Study On The Acaricidal Activity Of Neem Oil In Vitro And The Isolation, Purification And Identification Of Acaricidal 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,antimicrobial 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 veterinary ectozoon in recently.The most investigations relate to anti-tick activity. However,there is no information about neem oil against Sarcoptes scabiei.In order to develope a new botan-acaricide and utilize the resource of neem oil in China,this thesis focus on the acaricidal activity of neem oil against Sarcoptes scabiei var.cuniculi 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 follows:1.Study on the acaricidal activity of neem oil against S.scabiei var.cuniculi in vitro:The effect of temperature and medium on the survival of S.scabiei var.cuniculi was examed at 75%relative humidity(R.H) in vitro.Under the condition:the R.H was 75%,the temperature was 25℃,the medium was paraffin oil or peanut oil,the survival rate of mites was 90%in 24 h.This condition was benefit for acaricidal active test in vitro.The acaricidal bioassay of neem oil was performed according to a dipping method. The results showed that the undiluted neem oil killed all larvae in 25.00 min.The acaricidal activity of the undiluted neem oil was significantly higher than the pyrethrin of 500 mg/mL(825.00 min,P＜0.01),but not significantly different than the avermectin of 25 mg/mL(18.75 min,P＞0.05).The median lethal concentration value of neem oil was 2.908μL/mL,after 24 h.The median lethal time values were 0.991,2.176 and 5.33 h. respectively when the concentrations of neem oil were 500,250,125μL/mL,respectively. The acaricidal activiy of neem oil showed concentration and time relationship.These results demonstrated that neem oil had good effect on S.scabiei var.cuniculi larvae in vitro.2.Study on the aearieidal active site of neem oil:Neem oil was isolated by system solvent,and studied its active site by an acaricidal test in vitro.The result showed that the rates of petroleum ether,chloroform and acetic ether extracts were 56.82%,25.36%and 5.14%,respectively.The acaricidal activities of petroleum ether,chloroform and acetic ether extracts of 250μL/mL were not significantly different in both prophase(12 h) and anaphase(20 h) of treatment.In the metaphase(16 h) of treatment,the lethality rates of petroleum ether,chloroform and acetic ether extracts were 95.15%,80.28%,83.65%, respectively.It was observed that the acaricidal activity of petroleum ether extract was significantly higher(P＜0.05) than the other two in metaphase,while the activity of chloroform extract was not significantly different(P＞0.05) than acetic ether extract.These results showed that the petroleum ether,chloroform and acetic ether extracts were all effect on S.scabiei var.cuniculi larvae in vitro.The petroleum ether extract displayed relative better acaricidal activity than the other extracts,the chloroform and acetic ether extract showed the similar activity.3.Study on the isolation and purification of active compound from neem oil:A bioassay-directed fractionation of the petroleum ether and chloroform extracts of neem oil was conducted by the separation with silica gel column chromatography,and the active fraction was purified by re-crystallizing.Both petroleum ether and chloroform extracts were separated into four fractions.Fractionâ…¢of chloroform extract was demonstrated to be the most effective fraction when the concentration was 200 mg/mL with 100%lethal rate in 4.5 h against S.scabiei var.cuniculi larvae in vitro.The recrystallization with acetone resulted in the isolation of a white snowflake crystallization from fractionâ…¢.The results indicated that the acaricidal active component of neem oil was concentrated in fractionâ…¢of the chloroform extract,and a crystal compound was obtained from fractionâ…¢.4.Study on the active determination of the compound from nem oil:A complementary log-log model was used to analyze the biological activity of active compounds in vitro.The results showed that the concentration effect of the acaricidal toxicity of active compound was the correlation function of time effect,and the time effect of the acaricidal toxicity was the correlation function of concentraton effect,too.The median lethal concentration value of single compound was 0.082 mg/mL at 24 h.It was only one thirty third of the median lethal concentration value of neem oil.The median lethal time value of single compound was 15.33 h at the concentration of 7.500 mg/mL. The result revealed that the single compound has a good activity against S.scabiei var. cuniculi larvae in vitro.5.Study on the analysis of spectroscopy 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 2 D nuclear magnetic resonance spectroscopy.The molecular formula of the single compound was determined as C₄₀H₇₆O₅ by ESI-MS([M]⁺,at m/z 636),IRν_max^CHCl₃cm^-1:2917.40, 2849.33,1471.44,1381.38,717.67(alkyls),1735.84,1182.08,1256.82(—CO—O—), 1139(—O—);¹H NMR(CDCl₃,400 HZ,δ/ppm):0.86(—CH₃),1.25,1.63,2.35(—CH₂—),4.15(—CH₂—,—CH—);¹³C NMR(CDCl₃,400 HZ,δ/ppm):173.89,68.45,δ65.05, 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 atδ_H 0.86 with 1.25,δ_H 1.25 withδ_H 1.63,and signal atδ_H 1.63 withδ_H 2.35.¹H-detected heteronuclear multiple quantum coherence(HMQC):Signal atδ_C 14.09 was correlated with the signal atδ_H 0.86,δ_C 22.68 and 31.92 withδ_H 1.25,δ_C 24.90 withδ_H 1.63,δ_C 34.11 withδ_H 2.35,δ_C 65.05 and 68.45 withδ_H 4.15.¹H-detected heteronuclear multiple-bond coherence(HMBC):The cross-peaks were noted for correlation betweenδ_H 0.86/(δ_C 22.68, 31.92),δ_H 1.25/(δ_C31.92 and 24.92),δ_H 1.63/(δ_C 34.11 and 29.13),δ_H 4.15/(δ_C 68.45 and 65.05).From the above spectral data,the structure of the single compound was deduced to be Octadecanoic acid-tetrahydrofuran-3,4-diyl ester.