Method for extracting flavone from roses

Abstract

The invention provides a method for extracting flavone from roses, and belongs to the technical field of flavonoid extraction.The extraction method comprises the steps of rose pretreatment, crude extraction, glycosylation and hydrolysis; the hydrolysis method comprises the following steps: adding crude flavonoid glycoside into a NaOH solution, stirring until the crude flavonoid glycoside is completely dissolved, regulating the pH value to 3.8-4.5 by using an HCl solution, hydrolyzing at 55-65 DEG C for 5-7 hours, filtering, regulating the pH value of filtrate to be neutral, evaporating and concentrating at 85-95 DEG C until the volume is 8-12% of the original volume, adding ethyl acetate for extraction, and filtering to obtain the flavonoid glycoside. Adding the extracted ethyl acetate into the ethyl acetate in the step 3 for cyclic utilization, cleaning a filter cake, and drying the filter cake to constant weight to obtain flavone; the yield of the flavone obtained by the method disclosed by the invention is 14.11%-15.37%, and the purity of the flavone obtained by the method is 94.94%-95.83%.

Description

Technical Field

[0001] The invention belongs to the technical field of flavonoid extraction, and particularly relates to a method for extracting flavonoids from roses. Background Art

[0002] Flavonoids are a class of natural polyphenol compounds that are widely present in plants. They have diverse biological activities and application values, such as anti-aging, improving blood circulation, and anti-inflammation. They have wide application value in the fields of medicine, food and health care products.

[0003] Roses contain a high content of flavonoids. The extraction methods for flavonoids in roses include water extraction, organic solvent extraction, CO2 supercritical extraction, ultrasound-assisted extraction and microwave-assisted extraction. The separation and purification methods for flavonoids are more commonly used, including column chromatography, thin layer chromatography, macroporous resin adsorption, aqueous two-phase extraction and capillary electrophoresis.

[0004] At present, organic solvent extraction combined with organic solvent extraction is mainly used to extract, separate and purify flavonoids in roses. This method has high purity, low requirements for instruments and equipment, low energy consumption, and good stability, but requires multiple adsorption and elution, resulting in a large amount of organic solvent, cumbersome operation, large loss of flavonoids, and low yield.

[0005] Patent CN106668234B discloses a process for extracting and purifying total flavonoids from roses. The steps of the scheme are as follows: take dried rose medicinal materials, crush them, add ethanol for reflux extraction, filter the extract, and subject the obtained extract to multiple cycles of adsorption and elution through AB-8 macroporous resin, polyamide column, column chromatography polyamide, and C18 column to finally obtain total flavonoids. Although the flavonoids obtained by this method are of high purity, the amount of eluent used is large, the operation is cumbersome, and the elution rate cannot reach 100%, resulting in the loss of some flavonoids each time elution, which in turn leads to a low yield of the final product. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a method for extracting flavonoids from roses to achieve the following invention objectives: improving the purity and yield of flavonoids while reducing the amount of organic solvent used.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: A method for extracting flavonoids from roses comprises the following steps: 1. Rose pretreatment Wash the roses, dry them at 35-45°C, grind them into 0.18-0.28 mm powder to obtain rose powder, and store them at 3-7°C for later use.

[0008] 2. Rough extraction Add rose powder to deionized water, extract in a water bath at 60-75°C for 35-45 minutes, then filter, evaporate and concentrate the filtrate at 85-95°C to 15-25% of the original volume to obtain a flavonoid glycoside concentrate for later use; The filter cake is added to an ethanol solution, extracted in a water bath at 60-80°C for 100-130 minutes, and then transferred to a centrifuge, centrifuged at 4000-5000 r / min for 7-9 minutes. After the centrifugation, the supernatant is extracted, concentrated at 55-65°C, and dried to obtain crude flavonoids for later use; The mass ratio of the rose powder to deionized water is 0.8-1.2:45-55; The mass ratio of the filter cake to the ethanol solution is 0.8-1.2:17-23; The mass fraction of the ethanol solution is 65-75%.

[0009] 3. Glycosylation The crude flavonoids are added to the ethyl acetate mixture, mixed evenly, and then tetrabutylammonium bromide and bromoacetyl glucose are added, and the reaction is carried out at a stirring rate of 80-100 r / min for 14-16 hours. After the reaction is completed, the ethyl acetate is removed by rotary evaporation at 75-85° C., and then the mixture is evenly mixed with the flavonoid glycoside concentrate obtained in step 2, and rotary evaporation is carried out at 50-70° C. to dryness to obtain the crude flavonoid glycosides; The mass ratio of the crude flavonoids, ethyl acetate mixed solution, tetrabutylammonium bromide, bromoacetyl glucose, and flavonoid glycoside concentrate is 0.31-0.37:15-17:0.008-0.012:0.5-0.9:29-33; The ethyl acetate mixed solution comprises ethyl acetate and the ethyl acetate extracted in step 4, with a mass ratio of 4.7-5.3:0.8-1.2.

[0010] 4. Hydrolysis The crude flavonoid glycosides are added to deionized water, stirred for 25 to 35 minutes, and then insoluble impurities are filtered out. The pH value is adjusted to 3.8 to 4.5 with HCl solution, and then hydrolyzed at 55 to 65° C. for 5 to 7 hours, and then filtered. After the pH value of the filtrate is adjusted to neutral, it is evaporated and concentrated to 8 to 12% of the original volume at 85 to 95° C., and then ethyl acetate is added for extraction. The extracted ethyl acetate is added to the ethyl acetate mixed solution in step 3 for recycling, and the filter cake is cleaned with deionized water, and dried at 35 to 45° C. to constant weight to obtain flavonoids; The mass ratio of the crude flavonoid glycoside, deionized water and ethyl acetate is 0.8-1.2:37-44:8-10; The mass fraction of the HCl solution is 6-13%.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention utilizes the difference in solubility of flavonoids and flavonoid glycosides in different solvents, and through the mutual conversion between flavonoids and flavonoid glycosides, the final flavonoid yield is 14.11% to 15.37% and the purity is 94.94% to 95.83%; In the process of extracting flavonoids by the method of the present invention, the properties of flavonoid glycosides being easily soluble in water and flavonoids being easily soluble in organic solvents are utilized, and deionized water and ethanol are used to extract flavonoid glycosides and flavonoids respectively to obtain flavonoid glycoside concentrate and crude flavonoids. Compared with the conventional method of directly using ethanol for extraction, the method improves the utilization rate of flavonoid glycosides, thereby improving the yield of flavonoids. According to the method, crude flavonoids are dissolved in ethyl acetate, reacted with bromoacetyl glucose to generate flavonoid glycosides, mixed with flavonoid glycoside concentrate, concentrated and dried to obtain crude flavonoid glycosides, dissolved in deionized water, filtered out insoluble impurities, and adjusted to acidic pH value with HCl solution. Under acidic environment, flavonoid glycosides are hydrolyzed to generate flavonoids, which have low solubility in water. After crystallization, flavonoids are filtered to obtain flavonoids. Compared with separation and purification by macroporous resin, the flavonoids obtained by crystallization purification have higher purity and less organic solvents are used. Flavonoid glycosides are hydrolyzed to generate flavonoids. After filtration, the filtrate will still contain a small amount of unprecipitated flavonoids. After extraction, the obtained extract can be added to the ethyl acetate mixed solution in step 3 for recycling, thereby increasing the yield of flavonoids. DETAILED DESCRIPTION

[0012] Example 1 1. Rose pretreatment Wash the roses, dry them at 40°C, grind them into 0.21-0.25 mm powder to obtain rose powder, and store them at 5°C for later use.

[0013] 2. Rough extraction Add rose powder into deionized water, extract in a 70°C water bath for 40 minutes, then filter, evaporate and concentrate the filtrate at 90°C to 20% of the original volume to obtain a flavonoid glycoside concentrate for later use; The filter cake was added to the ethanol solution, extracted in a 70°C water bath for 120 min, and then transferred to a centrifuge and centrifuged at 4500 r / min for 8 min. After the centrifugation, the supernatant was extracted, concentrated at 60°C, and dried to obtain crude flavonoids for later use; The mass ratio of the rose powder to deionized water is 1:50; The mass ratio of the filter cake to the ethanol solution is 1:20; The mass fraction of the ethanol solution is 70%.

[0014] 3. Glycosylation The crude flavonoids were added to the ethyl acetate mixture, and after mixing, tetrabutylammonium bromide and bromoacetyl glucose were added, and the reaction was carried out at a stirring rate of 90 r / min for 15 hours. After the reaction was completed, the ethyl acetate was removed by rotary evaporation at 80° C., and then the mixture was evenly mixed with the flavonoid glycoside concentrate obtained in step 2, and rotary evaporation was carried out at 60° C. to dryness to obtain the crude flavonoid glycosides; The mass ratio of the crude flavonoids, ethyl acetate mixed solution, tetrabutylammonium bromide, bromoacetyl glucose, and flavonoid glycoside concentrate is 0.34:16:0.01:0.7:31; The ethyl acetate mixed solution comprises ethyl acetate and the ethyl acetate extracted in step 4, with a mass ratio of 5:1.

[0015] 4. Hydrolysis The crude flavonoid glycosides were added to deionized water, stirred for 30 minutes, and then insoluble impurities were filtered out. The pH value was adjusted to 4 with HCl solution, and then hydrolyzed at 60°C for 6 hours, and then filtered. After the pH value of the filtrate was adjusted to neutral, it was evaporated and concentrated to 10% of the original volume at 90°C, and then ethyl acetate was added for extraction. The extracted ethyl acetate was added to the ethyl acetate mixed solution in step 3 for recycling, and the filter cake was cleaned with deionized water and dried at 40°C to constant weight to obtain flavonoids; The mass ratio of the crude flavonoid glycoside, deionized water and ethyl acetate is 1:40:9; The mass fraction of the HCl solution is 9%.

[0016] Example 2 1. Rose pretreatment Wash the roses, dry them at 35°C, grind them into 0.18-0.21 mm powder to obtain rose powder, and store them at 3°C ​​for later use.

[0017] 2. Rough extraction Add rose powder to deionized water, extract in a 60°C water bath for 35 minutes, then filter, evaporate and concentrate the filtrate at 85°C to 15% of the original volume to obtain a flavonoid glycoside concentrate for later use; The filter cake was added to the ethanol solution, extracted in a 60°C water bath for 100 min, and then transferred to a centrifuge and centrifuged at 4000 r / min for 7 min. After the centrifugation, the supernatant was extracted, concentrated at 55°C, and dried to obtain the crude flavonoids for later use; The mass ratio of the rose powder to deionized water is 0.8:45; The mass ratio of the filter cake to the ethanol solution is 0.8:17; The mass fraction of the ethanol solution is 65.

[0018] 3. Glycosylation The crude flavonoids were added to the ethyl acetate mixture, and after mixing, tetrabutylammonium bromide and bromoacetyl glucose were added, and the reaction was carried out at a stirring rate of 80 r / min for 14 hours. After the reaction was completed, the ethyl acetate was removed by rotary evaporation at 75° C., and then the mixture was evenly mixed with the flavonoid glycoside concentrate obtained in step 2, and rotary evaporation was carried out at 50° C. to dryness to obtain the crude flavonoid glycosides; The mass ratio of the crude flavonoids, ethyl acetate mixed solution, tetrabutylammonium bromide, bromoacetyl glucose, and flavonoid glycoside concentrate is 0.31:15:0.008:0.5:29; The ethyl acetate mixed solution comprises ethyl acetate and the ethyl acetate extracted in step 4, with a mass ratio of 4.7:0.8.

[0019] 4. Hydrolysis The crude flavonoid glycosides were added to deionized water, stirred for 25 minutes, and then insoluble impurities were filtered out. The pH value was adjusted to 3.8 with HCl solution, and then hydrolyzed at 55°C for 5 hours, and then filtered. After the pH value of the filtrate was adjusted to neutral, it was evaporated and concentrated to 8% of the original volume at 85°C, and then ethyl acetate was added for extraction. The extracted ethyl acetate was added to the ethyl acetate mixed solution in step 3 for recycling, and the filter cake was cleaned with deionized water and dried at 35°C to constant weight to obtain flavonoids; The mass ratio of the crude flavonoid glycoside, deionized water and ethyl acetate is 0.8:37:8; The mass fraction of the HCl solution is 6%.

[0020] Example 3 1. Rose pretreatment Wash the roses, dry them at 45°C, grind them into 0.25-0.28 mm powder to obtain rose powder, and store them at 7°C for later use.

[0021] 2. Rough extraction Add rose powder to deionized water, extract in a 75°C water bath for 45 minutes, then filter, evaporate and concentrate the filtrate at 95°C to 25% of the original volume to obtain a flavonoid glycoside concentrate for later use; The filter cake was added to the ethanol solution, extracted in a water bath at 80°C for 130 min, and then transferred to a centrifuge and centrifuged at 5000 r / min for 9 min. After the centrifugation, the supernatant was extracted, concentrated at 65°C, and dried to obtain crude flavonoids for later use; The mass ratio of the rose powder to deionized water is 1.2:55; The mass ratio of the filter cake to the ethanol solution is 1.2:23; The mass fraction of the ethanol solution is 75%.

[0022] 3. Glycosylation The crude flavonoids were added to the ethyl acetate mixture, and after mixing, tetrabutylammonium bromide and bromoacetyl glucose were added, and the reaction was carried out at a stirring rate of 100 r / min for 16 hours. After the reaction was completed, the ethyl acetate was removed by rotary evaporation at 85° C., and then the mixture was evenly mixed with the flavonoid glycoside concentrate obtained in step 2, and rotary evaporation was carried out at 70° C. to dryness to obtain the crude flavonoid glycosides; The mass ratio of the crude flavonoids, ethyl acetate mixed solution, tetrabutylammonium bromide, bromoacetyl glucose, and flavonoid glycoside concentrate is 0.37:17:0.012:0.9:33; The ethyl acetate mixed solution comprises ethyl acetate and the ethyl acetate extracted in step 4, with a mass ratio of 5.3:1.2.

[0023] 4. Hydrolysis The crude flavonoid glycosides were added to deionized water, stirred for 35 minutes, and then insoluble impurities were filtered out. The pH value was adjusted to 4.5 with HCl solution, and then hydrolyzed at 65°C for 7 hours, and then filtered. After the pH value of the filtrate was adjusted to neutral, it was evaporated and concentrated to 12% of the original volume at 95°C, and then ethyl acetate was added for extraction. The extracted ethyl acetate was added to the ethyl acetate mixed solution in step 3 for recycling, and the filter cake was cleaned with deionized water and dried at 45°C to constant weight to obtain flavonoids; The mass ratio of the crude flavonoid glycoside, deionized water and ethyl acetate is 1.2:44:10; The mass fraction of the HCl solution is 13%.

[0024] Example 4 1. Rose pretreatment Wash the roses, dry them at 40°C, grind them into 0.21-0.25 mm powder to obtain rose powder, and store them at 5°C for later use.

[0025] 2. Rough extraction Add rose powder into deionized water, extract in a 70°C water bath for 40 minutes, then filter, evaporate and concentrate the filtrate at 90°C to 20% of the original volume to obtain a flavonoid glycoside concentrate for later use; The filter cake was added to the ethanol solution, extracted in a 70°C water bath for 120 min, and then transferred to a centrifuge and centrifuged at 4500 r / min for 8 min. After the centrifugation, the supernatant was extracted, concentrated at 60°C, and dried to obtain crude flavonoids for later use; The mass ratio of the rose powder to deionized water is 1:50; The mass ratio of the filter cake to the ethanol solution is 1:20; The mass fraction of the ethanol solution is 70%.

[0026] 3. Glycosylation The crude flavonoids were added to ethyl acetate, mixed evenly, and then tetrabutylammonium bromide and bromoacetyl glucose were added. The reaction was stirred at a rate of 90 r / min for 15 hours. After the reaction was completed, the ethyl acetate was removed by rotary evaporation at 80° C., and then the mixture was evenly mixed with the flavonoid glycoside concentrate obtained in step 2, and rotary evaporation was performed at 60° C. to dryness to obtain the crude flavonoid glycosides. The mass ratio of the crude flavonoids, ethyl acetate, tetrabutylammonium bromide, bromoacetyl glucose and flavonoid glycoside concentrate is 0.34:16:0.01:0.7:31.

[0027] 4. Hydrolysis The crude flavonoid glycoside was added into deionized water, stirred for 30 minutes, and then the insoluble impurities were filtered out. The pH value was adjusted to 4 with HCl solution, and then hydrolyzed at 60°C for 6 hours, and then filtered. The filter cake was washed with deionized water and dried at 40°C to constant weight to obtain flavonoids. The mass ratio of the crude flavonoid glycoside to deionized water is 1:40; The mass fraction of the HCl solution is 9%.

[0028] Example 5 1. Rose pretreatment Wash the roses, dry them at 40°C, grind them into 0.21-0.25 mm powder to obtain rose powder, and store them at 5°C for later use.

[0029] 2. Rough extraction Add rose powder into deionized water, extract in a 70°C water bath for 40 minutes, then filter, evaporate and concentrate the filtrate at 90°C to 20% of the original volume to obtain a flavonoid glycoside concentrate for later use; The filter cake was added to the ethanol solution, extracted in a 70°C water bath for 120 min, and then transferred to a centrifuge and centrifuged at 4500 r / min for 8 min. After the centrifugation, the supernatant was extracted, concentrated at 60°C, and dried to obtain crude flavonoids for later use; The mass ratio of the rose powder to deionized water is 1:50; The mass ratio of the filter cake to the ethanol solution is 1:20; The mass fraction of the ethanol solution is 70%.

[0030] 3. Separation and purification The crude flavonoids and the flavonoid glycoside concentrate were mixed and added into a 30%wt ethanol solution, stirred and dissolved, and then adsorbed by a macroporous resin, and the loading flow rate was controlled to be 1.5mL / min. After the loading was completed, the mixture was allowed to stand for 30min, and then eluted with a 70%wt ethanol solution at a flow rate of 30mL / min. The eluate was collected, concentrated at 60°C, and dried to obtain flavonoids; The mass ratio of the crude flavonoids, the flavonoid glycoside concentrate, the 30%wt ethanol solution, and the 70%wt ethanol solution is 1:10:60:240.

[0031] Example 6 The same extraction method as in Example 5 was used to obtain an eluent, except that the obtained eluent was mixed and diluted with deionized water, and then subjected to secondary adsorption by a macroporous resin, and the loading flow rate was controlled to be 1.5 mL / min. After the loading was completed, the mixture was allowed to stand for 30 min, and then eluted with a 70% wt ethanol solution at a flow rate of 30 mL / min. The secondary eluent was collected, concentrated at 60° C., and dried to obtain flavonoids. The mass ratio of the eluent, deionized water and 70%wt ethanol solution is 1:3:10.

[0032] Test example The flavonoids extracted from Examples 1 to 6 were tested, and the test items included flavonoid yield and flavonoid purity; The flavonoid yield test method is as follows: Flavonoid yield = flavonoid mass / rose powder mass; Using rutin as a control, the NaNO2-Al(NO3)3-NaOH colorimetric method was used to determine the flavonoid content and the purity of the flavonoid was obtained. The results of flavonoid yield and flavonoid content are shown in Table 1.

[0033] Table 1.

[0034]

[0035] It can be seen from Examples 1 to 6 that Example 4 does not recycle the filtrate after hydrolysis, which leads to a decrease in the yield of flavonoids; Example 5 uses a macroporous resin method to separate and purify crude flavonoids, and the flavonoid yield is not much different from that of the example, but the purity is lower. Therefore, when the macroporous resin method is used to separate and purify crude flavonoids, it is usually necessary to circulate and purify multiple times, but the yield of flavonoids will be reduced; Example 6 uses a macroporous resin method to circulate and separate and purify crude flavonoids twice. Compared with Example 5, the yield is reduced and the purity is improved.

Claims

1. A method for extracting flavonoids from rose, characterized in that: The method for extracting flavonoids comprises rose pretreatment, crude extraction, glycosylation, and hydrolysis; The glycosylation method comprises the following steps: adding the crude flavonoids obtained in the crude extraction step to an ethyl acetate mixture, mixing them evenly, adding tetrabutylammonium bromide and bromoacetyl glucose, stirring and reacting for 14 to 16 hours, and after the reaction is completed, removing the ethyl acetate by rotary evaporation at 75 to 85° C., and then mixing them evenly with the flavonoid glycoside concentrate obtained in the crude extraction step, and rotary evaporation and drying to obtain the crude flavonoid glycosides; The hydrolysis method comprises the following steps: adding crude flavonoid glycosides to deionized water, stirring for 25 to 35 minutes, filtering out insoluble impurities, adjusting the pH value to 3.8 to 4.5 using an HCl solution, hydrolyzing at 55 to 65° C. for 5 to 7 hours, filtering, adjusting the pH value of the filtrate to neutral, evaporating and concentrating the filtrate to 8 to 12% of the original volume at 85 to 95° C., adding ethyl acetate for extraction, adding the extracted ethyl acetate to the ethyl acetate mixed solution in the glycosidation step for recycling, cleaning the filter cake, and drying to constant weight to obtain flavonoids.

2. A method for extracting flavonoids from rose according to claim 1, characterized in that: In the glycosylation method, the mass ratio of crude flavonoids, ethyl acetate mixed solution, tetrabutylammonium bromide, bromoacetyl glucose, and flavonoid glycoside concentrate is 0.31-0.37:15-17:0.008-0.012:0.5-0.9:29-33, the ethyl acetate mixed solution comprises ethyl acetate and ethyl acetate extracted in the hydrolysis step, the mass ratio is 4.7-5.3:0.8-1.2, the stirring rate is 80-100 r / min, and the rotary evaporation drying temperature is 50-70°C.

3. A method for extracting flavonoids from rose according to claim 1, characterized in that: In the hydrolysis method, the mass ratio of crude flavonoid glycosides, deionized water and ethyl acetate is 0.8-1.2:37-44:8-10, and the mass fraction of the HCl solution is 6-13%.

4. A method for extracting flavonoids from rose according to claim 1, characterized in that: The rose pretreatment method comprises the following steps: cleaning the rose, drying the rose at 35-45°C, grinding the rose into 0.18-0.28 mm powder, obtaining rose powder, and storing the powder at 3-7°C for later use.

5. A method for extracting flavonoids from rose according to claim 1, characterized in that: The crude extraction method comprises the following steps: adding rose powder to deionized water, extracting in a water bath for 35 to 45 minutes, filtering, evaporating and concentrating the filtrate to 15 to 25% of the original volume at 85 to 95° C., obtaining a flavonoid glycoside concentrate for later use; adding the filter cake to an ethanol solution, extracting in a water bath for 100 to 130 minutes, transferring the filter cake to a centrifuge, centrifuging for 7 to 9 minutes, extracting the supernatant after the centrifugation, concentrating at 55 to 65° C., and drying the solution to obtain a crude flavonoid for later use.

6. A method for extracting flavonoids from rose according to claim 5, characterized in that: In the crude extraction method, the mass ratio of rose powder to deionized water is 0.8-1.2:45-55, the mass ratio of filter cake to ethanol solution is 0.8-1.2:17-23, and the mass fraction of ethanol solution is 65-75%.

7. A method for extracting flavonoids from rose according to claim 5, characterized in that: In the crude extraction method, the water bath extraction temperature of the rose powder is 60-75° C., the water bath extraction temperature of the filter cake is 60-80° C., and the centrifugal speed is 4000-5000 r / min.

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