Ultrasonic-assisted extraction method of dogwood extract containing ursolic acid

Abstract

The invention relates to the technical field of natural product extraction, in particular to an ultrasonic-assisted extraction method of a dogwood extract containing ursolic acid. The ultrasonic-assisted extraction method comprises the following steps: step 1, cleaning dried dogwood, removing stems and leaves, drying and grinding to obtain dried dogwood powder; step 2, adding the dried dogwood fruit powder into a solvent, carrying out ultrasonic treatment, centrifuging, and taking supernate, so as to obtain a dogwood extract containing ursolic acid; the solvent comprises ethanol and water. According to the method disclosed by the invention, the extraction rate of the dogwood extract containing ursolic acid can be obviously improved, the cost is low, large-scale application in the industry is facilitated, and important technical support and theoretical basis are provided for high-value development and utilization of dogwood resources.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction technology, and in particular to an ultrasound-assisted extraction method for Cornus officinalis extract containing ursolic acid. Background Technology

[0002] Ursolic acid (UA) is one of the main active components of Cornus officinalis, possessing various biological activities such as anti-inflammatory, antioxidant, and hypoglycemic effects, and has broad application prospects in functional foods and pharmaceuticals. Currently, the main extraction methods for ursolic acid include traditional solvent extraction, microwave-assisted extraction, and ultra-high pressure extraction. For example, Huang Minghui (2017) used microwave-assisted extraction to extract total pentacyclic triterpenoids from Cornus officinalis, achieving a yield of 34.11 mg / g; Lin Hai (2013) used ultra-high pressure extraction to extract ursolic acid, achieving a yield of 0.322%. Existing technologies suffer from low extraction efficiency, high energy consumption, and complex processes, especially regarding the selective extraction and process optimization of ursolic acid, which still have significant room for improvement. Although ultrasonic-assisted extraction has been used for natural product extraction, there are few studies on the systematic optimization of ursolic acid extraction from Cornus officinalis, particularly regarding the impact of multi-factor interactions on the extraction rate. Summary of the Invention

[0003] This invention aims to solve the problems of low extraction rate, unstable process, high energy consumption and complicated operation in existing methods for extracting ursolic acid from Cornus officinalis, and provides an efficient, stable and simple ultrasonic-assisted extraction method for Cornus officinalis extract containing ursolic acid.

[0004] To achieve the above objectives, the present invention provides the following solution: This invention provides an ultrasound-assisted extraction method for Cornus officinalis extract containing ursolic acid, comprising the following steps: Step 1: After washing, removing stems and leaves from the dried Cornus officinalis fruit, dry it and grind it to obtain Cornus officinalis fruit powder. Step 2: Add the dried fruit powder of Cornus officinalis to a solvent for ultrasonic treatment, centrifuge, and take the supernatant to obtain Cornus officinalis extract containing ursolic acid. The solvents include ethanol and water.

[0005] In a preferred embodiment of the present invention, in step 1, the drying temperature is 60 ℃ and the drying time is 12 h; after grinding, the material is passed through a 60-mesh sieve.

[0006] In a preferred embodiment of the present invention, in step 2, the solvent further includes ethyl acetate.

[0007] In a preferred embodiment of the present invention, in step 2, the solvent further includes ethyl acetate and glycerol.

[0008] In a preferred embodiment of the present invention, in step 2, the volume ratio of ethyl acetate, glycerol and water in the solvent is 4:1:1.

[0009] In a preferred embodiment of the present invention, in step 2, the volume concentration of ethanol in the solvent is 60% to 80%.

[0010] In a preferred embodiment of the present invention, in step 2, the ultrasonic power of the ultrasonic treatment is 200~300 W, the ultrasonic time is 40~60 min, and the ultrasonic temperature is 60~70 ℃.

[0011] In a preferred embodiment of the present invention, in step 2, the ratio of the dried fruit powder of Cornus officinalis to the solvent is 1:(20~40)g / mL.

[0012] In a preferred embodiment of the present invention, in step 2, the centrifugation conditions are set as follows: centrifugation at 4000 r / min for 15 min.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The method of this invention can significantly improve the extraction rate of Cornus officinalis extract containing ursolic acid. Furthermore, the method of this invention is low in cost and easy to apply on a large scale in industry, providing important technical support and theoretical basis for the high-value development and utilization of Cornus officinalis resources. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 Wavelength scanning results for water, blank control, Cornus officinalis test solution, and ursolic acid control solution.

[0016] Figure 2 For the linear standard curve of ursolic acid; ursolic acid standard curve: y =0.0107 x +0.0155, correlation coefficient R 2 =0.9994; where y is the absorbance value A; and x is the mass of ursolic acid, in μg.

[0017] Figure 3 The results are from a stability test (ursolic acid).

[0018] Figure 4The effect of different material-to-liquid ratios on the extraction rate (denoted as ursolic acid extraction rate) of Cornus officinalis extract containing ursolic acid.

[0019] Figure 5 The effect of different ethanol concentrations on the extraction rate of ursolic acid.

[0020] Figure 6 The effect of different ultrasonic powers on ursolic acid extraction rate.

[0021] Figure 7 The effect of different ultrasonic temperatures on the extraction rate of ursolic acid.

[0022] Figure 8 The effect of different ultrasound times on ursolic acid extraction rate.

[0023] Figure 9 The response surface and contour plot of the interaction of various factors on the ursolic acid extraction rate are shown. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] The method of the present invention has the following advantages: 1. High extraction efficiency: The ultrasonic-assisted extraction process was systematically optimized using response surface methodology. The order of influence of various factors on the extraction rate was clarified as ultrasonic power > solid-liquid ratio > ethanol concentration. Furthermore, a significant interaction between ethanol concentration and ultrasonic power was discovered for the first time. Under optimal process conditions, the extraction rate of ursolic acid reached 4.17%, higher than the extraction levels reported in existing literature.

[0030] 2. Stable and reliable process: The established ultraviolet spectrophotometric detection method has high accuracy. The relative standard deviation (RSD) of the precision test is 0.06%, the RSD of the repeatability test is 0.93%, and the recovery rate is 98.14%, indicating that the method and process have good reproducibility and the results are reliable.

[0031] 3. Low cost and easy to promote: The solvent composition of this invention is simple, and combined with conventional ultrasonic equipment, it avoids the use of expensive or special equipment, reduces production costs, and facilitates large-scale application in industry.

[0032] 4. Provides a theoretical basis for the deep processing of Cornus officinalis: The optimized extraction process and established detection method of this invention can provide important technical support and theoretical basis for the high-value development and utilization of Cornus officinalis resources.

[0033] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0034] The dried Cornus officinalis fruit used in this embodiment of the invention was provided by Shaanxi Changsheng Ecological Winery Co., Ltd.

[0035] 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.

[0036] Example 1 Step 1, Raw material pretreatment: After washing, removing stems and leaves from the dried Cornus officinalis fruit, place it in a 60 ℃ oven and dry for 12 h. After drying, put the dried Cornus officinalis fruit into a grinder and grind it thoroughly. Then, pass it through a 60-mesh sieve to obtain Cornus officinalis fruit powder. Seal and store it in a -4 ℃ refrigerator for later use.

[0037] Step 2: Weigh a certain amount of dried Cornus officinalis fruit powder, add a solvent of the corresponding concentration (composed of ethanol and water) according to a certain material-to-liquid ratio, and place the mixture in an ultrasonic instrument. Assist in the extraction of ursolic acid from Cornus officinalis by setting specific ultrasonic power, temperature, and time. After ultrasonic treatment, centrifuge the extract (4000 r / min for 15 min), collect the supernatant, and dilute it 10 times with an ethanol solution of the corresponding concentration to obtain the sample solution (Cornus officinalis test solution, i.e., the Cornus officinalis extract containing ursolic acid), for later testing.

[0038] Test case Establishment and validation of a detection method for Cornus officinalis extract containing ursolic acid 1.1 Preparation of ursolic acid reference solution Accurately weigh 5.0 mg of ursolic acid standard (purity ≥98%, Beijing Solarbio Science & Technology Co., Ltd.), dried to constant weight, and place it in a 50 mL volumetric flask. Add a small amount of anhydrous ethanol to dissolve it completely, then dilute to the mark with anhydrous ethanol and shake well to obtain a 0.1 mg / mL ursolic acid reference stock solution.

[0039] 1.2 Determination of the maximum absorption wavelength Pipette 0.5 mL of ursolic acid reference solution and 0.2 mL of Cornus officinalis test solution into separate test tubes. After evaporating the solvent in a boiling water bath, add 0.2 mL of 5% vanillin glacial acetic acid colorimetric reagent and 0.8 mL of perchloric acid sequentially. Mix thoroughly and place in a 60 ℃ constant temperature water bath for 15 min. Remove and rapidly cool to room temperature. Then add 5 mL of glacial acetic acid and mix well. Use the solution containing only 0.2 mL of 5% vanillin glacial acetic acid, 0.8 mL of perchloric acid, and 5 mL of glacial acetic acid as a blank control. Perform a full wavelength scan in the range of 450 nm to 700 nm. Plot wavelength (Wavelength) on the x-axis and absorbance (A) on the y-axis to obtain the visible light absorption diagrams of ursolic acid reference standard, Cornus officinalis test sample, and blank control.

[0040] The results are as follows Figure 1 As shown in the figure (A is water, B is blank control, C is Cornus officinalis test solution, and D is ursolic acid control solution wavelength scan results), both show a maximum absorption peak at 548 nm, and the blank does not interfere at this wavelength. Therefore, 548 nm is determined to be the measurement wavelength of this invention.

[0041] 1.3 Plotting the Standard Curve Accurately pipette 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mL of the ursolic acid reference stock solution prepared in 1.1 into 11 test tubes (corresponding to ursolic acid masses of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg, respectively). Process the solution according to the steps described in 1.2: "evaporation in boiling water bath → addition of colorimetric reagent → water bath heating → cooling → addition of glacial acetic acid". Finally, zero the absorbance (A) of each tube at 548 nm using a blank solution.

[0042] A standard curve was plotted with ursolic acid mass (x, μg) on ​​the x-axis and absorbance (y, A) on the y-axis. Linear regression yielded the equation: y = 0.0107x + 0.0155 (where y is the absorbance A; x is the mass of ursolic acid, μg), and the correlation coefficient R0 is [value missing]. 2 = 0.9994. The results show that ursolic acid exhibits good linearity in the range of 0-100 μg. Figure 2 ).

[0043] 1.4 Methodological Examination (1) Precision test One aliquot of the same ursolic acid reference solution and one aliquot of the Cornus officinalis test solution were precisely pipetted. After color development according to method 1.2, the absorbance was measured six times consecutively at a wavelength of 548 nm. The relative standard deviations (RSDs) of the absorbance of the reference standard and the test sample were calculated to be 0.15% and 0.06%, respectively, indicating that the instrument has good precision.

[0044] (2) Stability test Accurately pipette one portion each of the same ursolic acid reference solution and Cornus officinalis test solution. After color development according to method 1.2, measure the absorbance at 548 nm every 10 minutes for 2 hours. The calculated RSDs of the absorbance of the reference solution and the test solution were 2.29% and 0.93%, respectively, indicating that the sample solutions had good stability within 2 hours after color development.

[0045] (3) Repeatability test Six portions of dried Cornus officinalis fruit powder of the same mass were weighed and prepared into Cornus officinalis test solutions in test tubes according to method 1.1. Independent and repeated tests were performed. After treatment according to the colorimetric method in 1.2, the absorbance value was measured at the selected wavelength. The RSD of the ursolic acid content of the six samples was calculated to be 0.93%, indicating that the method has good repeatability.

[0046] (4) Recovery test Six 0.2 mL portions of the Cornus officinalis test solution with known concentration were taken, and 0.10 mL of ursolic acid reference solution with known concentration was added to each. After evaporating the solvent in a boiling water bath, the samples were processed according to the colorimetric method in section 1.2, and the absorbance (A) was measured at the selected wavelength. The RSD of the absorbance value was calculated. According to the formula: Recovery rate = (Measured value - Background value) / Spiked amount × 100%, the average recovery rate was calculated to be 98.14%, and the RSD was 1.76%, indicating that the method has high accuracy.

[0047] The results are as follows Figure 3 As shown in Table 1-3: Figure 3 Tables 1-3 show that the present invention establishes a reliable ultraviolet spectrophotometric detection method for Cornus officinalis extract containing ursolic acid.

[0048] Table 1. Precision test results (ursolic acid)

[0049] Table 2 Results of repeatability tests (ursolic acid)

[0050] Table 3 Results of the recovery test (ursolic acid)

[0051] 2. Research on the extraction process of ursolic acid from Cornus officinalis 2.1 Yield Calculation The extraction rate of Cornus officinalis extract containing ursolic acid is calculated using the following formula.

[0052]

[0053] In the formula, T u The extraction rate of Cornus officinalis extract containing ursolic acid, % . m 1 represents the mass of ursolic acid calculated by substituting into the standard curve, in μg; n represents the sample dilution factor. V 2 represents the total sample volume, in mL; V 1 represents the volume of the sample solution measured, in mL; m 2 represents the total mass of the sample, in g.

[0054] 2.2 Single-factor experiment The effects of five extraction conditions on the extraction rate of Cornus officinalis containing ursolic acid (denoted as ursolic acid extraction rate) were investigated: the ratio of Cornus officinalis to solvent (1:10, 1:20, 1:30, 1:40, 1:50 g / mL), the concentration of ethanol in the solvent (20%, 40%, 60%, 80%, 100%), the ultrasonic power (120, 200, 300, 400, 500 W), the ultrasonic temperature (30, 40, 50, 60, 70 ℃), and the ultrasonic time (10, 30, 50, 70, 90 min).

[0055] Using the controlled variable method, with other factors fixed, we examined the effects of the following five factors on the extraction rate, with each condition tested in triplicate.

[0056] The effect of different solid-liquid ratios (1:10, 1:20, 1:30, 1:40, 1:50 g / mL) was investigated, with fixed ethanol concentration of 60% (v / v), ultrasonic power of 300 W, ultrasonic temperature of 50℃, and ultrasonic time of 50 min. The results are as follows: Figure 4 As shown, the extraction rate was highest when the material-to-liquid ratio was 1:30.

[0057] Ethanol concentration: With a fixed material-to-liquid ratio of 1:30 g / mL, ultrasonic power of 300 W, ultrasonic temperature of 50℃, and ultrasonic time of 50 min, the effect of ethanol concentration (20%, 40%, 60%, 80%, 100%) was investigated. The results are as follows: Figure 5 As shown, the extraction rate was highest when the ethanol concentration was 80%.

[0058] Ultrasonic power: With a fixed material-to-liquid ratio of 1:30 g / mL, ethanol concentration of 60%, ultrasonic temperature of 50℃, and ultrasonic time of 50 min, the effect of ultrasonic power (120, 200, 300, 400, 500 W) was investigated. The results are as follows: Figure 6 As shown, the extraction rate was highest when the ultrasonic power was 300W.

[0059] Ultrasonic temperature: With a fixed material-to-liquid ratio of 1:30 g / mL, ethanol concentration of 60%, ultrasonic power of 300 W, and ultrasonic time of 50 min, the effect of ultrasonic temperature (30, 40, 50, 60, 70 ℃) was investigated. The results are as follows: Figure 7 As shown, the extraction rate was highest when the ultrasonic temperature was 70℃.

[0060] Ultrasonication time: With a fixed material-to-liquid ratio of 1:30 g / mL, ethanol concentration of 60%, ultrasonic power of 300 W, and ultrasonic temperature of 50℃, the effect of ultrasonication time (10, 30, 50, 70, 90 min) was investigated. Results are as follows: Figure 8 As shown, the extraction rate was highest when the ultrasonic time was 50 min.

[0061] 2.3 Response Surface Optimization Experiment Based on the results of the single-factor experiments, three factors with significant influence were selected: ethanol concentration (A), ultrasonic power (B), and solid-liquid ratio (C). Ursolic acid extraction rate (Y) was used as the response value. A three-factor, three-level response surface methodology optimization experiment was designed using Design-Expert 8.0.6 software. The experimental data were processed using IBM SPSS Statistics 26.0 software, and a one-way ANOVA test was used to analyze the variance of the results. A p-value < 0.05 was considered statistically significant.

[0062] The factors and levels are shown in Table 4.

[0063] The optimal conditions of 50 min for ultrasonic time and 70 ℃ for ultrasonic temperature were kept constant, and the effects of ethanol concentration (A), ultrasonic power (B), and solid-liquid ratio (C) were further investigated. The extraction process of ursolic acid from Cornus officinalis was optimized using Box-Behnken response surface methodology. The experimental design and results are shown in Table 5.

[0064] Table 4. Response surface methodology and level design (ursolic acid)

[0065] Table 5. Response surface methodology and results of ursolic acid extraction process from Cornus officinalis.

[0066] Table 6. Results of Variance Analysis of the Extraction Process of Usolic Acid from Cornus officinalis

[0067] Note: p-value < 0.05 indicates a significant effect, marked as "significant". A p-value < 0.01 indicates a highly significant effect, and is labeled as "". ".

[0068] The analysis results in Table 6 show that the model is highly significant (P = 0.0069 < 0.01), indicating its effectiveness. Analysis of variance shows that the order of influence of factors is: ultrasonic power (B) > feed-to-liquid ratio (C) > ethanol concentration (A), and the interaction between ethanol concentration and ultrasonic power (AB) is significant. (Combined with Table 6 and...) Figure 9 In summary, the order of influence of the interaction of the two factors on the ursolic acid extraction rate (Y) of Cornus officinalis is: AB>BC>AC.

[0069] 2.4 Validation of the optimal process Based on the response surface regression model, the optimal combination of ultrasonic-assisted ethanol extraction of ursolic acid from Cornus officinalis was determined to be 73.89% ethanol concentration, 200 W ultrasonic power, and a solid-liquid ratio of 1:31.13 g / mL, with a predicted ursolic acid extraction rate of 4.30%. To further verify the accuracy of this extraction process and considering its feasibility in actual production, the optimal extraction conditions were adjusted to 74% ethanol concentration, 200 W ultrasonic power, a solid-liquid ratio of 1:31 g / mL, ultrasonic time of 50 min, and ultrasonic temperature of 70 ℃. After three parallel verifications, the actual ursolic acid extraction rate was 4.17 ± 0.12%, or 41.68 ± 1.21 mg / g, indicating that the ursolic acid extraction process obtained after optimization by this model is feasible and has a relatively high yield.

[0070] Example 2 Step 1, Raw material pretreatment: After washing, removing stems and leaves from the dried Cornus officinalis fruit, place it in a 60 ℃ oven and dry for 12 h. After drying, put the dried Cornus officinalis fruit into a grinder and grind it thoroughly. Then, pass it through a 60-mesh sieve to obtain Cornus officinalis fruit powder. Seal and store it in a -4 ℃ refrigerator for later use.

[0071] Step 2: Weigh a certain amount of dried Cornus officinalis fruit powder, add a solvent of corresponding concentration (composed of ethanol and mixture A, which is a mixture of ethyl acetate and water in a volume ratio of 5:1) according to a certain material-to-liquid ratio, and place it in an ultrasonic instrument. Assist extraction of ursolic acid from Cornus officinalis is performed by setting a certain ultrasonic power, ultrasonic temperature, and ultrasonic time. After ultrasonic treatment, the extract is centrifuged (4000 r / min for 15 min), and the supernatant is taken. The supernatant is diluted 10 times with a corresponding concentration of ethanol solution to prepare the sample solution (Cornus officinalis test solution) for later testing. The parameters were: ethanol concentration 74%, ultrasonic power 200 W, material-to-liquid ratio 1:31 g / mL, ultrasonic time 50 min, and ultrasonic temperature 70 ℃. The calculated ursolic acid extraction rate in Example 2 was 49.82 ± 0.91 mg / g.

[0072] Example 3 Step 1, Raw material pretreatment: After washing, removing stems and leaves from the dried Cornus officinalis fruit, place it in a 60 ℃ oven and dry for 12 h. After drying, put the dried Cornus officinalis fruit into a grinder and grind it thoroughly. Then, pass it through a 60-mesh sieve to obtain Cornus officinalis fruit powder. Seal and store it in a -4 ℃ refrigerator for later use.

[0073] Step 2: Weigh a certain amount of dried Cornus officinalis fruit powder, add a solvent of corresponding concentration (composed of ethanol and mixture A, which is a mixture of glycerol and water in a volume ratio of 5:1) according to a certain material-to-liquid ratio, and place it in an ultrasonic instrument. Assist extraction of ursolic acid from Cornus officinalis is performed by setting a certain ultrasonic power, ultrasonic temperature, and ultrasonic time. After ultrasonic treatment, the extract is centrifuged (4000 r / min for 15 min), and the supernatant is taken. The supernatant is diluted 10 times with a corresponding concentration of ethanol solution to prepare the sample solution (Cornus officinalis test solution) for later testing. The parameters are: ethanol concentration 74%, ultrasonic power 200 W, material-to-liquid ratio 1:31 g / mL, ultrasonic time 50 min, and ultrasonic temperature 70 ℃. The calculated ursolic acid extraction rate in Example 3 is 33.49 ± 1.03 mg / g.

[0074] Example 4 Step 1, Raw material pretreatment: After washing, removing stems and leaves from the dried Cornus officinalis fruit, place it in a 60 ℃ oven and dry for 12 h. After drying, put the dried Cornus officinalis fruit into a grinder and grind it thoroughly. Then, pass it through a 60-mesh sieve to obtain Cornus officinalis fruit powder. Seal and store it in a -4 ℃ refrigerator for later use.

[0075] Step 2: Weigh a certain amount of dried Cornus officinalis fruit powder, add a solvent of corresponding concentration (composed of ethanol and mixture A, which is a mixture of ethyl acetate, glycerol, and water in a volume ratio of 4:1:1) according to a certain material-to-liquid ratio, and place it in an ultrasonic instrument. Assist extraction of ursolic acid from Cornus officinalis is performed by setting a certain ultrasonic power, ultrasonic temperature, and ultrasonic time. After ultrasonic treatment, the extract is centrifuged (4000 r / min for 15 min), and the supernatant is taken. The supernatant is diluted 10 times with a corresponding concentration of ethanol solution to prepare the sample solution (Cornus officinalis test solution) for later testing. The parameters were: ethanol concentration 74%, ultrasonic power 200 W, material-to-liquid ratio 1:31 g / mL, ultrasonic time 50 min, and ultrasonic temperature 70 ℃. The calculated ursolic acid extraction rate in Example 4 was 62.73 ± 1.16 mg / g.

[0076] Based on Example 1, this invention further determined the composition and optimal ratio of mixture A through single-factor experiments and response surface methodology. The results showed that the extraction rate of Cornus officinalis extract containing ursolic acid was highest when the extraction solvent consisted of ethanol and mixture A, and mixture A was a mixture of ethyl acetate, glycerol, and water in a volume ratio of 4:1:1. This may be because glycerol, acting as a "biocompatible hydrogen bond regulator," synergistically enhances the entire dissolution-diffusion-partition process of Cornus officinalis extract containing ursolic acid through a triple mechanism of δ-tuning, interfacial tension reduction, and crystallization inhibition.

[0077] The present invention also verified the application of the extract containing ursolic acid prepared in the examples. The results showed that: ① the extract containing ursolic acid has good protective effects on pancreatic β cells, improves insulin resistance, inhibits gluconeogenesis, and inhibits α-glucosidase; it can be used in functional foods or special medical foods for "prediabetes" and "decreased metabolic adaptability", and has potential therapeutic value for both type 1 and type 2 diabetes.

[0078] ② Extracts containing ursolic acid can act as effective antioxidants, directly scavenging free radicals in the body (such as superoxide anions and hydroxyl radicals) and upregulating the body's own antioxidant defense system (such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), thus reducing the damage caused by oxidative stress. Oxidative stress is a common basis for aging and many chronic diseases.

[0079] Therefore, the extract containing ursolic acid prepared by this invention can be used to develop high-end anti-aging skincare products.

[0080] ③ Extracts containing ursolic acid can target key inflammatory signaling pathways, such as the NF-κB and MAPK pathways, thereby reducing the production of various pro-inflammatory cytokines (such as TNF-α and IL-6).

[0081] ④ Extracts containing ursolic acid have inhibitory effects on the proliferation and apoptosis-inducing effects on various cancer cell lines, including inducing cancer cell apoptosis, inhibiting cancer cell proliferation and metastasis, and anti-angiogenesis.

[0082] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An ultrasound-assisted extraction method for Cornus officinalis extract containing ursolic acid, characterized in that, Includes the following steps: Step 1: After washing, removing stems and leaves from the dried Cornus officinalis fruit, dry it and grind it to obtain Cornus officinalis fruit powder. Step 2: Add the dried fruit powder of Cornus officinalis to a solvent for ultrasonic treatment, centrifuge, and take the supernatant to obtain Cornus officinalis extract containing ursolic acid. The solvents include ethanol and water.

2. The ultrasound-assisted extraction method for Cornus officinalis extract containing ursolic acid according to claim 1, characterized in that, In step 1, the drying temperature is 60 ℃ and the drying time is 12 h; after grinding, the material is passed through a 60-mesh sieve.

3. The ultrasound-assisted extraction method for Cornus officinalis extract containing ursolic acid according to claim 1, characterized in that, In step 2, the solvent also includes ethyl acetate.

4. The ultrasound-assisted extraction method for Cornus officinalis extract containing ursolic acid according to claim 1, characterized in that, In step 2, the solvent also includes ethyl acetate and glycerol.

5. The ultrasound-assisted extraction method for Cornus officinalis extract containing ursolic acid according to claim 4, characterized in that, In step 2, the volume ratio of ethyl acetate, glycerol, and water in the solvent is 4:1:

1.

6. The ultrasound-assisted extraction method for Cornus officinalis extract containing ursolic acid according to claim 1, characterized in that, In step 2, the volume concentration of ethanol in the solvent is 60%~80%.

7. The ultrasound-assisted extraction method for Cornus officinalis extract containing ursolic acid according to claim 1, characterized in that, In step 2, the ultrasonic power of the ultrasonic treatment is 200~300 W, the ultrasonic time is 40~60 min, and the ultrasonic temperature is 60~70℃.

8. The ultrasound-assisted extraction method for Cornus officinalis extract containing ursolic acid according to claim 1, characterized in that, In step 2, the ratio of the dried Cornus officinalis fruit powder to the solvent is 1:(20~40)g / mL.

9. The ultrasound-assisted extraction method for Cornus officinalis extract containing ursolic acid according to claim 1, characterized in that, In step 2, the centrifugation conditions are set as follows: centrifuge at 4000 r / min for 15 min.

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