Abstract
Background
Antimicrobial-resistant pathogenic microorganisms are disease causing microbes that cause several diseases in humans, animals, and plants. Currently, these microorganisms cause more than a quarter of the reported diseases worldwide. The other current global health risk is the imbalance generation of free radicals in human physiology, which exposes several millions to oxidative stress diseases like Alzheimer, Cancer, and Diabetes.
Objective
The objective of this study was to evaluate the antibacterial and antioxidant activities of Inula confertiflora roots, stems, and leaves extracts.
Method
The methanolic, ethyl acetate, and petroleum ether extracts of the roots, stems, and leaves of this plant were separately evaluated against the Gram-positive bacteria strains Enterococcus faecalis, Staphylococcus aureus, and Gram-negative bacteria Klebsiella pneumonia using the agar well diffusion method. Each crude extract was also evaluated by an invitro antioxidant activity test against DPPH free radical reagent.
Result
The crude extrats showed moderate to high activities against two Gram-positive bacterial strains, S. aureus and E. faecalis. The petroleum ether extract of the leaves showed the highest activity (39.97 ± 0.12 mm inhibition diameter) against S. aureus. All crude extracts also showed strong free radical scavenging activity, with a maximum percentage scavenging value. The root petroleum ether extracts showed 99.18 ± 0.06% scavenging activity at 1000 (µg/ml) concentration.
Conclusion
Extracts from this plant are potentially effective against S. aureus and E. faecalis; they also have strong free radical scavenging activities.
Keywords: Antioxidant, Antibacterial, Inula confertiflora
Introduction
Infectious diseases are diseases caused by pathogenic microorganisms like bacteria, fungi, protozoa, and viruses [1]. A worldwide reemergence of infections that cannot be effectively treated with monotherapy is on the horizon due to the growing threat of antimicrobial resistance [2, 3]. Among the bacterial pathogens E. faecalis, S. aureus, and K. pneumonia are mentioned as some of the major causes of bacterial infectious diseases [4, 5]. Out of these S. aureus is an opportunistic pathogen that commonly inhabits the skin and nasal regions of individuals. However, it can cause secondary pneumonia in hosts with compromised immune systems [6]. Patients infected with the influenza virus are at a very high risk of developing S. aureus infections because the viral infection damages the mucous lining of the respiratory tract, exposing the underlying soft tissue to the common microbial flora, including S. aureus [6]. In addition, a report by the United Nations Foundation suggests that in the years ahead, pathogenic microbial infection may result in significant global impacts on human health [7]. Various intervention activities were done against these pathogenic microbes, including antibiotic agents in human history. However, with the excessive use of these antibiotics to treat infectious diseases, the majority of the pathogenic microorganisms developed antibiotic resistance against conventional antibiotics, creating serious health problems worldwide [8]. The possible approach to solve this problem is using other alternatives, like plant sources that have whole-series activities and multi drug-resistance pattern-modifying effect [3, 9]. On the other hand, imbalanced generation of free radicals in the cells, which leads to oxidative stress, is a current serious issue [10]. Some of the free radicals include superoxide anion (O2−), perhydroxyl radical (HO2−), hydroxyl radical (OH), singlet oxygen (O2), peroxynitrite (ONOO−), nitrogen dioxide (NO2) and nitric oxide (NO) [11]. About ten to twenty thousand free radicals attack each cell every day; As a result, some of these free radicals have important functions like killing harmful microorganisms, controlling smooth muscles, and fighting inflammation [12]. However, when the numbers of free radicals are higher than their physiologically balanced value, free radicals can cause structural damage to cells, proteins, nucleic acids, membranes, and lipids. At high quantities, they cause a shift in oxidative metabolism towards pro-oxidants, which results in several diseases like Alzheimer, Cancer, Diabetes, Atherosclerosis, Arthritis, Asthma, Aging, Hypertension Cirrhosis Allergy, Cataract Receptor Alteration, reduced viscosity (synovial fluid) [4, 11].Today, the prevalence of diabetes and cancer, both of which are caused by oxidative stress diseases dramatically increased in the world [13]. The healing potential of plants was officially known before thousands of years, and they possess whole series of compounds capable of showing remarkable effects on animals, microorganisms, and plants themselves [9]. So, in response to the two major public health problems, natural products from plants, animals, and microorganisms could be the right approach [14]. Studies on the area witnessed that about 70–80% of people worldwide and 80% of the people of Ethiopia chiefly rely on traditional herbal medicines to heal their primary health problems [15]. The genus Inula is one of the plant genera in the largest Asteraceae family found in Africa, Asia, and Europe. They are known for their antimicrobial activities; hence, their phytochemicals are composed of steroids, terpenoids (Sesquiterpenes, diterpenes, and triterpenoids), phenolics, and flavonoids with significant antibacterial and antioxidant activity, I. viscosa and I. confertiflora are the best examples of this [16–19]. I. confertiflora also known as weinagift in local Amharic language, is an endemic plant and one member out of 100 species in the Inula genus, distributed in the highlands of Wollo, Shewa, Gonder and Gojam [18, 19]. Phytochemically it is composed of terpenoids, flavonoids, Phytosterols, tannins, and saponins; some of the secondary metabolites (1–10) previously reported from this plant are indicated in Fig. 1 [18].
Fig. 1.
Compounds reported from I. Confertiflora
Traditionally, this plant has been exhaustively used to treat different health problems of animals and humans in Ethiopia. For instance its fresh leaves are used to treat infected cattle eyes, while its flowers and roots are smoked during childbirth, and its leaves are commonly used to treat skin rashes of human beings locally [18]. Furthermore, a scientific study on the leaf extracts of I. confertiflora confirmed that, they are potentially effective in the prevention of viruses that cause influenza, avoid HSV-1 replication, and possess anti-inflammatory activity [20]. The other study conducted on this plant also reported that it is locally used to treat various acute medical conditions and has a 100% success rate in providing healing benefits [21]. Despite its widespread use as a primary health remedy, only a few scientific reports are present about the biological activities of this plant. Therefore, the aim of this study was to evaluate the antibacterial activities and antioxidant potential of different parts of this plant.
Materials and methods
Sample collection and preparation
The root, stem, and leaves of I. confertiflora were collected separately from the Ambassel district, 09 kebele, which is located at 110 21’ 21’’N and 390 34’ 95’’ E with an altitude of 2757 m, above sea level the sample view for each part is placed in Fig. 2.
Fig. 2.
Parts of I. Confertiflora plant root (Left), stem (Middle), leaves (Right), (Photo: Eshetu W., 02, March, 2023)
The plant sample was collected after getting a written consent letter from Wollo University Postgraduate Office. The specimen was submitted to Wollo University Department of Biology for identification and the National Herbarium Center for voucher number. The specimen was identified by Mr. Belay Melese (Assistant prof.) and Mr. Fassil Asafaw (MSc.) and assigned a voucher number (S1152). All parts were washed with tap water to remove soil and dust particles. Then the stems and roots were sliced uniformly into medium-size to make them appropriate for air drying and placed separately in a clean room for one week until completely dry. Then the air dried leaves, stems, and roots were ground into coarse powder solids by an electric grinder.
Chemicals and reagents
Analytical-grade chemicals and reagents were used to conduct each experiment. These are; methanol, ethyl acetate, petroleum ether, lead acetate, chloroform, hydrochloric acid, concentrated sulfuric acid, glacial acetic acid, distilled water, sodium hydroxide, ferric chloride, DMSO, muller hilton agar, nutrient broth, eosin methylene blue (EMB) agar, bile esculine agar, mannitol salt agar and DPPH.
Equipment
The equipment used for this study was a digital analytical balance and an electric grinder for sample preparation. Erlenmeyer flask, volumetric flask, bandage, what’s man filter paper, measuring cylinders, beaker, aluminum foil, and rotary evaporator were used for extraction and concentration purposes. Autoclave, petri dish (glass made), incubator, bacteriological safety cabinet, bunsen burner, inoculating loop, swab, micropipette, deep freezer, and test tubes were used for antimicrobial tests. A UV-spectrophotometer, micropipette and volumetric flask were used for the free radical scavenging activity test.
Extraction procedures
One of the major factors contributing to low achievement in the extraction and biological activity of plants is the effectiveness of the solvents used. In order to solve this problem in this study, the extraction mechanism considered the polarity gradient of solvents. The extraction was made with three organic solvents: methanol, ethyl acetate and petroleum ether separately. From each plant part, 200 g was soaked independently with three different solvents: methanol, ethyl acetate, and petroleum ether to get a total of nine different crude extracts. This non-sequential extraction process was done to obtain sufficient concentrations of the components in the crude extracts for each solvent system and plant part. The extraction method used was cold maceration for 48 h duration, with scheduled shaking intervals of three hours for each solvent system. Each soaked plant parts were first filtered with bandage followed by filtration with filter paper and concentrated using rotary evaporator at 35 °C. The extracts were placed in a cold and dark place until they were used for further analysis.
Phytochemical screening tests
The phytochemical screening experiments were conducted based on the previously conducted tests as indicated in (Table 1).
Table 1.
Phytochemical screening test procedures for the extracts
| Secondary metabolites | Procedures followed | Expected positive result |
|---|---|---|
| Alkaloids | 50 mg of plant crude extract was dissolved well in 2 mL of a 1% HCl solution, followed by slight heating until steaming. Then six drops of Wagner’s reagent were added to 1 mL of acidified extract. | Brown precipitate [22] |
| Cardiac glycosides | 0.5 gm. of the plant extract was dissolved in 0.4 mL of glacial acetic acid with trace amount of ferric chloride, and then 0.5 mL of conc. H2SO4 was added through the walls of the test tube. | Appearance of blue color in the acetic acid layer |
| Flavonoids | One mL of plant extract (50 mg/mL) was added to 1 mL of 10% lead acetate solution. | yellow precipitate [22] |
| Phenols | 1 mL of plant extract (50 mg/mL) was added to 0.5 mL of a 5% ethanolic solution of ferric chloride. | Formation of dark color [22] |
| Tannins | A few drops of 10% lead acetate were added to 2 mL of plant extract (50 mg/mL). | Precipitate formation [22] |
| Terpenoids | 2.5 mL of plant extract (50 mg/mL) was mixed with 1 mL of chloroform, and then 1.5 mL of conc. H2SO4 solution was added. | Appearance of reddish brown color [22] |
| AnthraquinonesAnthraquinon Anthraquinones | One mL of plant extract was placed on a test tube, and then 5 drops of 2% hydrochloric acid was added. | Appearance of red color |
| Phlobatannin test | Ten (10) drops of 1% hydrochloric acid were added to 1 mL of the crude extract in test tube. | Formation of red precipitate [22] |
| Phytosterols | (50 mg) of the methanolic crude extract was dissolved in 2 mL of acetic anhydride. Then two drops of concentrated sulfuric acid was added. | Formation of dark brown color [23] |
| Saponin | (50 mg) of the methanolic extract was diluted with distilled water and made up to 20 mL with a strong agitation. | The presence of foam which persists for about 15 min is a positive result [23]. |
Bacterial strain growth procedures
Antimicrobial activity tests were conducted on three bacterial strains, Gram-positive bacteria S. aureus, E. faecalis, and Gram-negative bacteria K. pneumoniae. These test strains were brought from the APHI (Amhara Public health Institute) Dessie branch. The test organisms were first placed on deep freezer, then thawed (placed at 37 °C to adapt the temperature for the test organisms), and then transferred to separate Media of nutrient broth for 24 h, with the same temperature. After 24 h incubation, these test strains were transferred to the selective Medias bile esculine agar, eosin methylene blue (EMB) agar and Mannitol salt agar for E. faecalis, K. pneumonia, and S. aureus, respectively. After an additional 24 h on the selective Media the test strains were cultured on nutrient agar for another 24 h. A few colonies of each test organism was taken and dissolved in normal saline solution separately. The turbidity of suspensions was adjusted equivalent with 0.5 McFarland standards. McFarland is a Barium Sulfate standard against which the turbidity of the test and control inoculum was compared. Out of the suspension 100 µL from each test organism were inoculated on the surface of Mueller Hinton agar plates (separate MH agar plates were used for each test).
Antibacterial susceptibility tests
The antibacterial tests were done according to Algasham and co-workers with minor modification [22]. Agar well diffusion method was used to test the antimicrobial capacity of the extracts. Based on this method, 0.1 mg/L (C1), 1 mg/L (C2) and 10 mg/L (C3) of extracts separately prepared in 5% DMSO solvent. Side to side muler Hilton agar was dispensed on sterilized glass Petri dishes aseptically. The test organisms were inoculated on the muler-Hilton agar dispensed Petri dishes which was further drilled with micro pipette tips. Then exactly 100 µL of resulting solution were transferred in to the holes prepared on the Muller- Hilton agar inoculated bacterial strains. These Petri dishes were incubated at 37 
for 18–24 h. After the incubation period, the plates were observed for a clearance zone around the holes which indicates a positive antibacterial activity of the respective plant extracts. The clear zone formed around each well was measured by Vernier caliper. Each experiment was carried out in triplicates and then mean of the inhibition zone of each test sample were taken for evaluating the antibacterial activity of the extract. Side to side the commercial standard antibiotics, Amoxicillin (10 µg/mL) was used as a positive control and 5% DMSO solvent was taken as a negative control [22].
Antioxidant activity test
The antioxidant potential of these plant extracts was conducted based on the method from previous researches [24, 25, 26], with some minor modifications. The free radical scavenging activity of the crude extracts was determined using a DPPH scavenging assay. Based on this principle, a series of concentrations, (62.5 µg/mL, 125 µg/mL, 250 µg/mL, 500 µg/mL and 1000 µg/mL) of the plant extracts were prepared in methanol. Parallely, 0.2 mM solutions of DPPH were also prepared, and 1 mL of the plant extract mixed with 2 mL of the DPPH solution, after 15 min at room temperature in a dark environment, absorbance was measured at 517 nm wavelength. Pure methanol was used as a blank and the methanolic solution of DPPH was used as control group. The percentage inhibition of DPPH free radical by the crude extracts was calculated as: (
), where, Ac-absorbance value for DPPH solution, As - stands for the absorbance of the test sample with DPPH [27–29].
Data analysis
Statistical data and graphics in this study were analyzed and drawn using Microsoft Excel. All the data were presented as means ± standard deviation for the triplicate trials for each item.
Results
Phytochemical profiles of the plant
According to the phytochemical data presented in (Table 2), this plant exhibits a high concentration of secondary metabolites. The presence of flavonoid, phenol, tannin, terpenoids, phytosterols, and saponins was confirmed through positive test results. However, the tests yielded negative results for alkaloids, anthraquinone, phlobatannin, and cardiac glycosides, indicating their absence in the plant. The (+) indicates the positive result for the respective test, and the (-) sign represents the absence of the secondary metabolite using the test we conducted.
Table 2.
Phytochemical profile of I. confertiflora
| Metabolites | Chemical test applied | Status (+/-) | ||
|---|---|---|---|---|
| Root | Stem | Leaves | ||
| Alkaloid | Hagere test | - | - | - |
| Flavonoid | Lead acetate test | + | + | + |
| Phenol | FeCl3 test | + | + | + |
| Tannin | Lead acetate test | + | + | + |
| Terpenoids | Salkowisk test | + | + | + |
| Anthraquinone | HCl test | - | - | - |
| Phlobatannin | HCl test | - | - | - |
| Cardiac glycosides | Acidic test | - | - | - |
| Phytosterols | Libermann-Burchard’s test | + | + | + |
| Saponins | Foam test | + | + | + |
Antibacterial activity of the crude extract
As reported in (Table 3) antibacterial activity of the plant extracts against three bacterial species, S. aureus, E. faecalis, and K. pneumonia was conducted, and varied activities were observed. Based on this data, the highest antibacterial activity was observed on S. aureus 39.97 ± 0.12 mm diameter of inhibition at 10 mg/mL concentration of the petroleum ether extracts of the leaves. The next highest inhibition diameter is also on S. aureus, which is about 34.03 ± 0.05 mm inhibition diameter from ethyl acetate extracts of the leaves. Medium growth inhibition activity was observed on E. faecalis species. The leaf crude extracts showed relatively good activity against Gram-positive bacteria E. faecalis 21.25 ± 0.25 mm, 20.11 ± 0.35 mm and 20.22 ± 0.20 mm for the methanol, ethyl acetate, and petroleum ether extracts, respectively, Whereas the growth inhibition capacity of this crude extract on the Gram-negative bacteria K. pneumonia is null (-) for these concentrations and this test.
Table 3.
Antibacterial activities of extracts
| Solvent | Plant part | Inhibition zones in mm | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Gram-positive | Gram-negative | ||||||||||
| S. aureus | E. faecalis | K. pneumoniae | |||||||||
| C1 | C2 | C3 | C1 | C2 | C3 | C1 | C2 | C3 | |||
| Methanol | Root | - | - | 10.03 ± 0.3 | - | - | 12.5 ± 0.03 | - | - | - | |
| Stem | - | - | - | - | - | 11.14 ± 0.31 | - | - | - | ||
| Leaf | - | 11.02±0.03 | 30.06 ± 0.23 | - | - | 21.25 ± 0.25 | - | - | - | ||
| Ethyl acetate | Root | - | - | 14.08 ± 0.31 | - | - | 14.17 ± 0.21 | - | - | - | |
| Stem | - | 10.4±0.2 | 25.4±0.2 | - | - | 15.12 ± 0.29 | - | - | - | ||
| Leaves | - | 16.65±0.25 | 34.03±0.05 | - | - | 20.11 ± 0.35 | - | - | - | ||
| Petroleum ether | Root | - | 9±0.34 | 29.04±0.06 | - | - | 13.2 ± 0.2 | - | - | - | |
| Stem | - | 12.35±0.21 | 29.2±0.32 | - | - | 14.27 ± 0.25 | - | - | - | ||
| Leaves | - | 13.45±0.35 | 39.97±0.12 | - | - | 20.22 ± 0.20 | - | - | - | ||
| Amoxicillin(10μg/mL) | 35.5±0.31 | 29.25±0.05 | 9.5±0.32 | ||||||||
| - | |||||||||||
| - | |||||||||||
| 5% DMSO | - | - | - | ||||||||
Antioxidant activity of the extracts
The results of percentage scavenging activity are presented as the mean of the triplicate trial plus/minus the standard deviation. The percentage scavenging activities of ascorbic acid standard group are placed together with the scavenging activities of extracts in (Table 4). The scavenging capacity of methanolic crude extract is indicated in (Table 4). From the three parts, the highest percentage scavenging activity was observed in the stem part, which is about 96.76 ± 0.3 at 1000 (µg/mL) concentration. As compared to the activities observed for the methanolic and petroleum ether extracts, the percentage free radical scavenging value for ethyl acetate is lower; except for the stem part, which is a little bit higher than the petroleum ether stem extract activity. The percentage scavenging activity of stem crude extract is 90.74 ± 0.02 at 1000 (µg/mL) concentration. At this concentration, the percentage free radical scavenging capacity of the root and the leaves is 88.20 ± 0.01 and 83.78 ± 0.03, respectively.
Table 4.
% DPPH scavenging capacity of extracts
| % DPPH inhibition | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Concentrations. in (µg/mL) | Ascorbic acid | Methanol | Ethyl acetate | Petroleum ether | ||||||
| Root | Stem | Leaves | Root | Stem | Leaves | Root | Stem | Leaves | ||
| 1000 | 99.87 ± 0.086 | 96.70 ± 0.2 | 96.76 ± 0.30 | 95.37 ± 0.02 | 88.20 ± 0.01 | 90.74 ± 0.02 | 83.78 ± 0.03 | 99.18 ± 0.06 | 82.71 ± 0.08 | 98.43 ± 0.18 |
| 500 | 96.68 ± 0.26 | 91.48 ± 0.2 | 93.39 ± 0.02 | 91.82 ± 0.02 | 80.42 ± 0.11 | 84.78 ± 0.02 | 75.96 ± 0.025 | 95.61 ± 0.16 | 77.74 ± 0.11 | 90.88 ± 0.09 |
| 250 | 92.50 ± 0.12 | 87.39 ± 0.03 | 90.30 ± 0.03 | 88.44 ± 0.01 | 74.63 ± 0.19 | 81.37 ± 0.01 | 72.23 ± 0.05 | 91.79 ± 0.03 | 73.56 ± 0.12 | 83.26 ± 0.05 |
| 125 | 89.24 ± 0.18 | 74.48 ± 0.09 | 86.89 ± 0.04 | 85.20 ± 0.01 | 70.40 ± 0.10 | 76.42 ± 0.015 | 68.70 ± 0.04 | 88.29 ± 0.025 | 69.87 ± 0.035 | 78.82 ± 0.08 |
| 62.5 | 85.94 ± 0.075 | 70.35 ± 0.05 | 82.96 ± 0.02 | 82.55 ± 0.012 | 64.75 ± 0.50 | 71.83 ± 0.01 | 63.36 ± 0.04 | 83.08 ± 0.06 | 65.76 ± 0.05 | 74.06 ± 0.06 |
Among the petroleum ether crude extracts, the highest scavenging activity was observed on the root part for this test. The percentage scavenging activity at 1000 (µg/mL) concentration is 99.18 ± 0.06, which is very close value to the ascorbic acid standard of 99.87 ± 0.086. At the same concentration, the lowest activity was relatively observed in the stem part, with a percentage free radical scavenging value of 82.71 ± 0.08. At a 62.5 µg/mL concentration, the lowest activity is registered in the stem, with a percentage free radical reading of 65.76 ± 0.05.
Discussions
The phytochemical data obtained from this test indicated in (Table 2) are the same for the root, stem, and leaves, which implies that the three parts have identical chemical contents. In addition, this study was able to verify that secondary metabolites found in this plant are more polar compounds by their nature, like hydroxyl groups containing phenols, tannins, terpenoids, and flavonoids, where all groups possess oxygen atoms that are capable of making the overall molecule more polar as well as having potent medicinal value. The phytochemical study result closely agreed with previously reported works in this plant [18, 28].
The petroleum ether extract of leaves showed the highest growth inhibition zone, 39.97 ± 0.12 mm, even greater than the positive control. This could be due to the fact that non-polar extract attributes specific components with significant concentrations in order to influence the test organism. For all extracts, the highest antibacterial activity was observed from the leaves part, in increasing order: methanol, ethyl acetate, and petroleum ether, which correspond with average values of inhibition zone 30.06 ± 0.23 mm, 34.03 ± 0.05 mm, and 39.97 ± 0.12 mm, respectively, against S. aureus. Previously, antimicrobial activity evaluation was conducted on the leaves only using two solvents, 80% methanol and petroleum ether, but the non-polar portion showed greater activity against S. aureus [30]. The phytochemical test part of this study confirmed that this plant is composed of phenols and flavonoids, and a study by [7] suggested that these secondary metabolites have an antibacterial effect associated with their ability to form complexes with the bacterial cell; As a result, the growth of bacteria is inhibited through multiple mechanisms of action. Bacterial growth and proliferation are suppressed by these metabolites through inhibition of microbial cell energy metabolism, interfere with nucleic acid synthesis, disrupt the function of the cytoplasmic membrane, affect membrane permeability, and cause leakage of cellular contents [7].
Although most extrats showed strong activity against the species S. aureus, methanol extracts of stem were inactive and showed almost zero growth inhibition this may be due to the fact that polar solvent consists of multiple components in a lower amount and components with antagonistic effects. However, it showed good activity against E. faecalis, an average of 11.14 ± 0.31 mm inhibition diameter was observed at 10 mg/mL. The leaves crude extracts showed relatively strong activity against Gram-positive bacteria E. faecalis 21.25 ± 0.25 mm, 20.11 ± 0.35 mm and 20.22 ± 0.20 mm for the methanol, ethyl acetate and petroleum ether extracts, respectively.
Relative to the activities observed in S. aureus, the inhibition diameter is lower for E. faecalis. The antimicrobial susceptibility analysis of this strain in the previous report indicates that the majority of the E. faecalis isolates (96.1%) have a drug resistant pattern [31]. Although the susceptibility of E. faecalis is incomparable with that of S. aureus, the plant extracts show encouraging activity, so crude extracts from this plant could be a good solution to reduce the resistant nature of E. faecalis. Hence, the previous report for this plant on its chemical constituents indicates the plant is rich in secondary metabolites like flavonoids, tannins, and terpenoids. The observed antimicrobial activity could be attributed to the presence of these phytochemicals. The work of Agyare and co-workers demonstrated that terpenoids play a great role for antibacterial activity of various plants because of their putative effect on the mevalonate pathway [32].
In contrast to their highest activities on the Gram-positive bacteria strains, the Gram-negative bacteria K. pneumonia is resistant to each of the nine extracts at these test concentrations. This could be due to biofilm formation among bacterial strains. Biofilms protect bacteria by increasing their resistance capability, allowing them to not only withstand harsh environments but also to resist antibiotics, potentially leading to multidrug-resistant, extensively drug-resistant, and completely drug-resistant bacteria [33].
From this study, we able to justify that S. aureus is more susceptible and next the extract is effective against E. faecalis but not active against K. pneumonia at these test concentrations. This is because Gram-negative bacteria strains are more resistant to the action of antimicrobials as compared with Gram-positive bacterias, due to the fact that Gram-negative bacteria strains have more complex cell wall [30]. Overall, these plant extracts showed varied activities against the test organisms; furthermore, our suggestion is that the growth inhibition will Increase if the concentration can be increased more than used here in this study.
%DPPH scavenging activity
In the DPPH free radical scavenging test, the concept is that the absorbance value decreases when the DPPH solution is mixed with the antioxidant plant material [34]. A single electron transfer from the antioxidant agent causes the violet DPPH free radical solution to change color to a yellow non radical DPPH solution [14]. The result obtained from this test confirms that the plant has a significant antioxidant nature in all solvents. The methanol extracts showed significant antioxidant activities as compared to the positive control ascorbic acid; the percentage DPPH scavenging activities for the root, stem, and leaves were 96.70 ± 0.2, 96.76 ± 0.3, and 95.37 ± 0.02, respectively, at a 1000 mg/mL concentration. This implies that the root and stem methanol extracts showed excellent percentage scavenging activity next to the ascorbic acid at this test concentration, as indicated in (Table 4). The antioxidant result directly fits with the phytochemical test result, which confirms the presence of tannins, flavonoids, and phenols. Plants with high concentrations of natural products like flavonoids, polyphenols, tannins, carotenoids, and terpenoids are good for their anti-carcinogenic and antioxidant capabilities [35]. A study by Asraoui and co-workers also elaborates that the Inula species have relatively strong radical scavenging ability due to the polyphenolic chemicals found in the species, which contribute more efficiently to the scavenging of free DPPH radicals [36]. The average percentage scavenging values of the methanolic crude extract data from Table 4 are presented in Fig. 3 for different ways of description.
Fig. 3.
Percentage DPPH free radical scavenging values of Methanolic extracts
Based on the data provided in Table 4; Fig. 4, it appears that the crude extracts obtained from the ethyl acetate section of the plant showed significant activity in terms of free radical scavenging activity. The activity of these extracts was found to be above 70% for most of the test concentrations, indicating their potential antioxidant properties.
Fig. 4.
%DPPH free radical scavenging activities of EtOAc extracts
Among the different parts of the plant, the stem extract exhibited the highest activity, while the leaf extract showed the least activity. However, when compared to the methanol and petroleum ether extracts, as well as the standard L-ascorbic acid (a known antioxidant), the scavenging activity of the ethyl acetate extracts from the root and leaf parts was relatively lower. Specifically, the percentage scavenging activity in ethyl acetate extracts for the root and leaves parts were found to be between 88.20 ± 0.01% and 63.36 ± 0.04%. Figure 4 presents the percentage free radical scavenging values of ethyl acetate extracts at different test concentrations.
The petroleum ether extracts of this plant showed the highest antioxidant activity for the root and leaf crude extracts this may be due to the phenolic group containing larger molecular weight compounds like sterols and tannins. In contrast, a review by Altemimi and co-workers indicates that highly polar organic solvents like methanol have a high effectiveness in extracting biomolecules for antioxidant purposes and also that acetone and N, N- dimethyl formamide (DMF) are also among the highly effective solvents for the extraction of antioxidants [37]. As indicated in (Table 4) the stem crude extracts in petroleum ether solvent show moderate activity as compared to the highest scavenging activity of root and leaf crude extracts in the same solvent. The average percentages of free radical scavenging values of the petroleum ether extracts at various concentrations are presented in Fig. 5.
Fig. 5.
%DPPH free radical scavenging activities of EtOAc extracts
The 50% inhibitory concentration for each extract was also calculated from the results obtained. The IC50 values indicated in Fig. 6 for each extract are lower as compared to the standard ascorbic acid at 90.96 µg/mL, which demonstrated the great potential of the extracts for their free radical scavenging activities.
Fig. 6.
IC50 values of extracts
Based on our previous findings on the chemical constituents, this plant is sufficiently rich in hydroxyl groups containing secondary metabolites like tannins, phenolic, and flavonoid; furthermore, the plant is composed of terpenoids and phytosterols group [18].The polyphenol concentration of plant extracts is known to have a strong relationship with their antioxidant activities. Tannins have the highest antioxidant activity among polyphenols, according to several studies conducted in vivo and in vitro using various biological models [37]. In addition to the relation of polyphenolic groups with their antioxidant properties, the polyphenolic compound containing plant extract has great value for their biological activities, and this study is able to strongly show this relation. To the best of our knowledge, this is the first report on the evaluation of the antioxidant activity of whole parts of I. confertiflora extracts.
Conclusion
Based on the findings of the present study, it is concluded that I. confertiflora is a highly potent plant against Gram-positive bacterial strains, including S. aureus and E. faecalis. Notably non-polar (Petroleum ether) and medium polar (Ethyl acetate) extracts exhibited greater activity against the Gram-positive bacterial strains compared to their polar (Methanol) counterparts. However, the extracts were found to be inactive against K. pneumonia at the tested concentration and under the conditions of the tests. Furthermore, the extracts demonstrated significant free radical scavenging activity and exhibited commendable antioxidant capacity.
Acknowledgements
The Authors would like to express deepest gratitude to Mr. Belay Melese and Mr. Fasil Asafaw for their contribution during authentication of the plant sample, Mr. Teklew Geto from LVM for his support during antimicrobial tests.
Authors’ contributions
EW and SA conducted the experimental work; GW contributed to the antimicrobial activity experiment; all authors participated in data interpretation and analysis; EW and SA wrote the original draft of the manuscript; and YA was involved in reviewing and editing the manuscript. All authors read and approved the final manuscript.
Funding
This study has not been offered any funding.
Data availability
The data that supports the findings of this study will be obtained from the corresponding Author’s email ( [eshewold457@gmail.com](mailto: eshewold457@gmail.com) ) up on the reasonable request.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Data Availability Statement
The data that supports the findings of this study will be obtained from the corresponding Author’s email ( [eshewold457@gmail.com](mailto: eshewold457@gmail.com) ) up on the reasonable request.