Antidiabetic activities of Bolanthus spergulifolius (Caryophyllaceae) extracts on insulin-resistant 3T3-L1 adipocytes

Diabetes mellitus (DM) is a metabolic disorder with chronic hyperglycemia featured by metabolic outcomes owing to insufficient insulin secretion and/or insulin effect defect. It is critical to investigate new therapeutic approaches for T2DM and alternative, natural agents that target molecules in potential signal pathways. Medicinal plants are significant resources in the research of alternative new drug active ingredients. Bolanthus spergulifolius (B. spergulifolius)is one of the generaof the family Caryophyllaceae. In this study,it was explored the potentialanti-diabetic effects in vitroof B. spergulifolius extractson 3T3-L1 adipocytes.The total phenolic contents (TPC) of methanolic (MeOH), ethyl acettate (EA) and aqueous extracts of B. spergulifoliuswere evaluated via Folin-Ciocateau. B. spergulifoliusextracts showing highly TPC (Aqueous< MeOH< EA) and their different concentrations were carried out on preadipocytes differentiated in to mature 3T3-L1 adipocytesto investigate their half-maximal (50%) inhibitory concentration (IC50) value by using Thiazolyl blue tetrazolium bromide (MTT) assay.  The IC50 of MeOH, EA and Aqueous extracts were observed as 305.7 ± 5.583 µg/mL, 567.4 ± 3.008 µg/mL, and 418.3 ± 4.390 µg/mL and used for further experiments.A live/dead assay further confirmed the cytotoxic effects of MeOH, EA and Aqueous extracts(respectively, 69.75 ± 1.70%, 61.75 ± 1.70%, 70 ± 4.24%, and for all p< 0.05). Also, effects of extracts on lipid accumulation in mature 3T3-L1 adipocyteswere evaluated by Oil-Red O staining assay. The extractseffectively decreasedlipid-accumulation compared to untreated adipocytes(for all p< 0.05).Moreover, effect of extracts on apoptosis regulated by the Baxand Bcl-2was investigated by quantitative reverse transcription polymerase chain reaction (qRT-PCR).Theextractssignificantly induced apoptosis by up-regulating pro-apoptoticBaxexpression but down-regulatedanti-apoptoticBcl-2gene expression compared to untreated adipocytes(for all p< 0.05).The Glut-4expression linked with insulin resistance was determined by qRT-PCR, Western-blot analysis, and immunofluorescence staining.In parallel, the expression of Glut-4 in adipocytes treated with extracts was significantlyhigher compared to untreated adipocytes(for all p< 0.05). Extracts significantly suppressed cell migration after 30 h of wounding in a scratch-assay(for all p< 0.05). Cell morphology and diameter were further evaluated by phase-contrast microscopy, scanning electron microscopy, Immunofluorescence with F-Actin and Giemsa staining. The adipocytes treated with extracts partially lost spherical morphology and showed smaller cell-diameter compared to untreated adipocytes (for all p< 0.05). In conclusion, these results suggest thatextracts of B. spergulifoliuscause to an induce apoptosis, decrease lipid-accumulation, woundhealing, up-regulating Glut-4 level and might contribute to reducing of insulin-resistance in DM.