Preparation method of hydroxysafflor yellow A

Abstract

The present invention belongs to the field of natural drug extraction, and specifically relates to a method for preparing hydroxysafflor yellow A. The method for preparing hydroxysafflor yellow A comprises the following steps: extraction and concentration; flocculation; HZ chromatography 3 # Resin column separation; HZ001 cationic resin exchange; gel column separation; concentration and drying. The preparation method of the present invention solves the problems of long process, low yield and high production cost in the current hydroxysafflor yellow A extraction process. The present invention provides a method for preparing hydroxysafflor yellow A with a short preparation process, low energy consumption and high product purity, which effectively improves the yield of hydroxysafflor yellow A.

Description

Technical Field

[0001] The invention belongs to the field of natural medicine extraction, and particularly relates to a preparation method of hydroxysafflor yellow A. Background Art

[0002] Safflower is the dried flower of Carthamus tinctorius L., a plant of the Asteraceae family. The flowers are picked when they turn from yellow to red in summer and dried in the shade or in the sun. It has the effects of promoting blood circulation, relieving menstruation, dispersing blood stasis and relieving pain. Safflower is a common Chinese medicine for promoting blood circulation and removing blood stasis, and can be used to treat many blood circulation disorders such as coronary heart disease and angina pectoris. It is also used to treat amenorrhea, dysmenorrhea, lochia retention, abdominal masses, chest pain, abdominal pain due to blood stasis, chest and flank pain, injuries from falls, and sores and swelling.

[0003] Safflower is rich in safflower yellow, and the proportion of safflower yellow in the safflower is 20%-30%. Hydroxysafflor yellow A is the main pharmacological substance of safflower yellow. Hydroxysafflor yellow A is a compound with a single chalcone glycoside structure. It is the most effective water-soluble part of the pharmacological effect of safflower. It can inhibit the platelet aggregation and release induced by platelet activating factor, and can competitively inhibit the binding of platelet activating factor to platelet receptors. It is the effective component of safflower yellow for promoting blood circulation and removing blood stasis. It has antithrombin-induced platelet aggregation activity, anti-inflammatory activity, cell protection activity, and anti-tumor activity, as well as anti-platelet and anti-myocardial ischemia effects. At the same time, hydroxysafflor yellow A can also be used to make health care and cosmetics, or it is a good food dye. Safflower yellow is a natural food colorant. Because it is bright yellow, has extremely low toxicity, strong tinting power, and stable color tone, it is widely used in food coloring such as juice-type beverages, candies, and cakes.

[0004] Although hydroxysafflor yellow A and its separation and purification methods have been disclosed in the prior art, such as Chinese patent application CN201010606888.5, which discloses a method for preparing hydroxysafflor yellow A, the method uses high-speed countercurrent chromatography to quickly and massively prepare high-purity hydroxysafflor yellow A from safflower, providing a material basis for the pharmacological and pharmacodynamic studies of safflower preparations and hydroxysafflor yellow A. Chinese patent application CN02132781.5 discloses a method for preparing hydroxysafflor yellow A, which prepares hydroxysafflor yellow A by extraction, separation, alcohol precipitation, and purification.

[0005] However, the purity of the existing hydroxy safflower yellow A product is not high. From the perspective of the purity of the existing safflower yellow, there are basically more than 10-20% impurities, so further purification is required after separation. The structural properties of these impurities have not been qualitatively determined, and there is a certain degree of quality uncontrollability. In addition, the current safflower yellow extraction process also has problems such as long process, high energy consumption, low yield, and high production cost.

[0006] The present invention aims to provide a preparation method of hydroxysafflor yellow A, which adopts a more environmentally friendly leaching method for extraction. Compared with the ultrasonic extraction method, the energy consumption level in the preparation process of hydroxysafflor yellow A is significantly reduced, thereby providing a low-energy consumption and environmentally friendly preparation method. The preparation method of hydroxysafflor yellow A provided by the invention has a short process flow, low energy consumption, environmental protection, high product purity, and effectively reduces the production cost of safflor yellow. Summary of the invention

[0007] In order to overcome the above technical problems, the present invention provides a method for preparing hydroxysafflor yellow A. The method for preparing hydroxysafflor yellow A obtained by optimizing the process extraction, separation and purification steps provides a method for quickly and low-costly preparing high-purity hydroxysafflor yellow A.

[0008] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0009] A method for preparing hydroxysafflor yellow A comprises the following steps:

[0010] (1) Extraction and concentration: extract safflower with 60-90% (volume concentration) ethanol, and concentrate the extract to obtain a concentrated solution;

[0011] (2) flocculation: flocculating the concentrated solution with a deacetylated chitosan solution to obtain a flocculated solution;

[0012] (3) HZ Chromatography 3 # Resin column separation: The flocculation treatment liquid is subjected to HZ chromatography 3 # Separate with resin column, elute with purified water at a rate of 0.5-1BV / h after loading, and collect the eluate;

[0013] (4) HZ001 cationic resin exchange: The eluate in (3) was exchanged with HZ001 cationic resin, and after loading, it was eluted with purified water at a rate of 0.5-1 BV / h, and the eluate was collected;

[0014] (5) Gel column separation: the eluate in (4) was separated by passing through an LH-20 gel column, loaded with the sample and eluted with purified water at a loading and elution rate of 0.2-0.4 BV / h, and the eluate was collected;

[0015] (6) Concentration and drying: Concentrate to dryness under reduced pressure, grind into fine powder, stir in ethanol at 50°C for 2 hours, filter, soak and wash the filter cake twice with ethanol, filter, and finally vacuum dry to obtain hydroxysafflor yellow A.

[0016] Preferably, in (1), the extraction is performed using 80-90% ethanol (volume concentration).

[0017] Preferably, in (1), the extraction temperature is 65-70°C.

[0018] Preferably, in (2), the reagent used for flocculation is a solution containing 2% deacetylated chitosan made of 1% glacial acetic acid.

[0019] Preferably, in (2), the flocculation treatment process is: firstly reduce the stirring speed to 15-60 rpm and stir, then add diatomaceous earth with a concentration of 4-6% of the safflower medicinal material and treat for 5-10 minutes, and let it stand for 1-3 hours.

[0020] Preferably, in (2), the flocculation treatment process is: firstly reduce the stirring speed to 15-60 rpm and stir, then add diatomaceous earth with a concentration of 5% of the safflower medicinal material and treat for 5 minutes, and let it stand for 2 hours.

[0021] Preferably, in (2), the flocculation treatment process is to add 2% deacetylated chitosan solution prepared by 1% glacial acetic acid with a medicinal material content of 3.0-3.5% at a stirring speed of 80 rpm, continue stirring for 1-5 minutes, reduce the stirring speed to 15-60 rpm, add diatomaceous earth with a medicinal material content of 5%, stir for 5 minutes, and let stand for 2 hours.

[0022] Preferably, in (3), the HZ chromatogram 3 # Resin column separation, after loading, elution with purified water at a speed of 0.5-1BV / h, when the yellow band of hydroxysafflor yellow A reaches the bottom of the column (when the eluent is monitored by an ultraviolet detector at an absorption wavelength of 403nm to detect strong absorption or when the Brix of the eluent decreases from 10°Bx to 8°Bx, or when the color of the eluent obviously changes from light yellow to light red), start collecting 5-10 portions of the eluent in sections of 0.02-0.04BV each, take 1μ of the sample and spot it on the same polyamide plate and develop it in 35-40% acetic acid, combine the eluents with obvious spots and continue to collect the eluent to 1-2BV.

[0023] Preferably, in (3), the HZ chromatogram 3 #Separate with resin column, elute with purified water at a rate of 0.5-1BV / h after loading. When the yellow band of hydroxysafflor yellow A reaches the bottom of the column, start collecting 8 portions of eluate in sections of 0.04BV each. Take 1μ of the sample and spot it on the same polyamide plate and develop it in 36% acetic acid. Combine the eluates with obvious spots and continue to collect the eluate to 1.5BV.

[0024] Preferably, in (4), the HZ001 cationic resin is exchanged, and after loading, purified water is used for elution at a rate of 0.5-1BV / h. When the effluent begins to turn yellow, collection begins. The collection volume is the sum of the loading volume and the column bed volume. The collected liquid is adjusted to pH 4.2-4.7 with ammonia water, and concentrated under reduced pressure below 45°C to a relative density of 1.07-1.10 at 20-30°C.

[0025] Preferably, in (5), the gel column separation is a LH-20 column, and purified water is used for elution after loading. The loading and elution rate is 0.2-0.4 BV / h. When the yellow band of hydroxysafflor yellow A reaches the bottom of the column, 6-8 portions are collected at 0.02-0.04 BV per portion, and then 0.02-0.04 BV per portion is collected, with a total collection amount of 0.3 BV. 1 μl of each sample is spotted on the same polyamide plate and developed in 30-40% acetic acid, and the eluates with obvious bright yellow spots are combined.

[0026] Preferably, in (5), the acetic acid is 36% acetic acid.

[0027] Preferably, in (6), the concentration and drying are carried out at 40-60°C, preferably 45°C, under reduced pressure.

[0028] Preferably, in (6), the product concentrated under reduced pressure is crushed into fine powder, and then ethanol in an amount of 8-10 times (V / W) of the fine powder is added, and the mixture is stirred at 40-60°C for 1-3 hours, filtered, and the filter cake is soaked and washed with ethanol for 1-3 times; then dried at 30-50°C and -0.090-0.050MPa in vacuum for 4-8 hours, and then phosphorus pentoxide with a thickness of 1 / 3-2 / 3 of the wet product is placed in a drying oven, and vacuum drying is continued at 30-50°C and -0.090-0.050MPa for 4-8 hours.

[0029] Preferably, in (6), the concentration and drying process is as follows: the sample separated by the gel column is concentrated to dryness under reduced pressure at below 45°C, crushed into fine powder, 9 times the amount (V / W) of ethanol as much as the fine powder is added, stirred at 50°C for 2 hours, filtered, and the filter cake is then washed twice with ethanol, each time with 2 times the amount (V / W) of the fine powder, filtered, and the wet product is placed in a tray to control the thickness to about 0.3g / cm 2After vacuum drying at 30°C and -0.090MPa for 5 hours, put 1 / 3 of the wet product in a drying oven with an equal thickness of phosphorus pentoxide, and continue vacuum drying at 50°C and -0.090MPa for 5 hours to obtain hydroxysafflor yellow A.

[0030] Compared with the prior art, the technical advantages of the present invention are:

[0031] (1) The present invention provides a new method for preparing hydroxysafflor yellow A compound. The method for preparing hydroxysafflor yellow A obtained by optimizing the process extraction, separation and purification steps provides a method for quickly and at low cost preparing highly hydroxysafflor yellow A.

[0032] (2) The preparation method provided by the present invention has a short process flow, low energy consumption, and high product purity, which effectively reduces the production cost of safflower yellow pigment and can quickly and low-cost prepare high-purity hydroxysafflower yellow pigment A. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : Liquid chromatogram of embodiment 1 product. DETAILED DESCRIPTION

[0034] The present invention is described below by specific examples to make the technical solution of the present invention easier to understand and grasp, but the present invention is not limited thereto. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0035] Example 1

[0036] 1. Extraction and concentration

[0037] Take safflower, extract the solution with 80% ethanol at 68°C twice, the first time with 12 times the amount (V / W) for 1.5 hours, the second time with 10 times the amount (V / W) for 1 hour, filter, centrifuge the residue, combine the liquid, and concentrate under reduced pressure below 45°C to a relative density of 1.03 (45°C).

[0038] 2. Flocculation

[0039] At a stirring speed of 80 rpm, 2% deacetylated chitosan solution prepared with 1% glacial acetic acid, which is 3.3% of the medicinal material, was added, and stirring was continued for 5 minutes. After reducing the stirring speed to 30 rpm, diatomaceous earth, which is 5% of the medicinal material, was added and stirred for 5 minutes, allowed to stand for 2 hours, and centrifuged.

[0040] 3.HZ Chromatography 3 # Resin column separation

[0041] The flocculated sample was loaded onto the HZ chromatograph at a rate of 0.5 BV / h. #Resin column (purchased from Shanghai Huazhen Technology Co., Ltd.), (100-150 mesh, column bed height 800mm, the amount of drug solution loaded per 1ml of resin is not more than [14.3 (mg / g) ÷ drug material hydroxysafflor yellow A content (mg / g) ÷ 90% (transfer rate) × 80%] g or not more than 11mg in terms of hydroxysafflor yellow A, the drug solution temperature is room temperature, and during regeneration, 1BV is eluted with 1% sodium hydroxide, purified water, and 70% ethanol at a speed of 1BV / h, and then about 3BV is eluted with purified water until there is no alcohol, After loading, use purified water to elute at a rate of 0.75 BV / h. When the yellow band of hydroxysafflor yellow A reaches the bottom of the column (when the eluent is monitored by an ultraviolet detector at an absorption wavelength of 403 nm to detect strong absorption or when the Brix of the eluent decreases from 10°Bx to 8°Bx, or when the color of the eluent obviously changes from light yellow to light red), start to collect 8 portions of the eluent in sections of about 0.04 BV each, take 1 μ of each spot on the same polyamide plate and develop it in 36% acetic acid, combine the eluents with obvious spots and continue to collect the eluent to 1.5 BV.

[0042] 4.HZ001 cationic resin exchange

[0043] The sample was loaded onto a HZ001 cationic resin exchange column (50-80 mesh, column bed height not less than 800 mm, the amount of drug solution loaded per 1 ml of resin was not more than 2.1 g in terms of drug material, the drug solution temperature was room temperature, 4 BV was eluted with 4% hydrochloric acid solution at a rate of 1 BV / h during regeneration, and then eluted with about 4 BV of purified water to a pH of about 4), after loading, the sample was eluted with purified water at a rate of 0.5 BV / h, and the effluent was collected when it began to turn yellow. The collected amount was the sum of the amount of drug solution loaded and the column bed volume. The collected liquid was adjusted to a pH of about 4.5 with ammonia water, and concentrated under reduced pressure below 45°C to a relative density of about 1.08 (25°C).

[0044] 5. Gel column separation

[0045] The sample was loaded onto an LH-20 column (Fine, column bed height 800 mm, the amount of drug solution loaded per 1 ml of gel was no more than 0.7 g in terms of drug material, and the volume of the drug solution was no more than 10% of the volume of the gel column, the loading drug solution temperature was room temperature, 0.2 BV was eluted with 0.3% sodium carbonate solution at a rate of 0.3 BV / h during regeneration, and then eluted to neutrality with about 1.5 BV of purified water), and eluted with purified water after loading, and the loading and elution rates were all 0.3 BV / h. When the yellow band of hydroxysafflor yellow A reached the bottom of the column, 7 portions were collected at a rate of about 0.02 BV per portion, and then at a rate of about 0.03 BV per portion, with a total collection of about 0.3 BV. 1 μl of the sample was spotted on the same polyamide plate and developed in 36% acetic acid, and the eluates with obvious bright yellow spots were combined.

[0046] 6. Concentration and drying

[0047] The sample separated by gel column was concentrated to dryness under reduced pressure below 45°C, crushed into fine powder, and 9 times (V / W) of ethanol as much as the fine powder was added, stirred at 50°C for 2 hours, filtered, and the filter cake was washed twice with ethanol, each time with 2 times (V / W) of the fine powder, filtered, and the wet product was placed in a tray to control the thickness to about 0.3g / cm 2 After vacuum drying at 30°C and -0.090 MPa for about 5 hours, 1 / 3 of the wet product with an equal thickness of phosphorus pentoxide was placed in a drying oven, and vacuum drying was continued at 50°C and -0.090 MPa for about 5 hours to obtain hydroxysafflor yellow A. The purity of hydroxysafflor yellow A is 98.7%.

[0048] Example 2

[0049] 1. Extraction and concentration

[0050] Take safflower, extract the solution with 80% ethanol at 68°C twice, the first time with 12 times the amount (V / W) for 1.5 hours, the second time with 10 times the amount (V / W) for 1 hour, filter, centrifuge the residue, combine the liquid, and concentrate under reduced pressure below 45°C to a relative density of 1.03 (45°C).

[0051] 2. Flocculation

[0052] At a stirring speed of 80 rpm, 2% deacetylated chitosan solution prepared with 1% glacial acetic acid, which is 3.3% of the medicinal material, was added, and stirring was continued for 5 minutes. After reducing the stirring speed to 30 rpm, diatomaceous earth, which is 5% of the medicinal material, was added and stirred for 5 minutes, allowed to stand for 2 hours, and centrifuged.

[0053] 3. HZ-816 resin column separation

[0054] The flocculated sample was loaded onto a HZ-816 resin column (100-150 mesh, column bed height 800 mm) at a rate of 0.5 BV / h. The amount of drug solution loaded per 1 ml of resin was no more than [14.3 (mg / g) ÷ drug hydroxysafflor yellow A content (mg / g) ÷ 90% (transfer rate) × 80%] g in terms of drug material or no more than 11 mg in terms of hydroxysafflor yellow A. The drug solution temperature was room temperature. After loading, the sample was washed with 30% ethanol at a rate of 0.5 BV / h. When the yellow band of hydroxysafflor yellow A reaches the bottom of the column (when the eluent detects strong absorption at an absorption wavelength of 403nm using an ultraviolet detector or the Brix of the eluent decreases from 10°Bx to 8°Bx, or the color of the eluent obviously changes from light yellow to light red), start to collect 8 portions of the eluate in sections of about 0.04BV each, take 1μ of each spot on the same polyamide plate and develop it in 36% acetic acid, combine the eluates with obvious spots and continue to collect the eluate to 1.5BV.

[0055] 4. 724 resin column separation

[0056] The sample was loaded onto a 724 resin column (50-80 mesh, column bed height not less than 800 mm, the amount of drug solution loaded per 1 ml of resin was not more than 2.1 g in terms of drug material, the drug solution temperature was room temperature, 4 BV was eluted with 4% hydrochloric acid solution at a rate of 1 BV / h during regeneration, and then eluted with about 4 BV of purified water to a pH of about 4), after loading, the sample was eluted with purified water at a rate of 0.75 BV / h, and the effluent was collected when it began to turn yellow. The collected amount was the sum of the amount of drug solution loaded and the column bed volume. The collected liquid was adjusted to a pH of about 4.5 with ammonia water, and concentrated under reduced pressure below 45°C to a relative density of about 1.07 (45°C).

[0057] 5. Gel column separation

[0058] The sample was loaded onto an LH-20 column (Fine, column bed height 800 mm, the amount of drug solution loaded per 1 ml of gel was no more than 0.7 g in terms of drug material, and the volume of the drug solution was no more than 10% of the volume of the gel column, the loading drug solution temperature was room temperature, and during regeneration, about 0.2 BV was eluted with 0.3% sodium carbonate solution at a rate of 0.3 BV / h, and then eluted with about 1.5 BV of purified water to neutrality), and after loading, purified water was used for elution, and the loading and elution rates were all 0.3 BV / h. When the yellow band of hydroxysafflor yellow A reached the bottom of the column, 7 portions were collected at about 0.02 BV per portion, and then at about 0.03 BV per portion, with a total collection of about 0.3 BV. 1 μl of the sample was spotted on the same polyamide plate and developed in 36% acetic acid, and the eluates with obvious bright yellow spots were combined.

[0059] 6. Concentration and drying

[0060] The sample separated by gel column was concentrated to dryness under reduced pressure below 45°C, crushed into fine powder, and 9 times (V / W) of ethanol as much as the fine powder was added, stirred at 50°C for 2 hours, filtered, and the filter cake was washed twice with ethanol, each time with 2 times (V / W) of the fine powder, filtered, and the wet product was placed in a tray to control the thickness to about 0.3g / cm 2 After vacuum drying at 30°C and -0.090 MPa for about 5 hours, 1 / 3 of the wet product with an equal thickness of phosphorus pentoxide was placed in a drying oven, and vacuum drying was continued at 50°C and -0.090 MPa for about 5 hours to obtain hydroxysafflor yellow A. The purity of hydroxysafflor yellow A is 90.1%.

[0061] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A method for preparing hydroxysafflor yellow A, comprising the following steps: (1) Extraction and concentration: Take safflower, extract it twice with 80% ethanol at 68℃, the first time with 12 times the amount V / W for 1.5 hours, the second time with 10 times the amount V / W for 1 hour, filter, centrifuge the residue, combine the liquid, and concentrate under reduced pressure below 45℃ to a relative density of 1.03 at 45℃; (2) Flocculation: At a stirring speed of 80 rpm, add 2% deacetylated chitosan solution prepared with 1% glacial acetic acid, which is 3.3% of the medicinal material, and continue stirring for 5 minutes. Reduce the stirring speed to 30 rpm, add diatomaceous earth, which is 5% of the medicinal material, and stir for 5 minutes. Let it stand for 2 hours and centrifuge. (3) HZ chromatography 3# resin column separation: The flocculated sample was loaded onto the HZ chromatograph at a rate of 0.5 BV / h. # After loading the sample on the resin column, use purified water to elute at a rate of 0.75 BV / h. When the yellow band of hydroxysafflor yellow A reaches the bottom of the column, start collecting 8 portions of the eluate in sections of 0.04 BV each. Take 1 μ of the sample and spot it on the same polyamide plate and develop it in 36% acetic acid. Combine the eluates with obvious spots and continue to collect the eluate to 1.5 BV. (4) HZ001 cationic resin exchange: Load the sample onto the HZ001 cationic resin exchange column at a rate of 0.5 BV / h, and then elute with purified water at a rate of 0.5 BV / h. When the effluent begins to turn yellow, start collecting. The collection volume is the sum of the loading volume and the column bed volume. Adjust the pH of the collected liquid to 4.5 with ammonia water, and concentrate under reduced pressure at below 45°C to a relative density of 1.08 at 25°C. (5) Gel column separation: The sample was loaded onto the LH-20 column and eluted with purified water. The loading and elution speed was 0.3 BV / h. When the yellow band of hydroxysafflor yellow A reached the bottom of the column, 7 portions were collected at 0.02 BV each, and then at 0.03 BV each. The total amount collected was 0.3 BV. 1 μl of the sample was spotted on the same polyamide plate and developed in 36% acetic acid. The eluates with obvious bright yellow spots were combined. (6) Concentration and drying: The sample separated by gel column was concentrated to dryness under reduced pressure below 45°C, crushed into fine powder, added 9 times the amount of fine powder V / W ethanol at 50°C, stirred for 2 hours, filtered, and the filter cake was soaked and washed twice with ethanol, each time with 2 times the amount of fine powder V / W, filtered, and the wet product was placed in a tray to control the thickness to 0.3g / cm 2 After vacuum drying at 30°C and -0.090MPa for 5 hours, put 1 / 3 of the wet product in a drying oven with an equal thickness of phosphorus pentoxide, and continue vacuum drying at 50°C and -0.090MPa for 5 hours to obtain hydroxysafflor yellow A.

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