Study On Isolation, Purification And Antibacterial Activity Of Antimicrobial Compound From Catalpa Ovata G.Don

Plants are a natural treasure of bioactive compounds. Antibacterial compounds widely exist in plants. In recent years, a great deal of experimental research was done on the extracts, screening, antibacterial properties and antibacterial mechanism of plant antibacterial compounds home and abroad. Great progress has been made and many antibacterial substances were isolated and identified from plants. There are many kinds of antibacterial ingredients, such as saponins, organic acids, volatile oils, flavonoids, terpenoids, steroids, alkaloids, quinones, etc.Catalpa ovata G.Don belongs to Bignoniaceae. Its bark is known as bark of Chinese catalpa and its fruit is known as fruit of Chinese catalpa. Both them can be used as medicine with the function of heat-clearing, detoxification, desinsection and diuresis. Although the reports on its Clinical medicine were a lot, these on its antibacterial and antimicrobial ingredients were little. In this paper, a systematical research was done on the antibacterial activity of catalpa fruit. We found the antibacterial activity of Catalpa ovata G.Don for the first time, and explored an easy method suitable to separation, purification and preparation the antibacterial compounds. And the antibacterial compoundsρ-hydroxybenzoic acid was isolated from this plant for the first time, too.Firstly, extract the powder of Catalpa ovata fruit with ethanol, and then concentrated to dryness and dissolve with methanol, finally obtained the crude extract. The antibacterial activity and stability of the crude extract were studied by means of cylinder-plate method using Bacillus subtilis as test bacteria. And the antibacterial activities of extract on the other six test bacteria were measured, such as Clavibacter michiganensis sub sp. michiganensis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Ralstonia solanacearum, Erwinia carotovora pv. Carotovora. Experiments showed that the crude extract of the Catalpa ovata fruit had a good inhibitory effect on Bacillus subtilis, and the relative concentration and the area of clearing zone showed a good gradient change. The crude extract inhibited significantly the growth of a wide range of the Gram-positive bacteria and the Gram-negative bacteria. Thermal treatment, lower temperature treatment, repeated freezing and thawing would not affect its activity. It can also inhibit the growth of Bacillus subtilis apparently.The main antibacterial compound was isolated from the crude extract by gel filtration Sephadex LH-20 and RP-HPLC combinations with bioassay-guided fractionation method. After separation by Sephadex LH-20 twice, it was found that the ingredient in tube 31~36 had the inhibitory effect on Bacillus subtilis. Then active parts were merged and several duplicates were collected. When separation conditions of the samples was explored on the analytical HPLC, we frond that using the ratio of methanol with 30%, the antibacterial compound was separated perfectly. There were three peaks. It was frond that the second peak had antibacterial activity. Subsequently semi-preparative HPLC separation was conducted. At last freeze-dried samples were obtained with purity of 99.926%The UV, MS, 1H NMR and 13C NMR of the antimicrobial compound were measured, and it was finally identified to beρ-hydroxybenzoic acid. Then we reconfirm the structure of the samples by contrastive analysis the HPLC and MS between the standard and the sample we got. The MIC of the standard against Bacillus subtilis , Staphylococcus aureus,Escherichia coli were tested, The results are respectively 3mg/mL, 1mg/mL, 1mg/mL.