CN104262231A - Method for extracting and separating L-tryptophan from nitraria tangutorum bobr seeds - Google Patents
Method for extracting and separating L-tryptophan from nitraria tangutorum bobr seeds Download PDFInfo
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- CN104262231A CN104262231A CN201410542537.0A CN201410542537A CN104262231A CN 104262231 A CN104262231 A CN 104262231A CN 201410542537 A CN201410542537 A CN 201410542537A CN 104262231 A CN104262231 A CN 104262231A
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- macroporous
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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 invention discloses a method for extracting and separating L-tryptophan from nitraria tangutorum bobr seeds. The method comprises operation steps as follows: (1), the nitraria tangutorum bobr seeds are taken and extracted by taking ethanol or hydrous ethanol as a solvent, extracting solutions are combined, ethanol is recovered, concentration is performed, petroleum ether is adopted for extraction, and an aqueous layer is collected; (2), the extraction liquor is taken to pass macroporous adsorption resin columns, water is adopted for elution, water eluent is collected, and a crude product of L-tryptophan is obtained after concentration and drying; and (3), the crude product of L-tryptophan is taken and purified to obtain a pure product of L-tryptophan. According to the research of the invention, the nitraria tangutorum bobr seeds are taken as raw materials, macroporous resin is adopted for washing, and then high-purity crude and pure products of L-tryptophan are obtained through separation and extraction. The eluting part is totally different from that in the prior art, the eluting technology only requires a washing liquid, the operation is simpler and more convenient, the cost is lower, and meanwhile, a new choice is provided for development and utilization of the nitraria tangutorum bobr seeds.
Description
Technical field
The present invention relates to the separating and extracting method of L-Trp.
Background technology
Nitraria tangutorum (Nitraria Tangutorum Bobr) is zygophyllaceae (Zygophyllaceae) Nitraria plant, and be a kind of plant of medicine-food two-purpose, primary growth is in desert or Semi-desert Area.Drought-resistant, the Salt And Alkali Tolerance that Nitraria tangutorum has, desertification the characteristic such as to bury and it are become check winds and fix drifting sand, conserve water and soil, improve the rare shrub kind such as soil.It is close that Nitraria tangutorum is called as card in " Chinese medicine voluminous dictionary ", gathers, dry and be used as medicine, have strengthening the spleen and stomach time ripe with fruit.The function of strengthening by means of tonics, regulating menstruation and activating blood, cures mainly asthenic body, deficiency of both QI and blood, incoordination between the spleen and the stomach, maldigestion, menoxenia, cold of insufficiency type lumbago.Describe in Compendium of Material Medica, " smell is pungent, cold, nontoxic for white thorn.Cure mainly stenocardia, the swollen purulence of bursting of carbuncle, pain relieving.Treat husband deficient, positive fistula essence goes out certainly, kidney tonifying gas, beneficial marrow etc. ".At the fruit of Nitraria tangutorum among the people also by the diet of lactating women, promote lactation.Research to show in berry of nitraria tangutorum bobr containing the composition such as anthocyanogen, total polyphenols, polysaccharide, amino acid, VITAMIN, flavones, alkaloid, mineral element, have hypoglycemic, reducing blood-fat significantly, immunity moderation, antifatigue, cold resistant, anti-oxidant, delay senility, hepatoprotective effect, the antitumor biological activity such as anticancer.
In order to deep development utilizes white thorn, Chinese patent 201010298758.x discloses a kind of method of extraction and isolation tryptophane from berry of nitraria tangutorum bobr, it is after being squeezed by berry of nitraria tangutorum bobr, remove seed skin, get after fruit juice crosses ceramic membrane removal of impurities, upper macroporous adsorptive resins, uses water, 40% ~ 100% ethanol elution successively, collect ethanol eluate, after ultrafiltration, can tryptophane be obtained.Seed and water elution liquid has been discarded in the method.
Yet there are no the method adopting macroporous adsorbent resin extraction and isolation L-Trp from white thorn seed.
Summary of the invention
The object of the present invention is to provide a kind of method of separation and Extraction L-Trp from white thorn seed.
The present invention studies discovery, the method for extraction and isolation L-Trp from white thorn seed, and it comprises following operation steps:
(1) get white thorn seed, with ethanol or aqueous ethanol for solvent extracts, united extraction liquid, reclaims ethanol, concentrated, with petroleum ether extraction, collects water layer;
(2) get the water layer that step (1) is collected, cross macroporous adsorptive resins, with water elution, collect water elution liquid, concentrated, dry, obtain L-Trp crude product;
(3) get L-Trp crude product, purifying, obtain L-Trp sterling.
Further, in step (1), described aqueous ethanol concentration is 60% ~ 90%.
Further, in step (1), described aqueous 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 adsorbent resin is nonpolar macroporous adsorption resin or low-pole macroporous adsorbent resin.
Closer, in step (2), described nonpolar macroporous adsorption resin is D101 type macroporous adsorbent resin, HPD100, HPD300, HP-10, X-5; Described low-pole macroporous adsorbent resin is AB-8 type macroporous adsorbent 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 the L-Trp of purity more than 90%.
In the present invention's embodiment, macroporous resin column specification is: diameter 5 centimetres, and post is high 1.2 meters.
Further, in step (3), described purifying adopts preparative liquid chromatography or semi-preparative liquid chromatography method.
Wherein, in described semi-preparative liquid chromatography method, determined wavelength is 254nm, and be moving phase with methanol-water, 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 semi-preparative liquid chromatography method, chromatographic column is the C18 post of 20*250mm.
In Chinese patent 201010298758.x, although be that raw material has carried out the extraction of tryptophane with nitraria sibirica pall juice, but what collect in its macroporous resin purification step is 40% ~ 100% alcohol elution, and washing position has been discarded; Unexpectedly, the present invention studies discovery, to sting seed in vain for raw material, after directly washing macroporous resin, obtains 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 other tryptophane silica gel, gel filtration chromatography technique at present, the present invention only uses macroporous resin, easy and simple to handle, and elutriant is without the need to an organic solvent, and technique is environmental protection more, and security is higher.
Accompanying drawing explanation
The liquid chromatogram of the direct eluted product of Fig. 1 macroporous resin, liquid-phase condition: 0-30min10%-45%B, mobile phase A: water B: methyl alcohol (during analysis, determined wavelength used is 254nm)
Fig. 2 semi-preparative liquid chromatography figure, main peak is L-Trp (in preparative chromatography, determined wavelength used is 254nm)
The liquid chromatogram of L-Trp after Fig. 3 purifying, chromatographic condition is as Fig. 1
Embodiment
Embodiment 1:L-tryptophane separating and extracting method
(1) ethanol adding 75% after getting the white thorn seed pulverizing of 5kg carries out heat extraction, Extracting temperature 60 DEG C, 2 hours extraction times in boiling machine, extracts three times altogether.First time, solid-liquid ratio was 1: 15, and second time solid-liquid ratio is 1: 12, and solid-liquid ratio is 1: 10 for the third time.United extraction liquid petroleum ether extraction 5 times after concentrating under reduced pressure 250mL, petroleum ether extraction ratio is that 250mL crude extract adds 750mL sherwood oil.
(2) macroporous resin column specification: diameter 5 centimetres, post is high 1.2 meters
Upper prop: get 1.5kg D101 macroporous adsorbent resin, load chromatography column, adsorb and carry out desorption process after three days, rush after each column volume of post connects sample with water and detect (column volume is about 2L), after rushing post to the 7th column volume, upper HPLC detects its composition and is mainly compound 1.Follow-up continuation rushes post, and concentrate on a rotary evaporator after being merged by 7-19 after having rushed altogether 19 column volumes and obtain 6.32g sample, be L-Trp crude product, its content accounts for more than 90% (see Fig. 1) that whole water rushes substances content
(3) redissolved by 6.32g sample in 200mL distilled water, upper semi-preparative liquid chromatography is further purified and refines, each loading 20mL, and sample HPLC after partly preparing purifying detects its purity and reaches 99.7%, obtains 5.88g high purity sample altogether.
Half preparation condition:
Semipreparative column: Dubhe C18 post (20*250mm) Han Bang Science and Technology Ltd..Detector: NU 3000SERIALS UV/VIS DetecTor, Han Bang Science and Technology Ltd..Pump: NP 7000SERIALS PUMP, 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 (according to preparative chromatography, same moving phase condition can be adopted)
Through NMR qualification, this compound is L-Trp.
NMR data are:
13c NMR (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, CH
2), 54.7 (d, CH), 170.3 (s, CO)
1H?NMR(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)
Embodiment 2:L-tryptophane separating and extracting method
(1) ethanol adding 75% after getting the white thorn seed pulverizing of 5kg carries out heat extraction, Extracting temperature 60 DEG C, 3 hours extraction times in boiling machine, extracts three times altogether.First time, solid-liquid ratio was 1: 12, and second time solid-liquid ratio is 1: 10, and solid-liquid ratio is 1: 8 for the third time.Get liquid and petroleum ether extraction 5 times after concentrating under reduced pressure 200mL, petroleum ether extraction ratio is that 200mL crude extract adds 700mL sherwood oil.
(2) macroporous resin column specification: diameter 5 centimetres, post is high 1.2 meters
Upper prop: get 1.5kg D101 macroporous adsorbent resin, load chromatography column, adsorb and carry out desorption process after three days, rush after each column volume of post connects sample with water and detect (column volume is about 2L), after rushing post to the 7th column volume, upper HPLC detects its composition and is mainly compound 1.Follow-up continuation rushes post, concentrates on a rotary evaporator and obtain L-Trp crude product after having rushed altogether 19 column volumes after being merged by 7-19;
(3) redissolved by crude product in 200mL distilled water, upper semi-preparative liquid chromatography is further purified and refines, each loading 20mL, and sample HPLC after partly preparing purifying detects its purity and reaches 99.7%.
Half preparation condition:
Semipreparative column: Dubhe C18 post (20*250mm) Han Bang Science and Technology Ltd..Detector: NU 3000SERIALS UV/VIS DetecTor, Han Bang Science and Technology Ltd..Pump: NP 7000SERIALS PUMP, 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 (according to preparative chromatography, same moving phase condition can be adopted).
Claims (10)
1., from white method of stinging extraction and isolation L-Trp seed, it is characterized in that: it comprises following operation steps:
(1) get white thorn seed, with ethanol or aqueous ethanol for solvent extracts, united extraction liquid, reclaims ethanol, concentrated, with petroleum ether extraction, collects water layer;
(2) get the water layer that step (1) is collected, cross macroporous adsorptive resins, with water elution, collect water elution liquid, concentrated, dry, obtain L-Trp crude product;
(3) get L-Trp crude product, purifying, obtain L-Trp sterling.
2. alcohol extraction procedure according to claim 1, is characterized in that: in step (1), and described aqueous ethanol concentration is 60% ~ 90%v/v.
3. alcohol extraction procedure according to claim 2, is characterized in that: in step (1), and described aqueous ethanol concentration is 70% ~ 80%v/v, is preferably 75%v/v.
4. alcohol extraction procedure according to claim 1, is characterized in that: in step (1), and Extracting temperature is 50 ~ 80 DEG C, is preferably 60 DEG C.
5. macroporous resin column separation method according to claim 1, is characterized in that: in step (2), and described macroporous adsorbent resin is nonpolar macroporous adsorption resin or low-pole macroporous adsorbent resin.
6. macroporous resin column separation method according to claim 5, is characterized in that: in step (2), and described nonpolar macroporous adsorption resin is D101 type macroporous adsorbent resin, HPD100, HPD 300, HP-10, X-5; Described low-pole macroporous adsorbent resin is AB-8 type macroporous adsorbent resin.
7. the macroporous resin column separation method according to claim 1,5 or 6, is characterized in that: the concrete operations of water elution are: wash 19 column volumes with water, collects the water elution liquid of 7th ~ 19 column volumes.
8. purification process according to claim 1, is characterized in that: in step (3), and described purifying adopts preparative liquid chromatography or semi-preparative liquid chromatography method.
9. purification process according to claim 8, is characterized in that: in described semi-preparative liquid chromatography method, determined wavelength is 254nm, and be moving phase with methanol-water, 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.
10. purification process according to claim 9, is characterized in that: in described semi-preparative liquid chromatography method, and chromatographic column is the C18 post of 20*250mm.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN114082222A (en) * | 2021-10-25 | 2022-02-25 | 青海大学 | Method for purifying free amino acid of Ribes nigrum |
| CN114646695A (en) * | 2020-12-21 | 2022-06-21 | 江阴天江药业有限公司 | Ultra-high performance liquid chromatography detection method for starwort standard decoction and application thereof |
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| CN101948420A (en) * | 2010-09-28 | 2011-01-19 | 中国科学院西北高原生物研究所 | Preparation method for separating and purifying tryptophan monomers from nitraria fruit juice |
| 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 |
| 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 |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101948420A (en) * | 2010-09-28 | 2011-01-19 | 中国科学院西北高原生物研究所 | Preparation method for separating and purifying tryptophan monomers from nitraria fruit juice |
| 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 |
Non-Patent Citations (1)
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN114646695A (en) * | 2020-12-21 | 2022-06-21 | 江阴天江药业有限公司 | Ultra-high performance liquid chromatography detection method for starwort standard decoction and application thereof |
| 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 |
| CN114082222A (en) * | 2021-10-25 | 2022-02-25 | 青海大学 | Method for purifying free amino acid of Ribes nigrum |
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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 |