A method for extracting and purifying phenolic substances from raspberry leaves

Abstract

The present invention relates to the field of biological extraction, and provides a method for extracting and purifying phenolic substances in raspberry leaves. The present invention extracts phenolic substances in raspberry leaves by a method combined with ultrasound (US), high-speed homogenization (HG) and steam explosion (SE), obtains a crude extract, and then purifies it by a macroporous resin column chromatography method to obtain a purified product. The results show that SE is first pretreated, and then combined with US+HG technology, the TPC value of the obtained crude extract (RLE) reaches up to 136.30-140.51 mgGAE / g, and after the first purification, the TPC value of the obtained purified product (RLP‑1) reaches 738.98 mg / g, and after the second purification, the TPC value of the obtained purified product (RLP‑2) is 905.27 mg / g, and the antioxidant activity of RLP‑2 and the inhibition of amylase and glucosidase activity are the highest.

Description

Technical Field

[0001] The invention relates to the technical field of biological extraction, and in particular to a method for extracting and purifying phenolic substances from raspberry leaves. Background Art

[0002] Raspberry (Rubus idaeus L.), also known as raspberry, is widely cultivated and planted in temperate and cold regions. Raspberry fruit is rich in various medicinal ingredients such as ellagic acid and flavonoids, and has many benefits for human health. Similarly, raspberry leaves are also rich in phenolic compounds. Katalinic et al. (V. Katalinic, et al. Screening of 70 medicinal plant extracts for antioxidant capacity and total phenols, Food Chemistry [J], 94 (2006): 550-557) found that the total phenol content (TPC) of raspberry leaves reached 848 mg CE / L (mg catechin equivalent per liter of injection), ranking fourth among 70 European medicinal plants. Since 1983, raspberry leaves have been registered in the British Pharmacopoeia and included in the European Community Herbal Medicines Monograph published by the European Medicines Agency in 2012. A comparison of 54 Chinese herbal medicines revealed that the TPC in raspberry leaves reached 123.93 mg / g, ranking ninth, indicating that raspberry leaves are a substance rich in bioactive ingredients and have high research value. In addition, the TPC content in raspberry leaf extract purified by macroporous resin exceeded 50%, mainly quercetin-3-glucuronide, free ellagic acid and tannin. After the extract was fed to mice, the mice gained less weight and the obesity rate decreased. Even in Eastern Europe, the young branches of raspberries have shown biological activity as traditional herbs.

[0003] Phenolic compounds have multiple medicinal values ​​such as antioxidant and anti-inflammatory, and are widely used in food and medicine. With the deepening of research on phenolic compounds, the requirements for the extraction of phenolic compounds are getting higher and higher, such as improving the extraction rate, shortening the extraction time, reducing energy consumption and reducing solvent consumption. Therefore, many studies have improved the conditions of many traditional extraction methods to obtain phenolic substances with higher purity. For example, Yang et al. (Jing Yang, et al, Ultrasound-homogenization-assisted extraction of polyphenols from coconut mesocarp: Optimization study, Ultrasonics Sonochemistry [J], 78 (2021): 105739) extracted polyphenols from coconut mesocarp by high-speed homogenization extraction and leaching respectively. The results showed that the polyphenol content obtained by high-speed homogenization extraction was nearly 50% higher than that by leaching. However, the time required for high-speed homogenization to crush the material is long, and the heat generation is too high, which can easily cause the destruction of the structure of polyphenolic compounds and reduce their activity. Therefore, how to improve the extraction rate of polyphenols in raspberry leaves while ensuring their activity is an urgent problem to be solved in this field. Summary of the invention

[0004] In view of this, the present invention provides a method for extracting and purifying phenolic substances from raspberry leaves. The present invention can quickly and effectively extract phenolic substances from raspberry leaves with a high extraction rate. After further purification, the content of phenolic substances in the obtained purified product is further increased, and the biological activity is high.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] A method for extracting phenolic substances from raspberry leaves comprises the following steps:

[0007] The raspberry leaf powder and the ethanol solution are mixed for composite extraction to obtain a crude extract of phenolic substances; the composite extraction method includes ultrasound, homogenization and steam explosion.

[0008] Preferably, the composite extraction comprises sequentially performing ultrasound, homogenization and steam explosion, or sequentially performing ultrasound, steam explosion and homogenization, or sequentially performing homogenization, ultrasound and steam explosion, or sequentially performing homogenization, steam explosion and ultrasound, or sequentially performing steam explosion, ultrasound and homogenization, or sequentially performing steam explosion, homogenization and ultrasound.

[0009] Preferably, in the composite extraction, the operating parameters of ultrasound include: power of 120-360 W, frequency of 40-80 Hz, temperature of 60-80° C., time of 30-60 min, and liquid-to-solid ratio of 20-40 mL / g; the operating parameters of homogenization include: rotation speed of 3000-7000 r / min, time of 0.5-1 min, and liquid-to-solid ratio of 20-40 mL / g; the operating parameters of steam explosion include: temperature of 105-125° C., time of 5-25 min, and liquid-to-solid ratio of 8-40 mL / g.

[0010] Preferably, the volume fraction of the ethanol solution is 40-80%.

[0011] The present invention also provides a method for purifying phenolic substances in raspberry leaves, comprising the following steps:

[0012] Extracting raspberry leaves according to the method described in the above scheme to obtain a crude extract of phenolic substances;

[0013] dissolving the crude extract of the phenolic substance in water to obtain a crude extract solution;

[0014] The crude extract solution is purified by column chromatography to obtain a purified product of phenolic substances; the resin used in the column chromatography is a macroporous resin, and the eluent used is an ethanol solution.

[0015] Preferably, the column chromatography includes sequential adsorption and desorption, and the operating parameters of the adsorption include: the concentration of total phenol in the crude extract solution is 0.1-2.5 mg / mL, the pH value of the crude extract solution is 1-6, the loading flow rate is 2-4 BV / h, and the loading volume is 18-20 BV.

[0016] Preferably, the desorption operation parameters include: the volume fraction of the ethanol solution is 40-80%, the flow rate is 4-6 BV / h, and the amount of the ethanol solution is 3-5 BV.

[0017] Preferably, the purification is performed 1 to 2 times.

[0018] Preferably, the macroporous resin is D101, D101C, LX12, LX83, XDA6 or AB8.

[0019] The present invention provides a method for extracting phenolic substances from raspberry leaves, comprising the following steps: mixing raspberry leaf powder and ethanol solution for composite extraction to obtain a crude extract (RLE) of phenolic substances; the composite extraction method includes ultrasound (US), homogenization (HG) and steam explosion (SE). The present invention extracts phenolic substances from raspberry leaves by a method combining ultrasound, homogenization and steam explosion, and the extraction rate of phenolic substances is high, and the total phenol content in the obtained crude extract is high. The results of the embodiment show that when the processes of SEHU (steam explosion + homogenization + ultrasound) and SEUH (steam explosion + ultrasound + homogenization) are adopted, the total phenol content (TPC) in the obtained crude extract is the highest, reaching 136.30±6.08mg / g to 140.51±1.67mg / g.

[0020] The present invention also provides a method for purifying phenolic substances in raspberry leaves. The present invention uses a column chromatography method to purify the crude extract obtained by the above scheme to obtain a purified product of phenolic substances. Further, the present invention optimizes the purification process by multiple factors such as macroporous resin screening, sample concentration, sample amount, pH, eluent, etc. to increase the content of phenolic compounds in the purified product. The results of the embodiment show that the medium-polarity XDA-6 is the best macroporous resin. After the first purification under the optimal conditions, the TPC value of the obtained purified product (referred to as RLP-1) reaches 738.98 mg / g. After the second purification, the TPC value of the obtained purified product (referred to as RLP-2) is 905.27 mg / g; and RLP-2 has the highest antioxidant activity and the highest inhibitory activity of amylase and glucosidase.

[0021] In summary, the present invention optimizes the extraction and purification process of the raw materials, which not only improves the utilization value of raspberry leaves, but also expands the development space of the raspberry industry, and has good theoretical significance and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The test results of TPC content in the supernatant obtained at different homogenization speeds;

[0023] Figure 2 is the effect of SE temperature on TPC value in supernatant;

[0024] Figure 3 is the effect of SE time on TPC value in supernatant;

[0025] Figure 4 The effect of liquid-to-solid ratio on TPC value in supernatant during SE treatment;

[0026] Figure 5 It is the response surface diagram of the effect of temperature and time on TPC content;

[0027] Figure 6It is the response surface diagram of the effect of temperature and liquid-to-solid ratio on TPC content;

[0028] Figure 7 It is the response surface diagram of the effect of time and liquid-to-solid ratio on TPC content;

[0029] Figure 8 The TPC values ​​of the supernatants obtained after the three processes were combined and extracted in different orders;

[0030] Fig. 9 The adsorption / desorption capacity and desorption ratio test results of six macroporous resins for phenolic compounds;

[0031] Fig.10 is the static adsorption isotherm of XDA-6 macroporous resin;

[0032] Fig.11 is the adsorption kinetic curve of XDA-6 macroporous resin;

[0033] Fig.12 The effect of pH value of sample solution on the adsorption capacity of XDA-6 macroporous resin;

[0034] Fig.13 The effect of ethanol concentration on the desorption capacity of XDA-6 macroporous resin;

[0035] Fig.14 Adsorption leakage curve (left) and desorption curves at different flow rates (right);

[0036] Fig.15 HPLC-MS chromatograms of RLE, RLP-1 and RLP-2;

[0037] Fig.16 The test results of the contents of different phenolic compounds in RLE, RLP-1 and RLP-2;

[0038] Fig.17 is the inhibition rate of RLE, RLP-1 and RLP-2 on α-amylase and α-glucosidase. DETAILED DESCRIPTION

[0039] The present invention provides a method for extracting phenolic substances from raspberry leaves, comprising the following steps:

[0040] The raspberry leaf powder and the ethanol solution are mixed for composite extraction to obtain a crude extract of phenolic substances; the composite extraction method includes ultrasound, homogenization and steam explosion.

[0041] In the present invention, the mesh size of the raspberry leaf powder is preferably 80 meshes; the volume fraction of the ethanol solution is preferably 40-80%, more preferably 60%.

[0042] In the present invention, the composite extraction is preferably divided into 6 methods, namely: sequentially performing ultrasound, homogenization and steam explosion (denoted as UHSE), or sequentially performing ultrasound, steam explosion and homogenization (denoted as USEH), or sequentially performing homogenization, ultrasound and steam explosion (denoted as HUSE), or sequentially performing homogenization, steam explosion and ultrasound (denoted as UHSE), or sequentially performing steam explosion, ultrasound and homogenization (SEHU), or sequentially performing steam explosion, homogenization and ultrasound (SEUH); in a specific embodiment of the present invention, the combination of SEUH and SEHU has the best effect, and the total phenol content in the obtained crude extract is high. In the present invention, steam explosion (SE) is to place the material in a high temperature and high pressure environment, and the generated steam will penetrate into the material, so that the tissue gap is filled with steam, and the high pressure generated by the saturated steam is released instantly, so that the steam in the tissue gap expands and further explodes, resulting in the formation of micropores on the cell wall, making it easier to release small molecules from the cells, which is beneficial to the extraction and conversion of phenolic compounds, thereby promoting the extraction of phenolic substances. Similarly, SE can cause degradation or polymerization of phenolic compounds to varying degrees, thereby changing the composition of phenolic compounds and their biological activity; the combination of SEUH and SEHU is better than other combination processes, indicating that the internal structure of the plant is efficiently destroyed after SE pretreatment, so that the subsequent U+H can better release the phenolic compounds in the plant.

[0043] In the present invention, in the composite extraction, the operating parameters of ultrasound preferably include: the power is preferably 120-360W, more preferably 120W, the frequency is preferably 40-80Hz, more preferably 40Hz, the temperature is preferably 60-80°C, more preferably 60°C, the time is preferably 30-60min, more preferably 30min, the liquid-to-solid ratio is preferably 20-40mL / g, more preferably 40mL / g; the operating parameters of homogenization preferably include: the speed is preferably 3000-7000r / min, more preferably 4000 r / min, the time is preferably 1 min, the liquid-to-solid ratio is preferably 20-40 mL / g, more preferably 40 mL / g; the homogenization is preferably carried out using a high-speed disperser; the operating parameters of the steam explosion preferably include: the temperature is preferably 105-125° C. (corresponding to a pressure of 0.168-0.23 MPa), more preferably 115° C., the time is preferably 5-25 min, more preferably 25 min, the liquid-to-solid ratio is preferably 8-40 mL / g, more preferably 29 mL / g; the steam explosion is preferably carried out in an autoclave.

[0044] In a specific embodiment of the present invention, when performing a composite extraction, after the previous extraction method is completed, the amount of ethanol in the extract is preferably adjusted by adding ethanol or rotary evaporation to meet the liquid-to-solid ratio requirement of the subsequent extraction.

[0045] In the present invention, after the composite extraction is completed, the ethanol in the obtained extract is preferably removed to obtain a crude extract (RLE) of phenolic compounds; in a specific embodiment of the present invention, the crude extract is directly purified after being obtained, then the ethanol in the extract is preferably removed by rotary evaporation, and the residue is purified; if the crude extract needs to be preserved and then purified, the extract is preferably freeze-dried and the obtained powder is stored.

[0046] The present invention also provides a method for purifying phenolic substances in raspberry leaves, comprising the following steps:

[0047] Extracting raspberry leaves according to the method described in the above scheme to obtain a crude extract of phenolic substances;

[0048] dissolving the crude extract of the phenolic substance in water to obtain a crude extract solution;

[0049] The crude extract solution is purified by column chromatography to obtain a purified product of phenolic substances; the resin used in the column chromatography is a macroporous resin, and the eluent used is an ethanol solution.

[0050] The present invention first extracts raspberry leaves according to the method described in the above scheme to obtain a crude extract of phenolic substances, and the specific extraction method is not repeated here.

[0051] After obtaining the crude extract of phenolic substances, the present invention dissolves the crude extract of phenolic substances in water to obtain a crude extract solution. In the present invention, the water is preferably deionized water.

[0052] After obtaining the crude extract solution, the crude extract solution is purified by column chromatography to obtain a purified product of phenolic substances. In the present invention, the resin used in the column chromatography is a macroporous resin, and the eluent used is an ethanol solution; the macroporous resin is preferably D101, D101C, LX12, LX83, XDA6 or AB8, more preferably XDA6; the macroporous resin is preferably pretreated before use, and the pretreatment is preferably: soaking the macroporous resin in an ethanol solution with a volume fraction of 95% for 12 hours, then removing the ethanol solution, washing with deionized water to neutrality, then soaking in 4% hydrochloric acid solution (v / v) and 4% NaOH solution (w / v), finally washing with deionized water to neutrality, and storing in 95% ethanol solution; the soaking time in the hydrochloric acid solution is preferably 4 to 5 hours, and the soaking time in the NaOH solution is preferably 4 to 5 hours.

[0053] In the present invention, the column chromatography includes sequential adsorption and desorption, and the operating parameters of the adsorption preferably include: the concentration of total phenol in the crude extract solution is 0.1-2.5 mg / mL, preferably 1-2 mg / mL, and more preferably 1.76 mg / mL, the pH value of the crude extract solution is 1-6, preferably 2-4, and more preferably 2, the loading flow rate is 2-4 BV / h, preferably 3 BV / h, and the loading volume is 18-20 BV, and more preferably 18 BV; in a specific embodiment of the present invention, it is preferred to use a hydrochloric acid solution to adjust the pH value of the crude extract solution to 2 before loading and adsorption.

[0054] In the present invention, the desorption operation parameters preferably include: the volume fraction of the ethanol solution is 40-80%, preferably 60%, the flow rate is 4-6BV / h, preferably 5BV / h, and the amount of ethanol solution is 3-5BV, preferably 5BV.

[0055] In the present invention, the purification is preferably performed 1 to 2 times, more preferably 2 times; when the purification is performed twice, after the first purification is completed, the ethanol in the purified product is preferably removed by rotary evaporation, and then the second purification is performed.

[0056] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0057] The raspberry leaves (R. idaeus L. cv. Heritage) used in the embodiment were collected from North China University, Taiyuan, Shanxi Province (38°01′71″N, 112°44′46″E) in October. The collected leaves were naturally air-dried and stored at room temperature. The raspberry leaf powder used in the embodiment was 80 mesh powder.

[0058] In the following examples, the total phenol content (TPC) of crude extracts and purified products was determined by the Folin-Ciocalteu method, as follows:

[0059] Take 100 μL of the extract, add 0.3 mL of Folin-Ciocalteu solution and 1.5 mL of 7% Na2CO3 solution, react for 2 h in the dark, and calculate according to the gallic acid standard curve (Formula (1)):

[0060] A = 1.808C + 0.0056 (1);

[0061] Where A is absorbance, C is gallic acid concentration (mg / mL);

[0062] After obtaining the concentration data, the TPC content is calculated according to TPC=CV / m, where V is the solution volume (mL); m is the sample weight (g), and the unit of TPC is mg / g.

[0063] In the following examples, each experiment was repeated at least 3 times, and the experimental results were expressed as mean ± standard deviation. SPSS software was used for variance analysis and statistical calculation of the data. P < 0.05 indicated significance.

[0064] Example 1 Homogenization-assisted extraction

[0065] Weigh 0.2 g of raspberry leaf powder, add 80 mL of 60% ethanol solution, and use a high-speed disperser to homogenize the sample at 3000, 4000, 5000, 6000 and 7000 r / min for 1 min. After centrifugation, measure the TPC in the supernatant.

[0066] Figure 1 The following are the test results of TPC content in the supernatant obtained at different homogenization speeds. Since too high a speed and too long a time during homogenization will cause excessive heat generation in the instrument, which may cause degradation of phenolic compounds and damage to the instrument, the TPC value in the extract after 1 minute of homogenization at different speeds was first determined. Figure 1 As shown in the figure, when the speed ranges from 3000 to 7000 r / min, the TPC content in the supernatant shows a trend of first increasing slightly and then decreasing sharply. There is no significant difference in the TPC content of the supernatant obtained under the conditions of 4000 and 5000 r / min, which are 101.96±1.11 mg / g and 103.58±1.76 mg / g respectively. This may be because the homogenization treatment is not thorough at too low a speed, and the plant tissue is not sufficiently destroyed, resulting in the failure to effectively release TPC. When the speed is too high, the polyphenol oxidase inside the plant cells is fully released, decomposing the phenols in the extract, and the phenol content will be reduced. Therefore, considering the influence of extraction rate and energy consumption, the optimal speed condition is 4000 r / min under the premise of 1 minute of homogenization.

[0067] Example 2 Steam Explosion (SE) Experiment

[0068] (1) Single factor experiment

[0069] Weigh 0.2 g of raspberry leaf powder, add 60% ethanol, and conduct a steam explosion single factor experiment at a certain temperature, time and solid-liquid ratio. After centrifugation, test the TPC in the supernatant. The single factor experimental design is shown in Table 1.

[0070] Table 1 Single factor experimental design

[0071]

[0072] When studying the effect of SE temperature on TPC value in raspberry leaves, other fixed factors were: liquid-to-solid ratio of 32 mL / g and pressurization time of 10 min.

[0073] Figure 2 The effect of SE temperature on TPC value in supernatant. Figure 2 It can be seen that as the temperature increases, the TPC value also increases, and at 115°C (0.168MPa), it reaches a maximum value of 103.72±2.21mg / g, indicating that appropriate pressure can destroy the internal structure of the plant and promote the release of phenolic compounds. When the temperature exceeds 115°C, the TPC value gradually decreases. Excessive pressure will destroy the structure of phenolic compounds and even carbonize the plant material.

[0074] When studying the effect of SE time on the TPC value of raspberry leaves, other fixed factors were: liquid-to-solid ratio of 32 mL / g and SE temperature of 110 °C.

[0075] Figure 3 The effect of SE time on TPC value in supernatant. Figure 3 It can be seen that with the increase of SE treatment time, TPC continued to increase. When the treatment time was 15 min, TPC reached the maximum value of 103.72 ± 2.21 mg / g. When the treatment time exceeded 15 min, TPC decreased rapidly. The possible reason is that when the SE time is short, although it will cause the decomposition of phenolic compounds, more phenolic compounds are released from the cells and depolymerization, so the TPC value will increase. After more than 15 min, fewer phenolic compounds are released from the cells and depolymerization, and more phenolic compounds are destroyed by SE, so the TPC value will decrease.

[0076] When studying the effect of different liquid-to-solid ratios on TPC values, other fixed factors were: SE time of 10 min and temperature of 110 °C.

[0077] Figure 4 The effect of liquid-to-solid ratio on TPC value in supernatant during SE treatment. Figure 4 It can be seen that with the increase of liquid-to-solid ratio, the TPC value of the supernatant obtained by extraction gradually increases. When the liquid-to-solid ratio is too low, the obtained TPC value is low. This may be because phenolic compounds are easily saturated when dissolved in less solvent and cannot continue to dissolve too many phenolic compounds. When the liquid-to-solid ratio is 24 mL / g, the TPC value reaches the maximum value. When the liquid-to-solid ratio continues to increase, the TPC value decreases. This may be because a large liquid-to-solid ratio is likely to cause the influence of mass transfer resistance, which reduces the dissolution of phenolic compounds and even causes solvent waste.

[0078] (2) Response surface experiment

[0079] According to the results of the single factor experiment, time A, temperature B, and liquid-to-solid ratio C were selected as the influencing factors of the response surface experiment. The TPC value was used as the response value. The Box-Behnken model was used and the different levels of each factor were coded with 1, 0, and -1. The specific design is shown in Table 2.

[0080] Table 2 Response surface experimental design

[0081]

[0082] The experimental results of the response surface experiment are shown in Table 3.

[0083] Table 3 Response surface experiment groups and results

[0084]

[0085]

[0086] The experimental results were analyzed using Design-Expert11. Let SE time be A, temperature be B, liquid-to-solid ratio be C, TPC value be response value Y, and the fitting equation is Y=113.69+4.70A+2.03B+7.80C-4.36AB+1.35AC-2.55BC-7.83A. 2 -14.00B 2 -15.31C 2 .

[0087] The experimental results were subjected to ANOVA variance analysis, and the analysis results are shown in Table 4. When p<0.05, it means that the model has reached a significant level, and when p<0.01, it means that the model has reached an extremely significant level. Comparing the F values ​​of the three factors, it can be seen that the influence of the three factors on the TPC value is ranked from large to small as follows: FC>FA>FB, indicating that the influence of SE processing time and liquid-to-solid ratio on the TPC value is greater than the influence of temperature. From the p value, it can also be concluded that the influence of time and liquid-to-solid ratio on the TPC value is extremely significant (p<0.01), while the influence of temperature on TPC content is significant (p<0.05). Among the first-order terms, the interaction of AB is extremely significant (p<0.01), the interaction of BC is significant (p<0.05), and the interaction of AC is not significant (p>0.05), while the quadratic terms are all significant. The R obtained by the model 2 The value is 0.9920, indicating that the model is consistent with the experimental results. In addition, the lack of fit term is not significant (p = 0.3815> 0.05), while AdjR 2 The value is 0.9818, Pred R 2 The value is 0.9299, and the difference is less than 0.2, which means that the model can well reflect the experimental data and can accurately predict and analyze the actual TPC value.

[0088] Table 4 ANOVA analysis of variance results

[0089]

[0090] Based on the above regression equation, a 3D surface plot can be drawn to further explore the impact of the interaction of the two factors on TPC extraction. The results are as follows: Figures 5 to 7 As shown, Figure 5 This is the response surface diagram of the effect of temperature and time on TPC content. Figure 6 This is the response surface diagram of the effect of temperature and liquid-to-solid ratio on TPC content. Figure 7 This is the response surface diagram of the effects of time and liquid-to-solid ratio on TPC content.

[0091] Depend on Figures 5 to 7 It can be seen that under the interaction of the two factors, the content of phenolic compounds shows a trend of first increasing and then decreasing, and the curvature of the curve can also indicate the strength of the influence of the interaction of the two factors on the RL-TPC content. The interaction between temperature and time, and temperature and liquid-to-solid ratio is strong, while the interaction between time and liquid-to-solid ratio is weak, which is the same as the results obtained in Table 4.

[0092] The results of response surface analysis show that the theoretical optimal extraction process of SE-assisted extraction of TPC from raspberry leaves is: time 25 min, temperature 113.9 °C, liquid-to-solid ratio 28.92 mL / g, and the theoretical maximum content of TPC obtained is 112.09 mg / g. Taking into account the actual operating conditions, the reaction conditions were slightly adjusted. The final process conditions were: SE time 25 min, temperature 115 °C, liquid-to-solid ratio 29 mL / g. Under these conditions, the actual TPC content obtained was 113.86 mg / g, which was close to the predicted value obtained under the theoretical process.

[0093] Example 3 Composite extraction experiment

[0094] Based on the three best processes of ultrasound (US), homogenization (HG) and steam explosion (SE), three processes were combined to compare the effects of six extraction combinations, including ultrasound + homogenization + steam explosion (UHSE), ultrasound + steam explosion + homogenization (USEH), homogenization + ultrasound + steam explosion (HUSE), homogenization + steam explosion + ultrasound (HSEU), steam explosion + ultrasound + homogenization (SEUH) and steam explosion + homogenization + ultrasound (SEHU), on the extraction of phenolics from raspberry leaves.

[0095] Among them, the optimal process conditions for ultrasound are: ultrasonic power 120W, frequency 40Hz, temperature 60℃, liquid-to-solid ratio 40mL / g, and ultrasonic time 30min; the optimal process conditions for homogenization are: speed 4000r / min, time 1min; the optimal process conditions for steam explosion are: time 25min, temperature 115℃, and liquid-to-solid ratio 29mL / g.

[0096] After the complex extraction is completed, the supernatant is centrifuged to test the TPC value.

[0097] Figure 8 Table 5 is the TPC value of the supernatant obtained after the three processes were combined in different orders; Table 6 is a summary of the results of the combined extraction and the extraction using US, HG and SE separately.

[0098] Table 5 Composite extraction experimental design and results

[0099]

[0100] The operating conditions in Table 5 are: HU: 4000 r / min, 1 min, 40 mL / g; SE: 25 min, 115°C, 29 mL / g; US: 60% ethanol, 40 mL / g, 120 W, 30 min.

[0101] according to Figure 8 As can be seen from Table 5, the TPC content of the supernatant obtained under the SEHU and SEUH process conditions was the highest, which were 136.30±6.08 mg / g and 140.51±1.67 mg / g, respectively, and there was no significant difference. They were all higher than the contents after single US (116.51 mg / g), HU (101.96 mg / g) and SE (113.86 mg / g) and the other two groups of composite process treatments, indicating that after pre-treatment with SE, hot steam destroyed the structure of the plant material from the inside, making its internal structure loose, so that the phenolic compounds in the material after subsequent US and HU treatments were more easily released and dissolved in the solvent, resulting in an increase in the TPC value.

[0102] The ethanol in the supernatant obtained by the SEUH process was removed by rotary evaporation, and the TPC concentration in the rotary evaporation residue was adjusted with water. The obtained crude extract solution was used in the purification process of the subsequent examples.

[0103] Example 4 Screening of macroporous resin

[0104] First, the macroporous resin was soaked in 95% ethanol overnight, and after removing the ethanol solution, it was washed with deionized water until neutral, and then soaked in 4% HCl (v / v) and 4% NaOH (w / v) for 4 to 6 hours, and the solution was removed, and after washing with deionized water until neutral, it was soaked in 95% ethanol solution for storage. The properties of the six macroporous resins are shown in Table 6.

[0105] Table 6 Properties of six macroporous resins

[0106]

[0107] Take 1g of macroporous resin and add it to a 250mL conical flask, add 20mL of crude extract solution (TPC concentration is 1.76mg / mL), and shake it in an air bath constant temperature shaking incubator at 25℃, 150rpm for 12h, and measure the TPC value in the extract of each conical flask. Calculate the adsorption amount according to formula (2). Then remove the extract, wash the macroporous resin with deionized water, add 30mL of 60% ethanol, and shake it at 25℃, 150rpm for 12h, calculate the desorption amount according to formula (3), and calculate its desorption ratio according to formula (4).

[0108]

[0109]

[0110]

[0111] In formula (2) to formula (4), q a and q b are the equilibrium adsorption capacity and the desorption amount, respectively; C0 is the initial concentration of total phenol in the extract; C a is the total phenol concentration in the solution after adsorption; C b is the total phenol concentration in the solution after analysis, V a is the volume of the extract, V b is the volume of ethanol, m is the mass of resin, and D is the analytical ratio.

[0112] Fig. 9 The following are the test results of the adsorption / desorption capacity and desorption ratio of six macroporous resins for phenolic compounds. Fig. 9It can be seen that although the desorption rates of XDA-6 and D101 are the highest, 87.46% and 83.26% respectively (P>0.05), the adsorption amount (58.43±2.95mg / g) and desorption amount (48.55±0.47mg / g) of D101 are much lower than those of XDA-6 (69.22±3.70mg / g) and desorption amount (60.41±0.98mg / g), so the selected macroporous resin model is XDA-6. The adsorption and desorption capacity of macroporous resin for phenolic compounds may be related to its polarity and specific surface area. XDA-6 macroporous resin has a larger specific surface area than D101, and the polarity of XDA-6 is medium polarity, which may be more conducive to the adsorption and desorption of phenolic compounds in raspberry leaves.

[0113] Example 5 Adsorption isotherm experiment

[0114] The crude extract solution was diluted with deionized water to a total phenol concentration of 0.32 mg / mL, 0.48 mg / mL, 0.64 mg / mL, 0.80 mg / mL, 0.96 mg / mL, 1.12 mg / mL, 1.28 mg / mL, 1.44 mg / mL, 1.76 mg / mL, and 1.92 mg / mL. 20 mL of the solution was added to a 250 mL conical flask, and 1 g of XDA-6 macroporous resin was added. After shaking at 25 ° C and 150 rpm for 12 hours, the total phenol concentration in the solution was determined and its adsorption amount was calculated.

[0115] In order to further study the adsorption characteristics of the resin, the Langmuir and Freundlich models were selected to analyze the data. The two models are shown in equations (5) and (6).

[0116] The Langmuir model is:

[0117] The Freundlich model is:

[0118] In formula (5) and formula (6): q m is the maximum theoretical adsorption capacity (mg), q e is the adsorption amount, Ce is the polyphenol concentration in the solution, K L is the Langmuir constant, n and K F is the Freundlich constant, K F represents the theoretical saturated adsorption capacity, n represents the driving force for adsorption, and n>1 indicates that it is conducive to adsorption.

[0119] Fig.10 is the static adsorption isotherm of XDA-6 macroporous resin. Fig.10It can be seen that when the total phenol concentration of the sample is lower than 1.76 mg / mL, the adsorption amount increases rapidly. When the sample concentration is higher than 1.76 mg / mL, the adsorption amount of phenolic compounds by XDA-6 resin remains basically unchanged, so the adsorption concentration is selected as 1.76 mg / mL.

[0120] The adsorption isotherms were fitted to the Langmuir and Freundlich models, and the results are shown in Table 7.

[0121] Table 7 Adsorption isotherm equation and its parameters

[0122]

[0123] The Langmuir adsorption model is described as a monolayer adsorption, that is, each adsorption site can only adsorb one adsorbed molecule. The Freundlich is an empirical equation, which describes that one adsorbed molecule can be adsorbed by multiple adsorption sites. Table 7 shows the relevant parameters of the two models. In the Langmuir adsorption model, q m The value of R2 of the Langmuir model and the two fitting equations was compared. The R2 of the Langmuir model was 1.36, which was in the range of 1-10. The R2 of the Langmuir model was 1.93 mg / g, which indicated that 1 g of XDA-6 macroporous resin could adsorb 51.93 mg of phenolic compounds. In the Freundlich model, the n value of 1.36 was in the range of 1-10, which indicated that XDA-6 resin could easily adsorb phenolic compounds in raspberry leaves. 2 =0.9872 value is greater than the Freundlich model R 2 =0.9721, indicating that the adsorption of phenolic compounds in raspberry leaves by XDA-6 macroporous resin tends to be monolayer adsorption.

[0124] Example 6 Adsorption kinetics experiment

[0125] Take 1g XDA-6 macroporous resin and add it into a 250mL conical flask, add 20mL of crude extract solution with a concentration of 1.76mg / mL, shake at 25℃ and 150rpm, and measure the total phenol concentration in the extract at 0min, 5min, 10min, 15min, 30min, 60min, 90min, 120min, and 180min respectively.

[0126] This embodiment selects two mathematical models, namely, pseudo first order and pseudo second order, as shown in equations (7) and (8):

[0127] Pseudo first order: q t =k i t 1 / 2 + C formula (7);

[0128] Pseudo second order: ln(qt -q e )=-k i t+lnq e Formula (8);

[0129] Where: q t is the adsorption capacity at interval t (mg), t represents the adsorption time (min), k i are pseudo-first-order and pseudo-second-order kinetic constants.

[0130] Fig.11 is the adsorption kinetics curve of XDA-6 macroporous resin. Fig.11 It can be seen that in the first 60 minutes of adsorption, there are many unadsorbed sites in the resin, and there is a concentration difference between the resin and the extract, which makes it easy for phenolic compounds to enter the resin and be absorbed more easily, so the adsorption of phenols by XDA-6 resin increases rapidly. After 60 minutes, the adsorption tends to be balanced. With the increase of adsorption time, the concentration of phenols in the resin gradually increases, the concentration difference becomes smaller, phenolic compounds are not easy to enter the resin, and the adsorption sites are also reduced, so the adsorption gradually reaches equilibrium.

[0131] The results of fitting the experimental data to the pseudo first-order model and the pseudo second-order model are shown in Table 8. The results show that the pseudo second-order kinetic model (R 2 =0.9988) is better than the pseudo first-order kinetic model (R 2 =0.9460), and compared with the actual maximum adsorption capacity (40.84 mg / g), the maximum adsorption capacity obtained by the pseudo-second-order model (43.80 mg / g) is closer to the actual maximum adsorption capacity than the pseudo-first-order model (29.13 mg / g), indicating that the kinetic model of the adsorption of phenolic compounds in raspberry leaves by XDA-6 macroporous resin is closer to the pseudo-second-order kinetic model, indicating that there may be multiple factors limiting the adsorption of phenolic compounds in raspberry leaves by XDA-6 macroporous resin.

[0132] Table 8 Adsorption kinetic equation and its parameters

[0133]

[0134] Example 7 Effect of Solution pH on Adsorption Capacity

[0135] The pH of the crude extract solution was adjusted to 1-8 with 4% HCl and 4% NaOH, respectively. 20 mL of the pH-adjusted solution was added to 250 mL conical flasks. 1 g of XDA-6 macroporous resin was added to each conical flask. The adsorption was carried out at 25 ° C and 150 rpm for 12 h, and the adsorption amount, desorption amount and desorption ratio were calculated.

[0136] Fig.12The effect of the pH value of the sample solution on the adsorption capacity of XDA-6 macroporous resin. The pH value of the sample solution will affect the adsorption of phenolic compounds by the macroporous resin, because the pH value will affect the ionization degree of polyphenols, thereby affecting the adsorption of phenolic compounds by the macroporous resin, so the desorption rate at different pH values ​​was determined. Fig.12 It can be seen that the desorption rate shows a trend of increasing first and then decreasing with the increase of pH value. When the pH value is less than 7, the desorption rate is high, and when the pH value is greater than 7, the desorption rate decreases rapidly. This is because phenol molecules are acidic. When in low pH environmental conditions, phenols exist in the form of molecules and are easily adsorbed by macroporous resins. When in alkaline conditions, phenol compounds exist in the form of ions and are not easily adsorbed by macroporous resins. When the sample solution pH = 2, the desorption rate is the highest, so the sample solution with pH = 2 is selected for subsequent adsorption experiments.

[0137] Example 8 Effect of ethanol concentration on desorption rate

[0138] Add 1 g of adsorption-saturated XDA-6 macroporous resin to each of 6 250 mL conical flasks, and add 20 mL of 0% (i.e., deionized water), 20%, 40%, 60%, 80%, and 100% ethanol, respectively. Oscillate at 25°C and 150 rpm for 12 h, and calculate the desorption amount by measuring the concentration of total phenol in the solution.

[0139] Fig.13 The effect of ethanol concentration on the desorption capacity of XDA-6 macroporous resin. Fig.13 It can be seen that when the ethanol concentration is less than 60%, the desorption rate gradually increases, and when the ethanol concentration is 60%, the desorption rate reaches the maximum value. When the ethanol concentration is greater than 60%, the total phenol content decreases, which may be because phenolic compounds are not easily soluble in low-concentration ethanol solutions, and when the ethanol concentration is too high, alcohol-soluble substances such as chlorophyll and lutein will be desorbed. Therefore, a 60% ethanol solution is selected as the best desorption solvent.

[0140] Example 9 Dynamic leakage curve and dynamic desorption curve

[0141] A glass column with an inner diameter of 1.5 cm and a length of 60 cm was selected for dynamic adsorption and desorption experiments. A pretreated macroporous resin with a height of 9 cm (diameter-to-height ratio of 1:6) was filled for dynamic adsorption and desorption experiments.

[0142] (1) Dynamic adsorption experiment

[0143] The crude extract solution (total phenol concentration was 1.76 mg / mL) was added at flow rates of 2 BV / h, 3 BV / h and 4 BV / h, respectively. The effluent of each column volume was collected and its total phenol concentration was determined.

[0144] (2) Dynamic desorption experiment

[0145] The chromatographic column after adsorption was washed with deionized water until the effluent was colorless. The optimal eluent obtained from the static desorption experiment was added at flow rates of 4 BV / h, 5 BV / h and 6 BV / h, and the eluents of different column volumes were collected and their total phenol concentrations were determined.

[0146] Fig.14 The adsorption leakage curve (left) and the desorption curve at different flow rates (right). At different flow rates, the sample solution with pH = 2 and phenolic compound concentration of 1.76 mg / mL was used to measure the adsorption leakage curve. When the phenolic compound concentration in the outflow solution reaches 10% of the initial solution, this point can be considered as the adsorption leakage point. Fig.14 As shown in the figure on the left, when the flow rate is 2, 3, and 4 BV / h, the leakage point appears at the 18th, 19th, and 20th BV respectively. Considering that when the flow rate is 2 BV / h, the adsorption time is longer and the adsorption leakage point is not reached, the flow rate of 3 BV / h shows a better adsorption effect, so the flow rate is 3 BV / h and the sample volume is 19 BV.

[0147] Using 60% ethanol solution as the elution solvent, the elution conditions at different flow rates (4BV / h, 5BV / h, 6BV / h) were measured. Fig.14 As shown in the figure on the right, when the flow rate is 6BV / h, the amount of eluent used is relatively large. At a faster flow rate, the elution solvent does not contact the adsorbed macroporous resin sufficiently, and the phenolic compounds cannot be fully desorbed from the macroporous resin, so the elution solvent used is relatively large. When the flow rate is 4BV / h and 5BV / h, the release efficiency is similar, but the desorption effect can be achieved faster when the flow rate is 5BV / h, so the selected elution solvent flow rate is 5BV / h and the amount is 3BV.

[0148] Example 10 Secondary Purification

[0149] The purified product obtained under the optimal conditions in Example 9 was recorded as the first purified product (RLP-1). After removing ethanol from the first purified product (RLP-1) using a rotary evaporator, a second purification was performed according to the obtained optimal conditions to obtain a second purified product (RLP-2); the crude extract that was not purified was recorded as RLE.

[0150] Example 11 Compound composition and biological activity test of RLE, RLP-1 and RLP-2

[0151] (1) Test on the types and contents of phenolic compounds in RLE, RLP-1 and RLP-2.

[0152] HPLC-MS was used to analyze the composition and content of compounds in RLE, RLP-1 and RLP-2. The method of HPLC-MS analysis was as follows: Raspberry leaf extract was analyzed by HPLC-MS using a Thermo Scientific QE Orbitrap mass spectrometer system (Thermo Fisher Scientific Inc, USA) equipped with an electrospray interface (ESI). The chromatographic column was Hypersil GOLD (C18 selective column, 100 mm × 2.1 mm, 3 μm). The sample injection volume was 1 μL, and the gradient process was divided into phase A (water + 0.1% formic acid) and phase B (acetonitrile). The flow rate was 0.3 mL / min, and the gradient was: 10% B, 0-1.5 min; 10-15% B, 1.5-2.5 min; 15% B, 2.5-4 min; 15-25% B, 4-6 min; 25% B, 6-8 min; 25-70% B, 8-9 min; 70-10% B, 9-10.5 min; 10% B, 10.5-12 min. The liquid entered the mass spectrometer through the electrospray interface. Nitrogen was used as nebulizing and drying gas. The working conditions of MS were: electrospray ionization (ESI) interface working in negative ion mode; source: 3.5 kV; sheath gas flow: 40 arb, auxiliary gas flow: 15 arb; capillary temperature: 320 ° C, heating temperature: 300 ° C. Take 1 mL of the supernatant and filter it through a 0.22 μm membrane, store it at 4 ° C, and perform HPLC-MS analysis.

[0153] Fig.15 HPLC-MS chromatograms of RLE, RLP-1 and RLP-2; Fig.16 These are the test results for the contents of different phenolic compounds in RLE, RLP-1 and RLP-2; among them, Q3GR is quercetin-glucuronide, K3R is kaempferol-3-rutinoside, Epc is epicatechin, ProcB1 is proanthocyanidin B1, EA is ellagic acid, GA is gallic acid, and CA is chlorogenic acid.

[0154] according to Figure 15-16 It can be seen that after purification, the contents of Q3GR, K3R, proanthocyanidin B1, and ellagic acid all increased significantly, which were 4.53, 3.07, 3.46, and 12.91 times before purification, respectively. The content of Q3GR was the highest before and after purification, which were 30.33 mg / g and 134.77 mg / g, respectively. The contents of epicatechin and chlorogenic acid did not change significantly. The above results show that purification can effectively increase the content of phenolic active ingredients in the sample.

[0155] (2) Antioxidant activity test of RLE, RLP-1 and RLP-2.

[0156] The antioxidant activity test includes the scavenging ability test of DPPH and the scavenging ability test of ABTS; the method of the scavenging ability test of DPPH is as follows: take 2mL of the extract and add 2mL of the prepared DPPH solution, react in the dark for 40 minutes, and measure its absorbance at 517nm, which is recorded as A1. The control is 2mL of DPPH solution added to 2mL of water, and the absorbance is recorded as A. The blank is 2mL of sample solution added to 2mL of 95% ethanol, and the absorbance is recorded as A0. The scavenging rate is calculated according to formula (9):

[0157]

[0158] The test method for the scavenging ability of ABTS is as follows: 1 mL of the reaction solution is added to 3 mL of the prepared ABTS solution, and the mixture is reacted for 6 min under light-proof conditions. The absorbance value is measured at 734 nm and recorded as A1. 1 mL of 70% ethanol is added to 3 mL of the prepared ABTS solution, and the absorbance value is recorded as A. The blank is 70% ethanol, and the scavenging rate is calculated according to formula (10):

[0159]

[0160] ICs of DPPH and ABTS scavenging by RLE, RLP-1 and RLP-2 50 The values ​​are shown in Table 9.

[0161] Table 9 IC of DPPH and ABTS clearance 50 Value (mg / g)

[0162]

[0163] The chemical structure and composition of phenolic compounds in plants are very complex, so the antioxidant activity varies greatly. There are many factors that affect the antioxidant activity of phenolic substances, such as the source, chemical structure, molecular weight, purity and processing technology of phenolic substances. According to the results in Table 9, the IC value of DPPH scavenging rate of RLE is 50 The value was 67.71 μg / mL. After primary purification and secondary purification, the IC values ​​of DPPH scavenging rate of RLP-1 and RLP-2 were 50 The values ​​were reduced to 33.99 μg / mL and 20.63 μg / mL, respectively. The IC values ​​of ABTS clearance of RLE 50 The value was 14.72 μg / mL. After primary purification and secondary purification, the IC values ​​of ABTS clearance of RLP-1 and RLP-2 were 50 The values ​​were reduced to 7.81μg / mL and 5.98μg / mL respectively. The above results show that the antioxidant activity of the products is improved after purification, which may be due to the increase in the content of phenolic compounds.

[0164] (4) The enzyme activity inhibition ability of RLE, RLP-1 and RLP-2 was tested, and abose was used as a positive control.

[0165] The method for determining the inhibition rate of α-amylase is as follows: add the sample solution, PBS solution (pH=6.8) and enzyme solution (5 mg / mL) to a 96-well plate, A: 60 μL PBS + 40 μL enzyme, B: 100 μL PBS, C: 35 μL PBS + 40 μL enzyme + 25 μL sample, D: 75 μL PBS + 25 μL sample, react at 37°C for 15 min after addition, then add 100 μL 1% starch solution, react at 37°C for 15 min, add 25 μL DNS, react at 85-90°C for 15 min, cool to room temperature, and measure the absorbance value at 540 nm, and calculate the inhibition rate according to formula 11:

[0166]

[0167] Method for determining the inhibition rate of α-glucosidase: add the sample solution, PBS solution (pH=6.8) and enzyme solution to a 96-well plate, A: 105μL PBS + 15μL enzyme, B: 120μL PBS, C: 85μL PBS + 15μL enzyme + 20μL sample, D: 100μL PBS + 20μL sample, react at 37°C for 15min after adding, then add 30μL PNPG (2.5mmol / L), react at 37°C for 20min, add 40μL Na2CO3 (0.2mol / L) to terminate the reaction, measure the absorbance value at 405nm, and calculate the inhibition rate according to formula 10.

[0168] Fig.17 is the inhibition rate of RLE, RLP-1 and RLP-2 on α-amylase and α-glucosidase.

[0169] The main function of digestive enzymes is to break down carbohydrates into small units for absorption by organisms. α-amylase is present in saliva and pancreas and is responsible for breaking down starch to produce disaccharides, which are then further broken down into glucose by α-glucosidase in the intestines and absorbed, raising blood sugar levels. Type 2 diabetes refers to an abnormal increase in blood sugar immediately after a meal. Inhibiting the activity of α-amylase and α-glucosidase can inhibit the intestinal absorption of glucose, thereby achieving the effect of lowering blood sugar. Fig.17It can be seen that the inhibition rates of RLE, RLP-1, and RLP-2 on α-amylase are 11.66%, 17.63%, and 27.78%, respectively, and the inhibition rates on α-glucosidase are 17.26%, 30.71%, and 31.04%, respectively, indicating that the biological activity of the samples was significantly improved after purification, and RLP-2 had the best inhibitory activity on amylase and glucosidase, which may be due to the increase in the biological activity caused by the increase in the TPC value of phenols after purification.

[0170] In summary, the present invention uses raspberry leaves as raw materials, and based on the results of single factor and response surface experiments, combines three extraction methods of ultrasound, high-speed homogenization and hot steam explosion to obtain the best extraction process. It is found that the TPC content obtained by SEHU and SEUH is 136.30±6.08mg / g~140.51±1.67mg / g, which is higher than that under single conditions and other combined processes, indicating that SE pretreatment efficiently destroys the internal structure of the plant, so that the subsequent U+H can better release the phenolic compounds in the plant. In addition, the present invention optimizes the purification process of phenolic compounds, and obtains that the medium-polarity XDA-6 macroporous resin is the best resin. After two purifications, the TPC values ​​of RLP-1 and RLP-2 are 738.98 mg / g and 905.27 mg / g, respectively. Among them, the highest content of Q3GR reaches 134.77 mg / g, and the contents of K3R, proanthocyanidin B1, and ellagic acid are also significantly increased, indicating that the process is suitable for purifying phenolic compounds in raspberry leaves and can obtain a purified product with a high TPC. Finally, the present invention tests the types of phenolic compounds in the extract and the purified product and the biological activity, and finds that after purification, the IC values ​​of the DPPH clearance rates of RLP-1 and RLP-2 are 50 The values ​​were reduced to 33.99 μg / mL and 20.63 μg / mL, respectively, and the IC 50 The values ​​were reduced to 7.81 μg / mL and 5.98 μg / mL, respectively, and the inhibition of amylase and glucosidase activities was improved compared with RLE, indicating that the purification treatment improved the activity of the sample.

[0171] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting crude extracts of phenolic substances from raspberry leaves, characterized in that: The following steps are involved: The raspberry leaf powder and the ethanol solution are mixed for composite extraction to obtain a crude extract of phenolic substances; the composite extraction method includes ultrasound, homogenization and steam explosion; the volume fraction of the ethanol solution is 40-80%; The composite extraction comprises steam explosion, ultrasound and homogenization in sequence, or comprises steam explosion, homogenization and ultrasound in sequence; In the composite extraction, the operating parameters of ultrasound include: power of 120-360 W, frequency of 40-80 Hz, temperature of 60-80° C., time of 30-60 min, and liquid-to-solid ratio of 20-40 mL / g; the operating parameters of homogenization include: rotation speed of 3000-7000 r / min, time of 0.5-1 min, and liquid-to-solid ratio of 20-40 mL / g; the operating parameters of steam explosion include: temperature of 105-125° C., time of 5-25 min, and liquid-to-solid ratio of 8-40 mL / g.

2. A method for purifying a crude extract of phenolic substances from raspberry leaves, characterized in that: The following steps are involved: Extracting raspberry leaves according to the method of claim 1 to obtain a crude extract of phenolic substances; dissolving the crude extract of the phenolic substance in water to obtain a crude extract solution; The crude extract solution is purified by column chromatography to obtain a purified product of phenolic substances; the resin used in the column chromatography is a macroporous resin, and the eluent used is an ethanol solution; The macroporous resin is XDA-6; The column chromatography includes sequential adsorption and desorption, and the operating parameters of the adsorption include: the concentration of total phenol in the crude extract solution is 0.1-2.5 mg / mL, the pH value of the crude extract solution is 1-6, the sample flow rate is 2-4 BV / h, and the sample volume is 18-20 BV; The desorption operation parameters include: the volume fraction of the ethanol solution is 40-80%, the flow rate is 4-6 BV / h, and the amount of the ethanol solution is 3-5 BV.

3. The purification method according to claim 2, characterized in that The purification is performed 1 to 2 times.

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