KR970003050B1 - Phospholipase a2 inhibitor - Google Patents

Abstract

The phospholipase A2 inhibitor useful as an anti-inflammatory analgesic agent was prepared. Thus, Bacillus subtillus PI-003 was cultured at 30 deg. C for 5 days in 4 l of a culture medium containing 2.5 % of glycerol, 0.5 % of beef extract, 1 % of enzyme extract, 0.05 % of MgSO4= 7H2O, 0.05 % of K2HPO4, 0.32 % of CaCO3 and 0.2 % of NaCl. A cell wall was destroyed by adding isopropyl alcohol, and vacuum concentrated, followed by dissolution of 50 % isopropyl alcohol after absorption in a hydrophobic resin HP-20 to give surfactin.

Description

Phospholipase A2 Inhibitors

1 is a preparative high performance liquid chromatography, and the conditions of preparative high performance liquid chromatography are as follows. :

Column: Connection column of Waters 製 uBondapack C ₁₈ , 30mm * 30cm and Supelco 製 SUPELEOSIL ^tm PLC-18, 21.2mm * 25cm

Flow rate: 20 ml / min

Mobile phase: 90% acetonitrile (pH 5.0)

Detection: uv220nm

Warranty time of the suspectin (R / T): 27.13 minutes

The present invention relates to a phospholipidase A2 (Phospholipase Az, PLA ₂ ) inhibitor.

Phospholipase Eto is arachidonic acid that is a precursor of lipids such as prostaglandin, which is collectively called eicosanoid and acts on phospholipids, which are cell membrane components, when reactive cells are stimulated by external signals and activated. It is an enzyme that releases arachidonic acid.

These chemical transporters play a central role in the pathology of various diseases such as inflammation and circulatory system diseases. In particular, platelet activating factor (PAF) is called PLA ₂ and acetyltransferase. Produced by the chain reaction of the two enzymes are reported to cause various inflammatory reactions, endotoxin shock, asthma and the like.

Inflammation-induced pathways caused by phospholipidase are as follows.

As a therapeutic agent for various inflammations, cerebral hemorrhages, and cardiovascular diseases known to date, nonsteroidal drugs that block only prostaglandins produced by the cyclooxygenase pathway from arachidonic acid are widely used. The drug cannot block the biosynthesis of leukotriene, a metabolite of the lipoxygenase pathway, another route of arachidonic acid, so only a partial therapeutic effect is expected, and it also causes various side effects for long-term administration. In addition, the steroidal drug that has been used as a PLA ₂ inhibitor has a serious side effect as much as the drug, which requires great attention.

Therefore, the emergence of new drugs of new mechanism is receiving hot attention worldwide. The present inventors separated PLA ₂ inhibitors from soil microorganisms in order to solve the above problems, and as a result of analysis of the physicochemical properties of the separated material, it was identified as a suspectin (Surfactin) which is a known compound having the following structural formula. PLA ₂ 50% inhibitory concentration (IC ₅₀ ) of the foreign material was 1.01 * 10 ^-5 M, showing a strong anti-inflammatory analgesic effect even in the in vivo test (in vivo test).

PLA ₂ used in the present invention, unlike the existing group I, II PLA ₂ (extracellualr PLA ₂ ) is intracellular PLA ₂ (cytosolic PLA ₂ ) having arachidonic acid specificity, molecular weight of about 100,000 Daltons (100kd) isolated from bovine platelets Was used.

The suspectin identified as a PLA ₂ inhibitor in the present invention is known as K Arima et al., Biochemical end biophysical research communication [Kei, Arima et al, Biochim. Biophys. Res. Comm. 31 (3), 488 (1968)], Yukio Kameda et al., Chemical and Pharmacuty Bulletin [Yukio Kameda et al, Chem. Pharm. Bul1. 20 (7), 1551 (1972), etc., and summarized in Table 1 for the pharmacological effects of the suspectin.

List of Pharmacological Effects of Suspectin

TABLE 1

However, no pharmacological effects of suspectin on PLA ₂ inhibition and anti-inflammatory analgesic have been reported. The inventors of the present invention isolated a strong inhibitor from Bacillus subtilis PI-003 (KFCC-10786) while searching for a substance having a PLA ₂ inhibitory activity using a fermentation product of a microorganism, which is known as The present invention was completed by confirming that the compound is a suspectin, and also confirming that the substance has anti-inflammatory and analgesic effects in an extension of PLA ₂ inhibitory activity. Hereinafter, the suspectin producing strain and its preparation method, PLA ₂ inhibition test and anti-inflammatory and analgesic test results are shown in the examples.

Example 1

Soils collected from all over the country were suspended and diluted in sterile water and plated on NA medium to separate microorganisms. The isolated microorganisms were inoculated in LB medium and incubated at 25 ° C. for 5 days. A portion of this culture was taken to isolate strains showing PLA ₂ inhibitory effects.

Among the strains isolated by the above method, the strain showing the best PLA ₂ inhibitory effect is the microorganism Bacillus subtilis PI-003 (KFCC-10786) of the present invention.

These microorganisms are gram-positive and motile at the beginning of growth, are rod-shaped, catalase-positive, oval endospores are produced, and acids are produced from glucose. Bacillus). The strain also grows at 55 ° C, grows in 7% salt, produces acids in L-arabinose, D-xylose, and D-mannitol, and releases carbon dioxide during glucose breakdown. Does not form, decomposes gelatin, starch, does not use citric acid, does not use propionate, reduces nitrate, does not deamine phenylalanine, In addition, the characteristics that produce acetoin (acetoin) during the glucose oxidation process prove that this strain is Bacillus subtilis. Therefore, this strain was named Bacillus subtilis PI-003 and deposited in KFCC. The accession number is KFCC-10786.

The characteristics of Bacillus subtilis PI-003 are shown in Table 2 below.

Morphological and Physiochemical Characteristics of Bacillus subtilis PI-003

TABLE 2

Example 2

Bacillus subtilis in medium 4L (pH 7.4) containing 2.5% of gglycerol, 0.5% of broth, 1% of yeast extract, 0.05% of MgSO ₄ 7H ₂ O, 0.05% of K ₂ HPO ₄ , 0.32% of CaCO ₃ and 0.2% of NaCl. Bacillus subtillus) PI-003 was incubated at 30 ° C for 5 days, the same amount of isopropyl alcohol was added to destroy the cell wall, concentrated under reduced pressure and adsorbed onto a hydrophobic resin, HP-20 (Samyang), followed by 50% isopropyl. Eluted with alcohol. The eluate was concentrated under reduced pressure, extracted with a mixed solvent of chloroform-methanol 4 to 1 ratio, and the extract was concentrated under reduced pressure again. This concentrate was aliquoted with 0.01 M Tris-HCl (pH 7.5) buffer. The fractions were concentrated under reduced pressure, extracted with chloroform, concentrated under reduced pressure, and the concentrate was dissolved in methanol, and 30 mg of pure material was isolated by preparative high performance liquid chromatography (Waters prep LC 2000) (FIG. 1). The separated material was white powder and identified as a known substance, suspectin, as a result of physicochemical and instrumental analysis.

Example 3

Experiments for measuring PLA ₂ inhibitory activity were carried out in accordance with the method of Kim, Dae-Kyung et al. The measurement method is as follows.

10 mM 1-acyl-2- [1- ¹⁴ C] arachidyl L-3-phosphatidylethanolamine, 50-60 mci / mmo (hereinafter, substrate), was dried with nitrogen gas and suspended in 1 ml of distilled water at room temperature. Ultrasonication was performed 4 times at 15 second intervals to prepare a reaction mixture of 10ul CaCl ₂ , 150mM Tris-HCl (pH 7.5), 50ul solution 110ul substrate consisting of 1mg / ml BSA, 20ul sample solution and 20ul 50μg / ml PLA ₂ enzyme solution. It is made to react at 60 degreeC for 60 minutes. 1.25 ml of isopropyl alcohol: heptane: 1N sulfuric acid (40:10: 1) solution was added to the reaction solution to stop the reaction. Here, 1.2 ml heptane and 1 ml of water were mixed and mixed well. Heptane and 150 mg of Biosil-A were mixed and centrifuged, and the supernatant was taken to measure the beta radiation dose of ¹⁴ C-arikidonic acid liberated from the substrate.

The 50% inhibitory concentration of ICectin (IC ₅₀ ) measured in this way was 1.01 * 10 ^-5 M.

Example 4

In the peritoneal cavity of male rats (weight 150-180 g), twice a dose of 20 mg / kg of serpentine was injected at two hours intervals. The hourly edema was then measured by Plethysumeta. Distilled water was administered to the control group and tested in the same manner. The edema rate was calculated by the following equation and the inflammation inhibition rate of the suspectin was calculated by comparing with the control group. Five rats were used for the suspectin administration group and the control group, respectively.

The edema inhibition rate of the suspectin measured by this method was about 50% inhibition 2 hours after the administration of the baseline agent.

Example 5

In the peritoneal cavity of an ISI male (ICR) male mouse (about 20 g body weight), a dose of 2 mg / kg was administered in a dose of 2 mg / kg, followed by 0.7% physiological saline solution intraperitoneally. Starting after 10 minutes, the number of riding symptoms (10 minutes) was measured and fertilized with the control group. In the control group, the number of riding symptoms was measured by experimenting with distilled water after the same method. Experimental and control groups used 10 mice each. Serectin showed an inhibition rate of about 80% compared to the control at a dose of 2 mg / kg.

Claims (1)

Phospholipase Az inhibitors and anti-inflammatory analgesics, characterized by the following molecular formula: