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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- CN104262231B CN104262231B CN201410542537.0A CN201410542537A CN104262231B CN 104262231 B CN104262231 B CN 104262231B CN 201410542537 A CN201410542537 A CN 201410542537A CN 104262231 B CN104262231 B CN 104262231B
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- 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
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- 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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Abstract
The present invention studies discovery, extracts the method that separates L-Trp from white thorn seed, and it comprises following operating procedure: (1) gets white thorn seed, extract taking ethanol or hydrous ethanol as solvent, merge extract, reclaim ethanol, concentrated, with petroleum ether extraction, collect water layer; (2) get the extract of step (1), cross large pore resin absorption column, with water elution, collect water elution liquid, concentrated, dry, obtain L-Trp crude product; (3) get L-Trp crude product, purifying, obtains L-Trp sterling. The present invention studies discovery, to sting in vain seed as raw material, after macroreticular resin is directly washed, has obtained highly purified L-Trp crude product and L-Trp sterling with regard to separation and Extraction. The wash-out position that the present invention uses is completely different from existing patented technology, and its elution processes only needs water lotion, and its operation is easier, and cost is more cheap, simultaneously also for the exploitation of white thorn seed provide new selection.
Description
Technical field
The present invention relates to the separating and extracting process of L-Trp.
Background technology
The Gu Tebaici of Tang (NitrariaTangutorumBobr) is zygophyllaceae (Zygophyllaceae) Nitraria plant, is a kind of plant of medicine-food two-purpose, is mainly grown in desert or Semi-desert Area. The characteristic such as that the Gu Tebaici of Tang has is drought-resistant, Salt And Alkali Tolerance, desertification bury becomes it and checks winds and fixes drifting sand, conserves water and soil, improves the rare shrub kinds such as soil. It is close that the Gu Tebaici of Tang is called as card in " Chinese medicine voluminous dictionary ", when ripe with fruit, gathers, and dries and be used as medicine, and has strengthening the spleen and stomach. The function of strengthening by means of tonics, regulating menstruation and activating blood, cures mainly gynecological ailments, qi-blood deficiency, incoordination between the spleen and the stomach, indigestion, irregular menstruation, cold of insufficiency type lumbago. In Compendium of Material Medica, describe, " smell is pungent, cold, nontoxic for white thorn. Cure mainly angina pectoris, the swollen purulence of bursting of carbuncle, pain relieving. Treat husband deficient, positive fistula essence goes out certainly, kidney tonifying gas, beneficial marrow etc. ". Also by women breast-feeding their children's diet, promote lactation at the fruit of the Gu Tebaici of Tang among the people. Research shows in berry of nitraria tangutorum bobr containing compositions such as anthocyanin, total polyphenols, polysaccharide, amino acid, vitamin, flavones, alkaloid, mineral matter elements, have hypoglycemic, reducing blood lipid significantly, adjusting immunity, antifatigue, cold resistant, anti-oxidant, delay senility, hepatoprotective effect, the antitumor biologically active such as anticancer.
For deep development utilization is stung in vain, Chinese patent 201010298758.x discloses a kind of method of extracting separating tryptophane from berry of nitraria tangutorum bobr, it is by after berry of nitraria tangutorum bobr squeezing, remove seed skin, get fruit juice and cross after ceramic membrane removal of impurities, upper large pore resin absorption column, successively water, 40%～100% ethanol elution, collect ethanol eluate, after ultrafiltration, can obtain tryptophan. Seed and water elution liquid in the method, are discarded.
Yet there are no and adopt macroporous absorbent resin from white thorn seed, to extract the method that separates L-Trp.
Summary of the invention
The object of the present invention is to provide a kind of from white thorn seed the method for separation and Extraction L-Trp.
The present invention studies discovery, extracts the method that separates L-Trp from white thorn seed, and it comprises following operating procedure:
(1) get white thorn seed, extract taking ethanol or hydrous ethanol as solvent, merge extract, reclaim ethanol, concentrated, with petroleum ether extraction, collect water layer;
(2) get the water layer that step (1) is collected, cross large pore resin absorption column, with water elution, collect water elution liquid, concentrated, dry, obtain L-Trp crude product;
(3) get L-Trp crude product, purifying, obtains L-Trp sterling.
Further, in step (1), described hydrous ethanol concentration is 60%～90%.
Further, in step (1), described hydrous ethanol concentration is 70%～80%, is preferably 75%.
Further, in step (1), extracting temperature is 50～80 DEG C, is preferably 60 DEG C.
Further, in step (2), described macroporous absorbent resin is nonpolar macroporous adsorption resin or low pole macroporous absorbent resin.
Closer, in step (2), described nonpolar macroporous adsorption resin is D101 type macroporous absorbent resin, HPD100, HPD300, HP-10, X-5; Described low pole macroporous absorbent resin is AB-8 type macroporous absorbent resin.
Further, the concrete operations of water elution are: wash 19 column volumes with water, collect the water elution liquid of 7th～19 column volumes. The present invention studies discovery, adopts above-mentioned type of elution, can obtain more than 90% L-Trp of purity.
In detailed description of the invention of the present invention, macroporous resin column specification is: 5 centimetres of diameters, post is high 1.2 meters.
Further, in step (3), described purifying adopts preparative liquid chromatography or half preparative liquid chromatography.
Wherein, in described half preparative liquid chromatography, detection wavelength is 254nm, and taking methanol-water as mobile phase, gradient condition is as follows: 0-10min, 10% methyl alcohol; 10-20min, 10%-15% methyl alcohol; 20-30min, 15% methyl alcohol; 30-50min, 15%-20% methyl alcohol; 50-55min, 20%-95% methyl alcohol; 55-65min, 95% methyl alcohol.
Further, in described half preparative liquid chromatography, the C18 post that chromatographic column is 20*250mm.
In Chinese patent 201010298758.x, although be the extraction of having carried out tryptophan taking nitraria sibirica pall juice as raw material, but what in its macroporous resin purification step, collect is 40%～100% alcohol elution, washing position has been discarded; Unexpectedly, the present invention studies discovery, to sting in vain seed as raw material, after macroreticular resin is directly washed, has obtained highly purified L-Trp crude product and L-Trp sterling with regard to separation and Extraction. As can be seen here, the wash-out position that the present invention uses is completely different from existing patented technology, and its elution processes only needs water lotion, and its operation is easier, and cost is more cheap, simultaneously also for the exploitation of white thorn seed provide new selection.
In addition, compare at present other tryptophan silica gel, gel filtration chromatography technique, the present invention only uses macroreticular resin, easy and simple to handle, and eluent is without with an organic solvent, and technique is environmental protection more, and security is higher.
Brief description of the drawings
The liquid chromatogram of the direct eluted product of Fig. 1 macroreticular resin, liquid-phase condition: 0-30min10%-45%B, mobile phase A: water B: methyl alcohol (when analysis, detection wavelength used is 254nm)
Fig. 2 half preparative liquid chromatography figure, main peak is L-Trp (in preparative chromatography, detection wavelength used is 254nm)
The liquid chromatogram of L-Trp after Fig. 3 purifying, chromatographic condition is as Fig. 1
Detailed description of the invention
Embodiment 1:L-tryptophan separating and extracting process
(1) the white thorn seed of getting 5kg adds 75% ethanol in boiling machine, to carry out heat to extract after pulverizing, extraction temperature 60 C, 2 hours extraction times, extracts three times altogether. Solid-liquid ratio is 1: 15 for the first time, and solid-liquid ratio is 1: 12 for the second time, and solid-liquid ratio is 1: 10 for the third time. Petroleum ether extraction 5 times after merging extract reduced pressure concentration 250mL, petroleum ether extraction ratio is that 250mL crude extract adds 750mL benzinum.
(2) macroporous resin column specification: 5 centimetres of diameters, post is high 1.2 meters
Upper prop: get 1.5kgD101 macroporous absorbent resin, pack chromatographic column into, adsorb and carry out desorption processing after three days, water rushes after each column volume of post connects sample and detects (column volume is about 2L), and on rushing after 7 column volumes of post to the, HPLC detects its composition and is mainly compound 1. Follow-uply continue to rush post, rushed altogether and concentratedly on Rotary Evaporators after 7-19 being merged after 19 column volumes obtained 6.32g sample, be L-Trp crude product, what its content accounted for that whole water rushes content of material more than 90% (sees Fig. 1)
(3) 6.32g sample is redissolved in 200mL distilled water, first preparative liquid chromatography is further purified with refining, each loading 20mL, and sample HPLC after partly preparing purifying detects its purity and has reached 99.7%, obtains altogether 5.88g high-purity sample.
Half preparation condition:
Semi-preparative column: DubheC18 post (20*250mm) Han Bang Science and Technology Ltd.. Detector: NU3000SERIALSUV/VISDetecTor, Han Bang Science and Technology Ltd.. Pump: NP7000SERIALSPUMP, Han Bang Science and Technology Ltd..
Partly prepare gradient condition: 0-10min, 10% methyl alcohol; 10-20min, 10%-15% methyl alcohol; 20-30min, 15% methyl alcohol; 30-50min, 15%-20% methyl alcohol; 50-55min, 20%-95% methyl alcohol; 55-65min, 95% methyl alcohol (if adopt preparative chromatography, can adopt same mobile phase condition)
Through NMR qualification, this compound is L-Trp.
NMR data are: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)
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)
Embodiment 2:L-tryptophan separating and extracting process
(1) the white thorn seed of getting 5kg adds 75% ethanol in boiling machine, to carry out heat to extract after pulverizing, extraction temperature 60 C, 3 hours extraction times, extracts three times altogether. Solid-liquid ratio is 1: 12 for the first time, and solid-liquid ratio is 1: 10 for the second time, and solid-liquid ratio is 1: 8 for the third time. Get after liquid reduced pressure concentration 200mL petroleum ether extraction 5 times, petroleum ether extraction ratio is that 200mL crude extract adds 700mL benzinum.
(2) macroporous resin column specification: 5 centimetres of diameters, post is high 1.2 meters
Upper prop: get 1.5kgD101 macroporous absorbent resin, pack chromatographic column into, adsorb and carry out desorption processing after three days, water rushes after each column volume of post connects sample and detects (column volume is about 2L), and on rushing after 7 column volumes of post to the, HPLC detects its composition and is mainly compound 1. Follow-uply continue to rush post, rushed altogether after 19 column volumes will 7-19 merge after on Rotary Evaporators the concentrated L-Trp crude product that obtains;
(3) crude product is redissolved in 200mL distilled water, first preparative liquid chromatography is further purified with refining, each loading 20mL, and sample HPLC after partly preparing purifying detects its purity and has reached 99.7%.
Half preparation condition:
Semi-preparative column: DubheC18 post (20*250mm) Han Bang Science and Technology Ltd.. Detector: NU3000SERIALSUV/VISDetecTor, Han Bang Science and Technology Ltd.. Pump: NP7000SERIALSPUMP, Han Bang Science and Technology Ltd..
Partly prepare gradient condition: 0-10min, 10% methyl alcohol; 10-20min, 10%-15% methyl alcohol; 20-30min, 15% methyl alcohol; 30-50min, 15%-20% methyl alcohol; 50-55min, 20%-95% methyl alcohol; 55-65min, 95% methyl alcohol (if adopt preparative chromatography, can adopt same mobile phase condition).
Claims (12)
1. from white thorn seed, extract the method that separates L-Trp, it is characterized in that: it comprises as followsOperating procedure:
(1) get white thorn seed, extract taking hydrous ethanol as solvent, merge extract, reclaim ethanol,Concentrated, with petroleum ether extraction, collect water layer;
(2) get the water layer that step (1) is collected, cross large pore resin absorption column, with water elution, collect waterEluent, concentrated, dry, obtain L-Trp crude product;
(3) get L-Trp crude product, purifying, obtains L-Trp sterling.
2. the method that separates L-Trp of extracting from white thorn seed according to claim 1, itsBe characterised in that: in step (1), described hydrous ethanol concentration is 60%～90%v/v.
3. the method that separates L-Trp of extracting from white thorn seed according to claim 2, itsBe characterised in that: in step (1), described hydrous ethanol concentration is 70～80%v/v.
4. the method that separates L-Trp of extracting from white thorn seed according to claim 3, itsBe characterised in that: in step (1), described hydrous ethanol concentration is 75%v/v.
5. the method that separates L-Trp of extracting from white thorn seed according to claim 1, itsBe characterised in that: in step (1), extracting temperature is 50～80 DEG C.
6. the method that separates L-Trp of extracting from white thorn seed according to claim 5, itsBe characterised in that: in step (1), extracting temperature is 60 DEG C.
7. the method that separates L-Trp of extracting from white thorn seed according to claim 1, itsBe characterised in that: in step (2), described macroporous absorbent resin is nonpolar macroporous adsorption resin or low poleMacroporous absorbent resin.
8. the method that separates L-Trp of extracting from white thorn seed according to claim 7, itsBe characterised in that: in step (2), described nonpolar macroporous adsorption resin is D101, HPD100, HP-10,X-5 type macroporous absorbent resin; Described low pole macroporous absorbent resin is AB-8 type macroporous absorbent resin.
9. separate L-look ammonia according to extracting described in claim 1,7 or 8 any one from white thorn seedThe method of acid, is characterized in that: the concrete operations of water elution are: wash 19 column volumes with water, collectThe water elution liquid of 7th～19 column volumes.
10. the method that separates L-Trp of extracting from white thorn seed according to claim 1, itsBe characterised in that: in step (3), described purifying adopts preparative liquid chromatography or half preparative liquid chromatography.
11. methods that separate L-Trp of extracting from white thorn seed according to claim 10,It is characterized in that: in described half preparative liquid chromatography, detection wavelength is 254nm, taking methanol-water as streamMoving phase, gradient condition is as follows: 0-10min, 10% methyl alcohol; 10-20min, 10%-15% methyl alcohol; 20-30Min, 15% methyl alcohol; 30-50min, 15%-20% methyl alcohol; 50-55min, 20%-95% methyl alcohol; 55-65min,95% methyl alcohol.
12. methods that separate L-Trp of extracting from white thorn seed according to claim 11,It is characterized in that: in described half preparative liquid chromatography, the C18 post that chromatographic column is 20*250mm.
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| CN111253298A (en) * | 2020-02-10 | 2020-06-09 | 南阳师范学院 | Method for separating L-tryptophan by using ultrahigh cross-linked adsorption resin |
| CN114646695B (en) * | 2020-12-21 | 2023-11-21 | 江阴天江药业有限公司 | Ultra-high performance liquid chromatography detection method of starwort root standard decoction and application thereof |
| CN114082222B (en) * | 2021-10-25 | 2023-03-17 | 青海大学 | Method for purifying free amino acids of Ribes nigrum L |
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| US20140148399A1 (en) * | 2012-11-26 | 2014-05-29 | Center Of Biotechnology Of Borj Cedria | Compositions and methods for treating or ameliorating obesity or for reducing diabetic hypercholesterolemia |
| CN103054021A (en) * | 2012-12-31 | 2013-04-24 | 青海省轻工业研究所有限责任公司 | Method for extracting compound from nitraria tangutorum peel |
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Effective date of registration: 20220818 Address after: Room 939-1, 9th Floor, Building 3, No. 1366 Hongfeng Road, Huzhou City, Zhejiang Province 313000 Patentee after: Zhejiang Zidi Technology Co., Ltd. Address before: 810001 No.23 Xinning road Xinning City Qinghai Province Patentee before: Northwest Institute of Plateau Biology, Chinese Academy of Sciences Patentee before: Qinghai University |