WO2009000149A1 - Use of notoginsenoside r1 in the preparation of the medicament for treating hepatic injuries - Google Patents

Use of notoginsenoside r1 in the preparation of the medicament for treating hepatic injuries Download PDF

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Publication number
WO2009000149A1
WO2009000149A1 PCT/CN2008/001169 CN2008001169W WO2009000149A1 WO 2009000149 A1 WO2009000149 A1 WO 2009000149A1 CN 2008001169 W CN2008001169 W CN 2008001169W WO 2009000149 A1 WO2009000149 A1 WO 2009000149A1
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liver
reperfusion
use according
ischemia
notoginsenoside
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PCT/CN2008/001169
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French (fr)
Chinese (zh)
Inventor
Jingyan Han
Weixing Chen
Fang Wang
Yuying Liu
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Tianjin Tasly Pharmaceutical Co. Ltd.
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Publication of WO2009000149A1 publication Critical patent/WO2009000149A1/en

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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
    • A61K31/7028—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
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses

Definitions

  • the present invention relates to a novel pharmaceutical use of Panax notoginseng saponin R1, an active ingredient of traditional Chinese medicine Sanqi, and particularly relates to the use of notoginsenoside R1 for the treatment and/or prevention of liver damage.
  • Background technique
  • PN Panax notoginseng (Burk. ) FH Chen
  • PN is a dry root of A liaceae (Sanqi), widely used in China, Korea, Japan and other Asian countries for the treatment of microcirculation.
  • Disorder-related diseases such as cardiovascular disease, cerebrovascular disease, and liver dysfunction.
  • Sanqi contains more than 30 different types of saponin compounds, of which ginsenoside Rgl (Rgl), ginsenoside Rbl (Rbl) and notoginsenoside Rl (R1) are more common.
  • the structure of R1 is as follows:
  • Liver injury is a common disease, and hepatitis virus, liver cirrhosis, drug abuse, alcohol abuse, and chemical poison contact are the main causes of liver damage.
  • the liver is the "life tower" of the human body.
  • the various metabolic, detoxification and immune functions of the human body are borne by the liver. Any adverse effects on hepatocytes and intrahepatic blood flow can damage liver tissue.
  • Common liver injuries include liver infection caused by viral infection or cirrhosis, drug liver injury, alcoholic liver injury, chemical liver injury, liver damage caused by ischemia-reperfusion, and the like. Liver damage caused by viral infection and cirrhosis mainly causes liver cell damage through the immune response of the human body.
  • liver injury is caused by the formation of oxygen free radicals by drug metabolites, lipid peroxidation, drugs Metabolism produces electrophilic products, superoxide ions, leading to liver cell damage. Alcoholic liver injury affects the blood supply to the liver and increases the burden on the liver, leading to alcoholic hepatitis, fatty liver, and cirrhosis. Chemical liver damage is caused by chemical poisons destroying liver cells, and liver function is abnormal.
  • Ischemia-reperfusion injury refers to the damage of tissues and organs caused by reperfusion of blood perfusion or oxygen supply in ischemic tissues or organs.
  • Hepatic ischemia-reperfusion injury Hepatic ischemia reperfusion injury (HIRI) can reduce liver metabolic detoxification ability and increase microcirculation resistance. In severe cases, liver failure can be caused. In liver transplantation, primary nonfunction (PNF) is observed. PNF is one of the leading causes of death in liver transplant patients.
  • Ischemia-reperfusion (I/R) can occur in many situations, such as trauma, vasoconstriction, PTCA, thrombolytic therapy, organ transplantation, and resuscitated hypovolemic shock.
  • Ischemia-reperfusion can cause microcirculation to be damaged, often accompanied by epithelial cell damage, enhanced granulocyte adhesion, macromolecular efflux, oxygen free radical production and mast cell degranulation, according to hepatic ischemia-reperfusion
  • the mechanism of injury can take many preventive and therapeutic methods.
  • Passive defense can be used during surgery, preoperative and postoperative, as well as adding drugs or active ingredients to the preservation solution.
  • Antioxidant free radical drugs such as allopurinol and glutathione Peptides, Ca 2+ antagonists, etc. have been used in clinical practice, and domestic researchers have also achieved good results by reasonably adding Chinese medicine preparations to the preservation solution.
  • notoginsenoside R1 can improve intestinal microcirculation disturbance induced by intestinal ischemia-reperfusion, thereby treating and/or preventing liver damage, and the present invention provides a novel therapeutic use of notoginsenoside R1. Summary of the invention
  • the present invention provides a novel therapeutic use of notoginsenoside R1.
  • the new therapeutic use is to treat and/or prevent liver damage, particularly ischemia-reperfusion-induced liver damage, with notoginsenoside R1.
  • the present invention provides a novel use of a drug, that is, the use of notoginsenoside R1 or a pharmaceutical composition thereof for the preparation of a medicament for treating and/or preventing liver damage.
  • the notoginsenoside R1 is obtained by extracting from the traditional Chinese medicine Panax notoginseng, the purity is >50%, preferably the purity is >90%, more preferably the purity is >98%.
  • the pharmaceutical composition of the present invention has notoginsenoside R1 as a pharmaceutically active ingredient, and the weight percentage in the preparation may be 0.1 to 99.9%, and the balance is a pharmaceutically acceptable carrier.
  • the notoginsenoside R1 in the present invention may be administered to a patient in the form of a pharmaceutical composition which is in the form of any pharmaceutically acceptable preparation, preferably an injection, which may be a solution or a lyophilized powder injection.
  • the liver injury is selected from liver damage caused by viral infection or cirrhosis, drug liver damage, alcoholic liver damage, chemical liver damage, liver damage caused by ischemia-reperfusion.
  • the liver injury is liver damage caused by ischemia-reperfusion.
  • the treatment and/or prevention of liver damage is achieved by ameliorating microcirculatory disorders including a decrease in venule diameter, a decrease in red blood cell flow rate, a decrease in the number of perfused sinusoids, and leukocyte Rolling and adhesion increase.
  • the treatment and/or prevention of liver damage is achieved by reducing the expression of hepatic vascular E-selectin and intercellular adhesion molecule-1.
  • the treatment and/or prevention of liver damage is achieved by reducing the expression of CD18 and CD1 lb on the surface of neutrophils.
  • FIG. 1 Chemical structure of notoginsenoside (R1).
  • Figure 2 R1 treatment of the diameter of the hepatic venules in the superior mesenteric artery ischemia-reperfusion mice, the detection time is 0 minutes before ischemia (basal value), 15 minutes and 30 minutes after reperfusion.
  • A The diameter of the terminal venule.
  • B Central vein diameter.
  • the abscissa represents the ratio of the diameter value to the base value at any point in time.
  • the results are the mean ⁇ SE values of 6 experimental animals. Compared with the control group, a P ⁇ 0.05; compared with the ischemia-reperfusion (I/R) group, e P ⁇ 0.05.
  • FIG. 3 R1 to the flow velocity of hepatic venule erythrocytes in the mesenteric artery ischemia-reperfusion mice Intervention, the time point of detection was 0 minutes before ischemia (basal value) and 15 and 30 minutes after reperfusion.
  • A The flow rate of red blood cells in the terminal venules.
  • B Red blood cell flow rate in the central vein.
  • the abscissa represents the ratio of the red blood cell flow rate value to the base value at any time point.
  • the results are the mean ⁇ SE values of 6 experimental animals.
  • a P ⁇ 0.05 compared with the ischemia-reperfusion (I/R) group, C P ⁇ 0.05.
  • FIG. 4 Intervention of R1 on perfusion of the sinusoids in the superior mesenteric artery ischemia-reperfusion mice at the 0th minute before ischemia (basal value) and 15 minutes and 30 minutes after reperfusion.
  • A sinusoidal perfusion in the terminal venule region.
  • B sinusoidal perfusion in the central venous area.
  • the abscissa represents the ratio of the sinusoids perfused at any point in time to the baseline value.
  • the results are the mean ⁇ SE values of 6 experimental animals. Compared with the control group, a P ⁇ 0.05; compared with the ischemia-reperfusion group (I/R), e P ⁇ 0.05.
  • Figure 5 Intervention of R1 on hepatic venous leukocyte rolling in the ischemic-reperfused mesenteric artery.
  • the time of detection was 0 minutes before ischemia (basal value) and 15 minutes and 30 minutes after reperfusion.
  • A The number of rolling granulocytes in the terminal venules.
  • B Number of rolling granulocytes in the central vein.
  • the abscissa represents the number of rolling granulocytes in each 200 ⁇ m 2 field of view.
  • the results are the mean ⁇ SE values of 6 experimental animals. Compared with the control group, a P ⁇ 0.05; compared with the ischemia-reperfusion group (I/R), e P ⁇ 0.05.
  • Figure 6 Intervention of R1 on hepatic venous leukocyte rolling in the ischemic-reperfused mesenteric artery.
  • the time of detection was 0 minutes before ischemia (basal value) and 15 minutes and 30 minutes after reperfusion.
  • A
  • ⁇ The number of granulocytes attached to the terminal venules.
  • ⁇ The number of granulocytes attached to the central vein.
  • the abscissa represents the number of granulocytes attached per 200 ⁇ 2 field of view.
  • the results are the mean ⁇ SE values of 6 experimental animals.
  • a P ⁇ 0.05 compared with the control group, a P ⁇ 0.05 ; compared with the ischemia-reperfusion (I/R) group, c P ⁇ 0.05 o Figure 8 R1 on leukocyte adhesion in the sinusoids of the superior mesenteric artery ischemia-reperfusion mice
  • the time point of detection is ischemia (basic value) before 0 minutes, after reperfusion 15 minutes and 30 minutes.
  • A The number of granulocytes attached to the sinusoids of the terminal venules.
  • B Number of granulocytes attached to the sinusoid in the central venous region.
  • the abscissa represents the number of granulocytes attached per 200 ⁇ 2 field of view.
  • the result was the mean SE value of 6 experimental animals.
  • a P ⁇ 0.05 compared with the control group, a P ⁇ 0.05; compared with the ischemia-reperfusion (I/R) group, c P ⁇ 0.05 o Figure 9 R1 on the hepatic vascular E-selectin and the mesenteric artery ischemia-reperfusion in mice Intervention of expression of intercellular adhesion molecule-1.
  • liver tissues were immunofluorescently stained.
  • the results are the mean ⁇ SE values of 6 experimental animals. Compared with the control group, a P ⁇ 0.05; compared with the ischemia-reperfusion (I/R) group, e P ⁇ 0.05.
  • Figure 10 Intervention of R1 on the expression of CD18 and CD1 lb on neutrophil surface in mice with superior mesenteric ischemia-reperfusion. At 30 minutes after reperfusion, neutrophils were isolated and subjected to flow cytometry.
  • the results are the mean ⁇ SE values of 6 experimental animals. Compared with the control group, a P ⁇ 0.05; compared with the ischemia-reperfusion (I/R) group, e P ⁇ 0.05.
  • Figure 12 in the serum of the superior mesenteric artery ischemia-reperfusion mice, tumor necrosis factor- ⁇ (TNF- ⁇ ), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) The concentration of intervention. At 30 minutes after reperfusion, serum was separated and subjected to flow cytometry. The results are the mean ⁇ SE values of 6 experimental animals. Compared with the control group, a P ⁇ 0.05; compared with the ischemia-reperfusion (I/R) group, e P ⁇ 0.05. detailed description
  • the notoginsenoside R1 of the present invention is a component of the traditional Chinese medicine Panax notoginseng, and is not particularly limited as long as it meets the medicinal standard.
  • the notoginsenoside R1 can be purchased from the market, such as products produced by Nanjing Qingze Pharmaceutical Technology Development Co., Ltd. and Shanghai Kangjiu Chemical Co., Ltd.; it can also be prepared according to the prior art, for example, Chinese patent 01136697.4 from Sanqisheng A method for extracting and separating notoginsenoside Rl, ginsenoside Re, and notoginseng by powder extraction.
  • the notoginsenoside R1 preferably has a purity of >50%, more preferably a purity of >90%, and most preferably a purity of >98%.
  • the medicament according to the present invention is a pharmaceutical composition prepared by using the above-mentioned notoginsenoside R1 as a pharmaceutically active ingredient.
  • the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier as needed, wherein notoginsenoside R1 is a pharmaceutically active ingredient, and the weight percentage in the preparation may be 0.1 to 99.9%, and the others are pharmaceutically acceptable. Carrier.
  • the pharmaceutical composition of the present invention is present in unit dosage form, which means a unit of the preparation, such as each tablet, each capsule, each vial of oral solution, granules per bag, each injection, and the like.
  • the pharmaceutical composition of the present invention may be in any pharmaceutically acceptable dosage form, including tablets (for example, sugar-coated tablets, film-coated tablets and enteric coated tablets), capsules (for example, hard capsules, soft capsules), orally.
  • the pharmaceutical composition of the present invention may contain a usual excipient such as a binder, a filler, a diluent, a tablet, a lubricant, a disintegrant, a coloring agent, a flavoring agent. And humectants, if necessary, the tablets can be coated.
  • Suitable fillers include cellulose, mannitol, lactose and other similar fillers.
  • Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate.
  • Suitable lubricants include, for example, magnesium stearate.
  • Suitable pharmaceutically acceptable wetting agents include sodium decyl sulfate.
  • the solid oral composition can be prepared by a conventional method such as mixing, filling, tableting or the like. The repeated mixing of the active ingredients allows the active ingredient to be distributed throughout the composition containing a large amount of filler.
  • the oral liquid preparation may be in the form of, for example, an aqueous or oily suspension, solution, emulsion, syrup or elixir, or may be a dry product which may be formulated with water or other suitable carrier before use.
  • Such liquid preparations may contain conventional additives such as suspending agents such as sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fat; Emulsifiers such as lecithin, sorbitan monooleate or gum arabic; non-aqueous carriers (which may include edible oils), such as almond oil, fractionated coconut oil, oily esters such as glycerides, propylene glycol or ethanol; preservatives, Such as p-hydroxybenzyl ester or pair Propyl hydroxybenzoate or sorbic acid, and if desired, may contain conventional flavoring or coloring agents.
  • the liquid unit dosage form prepared contains the active ingredient of the invention and a sterile vehicle.
  • This compound can be suspended or dissolved depending on the carrier and concentration.
  • the solution is usually prepared by dissolving the active ingredient in a carrier, sterilizing it by filtration prior to filling it into a suitable vial or ampoule, and then sealing. Excipients such as a local anesthetic, preservative and buffer may also be dissolved in such a carrier.
  • the composition can be frozen after filling the vial and the water removed under vacuum.
  • the pharmaceutical composition of the present invention may optionally be added to a suitable pharmaceutically acceptable carrier when prepared as a medicament, the pharmaceutically acceptable carrier being selected from the group consisting of: mannitol, sorbitol, sodium metabisulfite, sodium hydrogen sulfite , sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, calcium EDTA, monovalent alkali metal carbonate, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid , sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and Derivatives, alginate, gelatin, polyvinylpyrrolidone, glycerin
  • the injection is preferably a solution or a lyophilized powder injection.
  • the pharmaceutical composition of the present invention determines the usage amount according to the condition of the patient at the time of use.
  • the therapeutic use of the present invention will be described by taking the ischemia-reperfusion liver injury as an example, but the present invention is not limited to the embodiment.
  • Notoginsenoside R1 (referred to as Rl, purity >98%) was provided by Tianjin Tianshili Group (Tianjin, China).
  • mice (batch number: 6538, Santa Cruz Biotechnology, Inc., USA); mouse monocyte chemotactic protein-l (MCP-l) freely set (flex set) (batch number: 558342, BD bio Scientific System, USA); Mouse Tumor Necrosis Factor-a (TNF-a) Free Combination Pack (Batch No.: 558299, BD Bioscience System, USA); Mouse Interleukin-6 (IL-6) Free Combination Pack (Batch Number: 558301, BD Bioscience Systems, USA).
  • MCP-l mouse monocyte chemotactic protein-l
  • TNF-a Mouse Tumor Necrosis Factor-a
  • IL-6 Mouse Interleukin-6 Free Combination Pack
  • mice were purchased from the Experimental Animal Center of Peking University Medical School, weighing 22 ⁇ 26g, age 8 ⁇ 10 weeks, laboratory animal certificate number SCXK 2002-2001.
  • the experimental animals were housed in a temperature of 24 ⁇ 1 ° C, humidity of 50 ⁇ 5%, 12 hours of light and dark cycle environment, fasting for 12 hours before the test, free to drink water. All experimental animals were processed following the guidelines of the Peking University Laboratory Animal Management Committee. In vivo microscopy
  • mice were anesthetized with 20% urethane (10 ml/kg body weight).
  • the cannula was inserted into the left jugular vein and the test drug was perfused with a PE tube having a diameter of 0.96 mm.
  • the mice were placed in the lateral position on the observation table.
  • the liver was placed in a temperature-controlled (37 ° C) observation box on an adjustable Plexiglas microscope stage and carefully treated to reduce the effects of respiratory motion.
  • the left lobe of the liver was observed by an inverted biomicroscope (DM-IRB, Leica, Germany) through a 3CCD color camera (JK-TU53H, 3CCD camera, Toshiba Corporation, Japan).
  • physiological saline at 37 ° C was used to drip the liver surface to maintain the moisture of the liver, and the observation area around the liver surface was covered with physiologically wet cotton gauze.
  • the superior mesenteric artery was ligated with a polyethylene tube (1.00 mm in diameter) for 15 minutes. After ischemia, the ligation is gently released and reperfusion is performed. Immediately before ischemia (baseline), venule diameter, erythrocyte flow rate, sinusoidal reperfusion, and leukocyte rolling and adhesion were measured, and the above indicators were observed every 15 minutes within half an hour after reperfusion. Test plan
  • mice in the ischemia-reperfusion group were continuously perfused with physiological saline solution through the left neck vein.
  • Mice in the R1 + ischemia-reperfusion (I/R) group were continuously perfused with R1 (10 mg/kg/h) 10 minutes before ischemia-reperfusion to the end of observation.
  • Mice in the mock surgery control group were treated in the same manner as the blood-reperfusion group, but the superior mesenteric artery was not ligated. There were 6 mice in each group (3 males and 3 females).
  • the DVD image was replayed using Image-Pro Plus 5.0 software, and the terminal venule diameter and central vein diameter were measured.
  • the measurement results are expressed as the ratio of the measured value at the 15th minute or the 30th minute after reperfusion to the base value, and the results are shown in Fig. 2.
  • the monitor was tuned to a high-speed camera system (FASTCAM-ultima ⁇ , photon, Japan) by CCD, and the red blood cell flow rate in the terminal venules and central veins of the liver was recorded at 1000 frames per second, and the image was saved at 25 frames at high speed. / second speed replay.
  • Red blood cell flow rates were measured using Image-Pro Plus 5.0 software. The red blood cell flow rate is expressed in ⁇ ⁇ /sec.
  • the measurement results are expressed as the ratio of the measured value to the base value at the 15th or 30th minute after reperfusion.
  • the results are shown in Figure 3.
  • the number of liver sinusoids in the terminal venules and central veins of the liver was recorded.
  • the number of hepatic sinusoids is expressed as a perfused sinusoid in each field of view (250 ⁇ > 300 ⁇ ).
  • the measurement results are expressed as the ratio of the measured value to the base value at the 15th minute or the 30th minute after reperfusion, and the results are shown in FIG.
  • rhodamine 6G rhodamine 6G in physiological saline at a concentration of 0.5 mg/ml
  • rhodamine 6G in physiological saline at a concentration of 0.5 mg/ml rhodamine 6G in physiological saline at a concentration of 0.5 mg/ml
  • a 20-inch fluorescent objective lens, argon laser beam irradiation (wavelength 543 nm), recorded the rolling and adhesion of granulocytes in the terminal venules and central veins of the liver.
  • the results are shown in Figures 5 to 8.
  • the granulocytes that stayed in the terminal venules and central veins of the liver for less than 10 seconds were rolling granulocytes, expressed as the number observed per 200 ⁇ m 2 field of view, and the results are shown in Fig. 5. Scanning at a rate of 1 frame/second, a total of 10 consecutive frames are recorded at each time point, and the granulocytes that appear at the same position during the scanning of 10 consecutive frames are adherent granulocytes.
  • the granulocytes adhering to the terminal venules and central veins of the liver are expressed as the number of granulocytes observed per 200 ⁇ m 2 field of view, and the results are shown in Fig. 7.
  • the adherent granulocytes in the sinusoids of the liver are expressed in terms of the number of granulocytes observed per 200 ⁇ 2 field of view, and the results are shown in Fig. 8.
  • the liver was fixed by perfusion of 4% paraformaldehyde, the liver was taken out, frozen with liquid nitrogen, and then cut into 6 ⁇ thick slices using a cryostat (LEICACM1800, Leica Co., Germany). The sections were fixed again with 4% paraformaldehyde for 10 minutes at room temperature and finally rinsed with phosphate buffer solution. The samples were then subjected to conventional immunohistochemical staining. Goat anti-mouse ⁇ -selectin polyclonal antibody or goat anti-mouse intercellular adhesion molecule-1 polyclonal antibody was diluted (dilution factor 1: 50).
  • mice at 30 minutes and 60 minutes after reperfusion were taken from the inferior vena cava and anticoagulated with heparin (20 units/ml whole blood). Serum was separated by centrifugation (Allegra TM 64R Centrifuge, Beckman Coultertm, Germany) rotating at 4,000 revolutions / minute, placed in a 4 ° C 10 min, then stored at -20 ° C environment.
  • centrifugation Allegra TM 64R Centrifuge, Beckman Coultertm, Germany
  • Figure 3 shows the effect of R1 on the flow rate of red blood cells in the terminal venules and central veins of the liver after ischemia and reperfusion of the superior mesenteric artery in mice.
  • red blood cell flow rate in the blood vessels was significantly reduced in a time-dependent manner.
  • the decrease of red blood cell flow rate in the two types of blood vessels after ischemia-reperfusion was restored to some extent, and the effect was 30 minutes after ischemia-reperfusion.
  • the recovery of red blood cell flow in the terminal venules was more pronounced (see Figure 3A).
  • Figure 6 shows a typical image of microcirculation in the central venous region at 30 minutes after ischemia-reperfusion of the superior mesenteric artery. Some granulocytes can be observed to adhere to the central vein and sinusoids.
  • Figure 7 shows R1 adhesion to granulocytes induced by ischemia-reperfusion of superior mesenteric artery Intervention. Ischemia-reperfusion of the superior mesenteric artery greatly increases the amount of granulocyte adhesion in the terminal venules and central veins of the liver. After R1 treatment, the number of increased adherent granulocytes was reduced after ischemia-reperfusion, and the terminal venules were more significantly reduced (see Figure 7A).
  • Figure 8 shows the effect of R1 on leukocyte adhesion in the terminal venous and central sinusoids of the liver.
  • Figure 8 shows the effect of R1 on leukocyte adhesion in the terminal venous and central sinusoids of the liver.
  • Figure 8 shows the effect of R1 on leukocyte adhesion in the terminal venous and central sinusoids of the liver.
  • Figure 8 shows the effect of R1 on the expression of hepatic vascular E-selectin and intercellular adhesion molecule-1 in mice at 30 minutes after reperfusion.
  • E-selectin expression is significantly enhanced at the 30th minute after reperfusion, and has no effect on the expression of intercellular adhesion molecule-1.
  • R1 the expression of E-selectin increased after ischemia-reperfusion of the superior mesenteric artery completely disappeared, and the expression of intercellular adhesion molecule-1 was also reduced.
  • blood samples were taken to evaluate the effect of R1 on the expression of peripheral neutrophil adhesion molecules CD18 and CD1 lb in mice.
  • CD18 expression was significantly enhanced and CDl lb expression was slightly enhanced at 30 minutes after reperfusion.
  • R1 treatment significantly reduced the increase in mean fluorescence intensity of CD18 and CD1 lb induced by ischemia-reperfusion.
  • the present invention demonstrates that R1 treatment can significantly reduce hepatic microcirculatory disturbance induced by ischemia-reperfusion of the superior mesenteric artery, including a decrease in venule diameter, a decrease in red blood cell flow rate, and a decrease in the number of perfused sinusoids. As well as the rolling and adhesion of white blood cells.
  • the improved effect of R1 on microcirculatory disorders implies a protective effect on ischemia-reperfusion-induced liver injury.
  • the notoginsenoside Rl 5g is dissolved in water, and sodium chloride and ethyl p-hydroxybenzoate are dissolved in water by heating, mixed, and adjusted to pH.
  • the water for injection is diluted to 1000 ml, filtered through a hollow fiber membrane, filled, and sterilized.
  • the notoginsenoside Rl lg is dissolved in water, and sodium chloride and ethyl p-hydroxybenzoate are dissolved in water by heating, mixed, and adjusted to pH.
  • the water for injection is diluted to 1000 ml, filtered through a hollow fiber membrane, filled, and sterilized.

Abstract

The use of notoginsenoside R1 in the preparation of the medicament for treating hepatic injuries, especially the hepatic injuries caused by ischemia-reperfusion is provided.

Description

三七皂苷 R1在制备治疗肝损伤的药物中的应用  Application of notoginsenoside R1 in the preparation of drugs for treating liver injury
技术领域 Technical field
本发明涉及中药三七的有效成分三七皂苷 R1的新的药物用途， 特 别涉及三七皂苷 R1用于治疗和 /或预防肝损伤。 背景技术  The present invention relates to a novel pharmaceutical use of Panax notoginseng saponin R1, an active ingredient of traditional Chinese medicine Sanqi, and particularly relates to the use of notoginsenoside R1 for the treatment and/or prevention of liver damage. Background technique
中药三七 ( Panax notoginseng (Burk. ) F. H. Chen, PN) 为五力口科 植物三七 （A liaceae, 三七） 的干燥根， 在中国、 韩国、 日本及其它 亚洲国家广泛用于治疗微循环障碍相关疾病， 如心血管疾病、脑血管疾 病和肝功能障碍等。 三七含有 30多种不同类型的皂苷类化合物， 其中 人参皂苷 Rgl ( Rgl ) , 人参皂苷 Rbl (Rbl ) 和三七皂苷 Rl (R1 ) 较 为常见。 其中 R1的结构如下：  Panax notoginseng (Burk. ) FH Chen, PN is a dry root of A liaceae (Sanqi), widely used in China, Korea, Japan and other Asian countries for the treatment of microcirculation. Disorder-related diseases such as cardiovascular disease, cerebrovascular disease, and liver dysfunction. Sanqi contains more than 30 different types of saponin compounds, of which ginsenoside Rgl (Rgl), ginsenoside Rbl (Rbl) and notoginsenoside Rl (R1) are more common. The structure of R1 is as follows:
Figure imgf000003_0001
肝损伤是一种常见病， 肝炎病毒、 肝硬化、 滥用药物、 嗜酒、 化学 毒物接触是导致肝损伤的主要原因。 肝脏是人体的 "生命塔" ， 人体的各种代谢、解毒和免疫功能都靠 肝脏承担。 对肝细胞、 肝内血流的任何不利影响都会损伤肝组织。 常见 的肝损伤有病毒感染或肝硬化引起的肝损伤、药物肝损伤、酒精性肝损 伤、 化学性肝损伤、 缺血 -再灌注引起的肝损伤等。 病毒感染、肝硬化引起的肝损伤， 主要通过人体的免疫应答造成肝 细胞损伤， 感染人体后可刺激机体产生一系列抗体和细胞免疫反应， 如 果免疫反应不足以清除病毒， 病毒可持续存在， 造成肝细胞损伤， 进而 引起肝硬化， 使损伤加重。 药物肝损伤是因为药物代谢产物形成氧自由基使脂质过氧化、药物 代谢产生亲电子产物、 超氧化离子， 导致肝细胞损伤。 酒精性肝损伤影响肝脏供血状况，增加肝脏负担，导致酒精性肝炎、 脂肪肝， 直至肝硬化。 化学性肝损伤因化学毒物破坏肝细胞， 肝功呈功能异常表现。 缺血 -再灌注损伤 ( ischemia-reperfusion injury, I/R) 是指缺血组织 或器官重获血流灌注或供氧后对组织和器官所产生的损伤作用，肝脏缺 血 -再灌注损伤 （hepatic ischemia reperfusion injury, HIRI ) 可使肝代谢 解毒能力降低、 微循环阻力升高， 严重者可导致肝功能衰竭， 在肝移植 中则表现为移植物原发性无功能 （primary nonfunction, PNF ) , PNF是 肝移植患者死亡的主要原因之一。 缺血-再灌注 （I/R) 在很多情况都会发生， 例如发生创伤、 血管收 缩后返流、 血管成形术（PTCA)、 溶栓治疗、 器官移植和复苏型低血容 量性休克。 缺血 -再灌注可以导致微循环受到多种损伤， 常伴有上皮细 胞损伤、 粒性白细胞粘附增强、 大分子外流， 氧自由基产生和肥大细胞 脱颗粒作用， 根据肝脏缺血-再灌注的损伤机制可以采取诸多的预防和 治疗方法， 被动防御可在术中、术前和术后用药以及在保存液中加入药 物或活性成分， 其中抗氧自由基药物如别嘌呤醇、 谷胱甘肽、 Ca2+拮抗 剂等已应用于临床，国内学者在保存液中合理加入中药制剂也获得不错 的效果。 本发明者经过实验发现三七皂苷 R1 可以改善肠缺血 -再灌注诱导 的肝脏微循环障碍， 从而治疗和 /或预防肝损伤， 为此本发明提供一种 三七皂苷 R1新的治疗用途。 发明内容
Figure imgf000003_0001
Liver injury is a common disease, and hepatitis virus, liver cirrhosis, drug abuse, alcohol abuse, and chemical poison contact are the main causes of liver damage. The liver is the "life tower" of the human body. The various metabolic, detoxification and immune functions of the human body are borne by the liver. Any adverse effects on hepatocytes and intrahepatic blood flow can damage liver tissue. Common liver injuries include liver infection caused by viral infection or cirrhosis, drug liver injury, alcoholic liver injury, chemical liver injury, liver damage caused by ischemia-reperfusion, and the like. Liver damage caused by viral infection and cirrhosis mainly causes liver cell damage through the immune response of the human body. After infecting the human body, it can stimulate the body to produce a series of antibodies and cellular immune responses. If the immune response is insufficient to eliminate the virus, the virus can persist, resulting in Hepatocyte damage, which in turn causes cirrhosis, exacerbates the damage. Drug liver injury is caused by the formation of oxygen free radicals by drug metabolites, lipid peroxidation, drugs Metabolism produces electrophilic products, superoxide ions, leading to liver cell damage. Alcoholic liver injury affects the blood supply to the liver and increases the burden on the liver, leading to alcoholic hepatitis, fatty liver, and cirrhosis. Chemical liver damage is caused by chemical poisons destroying liver cells, and liver function is abnormal. Ischemia-reperfusion injury (I/R) refers to the damage of tissues and organs caused by reperfusion of blood perfusion or oxygen supply in ischemic tissues or organs. Hepatic ischemia-reperfusion injury ( Hepatic ischemia reperfusion injury (HIRI) can reduce liver metabolic detoxification ability and increase microcirculation resistance. In severe cases, liver failure can be caused. In liver transplantation, primary nonfunction (PNF) is observed. PNF is one of the leading causes of death in liver transplant patients. Ischemia-reperfusion (I/R) can occur in many situations, such as trauma, vasoconstriction, PTCA, thrombolytic therapy, organ transplantation, and resuscitated hypovolemic shock. Ischemia-reperfusion can cause microcirculation to be damaged, often accompanied by epithelial cell damage, enhanced granulocyte adhesion, macromolecular efflux, oxygen free radical production and mast cell degranulation, according to hepatic ischemia-reperfusion The mechanism of injury can take many preventive and therapeutic methods. Passive defense can be used during surgery, preoperative and postoperative, as well as adding drugs or active ingredients to the preservation solution. Antioxidant free radical drugs such as allopurinol and glutathione Peptides, Ca 2+ antagonists, etc. have been used in clinical practice, and domestic scholars have also achieved good results by reasonably adding Chinese medicine preparations to the preservation solution. The present inventors have found through experiments that notoginsenoside R1 can improve intestinal microcirculation disturbance induced by intestinal ischemia-reperfusion, thereby treating and/or preventing liver damage, and the present invention provides a novel therapeutic use of notoginsenoside R1. Summary of the invention
本发明提供一种三七皂苷 R1新的治疗用途。 所述新的治疗用途是 用三七皂苷 R1 治疗和 /或预防肝损伤， 特别是缺血 -再灌注引起的肝损 伤。 为此， 本发明提供一种药物新用途， 即三七皂苷 R1或其药物组合 物在制备治疗和 /或预防肝损伤的药物中的应用。 其中所述三七皂苷 Rl是从中药三七中提取获得的， 纯度 >50%， 优 选纯度 >90%， 更优选纯度 >98%。 本发明所述的药物组合物以三七皂苷 R1作为药物活性成分， 其在 制剂中所占重量百分比可以是 0.1〜99.9%， 其余为药学上可接受的载 体。 本发明中所述三七皂苷 R1可以药物组合物的形式给予患者， 所述 药物组合物为任何可药用的制剂形式，优选为注射剂，所述的注射剂可 以为溶液剂或冻干粉针剂。 根据本发明的应用，所述肝损伤选自病毒感染或肝硬化引起的肝损 伤、 药物肝损伤、 酒精性肝损伤、 化学性肝损伤、 缺血-再灌注引起的 肝损伤。 根据本发明的应用， 所述肝损伤是缺血-再灌注引起的肝损伤。 根据本发明的应用， 所述治疗和 /或预防肝损伤是通过改善微循环 障碍实现的，所述的微循环障碍包括微静脉直径降低、红细胞流速降低、 灌注的窦状隙数量减少以及白细胞的滚动和粘附增加。 根据本发明的应用， 所述治疗和 /或预防肝损伤是通过降低肝脏血 管 E-选择蛋白和细胞间粘附分子 -1的表达实现的。 根据本发明的应用， 所述治疗和 /或预防肝损伤是通过降低嗜中性 粒细胞表面 CD18和 CDl lb的表达实现的。 附图说明 The present invention provides a novel therapeutic use of notoginsenoside R1. The new therapeutic use is to treat and/or prevent liver damage, particularly ischemia-reperfusion-induced liver damage, with notoginsenoside R1. To this end, the present invention provides a novel use of a drug, that is, the use of notoginsenoside R1 or a pharmaceutical composition thereof for the preparation of a medicament for treating and/or preventing liver damage. Wherein the notoginsenoside R1 is obtained by extracting from the traditional Chinese medicine Panax notoginseng, the purity is >50%, preferably the purity is >90%, more preferably the purity is >98%. The pharmaceutical composition of the present invention has notoginsenoside R1 as a pharmaceutically active ingredient, and the weight percentage in the preparation may be 0.1 to 99.9%, and the balance is a pharmaceutically acceptable carrier. The notoginsenoside R1 in the present invention may be administered to a patient in the form of a pharmaceutical composition which is in the form of any pharmaceutically acceptable preparation, preferably an injection, which may be a solution or a lyophilized powder injection. According to the use of the present invention, the liver injury is selected from liver damage caused by viral infection or cirrhosis, drug liver damage, alcoholic liver damage, chemical liver damage, liver damage caused by ischemia-reperfusion. According to the use of the invention, the liver injury is liver damage caused by ischemia-reperfusion. According to the use of the present invention, the treatment and/or prevention of liver damage is achieved by ameliorating microcirculatory disorders including a decrease in venule diameter, a decrease in red blood cell flow rate, a decrease in the number of perfused sinusoids, and leukocyte Rolling and adhesion increase. According to the use of the present invention, the treatment and/or prevention of liver damage is achieved by reducing the expression of hepatic vascular E-selectin and intercellular adhesion molecule-1. According to the use of the present invention, the treatment and/or prevention of liver damage is achieved by reducing the expression of CD18 and CD1 lb on the surface of neutrophils. DRAWINGS
图 1三七皂苷 （R1 ) 的化学结构。 图 2 R1处理对肠系膜上动脉缺血 -再灌注小鼠肝脏微静脉直径的千 预作用， 检测时间为缺血 （基础值） 前第 0分钟， 再灌注后第 15分钟 和 30分钟。 A: 终末门微静脉直径。 B: 中央静脉直径。 横坐标代表在 任意时间点直径数值与基础值的比值。 结果为 6只实验动物的平均 ±SE 值。 与对照组比较， aP<0.05; 与缺血-再灌注 （I/R) 组比较， eP<0.05。 图 3 R1对肠系膜上动脉缺血-再灌注小鼠肝脏微静脉红细胞流速的 干预作用， 检测时间点为缺血 （基础值） 前第 0分钟， 再灌注后第 15 和 30分钟。 A: 终末门微静脉中的红细胞流速。 B : 中央静脉中的红细 胞流速。横坐标代表任意时间点红细胞流速值与基础值的比值。结果为 6只实验动物的平均 ±SE值。 与对照组比较， aP<0.05 ; 与缺血-再灌注 ( I/R) 组比较， CP<0.05。 图 4 R1对肠系膜上动脉缺血-再灌注小鼠灌注肝脏窦状隙的干预作 用， 检测时间点为缺血 （基础值） 前第 0分钟， 再灌注后第 15分钟和 30分钟。 A: 终末门微静脉区域中灌注的窦状隙。 B : 中央静脉区域中 灌注的窦状隙。 横坐标代表任意时间点灌注的窦状隙与基础值的比值。 结果为 6只实验动物的平均 ±SE值。 与对照组比较， aP<0.05 ; 与缺血- 再灌注组 （I/R) 比较， eP<0.05。 图 5 R1对肠系膜上动脉缺血-再灌注小鼠肝脏微静脉白细胞滚动的 干预作用， 检测时间点为缺血 （基础值） 前第 0分钟， 再灌注后第 15 分钟和 30分钟。 A: 终末门微静脉中的滚动粒性白细胞数量。 B: 中央 静脉中的滚动粒性白细胞数量。 横坐标代表每 200μιη2视野中的滚动粒 性白细胞数量。 结果为 6 只实验动物的平均 ±SE 值。 与对照组比较， aP<0.05 ; 与缺血-再灌注组 （I/R) 比较， eP<0.05。 图 6 肠系膜上动脉缺血 -再灌注后第 30分钟， 小鼠肝脏微静脉罗 丹明-标记粒性白细胞的典型图像。 观察中央静脉和肝脏窦状隙粒性白 细胞的粘附数量。 CV: 中央静脉。 S: 窦状隙。 箭头表示粘附的粒性白 细胞。 放大倍数： 20χ。 图 7 R1对肠系膜上动脉缺血-再灌注小鼠肝脏微静脉中白细胞粘附 的干预作用， 检测时间点为缺血 （基础值） 前第 0 分钟， 再灌注后第 15分钟和 30分钟。 Α: 终末门微静脉中粘附的粒性白细胞数量。 Β: 中央静脉中粘附的粒性白细胞数量。 横坐标代表每 200μιη2视野中粘附 的粒性白细胞数量。结果为 6只实验动物的平均 ±SE值。与对照组比较， aP<0.05 ; 与缺血-再灌注 ( I/R) 组比较， cP<0.05 o 图 8 R1对肠系膜上动脉缺血-再灌注小鼠肝脏窦状隙中白细胞粘附 的干预作用， 检测时间点为缺血 （基础值） 前第 0 分钟， 再灌注后第 15分钟和 30分钟。 A: 终末门微静脉区域窦状隙中粘附的粒性白细胞 数量。 B: 中央静脉区域窦状隙中粘附的粒性白细胞数量。 横坐标代表 每 200μιη2视野中粘附的粒性白细胞数量。 结果为 6只实验动物的平均 士 SE值。与对照组比较， aP<0.05 ;与缺血-再灌注（I/R)组比较， cP<0.05 o 图 9 R1 对肠系膜上动脉缺血-再灌注小鼠肝脏血管 E-选择蛋白和 细胞间粘附分子 -1表达的干预作用。 再灌注后第 30分钟， 对肝脏组织 进行免疫荧光染色。结果为 6只实验动物的平均值 ±SE值。 与对照组比 较， aP<0.05 ; 与缺血-再灌注 （I/R) 组比较， eP<0.05。 图 10 R1对肠系膜上动脉缺血-再灌注小鼠嗜中性粒细胞表面 CD18 和 CDl lb表达的干预作用。 再灌注后第 30分钟， 分离嗜中性粒细胞， 进行流式细胞计量术检査。结果为 6只实验动物的平均 ±SE值。与对照 组比较， aP<0.05 ; 与缺血-再灌注 （I/R) 组比较， eP<0.05。 图 11 R1 对肠系膜上动脉缺血-再灌注小鼠血清中乳酸脱氢酶 ( LDH)、 丙氨酸转氨酶 （ALT) 和天冬氨酸转氨酶 （AST) 浓度的干 预作用。 再灌注后第 30分钟和 60分钟， 分离血清进行酶测定。 结果为 6只实验动物的平均 ±SE值。 与对照组比较， aP<0.05 ; 与缺血-再灌注 ( I/R) 组比较， eP<0.05。 图 12 R1对肠系膜上动脉缺血-再灌注小鼠血清中的肿瘤坏死因子 -α ( TNF-α) 、 白介素 -6 ( IL-6 ) 和单核细胞趋化蛋白 -1 ( MCP-1 ) 浓 度的干预作用。 再灌注后第 30分钟， 分离血清， 进行流式细胞计量术 检查。 结果为 6只实验动物的平均值 ±SE值。 与对照组比较， aP<0.05 ; 与缺血-再灌注 （I/R) 组比较， eP<0.05。 具体实施方式 Figure 1. Chemical structure of notoginsenoside (R1). Figure 2 R1 treatment of the diameter of the hepatic venules in the superior mesenteric artery ischemia-reperfusion mice, the detection time is 0 minutes before ischemia (basal value), 15 minutes and 30 minutes after reperfusion. A: The diameter of the terminal venule. B: Central vein diameter. The abscissa represents the ratio of the diameter value to the base value at any point in time. The results are the mean ± SE values of 6 experimental animals. Compared with the control group, a P<0.05; compared with the ischemia-reperfusion (I/R) group, e P<0.05. Figure 3 R1 to the flow velocity of hepatic venule erythrocytes in the mesenteric artery ischemia-reperfusion mice Intervention, the time point of detection was 0 minutes before ischemia (basal value) and 15 and 30 minutes after reperfusion. A: The flow rate of red blood cells in the terminal venules. B : Red blood cell flow rate in the central vein. The abscissa represents the ratio of the red blood cell flow rate value to the base value at any time point. The results are the mean ± SE values of 6 experimental animals. Compared with the control group, a P<0.05 ; compared with the ischemia-reperfusion (I/R) group, C P<0.05. Figure 4 Intervention of R1 on perfusion of the sinusoids in the superior mesenteric artery ischemia-reperfusion mice at the 0th minute before ischemia (basal value) and 15 minutes and 30 minutes after reperfusion. A: sinusoidal perfusion in the terminal venule region. B: sinusoidal perfusion in the central venous area. The abscissa represents the ratio of the sinusoids perfused at any point in time to the baseline value. The results are the mean ± SE values of 6 experimental animals. Compared with the control group, a P<0.05; compared with the ischemia-reperfusion group (I/R), e P<0.05. Figure 5 Intervention of R1 on hepatic venous leukocyte rolling in the ischemic-reperfused mesenteric artery. The time of detection was 0 minutes before ischemia (basal value) and 15 minutes and 30 minutes after reperfusion. A: The number of rolling granulocytes in the terminal venules. B: Number of rolling granulocytes in the central vein. The abscissa represents the number of rolling granulocytes in each 200 μm 2 field of view. The results are the mean ± SE values of 6 experimental animals. Compared with the control group, a P<0.05; compared with the ischemia-reperfusion group (I/R), e P<0.05. Figure 6. Typical image of the hepatic venule rhodamine-labeled granulocytes in the 30th minute after ischemia-reperfusion of the superior mesenteric artery. The amount of adhesion of central venous and hepatic sinusoidal leukocytes was observed. CV: Central vein. S: sinusoidal gap. Arrows indicate adherent granulocytes. Magnification: 20 inches. Figure 7 Intervention of R1 on leukocyte adhesion in the hepatic venule of mice with superior mesenteric ischemia-reperfusion. The time of detection was 0 minutes before ischemia (basal value) and 15 minutes and 30 minutes after reperfusion. Α: The number of granulocytes attached to the terminal venules. Β: The number of granulocytes attached to the central vein. The abscissa represents the number of granulocytes attached per 200 μιη 2 field of view. The results are the mean ± SE values of 6 experimental animals. Compared with the control group, a P<0.05 ; compared with the ischemia-reperfusion (I/R) group, c P<0.05 o Figure 8 R1 on leukocyte adhesion in the sinusoids of the superior mesenteric artery ischemia-reperfusion mice With the intervention, the time point of detection is ischemia (basic value) before 0 minutes, after reperfusion 15 minutes and 30 minutes. A: The number of granulocytes attached to the sinusoids of the terminal venules. B: Number of granulocytes attached to the sinusoid in the central venous region. The abscissa represents the number of granulocytes attached per 200 μιη 2 field of view. The result was the mean SE value of 6 experimental animals. Compared with the control group, a P<0.05; compared with the ischemia-reperfusion (I/R) group, c P<0.05 o Figure 9 R1 on the hepatic vascular E-selectin and the mesenteric artery ischemia-reperfusion in mice Intervention of expression of intercellular adhesion molecule-1. At 30 minutes after reperfusion, liver tissues were immunofluorescently stained. The results are the mean ± SE values of 6 experimental animals. Compared with the control group, a P<0.05; compared with the ischemia-reperfusion (I/R) group, e P<0.05. Figure 10 Intervention of R1 on the expression of CD18 and CD1 lb on neutrophil surface in mice with superior mesenteric ischemia-reperfusion. At 30 minutes after reperfusion, neutrophils were isolated and subjected to flow cytometry. The results are the mean ± SE values of 6 experimental animals. Compared with the control group, a P<0.05; compared with the ischemia-reperfusion (I/R) group, e P<0.05. Figure 11 Intervention of R1 on serum lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) concentrations in the superior mesenteric artery ischemia-reperfusion mice. At 30 minutes and 60 minutes after reperfusion, serum was separated for enzyme assay. The results are the mean ± SE values of 6 experimental animals. Compared with the control group, a P<0.05; compared with the ischemia-reperfusion (I/R) group, e P<0.05. Figure 12 R1 in the serum of the superior mesenteric artery ischemia-reperfusion mice, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) The concentration of intervention. At 30 minutes after reperfusion, serum was separated and subjected to flow cytometry. The results are the mean ± SE values of 6 experimental animals. Compared with the control group, a P<0.05; compared with the ischemia-reperfusion (I/R) group, e P<0.05. detailed description
本发明所述三七皂苷 Rl， 是中药三七中的一种成分， 对其没有特 别的限制， 只要符合药用标准即可。 所述三七皂苷 R1可以从市场上买 到，例如南京青泽医药科技开发有限公司和上海康九化工有限公司生产 的产品； 也可以根据现有技术进行制备， 例如中国专利 01136697.4从 三七生粉提取分离三七皂苷 Rl、 人参皂甙 Re、 三七素的方法。 所述三 七皂苷 R1优选纯度>50%， 更优选纯度 >90%， 最优选纯度 >98%。 本发明所述的药物， 是用上述三七皂苷 R1作为药物活性成分制备 的药物组合物。 本发明的药物组合物， 根据需要可以含有药学上可接受的载体， 其 中三七皂苷 R1作为药物活性成分， 其在制剂中所占重量百分比可以是 0.1〜99.9%， 其余为药学上可接受的载体。 本发明的药物组合物， 以单 位剂量形式存在， 所述单位剂量形式是指制剂的单位， 如每片片剂、 每 粒胶囊、 每瓶口服液、 每袋颗粒剂、 每支注射剂等。 本发明的药物组合物可以是任何可药用的剂型， 包括片剂（例如糖 衣片剂、 薄膜衣片剂和肠溶衣片剂）、 胶囊剂 （例如硬胶囊剂、 软胶囊 剂）、 口服液、 口含剂、 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、 膏剂 （例如软膏剂、 硬膏剂）、 霜剂、 喷雾剂、 滴剂、 贴剂等。 本发明的药物组合物，其口服给药的药物组合物可含有常用的赋形 剂， 诸如粘合剂、 填充剂、 稀释剂、 压片剂、 润滑剂、 崩解剂、 着色剂、 调味剂和湿润剂， 必要时可对片剂进行包衣。 适宜的填充剂包括纤维素、 甘露糖醇、 乳糖和其它类似的填充剂。 适宜的崩解剂包括淀粉、聚乙烯吡咯垸酮和淀粉衍生物， 例如羟基乙酸 淀粉钠。 适宜的润滑剂包括， 例如硬脂酸镁。 适宜的药学上可接受的湿 润剂包括十二垸基硫酸钠。 可通过混合、 填充、 压片等常规方法制备固体口服组合物。迸行反 复混合可使活性成分分布在整个含有大量填充剂的组合物中。 口服液体制剂的形式例如可以是水性或油性悬浮剂、溶液剂、乳剂、 糖浆剂或酏剂，或者可以是一种在使用前可用水或其它适宜的载体复配 的干燥产品。 这种液体制剂可含有常规的添加剂， 诸如悬浮剂， 例如山 梨醇、 糖浆、 甲基纤维素、 明胶、 羟乙基纤维素、 羧甲基纤维素、 硬脂 酸铝凝胶或氢化食用脂肪； 乳化剂， 例如卵磷脂、 脱水山梨醇一油酸酯 或阿拉伯胶； 非水性载体（可包括食用油）， 例如杏仁油、 分馏椰子油、 诸如甘油酯的油性酯、 丙二醇或乙醇； 防腐剂， 例如对羟基苯甲酯或对 羟基苯甲酸丙酯或山梨酸， 并且如果需要， 可含有常规的香味剂或着色 剂。 对于注射剂，制备的液体单位剂型含有本发明的活性成分和无菌载 体。 根据载体和浓度， 可以将此化合物悬浮或者溶解。 溶液的制备通常 是通过将活性成分溶解在一种载体中，在将其装入一种适宜的小瓶或安 瓿前过滤消毒， 然后密封。 也可将辅料例如一种局部麻醉剂、 防腐剂和 缓冲剂溶解在这种载体中。为了提高其稳定性， 可在装入小瓶以后将这 种组合物冰冻， 并在真空下将水除去。 本发明的药物组合物，在制备成药剂时可选择性的加入适宜的药学 上可接受的载体， 所述药学上可接受的载体选自： 甘露醇、 山梨醇、 焦 亚硫酸钠、 亚硫酸氢钠、 硫代硫酸钠、 盐酸半胱氨酸、 巯基乙酸、 蛋氨 酸、 维生素 C、 EDTA二钠、 EDTA钙钠， 一价碱金属的碳酸盐、 醋酸 盐、 磷酸盐或其水溶液、 盐酸、 醋酸、 硫酸、 磷酸、 氨基酸、 氯化钠、 氯化钾、 乳酸钠、 木糖醇、 麦芽糖、 葡萄糖、 果糖、 右旋糖苷、 甘氨酸、 淀粉、蔗糖、 乳糖、甘露糖醇、 硅衍生物、 纤维素及其衍生物、 藻酸盐、 明胶、 聚乙烯吡咯垸酮、 甘油、 吐温 80、 琼脂、 碳酸钙、 碳酸氢钙、 表面活性剂、 聚乙二醇、 环糊精、 β-环糊精、 磷脂类材料、 高岭土、 滑 石粉、 硬脂酸钙、 硬脂酸镁等。 本发明优选注射剂， 所述注射剂是溶液剂或冻干粉针剂。 本发明的药物组合物在使用时根据病人的情况确定用法用量。 下面以缺血-再灌注肝损伤为例， 说明本发明所述的治疗用途， 但 本发明不限于该实施方式。 材料和方法 三七皂苷 R1和实验用药 The notoginsenoside R1 of the present invention is a component of the traditional Chinese medicine Panax notoginseng, and is not particularly limited as long as it meets the medicinal standard. The notoginsenoside R1 can be purchased from the market, such as products produced by Nanjing Qingze Pharmaceutical Technology Development Co., Ltd. and Shanghai Kangjiu Chemical Co., Ltd.; it can also be prepared according to the prior art, for example, Chinese patent 01136697.4 from Sanqisheng A method for extracting and separating notoginsenoside Rl, ginsenoside Re, and notoginseng by powder extraction. The notoginsenoside R1 preferably has a purity of >50%, more preferably a purity of >90%, and most preferably a purity of >98%. The medicament according to the present invention is a pharmaceutical composition prepared by using the above-mentioned notoginsenoside R1 as a pharmaceutically active ingredient. The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier as needed, wherein notoginsenoside R1 is a pharmaceutically active ingredient, and the weight percentage in the preparation may be 0.1 to 99.9%, and the others are pharmaceutically acceptable. Carrier. The pharmaceutical composition of the present invention is present in unit dosage form, which means a unit of the preparation, such as each tablet, each capsule, each vial of oral solution, granules per bag, each injection, and the like. The pharmaceutical composition of the present invention may be in any pharmaceutically acceptable dosage form, including tablets (for example, sugar-coated tablets, film-coated tablets and enteric coated tablets), capsules (for example, hard capsules, soft capsules), orally. Liquid, buccal, granules, granules, pills, powders, ointments, dandruffs, suspensions, powders, solutions, injections, suppositories, ointments (eg ointments, plasters), creams, sprays, drops Agents, patches, etc. The pharmaceutical composition of the present invention, which is orally administered, may contain a usual excipient such as a binder, a filler, a diluent, a tablet, a lubricant, a disintegrant, a coloring agent, a flavoring agent. And humectants, if necessary, the tablets can be coated. Suitable fillers include cellulose, mannitol, lactose and other similar fillers. Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate. Suitable lubricants include, for example, magnesium stearate. Suitable pharmaceutically acceptable wetting agents include sodium decyl sulfate. The solid oral composition can be prepared by a conventional method such as mixing, filling, tableting or the like. The repeated mixing of the active ingredients allows the active ingredient to be distributed throughout the composition containing a large amount of filler. The oral liquid preparation may be in the form of, for example, an aqueous or oily suspension, solution, emulsion, syrup or elixir, or may be a dry product which may be formulated with water or other suitable carrier before use. Such liquid preparations may contain conventional additives such as suspending agents such as sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fat; Emulsifiers such as lecithin, sorbitan monooleate or gum arabic; non-aqueous carriers (which may include edible oils), such as almond oil, fractionated coconut oil, oily esters such as glycerides, propylene glycol or ethanol; preservatives, Such as p-hydroxybenzyl ester or pair Propyl hydroxybenzoate or sorbic acid, and if desired, may contain conventional flavoring or coloring agents. For injection, the liquid unit dosage form prepared contains the active ingredient of the invention and a sterile vehicle. This compound can be suspended or dissolved depending on the carrier and concentration. The solution is usually prepared by dissolving the active ingredient in a carrier, sterilizing it by filtration prior to filling it into a suitable vial or ampoule, and then sealing. Excipients such as a local anesthetic, preservative and buffer may also be dissolved in such a carrier. To increase its stability, the composition can be frozen after filling the vial and the water removed under vacuum. The pharmaceutical composition of the present invention may optionally be added to a suitable pharmaceutically acceptable carrier when prepared as a medicament, the pharmaceutically acceptable carrier being selected from the group consisting of: mannitol, sorbitol, sodium metabisulfite, sodium hydrogen sulfite , sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, calcium EDTA, monovalent alkali metal carbonate, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid , sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and Derivatives, alginate, gelatin, polyvinylpyrrolidone, glycerin, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactant, polyethylene glycol, cyclodextrin, β-cyclodextrin, Phospholipid materials, kaolin, talc, calcium stearate, magnesium stearate, and the like. The injection is preferably a solution or a lyophilized powder injection. The pharmaceutical composition of the present invention determines the usage amount according to the condition of the patient at the time of use. Hereinafter, the therapeutic use of the present invention will be described by taking the ischemia-reperfusion liver injury as an example, but the present invention is not limited to the embodiment. Materials and Methods Notoginsenoside R1 and experimental drugs
三七皂苷 R1 (简称 Rl， 纯度 >98%) 由天津天士力集团 （天津， 中国） 提供。 试验中使用的其它试药包括： 罗丹明 6G (纯度 >99.0%， 批号： 2350994， Fluka公司， 瑞士） ； FITC-大鼠抗 -小鼠 CD18单克隆 抗体 （批号： 553293 , BD生物科学系统， 美国） ； FITC-大鼠抗 -小鼠 CDl lb单克隆抗体 （批号： 557396, BD生物科学系统， 美国） ； 山羊 抗小鼠 E-选择蛋白 （M-20 ) 多克隆抗体 （ sc-6939， Santa Cruz Biotechnology, Inc. , 美国） ； 山羊抗小鼠细胞间粘附分子 -1多克隆抗 体 （sc-1511， Santa Cruz Biotechnology, Inc., 美国） ； 罗丹明结合家 兔抗 -山羊 lgG-R (批号： B1006, Santa Cruz Biotechnology, Inc. ,美国）； Hoechst 33342 (批号： 6538， Santa Cruz Biotechnology, Inc. , 美国） ； 小鼠单核细胞趋化蛋白 -l ( MCP-l )自由组合装（flex set) (批号：558342， BD生物科学系统， 美国） ； 小鼠肿瘤坏死因子 -a ( TNF-a) 自由组合 装 （批号： 558299， BD生物科学系统， 美国） ； 小鼠白介素 -6 ( IL-6) 自由组合装 （批号： 558301 , BD生物科学系统， 美国） 。 实验动物 Notoginsenoside R1 (referred to as Rl, purity >98%) was provided by Tianjin Tianshili Group (Tianjin, China). Other reagents used in the trial included: Rhodamine 6G (purity > 99.0%, lot number: 2350994, Fluka, Switzerland); FITC-rat anti-mouse CD18 monoclonal antibody (batch number: 553293, BD Bioscience System, US); FITC-rat anti-mouse CDl lb monoclonal antibody (batch number: 557396, BD Bioscience Systems, USA); goat anti-mouse E-selectin (M-20) polyclonal antibody ( sc-6939, Santa Cruz Biotechnology, Inc., USA) ; Anti-mouse intercellular adhesion molecule-1 polyclonal antibody (sc-1511, Santa Cruz Biotechnology, Inc., USA); Rhodamine combined with rabbit anti-goat lgG-R (batch number: B1006, Santa Cruz Biotechnology, Inc. , USA); Hoechst 33342 (batch number: 6538, Santa Cruz Biotechnology, Inc., USA); mouse monocyte chemotactic protein-l (MCP-l) freely set (flex set) (batch number: 558342, BD bio Scientific System, USA); Mouse Tumor Necrosis Factor-a (TNF-a) Free Combination Pack (Batch No.: 558299, BD Bioscience System, USA); Mouse Interleukin-6 (IL-6) Free Combination Pack (Batch Number: 558301, BD Bioscience Systems, USA). Experimental animal
C57/BL小鼠购自北京大学医学部实验动物中心， 体重 22〜26g， 年龄 8〜10周， 实验动物证书号 SCXK 2002-2001。 实验动物饲养在温 度 24±1 °C， 湿度 50±5%， 12小时光暗循环周期环境中， 试验前禁食 12 小时， 可以自由饮水。 遵循北京大学实验动物管理委员会的指南， 处理 所有实验动物。 活体显微镜检査  C57/BL mice were purchased from the Experimental Animal Center of Peking University Medical School, weighing 22~26g, age 8~10 weeks, laboratory animal certificate number SCXK 2002-2001. The experimental animals were housed in a temperature of 24 ± 1 ° C, humidity of 50 ± 5%, 12 hours of light and dark cycle environment, fasting for 12 hours before the test, free to drink water. All experimental animals were processed following the guidelines of the Peking University Laboratory Animal Management Committee. In vivo microscopy
用 20%乌拉坦 （10ml/kg体重） 将 C57/BL小鼠麻醉。 将套管插入 左颈静脉， 用直径为 0.96mm的 PE管灌注试验药物。 剖腹后立即将小 鼠以侧卧位放置在观察台上。 将肝脏放置在可调节的 Plexiglas显微镜 载物台上的温控 （37°C ) 观察盒中， 小心处理， 降低呼吸运动的影响。 使用倒置生物显微镜 （DM-IRB, Leica, 德国） ， 通过 3CCD彩色摄像 机 （JK-TU53H, 3CCD相机， 日本东芝公司， 日本） 观察肝脏左侧叶。 选择包括终末门微静脉和中央静脉而不含粘附粒性白细胞的区域 400μηιχ320μιη进行观察。 经 20χ物镜观测肝脏表面微循环图像， 使用 DVD存储器 （DVR-R25 , 万利达， 中国） 将肝脏微循环动力学保存在 DVD光盘中。 在整个观测过程中， 使用 37°C生理盐水滴至肝脏表面以 保持肝脏的水分， 肝脏表面的观察区域周围覆盖生理盐水浸湿的棉纱 布。 缺血-再灌注操作步骤 C57/BL mice were anesthetized with 20% urethane (10 ml/kg body weight). The cannula was inserted into the left jugular vein and the test drug was perfused with a PE tube having a diameter of 0.96 mm. Immediately after laparotomy, the mice were placed in the lateral position on the observation table. The liver was placed in a temperature-controlled (37 ° C) observation box on an adjustable Plexiglas microscope stage and carefully treated to reduce the effects of respiratory motion. The left lobe of the liver was observed by an inverted biomicroscope (DM-IRB, Leica, Germany) through a 3CCD color camera (JK-TU53H, 3CCD camera, Toshiba Corporation, Japan). The observation included a region including terminal venules and central veins without adherent granulocytes, 400 μηιχ 320 μιη. Liver microcirculation images were observed through a 20-inch objective lens, and liver microcirculation dynamics were saved on a DVD disc using a DVD memory (DVR-R25, Malata, China). Throughout the observation process, physiological saline at 37 ° C was used to drip the liver surface to maintain the moisture of the liver, and the observation area around the liver surface was covered with physiologically wet cotton gauze. Ischemia-reperfusion procedure
在结扎肠系膜上动脉 （SMA) 前 10分钟， 观察肝脏表面， 确保所 有测量参数处于稳定状态。 用由聚乙烯管 （直径 1.00mm) 结扎肠系膜 上动脉 15分钟。 缺血后， 轻轻释放结扎， 进行再灌注。在局部缺血（基 线）前， 立即测量微静脉直径、 红细胞流速、 窦状隙再灌注以及白细胞 滚动和粘附， 再灌注后半小时内， 每 15分钟观察一次上述指标。 试验方案  Ten minutes before ligation of the superior mesenteric artery (SMA), the liver surface was observed to ensure that all measurements were stable. The superior mesenteric artery was ligated with a polyethylene tube (1.00 mm in diameter) for 15 minutes. After ischemia, the ligation is gently released and reperfusion is performed. Immediately before ischemia (baseline), venule diameter, erythrocyte flow rate, sinusoidal reperfusion, and leukocyte rolling and adhesion were measured, and the above indicators were observed every 15 minutes within half an hour after reperfusion. Test plan
缺血-再灌注前 10 分钟至观察结束， 缺血-再灌注组小鼠经左颈静 脉连续灌注生理盐水溶液。 缺血-再灌注前 10分钟至观察结束， 对 R1 +缺血-再灌注 （I/R) 组的小鼠连续灌注 Rl ( 10mg/kg/h) 。 按照与缺 血-再灌注组相同的方式处理模拟手术对照组的小鼠， 但不结扎肠系膜 上动脉。 每组分别有 6只小鼠 （3只雄性、 3只雌性） 。 微循环参数测量  From 10 minutes before ischemia-reperfusion to the end of observation, the mice in the ischemia-reperfusion group were continuously perfused with physiological saline solution through the left neck vein. Mice in the R1 + ischemia-reperfusion (I/R) group were continuously perfused with R1 (10 mg/kg/h) 10 minutes before ischemia-reperfusion to the end of observation. Mice in the mock surgery control group were treated in the same manner as the blood-reperfusion group, but the superior mesenteric artery was not ligated. There were 6 mice in each group (3 males and 3 females). Microcirculation parameter measurement
使用图像 -Pro Plus 5.0软件重新播放 DVD图像， 测量终末门微静 脉直径和中央静脉直径。测量结果以再灌注后第 15分钟或第 30分钟的 测量数值与基础值的比值表示， 结果参见图 2。 通过 CCD将监控器调至高速摄像系统 （FASTCAM-ultima ΑΡΧ, photon, 日本）， 以 1000 帧 /秒的速度记录肝脏终末门微静脉和中央静 脉中的红细胞流速， 将高速保存图像以 25帧 /秒的速度重新播放。 使用 图像 -Pro Plus 5.0软件测量红细胞流速。 红细胞流速以 μΐη/秒表示。 测 量结果表示成再灌注后第 15 分钟或第 30分钟测量数值与基础值的比 值， 结果参见图 3。 在重新播放 DVD时， 记录肝脏终末门微静脉和中央静脉中的肝脏 窦状隙数量。 其中， 肝脏窦状隙数量表示成每个视野 （250μπι> 300μιη) 中灌注的窦状隙。测量结果表示成再灌注后第 15分钟或第 30分钟的测 量数值与基础值的比值， 结果参见图 4。 为了评价白细胞滚动和粘附，经左颈静脉给予 0.2ml荧光标记剂罗 丹明 6G (罗丹明 6G在生理盐水中的浓度为 0.5mg/ml ) ， 有选择地将 体内白细胞染色。 使用倒置共聚焦激光扫描显微镜系统 （BIO-RAD , Radiance 2100, Axiovert 200， Carl Zeiss Shanghai Co, Ltd, 德国） ，The DVD image was replayed using Image-Pro Plus 5.0 software, and the terminal venule diameter and central vein diameter were measured. The measurement results are expressed as the ratio of the measured value at the 15th minute or the 30th minute after reperfusion to the base value, and the results are shown in Fig. 2. The monitor was tuned to a high-speed camera system (FASTCAM-ultima ΑΡΧ, photon, Japan) by CCD, and the red blood cell flow rate in the terminal venules and central veins of the liver was recorded at 1000 frames per second, and the image was saved at 25 frames at high speed. / second speed replay. Red blood cell flow rates were measured using Image-Pro Plus 5.0 software. The red blood cell flow rate is expressed in μΐ η/sec. The measurement results are expressed as the ratio of the measured value to the base value at the 15th or 30th minute after reperfusion. The results are shown in Figure 3. When the DVD was replayed, the number of liver sinusoids in the terminal venules and central veins of the liver was recorded. Among them, the number of hepatic sinusoids is expressed as a perfused sinusoid in each field of view (250 μπι> 300 μιη). The measurement results are expressed as the ratio of the measured value to the base value at the 15th minute or the 30th minute after reperfusion, and the results are shown in FIG. To evaluate leukocyte rolling and adhesion, 0.2 ml of the fluorescent labeling agent rhodamine 6G (rhodamine 6G in physiological saline at a concentration of 0.5 mg/ml) was administered via the left jugular vein to selectively stain the leukocytes in vivo. Using an inverted confocal laser scanning microscope system (BIO-RAD, Radiance 2100, Axiovert 200, Carl Zeiss Shanghai Co, Ltd, Germany),
20χ荧光物镜， 氩激光束照射 （波长 =543nm) ， 记录肝脏终末门微静脉 和中央静脉中的粒性白细胞滚动和粘附。 结果参见图 5〜图 8。 在肝脏终末门微静脉和中央静脉中停留 10秒以下的粒性白细胞为 滚动粒性白细胞， 以每 200μπι2视野中观测到的数量表示， 结果参见图 5。 以 1帧 /秒速度扫描， 每个时间点共记录 10个连续帧， 10个连续帧 的扫描过程中出现在同一位置的粒性白细胞即为粘附的粒性白细胞。肝 脏终末门微静脉和中央静脉中粘附的粒性白细胞以每 200μιη2视野中观 测到的粒性白细胞的数量表示， 结果参见图 7。 肝脏窦状隙中粘附的粒 性白细胞以每 200μπ2视野中观测到的粒性白细胞的数量表示， 结果参 见图 8。 肝脏上皮细胞粘附分子 Ε-选择蛋白和细胞间粘附分子 -1 的免疫荧光染 色分析 A 20-inch fluorescent objective lens, argon laser beam irradiation (wavelength = 543 nm), recorded the rolling and adhesion of granulocytes in the terminal venules and central veins of the liver. The results are shown in Figures 5 to 8. The granulocytes that stayed in the terminal venules and central veins of the liver for less than 10 seconds were rolling granulocytes, expressed as the number observed per 200 μm 2 field of view, and the results are shown in Fig. 5. Scanning at a rate of 1 frame/second, a total of 10 consecutive frames are recorded at each time point, and the granulocytes that appear at the same position during the scanning of 10 consecutive frames are adherent granulocytes. The granulocytes adhering to the terminal venules and central veins of the liver are expressed as the number of granulocytes observed per 200 μm 2 field of view, and the results are shown in Fig. 7. The adherent granulocytes in the sinusoids of the liver are expressed in terms of the number of granulocytes observed per 200 μπ 2 field of view, and the results are shown in Fig. 8. Immunofluorescence staining analysis of hepatic epithelial cell adhesion molecule Ε-selectin and intercellular adhesion molecule-1
再灌注后第 30分钟， 灌注 4%低聚甲醛将肝脏固定， 将肝脏取出， 用液氮冷冻， 然后使用恒冷切片机（LEICACM1800, Leica Co., 德国） 将其切成 6μηι厚的切片。 在室温条件下， 再次用 4%低聚甲醛固定切片 10 分钟， 最后用磷酸盐缓冲溶液冲洗。 然后， 对样品进行常规的免疫 组织化学染色。 山羊抗小鼠 Ε-选择蛋白多克隆抗体或山羊抗小鼠细胞 间粘附分子 -1多克隆抗体稀释备用 （稀释倍数 1: 50) 。 加入第二抗体 (罗丹明结合家兔抗 -山羊 lgG-R) (稀释倍数 1: 200) ) ， 在 37°C培 养 30分钟，然后用磷酸盐缓冲溶液冲洗，在室温条件下用 Hoechst 33342 (2μ§/ιτι1) 培养 3分钟。 磷酸盐缓冲溶液冲洗后， 将样本密封， 在共聚 焦激光扫描显微镜的 63χ物镜下观察。 测量荧光强度， 罗丹明的激发波 长为 543nm， Hoechst (核染剂） 的激发波长为 405nm。 评价肝脏微静 脉的五个视野 （每个视野 126μΐ 2) 。 使用图像 Pro Plus软件评价 Ε-选 择蛋白或细胞间粘附分子 -1 的荧光强度， 计算平均值， 结果以荧光强 度八 26μιη2表示， 结果参见图 9。 外周嗜中性粒细胞表面粘附分子 CDllb和 CD18表达评价 At 30 minutes after reperfusion, the liver was fixed by perfusion of 4% paraformaldehyde, the liver was taken out, frozen with liquid nitrogen, and then cut into 6 μη thick slices using a cryostat (LEICACM1800, Leica Co., Germany). The sections were fixed again with 4% paraformaldehyde for 10 minutes at room temperature and finally rinsed with phosphate buffer solution. The samples were then subjected to conventional immunohistochemical staining. Goat anti-mouse Ε-selectin polyclonal antibody or goat anti-mouse intercellular adhesion molecule-1 polyclonal antibody was diluted (dilution factor 1: 50). Add a second antibody (rhodamine combined with rabbit anti-goat lgG-R) (dilution factor 1: 200), incubate at 37 ° C for 30 minutes, then rinse with phosphate buffer solution, and use Hoechst 33342 at room temperature ( 2μ § /ιτι1) for 3 minutes. After rinsing with the phosphate buffer solution, the sample was sealed and observed under a 63 χ objective lens of a confocal laser scanning microscope. The fluorescence intensity was measured, the excitation wavelength of rhodamine was 543 nm, and the excitation wavelength of Hoechst (nuclear dye) was 405 nm. Five fields of view of the liver venules (126 μΐ 2 per field of view) were evaluated. The fluorescence intensity of Ε-selectin or intercellular adhesion molecule-1 was evaluated using Image Pro Plus software, and the average value was calculated. The result was expressed by fluorescence intensity of 八26 μηη 2 , and the results are shown in Fig. 9 . Evaluation of expression of CD11b and CD18 on peripheral neutrophil surface adhesion molecules
再灌注后第 30 分钟， 经下腔静脉采集血样， 加肝素 （20单位 /ml 全血） 抗凝。 样本加 l g FITC-标记的抗 CD18或 CDl lb抗体， 在室温 黑暗处培养 20分钟。 加溶血素将红细胞溶解， 用磷酸盐缓冲溶液洗涤 样品两次。 使用流式细胞仪 （FACS Calibur, B.D.Co, 美国） 测量每种 条件下 5,000个嗜中性粒细胞表面 CDl lb或 CD18的平均荧光强度，结 果参见图 10。 外周血液肝脏酶鉴定 At the 30th minute after reperfusion, blood samples were taken through the inferior vena cava and heparin was added (20 units/ml). Whole blood) anticoagulation. The samples were incubated with lg FITC-labeled anti-CD18 or CD1 lb antibody and incubated for 20 minutes at room temperature in the dark. The hemolysin was dissolved to dissolve the red blood cells, and the sample was washed twice with a phosphate buffer solution. The mean fluorescence intensity of 5,000 neutrophil surface CD1 lb or CD18 under each condition was measured using a flow cytometer (FACS Calibur, BDCo, USA), and the results are shown in FIG. Peripheral blood liver enzyme identification
取再灌注后第 30分钟和第 60分钟的部分小鼠，自下腔静脉抽取血 样， 加肝素 （20单位 /ml全血） 抗凝。 离心分离血清 （AllegraTM64R离 心机， Beckman Coultertm, 德国）， 转速为 4,000转 /分钟， 在 4°C放置 10分钟， 然后贮藏在 -20°C环境中。 遵循生产厂家的使用说明， 用全自 动酶分析仪（7170A全自动生化分析仪， Hitachi，日本），分别使用 Rate-A 乳酸脱氢酶 （LDH ) 试剂盒、 丙氨酸转氨酶 （ALT) 试剂盒和天冬氨酸 转氨酶（AST)试剂盒测量 LDH、 ALT和 AST的活性， 结果参见图 11。 外周血液 TNF-c IL-6和 MCP-1含量测量 A portion of the mice at 30 minutes and 60 minutes after reperfusion were taken from the inferior vena cava and anticoagulated with heparin (20 units/ml whole blood). Serum was separated by centrifugation (Allegra TM 64R Centrifuge, Beckman Coultertm, Germany) rotating at 4,000 revolutions / minute, placed in a 4 ° C 10 min, then stored at -20 ° C environment. Follow the manufacturer's instructions, using the automatic enzyme analyzer (7170A automatic biochemical analyzer, Hitachi, Japan), respectively, using the Rate-A lactate dehydrogenase (LDH) kit, alanine aminotransferase (ALT) kit The activity of LDH, ALT and AST was measured with an aspartate aminotransferase (AST) kit, and the results are shown in Fig. 11. Measurement of peripheral blood TNF-c IL-6 and MCP-1 levels
再灌注后第 30分钟， 自下腔静脉采集血样， 加肝素 （20单位 /ml 全血）抗凝。离心分离血清（AllegraTM64R离心机， Beckman Coultertm, 德国）， 转速为 4,000转 /分钟， 在 4X放置 10分钟， 然后贮藏在 -20Ό 环境中。使用流式细胞计量术，用 BD Cytometric Bead Array试剂盒（BD 生物科学系统， 美国） 测量 TNF-a、 IL-6 和 MCP-1 的浓度。 将 50μ1 液滴加入 50μ1血浆或标准物质中， 在室温黑暗处培养 1 小时。 然后加 入 50μ1 ΡΕ标记检测抗体， 在室温下培养 2小时， 形成夹心配合物。 培 养后， 用 lml清洗缓冲液（BD生物科学系统， 美国）将样品清洗干净。 使用流式细胞仪 （FACS Calibur, B.D.Co. , 美国） 测量 TNF-ot， IL-6 和 MCP-1的平均荧光强度，使用 BD Cytometric Bead Array分析软件分 析数据。 结果参见图 12。 统计分析 At the 30th minute after reperfusion, blood samples were taken from the inferior vena cava and anticoagulated with heparin (20 units/ml whole blood). Serum was separated by centrifugation (Allegra TM 64R Centrifuge, Beckman Coultertm, Germany) rotating at 4,000 revolutions / minute, placed in 4X 10 min, and then stored in -20Ό environment. The concentrations of TNF-a, IL-6 and MCP-1 were measured using flow cytometry using the BD Cytometric Bead Array Kit (BD Bioscience System, USA). 50 μl of the drop was added to 50 μl of plasma or standard material and incubated for 1 hour at room temperature in the dark. Then, 50 μl of the ruthenium detection antibody was added, and the mixture was incubated at room temperature for 2 hours to form a sandwich complex. After incubation, the samples were washed clean with 1 ml of wash buffer (BD Biosciences, USA). The mean fluorescence intensities of TNF-ot, IL-6 and MCP-1 were measured using a flow cytometer (FACS Calibur, BD Co., USA) and analyzed using BD Cytometric Bead Array analysis software. See Figure 12 for the results. Statistical Analysis
数值以平均士 S.E (N=6 ) 表示， 使用 SPSS 10.0统计软件进行 检 验。 P<0.05认为有统计显著性。 结果 Rl 对肠系膜上动脉缺血 -再灌注小鼠的肝脏终末门微静脉和中央 静脉直径的干预作用见图 2。 在整个观察期中， 对照组小鼠的终末门微 静脉和中央静脉的直径几乎保持不变。肠系膜上动脉缺血-再灌注（I/R) 后， 血管直径呈时间依赖性降低， 给予 R1后， 肠系膜上动脉缺血 -再灌 注导致的血管直径降低得到显著减弱。 图 3显示 R1对小鼠肠系膜上动脉缺血-再灌注后肝脏终末门微静脉 和中央静脉中红细胞流速的影响。 显然， 在整个观察期， 对照组中， 两 种类型血管中的红细胞流速没有出现显著变化。 肠系膜上动脉缺血-再 灌注后， 血管中的红细胞流速呈时间依赖性显著降低。 与缺血-再灌注 组比较， 经 R1处理后， 缺血-再灌注后两种类型的血管中红细胞流速的 降低得到一定程度的恢复，并且该作用对于缺血 -再灌注后第 30分钟时 终末门微静脉中的红细胞流速的恢复作用更为显著 （见图 3A) 。 Values are expressed as mean SE (N=6) and tested using SPSS 10.0 statistical software. P < 0.05 was considered statistically significant. result The effect of Rl on the hepatic terminal venules and central venous diameter of the superior mesenteric artery ischemia-reperfusion mice is shown in Figure 2. The diameters of the terminal venules and central veins of the control mice remained almost unchanged throughout the observation period. After ischemia/reperfusion (I/R) of the superior mesenteric artery, the diameter of the vessel decreased in a time-dependent manner. After R1 administration, the decrease in vessel diameter caused by ischemia-reperfusion of the superior mesenteric artery was significantly attenuated. Figure 3 shows the effect of R1 on the flow rate of red blood cells in the terminal venules and central veins of the liver after ischemia and reperfusion of the superior mesenteric artery in mice. Apparently, there was no significant change in red blood cell flow rates between the two types of blood vessels throughout the observation period. After ischemia-reperfusion of the superior mesenteric artery, the red blood cell flow rate in the blood vessels was significantly reduced in a time-dependent manner. Compared with the ischemia-reperfusion group, after R1 treatment, the decrease of red blood cell flow rate in the two types of blood vessels after ischemia-reperfusion was restored to some extent, and the effect was 30 minutes after ischemia-reperfusion. The recovery of red blood cell flow in the terminal venules was more pronounced (see Figure 3A).
R1 对肠系膜上动脉缺血 -再灌注后小鼠肝脏终末门微静脉和中央 静脉区域再灌注窦状隙数量的干预作用如图 4所示。对照组中， 终末门 微静脉区域或中央静脉区域的再灌注窦状隙数量没有出现显著变化。肠 系膜上动脉缺血-再灌注后的再灌注窦状隙数量呈时间依赖性降低。 与 对照组比较， 中央静脉区域再灌注后第 15分钟和终末门微静脉区域再 灌注后 30分钟出现统计显著性。 R1 处理后， 缺血 -再灌注后出现的中 央静脉区域再灌注窦状隙数量的降低显著减弱 （见图 4B )。 The intervention effect of R1 on the number of reperfusion sinus gaps in the terminal venules and central venous regions of the rat liver after ischemia-reperfusion of the superior mesenteric artery is shown in Fig. 4. In the control group, there was no significant change in the number of reperfusion sinusoids in the terminal venule region or central venous region. The number of reperfusion sinusoids in the superior mesenteric artery after ischemia-reperfusion decreased in a time-dependent manner. Compared with the control group, statistical significance was observed at 15 minutes after reperfusion in the central venous region and 30 minutes after reperfusion in the terminal venule region. After R1 treatment, the decrease in the number of reperfusion sinusoids in the central venous region after ischemia-reperfusion was significantly attenuated (see Figure 4B).
Rl 对肠系膜上动脉缺血 -再灌注后小鼠肝脏终末门微静脉和中央 静脉中的滚动粒性白细胞数量的干预作用见图 5。尽管在整个观察期内 可以观测到对照组出现少量滚动粒性白细胞， 但是与对照组比较， 缺血 -再灌注后， 终末门微静脉和中央静脉中滚动粒性白细胞的数量显著增 加。 R1处理后可以降低缺血-再灌注诱导的滚动粒性白细胞的增加量， 这种减弱作用对终末门微静脉和中央静脉均有统计显著性 （见图 5 )。 图 6显示肠系膜上动脉缺血 -再灌注后第 30分钟， 中央静脉区域微 循环的典型图像。 可以观测到一些粒性白细胞粘附在中央静脉和窦状 隙。 图 7显示 R1对肠系膜上动脉缺血 -再灌注诱导的粒性白细胞粘附的 干预作用。 肠系膜上动脉缺血 -再灌注极大地增加了肝脏终末门微静脉 和中央静脉中的粒性白细胞粘附数量。 R1处理后， 可以降低缺血 -再灌 注后的增加的粘附粒性白细胞数量，终末门微静脉出现更为显著地降低 (见图 7A)。 图 8显示 Rl对肝脏终末门微静脉和中央静脉窦状隙中白细胞粘附 的干预作用。 对于肝脏终末门微静脉和中央静脉， 对照组中， 终末门微 静脉区域或中央静脉区域窦状隙中只能观测到少量粘附的粒性白细胞。 肠系膜上动脉缺血-再灌注小鼠的两个区域窦状隙中的粘附的粒性白细 胞数量显著增加， R1 处理后， 可显著抑制粘附的粒性白细胞数量的增 力卩 （见图 8 )。 图 9显示 R1对再灌注后第 30分钟小鼠肝脏血管 E-选择蛋白和细 胞间粘附分子 -1表达的影响。可以看出再灌注后第 30分钟 E-选择蛋白 表达出现显著增强， 而对细胞间粘附分子 -1 表达没有任何影响。 使用 R1处理后，肠系膜上动脉缺血-再灌注后出现的 E-选择蛋白表达增强完 全消失， 并且细胞间粘附分子 -1的表达也得以降低。 再灌注后第 30分钟， 采集血样， 用于评价 R1 对小鼠外周嗜中性 粒细胞粘附分子 CD18和 CDl lb表达的干预作用。 如图 10所示， 与对 照组比较， 再灌注后第 30分钟， CD18表达显著增强， CDl lb的表达 也略微增强。 R1处理显著降低缺血-再灌注诱导的 CD18和 CDl lb平均 荧光强度的增加量。 总之，本发明表明 R1处理可以很好地降低肠系膜上动脉缺血 -再灌 注诱导的肝脏微循环障碍， 所述的微循环障碍包括微静脉直径降低、红 细胞流速降低、 灌注的窦状隙数量减少以及白细胞的滚动和粘附增加。 R1 对微循环障碍的改善作用意味着对缺血 -再灌注诱导的肝损伤的保 护作用。 实施例 制备实施例 1 片剂 The effect of Rl on the number of rolling granulocytes in the terminal venules and central veins of the liver in mice after ischemia-reperfusion of the superior mesenteric artery is shown in Fig. 5. Although a small amount of rolling granulocytes were observed in the control group throughout the observation period, the number of rolling granulocytes in the terminal venules and central vein was significantly increased after ischemia-reperfusion compared with the control group. R1 treatment reduced the increase in ischemia-reperfusion-induced granulocyte leukocytes, which was statistically significant for the terminal venules and central veins (see Figure 5). Figure 6 shows a typical image of microcirculation in the central venous region at 30 minutes after ischemia-reperfusion of the superior mesenteric artery. Some granulocytes can be observed to adhere to the central vein and sinusoids. Figure 7 shows R1 adhesion to granulocytes induced by ischemia-reperfusion of superior mesenteric artery Intervention. Ischemia-reperfusion of the superior mesenteric artery greatly increases the amount of granulocyte adhesion in the terminal venules and central veins of the liver. After R1 treatment, the number of increased adherent granulocytes was reduced after ischemia-reperfusion, and the terminal venules were more significantly reduced (see Figure 7A). Figure 8 shows the effect of R1 on leukocyte adhesion in the terminal venous and central sinusoids of the liver. For the terminal venules and central veins of the liver, only a small amount of adherent granulocytes were observed in the sinusoids of the terminal venules or central venous regions. The number of adherent granulocytes in the sinusoids of the two regions of the superior mesenteric ischemia-reperfusion mice was significantly increased. After R1 treatment, the number of adherent granulocytes could be significantly inhibited. 8 ). Figure 9 shows the effect of R1 on the expression of hepatic vascular E-selectin and intercellular adhesion molecule-1 in mice at 30 minutes after reperfusion. It can be seen that E-selectin expression is significantly enhanced at the 30th minute after reperfusion, and has no effect on the expression of intercellular adhesion molecule-1. After treatment with R1, the expression of E-selectin increased after ischemia-reperfusion of the superior mesenteric artery completely disappeared, and the expression of intercellular adhesion molecule-1 was also reduced. At 30 minutes after reperfusion, blood samples were taken to evaluate the effect of R1 on the expression of peripheral neutrophil adhesion molecules CD18 and CD1 lb in mice. As shown in Figure 10, compared with the control group, CD18 expression was significantly enhanced and CDl lb expression was slightly enhanced at 30 minutes after reperfusion. R1 treatment significantly reduced the increase in mean fluorescence intensity of CD18 and CD1 lb induced by ischemia-reperfusion. In conclusion, the present invention demonstrates that R1 treatment can significantly reduce hepatic microcirculatory disturbance induced by ischemia-reperfusion of the superior mesenteric artery, including a decrease in venule diameter, a decrease in red blood cell flow rate, and a decrease in the number of perfused sinusoids. As well as the rolling and adhesion of white blood cells. The improved effect of R1 on microcirculatory disorders implies a protective effect on ischemia-reperfusion-induced liver injury. EXAMPLES Preparation Example 1 Tablet
取三七皂苷 Rl 100g、硫酸钙 150g、微晶纤维素 50g、微粉硅胶 3g、 硬脂酸镁 1.5g Take notoginsenoside Rl 100g, calcium sulfate 150g, microcrystalline cellulose 50g, micronized silica gel 3g, Magnesium stearate 1.5g
取上述原料分别过 100目筛； 取三七皂苷、 硫酸钙、 微晶纤维素， 混匀， 用 60 % (v/v) 乙醇适量作为粘合剂， 制软材； 过 20目筛， 制粒; 6(TC干燥， 取出， 过 30目筛， 整粒； 加入微粉硅胶及硬脂酸镁， 混匀， 压片， 制成 1000片， 即得。 制备实施例 2 片剂  Take the above raw materials through a 100 mesh sieve; take notoginsenoside, calcium sulfate, microcrystalline cellulose, mix, use 60% (v / v) ethanol as a binder, make soft materials; through 20 mesh sieve, 6 (TC dry, take out, pass 30 mesh sieve, whole grain; add micro-powder silica gel and magnesium stearate, mix, tablet, make 1000 pieces, that is. Preparation Example 2 tablets
取三七皂苷 Rl 75g、硫酸钙 112g、微晶纤维素 37g、微粉硅胶 2.3g、 硬脂酸镁 l . lg  Take notoginsenoside Rl 75g, calcium sulfate 112g, microcrystalline cellulose 37g, micronized silica gel 2.3g, magnesium stearate l. lg
取上述原料分别过 100 目筛； 取三七皂苷、 硫酸钙、 微晶纤维素， 混匀， 用 60 % (v/v) 乙醇适量作为粘合剂， 制软材； 过 20目筛， 制粒； 60°C干燥， 取出， 过 30 目筛， 整粒； 加入适量微粉硅胶及硬脂酸镁， 混勾， 压片， 制成 1000片， 即得。 制备实施例 3 片剂  Take the above raw materials through a 100 mesh sieve; take notoginsenoside, calcium sulfate, microcrystalline cellulose, mix, use 60% (v / v) ethanol as a binder, make soft materials; through 20 mesh sieve, Granules; dried at 60 ° C, taken out, passed through a 30 mesh sieve, whole grain; add appropriate amount of micro-silica gel and magnesium stearate, mix hook, tablet, and make 1000 tablets, that is. Preparation Example 3 Tablet
取三七皂苷 Rl 133g、硫酸钙 200g、微晶纤维素 66g、微粉硅胶 4g、 硬脂酸镁 2g  Take notoginsenoside Rl 133g, calcium sulfate 200g, microcrystalline cellulose 66g, micronized silica gel 4g, magnesium stearate 2g
取上述原料分别过 100目筛； 取三七皂苷、 硫酸钙、 微晶纤维素， 混匀， 用 60% ( v/v) 乙醇适量作为粘合剂， 制软材； 过 20目筛， 制粒； 60°C干燥， 取出， 过 30目筛， 整粒； 加入适量微粉硅胶及硬脂酸镁， 混匀， 压片， 制成 1000片， 即得。 制备实施例 4 胶囊  Take the above raw materials through a 100 mesh sieve; take notoginsenoside, calcium sulfate, microcrystalline cellulose, mix, use 60% (v / v) ethanol as a binder, make soft materials; over 20 mesh sieve Granules; dried at 60 ° C, taken out, passed through a 30 mesh sieve, whole grain; add appropriate amount of micro-silica gel and magnesium stearate, mix and compress, and make 1000 tablets. Preparation Example 4 Capsules
取三七皂苷 Rl 50g, 加入适量淀粉、 硬脂酸镁等辅料， 制粒， 整 粒， 装入 1号胶囊， 即得。 制备实施例 5 口服液  Take the notoginsenoside Rl 50g, add appropriate amount of starch, magnesium stearate and other accessories, granulate, whole, and put into the No. 1 capsule, that is. Preparation Example 5 Oral solution
取三七皂苷 R1 5g， 加入适量蔗糖、 防腐剂， 加水到 1000ml, 分装 成 10ml/支， 即得口服液。 制备实施例 6 颗粒剂  Take notoginsenoside R1 5g, add appropriate amount of sucrose, preservative, add water to 1000ml, and pack into 10ml / support, that is, get oral liquid. Preparation Example 6 Granules
取三七皂苷 Rl 50g， 加入适量糊精、 甜菊素， 干式制粒， 整粒， 分装， 即得。 制备实施例 Ί 注射剂 Take 30 grams of notoginsenoside Rl, add appropriate amount of dextrin, stevioside, dry granulation, whole granules, and dispense. Preparation Example 注射 Injection
三七皂苷 Rl 5g加水溶解， 另将氯化钠、 对羟基苯甲酸乙酯通过加 热溶解于水， 混匀， 调 pH值。 注射用水稀释至 1000ml, 用中空纤维膜 滤过， 灌装， 灭菌， 即得。 制备实施例 8 注射剂  The notoginsenoside Rl 5g is dissolved in water, and sodium chloride and ethyl p-hydroxybenzoate are dissolved in water by heating, mixed, and adjusted to pH. The water for injection is diluted to 1000 ml, filtered through a hollow fiber membrane, filled, and sterilized. Preparation Example 8 Injection
三七皂苷 Rl lg加水溶解， 另将氯化钠、对羟基苯甲酸乙酯通过加 热溶解于水， 混匀， 调 pH值。 注射用水稀释至 1000ml, 用中空纤维膜 滤过， 灌装， 灭菌， 即得。  The notoginsenoside Rl lg is dissolved in water, and sodium chloride and ethyl p-hydroxybenzoate are dissolved in water by heating, mixed, and adjusted to pH. The water for injection is diluted to 1000 ml, filtered through a hollow fiber membrane, filled, and sterilized.

Claims

权利要求书 Claim
1 . 三七皂苷 R1在制备治疗和 /或预防肝损伤的药物中的应用。1. Use of notoginsenoside R1 in the preparation of a medicament for treating and/or preventing liver damage.
2. 如权利要求 1所述的应用， 其中所述三七皂苷 R1 是从中药三 七中提取获得的， 纯度〉 50%， 优选纯度>90%， 更优选纯度 >98%。 2. The use according to claim 1, wherein the notoginsenoside R1 is obtained by extracting from the traditional Chinese medicine 37, having a purity of > 50%, preferably a purity of > 90%, more preferably a purity of > 98%.
3. 如权利要求 1或 2所述的应用， 其中所述三七皂甙 R1 是含有 三七皂甙的药物组合物。  The use according to claim 1 or 2, wherein the notoginsenoside R1 is a pharmaceutical composition containing notoginsenoside.
4. 如权利要求 3所述的应用， 其中所述药物组合物以三七皂苷 R1 作为药物活性成分， 其在制剂中所占重量百分比可以是 0.1〜99.9%， 其余为药学上可接受的载体。  The use according to claim 3, wherein the pharmaceutical composition has notoginsenoside R1 as a pharmaceutically active ingredient, and the weight percentage in the preparation may be 0.1 to 99.9%, and the balance is a pharmaceutically acceptable carrier. .
5. 如权利要求 3所述的应用， 其中所述药物组合物为任何可药用 的制剂形式。  5. The use according to claim 3, wherein the pharmaceutical composition is in the form of any pharmaceutically acceptable formulation.
6. 如权利要求 5所述的应用， 其中所述药物组合物为注射剂。 6. The use according to claim 5, wherein the pharmaceutical composition is an injection.
7. 如权利要求 6所述的应用， 其中所述注射剂是溶液剂或冻干粉 针剂。 7. The use according to claim 6, wherein the injection is a solution or a lyophilized powder.
8. 如权利要求 1所述的应用， 其中所述肝损伤选自病毒感染或肝 硬化引起的肝损伤、 药物肝损伤、 酒精性肝损伤、 化学性肝损伤、 缺血 -再灌注引起的肝损伤。  8. The use according to claim 1, wherein the liver injury is selected from the group consisting of a liver infection caused by a viral infection or cirrhosis, a drug liver injury, an alcoholic liver injury, a chemical liver injury, and a liver caused by ischemia-reperfusion. damage.
9. 如权利要求 8所述的应用， 其中所述肝损伤是缺血 -再灌注引起 的肝损伤。  9. The use according to claim 8, wherein the liver injury is liver damage caused by ischemia-reperfusion.
10. 如权利要求 1 所述的应用， 所述治疗和 /或预防肝损伤是通过 改善微循环障碍实现的， 所述的微循环障碍包括微静脉直径降低、红细 胞流速降低、 灌注的窦状隙数量减少以及白细胞的滚动和粘附增加。  10. The use according to claim 1, wherein the treating and/or preventing liver damage is achieved by ameliorating a microcirculatory disorder comprising a decrease in venule diameter, a decrease in red blood cell flow rate, and a perfusion sinusoidal gap. The number is reduced and the rolling and adhesion of white blood cells is increased.
11 . 如权利要求 1 所述的应用， 所述治疗和 /或预防肝损伤是通过 降低肝脏血管 E-选择蛋白和细胞间粘附分子 -1的表达实现的。  The use according to claim 1, wherein the treatment and/or prevention of liver damage is achieved by reducing the expression of hepatic vascular E-selectin and intercellular adhesion molecule-1.
12. 如权利要求 1 所述的应用， 所述治疗和 /或预防肝损伤是通过 降低嗜中性粒细胞表面 CD18和 CDl lb的表达实现的。  12. The use according to claim 1, wherein said treating and/or preventing liver damage is achieved by reducing the expression of CD18 and CD1 lb on the surface of neutrophils.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111024471A (en) * 2019-12-17 2020-04-17 浙江殷欣生物技术有限公司 Liquid-based cell detection kit for cervical cancer screening and use method
CN114432494A (en) * 2022-01-12 2022-05-06 宁波市第一医院 Sanchinin-loaded hydrogel and preparation method thereof
CN115105492A (en) * 2021-03-17 2022-09-27 南方医科大学南方医院 New application of petroselinic acid

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105147753B (en) * 2015-10-23 2018-06-22 云南大学 Application of the arasaponin in direct anti hepatitis C virus drug is prepared
CN106511365A (en) * 2016-12-17 2017-03-22 郑州郑先医药科技有限公司 Western medicine composition for treating liver cirrhosis
CN109288853A (en) * 2018-11-21 2019-02-01 浙江中医药大学 Application of the notoginsenoside R in preparation prevention or treatment liver injury medicament

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LI Y. ET AL.: "The protective effect of panax notoginseng saponins on the transplanted liver during ischemia-reperfusion injury in rat with orthotopic liver transplantation", CHINESE JOURNAL OF GENERAL SURGERY, vol. 15, no. 7, 2006, pages 508 - 511 *
LI Z. ET AL.: "Pharmacological study and clinical application on Panax Notoginseng in the treatment of hepatic lesion", INNER MONGOLIA MED. J., vol. 36, no. 6, 2004, pages 426 - 428 *
ZHAO P. ET AL.: "Effect of saponin in protecting against alcoholic liver injury", CHINESE JOURNAL OF CLINICAL REHABILITATION, vol. 8, no. 30, 2004, pages 6686 - 6687 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111024471A (en) * 2019-12-17 2020-04-17 浙江殷欣生物技术有限公司 Liquid-based cell detection kit for cervical cancer screening and use method
CN115105492A (en) * 2021-03-17 2022-09-27 南方医科大学南方医院 New application of petroselinic acid
CN115105492B (en) * 2021-03-17 2023-08-25 南方医科大学南方医院 New use of petroselinic acid
CN114432494A (en) * 2022-01-12 2022-05-06 宁波市第一医院 Sanchinin-loaded hydrogel and preparation method thereof

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