentrated on a water bath to give a yield of 25.50 g representing a percentage yield of 5.10%.
Bench top centrifuge machine, refrigerator, micropipette, spectrophotometer, standard mouse cage, water Bath and sensitive weighing balance. Beaker (50 ml and 1000 ml), needle and syringe, measuring cylinder, spatula, dissecting kit, intubator, test tubes, test tube rack and reagent bottle.
Permanent marker, masking tape, disposable hand gloves, cotton wool, foil paper, tissue paper, EDTA bottles, plain bottles and lithium heparin bottle.
Distilled water, hand sanitizer, antiseptic, normal saline and aluminium chloride.
The bacterial strains used are Grams positive (Staphylococcus aureus, and Bacillus substilis) and Gram negative (Escherichia coli, Klebsiella pnuemoniae, Enterobacter aerogenes), was obtained from microbiology department, Adekunle Ajasin University, Akunga Akoko, Ondo state and was cultured aerobically at 37˚C in nutrient agar medium. Before experimental use, cultures from solid medium will be sub-cultivated in liquid media, incubated for 24 hours and was used as source of inoculums for each experiment. Antimicrobial activity was measured using agar-well diffusion method.
Antibacterial activities of the plant extracts of Massularia acuminata and fractionated constituents were tested by disc-diffusion method. The minimal inhibition concentration (MIC) of the extracts was determined. Five bacterial strains, Staphylococcus aureus, Bacillus substilis, Escherichia coli,Klebsiella pnuemoniae, and Enterobacter aerogenes were used in this study. The organisms were obtained from microbiology Department, Adekunle Ajasin University, Akungba Akoko, Ondo state and were cultured aerobically at 37˚C in nutrient agar medium. The bacteria were tested for purity by culturing on nutrient agar and maintained on nutrient agar slants.
Susceptibility of bacteria isolate to plant extract were determined following the BSAC Diffusion Method for Antimicrobial Susceptibility Testing Version 9.1 [
Five concentrations of each extract (40, 60, 80 and 100 mg/ml) was prepared. The antimicrobial effect of each concentration was measured. The various concentrations was loaded onto 6 mm disks which was then pressed onto already prepared Mueller-Hinton agar plates and SDA plates. The inoculated plates were incubated at 35˚C for 48 h. MICs were determined after 24 hours for the bacteria and after 48 hours for Massularia acuminata and its fractions extract zones of inhibition were measured at the end of the incubation period. The MICs was determined as the lowest concentrations of extracts inhibiting the visible growth of each organism on the agar plate.
The strains of microorganism obtained were inoculated in conical flask containing 100 ml of nutrient broth. These conical flasks were incubated at 37˚C or 24 h and were referred to as seeded broth. Media were prepared using Muller Hinton Agar (Himedia), poured on Petri dishes and inoculated with the test organisms from the seeded broth using cotton swabs. Sterile discs of six millimeter with had been impregnated with 30 µl of test extract and introduced onto the upper layer of the seeded agar plate. The plates were incubated overnight at 37˚C. The antimicrobial activity was recorded by measuring the width of the clear inhibition zone around the discs using zone reader (mm). Tetracycline (100 µg/disc) were used as standards.
Experimental results were expressed as mean ± standard error of mean (SEM). All measurements were replicated three times. The results were analyzed using one way analysis of variance (Anova). The level of significance used was P < 0.05.
| Fungi organism | Methanolic M. acuminata extract | Aqueous M. acuminata extract | Ethanolic M. acuminata extract | Nystatin (mm) |
|---|---|---|---|---|
| Aspergillus favus | 17.66 ± 0.19 | 10.16 ± 0.09 | 14.33 ± 0.19 | 21.2 ± 0.11 |
| Candida tropicalis | 16.55 ± 0.17 | 12.2 ± 0.11 | 14.5 ± 0.23 | 23.23 ± 0.13 |
| Candida albican | 16.26 ± 0.15 | 11.33 ± 0.19 | 22.1 ± 0.05 | 22.1 ± 0.05 |
| Rhyzopus stolonifer | 16.4 ± 0.15 | 12.43 ± 0.17 | 25.2 ± 0.24 | 26.73 ± 0.21 |
| Fusarium solani | 14.46 ± 0.16 | 12.5 ± 0.16 | 23.46 ± 0.26 | 23.33 ± 0.19 |
Sensitive (S) ≥ 21, Intermediate (I) ≤ 20 and resistant (R) ≤ 14.
| Bacterial Strain | Methanolic M. acuminata extract | Aqueous M. acuminata extract | Ethanolic M. acuminata extract | Tetracycline 30 µg |
|---|---|---|---|---|
| Gram positive | ||||
| Bacillus subtilis | 10.63 ± 0.18 | 0.00 ± 0.00 | 11.33 ± 0.50 | 12.66 ± 0.19 |
| Staphylococcus aureus | 13.16 ± 0.25 | 0.83 ± 0.03 | 12.16 ± 0.34 | 13.33 ± 0.19 |
| Gram negative | ||||
| Escherichia coil | 13.33 ± 0.50 | 0.86 ± 0.01 | 11.43 ± 0.25 | 13.66 ± 0.38 |
| Klebsiella pnuemoniae | 14.66 ± 0.38 | 11.66 ± 0.50 | 12.23 ± 0.13 | 14.26 ± 0.21 |
| Enterobacter aerogenes | 14.33 ± 0.50 | 0.86 ± 0.01 | 12.33 ± 0.19 | 14.33 ± 0.19 |
Sensitive (S) ≥ 21, intermediate (I) ≤ 20 and resistant (R) ≤ 14.
| Plant Extract | Conc mg/ml | B. subtilis | S. aureus | E. coli | K. pnuemoniae | E. aerogenes |
|---|---|---|---|---|---|---|
| Methanolic Extract | 100 | 10.53 ± 0.16 | 13.83 ± 0.25 | 13.63 ± 0.18 | 14.53 ± 0.16 | 14.76 ± 0.35 |
| 80 | 10.35 ± 0.17 | 0.86 ± 0.01 | 0.86 ± 0.01 | 0.76 ± 0.05 | 0.83 ± 0.03 | |
| 60 | 0.00 ± 0.00 | 10.55 ± 0.16 | 0.86 ± 0.01 | 11.16 ± 0.09 | 10.23 ± 0.13 | |
| 40 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | |
| Ethanolic Extract | 100 | 10.28 ± 0.13 | 11.7 ± 0.20 | 11.8 ± 0.35 | 11.23 ± 0.0.13 | 12.4 ± 0.17 |
| 80 | 0.83 ± 0.03 | 10.66 ± 0.19 | 0.86 ± 0.01 | 0.80 ± 0.03 | 11.16 ± 0.41 | |
| 60 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | |
| 40 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | |
| Aqueous Extract | 100 | 0.00 ± 0.00 | 0.86 ± 0.01 | 0.83 ± 0.03 | 0.83 ± 0.03 | 0.86 ± 0.01 |
| 80 | 0.83 ± 0.03 | 0.86 ± 0.01 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | |
| 60 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | |
| 40 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 |
Sensitive (S) ≥ 21, Intermediate (I) ≤ 20 and resistant (R) ≤ 14 Intermediate (I) ≤ 20.
People are aware that good oral health is very important. Consequently, daily brushing and flossing are taught to us from a very early age to keep our teeth as clean as possible. However, not everyone is aware that good oral health is more than pretty white of teeth. Both the upper and the lower parts of the human digestive tract harbor a complex ecosystem of bacteria, fungi, protozoa and viruses, referred to as the microbiome [
The results illustrated the inhibitory effect of the various fractions of Massularia acuminata extracts on the respective opportunistic infection found in the oral cavity as seen in the results. Opportunistic infections are infections that occur more often or are more severe in people with weakened immune systems than in people with healthy immune systems.
From the antifungal effect of the M. acuminata fractions, it could be inferred that the ethanolic and methanolic extracts were quite effective in the inhibition of the fungi infections and the methanolic extract proved very infective on Candida albican, Rhyzopus stolonifer and Fusarium solani. The degree of inhibition can be increased if, the concentrations of both extracts are increased. The aqueous extract was unable to inhibit the infections and probably, it effectiveness could be improved if its concentrations is increased. The standard drug, Nystatin, which has a broad spectrum in the inhibition of several opportunistic infections proved very much infective in the inhibition of the fungi infections.
From the result of the antibacterial effect of M. acuminata fractions, it could be inferred that the fractions of the extracts were unable to inhibit the growth of the strain of gram positive bacteria and probably, the potency could be increased, if the concentrations of the methanolic, ethanolic extracts and the standard drug, tetracycline are increased. Whereas, the methanolic extract and the standard drug were quite effective in the inhibition of some gram negative bacteria, Klebsiella pnuemoniae and Enterobacter aerogenes, and its effectiveness could be increased, if the concentrations are increased. The ethanolic and aqueous extracts were unable to inhibit the growth of the bacteria.
Massularia accuminata showed a high inhibitory activity against the bacteria species tested, since it exhibited the highest antibacterial inhibition. This was in conformity with the work by [
From the result, illustrating Minimum Inhibitory concentration (MIC) of methanolic extract, ethanolic extract and aqueous extract of Massularia acuminata against test microbes, it could be inferred that at high concentrations the inhibitory effects of various fractions of the extracts increased when compared with lower concentrations of the extracts. Probably, the effectiveness of the extracts could be improved, if the concentrations of the extracts are increased.
In conclusion, from the results obtained, it can be deduced that the ethanolic and methanolic extracts of Massularia acuminta possess antifungal and antibacterial potentials, when compared with the aqueous extract and its potency as an inhibitor of bacteria and fungi found in the oral cavity can be increased, if their respective concentrations are increased, provided the extracts have not gotten to their lethal doses, where they could prove fatal.
This research was funded by Tertiary Education Trust Fund (TET Fund) through its Institutional Based Research Grant program. We are also grateful to Adekunle Ajasin University Akungba Akoko, Ondo State, Nigeria, for providing the enabling environment for the sponsorship and success of the work.
The authors declare no conflicts of interest regarding the publication of this paper.
Olusola, A.O., Bakare, O.S., Ekun, O.E. and Olusola, A.O. (2020) A Comparative Study on the Effect of Massularia acuminata Fractions against Bacterial and Fungal Isolates from the Oral Cavity. Health, 12, 904-913. https://doi.org/10.4236/health.2020.127067