CN104262231B - From white thorn seed, extract the method that separates L-Trp - Google Patents
From white thorn seed, extract the method that separates L-Trp Download PDFInfo
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- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 title claims abstract description 77
- 238000000034 method Methods 0.000 title claims abstract description 27
- 239000000284 extract Substances 0.000 title claims abstract description 13
- 244000265913 Crataegus laevigata Species 0.000 title 1
- 235000013175 Crataegus laevigata Nutrition 0.000 title 1
- 229960004799 tryptophan Drugs 0.000 claims abstract description 41
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 36
- 241000159206 Nitraria Species 0.000 claims abstract description 35
- 239000011347 resin Substances 0.000 claims abstract description 29
- 229920005989 resin Polymers 0.000 claims abstract description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 28
- 238000001179 sorption measurement Methods 0.000 claims abstract description 23
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000012043 crude product Substances 0.000 claims abstract description 9
- 238000010828 elution Methods 0.000 claims abstract description 9
- 239000003208 petroleum Substances 0.000 claims abstract description 9
- 244000184734 Pyrus japonica Species 0.000 claims abstract description 7
- 239000012141 concentrate Substances 0.000 claims abstract description 7
- 239000000047 product Substances 0.000 claims abstract description 5
- 239000002904 solvent Substances 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 75
- 238000000605 extraction Methods 0.000 claims description 15
- 238000004262 preparative liquid chromatography Methods 0.000 claims description 10
- 238000000746 purification Methods 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 4
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 abstract description 5
- 239000002994 raw material Substances 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 2
- 238000005406 washing Methods 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 description 11
- 235000013399 edible fruits Nutrition 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 230000007812 deficiency Effects 0.000 description 3
- 238000004237 preparative chromatography Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 241001104824 Nitraria tangutorum Species 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000000287 crude extract Substances 0.000 description 2
- 238000003795 desorption Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000010298 pulverizing process Methods 0.000 description 2
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- 210000000952 spleen Anatomy 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 206010002383 Angina Pectoris Diseases 0.000 description 1
- 208000008035 Back Pain Diseases 0.000 description 1
- 206010006895 Cachexia Diseases 0.000 description 1
- 206010007247 Carbuncle Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010016717 Fistula Diseases 0.000 description 1
- 125000002707 L-tryptophyl group Chemical group [H]C1=C([H])C([H])=C2C(C([C@](N([H])[H])(C(=O)[*])[H])([H])[H])=C([H])N([H])C2=C1[H] 0.000 description 1
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- 208000025865 Ulcer Diseases 0.000 description 1
- 241000159213 Zygophyllaceae Species 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229930002877 anthocyanin Natural products 0.000 description 1
- 235000010208 anthocyanin Nutrition 0.000 description 1
- 239000004410 anthocyanin Substances 0.000 description 1
- 150000004636 anthocyanins Chemical class 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 238000009933 burial Methods 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
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- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 201000006549 dyspepsia Diseases 0.000 description 1
- 235000018927 edible plant Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 208000026500 emaciation Diseases 0.000 description 1
- 230000003890 fistula Effects 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
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- 230000005906 menstruation Effects 0.000 description 1
- 239000002366 mineral element Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
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- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
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- 229910002027 silica gel Inorganic materials 0.000 description 1
- 230000035943 smell Effects 0.000 description 1
- IFGCUJZIWBUILZ-UHFFFAOYSA-N sodium 2-[[2-[[hydroxy-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyphosphoryl]amino]-4-methylpentanoyl]amino]-3-(1H-indol-3-yl)propanoic acid Chemical compound [Na+].C=1NC2=CC=CC=C2C=1CC(C(O)=O)NC(=O)C(CC(C)C)NP(O)(=O)OC1OC(C)C(O)C(O)C1O IFGCUJZIWBUILZ-UHFFFAOYSA-N 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000036269 ulceration Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/04—Indoles; Hydrogenated indoles
- C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
- C07D209/18—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D209/20—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals substituted additionally by nitrogen atoms, e.g. tryptophane
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
本发明研究发现，从白刺种子中提取分离L-色氨酸的方法，它包括如下操作步骤：(1)取白刺种子，以乙醇或含水乙醇为溶剂进行提取，合并提取液，回收乙醇，浓缩，以石油醚萃取，收集水层；(2)取步骤(1)的萃取液，过大孔吸附树脂柱，以水洗脱，收集水洗脱液，浓缩，干燥，即得L-色氨酸粗品；(3)取L-色氨酸粗品，纯化，即得L-色氨酸纯品。本发明研究发现，以白刺种子为原料，对大孔树脂直接进行水洗后，就分离提取得到了高纯度的L-色氨酸粗品和L-色氨酸纯品。本发明使用的洗脱部位与现有专利技术完全不同，其洗脱工艺仅需要水洗液，其操作更简便，成本更为低廉，同时也为白刺种子的开发利用提供了新的选择。
The present invention finds that the method for extracting and separating L-tryptophan from Nitraria seeds comprises the following steps: (1) take Nitraria seeds, extract with ethanol or hydrous ethanol as a solvent, combine extracts, and reclaim ethanol , concentrated, extracted with petroleum ether, and collected the water layer; (2) take the extract of step (1), pass through a macroporous adsorption resin column, elute with water, collect the water eluate, concentrate, and dry to obtain L- The crude product of tryptophan; (3) take the crude product of L-tryptophan and purify it to obtain the pure product of L-tryptophan. The research of the present invention finds that the high-purity L-tryptophan crude product and L-tryptophan pure product are separated and extracted after the macroporous resin is washed directly with Nitraria seeds as raw materials. The elution part used in the present invention is completely different from the existing patented technology. Its elution process only needs water washing solution, its operation is simpler and its cost is lower, and it also provides a new choice for the development and utilization of Nitraria japonica seeds.
Description
技术领域 technical field
本发明涉及L-色氨酸的分离提取方法。 The invention relates to a method for separating and extracting L-tryptophan.
背景技术 Background technique
唐古特白刺(NitrariaTangutorumBobr)为蒺藜科(Zygophyllaceae)白刺属植物，是一种药食两用的植物，主要生长在荒漠或半荒漠地区。唐古特白刺所具有的耐干旱、耐盐碱、抗沙埋等特性使得其成为防风固沙、保持水土、改良土壤等不可多得的灌木品种。唐古特白刺在《中药大辞典》中被称为卡密，以果熟时采收，晒干入药，具有健脾胃。滋补强壮、调经活血的功能，主治身体瘦弱、气血两亏、脾胃不和、消化不良、月经不调、虚寒腰痛等症。《本草纲目》中记述，白刺“气味辛、寒、无毒。主治心绞痛、痈肿溃脓，止痛。疗丈夫虚损，阳瘘精自出，补肾气，益精髓等”。在民间唐古特白刺的果实还被于哺乳期妇女的日常饮食中，促进泌乳。研究表明白刺果实中含花色苷、总多酚、多糖、氨基酸、维生素、黄酮、生物碱、矿物质元素等成分，具有显著的降血糖、降血脂、调节免疫、抗疲劳、耐寒冷、抗氧化、延缓衰老、保肝作用、抗肿瘤抗癌等生物活性。 Nitraria Tangutorum Bobr (Nitraria Tangutorum Bobr) is a plant of the Nitraria genus Zygophyllaceae. It is a medicinal and edible plant that mainly grows in desert or semi-desert areas. Nitraria tangutica has the characteristics of drought resistance, salt and alkali resistance, and sand burial resistance, making it a rare shrub species for windbreak and sand fixation, water and soil conservation, and soil improvement. Tangut Nitraria is called Kami in the "Dictionary of Traditional Chinese Medicine". It is harvested when the fruit is ripe, dried and used as medicine, which has the effect of invigorating the spleen and stomach. Nourishes and strengthens, regulates menstruation and activates blood circulation, mainly treats diseases such as emaciation, deficiency of qi and blood, disharmony between spleen and stomach, indigestion, irregular menstruation, and low back pain due to deficiency and cold. According to the "Compendium of Materia Medica", Nitraria "smells pungent, cold, and non-toxic. It is mainly used to treat angina pectoris, carbuncle, ulceration and pus, and relieve pain. It can treat the deficiency of the husband, the essence of yang fistula will come out, nourish the kidney qi, and benefit the essence, etc." In folklore, the fruit of Nitraria tangutica is also used in the daily diet of lactating women to promote lactation. Studies have shown that the fruit of Nitraria thorn contains anthocyanins, total polyphenols, polysaccharides, amino acids, vitamins, flavonoids, alkaloids, mineral elements, etc. Oxidation, anti-aging, liver protection, anti-tumor and anti-cancer and other biological activities.
为了深度开发利用白刺，中国专利201010298758.x公开了一种从白刺果实中提取分离色氨酸的方法，它是将白刺果实压榨后，去除籽皮，取果汁过陶瓷膜除杂后，上大孔吸附树脂柱，依次用水、40％～100％乙醇洗脱，收集乙醇洗脱液，超滤后即可得到色氨酸。该方法中弃去了种子和水洗脱液。 In order to further develop and utilize Nitraria, Chinese patent 201010298758.x discloses a method for extracting and separating tryptophan from Nitraria fruit, which is to squeeze the Nitraria fruit, remove the seed skin, and take the juice through a ceramic membrane to remove impurities , put it on a macroporous adsorption resin column, elute with water and 40%-100% ethanol in sequence, collect the ethanol eluate, and obtain tryptophan after ultrafiltration. The seeds and water eluate were discarded in this method.
目前还未见采用大孔吸附树脂从白刺种子中提取分离L-色氨酸的方法。 There is no method for extracting and separating L-tryptophan from Nitraria seeds by adopting macroporous adsorption resin at present.
发明内容 Contents of the invention
本发明的目的在于提供一种从白刺种子中分离提取L-色氨酸的方法。 The object of the present invention is to provide a method for separating and extracting L-tryptophan from Nitraria japonica seeds.
本发明研究发现，从白刺种子中提取分离L-色氨酸的方法，它包括如下操作步骤： The present invention finds that the method for extracting and separating L-tryptophan from the seeds of Nitraria japonica comprises the following steps:
(1)取白刺种子，以乙醇或含水乙醇为溶剂进行提取，合并提取液，回收乙醇，浓缩，以石油醚萃取，收集水层； (1) Take Nitraria japonica seeds, extract with ethanol or hydrous ethanol as a solvent, combine the extracts, recover ethanol, concentrate, extract with petroleum ether, and collect the water layer;
(2)取步骤(1)收集的水层，过大孔吸附树脂柱，以水洗脱，收集水洗脱液，浓缩，干燥，即得L-色氨酸粗品； (2) Take the water layer collected in step (1), pass through a macroporous adsorption resin column, elute with water, collect the water eluate, concentrate, and dry to obtain the crude product of L-tryptophan;
(3)取L-色氨酸粗品，纯化，即得L-色氨酸纯品。 (3) Take the crude product of L-tryptophan and purify it to obtain the pure product of L-tryptophan.
进一步地，步骤(1)中，所述含水乙醇浓度为60％～90％。 Further, in step (1), the concentration of the aqueous ethanol is 60%-90%.
更进一步地，步骤(1)中，所述含水乙醇浓度为70％～80％，优选为75％。 Furthermore, in step (1), the concentration of the aqueous ethanol is 70%-80%, preferably 75%.
进一步地，步骤(1)中，提取温度为50～80℃，优选为60℃。 Further, in step (1), the extraction temperature is 50-80°C, preferably 60°C.
进一步地，步骤(2)中，所述大孔吸附树脂为非极性大孔吸附树脂或弱极性大孔吸附树脂。 Further, in step (2), the macroporous adsorption resin is a non-polar macroporous adsorption resin or a weakly polar macroporous adsorption resin.
更近一步地，步骤(2)中，所述非极性大孔吸附树脂为D101型大孔吸附树脂，HPD100，HPD300，HP-10，X-5；所述弱极性大孔吸附树脂为AB-8型大孔吸附树脂。 Further, in step (2), the non-polar macroporous adsorption resin is D101 type macroporous adsorption resin, HPD100, HPD300, HP-10, X-5; the weakly polar macroporous adsorption resin is AB-8 type macroporous adsorption resin.
进一步地，水洗脱的具体操作为：用水洗脱19个柱体积，收集第7～19个柱体积的水洗脱液。本发明研究发现，采用上述洗脱方式，即可得到纯度90％以上的L-色氨酸。 Further, the specific operation of water elution is as follows: 19 column volumes are eluted with water, and the water eluate of the 7th to 19th column volumes is collected. The research of the present invention finds that L-tryptophan with a purity of more than 90% can be obtained by adopting the above-mentioned elution method.
本发明一个具体实施方式中，大孔树脂柱规格为：直径5厘米，柱高1.2米。 In a specific embodiment of the present invention, the specifications of the macroporous resin column are: 5 cm in diameter and 1.2 m in height.
进一步地，步骤(3)中，所述纯化采用制备液相色谱法或半制备液相色谱法。 Further, in step (3), the purification adopts preparative liquid chromatography or semi-preparative liquid chromatography.
其中，所述半制备液相色谱法中，检测波长为254nm，以甲醇-水为流动相，梯度条件如下：0-10min，10％甲醇；10-20min，10％-15％甲醇；20-30min，15％甲醇；30-50min，15％-20％甲醇；50-55min，20％-95％甲醇；55-65min，95％甲醇。 Wherein, in the semi-preparative liquid chromatography, the detection wavelength is 254nm, and methanol-water is used as the mobile phase, and the gradient conditions are as follows: 0-10min, 10% methanol; 10-20min, 10%-15% methanol; 20- 30min, 15% methanol; 30-50min, 15%-20% methanol; 50-55min, 20%-95% methanol; 55-65min, 95% methanol.
进一步地，所述半制备液相色谱法中，色谱柱为20*250mm的C18柱。 Further, in the semi-preparative liquid chromatography, the chromatographic column is a 20*250mm C18 column.
中国专利201010298758.x中，虽然是以白刺果汁为原料进行了色氨酸的提取，然而，其大孔树脂纯化步骤中收集的是40％～100％乙醇洗脱部位，水洗部位被弃去了；令人意想不到的是，本发明研究发现，以白刺种子为原料，对大孔树脂直接进行水洗后，就分离提取得到了高纯度的L-色氨酸粗品和L-色氨酸纯品。由此可见，本发明使用的洗脱部位与现有专利技术完全不同，其洗脱工艺仅需要水洗液，其操作更简便，成本更为低廉，同时也为白刺种子的开发利用提供了新的选择。 In Chinese patent 201010298758.x, although the extraction of tryptophan is carried out with the juice of Nitraria japonica as raw material, the eluted part with 40% to 100% ethanol is collected in the macroporous resin purification step, and the washed part is discarded Unexpectedly, the present invention found that the Nitraria seeds were used as raw materials, and after the macroporous resin was directly washed with water, high-purity crude L-tryptophan and pure L-tryptophan were obtained through separation and extraction. Taste. It can be seen that the elution part used in the present invention is completely different from the existing patented technology. Its elution process only needs water washing solution, its operation is simpler, and the cost is lower. It also provides a new method for the development and utilization of Nitraria seeds s Choice.
另外，相比目前其他色氨酸硅胶、凝胶柱层析工艺而言，本发明仅仅只使用大孔树脂，操作简便，并且洗脱液无需使用有机溶剂，工艺更为环保，安全性更高。 In addition, compared with other current tryptophan silica gel and gel column chromatography processes, the present invention only uses macroporous resin, which is easy to operate, and the eluent does not need to use organic solvents, the process is more environmentally friendly and safer .
附图说明 Description of drawings
图1大孔树脂直接洗脱产物的液相色谱图，液相条件：0-30min10％-45％B，流动相A：水B：甲醇(分析时所用检测波长是254nm) The liquid chromatogram of Fig. 1 macroporous resin direct elution product, liquid condition: 0-30min10%-45% B, mobile phase A: water B: methyl alcohol (used detection wavelength is 254nm during analysis)
图2半制备液相色谱图，主峰为L-色氨酸(制备色谱中所用检测波长是254nm) Fig. 2 semi-preparative liquid chromatogram, main peak is L-tryptophan (detection wavelength used in preparative chromatography is 254nm)
图3纯化后L-色氨酸的液相色谱图，色谱条件如图1 The liquid chromatogram of L-tryptophan after Fig. 3 purification, chromatographic conditions are as shown in Fig. 1
具体实施方式 detailed description
实施例1：L-色氨酸分离提取方法 Embodiment 1: L-tryptophan separation and extraction method
(1)取5kg的白刺种子粉碎后加入75％的乙醇在煎药机中进行热提取，提取温度60℃，提取时间2小时，共提取三次。第一次料液比为1∶15，第二次料液比为1∶12，第三次料液比为1∶10。合并提取液并减压浓缩250mL后石油醚萃取5次，石油醚萃取比例为250mL粗提液加入750mL石油醚。 (1) After taking 5 kg of Nitraria seeds and pulverizing them, add 75% ethanol and carry out thermal extraction in a decoction machine at an extraction temperature of 60° C. and an extraction time of 2 hours, and extract three times altogether. The first feed-to-liquid ratio is 1:15, the second feed-to-liquid ratio is 1:12, and the third feed-to-liquid ratio is 1:10. The extracts were combined and concentrated under reduced pressure to 250 mL, then extracted 5 times with petroleum ether, and the extraction ratio of petroleum ether was 250 mL of crude extract and 750 mL of petroleum ether.
(2)大孔树脂柱规格：直径5厘米，柱高1.2米 (2) Specifications of macroporous resin column: diameter 5 cm, column height 1.2 m
上柱：取1.5kgD101大孔吸附树脂，装入层析柱，吸附三天后进行解吸附处理，用水冲柱每一个柱体积接样后检测(柱体积约为2L)，当冲柱到第7个柱体积后上HPLC检测其组成主要为化合物1。后续继续冲柱，一共冲了19个柱体积后将7-19合并后在旋转蒸发仪上浓缩得到6.32g样品，即为L-色氨酸粗品，其含量占整个水冲物质含量的90％以上(见图1) Loading the column: Take 1.5kg D101 macroporous adsorption resin, put it into the chromatography column, perform desorption treatment after three days of adsorption, wash the column with water for each column volume and collect samples (column volume is about 2L), when the column reaches the 7th After a column volume, it was detected by HPLC that its composition was mainly compound 1. Continue to flush the column. After flushing a total of 19 column volumes, combine 7-19 and concentrate on a rotary evaporator to obtain a 6.32g sample, which is the crude product of L-tryptophan, and its content accounts for 90% of the total content of the flushed substance. Above (See Figure 1)
(3)将6.32g样品复溶到200mL蒸馏水中，上半制备液相色谱进行进一步纯化与精制，每次上样20mL，样品经半制备纯化后HPLC检测其纯度达到了99.7％，共得到5.88g高纯度样品。 (3) 6.32g sample was redissolved in 200mL distilled water, and the first half of preparative liquid chromatography was further purified and refined, each time 20mL was loaded, and the purity of the sample was detected by HPLC after semi-preparative purification reached 99.7%, and a total of 5.88 g high-purity samples.
半制备条件： Semi-preparation conditions:
半制备柱：DubheC18柱(20*250mm)汉邦科技有限公司。检测器：NU3000SERIALSUV/VISDetecTor，汉邦科技有限公司。泵：NP7000SERIALSPUMP，汉邦科技有限公司。 Semi-preparative column: DubheC18 column (20*250mm) Hanbang Technology Co., Ltd. Detector: NU3000SERIALSUV/VISDetectecTor, Hanbang Technology Co., Ltd. Pump: NP7000SERIALSPUMP, Hanbang Technology Co., Ltd.
半制备梯度条件：0-10min，10％甲醇；10-20min，10％-15％甲醇；20-30min，15％甲醇；30-50min，15％-20％甲醇；50-55min，20％-95％甲醇；55-65min，95％甲醇(若采用制备色谱，可以采用同样的流动相条件) Semi-preparative gradient conditions: 0-10min, 10% methanol; 10-20min, 10%-15% methanol; 20-30min, 15% methanol; 30-50min, 15%-20% methanol; 50-55min, 20%- 95% methanol; 55-65min, 95% methanol (if using preparative chromatography, the same mobile phase conditions can be used)
经NMR鉴定，该化合物为L-色氨酸。 The compound was identified as L-tryptophan by NMR.
NMR数据为：13CNMR(DMSO-d6)δ124.1(d，C-2)，109.5(s，C-3)，118.4(d，C-4)，118.3(d，C-5)，120.9(d，6-C)，127.3(s，C-3a)，136.3(s，C-7a)，27.1(t，CH2)，54.7(d，CH)，170.3(s，CO) NMR data: 13 CNMR (DMSO-d 6 ) δ 124.1 (d, C-2), 109.5 (s, C-3), 118.4 (d, C-4), 118.3 (d, C-5), 120.9(d, 6-C), 127.3(s, C-3a), 136.3(s, C-7a), 27.1(t, CH2 ), 54.7(d, CH), 170.3(s, CO)
1HNMR(DMSO-d6)δ10.94(1H，s，NH)，7.21(1H，s，2-H)，7.56(1H，d，J＝8.0Hz，4-H)，6.97(1H，d，J＝8.0Hz，5-H)，7.04(1H，t，J＝8.0Hz，6-H)，7.33(1H，J＝8.0Hz，7-H)，3.32(1H，dd，J＝3.0，15.0Hz，CH2)，2.95(1H，dd，J＝9.0，15.0Hz，CH2)，3.46(1H，J＝5.0，8.0Hz，CH) 1 HNMR (DMSO-d 6 ) δ10.94 (1H, s, NH), 7.21 (1H, s, 2-H), 7.56 (1H, d, J=8.0Hz, 4-H), 6.97 (1H, d, J = 8.0Hz, 5-H), 7.04 (1H, t, J = 8.0Hz, 6-H), 7.33 (1H, J = 8.0Hz, 7-H), 3.32 (1H, dd, J = 3.0, 15.0Hz, CH 2 ), 2.95 (1H, dd, J=9.0, 15.0Hz, CH 2 ), 3.46 (1H, J=5.0, 8.0Hz, CH)
实施例2：L-色氨酸分离提取方法 Embodiment 2: L-tryptophan separation and extraction method
(1)取5kg的白刺种子粉碎后加入75％的乙醇在煎药机中进行热提取，提取温度60℃，提取时间3小时，共提取三次。第一次料液比为1∶12，第二次料液比为1∶10，第三次料液比为1∶8。取液并减压浓缩200mL后石油醚萃取5次，石油醚萃取比例为200mL粗提液加入700mL石油醚。 (1) Get 5kg of Nitraria japonica seeds and add 75% ethanol after pulverizing to carry out thermal extraction in a decoction machine. The extraction temperature is 60° C., and the extraction time is 3 hours, and the extracts are extracted three times. The first feed-to-liquid ratio is 1:12, the second feed-to-liquid ratio is 1:10, and the third feed-to-liquid ratio is 1:8. The liquid was taken and concentrated under reduced pressure to 200mL, and then extracted 5 times with petroleum ether, and the extraction ratio of petroleum ether was 200mL of crude extract and 700mL of petroleum ether.
(2)大孔树脂柱规格：直径5厘米，柱高1.2米 (2) Specifications of macroporous resin column: diameter 5 cm, column height 1.2 m
上柱：取1.5kgD101大孔吸附树脂，装入层析柱，吸附三天后进行解吸附处理，用水冲柱每一个柱体积接样后检测(柱体积约为2L)，当冲柱到第7个柱体积后上HPLC检测其组成主要为化合物1。后续继续冲柱，一共冲了19个柱体积后将7-19合并后在旋转蒸发仪上浓缩得到L-色氨酸粗品； Column loading: Take 1.5kg D101 macroporous adsorption resin, put it into the chromatography column, and perform desorption treatment after three days of adsorption, flush the column with water for each column volume and collect samples (column volume is about 2L), when the column is flushed to the 7th After a column volume, it was detected by HPLC that its composition was mainly compound 1. Continue to flush the column, after flushing a total of 19 column volumes, combine 7-19 and concentrate on a rotary evaporator to obtain crude L-tryptophan;
(3)将粗品复溶到200mL蒸馏水中，上半制备液相色谱进行进一步纯化与精制，每次上样20mL，样品经半制备纯化后HPLC检测其纯度达到了99.7％。 (3) The crude product was redissolved in 200 mL of distilled water, and further purified and refined by the first half of preparative liquid chromatography, each time 20 mL of sample was loaded, and the purity of the sample reached 99.7% by HPLC after semi-preparative purification.
半制备条件： Semi-preparation conditions:
半制备柱：DubheC18柱(20*250mm)汉邦科技有限公司。检测器：NU3000SERIALSUV/VISDetecTor，汉邦科技有限公司。泵：NP7000SERIALSPUMP，汉邦科技有限公司。 Semi-preparative column: DubheC18 column (20*250mm) Hanbang Technology Co., Ltd. Detector: NU3000SERIALSUV/VISDetectecTor, Hanbang Technology Co., Ltd. Pump: NP7000SERIALSPUMP, Hanbang Technology Co., Ltd.
半制备梯度条件：0-10min，10％甲醇；10-20min，10％-15％甲醇；20-30min，15％甲醇；30-50min，15％-20％甲醇；50-55min，20％-95％甲醇；55-65min，95％甲醇(若采用制备色谱，可以采用同样的流动相条件)。 Semi-preparative gradient conditions: 0-10min, 10% methanol; 10-20min, 10%-15% methanol; 20-30min, 15% methanol; 30-50min, 15%-20% methanol; 50-55min, 20%- 95% methanol; 55-65min, 95% methanol (if using preparative chromatography, the same mobile phase conditions can be used).
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