Background: Obesity as a developing global challenge can be characterized by increase in adipocyte number and size arising from adipogenesis. Control of adipogenesis, as a potential strategy, can prevent and manage obesity. So far, the effectiveness of herbal medicine and active ingredients therapies for obesity and metabolic syndrome treatment has been investigated. In this study, a novel combination of berberine, catechin, and capsaicin was developed, and their effect on 3T3-L1 adipocytes was investigated. Methods: The effect of active ingredient on the cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Adipocytes were treated with various concentrations of berberine (3 and 6.25 μM), catechin (6.25 and 12.5 μM), and capsaicin (6.25 and 12.5 μM) alone and in combination. Results: All active ingredients did not affect the cell viability by MTT assay at different concentrations. The dual and triple combinations of three active ingredients showed excellent potential as anti-obese without any toxicity. The inhibitory effect of berberine, catechin, and capsaicin on the differentiation of 3T3-L1 preadipocytes was found to be dose-dependent. These results indicate that catechin in both doses may have a stronger effect than the two other active ingredients on the intracellular lipid accumulation. Also, the triple combination of the aforementioned ingredients showed better responses than their dual combination. Conclusion: This work is the first report to simultaneously investigate these three active ingredients in a single, dual, and triple formats. The berberine, catechin, and capsaicin co-treatment inhibits the adipogenesis during the differentiation process. This compound can be a prospective therapy for obesity and relevant diseases such as dyslipidemia. 

Keywords: Berberine, Capsaicin, Catechin, Obesity, 3T3-L1 cells

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