Exploring the anti-inflammatory potential of the polyphenolic compounds in Moringa oleifera leaf: in silico molecular docking

ABSTRACT

A recent study conducted an in silico model to investigate the potential of Moringa oleifera’s ethanolic leaf extract as a natural anti-inflammatory agent. The HPLC analysis identified 7 phenolic acids (benzoic acid 28%, syringic acid 23%, ellagic acid 19%, chlorogenic acid 12%, catechol 11%, gallic acid 5%, and caffeic acid 2%) and 5 flavonoids (naringenin 31%, kaempferol 30%, apigenin 14%, rutin 13%, and quercetin 12%) that are known to have anti-inflammatory activities in vitro tested on human interleukin-1β (IL-1β) and interleukin-6 (IL-6) in a previously prepared study (Hamdy, 2023) The Moringa polyphenol compounds were docked against IL-1β, IL-6, and the human interleukin-1 receptor (IL1RA) using Auto-Dock Vina. Caffeic acid, chlorogenic acid, and kaempferol interacted with amino acid residues in IL-1β by creating hydrogen bonds and hydrophobic interactions. The co-crystalized ligand attached to IL-1β displayed an affinity score of −8.96 kcal/mol. Caffeic acid, chlorogenic acid, quercetin, rutin, and syringic acid had unique interaction patterns with IL-6, forming hydrogen bonds and being hydrophobic. The interactions’ binding energies ranged from −5.97 to 5.26 kcal/mol. Moringa’s polyphenolic compounds also docked with IL1RA, where rutin and chlorogenic acid exhibited binding energies of −8.96 kcal/mol and −7.12 kcal/mol, respectively, and created hydrophobic interactions and hydrogen bonds. In conclusion, Moringa oleifera polyphenols can interact with IL-1β, IL-6, and IL-1RA to reduce inflammation, making it a potential treatment strategy for improving recovery.

KEYWORDS:

• Moringa oleifera polyphenols
• immunomodulators
• molecular docking
• pro-inflammation markers

Introduction

The computational drug discovery programs are gaining popularity and have proven successful in predicting and discovering strong molecules prior to synthesis, ultimately saving time and reducing experimentation costs compared to traditional approaches [2]. In scientific studies, molecular docking analysis is an essential method that helps comprehend molecular interactions that are important for biotechnology, protein engineering, and drug development. It enhances understanding of ligand-receptor affinities and reaction mechanisms by predicting ideal configurations by simulating binding interactions between ligands and target proteins. This predictive power helps to optimize the interactions between enzymes and substrates, expedite the search for new drugs, and guide bio-molecular recognition [3]. Molecular docking’s adaptability and influences on scientific advancement are demonstrated by its application in areas other than drug creation, including environmental remediation, materials science, and agrochemical development. For example, the intersection of medicinal chemistry and polymerization catalysis is being explored in the field of bioinorganic coordination chemistry, where researchers utilize molecular operating environmental (MOE) docking to estimate the level of inhibition and assess the ability of specific chelates before screening. Further tests have been conducted to determine the potential applications of these chelates, including in-vitro studies to examine antimicrobial and antioxidant properties, the ability to bind with DNA, and cytotoxicity. These tests provide valuable insights into the potential benefits and limitations of these compounds [4]. Computer-assisted simulations have significantly advanced the field of synthetic chemistry, allowing researchers to study and explain the structural and biological aspects of newly synthesized chemical systems. Density functional theory (DFT) is the most widely used tool for molecular simulation and modeling. By utilizing DFT-based computer-assisted calculations, researchers gain a better understanding of electronic characteristics, reacting sites, chemical reaction processes, and stability. These calculations help identify non-covalent interactions that enhance the biological profile of bioactive substances within newly synthesized chemical systems [5]. Molecular docking is used to establish the inhibitory action of the compounds against a biological receptor, and the molecular structure of the complexes is theoretically optimized. Quantum chemical parameters are accurately calculated and correlated with their biological properties [6]. Natural products have a rich history of treating various diseases in developing countries and are recognized as an excellent source of lead compounds for developing conventional drugs. However, efforts in developing countries to discover natural product-based drugs mostly involve using crude extracts through the in vitro and/or in vivo assays, with limited attempts to isolate active principles for structure elucidation studies. Moreover, studies that isolate pure secondary metabolites and evaluate their bioactivity have been limited. Therefore, incorporating molecular docking techniques into natural product-based drug discovery programs can be a useful tool for predicting small-molecule interactions with drug targets and can significantly reduce the time and cost of drug discovery. Medicinal chemists can use this tool to predict and develop compounds with pharmacological activity and thus aid in the discovery of new drugs [7]. The study aims to evaluate the interaction of polyphenol compounds in the Moringa oleifera ethanolic extract against interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-1 receptor (IL1RA), providing insights for therapeutic intervention against inflammations.

Inflammation is caused by pathogens, damaged cells, and toxic compounds, triggering acute and/or chronic responses that damage organs. Inflammation plays a vital role in preserving overall health. Typically, when the body is exposed to acute and/or chronic inflammation, various cellular and molecular mechanisms collaborate to prevent damage and infection. This curative process ultimately resolves acute and/or chronic inflammation and restores tissue balance. Yet, if acute inflammation continues and goes uncontrolled, it can change into chronic inflammation, leading to a range of chronic inflammatory disorders [8]. Inflammation at the tissue level causes redness, swelling, heat, pain, and loss of tissue function due to local immune, vascular, and inflammatory cell responses to infection or injury. The process involves changes in vascular permeability, leukocyte enrollment and accumulation, as well as the release of inflammatory mediators [9]. In brief, inflammation depends on stimulus type and location. The process has four stages: 1) receptors detect stimuli; 2) pathways activate; 3) markers release; and 4) cells are enrolled [9]. When infectious microbes or other inflammatory factors enter the body, a complex series of intracellular signaling pathways is generated, leading to the production of inflammatory mediators. These mediators are primarily stimulated by antigens and cytokines such as IL-1β, IL-6, IL-1 R, and other mediators mediating the inflammatory response. The activation of these receptors initiates critical intracellular signaling pathways, including the mitogen-activated protein kinase (MAPK), nuclear factor kappa-B (NF-κB), and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways [10]. Cytokines are released by immune cells like monocytes, macrophages, and lymphocytes. Inflammatory cytokines convert leukocytes to the site of inflammation and regulate inflammation through a complex network of interactions. Excessive production of inflammatory cytokines can cause cytokine storms, which can lead to death [11]. A better understanding of how to regulate cytokine pathways would enable accurate identification of inflammation and treatment of inflammatory diseases.

Moringa oleifera is a treasured plant due to its nutritional and medicinal properties and is widely used in food, industry, agriculture, and medicine in developing countries. Its leaves contain active constituents like proteins, amino acids, polysaccharides, dietary fiber, phenols, flavonoids, phytosterols, and glycosides. They possess various pharmacological activities like antioxidant, anti-inflammatory, anti-hypertensive, hypoglycemic, hypolipidemic, liver and kidney protective, as well as anti-cancer effects [12]. A previous study by El-Sayed (2023) found that active constituents of M. oleifera leaves have significant enrichment applications in drug and functional food uses. He made moringa hard candy from M. oleifera leaf extract to enhance the immunity of the respiratory system against certain respiratory microbial infections [13]. However, the underlying anti-inflammatory mechanism is still unclear, which limits its application [14]. Here, the study was designed to investigate the M. oleifera leaf ethanolic extract key active components and their anti-inflammatory effects in silico, which may provide a theoretical basis for its application in the future as anti-inflammatory functional foods or drugs.

Methodology

Materials

The dried leaves of the Moringa Oleifera plant were obtained from National Research Center, Giza, Egypt. HPLC analysis for quantification of phenolic acids was done in the Laboratory of the Desert Research Center (DRC), Cairo, Egypt. Molecular docking analysis was conducted by using the Auto-Dock Vina program.

Methods

Preparation of Moringa oleifera ethanolic leaf extract

The preparation of Moringa oleifera leaf ethanolic extract was carried out based on a previous study conducted by Hamdy (2023) [1]. In detail, 100 g were soaked in 500 ml of 80% ethanol for 24 h at 60°C with stirring. The resulting extract was centrifuged at 6500 × g for 10 minutes, and the supernatant was filtered through Whatman filter paper (No. 3). The filtrate was then dried using a rotary evaporator under vacuum at 60°C to eliminate ethanol, and the yield was measured as weight/weight (w/w). The resulting sample was stored at 4°C until required.

HPLC analysis of Moringa oleifera leaf polyphenol identification

e Moringa leaf extract was analyzed using HPLC to identify polyphenolic compounds (one replicate only). Phenolic acid was evaluated by Goupy et al. (1999) [15], while flavonoids were quantified by Mattila et al. (2000) [16]. The dried sample was dissolved in methanol (HPLC). It was then injected into a thermo-hypersil reversed phase C18 column. The compounds were identified by comparing their retention times and UV absorption spectra to those of the standards.

In silico study

According to Biswas et al.. (2013) [17], a molecular docking study was conducted in which the compounds under investigation were subjected to docking against human interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and human interleukin-1 receptor (IL1RA) using the Auto-Dock Vina. Crystal proteins (PDB code: 6y8m, 1alu, and 1IRA) sourced from the RCSB were utilized. The targeted proteins underwent preparation procedures involving the removal of water molecules, the addition of missing amino acids, the correction of unfilled valence atoms, and the minimization of protein-peptide energy through the application of CHARMM force fields. The essential amino acids of the protein were selected and prepared for screening. The compounds under investigation were prepared by creating their 2D structures using Chem-Bio Draw Ultra17.0 and saving them in SDF file format. Subsequently, the tested ligands were protonated, and their energy was minimized using the MMFF94 force field with 0.1 RMSD kcal/mol. The minimized structures were then stored for molecular docking. The process of molecular docking was carried out using docking algorithms, with the target pocket being held rigid while the ligands were allowed to be flexible. Each molecule was permitted to produce twenty different interactions with the protein during the refinement. The docking scores, representing the affinity interaction energy, of the best-fitted poses with the active sites of the human interleukin-1β and interleukin-6 were recorded. The 3D orientation was generated using Discovery Studio 2016 visualizer software. To ensure the accuracy and reliability of molecular docking results, their validation was performed using Autodock Vina in two ways: redocking and blind techniques. This process aimed to verify that the predicted binding interactions between ligands and target proteins are consistent with the in vitro data obtained in the last study conducted by Hamdy (2023) [1].

Results

HPLC polyphenolic contents of moringa oleifera leaf extract

In the field of natural product drug discovery, extracting pharmacological compounds from medicinal plants requires the use of the right solvent system [18]. The HPLC analysis of the bioactive compounds in the ethanolic (80%) extract of Moringa leaves identified and quantified seven phenolic acid compounds, including benzoic acid (28%), syringic acid (23%), ellagic acid (19%), chlorogenic acid (12%), catechol (11%), gallic acid (5%), and caffeic acid (2%) as demonstrated in Figure 1. Furthermore, the HPLC analysis detected five flavonoids in the Moringa leaf ethanolic extract based on the set standards. The identified flavonoids were naringenin (31%), Kampferol (30%), apigenin (14%), rutin (13%), and quercetin (12%), as displayed in Figure 2. These findings provide a better understanding of the bioactive compounds present in the Moringa leaf ethanolic extract, which can have potential health benefits

Figure 1. Phenolic acids content in Moringa oleifera leaf extract (%).

Figure 2. Flavonoids content in Moringa oleifera leaf extract (%).

In silico findings and validation

Docking against (IL-1β)

Table 1 displays the binding modes and affinity energies of Moringa polyphenolic compounds with IL-1β, IL-6, and IL-1RA. Specifically, for IL-1β, caffeic acid, chlorogenic acid, and kaempferol exhibit energies of −5.01, −4.91, and −4.66 kcal/mol, respectively. Caffeic acid formed four hydrogen bonds with Asn108, Met148, Gln194, and Arg11, with bond lengths of 2.32, 2.26, 2.48, and 4.15 Å, respectively (Figure 3(a)). Chlorogenic acid is attached to IL1β by four hydrogen bonds and one hydrophobic interaction with Met148, Asn108, and Lys109 (Figure 3(b)). Similarly, kaempferol interacted with Asn108, Lys109, and Arg11 by forming four hydrogen bonds, with bond lengths of 2.30, 2.44, 2.59, and 4.41 Å, respectively (Figure 3(c)). The co-crystalized ligand attached to IL-1β showed an affinity score of −8.96 kcal/mol. It formed two hydrophobic interactions with Asn108 and Leu110 and four hydrogen bonds with Arg11, Met148, and Gln149. The distances between the ligand and the amino acid residues were 4.53, 2.08, 1.98, and 2.80 Å, respectively (as shown in Figure 3(d)). IL-1β is a key cytokine in the inflammatory response, playing a vital role in the host’s defense against pathogens while also contributing to tissue damage. Blocking its production and release active sides is of considerable interest. In the current in silico study, Moringa oleifera polyphenols may have a role in inhibiting IL-1β activity. In this study, the protein structures of IL-1β contain a co-crystalized ligand code (SX2), so re-docking of the co-crystalized ligand will be used in docking validation, as shown in Figure (3(e)).

Figure 3. (a) Mapping surface and 3d representation of caffeic acid docked in (IL-1β). (b) Mapping surface and 3d representation of chlorogenic acid docked in (IL-1β). (c) Mapping surface and 3d representation of kaempferol docked in (IL1β). (d) Mapping surface and 3d representation of the co-crystalized ligand docked in (IL1β). (e) 3D representation of the superimposition of the co-crystallized (orange) and the re-docking pose (green) of the ligand in IL-1β binding site RMSD value = 0.75.

Table 1. Molecular docking analysis of different moringa polyphenol compounds against IL-1β using auto dock vina.

Polyphenol compounds	IL-1β		IL-6		IL1RA
	RMSD value (Å)	Docking score (kcal/mol)	RMSD value (Å)	Docking score (kcal/mol)	RMSD value (Å)	Docking score (kcal/mol)
Naringenin	1.84	4.51-	0.89	4.75-	0.89	–5.17
Rutin	1.67	−4.21	1.98	−5.97	1.21	−8.96
Quercetin	1.13	−4.26	1.23	−5.55	1.23	−6.51
Kaempferol	0.93	4.66-	1.02	−4.25	0.84	−6.41
Apigenin	1.35	−4.43	1.28	4.89-	1.35	6.20-
Chlorogenic acid	1.34	−4.91	1.71	−5.26	1.51	−7.12
Caffeic acid	0.89	−5.01	1.56	−5.11	1.46	−5.11
Syringic acid	0.98	−4.24	1.33	−4.10	1.05	−5.21
Gallic acid	1.41	3.64-	1.36	−3.86	1.25	−4.83
Ellagic acid	2.18	−3.56	2.20	−3.21	1.10	−5.53
Catechol	1.16	−3.68	2.05	3.62-	–	–
Benzoic acid	2.25	−3.85	1.73	−3.36	0.89	5.17

Docking against (IL-6)

The interaction patterns of caffeic acid, chlorogenic acid, quercetin, rutin, and syringic acid with IL-6 were characterized by binding energies of −5.11, 5.26, −5.55, −5.97, and −4.10 kcal/mol, respectively (Table 1). Caffeic acid established three hydrogen bonds and one hydrophobic interaction with Arg30, Gln175, and Leu178 at distances of 2.93, 2.67, and 1.77 Å, respectively (Figure 4(a)). Chlorogenic acid formed five hydrogen bonds with Gln175, Arg182, and Arg179 at distances of 2.12, 2.25, 2.14, 2.39, and 3.39 Å, respectively, in its interaction with IL-6 (Figure 4(b)). Quercetin engages in three hydrogen bonds with Asp34, Gln175, and Arg30 at bond lengths of 2.37, 2.14, and 2.77 Å, respectively (Figure 4(c)). Rutin exhibited seven hydrogen bonds with IL-6, interacting with Asp34, Arg30, Arg182, Arg179, and Gln175 at distances of 1.81, 2.40, 2.88, 2.09, 2.21, 5.30, and 2.27 Å, and additionally forming two hydrophobic interactions with Arg30 and Arg179 (Figure 4(d)). Syringic acid established four hydrogen bonds and one hydrophobic interaction with Gln175, Arg182, Arg179, and Leu178 (Figure 4(e)). In this study, we used blind docking to assess molecular affinities of candidate proteins against Interleukin 6 (IL-6) protein structures since the ligand was not co-crystalized.

Figure 4. (a) Mapping surface and 3d representation of Caffeic acid docked in (IL-6). (b) Mapping surface and 3d representation of Chlorogenic acid docked in (IL-6). (c) Mapping surface and 3d representation of quercetin docked in (IL-6). (d) Mapping surface and 3d representation of rutin docked in (IL-6). (e) Mapping surface and 3d representation of syringic acid docked in (IL-6).

Docking against IL1RA

The results presented in Table 1 show the binding properties and strength of attraction of the polyphenolic compounds found in Moringa with IL1RA. The binding mechanism of rutin exhibited a binding energy of −8.96 kcal/mol toward IL1RA. The co-crystalized ligand formed three hydrophobic interactions with Ile10, Pro23, and Lys60, and additionally, it established eight hydrogen bonds with Glu126, Cys24, Glu59, Gln129, Leu26, Asp128, and Arg22 (refer to Figure 5(a)). Similarly, the binding mode of chlorogenic acid exhibited an energy binding of −7.12 kcal/mol against IL1RA. Chlorogenic acid engaged in two hydrophobic interactions with Ile89 and Pro25 and further formed four hydrogen bonds with Glu126, Gln129, and Ile10 (refer to Figure 5(b)). Due to the lack of a co-crystalized ligand in IL1RA, we utilized the blind docking technique to evaluate the molecular affinities of our candidate against the mentioned proteins.

Figure 5. (a) Mapping surface and 3d representation of rutin docked in (IL1RA). (b) Mapping surface and 3d orientation of Chlorogenic acid docked in (IL1RA).

Discussion

Bioactive polyphenolic compounds of moringa oleifera leaf extract

Moringa oleifera’s dried leaves are an excellent source of polyphenols [19]. Flavonoids and phenolic acids are the main types of polyphenols found in Moringa oleifera leaves [20]. Phenolic acids are plant-derived compounds that have well-documented health benefits. They are anti-inflammatory, anti-mutagenic, antioxidant, and anticancer [12]. Furthermore, in the present study, benzoic acid, syringic acid, and ellagic acid had the highest concentrations according to the standards used. Also, the leaves of Moringa oleifera are an excellent source of flavonoids, which are plant compounds with a benzo-pyrone structure. High flavonoid intake can protect against infectious and degenerative diseases, including cardiovascular diseases, cancers, and age-related illnesses [21]. Naringenin and kaempferol showed the highest levels based on study standards. The polyphenol concentration varies due to environmental conditions, plant genetics, and extraction methods used [22].

Benzoic acid is an organic compound that consists of a benzene ring and a carboxyl group, making it an aromatic carboxylic acid. It is a colorless, crystalline solid that has many important applications. Benzoic acid is used in phenol formulations, to make skin ointments for treating fungal infections, and as a preservative in cosmetic and food products. It is also a precursor to benzoyl chloride, which is utilized to produce various substances like drugs, perfumes, dyes, and herbicides. Syringic acid is a plant metabolite with potent antioxidant properties that benefit human health. It has therapeutic applications that include anti-inflammatory, antimicrobial, neuro- and hepato-protective effects [23]. Also, it reduces oxidative stress markers and acts as a free radical scavenger. Its positive effects may help prevent diabetes, cancer, and cerebral ischemia [24]. Ellagic acid is a natural bioactive substance found in plants. It has antioxidant, anti-inflammatory, anti-mutagenic, and anti-proliferative properties. It has revealed pharmacological effects in numerous model systems and has received attention for its cardio-, hepato-, nephron-, and neuro-protective properties [25]. HPLC analysis showed that Moringa leaves’ ethanolic extract contains moderate amounts of chlorogenic acid and catechol. Chlorogenic acid is a polyphenol with anti-inflammatory, anti-bacterial, and anti-carcinogenic properties. It also protects against oxidative stress-induced cell damage by scavenging radicals [26].

Catechol is a colorless substance; it is now primarily produced synthetically and used in the production of pesticides, flavors, and fragrances. Catechol can act as both pro-oxidants and antioxidants and can harm membrane functionality when used as an antimicrobial agent [27]. Gallic acid and caffeic acid were found in the HPLC analysis results in the lowest amounts. Gallic acid is a tri-phenolic compound with potential as an antioxidant and apoptosis inducer. It has been studied extensively for its mechanism of action, radical scavenging activity, inhibition of lipid peroxidation, maintenance of endogenous defense systems, and chelation of metal ions. Gallic acid derivatives have been found in various phyto-medicines that exhibit biological and pharmacological effects like inhibiting cell signaling pathways, attacking cancer cells through apoptosis, and radical scavenging [28]. Caffeic acid, found in Moringa and other plants, has anti-inflammatory, antioxidant, and anti-cancer properties. It acts as an antioxidant, prevents DNA damage caused by free radicals, and inhibits cancer cell growth [29].

Naringenin and kaempferol are two compounds with many health benefits. Naringenin is found in citrus fruits and can protect the heart, fight inflammation, and prevent cancer. Kaempferol is present in many plants and foods, can lower the risk of chronic diseases like cancer, and has additional health benefits like being antimicrobial, neuroprotective, and good for cardiovascular health [30]. Also, HPLC results show low concentrations of apigenin, rutin, and quercetin in the ethanolic extract of MO leaves. Apigenin is a bioflavonoid with potential benefits for anxiety reduction, immune health, hormone modulation, brain function, oxidative stress, and inflammation. Rutin has potential antioxidant and anti-inflammatory effects, and it may offer protection against cancer and other diseases [31]. Quercetin is found frequently in edible fruits and vegetables and has various functions, including antibacterial, antiviral, antioxidant, protein kinase inhibition, antineoplastic, phytoestrogen, radical scavenging, chelating, and Aurora kinase inhibition [32].

According to previous research, it has been established that Moringa oleifera leaf extract functions as an immune modulator from multiple scholarly sources due to its rich composition of polyphenol compounds. These constituents can influence the immune response by either stimulating or suppressing the proliferation and activation of immune cells, thereby modulating the overall functioning of the immune system within the body.

Molecular docking and therapeutic target prediction

Inflammation, especially prolonged types, can lead to serious diseases such as atherosclerosis, cancer, lung inflammation, asthma, and rheumatoid arthritis. Therefore, it is crucial to discover new anti-inflammatory drugs. Although there are many effective anti-inflammatory agents, such as mesalamine (Asacol HD, Delzicol, and others), balsalazide (Colazal), and olsalazine (Dipentum), they can have negative side effects [33]. To avoid such side effects, natural products have become an important alternative in this field. Computational methodologies, such as bioinformatics analysis and molecular docking, offer rapid and reliable solutions for drug discovery. They reduce the risks, side effects, and costs of discovering a new drug, and offer several advantages over traditional approaches [34]. In molecular docking studies, the lowest binding energy value indicates the most favorable conformational position of the ligand within the active region of the targeted protein [35]. In this study, molecular docking analysis was employed to elucidate the mechanism by which the anti-inflammatory components of M. oleifera (MO) act against IL-1β, IL-6, and IL1RA. MO is renowned for its high nutritional value and its ability to boost the immune system, as well as its diverse therapeutic properties. Previous in vitro investigation by Hamdy (2023) [1], showed that the ethanolic extract of Moringa oleifera leaves had significant anti-inflammatory properties. At 1, 10, and 100 µmol/ml concentrations, more than 85% of the cells were viable, indicating high cell viability. Compared to dexamethasone, a standard anti-inflammatory drug, the leaves’ ethanolic extract had higher cell viability (p < 0.05). Moreover, the extract showed an EC50 value of 0.79 µmol/ml and significantly suppressed the production levels of IL-1β (14.46 ± 1.92 mg/ml) and IL-6 (51.67 ± 2.0 mg/ml) in comparison to controls (p < 0.05). These findings confirm the traditional use of the M. oleifera plant as a potential source of bioactive compounds with antioxidant properties for inflammation-related diseases/disorders, particularly inflammation-related pneumonia. The findings demonstrated that M. oleifera contains phenols and flavonoids that have been scientifically proven to possess anti-inflammatory properties which confirmed with Shady et al. (2020) [36].

IL-1β

Interleukin 1 beta (IL-1β) is a vital cytokine that plays a central role in the immune response against infections and injuries. This protein is produced by immune system cells in response to specific molecular signals, and its release is a complex process that is still not completely defined. The secretion of IL-1β is dependent on the strength of the stimulus and the presence of extracellular IL-1β requirements [37]. The formation of inflammation can be triggered by the conventional pathway of protein secretion. When a cell is activated by a suitable stimulus, a series of interactions between adaptor molecules takes place, leading to the formation of inflammation. This process activates caspase-1, which then results in the secretion of IL-1β. Proteins that are conventionally secreted are transported into the endoplasmic reticulum (ER) and then pass through the ER and Golgi before reaching their final destination outside the cell. In the case of IL-1β, the secretion is further complicated by the fact that it is a pro-inflammatory cytokine that requires proteolytic cleavage to become biologically active. The inflammasome, a protein complex that is activated by pathogens and threat signals, plays a crucial role in the proteolytic cleavage and secretion of IL-1β. Furthermore, the regulation of IL-1β secretion is tightly controlled by various mechanisms, including transcriptional, translational, and post-translational modifications. These mechanisms influence the expression, translation, and stability of IL-1β mRNA, as well as the activity and post-transcriptional processing of IL-1β protein. Understanding the complex mechanism of IL-1β secretion is crucial for developing effective therapies for inflammatory diseases [38]. Scheme 1 represents how simply IL-1β works.

Scheme 1. The inflammatory response mediated by the release of IL-1β.

The inflammation treatments aim to either inhibit the activity of inflammatory cells or inhibit the production of inflammatory mediators. The majority of inflammatory conditions are managed using steroidal and non-steroidal anti-inflammatory drugs (NSAIDs), which work by reducing the levels of pro-inflammatory cytokines such as IL-1β. Steroids, such as prednisone or dexamethasone, mimic the action of cortisol, a hormone naturally produced by the adrenal gland that regulates the immune response and metabolism. Steroids can reduce inflammation by inhibiting the activity of immune cells, such as T cells and macrophages, and by decreasing the production of pro-inflammatory cytokines and chemokines. However, long-term use of steroids can result in undesirable side effects such as weight gain, mood changes, osteoporosis, and increased susceptibility to infections [39]. In addition, steroids can suppress the immune system, making the patient more susceptible to infections and cancers. NSAIDs, such as aspirin, ibuprofen, or naproxen, work by blocking the activity of cyclooxygenase (COX) enzymes, which are responsible for the synthesis of prostaglandins, lipid molecules that mediate inflammation, fever, and pain. By reducing the production of prostaglandins, NSAIDs can alleviate these inflammation signals (Scheme 2). However, long-term use of NSAIDs can also cause adverse effects such as stomach ulcers, bleeding, kidney damage, and cardiovascular events [40]. Therefore, the choice of anti-inflammatory treatment depends on the type, severity, and duration of the inflammatory condition, as well as the patient’s medical history, age, and lifestyle. In some cases, natural remedies such as curcumin, omega-3 fatty acids, or ginger may also have anti-inflammatory properties and be used as complementary or alternative therapies. Consequently, there is a growing interest in exploring alternative anti-inflammatory approaches, including those derived from natural sources. However, it is important to consult a healthcare professional before starting any new treatment or stopping the current treatment.

Scheme 2. Inhibition of IL-1β by synthetic inhibitors.

The study investigated the interaction of caffeic acid with IL-1β via molecular docking at four active sites: Asn108, Met148, Glutamine Gln194, and Arg11. The in silico investigation revealed that caffeic acid has anti-inflammatory properties against pro-inflammatory mediators. The molecular docking simulations confirmed that caffeic acid suppresses cytokines such as IL-1β, which contributes to the progression of chronic diseases. Caffeic acid has a significant inhibitory effect on the inflammation responses caused by IL-1β. This inhibition helps prevent the activation of other inflammatory signaling pathways, which are known to contribute to the progression of various chronic diseases. The results suggest that caffeic acid has the potential to be a promising anti-inflammatory agent that can be utilized for developing new treatments for inflammation-related conditions. Therefore, further research could explore the mechanisms of caffeic acid’s inhibitory effect on inflammation and evaluate its therapeutic potential. This result was confirmed by Kim et al. (2023) [33]. Chlorogenic acid affected IL-1β by interaction with 3 active sites: Met148, Asn108, and Lys109. The molecular docking result supports the enhancement of chlorogenic acid action. The molecular docking results strongly confirm the efficacy of chlorogenic acid, which is consistent with a prior in silico study by Santos-Álvarez et al. (2024) [41]. The identification of compounds with anti-inflammatory properties can be an effective approach to treat inflammatory disorders. Also, in this study, the effect of kaempferol on IL-1β activity was investigated. Results showed that kaempferol has anti-inflammatory activity, as demonstrated by the molecular docking studies, revealed that kaempferol has comparable binding energies and docking poses on target sites like Asn108, Lys109, and Arg11. Based on these findings, it can be concluded that kaempferol exhibits anti-inflammatory activity, by improving the expression levels of IL-1β. This result was compatible with Kadioglu et al. (2015) [42].

IL-6

IL-6 is a powerful inflammatory protein generated in reaction to infection or tissue injury and plays an essential role in regulating the immune response. It also plays a significant part in the development of inflammation. In the context of a stimulus, IL-6 is considered to be a key cytokine alongside IL-1 and TNF-α. Recent research indicates that the severity of inflammation outcomes among individuals is linked to the nature of their immune response. Elevated production of IL-6 can lead to an acute and severe systemic inflammatory response known as a cytokine storm. Numerous observations indicate that the degree and characteristics of the cytokine response are linked to the development of diseases. Consequently, it has been demonstrated that the unregulated and persistent release of IL-6 is associated with the degree of disease severity. Inhibiting IL-6 has proven to be effective in controlling these conditions [43]. This study involved an analysis of the interactions between 11 polyphenol compounds from ethanolic Moringa leaf extract and the targeted protein IL-6. The primary objective was to identify a potent and effective IL-6 blocker with anti-inflammatory properties among the selected polyphenol compounds while minimizing potential side effects. The interaction analysis and binding affinities demonstrated that all of the top 5 polyphenol compounds are effective against the targeted protein IL-6. Molecular docking studies of the active compounds indicated that these compounds are strong candidates as anti-inflammatory drugs against inflammation-related diseases.

The focus of this in silico study was to identify potential inhibitory properties of natural product-derived compounds on the pro-inflammatory cytokine IL-6 receptor. The inhibition of this receptor is crucial in the prevention of pain and inflammation, particularly in the context of chronic inflammation. Studies have shown that caffeic acid has anti-inflammatory properties in both in vivo and in vitro models. However, the exact mechanisms behind its anti-inflammatory effects remain unclear. In this study, it was discovered that caffeic acid treatment significantly lowered the protein levels of IL-6. This suggests that caffeic acid may act as an inhibitor of IL-6 protein synthesis, which could potentially lead to anti-inflammatory effects in acute and chronic inflammation. These findings suggest that caffeic acid might be a promising therapeutic agent for inflammatory-related diseases. This result was in agreement with Ezaouine et al. (2022) [44]. Through the molecular docking analysis, compounds such as kaempferol and chlorogenic acids from natural products were found to exhibit favorable binding energy and molecular interactions with the amino acids of the IL-6 target receptor, suggesting their potential suitability for anti-inflammatory purposes. Additionally, rutin was identified as having inhibitory potential against various inflammatory mediator targets, particularly IL-6, thus showing promise in preventing inflammatory diseases. The study also aimed to assess the potential of these compounds as successful drug candidates through molecular docking predictions. The results indicated that the three MO polyphenols may be a promising drug candidate for anti-inflammatory purposes, which was confirmed by Zulhendri et al.. (2022) [45].

However, it is important to note that these predictive findings were validated through the previous in vitro investigation by Hamdy (2023) [1], which confirms their efficacy in preventing inflammation. From the current molecular docking results, the interaction between quercetin and IL-6 May cause a decrease in IL-6 protein expression, which could potentially inhibit IL-6 functions. Previous studies have shown that quercetin can inhibit the release of inflammatory mediators such as histamine and tryptase, as well as the production of cytokines like IL-6, by regulating proinflammatory IL-6, inflammation-related signaling pathways, and activator of transcription pathways (Ezaouine et al. 2022) [44]. Depending on the in vitro findings obtained from our previous study [1] and these in silico results, the selected polyphenol compounds of ethanolic Moringa leaf extract show more promising results as therapeutic compounds rather than synthetic ones.

IL1RA

IL-1 serves as a significant mediator of inflammation and tissue damage in various organs, evident in both in vivo and in vitro models. The IL-1 family involves two agonists, IL-1α and IL-1β, two receptors, biologically active IL-1RI and inert IL-1RII, and a specific receptor antagonist, IL-1Ra. The balance between IL-1 and IL-1Ra within local tissues plays an important role in the susceptibility to and severity of numerous diseases. The IL-1Ra gene alteration is linked to various human diseases, especially those related to inflammation, which may result from an imbalance in the IL-1 system that characterized by increased production of IL-1β and reduced production of IL-1Ra [46]. Numerous epidemiological and experimental studies have reported the anti-inflammatory and immune-modulating effects of polyphenolic compounds, demonstrating their ability to influence the function and presence of immune cells and modulate the tissue balance between pro-inflammatory cytokines such as IL-1β, and anti-inflammatory cytokines such as IL-1Ra [47]. Through molecular docking results, rutin and chlorogenic acid were identified as potential regulators that maintain the balance between IL-1β and IL-1Ra, especially chlorogenic acid demonstrated a strong affinity for binding to both IL-1β and IL-1Ra.

At the end, Moringa leaf polyphenols have the ability to function in two different ways: as immunostimulants and immunosuppressants. As an immunostimulant, these polyphenols act as Mitogen-Activated Protein Kinase (MAPK), which triggers the phosphorylation response of various proteins, including activating transcription factor proteins like proinflammatory cytokines. This results in the up-regulation of cytokines’ expression, which in turn stimulates T and B cell proliferation and differentiation. This substance helps increase cytokine production in CD4+ T cell activity and cytotoxic T cells (CD8+). However, excessive dosages of these compounds can negatively impact T cells, leading to decrease NO generation and suppression of lymphocyte cell growth. On the other hand, Moringa polyphenols can also act as immuno-suppressants by preventing protein kinase from activating pro-inflammatory cytokines, which restrict T and B cell proliferation and differentiation that in turn prevents the stimulation of cytokine secretion [48].

Molecular docking validation

Molecular docking validation is a critical process that involves evaluating the accuracy and reliability of molecular docking results. The process is essential to ensuring that the predicted binding interactions between ligands and target proteins are consistent with experimental observations or known biological data [49]. There have been several methods developed to validate docking programs and scoring functions, one of which is pose selection. This process involves using docking programs to re-dock a compound with a known conformation and orientation into the target’s active site. The compound used is typically obtained from a co-crystal structure. Programs that can successfully return poses with an RMSD value below a preselected threshold (usually 1.5 or 2 Å, depending on the size of the ligand) from the known conformation are considered effective. Following pose selection, scoring and ranking are conducted to identify which scoring functions can most accurately rank the poses based on their RMSD values [50]. On the other hand, Blind Docking is a molecular docking technique that involves the docking of a ligand to the entire surface of a protein without any prior knowledge of the target pocket. This technique is widely used in drug discovery to identify potential ligands that can bind to the protein of interest. Blind docking involves multiple trials and energy calculations to find a favorable protein-ligand complex pose. The process of blind docking can be computationally intensive and time-consuming, as it requires the generation of multiple conformations of the ligand and the protein to identify the best possible binding pose. However, it has the advantage of being able to identify potential binding sites that may not be obvious from the protein structure alone. Overall, blind docking is a powerful tool in drug discovery that can help identify potential lead compounds for drug development [51].

In this study, the protein structures of interleukin 6 (IL-6) and human interleukin 1 receptor (IL1RA) were investigated to evaluate their molecular affinities with ligands. The data obtained indicates that these protein structures do not contain a co-crystallized bond, which means that we cannot use the traditional method of co-crystallization to evaluate the molecular affinities of the ligands against these proteins. To overcome this challenge, a blind docking technique was utilized, which is a computational method used to predict the binding affinity of a ligand to a protein. This technique was used to identify the potential binding sites of the ligands against the mentioned proteins. Then, docking simulations were performed to evaluate the molecular affinities of the targeted candidate against the mentioned proteins. On the other hand, Interleukin 1 Beta (IL-1β) contains the co-crystallized bonding codon (SX2), which makes it possible to use the traditional method of co-crystallization to evaluate the molecular affinities of the ligands against this protein. To validate the docking simulations for IL-1β, re-docking of the co-crystallized complex technique was used. This technique allowed us to assess the accuracy of the docking results and ensure that our predictions are consistent with experimental observations. By using these techniques, we hope to gain a better understanding of the molecular interactions between ligands and target proteins. This knowledge can be used to design more effective drugs that can treat various diseases.

Conclusions

The present in silico investigation aims to identify the therapeutic potential of Moringa leaves derived compounds in addressing inflammatory conditions as immune-modulators that can affect the immune response. Polyphenolic compounds, specifically phenolic acids and flavonoids, have been identified as having promising anti-inflammatory properties against pro-inflammatory markers IL-1β, IL-6, and IL-1Ra as antagonists to IL-1β. This study suggests that Moringa oleifera (MO) may be a valuable traditional medicinal plant, drawing attention to its phytochemical and pharmacological effects. The research demonstrates the anti-inflammatory properties of MO, indicating its potential to reduce IL-1β and IL-6 levels and regulate IL-1Ra levels. These findings suggest that the constituents of MO could serve as a natural source of anti-inflammatory agents, as evidenced by the previous in vitro research cited by Hamdy (2023) [1]. The research on the anti-inflammatory mechanism of Moringa oleifera polyphenols may serve as a theoretical foundation for the development of drugs and functional foods enriched with Moringa oleifera. However, the study was preliminary, and further clinical research is necessary to explore the in-depth mechanisms of MO polyphenols against inflammation, in addition to verifying the safety of these compounds on human health.

Abbreviations

HPLC	=	High-performance liquid chromatography
IL-1β	=	human interleukin-1β
IL-6	=	interleukin-6
IL1RA	=	human interleukin-1 receptor
STAT	=	activator of transcription pathway
JAK	=	Janus kinase
NF-κB	=	nuclear factor kappa-B
MAPK	=	mitogen-activated protein kinase
PDB	=	Protein data bank
ER	=	endoplasmic reticulum
NSAIDs	=	non-steroidal anti-inflammatory drugs
TNF-α	=	Tumor necrosis factor alpha
MO	=	Moringa Oleifera
PAH	=	pulmonary arterial hypertension

Statements and declaration

The authors report there are no competing interests to declare.

Acknowledgments

The author wish to express here sincere appreciation to Dr. Abdulrahman M.Saleh, Pharmaceutical Medicinal chemistry and Drug Design, Faculty of Pharmacy (Boys) Al-Azhar Uni., Cairo 11884, Egypt, [email protected], for his invaluable contribution in preparing essential molecular docking data.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The study was self-funded.