Preparation method of radix gardeniae and sojae decoction for treating alzheimer's disease and content determination method

Abstract

The application provides a preparation method and content determination method of Zhizi Chi Tang reference standard for treating Alzheimer's disease, and belongs to the field of traditional Chinese medicines.The preparation method comprises the following steps: a, weighing and proportioning raw medicinal materials;b, soaking gardenia with water, decocting, then adding light soybean, decocting for 30-60 minutes, filtering, and taking water extract;b, freezing and drying the water extract of step b.The application also provides the Zhizi Chi Tang reference standard prepared by the preparation method and the content determination method.The application screens out daidzein, genistein and quercetin as the effective components of Zhizi Chi Tang for treating Alzheimer's disease, establishes the preparation method of the Zhizi Chi Tang reference standard, establishes the content determination method by using high performance liquid chromatography, and applies the method to the product quality control of Zhizi Chi Tang for treating Alzheimer's disease, so as to ensure the effectiveness and stability of the product, and the method is simple and convenient, good in stability, high in precision, good in reproducibility, and easy to master.

Description

Technical Field

[0001] This invention relates to a method for preparing and determining the content of a standard sample of Gardenia and Fermented Soybean Decoction for treating Alzheimer's disease, belonging to the field of traditional Chinese medicine. Background Technology

[0002] Gardenia and Fermented Soybean Decoction is listed in the "Catalogue of Famous Ancient Prescriptions (Second Batch)" published by the State Administration of Traditional Chinese Medicine. It originates from Zhang Zhongjing's "Treatise on Febrile Diseases: Differentiation of Pulse and Symptoms in Taiyang Disease," written during the Han Dynasty. It consists of two herbs: gardenia and fermented soybean. It has the effects of clearing heat and purging fire, relieving irritability and depression. It is mainly used to treat exogenous febrile diseases, irritability, and chest tightness.

[0003] Alzheimer's disease (AD) is an insidious neurodegenerative disease of the central nervous system, mainly manifested as cognitive decline, memory and learning impairment, behavioral abnormalities, and dementia. Modern medicine believes that its pathogenesis is caused by the deposition of β-amyloid protein (Aβ) and the excessive phosphorylation of Tau protein in brain cells, forming neurofibrillary tangles (NFTs), which damage the neuronal cytoskeleton, affect cell transport and signal transduction, and lead to neuronal dysfunction and death. In traditional Chinese medicine, its pathogenesis is summarized as deficiency of the root and excess of the branch, and a mixture of deficiency and excess. The etiology is believed to be due to phlegm and blood stasis obstructing the brain's collaterals, resulting in malnourishment of the sensory orifices, deficiency of qi and blood, or insufficient kidney essence, leading to insufficient filling of the marrow sea and phlegm-induced dementia.

[0004] Gardenia and Fermented Soybean Decoction is a classic formula for treating "restlessness and insomnia after sweating, vomiting, and purging" and "fever due to overexertion." Its main effects are clearing heat and relieving restlessness, and dispersing stagnant heat. It is suitable for symptoms such as restlessness, insomnia, chest tightness, and fullness in the chest and abdomen caused by lingering heat after treatment with sweating, vomiting, and purging in exogenous diseases. It also has a good effect on fever and restlessness caused by relapse due to overexertion after illness. In modern times, it has significant efficacy in treating mental illnesses (Wang Fu. Thoughts on the formula and derivative formula of Gardenia and Fermented Soybean Decoction [J]. Journal of Traditional Chinese Medicine, 2015, 56(7):626-627; Xiao Qian, Yan Juntang, Wang Xueqian, et al. Professor Liu Duzhou's experience in treating mental illnesses using the "Gardenia and Fermented Soybean Decoction" formula [J]. National Medical Forum, 2015, 30(1):3-5.). It can treat depression, insomnia and anxiety. At the same time, Gardenia and Fermented Soybean Decoction also has a certain effect on other mental illnesses such as Alzheimer's disease (Fu Qing, Shan Qi, Zhou Yutong, et al. Research progress on the mechanism of action of iridoid ethers in Gardenia against Alzheimer's disease [J]. China Pharmacy, 2022, 33(01):123-128.). In addition, Gardenia and Fermented Soybean Decoction is a classic formula for treating chest and diaphragm heat stagnation (Wang Hui, et al., Application of Gardenia and Fermented Soybean Decoction and its series of formulas in respiratory diseases, Modern Chinese Medicine Clinical, Vol. 31, No. 5, September 2024).

[0005] Currently, there are relevant literature reports on the preparation method of Gardenia and Fermented Soybean Decoction, such as: Application No.: CN202311014978.9, Invention Title: An Extraction Process of Gardenia and Fermented Soybean Decoction, which discloses an extraction process for Gardenia and Fermented Soybean Decoction that can maximize the extraction of effective drug components, including the following steps: fermentation of light fermented soybean samples, and quantitative analysis of the iridoid glycosides geniposide and the soybean isoflavones daidzein in Gardenia and Fermented Soybean Decoction based on high performance liquid chromatography. The method of this invention can quantitatively detect the content of geniposide and daidzein in Gardenia and Fermented Soybean Decoction, and has clear control indicators compared with the traditional decoction method, which can more objectively evaluate the quality of Gardenia and Fermented Soybean Decoction related to efficacy. However, this patent literature does not involve the indications for Gardenia and Fermented Soybean Decoction.

[0006] Hu Zhongjiao, et al., Research on the Extraction Process of Gardenia and Fermented Soybean Decoction Based on Network Pharmacology and Quality by Design (QbD) Concept, Chinese Traditional and Herbal Drugs, April 2022, Vol. 53, No. 7. This study uses network pharmacology to explore the mechanism of action of Gardenia and Fermented Soybean Decoction in treating insomnia. Based on the QbD concept, the extraction process of Gardenia and Fermented Soybean Decoction was constructed and verified to be 8-11 times the amount of solvent, 30-84 min extraction time, and 1-2 extraction times.

[0007] Yang Xuejing, et al., Effects of Processing and Combination on the Effective Components of Gardenia and Fermented Soybean Decoction, Chinese Patent Medicine, July 2024, Vol. 46, No. 7. Appropriate amounts of raw, stir-fried, and charred products, as well as combinations of these three with light fermented soybeans, were taken. The contents of genipin, geniposide, genistein, crocin I, crocin II, daidzein, and genistein were simultaneously determined by HPLC, and the dissolution rate was calculated. The results showed that the seven components exhibited good linearity within their respective ranges (r > 0.9994), with an average recovery rate of 95.22%–104.94% and RSD ≤ 3.36%. After processing, the contents of geniposide, crocin I, and crocin II in the raw product decreased; after compounding, the dissolution rate of iridoid glycosides and diterpenoid pigments in the water extracts of both the raw and processed products decreased; ethanol extraction was beneficial to the dissolution of iridoid glycosides, and the extraction solvent had a greater impact on the dissolution of crocin.

[0008] Sun Simiao et al., Research on Optimization of Extraction and Preparation Processes of Gardenia and Fermented Soybean Decoction Oral Liquid using Orthogonal Experimental Method, *Chemistry and Adhesion*, 2022, Vol. 3, disclosed the optimal extraction and preparation processes for gardenia and fermented soybean decoction oral liquid. Using the gardenia and fermented soybean decoction content as evaluation indicators, the optimal extraction process was selected through orthogonal experimental design, considering factors such as water addition, extraction time, and extraction times. The content of gardenia and fermented soybean decoction was used as indicators for selecting water extraction conditions, and the clarity of the preparation after ethanol recovery was used as an indicator for selecting the alcohol precipitation process. The molding process was established by investigating the amount of sucrose used. The optimal preparation process for Gardenia and Fermented Soybean Decoction Oral Liquid was determined through experiments. The optimal process involves pulverizing 300g of gardenia and 200g of fermented soybean, adding 3000mL of water, soaking for 75min, reflux extraction three times for 1h each time, combining the three extracts, concentrating to a clear extract with a relative density of 1.05~1.15 (80℃), adding ethanol to achieve an ethanol concentration of 70%, performing alcohol precipitation, taking the supernatant, adding 150g of sucrose, adjusting the volume to 1000mL, filling, and sterilizing by moist heat sterilization to obtain the finished product.

[0009] Because the effects of Gardenia and Fermented Soybean Soup are complex, the reported processing methods lack specificity and have not been studied for specific effects.

[0010] Although there are literature reports on the use of Gardenia and Fermented Soybean Decoction in the treatment of Alzheimer's disease, the efficacy evaluation is still at the "model" stage, lacking quantifiable quality control benchmarks. Existing pharmacological studies mostly use water extracts or crude components, which are affected by place of origin, batch, and decoction parameters, and the content of gardenoside / genistein can vary by 3-5 times. Without a unified "benchmark standard", it is difficult to compare the results of different laboratories, let alone support the subsequent Phase I-III clinical dosage design.

[0011] Currently, there are no literature reports on the standard product for the use of Gardenia and Fermented Soybean Decoction in the treatment of Alzheimer's disease. Summary of the Invention

[0012] This invention provides a method for preparing a standard sample of Gardenia and Fermented Soybean Decoction for the treatment of Alzheimer's disease and a method for determining its content.

[0013] This invention provides a method for preparing a standard sample of Gardenia and Fermented Soybean Decoction for treating Alzheimer's disease, which includes the following steps:

[0014] a. Weigh the raw materials according to the following weight ratio: 1 part gardenia and 1 part fermented soybean;

[0015] b. Soak gardenia in water, decoct, then add light fermented soybeans and decoct for 30-60 minutes. Filter and extract the liquid.

[0016] c. Freeze-dry the aqueous extract from step b to obtain the standard sample of Gardenia and Fermented Soybean Soup;

[0017] The freeze-drying conditions are as follows:

[0018] (1) Pre-freezing: Temperature: -50℃, maintenance temperature: 8h; Pressure: atmospheric pressure;

[0019] (2) Sublimation drying: Temperature: -40℃, holding temperature: 2h;

[0020] Temperature: -30℃, temperature maintenance: 2h;

[0021] Temperature: -20℃, holding temperature: 2h;

[0022] Temperature: -10℃, temperature maintenance: 3h;

[0023] Temperature: 0℃, temperature maintenance: 3h;

[0024] (3) Drying: Temperature: 10℃, holding temperature: 3h;

[0025] Temperature: 20℃, holding temperature: 2h;

[0026] Temperature: 30℃, holding temperature: 2h;

[0027] Temperature: 40℃, temperature maintenance: 4h.

[0028] Preferably, the simmering method described in step b is to first simmer over high heat until boiling, and then simmer over low heat.

[0029] The present invention provides a standard sample of Gardenia and Fermented Soybean Soup prepared by the above method, which contains a total amount of daidzein, genistein and quercetin of not less than 0.75 mg / g; wherein the content of daidzein is not less than 0.27 mg / g, the content of genistein is not less than 0.08 mg / g and the content of quercetin is not less than 0.36 mg / g.

[0030] This invention provides a method for determining the content of gardenia and fermented soybean soup reference standard. It employs HPLC chromatography to detect the content of daidzein, genistein, and quercetin. The chromatographic conditions are as follows:

[0031] Octadecylsilane-bonded silica gel was used as the packing material; methanol was used as mobile phase A and 0.4% phosphoric acid solution was used as mobile phase B, and gradient elution was performed under the following conditions: 0–20 minutes, mobile phase A 40→80.

[0032] The column of the packing material has a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; the chromatographic conditions include a flow rate of 1.0 ml per minute, a column temperature of 30 °C, and a detection wavelength of 260 nm.

[0033] Specifically, it includes the following steps:

[0034] a. Preparation of the test solution:

[0035] Take the sample to be tested, place it in a stoppered conical flask, accurately add a methanol-25% hydrochloric acid mixed solution with a volume ratio of 4:1, heat under reflux for 60 minutes, cool, weigh it again, make up the lost weight with a methanol-25% hydrochloric acid mixed solution with a volume ratio of 4:1, shake well, filter, and take the filtrate to obtain the sample.

[0036] b. Detect according to the chromatographic conditions described.

[0037] It also includes the preparation of reference solutions, the preparation method of which is to take appropriate amounts of daidzein reference standard, genistein reference standard, and quercetin reference standard, and add methanol to prepare a mixed solution.

[0038] This invention provides a method for detecting qualified products of Gardenia and Fermented Soybean Decoction for the treatment of Alzheimer's disease, which includes the following steps:

[0039] a. Take a dried sample of the Gardenia and Fermented Soybean Decoction to be tested;

[0040] b. The contents of daidzein, genistein, and quercetin were determined according to the content determination method of the Gardenia and Fermented Soybean Soup reference standard.

[0041] c. If the total amount of daidzein, genistein, and quercetin is higher than 0.75 mg / g, and the content of daidzein is higher than 0.27 mg / g, the content of genistein is higher than 0.08 mg / g, and the content of quercetin is higher than 0.36 mg / g, then it is a qualified product.

[0042] The beneficial effects of this invention are:

[0043] This invention is based on screening the mechanism of action of Gardenia and Fermented Soybean Decoction in the clinical application of Alzheimer's disease. It screens out its active ingredients, daidzein, genistein, and quercetin, establishes a method for preparing Gardenia and Fermented Soybean Decoction reference samples, and establishes a method for determining their content using high performance liquid chromatography. This method is used for product quality control of Gardenia and Fermented Soybean Decoction in the treatment of Alzheimer's disease, ensuring the effectiveness and stability of the product. The invention method is simple, convenient, stable, precise, reproducible, and easy to master. Attached Figure Description

[0044] Figure 1 Select a mobile phase diagram;

[0045] Figure 2 Selection diagram for different wavelengths;

[0046] Figure 3 The results of the flow velocity study;

[0047] Figure 4 The results of the column temperature investigation;

[0048] Figure 5For the purpose of investigating the injection volume;

[0049] Figure 6 An investigation into the specificity of Gardenia and Fermented Soybean Decoction;

[0050] Figure 7 This is a standard curve of daidzein;

[0051] Figure 8 This is a standard curve diagram of lignin dyes;

[0052] Figure 9 This is a standard curve diagram for quercetin.

[0053] Figure 10 Venn diagram of the intersection targets for Gardenia and Fermented Soybean Decoction in the treatment of Alzheimer's disease;

[0054] Figure 11 For the regulation network of traditional Chinese medicine compound prescriptions;

[0055] Figure 12 PPI network diagram (A: Intersection target interaction network in STRIMG database; B: Intersection target interaction network; C: Intersection points screened once; D: Screened 10 key core genes);

[0056] Figure 13 The core genes and Degree values ​​for Gardenia and Fermented Soybean Decoction in the treatment of Alzheimer's disease;

[0057] Figure 14 For GO functional enrichment analysis;

[0058] Figure 15 For KEGG pathway enrichment analysis;

[0059] Figure 16 A thermal diagram of molecular docking binding energy;

[0060] Figure 17 Schematic diagram of partial molecular docking results (A: apigenin-ESR1; B: apigenin-SRAT3; C: daidzein-ESR1; D: genistein-ESR1; E: quercetin-ESR1).

[0061] Figure 18 Select the mobile phase. Detailed Implementation

[0062] Example 1: Preparation method of Gardenia and Fermented Soybean Soup Standard Sample

[0063] Gardenia and Fermented Soybean Decoction comes from the Treatise on Cold Damage by Zhang Zhongjing, a physician of the Han Dynasty. The prescription is: "Fourteen gardenia fruits (broken), four he of fermented soybeans (wrapped in cotton); boil the above two ingredients in four liters of water, first boil the gardenia fruits until two and a half liters remain, then add the fermented soybeans and boil until one and a half liters remain, remove the dregs, divide into two doses, take one dose warm, if vomiting occurs, stop taking the second dose." It consists of fourteen gardenia fruits (broken) and four he of fermented soybeans.

[0064] 1. Processing: According to the requirements of the classic prescription, each ingredient in Gardenia and Fermented Soybean Decoction is processed. Gardenia is processed to remove impurities; fermented soybean is processed to remove impurities.

[0065] 2. Weighing: Weigh 15g of gardenia and 15g of fermented soybean according to the prescription ratio.

[0066] 3. Preparation: Place the above-mentioned gardenia slices in a decoction pot, add 800ml of water, soak for 30 minutes, bring to a boil over high heat, then reduce to low heat and simmer until 500ml remains. Add fermented soybeans, bring to a boil over high heat, then reduce to low heat and simmer for 30-60 minutes until 300ml remains. Filter while hot, remove the residue, and extract the liquid.

[0067] 4. Drying: Prepare the Gardenia and Fermented Soybean Soup base material according to the following freeze-drying parameters. The freeze-drying program of the vacuum freeze dryer is as follows:

[0068] (1) Pre-freezing: Temperature: -50℃, maintenance temperature: 8h; Pressure: atmospheric pressure;

[0069] (2) Sublimation drying: Temperature: -40℃, holding temperature: 2h;

[0070] Temperature: -30℃, temperature maintenance: 2h;

[0071] Temperature: -20℃, holding temperature: 2h;

[0072] Temperature: -10℃, temperature maintenance: 3h;

[0073] Temperature: 0℃, temperature maintenance: 3h;

[0074] (3) Drying: Temperature: 10℃, holding temperature: 3h;

[0075] Temperature: 20℃, holding temperature: 2h;

[0076] Temperature: 30℃, holding temperature: 2h;

[0077] Temperature: 40℃, temperature maintenance: 4h.

[0078] Example 2: Screening test of process parameters for Gardenia and Fermented Soybean Soup of the present invention

[0079] 1. Examination of prescription ratios

[0080] According to the freeze-drying parameters of Example 1, the dosages of gardenia and fermented soybean in the prescription were mixed in a ratio of 1:1 and 1:4, and their contents were determined. The results are shown in the table below.

[0081] Table 1. Results of content determination of different prescriptions of Gardenia and Fermented Soybean Decoction

[0082]

[0083] To explore the application of Gardenia and Fermented Soybean Decoction in the treatment of Alzheimer's disease, three indicator components—daidzein, genistein, and quercetin—were screened to control the efficacy of this type of product. The results showed that the total content of daidzein, genistein, and quercetin was highest when the ratio of gardenia to fermented soybean in the prescription was 1:1.

[0084] 2. Evaluation of soaking and non-soaking

[0085] Using the total content of daidzein, genistein, and quercetin as the evaluation index, this study investigated the effect of soaking and non-soaking the medicinal slices on their content in the same prescription dosage and decoction conditions.

[0086] Table 2. Results of content determination of Gardenia and Fermented Soybean Soup with and without soaking

[0087]

[0088] The results showed that the content was higher in the sample after soaking and then decocting, compared to the sample without soaking. Therefore, soaking was the preferred method.

[0089] 3. Investigation of different freeze-drying parameters

[0090] Under the same decoction process, the total content of daidzein, genistein, and quercetin was used as an indicator to investigate the effect of different freeze-drying parameters on the content of these compounds. Freeze-drying parameter ① is shown in Table 3; freeze-drying parameter ② is shown in Table 4.

[0091] Table 3 Freeze-drying process

[0092]

[0093] Table 4 Freeze-drying process

[0094]

[0095] Table 5. Results of content determination for Gardenia and Fermented Soybean Soup without freeze-drying.

[0096]

[0097] The results showed that the content was higher under the freeze-drying parameter ①, so the freeze-drying parameter ① was used to prepare the sample.

[0098] Example 3: Quality Testing Method for Gardenia and Fermented Soybean Soup of the Present Invention

[0099] 1. Experimental instruments and materials

[0100] High Performance Liquid Chromatograph (HPLC): Thermo Fisher Scientific HPLC system;

[0101] Electronic balances: ME204E / 02, XPE26 (Mettler-Toledo Instruments Ltd.);

[0102] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0103] Ultrasonic cleaner: KQ600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0104] Column: ZORBAX Eclipse XDB-C18 4.6×250mm 5µm;

[0105] Methanol and phosphoric acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0106] Daidzein (China National Institutes for Food and Drug Control, batch number: 111502-202304, content calculated as 96.3%).

[0107] Genistein (China National Institutes for Food and Drug Control, batch number: 111704-202104, content calculated as 98.8%).

[0108] Quercetin (China National Institutes for Food and Drug Control, batch number: 100081-202411, content calculated as 100.0%).

[0109] Gardenia medicinal materials: ZZ-01, ZZ-02, ZZ-03, ZZ-04, ZZ-05, ZZ-06;

[0110] Lightly fermented soybean medicinal materials: DDC-01, DDC-02, DDC-03, DDC-04, DDC-05, DDC-06;

[0111] Gardenia and Fermented Soybean Soup Reference Samples: ZZCT-01, ZZCT-02, ZZCT-03, ZZCT-04, ZZCT-05, ZZCT-6;

[0112] 2 Experimental conditions

[0113] 2.1 Chromatographic conditions and system suitability test

[0114] The column was packed with octadecylsilane-bonded silica gel (250 mm column length, 4.6 mm inner diameter, 5 μm particle size); methanol was used as mobile phase A, and 0.4% phosphoric acid solution was used as mobile phase B, with gradient elution under the following conditions: 0–20 min, mobile phase A 40→80; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 260 nm. The theoretical plate number, calculated based on the daidzein peak, should be no less than 3000.

[0115] 2.2 Preparation of reference solution

[0116] Take appropriate amounts of daidzein reference standard, genistein reference standard, and quercetin reference standard, accurately weigh them, and add methanol to prepare a mixed solution containing 15 μg of daidzein, 15 μg of genistein, and 15 μg of quercetin per 1 ml.

[0117] 2.3 Preparation of the test solution

[0118] For the test solution, take an appropriate amount of this product, about 0.2 g, accurately weigh it, place it in a stoppered conical flask, accurately add 40 ml of methanol-25% hydrochloric acid (4:1) mixed solution, stopper tightly, weigh it, heat under reflux for 60 minutes, cool it, weigh it again, make up the lost weight with 7 ml of methanol-25% hydrochloric acid (4:1) mixed solution, shake well, filter it, and take the filtrate to obtain the test solution.

[0119] 2.4 Determination Method

[0120] Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0121] 3. Establishment of content determination method

[0122] 3.1 Chromatographic conditions and system suitability test

[0123] 3.1.1 Selection of mobile phase

[0124] Based on the above-planned experimental conditions, acetonitrile-0.4% phosphoric acid, acetonitrile-0.1% formic acid, and methanol-0.1% formic acid were selected for investigation. (See attached text.) Figure 1 .

[0125] The results showed that the target peak shape and separation effect were good when acetonitrile-0.4% phosphoric acid was used as the mobile phase. Therefore, acetonitrile-0.4% phosphoric acid solution was selected as the mobile phase for further investigation.

[0126] 3.1.2 Wavelength Selection

[0127] Based on the above-planned experimental conditions, the test solution was scanned across the entire wavelength range using a diode array detector, and chromatograms of the test solution at wavelengths of 230 nm, 260 nm, 280 nm, and 300 nm were extracted. See... Figure 2 .

[0128] By comparing the spectra, it was found that the peaks of quercetin and genistein both showed significant absorption at a detection wavelength of 260 nm. Considering all factors, the chromatogram baseline was more stable, so the detection wavelength was determined to be 260 nm.

[0129] 3.1.3 Flow velocity assessment

[0130] Under the proposed experimental conditions, the flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated. (See...) Figure 3 .

[0131] The results showed that the chromatogram peak shape and separation effect were better when the flow rate was 1.0 ml / min, and the proposed flow rate was 1.0 ml / min.

[0132] 3.1.4 Column Temperature Investigation

[0133] Based on the above-specified experimental conditions, the results were investigated at column temperatures of 25℃, 30℃, and 35℃. (See...) Figure 4 .

[0134] The results showed that the chromatogram peak shape and separation were better when the column temperature was 30℃. The column temperature was tentatively set at 30℃.

[0135] 3.1.5 Sample Injection Volume Examination

[0136] Based on the above-established experimental conditions, the effects were investigated at injection volumes of 5 μl, 10 μl, and 15 μl. Figure 5 As shown.

[0137] The results showed that the chromatogram peak shape was good and the resolution was moderate when the injection volume was 10 μl. Therefore, the injection volume was determined to be 10 μl.

[0138] The chromatographic conditions and system usability test for the determination of the content in the Gardenia and Fermented Soybean Soup reference sample are tentatively set as follows: Octadecylsilane-bonded silica gel is used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol is used as mobile phase A, and 0.4% phosphoric acid solution is used as mobile phase B, with gradient elution. Elution conditions are: 0–20 minutes, mobile phase A 40→80; flow rate 1.0 ml / min; column temperature 30℃; detection wavelength 260 nm. The theoretical plate number, calculated based on the daidzein peak, should not be less than 3000.

[0139] The assay involves precisely pipetting 10 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0140] 3.2 Methodological Examination

[0141] 3.2.1 Specificity Examination

[0142] Preparation of reference solution: Take appropriate amounts of daidzein reference standard, genistein reference standard, and quercetin reference standard, accurately weigh them, and add methanol to prepare a mixed solution containing 15 μg of daidzein, 15 μg of genistein, and 15 μg of quercetin per ml.

[0143] Preparation of the test solution: Take an appropriate amount of this product, about 0.2 g, accurately weigh it, place it in a stoppered conical flask, accurately add 40 ml of a mixed solution of methanol-25% hydrochloric acid (4:1), stopper tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with the mixed solution of methanol-25% hydrochloric acid (4:1), shake well, filter it, and take the filtrate to obtain the test solution.

[0144] Preparation of negative control solution for single herb deficiency: Prepare negative control solution according to the above-described method for preparing test solution. Results are shown below. Figure 6 .

[0145] As shown in the figure, daidzein and quercetin are derived from fermented soybean and gardenia, while genistein is mainly derived from fermented soybean.

[0146] 3.2.2 Precision test

[0147] A mixed solution of daidzein, genistein, and quercetin reference standards was injected five times consecutively. The peak areas were recorded, and the RSD values ​​were calculated. The results are shown in Table 6.

[0148] Table 6 Precision test results

[0149]

[0150] As can be seen from the above, the peak area RSD values ​​of each reference standard in the precision test were all less than 1.0%, indicating that the instrument precision was good.

[0151] 3.2.3 Linear Relationship

[0152] Precisely pipette the mixed stock solutions of daidzein, genistein, and quercetin reference standards to prepare reference solutions of different concentrations. Inject 10 μl of each solution into the liquid chromatograph and obtain the peak area. Plot a response curve with reference standard concentration (X, µg / ml) on the x-axis and peak area (Y) on the y-axis. The results are shown in Tables 7-9. Figures 7-9 .

[0153] Table 7. Results of the standard curve analysis of daidzein.

[0154]

[0155] Table 8. Analysis results of the standard curve of genistein

[0156]

[0157] Table 9. Results of Quercetin Standard Curve Analysis

[0158]

[0159] The results showed that the linear regression equation for daidzein was y = 0.9239x – 0.1012, with a correlation coefficient R² = 0.9999, indicating a good linear relationship between daidzein concentration and peak area within the concentration range of 5.361984 µg / ml to 107.23968 µg / ml.

[0160] The linear regression equation for genistein was y = 1.2476x – 0.019, with a correlation coefficient R² = 0.9999, indicating a good linear relationship between daidzein concentration and peak area within the concentration range of 4.864912 µg / ml to 97.29824 µg / ml.

[0161] The linear regression equation for quercetin was y = 0.5803x – 0.2376, with a correlation coefficient R² = 0.9997, indicating a good linear relationship between daidzein concentration and peak area within the concentration range of 4.648 µg / ml to 92.96 µg / ml.

[0162] 3.2.4 Repeatability

[0163] Take an appropriate amount of this product (batch number: SJGCT-01), about 0.2g, accurately weigh 6 portions, and have the same operator prepare the test solution according to the determined method. Accurately pipette 5μl of each test solution and inject it into the liquid chromatograph. Calculate the contents of daidzein reference standard, genistein reference standard and quercetin reference standard in the 6 samples. The results are shown in Table 10.

[0164] Table 10 Results of Repeatability Experiments

[0165]

[0166] The results showed that the RSD values ​​of each indicator were all less than 5%, indicating that the method had good repeatability.

[0167] 3.2.5 Intermediate Precision

[0168] Based on the above-established experimental conditions, different personnel conducted measurements at different times. An appropriate amount (approximately 0.2g) of the Gardenia and Fermented Soybean Soup reference sample was accurately weighed and used to prepare the test sample for analysis. The calculated contents of daidzein, genistein, and quercetin reference standards are shown in Table 11.

[0169] Table 11 Results of intermediate precision testing

[0170]

[0171] The results showed that the RSD values ​​of the contents were all less than 5.0%, indicating that the intermediate precision of this method was good.

[0172] 3.2.6 Recovery rate

[0173] Take approximately 0.1 g of the test sample (batch number: ZZCT-01, daidzein content 0.326 mg / g, genistein content 0.116 mg / g, quercetin content 0.593 mg / g), in six portions, accurately weigh them, and accurately add a certain amount of daidzein reference standard, genistein reference standard, and quercetin reference standard to each sample. Prepare the test solution according to the prescribed method and determine the recovery rate. The results are shown in Table 12. The calculation formula is as follows:

[0174]

[0175] Table 12 Results of the spiking recovery experiment

[0176]

[0177] The results showed that the recovery rates of all indicators were between 90% and 108%. The method demonstrated good accuracy.

[0178] 3.2.7 Stability Test

[0179] According to the experimental conditions proposed above, a test solution was prepared, and the peak area of ​​each index was measured at 0h, 2h, 4h, 8h, 12h and 24h respectively. The results are shown in Table 13.

[0180] Table 13 Stability test results

[0181]

[0182] The results showed that under the experimental conditions, the RSD% of each index was below 3%, and the test solution had good stability within 24 hours.

[0183] 3.3 Validation of Sample Content Determination

[0184] The six batches of standard samples of Gardenia and Fermented Soybean Soup were tested according to the proposed method, and the results are shown in Table 14.

[0185] Table 14. Results of content determination in six batches of Gardenia and Fermented Soybean Soup reference samples

[0186]

[0187] As shown above, the method for determining the content of Gardenia and Fermented Soybean Soup can effectively detect the content of Gardenia and Fermented Soybean Soup reference samples, and the method is stable and feasible. Based on the data analysis of 6 batches of reference samples, the preliminary limit ranges are set as follows: daidzein not less than 0.27 mg / g, genistein 0.08 mg / g, quercetin 0.36 mg / g, and total content not less than 0.75 mg / g.

[0188] To determine whether the selected preparation process and content range are appropriate, the beneficial effects of the present invention are demonstrated by the following pharmacological experiments.

[0189] Experimental Example 1 Exploration of the Mechanism of Action of "Zhizi Chi Decoction" in Treating Alzheimer's Disease Based on Network Pharmacology, Molecular Docking, and Molecular Dynamics Simulation

[0190] 1. Screening of Chemical Constituents and Action Targets of Zhizi Chi Decoction

[0191] In the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform TCMSP (https: / / old.tcmsp-e.com / tcmsp.php), using "Gardenia jasminoides Ellis" and "Semen Sojae Preparatum" as search terms respectively, chemical constituents with oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 were screened as the active ingredients and their action targets of the two traditional Chinese medicines. Further search and screening of the chemical constituents of "Semen Sojae Preparatum" were carried out in the herb database (https: / / herb.harvard.edu / ), removing ingredients without PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ) ID and with a component mass greater than 500, and using XLogP ≤ 5, H-BondDonorCount ≤ 5, and H-BondAcceptorCount ≤ 10 as screening conditions to conduct secondary screening of the above ingredients, and obtaining their targets for the obtained ingredients in the swiss target prediction database (http: / / www.swisstargetprediction.ch / ). The Uniprot database (http: / / UNIPROT.org / ) was used to standardize the above obtained targets, and at the same time, the chemical constituents and action targets of "Gardenia jasminoides Ellis" and "Semen Sojae Preparatum" were supplemented through literature retrieval.

[0192] 2. Collection and Screening of AD Genes

[0193] Using "Alzheimer’s disease, AD" as the keyword, potential disease-related genes were searched and screened in the OMIM database (http: / / OMIM.org / ) and the GeneCards database (http: / / GeneCards.org / ). The Venny2.1.0 (https: / / bioinfogp.cnb.csic.es / tools / venny / ) platform was used to screen the intersecting targets of AD and Zhizi Chi Decoction and conduct visual analysis. [[ID=十七]]

[0194] 3. Construction of the Regulatory Network of Traditional Chinese Medicine Compound

[0195] The components of Gardenia and Fermented Soybean Decoction were analyzed and organized in relation to AD genes, and the data were imported into Cytoscape 3.10.2 to establish a network diagram of "Traditional Chinese Medicine Compound - Components - Intersecting Targets - Disease". The Degree value was calculated using NetworkAnalyzer, and the core active components of Gardenia and Fermented Soybean Decoction were screened based on the results.

[0196] 4. Construction of PPI protein interaction network

[0197] The potential target of Gardenia and Fermented Soybean Decoction for AD treatment was imported into the STRING database (https: / / string-db.org / ), and the species "homosapiens" was selected for analysis to obtain a protein-protein interaction network. The Combinedscore was set to 0.9, and individual nodes unrelated to the network were hidden to obtain a PowerPoint network diagram. This diagram was imported into Cytoscape 3.10.2 software, and APPS (CytoNCA) was used to visualize and analyze the PPI network. Betweenness, closeness, Degree, Eigenvector, LAC, and Network values ​​were calculated. R language was used to screen for genes greater than the median of each item, and then the results were visualized. Betweenness represents the proportion of all shortest paths in the network that pass through this node; closeness represents the reciprocal of the average shortest path length from this node to all other nodes in the network; Degree represents the number of directly connected neighboring nodes; Eigenvector centrality represents the centrality calculated by weighting the importance of neighboring nodes; LAC (Local Average Connectivity) represents the average degree of a node's neighboring nodes; and Network represents the global characteristics of the entire PPI network, rather than a single node.

[0198] 5. Enrichment analysis of GO and KEGG pathways

[0199] Intersecting target points were input into the Metascape platform (http: / / meta-scape.org / gp / index.html), and the species were selected as "homosapiens" for searching. The GOMoleculrfunction, GOCellularComponents, GOBiologicalProcesses, and KEGGPathway files were downloaded. The top 20 entries and pathways were visualized using the microbioinformatics platform (http: / / www.bioinformatics.com.cn).

[0200] 6. Molecular docking analysis

[0201] The IDs of key protein targets under the "Entry" field were found using the Uniprot database and entered into the RCSBPDB website (https: / / www.rcsb.org / ) to filter and download "PDBFormat" files, obtaining the 3D structure of the protein receptor. Water molecules and small molecule ligands were removed using PyMOL software to optimize the protein receptor. The 3D or 2D structures of the chemical components were downloaded from the PubChem database in SDF format. Small molecule ligands were optimized using Chem3D software to obtain their minimum free energy 3D conformations, which were then imported into AutodockTools software for processing. Molecular docking was performed using VIA software with "energyrange=5" and "nummodes=20". Finally, the molecular docking results were visualized and analyzed using PyMOL software.

[0202] 7. Molecular dynamics verification

[0203] Molecular dynamics simulations were performed using Amber20 software to analyze the two ligand-protein complex systems with the lowest binding energies obtained from molecular docking screening. In the preprocessing stage, the system underwent a 10,000-step steepest descent energy minimization process, with positional constraints applied to the ligand molecules to optimize the system configuration. The entire simulation was conducted under isothermal and isobaric conditions. Temperature was controlled using the Langevin kinetic method (300 K), and pressure was adjusted using the Berendsen coupling method (1 bar), with appropriate time constants set to ensure the system slowly reached equilibrium. The formal simulation phase involved 100 ns of unconstrained molecular dynamics simulations, with trajectory data acquired every 10 ps. The "cpptraj" module was used to process and analyze the trajectory data. The stability and affinity of small molecule ligands to protein receptors were reflected by calculating indicators such as the number of hydrogen bonds (H-bonds), root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), and free energy surface (FEL).

[0204] 8. Results of a network pharmacology study on the treatment of Alzheimer's disease with Gardenia and Fermented Soybean Decoction

[0205] 8.1 Screening of chemical components and their target sites in Gardenia and Fermented Soybean Decoction

[0206] A total of 33 active ingredients from "gardenia" and 15 active ingredients from "fermented soybean" were screened, as shown in Tables 15 and 16. A total of 687 target sites were obtained.

[0207] Table 15 Active Ingredients of Gardenia

[0208]

[0209] Table 16 Active Ingredients of Fermented Soybeans

[0210]

[0211] 8.2 Collection of disease targets and screening of overlapping targets

[0212] A search of the GenenCards database yielded 12,586 genes. After filtering for Relevance scores ≥ 25, 806 genes were identified. An additional 52 genes were retrieved from the OMIM database. Combining these two databases resulted in a total of 858 genes. These genes were then intersected with the drug targets listed in section "3.1," and a Venn diagram was generated, as shown below. Figure 10 As shown, a total of 142 intersection targets were obtained, which are potential targets for the treatment of AD by Gardenia and Fermented Soybean Decoction.

[0213] 8.3 Construction of the "Traditional Chinese Medicine Compound-Component-Target-Disease" Regulatory Network Diagram

[0214] Use Cytoscape 3.10.2 drawing tools to draw a network diagram of active ingredients, targets, and diseases in traditional Chinese medicine compound prescriptions, such as... Figure 11 As shown, the degree values ​​of the nodes were calculated using AnalyzeNetwork and then sorted. The top five components were quercetin, genistein, apigenin, daidzein, and sinapicacid. It is preliminarily inferred that these components may be the key active ingredients in Gardenia and Fermented Soybean Decoction for treating Alzheimer's disease.

[0215] 8.4 Construction of PPI Network and Screening of Key Genes

[0216] A PPI protein interaction network was constructed according to the method described in section "2.4". Using R language, the initial medians were calculated as follows: Betweenness = 85.21; Closeness = 0.31; Degree = 4; Eigenvector = 0.032; LAC = 1.6; Network = 2.67. Genes with all values ​​greater than the median were selected, and the results were calculated again: Betweenness = 361.84; Closeness = 0.36; Degree = 16. Similarly, genes with all values ​​greater than the median were selected, resulting in 10 core genes: TP53, STAT3, AKT1, IL6, ESR1, NFKB1, TNF, MAPK1, MAPK3, and RELA. It is inferred that these targets may play an important role in the treatment of Alzheimer's disease with Gardenia and Fermented Soybean Decoction. See details below. Figure 12 , Figure 13 .

[0217] 8.5 Analysis of GO functional enrichment and KEGG signaling pathway enrichment results

[0218] The genes from the intersection obtained under section "3.2" were imported into the Metascape platform for Go Biological Analysis (GO). This analysis yielded 2083 Go Biological Processes, 240 Go Molecular Functions, and 135 Go Cellular Components. The Count value reflects the number of genes significantly enriched in a specific GO entry; a higher value indicates a wider range of genes involved in that function or pathway within the analyzed dataset. Therefore, this invention sorted the biological processes according to their Count values ​​and selected the top 20 for subsequent bar chart analysis. The results are shown below. Figure 14 The KEGG signaling pathway enriched 198 signaling pathways, mainly those involved in diabetic complications such as the AGE-RAGE, IL-17, prolactin, C-type lectin receptor, and HIF-1 signaling pathways. A bubble chart was created using the top 20 enrichment values. The volume of the dots in the chart was positively correlated with the number of enriched entries; darker colors indicated smaller p-values. Larger enrichment values ​​indicated more significant enrichment of the target gene in a specific pathway, suggesting that this pathway may play a key role in the treatment of Alzheimer's disease (AD). Figure 15 .

[0219] 8.6 Molecular docking results

[0220] After comprehensive analysis, quercetin, genistein, apigenin, daidzein, and coumarin were selected as small molecule ligands for molecular docking with the five core genes STAT3, IL6, ESR1, NFKB1, and TP53 selected from the initial screening. The most suitable binding sites for the ligands and receptors were then chosen, and the binding energies were as follows: Figure 16 As shown in the diagram. It is generally believed that the lower the binding affinity between the ligand and the receptor, the more stable the complex formed. In this invention, all five main active ingredients can spontaneously bind to the key targets. Specifically, daidzein binds to ESR1 with an energy of -9.0 kcal / mol; genistein binds to ESR1 with an energy of -9.5 kcal / mol; and quercetin binds to ESR1 with an energy of -8.9 kcal / mol. Partial molecular docking diagrams are shown in the diagram. Figure 17 Molecular docking results indicate that Gardenia and Fermented Soybean Decoction can exert its therapeutic effect by acting on AD targets.

[0221] 9 Conclusions

[0222] This invention yielded five key components of Gardenia and Fermented Soybean Soup, including quercetin, genistein, apigenin, daidzein, and coumarin. Molecular docking results showed that the main active components of Gardenia and Fermented Soybean Soup exhibited good docking activity with their core targets. Specifically, daidzein, genistein, and quercetin showed binding energies of -9.0 kcal / mol, -9.5 kcal / mol, and -8.9 kcal / mol with ESR1 protein, respectively. Apigenin's binding energies with both ESRI and STAT3 proteins were -7.7 kcal / mol. Daidzein, genistein, and quercetin had the highest binding energie scores.

[0223] In summary, this invention fully reveals the potential mechanism of Gardenia and Fermented Soybean Decoction in treating Alzheimer's disease (AD). The molecular docking and molecular dynamics simulation results of the active ingredients in Gardenia and Fermented Soybean Decoction regulating key targets and signaling pathways in the pathogenesis of AD further support the potential therapeutic effect of Gardenia and Fermented Soybean Decoction on AD.

[0224] The selection criteria for detecting the content of key components such as quercetin, genistein, and daidzein according to the method in Example 3

[0225] 1. Chromatographic conditions and system suitability test

[0226] The column was packed with octadecylsilane-bonded silica gel (250 mm column length, 4.6 mm inner diameter, 5 μm particle size); methanol was used as mobile phase A, and 0.4% phosphoric acid solution was used as mobile phase B, with gradient elution under the following conditions: 0–20 min, mobile phase A 40→80; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 260 nm. The theoretical plate number, calculated based on the daidzein peak, should be no less than 3000.

[0227] 2. Preparation of reference solution

[0228] Take appropriate amounts of daidzein reference standards, genistein reference standards, quercetin reference standards, coumarin reference standards, and apigenin reference standards, accurately weigh them, and add methanol to prepare a mixed solution containing 15 μg of daidzein, 15 μg of genistein, 15 μg of quercetin, 10 μg of coumarin, and 10 μg of apigenin per 1 ml.

[0229] Preparation of test solution

[0230] 3. Test solution: Take an appropriate amount of this product, about 0.2g, accurately weigh it, place it in a stoppered conical flask, accurately add 40ml of methanol-25% hydrochloric acid (4:1) mixed solution, stopper tightly, weigh it, heat under reflux for 60 minutes, cool it, weigh it again, make up the lost weight with 7ml of methanol-25% hydrochloric acid (4:1) mixed solution, shake well, filter, and take the filtrate to obtain the test solution.

[0231] 4. Determination method

[0232] Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0233] 5. Results of indicator component screening

[0234] Based on the above-established experimental conditions, the mixed control standard and the test sample were investigated separately, and the results are shown in the table below. Figure 18 .

[0235] Based on the above chromatographic analysis, combined with network pharmacology analysis, five chemical components—genistein, daidzein, quercetin, apigenin, and coumarin—were screened. In the chromatogram of the test sample, coumarin and apigenin were found to be present in extremely low amounts in the Gardenia and Fermented Soybean Decoction, close to the baseline. Furthermore, combined with the molecular kinetic analysis results from network pharmacology, genistein, daidzein, and quercetin had high binding energy scores, suggesting they may be key targets and signaling pathways in the active components of Gardenia and Fermented Soybean Decoction that regulate the pathogenesis of Alzheimer's disease. Moreover, genistein, daidzein, and quercetin are derived from fermented soybean and gardenia, respectively. Therefore, under the given conditions, genistein, daidzein, and quercetin are more suitable as the chemical indicators for content.

[0236] Experimental Example 2: Verification Experiment of Antioxidant Activity of Gardenia and Fermented Soybean Decoction of the Present Invention

[0237] Oxidative stress is not only one of the earliest events in Alzheimer's disease but also a catalyst that accelerates its progression. Oxidative stress directly promotes the production of Aβ and pathological changes in Tau protein, forming a positive feedback loop with Aβ, Tau, and neuroinflammation, continuously amplifying damage. Through the oxidation of lipids, proteins, and nucleic acids, it directly leads to neuronal dysfunction and death. Improving the brain's oxidative stress state is one of the intervention directions for treating Alzheimer's disease.

[0238] This invention identifies daidzein, genistein, and quercetin as key active ingredients in Gardenia and Fermented Soybean Decoction for treating Alzheimer's disease. These three components are all flavonoids with antioxidant, anti-inflammatory, and neuroprotective effects. Through the synergistic antioxidant and anti-inflammatory effects of the chemical components in Gardenia and Fermented Soybean Decoction, the "oxidative stress" state in the brain can be rapidly alleviated, playing an important role in reducing the patient's mental and behavioral state. Simultaneously, through long-term regulation at multiple targets, it can slow disease progression, protect neurons, and delay cognitive decline. Quercetin, as a potent antioxidant and anti-inflammatory component, assists and enhances the heat-clearing effect of Gardenia, jointly inhibiting microglia activation and reducing inflammatory damage. It possesses multiple biochemical effects such as antioxidant, apoptosis-inhibiting, and anti-inflammatory properties, and its outstanding neuroprotective effect makes it an emerging clinically applicable drug for treating AD. Studies have shown that moderate autophagy is a necessary condition for coping with cellular stress, inhibiting apoptosis, and promoting cell survival. It can intervene in the disease progression of Alzheimer's disease by interfering with the formation and deposition of β-amyloid protein and inhibiting the excessive phosphorylation of Tau protein, thereby playing a protective role for nerve cells.

[0239] In addition to their antioxidant and anti-inflammatory effects, daidzein and genistein possess unique phytoestrogen-like effects that can compensate for the decline in estrogen levels in postmenopausal women, providing additional protection for neurons. The mechanism of action of genistein in treating Alzheimer's disease (AD) is due to its estrogen-like structure, which allows it to mimic estrogen's effects, exhibiting multi-pathway targeting and low toxicity. Furthermore, it possesses multiple functions, including antioxidant, anti-inflammatory, anti-β-amyloid, cholinergic neurotransmission regulation, and inhibition of Tua protein phosphorylation.

[0240] In summary, daidzein, genistein, and quercetin in Gardenia and Fermented Soybean Decoction all contribute to the development and progression of Alzheimer's disease by combating oxidative stress in the brain through their antioxidant activity. Therefore, this invention uses the WST-8 assay and the thiobarbituric acid (TBA) assay to evaluate the total antioxidant effects of daidzein, genistein, and quercetin.

[0241] 1. Experiment Content

[0242] 1.1 Drug preparation

[0243] SPF-grade male C57BL / 6J mice, with 10-12 mice per group as needed;

[0244] Gardenia and fermented soybean soup reference sample (batch number: ZZCT-06, total content 0.878mg / g);

[0245] D-Galactose (batch number ST1218, Shanghai Beyotime Biotechnology Co., Ltd.)

[0246] Vitamin E (batch number RH653699, Ron Reagent);

[0247] The SOD assay kit (WST-8 method) (batch number S0101S) and the MDA assay kit (TBA method) (batch number S0131S) were both purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0248] Sodium carboxymethyl cellulose (batch number Y268573) and physiological saline (0.9% NaCl, sterile) (batch number ST341) were both purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0249] 1.2 Dosage Design

[0250] The total content of daidzein, genistein, and quercetin in the above batches, i.e., 100%, was used as the high dose in the experiment; 85% was used as the medium dose (i.e., 0.746 mg / g); and 70% was used as the low dose (i.e., 0.615 mg / g). The mixed drug solution was freshly prepared using 0.5% CMC-Na solution to ensure uniform drug suspension.

[0251] 1.3 Grouping and Modeling

[0252] Sixty C57BL / 6J mice were acclimatized to their new environment for one week and then randomly divided into six groups of ten mice each: a control group, an aging model group, a low-dose treatment group, a medium-dose treatment group, a high-dose treatment group, and a positive control group. Except for the control group (normal control), mice in the model and treatment groups were intraperitoneally injected with D-galactose daily for 6-8 weeks to establish an aging mouse model. Simultaneously, the treatment groups began gavage therapy once daily for 6-8 weeks. The control group received intraperitoneal injections of saline and gavage with an equal volume of 0.5% CMC-Na solution daily.

[0253] The specific groupings are shown in Table 17. Different control and experimental groups, A, B, C, D, E, and F, were set up respectively.

[0254] Table 17 Grouping Table

[0255]

[0256] 2. Sample Collection and Processing

[0257] Mice were fasted and deprived of water for 12 hours, then anesthetized. The hippocampus and part of the cerebral cortex were rapidly separated on an ice tray and rinsed with pre-cooled physiological saline. The tissue was blotted dry with filter paper and accurately weighed. Pre-cooled phosphate buffer was added at a weight-to-volume ratio (e.g., 1:9), and the mixture was mechanically homogenized under ice bath conditions. The homogenate was then centrifuged at 12000 rpm for 10-15 minutes at 4°C. The supernatant was carefully aliquoted into EP tubes and stored at -80°C for later analysis.

[0258] 3. Biochemical indicator testing

[0259] 3.1 SOD activity assay

[0260] Following the instructions of the SOD detection kit, add reagents, sample and distilled water to a 96-well plate in sequence, mix well and incubate at 37°C for 30 minutes. Measure the absorbance of each well at a wavelength of 450 nm.

[0261] 3.2 MDA content determination

[0262] Follow the instructions for the MDA detection kit. Add the sample, reagent 1, and reagent 2 to the centrifuge tube in sequence, mix well, and then incubate at 95°C for 30-40 minutes. After cooling under running water, centrifuge and take the supernatant to measure the absorbance at 532 nm.

[0263] 4. Effects of Gardenia and Fermented Soybean Decoction on MDA and SOD in Mouse Brain Tissue

[0264] As shown in the table below, compared with the blank control group, the MDA content in the brain tissue of mice in the aging model group was significantly increased (P<0.01), and the SOD activity was significantly decreased (P<0.01), indicating that D-galactose caused varying degrees of damage to the antioxidant system in mice. Compared with the model group, the MDA content in the brain tissue of mice in the positive drug treatment group and the high- and medium-dose Gardenia and Fermented Soybean Decoction treatment groups was significantly decreased, and the SOD activity was significantly increased.

[0265] Table 18 Effects of Gardenia and Fermented Soybean Decoction on MDA and SOD in Mouse Brain Tissue (x̄±s, n=10)

[0266]

[0267] Note: Compared with the blank control group, *P<0.05, **P<0.01; compared with the model group, # P<0.05, ## P<0.01.

[0268] The results showed that the model control group and the experimental group had significant differences in indicators at high doses, some differences at medium doses, but no significant differences at low doses. This indicates that the Gardenia and Fermented Soybean Decoction prepared according to the above method has a certain protective effect against oxidative damage caused by free radicals and lipid peroxidation in the body when the total content of daidzein, genistein, and quercetin is not less than 0.746 mg / g.

[0269] Experiments have shown that detecting the content of daidzein, genistein, and quercetin can be used to control the quality of Gardenia Soybean Decoction and its reference samples, and is directly related to the efficacy of Gardenia Soybean Decoction in treating Alzheimer's disease, thus ensuring the effectiveness and stability of Gardenia Soybean Decoction.

Claims

1. A reference standard of Zhizi Qili Decoction for treating Alzheimer's disease, characterized in that: The total content of daidzin, genistein and quercetin is not less than 0.75 mg / g; wherein the content of daidzin is not less than 0.27 mg / g, the content of genistein is not less than 0.08 mg / g, and the content of quercetin is not less than 0.36 mg / g; ​ The preparation method of the radix scrophulariae and sojae soup reference standard comprises the following steps: a. Weigh the raw medicinal materials according to the weight ratio: 1 part of radix scrophulariae and 1 part of sojae; b. Soak the radix scrophulariae in water, then add the sojae and boil for 30-60 minutes, filter and take the water extract; c. Freeze-dry the water extract of step b to obtain the radix scrophulariae and sojae soup reference standard; The freeze-drying conditions are as follows: (1) Pre-freezing: temperature: -50℃, temperature maintenance: 8h; Pressure: normal pressure; (2) Sublimation drying: temperature: -40℃, temperature maintenance: 2h; Temperature: -30℃, temperature maintenance: 2h; Temperature: -20℃, temperature maintenance: 2h; Temperature: -10℃, temperature maintenance: 3h; Temperature: 0℃, temperature maintenance: 3h; (3) Analysis drying: temperature: 10℃, temperature maintenance: 3h; Temperature: 20℃, temperature maintenance: 2h; Temperature: 30℃, temperature maintenance: 2h; Temperature: 40℃, temperature maintenance: 4h; The boiling method of step b is to first boil with strong fire until boiling, then boil with weak fire.

2. The method for determining the content of the reference standard of Zhizi Qiong Tang according to claim 1, characterized in that: It is to detect the contents of daidzin, genistein and quercetin by HPLC chromatography, and the chromatographic conditions are as follows: Use octadecylsilane-bonded silica gel as the filler, use methanol as the mobile phase A and 0.4% phosphoric acid solution as the mobile phase B, gradient elution, and the elution conditions are as follows: 0-20 minutes, mobile phase A 40→80.

3. The method of claim 2, wherein the amount of the Ziziphi and Ficus Linn. Decoction Reference Standard is determined by HPLC. The column length of the filler is 250mm, the inner diameter is 4.6mm, and the particle size is 5μm; the flow rate in the chromatographic conditions is 1.0ml per minute; the column temperature is 30℃; and the detection wavelength is 260nm.

4. The method for determining the content of the gardenia and fermented soybean soup reference standard according to claim 2 or 3, characterized in that: It comprises the following steps: a. Preparation of the test sample solution: Take the sample to be tested, add the methanol-25% hydrochloric acid mixed solution with a volume ratio of 4:1 into a conical flask with a plug, heat and reflux for 60 minutes, cool, reweigh, make up the weight loss with the methanol-25% hydrochloric acid mixed solution with a volume ratio of 4:1, shake well, filter, and take the filtrate to obtain the test sample solution; b. Detection according to the chromatographic conditions of claim 2 or 3.

5. The method of claim 4, wherein the amount of the Ziziphi and Fructus Gardeniae Decoction Reference Standard is determined by HPLC. It also comprises preparation of the control sample solution, and the preparation method is to take appropriate amounts of daidzin control sample, genistein control sample and quercetin control sample, and add methanol to prepare a mixed solution.

6. A method for detecting qualified products of Zhizi Chishih decoction for treating Alzheimer's disease, characterized in that: It comprises the following steps: a. Take the dried sample of the radix scrophulariae and sojae soup to be tested; b. Detect the contents of daidzin, genistein and quercetin according to the content determination method of the radix scrophulariae and sojae soup reference standard of any one of claims 2-5; c. If the total content of daidzin, genistein and quercetin is higher than 0.75 mg / g, the content of daidzin is higher than 0.27 mg / g, the content of genistein is higher than 0.08 mg / g, and the content of quercetin is higher than 0.36 mg / g, it is a qualified product.

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