Preparation method of pig bile fried spina date seed based on quality evaluation and application thereof in treatment of anxiety

Abstract

This invention discloses a method for processing jujube seeds stir-fried with pig bile based on quality evaluation and its application in the treatment of anxiety. Jujube seed saponin A, jujube seed saponin B, magnoflorine, spinosin, 6'''-feruloyl spinosin, and the alcoholic extract of jujube seeds stir-fried with pig bile are used as evaluation indicators. A comprehensive score is determined using the analytic hierarchy process (AHP)-entropy weight method. The processing method is optimized through combined experimental analysis. The jujube seeds stir-fried with pig bile obtained using this method show significant therapeutic effects in a rat anxiety model. The high-dose group of jujube seeds showed the most significant increase in the proportion of rats entering the open arm after administration, which was 596% higher than the model group. Simultaneously, jujube seeds stir-fried with pig bile can significantly improve problems such as reduced number and loose and irregular arrangement of hippocampal neurons in insomnia mice, demonstrating a significant therapeutic effect on anxiety symptoms.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine processing, specifically to a method for processing jujube seeds stir-fried with pig bile based on quality evaluation and its application in the treatment of anxiety disorders. Background Technology

[0002] Anxiety disorder is a common mental illness characterized by persistent and uncontrollable excessive worry, tension, and fear. It often presents with numerous complications, leading to a complex condition and a low rate of complete remission. It severely impacts patients' daily work and life and has become one of the leading causes of global disease burden. Statistics show that the global prevalence of anxiety disorders is currently 7.3%, and it is increasingly affecting younger people. Current clinical treatment primarily uses Western medicines, such as selective serotonin (5-HT) reuptake inhibitors (e.g., sertraline), 5-HT-norepinephrine reuptake inhibitors (e.g., venlafaxine extended-release), and benzodiazepines. However, long-term use of these medications can lead to drug tolerance and addiction, withdrawal symptoms, and rebound anxiety.

[0003] Anxiety disorder falls under the category of "depression syndrome" in Traditional Chinese Medicine (TCM). TCM theory holds that its onset is often related to internal damage caused by the seven emotions. Emotional imbalance leads to stagnation of Qi, which in turn generates pathogenic factors such as dampness, phlegm, fire, and blood stasis, damaging organs such as the heart, liver, spleen, and kidneys. This results in insufficient production of Qi and blood and dysfunction of organ systems (such as liver dysfunction), ultimately leading to depression syndrome. TCM treatment takes a holistic approach, featuring multiple components and multiple targets, with good safety and fewer adverse reactions, giving it a unique advantage in the treatment of anxiety disorder.

[0004] Stir-fried jujube seeds with pig bile is a specialty of the Longsha Medical School. The Longsha Medical School innovatively uses pig bile to stir-fry jujube seeds to treat different diseases in clinical practice, giving full play to their effects of purging liver and gallbladder fire, calming the mind and soothing the nerves, and soothing the nerves. It is used to treat different clinical diseases.

[0005] The literature "Research and Clinical Application of Pig Bile-Fried Jujube Seeds in the Special Processing Methods of the Longsha Medical School" briefly mentions the historical evolution of pig bile-fried jujube seeds, but it has not been applied to the field of anxiety treatment, nor has it conducted research on the optimization of processing techniques, quality standards, or efficacy. Currently, the processing technology for pig bile-fried jujube seeds still has many shortcomings, such as the fact that all processed products are manually fried, leading to batch-to-batch errors; and the lack of modern instrumental analysis to establish quality standards, etc., all of which require further improvement. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide a method for processing jujube seeds stir-fried with pig bile based on quality evaluation.

[0007] The second objective of this invention is to provide the application of a method for processing jujube seeds stir-fried with pig bile based on quality evaluation in the preparation of drugs for treating anxiety.

[0008] The final objective of this invention is to provide a traditional Chinese medicine for relieving anxiety.

[0009] Technical Solution: To solve the above-mentioned technical problems, this invention provides a method for processing jujube seeds stir-fried with pig bile based on quality evaluation, including the following steps:

[0010] A comprehensive score was established based on the anti-anxiety active ingredients of jujube seeds stir-fried with pig bile. The comprehensive score was applied to a combined experimental analysis method to obtain the processing method, and / or the quality of the product obtained by the processing method was evaluated based on the anti-anxiety active ingredients. The anti-anxiety active ingredients are jujube seed saponin A, jujube seed saponin B, magnoflorine, spinosin, 6'''-feruloyl spinosin, and alcoholic extract of jujube seeds stir-fried with pig bile. The processing method is as follows: 11-15 mL of pig bile is added to every 20 g of raw jujube seeds to be processed, stirred to mix evenly, and fully moistened for 0.5-1.5 h. Then, the mixture is placed in a pot and stir-fried at 100-120 ℃ for 3-5 min to obtain jujube seeds stir-fried with pig bile.

[0011] The preferred method is to add 12.4-12.8 mL of pig bile to every 20 g of raw jujube kernels to be processed, stir to mix evenly, soak thoroughly for 1.00-1.05 h, and then stir-fry in a pot at 109-111℃ for 3.9-4.1 min.

[0012] The comprehensive score is F, where F = [0.3485 × (content of jujube seed saponin A / maximum content of jujube seed saponin A) + 0.2156 × (content of jujube seed saponin B / maximum content of jujube seed saponin B) + 0.0564 × (content of magnoflorine / maximum content of magnoflorine) + 0.1671 × (content of spinosin / maximum content of spinosin) + 0.0793 × (content of 6'''-feruloyl spinosin / maximum content of 6'''-feruloyl spinosin) + 0.1331 × (content of alcoholic extract from jujube seeds stir-fried with pig bile / maximum content of alcoholic extract from jujube seeds stir-fried with pig bile)] × 100.

[0013] Preferably, the comprehensive score is obtained by combining the analytic hierarchy process (AHP) with the entropy weight method.

[0014] Preferably, the analytic hierarchy process (AHP) uses the content of anti-anxiety active ingredients as the evaluation index to calculate the weights.

[0015] Preferably, the entropy weight method calculates the entropy value of the content of the anti-anxiety active ingredient to calculate the weight.

[0016] Preferably, the combined experimental analysis method includes single-factor response surface methodology.

[0017] Preferably, the joint experimental analysis method further includes experimental analysis of convolutional neural network models.

[0018] Preferably, the quality evaluation includes detecting the content of the anti-anxiety active ingredient by high performance liquid chromatography.

[0019] This invention also provides the application of a method for processing jujube seeds with pig bile based on quality evaluation in the preparation of drugs for treating anxiety.

[0020] The present invention also provides an anti-anxiety traditional Chinese medicine, which comprises 0.2378~0.2395% of jujube seed saponin A, 0.0522~0.0538% of jujube seed saponin B, 0.0635~0.0666% of magnoflorine, 0.1057~0.1068% of spinosin, 0.0280~0.0286% of 6'''-feruloyl spinosin, and 18.61~19.65% of jujube seed extract stir-fried with pig bile.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0022] (1) The anti-anxiety active ingredients of traditional Chinese medicine provided by this invention are combined with the analytic hierarchy process (AHP) and entropy weight method to determine the comprehensive score. This score is applicable to the optimization of processing technology by multiple combined experimental analysis methods, including single-factor response surface methodology and convolutional neural network (CNN) model analysis. The correlation coefficient R of the regression equation of the comprehensive score of the single-factor response surface methodology provided by this invention is... 2 The coefficient of variation was 0.9473, and the difference between the two was less than 0.2, indicating that the model is reliable and can be used to analyze experimental results and predict the optimal processing technology. The CNN final model showed small prediction errors on the test samples, and the measured curves and predicted curves generally matched well. The full-sample RMSE was 0.3901, MAE was 0.2643, and R0.2 was 0.9473. 2 The value of 0.9927 indicates that the established model is stable and reliable.

[0023] (2) The pig bile-fried jujube seed prepared according to the optimized process of this invention has stable quality. The RSD value of the anti-anxiety active ingredient content in three batches of different optimized processes was less than 3%. The prepared pig bile-fried jujube seed has a significant therapeutic effect in rat anxiety model. In the open field test, the rats' time spent in the central area and the distance traveled in the central area were significantly increased after administration of pig bile-fried jujube seed compared with the model group. In the elevated cross maze test, the proportion of rats entering the open arm was significantly increased after administration of high dose of pig bile-fried jujube seed, which was 596% higher than that of the model group and significantly higher than that of the high dose of raw jujube seed, indicating that administration of pig bile-fried jujube seed can significantly improve the anxiety behavior of rats. At the same time, pig bile-fried jujube seed can significantly improve the problems of reduced number of neurons in the hippocampus and hypothalamus of insomnia mice, and loose and irregular arrangement, and has a significant therapeutic effect on anxiety symptoms. Attached Figure Description

[0024] Figure 1 Graph showing the HPLC results of the mixed standard and the jujube seed stir-fried with pig bile;

[0025] Figure 2 This is a graph showing the results of the single-factor comprehensive scoring.

[0026] Figure 3 The response surface plot shows the effect of the interaction of various factors on the comprehensive score of stir-fried jujube seeds with pig bile.

[0027] Figure 4 A comparison chart of test set metrics for three CNN parameter schemes;

[0028] Figure 5 This is a comparison chart of the measured and predicted values ​​of the optimal CNN solution.

[0029] Figure 6 Heatmap for optimizing CNN process parameters;

[0030] Figure 7 The results of the open field test are shown in the rat trajectory diagram.

[0031] Figure 8 The results of the elevated cross test and the rat trajectory diagram;

[0032] Figure 9 This is a diagram showing the results of the rat tail suspension test;

[0033] Figure 10 Pathological sections of the hippocampus from different groups of rats;

[0034] Figure 11 These are pathological sections of the hypothalamus in different rat groups. Detailed Implementation

[0035] The technical solution provided by the present invention will be described in detail below with reference to the embodiments.

[0036] Example 1: Establishment of a method for processing jujube seeds stir-fried with pig bile based on their anti-anxiety active ingredients.

[0037] 1. A comprehensive score was established based on the anti-anxiety active ingredients in jujube seeds stir-fried with pig bile.

[0038] After consulting pharmacopoeias and literature such as "Research Progress on Sedative-Hypnotic Active Components and Pharmacological Effects of Ziziphus jujuba var. spinosa" and "Research Progress on Pharmacological Effects and Drug Pair Applications of Ziziphus jujuba var. spinosa", and seeking expert opinions, the contents of five components in Ziziphus jujuba var. spinosa (fried with pig bile), namely jujuboside A, jujuboside B, magnoflorine, spinosin, and 6'''-feruloyl spinosin, as well as the alcohol extract of Ziziphus jujuba var. spinosa (fried with pig bile), were used as evaluation indicators for the preparation of anti-anxiety drugs.

[0039] Using the Analytic Hierarchy Process (AHP), the priority order was established as follows: Jujuboside A > Jujuboside B = Spinosin > 6'''-Feruloyl Spinosin > Pig bile-fried jujube seed alcohol extract > Magnolia alkaloid. This order was used as a comparative advantage matrix, and the weight coefficients C for each indicator were calculated, as shown in Table 1. A consistency test was performed on the results, and the calculated CR = 0.05 < 0.1 and CI = 0.06 ≠ 0, indicating that the priority judgment matrix has good consistency and the weight coefficients are effective, making it suitable for indicator weighting.

[0040] Table 1 Priority Matrix for Comparison of Indicator Components

[0041]

[0042] Entropy weight method for determining weights: By processing the j evaluation indices of the i-th trial, a matrix X is constructed. ij And calculate the information entropy E according to formulas 1-4. j and weight W j The entropy values ​​of jujuboside A, jujuboside B, magnoflorine, spinosin, 6'''-feruloyl spinosin, and the alcohol extract were 0.9677, 0.9593, 0.9348, 0.9685, 0.9690, and 0.9088, respectively, with weights of 0.1107, 0.1393, 0.2235, 0.1080, 0.1063, and 0.3123, respectively.

[0043]

[0044] (Formula 1)

[0045]

[0046] (Formula 2)

[0047]

[0048] (Formula 3)

[0049]

[0050] (Formula 4)

[0051] Among them, X ij X is the original data or the original matrix. ij ' For the new matrix or new data after transformation, max(X) j ) and min(X j ) represent the maximum and minimum values ​​of the j-th indicator in all samples, respectively; m is the number of samples, p ij Let k be the proportion of the i-th sample on the j-th indicator; k = 1 / ln(m), E j d is the entropy value of the j-th index; j d is a redundant value. j =1-E j w j Let be the weight of the j-th indicator, and n be the number of indicators.

[0052] Anxiety treatment comprehensive score determination: The calculation formula is analyzed by combining the AHP method and the entropy weight method, and the comprehensive weight A is calculated according to formula 5. j C j The weights obtained from the analytic hierarchy process, W j Using the entropy weight method, the comprehensive weights of jujuboside A, jujuboside B, magnoflorine, spinosin, 6'''-feruloyl spinosin, and alcohol extract are 0.3485, 0.2156, 0.0564, 0.1671, 0.0793, and 0.1331, respectively. The comprehensive score F is calculated according to Formula 6.

[0053]

[0054] (Formula 5)

[0055] F = [0.3485 × (Jujube seed saponin A content / maximum jujube seed saponin A content) + 0.2156 × (Jujube seed saponin B content / maximum jujube seed saponin B content) + 0.0564 × (Magnolia alkaloid content / maximum magnolia alkaloid content) + 0.1671 × (Spinosin content / maximum spinosin content) + 0.0793 × (6'''-feruloylspinosin content / maximum 6'''-feruloylspinosin content) + 0.1331 × (Alcohol extract content / maximum alcohol extract content)] × 100

[0056] (Formula 6)

[0057] 2. Conduct a single-factor response surface experiment based on the comprehensive score.

[0058] The processing method for stir-fried jujube seeds with pig bile involves mixing fresh pig bile with jujube seeds, thoroughly moistening them, then stir-frying them in a pan until dry. The mixture is then removed, spread out, and allowed to cool. Single-factor analysis was conducted on four factors: stir-frying time, stir-frying temperature, moistening time, and amount of pig bile used. Five levels were set for each factor to conduct preliminary screening of the processing method. Groups included:

[0059] (1) Keep the three factors of stir-frying temperature, soaking time and amount of pig bile constant, and set the stir-frying time at five different levels of 3, 4, 5, 6 and 7 min.

[0060] (2) Keep the three factors of stir-frying time, soaking time and amount of pig bile constant, and set the stir-frying temperature at five different levels of 110, 120, 130, 140 and 150 ℃.

[0061] (3) Keep the three factors of stir-frying time, stir-frying temperature and amount of pig bile constant, and set the soaking time at five different levels: 0.5, 1.0, 1.5, 2.0 and 2.5 h.

[0062] (4) Keep the three factors of stir-frying time, stir-frying temperature and moistening time constant, and set five different levels of pig bile dosage: 5, 7, 9, 11 and 13 mL.

[0063] After preparing pig bile-fried jujube seeds according to the above grouping, the comprehensive score F was calculated. The content of each evaluation index was obtained by high-performance liquid chromatography (HPLC). The steps are as follows:

[0064] Sample preparation: Accurately weigh 1 g of pig bile-fried jujube seed powder that has passed through a No. 4 sieve, place it in a 150 mL Erlenmeyer flask, add 30 mL of 70% ethanol, sonicate at room temperature for 90 min, filter, wash the filter residue with 5 mL of 70% ethanol solution, combine the filtrate and washing liquid, recover the solvent to dryness, dissolve the residue in methanol, transfer to a 10 mL volumetric flask, dilute to the mark with methanol, shake well, filter through a 0.22 μm microporous membrane, and take the filtrate as the test solution, ready for sample loading.

[0065] Preparing Agilent Eclipse XBD-C 18 The chromatographic column (4.6 × 250 mm, 5 μm) was used with water (A) and acetonitrile (B) as the mobile phase, using gradient elution (0–10 min, 10%–20% B; 10–50 min, 20% B; 50–55 min, 20%–10% B) at a flow rate of 1.0 mL / min. -1 The column temperature was 30 ℃, the detection wavelength was 335 nm, and the injection volume was 10 μL. The contents of magnoflorine, spinosin, and 6'''-feruloyl spinosin were measured.

[0066] Prepare AkzoNobel Kromasil 100-5-C 18 The chromatographic column (4.6 × 250 mm, 5 μm) was used with a mobile phase of 0.2% glacial acetic acid (A) and acetonitrile (B), using gradient elution (0–2 min, 22% B; 2–8 min, 22%–26% B; 8–16 min, 26%–32% B; 16–20 min, 32%–35% B; 20–25 min, 35%–42% B; 25–30 min, 42%–48% B; 30–32 min, 48% B; 32–35 min, 48%–22% B; 35–41 min, 22% B), at a flow rate of 0.9 mL / min. -1 The column temperature was 25 ℃, and the injection volume was 10 μL. ELSD parameters were: evaporator temperature 80 ℃, nebulizer temperature 70 ℃, and gas flow rate 1.45 SLM. The contents of jujube seed saponins A and B were measured after sample loading.

[0067] The chromatographic peaks of the measured components showed good separation under the above conditions, such as Figure 1 As shown, Figure 1 In the diagram, A and B represent the results of the standard and the sample, respectively. Components 1, 2, 3, 4, and 5 represent magnoflorine, spinosin, 6'''-feruloyl spinosin, jujube seed saponin A, and jujube seed saponin B, respectively.

[0068] Determination of alcohol extract content: Accurately weigh approximately 2-4 g of sieved jujube seed powder (fried with pig bile) and place it in a 100-250 mL Erlenmeyer flask. Accurately add 50-100 mL of 95% ethanol, seal tightly, weigh, and let stand for 1 h. Then connect a reflux condenser, heat to boiling, and maintain a gentle boil for 1 h. After cooling, remove the Erlenmeyer flask, seal tightly, weigh again, and replenish the lost weight with 95% ethanol. Shake well, filter through a dry filter, accurately measure 25 mL of the filtrate, place it in an evaporating dish that has been dried to constant weight, evaporate to dryness on a water bath, dry at 105 ℃ for 3 h, cool in a desiccator for 30 min, and quickly and accurately weigh.

[0069] The comprehensive score F for each indicator was calculated according to Formula 6, and the results are shown in Table 2. Figure 2 As shown, based on the results, the frying time of 4 min, the frying temperature of 110 ℃, the moistening time of 1.0 h, and the amount of pig bile used of 13 mL were selected as the central levels of the subsequent response surface methodology.

[0070] Table 2 Single-factor experimental design and results

[0071]

[0072] Based on the results of the single-factor experiment, a four-factor, three-level experiment was set up (as shown in Table 3). The Box-Behnken response surface methodology was conducted using Design Expert 11 software. The method for determining the component content was the same as that used in the single-factor experiment. The results are shown in Table 4.

[0073] The data in Table 4 were fitted using Design-Expert 11 software. With A (frying time), B (frying temperature), C (soaking time), and D (amount of pig bile) as independent variables, and Y as the comprehensive score, the following bivariate polynomial regression equation was obtained: Y = 88.16 + 1.54A - 0.3550B - 0.5192C - 1.64D - 0.3275AB + 1.39AC - 1.79AD + 0.3550BC + 0.7425BD - 1.25CD - 7.45A 2 -4.37B 2 -6.11C 2 -6.44D 2 The results of the regression model variance analysis are shown in Table 5. The model P < 0.001, and the lack-of-fit term value is 0.1193, indicating that the model has extremely significant differences. The lack-of-fit term is not significant, and the correlation coefficient R of the comprehensive score regression equation is [value missing]. 2 The coefficient of variation was 0.9473, and the difference between the two was less than 0.2, indicating that the model is reliable and can be used to analyze experimental results and predict the optimal processing technology. Factors A and D had highly significant differences in the scores, while factors B and C had no significant effect. The order of influence of each factor on the processing technology of stir-fried jujube seeds with pig bile was D>A>C>B. Meanwhile, in the binomial formula, A... 2 B 2 C 2 D 2 All showed extremely significant effects.

[0074] Table 3 Experimental design factors and levels

[0075]

[0076] Table 4 Response Surface Experiment Scheme and Results

[0077]

[0078]

[0079]

[0080] Table 5. Results of ANOVA for the Regression Model

[0081]

[0082] Use Design-Expert 11 software to draw a response surface diagram of the interactions of various factors, see [link / reference]. Figure 3 Based on the model fitting results, the optimal processing technique was determined to be a stir-frying time of 4.12 min, a stir-frying temperature of 109.42 ℃, a soaking time of 0.9925 h, and a pig bile volume of 12.71 mL, resulting in a comprehensive score of 88.39. In conjunction with actual production practices, the optimal processing technique was determined to be a stir-frying time of 4 min, a stir-frying temperature of 109 ℃, a soaking time of 1 h, and a pig bile volume of 12.6 mL.

[0083] 3. Experimental analysis of Convolutional Neural Network (CNN) models based on comprehensive scoring.

[0084] (1) CNN parameter setting and final model establishment

[0085] A CNN model was constructed using Python software. The CNN model employs a one-dimensional convolutional structure to process the process input variables and outputs a comprehensive score prediction value through a fully connected layer. Process factors were selected as input variables during modeling, including roasting time, roasting temperature, soaking time, and the amount of pig bile used; the comprehensive score prediction value was used as the output variable; jujube seed saponin A, jujube seed saponin B, magnoflorine, spinosin, 6'''-feruloylspinosin, and the alcohol extract of jujube seeds roasted with pig bile were recorded as measured results in the full dataset table to illustrate sample quality characteristics. To compare the impact of training parameters on model performance, three schemes were set up: optimal parameters, moderate parameters, and poor parameters.

[0086] From Table 6, Table 7 and Figure 4 It can be seen that when the kernel size is 3, the number of kernels is 16, the fully connected nodes are 16, the Dropout value is 0.05, the number of training epochs is 500, and the learning rate is 0.010, the optimal parameter scheme for CNN can be obtained. This scheme has an RMSE of 0.3188 on the training set and a training set R... 2 The R² value of 0.9950 indicates that the model has a strong fitting ability to the training samples. Using this optimal parameter scheme for testing, the test set showed an RMSE of 2.1009, a MAE of 1.6128, a MAPE of 2.0657%, and an R² value of 0.9950. 2 It is 0.7977.

[0087] Table 6 CNN Three-Group Parameter Settings

[0088] Table 7 Comparison of Evaluation Indicators for Three CNN Parameter Schemes

[0089]

[0090] The final model was retrained using the optimal parameters of the CNN across all samples. The measured values ​​and CNN predictions were compared across 27 sets of data, and the results are as follows: Figure 5 As shown, the final CNN model exhibits a small prediction error on the test samples, and the measured curve generally matches the predicted curve well. The full-sample RMSE is 0.3901, the MAE is 0.2643, and the R² is [missing data]. 2 The value of 0.9927 indicates that the established model is stable and reliable.

[0091] Using the final CNN model with determined optimal parameters and retrained with all samples, the process parameters were optimized using a grid within the original response surface experimental range. The search range included frying time (3-5 min), frying temperature (100-120 ℃), soaking time (0.5-1.5 h), and pig bile volume (11-15 mL). The step sizes for frying time, frying temperature, soaking time, and pig bile volume were 0.1 min, 1 ℃, 0.05 h, and 0.2 mL, respectively. A total of 194,481 process combinations were predicted. Table 8 shows the top 10 process combinations predicted by the CNN model. It can be seen that the top 10 process combinations are mainly concentrated around frying time (3.9-4.1 min), frying temperature (109-111 ℃), soaking time (1.00-1.05 h), and pig bile volume (12.4-12.8 mL), indicating that this region is the high-scoring process region identified by the CNN model.

[0092] Table 8 Top 10 process combinations predicted by CNN models

[0093]

[0094] Combination Figure 6 The heatmap analysis results of the process parameters optimization shown (red stars indicate the comprehensive optimal values) show that the process combination with the highest prediction score by the CNN model is 4.1 min frying time, 110 ℃ frying temperature, 1.05 h soaking time, and 12.6 mL pig bile usage, with a comprehensive prediction score of 88.45.

[0095] (2) Verification of CNN model prediction process based on comprehensive scoring

[0096] The optimal process predicted by the final CNN model was verified by the comprehensive score F, while the comprehensive score F of the optimal process determined by the single-factor response surface experiment was used as a control.

[0097] (1) CNN predicts the optimal process: Take 20 g of raw jujube kernels to be processed, add 12.6 mL of pig bile, stir to mix evenly, and fully moisten for 1.05 h. Then place it in a pot and stir-fry at 110 ℃ for 4.1 min. After removing the slightly dried processed product, spread it out and let it cool.

[0098] (2) Optimal process of response surface methodology: Take 20 g of raw jujube kernels to be processed, add 12.6 mL of pig bile, stir to mix evenly, and fully moisten for 1.00 h. Then place it in a pot and stir-fry at 109 ℃ for 4.0 min. After removing the slightly dried processed product, spread it out and let it cool.

[0099] The results are shown in Table 9. The comprehensive score F of the three batches of experiments of the optimal process predicted by CNN and the optimal process of the single-factor response surface experiment is similar, indicating that the comprehensive score based on the anti-anxiety active ingredient can be used as the evaluation standard of the CNN prediction model.

[0100] Among them, the average comprehensive score of the three batches of the optimal process in the response surface methodology was 96.63, which was slightly higher than the average comprehensive score of the three batches of the optimal process obtained by CNN prediction. Finally, the optimal processing method for stir-fried jujube seeds with pig bile was determined to be as follows: take 20 g of raw jujube seeds to be processed, add 12.6 mL of pig bile, stir to mix evenly, and fully moisten for 1.00 h, then place in a pot and stir-fry at 109 ℃ for 4.0 min.

[0101] Table 9 Results of the three batches of verification tests

[0102]

[0103] 4. Quality evaluation of stir-fried jujube seeds with pig bile based on anti-anxiety active ingredients

[0104] Three batches of pig bile-fried jujube seeds prepared under optimal process were analyzed for their anti-anxiety active ingredients using HPLC detection steps described in the single-factor-response surface methodology. The results are shown in Table 10. The RSD values ​​of the measured contents were all less than 3%, indicating that the quality of the prepared products was stable.

[0105] Table 10 Results of content determination in three batches

[0106]

[0107] Example 2: Application of the method of processing jujube seeds with pig bile in the treatment of anxiety.

[0108] Male SD rats (weighing 180±20 g, provided by Jiangsu Qinglongshan Biotechnology Co., Ltd., license number: SCXK(Su)2024-0001) were acclimatized in an SPF experimental environment for 1 week and then randomly divided into a blank group (normal), a model group (model), a high-dose (ZSS_H) and low-dose (ZSS_L) group of raw jujube seed, a high-dose (ZSD_H) and low-dose (ZSD_L) group of jujube seed stir-fried with pig bile, and an estazolam positive administration group (EST), with 8 rats in each group.

[0109] Except for the control group, the remaining rats were subjected to chronic restraint combined with pain stimulation and intraperitoneal injection of DL-4-chlorophenylalanine (PCPA) to establish an anxiety model for 14 days. From day 15, the high-dose groups of raw jujube seed and jujube seed stir-fried with pig bile were administered by gavage at a dose of 8.10 g / kg / d, the low-dose groups of raw jujube seed and jujube seed stir-fried with pig bile were administered by gavage at a dose of 4.05 g / kg / d, the positive control group was administered estazolam by gavage at a dose of 0.5 mg / kg / d, and the remaining groups were administered the same dose of 0.9% saline by gavage.

[0110] The processing method for the raw jujube seed stir-fried with pig bile is as follows: Take 20 g of raw jujube seed to be processed, add 12.6 mL of pig bile, stir to mix evenly, and fully moisten for 1.00 h. Then, place it in a pot and stir-fry at 109 ℃ for 4.0 min. After removing the slightly dried processed product, spread it out and let it cool.

[0111] Preparation of samples for oral administration: After processing and preparing jujube seeds with pig bile according to the above-mentioned optimal process, take equal amounts of raw jujube seeds, crush them separately, add appropriate amounts of water to submerge them, soak for 30 min, heat to boiling, reflux for 1 h, filter, extract the residue again, combine the filtrates, and concentrate them to appropriate concentrations to obtain high- and low-dose solutions of raw jujube seeds and jujube seeds with pig bile; Estazolam tablets (National Drug Approval Number H32020699, Batch Number 20241122) are crushed and dissolved in an appropriate amount of 0.9% physiological saline to the required concentration.

[0112] After 14 days of continuous modeling, the rats were highly stressed, would adopt a defensive posture at the slightest sound, had anxious eyes, disheveled and dull fur, and a significant increase in the number of times they bit and attacked each other. They struggled violently when restrained and grabbed, making restraint difficult, and their cries were sharp and aggressive. They would escape or scratch or bite the experimenters if not careful. During restraint, they defecate frequently and their stool was loose. After restraint, when they were put back into the cage, they fought violently with other rats.

[0113] Anxiety treatment outcome assessment:

[0114] 1. Open Field Test (OFT)

[0115] On day 14 after gavage, the open field test was conducted. After the rats had fully acclimatized to the environment, they were gently placed in the center of the open field. The rats' activities in the open field box were recorded within 5 minutes. The following indicators were measured: distance traveled in the center area, number of center entries, time spent in the center, and percentage of time spent in the center. The experimental results are as follows: Figure 7 As shown, compared with the control group, the rats in the model group had significantly reduced time spent in the central area and distance traveled in the central area in the open field, with varying degrees of improvement after drug treatment. The low-dose group of jujube seed stir-fried with pig bile had significantly increased time spent in the central area and time spent in the central area compared with the low-dose group of raw jujube seed. The high-dose group of jujube seed stir-fried with pig bile had significantly increased distance traveled in the central area compared with the high-dose group of raw jujube seed. This indicates that the jujube seed stir-fried with pig bile group was more effective in improving anxiety behavior than the raw jujube seed group (n=3, Note: △ indicates significant difference between the raw jujube seed group and the jujube seed stir-fried with pig bile group (△ P<0.05); △△ P<0.01 indicates highly significant difference; # indicates significant difference between the model group and the control group (# P<0.05); ## P<0.01 indicates highly significant difference; * indicates significant difference between each treatment group and the model group (* P<0.05); ** P<0.01 indicates highly significant difference).

[0116] 2. Elevated Cross Maze Test (EPM)

[0117] Starting on day 14 after gavage, the elevated cruciate maze test was conducted. After the rats had fully adapted to the environment, they were gently placed in the center of the elevated cruciate maze with their heads facing the open arm. The rats' activity was recorded within 5 minutes, and the percentage of time spent in the open arm was measured. Figure 8 As shown, compared with the control group, the proportion of time spent entering the open arm in the elevated cross maze test was significantly reduced in the model group. The results were significantly improved after drug treatment, especially in the high-dose group of pig bile-fried jujube seed, which showed the most significant increase in the proportion of time spent entering the open arm, exceeding the model group by 596%. This indicates a significant improvement in anxiety behavior. The proportion of time spent entering the open arm in the high-dose group of pig bile-fried jujube seed was significantly higher than that in the high-dose group of raw jujube seed, indicating that the high-dose group of pig bile-fried jujube seed was more effective in treating anxiety than the high-dose group of raw jujube seed (n=3, Note: △ indicates a significant difference between the raw jujube seed group and the pig bile-fried jujube seed group (△ P<0.05); △△ P<0.01 indicates a highly significant difference; # indicates a significant difference between the model group and the control group (# P<0.05); ## P<0.01 indicates a highly significant difference; * indicates a significant difference between each treatment group and the model group (* P<0.05); ** P<0.01 indicates a highly significant difference).

[0118] 3. Tail Suspension Test (TST)

[0119] On day 14 after gavage, the rat tail suspension test was conducted. After the rats had fully adapted to the environment, the tail tip (about one-third of its length) was fixed 1 m off the ground, and the rats were hung upside down. After the rats had adapted for 2 minutes, their activity was recorded over a 4-minute period. Indicators such as immobility time (lasting more than 3 seconds of stillness was considered valid) and number of struggles were measured. Figure 9 As shown, compared with the blank group, the model group rats had a significantly increased resting time and a significantly reduced number of struggles. Compared with the model group, the estazolam positive group, the high and low dose groups of raw jujube seed, and the high and low dose groups of jujube seed stir-fried with pig bile all showed varying degrees of improvement, with the high dose group of jujube seed stir-fried with pig bile and the positive drug group showing relatively better effects. The resting time of rats in the low dose group of jujube seed stir-fried with pig bile was significantly lower than that in the low dose group of raw jujube seed, indicating that the treatment effect of the low dose group of jujube seed stir-fried with pig bile was better than that of the low dose group of raw jujube seed. (n=3, Note: △ indicates a significant difference between the raw jujube seed treatment group and the jujube seed treatment group stir-fried with pig bile, △ P<0.05; △△ P<0.01, highly significant difference; # indicates a significant difference between the model group and the blank group, # P<0.05; ## P<0.01, highly significant difference; * indicates a significant difference between each treatment group and the model group, * P<0.05; ** P<0.01, highly significant difference.)

[0120] 4. Histopathological HE staining

[0121] (1) HE staining of the hippocampus

[0122] Rats were given hippocampal tissue. The hippocampus was fixed in 4% paraformaldehyde solution for 24 h. The tissue was then removed from the fixative, trimmed, and placed in a dehydration box. The dehydration box was then placed in a dehydrator and dehydrated with ethanol of different concentrations. The tissue was then embedded in paraffin to prepare slides. The hippocampal paraffin sections were then dewaxed to water and subjected to HE staining: first, the sections were soaked in xylene for 30 min, then passed through anhydrous ethanol, 95% ethanol, 85% ethanol, and 70% ethanol for 5 min each. The sections were then immersed in hematoxylin staining solution for 2 min, rinsed with running water, treated with differentiation solution, re-blued with blue solution for 10-15 s, rinsed with running water, stained with eosin solution for 20 s, and then passed through 70% ethanol, 85% ethanol, 95% ethanol, anhydrous ethanol, and xylene for 5 s each for dehydration and clearing. The sections were then mounted with neutral resin.

[0123] The figure shows the hippocampus and its characteristic regions CA1 and DG. These two regions are key areas of the hippocampal tissue, rich in neurons and with high neuronal density. Figure 10It was found that, compared with the control group, the model group showed a decrease in the number of hippocampal neurons in the CA1 and DG regions, with loose and irregular arrangement, inconsistent cell morphology, some neurons shrunken, unevenly distributed, and deeply stained, condensed nuclei with reduced volume, exhibiting obvious nuclear pyknosis. In contrast, after drug intervention, the number of hippocampal neurons in the CA1 and DG regions of rats was greater, their structure more intact, their arrangement neat and compact, and their nuclei clear and centrally located. Furthermore, the high-dose group of pig bile-fried jujube seed showed partial improvement in hippocampal neuronal structural damage, with more intact cell structure, more orderly arrangement, abundant cytoplasm, and clear nucleoli in some neurons. The results indicate that pig bile-fried jujube seed can significantly improve the problems of reduced hippocampal neuron number, loose and irregular arrangement, inconsistent cell morphology, shrunken neurons, and uneven distribution in insomnia-prone rats.

[0124] (2) HE staining of the hypothalamus

[0125] Rat hypothalamus tissue was collected and fixed in 4% paraformaldehyde solution for 24 h. The tissue was then removed from the fixative, trimmed, and placed in a dehydration box. The dehydration box was then placed in a dehydrator and dehydrated with ethanol of different concentrations. The tissue was then embedded in paraffin to prepare slides. The paraffin sections of the hypothalamus were dewaxed to water and then stained with hematoxylin and eosin (HE): First, the sections were soaked in xylene for 30 min, then passed through anhydrous ethanol, 95% ethanol, 85% ethanol, and 70% ethanol for 5 min each. The sections were then immersed in hematoxylin staining solution for 2 min, rinsed with running water, treated with differentiation solution, re-blued with blue solution for 10-15 s, rinsed with running water, stained with eosin solution for 20 s, and then passed through 70% ethanol, 85% ethanol, 95% ethanol, anhydrous ethanol, and xylene for 5 s each for dehydration and clearing. The sections were then mounted with neutral resin.

[0126] like Figure 11 As shown, the hypothalamic nerve cells in the blank control group were abundant, clearly morphological, and evenly distributed. In the model group, nerve cells were deformed and shrunken, loosely and irregularly arranged, and even disappeared. These pathological changes were restored to varying degrees after treatment in the estazolam-positive group, the high- and low-dose raw jujube seed groups, and the high- and low-dose jujube seed groups stir-fried with pig bile.

Claims

1. A method for processing jujube seeds with pig bile based on quality evaluation, characterized in that, Includes the following steps: A comprehensive score was established based on the anti-anxiety active ingredients of jujube seeds stir-fried with pig bile. The comprehensive score was applied to a combined experimental analysis method to obtain the processing method, and / or the quality of the product obtained by the processing method was evaluated based on the anti-anxiety active ingredients. The anti-anxiety active ingredients are jujube seed saponin A, jujube seed saponin B, magnoflorine, spinosin, 6'''-feruloyl spinosin, and alcoholic extract of jujube seeds stir-fried with pig bile. The processing method is as follows: 11-15 mL of pig bile is added to every 20 g of raw jujube seeds to be processed, stirred to mix evenly, and fully moistened for 0.5-1.5 h. Then, the mixture is placed in a pot and stir-fried at 100-120 ℃ for 3-5 min to obtain jujube seeds stir-fried with pig bile.

2. The method for processing jujube seeds with pig bile according to claim 1, characterized in that, The comprehensive score is F, where F = [0.3485 × (content of jujuboside A / maximum content of jujuboside A) + 0.2156 × (content of jujuboside B / maximum content of jujuboside B) + 0.0564 × (content of magnoflorine / maximum content of magnoflorine) + 0.1671 × (content of spinosin / maximum content of spinosin) + 0.0793 × (content of 6'''-feruloyl spinosin / maximum content of 6'''-feruloyl spinosin) + 0.1331 × (content of alcoholic extract from jujubo seeds stir-fried with pig bile / maximum content of alcoholic extract from jujubo seeds stir-fried with pig bile)] × 100.

3. The method for processing jujube seeds with pig bile according to claim 2, characterized in that, The comprehensive score is obtained by combining the analytic hierarchy process (AHP) with the entropy weight method.

4. The method for processing jujube seeds with pig bile according to claim 3, characterized in that, The analytic hierarchy process (AHP) uses the content of anti-anxiety active ingredients as the evaluation index to calculate the weights.

5. The method for processing jujube seeds with pig bile according to claim 3, characterized in that, The entropy weight method calculates the entropy value of the anti-anxiety active ingredient and then calculates the weight.

6. The method for processing jujube seeds with pig bile according to claim 1, characterized in that, The combined experimental analysis method includes single-factor response surface methodology or convolutional neural network model analysis.

7. The method for processing jujube seeds with pig bile according to claim 1, characterized in that, The quality assessment includes detecting the content of the anti-anxiety active ingredient of claim 1 using high performance liquid chromatography.

8. The method for processing jujube seeds with pig bile according to claim 1, characterized in that, The preferred processing method is to add 12.4~12.8 mL of pig bile to every 20 g of raw jujube kernels to be processed, stir to mix evenly, soak thoroughly for 1.00~1.05 h, and then stir-fry in a pot at 109~111℃ for 3.9~4.1 min.

9. The application of the method for processing pig bile into stir-fried jujube seeds according to any one of claims 1 to 8 in the preparation of a drug for treating anxiety.

10. A traditional Chinese medicine for relieving anxiety, characterized in that, The anti-anxiety traditional Chinese medicine includes 0.2378~0.2395% of jujube seed saponin A, 0.0522~0.0538% of jujube seed saponin B, 0.0635~0.0666% of magnoflorine, 0.1057~0.1068% of spinosin, 0.0280~0.0286% of 6'''-feruloyl spinosin, and 18.61~19.65% of jujube seed extract stir-fried with pig bile.

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