CN101766643B - 霍山石斛萃取物及其制备方法 - Google Patents
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Abstract
本发明提供一种具有如式I结构的物质,式I其中R1与R2分别选自α-阿拉伯糖(α-Ara)、β-阿拉伯糖(β-Ara)和β-木糖(β-Xyl)其中之一。
Description
技术领域
本发明涉及一种具有生物活性的植物萃取物的制备方法及该萃取物,尤其涉及一种利用水溶性有机溶剂或水溶性有机溶剂与水的混合物而获得植物萃取物的制备方法。
背景技术
如美国专利号7,101,577所述,已知石斛属植物被视为可治疗眼科疾病的珍贵药材。从先前的研究结果中可发现,其中以石斛(Herba Dendrobii,传统上作为具有疗效的石斛植物的统称)最具生物活性。但是根据近年来的趋势,其茎部具有疗效的石斛植物通称为茎石斛(Caulis Dendrobii),而整株植物具有疗效的石斛植物则通称为石斛(Herba Dendrobii)。
根据上述美国专利中披露的内容,已知视网膜色素上皮(retinal pigment epithelium,RPE)细胞是在视网膜表层的单层细胞(Monolayer cell),位于布鲁赫氏膜(Bruch’s membrance)与光感受器(photoreceptors)之间。由于RPE细胞可有效地清除或输送脉络膜与视网膜神经层的有毒物质及代谢物,因此亦构成了很重要的视网膜血管屏障(blood-retinal barrier)。
此外,RPE细胞具有许多功能,如:吸收光线,吞噬杆状细胞及锥状细胞受光线刺激而脱落的外节(rod outer segments,ROS)、分解吞噬体(phagosome)、合成细胞外基质(extracellularmatrix)与黑色素(melanin)、药物的解毒、提供光感受器的外节再生时所必须的物质、维生素A的储存与运送、视紫质(Rodopsin)的合成、形成网膜的接着力等等。因为RPE细胞正常的吞吃作用对于维持视网膜中光感受器的健康具有相当大的关联性,因此,一旦吞吃机能降低,将造成光感受器的退化(degeneration)。虽然RPE细胞会随着年龄的增加而死亡或游离至他处,但老化的RPE细胞仍具有吞噬能力。且杆状细胞的损失较锥状细胞损失更多,此可能造成老年人视力减退及疾病的发生,因此RPE细胞功能的维持对于视觉系统是相当重要的。
在免疫系统中,一氧化氮扮演着防御的角色并对细胞具有毒性;在血管系统中,一氧化氮则是所谓的内皮细胞舒张因子(EDRF);而在中枢神经系统,一氧化氮则是作为神经传递质(neurotransmitter)。
一氧化氮是在一氧化氮合成酶(nitric oxide synthase;NOS)将L-精氨酸(L-arginine)转变成L-瓜氨酸(L-citrulline)的过程中被释放。一氧化氮合成酶有三种异构体(isoforms),分别为神经元NOS(neuronal NOS)、内皮NOS(endothelial NOS)与免疫NOS(immunologic NOS)。其中神经元NOS及内皮NOS为基本型(constitutive form),简称cNOS,其活性受到钙离子(Ca++)及调钙素(calmodulin)的调节,所释放一氧化氮的浓度属于nM(10-9M)等级,免疫NOS属于可诱导型(inducible form),简称iNOS,但其活性不受到Ca++及调钙素的调节,所释放一氧化氮的浓度属于mM(10-6M)等级,其中cNOS与iNOS的基因是位于不同染色体上。
现已发现在视网膜中的视网膜神经细胞(retinal neuron)、RPE细胞、无长突细胞及神经节细胞(amacrine and ganglion cells)、米勒细胞(Muller cells)等都有一氧化氮合成酶(Nitric oxidesynthase;NOS)的存在,且到目前为止,已有证据显示一氧化氮在生理及病理状态下,都扮演重要的角色,且与眼睛的机能关系密切。
已发现在基础状态(basal condition)或缺血状态(ischemia)下,一氧化氮具有控制视网膜血流量的能力。而且一氧化氮可能具有调节糖尿病所引起的视网膜内血管受损的程度。另外,当视网膜神经胶质细胞(retinal glial cells)及RPE细胞受到LPS、IFN-γ、TNF-α刺激,会促进一氧化氮合成酶的表现,进而大量产生一氧化氮,由此可知一氧化氮在视网膜处于发炎或受到感染的情况下,可能扮演着防御、保护的角色。
然而,截至目前为止,cNOS在视觉感受器的位置及特性仍不甚清楚,有文献指出在视觉感受器本体有cNOS活性,另有文献则指出在视觉感受器外节(photoreceptor outer segments)才有cNOS活性,而被释放的一氧化氮可调节光线的传导、神经突触的讯号传递、生理状态下或缺血状态下视网膜的血流量等。iNOS的活性亦表现在视网膜的一些细胞中,例如RPE细胞与米勒细胞(Muller cells)等,有文献指出在培养牛的视网膜色素上皮细胞中,若经过IFN-γ,LPS,TNF-α刺激后约12小时可产生大量一氧化氮,且可至少持续96小时。细胞激素(cytokines)在RPE细胞的iNOS活性上的影响是相当复杂的,例如,需LPS及IFN-γ或TNF-α刺激才能产生大量一氧化氮,bFGF则具有抑制NOS的作用,而TGF-β则具有些许促进NOS的作用。对人类RPE细胞而言,必须有白细胞介素-1β(Interleukin-1β)的刺激才能产生大量NO,LPS则无影响,TGF-β却能明显抑制其释放一氧化氮。
当受到细菌感染时,iNOS的表现可能是有益的,其所释放的一氧化氮可以杀死入侵的微生物;相反的,在某些状况下太大量产生一氧化氮反而会导致自身免疫疾病(autoimmune disease)或败血性休克(septic shock)。1994年首度有证据说明一氧化氮与眼底发炎反应有关,本发明申请人则发现iNOS抑制剂可以阻断由内毒素(endotoxin)所引起的葡萄膜炎。另一方面,以IFN-γ及LPS处理RPE使之产生大量一氧化氮,而此作用会被aFGF、bFGF所抑制,且指出,是抑制在iNOS的表现而非iNOS mRNA的稳定性(stability),所以本发明申请人推测FGF可保护RPE免于受到内毒素或细胞激素所引起的伤害。由此可见,iNOS在视网膜中亦扮演着免疫调节的角色。
常见的视网膜病变有糖尿病所引起的视网膜血管新生(proliferative diabetic retinopathy)、增殖性玻璃体视网膜病变(proliferative vitreoretinopathy)及老年性黄斑变性(Aged-maculardegeneration)等,而视网膜的病变是所有眼科疾病中最难治疗的。高血糖(hyperglycemia)加速糖化作用(glycation)的发生,产生高度糖化终产物(advanced glycation end products,AGE)一直被认为是与糖尿病末期所引发的各种血管、神经并发症息息相关。AGE的形成是还原糖(reducing sugar)的醛基(aldehyde group)或酮基(ketone group)与蛋白质的一级氨基酸(primary amino acid)经由非酵素(nonenzymatic)的作用产生不稳定的许夫氏碱(Schiffbase),再经由阿马多尔重排(Amadori rearrangement)产生阿马多尔产物(Amardori product)。已知非酵素糖化作用是一不可逆反应且多发生在半衰期长的蛋白质上,因AGE的形成造成蛋白质交联(cross-linking)后会使得蛋白质对于蛋白酶(protease)产生抗性,因此AGE的累积是一老化的标志。随着年龄的增加,脑部锥状神经(pyramidal neurons)、布鲁赫氏膜(Bruch’s membrane)、胶原蛋白(collagen)中AGE的含量也都随着上升。非酵素糖化作用速率呈初级反应,反应速率决定于还原糖与蛋白质的浓度。通常,糖尿病患者血糖浓度较一般正常人要高,因此更加速了糖化作用的发生。已知糖尿病加速动脉粥样硬化、肾脏损伤、血管受损、神经病变、视网膜病变及糖尿病患者较健康者易中风,这都与AGE有直接的关系。糖尿病造成红血球的聚集,主要是白蛋白(albumin)被糖化后造成其三级结构(tertiary structure)改变,导致丧失抗聚集(anti-aggregation)的功能,另外,研究显示糖尿病引发肾脏丝球体通透性的改变,是因为白蛋白的糖化而非肾小球基膜(glomerular basement membrane)的糖化。而且糖化的蛋白质会增加其通透血脑屏障(blood brain barrier)的能力。
AGE能够和细胞表面上的一些受体(receptor)、蛋白质结合,目前已知的有清道夫受体(scavenger receptors)第一型及第二型,AGE受体(RAGE,the receptor for AGE)、OST-48(AGE-R1)、80K-H磷蛋白质(phosphoprotein)(AGE-R2)及半乳凝素-3(galectin-3)(AGE-R3)。在单核球、巨噬细胞、内皮细胞、神经胶细胞等中都可发现AGE受体,当细胞被AGE活化时,常使得细胞外基质蛋白质(extracellular matrix protein)、血管附着分子(vascular adhesion molecules)、及生长因子(growth factor)表现量增加,随着不同的细胞种类及讯息传递,而伴随着趋化性(chemotaxis)、血管新生(angiogenesis)、氧化压力、细胞增生或细胞程式凋亡(programmed cell death)。已知人类脑部各种细胞表现不同的高度糖化终产物受体,可清除AGE,当此能力丧失时,造成细胞外AGE的累积,而诱发中枢神经系统的发炎反应。而AGE可使RPE细胞的视网膜血管内皮细胞生长因子(retinalvascular endothelial growth factor),及PDGF-β的表现量增加。AGE在老化过程中扮演重要角色,因此以糖化白蛋白作为一个病理模型(model)来进行新药开发便是十分重要。
HGF/SF肝细胞生长因子(Hepatocyte growth factor)为肝脏最重要的生长因子,是由60KDa的重链(heavy chain)(αchain)及30KDa的轻链(light chain)(βchain)以双硫键相接所构成。体内刚合成的HGF/SF为prepro HGF/SF须经酵素修饰之后成为异二聚体形式(heterodimeric form)才具有生物活性。HGF为一种多功能的生长因子,不只对多种细胞都有调节生长的能力,在组织修复(tissue repair)及器官再生(organ regeneration)中也扮演重要角色。HGF在体内分布十分广泛,以肝脏产量最大,其余还有胰脏、胸腺、血液、小肠、胎盘等。除此之外,HGF/SF或HGF/SF受体也存在于眼睛的分泌物及组织中,如泪液、泪腺、角膜等,可见HGF/SF在眼睛内可能有其调控的角色。视网膜色素上皮细胞不但同时具有HGF及HGF受体(c-Met),而且c-Met的酪氨酸磷酸化反应(tyrosine phosphorylation)是一直持续不断的在表现,所以HGF对视网膜色素上皮细胞而言可能是一个自体作用的生长因子,而且可能与视网膜发育、伤口愈合及视网膜血管新生有关。
由以上内容可知,RPE细胞在视网膜调控机制上扮演了非常重要的角色。也就是说,目前已知石斛属中草药能增强或抑制RPE细胞的某些功能或调控机制。更特别的事,石斛属植物能增强RPE细胞的吞吃能力、一氧化氮的形成以及HGF的基因表现。石斛属植物更能在正常条件或缺血状态下抑制RPE细胞中bFGF、VEGF及TGF-β的基因表现量。因此,与增强RPE细胞活性的因子相关的研究,对于促进人体健康是十分重要的。在过去的研究中,虽然已知石斛萃取物具有加速RPE细胞吞吃及增加AGE降解的功效,为了更进一步掌控其加速RPE吞吃的功效,仍须对于石斛萃取物有更深的了解。
发明内容
于是,申请人针对现有技术的不足,经过悉心试验与研究,研发出本发明的霍山石斛萃取物及其制备方法。
本发明提供了一种组合物,其包含具有如式I结构的物质,
式I
其中R1与R2是相同或不同的官能基。
根据上述构想,该组合物具有生理上活性。
根据上述构想,其中该组合物包含具有加速视网膜色素上皮细胞的吞吃作用的功效。
根据上述构想,其中该物质是选自芹菜苷配基6,8-二-C-α-L-吡喃阿拉伯糖苷(apigenin 6,8-Di-C-α-L-arabinopyranoside)、芹菜苷配基6-C-β-D-吡喃木糖基-8-C-α-L-吡喃阿拉伯糖苷(apigenin6-C-β-D-Xylopyranosyl-8-C-α-L-arabinopyranoside)、芹菜苷配基6-C-α-L-吡喃阿拉伯糖基-8-C-β-D-吡喃木糖苷(apigenin 6-C-α-L-Arabinopyranosyl-8-C-β-D-xylopyranoside)及其混合物其中之一。
本发明进一步提供一种具有如式I结构的物质,
式I
其中R1与R2分别选自α-阿拉伯糖(α-Ara)、β-阿拉伯糖(β-Ara)和β-木糖(β-Xyl)其中之一。
根据上述构想,该物质具有促进视网膜色素细胞活性的功效。
本发明还提供一种具有如式I结构的物质,
式I
其中R1与R2分别选自以下组成的群中:
根据上述构想,其中R1与R2为相同的或不同的官能基,并分别选自C-β-D-吡喃葡萄糖基(C-β-D-glucopyranosyl)、C-β-D-吡喃半乳糖基(C-β-D-galactopyranosyl)、C-β-D-吡喃木糖基(C-β-D-xylopyranosyl)、C-β-D-吡喃阿拉伯糖基(C-β-D-arabinopyranosyl)、C-α-L-吡喃阿拉伯糖基(C-α-L-arabinopyranosyl)、C-α-L 吡喃鼠李糖基(C-α-L-rhamnopyranosyl)和C-6去氧-木-己-4-酮糖基(C-6-deoxy-xylo-hexos-4-ulosyl)其中之一。
另一方面,本发明提供一种具有生理活性的组成物,包含上述含有式I结构的三种物质中的任何一种,该物质具有促进视网膜色素上皮细胞功能的作用。
根据上述构想,该组成物进一步包含具有生理可接受的载体(Physiologically acceptable carrier),优选为药理上载体。
根据上述构想,其中该具有生理活性的组成物是一种具有药理活性的组成物。
根据上述构想,其中该具有生理活性的组成物对于一病变具有药理活性,该病变是选自:视网膜色素上皮细胞退化、老年性黄斑变性、光感受器退化、糖尿病、动脉硬化、血管受损、视网膜神经细胞病变、无长突细胞病变、神经节细胞病变、米勒细胞病变、葡萄膜炎、视网膜血管新生、增殖性玻璃体视网膜病变、视网膜发炎和布鲁赫氏膜病变其中至少之一。
为了易于说明,本发明借助下述优选实施例及附图而得到充分了解,并使得本领域技术人员可以据此完成本发明,但是本发明的实施方式并不局限于下列实施例。
附图说明
图1为本发明优选实施例的石斛萃取物的分离流程图;
图2为本发明的石斛萃取物DCMPbL6,7D2H2H3以LC-MS柱层析进行分析所得的结果;
图3为本发明的石斛萃取物DCMPbL6,7D2H2H3以HPLC柱层析进行分析所得的结果;
图4为本发明的石斛萃取物DCMPbL6,7D2H2H3H2以LC-MS柱层析进行分析所得的结果;
图5为本发明的石斛萃取物DCMPbL6,7D2H2H3H2以LC-MS柱层析进行另一次分析所得的结果;
图6为本发明的石斛萃取物DCMPbL6,7D2H2H3H3以LC-MS柱层析进行分析所得的结果;
图7为本发明的石斛萃取物DCMPbL6,7D2H2H3H4溶解于甲醇溶液中的UV光谱图;
图8为本发明的石斛萃取物DCMPbL6,7D2H2H3对RPE细胞整体吞吃作用影响的柱形图;
图9为本发明的石斛萃取物DCMPbL6,7D2H2H3对RPE细胞细胞内吞吃作用影响的柱形图;
图10为本发明的石斛萃取物DCMPbL6,7D2H2H3H2对RPE细胞整体吞吃作用影响的柱形图;
图11为本发明的石斛萃取物DCMPbL6,7D2H2H3H2对RPE细胞细胞内吞吃作用影响的柱形图;
图12为本发明的石斛萃取物DCMPbL6,7D2H2H3H3对RPE细胞整体吞吃作用影响的柱形图;
图13为本发明的石斛萃取物DCMPbL6,7D2H2H3H3对RPE细胞细胞内吞吃作用影响的柱形图;
图14为本发明的石斛萃取物DCMPbL6,7D2H2H3H4对RPE细胞整体吞吃作用影响的柱形图;以及
图15为本发明的石斛萃取物DCMPbL6,7D2H2H3H4对RPE细胞细胞内吞吃作用影响的柱形图。
具体实施方式
本发明的石斛萃取物及其制备方法将可由以下的实施例说明而得到充分了解,并使得本领域技术人员可以据此完成本发明,但是本发明的实施方式并不局限于下列实施例。
(一)石斛萃取物的制备及纯化活性成分的分离。
请参照图1,其为本发明优选实施例的石斛萃取物的分离流程图。以下方法可分离出石斛中三种活性成分。将1.9公斤石斛以甲醇(methanol)萃取三次以获得石斛甲醇萃取物,并将石斛甲醇萃取物进行浓缩后完全干燥,以形成DCM(石斛醇类粗萃取物)标准品。将干燥的DCM标准品溶解于2L乙醇(EtOAc)中,并以2L的水进行分层,可获得一EtOAc层及第一水层。将第一水层再以2L EtOAc萃取两次,收集所有的EtOAc层后,进行浓缩并完全干燥,以取得乙醇萃取物。将干燥的乙醇萃取物分别以4L己烷(hexane)及2L甲醇分层三次,以获得一己烷层及一甲醇层。经过浓缩与干燥后,将干燥后的己烷层及甲醇层分别命名为DCMPe/h标准品(Pe/h)及DCMPe/m标准品(Pe/m)。除此以外,加入去离子水将第一水层调整为2公升,再以丁醇(buantol)2公升进行分配萃取,以获得丁醇层及第二水层,经减压浓缩抽干后将干燥的丁醇层及第二水层分别命名为DCMPb标准品(Pb)及DCMPw标准品(Pw)。PCMPb标准品以LH20胶进行分子柱层析(2.5×107cm,移动相为:甲醇∶水=50∶50),进行活性的筛选后,得到命名为PCMPbL6,7的标准品。接着再以Diaion SP-20SS进行吸附柱层析(1×30cm),当移动相为异丙醇(isorpopanol)∶水=20∶80时,可获得命名为DCMPbL6,7D2的洗提产物。当移动相为异丙醇(isorpopanol)∶水=30∶70时,可获得命名为DCMPbL6,7D3的洗提产物。当移动相为异丙醇(isorpopanol)∶水=40∶60时,可获得命名为DCMPbL6,7D4的洗提产物。接着,DCMPbL6,7D2再经过HPLC反相(reverse phase)C18层析柱(10×300mm)在移动相为甲醇∶水∶乙酸=35∶65∶1时洗提,收集命名为DCMPbL6,7D2H2的洗提产物。
根据先前研究的成果,已知DCMPbL6,7D2H2具有加速视网膜色素上皮细胞吞吃作用以及增加高度糖化终产物降解的功效。以下步骤是为了更进一步了解加速视网膜色素上皮细胞吞吃作用的功效。
DCMPbL6,7D2H2再经过HPLC反相(reverse phase)C 18层析柱(10×300mm)在移动相为甲醇∶水∶乙酸=40∶60∶1时洗提,可获得命名为DCMPbL6,7D2H2H3的洗提产物。接着,DCMPbL6,7D2H2H3再经过HPLC反相(reverse phase)C18层析柱(4.6×250mm)在移动相为甲醇∶水=35∶65时洗提,可获得命名为DCMPbL6,7D2H2H3H2的洗提产物。DCMPbL6,7D2H2H3也可以经过HPLC反相(reverse phase)C18层析柱(4.6×250mm)在移动相为甲醇∶水=40∶60时洗提,即可获得命名为DCMPbL6,7D2H2H3H3的洗提产物。或者,DCMPbL6,7D2H2H3也可以经过HPLC反相(reverse phase)C18层析柱(4.6×250mm)在移动相为甲醇∶水=45∶55时洗提,获得命名为DCMPbL6,7D2H2H3H4的洗提产物。
请参照图2,其为本发明的石斛萃取物DCMPbL6,7D2H2H3以LC-MS柱层析进行分析所得的结果。图2中的分析条件为:Mightysil RP-C18柱(4.6×250mm),5μm;移动相在第0-80分钟时为甲醇/水=20/80至100%甲醇的梯度,第80-100分钟为100%甲醇;注射体积50μl;注射重量5μg;流速为0.25ml/分钟,在UV波长337nm下。
请参照图3,其为本发明的石斛萃取物DCMPbL6,7D2H2H3以HPLC柱层析进行分析所得的结果。图3中的分析条件为:Mightysil RP-C18柱(4.6×250mm),5μm;移动相在第0-40分钟时为甲醇/水=35/65,第40-45分钟时为甲醇/水=35/65至100%甲醇的梯度,第45-60分钟时为100%甲醇;注射体积250μl;注射重量250μg;流速为0.8ml/分钟,在UV波长280nm、312nm及337nm下。其中H2代表DCMPbL6,7D2H2H3H2、H3代表DCMPbL6,7D2H2H3H3而H4代表DCMPbL6,7D2H2H3H4。
请参照图4,其为本发明的石斛萃取物DCMPbL6,7D2H2H3H2以LC-MS柱层析进行分析所得的结果。图4中的分析条件为:Mightysil RP-C18柱(4.6×250mm),5μm;移动相在第0-80分钟时为甲醇/水=20/80至100%甲醇的梯度,第80-100分钟为100%甲醇;注射体积100μl;注射重量5μg;流速为0.8ml/分钟,在UV波长220nm且质谱仪(MS)设定为APCI+模式的条件下。
请参照图5,其为本发明的石斛萃取物DCMPbL6,7D2H2H3H2以LC-MS柱层析进行另一次分析所得的结果。图5中的分析条件为:Mightysil RP-C18柱(4.6×250mm),5μm;移动相在第0-80分钟时为甲醇/水=20/80至100%甲醇的梯度,第80-100分钟为100%甲醇;注射体积100μl;注射重量5μg;流速为0.8ml/分钟,在UV波长220nm且质谱仪设定为APCI-模式的条件下。
请参照图6,其为本发明的石斛萃取物DCMPbL6,7D2H2H3H3以LC-MS柱层析进行分析所得的结果。图6中的分析条件为:Mightysil RP-C18柱(4.6×250mm),5μm;移动相在第0-80分钟时为甲醇/水=20/80至100%甲醇的梯度,第80-100分钟为100%甲醇;注射体积100μl;注射重量5μg;流速为0.8ml/分钟,在UV波长220nm且质谱仪设定为APCI+模式的条件下。
请参照图7,其为本发明的石斛萃取物DCMPbL6,7D2H2H3H4溶解于甲醇溶液中的UV光谱图。如图7所示,DCMPbL6,7D2H2H3H4在波长(λ)为334nm时具有第一宽带(bandI),在波长为275nm时具有第二宽带(band II),因此可推论该化合物属于一种类黄酮(flavonoid)化合物。
由以上结果可发现,DCMPbL6,7D2H2H3的分子量约在534,而且选自芹菜苷配基6,8-二-C-α-L-吡喃阿拉伯糖苷(apigenin 6,8-Di-C-α-L-arabinopyranoside)、芹菜苷配基-C-β-D-吡喃木糖基-8-C-α-L-吡喃阿拉伯糖苷(apigenin6-C-β-D-Xylopyranosyl-8-C-α-L-arabinopyranoside)、芹菜苷配基6-C-α-L-吡喃阿拉伯糖基-8-C-β-D-吡喃木糖苷(apigenin 6-C-α-L-Arabinopyranosyl-8-C-β-D-xylopyranoside)及其混合物其中之一。其中芹菜苷配基6-C-β-D-吡喃木糖基-8-C-α-L-吡喃阿拉伯糖苷的结构如下所示:
M.w.=534
另外,DCMPbL6,7D2H2H3的红外线分析结果(未显示于图示中)也显示DCMPbL6,7D2H2H3的结构特征与芹菜苷配基的6,8-二-C-糖苷的结构特征相符合。
除此以外,在DCMPbL6,7D2H2H3H2、DCMPbL6,7D2H2H3H3、DCMPbL6,7D2H2H3H2以及DCMPbL6,7D2H2H3H4中加入一些试剂(氯化铝、醋酸钠或醋酸钠与硼酸的混合溶液)后,以UV分析其结果。UV结果显示DCMPbL6,7D2H2H3H2、DCMPbL6,7D2H2H3H3、DCMPbL6,7D2H2H3H2以及DCMPbL6,7D2H2H3H4的结构特征与类黄酮化合物(在C-5及C-7位置为氢氧基(hydroxyl group)而C-3位置不是氢氧基)的结构特征相符合。
在经过NMR分析后,DCMPbL6,7D2H2H3、DCMPbL6,7D2H2H3H2、DCMPbL6,7D2H2H3H3以及DCMPbL6,7D2H2H3H4的氢谱(H-NMR)及碳谱(C-NMR)结果显示,其对应的结构特征与芹菜苷配基的6,8-二-C-糖苷的结构特征相符。
由上述结果可发现,来自DCMPbL6,7D2H2的物质,如:DCMPbL6,7D2H2H3、DCMPbL6,7D2H2H3H2、DCMPbL6,7D2H2H3H3以及DCMPbL6,7D2H2H3H4,可以是芹菜苷配基的6,8-二-C-糖苷或其衍生物,且具有以下结构:
其中R1与R2为相同的或不同的官能基并分别选自α-阿拉伯糖(α-Ara)、β-阿拉伯糖(β-Ara)和β-木糖(β-Xyl)其中之一。
由于天然的C-糖基支链的C-糖基类黄酮包括C-β-D-吡喃葡萄糖基(C-β-D-glucopyranosyl)、C-β-D-吡喃半乳糖基(C-β-D-galactopyranosyl)、C-β-D-吡喃木糖基(C-β-D-xylopyranosyl)、C-β-D-吡喃阿拉伯糖基(C-β-D-arabinopyranosyl)、C-α-L-吡喃阿拉伯糖基(C-α-L-arabinopyranosyl)、C-α-L-吡喃鼠李糖基(C-α-L-rhamnopyranosyl)和C-6-去氧-木-己-4-酮糖基(C-6-deoxy-xylo-hexos-4-ulosyl),而且上述实验已经显示DCMPbL6,7D2H2H3、DCMPbL6,7D2H2H3H2、DCMPbL6,7D2H2H3H3和DCMPbL6,7D2H2H3H4都具有如前段所示的C-糖苷的主结构,因此从上述七种天然的C-糖基类黄酮选出R1及R2时,将会从中获得48种对应的化合物。同样地,当R1及R2选自非天然的C-糖基支链时,将会从中获得482种对应的化合物。由此可知,如果位于C-4、C-5及C-7位置的官能基被其他取代,则对应衍生物的数目将变得十分庞大。表一中显示了R1及R2可能的天然官能基组合。
表一R1及R2可能的天然官能基组合
*代表主要产物。
R1及R2可能的非天然官能基组合如表二所示。
表二R1及R2可能的非天然官能基组合
请参照图8,其为本发明的石斛萃取物DCMPbL6,7D2H2H3对RPE细胞整体吞吃作用影响的柱形图。如图8所示,不同浓度的DCMPbL6,7D2H2H3(0.1μg/ml、1μg/ml、10μg/ml及100μg/ml)都能加速视网膜色素上皮细胞的吞吃作用,相关实验内容简述如下。将1×104RPE细胞置入96孔盘中,在含有5%胎牛血清的DMEM培养液中培养48小时后,更换培养液为含有2%胎牛血清的DMEM 培养液。然后,分别加入不同浓度的DCMPbL6,7D2H2H3,24小时后,加入50μl的2×107/ml标定FITC荧光的ROS细胞(FITC-ROS)到各培养格中,培养4小时后,以混合2%蔗糖的磷酸盐缓冲液将未附着于细胞表面的FITC-ROS洗掉。以发射波长485nm,侦测波长530nm经由1420Multilable counter(PE)测量系统侦测荧光强度,而所侦测到的荧光强度代表整体吞吃作用的结果。若加入荧光淬灭(fluro Quench)染剂,则所侦测到的荧光强度代表细胞内吞吃作用的结果。与处理2%胎牛血清的RPE细胞的吞吃作用比较,*代表P值小于0.05、**代表P值小于0.02、***代表P值小于0.01以及****代表P值小于0.001。另外,图9为本发明的石斛萃取物DCMPbL6,7D2H2H3对RPE细胞细胞内吞吃作用影响的柱形图。
请参照图10,其为本发明的石斛萃取物DCMPbL6,7D2H2H3H2对RPE细胞整体吞吃作用影响的柱形图。如图10所示,不同浓度的DCMPbL6,7D2H2H3H2(0.1μg/ml、1μg/ml、10μg/ml及100μg/ml)都能加速视网膜色素上皮细胞的吞吃作用,相关实验内容简述如下。将1×104RPE细胞置入96孔盘中,在含有5%胎牛血清的DMEM培养液中培养48小时后,更换培养液为含有2%胎牛血清的DMEM培养液。然后,分别加入不同浓度的DCMPbL6,7D2H2H3H2,24小时后,加入50μl的2×107FITC-ROS/ml到各培养格中,培养4小时后,以混合2%蔗糖的磷酸盐缓冲液将未附着于细胞表面的FITC-ROS洗掉。以发射波长485nm,侦测波长530nm经由1420Multilable counter(PE)测量系统侦测荧光强度,而所侦测到的荧光强度代表整体吞吃作用的结果。若加入fluro Quench染剂,则所侦测到的荧光强度代表细胞内吞吃作用的结果。与处理2%胎牛血清的RPE细胞的吞吃作用比较,*代表P值小于0.05、**代表P值小于0.02、***代表P值小于0.01以及****代表P值小于0.001。另外,图11为本发明的石斛萃取物DCMPbL6,7D2H2H3H2对RPE细胞细胞内吞吃作用影响的柱形图。
请参照图12,其为本发明的石斛萃取物DCMPbL6,7D2H2H3H3对RPE细胞整体吞吃作用影响的柱形图。如图12所示,不同浓度的DCMPbL6,7D2H2H3H3(0.1μg/ml、1μg/ml、10μg/ml及100μg/ml)都能加速视网膜色素上皮细胞的吞吃作用,相关实验内容简述如下。将1×104RPE细胞置入96孔盘中,在含有5%胎牛血清的DMEM培养液中培养48小时后,更换培养液为含有2%胎牛血清的DMEM培养液。然后,分别加入不同浓度的DCMPbL6,7D2H2H3H3,24小时后,加入50μl的2×107FITC-ROS/ml到各培养格中,培养4小时后,以混合2%蔗糖的磷酸盐缓冲液将未附着于细胞表面的FITC-ROS洗掉。以发射波长485nm,侦测波长530nm经由1420Multilable counter(PE)测量系统侦测荧光强度,而所侦测到的荧光强度代表整体吞吃作用的结果。若加入fluro Quench染剂,则所侦测到的荧光强度代表细胞内吞吃作用的结果。与处理2%胎牛血清的RPE细胞的吞吃作用比较,*代表P值小于0.05、**代表P值小于0.02、***代表P值小于0.01以及****代表P值小于0.001。另外,图13为本发明的石斛萃取物DCMPbL6,7D2H2H3H3对RPE细胞细胞内吞吃作用影响的柱形图。
请参照图14,其为本发明的石斛萃取物DCMPbL6,7D2H2H3H4对RPE细胞整体吞吃作用影响的柱形图。如图14所示,不同浓度的DCMPbL6,7D2H2H3H4(0.1μg/ml、1μg/ml、10μg/ml及100μg/ml)都能加速视网膜色素上皮细胞的吞吃作用,相关实验内容简述如下。将1×104RPE细胞置入96孔盘中,在含有5%胎牛血清的DMEM培养液中培养48小时后,更换培养液为含有2%胎牛血清的DMEM培养液。然后,分别加入不同浓度的DCMPbL6,7D2H2H3H4,24小时后,加入50μl的2×107FITC-ROS/ml到各培养格中,培养4小时后,以混合2%蔗糖的磷酸盐缓冲液将未附着于细胞表面的FITC-ROS洗掉。以发射波长485nm,侦测波长530nm经由1420Multilable counter(PE)测量系统侦测荧光强度,而所侦测到的荧光强度代表整体吞吃作用的结果。若加入fluro Quench染剂,则所侦测到的荧光强度代表细胞内吞吃作用的结果。与处理2%胎牛血清的RPE细胞的吞吃作用比较,*代表P值小于0.05、**代表P值小于0.02、***代表P值小于0.01以及****代表P值小于0.001。另外,图15为本发明的石斛萃取物DCMPbL6,7D2H2H3H4对RPE细胞细胞内吞吃作用影响的柱形图。
根据图2至图7,DCMPbL6,7D2H2H3、DCMPbL6,7D2H2H3H2、DCMPbL6,7D2H2H3H3以及DCMPbL6,7D2H2H3H4,可以是芹菜苷配基的6,8-二-C-糖苷或其衍生物,且具有以下结构:
其中R1与R2为相同的或不同的官能基并分别选自表一和表二中的组合其中之一。
根据图8至图15,DCMPbL6,7D2H2H3、DCMPbL6,7D2H2H3H2、DCMPbL6,7D2H2H3H3以及DCMPbL6,7D2H2H3H4可以加速视网膜色素上皮细胞的吞吃作用。如上所述,应可理解具有上述结构的物质能加速视网膜色素上皮细胞的吞吃作用。
基于先前文献的研究,包括可加速视网膜色素上皮细胞吞吃作用的物质的组合物,对于下列病变具有功效,该病变是选自:视网膜色素上皮细胞退化、老年性黄斑变性、光感受器退化、糖尿病(如:血管病变)、动脉硬化、血管受损、视网膜神经细胞病变、无长突细胞病变、神经节细胞病变、米勒细胞病变、葡萄膜炎、视网膜血管新生、增殖性玻璃体视网膜病变、视网膜发炎和布鲁赫氏膜病变其中至少之一。
另外,因为本发明的实际应用范围很大,并不局限于对视网膜色素上皮细胞的吞吃作用,而是对于视网膜上皮细胞生理功能的增强与受损功能的恢复,而且乃是相关萃取物中具有明确化学结构的首例,因此本发明具有显著的新颖性与创造性。即使本发明的相关实施方案可由本领域普通技术人员施匠思而诸般修饰,也应不脱离如所附的本申请权利要求书所要求保护的范围。
Claims (5)
1.选自芹菜苷配基6,8-二-C-α-L-吡喃阿拉伯糖苷或芹菜苷配基6-C-β-D-吡喃木糖基-8-C-α-L-吡喃阿拉伯糖苷的物质在制造用于治疗一病变的药物中的用途,其中所述病变选自由下列组成的组:视网膜色素上皮细胞退化、老年性黄斑变性、光感受器退化、糖尿病、动脉硬化、血管受损、视网膜神经细胞病变、无长突细胞病变、神经节细胞病变、米勒细胞病变、葡萄膜炎、视网膜血管新生、增殖性玻璃体视网膜病变、视网膜发炎和布鲁赫氏膜病变。
2.如权利要求1所述的用途,其中所述物质是从石斛分离的。
3.如权利要求1所述的用途,其中所述物质加速视网膜色素上皮的吞吃作用。
4.一种用于治疗和/或预防一病变的药物组合物,包含生理学上可接受的载体和物质,其中所述物质选自:
芹菜苷配基6,8-二-C-α-L-吡喃阿拉伯糖苷,或
芹菜苷配基6-C-β-D-吡喃木糖基-8-C-α-L-吡喃阿拉伯糖苷,
其中所述病变选自由下列组成的组:视网膜色素上皮细胞退化、老年性黄斑变性、光感受器退化、糖尿病、动脉硬化、血管受损、视网膜神经细胞病变、无长突细胞病变、神经节细胞病变、米勒细胞病变、葡萄膜炎、视网膜血管新生、增殖性玻璃体视网膜病变、视网膜发炎和布鲁赫氏膜病变。
5.如权利要求4所述的药物组合物,其中所述物质是由石斛分离的。
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