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Studies on Improving Drosophila Sleep and 5-HT Levelby Petroleum Extract of Valeriana amurensis
Ling Liu, Jiashuai Chen, Junkai Wu, Xiaowei Du
Pharmacognosy, Heilongjiang University of Chinese Medicine, Harbin, China
Abstract.
Objective: To study the sleep improvement of petroleum extract of Valeriana amurensis, observe its effect on the contents of 5-HT in the brain of drosophila, and preliminarily investigate the activity mechanism. Method: We used an Infrared Drosophila Activity Monitor System (DAMS; Trikinetics Ine.USA) to record sleep state of drosophila. The improved-sleeping mechanism of petroleum extract was determined by detecting the contents of 5-HT in the brains of drosophila with ELISA method. Result: (1). In the experiment of the sleeping time which affected drosophila: 8% drug substratum for dose, 4 days for delivery time is best to female(P<0.01); 4% drug substratum for dose, 2 days for delivery time is best.
to male(P<0.01).(2). 5-HT contents in the brain of drosophila were increased at five time points by treating with optimal dose of petroleum extract.
Key Words: Valeriana amurensis; petroleum extract; improving sleep; mechanism
1. Introduction
While sleep is essential for health and cognition, few data are available about the neural mechanisms that govern the timing, onset and maintenance of sleep. Drosophila melanogaster exhibits a sleep-like rest state, characterized by long periods of immobility,1 increased arousal thresholds,1 homeostatic rebound after sleep deprivation,2 modulation by drugs,3 and so is an ideal model organism for the study of sleep.In Drosophila, serotonergic neurons send projections to most brain regions4. Conserved effects of serotonin on the regulation of complex behaviors in flies and mammals were demonstrated in fly models of addiction, aggression.
We demonstrate the petroleum extract of Valeriana amurensis which have sedative and hypnotic effects by Drosophila model.
2.Materials and methods 2.1. Animals
Flies were cultured and tested at 25°C,50% -60%humidity, 12h:12h light:dark cycle (lights on at 7 AM), on yeast, dark corn syrup,and agar food. Fly lines included the CS line, w1118,which were obtained from College of life science at Peking University .Exposure of Drosophila to the dietary supplements used in our analyses was performed by allowing flies to lay eggs on supplemented food or by transferring first-instar larvae to supplemented food and allowing them to develop to the adult stage. Adult flies were then collected during eclosion and transferred to fresh supplemented food.Flies were 6 days old at the beginning of the experiment.
2.2. Sleep Monitoring
Newly eclosed adult flies were collected from culture vials daily under CO2anesthesia.Separating female and male individuals, then place into the corresponding culture vials. Five day after collection, flies were individually placed into 65-mm glass tubes (5-mm diameter) and sleep was evaluated using the Drosophila Activity Monitor System (DAMS) inside glass tubes (1 fly per tube) with enough food for recording. After 1 days of environmental adaptation in the monitors, data were collected for one day. Monitors were housed inside environmental chambers where temperature and humidity were kept constant. Each DAMS monitor contained 32 glass tubes. Sleep was defined as periods of 5 min without recorded activity.
2.3.Drug Treatment
For the Valeriana amurensis petroleum extract treatment, flies were maintained in food containing 0.5%,1%,2%,4% and 8% Valeriana amurensis petroleum extract as the treatment group, and in food containing vehicle alone as the control group during adjustment period and the data collection period.
2.4.ELISA
Drosophila placed in liquid nitrogen for one minute,and remove and shake vigorously to make Drosophila head and body separated. Rapid collection of thirty drosophila head, adding 500^1saline, homogenized in manual homogenizer.Centrifuge samples for 30 minutes at 3000*g.Following the steps of the instructions of Fruit Serotonin(5-HT) ELISA to determine5-HT content.
2.5.Data Analysis
All statistical analysis was conducted using SPSS18.0. Normally distributed data were analyzed with 3-tailed, unpaired Student ¿-tests or one-way analysis of variance (ANOVA) followed by the Tukey-Kramer HSD Test as the post hoc test. Data were presented as mean behavioral responses and error bars represent the standard error of the mean (SEM). Differences between groups were considered significant if the probability of error was less than 0.05 (P < 0.05).
3.Results and discussion In order to determine the optimal dose of valerian, the flies raised in five differentconcentrationsextract. In female Drosophila,containing 4% petroleum extract of valerian displayed a significant sleep increase in daytime and total sleeping time. Containing 8% petroleum extract of valerian exhibited a robust sleep increased to control. In contrast, flies treated with 0.5%,1% and 2% extract of valerian displayed no significant sleep increase(Figure1A). Since 8% of the group was more effective, which were chosen the optimal dose of female flies. In male Drosophila,2% ,4% and 8% of petroleum extract had a significant sleep increase(Figure1B). In addition, valerian petroleum which improves the role of male flies sleep showed a dose-effect relationship. Drug concentrations in the range of less than 4% efficacy increases with increasing dose, but more than 4% in the base will not increase efficacy. So 4% was chosen as the optimal dose of male flies.
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Figure1. Petroleum extract of Valeriana amurensis causes sleep increase in Drosophila.(A) The day sleeping time,night sleeping time and total sleeping time change after feeding with different drug as compared with controls in female flies (*P < 0.05,**P < 0.01). (B) The day sleeping time,night sleeping time and total sleeping time increase after feeding with different concentrationdrug as compared with controls in male flies (*P < 0.05,**P < 0.01).
When the administration of one day, the sleeping time of female Drosophila was significantly prolonged compared with the control group.From the administration of two days to the six day, sleep lasted longer with the administration of one day group. As from four days, the sleeping time was short, so choose administered four days as the best administration time in female Drosophila(Figure2.A). The Study on the Time-effect Relationship in the Treatment of valerian petroleum ether fractions of the fale flies, the administration two days ago, the sleep time was prolonged trend, in which administration of one day compared with the control group with a very significant difference, administered two days group and treatment group than one day there was significant difference , so administered two days for the male flies the best time of administration(Figure2.B)
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Figure2. Length of different administration affects drosophila sleep. (A) In female drosophila, one day for delivery time can improve sleep time compared with untreated control ( *P < 0.05,**P <
0.01.. Two days for delivery time has significant in total sleeping time compared with one day(#p<0.05,##p<0.01).It can increase sleep until seven days.(B) In male drosophila, one day for delivery time can also improve sleep time compared with untreated control( *P < 0.05,**P < 0.01). Two days for delivery time has significant in total sleeping time compared with one day(#p<0.05,##p<0.01).Since the five days, petroleum extract has no significant compared with control.
According to the steps of the instructions of Fruit 5-HTELISA,the standard curve was Y=0.0016X+0.1489(R2=0.9926).
From the experimental results, 5-HT content of the female flies headupward trend from 21:00 to 1:00, and followed by a gradual decline; the 5-HT content of male Drosophila head was gradually increased over time. Compared with the control group, at each time point Drosophila head 5-HT levels were significantly increased. Conclusions
Valeriana amurensis petroleum part could significantly prolong the sleep duration of drosophila. The improved-sleeping mechanism of Valeriana amurensis petroleum extract was verified to exist the relationship with the content of 5-HT in drosophila brain. References
1. HendricksJC, FinnSM, PanckeriKA, etal. Rest in Drosophila is a sleep-like state[J]. Neuron 2000;25:129-38.
2. Huber R, Hill SL, Holladay C, Biesiadecki M, Tononi G, Cirelli C. Sleep homeostasis in Drosophila melanogaster[J]. Sleep 2004;27:628-39
3. Andretic R, van Swinderen B, Greenspan RJ. Dopaminergic modulation of arousal in Drosophila[J]. CurrBiol 2005;15:1165-75.
4. Monastirioti, M. Biogenic amine systems in the fruit fly Drosophila melanogaster. Microsc[J]. Res. Tech. 1999, 45, 106-121.
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Figure 3-The 5-HT level of Drosophila brain changed in different time periods.(A) Different time periods female Drosophila brain 5-HT content have significant differences compared with the control group(*p<0.05,**p<0.01). (B)The fale flies have the same trend with the female flies.5-HT level displayed a significant compared with the control(*p<0.05,**p<0.01).
PAMD and its monomeric components on BxPC-3 Pancreas Tumor cells Proliferation Effect
Yang Li
HeilongjiangUniversity of Traditional Chinese Medicine,Haerbin,China
Abstracts:Objective:Explore PAMD, Dau, DS and proportion of group (Dau mixed 3:1 with DS ) drugs is proliferation of BxPC-3 pancreatic tumor cells in vitro. Method: BxPC-3 cells were subcultured, were added to low high school three doses PAMD, DS, Dau and proportion of group solution , while the control group an equal volume of saline, the role of 72h, by MTT assay absorbance (OD) calculate the group BxPC-3 cell proliferation inhibition rate. Result: The results showed that, PAMD high school low-dose group, the inhibition rates were 93.27%, 24.64%, 15.45%, Dau high school low-dose group, the inhibition rates were 67.18%, 24.09%, 12.00%, DS High School low dose inhibition rates were was 20.18%, 15.64%, 6.27 %, and the proportion of high school low -dose group, group inhibition rates were 30.18%, 41.27%, 17.36%. With the control group, in addition to the low-dose group of DS Dau groups were statistically significant (P <0.05). Conclusion: PAMD and its monomer component Dau and DS on BxPC-3 cell proliferation was inhibited, but Dau, DS and proportion group of drugs on BxPC-3 cell proliferation was weaker than PAMD group, indicating PAMD on BxPC-3 cells may also inhibit the proliferation of other ingredients with PAMD relationship.
Keywords:PAMD,Dauricine,BxPC-3, Proliferation
Chinese medicine is Menispermi Menispermaceous plants Menispermum dauricum (DC) of dry roots, while Phenolic alkaloids of Menispermum dauricum (PAMD) is extracted Menispermi soluble mixture of alkaloids[1]. PAMD, containing mainly Dauricine, (Dau) and Daurisoline,(DS), etc. Our previous studies have shown that, PAMD variety of tumor cells proliferation and
