Preparation method of cyclo(tyrosine-valine)

Abstract

The present invention provides a preparation method of a ring (tyrosine valine), characterized in that the following steps are adopted: step A: preparing a raw material extract using yellow water as raw material; step B: subjecting the raw material extract obtained by step A to reduced pressure distillation to obtain a ring (tyrosine valine) crude extract; step C: diluting the ring (tyrosine valine) crude extract obtained by step B with ethanol and water to obtain a diluent, subjecting the diluent to chromatography using a resin column to obtain a chromatographic liquid, concentrating the chromatographic liquid to obtain a ring (tyrosine valine) crude product; step D: subjecting the ring (tyrosine valine) crude product obtained by step C to reverse phase column separation to obtain a separation liquid, concentrating and crystallizing the separation liquid to obtain a ring (tyrosine valine) pure product. The raw materials of the present invention are easily available, the method is simple, and the product purity is more than 95%, which can provide a technical basis for the separation and preparation of other peptide compounds in yellow water.

Description

Technical Field

[0001] The present invention relates to the field of separation and purification of substances, and in particular to a method for preparing cyclo(tyrosine-valine). Background Art

[0002] Cyclo(Tyr-Val), CAS: 21754-25-6, is a cyclic dipeptide compound formed by cyclization of tyrosine and valine through a peptide bond.

[0003] Cyclic dipeptide is a stable six-membered ring structure with two hydrogen bond acceptors and two hydrogen bond donors. Hydrogen bond is one of the main ways for drugs to interact with receptors. Therefore, cyclic dipeptide is an important pharmacophore in medicinal chemistry, showing antibacterial, anti-tumor and other biological activities. Cyclic dipeptides are mostly derived from protein hydrolysis, microbial cells, animals and plants. The raw materials are not easy to obtain and the preparation cost is high.

[0004] Yellow water is a yellow or brownish-yellow viscous liquid produced during the solid-state brewing of liquor. It is a fermentation product of microorganisms using grain as raw materials and a by-product of liquor brewing. It is rich in sugars, proteins and liquor flavor components. The yellow water flavor components are recycled through distillation, and the remaining bottom pot yellow water is discharged as waste liquid after treatment. Among them, a large number of peptide compounds produced by hydrolysis of plant protein, microbial protein, etc. during the fermentation process of liquor brewing are not recycled. Extracting, separating, purifying yellow water active peptides, identifying their structures, and further conducting activity studies to clarify the activity of Wuliangye yellow water active peptides can firstly improve the recycling value of Wuliangye yellow water and have certain economic benefits. Second, it can reduce the burden of waste liquid treatment and have certain environmental benefits.

[0005] The invention separates and purifies cyclo(tyrosine-valine) from yellow water which is a byproduct of liquor brewing, but there is no related report at present. Summary of the invention

[0006] The object of the present invention is to provide a method for preparing cyclo(tyrosine-valine), which is characterized by adopting the following steps:

[0007] Step A: preparing a raw material extract: using yellow water as a raw material, allowing the yellow water to stand, extracting a supernatant of the standing yellow water, and extracting the supernatant of the yellow water with n-butanol to obtain a raw material extract;

[0008] Step B: preparing a crude cyclo(tyrosine-valine) extract: subjecting the raw material extract obtained in step A to reduced pressure distillation to obtain a crude cyclo(tyrosine-valine) extract;

[0009] Step C: preparing a crude cyclo(tyrosine-valine) product: diluting the crude cyclo(tyrosine-valine) extract obtained in step B with ethanol and water to obtain a diluent, subjecting the diluent to chromatography using a resin column to obtain a chromatographic solution, and concentrating the chromatographic solution to obtain a crude cyclo(tyrosine-valine) product;

[0010] Step D: Preparation of pure cyclo(tyrosine-valine): The crude cyclo(tyrosine-valine) obtained in step C is separated by a reverse phase column to obtain a separated liquid, and the separated liquid is concentrated and crystallized to obtain a pure cyclo(tyrosine-valine).

[0011] Furthermore, in step A, the yellow water is a yellow or brownish-yellow viscous liquid produced in the process of brewing solid liquor. Preferably, the yellow water is a by-product of brewing Wuliangye Luzhou-flavor liquor; and the volume ratio of n-butanol to yellow water is 1:1 to 2:1.

[0012] Furthermore, in step A, the extraction is performed 2 to 3 times.

[0013] Furthermore, in step B, the temperature of the reduced pressure distillation is 40-80°C.

[0014] Furthermore, in step C, the volume ratio of the ethanol to water is 1:20 to 1:40, and the water is purified water.

[0015] Furthermore, in step C, the resin column is a macroporous resin DA201 and / or a macroporous adsorption resin DA301, the eluent of the resin column is a mixed solution of ethanol and water, and the chromatography uses an eluent with an ethanol content of 5%, 20%, 40%, 60%, 80% and 100% for gradient chromatography.

[0016] Furthermore, in step C, the concentration temperature is 40°C to 80°C.

[0017] Furthermore, in step D, the reverse phase column is a preparative chromatography, the stationary phase of the reverse phase column is a C18 column, the eluent of the reverse phase column is a mixed solution of methanol and / or ethanol and water, the reverse phase column is separated by gradient elution to obtain a separation liquid, the flow rate of the preparative chromatography is 1 to 10 mL / min, and the injection volume is 1 to 10 mL. Preferably, the flow rate of the preparative chromatography is 3 mL / min, and the injection volume is 5 mL.

[0018] Furthermore, in step D, the concentration temperature is 40°C to 80°C, and the crystallization temperature is -10°C to 20°C.

[0019] The beneficial effects of the present invention are as follows: (1) The present invention provides a new method for preparing cyclo(tyrosine-valine): it is separated from yellow water, a byproduct of Wuliang Luzhou-flavor liquor. In the process of exploring the health function factors in yellow water, the inventors first discovered the presence of cyclodipeptides in yellow water, a byproduct of brewing, which provides a possibility for separating and preparing cyclo(tyrosine-valine) from yellow water, and also provides a new method for obtaining such compounds in large quantities. (2) Yellow water is a byproduct of liquor brewing, and is usually discharged after being treated as waste liquid. On the one hand, it creates a burden for waste liquid treatment, and on the other hand, a large number of peptide compounds in yellow water are not recycled, resulting in a waste of resources. The present invention explores and prepares cyclo(tyrosine-valine) from yellow water, which can not only improve the application value of yellow water, increase the use of yellow water, but also reduce the burden of waste liquid treatment, and has certain environmental benefits. (3) The present invention prepares and separates cyclo(tyrosine-valine) from yellow water for the first time, the raw materials are easily available, the preparation method is simple, and the product purity is high. The present invention creatively combines a variety of separation technologies such as liquid-liquid extraction, macroporous resin column chromatography, and reverse-phase preparative chromatography, and separates for the first time a ring (tyrosine-valine) with a purity of more than 95% from yellow water with complex components, which can provide a technical basis for the separation and preparation of other peptide compounds in yellow water. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the secondary mass spectrum of cyclic (tyrosine-valine) in the n-butanol extract of Example 1.

[0021] Figure 2 The total ion current TIC diagram and the cyclic (tyrosine-valine) extracted ion current EIC diagram of the n-butanol extract in Example 1 are shown.

[0022] Figure 3 The total ion current TIC diagram and the cyclic (tyrosine-valine) extracted ion current EIC diagram of the crude product obtained in Example 1 are shown.

[0023] Figure 4 This is the EIC graph of the cyclic (tyrosine-valine) extracted by different extraction agents in Example 2. DETAILED DESCRIPTION

[0024] The purpose of the present invention is to provide a method for preparing cyclo(tyrosine-valine) to solve the problems of difficult to obtain raw materials and complicated preparation process in the existing preparation methods.

[0025] The present invention provides a method for preparing cyclo(tyrosine-valine): isolating it from yellow water. The method comprises the following steps:

[0026] Step A: preparing a raw material extract: using yellow water as a raw material, allowing the yellow water to stand, extracting a supernatant of the standing yellow water, and extracting the supernatant of the yellow water with n-butanol to obtain a raw material extract;

[0027] Step B: preparing a crude cyclo(tyrosine-valine) extract: subjecting the raw material extract obtained in step A to reduced pressure distillation to obtain a crude cyclo(tyrosine-valine) extract;

[0028] Step C: preparing a crude cyclo(tyrosine-valine) product: diluting the crude cyclo(tyrosine-valine) extract obtained in step B with ethanol and water to obtain a diluent, subjecting the diluent to chromatography using a resin column to obtain a chromatographic solution, and concentrating the chromatographic solution to obtain a crude cyclo(tyrosine-valine) product;

[0029] Step D: Preparation of pure cyclo(tyrosine-valine): The crude cyclo(tyrosine-valine) obtained in step C is separated by a reverse phase column to obtain a separated liquid, and the separated liquid is concentrated and crystallized to obtain a pure cyclo(tyrosine-valine).

[0030] The invention firstly selects the most suitable n-butanol as the extraction reagent according to the principle of like dissolves like, which can effectively extract the ring (tyrosine-valine) and avoid extracting the macromolecular polysaccharides, proteins and non-polar compounds in the yellow water into the extraction solution.

[0031] The extract is then concentrated by vacuum distillation, and n-butanol in the extract is removed at the same time.

[0032] Then, macroporous resin column chromatography is used for separation. The principle is that based on the adsorption effect of the resin, organic compounds can be eluted and separated by a certain solvent according to their adsorption force and molecular weight to achieve the purpose of separation, purification and impurity removal.

[0033] Finally, a preparative chromatography reverse phase column is used for separation, the principle of which is to separate compounds according to their different polarities, thereby achieving the purpose of separating and purifying the ring (tyrosine-valine).

[0034] In order to separate high-purity ring (tyrosine-valine) from yellow water with complex components, the above separation methods must be combined, otherwise it is difficult to achieve the separation and preparation of high-purity ring (tyrosine-valine).

[0035] The present invention adopts n-butanol as an extraction solvent, which can effectively extract the cyclic (tyrosine-valine), and can avoid extracting macromolecular polysaccharides, proteins and non-polar compounds in yellow water into the extraction solution, thereby reducing the impurity components in the extraction solution. The polarity of n-butanol is similar to that of the cyclic (tyrosine-valine), and it can be separated from the water phase, without considering the macromolecular polysaccharides and proteins in the yellow water, and the extraction of small molecular cyclic dipeptides can be directly realized.

[0036] After investigation, it was found that when the volume ratio of n-butanol to yellow water is 1:1-2:1 and the number of extractions is 2-3 times, it can not only ensure that the n-butanol and yellow water phases are not suspended and the two phases are separated, but also effectively extract the ring (tyrosine-valine) in the yellow water into the n-butanol phase without causing solvent waste. The extraction temperature is 5-30℃. The low temperature environment can prevent the impurities in the yellow water, especially the weak polar small molecule impurities, from entering the n-butanol phase as much as possible, thereby reducing the impurities in the extract and preventing too many impurities from entering the extract, which will bring difficulties to the subsequent separation and purification.

[0037] When n-butanol is used as an extractant, most of the impurities in the yellow water can be removed, but there are still some impurities with similar chemical polarity dissolved in the n-butanol phase, that is, in the extract. Therefore, the macroporous resin is used to further separate the compounds according to the different adsorption forces and molecular weights of the compounds in the extract, remove the impurity part, and collect the part where the ring (tyrosine-valine) is located. The n-butanol extract cannot be directly loaded onto the macroporous resin column, because the n-butanol solvent will reduce the adsorption effect of the resin on the target ring (tyrosine-valine), so the n-butanol in the n-butanol extract is removed by vacuum distillation and evaporation. Many components in the obtained crude extract are insoluble in water, so the crude extract is first dissolved in ethanol. Similarly, because the high concentration of ethanol system will affect the resin adsorption effect, the ethanol-dissolved crude extract is diluted with pure water, so that the ethanol concentration in the loading system of the macroporous resin column chromatography is less than 5%, that is, the volume ratio of ethanol to pure water is 1:20 to 1:40, which can not only ensure that the ethanol concentration in the loading system is within 5%, but also ensure the loading solution concentration.

[0038] The crude product of cyclo(tyrosine-valine) was dissolved in methanol and tested by high performance liquid chromatography. The ultraviolet absorption peak at a wavelength of 220 nm contained not only the target compound cyclo(tyrosine-valine) but also a small amount of impurity peaks. Therefore, preparative chromatography was further performed on a reversed-phase C18 column with methanol and / or ethanol and water as elution reagents, and gradient elution was performed to collect the ultraviolet absorption peak at a wavelength of 220 nm.

[0039] Samples were collected by preparative chromatography and identified by high-resolution high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry based on the similarity of the compound's mass-to-charge ratio, isotope relative abundance, isotope ratio, and secondary mass spectrometry characteristic ions, and were ultimately determined to be the target compound ring (tyrosine-valine).

[0040] Example 1 Preparation of Cyclo(Tyrosine-Valine) Using the Method of the Present Invention

[0041] Step A: preparing a raw material extract: taking 20 L of yellow water produced during the brewing process of Wuliangye Luzhou-flavor liquor, standing for 1 day, extracting 18 L of the supernatant of the standing yellow water, extracting with 36 L of n-butanol twice to obtain 36 L of raw material extract, diluting the extract with methanol, injecting high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, 220 nm wavelength ultraviolet detector, the total ion current TIC diagram of the extract and the cyclic (tyrosine-valine) extracted ion current EIC diagram are as follows: Figure 2 As shown, the secondary mass spectrum of cyclic (tyrosine-valine) is as follows Figure 1 shown.

[0042] Step B: Preparation of cyclo(tyrosine-valine) crude extract: The raw material extract obtained in step A is subjected to reduced pressure distillation at 50°C to remove n-butanol from the extract to obtain a cyclo(tyrosine-valine) crude extract.

[0043] Step C: Preparation of cyclo(tyrosine-valine) crude product: Dissolve the cyclo(tyrosine-valine) crude extract obtained in step B with ethanol in a volume ratio of 1:4, then dilute and filter with purified water and ethanol in a volume ratio of 20:1 to obtain 21L of dilution, which is prepared for resin column chromatography. The resin column is DA201 macroporous adsorption resin. Soak 2Kg of DA201 macroporous adsorption resin in anhydrous ethanol for 24h, wet-pack the column, and wash with deionized water until the effluent is not turbid after being diluted with 3 times water, and then wash and set aside. Load the washed macroporous resin column with the dilution 21L at a flow rate of 10mL / min. Using ethanol-water as the eluent, 12 L of each ethanol content in the ratio of 5%, 20%, 40%, 60%, 80%, and 100% was sequentially used for gradient elution chromatography, and each section of the chromatographic solution was collected. Each section of the chromatographic solution was loaded with a high performance liquid chromatography-quadrupole time-of-flight mass spectrometer, and the mass spectrometer detector was used for simultaneous detection to determine the 20% ethanol elution section (eluent #8 to 10) where the target compound ring (tyrosine-valine) was located. The eluents #8 to 10 were combined and concentrated, and crystallized at 10°C to obtain 160 mg of a crude product. The total ion current TIC graph and the ring (tyrosine-valine) extracted ion current EIC graph of the crude product are shown as follows: Figure 3 shown.

[0044] Step D: Preparation of pure cyclo(tyrosine-valine): The crude product obtained in step C was dissolved in 50% methanol aqueous solution and injected into a reverse phase separation column. The reverse phase separation column was a preparative chromatography column with a stationary phase of C18 column and a specification of XB-C18, 21.2×250mm, 5μm, flow rate of 3mL / min, injection volume of 5mL, gradient elution chromatography with liquid chromatography grade methanol and ultrapure water as eluents, collecting the ultraviolet absorption peak at a wavelength of 220nm, concentrating, and obtaining 76mg of pure cyclo(tyrosine-valine).

[0045] Weigh 1 mg of the pure cyclo(tyrosine-valine) obtained in step D, prepare a mixed solution with methanol at a concentration of 0.1 mg / mL, load it on high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, and perform qualitative and quantitative analysis with cyclo(tyrosine-valine) standards. The result shows that the content of cyclo(tyrosine-valine) in the pure product is 97.58%.

[0046] Example 2

[0047] Step A: Take 2 mL of yellow water, a by-product of the brewing process of Wuliangye Luzhou-flavor liquor, and put them into 4 15 mL centrifuge tubes: tube No. 1 contains 2 mL of yellow water, tube No. 2 contains 2 mL of yellow water and 4 mL of n-hexane, tube No. 3 contains 2 mL of yellow water and 4 mL of ethyl acetate, and tube No. 4 contains 2 mL of yellow water and 4 mL of n-butanol.

[0048] Step B: The four centrifuge tubes prepared in step A were oscillated and extracted for 1 min, and then allowed to stand. 100 uL of the supernatant was taken respectively, and the supernatant was diluted with 900 uL of methanol to obtain a dilution. The dilution was filtered with a 0.45 μm filter membrane and then injected into a high performance liquid chromatography quadrupole time-of-flight mass spectrometer. The comparison of the ring (tyrosine-valine) extracted ion current EIC (263.1390) of the four samples is shown in the figure below. Figure 4 shown.

[0049] The results showed that the samples from tubes 2 and 3 could not extract the ring (tyrosine-valine) from the yellow water, while the sample from tube 4 could effectively extract the ring (tyrosine-valine), so n-butanol was the most suitable extraction agent.

Claims

1. A method for preparing cyclo(tyrosine-valine), characterized in that: Use the following steps: Step A: preparing a raw material extract: using yellow water as a raw material, allowing the yellow water to stand, extracting a supernatant of the standing yellow water, and extracting the supernatant of the yellow water with n-butanol to obtain a raw material extract; Step B: preparing a crude cyclo(tyrosine-valine) extract: subjecting the raw material extract obtained in step A to reduced pressure distillation to obtain a crude cyclo(tyrosine-valine) extract; Step C: preparing a crude cyclo(tyrosine-valine) product: diluting the crude cyclo(tyrosine-valine) extract obtained in step B with ethanol and water to obtain a diluent, subjecting the diluent to chromatography using a resin column to obtain a chromatographic solution, and concentrating the chromatographic solution to obtain a crude cyclo(tyrosine-valine) product; Step D: preparing a pure cyclo(tyrosine-valine) product: subjecting the crude cyclo(tyrosine-valine) product obtained in step C to reverse phase column separation to obtain a separated liquid, and concentrating and crystallizing the separated liquid to obtain a pure cyclo(tyrosine-valine) product; In step A, the yellow water is a yellow or brownish yellow viscous liquid produced in the process of solid-state brewing liquor, and the volume ratio of n-butanol to yellow water is 1:1 to 2:

1.

2. The method for preparing cyclo(tyrosine-valine) according to claim 1, characterized in that: In step A, the yellow water is a by-product produced by brewing Wuliangye Luzhou-flavor liquor.

3. The method for preparing cyclo(tyrosine-valine) according to claim 1, characterized in that: In step A, the extraction is performed 2 to 3 times.

4. The method for preparing cyclo(tyrosine-valine) according to claim 1, characterized in that: In step B, the temperature of the reduced pressure distillation is 40-80°C.

5. The method for preparing cyclo(tyrosine-valine) according to claim 1, characterized in that: In step C, the volume ratio of the ethanol to water is 1:20 to 1:40, and the water is purified water.

6. The method for preparing cyclo(tyrosine-valine) according to claim 1, characterized in that: In step C, the resin column is macroporous resin DA201 and / or macroporous adsorption resin DA301, the eluent of the resin column is a mixed solution of ethanol and water, and the chromatography uses eluents with ethanol contents of 5%, 20%, 40%, 60%, 80% and 100% for gradient chromatography.

7. The method for preparing cyclo(tyrosine-valine) according to claim 1, characterized in that: In step C, the concentration temperature is 40°C to 80°C.

8. The method for preparing cyclo(tyrosine-valine) according to claim 1, characterized in that: In step D, the reverse phase column is a preparative chromatograph, the stationary phase of the reverse phase column is a C18 column, the eluent of the reverse phase column is a mixed solution of methanol and / or ethanol and water, the reverse phase column is separated by gradient elution to obtain a separation liquid, the flow rate of the preparative chromatography is 1 to 10 mL / min, and the injection volume is 1 to 10 mL.

9. The method for preparing cyclo(tyrosine-valine) according to claim 8, characterized in that: The flow rate of the preparative chromatography was 3 mL / min, and the injection volume was 5 mL.

10. The method for preparing cyclo(tyrosine-valine) according to claim 1, characterized in that: In step D, the concentration temperature is 40°C to 80°C, and the crystallization temperature is -10°C to 20°C.

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