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High‐speed counter‐current chromatography (HSCCC) was utilized as an effective procedure for isolation of targeted three anthocyanins di‐glucosides from Syzygium cumini (SC) pulp by using an optimized biphasic successful combination of different solvents. The resulted fractions described by HPLC/ESI‐MS to be delphinidin 3,5‐diglucoside (DDG), petunidin 3,5‐diglucoside (PDG), and malvidin 3,5‐diglucoside (MDG). A weight of 150 mg of sample yielded 7.53, 22.68, and 39.09 mg for DDG, PDG, and MDG, respectively. It was stated that the target three anthocyanins possessed strong antioxidant activities. Furthermore, MDG exhibited definite advantages for inhibition of nitric oxide release and pro‐inflammatory mediators like mouse interleukin 6 (IL‐6), mouse interleukin (IL‐1β) and mouse tumor necrosis factor (TNF‐α) in lipopolysaccharide (LPS)‐induced RAW264.7 macrophages. The results propose that HSCCC can be utilized to separate highly antioxidative and anti‐inflammatory natural components from SC pulp. Practical applications Anthocyanins are water‐soluble pigments and considered one of the groups of bioactive compounds, which generally concentrate in the skin, and often also the flesh of some fruits and vegetables as glycosides like acylglycosides and aglycones of anthocyanidins. The fully ripe fruits of SC contain anthocyanins, like as delphinidin, cyanidin, and petunidin, which supply them a distinctive color and good antioxidant characteristics. HSCCC considers a system of liquid‐liquid extraction with separating large quantities of materials, using a minimum of solvent. The findings of the study would pave a separation strategy for potential large‐scale preparation of anthocyanins di‐glucosides standards for compounds detection and reduce the inflammation symptoms through declining the induction of pro‐inflammatory cytokines such as IL‐1β, TNF‐α and IL‐6, which will also enhance the future notification on the structure‐activity correlations of anthocyanins di‐glucosides.
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