1. Introduction
Plants have been a source of medicinal agents for treating illnesses since the beginning of human civilization [1]. Recently, there has been a lot of attention focused on producing medicines and products that are natural [2]. Several leaves, stems and their extracts have been found to exhibit antimicrobial and antioxidant activities [1]. Researches into traditional plants and herbs have received a major boost due to increasing resistance of microorganisms to orthodox medicine [3]. Muhammad et al. [3] suggested that there is a need to search for new organic molecules of plants with antimicrobial properties.
The presence of bioactive components in medicinal plants makes them effective in treating human infections. These components are naturally occurring compounds in the medicinal plants such as tannins, saponins, flavonoids and they exhibit various important pharmacological activities against microorganisms, inflammation and cancer [4]. The World Health Organization (WHO) has categorized more than 20,000 plant species with medicinal properties and their use is gaining attention due to their availability, cost effectiveness, proven nature of specificity, biodegradability, low toxicity and minimum residual toxicity in the environment [5] [6] [7].
Guava (Psidium guajava) belongs to the family Myrtaceae and it is believed to have originated from South America. Guava tree grow in tropical and sub tropical area of the world like Asia and Africa. Guava has been reported by several researchers to be rich in tannins, phenols, flavonoids, essential oils, saponins. Birdi et al. [8] opined that P. guajava leaves have a broad spectrum of antimicrobial action against a wide range of pathogens. They further reported that guava leaf extract has analgesic, anti-inflammatory, antimicrobial, hepato protective and antioxidant activities.
Although, scientists have identified several phytochemicals in guava, only small fractions have been studied closely and each one works differently [9]. Begum et al. [10] reported the extraction of triterpenoids guavanoic acid and guavacouremic acid from the leaves of guava. Four flavonoids were isolated and identified by Arima and Danno [11] to inhibit the growth of Salmonella enteritidis and Bacillus cereus. The study was aimed at determining the phytochemical constituents and in vitro antibacterial activity of methanol and aqueous extracts from Psidium guajava leaves on Escherichia coli,Salmonella typhi,Staphylococcus aureus and Proteus spp in Ugbokolo, Nigeria.
2. Materials and Methods
Test Isolates
The study was conducted between July and December, 2019. The test organisms for the study; pure isolates of Salmonella typhi,Staphylococcus aureus,Escherichia coli and Proteus spp were obtained from stool and blood samples of patients attending health centres in Ugbokolo, Benue State, Nigeria. The isolates were confirmed at the Microbiology Laboratory, Benue State Polytechnic, Ugbokolo using morphological and appropriate biochemical tests such as Indole, motility, Triple sugar iron, catalase and coagulase and stored in refrigerator at 4˚C.
Collection of Plant Materials
Leaf and Stem bark of Psidium guajava were collected from residential areas in Ugbokolo, Benue State. Identification and authentication of the plant materials was done at the Department of Botany, University of Agriculture, Makurdi, by a botanist, Dr Okoh. S.
Preparation of Extracts
Aqueous and methanol extracts of Psidium guajava leaves and stem barks were prepared separately. The leaves and stem barks were washed in water and air dried on the desk in the laboratory for two weeks. They were frequently turned to achieve uniform dryness. The dried leaves were then crushed and pulverized by grinding using local mortar and pestle. 50 g powder of the leaves and stem barks was soaked in 500 ml each of distilled water and methanol respectively. The flasks were kept at room temperature for 3 days with intermittent shaking. Filtration was done using Whatman filter paper. The extracts were evaporated at 70˚C in water bath with shaker until dried extract samples were obtained. The extracts were purified and separated based on polarity into discrete fractions of bioactive components by column chromatography by a method described by [12].
Phytochemical Screening
The screening of the plant materials for bioactive components was done by a method decribed by Sofowora [13] and Trease and Evans [12]. The extract was tested for the presence of the following bioactive compounds:
Test for Phenol:
2 ml of the extract was measured and dispensed into a test tube, subsequently followed by 3 drops of ferric chloride. Dark blue or dark green colouration indicated the presence of phenol.
Test for Saponins
Frothing method was used to determine the presence of saponins in the extract. 3 ml of distilled water was measured and added to 1ml of the extract in a test tube and agitated vigorously for stable persistent froth. Formation of froth that is stable for up to 10 min is an indicative of the presence of saponins.
Test for Flavonoids
The ethanol and water extracts (5 ml) each was added differently to a concentrated sulphuric acid (1 ml) and 0.5 g of Mg. A pink or red coloration that disappear on standing (3 min) indicates the presence of flavonoids.
Test for Tannins
1 ml of the ethanol extract was added in 2 ml of water in a test tube. 2 to 3 drops of diluted ferric chloride solution was added and observed for green to blue-green (catechic tannins) or blue-black (garlic tannins) coloration.
Test for Alkaloids
20 ml of ethanol extract was evapourated and the dry residue dissolved in 5 ml of HCl (2N) and filtered. A few drops of Mayer’s reagent and Wagner were added, the presence of precipitate indicates the alkaloids.
Antibacterial Susceptibility test
The sensitivity of each extract was determined by using the agar well diffusion method as described by Ahmed and Beg [14] and Arora and Arora [15]. The concentration of the test organisms was adjusted to 0.5 McFarland standard using Phosphate buffered saline (PBS). The inoculums were spread over the entire sterile surface of sterile Mueller-Hinton agar plate, rotating the plate to ensure an even distribution using a sterile cotton swab dipped into the suspension. A sterile 6 mm diameter cork borer was used to bore 6 wells in the agar medium. 0.1 ml of the extract were dispensed into the wells at different concentrations of 50, 100, 150 and 200 mg/ml with utmost care to prevent the spillage onto the surface of the agar. DMSO was dispensed into the fifth hole and Amoxicillin into the sixth hole as negative and positive control respectively. The plates were allowed to stand on the laboratory bench for 1 hour for proper diffusion of the extract into the medium. The plates were then incubated at 37˚C for 24 hours. The zones of inhibition were observed and measured at the end of the incubation period around the agar well to the nearest millimeter using a ruler.
Determination of Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration (MIC) of the extracts was determined using broth dilution technique as described by Ali et al. [16]. Two fold serial dilutions of the extracts were prepared by adding 5 ml of 100 mg/ml of the extract into a test tube containing 5 ml of Nutrient broth and mixed vigorously, thus producing a solution containing 50 mg/ml of the extract. The process continued serially up to the fifth test tube and the last 5 ml was discarded leaving equal volume in the tubes, hence producing the following concentrations; 50, 25, 12.5, 6.25 and 3.125 mg/ml. The sixth test tube does not contain extracts and therefore serves as the negative control. 50 µl of 0.5 McFarland standards of test organisms were introduced into the test tubes and incubated at 37˚C for 24 hours. The test tubes were observed for turbidity. The least concentration with no turbidity was taken as the MIC.
Determination of Minimum Bactericidal Concentration (MBC)
The minimum inhibitory concentration (MBC) of the extracts was determined by a process described by Ali et al. [16].
Minimum bactericidal concentration (MBC) was performed to determine if the test microorganisms in the tubes without turbidity were killed or inhibited. 10 µl was aseptically transferred from tubes with different concentrations that showed no turbidity onto the solidified Mueller-Hinton agar medium and incubated at 37˚C for 24 hours. The MBC was recorded as the lowest concentration of the extract that had less than 99% growth on the agar plates.
Statistical Analysis
The data of average zone of inhibition produced by the isolates against the extracts used was analyzed using One-way ANOVA on the Statistical Package for Social Sciences (SPSS 20). Significance level for the differences was set as P < 0.05.
3. Results
Phyto-chemical screening
The phytochemical constituents of P. guajava leaf and stem bark are as presented in Table 1. The result showed the presence of saponin, flavonoid, tannin and phenol.
4. Antibacterial Activity of the Extracts
Leaf Extracts
The antibacterial activity of aqueous and methanol leaf extracts of Psidium guajava on the isolates is as shown in Table 2. The result showed that Staphylococcus aureus is the most susceptible isolate at 200 mg/ml concentration with zone of inhibition of 11.9 mm (ALE) and 19.0 mm (MLE) and Proteus sp. is the least susceptible with zone of inhibition of 8.5 mm (ALE) and 12.4 mm (MLE).
Stem Bark Extract
The antibacterial activity of aqueous and methanol stem bark extract of Psidium guajava on the isolates is as presented in Table 3. The result showed that Staphylococcus aureus is the most susceptible isolate at 200 mg/ml concentration with zone of inhibition of 14.1 mm (ASE) and 22.3 mm (MSE). Proteus sp. is the least susceptible with zone of inhibition of 13.6 mm (ASE) and 17.4 (MSE).
MIC and MBC
Minimum Inhibitory Concentration and Minimum Bactericidal Concentration of aqueous and methanol extract of P. guajava leaf and stem bark is represented in Table 4. The result showed dilutions of various concentrations of aqueous
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Table 1. Phytochemical constituents of P. guajava leaf and stem bark extracts.
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Table 2. Antibacterial activity of aqueous and methanol leaf extracts of Psidium guajava.
χ = 10.731, P < 0.05. Key: ALE: Aqueous Leaf extract, MLE: Methanol Leaf Extract.
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Table 3. Antibacterial activity of aqueous and methanol stem bark extracts of Psidium guajava on Isolates.
χ = 26.036, P < 0.05. Key: ASE: Aqueous Stem Bark Extract. MSE: Methanol Stem Bark Extract.
and methanol extracts can inhibit and/or kill the isolates. Lower MIC (3.125 mg/ml) was shown by methanol extract than aqueous extract. MBC of methanol extract ranges between 6.25 - 25.0 mg/ml.
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Table 4. MIC and MBC of Psidium guajava extracts.
5. Discussion
The findings of this study showed the presence of flavonoids, saponins, alkaloids, Tannins and Phenol as bioactive compounds from the phytochemical screening of leaf and stem bark extracts of P. guajava. This agrees with the findings of Uboh et al. [17] and Offor [18] who reported the presence of alkaloids, tannins, steroids and flavonoids as bioactive compound in the P. guajava extracts. Ibe and Maduagwu [4] and Nwanneka et al. [19] also reported the efficacy of Alkaloids as anti-imalaria, anticancer and antiasthma. Ngene et al. [20] reported that tannins are effective in treating intestinal disorders such as diarrhea and dysentery.
Results of the study showed that the zone of inhibition of the extracts on the isolates varied significantly. The extracts exhibited the highest antimicrobial activity at a concentration of 200 mg/ml against S. aureus as compared to E. coli, S. typhi and Proteus sp. This could be as a result of the susceptibility of Gram positive bacteria to therapeutic agents than Gram negative bacteria which may be attributed to inhibition of cell wall development by the extracts. This result is supported by findings of Aliyu et al. [21], Joseph and Priva [22] and Muhammad et al. [3] who revealed that the leaf and stem extracts of P. guajava had more antibacterial activity on Gram positive bacterium (Staphylococcus aureus) than Gram negative bacteria (Escherichia coli and Pseudomonas aeruginosa).
One of the important findings of this work is thatPsidium guajava methanol extracts showed higher antibacterial activity compared to aqueous extracts. This could be attributable to better solubility of the active component by the methanol than water. This corroborates the findings of Nwanneka et al. [19] who reported that ethanol extracts of P. guajava showed higher inhibition against bacteria and fungi than aqueous extract. However, this disagrees with the findings of Elekwa et al. [23] and Egharevba et al. [24] who reported that aqueous extracts of P. guajava had higher inhibitory effect than ethanol extract.
The results of the present research on the antibacterial activity of P. guajava extracts of leaf and stem on tested isolates revealed that stem bark extracts possessed higher antibacterial activity than the leaf extract. This agrees with/or holds true with the findings of Elekwa et al. [23] which showed that stem bark was more effective than leaf extract.
The minimum inhibitory concentrations (MICs) of the extract against the isolates showed that aqueous leaf and stem bark extracts inhibited the growth of S. aureus and S. typhi at 6.25 mg/ml while methanol leaf and stem bark extracts inhibited the growth of S. aureus at 3.125 mg/ml. This corroborates the findings of Sushmita et al. [2]. Ngene et al. [20] equally reported that Gram positive organisms exhibited smaller MIC when exposed to extracts of Psidium guajava than those of Gram negative organisms. The Minimum Bactericidal Concentration showed that the leaves and stem extracts of the plant can kill the isolates at 12.5 - 50 mg/ml. Methanol leaf and Stem bark extracts however, exhibited MBC at 6.25 mg/ml. S. aureus had the least MBC. This is consistent with the findings of Biswal et al. [25], El-Ahmady et al. [26] and Muhammad et al. [3].
6. Conclusion
The phytochemical screening of leaf and stem bark extract of P. guajava showed the presence of flavonoids, saponins, Tannins and Phenol as bioactive compounds. The antibacterial activity of the extracts showed that methanol extracts had higher antibacterial activity as compared to aqueous extract. The result equally showed that the Gram positive bacterium was more susceptible to the extracts than the Gram negative bacteria. The findings of this study justify the antibacterial efficacy of leaf and stem bark extracts of P. guajava in the treatment of infections of bacterial origin.
Limitations of the Study
Paucity of funds did not allow for quantitative analysis of the phytochemicals.
Authors Contributions
A.P. and E.E.T. designed the study, did the experimental studies, Bench work and data acquisition for analysis. A.J. did the data analysis and interpretation of results. A.P. did the drafting of the manuscript. Revision of the manuscript for critical intellectual content was done by A.P., O.G.E., A.Y. and H.A.O. All authors gave final approval of the version to be published.