The present review is centered on the characterization and biomedical applications of natural compounds, with a particular focus on aged garlic extract (AGE). Commercially available AGE is an odorless preparation obtained by immersing fresh garlic slices in an aqueous ethanol solution for several months at room temperature (1-5). A number of studies have supported the possible employment of AGE as an anti-inflammatory agent (6-10). In this context, the availability of simple experimental approaches for the screening and characterization of molecules that interfere with pro-inflammatory gene expression is crucial for advancing the knowledge on anti-inflammatory agents present within AGE. An introductory example of experimental cellular model systems of interest in the field of respiratory diseases is shown in Fig. 1. These experimental systems may be useful in generating datasets that support the possible employment of validated agents for the treatment of human pathologies characterized by a hyperinflammatory state. The study of pro-inflammatory genes is of notable importance since the expression of these genes is critical in the development of a number of diseases, such as COVID-19, cystic fibrosis and chronic obstructive pulmonary disease (COPD). Table I reports examples of relevant pro-inflammatory genes that have been targets for anti-inflammation therapy in pre-clinical or clinical studies (11-23). For instance, interleukin (IL)-8 antagonists and monoclonal antibodies against IL-8 have been proposed for the therapy of cystic fibrosis (11,12), COPD (13), severe asthma (14) and osteoarthritis (15). Additionally, anti-IL-6 therapy has been proposed for COVID-19, long-COVID and post-acute sequelae of SARS-CoV-2 infection (16-18). Furthermore, Toll-like receptor 4 (TLR4) inhibitors may be considered for treating COVID-19 (19-21), osteoarthritis (22) and other inflammation-related diseases, such as cystic fibrosis (23).

The present review will discuss experimental model systems and their application in characterizing the activity of AGE and selected components. Then, recent evidence regarding the possible effects and mechanism of action of two major a sulfur-containing AGE components, S-allyl cysteine (SAC) (24,25) and S-1-propenyl-l-cysteine (S1PC) (26), will be reviewed. In this respect, SAC and S1PC showed stable properties under cell culture conditions, and their acute/subacute toxicity was minor in mice and rats (24,26). In particular, the pharmacokinetics of SAC was investigated on human volunteers, indicating that SAC was rapidly absorbed from the gastrointestinal tract (24).

Finally, the possible application of AGE, SAC and S1PC in the development of protocols for possible therapeutic interventions will be discussed.

The cell models used for testing inhibitors of pro-inflammatory genes are typically mammalian cells infected with a microorganism, treated with a viral protein or treated with other agents stimulating pro-inflammatory genes. Table II provides a representative list of experimental models that have been used to characterize the effects of different classes of inhibitors of pro-inflammatory genes. One of the most used and well-characterized cell models is bronchial epithelial IB3-1 cells infected with Pseudomonas aeruginosa. Detailed descriptions of this experimental model system can be found in the publications by DiMango et al (27), who reported the origin of the IB3-1 cell line (derived from a cystic fibrosis patient with a DF508/W1282X genotype) and the protocols for infection with P. aeruginosa. The non-mucoid laboratory strain of P. aeruginosa, PAO1, is typically employed for infection. In the publication by DiMango et al (27), information can be found on PAO1 culturing and on the experimental conditions to prepare aliquots to stimulate IB3-1 cells. One of the most relevant effects of the P. aeruginosa infection of IB3-1 cells is the induction of a pro-inflammatory phenotype marked by the activation of several pro-inflammatory genes, the most relevant being the IL-8 genes. The role of transcription factors in the P. aeruginosa infection of IB3-1 cells was first reported by DiMango et al (27), who described NF-κB activation in this model. The cooperative action of the transcription factors, NF-κB, NF-IL6, activator protein 1, CHOP and cAMP responsive element binding protein, in the P. aeruginosa-dependent induction of IL-8 gene transcription in IB3-1 cells (27) was discussed in depth by Bezzerri et al (28). Notably, using this experimental model system, Finotti et al (29) demonstrated that decoy oligonucleotide molecules targeting NF-κB were able to inhibit the transcription of the IL-8 gene. Furthermore, using a chromatin immunoprecipitation assay, these authors found that the NF-κB decoy was able to strongly inhibit NF-κB recruitment to the IL-8 gene promoter (29). A microarray-based study that demonstrated the involvement of microRNAs in the P. aeruginosa infection of IB3-1 cells was reported by Fabbri et al (30), providing new information on possible employment of microRNA targeting for the development of therapeutic protocols for cystic fibrosis. Several studies have been performed using this system to demonstrate the potential anti-inflammatory properties of medicinal plant extracts or low-molecular weight drugs (31-33). For instance, Lampronti et al (31) reported the notable effects of Nigella arvensis extracts, which inhibit IL-8 expression in P. aeruginosa-infected IB3-1 cells. Furthermore, it was demonstrated that β-sitosterol is the compound responsible for this activity, while stigmasterol and campesterol are inactive in this context. These results support the conclusion that P. aeruginosa-infected IB3-1 cells serve as a suitable experimental model system to identify inhibitors of pro-inflammatory gene expression (30-33).

Similar experimental model systems have been proposed that are based on the P. aeruginosa infection of other cell lines, such as NuLi (30,31), CuFi (30,31), A549(34). Hawdon et al (34), Cerqueira et al (35) and Aval et al (36) demonstrated the potential anti-inflammatory effects of resveratrol and picetannol, using P. aeruginosa-infected A549 alveolar epithelial cells. In addition to P. aeruginosa infection, IB3-1 cells can be stimulated to upregulate pro-inflammatory genes by other stimuli, such as lipopolysaccharide (37), IL-1β (38), TNF-α (39-41) and the SARS-CoV-2 spike protein (42,43). Notably, the effects of the SARS-CoV-2 spike protein on a variety of cellular systems were reproduced by treatment with the spike-mRNA-based vaccine, BNT162b2 (44-46). Another experimental model system of particular interest in the context of the present review is the T-lymphoid Jurkat cell line, very useful to determine the effects of inhibitory agents on TLR4-dependent NFκB activation (47). Geng et al (48) used an electrophoretic mobility shift assay (EMSA) performed on nuclear extracts prepared from TNF-α stimulated Jurkat cells. This study demonstrated a powerful inhibitory effect of SAC on NF-κB activation, reinforcing the information on anti-inflammatory properties of this AGE component found using other experimental model systems (45).

In conclusion, several experimental model systems have been developed and validated for identifying agents that exhibit potent inhibitory activity on pro-inflammatory gene expression. Selected potential anti-inflammatory agents may also be tested in vivo using validated animal models of chronic P. aeruginosa lung infection that mimic cystic fibrosis, as proposed by Hoffmann et al (49). In the near future, we expect that the effects of AGE and its constituents will be analyzed using P. aeruginosa-infected primary bronchial epithelial cells. The results obtained should be considered with great attention, taking into account the anti-microbial effects of garlic-derived compounds as potential confounding factors (50,51) requiring adequate further control studies.

Among the plant extracts hypothesized to retain anti-inflammatory activities, AGE is of great interest (52,53). The preparation and characterization of AGE has been described in a number of publications such as those by Ohkubo et al (1) and Kurita et al (54). Briefly, AGE can be prepared by immersing fresh garlic in 15% aqueous ethanol solution for several months at room temperature (1). AGE is a commercial odorless preparation with antioxidant properties for scavenging reactive oxygen species (ROS) (54) and retains immunomodulatory and anticancer properties (54). It should be noted that interest in the preparation of AGE and AGE-related products is evidenced by the high number of patents that have been deposited over the years (a partial list of patents and patent applications is shown in Table III). This information should be considered with great attention for the development of biomedical approaches, despite the fact that the patenting of preparations for ‘aged garlic’ is outside the major objectives of the present short review. A specialized further review would be useful to describe this interesting activity, which precedes the industrial exploitation of AGE and AGE-related compounds, considering that few reports are available on the patents on garlic and garlic-related products (55). In this context, two notable components of AGE are SAC (25) and S1PC (26). SAC and S1PC showed stable properties under cell culture conditions, and their acute/subacute toxicity was minor in mice and rats (24,26). In particular, the pharmacokinetics of SAC was investigated on human volunteers, indicating that SAC was rapidly absorbed from the gastrointestinal tract (24). Other AGE components have been described by Kodera et al (56), including S-(2-propyl) 2-propen-1-sulfinothioate (allicin), 3-(allyltrisulfanyl)-2-aminopropanoic acid, S-allylmercaptocysteine, allixin and tetrahydro-β-carboline derivatives.

With respect to the anti-inflammatory activity of AGE, several studies are available on the effects of AGE components on the TLR4/NF-κB pathways, which play a central role in inflammatory processes (57-63). Regarding NF-κB, without external stimuli, an inactive trimer is formed in the cytoplasm that is composed of the inhibitory protein, IκB, and the p50/p65 NF-κB dimer. In these conditions, NF-κB is not translocated to the nucleus and the NF-κB regulated genes are mostly inactive, unless activated by other transcription factors (64,65). However, when external stimuli act on the corresponding receptors (for instance, when TLR4 is activated by a number of stimuli, some of which are listed in Table II), phosphorylation of IκB occurs, leading to dissociation of IκB from the trimer and its proteasome-mediated degradation, resulting in NF-κB activation. In these conditions, the p50/p65 NF-κB protein translocates to the nucleus and specifically interacts with NF-κB binding sites present in the promoters of NF-κB-regulated genes, such as the IL-6, IL-1β and IL-8 genes (66), all of which are involved in inflammatory processes that characterize several human diseases (as previously discussed in Table I). There is consensus that SAC interferes with NF-κB activation, thereby causing inhibition of the NF-κB-regulated genes (67,68). Table IV summarizes the evidence supporting the role of SAC in modulating the NF-κB pathway. Fig. 2 summarizes the biological effects of SAC (and of the analogue, S1PC) on different steps of the NF-κB pathway.

A notable study on the possible molecular targets of SAC has been reported by Park et al (57) and reviewed by Colín-González et al (58). In the study by Park et al (57), it was demonstrated that SAC is able to decrease IKKβ activity and the phosphorylation of IκBα, resulting in the attenuation of NF-κB. In another study, Shao et al (59) performed molecular docking analysis to determine that SAC may interact with the Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 complex, PPARγ, histone deacetylase (HDAC)1 and TLR4. The possible interactions with TLR4 is of particular interest since this receptor is an upstream regulator of NF-κB signaling (60-63).

In our laboratory, the interaction between SAC (and the analogue S1PC) and the intracellular domain of TLR4 has been simulated using the well-known docking software, AutoDock Vina (45,46). Furthermore, to confirm the reliability of the identified molecular interactions and to predict the possible impact on TLR4 function, the computed docking models underwent molecular dynamics simulation. These studies are reported in Gasparello et al (45) and Papi et al (46) and suggest that the complexes remained stable as the hydrogen bonds formed between SAC (and S1PC) and TLR4 were retained during the entire molecular dynamics simulation, confirming a plausible interaction between the small molecules and TLR4. In addition, the results obtained suggest that binding with SAC and S1PC reduces the intermolecular interaction between the TLR4 domains of the TLR4 dimer, supporting additional effects on NF-κB, as outlined in Fig. 2. The predicted interaction between SAC and the TLR4 complex is depicted in Fig. 2A. The binding mode predicted for S1PC with TLR4 was only slightly different from SAC/TLR4, and SAC and S1PC shared interactions with both ⁶⁵⁷Tyr and ⁷²²Arg (45,46). For both small molecules, the estimated interaction energy with TLR4 was approximately -65 Kcal/mol (sum of short-range Lennard-Jones and short-range Coulomb contributions over the molecular dynamics simulation), suggesting the two compounds may have highly comparable and favorable interactions with the protein target. This finding suggests that one effect of SAC and S1PC on target cells might involve inhibition of TLR4, causing a marked reduction in NF-κB signaling and a notable inhibition of the transcription of NF-κB-regulated genes (Fig. 2B and C).

SAC reduces ROS (48), exerting clear and expected anti-inflammatory effects, as an interactive role of TLR4 and ROS has been described (69) and inhibition of ROS generation attenuates the TLR4-mediated proinflammatory phenotype, as reported by Pi et al (70). In addition to these direct and indirect effects on TLR4 (summarized in Fig. 2C), SAC might inhibit the NF-κB activity via several mechanisms of action (summarized in Fig. 2B), including via a strong inhibitory effect on IKKβ kinase, which blocks IκBα phosphorylation (64,65). This has a notable consequence for NF-κB signaling. It is well established that the activation of NF-κB requires phosphorylation of the inhibitory subunit, IκBα, the ubiquitination of IκBα (71,72) and finally the proteasome-dependent degradation of IκBα (72,73). In these conditions the p65/p50 NF-κB complex translocates to the nucleus and activates NF-κB-regulated genes. In addition to an inhibitory effect on IKKβ kinase, SAC has been demonstrated to cause a MAP kinase-mediated inhibition of IκBα phosphorylation (64), with a marked inhibition of the formation of poly-ubiquitin IκBα, of IκBα degradation and of NF-κB nuclear translocation. Finally, SAC has been reported to enhance the expression of PPARγ (74) a recognized NF-κB inhibitor (75), and to modulate HDAC activity (76).

The interplay among TLR4, NF-κB and human pathologies has been reported by a number of investigations and reviewed by several articles (77-82). For instance, Aboudounya and Heads (77) reported that SARS-CoV-2 binds and activates TLR4 to increase angiotensin converting enzyme 2 expression, facilitating entry of the virus into cells and resulting in hyperinflammation. In a recent review, Asaba et al (21) described the interplay between TLR4 and SARS-CoV-2, focusing on inflammation and the severity SARS-CoV-2 infections, suggesting TLR4 as a possible target for anti-SARS-CoV2 agents. Sahanic et al (78) demonstrated that SARS-CoV-2 activates the TLR4/MyD88 pathway in human macrophages causing a strong pro-inflammatory response in severe COVID-19. In a notable study, Alamin et al (79) identified common molecular signatures characterizing the molecular effects of SARS-CoV-2 infections and different lung diseases. This study was based on the analysis of RNA-sequencing and microarray gene expression datasets (from the Gene Expression Omnibus platform of the National Center for Biotechnology Information) and considered the following lung diseases: COPD, idiopathic pulmonary fibrosis, ILD (interstitial lung disease), asthma, tuberculosis, cystic fibrosis, pneumonia, emphysema and bronchitis. The full information about these datasets and the description of the complete workflow of the study can be found in Alamin et al (79). Notably, TLR4 was identified in most of these diseases, among the top-ranked differentially expressed genes.

Since AGE (and its constituents, SAC and S1PC) might interfere with the TLR4 and NF-κB pathways (as outlined in Fig. 2), several pathologies caused by dysregulation of the TLR4/NF-κB axis might be affected by treatments with these natural products. Among these target pathologies, the most impactful for the worldwide health systems are COVID-19, lung infectious diseases, cystic fibrosis and COPD (https://www.healthdata.org/research-analysis/gbd; accessed February 20, 2025). Notably, and in agreement with the approach by Alamin et al (79), Hasan et al (80) reported using a differential gene expression pattern analysis, common pathophysiological processes highly similar in COVID-19 and cystic fibrosis, including the Toll-like receptor signaling pathway. Fig. 3 summarizes the pathologies with a dysregulated TLR4/NF-κB axis and/or oxidative stress, which leads to the activation of a hyperinflammatory state (81-94). In all these pathologies, novel anti-inflammatory molecules, such as AGE, SAC and S1PC, hold promise (81-94).

The main purpose of the present review was to discuss the possibility that AGE and its components, SAC and S1PC, might be employed in the treatment of several human pathologies that are characterized by a hyperinflammatory state. A partial list of published examples is reported in Fig. 3 (81-94). In most of these pathologies, dysregulation of the TLR4 and NF-κB pathways have been observed. In this respect, a large consensus has been reached sustaining the concept that AGE, and the most important constituents SAC and S1PC, inhibit TLR4 and NF-κB. SAC has been shown to be of interest for the treatment of lung pathologies (81,82), neurological diseases (83,84), osteoarthritis (85), muscular atrophy (86,87), cardiovascular diseases (88,89), diabetes (90,91) and cancer (92-95). In addition, in the possible use of AGE in the therapy of human pathologies, the antioxidative effects of AGE should be noted, whereby the oxidative stress of cells is altered in different pathologies. This is important for evaluating AGE and its constituents as treatments for hematological diseases characterized by oxidative stress of the erythroid cells. For instance, AGE and AGE components have been proposed for the treatment of sickle-cell diseases (95-97). In this respect, a Trade Mark for S-1-propenyl-l-cysteine (S1PC^TM) has been recently obtained by Wakunaga Holdings Co., Ltd., Japan (registered on July 9, 2024; https://branddb.wipo.int/; accessed on March 20, 2025).

To maximize the impact of the studies described in the present review, further research and development are necessary. Clinical trials are expected to verify possible applications on human pathologies in the real world. Taking advantage of the increase in genomic information, one notable issue is related to the design of personalized therapeutic intervention. In this context, pharmacogenomic studies should be considered and undertaken within clinical trials, to predict the possible response (or no response) to the proposed AGE-based treatments. In this respect, combined therapies using AGE (or its constituents) alongside established therapeutic drugs or RNA- and DNA-based drugs is expected to be an attractive area of investigation. For instance, Reyes-Soto et al (94) found that SAC exhibits cytotoxicity in glioblastoma cells and improves the effect of temozolomide through the regulation of oxidative responses. Regarding the effects on TLR4, NF-κB and pro-inflammatory gene expression, we anticipate development based on the co-treatment with other currently available molecular inhibitors, including RNA-based therapies using microRNAs, used to specifically downregulate TLR4 and NF-κB, as previously suggested (98,99).