Method for determining stewed radix puerariae processing technological parameters

Abstract

The invention relates to the technical field of traditional Chinese medicine preparation, and provides a method for determining stewed radix puerariae processing technological parameters, which comprises the following steps: taking processing temperature, processing time and bran dosage as investigated single factors; the method comprises the following steps: determining initial processing parameters by taking the contents of 6 ''-O-beta-D-glucoside, 3 '-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein and daidzein in a stewed radix puerariae sample as evaluation indexes; performing response surface experiment design by taking the processing temperature, the processing time and the bran dosage as independent variables; performing regression fitting on the obtained data to obtain a regression equation; performing variance analysis on the regression equation; and drawing a two-dimensional contour map and a three-dimensional response surface map of interaction of the factors, and determining optimal processing technological parameters. According to the technical scheme, the problem that the quality of stewed radix puerariae decoction pieces is unstable due to the fact that key parameters such as the processing time and the processing temperature in the stewed radix puerariae processing technology in the related technology are large in difference is solved.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine preparation technology, specifically to a method for determining the processing parameters of roasted kudzu root. Background Technology

[0002] Kudzu root is the wild kudzu plant of the legume family. Pueraria lobata The dried root of *Kudzu* (Willd.) Ohwi is sweet and pungent in taste, cool in nature, and enters the spleen, stomach, and lung meridians. It has the effects of relieving muscle tension and reducing fever, promoting body fluid production and quenching thirst, promoting rash eruption, raising yang and stopping diarrhea, clearing the meridians and activating collaterals, and detoxifying alcohol. Clinically, it is often used to treat symptoms such as fever and headache due to exogenous wind-cold, stiff neck and back pain, diabetes, and incomplete measles eruption. The chemical composition of kudzu root is complex, with flavonoids (such as puerarin and daidzein) being the main active ingredients, and it also contains organic acids, coumarins, and other components. Kudzu root can be processed in various ways, including stir-frying, baking in wet paper, and baking with wheat bran. Currently, clinical practice often uses wheat bran-baked kudzu root, believing that baking reduces its dryness and enhances its function of raising yang and stopping diarrhea. The 2020 edition of the Chinese Pharmacopoeia only includes processed kudzu root slices. While local processing standards do include the preparation of roasted kudzu root, they lack detailed descriptions of the degree of processing and its properties. The degree of processing is judged solely by sensory characteristics such as color, texture, and odor, without specifying concrete quality standards and quantitative parameters. Currently, the industry has not yet established a complete and unified processing procedure for roasted kudzu root. Furthermore, in practice, key parameters such as processing time and temperature vary significantly, directly leading to insufficient quality stability of roasted kudzu root slices and making it difficult to fully meet the stringent requirements of clinical use and market applications. Summary of the Invention

[0003] This invention proposes a method for determining the processing parameters of stewed kudzu root, which solves the problem of unstable quality of stewed kudzu root slices caused by large differences in key parameters such as processing time and temperature in the processing of stewed kudzu root in related technologies.

[0004] The technical solution of the present invention is as follows: This invention proposes a method for determining the processing parameters of stewed kudzu root, comprising the following steps: Using processing temperature, processing time, and bran dosage as single factors, kudzu root samples with different processing temperatures, processing times, and bran dosages were obtained. The initial processing parameters were determined by using the contents of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzein in the kudzu root samples as evaluation indicators. Based on the initial processing parameters, with processing temperature, processing time, and bran dosage as independent variables, response surface methodology was used to design a response surface experiment. The data obtained from the response surface methodology were fitted with regression, and the CRITIC weighted scores of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein and daidzein were used as the objective function to obtain the regression equation. Analysis of variance was performed on the regression equation to determine the order of influence of each factor on the quality of processed products; Two-dimensional contour plots and three-dimensional response surface plots of the interactions of various factors were drawn to determine the optimal processing parameters; Experimental verification was conducted based on the aforementioned optimal processing parameters.

[0005] As a further technical solution, the steps for obtaining kudzu root samples under different processing temperatures, processing times, and wheat bran dosages include: Heat the frying container to the processing temperature, sprinkle in the wheat bran according to the amount needed, add the kudzu root when it starts to smoke, fry and process for the required time, remove it, sift out the wheat bran, let it cool, and you will get stewed kudzu root.

[0006] As a further technical solution, when obtaining kudzu root samples with different processing temperatures, processing times, and bran dosages, the processing temperatures include 70℃, 80℃, 90℃, 100℃, 110℃, and 120℃; the processing times include 20min, 25min, 30min, 35min, 40min, and 50min; and the bran dosage includes 20g, 30g, 40g, 50g, and 60g of bran per 100g of kudzu root.

[0007] As a further technical solution, when using the content of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzein in the simmered kudzu root sample as evaluation indicators, the content of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzein in the simmered kudzu root sample is determined by HPLC.

[0008] As a further technical solution, mobile phase A is a 0.1% (w / w) aqueous solution of phosphoric acid, and mobile phase B is acetonitrile; a gradient elution program is adopted, which specifically includes the following steps: From 0 to 15 minutes, the volume fraction of fluidity A was 90% to 87%, and the volume fraction of fluidity B was 10% to 13%. At 15-20 min, the volume fraction of fluidity A was 87%, and the volume fraction of fluidity B was 13%. At 20–27 min, the volume fraction of fluidity A was 87%–75%, and the volume fraction of fluidity B was 13%–25%. At 27–37 min, the volume fraction of fluidity A was 75%–67%, and the volume fraction of fluidity B was 25%–33%. At 37-47 min, the volume fraction of fluidity A was 67%, and the volume fraction of fluidity B was 33%.

[0009] As a further technical solution, the flow rate is 0.8~1.2mL / min, the column temperature is 33~37℃, and the detection wavelength is 287nm.

[0010] As a further technical solution, the reference solution is a mixed reference solution of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein and daidzeinogen, and the test solution is a methanol solution of kudzu root.

[0011] Preferably, the solvent for the mixed reference solution is methanol, and the concentrations of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzein in the mixed reference solution are 25 μg / mL, 25 μg / mL, 100 μg / mL, 40 μg / mL, 25 μg / mL, and 25 μg / mL, respectively.

[0012] Preferably, the method for preparing the reference solution includes: taking appropriate amounts of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzeinogen, and placing them separately in 50 mL volumetric flasks containing 10 mL of 70 vt% methanol, and making up to volume with 70 vt% methanol to prepare a standard solution of 0.1 mg / mL. Taking an appropriate amount for dilution, a mixed reference solution with concentrations of 25 μg / mL for 6''-O-β-D-glucoside, 25 μg / mL for 3'-OH puerarin, 100 μg / mL for puerarin, 40 μg / mL for 3'-OCH3 puerarin, 25 μg / mL for daidzein, and 25 μg / mL for daidzeinogen is prepared.

[0013] The preparation method of the test solution includes: accurately weighing 0.2 g of kudzu root powder after passing it through a No. 4 sieve, accurately adding 25 mL of methanol with a volume fraction of 70%, placing it in a stoppered conical flask, weighing, sonicating for 30 min, cooling, weighing, replenishing the weight, taking the supernatant, and filtering it through a 0.45 μm microporous membrane to obtain the test solution.

[0014] As a further technical solution, the initial processing parameters include processing temperatures of 90℃, 100℃ and 110℃, processing times of 25min, 30min and 35min, and wheat bran amounts of 40g, 50g and 60g based on the mass of wheat bran added per 100g of kudzu root.

[0015] The regression equation is Y = -15.09950 + 0.166725 A + 0.250964 B + 0.041635 C + 0.000185 AB - 0.000039 AC + 0.000029 BC - 0.003165 A 2 -0.001315 B 2 -0.000436 C 2 In the formula, A represents the processing time, B represents the processing temperature, and C represents the amount of wheat bran used. This invention also proposes a processing method for stewing kudzu root, using the optimal processing parameters obtained from the method for determining the processing parameters of stewing kudzu root. The stewing kudzu root processing method includes: heating the stir-frying container to the optimal processing temperature, sprinkling wheat bran according to the optimal amount, adding kudzu root when it smokes, stir-frying for the optimal processing time, removing it, sifting out the wheat bran, and letting it cool to obtain the stewed kudzu root product. The optimal processing temperature is 100℃, the optimal processing time is 30 minutes, and the optimal amount of bran is 50g based on the mass of 100g of kudzu root added.

[0016] The working principle and beneficial effects of this invention are as follows: 1. The inventors discovered that during the processing of simmered kudzu root, the contents of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzein all change. When optimizing the processing parameters of simmered kudzu root using only the contents of puerarin, daidzein, and daidzein as evaluation criteria, the limited selection of effective components leads to incomplete evaluation of the details of the processing parameters, resulting in unstable quality of simmered kudzu root processed using the optimized processing parameters. In this invention, the contents of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzein are used as evaluation criteria. This evaluation criterion is more comprehensive, closer to the essential changes in the process, and allows for more precise monitoring of the processing. This improves the sensitivity and reliability of the processing optimization, thereby enhancing the quality stability of simmered kudzu root products obtained using the determined optimal processing parameters.

[0017] 2. In this invention, parameters such as processing temperature, processing time, and amount of bran in the kudzu root processing technology are quantified, making the kudzu root processing process more uniform in actual operation, thereby improving the quality stability of the kudzu root processed products. Attached Figure Description

[0018] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0019] Figure 1 This represents the results of a single-factor experiment in Example 1 of the present invention, with processing time as the variable. Figure 2 This represents the results of a single-factor experiment in Example 1 of the present invention, with processing temperature as the variable. Figure 3 This is the result of a single-factor experiment in Example 1 of the present invention, with the amount of bran used as the variable; Figure 4 The two-dimensional contour plot and three-dimensional response surface plot are shown in Embodiment 1 of the present invention, illustrating the interaction between processing time and processing temperature. Figure 5 The figures show the two-dimensional contour plot and three-dimensional response surface plot of the interaction between processing time and bran dosage in Embodiment 1 of the present invention. Figure 6 The two-dimensional contour plot and three-dimensional response surface plot are shown for the interaction between processing temperature and bran dosage in Embodiment 1 of the present invention. Figure 7 This is the HPLC chromatogram of the mixed reference standard in Example 3 of the present invention; In the figure, 1 is 6''-O-β-D-glucoside, 2 is 3'-OH puerarin, 3 is puerarin, 4 is 3'-OCH3 puerarin, 5 is daidzein, and 6 is daidzeinogen. Figure 8 This is the HPLC chromatogram of the simmered kudzu root sample in Example 3 of the present invention; In the figure, 1 is 6''-O-β-D-glucoside, 2 is 3'-OH puerarin, 3 is puerarin, 4 is 3'-OCH3 puerarin, 5 is daidzein, and 6 is daidzeinogen. Detailed Implementation

[0020] The technical solutions of the present invention will be clearly and completely described below with reference to 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0021] It should be understood that, unless the context clearly indicates otherwise, the terms “comprising,” “including,” or “having” as used herein refer to the presence of an element, but do not exclude the presence or addition of one or more other elements. Furthermore, as used herein, “comprising” and / or “including” indicate the presence of shapes, numbers, steps, operations, members, elements, and / or combinations thereof, and do not exclude the presence or addition of one or more other shapes, numbers, operations, elements, and / or combinations thereof.

[0022] In this application, the numerical range indicated by "~" refers to the range of values ​​specified as the lower and upper limits, respectively, before or after the term. When multiple values ​​for the upper or lower limit of any numerical range are mentioned, the range disclosed herein can be understood as a range with any one of the mentioned upper limits as its upper limit and any one of the mentioned lower limits as its lower limit.

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, a method for determining the processing parameters of simmered kudzu root according to an embodiment of this invention will be described in detail below.

[0024] According to one aspect of the present invention, a method for determining the processing parameters of stewed kudzu root is provided, comprising the following steps: Using processing temperature, processing time, and bran dosage as single factors, kudzu root samples with different processing temperatures, processing times, and bran dosages were obtained. The initial processing parameters were determined by using the contents of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzein in the kudzu root samples as evaluation indicators. Based on the above initial processing parameters, with processing temperature, processing time and bran amount as independent variables, response surface methodology was used to design a response surface experiment. The data obtained from the response surface methodology were fitted with regression, and the CRITIC weighted scores of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein and daidzein were used as the objective function to obtain the regression equation. Analysis of variance was performed on the regression equation to determine the order of influence of each factor on the quality of processed products; Two-dimensional contour plots and three-dimensional response surface plots of the interactions of various factors were drawn to determine the optimal processing parameters.

[0025] The advantages of response surface methodology (RSM) in optimizing the processing technology of traditional Chinese medicine (TCM) are mainly reflected in its ability to handle multi-factor, multi-level experimental designs, reveal the interaction effects between factors, and make predictions and optimizations through precise mathematical models. Compared with traditional single-factor experimental methods, RSM is more efficient and economical, while also providing more scientific and systematic optimization solutions, making it suitable for complex TCM processing technologies and industrial applications. In this invention, RSM is used to optimize the processing parameters of roasted kudzu root, making the determined processing parameters more reliable, thus ensuring good repeatability of the roasted kudzu root processing technology and facilitating the standardization of the quality of roasted kudzu root products.

[0026] In one embodiment of the present invention, the steps for obtaining kudzu root products with different processing temperatures, processing times, and wheat bran dosages include: Heat the frying container to the processing temperature, sprinkle in the wheat bran according to the amount needed, add kudzu root when it starts to smoke, fry and process for the required time, remove it, sift out the wheat bran, let it cool, and you will get the stewed kudzu root product.

[0027] In one embodiment of the present invention, when obtaining kudzu root samples with different processing temperatures, processing times, and bran dosages, the processing temperatures include 70°C, 80°C, 90°C, 100°C, 110°C, and 120°C; the processing times include 20 min, 25 min, 30 min, 35 min, 40 min, and 50 min; and the bran dosage includes 20 g, 30 g, 40 g, 50 g, and 60 g of bran per 100 g of kudzu root.

[0028] In one embodiment of the present invention, when the contents of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzein in the simmered kudzu root sample are used as evaluation indicators, the contents of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzein in the simmered kudzu root sample are determined by HPLC.

[0029] In this invention, to determine the content of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzein in simmered kudzu root samples, an HPLC method is provided for determining the content of these six components in simmered kudzu root samples. Methodological validation shows that this method exhibits good linearity, excellent repeatability, good stability, and strong robustness, enabling rapid and accurate detection of these six components in simmered kudzu root samples. This provides reliable data support for the quality control of simmered kudzu root products and the optimization of processing parameters.

[0030] In one embodiment of the present invention, when determining the contents of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzein in a simmered kudzu root sample using HPLC, mobile phase A is a 0.1% (w / w) aqueous solution of phosphoric acid, and mobile phase B is acetonitrile; a gradient elution program is used, specifically including the following steps: From 0 to 15 minutes, the volume fraction of fluidity A was 90% to 87%, and the volume fraction of fluidity B was 10% to 13%. At 15-20 min, the volume fraction of fluidity A was 87%, and the volume fraction of fluidity B was 13%. At 20–27 min, the volume fraction of fluidity A was 87%–75%, and the volume fraction of fluidity B was 13%–25%. At 27–37 min, the volume fraction of fluidity A was 75%–67%, and the volume fraction of fluidity B was 25%–33%. At 37-47 min, the volume fraction of fluidity A was 67%, and the volume fraction of fluidity B was 33%.

[0031] In this invention, when using HPLC to detect the content of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzein in processed kudzu root products, a 0.1% phosphoric acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, and a specific gradient elution program is employed to obtain chromatograms with high resolution and no tailing phenomenon, thereby making the detection results more accurate.

[0032] In one embodiment of the present invention, the flow rate is 0.5~1.5 mL / min, the column temperature is 30~40℃, and the detection wavelength is 287 nm.

[0033] Preferably, the flow rate is 1 mL / min, the column temperature is 35℃, and the detection wavelength is 287 nm.

[0034] Preferably, the chromatographic column is a Shiseido CAPCELL PAK C18 with a specification of 4.6 × 250 nm.

[0035] In one embodiment of the present invention, the reference solution is a mixed reference solution of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzeinogen, and the test solution is a methanol solution of kudzu root.

[0036] In one embodiment of the present invention, the initial processing parameters include processing temperatures of 90°C, 100°C, and 110°C, processing times of 25 min, 30 min, and 35 min, and the amount of bran added per 100 g of kudzu root is 40 g, 50 g, and 60 g.

[0037] In one embodiment of the present invention, the regression equation is Y = -15.09950 + 0.166725 A + 0.250964 B + 0.041635 C + 0.000185 AB - 0.000039 AC + 0.000029 BC - 0.003165A 2 -0.001315 B 2 -0.000436 C 2 In the formula, A is the processing time, B is the processing temperature, and C is the amount of wheat bran used.

[0038] According to another aspect of the present invention, the present invention also proposes a kudzu root processing method, which uses the above-mentioned method for determining the kudzu root processing parameters to obtain the optimal processing parameters. The kudzu root processing method includes: heating the frying container to the optimal processing temperature, sprinkling in the optimal amount of bran, adding kudzu root when it starts to smoke, frying for the optimal processing time, removing it, sifting out the bran, and cooling it to obtain the kudzu root product. The optimal processing temperature is 100℃, the optimal processing time is 30 minutes, and the optimal amount of wheat bran added is 50g based on the mass of 100g of kudzu root.

[0039] The following will describe in detail, with reference to examples, a method for determining the processing parameters of stewed kudzu root according to the present invention. The embodiments of the present invention described below can be modified in various forms; therefore, the scope of the present invention should not be construed as limited to the embodiments described in detail below. The embodiments are provided to help those skilled in the art to more easily understand the present invention.

[0040] The model numbers of instruments and reagents mentioned below are derived from the following: High-performance liquid chromatograph (Agilent Technologies 1260 Infinity); analytical balance: Mettler XPE26 (0.001 mg) (Shanghai Mettler Instruments Co., Ltd.); Mettler CPA224S (0.01 mg) (Shanghai Mettler Instruments Co., Ltd.); KH-800KDE ultrasonic cleaner (Kunshan Hechuang Ultrasonic Instruments Co., Ltd.); ultrapure water system (Millipore, USA); multi-functional mini roasting machine (Hunan Xiangjiang New Area Kuangyuxing Trading Co., Ltd.).

[0041] Reference standards: Puerarin (batch number: 110752-201514, content calculated as 95.5%, purchased from the National Institutes for Food and Drug Control); Daidzein (batch number: 111738-201904, content calculated as 93.4%, purchased from the National Institutes for Food and Drug Control); Daidzein (batch number: 1502-200101, content calculated as 97.2%, purchased from the National Institutes for Food and Drug Control); 6''-O-β-D-glucan Glycosides (batch number: 168060-90-0, content calculated at 96.96%, purchased from Chengdu Ruifen Endedan Biotechnology Co., Ltd.); 3'-OCH3 puerarin (batch number: 1117046-07-1, content calculated at 98.06%, purchased from Shanghai Chen Microbial Technology Co., Ltd.); 3'-OH puerarin (batch number: 117076-54-5, content calculated at 99.22%, purchased from Shanghai Chen Microbial Technology Co., Ltd.).

[0042] Four batches of kudzu root samples: all purchased from pharmacies, detailed information is shown in Table 1.

[0043] Table 14 Information on Kudzu Root Samples

[0044] Reagents: Methanol, acetonitrile (chromatographic grade, Merck, Germany), water was ultrapure water; all other reagents were analytical grade.

[0045] The chromatographic column was a Shiseido CAPCELL PAK C18 with a specification of 4.6 × 250 nm.

[0046] Example 1 A method for determining the processing parameters of stewed kudzu root includes: Single-factor experimental design: As shown in Tables 2 to 4 below, processing temperature, processing time, and amount of bran were investigated as single factors. Based on the processing method of the kudzu root sample, kudzu root samples with different processing temperatures, processing times, and amounts of bran were obtained. The processing method of the kudzu root sample was as follows: heat the frying container to the processing temperature, sprinkle in the bran according to the amount of bran, add kudzu root (batch number: 230301) when it smokes, fry for the processing time, remove, sift out the bran, and let cool to obtain the kudzu root sample.

[0047] Table 2 Single Factor Analysis—Processing Time

[0048] Table 3 Single Factors—Examination of Processing Temperature

[0049] Table 4 Single Factor Analysis—Examination of Bran Usage

[0050] The initial processing parameters were determined by using the contents of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein and daidzein in the simmered kudzu root samples as evaluation indicators. The contents of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzeinogen in stewed kudzu root samples were determined by HPLC. The specific steps are as follows: Preparation of reference solution: Take appropriate amounts of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzeinogen, and place them in 50 mL volumetric flasks containing 10 mL of 70 vt% methanol. Dilute to volume with 70 vt% methanol to prepare a 0.1 mg / mL standard solution. Take an appropriate amount to dilute and prepare a mixed reference solution. The concentrations of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzeinogen in this solution are 25 μg / mL, 25 μg / mL, 100 μg / mL, 40 μg / mL, 25 μg / mL, and 25 μg / mL, respectively. Preparation of the test solution: After passing the kudzu root powder through a No. 4 sieve, accurately weigh 0.2 g, accurately add 25 mL of 70 VT% methanol into a stoppered conical flask, weigh, sonicate for 30 min, cool, weigh, add 70 VT% methanol to make up the weight, take the supernatant, and filter it through a 0.45 μm microporous membrane to obtain the test solution.

[0051] Chromatographic conditions: The column type was Shiseido CAPCELL PAK C18, with a specification of 4.6 × 250 nm; Mobile phase A is a 0.1% (w / w) aqueous solution of phosphoric acid, and mobile phase B is acetonitrile; Perform gradient elution according to the gradient elution procedure in Table 5: Table 5 Gradient elution program

[0052] Flow rate: 1 mL / min, column temperature: 35℃, injection volume: 10 μL, detection wavelength: 287 nm.

[0053] The content determination results are shown in Table 6: Table 6. Content Determination Results

[0054] The data in Table 6 is standardized using the following formula: This represents the minimum value. (This represents the maximum value), and the results are shown in Table 7: Table 7 Results after standardization

[0055] The standardized results were subjected to correlation analysis to obtain the correlation coefficients, as shown in Table 8. Table 8 Correlation Coefficients

[0056] According to the formula and the calculated weight (where... C j Indicates the first j The amount of information contained in each evaluation indicator, σ j This represents the standard deviation of the standardized column vector. r tj Indicators t and j The correlation coefficient between them W j For the first j The objective weights of each indicator are shown in Table 9: Table 9 Weighting coefficients for different components

[0057] The results of the single-factor experiment are as follows Figures 1-3 As shown: from Figures 1-3 The initial processing parameters were determined as follows: processing time: 25 min, 30 min and 35 min; processing temperature: 90℃, 100℃ and 110℃; and amount of wheat bran (based on the mass of wheat bran added to 100g of kudzu root): 40g, 50g and 60g.

[0058] Based on the above initial processing parameters, with processing temperature, processing time, and bran dosage as independent variables, a response surface methodology was used to design a response surface experiment (kudzu root sample batch number: CP032230601), as shown in Table 10. Table 10 Response Surface Design

[0059] The contents of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzein in the kudzu root samples were determined by HPLC in response surface methodology. The results are shown in Table 11. Table 11 Results of response surface methodology content determination

[0060] The data in Table 11 is standardized using the following formula: This represents the minimum value. (This represents the maximum value), and the results are shown in Table 12: Table 12 Standardization Results

[0061] Using software for regression fitting, the regression equation with the CRITIC weighted score of the six components of kudzu as the objective function is Y = -15.09950 + 0.166725 A + 0.250964 B + 0.041635 C + 0.000185 AB -0.000039 AC + 0.000029 BC -0.003165 A² -0.001315 B² -0.000436 C², where A is the processing time, B is the processing temperature, and C is the amount of wheat bran used.

[0062] An analysis of variance was performed on the regression equation to determine the order of influence of each factor on the quality of processed products; the results of the analysis of variance are shown in Table 13. Table 13 Results of Analysis of Variance

[0063] Note:** P <0.01 indicates a highly significant difference;* P <0.05 indicates a significant difference; P A value >0.05 indicates that the difference is not significant.

[0064] As can be seen from Table 13: (1) The model shows that P(0.0005) < 0.001, indicating that the model has a highly significant difference and the model establishment is meaningful. Its lack-of-fit term P(0.1002) > 0.05, indicating that the model has a good fit in the regression region, the abnormal error is small in the actual experiment, the experimental method is reliable, and it can predict and analyze the optimal processing parameters of kudzu root. (2) Among the three single factors, in the three linear terms A (processing time), B (processing temperature), and C (wheat bran dosage), the partial regression coefficient of A (processing time) is less than 0.05, indicating that processing time has a significant effect on the processing of kudzu root. The partial regression coefficient of B (processing temperature) is less than 0.01, indicating that processing temperature has a highly significant effect on the processing of kudzu root. The interaction term has a significant effect on A. 2 B 2 C 2 The partial regression coefficients P (0.0011, 0.0001, 0.0213) < 0.05, indicating that the three factors have a significant impact on the processing of simmered kudzu root. These results demonstrate that the influence of each level of factor on the response value is not a simple linear relationship. (3) By analyzing the corresponding F and P values, it can be seen that the interaction between the two factors is: AB>BC>AC; the influence of each factor is in the order of B>A>C. It can be seen that the processing temperature is the main factor affecting the processing technology of stewed kudzu root, followed by the processing time, and the least important factor is the amount of wheat bran.

[0065] The two-dimensional contour plot and three-dimensional response surface plot of the interactions of various factors are shown in the figure. Figures 4-6 .

[0066] from Figures 4-6 The optimal processing parameters for stewing kudzu root were determined to be: processing time 28.907 min, processing temperature 98.021℃, and bran dosage (based on the mass of bran added per 100g of kudzu root) 49.64g. Under these conditions, the model predicted a total score of 0.6435 for stewing kudzu root. Considering the actual experimental operation, the optimal processing parameters were finally determined to be: processing time 30 min, processing temperature 100℃, and bran dosage (based on the mass of bran added per 100g of kudzu root) 50g.

[0067] To further verify the accuracy of the response surface methodology (RSM) model, the optimal processing parameters were selected: processing time 30 min, processing temperature 100℃, and wheat bran dosage (based on the mass of wheat bran added per 100g of kudzu root) 50g. Three trials were conducted (kudzu root sample batch numbers: 230301, 232590401, and 2307001). The average total score of the simmered kudzu root under these conditions was measured to be 0.6405. This result is close to the predicted value, indicating a good fit of the equation and that the extraction process obtained through RSM optimization is stable and feasible. Furthermore, it demonstrates that the RSM-optimized model fits the actual situation well, and that the model is stable and reliable.

[0068] Example 2 The process of simmering kudzu root includes the following steps: Heat the frying container to 100℃, sprinkle in 50g of wheat bran, and when it starts to smoke, add 100g of kudzu root. Fry for 30 minutes until it turns yellowish-brown and releases a caramel aroma. Remove the container, sift out the wheat bran, and let it cool to obtain the stewed kudzu root product.

[0069] Example 3 The contents of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzeinogen in a simmered kudzu root sample (batch number: CP032230601) were determined by HPLC, using the same method as in Example 1. The HPLC chromatogram of the mixed reference standard is shown below. Figure 7 As shown, the HPLC chromatogram of the stewed kudzu root sample is as follows. Figure 8 As shown.

[0070] The methodological validation of this method is as follows: 1. Linearity test Accurately pipette a series of concentrations of 6''-O-β-D-glucoside (5.051 μg / mL, 10.102 μg / mL, 25.255 μg / mL, 50.51 μg / mL, 101.02 μg / mL), 3'-OH puerarin (5.021 μg / mL, 10.042 μg / mL, 25.105 μg / mL, 50.21 μg / mL, 100.42 μg / mL), puerarin (25.335 μg / mL, 50.67 μg / mL, 126.675 μg / mL, 253.35 μg / mL, 506.7 μg / mL), and 3'-OCH3 puerarin (7.644 μg / mL, 15.288 μg / mL, 10.102 μg / mL, 25.255 μg / mL, 50.51 μg / mL, 101.02 μg / mL), respectively. 10 µL of reference solutions of daidzein (5.127 μg / mL, 10.254 μg / mL, 25.635 μg / mL, 51.27 μg / mL, 102.54 μg / mL), daidzein (1.678 μg / mL, 3.356 μg / mL, 8.39 μg / mL, 16.78 μg / mL, 33.56 μg / mL), 38.22 μg / mL, 76.44 μg / mL, 152.88 μg / mL, daidzein (5.127 μg / mL, 10.254 μg / mL, 25.635 μg / mL, 51.27 μg / mL, 102.54 μg / mL), and daidzein (1.678 μg / mL, 3.356 μg / mL, 8.39 μg / mL, 16.78 μg / mL, 33.56 μg / mL) were injected into a high-performance liquid chromatograph (HPLC) for determination. Linear regression was performed with the reference concentration (μg / mL) as the x-axis and the peak area integral as the y-axis. The results are shown in Table 14, indicating that each component has a good linear relationship within its respective linear range.

[0071] Table 14 Standard curves and linear ranges of the six components

[0072] 2. Precision test A mixed standard solution containing 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzeinogen at concentrations of 25.26 μg / mL, 25.11 μg / mL, 126.68 μg / mL, 38.22 μg / mL, 25.10 μg / mL, and 25.64 μg / mL, respectively, was injected six times. The peak area RSDs of each component were 0.8%, 0.4%, 0.1%, 0.1%, 0.4%, and 1.2%, respectively, indicating good precision.

[0073] 3. Stability test The same test solution (kudzu root sample batch number: CP032230601) was taken and placed at room temperature. The samples were then injected and analyzed at 0h, 2h, 4h, 8h, 12h, and 24h. The RSDs of the peak areas of each component were 0.7%, 0.4%, 0.1%, 0.2%, 0.8%, and 0.9%, respectively. This indicates that the test solution was basically stable within 24h.

[0074] 4. Repeatability test Take the same batch of samples (batch number: CP032230601), grind them finely and pass them through a No. 4 sieve. Take three portions of 0.1g, 0.2g and 0.3g respectively, weigh them accurately, and prepare test solutions according to the method in Example 1. Inject the samples under the proposed chromatographic conditions, record the peak area integral value of each component, calculate the content and its RSD. The results are shown in Table 15, indicating that the method has good repeatability.

[0075] Table 15 Repeatability test results (mg / g)

[0076] 5. Recovery test Take nine samples (batch number: CP032230601) with known content, each approximately 0.2 g, accurately weighed, and add 25 mL of three mixed reference solutions of low, medium, and high concentrations prepared with 70 vt% methanol solution. The concentrations of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzein in the low-concentration mixed reference solution were 5.692 μg / mL, 19.096 μg / mL, 91.472 μg / mL, 23.668 μg / mL, 17.464 μg / mL, and 2.904 μg / mL, respectively; the concentrations of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, and daidzein in the medium-concentration mixed reference solution were... The concentrations of daidzein and genistein were 11.384 μg / mL, 38.192 μg / mL, 182.944 μg / mL, 47.336 μg / mL, 34.928 μg / mL, and 5.808 μg / mL, respectively. In the high-concentration mixed reference standard, the concentrations of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and genistein were 17.076 μg / mL, 57.288 μg / mL, 274.416 μg / mL, 71.004 μg / mL, 52.392 μg / mL, and 8.712 μg / mL, respectively. Test solutions were prepared according to the method in Example 1, and the samples were injected for determination. The recovery rates were calculated, and the results are shown in Table 16, indicating that the method has a good recovery rate.

[0077] Table 16 Results of the recovery test of kudzu root ( n =9)

[0078]

[0079] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for determining the processing parameters of stewed kudzu root, characterized in that, Includes the following steps: Using processing temperature, processing time, and bran dosage as single factors, kudzu root samples with different processing temperatures, processing times, and bran dosages were obtained. The initial processing parameters were determined by using the contents of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzein in the kudzu root samples as evaluation indicators. Based on the initial processing parameters, with processing temperature, processing time, and bran dosage as independent variables, response surface methodology was used to design a response surface experiment. The data obtained from the response surface methodology were fitted with regression, and the CRITIC weighted scores of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein and daidzein were used as the objective function to obtain the regression equation. Analysis of variance was performed on the regression equation to determine the order of influence of each factor on the quality of processed products; Two-dimensional contour plots and three-dimensional response surface plots of the interactions of various factors were drawn to determine the optimal processing parameters.

2. The method for determining the processing parameters of stewed kudzu root according to claim 1, characterized in that, The steps for obtaining kudzu root samples with different processing temperatures, processing times, and bran dosages include: heating the frying container to the processing temperature, sprinkling in bran according to the required amount, adding kudzu root when it starts to smoke, frying for the specified processing time, removing the container, sifting out the bran, and letting it cool to obtain the kudzu root product.

3. The method for determining the processing parameters of stewed kudzu root according to claim 1, characterized in that, When obtaining kudzu root samples with different processing temperatures, processing times, and bran dosages, the processing temperatures included 70℃, 80℃, 90℃, 100℃, 110℃, and 120℃; the processing times included 20min, 25min, 30min, 35min, 40min, and 50min; and the bran dosage included 20g, 30g, 40g, 50g, and 60g of bran per 100g of kudzu root.

4. The method for determining the processing parameters of stewed kudzu root according to claim 1, characterized in that, When using the contents of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzein in the simmered kudzu root sample as evaluation indicators, the contents of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzein in the simmered kudzu root sample are determined by HPLC.

5. The method for determining the processing parameters of stewed kudzu root according to claim 4, characterized in that, Mobile phase A is a 0.1% (w / w) aqueous solution of phosphoric acid, and mobile phase B is acetonitrile; a gradient elution program is used, which specifically includes the following steps: From 0 to 15 minutes, the volume fraction of fluidity A was 90% to 87%, and the volume fraction of fluidity B was 10% to 13%. At 15-20 min, the volume fraction of fluidity A was 87%, and the volume fraction of fluidity B was 13%. At 20–27 min, the volume fraction of fluidity A was 87%–75%, and the volume fraction of fluidity B was 13%–25%. At 27–37 min, the volume fraction of fluidity A was 75%–67%, and the volume fraction of fluidity B was 25%–33%. At 37-47 min, the volume fraction of fluidity A was 67%, and the volume fraction of fluidity B was 33%.

6. The method for determining the processing parameters of stewed kudzu root according to claim 4, characterized in that, The flow rate was 0.8~1.2 mL / min, the column temperature was 33~37℃, and the detection wavelength was 287 nm.

7. The method for determining the processing parameters of stewed kudzu root according to claim 4, characterized in that, The reference solution is a mixed reference solution of 6''-O-β-D-glucoside, 3'-OH puerarin, puerarin, 3'-OCH3 puerarin, daidzein, and daidzeinogen; the test solution is a methanol solution of kudzu root.

8. The method for determining the processing parameters of stewed kudzu root according to claim 1, characterized in that, The initial processing parameters include processing temperatures of 90℃, 100℃, and 110℃, processing times of 25min, 30min, and 35min, and the amount of wheat bran added per 100g of kudzu root is 40g, 50g, and 60g.

9. A method for determining the processing parameters of stewed kudzu root according to claim 1, characterized in that, The regression equation is Y = -15.09950 + 0.166725 A + 0.250964 B + 0.041635 C + 0.000185 AB -0.000039 AC + 0.000029 BC -0.003165 A 2 -0.001315 B 2 -0.000436 C 2 .

10. A process for preparing kudzu root by simmering, characterized in that, The optimal processing parameters are obtained by using the method for determining the processing parameters of stewed kudzu root as described in any one of claims 1 to 9. The stewed kudzu root processing process includes: heating the stir-frying container to the optimal processing temperature, sprinkling in the optimal amount of bran, adding kudzu root when it starts to smoke, stir-frying for the optimal processing time, removing the container, sifting out the bran, and cooling it to obtain the stewed kudzu root product. The optimal processing temperature is 100℃, the optimal processing time is 30 minutes, and the optimal amount of bran is 50g based on the mass of 100g of kudzu root added.

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