Pharmaceutical composition for treating sepsis and its use

Abstract

The present invention provides a pharmaceutical composition for treating sepsis and its use, which belongs to the technical field of chemical medicine. The present invention provides a three-component pharmaceutical composition containing hydroxysafflor yellow A, paeoniflorin and albiflorin, and also provides a seven-component pharmaceutical composition containing hydroxysafflor yellow A, paeoniflorin, albiflorin, paeoniflorin oxide, senkyunolide I, sodium danshensu and ferulic acid. The present invention verifies that the two groups of pharmaceutical compositions have the effect of effectively treating sepsis at the cell level and animal level, and at the same time, drug toxicity experiments show that the drugs have relatively high safety. The pharmaceutical composition provided by the present invention is the active ingredient of Xuebijing injection, so it can be used in the clinical treatment of sepsis.

Description

[Technical field]

[0001] This application claims priority to a Chinese patent application filed with the China Patent Office on March 1, 2021, bearing application number 202110225428.6 and entitled "Pharmaceutical composition for treating sepsis and its use", the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of chemical medicines, in particular to a pharmaceutical composition for treating sepsis and its use. [Background technology]

[0003] Sepsis is a fatal organ dysfunction caused by abnormal host response to infection, and its viciousness and high mortality rate are the main causes of death in critically ill patients. For many years, traditional sepsis treatment has used antibiotics, antiviral drugs, vasodilators, etc., but no specific drugs that are sufficient to address the pathogenic mechanism of sepsis have yet been introduced into clinical practice. In the onset and progression of sepsis, how to quickly correct systemic inflammatory response, coagulation dysfunction, and immune dysfunction, quickly restore the biological pro-inflammatory-anti-inflammatory dynamic balance, and effectively improve patient prognosis has become an important issue to be resolved in the research and development of drugs for treating sepsis. Xuebijing (Blood Purification) Injection is an intravenous injection developed based on the dialectical principle of "three symptoms and three methods", under the guidance of the theory of "combined treatment of bacteria, poison and inflammation", and based on "Xuefu Zhuo Tang" recorded in the book "YilinGaicuo" by Wang Qingren of the Qing Dynasty. It is prepared from five herbal medicines, namely safflower, red peony, Szechuan cucumber, red sage, and angelica, by the modern process of extraction, purification, drying and blending. It is suitable for systemic inflammatory response syndrome induced by sepsis and infection, and can also be used in combination with the treatment of organ failure stage of multiple organ dysfunction syndrome. However, Xuebijing (Blood Purification) Injection is prepared from Chinese herbal medicines, and the main active ingredients of sepsis are still unknown, so it is necessary to extract and purify various Chinese herbal medicines to prepare a drug composition for treating sepsis. Summary of the Invention [Problem to be solved by the invention]

[0004] In view of this, the present invention aims to provide a novel pharmaceutical composition for treating sepsis and its use, which directly contains an active compound for treating sepsis, thereby eliminating the troublesome process of extracting herbal medicines, simplifying the composition of the pharmaceutical composition, and improving the efficacy of the product. [Means for solving the problem]

[0005] The present invention includes hydroxysafflor yellow A, the structural formula of which is represented by formula I, paeoniflorin, the structural formula of which is represented by formula II, and albiflorin, the structural formula of which is represented by formula III. The mass ratio of the hydroxysafflor yellow A, paeoniflorin and albiflorin is 1-100:1-100:0.1-10.

[0006] [ka]

[0007] Preferably, the mass ratio of hydroxysafflor yellow A, paeoniflorin and albiflorin is 1-100:1-100:1-10.

[0008] Preferably, the pharmaceutical composition further comprises oxidized paeoniflorin, the structural formula of which is shown in formula IV, senkynolide I, the structural formula of which is shown in formula V, danshensu sodium, the structural formula of which is shown in formula VI, and ferulic acid, the structural formula of which is shown in formula VII; The mass ratio of the hydroxysafflor yellow A, paeoniflorin, albiflorin, paeoniflorin oxide, senkynolide I, sodium danshensu, and ferulic acid is 1-100:1-100:1-10:0.1-10:0.1-10:0.1-10:0.01-1.

[0009] [ka]

[0010] Preferably, the mass ratio of the hydroxysafflor yellow A, paeoniflorin, albiflorin, paeoniflorin oxide, senkyunolide I), sodium danshensu and ferulic acid is 1-100:1-100:1-10:1-10:1-10:1-10:0.1-1.

[0011] The present invention provides a use of the pharmaceutical composition in the preparation of a medicament for treating sepsis.

[0012] Preferably, said pharmaceutical composition is used for the preparation of a medicament for inhibiting the release of TF and / or TM in sepsis.

[0013] Preferably, the pharmaceutical composition is used for preparing a medicament for promoting apoptosis of sepsis regulatory T cells.

[0014] Preferably, when the pharmaceutical composition consists of three active compounds, namely, hydroxysafflor yellow A, paeoniflorin and albiflorin, the dosage of hydroxysafflor yellow A is 6-600 mg / kg, the dosage of paeoniflorin is 6-600 mg / kg, and the dosage of albiflorin is 0.6-60 mg / kg.

[0015] Preferably, when said pharmaceutical composition is composed of seven active compounds including hydroxysafflor yellow A, paeoniflorin, albiflorin, paeoniflorin oxide, senkyunolide I, danshensu sodium and ferulic acid, the dosage of hydroxysafflor yellow A is 6-600mg / kg, the dosage of paeoniflorin is 6-600mg / kg, the dosage of albiflorin is 0.6-60mg / kg, the dosage of paeoniflorin oxide is 0.6-60mg / kg, the dosage of senkyunolide I is 0.6-60mg / kg, the dosage of danshensu sodium is 0.6-60mg / kg and the dosage of ferulic acid is 0.06-6mg / kg.

[0016] The present invention further provides a method for treating sepsis by administrating a medicament prepared from the pharmaceutical composition. Preferably, the pharmaceutical composition can treat sepsis by inhibiting the expression of IL-6, Foxp3, CTLA-4 and / or HMGB1 in sepsis.

[0017] Preferably, the pharmaceutical composition is capable of treating sepsis by inhibiting the release of TF and / or TM in sepsis.

[0018] Preferably, the pharmaceutical composition achieves the treatment of sepsis by promoting apoptosis of sepsis regulatory T cells.

[0019] Preferably, when the pharmaceutical composition consists of three active compounds, namely, hydroxysafflor yellow A, paeoniflorin and albiflorin, the dosage of hydroxysafflor yellow A is 6-600 mg / kg, the dosage of paeoniflorin is 6-600 mg / kg, and the dosage of albiflorin is 0.6-60 mg / kg.

[0020] Preferably, when said pharmaceutical composition is composed of seven active compounds including hydroxysafflor yellow A, paeoniflorin, albiflorin, paeoniflorin oxide, senkyunolide I, danshensu sodium and ferulic acid, the dosage of hydroxysafflor yellow A is 6-600mg / kg, the dosage of paeoniflorin is 6-600mg / kg, the dosage of albiflorin is 0.6-60mg / kg, the dosage of paeoniflorin oxide is 0.6-60mg / kg, the dosage of senkyunolide I is 0.6-60mg / kg, the dosage of danshensu sodium is 0.6-60mg / kg and the dosage of ferulic acid is 0.06-6mg / kg. Effect of the Invention

[0021] The pharmaceutical composition for treating sepsis provided by the present invention comprises hydroxysafflor yellow A, paeoniflorin and albiflorin, and the mass ratio of said hydroxysafflor yellow A, paeoniflorin and albiflorin is 1-100:1-100:0.1-10. The present invention proves at the cell level and animal level that said pharmaceutical composition has the effect of suppressing the expression of macrophage IL-6, HMGB1, CTLA-4 and Foxp3 in sepsis, effectively suppresses the release of TF and TM in sepsis, and at the same time has the effect of promoting the apoptosis of regulatory T lymphocytes, showing that the pharmaceutical composition provided by the present invention has the effect of obviously correcting the immunosuppressive state of cells or somatic cells in sepsis, and achieves the purpose of treating sepsis.

[0022] At the same time, the results of acute toxicity tests of the pharmaceutical composition of the present invention by intravenous administration showed that no obvious abnormalities were observed in the animals after administration, and the body weight and weight gain rate of the animals were not significantly different from those of the control group. No obvious abnormalities were observed in the macroscopic anatomical structures, indicating that the pharmaceutical composition provided by the present invention has pharmaceutical safety.

[0023] In addition, the pharmaceutical composition provided by the present invention further comprises paeoniflorin oxide having the structural formula shown in formula IV, senkyunolide I having the structural formula shown in formula V, danshensu sodium having the structural formula shown in formula VI, and ferulic acid having the structural formula shown in formula VII, and the mass ratio of said hydroxysafflor yellow A, paeoniflorin, albiflorin, paeoniflorin oxide, senkyunolide I, danshensu sodium, and ferulic acid is 1-100:1-100:0.1-10:0.1-10:0.1-10:0.1-10:0.01-1. The present invention has revealed that, compared with the pharmaceutical composition consisting of three compounds, hydroxysafflor yellow A as the active ingredient of safflower for treating sepsis, paeoniflorin, albiflorin and oxidized paeoniflorin as the active ingredients of red peony root and white peony root for treating sepsis, senkyunolide I as the active ingredients of szechuan rhizome and angelica for treating sepsis, ferulic acid as the active ingredient of angelica for treating sepsis, danshensu as the active ingredient of danshen for treating sepsis, and ferulic acid as the active ingredient of angelica for treating sepsis, the pharmaceutical composition containing seven active ingredients in strict dosage and ratio has stronger inhibitory effect on the expression of IL-6, HMGB1, CTLA-4 and Foxp3 of macrophages in sepsis, has stronger inhibitory effect on the release of TF and TM in sepsis, and at the same time has stronger promoting effect on the apoptosis of regulatory T lymphocytes. The experiments of the present invention show that the pharmaceutical composition has stronger efficacy in treating sepsis and achieves better therapeutic effect.

[0024] At the same time, the present invention also conducted an intravenous acute toxicity test on the pharmaceutical composition consisting of seven active ingredients, and the results showed that no obvious abnormalities were observed in the animals after administration, the body weight and body weight gain rate of the animals were not significantly different from those of the control group, and no obvious abnormalities were observed in the macroscopic anatomical structures, indicating that the pharmaceutical composition has the pharmaceutical safety provided by the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present invention provides a pharmaceutical composition for treating sepsis, comprising hydroxysafflor yellow A having a structural formula represented by formula I, paeoniflorin having a structural formula represented by formula II, and albiflorin having a structural formula represented by formula III: The mass ratio of the hydroxysafflor yellow A, paeoniflorin and albiflorin is 1-100:1-100:0.1-10.

[0026] [ka]

[0027] In the present invention, the mass ratio of the hydroxysafflor yellow A, paeoniflorin and albiflorin is preferably 1-100:1-100:1-10, including 1-100:1:1, 1-100:10:1, 1-100:100:1, 1-100:1:0.01, 1-100:1:10, 1-100:10:10, 1-100:10:1, 1-100:10:0.01, specifically 1:10:10, 1:100:1, 10:1:10, 10:10:1, 10:100:0.1, 100:1:1, 100:10:0.1 and 10:10:1, most preferably 10:10:1. Hydroxysafflor yellow A is a compound represented by the molecular formula C 27 H3O 16 It is an active ingredient of the commonly used Chinese herbal medicine safflower with a molecular weight of 612.53. Hydroxysafflor Yellow A is a monochalcone glycoside compound, which is the most effective water-soluble part of the pharmacological action of safflower. It can inhibit platelet aggregation and release caused by platelet activating factor, and competitively inhibit the binding of platelet activating factor to platelet receptors, and has antiplatelet and anti-myocardial ischemia effects. It is an active ingredient of safflor yellow in promoting blood circulation and relieving swelling, and at the same time, it also has anti-inflammatory, cytoprotective and anti-tumor effects. Paeoniflorin has the molecular formula C 23 H 28 O 11Paeoniflorin is an active ingredient of the commonly used Chinese herbal medicines Red Peony Root and White Peony Root, with a molecular weight of 480.46. Paeoniflorin is a monoterpene glycoside compound with diverse pharmacological effects, including anti-free radical damage, inhibition of intracellular calcium overload, and anti-neurotoxicity. In vivo experiments have demonstrated that it has various biological effects such as reducing blood viscosity, dilating blood vessels, improving microcirculation, antioxidant, and anticonvulsant, with few adverse reactions. Albiflorin has the molecular formula C 23 H 28 O 11 , molecular weight is 480.46. Albiflorin is similar to paeoniflorin in chemical structure, is an isomer of paeoniflorin, and its content is lower than paeoniflorin. Albiflorin acts on the spleen and thymus of immune system and hematopoietic cytokines of blood system, and has obvious effect of enriching blood, and at the same time, it also acts on the HPA axis of nervous system and the neurotransmission of monoamines in brain, and has obvious antidepressant effect. The forms of said hydroxysafflor yellow A, albiflorin and paeoniflorin include not only their monomer compounds, but also preferably their pharmaceutically acceptable salts, solvates, polycrystals, enantiomers or racemic mixtures, etc.

[0028] In the present invention, the pharmaceutical composition preferably further comprises oxidized paeoniflorin having a structural formula represented by formula IV, senkyunolide I having a structural formula represented by formula V, danshensu sodium having a structural formula represented by formula VI, and ferulic acid having a structural formula represented by formula VII, and the mass ratio of said hydroxysafflor yellow A, paeoniflorin, albiflorin, oxidized paeoniflorin, senkyunolide I, danshensu sodium, and ferulic acid is 1-100:1-100:0.1-10:0.1-10:0.1-10:0.1-10:0.01-1.

[0029] [ka]

[0030] In the present invention, the mass ratio of the hydroxysafflor yellow A, paeoniflorin, albiflorin, paeoniflorin oxide, senkyunolide I, sodium danshensu, and ferulic acid is preferably 1-100:1-100:1-10:1-10:1-10:1-10:1-10:0.1-1, specifically preferably 1:10:1:1:1:1:0.1, 1:100:10:10:10:10:1, 10:1:0.1:1:1:10:1, 10:10:1:10:10:0.1:0.01, 10:100:10:0.1:0.1:1:0.1, 100:0.1:1:0.1:10:1:1, 100:10:0. 1:10:1:0.1:10:0.01, 100:100:0.1:10:1:0.1:1, 1:1:10:10:1:1:0.01, 1:10:0.1:0.1:10:10:1, 1:100:1:1:0.1:0.1:1, 10:1:1:0.1:1:0.1:0.1, 10:10:10:10: 0.1:0.1:1, 10:100:0.1:1:10:1:0.01, 100:1:10:1:10:0.1:1, 100:10:0.1:10:0.1:1:1, 100:100:1:0.1:1:1:1, 100:100:1:0.1:1:10:0.01, most preferred is 100:100:1:0.1:1:10:0.01. The content of oxidized paeoniflorin is relatively low in red peony and white peony, and there are few reports on its pharmacological action. Paeoniflorin has analgesic, anti-infection, antioxidant, anti-cancer and other effects, and has very high application value, so it is worthy of clinical promotion. Senkyunolide I is a phthalide compound isolated from extracts of traditional Chinese medicines such as Szechuan Qing Dynasty and Angelica Root. It has excellent fat-solubility and water-solubility and has a variety of pharmacological effects. Senkyunolide I can exert anti-inflammatory effects by inhibiting the NF-κB pathway. In vivo experiments have confirmed that it has a protective effect against ischemia-reperfusion injury. At the same time, it can reduce the deformation index and directional coefficient of red blood cells, reduce red blood cell aggregation, and have anticoagulant and antiplatelet aggregation activities.Tanshinol is the main active ingredient of the water-soluble components of Danshen, a kind of aromatic acid compound of the phenolic group. In order to enhance stability, it is prepared into Danshen sodium, which has the same effect as Danshen. Research shows that Danshen has the effect of reducing the scope and course of myocardial infarction, reducing myocardial ischemia-reperfusion injury, and protecting the myocardium. Danshen can further significantly inhibit platelet aggregation, reduce the viscosity of whole blood, and improve the microcirculatory disorders of various organs of the body (heart, liver, lungs, etc.) through its anticoagulant effect, contributing to the recovery of body tissues. At the same time, Danshen has a certain antibacterial effect and anti-inflammatory effect, and has the effect of enhancing the body's immunity. Ferulic Acid is one of the active ingredients of Angelica, belonging to phenolic acid compounds, and has the effects of antiplatelet aggregation, inhibition of platelet 5-hydroxytryptamine release, inhibition of platelet thromboxane A2 (TXA2) production, enhancement of prostaglandin activity, analgesia, and relief of vasospasm. Experiment proves that hydroxysafflor yellow A, paeoniflorin, albiflorin, paeoniflorin oxide, senkyunolide I, danshensu sodium, ferulic acid are the active ingredients in Xuebijing injection, and their role in treating sepsis has important practical significance and application value.In the present invention, the form of paeoniflorin oxide, senkyunolide I, danshensu sodium, ferulic acid not only includes their monomer compounds, but also preferably includes their pharmaceutically acceptable salts, solvates, polycrystals, enantiomers or racemic mixtures, etc.

[0031] In the present invention, the method for preparing the pharmaceutical composition preferably includes the following steps: Hydroxysafflor yellow A, paeoniflorin and albiflorin are mixed in the above weight ratio to obtain the drug composition.

[0032] When the drug composition contains 7 kinds, it can be obtained by mixing hydroxysafflor yellow A, paeoniflorin, albiflorin, paeoniflorin oxide, senkyunolide I, sodium danshensu and ferulic acid according to the above mass ratio.

[0033] The present invention provides a use of the pharmaceutical composition in the preparation of a medicament for treating sepsis.The present invention also provides a use of the pharmaceutical composition in the treatment of sepsis.

[0034] In the present invention, the pharmaceutical composition is preferably used for preparing a drug for suppressing the expression of IL-6, Foxp3, CTLA-4 and / or HMGB1 in sepsis. The experimental results show that the control group contains a small amount of IL-6 and HMGB1 at the cell level and animal level, the contents of IL-6, HMGB1, Foxp3 and CTLA-4 in the model group are significantly increased, and the plasma contents of IL-6, HMGB1, Foxp3 and CTLA-4 in the pharmaceutical composition administration groups (8 hours, 24 hours and 48 hours) are all significantly lower than those of the model group, suggesting that the three-component drug group in each dosage group can significantly reduce the expression of IL-6, HMGB1, Foxp3 and CTLA-4 in CLP sepsis, and has obvious inhibitory effect on the early inflammatory response of sepsis rat model. The pharmaceutical composition achieves the effect of treating the early inflammatory response of sepsis by inhibiting the expression of IL-6, Foxp3, CTLA-4 and / or HMGB1.

[0035] In the present invention, the pharmaceutical composition is preferably used for preparing a drug for inhibiting the release of TF and / or TM in sepsis. The experiment shows that at the cell level and animal level, monocyte TM and TF are expressed at a certain level in the control group, and the expression of TM and TF (12 hours, 24 hours, 48 ​​hours, and 72 hours after operation) in the model group is significantly higher than that in the control group (p<0.05), and gradually increases with the extension of postoperative time, while the expression of TM and TF (12 hours, 24 hours, 48 ​​hours, and 72 hours after operation) in the pharmaceutical composition administration group is significantly lower than that in the model group (p<0.05). It shows that the pharmaceutical composition has an obvious effect of inhibiting the release of coagulation factors and improving the hypercoagulable state in the septic rat model. The pharmaceutical composition achieves the therapeutic effect of septic coagulation disease by inhibiting the release of TF and / or TM in sepsis.

[0036] In the present invention, the pharmaceutical composition is preferably used to prepare a drug for promoting apoptosis of sepsis regulatory T cells (Tred). The experiment showed that the ratio of apoptotic cells of Treg cells in the model group was significantly lower than that in the control group (p<0.05), and the ratio of apoptotic cells in the herbal composition group was significantly higher than that in the model group (p<0.05). Compared with the model group, the three-component drug group and the seven-component drug group in each dosage group can promote the apoptosis of sepsis Treg and down-regulate the inhibitory effect on the proliferation and secretion function of T lymphocytes. It shows that the pharmaceutical composition can regulate the proliferation and secretion function of T lymphocytes by promoting Treg apoptosis.

[0037] In the present invention, the pharmaceutical composition is subjected to separate drug experiments at the cell level and animal level, and based on the animal dosage, the dosage to be administered to humans is calculated according to Formula A according to the body surface area conversion method between humans and animals in "Pharmacological Experimental Methodology" edited by Xu Shuyun. Human dose = Rat dose / 0.018 Formula A Here, the standard weight is 70 kg for an adult and 0.2 kg for a rat.

[0038] Preferably, when the pharmaceutical composition consists of three active compounds, namely, hydroxysafflor yellow A, paeoniflorin and albiflorin, the dosage of hydroxysafflor yellow A is 6-600 mg / kg, the dosage of paeoniflorin is 6-600 mg / kg, and the dosage of albiflorin is 0.6-60 mg / kg. Preferably, when said pharmaceutical composition is composed of seven active compounds including hydroxysafflor yellow A, paeoniflorin, albiflorin, paeoniflorin oxide, senkyunolide I, danshensu sodium and ferulic acid, the dosage of hydroxysafflor yellow A is 6-600mg / kg, the dosage of paeoniflorin is 6-600mg / kg, the dosage of albiflorin is 0.6-60mg / kg, the dosage of paeoniflorin oxide is 0.6-60mg / kg, the dosage of senkyunolide I is 0.6-60mg / kg, the dosage of danshensu sodium is 0.6-60mg / kg and the dosage of ferulic acid is 0.06-6mg / kg.

[0039] The present invention is not particularly limited to the dosage form of the prepared drug, and any pharmaceutical dosage form known in the art may be used, such as tablets, capsules, oral liquids, granules, pills, powders, ointments, elixirs, suspensions, powders, solutions, injections, suppositories, sprays, drops, etc. The present invention is not particularly limited to the preparation method of each dosage form of the drug, and any pharmaceutical dosage form preparation method known in the art may be used.

[0040] The pharmaceutical composition for treating sepsis provided by the present invention and its use are described in detail below with reference to examples, which cannot be construed as limiting the protection scope of the present invention. Example 1

[0041] 1. Experimental Materials Clean grade SD rats (half male and half female), weighing 180–220 g, hydroxysafflor yellow A, paeoniflorin, albiflorin, and danshensu sodium were purchased from Shanghai Tongtian Biotechnology Co., Ltd., and paeoniflorin oxide, senkyunolide I, and ferulic acid were purchased from Dingrui Chemical (Shanghai) Co., Ltd., and physiological saline (0.9% sodium chloride injection).

[0042] 2. Experimental Method Based on the animals' weight gain, diet, and activity during the adaptation period, 80 animals (half male and half female) were screened to participate in the study, and the rats were divided into four groups according to weight grouping, with 20 rats in each group, and half of each sex.

[0043] The concentrations of each component of the three-component drug were 30mg / ml of hydroxysafflower yellow A, 40mg / ml of paeoniflorin, and 4mg / ml of albiflorin. The drug solution was prepared by weighing 3.0g of hydroxysafflower yellow A, 4.0g of paeoniflorin, and 0.4g of albiflorin on an electronic balance (1 / 10000g), dissolving them in an appropriate amount of physiological saline (0.9% sodium chloride solution), making the solution 100mL, and sterilizing it by filtration to prepare the three-component drug.

[0044] The concentration of each component of the seven-component drug was 30mg / ml of hydroxysafflower yellow A, 40mg / ml of paeoniflorin, 4mg / ml of albiflorin, 4mg / ml of paeoniflorin oxide, 4mg / ml of senkyunolide I, 4mg / ml of danshensu sodium, and 0.5mg / ml of ferulic acid. The drug solution was prepared by weighing out 3.0g of hydroxysafflower yellow A, 4.0g of paeoniflorin, 0.4g of albiflorin, 0.4g of paeoniflorin oxide, 0.4g of senkyunolide I, 0.4g of danshensu sodium, and 0.05g of ferulic acid, dissolving them in an appropriate amount of physiological saline (0.9% sodium chloride solution), making the solution 100mL, and sterilizing it by filtration to prepare the seven-component drug.

[0045] The limit volume of intravenous injection in rats was 6 mL / kg, and in summary, the doses of the three component drugs were 180 mg / kg hydroxysafflower yellow A, 240 mg / kg paeoniflorin, and 24 mg / kg albiflorin.

[0046] The dosages of the seven-component drugs were hydroxysafflor yellow A 180 mg / kg, paeoniflorin 240 mg / kg, albiflorin 24 mg / kg, paeoniflorin oxide 24 mg / kg, senkyunolide I 24 mg / kg, danshensu sodium 24 mg / kg, and ferulic acid 3 mg / kg.

[0047] The experimental groups were slowly intravenously injected with 6 mL / kg of the above three-component and seven-component drugs in a single dose, and the control group was administered the same volume of normal saline.

[0048] After administration, the animals were observed continuously for more than 2 hours, and the results were recorded once each in the morning and afternoon on the first day after administration, and then once a day for a total of 14 days. During the observation period, the toxic reactions of the animals and the deaths of the animals in each group were observed and recorded, and dead or moribund animals were autopsied in a timely manner. On the 14th day after administration, all surviving animals were autopsied, and obvious abnormal changes in the major organs were observed with the naked eye. If there were any changes in the volume, color, texture, etc. of the organs, they should all be recorded and histopathological examinations should be performed.

[0049] 3. Experimental Results The results of slow intravenous injection of the three-component drug into rats show that (1) there were no abnormal symptoms after administration to the animals, (2) there was no significant difference in the body weight and body weight gain rate of the animals in the treatment group and the control group, and (3) no obvious abnormalities were found upon gross autopsy.

[0050] The results of slow intravenous injection of the seven-component drug into rats show that (1) there were no abnormal symptoms after administration to the animals, (2) there was no significant difference in the body weight and body weight gain rate of animals in the treatment group and the control group, and (3) no obvious abnormalities were found upon gross autopsy. Example 2

[0051] Evaluation of the effects of three- and seven-component drugs on IL-6 release from rat peritoneal macrophages stimulated with LPS

[0052] 1. Experimental Materials Male clean grade SD rats (180-220 g), endotoxin (LPS), hydroxysafflor yellow A, paeoniflorin, albiflorin, oxidized paeoniflorin, senkyunolide I, danshensu sodium, ferulic acid, RPMI-1640 medium, 24-well plate, IL-6 ELISA kit.

[0053] 2. Experimental Method Peritoneal macrophages from male SD rats were isolated. The specific method was as follows: male SD rats were fasted for 12 hours before surgery, and the abdominal cavity was opened after anesthesia, 10 mL of pre-cooled PBS solution was injected into the abdominal cavity, and the abdominal wall was gently pressed with a finger to allow the liquid to flow in the abdominal cavity. The liquid in the abdominal cavity was aspirated and injected into a sterile tube, and washed once more with 10 mL of pre-cooled PBS solution, and the operation was the same as above. The collected washing solution was centrifuged at 250 g for 10 minutes at 4 ° C, and the supernatant was discarded. 2 mL of red blood cell lysate was added to dissolve the red blood cells, and the mixture was gently shaken twice, 5 seconds each time, and left to stand for 5 minutes, and then 4 mL of D-Hanks solution was added to terminate the reaction. The mixture was centrifuged again according to the above method, and the supernatant was discarded. After washing the precipitate with medium, the cells were resuspended and washed at 2 × 10 6 The cell suspension was seeded into a 24-well plate and incubated at 37°C, 5% CO 2 The cells were cultured in a cell culture incubator at 4°C. Each group had six parallel wells. After culturing for 12 and 24 hours separately, the supernatants were collected and the cytokine contents were measured by ELISA (Enzyme-Linked Immunosorbent Assay).

[0054] The experiment was divided into a control group, a model group, and a treatment group. The treatment method for each group was as follows: after culturing the cells of each group overnight in a cell incubator at 37℃ and 5%CO2, the control group and the model group were added with the corresponding amount of culture medium, and the treatment group was added with an equal amount of the corresponding concentration of drug solution. After culturing in the incubator for 1 hour, the control group was not added with LPS, and the model group and the treatment group were stimulated by adding LPS (75ng / mL). After 12 hours and 24 hours, 0.5mL of cell culture supernatant was collected from each group and stored in a refrigerator at -20℃, and the corresponding cytokines were detected by concentration. Here, the treatment group adopted the orthogonal experimental method, and the orthogonal experimental grouping method is shown in Table 1 and Table 2.

[0055] [Table 1]

[0056] In Table 1, the three concentrations of hydroxysafflor yellow A were low 2 μM, medium 20 μM, and high 200 μM, the three concentrations of paeoniflorin were low 4 μM, medium 40 μM, and high 400 μM, the three concentrations of albiflorin were low 2 μM, medium 20 μM, and high 200 μM, the three concentrations of oxidized paeoniflorin were low 2 μM, medium 20 μM, and high 200 μM, the three concentrations of senkyunolide I were low 4 μM, medium 40 μM, and high 400 μM, the three concentrations of danshensu sodium were low 2 μM, medium 20 μM, and high 200 μM, and the three concentrations of ferulic acid were low 4 μM, medium 40 μM, and high 400 μM.

[0057] [Table 2]

[0058] The preparation method of the drug solution is to weigh out the appropriate weights of hydroxysafflower yellow A, paeoniflorin, and albiflorin with an electronic balance (1 / 10000g) according to the corresponding concentrations of each group in Table 1, combine them into different measuring flasks according to different concentration combinations, and dissolve them in the appropriate amount of medium (containing 10% fetal bovine serum) respectively to create 9 groups of three-component drugs with the corresponding concentrations; According to the concentrations of each corresponding group in Table 2, appropriate weights of hydroxysafflower yellow A, paeoniflorin, albiflorin, paeoniflorin oxide, senkyunolide, danshensu sodium, and ferulic acid were weighed out by electronic balance (1 / 10000g), combined into different volumetric flasks according to different concentration combinations, and dissolved in appropriate medium (containing 10% fetal bovine serum) respectively to produce 18 groups of seven-component drugs with corresponding concentrations.

[0059] 3. Experimental Results The experimental results (Table 3) showed that compared with the model group, the three-component drug group and the seven-component drug group in each dose group could both significantly reduce the IL-6 release level of rat peritoneal macrophages at 12 hours and 24 hours, indicating that the three-component drug group and the seven-component drug group had good inhibitory effects on the early inflammatory factors of the sepsis cell model.

[0060] [Table 3] Example 3

[0061] Evaluation of the effects of three- and seven-component drugs on the release of HMGB1 from rat peritoneal macrophages stimulated with LPS

[0062] 1. Experimental Materials Male clean grade SD rats (180-220 g), endotoxin (LPS), hydroxysafflor yellow A, paeoniflorin, albiflorin, oxidized paeoniflorin, senkyunolide I, danshensu sodium, ferulic acid, RPMI-1640 medium, 24-well plate, and HMGB1 ELISA kit.

[0063] 2. Experimental Method Peritoneal macrophages from male SD rats were isolated. The specific method was as follows: male SD rats were fasted for 12 hours before surgery, and the abdominal cavity was opened after anesthesia, 10 mL of pre-cooled PBS solution was injected into the abdominal cavity, and the abdominal wall was gently pressed with a finger to allow the liquid to flow in the abdominal cavity. The liquid in the abdominal cavity was aspirated and injected into a sterile tube, and washed once more with 10 mL of pre-cooled PBS solution, and the operation was the same as above. The collected washing solution was centrifuged at 250 g for 10 minutes at 4 °C, and the supernatant was discarded. 2 mL of red blood cell lysate was added to dissolve the red blood cells, and the mixture was gently shaken twice, 5 seconds each time, and left to stand for 5 minutes, and then 4 mL of D-Hanks solution was added to terminate the reaction. The mixture was centrifuged again according to the above method, and the supernatant was discarded. After washing the precipitate with medium, the cells were resuspended and washed at 2 × 10 6 The cell suspension was inoculated into a 24-well plate and incubated at 37° C., 5% CO 2 The cells were cultured in a cell culture incubator at 37 °C. Each group had six parallel wells. After 48 and 72 h of culture, the supernatants were collected and the cytokine contents were measured by ELISA.

[0064] The experiment was divided into a control group, a model group, and an administration group, and the treatment methods for each group were as follows: After culturing the cells of each group overnight in a cell incubator at 37℃ and 5%CO2, the control group and the model group were added with the corresponding amount of culture medium, and the treatment group was added with an equal amount of the corresponding concentration of drug solution. After culturing in the incubator for 1 hour, the control group was not added with LPS, and the model group and the treatment group were stimulated by adding LPS (75ng / mL). After 48 hours and 72 hours, 0.5mL of cell culture supernatant was collected from each group and stored in a refrigerator at -20℃, and the corresponding cytokines were detected by concentration. The experimental grouping and drug solution preparation methods were the same as in Example 2.

[0065] 3. Experimental Results The experimental results (Table 4) showed that compared with the model group, the three-component drug group and the seven-component drug group in each dose group could both significantly reduce the release level of HMGB1 in rat peritoneal macrophages at 48 hours and 72 hours, indicating that the three-component drug group and the seven-component drug group had good inhibitory effects on late inflammatory factors in the sepsis cell model.

[0066] [Table 4] Example 4

[0067] Evaluation of the effects of three-component and seven-component drugs on the release of TM from rat abdominal aortic endothelial cells stimulated by LPS

[0068] 1. Experimental Materials Male clean grade SD rats (180-220 g), LPS, hydroxysafflor yellow A, paeoniflorin, albiflorin, oxidized paeoniflorin, senkyunolide I, danshensu sodium, ferulic acid, ECM medium, 24-well plate, tissue factor (TM) ELISA kit.

[0069] 2. Experimental Method The rats were killed by cervical dislocation and then immersed in 75% ethanol for 5 minutes. The thoracic and abdominal cavities were opened layer by layer to completely expose the thoracic and abdominal aortas and separate the surrounding tissues. The aortas were separated from their proximal ends to the branches of the common iliac arteries and placed in culture dishes containing PBS. The adipose and fibrous tissues of the vascular adventitia were aseptically peeled off, and the vascular lumen was washed with PBS. The aorta was cut into small pieces of about 1.5 mm x 1.5 mm, placed in 6 mL of 0.25% type IV collagenase, digested at 37 ° C for 15 minutes, shaken every 5 minutes, carefully aspirated the digestive solution, and retained the tissue chunks, then added 6 mL of 1.0% neutral protease, digested at 37 ° C for 15 minutes, shaken every 5 minutes, aspirated the digestive solution, supplemented with 10 mL of ECM, repeatedly blown, centrifuged at 1000 r / min for 10 minutes, discarded the medium and digestive solution, retained the fragments, spread the tissue fragments on the bottom of a 10 cm culture dish, placed upside down in a 37 ° C oven for 2 hours, firmly attached the tissue chunks, added an appropriate amount of ECM, and preferably submerged the tissue fragments, and cultured in a CO2 saturated humidity incubator with a volume fraction of 5% at 37 ° C, and the medium was replaced every 3 days. At about 7 days, endothelial cells were seen to creep out from the edge of the tissue piece and gradually extend outward, assuming a short, flattened spindle or polygonal shape, at which point the tissue chunks were removed and digested with 0.25% trypsin and cut into 25 cm pieces. 2 The cells were subcultured at a ratio of 1:3 into a culture flask, and experiments were performed using cells from the third or fourth passage.

[0070] The rat abdominal aortic endothelial cells were cultured according to the above-mentioned method, and a cell suspension was prepared. 5 Cells were inoculated at 100 μg / mL, the cell suspension was inoculated into a 24-well plate and cultured at 37°C. After about 12 hours, the model group and the treatment group were stimulated with LPS, and after 1 hour, they were given different solutions to intervene, and the supernatants were collected 24 hours, 48 ​​hours, and 72 hours after stimulation.

[0071] The experiment was divided into a control group, a model group, and a treatment group. The treatment method for each group was as follows: after culturing the cells of each group overnight in a cell incubator at 37℃ and 5%CO2, the corresponding amount of culture medium was added to the control group and the model group, and an equal amount of drug solution of the concentration corresponding to the treatment group was added. After culturing in the incubator for 1 hour, LPS was not added to the control group, and LPS (75ng / mL) was added to the model group and the treatment group for stimulation. After 24 hours, 48 ​​hours, and 72 hours, 0.5mL of cell culture supernatant was collected from each group and stored in a refrigerator at -20℃, and the corresponding cytokines were detected by concentration. The experimental grouping and drug solution preparation method were the same as in Example 2.

[0072] 3. Experimental Results TM is commonly present on the surface of rat abdominal aortic endothelial cells, and by binding to thrombin, it activates the protein C system to exert anticoagulant effects. Under sepsis conditions, TM is released in large quantities into the surrounding blood, weakening the anticoagulant effect. The reduction of TM under drug intervention contributes to the balance of anticoagulant / procoagulant factors and is beneficial for promoting the recovery of the blood coagulation function of cells / organisms to the normal state.

[0073] The experimental results (Table 5) show that compared with the model group, the three-component drug group and the seven-component drug group in each concentration group can all reduce the release of TM in rat abdominal aortic endothelial cells at 24 hours, 48 ​​hours and 72 hours, and the three-component drug group and the seven-component drug group have the effect of restoring blood coagulation function to normal in the sepsis cell model.

[0074] [Table 5] Example 5

[0075] Evaluation of the effects of three-component and seven-component drugs on the release of TF from rat abdominal aortic endothelial cells stimulated with LPS

[0076] 1. Experimental Materials Male clean grade SD rats (180-220 g), LPS, hydroxysafflor yellow A, paeoniflorin, albiflorin, oxidized paeoniflorin, senkyunolide I, danshensu sodium, ferulic acid, ECM medium, 24-well plate, tissue factor (TF) ELISA kit.

[0077] 2. Experimental Method The experimental method and grouping processing method were the same as in Example 4.

[0078] The experimental grouping and the method of preparing the drug solution were the same as in Example 2.

[0079] 3. Experimental Results TF is an important procoagulant in rat abdominal aortic endothelial cells, and in the state of sepsis, the hypercoagulable state is expressed as a massive release of TF. The reduction of TF under drug intervention contributes to the balance of anticoagulant / procoagulant factors and is beneficial to promote the recovery of the blood coagulation function of cells / organisms to the normal state.

[0080] The experimental results (Table 6) show that compared with the model group, the three-component drug group and the seven-component drug group in each concentration group could all reduce the release of TF, a procoagulant factor, from rat abdominal aortic endothelial cells at 24 hours, 48 ​​hours, and 72 hours, and the three-component drug group and the seven-component drug group had the effect of restoring blood coagulation function to normal in the septic cell model.

[0081] [Table 6] Example 6

[0082] Evaluation of the effects of three-component and seven-component drugs on apoptosis of rat splenic regulatory T cells (Treg) stimulated by LPS

[0083] 1. Experimental Materials Male clean grade SD rat (180-220g), LPS, hydroxysafflor yellow A, paeoniflorin, albiflorin, oxidized paeoniflorin, senkyunolide I, sodium danshensu, ferulic acid, 1640 medium, 24-well plate, phycoerythrin (PE)-anti-rat CD25, fluorescein isothiocyanate (FITC)-labeled anti-rat CD4, fluorescein isothiocyanate (FITC)-labeled annexin V apoptosis kit, rat CD25-APC antibody kit, APC magnetic beads, CD4 magnetic beads, MACSBuffer, MiniMACS magnetic separator and separation column (LS), anti-rat CD3 monoclonal antibody, anti-rat CD28 monoclonal antibody.

[0084] 2. Experimental Method Male SD rats were killed by cervical dislocation, and the spleens were collected. The spleens were pulverized with a sterile syringe plunger, the suspension was transferred to a centrifuge tube with a pipette, centrifuged at 1200 rpm for 7 min, the supernatant was discarded, an appropriate amount of MACSBuffer (10 mL / each spleen) was added to resuspend the spleen, and the upper layer of lymphocyte separation solution (1:1) was added along the wall of the tube, centrifuged at 3000 rpm for 15 min, the middle layer was aspirated with a pipette, placed in another centrifuge tube, the washing solution was added, and the spleen was centrifuged at 1200 rpm for 7 min, the supernatant was discarded, and the spleen was resuspended by adding MACSBuffer and set aside.

[0085] 1×10 7 Anti-CD25-APC antibody was added at 1 μL per 10 monocytes, incubated at 4°C for 15 minutes, and washed with MACSBuffer. 7 Add 20 μL of anti-APC magnetic beads and 80 μL of MACSBuffer per cell, incubate at 4 °C for 15 min, wash with MACSBuffer, resuspend in an appropriate volume of MACSBuffer, and magnetically separate on an LS column to isolate CD25 + Cells were obtained.

[0086] CD25 + After counting the cells, 1 x 10 7Add 20 µL of dissociation agent per cell, incubate at 4 °C for 1 min, wash with MACSBuffer, and add 1 x 10 7 Add 20 μL of anti-CD4 magnetic beads and 30 μL of terminator per cell, incubate at 4 °C for 15 min, wash with MACSBuffer, resuspend in an appropriate amount of MACSBuffer, and magnetically separate on an LS column to isolate CD4 + CD25 + Cells were obtained.

[0087] Treg cells were resuspended in an appropriate amount of medium and the cell number was adjusted to 2.5 × 10 6 The CD3 / CD28 ratio was adjusted to 0.5 μg / 10 mL and 100 μL of cells were seeded in each well of a 96-well plate. The model group and the experimental group were divided into two groups: CD3 / CD28 (CD3 was 0.5 μg / 10 mL). 6 , CD28 is 1μg / 10 6 ) + LPS (1 μg / mL), and the experiment was divided into a control group, a model group, and an administration group. One hour later, the model group and the administration group were each given different drug solutions as interventions, and the cells were cultured in a CO2 incubator at 37°C for 72 hours.

[0088] The cells were harvested, washed with pre-chilled PBS, washed again with 1 ml of 1× Binding Buffer, centrifuged, the supernatant was removed to leave 100 μl, 5 μl of FITC Annexin V and 5 μl of PI were then added, incubated for 15 minutes in the dark at room temperature (25°C), and then 200 μl of 1× Binding Buffer was added and mixed uniformly, and detected by flow cytometry within 1 hour.

[0089] The grouping treatment methods were as follows: Model group: The cells were cultured overnight in a cell incubator, and then stimulated by adding LPS (75ng / mL); Treatment group: The corresponding concentration of drug solution was added and cultured for 1 hour, and LPS stimulation was performed. After 72 hours of intervention in the experimental groups with different concentrations, 0.5mL of the cell culture supernatant was collected and stored in a -20℃ refrigerator, and the corresponding cytokines were detected by concentration. The experimental grouping and the method of preparing the drug solution were the same as in Example 2.

[0090] 3. Experimental Results After the function of Treg cells is enhanced, they produce immunosuppressive factors such as IL-10, IL-35, and TGF-β to inhibit normal T cells, and kill target cells with granzyme B and perforin-1 to exert their immunosuppressive effects. They also induce DC dendritic cells to produce indoleamine 2,3-dioxygenase, which catalyzes the decomposition of tryptophan into kynurene, causing the death of surrounding cells, and induce DC cells to secrete other amino acid-related enzymes to inhibit the proliferation of effector T cells, thereby exerting their immunosuppressive effects. Therefore, the drug promotes the apoptosis of Treg cells at a certain time, exerting a very important effect on biological immune regulation. Compared with the model group, the three-component drug group and the seven-component drug group in each dose group can both promote the apoptosis of rat regulatory T cells (Treg) (Table 7).

[0091] [Table 7] Example 7

[0092] Evaluation of the effects of three-component and seven-component drugs on CTLA-4 and Foxp3 expression in splenic regulatory T cells of rats stimulated with LPS

[0093] 1. Experimental Materials Male clean grade SD rat (180-220g), LPS, hydroxysafflor yellow A, paeoniflorin, albiflorin, oxidized paeoniflorin, senkyunolide I, sodium danshensu, ferulic acid, 1640 medium, 24-well plate, phycoerythrin (PE) anti-rat CD25, fluorescein isothiocyanate (FITC)-labeled anti-rat CD4, rat anti-PE kit, CD4 magnetic beads, MiniMACS magnetic separator and separation column, PE-labeled Foxp3 kit, PE-labeled CTLA-4 kit, anti-rat CD3 monoclonal antibody, anti-rat CD28 monoclonal antibody.

[0094] 2. Experimental Method The experimental method and grouping processing method were the same as in Example 6.

[0095] The experimental grouping and the method of preparing the drug solution were the same as in Example 2.

[0096] 3. Experimental Results Foxp3 + Treg cells exert their immunosuppressive effects by inhibiting normal T cells through the production of immunosuppressive factors such as IL-10, IL-35, and TGF-β, and by killing target cells with granzyme B and perforin-1. CTLA-4 induces DC dendritic cells to produce indoleamine 2,3-dioxygenase, which catalyzes the decomposition of tryptophan to kynurene, causing the death of surrounding cells, and further induces DC cells to secrete other amino acid-related enzymes, thereby inhibiting the proliferation of effector T cells. Therefore, Treg cells play a very important role in the regulation of biological immunity through Foxp3 and CTLA-4, and the expression of Foxp3 is closely related to the function of regulatory T cells (Treg).

[0097] Compared with the model group, the three-component drug group and the seven-component drug group in each dose group can both reduce the expression levels of CTLA-4 and Foxp3 in rat regulatory T cells (Treg) at 72 hours, indicating that the three-component drug group and the seven-component drug group have the effect of reducing the expression levels of CTLA-4 and Foxp3 in regulatory T cells (Treg) of septic rats at 72 hours (Tables 8 and 9).

[0098] [Table 8]

[0099] [Table 9] Example 8

[0100] Evaluation of the effects of three-component and seven-component drugs on IL-6 expression in rats with CLP-induced sepsis

[0101] 1. Experimental Materials Male clean grade SD rats (180-220g) that had been adaptively reared for one week. Hydroxysafflor Yellow A, paeoniflorin, albiflorin, paeoniflorin oxide, senkyunolide I, danshensu sodium, ferulic acid, IL-6 ELISA detection kit.

[0102] 2. Experimental Method 2.1 Grouping and intervention 290 rats were randomly divided into 29 groups, including a control group, a model group, and a treatment group, with 10 rats in each group. The orthogonal experimental method was used for the treatment group, and the grouping method for the orthogonal experiment is shown in Tables 10 and 11.

[0103] [Table 10]

[0104] In Table 10, the three concentrations of hydroxysafflor yellow A are low 0.9 mg / kg, medium 9 mg / kg, and high 90 mg / kg, respectively, the three concentrations of paeoniflorin are low 0.9 mg / kg, medium 9 mg / kg, and high 90 mg / kg, the three concentrations of albiflorin are low 0.09 mg / kg, medium 0.9 mg / kg, and high 9 mg / kg, and the three concentrations of oxidized paeoniflorin are low 0.09 mg / kg, medium 0.9 mg / kg, and high 9 mg / kg. The three concentrations of senkyunolide I were low 0.09 mg / kg, medium 0.9 mg / kg, and high 9 mg / kg, the three concentrations of danshensu sodium were low 0.09 mg / kg, medium 0.9 mg / kg, and high 9 mg / kg, and the three concentrations of ferulic acid were low 0.009 mg / kg, medium 0.09 mg / kg, and high 0.9 mg / kg.

[0105] [Table 11]

[0106] The preparation method of the solution and the injection amount in the experiment are as follows: Each rat is calculated as 200g, and the injection amount in the experiment is 1.0mL / time / rat. According to the concentration corresponding to each group in Table 10, the appropriate weight of hydroxysafflower yellow A, paeoniflorin and albiflorin is weighed by electronic balance (1 / 10000g), and they are mixed into different measuring flasks according to different concentration combinations, and dissolved in an appropriate amount of saline (0.9% sodium chloride solution) to prepare 9 groups of three-component drugs with corresponding concentrations; according to the concentration corresponding to each group in Table 11, the appropriate weight of hydroxysafflower yellow A, paeoniflorin, albiflorin, paeoniflorin oxide, senkyunolide I, sodium danshensu and ferulic acid is weighed by electronic balance (1 / 10000g), and they are mixed into measuring flasks according to different concentration combinations, and dissolved in an appropriate amount of saline (0.9% sodium chloride solution) to prepare 18 groups of seven-component drugs with corresponding concentrations. Before the experiment, the rats were fasted for 12 hours, weighed, and divided into groups according to a random number table method. In the control group, only the cecum was exposed by laparotomy, the skin was sutured, and 10 mL of saline was injected subcutaneously to resuscitate, while the model group and the treatment group were subjected to cecal ligation and puncture (CLP) modeling, followed by 10 mL of saline injection subcutaneously. The CLP modeling process is as follows: Rats were anesthetized by intramuscular injection of a mixture prepared by ketamine injection + smianxin II injection in a volume ratio of 2:1, and the sepsis animal model was prepared by CLP. The junction of the cecum and ileum was ligated, and an 18-gauge needle was used to pierce the cecum twice to form an intestinal fistula, and two drainage strips (0.5 cm × 2.0 cm) were left in place to prevent the needle hole from healing, and then the skin was sutured layer by layer, and the rats were resuscitated by subcutaneous injection of 10 mL of saline immediately after the operation. After the rats were operated on with CLP, the treatment group was injected with the above different drug solutions through the tail vein at 2, 12, 24, 36, 48 and 60 hours after the operation, and the model and control groups were injected with saline through the tail vein at the corresponding times. The experiment was repeated until n=10.

[0107] 2.2 Blood sampling and testing At 8 h, 24 h, and 48 h after CLP, 3 mL of sterile blood was collected from the abdominal aorta of animals in each group after anesthesia, and the IL-6 content in plasma was detected by ELISA.

[0108] 3. Experimental Results The experimental results (Table 12) show that the plasma of the control group contains a small amount of IL-6, and the IL-6 content of the model group is significantly increased early after CLP surgery, the level of IL-6 further increases at 8 hours and gradually decreases at 24 hours, but is still higher than the control group at 48 hours after surgery, the difference is statistically significant (p<0.05). Meanwhile, the IL-6 plasma content of the treatment group is significantly lower than that of the model group at 8 hours, 24 hours, and 48 hours after surgery in the treatment group (p<0.05), and is close to the level of the control group.

[0109] The above findings suggest that compared with the model group, the three-component drug group and the seven-component drug group in each dose group can significantly reduce the expression of IL-6 in CLP septic rats, and have obvious inhibitory effects on the early inflammatory response of septic rat models. When converted to adult doses, the doses are approximately the three-component drug group (hydroxysafflor yellow A 6mg-600mg, paeoniflorin 6mg-600mg, albiflorin 0.6mg-60mg), the seven-component drug group (hydroxysafflor yellow A 6mg-600mg, paeoniflorin 6mg-600mg, albiflorin 0.6mg-60mg, paeoniflorin oxide 0.6mg-60mg, senkyunolide I 0.6mg-60mg, danshensu sodium 0.6mg-60mg, ferulic acid 0.06mg-6mg). [According to calculations based on the body surface area conversion method for humans and animals in Pharmacological Experimental Methodology edited by Xu Shuyun, human dose = rat dose / 0.018 (adult body weight 70 kg, rat body weight 0.2 kg)].

[0110] [Table 12] Example 9

[0111] Evaluation of the effects of three-component and seven-component drugs on the expression of HMGB1 in CLP-induced septic rats

[0112] 1. Experimental Materials Male clean grade SD rats (180-220g) that had been adaptively reared for one week. Hydroxysafflor Yellow A, paeoniflorin, albiflorin, paeoniflorin oxide, senkyunolide I, danshensu sodium, ferulic acid, HMGB1 ELISA detection kit.

[0113] 2. Experimental Method 2.1 Grouping and intervention See Example 8.

[0114] 2.2 Blood sampling and testing At 48 and 72 hours after CLP surgery, 3 mL of sterile blood was collected from the abdominal aorta of each anesthetized animal, and plasma HMGB1 content was detected by ELISA.

[0115] 3. Experimental Results The experimental results (Table 13) show that the plasma of the control group contains a small amount of HMGB1, and in the late period after CLP surgery, the HMGB1 content in the model group increased significantly, gradually increased at 48 hours, and was still higher than the control group at 72 hours after surgery, and the difference was statistically significant (p<0.05). Meanwhile, the plasma HMGB1 content of the treatment groups at 48 hours and 72 hours after surgery was both significantly lower than that of the model group (p<0.05), and close to the level of the control group. The above findings show that compared with the model group, the three-component drug group and the seven-component drug group in each dosage group can both significantly reduce the expression of HMGB1 in CLP septic rats, and have obvious inhibitory effects on the late inflammatory response of septic rat models. When converted to adult doses, it is about 3-component drug group (hydroxysafflor yellow A 6mg-600mg, paeoniflorin 6mg-600mg, albiflorin 0.6mg-60mg) and 7-component drug group (hydroxysafflor yellow A 6mg-600mg, paeoniflorin 6mg-600mg, albiflorin 0.6mg-60mg, paeoniflorin oxide 0.6mg-60mg, senkyunolide I 0.6mg-60mg, danshensu sodium 0.6mg-60mg, ferulic acid 0.06mg-6mg). [According to the calculation based on the body surface area conversion method of humans and animals in "Pharmacological Experiment Methodology" edited by Xu Shuyun, human dose = rat dose / 0.018 (adult body weight 70kg, rat body weight 0.2kg total)].

[0116] [Table 13] Example 10

[0117] Evaluation of the effects of three- and seven-component drugs on TF release in CLP-induced septic rats

[0118] 1. Experimental Materials Male clean grade SD rats (180-220g) that had been adaptively raised for 1 week, hydroxysafflor yellow A, paeoniflorin, albiflorin, oxidized paeoniflorin, senkyunolide I, danshensu sodium, ferulic acid, and TFELISA detection kit.

[0119] 2. Experimental Method 2.1 Grouping and intervention See Example 8.

[0120] 2.2 Blood collection and testing At 12, 24, 48, and 72 hours after CLP surgery, 5 mL of sterile blood was collected from the abdominal aorta of anesthetized animals in each group. TF content was detected by enzyme-linked immunosorbent assay (ELISA).

[0121] 3. Experimental Results The experimental results (Table 14) show that there was a certain TF expression in monocytes in the control group, and the expression of TF (12 hours, 24 hours, 48 ​​hours and 72 hours after operation) in the model group was significantly higher than that in the control group (p<0.05), and gradually increased with the extension of postoperative time. The expression of TF (12 hours, 24 hours, 48 ​​hours and 72 hours after operation) in the treatment group was significantly decreased compared with the model group (p<0.05). The above findings show that compared with the model group, the three-component drug group and the seven-component drug group in each dose group can both significantly reduce the release of TF in CLP septic rats, and the three-component drug group and the seven-component drug group have obvious effects on inhibiting the release of coagulation factors and improving the hypercoagulable state in the septic rat model. When converted to adult doses, they were approximately a three-component drug group (hydroxysafflor yellow A 6 mg-600 mg, paeoniflorin 6 mg-600 mg, albiflorin 0.6 mg-60 mg) and a seven-component drug group (hydroxysafflor yellow A 6 mg-600 mg, paeoniflorin 6 mg-600 mg, albiflorin 0.6 mg-60 mg, paeoniflorin oxide 0.6 mg-60 mg, senkyunolide I 0.6 mg-60 mg, danshensu sodium 0.6 mg-60 mg, and ferulic acid 0.06 mg-6 mg).

[0122] [Table 14] Example 11

[0123] Evaluation of the effects of three- and seven-component drugs on the release of TM in CLP-induced septic rats

[0124] 1. Experimental Materials Male clean grade SD rats (180-220g) that had been adaptively reared for one week. Hydroxysafflor Yellow A, paeoniflorin, albiflorin, paeoniflorin oxide, senkyunolide I, danshensu sodium, ferulic acid, TM ELISA detection kit.

[0125] 2. Experimental Method 2.1 Grouping and intervention See Example 8.

[0126] 2.2 Blood sampling and testing At 12, 24, 48, and 72 hours after CLP surgery, 5 mL of sterile blood was collected from the abdominal aorta of anesthetized animals in each group. TM content was detected by enzyme-linked immunosorbent assay (ELISA).

[0127] 3. Experimental Results The experimental results (Table 15) show that there was a certain TF expression in monocytes in the control group, and the expression of TM (12 hours, 24 hours, 48 ​​hours and 72 hours after operation) in the model group was significantly higher than that in the control group (p<0.05), and gradually increased with the extension of postoperative time. The expression of TM (12 hours, 24 hours, 48 ​​hours and 72 hours after operation) in the treatment group was significantly decreased compared with the model group (p<0.05). The above findings show that compared with the model group, the three-component drug group and the seven-component drug group in each dose group can significantly reduce the release of TM in CLP septic rats, and the three-component drug group and the seven-component drug group have obvious effects of inhibiting the release of coagulation factors and improving the hypercoagulable state in the rat model of sepsis. When converted to adult doses, it is about 3-component drug group (hydroxysafflor yellow A 6mg-600mg, paeoniflorin 6mg-600mg, albiflorin 0.6mg-60mg) and 7-component drug group (hydroxysafflor yellow A 6mg-600mg, paeoniflorin 6mg-600mg, albiflorin 0.6mg-60mg, paeoniflorin oxide 0.6mg-60mg, senkyunolide I 0.6mg-60mg, danshensu sodium 0.6mg-60mg, ferulic acid 0.06mg-6mg). [According to the calculation based on the body surface area conversion method of humans and animals in "Pharmacological Experiment Methodology" edited by Xu Shuyun, human dose = rat dose / 0.018 (adult body weight 70kg, rat body weight 0.2kg total)].

[0128] [Table 15] Example 12

[0129] Evaluation of the effects of three-component and seven-component drugs on sepsis regulatory T cell function induced by CLP

[0130] 1. Experimental Materials Male clean grade SD rats (180-220g), endotoxin (LPS), hydroxysafflor yellow A, paeoniflorin, albiflorin, oxidized paeoniflorin, senkyunolide I, sodium danshensu, ferulic acid, 1640 medium, 24-well plate, phycoerythrin (PE)-anti-rat CD25, fluorescein isothiocyanate (FITC)-labeled anti-rat CD4, fluorescein isothiocyanate (FITC)-labeled Annexin V apoptosis kit, rat anti-PE kit, CD4 magnetic beads, MiniMACS magnetic separator and separation column, PE-labeled Foxp3 kit, PE-labeled CTLA-4 kit, anti-rat CD3 monoclonal antibody, anti-rat CD28 monoclonal antibody.

[0131] 2. Experimental Method 2.1 Grouping and intervention See Example 8.

[0132] 2.2 Cell isolation and culture 72 hours after CLP surgery, rats were killed by cervical dislocation, and the chest and abdomen were disinfected with alcohol cotton balls three times, then the abdomen was opened, the intact peritoneum was preserved, and the alcohol cotton ball was disinfected again, and the peritoneum was opened with another pair of tweezers, and the spleen was taken out and placed in 15 ml PBS at 4 ° C. The spleen capsule was torn using the tweezers, placed in a filter, and PBS was added to wet it, the spleen was torn, the fascia was removed, and the spleen was crushed with a sterile syringe plunger, the suspension was transferred to a centrifuge tube with a pipette, centrifuged at 1200 rpm for 7 minutes, the supernatant was discarded, and an appropriate amount of MACSBuffer (10 mL / each spleen) was added to the upper layer of lymphocyte separation solution (1:1) along the wall of the tube, and centrifuged at 3000 rpm for 15 minutes, and the middle layer was aspirated with a pipette, placed in another centrifuge tube, the washing solution was added, and centrifuged at 1200 rpm for 7 minutes, the supernatant was discarded, and an appropriate amount of MACSBuffer was added to resuspend it, and it was prepared.

[0133] 1×10 7 1 μl of anti-CD25-APC antibody was added per monocyte, incubated at 4°C for 15 minutes, and washed with MACSBuffer. 7 Add 20 μl of anti-APC magnetic beads and 80 μl of MACSBuffer per cell, incubate at 4°C for 15 min, wash with MACSBuffer, resuspend in an appropriate volume of MACSBuffer, and magnetically separate on an LS column to isolate CD25 + Cells were obtained.

[0134] CD25 + After cell counting, 1 x 10 cells 7 Add 20 μl of dissociation agent per 1 x 10 cells, incubate at 4 °C for 10 min, wash with MACSBuffer, and discard. 7 Add 20 μl of anti-CD4 magnetic beads and 30 μl of terminator per cell, incubate at 4°C for 15 min, wash with MACSBuffer, resuspend in an appropriate amount of MACSBuffer, and magnetically separate using an LS column to isolate CD4 + CD25 + Cells were obtained.

[0135] 2.3 Detection and Analysis Detection of Treg apoptosis rate: Collect cells, wash cells with pre-cooled PBS, wash again with 1ml of 1×Binding Buffer, after centrifugation, remove the supernatant to leave 100 μl, then add 5 μl of FITCAnnexinV and 5 μl of PI, incubate for 15 minutes in the dark at room temperature (25°C), add 200 μl of 1×Binding Buffer, mix uniformly, and detect by flow cytometry within 1 hour.

[0136] Detection of Foxp3 and CTLA-4 expression: After collecting the cell supernatant, the cells were washed with PBS, the supernatant was removed, CTLA-4-PE fluorescent antibody was added, incubated at 4℃ in the dark for 30 min, and membrane disruption agent was added overnight. The next day, the cells were washed with membrane permeabilization buffer, the supernatant was removed, Foxp3-PE-Cy7 fluorescent antibody was added, incubated at room temperature in the dark for 30 min, washed with membrane disruption buffer, the supernatant was removed, fixed with 1% paraformaldehyde, and detected by flow cytometry.

[0137] 3. Experimental Results 3.1 Effects of three-component and seven-component drugs on regulatory T cell function in CLP-induced septic rats

[0138] The experimental results (Table 16) show that the apoptosis rate of Treg in the model group was significantly lower than that of the control group (p<0.05), and the apoptosis rate of Treg in the treatment group was significantly higher than that of the model group (p<0.05). The expression of Foxp3 and CTLA-4 in the model group was significantly higher than that of the control group (p<0.05), and both treatment groups were significantly lower than that of the model group (p<0.05). The above findings suggest that compared with the model group, the three-component drug group and the seven-component drug group in each dose group can both promote the apoptosis of septic Treg and down-regulate the inhibitory effect on the proliferation and secretion function of T lymphocytes. For the rat model of sepsis, the three-component drug group and the seven-component drug group in each dose group have obvious effects on correcting the immunosuppressive state of living cells. When converted to adult doses, there were about three component drug groups (hydroxysafflor yellow A 6mg-600mg, paeoniflorin 6mg-600mg, albiflorin 0.6mg-60mg) and seven component drug groups (hydroxysafflor yellow A 6mg-600mg, paeoniflorin 6mg-600mg, albiflorin 0.6mg-60mg, paeoniflorin oxide 0.6mg-60mg, senkyunolide I 0.6mg-60mg, danshensu sodium 0.6mg-60mg, ferulic acid 0.06mg-6mg). [According to the calculation based on the body surface area conversion method for humans and animals in "Pharmacological Experiment Methodology" edited by Xu Shuyun, human dose = rat dose / 0.018 (adult body weight 70kg, rat body weight 0.2kg total)].

[0139] [Table 16]

[0140] The above description of the embodiments is only intended to help understand the method of the present invention and its core idea. It should be noted that those skilled in the art can make some improvements and modifications to the present invention without departing from the principles of the present invention, and these also fall within the scope of protection of the claims of the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined in this specification can be realized in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to these embodiments shown in this specification, but is to be covered in the widest scope consistent with the principles and novel features shown in this specification.

Claims

1. Hydroxysafflor Yellow A, the structural formula of which is represented by Formula I; Paeoniflorin, the structural formula of which is represented by Formula II; and Albiflorin, the structural formula of which is represented by Formula III. A pharmaceutical composition for treating sepsis, characterized in that the mass ratio of said hydroxysafflor yellow A, paeoniflorin and albiflorin is 1-100:1-100:0.1-10 (excluding compositions containing albiflorin, paeoniflorin, oxypaeoniflorin, benzoylpaeoniflorin, benzoyloxypaeoniflorin, mudampioside J, galloylpaeoniflorin, mudampioside C, benzoic acid, gallic acid, ethyl gallate and catechin, as well as Xuebijing (blood purification) injection). 【Chemistry 1】

2. The pharmaceutical composition for treating sepsis according to claim 1, characterized in that the mass ratio of said hydroxysafflor yellow A, paeoniflorin and albiflorin is 1-100:1-100:1-10.

3. The pharmaceutical composition for treating sepsis according to claim 1, comprising: oxidized paeoniflorin having a structural formula represented by formula IV; senkyunolide I having a structural formula represented by formula V; danshensu sodium having a structural formula represented by formula VI; and ferulic acid having a structural formula represented by formula VII; A pharmaceutical composition for treating sepsis, characterized in that the mass ratio of said hydroxysafflor yellow A, paeoniflorin, albiflorin, paeoniflorin oxide, senkyunolide I, sodium danshensu and ferulic acid is 1-100:1-100:1-10:0.1-10:0.1-10:0.1-10:0.01-1 (excluding compositions containing albiflorin, paeoniflorin, oxypaeoniflorin, benzoyl paeoniflorin, benzoyl oxypaeoniflorin, mudampioside J, galloyl paeoniflorin, mudampioside C, benzoic acid, gallic acid, ethyl gallate and catechin, and Xuebijing (blood purification) injection). 【Chemistry 2】

4. The pharmaceutical composition for treating sepsis according to claim 3, characterized in that the mass ratio of said hydroxysafflor yellow A, paeoniflorin, albiflorin, paeoniflorin oxide, senkyunolide I, sodium danshensu and ferulic acid is 1-100:1-100:1-10:1-10:1-10:1-10:0.1-1.

5. The pharmaceutical composition for treating sepsis according to claim 1, characterized in that the pharmaceutical composition achieves the treatment of sepsis by inhibiting the release of TF and / or TM in sepsis.

6. The pharmaceutical composition for treating sepsis according to claim 1, characterized in that the pharmaceutical composition achieves the treatment of sepsis by promoting apoptosis of sepsis regulatory T cells.

7. 13. Use of the pharmaceutical composition according to claim 1 in preparing a medicament for treating sepsis.

8. The use of the pharmaceutical composition according to claim 7, characterized in that it is used in the preparation of a medicament for inhibiting the expression of IL-6, Foxp3, CTLA-4 and / or HMGB1 in sepsis.

9. The use of the pharmaceutical composition according to claim 7, characterized in that it is used in the preparation of a drug for inhibiting the release of TF and / or TM in sepsis.

10. The use of the pharmaceutical composition according to claim 7 in the preparation of a medicament for promoting apoptosis of sepsis regulatory T cells in sepsis.

11. The use according to claim 7, characterized in that, when the pharmaceutical composition is composed of three active compounds, namely, hydroxysafflor yellow A, paeoniflorin and albiflorin, the dosage of hydroxysafflor yellow A is 6-600 mg / kg, the dosage of paeoniflorin is 6-600 mg / kg, and the dosage of albiflorin is 0.6-60 mg / kg.

12. The use according to claim 7, characterized in that, when said pharmaceutical composition is composed of seven active compounds including hydroxysafflor yellow A, paeoniflorin, albiflorin, paeoniflorin oxide, senkyunolide I, danshensu sodium and ferulic acid, the dosage of hydroxysafflor yellow A is 6-600mg / kg, the dosage of paeoniflorin is 6-600mg / kg, the dosage of albiflorin is 0.6-60mg / kg, the dosage of paeoniflorin oxide is 0.6-60mg / kg, the dosage of senkyunolide I is 0.6-60mg / kg, the dosage of danshensu sodium is 0.6-60mg / kg and the dosage of ferulic acid is 0.06-6mg / kg.

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