Nanoencapsulated Syzygium aromaticum oil alleviates acetic acid-induced ulcerative colitis in rats by influencing critical redox, NF-κB/iNOS, and Keap1/Nrf2/HO-1 signaling pathways

Ulcerative colitis (UC) ultimately leads to widespread mucosal disruption and ulceration. It is regarded as a type of crippling inflammatory bowel disease caused by the autoimmune system (Niu et al. 2015). UC can result in significant and sometimes fatal consequences, including colon cancer (Olén et al. 2020), circulatory and respiratory issues (Chu et al. 2017). The elevated production of free radicals and the decreased antioxidant capacity are recognized characteristics of inflammatory bowel disease (IBD) despite the primary etiology of UC remaining unknown (Amirshahrokhi 2019). The pathophysiology of UC is significantly influenced by oxidative stress and excessive inflammation (Tahan et al. 2011). In animal models of colitis, the elevated production of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), is crucial (El-Akabawy and El-Sherif 2019). Nuclear transcription factor kappa B (NF-κB) plays a vital role in the induction of UC as it stimulates the production of various inflammatory mediators, including cyclooxygenase-2 (COX-2) (Ahmed et al. 2014). Through the metabolism of arachidonic acid, COX-2 triggers the production and release of prostaglandins in the metabolic pathway of IBD (Le Loupp et al. 2015). Indeed, the massive production of nitrogen and reactive oxygen species (ROS) metabolites impacts the defense mechanism of the intestinal mucosa (Balmus et al. 2016). The damage to the intestinal mucosa may occur through the stimulation of inducible nitric oxide synthase (iNOS) and COX-2, which raises free radicals and suppresses the antioxidant system (Wang and DuBois 2010).

An accessible, reasonably priced, well-researched, and popular experimental paradigm for IBD research is acetic acid (AA)-induced UC (Randhawa et al. 2014). This model resembles the pathophysiology of human IBD and is linked to increased vasopermeability, prolonged neutrophil infiltration, and an elevated inflammatory mediator profile (Thippeswamy et al. 2011). Therefore, AA-induced colitis could serve as a valuable model to evaluate the effectiveness of potential treatments that address the pathophysiology of oxidative stress and inflammation (Low et al. 2013). One significant preventative and therapeutic treatment for UC would be the inhibition of lipid peroxidation and the scavenging of oxygen free radicals (Kassab et al. 2022). Even though there is no known cure for UC, the FDA has approved some medications, including antibiotics, antimicrobials, and anti-inflammatory agents, such as corticosteroids, sulfasalazine, and aminosalicylates (Dubinsky, 2017). However, using these drugs is linked to several adverse side effects, such as nephrotoxicity, hepatotoxicity, headaches, nausea, and epigastric discomfort (Kayal and Shah 2019; Tripathi and Feuerstein 2019). Therefore, there is a growing need to investigate safer and more tolerable alternative medicine alternatives (such as herbal remedies) to treat this illness.

Recent studies have demonstrated that encapsulation-based drug delivery methods can enhance the therapeutic effects of medicines by increasing their stability in vivo, facilitating target accumulation, and improving cellular and tissue absorption (Patil and Deshpande 2018; Zhang et al. 2018). Syzygium aromaticum is a tree from the Myrtaceae family, originating in Asia and commonly known as clove (Abtahi-Eivari et al. 2021). Clove oil (CO) is an essential oil extracted from the flowers, buds, leaves, and stems of the clove tree. CO is well-known for its antiviral, antifungal, antibacterial, and antioxidant properties (Bakour et al. 2018). These therapeutic properties of S. aromaticum extract are due to the presence of eugenol, carvacrol, thymol, and cinnamon aldehyde (Chen et al. 2016), eugenol acetate, and β-caryophyllene (Milind and Deepa 2011), which are in charge of the oil's functional properties, including its antioxidant (Gülçin et al. 2012; Nisar et al. 2018), anti-inflammatory (Chaieb et al. 2007), anti-stress (Singh et al. 2009), and antimicrobial capabilities (Cui et al. 2015). The encapsulation of CO or eugenol, through methods, such as microencapsulation, nanoencapsulation, or liposomes, may mitigate the drawbacks of CO or eugenol, including toxicity, low aqueous solubility, poor environmental stability, high pungency, and diminished bioavailability (Cortés-Rojas et al. 2014; Sebaaly et al. 2015; Yadav and Balasubramanian 2016). The protective effects of clove oil nanoemulsion against titanium dioxide nanoparticle toxicity have been previously demonstrated through its enhancement of antioxidant and anti-apoptotic effects, as well as its prevention of genotoxicity (Mohamed et al. 2024). Eugenol makes up 89% of the essential oil found in clove flower buds (Cortés-Rojas et al. 2014). Eugenol has recently been shown to possess numerous biological properties, including anti-proliferative, antioxidant, anticancer, anti-inflammatory, and antimicrobial activities (Al-Trad et al. 2019; Purkait et al. 2020; Fangjun and Zhijia 2018). Polymeric nanoparticles have gained attention recently as a potential novel method for medicine delivery. Among all aliphatic polyester polymers, polycaprolactone (PCL) is the most suitable nano-drug delivery vehicle for various applications due to its distinct and adjustable physicochemical characteristics (Washington et al. 2017; El Yousfi et al. 2023). PCL's low glass transition and melting points, semi-crystallinity, and strong mechanical flexibility enable it to exhibit exceptional drug loading capacity and consistent controlled release characteristics (Sisson et al. 2013; Blázquez-Blázquez et al. 2019). Chitosan (CS) nanoparticles are an example of a functional nano-formulation that stabilizes synthetic medicinal plant essential oils and their active ingredients (Negi and Kesari 2022). For various pharmacological compounds, CS is an economical carrier (Piras et al. 2015). Its cationic charge, inherent antibacterial potential, availability, safety, biodegradability, and biocompatibility are key features that make it an excellent carrier system (Jamil et al. 2016). Another natural biopolymer that can be obtained from brown seaweeds is sodium alginate (ALG), which has potential applications in drug delivery due to its hydrophilicity, biocompatibility, and ease of gelation (Bhattacharyya and Ray 2015).

The desirable features of nanostructures with high surface-area-to-volume ratios boost the CO-releasing capacity of nanoparticles for UC applications. PCL was selected over other polyesters, such as poly(lactic-co-glycolic acid) (PLGA), because it is cost-efficient, lipophilic to enhance passive absorption, and biocompatible (Haas et al. 2005). These attributes, combined with the bioadhesive properties of CS and ALG, are believed to enhance drug absorption through mucosal tissues, such as the bladder wall, and improve cellular interaction. Coated PCLNPs were employed in this study for this purpose, as previous research demonstrated their effectiveness in mucosal drug delivery. Using in vitro characterization methods, including particle size, surface charge, encapsulation efficiency, controlled release, and cellular contact, this study aims to develop and optimize a nanoparticulate carrier based on cationic and anionic polymers for encapsulating CO. According to the existing literature review, no studies have evaluated the efficacy of CO-loaded PCL coated with CS and ALG (CONPs) against AA-induced colitis in rat models. This research was conducted to investigate the potential preventive effects of CONPs (PCL@CO(CS + ALG)NPs) on AA-mediated UC, while also exploring mechanisms related to oxidative stress, inflammation, and histological changes. These results provide new insights for creating safe and effective treatments for UC.

The prevalence of UC continues to rise worldwide, accompanied by increasing medical and social expenses. The projected yearly direct and indirect expenditures associated with UC in the United States alone range from $8.1 billion to $14.9 billion (Cohen et al. 2010). In addition, the prevalence of UC has been steadily increasing (Ng et al. 2017), particularly in Asia, where it was previously low (Ng et al. 2019). The fact that chronic UC raises the risk of colon cancer (Dulai and Jairath 2018) and can be fatal if left untreated, which makes this alarming (Chu et al. 2017).

Chronic conditions like UC are thought to be influenced by oxidative stress (Shen et al. 2020). The present investigation found that AA significantly increased MDA and NO levels. This could be because active neutrophils and macrophages created nitric oxide, which in turn caused lipid peroxidation and the generation of excess MDA. El-Akabawy and El-Sherif (2019), Abdelaziz and Mahmoud (2023), and Rehman et al. (2024) reported similar results of high levels of MDA after injection of AA. Proinflammatory cytokines, such as TNF-α, activate iNOS, leading to increased nitric oxide production in UC. This increase promotes the generation of free radicals and attracts neutrophils and macrophages, thereby contributing to the pathophysiology of UC. Moreover, features, such as edema, erythema, inflammation, and diarrhea, are linked to nitric oxide levels in both serum and colon tissues (Kamalian et al. 2020). Colitis significantly decreases the levels of SOD and CAT, which have an essential protective response against oxidative stress (Abdelmegid et al. 2019; Rehman et al. 2024). Moreover, ROS production in the intestinal mucosa impairs blood flow, resulting in desquamation and loss of epithelial cells (Coskun 2014). Our results showed that colon tissue in the UC group had significantly higher levels of MDA, a commonly used indicator of lipid peroxidation, than in the control group. In contrast, the levels of GSH, GPx, and GR, as well as the enzymatic activity of the antioxidants SOD and CAT, were significantly lower in the UC group than in the control group. The low concentration of those antioxidant enzymes, which are primarily found in epithelial cells, may make the gut more vulnerable to oxidative damage (Cetinkaya et al. 2005). Our findings are also supported by earlier research (Badr et al. 2020; Rafeeq et al. 2021). Our histological results demonstrate that excessive generation of ROS and nitrogen metabolites does impact the intestinal mucosa's defense mechanism and eventually encourages the subsequent development of further inflammation and ulceration (Kruidenier and Verspaget 2002).

The shift in the expression pattern of the genes being studied may be caused by the acetic acid-induced colitis model, where inflammatory mediators, such as reactive oxygen species, vasoactive amines, and eicosanoids, play a significant role (Shahid et al. 2022). The underlying pathophysiological mechanisms involve disruption of blood clotting, elevated inflammatory mediators, increased vascular permeability, damage to colon structure, and mucosal barrier breakdown caused by chemical stimulation, which promotes fibrin hydrolysis. This creates a vicious cycle that generates more reactive metabolites, depletes cellular antioxidants, and further promotes inflammation and ulceration. An increase in lipid peroxides in colonic tissue can initiate this cycle (Thippeswamy et al. 2011). During inflammation, cells generate ROS and reactive nitrogen species (RNS), which are highly reactive molecules capable of damaging proteins, lipids, and nucleic acids within cells. In the gastrointestinal tract, oxidative stress additionally promotes the increased expression of genes associated with immune responses (Pravda 2005; Alzoghaibi 2013). In the current study, the macroscopic appearance of the colon in the model group, injected intrarectally with 1.5 ml of AA (5%), displayed hyperemia and ulceration, with a significantly increased mean score of colitis (4.8 ± 0.2) compared to the control group. The exact etiology of UC remains unknown; several processes are known to be involved in its pathogenesis (Graham and Xavier 2020). In this study, UC is primarily characterized by the initiation of oxidative stress, depletion of antioxidants, and increases in pro-inflammatory markers. Moreover, in this context, the UC group shows an increase in NO and PGE levels in colonic tissue. Sheibanie et al. (2007) noted that in DC, PGE2 inhibits the production of IL-12/IL-27 and promotes the expression of IL-23. In vivo, treatment with PGE analogs leads to the expansion of Th17 cells in the inflamed gut and elevated levels of IL-23 and IL-17.

NF-κB and TNF-α are key pro-inflammatory mediators involved in the pathophysiology of UC. When a damaging stimulus occurs, TNF-α is released, subsequently activating the NF-κB pathway. This activation leads to the inflammatory cascade by increasing the production of inflammatory mediators (Salama et al. 2021). TNF-α stimulates macrophages and neutrophils in the colon wall. This causes widespread intracellular acidification, resulting in acetic acid-induced colitis that damages mucosal barriers, injures the colonic epithelium, and triggers the release of inflammatory cytokines, which promote the recruitment of monocytes and macrophages (Jang et al. 2023). According to Shapiro et al. (2007), inflammatory cytokines (NF-κB, IL-6, IL-1β, and iNOS) release several chemokines, which in turn encourage chemotaxis. Antioxidants protect by removing ROS from the environment, blocking their synthesis, or shielding transition metals needed for free radical production (Masella et al. 2005). Histological sections of the colon revealed inflammation that was parallel and confirmed by increased levels of pro-inflammatory markers in the serum of the UC group. It is well-known that TNF-α promotes the production of chemical mediators, proteases, and pro-inflammatory factors, which then induce chemotaxis and infiltration of inflammatory cells. Furthermore, it facilitates neutrophil infiltration by increasing the levels of adhesion molecules, leading to tissue damage (Muthas et al. 2017). Moreover, IL-6 has been linked to the severity of UC patients' disease and is essential for pro-inflammatory processes and immune function dysregulation (Bernardo et al. 2012), which can result in ulceration and bleeding by producing cytotoxic reactive oxygen species (Jainu et al. 2006; Munakata et al. 2003). As anticipated, we found that AA treatment significantly increased the levels of all pro-inflammatory cytokines and mediators, including TNF-α, IL-10, and IL-1β, in this investigation. Similarly, earlier research by Gu et al. (2017), Impellizzeri et al. (2018), and Zhu et al. (2019) showed that the colitis model significantly increased TNF-α, IL-1, and IL-17. Furthermore, following AA injection, Badr et al. (2020) noted a substantial increase in TNF-α, IL-1, IL-6, and IL-17.

The COX-2 enzyme is a key element in the complex network of biochemical mediators involved in UC's pathophysiology. It produces PGE2, which helps regulate apoptotic mediators and pro-inflammatory cytokines and may also play a role in the malignant consequences of UC. Our study found that COX-2 levels are increased in the inflamed colon (Kjærgaard et al. 2020). By encouraging neutrophil and macrophage infiltration into the colon wall, NF-κB is one of the central pro-inflammatory mediators that drives inflammation in UC (Laurindo et al. 2023). Additionally, NF-κB through IL-1β increases the expression of the COX-2 enzyme, which is previously known to play a role in inflammation (Chiba et al. 2012).

Neutrophils contain the enzyme myeloperoxidase, whereas monocytes and macrophages have considerably lower levels of this enzyme. Consequently, MPO activity is a quantitative and sensitive assay for acute intestinal inflammation and is directly correlated with the neutrophil concentration in the inflammatory region (Choudhary et al. 2001). In untreated rats, the colon exhibited elevated MPO levels, suggesting neutrophil infiltration and extracellular MPO release. This process contributes to mucin degradation, enhances mucosal permeability, and harms epithelial cells by catalyzing the oxidation of chloride with hydrogen peroxide, which generates hypochlorous acid, a reactive compound oxidant. These findings align with those reported by Cetinkaya et al. (2005), Kruidenier and Verspaget (2002), and Rafeeq et al. (2021). Histological colon sections showed thickened submucosa due to inflammation, cell infiltration, and edema, confirming the UC model. Mononuclear cells caused degeneration of the muscularis externa. Fibroblasts, leukocytes, and epithelial cells secrete VEGF, which promotes angiogenesis and increases vascular permeability (Bousvaros et al. 1999). In certain patients with colitis, VEGF was produced from epithelial mucosal cells (Taha et al. 2004).

Nrf2 is a crucial transcription factor that acts as an antioxidant. During oxidative stress, it translocates to the nucleus and activates, enhancing the production of various antioxidant enzymes downstream (Deng et al. 2022). It has been found that Nrf2 overexpression improves UC (Tan and Zheng 2018). The central inducible defense against oxidative stress is the Keap1-Nrf2 stress response system, which controls the production of cytoprotective genes (Baird et al. 2020). Under typical circumstances, the cullin-based E3 ubiquitin ligase, which impairs Nrf2 transcriptional activity through ubiquitination and proteasomal degradation, utilizes Keap1 as a substrate adapter (Kobayashi et al. 2004). This could account for the different expression patterns revealed by our analysis for the Keap1 and Nrf2 genes. The rate-limiting enzyme in the heme catabolic pathway is heme oxygenase (HO-1), which breaks down heme into equimolar quantities of free iron, biliverdin, and carbon monoxide (Consoli et al. 2021). It is recognized as a stress-responsive protein with anti-inflammatory, anti-apoptotic, and antioxidant properties, and it is hypothesized to carry out several protective functions against diverse stresses (Mohamed et al. 2022). Threonine/tyrosine phosphatase, which is involved in cell cycle regulation, is encoded by Cdc25c (Cho et al. 2015). It controls the transition from the G₂ to the M phase of the cell cycle, blockage results in cell cycle arrest and increased apoptosis. DNA repair involving non-homologous end joining, homologous recombination, and nucleotide excision is carried out by RNF8 (Lu et al. 2012). According to Aichinger et al. (Aichinger et al. 2022), the Alternaria toxin causes the rat colon to become positive for the DNA damage markers Cdc25c and RNF8. In the current study, rats exposed to acetic acid had their expression patterns for NFKB, IL-6, IL-8, and iNOS, as well as for antioxidant (Nrf2, Keap1, and HO-1) and DNA damage (Cdc25c and RNF8) genes, evaluated. Compared to the control group, our findings showed that the genes NF-κB, IL-6, IL-1β, iNOS, Keap1, HO-1, and Cdc25c were up-regulated. Nrf2 mRNA levels, on the other hand, decreased. According to Shahid et al. (2022), acetic acid increases the expression patterns of apoptotic (Bax and caspase-3) and inflammatory (TNF-α, IL-6, MPO, PGE2, COX-2, and NF-κB) indicators. But there was a decrease in the anti-apoptotic protein (Bcl-2). Furthermore, compared to the control group, rats treated with acetic acid showed a significant increase in colonic TLR-4 gene expression (Alanazi et al. 2023). Fawzy et al. (2022) also mentioned that in colitis caused by acetic acid, the expression profile of the IL-1β gene is up-regulated.

Poor treatment outcomes and the frequency of adverse effects have created a need for novel, practical therapeutic approaches (Yuan et al. 2006; Clark et al. 2007). As current immunosuppressants, sulphonamides and hormone agents used for the treatment of UC only control the UC symptoms, and unfortunately, induce side effects and hormone resistance (Gao et al. 2019). Recently, plant-based drugs have been gaining more attention due to their potential effect in UC (Liu et al. 2021; Patel et al. 2022). Besides having limited side effects, they are increasingly being utilized for the treatment of IBD due to the beneficial effects of their antioxidants on experimental colitis (Nosál'ová et al. 2000). This is the first study to compare the potential protective and therapeutic effects of clove oil versus clove oil coated with CS and alginate nanoparticles. In the current study, pretreatment with CO or CONPs exhibits powerful antioxidant and anti-inflammatory effects, thereby mitigating the occurrence of UC.

To the best of our knowledge, a limited number of studies have been conducted on the effects of essential oils on in vitro cell migration. In addition, CO's high volatility and susceptibility to deterioration from exposure to oxygen, heat, and light limit its use (El Asbahani et al. 2015). Various preparation techniques have been employed to create CO polymer carriers, including nanoparticles, to increase the application of CO (Alam et al. 2017; Cui et al. 2018). There are benefits to using nanoemulsions, including improved delivery efficacy, physical stability, and resistance to volatility and degradation (Singh et al. 2009). The CS-induced mucoadhesion effect was confined to the surface of each droplet. Due to the droplets' nanometric size, which resulted in an enhanced surface-to-volume ratio, the encapsulated oil was able to make greater contact with the mucosal surface. By improving the process of passive transport, pharmaceuticals enclosed in emulsions enable the absorption of medicinal ingredients with increased particle uptake. Additionally, nanoemulsions are anticipated to pass through biological barriers without encountering any obstacles (Alzorqi et al. 2016). For NPs loaded with CO to be transported to the colon's inflammation site and enhance epithelial permeability and retention effect (Xiao et al. 2017), biomaterials composed of ALG and CS can be broken down in the colon without being harmed by the gastric environment (Zhang et al. 2018).

Originally native to Asia, S. aromaticum is recognized for its high content of phenolic compounds. Cloves have significant potential in food, medicine, cosmetics, and agriculture. Due to its medicinal properties, the essential oil derived from clove is extensively utilized. (Ramadan et al. 2013). It contains eugenol, phenols, flavonoids, oleanolic acid, and tannins, which have been demonstrated to reduce oxidative stress-induced damage to the brain, liver, heart, kidneys, and testicles (Abtahi-Eivari et al. 2021; Barghi et al. 2021). In recent years, essential oils derived from various plant sources have been used to treat a wide range of illnesses. It has been stated that using CO, which is produced from the dried buds of S. aromaticum, can reduce pain and aid in the healing process (Chaieb et al. 2007). Eugenol, a primary phenolic component of CO, has been linked to several critical pharmacological advantages, including anti-inflammatory, antibacterial, and antifungal characteristics (Kalemba and Kunicka 2003). Given that CO is highly volatile, efforts were made to encapsulate phenolic CO in delivery vehicles that are nanoscale, biocompatible, and biodegradable (Anwer et al. 2014).

The uniform spherical nano-PCL@CO formulations produced by the emulsion-evaporation technique have diameters that progressively increase with increasing CO supplementation. Despite this, the CO coated with ALG/CS embedded through PCL had consistent particle distribution. A new peak emerged in the nano-formulations upon the addition of CO. According to Liverani and Boccaccin (2016), peaks corresponding to carbonyl (C = O) stretching and both symmetric and asymmetric stretching of C–O–C bonds were found at 1725.805, 1244.137, and 1166.250 cm⁻¹, respectively. Additionally, in earlier research, distinctive FT-IR peaks representing the C–C aromatic ring vibrations of eugenol were observed at 1637 cm⁻¹, 1606 cm⁻¹, and 1513 cm⁻¹ (Yadav and Saini 1994; Smith et al. 1988; Mohammed et al. 2016). As explained by Smith (1988) and Keawpeng et al. (Keawpeng et al. 2022), the stretching vibration of C = C aromatic bonds is responsible for the CO bands that were detected at 1514 cm⁻¹, which corresponds to the primary component of CO—eugenol. The GC–MS analysis was used to confirm the presence of the appropriate components in the clove oil. These peaks were retained, and the CO nano-formulations exhibited a discernible peak shift, indicating that the CO and nanoparticles were indeed present in the matrix. The interactions between the constituent parts of the bionanocomposite may explain the apparent shift of these peaks. Natural nanoparticle clustering was observed in SEM images; this is most likely the result of operations where the surfactant was not present, leading to inadequate steric stabilization. Drug carriers with a diameter of less than 400 nm are confirmed to be the ideal size for administering UC drugs. Smaller NPs (less than 400 nm) can therefore be utilized as UC delivery vehicles, with a high potential to enhance the effectiveness of UC treatment. Moreover, particle size affects the process of particle absorption by cells. Because PCL polymer has carboxylic end groups (Öcal et al. 2014) and anionic biopolymer ALG contains carboxylic and hydroxyl end groups, it is expected that all created NP formulations will have a negative surface charge. Conversely, when coating with the CS, the zeta potential values changed to positive ones. The amino groups in CS, which were present on the surface of the nanoparticles, cause the negative zeta potential to change into a positive one. Therefore, having a charge that may readily engage through an ionic adsorption process with the nexus charged cell membrane may result in increased cellular absorption. Polysaccharide-based NPs, including CSNPs, are known to shrink or lose form when exposed to electron bombardment during SEM investigation, in contrast to polymeric nanoparticles like PCL and PLGA.

After being suspended in water (w/v 2/1) for a short while, the nanoparticles were ultrasonically agitated. The infiltrative ability of the generated nano-PCL@CO formulations has been significantly enhanced using water as a potential nanoparticle carrier vehicle, which is crucial for effective particle penetration. The percentage of EE is substantially impacted by the degree of CO solubility in the material. Furthermore, the emulsion-evaporation process typically results in high encapsulation values for hydrophobic substances. Considering this, a maximum %EE of 96.85% and a %LC of 20.39% were attained. According to the absorption spectra of essential CO, the electronic transition from the type *n–π for the alcohol group that binds with the aromatic ring, * n–π of the group carbonyl binds with the aromatic ring of quinone, and * π–π of the group ketone that binds with the aromatic ring and gives these compounds their yellow color occurred at 210, 227, and 282 nm, respectively (Nahar and Sarker 2019). This demonstrated the presence of clove's primary bioactive ingredient, eugenol. Therefore, using alcohol as a blank, the absorbance of CO was measured at 282 nm.

The release of CO close to the surface of nano-PCL@CO formulations caused the in vitro cumulative CO-release to follow a biphasic pattern, with an initial fast CO-burst lasting 4 h (Sansdrap and Moës, 1997). According to earlier descriptions, CO, especially surfactant-like eugenol, has an attraction for surfactant molecules, which may cause some of it to migrate to the surface of the nanoparticle (Tchakalova et al. 2008). PCL's carboxyl groups may have reacted with CO's OH groups, including eugenol, during encapsulation (DeRuiter, 2005).

Over time, the medicine can be released from nanoparticles because this phenomenon is reversible. As a result, after 120 h, a continuous progressive release of 29%, 33.5%, 41.5%, and 48% of CO was seen, caused by a combination of hydrolysis, degradation, and polymer diffusion (Sahana et al. 2008). The retention of the remaining released CO in the PCL matrix may be the cause of the emission of such small amounts. Although the CO-release profile in this study was only measured for 120 h, the steadier, longer release pattern suggests that CO will likely be emitted for a more extended period. The presence of the adsorbed drug on the surface of nanoparticles containing excess amounts of necessary CO, which were quickly dissolved into the medium from the matrix PCL, may have caused the drug's first burst release. The most crystalline and hydrophobic polymers degrade at the slowest rate (Lemoine et al. 1996; Lamprecht et al. 2000). Despite being hydrophobic as well, vital CO was released far less from PCL nanoparticles. According to some researchers, the gradual breakdown of polymers in the release media may cause release from these polymers to last for several months (Malhotra and Majumdar 2001). The burst effect's strength and the total rate of CO release were both decreased by the CS and ALG coating. The existence and function of the outer ALG layer as an additional barrier for CO release may help explain this. To further limit drug release, CS and ALG can interact with biological fluids, salts, and various delivery media to which formulations are exposed. Because CS offers a closed layer of PCL@CONPs, which serves as a physical barrier to the diffusion of CO into the bulk solution, the slow release of CO may be explained. In the past, PCL-based NPs have been used to extend the release pattern of numerous drugs (Coffin and McGinity 1992). The drug connection near the particle surface, and upon contact with the dissolving solvent, may be the cause of the initial burst release of the drug from the nano-formulations. On the other hand, the drug's regulated release from the nano-formulations may be caused by the polymeric shell's obstructive effects and increased diffusion distance (Kalimouttou et al. 2009; Patel et al. 2014).

An in silico study revealed the molecular interaction abilities of eugenol, the most abundant bioactive compound of S. aromaticum, with NF-κB, IL-6, iNOS, Keap1, and HO-1 proteins. This study is a molecular screening conducted before an in vivo study. Administration of CO or CONPs attenuates oxidative stress induced by acetic acid. In the rat group with CO, a significant decline in colonic levels of MDA and NO was observed when compared to the untreated groups. Additionally, pretreatments restore the antioxidant equilibrium by inducing a considerable increase in the levels of SOD, catalase, GSH, GPx, and GR compared to the UC group and the carrier group. Notably, the glutathione group is involved in the synthesis and repair of DNA, blocking free radical damage (Chavan et al. 2005). Our findings are in correspondence with the studies of Hassanen (2010) and Soltani et al. (2023), which revealed a decline in MDA levels and an increase in GPx activity and GSH after administration of clove oil in diabetic and PCOS rats, respectively. This may be related to the high content of eugenol derivatives in clove oil. As an antioxidant, eugenol effectively inhibits protein denaturation, which may maintain the integrity of the cell membrane and prevent intracellular enzyme leakage (Ulanowska and Olas 2021), thereby illuminating the integrity of colon tissue in treated groups, especially those pretreated with CONPs.

Clove oil or in nano-form diminishes infiltration of large numbers of phagocytic leukocytes into the mucosal interstitial, confirmed microscopically in histological sections of treated groups (CO and CONPs) as compared to UC group in the current study, reflecting powerful anti- inflammatory effect of CO. This may also have attributed to eugenol derivatives compound, it has anti-inflammatory effect, and prevent prostaglandin synthesis. Our findings revealed a significant reduction in all measured inflammatory markers and mediators of the CO and CONPs groups when compared to UC, including MPO, TNF-α, IL-6, IL-1β, and IL-10. This confirms less infiltration of colon tissue by inflammatory cells. Moreover, PGE levels declined sharply in groups treated with clove CO or CONPs compared to the untreated UC group. Prostaglandins have been demonstrated to promote the production of inflammatory exudates during tenderness. Taher et al. (2015) found that clove oil has anti-inflammatory properties, reducing edema production three hours after carrageenan injection. This suggests that the oil's effects are likely mediated by interfering with the production of prostaglandins.

An in silico approach revealed that eugenol exhibited high binding affinities to NF-κB, IL-6, IL-1β, iNOS, Keap1, HO-1, and Cdc25c, which may consequently lead to a decrease in their expression. The gene expression approach was employed to investigate the ameliorative effects of clove oil, PCL@CS + ALG, or clove oil nanoparticles in acetic acid-induced colitis. Rats exposed to clove oil, PCL@CS + ALG, or clove oil nanoparticles elicited a significant downregulation of NF-κB, IL-6, IL-1β, iNOS, Keap1, HO-1, Cdc25c, and RNF8. However, Nrf2 genes were significantly up-regulated, as reported by Jabbari et al. (2020), who stated that eugenol encapsulated in CS nanoparticles reduces TGF-β gene expression in an aggressive form of rheumatoid arthritis. According to earlier research, clove oil acts as an anti-inflammatory on COX-2 and 5-LOX, altering the NF-κB pathway and reducing the synthesis of interleukin-6 (Karaji et al. 2023). By affecting the NF-κB pathway and interleukin-6 synthesis, which are crucial elements of inflammation, clove oil appears to reduce NLRP1 mRNA and protein expression (Wie et al. 1997). Research has demonstrated that the main component of clove oil, eugenol, essentially possesses immunomodulatory, DNA-protective, anti-genotoxic, antioxidant, and anti-inflammatory properties (Yogalakshmi et al. 2010; Sharma et al. 2011). Because it inhibits prostaglandin synthesis and suppresses neutrophil/macrophage chemotaxis, eugenol also has anti-inflammatory properties (Estevão-Silva et al. 2014). Furthermore, rheumatoid arthritis may be treated with CS nanoparticles, which Eugenol has identified as a potent antioxidant (Jabbari et al. 2020).

In general, the nano-formulation of clove oil was more effective in the prevention and treatment of UC than the natural form of the CO, where most of the parameters returned to normal levels. The co-treatment with CONPs restored colon catalase and GR activities to their normal values, like those of the control rats. Interestingly, the lower level of oxidative biomarkers and the higher level of antioxidant molecules were detected in rats treated with NCOPs. Additionally, only in the CONPs group does the number of goblet cells increase to the normal value. In comparison to the other groups, the higher goblet cell count was also linked to improvements in all colitis scores and intestinal mucosal regeneration. This could be explained by the fact that the encapsulated version of clove oil has more antioxidant activity than clove oil itself (de Oliveira and Tavares 2022). Because nanoparticles have larger surface areas than comparable masses of larger-scale materials, it may be possible to enhance the bioavailability and bio-effectiveness of essential oils. Additionally, this particle size makes it possible to lower the oil dosages required to provide antioxidant and antibacterial effects (Hossain et al. 2019; Tiwari et al. 2020). Additionally, the primary antioxidant in clove oil, eugenol, had its cytotoxic effect lessened by the addition of PLA nanoparticles (de Oliveira and Tavares 2022). This explains the superior effect of CONPs over natural oil in this study.

The model of UC in this study was confirmed through histopathological sections of the colon. In the current study, the ulcerative group exhibited extensive and diffuse mucosal ulceration, characterized by loss of the entire surface epithelium and crypts, as well as a significant increase in both the inflammation score and the inflammatory infiltration score compared to the control group. In addition, the submucosa was markedly expanded, primarily due to the importance of inflammation, infiltration with inflammatory cells, and edema of blood vessels. Scores were significantly increased compared to the control group. Previous investigators have also detected such results (Abd El-Galil et al. 2015; Lean et al. 2015; Amal et al. 2018).

A significant decrease in the number of goblet cells was also observed in the UC model of the current study. Goblet cells produce mucins to maintain the mucus barrier. It separates intestinal microbial flora from the epithelium and aids in immunological activities like antigen presentation and tolerance (Merga et al. 2014). Recent research has highlighted the role of the mucous barrier in IBD pathogenesis, which is accompanied by a deficiency in the differentiation of intestinal stem cells to goblet cells (Gersemann et al. 2009, 2011) and a deficiency in the intestinal mucous barrier. Only in the CONPs group was the number of goblet cells increased to the normal value. The increased number of goblet cells was concurrently associated with improvement of all colitis scores and the regeneration of the intestinal mucosa compared to the other groups.

VEGF is a cytokine produced by epithelial cells, leucocytes, and fibroblasts that promotes vascular permeability and enhances angiogenesis (Bousvaros et al. 1999). In certain patients with colitis, VEGF was produced from the epithelial mucosal cells (Taha et al. 2004). In the current study, the expression of VEGF in the mucosa of the colon from the colitis group was barely detectable, as the colitis group exhibited extensive and diffused mucosal ulceration, resulting in the loss of the entire thickness of the surface epithelium and crypts. On the other hand, its expression was significantly increased in the submucosa of the colon from the UC group, suggesting a role for mononuclear inflammatory cells in the submucosa in the secretion of VEGF, a finding similar to my own, as reported by Koch et al. (1986) investigated that activated macrophages are sufficient to secrete VEGF. About the role of VEGF in the pathogenesis of UC, VEGF increases the permeability of blood vessels and enhances the infiltration of inflammatory cells in the UC group (Tolstanova et al. 2010). The expression of VEGF was nearly returned to the normal level in the CONPs group, as was the mononuclear cell infiltration score.

Myofibroblasts are specific cells that share properties of smooth muscle cells and fibroblasts. Their ability to produce chemokines, cytokines, matrix components, and prostaglandins is believed to play essential roles in inflammation, growth, repair, and neoplasia (Adegboyega et al. 2002). Colonic myofibroblasts can be either subepithelial or interstitial. Subepithelial myofibroblasts are known as colonic stellate cells. These cells are located at the crypt base, between the mucosal epithelium and capillaries, and play a role in UC regeneration. Interstitial myofibroblasts, found throughout the lamina propria and submucosa, contribute to fibrosis (Okayasu et al. 2009, 2011). In the current study, immune expression was detected only in the myoepithelial fibroblasts of the control group and CONPs groups. The interstitial myofibroblast with positive α-SMA immune expression was detected in the submucosa of the UC group and lamina propria and submucosa of CO and PCL@CS + ALG groups (hardly detected in the mucosa of the colitis group as the colitis group exhibited extensive and diffused mucosal ulceration with loss of the entire thickness of surface epithelium and crypts. My findings are compatible with the results of Adegboyega et al. (2002), who demonstrated that subepithelial myofibroblasts with positive α-SMA immune expression were found in the normal colonic mucosa; meanwhile, the interstitial myofibroblast with positive α-SMA immune expression was detected in the colon of a patient with UC and significantly increased compared to the control colon (Andoh et al. 2002). In UC patients, there is an increase in interstitial myofibroblasts and a decrease in subepithelial myofibroblasts, which promote regeneration (Okayasu et al. 2009). Fibroblasts were activated into myofibroblasts under the effect of inflammatory cytokines (Stallmach et al. 1992). In the current study, the immune expression of α-SMA was assessed in parallel with the score of inflammatory cell infiltration in all groups.

In addition to that, α-SMA acts as a powerful protein in the identification of normal and pathological conditions of smooth muscle (Okayasu et al. 2009). α-SMA immune expression was also detected in the smooth muscle cells of the lamina muscularis and tunica muscularis of the colon. In the current study, the lamina muscularis appeared attenuated, and the tunica muscularis appeared degenerated in the UC group. Consequently, the α-SMA immune expression was significantly decreased in these layers compared to the control group. The normal histoarchitecture of the lamina and tunica muscularis was nearly detected in the CONPs group; therefore, the α-SMA immune expression was almost returned to the normal level in this group.

A limitation of the present study is that only a single dose level of clove oil and its nano-formulation (250 mg/kg) was tested, without the inclusion of a standard positive control drug. Although this dose was carefully selected based on OECD guidelines and previous toxicity data to ensure safety and biological efficacy, the absence of a dose–response design and therapeutic reference limits the full extrapolation of the findings. Future investigations will therefore be extended to evaluate multiple dose levels and to include a positive control (e.g., sulfasalazine or mesalamine) in order to comply with standard preclinical requirements and provide more comprehensive evidence of therapeutic efficacy.

In conclusion, CO-loaded PCLNPs coated with CS/ALG (CONPs) significantly reduced oxidative damage and inflammatory mediators in the colon damaged by AA. Presumably, this protection may be due to: (1) CO and CONPs induced regulation of Keap1/Nrf2/HO-1 signaling pathway, which lessens damage to the colon mucosa; (2) CO and CONPs mediated inhibition of NF-κB translocation to the nucleus, which stops the NF-κB-dependent pathway; and (3) CO and CONPs generated suppression of free radicals and enhancement of endogenous antioxidant defense systems. These findings have crucially clarified the therapeutic potential of coating CO with chitosan/alginate nanoparticles to cure UC mediated by AA.