β-asarone对Aβ_(1-42)激活的ACM所致PC12细胞损伤的保护作用研究

目的:探讨不同浓度β-淀粉样蛋白(Aβ_(1-42))激活的星形胶质细胞条件培养液(ACM)对PC12细胞存活率及脑源性神经营养因子(BDNF)、乙酰胆碱酯酶(ACh E)蛋白表达的影响,以及β-细辛醚(β-asarone)的保护作用。方法:首先,将对数生长期的PC12细胞随机分为正常对照组、ACM(Aβ1-423.3μmol/L)组、ACM(Aβ_(1-42)10μmol/L)组、ACM(Aβ1-4230μmol/L)组,分别采用实时无标记动态细胞分析技术(Real Time Cell Analysis,RTCA)和MTT法检测各组PC12细胞的存活率,Western blot检测PC12细胞BDNF、ACh E蛋白的表达;其次,将对数生长期的PC12细胞随机分为正常对照组、ACM(Aβ_(1-42)10μmol/L)组、β-asarone(18.5、55.5、166.7μg/mL)组,RTCA法检测PC12细胞存活率的改变,Western blot检测PC12细胞BDNF、ACh E蛋白表达的变化。结果:ACM作用24 h,各组PC12细胞存活率无统计学差异(P>0.05),ACM作用36 h,ACM(Aβ1-4210μmol/L)组、ACM(Aβ_(1-42)30μmol/L)组PC12细胞存活率显著降低(P<0.01);BDNF、ACh E蛋白表达随Aβ_(1-42)浓度增加显著升高(P<0.05或P<0.01);β-asarone(18.5、55.5、166.7μg/mL)能显著提高PC12细胞的存活率,β-asarone(55.5、166.7μg/mL)组ACh E表达相比于模型组显著下降(P<0.05或P<0.01),β-asarone(55.5μg/mL)组与模型组比较BDNF表达增加(P<0.05)。结论:β-asarone能抑制ACh E的上调,对BDNF的表达有促进作用,提示β-asarone对神经元细胞有一定的保护作用。

Effects of acrous gramineus and its component, alpha-asarone, on apoptosis of hippocampal neurons after seizure in immature rats

BACKGROUND： α-asarone and acrous gramineus have been shown to play a necessary function in enhancing the reactivity and convulsant threshold to electric stimulation of immature rats. They have also been shown to effectively suppress epileptic seizures induced by pentylenetetrazol in young rats. However, the mechanisms for these roles have been still unclear. OBJECTIVE： To observe the effects in immature rats of acrous gramineus and α -asarone on apoptosis of hippocampal neurons after epileptic seizure at the protein level, and to analyze the mechanism for these effects. DESIGN： A randomized controlled animal experiment. SETTINGS： Department of Pediatrics, First Hospital of Jilin University; Department of Histology and Embryology, Norman Bethune Medical School of Jilin University; Department of Internal Medicine, Children＇s Hospital of Changchun City; Department of Neurology, First Clinical Hospital affiliated to Harbin Medical University. MATERIALS： Fifty 3-week old Wistar rats, 34-40 g, irrespective of gender, were provided by Gaoxin Research Center of Medical Animal Experiment, Changchun. The animals were treated according to the animal ethical standards. The following chemicals were used for this study： acrous gramineus powders or infusion （Batch No, 0307113, Tianjiang Medicine Company Limited, Jiangyin）, α-asarone tablets （Batch No. 030219, Tianwei Pharmaceutical Factory, Shenyang）, and phenobarbital sodium tablets （Batch No. 020608, Xinya Medicine Company Limited, Shanghai）. The animals were divided into five groups randomly. First, ten rats were chosen as the normal controls. The remaining rats were treated with i.p. injections of pentylenetetrazol to stimulate an epileptic model. METHODS： The experiments were performed at the Neurological Laboratory of the First Hospital of Jilin University between October and December 2004. The rats were treated with i.p. injections of pentylenetetrazol （60 mg/kg） to establish an epileptic model. According to Racine＇ s standard, animals that reached stage 4 and 5 were chosen and randomly divided into 4 groups： model group, phenobarbital sodium, acrous gramineus, and a-asarone group. The normal control group was treated with an i.p. injection of physiological saline （0.5 mL）. After modeling, the model groups were intragastrically administrated 0.5 mL saline. The phenobarbital sodium, acrous gramineus, and α-asarone groups were intragastrically administrated 18 mg/kg/d phenobarbital sodium, 2 350 mg/kg/d acrous gramineus and 29 mg/kg/d α-asarone, respectively. The course of treatment was twice a day for 7 days. The normal group received intragastric administration of 0.5 mL saline at the same time. The rats were sacrificed and brain sections were prepared for light microscopy and electron microscopy. MAIN OUTCOME MEASURES： （1） Pathological changes of CA1 and CA3 hippocampal region neurons were observed by light microscopy and electronic microscopy. （2） Neuronal apoptosis in the CA1 and CA3 region was measured by TUNEL staining. （3） Bcl-2 and Bax expression in CA1 and CA3 region neurons was detected by immunohistochemistry and a ratio of Bcl-2/Bax was calculated. RESULTS： All 50 immature rats were included in the final analysis. （1） Pathological changes of CA1 and CA3 region hippocampal neurons： there were different pathological changes in all groups other than the normal control group. The number of damaged neurons in the model group was highest. The phenobarbital sodium, acrous gramineus, and α -asarone group exhibited different degrees of improvement. （2） Neuronal apoptosis in the CA1 and CA3 regions： there were less TUNEL-positive cells in the CA1 and CA3 regions in the normal control group. One week after PTZ-induced seizure, numerous TUNEL-positive cells were detected in the CA1 and CA3 regions in the remaining four groups. There was a significant difference between the normal control group and the remaining four groups （t = 12.089-19.162, P 〈 0.0 1）. The number of TUNEL-positive cells was less in the phenobarbital sodium, acrous gramineus, and α-asarone groups compared to the model group （t = 4.707-5.268, P 〈 0.01）. （3）Bcl-2 and Bax expression of neurons in the CA1 and CA3 regions： The number of Bcl-2- and Bax-positive cells was less in the normal control group. The Bax-positive cells exhibited a normal shape and had large round nuclei that were predominant. One week after PTZ-induced epilepsy, the number of Bcl-2- and Bax-positive cells in the CA1 and CA2 regions was significantly increased in the remaining four groups compared to the normal control group （t = 11.606-27.042, P 〈 0.01）. The Bax-positive cells exhibited a reduced size and nuclear pyknosis was predominant. However, there was no significant difference among the four groups （P 〉 0.05）. The number of Bcl-2-positive cells in the phenobarbital sodium, acrous gramineus, and a-asarone groups were significantly increased compared to the model group （t = 4.051-6.404, P 〈 0.01）. However, the number of Bax-positive cells was not significantly different among the four groups. The ratio of Bcl-2 to Bax expression was approximately 6.0 in the normal controls, 0.7 in the model group, and 1.0 in the remaining three groups. CONCLUSION： Acrous gramineus and a-asarone increased Bcl-2 expression and decreased Bax expression, and also reduced the number of apoptotic hippocampal neurons during PTZ-induced epileptic seizures in immature rats.

Effect of Different Processing Methods on Content ofβ-Asarone in the Zhuang Medicine Rhizoma Acori Tatarinowii

[Objectives]This paper aims to establish a method to simultaneously determine the content ofβ-asarone in Rhizoma Acori Tatarinowii dried by three different methods.[Methods]Reversed-phase high-performance liquid chromatography was used,and the chromatographic conditions were as follows:column,Thermo SCIENTIFIC Hypersil GOLD Dim.(mm);mobile phase,methanol-0.1%phosphoric acid(63∶37);column temperature,30℃;flow rate,1mL/min;detection wavelength,257 nm;sample size,10μL.[Results]The linear range of the injection volume ofβ-asarone was 49.28-246.40μg/mL(R=0.9993);the limit of quantification was 0.85 ng and the detection limit was 0.34 ng;the RSD values of precision,stability and reproducibility tests were all less than 3%;and the sample recovery rate was 98.53%-98.97%(RSD<3.00).The results show that the content ofβ-asarone was highest in shade-dried Rhizoma Acori Tatarinowii.The order ofβ-asarone content was as follows:Rhizoma Acori Tatarinowii dried in shade>Rhizoma Acori Tatarinowii dried at 55℃>Rhizoma Acori Tatarinowii dried at 60℃.[Conclusions]This method is sensitive,reliable,and reproducible.It can be used to simultaneously determine the content ofβ-asarone in Rhizoma Acori Tatarinowii dried by three different methods.

The rat bowel of β-asaron absorbs the research

Objective Study the β-asaron under the condition that the bowel each segment of rat and be worth in the diffent medicine density and pH of the absorption dynamics characteristic,as to it's the rat absorbs the part in the body and it absorbs the mechanism to carry on the study,for the further design β-asaron settle release the product to provide the living creature medicine learn the basis.Methods Apply the rat to the body to infuse to flow the bowel absorption experiment investigation and absorption dynamics characteristic;adopt the HPLC method measurement β-asaron is in rat body the bowel absorbs the medicine density within the reflux liquid.Results It absorb the quantity and β-asaron of the medicine in the reflux liquid,the density of β-asaron becomes the direct proption,the absorption speed constant of the medicine is basic and constant within the scope of the 19 μg·mL-1-57 μg·mL-1;In the pH is 5.6;6.9;8.0 three kinds of dissimilarities lie the absorption velocity constant of the quality and absorb the of percentage and also did not show the difference of salience;β-asaron is in the small intestines the lower part absorb better,absorbthe velocity to press to return to bowel,ileum,jejunum,duodenum,colon to descend one by one in order,absorb the velocity constant one by one in order is 0.402,0.396,0.385,0.325 h-1.Conclusions β-asaron absorbs to present a class absorption dynamics characteristic in the bowel way,absorbing the mechanism as passive absorption;in order to return to ileum and jejunums,main absorption part there is certain absorption in the colon,too.

Biotransformation ofα-asarone by Alternaria longipes CGMCC 3.2875

Biotransformation ofα-asarone by Alternaria longipes CGMCC 3.2875 yielded two pairs of new neolignans,(+)(7 S,8 S,7’S,8’R)iso-magnosalicin(1 a)/(-)(7 R,8 R,7’R,8’S)iso-magnosalicin(1 b)and(+)(7 R,8 R,7’S,8’R)magnosalicin(2 a)/(-)(7 S,8 S,7’R,8’S)magnosalicin(2 b),and four known metabolites,(±)acoraminol A(3),(±)acoraminol B(4),asaraldehyde(5),and 2,4,5-trimethoxybenzoic acid(6).Their structures,including absolute configurations,were determined by extensive analysis of NMR spectra,X-ray crystallography,and quantum chemical ECD calculations.The cytotoxic activity and Aβ_(42)aggregation inhibitory activity of all the compounds were evaluated.Compound 2 displayed significant anti-Aβ_(42)aggregation activity with an inhibitory rate of 60.81%(the positive control EGCG:69.17%).In addition the biotransformation pathway ofα-asarone by Alternaria longipes CGMCC 3.2875 was proposed.

Protective effects of components of the Chinese herb grassleaf sweetflag rhizome on PC12 cells incubated with amyloid-beta42

The major ingredients of grassleaf sweetflag rhizome are β-asarone and eugenol, which can cross the blood-brain barrier and protect neurons. This study aimed to observe the neuroprotective effects and mechanisms of β-asarone and eugenol, components of the Chinese herb grassleaf sweetflag rhizome, on PC12 cells. First, PC12 cells were cultured with different concentrations（between 1 × 10–10 M and 1 × 10–5 M） of β-asarone and eugenol. Survival rates of PC12 cells were not significantly affected. Second, PC12 cells incubated with amyloid-beta42, which reduced cell survival, were cultured under the same conditions（1 × 10–6 M β-asarone and eugenol）. The survival rates of PC12 cells significantly increased, while expression levels of the m RNAs for the pro-apoptotic protein Bax decreased, and those for the anti-apoptotic protein Bcl m RNA increased. In addition, the combination of β-asarone with eugenol achieved better results than either component alone. Our experimental findings indicate that both β-asarone and eugenol protect PC12 cells through inhibiting apoptosis, and that the combination of the two is better than either alone.

Research Progress on Acorus tatarinowii Schott and Its Active Ingredients Preventing and Controlling Alzheimer's Disease

It is hot point of research to prevent and control Alzheimer's disease( AD) by traditional Chinese medicine. Acorus tatarinowii Schott extract and its contained chemical compositions could play multiple pharmacological effects in central nervous system,in which the reports on main active parts and chemical compositions( such as volatile oil and β-asarone) from A. tatarinowii improving learning memory and preventing AD are the most in recent years. Based on referring to related literature at home and abroad,different pharmacodynamic actions of A. tatarinowii extract and main chemical compositions on AD and action mechanism are summarized.