In vitro assessment of antisteatotic and antifibrotic potentials of Abelmoschus esculentus extracts

Metabolic dysfunction-associated steatotic liver disease (MASLD) represents a prevalent liver disorder with increasing incidence worldwide, characterized by the abnormal accumulation of lipids within the liver. This condition often progresses to more severe stages, such as metabolic dysfunction-associated steatohepatitis (MASH) and hepatic fibrosis, significantly increasing morbidity and mortality rates. Given the complex pathophysiology and limited therapeutic options for MASLD and its progressive manifestations, there is a critical need for novel treatment strategies. In this study we explored the potential therapeutic properties of three different extracts of Abelmoschus esculentus (Okra), a widely consumed vegetable known for its multiple uses in traditional medicine attributed to its content of bioactive compounds, including polyphenols, flavonoids, and peptides. This study aimed to prepare extracts of Okra fruits and pods by means of different extraction methods and to assess their ability to mitigate hepatic lipid accumulation and attenuate the progression of liver fibrosis by using in vitro models. In this study the extraction of bioactive compounds was performed by means of two extraction methods: maceration of pods (OFEM) and seeds (OSEM) and ultrasound-assisted extraction of okra pods (OFE). The maceration process was done in ethanol for 24 h, followed by filtration, evaporation, and drying. Ultrasound-assisted extraction was done on okra pods (OFE) using methanol:water (1:1), applying ultrasonic waves to favor the extraction of bioactive compounds. We quantified the total phenolic and flavonoid contents in various okra extracts. The total phenolic content (TP) was assessed with a modified Folin–Ciocalteu method. The results, expressed in gallic acid equivalents (GAE), provided a quantitative measure of phenolic compounds. The total flavonoid content (TF) was evaluated by means of a spectrophotometric method using quercetin as standard. Flavonoid content was expressed in quercetin equivalents (QE). The results showed than all the three extracts have a significant phenolic and flavonoid content present, variable in relation to the type of extraction solvent and method, suggesting that they could exploit pharmacological effects related to these two classes of bioactive substances. To assess the antisteatotic and antifibrotic effects of Okra extracts, we used two cell lines: HepG2 cells treated with oleic acid and palmitic acid to induce steatosis, and LX-2 cells activated with TGF-β1 to simulate hepatic fibrogenesis. Treatment with Abelmoschus esculentus extracts at a concentration of 5mg/mL for 24 hours resulted in a dose-dependent reduction of the intracellular lipid droplets of steatotic HepG2 cells, suggesting antisteatotic properties. Additionally, cells treated with TGF-β1 (5 µg/mL) for 24 hours in the presence of OFE, OFEM, and OSEM at a concentration of 5 µg/mL showed a significant decrease in the expression of Acta2 in TGF-β1-activated LX-2 cells, suggesting the antifibrotic effects of the extracts. These findings highlight the therapeutic potential of Abelmoschus esculentus extracts in mitigating hepatic steatosis and fibrosis, providing preclinical evidence useful for a further pharmacological investigation for targeting MASLD and MASH.