CN1117766C - Preparation method of mannuronic acid with single degree of polymerization and its application - Google Patents
Preparation method of mannuronic acid with single degree of polymerization and its application Download PDFInfo
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- CN1117766C CN1117766C CN 00111368 CN00111368A CN1117766C CN 1117766 C CN1117766 C CN 1117766C CN 00111368 CN00111368 CN 00111368 CN 00111368 A CN00111368 A CN 00111368A CN 1117766 C CN1117766 C CN 1117766C
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- mannuronic acid
- polymerization
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- precipitate
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- AEMOLEFTQBMNLQ-VANFPWTGSA-N D-mannopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H]1O AEMOLEFTQBMNLQ-VANFPWTGSA-N 0.000 title claims abstract description 22
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 title claims abstract description 22
- 238000006116 polymerization reaction Methods 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 14
- 108090000790 Enzymes Proteins 0.000 claims abstract description 10
- 102000004190 Enzymes Human genes 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 10
- 239000003480 eluent Substances 0.000 claims abstract description 8
- 108010004131 poly(beta-D-mannuronate) lyase Proteins 0.000 claims abstract description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000002244 precipitate Substances 0.000 claims abstract description 6
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000009835 boiling Methods 0.000 claims abstract description 4
- 239000012535 impurity Substances 0.000 claims abstract description 4
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 4
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 4
- 239000000661 sodium alginate Substances 0.000 claims abstract description 4
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims abstract description 3
- 241000607598 Vibrio Species 0.000 claims abstract description 3
- 238000010438 heat treatment Methods 0.000 claims abstract description 3
- 239000001632 sodium acetate Substances 0.000 claims abstract description 3
- 235000017281 sodium acetate Nutrition 0.000 claims abstract description 3
- 239000000203 mixture Substances 0.000 claims description 5
- 238000004255 ion exchange chromatography Methods 0.000 claims description 2
- 206010047400 Vibrio infections Diseases 0.000 claims 1
- 239000003513 alkali Substances 0.000 claims 1
- 230000009849 deactivation Effects 0.000 claims 1
- 238000010612 desalination reaction Methods 0.000 claims 1
- 238000004108 freeze drying Methods 0.000 claims 1
- 238000001556 precipitation Methods 0.000 claims 1
- 229960004249 sodium acetate Drugs 0.000 claims 1
- 230000000259 anti-tumor effect Effects 0.000 abstract description 5
- 239000002246 antineoplastic agent Substances 0.000 abstract description 3
- 229940041181 antineoplastic drug Drugs 0.000 abstract description 3
- 238000001035 drying Methods 0.000 abstract description 3
- 239000000047 product Substances 0.000 abstract description 3
- 238000005342 ion exchange Methods 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108090000856 Lyases Proteins 0.000 description 1
- 102000004317 Lyases Human genes 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 239000012564 Q sepharose fast flow resin Substances 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
一种单一聚合度的甘露糖醛酸的制备方法，包括使褐藻酸钠溶于水，加入褐藻胶裂合酶进行反应，用沸水浴加热使酶失活，离心后除去沉淀杂质，将溶液调pH，离心除去产生的沉淀物，用碱液溶解并调pH，加入乙醇产生沉淀，干燥后用离子交换色谱柱进行分离，用洗脱液洗脱，其特征是所述的将溶液的调pH范围为2.5～3.2，所述的洗脱液为醋酸钠的梯度洗脱液，所述的酶是由弧菌产生的专一性褐藻胶裂合酶。本产品是具有抗肿瘤活性，用于开发抗肿瘤药物。A method for preparing mannuronic acid with a single degree of polymerization, comprising dissolving sodium alginate in water, adding alginate lyase to react, heating in a boiling water bath to inactivate the enzyme, centrifuging to remove precipitated impurities, and adjusting the solution to pH, centrifuge to remove the produced precipitate, dissolve it with lye and adjust the pH, add ethanol to produce a precipitate, separate it with an ion-exchange chromatographic column after drying, and elute with an eluent, which is characterized in that the pH of the solution is adjusted The range is 2.5-3.2, the eluent is a gradient eluent of sodium acetate, and the enzyme is a specific alginate lyase produced by Vibrio. This product has anti-tumor activity and is used for the development of anti-tumor drugs.
Description
技术领域technical field
本发明涉及一种有机聚合物，特别是涉及一种具有抗肿瘤活性的单一聚合度的甘露糖醛酸。The invention relates to an organic polymer, in particular to a mannuronic acid with a single degree of polymerization having antitumor activity.
技术背景technical background
在一日本专利：(06172375)中公开了一种用鲍鱼丙酮粉提取物制备甘露糖醛酸的方法，该鲍鱼丙酮粉提取物中含有甘露糖醛酸裂合酶。这是一种应用酸水解和酶解制备聚合度2～7的寡聚甘露糖醛酸的方法，其分离效果不佳。A Japanese patent: (06172375) discloses a method for preparing mannuronic acid from abalone acetone powder extract, which contains mannuronate lyase. This is a method for preparing oligomannuronic acid with a degree of polymerization of 2 to 7 by using acid hydrolysis and enzymatic hydrolysis, and its separation effect is not good.
发明内容Contents of the invention
本发明的目的是提供一种制备过程简单，分离效果好的单一聚合度的甘露糖醛酸的制备方法，并且该产品具有抗肿瘤活性，可用于开发抗肿瘤药物。The purpose of the present invention is to provide a method for preparing mannuronic acid with a single degree of polymerization with simple preparation process and good separation effect, and the product has antitumor activity and can be used for the development of antitumor drugs.
一种单一聚合度的甘露糖醛酸的制备方法，包括使褐藻酸钠溶于水，加入褐藻胶裂合酶进行反应，用沸水浴加热使酶失活，离心后除去沉淀杂质，将溶液调pH，离心除去产生的沉淀物，用碱液溶解并调pH，加入乙醇产生沉淀，干燥后用离子交换色谱柱分离各单一聚合度的甘露糖醛酸混合物，将甘露糖醛酸混合物用洗脱液洗脱，分步收集洗脱液组分，分别加入乙醇进行沉淀，脱盐后冻干，其特征是所述的将溶液调pH范围为2.5～3.2，所述的洗脱液为醋酸钠的梯度洗脱液，所用褐藻胶裂合酶是由弧菌产生的。A method for preparing mannuronic acid with a single degree of polymerization, comprising dissolving sodium alginate in water, adding alginate lyase to react, heating in a boiling water bath to inactivate the enzyme, centrifuging to remove precipitated impurities, and adjusting the solution to pH, centrifuge to remove the resulting precipitate, dissolve it with lye and adjust the pH, add ethanol to produce a precipitate, and after drying, use an ion-exchange chromatography column to separate the mannuronic acid mixture with a single degree of polymerization, and elute the mannuronic acid mixture with elution, the eluent components are collected step by step, ethanol is added to precipitate, and freeze-dried after desalting, it is characterized in that the pH range of the solution is adjusted to 2.5 to 3.2, and the eluent is sodium acetate Gradient elution, the alginate lyase used was produced by Vibrio.
本发明的制备方法分离效率高，制出的各种单一聚合度的甘露糖醛酸具有不同程度的抗肿瘤活性，可用于开发成抗肿瘤药物。The preparation method of the invention has high separation efficiency, and the prepared mannuronic acid with a single degree of polymerization has different degrees of antitumor activity, and can be used to develop antitumor drugs.
具体实施方式Detailed ways
下面通过实施例说明本发明。The present invention is illustrated by the following examples.
取5g褐藻酸钠溶于100ml水，加入5单位(褐藻胶裂合酶的酶活力单位为每min使0.2％的褐藻胶溶液吸光度增加1所需的酶量为一个酶活单位。)由弧菌产生的褐藻胶裂合酶液，该酶的生产方法已在00111178.7号在先申请中叙述过了。在28℃下反应，直到230nm吸光值不再变化，然后沸水浴加热使酶失活，冷却后离心去杂质，将上清液调pH2.85，离心去掉沉淀，用4mol/L的NaOH调pH到7，加入乙醇使产品沉淀，干燥后得到不同聚合度的甘露糖醛酸混合物。用Q-sepharose fast flow阴离子交换色谱柱分离各单一聚合度的甘露糖醛酸。色谱柱先用0.1mol/L pH7.5的醋酸钠缓冲液起始洗脱液平衡好，再将1g上述制备的甘露糖醛酸溶于起始缓冲液上色谱柱，用0.1～1的醋酸钠梯度洗脱液洗脱，用230nm的紫外检测器检测洗脱下来的甘露糖醛酸含量，分步收集各洗脱组分，脱盐后分别加入乙醇沉淀出甘露糖醛酸，干燥后得到2～12不同聚合度的甘露糖醛酸。Take 5g of sodium alginate and dissolve it in 100ml of water, add 5 units (the enzyme activity unit of alginate lyase is the enzyme amount required to increase the absorbance of 0.2% alginate solution by 1 per minute is one enzyme activity unit.) by arc The alginate lyase liquid produced by bacteria, the production method of this enzyme has been described in No. 00111178.7 prior application. React at 28°C until the absorbance at 230nm does not change, then heat in a boiling water bath to inactivate the enzyme, cool down and centrifuge to remove impurities, adjust the supernatant to pH 2.85, centrifuge to remove the precipitate, and adjust the pH with 4mol/L NaOH To 7, add ethanol to precipitate the product, and obtain a mixture of mannuronic acids with different degrees of polymerization after drying. A Q-sepharose fast flow anion-exchange column was used to separate mannuronic acid with a single degree of polymerization. The chromatographic column is first equilibrated with the initial eluent of 0.1mol/L sodium acetate buffer solution of pH7.5, and then 1g of the above-prepared mannuronic acid is dissolved in the initial buffer solution on the chromatographic column, and the chromatographic column is mixed with 0.1-1 acetic acid Elute with sodium gradient eluent, detect the content of eluted mannuronic acid with a 230nm ultraviolet detector, collect the eluted components step by step, add ethanol to precipitate mannuronic acid after desalting, and obtain 2 ~12 mannuronic acids with different degrees of polymerization.
采用本方法可以制备出聚合度为2～12的甘露糖醛酸。这些寡糖用高效阴离子交换色谱(high performance anion exchange chromatography HPAEC)检测为单峰，说明为单一聚合度。The method can be used to prepare mannuronic acid with a polymerization degree of 2-12. These oligosaccharides were detected as single peaks by high performance anion exchange chromatography (HPAEC), indicating a single degree of polymerization.
用噻唑蓝(TTM)实验方法测定了用本发明的方法制备的各单一聚合度的甘露糖醛酸的抗肿瘤活性。实验时将癌细胞以10％小牛血清(FCS)的RPMI1640培养基配成104个/ml悬液，于37℃，5％CO2，100μl/孔(96孔板)抚育24h，各组对应加三聚甘露糖醛酸，使其浓度分别为1，10，100ug/ml，空白组对应加培养基，每组5个平行孔，培养24h后加入5mg/ml的TTM 20ul，培养4h，去上清液加150ul二甲基亚砜(DMSO)，用酶标仪测定570nm，(630nm为设定波长)吸光值。所用的癌细胞株为Ovcar-3人卵巢癌细胞。用此方法测得三聚甘露糖醛酸100ug/ml的剂量时的抑瘤率为18.6％。其他各种聚合度的甘露糖醛酸也以此方法测定了抑瘤率，其结果类似。The antitumor activity of the mannuronic acid with each single degree of polymerization prepared by the method of the present invention was determined by the thiazolium blue (TTM) experimental method. During the experiment, the cancer cells were prepared as 10 4 cells/ml suspension in RPMI1640 medium with 10% calf serum (FCS), and incubated at 37°C, 5% CO 2 , 100 μl/well (96-well plate) for 24 hours, each group Add trimeric mannuronic acid so that the concentrations are 1, 10, and 100ug/ml respectively, and add culture medium to the blank group, with 5 parallel wells in each group, add 20ul of 5mg/ml TTM after 24 hours of culture, and cultivate for 4 hours. Remove the supernatant and add 150ul dimethyl sulfoxide (DMSO), and measure the absorbance at 570nm (630nm is the set wavelength) with a microplate reader. The cancer cell line used was Ovcar-3 human ovarian cancer cells. The tumor inhibition rate was 18.6% when the dose of trimeric mannuronic acid 100ug/ml was measured by this method. The tumor inhibition rate of mannuronic acid with various degrees of polymerization was also determined by this method, and the results were similar.
Claims (2)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN 00111368 CN1117766C (en) | 2000-09-07 | 2000-09-07 | Preparation method of mannuronic acid with single degree of polymerization and its application |
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| CN 00111368 CN1117766C (en) | 2000-09-07 | 2000-09-07 | Preparation method of mannuronic acid with single degree of polymerization and its application |
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| CN1341665A CN1341665A (en) | 2002-03-27 |
| CN1117766C true CN1117766C (en) | 2003-08-13 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7090856B2 (en) | 2003-06-19 | 2006-08-15 | Hong Kong University Of Science And Technology | Anti-fouling exopolysaccharides isolated from cultures of Vibrio alginolyticus and Vibrio proteolyticus |
| CN100508985C (en) * | 2004-03-24 | 2009-07-08 | 中国海洋大学 | Algin oligose and derivative thereof and producing method and use thereof |
| CN1562050A (en) | 2004-03-24 | 2005-01-12 | 中国海洋大学 | Use of oligose alginate in anti-dementia and anti-diabetes |
| CN103961365B (en) * | 2014-03-25 | 2016-06-29 | 青岛海洋生物医药研究院股份有限公司 | Oligomannuronic acid salt is in the application of preparation preventing and treating hepatic injury and various hepatitis, hepatic fibrosis or liver cirrhosis medicine |
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2000
- 2000-09-07 CN CN 00111368 patent/CN1117766C/en not_active Expired - Lifetime
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