WO2022142561A1 - 一种诺丽酵素中季铵盐生物碱类化合物及其制备方法与应用 - Google Patents
一种诺丽酵素中季铵盐生物碱类化合物及其制备方法与应用 Download PDFInfo
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- compound
- quaternary ammonium
- ammonium salt
- noni
- salt alkaloid
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- 244000131360 Morinda citrifolia Species 0.000 title claims abstract description 48
- 235000017524 noni Nutrition 0.000 title claims abstract description 48
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 38
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 38
- 229930013930 alkaloid Natural products 0.000 title claims abstract description 36
- -1 Quaternary ammonium salt alkaloid compound Chemical class 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 150000001875 compounds Chemical class 0.000 claims abstract description 13
- 102100024579 Tyrosyl-DNA phosphodiesterase 1 Human genes 0.000 claims abstract description 12
- 101710205182 Tyrosyl-DNA phosphodiesterase 1 Proteins 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims abstract description 7
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 5
- 229940041181 antineoplastic drug Drugs 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 24
- 239000012046 mixed solvent Substances 0.000 claims description 21
- 239000002808 molecular sieve Substances 0.000 claims description 18
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 18
- 229940125904 compound 1 Drugs 0.000 claims description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 14
- 229940125782 compound 2 Drugs 0.000 claims description 14
- 239000003480 eluent Substances 0.000 claims description 14
- 238000004587 chromatography analysis Methods 0.000 claims description 13
- 238000004809 thin layer chromatography Methods 0.000 claims description 13
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000008346 aqueous phase Substances 0.000 claims description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 8
- 238000004440 column chromatography Methods 0.000 claims description 8
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical group CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims description 6
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 5
- 239000012074 organic phase Substances 0.000 claims description 5
- 239000011347 resin Substances 0.000 claims description 5
- 229920005989 resin Polymers 0.000 claims description 5
- 239000003112 inhibitor Substances 0.000 claims description 4
- OAIVIYSBZFEOIU-UHFFFAOYSA-N chloroform;propan-2-one Chemical compound CC(C)=O.ClC(Cl)Cl OAIVIYSBZFEOIU-UHFFFAOYSA-N 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 2
- 101001098814 Dictyostelium discoideum 3',5'-cyclic-nucleotide phosphodiesterase regA Proteins 0.000 claims 1
- 102100024578 Tyrosyl-DNA phosphodiesterase 2 Human genes 0.000 abstract description 9
- 101710205181 Tyrosyl-DNA phosphodiesterase 2 Proteins 0.000 abstract description 9
- 230000002401 inhibitory effect Effects 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 18
- 238000005481 NMR spectroscopy Methods 0.000 description 8
- 235000013399 edible fruits Nutrition 0.000 description 8
- 230000005764 inhibitory process Effects 0.000 description 8
- 101000760764 Homo sapiens Tyrosyl-DNA phosphodiesterase 1 Proteins 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- OSASVXMJTNOKOY-UHFFFAOYSA-N chlorobutanol Chemical compound CC(C)(O)C(Cl)(Cl)Cl OSASVXMJTNOKOY-UHFFFAOYSA-N 0.000 description 4
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 101710183280 Topoisomerase Proteins 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 229960004926 chlorobutanol Drugs 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 244000144927 Aloe barbadensis Species 0.000 description 1
- 235000002961 Aloe barbadensis Nutrition 0.000 description 1
- 244000247812 Amorphophallus rivieri Species 0.000 description 1
- 235000001206 Amorphophallus rivieri Nutrition 0.000 description 1
- 241001474374 Blennius Species 0.000 description 1
- 240000006432 Carica papaya Species 0.000 description 1
- 235000009467 Carica papaya Nutrition 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 229920002752 Konjac Polymers 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 101150041570 TOP1 gene Proteins 0.000 description 1
- 101150107801 Top2a gene Proteins 0.000 description 1
- 235000011399 aloe vera Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 235000010485 konjac Nutrition 0.000 description 1
- 239000000252 konjac Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 230000000474 nursing effect Effects 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229930001910 pseudoalkaloid Natural products 0.000 description 1
- 150000003856 quaternary ammonium compounds Chemical class 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
Images
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/62—Oxygen or sulfur atoms
- C07D213/63—One oxygen atom
- C07D213/68—One oxygen atom attached in position 4
Definitions
- the invention belongs to the field of phytochemistry, and in particular relates to a quaternary ammonium salt alkaloid compound in noni enzyme and a preparation method and application thereof.
- Alkaloids are a class of nitrogen-containing alkaline organic compounds that exist in nature, mainly plants, but some also exist in animals. It has alkali-like properties, so it is also called pseudo-alkaloid. Most of the alkaloids have complex ring structures, and nitrogen is mostly contained in the ring, which has significant biological activity and is one of the important active ingredients in Chinese herbal medicine. For quaternary ammonium alkaloids, the unique molecular structure of quaternary ammonium compounds endows them with a series of practical functions such as emulsifying, dispersing, sterilizing, and antiseptic. These unique properties make them show good application prospects in nursing and other aspects.
- Noni is a lesser-known but medicinally effective fruit in the South Pacific. Noni has been used in Polynesia, China, and India for over two thousand years. Like aloe vera, seaweed, papaya, konjac, and others, noni has been shown to improve many different health conditions in people. At present, noni enzymes obtained by fermentation of noni fruits are mostly sold as nutritious foods, and there are very few medicinal development researches on the active ingredients in noni enzymes.
- the present invention proposes a quaternary ammonium salt alkaloid compound in noni enzyme and a preparation method and application thereof.
- the present invention provides a quaternary ammonium salt alkaloid compound in noni enzyme, the compound is compound 1 or compound 2, and its structural formula is as follows:
- a method for preparing a quaternary ammonium salt alkaloid compound in noni enzyme comprising the following steps: extracting noni enzyme with ethyl acetate to obtain an organic phase and an aqueous phase, and sequentially subjecting the aqueous phase to column chromatography, thin layer analysis and molecular sieve chromatography to obtain the target compound 1 and compound 2.
- volume ratio of the noni enzyme and ethyl acetate is 1:1.
- the conditions of the column chromatography are: a macroporous resin D201 chromatography column, and an ethanol-water mixed solvent with an ethanol volume percentage of 25-40% is used as the eluent.
- the conditions of the thin layer chromatography are as follows: a methanol-chloroform mixed solvent with a methanol volume percentage of 20%-40% is used as a developing solvent, or a chloroform-acetone mixed solvent with an acetone volume percentage of 22%-23%. for the expander.
- the molecular sieve chromatography conditions are: the molecular sieve is Sephadex LH-20, and the chloroform-methanol mixed solvent with a volume percentage of chloroform of 10%-30% is used as the eluent.
- the beneficial effects of the present invention are as follows: the quaternary ammonium salt alkaloid compounds in the new noni enzyme provided by the present invention are more effective for tyrosyl-DNA phosphodiesterase 1 and tyrosyl-DNA phosphate. Diesterase 2 has an obvious inhibitory effect.
- the quaternary ammonium salt alkaloid compounds can be obtained by extracting with ethyl acetate, and then by column chromatography, thin layer chromatography and molecular sieve chromatography. The preparation process is simple, low cost, Raw materials come from a wide range of sources.
- Fig. 1 is the hydrogen nuclear magnetic resonance spectrum H NMR of compound 1;
- Fig. 2 is the carbon nuclear magnetic resonance spectrum 13 C NMR of compound 1;
- Fig. 3 is the hydrogen nuclear magnetic resonance spectrum H NMR of compound 2;
- FIG. 4 is the carbon nuclear magnetic resonance spectrum of compound 213C NMR.
- the noni enzyme used in the present invention is commercially available noni enzyme produced by Hainan Dazhou Island Noni Food Co., Ltd. Its preparation method comprises the following steps:
- Embodiment 1- a kind of preparation method of quaternary ammonium salt alkaloid compound in noni ferment, comprises the following steps:
- the conditions are as follows: the chromatographic column is a macroporous resin D201 column, and the ethanol-water mixed solvent with the ethanol volume percentage of 25% is used as the eluent; the thin-layer chromatography conditions are: the methanol-chloroform mixed solvent with the methanol volume percentage of 20% is used as the eluent.
- the molecular sieve chromatography conditions are as follows: the molecular sieve is Sephadex LH-20, and the chloroform-methanol mixed solvent with a volume percentage of chloroform of 30% is used as the eluent.
- compounds 1 and 2 are new compounds.
- Compound 1 is a colorless crystal, easily soluble in methanol and DMSO, and appears as a dark spot under a 254 nm UV lamp.
- HRESI-MS [m/z 233.1285, calculated value 233.1283] deduces that the molecular weight of this compound is 232, the molecular formula is C 13 H 14 NO 3 + , and the degree of unsaturation is 8.
- 1 H NMR (400 MHz in DMSO) and 13 C NMR (100 MHz in DMSO) are shown in the table.
- Compound 2 is a colorless crystal, easily soluble in methanol and DMSO, and appears as a dark spot under a 254 nm UV lamp.
- HRESI-MS [m/z 230.2479, calculated value 230.2475] deduces that the molecular weight of this compound is 207, the molecular formula is C 13 H 14 N 2 O 2 + , and the degree of unsaturation is 8.
- 1 H NMR (400 MHz in DMSO) and 13 C NMR (100 MHz in DMSO) are shown in the table.
- the preparation method of quaternary ammonium salt alkaloid compound in embodiment 2-noni ferment comprises the following steps:
- Noni enzyme and ethyl acetate in a volume ratio of 1:1 extracting to obtain an organic phase and an aqueous phase, and concentrating the aqueous phase through column chromatography, thin-layer chromatography, and molecular sieve chromatography;
- the conditions are as follows: the chromatographic column is a macroporous resin D201 column, and the ethanol-water mixed solvent with the ethanol volume percentage of 40% is used as the eluent; the thin-layer chromatography conditions are: the methanol-chloroform mixed solvent with the methanol volume percentage of 20% is used as the eluent.
- the molecular sieve chromatography conditions are: the molecular sieve is Sephadex LH-20, and the chloroform-methanol mixed solvent with chloroform volume percentage of 30% is used as the eluent.
- the preparation method of quaternary ammonium salt alkaloid compound in embodiment 3-noni ferment comprises the following steps:
- Noni enzyme and ethyl acetate in a volume ratio of 1:1 extracting to obtain an organic phase and an aqueous phase, and concentrating the aqueous phase through column chromatography, thin-layer chromatography, and molecular sieve chromatography;
- the conditions are: the chromatographic column is a macroporous resin D201 column, and the ethanol-water mixed solvent with the ethanol volume percentage of 35% is used as the eluent; the thin-layer chromatography conditions are: the methanol-chloroform mixed solvent with the methanol volume percentage of 20% is used as the eluent.
- the molecular sieve chromatography conditions are: the molecular sieve is Sephadex LH-20, and the chloroform-methanol mixed solvent with chloroform volume percentage of 30% is used as the eluent.
- Example 4- The preparation method of quaternary ammonium salt alkaloid compounds in Noni enzyme, the difference from Example 1 is only: the thin layer chromatography conditions are: methanol-chloroform mixed solvent with methanol volume percentage of 40% for the expander
- Example 5-Noni enzyme The preparation method of the quaternary ammonium salt alkaloid compound in Example 5-Noni enzyme, the difference from Example 1 is only: the molecular sieve chromatography conditions are: the molecular sieve is Sephadex LH-20, and the chloroform volume percentage is 10% The chloroform-methanol mixed solvent is the eluent,
- Example 6-Noni ferment The preparation method of the quaternary ammonium salt alkaloid compound in Example 6-Noni ferment is only different from Example 1 in that the thin-layer chromatography conditions are: using an acetone-chloroform mixed solvent with an acetone volume percentage of 22% for the expander.
- Example 7-Noni ferment The preparation method of the quaternary ammonium salt alkaloid compound in Example 7-Noni ferment is only different from Example 1 in that the thin-layer chromatography conditions are: using an acetone-chloroform mixed solvent with an acetone volume percentage of 23% for the expander.
- Tyrosyl-DNA phosphodiesterase 1 (tyrosyl-DNA phosphodiesterase 1, TDP1) and tyrosyl-DNA phosphodiesterase 2 (TDP2) can specifically recognize and repair topoisomeric DNA damage mediated by topoisomerase 1 (Top1) or topoisomerase 2 (Top2) is a potential tumor therapy target, and its inhibitors can enhance the efficacy of chemotherapeutic drugs or radiotherapy.
- the inhibition rate of TDP1 was 20.62% and the inhibition rate of TDP2 was 18.33%.
- the concentration of compound 2 is 100uM, the inhibition rate of TDP1 is 71.62% and the inhibition rate of TDP2 is 21.11%. It can be used in tyrosyl-DNA phosphodiesterase 1 and tyrosyl-DNA phosphodiesterase 2 inhibitors. as an effective anticancer drug.
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Abstract
本发明提供一种诺丽酵素中季铵盐生物碱类化合物及其制备方法与应用，该化合物结构式如下。本发明还提供上述化合物的制备方法以及诺丽酵素中季铵盐生物碱类化合物在制备治抗肿瘤药物的应用。本发明提供的新的诺丽酵素中季铵盐生物碱类化合物，对酪氨酰-DNA磷酸二酯酶1和酪氨酰-DNA磷酸二酯酶2具有明显的抑制作用，制备工艺简单、成本低廉、原料来源广泛。
Description
本发明属于植物化学领域，特别涉及一种诺丽酵素中季铵盐生物碱类化合物及其制备方法与应用。
生物碱是存在于自然界中的一类含氮的碱性有机化合物，其主要以植物为主，但有的也存在于动物。其有似碱的性质，因此又称为赝碱。生物碱大多数有复杂的环状结构，氮素多包含在环内，有显著的生物活性，是中草药中重要的有效成分之一。对于季铵盐生物碱类化合物而言，季铵盐化合物特有的分子结构赋予其乳化、分散、杀菌、防腐等一系列实际作用，这些独特性能使其在护理等方面表现出良好的应用前景。
诺丽是一种在南太平洋地区较不为人知，但具有医疗效果的果实，诺丽的使用在波利尼西亚、中国及印度等地区已经有超过两千年的历史。就像芦荟、海草、木瓜、康蒺及其他等植物一样，诺丽已被证明能改善人们的许多不同的健康状况。目前大多将诺丽果实发酵得到诺丽酵素当成营养食品进行售卖，而极少对诺丽酵素中有效成分进行药用开发研究。
发明内容
鉴以此，本发明提出一种诺丽酵素中季铵盐生物碱类化合物及其制备方法与应用。
本发明的技术方案是这样实现的：
本发明提供一种诺丽酵素中季铵盐生物碱类化合物，所述化合物为化合物1或化合物2，其结构式如下：
进一步说明，所述化合物1的手性结构式如下：
进一步说明，所述化合物2的手性结构式如下：
一种诺丽酵素中季铵盐生物碱类化合物的制备方法，包括以下步骤：将诺丽酵素采用乙酸乙酯萃取，得到有机相和水相，将水相依次经柱层析、薄层层析、分子筛层析，制得目标化合物1和化合物2。
进一步说明，所述诺丽酵素和乙酸乙酯的体积比为1：1。
进一步说明，所述柱层析的条件为：大孔树脂的D201层析柱，以乙醇体积百分数为25～40％的乙醇-水混合溶剂为洗脱剂。
进一步说明，所述薄层层析的条件为：以甲醇体积百分数为20％-40％的甲醇-氯仿混合溶剂为展开剂，或以丙酮体积百分数为22％-23％的氯仿-丙酮混合溶剂为展开剂。
进一步说明，所述分子筛层析条件为：分子筛为Sephadex LH-20，以氯仿体积百分数为10％-30％的氯仿-甲醇混合溶剂为洗脱剂。
一种诺丽酵素中季铵盐生物碱类的化合物1和化合物2在制备治抗肿瘤药物的应用。
一种诺丽酵素中季铵盐生物碱类的化合物1和化合物2在制备酪氨酰-DNA磷酸二酯酶1和酪氨酰-DNA磷酸二酯酶2抑制剂的应用
与现有技术相比，本发明的有益效果是：本发明提供的新的诺丽酵素中季铵盐生物碱类化合物，对酪氨酰-DNA磷酸二酯酶1和酪氨酰-DNA磷酸二酯酶2具有明显的抑制作用，通过采用乙酸乙酯萃取，并依次经柱层析、薄层层析、分子筛层析即可得到季铵盐生物碱类化合物，制备工艺简单、成本低廉、原料来源广泛。
图1为化合物1的核磁共振氢谱图H NMR；
图2为化合物1的核磁共振碳谱
13C NMR；
图3为化合物2的核磁共振氢谱图H NMR；
图4为化合物2的核磁共振碳谱
13C NMR。
为了更好理解本发明技术内容，下面提供具体实施例，对本发明做进一步的说明。
本发明实施例所用的实验方法如无特殊说明，均为常规方法。
本发明实施例所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明使用的诺丽酵素为海南大洲岛诺丽食品有限公司生产市售的诺丽酵素。其制备方法包括以下步骤：
(1)把成熟诺丽果的表皮用刷子洗净；
(2)把瓶子、刀子与砧板洗净沥干备用；
(3)把诺丽果取出、沥干水份、切片；将所有的切片诺丽果按照1:3:10(1斤糖：3斤诺丽果:10斤水)放入干净的发酵瓶(20L白色塑料桶)。瓶中留有2成左右的空间供其发酵；
(4)盖紧瓶盖，贴上制作日期；
(5)诺丽果发酵期间，前一个月每天打开搅拌或摇动瓶子，使顶层没被液 体浸到的果子渗透；为防止酵素酒化，在一个月左右时间加少量醋；
(6)置于阴凉处发酵三个月，之后再过滤得到诺丽酵素。
实施例1-一种诺丽酵素中季铵盐生物碱类化合物的制备方法，包括以下步骤：
将诺丽酵素和乙酸乙酯按体积比为1：1进行混合，萃取，得到有机相和水相，将水相浓缩后经柱层析、薄层层析、分子筛层析；柱层析的条件为：色谱柱为大孔树脂D201柱，以乙醇体积百分数为25％的乙醇-水混合溶剂为洗脱剂；薄层层析条件为：以甲醇体积百分数为20％的甲醇-氯仿混合溶剂为展开剂；分子筛层析条件为：分子筛为Sephadex LH-20，以氯仿体积百分数为30％的氯仿-甲醇混合溶剂为洗脱剂。
得到季铵盐生物碱类化合物，分别为：
Nonialkaloid A(1)，Nonialkaloid B(2)；结构式如下：
其中，化合物1和2为新化合物。
各化合物理化性质、氢谱及质谱检测数据如下：
化合物1为无色晶体，易溶于甲醇、DMSO，254nm紫外灯下呈暗斑。HRESI-MS[m/z 233.1285,计算值233.1283]推出该化合物分子量为232，分子式为C
13H
14NO
3
+，不饱和度为8。
1H NMR(400MHz in DMSO)和
13C NMR(100MHz in DMSO)见表。
化合物2为无色晶体，易溶于甲醇、DMSO，254nm紫外灯下呈暗斑。HRESI-MS[m/z 230.2479,计算值230.2475]推出该化合物分子量为207，分子式为C
13H
14N
2O
2
+，不饱和度为8。
1H NMR(400MHz in DMSO)和
13C NMR(100MHz in DMSO)见表。
实施例2-诺丽酵素中季铵盐生物碱类化合物的制备方法，包括以下步骤：
将诺丽酵素和乙酸乙酯按体积比为1：1进行混合，萃取，得到有机相和水相，将水相浓缩后经柱层析、薄层层析、分子筛层析；柱层析的条件为：色谱柱为大孔树脂D201柱，以乙醇体积百分数为40％的乙醇-水混合溶剂为洗脱剂；薄层层析条件为：以甲醇体积百分数为20％的甲醇-氯仿混合溶剂为展开剂；分子筛层析条件为：分子筛为Sephadex LH-20，以氯仿体积百分数为30％的氯仿-甲醇混合溶剂为洗脱剂。
得到2个诺丽酵素中季铵盐生物碱类化合物，经检测与实施例1一致。
实施例3-诺丽酵素中季铵盐生物碱类化合物的制备方法，包括以下步骤：
将诺丽酵素和乙酸乙酯按体积比为1：1进行混合，萃取，得到有机相和水相，将水相浓缩后经柱层析、薄层层析、分子筛层析；柱层析的条件为：色谱柱为大孔树脂D201柱，以乙醇体积百分数为35％的乙醇-水混合溶剂为洗脱剂；薄层层析条件为：以甲醇体积百分数为20％的甲醇-氯仿混合溶剂为展开剂；分子筛层析条件为：分子筛为Sephadex LH-20，以氯仿体积百分数为30％的氯仿-甲醇混合溶剂为洗脱剂。
实施例4-诺丽酵素中季铵盐生物碱类化合物的制备方法，与实施例1的不同之处仅在于：薄层层析条件为：以甲醇体积百分数为40％的甲醇-氯仿混合溶剂为展开剂
实施例5-诺丽酵素中季铵盐生物碱类化合物的制备方法，与实施例1的不同之处仅在于：分子筛层析条件为：分子筛为Sephadex LH-20，以氯仿体积百分数为10％的氯仿-甲醇混合溶剂为洗脱剂、
得到2个诺丽酵素中季铵盐生物碱类化合物，经检测与实施例1一致。
实施例6-诺丽酵素中季铵盐生物碱类化合物的制备方法，与实施例1的不同之处仅在于：薄层层析条件为：采用丙酮体积百分数为22％的丙酮-氯仿混合溶剂为展开剂。
得到2个诺丽酵素中季铵盐生物碱类化合物，经检测与实施例1一致。
实施例7-诺丽酵素中季铵盐生物碱类化合物的制备方法，与实施例1的不同之处仅在于：薄层层析条件为：采用丙酮体积百分数为23％的丙酮-氯仿混合溶剂为展开剂。
得到2个诺丽酵素中季铵盐生物碱类化合物，经检测与实施例1一致。
试验例1
酪氨酰-DNA磷酸二酯酶1(tyrosyl-DNA phosphodiesterase 1,TDP1)和酪氨酰-DNA磷酸二酯酶2(tyrosyl-DNA phosphodiesterase 2,TDP2)可以分别特异性识别和修复由拓扑异构酶1(topoisomerase 1,Top1)或拓扑异构酶2(topoisomerase 2,Top2)介导的DNA损伤，是潜在的肿瘤治疗靶点，其抑制剂可增强化疗药物或放疗措施的功效。
经实验检测，不同浓度下化合物1和2对TDP1和TDP2的抑制率如下表所示：
表1.化合物的抑制率
| Compound | TDP1 Inhibition％(100uM) | TDP2 Inhibition％(100uM) |
| 化合物1 | 20.62％ | 18.33％ |
| 化合物2 | 71.62％ | 21.11％ |
其中，化合物1在浓度为100uM时对TDP1抑制率为20.62％对TDP2抑制率为18.33％。化合物2在浓度为100uM时对TDP1抑制率为71.62％对TDP2抑制率为21.11％，可应用于酪氨酰-DNA磷酸二酯酶1和酪氨酰-DNA磷酸二酯酶2抑制剂中，作为有效的治抗肿瘤药物。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
- 一种诺丽酵素中季铵盐生物碱类化合物，其特征在于：所述化合物为化合物1或化合物2，其结构式如下：
- 根据权利要求1所述的一种诺丽酵素中季铵盐生物碱类化合物，其特征在于：所述化合物1的手性结构式如下：
- 根据权利要求1所述的一种诺丽酵素中季铵盐生物碱类化合物，其特征在于：所述化合物2的手性结构式如下：
- 一种如权利要求1～3中任意一项所述的诺丽酵素中季铵盐生物碱类化合物的制备方法，其特征在于：包括以下步骤：将诺丽酵素采用乙酸乙酯萃取，得到有机相和水相，将水相依次经柱层析、薄层层析、分子筛层析，制得目标化合物1和化合物2。
- 根据权利要求4所述的一种诺丽酵素中季铵盐生物碱类化合物的制备方法，其特征在于：所述诺丽酵素和乙酸乙酯的体积比为1：1。
- 根据权利要求4所述的一种诺丽酵素中季铵盐生物碱类化合物的制备方法，其特征在于：所述柱层析的条件为：大孔树脂的D201层析柱，以乙醇体积百分数为25～40％的乙醇-水混合溶剂为洗脱剂。
- 根据权利要求4所述的一种诺丽酵素中季铵盐生物碱类化合物的制备方法，其特征在于：所述薄层层析的条件为：以甲醇体积百分数为20％-40％的甲醇-氯仿混合溶剂为展开剂，或以丙酮体积百分数为22％-23％的氯仿-丙酮混合溶剂为展开剂。
- 根据权利要求4所述的一种诺丽酵素中季铵盐生物碱类化合物的制备方法，其特征在于：分子筛层析条件为：分子筛为Sephadex LH-20，以氯仿体积百分数为10％-30％的氯仿-甲醇混合溶剂为洗脱剂。
- 一种如权利要求1～3中任意一项所述的诺丽酵素中季铵盐生物碱类化合物的应用，其特征在于：化合物1和化合物2在制备治抗肿瘤药物的应用。
- 根据权利要求9所述的一种诺丽酵素中季铵盐生物碱类化合物的应用，其特征在于：化合物1和化合物2在制备酪氨酰-DNA磷酸二酯酶1和酪氨酰-DNA磷酸二酯酶2抑制剂的应用。
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