CN1690056A - 三个化合物及其制备方法和用途 - Google Patents
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Abstract
本发明提供了3个化合物的结构,它们是从灌胃给药中药黄芪或复方补阳还五汤水煎液的动物血清中分离出来的。本发明所述类型的化合物具有抗脑细胞缺血和抗脑细胞缺氧的活性作用。本发明还提供了中药复方体内化学成分的研究方法。
Description
发明领域
本发明涉及化学领域,具体说是提供3个化合物及其制备方法和用途。
背景技术
“补阳还五汤”为清代的名方,出自名医王清任的《医林改错》。方中含黄芪、归尾、赤芍、地龙、川芎、桃仁、红花,具有补气、活血、通络的功效。现临床主要用于缺血性脑血管病、出血性脑血管病、中风后遗症、冠心病及肺心病等多种疾病的治疗。前人对于补阳还五汤的有效物质基础无报导。中药化学成分复杂多样,目前,中药活性成分的研究方法多采用分步提取、药效追踪的体外水平,但很多成分即使体外有活性,体内却无明显作用,或不能被吸收、或经代谢后才能产生活性物质。因此所确定的成分是否是药效的代表性成分尚待商榷。我们知道,药物必须经过由用药部位进入血液循环才能起作用,中药亦不例外。中药虽含有众多成分,但只有被吸收入血的成分才能产生作用,否则没有成为有效成分的可能。传统中药多为口服给药,口服给药后药物成分或经过消化道直接吸收入血;或经消化液、消化酶及肠内菌群的作用分解成次生代谢产物被吸收入血;或经肝微粒体酶(P450)代谢成有活性的代谢产物。无论经过上述何种途径,其有效物质必须以血液为介质输送到靶点,从而产生作用。因而给药后的血清才是真正起作用的“制剂”,中药的药效物质基础应在给药后的血清样品中进行探讨。基于此种研究思路,发明人在对补阳还五汤有效成分的研究中,利用反相柱色谱等分离手段及高效液相色谱-二极管阵列检测器(HPLC-DAD)的分析检测技术从灌胃给药补阳还五汤水煎液的动物血清样品中检测出若干个来源于补阳还五汤化学成分的高效液相色谱峰,并利用液相色谱-质谱检测技术(HPLC-MS)鉴定其中7个色谱峰的化学结构,从而完成了本发明。
发明目的
本发明的目的就是提供3个新化合物。
本发明的另外一个目的是提供所述化合物的制备方法。
本发明的又一个目的是提供所述化合物的制药用途。
发明内容
1、本发明所述的化合物,具有下列结构式:
R1、R2、R3和R4为H、OH、烷氧基、葡萄糖醛酸基及硫酸酯基。优选R1为葡萄糖醛酸基及硫酸酯基,R2、R3和R4为H,OH,甲氧基,最优选的化合物名称为:7,2′-二羟基-3′,4′-二甲氧基异黄烷-7-O-葡萄糖醛酸苷;7,2′,3′-三羟基异黄烷-7-O-硫酸酯;7,3′-二羟基-4′-甲氧基异黄烷-7-O-葡萄糖醛酸苷;7-羟基-4′-甲氧基异黄烷-7-O-葡萄糖醛酸苷;7,2′,4′-三羟基-3′-甲氧基异黄烷-7-O-硫酸酯;3-羟基-9,10-二甲氧基紫檀烷-3-O-葡萄糖醛酸苷及3-羟基-9,10-二甲氧基紫檀烷-3-O-硫酸酯。
2、本发明的技术路线:上述化合物是从灌胃给药中药黄芪及补阳还五汤复方水煎液的动物血清中分离得到。具体方法如下:将黄芪或补阳还五汤药材水煎液,按临床人日用量的1~10倍剂量给猪、兔或鼠连续灌胃给药3~7次,最后一次给药后1~2小时颈总动脉取血并分离血清,血清冷冻干燥成干粉。给药动物血清冷干粉用甲醇超声提取1~3次,每次20~40分钟。提取液合并,减压回收得浸膏。浸膏样品的水溶液经反相色谱柱分离,100%水→100%甲醇梯度洗脱,对比分析各洗脱流份与空白动物血清甲醇提取液的高效液相色谱(HPLC)图的差异,寻找给药血清特有峰所在洗脱流份,并据此合并各洗脱流份。各洗脱流份根据情况用同样方法反复精制并检测,将给药血清特有峰的相对含量提高。采用HPLC-DAD-MS技术对特有峰进行化学结构鉴定。HPLC流动相为:0.025%磷酸水-乙腈(100∶0→0∶100,20~60min线性梯度);色谱柱:ZORBAX SB-C18(5μm,4.6×250mm);DAD检测;流速:0.5~1.5ml/min。
本发明所述类型的化合物具有抗脑细胞缺血和抗脑细胞缺氧的活性作用。通过实验例对本发明进行更详细的说明。
实验例1.优选化合物的制备:将补阳还五汤药材水煎液,按临床人日用量的10倍剂量给猪或兔连续灌胃给药7次,最后一次给药后1小时颈总动脉取血并分离血清,血清冷冻干燥成干粉。给药动物血清冷干粉用甲醇超声提取3次,每次30分钟。提取液合并,℃减压回收得浸膏。浸膏样品的水溶液经反相色谱柱分离,100%水-100%甲醇梯度洗脱,对比分析各洗脱流份与空白动物血清甲醇提取液的高效液相色谱(HPLC)图的差异,寻找给药血清特有峰所在洗脱流份,并据此合并各洗脱流份。各洗脱流份根据情况用同样方法反复精制并检测,将给药血清特有峰的相对含量提高。采用HPLC-DAD-MS技术对特有峰进行化学结构鉴定。HPLC流动相为:0.025%磷酸水-乙腈(100∶0→0∶100,20min线性梯度);色谱柱:ZORBAX SB-C18(5μm,4.6×250mm);DAD检测;流速:1.0ml/min。柱温:20℃。
优选化合物的结构是通过UV光谱、ESI-MSn质谱数据鉴定的(见表1)。
表1.优选化合物的质谱及UV光谱数据
化合物名称 | ESI-MSn质谱离子 | UVλmax(nm) |
7,2′-二羟基-3′,4′-二甲氧基异黄烷-7-O-葡萄糖醛酸苷 | 477→301,175;301→286,271,179,147,135,109 | 225sh,280low |
7,2′,3′-三羟基异黄烷-7-O-硫酸酯 | 337→257;257→147,135,121,109 | 225sh,280low |
7,3′-二羟基-4′-甲氧基异黄酮-7-O-葡萄糖醛酸苷 | 459→441,283,268,175;283→268;268→240,211 | 215sh,250,300sh |
7-羟基-4′-甲氧基异黄酮-7-O-葡萄糖醛酸苷 | 443→267(28),175(55);267→252; | 215sh,250,300sh |
7,2′,4′-三羟基-3′-甲氧基异黄烷-7-O-硫酸酯 | 367→349,287;287→272,255,177,165,147,135,109 | 225sh,280low |
3-羟基-9,10-二甲氧基紫檀烷-3-O-葡萄糖醛酸苷 | 475→457,299,284,175 | 225sh,280low |
3-羟基-9,10-二甲氧基紫檀烷-3-O-硫酸酯 | 379→299;299→284,269;269→241 | 225sh,280low |
实验例2.芒柄花素、紫檀烷、毛蕊异黄酮几毛蕊异黄酮苷对大脑细胞膜损伤的保护作用:脑细胞细胞膜流动性可以作为脑中风病变细胞损伤的指标。补阳还五汤对脑中风损伤细胞膜具有保护作用。为了明确补阳还五汤产生此保护作用的物质基础,我们对芒柄花素、紫檀烷、毛蕊异黄酮几毛蕊异黄酮苷进行了该项药理学实验研究。
(1)实验动物:雄性Wistar大鼠,体重170~90g,由北京大学医学部实验动物中心提供。自旋标记物5DS(5-doxyl-stearlic acid methyleater)购自Sigma公司,使用终浓度为4×10-5mol/L。D-Hanks液作为细胞液,用时加入葡萄糖1g/L。
(2)实验药物:芒柄花素,毛蕊异黄酮,3-羟基-9,10-二甲基紫檀烷-3-O-葡萄糖苷,毛蕊异黄酮-7-O-葡萄糖苷本课题组从补阳还五汤水煎液中分得。补阳还五汤冷干粉用生理盐水(NS)配制成20mg/ml。川芎嗪用NS配制成两个浓度,即2×10-5mol/L和8×10-7mol/L。其余样品用NS配制成2×10-5mol/L。自旋标记物5DS(5-doxyl-stearlic acid methyleater)购自Sigma公司,使用终浓度为4×10-5mol/L。D-Hanks液作为细胞液,用时加入葡萄糖1g/L。
(3)细胞制备方法:将大鼠随机分成实验损伤组(D)与对照组(C),其中D组乌拉坦(1200mg/kg,i.p)麻醉后分离双侧颈总动脉并结扎30min后再灌流2h,然后开颅取全脑,冰浴环境下将脑组织纵向切开,一半用福尔马林固定制作组织切片,另一半剔除脑膜,D-Hanks液洗去血污置100目筛网中剪碎使分离成单细胞。倒置显微镜下细胞记数并经低温离心将细胞调至5~8×107个/mL。台盼蓝染色证实细胞成活良好。正常大鼠作为对照,在乌拉坦麻醉下直接取脑组织。余下步骤同上。
(4)电子顺磁共振实验方法:参照文献完成脑细胞自旋标记,对标记后的损伤组和对照组细胞600μL,分别加入NS及不同浓度的补阳还五汤240μL,D-Hanks缓冲液1560μL。并继续25℃温育150分钟,离心洗涤至上清液中无游离的自旋标记物,取沉淀装入石英毛细管内,用Bruker ESP 300电子顺磁共振波谱仪常温下检测。仪器参数设置如下:微波功率10mW,调制频率100kHz,调制幅度0.2mT,中心磁场349mT,扫宽20mT。从ESR波谱图上计算细胞膜的序参数S,它与流动性的倒数相关,实验重复5次。统计方法用组间t检验,X±SD表示。
(5)计算方法:如附图1(为典型的5DS自旋标记的脑细胞膜电子顺磁共振波谱)所示,
S=0.568×(AII-A⊥)/(AII-+2A⊥)/3
(6)实验结果:结果(表2)显示补阳还五汤和所含化学成分芒柄花素,毛蕊异黄酮,3-羟基-9,10-二甲基紫檀烷-3-O-葡萄糖苷,毛蕊异黄酮-7-O-葡萄糖苷对脑中风损伤细胞膜流动性具有影响(见表2),可使升高的序参数(S)降低,即对细胞膜损伤有保护作用。
损伤组(D)与对照组(C)的生理盐水组比较,S值具有显著性差异(P<0.001),表明细胞受到损伤后膜流动性减少。D组加入各种药物,各种药物均可使细胞膜的流动性增大(P<0.01),其流动性改变趋向于正常脑细胞的水平。但是各种成分对流动性的影响各不相同。D组S值越小,表明药物引起损伤细胞膜流动性变化越大,药效越强。药物诱导损伤细胞恢复至正常的能力(与生理盐水组比较),由大至小依次为低浓度补阳还五汤、毛蕊异黄酮苷、毛蕊异黄酮、紫檀烷苷和芒柄花素。C组各加药组细胞膜流动性无显著性变化(P>0.001)。提示各药物对正常细胞无明显影响而对损伤细胞膜流动性有改善作用。
表2.补阳还五汤化学成分对正常和缺血再灌大鼠脑细胞膜序参数S0和Sx的影响
样品 | 终浓度mol.L-1 | 序参数S0 | 序参数Sx | (Sx-S0)×10 |
生理盐水芒柄花素3-羟基-9,10-二甲氧基紫檀烷-3-O-葡萄糖苷毛蕊异黄酮毛蕊异黄酮-7-O-葡萄糖苷补阳还五汤 | /2×10-52×10-52×10-52×10-520mg/ml | 0.6839±0.00830.6773±0.01360.6815±0.00990.6794±0.00950.6781±0.00700.6788±0.0119 | 0.7384±0.0035**0.7238±0.0157*0.7188±0.0111**0.7144±0.0275*0.7106±0.022**0.7001±0.0179** | 0.5560.4650.3730.3500.3250.213 |
(*P<0.01和**P<0.001,用生理盐水作溶液,与造模组相比较。**P<0.001,用生理盐水作溶液,与对照组相比较。)
实验例3.芒柄花素、毛蕊异黄酮、3-羟基-9,10-二甲氧基紫檀烷-3-O-葡萄糖苷、芒柄花素-7-O-葡萄糖苷、毛蕊异黄酮-7-O-葡萄糖苷对氰化钾引起的大脑皮层细胞缺氧损伤的保护作用:
利用大鼠乳鼠大脑皮层细胞原代培养的方法培养细胞,细胞接种在24孔培养板,细胞浓度为5×105细胞/ml,37℃5%CO2饱和湿度条件下培养至细胞铺满单层,取细胞铺满单层的24孔培养板,弃原培养液,用D-Hank′s液洗两次,每孔加入无糖的Earle′s液1ml,各药的终浓度为20ug/ml,培养30min后,加入终浓度为1mmol/L的氰化钾制造的大脑皮层细胞缺氧模型,12小时模型组大部分胞体肿胀,失去折光性,胞质出现颗粒与空泡。24小时胞体进一步肿胀,颗粒增多,大部分胞膜破裂,大量碎片生成,仅少数细胞存活。测定各组LDH漏出率。结果见表3。
模型对照组大脑皮层细胞12小时后大部分胞体肿胀,失去折光性,胞质出现颗粒与空泡。24小时后胞体进一步肿胀,颗粒增多,大部分胞膜破裂,大量碎片生成,仅少数细胞存活。编号为2、7、8、10、12、14、15的化合物组在20ug/ml的浓度作用下细胞生长良好,抗缺氧作用较明显(P<0.01),使1mmol/L的氰化钾作用24小时的大脑皮层细胞的LDH漏出率降低,其中以毛蕊异黄酮的作用为明显,抑制率高达70%(阳性对照组尼莫地平为57%)。
以上结果表明:芒柄花素,毛蕊异黄酮,3-羟基-9,10-二甲基紫檀烷-3-O-葡萄糖苷、芒柄花素-7-O-葡萄糖苷、毛蕊异黄酮-7-O-葡萄糖苷5个成分具有抗脑缺氧作用,其中毛蕊异黄酮的作用最明显。5个成分均属黄酮或黄烷类,这说明补阳还五汤中具有相似结构的成分在发挥相似的作用。
表3.补阳还五汤化学成分对1mmol/L的氰化钾大脑皮层细胞缺氧模型细胞的
LDH漏出率的影响
样品 | n | 药物浓度(ug/ml) | LDH漏出率% |
芒柄花素毛蕊异黄酮3-羟基-9,10-二甲氧基紫檀烷-3-O-葡萄糖苷芒柄花素-7-O-葡萄糖苷氰化钾(模型对照组)空白组尼莫地平 | 4444444 | 202020205×10-6mol/L | 44.7±1.6**38.6±5.0**44.4±2.0**46.5±1.2**62.8±2.911.4±1.740.2±2.6** |
**与模型对照组相比,P<0.01
Claims (10)
1.本发明所述的化合物,具有下列3个结构式:
R1为葡萄糖醛酸基或硫酸酯基。R2、R3和R4为H、OH、烷氧基。
2.根据权利要求1所述化合物,R2、R3和R4为H、OH、甲氧基。
3.权利要求1~2任何一项所述的化合物的制备方法,它包括下列步骤:将黄芪或补阳还五汤药材水煎液,按临床人日用量的1~10倍剂量给猪、兔或鼠连续灌胃给药3~7次,最后1次给药后1~2小时颈总动脉取血并分离血清,血清冷冻干燥成干粉。给药动物血清冷干粉用甲醇超声提取1~3次,每次20~40分钟。提取液合并,减压回收得浸膏。浸膏样品的水溶液经反相色谱柱分离,100%水→100%甲醇梯度洗脱,对比分析各洗脱流份与空白动物血清甲醇提取液的高效液相色谱(HPLC)图的差异,寻找给药血清特有峰所在洗脱流份,并据此合并各洗脱流份。各洗脱流份根据情况用同样方法反复精制并检测,将给药血清特有峰的相对含量提高。采用HPLC-DAD-MS技术对特有峰进行化学结构鉴定。HPLC流动相为:0.025%磷酸水-乙腈(100∶0→0∶100,20~60min线性梯度);色谱柱:ZORBAX SB-C18(5μm,4.6×250mm);DAD检测;流速:0.5~1.5ml/min。
4.根据权利要求3所述的化合物的制备方法,但实验受试动物改为猪或兔。
5.根据权利要求4所述的化合物的制备方法,它包括下列步骤:将黄芪或补阳还五汤药材水煎液,按临床人日用量(2.375g/kg)的10倍剂量给猪或兔连续灌胃给药7次,最后1次给药后1小时颈总动脉取血并分离血清,血清冷冻干燥成干粉。给药动物血清冷干粉用甲醇超声提取3次,每次30分钟。提取液合并,减压回收得浸膏。浸膏样品的水溶液经反相色谱柱分离,100%水→100%甲醇梯度洗脱,对比分析各洗脱流份与空白动物血清甲醇提取液的HPLC图谱的差异,寻找给药血清特有峰所在洗脱流份,并据此合并各洗脱流份。各洗脱流份根据情况用同样方法反复精制并检测,将给药血清特有峰的相对含量提高,采用HPLC-DAD-MS技术对特有峰进行化学结构鉴定。HPLC流动相为:0.025%磷酸水-乙腈(100∶0→0∶100,20min线性梯度);色谱柱:ZORBAX SB-C18(5μm,4.6×250mm);DAD检测;流速:1.0ml/min;柱温:20℃。
6.根据权利要求5所述的化合物的制备方法,但将药材改为补阳还五汤药材水煎液。
7.根据权利要求6所述的化合物的制备方法,但实验受试动物改为猪。
8.根据权利要求1~2所述任何一项所述的化合物与缺血性脑中风相关活性方面的治疗作用。
9.权利要求1~2所述任何一项所述的化合物对缺血-再灌损伤大鼠脑细胞膜流动性的增大作用。
10.权利要求1~2所述任何一项所述的化合物对大脑皮层细胞缺氧损伤的保护作用。
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WO2007128725A1 (en) * | 2006-05-03 | 2007-11-15 | Symrise Gmbh & Co. Kg | 6h-benzofuro[3,2-c] [1]benzopyran and [2] benzopyrano [4,3-b] [1]benzopyran derivatives and wood extracts of these compounds as aryl hydrocarbon receptor (ahr) antagonists for the prevention of uv-b induced skin damage |
CN102382092A (zh) * | 2011-07-15 | 2012-03-21 | 北京大学 | 新的异戊烯基黄酮类化合物及其应用 |
WO2014048070A1 (zh) * | 2012-09-27 | 2014-04-03 | 香港科技大学 | 一种用于促进红细胞生成的组合物及其用途 |
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CN102382092A (zh) * | 2011-07-15 | 2012-03-21 | 北京大学 | 新的异戊烯基黄酮类化合物及其应用 |
CN102382092B (zh) * | 2011-07-15 | 2014-04-23 | 北京大学 | 新的异戊烯基黄酮类化合物及其应用 |
WO2014048070A1 (zh) * | 2012-09-27 | 2014-04-03 | 香港科技大学 | 一种用于促进红细胞生成的组合物及其用途 |
US10183939B2 (en) | 2014-09-12 | 2019-01-22 | Redx Pharma Plc | Fused bicyclic (hetero)aromatic compounds useful for the treatment of cancers |
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