Processing technology optimization method for stir-frying honeysuckle into charcoal

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a processing technology optimization method for stir-frying honeysuckle flowers into charcoal, which comprises the following steps: firstly, extracting active ingredients of the honeysuckle flowers to obtain a chlorogenic acid extract, a luteoloside extract, a water extract and an alcohol extract; then dynamically monitoring pyrolysis characteristics of the extract, a chlorogenic acid reference substance, a honeysuckle reference substance and honeysuckle raw medicinal material powder by adopting a TG-DSC (Thermal Gravity-Differential Scanning Calorimetry) combined technology, and taking a peak temperature range of a maximum thermal weight loss rate peak in a first pyrolysis stage of the honeysuckle raw medicinal material powder as a primary processing temperature range; then, respectively taking the processing temperature and the processing time as single factor variables, and taking the content of chlorogenic acid in the processed honeysuckle as an evaluation index to determine an optimization range of the processing time; and finally, further optimizing the honeysuckle flower processing technology by adopting a response surface method in combination with the temperature and time ranges, and determining the optimal processing temperature and time. According to the method, the interaction of temperature and time is comprehensively considered, and precise optimization of the honeysuckle flower processing technology is achieved.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to an optimized method for processing honeysuckle by charring. Background Technology

[0002] Honeysuckle, also known as Lonicera japonica, is a plant in the Caprifoliaceae family. It is produced in East my country, Central South China, Southwest China, and parts of Hebei, Shanxi, Liaoning, and Shaanxi provinces. It has the effects of clearing heat and detoxifying, and dispersing wind-heat. It is used for carbuncles, boils, sore throat, dysentery due to heat toxicity, wind-heat colds, and febrile diseases. However, honeysuckle is cold in nature, and may cause gastrointestinal irritation in patients with spleen and stomach deficiency, limiting its widespread clinical use. Therefore, charred honeysuckle is often used clinically.

[0003] Charring is a method of processing traditional Chinese medicine. By charring the herbs, their medicinal properties and efficacy can be altered. After charring, honeysuckle's cold nature is reduced, while its hemostatic and astringent effects are enhanced, making it more suitable for patients with spleen and stomach deficiency. Charred honeysuckle, as one of the classic processed products of honeysuckle, has reduced coldness after charring and possesses anti-inflammatory, hemostatic, and antioxidant effects. It is effective in clearing heat and toxins from the lower burner and blood, and is clinically used for dysentery, metrorrhagia, and hematemesis. Currently, research on charred honeysuckle still faces several unresolved issues, such as the standardization of processing techniques. The lack of unified standards for charring temperature and time parameters affects product quality stability. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an optimized processing method for charred honeysuckle. This invention employs a combination of TG-DSC, single-factor experiments, and response surface methodology to make the honeysuckle processing more scientific and reliable, providing a precise and effective method for charred honeysuckle processing.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: An optimized method for processing honeysuckle into charcoal includes the following steps: The effective components of honeysuckle were extracted to obtain chlorogenic acid extract, luteolin extract, water extract, and alcohol extract; The pyrolysis characteristics of chlorogenic acid extract, luteolin extract, water extract, alcohol extract, chlorogenic acid reference standard, honeysuckle reference standard, and honeysuckle raw material powder were dynamically monitored using TG-DSC coupled technology. Based on the pyrolysis characteristics, the processing temperature range of honeysuckle was preliminarily obtained. Using processing temperature and processing time as single-factor variables, and the content of organic acids in processed honeysuckle as the evaluation index, the optimal range of honeysuckle processing time was determined. Based on the preliminary determination of the processing temperature range of honeysuckle by combining the results of pyrolysis characteristics and the optimization range of processing time of honeysuckle determined by single-factor experiments, the processing technology of honeysuckle was further optimized by using response surface methodology to determine the optimal processing temperature and processing time.

[0006] Furthermore, the chlorogenic acid extract is extracted using a 75% (v / v) methanol solution, and the luteolin glycoside extract is extracted using a 70% (v / v) ethanol solution.

[0007] Furthermore, the water extract includes chlorogenic acid, luteolin, and saponins.

[0008] Furthermore, the alcohol extract is extracted using anhydrous ethanol, and the alcohol extract includes chlorogenic acid, luteolin, and caffeic acid.

[0009] Furthermore, when initially determining the processing temperature range of honeysuckle based on the pyrolysis characteristics, the peak temperature range of the maximum thermal weight loss rate peak in the first pyrolysis stage of the honeysuckle raw material powder is taken as the processing temperature range of honeysuckle.

[0010] Furthermore, the organic acid is chlorogenic acid.

[0011] Furthermore, the response surface methodology was used with processing temperature and processing time as independent variables and chlorogenic acid content as the response value, and a quadratic polynomial model was fitted using Design Expert 13.0.1.0 software.

[0012] Furthermore, the quadratic polynomial model of the response surface method is: chlorogenic acid content = 24.35 + 1.13A + 0.2608B - 0.0350AB - 2.61A² - 0.9279B²; In the formula, A is the processing temperature and B is the processing time.

[0013] The optimal processing method for charring honeysuckle obtained by the above method is 205 ℃ and the optimal processing time is 7 min.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention first extracts the effective components of honeysuckle to obtain chlorogenic acid extract, luteolin extract, water extract, and alcohol extract. Then, using TG-DSC coupled technology, the pyrolysis characteristics of the above extracts, chlorogenic acid reference standard, honeysuckle reference standard, and honeysuckle raw material powder are dynamically monitored. The peak temperature range of the maximum thermal weight loss rate peak in the first pyrolysis stage of honeysuckle raw material powder is used to initially determine the processing temperature range. This process scientifically locks the temperature range based on the thermal stability of the components. Then, using processing temperature and time as single-factor variables and chlorogenic acid content as the evaluation index, the optimal processing time range is determined. Through single-variable control, the approximate range of key parameters is precisely screened. Finally, combining the preliminary determined processing temperature range of honeysuckle based on the pyrolysis characteristics and the optimized processing time range determined by the single-factor experiments, a response surface methodology is used. With temperature and time as independent variables and chlorogenic acid content as the response value, a quadratic polynomial model is fitted using Design Expert software to further optimize and obtain the optimal processing temperature. This method comprehensively considers the interaction between temperature and time, achieving precise parameter optimization. On the one hand, the application of TG-DSC combined technology provides a scientific basis for determining the temperature range from the perspective of pyrolysis characteristics, ensuring that the effective components are well preserved within this temperature range, while reducing the coldness of honeysuckle and increasing its astringency. Single-factor experiments can intuitively reflect the influence of a single parameter on chlorogenic acid content, laying the foundation for subsequent optimization. The response surface methodology, through the establishment of a mathematical model, accurately predicts the optimal process parameters. Verification showed that the average chlorogenic acid content of three parallel experiments was 23.45 mg / g, close to the predicted value, proving that this optimization method can stably obtain honeysuckle charcoal with high chlorogenic acid content. On the other hand, the entire optimization method is progressive, from preliminary screening to precise determination, with rigorous logic and scientific reliability. It avoids the empirical and random nature of traditional processing parameter selection, improves the controllability and repeatability of the process, and provides an effective means for the standardization of honeysuckle charcoal processing technology. This ensures the stability of the quality of processed honeysuckle charcoal, and honeysuckle processed by this technology has reduced gastrointestinal irritation, making it more suitable for clinical application. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1The figures show the pyrolysis characteristic curves of the thermal analysis samples. Among them, A is the TG-DTG curve of honeysuckle water extract, B is the TG-DTG curve of honeysuckle alcohol extract, C is the TG-DTG curve of honeysuckle luteolin extract, D is the TG-DTG curve of honeysuckle chlorogenic acid extract, E is the TG-DTG curve of chlorogenic acid reference standard, F is the TG-DTG curve of honeysuckle reference medicinal material, and G is the TG-DTG curve of honeysuckle raw medicinal material powder.

[0017] Figure 2 The effect of roasting temperature and roasting time on chlorogenic acid content of honeysuckle charcoal is shown in Figure a, where a is the three-dimensional response surface plot and b is the contour plot.

[0018] Figure 3 This is the chromatogram of the chlorogenic acid reference solution.

[0019] Figure 4 Chromatogram of honeysuckle charcoal test solution.

[0020] Figure 5 This is the standard curve for chlorogenic acid.

[0021] Figure 6 The ratio of honeysuckle charcoal to raw honeysuckle charcoal powder is used to measure the yield.

[0022] Figure 7 The effects of charred and raw honeysuckle on TRPM8 protein expression in colon tissue were investigated. In the figure, a is a Western blot electrophoresis image of TRPM8 protein, and b is the relative expression level of TRPM8 protein.

[0023] Figure 8 The effects of charred and raw honeysuckle on the pathological effects of mouse small intestinal tissue.

[0024] Figure 9 The effects of charred and raw honeysuckle on the pathological effects of mouse colon tissue.

[0025] Figure 10 The effects of charred and raw honeysuckle on the pathological effects of gastric tissue in mice. Detailed Implementation

[0026] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific 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. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0027] Drugs and reagents: Honeysuckle, purchased from Shanxi Yuanhetang Pharmaceutical Co., Ltd., batch number 230101; reference standards chlorogenic acid (batch number MUST-17030620), luteolin (batch number MUST-23082218), and honeysuckle reference material (batch number 2022-042201) were purchased from Chengdu Pufeed Biotechnology Co., Ltd.; PBS buffer, purchased from Wuhan Pronosai Life Science Technology Co., Ltd., batch number 18G28C21; 4% paraformaldehyde, purchased from Beijing Regen Biotechnology Co., Ltd., batch number 1110A23; BCA detection working solution, purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd., batch number 18J09A97; 30% acrylamide, purchased from Merck Life Sciences Co., Ltd., batch number 2309008; ECL luminescence solution, purchased from Dorest Medical Technology Co., Ltd., batch number MA0186.

[0028] Experimental Instruments: Heating incubator, model HP-9052MBE, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory; Digital display thermostatic heating mantle, model SKM, Shandong Juancheng Guangming Instrument Co., Ltd.; Rapid low-temperature cooling cycler, model DLK-4007, Ningbo Xinzhi Biotechnology Co., Ltd.; Circulating water multi-purpose vacuum pump, model SHZ-D(Ш), Zhengzhou Keda Machinery Instrument Equipment Co., Ltd.; Digital display thermostatic water bath, model HWS24, Guohua Electric Co., Ltd.; Rotary evaporator, model N-1300, Shanghai Ailang Instrument Co., Ltd.; Electrophoresis apparatus, model DYC-ZY2, Bio-Rad Laboratories, USA; Low-temperature tissue homogenizer, model KZ-5F-3D, Wuhan Saiweier Biotechnology Co., Ltd.; High-speed quick-freezing centrifuge, model 5424R, Beckman Coulter Trading Co., Ltd., USA; Thermostatic shaker, model TS-211B, Shanghai Tiancheng Experimental Instrument Manufacturing Co., Ltd.; Inverted fluorescence microscope, model Ti2-U, Nikon Corporation; Developing system, model Amersham Imager. 600, Cytiva, USA; Leica UC7 ultramicrotome; STA449-F5 thermogravimetric-differential thermal analyzer, Netzsch GmbH, Germany; UltiMate3000 ultra-high performance liquid chromatograph, Thermo Fisher Scientific; 600 electromagnetic stir-frying (pharmaceutical) machine, Beijing Hualin Ruikong Technology Co., Ltd.; SB-5200DTDN ultrasonic cleaner, Ningbo Xinzhi Biotechnology Co., Ltd.; AX224ZH / E electronic balance, Ohaus Instruments (Changzhou) Co., Ltd.; UltiMate3000 ultra-high performance liquid chromatograph, Thermo Fisher Scientific.

[0029] Experimental animals: ICR mice, provided by Spiford Biotechnology Co., Ltd., 6-8 weeks old, weighing approximately 23g, housed at a temperature of 22±2℃ and humidity of 55±10%, with free access to food and water. Animal experiments were conducted at Shanxi University of Traditional Chinese Medicine.

[0030] Example 1 An optimized method for processing honeysuckle into charcoal includes the following steps: The effective components of honeysuckle were extracted to obtain chlorogenic acid extract, luteolin extract, water extract, and alcohol extract; (1) Preparation of honeysuckle raw material powder sample: Weigh 100g of honeysuckle slices, crush them, pass them through a 60-mesh sieve, and set aside.

[0031] (2) Preparation of honeysuckle chlorogenic acid extract sample: Take about 10 g of honeysuckle raw material powder, put it in a stoppered conical flask, accurately add 500 mL of 75% methanol, weigh it, sonicate for 30 min, filter, take the filtrate, concentrate it in a rotary evaporator, and freeze dry it under vacuum for 8 h to obtain chlorogenic acid extract.

[0032] (3) Preparation of honeysuckle luteolin extract: Take about 10 g of honeysuckle raw material powder, place it in a stoppered conical flask, accurately add 250 mL of 70% ethanol, weigh it, sonicate for 1 h, filter, take the filtrate, concentrate it in a rotary evaporator, and freeze dry it under vacuum for 8 h to obtain luteolin extract.

[0033] (4) Preparation of honeysuckle water extract sample: Weigh 10 g of honeysuckle, crush it, put it in a round bottom flask, add pure water solvent to make it submerge the medicinal material, soak for 40 min, reflux extract for 1 h, filter, collect the filtrate, add pure water solvent to the filter residue again to make it submerge the medicinal material, reflux extract for 40 min, filter, combine the filtrates, concentrate in a rotary evaporator, and freeze dry under vacuum for 8 h to obtain water extract.

[0034] (5) Preparation of honeysuckle anhydrous ethanol extract: Weigh 10 g of honeysuckle, crush it, place it in a round bottom flask, add anhydrous ethanol solvent to submerge the medicinal material, soak for 40 min, reflux for 1 h, filter, collect the filtrate, add anhydrous ethanol solvent to the residue again, reflux for 40 min, filter, combine the filtrates, concentrate in a rotary evaporator, and freeze dry under vacuum for 8 h to obtain the ethanol extract.

[0035] The pyrolysis characteristics of chlorogenic acid extract, luteolin extract, water extract, alcohol extract, chlorogenic acid reference standard, honeysuckle reference standard, and honeysuckle raw material powder were dynamically monitored using TG-DSC coupled technology. Based on the pyrolysis characteristics, the processing temperature range of honeysuckle was preliminarily obtained. Using a STA449-F5 TG-DSC, N2 and O2 (N2:O2 volume ratio = 4:1) in the air were simulated as carrier gases. The heating rate was 10℃ / min and the volumetric flow rate was 60 mL / min. After blank baseline testing, under these conditions, pyrolysis characteristic analysis was performed on reference standards and samples of different extracted components. The sample amount was (20±5) mg. The temperature was raised from room temperature to 610℃, and each sample was tested in parallel 3 times.

[0036] Analysis of pyrolysis characteristic curves of thermal analysis experimental samples Figure 1 The figures show the pyrolysis characteristic curves of the samples in the thermal analysis experiment. A represents the TG-DTG curve of the aqueous extract of honeysuckle, B represents the TG-DTG curve of the ethanolic extract of honeysuckle, C represents the TG-DTG curve of the luteolin-based extract of honeysuckle, D represents the TG-DTG curve of the chlorogenic acid extract of honeysuckle, E represents the TG-DTG curve of the chlorogenic acid standard, F represents the TG-DTG curve of the reference medicinal material of honeysuckle, and G represents the TG-DTG curve of the raw honeysuckle powder. Relevant pyrolysis characteristic parameters are shown in Table 1.

[0037] Table 1. Pyrolysis characteristics of honeysuckle medicinal material and different extracts The direct water extract contains chlorogenic acid, luteolin, saponins, and other components soluble in hot water. The anhydrous ethanol extract contains chlorogenic acid, luteolin, caffeic acid, and other components soluble in ethanol.

[0038] The pyrolysis characteristics of the reference standard and compounds separated by different extraction methods were analyzed. Comparing the combustion pyrolysis curves of the honeysuckle water extract, the main pyrolysis stage was observed in the range of 120.5℃ to 371.6℃, with a mass fraction reduction of 46.77%. This indicates that within this temperature range, some volatile or easily decomposed components in the water extract begin to be lost, possibly due to the evaporation of water molecules and the decomposition of some heat-sensitive components. The combustion pyrolysis curves of the honeysuckle sample showed that the main pyrolysis stage was observed in the range of 187.7℃ to 351.2℃, with a mass fraction reduction of 47.30%. This indicates that within this temperature range, some components in honeysuckle begin to decompose, possibly due to the thermal decomposition or oxidation of heat-sensitive components. Comparing the combustion pyrolysis curves of the honeysuckle reference standard, the main pyrolysis stage was observed in the range of 128.0℃ to 371.8℃, with a mass fraction reduction of 50.79%. This indicates that within this temperature range, the components in the reference standard begin to decompose, possibly due to the thermal decomposition or oxidation of heat-sensitive components. The combustion pyrolysis curves of honeysuckle ethanol extract show that the main pyrolysis stage is between 121.5℃ and 413.8℃, with the mass fraction decreasing to 58.35%. This indicates that within this temperature range, some components in the ethanol extract begin to decompose, possibly due to the thermal decomposition or oxidation of heat-sensitive components. The combustion pyrolysis curves of honeysuckle luteolin extract show that the main pyrolysis stage is between 113.7℃ and 395.7℃, with the mass fraction decreasing to 59.85%. This indicates that within this temperature range, some components in the luteolin extract begin to decompose, possibly due to the thermal decomposition or oxidation of heat-sensitive components. The combustion pyrolysis curves of honeysuckle chlorogenic acid extract show that the main pyrolysis stage is between 121.7℃ and 398.3℃. During this pyrolysis stage, most of the chlorogenic acid components completed the pyrolysis, with the mass fraction decreasing to 51.30%. Comparing the combustion pyrolysis characteristics of chlorogenic acid reference standard, it can be concluded that the 145.0℃~408.0℃ stage is the main pyrolysis stage, during which the mass fraction decreases to 49.20%. In this stage, chlorogenic acid completes pyrolysis.

[0039] The study found that within the main pyrolysis stage of honeysuckle raw material powder (the peak temperature range of the maximum thermal weight loss rate in the first pyrolysis stage), from 187.7℃ to 229.8℃, the mass fractions of the water extract, alcohol extract, luteolin glycoside extract, and chlorogenic acid extract decreased by 12.12%, 14.55%, 10.07%, and 12.08%, respectively. However, within the temperature range of 229.8℃ to 351.2℃, the mass fractions of the water extract, alcohol extract, luteolin glycoside extract, and chlorogenic acid extract decreased by 27.94%, 30.93%, 39.95%, and 28.06%, respectively. Comparatively, the decrease in the mass fraction of the extracts was less within the 187.7℃ to 229.8℃ temperature range, indicating that the effective components were relatively well preserved within this temperature range. Within the temperature range of 187.7℃ to 229.8℃, the peak values ​​of the DTG curves were 3.64% / min, 4.37% / min, 3.32% / min, and 3.60% / min, respectively. Within this temperature range, honeysuckle not only exhibits reduced coldness but also increased astringency. Therefore, it can be inferred that the optimal processing temperature range for charring honeysuckle is 187.7℃ to 229.8℃.

[0040] Using processing temperature and processing time as single-factor variables, and the content of organic acids in honeysuckle after processing as the evaluation index, the optimal range of honeysuckle processing time was determined. (1) Effect of roasting temperature on the processing of honeysuckle charcoal: Take 5 portions of honeysuckle, 100 g each, place them in a roasting machine, and roast them for 8 min at 190℃, 200℃, 210℃, 220℃, and 230℃ respectively. Spray a little water to extinguish the sparks, spread them out to cool, and honeysuckle charcoal is obtained. The contents of organic acids in honeysuckle charcoal were measured to be 23.68 mg / g, 24.44 mg / g, 22.25 mg / g, 23.28 mg / g, and 19.81 mg / g respectively. The highest organic acid content was found at 200℃, so the optimal roasting temperature is 200℃.

[0041] (2) Effect of roasting time on the processing of honeysuckle charcoal: Take 5 portions of honeysuckle, 100 g each, and put them into a roasting machine. Roast them at 210℃ for 4 min, 6 min, 8 min, 10 min, and 12 min respectively. Spray a little water to extinguish the sparks, spread them out to cool, and honeysuckle charcoal is obtained. The contents of organic acids in honeysuckle charcoal were measured to be 17.25 mg / g, 22.22 mg / g, 25.14 mg / g, 23.02 mg / g, and 23.10 mg / g respectively. The highest organic acid content was found to be 8 min, therefore the optimal roasting time is 8 min.

[0042] Based on the preliminary determination of the processing temperature range of honeysuckle by combining the results of pyrolysis characteristics and the optimization range of processing time of honeysuckle determined by the results of single-factor experiments, the Box-Behnken response surface methodology was used to further optimize the processing technology of honeysuckle and determine the optimal processing temperature and processing time. (1) Experimental design and results: Based on the results of the single-factor experiment, the response value was selected as chlorogenic acid content, and the independent variables were frying temperature and frying time. Using Design Expert 13.0.1.0 x 64 software, the method was to construct a multi-factor, multi-level experimental design, analyze the influence of each factor on the response value and the interaction relationship, and thus determine the optimal processing parameters. The results are shown in Table 2.

[0043] (2) Experimental Analysis: Based on the experimental results, we can establish the following quadratic equation model to describe the relationship between chlorogenic acid content and roasting conditions: Chlorogenic acid content = 24.35 + 1.13A + 0.2608B - 0.0350AB - 2.61A 2 -0.9279B 2 r 2 =0.9113, this model can explain 91.13% of the response value variation, indicating a good fit and relatively small experimental error. Therefore, we can use this model to analyze and predict the changes in chlorogenic acid content in honeysuckle charcoal under different roasting conditions. The analysis of variance is shown in Table 3.

[0044] Table 2 Factor Levels, Response Surface Experiment Design and Results Table 3 Analysis of Variance Note: *P<0.05 indicates a significant term, **P<0.01 indicates a highly significant term. (3) Response surface methodology results: Based on the 3D model, the effects of frying temperature and frying time on the processing technology of honeysuckle charcoal are plotted. Figure 2 . Figure 2 In the diagram, a represents the three-dimensional response surface plot, and b represents the contour plot. Through data optimization, it was predicted that the highest chlorogenic acid content in honeysuckle charcoal was achieved at a roasting temperature of 204.424℃ and a roasting time of 6.98672 min. Therefore, this roasting temperature and time were selected as the optimal processing parameters for honeysuckle charcoal.

[0045] Process Result Validation Analysis of the model revealed the optimal processing parameters as follows: a frying temperature of 205℃ and a frying time of 7 minutes. Under these conditions, the chlorogenic acid content in honeysuckle charcoal was highest.

[0046] To verify the reliability of the results, the experiment was conducted in parallel three times. The chlorogenic acid content in the three experiments was 23.27 mg / g, 23.77 mg / g, and 23.36 mg / g, respectively. The results showed that the average chlorogenic acid content was 23.45 mg / g, which was very close to the predicted value. This further demonstrates the effectiveness of response surface methodology in optimizing the processing technology of honeysuckle.

[0047] Based on the optimal processing parameters (processing temperature of 205℃ and processing time of 7min) obtained by the above optimization method, honeysuckle was charred to obtain honeysuckle charcoal, and the performance of honeysuckle before and after charring was tested.

[0048] 1. Determination of chlorogenic acid content in honeysuckle before and after charring 1.1 Chromatographic conditions A Hypersil GOLDAQ (250 mm × 4.6 mm) column was used; the mobile phase was acetonitrile-0.1% phosphoric acid solution, and gradient elution was performed according to the program in Table 4; the column temperature was kept constant at 20℃; the flow rate was set to 0.7 mL / min; the injection volume was 10 μL per sample (except for linearity); the detection wavelength was set to 327 nm. The total run time was 20 min, and the chromatograms were recorded.

[0049] Table 4 Gradient elution program 1.2 Sample Preparation (1) Preparation of chlorogenic acid reference solution: Add 7 mg of chlorogenic acid reference standard to a brown volumetric flask (25 mL), add 75% methanol, and prepare a reference solution with a concentration of 0.28 mg / mL. Protect from light and store in a refrigerator at 5°C.

[0050] (2) Preparation of test solution: Weigh about 0.5 g each of honeysuckle raw material and honeysuckle charcoal, put them into conical flasks, add 50 mL of 75% methanol respectively, weigh, sonicate, cool, weigh again, use 75% methanol to make up the weight loss, shake well, filter, and filter through a 0.45 μm microporous membrane to obtain the test solution.

[0051] 1.3 Methodological Examination (1) Linearity investigation: Different volumes of chlorogenic acid standard solution were taken for HPLC detection at a wavelength of 327 nm. Linear regression analysis was performed using the injection amount and peak area of ​​the analyte as the x-axis and y-axis, respectively.

[0052] (2) Precision test: Inject chlorogenic acid standard solution (6 times / 10 μL), record the peak area, calculate RSD, and perform precision detection.

[0053] (3) Stability test: Take honeysuckle charcoal sample to prepare test solution, and under room temperature conditions, the time interval is 0, 2, 2, 4, 4 and 12 h, 10 μL each time, repeat 6 times, record the peak area at each time point, calculate RSD, and perform stability test.

[0054] (4) Repeatability test: Take honeysuckle charcoal sample to prepare test solution, a total of 6 portions, inject (6 times / 10 μL), record the corresponding peak area, calculate RSD, and perform repeatability test.

[0055] (5) Spiking recovery test: Accurately weigh 6 portions of 0.05 g honeysuckle charcoal sample powder, add 1.2 mg chlorogenic acid standard solution to each portion, mix well, prepare the sample, inject (6 times / 10 μL), record the corresponding peak area, and calculate the spiking recovery rate and RSD.

[0056] (6) Sample content determination: Take fresh honeysuckle and honeysuckle charcoal samples to prepare test solution, inject the sample (6 times / 10 μL), record the corresponding peak area, and calculate the sample content.

[0057] 2. Experimental study on gastrointestinal irritation before and after charring honeysuckle 2.1 Preparation of raw honeysuckle and honeysuckle charcoal extract Extract 200 g of raw honeysuckle by reflux for 1 hour with 8 times its volume of water, then collect the filtrate. Repeat the extraction process with 6 times its volume of water for another hour, collecting the filtrate again. Combine the filtrates and concentrate by evaporation to 100 mL. The concentration is 2 g / mL. Prepare high-concentration (high dose) honeysuckle by adding distilled water in the corresponding proportions, resulting in high-concentration (high dose) honeysuckle at 1 g / mL, medium-concentration (medium dose) honeysuckle at 0.5 g / mL, and low-concentration (low dose) honeysuckle at 0.25 g / mL. The same method applies to charred honeysuckle.

[0058] 2.2 Mouse grouping and administration Seventy mice were randomly divided into seven groups: a blank control group, low-, medium-, and high-dose groups of raw honeysuckle, and low-, medium-, and high-dose groups of charred honeysuckle. Mice in the blank control group were administered physiological saline solution by gavage, while mice in each treatment group were administered the corresponding dose of the drug solution (0.1 mL / 10 g) by gavage once daily for one week. After the last administration, mouse feces were collected under sterile conditions. Blood was collected from the ocular venous plexus of mice, centrifuged (3000 rpm / min, 10 min), and serum was prepared. Stomach, large intestine, and small intestine were collected from mice; a portion was blotted dry with absorbent paper and fixed in 4% paraformaldehyde, and another portion was blotted dry with absorbent paper and stored at -80℃ as tissue samples.

[0059] 2.3 Mouse carbon powder propulsion experiment Forty-two mice were randomly divided into seven groups: a blank control group, low-, medium-, and high-dose groups of raw honeysuckle, and low-, medium-, and high-dose groups of charred honeysuckle. Mice in the blank control group were administered physiological saline solution by gavage, while mice in each treatment group were administered the corresponding dose of the drug solution (0.1 mL / 10 g) by gavage once daily for one week. After the last administration, the mice were fasted for 24 hours and then administered a 20% ink-infused physiological saline solution (0.1 mL / 10 g) by gavage. Twenty minutes later, the mice were immediately euthanized by cervical dislocation, and the small intestine was dissected. The total length of the small intestine (pylorus to ileocecal junction) and the distance the charcoal was propelled (pylorus to the leading edge of the charcoal) were measured. The charcoal propulsion rate (%) was calculated as: (distance the charcoal was propelled in the intestine / total length of the small intestine) × 100%.

[0060] 2.4 HE staining method for detecting pathological damage in the stomach, small intestine and large intestine of mice Stomach, small intestine, and large intestine fixed in 4% paraformaldehyde were removed and rinsed three times with PBS for 15 minutes each time. After dehydration with graded ethanol, clearing with xylene, and embedding in liquid paraffin, the sections were cooled and placed on a microtome to a thickness of 5 μM. Paraffin sections were dewaxed with xylene, then dewaxed again with graded ethanol to water. After rinsing with tap water, hematoxylin was stained for 3 min, followed by differentiation with hydrochloric acid-alcohol buffer for 2 s, then rinsed and dried. Eosin staining was performed for 1 s, followed by rinsing with water for 3 s and dehydration with 100% ethanol for 2 s. After drying, the sections were mounted with neutral resin. The results were observed and images were acquired using an optical microscope.

[0061] 2.5 Western blot analysis of TRPM8 protein expression in mouse small intestinal tissue (1) Tissue protein extraction and sample preparation: 20 mg of small intestinal tissue was placed in tissue lysis buffer (RIPA + 1% PMSF + protease inhibitor), homogenized, and centrifuged (13000 rpm / min, 15 min, 4℃). The supernatant was collected, and protein quantification was performed using the BCA method. Loading buffer was added, and the protein was denatured by heating at 95℃ for 5 min. The sample was cooled and stored in a freezer at -80℃ for later use.

[0062] (2) Electrophoresis: Prepare a 10% polyacrylamide gel, load 20 μg of sample, and perform electrophoresis at 80 V until the sample enters the separating gel. Then increase the voltage to 120 V and continue electrophoresis until the sample reaches the bottom of the gel.

[0063] (3) Transfer: Activate the PVDF membrane in methanol for 5 min, and transfer the sample on the gel to the PVDF membrane. Constant current (200mA), wet transfer for 90 min.

[0064] (4) Sealing: The PVDF membrane was sealed in a TBST solution containing 5% skim milk powder for 3 hours.

[0065] (5) Primary antibody incubation: According to the instructions of the antibody, dilute the primary antibody with WB-specific diluent (primary antibody: dilution = 1:1000) and incubate the diluted primary antibody with the PVDF membrane on a shaker overnight at 4°C.

[0066] (6) Secondary antibody incubation: According to the antibody instructions, dilute the secondary antibody with TBST (1:5000), and then incubate the PVDF membrane in the secondary antibody solution at room temperature for 90 min.

[0067] (7) Development and detection: According to the instructions of the ECL luminescence reagent kit, mix solution A and solution B in a 1:1 volume ratio and use a chemiluminescence imaging system for detection and analysis.

[0068] 3. Data Analysis Data analysis was performed using Imagej and GraphpadPrism software. Comparisons between two groups were conducted using t-tests; for comparisons among multiple groups, one-way ANOVA was used. Data are presented as mean ± standard deviation. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0069] Analysis of Experimental Results 1. Determination of chlorogenic acid content in honeysuckle before and after charring 1.1 HPLC Characteristic Chromatography Inject 10 μL each of the reference solution and the test solution, according to Figure 3 , Figure 4 As shown, the chromatographic peak of chlorogenic acid in the honeysuckle charcoal test solution is sharp, and its retention time is similar to that of the reference solution. Figure 1 To.

[0070] 1.2 Methodological Investigation Results (1) Linearity investigation: The results show that the linear relationship is good in the injection volume range of 2-12 μL. The regression equation is: Y = 15.538X + 4.1746 (correlation coefficient r = 0.9995). For details, please refer to Table 5 and see [link to table]. Figure 5 .

[0071] Table 5. Linear relationship data of chlorogenic acid Table 5 (Continued) - Linear Relationship Data of Chlorogenic Acid (0) Precision test: The results showed that the RSD of the chlorogenic acid peak area was 2.32% (n = 6), indicating that the instrument has good precision. See Table 6 for details.

[0072] Table 6 Precision data of chlorogenic acid reference standard (3) Stability test: The results showed that the RSD of the chlorogenic acid peak area was 2.85% (n = 6), indicating that the test solution had good stability within 24 h. See Table 7 for details.

[0073] Table 7. Stability data of honeysuckle (4) Repeatability test: The results showed that the RSD of the chlorogenic acid peak area was 2.84% (n = 6), indicating that the method has good repeatability. See Table 8 for details.

[0074] Table 8. Repeatability data of honeysuckle (5) Recovery test: The results showed that the average recovery rate of chlorogenic acid was 97.64%, and the RSD was 2.05%, indicating that the method was accurate. See Table 9 for details.

[0075] Table 9. Recovery rate test data of honeysuckle. (6) Sample content determination: The experimental results showed that the content of crude drug sample was 32.31 mg / g (n = 3); the content of charred drug sample was 23.47 mg / g (n = 3), indicating that the content of chlorogenic acid in honeysuckle decreased after charring. The results are shown in Table 10.

[0076] Table 10 Results of Sample Content Determination 2. Experimental study on gastrointestinal irritation before and after charring honeysuckle 2.1 Carbon Powder Propulsion Experiment Table 11 Carbon-powder propulsion and Figure 6 The bar chart (*P<0.05, **P<0.01 vs. blank group) shows that the higher the concentration, the greater the carbon powder propulsion rate. Compared with the blank group, the carbon powder propulsion rate of both the raw honeysuckle group (raw) and the honeysuckle charcoal (processed) group increased. At the same concentration, the carbon powder propulsion rate of the raw group was greater than that of the processed group.

[0077] Table 11 Results of carbon-powder propulsion 2.2 Effects of honeysuckle charring on TRPM8 protein expression in colon tissue The expression of GAPDH and TRPM8, related proteins in the large intestine of mouse samples, was detected by Western blot. The results are as follows: Figure 7As shown, *P<0.05, **P<0.01 vs. blank group. Figure 7 In the figure, a represents the Western blot electrophoresis image of TRPM8 protein, and b represents the relative expression level of TRPM8 protein. The figure shows that, compared to the control group, the dose-dependent increase in TRPM8 in the raw product group was significantly higher. The honeysuckle charcoal (processed product) group also significantly increased the dose-dependent increase in TRPM8. Higher concentrations resulted in a stronger dose-dependent increase in TRPM8. However, at the same concentration, the dose-dependent increase in TRPM8 in the processed product group was weaker than that in the raw product group.

[0078] 2.3 Effects of honeysuckle charring on the pathological characteristics of stomach, small intestine, and large intestine tissues 2.3.1 H&E stained sections of small intestine observe Figure 8 H&E staining (×100) sections of the small and medium intestine revealed that: the small intestinal mucosa of mice in the blank group was intact, the glandular structure in the lamina propria was clear, the arrangement was regular, the cells were abundant and neatly arranged; the small intestinal mucosa of mice in the raw product group was severely damaged, the glands were damaged or disappeared, and the number of intact cells was reduced, and the higher the concentration, the more obvious the damage; compared with the raw product group, the morphology of the small intestinal mucosa in the charred product group at the same concentration was improved.

[0079] 2.3.2 H&E staining sections of colon observe Figure 9 H&E staining (×100) sections of the mid-colon revealed that: the colonic mucosa of mice in the blank group was intact, the glandular structure in the lamina propria was clear, regularly arranged, and the cells were abundant and neatly arranged; the colonic mucosa of mice in the raw product group was severely damaged, the glands were damaged or disappeared, and the number of intact cells was reduced, with the damage being more obvious at higher concentrations; the colonic mucosa morphology of the charred product group was improved compared to the raw product group at the same concentration.

[0080] 2.3.3 H&E stained sections of the stomach Figure 10 The effects of honeysuckle charring on the pathological structure of mouse gastric tissue (H&E staining, ×100) were investigated. The results showed that the gastric cell structure of mice in the blank control group was intact, the cell structure of the charred group was partially intact and partially damaged, and the cell structure of the raw group was significantly damaged.

[0081] Modern research shows that the content of honeysuckle's main active ingredients changes after it is charred. For example, the content of chlorogenic acid decreases, while the content of hemostatic components such as caffeic acid, quercetin, and luteolin increases. This may be the pharmacological basis for improving intestinal irritation after charring. Therefore, studying the effect of charred honeysuckle on improving gastrointestinal irritation will not only help broaden the clinical application of honeysuckle but also provide a safer and more effective drug option for the treatment of patients with spleen and stomach deficiency. In addition, this research will also provide a scientific basis for the processing technology of traditional Chinese medicine, promote the modernization and standardization of traditional Chinese medicine processing techniques, and has important theoretical and practical significance.

[0082] This invention utilizes a scientifically optimized processing technique to determine the optimal parameters for charring honeysuckle. Honeysuckle processed using this technique exhibits a high and stable chlorogenic acid content, with an average of 23.45 mg / g from three parallel experiments, close to the predicted value. Furthermore, compared to raw honeysuckle, charred honeysuckle shows significant improvement in gastrointestinal irritation. At the same concentration, the carbon propulsion rate of the charred group is lower than that of the raw group; its effect on enhancing TRPM8 protein expression is weaker than that of the raw group; and in terms of pathological manifestations in the small intestine, large intestine, and stomach, damage to the mucosal morphology is alleviated compared to the raw group, making it more suitable for patients with spleen and stomach deficiency and cold, thus expanding the clinical application range of honeysuckle. In addition, this invention employs a combination of TG-DSC, single-factor experiments, and response surface methodology, making the honeysuckle processing technique more scientific and reliable, providing a precise and effective method for charring honeysuckle.

[0083] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the inventive concept of the present invention, can make other changes and modifications to these embodiments, all of which fall within the scope of the present invention.

[0084] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. A processing technology optimization method of honeysuckle roasted carbon, characterized in that, The method comprises the following steps: extracting effective components of honeysuckle to obtain chlorogenic acid extract, luteoloside extract, water extract, alcohol extract; TG-DSC is used to dynamically monitor pyrolysis characteristics of chlorogenic acid extract, luteoloside extract, water extract, alcohol extract, chlorogenic acid reference substance, honeysuckle reference substance and honeysuckle raw medicinal material powder, and a processing temperature range of honeysuckle is preliminarily obtained according to the pyrolysis characteristics; processing temperature and processing time are used as single factor variables, and the content of organic acid in processed honeysuckle is used as an evaluation index to determine an optimization range of processing time of honeysuckle; the processing temperature range of honeysuckle preliminarily determined according to the pyrolysis characteristics and the processing time optimization range of honeysuckle determined according to the single factor experiment results are combined, and response surface method is used to further optimize the processing technology of honeysuckle to determine the optimal processing temperature and processing time.

2. The processing technology optimization method of honeysuckle roasted charcoal according to claim 1, characterized in that, The chlorogenic acid extract is extracted by using a 75% methanol solution, and the luteoloside extract is extracted by using a 70% ethanol solution.

3. The processing technology optimization method of honeysuckle roasted charcoal according to claim 1, characterized in that, The water extract comprises chlorogenic acid, luteoloside and saponin.

4. The processing technology optimization method of honeysuckle roasted charcoal according to claim 1, characterized in that, The alcohol extract is extracted by using anhydrous ethanol, and the alcohol extract comprises chlorogenic acid, luteoloside and caffeic acid.

5. The processing technology optimization method of honeysuckle roasted charcoal according to claim 1, characterized in that, When the processing temperature range of honeysuckle is preliminarily determined according to the pyrolysis characteristics, the peak temperature range of the maximum thermal weight loss rate peak in the first pyrolysis stage of honeysuckle raw medicinal material powder is used as the processing temperature range of honeysuckle.

6. The processing technology optimization method of honeysuckle roasted charcoal according to claim 1, characterized in that, The organic acid is chlorogenic acid.

7. The processing technology optimization method of honeysuckle roasted charcoal according to claim 1, characterized in that, The response surface method uses processing temperature and processing time as independent variables, and uses the content of chlorogenic acid as a response value, and Design Expert 13.0.1.0 software is used to fit a quadratic polynomial model.

8. The processing technology optimization method of honeysuckle roasted charcoal according to claim 7, characterized in that, The quadratic polynomial model of the response surface method is: chlorogenic acid content = 24.35 + 1.13A + 0.2608B - 0.0350AB - 2.61A² - 0.9279B²; In the formula, A is processing temperature, and B is processing time.

9. The processing technology of honeysuckle carbonized by roasting is optimized by using the method in any one of claims 1-8, the optimal processing temperature is 205 ℃, and the processing time is 7 min.

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