Antimicrobial Activity of Guava Leaf (Psidium Guajava) Extracts Against Some Clinical Bacteria Isolates
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ISSN (Print): 2476-8316
ISSN (Online): 2635-3490
Dutse Journal of Pure and Applied Sciences (DUJOPAS), Vol. 7 No. 2b June 2021
Antimicrobial Activity of Guava Leaf
(Psidium Guajava) Extracts Against
Some Clinical Bacteria Isolates
1
M.S Kaware, 2M.S Ismail
1
Department of Community Medicine,
College of Medical Sciences,
Umaru Musa Yar'adua University Katsina,
PMB 2218, Katsina State, Nigeria.
2
Department of Microbiology,
Faculty Natural and Applied Sciences,
Umaru Musa Yar'adua University Katsina,
PMB 2218, Katsina State, Nigeria.
Email: msanik2000@mail.com
Abstract
Guava (Psidium guajava) leaf is commonly used as a medicine against gastroenteritis and child
diarrhea by those who cannot afford or do not have access to antibiotics. This study was aimed to
determine the antimicrobial activity of Guava leaf (Psidium guajava) extracts on Staphylococcus
aureus, Streptococcus pyogenes, Salmonella typhi and Escherichia coli using the agar well diffusion
method. Two different extracts were obtained from the guava leaf (aqueous-soluble and ethanol-
soluble extracts). The antimicrobial test results showed that the Psidium guajava ethanol and aqueous
extracts have potential antimicrobial activity against the Gram-positive and Gram-negative bacterial
strains. The Ethanolic extract showed the maximum mean zone of inhibition on Staphylococcus
aureus (21.3mm at 250mg/ml conc.) and the least inhibitory effect on Escherichia coli (13.5mm at a
concentration of 150mg/ml). Also, from the result, the aqueous extract of P. guajava produces the
highest mean zone of inhibition on Staphylococcusaureus (18.8mm at the concentration of 250mg/ml)
and the lowest mean inhibition zone on E. coli (12.4mm at 200mg/ml conc.). The aqueous extract at a
150mg/ml concentration, 100mg/ml were ineffective to both E. coli and S. Typhi. The minimum
inhibitory concentration (MIC) showed that S. aureus was the most sensitive to Ethanolic extract (at
the lowest concentration of 12.5mg/ml) and S. pyogenes for aqueous extract lowest concentration of
25mg/ml. The minimum bactericidal concentration MBC of aqueous extract was 25mg/ml for S.
pyogenes and ethanol extract for 12.5mg/ml for S. aureus. Conclusively, this result indicate that
Guava leaf extracts have antimicrobial activities against the test isolates, and the ethanolic extract is
more effective compared to the aqueous extract.
Keywords: Psidium guajava, Antimicrobial, Ethanolic, Aqueous Zone of inhibition,
INTRODUCTION
The Guava plant (Psidium guajava) is a tropical plant easily found in Nigeria (Nwinyi et al.,
2008). Many parts of this plant are utilized by humans, especially its fruits and leaves.
Notably, its fruit is commonly consumed as fresh fruit or processed food. Guava fruit
*Author for Correspondence
M.S. Kaware, M.S. Ismail, DUJOPAS 7 (2b): 245-252, 2021 245Antimicrobial Activity of Guava Leaf (Psidium Guajava) Extracts Against Some Clinical Bacteria Isolates contains tryptophan lysine, pectin, calcium, phosphor, minerals and vitamin (Naseer et al., 2018). Its fruit is currently used to treat diabetes mellitus patients and people who have high-level blood cholesterol (Nwinyi et al., 2008). Guava is said to contain a broad spectrum of phytochemicals which include polysaccharides, vitamins, essential oils (Smith and Siwatibau, 1975, Macleod and Troconis, 1975), minerals, enzymes, proteins (Deo and Shastri, 2003), sesquiterpenoid alcohols and triterpenoid acids (Smith et al., 1975; Wilson and Shaw, 1978; Begum et al., 2002), alkaloids, glycosides, steroids, flavonoids, tannins, saponins (Cho etal., 2003; Narayana et al., 2001; Geidam et al., 2007). P. guajava or guava is very rich in antioxidants and vitamins and high in lutein, zeaxanthin and lycopene (Hobert and Tietze, 1998). Guava is rich in tannins, phenols, triterpenes, flavonoids, essential oils, saponins, carotenoids, lectins, vitamins, fiber and fatty acids. Guava fruits are also a good pectin source - a dietary fiber. The flavonoids have demonstrated antibacterial activity. Quercetin is thought to contribute to guava's anti- diarrhea effect; it can relax intestinal smooth muscle and inhibit bowel contractions. Also, other flavonoids and triterpenes in guava leaves show anti-spasmodic activity. Guava also has antioxidant properties attributed to the polyphenols found in the leaves (Cho etal., 2003; Narayana et al., 2001; Geidam et al., 2007). The guava fruits are either eaten fresh or made into drinks, ice cream, and preserves. Its fruit is still enjoyed as a sweet treat by indigenous peoples throughout the rainforest. The leaves and bark of the guava tree have a long history of medicinal uses that are still employed today. This study was aimed to determine the antibacterial activity of ethanolic and aqueous extracts of Psidium guajava leaves against some clinical bacterial isolates. MATERIALS AND METHODS Collection and Identification of Plant Material The Fresh forms of Guava leaves were obtained from Lambun Sarki, Kofar Marusa Low- cost, Katsina, Nigeria, in August2016. It was identified and authenticated at the Herbarium of the Biology Department, Umaru Musa Yar'adua University, Katsina and processed as described by Olayemi and Opaleye (1999). Extraction of the Plant Material The powdered sample of the plant materials was extracted following Gupta et al. (2009). Fifty grams (50g) of each dried powder were weighed into a glass container and extracted exhaustively with 500ml sterile distilled water by percolation method for one week. The sealed bottle has undergone vigorous shaking at regular intervals. The mixture was sieved through a muslin cloth and then re-filtered by passing through Whatman's filter paper No. 1. The deposit was concentrated by complete evaporation of the solvent on a water bath at 45°C to yield the reddish-brown gummy solid extract, which was 11.8% of the total powder extracted. The extract was subsequently transferred into a clean, sterile, airtight glass container and stored in the refrigerator at 4°C until use. Preparation of Water and Ethanolic Extracts of Psidium guajava The method of Olayemi and Opaley (1999) was adopted for the extraction of the plant. This was carried out by measuring 20g of each fine grounded powder of P. guajava leaves on an electronic weighing balance. It was dispensed into two beakers, each containing 80ml of distilled water and soaked for 72hrs, after which the solution was carefully filtered with M.S. Kaware, M.S. Ismail, DUJOPAS 7 (2b): 245-252, 2021 246
Antimicrobial Activity of Guava Leaf (Psidium Guajava) Extracts Against Some Clinical Bacteria Isolates muslin cloth into a sterilized conical flask of 100ml and the filtrates obtained were stored in the refrigerator at a temperature of 4°C until required. For the ethanolic extract,20g of each fine grounded powder of Guava leaves was dispensed into a beaker containing 80ml of 95% ethanol. They were soaked for 72hours while the resulting supernatant was decanted into a conical flask of 100ml and kept in the refrigerator for further study (Olayemi and Opaleye's: 1999). Collection of Test Organisms The clinical isolates of bacteria (Escherichia coli, Staphylococcus aureus, Salmonella typhi and Streptococcus pyogenes) were obtained from the laboratory of the Microbiology department, Umaru Musa Yar'adua University. The test organisms were characterized using the methods of Cheesebrough (2002) by observing their cultural growth characteristics. Biochemical confirmatory tests were performed to further confirm each test organism's identity following standard methods described by Manga (2004). The bacteria's pure cultures were streaked on to Nutrient Agar slants and then incubated at 370C for 24 hours. The cultures were stored in the refrigerator at four-degree Celsius until needed for subsequent use. Standardization of inoculums Bacterial colonies from the 24hours overnight cultures were used to make direct suspension of each bacterial isolates in separate test tubes containing 1ml of sterile physiological saline. The suspensions were adjusted to match the 0.5 McFarland turbidity standards. Antimicrobial Susceptibility Tests of Psidium guajava Extracts The agar diffusion method of Olayemi and Opaleye (1999) was used. From the 48hrs old cultures of the test organisms with the aid of six sterile syringes, 0.5ml of each of the test organisms was inoculated into various sterile Petri dishes. In each dish 20ml of sterile media was poured aseptically. The dishes were gently rocked for a proper mixture, and the nutrient agar was allowed to set. Following that, five wells (two for the ethanolic extract, two for the water extract and one for ethanol as a control) were dug in the plates with a sterilized cork borer of 6mm diameter. With the proper labelling of wells, 0.5ml of each of the plant's ethanol extract was introduced into the first and third wells, while 0.5ml of ethanol was introduced into the fifth well as the control. They were allowed to stand for one hour for proper diffusion and then incubated at 37°C for 24hours. The test organisms' sensitivity to ethanol and water extract of P. guajava was indicated by a clear zone of inhibition around the wells. The clear zone diameter (Zone of inhibition) was measured to the nearest millimeter using a transparent ruler. This was taken as an index of the test organisms' degree of sensitivity to ethanol and water extracts. Positive and negative controls were set up with some broad-spectrum antibiotics. Determination of Minimum Inhibitory Concentration (MIC) Double dilution of guava extracts wares prepared to range from (100, 50, 25, 12.5 and 6.5 mg/ml). The MIC of the Guava leaf extracts was determined using the broth dilution technique. Six sterile test tubes were placed in a test tube rack and labelled appropriately. Using a pipette, 4ml of Nutrient broth was dispensed into each tube. About 0.1ml of the standardized inoculum was inoculated in the tubes using a sterile syringe, and 1ml each of M.S. Kaware, M.S. Ismail, DUJOPAS 7 (2b): 245-252, 2021 247
Antimicrobial Activity of Guava Leaf (Psidium Guajava) Extracts Against Some Clinical Bacteria Isolates
the varied concentrations were dispensed into the tubes containing the nutrient broth and
bacterial suspension.
The control tube contained nutrient broth and standardized test isolates. The tubes were
incubated at 37oC for 24hrs. The tubes were examined for visible signs of bacterial growth by
checking for turbidity. The dilution that first showed no sign of visibleturbidity was taken as
the minimum inhibitory concentration (Akinyemiet al., 2005, Abubakar, 2010)
Determination of the Minimum Bactericidal Concentration (MBC.)
The test tubes for MIC that showed no visible turbidity after incubation were sub-cultured
on nutrient agar plates and incubated at 37oC for 24hours. The concentration that showed no
visible growth after incubation was considered the minimum bactericidal concentration.
(Chesbrough, 2002). This was carried out for all the organisms using both aqueous and
ethanolic extracts, respectively.
RESULTS
The result obtained from the antimicrobial activities of the ethanol and aqueous extracts of
Psidium guajava showed varying degrees of inhibition. The ethanol extract of Psidium guajava
produced the highest mean zone of inhibition on Staphylococcus aureus (21.3mm at a
concentration of 250mg/ml) while the least inhibitory effect was observed on Escherichia coli
(13.5 mm) at a concentration of 150mg/ml, as shown in Table 1 below.
Also, the result revealed that the aqueous extract of Psidium guajava produced the highest
mean zone of inhibition on Streptococcus pyogenes (19.1 mm at 250mg/ml conc.), while
aqueous extract was ineffective against E. coli and S. typhi at the concentrations of 150mg/ml
and 100mg/ml whereas, for S. aureus it is ineffective at the concentration of 100mg/ml as
indicated in Table 1 below.
TABLE 1: Mean diameter of the zone of inhibition produced by Ethanolic and Aqueous
extracts of P. guajava
Extracts Concentration S. aureus S. pyogenes E. coli S. typhi
(mm) (mm) (mm) (mm)
Ethanolic 250mg/ml 21.3 19.8 19.1 17.2
200mg/ml 20.3 18.8 18.5 17
150mg/ml 18.7 17.1 13.5 -
100mg/ml 18.7 - - -
Aqueous 250mg/ml 18.6 19.1 13.2 17.1
200mg/ml 18.7 18.3 12.4 14.1
150mg/ml 18.6 16.3 - -
100mg/ml - 17.4 - -
Key: -: No inhibition
The inhibitory effect of ethanolic and aqueous extract of P. guajava was presented in Table 2.
Minimum Inhibitory Concentration (MIC) values for ethanolic and aqueous extracts
arranged from 100mg/ml to 3.125mg/ml for S. aureus, S. pyogenes, E. coli and S. typhi. The
result indicated that Staphylococcus aureus was the most sensitive to Psidium guajava leaf
extract at the lowest concentration of 12.5mg/ml (ethanolic extract) and 25mg/ml (aqueous
extract).
M.S. Kaware, M.S. Ismail, DUJOPAS 7 (2b): 245-252, 2021 248Antimicrobial Activity of Guava Leaf (Psidium Guajava) Extracts Against Some Clinical Bacteria Isolates
TABLE 2: Minimum Inhibitory Concentration (MIC) of P. guajava leaf extracts against S.
aureus, S. pyogenes, E. coli and S. typhi
Concentration (mg/ml)
Organisms Extract 100 50 25 12.5 6.25 3.125
mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml
S. aureus Ethanol _ _ _ _* + +
Aqueous _ _ _* + + +
S. pyogenes Ethanol _ _ _ _ _* +
Aqueous _ _ _* + + +
E. coli Ethanol _ _ _* + + +
Aqueous _ _* + + + +
S. typhi Ethanol _ _ _ _* + +
Aqueous _* + + + + +
Key: * = MIC value + = turbidity - = no turbidity
The result of minimum bactericidal concentration (MBC) was presented in Table 3. This is
the lowest concentration at which the Psidium guajava extract completely kill the bacterial
isolates. For ethanolic extract, it is found to be at 12.5mg/ml for Staphylococcus aureus. While
for aqueous extract, it was also effective aga5inst Staphylococcus aureus at 25mg/ml
concentration.
TABLE 3: Minimum Bactericidal Concentration of P. guajava leaf extracts on test isolates.
Test organisms Ethanolic extract Aqueous extract
(mg/ml) (mg/ml)
S. aureus
12.5 25
S. pyogenes
6.25 25
E. coli
25 50
S. typhi
12.5 100
DISCUSSION
The antimicrobial activity of the Ethanolic and Aqueous guava leaf extracts was measured
using varying concentrations of 250, 200, 150 and 100mg/ml. The result has shown that both
Guava leaf extracts have antibacterial activities against the bacterial isolates. Comparatively,
the highest mean zone of inhibition (21.3mm) was observed on S. aureus at concentration of
250mg/ml using ethanolic extract. This shows similarities with the findings of Nwanneka et
al (2013), who also found that the ethanolic Guava leaf extract is more effective against S.
aureus when compared to the aqueous extract. This has been attributed to the differences in
compounds extracted using the two different solvents.
The most effective concentration of the aqueous extract was 250mg/ml against S. pyogenes
with zone of inhibition of 19.1 Although the reason for this variation is not clear, the
resistance of the Gram-negative bacteria could be attributed to its cell wall structure
(Biswaset al., 2013). Gram-negative bacteria have an effective permeability barrier,
comprised of a thin lipopolysaccharide exterior membrane, which could restrict the
extruding plant extract's penetration. Gram-negative bacteria were reported to be more
resistant to antimicrobials originated from plants compared to Gram-positive bacteria
(Biswas et al., 2013; Cowan, 1999). Gram-positive bacteria have a mesh-like peptidoglycan
layer which is more accessible to permeation by the extracts (Rameshkumar et al., 2007.)
M.S. Kaware, M.S. Ismail, DUJOPAS 7 (2b): 245-252, 2021 249Antimicrobial Activity of Guava Leaf (Psidium Guajava) Extracts Against Some Clinical Bacteria Isolates
The MIC of Psidium guajava ethanolic and aqueous extract was determined, and the result is
shown in (Table 2). From the results, the inhibitory effect of P. guajava ethanolic and aqueous
extract ranged from MIC values of 3.125mg/ml to 100mg/ml for S. aureus, S. Pyogenes, E.
coliandS Typhi. The result indicated that S. aureus was the most sensitive with a MIC of
12.5mg/ml (ethanolic extract) and 25mg/ml (aqueous extract). This conforms with the work
obtained by Chen et al. (1987) that the minimum inhibitory concentration (MIC) of ethanolic
extract of P. guajava against S. aureus is 12.5.mg/ml.
The result of minimum bactericidal concentration (MBC), which is the lowest concentration
at which the P. guajava extract completely kill the bacterial isolates, showed that ethanolic
extract gave MBC. a value of 12.5mg/ml concentration against S. aureus. The aqueous extract
against S. aureus gave an MBC value of 25mg/ml concentration (Begum et al., 2002).
Results found in this study were supported andopposed in the data reported in the
literature. Nascimento et al. (1999) conducted a study that supports the finding of thepresent
study. The guava extract could haveinhibitory effects against Staphylococcus and
Streptococcus and noeffect on the Escherichia and Salmonella. In contrast, Chanda and Kaneria
(2011) oppose the findings concerning Gram-negativebacteria. (Mahfuzul Hoque et al., 2007),
found no antibacterial activity of ethanolic extracts of guava against E. coli and S. enteritidis;
however, Lakshmi and Sudhakar (2009) found guava sprout extracts were effective against
inhibiting E. coli. Sanches et al., 2001, found that guava's aqueous extract was effective
against Staphylococcus, Streptococcus and Bacillus (Begum et al., 2002).
CONCLUSION
This research demonstrates the antimicrobial potentialof Psidium guajava leaves extract by
using various solvents. The results indicate that ethanol is better thanwater for the extraction
of the antibacterialproperties of guava. The results also indicate that the plant extracts have a
less antibacterial effect on the Gram-negativebacteria, showing that they do not contain
active ingredientsagainst the organisms. The observed inhibition of Gram-positive bacteria,
Streptococcus pyogenes and Staphylococcus aureus suggests that guava possesses compounds
containing antibacterial properties that can effectively suppress the growth when extracted
using ethanol as the solvent.
Comparisons with related data from the literature indicatethat the most diverse outcomes
can be obtained according to the different studies on antibacterial activity. This study
provides scientific insight to determine the antimicrobial principles further and investigate
otherpharmacological properties of guava. Based on thepresent finding, P. guajava leaves
possess the capabilitiesof being a good candidate in the search for a naturalantimicrobial
agent against infections and diseases causedby S. pyogenesand S. aureus.
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