A total flavonoid extract from the leaves of *Hibiscus mutabilis*, its extraction method and application

Abstract

This invention relates to the field of flavonoid extract technology, specifically disclosing a total flavonoid extract from *Hibiscus mutabilis* leaves, its extraction method, and its applications. *Hibiscus mutabilis* leaf powder is added to disodium hydrogen phosphate-citric acid buffer solution, and a complex enzyme is added for enzymatic hydrolysis to obtain an enzymatic hydrolysate. The complex enzyme includes cellulase and xylanase. Ethanol is added to the enzymatic hydrolysate for ultrasonic extraction to obtain a first flavonoid extract. Ammonium sulfate is added to the first flavonoid extract for a first extraction, and the mixture is allowed to stand and separate into layers. The upper layer is collected to obtain a second flavonoid extract. The second flavonoid extract is desalted and concentrated to obtain the total flavonoid extract from *Hibiscus mutabilis* leaves. This invention employs a combination of enzymatic hydrolysis, ultrasonic extraction, and aqueous two-phase extraction. Through synergistic effects, it precisely extracts flavonoid components, improves the selectivity of aqueous two-phase extraction, effectively enhances the purity of the product, and increases the total flavonoid content in the extract. Furthermore, the extraction process is green and efficient, possessing high market application value.

Description

Technical Field

[0001] This invention relates to the field of flavonoid extract technology, and in particular to a total flavonoid extract from the leaves of *Hibiscus mutabilis*, its extraction method, and its application. Background Technology

[0002] Nature is rich in polyphenolic chemicals. Flavonoids, in particular, refer to a group of compounds with a C6-C3-C6 structure, consisting of two benzene rings linked by three carbon atoms. They exhibit diversity and complexity. Many flavonoids have medicinal value, used to prevent and treat cardiovascular diseases. For example, they can reduce vascular fragility, improve vascular permeability, lower blood lipids and cholesterol, and prevent hypertension, cerebral hemorrhage, coronary heart disease, angina pectoris, dilate coronary vessels, and increase coronary blood flow. Many flavonoid components possess antitussive, expectorant, antiasthmatic, and antibacterial activities, while also exhibiting hepatoprotective, hepatotoxic, antifungal, therapeutic, and antioxidant effects against acute and chronic hepatitis and cirrhosis. Furthermore, flavonoids possess effects similar to phytoestrogens. In livestock production, the application of flavonoids can significantly improve animal production performance, enhance disease resistance, and improve immune function. Flavonoids are widely distributed in the plant kingdom. Most exist within plants in the form of glycosides or glycosyl groups bound to sugars, while some exist in free form. *Hibiscus rosa-sinensis*, an annual or perennial herbaceous plant belonging to the Malvaceae family and the *Hibiscus* genus, has a flavonoid content that is among the highest reported in plants, dozens of times higher than that of commonly used flavonoid production raw materials such as soybeans and ginkgo.

[0003] Currently, the most common extraction method for flavonoids is using a 20%–80% ethanol-water solution. However, this method often results in low flavonoid content, leading to problems such as low efficiency, high energy consumption, environmental pollution, poor extract stability, low purity, and poor activity in practical formulations. Supercritical CO2 extraction is also available, but it involves high equipment costs, complex operation, and high risks. Therefore, there is an urgent need to develop a flavonoid extraction method that improves flavonoid quality, achieves high extraction efficiency, and is environmentally friendly. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a total flavonoid extract from *Hibiscus mutabilis* leaves, its extraction method, and its applications. High-quality flavonoid extracts are obtained through a combination of enzymatic extraction, ultrasonic ethanol extraction, and aqueous two-phase purification, exhibiting high extraction efficiency and being environmentally friendly.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for extracting total flavonoids from the leaves of *Hibiscus mutabilis*, comprising the following steps: S1, mix the powdered leaves of *Hibiscus mutabilis* with disodium hydrogen phosphate-citric acid buffer, add a complex enzyme for enzymatic hydrolysis, and obtain the hydrolysate; the complex enzyme includes cellulase and xylanase. S2, ethanol is added to the enzymatic hydrolysate for ultrasonic extraction to obtain the first flavonoid extract; S3, add ammonium sulfate to the first flavonoid extract for the first extraction, let stand and separate the layers, and take the upper layer to obtain the second flavonoid extract; S4, the second flavonoid extract is desalted and concentrated to obtain the total flavonoid extract of hibiscus leaves.

[0006] Compared to existing technologies, the extraction method for total flavonoids from *Hibiscus mutabilis* leaves provided by this invention uses disodium hydrogen phosphate-citric acid buffer as the solvent for enzymatic hydrolysis, which is beneficial for the process. The use of cellulase and xylanase as a combined enzyme for enzymatic hydrolysis results in good hydrolysis efficiency and increases the total flavonoid content in the hydrolysate. After enzymatic hydrolysis, ultrasonic extraction with ethanol is performed, which further increases the total flavonoid content in the extract and improves the extraction efficiency. Then, ammonium sulfate is used for aqueous two-phase extraction, which further increases the total flavonoid content in the first flavonoid extract. After desalting, the ethanol in the second flavonoid extract is removed by concentration to obtain a high-purity total flavonoid extract from *Hibiscus mutabilis* leaves.

[0007] This invention employs a combination of enzymatic hydrolysis, ultrasonic extraction, and aqueous two-phase extraction. Through synergistic effects, it enables precise extraction of flavonoids, improves the selectivity of aqueous two-phase extraction, effectively enhances product purity, and increases the total flavonoid content in the extract. Furthermore, the extraction process is green and efficient. The extraction method for total flavonoids from *Hibiscus mutabilis* leaves provided by this invention is carried out under mild reaction conditions, which helps maintain the original properties of flavonoids. The operation is simple and easy to implement, while reducing dependence on harmful solvents. This achieves a highly efficient production process with lower operating costs and has certain market application value.

[0008] Preferably, in S1, the particle size of the hibiscus leaf powder is ≤0.5mm.

[0009] Preferably, in S1, the mass-to-volume ratio of the *Hibiscus mutabilis* leaf powder, the disodium hydrogen phosphate-citric acid buffer solution, and the complex enzyme is 100g:(1~5)L:(1.2~6)g, and more preferably 100g:(3~5)L:(2~4.8)g.

[0010] Preferably, in S1, the pH of the disodium hydrogen phosphate-citric acid buffer solution is 3.5~5.5 (more preferably pH 4.5~5.5), and the concentrations of disodium hydrogen phosphate and citric acid are both 0.04M~0.06M.

[0011] Preferably, in S1, the mass ratio of the cellulase to the xylanase is 2:1 to 1:4.

[0012] Preferably, in S1, the enzymatic hydrolysis temperature is 35℃~45℃, and the enzymatic hydrolysis time is 1h~3h.

[0013] By limiting step S1, this invention can further improve the efficiency of enzymatic hydrolysis and increase the total flavonoid content in the hydrolysate. Simultaneously, the mild hydrolysis conditions help maintain the original properties of flavonoids and enhance the activity of flavonoids in the extract.

[0014] Preferably, in S2, the volume ratio of the enzymatic hydrolysate to the ethanol is 1:(0.5~1.5), more preferably 1:(0.75~1).

[0015] It should be noted that the ethanol refers to anhydrous ethanol. Through extensive experimentation, the inventors discovered that as the anhydrous ethanol content increases, more pigments and other impurities dissolve from the leaves, gradually negatively impacting the flavonoid content. When the volume ratio of the enzymatic hydrolysate to ethanol is 1:0.75, the first flavonoid extract exhibits a higher flavonoid content and the best flavonoid quality.

[0016] Preferably, in S2, the ultrasonic extraction power is 120W~240W (more preferably 150W~200W), and the extraction time is 5min~25min.

[0017] This invention further improves the efficiency of ultrasonic extraction by controlling the ratio of enzymatic hydrolysate to ethanol and the conditions of ultrasonic extraction, and significantly increases the total flavonoid content in the first flavonoid extract.

[0018] Preferably, in S2, the flavonoid content in the first flavonoid extract is ≥35mg / g.

[0019] Preferably, in S3, the mass concentration of ammonium sulfate in the first flavonoid extract after adding the ammonium sulfate is 17%~21%.

[0020] Preferably, in step S3, after standing and separating the layers, the process further includes: taking the lower layer after the first extraction, adding ethanol for a second extraction, standing and separating the layers, taking the upper layer and mixing it with the second flavonoid extract to obtain a third flavonoid extract; In step S4, the third flavonoid extract is desalted and concentrated to obtain the total flavonoid extract of *Hibiscus mutabilis* leaves.

[0021] More preferably, the volume ratio of the ethanol in S3 to the ethanol in S2 is (0.45~0.55):1.

[0022] This invention uses ethanol for a second extraction of the lower layer after the first extraction, which can further extract flavonoids from the leaves of *Hibiscus mutabilis*, thereby improving the content and quality of flavonoids in the total flavonoid extract of *Hibiscus mutabilis* leaves.

[0023] It should be noted that the lower layer after the second extraction in this invention can be mixed with the first flavonoid extract obtained by ultrasonic extraction in the next cycle for recycling.

[0024] Preferably, in step S4, the desalination temperature is 1°C to 6°C.

[0025] For example, in S4, the second flavonoid extract is desalted at low temperature in a refrigerator at 1°C to 6°C, then concentrated under reduced pressure and evaporated to dryness to obtain the total flavonoid extract of *Hibiscus mutabilis* leaves.

[0026] Secondly, the present invention provides a total flavonoid extract from the leaves of *Hibiscus mutabilis*, prepared by the extraction method described above.

[0027] Thirdly, the present invention provides the application of the total flavonoid extract of *Hibiscus mutabilis* leaves in the preparation of pharmaceuticals.

[0028] The total flavonoid extract of Hibiscus mutabilis leaves provided by this invention has a variety of biological activities such as anti-oxidation, antiviral, antitumor, anti-inflammatory, antibacterial, analgesic, lipid-lowering, and immunomodulatory effects, and has application prospects in pharmaceutical production. Attached Figure Description

[0029] Figure 1 This is a graph showing the stimulation index of mouse T lymphocytes by flavonoids from *Hibiscus mutabilis* leaves in this invention. Figure 2 This is a graph showing the stimulation index of mouse B lymphocytes by flavonoids from *Hibiscus mutabilis* leaves in this invention. In the figure, SI represents the stimulation index; different letters indicate significant differences, P < 0.05. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0031] This invention provides a method for extracting total flavonoids from the leaves of *Hibiscus mutabilis*, comprising the following steps: S1, mix the powdered leaves of *Hibiscus mutabilis* with disodium hydrogen phosphate-citric acid buffer, add a complex enzyme for enzymatic hydrolysis, and obtain the hydrolysate; the complex enzyme includes cellulase and xylanase. S2, ethanol is added to the enzymatic hydrolysate for ultrasonic extraction to obtain the first flavonoid extract; S3. Add ammonium sulfate to the first flavonoid extract for the first extraction, let it stand for layering, and obtain the first upper layer and the first lower layer. The first upper layer is the second flavonoid extract; S4. Take the first lower layer, add ethanol for the second extraction, let it stand for layering, and mix the obtained second upper layer with the second flavonoid extract (the first upper layer) to obtain the third flavonoid extract; S5. Desalt and concentrate the third flavonoid extract to obtain the total flavonoid extract from Abelmoschus manihot leaves.

[0032] In the embodiments of the present invention, if there is no special indication, the percentages all refer to mass percentages. In the embodiments of the present invention, the concentrations of disodium hydrogen phosphate and citric acid in the disodium hydrogen phosphate-citric acid buffer solution in S1 are both 0.04M to 0.06M, and the specific concentration is subject to the dosage and pH value; the ethanol in S4 is half of the volume of the ethanol in S2.

[0033] The mice used in the present invention are SPF-grade healthy male Balb / c mice, 4 to 8 weeks old, with a body weight of about 18 to 22 g. They are purchased from Hebei Medical University, and the production unit license number is SCXK(Hebei)2022-001. The animal experiments of the present invention have been reviewed and approved by the Experimental Animal Welfare Committee of Hebei University of Science and Technology, and the ethical review approval number is: 22322801D.

[0034] To better illustrate the present invention, further examples are given below through embodiments.

[0035] Example 1 This embodiment provides a method for extracting the total flavonoid extract from Abelmoschus manihot leaves, including the following steps: S1. Dry, crush the Abelmoschus manihot leaves, and pass through a 40-mesh sieve to obtain Abelmoschus manihot leaf powder; place the Abelmoschus manihot leaf powder in a conical flask, add the disodium hydrogen phosphate-citric acid buffer solution and a composite enzyme composed of cellulase and xylanase with a mass ratio of 1:3. The mass-volume ratio of the Abelmoschus manihot leaf powder, the disodium hydrogen phosphate-citric acid buffer solution and the composite enzyme is 100g:4L:2.4g, the pH of the disodium hydrogen phosphate-citric acid buffer solution is 5, and enzymolysis is carried out at 37.5°C for 2.5 h to obtain an enzymolysis solution.

[0036] S2. Add absolute ethanol to the enzymolysis solution, and the volume ratio of the enzymolysis solution to absolute ethanol is 1:0.75. Ultrasonic extraction is carried out at 180W for 15 min to obtain the first flavonoid extract.

[0037] S3. Add ammonium sulfate to the first flavonoid extract to make the mass concentration of ammonium sulfate in the mixed solution 20%, carry out the first extraction, let it stand for layering, and obtain the first upper layer (the second flavonoid extract) and the first lower layer.

[0038] S4, take the first lower layer, add anhydrous ethanol for a second extraction, let stand to separate the layers, take the obtained second upper layer and mix it with the first upper layer (second flavonoid extract) to obtain the third flavonoid extract.

[0039] S5, the third flavonoid extract was desalted in a refrigerator at 4°C, then concentrated under reduced pressure and evaporated to dryness to obtain the total flavonoid extract of Hibiscus mutabilis leaves.

[0040] To demonstrate the beneficial effects of aqueous two-phase extraction, the total flavonoid content in the first upper layer, first lower layer, second upper layer, and second lower layer of this embodiment was tested, and the recovery rate was calculated. The flavonoid content was determined using the NaNO2-Al(NO3)3-NaOH colorimetric method to plot a rutin standard curve, yielding the linear equation y = 6.5658x - 0.0028, R0. 2 =0.9984. Take 125 μL of flavonoid extract (sample for determination), add colorimetric reagent for color development, and measure the absorbance at a wavelength of 510 nm. Calculate the mass of flavonoids in the sample using a standard curve. Then, calculate the flavonoid content (and flavonoid recovery rate) of the sample according to the formula: Flavonoid content = Mass of flavonoids in the sample / Mass of *Hibiscus mutabilis* leaf powder. Repeat the calculation three times and calculate the average value. The test results are shown in Table 1.

[0041] Table 1. Results of total flavonoid content and recovery rate after aqueous two-phase extraction. As shown in Table 1, the flavonoid content in the first flavonoid extract was 45.64 mg / g. After adding ammonium sulfate to form an aqueous two-phase mixture, the total flavonoid content was 46.85 mg / g. The recovery rate of flavonoids after the first extraction was 66.81%, and the total recovery rate of flavonoids after the second extraction was 93.34%. This indicates that the two extractions can extract most of the flavonoids in the leaves of *Hibiscus mutabilis* to the upper layer.

[0042] Example 2 This embodiment provides a method for extracting total flavonoids from the leaves of *Hibiscus mutabilis*, including the following steps: S1. Dry and pulverize the leaves of *Hibiscus mutabilis*, and pass them through a 40-mesh sieve to obtain *Hibiscus mutabilis* leaf powder. Place the *Hibiscus mutabilis* leaf powder in an Erlenmeyer flask, add disodium hydrogen phosphate-citric acid buffer and a complex enzyme composed of cellulase and xylanase in a mass ratio of 1:2. The mass-volume ratio of *Hibiscus mutabilis* leaf powder, disodium hydrogen phosphate-citric acid buffer and complex enzyme is 100g:3L:3g. The pH of the disodium hydrogen phosphate-citric acid buffer is 4. Enzymatic hydrolysis is carried out at 40℃ for 2 hours to obtain the enzymatic hydrolysate.

[0043] S2, add anhydrous ethanol to the enzymatic hydrolysate, with a volume ratio of 1:1 between the enzymatic hydrolysate and anhydrous ethanol, and extract by ultrasonication at 150W for 10 min to obtain the first flavonoid extract with a content of 44.87 mg / g.

[0044] S3. Ammonium sulfate was added to the first flavonoid extract to make the mass concentration of ammonium sulfate in the mixture 19%, and the first extraction was performed. After standing and separating the layers, a first upper layer (second flavonoid extract) and a first lower layer were obtained. After testing, the total flavonoid content after the formation of the aqueous two-phase system was 46.06 mg / g, and the recovery rate of flavonoids after the first extraction was 63.2%.

[0045] S4, take the first lower layer, add anhydrous ethanol for a second extraction, let stand to separate the layers, take the obtained second upper layer and mix it with the first upper layer (second flavonoid extract) to obtain the third flavonoid extract.

[0046] S5, the third flavonoid extract was desalted in a refrigerator at 3°C, then concentrated under reduced pressure and evaporated to dryness to obtain the total flavonoid extract of Hibiscus mutabilis leaves.

[0047] Example 3 This embodiment provides a method for extracting total flavonoids from the leaves of *Hibiscus mutabilis*, including the following steps: S1. Dry and pulverize the leaves of *Hibiscus mutabilis*, and pass them through a 40-mesh sieve to obtain *Hibiscus mutabilis* leaf powder. Place the *Hibiscus mutabilis* leaf powder in an Erlenmeyer flask, add disodium hydrogen phosphate-citric acid buffer and a complex enzyme composed of cellulase and xylanase in a mass ratio of 1:1. The mass-volume ratio of *Hibiscus mutabilis* leaf powder, disodium hydrogen phosphate-citric acid buffer and complex enzyme is 100g:5L:3.8g. The pH of the disodium hydrogen phosphate-citric acid buffer is 4.5. Enzymatic hydrolysis is carried out at 42.5℃ for 1.5h to obtain the enzymatic hydrolysate.

[0048] S2, add anhydrous ethanol to the enzymatic hydrolysate, with a volume ratio of enzymatic hydrolysate to anhydrous ethanol of 1:1.5, and extract by ultrasonication at 210W for 20 min to obtain the first flavonoid extract with a content of 44.28 mg / g.

[0049] S3. Ammonium sulfate was added to the first flavonoid extract to make the mass concentration of ammonium sulfate in the mixture 21%, and the first extraction was performed. After standing and separating the layers, a first upper layer (second flavonoid extract) and a first lower layer were obtained. After testing, the total flavonoid content after the formation of the aqueous two-phase system was 45.34 mg / g, and the recovery rate of flavonoids after the first extraction was 61.0%.

[0050] S4, take the first lower layer, add anhydrous ethanol for a second extraction, let stand to separate the layers, take the obtained second upper layer and mix it with the first upper layer (second flavonoid extract) to obtain the third flavonoid extract.

[0051] S5, the third flavonoid extract was desalted in a refrigerator at 5°C, then concentrated under reduced pressure and evaporated to dryness to obtain the total flavonoid extract of Hibiscus mutabilis leaves.

[0052] Example 4 This embodiment provides a method for extracting total flavonoids from the leaves of *Hibiscus mutabilis*, including the following steps: S1. Dry and pulverize the leaves of *Hibiscus mutabilis*, then pass them through a 40-mesh sieve to obtain *Hibiscus mutabilis* leaf powder. Place the *Hibiscus mutabilis* leaf powder in an Erlenmeyer flask, add disodium hydrogen phosphate-citric acid buffer and a complex enzyme composed of cellulase and xylanase in a mass ratio of 2:1. The mass-volume ratio of *Hibiscus mutabilis* leaf powder, disodium hydrogen phosphate-citric acid buffer and complex enzyme is 100g:2.5L:1.2g. The pH of the disodium hydrogen phosphate-citric acid buffer is 5.5. Enzymatic hydrolysis is carried out at 45℃ for 1 hour to obtain the enzymatic hydrolysate.

[0053] S2, add anhydrous ethanol to the enzymatic hydrolysate, with a volume ratio of enzymatic hydrolysate to anhydrous ethanol of 1:1.25, and extract by ultrasonication at 120W for 25 min to obtain a first flavonoid extract with a content of 40.87 mg / g.

[0054] S3. Ammonium sulfate was added to the first flavonoid extract to make the mass concentration of ammonium sulfate in the mixture 18%, and the first extraction was performed. After standing and separating the layers, a first upper layer (second flavonoid extract) and a first lower layer were obtained. After testing, the total flavonoid content after the formation of the aqueous two-phase system was 42.93 mg / g, and the recovery rate of flavonoids after the first extraction was 61.4%.

[0055] S4, take the first lower layer, add anhydrous ethanol for a second extraction, let stand to separate the layers, take the obtained second upper layer and mix it with the first upper layer (second flavonoid extract) to obtain the third flavonoid extract.

[0056] S5, the third flavonoid extract was desalted in a refrigerator at 1°C, then concentrated under reduced pressure and evaporated to dryness to obtain the total flavonoid extract of Hibiscus mutabilis leaves.

[0057] Example 5 This embodiment provides a method for extracting total flavonoids from the leaves of *Hibiscus mutabilis*, including the following steps: S1. Dry and pulverize the leaves of *Hibiscus mutabilis*, and pass them through a 40-mesh sieve to obtain *Hibiscus mutabilis* leaf powder. Place the *Hibiscus mutabilis* leaf powder in an Erlenmeyer flask, add disodium hydrogen phosphate-citric acid buffer and a complex enzyme composed of cellulase and xylanase in a mass ratio of 1:3.5. The mass-volume ratio of *Hibiscus mutabilis* leaf powder, disodium hydrogen phosphate-citric acid buffer and complex enzyme is 100g:2L:5g. The pH of the disodium hydrogen phosphate-citric acid buffer is 3.5. Enzymatic hydrolysis is carried out at 35℃ for 3 hours to obtain the enzymatic hydrolysate.

[0058] S2, add anhydrous ethanol to the enzymatic hydrolysate, with a volume ratio of enzymatic hydrolysate to anhydrous ethanol of 1:0.5, and extract by ultrasonication at 240W for 5 min to obtain a first flavonoid extract with a content of 40.41 mg / g.

[0059] S3. Ammonium sulfate was added to the first flavonoid extract to make the mass concentration of ammonium sulfate in the mixture 17%, and the first extraction was performed. After standing and separating the layers, a first upper layer (second flavonoid extract) and a first lower layer were obtained. After testing, the total flavonoid content after the formation of the aqueous two-phase system was 41.57 mg / g, and the recovery rate of flavonoids after the first extraction was 62.3%.

[0060] S4, take the first lower layer, add anhydrous ethanol for a second extraction, let stand to separate the layers, take the obtained second upper layer and mix it with the first upper layer (second flavonoid extract) to obtain the third flavonoid extract.

[0061] S5, the third flavonoid extract was desalted in a refrigerator at 6°C, then concentrated under reduced pressure and evaporated to dryness to obtain the total flavonoid extract of Hibiscus mutabilis leaves.

[0062] Example 6 This embodiment provides a method for extracting total flavonoids from Hibiscus mutabilis leaves, similar to Example 1, except that in S1, the mass ratio of cellulase to xylan is replaced with 4:1. The remaining conditions are the same as in Example 1 and will not be repeated.

[0063] The test showed that the flavonoid content in the first flavonoid extract was 39.39 mg / g; after adding ammonium sulfate to form an aqueous two-phase mixture, the total flavonoid content was 40.65 mg / g, and the recovery rate of flavonoids after the first extraction was 60.1%.

[0064] Example 7 This embodiment provides a method for extracting total flavonoids from *Hibiscus mutabilis* leaves, similar to Example 1, except that in S1, the pH of the disodium hydrogen phosphate-citric acid buffer solution is 6.5. All other conditions are the same as in Example 1 and will not be repeated.

[0065] The test showed that the flavonoid content in the first flavonoid extract was 38.4 mg / g; after adding ammonium sulfate to form an aqueous two-phase mixture, the total flavonoid content was 39.59 mg / g, and the recovery rate of flavonoids after the first extraction was 60.7%.

[0066] Example 8 This embodiment provides a method for extracting total flavonoids from Hibiscus mutabilis leaves, similar to Example 1, except that in S1, the enzymatic hydrolysis temperature is replaced with 20°C. The remaining conditions are the same as in Example 1 and will not be repeated.

[0067] The test showed that the flavonoid content in the first flavonoid extract was 37.3 mg / g; after adding ammonium sulfate to form an aqueous two-phase solution, the total flavonoid content was 38.51 mg / g, and the recovery rate of flavonoids after the first extraction was 61.0%.

[0068] Example 9 This embodiment provides a method for extracting total flavonoids from Hibiscus mutabilis leaves, similar to Example 1, except that in S2, the volume ratio of enzymatic hydrolysate to anhydrous ethanol is replaced with 1:0.3. All other conditions are the same as in Example 1 and will not be repeated.

[0069] The test showed that the flavonoid content in the first flavonoid extract was 37.5 mg / g; after adding ammonium sulfate to form an aqueous two-phase solution, the total flavonoid content was 38.82 mg / g, and the recovery rate of flavonoids after the first extraction was 61.1%.

[0070] Example 10 This embodiment provides a method for extracting total flavonoids from *Hibiscus mutabilis* leaves, similar to Example 1, except that in S3, the mass concentration of ammonium sulfate in the first flavonoid extract (i.e., the mixed solution) after adding ammonium sulfate is 25%. The remaining conditions are the same as in Example 1 and will not be repeated.

[0071] The test showed that the flavonoid content in the first flavonoid extract was 36.9 mg / g; after adding ammonium sulfate to form an aqueous two-phase solution, the total flavonoid content was 38.03 mg / g, and the recovery rate of flavonoids after the first extraction was 60.8%.

[0072] Comparative Example 1 This comparative example provides a method for extracting total flavonoids from the leaves of *Hibiscus mutabilis*, employing an ethanol ultrasonic extraction followed by an aqueous two-phase extraction method, including the following steps: S1. Dry the leaves of *Hibiscus mutabilis*, pulverize them, and pass them through a 40-mesh sieve to obtain *Hibiscus mutabilis* leaf powder. Add the *Hibiscus mutabilis* leaf powder to a 75% ethanol aqueous solution with a mass-to-volume ratio of 100g:4L. Extract the powder by ultrasonication at 180W for 15min to obtain the first flavonoid extract.

[0073] S2~S4 are the same as S3~S5 in Example 1, and will not be described again.

[0074] Comparative Example 2 This comparative example provides a method for extracting total flavonoids from the leaves of *Hibiscus mutabilis*, employing an enzymatic hydrolysis + aqueous two-phase extraction method, including the following steps: S1 is the same as S1 in Example 1, and will not be described again.

[0075] S2, add ammonium sulfate to the enzymatic hydrolysate to make the mass concentration of ammonium sulfate in the mixture 20%, perform the first extraction, let stand and separate the layers to obtain the first upper layer and the first lower layer.

[0076] S3~S4 are the same as S4~S5 in Example 1, and will not be described again.

[0077] Comparative Example 3 This comparative example provides a method for extracting total flavonoids from the leaves of *Hibiscus mutabilis*, employing an aqueous two-phase extraction method, including the following steps: S1. Dry the leaves of *Hibiscus mutabilis*, pulverize them, and pass them through a 40-mesh sieve to obtain *Hibiscus mutabilis* leaf powder. Add ammonium sulfate to the *Hibiscus mutabilis* leaf powder to make the mass concentration of ammonium sulfate in the mixture 20%, perform the first extraction, and allow it to stand to separate into layers to obtain the first upper layer and the first lower layer.

[0078] S2~S3 are the same as S4~S5 in Example 1, and will not be described again.

[0079] Comparative Example 4 This comparative example provides a method for extracting total flavonoids from the leaves of *Hibiscus mutabilis* using an ethanol extraction method, comprising the following steps: S1. Dry the leaves of *Hibiscus mutabilis*, pulverize them, and pass them through a 40-mesh sieve to obtain *Hibiscus mutabilis* leaf powder. Add the *Hibiscus mutabilis* leaf powder to an 80% ethanol aqueous solution at a mass-to-volume ratio of 100g:4L, and extract at 80℃ for 2 hours to obtain a flavonoid extract.

[0080] S2, the flavonoid extract was subjected to low-temperature desalting, vacuum concentration, and rotary evaporation to dryness to obtain the total flavonoid extract of Hibiscus mutabilis leaves.

[0081] Comparative Example 5 This comparative example provides a method for extracting total flavonoids from *Hibiscus mutabilis* leaves, similar to Example 1, except that in S1, xylanase is replaced with an equal mass of pectinase, i.e., the complex enzyme consists of cellulase and pectinase in a mass ratio of 1:3. All other conditions are the same as in Example 1 and will not be repeated.

[0082] Comparative Example 6 This comparative example provides a method for extracting total flavonoids from *Hibiscus mutabilis* leaves, similar to Example 1, except that in S1, xylanase is replaced with an equal mass of cellulase, i.e., only cellulase is used for enzymatic hydrolysis. The remaining conditions are the same as in Example 1 and will not be repeated.

[0083] Comparative Example 7 This comparative example provides a method for extracting total flavonoids from *Hibiscus mutabilis* leaves, similar to Example 1, except that in S3, ammonium sulfate is replaced with an equal mass of dipotassium hydrogen phosphate. The remaining conditions are the same as in Example 1 and will not be repeated.

[0084] It should be noted that this invention also replaced ammonium sulfate with anhydrous sodium carbonate, potassium dihydrogen phosphate, and sodium chloride, respectively, and the results are shown in Table 2. In the aqueous two-phase system containing ethanol and sodium carbonate, due to the strong alkalinity, flavonoids are easily destroyed under alkaline conditions; furthermore, in the aqueous two-phase system containing ethanol and sodium carbonate, the mass fractions of ethanol and inorganic salts are relatively small during separation, which is not conducive to the extraction of flavonoids. As can be seen from Table 2, the aqueous two-phase system containing ethanol-K₂HPO₄ does not selectively distribute flavonoids; ammonium sulfate has low solubility in ethanol and a strong ability to compete with ethanol for water molecules, achieving a good separation effect.

[0085] Table 2. Total flavonoid extracts from *Hibiscus mutabilis* leaves (Examples and Comparative Examples) Ingredient and activity identification 1. Component identification The total flavonoid extracts of *Hibiscus mutabilis* leaves prepared in Examples 1-10 were analyzed by UPLC-Orbitrap-MS, yielding a total of 50 flavonoid compounds (see Table 3). The main components were rutin, myricetin, quercetin, quercetin 3-O-glucoside, and hyperoside. Four new flavonoid compounds, namely genistein, geraniol, sagerol, and (+)-demethoxygeranium pelargonidin, were also identified for the first time.

[0086] The UPLC-Orbitrap-MS method was performed using a Thermo Vanquish (Thermo Fisher Scientific, USA) ultra-high performance liquid chromatography system, an ACQUITY UPLC HSS T3 column, a flow rate of 0.3 mL / min, a column temperature of 40 °C, and an injection volume of 2 μL. The mobile phase in positive ion mode was 0.1% formic acid acetonitrile (B2) and 0.1% formic acid water (A2), and the gradient elution program was as follows: 0~1min, 8% B2; From 1 min to 8 min, B2 content increased from 8% to 98%. 8-10 minutes, 98% B2; 10min~10.1min, 98%→8% B2; 10.1 min ~ 12 min, 8% B2.

[0087] The mobile phase in negative ion mode was acetonitrile (B3) and 5 mM ammonium formate aqueous solution (A3), and the gradient elution program was as follows: 0~1min, 8% B3; From 1 min to 8 min, 8% → 98% B3; 8-10 minutes, 98% B3; 10min~10.1min, 98%→8% B3; 10.1 min ~ 12 min, 8% B3.

[0088] Table 3 50 Flavonoids 2. Antioxidant activity identification DPPH free radical scavenging capacity determination: The total flavonoid extract of *Hibiscus mutabilis* leaves (Example 1) was dissolved in 60% ethanol aqueous solution to prepare sample solutions with mass concentrations of 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL, respectively. Vitamin C solution was used as a positive control, with mass concentrations of 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL, respectively. A certain amount of DPPH was weighed and prepared into a 0.2 mmol / L DPPH solution using 95% ethanol aqueous solution. 0.4 mL of each sample solution of different concentrations was added to 0.4 mL of the DPPH solution, mixed well, and incubated at room temperature in the dark for 30 min. The absorbance was then measured at 517 nm. The test results are shown in Table 4.

[0089] DPPH radical scavenging rate = [1 - (A1 - A2) / A0] × 100%; In the formula, the absorbance of the sample solution and the DPPH mixture is A1, the absorbance of the 95% ethanol aqueous solution and the sample mixture is A2, and the absorbance of DPPH and water is A0.

[0090] Table 4. Antioxidant activity results of total flavonoid extracts from *Hibiscus mutabilis* leaves in the examples and comparative examples. Note: Different letters indicate significant differences, P < 0.05.

[0091] 3. Immunomodulatory effects Using RPMI-1640 medium as the solvent, a solution with a mass concentration of 1×10⁻⁶ was prepared. 5 The total flavonoid extract of *Hibiscus mutabilis* leaves (Example 1) was diluted to prepare sample solutions of 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. Spleen cells were extracted from healthy, well-proportioned male Balb / c mice, ensuring a cell viability of over 95%, and the cell concentration was adjusted to 2 × 10⁻⁶.6 Cells / mL. Cell suspension was accurately aliquoted into 96-well cell culture plates, with 5 replicates per group. Experimental groups received 100 μL cell suspension, 50 μL of sample solutions at different concentrations, 20 μL of concanavalin A (ConA, final concentration 5 μg / mL) or 20 μL of lipopolysaccharide (LPS, final concentration 10 μg / mL), and 30 μL of RPMI-1640 complete medium. Control groups received 100 μL cell suspension, 50 μL of RPMI-1640 medium, 20 μL of ConA, and 30 μL of RPMI-1640 complete medium. The 96-well plates were incubated at 37.5℃ and 5% CO2 for 44 h. After 44 h, 10 μL of CCK-8 was added to each well, and the plates were incubated for another 2-4 h. The absorbance (OD) at 450 nm was measured using a microplate reader as an indicator of mouse splenic lymphocyte proliferation. The test results are shown below. Figures 1-2 As shown. The formula for calculating the Stimulus Index (SI) is as follows: As shown in Table 4, the antioxidant activity of the total flavonoid extract from *Hibiscus mutabilis* leaves gradually increased with the increase of flavonoid yield. Figures 1-2 As can be seen, with the increase of the mass concentration of the total flavonoid extract of Hibiscus mutabilis leaves, the stimulation index of the total flavonoid extract of Hibiscus mutabilis leaves on T lymphocytes and B lymphocytes increases. When the mass concentration of the total flavonoid extract of Hibiscus mutabilis leaves reaches 100 μg / mL, the stimulation index can reach 6.66 and 6.67, respectively, indicating that the total flavonoid extract of Hibiscus mutabilis leaves has the effect of improving immunity.

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting total flavonoids from the leaves of *Hibiscus mutabilis*, characterized in that, Includes the following steps: S1, mix the hibiscus leaf powder with disodium hydrogen phosphate-citric acid buffer, add a compound enzyme for enzymatic hydrolysis, and obtain the enzymatic hydrolysate; the compound enzyme is composed of cellulase and xylanase in a mass ratio of 2:1 to 1:4; the mass-volume ratio of the hibiscus leaf powder, the disodium hydrogen phosphate-citric acid buffer and the compound enzyme is 100g:(1~5)L:(1.2~6)g; S2, ethanol is added to the enzymatic hydrolysate for ultrasonic extraction to obtain a first flavonoid extract; the volume ratio of the enzymatic hydrolysate to the ethanol is 1:(0.5~1.5); the power of the ultrasonic extraction is 120W~240W; S3, add ammonium sulfate to the first flavonoid extract for a first extraction, allow it to stand and separate into layers, take the upper layer to obtain the second flavonoid extract; the mass concentration of ammonium sulfate in the first flavonoid extract after adding the ammonium sulfate is 17%~21% S4, the second flavonoid extract is desalted and concentrated to obtain the total flavonoid extract of Hibiscus mutabilis leaves.

2. The extraction method for total flavonoids from *Hibiscus mutabilis* leaves as described in claim 1, characterized in that, In S3, after standing and separating the layers, the process further includes: taking the lower layer after the first extraction, adding ethanol for a second extraction, standing and separating the layers, taking the upper layer and mixing it with the second flavonoid extract to obtain a third flavonoid extract. In step S4, the third flavonoid extract is desalted and concentrated to obtain the total flavonoid extract of *Hibiscus mutabilis* leaves.

3. The extraction method for total flavonoids from *Hibiscus mutabilis* leaves as described in claim 1 or 2, characterized in that, In S1, the pH of the disodium hydrogen phosphate-citric acid buffer solution is 3.5~5.5, and the concentrations of disodium hydrogen phosphate and citric acid are both 0.04M~0.06M.

4. The extraction method for total flavonoids from *Hibiscus mutabilis* leaves as described in claim 1 or 2, characterized in that, In S1, the enzymatic hydrolysis temperature is 35℃~45℃, and the enzymatic hydrolysis time is 1h~3h.

5. The extraction method for total flavonoids from *Hibiscus mutabilis* leaves as described in claim 1 or 2, characterized in that, In S2, the ultrasonic extraction time is 5 min to 25 min.

6. The extraction method for total flavonoids from *Hibiscus mutabilis* leaves as described in claim 2, characterized in that, The volume ratio of the ethanol in S3 to the ethanol in S2 is (0.45~0.55):1; and / or In S4, the desalination temperature is 1℃~6℃.

7. A total flavonoid extract from the leaves of *Hibiscus mutabilis*, characterized in that, It was prepared by the extraction method of total flavonoids from the leaves of *Hibiscus mutabilis* as described in any one of claims 1 to 6.

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