WO2022183539A1 - 一种用于治疗脓毒症的药物组合物及其应用 - Google Patents

一种用于治疗脓毒症的药物组合物及其应用 Download PDF

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WO2022183539A1
WO2022183539A1 PCT/CN2021/082244 CN2021082244W WO2022183539A1 WO 2022183539 A1 WO2022183539 A1 WO 2022183539A1 CN 2021082244 W CN2021082244 W CN 2021082244W WO 2022183539 A1 WO2022183539 A1 WO 2022183539A1
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paeoniflorin
sepsis
pharmaceutical composition
group
dosage
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French (fr)
Inventor
于洋
王建立
蒋春亮
王向成
李玉坤
李宝齐
张桂萍
董凯
姚小青
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Tianjin Chase Sun Pharmaceutical Co Ltd
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Tianjin Chase Sun Pharmaceutical Co Ltd
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Priority to JP2023538107A priority Critical patent/JP7612025B2/ja
Priority to US18/272,466 priority patent/US12616709B2/en
Priority to EP21928620.0A priority patent/EP4248964A4/en
Publication of WO2022183539A1 publication Critical patent/WO2022183539A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • the invention belongs to the technical field of chemical drugs, and particularly relates to a pharmaceutical composition for treating sepsis and its application.
  • Sepsis is a fatal organ dysfunction caused by a dysregulated host response to infection.
  • the dangerous condition and high fatality rate are the main causes of death in critically ill patients.
  • antibiotics, antiviral drugs, and vasopressors have been used in the traditional treatment of sepsis, but there are not enough specific drugs for the pathogenesis of sepsis put into clinical practice.
  • How to timely correct the systemic inflammatory response, coagulation dysfunction and immune dysfunction during the occurrence and development of sepsis, restore the body's pro-inflammatory-anti-inflammatory dynamic balance as soon as possible, and effectively improve the prognosis of patients has become an urgent need in the development of sepsis drugs. important issues to be addressed.
  • Xuebijing injection is based on the principle of “three syndromes and three methods", and under the theoretical guidance of “combined treatment of bacteria and toxins", it is based on “Xuefu Zhuyu Decoction” contained in "Yilin Correction” written by Wang Qingren in the Qing Dynasty. It is an intravenous injection developed based on modern technology such as safflower, red peony, chuanxiong, salvia, and angelica through extraction, refining, drying, and blending. It is suitable for sepsis/infection induced by systemic inflammatory response syndrome; it can also be used in conjunction with the treatment of organ dysfunction in multiple organ dysfunction syndrome.
  • the object of the present invention is to provide a novel pharmaceutical composition for the treatment of sepsis and application thereof, the pharmaceutical composition is directly composed of the active compound of sepsis, avoiding the cumbersome steps of Chinese medicine extraction, and The components of the pharmaceutical composition are simplified and the efficacy of the product is improved.
  • the present invention provides a pharmaceutical composition for treating sepsis, comprising hydroxysafflor yellow A with structural formula shown in formula I, paeoniflorin with structural formula shown in formula II and paeoniflorin with structural formula shown in formula III Lactone glycosides;
  • the mass ratio of the hydroxysafflor yellow A, paeoniflorin and paeoniflorin is 1-100:1-100:0.1-10;
  • the mass ratio of the hydroxysafflor yellow A, paeoniflorin and paeoniflorin is 1-100:1-100:1-10.
  • the pharmaceutical composition further comprises oxypaeoniflorin whose structural formula is shown in formula IV, Yangchuanxiong lactone I whose structural formula is shown in formula V, danshensu sodium whose structural formula is shown in formula VI, and danshensu sodium whose structural formula is shown in formula VII.
  • the mass ratio of the hydroxysafflor yellow A, paeoniflorin, paeoniflorin, oxypaeoniflorin, yangchuanxiong lactone I, danshensu sodium and ferulic acid is 1-100:1-100:1-10: 0.1 ⁇ 10: 0.1 ⁇ 10: 0.1 ⁇ 10: 0.01 ⁇ 1;
  • the mass ratio of the hydroxysafflor yellow A, paeoniflorin, paeoniflorin, oxypaeoniflorin, yangchuanxiong lactone I, danshensu sodium and ferulic acid is 1 ⁇ 100:1 ⁇ 100:1 ⁇ 10:1 ⁇ 10:1 ⁇ 10:0.1 ⁇ 1.
  • the present invention provides the application of the pharmaceutical composition in preparing a medicine for treating sepsis.
  • the pharmaceutical composition is used in the preparation of a drug for inhibiting the expression of IL-6, Foxp3, CTLA-4 and/or HMGB1 in sepsis.
  • the pharmaceutical composition is used in the preparation of a drug for inhibiting the release of TF and/or TM in sepsis.
  • the pharmaceutical composition is used in the preparation of a drug for promoting apoptosis of sepsis regulatory T cells.
  • the drug dosage of hydroxysafflor yellow A is 6-600 mg/kg
  • the drug of paeoniflorin is 6-600 mg/kg
  • the dosage is 6-600 mg/kg
  • the drug dosage of paeoniflorin is 0.6-60 mg/kg.
  • the pharmaceutical composition is composed of seven active compounds of hydroxysafflor yellow A, paeoniflorin, paeoniflorin, oxypaeoniflorin, yangchuanxiong lactone I, danshensu sodium and ferulic acid, hydroxysafflower
  • the dosage of yellow A is 6-600 mg/kg
  • the dosage of paeoniflorin is 6-600 mg/kg
  • the dosage of paeoniflorin is 0.6-60 mg/kg
  • the dosage of oxypaeoniflorin is 0.6-60 mg/kg kg
  • the dosage of Yangchuanxiong lactone I is 0.6-60 mg/kg
  • the dosage of Danshensu sodium is 0.6-60 mg/kg
  • the dosage of ferulic acid is 0.06-6 mg/kg.
  • the present invention also provides a method for treating sepsis by taking the medicine prepared by the pharmaceutical composition.
  • the pharmaceutical composition can treat sepsis by inhibiting the expression of IL-6, Foxp3, CTLA-4 and/or HMGB1 in sepsis.
  • the pharmaceutical composition achieves the treatment of sepsis by inhibiting the release of TF and/or TM in sepsis.
  • the pharmaceutical composition achieves the treatment of sepsis by promoting the apoptosis of sepsis regulatory T cells.
  • the drug dosage of hydroxysafflor yellow A is 6-600 mg/kg
  • the drug of paeoniflorin is 6-600 mg/kg
  • the dosage is 6-600 mg/kg
  • the drug dosage of paeoniflorin is 0.6-60 mg/kg.
  • the pharmaceutical composition is composed of seven active compounds of hydroxysafflor yellow A, paeoniflorin, paeoniflorin, oxypaeoniflorin, yangchuanxiong lactone I, danshensu sodium and ferulic acid, hydroxysafflower
  • the dosage of yellow A is 6-600 mg/kg
  • the dosage of paeoniflorin is 6-600 mg/kg
  • the dosage of paeoniflorin is 0.6-60 mg/kg
  • the dosage of oxypaeoniflorin is 0.6-60 mg/kg kg
  • the dosage of Yangchuanxiong lactone I is 0.6-60 mg/kg
  • the dosage of Danshensu sodium is 0.6-60 mg/kg
  • the dosage of ferulic acid is 0.06-6 mg/kg.
  • the pharmaceutical composition for treating sepsis provided by the present invention comprises hydroxysafflor yellow A, paeoniflorin and paeoniflorin; the mass ratio of the hydroxysafflor yellow A, paeoniflorin and paeoniflorin is: 1 to 100: 1 to 100: 0.1 to 10.
  • the present invention confirms at the cellular level and animal level that the pharmaceutical composition has an inhibitory effect on the expression of macrophage IL-6, HMGB1, CTLA-4 and Foxp3 in sepsis, and effectively inhibits TF and TM of sepsis release, and at the same time have a promoting effect on the apoptosis of regulatory T lymphocytes, indicating that the pharmaceutical composition provided by the present invention has an obvious effect of correcting the immunosuppressive state of cells or body cells in sepsis, thereby realizing the treatment of sepsis. Purpose.
  • the pharmaceutical composition of the present invention has undergone an acute toxicity test for intravenous administration, and the results show that no obvious abnormality is observed after the animal is administered, and there is no significant difference between the animal's body weight and body weight growth rate compared with the control group, and the gross anatomy shows no obvious abnormality. It is indicated that the pharmaceutical composition provided by the present invention has drug safety.
  • the pharmaceutical composition provided by the present invention also includes oxypaeoniflorin whose structural formula is shown in formula IV, yangchuanxiong lactone I whose structural formula is shown in formula V, danshensu sodium whose structural formula is shown in formula VI, and whose structural formula is such as formula Ferulic acid shown in VII;
  • the mass ratio of described hydroxysafflor yellow A, paeoniflorin, paeoniflorin, oxypaeoniflorin, yangchuanxiong lactone I, danshensu sodium and ferulic acid is 1 ⁇ 100: 1 to 100: 0.1 to 10: 0.1 to 10: 0.1 to 10: 0.01 to 1.
  • the present invention determines hydroxysafflor yellow A as the active ingredient of safflower for treating sepsis, paeoniflorin, paeoniflorin and oxypaeoniflorin as the active ingredient of red peony and white peony for treating sepsis, yangchuanxiong lactone I
  • Danshensu as the active ingredient of Salvia miltiorrhiza in the treatment of sepsis
  • ferulic acid as the active ingredient in the treatment of sepsis in Angelica sinensis
  • the pharmaceutical composition Compared with the pharmaceutical composition composed of three compounds, the pharmaceutical composition has stronger inhibitory effect on the expression of macrophage IL-6, HMGB1, CTLA-4 and Foxp3 in sepsis, and has a stronger inhibitory effect on the expression of IL-6, HMGB1, CTLA-4 and Foxp3 in sepsis.
  • the release of TF and TM has a stronger inhibitory effect, and at the same time has a stronger promotion effect on the apoptosis of regulatory T lymphocytes, the experiment of the present invention shows that the pharmaceutical composition has a stronger drug effect in the treatment of sepsis, achieve better treatment effect.
  • the present invention also carries out an acute toxicity test for intravenous administration of the pharmaceutical composition composed of seven active ingredients, and the results show that no obvious abnormality is observed after the animals are administered, and the animal body weight and body weight growth rate have no significant difference with the control group. No obvious abnormality was found in the anatomy, indicating that the pharmaceutical composition provided by the present invention has drug safety.
  • the present invention provides a pharmaceutical composition for treating sepsis, comprising hydroxysafflor yellow A with structural formula shown in formula I, paeoniflorin with structural formula shown in formula II and paeoniflorin with structural formula shown in formula III Lactone glycosides;
  • the mass ratio of the hydroxysafflor yellow A, paeoniflorin and paeoniflorin is 1-100:1-100:0.1-10;
  • the mass ratio of hydroxysafflor yellow A, paeoniflorin and paeoniflorin is preferably 1-100:1-100:1-10, including 1-100:1:1, 1-100 : 10: 1, 1 to 100: 100: 1, 1 to 100: 1: 0.01, 1 to 100: 1: 10, 1 to 100: 10: 10, 1 to 100: 10: 1, 1 to 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 molecular formula: C 27 H 3 O 16 , molecular weight: 612.53, is the medicinal component of safflower commonly used in traditional Chinese medicine.
  • Hydroxysafflor yellow A is a compound with a monochalcone glycoside structure.
  • the most effective water-soluble part of pharmacological efficacy can inhibit platelet aggregation and release induced by platelet activating factor, can competitively inhibit the binding of platelet activating factor and platelet receptor, has anti-platelet and anti-myocardial ischemia effects, is safflower yellow
  • the pigment is an active ingredient for promoting blood circulation and removing blood stasis, and also has certain anti-inflammatory, cytoprotective and anti-tumor activities.
  • Paeoniflorin, molecular formula: C 23 H 28 O 11 , molecular weight 480.46, is the medicinal component of commonly used traditional Chinese medicine red peony and white peony.
  • Paeoniflorin is a monoterpenoid glycoside compound with diverse pharmacological effects and anti-freeze properties. In vivo experiments have shown that it has various biological effects such as reducing blood viscosity, dilating blood vessels, improving microcirculation, anti-oxidation, and anti-convulsant, and has less toxic side effects.
  • Paeoniflorin and paeoniflorin have similar chemical structures, are isomers of paeoniflorin, and their content is lower than that of paeoniflorin.
  • Paeoniflorin can act on hematopoietic cytokines in the spleen, thymus and blood system in the immune system, and has It has a clear blood-replenishing effect, and can also act on the HPA axis in the nervous system and monoamine neurotransmitters in the brain, and has obvious antidepressant effects.
  • the forms of the hydroxysafflor yellow A, paeoniflorin and paeoniflorin not only include their compound monomers, but also preferably include their pharmaceutically acceptable salts, solvates, polymorphs, enantiomers or racemates body mixture, etc.
  • the pharmaceutical composition preferably further comprises oxypaeoniflorin whose structural formula is shown in formula IV, yangchuanxiong lactone I whose structural formula is shown in formula V, danshensu sodium whose structural formula is shown in formula VI, and whose structural formula is as shown in formula VI.
  • the ferulic acid shown in formula VII; the mass ratio of the hydroxysafflor yellow A, paeoniflorin, paeoniflorin, oxypaeoniflorin, yangchuanxiong lactone I, danshensu sodium and ferulic acid is 1 ⁇ 100 : 1 ⁇ 100: 0.1 ⁇ 10: 0.1 ⁇ 10: 0.1 ⁇ 10: 0.01 ⁇ 1;
  • the mass ratio of the hydroxysafflor yellow A, paeoniflorin, paeoniflorin, oxypaeoniflorin, yangchuanxiong lactone I, danshensu sodium and ferulic acid is preferably 1 ⁇ 100:1 ⁇ 100:1 ⁇ 10:1 ⁇ 10:1 ⁇ 10:0.1 ⁇ 1, more preferably 1:10:1:1:1:0.1, 1:100:10:10:10 :1, 10:1:0.1:1:1:10:1, 10:10:1:10:10:0.1:0.01, 10:100:10:0.1:0.1:0.1, 100:0.1:1 :0.1:10:1, 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, 1:100:1:1:0.1:1, 10:1:1:0.1:1:1:1:1:1:1:1:1:0:1:
  • Senkyunolide I is a kind of phthalide compound isolated from Chinese medicine extracts such as Chuanxiong and Angelica sinensis, has good fat solubility and water solubility, and has a variety of pharmacological effects. It can inhibit the NF- ⁇ B pathway, thereby exerting an anti-inflammatory effect.
  • Danshensu (Tanshinol) is the main active ingredient in the water-soluble components of Salvia miltiorrhiza. It is a phenolic aromatic acid compound. In order to increase the stability, it is made into Danshensu sodium. Its efficacy is the same as that of Danshensu.
  • Danshensu It can reduce the scope of myocardial infarction and reduce the course of disease, and reduce myocardial ischemia-reperfusion injury, and has a protective effect on the myocardium. Danshensu can also significantly inhibit platelet aggregation, reduce whole blood viscosity, and its anticoagulant effect can improve various organs of the body (such as heart, liver, lung and other organs) microcirculation disorders, help the recovery of body tissues, and Danshensu also has certain antibacterial, anti-inflammatory and immune-enhancing effects.
  • Ferulic acid is one of the medicinal components of Angelica sinensis. It belongs to phenolic acid compounds.
  • the forms of oxypaeoniflorin, yangchuanxiong lactone I, danshensu sodium, and ferulic acid not only include their compound monomers, but also preferably include their pharmaceutically acceptable salts, solvates, polymorphs, parabens Enantiomer or racemic mixture, etc.
  • the preparation method of the pharmaceutical composition preferably comprises the following steps:
  • the pharmaceutical composition includes 7 kinds, it is obtained by mixing hydroxysafflor yellow A, paeoniflorin, paeoniflorin, oxypaeoniflorin, yangchuanxiong lactone I, danshensu sodium and ferulic acid according to the above mass ratio.
  • the present invention provides the application of the pharmaceutical composition in preparing a medicine for treating sepsis.
  • the pharmaceutical composition is preferably used in the preparation of a drug for inhibiting the expression of IL-6, Foxp3, CTLA-4 and/or HMGB1 in sepsis.
  • the control group contained a small amount of IL-6, HMGB1; the model group IL-6, HMGB1, Foxp3, CTLA-4 content increased significantly, the pharmaceutical composition administration group (8h, HMGB1) 24h and 48h) plasma levels of IL-6, HMGB1, Foxp3 and CTLA-4 were significantly lower than those in the model group, and the three-component drug group in each dose group could significantly reduce IL-6, HMGB1, Foxp3 and CTLA in CLP sepsis
  • the expression of -4 indicates that it has a significant inhibitory effect on the early inflammatory response of the 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.
  • the pharmaceutical composition is preferably used in the preparation of a medicament for inhibiting the release of TF and/or TM in sepsis.
  • TM and TF in monocytes in the control group at the cellular and animal levels
  • the expression of TM and TF in the model group (12h, 24h, 48h and 72h after surgery) was significantly higher than that in the control group (p ⁇ 0.05).
  • the expressions of TM and TF (12h, 24h, 48h and 72h after operation) in the drug composition administration group were significantly lower than those in the model group (p ⁇ 0.05).
  • the pharmaceutical composition has obvious effects on inhibiting the release of coagulation factors and improving the hypercoagulable state on the sepsis rat model.
  • the pharmaceutical composition achieves the effect of treating sepsis coagulation by inhibiting the release of TF and/or TM.
  • the pharmaceutical composition is preferably used in the preparation of a drug for promoting apoptosis of regulatory T cells (Treg) in sepsis.
  • Treg regulatory T cells
  • the experiment showed that the apoptosis rate of Treg cells in the model group was significantly lower than that in the control group (p ⁇ 0.05), and the traditional Chinese medicine composition administration group was significantly higher than that in the model group (p ⁇ 0.05).
  • the three-component drug group and the seven-component drug group in each dose group could promote Treg apoptosis in sepsis and down-regulate the inhibitory effect on T lymphocyte proliferation and secretion function.
  • the pharmaceutical composition regulates T lymphocyte proliferation and secretion function by promoting Treg apoptosis.
  • the pharmaceutical composition is subjected to drug experiments at the cell level and animal level, respectively, and on the basis of the animal dose, according to the body surface area conversion method between humans and animals in "Pharmacological Experiment Methodology" edited by Xu Shuyun , and the human dose is calculated according to formula A:
  • the adult body weight is 70 kg and the rat body weight is 0.2 kg.
  • the pharmaceutical composition is preferably composed of three active compounds, hydroxysafflor yellow A, paeoniflorin and paeoniflorin
  • the dosage of hydroxysafflor yellow A is 6-600 mg/kg
  • the dosage of paeoniflorin is 6-600 mg/kg. 6 ⁇ 600mg/kg
  • the drug dose of paeoniflorin is 0.6 ⁇ 60mg/kg.
  • the pharmaceutical composition is preferably composed of seven active compounds of hydroxysafflor yellow A, paeoniflorin, paeoniflorin, oxypaeoniflorin, yangchuanxiong lactone I, danshensu sodium and ferulic acid, hydroxysafflor yellow pigment.
  • the dosage of A is 6-600 mg/kg
  • the dosage of paeoniflorin is 6-600 mg/kg
  • the dosage of paeoniflorin is 0.6-60 mg/kg
  • the dosage of oxypaeoniflorin is 0.6-60 mg/kg
  • the dosage of Yangchuanxiong lactone I is 0.6-60 mg/kg
  • the dosage of Danshensu sodium is 0.6-60 mg/kg
  • the dosage of ferulic acid is 0.06-6 mg/kg.
  • the present invention does not have any special restrictions on the dosage form of the prepared medicine, and any pharmaceutical dosage form well known in the art can be used, such as tablets, capsules, oral liquids, granules, granules, pills, powders, ointments, elixirs, suspensions , powder, solution, injection, suppository, spray, drops, etc.
  • the present invention has no particular limitation on the preparation method of each dosage form product of the medicine, and the preparation method of the pharmaceutical dosage form well known in the art can be adopted.
  • Clean-grade SD rats half male and female
  • 180-220 g hydroxysafflor yellow A, paeoniflorin, paeonolide and danshensu sodium were purchased from Shanghai Tongtian Biotechnology Co., Ltd.
  • 80 animals (half male and half male) were selected to enter the experiment according to the weight gain, diet, and activities of animals during the adaptation period, and were divided into 4 groups by weight group method, with 20 animals in each group, half male and half male.
  • the concentration of each component in the three-component drug hydroxysafflor yellow A 30mg/ml, paeoniflorin 40mg/ml, paeoniflorin 4mg/ml.
  • the preparation method of the medicinal liquid is as follows: Weigh (3.0 g of hydroxysafflor yellow A, 4.0 g of paeoniflorin, and 0.4 g of paeoniflorin) on a precision balance (1/10000 g) and dissolve them in an appropriate amount of normal saline (0.9% sodium chloride). water solution), dilute the volume to 100 mL, sterilize and filter to prepare a three-component medicine.
  • the concentration of each component in the seven-component medicine hydroxysafflor yellow A 30mg/ml, paeoniflorin 40mg/ml, paeoniflorin 4mg/ml, oxypaeoniflorin 4mg/ml, yangchuanxiong lactone I 4mg/ml , Danshensu sodium 4mg/ml, ferulic acid 0.5mg/ml.
  • the preparation method of the medicinal liquid is as follows: weighing 3.0g hydroxysafflor yellow A, 4.0g paeoniflorin, 0.4g paeoniflorin, 0.4g oxypaeoniflorin, 0.4g yangchuanxiong lactone I, 0.4g danshensu sodium, 0.05g g ferulic acid was dissolved in an appropriate amount of physiological saline (0.9% sodium chloride aqueous solution), and the volume was adjusted to 100 mL by sterile filtration to prepare a seven-component medicine.
  • Intravenous injection in rats is limited to 6 mL/kg.
  • the dosage of the three components is: hydroxysafflor yellow A 180 mg/kg, paeoniflorin 240 mg/kg, and paeoniflorin 24 mg/kg.
  • Seven-component drug dosage hydroxysafflor yellow A 180mg/kg, paeoniflorin 240mg/kg, paeoniflorin 24mg/kg, oxypaeoniflorin 24mg/kg, chuanxiongolide I 24mg/kg, danshensu Sodium 24mg/kg, ferulic acid 3mg/kg.
  • the experimental group was given a single dose of 6 mL/kg of the above-mentioned three-component drugs and seven-component drugs by intravenous slow injection; the control group was given the same dose of normal saline solution.
  • the patients were observed continuously for at least 2 hours, and each observation was recorded once in the morning and afternoon on the first day after administration, and was observed and recorded once a day thereafter, for a total of 14 days.
  • the toxic reactions of animals and the death of animals in each group were observed and recorded, and the dead or dying animals were necropsied in time.
  • 14 days after administration all surviving animals were necropsied, and the main organs were observed with naked eyes for obvious abnormal changes; if the organs showed changes in volume, color, texture, etc., they should be recorded and histopathological examinations were performed.
  • a single slow intravenous injection of the three-component drug to rats showed that: (1) the animals had no abnormal symptoms after administration; (2) there was no significant difference in the body weight and body weight growth rate of the animals in the administration group and the control group; (3) ) Gross necropsy showed no obvious abnormality.
  • a single slow intravenous injection of the seven-component drugs to rats showed that: (1) the animals had no abnormal symptoms after administration; (2) there was no significant difference in the body weight and body weight growth rate of the animals in the administration group and the control group; (3) ) Gross necropsy showed no obvious abnormality.
  • the peritoneal macrophages of male SD rats were isolated.
  • the specific method was as follows: male SD rats were fasted for 12 hours before operation, and the abdominal cavity was opened after anesthesia. flow. The liquid in the abdominal cavity was sucked into a sterilized tube, and then lavaged once with 10 mL of pre-cooled PBS solution, and the operation was the same as above. The combined collected lavage fluid was centrifuged at 250 g for 10 min at 4°C, and the supernatant was discarded. Add 2 mL of erythrocyte lysate to dissolve the red blood cells, shake twice for 5 s each time, and then add 4 mL of D-Hanks solution to stop the reaction after standing for 5 min.
  • the experiment was divided into a control group, a model group and an administration group.
  • the treatment methods for each group were as follows: after the cells in each group were placed in a cell incubator at 37 °C and 5% CO 2 overnight, the control group and the model group were added with the corresponding volume of culture medium. , the treatment group was added with an equal volume of the corresponding concentration of drug solution, and after 1 h of incubation in the incubator, the control group was not added with LPS, and the model group and the treatment group were stimulated with LPS (75ng/mL).
  • the culture medium supernatant 0.5mL was stored in a -20°C refrigerator, and the corresponding cytokine detection was carried out centrally.
  • the administration group adopts the orthogonal experiment method, and the grouping method of the orthogonal experiment is shown in Table 1 and Table 2.
  • the three concentrations of hydroxysafflor yellow A in Table 1 are respectively low 2 ⁇ M, medium 20 ⁇ M, high 200 ⁇ M; three concentrations of paeoniflorin are low 4 ⁇ M, medium 40 ⁇ M, high 400 ⁇ M; paeoniflorin three concentrations are low 2 ⁇ M , medium 20 ⁇ M, high 200 ⁇ M; three concentrations of oxypaeoniflorin were low 2 ⁇ M, medium 20 ⁇ M, high 200 ⁇ M; Yangchuanxiong lactone I three concentrations were low 4 ⁇ M, medium 40 ⁇ M, high 400 ⁇ M; Danshensu sodium three concentrations respectively Low 2 ⁇ M, medium 20 ⁇ M, high 200 ⁇ M; three concentrations of ferulic acid were low 4 ⁇ M, medium 40 ⁇ M, high 400 ⁇ M.
  • the preparation method of medicinal liquid is as follows: according to the corresponding concentration of each group in table 1, take by weighing appropriate weight of hydroxysafflor yellow A, paeoniflorin and paeoniflorin in a precision balance (1/10000g), and combine them according to different concentration combinations. In different volumetric flasks, dissolve them in appropriate medium (containing 10% fetal bovine serum) respectively to prepare 9 groups of three-component drugs with corresponding concentrations;
  • * means P ⁇ 0.05 compared with the model group.
  • the peritoneal macrophages of male SD rats were isolated.
  • the specific method was as follows: male SD rats were fasted for 12 hours before operation, and the abdominal cavity was opened after anesthesia. flow. The liquid in the abdominal cavity was sucked into a sterilized tube, and then lavaged once with 10 mL of pre-cooled PBS solution, and the operation was the same as above. The combined collected lavage fluid was centrifuged at 250 g for 10 min at 4°C, and the supernatant was discarded. Add 2 mL of erythrocyte lysate to dissolve the red blood cells, shake twice for 5 s each time, and then add 4 mL of D-Hanks solution to stop the reaction after standing for 5 min.
  • the experiment was divided into a control group, a model group and an administration group.
  • the treatment methods for each group were as follows: after the cells in each group were placed in a cell incubator at 37 °C and 5% CO 2 overnight, the control group and the model group were added with the corresponding volume of culture medium. , the treatment group was added with an equal volume of the corresponding concentration of drug solution, after 1 h of incubation in the incubator, the control group was not added with LPS, the model group and the treatment group were stimulated with LPS (75ng/mL), and the cells were collected after 48h and 72h in each group
  • the culture medium supernatant 0.5mL was stored in a -20°C refrigerator, and the corresponding cytokine detection was carried out centrally.
  • the experimental grouping and liquid preparation methods were the same as those in Example 2.
  • * means P ⁇ 0.05 compared with the model group.
  • the rats were killed by cervical dislocation, they were immersed in 75% ethanol for 5 min, the thoracic and abdominal cavities were opened layer by layer, the thoracic and abdominal aorta were fully exposed, the surrounding tissues were separated, and the aorta was separated from the proximal end to the iliac
  • the branch of the common artery was placed in a petri dish containing PBS, and the adipose tissue and fibrous tissue of the adventitia of the blood vessel were aseptically stripped, and the vascular lumen was washed with PBS.
  • the aorta was cut into small pieces of about 1.5mm ⁇ 1.5mm, placed in 6mL of 0.25% type IV collagenase, digested at 37°C for 15min, shaken every 5min, carefully aspirated the digestion solution, retained the tissue block, and then added 6mL of 1.0 % neutral protease, digested at 37°C for 15 min, shaken every 5 min, sucked off the digestion solution, supplemented with 10 mL of ECM, repeated pipetting, centrifuged at 1000 r/min for 10 min, discarded the medium and digestion solution, kept the fragments, and flattened the tissue blocks.
  • the cells were cultured according to the above-mentioned culture method of rat abdominal aortic endothelial cells, and the cell suspension was prepared. The cells were seeded at 1.2 ⁇ 10 5 /mL.
  • the LPS stimulation group and the administration group were given LPS stimulation, and different medicinal liquids were given for intervention after 1 h, and the supernatants were collected at 24 h, 48 h, and 72 h after stimulation.
  • the experiment was divided into a control group, a model group and an administration group.
  • the treatment methods for each group were as follows: after the cells in each group were placed in a cell incubator at 37 °C and 5% CO 2 overnight, the control group and the model group were added with the corresponding volume of culture medium. , the treatment group was added with an equal volume of the corresponding concentration of drug solution, and after 1 hour of incubation in the incubator, the control group was not added with LPS, and the model group and the treatment group were stimulated with LPS (75ng/mL). Collect 0.5 mL of the cell culture supernatant, store it in a -20°C refrigerator, and conduct the corresponding cytokine detection in a centralized manner.
  • the experimental grouping and liquid preparation methods were the same as those in Example 2.
  • TM is ubiquitously present on the surface of rat abdominal aortic endothelial cells, and exerts anticoagulant effect by binding to thrombin and activating the protein C system.
  • the reduction of TM under drug intervention is helpful for the balance of anticoagulation/procoagulation of coagulation factors, and it is beneficial to promote the recovery of cell/body coagulation function to a normal state.
  • * means P ⁇ 0.05 compared with the model group.
  • TF is an important procoagulant factor in rat abdominal aortic endothelial cells. In the state of sepsis, the hypercoagulable state is manifested by the massive release of TF. The reduction of TF under drug intervention is helpful for the balance of anticoagulation/procoagulation of coagulation factors, and it is beneficial to promote the recovery of cell/body coagulation function to a normal state.
  • * means P ⁇ 0.05 compared with the model group.
  • the Treg cells were resuspended with an appropriate amount of culture medium, the number of cells was adjusted to 2.5 ⁇ 10 6 /mL, 96-well plate, 100 ⁇ L of cells were inserted into each well, and CD3/CD28 (CD3 was 0.5 ⁇ g/10 6 , 0.5 ⁇ g/10 6 for CD3) was added to the model group and experimental group. CD28 was stimulated by 1 ⁇ g/10 6 ) + LPS (1 ⁇ g/mL), and the experiment was divided into control group, model group and administration group. Cultivated for 72h.
  • the grouping treatment method is as follows: Model group: After the cells were cultured in a cell incubator overnight, LPS (75ng/mL) was added to stimulate. Administration group: add the corresponding concentration of drug solution to incubate for 1 h, and then stimulate with LPS. After 72 h of intervention in the experimental group with different concentrations, collect 0.5 mL of cell culture supernatant, store it in a refrigerator at -20°C, and conduct centralized detection of corresponding cytokines.
  • Treg cell function After Treg cell function is enhanced, it suppresses ordinary T cells by producing immunosuppressive factors such as IL-10, IL-35, TGF- ⁇ , etc., and kills target cells through granzyme B and perforin-1, thereby exerting an immunosuppressive effect. It can also induce DC dendritic cells to produce indoleamine 2,3-dioxygenase, which catalyzes the decomposition of tryptophan into kynurenine, leading to the death of surrounding cells, and can also induce DC cells to secrete other amino acid-related enzymes, thereby inhibiting effector T cells. proliferation, thereby exerting an immunosuppressive effect.
  • immunosuppressive factors such as IL-10, IL-35, TGF- ⁇ , etc.
  • drugs can promote the apoptosis of Treg cells in a certain period of time, and play a very important role in the immune regulation of the body.
  • the three-component drug group and the seven-component drug group in each dose group could promote the apoptosis of regulatory T cells (Treg) in rats (Table 7).
  • * means P ⁇ 0.05 compared with the model group.
  • Treg cells inhibit ordinary T cells by producing immunosuppressive factors such as IL-10, IL-35, TGF- ⁇ , etc., and kill target cells through granzyme B and perforin-1, thereby exerting an immunosuppressive effect.
  • CTLA-4 can also induce DC dendritic cells to produce indoleamine 2,3-dioxygenase, which catalyzes the breakdown of tryptophan into kynurea leading to the death of surrounding cells, and also induces DC cells to secrete other amino acid-related enzymes, thereby inhibiting the
  • the proliferation of effector T cells plays a role in immunosuppression. Therefore, Treg cells play a very important role in immune regulation through Foxp3 and CTLA-4, and the expression of Foxp3 is closely related to the function of regulatory T cells (Treg).
  • the three-component drug group and the seven-component drug group in each dose group could reduce the expression levels of 72h CTLA-4 and Foxp3 in regulatory T cells (Treg) in rats, indicating that the three-component drug group and the seven-component drug group were significantly lower than the model group.
  • the component drug group had the effect of reducing the expression levels of CTLA-4 and Foxp3 in septic rat regulatory T cells (Treg) 72h (Table 8 and Table 9).
  • * means P ⁇ 0.05 compared with the model group.
  • * means P ⁇ 0.05 compared with the model group.
  • the three concentrations of hydroxysafflor yellow A in table 10 are respectively low 0.9mg/kg, medium 9mg/kg, high 90mg/kg; three concentrations of paeoniflorin are respectively low 0.9mg/kg, medium 9mg/kg, high 90mg /kg; the three concentrations of paeoniflorin were low 0.09mg/kg, medium 0.9mg/kg, high 9mg/kg; the three concentrations of oxypaeoniflorin were low 0.09mg/kg, medium 0.9mg/kg, high 9mg/kg; three concentrations of yangchuanxiong lactone I are respectively low 0.09mg/kg, middle 0.9mg/kg, high 9mg/kg; three concentrations of danshensu sodium are respectively low 0.09mg/kg, middle 0.9mg/kg , high 9mg/kg; three concentrations of ferulic acid were low 0.009mg/kg, medium 0.09mg/kg, high 0.9mg
  • each rat is calculated according to 200g and the experimental injection volume is 1.0mL/time/rat.
  • the corresponding concentrations of each group in Table 10 weigh appropriate weights of hydroxysafflor yellow A, paeoniflorin and paeoniflorin on a precision balance (1/10000g), and combine them into different volumetric flasks according to different concentration combinations.
  • the hydroxysafflor yellow A, paeoniflorin, paeoniflorin, oxypaeoniflorin, yangchuanxiong lactone, danshensu sodium and ferulic acid were combined into different volumetric flasks according to the combination of different concentrations, and dissolved in an appropriate amount of physiological In saline (0.9% sodium chloride aqueous solution), 18 groups of seven-component medicines with corresponding concentrations were prepared. The rats were fasted for 12 hours before the experiment, weighed and grouped according to the random number table method. In the control group, the skin was sutured after the cecum was exposed, and 10 mL of normal saline was injected subcutaneously for resuscitation.
  • the CLP modeling process is as follows: The rats were anesthetized by intramuscular injection of a mixture of ketamine injection + Sumianxin II injection in a volume ratio of 2:1, and CLP was used to prepare an animal model of sepsis. The junction between the cecum and the ileum was ligated, and an 18-gauge needle was used to penetrate the cecum twice to form an intestinal fistula, and two drainage strips (0.5cm ⁇ 2.0cm) were indwelled to prevent the needle hole from healing, and the skin was sutured layer by layer. Immediately after the operation, 10 mL of physiological Saline resuscitation.
  • the experimental results showed that the plasma of the control group contained a small amount of IL-6; in the early stage after CLP surgery, the IL-6 content of the model group increased significantly, 8h IL-6 level further increased, and gradually decreased at 24h, but 48h after the operation. Still higher than the control group, the difference was statistically significant (p ⁇ 0.05). However, the plasma levels of IL-6 in the administration group at 8h, 24h, and 48h after operation were significantly lower than those in the model group (p ⁇ 0.05), close to the level in the control group.
  • the inflammatory response has obvious inhibitory effect, and the dose converted into human body is about: three-component drug group (hydroxysafflor yellow A 6mg to 600mg, paeoniflorin 6mg to 600mg, paeoniflorin 0.6mg to 60mg), seven groups Sub-drug group (Hydroxysafflor yellow A 6mg to 600mg, Paeoniflorin 6mg to 600mg, Paeoniflorin 0.6mg to 60mg, Oxypaeoniflorin 0.6mg to 60mg, Yangchuanxiongolide I 0.6mg to 60mg, Danshensu sodium 0.6mg mg to 60 mg, ferulic acid 0.06 mg to 6 mg).
  • three-component drug group hydroxysafflor yellow A 6mg to 600mg, paeoniflorin 6mg to 600mg, paeoniflorin 0.6mg to 60mg
  • Sub-drug group Hydrosafflor yellow A 6mg
  • human dose rat dose/0.018 (calculated based on adult body weight of 70 kg and rat body weight of 0.2 kg)].
  • * means P ⁇ 0.05 compared with the model group.
  • the converted human dose is about: three-component drug group (hydroxysafflor yellow A 6mg to 600mg, paeoniflorin 6mg to 600mg, paeoniflorin 0.6mg to 60mg), seven-component drug group (hydroxysafflor yellow A 6mg to 600mg, Paeoniflorin 6mg to 600mg, Paeoniflorin 0.6mg to 60mg, Oxypaeoniflorin 0.6mg to 60mg, Chuanxiongolide I 0.6mg to 60mg, danshensu Sodium 0.6mg to 60mg, Ferulic acid 0.06mg to 6 mg).
  • human dose rat dose/0.018 (calculated based on adult body weight of 70 kg and rat body weight of 0.2 kg)].
  • * means P ⁇ 0.05 compared with the model group.
  • the converted human dose is about: three-component drug group (hydroxysafflor yellow A 6mg to 600mg, paeoniflorin 6mg to 600mg, paeoniflorin 0.6mg to 60mg), seven-component drug group (hydroxysafflor yellow pigment A 6mg to 600mg, Paeoniflorin 6mg to 600mg, Paeoniflorin 0.6mg to 60mg, Oxypaeoniflorin 0.6mg to 60mg, Chuanxiongolide I 0.6mg to 60mg, danshensu Sodium 0.6mg to 60mg, Ferulic acid 0.06 mg to 6 mg).
  • three-component drug group hydroxysafflor yellow A 6mg to 600mg, paeoniflorin 6mg to 600mg, paeoniflorin 0.6mg to 60mg
  • seven-component drug group hydroxysafflor yellow pigment A 6mg to 600mg, Paeoniflorin 6mg to
  • * means P ⁇ 0.05 compared with the model group.
  • TM content was detected by enzyme-linked immunosorbent assay (ELISA).
  • the converted human dose is about: three-component drug group (hydroxysafflor yellow A 6mg to 600mg, paeoniflorin 6mg to 600mg, paeonolide 0.6mg to 60mg), seven-component drug group (hydroxysafflor yellow pigment A 6mg to 600mg, Paeoniflorin 6mg to 600mg, Paeoniflorin 0.6mg to 60mg, Oxypaeoniflorin 0.6mg to 60mg, Chuanxiongolide I 0.6mg to 60mg, danshensu Sodium 0.6mg to 60mg, Ferulic acid 0.06 mg to 6 mg).
  • three-component drug group hydroxysafflor yellow A 6mg to 600mg, paeoniflorin 6mg to 600mg, paeonolide 0.6mg to 60mg
  • seven-component drug group hydroxysafflor yellow pigment A 6mg to 600mg, Paeoniflorin 6mg to 600m
  • * means P ⁇ 0.05 compared with the model group.
  • the rats were killed by neck dislocation, the chest and abdomen were sterilized with alcohol cotton balls for 3 times, the abdomen was opened, the intact peritoneum was preserved, and the alcohol cotton balls were sterilized again. Tear off the spleen capsule with tweezers, put it into a filter, add PBS to wet it, tear the spleen, remove the fascia, grind the spleen with a sterile syringe plunger, pipette the suspension into a centrifuge tube, centrifuge at 1200 rpm for 7 minutes, and discard it.
  • MACS Buffer (10ml/each spleen) to resuspend, add it along the tube wall to the upper layer of the lymphocyte separation solution (1:1), centrifuge at 3000 rpm for 15 minutes, suck the middle layer with a pipette, and put it into another In the centrifuge tube, add the washing solution and centrifuge at 1200 rpm for 7 minutes, discard the supernatant, and add MACS Buffer to resuspend for later use.
  • MACS Buffer 10ml/each spleen
  • Treg apoptosis rate Collect cells, wash cells with pre-cooled PBS, then wash them again with 1ml 1 ⁇ Binding Buffer, remove the remaining 100 ⁇ l of supernatant after centrifugation, then add 5 ⁇ l FITC Annexin V and 5 ⁇ l PI, room temperature (25°C) Incubate in the dark for 15 min, then add 200 ⁇ l of 1 ⁇ Binding Buffer to mix, and detect by flow cytometry within 1 h.
  • the converted human dose is about: three-component drug group (hydroxysafflor yellow A 6mg to 600mg, paeoniflorin 6mg to 600mg, paeoniflorin 0.6mg to 60mg), seven-component drug group (hydroxysafflor yellow pigment A 6mg to 600mg, Paeoniflorin 6mg to 600mg, Paeoniflorin 0.6mg to 60mg, Oxypaeoniflorin 0.6mg to 60mg, Chuanxiongolide I 0.6mg to 60mg, danshensu Sodium 0.6mg to 60mg, Ferulic acid 0.06 mg to 6 mg).
  • human dose rat dose/0.018 (calculated based on adult body weight of 70 kg and rat body weight of 0.2 kg)].
  • * means P ⁇ 0.05 compared with the model group.

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Abstract

本发明提供了一种用于治疗脓毒症的药物组合物及其应用，属于化学药物技术领域。本发明提供一种包含羟基红花黄色素A、芍药苷和芍药内酯苷三组分药物组合物，还提供了一种包含羟基红花黄色素A、芍药苷、芍药内酯苷、氧化芍药苷、洋川芎内酯I、丹参素钠和阿魏酸的七组分药物组合物，本发明在细胞水平和动物水平验证两组药物组合物均具有效治疗脓毒症的药效，同时药物毒性实验表明该药物具有较高的安全性。本发明提供的药物组合物是血必净注射液的药效成分，可用于临床上脓毒症的治疗。

Description

一种用于治疗脓毒症的药物组合物及其应用
本申请要求于2021年03月01日提交中国专利局、申请号为202110225428.6、发明名称为“一种用于治疗脓毒症的药物组合物及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于化学药物技术领域，具体涉及一种用于治疗脓毒症的药物组合物及其应用。
背景技术
脓毒症是针对感染的宿主反应失调引起的致命性器官功能障碍，病情凶险、致死率高是重症患者死亡的主要原因。多年以来，抗生素、抗病毒药物、血管升压药物等均用于脓毒症传统治疗，但尚未有足够的针对脓毒症发病机制的特异性药物投入临床实践。如何及时纠正脓毒症发生发展过程中的全身性炎症反应、凝血功能障碍和免疫功能紊乱，尽早恢复机体促炎-抗炎动态平衡，有效改善患者预后，成为了脓毒症治疗药物研发中亟待解决的重要课题。
血必净注射液是依据“三证三法”的辨证原则，在“菌毒炎并治”的理论指导下，以清代王清任《医林改错》所载“血府逐瘀汤”为基础，研制而成的一种静脉注射液，由红花、赤芍、川芎、丹参、当归五味药材经提取、精制、干燥、调配等现代工艺制备而成，适用于脓毒症/因感染诱发的全身炎症反应综合征；也可配合治疗多器官功能失常综合征的脏器功能受损期。但血必净注射液是以中药材为原料制备得到，目前对脓毒症的主要药效成分还不明确，仍需要对多种中药材进行提取精制制备用于治疗脓毒症的药物组合物。
发明内容
有鉴于此，本发明的目的在于提供一种新型的用于治疗脓毒症的药物组合物及其应用，该药物组合物是直接由脓毒症的活性化合物组成，避免中药提取繁琐步骤，且使药物组合物成分更加精简，提高产品药效。
本发明提供了一种用于治疗脓毒症的药物组合物，包括结构式如式I所示的羟基红花黄色素A、结构式如式II所示的芍药苷和结构式如式III所示的芍药内酯苷；
所述羟基红花黄色素A、芍药苷和芍药内酯苷的质量比为1～100：1～100：0.1～10；
Figure PCTCN2021082244-appb-000001
优选的，所述羟基红花黄色素A、芍药苷和芍药内酯苷的质量比为1～100：1～100：1～10。
优选的，所述药物组合物还包括结构式如式IV所示的氧化芍药苷、结构式如式V所示的洋川芎内酯I、结构式如式VI所示的丹参素钠、结构式如式VII所示的阿魏酸；
所述羟基红花黄色素A、芍药苷、芍药内酯苷、氧化芍药苷、洋川芎内酯I、丹参素钠和阿魏酸的质量比为1～100：1～100：1～10：0.1～10：0.1～10：0.1～10：0.01～1；
Figure PCTCN2021082244-appb-000002
优选的，所述羟基红花黄色素A、芍药苷、芍药内酯苷、氧化芍药苷、洋川芎内酯I、丹参素钠和阿魏酸的质量比为1～100：1～100：1～10：1～10：1～10：1～10：0.1～1。
本发明提供了所述药物组合物在制备治疗脓毒症的药物中的应用。
优选的，所述药物组合物在制备抑制脓毒症的IL-6、Foxp3、CTLA-4和/或HMGB1表达的药物中的应用。
优选的，所述药物组合物在制备抑制脓毒症的TF和/或TM释放的药物中的应用。
优选的，所述药物组合物在制备促进脓毒症调节性T细胞凋亡的药物中的应用。
优选的，所述药物组合物由羟基红花黄色素A、芍药苷和芍药内酯苷三种活性化合物组成时，羟基红花黄色素A的药物剂量为6～600mg/kg，芍药苷的药物剂量为6～600mg/kg，芍药内酯苷的药物剂量为0.6～60mg/kg。
优选的，所述药物组合物由羟基红花黄色素A、芍药苷、芍药内酯苷、氧化芍药苷、洋川芎内酯I、丹参素钠和阿魏酸七种活性化合物时，羟基红花黄色素A的药物剂量为6～600mg/kg，芍药苷的药物剂量为6～600mg/kg，芍药内酯苷的药物剂量为0.6～60mg/kg，氧化芍药苷的药物剂量为0.6～60mg/kg，洋川芎内酯I的药物剂量为0.6～60mg/kg，丹参素钠的药物剂量为0.6～60mg/kg，阿魏酸的药物剂量为0.06～6mg/kg。
本发明还提供了一种治疗脓毒症的方法，服用所述药物组合物制备的药物。
优选的，所述药物组合物通过抑制脓毒症的IL-6、Foxp3、CTLA-4和/或HMGB1表达实现治疗脓毒症。
优选的，所述药物组合物通过抑制脓毒症的TF和/或TM释放实现治疗脓毒症。
优选的，所述药物组合物通过促进脓毒症调节性T细胞凋亡实现治疗脓毒症。
优选的，所述药物组合物由羟基红花黄色素A、芍药苷和芍药内酯苷三种活性化合物组成时，羟基红花黄色素A的药物剂量为6～600mg/kg，芍药苷的药物剂量为6～600mg/kg，芍药内酯苷的药物剂量为0.6～60mg/kg。
优选的，所述药物组合物由羟基红花黄色素A、芍药苷、芍药内酯苷、氧化芍药苷、洋川芎内酯I、丹参素钠和阿魏酸七种活性化合物时，羟基红花黄色素A的药物剂量为6～600mg/kg，芍药苷的药物剂量为6～600mg/kg，芍药内酯苷的药物剂量为0.6～60mg/kg，氧化芍药苷的药物剂量为0.6～60mg/kg，洋川芎内酯I的药物剂量为0.6～60mg/kg，丹参素钠的药物剂量为0.6～60mg/kg，阿魏酸的药物剂量为0.06～6mg/kg。
本发明提供的用于治疗脓毒症的药物组合物包括羟基红花黄色素A、芍药苷和芍药内酯苷；所述羟基红花黄色素A、芍药苷和芍药内酯苷的质量比为1～100：1～100：0.1～10。本发明在细胞水平和动物水平上证实，所述药物组合物对脓毒症中巨噬细胞IL-6、HMGB1、CTLA-4和Foxp3的表达具有抑制作用，有效抑制脓毒症的TF和TM的释放，同时对调节性T淋巴细胞的凋亡有促进作用，表明本发明提供的药物组合物对脓毒症具有明显的纠正细胞或机体细胞免疫抑制状态的作用，从而实现治疗脓毒症的目的。
同时，本发明所述药物组合物经过静脉给药急性毒性试验，结果表明，动物给药后未见明显异常，动物体重和体重增长率与对照组无显著性差异，大体解剖未见明显异常，说明本发明提供的药物组合物具有药物安全性。
进一步的，本发明提供的药物组合物还包括结构式如式IV所示的氧化芍药苷、结构式如式V所示的洋川芎内酯I、结构式如式VI所示的丹参素钠、结构式如式VII所示的阿魏酸；所述羟基红花黄色素A、芍药苷、芍药内酯苷、氧化芍药苷、洋川芎内酯I、丹参素钠和阿魏酸的质量比为1～100：1～100：0.1～10：0.1～10：0.1～10：0.1～10：0.01～1。本发明确定羟基红花黄色素A作为红花治疗脓毒症的活性成分，芍药苷、芍药内酯苷和氧化芍药苷作为赤芍和白芍治疗脓毒症的活性成分，洋川芎内酯I作为川芎及当归中治疗脓毒症的活性成分，丹参素作为丹参治疗脓毒症的活性成分，阿魏酸作为当归治疗脓毒症的活性成分，7种活性成分进行严格剂量配比，制备的药物组合物与3种化合物组成的药物组合物相比，在对脓毒症中巨噬细胞IL-6、HMGB1、CTLA-4和Foxp3的表达方面具有更强的抑制作用，对脓毒症的TF和TM的释放具有更强的抑制作用，同时对调节性T淋巴细胞的凋亡有更强促进作用，本发明实验表明所述药物组合物在治疗脓毒症方面具有更强的药效，达到更优的治疗效果。
同时，本发明还对七种活性成分组成的药物组合物进行静脉给药急性毒性试验，结果表明，动物给药后未见明显异常，动物体重和体重增长率与对照组无显著性差异，大体解剖未见明显异常，说明本发明提供的药物组合物具有药物安全性。
具体实施方式
本发明提供了一种用于治疗脓毒症的药物组合物，包括结构式如式I所示的羟基红花黄色素A、结构式如式II所示的芍药苷和结构式如式III所示的芍药内酯苷；
所述羟基红花黄色素A、芍药苷和芍药内酯苷的质量比为1～100：1～100：0.1～10；
Figure PCTCN2021082244-appb-000003
在本发明中，所述羟基红花黄色素A、芍药苷和芍药内酯苷的质量比优选为1～100：1～100：1～10，包括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，具体为1：10：10、1：100：1、10：1：10、10：10：1、10：100：0.1、100：1：1、100：10：0.1和10：10：1，最优选为10：10：1。羟基红花黄色素A分子式：C 27H 3O 16，分子量：612.53，为常用中药红花的药效成分，羟基红花黄色素A是具有单查尔酮苷类结构的化合物，是红花药理功效的最有效水溶性部位，可抑制血小板激活因子诱发的血小板聚集与释放，可竞争性地抑制血小板激活因子与血小板受体的结合，具有抗血小板和抗心肌缺血作用，是红花黄色素的活血化瘀有效成分，同时还具有一定的抗炎、细胞保护和抗肿瘤活性。芍药苷(Paeoniflorin)，分子式：C 23H 28O 11，分子量为480.46，为常用中药赤芍 和白芍的药效成分，芍药苷是一种单萜类糖苷化合物，药理作用多样，具有抗自由基损伤、抑制细胞内钙超载和抗神经毒性等活性，体内实验证明其有降低血液黏度、扩张血管、改善微循环、抗氧化、抗惊厥等多种生物学效应，并且毒副作用较小。芍药内脂苷分子式：C 23H 28O 11，分子量：480.46。芍药内酯苷与芍药苷化学结构相似，是芍药苷的同分异构体，其含量较芍药苷低，芍药内酯苷可作用于免疫系统中脾脏、胸腺和血液系统中造血细胞因子，具有明确的补血作用，同时还可作用于神经系统中HPA轴和脑内单胺类神经递，具有明显的抗抑郁作用。所述羟基红花黄色素A、芍药内酯苷和芍药苷的形式不仅包含其化合物单体，还优选包括其药学上可接受的盐、溶剂化物、多晶体型、对映体或外消旋体混合物等。
在本发明中，所述药物组合物优选还包括结构式如式IV所示的氧化芍药苷、结构式如式V所示的洋川芎内酯I、结构式如式VI所示的丹参素钠、结构式如式VII所示的阿魏酸；所述羟基红花黄色素A、芍药苷、芍药内酯苷、氧化芍药苷、洋川芎内酯I、丹参素钠和阿魏酸的质量比为1～100：1～100：0.1～10：0.1～10：0.1～10：0.1～10：0.01～1；
Figure PCTCN2021082244-appb-000004
在本发明中，所述羟基红花黄色素A、芍药苷、芍药内酯苷、氧化芍药苷、洋川芎内酯I、丹参素钠和阿魏酸的质量比优选为1～100：1～100：1～10：1～10：1～10：1～10：0.1～1，具体优选为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、100:100:1:0.1:1：10:0.01，最优选为100:100:1:0.1:1：10:0.01。氧化芍药苷含量在赤芍和白芍中相对较小，其药理作用研究鲜有报导。芍药总苷有止痛、抗感染、抗氧化和抗癌等作用，有着极高的应用价值，因此值得在临床中推广。洋川芎内酯I(Senkyunolide I)为川芎及当归等中药提取物中分离得到的一种苯酞类化合物，具有很好的脂溶性和水溶性，并且具有多种药理作用，洋川芎内酯I能够抑制NF-κB通路，从而发挥抗炎作用，体内实验发现其对缺血性再灌注损伤具有一定的保护作用，同时能降低红细胞的变形指数和指向指数，降低红细胞的聚集性，具有抗凝和抗血小板聚集的活性作用。丹参素(Tanshinol)是丹参水溶性成分中的主要药效成分，是一种酚性芳香酸类化合物，为增 加稳定性，将其制成丹参素钠，功效与丹参素一致，研究表明丹参素能够缩小心肌梗死范围和减轻病程，并减少心肌缺血再灌注损伤，对心肌具有保护作用，丹参素还能明显抑制血小板的聚集，降低全血粘度，其抗凝作用能够改善全身各脏器(如心、肝、肺等器官)的微循环障碍，有助于机体组织的康复，同时丹参素还具有一定的抗菌消炎及增强机体免疫作用。阿魏酸(Ferulic Acid)是当归的药效成分之一，属于酚酸类化合物，具有抗血小板聚集，抑制血小板5-羟色胺释放，抑制血小板血栓素A2(TXA2)的生成，增强前列腺素活性，镇痛，缓解血管痉挛等作用。实验证明，羟基红花黄色素A、芍药苷、芍药内酯苷、氧化芍药苷、洋川芎内酯I、丹参素钠、阿魏酸是血必净注射液中的药效组分，在脓毒症治疗中的作用具有重要的现实意义和应用价值。在本发明中，氧化芍药苷、洋川芎内酯I、丹参素钠、阿魏酸的形式不仅包含其化合物单体，还优选包括其药学上可接受的盐、溶剂化物、多晶体型、对映体或外消旋体混合物等。
在本发明中，所述药物组合物的制备方法，优选包括以下步骤：
将羟基红花黄色素A、芍药苷、芍药内酯苷按上述质量比混合得到。
当药物组合物包括7种，将羟基红花黄色素A、芍药苷、芍药内酯苷、氧化芍药苷、洋川芎内酯I、丹参素钠和阿魏酸按上述质量比混合得到。
本发明提供了所述药物组合物在制备治疗脓毒症的药物中的应用。所述药物组合物在治疗脓毒症中的应用。
在本发明中，所述药物组合物优选在制备抑制脓毒症的IL-6、Foxp3、CTLA-4和/或HMGB1表达的药物中的应用。实验结果表明，在细胞水平和动物水平，对照组含有少量IL-6、HMGB1；模型组IL-6、HMGB1、Foxp3、CTLA-4含量即明显增加，所述药物组合物给药组(8h、24h和48h)的IL-6、HMGB1、Foxp3和CTLA-4血浆含量均显著低于模型组，各剂量组的三组分药物组可显著降低CLP脓毒症IL-6、HMGB1、Foxp3和CTLA-4的表达，提示对脓毒症大鼠模型早期炎症反应均具有明显的抑制作用。所述药物组合物通过抑制IL-6、Foxp3、CTLA-4和/或HMGB1表达达到治疗脓毒症早期炎症反应的效果。
在本发明中，所述药物组合物优选在制备抑制脓毒症的TF和/或TM释放的药物中的应用。实验表明，在细胞水平和动物水平上，对照组单核细胞TM和TF存在一定表达；模型组TM和TF(术后12h，24h，48h和72h)表达均显著高于对照组(p<0.05)，且随术后时间的延长而逐渐增高；药物组合物给药组TM和TF(术后12h，24h，48h和72h)表达与模型组相比均出现显著降低(p<0.05)。说明所述药物组合物对脓毒症大鼠模型具有明显的抑制凝血因子释放、改善高凝状态的作用。所述药物组合物通过抑制TF和/或TM释放，达到治疗脓毒症凝血症的效果。
在本发明中，所述药物组合物优选在制备促进脓毒症调节性T细胞(Treg)凋亡的药物中的应用。实验表明，模型组Treg细胞凋亡率明显低于对照组(p<0.05)，中药组合物给药组均显著高于模型组(p<0.05)。与模型组相比，各剂量组的三组分药物组和七组分药物组均可促进脓毒症Treg凋亡，下调对T淋巴细胞增殖和分泌功能的抑制效应。所述药物组合物通过促进Treg凋亡，调节T淋巴细胞增殖和分泌功能。
在本发明中，所述药物组合物在细胞水平和动物水平分别进行药物实验，在动物给药剂量的基础上，根据徐叔云主编的《药理实验方法学》中人和动物间按体表面积折算法，按照公式A计算人的给药剂量：
人体剂量＝大鼠剂量/0.018公式A
其中，以成人体重70kg、大鼠体重0.2kg计。
所述药物组合物优选由羟基红花黄色素A、芍药苷和芍药内酯苷三种活性化合物组成时，羟基红花黄色素A的药物剂量为6～600mg/kg，芍药苷的药物剂量为6～600mg/kg，芍药内酯苷的药物剂量为0.6～60mg/kg。所述药物组合物优选由羟基红花黄色素A、芍药苷、芍药内酯苷、氧化芍药苷、洋川芎内酯I、丹参素钠和阿魏酸七种活性 化合物时，羟基红花黄色素A的药物剂量为6～600mg/kg，芍药苷的药物剂量为6～600mg/kg，芍药内酯苷的药物剂量为0.6～60mg/kg，氧化芍药苷的药物剂量为0.6～60mg/kg，洋川芎内酯I的药物剂量为0.6～60mg/kg，丹参素钠的药物剂量为0.6～60mg/kg，阿魏酸的药物剂量为0.06～6mg/kg。
本发明对制备的药物的剂型没有特殊限制，采用本领域所熟知的药物剂型即可，例如片剂、胶囊剂、口服液、颗粒剂、冲剂、丸剂、散剂、膏剂、丹剂、混悬剂、粉剂、溶液剂、注射剂、栓剂、喷雾剂、滴剂等。本发明对所述药物的各剂型产品的制备方法没有特殊限制，采用本领域所熟知的药物剂型的制备方法即可。
下面结合实施例对本发明提供的一种用于治疗脓毒症的药物组合物及其应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
1.实验材料
清洁级SD大鼠(雌雄各半)，180～220g；羟基红花黄色素A、芍药苷、芍药内酯苷和丹参素钠购自上海同田生物技术股份有限公司；氧化芍药苷、洋川芎内酯I和阿魏酸购自鼎瑞化工(上海)有限公司，生理盐水(0.9％氯化钠注射液)。
2.实验方法
根据适应期动物体重增长、饮食、活动等筛选80只动物(雌雄各半)进入本试验，采用体重区组分组法分为4组，每组20只，雌雄各半。
三组分药物中各组分的浓度：羟基红花黄色素A 30mg/ml，芍药苷40mg/ml，芍药内酯苷4mg/ml。药液配制方法如下：在精密天平(1/10000g)分别称取(3.0g羟基红花黄色素A、4.0g芍药苷、0.4g芍药内酯苷溶解于适量生理盐水(0.9％的氯化钠水溶液)中，定容至100mL除菌过滤制成三组分药物。
七组分药物中各组分的浓度：羟基红花黄色素A 30mg/ml，芍药苷40mg/ml，芍药内酯苷4mg/ml，氧化芍药苷4mg/ml，洋川芎内酯I 4mg/ml，丹参素钠4mg/ml，阿魏酸0.5mg/ml。药液配制方法如下：称量3.0g羟基红花黄色素A、4.0g芍药苷、0.4g芍药内酯苷、0.4g氧化芍药苷、0.4g洋川芎内酯I，0.4g丹参素钠，0.05g阿魏酸溶解于适量生理盐水(0.9％的氯化钠水溶液)中，定容至100mL除菌过滤制成七组分药物。
大鼠静脉注射限量为6mL/kg，综上三组分药物给药剂量：羟基红花黄色素A 180mg/kg，芍药苷240mg/kg，芍药内酯苷24mg/kg。
七组分药物给药剂量：羟基红花黄色素A 180mg/kg，芍药苷240mg/kg，芍药内酯苷24mg/kg，氧化芍药苷24mg/kg，洋川芎内酯I 24mg/kg，丹参素钠24mg/kg，阿魏酸3mg/kg。
实验组单次给药，6mL/kg上述浓度的三组分药物和七组分药物，静脉缓慢注射；对照组给予相同剂量的生理盐水溶液。
给药后至少连续观察2小时，给药后第1天上下午各观察记录1次，以后每日观察记录1次，共观察14天。观察期内，观察记录动物毒性反应情况和各组动物的死亡情况，对死亡或濒死动物及时剖检。给药后14天，所有存活动物进行剖检，肉眼观察各主要脏器有无明显异常变化；脏器出现体积、颜色、质地等改变时，均应记录并进行病理组织学检查。
3.实验结果
大鼠单次缓慢静脉推注三组分药物，结果显示：(1)动物给药后未见异常症状；(2)给药组动物体重及体重增长率与对照组无显著性差异；(3)大体剖检未见明显异常。
大鼠单次缓慢静脉推注七组分药物，结果显示：(1)动物给药后未见异常症状；(2)给药组动物体重及体重增长率与对照组无显著性差异；(3)大体剖检未见明显异常。
实施例2
评价三组分药物、七组分药物对LPS刺激下大鼠腹腔巨噬细胞IL-6释放的影响
1.实验材料
雄性清洁级SD大鼠(180～220g)，内毒素(LPS)，羟基红花黄色素A，芍药苷，芍药内酯苷，氧化芍药苷，洋川芎内酯I，丹参素钠，阿魏酸，RPMI-1640培养基，24孔板，IL-6ELISA试剂盒。
2.实验方法
分离雄性SD大鼠腹腔巨噬细胞，具体方法如下：雄性SD大鼠术前禁食12h，麻醉后打开腹腔，向腹腔注入预冷PBS液10mL，用手指轻压揉腹壁，使液体在腹腔内流动。吸取腹腔中的液体注入灭菌管中，再用预冷PBS液10mL灌洗一次，操作同上。将合并收集到的灌洗液250g，4℃离心10min，弃上清。加入2mL红细胞裂解液溶解红细胞，轻微震荡两次，每次5s，静置5min后加入4mL D-Hanks溶液终止反应。按照上述方法再次离心，弃上清。用培养液洗涤沉淀，然后重悬细胞，制成2×10 6/mL细胞悬液。将细胞悬液接种于24孔板，置于37℃，5％CO 2的细胞培养箱培养。每组6个平行孔。分别培养12h和24h后收集上清，采用双抗体一步夹心法酶联免疫吸附试验(ELISA)测定细胞因子含量。
实验分为对照组、模型组和给药组，各组处理方法如下：各组细胞置于37℃，5％CO 2的细胞培养箱培养过夜后，对照组和模型组加入相应体积的培养液，给药组加入等体积的对应浓度药液，培养箱孵育1h后，对照组不加入LPS，模型组和给药组加入LPS(75ng/mL)刺激，各组在12h、24h后，收集细胞培养液上清0.5mL，-20℃冰箱保存，集中进行相应细胞因子检测。其中给药组采用正交实验法，正交实验分组方法见表1和表2。
表1三组分药物组正交实验分组
组别 羟基红花黄色素A 芍药苷 芍药内酯苷
1 低 低 低
2 低 中 高
3 低 高 中
4 中 低 高
5 中 中 中
6 中 高 低
7 高 低 中
8 高 中 低
9 高 高 高
表1中羟基红花黄色素A三个浓度分别为低2μM，中20μM，高200μM；芍药苷三个浓度分别为低4μM，中40μM，高400μM；芍药内酯苷三个浓度分别为低2μM，中20μM，高200μM；氧化芍药苷三个浓度分别为低2μM，中20μM，高200μM；洋川芎内酯I三个浓度分别为低4μM，中40μM，高400μM；丹参素钠三个浓度分别为低2μM，中20μM，高200μM；阿魏酸三个浓度分别为低4μM，中40μM，高400μM。
表2七组分药物组正交实验分组
Figure PCTCN2021082244-appb-000005
Figure PCTCN2021082244-appb-000006
药液配制方式如下：根据表1中对应的各组浓度，在精密天平(1/10000g)称取适当重量的羟基红花黄色素A、芍药苷和芍药内酯苷，按照不同浓度组合情况组合至不同容量瓶中，分别溶解于适量培养基(含10％胎牛血清)中，制成相应浓度的9组三组分药物；
根据表2中对应的各组浓度，在精密天平(1/10000g)称取适合重量的羟基红花黄色素A、芍药苷、芍药内酯苷、氧化芍药苷、洋川芎内酯、丹参素钠和阿魏酸，按照不同浓度组合情况组合至不同容量瓶中，分别溶解于适量培养基(含10％胎牛血清)中，制成相应浓度的18组七组分药物。
3.实验结果
实验结果(表3)表明：与模型组相比，各剂量组的三组分药物组和七组分药物组均可显著降低12h、24h大鼠腹腔巨噬细胞IL-6的释放水平，表明三组分药物组和七组分药物组对脓毒症细胞模型早期炎症因子具有很好的抑制效果。
表3三组分药物组和七组分药物组对LPS刺激下大鼠腹腔巨噬细胞IL-6表达的影响(
Figure PCTCN2021082244-appb-000007
n＝6，单位：pg/mL)
Figure PCTCN2021082244-appb-000008
Figure PCTCN2021082244-appb-000009
注： *，表示与模型组相比，P<0.05。
实施例3
评价三组分药物、七组分药物对LPS刺激下大鼠腹腔巨噬细胞HMGB1释放的影响
1.实验材料
雄性清洁级SD大鼠(180～220g)，内毒素(LPS)，羟基红花黄色素A，芍药苷，芍药内酯苷，氧化芍药苷，洋川芎内酯I，丹参素钠，阿魏酸，RPMI-1640培养基，24孔板，HMGB1ELISA试剂盒。
2.实验方法
分离雄性SD大鼠腹腔巨噬细胞，具体方法如下：雄性SD大鼠术前禁食12h，麻醉后打开腹腔，向腹腔注入预冷PBS液10mL，用手指轻压揉腹壁，使液体在腹腔内流动。吸取腹腔中的液体注入灭菌管中，再用预冷PBS液10mL灌洗一次，操作同上。将合并收集到的灌洗液250g，4℃离心10min，弃上清。加入2mL红细胞裂解液溶解红细胞，轻微震荡两次，每次5s，静置5min后加入4mL D-Hanks溶液终止反应。按照上述方法再次离心，弃上清。用培养液洗涤沉淀，然后重悬细胞，制成2×10 6/mL细胞悬液。将所述细胞悬液接种于24孔板，置于37℃，5％CO 2的细胞培养箱培养。每组6个平行孔。分别培养48h和72h后收集上清，采用双抗体一步夹心法酶联免疫吸附试验(ELISA)测定细胞因子含量。
实验分为对照组、模型组和给药组，各组处理方法如下：各组细胞置于37℃，5％CO 2的细胞培养箱培养过夜后，对照组和模型组加入相应体积的培养液，给药组加入等体积的对应浓度药液，培养箱孵育1h后，对照组不加入LPS，模型组和给药组加入LPS(75ng/mL)刺激，各组在48h、72h后，收集细胞培养液上清0.5mL，-20℃冰箱保存，集中进行相应细胞因子检测。实验分组及药液配制方式同实施例2。
3.实验结果
实验结果(表4)表明：与模型组相比，各剂量组的三组分药物组和七组分药物组均可显著降低48h、72h大鼠腹腔巨噬细胞HMGB1的释放水平，表明三组分药物组和七组分药物组对脓毒症细胞模型晚期炎症因子具有很好的抑制效果。
表4三组分药物组和七组分药物组对LPS刺激下大鼠腹腔巨噬细胞HMGB1表达的影响(
Figure PCTCN2021082244-appb-000010
n＝6，单位：pg/mL)
Figure PCTCN2021082244-appb-000011
Figure PCTCN2021082244-appb-000012
注：*，表示与模型组相比，P<0.05。
实施例4
评价三组分药物、七组分药物对LPS刺激下大鼠腹主动脉内皮细胞TM释放的影响
1.实验材料
雄性清洁级SD大鼠(180～220g)，LPS，羟基红花黄色素A，芍药苷，芍药内酯苷，氧化芍药苷，洋川芎内酯I，丹参素钠，阿魏酸，ECM培养基，24孔板，组织因子(TM)ELISA试剂盒。
2.实验方法
用颈椎脱臼法处死大鼠后，浸泡于体积分数为75％的乙醇中5min，逐层打开胸、腹腔，充分暴露胸、腹主动脉，分离其周围组织，从近心端分离主动脉至髂总动脉分支处，放入含有PBS的培养皿中，无菌剥离血管外膜的脂肪组织和纤维组织，PBS冲洗血管腔。将主动脉切成约1.5mm×1.5mm小块，置于6mL 0.25％的IV型胶原酶中，37℃消化15min，每5min震荡一次，小心吸走消化液，保留组织块，再加入6mL 1.0％的中性蛋白酶，37℃消化15min，每5min震荡一次，吸走消化液，补充ECM 10mL， 反复吹打，1000r/min离心10min，弃去培养基和消化液，保留碎块，将组织块平铺于10cm的培养皿底部，倒放于37℃烘箱中2h，使组织块粘牢，加入适量ECM，没入组织块为宜，放37℃、体积分数为5％的CO 2饱和湿度培养箱内培养，3天更换一次培养基。7天左右可见内皮细胞由组织块边缘爬出并逐渐向外延伸，呈扁平短梭形或多角形，去除组织块，此时用0.25％胰蛋白酶消化，按1：3的比例在25cm 2的培养瓶中传代，用第3～4代细胞进行实验。
按照上述中大鼠腹主动脉内皮细胞的培养方法培养细胞，制备细胞悬液，以1.2×10 5/mL接种细胞，将细胞悬液接种于24孔板中，37℃培养，约12h后模型组和给药组给予LPS刺激，1h后分别给予不同药液进行干预，分别于刺激后24h、48h、72h收集上清。
实验分为对照组、模型组和给药组，各组处理方法如下：各组细胞置于37℃，5％CO 2的细胞培养箱培养过夜后，对照组和模型组加入相应体积的培养液，给药组加入等体积的对应浓度药液，培养箱孵育1h后，对照组不加入LPS，模型组和给药组加入LPS(75ng/mL)刺激，各组在24h、48h、72h后，收集细胞培养液上清0.5mL，-20℃冰箱保存，集中进行相应细胞因子检测。实验分组及药液配制方式同实施例2。
3.实验结果
TM普遍存在于大鼠腹主动脉内皮细胞表面，通过与凝血酶结合和激活蛋白C系统发挥抗凝作用，在脓毒症状态下，TM大量释放到周围血液中，从而使抗凝作用减弱。药物干预下TM的降低有助于凝血因子抗凝/促凝的平衡，有利于促进细胞/机体凝血功能往正常状态恢复。
实验结果(表5)表明：与模型组相比，各浓度组的三组分药物组和七组分药物组均可降低24h、48h和72h大鼠腹主动脉内皮细胞TM的释放，表明三组分药物组和七组分药物组对脓毒症细胞模型有促进凝血功能恢复正常的效果。
表5三组分药物组和七组分药物组对LPS刺激下大鼠腹主动脉内皮细胞TM释放的影响(
Figure PCTCN2021082244-appb-000013
n＝6，单位：pg/mL)
Figure PCTCN2021082244-appb-000014
Figure PCTCN2021082244-appb-000015
注： *，表示与模型组相比，P<0.05。
实施例5
评价三组分药物、七组分药物对LPS刺激下大鼠腹主动脉内皮细胞TF释放的影响1.实验材料
雄性清洁级SD大鼠(180～220g)，LPS，羟基红花黄色素A，芍药苷，芍药内酯苷，氧化芍药苷，洋川芎内酯I，丹参素钠，阿魏酸，ECM培养基，24孔板，组织因子(TF)ELISA试剂盒。
2.实验方法
实验方法及分组处理方法同实施例4。
实验分组及药液配制方式同实施例2。
3.实验结果
TF是大鼠腹主动脉内皮细胞重要的促凝因子，在脓毒症状态下，高凝状态表现为TF的大量释放。药物干预下TF的降低有助于凝血因子抗凝/促凝的平衡，有利于促进细胞/机体凝血功能往正常状态恢复。
实验结果(表6)表明：与模型组相比，各浓度组的三组分药物组和七组分药物组均可降低24h，48h和72h大鼠腹主动脉内皮细胞促凝因子TF的释放，表明三组分药物组和七组分药物组对脓毒症细胞模型有促进凝血功能恢复正常的效果。
表6三组分药物组和七组分药物组对LPS刺激下大鼠腹主动脉内皮细胞TF释放的影响(
Figure PCTCN2021082244-appb-000016
n＝6，单位：pg/mL)
Figure PCTCN2021082244-appb-000017
Figure PCTCN2021082244-appb-000018
注： *，表示与模型组相比，P<0.05。
实施例6
评价三组分药物、七组分药物对LPS刺激下大鼠脾脏调节性T细胞(Treg)凋亡的影响
1.实验材料
雄性清洁级SD大鼠(180～220g)，LPS，羟基红花黄色素A，芍药苷，芍药内酯苷，氧化芍药苷，洋川芎内酯I，丹参素钠，阿魏酸，1640培养基，24孔板，藻红蛋白(PE)-抗大鼠CD25、异硫氰酸荧光素(FITC)标记抗大鼠CD4、异硫氰酸荧光素(FITC)标记膜联蛋白V凋亡试剂盒；大鼠CD25-APC抗体试剂盒、APC磁珠、CD4磁珠、MACS Buffer、MiniMACS磁性分离仪以及分选柱(LS)；抗大鼠CD3单克隆抗体、抗大鼠CD28单克隆抗体。
2.实验方法
将雄性SD大鼠断颈处死，取脾脏，无菌注射器活塞研磨脾脏，吸管吸取悬液到离心管，1200转×7分钟离心，弃上清，加入适量MACS Buffer(10mL/每个脾)重悬，沿着管壁加入到淋巴细胞分离液的上层(1：1)3000转×15分钟离心，用吸管吸取中层液，装入另一离心管中，加入清洗液1200转×7分钟离心，弃上清，加入MACS Buffer重悬备用。
每1×10 7单核细胞加入1μL抗CD25-APC抗体，4℃孵育15min，MACS Buffer洗涤。每1×10 7细胞加入20μL抗APC磁珠和80μLMACS Buffer，4℃孵育15min，MACS Buffer洗涤，加适量MACS Buffer重悬，LS柱磁性分选，即得CD25 +细胞。
CD25 +细胞计数后，每1×10 7细胞加入20μL解离剂，4℃孵育1min，MACS Buffer洗涤，每1×10 7细胞加入20μL抗CD4磁珠和30μL终止剂，4℃孵育15min，MACS Buffer洗涤，加入适量MACS Buffer重悬，LS柱磁性分选，即得CD4 +CD25 +细胞。
将Treg细胞用适量培养液重悬，调整细胞数为2.5×10 6/mL，96孔板，每孔接入细胞100μL，模型组和实验组加入CD3/CD28(CD3为0.5μg/10 6，CD28为1μg/10 6)+LPS(1μg/mL)刺激，实验分为对照组、模型组、给药组，1h后模型组和给药组分别给予不同药液进行干预，CO 2培养箱37℃培养72h。
收集细胞，细胞用预冷的PBS清洗，然后用1ml 1×Binding Buffer再次清洗，离心后去上清剩余100μl，然后加入5μl FITC AnnexinV和5μl PI，室温(25℃)避光孵育15min，然后加入200μl 1×Binding Buffer混匀，1h内流式细胞仪检测。
分组处理方法如下：模型组：细胞在细胞培养箱培养过夜后，加入LPS(75ng/mL)刺激。给药组：加入对应浓度药液孵育1h，进行LPS刺激，不同浓度实验组干预72h 后，收集细胞培养液上清0.5mL，-20℃冰箱保存，集中进行相应细胞因子检测。
实验分组及药液配制方式同实施例2。
3.实验结果
Treg细胞功能增强后通过产生免疫抑制因子如IL-10、IL-35、TGF-β等抑制普通T细胞，并通过颗粒酶B和穿孔素-1杀伤靶细胞，从而发挥免疫抑制作用。还能诱导DC树突状细胞产生吲哚胺2，3-双加氧酶，催化色氨酸分解为犬尿素导致周围细胞死亡，还能诱导DC细胞分泌其他氨基酸相关酶，从而抑制效应T细胞的增殖，从而发挥免疫抑制的作用。因此，药物在一定时期促进Treg细胞的凋亡，对机体免疫调控方面起着十分重要的作用。与模型组相比，各剂量组的三组分药物组和七组分药物组均能够促进大鼠调节T细胞(Treg)的凋亡(表7)。
表7三组分药物组和七组分药物组对LPS刺激下大鼠脾脏调节性T细胞凋亡的影响(
Figure PCTCN2021082244-appb-000019
n＝6，单位：％)
Figure PCTCN2021082244-appb-000020
注： *，表示与模型组相比，P<0.05。
实施例7
三组分药物组、七组分药物组对LPS刺激下大鼠脾脏调节性T细胞CTLA-4和 Foxp3表达的影响
1.实验材料
雄性清洁级SD大鼠(180～220g)，LPS，羟基红花黄色素A，芍药苷，芍药内酯苷，氧化芍药苷，洋川芎内酯I，丹参素钠，阿魏酸，1640培养基，24孔板，藻红蛋白(PE)-抗大鼠CD25、异硫氰酸荧光素(FITC)标记抗大鼠CD4；大鼠抗-PE试剂盒、CD4磁珠、MiniMACS磁性分离仪以及分选柱；PE标记Foxp3试剂盒、PE标记CTLA-4试剂盒、抗大鼠CD3单克隆抗体、抗大鼠CD28单克隆抗体。
2.实验方法
实验方法及分组处理方法同实施例6。
实验分组及药液配制方式同实施例2。
3.实验结果
Foxp3 +Treg细胞通过产生免疫抑制因子如IL-10、IL-35、TGF-β等抑制普通T细胞，并通过颗粒酶B和穿孔素-1杀伤靶细胞，从而发挥免疫抑制作用。CTLA-4还能诱导DC树突状细胞产生吲哚胺2，3-双加氧酶，催化色氨酸分解为犬尿素导致周围细胞死亡，还能诱导DC细胞分泌其他氨基酸相关酶，从而抑制效应T细胞的增殖,从而发挥免疫抑制的作用。因此，Treg细胞通过Foxp3和CTLA-4在机体免疫调控方面起着十分重要的作用，Foxp3的表达与调节性T细胞(Treg)功能密切相关。
与模型组相比，各剂量组的三组分药物组和七组分药物组均能够降低大鼠调节T细胞(Treg)72h CTLA-4和Foxp3的表达水平，表明三组分药物组和七组分药物组有对脓毒症大鼠调节T细胞(Treg)72h CTLA-4和Foxp3的表达水平降低的效果(表8和表9)。
表8三组分药物组和七组分药物组对LPS刺激下大鼠脾脏调节性T细胞CTLA-4表达的影响(
Figure PCTCN2021082244-appb-000021
n＝6，单位：％)
Figure PCTCN2021082244-appb-000022
Figure PCTCN2021082244-appb-000023
注： *，表示与模型组相比，P<0.05。
表9三组分药物组和七组分药物组对LPS刺激下大鼠脾脏调节性T细胞Foxp3表达的影响(
Figure PCTCN2021082244-appb-000024
n＝6，单位：％)
Figure PCTCN2021082244-appb-000025
注： *，表示与模型组相比，P<0.05。
实施例8
评价三组分药物、七组分药物对CLP诱导的脓毒症大鼠IL-6表达的影响
1.实验材料
雄性清洁级SD大鼠(180～220g)，适应性饲养1周。羟基红花黄色素A，芍药苷，芍药内酯苷，氧化芍药苷，洋川芎内酯I，丹参素钠，阿魏酸，IL-6ELISA检测试剂盒。
2.实验方法
2.1分组与干预
290只大鼠随机分为对照组、模型组和给药组共29组每组各10只，其中给药组采用正交实验法，正交实验分组方法见表10和表11。
表10三组分药物组正交实验分组
组别 羟基红花黄色素A 芍药苷 芍药内酯苷
1 低 低 低
2 低 中 高
3 低 高 中
4 中 低 高
5 中 中 中
6 中 高 低
7 高 低 中
8 高 中 低
9 高 高 高
表10中羟基红花黄色素A三个浓度分别为低0.9mg/kg，中9mg/kg，高90mg/kg；芍药苷三个浓度分别为低0.9mg/kg，中9mg/kg，高90mg/kg；芍药内酯苷三个浓度分别为低0.09mg/kg，中0.9mg/kg，高9mg/kg；氧化芍药苷三个浓度分别为低0.09mg/kg，中0.9mg/kg，高9mg/kg；洋川芎内酯I三个浓度分别为低0.09mg/kg，中0.9mg/kg，高9mg/kg；丹参素钠三个浓度分别为低0.09mg/kg，中0.9mg/kg，高9mg/kg；阿魏酸三个浓度分别为低0.009mg/kg，中0.09mg/kg，高0.9mg/kg。
表11七组分药物组正交实验分组
Figure PCTCN2021082244-appb-000026
Figure PCTCN2021082244-appb-000027
溶液配制方式与实验注射量如下：每只大鼠按照200g计算且实验注射量均为1.0mL/次/只。根据表10中对应的各组浓度，在精密天平(1/10000g)称取适合重量的羟基红花黄色素A、芍药苷和芍药内酯苷，按照不同浓度组合情况组合至不同容量瓶中，分别溶解于适量生理盐水(0.9％的氯化钠水溶液)，制成相应浓度的9组三组分药物；根据表11中对应的各组浓度，在精密天平(1/10000g)称取适合重量的羟基红花黄色素A、芍药苷、芍药内酯苷、氧化芍药苷、洋川芎内酯、丹参素钠和阿魏酸，按照不同浓度组合情况组合至不同容量瓶中，分别溶解于适量生理盐水(0.9％的氯化钠水溶液)中，制成相应浓度的18组七组分药物。实验前12h禁食，称重并按照随机数字表法进行分组。对照组仅开腹暴露盲肠后缝合皮肤，皮下注射10mL生理盐水复苏；模型组和给药组进行盲肠结扎穿孔术(CLP)造模后，皮下注射10mL生理盐水复苏。CLP造模过程如下：用氯胺酮注射液+速眠新II注射液按体积比2：1比例制备的混合液肌肉注射麻醉大鼠，采用CLP制备脓毒症动物模型。结扎盲肠与回肠连接处，用18号针头贯通盲肠2次形成肠瘘，并留置2条引流条(0.5cm×2.0cm)防止针孔愈合，后逐层缝合皮肤，术毕立即皮下注射10mL生理盐水复苏。大鼠CLP手术后，给药组在术后2h，12h，24h，36h，48h和60h经尾静脉分别注射含上述不同药液；模型组和对照组在相应时间经尾静脉注射生理盐水。重复该实验至n＝10。
2.2采血与检测
CLP后8h，24h，48h将各组动物麻醉后腹主动脉无菌采血3mL，利用ELISA法，检测血浆IL-6含量。
3.实验结果
实验结果(表12)表明：对照组血浆中含有少量IL-6；CLP手术后早期，模型组IL-6含量即明显增加，8h IL-6水平进一步提高，于24h逐渐下降，但术后48h仍高于对照组，差异均有统计学意义(p<0.05)。而给药组术后8h，24h，48h的IL-6血浆含量均明显低于模型组(p<0.05)，接近对照组水平。
上述发现表明，与模型组相比，各剂量组的三组分药物组、七组分药物组均可显著降低CLP脓毒症大鼠IL-6的表达，提示对脓毒症大鼠模型早期炎症反应均具有明显的抑制作用，折算成人体剂量约为：三组分药物组(羟基红花黄色素A 6mg至600mg，芍药苷6mg至600mg，芍药内酯苷0.6mg至60mg)、七组分药物组(羟基红花黄色素A6mg至600mg，芍药苷6mg至600mg，芍药内酯苷0.6mg至60mg，氧化芍药苷0.6mg至60mg，洋川芎内酯I 0.6mg至60mg，丹参素钠0.6mg至60mg，阿魏酸0.06mg至6mg)。[根据徐叔云主编的《药理实验方法学》中人和动物间按体表面积折算法计算，人体剂量＝大鼠剂量/0.018(以成人体重70kg、大鼠体重0.2kg计)]。
表12三组分药物组和七组分药物组对CLP诱导的脓毒症大鼠IL-6表达的影响(
Figure PCTCN2021082244-appb-000028
Figure PCTCN2021082244-appb-000029
n＝10，单位：pg/mL)
Figure PCTCN2021082244-appb-000030
Figure PCTCN2021082244-appb-000031
注： *，表示与模型组相比，P<0.05。
实施例9
评价三组分药物、七组分药物对CLP诱导的脓毒症大鼠HMGB1表达的影响
1.实验材料
雄性清洁级SD大鼠(180～220g)，适应性饲养1周。羟基红花黄色素A，芍药苷，芍药内酯苷，氧化芍药苷，洋川芎内酯I，丹参素钠，阿魏酸，HMGB1ELISA检测试剂盒。
2.实验方法
2.1分组与干预
见实施例8。
2.2采血与检测
CLP术后48h和72h，将各组动物麻醉后腹主动脉无菌采血3mL，利用ELISA法，检测血浆HMGB1含量。
3.实验结果
实验结果(表13)表明：对照组血浆中含有少量HMGB1；CLP手术后晚期，模型组HMGB1含量明显增加，48h逐渐升高，术后72h仍高于对照组，差异均有统计学意义(p<0.05)。而给药组术后48h和72h的HMGB1血浆含量均明显低于模型组(p<0.05)，接近对照组水平。上述发现表明，与模型组相比，各剂量组的三组分药物组和七组分药物组可显著降低CLP脓毒症大鼠HMGB1的表达，提示三组分药物组和七组分药物组对脓毒症大鼠模型晚期炎症因子有抑制作用。折算成人体剂量约为：三组分药物组(羟基红花黄色素A6mg至600mg，芍药苷6mg至600mg，芍药内酯苷0.6mg至60mg)、七组分药物组(羟基红花黄色素A 6mg至600mg，芍药苷6mg至600mg，芍药内酯苷0.6mg至60mg，氧化芍药苷0.6mg至60mg，洋川芎内酯I 0.6mg至60mg，丹参素钠0.6mg至60mg，阿魏酸0.06mg至6mg)。[根据徐叔云主编的《药理实验方法学》中人和动物间按体表面积折算法计算，人体剂量＝大鼠剂量/0.018(以成人体重70kg、大鼠体重0.2kg计)]。
表13三组分药物组和七组分药物组对CLP诱导的脓毒症大鼠HMGB1表达的影响(
Figure PCTCN2021082244-appb-000032
n＝10，单位：pg/mL)
Figure PCTCN2021082244-appb-000033
注： *，表示与模型组相比，P<0.05。
实施例10
评价三组分药物、七组分药物对CLP诱导的脓毒症大鼠TF释放的影响
1.实验材料
雄性清洁级SD大鼠(180～220g)，适应性饲养1周。羟基红花黄色素A，芍药苷，芍药内酯苷，氧化芍药苷，洋川芎内酯I，丹参素钠，阿魏酸，TF ELISA试剂盒。
2.实验方法
2.1分组与干预
见实施例8。
2.2采血与检测
CLP术后12h，24h，48h和72h，将各组动物麻醉后腹主动脉无菌采血5mL。利用酶联免疫吸附法(ELISA)，检测TF含量。
3.实验结果
实验结果(表14)表明：对照组单核细胞TF存在一定表达；模型组TF(术后12h，24h，48h和72h)表达均显著高于对照组(p<0.05)，且随术后时间的延长而逐渐增高。给药组的TF(术后12h，24h，48h和72h)表达与模型组相比均出现显著降低(p<0.05)。上述发现提示，与模型组相比，各剂量组的三组分药物组和七组分药物组均可显著降低CLP脓毒症大鼠TF的释放，表明三组分药物组和七组分药物组对脓毒症大鼠模型具有明显的抑制凝血因子释放、改善高凝状态的作用。折算成人体剂量约为：三组分药物组(羟基红花黄色素A 6mg至600mg，芍药苷6mg至600mg，芍药内酯苷0.6mg至60mg)、七组分药物组(羟基红花黄色素A 6mg至600mg，芍药苷6mg至600mg，芍药内酯苷0.6mg至60mg，氧化芍药苷0.6mg至60mg，洋川芎内酯I 0.6mg至60mg，丹参素钠0.6mg至60mg，阿魏酸0.06mg至6mg)。
表14.三组分药物组和七组分药物组对CLP诱导的脓毒症大鼠TF释放的影响(
Figure PCTCN2021082244-appb-000034
Figure PCTCN2021082244-appb-000035
n＝10，单位：pg/mL)
Figure PCTCN2021082244-appb-000036
Figure PCTCN2021082244-appb-000037
注： *，表示与模型组相比，P<0.05。
实施例11
评价三组分药物、七组分药物对CLP诱导的脓毒症大鼠TM释放的影响
1.实验材料
雄性清洁级SD大鼠(180～220g)，适应性饲养1周。羟基红花黄色素A，芍药苷，芍药内酯苷，氧化芍药苷，洋川芎内酯I，丹参素钠，阿魏酸，TM ELISA试剂盒。
2.实验方法
2.1分组与干预
见实施例8。
2.2采血与检测
CLP术后12h，24h，48h和72h，将各组动物麻醉后腹主动脉无菌采血5mL。利用酶联免疫吸附法(ELISA)，检测TM含量。
3.实验结果
实验结果(表15)表明：对照组单核细胞TM存在一定表达；模型组TM(术后12h，24h，48h和72h)表达均显著高于对照组(p<0.05)，且随术后时间的延长而逐渐增高。给药组的TM(术后12h，24h，48h和72h)表达与模型组相比均出现显著降低(p<0.05)。上述发现提示，与模型组相比，各剂量组的三组分药物组和七组分药物组均可显著降低CLP脓毒症大鼠TM的释放，表明三组分药物组和七组分药物组对脓毒症大鼠模型具有明显的抑制凝血因子释放、改善高凝状态的作用。折算成人体剂量约为：三组分药物组(羟基红花黄色素A 6mg至600mg，芍药苷6mg至600mg，芍药内酯苷0.6mg至60mg)、七组分药物组(羟基红花黄色素A 6mg至600mg，芍药苷6mg至600mg，芍药内酯苷0.6mg至60mg，氧化芍药苷0.6mg至60mg，洋川芎内酯I 0.6mg至60mg，丹参素钠0.6mg至60mg，阿魏酸0.06mg至6mg)。[根据徐叔云主编的《药理实验方法学》中人和动物间按体表面积折算法计算，人体剂量＝大鼠剂量/0.018(以成人体重70kg、大鼠体重0.2kg计)]。
表15三组分药物组和七组分药物组对CLP诱导的脓毒症大鼠TM释放的影响(
Figure PCTCN2021082244-appb-000038
Figure PCTCN2021082244-appb-000039
n＝10，单位：pg/mL)
Figure PCTCN2021082244-appb-000040
Figure PCTCN2021082244-appb-000041
注： *，表示与模型组相比，P<0.05。
实施例12
评价三组分药物、七组分药物对CLP诱导的脓毒症调节性T细胞功能的影响
1.实验材料
雄性清洁级SD大鼠(180～220g)，内毒素(LPS)，羟基红花黄色素A，芍药苷，芍药内酯苷，氧化芍药苷，洋川芎内酯I，丹参素钠，阿魏酸，1640培养基，24孔板，藻红蛋白(PE)-抗大鼠CD25、异硫氰酸荧光素(FITC)标记抗大鼠CD4、异硫氰酸荧光素(FITC)标记膜联蛋白V凋亡试剂盒；大鼠抗-PE试剂盒、CD4磁珠、MiniMACS磁性分离仪以及分选柱；PE标记Foxp3试剂盒、PE标记CTLA-4试剂盒、抗大鼠CD3单克隆抗体、抗大鼠CD28单克隆抗体。
2.实验方法
2.1分组与干预
见实施例8。
2.2细胞分离及培养
CLP术后72h大鼠断颈处死，酒精棉球消毒胸腹部3遍，开腹，保留完整腹膜，酒精棉球再次消毒，另外一副镊子开腹膜，取脾脏，置于4℃15ml PBS中。用镊子撕开脾脏包膜，放入滤网，加PBS润湿，撕碎脾脏，去除筋膜，无菌注射器活塞研磨脾脏，吸管吸取悬液到离心管，1200转×7分钟离心，弃上清，加入适量MACS Buffer(10ml/每个脾)重悬，沿着管壁加入到淋巴细胞分离液的上层(1：1)3000转×15分钟离心，用吸管吸取中层液，装入另一离心管中，加入清洗液1200转×7分钟离心，弃上清，加入MACS Buffer重悬备用。
每1×10 7单核细胞加入1μl抗CD25-APC抗体，4℃孵育15min，MACS Buffer洗涤。每1×10 7细胞加入20μl抗APC磁珠和80μl MACS Buffer，4℃孵育15min，MACS Buffer洗涤，加适量MACS Buffer重悬，LS柱磁性分选，即得CD25 +细胞。
CD25 +细胞计数后，每1×10 7细胞加入20μl解离剂，4℃孵育10min，MACS Buffer洗涤，每1×10 7细胞加入20μl抗CD4磁珠和30μl终止剂，4℃孵育15min，MACS Buffer洗涤，加入适量MACS Buffer重悬，LS柱磁性分选，即得CD4 +CD25 +细胞。
2.3检测与分析
Treg凋亡率检测：收集细胞，细胞用预冷的PBS清洗，然后用1ml 1×Binding Buffer再次清洗，离心后去上清剩余100μl，然后加入5μl FITC Annexin V和5μl PI，室温(25℃)避光孵育15min，然后加入200μl 1×Binding Buffer混匀，1h内流式细胞仪检测。
Foxp3和CTLA-4表达检测：细胞上清收集后，细胞用PBS清洗，去上清，加入CTLA-4-PE荧光抗体，4℃避光孵育30min，加入破膜剂过夜。第二天，加入破膜缓冲液清洗，去上清，加入Foxp3-PE-Cy7荧光抗体，室温避光孵育30min，加入破膜缓冲液清洗，去上清，1％多聚甲醛固定，流式细胞仪检测。
3.实验结果
3.1三组分药物、七组分药物对CLP诱导的脓毒症调节性T细胞功能的影响
实验结果(表16)表明：模型组Treg凋亡率明显低于对照组(p<0.05)，给药组均显著高于模型组(p<0.05)。模型组Foxp3、CTLA-4表达明显高于对照组(p<0.05)，给药组均显著低于模型组(p<0.05)。上述发现提示，与模型组相比，各剂量组的三组分药物组和七组分药物组均可促进脓毒症Treg凋亡，下调对T淋巴细胞增殖和分泌功能的抑制效应。表明各剂量组的三组分药物组和七组分药物组对脓毒症大鼠模型具有明显的纠正机体细胞免疫抑制状态的作用。折算成人体剂量约为：三组分药物组(羟基红花黄色素A 6mg至600mg，芍药苷6mg至600mg，芍药内酯苷0.6mg至60mg)、七组分药物组(羟基红花黄色素A 6mg至600mg，芍药苷6mg至600mg，芍药内酯苷0.6mg至60mg，氧化芍药苷0.6mg至60mg，洋川芎内酯I 0.6mg至60mg，丹参素钠0.6mg至60mg，阿魏酸0.06mg至6mg)。[根据徐叔云主编的《药理实验方法学》中人和动物间按体表面积折算法计算，人体剂量＝大鼠剂量/0.018(以成人体重70kg、大鼠体重0.2kg计)]。
表16三组分药物组和七组分药物组对脓毒症大鼠调节性T细胞凋亡率及Foxp3、CTLA-4表达的影响(
Figure PCTCN2021082244-appb-000042
n＝10，单位：％)
Figure PCTCN2021082244-appb-000043
Figure PCTCN2021082244-appb-000044
注： *，表示与模型组相比，P<0.05。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。对这些实施例的多种修改对本领域的专业技术人员来说是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (16)

  1. 一种用于治疗脓毒症的药物组合物，其特征在于，包括结构式如式I所示的羟基红花黄色素A、结构式如式II所示的芍药苷和结构式如式III所示的芍药内酯苷；
    所述羟基红花黄色素A、芍药苷和芍药内酯苷的质量比为1～100：1～100：0.1～10；
    Figure PCTCN2021082244-appb-100001
  2. 根据权利要求1所述用于治疗脓毒症的药物组合物，其特征在于，所述羟基红花黄色素A、芍药苷和芍药内酯苷的质量比为1～100：1～100：1～10。
  3. 根据权利要求1或2所述用于治疗脓毒症的药物组合物，其特征在于，还包括结构式如式IV所示的氧化芍药苷、结构式如式V所示的洋川芎内酯I、结构式如式VI所示的丹参素钠和结构式如式VII所示的阿魏酸；
    所述羟基红花黄色素A、芍药苷、芍药内酯苷、氧化芍药苷、洋川芎内酯I、丹参素钠和阿魏酸的质量比为1～100：1～100：1～10：0.1～10：0.1～10：0.1～10：0.01～1；
    Figure PCTCN2021082244-appb-100002
  4. 根据权利要求3所述用于治疗脓毒症的药物组合物，其特征在于，所述羟基红花黄色素A、芍药苷、芍药内酯苷、氧化芍药苷、洋川芎内酯I、丹参素钠和阿魏酸的质量比为1～100：1～100：1～10：1～10：1～10：1～10：0.1～1。
  5. 权利要求1～4任意一项所述药物组合物在制备治疗脓毒症的药物中的应用。
  6. 根据权利要求5所述应用，其特征在于，所述药物组合物在制备抑制脓毒症的IL-6、Foxp3、CTLA-4和/或HMGB1表达的药物中的应用。
  7. 根据权利要求5所述应用，其特征在于，所述药物组合物在制备抑制脓毒症的TF和/或TM释放的药物中的应用。
  8. 根据权利要求5所述应用，其特征在于，所述药物组合物在制备促进脓毒症调节性T细胞凋亡的药物中的应用。
  9. 根据权利要求5～8任意一项所述应用，其特征在于，所述药物组合物由羟基红花黄色素A、芍药苷和芍药内酯苷三种活性化合物组成时，羟基红花黄色素A的药物剂量为6～600mg/kg，芍药苷的药物剂量为6～600mg/kg，芍药内酯苷的药物剂量为0.6～60mg/kg。
  10. 根据权利要求5～8任意一项所述应用，其特征在于，所述药物组合物由羟基红花黄色素A、芍药苷、芍药内酯苷、氧化芍药苷、洋川芎内酯I、丹参素钠和阿魏酸七种活性化合物时，羟基红花黄色素A的药物剂量为6～600mg/kg，芍药苷的药物剂量为6～600mg/kg，芍药内酯苷的药物剂量为0.6～60mg/kg，氧化芍药苷的药物剂量为0.6～60mg/kg，洋川芎内酯I的药物剂量为0.6～60mg/kg，丹参素钠的药物剂量为0.6～60mg/kg，阿魏酸的药物剂量为0.06～6mg/kg。
  11. 一种治疗脓毒症的方法，其特征在于，服用权利要求1～4任意一项所述药物组合物制备的药物。
  12. 根据权利要求11所述治疗脓毒症的方法，其特征在于，所述药物组合物通过抑制脓毒症的IL-6、Foxp3、CTLA-4和/或HMGB1表达实现治疗脓毒症。
  13. 根据权利要求11所述治疗脓毒症的方法，其特征在于，所述药物组合物通过抑制脓毒症的TF和/或TM释放实现治疗脓毒症。
  14. 根据权利要求11所述治疗脓毒症的方法，其特征在于，所述药物组合物通过促进脓毒症调节性T细胞凋亡实现治疗脓毒症。
  15. 根据权利要求11～14任意一项所述治疗脓毒症的方法，其特征在于，所述药物组合物由羟基红花黄色素A、芍药苷和芍药内酯苷三种活性化合物组成时，羟基红花黄色素A的药物剂量为6～600mg/kg，芍药苷的药物剂量为6～600mg/kg，芍药内酯苷的药物剂量为0.6～60mg/kg。
  16. 根据权利要求11～14任意一项所述治疗脓毒症的方法，其特征在于，所述药物组合物由羟基红花黄色素A、芍药苷、芍药内酯苷、氧化芍药苷、洋川芎内酯I、丹参素钠和阿魏酸七种活性化合物时，羟基红花黄色素A的药物剂量为6～600mg/kg，芍药苷的药物剂量为6～600mg/kg，芍药内酯苷的药物剂量为0.6～60mg/kg，氧化芍药苷的药物剂量为0.6～60mg/kg，洋川芎内酯I的药物剂量为0.6～60mg/kg，丹参素钠的药物剂量为0.6～60mg/kg，阿魏酸的药物剂量为0.06～6mg/kg。
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Families Citing this family (1)

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Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1432391A (zh) * 2003-03-04 2003-07-30 天津红日药业股份有限公司 一种具有治疗和保健作用的中药组方
CN101317893A (zh) * 2008-07-11 2008-12-10 天津红日药业股份有限公司 一种血必净口服泡腾剂
CN105181848A (zh) * 2015-09-29 2015-12-23 天津宏仁堂药业有限公司 血府逐瘀汤及胶囊的uplc指纹图谱检测方法
CN105241980A (zh) * 2015-11-12 2016-01-13 陕西步长制药有限公司 一种脑心通胶囊快速分离液相色谱检测方法
CN106727649A (zh) * 2016-12-28 2017-05-31 天津红日药业股份有限公司 一种多组分注射液
CN106950307A (zh) * 2017-03-29 2017-07-14 中国科学院上海药物研究所 一种血必净注射液中五类化合物的检测方法
CN108508107A (zh) * 2018-03-22 2018-09-07 天津中医药大学 一种同时测定血浆中血必净注射液有效成分的方法
CN108743600A (zh) * 2018-07-04 2018-11-06 天津中医药大学 一种天然药物组合物和含有该天然药物的中药组合物及其应用
CN109260214A (zh) * 2018-10-19 2019-01-25 天津红日药业股份有限公司 芍药苷类化合物在制备治疗脓毒症药物中的应用
CN109394750A (zh) * 2018-10-19 2019-03-01 天津红日药业股份有限公司 羟基红花黄色素a在制备治疗脓毒症药物中的应用
CN109709244A (zh) * 2019-02-22 2019-05-03 天津红日药业股份有限公司 一种血必净注射液苯酞内酯类成分含量测定方法
CN110632230A (zh) * 2019-09-29 2019-12-31 中国科学院上海药物研究所 一种血必净注射液中多个小分子化学成分的检测方法
CN110907576A (zh) * 2019-11-06 2020-03-24 河北医科大学 同时测定冠心静胶囊中16个活性成分含量的方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104546832A (zh) * 2013-10-12 2015-04-29 烟台益诺依生物医药科技有限公司 一种药物组合物在制备治疗脓毒症药物中应用

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1432391A (zh) * 2003-03-04 2003-07-30 天津红日药业股份有限公司 一种具有治疗和保健作用的中药组方
CN101317893A (zh) * 2008-07-11 2008-12-10 天津红日药业股份有限公司 一种血必净口服泡腾剂
CN105181848A (zh) * 2015-09-29 2015-12-23 天津宏仁堂药业有限公司 血府逐瘀汤及胶囊的uplc指纹图谱检测方法
CN105241980A (zh) * 2015-11-12 2016-01-13 陕西步长制药有限公司 一种脑心通胶囊快速分离液相色谱检测方法
CN106727649A (zh) * 2016-12-28 2017-05-31 天津红日药业股份有限公司 一种多组分注射液
CN106950307A (zh) * 2017-03-29 2017-07-14 中国科学院上海药物研究所 一种血必净注射液中五类化合物的检测方法
CN108508107A (zh) * 2018-03-22 2018-09-07 天津中医药大学 一种同时测定血浆中血必净注射液有效成分的方法
CN108743600A (zh) * 2018-07-04 2018-11-06 天津中医药大学 一种天然药物组合物和含有该天然药物的中药组合物及其应用
CN109260214A (zh) * 2018-10-19 2019-01-25 天津红日药业股份有限公司 芍药苷类化合物在制备治疗脓毒症药物中的应用
CN109394750A (zh) * 2018-10-19 2019-03-01 天津红日药业股份有限公司 羟基红花黄色素a在制备治疗脓毒症药物中的应用
CN109709244A (zh) * 2019-02-22 2019-05-03 天津红日药业股份有限公司 一种血必净注射液苯酞内酯类成分含量测定方法
CN110632230A (zh) * 2019-09-29 2019-12-31 中国科学院上海药物研究所 一种血必净注射液中多个小分子化学成分的检测方法
CN110907576A (zh) * 2019-11-06 2020-03-24 河北医科大学 同时测定冠心静胶囊中16个活性成分含量的方法

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DONG, KAI: "Research Advances in Sepsis and Treatments Therefor", SICHUAN MEDICAL JOURNAL, vol. 32, no. 12, 1 December 2011 (2011-12-01), pages 1 - 3, XP055963981, DOI: 10.16252/j.cnki.issn1004-0501-2011.12.094 *
HUANG H., WANG J., FU J. Z., WANG L. Q., ZHAO H. Z., SONG S. Y., JI L. X., JIANG M., BAI G., LUO G. A.: "Simultaneous determination of thirteen main components and identification of eight major metabolites in Xuebijing Injection by UPLC/Q-TOF", JOURNAL OF ANALYTICAL CHEMISTRY., CONSULTANTS BUREAU. NEW YORK., US, vol. 68, no. 4, 1 April 2013 (2013-04-01), US , pages 348 - 356, XP055963987, ISSN: 1061-9348, DOI: 10.1134/S1061934813040023 *
HUANG HAO, JI LANXIN, SONG SHENGYOU, WANG JING, WEI NA, JIANG MIN, BAI GANG, LUO GUOAN: "Identification of the major constituents in Xuebijing injection by HPLC-ESI-MS", PHYTOCHEMICAL ANALYSIS, J. WILEY, vol. 22, no. 4, 1 July 2011 (2011-07-01), pages 330 - 338, XP055963989, ISSN: 0958-0344, DOI: 10.1002/pca.1284 *
LEI ZHANG; LIN ZHU; YUEFEI WANG; ZHENZUO JIANG; XIN CHAI; YAN ZHU; XIUMEI GAO; AIDI QI;: "Characterization and quantification of major constituents of Xue Fu Zhu Yu by UPLC-DADMS/MS", JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, ELSEVIER B.V., AMSTERDAM, NL, vol. 62, 21 December 2011 (2011-12-21), AMSTERDAM, NL , pages 203 - 209, XP028459647, ISSN: 0731-7085, DOI: 10.1016/j.jpba.2011.12.026 *
LIANG, QUN: "Research progress of Xuebijing Injection In the Treatment of Sepsis", TIANJIN JOURNAL OF TRADITIONAL CHINESE MEDICINE, vol. 36, 31 July 2019 (2019-07-31), pages 641 - 644, XP055963984, DOI: 10.11656/j.issn.1672-1519.2019.07.05 *
See also references of EP4248964A4 *

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