WO2008154794A1

WO2008154794A1 - The use of an extract of salvia miltiorrhiza for manufacture of a medicament for treatment of septicemia

Info

Publication number: WO2008154794A1
Application number: PCT/CN2008/000876
Authority: WO
Inventors: Jing-Yan Han; Jun Guo; Kai Su; Chuan-She Wang; Yu-Ying Liu
Original assignee: Tianjin Tasly Pharmaceutical Co. Ltd.
Priority date: 2007-06-21
Filing date: 2008-04-29
Publication date: 2008-12-24
Also published as: CN101327249A; CN101327249B

Abstract

The use of an extract of salvia miltiorrhiza , in particular a dihydroxyphenylactic acid or a salvianolic acid B for manufacture of a medicament for treatment of septicemia.

Description

Application of Salvia miltiorrhiza extract in preparing medicine for treating sepsis

Technical field

The present invention relates to new uses of traditional Chinese medicine products, and in particular to the use of Salvia miltiorrhiza extract for the treatment and/or prevention of sepsis. Background technique

Salvia miltiorrhiza is a traditional Chinese medicine in China. It was first seen in Shennong Bencaojing and was listed as top grade. Salvia miltiorrhiza is used as a dry root and rhizome of Salviami Itior2rhiza Bunge, a perennial herb of the genus Salicidae. Alias red ginseng, purple salvia miltiorrhiza Danshen is mainly produced in Shanxi, Sichuan, Anhui, Hebei, Jiangsu, Shandong, Zhejiang and other provinces. Danshen tastes bitter, slightly cold, into the heart, pericardium, liver. It has active phlegm, cool blood to eliminate phlegm and raise The efficacy of blood and nerves, the various pains caused by blood stasis, accumulation of symptoms, sore throat and palpitations, insomnia, etc., are commonly used in clinical medicine, especially for the treatment of coronary heart disease and ischemic cerebrovascular disease, and The curative effect is quite good, and it is mostly used for the treatment of coronary heart disease, angina pectoris, myocardial infarction, tachycardia, cerebral thrombosis, etc. It has been confirmed by modern clinical experiments and pharmacological experimental research to abundance of blood stasis and other pharmacological activities. Its main component is tanshinone. ( I , II A, II B) , Isotanshinone ( I , II ), cryptotanshinone, hydroxy tanshinone II A, cryptotanshinone, salvia miltiorrhiza, L-dihydrotanshinone I, methyl tanshinate, saponin Salvia phenol, acetate, propionate, β - sitosterol, 3, 4 - dihydroxybenzaldehyde, catechin, rutin, protocatechuic aldehyde, protocatechuic acid, lactic acid, and other vitamins Ε.

The water-soluble component of Salvia miltiorrhiza is mainly dihydroxyphenyl lactic acid (DLA, see Figure 1A).

AKA: Danshensu _D (+) f3 - (3, 4_Dihydroxyphenyl) lactic acid (1) is the basic structural component of the water-soluble various components of Salvia miltiorrhiza. It has been proven to have anti-oxidation and anti-fibrosis effects. .

Salvianolic acid B (SAB, see Figure 1B)

Salvianolic acid compounds have strong inhibition of lipid peroxidation, scavenging superoxide anion and hydroxyl free The role of the base. Among them, salvianolic acid A and B have the strongest activity. Salvianolic acid B significantly inhibited mitochondrial damage and neuronal apoptosis induced by cerebral ischemia-reperfusion injury. It can inhibit the apoptosis of PCI2 cells with high anti-Caspase-3 expression, and also inhibit the formation of B amyloid protein (A f3 1-40) and the mitochondrial damage and apoptosis of PC12 cells induced by A β 1-40. It is a strong antioxidant and can eliminate intracellular calcium overload.

Sepsis is an acute systemic infection caused by pathogenic bacteria or conditional pathogens invading the blood circulation, producing toxins and other metabolites. It is characterized by chills, fever, rash, joint pain and hepatosplenomegaly. There may be septic shock and migratory lesions. The causes of sepsis are:

First, the human factor:

1 When the skin and mucous membranes are damaged or have suppurative inflammation, the bacteria easily invade the body.

2 The human immune response can be divided into two types: non-specific immune response and specific immune response, and the latter can be divided into cellular immunity and humoral immunity. When the immune function of the body declines, the effect of phagocytosis and killing of bacteria cannot be fully exerted. Even if the amount of invading bacteria is small, the pathogenicity is not strong and can cause sepsis.

3 The iatrogenic infection caused by the conditional pathogens also gradually increased.

Second, the bacterial factors:

Mainly related to the virulence and quantity of pathogens. Pathogenic bacteria with high virulence or a large number of bacteria enter the body, which is more likely to cause sepsis.

The main symptoms of sepsis are:

1. Primary inflammation: The primary inflammation caused by various pathogens is related to its distribution in the human body. Primary inflammation is characterized by localized redness, swelling, heat, pain, and dysfunction.

2. Symptoms of Toxemia: The onset is more rapid. Often there are chills, high fever, and fever, which are mostly relaxation heat or intermittent heat. They can also be caused by heat retention, irregular heat and bimodal fever. The latter are caused by sepsis-negative bacilli sepsis. The fever is accompanied by varying degrees of symptoms of toxemia such as headache, nausea, vomiting, bloating, abdominal pain, generalizedness, and muscle and joint pain.

3. Rash: Seen in some patients, the most common defects are distributed in the trunk, limbs, conjunctiva, oral mucosa, etc., not many.

4. Joint symptoms: There may be large joint redness, swelling, heat, pain and limited mobility, even complicated with joint fluid and empyema. It is more common in the course of sepsis such as Gram-positive cocci, meningococcal, and Alcaligenes. .

5. Infectious shock: About 1/5~1/3 of patients with sepsis, manifested as irritability, rapid pulse rate, cold limbs, skin spots, decreased urine output and decreased blood pressure, etc., and DIC can occur; Severe toxemia To.

6. Hepatosplenomegaly: Generally only mild swelling.

According to the study, lipopolysaccharide (LPS) is one of the components of the cell wall of Gram-positive bacteria, causing multiple manifestations of human sepsis and septic shock caused by Gram-positive bacteria. The microcirculatory disorder that occurs during sepsis is a systemic inflammatory response caused by excessive release of LPS, mediated by a number of activated factors such as adhesion molecules, reactive oxygen species (R0S), and inflammatory precursor mediators such as tumors. Necrosis factor-a (TNF-α), interleukin-6 (IL-6) and interferon-Y (INF-γ). The microcirculatory disorder induced by LPS plays a key role in organ dysfunction during sepsis. During this process, white blood cells rotate along the endothelium and adhere to the vascular endothelium. Hydrogen peroxide and mast cell degranulation are produced in the vessel wall. The key steps of the report.

The main clinical treatments for current sepsis include volume recovery, the use of catecholamines, and antibiotic-compensatory treatment. These methods can increase the survival rate of patients with sepsis, but clinically lead to an increase in blood sugar and a decrease in blood pressure and blood calcium, and bleeding. Clinical studies suggest that anti-TNF-α therapy may be helpful in the treatment of sepsis. However, recent studies have shown that this method does not cure sepsis and increases mortality in patients with severe sepsis. Thus, finding a way to prevent severe microcirculation in LPS-induced sepsis remains a challenge.

The present inventors found that dihydroxyphenyl lactic acid and salvianolic acid B significantly improved lipopolysaccharide-induced rat mesenteric micros in the study of two important extracts of dihydroxyphenyl lactic acid and salvianolic acid B in Salvia miltiorrhiza. Circulatory disorders, and inhibition of lipopolysaccharide-induced CDl lb and CD18 expression and neutrophil production of Ό ₂ and H ₂ 0 ₂ , have significant therapeutic effects on sepsis. Summary of the invention:

The present invention provides a novel therapeutic use of Salvia miltiorrhiza extract. The new therapeutic use is the treatment of sepsis caused by microcirculatory disorders with salvia miltiorrhiza extract.

To this end, the present invention provides a new use of a medicament for preparing a medicament for treating and/or preventing sepsis using Salvia miltiorrhiza extract.

The Salvia miltiorrhiza extract of the present invention is selected from two important extracts, dihydroxyphenyl lactic acid and salvianolic acid B. The dihydroxyphenyl lactic acid and salvianolic acid B according to the present invention are prior art, are commercially available, can also be prepared according to the prior art, and can meet pharmaceutical standards. Preferably, the purity is > 50%, more preferably the purity is > 90%, Most preferably, the purity is > 98%.

The medicament prepared by the present invention is a pharmaceutical preparation composition prepared by using the above-mentioned salvia miltiorrhiza extract as a pharmaceutically active ingredient.

The medicinal composition of the present invention may comprise a pharmaceutically acceptable carrier, wherein the salvia miltiorrhizae extract may be 0. 1-99. Accepted carrier. The pharmaceutical preparation composition of the present invention is in the form of a unit dosage form, which means a unit of the preparation, such as each tablet of the tablet, each capsule of the capsule, each bottle of the oral solution, granules per bag, injection Every one of them.

The pharmaceutical preparation composition of the present invention may be in any pharmaceutically acceptable dosage form, and includes: a tablet, a sugar-coated tablet, a film-coated tablet, an enteric coated tablet, a capsule, a hard capsule, a soft capsule, orally. Liquid, buccal, granule, granule, pill, powder, ointment, dandruff, suspension, powder, solution, injection, suppository, ointment, plaster, cream, spray, drops, patch .

The traditional Chinese medicine preparation of the present invention, the preparation for oral administration may contain common excipients such as a binder, a filler, a diluent, a tablet, a lubricant, a disintegrant, a coloring agent, a flavoring agent and a wetting agent. , The tablets can be coated if necessary.

Suitable fillers include cellulose, mannitol, lactose and other similar fillers. Suitable disintegrating agents 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 usual method such as mixing, filling, tableting or the like. Repeated mixing allows the active material to be distributed throughout those compositions that use large amounts 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 or hydrogenated edible fats. 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 of esters such as glycerol, propylene glycol or ethanol; The agent, for example, p-hydroxybenzyl or propylparaben or sorbic acid, and if desired, may contain conventional flavoring or coloring agents.

For injection, the liquid unit dosage form prepared contains the active substance of the invention and a sterile vehicle. According to The carrier and the concentration can be suspended or dissolved. The solution is usually prepared by dissolving the active substance in a carrier, sterilizing it by filtration before 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 traditional Chinese medicine preparation of the present invention can be selectively added to a suitable pharmaceutically acceptable carrier when prepared as a medicament, and the pharmaceutically acceptable carrier is selected from the group consisting of: mannitol, sorbitol, sodium metabisulfite, sodium hydrogen sulfite, thio Sodium sulfate, 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 thereof , alginate, gelatin, polyvinylpyrrolidone, glycerin, earth temperature 80, agar, calcium carbonate, calcium bicarbonate, surfactant, polyethylene glycol, cyclodextrin, e-cyclodextrin, phospholipids, kaolin , talc, calcium stearate, magnesium stearate, etc.

The preparation of the present invention is used according to the condition of the patient at the time of use, and can be taken three times a day for 1 20 doses, such as: 1-20 bags or tablets or tablets, each dose of lmg-1000 mg. The therapeutic use of the present invention is demonstrated by the following experiments:

Materials and Method

Test drug

Both dihydroxyphenyl lactic acid and salvianolic acid B were purchased from China National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Lipopolysaccharide was obtained from E. coli serotype 055: B5, toluidine blue, 2, , 7'-dichlorofluorescein acetoacetate (DCFH-DA) and hydrogenated ethidine were purchased from Sigma (St. Louis, Missouri) . FITC-binding mouse anti-rat CD1 lb monoclonal antibody, FITC-binding mouse anti-rat CD18 monoclonal antibody, FITC-binding mouse IgA, κ and FITC-binding mouse IgG!, κ are purchased from BD Bioscience System (San Diego, CA). Hemolysin was purchased from the BD Bioscience Immunocytometer system (Silicon Valley, California, USA). The mono-Pol separation liquid was purchased from Dainippon Co., Ltd. (Osaka, Japan). RPMI 1640 and fetal calf serum were purchased from Hydone (Fagan, Utah). All other chemical reagents used in the test are the best commercial grade reagents.

Experimental animal Male Sprague-Dawley rats weighing 200-250 g were provided by the Experimental Animal Center of Peking University Medical School. All studies were approved and all experimental animals were processed following the guidelines of the Peking University Laboratory Animal Research Committee.

Mesenteric microcirculation

SD rats were fasted for 12 hours before the test and were given free access to water. Experimental animals were anesthetized with urethane (1.25 mg/kg body weight, intramuscularly). The catheter was inserted into the left jugular vein and the right femoral vein for injection of various experimental drugs. The ileocecal of the 20 cm mesenteric tail was gently removed from the abdomen by a 20- 30 mm incision through the midline of the abdomen and placed on a transparent plastic rat experimental bench. The mesentery was maintained at 37 ° C with a thermostat, and physiological saline was continuously dropped on the surface to maintain moisture. An inverted microscope (DM-IRB, Leica, Wetzlar, Germany) was used to observe the hemodynamics of the mesenteric microcirculation by transillumination. A camera (Jk-TU53H, Toshiba Corporation, Tokyo, Japan) is mounted on a microscope to reflect images to a color monitor (J2118A, TCL, Huizhou, China), using DVD (DVR-R25, Malata, Xiamen, China) ) Record images. The venules used for the measurement parameters in the study should be single, non-branched, without obvious bending, with a diameter range of 30μηι-50μηι and a length of 200μπι.

Lipopolysaccharide-induced microcirculatory disorders and drug infusion

Rats were randomly divided into 4 groups of 6 each. After observing the basic hemodynamics of the rat mesenteric microvascular system for 10 minutes, a vehicle (physiological saline solution) or a test drug was administered. From the beginning of the experiment (0th minute) to the end of the experiment (60th minute), the experimental animals were perfused with saline solution (8ml/kg body weight/hr) from the left jugular vein; from the start of the experiment (0th minute) to the end of the test (60th minute), lipopolysaccharide (2 mg/kg body weight/hrin saline solution) was continuously perfused from the left femoral vein of the experimental group of the lipopolysaccharide group. After infusion of lipopolysaccharide (2 mg/kg body weight/hr) from the left femoral vein of the experimental group of lipopolysaccharide + dihydroxyphenyl lactic acid, the left jugular vein was infused with dihydroxyphenyl lactic acid from 20 minutes before the end of the observation period to the end of observation. 5 mg/kg body weight/hrin saline solution). The lipopolysaccharide + salvianolic acid group B was treated in the same manner as the lipopolysaccharide + dihydroxyphenyl lactic acid group, and only the salvianolic acid B (5 mg/kg body weight / hrin saline solution) was used instead of the dihydroxyphenyl lactic acid. The total infusion volume was the same for the four experimental groups.

Vein diameter measurement

Using the image -Pro Plus 5.0 software to evaluate the venous dynamic image recorded by the system, at 0, 10,

The venule diameter was measured at 30 and 50 minutes.

Vein red blood cell flow rate measurement Record the venous red blood flow rate, adjust the monitor to the high speed camera system via CCD (FAST CAM-ultima APX, photron, San Diego, California), adjust the speed to 2000 frames per second (frames per second), replay at 25 frames per second Save images at high speed. The venous red blood cell flow rates at 0, 10, 30 and 50 minutes were measured using Image-Pro Plus 5.0 software.

Shear rate measurement

The venous rate (Y) was calculated according to the following formula: Y = 8 (average velocity of venule/venule diameter RBCs).

Granulocyte leukocyte adhesion measurement

A dynamic image of the granulocyte recorded by the system was observed to identify granulocytes adhering to the wall of the venule. Adherent granulocytes mean that granulocytes are attached to the same site for more than 10 seconds when the DVD image is replayed. The observed 0, 10, 30 and 50 minute images were randomly selected for 200 μπι venules, and then the number of adherent granulocytes was counted along the venules.

Chemotactic migratory white blood cell measurement

Examine the recorded images and measure the number of chemotactic leukocytes. Leukocyte chemotaxis is the number of granulocytes per 200 μιη η, and images were randomly selected at 0, 10, 30 and 50 minutes.

Mast cell degranulation measurement

After infusion of the lipopolysaccharide or saline solution for 60 minutes, the tissue was partially stained with 0.1% toluidine blue for 1 minute and then washed with physiological saline. The number of mast cells and degranulated mast cells without degranulation in the field of view was enlarged by 20 Χ, and 5 fields of view were evaluated for each experimental animal. The ratio of the number of degranulated mast cells to the total number of mast cells is the percentage of degranulated mast cells.

Measurement of CDl lb and CD18 expression on neutrophil surface

Blood samples from other groups (n=6) were collected from the abdominal aorta, and heparin was added for anticoagulation to detect CDl lb and CD18 expression on the surface of neutrophils. The samples were divided into four groups: control group, lipopolysaccharide group (lipopolysaccharide 2 g/ml), lipopolysaccharide + dihydroxyphenyl lactic acid group (lipid polysaccharide 2 g/ml plus dihydroxyphenyl lactic acid 0.2 mg/ml, 0.5 mg/ M or l.Omg/ml ) and lipopolysaccharide + salvianolic acid B group (lipopolysaccharide 2 g / ml plus salvianolic acid B 0.2 mg / ml, 0.5 mg / ml or 1.0 mg / ml). The samples were incubated in a 37 ° C water bath for 2 hours, then using ^g/ml FITC-binding mouse anti-rat CD1 lb antibody, lg/ml FITC-binding mouse anti-rat CD 18 antibody or FITC-binding The isotype (mouse IgA, Kfor CD 11 b, mouse IgG, KtheCD 18) was labeled for 20 minutes at room temperature. Then, according to the manufacturer (BD Bioscience System, US Plus State Silicon Valley) Description, red blood cells are dissolved with hemolysin, and phosphorus residual cells are washed twice with an acid buffer solution. According to the forward angle _/lateral angle "" scattering expression characteristics, neutrophils were classified using a FACS Calibur flow cytometer (BD Biosciences System, Silicon Valley, California, USA), and 5,000 singularities in each sample were evaluated. Granulocytes, measuring mean fluorescence intensity.

Intracellular 0 ₂ and '0 ₂ - measurements produced by neutrophils

Blood samples were taken from different groups of rats and supplemented with heparin. Neutrophils were separated using Mono-multiple separation solutions following the method described by Ting and then suspended in 0.1 M phosphate buffer solution. Intracellular production of Ό ₂ and 3⁄40 _{2 was} analyzed using FACSCalibur flow cytometry (BD Biosciences System, Silicon Valley, CA, USA). Briefly, collected neutrophils were incubated in 20 mM 2'-7'-dichlorofluorescein diacetate solution for 5 minutes at 37 ° C, then hydrogenated ethidium at 10 mM Cultivate for 15 minutes. The nitrate portion of 2'-V-dichlorofluorescein diacetate is cleaved by intracellular esterase, releasing 2',7'-dichlorofluorescein which cannot pass through the cell membrane and is oxidized to 2' by H ₂ 0 ₂ , 7'-dichlorofluorescein (DCF) emits fluorescence; on the other hand, Hydroethidium can be directly oxidized by '0 ₂ - to phenanthridine bromide (EB), which fluoresces after being embedded in nucleic acid. The cells were then treated with dihydroxyphenyl lactic acid (0.5 mg/ml) or salvianolic acid B (0.5 mg/ml) and stimulated with lipopolysaccharide (2 yg/ml). After incubation for 20 minutes in ice, the resulting H ₂ 0 ₂ and '0 ₂ were measured by flow cytometry, and the emission wavelengths were measured at 525 nm (FL1, forDCF) and 590 nm.

(FL2, forEB).

Statistical Analysis

Statistical significance was calculated using AN0VA and Fisher's post hoc test. The statistical significance level is p<0.05. The data is expressed as the average S. E. M.

Result

Intervention of dihydroxyphenyl lactic acid and salvianolic acid B on lipopolysaccharide-induced changes in venous red blood cell flow rate: The venous red blood cell flow rate in four experimental groups was measured at different time points (see Table 1). After 30 minutes of lipopolysaccharide infusion, the venous red blood cell flow rate was reduced to a 74% baseline level and this intervention was maintained until the end of the observation. After treatment with dihydroxyphenyl lactic acid or salvianolic acid B, the lipopolysaccharide-induced decrease in venous red blood cell flow rate was significantly reduced.

Intervention of dihydroxyphenyl lactic acid and salvianolic acid B on lipopolysaccharide-induced mesenteric venous shear rate changes: Table 2 shows the mesenteric venous shear rate observed in four experimental groups at different time points. Control group, lipopolysaccharide group, lipopolysaccharide + dihydroxyphenyl lactic acid group and lipopolysaccharide + salvianolic acid B group basal value venule There was no significant difference in shear rate. Mesenteric venules were significantly reduced after administration of lipopolysaccharide, becoming more pronounced after 30 minutes, and this decrease was maintained until 50 minutes after the end of lipopolysaccharide infusion. Treatment with dihydroxyphenyl lactic acid or salvianolic acid B attenuated lipopolysaccharide-induced reduction of mesenteric venous stenosis, and the decrease was small, so it was not significant. Lipopolysaccharide + dihydroxybenzene was detected after 50 minutes of lipopolysaccharide infusion. The rate of shear of the group of lactic acid or lipopolysaccharide + salvianolic acid B was decreased.

Interference of dihydroxyphenyl lactic acid and salvianolic acid B on lipopolysaccharide-induced adhesion of granulocytes on the wall of venules: evaluation of the number of granulocytes attached to the venule wall under different conditions over time The change (see Figure 2). During the observation period, the number of adherent granulocytes in the control group increased only slightly with time. However, the number of adherent granulocytes was significantly increased after lipopolysaccharide stimulation, increasing from the 10th minute of lipopolysaccharide infusion, and this increase was maintained for 50 minutes. Treatment with dihydroxyphenyl lactate or salvianolic acid B significantly inhibited lipopolysaccharide-induced leukocyte adhesion.

Interfering effects of dihydroxyphenyl lactic acid and salvianolic acid B on lipopolysaccharide-induced changes in the number of granulocytes in the venule wall: changes in the number of granulocytes in the venule wall See Figure 3. At all time points, there were little or no granulocytes in the control group with chemotactic migration. The administration of lipopolysaccharide caused a significant increase in the number of chemotactic leukocytes in 50 minutes, and almost no increase occurred after treatment with dihydroxyphenyllactate or salvianolic acid B.

Intervention of dihydroxyphenyl lactic acid and salvianolic acid B on lipopolysaccharide-induced degranulation of mast cells: Figure 4 shows the percentage of degranulated mast cells in the venules of the four experimental groups. A small amount of degranulated mast cells can be detected even in the control group. After 60 minutes of lipopolysaccharide infusion, the number of degranulated mast cells began to increase, and the dihydroxyphenyl lactate or salvianolic acid B treatment group significantly inhibited lipopolysaccharide-induced degranulation of mast cells.

Intervention of dihydroxyphenyl lactic acid and salvianolic acid B on neutrophil CDl lb and CD18 expression in vitro: This study used flow cytometry to analyze differently treated neutrophil surface adhesion molecules CDl lb And CD18 expression. Figure 5A shows that the fluorescence intensity of CDl lb on the surface of neutrophils after lipopolysaccharide treatment was significantly increased compared with the control group, and the fluorescence intensity of CDl lb induced by lipopolysaccharide was added after addition of salvianolic acid B or dihydroxyphenyllactate. 5mg/毫升。 The effective concentration of dihydroxyphenyl lactic acid can be as low as 0. 5mg / ml. Figure 5B shows a similar trend in the adhesion molecule CD18: the neutrophil surface CD18 fluorescence intensity is significantly increased after lipopolysaccharide treatment, using a concentration of 0.2 mg/ml, dihydroxyphenyl lactic acid or salvianolic acid This effect is inhibited after B treatment. In vitro intervention of dihydroxyphenyl lactic acid and salvianolic acid B on the production of '0 ₂ - and 0 ₂ in neutrophil cells: Detection of resistance of dihydroxyphenyl lactic acid and salvianolic acid B by flow cytometry oxidation. After the cells were stimulated by lipopolysaccharide, neutrophils produced a large amount of '0 ₂ —, and after adding 0.5 mg/ml of dihydroxyphenyllactate or salvianolic acid B, the cells were significantly inhibited from producing '0 ₂ — (see Figure 6A). . Compared with the control group, the neutrophil 0 ₂ fluorescence intensity almost doubled after lipopolysaccharide stimulation, and this effect was significantly reduced after treatment with 0.5 mg/ml dihydroxyphenyl lactic acid or salvianolic acid B (see Figure 6B). ).

The results showed that both dihydroxyphenyl lactic acid and salvianolic acid B could improve lipopolysaccharide-induced mesenteric microcirculation disturbance in rats. Including inhibition of leukocyte adhesion and chemotaxis, reducing red blood cell flow rate and venous shear rate reduction, inhibiting mast cell degranulation. Lipopolysaccharide-stimulated neutrophil adhesion molecules CD11b and CD18 expression and '0 ₂ and 3⁄40 ₂ production can be inhibited. These results provide a new alternative to the clinical treatment of LPS-induced sepsis.

Table 1

The venous red blood cell flow rate at different time points after infusion of saline solution or lipopolysaccharide.

The venous red blood cell flow rate was measured at 0, 10, 30 and 50 minutes after infusion of saline solution or lipopolysaccharide, with or without administration of dihydroxyphenyl lactic acid or salvianolic acid B, as described in Materials and Methods. Data are expressed as the mean S. E. M of 6 rats. Vein RBCsi velocity (mm/sec)

0 minutes 10 minutes 30 minutes 50 minutes control (n=6) 1.91 ±0.35 1.89 + 0.29 2.18 ±0.25 1.99±0.17 Lipopolysaccharide (n=6) 1.90±0.14 1.52±0.17 1.41 ±0.09* 1.35 ±0.11* Lipopolysaccharide+ Dihydroxyl L79±0.26 1.79±0.33 1.78 ±0.27 1.50±0.20 Phenyllactic acid (n=6)

Lipopolysaccharide + salvianolic acid 1.90 ± 0.33 1.93 ± 0.32 1.94 ± 0.35 1.78 ± 0.33 B (n = 6) Rats in the control group were perfused with vehicle (saline solution) (8 ml / kg body weight / hr) _; Polysaccharide (2 mg/kg body weight/hr); Lipopolysaccharide + dihydroxyphenyl lactic acid group Experimental animals were perfused with dihydroxyphenyl lactic acid (5 mg/kg body weight/hr) 20 minutes before infusion of lipopolysaccharide (2 mg/kg body weight/hr). ); lipopolysaccharide + salvianolic acid B, experimental animals infused with lipopolysaccharide (2 mg / kg body weight / hr) 20 minutes before infusion of Dan Phenolic acid B (5mg/kg body weight/hr) ·

*Compared with the control group p<0.05.

Table 2

The venous shear rate at different time points after infusion of saline solution or lipopolysaccharide.

The venous shear rate was measured at 0, 10, 30 and 50 minutes after the infusion of saline solution or lipopolysaccharide with or without the administration of dihydroxyphenyl lactic acid or salvianolic acid B according to the methods described in the materials and methods. . Data are average ± S.E.M of 6 rats. Said.

Ventrical shear rate (s-

0 minutes 10 minutes 30 minutes 50 minutes Control (η=6) 421.18±95·19 397.88±74.85 459.61 ±68.58 426.06±50.02 Lips Multi-sugar

394.95 ±31.90 330.87±44.96 295.52±24.36* 282.94±25.99*

(η=6)

Lipopolysaccharide + two

Hydroxyphenyl milk 417.35±64.11 402.50±69.90 410.05±59.77 338.48±48.86 acid (η=6)

Lipopolysaccharide + Dan

Phenolic acid 375 375.85±58.82 375.15±51.61 380.36±64.61 340.35±61.02

(η=6) Rats in the control group were perfused with vehicle (saline solution) (8 ml/kg body weight/hr); lipopolysaccharide group was perfused with lipopolysaccharide (2 mg/kg body weight/hr) _; lipopolysaccharide + dihydroxyphenyl lactic acid group Rat perfusion

(2mg/kg body weight/hr) 20 minutes before infusion of dihydroxyphenyl lactic acid (5mg/kg body weight / hr); monthly polysaccharide + salvianolic acid group B rats perfused with lipopolysaccharide (2mg / kg body weight / hr) 20 Minutes ago, infusion of salvianolic acid B (5 mg/kg body weight/hr).

*Compared with the control group p<0.05.

BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1. Chemical structure of dihydroxyphenyl lactic acid and salvianolic acid B.

Figure 2. Dihydroxyphenyl lactic acid and salvianolic acid B adhere to the venule wall after infusion of lipopolysaccharide in rats. Granule

Time course intervention for changes in the number of leukocytes. Rats in the control group were perfused with saline solution for 60 minutes (8 ml/kg body weight/hr), and rats in the lipopolysaccharide group were perfused with lipopolysaccharide (2 mg/kg body weight/hr) for 60 minutes. Following the method described in Materials and Methods, rats were infused with lipopolysaccharide (2 mg/kg body weight/hr) intravenously for 2 minutes before intravenous injection of dihydroxyphenyl lactate (5 mg/kg body weight/hr) or salvianolic acid B (5 mg/kg body weight). /hr), continuous infusion until the end of the observation. The total infusion volume was the same for the four experimental groups. The number of chemotactic leukocyte chemotaxis was counted and the results were expressed as the number of cells per 200 μπ venule. Data are mean SEM representation of 6 rats. * p<0.05 compared with the control group. , ^† and lipopolysaccharide ρ <0.05.

Figure 3. Time-course intervention of dihydroxyphenyl lactic acid and salvianolate on the changes in the number of granulocytes in the venules of rats after infusion of lipopolysaccharide. Rats in the control group were perfused with saline solution for 60 minutes (8 ml/kg body weight/hr), and rats in the lipopolysaccharide group were perfused with lipopolysaccharide (2 mg/kg body weight/hr) for 60 minutes. According to the methods described in the materials and methods, rats were injected with lipopolysaccharide (2 mg/kg body weight/hr) intravenously for 2 minutes before intravenous injection of dihydroxyphenyl lactate (5 mg/kg body weight/hr) or salvianolic acid B (5 mg/kg body weight). /hr), continuous infusion until the end of the observation. The total infusion volume was the same for the four experimental groups. The number of chemotactic leukocyte chemotaxis was counted and the results were expressed as the number of cells per 200 μm venule. Data The mean SE M of 6 rats is expressed. * p<0.05 compared with the control group. , ^† and lipopolysaccharide comparison p <0.05.

Figure 4. Intervention of dihydroxyphenyl lactic acid and salvianolic acid B on degranulation of microvessel mast cells after lipopolysaccharide in rats. Rats in the control group were perfused with saline solution for 60 minutes (8 ml/kg body weight/hr), and rats in the lipopolysaccharide group were perfused with lipopolysaccharide (2 mg/kg body weight/hr) for 60 minutes. Rats were infused with lipopolysaccharide (2 mg/kg body weight/hr) intravenously with dihydroxyphenyl lactate (5 mg/kg body weight/hr) or salvianolic acid B (5 mg/kg body weight) 20 minutes prior to the method described in Materials and Methods. /hr, continuous infusion until the end of observation. The total infusion volume of the four experimental groups was the same. The mast cells were degranulated after 60 minutes of lipopolysaccharide infusion. The data were averaged by SEM of 6 rats. *Compared with the control group p<0.05., ^† and lipopolysaccharide were compared p<0.05.

Figure 5. Intervention of dihydroxyphenyl lactic acid and salvianolic acid B on lipopolysaccharide-stimulated neutrophil adhesion molecules CD11b (A) and CD18 (B) expression in vitro. CD11b and CD18 expression were analyzed using flow cytometry. Blood samples were incubated with lipopolysaccharide (24 g/ml) with or without various concentrations of dihydroxyphenyl lactic acid or salvianolic acid B and then incubated with the corresponding antibodies. Red blood cells were lysed, neutrophils were classified, and evaluated by flow cytometry, and the average fluorescence intensity was measured. Data The average soil SE M of 6 rats is expressed. * p<0.05 compared with the control group. , t compared with lipopolysaccharide p <0.05. Figure 6. Intervention of dihydroxyphenyl lactic acid and salvianolic acid B on lipopolysaccharide-stimulated neutrophil production of '0 ₂ — (A) and H ₂ 0 ₂ (B). The neutrophils were incubated with 24 g/ml lipopolysaccharide with or without dihydroxyphenyl lactic acid (0.5 mg/ml) or salvianolic acid B (0.5 mg/ml). The resulting '0 ₂ and 0 ₂ were measured by flow cytometry. Data The average soil SE M of 6 rats is expressed. *Compared with the control group p<0.05. t<Comparative with lipopolysaccharide P<0.05. detailed description:

The invention is further illustrated by the following examples, which are not intended to limit the invention.

Example 1

Tablet

[Prescription] Dihydroxyphenyl lactic acid or salvianolic acid BlOOg Microcrystalline cellulose 50g Micropowder Silica gel 3g Magnesium stearate 1. 5g

[Preparation method] Take the original and auxiliary materials through 100 mesh sieves separately; take notoginsenoside, microcrystalline cellulose, mix well, use 60% ethanol as the binder to make soft materials, pass 20 mesh sieve particles, 60Ό dry, Take out, sift through 30 mesh, add micro-silica gel and magnesium stearate, mix and compress, and make 1000 pieces.

Example 2

[Prescription] Dihydroxyphenyl lactic acid or salvianolic acid B75g Microcrystalline cellulose 37g Microsilica gel 2. 3g Magnesium stearate l . lg

[Preparation method] Take the original and auxiliary materials through 100 mesh sieves separately; take notoginsenoside, calcium sulfate, microcrystalline cellulose, mix well, use 60% ethanol as the binder to make soft materials, and filter the particles through 20 mesh. Dry at 60 ° C, take out, sift through 30 mesh, add appropriate amount of micro-silica gel and magnesium stearate, mix and compress, and make 1000 pieces.

Example 3

[Prescription] Dihydroxyphenyl lactic acid or salvianolic acid B133g Microcrystalline cellulose 66g Microsilica gel

4g

Magnesium stearate 2g

[Preparation method] Take the original and auxiliary materials through 100 mesh sieves separately; take notoginsenoside, calcium sulfate, microcrystalline cellulose, mix well, use 60% ethanol as the binder to make soft materials, pass 20 mesh sieve particles, Dry at 60 ° C, Take out, sift through the 30 mesh sieve, add appropriate amount of micro-silica gel and magnesium stearate, mix the hook, and press to make 1000 pieces.

Example 4

Capsules, take dihydroxyphenyl lactic acid or salvianolic acid BlOOmg, add appropriate amount of starch, magnesium stearate and other accessories, granulate, whole, into the No. 1 capsule, that is.

Example 5

Oral solution, take dihydroxyphenyl lactic acid or salvianolic acid BlOOmg, add appropriate amount of sucrose, preservative, add water to 1000ml, dispense into 10ml - branch, that is, get oral liquid.

Example 6

Granules, taking dihydroxyphenyl lactic acid or salvianolic acid BlOOmg, adding appropriate amount of dextrin, stevioside, dry granulation, whole granules, and dispensing, that is, obtained.

Example 7

Injection, dihydroxyphenyl lactic acid or salvianolic acid B150g dissolved in water, another sodium chloride, ethyl p-hydroxybenzoate dissolved in water, mixed hook, tune

pH value. The water for injection is diluted to 1000 ml, filtered through a hollow fiber membrane, filled, and sterilized. Example 8

Application example, treatment of sepsis

Typical case (1)

Patient Sun X X, male, 30 years old, printer, has headache, nausea, vomiting, bloating, abdominal pain, general discomfort, muscle and joint pain, etc., diagnosed as sepsis

Start injection of dihydroxyphenyl lactic acid injection, size 0. lg / support, 2 times a day three times. After half a month, the symptoms were significantly improved. There were no adverse reactions during the medication.

Claims

Rights request

1. The use of Salvia miltiorrhiza extract for the preparation of a medicament for treating sepsis.

2. Use according to claim 1, characterized in that the Salvia miltiorrhiza extract is dihydroxyphenyl lactic acid.

3. The use according to claim 1, characterized in that the salvia miltiorrhiza extract is salvianolic acid 8.

The use according to claim 1, characterized in that the sepsis is an acute systemic infection caused by the incursion of pathogenic bacteria or conditional pathogens into the blood circulation to produce toxins and other metabolites.

The use according to claim 1, characterized in that the sepsis causes a microcirculatory disorder which is a systemic inflammatory reaction caused by excessive release of lipopolysaccharide.

6. The use according to claim 1, characterized in that the application is a treatment of dihydroxyphenyl lactic acid or salvianolic acid B, and the lipopolysaccharide-induced venous red blood cell flow rate is lowered.

7. Use according to claim 1, characterized in that the application is dihydroxyphenyl lactic acid or salvianolic acid B inhibiting lipopolysaccharide-induced leukocyte adhesion.

8. Use according to claim 1, characterized in that the application is dihydroxyphenyl lactic acid or salvianolic acid B inhibiting lipopolysaccharide-induced mast cell degranulation.

The use according to claim 1, characterized in that the Salvia miltiorrhiza extract is a pharmaceutical preparation composition containing Salvia miltiorrhiza extract.

10. The use according to claim 9, characterized in that the pharmaceutical preparation composition comprises salvia miltiorrhiza extract dihydroxyphenyl lactic acid and salvianolic acid B as pharmaceutically active ingredients, together with a pharmaceutically acceptable carrier, wherein salvia miltiorrhiza extract 9%。 The remainder is a pharmaceutically acceptable carrier.