Extraction process of fisetin from cotinus coggygria

Abstract

The present invention relates to the technical field of compound extraction, discloses a kind of extraction process of fisetin in cotinus coggygria, including: S1: take the branch and / or leaves of cotinus coggygria and crush them to obtain cotinus coggygria crushed material;S2: cotinus coggygria crushed material is added into a continuous countercurrent extraction tank, water is added, extraction is started, and extraction temperature is 90-95 DEG C to obtain extract;The extract is filtered and filtrate is taken, heated and concentrated to obtain a crude extract;S3: the crude extract is rinsed, filter and take filter cake, obtain solid, solid is added to ethanol for dissolution, after adding water and filtering, reduced pressure is concentrated to ethanol concentration of 60%-75%, crystallization, filter and take filter cake, obtain refined extract;S4: ethanol is added to the refined extract, heated to dissolve and continue 1-1.5h, filter and take filtrate, after reduced pressure is concentrated to ethanol concentration of 70%-75%, stand crystallization filtration and drying, obtain fisetin finished product. The extraction process of the present invention has high extraction efficiency, large conversion rate and is green and environmentally friendly.

Description

Technical Field

[0001] The invention relates to the technical field of compound extraction, in particular to an extraction process of fisetin from cotinus coggygria. Background Art

[0002] Cotinus coggygria (Cotinus coggygria scop.) is a plant of the genus Cotinus (Cotinus L.) in the Anacardiaceae family. According to the "Supplement to Materia Medica," it is bitter, cold, and non-toxic. It can relieve fever, alcohol-induced jaundice, and jaundice. It can be boiled and taken orally. It clears heat and detoxifies, dissipates blood stasis, and relieves pain. The rhizome of Cotinus coggygria is used for acute icteric hepatitis, chronic hepatitis, anicteric hepatitis, and measles; the leaves are used for erysipelas and lacquer sores. The main chemical components of Cotinus coggygria roots and stems are flavonoids (approximately 6.75%). Cotinus coggygria contains a variety of active ingredients, including flavonoids (primarily fisetin), anthocyanins, and tannins.

[0003] Fisetin, also known as fisetin, fisetin acid, and fisetin, is a high molecular weight natural compound with the molecular formula C 15 H 10 O6, with a molecular weight of 286.23. Fisetin is a yellow crystal that is soluble in ethanol, acetone, and acetic acid, but poorly soluble or insoluble in water, chloroform, and petroleum ether. Fisetin has excellent pharmacological properties, including promoting cell renewal and anti-aging; maintaining bone, skin, and cardiovascular health; and promoting weight loss.

[0004] The traditional extraction of fisetin from Cotinus coggygria requires pretreatment, including fermentation treatment, ethanol treatment, etc., and does not directly extract the Cotinus coggygria with water, which greatly prolongs the time. Secondly, the extraction process of fisetin from Cotinus coggygria often requires industrial reagents such as methanol, ammonia water, sodium hydroxide, n-butanol, etc., and the product safety is poor, and it cannot be used in functional food, cosmetics and other industries. In addition, the extraction of Cotinus coggygria mainly uses ethanol reflux extraction, which requires repeated extractions to completely extract fisetin from Cotinus coggygria, and the transfer rate of the target substance fisetin in each step is unknown. The existing methods for extracting fisetin from Cotinus coggygria are mostly to crush the Cotinus coggygria branches, treat them with organic solvents or enzymes for a period of time, and then extract them by reflux or solvent method, and then purify them by macroporous adsorption resin, ethanol, etc. Finally, n-butanol, ethyl acetate, etc. are required for extraction, recrystallization and other steps to finally obtain the product. The process is complicated, time-consuming and not suitable for mass production in factories. At the same time, most industrial production of flavonoids uses industrial reagents such as methanol and n-butanol, resulting in toxic reagent residues in the final product, which is unsafe and pollutes the environment. Summary of the Invention

[0005] In view of this, the present invention proposes an extraction process for fisetin from Cotinus coggygria to solve the problems of complex process, long time consumption, unsuitability for mass production in factories and certain pollution to the environment.

[0006] The following technical solution is currently adopted: A process for extracting fisetin from Cotinus coggygria, comprising:

[0007] S1: grinding the branches and / or leaves of Cotinus coggygria to obtain Cotinus coggygria powder;

[0008] S2: adding the crushed cotinus coggygria to a continuous countercurrent extraction tank, adding 8-10 times the mass of water of the crushed cotinus coggygria to the continuous countercurrent extraction tank, and starting extraction at an extraction temperature of 90-95° C. to obtain an extract;

[0009] The extract is filtered and the filtrate is collected, and the filtrate is concentrated by heating to a density of 1.02-1.06 g / cm3 to obtain a crude extract;

[0010] S3: washing the crude extract, filtering the filter cake to obtain a solid, dissolving the solid in 90%-95% ethanol, adding water, filtering, and concentrating under reduced pressure to an ethanol concentration of 60%-75%, crystallizing, and filtering to obtain a filter cake to obtain a refined extract;

[0011] S4: Add 95% ethanol to the refined extract, heat to dissolve and continue for 1-1.5 hours, filter the filtrate, and concentrate under reduced pressure to an ethanol concentration of 70%-75%, then stand for crystallization, filter, and dry to obtain the finished fisetin.

[0012] Furthermore, the extraction process in S2 lasts for 1-2 hours.

[0013] Furthermore, the temperature of the heating and concentration in S2 is 80°C.

[0014] Furthermore, the rinsing in S3 is specifically as follows: taking 1%-5% of the amount of the crushed cotinus coggygria to rinse the crude extract, and the water temperature is 80-90°C.

[0015] Furthermore, the amount of ethanol used in the dissolution in S3 is 23-32 times the mass of the solid.

[0016] Furthermore, the crystallization in S3 is allowed to stand at room temperature for at least 24 hours.

[0017] Furthermore, the concentration of ethanol added to the refined extract in S4 is 95%.

[0018] Furthermore, the standing crystallization in S4 is standing at room temperature for at least 24 hours.

[0019] The present invention discloses a process for extracting fisetin from cotinus coggygria. Compared with the prior art, the process has the following advantages:

[0020] In the process flow of the present invention, the extraction method used is continuous countercurrent extraction, which has the advantages of high efficiency, large-scale processing, only water as the extraction solvent, small amount of water used in the extraction process, and only one extraction required to extract flavonoids from cotinus coggygria with extremely high efficiency; the process of the present invention can directly perform water extraction on cotinus coggygria, greatly saving time; the process of the present invention only uses water and ethanol, and does not use any toxic industrial reagents, the product has high safety, and can be used in functional food, cosmetics and other industries; the process of the present invention is simple to operate, does not require repeated extraction, and the transfer rate of flavonoids during the process is high. DETAILED DESCRIPTION

[0021] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0022] The embodiment of the present invention provides a process for extracting fisetin from Cotinus coggygria, comprising:

[0023] Raw material crushing

[0024] The branches of Cotinus coggygria, a plant of the genus Cotinus in the Anacardiaceae family, are taken and crushed for later use. The raw materials of Cotinus coggygria used include Cotinus coggygria for landscape use, Cotinus coggygria with hair, Cotinus coggygria with red leaves, Cotinus coggygria with Sichuan leaves, Cotinus coggygria with dwarf leaves, etc., which are widely available and inexpensive.

[0025] Raw material extraction

[0026] The crushed cotinus coggygria is transferred to a continuous countercurrent extraction tank and extracted with water for 1-2 hours. The amount of water added is 8-10 times that of the raw material. The extraction temperature is 90-95°C. The extract is filtered to remove solid impurities. The filtrate is concentrated at 80°C to an extract with a density of 1.02-1.06 to obtain a crude product with a fisetin content of 17.6-21.8%.

[0027] refined

[0028] Hot water refining: The crude product obtained by extraction is washed with 1-5% of the raw material amount of 80-90°C hot water, and then filtered to obtain a filter cake to remove some water-soluble impurities, thereby obtaining a solid material with a fisetin content of 40%-45%. The fisetin transfer rate in this step reaches more than 93%;

[0029] Ethanol refining: The resulting solid is then dissolved in 23-32 times the concentration of 90-95% ethanol. After filtering to remove insoluble impurities, the filtrate is concentrated to an ethanol concentration of 60-75%. The filtrate is allowed to stand at room temperature for 24 hours to allow crystallization. The filter cake is filtered to obtain an extract. This purification step achieves a fisetin transfer rate exceeding 90%. The resulting extract is dried at 80°C to obtain a fisetin content exceeding 80%.

[0030] Add 25-35 times of 95% ethanol to dissolve 80% fisetin, heat at 80-90℃ to dissolve for 1 hour, filter to remove impurities, concentrate under reduced pressure to an alcohol content of 70-75%, let it stand for more than 24 hours to crystallize, filter and dry at 80℃ to obtain a finished product with a content of 98% fisetin.

[0031] Finished product inspection

[0032] Test Method: Prepare the sample solution to be tested, ensuring the appropriate concentration. This can be achieved through dilution or other methods. Prepare the required HPLC solution, including the mobile phase (acetonitrile: 0.1% phosphoric acid solution = 25:75). Check the chromatographic column for cleanliness according to the HPLC equipment requirements and equilibrate the column if necessary. Debug the HPLC instrument to ensure proper operation.

[0033] Turn on the HPLC instrument, start the system, and ensure that the instrument reaches a stable state. Set the temperature of the chromatograph to 30°C and maintain a stable column temperature. Set the flow rate to 1 mL / min and ensure a stable flow rate. Adjust the injection volume to 10 μL to ensure the injection volume is accurate. Set the detection wavelength to 368 nm to detect at the required wavelength. Adjust the initial mobile phase ratio according to the HPLC system used to ensure column equilibrium. Inject a certain amount of standard sample of known concentration for initial quantification and record the peak area. Inject the sample to be tested for detection and record the peak area.

[0034] Data processing: Based on the standard curve, plot the relationship between the concentration of the standard and the peak area. Using the standard curve, calculate the concentration of the sample to be tested based on the linear relationship between peak area and concentration.

[0035] Result analysis: Calculate the concentration of the sample to be tested based on the experimental results. Verify and quality control the data during the experiment to ensure the accuracy and reliability of the experimental results.

[0036] Cleaning and Maintenance: After the experiment, shut down the HPLC instrument and perform cleaning and maintenance. Clean the column and injector to ensure there is no cross-contamination of the sample. Maintain the HPLC instrument to keep it in normal operation.

[0037] Some examples and measurement data of the present invention are listed below to illustrate the significant progress of the present invention.

[0038] Example 1

[0039] 100 kg of cotinus coggygria branches were pulverized and subjected to continuous countercurrent extraction using 8 times the amount of water as the solvent at 90°C for 2 hours, yielding a crude extract with a 3.43% content and an 88.6% extraction yield. The extract was filtered through a ceramic membrane to remove impurities and concentrated at 80°C to obtain an extract (with a fisetin content of 18.2%). 80°C hot water was added to the extract with stirring, and the solids were filtered to obtain a fisetin content of 41.8% and a transfer efficiency of 93%. The solids were dissolved in 23 times 95% ethanol, filtered to remove impurities, and the filtrate was concentrated under reduced pressure at 70°C to 70% alcohol. The extract was allowed to crystallize, filtered, and dried at 80°C to obtain a crude fisetin product. HPLC analysis revealed a fisetin content of 83.5% and a transfer efficiency of 90%. The crude product was then added with 30 times 95% ethanol, heated at 80°C for 1 hour, filtered to remove impurities, and the filtrate was concentrated under reduced pressure to an alcohol content of 72%. It was allowed to stand at room temperature for 24 hours to crystallize, filtered, and dried at 80°C to obtain the finished product of fisetin. After HPLC detection, the fisetin content was 98.7% and the transfer rate was 66%.

[0040] Example 2

[0041] 100 kg of cotinus coggygria leaves were pulverized and subjected to continuous countercurrent extraction using 10 times the amount of water as the solvent at 95°C for 1 hour. The crude extract contained 2.91% of the crude extract and had an extraction yield of 90.8%. The extract was filtered through a ceramic membrane to remove impurities and concentrated at 80°C to obtain an extract (containing 19.3% fisetin). 85°C hot water was added to the extract with stirring, and the solids were filtered to obtain a fisetin content of 41.4% and a transfer efficiency of 94%. The solids were dissolved in 30 times 95% ethanol, filtered to remove impurities, and the filtrate was concentrated under reduced pressure at 70°C to 65% alcohol. The extract was allowed to crystallize, filtered, and dried at 80°C to obtain the fisetin product. HPLC analysis showed that the fisetin content of the product obtained by this method was 82.7% and the transfer efficiency was 92%. The crude product was then added with 30 times 95% ethanol, heated at 80°C for 1 hour, filtered to remove impurities, and the filtrate was concentrated under reduced pressure to an alcohol content of 70%. The product was allowed to stand at room temperature for 24 hours to crystallize, filtered, and dried at 80°C to obtain the finished product of fisetin. After HPLC detection, the fisetin content was 98.1% and the transfer rate was 68%.

[0042] Example 3

[0043] 100 kg of cotinus coggygria stems were pulverized and subjected to continuous countercurrent extraction using 9 times the amount of water as the solvent at 90°C for 2 hours. The crude extract contained 3.41% of the crude product and had an extraction yield of 89.3%. The extract was filtered through a ceramic membrane to remove impurities and concentrated at 80°C to obtain an extract (containing 20.8% fisetin). 90°C hot water was added to the extract with stirring, followed by filtration to obtain a solid with a fisetin content of 44.4% and a transfer efficiency of 94%. The solid was dissolved in 32 times 90% ethanol, filtered to remove impurities, and the filtrate was concentrated under reduced pressure at 70°C to 60% alcohol. The product was allowed to stand for crystallization, filtered, and dried at 80°C to obtain the fisetin product. HPLC analysis showed that the fisetin content of the product obtained using this method was 81.9% and the transfer efficiency was 92%. The crude product was then added with 30 times 95% ethanol, heated at 80°C for 1 hour, filtered to remove impurities, and the filtrate was concentrated under reduced pressure to an alcohol content of 74%. The product was allowed to stand at room temperature for 24 hours to crystallize, filtered, and dried at 80°C to obtain the finished product of fisetin. After HPLC detection, the fisetin content was 98.8% and the transfer rate was 68.7%.

[0044] Example 4

[0045] 100 kg of cotinus coggygria stems were pulverized and subjected to continuous countercurrent extraction using 9 times the amount of water as the solvent at 92°C for 1 hour. The crude extract contained 3.21% of the extract and had an extraction yield of 89.5%. The extract was filtered through a ceramic membrane to remove impurities and concentrated at 80°C to obtain an extract (containing 21.8% fisetin). The extract was stirred with 80°C hot water and then filtered to obtain a solid with a fisetin content of 42.7% and a transfer efficiency of 93%. The solid was dissolved in 30 parts 92% ethanol, filtered to remove impurities, and the filtrate was concentrated under reduced pressure at 70°C to 75% alcohol. The extract was allowed to crystallize, filtered, and dried at 80°C to obtain the fisetin product. HPLC analysis showed that the fisetin content of the product obtained by this method was 86.1% and the transfer efficiency was 91%. The crude product was then added with 30 times 95% ethanol, heated at 80°C for 1 hour, filtered to remove impurities, and the filtrate was concentrated under reduced pressure to an alcohol content of 73%. The product was allowed to stand at room temperature for 24 hours to crystallize, filtered, and dried at 80°C to obtain the finished product of fisetin. After HPLC detection, the fisetin content was 98.2% and the transfer rate was 70%.

[0046] Comparative Example 1

[0047] 100 kg of cotinus coggygria branches were crushed and the crushed cotinus coggygria was subjected to continuous countercurrent extraction with 9 times the amount of water as solvent. The extraction temperature was 60°C and the extraction time was 2 hours. The extract was filtered through a ceramic membrane to remove impurities and concentrated at 80°C to obtain an extract (the content of fisetin in the extract: 12.8%). 80°C hot water was added to the extract and stirred, and then filtered to obtain a solid. The solid was dissolved in 20 times 95% ethanol solvent, filtered to remove impurities, and the filtrate was concentrated under reduced pressure at 70°C to an alcohol content of 70%, allowed to stand for crystallization, filtered, and dried at 80°C to obtain a fisetin product. HPLC determination showed that the content of fisetin in the product obtained by this method was 68.6%. The crude product was then added with 25 times 95% ethanol, heated at 80°C for 1 hour, filtered to remove impurities, and the filtrate was concentrated under reduced pressure to an alcohol content of 72%. It was allowed to stand at room temperature for 24 hours to crystallize, filtered, and dried at 80°C to obtain the finished product of fisetin. After HPLC analysis, the fisetin content was 78.4%. The transfer rate was: 47.2%

[0048] Comparative Example 2

[0049] 100 kg of cotinus coggygria branches were crushed and subjected to continuous countercurrent extraction with 9 times the amount of water as solvent. The extraction temperature was 90 ° C and the extraction time was 2 hours. The extract was filtered through a ceramic membrane to remove impurities and concentrated at 80 ° C to obtain an extract (the content of flavonoids in the extract: 20.7%). 80 ° C hot water was added to the extract and stirred, and then filtered to obtain a solid. The solid was dissolved in 20 times 95% ethanol solvent, filtered to remove impurities, and the filtrate was concentrated under reduced pressure at 70 ° C to an alcohol content of 45%, allowed to stand for crystallization, filtered, and dried at 80 ° C to obtain a flavonoids product. After HPLC determination, the flavonoids content in the product obtained by this method was 77.67%. The crude product was then added with 30 times 95% ethanol, heated at 80°C for 1 hour, filtered to remove impurities, and the filtrate was concentrated under reduced pressure to 70% alcohol content. It was allowed to stand at room temperature for 24 hours to crystallize, filtered, and dried at 80°C to obtain the finished product of fisetin. After HPLC detection, the fisetin content was 92.42%. The transfer rate was: 93.8%

[0050] Comparative Example 3

[0051] 100 kg of cotinus coggygria branches were crushed and the crushed cotinus coggygria was subjected to continuous countercurrent extraction with 9 times the amount of water as solvent. The extraction temperature was 90°C and the extraction time was 2 hours. The extract was filtered through a ceramic membrane to remove impurities and concentrated at 80°C to obtain an extract (the content of fisetin in the extract: 21.2%). 80°C hot water was added to the extract and stirred, and then filtered to obtain a solid. The solid was dissolved in 20 times 95% ethanol solvent, filtered to remove impurities, and the filtrate was concentrated under reduced pressure at 70°C to an alcohol content of 80%, allowed to stand for crystallization, filtered, and dried at 80°C to obtain a fisetin product. HPLC determination showed that the fisetin content in the product obtained by this method was 87.47%. The crude product was then added with 30 times 95% ethanol, heated at 80°C for 1 hour, filtered to remove impurities, and the filtrate was concentrated under reduced pressure to an alcohol content of 70%. It was allowed to stand at room temperature for 24 hours to crystallize, filtered, and dried at 80°C to obtain the finished product of fisetin. After HPLC detection, the fisetin content was 98.5% and the transfer rate was 47.8%.

[0052] It can be seen from Comparative Example 1 that when the extraction temperature of the continuous countercurrent extraction tank is too low, the fisetin content in the product will be too low.

[0053] It can be seen from Comparative Examples 2 and 3 that when the alcohol content is too high, the final transfer rate will be low; when the alcohol content is too low, the content of fisetin in the product will be too low.

[0054] In summary, the present invention does not require pretreatment of the crushed cotinus coggygria, which can greatly save the total extraction time and improve the extraction efficiency. A continuous countercurrent extraction method is adopted. First, compared with the solvent extraction method, the present invention only uses water as a solvent for extraction, which has the advantages of being green, environmentally friendly, non-toxic and harmless. Moreover, the process of the present invention uses continuous countercurrent extraction and only uses 8-10 times the amount of raw material for 1-2 hours of solvent extraction, which can achieve an extraction rate of about 90% at a time, without the need for repeated extraction. This saves solvent and greatly improves the extraction efficiency. The process only uses water and ethanol throughout the process, and does not use methanol, n-butanol and other organic solvents commonly used in other processes. While maximizing the raw material extraction rate, it is more green, environmentally friendly and sustainable. The product can be widely used in food, cosmetics and other industries. In the refining step, the crude extract with a fisetin content of about 18-20% can be purified to a content of more than 80% through simple operations, and the transfer rate of purified fisetin in each step is high, and the loss of target product during the purification process is very small. Secondly, the present invention can produce fisetin with a purity of 98% in a short time using a simple, safe, and easy-to-use method. The purification process also features a high transfer rate, minimal loss of target product, and high economic benefits, making it highly suitable for industrialization. The raw material, Cotinus coggygria, is both a medicinal plant and a common ornamental plant.

[0055] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A process for extracting fisetin from Cotinus coggygria, characterized in that: include: S1: grinding the branches and / or leaves of Cotinus coggygria to obtain Cotinus coggygria powder; S2: adding the crushed cotinus coggygria to a continuous countercurrent extraction tank, adding 8-10 times the mass of water of the crushed cotinus coggygria to the continuous countercurrent extraction tank, and starting extraction at an extraction temperature of 90-95° C. to obtain an extract, and the extraction process lasts for 1-2 hours; The extract is filtered and the filtrate is collected. The filtrate is heated and concentrated to a density of 1.02-1.06 g / cm 3 , to obtain a crude extract; S3: Rinse the crude extract, filter and take the filter cake to obtain a solid, add the solid to 90%-95% ethanol for dissolution, filter and concentrate under reduced pressure to an ethanol concentration of 60%-75%, crystallize, filter and take the filter cake to obtain a refined extract, and the crystallization is allowed to stand at room temperature for at least 24 hours; the rinsing in S3 is specifically: take 1%-5% of the amount of the cotinus coggygria crushed material water to rinse the crude extract, and the water temperature is 80-90°C. S4: Add 95% ethanol to the refined extract, heat to dissolve and continue for 1-1.5 hours, filter the filtrate, and concentrate under reduced pressure to an ethanol concentration of 70%-75%, then stand for crystallization, filter, and dry to obtain the finished product of fisetin. The standing crystallization is standing at room temperature for at least 24 hours.

2. The extraction process of fisetin from Cotinus coggygria according to claim 1, characterized in that: The temperature for heating and concentrating in S2 is 80°C.

3. The extraction process of fisetin from Cotinus coggygria according to claim 1, characterized in that: The amount of ethanol used in the dissolution in S3 is 23-32 times the mass of the solid matter.