Study On The Extraction And Purification Of The Secondary Metabolites With Biological Activities From Sophora Japonica Roots

Sophora japonica L. also known as Huai, Zhong guo huai, Jia huai, is a arbor species belonging to the subfamily Faboideae of the pea family Fabaceae. This tree is grown native to China, and now extensively cultured all over the world. In traditional Chinese medicines,the flower, fruit, branch and leaf of S. japonica have long been used to cure various disorders, such as bleeding, conjunctivitis, hemorrhage and so on. Now S. japonica is used as an effevtive remedy in some cancer treatments.In this paper, the secondary metabolites of S. japonica roots were investigated. Dried and powdered roots of S. japonica were extracted by 95% ethanol (at room temperature).After combination, filtration and concentration in vacuo, the aqueous residue (crude extract)was yieled. The crude extract was suspended in water and then successfully fractionated by ligroine, EtOAc and n-BuOH on separation funnels. After concentration and freeze-drying,the resulted soluble fractions were obtained as four powders (ligroine soluble fraction, yield 0.82%; EtOAc soluble fraction, yield 2.45%; n-BuOH soluble fraction, yield 3.26%; H2O soluble fraction, yield 5.82%). The main secondary metabolites content in the extract were determined using UV spectrophotometry method. The main constituents were total polysaccharides (246.61 g) in all the extract, followed by total polyphenols (60.55 g),At least for the total tannin (8.51 g). The highest content of four kinds of component was the EtOAc soluble fraction.Antioxidant activity was evaluated by two in vitro antioxidant methods, including DPPH free radical scavenging and reducing capacity. As a whole, crude extract and soluble fractions showed significant scavenging activity and reducing capacity. Meanwhile,reducing capacity of EtOAc soluble fraction was the best, followed by ligroine and n-Butanol soluble fraction, water soluble was worst.Antibacterial activity was investigated using cylinder-plate method against Gram-positive bacteria (Staphylococous aureus and Bacillus subtilis) and Gram-negative bacteria (Escherichia coli and Salmonella enteric) on the crude extract and soluble fractions of Sophora japonica roots. Results suggested that the EtOAc soluble fraction and the crude extract exhibited strong antibacterial activity, and the ligroine and n-BuOH soluble fractions showed moderate antibacterial effect. While, the antibacterial activity of water soluble fraction was the weakest.The extraction phase of ligroine was selected to separate and purify the compounds.Repeated Silica gel Column Chromatography and Thin Layer Chromatography (TLC) over the ligroine soluble fraction led to the isolation and purification of five secondary metabolites, which were identified as Oleanolic acid (1), 3a-hydroxycativic acid (2),n-Heptanol (3),?-Amyrin (4) and Oleanolic acid 3-O-?-D-glucopyranoside (5). The structure of the compounds were mainly elucidated on the basis of Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS) techniques.