Use of safflower carbon in the preparation of liver-protecting drugs

Abstract

The application belongs to the technical field of medicine, and relates to medical use of safflower charcoal, in particular to use of the safflower charcoal in preparation of liver-protecting drugs.The safflower charcoal is prepared by a dry heat sterilization method, and animal experiments show that the dry heat sterilized safflower charcoal can significantly reduce the mortality of AIH model mice induced by ConA, liver tissue inflammation and cell apoptosis.The safflower charcoal has a good application prospect in liver protection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the medicinal uses of safflower charcoal, specifically its use in the preparation of hepatoprotective drugs. Background Technology

[0002] Autoimmune hepatitis (AIH) is an inflammatory disease characterized by liver parenchymal damage that can occur at any age and in any ethnic group. Without effective treatment, AIH can lead to progressive liver damage, gradually progressing to critical conditions such as cirrhosis or acute liver failure, ultimately resulting in death. The pathogenesis of AIH is complex, and current clinical treatment primarily relies on the use of glucocorticoids alone or in combination with immunosuppressants; however, this approach has been associated with intolerance and numerous side effects.

[0003] Safflower, a commonly used floral medicinal material in clinical practice, is the dried flower of the safflower plant (Carthamus tinctorius). Its effects include promoting blood circulation, regulating menstruation, dispersing blood stasis, and relieving pain. It is widely used in gynecology, orthopedics, dermatology, and other fields, with remarkable therapeutic effects. According to statistics, the 2020 edition of the Chinese Pharmacopoeia lists over 100 traditional Chinese medicine preparations containing safflower. More than 20 of these, including Zheng Tian Wan, Qi Li San, Tong Bi Capsules, Nao Xin Tong Capsules, Ba Wei Tan Xiang San, Die Da Qi Li Pian, and Fu Fang Yi Gan Wan, are directly formulated from safflower powder.

[0004] With the development of the modern Chinese medicine industry, the sterilization process of Chinese medicinal materials and their preparations, as a key link in ensuring product quality, has received increasing attention and research. In recent years, significant progress has been made in the research of sterilization processes for raw Chinese medicinal powders. Although traditional sterilization processes such as dry heat and moist heat can effectively kill microorganisms to a certain extent, they may also cause significant damage to the active ingredients, thus affecting efficacy. Therefore, how to maximize the preservation of the active ingredients of Chinese medicinal materials while ensuring sterilization effectiveness has become the key point in the selection of sterilization processes. Summary of the Invention

[0005] In the search for new and effective hepatoprotective drugs, the inventors creatively applied safflower charcoal to protect the liver, unexpectedly discovering its significant hepatoprotective effect. Therefore, this invention provides the use of safflower charcoal in the preparation of hepatoprotective drugs.

[0006] The safflower charcoal of this invention is prepared by dry heat sterilization.

[0007] In some embodiments of the present invention, the preparation method of the safflower charcoal is as follows: take an appropriate amount of safflower medicinal material, crush it, and sieve it to obtain safflower medicinal material raw powder; spread the raw powder evenly in a stainless steel tray, wrap it with kraft paper, place it in an electric heating forced-air drying oven, heat it at 130-180℃ for 2-3 hours, and then transfer it to a clean room for vacuum drying to obtain safflower charcoal after dry heat sterilization treatment.

[0008] In a preferred embodiment of the present invention, the preparation method of the safflower charcoal is as follows: Take an appropriate amount of safflower medicinal material, crush it, pass it through a No. 6 sieve to obtain raw safflower medicinal material powder; spread the raw powder evenly in a stainless steel tray, wrap it with kraft paper, place it in an electric heating forced-air drying oven, heat it at 180°C for 2 hours, and then transfer it to a clean room for vacuum drying (65°C ≤ -0.095MPa) for 30 minutes to obtain safflower charcoal after dry heat sterilization.

[0009] This invention provides the use of safflower charcoal in the preparation of hepatoprotective drugs.

[0010] This invention provides the use of safflower charcoal in the preparation of drugs for the prevention or treatment of autoimmune hepatitis.

[0011] Preferably, the safflower charcoal significantly reduces the mortality rate of patients with autoimmune hepatitis.

[0012] Preferably, the safflower charcoal significantly reduces ALT and AST levels in patients with autoimmune hepatitis.

[0013] Preferably, the safflower charcoal significantly reduces liver tissue inflammation in patients with autoimmune hepatitis.

[0014] Preferably, the safflower charcoal significantly reduces cell apoptosis in patients with autoimmune hepatitis.

[0015] In the uses described in this invention, the drug is a preparation made by adding pharmaceutically acceptable conventional excipients with safflower charcoal as the active ingredient.

[0016] Furthermore, the pharmaceutically acceptable excipients may be excipients, fillers, diluents, binders, disintegrants, solubilizers, adsorbents, pH buffers, preservatives, flavoring agents, or combinations thereof.

[0017] Furthermore, the preparation is an oral preparation, an injectable preparation, or a topical preparation.

[0018] Furthermore, the oral preparations are tablets, pills, powders, granules, capsules, or syrups; the injectable preparations include intravenous injections, intraperitoneal injections, intracavitary injections, irrigation solutions, or rinsing solutions; and the external preparations include patches, ointments, or gels.

[0019] To formulate the compositions of the present invention into tablets, a wide variety of excipients known in the relevant art can be used, including diluents, binders, wetting agents, disintegrants, lubricants, and flow aids. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropanol, etc.; binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; lubricants and flow aids can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0020] Tablets can also be further processed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0021] The present invention also provides a method for protecting the liver, including providing an effective amount of safflower charcoal to patients in need.

[0022] As used herein, the term "treatment" and other similar synonyms include relieving, reducing, or improving symptoms of a disease or condition; inhibiting a disease or condition, such as preventing its progression; alleviating a disease or condition; improving a disease or condition; relieving symptoms caused by a disease or condition; or stopping symptoms of a disease or condition; preventing other symptoms; improving or preventing the underlying metabolic causes of symptoms; and, moreover, the term includes preventative purposes. The term also includes achieving therapeutic and / or preventative effects. A therapeutic effect refers to the cure or improvement of the underlying disease being treated. Furthermore, the cure or improvement of one or more physiological symptoms associated with the underlying disease is also a therapeutic effect; for example, an improvement is observed in a patient even though they may still be affected by the underlying disease. In terms of preventative effects, the composition may be administered to patients at risk of developing a specific disease, or to patients exhibiting one or more physiological symptoms of a disease, even if no disease diagnosis has been made.

[0023] The beneficial effects of this invention are as follows:

[0024] This invention successfully induced an autoimmune hemorrhage (AIH) model in mice using ConA. Compared to the normal group, the model group mice showed mortality, significantly increased ALT and AST activity levels, and pathological results revealed extensive inflammatory cells and tissue necrosis in the liver tissue. Treatment with safflower prepared using various sterilization processes significantly reduced the mortality rate, liver inflammation, and cell apoptosis in ConA-induced AIH model mice. Simultaneously, safflower prepared using various sterilization processes significantly reduced serum ALT and AST levels in the AIH model. Among these, safflower charcoal prepared using dry heat sterilization showed the most significant therapeutic effect on AIH mice, followed by safflower prepared using irradiation and ultraviolet sterilization processes, while safflower prepared using moist heat sterilization showed the least effect.

[0025] The charred safflower of this invention significantly reduces ALT and AST in mice with autoimmune hepatitis and effectively improves liver damage, with effects significantly superior to raw safflower powder.

[0026] The safflower charcoal of this invention significantly reduces ALT and AST in mice with autoimmune hepatitis and effectively improves liver damage, with significantly better effects than peony bark charcoal and red peony charcoal.

[0027] The safflower charcoal of this invention significantly reduces ALT and AST levels in patients with autoimmune hepatitis.

[0028] The safflower charcoal of this invention has a promising application prospect in protecting and nourishing the liver. Attached Figure Description

[0029] Figure 1 Characteristics of safflower in various sterilization processes.

[0030] Figure 2 Comparison of HPLC chromatograms of safflower from different sterilization processes A. HPLC chromatogram of unsterilized safflower; B. HPLC chromatogram of reference standard: 1: protocatechuic acid, 2: hydroxysafflower yellow A, 3: chlorogenic acid, 4: rutin, 5: dehydrated safflower yellow B, 6: quercetin, 7: kaempferol; C. HPLC chromatogram of safflower processed by 5 different sterilization methods.

[0031] Figure 3 a: ALT activity in the serum of mice in each group; b: AST activity in the serum of mice in each group; compared with the normal group, there was a significant difference (*P<0.05) and a highly significant difference (**P<0.01); compared with the model group, there was a significant difference (#P<0.05) and a highly significant difference (##P<0.01).

[0032] Figure 4 Pathological damage in mice of each group. Detailed Implementation

[0033] Through extensive and in-depth research, the inventors of this invention have provided the use of safflower charcoal in the preparation of preventive or hepatoprotective drugs.

[0034] The following detailed description of specific embodiments further illustrates the content of the present invention, but it should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following embodiments. The following embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention shall still fall within the scope of protection of the present invention.

[0035] Preparation Example 1: Preparation of Dry Heat Sterilized Safflower (Safflower Charcoal)

[0036] Take an appropriate amount of safflower, crush it, and pass it through a No. 6 sieve to obtain raw safflower powder. Spread the raw powder evenly in a stainless steel tray, wrap it with kraft paper, and place it in an electric heating drying oven. Heat it at 180℃ for 2 hours, and then transfer it to a clean room for vacuum drying (65℃≤-0.095MPa) for 30 minutes to obtain safflower charcoal after dry heat sterilization.

[0037] Preparation Example 2: Preparation of Dry Heat Sterilized Safflower (Safflower Charcoal)

[0038] Take an appropriate amount of safflower, crush it, and pass it through a No. 6 sieve to obtain raw safflower powder. Spread the raw powder evenly in a stainless steel tray, wrap it with kraft paper, and place it in an electric heating drying oven. Heat it at 160℃ for 2 hours, and then transfer it to a clean room for vacuum drying (65℃≤-0.095MPa) for 30 minutes to obtain safflower charcoal after dry heat sterilization.

[0039] Preparation Example 3: Preparation of Dry Heat Sterilized Safflower (Safflower Charcoal)

[0040] Take an appropriate amount of safflower, crush it, and pass it through a No. 6 sieve to obtain raw safflower powder. Spread the raw powder evenly in a stainless steel tray, wrap it with kraft paper, and place it in an electric heating drying oven. Heat it at 130℃ for 3 hours, and then transfer it to a clean room for vacuum drying (65℃≤-0.095MPa) for 30 minutes to obtain safflower charcoal after dry heat sterilization.

[0041] Example 1: Comprehensive Analysis of Safflower Medicinal Materials under Different Sterilization Processes

[0042] 1. Experimental Materials

[0043] Safflower medicinal material (batch number 2203024) comes from Lunan Houpu Pharmaceutical Co., Ltd.

[0044] Take an appropriate amount of safflower, crush it, and pass it through a No. 6 sieve to obtain raw safflower powder (No. S1).

[0045] Take an appropriate amount of raw safflower powder and sterilize it by dry heat according to the method in Preparation Example 1 to obtain safflower charcoal after dry heat sterilization (number S2). Then, process the safflower powder according to the following wet heat sterilization, ultraviolet sterilization and irradiation sterilization processes.

[0046] The moist heat sterilization method is as follows: the raw powder of safflower is spread evenly in a stainless steel tray, wrapped with kraft paper, placed in a high-pressure steam sterilizer, heated at 121℃ for 0.5 hours, and then transferred to a clean room for vacuum drying (65℃, ≤ -0.095MPa) for 30 min to obtain safflower after moist heat sterilization (number S3).

[0047] The ultraviolet sterilization method is as follows: the raw powder of safflower is spread evenly in a stainless steel tray, wrapped in kraft paper, placed in an ultraviolet disinfection cabinet, irradiated at 254nm for 2 hours, and then transferred to a clean room for vacuum drying (65℃, ≤-0.095 MPa) for 30 min to obtain safflower after ultraviolet sterilization (number S4).

[0048] The irradiation sterilization method is as follows: Safflower raw powder is packaged in a PE composite film bag, placed in an electron irradiation processing system, and subjected to electron beam irradiation treatment at 9 kGy. After that, it is transferred to a clean room for vacuum drying (65 ℃, ≤ -0.095 MPa) for 30 min, and then used for later use to obtain safflower (number S5) after irradiation sterilization treatment.

[0049] 2. Comparison of traits

[0050] The properties of safflower after various sterilization processes were compared, including color, odor, and texture.

[0051] 3. Comparison of HPLC chromatograms of safflower during sterilization process

[0052] HPLC Chromatographic Conditions

[0053] Chromatographic column: Agilent SB-Aq (4.6×250nm, 5μm); mobile phase: 0.05% phosphoric acid aqueous solution-acetonitrile, gradient elution, elution gradient as shown in Table 1; flow rate: 0.8mL; column temperature: 30℃; injection volume: 10μL; detection wavelength: 264nm.

[0054] Table 1 Elution gradient

[0055] 4. Chemical composition analysis of safflower by UPLC-Q-Exactive Orbitrap-MS for each sterilization process

[0056] Safflower samples from various sterilization processes were collected, and the spectra of safflower groups from each sterilization process were analyzed and identified. The response values ​​of the identified compounds were recorded, and the results of unsterilized safflower were used as a reference for comparison. The changes in the response values ​​of the identified compounds were compared to provide a certain reference indicator for the changes in the content of each chemical component.

[0057] Chromatographic conditions

[0058] Waters ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm); mobile phase: 0.1% formic acid aqueous solution-acetonitrile, elution gradient as shown in Table 2; flow rate: 0.2 mL; column temperature: 30℃; injection volume: 1 mL. μ L.

[0059] Table 2 Elution gradient

[0060] Mass spectrometry conditions: Heated electrospray ionization source (HESI), positive and negative ion modes; scanning mode: Full MS / dd-MS2; scanning mass range: m / z 100~1500; Full MS resolution: 70,000; dd-MS2 scanning resolution: 17,500; auxiliary gas flow rate: 35 arb; sheath gas flow rate: 10 arb; capillary temperature: 350℃; auxiliary gas temperature: 350℃; physicochemical voltage: 3.0 kV; collision energy gradients: 20, 30, and 40 eV.

[0061] 5. Experimental Results

[0062] 5.1 Effects of various sterilization processes on safflower traits

[0063] (1) Moist heat sterilization: After moist heat sterilization, the color of safflower turns brownish-black and has a burnt smell. Figure 1 A).

[0064] (2) Dry heat sterilization: After dry heat sterilization, safflower turns dark brown, emits a strong burnt smell, and the powder becomes harder. Figure 1 B).

[0065] (3) Ultraviolet sterilization: After ultraviolet sterilization, safflower showed no significant changes in color or odor. Figure 1 C).

[0066] (4) Irradiation sterilization: After irradiation sterilization, safflower showed no significant changes in color or odor. Figure 1 D).

[0067] 5.2 Comparison of HPLC chromatograms of safflower from different sterilization processes

[0068] Unsterilized safflower and safflower processed by four different sterilization processes were collected and analyzed by HPLC, and the chromatograms were recorded. Data analysis was performed using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)". Using S1 as the reference chromatogram, with a time window width of 0.5 min, the average method was employed to perform full-spectrum peak matching, generating fingerprint chromatograms for each sterilization process and their corresponding control fingerprint chromatograms (common mode, R).

[0069] The HPLC chromatogram of unsterilized safflower is shown below. Figure 2 A, the reference standard chromatogram is shown in [reference]. Figure 2 B, HPLC chromatograms of safflower under five sterilization processes are shown in [reference]. Figure 2 C. A total of 9 common peaks were identified, while no common peaks were identified in the control, indicating significant quality differences among safflower samples sterilized using different methods. Compared to unsterilized safflower, the number of peaks was significantly reduced in the dry heat sterilized and wet heat sterilized safflower groups, suggesting that these two sterilization processes have a greater impact on the chemical composition of safflower.

[0070] Using the unsterilized safflower group (S1) as a reference, the similarity of the spectral data of safflower samples from each sterilization process was calculated to be 0.191, 0.434, 0.994, and 0.993, respectively. The results are shown in Table 3. Among them, the similarity of S4 and S5 is greater than 0.99, indicating that the ultraviolet and irradiation sterilization processes have almost no effect on the chemical composition of safflower. However, the similarity of S1 and S2 decreased significantly. The dry heat sterilization group had the lowest similarity, indicating that the chemical composition of safflower changed significantly after wet heat and dry heat sterilization, and the dry heat sterilization had the greatest impact.

[0071] Table 3. HPLC similarity of sterilized safflower species

[0072] 5.3 Chemical composition analysis of safflower by various sterilization processes using UPLC-Q-Exactive Orbitrap-MS

[0073] The results are shown in Table 4. Forty-one components were identified in unsterilized, irradiated, and UV-sterilized safflower; 34 components were identified in dry-heat-sterilized safflower; and 40 components were identified in moist-heat-sterilized safflower. Dry-heat sterilization significantly reduced the response values ​​of over 90% of the components, and moist-heat sterilization also reduced the response values ​​of most components. Irradiation and UV sterilization had relatively little impact on the response values ​​of most components, indicating that irradiation and UV sterilization are superior in preserving most components.

[0074] Table 4 Comparison of Response Value Changes

[0075]

[0076]

[0077] Concanavalin A (ConA) can induce autoimmune hepatitis (AIH), an acute liver injury model with a high mortality rate. Injection of ConA into the tail vein of mice leads to the activation of numerous immune cells, release of pro-inflammatory factors, elevated ALT and AST levels, immune cell infiltration, and liver tissue necrosis, similar to clinical AIH symptoms. Furthermore, its rapid modeling time, low cost, and reproducibility make it a representative AIH model.

[0078] Example 2: Hepatoprotective effect of safflower charcoal of the present invention on mice with autoimmune hepatitis.

[0079] 1. Laboratory animals Sixty SPF-grade male BALB / c mice (23–25 g, 7–8 weeks old) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. During the experiment, the animals were housed at room temperature (23 ± 2)℃ and humidity 40%–70%, with a 12-hour light-dark cycle and free access to food and water. The experiments were conducted after 3 days of acclimatization.

[0080] 2. Experimental Methods

[0081] 2.1 Processing of Safflower Medicinal Material

[0082] Safflower was processed using the four sterilization processes shown in Table 5. The sterilized safflower was then prepared into a 0.1 mg / mL suspension for later use. The dosage of safflower specified in the 2020 edition of the Chinese Pharmacopoeia was converted to the dosage for mice based on body surface area. The dosage for oral administration to mice is shown in Table 6.

[0083] Table 5. Sterilization process and parameters for raw powder of medicinal flowers

[0084] Table 6 Dosage settings for oral administration to mice

[0085] 2.2 Drug administration and model establishment in animals

[0086] SPF-grade male BALB / c mice were randomly assigned to 6 groups (n=10) according to body weight: control group, model group, moist heat sterilization group (SRMJ), dry heat sterilization group (GRMJ), ultraviolet sterilization group (ZWMJ), and irradiation sterilization group (FZMJ). Except for the control group and the model group, mice in each group were administered safflower suspension prepared according to the corresponding sterilization process by gavage (ig) at a dose of 0.1 mL / 10 g. Mice in the control group and the model group were given the same volume of purified water. The administration was carried out at the same time every day, once a day, for 14 consecutive days as a preventive measure.

[0087] One hour after the last administration, except for the normal group, mice in all groups were induced to develop an autoimmune hepatitis (AIH) model by intravenous injection of 14 mg / kg concanavalin A (ConA) solution via tail vein. Mice in the normal group were injected with an equal volume of physiological saline via tail vein. All mice were fasted but allowed free access to water, and experimental specimens were collected 24 hours later.

[0088] 2.3 Animal-based materials

[0089] The mortality rate and body weight of mice in each group were recorded. The mice were anesthetized with 4% chloral hydrate, and peripheral blood was collected from the main abdominal vein. After standing at room temperature for 1 hour, the blood was centrifuged at 4000 rpm for 10 minutes to separate the serum.

[0090] After collecting peripheral blood samples from mice, liver and spleen samples were quickly removed and weighed. Liver and spleen indices were calculated.

[0091] Liver index = liver weight × 1000 / mouse body weight; spleen index = spleen weight × 1000 / mouse body weight.

[0092] The liver was fixed in a 4% paraformaldehyde solution for histopathology.

[0093] 2.4 Serum level detection

[0094] The levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in the obtained mouse serum samples were detected using a BS-800 fully automated biochemical analyzer.

[0095] 2.5 Pathological analysis of mouse liver

[0096] Liver tissue fixed in 4% paraformaldehyde solution was removed, dehydrated by gradient alcohol, embedded, and then prepared into paraffin sections (5 μM) using a microtome. The sections were then baked at 60°C for 30 min.

[0097] 2.5.1 Specific operating steps

[0098] 1) Dewaxing: Xylene I 10 min → 100% ethanol 5 min → Xylene II 10 min → 100% ethanol 5 min → 95% ethanol 5 min → 80% ethanol 5 min → 70% ethanol 5 min → 70% ethanol 5 min → PBS washing, dewaxing.

[0099] 2) Hematoxylin and eosin (H&E) staining: The tissue was stained with HE using an automated staining machine (Leicast 5020) under the following conditions: hematoxylin staining for 3 min → rinsing with purified water for 5 min → differentiation with 1% hydrochloric acid ethanol for 10 s → rinsing with purified water for 5 min → eosin staining for 3 min → rinsing with purified water for 5 min → immersion in 95% ethanol I for 1 min → 95% ethanol II for 1 min → 100% ethanol I for 3 min → 100% ethanol II for 5 min → clearing with xylene for 10 min → mounting.

[0100] 3) Use 3D Histech Pannoramic Scan digital slides to perform system scanning and image analysis.

[0101] 2.6 Statistical Analysis

[0102] GraphPad Prism 9.5.0 software was used for plotting. One-way ANOVA was used for comparisons between groups. Experimental data are expressed as mean ± standard deviation. The statement indicates that the statistical significance of differences between multiple groups and two groups was assessed using one-way ANOVA and the T-test. P Results with a value <0.05 were considered statistically significant.

[0103] 3. Experimental Results

[0104] 3.1 Effects of different sterilization processes on organ indices of safflower in mice

[0105] Twenty-four hours after injection of ConA solution, one mouse in the model group died, while no mice in the other groups died. The liver and spleen indices of the mice in each group are shown in Table 7.

[0106] Compared to the Control group, the Model showed a significant increase in liver and spleen indices. P <0.001) indicates that the model was successfully created.

[0107] Regarding the spleen index, compared with the control group, all safflower groups under each sterilization process showed a significant increase, while compared with the model, all safflower groups under each sterilization process showed a decreasing trend. However, except for moist heat sterilization (SRMJ), there were no significant differences among the other groups.

[0108] Regarding liver indices, compared with the Control group, all safflower groups under different sterilization processes showed a significant increase, while compared with the Model group, there was no significant difference among the safflower groups under different sterilization processes.

[0109] The above results indicate that safflower from different sterilization processes had little effect on the spleen and liver indices of mice, with almost no significant difference.

[0110] Table 7. Liver and spleen indices of mice in each group.

[0111] The difference was statistically significant compared to the normal group (*). P <0.05), extremely significant difference (**) P <0.01); statistically significant compared with the model group ( # P <0.05), extremely significant difference ( ## P <0.01).

[0112] 3.2 Effects of different sterilization processes on serum biochemical parameters of safflower in mice

[0113] To investigate the protective effect of safflower against AIH after different sterilization methods, this study established a Con A-induced AIH mouse model.

[0114] The results are shown in Table 8 and Figure 3 As shown, compared with the control group, the levels of ALT and AST in the serum of the model increased ( P <0.01).

[0115] Compared to the Model, safflower treated with various sterilization methods showed statistically significant differences in ALT and AST levels, with dry heat sterilization (GRMJ) showing the highest ALT levels. P <0.01) and AST ( P The content of <0.01) was significantly reduced. Dry heat sterilization (GRMJ) had a better therapeutic effect than other sterilization methods. Ultraviolet sterilization and irradiation sterilization processes had better therapeutic effects than moist heat treatment sterilization.

[0116] The above results indicate that safflower treated with all four sterilization methods has a protective effect against Con A-induced liver function damage in AIH mice, and safflower charcoal sterilized by dry heat has a more significant protective effect.

[0117] Table 8. ALT and AST levels in each group of mice.

[0118] The difference was statistically significant compared to the normal group (*). P <0.05), extremely significant difference (**) P <0.01); statistically significant compared with the model group ( # P <0.05), extremely significant difference ( ##P <0.01).

[0119] 3.3 Effects of different sterilization processes on pathological damage of safflower in AIH mice

[0120] The liver tissue of experimental animals was pathologically examined using hematoxylin and eosin (H&E) staining techniques, and the morphological changes of the livers in different treatment groups were systematically observed.

[0121] See results Figure 4 Pathological examination results showed that the liver tissue structure of the control group mice was intact, the outline of the liver lobules was clearly visible, the hepatocytes around the central vein showed a regular radial arrangement, and the liver sinusoids were normal in morphology without dilation.

[0122] The liver tissue of the model mice showed typical pathological damage features, including significant inflammatory cell aggregation in the portal venous area, hepatocyte edema, severe sinusoidal congestion around the central vein, and punctate or patchy necrosis.

[0123] Safflower treated with different sterilization processes effectively reduced ConA-induced liver tissue damage and significantly alleviated pathological necrosis. Among them, the dry heat sterilization (GRMJ) treatment group showed the best intervention effect, which not only significantly reduced the infiltration of inflammatory cells in the liver parenchyma, but also effectively inhibited hepatocyte necrosis.

[0124] The results showed that safflower treated with different sterilization methods had a certain liver protective effect on mice with autoimmune hepatitis (AIH), and the safflower charcoal prepared by dry heat sterilization had the best therapeutic effect.

[0125] Safflower charcoal prepared by dry heat sterilization is more effective in treating autoimmune hepatitis than safflower treated by other sterilization methods.

[0126] The present invention describes the hepatoprotective effect of safflower charcoal and safflower raw powder on mice with autoimmune hepatitis.

[0127] 1. Laboratory animals

[0128] Forty SPF-grade male BALB / c mice (23–25 g, 7–8 weeks old) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. During the experiment, the animals were housed at room temperature (23 ± 2)℃ and humidity 40%–70%, with a 12-hour light-dark cycle, and free access to food and water. The experiments were conducted after 3 days of acclimatization.

[0129] 2. Experimental Methods

[0130] 2.1 Processing of Safflower Medicinal Material

[0131] Take an appropriate amount of safflower, pulverize it, and pass it through a No. 6 sieve to obtain raw safflower powder. Following the method in Preparation Example 1, dry heat sterilized safflower charcoal was prepared.

[0132] 2.2 Drug administration and model establishment in animals

[0133] SPF-grade male BALB / c mice were randomly assigned to four groups (n=10) based on body weight: Control group, Model group, Dry Heat Sterilization group (GRMJ, safflower charcoal after dry heat sterilization), and Safflower Herbal Medicine group (HHSW, safflower powder). Except for the Control and Model groups, mice in each group were administered the corresponding suspension by gavage (ig) at a dose of 0.1 mL / 10 g. Mice in the Control and Model groups were given the same volume of purified water. Administration was performed at a fixed time each day, once daily, for 14 consecutive days as a preventative measure.

[0134] One hour after the last administration, except for the normal group, mice in all groups were induced to develop an autoimmune hepatitis (AIH) model by intravenous injection of 14 mg / kg concanavalin A (ConA) solution via tail vein. Mice in the normal group were injected with an equal volume of physiological saline via tail vein. All mice were fasted but allowed free access to water, and experimental specimens were collected 24 hours later.

[0135] 2.3 Animal sampling and indicator testing

[0136] The mortality rate and body weight of mice in each group were recorded. The mice were anesthetized with 4% chloral hydrate, and peripheral blood was collected from the main abdominal vein. After standing at room temperature for 1 hour, the blood was centrifuged at 4000 rpm for 10 minutes to separate the serum.

[0137] The levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in the obtained mouse serum samples were detected using a BS-800 fully automated biochemical analyzer.

[0138] 2.6 Statistical Analysis

[0139] GraphPad Prism 9.5.0 software was used for plotting. One-way ANOVA was used for comparisons between groups. Experimental data are expressed as mean ± standard deviation. The statement indicates that the statistical significance of differences between multiple groups and two groups was assessed using one-way ANOVA and the T-test. P Results with a value <0.05 were considered statistically significant.

[0140] 3. Experimental Results

[0141] Effects on serum biochemical parameters in mice

[0142] The results are shown in Table 9. Compared with the control group, the serum ALT and AST levels in the Model increased ( P <0.01).

[0143] Compared to Model, ALT (in dry heat sterilization (GRMJ) P <0.01) and AST ( P The content of <0.01) was significantly reduced, and the therapeutic effect of dry heat sterilization (GRMJ) was better than that of the original safflower powder group.

[0144] Table 9. ALT and AST levels in each group of mice.

[0145] The difference was statistically significant compared to the normal group (*). P <0.05), extremely significant difference (**) P <0.01);

[0146] It is statistically significant compared with the model group. # P <0.05), extremely significant difference ( ## P <0.01);

[0147] The difference was statistically significant compared with the red peanut herb group. @ P <0.05), extremely significant difference ( @@ P <0.01).

[0148] This invention compares the hepatoprotective effects of safflower charcoal, peony bark charcoal, and red peony charcoal on mice with autoimmune hepatitis.

[0149] 1. Laboratory animals

[0150] Forty SPF-grade male BALB / c mice (23–25 g, 7–8 weeks old) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. During the experiment, the animals were housed at room temperature (23 ± 2)℃ and humidity 40%–70%, with a 12-hour light-dark cycle, and free access to food and water. The experiments were conducted after 3 days of acclimatization.

[0151] 2. Experimental Methods

[0152] 2.1 Preparation of Safflower Charcoal

[0153] Safflower charcoal was prepared by dry heat sterilization according to the method in Preparation Example 1.

[0154] Preparation of charcoal from peony bark

[0155] The peony bark was placed in a sealed crucible and calcined in a muffle furnace for 1 hour at a temperature of 350°C. After the muffle furnace cooled naturally to room temperature, the carbonized peony bark was removed, decocted with water, the filtrate was concentrated, and then dried.

[0156] Preparation of charred red peony root

[0157] The red peony root was placed in a sealed crucible and calcined in a muffle furnace for 1 hour at a temperature of 350°C. After the muffle furnace cooled naturally to room temperature, the carbonized red peony root was removed, decocted with water, the filtrate was concentrated, and then dried.

[0158] 2.2 Drug administration and model establishment in animals

[0159] SPF-grade male BALB / c mice were randomly assigned to four groups (n=8) based on body weight: Control group, Model group, Safflower Charcoal Group (HHT, safflower charcoal after dry heat sterilization), Moutan Bark Charcoal Group (DPT, moutan bark charcoal administered), and Red Peony Charcoal Group (CST, red peony charcoal administered). Except for the Control and Model groups, all mice were administered the corresponding drug at 1 g / kg via gavage (ig), with a dosage of 0.1 mL / 10 g. The Control and Model groups were given the same volume of purified water. The drugs were administered once daily at a fixed time for 14 consecutive days as a preventative measure.

[0160] One hour after the last administration, except for the normal group, mice in all groups were induced to develop an autoimmune hepatitis (AIH) model by intravenous injection of 14 mg / kg concanavalin A (ConA) solution via tail vein. Mice in the normal group were injected with an equal volume of physiological saline via tail vein. All mice were fasted but allowed free access to water, and experimental specimens were collected 24 hours later.

[0161] 2.3 Animal sampling and indicator testing

[0162] The mortality rate and body weight of mice in each group were recorded. The mice were anesthetized with 4% chloral hydrate, and peripheral blood was collected from the main abdominal vein. After standing at room temperature for 1 hour, the blood was centrifuged at 4000 rpm for 10 minutes to separate the serum.

[0163] The levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in the obtained mouse serum samples were detected using a BS-800 fully automated biochemical analyzer.

[0164] 2.6 Statistical Analysis

[0165] GraphPad Prism 9.5.0 software was used for plotting. One-way ANOVA was used for comparisons between groups. Experimental data are expressed as mean ± standard deviation. The statement indicates that the statistical significance of differences between multiple groups and two groups was assessed using one-way ANOVA and the T-test.P Results with a value <0.05 were considered statistically significant.

[0166] 3. Experimental Results

[0167] 3.1 Effects on serum biochemical parameters in mice

[0168] The results are shown in Table 10. Compared with the control group, the serum ALT and AST levels in the Model increased ( P <0.01).

[0169] Compared to the Model, the safflower charcoal group (HHT) had higher ALT content ( P <0.01) and AST ( P The content of <0.01) was significantly reduced, and the therapeutic effect of the safflower charcoal group (HHT) was better than that of the peony bark charcoal group (DPT) and the red peony charcoal group (CST).

[0170] Table 10. ALT and AST levels in each group of mice

[0171] The difference was statistically significant compared to the normal group (*). P <0.05), extremely significant difference (**) P <0.01);

[0172] It is statistically significant compared with the model group. # P <0.05), extremely significant difference ( ## P <0.01);

[0173] It was significantly different from the safflower charcoal group. @ P <0.05), extremely significant difference ( @@ P <0.01).

[0174] The safflower charcoal of this invention significantly reduces ALT and AST in mice with autoimmune hepatitis and effectively improves liver damage, with significantly better effects than peony bark charcoal and red peony charcoal.

[0175] All publications and patent applications cited in this application indicate the level of skill in the field to which this disclosure pertains. They are all incorporated herein by reference in their entirety.

[0176] The above embodiments are not intended to limit the present invention in any way. For those skilled in the art, any modifications or substitutions made within the scope of the present invention based on its technical essence shall still fall within the protection scope of the present invention.

Claims

1. The use of safflower charcoal in the preparation of hepatoprotective drugs.

2. Use of safflower charcoal in the preparation of drugs for the prevention or treatment of autoimmune hepatitis.

3. The use as described in claim 1 or 2, characterized in that, The safflower charcoal was prepared by dry heat sterilization.

4. The use as described in claim 3, characterized in that, The preparation method of the safflower charcoal is as follows: Take an appropriate amount of safflower medicinal material, crush it, and sieve it to obtain safflower medicinal material raw powder; spread the raw powder evenly in a stainless steel tray, wrap it with kraft paper, place it in an electric heating forced-air drying oven, heat it at 130-180℃ for 2-3 hours, and then transfer it to a clean room for vacuum drying to obtain safflower charcoal after dry heat sterilization.

5. The use as described in claim 4, characterized in that, The preparation method of the safflower charcoal is as follows: Take an appropriate amount of safflower medicinal material, crush it, pass it through a No. 6 sieve to obtain safflower medicinal material raw powder; spread the raw powder evenly in a stainless steel tray, wrap it with kraft paper, place it in an electric heating forced-air drying oven, heat it at 180℃ for 2 hours, and then transfer it to a clean room for vacuum drying for 30 minutes to obtain safflower charcoal after dry heat sterilization.

6. The use as described in claim 2, characterized in that, The safflower charcoal significantly reduced the mortality rate of patients with autoimmune hepatitis.

7. The use as described in claim 2, characterized in that, The safflower charcoal significantly reduced ALT and AST levels in patients with autoimmune hepatitis.

8. The use as described in claim 2, characterized in that, The safflower charcoal significantly reduced cell apoptosis in patients with autoimmune hepatitis.

9. The use as described in any one of claims 1-8, characterized in that, The drug is a formulation prepared by adding pharmaceutically acceptable conventional excipients, with safflower charcoal as the active ingredient. The pharmaceutically acceptable excipients may be excipients, fillers, diluents, binders, disintegrants, solubilizers, adsorbents, pH buffers, preservatives, flavoring agents, or combinations thereof.

10. The use as described in claim 9, characterized in that, The preparation is an oral preparation, an injection, or a topical preparation; the oral preparation is a tablet, pill, powder, granule, capsule, or syrup; the injection is an intravenous injection, intraperitoneal injection, intracavitary injection, irrigation solution, or rinsing solution; the topical preparation includes a patch, ointment, or gel.

Images