A method and device for optimizing extraction and characterizing components of plant polyphenols

Abstract

This invention discloses a method and apparatus for optimizing the extraction and characterizing the components of plant polyphenols, relating to the field of natural product extraction and component analysis technology. The method includes: selecting plant raw materials, drying, pulverizing, and sieving to obtain fine powder; determining the appropriate value range for ethanol concentration, material-to-liquid ratio, extraction time, and ultrasonic power through single-factor experiments, and detecting the total polyphenol content; based on this range, using response surface methodology to fit a regression model, and optimizing the extraction process parameters with the total polyphenol content as the response value; obtaining the plant polyphenol extract through ultrasonic extraction under optimal conditions; and then using UPLC-MS / MS to qualitatively and quantitatively characterize the polyphenolic components in the extract, thereby completing the entire process of efficient extraction and systematic component analysis of plant polyphenols. This invention achieves accurate qualitative and quantitative analysis of polyphenol components through high-sensitivity mass spectrometry analysis, solving the problem of insufficient data support for extract quality evaluation.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction and component analysis technology, and in particular to a method and apparatus for optimizing the extraction and characterizing the components of plant polyphenols. Background Technology

[0002] Goji berries (Lycium barbarum L.) are a traditional Chinese medicinal and edible plant. Their fruits are rich in nutrients and have significant health benefits, leading to large-scale application and industrial development in functional foods, dietary nutrition, and health products. Goji berry leaves, a substantial byproduct of goji berry cultivation and harvesting, are often underutilized and discarded in large quantities. However, they are rich in polyphenols, flavonoids, alkaloids, and other bioactive substances, possessing potential applications in antioxidation, anti-inflammation, and metabolic regulation, and exhibiting extremely high potential for deep processing and resource utilization.

[0003] With the deepening of the concept of comprehensive utilization and high-value transformation of agricultural and forestry by-products, the development and utilization of wolfberry leaves has gradually become a research hotspot. However, research on the efficient preparation, process system optimization, and accurate component identification of wolfberry leaf polyphenols is still relatively weak. Problems such as non-standard extraction methods, unclear process parameters, and incomplete component characterization are prominent, which greatly limit the industrial application and in-depth development of wolfberry leaf polyphenols.

[0004] Currently, the extraction of plant polyphenols mainly employs traditional solvent extraction, ultrasound-assisted extraction, and thermal reflux extraction. Studies often use total polyphenol content or extraction efficiency as a single evaluation index, and optimize the process through single-factor experiments or orthogonal design. However, plant polyphenols have diverse structures and complex components, and using only total phenol content as an indicator cannot fully reflect the chemical composition and quality characteristics of the extract. Furthermore, the effects of different extraction parameters on polyphenol monomer dissolution and component distribution lack systematic investigation, which can easily lead to a disconnect between process optimization and actual quality requirements.

[0005] Meanwhile, UPLC-MS / MS, with its high sensitivity, high resolution and strong qualitative ability, has become an important means for the precise analysis of plant polyphenol components. However, existing studies mostly use it as an independent follow-up detection method, and rarely combine it with the extraction process screening and parameter optimization process, making it difficult to form an integrated technical system of "extraction optimization - component characterization - quality evaluation".

[0006] Existing technologies struggle to balance extraction efficiency and component integrity of Goji Leaf Polyphenols (GLP), failing to provide standardized and systematic technical support for the high-value utilization of Goji Leaf by-products. Therefore, there is an urgent need to establish a GLP extraction method that couples response surface methodology optimization with UPLC-MS / MS system characterization to achieve standardized development of efficient preparation, accurate characterization, and quality control of Goji Leaf Polyphenols. Summary of the Invention

[0007] The main objective of this invention is to provide a method for optimizing the extraction and characterizing the components of plant polyphenols.

[0008] Another objective of this invention is to provide an apparatus for the extraction optimization and component characterization of plant polyphenols.

[0009] The third objective of this invention is to provide an electronic device.

[0010] A fourth objective of this invention is to provide a non-transitory computer-readable storage medium.

[0011] To achieve the above objectives, a first aspect of the present invention provides a method for optimizing the extraction and characterizing the components of plant polyphenols, comprising:

[0012] Plant raw materials are dried, pulverized and sieved in sequence to obtain plant powder; Using solvent concentration, material-to-liquid ratio, extraction time, and ultrasonic power as influencing factors, single-factor experiments were conducted based on plant powder to determine the range of influence of each factor on the extraction effect of plant polyphenols, and the total polyphenol content of the corresponding extract was measured. Based on the influence range determined by single-factor experiments, with solvent concentration, solid-liquid ratio, extraction time and ultrasonic power as independent variables and total polyphenol content as the response value, a regression model was established using response surface design, and the optimal combination of extraction parameters was determined through regression analysis. Based on the optimal combination of extraction parameters, plant powder was subjected to ultrasonic-assisted extraction to obtain plant polyphenol extracts. Ultra-high performance liquid chromatography-tandem mass spectrometry was used to characterize the chemical composition and content distribution of polyphenols, thereby achieving the optimization of plant polyphenol extraction and component characterization.

[0013] Optionally, the plant material is sequentially dried, pulverized, and sieved to obtain plant powder, including: The collected fresh plant materials are pre-frozen and freeze-dried. The processed raw materials are crushed to reduce the particle size and increase the contact area between the material and the solvent. The crushed material is sieved to remove coarse particles and obtain plant powder with uniform particle size.

[0014] Optionally, single-factor experiments were conducted based on plant powder, using solvent concentration, solid-liquid ratio, extraction time, and ultrasonic power as influencing factors, to determine the range of influence of each factor on the extraction effect of plant polyphenols, and to determine the total polyphenol content of the corresponding extracts, including: Using plant powder as the extraction substrate, solvent concentration, solid-liquid ratio, extraction time, and ultrasonic power were set as single variables. Under the premise of keeping other experimental conditions constant, the level of a single variable was changed step by step and the corresponding extraction operation was completed. Collect the extracts obtained from each group of experiments, determine the total polyphenol content of the extracts, and record the extraction effect under different variable levels; By comparing the differences in total polyphenol content under different factor levels, the influence trend and effective range of each factor on the extraction effect were determined.

[0015] Optionally, based on the influence range determined by single-factor experiments, a regression model is established using response surface design, with solvent concentration, solid-liquid ratio, extraction time, and ultrasonic power as independent variables and total polyphenol content as the response value. This model includes: The range of influence determined by single-factor experiments was used as the design interval, and solvent concentration, solid-liquid ratio, extraction time and ultrasonic power were selected as independent variables. The total polyphenol content was used as the response value to evaluate the extraction effect, and multiple combination experiments were carried out using response surface design. Collect measured data from each group of experiments, substitute the data into the model for fitting calculation, and establish a regression model between the independent variable and the response value.

[0016] Optionally, the optimal combination of extraction parameters can be determined through regression analysis, including: Statistical analysis was performed on the established regression model to determine the significance of the effects of each factor on the total polyphenol content. Analyze the interactions among various factors to determine the synergistic effects of different parameter combinations on the extraction results; The extreme value is solved based on the regression model to obtain the parameter conditions that make the total polyphenol content optimal. The optimal combination of extraction parameters was selected and determined based on the comprehensive model analysis results.

[0017] Optionally, the plant powder is subjected to ultrasound-assisted extraction based on the optimal combination of extraction parameters to obtain a plant polyphenol extract, including: Plant powder and extraction solvent are mixed in the optimal ratio to form an extraction system; Applying ultrasound under optimal solvent concentration, extraction time, and ultrasonic power conditions enhances the dissolution and diffusion of polyphenols. The complete extraction process is completed under the set extraction conditions, allowing the polyphenols in the plant powder to be fully released into the solvent; The extracted system was processed to obtain the target plant polyphenol extract.

[0018] Optionally, ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) is used to characterize the chemical composition and content distribution of polyphenols, including: Plant polyphenol extracts were used as the detection target, and ultra-high performance liquid chromatography was used to separate polyphenolic components. The chemical composition of polyphenols was determined by structural identification of the separated components using tandem mass spectrometry. Quantitative analysis was performed on each polyphenol component to calculate the specific content of each component; By integrating qualitative and quantitative results, the overall content distribution pattern of polyphenols was statistically analyzed, and the systematic component characterization of the extract was completed.

[0019] To achieve the above objectives, a second aspect of the present invention provides an extraction optimization and component characterization apparatus for plant polyphenols, comprising: The raw material preparation module is used to process plant raw materials by drying, crushing and sieving in sequence to obtain plant powder. Factor screening was used to determine the range of influence of each factor on the extraction effect of plant polyphenols by conducting single-factor experiments based on plant powder, with solvent concentration, material-liquid ratio, extraction time and ultrasonic power as influencing factors, and to determine the total polyphenol content of the corresponding extract. The parameter optimization module is used to establish a regression model based on the influence range determined by single-factor experiments, with solvent concentration, solid-liquid ratio, extraction time and ultrasonic power as independent variables and total polyphenol content as the response value. The optimal combination of extraction parameters is determined by response surface design and regression analysis. The component characterization module is used to perform ultrasonic-assisted extraction of plant powder based on the optimal combination of extraction parameters to obtain plant polyphenol extracts. Ultra-high performance liquid chromatography-tandem mass spectrometry is used to characterize the chemical composition and content distribution of polyphenols, thereby achieving the extraction optimization and component characterization of plant polyphenols.

[0020] Regarding the apparatus in the above embodiments, the specific manner in which each module performs its operation has been described in detail in the embodiments related to the method, and will not be elaborated upon here.

[0021] To achieve the above objectives, a third aspect of this application provides an electronic device, including a processor and a memory; wherein the processor reads executable program code stored in the memory to run a program corresponding to the executable program code, for implementing a method for extraction optimization and component characterization of plant polyphenols as described in the first aspect embodiment.

[0022] To achieve the above objectives, a fourth aspect of this application provides a non-transitory computer-readable storage medium storing a computer program thereon, which, when executed by a processor, implements a method for the extraction optimization and component characterization of plant polyphenols as described in the first aspect embodiment.

[0023] The embodiments of the present invention have the following beneficial effects: 1. By combining response surface methodology with UPLC-MS / MS component characterization, the optimal extraction process of GLP can be accurately obtained, and the composition and content distribution of polyphenols in the extract can be clearly defined. This breaks through the limitations of optimization based solely on the extraction rate, and improves the scientific nature and completeness of the process.

[0024] 2. By constructing a complete technical path of single-factor screening, response surface modeling, and parameter optimization, the stability and controllability of the GLP extraction process are significantly improved, ensuring the quality consistency of extracts from different batches and providing a reliable basis for industrial production.

[0025] 3. High-sensitivity, high-resolution mass spectrometry was used to perform qualitative and quantitative analysis of polyphenol components, clearly revealing the chemical composition characteristics of Lycium barbarum leaf polyphenols, providing solid data support for extract quality evaluation, standard establishment and functional development.

[0026] 4. Establish an integrated technical system from extraction optimization to component characterization, fully explore the utilization value of wolfberry leaf by-products, promote the high-value utilization of agricultural and forestry waste, and provide standardized technical solutions for the development of GLP-related functional products.

[0027] 5. The optimized process parameters have a reasonable adjustable range, which can be flexibly adapted to actual production needs, ensuring good extraction results while improving the applicability and on-site operability of the method. Attached Figure Description

[0028] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein: Figure 1 A flowchart illustrating a method for optimizing the extraction and characterizing the components of plant polyphenols, provided in an embodiment of the present invention; Figure 2 The regression standard curve of gallic acid provided in the embodiments of the present invention; Figure 3 The figure shows the results of a single-factor experiment on Lycium barbarum leaf polyphenols provided in an embodiment of the present invention. Figure 4 This is an example of the interaction diagram for the optimization of the response surface methodology of Lycium barbarum leaf polyphenols provided in this embodiment of the invention. Figure 5 This is a structural diagram of a plant polyphenol extraction optimization and component characterization device provided in an embodiment of the present invention. Detailed Implementation

[0029] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0030] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0031] The following describes, with reference to the accompanying drawings, a method and apparatus for the extraction optimization and component characterization of plant polyphenols according to embodiments of the present invention.

[0032] Example 1 This invention provides a method for optimizing the extraction and characterizing the components of plant polyphenols. Figure 1 This is a schematic flowchart illustrating a method for the extraction optimization and component characterization of plant polyphenols provided in an embodiment of the present invention. Figure 1 As shown, the method includes the following steps: Step S1: The plant raw materials are dried, pulverized and sieved in sequence to obtain plant powder.

[0033] In this embodiment, wolfberry leaves are used as a typical plant material. Fresh wolfberry leaves that are free from pests and diseases, free from rot and spoilage, and in good growth are selected as the raw material for GLP extraction. This ensures that the raw material is of uniform and pure quality, laying the foundation for the stability of the subsequent extraction process and the quality of the extract. The specific operation is as follows: First, the freshly collected goji berry leaves undergo pretreatment. The leaves are quickly rinsed 2-3 times with deionized water to remove surface dust, impurities, and residual soil particles. Then, the surface moisture is blotted dry with filter paper to prevent excessive moisture from affecting the subsequent pre-freezing and freeze-drying processes. Next, the cleaned goji berry leaves are placed in an ultra-low temperature freezer at -80℃ for 24 hours for pre-freezing. This rapid freezing at ultra-low temperatures causes the internal moisture of the goji berry leaves to quickly form small ice crystals, preventing large ice crystals from damaging the plant cell structure and thus reducing the loss and oxidative degradation of polyphenols within the cells.

[0034] After pre-freezing, the wolfberry leaves are transferred to a freeze dryer and freeze-dried at -60℃ and 0.1mbar for 48 hours. The ice crystals in the wolfberry leaves are gradually removed by sublimation, achieving low-temperature dehydration. Compared with traditional hot air drying, this method can effectively avoid the damage of polyphenols to polyphenols caused by high temperature, retain the activity of polyphenols in wolfberry leaves to the greatest extent, and prevent structural changes of polyphenols due to oxidation during the drying process, thus ensuring the quality of the extract.

[0035] After freeze-drying, the goji berry leaves are removed. At this point, the leaves are in a loose and porous dry state, facilitating subsequent pulverization. The dried goji berry leaves are then placed in a high-speed blender, where high-intensity mechanical force thoroughly breaks down the cell walls and cell membranes, pulverizing the leaves into fine powder. This reduces the particle size of the raw material, significantly increases the contact area between the material and the subsequent extraction solvent, accelerates the dissolution of polyphenols from the cells, improves extraction efficiency, and creates favorable conditions for the efficient dissolution of polyphenols.

[0036] After pulverization, the pulverized material is sieved using a 60-mesh standard sieve to remove large, incompletely pulverized particles and impurities. The wolfberry leaf powder that passes through the sieve is collected to ensure that the obtained wolfberry leaf powder has a uniform and fine particle size. This avoids the possibility that polyphenols in some particles cannot be fully dissolved during subsequent extraction due to uneven particle size, thus ensuring the consistency and repeatability of subsequent extraction experiments. This completes the entire raw material pretreatment step, providing stable and uniform experimental raw materials for subsequent single-factor experiments and response surface optimization experiments.

[0037] Step S2 involves conducting single-factor experiments based on plant powder, using solvent concentration, material-to-liquid ratio, extraction time, and ultrasonic power as influencing factors, to determine the range of influence of each factor on the extraction effect of plant polyphenols, and to measure the total polyphenol content of the corresponding extract.

[0038] In this embodiment, wolfberry leaf powder was used as the extraction substrate. Ethanol volume fraction, solid-liquid ratio, extraction time, and ultrasonic power were set as single variables. Under the premise of keeping other experimental conditions constant, the levels of these single variables were gradually changed to complete the corresponding extraction operations. The specific experimental design is as follows: To address the issue of ethanol volume fraction, 1.000g of wolfberry leaf powder was weighed and 25mL of ethanol solutions with volume fractions of 40%, 50%, 60%, 70%, and 80% were added respectively. The mixture was then extracted for 25min under ultrasonic power of 560W. To address the extraction time factor, 1.000g of wolfberry leaf powder was weighed and added to 25mL of 60% ethanol solution. The mixture was then extracted for 15, 20, 25, 30, and 35 minutes under ultrasonic power of 560W. To address the issue of the material-to-liquid ratio, 1.000g of wolfberry leaf powder was weighed and added to 20, 25, 30, 35, and 40mL of 60% ethanol solution, respectively, and extracted for 25min under ultrasonic power of 560W. To address the issue of ultrasonic power, 1.000g of wolfberry leaf powder was weighed and added to 25mL of 60% ethanol solution. The mixture was then extracted for 25min under ultrasonic power conditions of 480, 560, 640, 720, and 800W, respectively.

[0039] Collect the extracts obtained from each group of experiments and determine the total polyphenol content of the extracts. In this application example, the total polyphenol content was determined by the Folin-Ciocalteu colorimetric method. The specific operation refers to the method of GB / T8313-2018: Take 100 μL of sample, add 100 μL of Folin-Ciocalteu reagent, shake well and let stand for 2 min, then add 800 μL of 5% Na2CO3 solution, shake well and water bath in a 40℃ water bath for 20 min. After the water bath is completed, cool to room temperature and measure the absorbance at 760 nm.

[0040] A standard curve was plotted using gallic acid as a standard substance, such as... Figure 2 As shown, the regression curve equation for gallic acid is:

[0041] Among them, the coefficient of determination This indicates that the standard curve has a good linear relationship and can be used for accurate conversion of total polyphenol content. Total polyphenol content is calculated according to Formula 1:

[0042] Where X is the total polyphenol content in mg / g; C is the mass concentration of the sample solution converted from the gallic acid standard curve in mg / mL; V is the total volume of the sample extract in mL; n is the dilution factor; and m is the dry weight of the sample in g. The results are expressed as mgGAE / g.

[0043] Record the extraction results under different variable levels, compare the differences in total polyphenol content under different factor levels, determine the influence trend and effective range of each factor on the extraction effect, and the single-factor experimental results are as follows: Figure 3 As shown, where Figure 3 In the figure, A represents the effect of ultrasonic power on the total polyphenol content. Figure 3 In this context, B represents the effect of ethanol volume fraction on the total polyphenol content. Figure 3 C in the figure represents the effect of the solid-liquid ratio on the total polyphenol content. Figure 3 In this context, D represents the effect of ultrasound time on the total polyphenol content.

[0044] from Figure 3 It can be seen that within the ultrasonic power range of 480~640W, the total polyphenol content first increases and then decreases with increasing power, reaching a peak at 560W; within the ethanol volume fraction range of 40%~80%, the total polyphenol content first decreases and then increases with increasing volume fraction, reaching a maximum at 80%; within the material-to-liquid ratio range of 20~40mL / g, the total polyphenol content first increases and then decreases with increasing material-to-liquid ratio, reaching a peak at 30mL / g; within the ultrasonic time range of 15~35min, the total polyphenol content first increases and then decreases with increasing time, reaching a maximum at 20min.

[0045] Based on the results of the single-factor experiments, the suitable range of each factor was determined to be: ethanol volume fraction 80%~100%, solid-liquid ratio 25~35mL / g, extraction time 15~25min, and ultrasonic power 480~640W. The optimal extraction conditions for each factor were ethanol volume fraction 90%, solid-liquid ratio 1:30g / mL, extraction time 20min, and ultrasonic power 560W, providing a basic parameter range for subsequent response surface optimization experiments.

[0046] Step S3: Based on the influence range determined by the single-factor experiment, a regression model is established using response surface design with solvent concentration, material-liquid ratio, extraction time and ultrasonic power as independent variables and total polyphenol content as the response value. The optimal combination of extraction parameters is then determined through regression analysis.

[0047] In this embodiment, the influence range determined by the single-factor experiment is used as the design interval. Ethanol volume fraction (A), solid-liquid ratio (B), extraction time (C), and ultrasonic power (D) are selected as independent variables, and total polyphenol content (Y) is used as the response value to evaluate the extraction effect. A Box-Behnken design is used to conduct a four-factor, three-level response surface experiment. The level codes for each factor are shown in Table 1. Table 1

[0048] Among them, -1, 0, and 1 correspond to the low, medium, and high levels of each factor, respectively; the volume fraction of ethanol corresponds to 80%, 90%, and 100%; the solid-liquid ratio corresponds to 25 mL / g, 30 mL / g, and 35 mL / g; the extraction time corresponds to 15 min, 20 min, and 25 min; and the ultrasonic power corresponds to 480 W, 560 W, and 640 W.

[0049] The measured data for each group of experiments were collected, and the specific experimental data are shown in Table 2: Table 2

[0050] This table records in detail the coding levels of each factor and the corresponding measured values ​​of total polyphenol content for 27 response surface experiments, providing solid experimental data support for the subsequent fitting of the regression model.

[0051] Substitute the 27 sets of measured data from Table 2 into the model for fitting calculations to establish a regression model between the independent variable and the response value. The general formula of the established quadratic polynomial regression model is shown in Formula 2:

[0052] Where Y is the response value, i.e., the total polyphenol content; For constant terms; The coefficient of the linear term; The coefficient of the quadratic term; The coefficients of the interaction terms; and These are the coded values ​​for each influencing factor.

[0053] In this embodiment of the application, RSM analysis is performed using dedicated software, and regression fitting is performed on the response surface experimental data to obtain the regression equation for GLP extraction as follows: .

[0054] Statistical analysis was performed on the established regression model to determine the significance of the effects of each factor on the total polyphenol content. The results of the analysis of variance are shown in Table 3. Table 3

[0055] Note: P < 0.05 indicates a significant difference; P < 0.01 indicates that the difference is highly significant.

[0056] The model was highly significant overall (P<0.0001), with the first-order, interaction, and quadratic terms all reaching significant or highly significant levels. The lack-of-fit term had a P=0.8797 (>0.05), indicating no significant model failure and a good fit. The model's coefficient of determination R²=0.9962, and the adjusted coefficient of determination R²-adj=0.9918, with a small difference between the two, indicating good model fit and predictive ability. Based on the significance test of the regression coefficients, the order of significance of the four factors on the response value is as follows: A (ethanol volume fraction) > B (solid-liquid ratio) > D (ultrasonic power) > C (extraction time). Among them, ethanol volume fraction, solid-liquid ratio, and ultrasonic power had highly significant effects on the total polyphenol content, while extraction time had no significant effect.

[0057] The interactions between various factors were analyzed to determine the synergistic effects of different parameter combinations on the extraction results. The response surface methodology for the interactions of various factors is shown below. Figure 4 As shown, where Figure 4 In this context, A represents the interaction between the volume fraction of ethanol and the ultrasonic power. Figure 4 In this context, B represents the interaction between the material-to-liquid ratio and the extraction time. Figure 4 In this context, C represents the interaction between the feed-to-liquid ratio and the ultrasonic power. Figure 4 In this context, D represents the interaction between the volume fraction of ethanol and the feed-to-liquid ratio. Figure 4 In this context, E represents the interaction between the volume fraction of ethanol and the extraction time. Figure 4 F in the equation represents the interaction between extraction time and ultrasonic power.

[0058] from Figure 4It can be seen that the response surface changes of the A–B, A–C and A–D combinations are significant, indicating that there is a significant interaction between the ethanol volume fraction and other factors, and the ethanol volume fraction is the dominant factor affecting the GLP extraction effect; the B–C interaction term also shows a significant trend, indicating that there is a synergistic effect between the solid-liquid ratio and the extraction time; in contrast, the response surfaces of B–D and C–D are relatively flat, consistent with the non-significant statistical results.

[0059] The extreme value was solved based on the regression model to obtain the parameter conditions that maximize the total polyphenol content. The optimal combination of extraction parameters was screened and determined based on the comprehensive model analysis results. The response surface showed obvious curvature and reached the maximum response around A≈-0.21, B≈-0.30, C≈0, and D≈0.09. The corresponding optimal conditions were 88% ethanol volume fraction, a solid-liquid ratio of 1:28.5 g / mL, an ultrasonic time of 20 min, and a power of 560 W.

[0060] Under optimal prediction conditions, three repeated experiments were conducted, and the total phenol content obtained was 17.34±0.67 mg / g. The relative error between this result and the model prediction was 2.73%, indicating that the wolfberry leaf polyphenol extraction process optimized by response surface methodology has good feasibility and stability.

[0061] Step S4: Based on the optimal combination of extraction parameters, the plant powder is subjected to ultrasonic-assisted extraction to obtain plant polyphenol extract. The chemical composition and content distribution of polyphenols are characterized by ultra-high performance liquid chromatography-tandem mass spectrometry, thereby achieving the optimization of plant polyphenol extraction and component characterization.

[0062] In this embodiment, wolfberry leaf powder and extraction solvent are mixed according to the optimal parameter ratio to form an extraction system. Ultrasonic action is applied under the conditions of optimal solvent concentration of 88%, material-to-liquid ratio of 1:28.5 g / mL, extraction time of 20 min, and ultrasonic power of 560 W. By utilizing the cavitation, mechanical, and thermal effects of ultrasound, the dissolution and diffusion of polyphenols are enhanced. The complete extraction process is completed under the set extraction conditions, so that the polyphenols in wolfberry leaf powder are fully released into the solvent. The extracted system is then centrifuged and filtered to obtain the target GLP extract.

[0063] GLP extract was used as the detection target. Ultra-high performance liquid chromatography (UPLC-MS) was used to separate the polyphenolic components. The structures of the separated components were identified by tandem mass spectrometry (TMS) to determine the chemical composition of the polyphenols. The UPLC-MS / MS analytical conditions were as follows: Chromatographic conditions: A reversed-phase column (Agilent Technologies Eclipse Plus C18, 2.1 mm × 150 mm) was used. The mobile phase consisted of mobile phase A (0.1% formic acid in acetonitrile) and mobile phase B (0.5% formic acid in aqueous solution). The injection volume was 5 μL, the flow rate was 0.30 mL / min, the column temperature was 40 °C, and the elution gradient program was set as follows: For 0-2 minutes, mobile phase A is maintained at 5%; Within 2–12 minutes, mobile phase A increased linearly from 5% to 35%. Within 12–18 minutes, the mobile phase A increased linearly from 35% to 60%. Within 18–20 minutes, the mobile phase A increased linearly from 60% to 95%. For 20-23 minutes, mobile phase A is maintained at 95%. At 23-24 min, mobile phase A decreased linearly to 5%. Mobile phase A was maintained at 5% for 24-27 minutes to balance the column.

[0064] Mass spectrometry detection was performed using an electrospray ionization (ESI) source operating in negative ion mode. The source parameters were set as follows: sheath gas flow rate 40 L / min, auxiliary gas flow rate 10 L / min, spray voltage -3.0 kV, drying gas (N2) flow rate 10 L / min, drying gas temperature 350 °C, and nebulization pressure 35 psi. The acquisition method was a combination of full scan and two-stage fragmentation, with an m / z range of 100–1200. The first-stage mass spectrometry resolution was set to 60,000, and the second-stage mass spectrometry resolution was set to 30,000. The collision energy was a stepped collision energy set to 15 / 30 / 45 eV.

[0065] Quantitative analysis was performed on each polyphenol component, the specific content of each component was calculated, the qualitative and quantitative results were integrated, the overall content distribution pattern of polyphenols was statistically analyzed, and the systematic component characterization of the extract was completed. The results are shown in Table 4. Table 4

[0066] The polyphenols that can be detected and quantified in GLP mainly include phenolic acids (such as quinic acid, chlorogenic acid and its isomers) as well as flavonoids and their glycosides (such as rutin, kaempferol-3-O-rutin glycoside).

[0067] In terms of content and proportion, rutin and chlorogenic acid were the dominant polyphenol components in the GLP, accounting for 43.23% and 38.33% of the total detected phenol content, respectively. In addition, quinic acid accounted for 7.74%, kaempferol-3-O-rutinoside for 8.32%, and isorhamnetin-3-rutinoside for 2.00%. These components accounted for 99.92% of the total detectable polyphenols, indicating that the GLP prepared in this application has clear principal component characteristics in its polyphenol composition, and the component composition is concentrated and stable. Therefore, the UPLC-MS / MS results can provide quantifiable chemical fingerprints and key indicator component support for the GLP, which can be used for quality control, batch-to-batch consistency evaluation, and subsequent functional verification and application development of the product in this application.

[0068] This application utilizes a systematic approach combining process optimization and component characterization to achieve the rational determination of GLP extraction conditions and the clear characterization of its chemical composition. First, single-factor experiments were used to screen out the key factors affecting extraction efficiency. Based on this, a Box-Behnken response surface methodology was employed to establish a mathematical model relating ethanol volume fraction, solid-liquid ratio, extraction time, and ultrasonic power to the total polyphenol content, thus determining the optimal combination of extraction parameters. Subsequently, GLP extracts were prepared under the optimized conditions, and their chemical composition was systematically analyzed using UPLC-MS / MS to confirm the composition and content distribution characteristics of the main polyphenol components. Through these steps, this application constructs a complete technical system from extraction parameter screening and process optimization to component characterization. This system not only provides stable GLP extraction conditions but also offers a chemical compositional basis for the quality evaluation and subsequent applications of the extract, thereby achieving an organic combination of extraction process optimization and component characterization, demonstrating good industrial applicability.

[0069] Example 2 This invention provides an apparatus for optimizing the extraction and characterizing the components of plant polyphenols. Figure 5 This is a schematic flowchart of a plant polyphenol extraction optimization and component characterization device provided in an embodiment of the present invention. Figure 5 As shown, the device includes: The raw material preparation module 100 is used to sequentially dry, crush and sieve plant raw materials to obtain plant powder. Factor screening 200 was used to determine the range of influence of each factor on the extraction effect of plant polyphenols by using solvent concentration, material-liquid ratio, extraction time and ultrasonic power as influencing factors, and to determine the total polyphenol content of the corresponding extract. The parameter optimization module 300 is used to establish a regression model based on the influence range determined by single-factor experiments, with solvent concentration, material-liquid ratio, extraction time and ultrasonic power as independent variables and total polyphenol content as the response value. The optimal combination of extraction parameters is determined by response surface design and regression analysis. The component characterization module 400 is used to perform ultrasonic-assisted extraction of plant powder based on the optimal combination of extraction parameters to obtain plant polyphenol extracts, and to characterize the chemical composition and content distribution of polyphenols using ultra-high performance liquid chromatography-tandem mass spectrometry, thereby achieving the extraction optimization and component characterization of plant polyphenols.

[0070] Regarding the apparatus in the above embodiments, the specific manner in which each module performs its operation has been described in detail in the embodiments related to the method, and will not be elaborated upon here.

[0071] Example 3 To implement the methods of the above embodiments, the present invention also provides an electronic device, which includes a memory and a processor; wherein the processor reads executable program code stored in the memory to run a program corresponding to the executable program code, so as to implement the various steps of the methods described above.

[0072] Example 4 To implement the above embodiments, this application also proposes a non-transitory computer-readable storage medium storing a computer program thereon, which, when executed by a processor, implements the method described in the foregoing embodiments.

[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0074] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0075] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

Claims

1. A method for extraction optimization and characterization of ingredients of plant polyphenols, characterized in that, include: Plant raw materials are dried, pulverized and sieved in sequence to obtain plant powder; Using solvent concentration, material-to-liquid ratio, extraction time, and ultrasonic power as influencing factors, single-factor experiments were conducted based on plant powder to determine the range of influence of each factor on the extraction effect of plant polyphenols, and the total polyphenol content of the corresponding extract was measured. Based on the influence range determined by single-factor experiments, with solvent concentration, solid-liquid ratio, extraction time and ultrasonic power as independent variables and total polyphenol content as the response value, a regression model was established using response surface design, and the optimal combination of extraction parameters was determined through regression analysis. Based on the optimal combination of extraction parameters, plant powder was subjected to ultrasonic-assisted extraction to obtain plant polyphenol extracts. Ultra-high performance liquid chromatography-tandem mass spectrometry was used to characterize the chemical composition and content distribution of polyphenols, thereby achieving the optimization of plant polyphenol extraction and component characterization.

2. The method of claim 1, wherein, Plant raw materials are sequentially dried, pulverized, and sieved to obtain plant powder, including: The collected fresh plant materials are pre-frozen and freeze-dried. The processed raw materials are crushed to reduce the particle size and increase the contact area between the material and the solvent. The crushed material is sieved to remove coarse particles and obtain plant powder with uniform particle size.

3. The method of claim 2, wherein, Using solvent concentration, solid-liquid ratio, extraction time, and ultrasonic power as influencing factors, single-factor experiments were conducted based on plant powder to determine the range of influence of each factor on the extraction effect of plant polyphenols, and the total polyphenol content of the corresponding extracts was measured, including: Using plant powder as the extraction substrate, solvent concentration, solid-liquid ratio, extraction time, and ultrasonic power were set as single variables. Under the premise of keeping other experimental conditions constant, the level of a single variable was changed step by step and the corresponding extraction operation was completed. Collect the extracts obtained from each group of experiments, determine the total polyphenol content of the extracts, and record the extraction effect under different variable levels; By comparing the differences in total polyphenol content under different factor levels, the influence trend and effective range of each factor on the extraction effect were determined.

4. The method of claim 3, wherein, Based on the influence range determined by single-factor experiments, a regression model was established using response surface methodology with solvent concentration, solid-liquid ratio, extraction time, and ultrasonic power as independent variables and total polyphenol content as the response value. The model included: The range of influence determined by single-factor experiments was used as the design interval, and solvent concentration, solid-liquid ratio, extraction time and ultrasonic power were selected as independent variables. The total polyphenol content was used as the response value to evaluate the extraction effect, and multiple combination experiments were carried out using response surface design. Collect measured data from each group of experiments, substitute the data into the model for fitting calculation, and establish a regression model between the independent variable and the response value.

5. The method according to claim 4, characterized in that, The optimal combination of extraction parameters was determined through regression analysis, including: Statistical analysis was performed on the established regression model to determine the significance of the effects of each factor on the total polyphenol content. Analyze the interactions among various factors to determine the synergistic effects of different parameter combinations on the extraction results; The extreme value is solved based on the regression model to obtain the parameter conditions that make the total polyphenol content optimal. The optimal combination of extraction parameters was selected and determined based on the comprehensive model analysis results.

6. The method according to claim 5, characterized in that, Based on the optimal combination of extraction parameters, ultrasonic-assisted extraction of plant powder was performed to obtain plant polyphenol extracts, including: Plant powder and extraction solvent are mixed in the optimal ratio to form an extraction system; Applying ultrasound under optimal solvent concentration, extraction time, and ultrasonic power conditions enhances the dissolution and diffusion of polyphenols. The complete extraction process is completed under the set extraction conditions, allowing the polyphenols in the plant powder to be fully released into the solvent; The extracted system was processed to obtain the target plant polyphenol extract.

7. The method according to claim 6, characterized in that, The chemical composition and content distribution of polyphenols were characterized using ultra-high performance liquid chromatography-tandem mass spectrometry, including: Plant polyphenol extracts were used as the detection target, and ultra-high performance liquid chromatography was used to separate polyphenolic components. The chemical composition of polyphenols was determined by structural identification of the separated components using tandem mass spectrometry. Quantitative analysis was performed on each polyphenol component to calculate the specific content of each component; By integrating qualitative and quantitative results, the overall content distribution pattern of polyphenols was statistically analyzed, and the systematic component characterization of the extract was completed.

8. A device for the extraction optimization and component characterization of plant polyphenols, characterized in that, include: The raw material preparation module is used to process plant raw materials by drying, crushing and sieving in sequence to obtain plant powder. Factor screening was used to determine the range of influence of each factor on the extraction effect of plant polyphenols by conducting single-factor experiments based on plant powder, with solvent concentration, material-liquid ratio, extraction time and ultrasonic power as influencing factors, and to determine the total polyphenol content of the corresponding extract. The parameter optimization module is used to establish a regression model based on the influence range determined by single-factor experiments, with solvent concentration, solid-liquid ratio, extraction time and ultrasonic power as independent variables and total polyphenol content as the response value. The optimal combination of extraction parameters is determined by response surface design and regression analysis. The component characterization module is used to perform ultrasonic-assisted extraction of plant powder based on the optimal combination of extraction parameters to obtain plant polyphenol extracts. Ultra-high performance liquid chromatography-tandem mass spectrometry is used to characterize the chemical composition and content distribution of polyphenols, thereby achieving the extraction optimization and component characterization of plant polyphenols.

9. An electronic device, characterized in that, Including processor and memory; The processor runs a program corresponding to the executable program code stored in the memory to implement the method as described in any one of claims 1-7.

10. A non-transitory computer-readable storage medium having a computer program stored thereon, characterized in that, When the program is executed by the processor, it implements the method as described in any one of claims 1-7.

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