Application of natural-source compound monomer composition in preparation of injection medicine for preventing and / or treating pancreatitis

Abstract

The invention belongs to the technical field of medicines, and provides application of a natural-source compound monomer medicine composition in preparation of medicines for preventing and / or treating pancreatitis. The pharmaceutical composition comprises baicalin, paeoniflorin, geniposide, wogonin, baicalein, albiflorin, tetrahydropalmatine, rhein, wogonin, aloe-emodin, saikoside A, emodin, benzoyl paeoniflorin, chlorogenic acid, costunolide, or a pharmaceutically acceptable salt thereof. Compared with the prior art, the pharmaceutical composition disclosed by the invention has the advantages that the components of the composition are definite and are all natural sources, the safety is good, the pharmaceutical effect has a remarkable synergistic effect, the administration dosage is reduced by about 200 times under the condition that the treatment effect is better than that of a compound traditional Chinese medicine, the administration mode of the compound medicine for treating pancreatitis is innovated, and the pharmaceutical composition is suitable for clinical application. A new scheme is provided for the treatment of the disease.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a pharmaceutical composition for injection comprising baicalin, paeoniflorin, geniposide, wogonin, baicalein, paeoniflorin, tetrahydropalmatine, rhein, wogonin, aloe-emodin, saikosaponin A, emodin, benzoylpaeoniflorin, chlorogenic acid, costunolide, or salts thereof, and the use of the composition in preventing and / or treating acute pancreatitis. Background Art

[0002] Pancreatitis is an inflammatory disease caused by a variety of factors that lead to abnormal activation of trypsin, leading to autodigestion of pancreatic tissue. It is a common digestive system disease, often manifesting as severe or persistent upper abdominal pain. Acute pancreatitis is characterized by high rates of acute hospitalization and mortality. The main pathological manifestations are pancreatic edema, hemorrhage, and necrosis, which lead to an acute inflammatory response. Approximately 20% of patients develop severe disease, with systemic inflammatory response syndrome (SIRS) developing early on, accompanied by multi-organ dysfunction. If treatment is inadequate or delayed, it can lead to death. With improvements in living standards, the global annual incidence of pancreatitis continues to rise. Because its pathogenesis remains incompletely elucidated, the diagnosis and treatment of acute pancreatitis in clinical practice primarily focuses on symptomatic treatment and fluid replacement, aimed at reducing pancreatic secretions and secondary lesions, but the anti-inflammatory efficacy has not met expectations. The development of effective and safe therapeutic agents for the prevention and treatment of pancreatitis, especially severe acute pancreatitis, remains a pressing clinical need.

[0003] With the nationwide implementation of integrated Chinese and Western medicine for acute abdomen treatment, the treatment of pancreatitis has gradually formed a treatment plan that is mainly based on integrated Chinese and Western medicine internal medicine treatment, supplemented by timely surgery. According to the onset site and clinical characteristics of acute pancreatitis, with the concept of febrile disease as the main principle, the treatment concept of clearing the interior and attacking the lower part, clearing away heat and detoxifying is applied. Relying on the National Key Discipline / Specialty of Integrated Chinese and Western Medicine Clinical Practice of the First Affiliated Hospital of Dalian Medical University, the representative prescription Qingyi Fang (Qingyi Tang, Qingyi Granules) can achieve a relatively ideal anti-inflammatory effect for pancreatitis and has great application potential. Qingyi Fang is composed of 8 herbs: rhubarb, bupleurum, scutellaria, peony, costus root, corydalis, gardenia, and mirabilite. Research in the past three decades has found that Qingyi Fang has a good therapeutic effect on pancreatitis through multiple pathways and targets, and has now become one of the clinical treatment options in many hospitals in my country. The "Guidelines for the Diagnosis and Treatment of Acute Pancreatitis in China (2024, Shanghai)" has included Qingyi Fang and other prescriptions in the recommended treatment options. However, the complex and unclear composition of traditional Chinese medicine compounds, the difficulty in quality control, and the unclear composition of the active substances are the main problems limiting their modern application. Currently, there is no compound drug for the treatment of pancreatitis with clear ingredients and efficacy. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a natural source compound monomer composition for use in the preparation of an injectable drug for preventing and / or treating pancreatitis. The injectable drug composition is derived from a clinically effective compound, has clear ingredients after optimization, has synergistic anti-inflammatory effects, has an effective dose significantly lower than that of compound Chinese medicine, and has good safety. The injectable drug composition can significantly improve and treat pancreatitis (especially moderate to severe acute pancreatitis).

[0005] The present invention adopts the following technical solutions:

[0006] The present invention provides a use of a natural-source compound monomer composition in preparing a drug for preventing and / or treating pancreatitis. The composition comprises baicalin, paeoniflorin, geniposide, wogonin, baicalein, paeoniflorin, tetrahydropalmatine, rhein, wogonin, aloe-emodin, saikosaponin A, emodin, benzoylpeoniflorin, chlorogenic acid, costunolide, or pharmaceutically acceptable salts thereof.

[0007] In the above technical solution, further, the ratio of the pharmaceutical composition is optimized and screened based on the chemical component analysis of the clinically effective compound preparation and the results of multi-component in vivo component analysis, through ultra-high performance liquid chromatography-mass spectrometry tandem technology and cell-level activity evaluation. The mass ratio of baicalin, paeoniflorin, geniposide, wogonin, baicalein, paeoniflorin, tetrahydropalmatine, rhein, wogonin, aloe-emodin, saikosaponin A, emodin, benzoylpeoniflorin, chlorogenic acid, and costus oil in the pharmaceutical composition is 30-40:15-20:10-20:5-12:3-10:2-8:2-8:1.5-6:1.5-6:1.5-5:0.35-1.5:0.35-1.5:0.35-1.5:0.2-1:0.22-1. Preferably, the mass ratio of baicalin, paeoniflorin, gardenia jasminoidin, wogonin, baicalein, paeoniflorin, tetrahydropalmatine, rhein, wogonin, aloe-emodin, saikosaponin A, emodin, benzoylpeoniflorin, chlorogenic acid, and costunolide is 35.34:17.69:14.63:8.01:5.10:3.81:3.47:3.07:3.05:2.68:0.72:0.71:0.70:0.54:0.48, and the proportions are shown in the following table:

[0008]

[0009]

[0010] In the above technical solution, further, the baicalin has a structure shown in formula (I):

[0011] The paeoniflorin has a structure shown in formula (II):

[0012]

[0013] The geniposide has a structure shown in formula (III):

[0014]

[0015] The baicalin has a structure shown in formula (IV):

[0016] The baicalein has a structure shown in formula (V):

[0017]

[0018] The paeoniflorin has a structure shown in formula (VI):

[0019] The tetrahydropalmatine has a structure shown in formula (VII):

[0020] The rhein has a structure shown in formula (VIII):

[0021] The wogonin has a structure shown in formula (IX):

[0022] The aloe-emodin has a structure shown in formula (X):

[0023] Said saikosaponin A has the structure shown in formula (XI):

[0024] The emodin has a structure shown in formula (XII):

[0025]

[0026] The benzoylpenoside has a structure shown in formula (XIII):

[0027] The chlorogenic acid has a structure shown in formula (XIV):

[0028]

[0029]

[0030] The costunolide has a structure shown in formula (XV):

[0031]

[0032] In the above technical solution, the natural-source composite monomer composition is an injectable pharmaceutical composition, and the injectable pharmaceutical composition is administered intravenously. The solvent is a physiological saline solution containing 2-10% dimethyl sulfoxide. Preferably, the intravenous dosage of the composition is 10-20 mg / kg (rodents, mice; converted to a human dosage of 1.1-2.2 mg / kg).

[0033] In the above technical solution, further, the pharmaceutically acceptable salt includes hydrochloride, sulfate, hydrobromide, hydroiodide, formate, acetate, citrate or oxalate.

[0034] In the above technical solution, the composition further includes a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

[0035] In the above technical solution, further, the pancreatitis is an inflammatory lesion of the pancreatic tissue region caused by factors such as cholelithiasis, alcohol, blood vessels, trauma, infection, etc., manifested as abnormally increased serum amylase and (or) lipase concentrations and persistent pain in the upper abdomen, and the results of abdominal imaging examinations show imaging changes consistent with acute and chronic pancreatitis; preferably, the pancreatitis is moderate to severe acute pancreatitis.

[0036] The above-mentioned monomer combination has significant synergistic anti-inflammatory activity. After intravenous injection (including low doses) of the above-mentioned pharmaceutical composition, the area of ​​pancreatitis lesions in animals was significantly reduced, the pathological score was significantly improved, the level of pancreatic inflammation was significantly suppressed and basically close to the normal level, and the serum amylase and lipase levels were significantly reduced; no obvious liver and kidney toxicity was found. At the same time, the composition has a certain degree of improvement on liver function at a safe and effective dose.

[0037] The present invention is based on the chemical component analysis of clinically effective traditional Chinese medicine prescriptions for pancreatitis in the early stage and the results of multi-component in vivo component analysis at the animal level. By using ultra-high performance liquid chromatography-mass spectrometry tandem technology and cell-level activity evaluation and screening, the prescription is simplified and optimized. By simulating the exposure level of monomeric drug components in target tissues and changing the administration route according to the characteristics of their effects, a pharmaceutical composition containing baicalin, paeoniflorin, geniposide, wogonin, baicalein, paeoniflorin, tetrahydropalmatine, rhein, wogonin, aloe-emodin, saikosaponin A, emodin, benzoylpeoniflorin, chlorogenic acid, costunolide or pharmaceutically acceptable salts thereof is used to prepare a drug for preventing and / or treating pancreatitis. The ingredients in the above-mentioned composition are all of natural origin, and after combination, they have a synergistic anti-inflammatory effect that is superior to traditional Chinese medicine compounds. The effective dose is significantly lower than the oral dose of traditional Chinese medicine (about 200 times, 2g crude drug / kg→10mg / kg), has good safety, and an innovative administration method is an injectable preparation, providing a new solution for the treatment of this disease.

[0038] The experimental results showed that in the in vitro anti-inflammatory activity evaluation model of mouse acinar (266-6) cells and macrophage (RAW 264.7) cells, the anti-inflammatory activity of the pharmaceutical composition proposed in the present invention was superior to that of each monomer component at the same drug concentration, indicating that the composition has synergistic anti-inflammatory activity. BL / 6 In the mouse model, serum amylase and lipase concentrations were significantly higher than those in the normal control group, pancreatic islet cell fat-like vacuolar degeneration and fibrosis, pancreatic tissue loose edema, and the animal model produced (acute) pancreatitis symptoms that met the main clinical indications. After intravenous injection of the pharmaceutical composition proposed by the present invention, the fat-like vacuolar degeneration and fibrosis of pancreatic islet cells in the pancreatic tissue lesion area were improved at high and low doses, the degree of pancreatic tissue loose edema was alleviated, and the concentrations of amylase and lipase in serum were significantly reduced. The high-dose treatment group had a better therapeutic effect than the low-dose treatment group. In addition, compared to the oral compound Chinese medicine preparation (Qingyi Granules) treatment group far higher than the injection dose and the random combination treatment group of the two above-mentioned drug monomer components established at the same time, the composition proposed by the present invention had a better improvement effect on the pancreatic tissue lesion area and the therapeutic effect of lipase in serum. At the same time, the composition proposed by the present invention suggested that it has a certain protective effect on the liver. The above results illustrate that the pharmaceutical composition can synergistically exert its drug effect, effectively treat and improve the severity of pancreatitis.

[0039] Beneficial technical effects

[0040] The present invention proposes for the first time a pharmaceutical monomer composition comprising baicalin, paeoniflorin, geniposide, wogonin, baicalein, paeoniflorin, tetrahydropalmatine, rhein, wogonin, aloe-emodin, saikosaponin A, emodin, benzoylpaeoniflorin, chlorogenic acid, costunolide or their salts for the preparation of a drug for preventing and / or treating pancreatitis. Using two cell models and an animal model of pancreatitis induced by caerulein and lipopolysaccharide, this technical solution verified that the pharmaceutical composition can scientifically and synergistically exert its therapeutic effect on pancreatitis. Compared with the model group, intravenous injection of the pharmaceutical composition can significantly improve the fatty vacuolar degeneration and fibrosis of pancreatic islet cells in the pancreatic tissue lesion area, reduce the degree of pancreatic tissue loosening and edema, significantly reduce the levels of amylase and lipase in serum, and significantly improve the levels of pancreatic inflammatory factors interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha; in addition, the results of serum alanine aminotransferase and aspartate aminotransferase assays indicate that the drug combination has a certain protective effect on the liver, and no nephrotoxicity is shown. Furthermore, compared to oral Chinese herbal compound preparations with far higher injection doses and two randomized single-drug combinations, this pharmaceutical composition demonstrated superior efficacy in terms of pancreatitis pathology, amylase and lipase levels, and representative inflammatory factors such as interleukin-1beta and tumor necrosis factor-alpha. It also exhibited liver-protective effects similar to those of the Chinese herbal compound at both high and low doses, without exhibiting renal toxicity. Due to the complex etiology of pancreatitis, clinical treatments with single-target or single-ingredient drugs are ineffective, while multi-ingredient and multi-target approaches offer significant advantages. However, the composition of compound Chinese herbal medicines is unclear and difficult to control. Against this backdrop, the pharmaceutical composition proposed in this invention demonstrates outstanding technical benefits for the synergistic treatment / prevention of pancreatitis. The advantages of this invention lie in its anti-inflammatory and pancreatic pathology-modifying effects on pancreatitis that are far superior to those of currently clinically effective compound preparations and single-ingredient treatments. Furthermore, the ingredients of this pharmaceutical composition are all naturally derived, with a clear material basis, synergistic anti-inflammatory efficacy, a low onset dose (2g crude drug / kg → 10mg / kg), excellent efficacy, and good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The results show the expression level of inflammatory factor IL-6 in mouse acinar cells (266-6 cell line) after stimulation with sodium taurocholate and intervention with monomeric drug components.

[0042] Figure 2 The results show the expression level of inflammatory factor IL-1beta in mouse acinar cells (266-6 cell line) after stimulation with sodium taurocholate and intervention with monomeric drug components.

[0043] Figure 3The results show the expression level of inflammatory factor IL-6 in mouse macrophages (RAW 264.7 cell line) after stimulation with lipopolysaccharide and intervention with monomeric drug components.

[0044] Figure 4 The results show the expression level of inflammatory factor IL-1beta in mouse macrophages (RAW 264.7 cell line) after stimulation with lipopolysaccharide and intervention with monomeric drug components.

[0045] Figure 5 The results of pancreatic HE pathological sections of (acute) pancreatitis model mice after treatment with the pharmaceutical composition;

[0046] Figure 6 The statistical results of pancreatic tissue pathology scores of (acute) pancreatitis model mice after treatment with the pharmaceutical composition;

[0047] Figure 7 The results of serum amylase assay in (acute) pancreatitis model mice after treatment with the pharmaceutical composition;

[0048] Figure 8 The results of serum lipase assay in (acute) pancreatitis model mice after treatment with the pharmaceutical composition;

[0049] Figure 9 The results are for measuring the serum inflammatory factor IL-6 in (acute) pancreatitis model mice after treatment with the pharmaceutical composition;

[0050] Figure 10 The results are for measuring the serum inflammatory factor IL-1beta in (acute) pancreatitis model mice after treatment with the pharmaceutical composition;

[0051] Figure 11 The results are for measuring the serum inflammatory factor TNF-alpha in mice with (acute) pancreatitis model after treatment with the pharmaceutical composition;

[0052] Figure 12 The results of serum alanine aminotransferase assay in (acute) pancreatitis model mice after treatment with the pharmaceutical composition;

[0053] Figure 13 The results of serum aspartate aminotransferase assay in (acute) pancreatitis model mice after treatment with the pharmaceutical composition;

[0054] Figure 14 These are the results of serum creatinine measurement in (acute) pancreatitis model mice after treatment with the drug composition. DETAILED DESCRIPTION

[0055] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0056] Example 1. Evaluation of the synergistic effect of the drug composition using an in vitro cell model

[0057] 1. Cells

[0058] Mouse pancreatic acinar cell line 266-6 and mouse monocyte-macrophage RAW264.7 cell line.

[0059] 2. Analysis Method

[0060] Graphs were generated using GraphPad Prism, and the results are expressed as mean ± SD. One-way ANOVA was used to analyze the differences between groups. All statistical analyses were two-tailed. P < 0.05 was considered a significant difference, indicated by *; P < 0.01 was considered a very significant difference, indicated by **; and P < 0.001 was considered an extremely significant difference, indicated by ***.

[0061] 3. Experimental Design and Methods

[0062] Experimental design: This case is an in vitro cell experiment, which is one of the common methods for evaluating drug activity. 6266-6 cells were seeded in 6-well plates and cultured for 24 h. They were divided into control group, model group and drug treatment group. The drug treatment groups included: (1) drug combination treatment group, (2) baicalin group, (3) paeoniflorin group, (4) gardenia glycoside group, (5) wogonin group, (6) baicalein group, (7) paeoniflorin group, (8) tetrahydropalmatine group, (9) rhein group, (10) wogonin group, (11) aloe-emodin group, (12) saikosaponin A group, (13) emodin group, (14) benzoylpeoniflorin group, (15) chlorogenic acid group and (16) costunolide group. The mass ratios of baicalin, paeoniflorin, gardenia jasminoidin, wogonin, baicalein, paeoniflorin, tetrahydropalmatine, rhein, wogonin, aloe-emodin, saikosaponin A, emodin, benzoylpeoniflorin, chlorogenic acid and costunolide in the pharmaceutical composition are 35.34:17.69:14.63:8.01:5.10:3.81:3.47:3.07:3.05:2.68:0.72:0.71:0.70:0.54:0.48. The cell treatment method is as follows: discard the cell supernatant, add 2mL DMEM medium and 1μL dimethyl sulfoxide to the control group; add 2mL DMEM medium containing 0.4mg / mL sodium taurocholate (STC, purchased from MCE, product number: HY-N0545) and 1μL dimethyl sulfoxide to each well of the model group; add 2mL DMEM medium containing 0.4mg / mL sodium taurocholate and 1μL drug (combination or monomer) to each well of each drug treatment group, so that the final concentration of each drug monomer or combination in the culture medium except costunolide is 50μM (costunolide concentration is 5μM), incubate at 37℃ 5% CO2 for 24h, and then collect the cells. In addition, about 1×10 6 RAW264.7 cells were seeded in 6-well plates and cultured for 24 hours. The grouping and drug addition methods were the same as above. 2 mL of DMEM medium and 1 μL of dimethyl sulfoxide were added to the control group, and 2 mL of DMEM medium containing 1 μg / mL lipopolysaccharide (LPS, purchased from MCE, product number: HY-D1056) and 1 μL of dimethyl sulfoxide were added to each well of the model group; 2 mL of DMEM medium containing 1 μg / mL lipopolysaccharide and 1 μL of drug (composition or monomer) were added to each well of each drug treatment group, so that the final concentration of each drug monomer or composition in the culture medium except costunolide was 50 μM (costunolide concentration was 5 μM). After incubation at 37°C and 5% CO2 for 24 hours, the cells were collected.

[0063] In the RT-qPCR experiment, Trizol solution was used to extract RNA from each group of cells, and reverse transcription was performed according to the instructions of the All-in-One First-Strand Synthesis Master Mix (with dsDNase) kit to obtain cDNA. Prepare the qPCR system using the Green qPCR Premix (Universal) kit and perform the assay. The primers are listed below:

[0064]

[0065]

[0066] 4. Results

[0067] Interleukin-6 (IL-6) plays a key role in pancreatitis (especially acute pancreatitis). It is not only an important marker of the severity of pancreatitis, but also a core mediator that drives the inflammatory cascade and systemic inflammatory response. Figure 1 As shown in Table 1, the expression level of the inflammatory factor interleukin-6 in 266-6 cells was significantly increased after sodium taurocholate modeling (relative expression level increased to 5.1 times), indicating that the pancreatic acinar cell injury model was successfully established. In each monomer treatment group, baicalin, gardenia jasminoides, wogonin, baicalein, rhein, and wogonin showed an inhibitory effect on interleukin-6; in addition, the pharmaceutical composition significantly inhibited the expression of interleukin-6 (expression level decreased by 64.63%, P < 0.05). At the same dose (molar concentration), the inhibitory effect was stronger than that of baicalin, paeoniflorin, gardenia jasminoides, wogonin, baicalein, paeoniflorin, tetrahydropalmatine, rhein, aloe-emodin, saikosaponin A, rhein, benzoylpeoniflorin, chlorogenic acid, and costunolide.

[0068] Interleukin-1beta (IL-1β) plays a core pro-inflammatory role in pancreatitis (especially acute pancreatitis). Its role runs through the entire process of pancreatitis occurrence, progression and complication formation, and can aggravate pancreatic tissue edema, necrosis and neutrophil infiltration, driving local pancreatic inflammation. Figure 2As shown in Table 2, in 266-6 cells, the expression level of the inflammatory factor interleukin-1beta was significantly increased after sodium taurocholate modeling (the relative expression level increased to 2 times), indicating that the cell model was successfully established. In each monomer treatment group, baicalin, gardenia jasminoides, wogonin, baicalein, rhein, wogonin and aloe-emodin showed significant inhibitory effects on interleukin-1beta; in addition, the drug combination significantly inhibited the expression of interleukin-1beta (expression decreased by 90.04%, P < 0.001), and at the same dose (molar concentration), its inhibitory effect was stronger than that of all 15 monomer drug components mentioned above, including baicalin, paeoniflorin, gardenia jasminoides, wogonin, baicalein, paeoniflorin, tetrahydropalmatine, rhein, wogonin, aloe-emodin, saikosaponin A, emodin, benzoylpeoniflorin, chlorogenic acid and costunolide.

[0069] In the pathological process of pancreatitis, macrophages play an important role in regulating inflammatory mediators. Damaged acinar cells can be recognized by macrophage surface receptors, release cytokines, recruit neutrophils, amplify local inflammation or play an anti-inflammatory role in damage repair. In mouse macrophages, such as Figure 3 As shown in Table 3, the expression level of the inflammatory factor interleukin-6 was significantly increased after lipopolysaccharide modeling (the relative expression level increased by about 1500 times), indicating that the modeling was successful. In each treatment group, the pharmaceutical composition significantly inhibited the expression of interleukin-6 (the expression level decreased by 61.03%, P < 0.001), and at the same dose (molar concentration), the inhibitory effect was stronger than that of baicalin, paeoniflorin, jasminoidin, wogonin, baicalein, paeoniflorin, tetrahydropalmatine, rhein, aloe-emodin, saikosaponin A, emodin, benzoylpeoniflorin, chlorogenic acid and costunolide. Figure 4 As shown in Table 4, the expression level of the key inflammatory factor interleukin-1beta was significantly increased after modeling (the relative expression level increased to 1734 times), and in each monomer treatment group, the drug composition significantly inhibited the expression of interleukin-6 (reduced by 87.99%, P < 0.001). At the same dose (molar concentration), its inhibitory effect was stronger than that of all 15 monomer drug components mentioned above, including baicalin, paeoniflorin, gardenia glycoside, wogonin, baicalein, paeoniflorin, tetrahydropalmatine, rhein, wogonin, aloe-emodin, saikosaponin A, emodin, benzoylpeoniflorin, chlorogenic acid and costunolide.

[0070] It can be concluded that the pharmaceutical composition composed of the above 15 monomer components has synergistic anti-inflammatory activity in mouse acinar cell and macrophage models.

[0071] Table 1 Relative expression levels of interleukin-6 in 266-6 cells after stimulation with sodium taurocholate (n=3)

[0072]

[0073]

[0074] Table 2 Relative expression levels of interleukin-1beta, an inflammatory factor, in 266-6 cells after stimulation with sodium taurocholate (n=3)

[0075]

[0076] *Suspicious data are excluded from statistics.

[0077]

[0078] *Suspicious data are excluded from statistics.

[0079] Table 3 Relative expression of interleukin-6 in each group of RAW 264.7 cells after lipopolysaccharide stimulation (n=3)

[0080]

[0081]

[0082] *Suspicious data are excluded from statistics.

[0083] Table 4 Relative expression of interleukin-1beta in each group of RAW 264.7 cells after lipopolysaccharide stimulation (n=3)

[0084]

[0085] Example 2 Therapeutic effect of the pharmaceutical composition in a mouse model of pancreatitis induced by cerulein combined with lipopolysaccharide

[0086] Pancreatic HE pathological sections, serum amylase, serum lipase, and pancreatic tissue inflammatory factors IL-6, IL-1beta, and TNF-alpha are important indicators for evaluating the severity of pancreatitis. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine levels are important indicators for evaluating liver and kidney function in animals with pancreatitis.

[0087] 1. Experimental animals, instruments, and pharmaceutical reagents

[0088] C57 mice (8 weeks old) were purchased from Beijing Huafukang Biotechnology Co., Ltd. and housed in an SPF-grade environment (21 ± 2°C, 12-h light cycle). They had free access to food and water during the experiment. Baicalin, geniposide, wogonin, baicalein, paeoniflorin, rhein, wogonin, aloe-emodin, and rhein were purchased from Stend (purity ≥ 97%); saikosaponin A and costunolide were purchased from Shanghai Ronghe Biotechnology Co., Ltd. (purity ≥ 95%); tetrahydropalmatine, chlorogenic acid, benzoylpeoniflorin, and paeoniflorin were purchased from the National Institutes for the Control of Pharmaceutical Products (purity ≥ 99%). 4% tissue cell fixative and hematoxylin-eosin stain were purchased from Solebao Biotechnology Co., Ltd. Caerulein (98%) and lipopolysaccharide (95%) were purchased from MCE Biochemical Reagent Co., Ltd. Qingyi Granules were purchased from Dalian Hanfang Pharmaceutical Co., Ltd. Pathological section technical support was provided by Wuhan Savier Biotechnology Co., Ltd.

[0089] 2. Analysis Method

[0090] Hematoxylin-eosin (HE) staining of pancreatic tissue: The pancreatic head was fixed in 4% histiocytic fixative (4% paraformaldehyde) for 24 h, dehydrated in an automatic dehydrator for 16 h, embedded in paraffin embedding machine, sectioned, spread onto slides, dried, and stained with hematoxylin-eosin. After dehydration and transparency, the slides were mounted and examined under a microscope. Photos were recorded using an Olympus BX53 upright microscope.

[0091] Pathological scoring criteria for acute pancreatitis:

[0092] (1) Score of pancreatitis histological lesion severity

[0093] Slight edema and mild inflammatory cell infiltration of pancreatic tissue were scored as 1 point; obvious necrosis and inflammatory cell infiltration of pancreatic tissue were scored as 2 points; and more severe necrosis and inflammatory cell infiltration of pancreatic tissue were scored as 3 points.

[0094] (2) Pancreatic cell infiltration score

[0095] Normal glandular tissue is scored as 0 points; mild inflammatory cell infiltration, only a small amount of inflammatory cells infiltration, scored as 1 point; inflammatory cells, inflammatory cell infiltration is relatively obvious, scored as 2 points; severe inflammatory cell infiltration, inflammatory cell infiltration is relatively serious, scored as 3 points; very severe inflammatory cell infiltration, inflammatory cell infiltration is very serious, scored as 4 points.

[0096] (III) Pancreatic tissue necrosis score

[0097] 0 points were assigned if there was no necrosis in the pancreatic tissue and the tissue structure was intact; 1 point was assigned if there was mild necrosis in the pancreatic tissue and only a small amount of necrosis in the pancreatic tissue; 3 points were assigned if there was severe necrosis in the pancreatic tissue and the necrosis in the pancreatic tissue was relatively obvious; 4 points were assigned if there was very severe necrosis in the pancreatic tissue and the necrosis in the pancreatic tissue was very serious.

[0098] The pancreatitis pathology score is a cumulative score based on the three indicators above, assessing the extent of pancreatitis tissue lesions, inflammatory cell infiltration, and pancreatic tissue necrosis. A higher score indicates more severe pancreatitis and a more severe inflammatory response.

[0099] The pancreatic inflammatory factors interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α) were determined using qPCR detection reagents. Reverse transcription reagents and qPCR reagents were purchased from Jiangsu Yugong Biotechnology Co., Ltd. The specific methods are detailed in the instructions.

[0100] Serum α-amylase (starch-iodine colorimetric method), lipase (microplate method), alanine aminotransferase (ALT, Reiter method), aspartate aminotransferase (AST, microplate method), and creatinine (microplate method) were determined using test kits purchased from Nanjing Jiancheng Biotechnology Co., Ltd. For details, please refer to the instructions.

[0101] 3. Experimental Design and Animal Grouping

[0102] Experimental design: The experimental animals were divided into 7 groups, including a normal control group, an (acute) pancreatitis model group, a low-dose drug combination treatment group, a high-dose drug combination treatment group, an oral Qingyi granule treatment group, a random drug combination 1 treatment group, and a random drug combination 2 treatment group. Model establishment started with 8-week-old mice. After one week of adaptive feeding with ordinary feed, the model group and the drug treatment group were continuously modeled 10 times by intraperitoneal injection of caerulein (100 μg / kg), once every hour. One hour after the last caerulein modeling, lipopolysaccharide (10 mg / kg) was injected intraperitoneally. After the first caerulein injection and the lipopolysaccharide injection, the oral compound preparation or intravenous drug composition treatment was given, respectively. 12 hours after the lipopolysaccharide modeling, the animals were collected for fasting serum and pancreatic index measurement.

[0103] Animal Grouping:

[0104] (1) Normal control group: fed with normal feed + oral administration of 0.2 mL of normal saline;

[0105] (2) Acute pancreatitis model group: intraperitoneal injection of caerulein (0.1 mL / mouse, 100 μg / kg) combined with lipopolysaccharide (10 mg / kg) plus oral administration of 0.2 mL of normal saline;

[0106] (3) Low-dose drug combination treatment group: intraperitoneal injection of caerulein (0.1 mL / animal, 100 μg / kg) combined with lipopolysaccharide (0.1 mL / animal, 10 mg / kg) plus tail vein injection of low-dose drug combination (10 mg / kg, 0.1 mL / 20 g);

[0107] (4) High-dose drug combination treatment group: intraperitoneal injection of caerulein (0.1 mL / animal, 100 μg / kg) combined with lipopolysaccharide (0.1 mL / animal, 10 mg / kg) plus tail vein injection of high-dose drug combination (20 mg / kg, 0.1 mL / 20 g);

[0108] (5) Qingyi granule treatment group: intraperitoneal injection of caerulein (0.1 mL / mouse, 100 μg / kg) combined with lipopolysaccharide (0.1 mL / mouse, 10 mg / kg) plus oral Qingyi granule treatment group (0.2 mL / mouse);

[0109] (6) Randomized drug combination 1 treatment group: intraperitoneal injection of caerulein (0.1 mL / animal, 100 μg / kg) combined with lipopolysaccharide (0.1 mL / animal, 10 mg / kg) + tail vein injection of randomized combination 1 (drug concentrations are shown in the table below) (20 mg / kg, 0.1 mL / 20 g);

[0110] (7) Randomized drug combination 2 treatment group: intraperitoneal injection of caerulein (0.1 mL / animal, 100 μg / kg) combined with lipopolysaccharide (0.1 mL / animal, 10 mg / kg) + tail vein injection of randomized combination 2 (drug concentrations are shown in the table below) (20 mg / kg, 0.1 mL / animal);

[0111] Ratio of monomer components in the drug composition of each treatment group

[0112]

[0113]

[0114] Note*: The values ​​listed in the table are only the molar ratios of some ingredients in the Qingyi Granules compound. Other chemical components are not listed in this table.

[0115] 4. Results

[0116] The combined injection of cerulein and lipopolysaccharide is the most commonly used and stable method for modeling pancreatitis. Figure 5 , Figure 6As shown in Table 5, 24 hours after the first injection of caerulein, the pancreatic tissue of the pancreatitis model group mice showed obvious inflammatory cell infiltration, acinar cell edema, vacuolation, and necrosis. The pancreatic tissue of the normal control group showed no obvious pathological changes, indicating that the acute pancreatitis model was successfully established. HE pathological section staining and case score statistical results showed that after oral administration of Qingyi granules, the pathological changes of pancreatic tissue were alleviated to a certain extent compared with the model group (P < 0.05); in addition, after intravenous injection of random drug combination 1 and random drug combination 2, the pathological edema and vacuolation of pancreatic tissue were reduced; however, the pathological results showed that the efficacy of the above three treatment groups was not as good as that of the intravenous low-dose drug combination treatment group (P < 0.01, P < 0.001). The high-dose combination treatment group had a better efficacy than the low-dose treatment group, further proving the good efficacy of intravenous injection of the drug combination (compared with the model group) for pancreatitis.

[0117] like Figure 7 , Figure 8As shown in Tables 6 and 7, 24 hours after modeling, the serum amylase and lipase concentrations of mice in the model group were significantly higher than those in the normal control group (with the lipase level being more than three times the normal level), indicating that the animals presented with a pathological state of (moderate to severe) acute pancreatitis. The statistical results of amylase and lipase showed that the amylase and lipase concentrations in the low-dose composition treatment group decreased by 46.82% (P < 0.001) and 56.1% (P < 0.001), respectively, compared with the model group. The amylase and lipase concentrations in the high-dose composition treatment group decreased by 44.35% (P < 0.01) and 62.5% (P < 0.001), respectively, compared with the model group. Both treatment regimens had a certain effect on alleviating the severity of pancreatitis. In contrast, after oral treatment with Qingyi Granules, the concentration levels of amylase and lipase decreased by 35.32% (P < 0.05) and 27.44% (P < 0.05), respectively; after intravenous treatment with the same dose (high dose) of random drug combination 1, the concentration levels of amylase and lipase decreased by 40.66% (P < 0.01) and 52.44% (P < 0.001), respectively; after intravenous treatment with the same dose of random drug combination 2, the concentration levels of amylase and lipase decreased by 38.4% (P < 0.01) and 40.85% (P < 0.001), respectively. In the results of amylase and lipase levels, the therapeutic effect of the drug combination was better than that of the above-mentioned compound Chinese medicine and random drug combination, indicating the advantage of the compatibility ratio of the composition described in the present invention. This technical solution, using key pathological indicators of pancreatitis as evaluation criteria, demonstrated that intravenous administration of high- and low-dose combinations of drugs was more effective in alleviating the severity of pancreatitis than oral Qingyi Granules, randomized drug combination 1, and randomized drug combination 2. Furthermore, during the implementation of this case study, because pancreatitis itself is a progressive disease, and at the beginning of drug treatment, the model animals were already in a state of abnormal pancreatic secretion, which overlaps with the pathophysiological characteristics of chronic pancreatitis, researchers in this field believe that this drug combination also has certain effectiveness in preventing / treating chronic pancreatitis.

[0118] The expression level of inflammatory factors is an important indicator of local and systemic inflammation. Figure 9-11As shown in Tables 8-10, 24 hours after modeling, the expression levels of inflammatory factors (IL-1β, IL-6 and TNF-α) in the pancreatic tissue of mice in the model group were significantly higher than those in the normal control group, indicating that the pancreatitis model was successfully established. The statistical results of pancreatic tissue inflammatory factors showed that after intravenous injection of high and low doses of drug compositions, oral Qingyi granules, and intravenous injection of random drug combination 1 and random drug combination 2, the expression level of IL-6 in pancreatic tissue decreased compared with the model group. The intravenous injection of low-dose composition treatment group decreased by 82.52% (P < 0.001), the high-dose composition treatment group decreased by 59.54% (P < 0.01), and the Qingyi granule treatment group decreased by 56.62%. After treatment with random drug combination 1, the IL-6 level decreased by 81.19% (P < 0.001), and after treatment with drug combination 2, the IL-6 level decreased by 10. -6 level decreased by 79.56% (P < 0.001), among which the low-dose drug composition had the most significant effect in inhibiting IL-6 expression level; the results of IL-1β expression level determination showed that the low-dose intravenous injection composition treatment group decreased by 95.69% (P < 0.01) compared with the model group, and the high-dose composition treatment group decreased by 79.28% (P < 0.05), while the expression levels of the Qingyi granules and random drug composition treatment groups decreased by 73.76% (P < 0.05), 29.85% and 35.4%, respectively, among which the low-dose composition treatment group had a significant reduction effect. Results of TNF-α expression measurements showed that the low-dose intravenous injection group decreased by 91.78% (P < 0.01), the high-dose group decreased by 93.09% (P < 0.05), and the Qingyi Granule group decreased by 76.36% (P < 0.01). Randomized drug combination 1 and randomized drug combination 2 reduced IL-6 levels by 36.18% and 65.95%, respectively, with the low-dose drug combination group showing the most significant reduction. Combined pathology, amylase and lipase test results, and inflammatory factor test results indicate that pancreatic inflammation levels in mice significantly improved after treatment with the lower-dose drug combination, and its therapeutic effect in the animal model was superior to that of oral administration of a high-dose Qingyi Granule compound and the two randomized drug combinations.

[0119] Alanine aminotransferase and aspartate aminotransferase are important indicators for evaluating liver function. Figure 12 , Figure 13As shown in Tables 11 and 12, the serum alanine aminotransferase and aspartate aminotransferase test results of pancreatitis model mice were higher than those of the normal control group, suggesting that the liver function of pancreatitis mice may be impaired. After treatment with random drug combination 1 and random drug combination 2, there was no significant change in serum alanine aminotransferase and aspartate aminotransferase. After treatment with high and low doses of the drug combination, the serum alanine aminotransferase and aspartate aminotransferase activities were both lower than those of the model group (P < 0.05, P < 0.001); the improvement effect on liver function was similar to that of the Qingyi Granule compound. This suggests that in terms of liver function level, the low dose of the drug combination can achieve an improvement effect similar to that of the compound Qingyi Granule treatment, and the improvement effect is better than that of drug combination 1 and drug combination 2.

[0120] Creatinine level is an important indicator for evaluating renal function. Figure 14 As shown in Table 13, after pancreatitis model mice were treated with high and low doses of the composition and Qingyi granules, there was no significant change in the serum creatinine levels of the mice, and no nephrotoxicity was shown.

[0121] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

[0122] Table 5 Pancreatic HE pathological section scoring results of (acute) pancreatitis model mice after treatment with the pharmaceutical composition (n=6)

[0123]

[0124] Table 6 Serum amylase assay results of (acute) pancreatitis model mice after treatment with the pharmaceutical composition (U / L, n=6)

[0125]

[0126]

[0127] Table 7 Results of serum lipase assay in (acute) pancreatitis model mice after treatment with the pharmaceutical composition (U / L, n=6)

[0128]

[0129] Table 8 Results of serum interleukin-6 assay in (acute) pancreatitis model mice after treatment with the pharmaceutical composition (relative amount, n=6)

[0130]

[0131] Table 9 Results of serum interleukin-1beta assay in (acute) pancreatitis model mice after treatment with the pharmaceutical composition (relative amount, n=6)

[0132]

[0133]

[0134] Table 10 Results of serum inflammatory factor tumor necrosis factor-alpha determination in (acute) pancreatitis model mice after treatment with the pharmaceutical composition (relative amount, n=6)

[0135]

[0136] *Suspicious data are excluded from statistics.

[0137] Table 11 Results of serum alanine aminotransferase assay in (acute) pancreatitis model mice after treatment with the pharmaceutical composition (U / L, n=6)

[0138]

[0139] Table 12 Results of serum aspartate aminotransferase assay in (acute) pancreatitis model mice after treatment with the pharmaceutical composition (U / L, n=6)

[0140]

[0141] *Suspicious data are excluded from statistics.

[0142] Table 13 Serum creatinine measurement results of (acute) pancreatitis model mice after treatment with the pharmaceutical composition (μM, n=6)

[0143]

Claims

1. Use of a natural-source compound monomer composition in the preparation of a drug for preventing and / or treating pancreatitis, characterized in that: The composition comprises baicalin, paeoniflorin, geniposide, wogonin, baicalein, paeoniflorin, tetrahydropalmatine, rhein, wogonin, aloe-emodin, saikosaponin A, emodin, benzoylpaeoniflorin, chlorogenic acid, costunolide, or pharmaceutically acceptable salts thereof; The baicalin has a structure shown in formula (I): The paeoniflorin has a structure shown in formula (II): The geniposide has a structure shown in formula (III): The baicalin has a structure shown in formula (IV): The baicalein has a structure shown in formula (V): The paeoniflorin has a structure shown in formula (VI): The tetrahydropalmatine has a structure shown in formula (VII): The rhein has a structure shown in formula (VIII): The wogonin has a structure shown in formula (IX): The aloe-emodin has a structure shown in formula (X): Said saikosaponin A has the structure shown in formula (XI): The emodin has a structure shown in formula (XII): The benzoylpenoside has a structure shown in formula (XIII): The chlorogenic acid has a structure shown in formula (XIV): The costunolide has a structure shown in formula (XV):

2. The use according to claim 1, characterized in that The mass ratios of baicalin, paeoniflorin, gardenia jasminoidin, wogonin, baicalein, paeoniflorin, tetrahydropalmatine, rhein, wogonin, aloe-emodin, saikosaponin A, emodin, benzoylpeoniflorin, chlorogenic acid and costusin in the composition are 30-40:15-20:10-20:5-12:3-10:2-8:2-8; 1.5-6:1.5-6:1.5-5:0.35-1.5:0.35-1.5:0.2- 1:0.22~1; preferably, the mass ratio of baicalin, paeoniflorin, gardenia jasminoidin, wogonin, baicalein, paeoniflorin, tetrahydropalmatine, rhein, wogonin, aloe-emodin, saikosaponin A, emodin, benzoylpeoniflorin, chlorogenic acid, and costunolide is 35.34:17.69:14.63:8.01:5.10:3.81:3.47:3.07:3.05:2.68:0.72:0.71:0.70:0.54:0.

48.

3. The use according to claim 1, characterized in that For rodents, the dosage of the composition is 10 mg-20 mg / kg; for humans, the dosage of the composition is 1.1-2.2 mg / kg.

4. The use according to claim 1, characterized in that The natural source compound monomer composition is an injectable pharmaceutical composition.

5. The use according to claim 4, characterized in that The administration route of the injection pharmaceutical composition is intravenous injection, and the solvent is a physiological saline solution containing 2-10% dimethyl sulfoxide.

6. The use according to claim 1, characterized in that The pharmaceutically acceptable salts include hydrochloride, sulfate, hydrobromide, hydroiodide, formate, acetate, citrate or oxalate.

7. The use according to claim 1, characterized in that The composition further comprises a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

8. The use according to any one of claims 1 to 7, characterized in that The pancreatitis is an inflammatory lesion of the pancreatic tissue region caused by cholelithiasis, alcohol, blood vessels, trauma, and infection, manifested by abnormally elevated serum amylase and (or) lipase and persistent pain in the upper abdomen. Abdominal imaging examination results show imaging changes consistent with pancreatitis; preferably, the pancreatitis is moderate to severe acute pancreatitis.

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