ANTIBACTERIAL ACTIVITIES OF DRIED LEAF EXTRACTS OF CARICA PAPAYA, PTEROCARPUS SOYAUXII, AND VERNONIA AMYGDALINA ON CLINICAL ISOLATES OF ESCHERICHIA COLI, KLEBSIELLA PNEUMONIAE, STAPHYLOCOCCUS AUREUS AND BACILLUS SUBTILIS

Abstract

The antibacterial activities of cold and hot ethanol extracts of air-dried leaves of Carica papaya, Pterocarpus soyauxii, and Vernonia amygdalina on clinical isolates of Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Bacillus subtilis were investigated. The cup-plate agar method was used to determine bacterial susceptibility. All the plant extracts screened were potent on the entire clinical isolates tested. However, there was no significant difference in the inhibition zone diameters of the plant extracts screened (on all the test isolates). There was also no significant difference in the inhibition zone diameters between the cold and hot ethanol extracts of each plant. Phytochemical analysis revealed the presence of alkaloids, anthroquinone, flavonoids, saponins, steroids, tannins, terpenoids and glycosides in all leaf samples. The results obtained here reveal the antibacterial potentials of the leaf extracts of Carica papaya, Pterocarpus soyauxii, and Vernonia amygdalina, and suggests their possible exploitation for the development of novel herbal-based antimicrobials.

Headnote

ABSTRACT

The antibacterial activities of cold and hot ethanol extracts of air-dried leaves of Carica papaya, Pterocarpus soyauxii, and Vernonia amygdalina on clinical isolates of Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Bacillus subtilis were investigated. The cup-plate agar method was used to determine bacterial susceptibility. All the plant extracts screened were potent on the entire clinical isolates tested. However, there was no significant difference in the inhibition zone diameters of the plant extracts screened (on all the test isolates). There was also no significant difference in the inhibition zone diameters between the cold and hot ethanol extracts of each plant. Phytochemical analysis revealed the presence of alkaloids, anthroquinone, flavonoids, saponins, steroids, tannins, terpenoids and glycosides in all leaf samples. The results obtained here reveal the antibacterial potentials of the leaf extracts of Carica papaya, Pterocarpus soyauxii, and Vernonia amygdalina, and suggests their possible exploitation for the development of novel herbal-based antimicrobials.

KEY WORDS: Carica papaya, Pterocarpus soyauxii, Vernonia amygdalina, sensitivity, clinical isolates

INTRODUCTION

The exploitation of plants for medicinal purposes is an age-long practice common to all societies. It is on this basis that researchers continue working on medicinal plants with a view to developing novel and improved therapeutic products (Usman & Osuji, 2007). Studies have it that plants play an indispensable role in the maintenance of healthy living (Edeoga & Eriata, 2001). Plants have been extensively applied for the treatment of ailments worldwide, and according to WHO, 80% of world population depend on plant based medicine for their healthcare (Osuagwu & Akomas, 2013; WHO, 2001).

The renewed interest in herbal medicine and the growing preference for herb-based medicine over synthetically produced medicine may not be unconnected with: high cost, adulteration, and increasing toxic side effects of synthetic drugs; multiple drug resistance due to indiscriminate use of commercial antimicrobial drugs; and the safety and cost-effectiveness of using plant based medicines (Shariff, 2001; Koche et al, 2001; Gupta et al, 2008). Apart from the broad therapeutic potency of plant based medicine, antimicrobials from plants are not only highly effective in treating infectious diseases; they on the other hand mitigate many of undesirable side effects associated with synthesized antimicrobials (Werner et al, 1999; Perulmalsamy & Ignacimuthus, 2000).

According to researchers, the medicinal property of plants is due to the presence of secondary metabolites, such as: alkaloids, tannins, flavonoids, phenolics, steroids, resins, and others (Lohiya et al, 2008; Ianovici et al., 2010). Antimicrobial activities are either due to single or combined effects of these bioactive substances (Frel et al, 1998).

The antimicrobial potentials of a wide array of plants have been investigated by numerous workers. Carica papaya, Pterocarpus soyauxii, and Vernonia amygdalina are common plants of the tropics. While the leaves of P. soyauxii and V. amygdalina are commonly used as vegetables in food preparation, the fruit of C. papaya is a soft fruit commonly used as a snack or desert (Osuagwu, 2008). For therapeutic purposes: C. papaya leaves are made into tea for the treatment of malaria, indigestion, obesity, and high blood pressure, while the fresh leaf poultice is used to treat sores (Verpoorte et al, 2002; Alabi et al, 2012). The leaves of V. amygdalina are employed in the treatment of malaria, hiccups, stomach discomforts, and kidney diseases (Hamowia & Saffaf, 1994). This study explored the phytochemistry of the crude ethanol leaf extracts of C. papaya, P. soyauxii and V. amygdalina and measured the in vitro effects of these crude extracts on bacteria of medical importance, namely: Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Bacillus subtilis.

MATERIALS AND METHODS

Collection and authentification of plant samples. Leaves of Carica papaya, Pterocarpus soyauxii, and Vernonia amygdalina were collected from a household backyard garden in Mbaise, (Imo State, Nigeria) at midday. The leaves were authenticated and labeled by the Forestry Department, Ministry of Agriculture and Natural Resources, Owerri (Imo State, Nigeria).

Preparation of plant extracts. The leaves were thoroughly rinsed in running tap water and then in sterile distilled water. After which they were air-dried at room temperature (29±20C) to constant weight and then pulverized into fine powder with an electric mill. The pulverized leaves were labeled and stored in clean sealed containers at 40C till required. 100g of each powdered plant sample was soaked and thoroughly mixed in 500mL of 70% ethanol and left to stand for 48 hours for the cold extraction, while for the hot extraction a similar setup was maintained at 600C in a water-bath for 3 hours. The slurries were then aseptically filtered using folded white cotton handkerchiefs. The filtrates were evaporated to dryness using a rotary evaporator at 400C. The dried residues were dissolved in sterile distilled water using two-fold serial dilution method to obtain 100mg/mL, 50mg/mL, 25mg/mL, and 12.5mg/mL concentrations.

Collection and preparation of test organisms. Stock cultures of clinical isolates of Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Bacillus subtilis were obtained from the Microbiology Diagnostic Laboratory of Imo State University Teaching Hospital (IMSUTH) Orlu (Imo State, Nigeria). Viability test was carried out for each isolate by resuscitating the organism in buffered sterile peptone broth and then sub-cultured into soybean casein digest broth and incubated at 370C for 24 hours.

Antimicrobial activity test. Plant extracts were screened for antimicrobial activity on the test isolates using a modified method of the cup-plate agar diffusion method (Ebi & Ofoefule, 1997). 0.2mL of 24 hour broth culture of the test organism was used to aseptically seed 20mL of sterile molten Mueller Hinton agar in a sterile Petri dish. The contents were thoroughly mixed by rotating the Petri dish slowly, to ensure even distribution of the test organism, and then left to solidify. Cups of 4mm diameter were made in the solidified agar using a sterile cork borer. 0.04mL of each concentration of each plant extract screened was aseptically introduced into the cup and labeled accordingly. The dishes were left to stand for one hour at room temperature for proper diffusion of the extracts before incubating at 370C for 24 hours. Control experiments were also set up using sterile soybean casein digest broth. After incubation the inhibition zone diameters (IZD) were measured using a micrometer caliper. All tests were done in triplicate and the results presented as mean±SEM.

Minimum inhibitory concentration (MIC) test. The MIC of each extract was computed by plotting the logarithm of concentration (logConc) against the square of the inhibition zone diameters (IZD2). The antilogarithm of the intercept on the logConc axis gave the MIC value (Osadebe & Ukwue, 2004).

Phytochemical analysis. The powdered plant samples were used to carry out phytochemical tests using standard methods (Agomuo, 2002).

Statistical analysis. All statistical analyses were determined using Microsoft® Excel 2003.

RESULTS AND DISCUSSIONS

The sensitivities of test organisms to different plant extracts at various concentrations are displayed in Table 1. Inhibition zone diameters were directly proportional to the concentration of plant extract tested, however ANOVA revealed that among all the plants screened there was no significant difference in the IZD of the plant extracts per concentration per test isolate; and also no significant difference between the cold and hot ethanol extracts per concentration per test isolate. The IZD values obtained here for air-dried leaf samples appear to be lower than those obtained for fresh green leaves, but higher than those obtained for dried leaves with cold aqueous extraction as reported by Khan et al. (2012). This variation may be due to differences in the concentrations of bioactive substances in fresh and dried leaves, and the fact that the bioactive components may be more soluble in ethanol than in cold water (Doughari & Sunday, 2008). On the other hand values obtained here for P. soyauxii were higher than those obtained by Osuagwu and Akomas for ethanol extracts of dried leaves (Osuagwu & Akomas, 2013). This variation may be accounted for by the ages of the leaves tested. Younger leaves contain a relatively higher concentration of secondary metabolites than older leaves (Osuagwu et al, 2007).

The closeness in the values of the MIC in Table 2 corroborates the no significant difference in the IZD of the plant extracts on the test organisms as earlier pointed out by ANOVA. The relatively high MIC for cold ethanol extracts of Carica papaya and Pterocarpus soyauxii on Klebsiella pneumoniae and Escherichia coli respectively attests to the claim that gram negative bacteria have higher resistance to plant extracts, which is said to be related to the thick murein layer in their outer membrane which prevents the entry of inhibitor substances (Suresh et al, 2008). On the other hand disparity in MIC may also be due to variable sensitivity to chemical substances related to different resistance levels among strains (Cetin & Gurler, 1989).

From Table 3, all leaf samples analyzed appear to have similar phytochemical compositions. This may account for their analogous antimicrobial potencies as revealed in Tables 1 and 2. The presence of bioactive substances has been reported by different authors to confer antimicrobial activity to plant extracts. Draughon have specifically attributed antibacterial effects of plant extracts on the presence alkaloids, tannins and flavonoids.24 Despite the fact that the mechanism of action of these bioactive agents are not thoroughly understood, Hassan et al. (2007) suggested that the antimicrobial activity of these secondary metabolites may be due to the disruption of cell wall formation which consequently causes the leakage of cytoplasmic constituents.Tannins coagulate wall proteins; flavonoids inhibit enzyme activity, thus disrupting cell wall or cell membrane integrity at very low concentrations; while saponins disrupts cell wall porosity hence facilitating the entry of toxic components or the leakage of vital constituents (Dathak & Iwu, 1991; Kurta et al, 1994; Tsuchiya et al, 1996; Onwuliri & Wonang, 2005).

CONCLUSIONS

Each plant extract screened recorded antibacterial potencies on the entire test organisms surveyed in this work. Data obtained here also showed that the hot ethanol extracts showed more predictable and stable results than the cold extracts, thus suggesting hot ethanol extraction as a more efficient process especially when considered alongside the time saved in carrying out hot ethanol extraction. The results obtained here reveal the antibacterial potencies of these plants commonly found in the tropics. Hence, suggesting possible exploitation of these plants for their antimicrobial active principles for the development of novel herbal-based antimicrobials.

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