摘要
目的以橄榄总黄酮(按芦丁折算)提取物为有效成分,筛选出最佳提取条件,用大孔树脂法对橄榄总黄酮进行纯化,确定最佳纯化条件。采用大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)模型,研究TFCO对局灶性脑缺血再灌注损伤(CIRI)的保护作用,并探讨其机制。
方法(1)以橄榄总黄酮(按芦丁折算)提取物为有效成分,采用可见分光光度法,对TFCO的含量进行控制,采用乙醇作为溶剂对不同浓度、不同用量、不同提取时间3个因素按正交实验法设计优选出最佳提取工艺。用大孔树脂法对TFCO进行纯化,筛选了大孔树脂的种类,综合考察了树脂的吸附率,解析率,上样流速,上样液pH,洗脱剂浓度,洗脱体积,流速等各项指标。
(2)线栓法制备大鼠大脑中动脉闭塞2h/再灌注24h模型,于再灌注前4天灌胃给予橄榄总黄酮30、60 mg.kg~(-1)。神经病学评分观察神经功能缺失症状;TTC染色法观察脑梗死体积;AutoCAD图像分析软件计算脑水肿程度;光镜观察脑组织形态学变化;免疫组化法检测脑组织iNOS、TNF-α表达;生化法检测脑组织中MPO、GSH活性及T-AOC含量。
结果(1)超声提取的最佳工艺是:取橄榄适量,加4倍量95%乙醇,超声提取60分钟;(2)纯化的最佳工艺是:使用D101型大孔树脂,配制pH4.5吸附液以2BV/h上柱,至泄漏。用3BV纯水以2BV/h洗柱后,再用3BV 60%乙醇洗脱,流速为1BV/h,将洗脱液减压浓缩,干燥得橄榄总黄酮,纯度为90.1%。
(2)脑缺血2h/再灌注24h后,神经功能缺失,脑组织出现白色梗死灶,脑水肿明显,形态学呈病理改变,脑组织iNOS、TNF-α蛋白表达增强,MPO活性增高,GSH活性降低,T-AOC含量降低;
(3)TFCO 30、60 mg.kg~(-1)均能改善神经功能缺失症状,有效减小脑梗死灶,减轻脑水肿,抑制脑组织iNOS蛋白表达,增高脑组织GSH活性,提高T-AOC含量,还可缓解MPO升高,并能减轻脑组织病理变化,抑制脑组织TNF-α蛋白表达。
结论(1)大孔吸附树脂纯化橄榄总黄酮,使其纯度达到90%以上;(2)再灌注前4天分别给予TFCO30、60mg.kg~(-1),减轻脑损伤;(3)TFCO对CIRI的保护作用,可能与减少iNOS和TNF-α蛋白表达、抗炎、抗脂质过氧化有关。
OBJECTIVE To determine of total flavone ( rutin as standard ) of the extraction is controlled by visible spectrophotometry and to establish the optimal condition for ultrasonic extraction of total flavone from Chinese olive and investigate appropriate macroporous resins ;The optimal technological parameters of the purification process of TFCO are found ; To study the protective effects and possible mechanisms of TFCO on focal cerebral ischemia-reperfusion injury (CIRI) in rats by reversible middle cerebral artery occlusion (MCAO) model.
METHODS The ultrasonic extraction and orthogonal test were used to choose optimum extractive method which was as follows: optimum extractive solvent’s concentration; solvent volume; extractive time .The purification process of total flavone with macroreticular resin was studied and the type of macroporous resin, review the absorption ratio; desorption ration; liquor’s pH; ethanol’s concentration, volume, velocity were established.
MCAO model was produced by intraluminal suture technique, and reperfusion 24h was begun after 2h of occlusion. TFCO was intragastrically administrated at 1d,2d,3d and 4d of the reperfusion. After reperfusion 24h, the extent of neurological deficits was evaluated by Longer’s method; the infarct size of the brain and the degree of cerebral edema were measured by Auto CAD image analysis soft ware; the morphological changes of the brain tissue were observed; the expressions of iNOS and TNF-αproteins were measured;T-AOC、MPO and GSH level in the brain tissue were detected.
RESULTS (1) The optimum extraction procedures was as follows:4times of 95% ethanol ;extractive time :40 minutes .the best technics of purification is as follows: the type of macroporous resin was D101,the sample fluid(pH4.5),the velocity is 2BV/h,the water is 3BV with velocity of 2BV/h,60% ethanol is 3BV with velocity of 1BV/h.
(2)After 2h of occlusion and 24h of reperfusion, the neurological deficits were evidently observed;the brain infarct size and edema degree were increased; the changes of the brain histology were appeared and the expressions of iNOS and TNF-αproteins were increased;the activity of MPO were increased, the activity of GSH was decreased, the contents of T-AOC were increased.
(3)Both 30 and 60 mg.kg~(-1) doses of TFCO can dramatically reduce neurological score, the infarct size and degree of cerebral edema comparing with ischemia reperfusion group. Both 30 and 60 mg.kg~(-1) doses of TFCO can increase the activity of GSH,decrease the activity of MPO and the contents of T-AOC,inhibit the expression of iNOS protein and TNF-αprotein.
CONCLUSION (1) The total flavones’s purity could reach 90.1%.(2) TFCO 30、60 mg.kg~(-1) can play a protective role in the brain injury. TFCO could protect the focal CIRI by depressing permeability of the blood brain barrier, inhibiting the expressions of iNOS and TNF-αproteins, depressing inflammation reaction, decreasing lipid peroxidation.
引文
[1] 商洪才,张伯礼.缺血性中风急性期治疗现状及中医药的前景和策略[J].天津中医药,2005,22(6):523-525.
[2] HEUSCHMANNPU,KOLOMINSKY-RAVASPL,ROETHER J,et al.Predictors of in-hospital mortality in patients with acute ischemic stroke treated with thrombolytie therapy[J].JAMA,2004,292(15):1831-1838.
[3] GRAF BA,MILBURY PE,BLUMBERG JB.Flavonols,flavones,flavanones,and human health:epidemiological evidence [J].J Med Food,2005,8(3):281-290.
[4] HERTOG MG,HOLLMAN PC,KATAN MB, et al.Intake of potentially anticarcinogenic flavonoids and their determinants in adults in The Netherlands[J].Nutr Cancer,1993,20(1):21-29.
[5] HAVSTEEN B.Flavonoids,a class of natural products of high pharmacological potency[J].Biochem Pharmacol,1983,32(7):1141-1148.
[6] 吴修仁.广东药用植物简编.广东高等教育出版社.1992:226-227
[7] 项昭保,何从林,任绍光,唐春红.野生橄榄抑菌效应及其在牛奶保鲜中的应用研究[J].食品工业科技,2004,25(10):69-70
[8] 袁剑刚,刘听,汤展球.橄榄的抑菌效应及其药效成分的初步研究[J].食品科学,2001,22(3):82-84
[9] 唐春红.野生橄榄总黄酮提取方法研究[J].渝州大学学报(自然科学版),2000,17(4):70-72.
[10] 董嘉德.超声技术在中药提取中的应用[J].传统医药,2002,11(11):55~56.
[11] 丘树毅,姚汝华,宗敏华.超声波在生物工程中的应用.生物工程进展,1999, 19(3):45-48
[12] 李冬菊.山楂总黄酮的提取及鉴别[J].辽宁中医杂志,2003,30(7):578
[13] 陶诗清,吴先琪,胡伯材.镰形棘豆总黄酮甙元的含量测定[J].中草药,1980,11(11):493-496.
[14] 杨书斌,王琦,张黄,等.薄层析-分光光度法测定壳中的橙皮甙和柚甙[J].中国中药杂志,1994,19(6):359-360.
[15] 葛正华,康毅华,贾玉良,等.没产地红旱莲总黄酮的含量分析[J].中医药学报,1995,(6):36-37.
[16] 屠鹏飞,贾存秦,张洪全.大孔吸附树脂在中药新药研究和生产中的应用[J].世界科学技术—中医药现代化.高新技术应用,2004,6(3):22-28
[17] 米靖宇,宋纯清.大孔吸附树脂在中草药研究中的应用进展[J].中成药,2002,23(12):914一927
[18] 庄向平.银杏叶中总黄酮含量的测定和提取方法[J].中草药,1992,23(3):122-124
[19] 任顺成,丁霄霖.大孔树脂对玉米须类黄酮的吸附分离特性研究[J].食品与发酵工业,2003,29(12):17-21
[20] FANG XD.The chemical constituents of Citrus teticrlata[J].Zhong Yao Tong Bao,1985,10(2):77-78。
[21] MIDDLETON E JR,KANDASWAMI C,THEOHARIDES TC.The effects of plant flavonoids on mammalian cells:implications for inflammstion,heart disease,and cancer[J].Pharmacol Rev,2000,52(4):673-751.
[22] WAKUI Y,YANAGISAWA E,ISHIBASHI E,et al.Determination of baicalin and baicalein in rat plasma by high-performance liquid chromatography with electrochemical detection [J].J Chromatogr,1992,575(1):131-136.
[23] AHLEMEYER B,KRIEGLSTEIN J,Pharmacological studies supporting the therapeutic use of Ginkgo biloba extract for Alzheimer’s disease[J].Pharmacopsychiatry,2003,36 Suppl 1:S8-14.
[24] KANADAA,NISHINURA Y,YAMAGUCHI JY,et al.Extract of Ginkgo biloba leaves attenuates kainite-induced increase in intracellular Ca2+ concentration of rat cerebellar granule neurons [J].Biol Pharm Bull,2005,28(5):934-936.
[25] OYAMA Y,FUCHS PA,KATAYAMA N,et al.Myricetin and quercetin,the flavonoid constituents of Ginkgo biloba extract,greatly reduce oxidative metabolism in both resting and Ca2+-losded brain neurons [J].Brain Res,1994,635(1-2):125-129.
[26] ROBAK J,GRYGLEWSKI RJ.Flavonoids are scavengers of superoxide anions [J].Biochem Pharmacol,1988,37(5):837-841.
[27] CHEN C,WEI T,GAO Z, et al.Different effects of the constituents of EGb761 on apoptosis in rat cerevellar granule cells induced by hydroxyl radicals [J].Biochem Mol Biol Int,1999,47(3):397-405.
[28] CHENG XP,QIN S,DONG LY, et al. Inhibitory effect of Total Flavone of Abelmoschus manihot L.Medic on NMDA receptor-mediated current in cultured rat hippocampal meurons [J].Neurosci Res,2006.
[29] DATLA KP,CHRISTIDOU M,WIDMER WW, et al.Tissue distribution and neuroprotective effects of citrus flavonoid tangeretin in a rat model of Parkinson’s disease [J].Neuroreport,2001,122(17):3871-3875.
[30] CHOI J,CONRAD CC,MALAKOUSKY CA,et al.Flavones from Scutellaria baicalensis Georgi attenuate apoptosis and protein oxidantion in neuronal cell lines[J].Biochim Biophys Acta,2002,1571(3):201-210.
[31] YANAMOTO H,NAGATA I,NUTSU Y,et al.Evaluation of MACO stroke models in normotensive rats:standardized neocortical infarction by the 3VO technique [J].Exp Neurol,2003,182(2):261-274.
[32] MAIER CM,YU F,NISHI T,et al.Interferon-beta fails to protect in a model of transient focal stroke[J].Stroke,2006,37(4):1116-1119.
[33] MHAIRI M,I.New models of focal cerebral ischaemia [J].Br J Clin Pharmacol,1992,34(4):302-308.
[34] 王瑜,阎也.中药治疗脑缺血及对脑保护作用的研究[J].中医药学刊,2002,20(3):371-372.
[35] Memezawa H,Smith ML,Siesio BK. Penumbral tissues salvaged by reperfusion following middle cerebral artery occlusion in rats[J].Stroke,1992,23(4):552-559.
[36] 曾玲晖,张纬萍,王仁定,等.白三烯拮抗剂 ONO-1078 对小鼠局灶性脑缺血的保护作用[J].药学学报,2001,36(2):148-150
[37] Kimelberg HK.Current concepts of brain edama:review of laboratory investigations.J Neurosurg,1995;83:105 1-1059.
[38] Gong C,Qin Z,Betz AL,et a1.Cellular localization of tumor necrosis factoralpha following focal cerebral ischemia in mice.Brain Res,1998;80l:l-8.
[39] Zhang JP,Sun GY.Free fatty acids neutral glycerides and phoshoglycerides in transient focal cerebral ischemia[J].Neurochem,1995,64:1688-1695
[40] 种兆忠,冯亦璞.脑损伤过程中血脑屏障通透性的变化及其调节机制[J].中国药理学通报,1999,15(3):204-207
[41] Feelish M. Tepoel M.Zamora R,et al. Understanding the controversy over the identity of EDRF [J].Nature,1994,368(6466):62.
[42] 闻名,蔡定芳. 急性脑缺血损伤的微循环障碍机制[ J ]. 国外医学脑血管疾病分册. 2004, 12 (4): 289-291
[43] 徐丽荣,马世彬,李澎涛,等. 清开灵有效组分对MCAO大鼠脑微血管内皮细胞的影响[ J ]. 中药材. 2004;5 (27): 348-351
[44] 于永发, 董国淑, 黄湘楠. 急性脑梗塞脑微循环障碍研究进展[ J ]. 中国微循环. 2002, 6 (4): 251-253
[45] 陈友岱, 周总光. 血小板活化因子与局部微循环障碍[J].中国微循环.2001,5 (3):241-243
[46] Stanimirovic D, Satoh k. Inflammatory mediators of cerebral endothelium:a role in ischemic brain inflammation[ J ]. Brain Patho1. 2000, 10: 113-126
[47] Krawisz J E, Sharon P, Stenson W F. Quantitative assay for acute intestinal inflammation based on myeloperoxide activity[J],Gastroenterology,1994,87:1344
[48] 吴伟,史继新. 脑缺血再灌注损伤后神经元损伤机制及治疗研究进展[J].中国微循环,2003,12(7):391-394
[49] 吴立克,张茁,毕齐等.大鼠脑缺血后脑内 MPO、NSE 活性变化[J].北京医学,2006;18(11):670-672
[50] 曲友直 赵燕玲 赵振伟等.黄芪对脑缺血再灌注后脑组织 IL-1 含量及 MPO 活性的影响[J].中国中医急症,2006;15(2):176-178
[51] 叶心国 余绍祖 李承晏。亚低温对脑缺血再灌注大鼠的 ICAM-1 表达 MPO活性和脑梗死体积的影响[J].中华物理医学与康复杂志,2005;27(7):442-444
[52] 徐隽、余涓、王晶等.蕲蛇酶减轻大鼠局灶性脑缺血再灌注损伤的实验研究[J].中国临床药理学与治疗学,2004,9(3):294-298.
[53] 郑燕华,陈崇宏.大蒜素对大鼠急性脑缺血再灌注损伤保护作用的研究[J].中国药理学通报,2004,20(7)821-823.
[54] Deng XL, Qian ZY. Antagonistic effect of 3,6-dimethamid odibenzopyriodonium gluconate on lipid peroxidation in cerebral cortical neuronal cultures and rat brains during focal cerebral ischemia reperfusion [J]. Acta Pharmacol Sin.2000, 21(5):460-462
[55] 邱丽颖,余涓,陈崇宏,et al.阿司匹林不同治疗时间窗对缺血再灌注的保护作用及对脑 ATP 含量的影响[J].中国药学杂志,2004,39(7):509-510.
[56] 邱丽颖,余涓,陈崇宏,et al.阿司匹林对大鼠脑缺血再灌注损伤细胞凋亡的影响[J].中国药理学通报, 2004, 20(2):177-180.
[57] 邱丽颖、余涓、周宇,et al.阿司匹林对大鼠局灶性脑缺血再灌注损伤的保护作用及机制[J].药学学报,2003,38(8):561-564.
[58] Hayshi A,Ito H,Shomori K, et al. Frequent occurrence of hepatocytic apoptosis in acute rejection of the grafted rat liver. Pathol Int, 1997,47(8):518-523.
[59] Lewis SE, Boyle PM, Mckinney KA, et al. Total antioxidant capacity of seminial plasma is different in fertile and infertile men[J]. Fertil Steril, 1995, 64 (4):868-870.
[60] Wang YH, Wang WY, Chang CC, et al. Taxifolin ameliorates cerebral ischemia-reperfusion injury in rats through its anti-oxidative effect and modulation of NF-kappa B activation[J]. J Biomed Sci,2006,13(1):127-141.
[61] Zeng R, Li YJ. Effects of propyl gallate on myocardium mitochondrial induced by ischemia/reperfusion in rats[J]. J TCM Univ of Hunan,2005, 25(4):7-12.
[62] Jean WC, Spellman SR, Nussbaum ES, et al. Reperfusion injury after focal cerebral ischemia: the role of inflammation and the therapeutic horizon. Neurosurgery,1998,43:1382-1397.
[63] Buttini M, Matsuvama T, A, et al . Expression of tumor necrosis factor alpha after focal cerebral ischaemis in the the rat. Neuroscience,1996,71:1-16.
[64] Uno H, Matsuvama T,Akita H, et al. Induction of tumor necrosis factor alpha in the hippocampus following transient forebrain ischemia. J Cereb Blood Flow Metab,1997,17:491-499.
[65] Lavine SD, Hofman FM, Zlokovic BV, et al. Circulating antibody against tumornecrosis factor-alpha protects rat brain from reperfusion injury[J]. Jcere Blood Flow Metab,1998;18(1) 52-58
[66] Small D L,Buchan A M. Mechanisms of cerebral ischemis : intracellular cascades and therapeutic interventions [J]. J Cardiothorac Vasc Anesth,1996,10(1):139.
[67] Samdani A F, Dawson T M, Dawon V L. Nitrc oxide sythase in models of focal ischemia [J]. Stroke,1997,28(6):1283.
[68] Vanughan C J, Delanty N, Neuroprotective properties of statins in cerebral inschemia and stroke [J]. Stroke,1990,30:1969.
[69] Fagni L,Qlivirt M,Lafon-Cazal M, et al . Involvenment of divalent inos in the nitric oxide- induced blockage of N- methyl –D- aspartate receptors in cerebellar granule cells [J]. Mol Pharmacol,1995,47:1239.
[70] Bonroco E,Krainc D,Ankarcrona M, et al. Apoptosis and necrosis:two distinct events induced,respectively, by mild and intense insults with N-methyl-D-aspartate or nitrc/superoxide in cortical cell culture[J]. Proc Natl Acal Sci USA,1995,92:7162.
[71] Montague P R,Gancayco C O, Winn M J,et,al. Role of NO production in NMDA receptor-mediated neurotransmitter release in cerebral cortex[J]. Science, 1994, 263:973.
[72] Moro M A, Cardensa A, Hurtado O, et al. Role of metric oxide after brain ischemia[J]. Cell Calcium,2004,36(3-4):265-275.
[73] 张敏君,房玉珍,夏作理,等。L-NNA 对脑缺血再灌注后海马区细胞凋亡的影响[J].中国药理学通报,2000,16(3):357-358.
[74] Longa E Z,Weinstein P R,Carlson S,et al. Rwversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke,1989,20(1):84-91.
[75] Sekhon B,Sekhon C,Khan M, et al. N-Acetyl cysteine protects against injury in a rat model of focalcerebral ischemia[J].Brain Res,2003,971(1):1-8.
[76] 张会欣,张建新。一氧化氮在缺血性脑损伤中作用的实验研究进展[J].中国药理学通报,2004,20(10):1094-1097.
[77] Huang z, Huang P L, Ma J,et al. Enlarged infarcts in eddothelial nitric oxide synthase knockout mice are attenuated by nitro L-arginine [J]. J Cereb BloodFlow Metab,1996,16:981.
[78] Iadecola C, Zhang F,Casey R, et al. Knockout mice lacking the inducible nitric oxide synthase gene are resistant to cerebral ischemia [J]. Soc Neurosci Abatract,1996,22:169.
[1] 谭仁祥.植物成分[M]. 北京:科学出版社,2002:486—502.
[2] HU CH(胡春).Antioxidative property of flavonoid[J].China Oils and Fat(s中国油脂),1996,21(4):18-21(in Chinese)
[3] KUO S M,LEAVITT P S,LIN C P.Dinetary flavonoida interact with trace metals and affect metallothionein level in human intestinal dells[J].Biol.Trace Elem.Res.,1998,6,62(3):135-153.
[4] ZHANG Y W(张彦文).The pharmacology and the structure-activity relationship of the chalcones[J].Foreign Medical Sciences Section on Pharmacy(国外医学药学分册),1996,8,(23):218-223(in Chinese).
[5] HUANG H Y (黄华艺),CHA X L(查锡良).Development in research of antitumor effect of flavones compounds[J].Chinese Journal of New Drugs and Clinical Remedies(中国新药与临床杂志),2002,21(7):428—433(in Chinese).
[6] BROWNSON D M,AZIOS N G,FUQUA B K,etal.Flavonooid effects relevant to cancer[J].J.Nutr.2002,11,132(11 suppl):3482s-3489s.
[7] JOYEUX M,LOBATEIN A,ANTOM R.Comparative antilipoperoxidant,antinecrotic and scavenging properties of terpenes and biflavones from Ginkgo and some flavonoids[J].Planta Med.,1994,61(2):66-69.
[8] CHEN J W,ZHU Z Q,HU T X,ZHU D Y.Structure-activity relationship of natural flavonoids in hydroxyl radical-savenging effects[J].Acta Pharmacol.Sin.,2002,7,23(7):667-72.
[9] 陈新谦,金有豫,新编药物学(第 14 版)[M].北京:人民卫生出版社,1998:271-276.
[10] HUA JH(华辉),GUO Y(郭勇).Development in pharmacological research of flavonoids[J].Guangdong Pharmaceutical Journal(广东药学),1999,9(4):10-14(in Chinese).
[11] BALMIR F.An extract of soy flour influences serum cholesterol and thyroid hormones in rats an hamsters[J].J.Nutr.,1996,126:3046-3053.
[12] ZHU Q L(朱向磊),LU X R(吕欣然).Development in pharmacological and clinical apply research of puerarin[J].Chinese Traditional and Herbal Drugs(中草药),1997,28(11):693-696(in Chinese)
[13] DUAN CH G(段重高),LI H W(李宏伟),XU L N(徐理纳).The Affection of brain tiny surroundings of Syrian hamsters[J].Chinese Medical Journal(中华医学杂志),1991,71(9):516-517(in Chinese).
[14] ZHU X M(朱向明),ZHAO ZH D(赵向东).The progress of prevention and cure atherosclerosis research by green tea polyphenoles from tea extracts[J].Clinical Focus(临床荟萃),1996,11(9):416-418(in Chinese).
[15] SONG ZH J(宋芝娟),LIU W(刘文),LIANG N C(梁念慈),YU J(余戟),MO L E(莫丽儿).Effect of disodium quercetin disulfate on the formation of f-action pig platelet induced by thrombin[J].Chinese Traditional and Herbal Drugs(中草药),1997,28(8):477-479(in Chinese).
[16] CHAN E C,PANNANGPETCH P,WOODMAN O L.Relaxation to flavones and flavonls in rat isolated thoracic aorta: mechanism of action and structure-activity relationships[J].Cardiovasc Pharmacol.,2000,2,35(2):326-333.
[17] 范丽,董六一,江勤,等.黄蜀葵花总黄酮抗炎解热作用.安徽医科大学学报,2003,38(1):25-28
[18] Della Loggia R,Sosa S,Tubaro A,etal.Anti-inflammatory activity of Ginkgo biloba flavonoids.Planta Med,1993,(59):A588
[19] 刘金霞,邓淑华,杨贺松,等.黄芩茎叶总黄酮的抗炎作用机制的研究.中国药理学通报,2002,18(6):713-714
[20] 石新兰,凌允礼,侯建存.亚硒酸钠及抗氧化剂抑制小鼠肺泡 Arthus 血管炎发生的机制.中国医学科学院学报,1999,(4):257-26
[21] 张逸凡,于庆海.淫羊藿总黄酮的抗炎作用.沈阳医科大学学报,1999,16(2)
[22] ZHANG A L,LIU G Q,MA Q,GAO J M.Structure-activity relationship of flavonoids[J].Journal of Northwest Forestry University,2001,16(2):75-79
[23] SARTOR L,PEZZATO E,DELL’AICA I,CANIATO R,BIGGIN S,GARBISA S.Inhibition of martixproteases by polyphenols:chemical insights for anti-inflammatory and anti-invasion drug design[J].Biochem.Pharmacol,2002,7,64(2):229-237
[24] ZHONG F,JIANG Y,WU F F,SHU R H,CAI X D,TAN J P.Effect of total flavonoid of Hippophae rhamnoides on immune function in mice[J].Chinese Traditional and Herbal Drugs(中草药),1989,20(7):43-45
[25] YANG J Y,YU Q H,LI SH,XU J H.Immunopotentiation of total Epimedium on immunodepressant mice[J].Journal of Shenyang Pharmaceutical University,1998,15(2):94-97
[26] WANG Y P,DING X Y,PENG H,GUO Z ZH,ZUO J L,MA K Q.Quercetin regulation on cellular immunology[J].Journal of Hebei Medical University,1998,19(3):143-145
[27] CAO M F,LIN ZH H,YU Y Y,YANG X Q. Effect of tea polyphenol on cellular immune function and antitumor activity[J]. Modern Applied Pharmacy,1994,8,11(4):53-59
[28] ZHANG X P,LI ZH F,LIU X G. Review in pharmacological study of baicalein[J].Chinese Pharmacological Bulletin,2001,17(6):711-713
[29] YU L CH,GU CH H. Development in pharmacological response research of silymarin[J].Chinese Journal of Hospital Pharmacy,2001,8(21):493-494
[30] ZHANG R Q,HAN ZH K,CHEN J,ZHANG CH L. Daidzein diet promotes mammary gland development and lactation in pregnant rat[J].Acta Zoologica Sinica,1995,4(41):414-419
[31] JI H,LIU K,GONG X J,LI SH P,ZHANG M F. Effect of epimedium koreanum flavonoids on osteoporosis in ovariectomized rats[J].Chinese Journal of Osteoporosis,2001,7(1):4-8
[32] LI J,PAN X H,ZHONG W J,MI M. Development in research of prevention and cure osteoporosis by daidzein[J]. Shanghai Journal of Preventive Medicine,2002,12(4): 191-192
[33] ZHANG D Q,TAI J X,FU Q. Survey research and application on bioflavonoids[J].Food and Fermentation Industries,1999,25(6):52-57
[34] XU H X,LEE S F. Activity of plant flavonoids against antibiotic-resistant bacteria[J]. Phytother.Res.,2001,15(1):39-43
[35] ODAKA K,TAMURA Y,YAMAMOTO M. Identification of antimicrobial and antioxidant constituents from licorice of Russian and Xinjing origin[J].Chem.Pharm,Bull.,1987,37(9): 2528- 2531
[36] SHI Y,SHI R B,LIU B,LU Y R. Studies on antiviral flavonoides in yinqiaosan powder[J]. China Journal of Chinese Materia Medica,2001,5,26(5):320-323
[37] WU J A,AATTELE A S,ZHANG L,et al. Anti-HIV activity of medicinal herbs usage and potential development[J].Am.J.Chin.Med.,2001,29(1):69-81
[38] GASTRILLO J L. Action of 3-methylquercetin on poliovirus RNA replication[J].J. Virol,1987,61(10):3319-3321
[39] ALCARAZ L E,BLANCO S E,PUIG O N, TOMAS F,FERRETTI F H.Antivacterial activity of flavonoids against methicillin-resistant staphylococcus aureus strains[J].J.Theor.Biol,2000,21;205(2):231-240
[40] HU C Q,CHEN K,SHI Q,KILKUSKIE R E,CHENG Y C,LEE K H.Anti-AIDS agents,10. Acacetin-7-O-beta-D-galactopyranoside,an anti-HIV principle from Chrysanthemum morifolium and a structure-activity correlation with some related flavonoids[J].J.Nat.Prod.,1994,7,57(1):41-51
[41] WU G ZH,YANG Q J,WANG T M. Development in research of the mechanism of lipofusein (senileplaques)shaped and Chinese traditional medicine dispel lipofusein[J].Research of Traditional Chinese Medicine,1997,2,13(1):60-62
[41] ZHAO X Y,GU ZH L,SU L Y. Preliminary research of anti-radiation effect of quercetin [J].Acta Academiae Medicinae Suzhou,1998,18(12):1233-1234
[42] 孟宪丽,张艺,李建亚,等.淫羊藿总黄酮对老年大鼠神经分泌免疫调节作用的研究.中药药理与临床,1998,14(4):10-13
[43] 周兰兰,明亮,李前进,等.银杏叶提取物改善老龄前期小鼠学习记忆的作用.安徽医科大学学报,1998,33(5):337-339
[44] 张艳,明亮,李京培,等.银杏叶提取物对神经凋亡的保护作用.中国临床药理学与治疗学,2001,6(1):25-27
[45] Kanowski S,Herrmann WM,Stephan K,et al. Proof of efficacy of the Ginkgo biloba special extract EGB761 in outpatients suffering from mild to moderate primary degenerative dementia of Alzheimer type or multi-infant dementia.Pharmacopsychiatry,1996,29(2):47-56
[46] Oishi M,Mochizuki Y,Takasu Y,et al. Effectiveness of traditional Chinese medicine in Alzheimer disease. Alzheimer Dis Assoc Disord,1998,12(3):247-250
[47] 魏桂荣,肖克娜,胡波,等.银杏叶提取物对大鼠皮质缺血再灌注后自由基系统动态平衡的影响.卒中与神经疾病,2002,9(l):3-6
[48] 韩京艳,邢东明,孙虹等。银杏叶黄酮内醋及天然配比抗氧化作用的比较。中国药理学通报,2002;18(l):115-117
[49] Chavez,Mary L , Chavez,Pedro I.Ginkgo(Part1):History,Use,and Pharmacologic Properties.Hospital Pharmcy,1998,33(6):658-660
[50] Hu Bo,Sun Shenggang,Mei Guangwu,et al.Protective effects of Ginkgo biloba extract on rats during cerebral ischemia/reperfusion. Chinese Medical journal,2002,115(9):1316-1318.
[51] 徐江平,孙莉莎,杨雪梅.银杏内醋对大鼠脑缺血一再灌注损伤的保护机制.中国中西医结合急救杂志,2003,10(1):31-34
[52] 黄叶梅,黎霞,张丽.苦荞黄酮对大鼠脑缺血再灌注损伤的保护作用 [J].四川师范大学学报(自然科学版),2006,29(4):499-501
[53].Baba T,Black KL,Ikezaki K, et al. Intracarotid infusion of Leukotrience C4 seletively increase blood brain barrier permeability after focal ischemia in rats [J].J Cereb Blood Flow Metab,1991,11(4):638
[54].Bicker PE,Gallego SM,Hansen BM. Development changesin intracellar calcium reglulation in rat cerebral cortex during hypoxia [J].J Cereb Blood Flow Metab,1993,13(5):811
[55] Liu P.Calcium and cerebral ischemical injury[J].Foreign Medical Sciences Section on Neurology & Neurosurgery,2002,29(3):274一276
[56] Kobayashi M S ,Han D,Packer L,et al .Antioxidants and hebal extracts protect HT-4 neuronal cells against glutamate-induced cytotocicity[J].Free Radic Res,2000,32(2):115
[57] 王雪松,阮旭中,王伟.银杏叶提取物对缺血再灌注致鼠脑损伤的脑保护作用研究[J].华中科技大学学报,2003,6(5):278一280
[58] Matsuda T,Arakawa N,Takuma K,et al.SEA0400,a novel and selective inhibitor of the Na+-Ca2+ exchanger,attenuates reperfusion injury in vitro and in vivo cerebral ischemic models[J].J Pharmacol Exp Ther,2001,298(1):222-225
[59] Xiong Z G, Zhu X M, Chu X P, et al. Neuroprotection in ischemia:blocking calcium-permeable acid-sensing iron channels[J].Cell,2004,118(6):687一698
[60] 曲玲,王维亭,郭莲军.DDPH对大鼠缺血性脑损伤的保护作用[J].药学学报,2003,38(10):725-727
[61] 颜景芝,张光毅等 矾酸钠和染料木黄酮对脑缺血/再灌注诱导海马神经元损伤的作用及分子机制[J].徐州医学院学报,2003,23(3):189-193
[62] 王芳,余恩欣,刘惟莞.水杉总黄酮对大鼠脑缺血再灌注损伤的保护作用 [J].中国中药杂志,2004,29(2):179-181
[63] 王彤宇,炎症反应与缺血性卒中关系的研究发展[J].中国厂矿医学,2005,1(18):71-73
[64] Tan CB,Gong QY,Yao MH, et al.Effect of Ginkgo biloba extract on inflammation following focal ischemic brain injury in rats[J].Chi J New Drug Clin Rem,2002,7(21):385-389
[65] Zhang DL,Zhang YT,Yin JJ,et al. Oral administration of Crataegus flavonoids protects against ischemia/reperfusion brain damage in gerbils [J].J Nerrochem,2004,90(1):211-219
[66] Torregrosa G,Burguete MC,Perez-Asensio FJ, et al. Pharmacological profile of phytoestrogens in cerebral vessels :in vitro study with rabbit basilar artery[J]. Eur J Pharmacol,2003,15,482(1-3):227-234
[67] Sobey CG,Weiler JM,Boujaoude ML, et al. Effect of short-term phytoestrogen treatment in male rats on nitric oxide-mediated responses of carotid and cerebral arteries: comparison with 17beta-estradiol[J].J Pharmacol Exp Ther,2004,310(1):135-140.