Licorice residue extraction algae inhibitor and preparation method and application thereof

Abstract

The application provides liquorice residue extraction algae inhibitor and a preparation method and application thereof, and belongs to the field of algae inhibitors. The preparation method comprises the following steps: mixing liquorice residue and ethyl acetate solution, water bath, filtering to obtain filtrate 1 and residue 1; drying the filtrate 1 to obtain total flavone 1; mixing the residue 1 with water, water bath, filtering to obtain filtrate 2 and residue 2; mixing the filtrate 2 with ethanol solution, standing for 12-24 hours in a 4 DEG C environment, and filtering; precipitating and drying to obtain polysaccharide 1; or diluting the liquorice residue with water, placing in a water bath for heat preservation, filtering to obtain filtrate 3 and residue 3; adding ethanol to the filtrate 3, standing in a 3-5 DEG C environment, filtering after 12-24 hours, and precipitating and drying to obtain polysaccharide 2; adding ethyl acetate solution to the residue 3, water bath, filtering to obtain residue 4 and filtrate 4; and drying the filtrate 4 to obtain total flavone 2. The liquorice residue extraction algae inhibitor prepared by the method can effectively inhibit the growth of algae, and solves the problem of liquorice residue waste.

Description

Technical Field

[0001] This invention belongs to the technical field of algae inhibitors, and particularly relates to algae inhibitors extracted from licorice residue, their preparation methods, and applications. Background Technology

[0002] Licorice is one of the oldest and most commonly used herbs in Traditional Chinese Medicine (TCM), widely used in prescriptions and often referred to as the "King of Herbs." It is listed as a superior-grade herb in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and has been known since ancient times as a herb in nine out of ten prescriptions. Besides its clinical applications, licorice also has wide uses in the food and cosmetic industries.

[0003] Studies have shown that licorice has nine main pharmacological activities: antitumor, anti-inflammatory, antiviral, immunomodulatory, antibacterial, antidiabetic, hepatoprotective, adrenocorticotropic hormone-like function, memory enhancement, and neuroprotective effects. Secondary metabolites in licorice related to these pharmacological activities include triterpenoids, saponins, flavonoids, and phenolic compounds.

[0004] Previous studies revealed that the licorice residue was the largest remaining component in the preparation of licorice extract. Further research showed that this residue had no anti-algae effect and had to be discarded, resulting in resource waste. Therefore, further exploration of the uses of licorice residue is needed to reduce waste and achieve resource utilization. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an algae inhibitor extracted from licorice residue and its preparation method. The algae inhibitor extracted from licorice residue prepared by this method can effectively inhibit the growth of algae and realize the reuse of licorice residue.

[0006] This invention provides a licorice residue extract that inhibits the growth of Microcystis aeruginosa, the extract comprising: licorice residue polysaccharide extract or licorice residue flavonoid extract.

[0007] Preferably, the effective concentration of the licorice residue polysaccharide extract is 0.02% to 0.07%.

[0008] Preferably, the effective concentration of the flavonoid extract from licorice residue is 0.02% to 0.07%.

[0009] Preferably, the licorice residue polysaccharide extract and the licorice residue flavonoid extract are used separately.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0011] a. Mix licorice residue with ethyl acetate solution at a volume ratio of 1:5-15, bathe in a water bath at 50-70℃ for 45-75 minutes, filter, and obtain filtrate 1 and filter residue 1;

[0012] b. Dry filtrate 1 to constant weight to obtain total flavonoids 1;

[0013] c. Mix filter residue 1 with 5 to 10 times its volume of water, bathe in a water bath at 50 to 70°C for 20 to 40 minutes, filter, and obtain filtrate 2 and filter residue 2;

[0014] d. Mix filtrate 2 with 3 to 5 times its volume of 90% to 100% ethanol solution, let stand at 3 to 5°C for 12 to 24 hours, filter, and collect the precipitate;

[0015] The precipitate was dried to obtain polysaccharide 1;

[0016] Or a'. Mix the licorice residue with 5 to 15 times its volume of water, bathe in a water bath at 50 to 70°C for 20 to 40 minutes, filter, and obtain filtrate 3 and residue 3;

[0017] b'. Add 3 to 5 times the volume of 90 to 100% ethanol to filtrate 3, let stand at 3 to 5°C for 12 to 24 hours, filter and dry the precipitate to obtain polysaccharide 2;

[0018] c'. Add 5 to 15 times the volume of ethyl acetate solution to the filter residue 3, place it in a water bath at 50 to 70°C for 40 to 80 minutes, filter, and obtain filter residue 4 and filtrate 4;

[0019] d'. Dry filtrate 4 to constant weight to obtain total flavonoids 2.

[0020] Preferably, the ethyl acetate solution is an ethanol solution of ethyl acetate, wherein the volume ratio of ethyl acetate to ethanol is 4 to 5:1.

[0021] This invention provides the application of licorice residue extract in inhibiting the growth of Microcystis aeruginosa.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention further investigated the inhibitory effect of licorice residue on Microcystis aeruginosa, and found that licorice residue alone had no or low inhibitory effect on the growth of Microcystis aeruginosa. Through a large number of experiments, this invention extracted the components of licorice residue and found that the polysaccharides and flavonoids of licorice residue alone have a good algae-inhibiting effect, realizing the reuse of licorice residue and solving the problem of excessive licorice residue and resource waste. Attached Figure Description

[0024] Figure 1 Separation of algae-inhibiting components from licorice residue extract (extract);

[0025] Figure 2 The effect of licorice powder and licorice residue on the growth of Microcystis aeruginosa was investigated (0.1 g of licorice powder and licorice residue were added to 50 ml of algal cells (initial cell density was 1.41 × 10⁻⁶).6 cells ml -1 (, count continuously for 7 days);

[0026] Figure 3 Effects of flavonoids and polysaccharides in licorice residue on the inhibition rate of Microcystis aeruginosa and pH (7 days) (mean ± standard deviation);

[0027] Figure 4 Effects of flavonoids and polysaccharides in licorice residue on the inhibition rate of Pseudomonas aeruginosa microcapsules (14 days) (mean ± standard deviation). Detailed Implementation

[0028] This invention provides a licorice residue extract that inhibits the growth of Microcystis aeruginosa.

[0029] In this invention, the licorice residue polysaccharide extract or licorice residue flavonoid extract is used. When the licorice residue extract is licorice residue polysaccharide extract, the preferred concentration is 0.06-0.07%, and when the licorice residue extract is licorice residue flavonoid extract, the preferred concentration is 0.05-0.07%.

[0030] This invention also provides a method for extracting licorice residue extract, the method being as follows:

[0031] a. Mix licorice residue with ethyl acetate solution at a volume ratio of 1:5-15, bathe in a water bath at 50-70℃ for 45-75 minutes, filter, and obtain filtrate 1 and filter residue 1;

[0032] b. Dry filtrate 1 to constant weight to obtain total flavonoids 1;

[0033] c. Mix filter residue 1 with 5 to 10 times its volume of water, bathe in a water bath at 50 to 70°C for 20 to 40 minutes, filter, and obtain filtrate 2 and filter residue 2;

[0034] d. Mix filtrate 2 with 3 to 5 times its volume of 90% to 100% ethanol solution, let stand at 3 to 5°C for 12 to 24 hours, filter, and collect the precipitate;

[0035] e. The precipitate was dried to obtain polysaccharide 1.

[0036] In this invention, licorice residue and ethyl acetate solution are mixed at a volume ratio of 1:5 to 15, bathed in a water bath at 50 to 70°C for 45 to 75 minutes, and then filtered to obtain filtrate 1 and filter residue 1. In this invention, the volume ratio of licorice residue to ethyl acetate solution is preferably 1:5 to 15, more preferably 1:10; the water bath temperature is preferably 50 to 70°C, more preferably 60°C; the water bath time is preferably 50 to 70 minutes, more preferably 60 minutes; the filtration method is vacuum filtration; the ethyl acetate solution is an ethanol solution of ethyl acetate, wherein the volume ratio of ethyl acetate to ethanol is preferably 4 to 5:1, more preferably 4:1.

[0037] After obtaining filtrate 1 in this invention, filtrate 1 is dried to constant weight to obtain total flavonoids 1; in this invention, the drying temperature is 38-42°C, preferably 40°C; in this invention, the total flavonoids 1 is the aforementioned licorice residue flavonoid extract.

[0038] In this invention, the obtained filter residue 1 is mixed with water at a volume ratio of 1:5 to 15, bathed in a water bath at 50 to 70°C for 20 to 40 minutes, and then filtered to obtain filtrate 2 and filter residue 2. In this invention, the volume ratio of filter residue 1 to water is preferably 1:5 to 15, more preferably 1:10; the water bath temperature is preferably 50 to 70°C, more preferably 60°C; the water bath time is preferably 20 to 40 minutes, more preferably 30 minutes; and the filtration method is vacuum filtration.

[0039] After obtaining filtrate 2 in this invention, filtrate 2 is mixed with an ethanol solution at a volume ratio of 1:3 to 5, allowed to stand at 3 to 5°C for 12 to 24 hours, filtered, and dried to constant weight to obtain polysaccharide 1. In this invention, the volume ratio of filtrate 2 to ethanol solution is preferably 1:3 to 5, more preferably 1:4; the ethanol solution is an aqueous solution of ethanol, wherein the ethanol content is preferably 90% to 100%, more preferably 95%; the standing temperature is preferably 3 to 5°C, more preferably 4°C; the filtration method is vacuum filtration; in this invention, the drying temperature is 34 to 36°C, preferably 35°C. In this invention, polysaccharide 1 is the aforementioned licorice residue polysaccharide extract.

[0040] This invention also provides another method for extracting licorice residue extract, the steps of which are as follows:

[0041] a'. Mix the licorice residue with 5 to 15 times its volume of water, bathe in a water bath at 50 to 70°C for 20 to 40 minutes, filter, and obtain filtrate 3 and residue 3;

[0042] b'. Add 3 to 5 times the volume of 90 to 100% ethanol to filtrate 3, let stand at 3 to 5°C for 12 to 24 hours, filter and dry the precipitate to obtain polysaccharide 2;

[0043] c'. Add 5 to 15 times the volume of ethyl acetate solution to the filter residue 3, place it in a water bath at 50 to 70°C for 40 to 80 minutes, filter, and obtain filter residue 4 and filtrate 4;

[0044] d'. Dry filtrate 4 to constant weight to obtain total flavonoids 2.

[0045] In this invention, licorice residue and water are mixed at a volume ratio of 1:5 to 15, bathed in a water bath at 50 to 70°C for 20 to 40 minutes, and then filtered to obtain filtrate 3 and filter residue 3. In this invention, the preferred volume ratio of licorice residue to water is 1:5 to 15, more preferably 1:10; the preferred water bath temperature is 50 to 70°C, more preferably 60°C; the preferred heat treatment time is 20 to 40 minutes, more preferably 30 minutes; and the filtration method is vacuum filtration.

[0046] After obtaining filtrate 3 in this invention, filtrate 3 is mixed with an ethanol solution at a volume ratio of 1:3 to 5, allowed to stand at 3 to 5°C for 12 to 24 hours, filtered, and dried to constant weight to obtain polysaccharide 2. In this invention, the volume ratio of filtrate 3 to ethanol solution is preferably 1:3 to 5, more preferably 1:4; the ethanol solution is an aqueous solution of ethanol, wherein the ethanol content is preferably 90% to 100%, more preferably 95%; the standing temperature is preferably 3 to 5°C, more preferably 4°C; the filtration method is vacuum filtration; in this invention, the drying temperature is 34 to 36°C, preferably 35°C. In this invention, polysaccharide 2 is the aforementioned licorice residue polysaccharide extract.

[0047] After obtaining filter residue 3 in this invention, filter residue 3 is mixed with ethyl acetate solution at a volume ratio of 1:5 to 15, and then subjected to a water bath at 50 to 70°C for 45 to 75 minutes, followed by filtration to obtain filtrate 4 and filter residue 4. In this invention, the volume ratio of filter residue 3 to ethyl acetate solution is preferably 1:5 to 15, more preferably 1:10; the water bath temperature is preferably 50 to 70°C, more preferably 60°C; the water bath time is preferably 45 to 75 minutes, more preferably 60 minutes; in this invention, the ethyl acetate solution is an ethanol solution of ethyl acetate, wherein the volume ratio of ethyl acetate to ethanol is preferably 4 to 5:1, more preferably 4:1; the filtration method is vacuum filtration.

[0048] In this invention, the filtrate 4 is dried to constant weight to obtain total flavonoids 2; the total flavonoids 2 is the aforementioned licorice residue flavonoid extract.

[0049] The present invention also provides the application of the licorice residue extract in inhibiting the growth of Microcystis aeruginosa.

[0050] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1

[0052] Licorice residue: The precipitate produced during the filtration of licorice extract, which is dried to constant weight.

[0053] The licorice extract is prepared by boiling licorice powder and distilled water at a mass ratio of 1:20 for 90 minutes, filtering, and concentrating the filtrate to achieve a licorice powder to distilled water ratio of 1:10, denoted as 10% (w / v).

[0054] The formula for calculating the inhibition rate is:

[0055] Algal cell counting was performed under a microscope using a 0.1 ml counting chamber.

[0056] The inhibition rate (IR) is calculated using the following method.

[0057] IR (%) = (N0 - N S ) / N0×100

[0058] Where N0 = algal cell density of the control group (cells / ml) -1 Ns = Treatment algal cell density (cells / ml) -1 ).

[0059] A negative number indicates that the algae have not been suppressed, but have instead grown in large numbers.

[0060] Licorice residue was mixed with ethyl acetate solution (the volume ratio of ethyl acetate to ethanol was 1:10, 4:1), and the mixture was heated in a water bath at 60°C for 60 minutes. After filtration, filtrate 1 and residue 1 were obtained. Filtrate 1 was dried to constant weight to obtain total flavonoids 1. Residue 1 was mixed with 10 times its volume of water, heated in a water bath at 60°C for 30 minutes, and then filtered to obtain filtrate 2 and residue 2. Filtrate 2 was mixed with 4 times its volume of 95% ethanol solution, allowed to stand at 4°C for 12 hours, and then filtered. The precipitate was dried to obtain polysaccharide 1.

[0061] Example 2

[0062] Licorice residue was soaked in 10 times its volume of water in a 60°C water bath for 30 minutes, then filtered to obtain filtrate 3 and residue 3. 4 times its volume of 95% ethanol was added to filtrate 3, and the mixture was allowed to stand at 4°C for 12 hours. After filtration, the precipitate was dried to obtain polysaccharide 2. 10 times its volume of ethyl acetate solution (ethyl acetate: ethanol = 4:1) was added to residue 3, and the mixture was kept in a 60°C water bath for 60 minutes. After filtration, residue 4 and filtrate 4 were obtained. Filtrate 4 was dried to constant weight to obtain total flavonoids 2.

[0063] Comparative Example 1

[0064] Licorice powder and distilled water were boiled at a mass ratio of 1:20 for 90 minutes, filtered, and the filtrate was concentrated to a licorice powder to distilled water ratio of 1:10, denoted as 10% (w / v). Figure 1 The method was used to separate the algae-inhibiting components in licorice residue: glycyrrhizic acid, alkaloids and flavonoids.

[0065] will be pressed Figure 1 Crude products of glycyrrhizic acid, alkaloids and flavonoids isolated from licorice extract and their production process. Figure 2 The effects of flavonoid 1, flavonoid 2, polysaccharide 1 and polysaccharide 2 obtained from licorice residue using two different methods on the growth of Microcystis aeruginosa were investigated.

[0066] As shown in Table 1, the inhibition rate of flavonoid 1 in licorice residue was consistently >90% after day 5, and the inhibition rate of polysaccharide 1 was consistently >90% after day 4. On day 7, the crude products with inhibition rates >90% included flavonoid 1, flavonoid 2, and polysaccharide 1, but there was no significant difference among the three (P>0.05).

[0067] Table 1. Effects of various active ingredients in licorice on the growth of Microcystis aeruginosa (inhibition rate %) (mean ± standard deviation)

[0068]

[0069] Polysaccharide 1 (the polysaccharide extracted after flavonoid extraction) showed a much better inhibitory effect on Microcystis aeruginosa than polysaccharide 2 (the polysaccharide directly extracted from licorice residue).

[0070] Although there was no statistically significant difference in the extraction of flavonoids from the filter residue whether it was done first or later (P>0.05), flavonoid 2 (the flavonoid extracted from the filter residue after the polysaccharide extraction) showed better inhibitory effects on Microcystis aeruginosa. The flavonoids in the licorice extract did not inhibit Microcystis aeruginosa as effectively as the flavonoid mixture in the filter residue.

[0071] The algae-inhibiting effects of flavonoid 1, flavonoid 2, and polysaccharide 1 in this invention are significantly higher than those of glycyrrhizic acid, alkaloids, and flavonoids in Comparative Example 1. The effects of polysaccharide 2 in this invention are not significantly different from those in Comparative Example 1. It can be seen that the algae-inhibiting effect of the licorice residue extract in this invention is equal to or better than that in Comparative Example 1, and good technical progress has been achieved.

[0072] Detection Example 1

[0073] Prepare licorice extract separately ( Figure 1 The yields of licorice extract and licorice residue were measured after three parallel experiments. The yields were (23.26±5.67)% and (57.57±2.99)% respectively (see Table 2 for details). This indicates that treating licorice residue as waste is a huge waste.

[0074] Each component of licorice extract was prepared separately and dried to constant weight. The content of each component in licorice extract was calculated. The results showed that glycyrrhizic acid was the most abundant component in licorice extract, followed by alkaloids, and then flavonoids (Table 2).

[0075] Table 2. Content of each component in licorice extract (mean ± standard deviation)

[0076]

[0077] Detection Example 2

[0078] Each component in the licorice residue was prepared separately, and the content of each component was calculated. The results showed that the recovery rates of flavonoids and polysaccharides obtained by the method of first extracting polysaccharides and then extracting flavonoids were higher than those obtained by the method of first extracting flavonoids and then extracting polysaccharides (Table 3).

[0079] Table 3 Recovery rates of active ingredients from licorice residue extracted by different extraction sequences (mean ± standard deviation)

[0080]

[0081] Detection Example 3

[0082] Weigh out 0.1g of licorice powder and 0.1g of licorice residue and add them to 50ml of algal cells (initial algal cell density is 1.41×10⁻⁶). 6 cells ml -1 ), counted continuously for 7 days. Results are shown in Figure 2 . Figure 2The results showed that licorice powder and licorice residue had completely different effects on algal cell growth (P<0.05). Licorice powder exhibited significantly better algal inhibition than licorice residue, showing inhibitory effects on days 1 and 3–7, while the licorice residue group only showed inhibition on days 1–2. The licorice powder group showed the highest inhibition rate (approximately 86%) on day 6, while the highest inhibition rate in the licorice residue group occurred on day 2, at only about 32%, suggesting that the licorice residue contained no effective algal inhibitory components.

[0083] Detection Example 4

[0084] Two-way ANOVA analysis and Tukey's Multiple Comparison Test were used to determine significant differences between the control and experimental groups. All statistical analyses were performed using Graphpadprism 7.0.

[0085] The results of the combined algae suppression experiment were calculated based on the formula of the intermediate-efficiency principle (Chou, 2006).

[0086] The equation for the intermediate efficiency principle is: fa / fu=(D / Dm) m , log[(fa) -1 -1] -1 = m log(D) - m log(Dm) (or (D / Dm) = (fa / fu)) 1 / m ); where D is the drug dose, Dm is EC50, m is the kinetic series (m=1 represents a hyperbola, m<1 represents an sigmoid curve, m>1 represents a flat hyperbola), fa is the inhibition rate, and fu is the cell survival rate (fa+fu=1).

[0087] When the two drugs are used in combination, CI = (D)1 / (D) x )1+(D)2 / (D x )2; CI is the drug combination index (CI<1 indicates a synergistic effect when the two drugs are used together, CI=1 indicates an additive effect when the two drugs are used together, and CI>1 indicates an antagonistic effect when the two drugs are used together), (D)1 and (D)2 represent the individual doses of the two drugs required to produce the same effect when used together, respectively. x )1、(D x )2 represents the drug dosage required to produce effect x when the two drugs are used alone.

[0088] The formula for calculating DRI is: (DRI)1 = (D x )1 / (D)1,(DRI)2=(D x )2 / (D)2, and so on (DRI) n =(D x ) n / (D) n DRI stands for Dose Reduction Index. A higher DRI value indicates that a lower dose can be used to reduce toxic side effects for a given therapeutic effect.

[0089] To investigate the effects of various components in licorice filter residue, the dose-response parameters of flavonoids, polysaccharides, and filter residue on Microcystis aeruginosa were calculated based on the inhibition rate of each component at different doses and the intermediate-efficacy principle, as shown in Tables 4-5.

[0090] Table 4. Dose-response parameters of flavonoids, polysaccharides, and filter residue on Microcystis aeruginosa (6 days)

[0091]

[0092] D – Dosage of the drug;

[0093] Dm——EC50;

[0094] m — dynamic series, m = 1, <1 or >1 represent hyperbola, S-curve and flat hyperbola respectively;

[0095] r—correlation coefficient

[0096] Table 5. Combined index of algae inhibition by flavonoids, polysaccharides and filter residue.

[0097]

[0098] Note: CI < 1, synergistic effect; CI > 1, antagonistic effect; CI = 1.0, additive effect.

[0099] Flavonoid 1, polysaccharide 1, and the residue after removing the first two (residue 2 after polysaccharide extraction) from licorice filter residue were added to algal cells in the logarithmic growth phase. The final concentrations of flavonoids in the algal solution were 0.02%, 0.03%, 0.04%, 0.05%, and 0.06%, respectively; the final concentrations of polysaccharide 1 were 0.02%, 0.03%, 0.04%, and 0.05%, respectively; and the final concentrations of residue 2 were 0.03%, 0.04%, 0.05%, and 0.06%, respectively. Microcystis aeruginosa were counted and EC50 was calculated after 6 days.

[0100] As shown in Table 5, the CI values ​​of flavonoids combined with polysaccharides and flavonoids combined with filter residue are all >3, indicating antagonistic effects. The CI value of polysaccharides combined with filter residue is around 1, indicating an approximately additive effect. The CI value of flavonoids, polysaccharides, and filter residue combined is also >3 (except for the group with 0.030% flavonoids and 0.030% filter residue), therefore, the three groups have antagonistic effects. Figure 2 In this study, licorice residue did not show the same good algae-inhibiting effect as licorice powder.

[0101] Case 5

[0102] The flavonoids and polysaccharides from licorice residue were then added to the algal solution, resulting in an algal solution with an initial density of 4.7 × 10⁻⁶. 6 cells ml -1 The final concentrations of flavonoids and polysaccharides in the sample were 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, and 0.07%, respectively. Inhibition rate and pH were measured on day 7. Results are shown below. Figure 3 .

[0103] from Figure 3 As shown in Table 1, at the same concentration, the inhibitory effect of flavonoids on the growth of Microcystis aeruginosa was numerically better than that of polysaccharides, but there was no significant difference (P>0.05). The pH trend showed that when the concentration was higher than 0.06%, although the inhibitory effect of the flavonoid group on Microcystis aeruginosa was still better than that of the polysaccharide group, the pH value increased slightly. When the concentration was between 0.02% and 0.07%, the pH value difference between the flavonoid group and the polysaccharide group was very significant (P<0.01).

[0104] Case 6

[0105] from Figure 3 and Figure 4 It can be seen that there was no difference in the inhibitory effect of licorice flavonoids and licorice polysaccharides on Microcystis aeruginosa at 7 days (P>0.05). However, at 14 days, the inhibitory effect of the licorice polysaccharide group disappeared and the number of algal cells began to rebound, while the flavonoid group still inhibited the growth of algal cells. The differences between the flavonoid group and the polysaccharide group at each concentration point were very significant (P<0.01).

[0106] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of licorice residue extract in inhibiting the growth of Microcystis aeruginosa, characterized in that, The extract is: licorice residue polysaccharide or licorice residue flavonoid; The effective concentration of licorice residue polysaccharide is 0.02%~0.07%; The effective concentration of flavonoids from licorice residue is 0.02%~0.07%; Licorice residue polysaccharides and licorice residue flavonoids should be used separately. The extraction method for the licorice residue extract includes the following steps: a. Mix licorice residue with ethyl acetate solution at a volume ratio of 1:5~15, bathe in a water bath at 50~70℃ for 45~75 min, filter, and obtain filtrate 1 and filter residue 1; b. Dry filtrate 1 to constant weight to obtain total flavonoids 1; c. Mix filter residue 1 with 5 to 10 times its volume of water, bathe in a water bath at 50 to 70°C for 20 to 40 minutes, filter, and obtain filtrate 2 and filter residue 2; d. Mix filtrate 2 with 3 to 5 times its volume of 90% to 100% ethanol solution, let stand at 3 to 5°C for 12 to 24 hours, filter, and collect the precipitate; e. Dry the precipitate to obtain polysaccharide 1; Or a'. Mix the licorice residue with 5 to 15 times its volume of water, bathe in a water bath at 50 to 70°C for 20 to 40 minutes, filter, and obtain filtrate 3 and residue 3; b'. Add 3 to 5 times the volume of 90 to 100% ethanol to the filtrate 3, let it stand for 12 to 24 hours at 3 to 5°C, filter and dry the precipitate to obtain polysaccharide 2; c'. Add 5 to 15 times the volume of ethyl acetate solution to the filter residue 3, place it in a water bath at 50 to 70°C for 40 to 80 minutes, filter, and obtain filter residue 4 and filtrate 4; d'. Dry filtrate 4 to constant weight to obtain total flavonoids 2; The licorice residue is a precipitate produced during the filtration of licorice water extract; The licorice residue polysaccharide is polysaccharide 1; The flavonoids in the licorice residue are total flavonoid 1 or total flavonoid 2; The ethyl acetate solution is an ethanol solution of ethyl acetate, wherein the volume ratio of ethyl acetate to ethanol is 4~5:1.

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