Processing method and quality control method for toxicity reduction and efficacy enhancement of Lingshi

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to a processing method and a quality control method for toxicity reduction and efficacy enhancement of radix cynanchi bungei. According to the method, the content of alangiine, the content of ellagic acid, the common peak area of characteristic peaks and the content of water-soluble extracts in the wine-roasted berberis scenting decoction pieces are measured by adopting a high performance liquid chromatography (HPLC) method, and subjective assignment is carried out on the characters of the wine-roasted berberis scenting decoction pieces by combining Delphi evaluation to serve as evaluation indexes; the method comprises the following steps: determining the weight coefficient of each index by adopting an analytic hierarchy process-index repeatability correlation mixed weighting method (AHP-CRITIC), and screening the optimal wine roasting process of the Zhuang medicine Tibetan scene by combining the three factors of the frying temperature, the frying time and the yellow wine amount with star point design (CCD)-effect surface (RSM) according to the coefficients. And finally determining the optimal process of stir-frying the radix berberidis with wine as follows: the stir-frying temperature is 150 DEG C, the stir-frying time is 16min, the amount of yellow rice wine is 10%, and moistening is performed for 24h.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and particularly to a processing method and a quality control method for reducing toxicity and enhancing efficacy of Goingz (Alangium chinense). Background Art

[0002] The Zhuang medicine Goingz (Alangium chinense) is the dried lateral roots and fibrous roots of Alangium chinense (Lour.) Harms of the family Alangiaceae. Goingz is also used in the medicine of ethnic minorities such as the Yao, Miao, Yi, and Dong ethnic groups, and its Chinese medicine name is Alangium root-bark. Alangium root-bark was first recorded in "Ben Cao Cong Xin" in the Qing Dynasty, and was included in "Chinese Pharmacopoeia" (1977 edition). In Zhuang medicine, Alangium root-bark is bitter, spicy, slightly warm, and poisonous. It dredges the "dragon meridian" and "fire meridian", dispels wind toxin, and disperses stasis and relieves pain. It is used for "Fa Wang" (rheumatic arthralgia), "Ma Mo" (limb numbness), "Bang Ba Yin" (scapulohumeral periarthritis), "Huo Yao Yin" (cervical spondylosis), "Lin De Ding Xiang" (traumatic injury), and "He Yin" (low back pain). In modern Chinese medicine, Alangium root-bark is pungent, slightly warm, and slightly poisonous, and belongs to the liver and kidney meridians. There are many compatibility formulas with Alangium root-bark. For example, the Zhuang medicine Longzuan Tongbi Formula with it as an assistant medicine has the effects of dispelling wind and dampness, reducing swelling and relieving pain. The Fengshiding Capsule with Alangium root-bark as the monarch drug has the effects of promoting blood circulation to dredge collaterals and relieving pain due to arthralgia. The "Xiaobi Ling" mixture with Alangium root-bark as the main drug dispels wind and dredges collaterals, disperses stasis and relieves pain.

[0003] In recent years, pharmacological experiments and clinical experiments have proved that Alangium root-bark has the effects of anti-rheumatism, muscle relaxation, and anti-cancer. However, it is poisonous, especially the fibrous roots are more toxic. Taking an overdose or using the unprepared product can cause poisoning, which restricts the clinical use of Alangium root-bark to a certain extent. Therefore, it is necessary to find a processing method for reducing toxicity and enhancing efficacy of Goingz. Summary of the Invention

[0004] The purpose of the present invention is to provide a processing method and a quality control method for reducing toxicity and enhancing efficacy of Goingz to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above purpose, the present invention provides the following scheme:

[0006] The present invention provides a processing method for reducing toxicity and enhancing efficacy of Goingz, including the steps of mixing Goingz and yellow rice wine, then performing moistening, frying, and drying in sequence to obtain the wine-fried Goingz slices;

[0007] The dosage of the yellow rice wine is 10% of the mass of Goingz;

[0008] The frying time is 16 min, and the temperature is 150 °C.

[0009] Preferably, the moistening time is 24 h.

[0010] Preferably, the rotation speed of frying is 40 r / min, and the frying frequency is 120 times / min.

[0011] This invention provides a wine-processed Kejing herbal slice prepared using the above-described processing method.

[0012] This invention provides a quality control method for the above-mentioned wine-processed Kejing medicinal slices, comprising:

[0013] (1) Identify the physical characteristics of the wine-processed Kejing slices;

[0014] (2) Perform powder microscopic identification on the wine-processed Kejing medicinal slices;

[0015] (3) Thin-layer chromatography was used to identify the roasted and processed Kejing medicinal slices;

[0016] (4) The moisture, ash, acid-insoluble ash and extract content of the roasted sage slices were determined; the moisture content of the roasted sage slices shall not exceed 9.0%, the total ash content shall not exceed 6.00%, the acid-insoluble ash content shall not exceed 0.30%, and the extract content shall not be less than 14.0%;

[0017] (5) The content of star anise alkaloid in the wine-processed Kejing slices was determined by high performance liquid chromatography; the content of star anise alkaloid in the wine-processed Kejing slices shall not be less than 0.350%.

[0018] Preferably, the thin-layer chromatography identification method includes the steps of spotting the test solution and the reference solution onto a silica gel G thin-layer plate, using a mixed solution of toluene, ethyl acetate and formic acid as the developing solvent, and observing under a 365 nm ultraviolet lamp;

[0019] The method for preparing the test solution includes the steps of mixing the sample powder to be tested and ethanol at a ratio of 1g:10mL, and ultrasonically treating for 60min to obtain the test solution.

[0020] The method for preparing the reference solution includes mixing Illicium verum reference standard and ethanol at a ratio of 1g:10mL, and ultrasonically treating for 60min to obtain the reference solution.

[0021] Preferably, the volume ratio of toluene, ethyl acetate and formic acid in the mixed solution is 4:1:0.5.

[0022] Preferably, the high-performance liquid chromatography detection includes the steps of aspirating the reference solution and the test solution, injecting them into the liquid chromatograph, and performing chromatographic detection;

[0023] The preparation method of the test solution includes mixing the sample powder to be tested and an ethanol solution containing acetic acid at a ratio of 1g:25mL, and ultrasonically treating for 30min to obtain the test solution.

[0024] The method for preparing the reference solution includes the step of mixing Illicium verum reference standard and an ethanol solution containing acetic acid to obtain the reference solution; the concentration of Illicium verum in the reference solution is 1.3812 mg / mL.

[0025] Preferably, the chromatographic column used for the chromatographic detection is ECOSIL C10. 18 Chromatographic column; with acetonitrile as mobile phase A and buffer solution as mobile phase B;

[0026] The buffer solution comprises sodium heptanesulfonate and potassium dihydrogen phosphate; the concentration of sodium heptanesulfonate in the buffer solution is 0.2 g / L and the concentration of potassium dihydrogen phosphate is 3.5 g / L.

[0027] Preferably, the chromatographic detection conditions are: column temperature 25℃, flow rate 1.0 mL / min, injection volume 10 μL, and detection wavelength 254 nm.

[0028] The present invention discloses the following technical effects:

[0029] This invention uses high-performance liquid chromatography (HPLC) to determine the content of quinquefoliine, ellagic acid, and common peak area of ​​characteristic peaks in wine-processed *Gynostemma pentaphyllum*. The content of water-soluble extracts was also determined. The Delphi evaluation was used to subjectively score the characteristics of the wine-processed *Gynostemma pentaphyllum* slices as evaluation indicators. An analytic hierarchy process (AHP-CRITIC) was employed to determine the weight coefficients of each indicator. Based on these coefficients, a star-point design (CCD)-response surface methodology (RSM) was used to screen the optimal wine-processing technique for *Gynostemma pentaphyllum*. The optimal process was determined to be: a stir-frying temperature of 150℃, a stir-frying time of 16 min, a rice wine amount of 10%, and a soaking time of 24 h. In the processing verification (n=3), the average comprehensive score was 95.76 (RSD=1.02%), with a relative error of 8.61% compared to the predicted value.

[0030] Afterwards, according to the methods specified in the appendix of the 2020 edition of the Chinese Pharmacopoeia (Volume IV), the character, microscopic and thin-layer identifications of 14 batches of wine-fried Kejing slices from different producing areas were carried out, and the moisture content, ash content, extractives and the content of alangicine were determined. The results showed that the specificity of each identification method for wine-fried Kejing slices was strong. The moisture content, total ash content, acid-insoluble ash content, water-soluble extractives and the content of alangicine in wine-fried Kejing were 5.11% - 9.53%, 1.99% - 12.05%, 0.01% - 1.78%, 7.34% - 14.74% and 0.091% - 0.753% respectively. It was preliminarily determined that the moisture content of wine-fried Kejing slices should not be higher than 12.0%, the total ash content should not be higher than 6.0%, the acid-insoluble ash content should not be higher than 2.1%, the water-soluble extractives should not be lower than 6.0%, and the alangicine should not be lower than 0.35%. To sum up, based on the AHP-CRITIC hybrid weighting method combined with the star point design - response surface method, the optimized wine-frying process of the Zhuang medicine Kejing is stable and reliable, and can be used for the wine-frying process of Kejing. The quality standard of wine-fried Kejing slices established in this invention is stable and reliable, and can be used for the quality control of wine-fried Kejing slices. Description of the Drawings

[0031] Figure 1 It is the HPLC chromatogram of ellagic acid reference substance and the test sample of wine-fried Kejing; among which, 1 is ellagic acid, A is the ellagic acid reference substance, and B is the test sample solution of wine-fried Kejing;

[0032] Figure 2 It is the HPLC chromatogram of alangicine reference substance and the test sample of wine-fried Kejing; among which, 2 is alangicine, C is the alangicine reference substance, and D is the test sample solution of wine-fried Kejing;

[0033] Figure 3 It is the HPLC characteristic chromatogram of 14 batches of wine-fried Kejing; among which, 3 is ellagic acid;

[0034] Figure 4 It is the response surface of frying temperature;

[0035] Figure 5 It is the response surface of frying time;

[0036] Figure 6 It is the response surface of the amount of yellow rice wine;

[0037] Figure 7 It is the contour map of frying temperature;

[0038] Figure 8 It is the contour map of frying time;

[0039] Figure 9 It is the contour map of the amount of yellow rice wine;

[0040] Figure 10 It is the picture of raw Kejing and wine-fried Kejing;

[0041] Figure 11 An image showing the appearance of roasted and processed Kejing medicinal slices;

[0042] Figure 12 The results are for the identification of powdered *Gnaphalium affine*; where A is starch granules; B is cork cells; C is crystal sheath fibers; D is calcium oxalate clusters; E is fibers; F is bordered vessels; and G is sclereids.

[0043] Figure 13 The results are thin-layer chromatograms of the test solution and the reference solution at 365 nm; where 1-14 are the test solution and K is the reference solution.

[0044] Figure 14 Image of cardiac light microscopy, ×100; where A, C, represent blank group, wine-processed Kejing group, and raw Kejing group, respectively.

[0045] Figure 15 Image of cardiac light microscopy, ×400; where A, C, represent blank group, wine-processed Kejing group, and raw Kejing group, respectively.

[0046] Figure 16 The image shows a light microscopic observation of the liver, ×100; where A, C, represent the blank group, the wine-processed skewer group, and the raw skewer group, respectively; the green arrows indicate hepatocyte degeneration.

[0047] Figure 17 The image shows a light microscopic observation of the liver, ×400; where A and C represent the blank group, the wine-processed liver group, and the raw liver group, respectively.

[0048] Figure 18 Image of spleen under light microscopy, ×100; where A, C, represent blank group, wine-processed spleen group, and raw spleen group, respectively.

[0049] Figure 19 Image of spleen under light microscopy, ×400; where A, C, represent blank group, wine-processed spleen group, and raw spleen group, respectively.

[0050] Figure 20 The image shows a light microscopic observation of the lungs, ×100; where A, C, represent the blank group, the wine-processed Kejing group, and the raw Kejing group, respectively.

[0051] Figure 21 The image shows a light microscopic observation of the lungs, ×400; where A, C, represent the blank group, the wine-processed Kejing group, and the raw Kejing group, respectively.

[0052] Figure 22 The image shows a light microscopic observation of the kidney, ×100; where A, C, represent the blank group, the wine-processed koji group, and the raw koji group, respectively.

[0053] Figure 23The image shows a light microscopic observation of the kidney, ×400; where A, C, represent the blank group, the wine-processed koji group, and the raw koji group, respectively.

[0054] Figure 24 Images are taken using a brain microscope, ×100; where A and C represent the blank group, the wine-processed Kejing group, and the raw Kejing group, respectively.

[0055] Figure 25 Images are taken using a optic endoscopic microscope, ×400; where A, C, represent the blank group, the wine-processed oyster shell group, and the raw oyster shell group, respectively. Detailed Implementation

[0056] The instruments are as follows:

[0057] Waters Alliance e2695 UV-vis high-performance liquid chromatograph (Waters Corporation, USA); CAMAG semi-automatic spotter (CAMAG, Switzerland); Reprostar 3 CAMAG thin-layer chromatography scanning imaging system (CAMAG, Switzerland); Olympus BX53 microscope (Olympus (China) Co., Ltd., Japan); 0.0001 g electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); Milli-Q Reference laboratory ultrapure water system (Millipore Corporation, USA); Type 25B electric roasting machine (Shenma Machinery Factory, Ruian, Zhejiang); non-contact infrared thermometer (HIM Instruments Co., Ltd., Hong Kong).

[0058] The following drugs were tested:

[0059] Illicium verum alkaloids (MedChemExpress, USA, batch number 129513, mass fraction ≥98%); Illicium verum reference material (China National Institute for Food and Drug Control, batch number: R5G2-HRA7); acetonitrile and methanol were chromatographically pure (Thermo-Fisher, USA); phosphoric acid was chromatographically pure (Tianjin Damao Chemical Reagent Factory); sodium 1-heptanesulfonate was analytically pure (Shanghai Maclean Biochemical Technology Co., Ltd., batch number: C12673682); potassium dihydrogen phosphate was analytically pure (Chengdu Jinshan Chemical Reagent Co., Ltd., batch number: 20210302); silica gel G plate (Qingdao Ocean Chemical Co., Ltd., batch number: 20220703); water was self-made ultrapure water.

[0060] The medicinal materials are as follows:

[0061] Fourteen batches of *Alangium chinense* medicinal materials were purchased and harvested. These were identified by Senior Experimentalist Zhu Yilin of Guangxi University of Traditional Chinese Medicine as dried lateral roots and fibrous roots of *Alangium chinense* (Lour.) Harms, a plant of the Illicaceae family. Origin information is shown in Table 1. The *Alangium chinense* medicinal materials in Table 1 were processed with rice wine to obtain rice wine-processed *Alangium chinense* medicinal materials. The specific steps of rice wine processing were as follows: 200g of *Alangium chinense* medicinal materials were taken, and rice wine was sprayed onto the medicinal slices. The total amount of rice wine used was 10% of the weight of the *Alangium chinense* medicinal materials. The mixture was left to moisten at room temperature for 24 hours. Then, it was placed in a temperature-controlled stir-frying machine (40 rpm, 120 stirs / min) and stir-fried at 150℃ for 16 minutes. Afterward, it was removed and air-dried, resulting in 14 batches of rice wine-processed *Alangium chinense* medicinal materials, numbered S1-S2. 14 .

[0062] Table 1. Source information for 14 batches of Kejing (a type of ornamental plant).

[0063]

[0064] Example 1: Establishment of an HPLC method for the determination of ellagic acid and cinnamic acid content.

[0065] 1. Chromatographic conditions

[0066] Ellagic acid conditions: The chromatographic column was a Shim-pack VP-ODS (250 mm × 4.6 mm, 5 μm); the mobile phase was acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B); the elution method was gradient elution (0~30 min: 7%~23% B; 30~45 min: 23%~35% B); the flow rate was 1 mL / min; the column temperature was 25℃; the detection wavelength was 254 nm; and the injection volume was 10 μL.

[0067] Illicium verum alkaloid conditions: chromatographic column: ECOSIL C 18 (250mm×4.6mm, 5μm), mobile phase was 9% acetonitrile (A)-91% buffer salt (0.2g sodium heptanesulfonate + 3.5g potassium dihydrogen phosphate to 1000mL, B), elution method: isocratic elution; flow rate was 1mL / min; column temperature was 35℃; detection wavelength was 254nm; injection volume was 10μL.

[0068] 2. Preparation of reference solution

[0069] Take appropriate amounts of ellagic acid and cinnamon alkaloid, weigh them accurately, and place them in 25 mL volumetric flasks respectively. Dissolve the ellagic acid in 50% ethanol, and dilute the cinnamon alkaloid with 30% methanol containing 5% acetic acid to the mark to prepare a reference solution containing 1.200 mg / mL ellagic acid and 1.3812 mg / mL cinnamon alkaloid.

[0070] 3. Preparation of the test solution for roasted and processed *Gynostemma pentaphyllum*

[0071] Take 1g of the roasted sage powder (passed through a No. 4 sieve), place it in a 50mL stoppered conical flask, accurately measure 25mL of 30% methanol containing 5% acetic acid, weigh it, sonicate for 30min (power 200W, 40Hz), cool, make up the weight, shake well, and filter through a 0.22μm microporous membrane to obtain the final product.

[0072] 4. HPLC analysis was performed on ellagic acid reference standard, cinnamon alkaloid reference standard, and wine-processed *Illicium verum* sample.

[0073] The HPLC chromatograms of ellagic acid reference standard, cinnamon alkaloid reference standard, and wine-processed *Gynostemma pentaphyllum* sample are shown below. Figure 1 and Figure 2 .

[0074] 5. Plotting the standard curve

[0075] Ellagic acid was used to prepare six reference solutions with concentrations of 0.0300 mg / mL, 0.1200 mg / mL, 0.2400 mg / mL, 0.4800 mg / mL, 0.6000 mg / mL, and 1.2000 mg / mL, respectively. Illicium verum alkaloid was used to prepare six reference solutions with concentrations of 0.0575 mg / mL, 0.1151 mg / mL, 0.2302 mg / mL, 0.4604 mg / mL, 0.9208 mg / mL, and 1.3812 mg / mL, respectively. The reference solutions were accurately pipetted, and the peak areas were recorded.

[0076] Standard curves were plotted with the mass of each reference standard as the abscissa (X) and the peak area as the ordinate (Y). Regression analysis was performed to obtain the regression equations, correlation coefficients, and linear ranges for each index component, namely: ellagic acid Y1=5634468X-61268, R²=0.9999, 0.0375mg / mL-1.2000mg / mL; and cinnamic acid Y2=669424X-20846, R²=0.9998, 0.0575mg / mL~1.3812mg / mL.

[0077] 6. Precision test

[0078] Accurately pipette 10 μL of the reference solution and inject it six times consecutively under the chromatographic conditions of this embodiment. The RSDs of the peak areas of ellagic acid and cinnamon alkaloid were measured to be 0.74% and 0.06%, respectively, indicating that the instrument has good precision.

[0079] 7. Stability test

[0080] The same wine-processed herb sample solution was precisely pipetted into 10 μL at 0, 4, 8, 12, 20, and 24 h. The RSDs of the peak areas of ellagic acid and star anise alkaloid were calculated to be 0.77% and 2.72%, respectively, indicating that the sample solution was stable within 24 h.

[0081] 8. Repeatability test

[0082] Six samples of the wine-processed *Illicium verum* powder (passed through a No. 4 sieve, designated S1) were prepared according to the aforementioned method. The mass fractions of ellagic acid and cinnamon alkaloids were determined to be 0.105 and 6.53 mg / g, respectively, with RSDs of 1.58% and 2.31%, respectively, under the chromatographic conditions of this embodiment, indicating that the experiment had good repeatability.

[0083] 9. Spiking recovery test

[0084] Six portions of the previously tested processed *Acer buergerianum* powder (number S1), each weighing 1 g, were accurately weighed. The corresponding amounts of reference standards were added to each portion at 100% concentration, and test solutions were prepared according to the aforementioned method. The recoveries of ellagic acid and *Acer buergerianum* were determined to be 101.6% and 95.77%, respectively, with RSDs of 0.34% and 1.34%, respectively, under the chromatographic conditions of this embodiment.

[0085] 10. Establishment and analysis of HPLC characteristic peak chromatograms

[0086] Fourteen batches of wine-processed *Clerodendrum trichotomum* medicinal materials were taken, and fourteen batches of wine-processed *Clerodendrum trichotomum* test solutions were prepared according to the preparation method in this embodiment. Ellagic acid was determined according to the chromatographic conditions in this embodiment. The chromatograms of each batch of samples were recorded, and the CDF format data files of the 14 chromatograms were exported and imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (Version 2012.1)" software for common peak identification. The results showed that the characteristic chromatograms of the 14 batches of wine-processed *Clerodendrum trichotomum* medicinal materials had the same seven common peaks (…). Figure 3 ), of which 3 is ellagic acid.

[0087] 11. Evaluation of appearance characteristics (traditional experience-based evaluation)

[0088] The Delphi evaluation method is applicable to the inheritance, collation, and standardization of various empirical evaluation index systems. In this embodiment, referring to the Delphi method, professional colleagues are arranged to evaluate the appearance characteristics of the wine-processed medicinal materials.

[0089] 11.1 Selection of Experts

[0090] Select several professors, veteran pharmacists, and graduate students with experience in processing medicinal materials.

[0091] 11.2 Drafting the questionnaire

[0092] The questionnaire consists of four items, each with a maximum score of 5 points, for a total score of 20 points, as shown in Table 2.

[0093] Table 2. Scoring criteria for the appearance characteristics of roasted and processed *Gynostemma pentaphyllum*.

[0094]

[0095] 11.3 Sensory evaluation of Kejing medicinal materials

[0096] Raw and processed products were placed in the same space, and the expert group members conducted blind evaluation and centralized evaluation, and integrated the scores of each processed product.

[0097] 12. Determination of water-soluble extractives

[0098] The determination was performed according to the hot immersion method in Section 2201 of General Chapter IV of the 2020 edition of the Chinese Pharmacopoeia.

[0099] 13. Establishing Indicator Weights

[0100] 13.1 Weight Calculation Using the AHP Method

[0101] The decision-making objective of evaluating the processing technology of processed *Illicium verum* can be reflected by eight indicative components (secondary objectives). A hierarchical structure model is established, and a comprehensive evaluation is conducted based on the toxic components, efficacy components, and effects on the quality of the processed *Illicium verum*. The content of *Illicium verum* alkaloids, the common peak area of ​​characteristic peaks, water-soluble extracts, traditional experience evaluation, and ellagic acid content are used as weighted indicators for quantification. The five indicators are divided into four levels, and the priority order of each indicator is determined as follows: *Illicium verum* alkaloids content > traditional experience evaluation > common peak area of ​​characteristic peaks > water-soluble extracts > ellagic acid content. The traditional experience evaluation includes four indicators divided into four levels, with the priority order as follows: color > texture > odor > shape. A priority judgment matrix for comparison is constructed, and relative scores are assigned to each indicator, as shown in Tables 3 and 4.

[0102] Table 3 First Priority Judgment Matrix for Paired Comparison of Indicators

[0103]

[0104] Table 4. Second Priority Judgment Matrix for Paired Comparison of Traditional Empirical Evaluation Indicators

[0105]

[0106] Based on the judgment matrix scores, AHP hierarchical analysis was performed using SPSSAU (https: / / www.spssau.com) software. The weight coefficients for the content of star anise alkaloid, traditional experience evaluation, common peak area of ​​characteristic peaks, water-soluble extract, and ellagic acid were determined to be 0.4165, 0.2619, 0.1611, 0.0986, and 0.0624, respectively, with a consistency ratio (CR) of 0.067 < 0.10. The weight coefficients for shape, color, odor, and texture were 0.0920, 0.4124, 0.1735, and 0.3221, respectively, with a CR of 0.084 < 0.10. The consistency test was passed, and the weight coefficients were valid.

[0107] 13.2 Calculation of weights using the CRITIC method

[0108] First, the data were normalized and then CRITIC analysis was performed using SPSSAU software. The weighting coefficients for the content of quinquefoliine, ellagic acid, common peak area of ​​characteristic peaks, water-soluble extract, and traditional empirical evaluation were 0.1985, 0.1617, 0.1316, 0.3412, and 0.1671, respectively. The weighting coefficients for texture, color, odor, and shape were 0.2257, 0.2208, 0.3213, and 0.2322, respectively.

[0109] 13.3 Calculation of weights using the CRITIC-AHP composite weighting method

[0110] AHP is more subjective than CRITIC in calculating weighting coefficients, while CRITIC can more objectively evaluate the weighting coefficients of each indicator compared to AHP. Combining the two methods can more reasonably calculate the comprehensive weight. The weighting coefficients (ω) of the following indicators calculated by AHP-CRITIC are 0.3074, 0.0340, 0.0679, 0.0530, 0.0594, 0.1463, 0.2198, and 0.1120, respectively, for the content of star anise alkaloids, texture, color, odor, uniformity, common peak area of ​​characteristic peaks, water-soluble extract, and ellagic acid content.

[0111] The calculation formula is as follows:

[0112] ;

[0113] Where, ω AHP ω represents the weighting coefficients calculated by the AHP method. CRITIC This represents the weighting coefficients calculated using the CRITIC method;

[0114] The formula for calculating the overall score is as follows:

[0115] .

[0116] 14. CCD-RSM Preferred Kejing Wine Processing Technology

[0117] Take 200g of *Gynostemma pentaphyllum* slices, spray them with rice wine, let them soak for 24 hours, then put them into a temperature-controlled stir-frying machine (40r / min, 120 stirs / min) and stir-fry for the appropriate time. Remove and let them air dry.

[0118] The frying temperature (A), frying time (B), and amount of rice wine (C) were selected as the subjects of study. Using Design-Expert 8.0.6 statistical analysis software, and with the comprehensive score of the content of star aniseine, the common peak area of ​​characteristic peaks, the content of water-soluble extracts, traditional experience evaluation, and ellagic acid content as the response value, a star-point design-response surface methodology was conducted to optimize the extraction process parameters. The optimal extraction process parameters were selected. The specific experiments are as follows:

[0119] The experimental factors and levels of CCD-RSM are shown in Table 5, and the experimental design and results of CCD-RSM are shown in Table 6. Figures 4-9 .

[0120] Table 5. Experimental Factors and Levels of CCD-RSM

[0121]

[0122] Table 6. Experimental Design and Results of CCD-RSM (n=2)

[0123]

[0124] 15. Data Processing and Analysis

[0125] Using Design-Expert 8.0.6 software, with frying temperature (A), frying time (B), and amount of rice wine (C) as independent variables and experimental scores as dependent variables, the results of multiple linear regression and binomial fitting are as follows: Multiple linear regression equation: R1 = 64.62 - 1.25 × A - 1.6 × B - 4.71 × C (R1) 2 =0.3860, P=0.0454<0.05), binomial fitting equation: R2=58.72-1.25×A-1.60×B-4.71×C+0.48×AB+2.67×AC+1.10×BC+3.28×A 2 +2.49×B 2 +2.77×C 2 (R2) 2 =0.7848, P=0.0199<0.05).

[0126] R1 of the multiple regression fitting equation 2 =0.3860 and the binomial fitting equation R2 2 The quadratic fitting equation (0.784) has a higher correlation coefficient and better fitting effect compared to the binomial fitting equation, thus the optimal process parameters were chosen. The F-test results of the model are shown in Table 7.

[0127] Table 7. Results of ANOVA on the Processing Technology of Wine-Fried Kejing

[0128]

[0129] Analysis of variance showed that when the overall score was used as the response value, P < 0.05, indicating that the binomial equation model was significant. The lack-of-fit term, P = 0.3874, was not significant, indicating that the unknown factors had little interference with the experiment. The regression equation effectively reflected the relationship between the frying temperature, frying time, amount of rice wine, and the overall score. The order of influence of each factor on the overall score was: amount of rice wine (C) > frying time (B) > frying temperature (A). The contour lines and response surface plots of the quadratic regression equation were obtained using Design-Expert 8.0.6 software (see...). Figures 4-9 The model fit test was significant, indicating that the equation fits the actual situation well and can reflect the relationship between the comprehensive score of the evaluation index and A, B, and C, thus it is a successful model. The quadratic term A 2 C 2 The interaction terms have a significant impact on the overall score. The interaction terms are ordered from largest to smallest impact as follows: AC, BC, AB.

[0130] Based on the model fitting results, the optimal processing technology for roasted Kejing slices is predicted to be: a roasting temperature of 150℃, a roasting time of 16 min, and the amount of rice wine used being 10% of the Kejing slices' mass, with a weighting coefficient score of 104.78.

[0131] Meanwhile, the three weighting coefficients obtained above are used to comprehensively score the CCD-RSM results, as shown in Table 8.

[0132] Table 8. Comprehensive scoring results of the three weighting methods

[0133]

[0134] 16. Verification of processing methods

[0135] Based on the optimal conditions fitted by the software and considering actual operation, the processing method parameters were adjusted to the optimal processing technology for wine-roasted Kejing slices as follows: stir-frying temperature 150℃, stir-frying time 16min, and the amount of yellow wine used is 10% of the Kejing mass. The processing method was repeated 3 times under these conditions, and the experimental results are shown in Table 9.

[0136] Table 9. Star-shaped design process verification results

[0137]

[0138] According to the model, the theoretical predicted experimental score was 104.78, and the actual experimental score was 95.76 (RSD=1.05%). The deviation rate [deviation rate = (predicted value - measured value) / predicted value] was -8.61% (within ±20%), indicating that the model obtained by the star-shaped design effect surface methodology is accurate, reliable, and has good predictive ability. The process is stable, reasonable, and feasible. The results show that the raw Kejing (a type of medicinal herb) and the wine-processed Kejing slices (wine-processed product) are similar. Figure 10 As shown.

[0139] 17. Identification of physical characteristics

[0140] The processing method for processing *Clerodendrum trichotomum* (a type of medicinal herb) is described according to the "Identification Methods for Medicinal Materials and Processed Slices" in the *Chinese Pharmacopoeia*. Using the optimal processing method selected in this embodiment, *Clerodendrum trichotomum* was prepared at different locations and times, and its appearance is as follows. Figure 11 As shown. The results show that the wine-processed Kejing slices prepared using the optimal processing method selected in this embodiment are irregular thick slices or segments, with a yellowish-brown or yellowish-brown outer skin, fine longitudinal striations, and some with longitudinal cracks; the cut surface is grayish-brown or dark yellowish-brown, with slight scorch spots, and is powdery; the fibrous roots are slender, hard and brittle; it has a wine aroma and a bland taste.

[0141] 18. Microscopic identification

[0142] The powder of the wine-processed Kejing slices prepared using the optimal processing method selected in this embodiment is yellow or yellowish-brown. It contains numerous starch granules, each 4-5 μm in diameter, with compound granules consisting of 2-3 subgranules. The fibers are scattered individually, yellow, elongated spindle-shaped, with thick walls and distinct pits, measuring 100-250 μm in length. Calcium oxalate clusters are present, 10-50 μm in diameter. Stone cells are yellow, sub-round, sub-square, or sub-polygonal, 8-70 μm in diameter, with thick walls and fine pits. The vessels are mostly bordered pits, often broken, with regularly spaced pits; cork cells are occasionally seen, containing crystal sheath fibers. Figure 12 .

[0143] 19. Identification by Thin-Layer Chromatography

[0144] The wine-processed Kejing medicinal slices (numbered S1-S) were prepared using the optimal processing method obtained in this embodiment. 14 Take 1g of powder, add 10mL of anhydrous ethanol, sonicate for 60min, filter, and use as the test solution. Separately, take 1g of *Illicium verum* powder, add 10mL of anhydrous ethanol, sonicate for 60min, filter, and use as the reference solution. Perform the test according to the thin-layer chromatography method (General Rule 0502) of the 2020 Chinese Pharmacopoeia (Part IV). Apply 15μL of each of the above two solutions to the same silica gel G thin-layer plate. Use toluene-ethyl acetate-formic acid (volume ratio 4:1:0.5) as the developing solvent, develop to 8cm, remove, air dry, and observe under ultraviolet light (365nm). The fluorescent spots at corresponding positions in the chromatograms of the test solution and the reference solution are consistent in color. The results are as follows: Figure 13 As shown.

[0145] 20. Determination of moisture, ash and leachate

[0146] Moisture, ash, and extractives were determined according to the methods described in the Chinese Pharmacopoeia: "Moisture Determination (Drying Method)," "Ash Determination," and "Extractives Determination (Water-soluble Extractives, Hot Extraction Method)." The results showed that the moisture, total ash, acid-insoluble ash, and water-soluble extractives of 14 batches of wine-processed Kejing slices (processed using the optimal processing method selected in this example) were 5.11%–9.53%, 1.99%–12.05%, 0.01%–1.78%, and 7.34%–14.74%, respectively. The results are shown in Table 10.

[0147] The limits are set based on a 20% reduction from the average value and a 20% increase from the average value. The proposed limits are that the moisture content of Kejing and wine-processed Kejing slices shall not exceed 9.0%, the total ash content shall not exceed 6.00%, the acid-insoluble ash content shall not exceed 0.30%, and the water-soluble extract content shall not be less than 14.0%.

[0148] Table 10. Moisture, total ash, acid-insoluble ash, and water-soluble extract content in 14 batches of roasted koji (n=3)

[0149]

[0150] 21. Determination of Illicine Content

[0151] 21.1 Chromatographic conditions

[0152] ECOSIL C 18 The chromatographic column was 250 mm × 4.6 mm, 5 μm. The mobile phase was acetonitrile-buffered buffer (0.2 g sodium heptanesulfonate + 3.5 g potassium dihydrogen phosphate, diluted to 1000 mL). The flow rate was 9% acetonitrile-91% buffer, the column temperature was 35 °C, the detection wavelength was 254 nm, and the injection volume was 10 μL.

[0153] 21.2 Preparation of reference solution

[0154] Take an appropriate amount of cinnamon alkaloid, accurately weigh it, place it in a 25 mL volumetric flask, add 30% methanol containing 5% acetic acid to dissolve it, and dilute to the mark to prepare a reference solution containing 1.3812 mg / mL of cinnamon alkaloid.

[0155] 21.3 Preparation of the test solution

[0156] Take 1g of roasted sage powder (passed through a No. 4 sieve), weigh it accurately, place it in a stoppered conical flask, add 25mL of 30% methanol containing 5% acetic acid, weigh it, sonicate for 30min (power 200W, 40Hz), cool it, make up the weight, shake it well, and filter it through a 0.22μm microporous membrane to obtain the product.

[0157] 21.4 Examination of Linear Relationships

[0158] Six reference solutions were prepared with mass concentrations of 0.0575 mg / mL, 0.1151 mg / mL, 0.2302 mg / mL, 0.4604 mg / mL, 0.9208 mg / mL, and 1.3812 mg / mL, respectively. These solutions were injected and analyzed under the aforementioned chromatographic conditions, and the peak areas were recorded. Regression analysis was performed with the injection volume as the abscissa (X) and the peak area as the ordinate (Y). The regression equation for the alkaloid component was obtained as: Y = 669424X - 20846, with a correlation coefficient of R² = 0.9998, indicating a good linear relationship for alkaloids in the range of 0.0575 mg / mL to 1.3812 mg / mL.

[0159] 21.5 Precision Test

[0160] Accurately pipette 10 μL of the reference solution and inject it six times consecutively under the aforementioned chromatographic conditions. The RSD of the peak area of ​​Illicium verum alkaloid was measured to be 0.06%, indicating that the instrument has good precision.

[0161] 21.6 Stability Test

[0162] The same test solution (numbered S1) prepared according to the aforementioned method was precisely pipetted into the sample at 0, 4, 8, 12, 20 and 24 h. The RSD of the peak area of ​​Illicium verum alkaloid was calculated to be 2.72%, indicating that the test solution was stable within 24 h.

[0163] 21.7 Repeatability Test

[0164] Six test solutions were prepared by taking the powder of *Illicium verum* (passed through a No. 4 sieve, designated S1) according to the aforementioned preparation method. The average mass fraction of *Illicium verum* alkaloid was determined to be 5.70 mg / g under the aforementioned chromatographic conditions, with an RSD of 2.31%, indicating that the experiment had good repeatability.

[0165] 21.8 Recovery Test

[0166] Six portions of 1g each of the wine-processed *Illicium verum* powder (passed through a No. 4 sieve, designated S1) were accurately weighed. Appropriate amounts of reference standards were added to each portion, and test solutions were prepared according to the aforementioned preparation method. The average recovery rate of quinquefoliine in *Illicium verum* was determined to be 95.77% under the aforementioned chromatographic conditions, with an RSD of 1.34%.

[0167] 21.9 Determination of Sample Content

[0168] Fourteen batches of processed scallion greens were prepared into test solutions and analyzed under the aforementioned chromatographic conditions. The content of argan oil in the scallion greens was calculated, and the results showed that the content of argan oil in the scallion greens was 0.091%~0.753%.

[0169] Based on a 20% reduction from the average content of quinquefoliine, a limit standard was established, stipulating that the content of quinquefoliine in wine-processed Kejing slices should not be less than 0.350%, which meets the relevant requirements in the "Technical Requirements for Research and Formulation of Quality Standards for Traditional Chinese Medicine". The results are shown in Table 11.

[0170] Table 11. Content of Acerophora tinctoriae in 14 batches of wine-processed Kejing liquor (%, n=3)

[0171]

[0172] Example 3: Preliminary study on the detoxification and efficacy enhancement of Zhuang medicinal wine-processed Kejing slices before and after processing.

[0173] I. Safety Experiment

[0174] 1. Experimental Design

[0175] Following the methods specified in the "Technical Guidelines for Single-Dose Toxicity Studies of Drugs" and the "Technical Guidelines for Pharmacological Studies of Drug Safety," KM mice were used as the research subjects, and the drug was administered via gavage. Acute toxicity experiments were conducted to determine the median lethal dose (LD50) of the water extract of the processed *Clerodendrum trichotomum* (a type of medicinal herb) before and after processing. 50 The organ indices of the five internal organs and the whole brain were measured, and their pathological sections were observed. The hot plate analgesia, acetic acid writhing, xylene-induced ear swelling test were used to measure the levels of interleukin-6 (IL-6), interleukin-9 (IL-9), and tumor necrosis factor-α (TNF-α) inflammatory mediators in their serum.

[0176] The preparation method of the water extract of the wine-processed Kejing slices (No. S1) is as follows: take Kejing and wine-processed Kejing slices, place them in a clay pot, add 10 times the amount of water to soak for 30 minutes, keep boiling for 30 minutes, filter while hot, add 8 times the amount of water to keep boiling for 20 minutes, filter while hot, combine the two filtrates, concentrate under reduced pressure, and set aside.

[0177] The preparation method of the water extract of raw *Gynostemma pentaphyllum* is as follows: the same as that of wine-processed *Gynostemma pentaphyllum* slices;

[0178] KM mice treated with the aqueous extract of processed *Gynostemma pentaphyllum* were designated as the processed *Gynostemma pentaphyllum* group, and KM mice treated with the aqueous extract of raw *Gynostemma pentaphyllum* were designated as the raw *Gynostemma pentaphyllum* group. KM mice that did not undergo any treatment served as the control group.

[0179] 2. Results

[0180] 2.1 General Morphological Observation

[0181] In LD 50In the assay, mice in all groups exhibited muscle relaxation, prone position, reduced spontaneous activity, rapid but shallow breathing, ruffled fur, and cold skin after administration. Severe cases showed cyanosis (purple-blue discoloration of the tail, footpads, and mouth), and some even died. Most deaths occurred within 1 hour, with the carcasses exhibiting stiffness in the tail and purplish-black eyes. Bowel movements, urinary and reproductive systems, and eyes remained normal. Except for the 37g / kg raw *King Kong* group, the 75g / kg wine-processed *King Kong* group, and the control group, all other dosage groups experienced mortality. Mice that did not die recovered to normal after 2 days of administration. Mortality details are shown in Table 12.

[0182] 2.2. Preparation and processing of roasted and processed herbs (LD) 50 Comparison

[0183] Using SPSS 25 software, the number of deaths, total number, and dosage of mice in both the raw product group and the wine-processed group were imported together. The acute LD50 of the *King Kong* group and the wine-processed *King Kong* group was calculated using the "Analysis-Regression-Probit" method. 50 The values ​​were 96.7 g / kg and 141.9 g / kg, respectively, as shown in Table 12.

[0184] Table 12 Results of acute toxicity tests on aqueous extracts of *Gynostemma pentaphyllum* and *Gynostemma pentaphyllum* processed with alcohol (n=10)

[0185]

[0186] 2.3 Organ Index

[0187] The organ indices (organ index (g / g) = organ weight (g) / body weight (g)) of the heart, liver, spleen, lung, kidney and whole brain in the raw Kejing product group, wine-processed Kejing group and blank group were analyzed. There were no statistically significant differences in the organ indices of the heart, liver, spleen, lung, kidney and whole brain in the drug-treated groups compared with the blank group (P>0.05). The results are shown in Table 13.

[0188] Table 13 Acute toxicity organ index of *Kochia scoparia* before and after processing (x̅±s, n=10)

[0189]

[0190] 2.4. Histopathological effects of processing *Clerodendrum trichotomum* slices before and after processing on the five internal organs and whole brain of mice.

[0191] Compared with the control group, no significant pathological changes were observed in the five internal organs and the entire brain parenchyma of mice in the drug-treated group upon gross observation. LD 100 The raw and roasted succulent plants, along with the control group, were observed under a light microscope. The results are as follows: Figures 14-25In this experiment, mild degeneration of hepatocytes was observed in some liver tissues of the animals. This may be due to insufficient fasting during dissection, resulting in excessively high glycogen content in the liver. Since this was also observed in the control group, it is considered unrelated to the treatment (drug administration) in this experiment. No significant pathological changes related to drug administration were observed in the five internal organs and the whole brain of the mice in this experiment.

[0192] II. Verification of the synergistic effect of wine-processed Kejing medicinal slices

[0193] 1. Hot plate pain test and results

[0194] 1.1 Experimental Design

[0195] The hot and cold plate analgesia device was set to 55±1℃, and female mice with a pain response latency of 5-30 seconds were selected for the experiment. A total of 80 qualified female mice were selected and randomly divided into 8 groups. The average of two qualified pain response latencies was taken as the pre-drug administration pain response latency. The groups were: blank group (physiological saline), Tripterygium wilfordii polyglycoside tablet group (13.65mg / kg, converted to adult 1.5mg / kg·d), high-dose group (2.66g / kg), medium-dose group (1.33g / kg), and low-dose group (0.61g / kg), with 10 mice in each group. After the baseline pain threshold of the mice was determined, they were continuously administered the drug by gavage for 7 days, twice a day. The pain threshold of the mice was measured at 15min, 30min, 60min, 90min, and 120min after the last administration.

[0196] 1.2 Experimental Results

[0197] The experimental results are shown in Table 14. The results showed no significant difference in the baseline pain response latency among the groups (P>0.05), indicating that the pain response latency was similar in all groups before administration. After 7 consecutive days of gavage, there was no significant difference in pain response latency at 15 min and 30 min after the last administration (P>0.05). The high-dose raw product group, the low-dose wine-processed group, and the medium-dose wine-processed group showed significantly longer pain response latency at 60 min and 90 min after the last administration compared to before administration (P<0.05), while the high-dose wine-processed group showed a highly significant increase compared to before administration (P<0.01). The white group also showed a significant improvement (P<0.05); the Tripterygium wilfordii polyglycoside tablet group showed a highly significant improvement in the latency of pain response only 90 minutes after the last administration compared to before administration (P<0.01), and a significant improvement compared to the blank group (P<0.05); except for the high-dose alcohol-processed group, which still showed a highly significant improvement compared to before administration (P<0.01) and a significant improvement compared to the blank group (P<0.05) at 120 minutes, and even better than the Tripterygium wilfordii polyglycoside tablet group (P<0.05), the latency of pain response in the other groups returned to a level similar to that before administration after 120 minutes (P>0.05).

[0198] Table 14 Effects of *Clerodendrum chinense* wine-processing on hot plate pain in mice (x̅±s, n=10)

[0199]

[0200] Note: * This indicates a significant difference in pain threshold compared to the control group (P<0.05). ** The value indicated a highly significant difference in pain threshold compared to the control group (P<0.01); # indicated a significant difference in pain threshold compared to pre-drug administration (P<0.05); and ## indicated a highly significant difference in pain threshold compared to pre-drug administration (P<0.01). △ This indicates a significant difference in pain threshold compared to the Tripterygium wilfordii polyglycosides group (P<0.05). △△ This indicates a highly significant difference in pain threshold compared to the Tripterygium wilfordii polyglycosides group (P<0.01).

[0201] 2. Acetic acid writhing test and results

[0202] 2.1 Experimental Design

[0203] Animals were grouped (half male and half female) and the dosage was the same as in step "1.1". 30 minutes after the last administration, 0.1 mL of 0.7% acetic acid solution was injected intraperitoneally into each 10 g mouse. 5 minutes later, the number of writhing movements of the mice within 15 minutes (each instance was counted as an abdominal indentation and tightening) was observed and recorded. The formula for calculating the analgesic inhibition rate is as follows:

[0204] .

[0205] 2.2 Experimental Results

[0206] The experimental results are shown in Table 15. The results showed that after 7 consecutive days of gavage, all drug groups reduced the number of acetic acid writhing movements in mice compared with the blank group (P<0.01). The inhibitory effect of the drug groups on the number of writhing movements was more obvious with increasing dosage, and at the same dosage, the inhibition rate of the wine-processed group was slightly higher than that of the raw group. The inhibition rate of the high-dose wine-processed group was better than that of the Tripterygium wilfordii polyglycoside tablet group.

[0207] Table 5 Effects of *Clerodendrum chinense* on acetic acid-induced writhing in mice before and after wine processing (x̅±s, n=10)

[0208]

[0209] Note: * This indicates a significant difference in pain threshold compared to the control group (P<0.05). **The value indicated a highly significant difference in pain threshold compared to the control group (P<0.01); # indicated a significant difference in pain threshold compared to pre-drug administration (P<0.05); and ## indicated a highly significant difference in pain threshold compared to pre-drug administration (P<0.01). △ This indicates a significant difference in pain threshold values ​​compared to the Tripterygium wilfordii polyglycosides group (P<0.05). △△ This indicates a highly significant difference in pain threshold compared to the Tripterygium wilfordii polyglycosides group (P<0.01).

[0210] 3. Experiment and results of the xylene-induced ear swelling model

[0211] 3.1 Experimental Design

[0212] Animals were grouped (half male and half female) and the dosage was the same as in step "2.3". 0.1 mL of xylene (100%) was evenly applied to both the inner and outer surfaces of the right ear of mice. One hour later, the right ear and the untreated left ear were used as controls. Blood was collected from the eyeballs and tested. The blood was centrifuged on ice for two hours (4000 r / min, 15 min). The serum was carefully collected, aliquoted, and immediately placed in a -80℃ freezer for later use to assess the degree of edema stress response induced by xylene. After the mice bled to death, both ears were cut off along the base of the ear. Ear pieces were simultaneously removed from the same position on both ears using a 6 mm diameter punch, weighed, and the ear swelling degree and ear swelling inhibition rate were calculated sequentially using the following formula.

[0213] Ear swelling (mg) = weight of right earpiece (mg) - weight of left earpiece (mg);

[0214] .

[0215] 3.2 Experimental Results

[0216] The experimental results are shown in Table 16. The results showed that after 7 consecutive days of gavage, the degree of xylene-induced ear swelling in each treatment group was reduced to varying degrees compared with the control group (P < 0.01). The ear swelling inhibition rate of the treatment groups decreased with increasing dosage. Moreover, the ear swelling inhibition rate of the high-dose raw product group and the high-dose wine-processed product group was better than that of the Tripterygium wilfordii polyglycoside tablet group, and the wine-processed product group was better than that of the raw product group.

[0217] Table 16 Effects of *Symplocos edulis* on xylene-induced ear swelling in a mouse model (x̅±s, n=10)

[0218]

[0219] Note: * This indicates a significant difference in ear swelling compared to the control group (P<0.05). ** This indicates a highly significant difference in ear swelling compared to the control group (P<0.01).△ This indicates a significant difference in ear swelling compared to the Tripterygium wilfordii polyglycosides group (P<0.05). △△ This indicates a highly significant difference in ear swelling compared to the Tripterygium wilfordii polyglycosides group (P<0.01).

[0220] 4. Measurement and results of inflammatory mediators IL-6, TNF-α, and IL-9 levels.

[0221] After 7 consecutive days of gavage, the IL-6 levels in all treatment groups showed a highly significant decrease compared to the control group (P<0.01). Except for the low-dose group, the inhibition rate of IL-6 levels in the treatment groups was significantly lower than that in the positive control group. The high-dose group showed a significant difference in IL-6 level decrease compared to the positive control group (P<0.05), with the highest IL-6 level observed in the high-dose group treated with wine-processed products. Except for the low-dose group treated with wine-processed products, the TNF-α levels in the treatment groups showed a highly significant decrease compared to the control group (P<0.01). The dosage was inversely proportional to TNF-α levels. Except for the low-dose group, the plasma TNF-α levels in mice were comparable between the raw product group and the wine-processed group at the same dosage. The dosage was inversely proportional to IL-9 levels, and there was no significant difference in IL-9 levels among the treatment groups compared to the control group (P>0.05). The results are shown in Table 17.

[0222] Table 17 Comparison of IL-6 levels before and after alcohol-processed herb treatment (x̅±s, n=10)

[0223]

[0224] Note: * This indicates a significant difference in factor levels compared to the control group (P<0.05). ** This indicates that the factor levels were significantly different from the control group (P<0.01). △ This indicates a significant difference in factor levels compared to the Tripterygium wilfordii polyglycosides group (P<0.05). △△ This indicates that the factor levels were significantly different compared with the Tripterygium wilfordii polyglycosides group (P<0.01).

[0225] In summary, the LD50 of the water extract of Kejing slices before and after wine processing is... 50The effective values ​​were 96.75±0.97 g / kg and 141.94±1.50 g / kg, respectively, with 95% confidence intervals of 78.29-123.02 g / kg and 123.67-165.88 g / kg. There were no significant differences in the five internal organs, whole brain index, and pathological sections. In hot plate analgesia, acetic acid-induced writhing, and xylene-induced ear swelling tests, the wine-processed group showed improved anti-inflammatory and analgesic effects compared to the raw group. Regarding inflammatory factors, except for IL-9 levels, the levels of IL-6 and TNF-α showed a significant decreasing trend in the wine-processed group compared to the raw group. Therefore, the wine-processing of *Clerodendrum trichotomum* slices reduces the toxicity of the water extract to a certain extent while largely retaining its anti-inflammatory and analgesic effects.

[0226] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for processing *Gynostemma pentaphyllum* to reduce toxicity and enhance efficacy, characterized in that: The process includes mixing sage and rice wine, followed by soaking, stir-frying, and drying to obtain wine-roasted sage slices. The amount of yellow wine used is 10% of the weight of the stalks. The stir-frying time is 16 minutes and the temperature is 150℃.

2. The processing method according to claim 1, characterized in that, The soaking time is 24 hours.

3. The processing method according to claim 1, characterized in that, The stir-frying speed is 40 r / min, and the stirring frequency is 120 times / min.

4. Wine-processed Kejing slices prepared using the processing method according to any one of claims 1-3.

5. The quality control method for the wine-processed *Gynostemma pentaphyllum* slices according to claim 4, characterized in that, include: (1) Identify the physical characteristics of the wine-processed Kejing slices; (2) Perform powder microscopic identification on the wine-processed Kejing medicinal slices; (3) Thin-layer chromatography was used to identify the roasted and processed Kejing medicinal slices; (4) The moisture, ash, acid-insoluble ash and extract content of the roasted sage slices were determined; the moisture content of the roasted sage slices shall not exceed 9.0%, the total ash content shall not exceed 6.00%, the acid-insoluble ash content shall not exceed 0.30%, and the extract content shall not be less than 14.0%; (5) The content of star anise alkaloid in the wine-processed Kejing slices was determined by high performance liquid chromatography; the content of star anise alkaloid in the wine-processed Kejing slices shall not be less than 0.350%.

6. The quality control method according to claim 5, characterized in that, The thin-layer chromatography identification method includes the steps of spotting the test solution and the reference solution onto a silica gel G thin-layer plate, using a mixed solution of toluene, ethyl acetate and formic acid as the developing solvent, and observing under a 365 nm ultraviolet lamp; The method for preparing the test solution includes the steps of mixing the sample powder to be tested and ethanol at a ratio of 1g:10mL, and ultrasonically treating for 60min to obtain the test solution. The method for preparing the reference solution includes mixing Illicium verum reference standard and ethanol at a ratio of 1g:10mL, and ultrasonically treating for 60min to obtain the reference solution.

7. The quality control method according to claim 6, characterized in that, The volume ratio of toluene, ethyl acetate, and formic acid in the mixed solution is 4:1:0.

5.

8. The quality control method according to claim 5, characterized in that, The high-performance liquid chromatography detection includes the steps of injecting the reference solution and the test solution into the liquid chromatograph for chromatographic detection. The preparation method of the test solution includes mixing the sample powder to be tested and an ethanol solution containing acetic acid at a ratio of 1g:25mL, and ultrasonically treating for 30min to obtain the test solution. The method for preparing the reference solution includes the step of mixing Illicium verum reference standard and an ethanol solution containing acetic acid to obtain the reference solution; the concentration of Illicium verum in the reference solution is 1.3812 mg / mL.

9. The quality control method according to claim 8, characterized in that, The chromatographic column used for the chromatographic detection was ECOSIL C10. 18 Chromatographic column; with acetonitrile as mobile phase A and buffer solution as mobile phase B; The buffer solution comprises sodium heptanesulfonate and potassium dihydrogen phosphate; the concentration of sodium heptanesulfonate in the buffer solution is 0.2 g / L and the concentration of potassium dihydrogen phosphate is 3.5 g / L.

10. The quality control method according to claim 8, characterized in that, The chromatographic detection conditions were as follows: column temperature 25℃, flow rate 1.0 mL / min, injection volume 10 μL, and detection wavelength 254 nm.

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