JPH0449240A - Antipeptic ulcer agent and antipeptic ulcer agent for animal - Google Patents
Antipeptic ulcer agent and antipeptic ulcer agent for animalInfo
- Publication number
- JPH0449240A JPH0449240A JP2155429A JP15542990A JPH0449240A JP H0449240 A JPH0449240 A JP H0449240A JP 2155429 A JP2155429 A JP 2155429A JP 15542990 A JP15542990 A JP 15542990A JP H0449240 A JPH0449240 A JP H0449240A
- Authority
- JP
- Japan
- Prior art keywords
- genus
- family
- plant
- peptic ulcer
- ulcer agent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Abstract
Description
【発明の詳細な説明】
消化性潰瘍治療の２本柱は過去、現在を通じて［産業上
の利用分野］
本発明は、抗消化性潰瘍剤、動物用抗消化性潰瘍剤に間
する。DETAILED DESCRIPTION OF THE INVENTION The two pillars of peptic ulcer treatment have been in the past and present [Industrial Application Fields] The present invention relates to an anti-peptic ulcer agent and an anti-peptic ulcer agent for animals.
［従来の技術］
消化性潰瘍は、直接胃液と接する消化管に発生する、少
なくとも粘膜筋板をこえた、境界明瞭な限局性組織欠損
であり、人畜共通な疾患である。[Prior Art] Peptic ulcer is a well-defined, localized tissue defect that occurs in the gastrointestinal tract that is in direct contact with gastric fluid, extending at least beyond the muscularis mucosae, and is a common disease among humans.
臨床的にはしばしば、その発生部位に応して胃潰瘍、十
二指腸潰瘍、食道潰瘍という病名が使用されている。Clinically, disease names such as gastric ulcer, duodenal ulcer, and esophageal ulcer are often used depending on the site of occurrence.
消化性潰瘍の原因は、直接的には、胃酸やペプシンの異
常な分泌亢進、粘膜の防御機構の６弓化、局所的貧血等
であるが、現在、これらはストレスにより誘発されるこ
とが多い。動物では、牛、豚、鶏、犬、猫等で発症が報
告されている。（昭和６４年に養賢堂から発行された、
吐山豊秋著の「新編家畜薬理学」の２２４〜２２６頁）
。The direct causes of peptic ulcers include abnormally increased secretion of gastric acid and pepsin, deterioration of the mucosal defense mechanism, and local anemia, but these are now often induced by stress. . In animals, the disease has been reported in cows, pigs, chickens, dogs, cats, etc. (Published by Yokendo in 1986,
(pages 224-226 of “New Edition of Livestock Pharmacology” by Toyoaki Toyama)
.
制酸剤、自律神経遮断剤である。It is an antacid and an autonomic nerve blocker.
［発明が解決しようとする課題］
消化性潰瘍は、−旦発生すると治癒後も生涯にわたって
再発治癒をくりかえすので、連続投与しても副作用の発
生のない薬剤が望まれている。この点で、現在使用され
ている自律神経遮断剤はいずれも満足すべきものではな
い、又、消化性潰瘍は、前述の通り、ストレスから誘発
されることが多いので、日常的に摂取される食品にも配
合可能な予防効果がある薬剤の開発が強く望まれている
。[Problems to be Solved by the Invention] Once a peptic ulcer occurs, it repeatedly recurs and heals throughout life even after it has healed, so there is a desire for a drug that does not cause side effects even when administered continuously. In this respect, none of the currently used autonomic nerve blockers are satisfactory, and as mentioned above, peptic ulcers are often induced by stress, so foods that are consumed on a daily basis There is a strong desire to develop a drug with preventive effects that can be used in combination with other patients.
かかる現状に鑑み、本発明は、抗消化性潰瘍効果が高く
て副作用が少なく、従って化学療法係数が高く、かつ、
生産コストが低く、しかも、経口、経皮、注射での投与
が可能であり、かつ大量に供給可能であり、その上、毎
日食すことが可能な食品の内に配合可能な新規な抗消化
性潰瘍剤、動物用抗消化性潰瘍を提供するために完成さ
れたものである。In view of the current situation, the present invention has a high anti-peptic ulcer effect, few side effects, and therefore a high chemotherapeutic coefficient, and
A novel anti-digestive property that has low production costs, can be administered orally, transdermally, or by injection, can be supplied in large quantities, and can be incorporated into foods that can be eaten every day. It was developed to provide an anti-peptic ulcer agent for animals.
［課題を解決するための手段］
本発明により、ＬＰＳを含む抗消化性潰瘍剤、動物用抗
消化性潰瘍剤が提供される。この抗消化性潰瘍剤、動物
用抗消化性ｆ！Ｉ瘍剤には、インビトロで培養されるマ
クロファージのＴＮＦ産生能を活性化するＬＰＳのマク
ロファージ活性化能を指標とし、
縦軸に、そのＬＰＳを添加しないときのマクロファージ
のＴＮＦ産生量を与えるマクロファージ活性化能を０％
、マクロファージのＴＮＦ産生量を最大かつ一定のｌｆ
ｔ！ （本明細書の他の箇所においては、「最大恒量」
と称す）にする時のＬＰＳのマクロファージ活性化能を
１００％とするマクロファージ活性化能（％）を表し、
横軸に、そのＬＰＳのリムラステスト陽性ＬＰＳ含有量
を対数尺で表すシグモイド曲線を描くとき、
マクロファージ活性化能のＥＤｓｓを与えるリムラステ
スト陽性ＬＰＳ含有量が０．４〜１００ｎｇ／培養液ｍ
［あるＬＰＳの少なくとも１種が含まれる。[Means for Solving the Problems] The present invention provides an anti-peptic ulcer agent containing LPS and an anti-peptic ulcer agent for animals. This anti-peptic ulcer agent, anti-peptic f! For the I tumor agent, 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 macrophage activity, which gives the amount of TNF production by macrophages when the LPS is not added. 0%
, the amount of TNF produced by macrophages is maximized and constant lf
T! (Elsewhere in this specification, "maximum constant weight"
It represents the macrophage activation ability (%) when the macrophage activation ability of LPS is taken as 100%,
When a sigmoid curve is plotted on the horizontal axis representing the Limulus test-positive LPS content of that LPS 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.
[Contains at least one type of certain LPS.]
ここて「少なくとも１種を含む」とは、本発明のＬＰＳ
は各別に使用できることはもちろん、そのｆ図される用
途が阻害されない限り、それらの２種以上を任意に朝み
合わせて、又、更には他のいずれの物質とも組み合わせ
て使用できることを意味する。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.
「マクロファージ」は、免疫担当細胞の一種であり、動
物体内のほとんど全ての組織に分布し、粒子状の異物や
体内の老廃細胞などを捕食して消化する大型のアメーバ
状細胞の総称である。「ＴＮＦＪは、マクロファージに
より産生される腫瘍障害因子（Ｔｕｍｏｒ Ｎｅｃｒ
ｏｓｉｓＦａｃｔｏｒ）の総称であり［１９８５年に発
行された ザ ジャーナル オブ バイオロジカルケミ
ストリー（Ｔｈｅ Ｊｏｕｒｎａｌ ｏｆＢｊｏｌ
、Ｃｈｅｍ、 、２６０．２３４５〜２３５４頁コ、マ
クロファージの活性が高まるにつれてその産生量は増し
ていく。"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.
「リムラステスト」は、１９６８年にレヴイン（ｌｅｖ
ｉｎ）によりｉＩＪ案された、カブトガニ血球抽出液と
発色合成基質を用いたエンＦ’ ）キシン定量法である
。The "Limulus Test" was developed in 1968 by Lev
This is a method for quantifying enF')xin using a horseshoe crab blood cell extract and a chromogenic synthetic substrate, which was proposed by iIJ.
本発明の抗消化性潰瘍剤、動物用抗消化性潰瘍剤の活性
成分として使用できるＬＰＳは、特にその採取源、生産
方法、精製方法を限定されることはない０例えば、ＩＩ
Ｍや植物から採取されるＬＰＳであっても、或は合成リ
ビドＡのような合成品であってもよい、なお、本明細書
、特にその特許請求の範囲において、採取源は特に名称
で特定されたそのものに限定されることなく、その採取
源の成長、保存、流通の過程で付着、共存する細菌その
他の全てのものが含まれる０例えば、「小麦ＬＰＳＪと
特定された場合には、小麦そのものから採取されたＬＰ
Ｓのみならず、小麦の成長、保存、流通の過程で付着、
共存する細菌その他の全てのものが含まれるものと理解
されたい。なぜならば、特に寄生Ｍ物、寄生動物という
関係が解明されているもの以外にも、特定の植物、動物
、菌界生物、地衣界生物に、それらにより付着、共存を
許されたものが棲息している例が多く存在し得ることは
当業界で良く知られていることであるからである。LPS that can be used as an active ingredient in the anti-peptic ulcer agent and anti-peptic ulcer agent for animals of the present invention is not particularly limited in its collection source, production method, or purification method. For example, II
It may be LPS collected from plants such as M. This includes all bacteria and other substances that adhere to or coexist with the source during the growth, preservation, and distribution process, without being limited to the source of the wheat.For example, if it is identified as wheat LPSJ, LP taken from that
Not only S, but also adheres to wheat during the growth, storage, and distribution process.
It should be understood that all coexisting bacteria and other substances are included. This is because, in addition to parasites and animals whose relationships have been elucidated, there are also organisms that are allowed to adhere to and coexist with specific plants, animals, fungi, and lichens. This is because it is well known in the art that there may be many examples of such cases.
これらＬＰＳのうちから、本発明の抗消化性潰瘍剤、動
物用抗消化性潰瘍剤の活性成分として使用できるＬＰＳ
を選択するには、
インビトロで培養されるマクロファージのＴＮＦ産生能
を活性化するＬＰＳのマクロファージ活性化能を指標と
し、
縦軸に、そのＬＰＳを添加しないときのマクロファージ
のＴＮＦ産生量を与えるマクロファージ活性化能を０％
、マクロファージのＴＮＦ産生量を最大恒量にする時の
ＬＰＳのマクロファージ活性化能を１００％とするマク
ロファージ活性化能（％）を表し、横軸に、そのＬＰＳ
のリムラステスト陽性ＬＰＳ含有量を対数尺で表すシグ
モイド曲線を描くとき、
マクロファージ活性化能のＥＤｓ＠を与えるリムラステ
ス［１性ＬＰＳ含有量が０．４〜１０００ｇ／培１！Ｈ
ｍ ｃであるものを選択すればよい。Among these LPS, LPS that can be used as an active ingredient of the anti-peptic ulcer agent of the present invention and the anti-peptic ulcer 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 positive LPS content of Limulus test on a logarithmic scale, Limulus test that gives the EDs@ of macrophage activation ability [with monogenic LPS content of 0.4 to 1000 g/1 culture! H
What is necessary is to select one that is m c.
ｌムラステスト陽性纏１１ｉｉ１！ＬＰＳ従来より知ら
れている大＆！ｌ菌ＬＰＳ、百日咳菌ＬＰＳ、リピトＡ
等が該当する。l Muras test positive 11ii1! LPS has been known for a long time and! Bordetella LPS, Bordetella pertussis LPS, Lipito A
etc. are applicable.
大腸菌ＬＰＳは、例えば、米国デイフコ（Ｄ−ｉｆｃｏ
）社から市販されている。Escherichia coli LPS is, for example, available from D-ifco (USA).
) is commercially available from the company.
百日咳菌ＬＰＳは、例えば、フナコシ薬品から市販され
ている。又、公知の百日咳菌、例えば、東浜株Ｉ相菌の
死菌体から、例えば、下記文献２聴の公知方法により調
製することもてきる。Bordetella pertussis LPS is commercially available from Funakoshi Pharmaceutical Co., Ltd., for example. It can also be prepared from the killed cells of known Bordetella pertussis, for example, Higashihama strain I phase bacteria, by the known method described in Document 2 below.
ウェブスター（Ｗｅｂｓｔｅｒ）等著の「ジエイ、イミ
ュノル（Ｊ、Ｉｍｍｕｎｏ １．）、７４４．５５ （
１９５５）；
ウエストファル（Ｗｅ−ｓｔｐｈａｌ）等著の「ツエト
、ナツールフオルシュ（Ｚ、Ｎａｔｕｒｆｏｒｓｃｈ）
Ｊ、７６．１４８ （１９５２）。Webster et al., J. Immuno 1., 744.55 (
1955); “Z, Naturforsch” by We-stphal et al.
J, 76.148 (1952).
リビドＡは、例えば、第一化学薬品から市販されている
。Libido A is commercially available, for example, from Daiichi Chemical.
リムラステスト陽性植物１１１ＬＰｓ
原料植物として使用できるものを下記に例示する。なお
、水閘＊ｔに記載した植物が帰属する科名、１名は、次
の文献の記載を照合して決定された。Limulus test positive plant 111LPs Examples of plants that can be used as raw material plants are listed below. In addition, the family name and name to which the plants described in Mizusho*t belong were determined by comparing the descriptions in the following literature.
課子植物、単子葉類、双子葉類、シダ植物、ソウ類：昭
和５７年（正編）、昭和５８年（続編ンに北隆館から発
行された「原色牧野植物大図鑑」の記載を照合して所属
を決定した。但し、「燕麦」は、昭和４５年に女子栄養
大学出版部から発行された「食用植物図説」と、昭和５
８年に至文堂から発行された「新日本植物誌顕花簡」の
記載を照合し、［裸麦）は、昭和４６年に東京同文書院
から発行された「総合食品事典」の記載を照合し、「鳩
麦」、「カラスビシャク」、「ジャノヒゲ」、「ウコン
」、「マタタビ」、「アマチャヅル」、「ドクダミ」、
「胡椒」、「トウガラシＪ、「ダイウィキョウ」、「ダ
イダイ」、「クズ」、「ナンキンカンゾウ」、「オタネ
ニンジン」、［ボウフウＪ、「オオツヅラフジ」、「ウ
ンカリア・ヒルスタ」は、昭和６３年に北隆館から発行
された「原色牧野和漢薬草大図鑑」の記載を照合し、「
アボガＦ’ Ｊは、昭和５３年に財団法人農林統計協会
から発行された熱帯農業技術叢書第１５号「ブラジルの
果実」の記載を照合し、「カイワレダイコンＪは、昭和
５９年に北隆館から発行された「原色園芸植物大図鑑」
の記載を照合し、「ニクズク」は、昭和４４年に廣用書
店から発行された「図説熱帯植物集成」の記載を明合し
、「クロレラ」は、財団法人日本健康食品協会が昭和６
１年に公示した、「クロレラ規格基準」の記載を照合し
て所属を決定した。Section plants, monocotyledons, dicotyledons, ferns, and grasses: 1981 (main edition), 1981 (sequel edition includes descriptions of the ``Illustrated Encyclopedia of Primary Colored Makino Plants'' published by Hokuryukan) The affiliation was determined by cross-checking.However, ``oat'' was used in the ``Illustrated Guide to Edible Plants'' published by the Women's Nutrition University Press in 1970, and in 1973.
Comparing the descriptions in the "New Japanese Botanical Journal of Flowers" published by Shibundo in 1972, and comparing the descriptions for "Naked Mugi" in the "Comprehensive Food Encyclopedia" published by Tokyo Doubunshoin in 1971. ``Hatomugi'', ``Crowbishaku'', ``Janohige'', ``Turmeric'', ``Actella japonica'', ``Gyomachagulan'', ``Dokudami'',
``Pepper'', ``Capsicum J'', ``Daiwikyou'', ``Daidai'', ``Kudzu'', ``Nankin Licorice'', ``Panella ginseng'', ``Bofuu J'', ``Ootsudurafuji'', and ``Uncaria hirsuta'' were cultivated in northern Japan in 1986. We compared the descriptions in the ``Primal Makino Japanese and Chinese Medicinal Herb Illustrated Encyclopedia'' published by Ryuukan, and found ``
Aboga F'J collated the description in Tropical Agricultural Technology Series No. 15 "Fruits of Brazil" published by the Agricultural and Forestry Statistics Association in 1973, and found that "Kaiware radish J was acquired from Hokuryukan in 1980. Published "Illustrated Encyclopedia of Primary Colored Garden Plants"
"Nikuzuku" was found in the "Illustrated Collection of Tropical Plants" published by Hiroyo 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.
菌類：昭和６２年に保育社から発行された「原色日本新
菌類図鑑」の記載を照合して所属を決定した。但し、酵
母は、昭和３７年に技報堂から発行された「微生物ハン
ドブック」の記載を照合し、「冬虫夏草」は、前掲の「
原色牧野和漢薬草大図鑑」の記載を照合して所属を決定
した。Fungi: The affiliation was determined by comparing the descriptions in the "Primary Colors of Japan's New Illustrated Guide to Fungi," published by Yokusha in 1986. However, regarding yeast, we compared the description in the "Microorganism Handbook" published by Gihodo in 1963, and "Cordyceps sinensis"
The affiliation was determined by comparing the entries in the "Makino Japanese and Chinese Medicinal Herb Illustrated Encyclopedia."
本発明で使用できる原料植物は、例えば、裸子植物、単
子葉類、双子葉類、シダ植物、ソウ類、菌類の植物であ
り、これらは個別に或は混合して使用できる。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.
裸子植物としては、例えば、マツ科マツ属植物であるマ
ツを使用できる。As the gymnosperm, for example, pine, which is a plant of the Pinaceae family and genus Pinus, can be used.
草子葉類植物としては、例えば、イネ科イネ属植物であ
るイネ、イネ科コムギ属植物である小麦、イネ科オオム
ギ属植物である大麦、裸麦、イネ科カラス麦属植物であ
る烏麦、燕麦、イネ科ササ属植物であるクサ笹、イネ科
ジュズダマ属植物である鳩麦、アヤメ科アヤメ属植物で
あるアヤメ、ユリ科ネギ属植物であるニンニク、ユリ科
キジカクシ属植物であるアスパラガス、ユリ科ジャノヒ
ゲ属植物であるジャノヒゲ、ショウガ科ショウガ属植物
であるミョウガ、ショウガ科ウコン属植物であるウコン
、サトイモ科ハンゲ属植物であるカラスビシャクを使用
できる。Examples of grass cotyledonous 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, a plant belonging to the genus Barley, and oats and oats belonging to a plant belonging to the genus Oats, a family Poaceae. , 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 Zingiberaceae, myoga, a plant belonging to the genus Zingiberaceae, turmeric, a plant belonging to the genus Curcuma, belonging to the family Zingiberaceae, and a plant belonging to the genus Hange, belonging to the family Araceae.
双子葉類植物としては、マメ科ダイズ属植物である大豆
、マメ科インゲンマメ属植物である小豆、マメ科ソラマ
メ属植物であるそら豆、マメ科クズ属植物であるクズ、
マメ科カンゾウ属植物であるナンキンカンゾウ、ナス科
ナス属植物であるジャガイモ、トウガラン、ナス科トマ
ト属植物であるトマト、ナス科トウガラン［１物である
トウガラン、バラ科ビワ属Ｍ＃！Ｉであるビワ、バラ科
サクラ属Ｍ物であるモモ、クスノキ科アボガド属植物で
あるアボガド、クルミ科クルミ属植物であるクルミ、ウ
リ科トウナス属植物であるカボチャ、ウリ科アマチャヅ
ル属植物であるアマチャヅル、アブラナ科ダイコン属植
物であるカイワレダイコン、マタタビ科マタタビ属植物
であるマツタビ、ドクダミ科ドクダミ属植物であるドク
ダミ、コシヨウ科コシヨウ属植物である胡椒、シキミ科
シキミ属植物であるダイウィキョウ、ニクズク科ニクズ
ク属植物であるニクズク、ミカン科ミカン属ＶＬ物であ
るダイダイ、ウコギ科オタネニンジン属植物であるオタ
ネニンジン、セリ科サポシュニコビア属植物であるボウ
フウ、ツヅラフジ科オオツヅラフジ属植物であるオオツ
ヅラフジ、アカネ科カギカズラ属植物であるウンカリア
・ヒルスタを使用できる。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.
Daylily licorice, a plant of the genus Licorice of the Fabaceae family; Potato, a chili pepper of a plant of the genus Solanaceae of the family Solanaceae; Tomato, a plant of the genus Tomato of the family Solanaceae; Chili pepper of the Solanaceae family [1 species of Capsicum, genus Loquat of the Rosaceae family M#! loquat which is I, peach which is Prunus genus M of Rosaceae, avocado which is a plant of the genus Avocado of the Lauraceae family, walnut which is a plant of the genus Walnut of the family Cucurbitaceae, pumpkin which is a plant of the genus Tolanum of the Cucurbitaceae family, and Jiaogulan a plant of the genus Jiaogulan of the Cucurbitaceae family. , Daikon radish, a plant of the genus Radish in the family Brassicaceae, Matsutabi, a plant of the genus Actiniaceae, genus Actinaceae, genus Hekudami, a plant of the genus Actinaceae, genus Heterinaceae, pepper, a plant of the genus Koshiyo, genus Koshiyo, in the family Prunusaceae, daikon radish, a plant of the genus Shikimi, genus Squirrel, The plant Nikuzuku, the Rutaceae family Rutaceae genus VL plant Daidai, the Araliaceae family Araliaceae genus Panax ginseng plant, the Apiaceae family Sapochnicobia genus plant Boufuu, the Apiaceae family Otsuzurafuji plant genus Otsuzurafuji, the Rubiaceae family Rubiaceae genus Kagikasula plant. A certain Uncaria hirsuta can be used.
シダ植物としては、例えば、トクサ科トクサ属植物であ
るスキナ、ゼンマイ科ゼンマイ属植物であるゼンマイを
使用できる。As the fern plant, for example, Sukina, which is a plant of the genus Horsetail in the family Equisetaceae, and Zenmai, which is a plant of the genus Helicinza, in the family Medicinalaceae, can be used.
ソウ類植物としては、例えば、カッソウ類植物、紅ゾウ
ＭＭ物、緑ソウ類植物、ランソウｎ植物を使用できる。As the grass-like plants, for example, grass-like plants, red grass-like plants, green grass-like plants, and orchid-like plants can be used.
カッソウ類植物としては、例えは、コンブ科ワカメ属植
物であるワカメ、コンブ科コンブ属植物であるコンブ、
ホンダワラ科ヒジキ属植物であるヒジキを使用できる。Examples of cassous plants include wakame, which is a plant of the genus Wakame in the family Laminaceae, kelp, a plant of the genus Laminaria, and kelp, a genus Laminaria plant.
Hijiki, a plant belonging to the genus Hijiki in the Sargassum family, can be used.
紅ソウ類植物としては、例えば、ウシケノリ科アマノリ
属植物であるアサクサノリを使用できる。緑ソウ類植物
としては、例えば、オオシスティス科クロレラ属植物で
あるクロレラを使用できる。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.
菌類植物としては、例えば、担子菌類植物、子ノウ菌類
植物を使用できる。担子菌類植物としては、例えば、ヒ
ラタケ科マッオウジ属植物である椎茸、キシメジ科エノ
キタケ属植物であるエノキ茸、キシメジ科シメジ属植物
であるシメジ、タコウキン科マイタケ属植物であるマイ
茸、サルノコシカケ科ポリポラス属植物であるアワビ茸
、ハラタケ科ハラタケ属植物であるマツシュルーム、キ
クラゲ科キクラゲ属植物であるキクラゲ、モエギタケ科
スギタケ属植物であるナメコを使用できる。As the fungal plant, for example, a basidiomycete plant or an ascomycete plant can be used. Basidiomycete plants include, for example, shiitake mushrooms, which are plants of the genus Pleurotus in the family Pleurophyllaceae, enoki mushrooms, which are plants in the genus Enokitake, genus Enokitake, genus Enokitake, genus Enokitake, genus Enokitake, genus Enokitake, genus Enokitake, genus Enokitake, genus Enokitake, genus Enokitake, genus Enokitake, plants genus Enokitake, genus Enokitake, genus Enokitake, genus Enokitake, genus Enokitake, genus Enokitake, plants belonging to the genus Enokitake, genus Enokitake, genus Enokitake, genus Enokitake, genus Enokitake, plants belonging to the genus Elephantaceae, genus Polyporus, genus Polyporus, genus Eurasianaceae, Abalone mushrooms, which are plants, pine mushrooms, which are plants of the genus Agaricaceae, of the family Agaricaceae, wood ear mushrooms, which are plants of the genus Aeolus, of the family Asteraceae, and nameko, which are plants of the genus Sugitake, family Agaricaceae, can be used.
子ノウＷ類植物としては、例えば、エントミセタセア科
サツカロミセス属植物であるパン酵母、醸造用酵母を使
用できる。ｌＩ造用酵母にはビール酵母、清酒酵母、葡
萄酒酵母、醤油酵母、味噌酵母等の他、サツカロミセス
セレヴイシトに属する多くの酵母（例えば、ウィスキ
ーや老酒の製造に使用される酵母）が含まれる。又、バ
ッカクキン科ノムシタケ属植物である冬虫夏草も使用で
きる。For example, baker's yeast and brewer's yeast, which are plants of the genus Satucharomyces of the Entomycetaceae family, can be used as the W-class plants. II brewing yeast includes beer yeast, sake yeast, wine yeast, soy sauce yeast, miso yeast, etc., as well as many yeasts belonging to Satucharomyces cerevisito (for example, 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.
以上に述べた原料植物中のリムラステスト陽性ＬＰＳの
検出、含量測定は、例えば、生化学工業株式会社からト
キシカラーシステムという名称で市販されている試薬セ
ットを使用して実施できる。The above-mentioned detection and content measurement of Limulus test-positive LPS in raw material plants can be carried out using, for example, a reagent set commercially available from Seikagaku Corporation under the name Toxicolor System.
即ち、原料植物を同システムのＬＳ−１セツトと合わせ
て発色させ、その発色の強さを、同しく同セットのＥｔ
−２セツトを使用して作成した検量線と対比させればよ
い。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 determined by combining it with the Et set of the same system.
It is only necessary to compare it with a calibration curve created using -2 sets.
植物源ＬＰＳは、以下に述べる方法で分離、精製できる
。Plant-sourced LPS can be separated and purified by the method described below.
■原料植物を必要に応して適宜細切、乾燥、粉砕した後
に蒸留水によく懸濁し、上清を回収する。■After cutting, drying, and crushing the raw material plants as necessary, thoroughly suspend them in distilled water and collect the supernatant.
例えば、原料植物が穀類の種子である場合は、種皮をつ
けたまま、或は、種皮を除いた後に簡単に砕くか、又は
、食用に供せられている程度の粉末になるまで粉砕し、
得られた粉末に水を加えて分散液とし、攪拌した後に沈
降物を静置又は遠心分離により除去するか、粉末に水を
加えて練って得られるドウなミキサー中でゆるやかに水
洗し、沈降物を除去すればよい。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 form a dispersion, and after stirring, the sediment is left to stand or removed by centrifugation, or water is added to the powder and kneaded, then gently washed with water in a doughy mixer and allowed to settle. Just remove things.
原料植物がクロレラである場合には、まず細胞膜を破砕
し、エタノール洗浄により脂溶性物質を除去した後に水
抽出するとよい。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.
この水抽出の際の原料植物の粒度、水の温度、液性、添
加量、攪拌の速度、時間、遠心分離の際の条件等は特に
制限する必要はなく、原料植物の種類に応じて適宜調整
すればよい。又、抽出水の温度は高い方がＬＰＳの採取
量、純度ともに高い傾向があるが、操作の便宜上、原料
植物に含まれる澱粉の糊化を招来しない５０℃以下とす
ることが好ましい。又、水の添加量は、原料植物の種類
、粒度により異なるが、穀類種子の場合にはその割合が
７０ ｗ ／ ｖ％以下、望ましくは２０〜５０ｗ／Ｖ
％程度とすると操作上便宜である。更に、攪拌の速度は
、起泡を引き起こさない程度のものとすることが好まし
い、なお、この段階の操作塩で、本発明のリムラステス
ト陽性植物ＬＰＳの純度は、リムラステスト活性データ
から判断して、例えば小麦種子の場合には約３０倍に上
昇する。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 plant, but in the case of grain seeds, the ratio is 70 w/v% or less, preferably 20 to 50 w/v
It is convenient for operation to set it to about %. Further, the stirring speed is preferably set to a level that does not cause foaming. Furthermore, at this stage of the operation salt, the purity of the Limulus test-positive plant LPS of the present invention is judged from the Limulus test activity data, for example. In the case of wheat seeds, it increases about 30 times.
以下、穀類種子を原料として使用する場合を例にとり説
明するが、いわゆる当業者であれば、以下の記載を参考
にして、他植物から夾雑する糖、蛋白等を除去してリム
ラステスト陽性ＬＰＳを高純度で回収する方法を実施す
ることは極めて容易である。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 increase Limulus test positive LPS by referring to the description below. It is very easy to carry out the method of recovery in purity.
■純度を更に上げるためには、上記■て得られた上清を
常法に従って限外濾過に付して分子量５０００以下の画
分を除去すればよい。(2) In order to further increase the purity, the supernatant obtained in (1) above may be subjected to ultrafiltration according to a conventional method to remove fractions with a molecular weight of 5,000 or less.
■得られた乾燥品を、５０ ｍ ｇ ／ ｍ ｌ（こな
るように蒸留水に＠濁し、遠心分離操作に付して上溝を
回収する。(2) The obtained dried product was suspended in distilled water to a concentration of 50 mg/ml, and the upper layer was collected by centrifugation.
■この上清を氷水で冷却し、酸を添加して酸性にすると
沈殿が生しる。この際使用する酸は特定のものである必
要はなく、例えば、トリクロロ酢＃（以下、ＴＣＡと称
す）、過塩素酸、トリフルオロ酢酸、酢酸、ジクロロ酢
酸を使用できる。■ Cool this supernatant with ice water and add acid to make it acidic, forming a precipitate. The acid used at this time does not need to be a specific one, and for example, trichloroacetic acid # (hereinafter referred to as TCA), perchloric acid, trifluoroacetic acid, acetic acid, and dichloroacetic acid can be used.
■次いて、遠心分離操作に付して沈殿を回収して蒸留水
で洗浄し、再度遠心分離操作に付して沈殿を回収する。(2) Next, the precipitate is collected by centrifugation, washed with distilled water, and then centrifuged again to collect the precipitate.
■沈殿を蒸留水に懸濁し、沈殿が溶解するまでアルカリ
を加える。この際使用するアルカリも特定のものである
必要はなく、例えば水酸化ナトリウム、水酸化カリウム
、アンモニア、炭酸ナトリウム、酢酸ナトリウムを使用
できる。沈殿の溶解時に塩基性がｐＨＩ ｌより大きく
なると目的のＬＰＳが失活するので注意が必要である。■ 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. Care must be taken when dissolving the precipitate, as the target LPS will be inactivated if the basicity becomes greater than pHI.
■次いて酸を加えてｐＨ８としてから３７℃に加温し、
更に酸を加えて酸性にすると沈殿が生ずるので、３７℃
に保温した遠心分離器を使用して遠心分離操作に付す。■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.
なお、この際使用する酸も特定のものである必要はない
。Note that the acid used at this time does not need to be a specific one.
■上清を回収して水冷し、４℃で再び遠心分層操作に付
す゛
■上清を回収し、アルカリを添加して中和し、常法に従
って限外濾過て濃縮する。この際使用するアルカリも特
定のものである必要はない。(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.
［相］次いて常法に従ってゲル［過に付して、リムラス
テスト陽性画分を回収して併せる。ゲル濾適用の担体と
しては、例えばセファデックス（Ｓｅｐｈａｄｅｘ）Ｇ
−７５、Ｇ−１００、セファクリル（Ｓｅｐｈａｃｒｙ
ｌ）Ｓ−２００、セファロース（Ｓｅｐｈａｒｏｓｅ）
６Ｂ ［以上は米国ファルマシア社（Ｐｈａｒｍａｃｉ
ａＩｎｃ、）！！］、バイオゲル（Ｂｉｏｇｅｉ）Ｐ−
１００［米国バイオラッド（Ｂｉｏｒａｄ［ｎｃ、）社
製］、トーヨーバールＨＷ−５０、ＨＷ−５５（東洋曹
達工業社製）を使用できる。[Phase] Next, the gel is filtered 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, )! ! ], Biogei P-
100 [manufactured by Biorad (NC, USA)], Toyovar HW-50, and HW-55 (manufactured by Toyo Soda Kogyo Co., Ltd.) can be used.
緩衝液はｐＨ３〜１０のものならいずれてもよい。Any buffer having a pH of 3 to 10 may be used.
例えば、トリス−ＨＣＩＬ又はリン酸緩衝液を使用でき
る。For example, Tris-HCIL or phosphate buffer can be used.
０次いてこの両分に蛋白分解ＷＶＩ素を加え、３７℃て
２時間以上インキユヘーションして残存蛋白質を分解し
、得られた酵素処理液を常法に従って限外濾過により濃
縮する。なお、この際に使用する蛋白分解酵素も特定な
ものである必要はなく、例えば、Ｖ８プロテアーゼ、キ
モトリプシン、トリプシン、サーモライシンを単独で、
或は任意に朝み合わせて使用できる。市販品としては、
例えば、プロナーゼＥ（科研化学社）、プロティネース
Ｋ（メルク社）を使用できる。Next, proteolytic WVI element is added to both portions and 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 protease used at this time does not need to be specific; for example, V8 protease, chymotrypsin, trypsin, thermolysin alone,
Or you can use it in the morning at any time. As a commercially available product,
For example, Pronase E (Kaken Kagaku Co., Ltd.) and Proteinase K (Merck & Co., Ltd.) can be used.
０次いでこの両分を常法に従って、例えば、米国ファル
マシア社製のＦＰＬＣシステムでファルマシア社製のモ
ノＱ−セファロース（Ｓｅｐｈ−ａｒｏｓｅ）、Ｑ−セ
フｙａ−ス（Ｓｅｐｈａ−ｒｏｓｅ）を使用して陰イオ
ン交換クロマトグラフィーに付してリムラステスト陽性
画分を得る。Then, both of these aliquots are analysed, according to a conventional method, using, for example, Mono-Q-Sepharose and Q-Sepha-rose, both manufactured by Pharmacia, in an FPLC system manufactured by Pharmacia, USA. A Limulus test positive fraction is obtained by ion exchange chromatography.
０次いて、常法に従って脱塩のためにゲル濾過に付して
リムラステスト陽性画分を回収する。Next, the limulus test positive fraction is collected by gel filtration for desalting according to a conventional method.
以上の操作により、小麦種子の場合には、当初のリムラ
ス活性の約２０％が回収され、純度的９５％のＩＩ＃製
標品が得られる。又、段階の終了時の純度に比へ約１０
００１ｔ：の純度（小麦種子の場合）になる。By the above operations, in the case of wheat seeds, approximately 20% of the original Limulus activity is recovered, and a II# product standard with a purity of 95% is obtained. Also, the purity at the end of the step has a ratio of about 10 to
001t: purity (for wheat seeds).
以上の方法によって得られたリムラステスト陽性植物Ｌ
ＰＳはそのまま、或いは任意の程度に濃縮した形て提供
てきる。又、促存性を高めるために、凍結乾燥や噴霧乾
燥なとの任意の手段により乾燥粉末として提供すること
もてきる。これらはいずれも常法で生産できる。Limulus test positive plant L obtained by the above method
PS can be provided neat or in any degree of concentrated form. In addition, in order to enhance the shelf life, it can 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.
動物体内にＴＮＦを産生させるためには、産生前駆（ブ
ライミング）段階と産生開始（トリガリング）段階とが
必要であることは、カーズウエル（Ｃａｒｓｗｅｌｌ）
らにより、プロシーディング オブ ナショナル アカ
デミ−サイエンスオブ ニーニスニー［Ｐｒｏｃ、Ｎａ
ｔ Ｉ。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.
tI.
Ａｃａｄ、Ｓｃ ｉ、ＵＳＡ、、７２．３６６６−３６
７０頁（１９７５年）］に報告されている。Acad, Sci, USA, 72.3666-36
70 (1975)].
ブライミング段階開始のために投与される薬剤が「ブラ
イマー」 （内因性ＴＮＦ産生促進剤）であり、トリカ
リング段階開始のために投与される薬剤が「トリガー」
（内因性ＴＮＦ産生剤）である。The drug administered to initiate the briming phase is a ``brimer'' (endogenous TNF production promoter), and the drug administered to initiate the culling phase is a ``trigger''.
(endogenous TNF producing agent).
ＬＰＳがマクロファージのインビトロＴＮＦ産生能を活
性化する能力を測定するには、マウスのマクロファージ
腹腔常在細胞を採取し、これにブライマーとしての絹み
換えマウスＩＦＮ−γを添加し、次いて、トリガーとし
てのＬＰＳを添加し、そのＴＮＦ活性を測定すればよい
。To measure the ability of LPS to activate the in vitro TNF production capacity of macrophages, mouse macrophage resident peritoneal cells were harvested, resilked mouse IFN-γ was added as a primer, and then the trigger What is necessary is just to add LPS as a solution and measure its TNF activity.
ＴＮＦ活性は、Ｌ−９２９庸胞Ｃブａシーデイング オ
ブ ナショナル アカデミ−サイエンス オブ ニーニ
スニー ７２、３６６６〜３６７０頁］に対する細胞毒
性を基にして、次のようにして測定する。TNF activity is measured in the following manner based on the cytotoxicity against L-929 cells.
Ｌ９２９細胞を、５％仔牛脂児血清を加えたイーグルミ
ニマムエツセンシャル培地（以下、ＭＥＭ培地と表す）
て育成し、８Ｘｌ（１個の細胞が１００μ化の同上培地
に含まれる様にし、９６大の平底プレートて育種する。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 8Xl (one cell is contained in the same medium as 100μ) and bred in a 96-sized flat-bottomed plate.
育種条件は３７℃、２時間、５％Ｃ０２，１００％Ｈ２
０であり、通常の細胞培養に用いられる方法でよい。そ
の後、アクチノマイシンＤを培地中に終濃度ｌμｇ ／
ｍλとなるように加え、培ｌＩ液の液量を１５０μλ
とする。即座に、検体を適当にＭＥＭ培地で稀釈したも
のを５０μλ加える（この際稀釈率を適宜調製し、ＥＤ
５＠を求める事ができる）。更に、最終液量２００μｔ
となったＬ９２９纏胞を上記条件で１８時閉環養する。Breeding conditions are 37℃, 2 hours, 5% CO2, 100% H2
0, and any method used for normal cell culture may be used. Thereafter, actinomycin D was added to the medium at a final concentration of lμg/
mλ, and adjust the volume of the culture medium to 150 μλ.
shall be. Immediately, add 50 μλ of the sample appropriately diluted with MEM medium (at this time, adjust the dilution rate appropriately and add ED
5@ can be found). Furthermore, the final liquid volume is 200 μt.
The resulting L929 cysts were cultured at 18:00 under the above conditions.
細胞障害活性を測定するには、まず全培地を除去し、つ
いで０．１％クリスタルバイオレットを含む１％メチル
アルコール溶液を加えて固定染色する。クリスタルバイ
オレットは全有核細胞を染色するが、死細胞は染色後に
プレート底面より水洗で除去されるので、生存細胞の結
果から細胞障害活性を直接測定できる。この染色度を０
Ｄ６９１１ｎ＋＋での吸光度を指標として測定し、対照
群に対する染色度と比較する事で細胞障害活性を測定す
る。活性の定義は次の様に行う。To measure cytotoxic activity, first remove all the medium, then add a 1% methyl alcohol solution containing 0.1% crystal violet for fixation staining. 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. This staining degree is 0
The absorbance of D6911n++ is measured as an index, and the cytotoxic activity is measured by comparing the staining intensity with that of the control group. The definition of activity is as follows.
Ｌ９２９細胞が５０％生存できる検体の稀釈率（Ｎ）を
求める。対照としてウサギＴＮＳ ［腫瘍障害血清（Ｔ
ｕｍｏｒ Ｎｅｃｒｏｓｉｒ。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
umor Necrosir.
Ｓｅｔｕｍ）］を使用し、このウサギＴＮＳの活性ｎ（
単位／ｍ免）を２．４Ｘ１０６単位／ｍｇ／ｍ１ｈ（Ｄ
ＴＮＦ−αを用いて決定する。このウサギＴＮＳのＥＤ
ｓ＠を与える稀釈率（Ｃ）を求める。The rabbit TNS activity n(
unit/m) to 2.4X106 units/mg/m1h (D
Determined using TNF-α. Kono Usagi TNS ED
Find the dilution rate (C) that gives s@.
検体活性（単位／ｍλ）は − Ｘｎ で計算する。Analyte activity (units/mλ) is calculated as −Xn.
提供できる剤の製造方法
本発明の抗消化性潰瘍剤は、常法の製剤技術により、散
剤、顆粒剤、火剤、錠剤、トローチ剤、カプセル剤、液
剤、貼付剤、軟膏剤、リニメント剤、ローション剤、串
刺、注射剤等の形態で提供できる。又、動物用としては
、更に、飼料添加剤、プレミックス製剤、飲水添加剤と
してｔＩ製することもできる。飼料添加剤とする場合に
は、粉剤か顆粒剤とすることが好ましい。又、プレミッ
クス製剤とは、飼料との混合を容易にするために澱粉な
どの飼料成分で希釈されたものを指す。本発明の抗消化
性潰瘍剤を飼料添加剤、ブレミ・７クス製剤として添加
できる飼料は市販されている飼料のいずれでもよい、又
、ミネラル、ビタミン、アミノ酸等の飼料添加物を含Ｃ
飼料であってもよい。Method for manufacturing the agent that can be provided The anti-peptic ulcer agent of the present invention can be prepared as a powder, granule, gunpowder, tablet, troche, capsule, liquid, patch, ointment, liniment, It can be provided in the form of lotion, skewer, injection, etc. Furthermore, for animals, it can also be manufactured 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 to facilitate mixing with feed. The feed to which the anti-peptic ulcer agent of the present invention can be added as a feed additive or Bremi 7x formulation may be any commercially available feed, and may also contain feed additives such as minerals, vitamins, and amino acids.
It may also be feed.
これら製剤には、所望ならば、保存性、均質性を保持す
るために、常法により、賦形剤、保存剤、緩衝剤等の添
加剤を加えることもてきる。更に、矯味剤、矯臭剤、着
色剤を含めることもてきる。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.
賦形剤としては、例えば、乳糖、デンプンを使用できる
。保存剤としては、例えば、パラオキシ安息香酸メチル
、パラオキシ安息香酸エチル、バラオキシ安息香酸ブＣ
ピル等のバラオキシ安息香酸エステル類、デヒトａ酢酸
ナトリウム、フェノール、メチルパラベン、エチルパラ
ベン、プロピルバラヘン等を使用できる。緩衝剤として
は、例えば、クエン酸塩、酢酸塩、リン酸塩等が使用で
きる。As excipients, for example, lactose and starch can be used. Examples of preservatives include methyl paraoxybenzoate, ethyl paraoxybenzoate, and paraoxybenzoate.
Paraoxybenzoic acid esters such as pill, sodium dehyde acetate, phenol, methylparaben, ethylparaben, propylparaben, etc. can be used. As the buffer, for example, citrate, acetate, phosphate, etc. can be used.
以下、製造例、実施例、実験例により本発明を例示する
。The present invention will be illustrated below with reference to production examples, working examples, and experimental examples.
製造例Ｉ（小麦ＬＰＳの製造）
■小型ニーダに、１．０９％の成分を含む硬質小麦粉（
アメリカ又はカナダ産のハートレットスプリング）（３
，，１２０ｇ）を入れ、２．０３地の蒸留水を加えて１
０分間練ってトウとした。１５分間の静置後に１０ｕの
水を加えてゆるやかに攪拌してデンプン乳液を洗い出し
、同時に可溶性成分を溶出させた。この溶出液を５℃の
冷蔵庫中で１２時間静置した後、デンプン等の沈降部を
除去した。上澄み液を凍結乾燥して２０１．１ｇの粉末
を得た（粉末Ａ）。Production Example I (Production of wheat LPS) ■In a small kneader, hard flour containing 1.09% of ingredients (
American or Canadian Hartlett Spring) (3
,,120g), add 2.03 ounces of distilled water and 1
It was kneaded for 0 minutes and made into a dough. After standing still for 15 minutes, 10 u of water was added and gently stirred to wash out the starch emulsion and at the same time elute the soluble components. 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).
更に、残留ドウに５ｉｋの蒸留水を加えてゆるやかに攪
拌し、以下、上記と同様に処理して４０゜１ｇの粉末を
得た（粉末Ｂ）。Further, 5 ik of distilled water was added to the remaining dough and gently stirred, followed by treatment in the same manner as above to obtain 1 g of 40° powder (powder B).
■これら粉末Ａ、Ｂを米国アミコン社製限外濾過１１Ｈ
Ｆ−Ｌａｂｌに供し、分子量画分５．ｏｏｏについては
中空系カートリッジＨＦ−ＬａｂｌＰＭ５を、分子量画
分１０，０００については中空系カートリッジＨＦ−Ｌ
ａｂｌＰＭｌｏを取り付けて限外濾過を行った［温度５
〜１０℃。入圧２５ｐｓ ＋ （１，７６ｋｇ／ｃｍ２
）−出圧１５ｐｓ ｉ （１，０６ｋｇ／ｃｍＱコ。そ
の結果に基づき、各部分を次のように命名した。■These powders A and B were filtered through ultrafiltration 11H manufactured by Amicon Corporation in the United States.
The molecular weight fraction 5. For ooo, use the hollow cartridge HF-LablPM5, and for the molecular weight fraction 10,000, use the hollow cartridge HF-L.
Ultrafiltration was performed with ablPMlo attached [temperature 5
~10℃. Input pressure 25ps + (1,76kg/cm2
) - Output pressure 15 ps i (1,06 kg/cmQ).Based on the results, each part was named as follows.
粉末Ａ１分子量５，０００以下の部分をａ。The part of powder A1 with a molecular weight of 5,000 or less is a.
分子ｉ１５，０００以上の部分をａ２
粉末Ｂ：分子ｆｆ１５，０００以下の部分をｂ１分子量
５，０００以上の部分をｂ２
粉末Ａ：分子量１０，０００以下の部分をａ３３分子量
１，０００以上の部分をａ４
粉末Ｂ：分子量１０，０００以下の部分をｂ３３分子量
１，０００以上の部分をｂ４
これら各両分を後記実験例１に詳述する方法に準拠して
リムラステストに付したら、分子量５゜０００以上の両
分には多量のリムラステスト閣性成分が存在するが、分
子量５，０００以下の両分にはほとんど存在しないこと
が確認された。Powder B: The part with molecular weight i15,000 or more is a2 Powder B: The part with molecular weight ff15,000 or less b1 The part with molecular weight 5,000 or more is b2 Powder A: The part with molecular weight 10,000 or less is a33 The part with molecular weight 1,000 or more a4 Powder B: The part with a molecular weight of 10,000 or less is b33 The part with a molecular weight of 1,000 or more is b4 When these two parts are subjected to the Limulus test according to the method detailed in Experimental Example 1 below, the molecular weight is 5°000 or more. It was confirmed that a large amount of limulus test-specific components were present in both fractions, but almost none were present in both fractions with a molecular weight of 5,000 or less.
■上記粉末ａ２の３０ｇを１隻三角フラスコに入れ、６
００ ｍ ｌの蒸留水を注いで、６０分間スターラーで
攪拌した後、日立冷却高速遠心機５ＣＲ−２０Ｂ （ロ
ーターＲＰＲｌ ６を事前に４℃に冷却しておいた）で
４℃で遠心分離操作（１０，０００ｇＸ１０分）に付し
て上清を回収した。■Pour 30g of the above powder a2 into one Erlenmeyer flask, and
After pouring 00 ml of distilled water and stirring with a stirrer for 60 minutes, centrifugation was performed at 4°C using a Hitachi refrigerated high-speed centrifuge 5CR-20B (rotor RPRl 6 was pre-cooled to 4°C). 10,000 g for 10 minutes) and the supernatant was collected.
■この上清を１１三角フラスコに入れ、水冷下（液温的
２℃）、スターラーで攪拌しながら、事前に２℃に冷却
してあった１００％ＴＣＡ水溶液２０．５ｍ１Ｌを滴下
し、滴下終了後氷水中に１゜分間放置した。■Pour this supernatant into a No. 11 Erlenmeyer flask, and while stirring with a stirrer under water cooling (liquid temperature: 2°C), drop 20.5ml of 100% TCA aqueous solution that had been previously cooled to 2°C, and complete the dropwise addition. Afterwards, it was left in ice water for 1°.
■次いで前記と同様にして４℃で遠心分離操作（１０，
ＯＯＯｇｘｌＯ分）に付して沈殿を回収し、氷水中で冷
却下、３００ｍ１Ｌの蒸留水と共に５００ｍ１のビーカ
ーに入れて懸濁し、氷水中で冷却し、前記と同様にして
４℃で遠心分離操作（１０，０００ｇＸ１０分）に付し
て沈殿を回収した。■Next, centrifugation at 4°C in the same manner as above (10,
Collect the precipitate, cool in ice water, suspend in a 500 ml beaker with 300 ml of distilled water, cool in ice water, and centrifuge at 4°C in the same manner as above ( 10,000g x 10 minutes) to collect the precipitate.
■この沈殿をｌａヒビ−−に入れ、蒸留水５゜Ｏｍ ｌ
で懸濁し、ＩＮ水酸化ナトリウム溶液約３゜５ ｍ Ｉ
Ｌを使用して中和（ｐＨ７）Ｌ／、ついで、氷水中で冷
却しながら、ＩＮ水酸化ナトリウム溶液約２ｍｌを添加
して０．０２Ｎ水酸化ナトリウム溶液になるようにして
沈殿を溶解した。■Pour this precipitate into a la cracker and add 5°Oml of distilled water.
Suspend in approximately 3°5 m I of sodium hydroxide solution.
Neutralize (pH 7) using L/L and then, while cooling in ice water, add about 2 ml of IN sodium hydroxide solution to make a 0.02N sodium hydroxide solution to dissolve the precipitate.
■ＩＮ塩酸塩酸約１．５壱ｌえてｐＨ８とし、次いて１
００ｍ１の蒸留水を加えた後にＩＩＬ三角フラスコに移
して３７℃のインキュヘーター内で３０分間ゆっくり振
盪した。■ Add about 1.5 drops of IN hydrochloric acid to pH 8, then 1.
After adding 00ml of distilled water, the mixture was transferred to an IIL Erlenmeyer flask and slowly shaken in an incubator at 37°C for 30 minutes.
■１００％ＴＣＡ水溶液３０ｍ１を加えて混合した後、
３７℃のインキュヘーター内で１０分間ゆっくり振盪し
てから、約３７℃に保温した遠心分離器トミーＣＤ Ｉ
ＯＯＲ（）ミー精器社製）を使用して遠心分離操作（
３，０００ｇＸ１０分）に付した。■After adding and mixing 30ml of 100% TCA aqueous solution,
After shaking slowly for 10 minutes in an incubator at 37°C, centrifuge Tommy CD I kept at about 37°C.
Centrifugation operation (
3,000g×10 minutes).
■上清を回収して氷冷し、４℃で遠心分離操作（１０，
０００ｇＸ１０分）に付した。■ Collect the supernatant, cool it on ice, and centrifuge it at 4°C (10,
000g x 10 minutes).
［相］上清を回収してＩＯＮ水酸化ナトリウム溶液約３
．６ｍ１Ｌで中和してｐＨ７とし、限外ａｉ３１ｊＩ器
（東洋ＷＩＷｉＵＨＰ−１５０，１イル９−：ＵＫ −
１０、Ｎ２圧：４．０ｋｇ／ｃｍ２）で濃縮した。[Phase] Collect the supernatant and add ION sodium hydroxide solution to approx.
．． Neutralize to pH 7 with 6ml of 1L, and add to ultra-ai31jI apparatus (Toyo WIWiUHP-150, 1il9-:UK-
10, N2 pressure: 4.0 kg/cm2).
■得られた濃縮液６０ｍｌを、セファロース（Ｓｅｐｈ
ａｒｏｓｅ）６Ｂカラム［米国ファルマシア社（Ｐｈａ
ｒｍａｃｉａ Ｉｎｃ、）製、カラムサイズ：５ｃｍ
（内径）Ｘ１００ｃｍ（２０コを使い、ゲルｍＪｌ！！
［緩衝液：１０ｍＭトリス−ＨＣｌ／ｌ０ｍＭＮａＣ１
（ｐＨ７，５）、流速：６０ｍ１／時］に付して、各２
０ ｍ ｌの画分を得た。■ 60 ml of the obtained concentrate was added to Sepharose (Seph).
arose) 6B column [Pharmacia (USA)
manufactured by rmacia Inc., column size: 5cm
(Inner diameter) x 100cm (use 20 pieces, gel mJl!!
[Buffer: 10mM Tris-HCl/10mM NaCl
(pH 7,5), flow rate: 60 m1/hour], each 2
A fraction of 0 ml was obtained.
０初めから４３番目から５６番目迄の両分２８０ｍｌを
併せ、プロナーゼＥ（科研化学社）４５０μｇを加え、
振盪下、３７℃に２時間保温した後に、限外濾過器（東
洋濾紙ＵＨＰ−６２、フィルター： ＵＫ−１０，Ｎ２
圧：４．Ｏｋｇ／ｃｍ２）て濃縮した。次いて、ファル
マシア社製ＦＰＬＣシステム（カラム：モ／ＱＨＲ１０
／１０）を使って陰イオン交換クロマトグラフィーに付
した。即ち、１０ｍＭ）リス−ＨＣａ（ｐＨ７゜５）と
ｌ０ｍＭのＮａＣＩＬを含む緩衝液で試料をカラムに付
した後、上記緩衝液でＮａＣｌ１量が１６５ｍＭに増加
された結成を持つ緩衝液（２００ｍｌＬ）てカラムを洗
った。次いで、ＮａＣｌ濃度を、１６５ｍＭからＩＭの
Ｎ ａ ＣｆＬｌ１度勾配になるように増加させながら
全量４００ｍｌで目的ＬＰＳを溶出させ、各２ｍ１Ｌの
両分を回収した。リムラステスト陽性が確認された、濃
度勾配をかけてから５〜８番目の両分を併せて°、ＬＰ
Ｓ純度約９２％のｓｍｆＬｃＬｐｓ ： ３．０３ｍｇ
（リムラステストによる大腸菌ＬＰＳ換算値である。Combine 280 ml of both parts from 0 to 43rd to 56th, add 450 μg of Pronase E (Kaken Kagaku Co., Ltd.),
After incubating at 37°C for 2 hours with shaking, use an ultrafilter (Toyo Roshi UHP-62, filter: UK-10, N2).
Pressure: 4. The solution was concentrated at Next, Pharmacia FPLC system (column: MO/QHR10
/10) was used for anion exchange chromatography. That is, after applying the sample to the column with a buffer containing 10mM) Lys-HCa (pH 7°5) and 10mM NaCIL, a buffer (200ml) with the above buffer in which the amount of NaCl1 was increased to 165mM was added. The column was washed. Next, the target LPS was eluted in a total volume of 400 ml while increasing the NaCl concentration from 165 mM to IM Na CfLl 1 degree gradient, and both 2 ml and 1 L aliquots were collected. The 5th to 8th portions after applying the concentration gradient, in which a positive Limulus test was confirmed, are combined °, LP
smfLcLps with S purity of about 92%: 3.03mg
(Escherichia coli LPS conversion value by Limulus test.
以下のＬＰＳ量も全てこの換算値である）、糖：０．２
３ｍｇ、１！ｒ白：０．０４ｍｇ）を回収した。All LPS amounts below are also this conversion value), Sugar: 0.2
3mg, 1! r white: 0.04 mg) was collected.
０次いてその８ｍλを、セファデックス（Ｓｅｐｈａｄ
ｅｘ）Ｇ−２５Ｃカラム：２．Ｏｃｍ（内径）Ｘ２０．
２ｃｍ （６６ｍｌ）コを使ってゲル濾過（緩衝液：水
）に付して各３ ｍ ｐ、の画分を回収した。リムラス
テスト陽性の確認された第９〜１２番目の画分を併せて
、ＬＰＳ純度約９５％の１２ｍ１（ＬＰＳ ： ２．７
ｍｇ、糖：０゜１８ｍｇ、蛋白：０．０３ｍｇ）を回収
した。糖はフェノール−硫酸法で、蛋白はローリ−法で
測定した。なお、この両分は、陰イオン交換クロマトグ
ラフィーにより酸性であることを確認した。0th order, the 8mλ is treated with Sephadex (Sephadex).
ex) G-25C column: 2. Ocm (inner diameter) x 20.
The mixture was subjected to gel filtration (buffer: water) using a 2 cm (66 ml) tube, and fractions of 3 ml each were collected. Together with the 9th to 12th fractions that were confirmed to be positive in the Limulus test, 12 ml of LPS purity of about 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.
又、ＳＤＳゲル電気泳動法による分子量は６，０００〜
１０，０００だった。In addition, the molecular weight by SDS gel electrophoresis is 6,000 ~
It was 10,000.
［株］上記画分を一８０℃で凍結後に恒量になるまで凍
結乾燥し、重量を測定したら０．７５ｍｇあった。（以
下、この凍結乾燥標品を小麦ＬＰＳと称す）
この小麦ＬＰＳのリムラス活性を後記実験例１記載の方
法で測定したら２．７ｍｇに相当するので、その比活性
は
２．７÷０．７５＝３．６
になる。[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.
また、夾雑物として存在し得る単独の糠は、以上の精製
により実質上全て除去されたと考えられるので、検出さ
れた糖は全て、小麦ＬＰＳを構成している糖と考えられ
る。従って、この段階での小麦ＬＰＳの純度を重量に基
づいて計算すると、蛋白＝０．０３ｍｇ
ＬＰＳ＝０．７５−０．０３＝０．７２ｍｇだから、
０．７２÷０．７５ｘ１００＝９６ （％）である。Furthermore, since it is considered that substantially all of the bran that may exist as a contaminant was removed by the above purification, all of the detected sugars are considered to be sugars that constitute wheat LPS. Therefore, when calculating the purity of wheat LPS at this stage based on weight, protein = 0.03 mg LPS = 0.75 - 0.03 = 0.72 mg, so 0.72 ÷ 0.75 x 100 = 96 (%) It is.
小麦ＬＰＳの物性 ■分子量 小麦ＬＰＳを蒸留水に溶解して１ｍｇ／ｍＱ。Physical properties of wheat LPS ■Molecular weight Wheat LPS was dissolved in distilled water at 1 mg/mQ.
溶液を調製し、その４μ党を１．５ｍ化の卜しフチュー
ブに入れた。これに、別途、１ｍＭのＥＤＴＡに２．５
％ＳＤＳ、５％メルカプトエタノール、１０ｍＭ）リス
塩Ｍ （ｐＨ８，０）を加えて調製したＳＤＳ処理ｆｉ
１μ免を加え、この混液を３分間沸騰水に浸した。ファ
ルマシア社製のファストシステム（Ｐｈａｓｔ Ｓｙ
ｓｔｅｍ）を使用し・電極との間に５ＤＳ−バッファー
ストリップ（Ｂｕｆｆｅｒ ５ｔｒｉｐ）（ファル
マシア社製）が介在せられた１μ艷の上記混液をゲル［
ファルマシア社製のファスト ゲル グラデイエン）（
ＰｈａｓｔＧｅｌＧｒａｄｉｅｎｔ８−２５）に塗付し
、最大電圧２５０ｖ、最大電流１０ｍＡにセットして泳
動を開始させた。泳動終了後、クマシー染色と銀染色に
おける挙動を観察した。A solution was prepared, and 4μ of the solution was placed in a 1.5m tube. To this, separately add 2.5 to 1mM EDTA.
SDS-treated fi prepared by adding %SDS, 5% mercaptoethanol, 10mM) lithium salt M (pH 8,0)
1 μm of water was added and the mixture was immersed in boiling water for 3 minutes. Phast Sy manufactured by Pharmacia
5DS-buffer strip (Buffer 5trip) (manufactured by Pharmacia) was inserted between the gel and the electrode.
Fast Gel Gladien (manufactured by Pharmacia) (
PhastGel Gradient 8-25), and the maximum voltage was set to 250 V and the maximum current was set to 10 mA to start migration. After completion of electrophoresis, behavior in Coomassie staining and silver staining was observed.
クマシー染色では、染色液としてファルマシア製の０．
１％ファスト ゲル ブルー （Ｐｈ−ａｓｔ Ｇｅ
ｌ Ｂｌｕｅ） Ｒを、脱色液として、メタノール
：酢Ｍ：蒸留水（容量比３：１：６）混液を使用し、次
の順序で染色・脱色を行った。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: vinegar M: distilled water (volume ratio 3:1:6) as a decolorizing solution.
■５０℃で８分間中色
２）５０℃で５分間脱色
３〕５０℃で８分間中色
４）５０℃で１０分間脱色
５）５０℃で５分閏保護（グリセロール、酢酸、蒸留水
の容量比５：１０：８５混液）
６〕乾燥
銀染色は、次の順序で行った。■Medium color for 8 minutes at 50℃ 2) Decolorization for 5 minutes at 50℃ 3) Medium color for 8 minutes at 50℃ 4) Decolorization for 10 minutes at 50℃ 5) Crow protection for 5 minutes at 50℃ (glycerol, acetic acid, distilled water 6) Dry silver staining was performed in the following order.
１〕５０℃で２分間、洗浄液（エタノール、酢酸、蒸留
水の容量比５：１：４混液）で処理２〕５０℃で２分間
、洗浄液（エタノ−・ル、酢酸、蒸留水の容量比１０
： ５ ： ８５ 混液）で処理３３５０℃で４分間、
洗ＩｆＩ液（エタノール、酢酸蒸留水の容量比１０：５
：８５混液）で処理４）５０℃で６分間、増惑潰（８，
３％グルタルジアルデヒド）で処理
５）５０℃で３分間、洗浄液（エタノール、酢酸蒸留水
の容量比１０：５：８５混液）て処理６】５０℃で５分
間、洗浄液（エタノール、酢酸蒸留水の容量比１０：５
：８５混液）で処理７）５０℃で２分間、洗浄液（脱イ
オン水）で処理
８）５０℃で２分間、洗浄液（脱イオン水）て処理
９）４０℃で１３分間、０．２５ｗ／ｖ％硝酸銀で処理
１０１３０℃で３０秒間、洗浄液（脱イオン水）で処理
１１１３０℃で３０秒間、洗浄液（脱イオン水〉て処理
＋２＋３０℃で３０秒間、現像液（０，０４ｖ／ｖ％ホ
ルムアルデヒド＋２．５ｗ／ｖ％炭酸ナトリウム洗浄液
）で処理
１３］３０℃で４分間、現像液（０，０４ｖ／ｖ％ホル
ムアルデヒド＋２．５ｗ／ｖ％炭酸ナトリウム洗浄液）
で処理
１４１５０℃で２分間、反応停止液（５％ｖ／ｖ％酢酸
）で処理
１５１５０℃で３分間、保護液（酢酸、グリセロル、蒸
留水の容量比１０：８：８５混液）で処理
１６〕乾燥
ＬＰＳは銀染色に染まるが、クマシー染色には染まらな
い性質を利用して染色帯を観察したら、分子量８，００
０±１，０００の位置に小麦ＬＰＳの主要染色帯が検出
された。1] Treated at 50℃ 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 with a cleaning solution (a mixture of ethanol, acetic acid, and distilled water in a volume ratio of 5:1:4) at 50℃ for 2 minutes. 10
: 5 : 85 mixture) at 3350°C for 4 minutes,
Washing IfI solution (volume ratio of ethanol and acetic acid distilled water 10:5)
: 85 mixture) 4) At 50°C for 6 minutes, amplified crush (8,
3% glutardialdehyde) 5) Treated at 50°C for 3 minutes with a cleaning solution (a 10:5:85 mixture of ethanol and acetic acid-distilled water) 6) Treated at 50°C for 5 minutes with a cleaning solution (ethanol and acetic acid-distilled water) Capacity ratio of 10:5
: 85 mixed solution) 7) 50°C for 2 minutes, cleaning solution (deionized water) 8) 50°C for 2 minutes, cleaning solution (deionized water) 9) 40°C for 13 minutes, 0.25w/ Treated with v% silver nitrate 10 for 30 seconds at 30°C, treated with cleaning solution (deionized water) 11 Treated for 30 seconds at 130°C, treated with cleaning solution (deionized water) +2 + 30 seconds at 30°C, developer (0.04v/v% formaldehyde +2 .5 w/v% sodium carbonate washing solution) 13] At 30°C for 4 minutes, developing solution (0.04v/v% formaldehyde + 2.5w/v% sodium carbonate washing solution)
14 Treated with reaction stop solution (5% v/v% acetic acid) for 2 minutes at 150°C 15 Treated with protective solution (mixture of acetic acid, glycerol, and distilled water in a volume ratio of 10:8:85) for 3 minutes at 150°C 16 [Dried LPS stains with silver staining but does not stain with Coomassie staining. When we observed the dyed band, we found that the molecular weight was 8.00.
The main staining zone of wheat LPS was detected at the 0±1,000 position.
［株］リン含有量
チェンートリバラ（Ｃｈｅｎ−Ｔｏｒ ｉ ｂａ−ｒａ
）法［チエン等著、「アナリティカル ケミストリ（Ａ
ｎａｌｙｔｉｃａｌ Ｃｈｅｍｉｓ−ｔ ｒｙ） 、
ｖｏ １．２８．１７５６〜１７Ｆ’；８頁（１９５６
年）に準拠して次の通りに行った。[Co., Ltd.] Phosphorus Content Chen-Tor iba-ra
) method [by Chien et al., “Analytical Chemistry (A
(analytical chemistry),
vo 1.28.1756-17F'; 8 pages (1956
The following procedure was carried out in accordance with the following:
小麦ＬＰＳを蒸留水に溶解して、２５μｇの小麦ＬＰＳ
を含む２０μ琵の溶液を調製し、小試験管に入 れた。Dissolve wheat LPS in distilled water to obtain 25 μg of wheat LPS.
20 microliters of a solution containing the solution was prepared and placed in a small test tube.
２０μ（の５０ｖ／ｖ％硫酸を添加し、１６０℃で２時
間加熱した。次いて、２０μ化の１０ｖ／ｖ％過塩素酸
を添加した後にガスバーナーで１分間加熱して灰化させ
た。その後に０゜５ｍＱ、の蒸留水、次いて０．５ｍｌ
の反応試薬（１ｍｉの６ Ｎ ｔ＆ 酸、２ ｍ １１
の蒸留水、２ ｍ ｌの２．５Ｖ／Ｗ％モリブデン酸ア
ンモニウム及び１ ｍ Ｌの］Ｏｖ／ｗ％のアスコルビ
ン酸を混合して調製し、その０．５ｍ＋ＩＬを使用）を
添加して室温で３０分間放置した後に、８２００ｍでの
吸光度（ＯＤ ８２１１．、−）を測定した。なお、検
量線作製用の試料としては、リン酸二水素カリウム（和
光純薬社製）を蒸留水で希釈し、リン重量としてそれぞ
れ２．５μｇ、１μｇ・０，２５μｇ・０μｇを含む０
．５ｍＱ、の溶液を調製して使用した。なお、リンＩｇ
はリン酸二水素カリウム４．３９ｇに相当する。得られ
た結果を次表１に示す。50 v/v% sulfuric acid of 20 μm was added and heated at 160° C. for 2 hours. Next, 10 v/v% perchloric acid of 20 μm was added and then heated with a gas burner for 1 minute to incinerate. Then add 0.5 mQ of distilled water, then 0.5 ml.
of reaction reagents (1 mi of 6 N t&acid, 2 m 11
of distilled water, 2 ml of 2.5 V/W% ammonium molybdate, and 1 mL of ]Ov/w% ascorbic acid (0.5 m+IL) was added and incubated at room temperature. After standing for 30 minutes, the absorbance at 8200 m (OD 8211., -) was measured. In addition, as samples for preparing the calibration curve, potassium dihydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) was diluted with distilled water, and the phosphorus weight was 2.5 μg, 1 μg, 0, 25 μg, and 0 μg, respectively.
．． A solution of 5 mQ was prepared and used. In addition, phosphorus Ig
corresponds to 4.39 g of potassium dihydrogen phosphate. The results obtained are shown in Table 1 below.
表 １
注：小麦ＬＰＳのデータは、漸機リンの混入（例えば、
リン酸緩衝液に由来する）による誤差を避けるために、
加熱処理をしていない対間のデータを減した値である。Table 1 Note: Wheat LPS data is based on gradual phosphorus contamination (e.g.
To avoid errors caused by (derived from phosphate buffer)
This is the value obtained by subtracting the data between pairs that were not heat treated.
小麦ＬＰＳの分子量を８，０００と仮定し、上表の結果
に基づいてその１分子当たりのリン数を次式により計算
すると１〜４になる。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.
上記実験でリン数がｌ〜４と変動している原因の１つと
しては、精製段階でのモノフォスフオニステラーゼの混
入により、リン酸が脱離したことも考えられる。One of the reasons why the phosphorus number varied from 1 to 4 in the above experiment may be that phosphoric acid was removed due to the contamination of monophosphonisterase during the purification step.
Ｏヘキソサミン含有量
エルソンーモルガン（Ｅｌｓｏｎ−Ｍａｒ −ｇａｎ）
法（東京化学同人出版「生化学実験講座」Ｎｏ、４の３
７７〜３７９頁）に準拠して次の通りに行った。O hexosamine content Elson-Mar-gan
Law (Tokyo Kagaku Doujin Publishing “Biochemistry Experiment Course” No. 4-3
77-379) as follows.
小麦ＬＰＳを蒸留水に溶解して１ ｍ ｇ ／ ｍ ｌ
の溶液を調製し、その１００μ露をスクリューキャップ
付きスピッツ（イワキガラス社製）に入れ、これに１０
０μ化の８ＮＨＣａを添加して１１０℃で１６時間加熱
した。４ＮＮａＯＨを約２００μを添加してｐＨ７とし
た。その１００μλを分取し、別のスクリューキャップ
付きスピッツに入れ、２００μｔの下記試薬Ａを加えた
後に、１０５℃で１．５時間加熱し、次いて流水で冷却
した。次いて、】００μ丈を分取し、６７０μ込の９６
％エタノールを加え、更に、６７μ艷の下記試薬Ｂを加
えた後に室温で１時間放置し、５３５ｎｍで吸光度を測
定した。検量線作製用試料としては０．２０〜２００μ
ｇ ／ ｍ ＩＬのＮ−アセチル グルコサミン（和光
純薬社８１）を使用した。Wheat LPS was dissolved in distilled water at 1 mg/ml.
Prepare a solution of
8NHCa of 0 μm was added and heated at 110° C. for 16 hours. Approximately 200μ of 4N NaOH was added to bring the pH to 7. 100 μλ of the mixture was collected, placed in another spitz with a screw cap, and 200 μt of Reagent A below was added thereto, heated at 105° C. for 1.5 hours, and then cooled with running water. Next, extract the length of ]00μ, and collect 96 pieces containing 670μ.
% ethanol was added thereto, and 67 μm of Reagent B below was added thereto, and the mixture was left to stand at room temperature for 1 hour, and the absorbance was measured at 535 nm. 0.20 to 200 μ as a sample for preparing a calibration curve
g/m IL of N-acetyl glucosamine (Wako Pure Chemical Industries, Ltd. 81) was used.
（試薬Ａ）７５μ役のアセチルアセトンと２．５ｍｊｌ
の１．２５Ｎ炭酸ナトリウムを混合してｖ４Ｉ！。(Reagent A) Acetylacetone as 75 μ and 2.5 mjl
Mix 1.25N sodium carbonate to v4I! .
（試薬Ｂ）１．６ｇのｐ−ジメチルベンスアルデヒドと
３０ｍｕの濃塩酸と３０ｍ地の９６％エタノールを混合
して調製。(Reagent B) Prepared by mixing 1.6g of p-dimethylbenzaldehyde, 30mu of concentrated hydrochloric acid, and 30mu of 96% ethanol.
結果、小麦ＬＰＳのへキソサミン数は６±２７分子（仮
定分子量８，０００）だった。As a result, the number of hexosamine molecules in wheat LPS was 6±27 molecules (assumed molecular weight 8,000).
０脂肪酸含有量
９０μ＆の小麦ＬＰＳ蒸留水溶液（１ｍｇ／ｍ１Ｌ）に
１０．ｕｌの内部標準（０，５５ｍＭのマルガリンＭ）
を加えた。１．０ｍλの０．５Ｍナトノウムメチラート
を加えて脂肪酸エステルの加水分解とエステル化を行っ
た。室温で】時間放置後に９６０μ免の０．５ＮＨＣｌ
を加えて中和した。10.0 to a wheat LPS distilled aqueous solution (1 mg/ml 1 L) with a fatty acid content of 90μ. ul internal standard (0.55mM Margarine M)
added. Hydrolysis and esterification of fatty acid esters were carried out by adding 1.0 mλ of 0.5M sodium methylate. After standing for a period of time at room temperature, add 960μ of 0.5N HCl.
was added to neutralize it.
これに２ｒｒｌｌのへキサンを加えて１５分間激しく攪
拌した。次いて、１，０００ｇで５分間遠心分離を行い
ヘキサン層を分取した。窒素カスてヘキサンを蒸発させ
て、約２０μ（になるまで濃縮した。To this was added 2rrll of hexane, 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. The hexane was evaporated under a nitrogen sparge and concentrated to about 20 μm.
このサンプルをガスクロマトグラフィー［本体：島津社
製のＧＣ８ＡＰＦ、キャピラリーカラム：カナダのスベ
ルコ（Ｓｐｅｌｃｏ）社製ＦＳＣＡＰ ５ｐ２３３０
、キャリヤーガス：望素コに付して脂肪酸量を測定した
。脂肪酸量測定の基準としては、第一化学薬品社製の合
成リピドＡである大Ｉｌｌ菌型ＬＡ−１５−ＰＰ （分
子量２，０００で、１分子中の脂肪酸数は６であること
が知られている）を用いた。This sample was subjected to gas chromatography [Main body: GC8APF manufactured by Shimadzu Corporation, capillary column: FSCAP 5p2330 manufactured by Spelco of Canada.
, carrier gas: The amount of fatty acids was measured. The standard for measuring the amount of fatty acids was Bacillus major 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.
結果、小麦ＬＰＳの脂肪酸数は６±２７分子（仮定分子
量８，０００）であると推定された。As a result, the number of fatty acids in wheat LPS was estimated to be 6±27 molecules (assumed molecular weight 8,000).
上記ガスクロマトグラフィーで観察されたチャートを添
付図面第１〜３図に示す。′ｓＩ図は小麦ＬＰＳの、第
２図は大１１１ｉｉｉＬ Ｐ Ｓの、第３図は百日咳菌
ＬＰＳのチャートである。Charts observed by the above gas chromatography are shown in Figures 1 to 3 of the accompanying drawings. 'sI diagram is a chart of wheat LPS, Figure 2 is a chart of large 111iii LPS, and Figure 3 is a chart of Bordetella pertussis LPS.
第１〜３図において、図示されている主要ピーク番号に
対応する保持時間（分）は次の通りであった。In FIGS. 1 to 3, the retention times (minutes) corresponding to the main peak numbers shown were as follows.
第１図： ビーク番号 保持時間（分）１
２ 。 ４５０２
２、 ７５８第２図： ビーク番号 保持時
間（分）１ ２．４１７
２ ２．７４２
第３図： ビーク番号 保持時間（分）１
２．４３３
２ ３．０２８
第１〜３図の比較により、小麦ＬＰＳのチャドは大腸菌
ＬＰＳのチャートに似ているが、百日咳菌ＬＰＳのもの
とは大きく異なることは明白である。Figure 1: Beak number Retention time (min) 1
2. 4502
2, 758 Figure 2: Beak number Retention time (min) 1 2.417 2 2.742 Figure 3: Beak number Retention time (min) 1
2.433 2 3.028 By comparing Figures 1 to 3, it is clear that the wheat LPS chad is similar to the E. coli LPS chart, but is significantly different from that of the Bordetella pertussis LPS.
＠ＫＤＯ含有量
ＫＤＯ（２−ケト−３−デオキシオクトネート）含有量
をジフェニルアミン法［シャビ アール（Ｓｈａｂｙ
Ｒ，）等著、アナリティカルバイオケム（Ａｎａｌｙ
ｔｊｃａｌ Ｂｊｏ −ｃｈｅｍ、） 、５８ （１
） 、１２３〜１２９頁（１９７４年）］に４拠して次
の通りに行フた。@KDO content The KDO (2-keto-3-deoxyoctonate) content was determined by the diphenylamine method [Shaby
R,) et al., Analytical Biochem (Analy
tjcal Bjo-chem, ), 58 (1
), pp. 123-129 (1974)].
５００ ｍ ｇのジフェニルアミン、５ ｍ ｌのエタ
ノール、４５ｍ１の氷酢酸、５ ｏｍａの製塩ｗＩ（全
て和光純薬社＆りを合わせてＫＤＯ検出試薬を調製した
。その５００ ｕｆＬＩこ、１．０５ｍｇ／ｍｌの小麦
ＬＰＳを含む蒸留水２５０ ｐＬを合わせ、１００℃の
沸騰水浴中で３０分間加熱後に冷水（２３℃）中で３０
分間冷却し、ついて日立分光光度計３２０を使って４２
０，４７０．６３０．６６０ｎｍての紫外部吸収を測定
した（それぞれＡ４２ｅ、Ａ ａ ？ ＠、Ａ６３９％
Ａ６５！１とする）。標準試料としては、１２７４ｇ
／ｍｌのＫＤＯアンモニウム塩［米国シグマ（Ｓ ｉ
ｇｍａ）社製］を含む蒸留水２５０μ北を使用した。The KDO detection reagent was prepared by combining 500 mg of diphenylamine, 5 ml of ethanol, 45 ml of glacial acetic acid, and 5 oma of salt wI (all from Wako Pure Chemical Industries, Ltd.). Combine 250 pL of distilled water containing wheat LPS and heat in a boiling water bath at 100 °C for 30 minutes, then boil in cold water (23 °C) for 30 min.
Cool for 40 minutes and then analyze using a Hitachi spectrophotometer 320.
The ultraviolet absorption at 0,470.630.660 nm was measured (A42e, A a ? @, A639%, respectively).
A65!1). As a standard sample, 1274g
/ml KDO ammonium salt [US Sigma (S i
250 μm of distilled water was used.
検体試料、標準試料それぞれについて、次式の値を求め
た。The values of the following equations were determined for each of the specimen sample and standard sample.
Ｓ ＝＝ Ａ ｔ２１Ｉ−Ａ ａ−、ｌＩ＋Ａ ６３ｉ
ＩＡ ａｓｓ検体試料” ｔｌｉ （Ｓ Ｔ）は０．３
７９． ＩＫｆｆｉ試１１のＩＩ（Ｓｓ）は０．２９４
であった。この値の比較により、小麦ＬＰＳには５±１
モル／分子ｆＩ１８千のＫＤＯが含まれると推定された
。S == A t21I-A a-, lI+A 63i
IAass specimen sample” tli (S T) is 0.3
79. IKffi Exam 11 II (Ss) is 0.294
Met. By comparing these values, wheat LPS has 5 ± 1
It was estimated to contain mol/molecule fI of 18,000 KDO.
製造例２（クロレラＬＰＳの製造）
■繕胞膜破砕クロレラ（■マンナンフーズ社製）３０ｇ
を、洗浄液が緑色に着色しなくなるまでエタノールで洗
浄した。Production Example 2 (Production of Chlorella LPS) ■ 30 g of crushed spore membrane chlorella (■ Manufactured by Mannan Foods)
were washed with ethanol until the washing solution was no longer colored green.
■この洗浄残渣２６ｇを１００ｍｇ／ｍλの濃度で蒸留
水に溶かし、４５℃で２時間振盪後に遠心分離操作（４
℃、１０，０００ｇＸ３０分）に付した。■26 g of this washing residue was dissolved in distilled water at a concentration of 100 mg/mλ, and after shaking at 45°C for 2 hours, centrifugation was performed (4
℃, 10,000 g x 30 minutes).
■上溝を回収し、東洋濾紙Ｎｏ、２で１１１過し、次い
て蒸留水で抽出した。(2) The upper groove was collected, filtered through Toyo Roshi No. 2, and then extracted with distilled water.
■抽出液２９０ｍｌを下記条件で陰イオン交換クロマト
グラフィーに付した。(2) 290 ml of the extract was subjected to anion exchange chromatography under the following conditions.
カラム、Ｑ−セラフ０−ス（φ３ｃｍＸ２３ｃｍ、容量
約１８０ｍ込）
緩衝剤：］ＯｍＭ）リス−ＨＣａ（ｐＨ７，５）、Ｎａ
ＣＩＪ度勾配：］ＯｍＭ、４００ｍＭ、１Ｍ
流速： ］ ＯＯ〜２００ｍ１Ｌ／時
温度：室温
０２通りした画分３１０ｍ１Ｌをグルコアミラーゼで処
理して澱粉を分解した（ｐＨ５，０，４０℃、約２時間
）。澱粉の分解は、ヨウ素澱粉反応で着色が生しないこ
とにより確認した。Column, Q-Seraph 0-su (φ3cm x 23cm, capacity approximately 180m included) Buffer: ]OmM) Lith-HCa (pH 7,5), Na
CIJ gradient:] OmM, 400mM, 1M Flow rate: ] OO~200ml/hour Temperature: Room temperature 310ml of the fraction passed through 02 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 coloring occurred in the iodine-starch reaction.
■遠心分離（１０，０００ｇＸ１０分）に付して上清を
回収し、１０ＮＮａＯＨ溶液で中和してｐＨ７とし、分
子量２ｏ万カツトのポアサイズを有するウルトラフィル
ターを使って限外濾過して、分解物の除去及び濃縮を行
った。■ Collect the supernatant by centrifugation (10,000g x 10 minutes), neutralize it with 10N NaOH solution to pH 7, and ultrafiltrate it using an ultrafilter with a pore size of molecular weight 20,000 to remove the decomposed product. was removed and concentrated.
■得られた濃縮液３０ｍｇ、をファルマシア社製ＦＰＬ
Ｃシステム（カーｆｔム： モ／ＱＨＲｌ Ｏ／ １０
）を使って陰イオン交換クロマトグラフィーに付した。■ 30 mg of the obtained concentrate was added to FPL manufactured by Pharmacia Co., Ltd.
C system (Car ft: Mo/QHRl O/ 10
) was used for anion exchange chromatography.
即ち、Ｉ Ｏｍ Ｍ ）リスーＨＣ琵とｌ０ｍＭのＮａ
Ｃ１を含む！Ｉ衝１（ｐＨ７，５＞で試料をカラムに付
した後、上記緩Ｉｉ液でＮａｃａ量が１６５ｍＭに増加
された！Ｉ１１成をしたＭ（２００ｍ史）てカラムを洗
った。次いて、目的ＬＰＳを、ｉ出するため、１６５ｍ
Ｍｂ）らＩＭのＮ ａ ＣＩＪ１度勾正勾配るようにＮ
ａ ＣａＪ度を増加させながら全量４００ｍλてカラ
ムを洗い、各２ｍｉの両分を回収した。リムラステスト
陽性が確認された、濃度勾配をかけてから５〜８番目の
両分を併せた。That is, I Om M) Lithium HC and 10 mM Na
Including C1! After applying the sample to the column at I buffer 1 (pH 7,5), the Naca amount was increased to 165 mM using the buffer solution I1. 165m to emit LPS
Mb) et al. IM N a CIJ 1 degree gradient N
a The column was washed with a total volume of 400 mλ while increasing the CaJ degree, and both 2 mi portions were collected. The 5th to 8th samples after applying the concentration gradient, which were confirmed to be positive in the Limulus test, were combined.
■イ欠いてその８ｍ１ｌを、セフ７デツクズ（Ｓｅｐｈ
ａｄｅｘ）Ｇ−２５［カラム：２．。■ Seph 7 decks (Seph 7 decks)
adex) G-25 [Column: 2. .
ｃｍ（内径）Ｘ２０．２ｃｍ （６６ｍｌｌ）コを使っ
てゲル濾過（Ｍ衝液：水ンに１寸して各３ｍ兄の画分を
回収した。リムラステスト陽性の確認された第９〜１２
番目の両分を併せて１２ｍ１Ｌｅ回収した（ＬＰＳ ：
１４．３ｍｇ、糖：２．０ｍｇ、蛋白： ０．５３ｍ
ｇ）、ＬＰＳは後記実験例１記載の方法で、糖はフェノ
ール−＠酸性て、蛋白はローリ−法で測定した。cm (inner diameter) x 20.2 cm (66 ml) was used for gel filtration (M solution: diluted 1 inch in water and collected fractions of 3 m each.
A total of 12mlLe was recovered from both volumes (LPS:
14.3mg, sugar: 2.0mg, protein: 0.53m
g), LPS was measured by the method described in Experimental Example 1 below, sugar was measured using phenol @ acidic method, and protein was measured using the Lowry method.
■上記画分を一８０’Ｃて凍結後に恒星ζこなるまてＩ
Ｉ凍結燥し、重量を測定したら５．８ｍｇあった。（以
下、この凍結乾燥標品をクロレラＬＰＳと称す）
このクロレラＬＰＳのリムラス活性は１４．３ｍｇに相
当するので、その比活性は
１４．３÷５．８＝２．５
になる。■ After freezing the above fraction at -80'C, it becomes a star
I freeze-dried 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 14.3÷5.8=2.5.
また、以上の精製で、夾雑物として存在し得る単独の糖
は実賞上全て除去されたと考えられるので、検出された
糖は全て、クロレラＬＰＳを構成している糖と考えられ
る。従って、この段階でのクロレラＬＰＳの純度を重量
に基づいて計算すると、
蛋白＝０．５３ｍｇ
ＬＰＳ＝５． ８−０． ５３＝ ６．２７ｍｇだか
ら、
５．２７÷５．５ｘ１０ｏ＝９１ （％）である。In addition, it is considered that all single sugars that may exist as impurities have been virtually removed through the above purification, so all the detected sugars are considered to be sugars constituting Chlorella LPS. Therefore, the purity of Chlorella LPS at this stage is calculated based on weight: Protein = 0.53 mg LPS = 5. 8-0. Since 53=6.27mg, 5.27÷5.5x10o=91 (%).
クロレラＬＰＳの物性
製造例１に記載の方法と同様にして、次の値が得られた
。Physical Properties of Chlorella LPS The following values were obtained in the same manner as in Production Example 1.
分子量＝４０，０００〜９０．０００
リン数＝４±ｌ／分子量１万
ヘキソサミン数＝７±ｌ／分子量１万
脂肪酸数＝６±ｌ／分子量１万
ＫＤＯ数＝２±ｌ／分子量１万
製造例３（百日咳菌ＬＰＳの製造）
千葉県血清研究所から人手した試験用百日咳菌液（２，
ＯＸ １０１” ！Ｉ胞／ｒｒｌＬ）を死菌体として用
いた。Molecular weight = 40,000 to 90,000 Phosphorus number = 4 ± l / molecular weight 10,000 Hexosamine number = 7 ± l / molecular weight 10,000 Number of fatty acids = 6 ± l / molecular weight 10,000 KDO number = 2 ± l / molecular weight 10,000 manufactured Example 3 (Production of B. pertussis LPS) Test B. pertussis solution (2,
OX 101''!I cells/rrlL) were used as dead cells.
上記死菌体を２５ｍｇ（乾燥重量） ／ｍｌとなるよう
に滅菌水に懸濁した。これに等量の９０％熱フェノール
液（６８〜７０℃）を添加し、６８℃で１時間振盪しな
がら抽出した。ｓ、０００ｇ、４℃で２０分間遠心分離
して水層を分取した。残りのフェノール層に、上記水層
と等量の滅菌水を加えて同様の抽出を行った。得られた
水層を先の水層と合わせて流水中で一晩透析後に、ロー
タリーエバポレータでｌ／１０に濃縮した。これを８゜
０００ ｇ、４℃で２０分間遠心分離した。上清を分取
し、酢酸ナトリウムを少量加え、０〜４℃の冷エタノー
ルを６倍量加えて一２０℃で一晩放置した。４，０００
ｇ、４℃で３０分間遠心分離して回収した沈殿物をエタ
ノールで２回、次いてアセトンで１回遠心洗浄し、アス
ピレータで乾燥させた。The dead cells were suspended in sterilized water at a concentration of 25 mg (dry weight)/ml. 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 for 20 minutes at 4°C, 000g, and 4°C. The same amount of sterilized water as the aqueous layer was added to the remaining phenol layer, and the same extraction was performed. The resulting aqueous layer was combined with the previous aqueous layer, dialyzed in running water overnight, and then concentrated to 1/10 using a rotary evaporator. This was centrifuged at 8°000 g 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. 4,000
g, The precipitate collected by centrifugation at 4°C for 30 minutes was centrifugally washed twice with ethanol and once with acetone, and dried with an aspirator.
残さを、２０ ｍ ｇ ／ ｍ ＩＬとなるように蒸留
水に懸濁し、米国ブランマン（Ｂｒａｎｓｏｎ）社製の
ソニファイア１８５型で超音波処理（出力コントロール
５．１５分、室温）に付した。次いて２゜５００ｇ、４
℃で１０分間遠心分離し、上清を分取した。The residue was suspended in distilled water to give an IL of 20 mg/m, and subjected to ultrasonication (output control 5.15 minutes, room temperature) using Sonifier Model 185 manufactured by Branson, USA. Next, 2゜500g, 4
The mixture was centrifuged at ℃ for 10 minutes, and the supernatant was collected.
この上清を４℃で、米国シグマ（Ｓ １ｇｍａ）社製の
核酸分解酵素ＤＮａｓｅ １．ＲｎａｓｅＡて１５〜
１６時間処理した（最終的には１０℃ｇ／ｍｌＬのＤＮ
ａｓｅ Ｉ と、２０℃ｇ／ｍｌＬのＲｎａｓｅＡ
を使用した）。更に同じ濃度の核酸分解酵素を加えて３
７℃で２時間加温した。次いで２．５００ｇ、４℃で１
０分間遠心分離し、上清を分取した。This supernatant was heated at 4°C using DNase 1, a nucleic acid degrading enzyme manufactured by Sigma (USA). RnaseAte15~
Treated for 16 hours (finally 10°C g/ml DN)
ase I and Rnase A at 20°C g/ml
It was used). Furthermore, add the same concentration of nucleolytic enzyme and
It was heated at 7°C for 2 hours. Then 2.500 g, 1 at 4°C
The mixture was centrifuged for 0 minutes, and the supernatant was collected.
この上清を米国ゲルマン（Ｇｅｌｍａｎ）社のアクロデ
ィスク（ＡｃｒｏｄｉｓＣ）を使い、孔径０．２μｍで
濾過した。ｍ液を分子篩にかけ［樹脂：米国ファルマシ
ア（Ｐｈａｒｍａｃｉａ）社製セファロース（Ｓｅｐｈ
ａｒｏｓｅ）６Ｂ、カラムサイズ＝内径５ ｃ ｍ Ｘ
長さ１００ｃｍ、緩衝Ｗ＝１０ｍＭのトリス−ＨＣｌ、
１０ｍＭのＮａｃＱ、（ｐＨ７，５） 、流速＝約３ｍ
ｌ／ｃｍ２／時）、生化学工業社製のＬＳ−１キツトを
用いてツムラス活性陽性画分を調べて合わせ、上記ゲル
マン社のアクロディスクを使い、孔径０，２μｍで濾過
した。濾液をイオン交換にかけ［装置：米国ファルマシ
ア（Ｐｈａｒｍａｃｉａ）社製ＦＰＬＣ１樹脂：米国フ
ァルマシア社製モノＱ ＨＲｌ ０／ １０．緩衝液
＝１０ｍＭのトリス−ＨＣａ＋１０ｍＭのＮａＣ１（ｐ
Ｈ７，５）で１５分洗浄し、次いて、ＮａＣＩＬ量を１
６５ｍＭに増加して３０分洗浄し、次いて、２０分かけ
て、ＮａＣ１量が１６５ｍＭからＩＭの濃度勾配になる
ようにＮａＣｌ量を増加させながら洗浄し、次いて、Ｉ
ＭのＮ ａ ＣＩＬｆｌで３０洗浄する、流速＝２ｍ＋
Ｌ／分］、生化学工業社製のＬＳ−１キツトを用いてリ
ムラス活性陽性画分を調べて合わせた。This supernatant was filtered using an AcrodisC manufactured by Gelman, USA, with a pore size of 0.2 μm. M solution was passed through a molecular sieve [Resin: Sepharose (Seph, manufactured by Pharmacia, USA)]
arose) 6B, column size = inner diameter 5 cm
Length 100 cm, buffer W = 10 mM Tris-HCl,
10mM NacQ, (pH 7,5), flow rate = approx. 3m
fractions positive for Tumulus activity were examined using an LS-1 kit manufactured by Seikagaku Corporation, combined, and filtered using the above-mentioned Acrodisc manufactured by Gelman Co., Ltd. with a pore size of 0.2 μm. The filtrate was subjected to ion exchange [Apparatus: FPLC1 manufactured by Pharmacia, USA Resin: MonoQ HRl 0/10, manufactured by Pharmacia, USA. Buffer = 10mM Tris-HCa + 10mM NaCl (p
Wash with H7,5) for 15 minutes, then add 1 amount of NaCIL.
The NaCl concentration was increased to 65 mM and washed for 30 minutes, then the NaCl concentration was increased over 20 minutes to create a concentration gradient from 165 mM to IM, and then the I
30 washes with Na CILfl of M, flow rate = 2 m+
Limulus activity positive fractions were examined using an LS-1 kit manufactured by Seikagaku Corporation and combined.
合わせた画分をカラムで脱塩し［樹脂：米国ファルマシ
ア（Ｐｈａｒｍａｃ ｊ ａ）社製セファデックスＧ−
２５フアイン（ｆｉｎｅ）、カラムサイズ；内径２ｃｍ
Ｘ長さ２５ｃｍ、溶出液＝蒸留水］、次いて凍結乾燥し
た。The combined fractions were desalted using a column [Resin: Sephadex G-
25 fine, column size; inner diameter 2cm
x length 25 cm, eluent=distilled water] and then freeze-dried.
この凍結乾燥標品（４，５０ｍｇ）に混入している可能
性の最も高い物質は核酸である。そこで、紫外吸収曲線
（２００〜４００ｎｍ）をとり、２６０ｎｍでの吸光度
を求めた。吸光度１のときの核ｅＩａ度が５０μｇ／ｍ
ｌであることを用いて上記吸光度から核酸濃度を算出し
たら１％以下であった。又、ＳＤＳ電気泳動ては蛋白質
は明確には検出されなかった。従って、検出感度を考慮
すると、上記凍結乾燥標品に混入している蛋白質は高／
７０〜３％と推定される。従って、上記凍結乾燥標品の
純度は９６％以上と推定された。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. Nuclear eIa degree when absorbance is 1 is 50μg/m
When the nucleic acid concentration was calculated from the above absorbance using the fact that 1%, it was 1% or less. Furthermore, no protein was clearly detected by SDS electrophoresis. Therefore, considering the detection sensitivity, the protein contained in the freeze-dried sample mentioned above is highly
It is estimated to be 70-3%. Therefore, the purity of the freeze-dried sample was estimated to be 96% or more.
製造例１に記載の方法と同様にして測定されたこの百日
咳菌ＬＰＳの物性は次の通りであった。The physical properties of this Bordetella pertussis LPS measured in the same manner as described in Production Example 1 were as follows.
百日咳菌ＬＰＳの物性
分子量＝６，０００±１，０００．
９．０００±１，０００
（複数観察されたクマシー染色帯のうち、染色強度が最
高の２つの染色帯の値である。）
リン数＝５７分子量８千
ヘキソサミン数＝１６±２７分子量８千脂肪酸数＝５７
分子量８千
ＫＤＯ数＝２±１７分子量８千
なお、製造例１に記載の方法と同様にして測定された大
腸菌ＬＰＳ ［米国デイフコ（Ｄｉｒ：ｏ）社！！０１
２８ ： ８８］の物性は次の通りであった。Physical properties of Bordetella pertussis LPS Molecular weight = 6,000±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 measured in the same manner as described in Production Example 1 [Dir:o, USA!] ! 01
28:88] had the following physical properties.
大腸菌ＬＰＳの物性
分子量＝３０．ＯＯＯ＋５．０００
（階段状に連続したクマシー染色帯のうち、染色強度が
最高のものの値である。）リン教＝１２／分子ｔ３万
ヘキソサミン数＝４５±６／分子１１３万脂肪酸数＝１
８／分子量３万
ＫＤＯ数＝５±１７分子１ｉ３万
以下は、本発明のＬＰＳを含む製剤の処方例である。な
お、ＬＰＳ量は、リムラステストによる大Ｉｌ！菌ＬＰ
Ｓ換算量である。Physical properties of E. coli LPS Molecular weight = 30. OOO+5.000 (This is the value of the one with the highest staining intensity among the continuous step-like Coomassie staining bands.) Lint = 12/molecule t30,000 Number of hexosamines = 45±6/molecule 1,130,000 Number of fatty acids = 1
8/molecular weight 30,000 KDO number = 5±17 molecules 1i 30,000 or less is a formulation example of a preparation containing LPS of the present invention. In addition, the amount of LPS is determined by the Limulus test. Bacteria LP
This is the S equivalent amount.
実施例１ （錠剤）
小麦ＬＰ３ ０．０４ｇ６％ＨＰＣ乳糖
１７８ｇステアリン酸タルク
８ｇバレイショデンブン １４ｇ以
上を混和し、打錠して、０．１ｍｇの小麦ＬＰＳを含む
０．５ｇの錠剤４００個を調製した。Example 1 (Tablet) Wheat LP3 0.04g 6% HPC lactose 178g Talc stearate
At least 14 g of 8 g potato starch was mixed and tableted to prepare 400 0.5 g tablets containing 0.1 mg of wheat LPS.
実施例２（内用液剤）
クロレラＬＰＳ
１ ｍ ｇ
精製水
１００ ｍ 党
実施例３（軟膏剤）
小麦ＬＰＳ
０ 、１ ｇ
精製ラノリン
０ｇ
０００ｇ
実施例４（注射剤）
小麦ＬＰＳ
０、 ５ｍｇ
合計
１０００ｍ 弛
実験例１（リムラステスト陽性植物ＬＰＳの定Ｉ）各種
植物に含まれるリムラステスト陽性ＬＰＳの定量を、生
化学工業株式会社のトキシカラーシステムを使って行っ
た。Example 2 (oral solution) Chlorella LPS 1 mg Purified water 100 m Example 3 (ointment) Wheat LPS 0, 1 g Purified lanolin 0 g 000 g Example 4 (injection) Wheat LPS 0, 5 mg Total 1000 m Experimental Example 1 (Determination of LPS in Limulus Test-Positive Plants I) Quantification of Limulus Test-positive LPS contained in various plants was carried out using the Toxicolor System manufactured by Seikagaku Corporation.
■９６大の平底または丸底プレートに注射用蒸留水を１
穴当たり１８０μ免入れた。ＫＦ４２ｏμ２（試料が固
体の場合には注射用蒸留水に溶解して調製した）をプレ
ートの穴の１つに加えた。プレートミキサーで攪拌しな
がらピペッティングを行って１０倍希釈淑をＳｒ１製し
た。（以後、順次希釈試料を２０μλずつとり、同様に
処理することて１００倍、Ｉ ０００倍、・・・と１θ
倍希釈系列液を調製できる。また、注射用蒸留水と試料
の量比を変えることにより希釈率は任意に設定できる。■Pour 1 cup of distilled water for injection into a 96 sized flat bottom or round bottom plate.
I inserted 180 μm per hole. KF42oμ2 (prepared by dissolving in water for injection if the sample was a solid) was added to one of the wells of the plate. Pipetting was performed while stirring with a plate mixer to prepare Sr1 diluted 10 times. (Hereafter, 20 μλ of diluted samples were taken and treated in the same way as 100 times, I 000 times, etc., and 1θ
A series of two-fold dilutions can be prepared. Further, the dilution rate can be set arbitrarily by changing the ratio of the amount of distilled water for injection to the sample.
）■内部標準として１．５μｇ／ｍｌの大腸菌ＬＰＳ溶
液の１００，０００倍希釈液を調製し、希釈やリムラス
テスト発色が正常であることを確認した。) ■ A 100,000-fold dilution of a 1.5 μg/ml E. coli LPS solution was prepared as an internal standard, and it was confirmed that the dilution and Limulus test color development were normal.
■上記■の１０倍希釈液３５μ琵を別のプレートの穴に
とり、生化学工業株式会社のトキシカラーシステムのＬ
Ｓ−１セツト３５μ地を添加し、３７℃で３０分間放置
した。ついて１０５μｉの１Ｍ酢酸水を加えて攪拌して
反応を停止させた。■Take 35 μl of the 10-fold diluted solution from ■ above into the well of another plate, and use Seikagaku Corporation's Toxicolor System L.
A 35μ substrate of S-1 set was added and left at 37°C for 30 minutes. Then, 105 μi of 1M acetic acid water was added and stirred to stop the reaction.
この試料液の波長４１５ｎｍでの吸光度を、９６穴用吸
光度計プレートリーダーＭＴＰ−１００（コロナ！気株
式会社製）で測定した。ハックグランドとしては蒸留水
を、検量線作成用としては４２ｐｇ／ｍａの生化学工業
株式会社のトキシカラーシステムのＥＴ−１セツトを使
用して検量線を作成し、この検量線を基準にして各試料
中のリムラステスト陽性ＬＰＳの定量を行った。（試料
が蒸留水である場合の吸光度をＯとした。、）なお、こ
の方法で前記ＬＳ−１セットを使用した場合にはＩＯ〜
４５ ｐ ｇ／ｍ９．の範囲内て発色に定量性があるこ
とが確認されたので、この範囲に入らないときは、希釈
率を変えて再実験した。The absorbance of this sample solution at a wavelength of 415 nm was measured using a 96-well absorbance meter plate reader MTP-100 (manufactured by Corona!Ki Co., Ltd.). A calibration curve was created using distilled water as a hack ground and the 42 pg/ma Toxicolor System ET-1 set from Seikagaku Corporation for creating a calibration curve. Limulus test positive LPS in the sample was quantified. (The absorbance when the sample is distilled water is O.) In addition, when using the LS-1 set in this method, IO ~
45 pg/m9. It was confirmed that the color development was quantitative within this range, so if it was not within this range, the dilution rate was changed and the experiment was repeated.
希釈試料の定量値は、
（検ＩＩ！から読み取った値）×（希釈率）で計算した
。The quantitative value of the diluted sample was calculated by (value read from Test II!) x (dilution rate).
得られた結果を、固体試料の場合にはｎｇ／ｇ単位で、
液体試料の場合にはｎｇ／ｒｎλ単位て次表１に示す。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 ng/rnλ in Table 1 below.
なお、表中の試料の欄の会社名、地名等は、当該試料の
入手先、産地をさす。かかる記載がない品はスーパース
トアー忠実屋の神奈川県津久井郡中野町店で購入した品
で、製造者が不明なものを指す。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.
表 １
ツムラステスト陽性
試料（固体） ＬＰＳ量 （ｎ ）裸子植
物
松の実（興南貿易） １２５単子葉
類
硬質系小麦種子（千葉製粉） ２，２５０硬質
系小麦種子（千葉製粉）
（分子１ｉ５０００以上） １，０００，０００硬
質系小麦粉（千葉製粉） ７，５００小麦ふす
ま（千葉製粉）
（分子ｆｆ１５０００以上） ３００
小麦胚芽（千葉製粉） １，６００小麦
胚芽（千葉製粉）
（分子量５０００以上） ＜１０，０００玄米
１，１００米粉（日の
本穀粉）
（分子量５０００以上） ３
米ぬか
米ぬか（分子量５０００以上）
コーンフラワー（大洋飼料）
（分子量５０００以上）
コーングリッツ（大洋飼料）
（分子ｊ１５０００以上）
コーン（和光食ｔり
クマ笹（開本物産）
アヤメ（種子）
ニンニク（鱗茎）
アスパラガス（芽）
ミョウガ（花房）
ヨクイニン（ウチダ和漢ｇり
（原Ｍ物は鳩麦）
ハンゲ（松浦薬業）
（原植物はカラスビシャク）
バクモントウ（栃木天海掌）
（原植物はジャノヒゲ）
１、 ０００． ０００
２９、 ０００
５００、 ０００
〈 ０
１５．０００
３、 ３００
４、 ５００
４１、 ０００
２、 ３００
５、 ５００
４、 ０００
ターメリック（エスピー食品ン １９５，０００（原
植物はウコン）
双子葉類
大豆（王女食品ン １５０大豆
（はくれん）（分子１１５０００以上）４００丹波黒大
豆（和光食糧）８５
小豆く和光食糧） ４５０小豆
（和光食糧）
（分子量５０００以上’） ３６，０００，０００ひ
たし豆（和光食糧） ８００大正金時
（和光食糧） ５５０大福豆（和光
食糧） ３５０そら豆（生）
７５０ジヤガイモ（はぐれん）
（分子量５０００以上） ＜０．３ビワ
（種子） ８００アボガド
（種子） ９５０モモ（種子）
４，５００クルミ（種子）
１，９００ソラ豆（種子）
７５０カポチヤ（種子）
トマト（生の実）
カイワレダイコン（根を除く）
マツタビ（丸久物産）
アマチ十ズル（Ｋ、に、桜井）
ドクダミ（湿潤重量当たり）
（帝京大学薬用植物園）
胡＃Ｉ（白）（エスピー食品）
トウガラシ（真南貿易）
六角（真南貿易）
ナツメグ（ライオン）
（［ｉ物はニクズク）
トウヒ（ウチダ和漢薬）
（原植物はダイダイ）
カッコン（栃木天海堂）
（原植物はクズ）
ナンキンカンゾウ（ウチダ和漢薬）
オタネニンジン（ウチダ和漢薬）
ボウフウ（栃木天海堂）
１０、 ０００
！０． ５００
５０、 ０００
４０、 ０００
７３、 ０００
１．２００
２、 ３００
２、 ３００
５、 ５００
２、 ０００
８、 ０００
３、 ０００
１８、 ０００
４ δ 、 ０００
５０、 ０００
カンボウィ（栃木天海堂） ６００，０００（原
植物はオオッヅラフジ）
チョウトウコラ（ウチダ和漢薬） ７，０００（原
植物はウンカリア・ヒルスタ）
八味地黄丸（カネボウ薬品） １７，０００小柴
胡濁（ツムラ） １３．０００五苓１
（ツムラ） １２，０００猪苓湯（
ツムラ） １４．０００十全大補′
ＩＢ（ツムラ） ８．ｏｏ。Table 1 Tumulus test positive sample (solid) LPS amount (n) Gymnosperm pine nuts (Konan Boeki) 125 Monocot hard wheat seeds (Chiba Flour Milling) 2,250 Hard wheat seeds (Chiba Flour Milling) (Molecular 1i5000 1,000,000 Hard wheat flour (Chiba Flour Milling) 7,500 Wheat bran (Chiba Flour Milling) (Molecular ff 15,000 or more) 300
Wheat germ (Chiba flour) 1,600 Wheat germ (Chiba flour) (Molecular weight 5000 or more) <10,000 Brown rice 1,100 Rice flour (Hinomoto flour) (Molecular weight 5000 or more) 3 Rice bran (Molecular weight 5000 or more) Corn flour (Ocean Feed) (Molecular weight 5000 or more) Corn grits (Ocean Feed) (Molecular weight 15000 or more) Corn (Wako Shokudo) Iris (seeds) Garlic (scales) Asparagus (buds) Myoga (flower clusters) Yokuinin (Uchida Japanese and Chinese varieties (original M product is Hatomugi) Hange (Matsuura Yakugyo) (Original plant is Karasbishaku) Bakumontou (Tochigi Tenkaisho) (Original plant is Janohige) 1, 000. 000 29, 000 500, 000 〈 0 15 .000 3, 300 4, 500 41, 000 2, 300 5, 500 4, 000 Turmeric (SP Foods 195,000 (original plant is turmeric) Dicotyledonous soybeans (Princess Foods 150 Soybeans (Hakuren) (molecules 115,000 or more) 400 Tanba black soybeans (Wako Foods) 85 Red beans (Wako Foods) 450 Azuki beans (Wako Foods) (Molecular weight 5,000 or more) 36,000,000 Hitashi beans (Wako Foods) 800 Taisho Kintoki (Wako Foods) 550 Dai Fukumame (Wako Foods) 350 broad beans (raw)
750 Ginger (molecular weight 5000 or more) <0.3 Loquat (seed) 800 Avocado (seed) 950 Peach (seed)
4,500 walnuts (seeds)
1,900 fava beans (seeds)
750 Kapochaya (seed) Tomato (raw fruit) Daikon daikon (root excluded) Matsutabi (Marukyu Bussan) Amachi Juzuru (K, Ni, Sakurai) Hokudami (per wet weight) (Teikyo University Medicinal Botanical Garden) Hu #I (white) (SP Foods) Chili pepper (Shinan Trading) Hexagon (Shinan Trading) Nutmeg (Lion) ([i-thing is Nikuzuku] Spruce (Uchida Japanese and Chinese Medicine) (Original plant is Daidai) Kakkon (Tochigi Tenkaido) (Original plant is Kuzu ) Glycyrrhiza (Uchida Japanese and Chinese Medicine) Panax Ginseng (Uchida Japanese and Chinese Medicine) Boufuu (Tochigi Tenkaido) 10,000! 0. 500 50, 000 40, 000 73, 000 1.200 2, 300 2, 300 5, 500 2, 000 8, 000 3, 000 18, 000 4 δ, 000 50, 000 Kambowi (Tochigi Tenkaido) 600,000 (Original plant is Oozurafuji) Chotoukola (Uchida Japanese and Chinese Medicine) 7,000 (Original plant is Uncaria hirsuta) Hachimijiogan (Kanebo Pharmaceutical) 17,000 Koshiba Kokoku (Tsumura) 13,000 Gorei 1
(Tsumura) 12,000 Ireito (
Tsumura) 14.000 Juzen Taishu'
IB (Tsumura) 8. oo.
八味地黄丸（ツムラ＞ ｓ、ｏｏ。Hachimijiomaru (Tsmura> s, oo.
ローヤルゼリー １．０００［ペキ
ン ローヤル ゼリー
（Ｐｅｋｉｎ Ｒｏｙａｌ Ｊｅｌ Ｉｙ）ハチミ
ツ（加藤美峰園本舗） ８００シダ植物
スギナ（湿潤重量当たり） ７００（
帝京大学薬用植物ＩＩ）
ゼンマイ（開本物産） １０，０００ソ
ウ類
わかめ（三陸天然品） １１，０００わ
かめ芽株（４谷健康食品〕
ひしき（生）
芽ひしきく小善本店）
コブ（ヤマトタ力ハシ）
アサクサノリ（乾燥生ノリ）
クロレラ
（■ヘルスタージャパンＹＳ）
クロレラ
（■マンナンフーズＹ５）
菌類
椎茸（下仁田産ン
えのき茸（長野県中懸重）
しめしく勢多郡宮城町）
まいたけ（大利根）
あわび茸（羽生）
マツシュルーム
きくらげ
ナメコ
エビオス（アサヒビール社製
ビール酵母）
２００ 。Royal Jelly 1.000 [Pekin Royal Jel Iy] Honey (Kato Bihoen Honpo) 800 Horsetail Fern (per wet weight) 700 (
Teikyo University Medicinal Plants II) Zenmai (Kaibutsu) 10,000 Japanese seaweed (Sanriku Tennen Products) 11,000 Wakame bud stock (Yutani Health Foods) Hishiki (raw) Mehishikiku Kozen Main Store) Cob (Yamatota) Chikaraashi) Asakusanori (dried raw nori) Chlorella (■ Helstar Japan YS) Chlorella (■ Mannan Foods Y5) Fungal Shiitake mushrooms (Enoki mushrooms from Shimonita (Nakakakeju, Nagano Prefecture, Miyagi Town, Shimeshiku Seta District) Maitake mushrooms (Otone) Abalone mushroom (Hanyu) Pine mushroom mushroom namekoebios (brewery yeast manufactured by Asahi Breweries) 200.
８５ 。85.
１０５ 。105.
２３５ 。235.
１３０ 。130.
１、 ９００゜ １、 ０００゜ 】 ６ 。1, 900° 1,000° ] 6.
２０ 。20.
４０ 。40.
２０５ 。205.
８ 。8.
２０ 。20.
７５ 。75.
２１ 。21.
２５０ 。250.
ＯＯ
冬虫夏＊ ２４０，０００その
他
雪印ナチュレヨーグルト（■１印） ５，０００グリ
コビフイズスヨーグルト（■グリコ）５０試料（液体）
リムラステスト陽性
ＬＰＳ量 （ｎ ）
ビール
キリン
アサヒ
ファインビルスナー
ラガービール
ハートラント
ファイントラフト
スーパーイースト
ワイン
１、 １５０
１、 ２５０
１、 ５５０
１、 ４００
サントリー
サントネージュ（白）
（赤）
ジードル（アップル）
日本酒
大間−級（大間酒造）
黄桜二級（黄桜酒造）
２ 、４
】 、 ７
大寒吟醜二級（玉泉窒酒造）
玄米イ酉
日々−献（大間酒造）
薬味酒
陶陶酒デルカップ（陶陶酒本１ｍ）
宝焼耐（宝酒造）
その他
キョーしオピン（湧水製薬）
ニンニク抽出液（湧水製薬）
グロスキュー（クロレラ工業）
大麦健康メツコール（韓国・−和）
サクロンハーブ液（エーザイ）
ヘチマ水（自家製）
パイオアルゲン（クロレラ工業）
パンシロン内服液（ロート製薬）
ユンケルファンティー（佐原製薬）
コリホグス（小林製薬）
ツディ（二接）
ミオＤコーワ１００（コーリ）
１．２
＜２．０
６、 ０００
２、 ０００
１、 ０００
ノゲイン（二接） ９０ブレ
ン５０（第−Ｉ！薬） ７ソルマソク
（大Ｓ製藁） ６０−ゼリーゴー
ルト（中外製薬） ５バスビタン３０（常盤
製薬） ５チオビタ（太鵬製薬）
５未満リボビタン（大正製菓）
５未満アスハラゴールト（田辺製薬）
５未満実験例２（マクロファージのインビト０ＴＮＦ
産生能を活性化する際のＥＤ５０を与えるリムラステス
ト陽性ＬＰＳの含有量が０゜
４〜１１００ｎ／培養液ｍｌであるＬＰＳの選択方法）
９週齢の、平均体重２９ｇの各群３匹のオスのＣ３Ｈ／
Ｈｅマウスのマクロファージ腹腔常在細胞２００μ見（
２Ｘ１０５個）／穴を９６穴の平底プレートに入れ、ブ
ライマーとしての組換えマウス１．ＦＮ−７（１００単
位／ ｍ ｌ）を各式に１０μλ宛加えた。別途、各種
ＬＰＳＲを６５℃の熱水（ｇ／ｍｃ）で５時間抽出して
７１製した抽出液を各鵠希釈し、その１Ｏｕ９／穴をブ
ライマー投与の３時間後にトリガーとして加えた。２時
間培Ｍ後に遠心分離操作に付した（３０００ｇ、２０分
）。各式から得られた１３０μλの、ＴＮＦ活性はＬ９
２９纏胞に対する毒性に基づいて測定し、又、リムラス
テスト陽性ＬＰＳ含有量は生化学工業株式会社のトキシ
カラーシステムを使用して測定した。OO Caterpillar Summer* 240,000 Other Snow Brand Nature Yogurt (■1 mark) 5,000 Glycobifuzu Yogurt (■Glico) 50 samples (liquid) Limulus test positive LPS amount (n) Beer Kirin Asahi Fine Birsunner Lager Beer Heart Land Fein Traft Super Yeast Wine 1, 150 1, 250 1, 550 1, 400 Suntory Sainte Neige (white) (red) Siedle (apple) Sake Oma-grade (Oma Sake Brewery) Kizakura 2nd grade (Kizakura Sake Brewery) 2, 4 ] , 7 Daikan Ginbu 2nd Class (Gyokusen Nitshu Brewery) Brown Rice Tori Hibi-Ken (Oma Sake Brewery) Spicy Sake Ceramic Sake Del Cap (Ceramic Sake Book 1m) Takara Yaki Tai (Takara Shuzo) Other Kyoshi Opin (Yusui Pharmaceutical) Garlic extract (Yusui Pharmaceutical) Groscue (Chlorella Industries) Barley Health Metcol (Korea/Japan) Saclone Herb Liquid (Eisai) Luffa water (homemade) Paio Algen (Chlorella Industries) Pancilon oral solution (Rohto Pharmaceutical) Yunkerfan tea (Sahara Pharmaceutical) Corihogus (Kobayashi Pharmaceutical) Tsudi (Second) Mio D Kowa 100 (Koli) 1.2 <2.0 6, 000 2, 000 1, 000 Nogain (Second) 90 Bren 50 (Part-I! Medicines) 7 Solmasoku (Daisy S Straw) 60 Jelly Gault (Chugai Pharmaceutical) 5 Basvitan 30 (Tokiwa Pharmaceutical) 5 Tiovita (Taiho Pharmaceutical)
Ribovitan less than 5 (Taisho Seika)
Less than 5 Ashara Gold (Tanabe Pharmaceutical)
Experimental Example 2 (in vitro 0 TNF of macrophages)
Method for selecting LPS with a Limulus test-positive LPS content of 0°4 to 1100n/ml of culture solution that gives the ED50 when activating production ability) 9-week-old males with an average weight of 29g, 3 in each group. C3H/
He mouse macrophage peritoneal resident cells 200 μm (
Recombinant mice 1. 10 μλ of FN-7 (100 units/ml) was added to each formula. Separately, various LPSRs were extracted with hot water (g/mc) at 65° C. for 5 hours to prepare an extract solution, which was diluted to each mouse, and 1 Ou9/well of the diluted solution was added as a trigger 3 hours after the administration of Brimer. After 2 hours of culture, the mixture was subjected to centrifugation (3000g, 20 minutes). Of the 130μλ obtained from each formula, the TNF activity is L9
The measurement was based on the toxicity to No. 29 phthisis, and the Limulus test positive LPS content was measured using the Toxicolor system manufactured by Seikagaku Corporation.
測定値を、縦軸にＴＮＦ産生量（単位／培養液ｍｌ）を
、横軸（対数尺）に対応リムラステスト陽性ＬＰＳ含有
量（ｎｇ／培養液ｍｌ）を表す座標にプロットし、プロ
ットされた各点から推定されるシグモイド曲線を描いた
。トリガーを投与しなかった場合のＴＮＦ産生量を与え
る各トリガーのマクロファージ活性化能を０％とし、ト
リガー投与の効果として増大するＴＮＦ産生量が最大恒
量に達したときの各トリガーのマクロファージ活性化能
を１００％とし、その５０％に相当するマクロファージ
活性化能を与えるリムラステスト陽性ＬＰＳ含有量を曲
線から読み取った。The measured values are plotted on coordinates representing the TNF production amount (unit/mL of culture solution) on the vertical axis and the corresponding Limulus test positive LPS content (ng/mL of culture solution) on the horizontal axis (logarithmic scale). A sigmoid curve estimated from the points 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%, and the macrophage activation ability of each trigger when the amount of TNF production that increases as an effect of trigger administration reaches the maximum constant amount. was set as 100%, and the Limulus test-positive LPS content that gave the macrophage activation ability corresponding to 50% was read from the curve.
マクロファージ活性化能とリムラステスト陽性ＬＰＳ含
有量との相ｒＩ！Ｊ間係が上記条件を満たしたＬＰＳ採
取源の結果を次の表２に示す。表中で、ｒＴＮＦＪはＴ
ＮＦ産生量（単位／培養液ｍｕ）を、「活性化能」はマ
クロファージ活性化能（％）を、「ＬＰＳ」はリムラス
テスト陽性ＬＰＳ含有量（ｎｇ／培！Ｉ液ｍλ）を表す
。なお、トリガー漸添加時のＴＮＦ産生量は０．７５単
位／ ｍ ｌであったので、ＴＮＦ産生量が０．７５単
位／ｍＱ。Correlation between macrophage activation ability and Limulus test positive LPS content! Table 2 below shows the results of the LPS collection sources for which the J staff met the above conditions. In the table, rTNFJ is T
NF production amount (unit/culture fluid mu), "activation ability" represents macrophage activation ability (%), and "LPS" represents Limulus test positive LPS content (ng/culture medium! I fluid mλ). In addition, the TNF production amount at the time of gradual addition of the trigger was 0.75 units/ml, so the TNF production amount was 0.75 units/mQ.
以下である場合をマクロファージ活性化能θ％とし、マ
クロファージ活性化能（％）は次式により計算した。If the following is the case, the macrophage activation ability θ% was defined as the macrophage activation ability (%), and the macrophage activation ability (%) was calculated using the following formula.
表 ２ 表２に示された結果を第４〜７図に示す。Table 2 The results shown in Table 2 are shown in Figures 4-7.
１．４〜７図において、縦軸はマクロファージ活性化能
（％）を表し、横軸（対数尺）はリムラステスト陽性Ｌ
ＰＳ含有量（ｎｇ／培養液ｍ１Ｌ）を表している。In Figures 1.4 to 7, the vertical axis represents macrophage activation ability (%), and the horizontal axis (logarithmic scale) represents Limulus test positive L.
PS content (ng/mL of culture solution) is expressed.
第４図において、Ｑはターメリックの、・はカンボーイ
の、口はコンブの、■はアサクサノリのデータを示す。In Fig. 4, Q indicates data for turmeric, * indicates data for Kanboy, mouth indicates data for kelp, and ■ indicates data for Asakusanori.
第Ｓ図において、○はワカメ芽株エキスの、口は芽ヒジ
キの、■はエビオスのデータを示す。In FIG. S, ○ indicates data for wakame bud extract, mouth indicates data for Me Hijiki, and ■ indicates data for Ebios.
第６図において、○は冬虫夏草の、・はワカメ芽株の、
口はクロレラのデータを示す。In Figure 6, ○ indicates cordyceps sinensis, ・ indicates wakame bud stock,
The mouth shows data on chlorella.
第７図において、○は大腸菌ＬＰＳの、・は小麦ＬＰＳ
の、口は百日咳菌ＬＰＳの、■はリピトＡのデータを示
す。In Figure 7, ○ indicates E. coli LPS, ・ indicates wheat LPS.
, Mouth indicates data for Bordetella pertussis LPS, and ■ indicates data for LipitoA.
実験例３（抗消化性潰瘍効果の測定）
各群３匹のＣ３Ｈ／Ｈｅマウス（１２週齢の雄、平均体
重２５ｇ）を２４時閏絶食させた。但し、水は自由に摂
取させた。Experimental Example 3 (Measurement of anti-peptic ulcer effect) Three C3H/He mice in each group (12-week-old male, average weight 25 g) were fasted for 24 hours. However, water was available ad libitum.
各群にそれぞれ５０ｍｇ／匹の、製造例１の■て得られ
た粉末Ａ ａ ２ （９００μｇ／ｇのリムラステス
ト陽性ＬＰＳを含む）、■マンナンフーズＹＳから市販
されている纏胞膜破砕クロレラ（３ｍｇ／ｇのリムラス
テスト陽性ＬＰＳを含む）、蒸留水をゾンデて経口投与
し、その１時間後に１．５ｍｇのインドメタシンを各マ
ウスに皮下投与し、腺胃に胃ａｇｉを発生させた。Each group received 50 mg/mouse of the powder A a 2 (containing 900 μg/g of Limulus test-positive LPS) obtained in Production Example 1, and ■ 3 mg/mouse of crushed chlorella commercially available from Mannan Foods YS. (containing Limulus test-positive LPS) and distilled water were administered orally through a probe, and 1 hour later, 1.5 mg of indomethacin was subcutaneously administered to each mouse to induce gastric agi in the glandular stomach.
インドメタシン投与の７時間後に胃を摘出し、２％ホル
マリンで固定した。病変部を切開し、潰瘍の数、長さ、
面積を測定した。結果を各群３匹の平均として次表３に
表す、なお、カッコ内のデータは、蒸留水投与群の値を
１００％とした場合の割合を表している。なお、製造例
１て得られた小麦ＬＰＳを３μｇ静注した場合には潰瘍
発生はほぼ１００％予防された。Seven hours after indomethacin administration, the stomach was removed and fixed in 2% formalin. The lesion is incised and the number, length, and number of ulcers are determined.
The area was measured. The results are shown in the following Table 3 as the average of three animals in each group. Furthermore, the data in parentheses represents the percentage when the value for the distilled water administration group is taken as 100%. In addition, when 3 μg of wheat LPS obtained in Production Example 1 was intravenously injected, almost 100% of ulcer development was prevented.
表 ３
本発明のＬＰＳを抗消化性潰瘍剤、動物用抗消化性ｉｌ
ｌ 瘍剤として投与するさいの量、投与間隔は、当然、
担当医師或いは獣医師の厳重な管理下、投与対象の年齢
、症状、体重、投与効果を勘案して個別に決定されるが
、人臣の成人（６０ｋ ｇ）で、経口投与で１μｇ −
１００ｍ ｇ、静脈投与でｌＯｒｌｇ−１ｍｇ、経皮投
与で１００ ｎ ｇ −１ｍ ｇが１８１回の投与量の
一応の目安となる。なお、動物では、牛、馬等の大型動
物は上記の量の６０分の１を体重１ｋｇ当たりの量の目
安とし、豚、犬、猫等の中型、小型の動物ではその２倍
量を体重１ｋｇ当たりの量の目安とし、鶏等の鳥類では
更にその２倍量を体重１ｋｇ当たりの量の目安とし投与
できる。Table 3 The LPS of the present invention is used as an anti-peptic ulcer agent and as an anti-peptic il for animals.
l When administering as an anti-cancer drug, the amount and interval of administration are, of course, as follows:
It is determined individually under the strict supervision of the attending physician or veterinarian, taking into account the age, symptoms, body weight, and administration effects of the subject, but oral administration of 1 μg for an adult human subject (60 kg).
100mg, lOrlg-1mg for intravenous administration, and 100ng-1mg for transdermal administration are tentative guidelines for the 181 doses. In addition, for large animals such as cows and horses, use 1/60th of the above amount per kg of body weight, and for medium-sized and small animals such as pigs, dogs, and cats, double the amount per kg of body weight. The amount can be administered per 1 kg of body weight, and for birds such as chickens, double the amount can be administered per 1 kg of body weight.
又、小麦ＬＰＳ （製造例］）、クロレラＬＰＳ（製造
例２）、大腸菌ＬＰＳ ［米国デイフコ＜Ｄｉｒｃｏ）
？ｆ製０１２８：Ｂ８＞、百日咳菌ＬＰＳ （製造例３
）の毒性値ＬＤ５９（１群２匹の雄ＢＡＬＢ／Ｃマウス
、平均体重４５ｇ、における平均値）は次の通りであっ
た。In addition, wheat LPS (manufacturing example)), chlorella LPS (manufacturing example 2), Escherichia coli LPS [Dirco, USA]
? f0128:B8>, Bordetella pertussis LPS (Production Example 3
) toxicity value LD59 (average value in 2 male BALB/C mice per group, average weight 45 g) was as follows.
［発明の効果］
本発明により、抗消化性潰瘍効果が高くて副作用が少な
く、従って化学療法係数が高く、かつ、生産コストが低
く、しかも、経口、経皮、注射ての投与が可能であり、
かつ大量に供給可能であり、その上、毎日食すことが可
能な食品の内に配合可能な新規な抗消化性［１剤、動物
用抗消化性Ｊ１瘍が提供される。[Effects of the Invention] The present invention has a high anti-peptic ulcer effect, few side effects, high chemotherapy coefficient, low production cost, and can be administered orally, transdermally, or by injection. ,
Moreover, a novel anti-digestive agent [1 agent, anti-peptic J1 ulcer for animals] which can be supplied in large quantities and can be incorporated into foods that can be eaten every day is provided.
第１図は、小麦ＬＰＳをガスクロマトグラフィにかけて
得られる、分子中における脂肪酸の存在を示すピークを
図示したチャートである。
第２図は、大Ｉ細菌ＬＰＳをガスクロマトグラフィーに
かけて得られる、分子中における脂肪酸の存在を示すピ
ークを図示したチャートである。
第３図は、百日咳菌ＬＰＳをガスクロマトグラフィーに
かけて得られる、分子中における脂肪酸の存在を示すピ
ークを図示したチャートである。
第４〜７図は、マクロファージ活性化能とリムラステス
ト陽性ＬＰＳ含有量との相間間係が本発明の条件を満た
している各！ＬＰＳの当該相Ｒ間係を示すグラフである
。
第４〜７図において、縦軸はマクロファージ活性化能（
％）を表し、横軸（対数尺）はリムラステスト陽性ＬＰ
Ｓ含有量（ｎｇ／培養液ｍｌ）を表している。
第４図において、○はターメリックの、・は力ンボーイ
の、口はコンブの、層はアサクサノリのデータをボす。
第５図二二おいて、○はワカメ芽株エキスの、・は芽ヒ
ジキの、口はエビオスのデータを示す。
第６図において、○は冬虫夏草の、・はワカメ芽株の、
口はクロレラのデータを示す。
第７図において、Ｏは大腸菌ＬＰＳの、・は小麦ＬＰＳ
の、口は百日咳＠ＬＰＳの、■はリピドＡのデータを示
す。FIG. 1 is a chart showing 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 the molecule, which are obtained by gas chromatography of Bacterium LPS. 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. Figures 4 to 7 show each case where the correlation between macrophage activation ability and Limulus test positive LPS content satisfies the conditions of the present invention. It is a graph which shows the relationship between the said phase R of LPS. 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 the S content (ng/ml of culture solution). In Figure 4, ○ indicates turmeric, ・ indicates Riki-n-Boy, mouth indicates kelp, and layer indicates Asakusanori. In Fig. 5-22, ○ 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.
, Mouth indicates pertussis @LPS data, ■ indicates lipid A data.
Claims (1)
で培養されるマクロファージのＴＮＦ産生能を活性化す
るＬＰＳのマクロファージ活性化能を指標とし、 縦軸に、そのＬＰＳを添加しないときのマクロファージ
のＴＮＦ産生量を与えるマクロファージ活性化能を０％
、マクロファージのＴＮＦ産生量を最大恒量にする時の
ＬＰＳのマクロファージ活性化能を１００％とするマク
ロファージ活性化能（％）を表し、横軸に、そのＬＰＳ
のリムラステスト陽性ＬＰＳ含有量を対数尺で表すシグ
モイド曲線を描くとき、 マクロファージ活性化能のＥＤ＿５＿０を与えるリムラ
ステスト陽性ＬＰＳ含有量が０．４〜１００ｎｇ／培養
液ｍｌであるＬＰＳの少なくとも１種を含む抗消化性潰
瘍剤。 （２）ＬＰＳが、植物から得られるＬＰＳ、細菌から得
られるＬＰＳ及びリピドＡからなる群から選択される、
請求項１記載の抗消化性潰瘍剤。 （３）植物が裸子植物、単子葉類植物、双子葉類植物、
シダ植物、ソウ類植物、菌類植物及びそれらの混合物か
らなる群から選択されるものである、請求項２記載の抗
消化性潰瘍剤。 （４）裸子植物がマツ科マツ属植物である、請求項３記
載の抗消化性潰瘍剤。 （５）マツ科マツ属植物がマツである、請求項４記載の
抗消化性潰瘍剤。 （６）単子葉類植物がイネ科のイネ属植物、コムギ属植
物、オオムギ属植物、カラス麦属植物、ササ属植物、ジ
ュズダマ属植物、アヤメ科のアヤメ属植物、ユリ科のネ
ギ属植物、キジカクシ属植物、ジャノヒゲ属植物、ショ
ウガ科のショウガ属植物、ウコン属植物、サトイモ科ハ
ンゲ属植物及びそれらの混合物からなる群から選択され
るものである、請求項３記載の抗消化性潰瘍剤。 （７）イネ科イネ属植物がイネである、請求項６記載の
抗消化性潰瘍剤。 （８）イネ科コムギ属植物が小麦である、請求項６記載
の抗消化性潰瘍剤。 （９）イネ科オオムギ属植物が大麦、裸麦及びそれらの
混合物からなる群から選択されるものである、請求項６
記載の抗消化性潰瘍剤。 （１０）イネ科カラス麦属植物が烏麦、燕麦及びそれら
の混合物からなる群から選択される、請求項６記載の抗
消化性潰瘍剤。 （１１）イネ科ササ属植物がクサ笹である、請求項６記
載の抗消化性潰瘍剤。 （１２）イネ科ジュズダマ属植物が鳩麦である、請求項
６記載の抗消化性潰瘍剤。 （１３）アヤメ科アヤメ属植物がアヤメである、請求項
６記載の抗消化性潰瘍剤。 （１４）ユリ科ネギ属植物がニンニクである、請求項６
記載の抗消化性潰瘍剤。 （１５）ユリ科キジカクシ属植物がアスパラガスである
、請求項６記載の抗消化性潰瘍剤。 （１６）ユリ科ジャノヒゲ属植物がジャノヒゲである、
請求項６記載の抗消化性潰瘍剤。（１７）ショウガ科シ
ョウガ属植物がミョウガである、請求項６記載の抗消化
性潰瘍剤。（１８）ショウガ科ウコン属植物がウコンで
ある、請求項６記載の抗消化性潰瘍剤。 （１９）サトイモ科ハンゲ属植物がカラスビシャクであ
る、請求項６記載の抗消化性潰瘍剤。 （２０）小麦から得られるＬＰＳが次の物性を有するも
のである、請求項８記載の抗消化性潰瘍剤。 分子量：８，０００±１，０００（ＳＤＳ電気泳動法） リン数：１以上／分子量８千 ヘキソサミン数：６±２／分子量８千 脂肪酸数：６±２／分子量８千 ＫＤＯ数：５±１／分子量８千 （２１）双子葉類植物がマメ科のダイズ属植物、インゲ
ンマメ属植物、ソラマメ属植物、クズ属植物、カンゾウ
属植物、ナス科のナス属植物、トマト属植物、トウガラ
シ属植物、バラ科のビワ属植物、サクラ属植物、クスノ
キ科アボガド属植物、クルミ科クルミ属植物、ウリ科の
トウナス属植物、アマチャヅル属植物、アブラナ科ダイ
コン属植物、マタタビ科マタタビ属植物、ドクダミ科ド
クダミ属植物、コショウ科コショウ属植物、シキミ科シ
キミ属植物、ニクズク科ニクズク属植物、ミカン科ミカ
ン属植物、ウコギ科オタネニンジン属植物、セリ科サポ
シュニコビア属植物、ツヅラフジ科オオツヅラフジ属植
物、アカネ科カギカズラ属植物及びそれらの混合物から
なる群から選択されるものである、請求項３記載の抗消
化性潰瘍剤。 （２２）マメ科ダイズ属植物が大豆である、請求項２１
記載の抗消化性潰瘍剤。 （２３）マメ科インゲンマメ属植物が小豆である、請求
項２１記載の抗消化性潰瘍剤。 （２４）マメ科ソラマメ属植物がそら豆である、請求項
２１記載の抗消化性潰瘍剤。 （２５）マメ科クズ属植物がクズである、請求項２１記
載の抗消化性潰瘍剤。 （２６）マメ科カンゾウ属植物がナンキンカンゾウであ
る、請求項２１記載の抗消化性潰瘍剤。 （２７）ナス科ナス属植物がジャガイモ、トウガラシ及
びそれらの混合物からなる群から選択されるものである
、請求項２１記載の抗消化性潰瘍剤。 （２８）ナス科トマト属植物がトマトである、請求項２
１記載の抗消化性潰瘍剤。 （２９）ナス科トウガラシ属植物がトウガラシである、
請求項２１記載の抗消化性潰瘍剤。 （３０）バラ科ビワ属植物がビワである、請求項２１記
載の抗消化性潰瘍剤。 （３１）バラ科サクラ属植物がモモである、請求項２１
記載の抗消化性潰瘍剤。 （３２）クスノキ科アボガド属植物がアボガドである、
請求項２１記載の抗消化性潰瘍剤。 （３３）クルミ科クルミ属植物がクルミである、請求項
２１記載の抗消化性潰瘍剤。 （３４）ウリ科トウナス属植物がカボチャである、請求
項２１記載の抗消化性潰瘍剤。 （３５）ウリ科アマチャヅル属植物がアマチャヅルであ
る、請求項２１記載の抗消化性潰瘍剤。 （３６）アブラナ科ダイコン属植物がカイワレダイコン
である、請求項２１記載の抗消化性潰瘍剤。 （３７）マタタビ科マタタビ属植物がマタタビである、
請求項２１記載の抗消化性潰瘍剤。 （３８）ドクダミ科ドクダミ属植物がドクダミである、
請求項２１記載の抗消化性潰瘍剤。 （３９）コショウ科コショウ属植物が胡椒である、請求
項２１記載の抗消化性潰瘍剤。 （４０）シキミ科シキミ属植物がダイウイキョウである
、請求項２１記載の抗消化性潰瘍剤。 （４１）ニクズク科ニクズク属植物がニクズクである、
請求項２１記載の抗消化性潰瘍剤。 （４２）ミカン科ミカン属植物がダイダイである、請求
項２１記載の抗消化性潰瘍剤。 （４３）ウコギ科オタネニンジン属植物がオタネニンジ
ンである、請求項２１記載の抗消化性潰瘍剤。 （４４）セリ科サポシュニコビア属植物がボウフウであ
る、請求項２１記載の抗消化性潰瘍剤。 （４５）ツヅラフジ科オオツヅラフジ属植物がオオツヅ
ラフジである、請求項２１記載の抗消化性潰瘍剤。 （４６）アカネ科カギカズラ属植物がウンカリア・ヒル
スタである、請求項２１記載の抗消化性潰瘍剤。 （４７）シダ植物がトクサ科トクサ属植物、ゼンマイ科
ゼンマイ属植物及びそれらの混合物からなる群から選択
されるものである、請求項３記載の抗消化性潰瘍剤。 （４８）トクサ科トクサ属植物がスギナである、請求項
４７記載の抗消化性潰瘍剤。 （４９）ゼンマイ科ゼンマイ属植物がゼンマイである、
請求項４７記載の抗消化性潰瘍剤。 （５０）ソウ類植物がカッソウ類植物、紅ソウ類植物、
緑ソウ類植物、ランソウ類植物及びそれらの混合物から
なる群から選択されるものである、請求項３記載の抗消
化性潰瘍剤。 （５１）カッソウ類植物がコンブ科のワカメ属植物、コ
ンブ属植物、ホンダワラ科ヒジキ属植物及びそれらの混
合物からなる群から選択されるものである、請求項５０
記載の抗消化性潰瘍剤。 （５２）コンブ科ワカメ属植物がワカメである、請求項
５１記載の抗消化性潰瘍剤。 （５３）コンブ科コンブ属植物がコンブである、請求項
５１記載の抗消化性潰瘍剤。 （５４）ホンダワラ科ヒジキ属植物がヒジキである、請
求項５１記載の抗消化性潰瘍剤。（５５）紅ソウ類植物
がウシケノリ科アマノリ属植物である、請求項５０記載
の抗消化性潰瘍剤。 （５６）ウシケノリ科アマノリ属植物がアサクサノリで
ある、請求項５５記載の抗消化性潰瘍剤。 （５７）緑ソウ類植物がオオシスティス科クロレラ属植
物である、請求項５０記載の抗消化性潰瘍剤。 （５８）オオシスティス科クロレラ属植物がクロレラで
ある、請求項５７記載の抗消化性潰瘍剤。 （５９）クロレラから得られるＬＰＳが次の物性を有す
るものである、請求項５８記載の抗消化性潰瘍剤。 分子量＝４０，０００〜９０，０００（ＳＤＳ電気泳動
法） リン数＝４±１／分子量１万 ヘキソサミン数＝７±１／分子量１万 脂肪酸数＝６±１／分子量１万 ＫＤＯ数＝２±１／分子量１万 （６０）菌類植物が担子菌類植物、子ノウ菌類植物及び
それらの混合物からなる群から選択されるものである、
請求項３記載の抗消化性潰瘍剤。 （６１）担子菌類植物がヒラタケ科マツオウジ属植物、
キシメジ科のエノキタケ属植物、シメジ属植物、タコウ
キン科マイタケ属植物、サルノコシカケ科ポリポラス属
植物、ハラタケ科ハラタケ属植物、キクラゲ科キクラゲ
属植物、モエギタケ科スギタケ属植物及びそれらの混合
物である、請求項６０記載の抗消化性潰瘍剤。 （６２）ヒラタケ科マツオウジ属植物が椎茸である、請
求項６１記載の抗消化性潰瘍剤。（６３）キシメジ科エ
ノキタケ属植物がエノキ茸である、請求項６１記載の抗
消化性潰瘍剤。 （６４）キシメジ科シメジ属植物がシメジである、請求
項６２記載の抗消化性潰瘍剤。 （６５）タコウキン科マイタケ属植物がマイ茸である、
請求項６１記載の抗消化性潰瘍剤。 （６６）サルノコシカケ科ポリポラス属植物がアワビ茸
である、請求項６１記載の抗消化性潰瘍剤。 （６７）ハラタケ科ハラタケ属植物がマッシュルームで
ある、請求項６１記載の抗消化性潰瘍剤。 （６８）キクラゲ科キクラゲ属植物がキクラゲである、
請求項６１記載の抗消化性潰瘍剤。 （６９）モエギタケ科スギタケ属植物がナメコである、
請求項６１記載の抗消化性潰瘍剤。 （７０）子ノウ菌類植物がエンドミセタセア科サッカロ
ミセス属植物、バッカクキン科ノムシタケ属植物及びそ
れらの混合物である、請求項６０記載の抗消化性潰瘍剤
。 （７１）エンドミセタセア科サッカロミセス属植物が、
パン酵母、醸造用酵母及びそれらの混合物である、請求
項７０記載の抗消化性潰瘍剤。 （７２）バッカクキン科ノムシタケ属植物が冬虫夏草で
ある、請求項７０記載の抗消化性潰瘍剤。 （７３）細菌が大腸菌、百日咳菌及びそれらの混合物か
らなる群から選択されるものである、請求項３記載の抗
消化性潰瘍剤。 （７４）大腸菌から得られるＬＰＳが次の物性を有する
ものである、請求項７３記載の抗消化性潰瘍剤。 分子量＝３０，０００±５，０００（ＳＤ Ｓ電気泳動法） リン数＝１２／分子量３万 ヘキソサミン数＝４５±６／分子量３万 脂肪酸数＝１８／分子量３万 ＫＤＯ数＝５±１／分子量３万 （７５）百日咳菌から得られるＬＰＳが次の物性を有す
るものである、請求項７３記載の抗消化性潰瘍剤。 分子量＝６，０００±１，０００ ９，０００±１，０００ （ＳＤＳ電気泳動法） リン数＝５／分子量８千 ヘキソサミン数＝１６±２／分子量８千 脂肪酸数＝５／分子量８千 ＫＤＯ数＝２±１／分子量８千 （７６）ＬＰＳを含む動物用抗消化性潰瘍剤であり、 インビトロで培養されるマクロファージのＴＮＦ産生能
を活性化するＬＰＳのマクロファージ活性化能を指標と
し、縦軸に、そのＬＰＳを添加しないときのマクロファ
ージのＴＮＦ産生量を与えるマクロファージ活性化能を
０％、マクロファージのＴＮＦ産生量を最大恒量にする
時のＬＰＳのマクロファージ活性化能を１００％とする
マクロファージ活性化能（％）を表し、横軸に、そのＬ
ＰＳのリムラステスト陽性ＬＰＳ含有量を対数尺で表す
シグモイド曲線を描くとき、 マクロファージ活性化能のＥＤ＿５＿８を与えるリムラ
ステスト陽性ＬＰＳ含有量が０．４〜１００ｎｇ／培養
液ｍｌであるＬＰＳの少なくとも１種を含む動物用抗消
化性潰瘍剤。[Scope of Claims] (1) An anti-peptic ulcer agent containing LPS, with the macrophage activation ability of LPS, which activates the TNF production ability of macrophages cultured in vitro, as an index, and the vertical axis indicates the LPS The macrophage activation ability that gives the amount of TNF production by macrophages when not added is 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 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. Peptic ulcer agent. (2) the LPS is selected from the group consisting of LPS obtained from plants, LPS obtained from bacteria, and lipid A;
The anti-peptic ulcer agent according to claim 1. (3) The plant is a gymnosperm, a monocot, a dicot,
The anti-peptic ulcer agent according to claim 2, which is selected from the group consisting of fern plants, grass plants, fungal plants, and mixtures thereof. (4) The anti-peptic ulcer agent according to claim 3, wherein the gymnosperm is a plant of the genus Pinus of the family Pinaceae. (5) The anti-peptic ulcer agent according to claim 4, wherein the plant of the genus Pinus of the family Pinaceae is pine. (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-peptic ulcer agent according to claim 3, which is selected from the group consisting of plants of the genus Porphyra, plants of the genus Zingiber, 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 anti-peptic ulcer agent according to claim 6, wherein the plant of the Gramineae family, Poaceae, is rice. (8) The anti-peptic ulcer agent according to claim 6, wherein the plant of the Gramineae family, Triticum genus, 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-peptic ulcer agent as described. (10) The anti-peptic ulcer 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-peptic ulcer agent according to claim 6, wherein the plant of the genus Bamboo, belonging to the family Poaceae, is bamboo grass. (12) The anti-peptic ulcer agent according to claim 6, wherein the plant of the genus Juzdama, belonging to the family Poaceae, is pigeon wheat. (13) The anti-peptic ulcer 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-peptic ulcer agent as described. (15) The anti-peptic ulcer agent according to claim 6, wherein the plant of the family Liliaceae, belonging to the genus Acanthus, is asparagus. (16) A plant of the genus Janohige of the family Liliaceae is Janohige,
The anti-peptic ulcer agent according to claim 6. (17) The anti-peptic ulcer agent according to claim 6, wherein the ginger plant of the family Zingiberaceae is ginger. (18) The anti-peptic ulcer agent according to claim 6, wherein the plant of the genus Curcuma of the family Zingiberaceae is turmeric. (19) The anti-peptic ulcer agent according to claim 6, wherein the plant of the family Araceae, belonging to the genus Araceae, is Araceae. (20) The anti-peptic ulcer 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-peptic ulcer 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-peptic ulcer agent as described. (23) The anti-peptic ulcer agent according to claim 21, wherein the plant of the Fabaceae family, Phaseolus genus, is azuki bean. (24) The anti-peptic ulcer agent according to claim 21, wherein the plant of the Fabaceae family, Faba genus, is broad bean. (25) The anti-peptic ulcer agent according to claim 21, wherein the plant of the genus Kudzu of the family Fabaceae is kudzu. (26) The anti-peptic ulcer agent according to claim 21, wherein the plant of the Fabaceae family, Licorice genus is Glycyrrhiza. (27) The anti-peptic ulcer agent according to claim 21, wherein the Solanaceae plant is selected from the group consisting of potato, chili pepper, and mixtures thereof. (28) Claim 2, wherein the plant of the genus Tomato of the family Solanaceae is a tomato.
The anti-peptic ulcer agent according to 1. (29) A plant of the Capsicum genus of the Solanaceae family is Capsicum,
The anti-peptic ulcer agent according to claim 21. (30) The anti-peptic ulcer 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-peptic ulcer agent as described. (32) Avocado is a plant of the genus Avocado of the Lauraceae family,
The anti-peptic ulcer agent according to claim 21. (33) The anti-peptic ulcer agent according to claim 21, wherein the plant of the family Walnut family, genus Walnut, is walnut. (34) The anti-peptic ulcer agent according to claim 21, wherein the plant of the Cucurbitaceae family, genus Tonus, is pumpkin. (35) The anti-peptic ulcer agent according to claim 21, wherein the plant of the genus Jiaogulan of the Cucurbitaceae family is Jiaogulan. (36) The anti-peptic ulcer 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 anti-peptic ulcer agent according to claim 21. (38) A plant of the genus Dokudami of the family Dokudamiaceae is Dokudami,
The anti-peptic ulcer agent according to claim 21. (39) The anti-peptic ulcer agent according to claim 21, wherein the plant of the genus Piper of the family Piperaceae is pepper. (40) The anti-peptic ulcer agent according to claim 21, wherein the plant of the genus Shikimi in the family Shikimi family is Rhizophyllaceae. (41) A plant of the genus Chrysanthemum of the family Chrysanthemum is a stinging owl,
The anti-peptic ulcer agent according to claim 21. (42) The anti-peptic ulcer agent according to claim 21, wherein the plant of the Rutaceae family, Rutaceae, genus Rutaceae is Lamium daisies. (43) The anti-peptic ulcer agent according to claim 21, wherein the plant of the genus Panax of the family Araliaceae is Panax ginseng. (44) The anti-peptic ulcer agent according to claim 21, wherein the plant of the genus Saposchnicobia of the family Umbelliferae is Bofu. (45) The anti-peptic ulcer agent according to claim 21, wherein the plant of the genus Ophthalmia of the family Ophthalmiaceae is Ophthalmia occidentalis. (46) The anti-peptic ulcer agent according to claim 21, wherein the plant of the family Rubiaceae, belonging to the genus Rubiaceae, is Uncaria hirsuta. (47) The anti-peptic ulcer 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 Helium of the family Heliumaceae, and a mixture thereof. (48) The anti-peptic ulcer 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 anti-peptic ulcer agent according to claim 47. (50) The grass plant is a grass plant, a red grass plant,
The anti-peptic ulcer 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-peptic ulcer agent as described. (52) The anti-peptic ulcer agent according to claim 51, wherein the plant of the genus Wakame of the family Laminariaceae is wakame. (53) The anti-peptic ulcer agent according to claim 51, wherein the plant of the Laminaria family, Laminaria genus is Laminaria laminaria. (54) The anti-peptic ulcer agent according to claim 51, wherein the plant of the genus Hijiki of the Sargassum family is Hijiki. (55) The anti-peptic ulcer agent according to claim 50, wherein the ruboraceae plant is a plant of the genus Porphyra of the family Prunusaceae. (56) The anti-peptic ulcer agent according to claim 55, wherein the plant of the genus Porphyra of the family Ceramicaceae is Prunus elegans. (57) The anti-peptic ulcer agent according to claim 50, wherein the green grass plant is a Chlorella plant of the family Oocystis family. (58) The anti-peptic ulcer agent according to claim 57, wherein the plant of the genus Chlorella of the family Oocystis family is chlorella. (59) The anti-peptic ulcer 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-peptic ulcer 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-peptic ulcer agent as described. (62) The anti-peptic ulcer agent according to claim 61, wherein the plant of the genus Pyrophylla of the family Oysteraceae is a shiitake mushroom. (63) The anti-peptic ulcer agent according to claim 61, wherein the plant of the genus Enokitake of the family Asteraceae is Enoki mushroom. (64) The anti-peptic ulcer 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-peptic ulcer agent according to claim 61. (66) The anti-peptic ulcer agent according to claim 61, wherein the plant of the genus Polyporus of the family Salmonaceae is an abalone mushroom. (67) The anti-peptic ulcer agent according to claim 61, wherein the plant of the family Agaricaceae, genus Agaricaceae, is a mushroom. (68) A plant of the genus Fungus of the family Anoraceae is a fungus.
The anti-peptic ulcer agent according to claim 61. (69) A plant of the genus Sugitake of the family Moegitake is nameko.
The anti-peptic ulcer agent according to claim 61. (70) The anti-peptic ulcer agent according to claim 60, wherein the ascomycete 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-peptic ulcer agent according to claim 70, which is baker's yeast, brewer's yeast, or a mixture thereof. (72) The anti-peptic ulcer agent according to claim 70, wherein the plant of the genus Cordyceps of the family Buccaneaceae is Cordyceps sinensis. (73) The anti-peptic ulcer 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-peptic ulcer 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) Number of phosphorus = 12 / molecular weight 30,000 Number of hexosamine = 45 ± 6 / molecular weight 30,000 Number of fatty acids = 18 / molecular weight 30,000 Number of KDO = 5 ± 1 / molecular weight 75. The anti-peptic ulcer 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-peptic ulcer agent for animals containing LPS, and 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 Macrophage activation, where the macrophage activation ability that gives the amount of TNF production by macrophages when LPS is not added is 0%, and the macrophage activation ability of LPS that gives the maximum constant amount of TNF production by macrophages is 100%. capacity (%), 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_8 of macrophage activation ability is 0.4 to 100 ng/ml of culture solution. Anti-peptic ulcer agent for animals.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2155429A JPH0449240A (en) | 1990-06-15 | 1990-06-15 | Antipeptic ulcer agent and antipeptic ulcer agent for animal |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2155429A JPH0449240A (en) | 1990-06-15 | 1990-06-15 | Antipeptic ulcer agent and antipeptic ulcer agent for animal |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0449240A true JPH0449240A (en) | 1992-02-18 |
Family
ID=15605823
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2155429A Pending JPH0449240A (en) | 1990-06-15 | 1990-06-15 | Antipeptic ulcer agent and antipeptic ulcer agent for animal |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0449240A (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP0616810A1 (en) * | 1993-02-19 | 1994-09-28 | SOKEN Co., Ltd. | Anti-cancer agent |
| EP0620007A1 (en) * | 1993-02-16 | 1994-10-19 | SOKEN Co., Ltd. | Anti-ulcer agent |
| JP2006124370A (en) * | 2004-09-28 | 2006-05-18 | Okumoto Seifun Kk | Anti-ulcer agent |
| JP2010150216A (en) * | 2008-12-26 | 2010-07-08 | Shiseido Co Ltd | Stress hormone action demulcent |
| US8075928B2 (en) | 2003-09-26 | 2011-12-13 | Gen-Ichiro Soma | Method for fermentation and cultivation, fermented plant extract, fermented plant extract powder, and composition containing the extract of fermented plant |
| JP2013136787A (en) * | 2005-11-28 | 2013-07-11 | Genichiro Soma | Lipopolysaccharide, method for producing lipopolysaccharide, and lipopolysaccharide composition |
| JP2018044038A (en) * | 2016-09-12 | 2018-03-22 | 有限会社バイオメディカルリサーチグループ | Lipopolysaccharides, lipopolysaccharide production method and lipopolysaccharide compound |
| CN109331050A (en) * | 2018-12-07 | 2019-02-15 | 广东省林业科学研究院 | Application of the halogen fern water extract in preparation treatment acute gastric ulcer drug |
-
1990
- 1990-06-15 JP JP2155429A patent/JPH0449240A/en active Pending
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| EP0620007A1 (en) * | 1993-02-16 | 1994-10-19 | SOKEN Co., Ltd. | Anti-ulcer agent |
| EP0616810A1 (en) * | 1993-02-19 | 1994-09-28 | SOKEN Co., Ltd. | Anti-cancer agent |
| US8075928B2 (en) | 2003-09-26 | 2011-12-13 | Gen-Ichiro Soma | Method for fermentation and cultivation, fermented plant extract, fermented plant extract powder, and composition containing the extract of fermented plant |
| EP2444480A1 (en) | 2003-09-26 | 2012-04-25 | Gen-Ichiro Soma | Method for fermentation and cultivation, fermented plant extract, fermented plant extract powder, and composition containing the extract of fermented plant |
| US9394513B2 (en) | 2003-09-26 | 2016-07-19 | Gen-Ichiro Soma | Method for fermentation and cultivation, fermented plant extract, fermented plant extract powder, and composition containing the extract of fermented plant |
| JP2006124370A (en) * | 2004-09-28 | 2006-05-18 | Okumoto Seifun Kk | Anti-ulcer agent |
| JP2013136787A (en) * | 2005-11-28 | 2013-07-11 | Genichiro Soma | Lipopolysaccharide, method for producing lipopolysaccharide, and lipopolysaccharide composition |
| JP2016222923A (en) * | 2005-11-28 | 2016-12-28 | 源一郎 杣 | Lipopolysaccharide, method for producing lipopolysaccharide, and lipopolysaccharide formulation |
| US10017727B2 (en) | 2005-11-28 | 2018-07-10 | Gen-Ichiro Soma | Method for fermentation and culture, fermented plant extract, fermented plant extract composition, method for producing lipopolysaccharide and lipopolysaccharide |
| JP2010150216A (en) * | 2008-12-26 | 2010-07-08 | Shiseido Co Ltd | Stress hormone action demulcent |
| JP2018044038A (en) * | 2016-09-12 | 2018-03-22 | 有限会社バイオメディカルリサーチグループ | Lipopolysaccharides, lipopolysaccharide production method and lipopolysaccharide compound |
| CN109331050A (en) * | 2018-12-07 | 2019-02-15 | 广东省林业科学研究院 | Application of the halogen fern water extract in preparation treatment acute gastric ulcer drug |
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