The organic solvents including acetone, ethanol, deionized water, and chloroform were used in this research work for soaking, extracting, and other purposes. Moreover, the chemicals used for the preparation of reagents to investigate secondary metabolites like tannins, alkaloids, saponins, flavonoids, phenols, and terpenoids are: vanillin, sulphuric acid, hydrochloric acid, potassium hydroxide (85% Mumbai, India), acetic anhydrous (British Drug House Ltd., UK), ferric chloride (British Drug House Ltd., England), potassium iodide, and iodine. All these chemicals were of analytical grade and were directly employed in the study without any purification procedures.
Plant material collection and preparation of extracts
Samples of Inula confertiflora were obtained from the Gettila basin, which is situated in Debre Markos, Ethiopia, for the current study. After that, a voucher specimen was carefully processed and kept in the biology lab at Debre Markos University's College of Natural and Computational Science. Following collection and vouchering, the leaf sections of the Inula confertiflora plant were thoroughly cleaned with deionized water to remove any remaining dirt, and they were then allowed to dry for seven days under the shade in the chemistry lab. The dried leaf parts of Inula confertiflora were then grinded into fine powder forms using a grinder and immersed in three analytical-grade solvents (acetone, ethanol, and deionized water) to extract the powdered plant material using a slightly modified maceration procedure. In detail, the dried and powdered leaf of I. confertiflora, about 50 g in a two-neck volumetric flask, were soaked and extracted with 200 mL of acetone, ethanol, and water through stirring using a magnetic stirrer for about three days. Taking into account the solvents' tendency to boiling point, the extracted crudes were then centrifuged once more using a rotary evaporator for sample drying. The centrifuged and dried extracts of the plant were meticulously calculated to investigate the yield of extracts with the respective solvents using the following formula:
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)
Finally, the centrifuged and dried extracts were kept in the refrigerator for further study.
Phytochemical Screening-Qualitative test
The preliminary qualitative phytochemical analysis taste was carried out to investigate the presence or absence of secondary metabolites in acetone, ethanol, and water leaf extracts of Inula confertiflora. To study the phytochemical screening test qualitatively, around 400 mg of the freshly extracted Inula confertiflora leaf by each solvents was diluted in 100 ml of each solvent, respectively, to make a stock concentrated solution of 4 mg/ml. Accordingly, the following methods (Cho et al., 2003; Clarke, 1975) were applied to the conventional phytochemical screening tests in order to perform the preliminary phytochemical screening test of I. confertiflora leaf extracts:
Tannins-using Ferric Chloride Test
Two milliliters of 5% ferric chloride were added to one milliliter of I. confertiflora leaf extract. The development of a greenish-black or dark blue hue suggests the presence of tannins.
Alkaloid-using Mayer's Test
Two milliliters of strong hydrochloric acid were added to two milliliters of I. confertiflora extract. Next, a couple of drops of Mayer's reagent were introduced. Alkaloids are present when a precipitate that is either green or white is present.
Saponins-Foam Test
In a graduated cylinder, 2 ml of I. confertiflora extract and 2 ml of distilled water were combined and shaken for 15 minutes lengthwise. Saponins are present when a layer of foam forms that is almost one centimeter thick.
Flavonoids-Sodium Hydroxide Test
One milliliter of 2N sodium hydroxide was added to two milliliters of I. confertiflora extract. The appearance of a yellow color indicates the presence of flavonoids.
Phenols-Ferric Chloride Test
Three drops containing the mixture of 10% ferric chloride and 10% ferrocyanide were added to three milliliters of I. confertiflora extract. The presence of phenolics is indicated by the formation of an orange-brown precipitate.
Terpenoids-Salkowski’s Test
After adding about 5 ml of I. confertiflora extract to the test tube, 2 ml of chloroform and 2 ml of concentrated H2SO4 were added. The presence of terpenoids is indicated by the formation of a reddish-brown ring.
Phytochemical Screening-Quantitative Test
The total contents of phenolic, and flavonoids in leaf extracts of I. Confertiflora were determined using the following standard procedures (Chatatikun & Chiabchalard, 2013; Sandip, Shreewardhan, Sandeephan, Abhay, & Debjani, 2014):
Total phenolic content of leaf extracts of I. Confertflora
The modified calorimetric Folin-Ciocalteu technique was utilized to assess the total phenolic content of I. confertiflora leaf extracts extracted by each solvent. In this experimental methodology, Gallic acid acts as a positive control. To be more precise, 50 mL of 50% methanol (v/v) was used to dissolve 50 mg of tannic acid to prepare 1 mg/mL of the positive control's stock solution. After serially diluting the stock solution with a concentration of 1 mg/mL to the following concentrations: 0, 20, 40, 80, 160, and 320 μg/mL, a solution containing 10 mL of I. confertiflora leaf extracts in 50% methanol at a concentration of 1 mg/mL was prepared. After combining an aliquot of 1 mL of I. confertiflora extracts or a positive control separately with 2.5 mL of Folin-Ciocalteu (10% v/v in water) solution and adding 5 mL of 7.5% sodium carbonate (w/v) solution, the mixture was vortexed and left to stand at room temperature for two hours. The UV-Vis absorbance of the reaction mixtures was measured at 760 nm with respect to a blank solution consisting of 50% methanol. Gallic acid equivalents (GAE) were calculated as the total phenolic content (TPC) of the I. confertiflora extract based on the calibration curve obtained from the tannic acid solution. The results were represented as TPC per milligram of dry weight of I. confertiflora extracts.
Total Flavonoid Content of Leaf Extracts of I. Confertiflora
The total flavonoid contents found in I. confertiflora leaf extracts were determined by applying a slight modification to the calorimetric aluminum chloride method. The positive control in this experiment was quercetin, which was dissolved in 5 mL of 50% methanol to produce a 1 mg/mL concentration for the stock solution. From the quercetin stock solution, serial dilutions of 0, 20, 40, 80, 160, and 320 μg/mL were prepared, and a 10 mL solution containing a concentration of 1 mg/mL of I. confertiflora extracts was prepared. After mixing 1 mL of 5% sodium nitrite with around 1 mL of standard solution, 5 minutes later, 1 mL of 10% aluminum chloride was added. Following a two-minute waiting period, 2 mL of 1.0 M sodium hydroxide was added to the solution. After completely mixing and diluting the mixture with 4 mL of distilled water, the UV-Vis absorbance was measured at 510 nm. Based on the quercetin calibration curve, the total flavonoid content of the each-extracted Inula confertiflora extracts was calculated, and the results are given in milligrams of quercetin equivalent (QE) per dry weight of the leaf extracts.
Antioxidant activity of I. confertiflora extracts using the DPPH method
With a few minor adjustments to the protocol used by Roberta et al. and Blois(Blois, 1958; Roesler, Malta, Carrasco, & Pastore, 2006), the antioxidant activity of Inula confertiflora leaf extracts against radical scavenging activity was examined using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay methods. In particular, 2 mL of I. confertiflora extracted using acetone, ethanol, and water extracts was dissolved in 0.2 mM of DPPH solution, which was the equivalent concentration. Once each extract had been incubated for 20 minutes at physiological temperature, the scavenging efficiency was evaluated against the DPPH solution using UV-Vis spectrophotometer at an absorbance of 517 nm. The ascorbic acid standard solution (2 mM) exhibited a maximum absorbance intensity of 1.37 a.u and served as the reference solution for this DPPH antioxidant experiment.
The percent scavenging efficiency of extracts against DPPH was calculated using the following formula:
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)
Bacterial strains
The antibacterial potency of the leaf extracts of the Inula confertiflora was investigated using two gram-positive (Staphylococcus aureus and Bacillus cereus) and gram-negative (Escherichia coli and pseudomonas) bacterial strains. All the bacterial strains were prepared in Bahir-Dar university, College of natural science at the department of Biology.
In-vitro antibacterial activity of extracts
All the extracts of I. confertiflora was assessed using disc-diffusion methods with little modification that was assayed with the following methodology. In detail, the dried crude extracts of leaf I. confertiflora using acetone, ethanol, and water were dissolved in distilled water to obtain a yield 1 mg/mL concentrated stalk solution. Furthermore, the each extracted crudes were inoculated at the required concentrations of 200 µg/ml on inoculums of bacteria with disc diameters of about 6 mm and placed on plates after being incubated at 37 ºC for 24 hours. Based on the above concentrations, the antibacterial activity of each extracts were investigated by measuring the zone of inhibition in millimeter against gram-positive and gram-negative bacterial strains. In these activity to the common drug gentamycin, which was used as a positive control antibiotic agent and DMSO, which was used as a negative control. Finally, the diameters of the inhibition zones discovered around the discs represent antibacterial efficiency of the leaf extracts of the I. confertiflora.