Preparation method of Tricin

Abstract

The present invention belongs to the technical field of chemical analysis, and relates to a method for preparing Tricin. Aiming at the problem that raw materials are not easy to obtain and the preparation process is complicated in the existing preparation method, the present invention provides a method for preparing Tricin, which comprises the following steps: a. taking distiller's grains, extracting with an organic solvent, and concentrating to obtain a Tricin extract; b. extracting the Tricin extract with petroleum ether and ethyl acetate in turn, collecting the ethyl acetate extract, and concentrating to obtain an ethyl acetate extract; c. subjecting the ethyl acetate extract to silica gel normal phase column chromatography, collecting the eluate containing Tricin, and concentrating to obtain a crude product; d. subjecting the crude product to gel column chromatography, collecting the eluate containing Tricin, and concentrating to obtain a pure Tricin product. The present invention combines liquid-liquid extraction, normal phase column chromatography, and gel column chromatography separation technology to separate and prepare Tricin from distiller's grains, with high separation degree and high product purity.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and particularly relates to a preparation method of Tricin. Background Art

[0002] Tricin, whose chemical name is 5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)benzopyran-4-one, is a natural flavonoid widely distributed in plants of the Poaceae and Euphorbiaceae families. It has significant anti-inflammatory, antioxidant, anti-tumor, antiviral and immunomodulatory effects.

[0003] There are two main ways to prepare Tricin. One is through chemical synthesis, using phloroglucinol and chloroacetonitrile as raw materials to condense under the action of a catalyst to obtain 2-(2-chloro-1-iminoethyl)benzene-1,3,5-triphenol, and then hydrolyzing to obtain 2-chloro-1-(2,4,6-trihydroxyphenyl)ethanone, and then cyclizing with syringaldehyde under the action of ultrasound and the presence of a phase transfer catalyst tetrabutylammonium bromide (TBAB). This chemical synthesis method requires the use of a variety of chemical reagents and has high requirements for synthesis conditions. The other is to use a preparation method of natural medicines and extract from plants. Currently, most of the commercially available Tricin is extracted from plants, which has a wider applicability. However, the available plant raw material resources are limited, the cost is high, and the price is expensive.

[0004] Wine lees are the mash before and after the solid-state brewing of liquor is distilled. Especially for Wuliang Luzhou-flavor liquor, the wine lees before distillation are made of five kinds of grains, rice, glutinous rice, wheat, sorghum and corn, and Daqu is used as the fermentation agent. After about 70 days of solid natural fermentation, it is rich in wine-making raw materials, fermentation agents and flavor compounds formed by microbial decomposition and metabolism. Bioactive components. Wine lees after distillation belong to the by-product of wine-making, which has an annual output of tens of millions of tons in my country. It can only be thrown away as waste, which not only pollutes the environment, but also causes a huge waste of resources. Wine lees are the products of long-term composite microbial fermentation of grain raw materials. They contain grain raw materials, as well as active functional factors produced by microbial metabolism and enzymolysis. Therefore, exploring the active functional factors of flavonoids contained in wine lees can provide a basis for the existence of flavonoid compounds in the liquor brewing process, improve the application value of wine lees, reduce the treatment cost of waste wine lees, and reduce carbon emissions, which has great economic and environmental benefits.

[0005] At present, there is no research report on the separation and preparation of Tricin from wine grains. Summary of the invention

[0006] In view of the fact that there are currently no relevant research reports on the separation and preparation of Tricin from distiller's grains, the present invention provides a method for preparing Tricin, and more specifically provides a method for separating and preparing Tricin from distiller's grains, so as to solve the problems of the existing preparation methods such as the difficulty in obtaining raw materials and the complex preparation process.

[0007] The invention provides a preparation method of Tricin: the Tricin is separated from wine lees.

[0008] Furthermore, the preparation method comprises the following steps:

[0009] a. Take vinasse, extract with organic solvent, and concentrate to obtain Tricin extract;

[0010] b. Extract the Tricin extract with petroleum ether and ethyl acetate in turn, collect the ethyl acetate extract, and concentrate it to obtain ethyl acetate extract;

[0011] c. Subjecting the ethyl acetate extract to silica gel normal phase column chromatography, collecting the eluate containing Tricin, and concentrating it to obtain a crude product;

[0012] d. Subject the crude product to gel column chromatography, collect the eluate containing Tricin, and concentrate it to obtain pure Tricin.

[0013] Furthermore, in step a, the organic solvent is an alcohol solvent.

[0014] Furthermore, in step a, the organic solvent is methanol and / or ethanol.

[0015] Furthermore, the amount of methanol or ethanol used is 3 to 5 times the weight of the lees.

[0016] Furthermore, in step a, the extraction temperature is 15-40°C.

[0017] Furthermore, in step a, the extraction times are 2 to 4 times.

[0018] Furthermore, in step a, the extraction time is 1 to 2 days each time.

[0019] Furthermore, in step b, the volume ratio of petroleum ether to the extract is (1:1) to (3:1).

[0020] Furthermore, in step b, the volume ratio of ethyl acetate to the extract is (1:1) to (3:1).

[0021] Furthermore, in step b, the extraction temperature is 15-40°C.

[0022] Furthermore, in step b, the extraction times are 2 to 4 times.

[0023] Furthermore, in step c, during the silica gel normal phase column chromatography, the silica gel is 100-300 mesh normal phase silica gel.

[0024] In the silica gel normal phase column chromatography process, petroleum ether-ethyl acetate is used as the eluent.

[0025] Furthermore, in step c, gradient elution is performed with petroleum ether / ethyl acetate, v / v, ratios of 50:1, 10:1, 5:1, 1:2, 1:5.

[0026] Furthermore, in step d, gel column chromatography is used.

[0027] Furthermore, in step d, the gel is sephadex LH-20.

[0028] Furthermore, in step d, methanol-water is used as the elution reagent.

[0029] Furthermore, in step d, gradient elution is performed with methanol / water at a ratio of methanol (% vol) of 20%, 40%, 60%, 80%, and 100%.

[0030] Furthermore, the preparation method also includes a recrystallization step.

[0031] Furthermore, the recrystallization solvent is methanol or ethanol.

[0032] The number of recrystallizations is 2 to 4 times.

[0033] Furthermore, the concentration temperature is 40-80°C.

[0034] Furthermore, the lees are liquor lees, preferably Wuliang Luzhou-flavor liquor lees.

[0035] Furthermore, the Wuliang Luzhou-flavor liquor lees are made from five kinds of grains, namely rice, glutinous rice, corn, sorghum and wheat, with the addition of Daqu fermentation agent and network brewing environment microorganisms, and are made through solid-state natural fermentation. They are the mash before distillation and / or the discarded lees after distillation.

[0036] Beneficial effects:

[0037] 1. The present invention provides a new method for preparing Tricin: separating it from wine lees. In the process of exploring the health function factors in liquor, it was first discovered that Tricin exists in liquor lees, more specifically in Wuliang Luzhou-flavor liquor lees, which provides the possibility of separating and preparing Tricin from wine lees and also provides a new method for obtaining such compounds in large quantities.

[0038] 2. The lees before distillation are fermented grains, and the flavor components and health function factors in them enter the liquor through the distillation process. The present invention explores and prepares Tricin from fermented grains, which can provide technical support for exploring how to increase the content of such health function factors in liquor and enhance the health value of liquor.

[0039] 3. The lees after distillation are waste lees, which are by-products of winemaking. They are produced in large quantities and are often discarded as waste, which not only pollutes the environment but also causes a huge waste of resources. The present invention explores and prepares Tricin from the waste lees, which can improve the application value of the waste lees.

[0040] 4. The present invention separates Tricin from Wuliang Luzhou-flavor liquor lees for the first time, and the preparation and separation method is simple and the product has high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The secondary mass spectrum of Tricin obtained in Example 1;

[0042] Figure 2 This is the 1H-NMR spectrum of Tricin obtained in Example 1;

[0043] Figure 3 The 13C-NMR spectrum of Tricin obtained in Example 1;

[0044] Figure 4 The total ion chromatogram (TIC) of the combined solution in Example 2 and the extracted ion chromatogram (EIC) of Tricin. DETAILED DESCRIPTION

[0045] The purpose of the present invention is to provide a method for preparing Tricin, so as to solve the problems existing in the existing preparation methods, such as the difficulty in obtaining raw materials and the complicated preparation process.

[0046] The present invention provides a method for preparing Tricin: Tricin is separated from wine lees, and comprises the following steps:

[0047] a. Take vinasse, extract with organic solvent, and concentrate to obtain Tricin extract;

[0048] b. Extract the Tricin extract with petroleum ether and ethyl acetate in turn, collect the ethyl acetate extract, and concentrate it to obtain ethyl acetate extract;

[0049] c. Subjecting the ethyl acetate extract to silica gel normal phase column chromatography, collecting the eluate containing Tricin, and concentrating it to obtain a crude product;

[0050] d. Subject the crude product to gel column chromatography, collect the eluate containing Tricin, and concentrate it to obtain pure Tricin.

[0051] The invention firstly selects suitable alcohol organic solvents: ethanol and / or methanol to extract trace target substances in vinasse, and at the same time prevents macromolecules such as starch, sugar, protein and the like from entering into the extract. According to the principle of like dissolves like, non-polar petroleum ether is selected to extract and remove non-polar and weakly polar higher fatty acids and esters thereof, and further extracts with medium-polar ethyl acetate to obtain a crude extract of Tricin.

[0052] Then, silica gel normal phase column chromatography is used. The principle is that according to the adsorption effect of silica gel, organic compounds can be eluted through a certain solvent according to the adsorption force, and the compounds flow out in order from small to large polarity, thereby achieving the purpose of separation, purification and impurity removal.

[0053] Finally, dextran gel column chromatography was used. In the reverse phase elution solvent system, dextran gel sephadex LH-20 has both molecular sieving and reverse phase distribution effects. Due to the molecular sieving effect, the molecular weights flow out in order from large to small; at the same time, reverse phase solvent elution is used to separate the compounds according to their polarity, and the compounds flow out in order from large to small polarity. The dual effects achieve the purpose of separating and purifying Tricin. In order to separate high-purity Tricin from the complex lees, the above separation methods must be combined. The above four steps a, b, c, and d are indispensable. After a large number of experiments, the applicant found that if any of the steps is missing, it is difficult to obtain pure Tricin.

[0054] The present invention uses an alcohol solvent as an extraction solvent, which can fully extract Tricin from the lees, while preventing starch, sugars, proteins and other components from entering the extract. In a preferred embodiment, methanol and ethanol have low boiling points, and there is no need to set too high a temperature during reduced pressure concentration, which can prevent the target compound from being destroyed by high temperature, save time, and facilitate solvent recovery. Therefore, in step a of the preparation method of the present invention, the organic solvent A is an alcohol solvent. Preferably, the alcohol solvent is methanol and / or ethanol.

[0055] After investigation, it is found that the best amount of methanol and / or ethanol is 3 to 5 times the weight of the lees, which can fully extract Tricin from the lees without causing a waste of solvent. The extraction temperature is 15 to 40°C, which can fully extract Tricin from the lees without causing serious solvent volatilization and excessive loss. The number of extractions is 2 to 4 times, and the best time for each extraction is 1 to 2 days, which can fully extract Tricin from the lees and avoid excessive impurities entering the extract, which will bring difficulties to subsequent separation and purification. Therefore, in step a of the preparation method of the present invention, the organic solvent is methanol and / or ethanol, and the amount of methanol and / or ethanol is 3 to 5 times the weight of the lees; the extraction temperature is 15 to 40°C; the number of extractions is 2 to 4 times; and the extraction time is 1 to 2 days each time.

[0056] When methanol or ethanol is used as the extraction solvent, the target Tricin and various impurities of different polarities in the lees can be dissolved therein. Therefore, the present invention uses non-polar petroleum ether to extract and remove the higher fatty acid esters therein, and then extracts with ethyl acetate according to the principle of like dissolves like, which can not only reduce the content of non-polar impurities in the ethyl acetate extraction phase, but also ensure that Tricin effectively enters the ethyl acetate extraction liquid. After investigation, the volume ratio of petroleum ether or ethyl acetate to the extraction liquid is (1:1) to (3:1); the extraction temperature is 15 to 40°C; and the number of extractions is 2 to 4 times.

[0057] Furthermore, in step c, the silica gel is 100-300 mesh normal phase silica gel; the purpose of selecting 100-300 mesh normal phase silica gel in the present invention is to achieve a better purification effect and ensure the separation speed.

[0058] In step c of the preparation method of the present invention, petroleum ether-ethyl acetate is used as an eluent; gradient elution is performed at a ratio of petroleum ether / ethyl acetate, v / v, 50:1, 10:1, 5:1, 1:2, and 1:5. The purpose is to use petroleum ether-ethyl acetate as an elution reagent and adjust the gradient of the eluent to gradually elute different compounds of the crude extract of Tricin from weak polarity to strong polarity.

[0059] In step d of the preparation method of the present invention, gel column chromatography is used; the preferred gel of the present invention is dextran gel sephadex LH-20, methanol-water is used as an elution reagent, and reverse phase gradient elution is performed with methanol / water, methanol (%vol) 20%, 40%, 60%, 80%, 100%, which can not only play the role of molecular sieve gel filtration, but also realize the role of reverse phase column chromatography, so as to achieve the purpose of separating and purifying Tricin.

[0060] Furthermore, the preparation method also includes a recrystallization step, and the recrystallization solvent is methanol or ethanol.

[0061] The number of recrystallizations is 2 to 4 times.

[0062] The lees in the preparation method of the present invention are selected from liquor lees, preferably five-grain Luzhou-flavor liquor lees, which are made from five kinds of grains, namely rice, glutinous rice, corn, sorghum and wheat, and are naturally fermented by adding a Daqu fermentation agent and microorganisms in a network brewing environment. They are the mash before distillation and / or the discarded lees after distillation.

[0063] The present invention provides a method for preparing Tricin, which combines multiple chromatography techniques to separate and prepare Tricin from wine lees for the first time, and mainly has the following advantages:

[0064] 1. The present invention provides a new method for preparing Tricin: separating it from Wuliang Luzhou-flavor liquor lees. In the process of exploring the health function factors in liquor, the inventors first discovered the existence of Tricin in Wuliang Luzhou-flavor liquor lees, which provides the possibility of separating and preparing Tricin from lees and also provides a new method for obtaining a large amount of this type of compound.

[0065] 2. The lees before distillation are fermented grains, and the flavor components and health function factors in them enter the liquor through the distillation process. The present invention explores and prepares Tricin from fermented grains, which can provide technical support for exploring how to increase the content of such health function factors in liquor and enhance the health value of liquor.

[0066] 3. The lees after distillation are waste lees, which are by-products of winemaking. They are produced in large quantities and are often discarded as waste, which not only pollutes the environment but also causes a huge waste of resources. The present invention explores and prepares Tricin from the waste lees, which can improve the application value of the waste lees.

[0067] 4. The present invention separates Tricin from Wuliang Luzhou-flavor liquor lees for the first time, and the preparation and separation method is simple and the product has high purity.

[0068] At present, there is no report on Tricin in wine lees, let alone the separation and preparation of Tricin from wine lees.

[0069] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0070] Example 1 Preparation of Tricin from Fermented Grains Using the Method of the Present Invention

[0071] (1) Take 10 kg of Wuliang Luzhou-flavor liquor before distillation, add 50 kg of anhydrous ethanol, extract twice at 20°C, each time for 2 days, filter the extract, concentrate under reduced pressure at 60°C to obtain 2 L of extract, extract the extract twice with 6 L of petroleum ether, and then extract 4 times with 6 L of ethyl acetate, collect the ethyl acetate phase, and concentrate under reduced pressure at 40°C to obtain 240 g of ethyl acetate extract.

[0072] (2) The ethyl acetate extract was uniformly stirred with 100-200 mesh normal phase silica gel, and the silica gel column was loaded with 200-300 mesh for silica gel normal phase column chromatography. Petroleum ether / ethyl acetate was used as the elution reagent, and gradient elution was performed in the ratios of 50:1, 10:1, 5:1, 1:2, and 1:5 (petroleum ether / ethyl acetate, v / v). 8 L was eluted at each ratio; the 1:2 elution portion was collected in sections and subjected to thin layer chromatography (petroleum ether / ethyl acetate, 1:1, v / v), and the samples with an Rf value of 0.42 were combined and dried under reduced pressure to obtain component E6.

[0073] (3) Dissolve the component E6 obtained in step (2) in methanol, dilute it with deionized water 5 times the volume of methanol, load it on a Sephadex LH-20 chromatography column, use methanol / water as the elution reagent, and perform reverse phase gradient elution with methanol (%vol) in the ratio of 20%, 40%, 60%, 80%, and 100%. Collect the eluate in sections, take 100uL of each section, and use high performance liquid chromatography-quadrupole time-of-flight mass spectrometry to determine the 4-6# eluates of the 60% methanol elution section where the target compound Tricin is located, combine them, and concentrate the 4-6# eluates under reduced pressure at 80°C to obtain the pure target compound G46.

[0074] (4) The target compound obtained in step (3) was dissolved in methanol and recrystallized twice to obtain 125 mg of the pure compound. The content of the pure compound was 98.67% after quantitative analysis by HPLC-QTOF using Tricin standard.

[0075] The pure product obtained in step (4) was analyzed by mass spectrometry (HPLC-QTOF). The results showed that the molecular ion of the compound was [M+H] + is 331.0812, and the secondary mass spectrum is shown in Figure 1 .

[0076] The pure product obtained in step (4) was analyzed by 1H-NMR (400 MHz, DMSO-d6). The data showed that δ12.97 (1H, s) was the signal of the hydroxyl proton at position 5 of flavonoids, δ7.32 (2H, s) was the two proton signals at positions 2' and 6', δ6.96 (1H, s), δ6.52 (1H, s), and δ6.17 (1H, s) were the proton signals at positions 3, 8, and 6 on the flavonoid skeleton, respectively, and δ3.88 (6H, s) was the two groups of methoxy proton signals. The 1H-NMR results of the pure product are detailed in the attached Figure 2 .

[0077] 13C-NMR (100MHz, DMSO-d6) δ182.04 (C-4), δ163.89 (C-7), δ161.82 (C-2), δ158.91 (C-5), δ157.89 (C-9), δ148.67 (C-3', C-5'), δ140.4 7(C-4'), δ120.73(C-1'), δ104.75(C-2',C-6'), δ103.89(C-1), δ103.68(C-3), δ99.61(C-6), δ94.84(C-8), δ56.80(-OCH3×2), pure NMR 13 C-NMR results are shown in the attached Figure 3 The above spectral data prove that the compound is Tricin, that is, 5,7-dihydroxy-2-(4-hydroxy-3,5-dimethoxyphenyl)benzopyran-4-one.

[0078] Example 2 Preparation of Tricin from Waste Slag Using the Method of the Present Invention

[0079] (1) 10 kg of the waste grains after distillation of Wuliang Luzhou-flavor liquor were taken, 30 kg of anhydrous ethanol was added, and the mixture was extracted at 40° C. for 4 times, each time for 1 day. The extract was filtered and concentrated under reduced pressure at 60° C. to obtain 1.6 L of extract. The extract was extracted twice with 1.6 L of petroleum ether, and then extracted twice with 1.6 L of ethyl acetate. The ethyl acetate phase was collected and concentrated under reduced pressure at 40° C. to obtain 180 g of ethyl acetate extract.

[0080] (2) The ethyl acetate extract was uniformly stirred with 100-200 mesh normal phase silica gel, and 200-300 mesh silica gel was loaded into a column for silica gel normal phase column chromatography. Petroleum ether / ethyl acetate was used as the elution reagent, and gradient elution was performed at a ratio of 50:1, 10:1, 5:1, 1:2, 1:5 (petroleum ether / ethyl acetate, v / v), and 6 L of each ratio was eluted; the 1:2 elution portion was collected in sections and subjected to thin layer chromatography (petroleum ether / ethyl acetate, 1:1, v / v), and the samples with Rf value of 0.42 were combined, and 100 uL of the combined solution was taken for detection by high performance liquid chromatography quadrupole time-of-flight mass spectrometry. The total ion chromatogram (TIC) of the combined solution and the extracted ion chromatogram (EIC) of Tricin are shown in the attached figure. Figure 4 , HPLC-QTOF was used for drying under reduced pressure to obtain component D7.

[0081] (3) Dissolve the component D7 obtained in step (2) in methanol, dilute with deionized water, and load onto a Sephadex LH-20 chromatography column. Use methanol / water as the elution reagent and perform reverse phase gradient elution at a methanol (% vol) content of 20%, 40%, 60%, 80%, and 100%. Collect the 60% methanol elution portion in sections and perform thin layer chromatography (petroleum ether / ethyl acetate, 1:1, v / v). Combine the samples with an Rf value of 0.42 and dry under reduced pressure to obtain the target pure product H5.

[0082] (4) The target compound H5 obtained in step (3) was dissolved in ethanol and recrystallized twice to obtain 69 mg of the pure compound. The content of the pure compound was 98.09% after quantitative analysis by HPLC-QTOF using Tricin standard.

Claims

1. The preparation method of Tricin is characterized by: Separation from wine lees includes the following steps: a. Take vinasse, extract with organic solvent methanol and / or ethanol, and concentrate to obtain Tricin extract; b. Extract the Tricin extract with petroleum ether and ethyl acetate in turn, collect the ethyl acetate extract, and concentrate it to obtain ethyl acetate extract; c. The ethyl acetate extract was subjected to silica gel normal phase column chromatography, eluted with petroleum ether-ethyl acetate as eluent, the eluate containing Tricin was collected and concentrated to obtain a crude product; d. The crude product was subjected to gel column chromatography, eluted with methanol-water, and the eluate containing Tricin was collected and concentrated to obtain pure Tricin.

2. The preparation method of Tricin according to claim 1, characterized in that: In step a, at least one of the following is met: The organic solvent is methanol or ethanol; the amount of methanol or ethanol is 3 to 5 times the weight of the lees; The extraction temperature is 15~40℃; The number of extractions is 2 to 4 times; The extraction time is 1 to 2 days each time.

3. The preparation method of Tricin according to claim 1, characterized in that: In step b, at least one of the following is satisfied: The volume ratio of petroleum ether to the extract is (1:1)~(3:1); The volume ratio of ethyl acetate to extract is (1:1)~(3:1); The extraction temperature is 15~40℃; The number of extractions is 2 to 4 times.

4. The preparation method of Tricin according to claim 1, characterized in that: In step c, at least one of the following is satisfied: In the silica gel normal phase column chromatography process, the silica gel is 100-300 mesh normal phase silica gel; Gradient elution was performed with petroleum ether / ethyl acetate, v / v, 50:1, 10:1, 5:1, 1:2, 1:

5.

5. The preparation method of Tricin according to claim 1, characterized in that: In step d, at least one of the following is satisfied: The gel is a dextran gel, sephadex LH-20; Gradient elution was performed with methanol / water in the ratio of 20%, 40%, 60%, 80%, and 100% methanol by volume.

6. The method for preparing Tricin according to any one of claims 1 to 5, characterized in that: The preparation method also includes a recrystallization step.

7. The method for preparing Tricin according to claim 6, characterized in that: The solvent in the recrystallization step is methanol or ethanol.

8. The method for preparing Tricin according to claim 7, characterized in that: The number of recrystallizations is 2 to 4 times.

9. The method for preparing Tricin according to any one of claims 1 to 5, characterized in that: The concentration temperature is 40-80°C.

10. The method for preparing Tricin according to any one of claims 1 to 5, characterized in that: The lees are white wine lees.

11. The method for preparing Tricin according to claim 10, characterized in that: The lees are Wuliang Luzhou-flavor liquor lees.

12. The method for preparing Tricin according to claim 11, characterized in that: The Wuliang Luzhou-flavor liquor lees are made from five kinds of grains, namely rice, glutinous rice, corn, sorghum and wheat, with the addition of Daqu fermentation agent and network brewing environment microorganisms, and are made through solid-state natural fermentation. They are the mash before distillation and / or the discarded lees after distillation.

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