|Year : 2016 | Volume
| Issue : 2 | Page : 64-68
A comparative study on the effect of Massularia acuminata and mouthwash against isolates from the oral cavity
Emudainohwo Joseph Oghenebrorie Tedwins1, Owhe-Ureghe Ubreye Benjamin2, Ewharieme Daniel Ayobola2, Moke Emuesiri Goodies1, Erhirhie Earnest Oghenesuvwe1
1 Department of Pharmacology and Therapeutics, Delta State University, Abraka, Delta State, Nigeria
2 Department of Microbiology, Delta State University, Abraka, Delta State, Nigeria
|Date of Web Publication||21-Apr-2016|
Dr. Emudainohwo Joseph Oghenebrorie Tedwins
Department of Pharmacology and Therapeutics, Delta State University, Abraka, Delta State
Source of Support: None, Conflict of Interest: None
Aim: This research determines the antimicrobial activity of 'PakoIjebu' chewing stick (Massularia acuminata) and some common mouthwashes (Hexedene®, Brett®, and Listerine®) against some isolates of the oral cavity.Settings and Design: The samples were obtained by the use of sterile swab stick to isolate the microorganisms (Streptococcus oralis, Staphylococcus aureus, Lactobacillus species, Klebsiella species, Neissera sicca, Micrococcus species, Muccor, Aspergillus flavus and Giosporium) from the oral cavity of healthy undergraduate students of Delta State University, Abraka, Nigeria using standard microbiological techniques. The sensitivity test, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) were determined.Results: The aqueous and ethanol extract of Massularia acuminata had an MIC of 25 mg/mL which was similar to those of commercially available mouth wash analyzed in the study. The ethanol extract of Massularia acuminata was bactericidal against Lactobacilus species, Streptococcus oralis and Neissera sicca at a concentration of 50 mg/mL which suggests that the extract is more effective compared to the tested mouth wash. Among the mouth wash, only Listerine was bactericidal against Neissera sicca at an MBC of 50 mg/mL. Conclusion: This study confirmed the age long belief that those who use Massularia acuminate regularly for their oral care has a low rate of oral infections; therefore, extract of Massularia acuminata could be incorporated into tooth paste or used as mouth wash for periodontal infections.
Keywords: Brett, Chewing stick, hexedene, listerene, Massularia accuminata, mouth wash, periodontal infections
|How to cite this article:|
Tedwins EJ, Benjamin OUU, Ayobola ED, Goodies ME, Oghenesuvwe EE. A comparative study on the effect of Massularia acuminata and mouthwash against isolates from the oral cavity. J Res Dent 2016;4:64-8
|How to cite this URL:|
Tedwins EJ, Benjamin OUU, Ayobola ED, Goodies ME, Oghenesuvwe EE. A comparative study on the effect of Massularia acuminata and mouthwash against isolates from the oral cavity. J Res Dent [serial online] 2016 [cited 2020 Jul 2];4:64-8. Available from: http://www.jresdent.org/text.asp?2016/4/2/64/180995
| Introduction|| |
The most popular and commonly used oral hygiene kits remain the regular tooth brushes and a plethora of tooth pastes. However, Shada and Shada (2010) stated that individuals either by medical advice or by personal preference have adapted to other oral hygiene kits that includes traditional chewing sticks. Hooda et al. (2009) opined that chew sticks has become a favorite amongst not just local African folks, but amongst persons of affluence. This they attributed to the fact that local chewing sticks are not only available and affordable, but have been shown, based on their age-long traditionally recognized ethno-medical values, to possessing effective agents for oral plaque control.,
In Nigeria, almost everyone has at one time or the other used locally sourced chewing sticks for oral hygiene. This has given rise to several studies on the efficacy of local Nigerian chewing sticks against oral bacteria, such as Streptococcus and oral anaerobes,,Staphylococcus,Lactobacilus specie, Micrococcus specie, N. sicca, Streptococcus oralis, Staphlococcus aureus, Klebsiella specie, Muccor, Giosporium and Aspergillus flavus, which are the organisms frequently associated with oro-dental infections as well as dental caries.
In recent times, there has been an increase in the brands of tooth pastes and upsurge in advertorials for mouth washes with claimed high efficacy in their antimicrobial, antibacterial, and antiplaque properties. While chewing sticks and many of the toothpastes are available and affordable, many of these branded mouthwashes such as Hexedene ®, Brett ®, and Listerine ® are quite expensive. This has sort of conferred on these mouthwashes an ostentatious status, with many local folks believing they are not as effective as their manufacturers claim. This has led many local folks to still prefer the chewing sticks to branded mouthwashes.
Massularia acuminata (G. Don) Bullock ex Hoyl. (Rubiaceae) is one local Nigerian chewing stick that has been highly researched.,,M. acuminate popularly referred to by the Yoruba people of Western Nigeria as Pako Ijebu or Orini jebu is a plant (herb) that grows up to 5m in height. Apart from Nigeria, it is also found in Sierria Leone and the Democratic Republic of Congo. Previous phytochemical studies by Yakubu et al. (2008) have shown that aqueous extract of M. acuminata stem included alkaloids (0.22%), saponins (1.18%), anthraquinones (0.048%), flavonoids (0.032%), tannins (0.75%), and phenolics (0.066%). Apart from its antibacterial and antimicrobial properties,, it has also been shown to have anti-inflammatory properties,, as well as aphrodisiac, androgenic, and gonadotropic properties.
Despite all these studies, presently there is a dearth of research data on the antimicrobial properties of M. acuminata in comparison with some branded mouthwashes. This study therefore, was designed to provide data on the antimicrobial effect of aqueous stem extract of M. acuminate compared with Hexedene ®, Brett ®, and Listerine ®.
| Materials and Methods|| |
Source and collection of samples
Samples of M. acuminata (Pako Ijebu chewing stick) were obtained from Tejuoso Market, Lagos, Nigeria. Branded mouthwashes (Hexedene ®, Brett ®, and Listerine ®) were procured from Humphrey Pharmacy Abraka, Nigeria.
Isolates from the oral cavity were collected from undergraduate students of the Delta State University, Abraka, Nigeria, and were taken to the main laboratory of the Department of Microbiology of the same university within an hour of collection. These were cultured to obtain pure clinical bacterial and fungal isolates using standard streak plate techniques of Cowan and Steel, (2004) and (Harrigan and McCance, 1982).
Preparation of extracts
The method of Okigbo and Omodamiro (2006) which is a modification of that of Al-magboul, et al. (1997) was used to obtain the aqueous extracts. One hundred and fifty gram of the air-dried blended chewing stick (M. acuminata) was soaked in 750 mL of cold water for 72 hours at room temperature and stirring was done every 6 h. It was filtered with a sterile filter paper inserted in a sterile glass funnel into a 250-mL conical flask. The extract was then evaporated under reduced pressure using a rotary evaporator. The standard extract obtained was stored in a refrigerator at 4° C until required for use.
Ethanol extract was prepared by using a slightly modified method of Adekunle and Odukoya (2006). One hundred and fifty gram of air dried blended chewing stick was soaked in 750 mL of 80% ethanol for 72 h at room temperature and stirring was done every 6 h. The content was filtered with a sterile Whatman's filter paper inserted in a sterile glass funnel into a 250-mL conical flask. The extract was then evaporated under pressure using a rotary evaporator. The standard extract obtained was stored in a refrigerator at 4° C until required for use.
Chemicals and reagents
Hexedene ® (made by Pharma Deko Plc (RC6711) — Batch Number 086), Brett ® (made by Pharma Deko Plc (RC6711) — Batch Number 170) and Listerine ® (made by Johnson and Johnson Limited, Maidenhead, United Kingdom — Batch Number 583890), were all procured from Humphry Pharmacy, Abraka, Nigeria, for the purpose of the experiment.
Standardization of inoculum
The McFarland standard (1907) was used for the standardization of bacteria and fungi isolates. For the preparation of McFarland standard 1% of anhydrous barium chloride (BaCl2) was mixed with 1% (vol/vol) cold pure sulfuric acid (H2 SO4). The screw cap was tightly sealed and the suspension was stored in dark at room temperature.
Bacterial and fungal growths in broth were then adjusted to be visually comparable with the McFarland's standard.
Assay of antimicrobial extracts
Antimicrobial activity of all the extracts were evaluated by “zones of inhibition” using the agar well diffusion method for bacteria and disc diffusion method for fungi. The prepared nutrient agar plates were allowed to dry, after that standardized broth culture were used to inoculate sterile nutrient agar plate in order to obtain a confluent growth on the agar surface. Sterile cork borer was used for the bacterial study, while impregnated discs were used for the fungal study.
For the bacteria, a sterile cork borer was used to make five wells in each plate and different concentrations of an extract were introduced into each plate. In a plate, the same solution but of different concentration was introduced into each well using sterile pipette. These were then left on the bench for 1 h for adequate diffusion of the extracts and were incubated at 37°C for 24 h. After incubation, the diameter of the zones of inhibition around each well was measured in mm and recorded.,
For the fungi, the disc diffusion technique according to Singh et al., (2008) with minor modification, was used to determine the antimicrobial sensitivity pattern of the fungi isolates. The disc was prepared from Whatman's filter paper by puncturing with a cork borer of 6-mm diameter. The disc was sterilized by autoclaving at 121°C for 15 mi using a sterile pipette. Different concentrations of each extract were impregnated on the disc and allowed to dry on the bench for 1 h before introducing it on the inoculated plate and was kept for 24 h at normal atmospheric temperature. After which the diameter of the zones of inhibition around each disc was measured in mm and recorded.
Determination of minimum inhibitory concentration (MIC)
The MIC helps to measure the concentration of an antibiotic necessary to inhibit growth of standardized inoculums more accurately under defined conditions. The MIC of active extracts was determined by the tube dilution method. Twofold serial dilutions of the extracts with peptone water were prepared. These were then challenged with small inoculum (0.1 mL) of an overnight sterile diluted broth culture of the test organisms as described previously by Owhe-Ureghe et al. 2010.
The culture was then covered and incubated at 37°C for 24 h. The tubes were examined for turbidity. The lowest concentration that inhibited the growth of the test organism (tubes lacking visual turbidity matching the negative growth control) was taken as MIC.
Determination of minimum bactericidal concentration (MBC)
The minimum bactericidal concentration (MBC) is the lowest concentration of the antimicrobial agent that kills at least 99.9% of the test organism. The tubes that showed no visible growth in the MIC test tubes were subcultured onto nutrient agar plate and incubated at 37°C for 24 h. The lowest concentration of extracts that yielded no growth was recorded as the MBC.
| Results and Discussion|| |
The ethanol extract of M.A. was bactericidal for N.S and S.O, while it was not for other test bacterial species, while the aqueous extract of MA. Was bactericidal for N.S. only. Two of the synthetic mouth wash (Hexedene and Listerine) showed similar trend for the test organisms, i.e bactericidal for N.S., but were not for other test organisms. However, Listerine (0.5 ml) was also bactericidal for Micrococcus sp [Table 1]. The result of the MIC and the MBC of MA for all the tested bacterial isolates in [Table 2] were 25.0 mg/ml and >50.0 mg/ml respectively.
|Table 1: Zones of inhibition (mm) of extract and mouthwashes on test isolates of bacteria species|
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|Table 2: Minimum inhibitory and minimum bactericidal concentration (MIC and MBC) of extracts on bacteria species|
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The antimycotic activity of MA is shown in [Table 3] and [Table 4] below. Mucor sp was most sensitive to MA and Listerine (synthetic mouth wash) with zones of inhibition of 23.0 mm and 20.0 mm respectively. Their respective MIC and MBC were 25.0 mg/ml and 50.0 mg/ml.
|Table 3: Zones of inhibition (mm) of extracts and mouthwashes on test isolates of fungi species|
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|Table 4: Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of extracts on fungi species|
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The antibacterial and antifungal activity of M. acuminata can be attributed to the presence of phytochemical compounds (glycosides, saponin, tannins, flavonoids, anthocyanin, anthocyanins, anthraquinones, and phlobatannin) present in it. The extract of M. acuminata showed a high inhibitory activity against the bacteria species tested, since it exhibited the highest antibacterial inhibition among the extract, this shows that this research work is in conformity with the work by Barnabas and Nagarajan (1998). The result obtained showed that ethanol was a better and more powerful solvent than water. This is in agreement with the report of Kareen et al. (2008) which stated that active components of plants are more soluble in organic solvents. The great antibacterial effect showed by the chewing stick may have been as a result of tannin present in them.
Listerine mouthwash showed a high inhibitory activity against the test organism, and it showed the highest inhibitory activity against the test organisms for fungal species [Table 3]. This may be due to the presence of essential oils, thymol, methyl salicylate, and encalyptol. The result obtained shows that all the extracts were bacteriostatic but not all were bactericidal.
| Conclusion|| |
In vitro studies in this research work showed that the aqueous and ethanol extract of M. acuminate had an MIC of 25 mg/mL against all the test isolates. In conclusion, based on the above findings, all the extracts contain potential antimicrobial substance that can be used in the control of bacterial and fungal infections. This provides scientific support for the use of these natural antimicrobials in the treatment of pero-dental infections.
Based on the result obtained, M. acuminata when properly used will be more effective in the treatment of oro-dental infections compared to the mouthwash (Hexedene ®, Brett ®, and Listerine ®) used in this research. This is because most of the pero-dental infections are caused by bacteria. As a result, it is suggested that more research should be carried out on the antimicrobial effect of the leaf, stem, and root extract of this plant to ascertain the pharmacokinetics and its mechanism of action.
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[Table 1], [Table 2], [Table 3], [Table 4]