Study On The Antibacterial Active Component And Mechanism Of Ainsliaea Bonatii Beauv

This thesis consists of four chapters. The first chapter summarizes the pathogenic characteristics and pathogenicity of Staphylococcus aureus, antibacterial ingredients in Chinese herbal medicine, method and mechanism of antimicrobial activity. The second chapter studies the antibacterial activity of hydroquinone and arbutin against six kinds of bacteria in vitro. The third chapter by studying the changing of alkaline phosphatase content, electrical conductivity, the soluble protein concentration, SDS-PAGE and SEM, investigate the mechanism of hydroquinone against SA, MRSA and β-Lactamase producing S. aureus. The fourth chapter determinate arbutin and hydroquinone in whole plant of Ainsliaea bonatii with HPLC, after established ionic liquid were selected as extraction solvent, determinate arbutin and hydroquinone in different part of A. bonatii.Chapter 1 Advances of antibacterial ingredients and mechanism of chinese medicinePathogenic characteristics and pathogenicity of S. aureus, antibacterial ingredients in Chinese herbal medicine, method in vitro and vivo and mechanism of antimicrobial activity were summarized in vitro.Chapter 2 The antibacterial activity of hydroquinone and arbutin in vitro of A. bonatiiDisc diffusion method(K-B method) was used for the determination of the inhibition zone size(IZ) and the minimum inhibitory concentration(MIC) of hydroquinone and arbutin antimicrobial activity in A. bonatii. Half inhibitory concentration(IC50) was determined by liquid culture method. Liquid culture method and streak plate method to determine the minimum bactericidal concentration(MBC). The results showed that hydroquinone and arbutin had a relatively strong antibacterial activity against S. aureus(SA), methicillin-resistant S. aureus(MRSA) and β-Lactamase producing S. aureus, but for EC, ESBLs-EC and PA no showed antimicrobial activity. MIC value of hydroquinone were 0.31, 1.25 and 0.63 mg/mL against SA, MRSA and β-Lactamase producing S. aureus respectively. MIC value of arbutin were 2.5, 10.0 and 10.0 mg/mL against SA, MRSA and β-Lactamase producing S. aureus respectively. IC50 value of hydroquinone were 1.09±0.05, 1.74±0.06 and 1.17±0.01 mg/mL against SA, MRSA and β-Lactamase producing S. aureus respectively; IC50 value of arbutin were 8.42±0.02, 16.74±0.04 and 18.14±0.02 mg/mL against SA, MRSA and β-Lactamase producing S. aureus respectively.Chapter 3 The antibacterial mechanism of hydroquinoneThe mechanism of hydroquinone against SA, MRSA and beta-Lactamase producing S. aureus was assayed by studying the changing of alkaline phosphatase content, electrical conductivity, the soluble protein concentration, SDS-PAGE and SEM.Results showed that bacteria were reacted by hydroquinone, could lead to the destruction of the bacterial cell wall and cell membrane, permeability increased, a large number of intracellular material leak, at the same time, the synthesis of protein and the expression of related genes are affected, reduce the quality of the corresponding protein.Chapter 4 Simultaneous determination of arbutin and hydroquinone in Ainsliaea fragrans by ionic liquid-based HPLCThe chromatographic column was discovery Purospher star RP-C18(250 mm×4.6 mm, 5μm), UV detection wavelength was set at 286 nm, a mixture of methanol and 0.4% phosphoric acid-water was used as mobile phase by low pressure elution at a flow rate 0.65 mL/min. The result display that arbutin at the sample size of 0.35~3.5 μg showed good linearity(n=6, r=1.0000), the average recovery was 99.75%; hydroquinone at the sample size of 0.025~0.25 μg showed good linearity(n=6, r=1.0000), the average recovery was 98.04%.A novel ionic liquid-based ultrasonic-assisted extraction(ILUAE) combined with reverse-phase high performance liquid chromatography tandem UV detection(HPLC-UV) was developed to isolate and determinate arbutin and hydroquinone in different part of A. bonatii. The 1-Butyl-3-methylimidazolium bromide([BMIM]Br), 1-Butyl-3-methylimidazolium tetrafluoroborate([BMIM]BF4), 1-Butyl-3-methylimidazolium hexafluorophosphate([BMIM]PF6) and 1-Hexyl-3-methylimidazolium hexafluorophosphate([HMIM]PF6) were selected as extraction solvent. Methanol, acetonitrile and ethanol were selected as dispersant. UV detection wavelength was set at 286 nm, a mixture of methanol and 0.4% phosphoric acid-water was used as mobile phase by low pressure gradient elution at a flow rate 0.65 mL/min. The result showed it had the highest extraction yield that the conditions were 50 mesh sieve powder, solid-liquid ratio is 1:60(g/mL), and the [BMIM]BF4 methanol solution concentration was 0.7 mol/L. Under the optimal extraction conditions, arbutin at the sample size of 1.4~14.0 μg showed good linearity(n=6, r=1.0000), the average recovery was 99.74%, hydroquinone at the sample size of 0.1~1.0 μg showed good linearity(n=6, r=1.0000), the average recovery was 97.20%.Methanol extracted solution and [BMIM]BF4 methanol extracted solution of A. fragrans root, petiole and leaf blade were compared for antimicrobial activity. To study the antibacterial activity of different extracted solution. The results display that the three parts of the methanol extracted solution are inactive, the three parts of the [BMIM]BF4 methanol extracted solution activity are very strong. From the three parts of the [BMIM]BF4 methanol extracted solution on MIC values against MRSA, it can be seen that the activity is better than streptomycin.