Anti-adipogenic potential of hot- extracted Maclura pomifera (Raf.) C.K.Schneid. fruit peel Ömer Şen 1 , Sıla Özlem Şener 2 PS-036 Quality of Herbal Products Istanbul · 16–17 October 2026 1 PhD Program in Pharmacognosy, Gulhane Institute of Health Sciences, University of Health Sciences, Ankara, Türkiye 2 Department of Pharmacognosy, Gulhane Faculty of Pharmacy, University of Health Sciences, Ankara, Türkiye beyomersen@gmail.com Introduction / Objective Obesity is a chronic and increasingly prevalent metabolic disease characterized by excessive adipose tissue accumulation [1]. Adipose tissue expansion occurs through adipocyte hypertrophy and adipogenesis, accompanied by increased lipid storage [2]. Despite advances in pharmacological treatment, limitations in tolerability and long-term management support continued investigation of natural products as potential anti-obesity agents [3,4]. AIM This study aimed to evaluate the anti-adipogenic potential of a hot-extracted Maclura pomifera (Raf.) C.K.Schneid. fruit peel extract in 3T3-L1 cells, using the MTT assay to determine non- cytotoxic concentrations and Oil Red O staining to assess intracellular lipid accumulation. Materials and Methods 1 Extraction Maclura pomifera fruit peels were extracted with hexane using a hot extraction technique, and the solvent was subsequently removed by rotary evaporation to yield a dry hexane extract. 2 Cytotoxicity (MTT) Cytotoxicity in 3T3-L1 preadipocytes was evaluated using the MTT assay at 25, 50, 100, and 200 μg/mL to determine non-toxic concentrations. 3 Differentiation & treatment Adipocyte differentiation was induced according to the protocol, and cells were subsequently treated with the extract at 25 μg/mL only (MP25), with untreated differentiated cells in plain DMEM as negative control (NC) and orlistat as positive control (PC). 4 Oil Red O staining Intracellular lipid accumulation was measured by Oil Red O staining, with lipid droplets examined microscopically and quantified spectrophotometrically [5]. Results 91.8% Cell viability at 25 μg/mL (± 5.3%), the only non-toxic concentration 18.47% Reduction in intracellular lipid accumulation at 25 μg/mL (p<0.001) 33.40% Reduction with orlistat, the positive control Cytotoxicity MTT assay Cell viability decreased in a concentration-dependent manner, ranging from 91.8% ± 5.3% at 25 μg/mL to 45.4% ± 5.1% at 200 μg/mL. According to the 70% viability threshold (ISO 10993-5), only the 25 μg/mL concentration (91.8% ± 5.3%) was considered non-toxic [6]. This concentration was therefore used in the differentiation assay. Cell viability (%) 100 75 50 25 0 91.8% 70.5% 51.2% 45.4% 70% 25 50 100 200 Extract concentration (μg/mL) Dose used in the differentiation assay Figure 1. Cytotoxicity of M. pomifera extract in 3T3-L1 preadipocytes (MTT assay). Dashed line: 70% viability threshold (ISO 10993-5). Lipid accumulation Oil Red O staining At this concentration, the extract reduced intracellular lipid accumulation by 18.47%, as quantified spectrophotometrically, compared with differentiated control cells (p<0.001), versus a 33.40% reduction with orlistat as the positive control. NC MP25 PC Figure 2. Oil Red O staining of 3T3-L1 adipocytes treated with M. pomifera extract. NC: negative control (untreated) · MP25: 25 μg/mL extract · PC: positive control (orlistat) Conclusion The M. pomifera fruit peel extract demonstrated anti-adipogenic activity in 3T3-L1 cells while maintaining cell viability at active concentrations. These findings support its potential as a natural anti-obesity agent and indicate the need for further investigation. Keywords 3T3-L1 preadipocytes adipogenesis anti-obesity activity hot extraction Maclura pomifera References [1] NCD Risk Factor Collaboration (NCD-RisC). Worldwide trends in underweight and obesity from 1990 to 2022: a pooled analysis of 3663 population-representative studies with 222 million children, adolescents, and adults. Lancet. 2024;403(10431):1027–1050. [2] Ghaben AL, Scherer PE. Adipogenesis and metabolic health. Nat Rev Mol Cell Biol. 2019;20:242–258. [3] Gudzune KA, Kushner RF. Medications for Obesity: A Review. JAMA. 2024;332(7):571–584. [4] Shaik Mohamed Sayed UF, Moshawih S, Goh HP, et al. Natural products as novel anti-obesity agents: insights into mechanisms of action and potential for therapeutic management. Front Pharmacol. 2023;14:1182937. [5] Kaczmarek I, Suchý T, Strnadová M, Thor D. Qualitative and quantitative analysis of lipid droplets in mature 3T3- L1 adipocytes using oil red O. STAR Protoc. 2024;5(2):102977. [6] Gruber S, Nickel A. Toxic or not toxic? The specifications of the standard ISO 10993-5 are not explicit enough to yield comparable results in the cytotoxicity assessment of an identical medical device. Front Med Technol. 2023;5:1195529. Scan for the digital version QR