Processing method of four kinds of rhizoma coptidis pieces, and fingerprint spectrum construction method and application thereof

Abstract

This invention discloses a method for processing four-stage Coptis chinensis slices, its fingerprint spectrum, and its applications. The processing method includes two steps: preparation of excipients and processing. First, ginger juice, Evodia rutaecarpa juice, and licorice juice are prepared and mixed with rice wine in equal proportions to form a mixed medicinal juice. Then, the cleaned Coptis chinensis slices are mixed with the mixed medicinal juice and absorbed completely. After fermentation at 5℃-15℃ for 5-6 days, steaming for 1-2 hours, and drying at 70℃, the final product is obtained. This method is stable and feasible, and can significantly increase the content of alkaloid active ingredients in the slices. It also provides a method for establishing a fingerprint spectrum of the four-stage Coptis chinensis slices prepared by the above processing method. The fingerprint spectrum has been verified by precision, stability, and repeatability tests, showing high reliability. It can be used for quality evaluation of four-stage Coptis chinensis slices and for identification with Coptis chinensis slices processed using other methods. This invention constructs an integrated "processing-quality control" system, providing a scientific basis for standardized production, quality supervision, and standardized clinical application.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine processing and component analysis technology, specifically to a method for processing four-stage Coptis chinensis slices, its fingerprint spectrum construction method, and its application. Background Technology

[0002] Coptis chinensis is the dried rhizome of *Coptis chinensis*, *Coptis triangularis*, or *Coptis yunnanensis*, all belonging to the Ranunculaceae family. It is bitter and cold in nature, and enters the heart, spleen, stomach, liver, gallbladder, and large intestine meridians. It has the effects of clearing heat and drying dampness, purging fire and detoxifying. Clinically, it is widely used for damp-heat stagnation, vomiting and acid regurgitation, diarrhea, jaundice, high fever with delirium, excessive heart fire, red eyes, toothache, and diabetes. Modern pharmacological studies have shown that the main active components of Coptis chinensis are alkaloids such as palmatine, epiberberine, berberine hydrochloride, berberine root alkaloids, and coptisine, which have various pharmacological effects including vascular protection, hypoglycemia, anti-inflammation, anti-tumor, antimicrobial, and anti-anxiety.

[0003] "Using raw and processed herbs in medicine" is one of the core characteristics of traditional Chinese medicine. Coptis chinensis, after being processed with different excipients, undergoes targeted changes in its medicinal properties and efficacy to suit different clinical needs: wine-processed Coptis chinensis can mitigate its cold nature and guide the medicine upwards; ginger-processed Coptis chinensis can enhance its stomach-soothing and antiemetic effects; Evodia rutaecarpa-processed Coptis chinensis can soothe the liver and stomach; and licorice-processed Coptis chinensis can harmonize its medicinal properties. Four-processed Coptis chinensis, as a distinctive processed variety of traditional Chinese medicine, is fermented and processed with four excipients: ginger, rice wine, Evodia rutaecarpa, and licorice. This process retains the core efficacy of Coptis chinensis in clearing heat and purging fire, while also harmonizing its bitter and cold nature through the principle of "pungent opening and bitter descending," giving it unique advantages in the clinical adjuvant treatment of diseases such as diabetes.

[0004] However, the current processing techniques for four-stage Coptis chinensis are mostly used in folk medicine, lacking systematic and standardized research and formulation. Key process parameters such as fermentation time, fermentation temperature, and steaming time lack unified standards, and the process parameters vary significantly among different manufacturers. This results in inconsistent quality of the processed Coptis chinensis slices, with significant fluctuations in the content of alkaloid active ingredients, seriously affecting the efficacy and safety of clinical use. Furthermore, quality control methods for four-stage Coptis chinensis slices are relatively simplistic, often relying solely on the determination of single component content, which cannot comprehensively reflect the overall quality of the slices. Existing technologies have not yet established a specific fingerprint spectrum for four-stage Coptis chinensis slices, making it difficult to achieve a comprehensive evaluation of its quality and effectively distinguish it from Coptis chinensis slices processed using other methods. In addition, research on the correlation between the processing techniques of four-stage Coptis chinensis slices and their hypoglycemic activity is insufficient, and there is a lack of quality control methods to assist in evaluating its hypoglycemic activity.

[0005] Therefore, it is urgent to develop a stable and parameter-defined processing method for four-stage Coptis chinensis slices, establish a fingerprint spectrum that can comprehensively reflect its quality, and clarify the application scenarios of this fingerprint spectrum. This will provide a scientific basis for the standardized production, quality control, authenticity identification, and auxiliary evaluation of pharmacological activity of four-stage Coptis chinensis slices, and promote the industrialization and standardized clinical application of four-stage Coptis chinensis slices. Summary of the Invention

[0006] In view of the above-mentioned shortcomings of the existing technology, the purpose of this invention is to provide a method for processing four-processed Coptis chinensis slices, a method for constructing its fingerprint spectrum, and its application. This solves the problems of insufficient research on the correlation between the processing technology of four-processed Coptis chinensis slices and its hypoglycemic activity, and the lack of quality control methods to assist in evaluating its hypoglycemic activity in the existing technology.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for processing Coptis chinensis slices using four different methods includes the following steps:

[0009] S1. Preparation of excipients: Prepare ginger juice, evodia juice, and licorice juice, and mix them with rice wine in equal proportions to form a mixed medicinal juice for later use;

[0010] S2. Take clean Coptis chinensis slices, add mixed medicinal juice, stir until the medicinal juice is absorbed, and ferment in a stability test chamber for 5-6 days at a temperature of 5℃-15℃. After taking out the Coptis chinensis slices, steam them in a water bath for 1-2 hours. After steaming, dry them to obtain the four-processed Coptis chinensis slices.

[0011] Preferably, the fermentation time is 5 days, the fermentation temperature is 14℃, the steaming is done for 2 hours, and the drying temperature is 70℃.

[0012] This invention also provides a method for constructing a fingerprint spectrum of Coptis chinensis slices, comprising the following steps:

[0013] S1. Preparation of test solution: Take the Coptis chinensis slices prepared by the processing method described in claim 1 as the sample, and use methanol-hydrochloric acid mixed solution as the extraction solvent to prepare the test solution.

[0014] S2. Preparation of reference solution: Select a reference standard and prepare a reference solution using methanol as the solvent.

[0015] S3. Chromatographic determination: The test solution and the reference solution are injected into a high-performance liquid chromatograph and measured under preset chromatographic conditions to obtain chromatograms of the test solution and the reference solution, respectively.

[0016] S4. Fingerprint chromatogram construction: The obtained chromatogram is imported into the preset Chinese medicine chromatographic fingerprint chromatogram similarity evaluation system. Through multi-point correction and Mark peak matching, a fingerprint chromatogram containing 9 common peaks is obtained, that is, the fingerprint chromatogram of the four-processed Coptis chinensis slices is constructed.

[0017] Preferably, the reference standard is berberine hydrochloride, palmatine, berberine, epiberberine, or cypermethrin, and the reference standard solution contains 416 μg / mL berberine hydrochloride, 314 μg / mL palmatine, 213 μg / mL berberine, 121 μg / mL epiberberine, and 120 μg / mL cypermethrin.

[0018] Preferably, the preset chromatographic conditions of the high performance liquid chromatograph include: a Kromasil C18 column with dimensions of 250 mm × 4.6 mm and 5 μm; mobile phase A being an aqueous solution of ammonium bicarbonate containing ammonia and triethylamine; mobile phase B being acetonitrile; gradient elution; detection wavelength of 270 nm; column temperature of 30 °C; and flow rate of 1 mL / min.

[0019] Preferably, the mobile phase A is a 30 mmol / L ammonium bicarbonate aqueous solution containing 0.7% ammonia and 0.1% triethylamine; the gradient elution conditions are: 0-15 min, mobile phase B accounts for 10%-25%; 15-23 min, mobile phase B accounts for 25%-30%; 23-35 min, mobile phase B accounts for 30%-40%; 35-40 min, mobile phase B accounts for 40%.

[0020] Preferably, in step S4, among the nine common peaks, peak 5 is berberine, peak 6 is epiberberine, peak 7 is berberine, peak 8 is palmatine, and peak 9 is berberine hydrochloride, with peak 9 corresponding to berberine hydrochloride as the reference peak.

[0021] Preferably, the preparation steps of the test solution in step S1 are as follows: Take 0.2g of four-processed Coptis chinensis powder, accurately weigh it, place it in a 50mL stoppered conical flask, add 50mL of methanol-hydrochloric acid mixed solution with a volume ratio of 100:1, weigh it again, extract it by ultrasonication for 30min, weigh it after cooling and make up for the weight loss, filter it through a 0.45μm filter membrane to obtain the test solution;

[0022] The preparation step of the reference solution in step S2 is as follows: Dissolve the reference standard in methanol, make up to a certain volume, and mix to prepare a reference solution containing 416 μg / mL berberine hydrochloride, 314 μg / mL palmatine, 213 μg / mL berberine, 121 μg / mL epiberberine, and 120 μg / mL cypermethrin.

[0023] The present invention also provides an application of the fingerprint spectrum of four-processed Coptis chinensis slices, which is used for the quality evaluation of four-processed Coptis chinensis slices, the identification of Coptis chinensis slices with different processing techniques, and the auxiliary evaluation of the hypoglycemic activity of four-processed Coptis chinensis slices.

[0024] Preferably, the four-processed Coptis chinensis slices to be tested are subjected to similarity analysis. If the similarity is ≥0.9, the quality uniformity of the four-processed Coptis chinensis slices to be tested is determined to be in line with the requirements.

[0025] If the fingerprint spectrum of the Coptis chinensis slices to be identified shows the nine common peaks and the similarity is ≥0.9, it is determined to be the four-processed Coptis chinensis slices prepared by the processing method described in claim 1. If the corresponding nine common peaks are not shown or any peak is missing, it is determined to be raw Coptis chinensis slices, wine-processed Coptis chinensis slices, ginger-processed Coptis chinensis slices or Coptis chinensis slices processed with a single auxiliary ingredient.

[0026] The higher the similarity of the four-processed Coptis chinensis slices to be tested, the stronger their hypoglycemic activity.

[0027] The four-processed Coptis chinensis slices prepared by this invention are used to lower blood sugar and treat diabetes.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. The processing method of the four-processed Coptis chinensis slices provided by this invention clarifies the concentration standard of excipient preparation, the material-liquid ratio of clean Coptis chinensis slices to mixed medicinal juice, the key process parameter range of fermentation and steaming, and optimizes the optimal process parameters. The process is stable, feasible, and has good repeatability. The prepared four-processed Coptis chinensis slices can effectively retain and improve the content of alkaloid active ingredients such as palmatine, epiberberine, and berberine hydrochloride. Compared with traditional folk processes, the total alkaloid content is increased by an average of more than 24%, and the quality of the slices is uniform. This provides a unified process basis for the standardized production of four-processed Coptis chinensis slices.

[0030] 2. The fingerprint spectrum of the four-processed Coptis chinensis slices constructed in this invention uses characteristic alkaloids as reference standards, identifies 9 common peaks and completes the identification of characteristic peaks, which can comprehensively and holistically reflect the overall quality of the four-processed Coptis chinensis slices and make up for the shortcomings of single component content determination; the method of establishing the fingerprint spectrum is simple to operate, has high precision, good stability and good repeatability, and is suitable for widespread application.

[0031] 3. This invention clarifies multiple application scenarios of the fingerprint spectrum of four-processed Coptis chinensis slices. It can not only realize the overall quality evaluation of four-processed Coptis chinensis slices, but also effectively distinguish four-processed Coptis chinensis slices from raw Coptis chinensis, Coptis chinensis processed with other single-herb excipients, etc. At the same time, it can assist in evaluating the hypoglycemic activity of four-processed Coptis chinensis slices, providing new technical means for the quality supervision, authenticity identification and pharmacological activity research of four-processed Coptis chinensis slices.

[0032] 4. The processing method and the fingerprint spectrum establishment method of the present invention complement each other. The processing method provides a standardized sample basis for the fingerprint spectrum, and the fingerprint spectrum provides a scientific basis for the effectiveness verification and process optimization of the processing technology. This forms a complete technical system of "processing-quality control-application" for the four-processed Coptis chinensis slices, which promotes the industrialization of the four-processed Coptis chinensis slices and the standardized clinical use of the medicine. Attached Figure Description

[0033] Figure 1 This is a graph showing the changes in fermentation time and OD value in this invention;

[0034] Figure 2 This is a graph showing the changes in temperature and OD value during fermentation in this invention;

[0035] Figure 3 This is a graph showing the change in steaming time and OD value in this invention;

[0036] Figure 4 This is the interaction response surface diagram of various factors in the four-process Coptis chinensis processing method of the present invention;

[0037] Figure 5 This is a contour map showing the interaction of various factors in the four-process Coptis chinensis preparation method of this invention;

[0038] Figure 6 These are Coptis chinensis slices that were not processed using the method of this invention;

[0039] Figure 7 It is a four-processed Coptis chinensis slice made using the processing method of this invention;

[0040] Figure 8 These are the fingerprint spectra of 10 batches of four-processed Coptis chinensis samples in this invention;

[0041] Figure 9 This is the chromatogram of the control sample solution in this invention;

[0042] Figure 10 This is the chromatogram of the four-processed Coptis chinensis sample solution in this invention;

[0043] Figure 11 This is a graph showing the relationship between the inhibitory effects of four-processed Coptis chinensis, Coptis chinensis slices, and acarbose on α-glucosidase activity;

[0044] Figure 12 This is a graph showing the relationship between the inhibitory effects of four-processed Coptis chinensis, Coptis chinensis slices, and acarbose on α-amylase activity. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] The instruments, equipment, reagents, and materials used in the embodiments of this invention are all commercially available conventional products, as detailed below:

[0047] 1. Instruments and Equipment: Waterse2695 high-performance liquid chromatograph (with UV detector); ATX224 0.0001 g electronic analytical balance; ME55 0.0001 g analytical balance; KQ-5200DE ultrasonic cleaner; DHG-9053A electric thermostatic drying oven; GCZX-025 drug stability test chamber; SpectraMaxiD3 spectrophotometer; HH-6 electric thermostatic water bath; 0.45 μm organic filter membrane; No. 2 drug sieve; DesignExpert13 data analysis software.

[0048] 2. Reagents: Methanol, hydrochloric acid, acetonitrile, ammonium bicarbonate, ammonia, and triethylamine were all of chromatographic grade; water was ultrapure water prepared by Milli-Q; α-glucosidase, α-amylase, p-nitrophenyl-α-D-glucopyranoside (PNPG), and acarbose; other reagents were of analytical grade.

[0049] 3. Materials: Berberine hydrochloride, palmatine, coptisine, epiberberine, and cypermethrin reference standards (all batch numbers 20240614, mass fraction ≥98%); Coptis chinensis slices (origin: Shizhu, Chongqing, batch number: C240403, dried rhizome of Coptis chinensis plant, Ranunculaceae family, cleaned and sliced); ginger, Evodia rutaecarpa (origin: Zhangshu, Jiangxi, batch number: 240701), and licorice (origin: Ordos, Inner Mongolia, batch number: 240901) were all commercially available qualified Chinese medicinal materials; rice wine (manufacturer: Chengdu Julong Biotechnology Co., Ltd., batch number: 240901, alcohol content ≥10%vol) was commercially available edible grade rice wine.

[0050] First aspect: Single-factor experiments and response surface optimization of the processing methods of four-stage Coptis chinensis slices.

[0051] Using the total normalized value (OD) of the contents of five alkaloids—berberine, epiberberine, berberine hydrochloride, berberine, and coptisine—as the evaluation index, the effects of fermentation time, fermentation temperature, and steaming time on the quality of processed Coptis chinensis slices were investigated.

[0052] 1.1 Calculation of the normalized value of the overall score

[0053] The content of five alkaloids was normalized using the Hassan method; a larger response value indicates a better result. The formula for calculating the normalization value is: d i =(Y i -Y min ) / (Y max -Y min );

[0054] Among them, Y i This represents the i-th observation in the original data;

[0055] Y max The highest response value;

[0056] Y min This is the lowest response value.

[0057] Then, the geometric mean of the normalized values ​​is calculated to obtain the overall normalized value (OD).

[0058] 1.2 Single-factor experiment

[0059] 1.2.1 Investigation of fermentation time: 1000g of Coptis chinensis slices were added to 100mL of mixed medicinal juice and fermented at 15℃ for 5, 7, 9, 11, and 13 days, followed by steaming for 3 hours. The OD value was measured, and the results are shown below. Figure 1 As fermentation time increases, the OD value first rises and then falls, with 5-6 days being the optimal fermentation time.

[0060] 1.2.2 Investigation of fermentation temperature: 1000g of Coptis chinensis slices were added to 100mL of mixed herbal decoction and fermented at 5, 10, 15, 20, and 25℃ for 6 days, followed by steaming for 3 hours. The OD value was measured, and the results are shown below. Figure 2 As the fermentation temperature increased, the OD value first decreased and then increased, reaching its maximum at 15℃. The sample fermented at 25℃ began to mold on the fourth day of fermentation, so the alkaloid content of this group of samples was not measured.

[0061] 1.2.3 Investigation of steaming time: 1000g of Coptis chinensis slices were added to 100mL of mixed medicinal juice and fermented at 15℃ for 6 days. The mixture was steamed for 1, 2, 3, 4, and 5 hours respectively, and the OD values ​​were measured. The results are shown below. Figure 3 As the steaming time increases, the OD value first rises and then falls, with 1 to 2 hours being the optimal steaming time.

[0062] 1.3 Response Surface Experiment

[0063] 1.3.1 Experimental Design and Results: Based on the single-factor results, fermentation time (A, 5-9 days), fermentation temperature (B, 5-15℃), and steaming time (C, 1-3h) were selected as independent variables, with OD value as the response value. A three-factor, three-level BBD (Box-Behnken Design with Central Composite) design was adopted. The experimental factors and levels are shown in Table 1, and the BBD experimental design and results are shown in Table 2.

[0064] Table 1. Response surface methodology factors and levels for the four-stage processing of Coptis chinensis.

[0065]

[0066] Table 2 BBD Experimental Design and Results

[0067]

[0068] 1.3.2 Response Surface Model Fitting and Analysis of Variance

[0069] Seventeen groups of experiments were conducted based on the above factors and levels. According to the experimental design results, DesignExpert 13 software was used to perform multivariate regression fitting on the experimental data, yielding the equation: OD = 0.198 - 0.1875A + 0.05B ​​- ​​0.035C + 0.0375AB - 0.1825AC + 0.0575BC + 0.34475A. 2 +0.27475B 2 -0.03025C 2 Analysis of variance was performed on the regression model, and the results are shown in Table 3.

[0070] A in the model 2 B 2 The p-values ​​were all less than 0.05, indicating statistical significance. Comparing the F-values ​​of factors A (fermentation days), B (fermentation temperature), and C (steaming time), we can see that A > B > C. Table 3 shows the results of the ANOVA: model p = 0.0004 < 0.01, indicating statistical significance. The lack-of-fit term p = 0.1271 > 0.05, indicating that the constructed model has a small error and good consistency with reality. Therefore, the model established in this study has a good fit. The F-values ​​show that the influence of each factor on the OD value is: fermentation time (A) > fermentation temperature (B) > steaming time (C).

[0071] Table 3 Results of ANOVA for the Regression Model

[0072]

[0073] 1.3.3 Response Surface Analysis and Process Prediction

[0074] Based on the model regression equation, a three-dimensional spatial surface and contour map were constructed, representing the interaction of multiple factors such as fermentation days, fermentation temperature, and steaming time. A steeper response surface curve indicates a greater influence of the interaction between the two factors on the response value, and vice versa; elliptical contour lines indicate a significant interaction effect between the two factors. Figure 4 and Figure 5 As shown, the interaction between fermentation time and steaming time, and between fermentation temperature and steaming time, is strong and significantly affects the content of the investigated indicators. Analysis using DesignExpert13 software revealed the optimal processing conditions for the four-stage Coptis chinensis preparation to be: fermentation time 5.29 days, fermentation temperature 13.88℃, steaming time 1.82 h, and theoretical OD value 1.0543.

[0075] 1.4 Process Validation Test

[0076] To facilitate experimental operation, the optimal processing technique for four-stage Coptis chinensis was adjusted. The optimal process parameters were adjusted to: 5 days of fermentation, 14℃ fermentation temperature, 2 hours of steaming, 70℃ oven drying, and powdering followed by sieving through a No. 2 sieve. The contents of five alkaloids in the four-stage Coptis chinensis slices and raw Coptis chinensis slices prepared by the optimal process were determined, and the results are shown in Table 4.

[0077] Table 4. Content of five alkaloid components in Coptis chinensis before and after four processing steps (mg / g)

[0078]

[0079] Three experiments verified that the results were consistent with expectations, indicating that the processing technique is reasonable and stable. After four processing steps, the content of five alkaloids in Coptis chinensis slices increased by an average of 24.8%, which can significantly improve the intrinsic quality of Coptis chinensis slices.

[0080] Secondly, this invention provides a method for processing four types of Coptis chinensis slices.

[0081] Includes the following steps:

[0082] S1. Preparation of excipients: Wash and juice fresh ginger, adjust the concentration to a mass-to-volume ratio of ginger to ginger juice of 1:1 to obtain ginger juice; decoct Evodia rutaecarpa twice with water, each time adding 10 times the mass of Evodia rutaecarpa, decoct for 1 hour each time, combine the two decoctions and concentrate to a mass-to-volume ratio of Evodia rutaecarpa to medicinal juice of 1:1 to obtain Evodia rutaecarpa juice; decoct licorice root twice with water, each time adding 10 times the mass of licorice root, decoct for 1 hour each time, combine the two decoctions and concentrate to a mass-to-volume ratio of licorice root to medicinal juice of 1:1 to obtain licorice root juice; mix the ginger juice, Evodia rutaecarpa juice, licorice root juice and rice wine in equal volume proportions, stir evenly to obtain mixed medicinal juice for later use;

[0083] S2. Take clean Coptis chinensis slices, add 100mL of mixed medicinal juice, stir until the medicinal juice is absorbed, and ferment in a stability test chamber for 5-6 days at a temperature of 5℃-15℃. After taking out the Coptis chinensis slices, steam them in a water bath for 1-2 hours. After steaming, place the Coptis chinensis slices in a drying equipment and dry them at 70℃ to obtain the four-processed Coptis chinensis slices.

[0084] Please see Figure 6 and Figure 7 Based on the above-mentioned optimized design and verification of process parameters, the preferred fermentation process parameters are: fermentation time of 5 days and fermentation temperature of 14℃; steaming time of 2 hours; and drying temperature of 70℃. These optimal process parameters can maximize the retention and enhancement of the content of alkaloid active ingredients in the four-processed Coptis chinensis slices, resulting in the best uniformity of slice quality.

[0085] The third aspect: Construction of fingerprint spectrum of Coptis chinensis slices processed in four ways

[0086] Using the above-mentioned four-processed Coptis chinensis slices as samples, fingerprint chromatograms were established according to the following steps:

[0087] S1. Preparation of the test solution

[0088] Take the above-mentioned four-processed Coptis chinensis slices, pulverize and sieve them. Take 0.20g of the powder, accurately weigh it, and place it in a 50mL stoppered conical flask. Accurately add 50mL of a methanol-hydrochloric acid mixed solution with a volume ratio of 100:1, weigh it, and place it in a KQ-5200DE ultrasonic cleaner. Extract it by ultrasonication at 250W and 40kHz for 30min. Cool it to room temperature, make up the weight loss with methanol, shake it well, filter it through a 0.45μm organic filter membrane, and take the filtrate to obtain the test solution.

[0089] S2. Preparation of reference solution

[0090] Accurately weigh 41.6 mg of berberine hydrochloride, 31.4 mg of palmatine, 21.3 mg of berberine, 12.1 mg of epiberberine, and 12.0 mg of cypermethrin, place them in a 100 mL volumetric flask, dissolve them in methanol and dilute to the mark, shake well to obtain a mixed reference solution (berberine hydrochloride 416 μg / mL, palmatine 314 μg / mL, berberine 213 μg / mL, epiberberine 121 μg / mL, cypermethrin 120 μg / mL), and store at 4 °C.

[0091] S3, Chromatographic determination

[0092] Accurately pipette 10 μL each of the test solution and the mixed reference solution into a Waterse2695 high-performance liquid chromatograph, and determine the chromatographic conditions as follows:

[0093] Chromatographic column: Kromasil C18 column (250mm × 4.6mm, 5μm);

[0094] Mobile phase: Mobile phase A is a 30 mmol / L ammonium bicarbonate aqueous solution (containing 0.7% ammonia and 0.1% triethylamine), and mobile phase B is acetonitrile;

[0095] Gradient elution: 0-15 min, mobile phase B accounts for 10%-25%; 15-23 min, mobile phase B accounts for 25%-30%; 23-35 min, mobile phase B accounts for 30%-40%; 35-40 min, mobile phase B accounts for 40%.

[0096] Detection wavelength: 270 nm; Column temperature: 30℃; Flow rate: 1.0 mL / min; Injection volume: 10 μL;

[0097] Record the chromatograms of the test sample and the reference sample separately.

[0098] S4, Fingerprint Map Construction

[0099] Ten batches of processed Coptis chinensis slices prepared using the above-described processing method were taken. Test solutions were prepared and analyzed according to steps S1-S3, yielding chromatograms for the ten batches of samples. The chromatogram data were imported into the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 version). A time window width of 0.1 was set. After multi-point correction and Mark peak matching, a fingerprint chromatogram containing nine common peaks was obtained, which is the fingerprint chromatogram of the processed Coptis chinensis slices of this invention. Please refer to [link to relevant documentation]. Figure 8 Please refer to the chromatogram comparison with the reference standard. Figure 9 Peak 5 was identified as berberine, peak 6 as epiberberine, peak 7 as berberine, peak 8 as palmatine, and peak 9 as berberine hydrochloride, with peak 9 (berberine hydrochloride) as the reference peak.

[0100] Fourthly: Verification of the constructed fingerprint spectrum of the four-processed Coptis chinensis slices.

[0101] Using the prepared Coptis chinensis slices obtained by the above processing method as samples, test solutions and reference solutions were prepared according to the above method. Precision, stability, and repeatability tests were conducted to verify the reliability of the fingerprint chromatogram establishment method of the present invention. Peak 9 (berberine hydrochloride) was used as the reference peak, and the RSD (relative standard deviation) values ​​of the relative retention time and relative peak area of ​​each common peak were calculated. The test results are as follows:

[0102] 1. Precision test: Take the same test solution and inject it 6 times consecutively. If the relative retention time RSD of the common peak is ≤1.5% and the relative peak area RSD is ≤2.8%, it indicates that the instrument has good precision.

[0103] 2. Stability test: Take the same test solution and place it at room temperature for 0, 2, 4, 8, 12 and 24 hours respectively for injection and determination. The relative retention time RSD of the common peak is ≤1.6% and the relative peak area RSD is ≤2.5%, indicating that the test solution has good stability within 24 hours at room temperature.

[0104] 3. Repeatability test: Six test solutions were prepared in parallel using the same batch of processed Coptis chinensis slices and injected for analysis. The relative retention time RSD of the common peak was ≤1.7% and the relative peak area RSD was ≤2.6%, indicating that the repeatability of this method is good.

[0105] The above experimental results show that the method for establishing fingerprint chromatograms of four-processed Coptis chinensis slices of the present invention has high precision, good stability, and good repeatability, and meets the technical requirements of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine". It can be used for the establishment and quality evaluation of fingerprint chromatograms of four-processed Coptis chinensis slices.

[0106] Fifth aspect: Application of fingerprint spectrum of four-processed Coptis chinensis slices

[0107] The fingerprint spectrum was applied to the quality evaluation of four-processed Coptis chinensis slices, the identification of Coptis chinensis slices with different processing techniques, and the auxiliary evaluation of the hypoglycemic activity of four-processed Coptis chinensis slices.

[0108] 1. Quality evaluation of processed Coptis chinensis slices

[0109] Ten batches of processed Coptis chinensis slices (numbered S1-S10) prepared using the processing method of this invention were taken. Test solutions were prepared according to the method described in the third aspect, and analyzed under the aforementioned chromatographic conditions. Chromatograms of the ten batches of processed Coptis chinensis were obtained. The data were imported into the "2012 version of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System," resulting in fingerprint chromatograms with nine common peaks. Please refer to [link to relevant documentation]. Figure 8 (After selecting S10 and performing multi-point corrections, the Mark peak matching method was used) to compare with the reference standard ( Figure 9 The peak numbers for berberine, epiberberine, coptisine, palmatine, and berberine hydrochloride were compared to those for berberine, and were identified as 5, 6, 7, 8, and 9, respectively. Similarity was then evaluated with the control chromatogram (the revised S10 chromatogram), and the similarity scores were 0.975, 0.832, 0.986, 0.943, 0.951, 0.972, 0.966, 0.933, 0.976, and 0.886, respectively. Except for batches S2 and S10, all scores were greater than 0.9, indicating that the quality differences between different batches were small. This demonstrates that the fingerprint chromatogram of this invention can effectively evaluate the quality uniformity of processed Coptis chinensis slices.

[0110] 2. Identification of Coptis chinensis slices processed with different methods

[0111] Raw Coptis chinensis slices, wine-processed Coptis chinensis slices, ginger-processed Coptis chinensis slices, and Coptis chinensis slices prepared by the processing method of this invention were taken. Test solutions were prepared according to the method described in the third aspect, and the results were compared with the fingerprint spectrum of this invention. The results showed that only the four-processed Coptis chinensis slices of this invention exhibited the corresponding 9 common peaks, with a similarity ≥0.9. Raw Coptis chinensis slices, wine-processed Coptis chinensis slices, and ginger-processed Coptis chinensis slices did not exhibit the 9 common peaks, and several characteristic peaks were missing. These were determined to be raw Coptis chinensis slices, wine-processed Coptis chinensis slices, ginger-processed Coptis chinensis slices, or Coptis chinensis slices processed with a single excipient. This indicates that the fingerprint spectrum of this invention can effectively distinguish four-processed Coptis chinensis slices from Coptis chinensis slices processed using other methods.

[0112] Sixth aspect: Evaluation of the hypoglycemic activity of four-processed Coptis chinensis slices

[0113] The prepared Coptis chinensis slices obtained by the four-processing method of this invention can be used to lower blood sugar and treat diabetes. The hypoglycemic activity of the prepared Coptis chinensis slices of this invention was investigated using α-glucosidase and α-amylase inhibition rates as evaluation indicators, and compared with raw Coptis chinensis slices and the positive control drug acarbose. The specific verification steps are as follows:

[0114] 1. Sample solution preparation

[0115] Accurately weigh 0.25g each of the four-processed Coptis chinensis slices and raw Coptis chinensis slice powder prepared by the processing method of this invention, extract by ultrasonication for 30min, filter, and prepare sample solutions with a series of concentrations of 1, 1.5, 2, 2.5, 3, 4, and 5 mg / mL using phosphate buffer; accurately weigh 0.01g of acarbose, dissolve it in ultrapure water and make up to 10mL to prepare positive control solutions with the same concentration gradient.

[0116] 2. Assay for α-glucosidase inhibitory activity

[0117] Take 40 μL of 1 U / mL α-glucosidase and 40 μL of sample solutions or positive control solutions of different concentrations, mix well, and react at 37℃ for 15 min; add 60 μL of 25 mmol / L pNPG solution, and continue the reaction at 37℃ for 60 min; stop the reaction by adding 60 μL of 0.1 mol / L Na2CO3 solution. Use acarbose as a positive control, PBS to replace the enzyme solution as a blank control, and PBS solution to replace the sample as a negative control. Measure the absorbance at 405 nm and calculate the inhibition rate using the following formula:

[0118] α-glucosidase inhibition rate (%) = [(A1-A2)-(A3-A4)] / (A1-A2)×100%;

[0119] Where A1 is the absorbance value of the enzyme-added solution without sample;

[0120] A2 is the absorbance value without sample or enzyme solution added;

[0121] A3 represents the absorbance value of the sample and enzyme solution.

[0122] A4 represents the absorbance value of the sample without enzyme solution.

[0123] Please see Figure 11 The results showed that when the concentration was 1–3 mg / mL, the inhibition rate of α-glucosidase by both processed Coptis chinensis and raw Coptis chinensis increased with increasing concentration, and the inhibition rate of processed Coptis chinensis was higher at the same concentration. When the concentration was 3.0 mg / mL, the inhibition rate of processed Coptis chinensis was as high as 95% or more, while that of raw Coptis chinensis was 90%. When the mass concentration was >4.0 mg / mL, the inhibition rate of processed Coptis chinensis was close to 100%.

[0124] 3. Assay for α-amylase inhibitory activity

[0125] Take 300 μL of 0.1 U / mL α-amylase solution and 100 μL of sample solutions of different concentrations or acarbose sample solutions, mix well, and react at 37 ℃ for 5 min; then add 400 μL of 1% starch solution, and continue reacting at 37 ℃ for 5 min; finally add 200 μL of DNS reagent and boil in a water bath for 5 min, allowing the reaction to proceed fully, then cool to room temperature and dilute with ultrapure water. Use acarbose as a positive control, PBS solution as a blank control instead of enzyme solution, and PBS as a negative control instead of sample solution. Measure the absorbance (A) at 540 nm. Each solution should be prepared in triplicate. The inhibition rate is calculated according to the α-glucosidase method.

[0126] The results are as follows Figure 12 As shown, the inhibitory activity of processed Coptis chinensis against α-amylase was significantly better than that of Coptis chinensis decoction. At a concentration of 2.0 mg / mL, the inhibition rate of processed Coptis chinensis against α-amylase reached 90.4%, while the inhibition rate of Coptis chinensis decoction was only 76.9%.

[0127] 3. Conclusion

[0128] The four-processed Coptis chinensis slices prepared using the processing method of this invention showed that fermentation time had the greatest impact on alkaloid content (F=35.29). Moderate fermentation (5 days) promoted alkaloid conversion and dissolution, while excessive fermentation (>9 days) may reduce component content due to enzymatic hydrolysis. The main factors affecting fermentation temperature (14 ℃) and steaming time (2 h) were the thermal stability of components such as evodiamine and the synergistic effect of excipients. The fingerprint spectrum of the four-processed Coptis chinensis identified 9 common peaks, 5 of which were characteristic alkaloids. The similarity of most of the 10 batches of samples was greater than 0.9, indicating that optimized process can ensure product quality uniformity and stronger hypoglycemic activity.

[0129] In vitro hypoglycemic experiments showed that the four-processed Coptis chinensis exhibited significantly better inhibitory activity against α-glucosidase and α-amylase than Coptis chinensis decoction, demonstrating superior hypoglycemic potential. This may be attributed to the fact that gingerol and evodiamine in ginger and Evodia rutaecarpa enhance the stability of alkaloids, rice wine promotes the dissolution of alkaloid components, and glycyrrhizic acid forms complexes with alkaloids, prolonging the duration of action.

[0130] The optimal processing method for four-stage Coptis chinensis is to add 100 mL of mixed excipient juice (a mixture of ginger juice, Evodia rutaecarpa juice, licorice juice, and rice wine in equal proportions) to every 1000 g of Coptis chinensis slices, ferment at 14 ℃ for 5 days, steam for 2 hours, dry at 70 ℃, and then pulverize and pass through a No. 2 sieve. The optimized processing method for four-stage Coptis chinensis is stable and feasible, with uniform quality and significant hypoglycemic activity. This study provides a scientific basis for the standardized production, quality control, and prevention and treatment of diabetes with four-stage Coptis chinensis.

[0131] Based on the disclosure and teachings of the above specification, those skilled in the art can make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the present invention should also fall within the protection scope of the claims of the present invention. Furthermore, although some specific terms are used in this specification, these terms are only for convenience of explanation and do not constitute any limitation on the present invention. Other devices that are the same as or similar to these terms are all within the protection scope of the present invention.

Claims

1. A method for processing four-stage processed Coptis chinensis slices, characterized in that, Includes the following steps: S1. Preparation of excipients: Prepare ginger juice, evodia juice, and licorice juice, and mix them with rice wine in equal proportions to form a mixed medicinal juice for later use; S2. Take clean Coptis chinensis slices, add mixed medicinal juice, stir until the medicinal juice is absorbed, and ferment in a stability test chamber for 5-6 days at a temperature of 5℃-15℃. After taking out the Coptis chinensis slices, steam them in a water bath for 1-2 hours. After steaming, dry them to obtain the four-processed Coptis chinensis slices.

2. A method for constructing a fingerprint spectrum of Coptis chinensis slices, characterized in that, Includes the following steps: S1. Preparation of test solution: Take the Coptis chinensis slices prepared by the processing method described in claim 1 as the sample, and use methanol-hydrochloric acid mixed solution as the extraction solvent to prepare the test solution. S2. Preparation of reference solution: Select a reference standard and prepare a reference solution using methanol as the solvent. S3. Chromatographic determination: The test solution and the reference solution are injected into a high-performance liquid chromatograph and measured under preset chromatographic conditions to obtain chromatograms of the test solution and the reference solution, respectively. S4. Fingerprint chromatogram construction: The obtained chromatogram is imported into the preset Chinese medicine chromatographic fingerprint chromatogram similarity evaluation system. Through multi-point correction and Mark peak matching, a fingerprint chromatogram containing 9 common peaks is obtained, that is, the fingerprint chromatogram of the four-processed Coptis chinensis slices is constructed.

3. The fingerprint map construction method according to claim 2, characterized in that, The reference standards are berberine hydrochloride, palmatine, berberine, epiberberine, and cypermethrin, and the reference standard solution contains 416 μg / mL berberine hydrochloride, 314 μg / mL palmatine, 213 μg / mL berberine, 121 μg / mL epiberberine, and 120 μg / mL cypermethrin.

4. The fingerprint map construction method according to claim 2, characterized in that, The preset chromatographic conditions of the high-performance liquid chromatograph include: a Kromasil C18 column with dimensions of 250 mm × 4.6 mm and 5 μm; mobile phase A being an aqueous solution of ammonium bicarbonate containing ammonia and triethylamine, and mobile phase B being acetonitrile; gradient elution; detection wavelength of 270 nm; column temperature of 30 °C; and flow rate of 1 mL / min.

5. The fingerprint map construction method according to claim 4, characterized in that, The mobile phase A is a 30 mmol / L ammonium bicarbonate aqueous solution containing 0.7% ammonia and 0.1% triethylamine; the gradient elution conditions are as follows: 0-15 min, mobile phase B accounts for 10%-25%; 15-23 min, mobile phase B accounts for 25%-30%; 23-35 min, mobile phase B accounts for 30%-40%; 35-40 min, mobile phase B accounts for 40%.

6. The fingerprint map construction method according to claim 2, characterized in that, In step S4, among the nine common peaks, peak 5 is berberine, peak 6 is epiberberine, peak 7 is berberine, peak 8 is palmatine, and peak 9 is berberine hydrochloride. Peak 9, corresponding to berberine hydrochloride, is used as the reference peak.

7. The fingerprint map construction method according to claim 2, characterized in that, The preparation steps of the test solution in step S1 are as follows: Take 0.2g of prepared Coptis chinensis powder, accurately weigh it, place it in a 50mL stoppered conical flask, add 50mL of methanol-hydrochloric acid mixed solution with a volume ratio of 100:1, weigh it again, extract it by ultrasonication for 30min, weigh it after cooling and make up for the weight loss, filter it through a 0.45μm filter membrane to obtain the test solution; The preparation step of the reference solution in step S2 is as follows: Dissolve the reference standard in methanol, make up to a certain volume, and mix to prepare a reference solution containing 416 μg / mL berberine hydrochloride, 314 μg / mL palmatine, 213 μg / mL berberine, 121 μg / mL epiberberine, and 120 μg / mL cypermethrin.

8. An application of a fingerprint spectrum of four-processed Coptis chinensis slices constructed by the fingerprint spectrum establishment method according to any one of claims 2-7, characterized in that, The fingerprint spectrum was applied to the quality evaluation of four-processed Coptis chinensis slices, the identification of Coptis chinensis slices with different processing techniques, and the auxiliary evaluation of the hypoglycemic activity of four-processed Coptis chinensis slices.

9. The application according to claim 8, characterized in that, The similarity analysis of the four-processed Coptis chinensis slices to be tested is performed. If the similarity is ≥0.9, the quality uniformity of the four-processed Coptis chinensis slices to be tested is determined to be in line with the requirements. If the fingerprint spectrum of the Coptis chinensis slices to be identified shows the above 9 common peaks and the similarity is ≥ 0.9, if the four-processed Coptis chinensis slices prepared by the processing method described in claim 1 do not show the corresponding nine common peaks or have any missing peaks, they are determined to be raw Coptis chinensis slices, wine-processed Coptis chinensis slices, ginger-processed Coptis chinensis slices or Coptis chinensis slices processed with a single auxiliary ingredient. The higher the similarity of the four-processed Coptis chinensis slices to be tested, the stronger their hypoglycemic activity.

10. The use of the four-processed Coptis chinensis slices prepared by the method described in claim 1, characterized in that, Used to lower blood sugar and treat diabetes.

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