Antimicrobial Activity of Lippia adoensis var. koseret Against Human Pathogenic Bacteria and Fungi
Gemechu Ameya Buli1, *, Abdella Gure Duga2, Engda Dessalegn3
1Department of Medical Laboratory Science, College of Medicine and Health Sciences, Arba Minch University, Arba Minch, Ethiopia
2Wondo Genet Colleges of Forestry and Natural Resources, Hawassa University, Hawassa, Ethiopia
3Department of Chemistry, Hawassa College of Education, Hawassa, Ethiopia
Gemechu Ameya Buli, Abdella Gure Duga, Engda Dessalegn. Antimicrobial Activity of Lippia adoensis var. koseret Against Human Pathogenic Bacteria and Fungi. American Journal of Clinical and Experimental Medicine. Vol. 3, No. 3, 2015, pp. 118-123. doi: 10.11648/j.ajcem.20150303.18
Abstract: Background: Lippia adoensis var. koseret is a well known medicinal plant endemic to Ethiopia. It has been traditionally used to treat different infectious diseases and also in food preparation as condiment. The aim of the current study was to evaluate antibacterial and antifungal activities of water, ethanol and methanol based crude extracts of L. adoensis var. koseret against selected human pathogenic bacteria and fungi. Methods: Crude extracts of L. adoensis var. koseret were extracted by maceration method. Disc diffusion assay of the extracts were carried out in four different concentrations against three different bacteria species (Staphylococcus aureus, Enterococcus faecalis and Escherichia coli) and two clinical isolated fungal species (Candida albicans and Aspergillus flavus) by using Kirby- Baur disk diffusion method. Agar dilution method was used to determine the minimum inhibitory concentration, the minimum bactericidal and fungicidal concentrations of the extracts against similar microorganisms. Results: Water-based extract of L. adoensis var. koseret exhibited significantly less antimicrobial activity as compared to ethanol and methanol crude extract against tested isolates of bacteria and fungi (P < 0.05); while, there was no significant difference between ethanol and methanol extracts. Among the tested microorganism S. aureus, was the most sensitive of all whereas C. albicans was the most resistant microorganism to alcohol based extract of L. adoensis var. koseret. The minimum inhibitory concentration of L. adoensis var. koseret ranged from 3.12 to 25mg/ml in the alcohol based extracts but it was higher in the water-based extract. The lower bactericidal concentration (5.20 mg/l) and the highest fungicidal concentration (37.50 mg/ml) were observed in methanol based extracts against S. aureus and C. albicans, respectively. Conclusions: Antimicrobial activity of L. adoensis var. koseret varies with extraction solvent and tested microorganisms.
Keywords: Antibacterial Activity, Antifungal Activity, Crude Extract, L. adoensis var. koseret
Medicinal plants are the oldest form of healthcare known to mankind . They had been used by all cultures throughout history and were essential part of the advance of modern civilization . Regardless of the enormous advances observed in modern medicine, plants still play important role to in health care and considerable percent of modern pharmaceutical drugs contain plant ingredients. However, only a small proportion of medicinal plants are examined for bioactive compounds .
Lippia adoensis is one of the five indigenous Lippia species in Ethiopia where it occurs as an erect woody shrub up to 1-3m tall. It is endemic medicinal plant and cultivated variety commonly found in home gardens in different regions of Ethiopia with altitudinal range of 1600-2200m . Two varieties are recognized in Ethiopia, the wild variety (var. adoensis) and the cultivated variety (var. koseret sebsebe). L. adoensis var. koseret sebsebe, locally known as koseret, is widely grown in the central and southern highlands of the country. Traditionally, the dried leaves are used as one of the ingredients in the preparation of spiced butter  and also for food flavoring agent and preservative .
The leaves of L. adoensis are used in Ethiopian traditional medicine for the treatment of various skin diseases including eczema and superficial fungal infections . The dried leaves powdered together with barely eaten to get relief from stomach complaints . A previous study showed that essential oils of this medicinal plant possess a significant radical scavenging property. Its free radical oxidative stress is implicated in the inhibition of pathogenesis of a variety of inflammatory diseases . The chemical compositions of L. adoensis var koseret, investigated so far are essential oils. Lonalool is the major component, and appreciable amount of sesquiterpene hydrocarbons (germacrene, α-copaene, β-cadinrne, and, β- caryophyllene) and uncommon monoterpene ketone, 2-methyl-6-methylene-2, 7-octadien-4-one (ipsdienone), were also found in the essential oil .
Due to the increasing incidence of antibiotic resistance in bacteria and fungi, there always is a need for new and effective therapeutic drugs . This necessitates continued efforts to look for alternative antimicrobial drugs. The purpose of the current study was to evaluate antibacterial and antifungal activities of water, ethanol and methanol based crude extracts of L. adoensis var. koseret against selected human pathogenic bacteria and fungi.
2.1. Collection and Extraction of Plant Materials
Fresh leaves of L. adoensis var. koseret were collected from Botanical garden of Hawassa College of Education, Hawassa, South Ethiopia in October, 2013 and identified by taxonomist at the Biology Department, College of Natural Sciences of Addis Ababa University, Ethiopia. The voucher specimen was deposited at the Herbarium, Biology department Addis Ababa University. The leaves were transported by polyethylene bag followed by sprinkling water on the leaves and piercing the bag at several points in order to allow air circulation. In the laboratory, the leaves were washed three times under running tap water followed by rinsing once with sterile distilled water and then air-dried.
The leaves were then ground into fine powder using electric grinder followed by the preparation of suspensions in ethanol, methanol and water. Twenty five grams of the fine powder was suspended in 250ml of each of three solvents separately in sterilized screw capped 500ml glass beakers. The suspensions were kept in orbital shaker for 12hrs at room temperature. The suspensions were filtered using Whatman No. 1 filter paper and were evaporated to remove the solvent under vacuum in Rotary evaporator kept at 40 ºC. The powdered crude extracts obtained from the three solvents were weighed and separately dissolved in distilled water to prepare the respective stock solutions of 200mg/ml and stored in deep freezer at a temperature of -20 ºC until further use [12,13].
2.2. Bacterial and Fungal Isolates
The screening for antibacterial and antifungal activities of L. adoensis var. koseret leaf crude extracts was carried out by using three bacterial pure cultures: Staphylococcus aureus (ATCC-25923), Enterococcus faecalis (ATCC-29212) and Escherichia coli (ATCC-25922) and two clinical fungal isolates of Candida albicans and Aspergillus flavus, obtained from Ethiopia Public Health Laboratory. Mueller Hinton agar (MHA) medium and Sabouraud’s dextrose agar (SDA) were used to carry out the screening for antibacterial and antifungal activity, respectively.
2.3. Disc Diffusion Assay
Suspensions of the leaf crude extracts of L. adaoensis var. koseret obtained using the aforementioned extraction solvents were prepared in distilled water at concentrations of 10, 20, 40 and 80 mg/ml for disc diffusion assay. Diffusion discs of approximately 6mm diameter were prepared from Whatman No. 1 filter paper by using puncher and sterilized by autoclaving and dried in oven. Then 10µl of the extracts from each of concentrations was impregnated on sterile disc using sterile micropipette tips followed by air drying and then stored at 4oC in separate sterile containers. Then disc diffusion assay was carried out using Kirby- Baur disk diffusion method according to . Gentamycin (10 μg mlG) and ketoconazole disc (10 μg mlG) were used as positive control for bacteria and fungi, respectively while blank discs impregnated with each solvents were used as negative control. All the tests were conducted in triplicate and the average of the three measurements was used to present the results.
2.4. Determination of Minimum Inhibitory Concentration
Agar dilutions method was used to determine the minimum inhibitory concentration (MIC) of L. adoensis var. koseret extracts. From 200mg/ml of stock plant extract, two fold serial dilutions was used to prepare suspensions of 100mg/ml, 50mg/ml, 25mg/ml, 12.5mg/ml, 6.25mg/ml, 3.125mg/ml and 1.56mg/ml concentrations in molten MHA and SDA after cooling to 45oC. The resulting suspensions were poured into sterile Petri dishes under aseptic condition and let to solidify. After the plates were solidified, they were inoculated with diluted suspension of each microorganism at 0.5 McFarland standards. The plates were then incubated at 37 oC for 24 hrs, and 27 oC for 48hrs in the case of bacteria and fungi, respectively. The minimum dilution of the leaf crude extracts completely inhibiting the growth of each organism was taken as the MIC .
2.5. Determination of the Minimum Bactericidal and Fungicidal Concentration
In order to determine minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC); the MIC, the concentration preceding to MIC and one more concentration between this two concentrations were used. Then, those three concentrations were adjusted in nutrient broth and Sabouraud’s dextrose broth in 250ml conical flask and inoculated with the respective test microorganism in test tube and incubated at 37 ºC for 24 hrs and 48hrs at 27 ºC for bacteria and fungi, respectively. Then, sterile plates of MHA and SDA were inoculated by transferring a loopful of broth medium inoculated with the respective test microorganism followed by incubation at 37ºC for 48hrs and at 27ºC for 72hrs for bacteria and fungi, respectively. MHA and SDA plates without the leaf extract and inoculated with the respective test microorganism served as control. All the tests were done in triplicates .
2.6. Statistical Analysis
For each assay, all the measurements were replicated three times and the results were presented as mean SD. One way ANOVA followed by Tukey’s test was used to compare extraction solvents and the difference in the sensitivity of the test microorganisms using the statistical package for social sciences (SPSS) version 16 and P-value ≤ 0.05 was considered as statistically significant.
In the disc diffusion assay of L. adoensis var. koseret, water-based extracts had antimicrobial activity only against S. aureus and E. faecalis at the concentration of 80 mg/ml. E. coli and both the tested fungal species (C. albicans and A. flavus) were resistant to water-based extracts at 80mg/ml. In general, water-based extract of L. adoensis var. koseret showed significantly (p < 0.5) lower antimicrobial activity than the ethanol and methanol based extracts (Table 1).
Disc diffusion assay of ethanol based extracts exhibited antimicrobial activity against all the tested organisms at 80 mg/ml. S. aureus exhibited antimicrobial activity at 20 mg/ml while C. albicans was only inhibited at the concentration of 80mg/ml while E. coli, E. faecalis and A. flavus were inhibited by ethanol based extract at 40mg/ml and above. In the methanol based extract, high antimicrobial activities were observed. The highest average (20.33mm) zone of inhibition was observed in methanol based extract at the concentration of 80 mg/ml against S. aureus (Figure 1). On the other hand, more or less similar results were obtained against the other tested microorganisms. Positive and negative controls were also carried out to monitor antimicrobial activity of the medicinal plant. All negative controls showed no antimicrobial activities whereas positive gives different zone of inhibition for different microorganisms (Table 1).
|Extraction solvents||Extract concentration||Inhibition zone (mm)|
|Bacterial species||Fungal Species|
|S. aureus||E. coli||E. faecalis||A. flavus||C. albicans|
|80 mg/ml||9.00 ± 1.00||-||8.33 ± 0.57||-||-|
|20 mg/ml||8.33 ± 0.57||-||-||-||-|
|40 mg/ml||9.66 ± 0.57||8.33 ± 0.57||8.33 ± 0.57||8.33 ± 0.57||-|
|80 mg/ml||13.00 ± 1.00||9.00 ± 1.00||10.33 ± 0.57||12.00 ± 1.00||8.66 ± 1.15|
|Methanol||10 mg/ml||11.33 ± 1.15||-||-||-||-|
|20 mg/ml||13.00 ± 1.00||-||-||-||-|
|40 mg/ml||17.66 ± 0.57||8.33 ± 0.57||10.00 ± 1.00||9.66 ± 1.52||-|
|80 mg/ml||20.33 ± 0.57||11.33 ± 1.15||13.33 ± 0.57||12.33 ± 0.57||8.33 ± 0.57|
|Positive control||22.33 ± 0.57||20 ±1.00||17.00 ± 1.00||22.33±0.57||23.66±0.57|
(-) = No antimicrobial activity; Values are mean of inhibition zone (mm) ± S.D of three replicates
In the determination of MIC against the tested microorganisms, E. coli and C. albicans were not inhibited by water extract at 100mg/ml. In the contrary, the other tested microorganisms were inhibited at 100 mg/ml in water extract. Like in the case of disc diffusion assay, the tested microorganisms were more resistant to water-based extracts than to the ethanol and methanol based extracts.
The MIC of ethanol based extract was 6.25mg/ml against S. aureus while it was 25mg/ml in the case of E. coli and C. albicans. The MIC for E. faecalis and A. flavus was 12.50mg/ml. In the methanol based extract, the MIC was 3.12 mg/ml in the case of S. aureus while C. albicans was the most resistant microorganism. In the case of the other tested microorganisms, there was no significant difference between alcoholic extracts (Table 2).
|Assay methods||Extract solvent||MIC, MBC and MFC (mg/ ml)|
|Bacterial species||Fungal species|
|S. aureus||E. coli||E. faecalis||A. flavus||C. albicans|
(*) = No inhibitory or bactericidal/fungicidal activity at 100mg/ml, MIC=Minimum inhibition concentration, MBC=Minimum bactericidal concentration, MFC= Minimum fungicidal concentration
Like in the case of disc diffusion and MIC assay, the extracts exhibited bactericidal and fungicidal activities to varying extents. Water-based extracts had exhibited bactericidal effects only against S. aureus at the concentration of 100mg/ml (Table 2). The lowest MBC (5.20 mg/ml) was recorded in the case of methanol based extracts against S. aureus while higher values were recorded in all the tests against all other microorganisms included in the screening (Table 2).
In current study, of all the extracts obtained using the three extraction solvents at four different concentrations, methanol based extracts at the concentration of 80 mg/ml exhibited the highest antimicrobial effects against all the tested microorganisms although the extent of inhibition across the microorganisms was variable. Among the tested bacterial species, S. aureus was the most sensitive while E. coli was the most resistant to the extracts obtained using all the three solvents. Likewise, from the two fungal species, A. falvus was more sensitive than C. albicans in all cases. Similar results were observed by 80% methanol extract of this medicinal plant against similar bacteria species but with a little difference [7,15]. Among the extraction solvents, methanol was the best followed by ethanol.
One of the differences between the current study and those studies may be the concentration of methanol. In the previous two studies, 80% methanol was used while in the current study absolute methanol was used. So, the results may have been influenced due to the differences in the polarity and concentrations of the alcohol. Penetration of alcohols increases with dilution with water which in turn improves the phytochemical extraction power of the alcohol . Besides, the amount of active ingredient in a plant can vary with factors like the variety of plant, the geographic location, the season and time of harvest, soil conditions, storage conditions, and the method of preparation.
Extracts in both ethanol and methanol had significantly higher antimicrobial activities against tested microbes than water extract. This is due to alcohols are more efficient in cell walls degradation which have non-polar character and cause polyphenols to be released from cells. Enzyme polyphenol oxidase is also inactivated in methanol and ethanol extract . In addition, alcohols were found easier to penetrate the cellular membrane to extract the intracellular ingredients from the plant material  and nearly all of the identified components from plants active against microorganisms are aromatic or saturated organic compounds which are most often obtained through initial alcoholic extraction .
E. coli, Gram negative bacteria are more resistance to tested microorganism because they have an outer membrane that is made up of high density lipopolysaccharides that serves as a barrier to many environmental substances including antibiotics . They also have innate multidrug resistance to many antimicrobial compounds owing to the presence of efflux pumps in meticulous . But successively numerous phytochemicals were shown to act as potential efflux pump inhibitors with antimicrobials for Gram-positive bacteria [22,23]. Hence, these are the possible reason why all selected medicinal plant showed lower antimicrobial activity than the others.
Phytochemical screening tests carried out on L. adoensis also indicated the presence of tannins, flavonoids, polyphenols, alkaloids and saponins which are the main antimicrobial phytochemical . Mechanism of action of chemical compounds of medicinal plants mediate their effects on the microorganisms is almost the same to conventional drugs those already well understood for the chemical compounds. This indicates herbal medicines do not differ greatly from conventional drugs in terms of how they work and medicinal plants can be as effective as conventional .
In the current study, antimicrobial activity of L. adoensis var. koseret extract in methanol exhibited high antimicrobial activity against S. aureus. At concentration of 80 mg/ml this extract showed comparable antimicrobial activity with positive control, Gentamycin (10 μg mlG) against S. aureus while no growth inhibition was observed in the case of all the negative controls. On the other hand this medicinal plant showed a low antimicrobial activity against C. albicans in all assay methods and similar finding was observed with Yared et al., (2014). Generally, all three tested assay methods were support each others in current study.
Medicinal plants are commonly used in treatment of various diseases and scientific verification should be done to enhance trust of consumer and to show as they are effective for what they needed. Then this study is expected to fill knowledge gab and provide base line evidence based information about antimicrobial activity of this medicinal plant against common pathogenic and opportunistic microorganisms. In addition to this it also gives some information for traditional medicine practitioners to develop their profession that improve the quality of the health service they render. Traditional use of L. adoensis var. koseret by the local people in treating various types of infectious diseases specially caused by S. aureus was supported by this study. This study has some limitation like lack of phytochemical analysis, fractional extraction and addition pharmaceutical tests that should be carried out to confirm patient safety. Therefore, other additional studies are needed to acknowledge traditional medicine practitioners practice.
Antimicrobial activity of L. adoensis var. koseret differs based on the extraction solvent used, the concentration of the extract and the species of the tested microorganism. Ethanol and methanol extracts showed significant antimicrobial activity against S. aureus while E. coli and Candida albicans were the most resistant microorganisms in all assay methods.
ATCC: American type culture collection; MBC: Minimum bactericidal concentration; MFC: Minimum fungicidal concentration; MHA: Mueller Hinton agar; MIC: Minimum inhibition concentration; SD: Standard deviation; SDA: Sabouraud’s dextrose agar
GA: participated in the design of the study, coordinate and involved in data collection and experimental work, analyzed the data and drafted the paper. AG and ED participated in the analysis and revised subsequent drafts of the paper. All authors read and approved the final manuscript.
The authors would like to acknowledge Hawassa University for financial support and Hawassa University referral hospital laboratory, department of Biology and Chemistry, Wondo Genet College of forestry and natural resources of Hawassa University for their technical assistance while the experiment was carried out. We also acknowledge Professor Sebsebe Demisew of Biology Department of Addis Ababa University for identifying the plant material.