JPH0449241A - Antirheumatic agent and antirheumatic agent for animal - Google Patents

Abstract

PURPOSE:To obtain an antirheumatic agent, containing a lipopolysaccharide (LPS) of a specific macrophage activating ability, having a high chemotherapeutic coefficient with hardly any side effects and suppliable in a large amount. CONSTITUTION:An antirheumatic agent is obtained by containing an LPS in which the content of an LPS, positive to limulus tests and providing an ED50 of the macrophage activating ability is 0.4-100ng/ml culture solution when a sigmoid curve indicating the macrophage activating ability (%) by standarizing the activating ability to provide a TNF production of the macrophage without addition of the LPS to be 0% and the activating ability to afford the maximum constant amount of the TNF production to be 100% in the ordinate axis, and the LPS content positive to the limulus tests in the LPS on a logarithmic scale in the abscissa axis is plotted using the macrophage activating ability of the LPS to activate the TNF productivity of the macrophage cultured in vitro as an index. An LPS collected from bacteria or plants or synthetic lipid A, etc., can be used as the LPS.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an anti-rheumatic agent and an anti-rheumatic agent for animals.

[Prior Art] Rheumatoid arthritis is a representative inflammatory disease that is pathologically classified as a collagen disease and is common to both humans and humans.

In animals, it occurs relatively frequently in cattle, especially housed cattle, followed by dogs and goats, and rarely in horses and pigs. Small animals can be kept alive if appropriate treatment is continued, but large animals tend to suffer from malnutrition and die due to secondary infection. (pages 213-214 of “New Veterinary Handbook” by et al.)
.

Since rheumatoid arthritis is a stubborn and intractable systemic disease, it is necessary to provide appropriate treatment over a long period of time to maintain daily life and social life.

As one of the basic treatments for rheumatoid arthritis, drug therapy occupies a fairly large scope, and aspirin, nonsteroidal anti-inflammatory drugs, corticosteroids, and the like are used. Among these, non-steroidal anti-inflammatory drugs are most commonly used.

[Problems to be Solved by the Invention] However, all conventional drugs have problems with side effects, and great care is required in administration. (■Metal Publishing, edited by Yasuhito Itami et al., Orthopedics MOOK 11 Jo, 37r Treatment of Chronic Articular Rheumatoid Arthritis, pp. 5-19, 1984).

In view of the current situation, the present invention has a high anti-rheumatic effect, few side effects, and therefore has a high chemotherapeutic coefficient, and
This was completed in order to provide a new anti-rheumatic agent and an anti-rheumatic agent for animals that is low in production cost, can be administered orally, transdermally, or by injection, and can be supplied in large quantities.

[Means for Solving the Problems] The present invention provides an anti-rheumatic agent containing LPS and an anti-rheumatic agent for animals. This anti-rheumatic agent and anti-rheumatic agent for animals uses the macrophage activation ability of LPS, which activates the TNF production ability of macrophages cultured in vitro, as an index, and the vertical axis shows the macrophage activation ability when the LPS is not added. 0% macrophage activation ability that gives TNF production
, the amount of TNF produced by macrophages is maximized and constant fl
The ability of LPS to activate macrophages at 10%
When drawing a sigmoid curve that represents the macrophage activation ability (%) with 0% and represents the Limulus test positive LPS content of that LPS on a log scale on the horizontal axis, the Limulus test positive LPS content that gives the ED5s of the macrophage activation ability. is included in the range of 0.4 to b.

Here, "containing at least one" means the LPS of the present invention.
This means that not only can they be used individually, but also two or more of them can be used in any combination, or even in combination with any other substance, as long as the intended use is not hindered. .

"Macrophage" is a type of immunocompetent cell, and is a general term for large amoeboid cells that are distributed in almost all tissues in the animal body and prey on and digest particulate foreign substances and waste cells in the body. . ``TNFJ is a tumor-damaging factor produced by macrophages.
osisFactor) [The Journal of Biological Chemistry, published in 1985]
, Chem, pp. 2345-2354, as the activity of macrophages increases, the amount of macrophages produced increases.

The Limulus Test was developed in 1968 by Lev
This is a method for quantifying endotoxin using a horseshoe crab blood cell extract and a chromogenic synthetic substrate, which was invented by Dr.

LPS that can be used as an active ingredient in the anti-rheumatic agent and anti-rheumatic agent for animals of the present invention is not particularly limited in its collection source, production method, or purification method. For example, LPS collected from bacteria or plants may be used. Alternatively, it may be a synthetic product such as synthetic lipid A. In addition, in this specification,
In particular, within the scope of the claims, the collection source is not limited to those specifically identified by name, but includes all bacteria and other things that adhere to or coexist with the collection source during the growth, preservation, and distribution processes. 9 For example, "If wheat LPSJ is identified, LPS collected from wheat itself"
In addition, it should be understood that it includes all bacteria and other substances that adhere to or coexist with wheat during the growth, storage, and distribution processes. This is because, in addition to those whose relationships have been elucidated, such as parasitic plants and animals, there are also certain plants, animals,
This is because it is well known in the art that there are many examples of organisms living in the fungal and lichen kingdoms that are allowed to adhere to and coexist with them.

Among these LPS, LPS that can be used as an active ingredient of the anti-rheumatic agent of the present invention and the anti-rheumatic agent for animals
To select the macrophage activity, the macrophage activation ability of LPS, which activates the TNF production ability of macrophages cultured in vitro, is used as an index, and the vertical axis is the macrophage activity, which gives the amount of TNF production by macrophages when the LPS is not added. 0%
, represents the macrophage activation ability (%) with the macrophage activation ability of LPS taken as 100% when the macrophage produces the maximum amount of TNF, and the horizontal axis shows the LPS
When drawing a sigmoid curve expressing the Limulus test-positive LPS content on a logarithmic scale, the Limulus test-positive LPS content that gives the EDss of macrophage activation ability is 0.4 to 100 ng/m of culture solution.
What is necessary is to select the one that is l.

Limulus test positive bacterial source LPS: previously known Escherichia coli LPS, Bordetella pertussis LPS,
This applies to lipids, etc.

The large 1111iLPS is, for example, the American Difco (D-i
It is commercially available from fco.

Pertussis 1iLPS is commercially available from Funakoshi Pharmaceutical Co., Ltd., for example. It can also be prepared from killed cells of a known Bordetella pertussis, for example, Higashihama strain II, by a known method described in the following literature.

Webster et al., J. Immuno 1., 744.55
(1955): “Z, Naturforsch” by We-stphal et al.
”, 76.14B (1952).

Lipid A is commercially available, for example, from Daiichi Chemical.

Limulus test positive plants tTjLPs Examples of plants that can be used as raw material plants are listed below. The family and genus names to which the plants described in this specification belong were determined by comparing the descriptions in the following documents.

Tricots, monocots, dicotyledons, ferns, and grasses: descriptions from the ``Illustrated Encyclopedia of Primary Colored Wild Plants'' published by Hokuryukan in 1982 (Sho & I) and 1988 (Subsequent) The affiliation was determined by cross-checking. However, "Oat" is referred to in the "Illustrated Guide to Edible Plants" published by the Women's Nutrition University Press in 1971, and the "New Japanese Botany Flower Edition" published by Shibunkyo in 1981.
``7x barley'' was determined by comparing the description in the R Comprehensive Food Encyclopedia J published by the Tokyo Dobun Shoin in 1971.
``Hatomugi'', ``Crowbishaku'', ``Janohige'', ``Turmeric'', ``Actinifolia'', ``Gynostemma'', ``Dokudami'', ``Pepper'', ``Capsicum'', ``Daiwikyou'',
``Daidai'', ``Kuzu'', ``Nankin Glory'', ``Otanene Carrot'', ``Bofuu J'', ``Oozurafuji J'',
``Uncaria hirsuta'' was determined by checking the description in ``Primal Makino Illustrated Encyclopedia of Japanese and Chinese Medicinal Herbs'' published by Hokuryukan in 1986, and ``Avocado'' was determined by the information published by the Agricultural and Forestry Statistics Association in 1978. By comparing the description in Tropical Agricultural Technology Series No. 15, "Fruits of Brazil,""Kaiwareradish" was found in the "Illustrated Encyclopedia of Primary Colored Garden Plants" published by Hokuryukan in 1981.
``Nikuzuku'' was compared with the description in ``Illustrated Tropical Plants Collection'' published by Kyusei Shoten in 1962, and ``Chlorella'' was published by the Japan Health Food Association in 1939.
The affiliation was determined by comparing the descriptions in the ``Chlorella Standards'' published in 2017.

Class III: The affiliation was determined by comparing the descriptions in the ``Primary Colored Japanese New Fungi Illustrated Book'' published by Yokusha in 1986. However, the affiliation of yeast was determined by checking the descriptions in the ``Microbiology Handbook'' published by Gihodo in 1963, and the affiliation of ``Cordyceps sinensis'' was determined by checking the descriptions in the ``Prisei Makino Encyclopedia of Japanese and Chinese Medicinal Herbs'' mentioned above. .

The raw material plants that can be used in the present invention include, for example, gymnosperms, monocots, dicots, ferns, grasses, and fungi, and these can be used individually or in a mixture.

As the gymnosperm, for example, pine, which is a plant of the Pinaceae family and genus Pinus, can be used.

Examples of monocotyledonous plants include rice, a plant belonging to the genus Poaceae, wheat, a plant belonging to the genus Triticum, a plant belonging to the genus Triticum, barley, barley, a plant belonging to the genus Barley, a plant belonging to the genus Barley; , Kusa bamboo, which is a plant of the genus Bamboo in the family Poaceae, Hatomugi, a plant of the genus Juzdama, genus Graine, Iris, a plant of the genus Iris, genus Iris, garlic, Garlic, a plant of the genus Allium, genus Liliaceae, Asparagus, a plant of the genus Acanthus, genus Liliaceae It is possible to use a plant belonging to the genus Araceae, myoga, a plant belonging to the genus Zingiberaceae, a plant belonging to the genus Zingiberaceae, turmeric, and a plant belonging to the genus Hange, belonging to the family Araceae.

Examples of dicotyledonous plants include soybeans, which belong to the genus Soybean, which belongs to the Fabaceae family, adzuki beans, which belong to the genus Phaseolus, which belongs to the Fabaceae family, fava beans, which belong to the genus Fabaceae, and kudzu, which belongs to the genus Kudzu, which belongs to the Fabaceae family.
Glycyrrhiza, a plant of the genus Licorice of the Fabaceae family; Potato, a capsicum, a plant of the genus Solanum of the family Solanaceae; Tomato, a plant of the genus Tomato of the family Solanaceae; Capsicum, a plant of the genus Capsicum of the Solanaceae family; Loquat, a plant of the genus Loquat of the Rosaceae family; Rosaceae Peach, which is a plant belonging to the Prunus genus, avocado, which is a plant belonging to the Lauraceae family, Avocado lll1M, and walnut, which is a plant belonging to the Walnut family,
Kapocha, a plant of the genus Solanum of the Cucurbitaceae family, Jiaogulan a plant of the genus Jiaogulan of the family Cucurbitaceae, daikon radish a plant of the genus Radish of the family Brassicaceae, plant of the genus Actinidia of the genus Actinidia of the family Actinaceae, Houtumia genus of a plant of the genus Polygonum of the family Hemoraceae; Hukai, which is a plant of the genus Shikimi, which is a plant of the genus Shikimi, a plant of the genus Shikimi, which is a plant of the genus Shikimi, a plant of the genus Shikimi, which is a plant of the genus Shikimi, which is a plant of the genus Shikimi, which is a plant of the genus Shikimi, which is a plant of the genus Shikimi, which is a plant of the genus Shikimi, which is a plant of the genus Shikimi of the family Apiaceae; It is possible to use Uncaria hirsuta, which is a plant belonging to the genus Occidentalis, belonging to the family Rubiaceae, and Uncaria hirsuta, which is a plant belonging to the genus Occidentalis, family Rubiaceae.

As the fern plant, for example, horsetail, which is a plant of the genus Horsetail of the family Equisetaceae, and japonica, which is a plant of the genus Centrum of the family Medicinalaceae, can be used.

Examples of grass-like plants that can be used include grass-like plants, red grass-like plants, red-flowered grass plants, green grass-like plants, and orchid-like plants.

Examples of daylily plants include wakame, a plant belonging to the genus Wakame of the family Laminaceae, kelp, a plant belonging to the genus Laminaria of the family Laminaria,
A plant belonging to the genus Sargassum, belonging to the Sargassum family, can be used.

As the plant belonging to the genus Porphyra, for example, Porphyra elegans, which is a plant belonging to the genus Porphyra in the family Prunusaceae, can be used. As the green grass plant, for example, chlorella, which is a plant of the genus Chlorella in the family Oocystis family, can be used.

As the fungal plant, for example, a basidiomycete plant or an ascomycete plant can be used. Basidiomycete plants include, for example, Shiitake, which is a plant of the genus Chrysanthemum in the family Pleurophyllaceae, L. tsukitake, a plant of the genus Enokitake in the family Chrysanthemum, shimen, a plant of the genus Shimeji in the family Chrysanthemum, maitake, a plant of the genus Maitake in the family Chrysanthemum, and Polyporus, a plant belonging to the genus Maitake, family Salmonaceae. Abalone mushroom, which is a plant of the genus Agaricaceae, pine mushroom, which is a plant of the genus Agaricaceae, of the family Agaricaceae, wood ear fungus, a plant of the genus Aeolus of the family Asteraceae, and nameko, a plant of the genus Sugitake of the family Araceae, can be used.

As the Ascomycete plant, for example, baker's yeast and brewer's yeast, which belong to the family Endomycetaceae and the genus Saccharomyces, can be used. Brewing yeasts include beer yeast, clear yeast, wine yeast, soy sauce yeast, miso yeast, etc., as well as many yeasts belonging to the genus Saccharomyces cerevisito (e.g., yeast used in the production of whiskey and aged sake). . Cordyceps sinensis, which is a plant of the genus Cordyceps of the family Acanthaceae, can also be used.

The above-described detection and content measurement of Limulus test-positive LPS in raw material plants can be carried out using, for example, a medicine set commercially available from Seikagaku Corporation under the name Toxicolor System.

That is, the raw material plant is colored by combining it with the LS-1 set of the same system, and the strength of the color development is measured by combining it with the E of the same set.
It may be compared with a calibration curve created using the t-2 set.

Plant ffLPs can be separated and purified by the method described below.

■After cutting, drying, and pulverizing the raw material plants as necessary, thoroughly suspend them in distilled water and collect the top layer.

For example, if the raw material plant is a grain seed, it can be easily crushed with the seed coat still attached, or after the seed coat has been removed, or it can be crushed until it becomes an edible powder.
Water is added to the resulting powder to make a dispersion, and after stirring, the sediment is left to stand or removed by centrifugation, or water is added to the powder and kneaded, and the resulting tow is gently washed with water in a mixer and allowed to settle. Just remove things.

When the raw material plant is chlorella, it is preferable to crush the cell membrane first, remove fat-soluble substances by washing with ethanol, and then extract with water.

There is no need to particularly limit the particle size of the raw material plant, water temperature, liquid properties, amount added, stirring speed, time, conditions for centrifugation, etc. during this water extraction, and can be adjusted as appropriate depending on the type of raw material plant. Just adjust it. In addition, the higher the temperature of the extracted water, the higher the LPS collection amount and purity, but for convenience of operation, it is preferably set to 50° C. or lower so as not to cause gelatinization of the starch contained in the raw material plants. The amount of water added varies depending on the type and particle size of the raw material Mγ, but in the case of grain seeds, the ratio is 70 w/v% or less, preferably 20-50 w.
/V% is convenient for operation. Furthermore, it is preferable that the stirring speed is at a level that does not cause foaming. Furthermore, up to this stage of operation, the purity of the Limulus test-positive M product LPS of the present invention is determined based on the Limulus test activity data. For example, in the case of wheat seeds, it increases about 30 times.

The following will explain the case where grain seeds are used as a raw material, but a person skilled in the art would be able to remove contaminant sugars, proteins, etc. from other plants and produce Limulus test positive LPS by referring to the following description. It is extremely easy to implement a method for recovering with high purity.

(2) In order to further increase the purity, the upper layer obtained in (2) above may be subjected to ultrafiltration according to a conventional method to remove components having a molecular weight of 5,000 or less.

(2) The obtained dried product is suspended in distilled water to a concentration of 50 mg/m SL, and the supernatant is collected by centrifugation.

■ Cool this supernatant with ice water and add acid to make it acidic, forming a precipitate. The acid used at this time does not have to be a specific acid, for example, trichloroacetic acid #1 (hereinafter referred to as TCA
), perchloric acid, trifluoroacetic acid, FIR, dichloroacetic acid can be used.

(2) Next, the precipitate is collected by centrifugation, washed with distilled water, and then centrifuged again to collect the precipitate.

■ Suspend the precipitate in distilled water and add alkali until the precipitate is dissolved. The alkali used at this time does not need to be a specific one, and for example, sodium hydroxide, potassium hydroxide, ammonia, sodium carbonate, and sodium acetate can be used. If the basicity becomes higher than pH 11 when dissolving the precipitate, the target LPS will be deactivated, so care must be taken.

■Next, add acid to adjust the pH to 8, then warm to 37°C.
Adding more acid to make it acidic will cause precipitation, so at 37℃
Centrifuge using a centrifuge kept warm.

Note that the acid used at this time does not need to be a specific one.

(2) The supernatant is collected, cooled with water, and centrifuged again at 4°C. (2) The supernatant is collected, neutralized by adding alkali, and concentrated by ultrafiltration according to a conventional method. The alkali used at this time does not need to be a specific one either.

[Phase] Next, gel filtration is performed according to a conventional method, and the Limulus test positive fractions are collected and combined. As a carrier for gel filtration, for example, Sephadex G
-75, G-100゜Sephacryl
l) S-200, Sepharose
6B [The above is a product of Pharmacia, Inc.
aInc, ), Biogel P-1
00 [manufactured by Biorad Inc., USA], Toyo Pearl HW-50, and HW-55 (manufactured by Toyo Soda Kogyo Co., Ltd.) can be used.

Any buffer solution having a pH of 3 to 10 may be used.

For example, Tris-HCt or phosphate buffer can be used.

Next, a protease is added to both portions, and the mixture is incubated at 37° C. for 2 hours or more to decompose the remaining proteins, and the resulting enzyme-treated solution is concentrated by ultrafiltration according to a conventional method. Note that the proteolytic enzyme used at this time does not need to be specific; for example, v8 protease, chymotrypsin, trypsin, and thermolysin can be used alone or in any combination. Commercially available products include, for example, Pronase E (Kaken Chemical Co., Ltd.) and Proteinase K.
(Merck) can be used.

@Then, this fraction is treated in accordance with a conventional method, for example, using a FPLC system manufactured by Pharmacia, Inc., in the United States, using MonoQ-Seph-arose, Q-Sepharose, manufactured by Pharmacia, Inc. A Limulus test positive fraction is obtained by subjecting it to anion exchange chromatography.

@Next, the Limulus test positive fraction is collected by gel filtration for desalting according to a conventional method.

By the above operations, in the case of wheat seeds, about 20% of the initial limulus activity is recovered and a purified sample with a purity of about 95% is obtained. In addition, the purity at the end of stage ■ is about 1000%
twice as pure (for wheat seeds).

Limulus test positive plant L obtained by the above method
PS can be provided neat or in any degree of concentrated form. Furthermore, in order to improve storage stability, it can also be provided as a dry powder by any means such as freeze-drying or spray-drying. All of these can be produced using conventional methods.

Carswell said that in order to produce TNF in an animal body, a production precursor (briming) stage and a production initiation (triggering) stage are necessary.
et al., Proceedings of the National Academy of Sciences of Ninisny [Proc.
tl.

Acad, Sc], USA, 72.3666-36
70 (1975)].

The drug administered to initiate the briming phase is a ``brimer'' (endogenous TNF production promoter), and the drug administered to initiate the triggering phase is a ``triker''.
(endogenous TNF producing agent).

To measure the ability of LPS to activate the in vitro TNF production capacity of macrophages, mouse macrophage resident peritoneal cells were collected, fresh mouse IF N-r was added as a primer, and then , LPSfe may be added as a trigger, and the TNF activity thereof may be measured.

TNF activity is measured as follows based on cytotoxicity to L-929 cells [Proceedings of the National Academy of Sciences of Ninisny 72, pp. 3666-3670].

L929 cells were grown in Eagle's minimum essential medium (hereinafter referred to as MEM medium) supplemented with 5% calf fat serum.
The cells are grown in 96-sized flat-bottomed plates so that 8XIOJ cells are contained in 100 μl of the same medium.

Breeding conditions are 37℃, 2 hour leap, 5% CO2, 100% H2
0, and any method used for normal cell culture may be used. Then, actinomycin D was added to the medium at a final concentration of 1 μg/m.
λ, and make the volume of the culture medium 150 μ9. Immediately add 50 μl of the sample appropriately diluted with MEM medium (at this time, adjust the dilution rate appropriately and add ED5a
The final volume of the solution was reduced to 200 μm, and the 929 cells were cultured under the above conditions for 18 hours.

To measure cytotoxic activity, first remove all the medium, then add a 1% methyl alcohol solution containing 0.1% cristalviolet to fix and stain. Crystal violet stains all nucleated cells, but since dead cells are removed from the bottom of the plate by washing with water after staining, cytotoxic activity can be directly measured from the results for viable cells. The degree of staining is measured using the absorbance at OD 59111111 as an index, and the cytotoxic activity is determined by comparing the degree of staining with that of the control group. The definition of activity is as follows.

The dilution rate (N) of the sample at which 50% of L929 cells can survive is determined. As a control, rabbit TNS [tumor-damaged serum (T
The activity n (units/m9) of this rabbit TNS is determined using 24X106 units/mg/m LcD TN F-α. The dilution rate (C) that gives the EDs@ of this rabbit TNS is determined.

Analyte activity (units/ml) is calculated as −× n .

Method for manufacturing the agent that can be provided The antirheumatic agent of the present invention can be prepared as a powder, granule, gunpowder, tablet, troche, capsule, liquid, patch, ointment, liniment, or lotion using conventional formulation techniques. It is available in the form of , skewers, injections, etc. Furthermore, for animals, it can also be prepared as a feed additive, premix preparation, or drinking water additive. When used as a feed additive, it is preferably in the form of powder or granules. Moreover, a premix preparation refers to a preparation diluted with feed ingredients such as starch in order to facilitate mixing with feed. The feed to which the anti-rheumatic agent of the present invention can be added as a feed additive or premix formulation may be any commercially available feed, or may be a feed containing feed additives such as minerals, vitamins, and amino acids.

If desired, additives such as excipients, preservatives, buffers, etc. can be added to these preparations by conventional methods in order to maintain preservability and homogeneity. Additionally, flavoring agents, flavoring agents, and coloring agents may also be included.

As excipients, for example, lactose and starch can be used. As the preservative, for example, paraoxybenzoic acid esters such as methyl paraoxybenzoate, ethyl paraoxybenzoate, and propyl paraoxybenzoate, sodium dehydroacetate, phenol, methylparahen, ethylparaben, propylparaben, and the like can be used. As the buffer, for example, citrate, acetate, phosphate, etc. can be used.

The present invention will be illustrated below with reference to production examples, working examples, and experimental examples.

Production Example 1 (Production of wheat LPS) ■In a small kneader, hard flour containing 1.09% ash (
American or Canadian hardlet spring) (3
, 120g), add 2.032 g of distilled water and
Knead for a minute and make a dough. After standing still for 15 minutes, add 101 water and gently stir to wash out the starch emulsion.
At the same time, soluble components were eluted. This eluate was allowed to stand in a refrigerator at 5° C. for 12 hours, and then precipitated portions such as starch were removed. The supernatant liquid was freeze-dried to obtain 201.1 g of powder (powder A).

Furthermore, 52 distilled water was added to the remaining tow, and the mixture was gently stirred, followed by treatment in the same manner as above to obtain 40.degree. 1 g of powder (powder day).

■These powders A and B are filtered through an ultrafilter HF manufactured by Amicon Corporation in the United States.
-Labl, the hollow cartridge HF-LablPM5 was used for the molecular weight fraction 5,000, and the hollow cartridge HF-Labl PM5 was used for the molecular weight fraction 10,000.
Ultrafiltration was performed with blPMlo attached [temperature 5~
10℃. Input pressure 25 ps i (1,76 kg/cm2
), specific pressure 15 ps'i (1,06 kg/cm2)]
Based on the results, each part was named as follows.

Powder A2 The portion with a molecular weight of 5,000 or less is a molecular weight of 5. o
The part with a molecular weight of oo or more is a2. Powder B: The part with a molecular weight of 5,000 or less is b + molecular weight 5.
The part with a molecular weight of 10,000 or more is b2 Powder A: The part with a molecular weight of 10,000 or less is a3 Molecular weight 1
Part with a molecular weight of 0,000 or more is a1 Powder B: The part with a molecular weight of 10,000 or less is b3 Molecular weight 1
0,000 or more.

When each of these fractions was subjected to the Limulus test according to the method detailed in Experimental Example 1 below, a large amount of Limulus test positive components were present in the fractions with a molecular weight of 5,000 or more, but in the fractions with a molecular weight of 5,000 or less. It was confirmed that there are almost no

■Pour 30g of the above powder A2 into an IQ Erlenmeyer flask,
After pouring 600 mλ of distilled water and stirring with a stirrer for 60 minutes, it was heated to 4°C using a Hitachi refrigerated high-speed centrifuge 5CR-20B (rotor RPR16 was pre-cooled to 4°C).
The supernatant was collected by centrifugation (10,000 g x 10 minutes).

■Pour this supernatant into a 19-Erlenmeyer flask, and while stirring with a starter under water cooling (liquid temperature approximately 2°C), drop 20.5 m + 1 of a 100% TCA aqueous solution that had been previously cooled to 2°C to complete the dropwise addition. After that, it was left closed in ice water for 10 minutes.

■Next, perform centrifugation at 4℃ in the same manner as above (10,
000 g for 10 minutes), cooled in ice water, suspended in a 500 mλ beaker with 100 ml of distilled water, cooled in ice water, and centrifuged at 4°C in the same manner as above. The precipitate was collected by operation (10,000 g x 10 minutes).

■Pour this precipitate into an IT crack and add 500 m of distilled water.
Neutralize (pH 7) using about 3.5 mλ of IN sodium hydroxide solution, add about 2 ml of IN sodium hydroxide solution to 0.02 N water while cooling in ice water. The precipitate was dissolved to become a sodium oxide solution.

(2) Approximately 1.5 ml of IN hydrochloric acid was added to adjust the pH to 8, and then 100 ml of distilled water was added, and the mixture was transferred to a 12-erlenmeyer flask and slowly shaken for 30 minutes in an incubator at 37°C.

■After adding and mixing 100% TCA water iM30ma,
After shaking slowly for 10 minutes in an incubator at 37°C, centrifuge Tommy CD10 kept at about 37°C.
Centrifugation operation (3,
000g x 10 minutes).

■ Collect the supernatant, cool it with water, and centrifuge it at 4°C (10,
000gXIO min).

[Phase] Collect the supernatant and add ION sodium hydroxide solution to approx.
．． The mixture was neutralized to pH 7 with 6ml of 1L, and concentrated using an ultrafilter (Toyo Roshi UHP-150, filter: UK-10, N2 pressure: 4.0kg/cm2).

■ Obtained concentrated f160ml + E, Sepharose (Se
pharose) 6B column [Pharmacia (USA)
manufactured by harmacia Inc. Column size: 5
cm (inner diameter)
M N a CIL (pH 7, 5), flow rate: 60 m
Both portions of 20 mQ each were obtained at 1/hour.

■ Combine 280 mλ from the 43rd to 56th portions, add 450 μg of Bronase E (Kaken Chemical Co., Ltd.),
After incubating at 37°C for 2 hours with shaking, filter the filter using an ultrafilter (Toyo Roshi UHP-62, filter: UK-10,
The mixture was concentrated under N2 pressure: 4.0 kg/cm2. Next, Pharmacia FPLC system (column: MonoQHR
It was subjected to anion exchange chromatography using IO/10). i.e. 10mM) Lis-HCQ [pH 7°5
) and 10 mM NaCa were applied to the column, and then the column was washed with a buffer (200 mfL) having the composition of the above buffer with the NaCl content increased to 165 mM. Next, the target LPS was eluted in a total volume of 400 ml while increasing the Na Cl m 1 degree gradient from 165 mM to IM, and both 2 mλ portions were collected. The 5th to 8th fractions after applying the concentration gradient, which were confirmed to be positive in the Limulus test, were combined and combined into LP.
8ml with S purity of approximately 92% [LPS: 3.03mg
(This is a large@@LPS conversion value based on the Limulus test. All LPS amounts below are also this conversion value.) Sugar: 0.
23 mg, protein: 0.04 mg) was recovered.

■Next, add the 8rnl to Sephadex (Sephadex).
dex) G-25 [Column: 2. Ocm (inner diameter) x 20
．． 2 cm (66 ml)] and subjected to gel regression (buffer: water) to collect both 3 mλ portions. Together with the 9th to 12th cases that were confirmed to have positive Limulus tests,
12ml 1L with LPS purity of approximately 95% (LPS: 2.7
mg, sugar: 0.18 mg, protein: 0.03 mg). Sugar was measured by the phenol-sulfuric acid method, and protein was measured by the Lowry method. It was confirmed that both of these components were acidic by anion exchange chromatography.

In addition, the molecular weight by SDS gel electrophoresis is 6,000 ~
10,000).

[Co., Ltd.] The above fraction was frozen at -80° C., then lyophilized to a constant weight, and the weight was 0.75 mg. (Hereinafter, this freeze-dried sample will be referred to as wheat LPS) When the limulus activity of this wheat LPS was measured by the method described in Experimental Example 1 below, it was equivalent to 2.7 mg, so the specific activity was 2.7 ÷ 0.75 =3.6.

Furthermore, since it is considered that any single sugar that may exist as a contaminant was substantially removed by the above purification, all of the detected sugars are considered to be sugars constituting wheat LPS. Therefore, when calculating the purity of wheat LPS at this stage based on weight, protein = 0.03 mg LPS amount 0. 75-0. 03=0. Since it is 72mg, 0.72÷0.75X100=96 (%).

Physical properties of wheat LPS ■ Molecular weight Wheat LPS was dissolved in distilled water to prepare a 1 mg/m51 solution, and the solution was placed in a 1.5 mλ tref tube. To this, separately, 2.5% SDS in 1mM EDTA,
5% mercaptoethanol, 10mM) lithium-hydrochloric acid (pH
Add 1μ of the SO5 treatment solution prepared by adding 8,0)
This mixture was immersed in boiling water for 3 minutes. Using Pharmacia's Phast System, a 5DS-buffer strip (
Buffer 5 trip) (interposed with Pbast gel)
Gel Gradient 8-2
5), maximum voltage 250v, maximum current 10mAE
I set Z and started electrophoresis. After the electrophoresis was completed, the behavior in Coomassie staining and silver staining was observed.

For Coomassie staining, Pharmacia's 0.
1% Fast Gel Blue (Ph-ast Ge
1 Blue) R was dyed and decolorized in the following order using a mixture of methanol:acetic acid:distilled water (volume ratio 3:1:6) as a decolorizing solution.

1) Staining at 50℃ for 1 day 2) Decolorizing at 50℃ for 5 minutes 3) Staining for 8 minutes at 50℃ 4) Closing and releasing color at 50℃ for 10 minutes 5) Protecting at 50℃ for 5 minutes (glycerol, acetic acid, distilled water) 6) Dry silver staining was performed in the following order.

1) Treated at 50°C for 2 minutes with a cleaning solution (a mixture of ethanol, acetic acid, and distilled water in a volume ratio of 5:1:4) 2) Treated at 50°C for 2 minutes with a cleaning solution (a mixture of ethanol, acetic acid, and distilled water in a volume ratio of 10:5)
3) Treated with a cleaning solution (mixture of ethanol and acetic acid distilled water 10:5:85 by volume) at 50°C for 4 minutes. 4) Treated with a sensitizing solution (8.3% glutaric acid mixture) at 50°C for 6 minutes. 5) Treated with a cleaning solution (mixture of ethanol and distilled acetic acid in a volume ratio of 10:5:85) at 50°C for 3 minutes. 6) Treated with a cleaning solution (mixture of ethanol and distilled acetic acid in a volume ratio of 10:5:85) at 50°C for 5 minutes. 10:5
: 85 mixture) 7) 50°C for 2 minutes and a cleaning solution (deionized water) 8) 50°C for 2 minutes and a cleaning solution (deionized water) 9) 40°C for 133 minutes 0.25w/v Treated with % silver nitrate, washed at 10130°C for 30 seconds? & (deionized water) 11 Treated with cleaning solution (deionized water) at 30 °C for 30 seconds 12 Treated with developer solution (0.04 v/v % formaldehyde + 2.5 w/v % sodium carbonate cleaning solution) for 30 seconds at 12 130 °C 13 Treated with developer solution (0.04 v/v% formaldehyde + 2.5 w/v% sodium carbonate wash solution) for 4 minutes at 30°C 14 Treated with stop solution (5% v/v% acetic acid) for 2 minutes at 150°C + 5150 Protective solution (acetic acid, glycerol,
16) Dried LPS is stained by silver staining, but not by Coomassie staining, and when the dyed band was observed, it was found that the molecular weight was 8.00.
The main staining zone of wheat LPS was detected at the 0±1,000 position.

[Phase] Phosphorus content Chen-Torjba-ra
Law [Chen et al., “Analytical Chemistry”
analytic Chemistry), vo
1.2B, pp. 1756-175B (1956), as follows.

Dissolve wheat LPS in distilled water to obtain 25 μg of wheat LPS.
A solution containing 20 ttfL was prepared and placed in a small test tube. Add 20 μL of 50 v/v% sulfuric acid,
Heated at ℃ for 2 hours. Then 10 v/of 20μ Qi
After adding v% perchloric acid, it was preheated with a gas burner for 1 minute to incinerate. Then add 0.5 ml of distilled water,
Then 0.5 mfL of reaction reagent (1 ma of 6N sulfuric acid, 2
m-distilled water, 2 mL of 2.5 V/W% ammonium molybdate and 1 mL of 10 V/W % ascorbic acid (0.5 mλ of which was used) was added and left at room temperature for 30 minutes. Afterwards, the absorbance at 820 nm (0D821In) was measured. In addition, as a sample for preparing the calibration curve, potassium dihydrogen phosphate (Wako Pure Chemical Industries & Co., Ltd.) was diluted with distilled water, and the phosphorus weight was 2.6%.
0.5 containing μg, 1μg, o, 2su-g, 0μg
ml of solution was prepared and used. Note that phosphorus Ig corresponds to 4.39 g of potassium dihydrogen phosphate. The results obtained are shown in Table 1 below.

Table 1 Note: Wheat LPS data is based on the presence of Kenkirin contamination (e.g.
In order to avoid errors caused by phosphorus vi buffer), the data for the control without heat treatment is subtracted.

Assuming that the molecular weight of wheat LPS is 8,000, the number of phosphorus per molecule is calculated from the following formula based on the results in the above table to be 1 to 4.

25XlO-32 One of the reasons why the number of runs varied from 1 to 4 in the above experiment may be that phosphate was eliminated due to the contamination of monophosphonisterase during the purification step.

0 Hexosamine Content Elson-Mar-gan
Law (Tokyo Kagaku Doujin Publishing “Biochemistry Experiment Course” No. 4-3
77-379) as follows.

Wheat LPS was dissolved in distilled water to prepare a 1 mg/m solution, and 100 μg of the solution was placed in a Spitz with a screw cap (manufactured by Iwaki Glass Co., Ltd.).
8N HCa of μm was added and heated at 110° C. for 16 hours. Approximately 200 μi of 4N NaOH was added to bring the pH to 7. 100μλ of the mixture was collected, placed in another spitz with a screw cap, and after adding the following sample 1iA of 200μ, it was heated at 105°C for 1.5 hours, and then cooled with running water. Next, 100 μλ was collected and 96 of 670 μ
% ethanol was added thereto, and the following reagent B at 67 μm was added thereto, and the mixture was allowed to stand at room temperature for 1 hour, and the absorbance was measured at 535 μm. 0.20 to 200 as a sample for preparing a calibration curve
μg/ml N-acetyl glucosamine (Wako Pure Chemical Industries, Ltd.) was used.

(Reagent A) Prepared by mixing 75μt of acetylacetone and 2.5mλ of 1.25N sodium carbonate.

(Reagent B) Mix 1.6 g of p-dimethylhenzaldehyde, 30 mfL of concentrated hydrochloric acid, and 30 mA of 96% ethanol to FAI! .

As a result, the number of hexosamine molecules in wheat LPS was 6±27 molecules (assumed molecular weight 8,000).

An internal standard (0.55mM margaric acid) with a concentration of 10μ was added to a wheat LPS distilled aqueous solution (1 mg/ml 1 L) with a fatty acid content of 90μ. 1.0 ml of 0.5M sodium methylate was added to perform hydrolysis and esterification of the fatty acid ester. After leaving at room temperature for 1 hour, 960μ(0.5NHC+L)
was added to neutralize it.

2 ml of hexane was added to this and the mixture was vigorously stirred for 15 minutes. Next, centrifugation was performed at 1,000 g for 5 minutes to separate the hexane layer. Hexane was evaporated using nitrogen gas, and the mixture was concentrated to about 20 μm of cod.

This sample was subjected to gas chromatography [Main body: GC8APF manufactured by Shimadzu Corporation, capillary column: FSCAP 5p2330 manufactured by Spelco (Canada).
, carrier gas: U element] to measure the amount of fatty acids. The standard for measuring the amount of fatty acids is E. coli type LA-15-PP, a synthetic lipid A manufactured by Daiichi Chemical Co., Ltd. (it is known that the molecular weight is 2,000 and the number of fatty acids in one molecule is 6). ) was used.

As a result, the number of fatty acids in wheat LPS was estimated to be 6±27 molecules (assumed molecular weight 8,000).

Charts observed by the above gas chromatography are shown in Figures 1 to 3 of the accompanying drawings. Figure 1 shows wheat LPS,
The figure is a chart of Escherichia coli LPS, and FIG. 3 is a chart of Bordetella pertussis LPS.

In FIGS. 1 to 3, the retention times (minutes) corresponding to the main numbers shown were as follows.

Figure 1: Beak number Retention time (min) 1
2.450 2 2.758 Figure 2: Beak number Retention time (min) 1
2.4172
2, 742 Figure 3: Beak number Retention time (minutes) 1 2, 43
32 3, 028 1st~
By comparing the three figures, the wheat LPS chart is the E. coli LP chart.
Although similar to the chart of S., it is clear that it is significantly different from that of B. pertussis LPS.

@KDO content The KDO (2-keto-3-deoxyoctonate) content was determined by the diphenylamine method [Shaby
R,) et al.
tical Bio-chem,), 5B(1),
123-129 (1974)] as follows.

500mg of diphenylamine, 5m of ethanol,
A KDO detection reagent was prepared by combining glacial acetic acid at 45° C. and 50 ml of concentrated hydrochloric acid (all manufactured by Wako Pure Chemical Industries, Ltd. 11). Part 50
Combine 250μ of distilled water containing 1.05mg/mt of wheat LPS, heat in a boiling water bath at 100°C for 30 minutes, cool in cold water (23°C) for 30 minutes, and add to a Hitachi spectrophotometer. 420.470 using 320
．． Ultraviolet absorption at 630.650 nm was measured (AJ211, A 4?11% A 639s A, respectively)
859). As a standard sample, 127μg/
m fL of KDO ammonium salt [US Sigma (S i
250 µλ of distilled water containing 250 µλ of GMA) was used.

The values of the following formula were determined for each of the specimen sample and the four standard samples.

5=IIN(ST) of AJ211-Aa711+Ae3e-A650 specimen sample is 0.379, 1Ii of standard 4 samples
(Ss) was 0.294. By comparing this value,
Wheat LPS was estimated to contain 5±1 mol/molecular weight of KDO of 8,000.

Production example 2 (manufacture of chlorella LPS) ■Cell membrane crushed chlorella (■Mannanzu's 81) 30
g was washed with ethanol until the washing solution was no longer colored green.

■26g of this washing residue was dissolved in distilled water at a concentration of 100mg/mfL, and after shaking at 45°C for 2 hours, centrifugation was performed (
4° C., 10.00θg×30 minutes).

(2) The upper groove was collected, filtered through Toyo Roshi N082, and then extracted with distilled water.

(2) 290 mm of the extract was subjected to anion exchange chromatography under the following conditions.

Column: Q-Sepharose (φ3 cm x 23c
m, volumetric 180 ml) Buffer: 10mM) Lis-ICIL (p) (7,5),
Na CILs gradient: 10mM, 400mM, 1M Flow rate: 100-200mN 1 hour Temperature: room temperature 310mλ of the fraction that passed through was treated with glucoamylase to decompose starch (pH 5, 0, 40℃, about 2 hours)
. Decomposition of starch was confirmed by the fact that no coloring occurred in the iodine-starch reaction.

■Recover the upper groove by centrifugation (10,000g x 10 minutes), neutralize with 10N NaOH solution to pH 7, and ultrafiltrate using an ultrafilter with a pore size of 200,000 molecular weight to remove the decomposed product. Removal and concentration were performed.

■30ml of the obtained concentrate was analyzed using FPLC manufactured by Pharmacia.
It was subjected to anion exchange chromatography using a system (column: MonoQHRIO/10). That is, IOm
M) Buffer containing Lisu-HC and 10mM NaC (
After applying the sample to the column at pH 7.5), M(2
The column was washed at 00°C. Next, in order to elute the target LPS, the total amount of LPS was increased by increasing the NaC concentration to 111 degrees gradient from 165 mM to IM.
The column was washed with 00mt and each 2m-fraction was collected. The 5th to 8th samples after applying the concentration gradient, which were confirmed to be positive in the Limulus test, were combined.

Next, 8ml of the 1L was treated with Sephadex (Sephadex).
dex) G-25 [Column: 2.0cm (inner diameter) x 20
．． 2 cm (66 ml)] was used to collect both 3 ml fractions (buffer solution: 1 sushi in water).The 9th to 12th fractions, which were confirmed to be positive for the Limulus test, were combined to make 12 ml. was recovered (LPS: 14.3 mg
, sugar: 2.0 mg, protein: 0.53 mg), LPS was measured by the method described in Experimental Example 1 below, sugar was measured by the phenol-RWII method, and protein was measured by the Lowry method.

(2) The above fraction was frozen at -80°C, freeze-dried to a constant weight, and weighed 5.8 mg. (Hereinafter, this freeze-dried sample will be referred to as Chlorella LPS.) Since the limulus activity of this Chlorella LPS is equivalent to 14.3 mg, its specific activity is l 4 , 3 ÷ 5. 8=2. It becomes 5.

In addition, in the above-mentioned W product, it is considered that single sugars that may exist as impurities have been substantially removed, so all the detected sugars are considered to be sugars constituting Chlorella LPS. Therefore, when calculating the purity of Chlorella LPS at this stage based on weight, protein = 0.53 mg LPS = 5.8 - 0.53 = 5.27 mg, so 5.27 ÷ 5.8 x 100 = 91 (%) It is.

Physical Properties of Chlorella LPS The following values were obtained in the same manner as in Production Example 1.

Molecular weight = 40,000 to 90,000 Phosphorus number = 4 ± 1 / molecular weight 10,000 Hexosamine number = 7 ± 1 / minute 10,000 fatty acid number = 6 ± 1 / minute 10,000 KDO number = 2 ± 1 / molecular weight 10,000 Production Example 3 (Manufacture of B. pertussis LPS) Test B. pertussis solution (2,
OX 101''!I cells/m IL) were used as dead cells.

The dead cells were suspended in sterilized water at a concentration of 25 mg (dry weight)/mfL. An equal amount of 90% hot phenol solution (68-70°C) was added to this, and the mixture was extracted with shaking at 68°C for 1 hour. The aqueous layer was separated by centrifugation at 8,000g and 4°C for 20 minutes. To the remaining phenol layer, the same amount of W&bacteria water as the aqueous layer was added and the same extraction was performed.

The resulting aqueous layer was combined with the previous aqueous layer, dialyzed overnight in running water, and then concentrated to 1/10 using a rotary evaporator. This was centrifuged at 8°000g and 4°C for 20 minutes. The supernatant was collected, a small amount of sodium acetate was added, 6 times the amount of cold ethanol at 0 to 4°C was added, and the mixture was left at -20°C overnight. 4.
The precipitate collected by centrifugation at 00-0g for 30 minutes at 4°C was centrifugally washed twice with ethanol and once with acetone, and dried with an aspirator.

The residue was suspended in distilled water to a concentration of 20 mg/ml, and subjected to ultrasonic treatment (output control 5) using Sonifier Model 185 manufactured by Branson, USA.
．． 15 minutes at room temperature). Next, 2゜500g, 4℃
The mixture was centrifuged for 10 minutes, and the supernatant was collected.

The supernatant was heated at 4°C using a nucleolytic enzyme DNase 15RnaseA manufactured by Sigma, USA.
treated for 6 hours (finally 10 μg/ml DNAs)
e + and 20 ug/mg of Rnase A). Furthermore, add the same concentration of nuclease and heat at 37°C.
The mixture was heated for 2 hours. The mixture was then centrifuged at 2.500g for 10 minutes at 4°C, and the supernatant was collected.

This supernatant was filtered using an Acrodisc manufactured by Gelman, USA, with a pore size of 0.2 μm. The filtrate was passed through a molecular sieve [Resin: Sepharose 6B manufactured by Pharmacia, USA, column size = inner diameter 5 cm x length 100 cm, buffer = IOmM Tris-HCfL, 10mM NaCl (pH 7,5)
, flow rate = approximately 3 ma/cm2/hour), Limulus activity-positive fractions were examined and combined using an LS-1 kit manufactured by Seikagaku Corporation, and filtered using the above-mentioned Acrodisc manufactured by Gelman Inc. with a pore size of 0.21 m. . The filtrate was subjected to ion exchange [equipment: F manufactured by Pharmacia, USA]
PLC1 resin: MonoQ HR1 manufactured by Pharmacia, USA
0/10, buffer = 10mM Tris-HCu + 10m
IM NaCQ, (pH 7,5) for 15 min, followed by a 30 min wash with increasing NaCl amount to 165 mM, then IM NaCQ from 165 mM to IM over 20 min.
Wash with increasing amounts of NaCl so as to obtain a concentration gradient of Positive fractions were examined and combined.

The combined fractions were desalted using a column [Resin: Sephadex G-25 manufactured by Pharmacia, USA].
fine, column size = inner diameter 2 cm x length 25 cm, eluent = distilled water, and then freeze-dried.

The substance most likely to be contaminated in this freeze-dried sample (4.50 mg) is a nucleic acid. Therefore, an ultraviolet absorption curve (200 to 400 nm) was taken, and the absorbance at 260 nm was determined. Nucleic acid concentration at absorbance 1 is 50ug/mλ
When the nucleic acid concentration was calculated from the above absorbance using the above equation, it was found to be 1% or less. Furthermore, no protein was clearly detected by SDS electrophoresis. Therefore, considering detection sensitivity, the amount of protein contained in the freeze-dried sample is at most 0.
Estimated to be ~3%. Therefore, the purity of the freeze-dried sample was estimated to be 96% or more.

I! The physical properties of this pertussis 1fLPs measured in the same manner as described in Preparation Example 1 were as follows.

Physical properties of Bordetella pertussis LPS molecule t=6. ooo±1.000. 9.000±1,000 (Among the multiple Coomassie-stained bands observed, this is the value of the two staining bands with the highest staining intensity.) Phosphorus number = 57 Molecular weight 8,000 Hexosamine number = 16 ± 27 Molecular weight 8,000 Fatty acid Number = 57
Molecular weight: 8,000 KDO number = 2 ± 17 Molecular weight: 8,000 Escherichia coli LPS [manufactured by Dirco, USA, 012]
The physical properties of 8:B8:l were as follows.

Physical properties of Dai Illono LPS Molecular weight = 30,000 ± 5,000 (This is the value of the one with the highest staining intensity among the Coomassie staining zones continuous in PJ steps.) Phosphorus number = 127 Molecular weight 30,000 Hexosamine number = 45 ±6/molecular weight 30,000 Number of fatty acids = 18
/molecular weight 30,000 KDO number = 5+1/molecular weight 30,000 or less is a formulation example of a preparation containing LPS of the present invention. Note that the LPS amount is the E. coli LPS equivalent amount determined by Limulus test.

Example 2 (internal liquid) Chlorella LPS 1 mg Purified water 100 m Example 13 (ointment) Wheat LPS O, 1 g Purified lanolin 0 g 000 g Example 4 (injection) Wheat LPS 0.5 mg Example 1 (tablet) Wheat LP5 0.04g 6% RPC lactose 178g stearic acid talc
8g potato starch Mix 14g or more and press into a tablet to obtain O,0 containing 1mg of wheat LPS.
．． 400 5g tablets were prepared.

Total: 1000 m Experimental Example 1 (Quantification of LPS from Limulus test positive plants) Quantification of Limulus test positive LPS contained in various plants was carried out using Seikagaku Corporation's Toxicolor System.

■Pour 1 cup of distilled water for injection into a 96-hole flat bottom or round bottom plate.
I inserted 180μ per hole. 20μ2 of the sample (if the sample was a solid, it was dissolved in distilled water for injection) was added to one of the wells of the plate. A 10-fold dilution was prepared by pipetting while stirring with a plate mixer. (Then, by sequentially taking 20μ aliquots of the diluted sample and treating it in the same way, you can prepare a 10-fold dilution series such as 100-fold, 1000-fold, etc. Also, change the ratio of the sample to distilled water for injection. (This allows the dilution rate to be set arbitrarily.)
■Escherichia coli L at 1.5 μg/m Q, as an internal standard
Ioo and ooo fold dilutions of the PS solution were prepared, and it was confirmed that the dilution and limulus test color development were normal.

■Take 35μ of the 10-fold diluted solution from ■ above into the wells of another plate, and use Seikagaku Corporation's Toxicolor System L.
35 microliters of S-1 set was added and left at 37°C for 30 minutes. Then, 105 μt of 1M acetic acid water was added and stirred to stop the reaction.

The absorbance of this sample solution at a wavelength of 415 nm was measured using a 96-hole absorbance meter plate reader MTP-100 (Corona! Co., Ltd.). Distilled water was used as the background, and 42 pg/mλ was used to create a calibration curve. A calibration curve was created using the ET-1 set of Toxicolor System from Seikagaku Corporation, and the amount of Limulus test-positive LPS in each sample was determined based on this calibration curve. (The sample was distilled water. The absorbance when
It was confirmed that the color development was quantitative within the range of 5 pg/ml, so if it was not within this range, the experiment was repeated by changing the dilution rate.

The quantitative value of the diluted sample was calculated by (value read from the calibration curve) x (dilution!$).

The obtained results are expressed in ng/g in the case of solid samples;
In the case of liquid samples, the values are shown in Table 1 below in units of n g/mλ.

In addition, the company name, place name, etc. in the sample column in the table refer to the place where the sample was obtained or produced. Items without such a description are items purchased at Superstore Chujitsuya's Nakanomachi store in Tsukui District, Kanagawa Prefecture, and the manufacturer is unknown.

Table 1 Limulus test positive test N (solid) LPSI (ng) Gymnosperm pine nuts (Koshinan Boeki) 125 Monocot hard wheat seeds (Chiba Flour Milling) 2,250 Hard wheat seeds (Chiba Flour Milling) (Molecular weight 5000 or more ) 1,000.000 Hard wheat flour (Chiba Flour Milling) '7,500 Wheat bran (Chiba Flour Milling (molecular @ 5000 or more) 100 Wheat Germ (1,000# milling) 1,600 Wheat Germ (1,000 M milling) (Molecular weight 5,000 or more) <10,000 brown rice 1,100 Japanese grain flour) (molecular weight 5,000 or more) 3 Rice bran (molecular weight 5,000 or more) Corn flour (ocean feed) (molecular weight 5,000 or more) Corn grits (ocean feed) (molecular weight 15,000 or more) Above) Corn (Wako Shokuryo) Kumazasa (Kaimotosan) Iris (seed) Garlic (scale) Asparagus (bud) Myoga (flower cluster) Yokuinin (Uchida Japanese and Chinese Medicine) (Original M product is pigeon barley) Hange (Matsuura Pharmaceutical) (Original plant is crow bishaku) Bakumontou (Tochigi dog? II room) (Original plant is shirt and ge) 1,000. 000 29, 000 500, 000 <0.3 15.000 3, 100 4, 100 41, 000 2, 100 5, 500 4, 000 Turmeric (SP Foods) 195,000 (original plant is turmeric) Dicotyledonous soybean (Princess Foods) 150 Soybeans (Haguren) (Molecular weight 5000 or more) 400 Tamba Black Soybeans (Wako Foods) 85 Azuki Beans (Wako quantity [) 450 Azuki Beans (Wako quantity $1 (Molecular weight 5000 or more) 36,000,000 Hitashi Beans ( Wako Food) 800 Taisho Kintoki (
Wako Foods) 550 Daifuku Beans (Wako amount fM) 350 Broad Beans (Raw)
750 Potato (Hakuren) (Molecular weight 5000 or more) <0.3 Loquat (seed) 800 Avocado (seed) 950 Peach (seed)
4,5001.900 Walnuts (seeds) Broad beans (seeds) Kapocha (fi ko) Tomatoes (raw fruit) Japanese radish (root removed) Matatabi (Marukyu Bussan) Amachazuru (K, Ni, Sakurai) Dokudami (Ni Jun Iit) (Teikyo University Medicinal Botanical Garden) Hukai (white) (SP Foods) Capsicum (Konan Boeki) Hexagon (Konan Boeki) Nutmeg (Lion) (Original plant is Nikuzuku) Spruce (Uchida Japanese and Chinese Medicine) (Original plant is Daidai) Kakkon (Tochigi Tenkaido) (Original plant is scum) Glycyrrhiza (Uchida Japanese and Chinese medicine) Panax ginseng (Uchida Japanese and Chinese medicine) Bofuu (Tochigi Tenkaido) 10, 000 10, 500 50, 000 40.000 73, 000 1.200 2, 100 2, 100 5, 500 2, 000 8, 000 3, 000 1B, COO 50,000 Kanboui (Tochigi Tenmando) 600,000 (Original plant is Otsuzurouji) Choto kola (Uchida Japanese and Chinese medicine) 7,000 (Original plant Uncaria hirsuta) Hachimijiogan (Kanebo Pharmaceutical) 17,000 small saiko! (Tsumura) 13,000 Gorei!
(Tsumura) 12,000 Ireito (
Tsumura'I 14,000 Juzen Taisho 11! (Tsumura) 8,000 Hachimijiogan (Tsumura) 8,000 Royal Jelly 1,000 [Pekin Royal Jelly] Honey (Kato Bihoen Honpo) 800 Cedar fern (per wet weight) 700 (Teikyo University Medicinal) Botanical Garden) Zenmai (Kaibutsu) 10,000 Japanese seaweed (Sanriku Tennen Products) 11,000 seaweed sprouts (4tani Health Foods) Hijiki (raw) Mehiki (Kozen Main Store) Cob (Yamato Takahashi) Asakusanori ( Dried fresh seaweed) Chlorella (■ Helstar Japan YS) Chlorella (■ Mannan Foods Y5) ■ Shiitake mushrooms (from Shimonita) Enoki mushrooms (Nakakakeju, Nagano Prefecture) Miyagi-cho, Shimeshiku Seta-gun) Maitake mushrooms (Otone) Abalone mushrooms (Hanyu) Pine mushroom mushroom namekoebios (brewery yeast manufactured by Asahi Breweries) 200, 000 B5.1) 00 105, 000 2 3 FJ, 0 0 0 130, (100 1, 900,000 1,000,000 16,000 20,000 40,000 205,000 s, oo.

20, 000 75, 000 21.000 250, 000 Cordyceps sinensis 240. 000 Others Snow Brand Nature Yogurt (■1 mark) 5,000 Glyco Bifuzu Yogurt (■ Glyco) 50 Limulus test positive sample (liquid) LPS amount (ng) Beer Kirin Asahi Fine Birsner Lager Beer Heartland Fine Draft Super Yeast Wine 1 , 150 1.250 1.550 1.400 Suntory Sainte Neige (white) (red) Siedle (apple) Sake Oma-grade Kizakura 2nd grade (Oma Sake Brewery) (Kizakura Sake Brewery) 2 , 4 1.7 Daikan Ginjo 2 Class (Gyokusendo Sake Brewery) Genmai Sake Hibi-Ken (Oma Sake Brewery) Spicy Sake Totoshu Delcap (Tai Toshu Honpo) Takarayaki Tai (Takara Shuzo) Other Kyoreopin (Yusui Seiyaku) Garlic Extract (Yusui Seiyaku) Groscue (Chlorella Industries) ) Barley Health Metcol (Korea/Japan) Saclone Herbal Liquid (Eisai) Loofah Water (In-house! Ripaio Algen (Chlorella Industries) Panshiron Oral Solution (Rohto Pharmaceutical) Yunkerfan Tea (SaMH Pharmaceutical) Kolihogus (Kobayashi Pharmaceutical) Tsudi ( Sankyo) Mio D Kowa 100 (Coates) 2, 1] 2 1.2 <2, 0 s, oo.

2, 0. ) 0 1, 000 Nogain (Sankyo) 90 Bren 50 (No.-IIM 7 Solmatsu (large mu drug) 60-Zeligo ~
Ruto (Chugai Pharmaceutical) 5 Busvitan 30 (T,
Ban Pharmaceutical) 5 Tiovita (Daii!! Medicine)
Ribovitan less than 5 (Taisho Pharmaceutical)
Less than 5 Asparagus Gold (Tanabe Pharmaceutical)
Less than 5 Experimental Example 2 (Method for selecting LPS with a Limulus test-positive LPS content of 0°4 to 1100 n/mt of culture medium that gives EDss when activating the in vitro TNF production ability of macrophages) 9 weeks old, average C3H/3 males in each group weighing 29 g.
He mouse macrophage peritoneal resident cells 200 μλ (
2×105 cells/well were placed in a 96-well flat-bottomed plate, and 10 μl of silk recombinant mouse IFN-r (100 units/m a) was added as a brimer. Separately, various LPSil! The extract was prepared by extracting with hot water (g/ml) at 65° C. for 5 hours and diluted in various ways, and 10 μl of the diluted solution was added as a trigger 3 hours after the administration of Brimer. 2
After incubation for an hour, centrifugation was performed for 1 w (1000 g, 2
0 minutes). The TNF activity of the 130 μL obtained from Jindai was determined based on toxicity to L929″m cells, and the Limulus test positive LPS content was determined using the Seikagaku Corporation's Toxicolor system.

The measured value is plotted on the vertical axis as the TNF production amount (unit/mL of culture solution).
was plotted on the horizontal axis (logarithmic scale) at coordinates representing Limulus test positive LPS content (ng/ml of culture solution), and a sigmoid curve estimated from each plotted point was drawn. The macrophage activation ability of each trigger that gives the amount of TNF production when no trigger is administered is set as 0%,
The macrophage activating ability of each trigger when the increased TNF production amount reaches the maximum constant level as the effect of trigger administration is set as 100%, and the Limulus test positive LPS content that gives the macrophage activating ability equivalent to 50% is calculated from the curve. I read it.

Table 2 below shows the results of LPS collection sources that satisfied the above conditions in terms of the correlation between macrophage activation ability and Limulus test positive LPS content. In the table, "TNF" indicates the amount of TNF produced (unit/mL of culture solution), "activation ability" indicates macrophage activation ability (%), and rLPsJ indicates the Limulus test-positive LPS content (ng/mL of culture solution). represent. In addition, the amount of TNF produced during gradual addition of the trigger was 0.75 units/
ml, the amount of TNF produced was 0.75 units/ml.
If the following is the case, the macrophage activation ability is defined as O%, and the macrophage activation ability (%) was calculated using the following formula.

Table 2 The results shown in Table 2 are shown in Figures 4-7.

In Figures 4 to 7, the vertical axis represents macrophage activation ability (
%), and the horizontal axis (logarithmic scale) is Limulus test positive LP.
It represents Sf_M(ng/sgamma).

In Figure 4, ○ indicates data on turmeric, - indicates data on Riki-n-Boy, mouth indicates data on kelp, and ■ indicates data on Asakusanori.

In FIG. 5, ◯ indicates data for wakame bud extract, mouth indicates data for Mehijiki, and ■ indicates data for Ebios.

In Figure 6, ○ indicates Cordyceps sinensis, ・ indicates wakame bud stock,
The mouth shows data on chlorella.

In Figure 7, O is E. m. LPS and . is wheat LPS.
, mouth indicates data for Bordetella pertussis LPS, and ■ indicates data for lipid A.

Experimental Example 3 (Measurement of anti-rheumatic effect) Complete Freund's adjuvant (001 ml/rat) was intradermally administered to four Wistar rats (10 week old male, average weight 400 g) in each group (Difco, USA). (Djf
cm) was administered, and the anti-rheumatic effect of the wheat LPS produced in Production Example 1 was measured using the degree of prevention of foot swelling as an index.

As a positive control, phenylbutacin, a representative non-steroidal anti-inflammatory drug whose anti-inflammatory properties are highly evaluated, was administered. The dosage of wheat LPS was 1 μg/mouse (the amount of LPS positive in Limulus test), and the phenylbutacin was administered at 1 mg/mouse.

The experiment was conducted in two ways. In one method A, the specimen was administered every other day starting 4 days before adjuvant administration, and in the other method B, the specimen was administered every other day after adjuvant administration (for both methods, the specimen was administered 6 days after adjuvant administration). (administered intravenously to the eyes). The results are based on a relative evaluation assuming that the thickness of the foot joint before adjuvant administration is 0%.
Figure 8.9 shows the average of animals. FIG. 8 shows the results of method A, and FIG. 9 shows the results of method B.

In both figures, ◯ indicates data for physiological saline, △ indicates data for phenylbutacin, and ◯ indicates data for wheat LPS.

From the results shown in Figure 8.9, the LPS of the present invention shows an anti-inflammatory effect equal to or greater than that of phenylbutacin, and method A, in which the sample is administered before the administration of the adjuvant, is more effective than the adjuvant. The inhibitory effect of the LPS of the present invention lasts longer than method B in which administration of the sample is started again.
It is clear that it has both therapeutic and preventive effects on rheumatism.

Dosage, administration interval, toxicity value When administering the LPS of the present invention as an anti-rheumatic agent or an anti-rheumatic agent for animals, the amount and interval of administration must be determined under the strict control of the attending physician or veterinarian, depending on the age of the subject to be administered. , symptoms, body [adult human <60 kg, although determined individually taking into account the administration effect], 1 μg -100 m after oral administration.
g, 10 ng to 1 mg for intravenous administration, 100 for transdermal administration
n g −1 mg is a tentative guideline for the once-daily dosage. For animals, the recommended amount is 1/60th of the above amount per kg of body weight for large animals such as cows and horses, and double the amount for medium and small animals such as pigs, dogs, and cats. weight 1
The amount per kg is used as a guideline, and for birds such as chickens, the second
Double doses can be administered per 1 kg of body weight.

In addition, wheat LPS (%1 production example 1), chlorella LPS (production example 2), large bacterial LPS [Difco (USA)
fco) Company 110128:B8), Bordetella pertussis LPS
(Production Example 3) Toxicity 11iLD5s (2 male B per group)
Average value for ALB/C mice, average weight 45 g)
was as follows.

[Effects of the Invention] The present invention has a high anti-rheumatic effect, few side effects, high chemotherapy coefficient, low production cost, and can be administered orally, transdermally, or by injection, and can be supplied in large quantities. A new anti-rheumatic agent and an anti-rheumatic agent for animals are provided.

[Brief explanation of the drawing]

FIG. 1 is a chart illustrating peaks indicating the presence of fatty acids in the molecule, obtained by subjecting wheat LPS to gas chromatography. FIG. 2 is a chart illustrating peaks indicating the presence of fatty acids in molecules obtained by subjecting E. coli LPS to gas chromatography. FIG. 3 is a chart illustrating peaks indicating the presence of fatty acids in the molecule, obtained by subjecting Bordetella pertussis LPS to gas chromatography. FIGS. 4 to 7 are graphs showing the phase rI11 relationship of various LPSs in which the phase relationship between macrophage activation ability and Limulus test positive LPS content satisfies the conditions of the present invention. FIG. 8 shows the anti-rheumatic effect of wheat LPS when wheat LPS was continuously given before administration of the adjuvant. FIG. 9 shows the anti-rheumatic effect of wheat LPS when wheat LPS was continuously given after administration of the adjuvant. In Figures 4 to 7, the vertical axis represents macrophage activation ability (
%), and the horizontal axis (logarithmic scale) is Limulus test positive LP.
Contains S! (ng/culture! I solution mi). In Fig. 4, ○ indicates data on turmeric, * indicates data on Riki-n-Boy, mouth indicates data on kelp, and ■ indicates data on Asakusanori. In FIG. 5, ◯ indicates data for wakame bud extract, . indicates data for bud hijiki, and mouth indicates data for ebios. In Figure 6, ○ indicates cordyceps sinensis, ・ indicates wakame bud stock,
The mouth shows data on chlorella. In Figure 7, O is E. coli LPS, . is wheat LPS.
The mouth shows pertussis MLPS data, and the intestine shows lipid A data. In Figure 8.9, ◯ indicates data for physiological saline, △ indicates data for phenylbutacin, a known non-steroidal anti-inflammatory agent, and ◯ indicates data for wheat LPS.

Claims (1)

[Scope of Claims] (1) An anti-rheumatic agent containing LPS, which activates the TNF-producing ability of macrophages cultured in vitro.The macrophage activation ability of LPS is used as an index, and the LPS is added on the vertical axis. 0% macrophage activation ability that gives the amount of TNF produced by macrophages when not
, represents the macrophage activation ability (%) with the macrophage activation ability of LPS taken as 100% when the macrophage produces the maximum amount of TNF, and the horizontal axis shows the LPS
When drawing a sigmoid curve representing the Limulus test-positive LPS content on a logarithmic scale, an antimicrobial agent containing at least one type of LPS whose Limulus test-positive LPS content is 0.4 to 100 ng/ml of culture solution, giving an ED_5_0 of macrophage activation ability. Rheumatism medicine. (2) the LPS is selected from the group consisting of LPS obtained from plants, LPS obtained from bacteria, and lipid A;
The antirheumatic agent according to claim 1. (3) The plant is a gymnosperm, a monocot, a dicot,
The antirheumatic agent according to claim 2, which is selected from the group consisting of fern plants, grass plants, fungal plants, and mixtures thereof. (4) The antirheumatic agent according to claim 3, wherein the gymnosperm is a plant of the genus Pinus of the family Pinaceae. (5) The anti-rheumatic agent according to claim 4, wherein the plant of the Pinaceae family, Pinus genus, is Pinus. (6) Monocotyledonous plants include plants of the genus Poaceae of the Poaceae family, plants of the genus Triticum, plants of the genus Barley, plants of the genus Oatus, plants of the genus Sasa, plants of the genus Juzdama, plants of the genus Iris of the Iridaceae family, plants of the genus Allium of the genus Liliaceae, 4. The anti-rheumatic agent according to claim 3, which is selected from the group consisting of plants of the genus Porphyra, plants of the genus Zingiberaceae, plants of the genus Zingiber of the ginger family, plants of the genus Curcuma, plants of the genus Araceae of the family Araceae, and mixtures thereof. (7) The antirheumatic agent according to claim 6, wherein the plant of the genus Poaceae of the family Poaceae is rice. (8) The anti-rheumatic agent according to claim 6, wherein the plant of the genus Triticum, belonging to the family Poaceae, is wheat. (9) Claim 6, wherein the plant of the genus Barley, belonging to the family Poaceae, is selected from the group consisting of barley, naked wheat, and mixtures thereof.
Anti-rheumatic agents listed. (10) The anti-rheumatic agent according to claim 6, wherein the plant of the genus Avitae of the family Poaceae is selected from the group consisting of oats, oats, and mixtures thereof. (11) The anti-rheumatic agent according to claim 6, wherein the plant of the genus Bamboo in the family Poaceae is grass grass. (12) The anti-rheumatic agent according to claim 6, wherein the plant of the genus Juzdama, belonging to the family Poaceae, is sagebrush. (13) The anti-rheumatic agent according to claim 6, wherein the plant of the Iridaceae family, Iris genus, is Iris. (14) Claim 6, wherein the plant of the genus Allium of the family Liliaceae is garlic.
Anti-rheumatic agents listed. (15) The anti-rheumatic agent according to claim 6, wherein the plant of the Liliaceae family, the genus Acanthus, is asparagus. (16) A plant of the genus Janohige of the family Liliaceae is Janohige,
The anti-rheumatic agent according to claim 6. (17) The anti-rheumatic agent according to claim 6, wherein the ginger plant is ginger. (18) The anti-rheumatic agent according to claim 6, wherein the plant of the family Zingiberaceae and genus Curcuma is turmeric. (19) The anti-rheumatic agent according to claim 6, wherein the plant of the family Araceae, belonging to the genus Araceae, is Araceae. (20) The anti-rheumatic agent according to claim 8, wherein the LPS obtained from wheat has the following physical properties. Molecular weight: 8,000 ± 1,000 (SDS electrophoresis method) Number of phosphorus: 1 or more / molecular weight 8,000 Hexosamine number: 6 ± 2 / molecular weight 8,000 Number of fatty acids: 6 ± 2 / molecular weight 8,000 KDO number: 5 ± 1 / molecular weight 8,000 (21) Dicotyledonous plants include plants of the genus Soybean, plants of the genus Phaseolus, plants of the genus Broad bean, plants of the genus Kudzu, plants of the genus Licorice, plants of the genus Solanum of the Solanaceae family, plants of the genus Tomato, plants of the genus Capsicum, Plants of the genus Loquat of the Rosaceae, plants of the genus Prunus, plants of the genus Avocado of the Lauraceae family, plants of the genus Walnut of the Walnut family, plants of the genus Tolanaceae of the Cucurbitaceae family, plants of the genus Jiaogulan, plants of the genus Brassicaceae, plants of the genus Actus of the Brassicaceae family, plants of the genus Actinidia of the family Actinaceae, genus Heterinaceae Plants, Piperaceae plants of the genus Piper, plants of the genus Shikiminaceae of the family Chrysanthemum, plants of the genus Rutaceae of the genus Rutaceae, plants of the genus Panax of the genus Araliaceae, plants of the genus Apiaceae of the genus Saposhnicobia, plants of the genus Sapochnicobia of the family Araliaceae, plants of the genus Rubiaceae of the family Rubiaceae The anti-rheumatic agent according to claim 3, which is selected from the group consisting of: and mixtures thereof. (22) Claim 21, wherein the plant of the genus Soybean of the Fabaceae family is soybean.
Anti-rheumatic agents listed. (23) The anti-rheumatic agent according to claim 21, wherein the plant of the genus Phaseolus of the Fabaceae family is azuki bean. (24) The anti-rheumatic agent according to claim 21, wherein the plant of the Fabaceae family, Faba genus, is broad bean. (25) The anti-rheumatic agent according to claim 21, wherein the plant of the Fabaceae family, the genus Kudzu, is kudzu. (26) The anti-rheumatic plant according to claim 21, wherein the plant of the genus Licorice of the family Fabaceae is Glycyrrhiza. The anti-rheumatic agent according to 21. (28) Claim 2, wherein the plant of the genus Tomato of the family Solanaceae is a tomato.
The anti-rheumatic agent according to 1. (29) A plant of the Capsicum genus of the Solanaceae family is Capsicum,
The antirheumatic agent according to claim 21. (30) The anti-rheumatic agent according to claim 21, wherein the plant of the Rosaceae genus Loquat is Loquat. (31) Claim 21, wherein the Rosaceae Prunus genus plant is a peach.
Anti-rheumatic agents listed. (32) Avocado is a plant of the genus Avocado of the Lauraceae family,
The antirheumatic agent according to claim 21. (33) The anti-rheumatic agent according to claim 21, wherein the plant of the family Walnut family, genus Walnut, is walnut. (34) The anti-rheumatic agent according to claim 21, wherein the plant of the Cucurbitaceae family, genus Tuscanthus, is pumpkin. (35) The anti-rheumatic agent according to claim 21, wherein the plant of the genus Jiaogulan of the Cucurbitaceae family is Jiaogulan. (36) The anti-rheumatic agent according to claim 21, wherein the plant of the genus Radish in the family Brassicaceae is Radish radish. (37) A plant of the Actiniaceae family, Actinidia genus is Actinidia,
The antirheumatic agent according to claim 21. (38) A plant of the genus Dokudami of the family Dokudamiaceae is Dokudami,
The antirheumatic agent according to claim 21. (39) The anti-rheumatic agent according to claim 21, wherein the plant of the genus Piper of the family Piperaceae is pepper. (40) The anti-rheumatic agent according to claim 21, wherein the plant of the genus Shikimi of the family Shikimi family is Rheumatoidum. (41) A plant of the genus Chrysanthemum of the family Chrysanthemum is a stinging owl,
The antirheumatic agent according to claim 21. (42) The anti-rheumatic agent according to claim 21, wherein the plant of the Rutaceae family, Rutaceae, genus Rutaceae is Lamium daisies. (43) The anti-rheumatic agent according to claim 21, wherein the plant of the genus Panax of the family Araliaceae is Panax ginseng. (44) The anti-rheumatic agent according to claim 21, wherein the plant of the genus Saposchnicobia of the family Apiaceae is sapochnicobia. (45) The anti-rheumatic agent according to claim 21, wherein the plant of the genus Ophthalmia of the family Ophthalmidae is Ophthalmia vulgaris. (46) The anti-rheumatic agent according to claim 21, wherein the plant of the family Rubiaceae, belonging to the genus Capricornis, is Uncaria hirsuta. (47) The anti-rheumatic agent according to claim 3, wherein the fern plant is selected from the group consisting of a plant of the genus Horsetail of the family Equisetaceae, a plant of the genus Horsetail of the family Heliumaceae, and a mixture thereof. (48) The anti-rheumatic agent according to claim 47, wherein the plant of the genus Horsetail of the family Equisetaceae is horsetail. (49) A plant of the genus Chrysanthemum of the family Chrysanthemum is a windworm.
The antirheumatic agent according to claim 47. (50) The grass plant is a grass plant, a red grass plant,
The anti-rheumatic agent according to claim 3, which is selected from the group consisting of green grass plants, orchid plants, and mixtures thereof. (51) Claim 50, wherein the Cassowaceae plant is selected from the group consisting of plants of the genus Wakame of the Laminaceae family, plants of the genus Laminaria, plants of the genus Hijiki of the Sargassum family, and mixtures thereof.
Anti-rheumatic agents listed. (52) The anti-rheumatic agent according to claim 51, wherein the plant of the genus Wakame in the family Laminaria family is wakame. (53) The anti-rheumatic agent according to claim 51, wherein the plant of the Laminaria family, Laminaria genus is Laminaria laminaria. (54) The anti-rheumatic agent according to claim 51, wherein the plant of the genus Hijiki of the Sargassum family is Hijiki. (55) The anti-rheumatic agent according to claim 50, wherein the ruboraceae plant is a plant of the genus Porphyra of the family Ceramicaceae. (56) The anti-rheumatic agent according to claim 55, wherein the plant of the genus Porphyra of the family Ceramicaceae is Porphyra spp. (57) The anti-rheumatic agent according to claim 50, wherein the green grass plant is a Chlorella plant of the Oocystis family. (58) The antirheumatic agent according to claim 57, wherein the plant of the genus Chlorella of the family Oocystis family is Chlorella. (59) The anti-rheumatic agent according to claim 58, wherein the LPS obtained from chlorella has the following physical properties. Molecular weight = 40,000 to 90,000 (SDS electrophoresis method) Number of phosphorus = 4 ± 1 / molecular weight 10,000, number of hexosamine = 7 ± 1 / molecular weight 10,000, number of fatty acids = 6 ± 1 / molecular weight 10,000, number of KDO = 2 ± 1/molecular weight 10,000 (60) fungal plants are selected from the group consisting of basidiomycete plants, ascomycete plants and mixtures thereof;
The anti-rheumatic agent according to claim 3. (61) The basidiomycete plant is a plant of the genus Pyrophyllaceae,
Claim 60, which is a plant of the genus Enokitake of the family Asteraceae, a plant of the genus Shimeji, a plant of the genus Maitake of the family Orientalaceae, a plant of the genus Polyporus of the family Agaricaceae, a plant of the genus Agaricaceae of the family Agaricaceae, a plant of the genus Auricularia of the family Acanthaceae, a plant of the genus Sugitake of the family Moegitake, and a mixture thereof. Anti-rheumatic agents listed. (62) The anti-rheumatic agent according to claim 61, wherein the plant of the genus Pyrophylla of the family Pleurophyllaceae is a shiitake mushroom. (63) The anti-rheumatic agent according to claim 61, wherein the plant of the genus Enokitake of the family Asteraceae is Enoki mushroom. (64) The anti-rheumatic agent according to claim 62, wherein the plant of the genus Shimeji in the family Shimediaceae is Shimeji. (65) Maitake is a plant of the genus Maitake of the family Takokinaceae,
The anti-rheumatic agent according to claim 61. (66) The anti-rheumatic agent according to claim 61, wherein the plant of the genus Polyporus of the family Salmonaceae is an abalone mushroom. (67) The anti-rheumatic agent according to claim 61, wherein the plant of the genus Agaricaceae of the family Agaricaceae is a mushroom. (68) A plant of the genus Fungus of the family Anoraceae is a fungus.
The anti-rheumatic agent according to claim 61. (69) A plant of the genus Sugitake of the family Moegitake is nameko.
The anti-rheumatic agent according to claim 61. (70) The anti-rheumatic agent according to claim 60, wherein the Ascomycetes plant is a plant of the genus Saccharomyces of the family Endomycetaceae, a plant of the genus Nomushitake of the family Buccaneaceae, or a mixture thereof. (71) A plant of the genus Saccharomyces in the family Endomycetaceae,
The anti-rheumatic agent according to claim 70, which is baker's yeast, brewer's yeast, and mixtures thereof. (72) The anti-rheumatic agent according to claim 70, wherein the plant of the genus Cordyceps of the family Acanthaceae is Cordyceps sinensis. (73) The anti-rheumatic agent according to claim 3, wherein the bacteria are selected from the group consisting of Escherichia coli, Bordetella pertussis, and mixtures thereof. (74) The anti-rheumatic agent according to claim 73, wherein the LPS obtained from E. coli has the following physical properties. Molecular weight = 30,000 ± 5,000 (SD S electrophoresis method) Phosphorus number = 12 / molecular weight 3-way hexosamine number = 45 ± 6 / molecular weight 30,000 fatty acid number = 18 / molecular weight 3-way KDO number = 5 ± 1 / molecular weight 75. The antirheumatic agent according to claim 73, wherein the LPS obtained from Bordetella pertussis has the following physical properties. Molecular weight = 6,000 ± 1,000 9,000 ± 1,000 (SDS electrophoresis method) Number of phosphorus = 5 / molecular weight 8,000 Hexosamine number = 16 ± 2 / molecular weight 8,000 Number of fatty acids = 5 / molecular weight 8,000 KDO number = 2±1/molecular weight 8,000 (76) It is an anti-rheumatic agent for animals containing LPS, and the macrophage activation ability of LPS that activates the TNF production ability of macrophages cultured in vitro is used as an index, and the vertical axis is The macrophage activating ability that gives the amount of TNF production by macrophages when LPS is not added is 0%, and the macrophage activating ability of LPS that gives the amount of TNF production by macrophages to the maximum constant amount is 100% ( %), and the horizontal axis shows the L
When drawing a sigmoid curve expressing the Limulus test positive LPS content of PS on a logarithmic scale, at least one type of LPS whose Limulus test positive LPS content giving ED_5_0 of macrophage activation ability is 0.4 to 100 ng/ml of culture solution. Antirheumatic agent for animals.