Phytochemical screening and in vitro antibacterial activity of Echinops kebericho Mesfin tuber extracts: experimental studies

Description of the plant collection areas

We conducted the study at Ambo University after collecting plants from two districts of the West Shewa zone of the Oromia regional state in Ethiopia, namely, the Ambo and Toke Kutaye districts. Administratively, the Ambo district belongs to the West Shewa Zone, Oromia Regional State of Ethiopia. The Ambo district is located at a latitude of 8°59′N and 8°98.3′N and longitudes of 37°51′E and 37°85′E with an elevation range of 1,900–2,275 m above sea level. The Toke Kutaye district is located in the west Shewa zone of the Oromia regional state, 126 kilometers west of Addis Ababa and 12 km from Ambo. Geographically, it is situated between latitudes 8°47′N and 9°21′N and longitudes 37°32′E and 37°03′E.

We designed the in vitro experimental study to be carried out from November 2022 to September 2023. All the experimental tests were performed in triplicate along with the positive and negative controls to determine the antibacterial activity of the plant.

Plant material collection and identification

Mr. Fikadu Lebeta, a botanist in the Plant Science Department at Ambo University’s Guder Mamo Mezemir Campus, collected tubers of E. kebericho Mesfin from the Toke Kutaye and Ambo districts and authenticated them. He collected and transported the plant materials using standard storage protocols and wrapped them in plastic sheets. Then, he deposited voucher numbers (AU2202) on the specimens of E. kebericho Mesfin in the Ambo University plant science laboratory. We washed tubers of E. kebericho Mesfin plants with clean water to remove dirt and rinsed them with distilled water. We cut the rinsed plant materials into pieces and dried them in the shade at room temperature. We ground the dried plant samples using a mortar and pestle. We stored powders of the plant material in clean and dry plastic bags.

Plant extraction

We extracted the powder part of the study plant material utilizing absolute methanol (99.8%) and petroleum ether (99.8%) through the maceration method. They prepared the crude plant extracts separately using methanol (99.8%) and petroleum ether (99.8%). A sensitive digital weighing balance weighed out two hundred grams of air-dried powdered plant material. Then, it placed the material in Erlenmeyer flasks and filled them with 800 mL of extraction solvent (absolute methanol). The plant material was macerated for 72 h over an HZ rotary shaker at 121 rpm at room temperature, following the method described by Crăciun & Gutt (2023) for the solid–liquid ratio of plant material and solvent. The extract was separated from the marc using gauze, and the resulting suspension was filtered with Whatman™ filter paper number 1. The resulting filtrate was concentrated under reduced pressure in a rotary evaporator at 40 °C and 121 rpm, followed by heating in an oven at 40 °C (Kifle, Anteneh & Atnafie, 2020; Marami et al., 2021).

We soaked approximately 200 g of sieved plant powder in petroleum ether (99.8%) in separate 800 mL Erlenmeyer flasks on an HZ rotary shaker at 121 rpm at room temperature for 72 h (Abubakar & Haque, 2020; Ali & Awoke, 2021). The extracts were filtered using gauze followed by Whatman™ filter paper number 1. Concentrated the filtrate under reduced pressure in a rotary evaporator at 40 °C and 121 rpm, and then heated it in a hot air oven at 40 °C (Yasien et al., 2022). Transferred the dried crude extract of each solvent to a vial, measured the concentration of each plant extract, and stored the sample in a refrigerator until further processing. The percentage yields of all the plant extracts calculated using the following formula (Abbas et al., 2021; Gizaw et al., 2022). ${\displaystyle \text{Yield}\left(\text{\%}\right):=\frac{\text{Weights of extract}\left(\mathrm{g}\right)}{\text{Weight of plant material}\left(\mathrm{g}\right)}\ast 100.}$

Preliminary phytochemical screening of the extract

We conducted a standard preliminary phytochemical analysis to determine the presence or absence of secondary metabolites in the methanol and petroleum ether crude extracts of each plant using standard phytochemical screening methods. We identified the presence of bioactive compounds such as flavonoids, saponins, alkaloids, tannins, phenols, cardiac glycosides, and phytosterols in crude tuber extracts of E. kebericho through standard testing methods.

Test for alkaloids

Wagner’s reagent used to test the alkaloid content. Briefly, 15 mg of crude plant extract was mixed from each of the two solvents in a water bath for 5 min before filtering. A few drops of Wagner’s reagent were added to the plant extract filtrates, and the presence of brown or reddish precipitates indicated the presence of alkaloids in the extract (Joshi, Bhobe & Sattarkar, 2013; Iqbal, Salim & Lim, 2015).

Test for flavonoids

A portion of the powdered crude plant (0.25 g) sample was heated for 3 min in the presence of ten milliliters of ethyl acetate for every plant extract. After the mixtures were filtered, 1 ml of diluted ammonia solution was shaken with 4 ml of the filtrate. The solution was yellow, which is indicative of the presence of flavonoids (Edeoga, Okwu & Mbaebie, 2005; Lalitha & Gayathiri, 2013).

Test for saponins

We boiled approximately 2 g of the crude sample extract in 20 ml of distilled water in a water bath and filtered it. They then mixed 10 milliliters of the filtrate with 5 ml of distilled water, shook it vigorously for 2–3 min, and checked for stable persistent froth, which indicated saponins in the sample extract (Edeoga, Okwu & Mbaebie, 2005; Dhayalan et al., 2018)

Tannin test

We used a bromine water test to determine the tannins in the plant extracts. They dissolved 5 mg of the crude extracts from the plants in 5 ml of 95% ethanol and filtered the solution. They then added three or four drops of bromine water to the filtered solution. They detected condensed tannins in the solution by observing buff-colored precipitates; the absence of color showed hydrolyzable tannins (Ali et al., 2018; Salehdeen, Abdulrazaq & Osinlu, 2019).

Test for phenols

Phenol was detected in the plant extracts using the ferric chloride test. Briefly, 0.25 gm of the crude extract was treated with 4 drops of FeCl₃ to afford a blue −black color, which indicates the presence of phenols in the plant extracts (Gizaw et al., 2022; Hasan et al., 2017).

Test for phytosterols

We applied chloroform to 0.25 grams of plant extract and filtered the resulting solution to identify the presence of phytosterols through the Szarkowski test. The filtrates were mixed with concentrated H₂SO₄, vigorously shaken, and then allowed to stand for a brief period of time. The ability of the extracts to produce a golden yellow hue suggested the presence of phytosterols (Abubakar & Haque, 2020; Hasan et al., 2017).

Tests for cardiac glycosides

We tested the extract for cardiac glycosides using the Keller-Killani test. We dissolved 0.2 grams of the extract by mixing two milliliters of glacial acetic acid with one drop of a 5% ferric chloride solution. We filled the test tube containing the solution with a few drops of concentrated H₂SO₄. A cardiac glycoside is present in a solution that gradually becomes bluish-green in color (Oloya et al., 2022).

Bacterial strains

The standard reference bacterial species used were the American Type Culture Collection (ATCC) of Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 43816), Staphylococcus aureus (ATCC 25923) and Pseudomonas auroginosa auroginosa (ATCC 27853), which were collected from the Bacteriology Laboratory of the Ethiopian Public Health Institute (EPHI). We cultured and maintained these bacterial strains in appropriate culture media before performing antimicrobial tests. We performed all laboratory work according to CLSI guidelines (CLSI, 2022).

Determination of the antibacterial activity of the extracts

Inoculum preparation

Differential and selective media were used to streak standard test organisms. Bacterial strains were verified and confirmed after biochemical analysis, Gram staining and growth on selective media. We used mannitol salt agar for S. aureus, eosin-methylene blue (EMB) agar for E. coli, cetrimide agar for P. aeruginosa, and MacConkey agar for K. pneumoniae. We prepared the inoculum according to the Clinical and Laboratory Standards Institute (CLSI) guidelines and standardized it to a 0.5 McFarland turbidity standard equivalent. The bacterial strains were cultured on selective media or differential media at 37 °C for 24 h. We transferred sterile nutrient agar plates to the colonies for subculture. We transferred sterile nutrient agar plates to the colonies for subculture. Four to five bacterial colonies removed from the subcultured agar and inoculated into a sterile physiological saline solution (Cavaleri et al., 2005). We emulsified the colony suspensions in sterile saline and diluted them until the turbidity matched that of a 0.5 McFarland turbidity standard, with a concentration of (1.5 × 108 CFU/ml) (CLSI, 2012 A Wickerham Card was used to visualize turbidity regarding the 0.5 McFarland turbidity standard after the suspension was vortex mixed (Fadahunsi & Babalola, 2021).

Agar well diffusion

The agar well diffusion method used to evaluate the antimicrobial activity of the plant extracts. Agar well diffusion method was used to evaluating the extracts antibacterial activity. The media cooled immediately after autoclaving, at temperatures between 45 to 50 °C. The freshly prepared and cooled media were poured into 90 mm diameter Petri dishes and set on a level and horizontal surface to achieve a uniform depth of nearly four mm. The agar media cooled and solidified at room temperature (Cavaleri et al., 2005).

McFarland standard (0.5) used to adjust vortexed bacterial suspension (equivalent to 1.5 × 10⁸ CFU/ml) by contrast against Wickerham Card (black and white) stripped paper to visualize turbidity to the standard level. The bacterial suspension was prepared in a sterile physiological saline solution tube within 15 min. The aliquot spread evenly onto MHA with a sterile cotton swab (Wiegand, Hilpert & Hancock, 2008).

The swabbed Mueller Hinton agar was left for 15 min to allow bacteria to attach to the media. A sterilized cork borer 6 mm in diameter was used to create a well on swabbed MHA medium to form 6 mm diameter wells. Five equidistant wells 6 mm in diameter and 4 mm in depth were made on the agar plate. With a sterile needle, we discarded the circle created by the cork borer to make a well. The created wells filled with 50 µL of plant extracts at different concentrations (200 mg/ml, 100 mg/ml, 50 mg/ml, and 25 mg/ml) or with 5% DMSO as a negative control (Jamal et al., 2011; Manandhar, Luitel & Dahal, 2019). After all the agar wells were filled and a positive control was placed, the plates were kept at room temperature for approximately 40 min to allow the extracts to diffuse into the media (Taye et al., 2011).

We allowed the agar plates to incubate at 37 °C for 24 h. Then we placed gentamycin (30 µg) discs as positive controls on the media and dispensed methanol and petroleum ether (5% DMSO) in the negative control wells. The positive control was selected based on the susceptibility of the bacterium used (CLSI, 2012). An agar well (6 mm) showing no zone of inhibition was considered to have no antimicrobial activity. We measured the diameters of the inhibitory growth zones using a ruler and recorded the measurements in millimeters after incubation. We conducted the experiment three times.

Determination of the minimum inhibitory concentration by the agar dilution method

The minimum inhibitory concentration is the lowest concentration of an antibacterial agent that completely prevents visible growth of the test strain of an organism (Cock et al., 2022). For the agar dilution method, plant extracts serially diluted twice in molten MHA and cooled to 50 °C in a water bath to achieve the desired final concentrations. A twofold serial dilution of stock solution containing 200 mg/ml of each plant extract in 5% DMSO used to achieve the desired concentration. The test concentrations were 100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml, 6.25 mg/ml, and 3.12 mg/ml.

The MHA sterilized for 15 min at 121 °C in an autoclave before allowed cooling to 50 °C in a water bath. One milliliter of each twofold serial dilution of plant extract was added to 19 mL of liquefied MHA, which was carefully swirled, poured into sterile petri dishes and allowed to set (Mazhangara et al., 2020; Kowalska-Krochmal & Dudek-Wicher, 2021).

Standardization of inocula for agar dilution

A bacterial suspension equivalent to the 0.5 McFarland standard (1.5 × 10⁸ CFU/ml) made in sterile saline solution for each ATCC reference. We adjusted the final inoculum for MIC to 1 × 10⁴ CFU/spot to get the desired cell density. To obtain the final desired cell density of each bacterial suspension, a standardized inoculum of 10⁸ CFU/ml was further diluted 1:10 in sterile saline solution. Using a micropipette, 0.1 ml of the 10⁸ CFU/ml bacterial inoculum transferred to a sterile Eppendorf tube containing 0.9 ml of sterile saline solution to obtain 10⁷ CFU/ml. By the same method, the inoculum was further diluted to obtain a standard inoculum of 1 × 10⁴ CFU/spot (Wiegand, Hilpert & Hancock, 2008; Kowalska-Krochmal & Dudek-Wicher, 2021).

Approximately 1 µL of the prepared inoculum was dispensed on MHA containing different concentrations of plant extracts via a 5 µL micropipette. A bacterial suspension was used to inoculate MHAs without plant extract, serving as control plates in the experiment (Wiegand, Hilpert & Hancock, 2008). After allowing the spotted inoculum to dry at room temperature for a few minutes, we invert the plates for incubation. The plant extract inhibited visible growth completely at the lowest concentration, which was recorded as the MIC.

Statistical analysis

All the investigations stored the gathered data in Microsoft Excel 2016 spreadsheets. We conducted all measurements in triplicate to test the reproducibility of the experimental data. We utilized IBM SPSS version 20 software (IBM, Armonk, NY, USA) to perform statistical analysis of the results. A one-way ANOVA, followed by Tukey’s post hoc multiple comparison test, determined the significance of the difference between the means of the concentrations. The experimental antibacterial activity data express the mean ± standard deviation. A statistical significance level of P < 0.05 indicated significance.

Percentage yield of the plant extract

The present study presented the percentage yield of methanol extract is 7.85%. We extracted the lowest mass of petroleum ether (6.25%) roots of the plants. Methanol-extracted plants produce a higher yield than petroleum ether-extracted plants.

In vitro antibacterial activity of the plant extract

We evaluated the antibacterial activity of the tuber extract of E. kebericho Mesfin through agar well diffusion. We screened methanol and petroleum ether extracts of the plants against four standard bacterial species in triplicate, at concentrations of 200 mg/ml, 100 mg/ml, 50 mg/ml, and 25 mg/ml. The evaluation of antibacterial activity by the agar well dilution method indicated that all the bacteria tested inhibited the growth of the plant extracts of both solvents at a concentration of 200 mg/ml, which ranged from 16.67 ± 0.58 mm to 7.0 ± 0.0 (mm) standard deviation (SD) of the mean inhibition zone diameter, as indicated in Table 1. The observed mean inhibition zone diameters of plant extracts at different concentrations against the tested bacteria were significantly different (P < 0.05). The results showed that the extracts have good dose-dependent antibacterial activity.

The maximum activity of the methanol fraction was conferred against S. aureus (ATCC 25923) (16.67 ± 0.58 mm), while the minimum activity was observed against P. aeruginosa (ATCC 27853) and K. pneumoniae (ATCC 43816), with mean inhibition zone diameters of 9.0 ± 1.75 mm and 7.3 ± 0.58 mm, respectively, at the lowest concentration (200 mg/ml), as shown in Table 1. The petroleum ether extract of the plant showed moderate inhibition, with a maximum inhibition of 14.3 ± 1.52 mm against S. aureus (ATCC 25923) (Table 1). In general, the extent of bacterial growth inhibition increased as the concentration of the extracts increased. At the concentration of 25 mg/ml, no bacteria inhibited (Table 1). The results obtained for methanol extracts of E. kebericho Mesfin showed that there was a significant difference (P < 0.05) in the inhibition of all the test organisms at different concentrations, indicating that the antibacterial activities were concentration dependent. The positive control gentamycin strongly inhibited the growth of the selected bacteria.

Minimum inhibition concentration of the plant extract

The determination of the MIC by agar dilution of the extract revealed that the extract had an inhibitory effect on S. aureus MIC at a concentration of 25 mg/ml (ATCC 25923). The MIC of the methanol extract of E. kebericho Mesfin against S. aureus was the lowest (25 mg/ml) (Table 2). The agar dilution demonstrated that S. aureus showed complete growth inhibition at the lowest MIC values compared to the other tested bacteria. The current study confirmed that the methanol extract exhibited the lowest MIC values when compared with the petroleum ether extract.

Phytochemical constituents of the plant extracts

Phytochemical screening of the methanol and petroleum ether extracts of E. kebericho revealed phytochemicals such as alkaloids, flavonoids, tannins, and cardiac glycosides in both solvent extracts (Table 3).