CN102885315A - 一种制造用于治疗或预防摄护腺肥大或肾功能疾病的类茄红素组合物 - Google Patents
一种制造用于治疗或预防摄护腺肥大或肾功能疾病的类茄红素组合物 Download PDFInfo
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Abstract
本发明涉及一种用于治疗或预防摄护腺肥大的类茄红素组合物，所述类茄红素组合物包含有效剂量的类茄红素萃取物。本发明也涉及一种用于治疗或预防肾功能疾病的类茄红素组合物，所述类茄红素组合物包含有效剂量的类茄红素萃取物。
Description
技术领域
本发明涉及一种于制造治疗或预防摄护腺肥大或肾功能疾病的类茄红素组合物。
背景技术
西红柿红素(Lycopene)，简称茄红素，是类胡萝卜素的一种。作为一种天然色素存在于自然界中，呈红色，因最早发现于西红柿中而得名。在西红柿由绿转红时其含量增加，西红柿愈红，茄红素的含量愈多。一般营养素易受热而破坏，但是茄红素在食物中相当稳定，不会因烹调而流失，反而是经过加热加工后，人体吸收率会更好，因为茄红素属于脂溶性，在和油脂一起烹调之下成份会溶入脂肪中，较易被人体所吸收而增加利用率。
摄护腺又称前列腺，影响摄护腺肥大的因素有很多，摄护腺会随着年龄的增加而增大。前列腺过大会压迫尿道，影响正常排尿并危及肾脏。肾脏将受压迫力损害及被尿里的细菌感染。当肾脏充斥着这些细菌，很可能发生肾脏炎。前列腺炎也可能引起膀胱炎。摄护腺肥大是一种扰人的病变，40岁以上的男性几乎都有这项困扰，80岁以上饱受摄护腺肥大之苦者，更高达九成以上，其症状包括有频尿、排尿困难、尿流减弱和排尿不净等。传统治疗摄护腺肥大的药物，多是采用甲型交感神经阻断剂，不过易出现副作用，约有一至二成的患者，在服用后会出现血压低、头晕等症状，对于上了年纪的老人而言，一不小心还会导致跌倒等意外；因此，没有副作用的茄红素，可说是提供一个极佳的治疗方式。
尽管茄红素对人体具有上述之效益，而目前由西红柿中所取得的茄红素的量及速度仍属有限，如台湾发明专利第100125866号申请专利案，其揭示一种突变可产生类茄红素的菌种，有效的快速萃取出大量的类茄红素，该类茄红素产生抗发炎、抗氧化、美白、抑制胶原蛋白分解或促进胶原蛋白增生之多种功效。因此，茄红素可用于治疗摄护腺肥大或肾功能具有相当大的潜力，然而却未有研究发现利用突变的菌种产生类茄红素治疗或预防摄护腺肥大或肾功能疾病，而市面上对于治疗或预防摄护腺肥大或肾功能的类茄红素产品，亦不多见。因此，本发明将有助于护腺肥大或肾功能相关疾病的治疗或预防。
发明内容
本发明提供一种新的类茄红素(Lycogen)组合物，所述组合物中的类茄红素是由光合菌所萃取而来，所述光合菌为球型红杆菌(Rhodobacter sphaeroides)，由野生或培养的原生光合菌经紫外线照射使其变性而来，已保藏于布达佩斯条约的德国微生物与细胞搜集中心(German Collection of Microorganisms and Cell Cultures)，地址位于德国不伦瑞克(DSMA GmbH，Inhoffen街7B，38124 Braunschweig，Germany)，保藏编号为WL-APD911，保藏日期2011年8月1日；同时也保藏于台湾新竹食品工业发展研究所，保藏编号为BCRC910406，光合菌的CrtC酵素DNA序列如SEQ ID NO：1所示，光合菌CrtC酵素氨基酸序列如SEQ ID NO：2所示。所述类茄红素是利用有机溶剂萃取，所述有机溶剂可为甲醇、乙醇、丙酮、正丁醇、正己烷、二氯甲烷或乙酸乙酯等。
本发明利用一种类茄红素萃取物，其所含活性成分选自ζ-胡萝卜素(ζ-carotene)、链孢红素(neurosporene)、球形烯醇(spheroidenone)和/或甲氧基链孢红素(methoxyneurosporene)。在本发明的一具体实施例中，所述活性成分萃取自突变的光合菌。在另一具体实施例中，所述突变的光合菌是保藏编号为WL-APD911的球形红杆菌(Rhodobacter sphaeroides)。在另一具体实施例中，所述突变的光合菌之CrtC酵素(hydroxyneurosporene dehydrogenase)的DNA序列如SEQ ID NO：1所示；而野生型光合菌的CrtC酵素(hydroxyneurosporene dehydrogenase)的DNA序列如SEQ ID NO：3所示。在另一具体实施例中，所述突变之光合菌的CrtC酵素(hydroxyneurosporene dehydrogenase)之氨基酸序列如SEQ ID NO：2所示；而野生型光合菌之CrtC酵素的氨基酸序列如SEQ ID NO：4所示。
本发明提供一种用于制造治疗或预防摄护腺肥大的类茄红素组合物，所述的类茄红素组合物包含有效剂量的类茄红素萃取物。其中所述的类茄红素组合物的有效剂量为25～150毫克/公斤。
根据本发明之具体实施例，所述的类茄红素组合物所含活性成分选自ζ-胡萝卜素(ζ-carotene)、链孢红素(neurosporene)、球形烯醇(spheroidenone)及/或甲氧基链孢红素(methoxyneurosporene)。在本发明的一具体实施例中，所述活性成分萃取自突变的光合菌。在另一具体实施例中，所述突变的光合菌是保藏编号为WL-APD911的球形红杆菌(Rhodobacter sphaeroides)。在尚有另一具体实施例中，所述突变之光合菌的CrtC酵素(hydroxyneurosporene dehydrogenase)的DNA序列如SEQ ID NO：1所示。
本发明另提供一种用于制造治疗或预防肾功能疾病的类茄红素组合物，所述的类茄红素组合物包含有效剂量的类茄红素萃取物。其中所述的类茄红素组合物的有效剂量为25～150毫克/公斤。
根据本发明之具体实施例中，所述的类茄红素组合物所含活性成分选自ζ-胡萝卜素(ζ-carotene)、链孢红素(neurosporene)、球形烯醇(spheroidenone)及/或甲氧基链孢红素(methoxyneurosporene)。在本发明的一具体实施例中，所述活性成分萃取自突变的光合菌。在另一具体实施例中，所述突变的光合菌是保藏编号为WL-APD911的球形红杆菌(Rhodobacter sphaeroides)。在另一具体实施例中，所述突变的光合菌的CrtC酵素(hydroxyneurosporene dehydrogenase)的DNA序列如SEQ IDNO：1所示。
根据本发明所述组合物的具体实施例，所述组合物可用作于食物增补剂、动物饲料、人类食物产品或医药品或化妆品组合物。
根据本发明所述组合物的另一具体实施例中，其可被制成适合用于非经肠道、局部或口服投药剂型。优选地，本发明的组合物可被制成溶液、乳剂、粉末、锭剂、胶囊或供用于口服投药的其它形式。
本发明的具体实施例中，所述组合物可作为包括，但不限于：膳食补充剂、食品、饮料、乳品或上述形式的组合；其中乳品可为流体乳品(如：牛奶与浓缩牛奶)、发酵乳品(酸奶、酸乳、冷冻优格、乳酸菌发酵饮料)、奶粉、冰淇淋、奶酪、干酪、豆奶与发酵豆奶等。饮料可为蔬果汁、果汁及运动饮料等。食品可为甜点、糖果、婴儿食品、动物饲料等。
以下列实施方式予以详细说明本发明，但并不意味本发明仅局限于此等实施方式所揭示的内容。
关于本发明涉及的菌种说明：
球型红杆菌(Rhodobacter sphaeroides)，由野生或培养的原生光合菌经紫外线照射使其变性而来，已保藏于布达佩斯条约的德国微生物与细胞搜集中心(German Collection ofMicroorganisms and Cell Cultures)，地址位于德国不伦瑞克(DSMA GmbH，Inhoffen街7B，38124 Braunschweig，Germany)，保藏编号为WL-APD911，保藏日期2011年8月1日。
附图说明
图1为根据本发明的一具体实施例，产生茄红菌之流程图。
图2为原生光合菌之代谢途径。
图3(A-B)为根据本发明的一具体实施例，本发明之茄红菌与野生光合菌之CrtC酵素DNA序列比较图。
图4为根据本发明的一具体实施例，本发明之茄红菌与野生光合菌之CrtC酵素氨基酸序列比较图。
图5为根据本发明的一具体实施例之高效液相层析法(HPLC)分析图；(A)至(F)为以不同有机溶剂萃取本发明茄红菌的萃取物；图5(G)为茄红素标准品；
图5(H)为本发明茄红菌的乙醇丙酮类茄红素萃取物。
图6显示(A)茄红素；(B)ζ-胡萝卜素；(C)链孢红素；(D)球形烯醇及(E)甲氧基链孢红素之分子结构。
图7显示(A)ζ-胡萝卜素；(B)链孢红素；(C)球形烯醇及(D)甲氧基链孢红素的预测NMR光谱；该些经标记的波峰代表该些可与类茄红素萃取物之COSY NMR光谱所示的波峰相对应的波峰。
图8显示类茄红素萃取物之COSY NMR光谱；标记为「a」的波峰代表可能的杂质；标记为「b」的波峰代表球形烯醇及甲氧基链孢红素；标记为「c」的波峰代表ζ-胡萝卜素及链孢红素；且标记为「d」的波峰代表球形烯醇。
图9显示根据本发明的一具体实施例的ζ-胡萝卜素纯化物(A)及链孢红素纯化物(B)1H NMR光谱；箭头所指处表示ζ-胡萝卜素纯化物及链孢红素纯化物皆会在5.1ppm处产生一波峰。
图10显示根据本发明的一具体实施例的类茄红素萃取物1H NMR光谱(A)及其部分放大图(B及C)。
图11显示根据本发明的一具体实施例的用于定量分析的类茄红素萃取物1H NMR光谱。
图12为根据本发明的一具体实施例，进行效用测试实验之实验流程图。
图13为试验实验中经过本发明组合物处理的BPH老鼠的摄护腺指数。A：空白组；B：载体(南瓜子油)；C：类茄红素。
图14为BPH老鼠经由不同剂量的组合物处理后整体体重的改变。
图15为BPH老鼠的摄护腺指数经由注射不同剂量的组合物的改变情形。患有BPH的实验老鼠经由喂食不同剂量的组合物且持续28天。图15(A)为摄护腺指数的结果，其计算方式为摄护腺的总重占整体体重的比率。每个数据接取自于来自同一组别的8只老鼠的结果平均加减标准偏差，且经邓肯多变域检定发现有统计上的意义(P＜0.05)。1：空白对照组；2：BPH对照组；3：50毫克/公斤(低浓度)；4：50毫克/公斤(中浓度)；5：150毫克/公斤(高浓度)。图15(B)为摄护腺抑制效果，将空白组的摄护腺指数设定为0％，BPH对照组则为100％，而在观察喂食不同剂量的组合物的老鼠其抑制摄护腺肿大的情形。A：50毫克/公斤(低浓度)；B：50毫克/公斤(中浓度)；C：150毫克/公斤(高浓度)。
图16为H&E染色法，其显示喂食不同浓度Locogen(LyG)的老鼠摄护腺组织情况。
图17显示随着Lycogen的增加(50～150mg/kg)对腹侧及背侧的摄护腺肥大改善效果有递增的趋势。
图18为利用腹侧前列腺与背侧前列腺的平均值表示整体摄护腺肥大的改善效果。
图19为不同剂量的组合物对于血中尿素氮的结果。A：空白对照组；B：25毫克/公斤(低剂量)；C：100毫克/公斤(高剂量)。
图20为不同剂量的组合物对于血清肌酸酐的结果。A：空白对照组；B：25毫克/公斤(低剂量)；C：100毫克/公斤(高剂量)。
图21为不同剂量的组合物对于血中尿酸的结果。A：空白对照组；B：25毫克/公斤(低剂量)；C：100毫克/公斤(高剂量)。
具体实施方式
首先，请参阅图1的流程图，本发明提供一种生成类茄红素的微生物，其主要是将野生或培养的原生光合菌，利用紫外线分别予以照射5、10、20、30、40秒后，使其产生变性，而当光合菌经由紫外线的照射而变成含有丰富茄红素之突变光合菌(命名为茄红菌)时，将该突变的光合菌(茄红菌)筛选取出，并予以培养繁殖，再以有机溶剂予以萃取出茄红素及类茄红素。
请参阅图2，原生光合菌生成茄红素的代谢途径中，由甲基辛烯二甲基辛烯焦磷酸(Geranylgeranyl diphosphate)起始，其经由CrtB酵素作用转变成八氢西红柿红菌(Phytoene)，接着藉由CrtI酵素作用转变成六氢西红柿红菌(Phytofluene)，该六氢西红柿红菌则接着经由CrtI酵素变化成ζ-胡萝卜素(ζ-carotene)，而ζ-胡萝卜素会再经由CrtI酵素作用变化成链孢红素(Neurosporene)；接着，链孢红素经由CrtI酵素作用转变成茄红素(Lycopene)。该茄红素生成后被CrtC酵素转化成紫菌红素(Rhodopin)，该紫菌红素经由CrtD酵素作用转化成3，4-二脱氢紫菌红素(3，4-didehydrorhodopin)，该3，4-二脱氢紫菌红素会经CrtF酵素变化成脱水紫菌红醇(Anhydro-rhodovibrin)，该脱水紫菌红醇会经CrtC酵素变化成紫菌红醇(Rhodovibrin)；接着该紫菌红醇再经由CrtD酵素变化成单脱甲基螺菌黄素(Monodemethyl-spirilloxanthin)，该单脱甲基螺菌黄素最后由CrtF酵素变化成螺菌黄素(Spirilloxanthin)。
上述的代谢过程中，该链孢红素(Neurosporene)还有可能会由CrtC酵素变化成羟基链孢红素(hydroxyneurosporene)，该羟基链孢红素再经由CrtF酵素转变成甲氧基链孢红素(Methoxyneurosporene)，然后该甲氧基链孢红素由CrtD酵素变化成球形烯(Spheroidene)；其中，该球形烯可能由CrtC酵素作用生成羟基球形烯羟(Hydroxyspheroidene)，或由CrtA酵素作用生成球形烯醇(Spheroidenone)。
其中，本发明的原生光合菌经由紫外线之照射后，其颜色会产生变化，当其变化成鲜红色的突变光合菌(茄红菌)时，将菌液筛选出至培养皿中进行培养繁殖，经由培养繁殖的茄红菌的CrtC酵素的DNA序列如SEQ ID NO：1所示，CrtC酵素的氨基酸序列如SEQ ID NO：2所示。比较该突变的光合菌(茄红菌)的CrtC酵素的DNA序列与野生(原生)的光合菌的CrtC酵素的DNA序列时，如图3所示，茄红菌于第76、88、147、214、274、282、310、496、694、707、708及715个序列会产生变异；藉此，可确定于CrtC酵素DNA序列的第76、88、147、214、274、282、310、496、694、707、708及715个核苷酸具有变异的突变菌株即为茄红菌。此外，该茄红菌的CrtC酵素胺基酸序列则如SEQ ID NO：2所示。该茄红菌之CrtC酵素胺基酸序列与原生之光合菌比较时，如图4所示，可于第45、46、47、190及239个氨基酸位置发现变异；藉此，可确定于CrtC酵素氨基酸序列第45、46、47、190及239个氨基酸位置具有变异的突变菌株即为茄红菌。接着将该等茄红菌筛选至培养皿中予以培养繁殖，再利用有机溶剂萃取培养所得之茄红菌，而当采用该有机溶剂时，可利用脂溶性有机溶剂。
本发明通过采用物理性的紫外线照射，将野生或培养的原生光合菌以不同时间照射处理，使该光合菌产生突变菌株，并筛选出颜色呈鲜红色的茄红菌，将该茄红菌筛选至培养皿中予以培养繁殖，再以有机溶剂进行萃取，藉此，无须由西红柿中萃取，即可达到快速的产生大量茄红素与类茄红素之功效。
类茄红素之提取与分离
1.甲醇和丙酮萃取物
菌体以甲醇于室温下震荡后离心8000rpm，10分钟，取上清液后过滤，重复此抽取步骤三次，合并三次滤液，并减压浓缩得到甲醇萃取物(Rs-M)。经甲醇萃取后的菌体残余物再以丙酮萃取，重复以上步骤，减压浓缩后得到丙酮萃取物(Rs-M/A)。
2.乙醇和丙酮萃取物
同上步骤1，分别得乙醇萃取物(Rs-E)和丙酮萃取物(Rs-E/A)。
3.丙酮萃取物
同上步骤1，得丙酮萃取物(Rs-A)
4.萃取物的分离
萃取物加入蒸馏水使之成悬浮液，再以正己烷萃取三次，萃取液合并减压浓缩至干，得到正己烷层(Fr，Rs-H)，同上法再将水层依序增加极性以二氯甲烷、乙酸乙酯、水饱和的正丁醇萃取之，分别得二氯甲烷层(Fr，Rs-C)，乙酸乙酯层(Fr，Rs-E)，正丁醇层(Fr，Rs-B)，余者为水层(Fr，Rs-W)。
HPLC分析法
菌色素含量分析，取菌体萃取粉末以适量DMSO回溶，全程避光。HPLC条件如下：采用C18管柱，波长470nm，移动相为甲醇/异丙醇(95/5，v/v)，流速1毫升/分钟，茄红素标准品滞留时间为50±2分钟。
甲醇萃取物(Rs-M)颜色为绿色，乙醇萃取物(Rs-E)颜色为茶褐色，丙酮萃取物(Rs-A)颜色为深红色。以茄红素标准品为比较，经HPLC分析发现，Rs-A的茄红素含量最多(RT＝50分钟)，Rs-E微量，而Rs-M则完全没有茄红素；推测使用甲醇和乙醇萃取的残余菌体仍含有茄红素，故将甲醇与乙醇处理后的菌体残余物分别使用丙酮再进行萃取，结果分别得到甲醇残余物的丙酮萃取物(Rs-M/A)和乙醇残余物的丙酮萃取物(Rs-E/A)，二者颜色均为红色。Rs-E/A和Rs-M/A亦进行HPLC分析，如图5(A)至(F)所示，箭头所指处为茄红素吸收峰。
菌体的乙醇丙酮萃取物溶于二甲亚砜(DMSO)后经HPLC分析，并与茄红素标准品(L-9879，Sigma)比对其滞留时间(图5(G))，发现菌体萃取物之HPLC分析图谱显示位于标准品相同滞留时间(50分钟)的波峰极小，最大的波峰出现在滞留时间约21分钟左右(约21.7～24.19分钟，称为未知波峰)(图5(H))。表示菌体茄红素在波长470nm下吸光的主要成分以21分钟出现之物质为主，可能是菌体本身合成茄红素过程之中间代谢产物，因此称之为「类茄红素」。以波峰面积来计算的话，其中的茄红素面积仅为类茄红素的3.3％左右。
NMR分析
以下实验中所使用的样本系利用乙醇丙酮萃取本发明的光合菌所得的类茄红素萃取物。
A：样本制备
秤取约15mg样本并将其溶解于D-三氯甲烷中。将溶解完成之样品装入常规液态NMR tube，装入的体积大约在500uL左右。装入后使用parafilm(石腊膜)将管口密封，并置于冰箱中保存(～4℃)，以防止溶剂的挥发与样品的变质。
B：NMR实验
将配置好的样品放入液态500NMR(Varian公司产品，磁场强度为11.74T)。将样品进行20rps的转动(仪器内建功能)，以消除磁场不均匀性，提升光谱分辨率。呼叫仪器内部脉冲序列信息，分别进行一维1H NMR、13C NMR、DEPT NMR、COSYNMR、NOESY NMR实验。实验完成后，调整得到的光谱(基线调整、相位调整、施加窗函数)。上述这些调整主要目的在于利于后续的分析。本发明所属领域技术人员可知，这些调整不会对光谱产生严重性失真，因此被广为应用。
C：光谱分析
将NMR实验中得到的一维13C谱、DEPT谱、二维COSY谱(图8)及二维NOESY谱进行分析比较。一维13C谱展示所有13C信息。DEPT谱可以用来区分甲基、乙基及丙基碳。COSY和NOESY谱则可以用来鉴定分子结构。
将COSY谱与预测所得的光谱(图7，由NMR预测器(nmrdb.org)产生)进行比较以辨识样本中所含的各种成分。为了进行NMR光谱预测，自KEGG网站下载各化合物(请参照图6)的分子表达谱(molecular profiles)，并以NMR预测器进行数据处理及预测。利用上述光谱得到的各项信息，进行样品定性以及定量的判断。接着请参照图8，标记为「a」的波峰代表可能的杂质；标记为「b」之波峰代表球形烯醇(spheriodenone)及甲氧基链孢红素(methoxyneurosporene)；标记为「c」之波峰代表ζ-胡萝卜素(ζ-carotene)及链孢红素(neurosporene)；且标记为「d」之波峰代表球形烯醇(spheriodenone)。根据标记为「c」之波峰的低强度，相较于在3-4ppm之间的两个波峰(标记为「b」)，可得知球形烯醇(spheriodenone)及甲氧基链孢红素(methoxyneurosporene)为样本中的主要成分，且其比例约为一比一。所述球形烯醇含量多于样本的30重量百分比，而甲氧基链孢红素含量亦多于样本的30重量百分比。
ζ-胡萝卜素纯化物、链孢红素纯化物及类茄红素萃取物的1H NMR光谱如图9至11所示。以前述信息为基础，藉由将图10(A)中所示的光谱与图9(A)及(B)中所示的光谱进行比较，以进一步辨识类茄红素萃取物中的活性成分及其所占比例。图10(A)光谱中各波峰所代表的化合物被标示于图10(B)及(C)中。图10(C)虚线长方型区域内的波峰均可与ζ-胡萝卜素纯化物(图9(A))及链孢红素纯化物的1H NMR光谱中的波峰相对应(图9(B))。由图9(A)及(B)可得知，ζ-胡萝卜素纯化物及链孢红素纯化物在进行1H NMR时，均会产生一个在5.1ppm处的波峰(箭头标示)。
由一化合物在NMR光谱中波峰的强度及/或波峰下的面积可推知该化合物的含量。图9(A)及(B)中，箭头所指波峰的强度比为1∶1.273，代表ζ-胡萝卜素及链孢红素之比例为1∶1.273。图11中的波峰a代表ζ-胡萝卜素及链孢红素；波峰b代表球形烯醇；而波峰c代表甲氧基链孢红素。上述波峰a、b及c的面积比为1∶1.553∶1.604。根据上述所有实验资料，可计算得知本发明类茄红素萃取物中所含的ζ-胡萝卜素约为10.58重量百分比，链孢红素约为13.47重量百分比，球形烯醇约为37.37重量百分比，而甲氧基链孢红素约为38.58重量百分比。
恶性摄护腺肥大的动物实验
本发明购自六只正常和28只经历睪丸摘除手术之公鼠，其鼠龄均约为6-7个礼拜，体重介于180-220公克之间，购买后隔离饲养1个礼拜。饲养公鼠的环境设定为为摄氏23±1℃且为12小时日夜循环。该些公鼠皆能自行饮食及喝水；另一方面，亦准备三种浓度的试剂(表一)。本实验步骤已提交动物保护协会及生技发展中心审查且得到认可。
表一、测试实验准备
试验性实验：将具有预防效果的类茄红素组合物以10只经睪丸切除的老鼠进行前测(pretest)。睪丸切除的老鼠随机分为两组，1)载体组，2)加入LyG 50毫克/公斤。在睪丸摘除的老鼠的肌肉内注射每公斤10毫克的睾酮环戊丙酸，每礼拜注射两次且持续四个礼拜，使其患有摄护腺肥大(BPH)。其分组如表二。
表2、试验研究分组
本发明使用巴比妥(体重每一公斤使用60毫克)将老鼠安乐死，且从其股静脉取血液样本。在分析样本前，将血液凝固后使用3000g离心10分钟，以得到血清后在摄氏-20℃下保存。另外也从老鼠取下摄护腺，且将邻近的血管及结缔组织清除。经过假手术的对照组也是经由上述相同的方式取下其摄护腺。摄护腺指数的计算方式为摄护腺的总重占整体体重的比重，如式一
摄护腺指数＝(摄护腺总重/整体体重)x100％ (式一)
实验结果分析：
数据结果是使用平均±标准偏差的方式呈现。而统计分析则使用单因子变量分析，其根据邓肯多变域检定(SPSS软件包)。P值小于0.05是有统计上的意义。
效用测试实验：
A.诱导阶段：做为空白组的实验生物为睪丸并未被摘除的假手术。而睪丸被摘除的老鼠则随机分配至四组，分别为(a)BPH对照组；(b)低浓度-注射每公斤50毫克的LyG；(c)中浓度-注射每公斤100毫克的LyG；(d)高浓度-注射每公斤150毫克的LyG，与先前所述的试验性研究的研究条件相同。
B.测试阶段：所有的实验组及对照组皆使用灌食的方式喂食且持续28天，其实验细节于图12及表三。
表三、实验分组
C.评估老鼠的摄护腺指数及体重：使用巴比妥(体重每一公斤使用60毫克)将老鼠安乐死，且从其股静脉取血液样本。在分析样本前，将血液凝固后使用3000g离心10分钟，以得到血清后在摄氏-20℃下保存。另外也从老鼠取下摄护腺，且将邻近的血管及结缔组织清除。经过假手术的对照组也是经由上述相同的方式取下其摄护腺。摄护腺指数的计算方式为摄护腺的总重占整体体重的比重，如式一。
切片实验
利用上述实验的老鼠，进行切片实验，为了评估前列腺组织的型态变化，本实验结果由国家实验室动物中心(NLAC，台湾)进行评估。
一开始先收集上述实验中每只老鼠的前列腺组织，并将前列腺组织浸泡于10％甲醛缓冲液中，并进行组织石蜡包埋、组织切片，切片厚度约3-5μm并利用苏木精和伊红(Hematoxylin & Eosin)染色法染色，最后利用显微镜观察组织情况。
实验结果分析：
数据结果是使用平均±标准偏差的方式呈现。而统计分析则使用单因子变量分析，其根据邓肯多变域检定(SPSS软件包)。P值小于0.05是有统计上的意义。
实验结果：
在试验性实验中，图13显示有摄取类茄红素(50毫克/公斤)的老鼠，其摄护腺的指数有下降的趋势，然而其结果中经由t测试发现并无统计上的意义(P＞0.05)。因此在效用实验终将类茄红素的剂量提高。
在效用实验中，每礼拜两次监控试验中老鼠的体重，图14显示患有BPH的实验老鼠经由喂食不同剂量的Lcogen(LyG)且持续28天，且每礼拜两次的监控其体重变化。另一方面，结果显示喂食后的体重将高于尚未喂食时的体重，对照组及有食用组合物的老鼠的体重浮动百分比并无明显的差异。
在评估摄护腺指数上，睪固酮可以成功地诱导BPH对照组的摄护腺肿大，其增加的比例约为0.95％，相较于空白组的0.50％。所有经由Lcogen(LyG)处理的BPH老鼠皆呈现抑制摄护腺肿大的情形，且呈现剂量依赖性(图15A)。在施打低浓度、中浓度和高浓度的Lcogen(LyG)的BPH老鼠相较于BPH老鼠对照组而言，其抑制摄护腺肿大分别呈现6.8％、12.3％和13.9％(图15B)。此外，在中浓度和高浓度的Lcogen(LyG)处理的老鼠实验结果中，有统计上的显着相关(P＜0.05)。上述结果指出Lcogen(LyG)具有抑制摄护腺肿大的效果，且呈现剂量依赖性。
另一方面，图16为H&E染色法，其显示喂食不同浓度Locogen(LyG)的老鼠摄护腺组织情况，图16(A)为无摄护腺肥大的正常组织。图16(B)为睪固酮诱发的摄护腺肥大组织。图16(C)为媒介物处理的对照组；图16(D)-(F)为不同剂量的Lycogen治疗处理组。由图16(B)显示睪固酮的处理成功的诱发腺泡腔区的肥厚与上皮细胞增生(箭头指处)。图16(C)显示媒介物(oil)的处理并无明显改善效果。由图16(D)-(F)可以发现将老鼠喂食不同浓度(100～150mg/kg)的Lycogen(LyG)时即有明显改善之效果(箭头指处)。
图17显示随着Lycogen的增加(50～150mg/kg)对腹侧及背侧之摄护腺肥大改善效果有递增的趋势，显示出Lycogen确实有改善摄护腺肥大的效果。黑色：腹侧前列腺(Ventral Prostate，VP)；灰色：背侧前列腺(Dorsolateral Prostate，DP)。
图18为利用VP与DP的平均值表示整体摄护腺肥大的改善效果，图18显示Lycogen在50、100、150mg/kg处理下，分别产生约7％，12％，及25％的改善摄护腺肥大效果，显示出随着Lycogen的增加(50～150mg/kg)对摄护腺肥大改善效果有递增的趋势
由上述结果进一步证实本发明的Lycogen(LyG)确实有改善摄护腺肥大的效果。
肾功能之生化数值分析
类茄红素Lycogen对于肾功能之生化数值分析
A：血中尿素氮(BUN)分析结果
血中尿素氮主要经肾小球滤过，从小便中排出体外，当肾小球受损时滤过率降低，血中BUN升高。因此BUN是反映肾小球滤过功能的重要指针。若BUN升高时，会造成以下肾脏疾病；然而，若是严重肝脏疾患，BUN可能亦会降低。
1)如慢性肾炎，肾动脉硬化症，严重肾盂肾炎、肾结核和肾肿瘤的晚期，尤其是在肾功能衰竭尿毒症时。
2)肾前性疾病如脱水、水肿、腹水、循环功能不全等。
3)肾后性疾病如尿路结石或前列腺肿大引起的尿路梗阻。
4)体内蛋白质分解过盛时，如上消化道出血、大面积烧伤、甲状腺功能亢进等。
本发明将ICR老鼠分为三组，各组老鼠为6只，分别为对照组(control；仅为安慰剂)、低剂量组(25mg/kg；Low dose)及高剂量组(100mg/kg；High dose)，以管灌喂食之方式进行，每天于固定时间喂食一次，为期30天，以观察血中尿素氮(BUN)代谢情况。
图19的结果显示对照组、低剂量组到高剂量组随着剂量的增加血中尿素氮(BUN)的含量递减，可以说明本发明组合物(Lycogen)似乎可以增加肾脏对血中尿素氮(BUN)的代谢速率，且高剂量组更可达显着差异(表五；p＝0.049)。
B.血清肌酸酐(Creatinine)分析
由图20的结果显示随着剂量的增加，Creatinine的含量递减，可以说明本发明组合物似乎可以增加肾脏对Creatinine(Creatinine)的代谢速率，各组间并未达显着差异(表五)。
C.血中尿酸(Uric Acid)分析
由图21的结果显示随着剂量的增加，血中尿酸(Urin Acid)的含量逐渐递减，可以说明Lycogen似乎可以增加肾脏对Urin Acid的代谢速率，各组间并未达显着差异(表五)。
表五、各组间之肾功能生化统计分析表
(低剂量：25mg/kg；高剂量：100mg/kg)
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Claims (12)
1.一种用于制造治疗或预防摄护腺肥大的类茄红素组合物，其特征在于所述的类茄红素组合物包含有效剂量的类茄红素萃取物。
2.如权利要求1所述的用于制造治疗或预防摄护腺肥大的类茄红素组合物，其特征在于所述的类茄红素萃取物所含活性成分选自z-胡萝卜素（z-carotene）、链孢红素（neurosporene）、球形烯醇（spheroidenone）和/或甲氧基链孢红素（methoxyneurosporene）。
3.如权利要求1所述的用于制造治疗或预防摄护腺肥大的类茄红素组合物，其特征在于所述的类茄红素萃取物是萃取自突变的光合菌。
4.如权利要求3所述的用于制造治疗或预防摄护腺肥大的类茄红素组合物，其特征在于所述的突变的光合菌是保藏于布达佩斯条约的德国微生物与细胞搜集中心(German Collection of Microorganisms and Cell Cultures)，保藏编号为WL-APD911的球形红杆菌（Rhodobacter sphaeroides），保藏日期2011年8月1日。
5.如权利要求1所述的用于制造治疗或预防摄护腺肥大的类茄红素组合物，其特征在于所述类茄红素组合物用作食物增补剂、动物饲料、人类食物产品或医药品或化妆品组合物。
6.一种用于制造治疗或预防肾功能疾病的类茄红素组合物，其特征在于所述的类茄红素组合物包含有效剂量的类茄红素萃取物。
7.如权利要求6所述的用于制造治疗或预防肾功能的类茄红素组合物，其特征在于所述的类茄红素萃取物所含活性成分选自z-胡萝卜素（z-carotene）、链孢红素（neurosporene）、球形烯醇（spheroidenone）和/或甲氧基链孢红素（methoxyneurosporene）。
8.如权利要求6所述的用于制造治疗或预防肾功能的类茄红素组合物，其特征在于所述的类茄红素萃取物是萃取自突变的光合菌。
9.如权利要求8所述的用于制造治疗或预防肾功能的类茄红素组合物，其特征在于所述的突变的光合菌保藏于布达佩斯条约的德国微生物与细胞搜集中心(German Collection of Microorganisms and Cell Cultures)，保藏编号为WL-APD911的球形红杆菌（Rhodobacter sphaeroides），保藏日期2011年8月1日。
10.如权利要求6所述的用于制造治疗或预防肾功能的类茄红素组合物，其特征在于所述类茄红素组合物用作食物增补剂、动物饲料、人类食物产品或医药品或化妆品组合物。
11.一种光合菌，保藏于布达佩斯条约的德国微生物与细胞搜集中心(German Collection of Microorganisms and Cell Cultures)，保藏编号为WL-APD911，保藏日期2011年8月1日。
12.如权利要求11所述的光合菌在生产茄红素或类茄红素方面的用途。
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| TWI459950B (zh) * | 2012-12-22 | 2014-11-11 | Asia Pacific Biotech Developing Inc | 一種治療或預防糖尿病或大腸直腸癌的醫藥組合物 |
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| CN108904435A (zh) * | 2018-08-28 | 2018-11-30 | 上海能相旭生物科技有限公司 | 一种球形酮的应用及其制备方法 |
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| TWI406942B (zh) | 2013-09-01 |
| TW201209162A (en) | 2012-03-01 |
| US8563267B2 (en) | 2013-10-22 |
| CN102885315B (zh) | 2016-02-10 |
| US20120027707A1 (en) | 2012-02-02 |
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