A kind of processing technology of honey-roasted mulberry leaves

Abstract

The present invention relates to the field of traditional Chinese medicine processing technology. The present invention quantifies the three factors of frying temperature, frying time, and auxiliary material dosage, establishes an objective evaluation method, optimizes the processing technology of honey mulberry leaves using the Box-Behnken design-response surface method, detects the index components of honey mulberry leaves using modern instrumental analysis methods, calculates the weight coefficients of seven indicators including chlorogenic acid, rutin, isoquercetin, astragalin, total flavonoids, alcohol extract, and moisture using the CRITIC-AHP composite weighting method, and calculates the overall evaluation "normalization value" to obtain the optimal processing technology results. The final optimized processing process steps are as follows: take 7.5g of refined honey, dilute it with boiling water (mulberry leaf: boiling water = 1:2), pour it into 30g of clean mulberry leaf medicinal materials, mix well, simmer for 4 minutes, place it in a hot pot, fry it at 170°C for 10 minutes, remove it and cool it, and then seal it for storage.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine processing, and in particular to a processing technology for roasting mulberry leaves with honey. Background Art

[0002] Mulberry leaf, the dried leaves of Morus alba L. (Moraceae), has the effects of dispelling wind-heat, clearing the lungs and moistening dryness, and clearing the liver and improving eyesight. It contains a variety of active ingredients, including flavonoids, polyphenols, alkaloids, polysaccharides, and sterols. It has antioxidant, blood sugar-lowering, cholesterol-lowering, anti-aging, cancer-preventing, anti-inflammatory, liver-protecting, and intestinal flora-regulating effects, and is widely used in food, medicine, and other fields. Mulberry leaf processing and application has a long history, with various methods throughout history, such as external application of mulberry leaf ash, boiling mulberry leaf in vinegar, steaming mulberry leaf with wine, steaming and drying mulberry leaf nine times and drying it nine times, and steaming mulberry leaf with sesame seeds. Modern methods primarily involve roasting mulberry leaf with honey (hereinafter referred to as honeyed mulberry leaf).

[0003] The use of honey mulberry leaf was first recorded in the "Taiping Shenghui Fang" during the Song and Yuan dynasties. Its moist nature enhances its lung-moistening effect compared to raw herbs and is often used for dry coughs. The "Pharmacopoeia of the People's Republic of China" (1963 edition), the "Hubei Province Traditional Chinese Medicine Preparation Specifications" (2018 edition), and the "Shandong Province Traditional Chinese Medicine Preparation Specifications" (2012 edition) include honey mulberry leaf, but none of them provide specific processing parameters for honey mulberry leaf. Most descriptions use the phrase "fry over low heat until the surface is dark yellow, slightly shiny, and not sticky." Furthermore, the moisture content and index component content of honey mulberry leaf pose challenges in production, processing, and quality control, and there are few literature reports on the processing technology of honey mulberry leaf. Therefore, clarifying the processing parameters of honey mulberry leaf is of great reference value for its production, processing, and quality control. Summary of the Invention

[0004] The purpose of the present invention is to propose a method for optimizing the processing technology of honey-roasted mulberry leaves, optimize the processing technology of honey-roasted mulberry leaves by Box-Behnken design-response surface methodology, calculate the weight coefficients of seven indicators including chlorogenic acid, rutin, isoquercetin, astragalin, total flavonoids, alcohol extract and moisture by CRITIC-AHP composite weighting method, and calculate the overall evaluation "normalized value" (OD) to obtain the optimal processing technology result.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for optimizing the processing technology of honey-roasted mulberry leaves, comprising the following steps:

[0007] 1) Determination of chlorogenic acid, rutin, isoquercetin and astragalin in honey mulberry leaves by UPLC ultra high performance liquid chromatography

[0008] 2) Determination of total flavonoid content

[0009] The total flavonoids concentration in the sample was determined according to the standard curve constructed with rutin standard, and the total flavonoids content in mulberry leaves was calculated according to the following formula:

[0010]

[0011] Where c is the total flavonoid concentration of the test solution (mg·mL -1 ); n is the dilution factor; V is the sample volume (mL); M is the sample mass (g);

[0012] 3) Determination of alcohol extract content

[0013] Determined according to the alcohol extract content determination method;

[0014] 4) Determination and scoring of moisture

[0015] After measuring the moisture content, the moisture content of 7% to 13% was selected as the best value, with 5 points; 0% to 7% or 13% to 15% was selected as the excellent value, with 4 points; 15% to 20% was selected as the medium value, with 3 points; 20% to 30% was selected as the poor value, with 2 points; and above 30% was selected as the worst value, with 1 point.

[0016] 5) Determine weights using the CRITIC entropy weight method

[0017] The CRITIC entropy weight method was used to objectively assign weights to seven indicators, including chlorogenic acid, rutin, isoquercetin, astragalin, total flavonoids, alcohol extract, and moisture. Positive indicators were used for data standardization. After obtaining the standardized values, the correlation of each data was analyzed to obtain the correlation matrix, and the weights of the seven indicators were calculated respectively.

[0018] 6) Determine weights using AHP

[0019] For the seven indicators, a pairwise comparison discriminant matrix was constructed to assign importance and calculate weights. According to the priority order, the weight coefficients of rutin, alcohol extract, water, total flavonoids, isoquercetin, astragalin, and chlorogenic acid were calculated to be 0.31, 0.19, 0.19, 0.11, 0.06, 0.06, and 0.06, respectively. The consistency ratio CI of the matrix was 0.007 and the consistency ratio CR was 0.005, both less than 0.10.

[0020] 7) Determination of composite weight and comprehensive score

[0021] The objective weight w calculated by the CRITIC method i And the objective weight w calculated by AHP method A The composite weight w is obtained by comprehensive calculation, and the OD value is calculated:

[0022] Compound weight calculation formula:

[0023] OD value calculation formula:

[0024] 8) Single-factor experiment

[0025] The frying temperature, frying time and auxiliary material dosage were used as independent variable factors, and the overall OD value was obtained by comprehensive weighting according to the CRITIC-AHP composite weighting method, and the center points corresponding to the highest overall OD value were obtained;

[0026] 9) Response surface methodology to optimize process experiments

[0027] The three factors of frying temperature (X1), frying time (X2) and auxiliary material dosage (X3) were selected as independent variables, and three levels were selected for each factor. The OD values of chlorogenic acid, rutin, isoquercetin, astragalin, total flavonoids, alcohol extract and moisture content were used as response values. A three-factor three-level experiment was conducted. The frying temperature, frying time and auxiliary material dosage corresponding to the highest theoretical OD value were calculated, which were the optimized processing parameters of honey-roasted mulberry leaves.

[0028] The optimized processing steps of honey-roasted mulberry leaves are as follows: take 7.5g of refined honey, dilute it with boiling water (mulberry leaves: boiling water = 1:2), pour it into 30g of clean mulberry leaves and mix well, simmer for 4 minutes, place it in a hot pot, stir-fry at 170℃ for 10 minutes, take it out and cool it, and then seal it for storage.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1) Our research team previously conducted fingerprint studies on mulberry leaves from ten different origins, including Bozhou, Anhui; Hefei, Anhui; Lintong, Shaanxi; Nanjing, Jiangsu; and Qingyuan, Guangdong. Results showed that mulberry leaves from Lintong, Shaanxi, had the highest levels of chlorogenic acid, rutin, isoquercetin, and astragalin, with more peaks and better resolution. Therefore, mulberry leaves from Lintong, Shaanxi, were selected as the experimental material for this experiment. Based on preliminary results from previous experiments, a 1:2 ratio of mulberry leaves to boiling water was found to be optimal for diluting the honey in this experiment. Excessive water content can lead to excess moisture in the leaves, prolonging the stir-frying time and increasing the amount of honey lost due to sticking to the bottom of the pan, affecting the accuracy of the results. Excessive water content prevents the honey from fully absorbing the leaves, making it difficult to mix evenly. Uneven water absorption also leads to uneven heating during stir-frying, further impacting the accuracy of the results. Therefore, a 1:2 ratio of mulberry leaves to boiling water was selected for dilution.

[0031] 2) For the selection of chromatographic conditions, preliminary experiments were conducted to screen for the optimal conditions. Different mobile phase systems (acetonitrile-0.2% phosphoric acid, acetonitrile-0.5% phosphoric acid, methanol-0.5% formic acid, and acetonitrile-0.5% formic acid) were investigated, and the peak shape and separation were optimal when using acetonitrile-0.5% formic acid aqueous solution. Two extraction methods, ethanol reflux and methanol ultrasonic extraction, were investigated, and the results showed no significant difference in chromatographic information between the two. However, the methanol ultrasonic extraction method was simple to operate, so the methanol ultrasonic extraction method was selected to prepare the test solution.

[0032] 3) The CRITIC entropy weight method can objectively reflect the importance of various factors, but it cannot reflect the requirements of human-defined codes and standards. The AHP (Analytical Hierarchy Process) assigns weights through subjective decision-making analysis. This experiment uses a composite weighting method that combines these two weighting methods. This method can both take into account the requirements of human regulations and avoid errors caused by excessive subjectivity. Taking into account the advantages of both objective indicator evaluation and subjective human regulations, it avoids the situation where a single objective evaluation results in a failure to meet code requirements, and also avoids the situation where a single subjective evaluation reduces the stability and repeatability of the results.

[0033] 4) The present invention quantifies the three factors of frying temperature, frying time, and auxiliary material dosage, establishes an objective evaluation method, optimizes the processing technology of honey mulberry leaves by Box-Behnken design-response surface method, detects the index components of honey mulberry leaves by modern instrumental analysis methods, calculates the weight coefficients of chlorogenic acid, rutin, isoquercetin, astragalin, total flavonoids, alcohol extract, and moisture by CRITIC-AHP composite weighting method, and calculates the overall evaluation "normalized value" (OD) to obtain the optimal processing process results, in order to provide a basis for the standardized production of honey mulberry leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Figure 2 is the UPLC chromatogram of blank control (A), mixed control (B) and test sample (C). Peaks 1, 2, 3, and 4 in the figure correspond to chlorogenic acid, rutin, isoquercetin, and astragalin, respectively.

[0035] Figure 2 This is a bar chart showing the content changes of four components in 10 batches of raw and honey mulberry leaves. S represents the average content of the 10 batches of raw product, and P represents the average content of the 10 batches of processed product. Groups 1, 2, 3, and 4 correspond to chlorogenic acid, rutin, isoquercitrin, and astragalin, respectively. *P < 0.05, **P < 0.01, ***P < 0.001 for comparisons between the two groups.

[0036] Figure 3 This is the curve of the influence of frying temperature on OD value.

[0037] Figure 4 This is the curve of the effect of frying time on OD value.

[0038] Figure 5 This is the curve showing the effect of excipient dosage on OD value.

[0039] Figure 6 The interaction response surface (A) and contour map (B) of each factor. DETAILED DESCRIPTION

[0040] The method for optimizing the processing technology of honey-roasted mulberry leaves of the present invention is further described in detail below with reference to the examples and drawings.

[0041] Example 1

[0042] 1. Instruments and Materials

[0043] Waters Acquity ultrahigh performance liquid chromatograph (Waters); 1510 full-wavelength microplate reader (ThermoFisher Scientific); DHG-9202-2 electric constant-temperature drying oven (Shanghai Sanfa Scientific Instrument Co., Ltd.); DK-S24 electric constant-boiling water bath (Shanghai Sanfa Scientific Instrument Co., Ltd.); KQ-300DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); XY-100MW-T moisture meter (Changzhou Lucky Electronic Equipment Co., Ltd.); BS200S electronic balance (Sartorius, Germany).

[0044] Rutin standard (batch number: PU0001-0025, purity ≥98%), chlorogenic acid standard (batch number: PU0100-0025, purity ≥98%), isoquercetin standard (batch number: PS011802, purity ≥98%), and astragalin standard (batch number: PS011379, purity ≥98%) were purchased from Chengdu Pusi Biotechnology Co., Ltd.; sodium nitrite (batch number: 0101020) was purchased from Shanghai Suyi Chemical Reagent Co., Ltd. Aluminum nitrate (batch number: C10792299) was purchased from Shanghai McLean Biochemical Technology Co., Ltd.; sodium hydroxide (batch number: 900720) was purchased from Tianjin Jinbei Fine Chemical Co., Ltd.; acetonitrile (chromatographic grade, batch number: 22125324) and methanol (chromatographic grade, batch number: 22075293) were purchased from Anhui Tiandi High Purity Solvent Co., Ltd.; formic acid (analytical grade, 20191115) was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; water was Watsons purified water.

[0045] The mulberry leaf samples were collected from Lintong, Xi'an, Shaanxi on November 12, 2022, dried and set aside. They were identified by Peng Tangyi, deputy director of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, as dried leaves of Morus aLba L., a plant of the Moraceae family.

[0046] Baihua brand jujube flower honey (batch number: 20221220) was purchased from Beijing Baihua Beekeeping Technology Development Co., Ltd.

[0047] 2. Methods and Results

[0048] 2.1 Sample preparation

[0049] 2.1.1 Preparation of raw mulberry leaves:

[0050] Remove impurities, crush, remove stems, and sieve out ash. Take 30g of clean mulberry leaves, crush them, pass through a 50-mesh sieve, and set aside.

[0051] 2.1.2 Honey refining method:

[0052] Take honey and add water and mix well. Pour the mixed honey into the pot, stir while adding, heat it on medium heat until it boils, skim off the foam, then heat it on low heat to 116-118℃ and keep it slightly boiling. When light yellow, shiny and uniform fish-eye bubbles appear, refine it to the flag-hanging state. When the honey is brown, it feels sticky when you twist it with your hands, and no long white threads appear between two fingers, quickly remove it from the pot and set aside.

[0053] 2.1.3 Preparation of honey mulberry leaves:

[0054] Prepare according to traditional standards. Take 30g of cleaned mulberry leaves and stir-fry them using the honey-roasting method until they are no longer sticky. Take another 7.5g of refined honey and dilute it with boiling water (mulberry leaves: boiling water = 1:2). Pour it into the herbs and mix thoroughly. Let it simmer for a while. Place in a hot pot and stir-fry over low heat (120-160°C) until the surface is dark yellow and slightly shiny. When it is no longer sticky, remove from the heat, dry, crush, and pass through a 50-mesh sieve. Set aside.

[0055] 2.2 Solution preparation

[0056] 2.2.1 Preparation of standard solution:

[0057] Weigh appropriate amounts of chlorogenic acid, rutin, isoquercetin, and astragalin standards respectively, accurately weigh them, and place them in 10 mL volumetric flasks. Add methanol until dissolved at the scale line to prepare a solution containing 0.816 mg·mL chlorogenic acid. -1 , rutin 0.581 mg·mL -1 , isoquercetin 0.489 mg·mL -1 , astragaloside 0.435 mg·mL -1 0.74 mL, 0.55 mL, 0.8 mL, and 0.33 mL of the standard stock solution were respectively drawn into a 5 mL volumetric flask, and methanol was added to the volume up to the mark. Ultrasonication was performed for 5 minutes to obtain a mass concentration of 0.121 mg·mL of chlorogenic acid. -1 , rutin 0.064 mg·mL -1 , isoquercetin 0.078 mg·mL -1 , astragaloside 0.029 mg·mL-1 The mixed reference solution was stored in a 4°C refrigerator for later use.

[0058] 2.2.2 Preparation of test solution:

[0059] Accurately weigh 1.0 g of sample powder (passed through No. 3 sieve) and place it in a 10 mL volumetric flask. Add methanol and dilute to the mark. Stopper the flask, weigh it, and ultrasonically treat it for 30 min (40 kHz, 300 W). Take it out, let it cool, make up for the lost mass, shake it well, filter it, and spin it at 12000 r / min. -1 Centrifuge (centrifugal radius 3 cm) for 20 min, filter the supernatant through a 0.22 μm microporous filter membrane, and take the filtrate to obtain the test solution.

[0060] 2.3 Determination of index component content

[0061] 2.3.1 Chromatographic conditions:

[0062] EcLipse PLus C 18 Chromatographic column (4.6×250 mm, 5 μm); mobile phase: acetonitrile (A)-0.5% formic acid aqueous solution (B); gradient elution program: 10%–15% A (0–3 min), 15%–15% A (3–8 min), 15%–25% A (8–15 min), 25%–45% A (15–25 min), 45%–70% A (25–27 min), 70%–100% A (27–30 min), 100%–10% A (30–32 min), 10%–10% A (32–35 min); injection volume: 5 μL; detection wavelength: 358 nm; flow rate: 1.0 mL min -1 ; Column temperature is 30℃.

[0063] 2.3.2 System suitability test:

[0064] The chromatogram is obtained by detecting under the chromatographic conditions (such as Figure 1 Under these conditions, the theoretical plate number (n) calculated based on the chlorogenic acid peak was no less than 5,000, based on the rutin and isoquercetin peaks no less than 15,000, and based on the astragalin peak no less than 25,000. The resolution between the four measured chromatographic peaks and adjacent peaks was greater than 1.5, indicating good resolution. The signal-to-noise ratios for all four components were greater than 10, meeting the requirements for quantitative determination. The tailing factors for all four components were between 0.95 and 1.05, indicating good chromatographic peak symmetry and meeting the system suitability requirements.

[0065] 2.3.3 Specificity inspection:

[0066] according to Figure 1It can be seen that under the chromatographic conditions of this experiment, the separation of chlorogenic acid, rutin, isoquercetin and astragaloside was good, and the methanol solution in the blank group had no interference, and the test specificity was good.

[0067] 2.3.4 Linear relationship investigation:

[0068] Accurately pipette 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 μL of the mixed standard solution prepared under "2.2.1" into the sample and analyze according to the chromatographic conditions under "2.3.1." Plot a standard curve using the injected standard volume (μg) as the abscissa (x) and the corresponding peak area as the ordinate (Y). Calculate the regression equation. The results (see Table 1) indicate that each component exhibits a good linear relationship within the corresponding mass concentration range.

[0069] Table 1 Results of linear relationship investigation of the four components in the sample

[0070]

[0071] 2.3.5 Precision inspection:

[0072] Accurately pipette 5 μL of the test solution and inject it continuously for a total of 6 times. Measure according to the chromatographic conditions in "2.3.1" and calculate the RSD values of the common peak area and retention time. The results show that the RSD values of the peak area are 8.21% to 174.32%, and the RSD values of the retention time are 1.05% to 13.16%, indicating good instrument precision.

[0073] 2.3.6 Stability Study: Take the same test sample solution and, according to the chromatographic conditions in 2.3.1, inject the sample at 0, 4, 8, 12, 16, and 24 hours. Record the peak area and calculate the RSD of each peak area. The results show that the RSD values range from 22.23% to 789.07%, indicating that the test sample is stable within 24 hours.

[0074] 2.3.7 Repeatability inspection:

[0075] Take 6 portions of the sample powder, accurately weigh them, and prepare the test solution according to the method in "2.2.2". Detect the solution using the chromatographic conditions in "2.3.1". Record the peak area of each component and calculate the RSD of the peak area of each component. The results show that the RSD values range from 10.17% to 232.15%, indicating good reproducibility under the conditions.

[0076] 2.3.8 Sample recovery test:

[0077] Six samples (0.5 g each) of the four components whose contents had been determined were collected. Standard solutions with concentrations close to those in the samples were added. Test solutions were prepared according to the method in "2.2.2." Samples were injected and tested according to the chromatographic conditions in "2.3.1." The peak area of each component was recorded. The results showed that the average recoveries of chlorogenic acid, rutin, isoquercetin, and astragalin were 100.22%, 99.84%, 100.27%, and 99.65%, respectively. The RSDs were 0.41%, 0.71%, 0.36%, and 0.76%, respectively, indicating good recovery.

[0078] 2.3.9 Determination of index components before and after roasting mulberry leaves with honey:

[0079] Prepare 10 portions of raw mulberry leaves and honeyed mulberry leaves according to the methods in "2.1.1" and "2.1.3", and record them as S1~S10 and P1~P10 respectively. Accurately weigh the powder and prepare the test solution according to "2.2.2". Accurately aspirate 5μL of the test solution and inject it into the ultra-high performance liquid chromatography under the chromatographic conditions in "2.3.1". Calculate the content of each component. The results are as follows: Figure 2 The results showed that the chlorogenic acid content of mulberry leaves decreased significantly after honey roasting (P < 0.01), while the contents of rutin, isoquercetin and astragalin increased significantly (P < 0.01).

[0080] 2.4 Determination of total flavonoid content

[0081] 2.4.1 Preparation of standard solution:

[0082] Accurately weigh 10.11 mg of rutin standard, place it in a 25 mL volumetric flask, dilute to the mark with 95% ethanol, and shake well to obtain a concentration of 0.4044 mg mL -1 The standard stock solution was stored at 4°C until use.

[0083] 2.4.2 Preparation of test solution:

[0084] Take 0.5g of sample powder and place it in a 25mL stoppered conical flask. Add 15mL of 55% ethanol, weigh and record the weight. Ultrasonicate at 50℃ for 17min. Take it out, let it cool, make up the lost weight, shake it well, filter it, take 2mL into a centrifuge tube, and centrifuge it at 12000r·min. -1 Centrifuge (centrifugal radius 3 cm) for 20 minutes and take the supernatant.

[0085] 2.4.3 Linear relationship investigation:

[0086] Accurately pipette 6 portions of the above standard solution, 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL each, into 2 mL centrifuge tubes. Then, add 1.0, 0.9, 0.8, 0.6, 0.4, 0.2, and 0 mL of 95% ethanol solution, respectively. Seal and mix to obtain rutin standard solutions of different concentrations. Add 150 μL of 20% NaNO2 solution to the system, mix thoroughly, and let stand for 5 minutes; add 200 μL of 40% Al(NO3)3 solution, mix thoroughly, and let stand for 5 minutes; finally, add 500 μL of 20% NaOH solution, mix thoroughly, and let stand for 15 minutes. The concentrations obtained are 0, 0.022, 0.044, 0.087, 0.131, 0.175, and 0.219 mg·mL -1 95% ethanol solution was used as blank control solution. 200 μL of each sample was added to a 96-well plate. The concentration of rutin stock solution in each well was recorded. The plate was placed in an automatic microplate reader, the wavelength was set to 510 nm, and the absorbance value was measured. The mass concentration of rutin standard (mg mL -1 ) as the X-axis and the Y-axis as the absorbance (A) to draw a standard curve, and the linear equation Y = 5.5311x + 0.0721, r 2 =0.9995, linear range 0~0.219mg·mL -1 .

[0087] 2.4.4 Detection and calculation of total flavonoid content:

[0088] Accurately pipette 1.0 mL of the test solution into a centrifuge tube, determine the flavonoid concentration in the sample according to the standard curve, and calculate the total flavonoid content of mulberry leaves (mg g) according to the following formula: -1 ), repeated three times and took the average value. The total flavonoids content of mulberry leaves is:

[0089]

[0090] Note: c is the total flavonoid concentration of the test solution (mg·mL -1 ); n is the dilution factor; V is the sample volume (mL); M is the sample mass (g)

[0091] 2.5 Determination of alcohol extract content

[0092] According to the pharmacopoeia method, place 1.000g of sample into a 50mL stoppered conical flask. Accurately measure 25mL of anhydrous ethanol, seal, weigh, and record the weight. Let it stand for 1 hour. Slightly boil in a waterbath for 1 hour. After cooling, seal, weigh, adjust the weight, and filter under reduced pressure. Accurately measure 10mL of the filtrate into a steaming dish that has been dried to constant weight. Evaporate the solvent in a waterbath, then dry in a 105°C oven for 3 hours to constant weight. Cool to room temperature in a desiccator. Quickly and accurately weigh and record the weight. Repeat three times and take the average.

[0093] 2.6 Moisture determination

[0094] 2.6.1 Determination method:

[0095] Take 3.000g of sample and spread it evenly on the sample tray of the moisture analyzer with a thickness not exceeding 5mm. Start the instrument and dry it until the reading remains unchanged for 1 minute. Record the reading.

[0096] 2.6.2 Moisture determination scoring method:

[0097] Moisture content is a key indicator for evaluating the quality of medicinal slices, directly impacting the intrinsic quality of the material. Due to the moisture content requirements of the Pharmacopoeia of the People's Republic of China (hereinafter referred to as the "Pharmacopoeia") and the General Rules for Quality Standards of Chinese Herbal Medicines (Trial Implementation) (hereinafter referred to as the "General Rules"), this experiment selected a moisture content of 7% to 13% as the optimal value, assigned 5 points; 0% to 7% or 13% to 15% as the excellent value, assigned 4 points; 15% to 20% as the median value, assigned 3 points; 20% to 30% as the poor value, assigned 2 points; and above 30% as the worst value, assigned 1 point. Directly using moisture content for scoring does not reflect the moisture content requirements of the Pharmacopoeia and the "General Rules," and moisture content is negatively correlated with medicinal material quality. Therefore, a scoring method was used in conjunction with relevant regulations to score moisture content, as shown in Table 2.

[0098] Table 2 Moisture content scoring criteria

[0099]

[0100] 2.7 Comprehensive scoring method

[0101] 2.7.1 CRITIC entropy weight method to determine weights:

[0102] In this experiment, moisture content directly affects the determination of component content. The alcohol extract contains total flavonoids, which include rutin, astragalin, and isoquercetin. The contents of the four selected chemical components increased and decreased before and after processing, suggesting potential mutual conversion or decomposition. Therefore, the seven selected indicator components are correlated rather than independent of each other. Therefore, the CRITIC entropy weight method was used for objective weighting. Higher scores for indicator component content indicate better processing parameters, so positive indicators were used for data standardization.

[0103] Right now:

[0104] After obtaining the standardized values, the correlation of each data was analyzed to obtain a correlation matrix (see Table 3). According to the CRITIC method formula, the weights of the seven indicators of chlorogenic acid, rutin, isoquercetin, astragalin, total flavonoids, alcohol extract, and water content were calculated (see Table 4).

[0105] Table 3 Correlation matrix of CRITIC entropy weight index

[0106]

[0107] Table 4 Calculation results of indicator weights

[0108]

[0109]

[0110] 2.7.2 Determine weights using AHP:

[0111] The Analytic Hierarchy Process (AHP) constructs an analysis matrix based on subjective judgment, thereby classifying factors into an ordered hierarchy for analysis. The processing technology of honey-roasted mulberry leaves was used as the decision-making objective, which can be reflected by seven indicators (sub-objectives) to establish a hierarchical model. Referring to the "Hubei Province Traditional Chinese Medicine Preparation Specifications" (2018 edition) and the "Shandong Province Traditional Chinese Medicine Preparation Specifications" (2012 edition), rutin, alcohol extract and moisture content, total flavonoids, and isoquercitrin, astragalin, and chlorogenic acid were selected as control indicators based on their contribution to the quality control of honey-roasted mulberry leaves (rutin > alcohol extract = moisture content > total flavonoids > isoquercitrin = astragalin = chlorogenic acid). A pairwise comparison discriminant matrix was constructed (see Table 5), and importance was assigned and weights were calculated. According to the priority order, the weight coefficients of rutin, alcohol extract, water, total flavonoids, isoquercetin, astragalin and chlorogenic acid were calculated to be 0.31, 0.19, 0.19, 0.11, 0.06, 0.06 and 0.06, respectively. The consistency proportional factor CI of the matrix was 0.007 and the consistency ratio CR was 0.005, both of which were less than 0.10, indicating that the constructed priority judgment matrix has good consistency and the corresponding weight coefficients are valid, which can be used to weight the above seven indicators.

[0112] Table 5 Judgment matrix of pairwise comparison of AHP indicators

[0113]

[0114] Note: CI=0.007<0.10, CR=0.005<0.10

[0115] 2.7.3 Determination of composite weights and comprehensive scores:

[0116] The objective weight w calculated by the CRITIC method i And the objective weight w calculated by AHP method A The composite weight w is calculated comprehensively and the OD value is calculated. (See Table 6)

[0117] Compound weight calculation formula:

[0118] OD value calculation formula:

[0119] Among them, x i Represents a single data, x i(max) Represents the maximum value in a set of data.

[0120] Table 6 Comprehensive weight coefficients of various indicators

[0121]

[0122] 2.8 Single-factor experiment

[0123] 2.8.1 Stir-frying temperature:

[0124] Weigh 6 portions of mulberry leaves, 30g each, and dissolve 7.5g (25%) of refined honey in boiling water (mulberry leaves: boiling water = 1:2), let it sit for 4 minutes, and stir-fry at 120, 140, 160, 180, 200, and 220℃ for 7 minutes. Prepare the test samples and determine their rutin, alcohol extract, moisture, total flavonoids, chlorogenic acid, isoquercetin, and astragalin, and use the CRITIC-AHP composite weighting method to obtain the overall OD value. The results show that when the stir-frying temperature is 160℃, the content of each index is higher and the overall OD value is the highest. Therefore, 160℃ is selected as the center point of the stir-frying temperature in the response surface experiment. The results are shown in Figure 2. Figure 3 .

[0125] 2.8.2 Stir-frying time:

[0126] Weigh 5 portions of mulberry leaves, 30g each, dissolve 7.5g (25%) of refined honey in boiling water (mulberry leaves: boiling water = 1:2), let it sit for 4 minutes, and stir-fry at 160℃ for 3, 5, 7, 9, and 11 minutes respectively. Prepare the test samples and determine their rutin, alcohol extract, water, total flavonoids, chlorogenic acid, isoquercetin, and astragalin, and use the CRITIC-AHP composite weighting method to obtain the total score OD value. The results show that the total score OD value is the highest when the stir-frying time is 9 minutes, so 9 minutes is selected as the center point of the stir-frying time in the response surface experiment. The results are shown in Figure 2. Figure 4 .

[0127] 2.8.3 Auxiliary material (refined honey) dosage:

[0128] Weigh 5 portions of mulberry leaves, 30 g each, and dissolve 4.5 (15%), 6.0 (20%), 7.5 (25%), 9.0 (30%), and 10.5 (35%) g of refined honey in boiling water (mulberry leaves: boiling water = 1:2), simmer for 4 minutes, and stir-fry at 160°C for 7 minutes. Prepare the test samples and determine their rutin, alcohol extract, moisture, total flavonoids, chlorogenic acid, isoquercetin, and astragalin, and obtain the overall OD value by comprehensive weighting according to the CRITIC-AHP composite weighting method. The results show that the overall OD value is the highest when the auxiliary material dosage is 25%, so 25% is selected as the center point of the auxiliary material dosage in the response surface experiment. The results are shown in Figure 2. Figure 5 .

[0129] 2.9 Response surface methodology for process optimization

[0130] 2.9.1 Experimental Design:

[0131] According to the results of the single-factor experiment and the principle of response surface experimental design, three factors were selected as independent variables, namely, frying temperature (X1), frying time (X2), and auxiliary material dosage (X3). Three levels were selected for each factor, and the OD values of chlorogenic acid, rutin, isoquercetin, astragalin, total flavonoids, alcohol extract, and moisture content were used as response values. Design Expert DX13 software was used to perform a three-factor, three-level experimental design. The factors and levels are shown in Table 7, and the experimental arrangement and results are shown in Table 8.

[0132] Table 7 Design experimental factors and levels

[0133]

[0134] Table 8 Experimental arrangement and results of optimizing mulberry leaf honey roasting process using Box-Behnken response surface method

[0135]

[0136] 2.9.2 Model fitting and significance analysis:

[0137] Using Design-Expert 13 software, the data were subjected to quadratic polynomial regression and fitting, and the fitted comprehensive score regression equation was obtained: Y = 93.99 + 17.88X1 + 5.98X 2+ 1.58X3-3.96X1X2-1.17X1X3+2.07X2X3-31.49X1 2 -4.23X2 2 -5.45X3 2 , R 2=0.9911 indicates that the model has a good fit for this experiment and the difference is statistically significant within the scope of this experimental study. The results are shown in Table 9. As can be seen from the table: P < 0.0001 for the fitted model, indicating that the fit of this model is extremely significant and the fitted equation is consistent with the actual operation of honey-roasted mulberry leaves; the F value of the lack of fit term is 3.05, P = 0.1549 > 0.1 is extremely insignificant, indicating that this model fits well and there is no lack of fit term influence. The adjustment coefficient R 2 adj =0.9796, RSD is 4.17%, that is, the model can explain 97.96% of the response value changes, and the model fitting degree is high, indicating that the regression equation can make a good prediction of the processing technology. 2 The P≤0.001 has a very significant impact on the model, that is, X1 and X2 are very significant terms, which are the main factors affecting the quality of processed products. There are no significant differences in the interaction terms X1X2, X1X3, and X2X3, indicating that the interaction between the factors is not obvious. The quadratic term X1 2 、X2 2 、X3 2 There are statistical differences, indicating that the influence of each factor on the comprehensive score is not a simple linear relationship. According to the F value and equation analysis, the factors affecting the quality of honey-roasted mulberry leaves are ranked as follows: X1 (roasting temperature) > X2 (roasting time) > X3 (amount of auxiliary materials).

[0138] Table 9 Response surface regression simulation variance analysis of honey-roasted mulberry leaves processing technology

[0139]

[0140] Note: “-” indicates no significant difference, *P < 0.05 indicates statistical difference, **P < 0.01 indicates significant difference, ***P ≤ 0.001 indicates extremely significant difference.

[0141] 2.9.3 Results and Analysis:

[0142] The results of the response surface experiment are shown in Figure 6 The interactive effects of various factors on the processing of honey-roasted mulberry leaves show that the slopes for X1X2 and X1X3 are steeper, indicating that their interactions have a greater impact on the OD value. Before the frying temperature reaches its peak in the range of approximately 160-170°C, the OD value shows a rapid, linear upward trend, followed by a downward trend. This indicates that the active ingredient content of honey-roasted mulberry leaves increases with increasing processing temperature within a certain range, then decreases after reaching a peak, showing a trend of initially increasing and then decreasing. The slope for X2X3 is gentler, indicating that their interactions have a smaller impact on the OD value.

[0143] 2.9.4 Optimization of the best preparation process:

[0144] Using Design-Expert 13 software and the resulting fitting equation, we optimized the predicted processing conditions for honey-roasted mulberry leaves. We found that the highest theoretical OD value was 98.019, corresponding to a frying temperature of 170°C, a frying time of 10 minutes, and an auxiliary material dosage of 25%. The optimized processing conditions for honey-roasted mulberry leaves are as follows: 7.5g of refined honey was diluted with boiling water (mulberry leaves: boiling water = 1:2), poured into 30g of cleaned mulberry leaves, mixed thoroughly, and allowed to simmer for 4 minutes. The mixture was then placed in a hot pot and fried at 170°C for 10 minutes. The mixture was removed, cooled, and then sealed for storage.

[0145] Example 2

[0146] Process validation test:

[0147] Three validation tests were conducted according to the optimized process under "2.9.4". Rutin, alcohol extract, moisture, total flavonoids, chlorogenic acid, isoquercetin, and astragalin were measured, and weighted scores were assigned according to the method under "2.7.3" to obtain the overall OD value. The results are shown in Table 9. The experimental results show that the OD values of the three batches of honey-roasted mulberry leaves were 97.405, 96.865, and 98.465, respectively, with an average comprehensive score of 97.578. The deviations between the results of each group and the predicted values were 0.627%, 1.177%, and -0.455%, respectively, with an RSD of 0.834%, indicating that the experimental results were stable and had no significant differences, indicating that the honey-roasted mulberry leaf processing technology under the predicted value conditions was stable and controllable.

[0148] Table 10 Process verification test results

[0149]

[0150] The above content is merely an example and explanation of the concept of the present invention. Those skilled in the art may make various modifications or additions to the described specific embodiments or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the scope of protection of the present invention.

Claims

1. A process for preparing honey-roasted mulberry leaves, characterized in that: Take 7.5 g of refined honey, dilute it with 60 g of boiling water, pour it into 30 g of clean mulberry leaves, mix well, simmer for 4 minutes, place it in a hot pot, stir-fry at 170℃ for 10 minutes, take it out and cool it, then seal it for storage.

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