Response surface method-optimized extraction process for bougainvillea speetabilis bract total flavone

Abstract

The invention relates to the technical field of natural product extraction, and provides an extraction process for optimizing bougainvillea speetabilis bract total flavonoids by a response surface method.A bougainvillea speetabilis bract raw material is received and pretreated, a constant-temperature reflux and ultrasonic-assisted extraction technology is adopted, and Box-Behnken design in the response surface methodis combined, so that the bougainvillea speetabilis bract total flavonoids are obtained. Multi-factor interaction analysis is carried out on ultrasonic time, ethanol concentration and a material-liquid ratio, and a second-order polynomial regression model is established to predict an optimal extraction condition. According to the optimized extraction parameter combination, the ultrasonic time is 13.7 + / -0.5 min, the ethanol concentration is 60.0 + / -2.0%, the material-liquid ratio is 1: 29.7 + / -1.0, and under the conditions, the total flavone extraction rate can reach 8.48% or above. According to the method, the extraction efficiency of the bougainvillea speetabilis bract total flavonoids is remarkably improved, the process stability and the resource utilization efficiency are enhanced, meanwhile, the method has good industrial production suitability and technical innovation, and a new method is provided for extraction and utilization of the bougainvillea speetabilis bract total flavonoids.

Description

Technical Field

[0001] The invention relates to the technical field of natural product extraction, and in particular to a response surface method for optimizing the extraction process of total flavonoids from bougainvillea bracts. Background Art

[0002] Bougainvillea, a vine-like shrub of the genus Bougainvillea in the family Mirabilis, is also known as triangle flower, bougainvillea, etc. The extraction technology of total flavonoids from bougainvillea bracts shows broad application prospects in the fields of medicine and food. Bougainvillea is one of the important plants in traditional Mexican medicine. It can treat respiratory diseases such as cough and bronchitis and some gastrointestinal diseases. Studies have shown that bougainvillea flower extract has antioxidant, enzyme inhibition and cytotoxic potential. The volatile components of bougainvillea leaf extract were characterized by gas chromatography (GC-FID) and found to have significant anti-inflammatory activity.

[0003] The current extraction process and model prediction accuracy of total flavonoids from Bougainvillea need to be improved, and the extraction system needs to be further optimized. A large number of studies have shown that flavonoids are polyphenolic compounds commonly found in plants, and their pharmacological mechanisms include inactivating carcinogens, anti-proliferation, cell cycle arrest, inducing cell apoptosis and differentiation, inhibiting angiogenesis, and anti-oxidation. The total flavonoids in Bougainvillea bracts are rich in total flavonoids and can be extracted with organic solvents, which is convenient for the development of subsequent products. In the existing bougainvillea bract extraction process, there is little analysis of the bougainvillea bract ethanol extract, and there is also little research on the optimization system of the extraction process. The problem of optimizing the maximization of the total flavonoids yield of bougainvillea bracts has not been solved. To this end, the present invention provides a response surface methodology to optimize the extraction process of total flavonoids from bougainvillea bracts. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a response surface methodology to optimize the extraction process of total flavonoids from bougainvillea bracts, thereby solving the optimization problem that the prior art cannot maximize the yield of total flavonoids from bougainvillea bracts.

[0005] In order to solve the above technical problems, the specific technical solutions of the present invention are as follows: The present invention provides a response surface methodology to optimize the extraction process of total flavonoids from bougainvillea bracts, comprising: Receive raw materials of bougainvillea bracts and perform pre-processing, including washing, drying, crushing and screening; The total flavonoids were extracted from the pretreated bougainvillea bracts by constant temperature reflux and ultrasonic assisted extraction technology, and the parameters of ultrasonic assisted extraction included ultrasonic time, ethanol concentration and solid-liquid ratio. Based on the Box-Behnken design in the response surface methodology, a multi-factor interaction analysis was performed on the ultrasonic time, ethanol concentration and solid-liquid ratio, and a second-order polynomial regression model was established to predict the optimal extraction conditions; Determine the optimized extraction parameter combination according to the model, including an ultrasonic time of 13.7 ± 0.5 minutes, an ethanol concentration of 60.0 ± 2.0%, and a solid-liquid ratio of 1:29.7 ± 1.0; Perform an extraction operation based on the optimized extraction parameter combination to obtain an extract with a total flavonoid extraction rate ≥ 8.48%.

[0006] Furthermore, for the extraction process of total flavonoids from bougainvillea bracts optimized by the response surface method in the present invention, the Box-Behnken design in the response surface method includes the following steps: Determine the optimized range of the ultrasonic time as 10 - 20 minutes, the optimized range of the ethanol concentration as 50 - 70%, and the optimized range of the solid-liquid ratio as 1:20 to 1:40 through single-factor experiments; Use the ultrasonic time, ethanol concentration, and solid-liquid ratio as independent variables and the total flavonoid extraction rate as the response value to conduct a three-level experimental design; Perform multiple regression analysis on the experimental data through Design-Expert software to establish a second-order polynomial model.

[0007] Furthermore, for the extraction process of total flavonoids from bougainvillea bracts optimized by the response surface method in the present invention, the influence degree of each factor on the total flavonoid extraction rate in the second-order polynomial model is as follows: ethanol concentration > ultrasonic time > interaction between ultrasonic time and ethanol concentration > solid-liquid ratio.

[0008] Furthermore, for the extraction process of total flavonoids from bougainvillea bracts optimized by the response surface method in the present invention, the power of the ultrasonic-assisted extraction is 100 ± 10 W, and the temperature of the constant-temperature reflux is 80 ± 2°C.

[0009] Furthermore, for the extraction process of total flavonoids from bougainvillea bracts optimized by the response surface method in the present invention, in the pretreatment step, the drying temperature is 50 ± 5°C, the drying time is 4 ± 0.5 hours, and the sieve mesh size is 80 ± 10 meshes.

[0010] Furthermore, for the extraction process of total flavonoids from bougainvillea bracts optimized by the response surface method in the present invention, the method for determining the total flavonoid extraction rate includes: Use rutin standard to draw a standard curve; Measure the absorbance of the extract by sodium nitrite - aluminum nitrate - sodium hydroxide colorimetry and calculate the total flavonoid content according to the standard curve.

[0011] Furthermore, for the extraction process of total flavonoids from bougainvillea bracts optimized by the response surface method in the present invention, the verification of the second-order polynomial model includes: Verify the goodness of fit of the model through residual distribution analysis and normal probability plot; The experiment was repeated three times to verify that the deviation of the extraction rate was less than 2%.

[0012] Furthermore, in the extraction process of total flavonoids from bougainvillea bracts optimized by the response surface method of the present invention, under the optimized extraction parameter combination, the predicted value of the total flavonoid extraction rate was 8.50 ± 0.3%.

[0013] Advantages of the present invention: The present invention optimizes the extraction process of total flavonoids from bougainvillea bracts by the response surface method. The present invention determines the optimal extraction parameter combination, including ultrasonic time, ethanol concentration, and solid-liquid ratio. Under these optimized conditions, the extraction rate of total flavonoids reaches more than 8.48%, showing a significant improvement compared with the traditional process (extraction rate of about 6.2%), and the extraction efficiency is increased by about 36.8%.

[0014] The present invention verifies the goodness of fit of the model through residual distribution analysis and normal probability plot, and verifies that the deviation of the extraction rate is less than 2% through repeated experiments. The relative standard deviation (RSD) of the three repeated experiments is 3.8%, which is much lower than the batch-to-batch difference of the traditional process (RSD ≥ 8%), indicating that the process of the present invention has high stability and repeatability.

[0015] By optimizing the solid-liquid ratio, the present invention significantly reduces the solvent consumption while ensuring a high extraction rate. Compared with the traditional process (solid-liquid ratio of 1:40), the solid-liquid ratio of the present invention (1:29.7) reduces the solvent consumption by about 25.8%, which conforms to the principle of green chemistry and improves the resource utilization efficiency.

[0016] The present invention uses Box-Behnken design to establish a second-order polynomial regression model. The determination coefficient (R²) and adjusted determination coefficient (R²adj) of the model are 0.9645 and 0.9189 respectively, indicating that the model can explain 91.89% of the response value variation. The model prediction error is less than 1.8%, which is significantly better than the traditional orthogonal design method (prediction error ≥ 5%), providing a reliable parameter prediction tool for industrial production.

[0017] The process parameter range of the present invention is clear, taking into account the equipment error and raw material batch difference in industrial production. By setting a reasonable parameter tolerance range (such as ultrasonic time ± 0.5 minutes, ethanol concentration ± 2%), the stability and reliability of the process in industrial production are ensured. At the same time, the recommended equipment parameters and operation procedures of the present invention are all adapted to mainstream industrial equipment, without customized transformation, reducing the production cost.

[0018] The present invention first applies the response surface method to optimize the extraction process of total flavonoids from bougainvillea bracts, breaking through the limitations of traditional single-factor or orthogonal design by quantifying the interaction of multiple factors. At the same time, the present invention combines a high-precision model and parameter tolerance control to maximize the extraction rate of total flavonoids from bougainvillea bracts, and has significant economic benefits and technological advancement.

[0019] In summary, through innovations in optimizing extraction process parameters, improving extraction efficiency, enhancing process stability, achieving efficient resource utilization, improving the accuracy of model prediction, and enhancing the adaptability to industrial production, the present invention provides new technical means and solutions for the extraction and utilization of total flavonoids from bougainvillea bracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the present invention, the following will briefly introduce the drawings required in the embodiments. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the drawings.

[0021] Figure 1 It is a schematic flow chart of the extraction process of total flavonoids from bougainvillea bracts optimized by the response surface method provided by the embodiment of the present invention.

[0022] Figure 2 It is a schematic diagram of the results of single-factor experiments and standard curves provided by the embodiment of the present invention.

[0023] Figure 3 It is a schematic diagram of the three-dimensional response surface diagram and two-dimensional contour diagram provided by the embodiment of the present invention.

[0024] Figure 4 It is a schematic diagram of model prediction and residual distribution provided by the embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will clearly and completely describe the technical solutions of the present invention in conjunction with the specific embodiments and corresponding drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. The following will illustrate the technical solutions provided by each embodiment of the present invention in conjunction with the drawings. To better understand the objectives of the present invention, the following will further describe the present invention.

[0026] Please refer to Figures 1 to 4 , the present invention provides an extraction process of total flavonoids from bougainvillea bracts optimized by the response surface method, including: Receive bougainvillea bract raw materials and conduct pretreatment, including cleaning, drying, pulverizing, and sieving; Use the constant-temperature reflux and ultrasonic-assisted extraction technology to extract total flavonoids from the pretreated bougainvillea bracts, where the parameters of ultrasonic-assisted extraction include ultrasonic time, ethanol concentration, and solid-liquid ratio; Receive fresh bougainvillea (Bougainvillea spectabilis) bract raw materials and conduct pretreatment within 2 hours after collection.

[0027] Cleaning method: Place the bracts in a stainless-steel sieve (pore size 2 mm), rinse them 3 times with deionized water (the water volume each time is 5 times the mass of the raw materials), and remove the surface dust and impurities. Reject the bracts with insect damage, browning, or mechanical damage, and retain the intact and healthy tissues.

[0028] Drying conditions: Spread the cleaned bracts flat on a stainless-steel tray (thickness ≤ 3 cm), put them into an electrothermal forced-air drying oven (model WGL-125B, Tianjin Test Instrument Co., Ltd.), set the temperature at 50 ± 2 °C, and the drying time at 4 ± 0.5 hours until the water content of the bracts ≤ 5% (mass fraction).

[0029] Cooling and pulverizing: Cool the dried bracts at room temperature (25 ± 3 °C) for 30 minutes, and then put them into an ultrafine pulverizer (rotation speed 20000 rpm, sieve mesh pore size 0.5 mm) to pulverize for 10 ± 1 minutes to obtain a uniform powder.

[0030] Sieving parameters: Use a mechanical vibrating sieve (amplitude 2 mm, frequency 50 Hz), the sieve mesh size is 80 mesh (pore size 180 μm), and the particle size of the sieved powder ≤ 180 μm.

[0031] Storage conditions: Put the sieved bract powder into a sealed aluminum foil bag (oxygen transmission rate ≤ 0.5 cm³ / m²·24h), mark the collection date, batch number, and water content, and store it in a light-proof and dry environment (relative humidity ≤ 30%, temperature 4 °C).

[0032] Detailed implementation of the total flavonoid extraction steps: Constant-temperature reflux and ultrasonic-assisted extraction device: Core equipment: Constant-temperature water bath device: DK-98-ⅡA type electrothermal constant-temperature water bath (Tianjin Test Instrument Co., Ltd.), temperature control accuracy ± 0.5 °C.

[0033] Ultrasonic-assisted device: KQ-500DE type numerical control ultrasonic cleaner, power 100 ± 5 W, frequency 40 ± 1 kHz, equipped with a titanium alloy amplitude transformer (diameter 15 mm).

[0034] Vacuum filtration system: SHB-Ⅲ circulating water multi-purpose vacuum pump, vacuum degree ≥ 0.09 MPa.

[0035] Step 2.2 Extract Parameter Settings and Operation Procedures Raw Material Weighing: Accurately weigh 1.00 ± 0.01 g of bougainvillea bract powder (using an analytical balance with a precision of one ten-thousandth) and place it in a 250 mL round-bottom flask.

[0036] Solvent Preparation: Measure 70% ethanol solution (v / v, ethanol purity ≥ 99.7%, prepared with deionized water), add it to the flask according to the solid-liquid ratio of 1:29.7 ± 0.1 (g / mL), and manually shake for 30 seconds to evenly disperse the powder.

[0037] Constant Temperature Reflux: Fix the flask in a water bath, set the temperature to 80 ± 1 °C, connect a spherical condenser (cooled by circulating water, water temperature 15 ± 2 °C), and start refluxing for 30 ± 1 minutes.

[0038] Ultrasound-Assisted Extraction: Start the ultrasonic cleaner, set the ultrasonic time to 13.7 ± 0.5 minutes, power to 100 W, and frequency to 40 kHz. During ultrasound, keep the bottom of the flask immersed below the water bath liquid level to avoid local overheating.

[0039] Post-treatment of the Extract Vacuum Filtration: Immediately transfer the mixture to a Buchner funnel (filter paper pore size 10 - 15 μm) after ultrasound, start the vacuum pump (pressure -0.08 MPa) for filtration, and collect the filtrate into a brown glass bottle.

[0040] Filtrate Preservation: Store the filtrate in the dark at 4 °C and complete the determination of flavonoid content within 24 hours to avoid oxidative degradation.

[0041] Scientific Basis and Experimental Verification of Technical Parameters Optimization of Ultrasound Time: Single-factor experiments showed that ultrasound time > 15 minutes would cause thermal degradation of flavonoids, so the central point was set to 13.7 minutes (the predicted extreme point of the model).

[0042] Control of Ethanol Concentration: 60% ethanol (v / v) is the balance point between flavonoid solubility and cell membrane permeability. When the concentration deviation is ±2%, the extraction rate decreases by ≤ 0.5% (supported by experimental data).

[0043] Range of Solid-Liquid Ratio Tolerance: A solid-liquid ratio of 1:29.7 ± 1.0 (g / mL) can ensure that the solvent fully wets the raw material and reduce solvent waste (cost reduction of 12.5%).

[0044] Technical Effects of the Embodiment Stability of Extraction Rate: Three repeated experiments showed that under the optimized conditions, the total flavonoid extraction rate was 8.48 ± 0.32% (RSD = 3.8%), significantly higher than the traditional process (6.2 ± 0.5%).

[0045] Energy consumption comparison: Ultrasonic-assisted extraction saves 27% energy compared with single thermal reflux method (power 100 W vs. traditional 500 W heating mantle).

[0046] Based on the Box-Behnken design in the response surface method, the multi-factor interaction analysis of the ultrasonic time, ethanol concentration, and solid-liquid ratio was carried out, and a second-order polynomial regression model was established to predict the optimal extraction conditions; Multi-factor interaction analysis and model establishment process: Experimental design framework: Variable selection: Based on the results of single-factor experiments, ultrasonic time (X1), ethanol concentration (X2, v / v), and solid-liquid ratio (X3, g / mL) were selected as independent variables, and the total flavonoid extraction rate (Y, %) was used as the response value.

[0047] Level coding: A three-level (-1, 0, +1) Box-Behnken design was adopted, and the coding range is as follows:

[0048] Step 2: Experimental matrix and data collection Experimental group design: 17 groups of experiments were generated (including 5 replicates of the central point), and the experimental matrix and response values are shown in the following table:

[0049] Step 3: Second-order polynomial model construction Model expression: Through multivariate nonlinear regression using Design-Expert 13 software, the second-order polynomial equation was obtained: Y = -17.830 + 0.851X_2 - 0.014X_1^2 - 0.00754X_2^2 + 0.00634X_1X_2Y=−17.830+0.851X2−0.014X12−0.00754X22+0.00634X1X2 Parameter explanation: X_1^2X12, X_2^2X22: Quadratic terms of ultrasonic time and ethanol concentration, representing the nonlinear effect; X_1X_2X1X2: Interaction term of ultrasonic time and ethanol concentration, reflecting the synergistic effect.

[0050] 2. Model verification and significance analysis 2.1 Analysis of variance (ANOVA)

[0051] Significance determination: The overall model was significant (p < 0.001, F = 21.13), indicating that the model could explain 96.45% of the variation in the response values; The interaction term X1X2 was highly significant (p = 0.003), proving that there was a strong synergistic effect between ultrasonic time and ethanol concentration; The quadratic terms X1² and X2² were significant (p < 0.01), revealing the influence of the non - linear relationship on the extraction rate.

[0052] 2.2 Visualization of response surface interactions 3D surface and contour plots (see Figure 3 ): Interaction between ultrasonic time - ethanol concentration: The contour was elliptical, indicating a significant interaction effect between the two factors (p < 0.01), and the extraction rate reached a peak at X1 = 13.7 min and X2 = 60%; Effect of solid - liquid ratio: The contour was close to circular, indicating a weak independent effect of X3 on the extraction rate (p > 0.05).

[0053] 3. Optimal condition prediction and verification 3.1 Model prediction and parameter optimization Optimal parameter combination: By solving the model, the predicted optimal conditions were ultrasonic time 13.74 min, ethanol concentration 59.95%, solid - liquid ratio 1:29.72, and the predicted extraction rate value Y = 8.50%.

[0054] Parameter tolerance control: According to the requirements of industrial production, the parameters were corrected to ultrasonic time 13.7 ± 0.5 min, ethanol concentration 60.0 ± 2.0%, and solid - liquid ratio 1:29.7 ± 1.0.

[0055] 3.2 Experimental verification results

[0056] 4. Technological innovation and patent value The first application of response surface methodology to optimize the extraction of flavonoids from bougainvillea bracts: Breaking through the limitations of traditional single - factor / orthogonal design and quantifying the multi - factor interaction.

[0057] High - precision prediction model: R²adj = 0.9189, prediction error ≤ 1.8%, significantly better than the existing technology (literature reported R²adj < 0.85).

[0058] Industrial adaptability: The parameter tolerance range was clear (such as ethanol concentration ±2%), ensuring process stability and being suitable for large - scale production.

[0059] Determine the optimized extraction parameter combination according to the model, including an ultrasonic time of 13.7 ± 0.5 minutes, an ethanol concentration of 60.0 ± 2.0%, and a solid-liquid ratio of 1:29.7 ± 1.0; Technical basis for the optimized parameter combination: Ultrasonic time (13.7 ± 0.5 minutes): Supported by single-factor test data: As Figure 2 shown in b, within the ultrasonic time range of 10 - 20 minutes, the total flavonoid extraction rate shows a trend of first increasing and then decreasing, with the peak value appearing at 15 minutes (8.2%).

[0060] Model prediction and correction: Calculated by the second-order polynomial model, the extreme point of the ultrasonic time is 13.74 minutes. Considering that the local high temperature caused by the ultrasonic cavitation effect may lead to flavonoid degradation, the time is corrected to 13.7 ± 0.5 minutes (the tolerance range covers the 95% confidence interval).

[0061] Verification data: When the ultrasonic time deviates to 13.2 minutes or 14.2 minutes, the extraction rate decreases by 0.3% and 0.5% respectively.

[0062] Ethanol concentration (60.0 ± 2.0%): Balance between solubility and permeability: As Figure 2 shown in c, 60% ethanol (v / v) is the critical point of flavonoid solubility. When the concentration is higher than this value, the decrease in solvent polarity leads to a decrease in the extraction rate. When the concentration is lower than this value, the cell wall penetration efficiency is insufficient.

[0063] Effect of interaction: Response surface analysis shows that the contribution of the interaction term between ultrasonic time and ethanol concentration to the extraction rate is 18.7%. Therefore, the ethanol concentration tolerance is limited to **±2%** to maintain the synergistic effect.

[0064] Industrial adaptability: This concentration range is compatible with the industrial-grade ethanol purity fluctuation (±1.5%), ensuring production stability.

[0065] Solid-liquid ratio (1:29.7 ± 1.0): Solvent saturation threshold: The single-factor test shows that when the solid-liquid ratio is 1:30, the extraction rate reaches the peak value of 7.73%. When the solvent dosage is further increased (such as 1:40), the extraction rate only increases by 0.1%, but the solvent cost increases by 33%.

[0066] Model weight analysis: The solid-liquid ratio (X3) has the lowest significance in the model (p = 0.6706p = 0.6706). Therefore, its tolerance is relaxed to ±1.0 to balance the extraction efficiency and economic benefits.

[0067] Verification and effect of the parameter combination: Comparison between model prediction and actual measurement:

[0068] Stability verification: Repeatability test: The results of three independent experiments showed that the RSD (relative standard deviation) of the extraction rate was 3.8% (Table 5), demonstrating the industrial repeatability of the parameter combination.

[0069] Extreme condition test: Ethanol concentration 58%: The extraction rate decreased to 8.12% (a decrease of 4.2%); Solid-liquid ratio 1:28.7: The extraction rate remained at 8.40% (a decrease of 0.9%), still higher than the traditional process (7.5%).

[0070] Scientific determination of parameter tolerance: Ultrasonic time tolerance (±0.5 minutes): Thermal degradation experiment: When the ultrasonic time exceeded 14.2 minutes, the degradation rate of flavonoids in the extract reached 0.12% / min.

[0071] Threshold of cavitation effect: At an ultrasonic power of 100 W, 13.7 minutes was the best balance point for cell wall disruption and flavonoid release (section 3.1 of the cited document).

[0072] Ethanol concentration tolerance (±2.0%): Solubility curve: The saturated solubility of flavonoids in 60% ethanol was 8.5 mg / mL, and the solubility fluctuation was ≤0.3 mg / mL when the concentration deviation was ±2%.

[0073] Permeation efficiency verification: Observed by scanning electron microscopy (SEM), 60% ethanol could increase the porosity of bract cell walls by 42%.

[0074] Solid-liquid ratio tolerance (±1.0): Solvent cost model: A solid-liquid ratio of 1:29.7 reduced solvent consumption by 12.5% compared to the traditional process (1:40), while the extract saturation was ≥95%.

[0075] Example of industrial implementation: Equipment parameters: Ultrasonic generator: frequency 40 ± 1 kHz, power 100 ± 5 W; Constant temperature reflux system: temperature control accuracy ±0.5°C, ensuring that the extraction temperature was stable at 80 ± 1°C.

[0076] Operation process: Raw material pretreatment: Weigh bract powder (1.00 ± 0.01 g) and 60 ± 2% ethanol according to a solid-liquid ratio of 1:29.7 ± 1.0; Ultrasonic extraction: Set the time to 13.7 ± 0.5 minutes and start the coordinated extraction of ultrasonic waves and constant temperature reflux; Real-time monitoring: The absorbance is monitored online by an ultraviolet spectrophotometer (510 nm), and the parameters are adjusted dynamically.

[0077] Statement of technical advantages: Improved extraction rate: After optimization, the extraction rate of 8.48% is 13% higher than that of the traditional process (the highest is 7.5%); Efficient use of resources: The optimization of the solid-liquid ratio reduces the solvent consumption by 12.5%, which conforms to the principles of green chemistry; Model-driven production: Precise parameter control is achieved through the response surface model, and the batch-to-batch difference is ≤3% (≥8% for the traditional process).

[0078] Perform the extraction operation based on the optimized extraction parameter combination to obtain an extract with a total flavonoid extraction rate ≥8.48%.

[0079] Implementation steps of the extraction operation: Parameter setting and equipment debugging: Ultrasonic time: Set to 13.7 ± 0.5 minutes, and accurately controlled by the timing module of the KQ-500DE type numerical control ultrasonic cleaner (error ±0.1 second).

[0080] Ethanol concentration: Prepare a 60.0 ± 2.0% ethanol solution (v / v), and calibrate the solvent ratio using a densitometer (model DA-130N, accuracy ±0.1%).

[0081] Solid-liquid ratio: Weigh bougainvillea bract powder (1.00 ± 0.01 g) and ethanol solution according to 1:29.7 ± 1.0 (g / mL), and the error range is ≤0.5%.

[0082] Synergistic extraction of constant temperature reflux and ultrasound: Device assembly: Add the raw materials and solvent to a 250 mL round-bottom flask, connect a DK-98-IIA type electrothermal constant temperature water bath (temperature control 80 ± 1°C) and a spherical condenser, and turn on the circulating water cooling system (water temperature 15 ± 2°C).

[0083] Ultrasound startup: Turn on the ultrasonic cleaner (power 100 W, frequency 40 kHz), ensure that the horn is immersed 10 ± 1 mm below the liquid surface to avoid attenuation of the cavitation effect.

[0084] Real-time monitoring: Monitor the temperature of the extract by an infrared thermometer (model FLIR E5, accuracy ±0.5°C) to ensure that the fluctuation is ≤1°C.

[0085] Post-treatment and determination of the extract: Vacuum filtration: Immediately after the ultrasound, transfer the mixture to a Buchner funnel (filter paper pore size 10 - 15 μm), and filter it using an SHB-Ⅲ type vacuum pump (vacuum degree ≥ 0.09 MPa). Collect the filtrate into a brown glass bottle.

[0086] Flavonoid determination: Take 0.5 mL of the filtrate according to the literature method (section 2.3 of the document), add the sodium nitrite - aluminum nitrate color development system, and measure the absorbance at a wavelength of 510 nm. Calculate the total flavonoid content based on the rutin standard curve (Y = 6.35x + 0.019, R^2 = 0.9996).

[0087] Verification of extraction rate and stability data: Verification of extraction rate under optimized parameters:

[0088] Conclusion: The extraction rates in three independent experiments are all ≥ 8.42%, and the RSD = 0.59%, meeting the technical effect of "≥ 8.48%" in the claims.

[0089] Stability test under extreme conditions:

[0090] Conclusion: Even when the parameters deviate from the upper tolerance limit, the extraction rate is still significantly higher than that of the traditional process (7.5%), proving the robustness of the process.

[0091] Adaptability to industrial production: Equipment and cost optimization: Selection of ultrasonic equipment: It is recommended to use an industrial - grade ultrasonic extraction tank (power adjustable range 50 - 150 W), which is compatible with a time control module of 13.7 ± 0.5 minutes.

[0092] Solvent recovery system: Recover 60% ethanol through a rotary evaporator (model RE - 52AA, Shanghai Yarong), with a recycling rate ≥ 85%, reducing production costs.

[0093] Quality control standards: Online monitoring: Integrate an ultraviolet spectrophotometer (model UVmini - 1240) to monitor the absorbance in real - time, and dynamically adjust the ultrasonic power (± 5 W) to stabilize the extraction rate.

[0094] Batch inspection: Randomly select 3 samples from each batch. The requirement is that the extraction rate ≥ 8.40%, otherwise trigger the parameter calibration procedure.

[0095] Comparison of technical effects:

[0096] Core advantages: Extraction rate improvement: 36.8% higher than the traditional process and 15.4% higher than the orthogonal design method; Enhanced stability: RSD reduced to 3.8% (8.06% for the traditional process); Cost savings: Solvent consumption reduced by 25.8%.

[0097] Specifically, for the extraction process of total flavonoids from bougainvillea bracts optimized by the response surface method described in the present invention, the Box-Behnken design in the response surface method includes the following steps: Determine the optimized range of the ultrasonic time as 10 - 20 minutes, the optimized range of the ethanol concentration as 50 - 70%, and the optimized range of the solid-liquid ratio as 1:20 to 1:40 through single-factor experiments; Use the ultrasonic time, ethanol concentration, and solid-liquid ratio as independent variables, and the total flavonoid extraction rate as the response value for a three-level experimental design; Conduct multiple regression analysis on the experimental data through Design-Expert software to establish a second-order polynomial model.

[0098] Detailed description of the implementation steps of the Box-Behnken design: Determine the optimized range through single-factor experiments: Optimized range of ultrasonic time (10 - 20 minutes): Experimental basis: Single-factor experiments showed that within the ultrasonic time of 10 - 20 minutes, the total flavonoid extraction rate increased from 7.35% to the peak value of 8.20% (15 minutes), and then decreased to 7.37% (20 minutes) due to thermal degradation. Therefore, this interval was selected for response surface optimization.

[0099] Critical point verification: Monitor flavonoid degradation products (such as kaempferol derivatives) by HPLC to confirm that the degradation rate > 0.1% / min when the ultrasonic time > 15 minutes.

[0100] Optimized range of ethanol concentration (50% - 70%): Solubility curve: Within the ethanol concentration range of 50% - 70%, the solubility of total flavonoids increased from 6.87 mg / mL (50%) to 8.13 mg / mL (60%), and then decreased to 7.45 mg / mL (70%) due to the decrease in solvent polarity.

[0101] Permeation efficiency verification: Scanning electron microscopy (SEM) observations showed that 50% - 70% ethanol could increase the porosity of bract cell walls from 35% to 58% (section 3.1 of the document).

[0102] Optimized range of solid-liquid ratio (1:20 - 1:40): Solvent saturation analysis: When the material-liquid ratio is 1:20, the saturation of the extract is only 72%, while when it is 1:40, the saturation reaches 98%, but the solvent consumption increases by 100%. Based on the cost-benefit balance, the range of 1:20 - 1:40 is selected as the optimization range.

[0103] Box-Behnken three-level experimental design: Variable coding and experimental matrix: Variable coding table:

[0104] Experimental matrix generation: Using Design-Expert 13 software, 17 groups of experiments are generated (including 5 replicates of the central point), and the experimental design is shown in the following table (partial examples):

[0105] Experimental execution and data collection: Repeatability control: Each group of experiments is repeated 3 times, and the average value is taken as the response value (total flavonoid extraction rate). The experimental data refers to Table 3 (Box-Behnken design results) in the document.

[0106] Error control: The RSD of the extraction rate for the central point repeated experiments (experiment numbers 13 - 17) is 1.2%, indicating that the experimental systematic error is controllable.

[0107] Second-order polynomial model establishment and verification: Model construction process: Data input and regression analysis: Import the 17 groups of experimental data into Design-Expert 13 software, select the "Quadratic (second-order)" model type, and perform multivariate nonlinear regression analysis.

[0108] Model equation: The finally fitted second-order polynomial equation is: Specifically, for the extraction process of total flavonoids from bougainvillea bracts optimized by the response surface method of the present invention, the influence degree of each factor on the total flavonoid extraction rate in the second-order polynomial model is as follows: ethanol concentration > ultrasonic time > interaction between ultrasonic time and ethanol concentration > material-liquid ratio.

[0109] According to the second-order polynomial model established by the response surface method and the variance analysis, the influence degree of each factor on the total flavonoid extraction rate is as follows: Ethanol concentration (X2) > ultrasonic time (X1) > interaction between ultrasonic time and ethanol concentration (X1X2) > material-liquid ratio (X3), and the specific basis is as follows: Specifically, for the extraction process of total flavonoids from bougainvillea bracts optimized by the response surface method of the present invention, the power of the ultrasonic-assisted extraction is 100 ± 10 W, and the temperature of the constant-temperature reflux is 80 ± 2°C.

[0110] Power-temperature collaborative optimization: For the first time, the matching relationship between the ultrasonic power of 100 ± 10 W and the temperature of 80 ± 2 °C is clearly defined, avoiding the flavonoid degradation caused by power-temperature mismatch in traditional processes.

[0111] Specifically, for the extraction process of total flavonoids from bougainvillea bracts optimized by the response surface method of the present invention, in the pretreatment step, the drying temperature is 50 ± 5 °C, the drying time is 4 ± 0.5 hours, and the sieving mesh number is 80 ± 10 meshes.

[0112] The optimized pretreatment process increases the extraction rate of total flavonoids by 17.8% compared with the traditional process (7.2%); within the parameter tolerance range, the RSD of the extraction rate between batches ≤ 2.5%.

[0113] Specifically, for the extraction process of total flavonoids from bougainvillea bracts optimized by the response surface method of the present invention, the method for measuring the extraction rate of total flavonoids includes: Drawing a standard curve using rutin standard; Measuring the absorbance of the extract by sodium nitrite-aluminum nitrate-sodium hydroxide colorimetric method and calculating the total flavonoid content according to the standard curve.

[0114] Technical details of the method for measuring the extraction rate of total flavonoids: Drawing of the rutin standard curve: Preparation of the standard: Precisely weigh 10.0 mg of rutin standard (purity ≥ 98%, National Institute for the Control of Pharmaceutical and Biological Products), dissolve it in 70% ethanol solution and make up to 100 mL to prepare a 0.1 mg / mL stock solution; Successively dilute the stock solution to obtain rutin standard solutions with concentration gradients of 0.03, 0.05, 0.07, 0.09, 0.11, and 0.13 mg / mL.

[0115] Color reaction and measurement: Take 0.5 mL of each concentration standard solution and add it to a 25 mL volumetric flask; Successively add 0.5 mL of 5% sodium nitrite solution, shake well and let stand for 6 minutes; Add 0.5 mL of 10% aluminum nitrate solution, shake well and let stand for 6 minutes; Add 5 mL of 1 mol / L sodium hydroxide solution, make up to the mark with 70% ethanol, shake well and let stand for 15 minutes; Measure the absorbance at a wavelength of 510 nm using a UV-visible spectrophotometer (model UVmini-1240, Shimadzu, Japan).

[0116] Standard curve equation: Through linear regression analysis, the regression equation between rutin concentration (x, mg / mL) and absorbance (y) was obtained as follows: y = 6.35x + 0.019 \quad (R^2 = 0.9996)y=6.35x+0.019(R2=0.9996) Linear range: 0.03–0.13 mg / mL, meeting the requirements for quantitative analysis (Section 3.3 of the document).

[0117] Operating procedure for the determination of total flavonoid content: Color development treatment of the extract: Sample dilution: Take 0.5 mL of the extract from bougainvillea bracts, add it to a 25 mL volumetric flask, and dilute it to the mark with 70% ethanol; Color reaction: Add sodium nitrite, aluminum nitrate, and sodium hydroxide solutions successively according to the steps of the above standard curve, and strictly control the standing time at 6 ± 0.5 minutes; Blank control: Use 0.5 mL of 70% ethanol to replace the extract, and prepare a blank sample according to the same steps.

[0118] Specifically, for the extraction process of total flavonoids from bougainvillea bracts optimized by the response surface method described in the present invention, the verification of the second-order polynomial model includes: Verifying the goodness of fit of the model through residual distribution analysis and normal probability plot; Repeating the experiment three times to verify that the deviation of the extraction rate is less than 2%.

[0119] Specifically, for the extraction process of total flavonoids from bougainvillea bracts optimized by the response surface method described in the present invention, under the optimized extraction parameter combination, the predicted value of the total flavonoid extraction rate is 8.50 ± 0.3%.

[0120] The present invention systematically solves the problem that the traditional method cannot maximize the yield in the extraction of total flavonoids from bougainvillea bracts through the following innovative technical means: Defects of the prior art: The traditional process relies on single-factor experiments or orthogonal designs, and cannot quantify the interaction between ultrasonic time, ethanol concentration, and solid-liquid ratio, resulting in one-sided parameter selection and insufficient accuracy of the prediction model (the reported coefficient of determination R^2 of the model < 0.85).

[0121] Solution of the present invention: Box-Behnken experimental design: Adopt the three-factor three-level response surface method, and comprehensively analyze the influence of each factor and its interaction on the extraction rate through 17 groups of experiments (including 5 groups of center point repetitions).

[0122] Second-order polynomial model: Establish a high-precision regression equation, and clarify that the order of the influence degree of each factor is ethanol concentration > ultrasonic time > interaction > solid-liquid ratio. Prioritize the optimization of key parameters.

[0123] Interaction verification: Through the three-dimensional response surface diagram ( Figure 3 ), and analysis of variance (ANOVA) to prove that the synergistic effect between ultrasonic time and ethanol concentration is extremely significant (p < 0.01), and the extraction rate after optimization is increased by 13% (from 7.5% to 8.48%).

[0124] Precise parameter optimization and tolerance control: Defects of the existing technology: The traditional process parameter range is wide (such as ethanol concentration ±5%), lacking scientific limitation on parameter fluctuations, resulting in poor stability in industrial production (batch-to-batch difference ≥8%).

[0125] Solution of the present invention: Optimal parameter combination: Determine the ultrasonic time of 13.7±0.5 minutes, ethanol concentration of 60.0±2.0%, and solid-liquid ratio of 1:29.7±1.0 through model prediction, covering a 95% confidence interval.

[0126] Scientific basis for tolerance: Ultrasonic time: When it exceeds 14.2 minutes, the flavonoid degradation rate > 0.1% / min (verified by HPLC); Ethanol concentration: 60% is the solubility inflection point, and the extraction rate fluctuation is ≤0.5% under a deviation of ±2%; Solid-liquid ratio: 1:29.7 balances the solvent saturation (≥95%) and cost (saving 12.5%).

[0127] Industrial adaptability: The parameter tolerance range is compatible with equipment errors (such as ultrasonic power ±10 W), ensuring production stability (RSD≤3.8%).

[0128] The present invention quantifies the multi-factor interaction by the response surface method, combines a high-precision model and parameter tolerance control, systematically solves the deficiencies of the traditional process in extraction efficiency, stability and industrial adaptability, realizes the maximization of the total flavonoid extraction rate from bougainvillea bracts (≥8.48%), and has significant economic benefits (cost reduction of 25.8%) and technological advancement (model error ≤1.8%).

Claims

1. Optimization of the extraction process of total flavonoids from bougainvillea bracts by response surface methodology, characterized in that: include: Receive raw materials of bougainvillea bracts and perform pre-processing, including washing, drying, crushing and screening; The total flavonoids were extracted from the pretreated bougainvillea bracts by constant temperature reflux and ultrasonic assisted extraction technology, and the parameters of ultrasonic assisted extraction included ultrasonic time, ethanol concentration and solid-liquid ratio. Based on the Box-Behnken design in the response surface methodology, a multi-factor interaction analysis was performed on the ultrasonic time, ethanol concentration and solid-liquid ratio, and a second-order polynomial regression model was established to predict the optimal extraction conditions; The optimized extraction parameter combination was determined according to the model, including ultrasonic time of 13.7±0.5 minutes, ethanol concentration of 60.0±2.0%, and solid-liquid ratio of 1:29.7±1.0; The extraction operation is performed based on the optimized extraction parameter combination to obtain an extract having a total flavonoid extraction rate of ≥8.48%.

2. The response surface methodology for optimizing the extraction of total flavonoids from bougainvillea bracts according to claim 1, characterized in that: The Box-Behnken design in the response surface methodology includes the following steps: Through single factor experiments, it was determined that the optimal range of the ultrasonic time was 10-20 minutes, the optimal range of the ethanol concentration was 50-70%, and the optimal range of the solid-liquid ratio was 1:20 to 1:40; The ultrasonic time, ethanol concentration and solid-liquid ratio were used as independent variables, and the total flavonoids extraction rate was used as the response value to conduct a three-level experimental design; The experimental data were analyzed by multiple regression using Design-Expert software, and a second-order polynomial model was established.

3. The response surface methodology for optimizing the extraction of total flavonoids from bougainvillea bracts according to claim 2, characterized in that: The influence of each factor on the total flavonoids extraction rate in the second-order polynomial model is in the following order: ethanol concentration > ultrasonic time > interaction between ultrasonic time and ethanol concentration > solid-liquid ratio.

4. The response surface methodology for optimizing the extraction of total flavonoids from Bougainvillea bracts according to claim 1, characterized in that: The power of the ultrasonic assisted extraction is 100±10 W, and the temperature of the constant temperature reflux is 80±2°C.

5. The response surface methodology for optimizing the extraction of total flavonoids from bougainvillea bracts according to claim 1, characterized in that: In the pretreatment step, the drying temperature is 50±5° C., the drying time is 4±0.5 hours, and the sieving mesh number is 80±10 meshes.

6. The response surface methodology for optimizing the extraction of total flavonoids from bougainvillea bracts according to claim 1, characterized in that: The method for determining the total flavonoid extraction rate comprises: The standard curve was drawn using rutin standards; The absorbance of the extract was determined by sodium nitrite-aluminum nitrate-sodium hydroxide colorimetry, and the total flavonoid content was calculated according to the standard curve.

7. The response surface methodology for optimizing the extraction of total flavonoids from bougainvillea bracts according to claim 1, characterized in that: The validation of the second-order polynomial model includes: The goodness of fit of the model was verified through residual distribution analysis and normal probability plot; The experiment was repeated three times to verify that the deviation of the extraction rate was less than 2%.

8. The response surface methodology for optimizing the extraction of total flavonoids from bougainvillea bracts according to claim 1, characterized in that: Under the optimized extraction parameter combination, the predicted value of total flavonoid extraction rate is 8.50±0.3%.

Images