Method for extracting and purifying phellinus igniarius flavone

Abstract

The invention discloses an extraction and purification method of phellinus igniarius flavone. The extraction and purification method of phellinus igniarius flavone comprises the following steps: drying and crushing phellinus igniarius mycelium to obtain phellinus igniarius mycelium dry powder; extracting phellinus igniarius total flavonoids in the phellinus igniarius mycelium dry powder by adopting an ethanol heating reflux method, and extracting the phellinus igniarius mycelium dry powder at least once to obtain an extracting solution; concentrating the extracting solution, and purifying by adopting + 3'macroporous resin; and concentrating and freeze-drying the purified flavone extracting solution to obtain the purified phellinus igniarius flavone. The method for purifying the phellinus igniarius flavone by adopting the + 3'macroporous resin is convenient to operate, low in cost and remarkable in enrichment and purification effect, and the purity of the obtained phellinus igniarius flavone is improved to be higher than or equal to that of the phellinus igniarius flavone.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction and purification of flavonoid active substances, and specifically relates to a method for extracting and purifying mulberry flavonoids. Background Art

[0002] Sanghuangporus sanghuang is a large and rare edible and medicinal fungus, which was first recorded as "mulberry ear" in Shennong's Materia Medica, a Chinese traditional medicine monograph more than 2,000 years ago. Sanghuangporus sanghuang is rich in various polysaccharides, polyphenols, flavonoids, terpenes and other active substances, and has anti-tumor, hypoglycemic, immunomodulatory, antioxidant, liver protection and liver protection effects, and has good application prospects.

[0003] At present, the research on Phellinus igniarius is concentrated on its polysaccharides, polyphenols and bacterial fermentation culture. Since the species relationship of Phellinus igniarius was not clearly understood until 2016, the research on the biological activity or medicinal function of Phellinus igniarius in recent years has actually included multiple species. In addition, the research on the biological activity of Phellinus igniarius flavonoids is mainly based on crude flavonoids, and there are relatively few studies on purified Phellinus igniarius flavonoids. Summary of the invention

[0004] In order to solve the deficiencies in the prior art, the present invention aims to provide a method for extracting and purifying mulberry flavonoids.

[0005] The specific technical solutions of the present invention are as follows:

[0006] The present invention provides a method for extracting and purifying phellin flavonoids, comprising the following steps:

[0007] (1) drying and crushing the mycelium of Phellinus linteus to obtain a dry powder of the mycelium of Phellinus linteus;

[0008] (2) extracting the total flavonoids of Phellinus igniarius in the dry powder of Phellinus igniarius mycelium by ethanol heating and reflux method, extracting the dry powder of Phellinus igniarius mycelium at least once to obtain an extract;

[0009] (3) concentrating the extract and purifying it using HPD-100 macroporous resin;

[0010] (4) The purified flavonoid extract is concentrated and freeze-dried to obtain purified mulberry flavonoids.

[0011] Furthermore, in step (1), the mycelium of Phellinus igniarius is dried at 40°C to 60°C for 12 to 24 hours, and the moisture content shall not exceed 8%.

[0012] Furthermore, in step (1), the crushed Phellinus linteus mycelium is sieved through an 80-mesh sieve to obtain Phellinus linteus mycelium dry powder.

[0013] Furthermore, in step (2), the specific conditions for extracting the total flavonoids of Phellinus igniarius in the dry powder of Phellinus igniarius mycelium by ethanol heating and reflux method are: the volume concentration of ethanol is 55% to 75%, the solid-liquid ratio is 1:30 to 1:60, and the extraction is performed twice at 65°C to 95°C, each time for 0.5 to 2.5 hours; preferably, the specific conditions for extracting the total flavonoids of Phellinus igniarius in the dry powder of Phellinus igniarius mycelium by ethanol heating and reflux method are: the volume concentration of ethanol is 70%, the solid-liquid ratio is 1:50, and the extraction is performed twice at 80°C, each time for 1.5 hours.

[0014] Furthermore, in step (3), the specific conditions for purification using HPD-100 macroporous resin are: adsorption time of 3 to 6 hours, desorption time of 1.5 to 3.5 hours; the sample volume is 2.5 to 5.5 BV, the sample concentration is 1.0 to 3.0 mg / mL, 50 to 90% ethanol is used for desorption, and the eluent volume is 2.5 to 5.5 BV; preferably, the specific conditions for purification using HPD-100 macroporous resin are: adsorption time of 5 hours, desorption time of 2 hours; the sample volume is 3.5 BV, the sample concentration is 3.0 mg / mL, 70% ethanol is used for desorption, and the eluent volume is 4 BV.

[0015] Furthermore, in step (3), the HPD-100 macroporous resin is pretreated before purification by using the HPD-100 macroporous resin, specifically, the HPD-100 macroporous resin is soaked in anhydrous ethanol for 12 to 16 hours, and then washed with distilled water for multiple times until there is no alcohol smell and it is ready for use.

[0016] Furthermore, in step (4), the purified flavonoid extract is concentrated to 1 / 10 to 1 / 5 of the original volume.

[0017] Furthermore, in step (4), the purified flavonoid extract is concentrated by rotary evaporation, specifically: the purified flavonoid extract is placed in a rotary evaporator for rotary evaporation, and the rotary evaporation conditions are 30-60° C., 80-120 rpm. The vacuum degree is controlled at 0.06-0.1 MPa; preferably, the purified flavonoid extract is placed in a rotary evaporator for rotary evaporation, and the rotary evaporation conditions are 50° C., 80 rpm, and the vacuum degree is 0.8 MPa.

[0018] Furthermore, in step (4), the specific operation of freeze drying is: freezing at -80°C for 8 to 10 hours, and then vacuum freeze drying to obtain purified mulberry flavonoids.

[0019] The beneficial effects of the present invention are:

[0020] The present invention screened and compared four commonly used macroporous resins for purifying mulberry flavonoids, and found that HPD100 macroporous resin had the best effect in purifying mulberry flavonoids, and obtained the optimal process parameters for purifying mulberry flavonoids with HPD-100 macroporous resin, which provided a reference for further enrichment, purification and efficient utilization of mulberry flavonoids. The present invention found that the adsorption rate, desorption rate and recovery rate of HPD100 resin were significantly higher than those of D101 and AB-8 resins, which enabled HPD100 resin to more effectively elute the adsorbed flavonoids from the resin, and the final amount of flavonoids obtained was more and the loss was less than that of D101 and AB-8 resins.

[0021] In addition, by comparing the physical parameters of the four resins, it can be seen that HPD100 macroporous resin has better selectivity for mulberry flavonoids than D101 and AB-8. HPD100, D101, and AB-8 macroporous resins are all styrene-type copolymers. Since there are certain ester groups on the surface of AB-8 macroporous resin, the hydrophilicity is improved, and its adsorption mechanism is still hydrophobic adsorption. Compared with HPD100, its adsorption selectivity for mulberry flavonoids is weaker. The specific surface area of ​​HPD100 is 650-700m 2 / g, the larger specific surface area and suitable pore size can provide more adsorption sites for mulberry flavonoids, so that mulberry flavonoids can fully contact with the resin and be adsorbed, thereby improving the adsorption amount and efficiency. The regeneration process of AB-8 and D101 macroporous resins requires the use of acid and alkali solutions for treatment, and the regeneration process is relatively cumbersome. The HPD100 macroporous resin is easy to regenerate, and the regeneration process is relatively simple. The regenerated resin can still maintain good adsorption performance, which means that under the same processing volume, the amount of HPD100 resin used is relatively small, which reduces the cost of resin regeneration and the cost of frequent resin replacement. The HPD100 macroporous resin screened out in the present invention has the characteristics of high adsorption rate, large adsorption capacity and fast desorption speed for mulberry igniarin. Compared with the mulberry igniarin purification methods using AB-8, D101 and X-5 macroporous resins, the method has the characteristics of convenient operation, low cost and significant enrichment and purification effect. The purity of the purified mulberry igniarin is increased from 56% to more than 95%, and the purified mulberry igniarin has stronger DPPH and ABTS oxygen free radical scavenging effect than the unpurified mulberry igniarin. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the standard curve of rutin;

[0023] Figure 2 This is the static adsorption effect diagram of HPD-100 macroporous resin;

[0024] Figure 3 This is the static desorption effect diagram of HPD-100 macroporous resin;

[0025] Figure 4is the ethanol concentration screening graph;

[0026] Figure 5 This is the sample concentration screening graph;

[0027] Figure 6 Screening plot for elution volume;

[0028] Figure 7 This is a diagram showing the scavenging effect of DPPH oxygen free radicals before and after purification of mulberry flavonoids;

[0029] Figure 8 This is a diagram showing the ABTS oxygen free radical scavenging effect before and after purification of mulberry flavonoids. DETAILED DESCRIPTION

[0030] In order to understand the present invention more clearly, the present invention is further described with reference to the following examples and accompanying drawings. The examples are only used for explanation and are not intended to limit the present invention in any way. In the examples, each raw material reagent is commercially available, and the experimental methods without specifying specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0031] Example 1

[0032] This example screens the purification effect of mulberry flavonoids by using four commonly used macroporous resins on the market, namely AB-8, D101, HPD100, and X-5, and finally uses HPD100 macroporous resin to enrich and purify mulberry flavonoids. By conducting single-factor experiments on the adsorption time, desorption time, sample concentration, elution volume, ethanol concentration and other conditions during HPD100 purification, the optimal process parameters for purifying mulberry flavonoids with HPD-100 macroporous resin are obtained, thereby providing an optimal method for extracting and purifying mulberry flavonoids. The details are as follows:

[0033] 1. Preparation of crude extract of total flavonoids from Phellinus igniarius

[0034] (1) drying the mycelium of Phellinus igniarius at 50° C. for 24 hours, with a moisture content of no more than 8%, crushing the mycelium, and passing the crushed mycelium through an 80-mesh sieve to obtain a dry powder of the mycelium of Phellinus igniarius;

[0035] (2) The total flavonoids from Phellinus igniarius were extracted by ethanol heating reflux method. The volume concentration of the ethanol solution was 70%, the solid-liquid ratio was 1:50, the extraction temperature was 80°C, the extraction time was 1.5 hours, the number of extractions was 2 times, and the filtrates were collected twice to obtain the crude extract of the total flavonoids from Phellinus igniarius.

[0036] 2. Screening of the optimal purification conditions for the crude extract of Phellinus igniarius total flavonoids

[0037] (1) By comparing the static adsorption and desorption effects of four macroporous resins, AB-8, D101, HPD100, and X-5, on total flavonoids from Phellinus igniarius, the most suitable resin for the purification of total flavonoids from Phellinus igniarius was screened out.

[0038] The extract was concentrated to 3 mg / mL (the total flavonoid content was measured by NaNO 2 -Al(NO 3 ) 3 -NaOH colorimetric determination) Weigh 5.0 g of pretreated AB-8, D101, HPD100, and X-5 macroporous resins, place them in a conical flask, and oscillate at a constant temperature for 24 hours. Take the supernatant, determine the mass concentration at adsorption equilibrium, and calculate the adsorption amount and adsorption rate of each resin on the mulberry flavonoids extract; filter the resin after adsorption equilibrium, add 70% ethanol, oscillate at a constant temperature for 12 hours, then filter, determine the mass concentration of flavonoids in the eluate, and calculate the static desorption rate and recovery rate of each resin.

[0039] The physical parameters of AB-8, D101, HPD100 and X-5 are shown in Table 1, and the static adsorption and desorption effects are shown in Table 2. The results show that the HPD100 macroporous resin has the characteristics of fast adsorption speed, large adsorption capacity and fast desorption speed for phellinus flavonoids, and HPD100 has the best adsorption and desorption effects. It is determined that the most suitable resin for the purification of phellinus flavonoids is HPD100.

[0040] Table 1. Physical parameters of AB-8, D101, HPD100, and X-5

[0041]

[0042] Figure 2 .Static adsorption and desorption effects of AB-8, D101, HPD100, and X-5 resins

[0043]

[0044] (2) The crude flavonoid extract obtained by extraction was purified by adsorption using HPD100 macroporous resin, and various factors during purification were investigated to screen out the best conditions. Rutin was used as a standard to calculate the purity of the purified mulberry flavonoids.

[0045] 1) Weigh 5.0 g of HPD100 macroporous resin and place it in a conical flask, add 10 mL (3 mg / mL) of mulberry flavonoids concentrate, shake on a horizontal shaker for 24 hours, filter, and use 10 mL of 50%, 60%, 70%, 80%, and 90% ethanol for desorption. Collect the eluate and determine the flavonoid concentration, calculate the desorption rate, and determine the optimal concentration of the eluate.

[0046] 2) The concentration of the flavonoid extract was adjusted to 1.0, 1.5, 2.0, 2.5, 3.0 (mg / mL), 10 mL of the above concentration solution was taken, 5.0 g of HPD100 macroporous resin was added to a conical flask, and the solution was shaken on a horizontal shaker for 24 hours, filtered, and the concentration of the filtrate was determined. 10 mL of 70% ethanol was taken, shaken on a horizontal shaker for 24 hours, filtered, and the concentration of the filtrate was determined to determine the optimal loading mass concentration.

[0047] 3) 5.0 g of HPD100 macroporous resin was loaded into the column, 3.0 mg / mL was loaded, 70% ethanol was used for elution, and the eluate was collected every 0.5 BV. The flavonoid content in each eluate was detected, and the elution curve was drawn to determine the optimal volume of the eluate.

[0048] The results are as follows Figure 2-6 As shown in the figure, the optimal purification conditions of HPD100 macroporous resin screened out are: adsorption time of 5 hours, desorption time of 2 hours, sample volume of 3.5BV, sample concentration of 3.0 mg / mL. 70% ethanol was used for desorption, and the eluent volume was 4BV.

[0049] 4) The flavonoid extract was enriched and purified using the optimal purification conditions of HPD100 macroporous resin, and the eluate was collected and placed in a rotary evaporator for rotary evaporation. The rotary evaporation conditions were 50°C, 80 rpm, and a vacuum degree of 0.8 MPa. After being concentrated to 1 / 10 of the original volume, it was frozen at -80°C for 10 hours and vacuum freeze-dried to obtain purified mulberry flavonoids. The purity of the obtained mulberry flavonoids was increased from 56% to more than 95%.

[0050] The purified and freeze-dried mulberry flavonoids were prepared into a certain concentration of test sample solution for the study of DPPH and ABTS oxygen free radical scavenging ability. Specifically: the purified and freeze-dried mulberry flavonoids were prepared into 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mg / mL test sample solutions with 80% ethanol, 0.2 mL of the test sample solution with different concentration gradients was added, 2 mL of 0.2 mmol / L DPPH·ethanol solution was added, mixed and kept away from light for 30 minutes, and the absorbance was measured at 517 nm; 20 μL of the test sample solution with different concentration gradients was added, 400 μL of 80% ABTS ethanol solution was added, mixed and kept away from light for 6 minutes, and the absorbance was measured at a wavelength of 734 nm. Figure 7 This is the DPPH oxygen free radical scavenging effect diagram of mulberry flavonoids before and after purification. Figure 8 This is a diagram of the ABTS oxygen free radical scavenging effect of mulberry flavonoids before and after purification. When the concentration of mulberry flavonoids after purification was 0.8 mg / mL, the DPPH and ABTS oxygen free radical scavenging rates were 1.4 times and 1.5 times that of mulberry flavonoids before purification, respectively.

[0051] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for extracting and purifying phellin flavonoids, characterized in that: The following steps are involved: (1) drying and crushing the mycelium of Phellinus linteus to obtain a dry powder of the mycelium of Phellinus linteus; (2) extracting the total flavonoids of Phellinus igniarius in the dry powder of Phellinus igniarius mycelium by ethanol heating and reflux method, extracting the dry powder of Phellinus igniarius mycelium at least once to obtain an extract; (3) concentrating the extract and purifying it using HPD-100 macroporous resin; (4) The purified flavonoid extract is concentrated and freeze-dried to obtain purified mulberry flavonoids.

2. The extraction and purification method according to claim 1, characterized in that: In step (1), the mycelium of Phellinus igniarius is dried at 40° C. to 60° C. for 12 to 24 hours, and the moisture content shall not exceed 8%.

3. The extraction and purification method according to claim 1, characterized in that: In step (1), the crushed Phellinus linteus mycelia are passed through an 80-mesh sieve to obtain Phellinus linteus mycelia dry powder.

4. The extraction and purification method according to claim 1, characterized in that: In step (2), the specific conditions for extracting the total flavonoids of Phellinus igniarius from the dry powder of Phellinus igniarius mycelium by ethanol heating reflux method are: the volume concentration of ethanol is 55% to 75%, the solid-liquid ratio is 1:30 to 1:60, and the extraction is performed twice at 65°C to 95°C, each time for 0.5 to 2.5 hours.

5. The extraction and purification method according to claim 1, characterized in that: In step (3), the specific conditions for purification using HPD-100 macroporous resin are: adsorption time is 3 to 6 hours, desorption time is 1.5 to 3.5 hours; the sample volume is 2.5 to 5.5 BV, the sample concentration is 1.0 to 3.0 mg / mL, 50 to 90% ethanol is used for desorption, and the eluent volume is 2.5 to 5.5 BV.

6. The extraction and purification method according to claim 1, characterized in that: In step (3), the specific conditions for purification using HPD-100 macroporous resin are: adsorption time is 5 hours, desorption time is 2 hours; the loading volume is 3.5BV, the loading concentration is 3.Omg / mL, 70% ethanol is used for desorption, and the eluent volume is 4BV.

7. The extraction and purification method according to claim 1, characterized in that: In step (3), the HPD-100 macroporous resin is pretreated before purification by using the HPD-100 macroporous resin, specifically, the HPD-100 macroporous resin is soaked in anhydrous ethanol for 12 to 16 hours, and then washed with distilled water for multiple times until there is no alcohol smell and it is ready for use.

8. The extraction and purification method according to claim 1, characterized in that: In step (4), the purified flavonoid extract is concentrated to 1 / 10 to 1 / 5 of the original volume.

9. The extraction and purification method according to claim 1, characterized in that: In step (4), the purified flavonoid extract is concentrated by rotary evaporation, specifically, the purified flavonoid extract is placed in a rotary evaporator for rotary evaporation, and the rotary evaporation conditions are 30-60° C., 80-120 rpm, and the vacuum degree is controlled at 0.06-0.1 MPa.

10. The extraction and purification method according to claim 1, characterized in that: In step (4), the specific operation of freeze drying is: freezing at -80°C for 8 to 12 hours, and then vacuum freeze drying to obtain purified mulberry flavonoids.

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