CN1371426A - 通过酶水解制备假叶树甾类糖苷衍生物的方法 - Google Patents

通过酶水解制备假叶树甾类糖苷衍生物的方法 Download PDF

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CN1371426A
CN1371426A CN00808244A CN00808244A CN1371426A CN 1371426 A CN1371426 A CN 1371426A CN 00808244 A CN00808244 A CN 00808244A CN 00808244 A CN00808244 A CN 00808244A CN 1371426 A CN1371426 A CN 1371426A
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ruscus aculeatus
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hydrolysis
enzymatic hydrolysis
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CN1198941C (zh
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C·庞佐恩
M·德罗萨
A·莫拉纳
A·迪拉加罗
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Abstract

制备去葡萄糖去鼠李糖假叶树素的方法,包括使用得自黑曲霉的粗水解酶将假叶树甾类糖苷(假叶树皂甙)进行酶水解。

Description

通过酶水解制备假叶树甾类糖苷衍生物的方法
本发明涉及制备假叶树甾类糖苷衍生物(假叶树皂苷(ruscosaponins))的方法。
更具体来说，本发明涉及通过部分酶水解假叶树素(ruscin)、去葡萄糖假叶树素(desglucoruscin)或假叶树糖苷(ruscoside)来制备去葡萄糖去鼠李糖假叶树素(desglucodesrhamnoruscin)的方法。
本发明酶水解是使用基于从黑曲霉(Aspergillus niger)提取的水解酶的酶粗品制备物进行的。所述制备物是商购获得的，并且用于葡萄酒和饮食工业中。特别优选使用商品名为Cytolase PCL5的从Gist Brocades购得的市售制备物。
所述酶制备物同时具有β-吡喃葡糖苷酶和α-吡喃鼠李糖苷酶活性，因此能依次除去β-葡糖苷残基和α-鼠李糖苷残基以获得所需产物。
本发明酶水解是在缓冲至pH5的水溶液中或者含有至多约30％体积乙醇的水中进行的。已经发现，乙醇的存在对酶活性无不利影响：β-吡喃葡糖苷酶活性在乙醇存在下会增加，而与此同时α-吡喃鼠李糖苷酶活性会稍微下降，但是下降的程度无论如何也不会影响本发明反应。为了将去葡萄糖假叶树素完全溶解，需要大约30％v/v乙醇，而对于假叶树糖苷，需要约17％v/v的较低百分比。因此使用后者有时是优选的，这还因为其不需要特殊预处理，并且由于其表面活性特性可将得自第一个酶水解步骤的去葡萄糖假叶树素溶解，之后可更有效地将其转化成α-吡喃鼠李糖苷酶。
假叶树皂苷的浓度为约5-约40％w/v，并且水解是在20-50℃的温度下进行3-7天。
在生物转化结束时，不溶于反应体系的去葡萄糖去鼠李糖假叶树素终产物可通过离心、随后用甲醇洗涤而方便地回收。此外，本发明方法可连续进行，即，在固定时间除去水解产物，并连续加入新鲜底物，这是由于所用酶的稳定性所致。对于该目的，优选使用装配有超滤膜的生物反应器以防止抑制剂和变性分子积聚。
下述实施例更详细地举例说明本发明。
实施例
使用前，在装配有XM膜(截留分子量为50kDa)的Amicon仪器中用0.1M柠檬酸盐-磷酸盐缓冲液pH5将酶制备物Cytolase PCL5(GistBrocades)渗滤(diafilitered)，以除去变性剂和抑制剂。
培养混合物是通过以下述顺序进行的加入而制得的：底物(去葡萄糖假叶树素或假叶树糖苷)、0.1M柠檬酸盐-磷酸盐缓冲液pH5.0、Cytolase PCL5、和在搅拌下缓慢地加入乙醇。底物浓度为5-40％(w/v)、乙醇浓度为0-30％(v/w)。反应通过在硅胶板(60 F250Merck)上的TLC进行监测，用乙酸乙酯/甲醇/水100∶15∶10作为展开剂。该酶制备物的活性为38单位β-吡喃葡糖苷酶和2.3单位α-吡喃鼠李糖苷酶/毫升培养混合物。
本发明生物转化在25℃进行。从去葡萄糖假叶树素开始，在约72小时内完全转化成去葡萄糖去鼠李糖假叶树素，从假叶树糖苷开始，在约96小时内完全转化成去葡萄糖去鼠李糖假叶树素。
然后将该培养混合物在4℃以12,100g离心30分钟。用甲醇洗涤沉淀物，并在相同条件下再次离心。将该步骤重复5次，并合并上清液。然后将甲醇溶液真空蒸发，研制所得产物，以获得含有20％去葡萄糖去鼠李糖假叶树素(HPLC分析)的深黄色细粉末。

Claims (6)

1.制备去葡萄糖去鼠李糖假叶树素的方法，该方法包括使用得自黑曲霉的粗水解酶将假叶树甾类糖苷(假叶树皂苷)进行酶水解。
2.权利要求1的方法，其中所述粗水解酶是制备物Cytolase PCL5。
3.前述权利要求任一项的方法，其中所述水解是在pH5的水溶液中或含有至多30％体积乙醇的水-乙醇混合物中进行的。
4.前述权利要求任一项的方法，其中假叶树皂苷的浓度为5-40％w/v。
5.前述权利要求任一项的方法，其中所述水解在20-40℃的温度下进行3-7天。
6.前述权利要求任一项的方法，其中所述终产物是通过离心并用甲醇洗涤而回收的。
CNB008082448A 1999-06-01 2000-05-29 通过酶水解制备假叶树甾类糖苷衍生物的方法 Expired - Fee Related CN1198941C (zh)

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IT1999MI001222A ITMI991222A1 (it) 1999-06-01 1999-06-01 Processo per la preparazione di derivati di glicosidi steroidei di ruscus aculeatus mediante idrolisi enzimatica
ITMI99A001222 1999-06-01

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CN103588852A (zh) * 2013-11-26 2014-02-19 陕西嘉禾植物化工有限责任公司 从假叶树中提取分离鲁斯可皂苷元的方法

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JP2013516963A (ja) 2010-01-19 2013-05-16 ディーエスエム アイピー アセッツ ビー．ブイ． ステビオシドからステビオールを酵素的に調製する方法
CN102205071B (zh) * 2011-05-28 2012-07-04 吴汉利 一种治疗甲状腺囊肿的中药组合物
RU2484839C1 (ru) * 2012-02-06 2013-06-20 Михаил Михайлович Шашурин Способ получения экстракта рододендрона золотистого с пониженным содержанием стероидных (сердечных) гликозидов
CA3203278A1 (en) * 2020-12-09 2022-06-16 Glaxosmithkline Biologicals Sa Saponins

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JPS59192075A (ja) * 1983-04-12 1984-10-31 Dainippon Ink & Chem Inc アロエ抽出物の苦味改良法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103588852A (zh) * 2013-11-26 2014-02-19 陕西嘉禾植物化工有限责任公司 从假叶树中提取分离鲁斯可皂苷元的方法
CN103588852B (zh) * 2013-11-26 2016-03-30 陕西嘉禾生物科技股份有限公司 从假叶树中提取分离鲁斯可皂苷元的方法

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SK17472001A3 (sk) 2002-04-04
WO2000073483A2 (en) 2000-12-07
KR20020011427A (ko) 2002-02-08
AU5073200A (en) 2000-12-18
JP4603220B2 (ja) 2010-12-22
JP2003501044A (ja) 2003-01-14
RU2249042C2 (ru) 2005-03-27
CA2375090A1 (en) 2000-12-07
ITMI991222A0 (it) 1999-06-01
HK1048338B (zh) 2005-11-11
ITMI991222A1 (it) 2000-12-01
DE60030711D1 (de) 2006-10-26
PL352148A1 (en) 2003-07-28
ATE339513T1 (de) 2006-10-15
AU773232B2 (en) 2004-05-20
CN1198941C (zh) 2005-04-27
NO326846B1 (no) 2009-03-02
EP1180161A2 (en) 2002-02-20
NO20015823D0 (no) 2001-11-29
DE60030711T2 (de) 2007-09-20
KR100685529B1 (ko) 2007-02-22
NO20015823L (no) 2001-11-29
HK1048338A1 (en) 2003-03-28
DK1180161T3 (da) 2007-01-08
SK286062B6 (sk) 2008-02-05
US6911325B1 (en) 2005-06-28
PL200909B1 (pl) 2009-02-27
CZ20014282A3 (cs) 2002-04-17
CZ301179B6 (cs) 2009-11-25
ES2272286T3 (es) 2007-05-01
PT1180161E (pt) 2007-01-31
EP1180161B1 (en) 2006-09-13
HUP0201379A3 (en) 2007-10-29
CA2375090C (en) 2011-05-03
HUP0201379A2 (en) 2002-08-28
WO2000073483A3 (en) 2001-05-10
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