Antibacterial, antioxidant, cytotoxicity, and phytochemical screening of Moringa oleifera leaves

Antibiotic-resistant bacterial infections increasing from acquired resistance and/or during biofilm formation necessitate the development of innovative ‘outside of the box’ drugs. Plant-based natural products have historically been used in pharmaceuticals, food, and cosmetics industries and are documented in many countries. Ancient civilizations used these items as plant food and as a source of medical therapy¹. However, in recent decades, there has been a significant increase in the interest of researchers to investigate the details of their compositions and to investigate and establish their potential applications in a variety of fields. We should employ the fundamental power of nature to address the proliferation of diseases such as cancer, diabetes, obesity, heart attacks, microbial infections, accelerated skin aging, and forthcoming varieties of new alarming health concerns². Using natural materials has advantages over medication derived from synthetic sources. Due to their minimal adverse effects, this approach is usually chosen as the most beneficial when comparing the toxicological and pharmacological activity of these compounds with those of medications derived from chemical sources. A huge pharmacological activities of natural product extracts have been discovered, such as antibacterial, antioxidant, antidiabetic, anti-inflammatory anti-aging, cardio-protective, neuroprotective, immunomodulatory, antiparasitic, and antiviral¹. Various plant parts, including the seeds, roots, stem, bark, leaves, flowers, and fruits, each contain unique phytochemical compositions and perhaps medicinal benefits.

Moringa oleifera known as the drumstick tree belongs to the genus Moringa, Moringaceae family³. There are several species of Moringa throughout the world that are renowned for their range of applications. Because of its many medical benefits, the Moringa oleifera is regarded as one of the magical plants⁴. Moringa oleifera’s antimicrobial components have been utilized to treat a variety of bacteria. Aqueous extracts of Moringa oleifera were shown to have antibacterial action against various harmful bacteria, including Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli⁵. Sayeed et al.⁶, showed that the fruit extract of Moringa oleifera exhibits a broad-spectrum activity against many microbes. The flavonoids extracted from Moringa oleifera seed exhibited antimicrobial and antibiofilm activities against biofilm-forming Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans⁷. Moringa oleifera extract formulation led to the healing of wounds infected with both P. aeruginosa and MRSA⁸.

Moringa species especially Moringa oleifera are high in protein and important amino acids, including tryptophan, methionine, cysteine, and lysine. These amino acids are essential for many body activities, including protein synthesis, enzyme manufacturing, and general growth and development. Moringa has a wide range of phytochemicals. These include flavonoids, which are known for their antioxidant and anti-inflammatory effects; terpenoids, which have been shown to have anti-cancer and anti-microbial properties; tannins, which are effective in reducing inflammation and fighting infections; anthocyanins, which are known for their antioxidant and anti-inflammatory benefits; and proanthocyanidins, which contribute to cardiovascular health and have strong antioxidant properties. These chemicals provide Moringa with powerful antioxidant, antigenotoxic (DNA-protective), and immunostimulant (immune-boosting) characteristics, making it an important plant for improving health and avoiding illness^9,10,11. Furthermore, Moringa has a high concentration of vital nutrients. It includes a diverse spectrum of minerals, including phosphorus (P), nitrogen (N), potassium (K), zinc (Zn), calcium (Ca), iron (Fe), and magnesium (Mg), all of which are essential for body activities such as bone health, oxygen transfer, enzyme activation, and muscle function. Moringa contains several antioxidants, including quercetin, chlorogenic acid, gallic acid, vanillic acid, syringic acid, ferulic acid, coumaric acid, and sinapic acid. These antioxidants serve to battle oxidative stress, decrease inflammation, and protect cells from harm. Additionally, Moringa contains high-quality proteins that are required for muscle repair, immunological function, and general growth and development^10,12. Flavonoids and phenolic acids are the most efficacious polyphenols. The highest antioxidants are flavonoids, which neutralize ROS production, inhibit various enzymes, and chelate all metals used in radical chain reactions^13,14,15,16. The flavonoid quercetin was discovered to be the most dominant component, with a well-known ability to bind numerous metal ions (Cu and Fe) and limit free radical production. Phenolic acids provide a range of beneficial pharmacological properties such as antimutagenic, antidiabetic, antioxidant, anti-inflammatory, and antimicrobial¹⁷. The potent antioxidant properties Moringa oleifera leaf extract exhibits a linear relationship with phenols, including ferulic acid, which is involved in the protection of lipids from peroxidation, the scavenging of free radicals, and the binding of iron and copper^18,19. This drumstick tree is well-known in conventional medicine for cancer treatment²⁰, with anti-inflammatory effects²¹, and antidiabetics²². Moringa oleifera is a valuable source of secondary metabolites and has made significant contributions to therapeutic, biological, and pharmacological qualities³. Modern computational tools, such as ADMET lab 2.0, have completely altered the way pharmacokinetic characteristics are assessed throughout the drug development process. The capacity to anticipate how chemicals will act in biological systems is greatly improved by this platform, which offers a thorough and fast way to evaluate several ADMET characteristics concurrently. With an increase of 27 toxicity endpoints and 8 toxicophore rules, ADMET lab 2.0 covers a vast diversity of endpoints, including 17 physicochemical qualities, 13 medicinal chemistry properties, 23 ADME properties, and a whopping 58 evaluations in all. Medicinal chemists may optimize lead compounds efficiently and eliminate unwanted candidates early on in the drug discovery process with the help of a multi-task graph attention framework that guarantees robust and accurate predictions²³. Because it can do batch screens as well as single-molecule assessments, this computational tool is suitable for use in high-throughput drug discovery applications. Users can swiftly get prediction estimates for different pharmacokinetic parameters by standardizing input via chemical structure drawings or SMILES strings. Researchers can make better judgments throughout the drug development pipeline because of the data presentation in an easy-to-understand style, which includes radar plots that describe the physicochemical quality of compounds. By tackling the critical aspects of absorption, distribution, metabolism, excretion, and toxicity early on in the process, computational tools like ADMET lab 2.0 are essential for improving pharmacokinetic evaluations, which in turn leads to more successful drug development outcomes²⁴.

Despite the widespread usage of Moringa oleifera to treat a variety of illnesses, there is no scientific evidence to support the safety of this medicine, and no draft has been discovered to determine the biosafety index of this herbal medication preparation. The present study will investigate the phytochemical analysis of Moringa oleifera extract to determine its medicinal efficacy and potential uses in a variety of industries such as cosmetics and food.

Materials and methods

Chemicals and reagents

All antibacterial, antioxidant, and standard chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Himedia (Mumbai, India). Culture media was purchased from Himedia. Each additional reagent that was utilized was an analytical grade.

Plant material

Moringa oleifera leaves were collected from Horbit Village, El-Sharkyea Governorate, Egypt. It was identified and authenticated taxonomically by Dr. El-Baraa Mohammed El-Saied, “assistant professor of Plant Ecology, A specimen was kept at Botany and Microbiology Department, Faculty of Science, Al-Azhar University. Egypt. Moringa oleifera was ground to a powder using an electrical blender after thoroughly washing with distilled water and drying at room temperature. Subsequently, the powdered substance was extracted by percolation through a Soxhlet extractor (3840; Borosil Glass Works Ltd., Mumbai, India) with methanol. The extracts were then dried and concentrated by rotary vacuum evaporation (Yamato BO410, Yamato Scientific Co., Ltd., Tokyo, Japan). Using different in vitro assays, the resulting extracts were subsequently assessed for their, antibacterial, antioxidant, cytotoxicity, and phytochemical composition.

Antibacterial activity of the extract of Moringa oleifera

The antibacterial activity of Moringa oleifera extract was assessed by disc diffusion assay using the following bacteria: Salmonella typhimurium, Staphylococcus aureus, Listeria monoctogens, Klebsiella pneumoniae, Escherichia coli and standard strain Bacillus subtilis ATCC 6633. All these isolates previously isolated from food, were identified and obtained from the bacteriology laboratory at the Faculty of Science, Al-Azhar University. The investigated bacteria were cultured in Muller Hinton broth (MHB) and incubated for 24 h at 37 °C. A 0.1 ml bacterial suspension (in a McFarland turbidity of 0.5) was then swirled over the Mueller Hinton agar medium, followed by loading of the filter paper discs with 50 µl of extract from Moringa oleifera in the surface medium, a chloramphenicol 30 µg/ml, erythromycin 15 µg/ml gentamicin (10 µg/ml), neomycin 30 µg/ml, and amoxicillin/clavulanic acid 30 µg/ml (Oxoid, England), were used as controls. All plates were incubated at 4° C for 2 h and then transferred to an incubator for incubation at 37 ° C for 24 h. The inhibitory zone that developed around the discs was measured in millimeters (mm). The testing was carried out in triplicate^25,26.

Determination of the MIC values of Moringa oleifera

Using the microdilution broth method in a 96-well microplate, the MIC of Moringa oleifera extract has been determined against the above-mentioned bacterial strains. Gentamicin was used as the positive control in the MIC assay. Each well of the microplate was loaded with 200 µl of Muller-Hinton broth (MHB) medium. A two-fold serial dilution was utilized to examine the extract of Moringa oleifera and gentamicin. The concentrations of gentamicin were 0.1, 0.2, 0. 0.4, 0.6, 1.2, 2.4, 4.8, 9.6, 10,0.0 and 15.0 µg/ml, whereas those of Moringa oleifera extract were 0.0, 5.0, 10.0, 25.0, 20.0, 25.0, 30.0, 35.0, 40,0, 45.0, 50.0, 55.0, 60.0, 65.0, and 70.0 µg/ml. After that, 10 µl of a bacterial cell suspension that had been adjusted to 10⁶ CFU/ml and added to each well. The microplates were incubated at 37 °C for 24 h. The microplates were scanned at an OD of 630 nm after the incubation time. Gentamicin or Moringa oleifera extract’s MIC was thought to be the lowest concentration at which detectable bacterial growth was inhibited according to CLSI²⁷, .

The effects of Moringa oleifera extract observation on bacterial cells under transmission electron microscopic (TEM)

To determine the impact of Moringa oleifera extract on bacterial cells, TEM observations were conducted on K. pneumoniae, E. coli, and S. aureus, representing models among the tested bacteria. Standards suspensions (0.5 McFarland) of the tested bacteria were inoculated (5% V/V) in a 100 ml conical flask that contained 20 ml of nutrient broth medium. Moringa oleifera extract at a sublethal concentration (one-half MIC value) corresponding to each bacterial type was added to the prepared media. Additionally, flasks that contained solely media were prepared with the same volume (control) and inoculated with the bacterial suspension that had been prepared. The controls and the treated cells were incubated on a rotary agitator at 120 rpm at 37 °C for 18 h. The ultrathin sections were prepared by following the procedures outlined by Sharaf et al.²⁶, with a distinct collection of control and treated cells following incubation. These sections were subsequently examined at 80 KV using a JEOL 1010 Transmission Electron Microscope at The Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt.

Antioxidant activity of Moringa oleifera extract

Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the Moringa oleifera extract

The methanolic extract of Moringa oleifera leaves was subjected to radical scavenging activity using the DPPH radical scavenging method, as illustrated in the study by Elbestawy et al.¹. Various concentrations of Moringa oleifera extract were prepared (1000, 500, 250, 125, 62.5, 31.25, 15.62, and 7.81 µg/ml). A 0.1 mmol/l ethanol solution of DPPH was prepared. To each concentration of the extract, 5 ml of the DPPH solution was added, and the mixture was vigorously agitated. Ascorbic acid was used as the standard control. The reaction mixtures were allowed to rest for 20 min at 27 °C. After the incubation period, the absorbance of the samples was measured at 517 nm. The IC₅₀ values for ascorbic acid and Moringa oleifera extract, indicating the concentration required to reduce the initial DPPH concentration by 50%, were determined. The antioxidant activity of the Moringa oleifera extract was evaluated using the following formula:

ABTS cation radical scavenging activity

To evaluate the antioxidant effectiveness of Moringa oleifera extract, the ABTS (2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) cation radical decolorization technique was implemented. This method was performed according to the procedure described by Pellegrini et al.²⁸. The concentrations of ascorbic acid and Moringa oleifera extract samples were established as follows: 1000, 500, 250, 125, 62.5, 31.25, 15.62, and 7.81 µg/ml. A 7 mmol/l ABTS solution was reacted with 2.4 mmol/l potassium persulfate in the dark for 12–16 h at 25 °C to generate the ABTS cation radical. This ABTS radical solution was subsequently diluted in ethanol (1:89, V/V) to obtain an absorbance of approximately 0.70 ± 0.02 at 734 nm. The assay involved the mixing of 1 ml of the diluted ABTS radical solution with 1 ml of each concentration of Moringa oleifera extract or ascorbic acid. A spectrophotometer was employed to measure the absorbance at 734 nm after the reaction mixtures were allowed to equilibrate at 30 °C. The antioxidant activity was determined by comparing the absorbance decrease of the samples to that of the control (ABTS solution without extract or ascorbic acid). The following formula was employed to express the results as the percentage of ABTS radical scavenging activity:

In vitro, cytotoxicity of Moringa oleifera extract against normal cells

The cytotoxic effects of Moringa oleifera extract were assessed in vitro using the HFB-4 (normal human melanocytes) cell lines, following the methodology outlined by El-Sherbiny et al.²⁹. The experiment was conducted in triplicate, and cell viability and proliferative potential were evaluated through the MTT assay, which measures metabolic activity. The culture medium was replaced with varying concentrations of Moringa oleifera extract (ranging from 0.0 to 1200 µg/ml), and the cells were incubated for 24 h. After incubation, the cells were washed with fresh medium or cold PBS, followed by incubation with 0.5 mg/ml MTT solution for 2–5 h. The MTT solution was then discarded, and 200 µl of DMSO was added to each well. The optical density (OD) of each treatment was measured at 570 nm using a microplate reader. The percentage of cell viability and cell death were calculated using the following formulas:

Quantification of the total content of phenolics and flavonoids

Determination of the total phenolic constituents

The total phenolic content of Moringa oleifera leaves was evaluated using the Folin-Ciocalteu reagent technique. The reaction mixture included 200 µL of the extract, 750 µL of newly made 1:10 diluted Folin-Ciocalteu reagent, and 2 mL of 7.5% sodium carbonate. The final amount was completed up to 7 mL using distilled water. The mixes were left in the dark at room temperature for 2 h to enable the reaction to finish. The absorbance was then measured at 765 nm using a Perkin-Elmer Lambda-2 spectrophotometer and a 1 cm cell. Each experiment was carried out in triplicate, using gallic acid as the calibration standard, and the findings were represented as gallic acid equivalents (g/100 g) of the extract³⁰.

Determination of the total flavonoid constituents

Briefly, 1 mL of the methanolic extract was diluted and transferred to a 10 mL volumetric vial that contained 4 mL of distilled water. Afterward, each flask was supplemented with 0.3 mL of 5% NaNO₂. 0.3 mL of 10% AlCl₃ was added after 5 min, and 2 mL of 1 M NaOH was added after 6 min. Each reaction mixture was promptly diluted with 2.4 mL of distilled water and thoroughly mixed. The absorbance of the compounds was verified at 510 nm. The epicatechin equivalents (mg/g) were used to determine the total flavonoid content of the samples. The average value was recorded after each sample was measured three times³⁰.

Identification of Moringa oleifera extract components by HPLC

Sample preparation for HPLC

validated HPLC technique was used to evaluate the Moringa oleifera extract. A precisely measured portion of the extract (100 mg) was dissolved in 10 mL of 50% methanol. The solution underwent triple analysis after passing through a nylon membrane filter with a pore size of 0.2 μm.

HPLC apparatus and chromatographic conditions

The extract of Moringa oleifera was loaded into a HPLC system (Shimadzu SPD-10 A, Kyoto, Japan) that included a SIL-10AD auto-injector, an SPD-10AV UV-Vis detector (280 nm), a DGU-10 A degasser, and an LC-10AD pump. For the separation, a Shim-pack CLC-ODS (C-18) (2 cm, 4.6 mm, 5 μm) from Cheshire, UK was used, along with a C18 guard column. The elution was performed using a gradient solvent system with 1% acetic acid (solvent A) and acetonitrile (solvent B) as the mobile phases. The gradient was A (H₂O: AA 94:6, pH = 2.27), B (ACN 100%), with 0–15 min representing 15% B, 15–30 representing 45% B, and 30–45 representing 100% B. One milliliter per minute was the flow rate at room temperature. The peak width from each compound’s HPLC analysis was considered¹².

Pharmacokinetic study and anticipation of drug-likeness

According to Xiong et al.³¹, the open-source ADMET Lab 2.0 application from the computational Biology & Drug Design Group (https://admetmesh.scbdd.com/, accessed on June 8, 2024) was used to calculate the parameters associated with quercetin, including absorption, distribution, metabolism, elimination, and toxicity (ADMET).

Statistical analysis

Data are presented as the mean ± SD value, which was computed with Minitab 18 software extended with a statistical package and Microsoft Excel 365.

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• Source: https://www.nature.com/articles/s41598-024-80700-y