| Objective:The specification sleep deprivation model of rats caused by level set method of the replication and acanthopanax aggregation based on the study of the preparation and quality control standards, observation of acanthopanax aggregation glycosides on sleep deprivation in rats sleep time and sleep phase.Methods:1. Level set method research on copy related factors of sleep deprivation model of ratsIn male Wistar rats as experimental object, the selection of weight (190g±30g、250g±30g、310g±30g) and sleep deprivation time (one day, three days, five days) for the influencing factors, and by orthogonal experiment design method, according to determine the experimental scheme, by the method of improved multi-platform sleep deprivation, sleep deprivation experimental animals, and within24after deprived eeg, and analysis of the cortical eeg, in order to determine the weight of experimental animals and sleep deprivation time of level set method to copy the effects of sleep deprivation model of rats.2. The preparation of acanthopanax aggregation glycosidesSelection of acanthopanax water alcohol immersed ointment, with D101macroporous adsorption resin method, extraction, separation and purification of acanthopanax aggregation glycosides, in acanthopanax glycosides B and acanthopanax nucleoside E rate as index, the preparation of acanthopanax aggregation glycosides.On this basis, the use of high-efficiency chromatographic analysis, of acanthopanax aggregation glycosides of acanthopanax glycosides B and acanthopanax nucleoside E quantitative analysis, establish a acanthopanax glycosides B and acanthopanax glycosides E content as an index of quality control standards.3. The quality control of acanthopanax aggregation glycosidesBased on the preparation of acanthopanax aggregation glycosides, using high-efficiency chromatographic analysis, of acanthopanax aggregation glycosides of acanthopanax glycosides B and acanthopanax nucleoside E quantitative analysis, establish a acanthopanax glycosides B and acanthopanax glycosides E content as an index of quality control standards.4. Acanthopanax aggregation glycosides of level set method in rats causedby sleep deprivation sleep time and sleep Selection of weight (250g±30g)16male Wistar rats, were randomly divided into acanthopanax aggregation glycosides to medicine group and blank control group.Acanthopanax aggregation nucleoside drugs group according to5.6g·kg-1dose lavage give medicine seven consecutive days.Experimental animals, respectively, using modified method of multiplatform sleep deprivation sleep deprivation, deprived of three days, within12hours after sleep deprivation, to trace and analysis of the experimental animal cortex eeg, study of acanthopanax aggregation glycosides on levels of sleep deprivation in rats sleep time and sleep.Results:1. Level set method research on copy related factors of sleep deprivation model of ratsAfter compared with before sleep deprivation, sleep deprivation, plus or minus (190g±30g) in the rat sleep deprivation one day, sleep deprivation effect is not obvious, there was no significant difference compared with before sleep deprivation.Plus or minus (190g±30g) in the rat sleep deprivation three days, obvious effects of sleep deprivation, compared with before sleep deprivation has a significant difference (P<0.01).Plus or minus (190g±30g) in the rat sleep deprivation for five days, sleep deprivation effect extremely obvious, compared with before sleep deprivation has extremely significant difference (P <0.01).Plus or minus (250g±30g) in the rat sleep deprivation one day, sleep deprivation effect is not obvious, there was no significant difference compared with before sleep deprivation.Plus or minus (250g±30g) in the rat sleep deprivation three days, obvious effects of sleep deprivation, compared with before sleep deprivation has extremely significant difference (P <0.01).Plus or minus (250g±30g) in the rat sleep deprivation for five days, sleep deprivation effect extremely obvious, compared with before sleep deprivation has extremely significant difference (P <0.01).Plus or minus (310g±30g) in the rat sleep deprivation one day, sleep deprivation effect is not obvious, there was no significant difference compared with before sleep deprivation.Plus or minus (310g±30g) in the rat sleep deprivation three days, obvious effects of sleep deprivation, compared with before sleep deprivation has a significant difference (P <0.01).Plus or minus(310g±30g) in the rat sleep deprivation for five days, sleep deprivation effect extremely obvious, compared with before sleep deprivation has extremely significant difference (P <0.01).2. The preparation of acanthopanax aggregation glycosidesAcanthopanax extract mixture on the0.5g-mL-1sample solution, the adsorption velocity of2BV·h-1,3hours after the completion of the static adsorption,30%ethanol elution liquid elution, elution velocity1BV·h-1. In order to improve the purity of acanthopanax aggregation glycosides, on the basis of the existing preparation increased dialysis bag processing, condensed the eluent collected dry ethanol, until the volume for a third of the volume before enrichment, about200ml.Pour200ml concentrate into dialysis bag, air bag, seal is placed within the containers of1200ml of distilled water, after with a plastic wrap closed container, in the shade rest for48hours.Within48hours after all active ingredients to infiltrate distilled water, distilled water for HPLC detection spin recovery section, abandon to dialysis bag solution. Determines the acanthopanax glycosides B and chromatographic method for the determination of acanthopanax nucleoside E at the same time, chromatographic column, C18(4.6mm x250mm);Detection wavelength of220nm;Mobile phase:acetonitrile-water (0~10min,90:10;10~30min,90~80;10~20); Flow rate:1.0mL·min-1;Column temperature25°C.3-. The quality control of acanthopanax aggregation glycosidesAcanthopanax aggregation of quality standard, the content of acanthopanax nucleoside B not less than301.4mg· g-1, the content of acanthopanax nucleoside E not less than123.6mg· g-1, acanthopanax glycosides B and acanthopanax glycosides E total content of not less than425.0mg· g-1.4. Acanthopanax aggregation glycosides of level set method in rats caused by sleep deprivation sleep time and sleepCompared with the blank control group, acanthopanax aggregation glycosides give medicine group of level set method caused by sleep deprivation sleep significantly shorten the total time in rats, compared both have significant difference (P<0.05).Fast rem sleep time of sleep-deprived rats significantly shorten the role, compared with the blank group was extremely significant difference (P<0.01).On sleep deprivation in rats not fast rem sleep significantly shorten the time, compared with the blank group had significant difference (P<0.05).Conclusion:1. Experimental animals to choose males, weight (250g±30g), sleep deprivation for three days, the most obvious effects on experimental animals sleep.2. Preparation technology of acanthopanax aggregation glycosides, water alcohol sink acanthopanax extract right amount, type D101macroporous adsorption resin separation and purification, the sample liquid concentration is0.5g·mL-1,2BV·h "’adsorption velocity,3hours after the completion of the static adsorption,30%ethanol elution liquid, elution velocity1BV·h-’.Dialysis bag processing, will be collected by the eluent concentration dry ethanol, until the volume for a third of the volume before enrichment, about200ml.Pour200ml concentrate into dialysis bag, air bag, seal is placed within the containers of1200ml of distilled water, after with a plastic wrap closed container, in the shade rest for48hours.Within48hours after all active ingredients to infiltrate distilled water, distilled water for HPLC detection spin recovery section, abandon to dialysis bag solution.Acanthopanax aggregation of quality standard, the content of acanthopanax nucleoside B not less than301.4mg· g-1, the content of acanthopanax nucleoside E not less than123.6mg· g-1, acanthopanax glycosides B and acanthopanax glycosides E total content of not less than425.0mg· g-1.3. Acanthopanax aggregation of quality standard, the content of acanthopanax nucleoside B not less than301.4mg· g-1, the content of acanthopanax nucleoside E not less than123.6mg· g-1, acanthopanax glycosides B and acanthopanax glycosides E total content of not less than425.0mg· g-1.4. Acanthopanax aggregation glycosides can obviously improve total sleep time and sleep in rats. |
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