Study On The Antibacterial Activity Of Neem Oil In Vitro And The Isolation, Purification And Identification Of Antibacterial Active Component From Neem Oil

Neem oil, extracted from the seeds of Azadirachta indica A. juss, has been well known in the Indian, as a traditional medicine product possessing a wide pharmaco-activity, including anti-parasite activity, antibacterial activity, antipyretic activity, anti-inflammatory activity, immunostimulant activity, antiulcer activity and promotion of wound healing, etc. It has prompted the screening of neem oil for the biological effects on plant pathogenic bacteria recently. Moreover, the most investigations were related to antibacterial activity with neem crude oil. However, there is no information about neem oil against animal pathogenic bacteria and isolation, purification and identification of antibacterial active component from neem oil. So this thesis focus on the antibacterial activity of neem oil, extracts of neem oil against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Salmonella sp CMCC (B) 50041 in vitro. The screening for active part, isolation, purification and identification for active component of neem oil were studied by a bioassay-directed fractionation. The main results as following:1. Study on the antibacterial active site of neem oil:Neem oil was successively extracted with petroleum ether, chloroform and n-butanol by system solvent, and the activities of extracts against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Salmonella sp CMCC (B) 50041 were studied by agar dilution method in vitro. The result showed that the rates of petroleum ether, chloroform and n-butanol extracts were 65.30%,10.10% and 18.80%, respectively. The rate of petroleum ether extract was the highest. The MICs of petroleum ether and chloroform extracts against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Salmonella sp CMCC (B) 50041 were the same,50,100 and 100 mg/mL, respectively. The MICs of n-butanol extract against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Salmonella sp CMCC (B) 50041 were 25,50 and 50 mg/mL. The petroleum ether extract of neem oil was studied further concerning rate and bacteriostatic effect of three extracts of neem oil.2. Study on the isolation and purification of active compound of petroleum ether extract from neem oil:A bioassay-directed fractionation of the petroleum ether extract of neem oil was conducted by the separation with silica gel column chromatography, each fraction was studied its activity against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Salmonella sp CMCC (B) 50041 by tube dilution method, and the active fraction was purified by re-crystallizing. Petroleum ether extract was separated into three fractions, A, B and C, and the rates were 45.94%,19.16%,6.52%, respectively. The MIC of fraction A were 100 mg/mL against three bacteria strains, and the MICs of fraction B and C were 50 mg/mL against the same strains. The results indicated that strains were more sensitive to fraction B and C than to fraction A, and two white active compounds (compound 1 and compound 2) and a white active mixture 3 were obtained from fraction B.3. Study on the active determination of the crystal (from acetone) of petroleum ether extract from neem oil:The activity of compound 1, compound 2 and mixture 3 against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Salmonella sp CMCC (B) 50041 were determined by broth microdilution method, and time-kill curves of three drugs were developed in order to study the correlation of the concentration and bacteriostatic effect of drugs. The MICs and MBCs of compound 1 against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Salmonella sp CMCC (B) 50041 were 20,5,10 mg/mL and 20,20,10 mg/mL, respectively. The MICs and MBCs of compound 2 against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Salmonella sp CMCC (B) 50041 were 10,10, 2.5 mg/mL and 20,20,20 mg/mL; respectively. The MICs and MBCs of mixture 3 against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922 and Salmonella sp CMCC (B) 50041 were 5,10,20 mg/mL and 10,20,20 mg/mL, respectively. In conclution, the antibacterial activity of compound 1 against Escherichia coli ATCC 25922 was the best, the antibacterial activity of compound 2 against Salmonella sp CMCC (B) 50041 was the best, the antibacterial activity of mixture 3 against Staphylococcus aureus ATCC 25923 was the best. Time-kill test indicated that compound 1 and compound 2 were drugs depended on concentration, mixture 3 was the drug depended on time.4. Study on the analysis of spectroscopy and identification of structure of the compound from neem oil:The chemical structure of the single compound was analyzed by spectroscopy including Infrared spectroscopy (IR), Electron spray ionization mass spectrometry (ESI-MS),1H nuclear magnetic resonance spectroscopy (1H NMR),13C nuclear magnetic resonance spectroscopy (13C NMR), Distortionless enhancement by polarization transfer and 2D nuclear magnetic resonance spectroscopy. The results as following:The molecular formula of compound 1 was determined as C38H70O5 by ESI-MS ([M]+, at m/z 606), IRνmas CHCl 3 cm-1:3004.56,659.68,1069.28 (-CH=CH-),2917.56,2851.72, 1466.48,1381.38,717.67 (alkyls),1734.79,1182.37,1295.82(-CO-O-),1139(-O-); 1H NMR (CDCl3,400 HZ, d/ppm):0.87 (-CH3),1.29,1.62,2.32 (-CH2-),4.15 (-CH2-,-CH-),5.34(-CH=CH-); 13C NMR (CDCl3,400 HZ, d/ppm):173.88,129.67, 68.33,65.00,14.06,22.64,24.83,27.15,29.11,31.86,34.07.'H-'H chemical shift correlation spectroscopy (1H-1H COSY):The'H-'H COSY spectrum showed the connectivities of signal at dH 0.87 with dH 1.29, dH 1.29 with dH 1.62, and signal at dH 1.62 with dH 2.35.1H-detected heteronuclear multiple quantum coherence (HMQC):Signal at dc 14.06 was correlated with the signal at dH 0.87, dC 22.64,29.11 and 31.86 with dH 1.29, dC 24.83 with dH 1.62, dC 27.15 with dH 1.98, dC 34.07 with dH 2.35, dC 65.00 and 68.33 with dH 4.15, dC 129.67 with dH 5.34.1H-detected heteronuclear multiple-bond coherence (HMBC):The cross-peaks were noted for correlation between dH 0.87/(dc 22.64,31.86), dH 1.29/(dc31.86 and 24.83), dH 1.62/(dc 34.07 and 29.11), dH 4.15/(dc 68.33 and 65.00), dH 1.98/dC 129.67, dH 5.34/dc 27.15. From the above spectral data, the structure of compound 1 was deduced to be 9-octadecanoic acid-hexadecanoic acid-3,4-tetrahydrofuran diester。The molecular formula of compound 2 was determined as C40H76O5 by ESI-MS ([M]+, at m/z 636),IRνmax CHCl 3 cm-1:2917.40,2849.33,1471.44,1381.38,717.67 (alkyls),1735.84, 1182.08,1256.82 (-CO-O-),1139 (-O-); 1H NMR (CDC13,400 HZ, d/ppm):0.86 (-CH3),1.25,1.63,2.35 (-CH2-),4.15 (-CH2-,-CH-); 13C NMR (CDC13,400 HZ, d/ppm):173.89,68.45,65.05,14.09,22.68,24.90,29.13,31.92,34.11. 1H-1H chemical shift correlation spectroscopy (1H-1H COSY):The 1H-1H COSY spectrum showed the connectivities of signal at dH 0.86 with dH 1.25, dH 1.25 with dH 1.63, and signal at dH 1.63 with dH 2.35.1H-detected heteronuclear multiple quantum coherence (HMQC): Signal at dc 14.09 was correlated with the signal at dH 0.86, dC 22.68 and 31.92 with dH 1.25, dC 24.90 with dH 1.63, dC 34.11 with dH 2.35, dC 65.05 and 68.45 with dH 4.15. 1H-detected heteronuclear multiple-bond coherence (HMBC):The cross-peaks were noted for correlation between dH 0.86/(dC 22.68,31.92), dH 1.25/(dC 31.92 and 24.92), dH 1.63/(dc 34.11 and 29.13), dH 4.15/(dc 68.45 and 65.05). From the above spectral data, the structure of compound 2 was deduced to be octadecanoic acid-3,4-tetrahydrofuran diester.The mixture 3 was mainly composed of palmitic acid, octadecanoic acid, erucamide, docosane and contained a little of oleinic acid, diethyl phthalate and tetratetracontane, based on gas chromatography and mass spectrogram analysis.