Effects of Black Garlic (Allium sativum) Supplementation on Oxidative Stress and Leptin Level in Male Obese Rats

774 To cite this paper: Ibrahim, Yerizel E, Endrinaldi, and Revilla G (2025). Effects of Black Garlic (Allium sativum) Supplementation on Oxidative Stress and Leptin Level in Male Obese Rats. World Vet. J., 15(3): 774-781. DOI: https://dx.doi.org/10.54203/scil.2025.wvj79 2025, Scienceline Publication World’s Veterinary Journal World Vet J, 15(3): 774-781. ISSN 2322-4568 Effects of Black Garlic (Allium sativum) Supplementation on Oxidative Stress and Leptin Level in Male Obese Rats Ibrahim1,2 , Eti Yerizel3  , Endrinaldi4, and Gusti Revilla5 ¹Program Doctoral of Biomedical Science, Faculty of Medicine, Andalas University, Limau Manis, Padang, Sumatra Barat 25175, Indonesia ²Departement of Medical Laboratory Technology, Syedza Saintika University, Jl. Prof. Dr. Hamka No.228, Air Tawar Tim., Padang, Sumatera Barat 25132, Indonesia ³Departement of Biochemistry, Faculty of Medicine, Andalas University, Limau Manis, Padang, Sumatra Barat 25175, Indonesia ⁴Departement of Biomedical, Faculty of Medicine, Andalas University, Limau Manis, Padang, Sumatra Barat 25175, Indonesia ⁵Departement of Anatomy, Faculty of Medicine, Andalas University, Limau Manis, Padang, Sumatra Barat 25175, Indonesia *Corresponding author's Email: [email protected] ABSTRACT Obesity is characterized by excessive fat accumulation in tissues, leading to leptin resistance and chronic inflammation. Black garlic is a well-documented natural remedy capable of improving leptin resistance and oxidative stress through its antioxidant compounds, such as S-Allyl cysteine and polyphenol flavonoids. The present study aimed to evaluate the effects of black garlic on oxidative stress and leptin levels in obese male rats. Twentyfive adult male Wistar rats, aged 10 to 12 weeks and weighing approximately 200 g, were divided into five experimental groups, each comprising five rats. These groups included a negative control group fed a standard diet (K1), a positive control group given a high-fat diet (K2), Treatment group 1 receiving a high-fat diet plus 200 mg of black garlic (P1), Treatment group 2 receiving a high-fat diet plus 400 mg of black garlic (P2), and Treatment group 3 receiving a high-fat diet plus 800 mg of black garlic (P3). The rats received black garlic orally for 28 days. The blood samples were analyzed for markers of oxidative stress, specifically malondialdehyde, as well as leptin levels. The present findings indicated that black garlic significantly decreased malondialdehyde and leptin concentrations in groups P1, P2, and P3 compared to the positive control group. Black garlic, administered at doses of 200, 400, and 800 mg, demonstrated the capacity to diminish oxidative stress and leptin levels in obese male rats. Keywords: Antioxidant, Black garlic, Leptin, Malondialdehyde, Obesity, Oxidative stress, Rat ORIGINAL ARTICLE Received: June 24, 2025 Revised: July 28, 2025 Accepted: August 27, 2025 Published: September 30, 2025 INTRODUCTION Obesity leads to lipid accumulation in adipose tissue (Aziz et al., 2020). It is a multifactorial disease caused by genetic, cultural, hormonal, psychological, and social factors (Lin and Li, 2021; Panuganti et al., 2023). Obesity is influenced by hormones that regulate food intake, including leptin, ghrelin, adiponectin, and cholecystokinin, among other mediators (Sumadewi et al., 2016). In obesity, leptin resistance often develops, leading to increased hunger and food consumption (Subarjati and Nuryanto, 2015; Lin and Li, 2021). Leptin is a hormone synthesized by adipose cells that reduces food intake, increases energy expenditure through specific signals in the hypothalamus, and maintains body weight homeostasis (Amos et al., 2017; Xu et al., 2018). Excessive lipid accumulation associated with obesity can lead to an elevated production of reactive oxygen species (ROS) and free radicals, both in circulating blood and adipocyte cells (Fajrani et al., 2021). Oxidative stress is characterized by increased lipid peroxides, as indicated by elevated levels of malondialdehyde (MDA; Jovie et al., 2022). Management of free radicals and oxidative stress has been studied using different medications, including both chemical medicines from the statin class and natural remedies such as Camellia sinensis, Allium sativum, Syzygium polyanthum, Uncaria, and botanicals rich in antioxidant compounds, including polyphenols, flavonoids, phenolic acids, and vitamins. One of the traditional plants used as herbal medicine is black garlic (Sembiring and Iskandar, 2019). The active organosulfur compounds in black garlic have a higher antioxidant concentration than regular garlic (Ahmed and Wang, 2021). Black garlic is produced from regular garlic and is popular due to its low cost and minimal side effects. In both in vitro and in vivo antioxidant tests, black garlic extract exhibited dose-dependent free radical scavenging activity. Black garlic significantly extended the lifespan of Drosophila melanogaster, increased the activities of superoxide dismutase (SOD), including CuZn-SOD and Mn-SOD, and catalase (CAT) as its concentration increased, and lowered MDA levels. These findings suggested that black garlic extract may prolong the lifespan of Drosophila melanogaster by activating SOD and CAT and reducing MDA production (Lei et al., 2014). Black garlic comprises slightly different compounds compared to white garlic. Fermentation conducted at temperatures between 60-90°C induces the conversion of γ-glutamyl-S-allylcysteine to S-allyl cysteine, resulting in a 4-8 times increase compared to fresh garlic (Chae et al., 2023). Black garlic contains 106.1 mg/kg of S-allyl cysteine, which DOI: https://dx.doi.org/10.54203/scil.2025.wvj79 PII: S232245682500079-15 World Vet. J., 15(3): 774-781, 2025 775 is five times higher than the S-allyl cysteine content of white garlic, measured at 21.0 mg/kg (Lee et al., 2009; Zhao et al., 2021). This fermentation process triggers numerous chemical reactions in garlic, including enzymatic browning and Maillard reactions, which cause the color to change from white and yellow to dark brown (Lei et al., 2014; Ha and Kim, 2017). Studies on black garlic and its components against different diseases have been extensively conducted in recent years. However, investigations to establish the therapeutic effects of black garlic remain substantially limited. Accordingly, the present study aimed to evaluate the impacts of black garlic supplementation on oxidative stress markers and leptin levels in male Wistar rats with obesity. MATERIALS AND METHODS Ethical approval The present study was conducted with the approval of the Ethics Committee of the Faculty of Medicine, Andalas University, Indonesia, and in accordance with ethical standards in medical research (ethics permit number: 520/UN.16.2/KEP-FK/2024, issued on October 4, 2024). Animals The present study was conducted in the animal house at the Biomedical Laboratory, Faculty of Medicine, Andalas University, Indonesia. Thirty-five Wistar rats, aged 10-12 weeks and weighing 200 g, were obtained from the Biomedical Laboratory of Andalas University, Indonesia. The rats were kept in a controlled environment with a 12-hour light-dark cycle, a room temperature of 22 ± 4°C, and a humidity level of 5 ± 10%. Each rat was allocated 125 cm² of floor space (approximately 49.2 in²) within a cage measuring 14 cm (5.5 inches) in height. During a seven-day adaptation period, the rats were given unlimited access to water and commercial pellet feed. The commercial pellet feed contained a metabolizable energy of 3.4 kcal/g, comprising 60% carbohydrate, 20% protein, 10% fat, and 6% fiber. Plant collection and identification Black garlic is a local Indonesian variety indigenous to Bogor Regency, West Java, and its surrounding regions. The black garlic is produced by fermenting Allium sativum garlic for 21 days at a temperature of 70°C and a humidity of 80%. The black garlic utilized in the present study was a product of Serambi Botani IPB, MD 655628, and was weighed according to the respective doses for each rat, namely 200 mg, 400 mg, and 800 mg. The black garlic was ground into a smooth paste and mixed with distilled water to facilitate administration to the rats. Experimental design Creating an obesity model in rats Male Wistar rats weighing 200 g were fed a high-fat diet (HFD; 2 mL goat oil and 1 mL egg yolk) for 12 weeks, administered via a tube every morning between 9:00 and 10:00 a.m. After 16 weeks, the body weight and body length of the rats were measured to determine the Lee obesity index in rats, which is similar to BMI in humans. Rats with a Lee index of 0.300 or higher were considered obese (Fajrani et al., 2021). Experiment procedure Based on the sample calculation utilizing Federer's (1991) formula, the minimum sample size required was five for each treatment group. Twenty-five rats were divided into five groups, each consisting of five rats, including the negative control group (K1), which received a regular diet with distilled water, the positive control group (K2), which received an HFD with distilled water, Treatment group 1 (P1), which received an HFD and a black garlic at a dose of 200 mg/rat, Treatment group 2 (P2), which received an HFD and a black garlic at a dose of 400 mg/rat, and Treatment group 3 (P3), which received an HFD and a black garlic at a dose of 800 mg/rat. These treatments were administered orally for 28 days. Body weight, body length, and activity levels of rats were monitored weekly. The body weight of male rats was assessed utilizing a precision digital scale (Sartorius, Germany), while the body length was determined with an animal measuring board. Body length was measured from the nose to the anus using a tape measure after administering chloroform anesthesia to the rats (Fajrani et al., 2021). To measure activity, the rats were observed for aggressive behavior and sedentary behavior. Malondialdehyde and leptin analysis The MDA and leptin parameters were measured on the final day of the experiment. Blood samples were collected from the orbital sinuses of the rats and then centrifuged to separate plasma and serum. The serum was utilized to determine the MDA and leptin levels. The MDA levels were analyzed using enzymatic samples (UV-Vis Spectrophotometry, Italy) at a wavelength of 532 nm, and the results were calculated based on a curve equation, yielding Ibrahim et al., 2025 776 MDA levels expressed in nmol/mL (Fajrani et al., 2021). Additionally, leptin hormone levels were assessed using serum samples via the enzyme-linked immunosorbent assay (ELISA). The ELISA leptin assay kit, specifically the SandwichELISA rat leptin (Elabscience®, Catalogue number: E-EL-R0582, England), was employed, and the results indicated leptin levels as expressed in ng/mL. Statistical analysis The present data are presented as mean ± standard deviation (SD). Since the data for MDA and leptin were not normally distributed, the Kruskal-Wallis test was used to find significant differences (p < 0.05) between the control and treatment groups. Statistical analysis was done with IBM SPSS Statistics version 22.0. RESULTS Table 1 displays that there was a significant difference in body weight in Wistar rats fed HFD for 16 weeks (p < 0.05). The body weight was the highest in P3 (326.80 ± 3.768) and the lowest in the negative control group (240.20 ± 12.194). In addition, the body weights were 319.20 ± 4.658, 318.20 ± 4.919, and 325.00 ± 5.244 in the positive control group, P1, and P2, respectively. After 28 days of feeding an HFD supplemented with black garlic, the body weight in Group P3 was lower (300.20 ± 14.923) than that of the positive control group (327.80 ± 6.686), Group P1 (307.20 ± 7.225), and Group P2 (310.00 ± 9.935). However, the concentration was still higher than the negative control group (254.80 ± 12.677). A significant difference was observed among the negative control group, the positive control group, and groups P1, P2, and P3 (p < 0.05; Table 2). Table 3 illustrates the levels of leptin hormone following the administration of an HFD supplemented with black garlic over a period of 28 days in Wistar rats. In Group P3, the leptin level was observed to be lower (1.12560 ± 0.160120) in comparison to the negative control group (1.51440 ± 0.241909), the positive control group (3.69020 ± 2.372940), Group P1 (1.41720 ± 0.238614), and Group P2 (1.54500 ± 0.195533). A statistically significant difference was identified among the negative control group, the positive control group, and groups P1, P2, and P3 (p < 0.05). The Effects of black garlic on MDA levels in Wistar rats at doses of 200 mg/rat, 400 mg/rat, and 800 mg/rat given for 28 days are illustrated in Table 4. The MDA levels observed after administering an HFD enriched with black garlic for 28 days indicated that Group P3 exhibited reduced MDA levels (2.2320 ± 0.24793) in comparison to the positive control group (3.9760 ± 0.38927), Group P1 (2.5280 ± 0.16514), and Group P2 (2.4280 ± 0.12337). Nonetheless, these levels remained higher than those observed in the negative control group (1.9300 ± 0.06364). Significant differences were observed among the negative control group, the positive control group, and groups P1, P2, and P3 (p < 0.05). Table 1. Effects of feeding a high-fat diet for 16 weeks on body weight in Wistar rats Group Rats (Number) Minimum body weight (g) Maximum body weight (g) Mean ± SD Body weight of rats (g) Negative control 5 221 253 240.20 ± 12.194 Positive control 5 314 326 319.20 ± 4.658 Treatment 1 5 312 324 318.20 ± 4.919 Treatment 2 5 319 332 325.00 ± 5.244 Treatment 3 5 321 331 326.80 ± 3.768 Total 25 221 332 305.88 ± 34.264 Significant 0.002 Data are expressed as minimum, maximum, mean ± Standard deviation (SD). Negative control: Rats that received regular diet with distilled water, positive control: Rats that received an HFD with distilled water, Treatment 1: An HFD and a black garlic at a dose of 200 mg/rat, Treatment 2: An HFD and a black garlic at a dose of 400 mg/rat, Treatment 3: An HFD and a black garlic at a dose of 800 mg/rat. Table 2. Effects of black garlic on body weight in Wistar rats at doses of 200 mg/rat, 400 mg/rat, and 800 mg/rat given for 28 days Group Rats (Number) Minimum body weight (g) Maximum body weight (g) Mean ± SD Body weight of rats (g) Negative control 5 235 266 254.80 ± 12.677 Positive control 5 320 336 327.80 ± 6.686 Treatment 1 5 297 315 307.20 ± 7.225 Treatment 2 5 298 321 310.00 ± 9.935 Treatment 3 5 279 316 300.20 ± 14.923 Total 25 235 336 300.04 ± 26.751 Significant 0.001 Data are expressed as minimum, maximum, mean ± Standard deviation (SD). Negative control: Rats that received regular diet with distilled water, positive control: Rats that received an HFD with distilled water, Treatment 1: An HFD and a black garlic at a dose of 200 mg/rat, Treatment 2: An HFD and a black garlic at a dose of 400 mg/rat, Treatment 3: An HFD and a black garlic at a dose of 800 mg/rat. World Vet. J., 15(3): 774-781, 2025 777 Table 3. Effects of black garlic on leptin levels in Wistar rats at doses of 200 mg/rat, 400 mg/rat, and 800 mg/rat given for 28 days Group Rats (Number) Minimum level of leptin Maximum level of leptin Mean ± SD Leptin hormone (ng/mL) Negative control 5 1.139 1.763 1.51440 ± 0.241909 Positive control 5 1.964 7.859 3.69020 ± 2.372940 Treatment 1 5 1.134 1.790 1.41720 ± 0.238614 Treatment 2 5 1.294 1.728 1.54500 ± 0.195533 Treatment 3 5 0.955 1.383 1.12560 ± 0.160120 Total 25 0.955 7.859 1.85848 ± 1.365638 Significant 0.008 Data are expressed as minimum, maximum, and Mean ± standard deviation (SD). Negative control: Rats that received regular diet with distilled water, positive control: Rats that received an HFD with distilled water, Treatment 1: An HFD and a black garlic at a dose of 200 mg/rat, Treatment 2: An HFD and a black garlic at a dose of 400 mg/rat, Treatment 3: An HFD and a black garlic at a dose of 800 mg/rat. Table 4. Effects of black garlic on malondialdehyde levels in Wistar rats at doses of 200 mg/rat, 400 mg/rat, and 800 mg/rat given for 28 days Group Rats (Number) Minimum level of MDA Maximum level of MDA Mean ± SD MDA (nmol/mL) Negative control 5 1.85 2.02 1.9300 ± 0.06364 Positive control 5 3.59 4.58 3.9760 ± 0.38927 Treatment 1 5 2.34 2.72 2.5280 ± 0.16514 Treatment 2 5 2.29 2.61 2.4280 ± 0.12337 Treatment 3 5 1.85 2.45 2.2320 ± 0.24793 Total 25 1.85 4.58 2.6188 ± 0.75254 Significant 0.001 Data are expressed as minimum, maximum, and Mean ± Standard deviation (SD). MDA: Malondialdehyde, Negative control: Rats that received regular diet with distilled water, Positive control: Rats that received an HFD with distilled water, Treatment 1: An HFD and a black garlic at a dose of 200 mg/rat, Treatment 2: An HFD and a black garlic at a dose of 400 mg/rat, Treatment 3: An HFD and a black garlic at a dose of 800 mg/rat. DISCUSSION An HFD is a dietary regimen in which the majority of daily caloric intake is derived from fat, typically contributing around 40-60% of total energy over a period of 8-16 weeks. Obesity is defined as the excessive accumulation of adipose tissue (Panuganti et al., 2023). In numerous cases involving obesity, leptin levels are elevated concurrently with the development of leptin resistance (Sharebiani et al., 2023). Additionally, obesity is associated with heightened systemic oxidative stress, which results from an imbalance between free radicals and antioxidants, often marked by increased MDA levels (Ashar, 2023). In the study conducted by Chang et al. (2017), rats administered an HFD comprising 2 mL of goat fat per 200 g of body weight and 1 mL of egg yolk per 200 g of body weight over a period of 16 weeks via oral gavage exhibited an approximate 62.9% increase in body weight. Similar findings have been reported in a study conducted by Kim et al. (2017), which demonstrated that a diet comprising 60% fat, administered over six weeks, induced obesity in rats. Additionally, an HFD consisting of 45% kcal over ten weeks resulted in notable weight gain. Likewise, Chae et al. (2023) induced obesity in animals using an HFD at 60% over 16 weeks. Jahan et al. (2024) reported weight gain in rats fed a combination of 90% standard pellets and 10% goat fat oil. Sukmawati and Asgap (2017) developed a hyperlipidemia and obesity model using an HFD comprising 50% goat fat mixed with butter, coconut oil, and cholesterol. Furthermore, Maigoda et al. (2016) confirmed the efficacy of goat fat mixtures in inducing obesity. Additionally, Yu et al. (2019) demonstrated that prolonged consumption of egg yolk resulted in increased adiposity in rats. A study by Nasution (2023) revealed that Sprague-Dawley rats aged 20-24 weeks and weighing in the range of 250 to 300 g, when subjected to a diet comprising a mixture of beef fat, egg yolk, and palm oil for a duration of 30 days, demonstrated a considerable increase in weight gain in comparison to the control groups. Additionally, these subjects exhibited elevated levels of total cholesterol, triglycerides, and low-density lipoprotein (LDL), alongside a reduction in high-density lipoprotein. Aguila et al. (2003) observed that rats fed egg yolks experienced an average weight gain of 32.6 ± 1.3 g over four weeks, surpassing the gains observed in rats fed oats (28.6 g) or wheat bran (25.7 g). Similarly, Omole and Ighodaro (2013) reported that mice subjected to an HFD gained approximately 40% more body weight, along with an increase in total cholesterol by 30%, triglycerides by 11.9%, and LDL from 56.9 to 81 mg/dL. Yu et al. (2019) investigated the effects of a diet incorporating egg yolk powder combined with vanadium, where the HFD (containing 301.2 g per kg of ether extract derived from egg yolk) over five weeks remarkably elevated body weight and markers of Ibrahim et al., 2025 778 oxidative stress. Feeding rats utilizing an HFD was linked to the development of obesity, caused by excessive energy intake and disrupted regulation of satiety and hunger signals, which subsequently led to leptin resistance and chronic inflammation. Black garlic is a fermented form of garlic characterized by a soft, savory texture, mild aroma, slightly sweet or tangy flavor, and a chewy or jelly-like consistency (Choi et al., 2014; Sukrianto et al., 2022). According to Sailah (2021), raw garlic can be processed through fermentation or prolonged heating. The fermentation process is most effective at a temperature of 70-80°C with a controlled humidity of 70-90% for 21 days, without adding any extra ingredients (Lei et al., 2014). This process leads to an increase in total acid content, amino nitrogen, polyphenols, flavonoids, and certain bioactive compounds such as lactic acid, gly-pro-glu, sorbase, and 3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1benzopyran-2-propanoate (CEHC), while reducing 5-hydroxymethyl furfural levels and enhancing grassy, floral, and fruity aromas (Si et al., 2019). During the present study, rats fed an HFD and supplemented with black garlic for 28 days exhibited different results across the different treatment groups. The rats that received 800 mg of black garlic per rat demonstrated a lower body weight compared to the positive control group and the group receiving 400 mg per rat. The present results support the findings of other studies that highlighted the anti-obesity properties of black garlic. Chang et al. (2017) reported that black garlic supplementation improved HFD-induced obesity, while Chae et al. (2023) demonstrated that aged black garlic mitigates obesity and prevents muscle atrophy. Xu et al. (2018) found that aged garlic extract improved inflammatory and immune responses in obese adults, and Lei et al. (2014) confirmed its role in reducing oxidative stress. Similarly, Kimura et al. (2017) analyzed saccharides and antioxidant activity in black garlic, linking them to weight reduction. In contrast, Sembiring and Iskandar (2019) emphasized the antioxidant and antiinflammatory effects of black garlic in decreasing both weight and oxidative stress. Other studies, by Nasr and Saleh (2014) and Si et al. (2019), confirmed its pharmacological activity as an antioxidant and anti-obesity agent. Additionally, Ahmed and Wang (2021) demonstrated that black garlic supplementation reduced body weight by 18% and fat mass by 44-63% in obese mice. Also, Chang et al. (2017) exhibited that Wistar rats fed an HFD supplemented with 0.2%, 0.6%, or 1.2% black garlic for six weeks demonstrated significant weight reductions of 4.8-8.9% compared to unsupplemented HFD rats. The beneficial effects of black garlic are attributed to its high antioxidant content, including S-allylcysteine, flavonoids, and polyphenols, which lower oxidative stress and elevate adiponectin levels, a protective hormone secreted by adipose tissue (Setiawan et al., 2021). Mechanistically, black garlic modulates fat metabolism by downregulating the expression of fatty acid synthase (FAS) and inflammatory cytokines, such as interleukin-6 (IL-6; Xu et al., 2018), while increasing the activity of lipolytic enzymes, including ATGL, CPT-1, and HSL, in adipose tissue and the liver. Furthermore, black garlic inhibits lipogenesis by downregulating genes such as SREBP-1c, ACC, FAS, G6PDH, HMGCoA reductase, and ACAT, while concurrently upregulating CPT-1 expression, thereby promoting fatty acid oxidation (Ha et al., 2015; Lestari and Santika, 2023). Leptin is a protein hormone composed of 167 amino acids, primarily secreted by adipose tissue (Amos et al., 2017; Nasution, 2023), acting on the central nervous system (CNS) to suppress appetite and increase energy expenditure (Amor et al., 2019; Kim and Kim, 2023). In the present study, supplementation with 800 mg/rat of black garlic resulted in the most significant reduction in leptin levels in rats fed an HFD. These findings are consistent with those of Chae et al. (2023), who reported that black garlic supplementation reduced obesity in HFD-fed mice and lowered leptin levels. Similarly, Chang et al. (2017) indicated that black garlic improved leptin regulation in obese mice, while Xu et al. (2018) demonstrated that aged garlic extract reduced inflammation and modulated leptin and other adipokines in obese individuals. Additionally, Amor et al. (2019) confirmed that HFD-fed mice supplemented with aged black garlic exhibited a marked decrease in plasma leptin concentrations from 19.4 to 9.4 ng/dL. Moreover, black garlic enhances leptin sensitivity, primarily due to its high Sulfur-containing compounds such as Sallylcysteine and other antioxidant compounds, which reduce oxidative stress and inflammation in the hypothalamus (Amor et al., 2019; Halim and Suzan, 2020). Given that hypothalamic inflammation contributes to leptin resistance, its reduction improved leptin responsiveness and signaling efficiency. Additionally, black garlic has demonstrated effects in modulating the gut microbiota by reducing lipopolysaccharide (LPS), a principal inducer of systemic inflammation. The reduction of LPS levels combined with an increase in short-chain fatty acids contributes to enhanced leptin sensitivity and a decrease in leptin resistance (Ha et al., 2015; Amor et al., 2019; Kim and Kim, 2023; Morawati et al., 2024). Numerous individuals with obesity exhibited high leptin levels without the anticipated decrease in fat mass, which signifies leptin resistance. This condition occurs when increased leptin, caused by greater adiposity, fails to stimulate adequate satiety or energy expenditure. Leptin resistance is frequently linked to chronic inflammation in adipose tissue and the brain. Common signs of leptin resistance include lower levels of soluble leptin receptors, decreased proteinbound leptin, and higher levels of circulating free leptin (Subarjati and Nuryanto, 2015; Halim and Suzan, 2020; Amudi et al., 2021). Black garlic possesses antioxidant and anti-inflammatory properties that aid in leptin resistance by inhibiting the release of pro-inflammatory cytokines such as TNF-α and IL-6, which are recognized for their role in World Vet. J., 15(3): 774-781, 2025 779 impairing leptin signaling (Salsabila and Busman, 2021). Overall, leptin is crucial in regulating body weight and lipid metabolism. Black garlic directly enhances leptin sensitivity by reducing oxidative stress, inflammation, and regulating fat metabolism, helping to alleviate leptin resistance and support metabolic homeostasis (Amor et al., 2019; Nasution, 2023). Malondialdehyde, with the chemical formula CH₂(CHO)₂, is a reactive dialdehyde widely recognized as a reliable marker of oxidative stress. It is formed as a terminal product of lipid peroxidation, a process that damages cell membranes. Given that MDA is chemically more stable than numerous other peroxidation byproducts, it is frequently employed in experiments as a marker of oxidative damage (Mulianto, 2020; Huang et al., 2023; Widhiani et al., 2023). An HFD facilitates the production of ROS, thereby accelerating lipid peroxidation and elevating MDA levels, which serve as indicators of oxidative stress. Black garlic, rich in antioxidant and anti-inflammatory compounds, can neutralize ROS, decrease oxidative stress, reduce MDA concentrations, and promote cellular health repair. In the present study, rats given the highest dose (800 mg/rat) exhibited significantly lower MDA values compared to the positive control group and other treatment groups. These findings are consistent with the study conducted by Saravanan and Ponmurugan (2010), which demonstrated that S-allyl cysteine, a key component of garlic and black garlic, decreased MDA and oxidative stress in diabetic rats. Si et al. (2019) highlighted the strong antioxidant potential of black garlic in lowering MDA, while Chae et al. (2023) confirmed its ability to mitigate oxidative stress biomarkers. Chang et al. (2017) demonstrated that black garlic supplementation reduced hepatic oxidative stress in obese rats, and Ha and Kim (2017) reported that supplementation up to 1.5% significantly inhibited lipid peroxidation. Furthermore, Ahmed and Wang (2021) found that garlic fermentation reduced MDA levels, supporting its role as a potent antioxidant intervention. Obesity in the United States has been linked to elevated oxidative stress. According to a study by Lee et al. (2009), MDA levels in the control group were reported as 2.33 ± 0.36. In contrast, rats receiving a diet supplemented with 5% black garlic exhibited notably lower levels at 1.32 ± 0.26, representing an approximate reduction of 43.3%. The reduction in MDA with black garlic supplementation is attributed to its S-allylcysteine, flavonoids, polyphenols, and other organosulfur constituents, which act as scavengers of reactive oxygen and nitrogen species (ROS and RNS). By neutralizing these radicals, black garlic suppresses lipid peroxidation, thereby lowering MDA concentrations (Lee et al., 2009; Dampati and Veronica, 2020; Sukrianto et al., 2022). Black garlic enhances the function of superoxide dismutase, which converts superoxide into hydrogen peroxide (H₂O₂), and CAT and glutathione peroxidase, which break down H₂O₂ into water. This enzymatic defense prevents the formation of hydroxyl radicals, one of the most damaging reactive species responsible for lipid peroxidation. Furthermore, black garlic reduces inflammation and inhibits NF-κB signaling (Xu et al., 2018; Salsabila and Busman, 2021). Chronic inflammation contributes to increased ROS through macrophage activation and NF-κB pathway stimulation. By suppressing pro-inflammatory cytokines such as IL-6 and TNF-α, black garlic lowers ROS generation from inflammatory processes, protecting lipids from oxidative damage, and reducing MDA formation (Lei et al., 2014; Kusriani et al., 2022; Morawati et al., 2024). CONCLUSION In the present study, leptin levels were assessed after 28 days of administering an HFD supplemented with black garlic at doses of 200 mg, 400 mg, and 800 mg per rat. The present results suggested that black garlic effectively contributes to weight reduction, lowers leptin concentrations, and decreases MDA levels as an indicator of oxidative stress. The 800 mg/rat dose resulted in a significantly greater reduction compared to the positive control group that received only an HFD. A larger sample size is required to enhance the statistical power and generalizability of the findings. Utilizing animals of different sexes or species could yield a more comprehensive understanding of the effects of black garlic. Furthermore, testing different dosages and determining the optimal dose of administering black garlic are essential to establish the boundaries of efficacy and safety. DECLARATIONS Acknowledgments The authors express their gratitude to the Department of Biomedical Laboratory and the Biochemistry Laboratory, Faculty of Medicine, Andalas University, Indonesia, for their assistance with sample analysis. Authors’ contributions Ibrahim, Eti Yerizel, Endrinaldi, and Gusti Revilla were responsible for conceptualizing the article. Ibrahim collected the data and the manuscript, while Eti Yerizel reviewed it. All authors considered and agreed on the final edition of the manuscript for publication. Funding The present study was privately funded by the authors. Ibrahim et al., 2025 780 Availability of data and materials The data are available upon reasonable request from the corresponding author. Competing interests The authors declared no conflicts of interest. Ethical considerations Ethical issues, including plagiarism, consent to publish, misconduct, data fabrication and/or falsification, double publication and/or submission, and redundancy, have been checked by all the authors. REFERENCES Aguila MB, Pinheiro ADR, Parente LB, and Mandarim LCA (2003). Dietary effect of different high-fat diet on rat liver stereology. Liver International: Official Journal of the International Association for the Study of the Liver, 23(5): 363-370. DOI: https://www.doi.org/10.1034/j.14783231.2003.00858.x Ahmed T and Wang CK (2021). Black garlic and its bioactive compounds on human health diseases: A review. Molecules, 26(16): 2508. DOI: https://www.doi.org/10.3390/molecules26165028 Amor S, Gonzalez HD, Martín CB, Inarejos GAM, Almodovar P, Prodanov M, Garcia VAL, and García MG (2019). Beneficial effects of an aged black garlic extract in the metabolic and vascular alterations induced by a high fat/sucrose diet in male rats. Nutrients, 11(1): 153. DOI: https://www.doi.org/10.3390/nu11010153 Amos DL, Robinson T, Massie MB, Cook C, Hoffsted A, Crain C, and Santanam N. (2017). Catalase overexpression modulates metabolic parameters in a new ‘stress-less’ leptin deficient mouse model. BBA-Molecular Basis of Disease, 1863(9): 2293-2306. DOI: https://www.doi.org/10.1016/j.bbadis.2017.06.016 Amudi T, Pandelaki K, and Palar S. (2021). The relationship between HS-CRP, adiponectin, fetuin a and insulin resistance in young adult men with central obesity. E-CliniC, 9(1): 231-237. DOI: https://www.doi.org/10.35790/ecl.v9i1.32476 Ashar YK (2023). Serum malondialdehyde in obese and non-obese patients. Jurnal Kesehatan Tambusai, 4(4): 7053-7057. DOI: https://www.doi.org/10.31004/jkt.v4i4.22473 Aziz S, Adliani N, and Sukrasno (2020). Molecular docking of oryzanol derivative compounds to enzymes 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reduktase. Journal of Science and Applicative Technology, 4: 43-48. Available at: https://journal.itera.ac.id/index.php/jsat/%0AReceived Chae J, Lee E, Oh SM, Ryu HW, Kim S, and Nam JO (2023). Aged black garlic (Allium sativum L.) and aged black elephant garlic (Allium ampeloprasum L.) alleviate obesity and attenuate obesity induced muscle atrophy in diet induced obese C57BL/6 mice. Biomedicine and Pharmacotherapy, 163: 114810. DOI: https://www.doi.org/10.1016/j.biopha.2023.114810 Chang WT, Shiau DK, Cheng MC, Tseng CY, Chen CS, Wu MF, and Hsu CL (2017). Black garlic ameliorates obesity induced by a high-fat diet in rats. Journal of Food and Nutrition Research, 5(10): 736741. DOI: https://www.doi.org/10.12691/jfnr-5-10-3 Choi IS, Cha HS, and Lee YS (2014). Physicochemical and antioxidant properties of black garlic. Molecules, 19(10): 16811-16823. DOI: https://www.doi.org/10.3390/molecules191016811 Dampati PS and Veronica E (2020). The Potential of black garlic extract as a sunscreen against ultraviolet ray exposure. KELUWIH: Journal of Health and Medicine, 2(1): 23-31. DOI: https://www.doi.org/10.24123/kesdok.v2i1.3020 Fajrani AM, Sulchan M, and Muis SF (2021). Effect of black garlic on visceral fat, oxidative stress and insulin resistance in nonalcoholic fatty liver disease rats. Journal Of Nutrition and Food Science, 51(7): 1084-1095. DOI: https://www.doi.org/10.1108/NFS-11-2020-0439 Ha AW and Kim WK (2017). Antioxidant mechanism of black garlic extract involving nuclear factor erythroid 2-like factor 2 pathway. Nutrition Research and Practice, 11(3): 206-213. DOI: https://www.doi.org/10.4162/nrp.2017.11.3.206 Ha AW, Ying T, and Kim WK (2015). The effects of black garlic (Allium satvium) extracts on lipid metabolism in rats fed a high fat diet. Nutrition Research and Practice, 9(1): 30-36. DOI: https://www.doi.org/10.4162/nrp.2015.9.1.30 Halim R and Suzan R (2020). Correlation of fat mass and visceral fat with serum leptin levels in overweight and obese adolescents. Jambi Medical; Journal of Medicine and Health, 8(1): 102-110. DOI: https://www.doi.org/10.22437/jmj.v8i1.9477 Huang Y, Chen H, Liu Q, Hu J, Hu D, Huang Z, Xu Z, and Wan R (2023). Obesity difference on association blood malondialdehyde level and diastolic hypertension in the elderly population: A cross sectional analysis. European Journal of Medical Research, 28(1): 44. DOI: https://www.doi.org/10.1186/s40001-022-00983-7 Jahan MS, Haque MI, Gautam M, and Bhuiyan MER (2024). Comparative analysis of high-fat diets: Effects of mutton, beef, and vegetable fats on body weight, biochemical profiles, and liver histology in mice. Journal Of Heliyon, 10(20): e39349. DOI: https://www.doi.org/10.1016/j.heliyon.2024.e39349 Jovie IF, Alioes Y, and Irrahmah M (2022). The relationship between the degree of obesity and malondialdehyde levels in medical students of the faculty of medicine, Andalas University. Indonesian Journal of Health Sciences, 2(3): 135-141. DOI: https://www.doi.org/10.25077/jikesi.v2i3.346 Kim MR, Kim JW, Park JB, Hong YK, Ku SK, and Choi JS (2017). Anti-obesity effects of yellow catfish protein hydrolysate on mice fed a 45% kcal high-fat diet. International Journal of Molecular Medicine, 40(3): 784-800. DOI: https://www.doi.org/10.3892/ijmm.2017.3063 Kim HJ and Kim M (2023). Diallyl disulfide alleviates hypercholesterolemia induced by a western diet by suppressing endoplasmic reticulum stress in apolipoprotein E-deficient mice. BMC Complementary Medicine and Therapies, 23(1): 141. DOI: https://www.doi.org/10.1186/s12906-02303920-1 Kimura S, Tung YC, Pan MH, Su NW, Lai YJ, and Cheng KC (2017). Black garlic: A critical review of its production, bioactivity, and application. Journal of Food and Drug Analysis, 25(1): 62-70. DOI: https://www.doi.org/10.1016/j.jfda.2016.11.003 Kusriani RH, Kurnia D, and Umaro SQ (2022). Effects of garlic (allium sativum l) and black garlic extract on malondialdehyde (MDA) levels in rats blood. Galenika Pharmaceutical Journal, 8(3): 168-181. DOI: https://www.doi.org/10.70410/jfg.v8i3.199 Lee YM, Gweon OC, Seo YJ, Im J, Kang MJ, Kim MJ, and Kim JI (2009). Antioxidant effect of garlic and aged black garlic in animal model of type 2 diabetes mellitus. Nutrition Research and Practice, 3(2): 156-161. DOI: https://www.doi.org/10.4162/nrp.2009.3.2.156 World Vet. J., 15(3): 774-781, 2025 781 Lei M, Xu M, Zhang Z, Zhang M, and Gao Y (2014). The analysis of saccharide in black garlic and its antioxidant activity. Advance Journal of Food Science and Technology, 6(6): 755-760. DOI: https://www.doi.org/10.19026/ajfst.6.106 Lestari GAPW and Santika IWM (2023). Anti-cholesterol potential of garlic (Allium sativum): Systematic review. Proceedings of the National Pharmacy Workshop and Symposium, 2: 44-60. DOI: https://www.doi.org/10.24843/wsnf.2022.v02.p04 Lin X and Li H (2021). Obesity: Epidemiology, pathophysiology, and therapeutics. Article review. Frontiers in Endocrinology, 12: 706978. DOI: https://www.doi.org/10.3389/fendo.2021.706978 Maigoda TC, Sulaeman A, Setiawan B, and Wibawan IWT (2016). Effects of red dragon fruits (Hylocereus polyrhizus) powder and swimming exercise on inflammation, oxidative stress markers, and physical fitness in male obesity rats (Sprague dawley). International Journal of Sciences: Basic and Applied Research, 25(1): 123-141. Available at: https://gssrr.org/JournalOfBasicAndApplied/article/view/5111 Morawati S, Dewi L, Angica PR, Adelin P, and Ashan H (2024). Beneficial effects of black garlic on plasma atherogenic index values in obese patients. Scientific Journal, 3(3): 174-184. DOI: https://www.doi.org/10.56260/sciena.v3i3.145 Mulianto N (2020). Malondialdehyde as a marker of oxidative stress in various skin diseases. Mirror of the World of Medicine, 47(1): 39-44. DOI: https://www.doi.org/10.55175/cdk.v47i1.13 Nasr AY and Saleh HAM (2014). Aged garlic extract protects against oxidative stress and renal changes in cisplatin treated adult male rats. Research. Journal of Experimental and Clinical Cancer Cell, 14(1): 92. DOI: https://www.doi.org/10.1186/s12935-014-0092-x Nasution SD (2023). The role of leptin in metabolism. Journal Pandu Husada, 4(2): 35-40. Available at: https://jurnal.umsu.ac.id/index.php/JPH/article/view/16449 Omole JO and Ighodaro OM (2013). Comparative studies of the effects of egg yolk, oats, apple, and wheat bran on serum lipid profile of Wistar rats. ISRN Nutrition, 2013: 730479. DOI: https://www.doi.org/10.5402/2013/730479 Panuganti KK, Nguyen M, and Kshirsagar RK (2023). Obesity. National Library of Medicine. Statpearls, Agustus. Available at: https://www.ncbi.nlm.nih.gov/books/NBK459357/ Sailah I (2021). Physicochemical properties changes during the processing of single garlic into black garlic using a rice cooker. Journal of Agricultural Industrial Technology, 31(1): 88-97. DOI: https://www.doi.org/10.24961/j.tek.ind.pert.2021.31.1.88 Salsabila Q and Busman H (2021). Anti-inflammatory activity of black garlic (Allium sativum L.). Jurnal Ilmiah Kesehatan Sandi Husada, 10(1): 4147. DOI: https://www.doi.org/10.35816/jiskh.v10i1.502 Sembiring N and Iskandar Y (2019). A review of component and pharmacology activities of black garlic. Traditional Medicine Journal, 24(3): 178183. DOI: https://www.doi.org/10.22146/mot.45277 Setiawan AA, Purnomo FA, Karlowee V, and Wijayahadi N (2021). The effect of black garlic (Allium sativum Linn) on cardiac and aortic histopathology in experimental studies in obesity rats. Journal of Biomedicine and Translational Research, 7(2): 62-68. DOI: https://www.doi.org/10.14710/jbtr.v7i2.11686 Sharebiani H, Keramat S, Chavoshan A, Fazeli B, and Stanek A (2023). The influence of antioxidants on oxidative stress induced vascular aging in obesity. Journal Antioxidants, 12(6): 1295. DOI: https://www.doi.org/10.3390/antiox12061295 Si L, Lin R, Jia Y, Jian W, Yu Q, and Wang M (2019). Lactobacillus bulgaricus improves antioxidant capacity of black garlic in the prevention of gestational diabetes mellitus : A randomized control trial. Bioscience Reports, 39: 1-19. Available at: https://pmc.ncbi.nlm.nih.gov/articles/PMC6689107/pdf/bsr-39-bsr20182254.pdf Subarjati A and Nuryanto N (2015). The relationship between body mass index and leptin and adiponectin levels. Journal of Nutrition College, 4(4): 428-434. DOI: https://www.doi.org/10.14710/jnc.v4i4.10121 Sukmawati S and Asgap ARP (2017). Test of the effects of bean sprout juice on cholesterol levels of male hyperlipidemic rats (Rattus norvegicus). Jurnal Ilmiah As-Syifaa, 9(2): 188-194. DOI: https://www.doi.org/10.33096/jifa.v9i2.301 Sukrianto, Harianto LI, Rizqiya F, Muzadid AFH, and Akbar J (2022). Black garlic products and consumption for community immunity. Seminar Nasional Pengabdian Masyarakat LPPM UMJ, Jakarta, pp. 1-9. Available at: https://jurnal.umj.ac.id/index.php/semnaskat/article/view/14354/7495 Widhiani NPV, Widiastuti IAE, and Zubaidi FF (2023). The difference in malondialdehyde (MDA) levels among normoweight, overweight, and obesity in student of medicine program, Faculty of Medicine, Mataram University. Jurnal Biologi Tropis, 23(3): 595-599. DOI: https://www.doi.org/10.29303/jbt.v23i3.5429 Xu C, Mathews AE, Rodrigues C, Eudy BJ, Rowe CA, O’Donoughue A, and Percival SS (2018). Aged garlic extract supplementation modifies inflammation and immunity of adults with obesity: A randomized, double blind, placebo controlled clinical trial. Clinical Nutrition ESPEN, 24: 148-155. DOI: https://www.doi.org/10.1016/j.clnesp.2017.11.010 Yu Z, Wang N, Hu G, and Ma M (2019). Comparison of long term effects of egg yolk consumption under normal and high fat diet on lipid metabolism and fatty acids profile in mice. Food Science and Biotechnology, 28(4): 1195-1206. DOI: https://www.doi.org/10.1007/s10068-018-00545-w Zhao XX, Lin FJ, Li H, Li HB, Wu DT, Geng F, Ma W, Wang Y, Miao BH, and Gan RY (2021). Recent Advances in bioactive compounds, health functions, and safety concerns of onion (Allium cepa L.). Frontiers in Nutrition, 8: 669805. DOI: https://www.doi.org/10.3389/fnut.2021.669805 Publisher’s note: Scienceline Publication Ltd. remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access: This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit https://creativecommons.org/licenses/by/4.0/. © The Author(s) 2025