Bacterial infections are responsible for a large number of deaths every year worldwide. On average, 80% of the African population cannot afford conventional drugs. Moreover, many synthetic antibiotics are associated with side effects and progressive increase in antimicrobial resistance. Currently, there is growing interest in discovering new antibacterial agents from ethnomedicinal plants. About 60% of the population living in developing countries depends on herbal drugs for healthcare needs. This study involved the screening of
Infectious diseases are a major cause of morbidity and mortality worldwide [
In addition, many bacterial pathogens form biofilms when they come in contact with a hydrated surface [
About 80% of the population living in developing countries uses medicinal plants for their health care needs due to their inability to maintain a steady supply of conventional medicines [
The World Health Organization (WHO) emphasizes the need to compliment conventional treatment with herbal medicines [
Bacterial resistance to currently used antibiotics necessitates the search for effective therapeutic agents. The use of medicinal plants presents a great potential as a source of antimicrobial compounds against resistant pathogenic microorganisms [
Medicinal plants have been used for a long time [
A large number of people in developing countries depend on medicinal plants as their primary source of medication [
Picture of
Whole plant samples of
Two hundred grams (200 g) of the plant sample powder was weighed and soaked in a cold 600 ml mixture of dichloromethane: methanol (1 : 1) for 48 h in a flask to obtain the extract [
A completely randomized design was applied in this study.
Microorganisms used in the experiment were standard reference strains sourced from the Microbiology laboratory, Kenyatta University, namely,
The stock cultures of bacterial strains were cultured on Mueller Hinton agar and incubated at 37°C for 24 h to obtain fresh growing colonies [
The plant extract stock solution was reconstituted using 0.2% DMSO by weighing 1 g of the extract in a sterile sample bottle and dissolved in 1 ml 0.2% DMSO to make a concentration of 1 g/ml. The stock solution was then diluted serially in a two-fold dilution starting from 500 mg/ml. Paper discs of 6 mm in diameter were prepared from Whatman No. 1 filter paper using a paper punch and sterilized at 121°C for 15 min. Twenty microlitres from each of the different concentrations was used to impregnate the paper discs. The impregnated discs were air dried in a laminar flow hood for 3 h and immediately used for the sensitivity tests.
The assays for antimicrobial activity of the DCM: methanolic extract of
The broth microdilution method was used to determine minimum inhibitory concentrations (MICs) of the plant extract against the test bacterial cultures using 96-well microtiter plates [
Ten microlitre (10
The MIC was recorded as the lowest concentration of the extract that inhibited visible growth of the test bacteria as observed by an unaided eye. Each experiment was performed in triplicate.
Samples of 10
Time-kill kinetics was carried out to indicate the rate and extent of bacterial killing by the antibacterial agent [
Twenty-four hour cultures of the test microorganisms on MHA were suspended in MHB to obtain bacterial suspension of 1 × 106 CFU/ml. One microlitre (1 ml) of the bacterial suspension was added to 9 ml of MHB containing the extract.
The
Qualitative phytochemical screening was performed on the plant extract to determine the presence or absence of bioactive compounds [
Experimental data on zones of inhibition readings, MIC, MBC, and time-kill kinetics were recorded and tabulated on a broad spread sheet using MS excel program. Statistical analysis of the data was performed using Minitab statistical software version 17.0.
In order to test for parametric assumptions, results were expressed as mean ± standard error of the mean (SEM). One-way ANOVA and Tukey’s post-hoc test were used for separation and comparison of means to obtain the specific significance difference. The values of
In this study, the dichloromethane: methanolic extracts of
Zones of inhibition produced by the
Group | Zones of inhibition (mm) | |||||
---|---|---|---|---|---|---|
Treatment | ||||||
Negative control | DMSO | 6.00 ± 0.00d | 6.00 ± 0.00f | 6.00 ± 0.00e | 6.00 ± 0.00d | 6.00 ± 0.00d |
Positive control | Tetracycline | 29.33 ± 0.67a | 26.67 ± 0.33a | 27.67 ± 0.33a | 22.67 ± 0.33a | 25.33 ± 0.33a |
15.625 mg/ml | 6.00 ± 0.00d | 6.00 ± 0.00f | 6.00 ± 0.00e | 6.00 ± 0.00d | 6.00 ± 0.00d | |
31.25 mg/ml | 6.00 ± 0.00d | 6.00 ± 0.00f | 6.00 ± 0.00e | 6.00 ± 0.00d | 6.00 ± 0.00d | |
62.5 mg/ml | 6.00 ± 0.00d | 7.33 ± 0.33e | 6.00 ± 0.00e | 6.00 ± 0.00d | 6.00 ± 0.00d | |
125 mg/ml | 6.00 ± 0.00d | 8.67 ± 0.33d | 8.00 ± 0.33d | 6.00 ± 0.00d | 7.33 ± 0.33d | |
250 mg/ml | 9.67 ± 0.33c | 13.67 ± 0.33c | 10.67 ± 0.33c | 7.67 ± 0.33c | 9.00 ± 0.58c | |
500 mg/ml | 12.00 ± 0.58b | 16.33 ± 0.33b | 13.00 ± 0.58b | 9.67 ± 0.33b | 15.67 ± 0.33b |
Values are expressed as mean ± standard error of the mean (SEM) for triplicate reading. Values with the same superscript letter in the columns are not significantly different by one-way ANOVA followed by Tukey’s post-hoc test.
The MIC values obtained for the DCM : MeOH extract of
Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) for bacteria test cultures in mg/ml.
Concentration | ||||
---|---|---|---|---|
Tetracycline ( | ||||
MIC | MBC | MIC | MBC | |
62.50 ± 0.00a | 125.00 ± 0.00a | 1.96 ± 0.22b | 3.91 ± 0.00b | |
52.1 ± 10.40ab | 62.50 ± 0.00b | 4 ± 0.00a | 32 ± 0.00a | |
52.1 ± 10.40ab | 62.50 ± 0.00b | 1.96 ± 0.22b | 15.63 ± 0.1a | |
62.50 ± 0.00a | 125.00 ± 0.00a | 0.98 ± 1.0c | 3.91 ± 0.00b | |
26.04 ± 5.21b | 52.10 ± 10.4b | 0.25 ± 0.00c | 1.96 ± 0.00c |
Values are expressed as mean ± standard error of the mean (SEM) for triplicate reading. Values with the same superscript letter in the columns are not significantly different by one-way ANOVA followed by Tukey’s post-hoc test.
The time-kill kinetics of the extract concentrations of
Time-kill kinetics activities of the dichloromethane: methanolic extract of
Time-kill kinetics activities of the dichloromethane: methanolic extract of
Time-kill kinetics activities of the dichloromethane: methanolic extract of
Time-kill kinetics activities of the dichloromethane: methanolic extract of
Time-kill kinetics activities of the dichloromethane: methanolic extract of
Qualitative phytochemical screening of the dichloromethane: methanolic extract of
Phytochemical composition of the dichloromethane:methanolic extract of
Phytochemical | |
---|---|
Alkaloids | + |
Flavonoids | + |
Steroids | − |
Saponins | + |
Cardiac glycosides | + |
Phenolics | + |
Terpenoids | + |
Tannins | + |
Present phytochemicals are denoted by the (+) sign, while the absent phytochemicals are denoted by the (−) sign.
The inhibition of
The results obtained showed that the extract contained different phytochemicals that included alkaloids, cardiac glycosides, saponins, tannins, flavonoids, terpenoids, and phenols.
Phenolic compounds have been reported to have an antibacterial activity against
Alkaloids such as ramiflovines A and B extracted from
Where similar MIC and MBC values against test bacteria were obtained, MIC indicated a bactericidal activity, while those having MBC greater than MIC, the MIC of the extract, in this case, indicated bacteriostatic activity. From all the tests, the values of MBCs obtained were not more than 4 times higher than those of MICs on the corresponding test bacteria indicating that the extracts tested had an antimicrobial activity [
Time-kill kinetics studies of the dichloromethane: methanolic extract of
The existence of viable bacteria colonies after 24 h of incubation with the antibacterial agent present could be due to the occurrence of mutant forms which resist and grow in the presence of extract concentrations and change to vegetative forms when antibacterial agents are withdrawn. Mutant bacterial cells devoid of cell wall can be produced by both Gram-negative and Gram-positive bacteria [
The dichloromethane: methanolic extract of
Based on the abovementioned conclusion, the following recommendations are forwarded: The plant part should be extracted with other solvent types to get enough of bioactive molecules The different fractions of the plant extract to be evaluated for antibacterial activities
Original
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that they have no conflicts of interest.
It is out of the invaluable guidance and mentorship from George Isanda Omwenga and Mathew Piero Ngugi and the inspirational support from Rachael Kitondo Wambua and Judith Chemutai Samoei that this study was a success.
The authors are grateful for the technical support provided by Catherine Muthoni and Joseph Maingi, both in the Microbiology laboratory, Kenyatta University. The authors’ heartfelt thanks also go to Kenyatta University for resource support.