Effective Separation of Granaticins From <i>Streptomyces Vilmorinianum</i> YP1 Using High‐Speed Countercurrent Chromatography and Their Characteristic Evaluation

Granaticins and their derivatives are a series of benzoisochromanequinones (BIQs) compounds with remarkable biological activities, but their practical applications have been restricted due to the low separation efficiency of traditional methods. In this work, a high-speed countercurrent chromatography (HSCCC) method was developed for the first time to separate granaticins from the fermentation broth of Streptomyces vilmorinianum YP1. Considering the similar structures, polarities, and dissociation constants of granaticins, an appropriate solvent system for their complete separation by HSCCC was determined. This system consisted of n-hexane: ethyl acetate: methanol: water in volume ratios of 2: 5: 3: 4 and 1: 4: 1: 4. Five granaticins with high purity were obtained: a granaticin analogue (29.5 mg); granaticin acid (111.3 mg; purity: 99.2%), MM44785 (49.4 mg; purity: 99.1%), granaticin (0.8 mg; purity: 98.7 %), and granaticin B (73.3 mg; purity: 99.4%) from 2.7 g of crude granaticins extract. The antibacterial activity and stability characteristics of the two main compounds were further evaluated. Granaticin B exhibited excellent antibacterial effects on Gram-positive bacteria, while granaticin acid showed better thermal stability. This is the first time to develop a general separation strategy for granaticins that are potentially applicable in industry production.