JPH06263650A - Lps-containing antitumor agent and antitumor agent for animal - Google Patents

Abstract

PURPOSE:To obtain an antitumor agent having high antitumor effect, usable for a long period of time, capable of being administered orally, percutaneously or by injection and an antitumor agent for animal. CONSTITUTION:An antitumor agent and an antitumor agent for animal contains at least one of the following LPSs. When macrophage activating ability of LPS to activate TNF producing ability of macrophage to be cultured in vitro of LPS is used as an index and a sigmoid curve is drawn by showing macrophage activating ability (%) wherein macrophage activating ability to provide a produced amount of TNF of macrophage when LPS is not added is 0% and macrophage activating ability of LPS to make a produced amount of TNF of macrophage at the maximum amount is 100% in ordinate and exhibiting a content of LPS positive in a limulus test of the LPS on a logarithmic scale in abscissa, the LPSs have 0.4-100ng/ml culture solution of the content of LPS positive in a limulus test to provide ED50 of macrophage activating ability.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antitumor agent and an antitumor agent for animals. More specifically, the present invention relates to an antitumor agent containing LPS and an antitumor agent for animals.

[0002]

2. Description of the Related Art Tumors are a collection of cells that exhibit autonomous hyperproliferation ("Iwanami Biology Encyclopedia, 3rd Edition, pp. 571, 1".
Published by Iwanami Shoten in 983). There are many exceptions, and it is difficult to make a strict distinction between malignant tumors (so-called cancers) that can cause tumor-bearing animals to die and so-called benign tumors (see Iwanami Biology Encyclopedia above). 3rd edition, page 4).
Depending on the type, cancer treatment methods have progressed to the point where 90% is said to be completely cured by early detection and early treatment, but in Japan since 1981, it remains the leading cause of mortality (Cancer statistics published by the Cancer Research Promotion Foundation in 1991). Currently, the three pillars of cancer treatment methods are surgical therapy, drug therapy, and radiation therapy, and in reality, multimodal therapy combining these with laser therapy or the like is widely practiced. However, given the pain and metastasis associated with treatment, it is natural that expectations for drug therapy are high, and many anti-tumor agents have been developed and used in response to this (Translated by Motohara Fujiwara et al. “Goodman Gilman”). Pharmacology book, 7th edition, second volume, 1
(Pp. 527 to 1607, published by Hirokawa Shoten in 1988), none of them are effective against all types of tumors, and many cannot exert sufficient effects alone, and many cannot be used for a long time because of strong side effects. , Multidrug therapy has become common. Therefore, there is a strong hope for the development of new antitumor agents, particularly those having a high antitumor effect and which can be used for a long time by a simple method.

[0003]

DISCLOSURE OF THE INVENTION The present invention aims to provide a novel antitumor agent and an antitumor agent for animals.

[0004]

[Means for Solving the Problems] The above technical problems have an excellent antitumor effect, can be used for a long period of time, have a low production cost, and can be administered by any of the oral, transdermal and injection routes. This can be achieved by providing an antitumor agent containing LPS, which can be supplied in a large amount, and an antitumor agent for animals.
The antitumor agent and the animal antitumor agent are LPs that activate the TNF-producing ability of macrophages cultured in vitro.
Using the macrophage activating ability of S as an index, the vertical axis indicates the macrophage activating ability that gives the TNF production amount of macrophages when LPS is not added to 0%, and the macrophage TNF production amount is the maximum and constant value (the present specification). In other parts of the book, "L" is the "maximum constant"
When the sigmoid curve showing the limulus test-positive LPS content of the LPS on a logarithmic scale is drawn on the horizontal axis, the ED of the macrophage activating ability is shown, which represents the macrophage activating ability (%) with the macrophage activating ability of PS as 100%.
Limulus test positive LPS content giving ₅₀ is 0.4
~ 100 ng / ml of culture medium of at least 1 of LPS
Seeds are included. Here, "containing at least one kind" means
It means that the LPS of the present invention can be used separately, and as long as its intended use is not impaired, two or more thereof can be used in any combination, and further, can be used in combination with any other substance. . For example,
It can also be combined with other antitumor agents and the like.

[0005] "Macrophage" is a type of immunocompetent cell, and is a generic term for large amoebic 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. Is. "TNF" is a tumor-damaging factor (Tumor) produced by macrophages.
Necrosis Factor) [1
The Journal of Biological Chemistry, published in 985 (The Journal o
f Biological Chemistry. , 2
60 , 2345-2354], the production amount increases as the activity of macrophages increases. The "limulus test" is an endotoxin quantification method, which was invented by Levin in 1968, using a horseshoe crab blood cell extract and a chromogenic synthetic substrate. LPS that can be used as an active ingredient of the antitumor agent or the animal antitumor agent of the present invention is not particularly limited in its collection source, production method and purification method. For example, it may be LPS collected from bacteria or plants, or a synthetic product such as synthetic lipid A.
Incidentally, in the present specification, particularly in the claims thereof, the collection source is not particularly limited to the one specified by the name, but the growth source of the collection source, storage, attachment of bacteria in the process of distribution, other coexisting bacteria, etc. Everything is included. For example,
When "wheat LPS" is specified, it is understood that it includes not only LPS collected from wheat itself, but also all bacteria and other substances that adhere and coexist during the growth, storage, and distribution processes of wheat. I want to. This is because, in particular, other than those for which the relationship of parasitism, symbiotic plants, parasitism, and symbiotic animals has been elucidated, those that are allowed to adhere to or coexist with specific plants, animals, fungal organisms, lichen organisms. This is because it is well known in the art that there can be many examples in which

Of these LPS, LPS which can be used as an active ingredient of the antitumor agent or the animal antitumor agent of the present invention
To select TNF production capacity of macrophages cultured in vitro, the macrophage activation ability of LPS is used as an index, and the vertical axis indicates the TNF production amount of macrophages when LPS is not added. The ability to activate macrophage (%) is defined as 0%, and the ability to activate macrophage to 100% is the ability to activate macrophage when the amount of TNF produced by macrophage is maximally constant. When drawing a sigmoid curve that represents the amount on a logarithmic scale, a limus test-positive LPS content that gives an ED ₅₀ of macrophage activating capacity of 0.4 to 100 ng / ml of culture medium may be selected.

Examples of limulus test-positive plant-source LPS raw material plants that can be used are shown below. The family name and genus to which the plants described in this specification belong are
It was decided by collating the descriptions in the following documents. Gymnosperms, monocotyledons, dicotyledons, ferns, and sows: Matched to "Primary color Makino Botanical Encyclopedia" issued by Kitatakakan in 1982 (original edition) and 1983 (sequel) And decided the affiliation.
However, for "tsubame," the "edible plant illustrations" published by the Women's Nutrition University Press in 1965 and the "New Japan Botanical Magazine Hana Hana" published by Sobundou in 1983 are checked. However, "bare barley" matches the "General food encyclopedia" issued by Tokyo Doshoin in 1946, and shows "Pigeon barley", "Karabisubikaku", "Janohigashi", "Turmeric", "Matatabi", "Amateur crane", "Dokudami", "Pepper",
"Pepper", "Dai Feng", "Daidai",
"Kudzu", "Nankinkanzo", "Garden carrot", "Bowfu", "Otsudurafuji", "Uncaria hirsuta" are the descriptions of "Primary color Makino Japanese and Chinese herbal encyclopedia" issued by Kitatakakan in 1988. "Avogado" is the tropical agricultural technology series No. 15 "Brazilian fruits" published by the Japan Agricultural and Forestry Statistics Association in 1978, and "Kaiware radish" is the north in 1984. Matching the description of "Primary color garden plant large picture book" issued by Ryukan, "Nikuzuku" matches the description of "Illustrated tropical plant compilation" issued by Hirokawa Shoten in 1969, "Chlorella" is , Japan Health Food Association announced in 1986, the "Chlorella Standards and Standards" description was collated to determine the affiliation. Fungi: The affiliation was determined by collating the description in the "Primary color Japanese new fungus pictorial book" issued by a nursery company in 1987.
However, for yeast, check the description in the "Microbiology Handbook" issued by Gihodo in 1937, and confirm that it is "caterpillar".
Decided the affiliation by collating the description in the “Primary color Makino Wakahan herb encyclopedia” above. The raw material plants that can be used in the present invention are, for example, gymnosperms, monocotyledons, dicotyledons, ferns, sows, and fungi, which can be used individually or as a mixture. As the gymnosperm, for example, pine which is a pine plant of the pine family can be used. The monocotyledonous plants include, for example, rice, which is a plant of the genus Gramineae, wheat, which is a plant of the genus Wheat, barley, which is a plant of the genus Barley of the family Gramineae, barley, and crow, which is a genus of oats, and oats. , Bear grass, which belongs to the genus Gramineae, pigeon barley, which belongs to the genus Juzudama, iris, which belongs to the family Iridaceae, garlic, which belongs to the genus Liliaceae, and asparagus, which belongs to the family Liliaceae, to the family Liliaceae. It is possible to use Janohige, which is a plant of the genus Jardinaceae, ginger, which is a plant of the genus Ginger of the ginger family, turmeric which is a plant of the genus Turmeric of the ginger family, and crow bishaku, which is a plant of the genus Araceae family of the genus Hange. Examples of the dicotyledonous plant include soybean, which is a leguminous soybean plant, adzuki bean, which is a leguminous kidney bean plant, broad bean, which is a leguminous broad bean plant, kudzu, which is a leguminous plant, and legume licoriceae plant. Pomegranate liquorice, potato which is a Solanaceae plant of the genus Solanaceae, capsicum, tomato which is a plant of the genus Tomato of the Solanaceae family, capsicum which is a plant of the genus Capsicum family, loquat which is a plant of the genus Rosaceae, peach which is a plant of the genus Rosaceae, Lauraceae avocado plant Avocado, walnut family walnut plant walnut, cucurbitaceae Touna plant pumpkin, cucurbitaceae plant genus Amachatsuru, Brassicaceae plant radish plant Kaiware radish, caterpillar plant Matatabi, which is a plant of the genus Lentilaceae Cucumber, which is a genus of genus, Daiwikyo, which is a genus of genus Shikimi, squid, which is a genus of narcotics, daisies, which is a plant of citrus citrus, citrus ginseng, ginseng, which is a genus of ginseng genus, and saposi nicosia spp. Can be used, and Uncaria hilsta, which is a plant belonging to the family Rubiaceae, Kazikazura, can be used. As the fern plant, for example, horsetail, which is a plant belonging to the genus Astragalus, belonging to the family Equisetum, and mainspring, which is a plant belonging to the genus Spiral sp. Examples of the plants belonging to the genus Caudaceae include plants belonging to the genus Duckweed, plants belonging to the genus Rhodophyta, plants belonging to the genus Rhododendron and plants belonging to the genus Ranunculus. As the Cassowaceae plant, for example, wakame, which is a plant belonging to the kelp family Wakame, kelp which is a plant belonging to the kelp family kelp, and Hijiki which is a plant belonging to the genus Rhizophoraceae. As the red sour plant, for example, Asakusanori, which is a plant belonging to the genus Amanori of the family Ushikenori can be used. As the green grass plant, for example, chlorella which is a plant of the genus Chlorella of the family Oleocystis can be used. As the fungal plant, for example, a basidiomycete plant or an aspergillus fungus plant can be used. Examples of basidiomycete plants include shiitake mushrooms that are genus Pleurotus genus plants, enoki mushrooms that are enokitake plants of the family Kishimeji family, shimeji mushrooms that are genus plants of the genus Chidaceae, maitake mushrooms that belong to the genus Physcomitaceae of the family Oleaceae, genus Polyporus Abalone mushrooms, which are plants, mushrooms, which belong to the genus Agaricaceae, mushrooms, which belong to the genus Asteraceae, and nameko, which belongs to the genus Asteraceae, can be used. As an ascomycete plant, for example, baker's yeast, which is a plant of the genus Saccharomyces of the family Endomyceteaceae, or yeast for brewing can be used. Brewing yeasts include brewer's yeast, sake yeast, wine yeast, soy sauce yeast,
In addition to miso yeast and the like, many yeasts belonging to Saccharomyces cerevisiae (for example, yeasts used in the production of whiskey and sake) are included. Moreover, Cordyceps sinensis, which is a plant of the genus Nostoc, belonging to the family Baccinaceae can be used. Plant source LPS
Can be separated and purified by the method described below. 1) If necessary, the raw material plant is appropriately shredded, dried, pulverized, and then well suspended in distilled water, and the supernatant is recovered. For example, if the source plant is cereal seed, leave the seed coat attached,
Alternatively, after removing the seed coat, it is easily crushed, or crushed until it becomes a powder that is about to be used for food, water is added to the obtained powder to make a dispersion liquid, and the precipitate is stirred and then precipitated. It may be removed by standing or centrifugation, or the dough obtained by adding water to the powder and kneading may be gently washed with water in a mixer to remove the precipitate. When the starting plant is Chlorella, it is advisable to first disrupt the cell membrane, remove the fat-soluble substance by washing with ethanol, and then extract with water. The particle size of the raw material plant at the time of this water extraction, the temperature of water, the liquidity, the addition amount, the stirring speed, the time, the conditions at the time of centrifugation, etc. need not be particularly limited, and may be appropriately selected depending on the type of the raw material plant. Adjust it.
Further, the higher the temperature of the extracted water, the higher the amount of LPS collected and the higher the purity tend to be. However, for convenience of operation, it is preferably 50 ° C. or lower, which does not cause gelatinization of the starch contained in the raw material plant. The amount of water added varies depending on the type of raw material plant and particle size, but in the case of cereal seeds, the ratio is 70 w / v%.
Hereafter, it is desirable for the operation to be about 20 to 50 w / v%, which is convenient for operation. Furthermore, it is preferable that the stirring speed be such that foaming does not occur. By the operation up to this stage, the purity of the limulus test-positive plant LPS of the present invention is judged to be about 30 times higher in the case of wheat seeds, as judged from the limulus test activity data. Hereinafter, the case where grain seeds are used as a raw material will be described as an example,
It is extremely easy for a person skilled in the art to carry out a method for removing high-purity limulus test-positive LPS by removing contaminating sugars, proteins and the like from other plants by referring to the following description. 2) To further raise the purity, the supernatant obtained in 1 above may be subjected to ultrafiltration according to a conventional method to remove a fraction having a molecular weight of 5000 or less. 3) The dried product thus obtained is suspended in distilled water so as to have a concentration of 50 mg / ml, and subjected to a centrifugation operation to collect a supernatant. 4) The supernatant is cooled with ice water, and acid is added to make the solution acidic to cause precipitation. The acid used at this time does not have to be a specific one, and for example, trichloroacetic acid (hereinafter referred to as TCA), perchloric acid, trifluoroacetic acid, acetic acid, dichloroacetic acid can be used. 5) Next, it is subjected to a centrifugation operation to collect the precipitate, washed with distilled water, and again subjected to a centrifugation operation to recover the precipitate. 6) Suspend the precipitate in distilled water and add alkali until the precipitate dissolves. The alkali used at this time does not have to be a specific one, and for example, sodium hydroxide, potassium hydroxide, ammonia, sodium carbonate, sodium acetate can be used. It should be noted that the target LPS will be inactivated if the basicity becomes higher than pH 11 when the precipitate is dissolved. 7) Next, add an acid to adjust the pH to 8 and then heat to 37 ° C, and add an acid to acidify the product.
Centrifuge using a centrifuge kept at 7 ° C. The acid used at this time does not have to be a specific one. 8) Collect the supernatant, cool with ice, and centrifuge again at 4 ° C. 9) The supernatant is collected, neutralized by adding an alkali, and concentrated by ultrafiltration according to a conventional method. The alkali used at this time does not have to be a specific one. 10) Then, subject to gel filtration according to a conventional method to collect and combine the rimus test positive fractions. As a carrier for gel filtration, for example, Sephadex G
-75, G-100, Sephacryl
l) S-200, Sepharose
6B [The above is Pharmacia, USA (Pharmacia
Inc. )], Biogel (Biogel) P-1
00 [manufactured by US Biorad Inc.], Toyo Pearl HW-50, HW-55 (manufactured by Toyo Soda Kogyo Co., Ltd.) can be used. Any buffer may be used as long as it has a pH of 3 to 10. For example, Tris-HCl or phosphate buffer can be used. 11) Then, a proteolytic enzyme is added to this fraction and the mixture is incubated at 37 ° C. for 2 hours or more to decompose the residual protein, and the obtained enzyme-treated solution is concentrated by ultrafiltration according to a conventional method. The proteolytic enzyme used at this time does not have to be a specific one. For example, V8 protease,
Chymotrypsin, trypsin, thermolysin can be used alone or in any combination. As commercially available products, for example, Pronase E (Kaken Kagaku) and Proteinase K (Merck) can be used. 12) Then, this fraction was subjected to a conventional method, for example, using a FPLC system manufactured by Pharmacia, USA, mono-Q-Sepharose, Q-, manufactured by Pharmacia.
Anion exchange chromatography is performed using Sepharose to obtain a limulus test positive fraction. 13) Next, gel filtration is carried out for desalting according to a conventional method to collect a limulus test positive fraction. By the above operation, 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. Also, the purity is about 100 compared to the purity at the end of step 1).
It is 0 times more pure (for wheat seeds). The limulus test-positive plant LPS obtained by the above method can be provided as it is or in a concentrated form to an arbitrary degree. or,
In order to improve storage stability, it can be provided as a dry powder by any means such as freeze-drying or spray-drying. Any of these can be produced by a conventional method.

Limulus Test Positive Bacterial Source LPS E. coli LPS, Alcaligenes, which has been conventionally known
LPS obtained from A. radiobacterium [P. H. Graham (PH
Graham), Em. A. Aubrien (MA
Co-authored by O'Brien, "Antonic van Leeuwock
enhock), vol. 34, 326-330 pages (1
968); elsewhere in this specification, A. [Radiobacter LPS]], B. pertussis LPS, Lipid A, etc., as well as bacterial sources LPS1, LPS described in detail later in this specification.
2, LPS3 and their synthetic LPS are relevant. E. coli LPS is commercially available from, for example, Difco, USA. B. pertussis LPS is commercially available from, for example, Funakoshi Yakuhin (Japan). It can also be prepared from known pertussis bacteria, for example, killed cells of Tohama strain Phase I bacteria, by a known method described in the following documents, for example. "Journal of Immunity" by Webster and others.
, 744, 55 (1955); Westphal et al., "Z. Naturforsch," 76, 1.
48 (1952). Lipid A is commercially available, for example, from Daiichi Pure Chemicals. The above-mentioned bacterial sources LPS1, LPS2, L
The three types of bacteria that produce PS3, respectively, have been isolated from wheat examined by the present inventors, regardless of their origin and type. Therefore, it is presumed that it is separated from wheat and wheat products of all production areas and types. The present inventors
The flour production areas and types confirmed to be able to separate the bacteria of the species are as follows. The name of dark wheat, the origin of wheat flour Dark Northern Springs USA 1. Canadian Wheat Canada Hard Red Winter Semi Hard USA Australian Standard Wheat Australia Horosiri Japan To isolate LPS1, LPS2 and LPS3 from the above bacteria , Westphal, et al., “Methods in Carbohydrate Chemistry (Me
ways in Carbohydrate Che
(mistry) vol. V [published by Academic Press, Inc., New York, USA, 1965], page 83, using the hot phenol method and further purification with an anion exchange resin. That is, after suspending the bacterial cells in distilled water, stirring with distilled water and an equal volume of hot phenol, then collecting the aqueous layer by centrifugation, the aqueous layer is dialyzed to remove the phenol,
A crude LPS fraction was obtained by concentration by ultrafiltration, and this fraction was subjected to a conventional method, for example, using a Pharmacia FPLC system to produce Pharmacia Mono Q-Sepharose (Se).
pharose), Q-sepharose (Sepharo)
Se) may be used for purification by anion exchange chromatography, and desalting may be performed according to a conventional method. By the above operation, a purified sample with a purity of 96% or more can be obtained.
The limulus test positive LPS in the raw material can be detected and the content thereof can be measured using a reagent set commercially available from Seikagaku Corporation under the name Toxicolor System, as described in detail in Experimental Example 1 below. That is, the raw material plants may be colored together with the LS-1 set of the same system, and the intensity of the color development may be compared with the calibration curve prepared using the Et-2 set of the same set. The sugar is a phenol-sulfuric acid method [M. Deuboisu (M. Dubo
is) et al., Analytical Chemistry (Analy)
mechanical Chemistry), vol. 28, 3
50, 1956], the protein is the Lowry method [Oh. H. OH Lowry et al., Journal of Biological Chemistry (Journa)
l of Biological Chemistr
y)], vol. 193, 65, 1951].

In vitro TN of macrophages with LPS
Method for measuring the ability to activate F-producing ability It is necessary to have a production precursor (priming) step and a production initiation (triggering) step in order to produce TNF in the animal body.
The Proceeding of National Academy Science of USA [Proce
eding of NationalAcademy
Science of USA. , 72, 3666-3
670 (1975)]. The drug administered to initiate the priming phase is the "primer"
(Endogenous TNF production promoter), the drug administered to initiate the triggering phase is the "trigger" (endogenous TNF).
NF producer). To measure the ability of LPS to activate the in vitro TNF-producing ability of macrophages, mouse macrophage peritoneal resident cells were collected, to which recombinant mouse IFN-γ as a primer was added, and then as a trigger. LPS may be added and its TNF activity may be measured. TNF activity was measured in L-929 cells [Proceeding of National Academy].
Science of USA 72 , 3666-
Based on the cytotoxicity to [3670 page], it is measured as follows. L929 cells were prepared from Eagle Minimum Essential Medium (hereinafter, ME) supplemented with 5% fetal bovine serum.
M medium), and 8 × 10 ⁴ cells are 100
It is made to be contained in μl of the same medium and bred in a 96-well flat bottom plate. Breeding conditions are 37 ° C., 2 hours, 5% CO _2.
The method used in ordinary cell culture may be used. After that, actinomycin D was added to the medium at a final concentration of 1 μg / ml.
And the volume of the culture solution is 150 μl.
Immediately, dilute the sample appropriately with MEM medium to 50
Add μl (at this time, adjust the dilution ratio appropriately to obtain the ED ₅₀ ). Furthermore, the final volume of L92 was 200 μl
Nine cells are cultured under the above conditions for 18 hours. To measure cytotoxic activity, first remove all medium, then 0.1%
Fix and stain by adding 1% methyl alcohol solution containing crystal violet. Although crystal violet stains all nucleated cells, dead cells are removed by washing with water from the bottom of the plate after staining, and therefore cytotoxic activity can be directly measured from the results of viable cells. This dyeing degree is OD (590n
The cytotoxic activity is measured by measuring the absorbance in m) as an index and comparing it with the staining degree for the control group. The definition of activity is as follows. The dilution ratio (N) of the sample in which L929 cells can survive 50% is determined. Rabbit TNS as a control
[Tumor Necrosis Se
Rum)], the activity n (unit / ml) of this rabbit TNS is 2.4 × 10 ⁶ units / mg / ml of TNF-.
α (recombinant human TNF obtained from Asahi Kasei Corporation)
To determine. The dilution ratio (C) giving the ED _{50 of} this rabbit TNS is determined. Sample activity (unit / ml) is N
Calculate with / C × n.

Method of Producing Agent that can be Provided The antitumor agent of the present invention can be prepared into powders, granules, pills, tablets, troches, capsules, liquids, patches, ointments, liniments by conventional formulation techniques. , Lotion, suppository, injection and the like. For animals, it can be further prepared as a feed additive, a premix preparation, and a drinking water additive. When used as a feed additive, it is preferably a powder or granules. Further, the premix formulation refers to a formulation diluted with a feed ingredient such as starch in order to facilitate mixing with a feed. The feed to which the antitumor agent of the present invention can be added as a feed additive or a premix preparation may be any commercially available feed. It may also be a feed containing feed additives such as minerals, vitamins and amino acids. If desired, additives such as an excipient, a preservative and a buffer may be added to these preparations by a conventional method in order to maintain the storability and homogeneity. Furthermore, flavoring agents, flavoring agents,
Colorants can also be included. As the excipient, for example, lactose or starch can be used. As the preservative, for example, paraoxybenzoic acid esters such as methyl paraoxybenzoate, ethyl paraoxybenzoate and propyl paraoxybenzoate, sodium dehydroacetate, phenol, methylparaben, ethylparaben, propylparaben and the like can be used. As the buffer, for example, citrate, acetate, phosphate and the like can be used.

Confirmation of antitumor effect The antitumor effect of the present invention was confirmed by administration to mice. Hereinafter, the present invention will be described in more detail with reference to Examples, Production Examples, and Experimental Examples. In addition, "E. coli LP used in them
S] is O128: B manufactured by Difco, USA
8

Production Example 1 ( Production of wheat LPS) 1) Hard wheat flour containing 1.09% ash in a small kneader (hardlet spring produced in the United States or Canada)
(3,120 g) was added, 2.03 liters of distilled water was added, and the mixture was kneaded for 10 minutes to give a dough. After standing for 15 minutes, 10 liters of water was added and gently stirred to wash out the starch emulsion, and at the same time, soluble components were eluted. The eluate was allowed to stand in a refrigerator at 5 ° C for 12 hours, and then the sedimented portion such as starch was removed. Lyophilize the supernatant to 201.
1 g of powder was obtained (powder A). Further, 5 liters of distilled water was added to the residual dough, and the mixture was gently stirred, and then treated in the same manner as above to obtain 40.1 g of powder (powder B). 2) These powders A and B are ultrafilter H manufactured by Amicon Inc.
F-Lab1, and the hollow cartridge HF-Lab1PM5 for the molecular weight fraction of 5,000 and the hollow cartridge HF-L for the molecular weight of 10,000.
ab1PM10 was attached and ultrafiltration was performed [Temperature 5
-10 ° C. Input pressure 25 psi (1.76 kg / cm ² ),
Output pressure 15 psi (1.06 kg / cm ² )]. Based on the result, each part was named as follows. Powder A: a portion having a molecular weight of 5,000 or less a ₁ powder having a molecular weight of 5,000 or more a ₂ powder B: portion having a molecular weight of 5,000 or less b _{1 having a} molecular weight of 5,000 or more b ₂ powder A: molecular weight A portion having a molecular weight of 10,000 or less is a ₃ A portion having a molecular weight of 10,000 or more is a ₄ Powder B: A portion having a molecular weight of 10,000 or less is b ₃ A portion having a molecular weight of 10,000 or more is b ₄ When subjected to the limulus test according to the method described in detail in Section 1, a large amount of the limulus test positive component is present in the fraction having a molecular weight of 5,000 or more, but it is almost absent in the fraction having a molecular weight of 5,000 or less. confirmed. 3) Put 30 g of the above powder a _{2 in} a 1-liter Erlenmeyer flask, pour 600 ml of distilled water, stir with a stirrer for 60 minutes, and then use Hitachi Cooling High Speed Centrifuge SCR-20B.
(Rotor RPR16 was previously cooled to 4 ° C)
The mixture was subjected to a centrifugation operation (10,000 G × 10 minutes) at 4 ° C. to collect the supernatant. 4) Put this supernatant in a 1 liter Erlenmeyer flask and stir with a stirrer under ice-cooling (liquid temperature about 2 ° C.) in advance.
20.5 ml of 100% TCA aqueous solution that had been cooled to ℃
Was added dropwise, and after completion of the addition, the mixture was left in ice water for 10 minutes. 5) Then, in the same manner as above, the centrifugation operation (1
The precipitate is recovered by subjecting it to 50,000 G × 10 minutes), put in a beaker of 500 ml together with 300 ml of distilled water under cooling in ice water, and then cooled in ice water at 4 ° C. in the same manner as above. The precipitate was collected by centrifugation (10,000 G × 10 minutes). 6) Place this precipitate in a 1-liter beaker and add distilled water 50
Suspended with 0 ml, 1N sodium hydroxide solution about 3.5 m
It was neutralized (pH 7) using 1 and then while cooling in ice water, about 2 ml of 1N sodium hydroxide solution was added to dissolve the precipitate to a 0.02N sodium hydroxide solution. 7) About 1.5 ml of 1N hydrochloric acid was added to adjust the pH to 8, then 100 ml of distilled water was added, and the mixture was transferred to a 1 liter Erlenmeyer flask and shaken slowly in an incubator at 37 ° C. for 30 minutes. 8) After adding 30 ml of 100% TCA aqueous solution and mixing, slowly shake in an incubator at 37 ° C for 10 minutes, and then centrifuge Tommy C kept at about 37 ° C.
D100R (manufactured by Tommy Seiki Co., Ltd.) was used for centrifugation (3,000 G × 10 minutes). 9) Collect the supernatant, cool with ice, and centrifuge at 4 ℃ (1.
10,000 G × 10 minutes). 10) The supernatant was recovered, neutralized with about 3.6 ml of 10N sodium hydroxide solution to pH 7, and then ultrafiltered (Toyo Roshi UHP-150, filter: UK-10, N ₂ pressure:
It concentrated at 4.0 kg / cm < ² >. 11) 60 ml of the obtained concentrate was added to Sepharose (Se).
pharose) 6B column [Pharmacia, USA (P
farm Inc. ), Column size: 5c
m (inner diameter) x 100 cm (2 liters)] and gel filtration [buffer: 10 mM Tris-HCl / 10 mM Na]
Cl (pH 7.5), flow rate: 60 ml / hour],
Fractions of 20 ml each were obtained. 12) Fraction 280m from the 43rd to the 56th from the beginning
l, combined with 450 μg of Pronase E (Kaken Kagaku Co., Ltd.), and kept under shaking at 37 ° C. for 2 hours, and then filtered with an ultrafilter (Toyo Roshi UHP-62, filter: UK-10,
The mixture was concentrated under N ₂ pressure of 4.0 kg / cm ² . Next, Pharmacia FPLC system (column: Mono QHR
10/10) and subjected to anion exchange chromatography. That is, 10 mM Tris-HCl (pH 7.
After the sample was applied to the column with a buffer containing 5) and 10 mM NaCl, the column was washed with a buffer (200 ml) having a composition in which the amount of NaCl was increased to 165 mM with the above buffer. Then, the NaCl concentration was increased to 165 mM to 1 M NaCl concentration gradient, and the total amount was 40%.
The target LPS was eluted with 0 ml, and fractions of 2 ml each were collected. The limulus test was confirmed to be positive, and the LPS purity of about 92 was calculated by combining the 5th to 8th fractions after applying the concentration gradient.
% 8 ml [LPS: 3.03 mg (the limulus test positive LPS conversion value measured by the method described in Experimental Example 1 below. All LPS amounts below are also this conversion value), sugar:
0.23 mg, protein: 0.04 mg] was recovered. 13) Then, 8 ml of the same is applied to Sephadex (Seph).
adex) G-25 [column: 2.0 cm (inner diameter) x 2
0.2 cm (66 ml)] using gel filtration (buffer:
Water) to collect 3 ml fractions. Combine the 9th to 12th fractions with positive limulus test and
12 ml of PS purity about 95% (LPS: 2.7 mg,
Sugar: 0.18 mg, protein: 0.03 mg) was recovered.
It was confirmed by anion exchange chromatography that this fraction was acidic. 14) The above fraction was frozen at −80 ° C. and then freeze-dried until a constant weight was obtained, and the weight was measured and found to be 0.75 mg. (Hereinafter, this freeze-dried preparation is referred to as wheat LPS) This wheat L
When the limulus activity of PS was measured by the method described in Experimental Example 1 below, it corresponded to 2.7 mg, so its specific activity was 2.7 ÷
0.75 = 3.6. In addition, since it is considered that substantially all the single sugar that may be present as a contaminant has been removed by the above purification, all the detected sugars are considered to be sugars that compose wheat LPS. Therefore, when the purity of wheat LPS at this stage is calculated based on weight, protein = 0.03 mg LPS = 0.75-0.03 = 0.72 mg, so 0.72 ÷ 0.75 × 100 = 96 (% ) Is.

Physical Properties of Wheat LPS 15) Molecular Weight Wheat LPS was dissolved in distilled water to prepare a 1 mg / ml solution, and 4 μl of the solution was placed in a 1.5 ml tref tube. Separately, add 2.5 mM SD to 1 mM EDTA.
1 μl of an SDS-treated solution prepared by adding S, 5% mercaptoethanol, 10 mM Tris-hydrochloric acid (pH 8.0) was added, and this mixed solution was immersed in boiling water for 3 minutes. Pharmacia Fast System (Phast System)
And SDS-buffer strip (Buffer Strip) (manufactured by Pharmacia) between the electrodes.
1 μl of the above mixed solution in which a gel was interposed was used as a gel [Pharmacia Fast Gel Gradient (Phast
Gel Gradient 8-25)], the maximum voltage was set to 250 v and the maximum current was set to 10 mA to start the migration (this migration method is referred to as SDS-1 method in the present specification). After the electrophoresis, the behaviors of Coomassie staining and silver staining were observed. For Coomassie staining, 0.1% Fast Gel Blue (Phast) made by Pharmacia
Gel Blue) R was dyed and decolorized in the following order using a mixed solution of methanol: acetic acid: distilled water (volume ratio 3: 1: 6) as a decolorizing solution. 1: Staining at 50 ° C. for 8 minutes 2: Decoloring at 50 ° C. for 5 minutes 3: Staining at 50 ° C. for 8 minutes 4: Decoloring at 50 ° C. for 10 minutes 5: Protection at 5 ° C. for 5 minutes (volume ratio of glycerol, acetic acid, and distilled water) 5:10:85 mixed solution) 6: Drying Silver dyeing was performed in the following order. Treatment with washing liquid (ethanol, acetic acid, distilled water volume ratio 5: 1: 4 mixture) at 1: 50 ° C. for 2 minutes, 2: 50 ° C. for 2 minutes washing liquid (ethanol, acetic acid, distilled water volume ratio 10: 5 : 85 mixed solution) 3: 50 ° C. for 4 minutes, washed solution (ethanol, acetic acid, distilled water volume ratio 10: 5: 85 mixed solution) treated at 4: 50 ° C. for 6 minutes, sensitizing solution (8.3%) Treatment with glutardialdehyde) 5: 50 ° C. for 3 minutes, cleaning solution (mixture of ethanol, acetic acid and distilled water at a volume ratio of 10: 5: 85) Treatment with 6: 50 ° C. for 5 minutes, cleaning solution (ethanol, acetic acid, distilled water) Volume ratio of 10: 5: 85) 7: 50 ° C for 2 minutes, cleaning solution (deionized water) 8: 50 ° C for 2 minutes, cleaning solution (deionized water) 9: 40 ° C 13 Treatment with 0.25 w / v% silver nitrate for 10 minutes, washing at 10: 30 ° C. for 30 seconds Treatment with (deionized water) 11: 30 ° C. for 30 seconds, treatment with cleaning solution (deionized water) 12: 30 ° C. for 30 seconds, developing solution (0.04 v / v% formaldehyde + 2.5 w / v% sodium carbonate washing solution) ) 13:30 for 4 minutes, developer (0.04 v / v% formaldehyde + 2.5 w / v% sodium carbonate wash)
Treatment with 14: 50 ° C. for 2 minutes, reaction stop solution (5% v / v% acetic acid) 15: 50 ° C. for 3 minutes, protection solution (acetic acid, glycerol,
Treatment with distilled water volume ratio of 10: 8: 85) 16: Dry LPS dyes silver, but does not dye Coomassie.
A major stained band of wheat LPS was detected at a position of 0 ± 1,000. Separately, dissolve wheat LPS in distilled water to 2 mg /
A ml solution was prepared and 10 μl thereof was placed in a 1.5 ml plastic tube. Separately, 180 μl of 10% (w / v) SDS, 45 μl of 5% β-mercaptoethanol, 90 μl of CBB dye solution, 112.5
μl 0.5 M Tris-HCl (pH 6.8) and 22.5
10 μl of an SDS-treated solution prepared by adding μl of distilled water was added and mixed well, then immersed in a boiling water bath for 5 minutes, immediately after this heating, immediately immersed in ice water and rapidly cooled. 10 ml 1
2. 0% (w / v) SDS, 17.9 g tricine and 3.
The migration buffer prepared by dissolving 03 g of Tris in 1 liter of distilled water was placed in a slab gel electrophoresis tank manufactured by Marisol. A 20% polyacrylamide gel was fixed in the electrophoresis tank, the sample was put in the sample groove, and the voltage was set to 50 V for 1 hour.
Then, the sample was fixed at 150 v, and electrophoresis was continued until the dye was eluted from the gel (this electrophoresis method was referred to as SD herein).
S-2 method). After completion of the electrophoresis, silver staining was performed at room temperature using Silver Staining Kit 161-0443 manufactured by Bio-Rad to confirm the behavior. As a result, the molecular weight is 8,000 ± 1,
A major staining band of wheat LPS was detected at the positions of 000, 5,000 ± 2,000. The SDS-1 method and SDS
-2 method, protein molecular weight markers electrophoresed with wheat LPS were LMW kit E manufactured by Pharmacia (phosphorylase b (94k), albumin (67k), ovalbumin (43k), carbonic anhydrase (3
0k), trypsin inhibitor (20k), α-lactalbumin (14k)], and peptide molecular weight markers are 1860-101 molecular weight markers [myoglobin (16.9k), myoglobin I & I manufactured by Pharmacia.
I (14.4k), myoglobin I (8.2k), myoglobin II (6.0k), myoglobin IV (2.5
k)]. 16) Phosphorus content Chen-Toribara method [Chen et al., "Analytical Chemistry (Ana
(Lytical Chemistry), vol. Two
8, 1756-1758 (1956). Dissolve wheat LPS in distilled water and add 25
A 20 μl solution containing μg wheat LPS was prepared and placed in a small test tube. Add 20 μl of 50 v / v% sulfuric acid,
Heated at 160 ° C. for 2 hours. Then 20 μl of 10v
After adding / v% perchloric acid, it was incinerated by heating with a gas burner for 1 minute. Then 0.5 ml distilled water, then 0.5 ml reaction reagent (1 ml 6N sulfuric acid, 2 ml distilled water, 2 ml 2.5 v / w% ammonium molybdate and 1 ml 10 v / w% ascorbic acid were mixed. Prepared, and 0.5 ml of the solution was added, and the mixture was allowed to stand at room temperature for 30 minutes, and then the absorbance at 820 nm (OD
_{820 nm} ) was measured. In addition, as a sample for preparing a calibration curve, potassium dihydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) was diluted with distilled water, and the phosphorus weights were 2.5 μg and 1 μ, respectively.
A 0.5 ml solution containing g, 0.25 μg, 0 μg was prepared and used. In addition, 1 g of phosphorus corresponds to 4.39 g of potassium dihydrogen phosphate. The results obtained are shown in Table 1.

[0014]

[Table 1]

In Table 1, the data of wheat LPS are the values obtained by subtracting the data of the control which has not been heat-treated in order to avoid an error due to contamination with inorganic phosphorus (for example, derived from a phosphate buffer solution). . The molecular weight of wheat LPS is 8,000
Assuming 0, the number of phosphorus per molecule is calculated by the following formula based on the results in the above table, and becomes 1 to 4.

[0016]

[Equation 1]

It is considered that one of the causes that the number of phosphorus fluctuates from 1 to 4 in the above experiment is that the phosphate is eliminated due to the contamination with monophosphoesterase in the purification step. 17) Hexosamine content Elson-Morgan method (Tokyo Kagaku Dojin Shuppan "Biochemistry Experiment Course" No. 4 37)
7 to 379) and performed as follows. Wheat LP
S was dissolved in distilled water to prepare a 1 mg / ml solution, 100 μl of which was placed in a Spitz with screw cap (Iwaki Glass Co., Ltd.), and 100 μl of 8N HCl was added thereto.
And heated at 110 ° C. for 16 hours. 4N NaOH
Was added to adjust the pH to 7. 100 μl
Was collected, placed in another Spitz with a screw cap, added with 200 μl of the following reagent A, heated at 105 ° C. for 1.5 hours, and then cooled with running water. Then 1
Aliquot 00 μl, add 670 μl of 96% ethanol, and further add 67 μl of the following reagent B.
After standing for a time, the absorbance was measured at 535 nm. As a sample for preparing a calibration curve, 0.20 to 200 μg / ml N-acetylglucosamine (manufactured by Wako Pure Chemical Industries, Ltd.) was used. (Reagent A) Prepared by mixing 75 μl of acetylacetone and 2.5 ml of 1.25N sodium carbonate. (Reagent B) Prepared by mixing 1.6 g of р-dimethylbenzaldehyde, 30 ml of concentrated hydrochloric acid and 30 ml of 96% ethanol. As a result, the hexosamine number of wheat LPS was 6 ± 2 / molecule (the assumed molecular weight was 8,000). 18) Fatty acid content 1 in 90 μl wheat LPS distilled aqueous solution (1 mg / ml)
0 μl of internal standard (0.55 mM margaric acid) was added. The fatty acid ester was hydrolyzed and esterified by adding 1.0 ml of 0.5 M sodium methylate.
After standing at room temperature for 1 hour, 960 μl of 0.5N HCl was added to neutralize. To this, 2 ml of hexane was added and stirred vigorously for 15 minutes. Then, centrifugation was performed at 1,000 g for 5 minutes to separate the hexane layer. Hexane was evaporated with nitrogen gas and concentrated to about 20 μl. This sample was gas chromatographed [Main body: Shimadzu G
C8APF, capillary column: FSCAP Sp2330 manufactured by Spelco of Canada, carrier gas: nitrogen], and the amount of fatty acid was measured. As a standard for measuring the amount of fatty acid, Escherichia coli type LA-15-PP (molecular weight of 2,000) which is a synthetic lipid A manufactured by Daiichi Pure Chemicals, Ltd.
0, the number of fatty acids in one molecule is known to be 6). As a result, the number of fatty acids in wheat LPS is 6 ± 2 /
It was estimated to be a molecule (assumed molecular weight 8,000). The charts observed by the above gas chromatography are shown in the attached FIGS. 1 to 3. FIG. 1 is a chart of wheat LPS, FIG. 2 is a chart of E. coli LPS, and FIG. 3 is a chart of B. pertussis LPS.
The retention times (minutes) corresponding to the major peak numbers shown in FIGS. 1 to 3 were as follows. By comparison of Figures 1-3, it is clear that the wheat LPS chart is similar to the E. coli LPS chart, but is significantly different from that of B. pertussis LPS. 19) KDO content The KDO (2-keto-3-deoxyoctonate) content was measured by the diphenylamine method [Shabyal (Shaby
R. ) Et al., Analytical Biochem (Analy
mechanical Biochem. ), 58 (1), 123
~ 129 (1974)]. A KDO detection reagent was prepared by combining 500 mg of diphenylamine, 5 ml of ethanol, 45 ml of glacial acetic acid, and 50 ml of concentrated hydrochloric acid (all manufactured by Wako Pure Chemical Industries, Ltd.). Part 5
250 μl of distilled water containing 1.05 mg / ml of wheat LPS was combined with 00 μl, and the mixture was heated in a boiling water bath at 100 ° C. for 30 times.
After heating for 1 minute, cool in cold water (23 ° C) for 30 minutes, then use Hitachi spectrophotometer 320 to 420, 470, 63
The ultraviolet absorption at 0 and 650 nm was measured (measured values are A420, A470, A630, and A650, respectively). As a standard sample, 250 μl of distilled water containing 127 μg / ml of KDO ammonium salt [manufactured by Sigma, USA] was used. The value of the following equation was determined for each of the specimen sample and the standard sample. S = A420-A470 + A630-A650 The value (S _T ) of the specimen sample is 0.379, and the value (S _T ) of the standard sample
_S ) was 0.294. By comparing these values, it was estimated that wheat LPS contained 5 ± 1 mol / KDO of 8,000 molecular weight.

Production Example 2 ( Production of Chlorella LPS) 1) Cell membrane disruption Chlorella (mannan foods Co., Ltd.)
30 g was washed with ethanol until the washings did not turn green. 2) 26 g of this washing residue was dissolved in distilled water at a concentration of 100 mg / ml, shaken at 45 ° C. for 2 hours, and then subjected to centrifugation (4 ° C., 10,000 G × 30 minutes). 3) The supernatant was collected, and Toyo Roshi No. It was filtered through 2 and then extracted with distilled water. 4) 290 ml of the extract was subjected to anion exchange chromatography under the following conditions. Column: Q-Sepharose (φ3 cm × 23 cm, capacity about 180 ml) Buffer: 10 mM Tris-HCl (pH 7.5), Na
Cl concentration gradient: 10 mM, 400 mM, 1 M Flow rate: 100 to 200 ml / hour Temperature: room temperature 5) 310 ml of the passed-through fraction was treated with glucoamylase to decompose starch (pH 5.0, 40 ° C., about 2 hours). Decomposition of starch was confirmed by the fact that no coloration occurred in the iodine starch reaction. 6) Centrifuge (10,000 G × 10 minutes) to collect the supernatant, neutralize with 10N NaOH solution to pH 7,
Ultrafiltration was carried out using an ultrafilter having a pore size of 200,000 molecular weight cut to remove and concentrate decomposed products. 7) Add 30 ml of the obtained concentrate to FPL manufactured by Pharmacia.
Anion exchange chromatography was carried out using the C system (column: Mono QHR 10/10). That is, 10
After the sample was applied to the column with a buffer solution (pH 7.5) containing mM Tris-HCl and 10 mM NaCl, the column was washed with a solution (200 ml) having a composition in which the NaCl amount was increased to 165 mM with the above buffer solution. . Then the target LPS
To elute the total amount of 40% while increasing the NaCl concentration to obtain a 165 mM to 1 M NaCl concentration gradient.
The column was washed with 0 ml and each 2 ml fraction was collected. The 5th to 8th fractions after the concentration gradient was applied, where the positive limulus test was confirmed, were combined. 8) Next, 8 ml of the same was added to Sephadex (Serha
dex) G-25 [column: 2.0 cm (inner diameter) x 2
0.2 cm (66 ml)] using gel filtration (buffer:
Water) to collect 3 ml fractions. Combined with the 9th to 12th fractions with positive limulus test
ml was collected (LPS: 14.3 mg, sugar: 2.0 m)
g, protein: 0.53 mg). LPS was measured by the method described in Experimental Example 1 below. 9) The above fraction was freeze-dried at −80 ° C. and then freeze-dried to a constant weight, and the weight was measured and found to be 5.8 mg. (Less than,
This freeze-dried preparation is referred to as Chlorella LPS) Since the limulus activity of this Chlorella LPS corresponds to 14.3 mg, its specific activity is 14.3 ÷ 5.8 = 2.5. In addition, it is considered that the above-described purification removed substantially all the single sugars that could be present as contaminants, and therefore all the detected sugars are considered to be sugars that make up Chlorella LPS. Therefore, when the purity of Chlorella LPS at this stage was calculated based on the weight, it was: Protein = 0.53 mg LPS = 5.8-0.53 = 5.27 mg, so 5.27 ÷ 5.8 × 100 = 91 ( %). Physical Properties of Chlorella LPS The following values were obtained by the same method as in Production Example 1. The molecular weight was measured by the SDS-2 method. Main molecular weight = 40,000 to 90,000 Phosphorus number = 4 ± 1 / molecular weight 10,000 Hexosamine number = 7 ± 1 / molecular weight 10,000 Fatty acid number = 6 ± 1 / molecular weight 10,000 KDO number = 2 ± 1 / molecular weight 10,000

Production Example 3 ( Production of Bordetella pertussis LPS) A pertussis solution for test (2.
0 × 10 ¹⁰ cells / ml) were used as dead cells. The dead cells were suspended in sterile water to a concentration of 25 mg (dry weight) / ml. To this, an equal amount of 90% hot phenol solution (68 to 70 ° C) was added, and extraction was performed while shaking at 68 ° C for 1 hour. The aqueous layer was separated by centrifugation at 8,000 G and 4 ° C. for 20 minutes. To the remaining phenol layer, the same amount of sterilized water as the above aqueous layer was added, and the same extraction was performed. The obtained aqueous layer was combined with the previous aqueous layer, dialyzed in running water overnight, and then concentrated to 1/10 with a rotary evaporator. This is 8,0
Centrifuge for 20 minutes at 00G, 4 ° C. Collect the supernatant,
A small amount of sodium acetate was added, 6 volumes of cold ethanol at 0 to 4 ° C was added, and the mixture was left at -20 ° C overnight. 4,000G,
The precipitate recovered by centrifugation at 4 ° C. for 30 minutes was washed twice with ethanol, then once with acetone, and dried with an aspirator. The residue was suspended in distilled water so as to have a concentration of 20 mg / ml, and the solution was transferred to Branson, USA.
It was subjected to ultrasonic treatment (power control 5, 15 minutes, room temperature) with a Sonifier 185 manufactured by the same company. Then 2,500
G, centrifuged at 4 ° C. for 10 minutes, and the supernatant was collected. The supernatant was treated at 4 ° C. for 15 to 16 hours with DNase I and RNase A, nucleolytic enzymes manufactured by Sigma, USA (finally 10 μg / ml DNase I).
And 20 μg / ml RNase A was used). Further, the same concentration of nucleolytic enzyme was added and the mixture was heated at 37 ° C. for 2 hours. Then, the mixture was centrifuged at 2,500 G and 4 ° C. for 10 minutes, and the supernatant was collected. This supernatant is
(manufactured by Man) Co., Ltd., and filtered with a pore size of 0.2 μm. The filtrate was subjected to molecular sieve [Resin: Sepharose 6B manufactured by Pharmacia, USA, column size = internal diameter 5 cm × length 100 cm, buffer = 10 mM Tris-HCl, 10 mM NaCl (pH 7.5),
Flow rate = about 3 ml / cm ² / hour)], L manufactured by Seikagaku Corporation
Fractions positive for limulus activity were examined and combined using the S-1 kit, and filtered with an acrodisc of Gelman Co. at a pore size of 0.2 μm. The filtrate is subjected to ion exchange [apparatus: FP manufactured by Pharmacia, USA]
LC, Resin: Mono Q HR10 / manufactured by Pharmacia, USA
10. Wash with buffer = 10 mM Tris-HCl + 10 mM NaCl (pH 7.5) for 15 minutes, then Na
Increase the amount of Cl to 165 mM and wash for 30 minutes, then
Washing is carried out for 20 minutes while increasing the NaCl amount so that the NaCl amount becomes a concentration gradient from 165 mM to 1 M,
Then wash with 1 M NaCl for 30 minutes, flow rate = 2 m
1 / min], limus activity positive fractions were examined and combined using the LS-1 kit manufactured by Seikagaku Corporation. The combined fractions were desalted on a column [resin: Pharmacia, USA (Pharm
Acea Sephadex G-25 Fine (fi
ne), column size = inner diameter 2 cm × length 25 cm, eluent = distilled water], and then freeze-dried. The substance most likely contaminated with this freeze-dried preparation (4.50 mg) is nucleic acid. Therefore, the ultraviolet absorption curve (200-40
0 nm) was taken and the absorbance at 260 nm was determined. Using the fact that the nucleic acid concentration at the absorbance of 1 was 50 μg / ml, the nucleic acid concentration was calculated from the above absorbance to be 1% or less. Moreover, the protein was not clearly detected by either the SDS-1 method or the SDS-2 method. Therefore, considering the detection sensitivity, it is estimated that the protein mixed in the freeze-dried preparation is 0 to 3% at most. Therefore, the purity of the freeze-dried preparation was estimated to be 96% or higher. The physical properties of this B. pertussis LPS measured in the same manner as in Production Example 1 were as follows. The molecular weight was measured by the SDS-2 method. Physical properties of B. pertussis LPS Main molecular weight = 6,000 ± 1,000 Phosphorus number = 4 / molecular weight 6,000 Hexosamine number = 12 / molecular weight 6,000 Fatty acid number = 4 / molecular weight 6,000 KDO number = 2 ± 1 / molecular weight 6,000

Escherichia coli LPS measured in the same manner as in Production Example 1 [Difco, USA]
The physical properties of O128: B8] manufactured by the company were as follows. The molecular weight was measured by the SDS-2 method. Physical properties of Escherichia coli LPS Main molecular weight = 40,000 ± 10,000 8,000 ± 4,000 Phosphorus number = 12 / molecular weight 30,000 Hexosamine number = 45 ± 6 / molecular weight 30,000 Fatty acid number = 18 / molecular weight 30,000 KDO number = 5 ± 1 / molecular weight 30,000

Production Example 4 (Production of LPS1, LPS2, LPS3) 1) Into a 50 ml Corning tube, weigh 1.04 g of hard wheat flour (Canadian wheat from Canada) containing 1.09% ash. Then, 20 ml of distilled water was added thereto to prepare a 50 mg / ml flour liquid. 2) This solution was subjected to shaking culture in a water bath at 37 ° C., and 0 hours, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours after the start of the culture.
8 hours, 10 hours, 12 hours, 20 hours, 24 hours, 4
At 5 hours, 0.5 ml of each was collected, diluted 10 ⁰ to 10 ⁵ times, and 100 μl was spread on a standard agar medium (medium manufactured by Nissui Pharmaceutical Co., Ltd. having the following composition) to measure the viable cell count, The colonies were observed. Standard agar medium (Nissui Pharmaceutical Co., Ltd. code number: 05618) In 1 liter Yeast extract 2.5 g Peptone 5.0 g Glucose 1.0 g Agar 15.0 g pH 7.1 ± 0.1 3) Different types were considered. , Yellow to cream opaque colonies (colony 1), cream opaque colonies (colony 2), yellow translucent colonies (colony 3), milky white opaque colonies (colony 4) observed at the 8th and 10th hours of elapsed culture time. ), Small white opaque colonies (colony 5) were seeded on another standard agar medium of the same species as above and subcultured, while the colony 1 to 5 bacteria were examined for Gram stainability and limulus activity. Of the above colonies, colony 4 and colony 5 (both Gram stain +)
Limulus activity was significantly lower than that of colonies 1 to 3 (both Gram stainability −), and therefore was excluded from the subsequent examination, using Nissui Pharmaceutical's medium and ID test EB-20, The morphology and biochemical properties of 3 were observed. The following results were obtained.

Bacteria forming colony 1 (identification number: 9
(0081-1) (Ministry of International Trade and Industry, Institute of Industrial Science and Technology, Institute of Microbial Technology)
Deposited in Japan as Microbiology Research Institute Contribution No. 11664 from August 17, 1950
It was presumed that the bacterium belongs to the genus Serratia of the family Enterobacteriaceae based on the morphology and biochemical properties described below (which was transferred to the international deposit under the Budapest Treaty as No. 9). (A) Morphology 1) Short rods 2) No motility 3) Gram stainability :-( b) Growth state 1) Standard agar medium: Yellow to cream colored, round opaque colonies are formed. 2) SS agar medium: white and translucent colonies are formed. [SS agar medium: Nissui Pharmaceutical Co., Ltd. code number: 05031] In 1 liter composition, meat extract 5.0 g bile salt 9.0 g peptone 7.5 g lactose 10.0 g sodium citrate 8.5 g sodium thiosulfate 5.5 g citric acid Ferric acid 1.0 g Neutral red 0.025 g Brilliant gulin 0.033 g Agar 13.5 g pH: 7.1 ± 0.1 3) TSI agar medium: No change on the slope, but yellow on the upper part To do. Produces gas. [TSI agar medium: Nissui Pharmaceutical Co. code number: 0510
3] In a composition of 1 liter Meat extract 5.0 g NaCl 5.0 g Peptone 15.0 g Lactose 10.0 g Sucrose 10.0 g Glucose 1.0 g Ferric citrate 0.2 g Sodium thiosulfate 0.2 g Phenol red 02g agar 15.0g pH: 7.6 ± 0.1 (c) Physiological properties 1) Forges-Proscher's reaction: + 2) Indole formation: -3) Hydrogen sulfide formation: -4) Use of citric acid : + 5) Urease: -6) Oxidase: -7) OF test: + (d) Availability of carbon source 1) Lactose: + 2) Adnite: -3) Rhamnose: + 4) Mannitol: +5 ) Esculin: + 6) Inosit: -7) Sorbit: + 8) Arabinose: + 9) Raffinose: + 10) Sucrose: + (e) 1) Decarboxylation of lysine: - 2) Use of malonic acid: - 3) decomposition of arginine: - 4) deamination of phenylalanine: - 5) Decarboxylation of ornithine: -

Bacteria forming colony 2 (identification number: 9
(Headed to the Institute of Microbial Technology, Ministry of International Trade and Industry, Institute of Industrial Technology, Heisei 2)
Deposited in Japan as Microbiology Research Institute No. 11665 from August 17, 1996, and Microbiology Research Article No. 351 from August 12, 1991.
No. 0 was transferred to the international deposit in accordance with the Budapest Treaty) Based on the morphology and biochemical properties described below, the bacterium is presumed to belong to the genus Enterobacter of the Enterobacteriaceae family. (A) Morphology 1) Short rods 2) No motility 3) Gram stainability :-( b) Growth state 1) Standard agar medium: A cream-colored and opaque colony is formed. 2) SS agar medium: Red and opaque colonies are formed. 3) TSI agar medium: There is no change on the slope, but the upper part turns yellow. Produces gas. (C) Physiological properties 1) Forges-Proschauer reaction: +2) Production of indole: -3) Production of hydrogen sulfide: -4) Utilization of citric acid: +5) Urease: -6) Oxidase: -7) OF test: + (d) Availability of carbon source 1) Lactose: + 2) Adnite: -3) Rhamnose: + 4) Mannitol: + 5) Esculin: + 6) Inosit: -7) Sorbit: + 8) Arabinose: + 9) Raffinose: + 10) Sucrose: + (e) Others 1) Decarboxylation of lysine: -2) Utilization of malonic acid: + 3) Decomposition of arginine: + 4) Deamination of phenylalanine Reaction: -5) Decarboxylation of ornithine: +

Bacteria forming colony 3 (identification number: 9
(Heisei 2 in the Institute of Microbial Technology, Ministry of International Trade and Industry
Deposited in Japan as Microbiology Research Institute No. 11666 from August 17, 1996, and Microbiology Research Article No. 351 from August 12, 1991.
No. 1 was transferred to the international deposit in accordance with the Budapest Treaty) Based on the morphology and biochemical properties described below, this bacterium is presumed to belong to the genus Pantoea of the Enterobacteriaceae family. (A) Morphology 1) Short rods 2) No motility 3) Gram stainability :-( b) Growth state 1) Standard agar medium: Yellow, round translucent colonies are formed. 2) SS agar: does not form colonies. 3) TSI agar medium: There is no change on the slope, but the upper part turns yellow. Does not generate gas. (C) Physiological properties 1) Forges-Proscher's reaction: +2) Indole formation: -3) Hydrogen sulfide formation: -4) Citric acid utilization: +5) Urease: -6) Oxidase: -7) OF test: + (d) Availability of carbon source 1) Lactose: + 2) Adnite: − 3) Rhamnose: + 4) Mannitol: + 5) Esculin: + 6) Inosit: − 7) Sorbit: − 8) Arabinose: + 9) Raffinose: -10) Sucrose: + (e) Others 1) Decarboxylation of lysine: -2) Utilization of malonic acid: + 3) Decomposition of arginine: -4) Deamination of phenylalanine Reaction: -5) Decarboxylation of ornithine:-

4) Each of the colonies 1, 2 and 3 was transferred to 1 liter of L-broth medium and shaken at 37 ° C. overnight,
The cells were collected by centrifugation at 5,000 G for 20 minutes at 4 ° C.
The L-broth medium was prepared by adding 10 g of polypeptone manufactured by Difco, 5 g of yeast extract manufactured by Difco, and special grade NaCl (5 g) manufactured by Wako Pure Chemical Industries, Ltd.
Was adjusted to pH 7.5, autoclaved, and separately prepared by adding a 40% solution of a special grade glucose of Wako Pure Chemical Industries, which had been prepared in advance, diluted 400 times. 5) Each cell was suspended in 50 ml of distilled water, and 50 ml of 90% hot phenol was added to the suspension to add 65-70 ° C.
The mixture was stirred for 20 minutes at 20 ° C., cooled, and then centrifuged at 10,000 G, 4 ° C. for 20 minutes to collect an aqueous layer. The phenol layer was subjected to the same operation as above twice more. The three aqueous layers were combined and dialyzed overnight to remove phenol, and the internal solution was advantech toyo (ADVANTEC TOY
O-) of UK-200 was used for ultrafiltration and concentrated by a molecular weight cut-off of 200,000 (N ₂ pressure: 2 atm). 6) The concentrated sample was used as Q-Sepharose Fast Flow (Q-Sepharose) manufactured by Pharmacia.
Fast Flow) and subjected to anion exchange chromatography. That is, 10 mM Tris-HCl (p
After the sample was applied to the column with a buffer solution containing H7.5) and 10 mM NaCl, the limulus active fraction was eluted with 400 mM NaCl / 10 mM Tris-HCl (pH 7.5). This eluate was subjected to ultrafiltration under the same conditions as described above, desalted and concentrated to obtain LPS having a purity of 96% or more. The nucleic acid was 1M NaCl / 10 mM Tris-HCl (p
It was eluted with H7.5).

The results of each bacterial cell are shown in Tables 2 to 4 below. The amount of nucleic acid was calculated based on the measured value at OD (260 nm) (10D = 50 μg), and the purity (%) was calculated based on the following formula.

[0027]

[Equation 2]

[0028]

[Table 2]

[0029]

[Table 3]

[0030]

[Table 4]

7) Molecular weight The molecular weight of each bacterial cell LPS was changed to SDS in the same manner as in Production Example 1.
-5,000 as measured by the -2 method (LPS1 derived from bacterial cell 900814-1), 6,500
± 2,500 (LPS2 derived from the same-2 and LPS3 derived from the same-3). Those dyed bands in silver staining are shown in FIG. In FIG. 4, numbers 1 to 3 are L, respectively.
Corresponds to PS1 to LPS3. As shown in FIG.
LPS1 showed a slightly aggregated staining band even at a molecular weight of around 30,000. Staining bands of LPS2 are also observed between 30,000 and 43,000, but it is estimated that there are very few high molecular weight compounds as compared with the staining degree of staining bands of 14,000 or less. Judging from the amount of sugar and the amount of hexosamine described later, LPS2 has the lowest sugar content.
3 and LPS1 increase in this order, which is considered to be consistent with the pattern observed by electrophoresis. Further, the ratio of the amount of LPS / the total dry yield also decreases in the order of LPS2, LPS3, and LPS1. From the above observation results, it is estimated that LPS2 has a relatively large amount of low-molecular-weight LPS, and then the ratio of LPS3 and LPS1 decreases in that order.

8) Phosphorus content Chen-Toribara method [Chen et al., "Analytical Chemistry (Ana)"]
(Lytical Chemistry), vol. Two
8, 1756-1758 (1956). LPS1, LPS2 and LPS3 were separately dissolved in distilled water to give 31.6 μg and 57.
A 20 μl solution containing 6 μg, 103.6 μg LPS was prepared and placed in a small test tube. 20 μl of 50 v / v%
Sulfuric acid was added and heated at 160 ° C. for 2 hours. Then 2
After adding 0 μl of 10 v / v% perchloric acid, the mixture was heated with a gas burner for 1 minute to incinerate. Then 0.5 ml
Distilled water, then 0.5 ml of reaction reagent (1 ml of 6N
Sulfuric acid, 2 ml of distilled water, 2 ml of 2.5 v / w% ammonium molybdate and 1 ml of 10 v / w% ascorbic acid were mixed and prepared, 0.5 ml of which was used) and added for 30 minutes at room temperature. After standing, the absorbance OD at 820 nm (820 nm) was measured. In addition, as a sample for preparing a calibration curve, potassium dihydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) was diluted with distilled water, and phosphoric acid weights of 2.5 μg, 1 μg, 0.25 μg, and 0 μg were included, respectively.
A 5 ml solution was prepared and used. In addition, 1 g of phosphorus corresponds to 4.39 g of potassium dihydrogen phosphate. The results are shown in Table 5.
Shown in. The numerical value indicating the absorbance is a value obtained by subtracting the data of the control which has not been heat-treated in order to avoid an error due to the mixing of inorganic phosphorus (for example, derived from a phosphate buffer solution). The phosphorus amount (μg) is a value calculated from the light absorption amount. The phosphorus amount (% by weight) was calculated by the following formula. In the formula, “0.67” indicates the OD value of 1 μg of standard phosphorus, and the sample concentration indicates the concentration (mg / ml) of each LPS dissolved in distilled water.

[0033]

[Equation 3]

The phosphorus number is a conversion number per 5,000 molecular weight calculated by the following formula.

[0035]

[Equation 4]

[0036]

[Table 5]

9) Hexosamine content Elson-Morgan method (Tokyo Kagaku Dojin Shuppan "Biochemistry Experiment Course" No. 37)
7 to 379) and performed as follows. Dissolve LPS in distilled water to give 1.58 mg (LPS1), 2.88
A solution of mg (LPS2), 5.18 mg (LPS3) / ml was prepared, and 100 μl thereof was put into a Spitz (manufactured by Iwaki Glass Co., Ltd.) with a screw cap, and 100
μl 8N HCl was added and heated at 110 ° C. for 16 hours. About 200 μl of 4N NaOH was added to adjust the pH to 7. A 100 μl portion thereof was taken, placed in another Spitz equipped with a screw cap, and after adding 200 μl of the following reagent A, the mixture was heated at 105 ° C. for 1.5 hours and then cooled with running water. Then 100 μl was taken and 670 μl of 96
% Ethanol was added, and 67 μl of the following reagent B was added, and the mixture was allowed to stand at room temperature for 1 hour, and the absorbance was measured at 535 nm. 0.20 to 200μ as a sample for preparing a calibration curve
g / ml N-acetylglucosamine (manufactured by Wako Pure Chemical Industries, Ltd.) was used. (Reagent A) It was prepared by mixing 75 μl of acetylacetone and 2.5 ml of 1.25N sodium carbonate. (Reagent B) It was prepared by mixing 1.6 g of p-dimethylbenzaldehyde, 30 ml of concentrated hydrochloric acid and 30 ml of 96% ethanol. As a result, the number of hexosamines in LPS1, LPS2, and LPS3 was 9 ± 1 / molecular weight 5,000 and 7 ± 1 / molecular weight 5, respectively.
It was 5,000, 5 ± 1 / molecular weight 5,000.

10) KDO content The KDO (2-keto-3-deoxyoctonate) content was determined by the diphenylamine method [Shabyal.
R. ) Et al., Analytical Biochem (Analy
mechanical Biochem. ), 58 (1), 123
~ 129 (1974)]. A KDO detection reagent was prepared by combining 500 mg of diphenylamine, 5 ml of ethanol, 45 ml of glacial acetic acid, and 50 ml of concentrated hydrochloric acid (all manufactured by Wako Pure Chemical Industries, Ltd.). Part 5
(1) 0.505 mg / ml LPS1 in 250 μl distilled aqueous solution in 00 μl; (2) 0.576 mg / m
250 μl distilled aqueous solution containing 1 l of LPS2; (3) 0.
250 μl distilled aqueous solution containing 518 mg / ml LPS3; and 33 in a boiling water bath at 100 ° C.
After heating for 1 minute, cool in cold water (24.5 ° C) for 30 minutes,
Then, using Hitachi spectrophotometer 320, 420, 470, 6
Ultraviolet absorption was measured at 30 and 650 nm (measured values are A420, A470, A630, and A650, respectively). As a standard sample, 0.5 μmol / ml KDO
250 μl of distilled water containing ammonium salt [manufactured by Sigma, USA] was used. The value of the following equation was determined for each of the specimen sample and the standard sample. S = A420-A470 + A630-A650 The sample value (S _T ) is 0.109 for LPS1, LP
It was 0.078 for S2 and 0.099 for LPS3. The value of the standard sample _{(S S)} is 0.246, the value of the distilled water alone was 0.005. By comparison of these values, it was estimated that LPS1 contained 2 ± 1, LPS2 contained 1-2, and LPS3 contained 2 ± 1 KDO per 5,000 molecular weight. Note that these values are calculated as follows, taking LPS1 as an example. KDD contained in solution
Let χ (μmol / ml) be the concentration of

[0039]

[Equation 5]

From the above equation, χ = 0.221. Therefore, letting y be the number of moles of KDD contained in 1 mole of LPS1 (assuming 5,000), y = 2.1
It becomes 9.

[0041]

[Equation 6]

The following is a formulation example of a preparation containing the LPS of the present invention. The LPS amounts in Examples 1 to 4 are E. coli LPS-equivalent amounts by the limulus test. Example 1 (Tablet) Wheat LPS 0.04 g 6% HPC Lactose 178 g Talc stearate 8 g Potato starch 14 g The above ingredients were mixed and tableted to prepare 400 0.5 g tablets containing 0.1 mg wheat LPS. did. Example 2 (internal liquid formulation) Chlorella LPS 1 mg Purified water 100 ml Example 4 (Injection) LPS3 0.5 mg Distilled water for injection Total amount 1000 ml

Experimental Example 1 (Quantification of limulus test positive plant LPS) The limulus test positive LPS contained in various plants was quantified using a Toxicolor system manufactured by Seikagaku Corporation. 1 well of 96-well flat bottom or round bottom plate with distilled water for injection
180 μl was added per hole. 20 μl of sample (prepared by dissolving in distilled water for injection if the sample was a solid) was added to one of the holes in the plate. A 10-fold diluted solution was prepared by pipetting while stirring with a plate mixer.
(Hereafter, by taking 20 μl of each diluted sample and treating in the same manner, a 10-fold, 1000-fold, ..., 10-fold diluted series solution can be prepared. Dilution is performed by changing the ratio of distilled water for injection and sample. The rate can be set arbitrarily.) As an internal standard, a 100,000-fold diluted solution of 1.5 μg / ml E. coli LPS solution was prepared, and it was confirmed that the dilution and the color development of the limulus test were normal. 35 μl of the 10-fold diluted solution described above is put in a hole of another plate, and L of Tokicolor System of Seikagaku Corporation
35 μl of S-1 set was added and left at 37 ° C. for 30 minutes. Then, 105 μl of 1 M 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 an absorptiometer for 96-well plate reader-MTP-1.
00 (manufactured by Corona Electric Co., Ltd.). Distilled water as background, 42 pg for making calibration curve
/ Ml Seikagaku Corporation's Toxicolor System ET-1 set was used to create a calibration curve, and the limulus test positive LPS in each sample was quantified based on this calibration curve. (When the sample was distilled water, the absorbance was set to 0.) When the LS-1 set was used by this method, it was confirmed that the color development was quantitative within the range of 10 to 45 pg / ml. Therefore, if it did not fall 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 calibration curve) x (dilution rate). The result obtained is ng in the case of a solid sample.
/ G unit, and in the case of a liquid sample, ng / ml unit is shown in the following Tables 6 to 11. The company name in the sample column in the table,
The place name, etc. refer to the place where the sample is obtained and the place of origin. Items without such a description are those purchased at the Superstore Faithful store in Nakano-cho, Tsukui-gun, Kanagawa prefecture, and the manufacturer is unknown.
“Hokuren” is an abbreviation for the Hokkaido Federation of Agricultural Cooperatives.

[0044]

[Table 6]

[Table 7]

[Table 8]

[Table 9]

[Table 10]

[Table 11]

Experimental Example 2 (In vitro T of macrophages
Selection method of LPS in which the content of limulus test-positive LPS that gives an ED ₅₀ for activating NF production is 0.4 to 100 ng / ml of culture solution) 9 weeks old, average weight 29 g Male C3H /
200 μl of macrophage peritoneal cells of He mouse
(2 × 10 ⁵ pieces) / Put holes in a 96-well flat bottom plate,
Recombinant mouse IFN-γ (100 units / ml) as a primer was added to each well in an amount of 10 μl. Separately, various LP
Extraction solutions prepared by extracting the S source with hot water (g / ml) at 65 ° C. for 5 hours were diluted variously, and 10 μl / well thereof was added as a trigger 3 hours after the primer administration. After culturing for 2 hours, centrifugation was performed (3000 G, 20 minutes). The TNF activity of 130 μl obtained from each well was measured based on the toxicity to L929 cells, and the limulus test positive LPS content was measured using Toxicolor System manufactured by Seikagaku Corporation. The measured value is plotted on the vertical axis as TNF
Amount of production (unit / ml of culture solution) corresponds to the horizontal axis (logarithmic scale) Limuras test positive LPS content (ng / ml of culture solution)
Was plotted on the coordinates representing the sigmoid curve estimated from each plotted point. The macrophage activating ability of each trigger that gives the TNF production amount when the trigger is not administered is 0%, and the macrophage activating ability of each trigger when the TNF production amount that increases as the effect of the trigger administration reaches the maximum constant amount To 100%, 5
The limulus test positive LPS content giving a macrophage activating capacity corresponding to 0% was read from the curve. Tables 12 to 14 show the results of LPS collection sources in which the correlation between the macrophage activating ability and the limulus test positive LPS content satisfied the above conditions. In the table, "TNF" is TNF
The production amount (unit / ml of culture solution), the "activation ability" represents the macrophage activation ability (%), and the "LPS" represents the limulus test positive LPS content (ng / culture medium ml). The amount of TNF produced without addition of trigger was 0.75 units /
Since it was ml, the TNF production amount was 0.75 unit / ml
The following cases were defined as 0% macrophage activating ability, and the macrophage activating ability (%) was calculated by the following formula.

[0046]

[Equation 7]

[0047]

[Table 12]

[Table 13]

[Table 14]

Experimental Example 3 (Antitumor effect in experimental animals-1) MethA fibrosarcoma cells, which are syngeneic tumors, were injected into the right abdominal skin of 9-week-old BALB / C male mice (body weight 25 ± 2 g). 1.2 × 10 ⁴ cells were inoculated. One day later, the following was administered intradermally in the left abdomen of each mouse. Physiological saline solution 50 μl (6 animals) (hereinafter referred to as control group) LPS3 (Production Example 4) 15 μg physiological saline solution 5
0 μl (7 animals) (hereinafter referred to as 15 μg administration group) LPS3 (Production Example 4) 30 μg physiological saline solution 5
0 μl (7 animals) (hereinafter referred to as 30 μg administration group) LPS3 (Production Example 4) 60 μg physiological saline solution 5
0 μl (7 animals) (hereinafter referred to as 60 μg administration group) Short diameter (mm) of each tumor on the 7th day, 10th day, 13th day, 16th day, 19th day, and 22nd day after MethA inoculation And the major axis (mm) were measured, and the geometric mean (square root of the product of the minor axis and the major axis) was recorded as the tumor diameter (mm). The results are shown in Table 15 below as the average value (/ animal) of each group.

[0049]

[Table 15]

FIG. 5 is a graph of the results shown in Table 15. From Table 15 and FIG. 5, it is clear that the LPS of the present invention shows a significant antitumor effect.

Experimental Example 4 (Anti-tumor effect in experimental animals-2) Experimental animals were used as 13 week-old C3H / He male mice (body weight: 30).
The experimental example 3 was repeated by changing the tumor cells to MM46 breast cancer cells and the drug to be administered to the following (± 2 g). Physiological saline 50 μl (7 animals) (hereinafter referred to as control group) LPS3 (Production Example 4) 15 μg physiological saline solution 5
0 μl (7 animals) (hereinafter referred to as 15 μg administration group) LPS3 (Production Example 4) 30 μg physiological saline solution 5
0 μl (6 animals) (hereinafter referred to as 30 μg administration group) LPS3 (Production Example 4) 60 μg physiological saline solution 5
0 μl (6 animals) (hereinafter, referred to as 60 μg administration group) The tumor diameter of each tumor was recorded on the 7th day, 10th day, 13th day, 16th day, and 19th day after tumor cell inoculation. The results are shown in Table 16 below as the average value (/ animal) of each group.

[0052]

[Table 16]

FIG. 6 is a graph of the results shown in Table 16. From Table 16 and FIG. 6, it is clear that the LPS of the present invention shows a significant antitumor effect.

Experimental Example 5 (Anti-tumor effect in experimental animals-Part 3) Experimental animals were used as 12 week-old C3H / He male mice (body weight: 29).
Experimental Example 4 was repeated by changing the tumor cells to MH134 hepatoma cells and the drugs to be administered to the following ± 2 g). Physiological saline solution 50 μl (6 animals) (hereinafter referred to as control group) LPS3 (Production Example 4) 15 μg physiological saline solution 5
0 μl (6 animals) (hereinafter referred to as 15 μg administration group) LPS3 (Production Example 4) 30 μg physiological saline solution 5
0 μl (6 animals) (hereinafter referred to as 30 μg administration group) LPS3 (Production Example 4) 60 μg physiological saline solution 5
0 μl (6 animals) (hereinafter referred to as 60 μg administration group) The results are shown in Table 17 below as the average value (/ animal) of each group.

[0055]

[Table 17]

FIG. 7 is a graph of the results shown in Table 17. From Table 17 and FIG. 7, it is clear that the LPS of the present invention shows a significant antitumor effect. Experimental Example 6 (Antitumor effect in experimental animals-Part 4) 2 × 10 ⁵ MH134 hepatoma cells were inoculated into the right abdominal skin of 10-week-old C3H / He male mice (weight: 29 ± 2 g). Seven days later, the following was injected through the tail vein. Physiological saline 50 μl (5 animals) (hereinafter referred to as control group) LPS3 (Production Example 4) 1 μg physiological saline solution 50
μl (5 animals) (hereinafter referred to as 1 μg administration group) LPS3 (Production Example 4) 4 μg physiological saline solution 50
μl (5 animals) (hereinafter referred to as 4 μg administration group) LPS3 (Production Example 4) 16 μg physiological saline solution 5
0 μl (5 animals) (hereinafter referred to as 16 μg administration group) LPS3 (Production Example 4) 64 μg physiological saline solution 5
0 μl (5 animals) (hereinafter referred to as 64 μg administration group) The tumor diameter was recorded immediately after inoculation of the tumor cells, and 10 days, 13 days, and 16 days after the inoculation. The results are shown in Table 18 below as the average value (/ animal) of each group.

[0057]

[Table 18]

FIG. 8 is a graph of the results shown in Table 18. From Table 18 and FIG. 8, it is clear that the LPS of the present invention shows a significant antitumor effect.

Dosage amount, administration interval, toxicity value When the LPS of the present invention is administered as an antitumor agent or an animal antitumor agent, the amount and administration interval are, of course, under strict control of the attending physician or veterinarian. It is decided individually considering the age, symptoms, weight, and administration effect of the subject, but
kg), 1 μg to 100 mg for oral administration, 10 ng to 10 mg for intravenous administration, and 100 ng to 1 mg for transdermal administration are tentative guidelines for once daily administration. For animals, for large animals such as cows and horses, 1/60 of the above amount should be used as a guideline for the amount per 1 kg of body weight. The dose per kg can be used as a standard, and for birds such as chickens, double the dose can be administered as a guide for the amount per kg of body weight. Behrens Kerber

[Outer 1] rber) method, 7-week-old average body weight 22 g
, LPS1, LPS2 in C3H / He male mice
LD ₅₀ of LPS3 is 150, 180, 18 respectively.
0 μg / animal, E. coli LPS [US Difco (Di
fco) 0128: B8] value 300 μg / animal 6
It was 0% or less. In addition, the toxicity value LD ₅₀ (average value in 2 male BALB / C mice per group, average weight 45 g) of wheat LPS (Production Example 1), Escherichia coli LPS (same as above), and B. pertussis LPS (Production Example 3) was The intravenous administration was 3.2, 3.4, and 11 mg / kg, and the intradermal administration was 16, 16, and 32 mg / kg, respectively.

[0060]

EFFECTS OF THE INVENTION According to the present invention, it has an excellent antitumor effect, can be used for a long period of time, has a low production cost, and can be administered by oral, transdermal, or injection routes in large amounts. An antitumor agent that can be supplied and an antitumor agent for animals are provided.

[Brief description of drawings]

FIG. 1 is a chart showing peaks showing the presence of fatty acids in a molecule, obtained by subjecting wheat LPS to gas chromatography.

FIG. 2 is a chart showing peaks showing the presence of fatty acids in the molecule obtained by subjecting Escherichia coli LPS to gas chromatography.

FIG. 3 is a chart showing peaks showing the presence of fatty acids in the molecule obtained by subjecting B. pertussis LPS to gas chromatography.

FIG. 4 shows SDS-2 of LPS1, LPS2, and LPS3.
It is a figure which shows the pattern in a method.

FIG. 5 is a graph showing the antitumor effect of LPS of the present invention.

FIG. 6 is a graph showing the antitumor effect of LPS of the present invention.

FIG. 7 is a graph showing the antitumor effect of LPS of the present invention.

FIG. 8 is a graph showing the antitumor effect of LPS of the present invention.

[Explanation of symbols]

In FIG. 4, 1 shows the pattern of LPS1, 2 shows the pattern of LPS2, and 3 shows the pattern of LPS3. 5, 6, and 7, ◯ is a physiological saline administration group, ● is a LPS 15 μg administration group of the present invention, □ is a LPS 30 μg administration group of the present invention, and a black square is a book. The data of the 60-microgram administration group of LPS of the invention are shown. In FIG. 8, ◯ is a physiological saline administration group, ● is a 1 μg administration group of the LPS of the present invention, Δ is a 4 μg administration group of the LPS of the present invention, and ▲ is 16 of the LPS of the present invention.
In the μg administration group, □ represents data of the LPS 64 μg administration group of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication location A61K 35/82 7167-4C (72) Inventor Daisuke Tsukioka 1-21-17 Kasuga, Chiba, Chiba ( 72) Inventor Denichi Mizuno 18 Okamoto, Kamakura City, Kanagawa Prefecture (72) Inventor Haruyuki Oshima 1097 Tatemachi Tachimachi, Hachioji City, Tokyo 2-1 -513

Claims (11)

[Claims] 1. An antitumor agent containing LPS, wherein the macrophage activating ability of LPS that activates the TNF producing ability of macrophages cultured in vitro is used as an index, and the vertical axis shows the macrophages when the LPS is not added. Of macrophage activation that gives TNF production
%, The macrophage activating ability (%) is defined as 100% of the macrophage activating ability of LPS when the TNF production amount of the macrophage is maximally constant, and the horizontal axis shows the LP.
When a sigmoid curve representing the limulus test positive LPS content of S on a logarithmic scale is drawn, the limus test positive LPS content giving an ED ₅₀ of macrophage activating capacity is 0.4 to 100 ng / medium
An antitumor agent comprising at least one LPS which is 1. 2. LPS is an LPS obtained from a plant, an LPS obtained from a bacterium, a lipid A, or a synthetic LPS thereof.
The antitumor agent according to claim 1, which is selected from the group consisting of: and a mixture thereof. 3. The antitumor agent according to claim 2, wherein the LPS obtained from a plant is an LPS obtained from wheat and having the following physical properties. Main molecular weight: 8,000 ± 1,000 (by SDS-1 method) 8,000 ± 1,000 (by SDS-2 method) 5,000 ± 2,000 (by SDS-2 method) Phosphorus number: 1 to 1 4 / Molecular weight 8,000 Hexosamine number: 6 ± 2 / Molecular weight 8,000 Fatty acid number: 6 ± 2 / Molecular weight 8,000 KDO number = 5 ± 1 / Molecular weight 8,000 4. The antitumor agent according to claim 2, wherein the LPS obtained from a plant is an LPS obtained from Chlorella and having the following physical properties. Main molecular weight = 40,000 to 90,000 (SDS-2
Phosphorus number = 4 ± 1 / molecular weight 10,000 Hexosamine number = 7 ± 1 / molecular weight 10,000 Fatty acid number = 6 ± 1 / molecular weight 10,000 KDO number = 2 ± 1 / molecular weight 10,000 5. The antitumor agent according to claim 2, wherein LPS obtained from bacteria is LPS obtained from Escherichia coli and having the following physical properties. Main molecular weight = 40,000 ± 10,000 8,000 ± 4,000 (by SDS-2 method) Phosphorus number = 12 / molecular weight 30,000 Hexosamine number = 45 ± 6 / molecular weight 30,000 Fatty acid number = 18 / molecular weight 30,000 KDO number = 5 ± 1 / molecular weight 30,000 6. The antitumor agent according to claim 2, wherein the LPS obtained from bacteria is LPS having the following physical properties. Main molecular weight: 5,000 ± 1,000 (by SDS-2 method) Phosphorus number: 2 ± 1 / molecular weight 5,000 Hexosamine number: 9 ± 1 / molecular weight 5,000 KDO number: 2 ± 1 / molecular weight 5,000 7. The antitumor agent according to claim 2, wherein the LPS obtained from bacteria is an LPS having the following physical properties. Main molecular weight: 6,500 ± 2,500 (by SDS-2 method) Phosphorus number: 1-2 / molecular weight 5,000 Hexosamine number: 7 ± 1 / molecular weight 5,000 KDO number: 1-2 / molecular weight 5,000 8. The antitumor agent according to claim 2, wherein the LPS obtained from bacteria is an LPS having the following physical properties. Main molecular weight: 6,500 ± 2,500 (by SDS-2 method) Phosphorus number: 2 ± 1 / molecular weight 5,000 Hexosamine number: 5 ± 1 / molecular weight 5,000 KDO number: 2 ± 1 / molecular weight 5,000 9. The antitumor agent according to claim 2, wherein the LPS obtained from bacteria is LPS obtained from B. pertussis and having the following physical properties. Main molecular weight = 6,000 ± 1,000 (by SDS-2 method) Phosphorus number = 4 / molecular weight 6,000 Hexosamine number = 12 / molecular weight 6,000 Fatty acid number = 4 / molecular weight 6,000 KDO number = 2 ± 1 / molecular weight 6 thousand 10. The LPS obtained from bacteria is A. The antitumor agent according to claim 2, which is Radiobacter LPS. 11. An animal antitumor agent containing LPS, wherein the macrophage activating ability of LPS that activates the TNF producing ability of macrophages cultured in vitro is used as an index, and the LPS is not added to the vertical axis. The macrophage activating ability that gives TNF production of macrophages is 0%, and the macrophage activating ability (%) is 100%, which is the macrophage activating ability of LPS when the TNF production of macrophages is maximally constant. The L on the axis
When drawing a sigmoid curve showing the limulus test positive LPS content of PS on a logarithmic scale, the limus test positive LPS content that gives an ED ₅₀ of macrophage activation ability is 0.4 to 100 ng / medium
An antitumor agent for animals, which comprises at least one LPS which is 1.