Ultrasonic composite extraction method of trollflower stem flavone

Abstract

The invention discloses an ultrasonic composite extraction method of trollius chinensis stem flavone, which comprises the following steps: pretreating trollius chinensis stems, extracting the trollius chinensis stem flavone by adopting composite extraction methods of enzymolysis, ultrasonic wave, microwave and the like, and taking active functions such as oxidation resistance, moisture retention and the like as response indexes; the optimal extraction method is determined by combining a variable coefficient method, the ultrasonic time is 40 min, the ultrasonic power is 400 W, the liquid-material ratio is 50 mL / g, and the enzyme content is 1.0%. Experimental verification shows that the flavone yield of the extraction method is increased by 1.85 times compared with that of an ultrasonic wave and ethanol extraction method (UA); in the aspect of the antioxidant effect, the hydroxyl free radical scavenging capacity is improved by 2.1 times most remarkably, and the scavenging rates of DPPH and ABTS free radicals are improved by 1.15 times and 1.2 times respectively.

Description

Technical Field

[0001] The present invention relates to the field of chemical engineering and technology. Specifically, it relates to an ultrasonic composite extraction method for flavonoids from the stems of Trollius chinensis, combining various extraction techniques such as ultrasonic, enzymatic hydrolysis, and microwave, aiming to improve the application effect of flavonoids from the stems of Trollius chinensis. Background Art

[0002] Trollius chinensis, a plant of the Ranunculaceae family, is mostly distributed in alpine areas with an altitude of 1000 - 2000 m in the north such as Inner Mongolia, Hebei, and Heilongjiang. As one of the traditional Chinese medicinal materials, the medicinal records of Trollius chinensis have a long history. It was once recorded in "Supplements to Compendium of Materia Medica" that it has the effects of "treating oral ulcers, throat swelling, floating heat in the teeth, and ear and eye pain". Through research, it is found that the plant height of Trollius chinensis is about 80 cm, and the flower stems account for about 90% of the total weight of the plant, having potential application value. However, currently, they are mostly treated as waste and have not been effectively developed and utilized.

[0003] There are about more than 100 compounds in Trollius chinensis, mainly including flavonoids, polysaccharides, organic acids, alkaloids, etc. Among them, flavonoids and polysaccharides are the main effective active components. Modern pharmacological research shows that both flavonoid and polysaccharide components in Trollius chinensis have good active functions such as anti - cold pharyngitis, anti - respiratory tract infection, antibacterial and anti - inflammatory effects, and can be used to treat tonsillitis, colds, blood pressure reduction and other diseases. They can improve the body's immunity by regulating different signal pathways of the immune system; they can scavenge free radicals in the body and reduce the damage of oxidative stress to cells, having good antioxidant effects.

[0004] Existing technical research shows that: the composition and functions of active substances in the stems and leaves of Trollius chinensis are similar to those in the petals. Among them, flavonoids from the stems of Trollius chinensis have very excellent antioxidant and moisturizing effects and have great potential for industrial application. However, the stems and leaves of Trollius chinensis are mostly treated as waste, causing huge waste. Based on the above situation, the present invention mainly uses ultrasonic - assisted extraction technology suitable for large - scale industrial production, and coordinates with other extraction methods such as enzymatic hydrolysis extraction and microwave assistance. By comparing the functional evaluations such as the scavenging rates of various free radicals and the flavonoid yield of ultrasonic combination with different extraction methods as indicators, and combining the coefficient of variation weight analysis method, the extraction method of flavonoids from the stems and leaves of Trollius chinensis with the best application effect is obtained. Summary of the Invention

[0005] The present invention aims to provide an ultrasonic composite extraction method for the high - efficient application effect of flavonoids from the stems of Trollius chinensis. Taking in vitro antioxidant and flavonoid yield as multiple response indicators, and using an extraction method combining ultrasound and enzymatic hydrolysis, the yield and application effect are significantly improved compared with other extraction methods.

[0006] An ultrasonic composite extraction method for flavonoids from Trollius chinensis Bunge stems. The Trollius chinensis Bunge stems are crushed, and an ethanol aqueous solution is used as a solvent, followed by enzymatic hydrolysis with cellulase. After enzymatic hydrolysis, it is placed in an ultrasonic extractor for extraction, and then freeze-dried.

[0007] Preferably, the method specifically includes the following steps:

[0008] (1) Dry the Trollius chinensis Bunge stems to constant weight, crush them, sieve them, and store for later use;

[0009] (2) Weigh 10.0 g of the Trollius chinensis Bunge stem powder obtained in step (1), add an ethanol aqueous solution and cellulase, and carry out enzymatic hydrolysis;

[0010] (3) After extracting the enzymatic hydrolysis solution of the Trollius chinensis Bunge stems obtained in step (2), place it in an ultrasonic extractor for extraction. After the sample cools, carry out suction filtration to obtain the enzymatic hydrolysis and ultrasonic extraction solution of the Trollius chinensis Bunge stems;

[0011] (4) Put the enzymatic hydrolysis and ultrasonic extraction solution of the Trollius chinensis Bunge stems in step (3) into a refrigerator and let it stand at 4 - 5 °C for about 4 h, then carry out centrifugation, take the supernatant to obtain the crude flavonoid solution of the Trollius chinensis Bunge stems;

[0012] (5) Carry out rotary evaporation on the crude flavonoid solution of the Trollius chinensis Bunge stems obtained in step (4) to obtain a concentrated solution. Place it in an ultra-low temperature freezer and freeze it at -80 °C for 12 h, then put it into a vacuum dryer for freeze-drying, repeat 2 - 3 times to obtain the crude flavonoid powder of the Trollius chinensis Bunge stems.

[0013] Preferably, the method further includes the following steps:

[0014] (6) Measure the DPPH free radical scavenging rate, ABTS free radical scavenging rate, and hydroxyl free radical scavenging rate of the crude flavonoids of the Trollius chinensis Bunge stems obtained in step (5), as well as the yield;

[0015] (7) Utilize the test results of each property index obtained in step (6), determine the weight coefficients of each functional evaluation index of the crude flavonoids of the Trollius chinensis Bunge stems by the coefficient of variation method, normalize the data of each functional evaluation index of the Trollius chinensis Bunge flavonoids by the Z-score normalization method, and sum the products of the normalized data of each index and the corresponding weight coefficients to obtain the comprehensive evaluation total score.

[0016] Preferably, in step (2), the solid-liquid ratio is 1:20 - 1:60 g / mL, the ethanol volume fraction is 55 - 70 V%, and the cellulase addition amount is 0.1 - 1.5 wt% of the Trollius chinensis Bunge stem powder; more preferably, the solid-liquid ratio is 1:50 g / mL, the ethanol volume fraction is 70 V%, and the cellulase addition amount is 1.0 wt%;

[0017] Preferably, in step (3), the ultrasonic power is set in the range of 200 - 600 W, and the ultrasonic time is 20 - 60 min; preferably, the ultrasonic power is 400 W and the ultrasonic time is 40 min;

[0018] The beneficial effects of the present invention are as follows:

[0019] (1) Compared with other extraction methods, the flavonoid yield of Trollius chinensis Bunge stems is significantly increased;

[0020] (2) Compared with other extraction methods, attention is paid to the high - efficiency application effect of flavonoids from Trollius chinensis Bunge stems. Taking its multi - active functions such as in vitro antioxidant and moisturizing as response indicators, the coefficient of variation method is used to determine the optimal extraction method of flavonoids from Trollius chinensis Bunge stems, rather than taking the yield as the single response indicator;

[0021] (3) Compared with the single ultrasonic extraction method, the antioxidant effect and yield of flavonoids from Trollius chinensis Bunge stems are significantly improved. Among them, the flavonoid yield is 1.85 times higher than that of the ultrasonic + ethanol extraction method; in terms of antioxidant effect, the scavenging ability of hydroxyl radicals is most significantly improved by 2.1 times, while the scavenging rates of DPPH and ABTS radicals are also increased by 1.15 times and 1.2 times respectively. Description of the Drawings

[0022] Figure 1 It is a comparison chart of flavonoid extraction rates of different extraction methods;

[0023] Figure 2 It is the IC of antioxidant evaluation indicators of flavonoids and Vc extracted by different methods 50 Figure;

[0024] Figure 3 It is a graph of flavonoid loss rates obtained by different methods;

[0025] Figure 4 It is a comprehensive score graph of the antioxidant effect and loss rate of flavonoids obtained by different extraction methods;

[0026] Figure 5 It is the comprehensive score of the antioxidant effect and loss rate of flavonoids obtained by different single factors;

[0027] (a) It is a comprehensive score graph of the antioxidant effect, yield and loss rate of flavonoids obtained with different ultrasonic powers;

[0028] (b) It is a comprehensive score graph of the antioxidant effect, yield and loss rate of flavonoids obtained with different ultrasonic times;

[0029] (c) It is a comprehensive score graph of the antioxidant effect, yield and loss rate of flavonoids obtained with different enzyme addition amounts;

[0030] (d) It is a comprehensive score graph of the antioxidant effect, yield and loss rate of flavonoids obtained with different liquid - to - material ratios;

[0031] (e) Comprehensive score chart of the antioxidant effect, yield, and loss rate of flavonoids obtained at different ethanol concentrations. Detailed implementation mode

[0032] The terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified. The present invention will be described in further detail below with reference to specific examples and data. It should be understood that these examples are only for illustrative purposes and do not limit the scope of the present invention in any way.

[0033] Experimental materials:

[0034] 1. Trollius chinensis Bunge stems and leaves, purchased from Tianxiang Linyuan Specialties Company in Chengde, Hebei; DPPH (1,1-diphenyl-2-picrylhydrazyl, product number AS395541-1g, CAS number: 1898-66-4), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, product number AB009301-1g, CAS number: 30931-67-0) were purchased from Anhui Cool Biological Engineering Co., Ltd.; cellulase was purchased from Nanning Pangbo Biological Engineering Co., Ltd. (enzyme activity: 10,000 - 35,000 u / g, CAS number: 9012-54-8, purity 99%).

[0035] Main experimental instruments: Ultrasonic extractor (JY92-ⅡN, Ningbo Xinzhi Biotechnology Co., Ltd.); ultra-low temperature refrigerator (DW-861L416G, Haier Group); vacuum freeze dryer (LGJ-12, Beijing Songyuan Huaxing Technology Development Co., Ltd.); high-speed desktop centrifuge (TG16-WS, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); spectrophotometer (T6 new generation, Beijing Purkinje General Instrument Co., Ltd.); rotary evaporator (RE-5220A, Nantong Puri Technology Co., Ltd.), etc.

[0036] Extraction template conditions for crude flavonoids from Trollius chinensis Bunge

[0037] Pretreatment process: Dry the Trollius chinensis Bunge stems to constant weight, crush them, pass through a 40-mesh sieve, seal them in a glass bottle, and store them at room temperature for later use;

[0038] Enzymolysis process: For the pretreated Trollius chinensis Bunge stem powder, use ethanol with a volume fraction of 55 - 70V% at a solid-liquid ratio of 1:20 - 1:60 g / mL, and the cellulase addition amount is 0.1 - 1.5 wt% (calculated based on the Trollius chinensis Bunge stem powder). Enzymolyze in a constant temperature water bath at 50 °C for 70 min, and then extract in a constant temperature water bath at 70 °C for 1 h;

[0039] Ultrasonic process: Place the enzymolyzed solution of Trollius chinensis Bunge stems in an ultrasonic extractor for extraction. Set the ultrasonic power to 200 - 600 W and the ultrasonic time to 20 - 60 min to obtain the enzymolyzed and ultrasonically extracted solution of Trollius chinensis Bunge stems;

[0040] Filter the obtained enzymolyzed and ultrasonically extracted solution of Trollius chinensis Bunge stems, place it in a refrigerator at 4 - 5 °C for about 4 h, then centrifuge it. Take the supernatant to obtain the crude flavonoid solution of Trollius chinensis Bunge. Rotavaporize the extracted crude flavonoid solution to obtain a concentrated solution. Freeze it in a ultra-low temperature freezer at -80 °C for 12 h, then place it in a vacuum dryer for freeze-drying. Repeat this process 2 - 3 times to obtain the crude flavonoid powder.

[0041] Testing of the crude flavonoid content of Trollius chinensis Bunge

[0042] Use the rutin standard curve method to test the flavonoid concentration in Trollius chinensis Bunge stems. With the rutin concentration (C) as the abscissa and the absorbance value (A) as the ordinate, plot the standard curve to obtain the regression equation and calculate the correlation coefficient. The regression equation of the rutin standard curve is: y = 0.01389x - 0.01271, and the correlation coefficient R2 = 0.99901. The regression equation has good linearity and can be used for calculating the flavonoid content. Precisely pipette 10 mL of the test solution into a 25 mL volumetric flask. According to the preparation method and determination conditions of the standard curve, measure the absorbance at a wavelength of 510 nm, and calculate the total flavonoid content based on the standard curve and the absorbance of the sample solution. The content of flavonoids is calculated by Equation (1):

[0043]

[0044] In the formula: m1—the mass of the total flavonoids in the sample solution calculated from the working curve, g;

[0045] V—the extraction volume of the sample, mL;

[0046] M—the mass of the sample, g;

[0047] V1—the volume of the sample taken during determination, mL.

[0048] Evaluation of the functional activity of polysaccharides

[0049] 1. In vitro antioxidant evaluation

[0050] (1) Determination of the DPPH free radical scavenging rate.

[0051] Weigh 0.0394 g of DPPH and make up the volume to 500 mL with absolute ethanol to prepare a 0.2 mmol / L DPPH-absolute ethanol solution.

[0052] Take 2 mL of DPPH-anhydrous ethanol solution, add 2 mL of anhydrous ethanol, place it in the dark for 30 min, zero it with anhydrous ethanol, and measure its absorbance value A0 at a wavelength of 517 nm; take 2 mL of flavonoid solutions with different mass concentrations (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0 mg / mL), the control group is Vc solution with the same concentration, add an equal volume of 0.2 mmol / L DPPH-anhydrous ethanol solution, place it in the dark for 30 min, zero it with anhydrous ethanol, and measure its absorbance value A1 at a wavelength of 517 nm; take 2 mL of flavonoid solutions with different mass concentrations (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0 mg / mL), add an equal volume of anhydrous ethanol, place it in the dark for 30 min, zero it with anhydrous ethanol, and measure its absorbance value A2 at a wavelength of 517 nm. Each sample is repeated 3 times and the average value is taken. Calculate the scavenging rate of the sample on DPPH free radicals according to formula (2).

[0053]

[0054] In the formula: A0 is the absorbance value of 2 mL of DPPH solution + 2 mL of anhydrous ethanol;

[0055] A1 is the absorbance value of 2 mL of DPPH solution + 2 mL of sample solution;

[0056] A2 is the absorbance value of 2 mL of sample solution + 2 mL of anhydrous ethanol;

[0057] A is the absorbance value of 70% ethanol.

[0058] (2) Determination of ABTS free radical scavenging rate.

[0059] Weigh 0.2 g of ABTS, make up the volume to 50 mL with distilled water to prepare 7.4 mmol / L ABTS; weigh 0.035 g of potassium persulfate, make up the volume to 50 mL with distilled water to prepare 2.6 mmol / L potassium persulfate. Mix 7.4 mmol / L ABTS and 2.6 mmol / L potassium persulfate in a 1:1 ratio and place it in the dark at room temperature for 12 h to form an ABTS free radical stock solution. Dilute the stock solution with anhydrous ethanol to make its absorbance value at 734 nm be 0.70 ± 0.02 to obtain the ABTS free radical working solution.

[0060] Take 3.0 mL of ABTS radical working solution and add it to 1.0 mL of samples with different concentrations (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0 mg / mL) or the positive control Vc solution. Shake well, let it stand at room temperature for 7 min, measure the absorbance at a wavelength of 734 nm. Repeat each sample 3 times and take the average value. Calculate the scavenging rate of the samples on ABTS radicals according to formula (3).

[0061]

[0062] In the formula: A0 is the absorbance value of 3 mL of ABTS solution + 1 mL of absolute ethanol;

[0063] A1 is the absorbance value of 3 mL of ABTS solution + 1 mL of sample solution;

[0064] A2 is the absorbance value of 1 mL of sample solution + 3 mL of absolute ethanol;

[0065] A is the absorbance of 70% ethanol.

[0066] (3) Determination of hydroxyl radical scavenging rate. First, prepare 9 mmol / L salicylic acid, 9 mmol / L FeSO4 and 8.8 mmol / L H2O2.

[0067] Add 2 mL of samples with different concentrations (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0 mg / mL) to a test tube, then add 1 mL of salicylic acid and 1 mL of FeSO4 respectively, and finally add 2 mL of H2O2. Mix well, place it in a water bath at 37 °C for 30 min, and measure the absorbance A1 at 510 nm; use the same volume of water to replace salicylic acid and measure its absorbance A2; then use the same volume of water to replace the sample solution and measure its absorbance A3. Repeat each sample 3 times and take the average value. Use Vc as the positive control and calculate the hydroxyl radical scavenging rate according to formula (4).

[0068]

[0069] In the formula: A1 is the absorbance value of 2 mL of sample solution + 1 mL of salicylic acid + 1 mL of FeSO4 + 1 mL of H2O2;

[0070] A2 is the absorbance value of 2 mL of sample solution + 1 mL of water + 1 mL of FeSO4 + 1 mL of H2O2;

[0071] A3 is the absorbance value of 2 mL of water + 1 mL of salicylic acid + 1 mL of FeSO4 + 1 mL of H2O2.

[0072] 2. Determination of flavonoid yield

[0073] The extracted crude flavone solution was rotary evaporated to obtain a concentrated solution. After freezing in an ultra-low temperature freezer for 12 h, it was freeze-dried in a vacuum dryer, and this was repeated 2 - 3 times to obtain crude flavone powder. The mass of the powder was weighed, and the flavone yield was calculated using formula (5):

[0074]

[0075] In the formula: m——mass of crude flavone powder, g;

[0076] W——mass of Trollius chinensis Bunge stem sample, g.

[0077] 3. Determination of other active functions

[0078] (1) Determination of α-glucosidase activity inhibition rate.

[0079] The flavone sample from Trollius chinensis Bunge stem was dissolved in phosphate buffer solution (PBS, pH 6.8) to prepare sample solutions with different concentrations (0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, etc.). Add 50 μL of PBS (blank control) or flavone solutions with different concentrations from Trollius chinensis Bunge stem into an ELISA plate or a test tube; add 50 μL of 1 U / mL α-glucosidase solution prepared with PBS into another ELISA plate or test tube. The mixture was reacted in a constant temperature water bath at 37 °C for 10 min. After the reaction, add 100 μL of sodium carbonate solution to terminate the reaction. Measure the absorbance at 405 nm, and each sample was parallelly experimented three times. The inhibition rate was calculated as formula (6):

[0080]

[0081] In the formula: Acontrol is the absorbance of the blank control, and Asample is the absorbance of the sample containing flavone.

[0082] (2) Inhibitory effect on pancreatic lipase

[0083] The inhibitory effect of flavone from Trollius chinensis Bunge stem on pancreatic lipase was determined. Edible olive oil, phosphate buffer solution, and pancreatic lipase solution (prepared with phosphate buffer solution) were successively added into several 250 mL conical flasks, and distilled water was used as a blank control; in the inhibition tubes, different concentrations of flavone solutions from Trollius chinensis Bunge stem and pancreatic lipase solution were successively added, and at the same time, distilled water was used to replace the flavone solution from Trollius chinensis Bunge stem as a background control. After water bath at 37 °C for 5 min, add ethanol (95%) to terminate the reaction, add 3 - 4 drops of 1% phenolphthalein, and titrate with 0.025 mol / L NaOH

[0084] solution, and at the same time, a blank control was made. Calculate the inhibition rate (I) according to formula (7).

[0085]

[0086] In the formula, V1 is the volume of NaOH solution consumed by the sample tube, in mL; V2 is the volume of NaOH solution consumed by the background control, in mL; V3 is the volume of NaOH solution consumed by the blank tube, in mL; V4 is the volume of NaOH solution consumed by the blank control tube.

[0087] (3) Moisturizing activity detection

[0088] To evaluate the moisturizing property, the sample was added to 20 mL of distilled water and placed in a silica gel (43% RH) desiccator at 25 °C. The moisturizing rate (Rh) was measured by the following formula:

[0089]

[0090] In the formula: H o is the weight of the sample before the test, H n is the weight of the sample after being added to the desiccator.

[0091] Coefficient of variation weight analysis method

[0092] The coefficient of variation method was used to determine the weight coefficients of various functional evaluation indicators of flavonoids from Trollius chinensis Bunge stems. The coefficient of variation calculation formula is:

[0093]

[0094] In the formula: V i represents the coefficient of variation of the i-th indicator; σ i represents the standard deviation of the i-th indicator; X i represents the arithmetic mean of the i-th indicator.

[0095] The weight calculation formula for each functional evaluation indicator is:

[0096]

[0097] In the formula: Wi represents the weight of the i-th indicator; Vi represents the coefficient of variation of the i-th indicator.

[0098] Data normalization

[0099] The Z-score normalization method was used to normalize the data of various functional evaluation indicators of flavonoids from Trollius chinensis Bunge stems. The calculation formula is:

[0100]

[0101] In the formula: W i is the standardized variable value; X max is the maximum value of the same group of indicators; X i represents the value of the i-th indicator; X minis the minimum value of the same group of indicators.

[0102] Comprehensive score calculation

[0103] The sum of the standardized data of each indicator multiplied by the weight coefficient is the total comprehensive evaluation score. According to the formula calculation, the higher the comprehensive score, the stronger the comprehensive evaluation of the flavonoid activity extracted from Trollius chinensis Bunge stems.

[0104] Response surface process optimization for the extraction of flavonoids from Trollius chinensis Bunge stems

[0105] After determining the co-extraction method, taking the ultrasonic extraction power, liquid-to-material ratio, ultrasonic time, etc. as single-factor investigation indicators, and using the comprehensive evaluation score of the function evaluation of Trollius chinensis Bunge stems as the response index, the response surface optimization method (Response Surface Methodology, RSM) is used to determine the optimal extraction process.

[0106] Example 1: Determination of the extraction method of flavonoids from Trollius chinensis Bunge stems

[0107] Based on the previous research foundation and relevant literature, five extraction methods such as ultrasonic + ethanol extraction method, ultrasonic + enzymatic hydrolysis method, ultrasonic + microwave method, ultrasonic + enzymatic hydrolysis + ethanol extraction method, and ultrasonic + enzymatic hydrolysis + microwave extraction method are proposed. The steps of each extraction method are as follows:

[0108] (1) Ultrasonic + ethanol extraction method (UA). Weigh 10 g of pretreated Trollius chinensis Bunge, add 20 times the weight of 70% ethanol, stir evenly with a glass rod and place it in a 70 °C constant temperature water bath. After extraction for 1 h, perform ultrasonic-assisted extraction. The ultrasonic power is 400 W, the ultrasonic temperature is 70 °C, and the ultrasonic time is 40 min. After the sample cools, perform suction filtration, place it in the refrigerator for about 4 h, then centrifuge, take the supernatant to obtain the crude flavonoid solution of Trollius chinensis Bunge, measure the volume of the crude flavonoid and set it aside.

[0109] The process for obtaining the crude flavonoid powder of Trollius chinensis Bunge stems and testing each index is as follows: Filter the obtained crude flavonoid solution of Trollius chinensis Bunge, place it in the refrigerator for about 4 h, then centrifuge, take the supernatant to obtain the crude flavonoid solution of Trollius chinensis Bunge. Rotate and evaporate the extracted crude flavonoid solution to obtain a concentrated solution. Freeze it in an ultra-low temperature freezer for 12 h, then put it into a vacuum dryer for freeze-drying, repeat 2 - 3 times to obtain the crude flavonoid powder. According to the test methods of DPPH free radical scavenging rate, ABTS free radical scavenging rate, and hydroxyl free radical scavenging rate, test the in vitro antioxidant activity of the crude flavonoid of Trollius chinensis Bunge. According to the yield test method, test the yield of the crude flavonoid of Trollius chinensis Bunge. According to the test results of each index obtained above, use the coefficient of variation method to determine the weight coefficients of each function evaluation index of the flavonoids in Trollius chinensis Bunge stems, and use the Z-score normalization method to normalize the data of each function evaluation index of the polysaccharides in Trollius chinensis Bunge stems. The sum of the normalized data of each index multiplied by the corresponding weight coefficient is used to obtain the total comprehensive evaluation score.

[0110] (2) Ultrasonic + enzymatic hydrolysis method (UE). Weigh 10.0 g of pretreated Trollius chinensis, add 20 times the weight of 70% ethanol by volume, the addition amount of cellulase is 0.20 wt% of the Trollius chinensis stem, and enzymatically hydrolyze in a constant temperature water bath at 50 °C for 70 min. The ultrasonic extraction conditions, the method for obtaining the crude flavonoid powder of the Trollius chinensis stem, and the test procedures for each index are the same as above.

[0111] (3) Ultrasonic + microwave method (UM). Weigh 10.0 g of pretreated Trollius chinensis, add 20 times the weight of 70% ethanol by volume, the microwave power is 640 W, and the microwave time is 10 min. The ultrasonic extraction conditions, the method for obtaining the crude flavonoid powder of the Trollius chinensis stem, and the test procedures for each index are the same as above.

[0112] (4) Ultrasonic + enzymatic hydrolysis method + ethanol extraction method (UEA). Weigh 10.0 g of pretreated Trollius chinensis, add 20 times the weight of 70% ethanol by volume, the addition amount of cellulase is 0.20 wt% of the Trollius chinensis stem, and enzymatically hydrolyze in a constant temperature water bath at 50 °C for 70 min; then place it in a constant temperature water bath at 70 °C for extraction for 1 h, and then perform ultrasonic-assisted extraction. The ultrasonic extraction conditions, the method for obtaining the crude flavonoid powder of the Trollius chinensis stem, and the test procedures for each index are the same as above.

[0113] (5) Ultrasonic + enzymatic hydrolysis method + microwave extraction method (UEM). Weigh 10.0 g of pretreated Trollius chinensis, add 20 times the weight of 70% ethanol by volume, the addition amount of cellulase is 0.20 wt% of the Trollius chinensis stem, and enzymatically hydrolyze in a constant temperature water bath at 50 °C for 70 min; then perform microwave-assisted (microwave power 640 W, microwave time 10 min) and ultrasonic-assisted extraction in sequence. The ultrasonic extraction conditions, the method for obtaining the crude flavonoid powder of the Trollius chinensis stem, and the test procedures for each index are the same as above.

[0114] Example 2: Determination of the flavonoid yield of Trollius chinensis stem

[0115] For the determination of the yield of flavonoids from the stems of Trollius chinensis in Example 1, as can be seen from Table 1, the yield of the ultrasonic + ethanol extraction method is relatively low, indicating that this method may not fully utilize the auxiliary effect of ultrasound to improve the extraction efficiency; the yield of the ultrasonic + enzymolysis extraction method is the highest, indicating that the auxiliary effect of the enzyme significantly increases the yield, which may be because cellulase can decompose the cell wall, making it easier for the solvent to contact flavonoid compounds; the yield of the ultrasonic + microwave extraction method is higher than that of the ultrasonic + ethanol extraction method, but lower than that of the ultrasonic + enzymolysis and ultrasonic + enzymolysis + ethanol extraction methods. Microwave may help increase the contact area between the solvent and the raw material, but the extraction effect is still limited; the yield of the ultrasonic + enzymolysis + ethanol extraction method is relatively high, close to that of the ultrasonic + enzymolysis method, indicating that combining cellulase treatment and ethanol solvent can effectively increase the yield of flavonoid compounds; the yield of the ultrasonic + enzymolysis + microwave method is lower than that of the ultrasonic + enzymolysis and ultrasonic + enzymolysis + ethanol extraction methods, which may be due to some limitations during the microwave process, or the combination of cellulase and microwave does not produce a synergistic effect.

[0116] Table 1 Flavonoid yields obtained by different extraction methods

[0117]

[0118]

[0119] Note: Different lowercase letters represent significant differences at the 0.05 level.

[0120] In summary, from the perspective of yield, the UE method is the most efficient, followed by the UEA extraction method. Considering the cost, the cost of cellulase is moderate and the treatment process is relatively simple.

[0121] Example 3: Evaluation of the functional activities of flavonoids from the stems of Trollius chinensis

[0122] Referring to the extraction template conditions of crude flavonoids from Trollius chinensis,

[0123] The yields and antioxidant effects of flavonoids obtained by different extraction methods were evaluated, and the synergistic extraction method was finally determined. In view of the evaluation of the antioxidant effect based on the IC 50 , IC 50 The smaller the value, the better the antioxidant effect. Therefore, in order to more conveniently calculate the comprehensive score based on the coefficient of variation, the yield in the above examples was replaced with the loss rate (calculation formula: 1 - yield %). That is, the smaller the loss rate, the higher the yield.

[0124] After calculating the mean value of the IC 50 of the antioxidant index and the mean value of the loss rate, the weight coefficient results of each functional evaluation index calculated by the coefficient of variation method are shown in Table 2:

[0125] Table 2 Calculation of weight coefficients

[0126]

[0127]

[0128] According to Figure 2 it can be seen that the DPPH and ABTS free radical scavenging IC 50 values of the UM and UE extraction methods are relatively low, showing good antioxidant effects; the hydroxyl radical scavenging IC 50 values of the UM and UMA extraction methods are relatively low, showing good antioxidant effects. From Figure 3 it can be seen that the loss rate of the UE method is the lowest and the product yield is the highest. According to the weight coefficients of each index calculated in Table 2, the sum of the normalized data of each index multiplied by the weight coefficient can be calculated, which is the total comprehensive evaluation score. The comprehensive scores of the antioxidant effects and loss rates of flavonoids obtained by different extraction methods are as Figure 4 shown. From the results in the figure, it can be seen that the comprehensive score of the UE extraction method is significantly higher than (P<0.05) that of other extraction methods. Therefore, the final extraction scheme is determined to be the UE extraction method.

[0129] Example 4: Single-factor experiment

[0130] In order to further determine the optimal process for flavonoid extraction by the ultra-enzyme (UE) method, five single factors, namely ultrasonic time (A), ultrasonic power (B), liquid-to-solid ratio (C), enzyme addition amount (D), and ethanol volume fraction (E), were set respectively, and single-factor experiments were carried out. The experimental results are as Figure 5 (a)- Figure 5 (e) shown.

[0131] From Figure 4 analysis, it can be seen that in the first single factor of ultrasonic time, the comprehensive score is the highest when the ultrasonic time is 40 minutes, indicating that the treatment time of 40 minutes of ultrasonic time may be more optimal under the test conditions. When the ultrasonic time is 50 minutes, the comprehensive score decreases, indicating that too long treatment time may lead to component degradation or other adverse effects. When the ultrasonic time is 60 minutes, the comprehensive score is about 0.8, which is still relatively low, further supporting the trend observed at 50 minutes of ultrasonic time.

[0132] In the second single factor of ultrasonic power, the comprehensive score is the highest when the power is 400W, indicating that the power condition of 400W may be more suitable for extraction. When the power is 500W, the comprehensive score drops to about 0.15, probably because too high power causes damage to the active ingredients. When the ultrasonic power is 600W, the comprehensive score is about 0.5, further confirming that high power may be unfavorable for extraction.

[0133] In the third single-factor liquid-to-solid ratio, the comprehensive score of the liquid-to-solid ratio of 50 mL / g is the highest, about 0.95. The comprehensive score of the liquid-to-solid ratio of 40 mL / g is about 0.16, showing a significant decrease. However, the comprehensive score of the liquid-to-solid ratio of 60 mL / g is about 0.75, showing a recovery. Therefore, it is judged that the error may be caused by improper experimental operation.

[0134] In the fourth single-factor enzyme addition amount, the comprehensive score is the highest when the enzyme dosage is 1.0%, indicating that the enzyme dosage of 1.0% may be the most effective. The comprehensive score drops to about 0.78 when the enzyme addition amount is 1.5%, indicating that too high an enzyme dosage may cause adverse effects. The comprehensive score is the lowest when the enzyme dosage is 0.1%, indicating that too low an enzyme addition amount has a poor effect on the extraction of flavonoids.

[0135] In the fifth single-factor volume fraction of ethanol, the comprehensive scores of 55%, 60%, 65% and 70% are similar, and the difference is not significant. The comprehensive score of 75% drops significantly to 0.6, which may be due to the fact that too high an ethanol concentration will significantly reduce the extraction effect, or it may be due to errors caused by improper experimental operation. After comprehensive consideration, this factor is discarded.

[0136] A four-factor and three-level response surface analysis factor and level table is established, as shown in Table 3.

[0137] Table 3 Response surface analysis factor and level table

[0138]

[0139]

[0140] Example 5: Results of Box-Behnken experimental design

[0141] According to the single-factor test results in Example 4, taking the comprehensive scores of the antioxidant index IC 50 and the loss rate as the response values, the effects of 4 factors, namely ultrasonic time (A), ultrasonic power (B), liquid-to-solid ratio (C), and enzyme addition amount (D), on the comprehensive score (Y) were investigated.

[0142] Table 4 Box-Behnken test scheme and results

[0143]

[0144]

[0145] As can be seen from Table 4, there are a total of 29 groups in the experimental design, including 5 zero-point tests and 24 factorial tests.

[0146] Example 6: Establishment and significance test of the regression equation

[0147] The response surface was analyzed using Design Expert 10.0.1 software, and the quaternary quadratic regression model equation is as follows:

[0148] y = 0.96168A + 0.002875B - 0.00445C + 0.012433333D + 0.029425AB + 0.00555AC + 0.0291AD + 0.010125BC - 0.033925BD + 0.070575CD - 0.071060833A 2 - 0.074535833B 2 - 0.059823333C 2 - 0.091873333D 2

[0149] The results of the regression analysis are shown in Table 5.

[0150] Table 5 Results of Response Surface Regression Analysis

[0151]

[0152] The P - value in the table can represent the significance of the influence of each variable in the regression equation on the response value, and the significance level of the corresponding variable is negatively correlated with the P - value, that is, the smaller the P - value, the higher the significance of the variable.

[0153] It can be seen from Table 5 that the model reaches an extremely significant level and the lack - of - fit term is not significant, indicating that this model has a good fit with the actual situation. The linear term of ultrasonic time (A) has an extremely significant influence on the model (P < 0.01), and the enzyme addition amount (D) has a significant influence on the model (P < 0.05); the interaction term CD (liquid - to - material ratio and enzyme addition amount) has an extremely significant interaction effect (P < 0.01), and the interaction terms AB (ultrasonic time and ultrasonic power), AD (ultrasonic time and enzyme addition amount), BD (ultrasonic power and enzyme addition amount) have significant interaction effects (P < 0.05); the influence of the quadratic terms of each factor A2, B2, C2, D2 all reaches an extremely significant level (P < 0.01), further indicating that the influence of each factor on the response surface is not a simple linear relationship. Comparing the influence degrees of each factor on the comprehensive score, the order from large to small is ultrasonic power > liquid - to - material ratio > enzyme addition amount > ultrasonic time.

[0154] Combined with the regression mathematical model analysis, solving the above - mentioned quadratic regression equation, the optimal extraction process conditions can be obtained: ultrasonic time 42.444 min, ultrasonic power 404.421 W, liquid - to - material ratio 50.459 mL / g, enzyme content 1.058%. Considering the operability of the process, the parameters are adjusted to: ultrasonic time 40 min, ultrasonic power 400 W, liquid - to - material ratio 50 mL / g, enzyme content 1.0%. Under these conditions, the comprehensive score is 0.966.

[0155] Example 7: Response surface interaction analysis and verification test under the optimal process conditions

[0156] According to the response surface interaction analysis, there are significant non-linear effects of various factors on the comprehensive score of flavonoid extraction rate. Through the three-dimensional response surface and contour line analysis, it can be seen that all response surfaces show the characteristics of a downward-opening parabola, indicating that the comprehensive score first increases and then decreases with the increase of each factor level, and there is a clear extreme point corresponding to the optimal extraction conditions. Among them, the interaction between the liquid-to-solid ratio and the enzyme content is the most significant, with the steepest slope of its response surface and obvious elliptical contour lines, indicating that the synergistic effect of the two has a prominent impact on the extraction rate.

[0157] Further comparison of the interaction between various factors reveals that in the interaction between ultrasonic time and liquid-to-solid ratio, the contribution of ultrasonic time is higher; while in the interaction between ultrasonic time and enzyme content, the enzyme content is the dominant factor. In the interactions between ultrasonic power and the liquid-to-solid ratio and enzyme content respectively, the contribution degrees of both sides are similar. The contour line morphology analysis confirms that the interaction of the factor combinations corresponding to the elliptical contour lines (such as liquid-to-solid ratio - enzyme content) is significant, while the combined effect of the nearly circular contour line combinations is weak.

[0158] By comprehensively evaluating the influence intensity of each single factor, their importance ranking is: ultrasonic power > liquid-to-solid ratio > enzyme addition amount > ultrasonic time. This result reveals that in the flavonoid extraction process, the energy input parameter (ultrasonic power) and the solvent system parameter (liquid-to-solid ratio) have a stronger regulatory effect on the extraction efficiency, providing a key theoretical basis for process optimization. To verify the effectiveness of the model, a repeated verification test was carried out. The comprehensive score of the flavonoids from Trollius chinensis Bunge stems obtained was 0.971, higher than the theoretical predicted value of 0.966, indicating that there is a good fit between the predicted value and the actual value, thus indicating that this regression mathematical model can well predict the yield under different conditions and has certain practical value.

[0159] Example 8: Evaluation and comparison of the antioxidant effects of flavonoids from Trollius chinensis Bunge stems obtained after optimizing the process

[0160] The antioxidant effects of the flavonoids from Trollius chinensis Bunge stems extracted by the optimal process (ultrasonic time 40 min, ultrasonic power 400 W, liquid-to-solid ratio 50 mL / g, enzyme content 1.0%) in the UE method obtained in Example 6 were compared with those of the crude flavonoids from Trollius chinensis Bunge stems extracted by the ultrasonic + ethanol extraction method (UA).

[0161] The specific process of ultrasonic + ethanol extraction method is as follows: The powder of Trollius chinensis Bunge stems after crushing and sieving is placed in hot water at 70°C with a solid-liquid ratio of 1:20 (g / mL) for extraction. After 1 hour of extraction, ultrasonic-assisted extraction is carried out. The ultrasonic power is 400 W, the ultrasonic temperature is 70°C, and the ultrasonic time is 40 minutes. After the sample is cooled, filtration is carried out, and centrifugation is carried out after placing it in the refrigerator for about 4 hours. The supernatant is taken to obtain the crude flavonoid solution of Trollius chinensis Bunge stems. The obtained crude flavonoid solution of Trollius chinensis Bunge stems is rotary evaporated to obtain a concentrated solution, which is placed in an ultra-low temperature freezer and frozen at -80°C for 12 hours, and then freeze-dried in a vacuum dryer, repeating 2 - 3 times to obtain the crude flavonoid powder of Trollius chinensis Bunge stems. The comparison results are shown in Table 6.

[0162] Table 6 Comparison of flavonoids from Trollius chinensis Bunge stems and Vc IC 50 Data comparison

[0163]

[0164] As can be seen from Table 6, the antioxidant ability of flavonoids extracted from Trollius chinensis Bunge stems after optimizing the process is weaker than that of Vc. However, the IC 50 of flavonoids from Trollius chinensis Bunge stems against DPPH is 0.141 mg / mL, while the IC 50 of Vc against DPPH is 0.023 mg / mL. It can be seen that flavonoids from Trollius chinensis Bunge stems have good DPPH scavenging ability. Compared with the ultrasonic + ethanol extraction method, the IC 50 of flavonoids from Trollius chinensis Bunge stems obtained after optimizing the process against DPPH decreased from 0.162 mg / mL to 0.141 mg / mL, and the scavenging ability increased by 1.15 times; compared with the total flavonoids from the stems and leaves of Astragalus membranaceus (IC 50 is 0.933 mg / mL) (Food Research and Development, 2025, 46(05): 94 - 101), which shows outstanding antioxidant performance among flavonoids of the same kind of plants, its antioxidant effect also increased by 6.6 times. Compared with flavonoids from Olea europaea leaves (IC 50 is 0.64 mg / mL) (Chemistry and Industry of Forest Products, 2024, 44(06): 167 - 172), its antioxidant effect also increased significantly.

[0165] As can be seen from Table 6, the IC 50 of Vc for the scavenging rate of ABTS free radicals is 0.016 mg / mL, while the IC 50 of flavonoids from Trollius chinensis Bunge stems against ABTS is 0.041 mg / mL. Although the ability to scavenge ABTS is weaker than that of Vc, compared with the ultrasonic + ethanol extraction method, the IC 50 of flavonoids from Trollius chinensis Bunge stems obtained by UE extraction against ABTS decreased from 0.049 mg / mL to 0.041 mg / mL, and the scavenging ability increased by 1.2 times; compared with the natural flavonoid daphnetin (IC 50Compared with the value of 0.148 mg / mL (Modern Chinese Medicine Research and Practice, 2024, 38(06): 27 - 31), its clearance rate increased by nearly 3.6 times.

[0166] Regarding the scavenging rate of hydroxyl radicals, Vc still showed better scavenging effect, and its IC 50 was 0.092 mg / mL, while the IC 50 of flavonoids from Trollius chinensis Bunge stems extracted by UE was 0.330 mg / mL. Compared with the ultrasonic + ethanol extraction method (IC 50 was 0.693 mg / mL), the antioxidant effect was increased by 2.1 times. Compared with the scavenging ability of total flavonoids extract from Artemisia argyi Levl. et Vant. on hydroxyl radicals (IC 50 was 0.185 mg / mL) (Chemical Engineer, 2024, 38(10): 95 - 99), its antioxidant effect was also significantly improved.

[0167] Example 9: Comparison of other functional activity effects of flavonoids from Trollius chinensis Bunge stems extracted by different methods

[0168] The flavonoids from Trollius chinensis Bunge stems extracted by the optimal process (ultrasonic time 40 min, ultrasonic power 400 W, liquid - to - material ratio 50 mL / g, enzyme content 1.0%) in the UE method obtained in Example 6 were compared with the crude flavonoids from Trollius chinensis Bunge stems extracted by other methods in terms of hypoglycemic, hypolipidemic and moisturizing effects. The comparison results are shown in Table 7.

[0169] Table 7 IC 50 Data comparison between flavonoids from Trollius chinensis Bunge stems and Vc

[0170]

[0171] In terms of hypoglycemic activity, the activities of the UE sample (0.023 mg / mL) and the UA sample (0.028 mg / mL) were relatively weak, which might be related to the degradation of heat - sensitive components during the extraction process; compared with the UM, UEA, and UEM samples, they had better α - glucosidase inhibitory ability, suggesting that its optimized process might enhance the regulation of glucose metabolism by retaining specific flavonoid glycoside structures. The sample prepared by the UE method was compared with the flavonoids from Actinidia arguta (Sieb. & Zucc.) Planch. ex Miq. with better reported hypoglycemic effect (IC 50 was 1.87 mg / mL) (Yao Jiaqi. Research on the Optimization of Extraction Process and Hypoglycemic Activity of Flavonoids from Actinidia arguta (Sieb. & Zucc.) Planch. ex Miq. [D]. Liaoning University, 2023. DOI: 10.27209 / d.cnki.glniu.2023.000017.), and it had an extremely significant advantage in the inhibitory effect on α - glucosidase (the hypoglycemic IC 50 of the sample prepared by this method was better than the hypoglycemic IC 50 of the sample in the comparative literature by nearly 82 times).

[0172] In terms of hypolipidemic activity, the IC of pancreatic lipase inhibition of UEA (3.040 mg / mL) and UE (3.044 mg / mL) 50 value was significantly lower than that of other samples (P < 0.05), and the hypolipidemic effect was significantly improved, indicating that the synergistic extraction process (such as enzyme-assisted) may be more conducive to releasing hydrophobic flavonoid components, thereby enhancing the lipid-binding efficiency; the UEM sample (3.592 mg / mL) had the lowest activity and the worst hypolipidemic effect, presumably because the composite process introduced impurities that interfered with the flavonoid-enzyme interaction. Among them, the UE sample and the raspberry seed flavonoids with better lipid-regulating efficacy (the IC of sodium glycocholate, sodium taurocholate, and sodium cholate were 2.911, 3.779, and 3.257 mg / mL respectively) (Journal of Chinese Institute of Food Science and Technology, 2019, 19(10): 172-178.) 50 In comparison, UE also showed better lipid-regulating efficacy.

[0173] In terms of moisture retention performance, the UE sample (98.92%) was the best. The proportion of its hydroxy / glycosylated modified flavonoids may be higher, enhancing the water molecule retention ability through the hydrogen bond network, which was 1.1 percentage points higher than that of the traditional process (UA: 97.82%), approaching the hyaluronic acid benchmark value (>99%).

[0174] In summary, the UE process is outstanding in comprehensive functional activity. It balances the polarity and stability of flavonoid components, providing a theoretical basis for the development of natural products with both metabolic regulation and skin barrier repair functions.

[0175] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. An ultrasonic composite extraction method for flavonoids from Trollius chinensis Bunge stems, comprising the following steps: Crush the Trollius chinensis Bunge stems, use an ethanol aqueous solution as a solvent, and carry out enzymatic hydrolysis with cellulase; after enzymatic hydrolysis, place it in an ultrasonic extractor for extraction, and then carry out freeze-drying.

2. The method according to claim 1, comprising the following steps: (1) Dry the Trollius chinensis Bunge stems to a constant weight, crush them, sieve them, and store for later use; (2) Add an ethanol aqueous solution and cellulase to the Trollius chinensis Bunge stem powder obtained in step (1) for enzymatic hydrolysis; (3) Place the enzymatic hydrolysate of Trollius chinensis Bunge stems obtained in step (2) in an ultrasonic extractor for extraction. After the sample cools, carry out suction filtration to obtain the enzymatic hydrolysis and ultrasonic extraction solution of Trollius chinensis Bunge stems; (4) Let the enzymatic hydrolysis and ultrasonic extraction solution of Trollius chinensis Bunge stems obtained in step (3) stand at 4 - 5 °C and then carry out centrifugation. Take the supernatant to obtain the crude flavonoid solution of Trollius chinensis Bunge; (5) Carry out rotary evaporation on the crude flavonoid solution of Trollius chinensis Bunge obtained in step (4) to obtain a concentrated solution. After ultra-low temperature freezing, put it into a vacuum dryer for freeze-drying, repeat 2 - 3 times to obtain the crude flavonoid powder.

3. The method according to claim 2, further comprising the following steps: (6) Measure the DPPH free radical scavenging rate, ABTS free radical scavenging rate, and hydroxyl free radical scavenging rate of the three in vitro antioxidant activities and the yield of the crude flavonoids from Trollius chinensis Bunge stems obtained in step (5); (7) Use the test results of each property index obtained in step (6), determine the weight coefficients of each functional evaluation index of the flavonoids from Trollius chinensis Bunge stems by the coefficient of variation method, use the Z-score normalization method to normalize the data of each functional evaluation index of the flavonoids from Trollius chinensis Bunge stems, and sum the products of the normalized data of each index and the corresponding weight coefficients to obtain the total comprehensive evaluation score.

4. The method according to claim 2 or 3, In step (2), the material-liquid ratio is 1:20 - 1:60 g / mL, the ethanol volume fraction is 55 - 70 V%, and the cellulase addition amount is 0.1 - 1.5 wt% of the Trollius chinensis Bunge stem powder.

5. The method according to claim 4, in step (2), the ethanol volume fraction is 70 V%, the cellulase addition amount is 1.0 wt% of the Trollius chinensis Bunge stem powder, and the material-liquid ratio is 1:50 g / mL.

6. The method according to claim 2 or 3, wherein in step (3), the ultrasonic power setting range is 200 - 600 W and the ultrasonic time is 20 - 60 min.

7. The method according to claim 6, wherein in step (3), the ultrasonic power setting range is 400 W and the ultrasonic time is 40 min.

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