Antibacterial Activities of Psidium guajava (Guava) and Velvet tamarin (Icheku) Local Chewing Sticks on Streptococcus mutans Isolated from Human Mouth

Globally dental diseases are mainly caused by Streptococcus mutans, it is one of the leading causative agents of dental caries worldwide, because of its resistance to conventional antibacterial agents, alternative therapies are used to control resistance of oral pathogens. This research was done to determine the antibacterial activities of Psidium guajava (guava) and Velvet tamarin (Icheku) chewing sticks on Streptococcus mutans isolated from the oral cavity. The study was conducted in Owerri Imo State Nigeria during November-December period. Phytochemical analysis of the plant extracts was done using appropri-ate techniques. The procedure used for antimicrobial susceptibility test was disk diffusion method. Serial dilutions of Psidium guajava (guava) and Velvet tamarind (Icheku) extracts were prepared, Muller-Hinton media was used to put together the extract of serial dilutions of Psidium guajava (guava) and Velvet tamarin (Icheku) and a microbiological procedure were used for vi-sually determining the minimum inhibitory concentration as well as tans and can be used in oral hygiene. There is a need for further investigation on the plant extracts as the rural poor make use of it because it is cheap, rea-dily available and the rich also use it once they are in the village mostly in Eastern Nigeria. Similarly chewing sticks has been reported to be practiced by 90 of rural population in Nigeria.


Introduction
Nowadays, 60% -90% of the young people worldwide suffer from dental caries [1]. The scientific name for cavities or tooth decay is dental caries, it is the breakdown of teeth caused by acid made by bacteria, the acid they make destroys the tooth hard tissues (enamel, dentin and cementum). This acid is produced by the bacteria when they break down food debris or sugar on the tooth surface and thus a diet high in simple sugar is a risk factor. If mineral breakdown is greater than build up from sources such as saliva, caries results. Proper oral hygiene habits are needed for the control of dental caries due to their multifactorial etiology [2] and [3].
Streptococcus mutans is a facultative anaerobic gram positive coccus commonly found in the oral cavity and is a significant contributor to dental caries and the main microorganism associated with caries and dental plaque [4]. This organism splits the sucrose in food and uses one of the sugars to build its capsule which sticks tightly to the tooth. The bacteria that are trapped in the capsule use the sugar to fuel metabolism [5].
Chewing sticks are often used in Nigeria and Africa in general in maintaining oral hygiene, they are made from roots, twigs or stems of a plant. The preferred are cleaned with water to remove dirt, cut to convenient length which varies In Middle East and Africa shrubs and local trees with good taste, bitter and bristle are chosen as chewing sticks for their beneficial effects on the supporting tissues and teeth [6]. The comparative benefit and popularity of chewing sticks in the world as an oral hygiene alternative make it a cheap agent for plaque control in our environment. Their taste is having anti-plaque and many other pharmacological properties [7]. Most of these plants' species have antibacterial properties, good flavor, foaminess, hardness and a texture that is friendly on the teeth ids and alkaloids, these chemicals are responsible for their effectiveness when used as twig [9]. In Nigeria Psidium guajava chewing sticks are used in mechanical and chemical cleaning of oral tissue and it is efficient and effective.
The teeth cleaned with Psidium guajava chewing sticks are usually devoid of tartar and other stains from the teeth, provide enamel barrier, whitens teeth, mineralize dental tissue, increase salivary flow, fresh and devoid of dental plaques and caries [10].
The present study was set to determine the antibacterial activities of Psidium guajava (guava) and Velvet tamarin (Icheku) chewing sticks on Streptococcus mutans isolated from human mouth.

Study Area
Owerri municipal is a local government area in Imo State, Nigeria. It has an es-

Ethical Approval
Scientific and Ethical permit/clearances were obtained from Medical Centre Federal Polytechnic Nekede in Owerri municipality. Written informed consent was given to parents or guardians of children that participated in the study.

Study Population
The study population includes human of all ages and sex. The saliva samples

Samples Collection
Saliva Samples was collected from a consenting adult volunteer at Federal medical Centre Owerri, Imo State. Criteria used in the collection of the saliva samples include: 1) The patient was told when to collect the saliva (which is from 8 -10 a.m. if possible) and the subject was asked to refrain from drinking, eating and oral hygiene procedures for at least an hour prior to the collection.
2) The subject was given distilled drinking water and be asked to rinse their mouth well for a minute and then expectorate or swallow the water.
3) Five minutes after this oral rinse, the subject was asked to spit into a 2ml sterile tube. Encourage the subjects to place the tube on ice while collecting the saliva.
4) Approximately 2 ml volume of saliva was collected.
5) The specimen was returned to the laboratory immediately for processing.
Processing was done within an hour window of time [11].

Isolation of the Test Bacterium
The media used for isolation of the organism is Mitis-salivarius Bacitracin (MSBA) and it was prepared according to the manufacturer's instruction.
A milliliter of each sample collected was spread on MS-agar plates using sterile spreading glass. Cultures were incubated anaerobically, using anaerobic candle jar, for 48 hrs. at 37˚C.

Identification of Isolates
Colonies grown on MS-agar medium were spread on the Mitis-salivarius Bacitracin agar (MSBA) plates and incubated anaerobically for two days. Subcultures were repeated several times in order to obtain pure cultures. The isolates will also be identified and characterized using the following tests: gram staining, catalase and haemolysis tests [12] and [13]. Results will be compared to those of ref-

Plant Twigs Collection and Identification
Local identification of the plants Psidium guajava (guava tree) and Velvet tamarin (Icheku tree) and collection of their twigs were done during field walks at Okwelle Irete Owerri, Imo State with the help of key informants and a guide.
Taxonomical identification was done with the help of a botanist and voucher specimens stored at the Federal Polytechnic Nekede Owerri.

Extraction and Sterilization of Aqueous Extracts of the Plant Twigs
Preparation of extracts was done according to the method of (14), with a few modifications as follows: fresh twigs from Psidiumguajava (guava tree) and Velvet tamarin (Icheku tree) was crushed using a wooden mortar and pestle and allowed to dry under shade for 3 weeks and a sterile manual grinder was used to crush the twigs into powder.

Soxhlet Extraction
This was done using 95% ethanol in a Soxhlet apparatus for the two plants [15].

Phytochemical Analysis of Psidium guajava (Guava) and Velvet tamarin (Icheku)
The aqueous and soxhlet extracts obtained from the twig of the plant were subjected to phytochemical test using standard methods [15].

Test for Saponins (Frothing Test)
3 ml of each extract was shaken vigorously for about 5 min; it was allowed to stand for 30 sec and observed for frothing which is indicative of the presence of saponins.

Test for Tannins (Ferric Chloride Test)
2 drops of 5% FeCl 3 were added to 1 ml of the extract. A greenish precipitate indicated the presence of tannin in the four extracts. showed the presence of Flavonoids in each extract.

Test for Phlobatannins (Hydrochloric Acid Test)
2 ml of the extract was added to dilute hydrochloric acid and observed for a red aprecipitate formation that indicated the presence of phlobatannins.

Preparation of Culture Plates
Ampoule containing pure forms of S. mutans were obtained. Culture plates for S mutans were prepared, by inoculating the content of the ampoule in nutrient agar at 37˚C for 12 h. Growth obtained from agar plates was transferred to nutrient agar for testing the antimicrobial activity of the extracts and mouthwash.

Antimicrobial Susceptibility Testing
The disc diffusion (agar well) technique as described by (16) was adopted for this study to evaluate the antibacterial activity of the plant extracts. 0.2 ml aliquot of each of each of the extract was asceptically dropped into agar wells (of 6 millimetres in diameter) bored on already inoculated nutrient agar plates containing the test organism (Streptococcus mutans) and appropriately labelled.
The nutrient agar plates were then incubated at 37˚C for 24 hours for the development of zones of inhibition or its absence. The zones of inhibitions were measured with a meter rule.

Determination of Minimum Inhibitory Concentration (MIC)
For the MIC test, 2 g of each of the extract was dissolved in four millilitre (4 ml) of peptone water; this gives 500 mg/ml. Also, 0.8 g of the same exudate was

Test for Minimium Bacteriocidal Concentration of the Extracts
Tubes showing no visible growth from the MIC test were sub-cultured onto sterile nutrient agar plates and incubated at 37˚C for 24 hours. The lowest concentration of the extracts yielding no growths recorded as the MBC. Table 1 reported the isolation of Streptococcus mutans and the result is in line with the finding of Ryan and Ray, (2010) which states that Streptococcus mutans is a facultative anaerobic cocci-shaped, gram positive bacteria commonly found in the oral cavity and has a major role in tooth decay formation. Thus, Streptococcus mutans are considered the main causative microorganism associated with dental caries that plays a major role in tooth decay [18].

Phytochemical Analysis of the Plant Extracts
Phytochemical analysis of the plants extracts revealed the presence of tannins, saponins glycosides, alkaloid, steriods, and phenol (Table 2). These phytochemical constituents depicts the antimicrobial effects of the plants and these results support earlier findings on the efficacy of the plant extracts and also confirmed    the rationale for the medicinal use of the studied plants [19]. The results from this study reveals that the ethanol extract of plants extracts contain more of the constituents when compared with the aqueous extracts. Table 3 shows  [20] and [21].

Minimum Inhibitory Concentrations (MIC)
The Minimum Inhibitory Concentration (MIC) of the extracts as presented in Table 4 shows that the aqueous extract of guava twig has the least inhibitory ef- This simply implies that the plant extracts is efficient in inhibiting visible microbial growth but the ethanol extract of guava, icheku and their combined form inhibits the organism at a lower concentration followed by the aqueous extract of guava and icheku and then the aqueous extract of Guava will come last.

Conclusion and Recommendations
Findings from this work support the use of Psidium guajava (Guava) and Velvet tamarin (Icheku) twigs in oral hygiene since their potential anti-plaque effect is likely to complement the mechanical plaque-removing property of chewing-sticks and suggests that Psidium guajava (Guava) and Velvet tamarin (Icheku) twigs contain compounds that are active against S. mutans, and merit further investigation as they are possible sources of cheap dental health care for the rural poor.