Preparation method of peony seed shell flavone extract

Abstract

The invention discloses a preparation method of a peony seed shell flavone extract, which comprises the following steps: extracting peony seed shell flavone (ZKHT) by adopting enzymolysis-ultrasonic extraction, and separating and purifying by using a resin adsorption combined membrane separation technology, so that various flavonoid active compounds such as kaempferol, luteolin and apigenin with high content in peony seed shells can be effectively extracted at the same time; the method provided by the invention provides technical support for reutilization of the peony seed shell waste, and realizes efficient and comprehensive utilization of the peony seeds and by-products thereof.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of traditional Chinese medicine extracts, and particularly relates to a method for preparing a peony seed shell flavonoid extract. Background Art

[0002] Peony (Paeonia suffruticosa Andr.) is a woody plant of the genus Paeonia in the family Ranunculaceae, and is known as the "king of flowers". Oil-producing peony is a type of peony with the ability to bear fruit. Its mature seeds, namely peony seeds, can be used to extract oil, with an oil yield of more than 20%. Peony seed shells and peony seed meal are by-products produced during the production and processing of peony seed oil. Among them, peony seed shells contain rich active substances. Flavonoids are one of the many active substances that continue to receive attention. Their efficient extraction and application in the medical field have been proven to have extensive and potential benefits. Studies have found that peony seed shells contain a class of antioxidant and anti-cancer flavonoid compounds - pine pine.

[0003] However, in the current production and processing process, peony seed shells are mostly treated as garbage and cannot be effectively utilized, resulting in a waste of resources. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a peony seed shell flavonoid extract that can turn peony seed shells into valuables. The method uses enzymatic hydrolysis-assisted ultrasonic extraction of total flavonoids from peony seed shells, and separates and purifies by macroporous resin-membrane separation technology, which can effectively extract multiple flavonoid active compounds from peony seed shells at the same time, and the total flavonoid yield is high, thereby providing technical support for the recycling of peony seed shell waste and realizing efficient and comprehensive utilization of peony seeds and their by-products.

[0005] The purpose of the present invention is specifically achieved in the following ways.

[0006] A method for preparing a peony seed shell flavonoid extract, the method comprising the following steps:

[0007] (1) preparing an ethanol-enzyme solution of 8-10 mg / mL by taking pectinase, cellulase and ethanol solution, weighing peony seed shell powder, mixing the peony seed shell powder and the ethanol-enzyme solution, performing enzymolysis-ultrasound treatment at 250 W: the ultrasonic time is 80-100 min, the solid-liquid ratio of the peony seed shell powder and the ethanol-enzyme solution is 1:10-20 g / mL, the ethanol concentration is 30-50%, the pH value is 3-6, and the enzyme addition amount is 5-25 mg / mL; inactivating the enzyme, cooling, centrifuging, filtering, concentrating and re-dissolving with ethanol to obtain a crude extract;

[0008] (2) The crude extract was separated and purified by resin adsorption combined with membrane separation: the resin adsorption / desorption process conditions were as follows: a sample volume of 69-70 mL, a sample flow rate of 1-3 mL / min, adsorption for 1-1.5 h, and elution with 65-75% ethanol at a flow rate of 1-1.5 mL / min.

[0009] Preferably, the enzymatic hydrolysis-ultrasonic treatment conditions in step (1) are: solid-liquid ratio 1:10 g / mL, ethanol concentration 40%, pH 6, enzyme addition amount 20 mg / mL, ultrasonic temperature 50°C, and ultrasonic time 90 min.

[0010] Preferably, in step (2), the resin adsorption / desorption process conditions are: sample volume 70 mL, sample liquid flow rate 2 mL / min, adsorption 1 h, 70% ethanol elution at a flow rate of 1 mL / min.

[0011] Preferably, the adsorption resin model used in step (2) is S-8, D101, HPD600 or NKA-9.

[0012] Preferably, the adsorption resin model used in step (2) is D101.

[0013] Preferably, the centrifugal condition in step (1) is 1000 r / min.

[0014] Preferably, the re-dissolution in step (1) uses ethanol with a mass concentration of 40%.

[0015] Preferably, the pectinase and cellulase are weighed in a mass ratio of 1:2.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: the preparation method of the peony seed shell flavonoid extract of the present invention adopts enzymatic hydrolysis-ultrasonic extraction of peony seed shell flavonoids (ZKHT), and uses resin adsorption combined with membrane separation technology for separation and purification. Optimal extraction process: Under the conditions of solid-liquid ratio of 1:10, ethanol concentration of 40%, pH value of 6, enzyme addition of 20 mg / mL, ultrasonic temperature of 50°C, and ultrasonic time of 90 min, the ZKHT yield is 14.32%. The optimal resin adsorption / desorption process conditions are as follows: the sample amount is 70 mL, the sample flow rate is 2 mL / min, adsorption is 1 h, 70% ethanol is eluted at a flow rate of 1 mL / min, the ZKHT yield is 24.66%, and the relative contents of kaempferol, luteolin and apigenin are 4.34%, 6.79% and 0.82% respectively. The method of the present invention can effectively extract a variety of flavonoid active compounds in peony seed shells at the same time, and the total flavonoid yield is high, so that the peony seed shells are turned into treasures, and have an effective utilization prospect in the food industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1The influence of various factors on ZKHT yield is shown in the figure

[0018] Figure 2 Figure 2 shows the adsorption and desorption performance of ZKHT by different resins.

[0019] Figure 3 This is the leakage curve of ZKHT

[0020] Figure 4 The influence of each single factor on the mass concentration of ZKHT in the resin eluent is shown in the figure

[0021] Figure 5 HPLC chromatogram of standard product

[0022] Figure 6 HPLC chromatogram of the sample (a, before purification; b, after purification; 1, kaempferol; 2, luteolin; 3, apigenin) DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and drawings. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0024] The experimental methods described in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. described in the following examples are all commercially available unless otherwise specified.

[0025] Test Example 1

[0026] 1.1 Materials and reagents

[0027] Peony seed shell: Taizhou Qinghe Agricultural Technology Co., Ltd.; cellulase, pectinase, rutin: Hefei Bomei Biotechnology Co., Ltd.; macroporous adsorption resin: Beijing Solebow Technology Co., Ltd.; acetonitrile, methanol, formic acid, sodium nitrite, aluminum nitrate, sodium hydroxide: Xilong Science Co., Ltd.

[0028] 1.2 Instruments and Equipment

[0029] Practum612-1CN electronic balance: Beijing Sartorius Scientific Instrument Co., Ltd.; KQ-250B ultrasonic cleaner: Hangzhou Blancpain Ultrasonic Technology Co., Ltd.; T6 UV-visible spectrophotometer: Beijing Puxi General Instrument Co., Ltd.; MSM2013 Membrane Separation Device: Shanghai Mosu Scientific Instrument Co., Ltd.; LabconooFreeZone 6L desktop freeze dryer: Beijing Yaxing Instrument Technology Development Co., Ltd.; LC-2050 liquid chromatograph: Shimadzu (Shanghai) Laboratory Equipment Co., Ltd.

[0030] Extraction technology of peony seed shell flavonoids (ZKHT)

[0031] Peony seed shell → drying → crushing and sieving → enzymatic hydrolysis-ultrasonic extraction → enzyme inactivation → filtration → concentration → re-dissolution → ZKHT crude extract.

[0032] After drying, the peony seed shell is cooled, crushed, and sieved. Accurately weigh 5.00 g of peony seed shell powder, weigh pectinase, cellulase, and ethanol solution in a ratio of 1:2 to prepare a 10 mg / mL ethanol-enzyme solution, add the peony seed shell powder and ethanol-enzyme solution into a conical flask, shake well, put into an ultrasonic cleaner, enzymolysis-ultrasound treatment at 250W and 50°C, inactivate the enzyme, cool, centrifuge at 1000r / min, filter, concentrate and divide into two parts, one of which is freeze-dried; the other is re-dissolved in 40% ethanol, that is, ZKHT crude extract, for standby use.

[0033] 1.3.1 Optimization of ZKHT extraction process

[0034] (1) Single factor experiment: The effects of ethanol concentration (20, 30, 40, 50, 60%), solid-liquid ratio (i.e. the ratio of peony seed shell powder and ethanol-enzyme hydrolysate) (1:5, 1:10, 1:15, 1:20, 1:25 g / mL), enzyme addition amount (5, 10, 15, 20, 25 mg / mL), pH value (3.0, 4.0, 5.0, 6.0, 7.0), ultrasonic temperature (30.0, 40.0, 50.0, 60.0, 70.0℃) and ultrasonic time (30, 60, 90, 120, 180 min) on the ZKHT yield were investigated.

[0035] (2) Orthogonal test: Taking ZKHT yield as the evaluation index, the solid-liquid ratio, ultrasonic time and ethanol concentration were selected to perform L 9 (3 3 ) was used to optimize the extraction process of ZKHT. The orthogonal test factors and level design are shown in Table 1.

[0036] Table 1 Orthogonal test design table

[0037]

[0038] 1.3.2 ZKHT determination

[0039] Draw the standard curve, and the standard curve is y = 0.721x + 0.057, R 2 =0.9997. The ZKHT yield in the crude extract was measured and the ZKHT yield was calculated according to formula (1).

[0040]

[0041] Wherein: Y is the ZKHT yield / %; C is the ZKHT mass concentration / (mg / mL); V is the volume of the extract / mL, and m is the mass of the weighed peony seed shell powder / g.

[0042] 1.3.3 Macroporous resin experiment

[0043] (1) Static adsorption and desorption test: Weigh 2.5g of each of the five resins into a conical flask, add 50mL of 0.51mg / mL crude extract, oscillate at 30℃, 150r / min for 12h, filter the filtrate to detect the ZKHT mass concentration. Calculate the adsorption rate X according to formula (2); rinse the resin with pure water until the filtrate is colorless, drain and pour it back into a conical flask, add 50mL of 70% ethanol, fully desorb under the same conditions, and calculate the desorption rate J according to formula (3):

[0044]

[0045]

[0046] Where: X is ZKHT adsorption rate / %; J is ZKHT desorption rate / %; A 0 is the mass concentration of ZKHT measured before adsorption, A 1 is the mass concentration of ZKHT measured after adsorption, A 2 is the mass concentration of ZKHT measured after desorption.

[0047] (2) Dynamic adsorption and desorption process: A chromatography column (30 mm × 300 mm) was fixed vertically, and the pretreated resin was dry loaded onto the column. After rinsing with pure water for 2-3 BV, it was activated with 95% ethanol for 12 h. The pH value of the ZKHT crude extract was adjusted, and the crude extract was loaded through a constant flow pump. The adsorption was allowed to stand, and the eluate was loaded through a constant flow pump. The eluate was collected, and each 4 mL was tested according to the steps in 1.3.2. The mass concentration of ZKHT in the eluate was calculated and used as the ordinate, and the loading volume was used as the abscissa to draw a curve.

[0048] (3) Dynamic process optimization

[0049] Determination of loading volume: vertically fix the chromatography column (30mm×300mm), dry-load the pretreated resin onto the column, rinse with pure water for 2-3BV, and then load the crude extract at a certain loading flow rate through a constant flow pump, collect the effluent, 2mL as 1 tube, and calculate the ZKHT mass concentration of the effluent as the ordinate according to the above steps, and the volume of the loaded crude extract as the abscissa. After the resin is saturated with adsorption, record the loading volume of the crude extract and draw a leakage curve.

[0050] Process: According to the optimal loading amount, the pH value of the crude extract was adjusted to 4, 5, 6, 7, and 8, and then loaded at a flow rate of 1, 2, 3, 4, and 5 mL / min. After static adsorption for 1, 2, 3, 4, and 5 hours, 40, 50, 60, 70, and 80% ethanol was used to elute through a constant flow pump at a flow rate of 1, 2, 3, 4, and 5 mL / min. The eluate was collected in 1 tube of 4 mL to determine the optimal process conditions.

[0051] 1.3.4 Membrane separation

[0052] After testing according to the optimal macroporous resin adsorption elution conditions, the eluate was collected, filtered through a 50 mm filter membrane, and then filtered and impurities were removed using ultrafiltration (pore size between 10-20 nm, operating pressure of 0.3-0.5 MPa) at room temperature. The filtered permeate was collected, rotary evaporated, freeze-dried (-25°C, 5-10 Pa), and set aside.

[0053] 1.3.5 Detection and Analysis

[0054] Sample and standard treatment: Weigh 10 mg of the lyophilized sample and standard prepared in the above steps respectively, dissolve in 100 mL of 70% methanol (chromatographic grade), shake and dilute to volume, centrifuge, take the supernatant, filter through a membrane, and set aside.

[0055] HPLC chromatographic conditions: using Wonda Sil C18-WR (4.6×250mm) chromatographic column, flow rate 1.0mL / min, column temperature 40℃, injection volume 10uL, mobile phase 0.05% acetic acid (A)-methanol (B), UV detection wavelength 330nm. Gradient elution program is 0~1min, 20%B; 1.01~1.5min, 20%~50%B; 1.51~2.0min, 50%~95%B; 2.01~10.0min, 95%B; 10.01~12.0min, 95%~50%B; 12.01~14.0min, 50%~20%B; 14.01~20.0min, 20%B.

[0056] Data analysis

[0057] The experiment was repeated 3 times and the results are the average values. Data were analyzed using Excel 2019 and plotted using Origin 2018.

[0058] Results and Analysis

[0059] 2.1ZKHT process optimization

[0060] 2.2.1 Single factor test results

[0061] Depend on Figure 1 It can be seen that with the continuous increase in the volume of the solvent (ethanol-water solution), the ZKHT yield first increases and then decreases. When the solid-liquid ratio is 1:10, the ZKHT yield reaches the highest, which is 13.62%. The ethanol concentration has a significant effect on the ZKHT yield. When the ethanol concentration is 40%, the ZKHT yield reaches a maximum of 13.31%. With the increase of pH value, the ZKHT yield first increases and then decreases. When the pH value is 6, the ZKHT yield is the highest, which is 13.32%. The enzyme is most active and has the best effect in the optimal pH range, that is, under weakly acidic conditions, so the ZKHT yield shows an increasing trend. When the enzyme addition amount is 20 mg / mL, the ZKHT yield is as high as 13.77%. When the addition amount increases from 5 to 20 mg / mL, the enzyme reacts more fully with the substrate until saturation. The ZKHT yield is the highest at an ultrasonic temperature of 50.0℃, which is 13.75%. Ultrasonic time has a significant effect on the yield of ZKHT. When the ultrasonic treatment is carried out for 90 minutes, the highest yield is 13.83%. When the ultrasonic treatment is carried out for 30 to 90 minutes, the ultrasonic time is prolonged, the ultrasonic and enzymatic hydrolysis are fully effective, and the ZKHT yield is improved. However, when the ultrasonic treatment time is as long as 90 minutes, the ultrasonic treatment maintains a high temperature, which will destroy and degrade some flavonoids, and the ZKHT yield will decrease.

[0062] 2.2.2 Orthogonal test results

[0063] As shown in Table 2, among the three factors A, B, and C, A has the greatest effect on the ZKHT yield, followed by C, and B has the least effect, that is, A (solid-liquid ratio) > C (ethanol concentration) > B (ultrasonic time). According to the K value, the optimal solution for ZKHT extraction is A. 1 B 2 C 2 , that is, the solid-liquid ratio is 1:10 g / mL, the ultrasonic time is 90 min, and the ethanol concentration is 40%.

[0064] Table 2 Orthogonal test results

[0065]

[0066]

[0067] 2.2 Resin screening

[0068] There are certain differences in the adsorption and desorption performance of ZKHT by different types of macroporous resins. Figure 2 It can be seen that in terms of adsorption effect, the adsorption rates of S-8, D101, HPD600, NKA-9 and AB-8 are 96.26%, 94.98%, 94.12%, 94.22% and 72.44% respectively. The adsorption rates of S-8, D101, HPD600 and NKA-9 are all over 94%, among which S-8 has the best adsorption effect. Within a certain range, the larger the specific surface area of ​​the resin, the better the adsorption effect. The specific surface areas of S-8, D101, HPD600 and NKA-9 resins are all over 100m 2 / g, adsorption rate. In terms of analytical effect, D101 and AB-8 have better desorption rates, which are 93.60% and 83.67% respectively. D101 has the highest desorption rate, exceeding 93%, and has the best analytical effect. The average particle size comprehensively considers the static adsorption rate and desorption rate of the resin, and finally D101 resin is selected as the chromatography column filling resin used.

[0069] 2.3 Dynamic univariate analysis

[0070] As the volume of ZKHT crude extract increases, the mass concentration of the effluent increases slowly at first, until an inflection point appears and then gradually stabilizes. Figure 3 It can be seen that when the volume of ZKHT crude extract reaches 69mL, the mass concentration of the effluent begins to show an inflection point. The reason may be that the adsorption of ZKHT by D101 resin has reached saturation. If the sample is continued, the resin will not adsorb any more. Therefore, the sample volume of ZKHTZ crude extract is selected to be 70mL.

[0071] Figure 4 From left to right, the first row is a, b, c, and from left to right, the second row is d, e. Figure 4 a It can be seen that with the continuous increase of the sample volume, different sample flow rates have a certain effect on the effect of D101 resin adsorbing ZKHT. When the sample flow rate is 1, 2, 3, 4, and 5 mL / min, the mass concentration of ZKHT in the eluent can reach 0.96, 0.99, 0.95, 0.68, and 0.69 mg / mL, respectively. The sample flow rate of 2 mL / min is the highest, which is 0.99 mg / mL. The mass concentration of ZKHT adsorbed and eluted at 4 and 5 mL / min is much lower than that of 1, 2, and 3 mL / min. This is because the flow rate is too fast, and D101 does not completely adsorb ZKHT, resulting in unsatisfactory results. Therefore, the sample flow rate is determined to be 2 mL / min.

[0072] from Figure 4b It can be seen that the different pH values ​​of the loading solution will affect the mass concentration of the eluent ZKHT. When the pH value of the loading solution is 4, 5, 6, 7 and 8, the mass concentration of ZKHT in the eluent can reach 0.83, 0.90, 0.94, 0.99 and 0.75 mg / mL respectively. The highest concentration is 0.99 mg / mL when the pH value of the loading solution is 7. The reason may be that ZKHT itself, flavonoids may react under acidic conditions; it may also be the resin, the adsorption capacity of the resin under different pH conditions is different. Therefore, in subsequent experiments, the pH value of the sample solution is 7.

[0073] Figure 4 c shows that the adsorption time has a significant effect on the ZKHT mass concentration of the eluent. When the adsorption time is 1h, the ZKHT mass concentration is the highest at 0.96mg / mL. If the adsorption time is too long, the adsorption performance of the resin on ZKHT will decrease. Figure 4 c It can be seen that when the adsorption time is 5 h, the minimum mass concentration of ZKHT is 0.65 mg / mL.

[0074] from Figure 4 As can be seen in Figure d, the eluent concentration has a significant effect on the ZKHT mass concentration. When the eluent concentration is 40%, the maximum ZKHT mass concentration of the eluent is 0.25 mg / mL, and when the eluent concentration is 70%, it can reach a maximum of 0.92%. When the eluent volume of the 40% ethanol solution is 150-200 mL, the ZKHT mass concentration is more than double that of other concentrations, indicating that the eluent concentration is too low, the elution of flavonoids is incomplete, and the elution effect is poor.

[0075] Figure 4 As can be seen in Figure e, when the eluent flow rate is 1, 2, 3, 4, and 5 mL / min, the mass concentration of ZKHT in the eluent can reach 0.98, 0.92, 0.88, 0.82, and 0.79 mg / mL, respectively. The eluent flow rate is 1 mL / min, which is the highest, at 0.98 mg / mL. Too high a flow rate will shorten the time the eluent contacts ZKHT, making it difficult to displace the desired target substance, affecting the elution effect.

[0076] 2.4 Effect of purification process on ZKHT

[0077] Table 3 Comparison of the contents of kaempferol, luteolin and apigenin before and after purification

[0078]

[0079] The ZKHT yield in the crude extract was 14.32%. Under the optimal conditions, after resin adsorption elution and membrane separation, the ZKHT yield was 24.66%. The crude extract sample and the purified sample were analyzed by HPLC to obtain the liquid chromatogram of the ZKHT component before and after purification ( Figure 6 ), a total of 22 components. From Table 3, Figure 5 and Figure 6 It can be seen that the retention times of the three components in the sample are 3.208, 7.182 and 8.413 min, respectively, which are basically consistent with the peak time of the standard, so they are determined to be kaempferol, luteolin and apigenin. Figure 6 It can be seen that the relative content of kaempferol increased from 2.95% to 4.34%, the relative content of luteolin increased from 5.17% to 6.79%, and the relative content of apigenin increased from 0.33% to 0.82%, and the relative contents of the three components increased to a certain extent. After purification, the ZKHT yield increased by 72.21%, and the relative contents of kaempferol, luteolin and apigenin also increased by 47.19%, 31.19% and 146.47%, respectively. The flavonoid yield and the content of active substances were improved to a certain extent, which shows that the purification effect of the resin-membrane separation technology of the present invention is significant.

[0080] The method of the invention can effectively and simultaneously extract high-content kaempferol, luteolin and apigenin and has a high yield of total flavonoids.

[0081] Example 1

[0082] (1) Pectinase, cellulase and ethanol solution are prepared into 10 mg / mL ethanol-enzyme hydrolysate, peony seed shell powder is weighed, peony seed shell powder and ethanol-enzyme hydrolysate are mixed, and enzymolysis-ultrasound treatment is performed under 250W conditions: the solid-liquid ratio of peony seed shell powder and ethanol-enzyme hydrolysate is 1:10 g / mL, the ethanol concentration is 40%, the pH value is 6, the enzyme addition amount is 20 mg / mL, the ultrasonic temperature is 50°C, and the ultrasonic time is 90 min; the enzyme is inactivated, cooled, centrifuged at 1000 r / min, filtered, concentrated and then re-dissolved with 40% ethanol to obtain a crude extract; wherein pectinase and cellulase are weighed in a mass ratio of 1:2.

[0083] (2) The crude extract was separated and purified by resin adsorption combined with membrane separation: the resin adsorption / desorption process conditions were: sample volume 70 mL, sample flow rate 2 mL / min, adsorption 1 h, 70% ethanol elution at a flow rate of 1 mL / min. The adsorption resin model was D101.

[0084] (3) The eluate obtained after elution by resin adsorption was filtered through a 50 mm filter membrane, filtered and impurities were removed by ultrafiltration at room temperature, and the permeate was collected, rotary evaporated, and freeze-dried (-25°C, 5-10 Pa). Under this condition, the flavonoid yield was 24.66%, the relative content of kaempferol was 4.34%, the relative content of luteolin was 6.79%, and the relative content of apigenin was 0.82%.

Claims

1. A method for preparing a peony seed shell flavonoid extract, characterized in that The method comprises the following steps: (1) preparing an ethanol-enzyme solution of 8-10 mg / mL by taking pectinase, cellulase and ethanol solution, weighing peony seed shell powder, mixing the peony seed shell powder and the ethanol-enzyme solution, performing enzymolysis-ultrasound treatment at 250 W: the ultrasonic time is 80-100 min, the solid-liquid ratio of the peony seed shell powder and the ethanol-enzyme solution is 1:10-20 g / mL, the ethanol concentration is 30-50%, the pH value is 3-6, and the enzyme addition amount is 5-25 mg / mL; inactivating the enzyme, cooling, centrifuging, filtering, concentrating and re-dissolving with ethanol to obtain a crude extract; (2) The crude extract was separated and purified by resin adsorption combined with membrane separation: the resin adsorption / desorption process conditions were as follows: a sample volume of 69-70 mL, a sample flow rate of 1-3 mL / min, adsorption for 1-1.5 h, and elution with 65-75% ethanol at a flow rate of 1-1.5 mL / min.

2. The method for preparing the peony seed shell flavonoid extract according to claim 1, characterized in that The enzymatic hydrolysis-ultrasonic treatment conditions in step (1) are: solid-liquid ratio 1:10 g / mL, ethanol concentration 40%, pH value 6, enzyme addition amount 20 mg / mL, ultrasonic temperature 50°C, and ultrasonic time 90 min.

3. The method for preparing the peony seed shell flavonoid extract according to claim 1, characterized in that In step (2), the resin adsorption / desorption process conditions are: sample volume 70 mL, sample liquid flow rate 2 mL / min, adsorption 1 h, 70% ethanol elution at a flow rate of 1 mL / min.

4. The method for preparing the peony seed shell flavonoid extract according to claim 1, characterized in that The adsorption resin model used in step (2) is S-8, D101, HPD600 or NKA-9.

5. The method for preparing the peony seed shell flavonoid extract according to claim 4, characterized in that The adsorption resin model used in step (2) is D101.

6. The method for preparing the peony seed shell flavonoid extract according to claim 1, characterized in that The centrifugal condition in step (1) is 1000 r / min.

7. The method for preparing the peony seed shell flavonoid extract according to claim 1, characterized in that In the step (1), ethanol with a mass concentration of 40% is used for re-dissolution.

8. The method for preparing the peony seed shell flavonoid extract according to claim 1, characterized in that The pectinase and cellulase are weighed in a mass ratio of 1:

2.

9. The method for preparing the peony seed shell flavonoid extract according to claim 1, characterized in that The membrane separation is to obtain the eluate after elution by resin adsorption, filter it through a 50mm filter membrane, filter and remove impurities by ultrafiltration at room temperature, collect the permeate, rotary evaporate and freeze-dry.

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