Purification of Salvianolic Acid B

Dried danshen roots (5.4 kg) were extracted three times with methanol (MeOH) at room temperature and the solvent was removed with a vacuum. The extract was partitioned between dichloromethane and water (H₂O). The aqueous layer was adjusted to pH 2.0 with addition of trifluoroacetic acid and was further partitioned with ethyl acetate (EtOAc). The EtOAc-soluble layer was fractionated by flash silica column chromatography and eluted with a step gradient of chloroform, MeOH and H₂O (3:1:0.1→1:1:0.1, v/v/v). The fraction containing the mixture of salvianolic acids was further isolated by reversed-phase HPLC to isolate salvianolic acid B (100 mg). Based on a comparison of the MS and NMR data with previously reported data, compound 1 was identified as salvianolic acid B [16]. The purity of SalB was 96.7 ± 0.2% using area normalization methods by HPLC-UV.