CN104523766A - 山苏萃取物 - Google Patents
山苏萃取物 Download PDFInfo
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- CN104523766A CN104523766A CN201410689932.1A CN201410689932A CN104523766A CN 104523766 A CN104523766 A CN 104523766A CN 201410689932 A CN201410689932 A CN 201410689932A CN 104523766 A CN104523766 A CN 104523766A
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- extract
- soviet union
- mountain
- prostate
- mountain soviet
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- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
一种山苏萃取物,供制备改善摄护腺疾病的药物或保健食品,包含焦脱镁叶绿酸-a甲酯、脱镁叶绿酸-a甲酯、1-亚麻油酸-3-亚麻酸-甘油酯、1-亚麻酸-2,3-双棕梠酸-甘油酯,以及1,3-双棕梠酸-甘油酯。该山苏萃取物通过一包含下列步骤的方法所制得:一溶剂萃取步骤,即使用一萃取溶剂,对一山苏样品进行萃取处理,使该山苏样品与该萃取溶剂的重量体积比(w/v,g/ml)为50至60mg/ml,并回收得一萃取物;以及一管柱层析步骤,利用水和乙醇作为流动相,进行该萃取物的层析分离处理,以获得数个区分物,冲提先后而编号为区段a至区段i,撷选该区段b、c、d或其组合物经减压浓缩及冷冻干燥后,即得该山苏萃取物。
Description
技术领域
本发明关于一种植物萃取物及其用途,特别是关于一种山苏萃取物以及其在改善摄护腺疾病的用途。
背景技术
摄护腺又称***(Prostate),位于膀胱出口处,包围着尿道,为男性特有的生殖器官之一。
良性摄护腺肥大(Benign Prostatic Hyperplasia,简称BPH)为男性常见的良性肿瘤,好发于五十岁以上的男性,其致病机转仍未臻明了,目前推测与年龄增长及荷尔蒙分泌相关。一般而言,摄护腺的扩大会直接压迫尿道,导致尿道过度收缩、内压上升而引起排尿障碍,如尿路中断、频尿、漏尿及残尿感等症状,严重时可导致***或肾衰竭。良性摄护腺肥大虽不致提高罹患摄护腺癌的危险率,然良性摄护腺肥大与摄护腺癌早期的症状相近,不易区分,恐误导摄护腺癌的正确诊断,延误就诊或影响患者心情。
现有良性摄护腺肥大的治疗可仰赖手术切除或药物,手术切除的预后期较长且风险略高,因此内科用药仍为主要的治疗手段,常见的现有药物包含甲型交感神经阻断剂(alpha adrenergic blockers),如Minipress、Dibenyline、Hytrin、Doxaben、Xatral等,以及荷尔蒙抑制剂,但是,前述两种药物皆有衍生副作用的可能,可能引发姿势性低血压、尿失禁、鼻塞、疲倦以及性功能障碍等问题,又,该常用药物若与其它药物并服(如感冒药、心血管疾病用药或降血压药)更可能导致严重的医疗伤害,造成患者难忍的煎熬。此外,另有研究指出长期服用该常用药物会提高罹癌(严重的摄护腺癌及乳癌)的机率,于患者而言更是雪上加霜。
近年来,自欧美国家兴起自然回归与健康养生的概念,使得传统医学(traditional medicine,TM)或辅助替代医学(complementary and alternative medicine,CAM)渐为风行,因此,与摄护腺疾病相关的传统中草药与天然植物的开发备受重视,陆续有医用茄红素、南瓜子、锯棕榈(Saw Palmetto)、非洲刺李(Pygeum Africana)及黄体素等天然植物或其成分对于男性摄护腺保健具有明显疗效的报导,然而,前述天然植物多有疗效不明显(如茄红素、南瓜子对于摄护腺疾病的治疗效果有限),或是受限于原料产地或萃取效率等问题,如锯棕榈及非洲刺李仅产于非洲或大西洋、加勒比海沿岸,原料取得不易且萃取效率又随萃取原料的部位而多有变动,因而损及其应用层面;又,部分天然植物恐有衍生副作用的可能性,如锯棕榈易造成胃部不适、恶心、便秘或腹泻(节录自网络数据:http://liaozhai.pujia.com/thread-500003-1.html),虽不至造成严重的后遗症,但终会使服用者感到不适,难获一致性的认同。
因此,若能进一步找到对摄护腺肥大具有显着疗效的植物药物,应用于治疗及预防用药的开发,必然为各界所引领期盼,以有效改善人类摄护腺问题。
山苏(Asplenium nidus L.)属铁角蕨类,为多年生大型食用性植物,原产于中国台湾及东南亚一带,常生于阴湿的树干或岩石细缝上,目前在台湾各地皆已在平地农改大量量产栽种。传统医学记载,山苏富含多种营养成份及膳食纤维,山苏嫩叶为亚种食材适合于炒菜,长期食用安全性高,具有预防高血压、糖尿病等功效,是现代人预防便秘,大肠癌的健康蔬菜,却未见其在改善摄护腺疾病的相关报导与应用。
发明内容
本发明的主要目的是提供一种山苏萃取物,含有抑制雄性素的天然活性成分,可有效改善男性摄护腺相关的疾病。
本发明的次一目的是提供一种山苏萃取物,对于荷尔蒙受体调控具有显着调控功能,该山苏萃取物便于取得、分离而可应用于改善男性摄护腺的药物组成分或保健食品的开发。
本发明的再一目的是提供山苏萃取物在改善男性摄护腺疾病的用途,是将前述的山苏萃取物作为一活性成分,应用于治疗或预防男性摄护腺疾病的药物组成分或保健食品的开发。
为达到前述发明目的,本发明所运用的技术手段及借助该技术手段所能达到的功效包含有:
一种山苏萃取物,包含焦脱镁叶绿酸-a甲酯、脱镁叶绿酸-a甲酯、1-亚麻油酸-3-亚麻酸-甘油酯、1-亚麻酸-2,3-双棕梠酸-甘油酯,以及1,3-双棕梠酸-甘油酯。
本发明的山苏萃取物,其中,该山苏萃取物借助一包含下列步骤的方法所制得:一溶剂萃取步骤,是使用一萃取溶剂,对一山苏样品进行萃取处理,使该山苏样品与该萃取溶剂的重量体积比(w/v,g/ml)为50至60mg/mL,并回收得一萃取物;以及一管柱层析步骤,利用水和乙醇作为流动相,进行该萃取物的层析分离处理,以获得数个区分物,洗涤先后而编号为区段a至区段i,撷选该区段c经减压浓缩及冷冻干燥后,即得该山苏萃取物。
本发明的山苏萃取物,其中,该管柱层析步骤为撷选该区段b经减压浓缩及冷冻干燥后,即得该山苏萃取物。
本发明的山苏萃取物,其中,该管柱层析步骤为撷选该区段d经减压浓缩及冷冻干燥后,即得该山苏萃取物。
本发明的山苏萃取物,其中,该溶剂萃取步骤的萃取溶剂为95%的乙醇。
本发明的山苏萃取物,其中,该溶剂萃取步骤的萃取溶剂为水。
本发明的山苏萃取物,其中,该方法更包含一采选步骤,即选摘整株山苏花含根为该山苏样品。
以及,一种山苏萃取物的用途,是将该山苏萃取物作为一活性物质,应用于制备用以治疗摄护腺疾病的药物组成物。
本发明的山苏萃取物的用途,是将该山苏萃取物作为一活性物质,应用于制备用以治疗摄护腺疾病的保健食品。
本发明的山苏萃取物的用途,该摄护腺疾病包含摄护腺肥大、摄护腺癌及摄护腺炎。
本发明的山苏萃取物的用途,其中,该山苏萃取物是以口服方式投予一生物个体。
本发明的山苏萃取物的用途,其中,该山苏萃取物的投予量为每天每单位体重(kg)的该生物个体投予10至50毫克,较佳为每天每单位体重(kg)的该生物个体投予50毫克。
本发明的山苏萃取物具有抑制雄性素的天然活性成分,因此,可有效改善由荷尔蒙受体失调所引发的疾病,特别是男性摄护腺相关的疾病,且不致引发副作用。该山苏萃取物可通过工业化或通过超临界萃取步骤而制得,而应用于治疗或预防男性摄护腺疾病的药物组成份或保健食品的开发,以改善男性摄护腺的问题,为本发明的功效。
附图说明
图1为本发明的焦脱镁叶绿酸-a甲酯的化学结构图。
图2为本发明的脱镁叶绿酸-a甲酯的化学结构图。
图3为本发明的1-亚麻油酸-3-亚麻酸-甘油酯的化学结构图。
图4为本发明的1-亚麻酸-2,3-双棕梠酸-甘油酯的化学结构图。
图5为本发明的1,3-双棕梠酸-甘油酯的化学结构图。
图6为本发明山苏萃取物的萃取方法流程图。
图7为本发明山苏萃取物的冲提区段图。
图8为本发明山苏萃取物的又一萃取方法流程图。
图9为本发明山苏萃取物对于雄性素的抑制效果示意图。
图10为本发明山苏萃取物对于雄性素的抑制效果示意图。
图11为本发明山苏萃取物对于摄护腺基层肌成纤维细胞的抑制作用示意图。
图12为本发明山苏萃取物对于摄护腺癌细胞株的抑制作用示意图。
图13为本发明山苏萃取物的剂量与抑制摄护腺癌细胞能力的关系图。
图14a为本发明山苏萃取物降低小鼠摄护腺指数示意图。
图14b为本发明山苏萃取物提升小鼠排尿量示意图。
图14c为本发明山苏萃取物降低小鼠饮水量示意图。
图15为本发明山苏萃取物抑制摄护腺肥大的病理特征示意图。
(本发明)
S1 溶剂萃取步骤
S2 管柱层析步骤
S3 采选步骤。
具体实施方式
为让本发明的上述及其它目的、特征及优点能更明显易懂,下文特举本发明的较佳实施例,并配合附图,作详细说明如下:
请参照图1至图5所示,本发明的山苏萃取物,包含该图1所示的焦脱镁叶绿酸-a甲酯(pyropheophorbide a methyl ester;C34H36N4O3,占该山苏萃取物总重量的5%)、该图2所示的脱镁叶绿酸-a甲酯(pheophorbide a methyl ester;C35H36N4O5,占该山苏萃取物总重量的 3.4%)、该图3所示的1-亚麻油酸-3-亚麻酸-甘油酯(1-linleoyl-3-linolenoyl-glycerol;C39H66O5,占该山苏萃取物总重量的2.9%)、该图4所示的1-亚麻酸-2,3-双棕梠酸-甘油酯(1-linleoyl-2,3-dipalmitoyl-rac-glycerol;C53H98O6,占该山苏萃取物总重量的5.4%),以及该图5所示的1,3-双棕梠酸-甘油酯(1,3-dipalmitoyl-sn-glycerol;C35H68O5,占该山苏萃取物总重量的7.3%)。该山苏萃取物可有效抑制雄性素的分泌及抗发炎,从而改善男性摄护腺疾病。
请参照图6所示,本发明的山苏萃取物是通过包含下列步骤的萃取方法所获得:溶剂萃取步骤S1以及管柱层析步骤S2。该溶剂萃取步骤S1,是使用95%的乙醇或水作为萃取溶剂,对一山苏样品进行萃取处理,而得一萃取物,其中该山苏样品与该萃取溶剂的重量体积比(w/v,g/ml)为50至60mg/mL。
该管柱层析步骤S2,是以硅胶管柱为固定相,水和乙醇作为流动相,进行该萃取物的层析分离处理,以获得数个区分物,该区分物依洗涤先后而编号为区段a至区段i,撷选区段b、c、d或其组合,经减压浓缩及冷冻干燥处理后,即得本发明的山苏萃取物。
更详言之,该管柱层析步骤S2是利用自动化中型管柱液相层析仪(Flash LC)进行该萃取物的层析分离处理,较佳是选用C18的蛋白纯化反应相柱(reverse phase column)作为固定相,以提升萃取率,再依序利用不同比例的流动相进行冲提,分段收集冲提液而得数个区分物,如该图7所示。该数个区分物依其冲提的先后而分别为区段a至i,撷取区段b、c、d或其组合物经减压浓缩及冷冻干燥后,即得本发明的山苏萃取物。其中,本发明的流动相是由乙醇及水混合成线性梯度,该乙醇及水的混合是由70%乙醇提升至100%乙醇(V/V)。
请参照图8所示,本发明的山苏萃取物更佳是于该溶剂萃取步骤S1之前,先操作一采选步骤S3,即选摘整株山苏花含根为该山苏样品,以获得活性成分较高的山苏萃取物,该山苏样品可选自于山苏花(Asplenium antiquum Makino;又称鸟巢蕨、巢蕨)或台湾山苏花(Asplenium nidus Linn;又称台湾巢蕨、台湾鸟巢蕨),较佳为采用台湾山苏花(Asplenium nidus Linn)的整株含根,该台湾山苏花在中国台湾的分布及栽种面积较广,可周年生产,较利于本发明山苏萃取物的制备及原料取得,再者,台湾山苏花多为无农业污染的清洁蔬菜,少病虫害或农药污染等问题,可提升本发明山苏萃取物的安全性。
本发明的山苏萃取物经科学验证而证实具有抗发炎及抑制雄性素的有效成分,包含焦脱镁叶绿酸-a甲酯、脱镁叶绿酸-a甲酯、1-亚麻油酸-3-亚麻酸-甘油酯、1-亚麻酸-2,3-双棕梠酸-甘油酯及1,3-双棕梠酸-甘油酯,因此,可调控于荷尔蒙受体发挥预期功能,进而改善由荷尔蒙受体失调所引发的疾病,如男性摄护腺疾病(包含良性摄护腺肥大、摄护腺癌及摄护腺炎)、男女尿失禁、男女雄性秃以及女性更年期障碍等。
又,该山苏萃取物可便于萃取、分离,而具有应用于新药开发的潜力。即将该山苏萃取物作为一活性成分,应用于治疗或预防前述疾病的药物组成分或保健食品的开发,特别是用以改善男性摄护腺疾病的药物组成分或保健食品,进而改善国人健康问题。本发明的山苏萃取物可以单独使用于相关药物的组成分或保健食品的开发,或者搭配医药学上可接受的载剂、赋形剂、盐类或其它营养成分等而组合一复合物。此外,该山苏萃取物更可以通过进一步加工而制备成任何适用于口服的剂型,如锭剂、胶囊、粉剂、丸剂、液剂或发酵物等,或者与其它食品或饮料组合以制成更适于取用的食品样态。
为证实本发明的山苏萃取物确实具有抑制雄性素的功能,特操作如 下方所示的试验,然其应用并不以此为限:
本试验是按前述的萃取方法,依序操作该采选步骤S3、该溶剂萃取步骤S1及该管柱层析步骤S2,利用95%酒精作为萃取溶剂进行山苏样品的萃取处理,再由该自动化中型管柱液相层析仪,以C18的蛋白纯化反应相柱进行分离所获得的九个区段(a至i)。
请参照图9及图10所示,该九个区段分别与摄护腺癌细胞22Rv/103E单独培养,或者与该摄护腺癌细胞22Rv/103E及摄护腺基层成肌纤维母细胞(stromal myofibroblast)-WPMY-1共同培养,再分别测定该九个区段对于雄性素的抑制活性。
其结果显示,区段b、c及d,在与摄护腺癌细胞22Rv/103E单独培养(如图9所示),或者与该摄护腺癌细胞22Rv/103E103E及摄护腺基层成肌纤维母细胞(stromal myofibroblast)-WPMY-1共同培养(如图10所示)的情况下,均对该雄性素具有明显的抑制效果,且该抑制效果与该区段b、c及d的剂量成正比。
请参照图11及图12所示,为该九个区段对该摄护腺基层成肌纤维母细胞-WPMY-1及摄护腺癌细胞株-LNCaP生长的影响。该九个区段分别与该摄护腺基层肌成纤维细胞-WPMY-1及该摄护腺癌细胞株-LNCaP细胞共同培养48小时,利用SRB染剂对培养后的细胞进行染色,再分别测定其细胞活性。其结果显示,该区段b、c及d确实可抑制该摄护腺基层成肌纤维母细胞-WPMY-1及该摄护腺癌细胞株-LNCaP的生长,且该抑制作用与该区段b、c及d的剂量成正本。
请参照图13所示,为该区段b、c及d的组合物对该摄护腺基层成肌纤维母细胞-WPMY-1(1)及该摄护腺癌细胞株-LNCaP(2)生长的影响。利用细胞群落生长分析法(colony formation assay)测定该组合物在不同剂量下,对该摄护腺基层成肌纤维母细胞-WPMY-1及该摄护腺癌细 胞株-LNCaP的抑制能力,其结果显示,该组合物的剂量与该组合物对摄护腺癌细胞生长的抑制能力为成正向关系,并且,较高剂量(如100μg/mL)的该组合物更会造成摄护腺癌细胞死亡。该组合物对于该摄护腺基层肌成纤维细胞-WPMY-1及该摄护腺癌细胞株-LNCaP的半抑制剂量(IC50值)分别为11.63μg/mL及13.19μg/mL。
此外,为证实本发明的山苏萃取物可以有效改善小鼠摄护腺肥大症状,遂进行以下试验,惟其应用范畴并不以此为限:
本试验选用C57BL/6Jnar1雄性小鼠(购自国家动物中心),该小鼠为饲养于中研院农业生物科技研究中心SPF动物房,饲养环境为22℃,光照时间与黑暗时间各为12小时。
本试验是以皮下注射脱氧肾上腺素(phenylephrine,简称PE,施用量为每单位体重公斤施予15毫克脱氧肾上腺素),以活化α1-肾上腺素受体,进而诱导小鼠产生摄护腺肥大现象(其诱导期间为每周注射五次脱氧肾上腺素,且连续注射五周);并且同时口服喂食不同剂量的本发明的山苏萃取物,观察受试小鼠的摄护腺指数(prostate index,简称PI,即摄护腺内侧部位湿重/小鼠体重)、小鼠排尿状况及饮水状况,分别纪录于图14a、图14b及图14c;并且于牺牲小鼠后,取其摄护腺进行切片染色,其结果如图15所示。
请参照图14a所示,相较于控制组,施予脱氧肾上腺素的小鼠(PE组)的摄护腺指数显着上升,表示该小鼠产生摄护腺内侧部位肿大的现象,该小鼠同时有排尿减少,以及饮水量减少等现象;而同时喂食山苏酒萃物(ANE组,未经管柱层析步骤,剂量为每天每单位体重公斤小鼠喂食100毫克)或本发明的山苏萃取物(Lo组,剂量为每天每单位体重公斤小鼠喂食10毫克;或Hi组,每天每单位体重公斤小鼠喂食50毫克)均可以有效减缓小鼠摄护腺指数的变化,也可以缓解小鼠排 尿减少及饮水量增加的问题(p<0.01,请参照图14b及图14c),其中,以每天每单位体重公斤小鼠喂食50毫克的本发明山苏萃取物(Hi组)的效果最佳。是以,本发明的山苏萃取物可以有效缓解生物体的摄护腺肥大现象。
接着,将上述五组小鼠(控制组、PE组、ANE组、Lo组及Hi组)牺牲后,取摄护腺进行切片,并进行苏木精-伊红染色(Hematoxylin-eosin staining,简称H&E staining),相较于控制组,施予脱氧肾上腺素的小鼠(PE组)可以观察到上皮细胞增生现象,使PE组小鼠泌尿道腔室面积显着减小,表示PE组小鼠的分泌腺体受到压缩(如图15的ii所示),而同时喂食山苏酒萃物(ANE组)或本发明的山苏萃取物(Lo组或Hi组)均可以有效减缓上述上皮细胞增生现象(如图15的iii、iv及v所示)。
此外,请参照图15的vii至ix所示,本试验更以梅生氏三色染色法(Masson’s trichrome staining),观察各组切片的胶原蛋白的分布状况,控制组的胶原蛋白分布范围较广(如图15的vii所示,黄色箭头所指蓝色区域即为胶原蛋白),更可以于摄护腺基质观察到网状纤维的分布(如橘色箭头所指区域),而PE组小鼠的切片则较难观察到此一现象(如图15的viii所示),而同时喂食本发明的山苏萃取物(Hi组)则可以有效回复上述现象(如图15的ix所示)。
是以,本发明的山苏萃取物可以有效舒缓脱氧肾上腺素造成的摄护腺肥大现象,增加小鼠排尿量,并且也可以减缓摄护腺肥大造成的病理特征,可以作为改善男性摄护腺问题的活性物质,应用于制备用以治疗摄护腺疾病的药物组成分或保健食品。
本发明山苏萃取物的实际应用如下方实施例所示,但其应用并不以此例为限:
本实施例按前述的萃取方法,依序操作该采选步骤S3、溶剂萃取步骤S1及管柱层析步骤S2,以制得该山苏萃取物。请参照第1表所示,该山苏萃取物提供给88名不同年龄层的摄护腺疾病患者服用,平均二至四星期内可改善患者的排尿状况,同时未引发任何副作用,有助于摄护腺肥大、摄护腺炎或摄护腺癌的治疗。
摄护腺肥大的病征之一就是频尿与夜尿,本发明的山苏萃取物可将频尿与夜尿的频率降至与正常人无异。将前述88名患者试验前后的情况,依据国际摄护腺症状评分表(International Prostate Symptom Score,I-PSS)进行分析,在2至4周内进步分数平均至少达约20至30分,远高于市售现用营养补充产品的平均2-3分,以及药物治疗的平均14分。
第1表:88名摄护腺疾病患的使用结果
a麻醉手术后,尿道沾黏并非摄护腺肥大或摄护腺癌的术后专有病征。严重者甚至延宕数个月后遗症之久,一般多用α-受体阻断剂进行缓和。然,本发明的山苏萃取物具有速效及作用持久等功效,可用以改善尿道沾黏。
承上所述,本发明的山苏萃取物具有抗发炎及抑制雄性素的天然活性成分,因此,可有效改善由荷尔蒙受体失调所引发的疾病,特别是男性摄护腺相关的疾病,且不致引发副作用。该山苏萃取物可通过工业化或通过超临界萃取步骤而制得,而应用于治疗或预防男性摄护腺疾病的药物组成份或保健食品的开发,以改善男性摄护腺的问题,为本发明的功效。
Claims (9)
1.一种山苏萃取物,包含: 焦脱镁叶绿酸-a甲酯; 脱镁叶绿酸-a甲酯; 1-亚麻油酸-3-亚麻酸-甘油酯; 1-亚麻酸-2,3-双棕梠酸-甘油酯;以及 1,3-双棕梠酸-甘油酯。
2.如权利要求1所述的山苏萃取物,其特征在于,该山苏萃取物通过一包含下列步骤的方法所制得:
一个溶剂萃取步骤,即使用一个萃取溶剂,对一个山苏样品进行萃取处理,使该山苏样品与该萃取溶剂的重量体积比(w/v, g/ml)为50至60 mg/ml,并回收得一个萃取物;以及
一个管柱层析步骤,利用乙醇和水混合成线性梯度作为流动相,进行该萃取物的层析分离处理,其中乙醇及水的混合是由70%乙醇提升至100%乙醇(V/V),以获得数个区分物,依冲提顺序而编号为区段a至i,撷选该区段c经减压浓缩及冷冻干燥后,即得该山苏萃取物。
3.如权利要求2所述的山苏萃取物,其特征在于,该管柱层析步骤是撷选该区段b经减压浓缩及冷冻干燥后,即得该山苏萃取物。
4.如权利要求2所述的山苏萃取物,其特征在于,该管柱层析步骤是撷选该区段d经减压浓缩及冷冻干燥后,即得该山苏萃取物。
5.如权利要求2所述的山苏萃取物,其特征在于,该溶剂萃取步骤的萃取溶剂是95%的乙醇。
6.如权利要求2所述的山苏萃取物,其特征在于,该溶剂萃取步骤的萃取溶剂是水。
7.如权利要求2所述的山苏萃取物,其特征在于,该方法更包含一采选步骤,是选整株山苏花含根为该山苏样品。
8.如权利要求7所述的山苏萃取物,其特征在于,该采选步骤,是选用山苏花(Asplenium antiquum Makino)的整株植物含根为该山苏样品。
9.如权利要求7所述的山苏萃取物,其特征在于,该采选步骤,是选用台湾山苏花(Asplenium nidus Linn)的整株植物含根为该山苏样品。
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