CN115385788B - Solvent-free preparation method of crocetin - Google Patents

Abstract

The invention discloses a solvent-free preparation method of crocetin, which comprises the following steps: 1) Putting gardenia yellow pigment and an alkaline reagent into a grinding tank, adding grinding beads, screwing the grinding tank, and putting the grinding tank into a grinder for grinding; 2) After finishing grinding in the step 1), taking down the grinding tank, adding an acidic reagent into the grinding tank, and continuing grinding in a grinder; 3) After finishing grinding in the step 2), pouring all the reaction mixture out of a grinding tank, taking out grinding beads, adding water into the reactant, carrying out suction filtration by using a Buchner funnel, collecting a filter cake, putting the filter cake into a vacuum drying oven for drying, and taking out after the drying is finished, thereby obtaining the crocetin.

Description

A solvent-free preparation method of crocetin

Technical field

The present invention relates to the technical field of preparation of crocetin, and in particular to a solvent-free preparation method of crocetin.

Background technique

The two main sources of crocetin are the fruits of gardenia and the stigmas of saffron. Specifically, crocin is obtained by extracting gardenia yellow from gardenia fruits or crocin from the stigma of saffron, and then further processing it to obtain crocin. Gardenia yellow pigment, also known as crocin, is a type of water-soluble carotenoid, consisting of a conjugated polyene skeleton and R groups at both ends, as shown in the following formula (I); depending on the different substituent groups at both ends, Divided into crocin-1, croncin-2, crocin-3, crocin-4, etc., among which crocin-1 is the most common, as shown in (II) below. When the R group is H, it is crocetin. Studies have shown that crocetin has various pharmacological effects such as anti-tumor, neuroprotection, cardioprotection, liver protection, antidepressant, anti-angiogenesis, healing burns, improving asthma, Alzheimer's disease, diabetes, colitis, etc. (Phytomedicine, 2015 ,22,36-44;Biomol.Ther. (Seoul),2016, 24,156-162; Fitoterapia, 2017, 121, 106-111; Eur. J. Pharmacol., 2017, 554, 191-195; Life Sci., 2017, 181, 9-16; J. Cell. Physiol. 2018, 233, 2170-2182;.), is widely used in medicine, food and other fields.

Since saffron has strict requirements on the growth environment, cultivation is difficult, and the yield per mu is low, making it expensive, gardenia has simple requirements on the growth environment, is easy to cultivate, and can be cultivated on a large scale. Therefore, it is practical to use gardenia fruits as a source to prepare crocetin. significance. After gardenia yellow pigment is extracted from gardenia, it needs to be further processed to obtain higher content of crocetin. Most of the methods for obtaining crocetin in existing literature and patents adopt alkali hydrolysis, acidification and recrystallization purification to obtain crocetin. During the preparation process, excessive amounts of strong alkaline reagents (such as sodium hydroxide, hydroxide, etc.) are required. Potassium), strong acid reagents (such as hydrochloric acid, phosphoric acid, sulfuric acid), and recrystallization reagents (such as pyridine, N,N-dimethylformamide, dimethyl sulfoxide, absolute ethanol, methanol, ethyl acetate, etc.), The entire preparation process is time-consuming, the steps are complicated, the waste liquid is large, and the final yield is not ideal (J. Agric. Food Chem., 2005, 53, 7302-7306; Biomed. Pharmacother., 2018, 98, 157-164; CN101225040A ; CN103539657A; CN103695494A; CN105063107A; CN105907807A; CN108218692A). In addition to the above-mentioned alkaline hydrolysis and acidification pathways, some researchers have obtained crocetin by repeatedly extracting the gardenia yellow pigment extracted from gardenia using a mixture of ethyl acetate and ethanol. Although the use of acid-base reagents is avoided, It requires multiple extractions, consumes a lot of solvent, and the final yield (20-26%) and content (69.5%-75%) of crocetin are low (CN105503968A). In addition, crocetin can also be obtained by genetically modifying the Saccharomyces cerevisiae strain that produces β-carotene. By introducing three heterologous genes CrtZ, CCD, and ALD into the Saccharomyces cerevisiae strain, β-carotene hydroxylase is produced after expression ( CrtZ), carotenoid cleavage dioxygenase (CCD) and aldehyde dehydrogenase (ALD). Under the catalysis of these three enzymes, β-carotene can be converted into crocetin. Under optimal conditions, crocin The yield is 6278 μg/L (Microb. Cell Factories, 2017, 16, 54.). This biocatalytic method requires high experimental skills of workers, the plasmid preparation process is complex, and the strain culture cycle is long. Subsequently, it needs to be Crocetin is separated from the fermentation broth, and only a small amount of product can be obtained on a large-scale fermentation scale.

In recent years, mechanochemistry has received widespread attention from chemists due to its green and efficient characteristics. Mechanical grinding technology has become an important way to promote chemical reactions. Compared with general solution reaction methods, mechanical grinding reactions are usually carried out by solvent-free grinding, which avoids the use of environmentally unfriendly solvents and reagents during the reaction process and can Significantly increase the reaction rate and shorten the reaction time. However, so far, there have been no reports of using mechanical grinding technology to prepare crocin.

Contents of the invention

In view of the problems existing in the prior art, the object of the present invention is to provide a solvent-free, easy-to-operate, environmentally friendly, fast and efficient solvent-free preparation method of crocetin.

The technical solution of the present invention is as follows:

The invention proposes a solvent-free preparation method of crocetin, which includes the following steps:

1) Put the gardenia yellow pigment and alkaline reagent into the grinding jar, add grinding beads, then tighten the grinding jar and put it into the grinder for grinding;

2) After the grinding in step 1) is completed, remove the grinding jar, add acidic reagent to it and continue grinding in the grinder;

3) After the grinding in step 2) is completed, pour out the entire reaction mixture from the grinding jar, take out the grinding beads, add water to the reactant, filter with a Buchner funnel, collect the filter cake, and put the filter cake into a vacuum Dry in the drying box and take it out after the drying is completed to obtain crocin.

Further, the color value of the gardenia yellow pigment in step 1) is 40 to 600.

Further, in step 1) the ratio of the amount of gardenia yellow pigment to the alkaline reagent is 1:2 to 10; the alkaline reagent is sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, carbonic acid. Potassium, sodium methoxide or sodium tert-butoxide.

Furthermore, the alkaline reagent in step 1) is preferably sodium hydroxide.

Further, in step 1) the grinding jar is a 45-80mL stainless steel jar, the grinding beads are stainless steel balls with a diameter of 4mm-8mm, the number of beads added is 40-360, and the grinding filling rate is 9%-27%; so The grinder is a planetary grinder, with a grinding speed of 100 to 600 rpm and a grinding time of 15 to 90 minutes.

Further, in step 2), the material mass ratio of gardenia yellow pigment to acidic reagent is 1:0.6-4, and the acidic reagent is citric acid, malic acid, tartaric acid, malonic acid or adipic acid.

Further, the acidic reagent is preferably citric acid.

Further, in step 2), the grinding speed is 100~600 rpm, and the grinding time is 15~90 min.

Further, the volume ratio of the mass of the reaction mixture to water in step 3) is 1:4~6, the mass unit is g, and the volume unit is mL; the vacuum drying oven temperature is 50°C, and the drying time is 4 h.

Beneficial effects of the present invention:

1) For the first time, the present invention adopts mechanochemical methods to prepare crocin acid quickly and efficiently. Using gardenia yellow pigment as raw material, solid-state alkaline hydrolysis and solid-state neutralization reaction are carried out through solvent-free grinding, and then freed by water washing to obtain crocetin acid. After vacuum drying, the product content is detected by high performance liquid chromatography;

2) Compared with the traditional solution method for preparing crocetin, the present invention does not require heating, takes a short time, has simple post-processing, and can obtain high-quality products without recrystallization and purification;

3) The reaction of the method of the present invention is carried out under solvent-free conditions. While reducing reaction costs, it can effectively eliminate the use of organic solvents and strongly acidic liquid reagents, reducing potential environmental pollution and safety hazards;

4) The preparation method of the present invention has the characteristics of short reaction time, simple system, low cost, easy operation, and environmental friendliness, and has good promotion prospects.

Description of the drawings

Figure 1 is a high performance liquid chromatogram of the crocetin reference substance (crocetin content ≥ 99.8%) in the present invention;

Figure 2 is a high performance liquid chromatogram of crocetin prepared in Example 1 of the present invention.

Detailed ways

The present invention will be further described below in conjunction with the examples, but the scope of protection of the present invention is not limited to the described scope.

The content of crocetin prepared in the following examples is measured and analyzed using high performance liquid chromatography, which specifically includes the following steps:

Weigh 1 to 2 mg of crocetin in a 10 mL volumetric flask, add a trace amount of DMSO to dissolve, and adjust the volume to the mark with chromatographic methanol. After appropriate dilution, filter through a 0.22 μm PTFE organic filter, and the filtrate is detected by high-performance liquid chromatography. Calculate the crocetin content of your product.

In the process of analysis using high-performance liquid chromatography (Shimadzu; SPD detector), the liquid phase conditions were based on the method used in the literature (J.Pharm. Biomed. Anal., 2011, 55, 563-568.). The liquid phase The conditions are: the chromatographic column is an ODS-3 C18 column (4.6×250 mm, 5 µm); the flow rate is 1.0 mL/min; the injection volume is 10 μL; the column temperature is 30°C; the mobile phase is methanol: water: glacial acetic acid = 75% : 24.5%: 0.5%; detection time 30 minutes; detection wavelength 440 nm.

The crocetin content in the final product is calculated using crocetin (≥99.8%) as the reference substance. The high-performance liquid chromatogram is shown in Figure 1 and is calculated using the external standard method.

Taking the peak area y as the ordinate and the solution concentration x (ppm) of the above reference substance as the abscissa, draw a standard curve. The regression equation is as follows:

y=202113x-104498，R ² =0.9996

Ⅲ,

The calculation formula for the final crocetin yield is:

Yield % = [(product mass * content calculated from liquid phase) / theoretical product mass] * 100%

Example 1

In a 45 mL stainless steel grinding jar, add gardenia yellow pigment (814.2 mg, 0.5 mmol) with a color value of 600, sodium hydroxide (200.0 mg, 5.0 mmol), and then add 280 stainless steel beads with a diameter of 4 mm (filling rate 21%), tighten the grinding jar, put it into the P7 planetary grinder, and grind at 100 rpm for 90 minutes; after grinding, remove the grinding jar and add citric acid (320.0 mg, 1.7 mmol) to it. Grind at 400 rpm for 45 minutes; after grinding, pour out the entire reaction mixture from the grinding jar, take out the stainless steel ball, add 5.3 mL of water to the reactant (material-to-liquid ratio is about 1:4), and use a Brinell filter Filter through a funnel, put the filter cake into a 50°C vacuum drying oven, and take it out after 4 hours to obtain 157.3 mg of red saffron acid;

Weigh 1 mg of crocin product into a 10 mL volumetric flask, add a trace amount of DMSO to dissolve, dilute to volume with chromatography-grade methanol, filter through a 0.22 μm PTFE organic filter, and use a Shimadzu liquid chromatograph to measure the content. When the retention time is 17.926 min, the height is 44179, the peak area is 1781368, the content is 97.3% calculated according to the regression equation of the above formula (III), and the yield is 93.2% calculated according to the yield calculation formula.

Red solid, melting point: 284-285 ℃, ¹ H NMR (500 MHz, DMSO-d6) δ 12.21 (s, 2H), 7.22 (d, J = 1.5, 1H), 7.20 (d, J = 1.5, 1H) , 6.86-6.70 (m, 4H), 6.63-6.58(m, 2H), 6.52-6.45 (m, 2H), 1.97 (s, 6H), 1.92 (s, 6H).

Example 2

In a 45 mL stainless steel grinding jar, add gardenia yellow pigment (814.2 mg, 0.5 mmol) with a color value of 600, sodium carbonate (424.0 mg, 4.0 mmol), and then add 280 stainless steel balls with a diameter of 4 mm (filling rate 21 %), tighten the grinding jar, put it into the P7 planetary grinder, and grind at 500 rpm for 45 minutes; after grinding, remove the grinding jar, add citric acid (320.0 mg, 1.7 mmol) to it, and grind in Grind for 90 minutes at 200 rpm; after grinding, pour out the entire reaction mixture from the grinding jar, take out the stainless steel ball, add 7.8 mL of water to the reactant (material-to-liquid ratio is about 1:5), and use a Buchner funnel to After suction filtration, the filter cake was placed in a 50°C vacuum drying oven and taken out after 4 hours to obtain 152.3 mg of red crocetin. The content was then measured. The operating steps were repeated in Example 1. The content was 96.9% and the yield was 89.8%.

Example 3

In a 45 mL stainless steel grinding jar, add gardenia yellow pigment (977.0 mg, 0.5 mmol) with a color value of 500, sodium hydroxide (200.0 mg, 5.0 mmol), and then add 120 stainless steel balls with a diameter of 4 mm (filling rate 9%), tighten the grinding jar, put it into the P7 planetary grinder, and grind at 300 rpm for 60 minutes; after grinding, remove the grinding jar and add adipic acid (73.0 mg, 0.5 mmol) to it. , grind at 400 rpm for 30 minutes; after grinding, pour out the entire reaction mixture from the grinding jar, take out the stainless steel ball, add 6.2 mL of water to the reactant (material-to-liquid ratio is about 1:5), and wipe with a cloth Filter through a funnel, put the filter cake into a 50°C vacuum drying oven, and take it out after 4 hours to obtain 98.9 mg of red saffron acid. Then perform content measurement and repeat the steps of Example 1; the content is 95.1% and the yield is 57.3%. .

Example 4

In a 45 mL stainless steel grinding jar, add gardenia yellow pigment (1221.2 mg, 0.5 mmol) with a color value of 400, sodium bicarbonate (420.0 mg, 5.0 mmol), and then add 200 stainless steel beads with a diameter of 4 mm (filling rate 15%), tighten the grinding jar, put it into the P7 planetary grinder, and grind at 400 rpm for 60 minutes; after grinding, remove the grinding jar and add malic acid (268.0 mg, 2.0 mmol) to it. Grind at 300 rpm for 45 minutes; after grinding, pour out the entire reaction mixture from the grinding jar, take out the stainless steel ball, add 9.5 mL of water to the reactant (material-to-liquid ratio is about 1:5), and use a Brinell filter Filter through the funnel, put the filter cake into a 50°C vacuum drying oven, and take it out after 4 hours to obtain 151.8 mg of red crocetin. Then perform content measurement and repeat the steps of Example 1; the content is 98.2% and the yield is 90.8%.

Example 5

In a 45 mL stainless steel grinding jar, add gardenia yellow pigment (1628.3 mg, 0.5 mmol) with a color value of 300, sodium hydroxide (40.0 mg, 1.0 mmol), and then add 200 stainless steel balls with a diameter of 4 mm (filling rate 15%), tighten the grinding jar, put it into the P7 planetary grinder, and grind at 400 rpm for 75 minutes; after grinding, remove the grinding jar, add tartaric acid (225.0 mg, 1.5 mmol) to it, and grind in Grind at 500 rpm for 15 minutes; after grinding, pour out the entire reaction mixture from the grinding jar, take out the stainless steel ball, add 7.6 mL of water to the reactant (material-to-liquid ratio is about 1:4), and use a Buchner funnel to After suction filtration, the filter cake was placed in a 50°C vacuum drying oven and taken out after 4 hours to obtain 56.8 mg of red crocetin. The content was then measured and the operation steps were repeated in Example 1; the content was 92.2% and the yield was 31.9%.

Example 6

In a 45 mL stainless steel grinding jar, add gardenia yellow pigment (2442.5 mg, 0.5 mmol) with a color value of 200, potassium hydroxide (112.0 mg, 2.0 mmol), and then add 360 stainless steel beads with a diameter of 4 mm (filling rate 27%), tighten the grinding jar, put it into the P7 planetary grinder, and grind at 600 rpm for 15 minutes; after grinding, remove the grinding jar and add citric acid (320.0 mg, 1.7 mmol) to it. Grind at 100 rpm for 75 min; after grinding, pour out the entire reaction mixture from the grinding jar, take out the stainless steel ball, add 14.4 mL of water to the reactant (material-to-liquid ratio is about 1:5), and use a Brinell filter Filter through a funnel, put the filter cake into a 50°C vacuum drying oven, and take it out after 4 hours to obtain 83.8 mg of red crocetin. Then perform content measurement and repeat the steps of Example 1; the content is 94.5% and the yield is 48.2%.

Example 7

In a 45 mL stainless steel grinding jar, add gardenia yellow pigment (814.2 mg, 0.5 mmol) with a color value of 600, potassium carbonate (414.0 mg, 3.0 mmol), and then add 200 stainless steel balls with a diameter of 4 mm (filling rate 15 %), tighten the grinding jar, put it into the P7 planetary grinder, and grind at 200 rpm for 30 minutes; after grinding, remove the grinding jar, add citric acid (64.0 mg, 0.3 mmol) to it, and Grind for 60 minutes at 600 rpm; after grinding, pour out the entire reaction mixture from the grinding jar, take out the stainless steel ball, add 5.2 mL of water to the reactant (material-to-liquid ratio is about 1:4), and use a Buchner funnel After suction filtration, the filter cake was placed in a 50°C vacuum drying oven and taken out after 4 hours to obtain 110.8 mg of red crocetin. The content was then measured and the operation steps were repeated in Example 1; the content was 93.7% and the yield was 63.2%.

Example 8

In a 45 mL stainless steel grinding jar, add gardenia yellow pigment (814.2 mg, 0.5 mmol) with a color value of 600, potassium bicarbonate (400.0 mg, 4.0 mmol), and then add 120 stainless steel balls with a diameter of 4 mm (filling rate 9%), tighten the grinding jar, put it into the P7 planetary grinder, and grind at 200 rpm for 75 minutes; after grinding, remove the grinding jar and add malonic acid (208.0 mg, 2.0 mmol) to it. , grind at 400 rpm for 45 minutes; after grinding, pour out the entire reaction mixture from the grinding jar, take out the stainless steel ball, add 7.1 mL of water to the reactant (material-to-liquid ratio is about 1:5), and wipe with a cloth Filter through a funnel, put the filter cake into a 50°C vacuum drying oven, and take it out after 4 hours to obtain 150.5 mg of red saffron acid. Then perform content measurement and repeat the steps of Example 1; the content is 97.3% and the yield is 89.2%. .

Example 9

In a 45 mL stainless steel grinding jar, add gardenia yellow pigment (814.2 mg, 0.5 mmol) with a color value of 600, sodium methoxide (108.0 mg, 2.0 mmol), and then add 200 stainless steel balls with a diameter of 4 mm (filling rate 15 %), tighten the grinding jar, put it into the P7 planetary grinder, and grind at 300 rpm for 90 minutes; after grinding, remove the grinding jar, add citric acid (128.0 mg, 0.7 mmol) to it, and grind in Grind for 30 minutes at 100 rpm; after grinding, pour out the entire reaction mixture from the grinding jar, take out the stainless steel ball and add 6.3 mL of water to the reactant (material-to-liquid ratio is about 1:6), and use a Buchner funnel to After suction filtration, the filter cake was placed in a 50°C vacuum drying oven and taken out after 4 hours to obtain 98.9 mg of red crocetin. The content was then measured and the operation steps were repeated in Example 1; the content was 93.7% and the yield was 60.4%.

Example 10

In a 45 mL stainless steel grinding jar, add gardenia yellow pigment (814.2 mg, 0.5 mmol) with a color value of 600, sodium tert-butoxide (288.0 mg, 3.0 mmol), and then add 200 stainless steel beads with a diameter of 4 mm (filled rate 15%), tighten the grinding jar, put it into the P7 planetary grinder, and grind at 400 rpm for 60 minutes; after grinding, remove the grinding jar and add tartaric acid (225.0 mg, 1.5 mmol) to it. Grind at 300 rpm for 45 minutes; after grinding, pour out the entire reaction mixture from the grinding jar, take out the stainless steel ball, add 6.6 mL of water to the reactant (material-to-liquid ratio is about 1:5), and use a Brinell filter Filter through a funnel, put the filter cake into a 50°C vacuum drying oven, and take it out after 4 hours to obtain 105.8 mg of red saffron acid. Then perform content measurement and repeat the steps of Example 1. The content is 95.6%, and the yield is 69.8%.

Example 11

In an 80 mL stainless steel grinding jar, add gardenia yellow pigment (8142.0 mg, 5.0 mmol) with a color value of 600, sodium hydroxide (2000.0 mg, 50.0 mmol), and then add 45 stainless steel balls with a diameter of 8 mm (filling rate 15%), tighten the grinding jar, put it into the P7 planetary grinder, and grind at 500 rpm for 75 minutes; after grinding, remove the grinding jar and add citric acid (3200.0 mg, 16.7 mmol) to it, Grind at 200 rpm for 45 minutes; after grinding, pour out the entire reaction mixture from the grinding jar, take out the stainless steel ball, add 80.0 mL of water to the reactant (material-to-liquid ratio is about 1:6), and use a Brinell filter Filter through the funnel, put the filter cake into a 50°C vacuum drying oven, and take it out after 4 hours to obtain 1552.0 mg of red crocetin. The content is then measured, and the operation steps are repeated in Example 1; the content is 99.2%, and the yield is 93.8%.

Example 12

Add gardenia yellow pigment with a color value of 600 (8142.0 mg, 5.0 mmol) and 40 mL of 10% NaOH (4000.0 mg, 100.0 mmol) solution into a 100 mL round-bottomed flask, and heat to reflux at 60°C for 2 h. After the reaction, use Adjust the pH to 2~3 with 1 mol/L hydrochloric acid solution to obtain a red precipitate, which is filtered, washed and dried to obtain a brick red solid; then dissolved in methanol to remove impurities, refluxed at 50°C for 30 minutes, filtered to obtain a red solid, and the obtained The solid was recrystallized with N,N-dimethylformamide, and finally 177.2 mg of red crocetin was obtained. Then the content was measured, and the operation steps were repeated in Example 1; the content was 98.2%, and the yield was 10.6%.

Comparing Example 11 and Example 12, it can be seen that on the basis of reducing the amount of alkaline reagents, the mechanochemical method for preparing crocetin has a short process route, a small amount of reagents, simple operation, low cost, less pollution, and does not use organic reagents and Strongly acidic liquid reagent, the final product content and yield are higher than the traditional solution method.

Claims (8)

1. A solvent-free preparation method of crocetin, characterized by comprising the following steps: 1) Put the gardenia yellow pigment and alkaline reagent into the grinding jar, add grinding beads, then tighten the grinding jar and put it into the grinder for grinding; 2) After the grinding in step 1) is completed, remove the grinding jar, add acidic reagent to it and continue grinding in the grinder; 3) After the grinding in step 2) is completed, pour out the entire reaction mixture from the grinding jar, take out the grinding beads, add water to the reactant, filter with a Buchner funnel, collect the filter cake, and put the filter cake into a vacuum Dry in the drying box and take it out after the drying is completed to obtain the crocin product; 1) In the step, the grinding jar is a 45-80mL stainless steel jar, the grinding beads are stainless steel balls with a diameter of 4mm-8mm, the number of balls added is 40-360, and the grinding filling rate is 9%-27%; the grinder It is a planetary grinder with a grinding speed of 100 to 600 rpm and a grinding time of 15 to 90 minutes; The alkaline reagent is sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate or potassium bicarbonate; The acidic reagent is citric acid, malic acid, malonic acid or adipic acid. 2. A solvent-free preparation method of crocetin according to claim 1, characterized in that the color value of gardenia yellow pigment in step 1) is 40 to 600. 3. A solvent-free preparation method of crocetin according to claim 1, characterized in that the ratio of the amounts of gardenia yellow pigment and alkaline reagent in step 1) is 1:2 to 10. 4. A solvent-free preparation method of crocetin according to claim 1, characterized in that the alkaline reagent in step 1) is sodium hydroxide. 5. A solvent-free preparation method of crocetin according to claim 1, characterized in that the ratio of the amounts of gardenia yellow pigment and acidic reagent in step 2) is 1:0.6-4. 6. A solvent-free preparation method of crocetin according to claim 1, characterized in that the acidic reagent is citric acid. 7. A solvent-free preparation method of crocetin according to claim 1, characterized in that in step 2), the grinding speed is 100~600 rpm, and the grinding time is 15~90 min. 8. A solvent-free preparation method of crocetin according to claim 1, characterized in that the volume ratio of the mass of the reaction mixture to water in step 3) is 1:4-6, the mass unit is g, and the volume unit is mL; the vacuum drying oven temperature is 50°C, and the drying time is 4 h.