Antimicrobial, Antiradical Activity, and X-Ray Fluorescence Spectroscopy Analysis of Aloe otallensis Plant Used in Traditional Medicine in Southern Ethiopia

Medicinal plants have a long history of treating diseases in animals and humans in Ethiopia. Nevertheless, not enough research has been done on the antibacterial properties and possible bioactive components of the majority of medicinal plants. Therefore, this study was concerned with the evaluation of the percentage yield, phytochemical, antimicrobial, antifungal, MIC, antiradical activities, phenolic content, and X-ray fluorescence spectroscopy (XRF) analysis of A. otallensis plant extracts. The mean values of antimicrobial, antifungal, MIC, antiradical, phenolic content, and XRF analysis were reported as mean ± standard deviation. The solvent methanol showed a higher degree of yield in leaf and root extract which was 8.45 (22.27%) and 3.12 g (15.58%), respectively, while distilled water extract of leaf and root showed less degree of yield which was 0.22 g (1.10%) and 0.42 g (2.1%), respectively. Qualitative phytochemical analyses of the plant parts have revealed the presence of various components of metabolites like alkaloids, flavonoids, phenol, saponins, tannins, steroids, steroids, terpenoids, triterpenoids, glycosides, anthraquinones, diterpenes, phytosterols, and phlobatannals. A. otallensis gel extracts had shown significant antibacterial and antifungal activity against the test bacterial and fungus, respectively. Moreover, the methanolic gel extracts of A. otallensis demonstrated notable antiradical activity than the leaf and the root. The highest value of phenolic content was obtained in A. otallensis; gel, leaf, and root extract which was 61.9 ± 0.5 mg/g, 53.6 ± 0.3 mg/g, and 51.6 ± 0.6 mg/g, respectively. In this study, twelve elements in the plant parts of A. otallensis were determined using XRF spectroscopy. Overall, this research contributes to the understanding of the pharmacological potential of A. otallensis and highlights the importance of further research into its medicinal properties. The results provide valuable insights into the use of medicinal plants to treat diseases and support the development of natural therapeutics.


Background
Ethnotherapeutic practice of plant species are believed to be one of the potential bases for the development of safe and efective treatments [1].Ethiopia has a long history of a traditional health care system, but studies on traditional medicinal plants have been limited in comparison to the country's multiethnic, cultural, and fora diversity [2].Moreover, the use of medicinal plants to treat infections is an old practice in large parts of Ethiopia to solve health problems for livestock and humans [3][4][5].Yet, most of these experiences are not documented and supported by scientifc experiments.Besides, antimicrobial resistance is spreading at an alarming rate, making treating bacterial infections in animals and humans more difcult.Even modern medicines have failed to treat those resistant bacteria since they are developing resistant behavior [6].Tough just 15% of the world's higher plants have been thoroughly studied for bioactivity, it makes sense to look for alternate approaches to treating infectious diseases.Additionally, plants have the potential to yield innovative antibiotics and medications [7].
Te genus Aloe had recorded history of mentioned and given high rank as multipurpose herbal plant.It was renowned for its medicinal and cosmetic properties that have been exploited over millennia in Ethiopia [8,9].Recent ethnobotanical studies have been reported the use of various

Collection and Identifcation of the Plant Material.
Fresh whole parts of A. otallensis (i.e., leaf, gel, and root) used for this experiment were collected following standard botanical procedures in February 2023.Plant materials and A. otallensis plant parts were collected from Yetnebershi which is found in Arba Minch, Ethiopia.Arba Minch is located at 6 °2″ N latitude and 37 °33″ E longitude, far about 500 km from Addis Ababa and at an elevation of 1285 m.Current estimated number of the zone total population (permanent residents) is 95373 people, including children under the age of 6-9478 people, teenagers (school children) aged 7 to 17 years-11314 people, young people from 18 to 29 years old-11385 people, adults aged 30 to 60 years-41070 people, elderly people over 60 years old-20791 people, and the centenarians Arba Minch over 80 years old-1335 people.Its total area has been estimated 10,000 km 2 lying within an elevation of 710 to 4200 m above sea level.Te identifcation of the plant sample was authenticated at Arba Minch University, College of Computational and Natural Sciences, Department of Biology.

Preparation of Plant for Extraction. Te fresh leaves of
A. otallensis were well washed with tap water, dried at 20-25 °C, and grinded, respectively.Te fresh roots of A. otallensis were also washed with tap water, dried at 20-25 °C, and grinded, respectively.Te fresh leaf of A. otallensis was cleaned with tap water, and then gel was extracted from the inner leaf pulp and homogenized aseptically using a sterile meal grinder [IKA.A11 BASIC, D-79219 Staufen].Te homogenized gels were squeezed using sterile cheesecloth.Te fltrate was kept in 15 ml of test tubes and was stored at 20 °C until use.Following that, 300 mg/ml of stock solution was prepared with 10% and 10 ml of DMSO solution which was considered as 100% in concentration.

Filtration, Evaporation, and Yield of Extracts.
Te extracts were fltered using Whatman No. 1 flter paper, the fltered extracts were concentrated by using a rotary evaporator, and the residual extracts were dried.Te percentage yield was obtained using dry weight, from equation (1).Te extracts were kept and stored in the refrigerator at 5 °C until use.
where W1 is the weight of the extract residue after solvent removal and W2 is the weight of dried plant powder.

Preliminary Qualitative Phytochemical Screening of Plant
Specimen.In diferent conical fasks, 5 g of powdered leaf and root sample was soaked for 72 hr with 50 ml of methanol, n-hexane, petroleum ether, and distilled water.A phytochemical screening method was used to identify and evaluate the presence of secondary metabolites, including alkaloids, favonoids, phenol, saponins, tannins, steroids, terpenoids, triterpenoids, glycosides, anthraquinones, diterpenes, phytosterols, and phlobannals.Te extracts were fltered.For the crude extracts of methanol, n-hexane, petroleum ether, and distilled water, a phytochemical screening was done.Standard protocols were followed in the screening process to look for secondary metabolites [15,16].

Inoculum Preparation and Preparation of Test
Solutions.Te tested microorganisms were separately cultured on sterilized Muller-Hinton agar (MHA) at 37 °C for 24 hr by using the streak plate method.Ten, well-isolated overnight cultured colonies of the same morphological type were selected from the cultured media.Each colony was touched with a famed wire loop, and the growth was transferred into a sterilized test tube containing 5 ml sterile normal saline solution.Te test tubes that contain the bacterial suspension were vortexed to be mixed well uniformly.Ten, the bacterial suspension was adjusted with 0.5 McFarland turbidity standards.Te adjustment and comparison of turbidity of inoculum tubes were performed by using spectrophotometer reading against 0.5 McFarland turbidity.

Assay of Antibacterial Activity by Agar Well Difusion
Method.Te agar well difusion method expresses the results as the width of the inhibition zone produced by the plant extract [20].Te plant extracts were tested against bacterial strains.For the agar well difusion method, 5 mm size well was prepared in the cultural strain swabbed plates with the help of a well cutter.Ten, 5 µl of plant extracts were added to the well by using a micropipette.5% of dimethyl sulfoxide (DMSO) was carefully added to the well as a control.Ten, plates were incubated at 37 °C for 24 hr.After the incubation period, the zone of inhibition was measured.A well was prepared in the plates with the help of a cork-borer (5 mm) [21].

Determination of Minimum Inhibitory Concentration (MIC) of Plant Extract by Agar Dilution Method.
Te MIC of all crude extracts was evaluated against S. aureus and E. coli.
A 5% DMSO was used to dilute crude plant extracts.Ten, after serial dilution, the crude extract of 2 ml was mixed with molten MHA 18 ml and poured into sterilized Petri dishes.
Te plate was inoculated with the standardized (0.5 McFarland standard) bacterial inoculum and incubated at 37 °C for 24 hr.Te result of bacterial inhibition was judged by comparison with growth in positive and negative controls [22].
International Journal of Microbiology

Assay of Antifungal Activity by Agar Well Difusion
Method.All the fungal isolates were checked for purity and maintained on SDA at 4 °C in the refrigerator until required for use.Antifungal activity of 1 : 1 was tested using the agar well method.Autoclaved distilled water was used for the preparation of fungal spore suspension and transferred aseptically into each SDA plate.Te doses of test extracts were prepared from 5 g/100 ml stock solution from which 30 µl was added to each well.All plates were incubated at 28 ± 2 °C for 24-48 hr, and after incubation, the diameter of zone of inhibition was measured [23].

DPPH Radical Scavenging
Assay.DPPH radical scavenging by A. otallensis plant extracts was estimated according to a previously reported method [24]. 2 ml of DPPH solution in methanol (0.004%, 0.102 mM) was mixed with 2 ml of extracts with diferent concentrations (200-800 mg/l).For blank solution, the extracts were substituted by methanol and used for the correction of the baseline at 515 nm.Te tubes were allowed to stand at 20-25 °C for 20 min.Te antiradical activity was based on the measurement of the reducing ability of the plant extract towards DPPH radical.Ascorbic acid was used as a standard in the range of (25-100 mg/l), and the scavenging efects of the leaf extracts were determined with a linear curve of the ascorbic acid standard.

Determination of Total Phenol by Folin-Ciocalteau
Reagent Method.Total phenol content was determined by the Folin-Ciocalteau reagent method with some modifcation.
From each crude extract, 1 mg was dissolved in 1 ml of methanol.A total of 10% Folin-Ciocalteau reagent was prepared by adding the Folin-Ciocalteau reagent (10 ml) in water (90 ml).Ten, 5% Na 2 CO 3 (3 g) was prepared by dissolving Na 2 CO 3 (3 g) in water (50 ml).200 µl of each crude extract were taken in a test tube, and 1.5 ml of 10% Folin-Ciocalteau reagent was added.Ten, all the test tubes were kept in a dark place for 5 min.Finally, 1.5 ml of 5% Na 2 CO 3 was added to the solutions and mixed well by hand.Again all the test tubes were kept in the dark for 2 hr.Te absorbance was measured for all solution by using a UV spectrophotometer at a constant wavelength of 750 nm.Gallic acid was used as a standard in the range of 2.5-100 mg/l, and the phenol contents of the plant extracts were determined with a linear curve of gallic acid standard [25].

X-Ray Fluorescence Spectroscopy (XRF) Analysis.
XRF analyses were performed using 100 g of the plant sample.Te XRF measuring system consisted of a multichannel analyzer ORTECR, semiconductor detector Si/Li (thickness of beryllium window � 0.25 mm, the diameter of beryllium window � 5 mm), and radionuclide source of radiation 238 Pu (A � 370 MBq, E � 12-22 keV, T � 86.4 years) made by Amersham in the form of the planar disk source.All the measurements were performed in the noncoaxial geometrical arrangement of source, sample, and detector, and the acquisition time was 2000 seconds [26].

Data Analysis.
All the experiments were performed in triplicates to minimize the experimental error, while data were reported as the mean ± SD (n � 3).Statistical analysis of assays results was performed using one way ANOVA, and statistical analysis was performed using the post hoc Tukey test of SPSS 25 statistical software.A p value less than or equal to 0.05 was adopted as the statistical signifcance level.

Results and Discussion
3.1.Percentage Yield of Extracts.Te solvent methanol showed a higher degree of yield in the leaf extract which is 8.45 (22.27%), while the distilled water extract of the leaf showed less degree of yield which is 0.22 g (1.10%) as shown in Table 1.Te solvent methanol showed a high degree of yield in the root extract which is 3.12 g (15.58%), while the petroleum ether extract showed less degree of yield which is 0.42 g (2.1%) as shown in Table 1.Te polarity of the various compounds that are found in the plant causes variations in the extraction yield, which have been reported in the literature on medicinal plants from Vietnam [27].To generate extracts with acceptable yields and potent antibacterial activity, solvent selection is a crucial step.

Phytochemical Screening.
Te phytochemical analysis of the A. otallensis gel extract demonstrates a notable presence of alkaloids, phenols, saponins, tannins, steroids, terpenoids, glycoside, anthraquinones, and phlobatannals.Te methanolic extract of the root contained glycoside, diterpenes, phytosterols, and phlobatannals, while the methanolic extract of the leaf showed the presence of triterpenoids, alkaloids, tannins, and glycoside.A photochemical analysis shows that the petroleum ether extract of the root included a presence of favonoids, saponins, glycosides, anthraquinones, diterpenes, and phytosterols, while the petroleum ether extract of the leaf contained alkaloids and anthraquinones.According to a photochemical analysis, terpenoids, anthraquinones, diterpenes, and phytosterols were remarkably found in n-hexane extract of the root, whereas alkaloids, favonoids, saponins, terpenoids, triterpenoids, and anthraquinones were detected in the n-hexane extract of the leaf.Likewise, the photochemical analysis indicates a notable existence of saponins, glycosides, anthraquinones, and terpenes in the distilled water extract of the root, whereas the distilled water extract of the leaf contained saponins, tannins, terpenoids, and anthraquinones.Table 2 shows the phytochemical composition of each extract.
It is known that the photochemical substances that were identifed have medicinal uses.Alkaloids are one such example of a potent toxin.Numerous alkaloids that are extracted from medicinal plants exhibit biological activity such as cytotoxicity, anti-infammatory, antimalaria, antimicrobial, and pharmacological properties [28][29][30][31].Likewise, plant-based steroids are recognized to have cardiotonic properties in addition to their antimicrobial and insecticidal attributes.Since their biological actions are widely established, they are frequently employed in therapeutics.Research has shown that tannins possess antiviral, anticancer, 4 International Journal of Microbiology and antibacterial properties [32].Congestive heart failure and cardiac arrhythmia have been treated with additional phytochemicals known as cardiac glycosides [33].Te biological activity exhibited by A. otallensis and the reason behind its use in traditional medicine may be attributed to these phytochemical substances found in the gel, root, and leaf extracts.Diferences in antimicrobial activity of medicinal plants are obviously related to diferences in their contents of active compounds [34].Available reports tend to show that alkaloids and favonoids are the responsible compounds for the antimicrobial activities in higher plants [35].Moreover, it is also claimed that secondary metabolites such as tannins and other compounds of phenolic nature are classifed as active antimicrobial compounds [36].Te primary bioactive components and antimicrobial agents found in plants are favonoids, phenolic compounds, tannins, and alkaloids [37].Some bioactive compounds inhibit microorganisms from carrying out their biological functions via altering their biochemical systems, attaching to their protein molecules, serving as chelating agents, or inducing cell infammation [38].According to [39], several plant extracts have been found to be inefective against specifc test organisms at lower concentrations; this may be because the extracts contain fewer antimicrobial agents.Consistent with our fndings, [40] found that the S. aureus ATCC25923 strain is susceptible to both aqueous and ethanol extracts in their research on Matricaria pubescens.

Minimum Inhibitory Concentration (MIC) of Plant
Extracts.Crude plant extract was serially diluted using 5% DMSO for the MIC test.A MIC calculation was performed for extracts that displayed a diameter of at least 6 mm within the growth inhibition zone at a concentration of 25 mg/ml.Te reference bacterial strains were treated with crude extracts of direct gel extract, root, and leaf extracts with different solvents' (i.e., methanol, chloroform, n-hexane, and distilled water) results are shown in Table 4. Te antibacterial International Journal of Microbiology activity was considered as signifcant when the MIC was less than 100 μg/mL, moderate when the MIC was between 100 and 625 μg/mL, and low when the MIC was greater than 625 μg/mL [41].Te results of MIC value of direct gel extract with leaf methanol and leaf petroleum ether noted the signifcant diference against S. aureus at (p � 0.0072) and (p � 0.0151), respectively.Te root extract of methanol and distilled water indicated positive association at (p � 0.0465), and also, the root extract of n-hexane and leaf methanol showed positive relationship at (p � 0.0262) against S. aureus.No signifcant diference was observed between direct gel and leaf petroleum ether extract on S. aureus at (p � 0.0787).On the other hand, MIC of direct gel extract and leaf methanol extract showed a signifcant diference at (p � 0.0200) and direct gel extract with leaf n-hexane extract at (p � 0.0052) against E. coli, respectively.Similarly, root distilled water extract and leaf methanol extract noted a signifcant relationship at (p � 0.0025) and also root distilled water extract with leaf n-hexane at (p � 0.0005) against E. coli, respectively.Unfortunately, the MIC of direct gel extract with root methanol extract and leaf petroleum ether extract with root n-hexane had showed no signifcant diference at (p � 0.6416) and (p � 0.7174) on E. coli, respectively.Te results of the MIC determination test showed that diferent plant parts had varying minimum concentrations of the crude extract that might inhibit the reference bacteria's growth.Te plant's antibacterial activity against the test bacterium lines up with the MIC value.Tis fnding was corresponding with the earlier report of Tadele [42], who reported 5 mg/ml against S. aureus.MIC that is extremely low indicates signifcant antibacterial activity [43].Tis is consistent with our fndings because [44] reported that the MIC of extracts against various strains varied depending on the strains tested and the extract in question.

Antifungal Activity of A. otallensis.
Antifungal activity of the A. otallensis root, leaf, and gel was checked against fungi (dandruf).During antifungal activity test, a signifcant diference was observed between gel and root extract at the (p � 0.0063), but there was no signifcant diference between leaf extract with gel and root extract at (p � 0.17).Te result as expressed in Table 5 showed that the root has no efect on fungi (dandruf).A diferent study reported that Aloe species possesses antifungal activity.Aqueous extract of A. otallensis can be used to treat infections from fungi (dandruf).
Our study investigated the antibacterial and antifungal activities of Commelina difusa Burm.F. extracts from diferent plant parts using various solvents.We observed signifcant variations in activity against the same microorganisms, which can be attributed to diferences in plant part composition and extraction solvents.Diferent plant parts (leaves, stems, and roots) contain varying concentrations of bioactive compounds.Extraction solvents also play a crucial role, infuencing which compounds are extracted and their overall antimicrobial efcacy.For example, polar solvents like methanol extract a wider range of compounds compared to nonpolar solvents like hexane, impacting antimicrobial activity diferently.

Antiradical Activity of Plant Extracts.
Te DPPH radical scavenging activities of the plant extracts were estimated by comparing the percentage scavenging activity of the DPPH with a standard, ascorbic acid Figure 1.In the present study, DPPH, a stable free radical with a characteristic absorption at 515 nm, was used to study the radical-scavenging efects.Ascorbic acid was used as a standard since ascorbic acid is considered to be a strong antiradical due to its ability to scavenge free radicals and bind transition metal ions.
Te results of DPPH free radicals scavenging are presented in Figure 2. Te decrease in absorption is taken as a measure of the extent of radical scavenging.Te radicalscavenging activity values were expressed as the ratio percentage of sample absorbance decrease and the absorbance of DPPH solution in the absence of extract at 515 nm.

International Journal of Microbiology
A. otallensis plant parts were proved to be inhibiting the DPPH free radical scavenging activity with IC 50 value as shown in Figure 2. Tis means that it shows considerable antiradical activity in quenching the free radical scavenging of DPPH.
In general, among all the plant extracts, the crude extracts obtained from A. otallensis gel extracts showed the best antiradical activity, and on the other hand, A. otallensis leaf and root extract shows lower antiradical activities, respectively.A correlation between the total phenolic content and antioxidant activity has been found by some authors [45].According to [46], antioxidant substances may have potent metal chelation activities.Because of their redox characteristics, they quench singlet and triplet oxygen, donate hydrogen and function as reducing agents [47].Te extract's antioxidant content, as well as the interactions and structures of these molecules, determine its antioxidant capacity [48].Te inhibition efect of diferent concentrations of the plant extract and ascorbic acid in scavenging of DPPH was evaluated.Te IC 50 values of the plant extracts are presented in Figure 3.
In general, among all the plant extracts, the crude extracts obtained from A. otallensis gel extracts showed the highest IC 50 , and on the other hand, A. otallensis leaf and root crude extract shows lower IC 50 , respectively.

Total Phenolic Content of Plant Extracts.
Te content of phenolic compounds in plant extracts of A. otallensis was determined from regression equation of calibration curve of gallic acid Figure 4 and expressed as milligrams, the equivalent of gallic acid per gram of dry extract (mg GAE/g) in Table 6.
Te total phenolic content calculated in this study is presented in Table 6.Te highest value of phenolic content was obtained in A. otallensis gel methanol extract followed by leaf extract, while A. otallensis root methanol extract shows lower phenolic content as shown in Table 6.
Strong chain-breaking antioxidants such chemical compounds called phenol are known to have direct efects on antioxidant activity [49,50].Tese phenolic compounds contribute to antioxidant activity due to the arrangement of functional groups (hydroxyl) in their nuclear structure for hydrogen donation to stabilize radical molecules [51].
In the leaf extract of A. otallensis, the concentrations of various elements were also determined as follows: Potassium (K) was found to be 3136.27  Te root extract of A. otallensis was analyzed for its elemental composition, revealing signifcant concentrations of various elements.Calcium (Ca) was found to be the most   7.
Tese fndings provide insights into the elemental profle of A. otallensis root extract, highlighting its potential nutritional and medicinal properties based on the presence of these elements.Te mineral composition of the A. otallensis plant revealed the presence of all the mineral elements, and the interaction of trace minerals composition present in the medicinal plants has great importance to understand their functions in the human body.

Conclusions
Te choice of the extraction method and solvent must be carefully considered in order to maximize extract yield and bioactivity.Te percentage yield of A. otallensis plant extract with various solvents has been compared, and among those solvents, methanol yields higher than other solvents of A. otallensis plant parts.Due to its high content of secondary metabolites, A. otallensis is used in traditional medicine to treat and prevent infections.Te antibacterial assay employed in this work revealed that E. coli was more susceptible to the plant extracts than S. aureus.Te minimum inhibitory concentration of gel showed list than root and leaf plant extract of A. otallensis against S. aureus and E. coli.Compared to other extracts, gel extracts exhibited superior MIC activity and strong antibacterial activity with antibiotics.Te study's fndings showed that the A. otallensis gel plant part's methanol extracts made using maceration techniques had a high antiradical and phenolic content and included a variety of three macro, eight micro, and one heavy metal components.

Figure 4 :
Figure 4: Total phenolic content for standard gallic acid, values expressed in terms of gallic acid.
[19] of Saponins.3ml of the extract in a test tube was mixed with 5 ml of distilled water and shaken vigorously for 2 minutes.Te formation of stable form or froths established the presence of saponins[19].
2.4.3.Test of Phenols. 1 ml of the aqueous extract and 3-4 drops of 5% FeCl 3 (w/v) were added.Formation of the bluish black color indicates the presence of phenol.

Table 3
Activity of A. otallensis.Te result of antibacterial evaluation of the diferent parts of A. otallensis is presented in at (p � 0.004) but no signifcant diference observed between direct gel extract with leaf methanol extract at (p � 0.072) and leaf petroleum ether extract at (p � 0.374) on S. aureus.

Table 1 :
Percentage yield of A. otallensis leaf and root extracts.

Table 2 :
Preliminary phytochemical compounds in A. otallensis plant from diferent extracts.
Key: a � mean values from triplicate, _b � No MIC.

Table 3 :
Antibacterial activity of A. otallensis plant extracts.

Table 6 :
Total phenolic content (mg GAE/g) of methanol extract of A. otallensis.