Analysis of the Antibacterial Activity and the Total Phenolic and Flavonoid Contents of the Moringa oleifera Leaf Extract as an Antimicrobial Agent against Pseudomonas aeruginosa

Pseudomonas aeruginosa is a bacterium that causes metal deterioration by forming biofilms on metal surfaces. This work was carried out to analyze the antibacterial activity and the phenolic and flavonoid contents of the Moringa oleifera leaf extract against Pseudomonas aeruginosa. M. oleifera leaves were extracted in a methanol solution at different concentrations. The M. oleifera leaf extract yields were 12.84%, 18.96%, and 19.64% for the 100%, 75%, and 50% methanol ratios, respectively. Extracts of M. oleifera leaves had a minimum inhibiting concentration (MIC) of approximately 6144 μg/mL against P. aeruginosa for a ratio of 100% methanol. In addition, no antibacterial activity was found for the 75% and 50% methanol ratios. The total phenolic levels were 16.26%, 12.73%, and 12.33% for the 100%, 75%, and 50% methanol solvent ratios, respectively. The total amounts of flavonoids were 23.32%, 3.40%, and 0.64% for the 100%, 75%, and 50% methanol solvents, respectively. The chemical structure of M. oleifera consists of kaemferol-3-O-rutinoside, quercimeritrin, kaempferol-3-O-β-D-glucopyranoside, stearidonic acid, trichosanic acid, pyrophaeophorbide A, and stigmastan-3,6-dione. The concentration of the solvent is essential in the extraction of plant constituents. Different concentrations indicate differences in antibacterial activity, phenolic and flavonoid contents, and chemical structure.


Introduction
Te main bacterium in the marine environment is Pseudomonas aeruginosa, which can produce a bioflm layer on metal surfaces [1].Te reaction between the surface metal and bioflm layer and the diferential aeration cell formation on the metal surface generate conditions that initiate and accelerate the corrosion rate [2].Tis type of bacteria in bioflms can cause severe corrosion damage to steel [1] and other metals [3][4][5].
Microbiologically afected corrosion is a form of destructive corrosion that is started, aided, and facilitated by the presence of microbe activities [6,7] and most generally manifests in holes localized on the surface material [8].Bacteria attach to the substrate and form a bioflm layer, creating conditions that promote metal corrosion [9].Te bacteria inside the bioflm accelerate and create diferential oxygen, resulting in corrosion and severe material damage [10].Terefore, a comprehensive strategy is needed to overcome these bioflm-forming bacteria.
Te creation of various bioflm layers on the metal substrate by bacteria results in the formation of diferential aeration cells, which causes local corrosion [11].Te cathode is above the steel substrate and enriched with oxygen, while the anode is underneath a bioflm layer and lacks oxygen.Te diference in the oxygen concentration between the anode and the cathode activates the electrochemical cells, resulting in localized corrosion in the form of pitting or crevices [12].P. aeruginosa strains have also been linked to the oxidation of iron to ferrous iron (Fe 2+ ), which exposes the steel to further dissolution because iron ions are more soluble than iron.Tis process attacked the protective layer of the steel surface [13].
Generally, the major bacterial corrosion mechanism has been classifed into the following three phases [14]: (a) diferential aeration cells caused by the formation of bioflms, which cause corrosion damage; (b) a reaction between a steel substrate and exopolysaccharide polymer substrate (EPS), in which EPS metal biominerals act as corrosion inducers; and (c) the role of siderophore bacteria in iron reduction.Corrosion control techniques due to bacteria are carried out physically and chemically; physically through regular cleaning of mucus and deposits in pipes [15], while chemically using biocides (chemical reagents) [16,17].Te limit for chemical biocides is that they are toxic and ecologically unfriendly [18].Recently, researchers have developed an ecofriendly biocide to address this issue [19][20][21].
Plants are a rich source of many active compounds, primarily secondary metabolites with antibioflm, antifungal, and antibacterial functions [21].Researchers have reported that some extraction plants are antimicrobial active and are used to control corrosion [22][23][24].Moringa oleifera extracts have been studied for mild steel corrosion as a potential corrosion inhibitor in acidic environments [25].Te M. oleifera extract may also act as a resistance modifer, increasing the efectiveness of various antibiotics against certain bacteria [26].As a result, the M. oleifera extract has the potential to inhibit bacterial growth in various environments.
Active compounds such as favonoids and phenols have been identifed to help inhibit the formation of bioflms [27,28].Terefore, an approach to inhibit the formation of bioflms involves identifying or extracting active compounds that act as inhibitors of bioflms.Maceration is one of the simple and inexpensive techniques for extracting the active substance from plants [29].In addition, maceration is recommended as an extraction method with diferent solvents to extract high-quality antioxidant raw materials from the M. oleifera leaf to obtain a total phenolic and a maximum of favonoids [30].Te M. oleifera methanol extract shows more antioxidant and antibacterial activities against foodborne pathogens than diethyl ether extracts [31].Tese phenolic and favonoid compounds have the potential to disrupt the bacterial cellular system by inactivating their receptors in the bacterial signaling pathway [27].
Tis study focused on extracting phenolic and favonoid compounds and their antibacterial activities against P. aeruginosa.M. oleifera plants were extracted at room temperature in various methanol concentration ratios.Te favonoid and phenolic contents in the extract were analyzed by the colorimetric aluminum chloride and Folin-Ciocalteu methods.In addition, extracts were assessed for their suitability as antimicrobials to inhibit P. aeruginosa.M. oleifera extracts were also evaluated for their polarity structure and distribution with liquid chromatography-mass spectrometry (LC-MS/MS) and thin layer chromatography (TLC).To the best of our knowledge, no M. oleifera extract has been reported as a microbial corrosion or biocide inhibitor against bioflm-forming bacteria.Tus, this study is the frst time as new green biocidal materials for antibiocorrosion additives would constitute new information.Te results of this research are expected to serve as initial guidelines before extracts are used as antibioflms and biocorrosion.

Preparation of the Extract.
Te leaves of M. oleifera were carefully cleaned with running water to eliminate soil particles and dust.Ten, this M. oleifera leaf was dried under the scorching sun for three days.M. oleifera dried leaf was ground to a size of <60 mesh.25 g of M. oleifera powdered leaf was added to 150 mL of solvent with ratio concentrations of 100%, 75%, and 50% (v/v) methanol water, respectively.Te extraction technique was performed by macerating at room temperature for 3 × 24 hours, and a new solution was replaced every 24 hours.After maceration, fltering is carried out and then concentrated in a rotating evaporator at 50 °C.Finally, the M. oleifera leaf extract was refrigerated until further processing.Te result of the extraction is determined according to the following formula: where W 1 represents the fnal weight of the M. oleifera leaf extract (concentrated extract) and while W 0 represents the initial weight of M. oleifera dried leaf (M.oleifera leaf powder).Te oblique bacteria culture was moved to the freshwater brain heart infusion (BHI) broth media and then transferred to the marine BHI broth media as a test material.

Antibacterial Test.
Te antibacterial activity and minimum inhibitory concentration (MIC) were assessed using the MTT methods [32,33].Te media was prepared in accordance with the manufacturing instructions.37 g of BHI were suspended in 1000 mL of distilled water and then mixed, heated, and boiled for 1 minute until fully dissolved.
It is then autoclaved at 121 °C for about 15 minutes.Furthermore, artifcial seawater was prepared with the following manufacturing instructions: 38 g of powders (Marine Art SF-1) were dissolved into 1000 mL of distilled water, followed by heating and mixing.Subsequently, the solution was autoclaved for 15 minutes at 121 °C.In summary, an aliquot of 100 μL (BHI-artifcial sea water in a ratio of 1 : 1) consisting of diferent concentrations of the extract was added to each plate of 96 wells.Te extract concentration varied from 0 μg/mL and 512 μg/mL up to 6144 μg/mL for this study.Each tube well is flled with a suspension of bacteria cells (2 μL) from a 24-hour culture.Microplates are incubated for 24 hours at room temperature.Furthermore, 10 μL of MTT solution (containing 5 mg/mL) was flled into the tube well and incubated for 1 hour.Te well was then flled with 10 μL of MTT solution (5 mg/mL) and incubated for 1 hour.Afterwards, each tube well was flled with 100 μL of propan-2-ol containing 0.04 M of HCl.A microplate reader measured cell suspension absorption at 595 nm (Bio-Rad xMark).All experiments were carried out in three replications.Te percentage of inhibition against viable cells was determined using the following equation [30]: where Abs t and Abs c are cell absorbance treated and cell absorbance control, respectively.

Total Phenolic and Flavonoid
Analysis.Te total phenolic compounds in the extract were estimated to follow the previously reported [34,35] using Folin-Ciocalteu methods with gallic acid as reference.Te sample solution and the reference solution of gallic acid are each placed into a test tube and then dried into 4 mL by adding aquades, Folin-Ciocalteau 250 μL, and shaken.After 8 minutes, 750 μL of 20% Na 2 CO 3 was added and shaken homogeneously.Tis mixture is then left for 2 hours at room temperature.Absorption was read by using a spectrophotometer at 765 nm.Te total favonoid compounds were analyzed by the colorimetric aluminum chloride method [36] with quercetin as a standard solution.4 mg of each quercetin was used, and the extracted sample was dissolved in 4 ml of methanol.Pipetting up to 250 μL of sample into the test tube allowed for accurate measurements.2 ml of aquades and 150 μL of 5% NaNO 2 were added to each test tube.150 μL of 10% AlCl 3 was added after 5 minutes.Six minutes later, 2 ml of NaOH 1 M was added, and the volume was calibrated to 5 mL with the addition of aquades.Te mixture was homogenized and measured using a UV-Vis spectrophotometer at 510 nm.

Analysis of Structural Compounds.
Te shape and active compounds in the M. oleifera leaf extract were determined using structural analysis.LC/MS-MS was used to examine the compound structure of the M. oleifera extract (Agilent Technologies 7890).For 17 minutes, structural compounds were measured using LC-MS-MS at 0.3 mL/min of fow rate.

Results and Discussion
Samples of M. oleifera leaves (Figure 1) used in this study have been identifed at the Botanical Laboratory (Herbarium Bogoriense) under the Directorate of Scientifc Collection Management, National Research and Innovation Agency, BRIN, with certifcate number B-1809/II.6.2/DI.05.07/6/ 2022.Te following are the results of determining the M. oleifera plant used in this work, as tabulated in Table 1.

Yield Extracts.
Te yield of M. oleifera leaf (25 g) with various methanol solvent ratios was 12.84, 18.96%, and 19.64% for 100%, 75%, and 50%, respectively (Table 2).According to Table 2, solvents with a higher polarity (i.e., a larger ratio of water solvents) extract more signifcant quantities.Conversely, extracts with a lower solvent polarity (100% methanol) also have a lower percentage of the extract.Fewer polar compounds can be extracted with a methanol ratio of 100%.In the maceration of the plant, the breakdown of cell walls and membranes was caused by pressure differences, so the secondary metabolites in the cytoplasm will be dissolved in the organic solvent [37].In addition, the maceration process can be carried out without heat, providing that the secondary compounds to be observed were not damaged [38].
Tese results are consistent with the recent report, which demonstrated that aqueous solvents produce extracts higher than methanol solvents [39].Other researchers reported that extracting M. oleifera leaves (1.5 g) with diferent polarity solvents resulted in dry extracts with a weight of 5%-36% (M.oleifera leaf hexane extract) [26].Hexane, ethyl acetate, and chloroform extracts generally produced a relatively low Scientifca mass of extracted material compared to water and methanol extracts [27].Hence, the type of solvent is critical in extracting the active content from the plant [28].
Te use of methanolic solvents in maceration was based on the fact that methanolic has a boiling point of 65 °C and a polarity index of 5.1.In comparison, ethanol has a boiling point of 78 °C and a polarity index of 4.9 [40].Terefore, the evaporation process uses a rotary evaporator to obtain a viscous extract using methanol faster than ethanol.Te temperature for evaporating the sample with methanolic solvent is not very high, minimizing the risk of overheating and destroying the secondary metabolite content in the sample.Consequently, the processing time needed to evaporate the sample is relatively quick.Furthermore, extraction studies using various solvents revealed that methanolic is the best solvent for extracting bioactive contents with the highest extract yield [41].

Tin Layer Chromatography (TLC)
. TLC identifcation results showed polarity behavior with an increase in the ratios of ethyl acetate at the ratios of extracts of 100% methanol and methanol 75% and 50%.Tis diference is due to the higher polarity of ethyl acetate solutions with respect to n-hexane solutions [40].Te chromatogram pattern resulting from the extract on the TLC plate was examined under visible light and UV light at 366 nm and 254 nm of wavelengths, as shown in Figure 2. Figure 2 shows the polarity spot distribution of the active metabolite in the M. oleifera leaf extract and the ratio efect of methanol solvents in the variation of n-hexane: ethyl acetate as the mobile phase.
Te value of the retention factor, R f (ratio of distance traveled by the substance to the solution), is an important parameter used for qualitative TLC analysis [42].Te two components are similar molecules if two points travel the same distance or have the same R f value.In this study, qualitative tests for all the three extract ratios revealed similar indications of metabolite.TLC profling of all the three extract ratios yielded impressive results, indicating the presence of several phytochemicals.In diferent solvent systems, diferent phytochemicals have diferent R f values [43].Pure compounds can be separated from plant extracts using diferent ratios of solvents with variable polarity.Only by analyzing the R f value of the compound in diferent solvent systems can the correct solvent system for a specifc plant extract be determined [43].Tese fndings will aid in selecting an appropriate solvent system for further compound separation from this plant extract.

Antibacterial Activity.
Te results of the antibacterial activity test of the M. oleifera leaf extract with variations in concentration are shown in Figure 3. Analysis of optical density data obtained for all experiment repetitions showed the average OD value (± standard deviation) against the control.
Te microdilution method determined the minimum inhibitory concentration (MIC) of the M. oleifera leaf extract.MIC is defned as the minimum concentration required to inhibit the bacterial growth.At a methanol concentration ratio of 100%, the initial inhibition of bacteria is indicated by a brighter color than other concentrations (75% and 50% of the methanol solvent) in the 6144 μg/mL concentration extract.At the same time, the M. oleifera leaf extract could not inhibit the growth of P. aeruginosa (dark blue color) at a concentration of 75% like at a methanol concentration of 50%.Te indicator of minimum inhibition was observed by the degradation of the blue color of the MTT seawater to be lighter.
Te test results confrmed that the M. oleifera leaf methanol extract has antibacterial activity against the bacteria tested (P.aeruginosa) with tabulated absorbance in the graph in Figure 4. Te percentage of the antibacterial activity of M. oleifera leaf extract against P. aeruginosa is presented in the graph in Figure 5.At a ratio of 100%, a concentration of 6144 ug/mL of the M. oleifera leaf extract can inhibit the growth of P. aeruginosa by about 43.84%; at the same  4 Scientifca concentration (6144 ug/mL), M. oleifera extracts only inhibit the growth of P. aeruginosa by 27.36% and 4.76% for the ratios of 75% and 50%, respectively.Te ability of active content from the M. oleifera leaf extract against P. aeruginosa was investigated in this study.Te antibacterial activity tests show that the M. oleifera leaf extract can inhibit bacterial growth, although it is still relatively low.Another report found that the M. oleifera leaf extract had varying antimicrobial activity in various microorganisms [44].Bacterial growth inhibition based on extract concentrations implies that increasing the concentration of the methanol extract increases inhibitory absorption.Tese fndings support previous research that M. oleifera leaf powder has antibacterial activity against negative bacteria at a low level [39].Kumar et al. [45] reported that the M. oleifera extract with the dilution method has an antibacterial activity with a minimum inhibitory concentration of 7.4 mg/mL and 2.4 mg/mL for S. aureus and E. coli, respectively.
Another researcher confrmed that the M. oleifera crude extract had no inhibition against P. aeruginosa ATCC 27853 at 175 μg/mL of concentration in the purifcation and characterization of phytocystatin isolated from M. oleifera [46].In addition, plants in various solvents showed diferent activities against P. aeruginosa bioflms [28].Another work found that the efcacy of 10 mL of the M. oleifera seed extract can cause Gram-positive (B.subtilis) and Gramnegative (E.coli) bacterial decay to a maximum of 93.2% and 96.2%, respectively [47].Te inhibitory activity of plant extracts against bacteria varies depending on the type of plant extracted [48,49], the method and solvent used [49,50], the type of target bacteria [39], and the content of active compounds in the extract [27].
Te efectiveness of this inhibition against bacteria was caused by bioactive contents in the extract, which can damage cell walls (DNA) so that their growth slows down and even causes bacterial death [50].Te inhibition of DNA replication will cause bacteria to be unable to divide themselves, thus inhibiting the growth of bacteria.Other  Scientifca sources stated that favonoids are one of the essential secondary metabolites identifed as potential antimicrobial agents against various pathogenic microorganisms [51].
Flavonoids have an antibacterial efect due to their many biological actions, which may seem not very specifc initially.However, prospective antibacterial favonoids efectively target bacterial cells and inhibit virulence factors and other types of bacterial risks, such as bioflm formation.

Total Phenolic and Flavonoid Contents.
Te efect of the ratio of methanol to the bioactive contents of the M. oleifera leaf extract is presented in Table 3. Tis study revealed a considerable diference in the percentage of bioactive compounds (favonoids) to the ratio of methanol solvents in M. oleifera extracts.Te resulting equation of the fault acid reference curve is y � 0.1032x + 0.0708; r 2 � 0.9996.Te total values of the phenolic content (TPC) are about 16.26%, 12.73%, and 12.33% for methanol ratios of 100%, 75%, and 50%, respectively, as described in Table 3. Te total value of the percentage of the phenolic content decreases ramps with a decrease in the solvent ratio of methanol.Tis reduction may be due to methanol attracting polar and nonpolar compounds while water attracts only polar compounds.Our results aligned with Elboughdiri [52], which found that the total phenolic content (TPC) decreased rampantly when the solvent concentration ratio was lowered.Te phenolic content also depends on the plant organs [53].For example, a study of extracts on diferent parts of M. oleifera reported that the total phenolic content was greater in the leaves than in other organ parts (whole seeds, kernels, mantle, and pods) [54].In addition, the phytochemical properties (phenolics and favonoids) of the M. oleifera leaf extract were also signifcantly afected by leaf age [49].
Meanwhile, in the standard curve of quercetin, the linear equation is y � 0.0073x − 0.0802 with r 2 � 0.995.According to the results, the TFC extract values were 23.32%, 3.40%, and 0.64% for methanol ratios of 100%, 75%, and 50%, respectively.Te TFC value of the M. oleifera extract difers markedly by diferent methanol ratios.Biochemical studies of the M. oleifera leaf extract reported that favonoids were obtained with methanol solvents [55].In addition, the signifcance of secondary metabolites in inhibiting P. aeruginosa bioflms was revealed by a strong connection between the favonoid concentration and antibioflm activity in the methanol extract [28].6 Scientifca Based on the total content of phenolics and favonoids, optimal conditions were obtained at a methanol ratio of 100% to extract bioactive components from the M. oleifera leaf.Te highest total phenolic content (about 16.26%) and favonoids (about 23.32%) were obtained in these conditions.At the same time, a 50% methanol solvent shows lower    Scientifca efciency in extracting phenolic compounds and favonoids by 12.33% and 0.64%, respectively.Tese results are confrmed by the data of the bacterial activity test results that the highest inhibition was obtained at a concentration ratio of 100% methanol (Figure 5).

Te
Structure of the M. oleifera Leaf Extract.Te chromatogram results with LC-MS/MS are presented in Figure 6.
Tese results illustrate the diference in the compound content of the methanol extract of M. oleifera leaf at diferent concentration ratios.Tis diference in active compounds is explained by the peak chromatograms of compounds of diferent molecular weights.Te bioactive content of all methanol concentration ratios has the same compound, trichonic acid (C 18 H 30 O 2 ), at a retention time of 9.31.Te results of molecular weight analysis with LC-MS/MS showed that the active compound was found at methanol ratios of 100%, 75%, and 50%, as shown in Figure 6. Figure 6 presents various compounds with varying degrees of peak intensity.In the compound at retention times 0.53, 0.68, 2.75, 3.26, 3.48, and 7.10, the peak intensity increases with a decrease in the methanol concentration ratio.In contrast, in the compound at a retention time of 9.31, 10.10, 10.42, and 12.89, peak intensities tend to get lower with a decrease in the concentration ratio of methanol solvents.Te polarity of the extracted active compounds can cause diferences in the intensity of such compounds.Te compounds in the 50% methanol extract may be more polar than those in the 75% and 100% ratio.While these compounds are only found in 100% methanol, such compounds tend to have a lower polarity than compounds at their other  8 Scientifca    7.
Te development of bioactive compounds in some plants can be used for various purposes, both in the medical feld and in others.Due to the bioactive content in the M. oleifera plant, this plant has the potential for antimicrobial and antioxidant activity.M. oleifera consists of a large number of secondary metabolites [57].Nizioł-Łukaszewska [59] concluded that the tested M. oleifera leaf extract contained high favonoid and phenolic compounds and antioxidant potential and positively afected cell proliferation and metabolism at concentrations up to 5%.According to other sources, the phytochemicals of M. oleifera had a high amount of terpenoids, tannins, favonoids, saponins, glycosides, alkaloids, and phenolic content [58].In our study, the result of the structure of the extracted compound of M. oleifera is polyphenol glycoside (favonoid glycoside), polyunsaturated fatty acid (PUFA), and alkaloid compounds, as shown in Figure 7. Flavonoids provide antibacterial activity by inhibiting nucleic acid production, cytoplasmic membrane function, energy metabolism, adhesion and bioflm development, suppression of porins in cell membranes, alterations in membrane permeability, and pathogenicity [60].Meanwhile, the antibacterial activity of polyphenols mostly depends on their interaction with the bacterial cell surface [61].

Figure 1 :
Figure 1: M. oleifera leaf was used in this work.

Table 1 :
Te determination results for the plant material used in this work.

Table 2 :
Te yield of M. oleifera extracts in 3 diferent ratios of methanol solvents.

Table 3 :
Te phenolic and favonoid contents in the M. oleifera leaf extract in various methanolic solvents.

Table 4 :
Te name of the active content component in the M. oleifera leaf extract.