Sea grapes were procured from ponds situated within Takalar Regency, South Sulawesi Province, Indonesia. The sea grapes were then transformed into sea grape powder using a vacuum evaporator operating at a temperature of 40 °C within the PAU Laboratory of IPB University. The liver was pre-primed using an ELISA kit (BioTek 800 TS, Sigma-Aldrich). TNF-α (RAB0480, USA) was placed on a microplate using a micropipette to analyze TNF-α-mediated inflammation in the rat liver, and the resulting signal was read using an ELISA reader (800 TS). Liver tissue preparation was conducted using a Biocare kit to describe the inflammatory histology in rat liver using the TNF-α immunohistochemistry method, which was then read using an Olympus Digital BX51-P microscope. The Randox RX Monza SD 125 kit was used to prepare rat liver organs before analysis using a spectrophotometer (PerkinElmer).

The analysis of bioactive compounds from sea grape powder administered to rats consisted of an examination of antioxidants, which consisted of beta-carotene, carotene, phenol, and flavonoid content using a colorimetric method. Beta-carotene analysis was conducted using HPLC (Waters Alliance e2695) at SIG, Bogor, Indonesia. Carotenoid, phenol, and flavonoid contents were analyzed using a spectrophotometer (PerkinElmer) at the Biochemistry Laboratory of the Faculty of Medicine and Health Sciences at Alauddin State Islamic University of Makassar.

The experimental research design employed a completely randomized design consisting of six treatment groups. The animal model employed was male Wistar rats (Rattus norvergicus), with an initial body weight of 150–250 g and an age of two (2) to two and a half (2.5) months. Rats were procured from PT Biomedical Technology, Indonesia. The sample size was determined using the Federer formula (1966), which yielded 24 rats. The rats were immobilized for 14 days in an environment that was alternately illuminated for12 hours per day. The subjects were provided with a high-fat diet (46% fat) for 60 days. Subsequently, the obese rats were administered SGPD for 21 days. The rats were anesthetized using a combination of ketamine (80 mg/kg bw) and xylazine (5 mg/ kg bw) on the day following the conclusion of the treatment phase, after which liver samples were collected to determine oxidative stress, antioxidant activity, and inflammatory markers. This study was approved by the Animal Ethics Committee of the School of Veterinary Medicine and Biomedical Sciences (SKHB), IPB University (reference number: 015/KEH/SKE/VI/2021).

During the acclimatization phase, all experimental groups were provided standard feed and water ad libitum. Subsequently, for a period of eight weeks, all groups were provided with a high-fat diet, except the standard feed group, until they reached an index value of greater than 300 g/cm. The rats were divided into three control groups: standard rations (SR), high-fat ration (HFR)+carboxymethyl cellulose (CMC), and HFR+ Orlistat 10.8 mg/ kg, as well as HFR, and three treatment groups: HFR+sea grape powder drink (SGPD) 1 g, HFR+SGPD 1.5 g, and HFR+SGPD 2 g. The control group received orlistat orally for 21 days, whereas the treatment groups received sea grape powder orally for the same duration.

The efect of sea grape powder drink on inflammatory data was analyzed using a completely randomized design (CRD) with a single factor and six treatments, with three replications for each treatment. Statistical analyses were performed using Microsoft Excel and SPSS Version 22, and the data are expressed as the mean ± standard error. Diferences among the treatment groups were evaluated using one-way analysis of variance (ANOVA). When a significant efect was detected (p<0.05), the analysis was followed by Duncan’s multiple range test to determine diferences between groups.

Antioxidants play a crucial role in protecting biological systems from oxidative stress, which is associated with various diseases. In this study, the antioxidant composition of SGPD administered to rats was analyzed to assess its potential protective efects. The results of this analysis are presented in Table 1. Antioxidants play a crucial role in protecting biological systems from oxidative stress, which is associated with various diseases. In this study, the antioxidant composition of SGPD administered to rats was analyzed to assess its potential protective efects. The results of this analysis are presented in Table 1.

The antioxidant properties of Caulerpa lentilifera, commonly known as sea grapes, have been extensively studied, revealing its potential as a functional food with significant health benefits. Various extraction methods and conditions have been shown to influence antioxidant activity, with findings indicating that diferent solvents yield varying levels of bioactive compounds. One study reported that the total phenolic content in methanol extracts was 1.54 mg GAE/g, while flavonoids were only detected in methanol extracts at 1.16 mg QE/g (Diharmi et al., 2024). Another study reported that the total phenolic content of Caulerpa lentillifera ranged from 1.30–2.04 mg GAE/g with diferent treatments (Nguyen et al., 2011) and the total flavonoid content ranged from 1.17–1.47 mg GAE/g (Peerakietkhajorn et al., 2024). The carotene compounds have diferent contents based on the specific compound in the carotene group. The highest compound yielded in Caulerpa lentillifera was β-carotene at 19.5 mg/g, followed by canthaxanthin and astaxanthin, which yielded 14.6 and 3.6 mg/g, respectively (Balasubramaniam et al., 2020).

The antioxidant compounds mentioned above play a crucial role in enhancing liver function by mitigating oxidative stress and promoting hepatoprotection. The liver, a primary detoxifying organ, is susceptible to damage by free radicals generated during metabolic processes. As the primary detoxifying organ, the liver is highly susceptible to damage by free radicals generated during metabolic processes. Antioxidants help restore oxidative balance by neutralizing free radicals, reducing lipid peroxidation, and enhancing intrinsic antioxidant defense systems, such as superoxide dismutase, thereby preventing hepatocyte damage and liver dysfunction (Patil et al., 2014; Casas-Grajales & Muriel, 2015). These compounds have also demonstrated clinical benefits, such as improving graft function and patient survival in liver transplantation by reducing ischemia-reperfusion injury (Patil et al., 2014). Furthermore, natural antioxidants from dietary sources, including fruits and vegetables, have been shown to be efective in supporting liver health and preventing diseases (Gheorghe et al., 2019). However, while antioxidants show promise in improving liver health, caution is warranted due to potential pro-oxidant efects, emphasizing the need for further research to clarify their clinical applications and interactions with other treatments (Das et al., 2024).

Phenolic acids and phenols have been shown to improve liver function through their antioxidant properties. A study demonstrated that phenolic acids protect liver cells from oxidative stress caused by iron overload by activating nuclear factor erythroid-2-related factor 2 (Nrf2), which regulates antioxidant genes (Kose et al., 2023). Another study found that phenolic acids upregulate hepatic antioxidant enzymes, such as superoxide dismutase (SOD) and glutathione peroxidase (GPx), enhancing the liver’s defense against oxidative damage (Machado et al., 2023). Additionally, another study suggested that their hydrophilic nature enhances their hepatoprotective efects, making them more bioavailable for liver protection (Kandalintseva et al., 2002). These findings indicate that dietary intake or supplementation with phenolic acids may serve as a natural therapeutic strategy for liver disorders caused by oxidative stress.

The antioxidant mechanisms of flavonoids are similar. Flavonoids exhibit strong antioxidant properties that contribute significantly to liver protection and regeneration. These compounds enhance hepatic antioxidant defenses by increasing the levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione-S-transferase, thereby reducing oxidative stress, which is a key factor in liver diseases (Wan & Jiang, 2018). Additionally, flavonoids regulate liver metabolism and inflammatory pathways by inhibiting tumor necrosis factoralpha (TNF-α) and interleukin-6 (IL-6) and modulating apoptosis and autophagy through the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. The hepatoprotective efects of flavonoids extend to their role in the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, where they activate antioxidant response elements (AREs), reducing liver inflammation and fibrosis and promoting mitochondrial function (Sharma et al., 2024). Studies on specific flavonoids, such as those extracted from Buchholzia coriacea, indicate their ability to improve liver biomarkers, reduce oxidative damage, and protect against metabolic disorders, such as diabetes-induced liver stress (Amaralam et al., 2025). Moreover, synthetic flavones, such as 6,3’-dimethoxy flavone (DMF), exhibit hepatoprotective efects against druginduced toxicity, comparable to standard treatments such as silymarin (Abou Baker, 2022). These findings underscore the potential of flavonoids as therapeutic agents for liver diseases, highlighting their promise for future drug development.

Table 2 illustrates that the administration of HFR + SGPD 2 g influenced the decrease in TNF-α levels in the livers of obese rats. The higher the dosage of sea grapes administered, the greater the decrease, comparable to the efect observed with HFR + Orlistat administration. This finding aligns with the results of another study, which showed that eight weeks of sea grape administration can improve TNF-α parameters in the liver and heart.

Table 2 illustrates that the administration of HFR+SGPD 2 g decreased TNF-α levels in the livers of obese rats. The higher the dosage of sea grapes administered, the greater the decrease, comparable to the efect observed with RTL + Orlistat administration. Statistical analysis showed that the HFR + SGPD 2 g treatment produced significantly diferent results compared to the other treatments (p < 0.05), demonstrating better MDA prevention and higher SOD activity. This finding aligns with the results of a study by Preez et al. (2020), who showed that eight weeks of sea grape administration can improve TNF-α parameters in the liver and heart. Epidemiological studies have demonstrated a negative correlation between body mass and plasma carotene levels (Gunanti et al., 2014). During obesity, carotene is sequestered in the adipose tissue, leading to a reduction in its plasma concentration (Okechukuwu et al., 2019). Consequently, the body requires carotene intake as an anti-obesity agent, which has been linked to pro-vitamin A and has the potential to mitigate oxidative stress (Hamulka et al., 2023). Table 2 shows that sea grape administration had no discernible efect on MDA and SOD levels across all treatment groups. However, there was a notable tendency for a decrease in MDA levels and an increase in SOD activity in the livers of obese rats. These results difer from those of previous studies, which showed a decrease in MDA levels following the administration of sea grapes for eight weeks in Wistar rats (Julyasih et al., 2017). This can be attributed to the longer treatment duration, which allowed more pronounced diferences to emerge.

The sea grape powder drink exhibited anti-inflammatory efects due to the presence of bioactive compounds, namely phenols and carotenes, which can act as antioxidants. A study revealed that the phenolic derivatives present in Caulerpa sp. include gallic acid, catechins, isoquercetin tannic acid, and quercetin (Yoojam et al., 2021). Phenolic compounds have been demonstrated to suppress inflammation and oxidative stress by suppressing the regulation of NFkB gene expression and modulating the control of antioxidative signals through Nrf2 regulation. Additionally, they have been shown to reduce de novo lipogenesis by suppressing the regulation of the SREB1c gene, thereby reducing the accumulation of excess fatty acids and, consequently, lipid peroxidation, which is also a factor causing inflammation (Rodriguez-Ramiro et al., 2016; Lu et al., 2023). Catechins and their derivatives can also reduce oxidative stress directly by functioning as traps and antidotes for free radicals or indirectly by increasing superoxide dismutase (SOD) levels and reducing malondialdehyde (MDA) levels in the liver (Bernatoniene & Kopustinskiene, 2018). Epigallocatechin is a treatment for fatty liver disease because it is a hepatoprotector (Chen et al., 2018).

Additionally, SGPD contains carotene, another antioxidant. Previous studies have demonstrated that carotene can reduce cytochrome P450 activity and the expression of pro-inflammatory cytokines at the mRNA level. Furthermore, it has been shown to impede IkB Kinase (IKK) activity, which is responsible for regulating NFkB activity and downregulating inflammatory genes such as TNF α (Tolares et al., 2019).

Carotene functions as a precursor of retinoic acid, which binds to nuclear receptors, namely, the retinoic acid receptor (RAR) and retinoid X receptor (RXR). These receptors form heterodimers with peroxisome proliferator-activated receptors (PPAR) and other nuclear receptors, thereby playing a role in whole-body glucose regulation. This occurs through two main mechanisms: increased insulin sensitivity and insulin release (Saeed et al., 2017; Harari et al., 2020). Additionally, carotene exerts protective efects through its antioxidant capacity (Jurnalis et al., 2014). Carotene can bind to reactive species, such as singlet oxygen and peroxyl radicals, and has been shown to increase Nrf2 levels (Luisa Bonet et al., 2015).

An imbalance between free radicals and antioxidants results in oxidative stress, characterized by increased MDA levels (Elsayed et al., 2019). The continuous occurrence of oxidative stress reactions results in an elevation of lipid peroxides, which impairs mitochondrial function and triggers the induction of TNF-α (Chernyavskij et al., 2023). Fat accumulation results from an imbalance between the intake, synthesis, degradation, and secretion of fat in the liver. Fatty acids for triacylglycerol synthesis are obtained from de novo formation in the liver, portal blood as free fatty acids, and circulating lipoproteins, especially chylomicrons (Sinulingga et al. 2024). Serum-free fatty acids are increased by accelerated lipolysis in the peripheral adipose tissue and visceral fat (Kucera et al., 2014).

Immunohistochemical images obtained at 40x magnification of liver tissue demonstrated the expression of TNF-α in hepatocytes A and B, as indicated by brown coloration within the cell nucleus and cytoplasm. Tumor necrosis factor-ɑ (TNF-α) is mainly produced by the monocytemacrophage lineage in the liver, with Kupfer cells representing the predominant population in this lineage (Jiang et al., 2016). The appearance of brown coloration, indicative of a positive immunoreactive result, is a consequence of the immunohistochemical (IHK) staining process. During this process, antigen in the form of the TNF-α cytokine in the cell cytoplasm binds to the primary antibody (Rat Anti TNF-α) and is then labeled by secondary antibodies.

Once the binding process was complete, the diaminobenzidine (DAB) substrate was added, resulting in the production of a brown coloration indicative of the presence of the cytokine (TNF-α) within the cell cytoplasm. The expression level of TNF-α in liver tissue is a crucial marker for evaluating both the normal and diseased states of hepatocytes (Mohallem et al., 2021). Tumor necrosis factor-α (TNF) is an endogenous soluble molecule that causes necrosis in solid tumors.

TNF is a major inflammatory marker cytokine linked to the immunopathogenesis of several autoimmune diseases. TNF causes hepatocyte death and necroptosis, as well as hepatic inflammation, regeneration, autoimmunity, and the progression of hepatocellular cancer in the liver (Tiegs & Horst, 2022).

Inflammation plays a crucial role in metabolic disorders, including obesity-induced liver dysfunction. Tumor necrosis factor-alpha (TNF-α) is a key proinflammatory cytokine involved in hepatic inflammation, and its expression can be assessed using immunohistochemistry. The presence of TNF-α in liver tissue was indicated by brown staining in the nucleus and cytoplasm of the cells. A reduction in TNF-α expression suggests a potential antiinflammatory efect of the treatment. In this study, the efect of sea grape powder on TNF-α expression in rat liver tissue was evaluated, and the results are shown in Fig. 1.

Figure 1 illustrates the efect of sea grape powder on TNF-α expression in rat liver tissue, as observed through immunohistochemical analysis. TNF-α presence was indicated by brown staining within the nucleus and cytoplasm, reflecting the level of inflammation. In the control group (a), a strong brown coloration was observed, suggesting high TNF-α expression and significant inflammation. Similarly, the commercial treatment groups (b and c) exhibited noticeable TNF-α staining, although with some reduction compared to the control. In contrast, the sea grape treatment groups (d, e, and f) showed visibly lower brown staining intensity, indicating a potential antiinflammatory efect. The reduction in TNF-α expression suggests that sea grape powder may mitigate inflammation in diet-induced obese rats, consistent with its known bioactive properties. These findings support the potential of sea grape powder as a functional food ingredient for managing inflammation.

TNF-α is a proinflammatory cytokine released by Kupfer cells, which are magrophage (m1) caused by fat overload in the liver (Su et al., 2018). In the liver, KCs respond to two main types of stimuli: intrahepatic danger-associated molecular patterns (DAMPs), which are released by infiltrated and damaged hepatocytes, and gut-derived bacterial antigens, also known as pathogen-associated molecular patterns (PAMPs), which travel from the gut to the liver owing to a damaged gut epithelial barrier (Vachliotis & Polyzos, 2023). A significant signaling mechanism for the transcription of TNF-α, as well as other cytokines and chemokines, DAMPs, and PAMPs, is binding to Toll-like receptors (TLRs) on the surface of KCs and activating the nuclear factor-kappa B (NF-κB) intracellular pathway (Liu et al., 2017). TNF-α can induce the accumulation of reactive oxygen species (ROS) in the liver, potentially resulting in hepatic damage (Yang, 2023).

The results of this study showed no visible expression of TNF-α in Figure 1F hepatocyte tissue. These results suggest that SGPD exerts an anti-inflammatory efect. According to the findings of earlier studies, SPGD possesses antioxidant properties and has the potential to serve as an alternative treatment for obesity (Damayati et al., 2023). Antioxidants have emerged as promising therapeutic agents because they mitigate oxidative stress and modulate inflammatory pathways (Bhol et al., 2024). Antioxidants reduce inflammation by inhibiting the expression of tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine. This inhibition occurs at the transcriptional level by blocking the activation of NF-κB, which regulates TNF-α transcription (Mucha et al., 2021). Green seaweed can also inhibit the release of cytokines and inflammatory mediators, such as AMPK, mTOR, IL4, and TNF-α (Prayogo et al., 2024).

The results of this study are consistent with those of a previous study, which showed that the administration of lycopene can downregulate the expression of TNF-α, thereby reducing fatty liver infiltration and improving histopathological changes, with the degree of improvement depending on the administered dose (Jiang et al., 2016). Lycopene is a carotene-derived antioxidant that is both acyclic and nonpolar (Bin-Jumah et al., 2022). It is postulated that the increased levels of antioxidant enzymes and decreased lipid peroxide content observed in the presence of lycopene are important mechanisms by which this compound prevents the development of liver damage caused by a high-fat diet (Jiang et al., 2016).