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| ORIGINAL ARTICLE |
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| Year : 2020 | Volume
: 5
| Issue : 3 | Page : 66-71 |
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Antimicrobial potential of extract fractions of ficus vogelii Miq.
Akataobi Uche Stephen
Department of Biochemistry, Faculty of Basic Medical Sciences, University of Calabar, Calabar, Cross River State, Nigeria
| Date of Submission | 03-Mar-2020 |
| Date of Decision | 07-Jul-2020 |
| Date of Acceptance | 04-Aug-2020 |
| Date of Web Publication | 30-Sep-2020 |
Correspondence Address:
Dr. Akataobi Uche Stephen
Department of Biochemistry, Faculty of Basic Medical Sciences, University of Calabar, P.M.B. 1115, Calabar, Cross River State
Nigeria
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/ed.ed_7_20
Purpose: Microorganisms are responsible for a host of infectious disease diagnosed in human. Studies have shown that extracts of medicinal plants contain antimicrobial properties effective in the treatment of these infectious diseases. This study evaluated the antimicrobial potency of ficus vogelii Miq. extract fractions on selected microorganisms.
Material and Method: The leave extract of the plant was prepared with n-hexane, crude, and methanol, serially diluted, and screened against Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, and Candida albicans for zones of inhibition, minimum inhibitory concentration, resistance, and sensitivity test at a concentration of 12.5, 25, 50, and 100 mg/ml.
Result: Crude and n-Hexane extract fractions inhibited E.coli and S. typhimurium effectively at different concentrations. Methanol fraction less inhibited E. coli only while all the extract fractions did not show any antimicrobial potency against S. aureus and Candida abicans.
Conclusion: This study concludes that crude and n-hexane extract of ficus vogelii is a potent antimicrobial agent against E. coli and S. typhimurium at a concentration of as low as 12.5 μg/ml.
Keywords: Candida albicans, Escherichia coli, Ficus vogelii Miq., Salmonella typhimurium, Streptococcus aureus
How to cite this article:
Stephen AU. Antimicrobial potential of extract fractions of ficus vogelii Miq. Environ Dis 2020;5:66-71 |
| Introduction | |  |
The presence of microorganisms or simply microbes in the human environment has resulted in a myriad of unpleasant infectious diseases.[1] These infections occur as a result of exposure followed by invasion of microorganisms such as bacteria and fungi into the human body and their ability to fix themselves to the host cells and replicate.[2] For them to do this, they are able to enter and overcome the body's (host) natural defense mechanisms.[3]
The infectious diseases caused can be deadly and in most cases fatal and can be transmitted in many ways such as sexual contacts, body contact (through body fluids), droplets (human droplets) such as coughing and sneezing, and low or poor personal and environmental hygiene.[2],[4] Bacteria and fungi such as Escherichia More Detailscoli,Staphylococcusaureus, Salmonella More Detailstyphimurium, and Candidaalbicans are pathogenic microbes (agents) that are responsible for all these infections including common fever, severe flu, skin infections (cellulitisand erysipelas), hepatitis, diarrhea, and blood infections.[5] These pathogenic microorganisms are found in various parts of the body where they colonize, grow, and exert their harmful effects and are grouped into two major parts, namely, the intracellular and the extracellular host.[6]
Pathogens of intracellular occupy their host cells for them to be able to replicate and cause disease condition on the process, among which are pathogens with the ability to replicate freely in the cell like Chlamydia, Rickettsia, and Listeria. The other group include those pathogens that can reproduce in the cellular vesicle[4] among which include Streptococcuspneumoniae.[7] Over the years, studies have been carried out, aimed at understanding the different antimicrobial and phytochemical properties of medicinal plants and a way in which they can be used in the prevention and treatment of disease conditions caused by these microbes, as an alternative to synthetic drugs.[8] This is because despite advances made in recent times in the study of microbiology there are increased incidence of epidemic been reported due to drug resistance as well as emergence of new pathogenic microorganisms that have raised the level of concerns about public health.[9]
It has also been reported that within the last few decades, the effectiveness of available antimicrobial drugs has reduced as a result of resistance of these microbes to synthetic drugs.[10] Which are expensive and not reliable to fight the harmful effect of microorganisms.[11] On the other hand, phytochemical properties isolated from medicinal plants are shown to have a positive effect and act as an alternative to the synthetic antibiotics in the control and treatment of microbial infections; which include Psidiumguajava and Mangiferaindica which contain phytochemicals like tannins, saponins, and alkaloids that have been reported to be biologically active for inhibiting microbial activities. Such as E.coli,Streptococcusfaecalis,S.aureus, and Pseudomonasaeruginosa activities.[12] In the search for alternative to existing synthetic antibiotics, hundreds of plant species have been evaluated though vast majority are yet to be considered as potential sources of antimicrobial and antifungal agents,[5] for the treatment of infectious diseases caused by most group of pathogens pathogens which colonizes certain parts of the body and exhibit their harmful effects. In most circumstances they cause superficial and systemic infections like Boils, Impetigo, Folliculitis and more seriously pneumonia, e.g., S.pneumonia,[13] and Streptococcuspyogenes (strep group A), responsiblefor causing bacterial pharyngitis (or simply strep throat) in humans which if left untreated can result to rheumatic fever, and glomerulonephrites.[10] The growth of these microorganisms in recent studies has been shown to be inhibited by extracts of medicinal plants such as ficusvogelii[9] that may act as an effective source of bioactive agents for the treatment of infections caused by these microbes, suggesting their use in the treatment of these infections.[8] Effective growth inhibition of S.aureus,Bacillussubtilis,P.aeruginosa,E.coli, and ScopulariopsisCandida using ethanolic and aqueous extracts of Sidaacuta rich in phytochemical components have been reported. Ohunayo et al.[14] reported that n-hexane extract fraction of ficusvogelii showed potency against E. coli at higher concentration.
FicusVogeliiMiq. among other medicinal plants has been evaluated for their antiviral potentials on infections that affect specific organs such as the liver.[15] And the result showed that ethanol extracts of FicusVogelii are bioactive in the reduction of hepatocellular degeneration.[16] Other microbial agents also considered include Candidatropicans known as the second most common of the Candida species that are responsible for about 5%–20% of blood infections from yeast and are the major agent responsible for causing Septicemia and disseminated Candidiasis.[17] Mostly in people with low immune system (people with Lymphoma, Leukemia and diabetes) and Candidaparapsilosis another species that are involve with up to 15%–30% of Candida infections responsible for attacking and causing fungal infection of the nail beds have also been showed to be effectively inhabited by the use of medicinal plants such as leave extracts of P. guajava and Allium sativum[8],[12] That may act as an alternative to available synthetic anti-biotic drugs.[18]
This has led to the study and use of medicinal plants with antimicrobial property for the prevention and treatment of these disease conditions such as Ficus of the family of Moracae. This genus species amounting to about 800 plants collectively referred to as Fig's. Most Ficus plants produce edible fruits, some are used as food sources by wide-life while others serve medicinal purposes.[13] Ficusvogelii belong to this family and are known for its ethnomedicinal application in the treatment of diseases such as diabetes,[11] anorexia, and anemia.[9] Studies that evaluated the therapeutic purpose of ficusvogelii indicated its use as an antiulcer agent,[19] anti-anemia, and antidiabetic.[11] This study is aimed at evaluating the antimicrobial potency of n-hexane, methanol, and crude extract fractions of FicusvogeliiMiq. on selected human pathogens.
| Materials and Methods | | |
Collection of plant
Fresh leaves of FicusvogeliiMiq. were harvested from a farm in Odukpani Local Government Area of Cross River State and authenticated by a botanist in the Department of Botany, University of Calabar, Nigeria. The was transported to the department of biochemistry where it was thoroughly washed with tap water and allowed to air-dry at room temperature for a period of 3 weeks in the department laboratory.
Preparation of plant extracts
The dried leaves were powdered using an electric blender and 150 g weighed using an analytical weighing balance which was then divided into 3 of 50 g each. 50 g of the prepared powder was suspended in crude solvent in the ratio of 1:3 sample-solvent, and the suspension thoroughly agitated using electric blender. Thereafter, the mixture was allowed to stand for 24 hours at room temperature, after which it was filtered first using a cheese material followed with a Whiteman No. 2 filter paper.
The filtrate was then concentrated in a rotary evaporator (45°C) to about 1/10th of its original volume and evaporated to dryness using a water bath (45°C). This procedure was then repeated using methanol and n-hexane as solvents and then the extracts (brown and green) were stored in a clean corked bottle for microbial analysis.
Dilution (concentrations) of the extracts for antibacterial and antifungal screening
Four test tubes for the aqueous leaf extract (crude) were placed on a test tube racks and 2 ml of the extract was placed in the 1st test tube. Whereas, 1 ml of distilled water was added to the remaining three test tubes. From the 1st test tube, 1 ml was transferred into the 2nd test tube and from the 2nd 1 ml was transferred into the 3rd, and from the 3rd 1 ml was transferred into the 4th test tube; this serial dilution technique was repeated for each of the other two extracts, respectively, (to get the concentrations of 100 mg/ml, 50 mg/ml, 25 mg/ml, and 12.5 mg/ml); each transfer was followed by thorough shaking of the test tube for complete mixing of the content.
Viability test for clinical isolates
Four clinical isolates (E.coli,S.aureus,Salmonellatyphimurium, and C.albicans) collected from the University of Calabar Teaching Hospital were first subjected to viability test to confirm whether they are still viable or not. To do this, 1.5 g of nutrient agar and 2.0 g of potato dextrose were each dissolved in 50 ml distilled water in 250 ml conical flask. The contents of the flask were thoroughly shaken to mix well. Next, the media were sterilized at 121°C in an oven for 15 min. At the end of sterilization, the media in the conical flask were allowed to cool for 45 min and later 20 ml of the medium was placed in properly labeled Petri dish More Detailses.
The media were further allowed to cool for 30 min in order to solidify. After solidification, stock culture of the clinical isolate was streaked on freshly prepared plates and then incubated at 37°C for 20 h. At the end of incubation, the clinical bacteria isolates were characterized using gram reaction, while lactophenol blue test was used to confirm Candidaalbicans viability. The clinical isolate so characterized were stored in stoppage bijou bottle placed in the refrigerator in preparation for the susceptibility (sensitivity) testing.[20]
Susceptibility test; the method used is the method of Kirby-Bauer disc diffusion technique. Broth dilution technique was used to determine the minimum inhibitory concentration (MIC) as described by Lynn S and Avery C.[20]
Description of the disc diffusion technique
Glass plates (Petri dishes) sterilized in a hot oven for each of the isolate to be tested were properly labeled accordingly. 1 ml of the clinical isolate was placed in each of the well-labeled Petri dishes according to the labels, after which 20 ml of already sterilized Mueller–Hinton agar held at a temperature of 45°C was added to each of the glass plates, and the contents were allowed to mix very well. The plates were further kept for 45 min for the agar to solidify. Then, different concentrations of n-hexane extract (0.1 ml) were placed with a dropping pipette at different positions on a dried filter paper disc (filter paper disk impregnated with different concentrations of the extract) and placed on the inoculated agar surface on the Petri dish using a sterile forceps. This procedure was repeated for crude and methanol extracts fractions, respectively. At the end of this exercise, the plates were incubated at 37°C for 24 h, and at the end of the incubation, zones of inhibition that developed around the disc were measured and recorded in millimeters.[20]
Agar diffusion (well method) technique
In this technique, the procedure for the disc diffusion method was followed except that after overlaying 1 ml of the clinical isolate with 20 ml of sterilized Muller–Hinton agar was used, followed by solidification of the agar. A cork borer was used to prepare four wells at specific locations on the agar surface. Each “well” representing a specific dilution or concentration of the extract. After the preparation of the wells, 0.1 ml of crude extract of a particular concentration was transferred (with a dropping pipette) into the corresponding well (location) on the Petri dish. This procedure was repeated for the n-hexane and methanol extracts, respectively. The plates were incubated at 37°C for 24 h. After incubation zones that developed around the wells was carefully measured with a transparent ruler and recorded in millimeter.[20]
Agar dilution method
Sterilized plates were labeled accordingly with each plate having the name of the clinical isolate and the concentration of the extract on it. 1 ml of the isolate (E.coli,S.aureus,andC.albicans) was placed in each Petri dish. Then, 20 ml of Muller–Hinton agar held at 45°C was added onto the isolate in the Petri dish, followed by swinging to allow content of the Petri dish to mix well. The agar was kept to cool to a semisolid state and over layed with 1 ml of the extract followed again by swinging of the Petri dish. Finally, the plates were incubated at 37°C for 24 hand MIC determined.[19]
| Results and Discussion | | |
Result obtained using the Agar disc diffusion method revealed that the microbes were inhibited at different concentrations of the extracts, and the highest zone of inhibition was recorded using n-Hexane extract followed by crude extract while methanol extract showed the least zone of inhibition.
Effect of n-hexane extracts on microbial growth
From the result [Table 1] n-Hexane extract fraction showed a more inhibitive potency against E.coli, with the highest zone of inhibition obtained at 50 mg/ml (43 mm), 31 mm and 24 mm recorded in 100 mg/ml and 25 mg/ml respectively while the lowest zone of inhibition was recorded in 12.5 mg/ml (23 mm), indicating a concentration dependent inhibitive potency of n-Hexane extract fraction of the plant. This is followed by Salmonellatyphimurium with the highest zone of inhibition of 20 mm (in 12.5 mg/ml) and 14 mm (in 100 mg/ml). Furthermore, the result showed that the extract fraction indicated no potency on S.aureus and Candidaalbicans, respectively, using Ampicillin and Amphotericin as reference drugs.
Effects of crude extracts on microbial growth
Crude extract result [Table 2] indicated a more inhibitory potency of FicusvogeliiMiq. on the tasted microorganisms inhibiting the growth both of E. coli and Salmonellatyphimurium strongly. The highest zone of inhibition against E. coli was recorded in 25 mg/ml (35 mm) 33 mm and 31 mm at 12.5 and 100 mg/ml, respectively, while 25 mm at 50 mg/ml was recorded as the least. This is followed by S.typhimurium with 29 mm at 100 mg/ml and 28 mm at 12.5 mg/ml, while S.aureus and Candida albicans were not inhibited, both with 6mm in all concentrations of the extract, when compared to ≥14 obtained in standard drug.
Inhibition of methanol extracts on microbial
Methanol extract fraction [Table 3] gave the least inhibitory potency among the extract fractions. E. coli was only inhibited (less when compared to other fraction of the plant) with the highest zone at 50 mg/ml (17 mm) and the lowest 10 mm at 12.5 mg/ml. The result further showed that the extract has no antimicrobial potency against the other tested microorganisms.
Sensitivity test results
n-Hexane
The results obtained from the sensitivity tests [Table 4] indicated that in n-hexane extract fractions E. coli was viable in 100 mg/ml and sensitive in other concentrations tested,Candidaalbican was sensitive in all the concentrations and Staphylococcusareurs were sensitive in all concentration except in 12.5 mg/ml of the extract while S.typhimurium indicated viable and resistance in 50 mg/ml and 25 mg/ml respectively.
Methanol extract
Sensitivity result of methanol extract [Table 5] in all concentrations showed that at E. coli was sensitive in all the concentrations, S.aureus with exception of 12.5 mg/ml were also sensitive in all the tested concentrations and Candidaalbican was also sensitive in all the concentrations tested while S.typhimurium indicate resistance to the extract in all concentrations except in 12.5 mg/ml.
Plants are important sources of useful raw material for the development of new antimicrobial agents.[21] Reports have revealed that a good number of these plants contain anti-fungi and anti-bacterial properties.[22] These report have helped in the identification of the active properties responsible for the inhibitory ability of these plants and in the development of new and more novel therapeutic and antibiotic drugs for human use.[4] It has been clearly understood that plants contain phytochemicals or bioactive compounds that are effective inhibitors of microbial growth;[9] these phytochemicals with recorded antimicrobial potency possess lipohic properties and are able to exhibit proton efflux as well as charged proton that promote their antimicrobial inhibitive activities.[6] Furthermore, studies have shown that microorganisms possess mechanisms that help them defend against plant phytochemicals dose dependently.[9] This means that growth of microorganisms can be inhibited only by certain bioactive compound and in certain quantity or they could overcome the sensitivity of the compound and exhibit resistance to their effects.[20] This is evidence to the result obtained in this current study which indicated that in all the extracts tested methanol extract showed no inhibitive potency against Salmonella typhiruium,S.aureus and C.albicans in line with the report that indicated the inability of the methanol extract to exhibit inhibitory potency against tested microorganisms.[9] But on like the report our current study showed that the extract were not completely ineffective against E. coli [Table 3] but were less inhibitive (25 mg/ml). This result suggests that methanol extract does not contain active inhibitive compounds against the tested microorganisms in 12.5, 25, 50, and 100 mg/ml[19] when compared with Streptomycin used as control in this study.
This study also showed that ficusvogeliiMiq. extracts are more potent against E. coli followed by Salmonellatyphimurium. The former showed zones of inhibition in all concentrations (12.5, 25, 50, and 100 mg/ml), especially in the crud extract fraction [Table 2]. Which indicated a higher inhibitive potency in 25 mg/ml (35 mm) against ≥14 obtained in Streptomycin used as standard. While Salmonellatyphimurium showed the highest zone of inhibition in 100 mg/ml (29 mm) suggesting a high potency of the plant extract.[22] The extract did not show any inhibitive potency against S. typhimurium andC.albicans which may be due to lack of bioactive properties in the plant against the microorganisms as suggested by Ohunayo et al.[9] n-Hexane extract like the crude extract inhibited E. coli effectively with the highest inhibitory potency in 50 mg/ml (43 mm) and less potent against Salmonellatyphimurium which were only inhibited at 12.5 mg/ml (20 mm) when compared with the against ≥14 obtained in standard drug. Furthermore, Salmonella typhimuriumandC.albicans growths in all the extract fractions were not affected which suggests either that ficusvogelii do not contain phytochemicals with inhibitive ability against both microorganisms or the solvents used in extraction were not able to extract the required properties with anti-microbial potency against both microorganisms.[6]
This also means that growth inhibition of microbes can be effectively achieved at different concentrations so mentioned of the extracts respectively and are the specific concentration at which infectious diseases caused by these pathogenic organisms could be arrested during treatment. Finally, it was observed that the highest concentration of the n-hexane and methanol extract of the Ficusvogelii extracts have more significant effect on the fungi isolates. They had higher zone of inhibition compared to the antifungal agents used as control a similar result has also been reported by Ekpo and Etim[9] in their anti-microbial study using ethanol and aqueous extracts of Sidaacuta.[8] Sensitivity and resistance patterns of microorganisms are constantly changing because of the ability of the microorganisms to develop resistance to the antibiotics.[19] Thus, testing these pathogenic microorganisms for an appropriate antimicrobial agent are important in determining an appropriate treatment of infectious diseases they cause.[9]
This study indicated that n-hexane and methanol extract fractions of ficusvogeliiMiq. contain phytochemical properties[19] which E. coli were sensitive to in all the concentration but S.typhimurium on the hand were not sensitive to the constituent of the plant in n-hexane extract except in 12.5 mg/ml.[9] Despite indicating the antimicrobial potency of the plant extract the result further showed the lowest concentration at which the pathogenic organisms studied maybe arrested using the respective extract fractions in MIC test. The MIC result [Figure 1] indicated minimum inhibitory potency of ficusvogelii.[11] 12.5 μg/ml in n-Hexane, 25 μg/ml and 50 μg/ml in crude and methanol extract fractions for E.coli, in S.typhimurium 12.5 μg/ml, 50 μg/ml and 25 μg/ml were recorded as the lowest inhibitory concentration in n-Hexane, crude and methanol extract fractions. S. aureus showed 25 μg/ml, 12.5 μg/ml and 100 μg/ml n-Hexane, crude and methanol extract fractions while C.albicans 12.5 μg/ml was recorded in n-Hexane and 50 μg/ml for both crude and methanol extract fractions. | Figure 1: MIC result. EC: Escherichia coli, ST: Salmonella typhimurium, SA: Staphylococcus aureus, CA: Candida albicans
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| Conclusion | | |
From the results obtained in this current study, it can be concluded that extract fractions of ficusvogelii possesses potent antimicrobial properties, effective in inhibiting microbial growth and activities indicated in its effect against E. coli andS.typhimurium but were less effective inhibitor of Staphyloccusaureus and C.albicans. In MIC the study concluded that growth of the microorganisms can be inhibited using the plant extract in concentration as low as 12.5 μg/ml. Based on this result, it is recommended that this result may be used as a baseline data in bacterial resistance that could be useful to policy makers in the treatment of microbial infections.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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| 13. | Egbuna PA, Joshua C, Parker E, Chigbo MU. Antihepatotoxic effects of Ficus Vogelii ethanol leaf extract on the liver function indices of ccl4 –induced hepatotoxicity in rats. Department of Biochemistry, University of Nigeria, Nsukka, Enugu, Nigeria. J Am Sci 2011;7,13:07-11. |
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| 16. | Sonibare O, Effiong I. Antibacterial activity and cytotoxicity of essential oil of Lantana Camara L. leaves from Nigeria. Afr J Biotechnol 2008;7:2618-20. |
| 17. | Sam GD, Khan K. Biochemical compositions and antibacterial activities of Lantana camara plants with yellow, lavender, red and white flowers. Eur Asian J BioSci 2009;3:69-77. |
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| 19. | Igile GO, Utin IC, Iwara IA, Mgbeje BI, Ebong PE. Ethanolic extract of ficus vogelii ameliorats dyslipdemia in diabetes albino wistar rats. Int J Curr Res Biosci Plant Biol 2015;2:87-96. |
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[Figure 1]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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