JPH11318445A - Production of neuraminidase inhibitory substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce the subject neuraminidase inhibitor that can be produced in a large amount independently of the natural conditions and is useful for prophylaxis of infections by adding elicitor or the like to the callus cells in a Daucus plant, accumulating the neuraminidase inhibitor in the callus cells and extracting the accumulated substance. SOLUTION: An elicitor such as an oligomer of galacturonic acid or its oligomer or a heavy metal such as copper sulfate is added to the callus cells of carrot subterranean stem in Daucus plant to accumulate the neuraminidase inhibitory substance in the callus cells, and the substance is extracted to give the objective inhibitor. In a preferred embodiment, the resultant neuraminidase inhibitory substance is purified through the reverse phase chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the field of biochemistry, medicine, and medical care, in which the neuraminidase present on the membrane surface of viruses such as influenza virus is studied to analyze the antigen structure of the virus, the mechanism of infection, and infection. The present invention relates to a mechanism for preventing and infecting pathogenic bacteria such as Salmonella, Vibrio cholerae and Clostridium, and a method for producing a neuraminidase inhibitor that can be used for preventing infection.

[0002] Neuraminidase inhibitors have a function of stopping the action of neuraminidase enzyme activity. Neuraminic acid is a sugar present on the cell surface of humans and microorganisms, and neuraminidase is an enzyme that breaks down glycoside bonds of neuraminic acid.

[0003] Neuraminidase is involved in influenza virus infecting humans, being released from infected cells and infecting other cells. In other words, neuraminidase is an essential enzyme when the virus infects human cells. Conversely, viruses without neuraminidase or viruses with weak neuraminidase activity are difficult to infect humans and animals. At present, development of antiviral agents is very difficult,
There is no effective treatment for the virus. Therefore, neuraminidase inhibitors hold promise as new treatments. Neuraminidase inhibitors inhibit viral infection in mucus at respiratory sites and are thought to be effective for early treatment. Further, since it is known that the virus invades the erythrocyte membrane by its neuraminidase and causes hemolysis, it can be protected by the substance obtained by the present production method.

[0004] On the other hand, bacterial neuraminidase has received less attention than viral neuraminidase, but bacteria that secrete neuraminidase include pathogens such as Salmonella, cholera and Clostridium (such as tetanus, botulinum, and welsh). Neuraminidase inhibitors are thought to be very effective in preventing infection and onset, since they are highly susceptible bacteria and are involved in the transmission of those bacteria to humans. Furthermore, since neuraminic acid is present on the surface of human cells, it is expected that neuraminidase inhibitors will be used as a very useful tool for cell studies, especially for cancer cell studies.

[0005]

2. Description of the Related Art As a method for producing a neuraminidase inhibitory active substance, a chemical synthesis method and a production method using a microorganism are already known. Staphylococcus, Actinomyces and Streptomyces are used as microorganisms. However, the neuraminidase inhibitor produced by these microorganisms has a drawback that its activity is low and extraction and separation procedures are very complicated. Further, the production method by the chemical synthesis method has a drawback that the activity and the yield of the synthesis are low.

[0006]

DISCLOSURE OF THE INVENTION The present invention overcomes the drawbacks of the conventional chemical synthesis method, such as poor yield and complexity of production, and is more effective than inhibitors obtained with conventional microorganisms. In a way that inhibitors can be produced easily and in large quantities,
The purpose of the present invention is to provide a production method advantageous for industrial implementation.

[0007]

Means for Solving the Problems In order to achieve the above-mentioned object of the method for producing a neuraminidase inhibitor, the present inventors have conducted intensive studies. Succeeded in producing and accumulating the target substance, and found that it was suitable for this purpose. The method of the present invention comprises producing a neuraminidase inhibitor by adding an elicitor or a salt thereof or a heavy metal or a salt thereof to cultured carrot callus cells, producing and accumulating a neuraminidase inhibitor in the callus cells, and then extracting the neuraminidase inhibitor. Consists of

[0008] Callus formation and culture methods of the genus Carrot have been studied for a long time, and the conditions are the simplest and most common among plants, and are therefore widely known. Moreover, since it grows fast, it is a material suitable for industrial production. Callus of carrot genus is a typical medium, Murashige-Skoog medium containing inorganic salts and vitamins, sucrose, inositol, auxin and kinetin or natural or synthetic plant hormones showing the same effect as those, and cultured It is common to do.

[0009] Plants sometimes produce secondary metabolites such as antibacterial substances and the like. Elicitors refer to substances that induce a defense response in plants as described above, and include oligomers of galacturonic acid or salts thereof. Oligomers of β-glucan, chitin and chitosan or salts thereof, jasmonic acid, methyl jasmonate, salicylic acid and derivatives thereof, cell walls of plants and microorganisms, and their degradation products are used.
As heavy metals, water-soluble copper salts (eg, copper sulfate), iron salts (eg, iron chloride), mercury salts, lead salts, tin salts, cadmium salts, barium salts, gold salts, silver salts, zinc salts, nickel salts , Cobalt salts, chromium salts, strontium salts and the like. When these elicitors, heavy metals or salts thereof are added to the culture medium of carrot cells of the genus Carrot, the concentration of elicitors is preferably in the range of several ppm to several thousand ppm, and the concentration of heavy metals is preferably several nM to several mM. In addition, the neuraminidase inhibitor is produced and accumulated in the cells several hours after the addition of the elicitor or heavy metal, so that the cells are collected when the concentration reaches the maximum. One method is to wash the collected callus cells with water to remove the components of the medium. Next, the cells are extracted with an organic solvent, for example, an alcohol such as methanol or ethanol, acetone, or ethyl acetate. Preferably, the cells are physically ground to increase extraction efficiency.

The target product produced in this way is characterized by a high activity. The resulting neuraminidase inhibitor reduces virus and bacterial neuraminidase activity by 100
% Inhibition. Also, the resulting inhibitors are thermostable,
Its activity is not reduced by the action of acids and alkalis.

Hereinafter, the present invention will be described in detail with reference to examples. However, this is merely for illustrative purposes, and the present invention is not limited to these examples.

[0012]

Example 1 A method for preparing an oligomer of galacturonic acid as an elicitor.

In a 500 ml Erlenmeyer flask, 0.5% polygalacturonic acid is dissolved in 200 ml of 50 mM acetate buffer (pH 4.5), and pectinase SE-150 which is an endo-type pectin-degrading enzyme is dissolved.
(Trade name, manufactured by Shikibo Co., Ltd.) (200 μg) was added and reacted at 30 ° C. for 30 minutes. Then, it was immediately transferred to a hot water bath at 100 ° C. and left for 30 minutes to stop the reaction. Next, this solution was passed through a cation exchange resin to remove pectin-degrading enzymes and sodium ions, concentrated under reduced pressure, and freeze-dried to obtain a powder of a galacturonic acid oligomer.

[0014]

Example 2 A method for culturing carrot cells of the genus Carrot.

[0015] Carrot cells of the genus Carrot were cultured by a conventional method. That is, after cartilage rhizome was sterilized with 70% ethanol and 5% sodium hypochlorite, a slice of about 5 mm in size was placed on Murashige-Skoog medium (pH 5.6-5.8) with 3% sucrose, 0.01% inositol and 5 μM as plant hormone
The cells were cultured at 25 ° C. on an agar medium supplemented with 2,4-dichlorophenoxyacetic acid and 0.5 μM benzyladenine. After 2-3 weeks, callus formation occurred, so the cells were subcultured to a new medium every 1-2 months and cultured.

[0016]

Example 3 A method for producing a neuraminidase inhibitor.

In 100 ml of Murashige-Skoog liquid medium containing the above plant hormones, etc.
Carrot cells of the genus Carrot of 10 months (fresh weight) were transferred and cultured with shaking at 25 ° C. for one week. One week later, the galacturonic acid oligomer prepared in Example 1 was adjusted to pH 6.0 with sodium hydroxide, and sterilized to 10 mg.
added. Thereafter, shaking culture was performed again at 25 ° C. Twenty-four hours later, the carrot callus cells were collected by centrifugation, washed three times with distilled water, sea sand was added to a mortar, and triturated with methanol. This solution was collected by filtration.

As a purification method, this solution is concentrated by a rotary evaporator and subjected to reverse phase chromatography.
Using 5 g of Sep-Pak C18 (trade name, Waters, USA), elution was performed with 30 ml, 80%, and 100% methanol, respectively, to obtain a 80% methanol fraction, which was concentrated. This was separated by high performance liquid chromatography using Ultrasphere C18 (3.9 mm × 150 mm: trade name, Waters, USA), a reverse phase column, with a linear gradient of acetonitrile and water. Detection was performed by measuring ultraviolet absorption. The eluent conditions were a flow rate of 1 ml / min, 0 minutes (acetonitrile 40%: water 60%), 5 minutes (acetonitrile 40%:
With a linear gradient from 60% water to 35 minutes (100% acetonitrile: 0% water), the substance of the present invention is separated at an elution position of about 15 minutes. If a similar process is performed on carrot calli without the addition of galacturonic acid oligomers, no material is separated at this 15 minute position. rear,
Only the substance eluted at the position of 15 minutes was collected, concentrated and dried by a rotary evaporator, and the yield was measured. As a result, it was 1.7 mg.

[0019]

Example 4 The oligomer of galacturonic acid prepared in Example 1 was replaced with a weakly basic anion exchange resin Sephadex A.
At -25, separation was achieved with a linear gradient of ammonium bicarbonate from 0.3M to 0.6M. The galacturonic acid having a polymerization degree of 4 separated here was collected, desalted by gel filtration, concentrated, and lyophilized to obtain a powder.

In Example 3, 1 mg of galacturonic acid having a polymerization degree of 4 was aseptically added to 100 ml of carrot cell liquid medium instead of an oligomer of galacturonic acid to obtain 2 g of galacturonic acid oligomer.
Four hours later, carrot callus cells were collected, extracted with methanol in the same manner as above, separated and purified by high performance liquid chromatography, and fractionated. The yield at that time was 1.9 mg.

[0021]

Example 5 In Example 3, instead of the galacturonic acid oligomer, copper sulfate pentahydrate was added aseptically to a carrot callus cell liquid medium (100 ml) at a final concentration of 10 μM.
After 4 hours, callus cells of carrot were collected, extracted with methanol in the same manner as in the above example, separated and purified by high performance liquid chromatography, and fractionated. The yield at that time is 1.4mg
Met.

[0022]

Example 6 Virus Inhibitory Activity of Neuraminidase Enzyme Reaction

Final concentration in 50 mM acetate buffer (pH 5.5)
0.5 mM 2 '-(4-methylumbelliferyl) -α-DN-acetylneuraminic acid ammonium (manufacturer, Malkin Shoyu Co., Ltd.) and Newcastle disease virus, Hitchner B1 strain
0.9μuni of Neuraminidase (Sigma, USA)
In addition, ts was added, and 5 μg of the product separated and purified by high performance liquid chromatography obtained in Examples 3 to 5 was dissolved in 75% dimethyl sulfoxide and added, and an enzymatic reaction was carried out in a total volume of 600 ml. The enzymatic reaction was determined by measuring 4-methylumbelliferone released as a result of the enzymatic reaction by measuring the fluorescence intensity at EX 365 nm and EM 450 nm.

As a result, the neuraminidase enzyme activity was reduced by 10%.
0% inhibition.

[0025]

Example 7 Bacterial neuraminidase enzyme reaction inhibitory activity.

The final concentration in 50 mM acetate buffer (pH 4.5)
Example 2 0.5 mM ammonium 2 '-(4-methylumbelliferyl) -α-DN-acetylneuraminate (manufacturer, Malkin Shoyu Co.) and 0.375 μg of Clostridium perfringens neuraminidase (Sigma, USA) were added. 3-5
The product separated and purified by high performance liquid chromatography obtained in was dissolved in 75% dimethyl sulfoxide, added to each concentration, and the enzyme reaction was carried out in a total volume of 600 ml. The enzymatic reaction is
EX 4-methylumbelliferone released as a result of enzymatic reaction
It was determined by measuring the fluorescence intensity at 365 nm and EM 450 nm.

As a result, the concentration I which inhibits the enzyme reaction by 50%
C50 was 21 ppb.

[0028]

Since the present invention is configured as described above, it has the following effects.

Since a cultured cell of a plant is used instead of a cultivated plant as a material, it can be produced throughout the year without being affected by natural conditions in production. Further, manufacturing conditions can be made uniform.

Since a carrot genus that grows fast is used as a plant of the cultured cells, it can be expected in terms of yield, and industrial mass production can be performed.

Further, extraction, separation and purification of the neuraminidase inhibitor can be performed very easily.

──────────────────────────────────────────────────続 き Continuing on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:91) (72) Inventor Norimitsu Hanamatsu 202-4 Ashiya, Aomori-shi, Aomori-shi Aomori Industrial Technology Development Inside the center (72) Inventor Kazu Matsue 202-4 Ashitani, Aoyamori, Aomori Pref.

Claims (2)

[Claims] 1. A method for producing a neuraminidase inhibitor, comprising adding an elicitor or a heavy metal to a callus cell of the genus Carrot, accumulating a neuraminidase inhibitor in the callus cell, and extracting from the callus cell. 2. The production method according to claim 1, wherein the target substance is purified by reverse phase chromatography.