JP4863870B2 - Antiallergic agent - Google Patents
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Description
本発明は、抗アレルギー剤に関する。本発明の抗アレルギー剤は、医薬品として投与することができるだけでなく、種々の形態、例えば、健康食品(好ましくは機能性食品)又は飼料として飲食物の形で与えることも可能である。なお、前記食品には飲料が含まれる。更には、オーラル衛生用組成物、例えば、口中に一時的に含むものの、そのほとんどを口中より吐き出す形態、例えば、歯磨剤、洗口剤、チューインガム、又はうがい剤の形で与えることも、あるいは、鼻から吸引させる吸入剤の形で与えることも可能である。 The present invention relates to an antiallergic agent. The antiallergic agent of the present invention can be administered not only as a pharmaceutical product, but also in various forms such as health food (preferably functional food) or feed as food and drink. The food includes a beverage. Furthermore, an oral hygiene composition, for example, temporarily contained in the mouth, but most of it is given in the form of exhaling from the mouth, for example, in the form of a dentifrice, mouthwash, chewing gum, or mouthwash, or It is also possible to give in the form of an inhalant to be aspirated from the nose.
多孔菌科のカワラタケ属に属する担子菌には種々の生理活性物質が含まれていることが知られている。例えば、特許文献1には、カワラタケ属に属するカワラタケに含有される糖タンパク質複合体が抗腫瘍作用、免疫調節作用、及び増殖因子阻害作用を有することが開示されている。また、特許文献2には、多孔菌科に属する菌、例えば、カワラタケが抗腫瘍性成分を含むことが開示されている。
本発明者は、カワラタケ属に属する担子菌に含まれる生理活性物質を見出すことを目的に鋭意研究したところ、カワラタケ由来画分が抗アレルギー活性を示すことを見出し、更に、その活性成分が2,3,8,9−テトラヒドロキシベンゾビス[1,2−b,4,5−b’]ベンゾフラン−6,12−キノン(2,3,8,9-Tetrahydroxybenzobis[1,2-b,4,5-b’]benzofuran-6,12-quinone;以下、テレホール酸と称する)であることを見出した。本発明は、このような知見に基づくものである。
本発明の課題は、新規の抗アレルギー剤を提供することにある。The present inventor has intensively studied for the purpose of finding a physiologically active substance contained in basidiomycetes belonging to the genus Kawaratake, and found that the fraction derived from Kawaratake exhibits anti-allergic activity. 3,8,9-tetrahydroxybenzobis [1,2-b, 4,5-b ′] benzofuran-6,12-quinone (2,3,8,9-Tetrahydroxybenzobis [1,2-b, 4, 5-b '] benzofuran-6,12-quinone; hereinafter referred to as teleholic acid). The present invention is based on such knowledge.
An object of the present invention is to provide a novel antiallergic agent.
前記課題は、本発明による、(1)一般式(I):
で表されるテレホール酸化合物若しくはその塩、あるいは、前記テレホール酸化合物若しくはその塩とタンパク質との結合物、
(2)(a)カワラタケ属に属する担子菌のアルカリ水溶液抽出液を酸性に調整することにより得られる、アルカリ水溶液抽出液の酸性条件下沈殿画分(以下、画分F1と称することがある)、
(b)前記画分(a)を極性有機溶媒存在下で還流して得られる、極性有機溶媒抽出画分(以下、画分F2と称することがある)、
(c)前記画分(b)から熱水可溶部及び非極性有機溶媒可溶部を除去して得られる、熱水可溶部及び非極性有機溶媒可溶部除去画分(以下、画分F3と称することがある)、
(d)前記画分(c)をアセチル化して得られる、アセチル化画分(又はアセチル化物)(以下、画分F4と称することがある)、
(e)前記画分(d)から酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部を除去して得られる、酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部除去画分(以下、画分F5と称することがある)、
(f)前記画分(e)を脱アセチル化して得られる、脱アセチル化画分(又は脱アセチル化物)(以下、画分F6と称することがある)、若しくは
(g)前記画分(f)の有機溶媒抽出画分(以下、画分F7と称することがある)、又は(3)(h)カワラタケ属に属する担子菌を極性有機溶媒により還流抽出した後、得られた極性有機溶媒抽出画分から非極性有機溶媒可溶部及び熱水可溶部を除去して得られる、極性有機溶媒抽出画分の熱水可溶部及び非極性有機溶媒可溶部除去画分(以下、画分F8と称することがある)、
(i)前記画分(h)をアセチル化して得られる、アセチル化画分(又はアセチル化物)(以下、画分F9と称することがある)、
(j)前記画分(h)から酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部を除去して得られる、酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部除去画分(以下、画分F10と称することがある)、
(k)前記画分(j)を脱アセチル化して得られる、脱アセチル化画分(又は脱アセチル化物)(以下、画分F11と称することがある)、若しくは
(l)前記画分(k)の有機溶媒抽出画分(以下、画分F12と称することがある)、又は
(4)(m)前記画分(a)〜(l)の少なくとも1つの画分と、タンパク質との結合物を有効成分として含有することを特徴とする、抗アレルギー剤により解決することができる。The object is achieved by the present invention in accordance with (1) the general formula (I):
A teleholic acid compound or a salt thereof represented by the above, or a conjugate of the teleholic acid compound or a salt thereof and a protein,
(2) (a) Precipitation fraction under acidic conditions of an aqueous alkaline solution obtained by adjusting the aqueous alkaline solution of a basidiomycete belonging to the genus Kawaratake (hereinafter sometimes referred to as fraction F1). ,
(B) a polar organic solvent extraction fraction obtained by refluxing the fraction (a) in the presence of a polar organic solvent (hereinafter sometimes referred to as fraction F2),
(C) Hot water soluble part and nonpolar organic solvent soluble part removal fraction (hereinafter referred to as fraction) obtained by removing the hot water soluble part and nonpolar organic solvent soluble part from the fraction (b). Sometimes referred to as minute F3),
(D) an acetylated fraction (or acetylated product) obtained by acetylating the fraction (c) (hereinafter sometimes referred to as fraction F4),
(E) an acidic aqueous solution soluble part, an alkaline aqueous solution soluble part obtained by removing an acidic aqueous solution soluble part, an alkaline aqueous solution soluble part, and a nonpolar organic solvent soluble part from the fraction (d), and Nonpolar organic solvent soluble part removal fraction (hereinafter sometimes referred to as fraction F5),
(F) a deacetylated fraction (or deacetylated product) (hereinafter sometimes referred to as fraction F6) obtained by deacetylating the fraction (e), or (g) the fraction (f ) Organic solvent extraction fraction (hereinafter sometimes referred to as Fraction F7), or (3) (h) basidiomycete belonging to the genus Kawaratake is extracted by refluxing with a polar organic solvent, and the obtained polar organic solvent extraction The fraction obtained by removing the non-polar organic solvent soluble part and the hot water soluble part from the fraction, and the fraction obtained by removing the hot water soluble part and the non-polar organic solvent soluble part of the polar organic solvent extraction fraction (hereinafter referred to as fraction). Sometimes called F8),
(I) an acetylated fraction (or an acetylated product) obtained by acetylating the fraction (h) (hereinafter sometimes referred to as fraction F9),
(J) An acidic aqueous solution soluble part, an alkaline aqueous solution soluble part obtained by removing an acidic aqueous solution soluble part, an alkaline aqueous solution soluble part, and a nonpolar organic solvent soluble part from the fraction (h), and Nonpolar organic solvent soluble part removal fraction (hereinafter sometimes referred to as fraction F10),
(K) a deacetylated fraction (or deacetylated product) (hereinafter sometimes referred to as fraction F11) obtained by deacetylating the fraction (j), or (l) the fraction (k ) Organic solvent extract fraction (hereinafter also referred to as fraction F12), or (4) (m) at least one fraction of the fractions (a) to (l) and a conjugate of protein Can be solved by an antiallergic agent characterized in that it contains as an active ingredient.
また、本発明は、
(a)カワラタケ属に属する担子菌のアルカリ水溶液抽出液を酸性に調整することにより得られる、アルカリ水溶液抽出液の酸性条件下沈殿画分、
(b)前記画分(a)を極性有機溶媒存在下で還流して得られる、極性有機溶媒抽出画分、
(c)前記画分(b)から熱水可溶部及び非極性有機溶媒可溶部を除去して得られる、熱水可溶部及び非極性有機溶媒可溶部除去画分、
(d)前記画分(c)をアセチル化して得られる、アセチル化画分(又はアセチル化物)、
(e)前記画分(d)から酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部を除去して得られる、酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部除去画分、
(f)前記画分(e)を脱アセチル化して得られる、脱アセチル化画分(又は脱アセチル化物)、
(g)前記画分(f)の有機溶媒抽出画分、
(h)カワラタケ属に属する担子菌を極性有機溶媒により還流抽出した後、得られた極性有機溶媒抽出画分から非極性有機溶媒可溶部及び熱水可溶部を除去して得られる、極性有機溶媒抽出画分の熱水可溶部及び非極性有機溶媒可溶部除去画分、
(i)前記画分(h)をアセチル化して得られる、アセチル化画分(又はアセチル化物)、
(j)前記画分(h)から酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部を除去して得られる、酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部除去画分、
(k)前記画分(j)を脱アセチル化して得られる、脱アセチル化画分(又は脱アセチル化物)、若しくは
(l)前記画分(k)の有機溶媒抽出画分、又は
(m)前記画分(a)〜(l)の少なくとも1つの画分と、タンパク質との結合物
に関する。The present invention also provides:
(A) a precipitate fraction under acidic conditions of an aqueous alkaline solution obtained by adjusting the aqueous alkaline solution of a basidiomycete belonging to the genus Kawaratake
(B) a polar organic solvent extraction fraction obtained by refluxing the fraction (a) in the presence of a polar organic solvent,
(C) A hot water soluble part and a nonpolar organic solvent soluble part removal fraction obtained by removing a hot water soluble part and a nonpolar organic solvent soluble part from the fraction (b),
(D) an acetylated fraction (or acetylated product) obtained by acetylating the fraction (c),
(E) an acidic aqueous solution soluble part, an alkaline aqueous solution soluble part obtained by removing an acidic aqueous solution soluble part, an alkaline aqueous solution soluble part, and a nonpolar organic solvent soluble part from the fraction (d), and Non-polar organic solvent soluble part removal fraction,
(F) a deacetylated fraction (or deacetylated product) obtained by deacetylating the fraction (e),
(G) an organic solvent extraction fraction of the fraction (f),
(H) A polar organic compound obtained by reflux-extracting basidiomycetes belonging to the genus Kawaratake with a polar organic solvent and then removing the nonpolar organic solvent soluble part and the hot water soluble part from the obtained polar organic solvent extract fraction Hot water soluble part and nonpolar organic solvent soluble part removal fraction of solvent extraction fraction,
(I) an acetylated fraction (or acetylated product) obtained by acetylating the fraction (h),
(J) An acidic aqueous solution soluble part, an alkaline aqueous solution soluble part obtained by removing an acidic aqueous solution soluble part, an alkaline aqueous solution soluble part, and a nonpolar organic solvent soluble part from the fraction (h), and Non-polar organic solvent soluble part removal fraction,
(K) a deacetylated fraction (or deacetylated product) obtained by deacetylating the fraction (j), or (l) an organic solvent extract fraction of the fraction (k), or (m) The present invention relates to a bound product of at least one of the fractions (a) to (l) and a protein.
また、本発明は、前記画分(a)〜(l)又は結合物(m)の少なくとも1つと、薬剤学的又は獣医学的に許容することのできる担体又は希釈剤とを含有する、医薬組成物に関する。
また、本発明は、前記物質(1)、画分(a)〜(l)、又は結合物(m)の少なくとも1つを、アレルギー抑制の必要な対象に、有効量で投与することを含む、アレルギー抑制方法に関する。
また、本発明は、前記物質(1)、画分(a)〜(l)、又は結合物(m)の少なくとも1つを、アレルギーに関連する疾病の治療又は予防の必要な対象に、有効量で投与することを含む、前記疾病の治療又は予防方法に関する。
また、本発明は、前記物質(1)、画分(a)〜(l)、又は結合物(m)の少なくとも1つの、抗アレルギー剤を製造するための使用に関する。The present invention also provides a pharmaceutical comprising at least one of the fractions (a) to (l) or the conjugate (m) and a pharmaceutically or veterinarily acceptable carrier or diluent. Relates to the composition.
Further, the present invention includes administering at least one of the substance (1), fractions (a) to (l), or the conjugate (m) in an effective amount to a subject in need of allergy suppression. , Allergy control method.
In addition, the present invention is effective for at least one of the substances (1), fractions (a) to (l), or the conjugate (m) for a subject in need of treatment or prevention of a disease associated with allergy. The present invention relates to a method for treating or preventing the above-mentioned diseases, which comprises administering in an amount.
The present invention also relates to the use of at least one of the substances (1), fractions (a) to (l) or the conjugate (m) for producing an antiallergic agent.
本発明の抗アレルギー剤によれば、アレルギーを抑制することができる。従って、本発明の抗アレルギー剤は、アレルギーに関連する疾病の治療及び/又は予防に有用である。 According to the antiallergic agent of the present invention, allergy can be suppressed. Therefore, the antiallergic agent of the present invention is useful for the treatment and / or prevention of diseases related to allergy.
本発明の抗アレルギー剤は、有効成分として、(1)一般式(I):
で表されるテレホール酸化合物若しくはその塩、あるいは、前記テレホール酸化合物若しくはその塩とタンパク質との結合物、又は(2)カワラタケ属に属する担子菌に由来する本発明の各種画分(F1〜F12)の少なくとも1つ、あるいは、前記画分F1〜F12の少なくとも1つの画分とタンパク質との結合物を含有する。The antiallergic agent of the present invention comprises (1) general formula (I):
Or a combination of the teleholic acid compound or a salt thereof and a protein, or (2) various fractions of the present invention derived from a basidiomycete belonging to the genus Kawaratake (F1 to F12) Or at least one fraction of the fractions F1 to F12 and a bound product of protein.
本発明の抗アレルギー剤における有効成分の1つであるテレホール酸は、一般式(I)においてR1、R2、R3、及びR4が全て水素原子である化合物である。テレホール酸は、例えば、J. Gripenberg, Tetrahedron, vol.10, pp. 135-143 (1960)に記載の方法により化学合成することもできるし、あるいは、天然のキノコ又は人工培養の菌糸体から分離することもできる。テレホール酸を含むキノコとしては、例えば、カワラタケ
(Fr.) Karst.)、又はシシタケ(Sarcodon imbricatum (L. ex Fr.) Karst.)]を挙げることができる。Teleholic acid, which is one of the active ingredients in the antiallergic agent of the present invention, is a compound in which R 1 , R 2 , R 3 , and R 4 are all hydrogen atoms in general formula (I). Teleholic acid can be chemically synthesized by, for example, the method described in J. Gripenberg, Tetrahedron, vol. 10, pp. 135-143 (1960), or separated from natural mushrooms or mycelium of artificial culture. You can also Examples of mushrooms containing teleholic acid include Kawaratake
(Fr.) Karst.), Or Shishitake (Sarcodon imbricatum (L. ex Fr.) Karst.)] Can be exemplified.
テレホール酸の溶解性は、
ピリジン、ジメチルスルホキシド(DMSO)、又はジオキサン>アセトン>メタノール、エタノール、酢酸エチル>エーテル(例えば、ジエチルエーテル)、エステル類>
水、ベンゼン、ヘキサン、四塩化炭素
であり、テレホール酸のアセチル化体の溶解性は、ピリジン、DMSO、又はジオキサンに溶解するが、それ以外の主要な溶媒には不溶である。The solubility of teleholic acid is
Pyridine, dimethyl sulfoxide (DMSO), or dioxane>acetone> methanol, ethanol, ethyl acetate> ether (for example, diethyl ether), esters>
Water, benzene, hexane, and carbon tetrachloride. The solubility of the acetylated form of teleholic acid is soluble in pyridine, DMSO, or dioxane, but insoluble in other major solvents.
一般式(I)における炭素数1〜4の低級アルキル基には、直鎖状アルキル基及び分枝状アルキル基が含まれ、例えば、メチル基、エチル基、n−プロピル基、イソプロピル基、n−ブチル基、イソブチル基、sec−ブチル基、又はtert−ブチル基を挙げることができ、メチル基又はエチル基が好ましい。 The lower alkyl group having 1 to 4 carbon atoms in the general formula (I) includes a linear alkyl group and a branched alkyl group. For example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, n -A butyl group, an isobutyl group, a sec-butyl group, or a tert- butyl group can be mentioned, A methyl group or an ethyl group is preferable.
本発明における有効成分の1つであるテレホール酸の塩は、薬剤学的に許容される塩である限り、特に限定されるものではないが、例えば、無機塩基又は有機塩基との塩を挙げることができる。塩の形成に適した無機塩基は、例えば、アンモニア、カリウム、ナトリウム、リチウム、カルシウム、マグネシウム、又はアルミニウム等の水酸化物、炭酸塩、又は重炭酸塩等である。有機塩基との塩としては、例えば、メチルアミン、ジメチルアミン、トリエチルアミンのようなモノ−、ジ−、若しくはトリ−アルキルアミン塩、モノ−、ジ−、若しくはトリ−ヒドロキシアルキルアミン塩、グアニジン塩、N−メチルグルコサミン塩、又はアミノ酸塩等を挙げることができる。 The salt of teleholic acid, which is one of the active ingredients in the present invention, is not particularly limited as long as it is a pharmaceutically acceptable salt. Examples thereof include salts with inorganic bases or organic bases. Can do. Inorganic bases suitable for salt formation are, for example, hydroxides such as ammonia, potassium, sodium, lithium, calcium, magnesium, or aluminum, carbonates, bicarbonates, and the like. Examples of salts with organic bases include mono-, di-, or tri-alkylamine salts such as methylamine, dimethylamine, and triethylamine, mono-, di-, or tri-hydroxyalkylamine salts, guanidine salts, N-methylglucosamine salt, amino acid salt, etc. can be mentioned.
本発明において抗アレルギー剤における有効成分として用いることのできる、カワラタケ属に属する担子菌に由来する画分F1〜F7及び画分F8〜F12は、それ自体、新規物質である。
カワラタケ属に属する担子菌としては、例えば、カワラタケ
Examples of basidiomycetes belonging to the genus Kawaratake include, for example, Kawaratake
本発明で用いるカワラタケ属に属する担子菌(好ましくはカワラタケ)としては、例えば、天然の担子菌の子実体若しくは菌糸体、又は培養により得られる担子菌の菌糸体若しくは培養物(Broth)を挙げることができる。前記担子菌として子実体又は菌糸体を用いる場合には、抽出効率が向上するように、破砕物又は粉体の状態に加工することが好ましい。 Examples of basidiomycetes (preferably Kawaratake) belonging to the genus Kawaratake used in the present invention include the fruit bodies or mycelia of natural basidiomycetes, or mycelia or cultures (Broth) of basidiomycetes obtained by culturing. Can do. When a fruit body or mycelium is used as the basidiomycete, it is preferably processed into a crushed material or powder so as to improve extraction efficiency.
本発明の酸性条件下沈殿画分(すなわち、カワラタケ属に属する担子菌のアルカリ水溶液抽出液を酸性に調整する工程により得られる、酸性条件下沈殿画分;画分F1)は、カワラタケ属に属する担子菌をアルカリ水溶液で抽出し、得られたアルカリ水溶液抽出液を酸性に調整し、酸性条件で放置することにより得ることができる。前記アルカリ水溶液としては、例えば、飽和ホウ酸ナトリウム水溶液を用いることができる。アルカリ水溶液による抽出は、例えば、pH8〜14のアルカリ条件下で、0〜100℃にて0.5〜72時間行うことができる。前記酸性条件は、例えば、pH1〜6とすることができ、例えば、0〜100℃にて0.5〜72時間放置した後、遠心分離することにより、沈殿を回収することができる。前記遠心分離は、例えば、10,000〜50,000×gで10〜30分間実施することができる。得られた沈殿は、適当な洗浄液(例えば水、好ましくは蒸留水)を用いて、洗浄液が中性(pH7)になるまで洗浄を繰り返すことが好ましい。
The precipitate fraction under acidic conditions of the present invention (that is, the precipitate fraction under acidic conditions obtained by the step of adjusting the alkaline aqueous solution extract of basidiomyceae belonging to the genus Kawaratake; fraction F1) belongs to the genus Kawaratake. The basidiomycete can be extracted with an alkaline aqueous solution, and the resulting alkaline aqueous solution extract is adjusted to be acidic and left under acidic conditions. As the alkaline aqueous solution, for example, a saturated sodium borate aqueous solution can be used. Extraction with an aqueous alkali solution can be performed, for example, at 0 to 100 ° C. for 0.5 to 72 hours under alkaline conditions of
本発明の極性有機溶媒抽出画分(すなわち、カワラタケ属に属する担子菌のアルカリ水溶液抽出液を酸性に調整する工程、及び前記工程により得られる酸性条件下沈殿画分を極性有機溶媒存在下で還流する工程により得られる、極性有機溶媒抽出画分;画分F2)は、前記酸性条件下沈殿画分に、極性有機溶媒を添加し、還流することにより得ることができる。前記極性有機溶媒は、水溶性の性質を示すものである限り、特に限定されるものではないが、例えば、アセトン、メタノール、エタノール、酢酸エチル、又はジメチルエーテルを用いることができ、アセトンが好ましい。還流は、有機溶媒抽出液の色が消えるまで実施することが好ましく、例えば、50〜150℃にて1〜24時間実施することができる。本発明の極性有機溶媒抽出画分には、還流した後の抽出液と、その乾燥物の両方が含まれる。 Extraction fraction of polar organic solvent of the present invention (that is, the step of adjusting the alkaline aqueous solution extract of basidiomycete belonging to the genus Kawaratake to acidity, and refluxing the precipitate fraction obtained by the above step in the presence of the polar organic solvent) The polar organic solvent extraction fraction; fraction F2) obtained by the above step can be obtained by adding a polar organic solvent to the precipitate fraction under the acidic condition and refluxing. The polar organic solvent is not particularly limited as long as it exhibits water-soluble properties. For example, acetone, methanol, ethanol, ethyl acetate, or dimethyl ether can be used, and acetone is preferable. The refluxing is preferably carried out until the color of the organic solvent extract disappears, and can be carried out, for example, at 50 to 150 ° C. for 1 to 24 hours. The polar organic solvent extraction fraction of the present invention includes both the extract after reflux and its dried product.
本発明の熱水可溶部及び非極性有機溶媒可溶部除去画分(すなわち、カワラタケ属に属する担子菌のアルカリ水溶液抽出液を酸性に調整する工程、前記工程により得られる酸性条件下沈殿画分を極性有機溶媒存在下で還流する工程、並びに前記工程により得られる極性有機溶媒抽出画分から熱水可溶部及び非極性有機溶媒可溶部を除去する工程により得られる、熱水可溶部及び非極性有機溶媒可溶部除去画分;画分F3)は、例えば、前記極性有機溶媒抽出画分と熱水とを接触させた後、熱水可溶部を、例えば、遠心分離により除去し、続いて、得られた熱水可溶部除去画分と非極性有機溶媒とを接触させた後、非極性有機溶媒可溶部を、例えば、遠心分離により除去することにより得ることができる。熱水との接触と非極性有機溶媒との接触は、この順序で実施することもできるし、逆の順序で実施することもできる。 Fraction removing hot water soluble part and non-polar organic solvent soluble part of the present invention (that is, a step of adjusting the aqueous alkaline solution extract of basidiomyceae belonging to the genus Kawaratake to acidic, a precipitate under acidic conditions obtained by the step A hydrothermally soluble part obtained by a step of refluxing the fraction in the presence of a polar organic solvent, and a process of removing the hot water soluble part and the nonpolar organic solvent soluble part from the polar organic solvent extraction fraction obtained by the process And non-polar organic solvent soluble part removal fraction; fraction F3), for example, after contacting the polar organic solvent extraction fraction and hot water, the hot water soluble part is removed, for example, by centrifugation Subsequently, after contacting the obtained hot water soluble part removal fraction and the nonpolar organic solvent, the nonpolar organic solvent soluble part can be obtained, for example, by removing by centrifugation. . The contact with hot water and the contact with the nonpolar organic solvent can be carried out in this order, or can be carried out in the reverse order.
熱水との接触は、例えば、50〜100℃で10〜60分間実施することができる。熱水との接触は、5〜10回繰り返すことが好ましい。
非極性有機溶媒との接触は、例えば、10〜60分間実施することができる。前記非極性有機溶媒としては、例えば、クロロホルム、ヘキサン、ベンゼン、四塩化炭素、又はメチレンクロライドを用いることができる。非極性有機溶媒との接触は、1つの非極性有機溶媒に関して、5〜10回繰り返すことが好ましい。また、1種類の非極性有機溶媒だけを用いることもできるし、異なる非極性有機溶媒を順次用いることもできる。Contact with hot water can be carried out, for example, at 50 to 100 ° C. for 10 to 60 minutes. The contact with hot water is preferably repeated 5 to 10 times.
The contact with the nonpolar organic solvent can be carried out, for example, for 10 to 60 minutes. As the nonpolar organic solvent, for example, chloroform, hexane, benzene, carbon tetrachloride, or methylene chloride can be used. The contact with the nonpolar organic solvent is preferably repeated 5 to 10 times for one nonpolar organic solvent. Moreover, only one type of nonpolar organic solvent can be used, or different nonpolar organic solvents can be used sequentially.
本発明のアセチル化画分(又はアセチル化物)[すなわち、カワラタケ属に属する担子菌のアルカリ水溶液抽出液を酸性に調整する工程、前記工程により得られる酸性条件下沈殿画分を極性有機溶媒存在下で還流する工程、前記工程により得られる極性有機溶媒抽出画分から熱水可溶部及び非極性有機溶媒可溶部を除去する工程、並びに前記工程により得られる熱水可溶部及び非極性有機溶媒可溶部除去画分をアセチル化する工程により得られる、アセチル化画分(又はアセチル化物);画分F4]は、前記熱水可溶部及び非極性有機溶媒可溶部除去画分をアセチル化することにより得ることができる。前記アセチル化は、例えば、前記熱水可溶部及び非極性有機溶媒可溶部除去画分に、無水酢酸と適当な有機溶媒[例えば、ピリジン、ジメチルスルホキシド(DMSO)、又はジオキサン]を添加し、0〜100℃にて1〜24時間加熱することにより実施することができる。 The acetylated fraction (or acetylated product) of the present invention [that is, the step of adjusting the alkaline aqueous solution extract of basidiomycetes belonging to the genus Kawaratake to acidity, the precipitate fraction obtained under the acidic conditions obtained by the above step in the presence of a polar organic solvent In which the hot water soluble part and the nonpolar organic solvent soluble part are removed from the polar organic solvent extraction fraction obtained by the process, and the hot water soluble part and the nonpolar organic solvent obtained by the process The acetylated fraction (or acetylated product); fraction F4] obtained by the step of acetylating the soluble portion-removed fraction is obtained by acetylating the hot water soluble portion and the nonpolar organic solvent soluble portion removed fraction. Can be obtained. In the acetylation, for example, acetic anhydride and a suitable organic solvent [for example, pyridine, dimethyl sulfoxide (DMSO), or dioxane] are added to the hot water soluble part and the nonpolar organic solvent soluble part removal fraction. It can be carried out by heating at 0 to 100 ° C. for 1 to 24 hours.
本発明の酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部除去画分(すなわち、カワラタケ属に属する担子菌のアルカリ水溶液抽出液を酸性に調整する工程、前記工程により得られる酸性条件下沈殿画分を極性有機溶媒存在下で還流する工程、前記工程により得られる極性有機溶媒抽出画分から熱水可溶部及び非極性有機溶媒可溶部を除去する工程、前記工程により得られる熱水可溶部及び非極性有機溶媒可溶部除去画分をアセチル化する工程、並びに前記工程により得られるアセチル化画分から、酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部を除去する工程から得られる、酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部除去画分;画分F5)は、前記アセチル化画分と酸性水溶液とを接触させた後、酸性水溶液可溶部を除去し、続いて、得られた酸性水溶液可溶部除去画分とアルカリ水溶液とを接触させた後、アルカリ水溶液可溶部を除去し、更に、得られた酸性水溶液可溶部及びアルカリ水溶液可溶部除去画分と非極性有機溶媒とを接触させた後、非極性有機溶媒可溶部を除去することにより得ることができる。酸性水溶液、アルカリ水溶液、及び非極性有機溶媒との接触は、この順序で実施することもできるし、これ以外の任意の順序で実施することもできる。 The acidic aqueous solution soluble part, the alkaline aqueous solution soluble part, and the nonpolar organic solvent soluble part removal fraction of the present invention (that is, the step of adjusting the alkaline aqueous solution extract of basidiomycetes belonging to the genus Kawaratake to the acid, A step of refluxing a precipitate fraction in the presence of a polar organic solvent under acidic conditions, a step of removing a hot water-soluble portion and a nonpolar organic solvent-soluble portion from the polar organic solvent extraction fraction obtained by the step, and the step From the step of acetylating the hot water soluble part and the nonpolar organic solvent soluble part removal fraction obtained by the above, and from the acetylated fraction obtained by the above step, an acidic aqueous solution soluble part, an alkaline aqueous solution soluble part, The acidic aqueous solution soluble part, the alkaline aqueous solution soluble part, and the nonpolar organic solvent soluble part removal fraction obtained from the step of removing the polar organic solvent soluble part; fraction F5) is the acetylated fraction. After contacting with the acidic aqueous solution, the acidic aqueous solution soluble part is removed, and then the obtained acidic aqueous solution soluble part removing fraction and the alkaline aqueous solution are contacted, and then the alkaline aqueous solution soluble part is removed. Furthermore, after making the acidic aqueous solution soluble part and alkaline aqueous solution soluble part removal fraction and nonpolar organic solvent which were obtained contact, the nonpolar organic solvent soluble part can be removed. Contact with the acidic aqueous solution, the alkaline aqueous solution, and the nonpolar organic solvent can be performed in this order, or can be performed in any other order.
酸性水溶液との接触は、例えば、0〜100℃で10〜60分間実施することができる。前記酸性水溶液としては、例えば、塩酸、硫酸、リン酸、又は硝酸を用いることができ、その濃度は、0.5〜2Nであることができる。酸性水溶液との接触は、5〜10回繰り返すことが好ましい。 Contact with acidic aqueous solution can be implemented at 0-100 degreeC for 10 to 60 minutes, for example. As said acidic aqueous solution, hydrochloric acid, a sulfuric acid, phosphoric acid, or nitric acid can be used, for example, The density | concentration can be 0.5-2N. The contact with the acidic aqueous solution is preferably repeated 5 to 10 times.
アルカリ水溶液との接触は、例えば、0〜100℃で10〜60分間実施することができる。前記アルカリ水溶液としては、例えば、飽和炭酸水素ナトリウム水溶液、10〜20%炭酸ナトリウム水溶液、又は飽和炭酸水素カリウム水溶液を用いることができ、そのpHは7.5〜10であることができる。酸性水溶液との接触は、5〜10回繰り返すことが好ましい。 The contact with the alkaline aqueous solution can be carried out, for example, at 0 to 100 ° C. for 10 to 60 minutes. As the alkaline aqueous solution, for example, a saturated sodium hydrogen carbonate aqueous solution, a 10-20% sodium carbonate aqueous solution, or a saturated potassium hydrogen carbonate aqueous solution can be used, and its pH can be 7.5-10. The contact with the acidic aqueous solution is preferably repeated 5 to 10 times.
非極性有機溶媒との接触は、例えば、0〜100℃で10〜60分間実施することができる。前記非極性有機溶媒としては、例えば、クロロホルム、酢酸エチル、ヘキサン、ベンゼン、四塩化炭素、又はメチレンクロライドを用いることができる。非極性有機溶媒との接触は、1つの非極性有機溶媒に関して、5〜10回繰り返すことが好ましい。また、1種類の非極性有機溶媒だけを用いることもできるし、異なる非極性有機溶媒を順次用いることもできる。 The contact with the nonpolar organic solvent can be carried out, for example, at 0 to 100 ° C. for 10 to 60 minutes. As the nonpolar organic solvent, for example, chloroform, ethyl acetate, hexane, benzene, carbon tetrachloride, or methylene chloride can be used. The contact with the nonpolar organic solvent is preferably repeated 5 to 10 times for one nonpolar organic solvent. Moreover, only one type of nonpolar organic solvent can be used, or different nonpolar organic solvents can be used sequentially.
本発明の脱アセチル化画分(又は脱アセチル化物)(すなわち、カワラタケ属に属する担子菌のアルカリ水溶液抽出液を酸性に調整する工程、前記工程により得られる酸性条件下沈殿画分を極性有機溶媒存在下で還流する工程、前記工程により得られる極性有機溶媒抽出画分から熱水可溶部及び非極性有機溶媒可溶部を除去する工程、前記工程により得られる熱水可溶部及び非極性有機溶媒可溶部除去画分をアセチル化する工程、前記工程により得られるアセチル化画分から、酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部を除去する工程、並びに前記工程から得られる酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部除去画を脱アセチル化する工程から得られる、脱アセチル化画分(又は脱アセチル化物);画分F6)は、前記酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部除去画分を脱アセチル化することにより得ることができる。前記脱アセチル化は、例えば、前記酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部除去画分に、アルコキシド(例えば、ナトリウムメトキシド)を適当な有機溶媒(例えば、無水エタノール、ビリジン、DMSO、又はジオキサン)に溶解した溶液を添加し、70〜100℃にて1〜24時間撹拌することにより実施することができる。 Deacetylated fraction (or deacetylated product) of the present invention (that is, a step of adjusting the alkaline aqueous solution extract of basidiomycetes belonging to the genus Kawaratake to acid, the precipitate fraction obtained under the acidic conditions obtained by the step described above as a polar organic solvent The step of refluxing in the presence, the step of removing the hot water soluble part and the nonpolar organic solvent soluble part from the polar organic solvent extraction fraction obtained by the step, the hot water soluble part and the nonpolar organic obtained by the step The step of acetylating the solvent-soluble portion removal fraction, the step of removing the acidic aqueous solution-soluble portion, the alkaline aqueous solution-soluble portion, and the nonpolar organic solvent-soluble portion from the acetylated fraction obtained by the above-mentioned step, and Deacetylation fraction (or deaeration) obtained from the step of deacetylating the acidic aqueous solution soluble portion, alkaline aqueous solution soluble portion, and nonpolar organic solvent soluble portion removal fraction obtained from the step Chill products); Fraction F6), the acidic aqueous solution-soluble portion, alkaline aqueous solution-soluble portion, and a non-polar organic solvent-soluble portion removed fraction may be obtained by deacetylation. In the deacetylation, for example, an alkoxide (for example, sodium methoxide) is added to an appropriate organic solvent (for example, sodium methoxide) in the acidic aqueous solution soluble part, the alkaline aqueous solution soluble part, and the nonpolar organic solvent soluble part removal fraction. It can carry out by adding the solution melt | dissolved in absolute ethanol, pyridine, DMSO, or a dioxane and stirring at 70-100 degreeC for 1 to 24 hours.
本発明の有機溶媒抽出画分(すなわち、カワラタケ属に属する担子菌のアルカリ水溶液抽出液を酸性に調整する工程、前記工程により得られる酸性条件下沈殿画分を極性有機溶媒存在下で還流する工程、前記工程により得られる極性有機溶媒抽出画分から熱水可溶部及び非極性有機溶媒可溶部を除去する工程、前記工程により得られる熱水可溶部及び非極性有機溶媒可溶部除去画分をアセチル化する工程、前記工程により得られるアセチル化画分から、酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部を除去する工程、前記工程から得られる酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部除去画を脱アセチル化する工程、並びに前記工程から得られる脱アセチル化画分を有機溶媒で抽出する工程により得られる、有機溶媒抽出画分;画分F7)は、前記脱アセチル化画分と有機溶媒とを接触させ、有機溶媒相を回収することにより得ることができる。前記有機溶媒は、疎水性の性質を示すものである限り、特に限定されるものではないが、例えば、ビリジン、DMSO、又は酢酸エチルを用いることができる。有機溶媒との接触による抽出は、有機溶媒相に着色が見られなくなるまで実施することが好ましい。 The organic solvent extraction fraction of the present invention (that is, the step of adjusting the alkaline aqueous solution extract of basidiomyceae belonging to the genus Kawaratake to acidity, the step of refluxing the precipitate fraction obtained under the acidic conditions in the presence of a polar organic solvent) , The step of removing the hot water soluble part and the nonpolar organic solvent soluble part from the polar organic solvent extraction fraction obtained by the step, the hot water soluble part and the nonpolar organic solvent soluble part removal fraction obtained by the step The step of acetylating the fraction, the step of removing the acidic aqueous solution soluble part, the alkaline aqueous solution soluble part and the non-polar organic solvent soluble part from the acetylated fraction obtained by the above process, the acidic aqueous solution obtained from the above process A step of deacetylating a soluble portion, an aqueous alkali solution soluble portion, and a non-polar organic solvent soluble portion removed fraction, and a step of extracting a deacetylated fraction obtained from the step with an organic solvent More obtained organic solvent extract fraction; fractions F7), the contacting the deacetylation fraction and an organic solvent, can be obtained by recovering an organic solvent phase. The organic solvent is not particularly limited as long as it exhibits hydrophobic properties. For example, viridine, DMSO, or ethyl acetate can be used. The extraction by contact with an organic solvent is preferably carried out until no coloration is observed in the organic solvent phase.
本発明の極性有機溶媒抽出画分の熱水可溶部及び非極性有機溶媒可溶部除去画分(すなわち、カワラタケ属に属する担子菌を極性有機溶媒により還流抽出した後、得られた極性有機溶媒抽出画分から非極性有機溶媒可溶部及び熱水可溶部を除去する工程により得られる、極性有機溶媒抽出画分の熱水可溶部及び非極性有機溶媒可溶部除去画分;画分F8)は、カワラタケ属に属する担子菌を極性有機溶媒により還流抽出し、例えば、得られた極性有機溶媒抽出画分と非極性有機溶媒とを接触させた後、非極性有機溶媒可溶部を除去し、続いて、得られた非極性有機溶媒除去画分と熱水とを接触させた後、熱水可溶部を除去することにより得ることができる。非極性有機溶媒との接触と熱水との接触は、この順序で実施することもできるし、逆の順序で実施することもできる。 The fraction obtained by removing the hot water soluble part and the nonpolar organic solvent soluble part of the fraction extracted from the polar organic solvent of the present invention (that is, the basidiomycete belonging to the genus Kawaratake was refluxed and extracted with a polar organic solvent, A hot water soluble part and a nonpolar organic solvent soluble part removal fraction obtained by a process of removing a nonpolar organic solvent soluble part and a hot water soluble part from a solvent extraction fraction; The fraction F8) is obtained by reflux-extracting basidiomycetes belonging to the genus Kawaratake with a polar organic solvent, for example, bringing the obtained polar organic solvent-extracted fraction into contact with a nonpolar organic solvent, and then a nonpolar organic solvent soluble part Subsequently, after the obtained nonpolar organic solvent removal fraction and hot water are brought into contact with each other, the hot water-soluble part can be removed. The contact with the non-polar organic solvent and the contact with hot water can be performed in this order, or can be performed in the reverse order.
前記極性有機溶媒は、水溶性の性質を示すものである限り、特に限定されるものではないが、例えば、アセトン、メタノール、エタノール、酢酸エチル、又はジメチルエーテルを用いることができ、アセトンが好ましい。還流は、有機溶媒抽出液の色が消えるまで実施することが好ましく、例えば、50〜150℃にて1〜24時間実施することができる。本発明の極性有機溶媒抽出画分には、還流した後の抽出液と、その乾燥物の両方が含まれる。 The polar organic solvent is not particularly limited as long as it exhibits water-soluble properties. For example, acetone, methanol, ethanol, ethyl acetate, or dimethyl ether can be used, and acetone is preferable. The refluxing is preferably carried out until the color of the organic solvent extract disappears, and can be carried out, for example, at 50 to 150 ° C. for 1 to 24 hours. The polar organic solvent extraction fraction of the present invention includes both the extract after reflux and its dried product.
非極性有機溶媒との接触は、例えば、10〜60分間実施することができる。前記非極性有機溶媒としては、例えば、クロロホルム、ヘキサン、ベンゼン、四塩化炭素、又はメチレンクロライドを用いることができる。非極性有機溶媒との接触は、1つの非極性有機溶媒に関して、5〜10回繰り返すことが好ましい。また、1種類の非極性有機溶媒だけを用いることもできるし、異なる非極性有機溶媒を順次用いることもできる。
また、熱水との接触は、例えば、50〜100℃で10〜60分間実施することができる。熱水との接触は、5〜10回繰り返すことが好ましい。The contact with the nonpolar organic solvent can be carried out, for example, for 10 to 60 minutes. As the nonpolar organic solvent, for example, chloroform, hexane, benzene, carbon tetrachloride, or methylene chloride can be used. The contact with the nonpolar organic solvent is preferably repeated 5 to 10 times for one nonpolar organic solvent. Moreover, only one type of nonpolar organic solvent can be used, or different nonpolar organic solvents can be used sequentially.
Moreover, a contact with hot water can be implemented at 50-100 degreeC for 10 to 60 minutes, for example. The contact with hot water is preferably repeated 5 to 10 times.
本発明のアセチル化画分(又はアセチル化物)[すなわち、カワラタケ属に属する担子菌を極性有機溶媒により還流抽出した後、得られた極性有機溶媒抽出画分から非極性有機溶媒可溶部及び熱水可溶部を除去する工程、並びに前記工程により得られる画分をアセチル化する工程により得られる、アセチル化画分(又はアセチル化物);画分F9]は、前記極性有機溶媒抽出画分の熱水可溶部及び非極性有機溶媒可溶部除去画分をアセチル化することにより得ることができる。前記アセチル化は、出発材料が異なること以外は、本発明の前記画分F4を調製するために実施するアセチル化と同様にして実施することができる。 Acetylated fraction (or acetylated product) of the present invention [that is, basidiomycetes belonging to the genus Kawaratake are extracted by refluxing with a polar organic solvent, and then a non-polar organic solvent soluble part and hot water are obtained from the obtained polar organic solvent extract fraction. The acetylated fraction (or acetylated product) obtained by the step of removing the soluble part and the step of acetylating the fraction obtained by the above step; the fraction F9] is the heat of the polar organic solvent extraction fraction. It can be obtained by acetylating the water-soluble part and the nonpolar organic solvent-soluble part removal fraction. The acetylation can be carried out in the same manner as the acetylation carried out to prepare the fraction F4 of the present invention except that the starting materials are different.
本発明の酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部除去画分(すなわち、カワラタケ属に属する担子菌を極性有機溶媒により還流抽出した後、得られた極性有機溶媒抽出画分から非極性有機溶媒可溶部及び熱水可溶部を除去する工程、前記工程により得られる画分をアセチル化する工程、並びに前記工程により得られるアセチル化画分から、酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部を除去する工程から得られる、酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部除去画分;画分F10)は、前記アセチル化画分と酸性水溶液とを接触させた後、酸性水溶液可溶部を除去し、続いて、得られた酸性水溶液可溶部除去画分とアルカリ水溶液とを接触させた後、アルカリ水溶液可溶部を除去し、更に、得られた酸性水溶液可溶部及びアルカリ水溶液可溶部除去画分と非極性有機溶媒とを接触させた後、非極性有機溶媒可溶部を除去することにより得ることができる。酸性水溶液、アルカリ水溶液、及び非極性有機溶媒との接触は、この順序で実施することもできるし、これ以外の任意の順序で実施することもできる。
酸性水溶液、アルカリ水溶液、及び非極性有機溶媒との各接触は、出発材料が異なること以外は、本発明の前記画分F5を調製するために実施する各接触と同様にして実施することができる。The acidic aqueous solution soluble part, the alkaline aqueous solution soluble part, and the nonpolar organic solvent soluble part removal fraction of the present invention (ie, the polar organic obtained after reflux extraction of basidiomycetes belonging to the genus Kawaratake with a polar organic solvent) The step of removing the non-polar organic solvent soluble part and the hot water soluble part from the solvent extraction fraction, the step of acetylating the fraction obtained by the above step, and the acidic aqueous solution soluble from the acetylated fraction obtained by the above step Fraction, acidic aqueous solution soluble part, alkaline aqueous solution soluble part, and nonpolar organic solvent soluble part removal fraction obtained from the step of removing the aqueous solution, alkaline aqueous solution soluble part, and nonpolar organic solvent soluble part; F10), after contacting the acetylated fraction and the acidic aqueous solution, the acidic aqueous solution soluble part was removed, and then the obtained acidic aqueous solution soluble part removing fraction was contacted with the alkaline aqueous solution. After, al The aqueous solution soluble part is removed, and after the acidic aqueous solution soluble part and alkaline aqueous solution soluble part removal fraction are brought into contact with the nonpolar organic solvent, the nonpolar organic solvent soluble part is removed. Can be obtained. Contact with the acidic aqueous solution, the alkaline aqueous solution, and the nonpolar organic solvent can be performed in this order, or can be performed in any other order.
Each contact with the acidic aqueous solution, the alkaline aqueous solution, and the nonpolar organic solvent can be carried out in the same manner as each contact carried out for preparing the fraction F5 of the present invention except that the starting materials are different. .
本発明の脱アセチル化画分(又は脱アセチル化物)(すなわち、カワラタケ属に属する担子菌を極性有機溶媒により還流抽出した後、得られた極性有機溶媒抽出画分から非極性有機溶媒可溶部及び熱水可溶部を除去する工程、前記工程により得られる画分をアセチル化する工程、並びに前記工程により得られるアセチル化画分から、酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部を除去する工程、並びに前記工程から得られる酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部除去画を脱アセチル化する工程から得られる、脱アセチル化画分(又は脱アセチル化物);画分F11)は、前記酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部除去画分を脱アセチル化することにより得ることができる。前記脱アセチル化は、出発材料が異なること以外は、本発明の前記画分F6を調製するために実施する脱アセチル化と同様にして実施することができる。 The deacetylated fraction (or deacetylated product) of the present invention (that is, basidiomyceae belonging to the genus Kawaratake) after reflux extraction with a polar organic solvent, from the obtained polar organic solvent extract fraction, From the step of removing the hot water soluble portion, the step of acetylating the fraction obtained by the above step, and the acetylated fraction obtained by the above step, the acidic aqueous solution soluble portion, the alkaline aqueous solution soluble portion, and the nonpolar organic Deacetylation obtained from the step of removing the solvent soluble part, and the step of deacetylating the acidic aqueous solution soluble part, the alkaline aqueous solution soluble part, and the nonpolar organic solvent soluble part removal fraction obtained from the above step The fraction (or deacetylated product); the fraction F11) is to deacetylate the acidic aqueous solution soluble part, the alkaline aqueous solution soluble part, and the nonpolar organic solvent soluble part removal fraction. Ri may be obtained. The deacetylation can be carried out in the same manner as the deacetylation carried out to prepare the fraction F6 of the present invention, except that the starting materials are different.
本発明の有機溶媒抽出画分(すなわち、カワラタケ属に属する担子菌を極性有機溶媒により還流抽出した後、得られた極性有機溶媒抽出画分から非極性有機溶媒可溶部及び熱水可溶部を除去する工程、前記工程により得られる画分をアセチル化する工程、並びに前記工程により得られるアセチル化画分から、酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部を除去する工程、並びに前記工程から得られる酸性水溶液可溶部、アルカリ水溶液可溶部、及び非極性有機溶媒可溶部除去画を脱アセチル化する工程、並びに前記工程から得られる脱アセチル化画分を有機溶媒で抽出する工程により得られる、有機溶媒抽出画分;画分F12)は、前記脱アセチル化画分と有機溶媒とを接触させ、有機溶媒相を回収することにより得ることができる。前記有機溶媒との接触は、出発材料が異なること以外は、本発明の前記画分F7を調製するために実施する有機溶媒との接触と同様にして実施することができる。 The organic solvent extract fraction of the present invention (that is, basidiomycetes belonging to the genus Kawaratake are extracted by refluxing with a polar organic solvent, and then the nonpolar organic solvent soluble part and the hot water soluble part are extracted from the obtained polar organic solvent extract fraction. The step of removing, the step of acetylating the fraction obtained by the above step, and the acetylated fraction obtained by the above step remove the acidic aqueous solution soluble portion, the alkaline aqueous solution soluble portion, and the nonpolar organic solvent soluble portion. A step of deacetylating the acidic aqueous solution soluble part, the alkaline aqueous solution soluble part, and the nonpolar organic solvent soluble part removal fraction obtained from the step, and the deacetylation fraction obtained from the step The organic solvent extraction fraction obtained by the step of extracting with an organic solvent; fraction F12) is obtained by contacting the deacetylated fraction with an organic solvent and recovering the organic solvent phase. Rukoto can. The contact with the organic solvent can be carried out in the same manner as the contact with the organic solvent carried out for preparing the fraction F7 of the present invention, except that the starting materials are different.
本発明の結合物は、本発明の前記画分F1〜F12の少なくとも1つの画分、又は一般式(I)で表されるテレホール酸化合物若しくはその塩と、タンパク質との結合物である限り、特に限定されるものではない。
前記タンパク質は、前記テレホール酸と複合体を形成することができ、且つ前記複合体が抗アレルギー活性を示す限り、特に限定されるものではないが、例えば、アルブミン又はグロブリンを挙げることができる。As long as the conjugate of the present invention is a conjugate of a protein with at least one fraction of the fractions F1 to F12 of the present invention, or a telephoric acid compound represented by the general formula (I) or a salt thereof, It is not particularly limited.
The protein is not particularly limited as long as it can form a complex with the teleholic acid and the complex exhibits antiallergic activity, and examples thereof include albumin and globulin.
カワラタケ属に属する担子菌に由来する本発明の画分F1〜F12は、例えば、不溶物が混在する状態でそのまま、あるいは、不溶物を除去した状態で、更には、適当な処理(例えば、濃縮若しくは希釈、乾燥、又は再溶解)を施した状態で、本発明の抗アレルギー剤の有効成分として用いることができる。本発明の画分F7の内、実施例1の手順に従って調製した画分は、テレホール酸のみを実質的に含有する。
なお、本発明において、有効成分として本発明の画分F1〜F12を用いた場合、前記画分中に含有されるテレホール酸が抗アレルギー活性を示すものと本発明者は考えているが、前記画分中のテレホール酸以外の成分も抗アレルギー活性を示すことが考えられ、本発明の範囲は前記機構に限定されるものではない。The fractions F1 to F12 of the present invention derived from basidiomycetes belonging to the genus Kawaratake are, for example, as they are in a state where insoluble matters are mixed, or in a state where insoluble matters are removed, and further suitable treatment (for example, concentration) Alternatively, it can be used as an active ingredient of the antiallergic agent of the present invention after being diluted, dried, or redissolved). Of the fraction F7 of the present invention, the fraction prepared according to the procedure of Example 1 substantially contains only teleholic acid.
In the present invention, when the fractions F1 to F12 of the present invention are used as active ingredients, the present inventor believes that the teleholic acid contained in the fraction exhibits antiallergic activity. It is considered that components other than teleholic acid in the fraction also exhibit antiallergic activity, and the scope of the present invention is not limited to the above mechanism.
本発明の抗アレルギー剤は、有効成分として、(1)一般式(I)で表されるテレホール酸化合物若しくはその塩、あるいは、前記テレホール酸化合物若しくはその塩とタンパク質との結合物、又は(2)カワラタケ属に属する担子菌に由来する本発明の各種画分(F1〜F12)の少なくとも1つ、あるいは、前記画分F1〜F12の少なくとも1つの画分とタンパク質との結合物を含有する。本発明の抗アレルギー剤は、前記有効成分のいずれか1つを含有することもできるし、2つ以上を任意の組み合わせで含有することもできる。本発明においては、前記有効成分をそれ単独で、あるいは、好ましくは薬剤学的又は獣医学的に許容することのできる通常の担体又は希釈剤と共に、アレルギー抑制、あるいは、アレルギーに関連する疾病の治療又は予防の必要な対象[例えば、動物、好ましくは哺乳動物(特にはヒト)]に有効量で投与することができる。また、前記有効成分は、抗アレルギー剤、又はアレルギーに関連する疾病の治療又は予防剤を製造するために使用することができる。 The antiallergic agent of the present invention comprises, as an active ingredient, (1) a teleholic acid compound represented by the general formula (I) or a salt thereof, a conjugate of the teleholic acid compound or a salt thereof and a protein, or (2 ) It contains at least one of the various fractions (F1 to F12) of the present invention derived from basidiomycetes belonging to the genus Kawaratake, or a conjugate of at least one fraction of the fractions F1 to F12 and a protein. The antiallergic agent of the present invention can contain any one of the above active ingredients, and can contain two or more in any combination. In the present invention, the above-mentioned active ingredient alone, or preferably together with a usual carrier or diluent that can be pharmaceutically or veterinarily acceptable, suppresses allergy or treats an allergy-related disease. Alternatively, it can be administered to a subject in need of prevention [eg, an animal, preferably a mammal (particularly a human)] in an effective amount. Moreover, the said active ingredient can be used in order to manufacture the antiallergic agent or the treatment or prevention agent of the disease relevant to allergy.
アレルギーに関連する疾病としては、例えば、喘息、アトピー性皮膚炎、化学物質過敏症、花粉症、食物アレルギー、ラテックスアレルギー、金属アレルギー、ダニアレルギー、薬かぶれ、気管支喘息、アレルギー性鼻炎、じんましん、アレルギー性気管支喘息、薬
剤によるアナフィラキシーショック、消化管アレルギー、アレルギー性亜敗血症、アレルギー性胃腸症、アレルギー性気管支肺アスペルギルス症、アレルギー性口内炎、アレルギー性紫斑病、アレルギー性肉芽腫、アレルギー性肺炎、アレルギー性肺好酸球症、アレルギー性肺胞炎、アレルギー性食中毒、又はアレルギー性肉芽腫性血管炎などを挙げることができる。Diseases related to allergy include, for example, asthma, atopic dermatitis, chemical sensitivity, hay fever, food allergy, latex allergy, metal allergy, tick allergy, drug rash, bronchial asthma, allergic rhinitis, hives, allergies Bronchial asthma, drug anaphylactic shock, gastrointestinal allergy, allergic subsepsis, allergic gastroenteropathy, allergic bronchopulmonary aspergillosis, allergic stomatitis, allergic purpura, allergic granulomas, allergic pneumonia, allergic Examples thereof include pulmonary eosinophilic disease, allergic alveolitis, allergic food poisoning, or allergic granulomatous vasculitis.
本発明の抗アレルギー剤の投与剤型としては、特に限定がなく、例えば、散剤、細粒剤、顆粒剤、錠剤、カプセル剤、懸濁液、エマルジョン剤、シロップ剤、エキス剤、若しくは丸剤等の経口剤、又は注射剤、外用液剤、軟膏剤、坐剤、局所投与のクリーム、若しくは点眼薬などの非経口剤を挙げることができる。 The dosage form of the antiallergic agent of the present invention is not particularly limited, and examples thereof include powders, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts, or pills. Or parenteral preparations such as injections, external solutions, ointments, suppositories, topical creams, or eye drops.
これらの経口剤は、例えば、アルギン酸ナトリウム、澱粉、コーンスターチ、白糖、乳糖、ぶどう糖、マンニット、カルボキシメチルセルロース、デキストリン、ポリビニルピロリドン、結晶セルロース、大豆レシチン、ショ糖、脂肪酸エステル、タルク、ステアリン酸マグネシウム、ポリエチレングリコール、ケイ酸マグネシウム、無水ケイ酸、又は合成ケイ酸アルミニウムなどの賦形剤、結合剤、崩壊剤、界面活性剤、滑沢剤、流動性促進剤、希釈剤、保存剤、着色剤、香料、矯味剤、安定化剤、保湿剤、防腐剤、又は酸化防止剤等を用いて、常法に従って製造することができる。 These oral preparations include, for example, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soybean lecithin, sucrose, fatty acid ester, talc, magnesium stearate, Excipients such as polyethylene glycol, magnesium silicate, anhydrous silicic acid, or synthetic aluminum silicate, binders, disintegrants, surfactants, lubricants, fluidity promoters, diluents, preservatives, colorants, It can be produced according to a conventional method using a fragrance, a flavoring agent, a stabilizer, a moisturizing agent, an antiseptic, or an antioxidant.
非経口投与方法としては、注射(皮下、静脈内等)、又は直腸投与等が例示される。これらのなかで、注射剤が最も好適に用いられる。
例えば、注射剤の調製においては、有効成分の他に、例えば、生理食塩水若しくはリンゲル液等の水溶性溶剤、植物油若しくは脂肪酸エステル等の非水溶性溶剤、ブドウ糖若しくは塩化ナトリウム等の等張化剤、溶解補助剤、安定化剤、防腐剤、懸濁化剤、又は乳化剤などを任意に用いることができる。
また、本発明の抗アレルギー剤は、徐放性ポリマーなどを用いた徐放性製剤の手法を用いて投与してもよい。例えば、本発明における前記有効成分をエチレンビニル酢酸ポリマーのペレットに取り込ませて、このペレットを治療又は予防すべき組織中に外科的に移植することができる。Examples of parenteral administration methods include injection (subcutaneous, intravenous, etc.) or rectal administration. Of these, the injection is most preferably used.
For example, in the preparation of an injection, in addition to the active ingredient, for example, a water-soluble solvent such as physiological saline or Ringer's solution, a water-insoluble solvent such as vegetable oil or fatty acid ester, an isotonic agent such as glucose or sodium chloride, Solubilizers, stabilizers, preservatives, suspending agents, or emulsifiers can be optionally used.
Further, the antiallergic agent of the present invention may be administered by using a method of sustained release preparation using a sustained release polymer or the like. For example, the active ingredient in the present invention can be incorporated into a pellet of ethylene vinyl acetate polymer and the pellet can be surgically implanted into the tissue to be treated or prevented.
本発明の抗アレルギー剤は、これに限定されるものではないが、本発明における前記有効成分を0.01〜99重量%、好ましくは0.1〜80重量%の量で含有することができる。
本発明の抗アレルギー剤を用いる場合の投与量は、病気の種類、患者の年齢、性別、体重、症状の程度、又は投与方法などに応じて適宜決定することができ、経口的に又は非経口的に投与することが可能である。The antiallergic agent of the present invention is not limited to this, but the active ingredient in the present invention can be contained in an amount of 0.01 to 99% by weight, preferably 0.1 to 80% by weight. .
The dosage in the case of using the antiallergic agent of the present invention can be appropriately determined depending on the type of disease, the age, sex, weight, symptom of the patient, or the administration method, orally or parenterally. Can be administered as a whole.
また、投与形態も医薬品に限定されるものではなく、種々の形態、例えば、健康食品(好ましくは機能性食品)又は飼料として飲食物の形で与えることも可能である。なお、前記食品には飲料が含まれる。
食品には、(1)栄養素としての働き(第一次機能)、(2)人間の五感に訴える働き(第2次機能)の他に、(3)人間の健康、身体能力、又は心理状態に好ましい影響を与える働き(第3次機能)、例えば、消化器系、循環器系、内分泌系、免疫系、又は神経系などの生理系統を調節して、健康の維持や健康の回復に好ましい効果を及ぼす働きがあることが知られている。本明細書において「健康食品」とは、健康に何らかの効果を与えるか、あるいは、効果を期待することができる食品を意味し、「機能性食品」とは、前記「健康食品」の中でも、前記の種々の生体調節機能(すなわち、消化器系、循環器系、内分泌系、免疫系、又は神経系などの生理系統の調節機能)を充分に発現することができるように設計及び加工された食品を意味する。Further, the administration form is not limited to pharmaceuticals, and various forms such as health food (preferably functional food) or feed can be given in the form of food and drink. The food includes a beverage.
In addition to (1) function as a nutrient (primary function), (2) function to appeal to the human senses (secondary function), (3) human health, physical ability, or psychological state For the maintenance of health and recovery of health by regulating physiological systems such as the digestive system, circulatory system, endocrine system, immune system, or nervous system. It is known to have an effect. In the present specification, “health food” means a food that has some effect on health or can be expected to have an effect, and “functional food” refers to the above-mentioned “health food”. Foods designed and processed to fully express various bioregulatory functions (i.e., regulatory functions of digestive, circulatory, endocrine, immune, or nervous systems) Means.
更には、オーラル衛生用組成物、例えば、口中に一時的に含むものの、そのほとんどを口中より吐き出す形態、例えば、歯磨剤、洗口剤、チューインガム、又はうがい剤の形で与えることも、あるいは、鼻から吸引させる吸入剤の形で与えることも可能である。例えば、本発明における前記有効成分を、添加剤(例えば、食品添加剤)として、所望の食品(飲料を含む)、飼料、歯磨剤、洗口剤、チューインガム、又はうがい剤等に添加することができる。 Furthermore, an oral hygiene composition, for example, temporarily contained in the mouth, but most of it is given in the form of exhaling from the mouth, for example, in the form of a dentifrice, mouthwash, chewing gum, or mouthwash, or It is also possible to give in the form of an inhalant to be aspirated from the nose. For example, the active ingredient in the present invention may be added as an additive (for example, a food additive) to a desired food (including beverage), feed, dentifrice, mouthwash, chewing gum, gargle, or the like. it can.
以下、実施例によって本発明を具体的に説明するが、これらは本発明の範囲を限定するものではない。 EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.
《実施例1:カワラタケ乾燥子実体からのテレホール酸画分の分離及び同定》
カワラタケの乾燥子実体粉末15gに、飽和ホウ酸ナトリウム水溶液2Lを加え、5L容のビーカー中、25℃で24時間攪拌抽出の後、遠心分離(12,000xg、30分間)して上清を回収した。塩酸にて前記上清のpHを2.0に調整し、室温で一晩放置の後、遠心分離(40,000xg、30分間)して沈殿部を回収した。沈殿部に蒸留水500mLを加え、充分に懸濁し、洗浄液のpHが7.0になるまで洗浄操作を繰り返した後、遠心分離(40,000xg、30分間)を行い、沈殿部を回収し、凍結乾燥して粉末1.2gを得た。<< Example 1: Separation and identification of teleholic acid fraction from dried fruit body of Kawaratake >>
Add 2 L of saturated sodium borate aqueous solution to 15 g of dried fruit body powder of Kawaratake and stir and extract in a 5 L beaker at 25 ° C. for 24 hours, and then centrifuge (12,000 × g, 30 minutes) to recover the supernatant did. The pH of the supernatant was adjusted to 2.0 with hydrochloric acid, allowed to stand at room temperature overnight, and then centrifuged (40,000 × g, 30 minutes) to recover the precipitate. Distilled water (500 mL) was added to the precipitation part, suspended sufficiently, and after repeating the washing operation until the pH of the washing solution reached 7.0, centrifugation (40,000 × g, 30 minutes) was performed to recover the precipitation part, Lyophilization gave 1.2 g of powder.
ジムロート(PYREX製)に凍結乾燥物1.2gを入れ、アセトン10mLを加えて、80℃にて3時間加熱還流した。抽出液の色が無くなるまで抽出を繰り返した後、抽出液を集め、エバポレーターを用いて濃縮乾固して乾固物を得た。この乾固物に蒸留水10mLを加え、80℃で5分間加熱し、遠心分離により熱水可溶部を除去した。この操作を10回繰り返した後、溶媒をヘキサン、次いでクロロホルムに代えて同様の操作を10回ずつ行い、青紫色の沈殿画分8mgを得た。 1.2 g of lyophilized product was added to Dimroth (manufactured by PYREX), 10 mL of acetone was added, and the mixture was heated to reflux at 80 ° C for 3 hours. Extraction was repeated until the color of the extract disappeared, and then the extract was collected and concentrated to dryness using an evaporator to obtain a dried product. Distilled water (10 mL) was added to the dried product, heated at 80 ° C. for 5 minutes, and the hot water soluble part was removed by centrifugation. After this operation was repeated 10 times, the same operation was performed 10 times each by replacing the solvent with hexane and then chloroform to obtain 8 mg of a blue-purple precipitate fraction.
この画分に無水酢酸1mLを加え、ピリジン1mLの入った50mL容のナスフラスコ中で70℃にて24時間加熱後、エバポレーターを用いて濃縮乾固した。次に、1N塩酸10mLを加え、塩酸可溶部を除去し、この操作を3回繰り返した。次いで、塩酸の代わりに、飽和炭酸水素ナトリウム水溶液、ヘキサン、クロロホルム、及び酢酸エチルを順に用いて、同様の操作を繰り返すことにより、各可溶部を除去した後、その不溶部をピリジンに溶解し、エバポレーターを用いて濃縮乾固してアセチル化画分5.9mgを得た。 1 mL of acetic anhydride was added to this fraction, heated in a 50 mL eggplant flask containing 1 mL of pyridine at 70 ° C. for 24 hours, and then concentrated to dryness using an evaporator. Next, 10 mL of 1N hydrochloric acid was added to remove the hydrochloric acid soluble part, and this operation was repeated three times. Then, instead of hydrochloric acid, using a saturated aqueous sodium bicarbonate solution, hexane, chloroform, and ethyl acetate in this order, the same operation was repeated to remove each soluble part, and then the insoluble part was dissolved in pyridine. Then, it was concentrated to dryness using an evaporator to obtain 5.9 mg of an acetylated fraction.
アセチル化画分に、CH3ONaを1Nとなるように無水エタノールに溶解した溶液1mLを加え、50mL容三角フラスコ中、室温で1時間攪拌した。次に、エバポレーターを用いて濃縮乾固した後、1N塩酸溶液20mLを加え、分液ロートに入れた。次に等量の酢酸エチルを加え、抽出操作を行った。酢酸エチル相に着色が見られなくなるまでこの操作を繰り返した。そして、酢酸エチル相を回収し、エバポレーターを用いて濃縮乾固して青色の脱アセチル化物4mgを得た。To the acetylated fraction, 1 mL of a solution in which CH 3 ONa was dissolved in absolute ethanol to 1 N was added, and the mixture was stirred in a 50 mL Erlenmeyer flask at room temperature for 1 hour. Next, after concentrating to dryness using an evaporator, 20 mL of 1N hydrochloric acid solution was added and put into a separatory funnel. Next, an equal amount of ethyl acetate was added, and extraction operation was performed. This operation was repeated until no coloration was observed in the ethyl acetate phase. Then, the ethyl acetate phase was recovered and concentrated to dryness using an evaporator to obtain 4 mg of a blue deacetylated product.
得られた画分を1H−NMR及び13C−NMR(UNITY INOVA 500;バリアン)により構造解析したところ、1H−NMR(図1)において、δ7.14、7.35ppmにベンゼン環由来の付け根のシグナル、δ9.81、10.08ppmに水酸基由来のシグナルが観察された。更に、13C−NMR(図2)において、δ98.49、104.82、146.43、149.24、150.79、151.326ppm付近にベンゼン環由来のシグナル、δ172.38ppmにキノン由来のシグナルが確認された。以上のことから、キノン骨格を有する芳香族の存在を確認した。The obtained fraction was subjected to structural analysis by 1 H-NMR and 13 C-NMR (
次に、IRスペクトル(FT/IR VALORIII;日本分光)において、3204cm−1に水酸基由来の吸収、1634cm−1にキノン由来のカルボニル吸収、867cm−1及び801cm−1にフラン環由来の吸収を確認した(図3)。MS解析(Hybrid Mstation JMS−700QQ;日本電子)では、m/zC18H8O8(M+)352.0240を示し、テレホール酸と一致した(図4)。Next, in the IR spectrum (FT / IR VALORIII; JASCO), 3204 cm −1 absorbs hydroxyl group, 1634 cm −1 absorbs quinone derived carbonyl, 867 cm −1 and 801 cm −1 absorbs furan ring. (FIG. 3). MS analysis (Hybrid Mstation JMS-700QQ; JEOL) showed m / zC 18 H 8 O 8 (M + ) 352.0240, which was consistent with teleholic acid (FIG. 4).
《実施例2:テレホール酸画分とタンパク質との結合物の調製》
実施例1で調製したテレホール酸画分をジメチルスルホキシド(DMSO)に溶解し、1mmol/Lに調整し、テレホール酸溶液を調製した。この溶液を、ヒトアルブミン又はヒトグロブリンのPBS溶解液(pH7.2)10mg/mLにそれぞれ加え、室温で3時間反応させた。得られた反応液を蒸留水に対して透析後、内容液を回収し、凍結乾燥して、テレホール酸・ヒトアルブミン結合物及びテレホール酸・ヒトグロブリン結合物を得た。<< Example 2: Preparation of a conjugate of a teleholic acid fraction and a protein >>
The teleholic acid fraction prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) and adjusted to 1 mmol / L to prepare a teleholic acid solution. This solution was added to 10 mg / mL of human albumin or human globulin in PBS (pH 7.2) and reacted at room temperature for 3 hours. The resulting reaction solution was dialyzed against distilled water, and the content solution was collected and lyophilized to obtain a telephoric acid / human albumin conjugate and a teleholic acid / human globulin conjugate.
《実施例3:PCA反応を指標とした抗アレルギー活性の評価》
(1)感作用試料液及び各投与液の調製
試料液として、サンプル(すなわち、実施例1で調製したテレホール酸画分)1mgを10mmol/Lホウ酸ナトリウム水溶液250μLに溶解し、超音波処理した後、蒸留水250μLを加え、試料液(1)とした。<< Example 3: Evaluation of antiallergic activity using PCA reaction as an index >>
(1) Preparation of sensitizing sample solution and each administration solution As a sample solution, 1 mg of a sample (that is, the teleholic acid fraction prepared in Example 1) was dissolved in 250 μL of a 10 mmol / L sodium borate aqueous solution and sonicated. Thereafter, 250 μL of distilled water was added to obtain a sample solution (1).
投与液I〜投与液Vは、以下の手順で調製した。なお、各投与液の調製は、用時に行った。
投与液Iは、前記試料液(1)と、10mmol/Lリン酸緩衝化生理食塩水(以下、PBSと称する)(pH7.2)で1000倍希釈した抗卵白アルブミン抗体(ウサギ抗血清)とを等量混和して調製した。
投与液IIは、蒸留水で2倍希釈した前記試料液(1)と、10mmol/LのPBS(pH7.2)で1000倍希釈した抗卵白アルブミン抗体とを等量混和して調製した。
投与液III及び投与液VIは、10mmol/Lホウ酸ナトリウム水溶液と、10mmol/LのPBS(pH7.2)で1000倍希釈した抗卵白アルブミン抗体とを等量混和して調製した。The administration liquid I to the administration liquid V were prepared by the following procedure. In addition, preparation of each administration liquid was performed at the time of use.
The administration liquid I comprises the sample liquid (1), an anti-ovalbumin antibody (rabbit antiserum) diluted 1000 times with 10 mmol / L phosphate buffered saline (hereinafter referred to as PBS) (pH 7.2), and Were mixed to prepare an equal amount.
The dosing solution II was prepared by mixing equal amounts of the sample solution (1) diluted 2-fold with distilled water and the anti-ovalbumin antibody diluted 1000-fold with 10 mmol / L PBS (pH 7.2).
The administration liquid III and the administration liquid VI were prepared by mixing equal amounts of a 10 mmol / L sodium borate aqueous solution and an anti-ovalbumin antibody diluted 1000 times with 10 mmol / L PBS (pH 7.2).
投与液IVは、ジフェンヒドラミン塩酸塩(陽性対照)1mgを10mmol/Lホウ酸ナトリウム水溶液250μLに溶解し、超音波処理した後、蒸留水250μLを加えて調製した試料液(2)と、10mmol/LのPBS(pH7.2)で1000倍希釈した抗卵白アルブミン抗体とを等量混和して調製した。
投与液Vは、蒸留水で2倍希釈した前記試料液(2)と、10mmol/LのPBS(pH7.2)で1000倍希釈した抗卵白アルブミン抗体とを等量混和して調製した。The administration liquid IV was prepared by dissolving 1 mg of diphenhydramine hydrochloride (positive control) in 250 μL of 10 mmol / L sodium borate aqueous solution, sonicating, and then adding 250 μL of distilled water, and 10 mmol / L An equal amount of an anti-ovalbumin antibody diluted 1000 times with PBS (pH 7.2) was prepared.
The dosing solution V was prepared by mixing equal amounts of the sample solution (2) diluted 2-fold with distilled water and the anti-ovalbumin antibody diluted 1000-fold with 10 mmol / L PBS (pH 7.2).
(2)感作方法
モルモットの背部(約15×8cm)をバリカンで刈毛した。各投与液0.1mLをそれぞれ3匹のモルモットの背部皮内に注射した。投与部位は背仲線を境に右側に頭部から投与液I〜III、左側に投与液IV〜VIとした。(2) Sensitization method The back of the guinea pig (about 15 × 8 cm) was shaved with a clipper. Each administration solution (0.1 mL) was injected into the back skin of three guinea pigs. The administration sites were administration liquids I to III from the head on the right side, and administration liquids IV to VI on the left side of the back-middle line.
(3)惹起方法
皮内注射の48時間後に、惹起抗原液を1%エバンスブルー(和光純薬)/生理食塩溶液と等量ずつ混和し、その1mLをモルモットの後肢後伏静脈内に注射した。(3) Induction method 48 hours after the intradermal injection, the inducing antigen solution was mixed with 1% Evans blue (Wako Pure Chemicals) / physiological saline solution in equal amounts, and 1 mL was injected into the hindlimb hind vein of the guinea pig. .
(4)判定方法
色素液注射から30分後にモルモットを屠殺し、注射部位周辺の表皮を剥がした。皮内注射部位を観察し、色素斑の長径及び短径を測定し、A[=長径×短径(mm2)]を算出した。次に、背中線を境に同側部のコントロール感作部位(部位III又はVI)A’に対する試料処置部位Aの比B[=A/A’]を求めた。
PCA(passive cutaneous anaphylaxis)反応抑制率は下記の式から算出した。
C=100−B×100(%)(4) Determination method Guinea pigs were sacrificed 30 minutes after the dye solution injection, and the epidermis around the injection site was peeled off. The site of intradermal injection was observed, the major axis and minor axis of pigment spots were measured, and A [= major axis x minor axis (mm 2 )] was calculated. Next, the ratio B [= A / A ′] of the sample treatment site A to the control sensitization site (site III or VI) A ′ on the same side with the back line as a boundary was determined.
PCA (passive cutaneous anaphylaxis) reaction inhibition rate was calculated from the following equation.
C = 100−B × 100 (%)
(5)結果
表1に示すように、実施例1で調製したサンプル(カワラタケ由来テレホール酸)はPCA反応を抑制した。
《実施例4:IgE産生を指標とした抗アレルギー活性の評価》
(1)実験方法
日本チャールズリバー社(神奈川)から購入した雌性BALB/cマウスを予備飼育し、8週齢で実験に供した(1群5匹)。
前記実施例1で調製したテレホール酸をジメチルスルホキシド(DMSO)に溶解し、ポリエチレングリコール(分子量400;シグマ−アルドリッチ社)にて所定濃度に調整し、5mg/kgの量で、抗原感作1週前から実験終了日まで、週3回、皮下注射した。コントロールとして、ポリエチレングリコール皮下注射群を設けた。
抗原感作のために、ジニトロフェニル結合キーホールリンペット・ヘモシアニン(以下、DNP−KLHと略称する;コスモバイオ、東京)をリン酸緩衝液に溶解し、10mg/mL濃度に調整した後、その2μLを20mg/mLのALUM(コスモバイオ)溶液1mLに加え、混合した。この混合液0.2mLをマウス腹腔内に注射することにより、抗原感作した。
抗原感作3、7、14、及び21日後、マウス眼窩血管叢をキャピラリーで軽く傷つけ、部分採血し、血清を分離した。血清中の血清IgEレベルを市販のELISAキットを用いて測定した。<< Example 4: Evaluation of anti-allergic activity using IgE production as an index >>
(1) Experimental Method Female BALB / c mice purchased from Charles River Japan (Kanagawa) were preliminarily raised and subjected to experiments at 8 weeks of age (5 per group).
The telephoric acid prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO), adjusted to a predetermined concentration with polyethylene glycol (
For antigen sensitization, dinitrophenyl-linked keyhole limpet hemocyanin (hereinafter abbreviated as DNP-KLH; Cosmo Bio, Tokyo) was dissolved in a phosphate buffer and adjusted to a concentration of 10 mg / mL. 2 μL was added to 1 mL of a 20 mg / mL ALUM (Cosmo Bio) solution and mixed. Antigen sensitization was performed by injecting 0.2 mL of this mixed solution into a mouse abdominal cavity.
After 3, 7, 14, and 21 days of antigen sensitization, the mouse orbital vascular plexus was lightly damaged with a capillary, a partial blood sample was collected, and the serum was separated. Serum IgE levels in the serum were measured using a commercially available ELISA kit.
遊離ヒスタミンの測定は、以下の手順で実施した。すなわち、感作14日後のマウスをと殺し、腹腔内をRPMI1640培地で洗浄及び回収し、遠心分離(700rpm、10分間)して腹腔細胞を得た。この腹腔細胞をRPMI1640培地中に懸濁し、ウシ血清アルブミン(BSA)/生理食塩水グラジエント液(d=1.07)で比重遠心分離にかけ、肥満細胞を得た。この細胞をRPMI1640培地で洗浄した後、1×105mLの濃度に調整し、DNP−KLHの250ng/mLを添加して、37℃の5%炭酸ガス培養容器中で30分間反応させた。培養上清中のヒスタミンレベルを市販のELISAキット(Immunotech社)を用いて定量した。ヒスタミン遊離率は下記式:
(DNP−KLH刺激群の総ヒスタミン量)×100/(DNP−KLH非刺激群の総ヒスタミン量)
により算出した。The measurement of free histamine was carried out by the following procedure. That is, mice 14 days after sensitization were killed, and the peritoneal cavity was washed and collected with RPMI 1640 medium, and centrifuged (700 rpm, 10 minutes) to obtain peritoneal cells. The peritoneal cells were suspended in RPMI 1640 medium and subjected to specific gravity centrifugation with bovine serum albumin (BSA) / saline gradient solution (d = 1.07) to obtain mast cells. The cells were washed with RPMI1640 medium, adjusted to a concentration of 1 × 10 5 mL, added with 250 ng / mL of DNP-KLH, and reacted in a 5% carbon dioxide culture vessel at 37 ° C. for 30 minutes. The histamine level in the culture supernatant was quantified using a commercially available ELISA kit (Immunotech). The histamine release rate is given by the following formula:
(Total amount of histamine in DNP-KLH-stimulated group) × 100 / (Total amount of histamine in non-stimulated DNP-KLH group)
Calculated by
(2)結果
DNP−KLH感作したコントロールマウスでは、感作3日後よりIgE産生が認められ、感作21日後まで持続していた。一方、実施例1で得られたサンプル(テレホール酸画分)を皮下投与(5mg/kg)した群では、観察期間を通し、コントロール群に比してIgE産生を有意に抑制した。
また、DNP−KLH感作14日後のマウスから腹腔内肥満細胞を分離し、DNP−KLHで再刺激した時のヒスタミン遊離量を測定したところ、実施例サンプル投与群のヒスタミン遊離量は、コントロール群に比し、有意に抑制されていた。
以上の結果、テレホール酸は、DNP−KLH感作マウスにおける血中IgE産生を抑制し、従って、肥満細胞の脱顆粒誘導を抑制すると考えられる。(2) Results In control mice sensitized with DNP-KLH, IgE production was observed from 3 days after sensitization, and persisted until 21 days after sensitization. On the other hand, in the group in which the sample (teleholic acid fraction) obtained in Example 1 was administered subcutaneously (5 mg / kg), IgE production was significantly suppressed throughout the observation period compared to the control group.
Further, when intraperitoneal mast cells were isolated from mice 14 days after sensitization with DNP-KLH, and the amount of histamine released when restimulated with DNP-KLH was measured, the amount of histamine released in the example sample administration group was It was significantly suppressed compared to.
As a result of the above, it is considered that teleholic acid suppresses blood IgE production in DNP-KLH-sensitized mice, and thus suppresses degranulation induction of mast cells.
《実施例5:好塩基球のヒスタミン遊離に及ぼすテレホール酸の影響》
本実施例では、ラット好塩基球性白血病細胞株RBL−2H3[Rintaro
Yamanishi et al., Bioscience, Biotechnology and Biochemistry, 61, 19-23 (2002)]をヒスタミン分泌細胞として用いた。なお、前記細胞株RBL−2H3は、東北大学・加齢研究所・医用細胞資源センター(仙台、宮城県)から入手し、呉羽化学工業株式会社生物医学研究所において、10%FBS添加RPMI1640培地中で継代・維持しているものである。48ウエルの培養用プレートに、前記細胞株RBL−2H3をウエル当たり2.5×105細胞(容量500μL)となるように蒔き、5%炭酸ガス培養器中で一晩培養した後、11μg/mL抗DNP−回虫(DNP-Ascaris)IgE(生化学工業、東京)溶液50μLを添加(終濃度1μg/mL)し、3時間培養した。Example 5 Effect of teleholic acid on histamine release from basophils
In this example, the rat basophilic leukemia cell line RBL-2H3 [Rintaro
Yamanishi et al., Bioscience, Biotechnology and Biochemistry, 61, 19-23 (2002)] were used as histamine-secreting cells. The cell line RBL-2H3 was obtained from Tohoku University, Aging Research Institute, Medical Cell Resource Center (Sendai, Miyagi Prefecture), and in Kureha Chemical Industry Co., Ltd. Biomedical Research Institute in 10% FBS-added RPMI1640 medium. It is the one that has been passaged and maintained. The cell line RBL-2H3 was seeded on a 48-well culture plate at 2.5 × 10 5 cells per well (
培養終了後、PBSで各ウエルを洗浄し、次いで、テレホール酸0、0.1、1、又は10μmol/Lを含む培地500μLを、各ウエルに分注して30分間培養した。次いで、40μg/mLのDNP−BSA(コスモ・バイオ、東京)溶液10μLを添加後(終濃度0.8μg/mL)、更に1時間培養した。そして、遠心分離により培養上清を回収して−20℃で凍結保存した。 After completion of the culture, each well was washed with PBS, and then 500 μL of a medium containing 0, 0.1, 1 or 10 μmol / L of teleholic acid was dispensed into each well and incubated for 30 minutes. Subsequently, 10 μL of a 40 μg / mL DNP-BSA (Cosmo Bio, Tokyo) solution was added (final concentration 0.8 μg / mL), followed by further culturing for 1 hour. Then, the culture supernatant was collected by centrifugation and stored frozen at -20 ° C.
培養上清中のヒスタミン含量は、市販ELISAキット(Histamine EIA
KIT; SPI-BIO社, Montigny Le Bretonneux, フランス)を用いて測定した。測定結果を表2に示す。対照群に比べると、テレホール酸添加群では上清中のヒスタミンレベルは有意に低かった。すなわち、テレホール酸がヒスタミン分泌を抑制することにより、アレルギーを抑制している可能性が示唆された。The histamine content in the culture supernatant was measured using a commercially available ELISA kit (Histamine EIA
KIT; SPI-BIO, Montigny Le Bretonneux, France). The measurement results are shown in Table 2. Compared with the control group, the histamine level in the supernatant was significantly lower in the teleholic acid addition group. That is, it was suggested that teleholic acid may suppress allergy by suppressing histamine secretion.
《実施例6:テレホール酸による受身PCA反応の抑制》
被検物質テレホール酸1mgは、10mmol/Lホウ酸ナトリウム水溶液250μLに溶解後、蒸留水250μLを加えて0.2mg/mLとした。陽性対照のクロモグリク酸ナトリウム(藤沢薬品工業、大阪)は、生理食塩水に溶解して0.2mg/mLとした。Example 6 Inhibition of Passive PCA Reaction by Teleholic Acid
1 mg of the test substance teleholic acid was dissolved in 250 μL of 10 mmol / L sodium borate aqueous solution, and then 250 μL of distilled water was added to make 0.2 mg / mL. The positive control sodium cromoglycate (Fujisawa Pharmaceutical, Osaka) was dissolved in physiological saline to a concentration of 0.2 mg / mL.
8週齢の雄性SDラット(日本クレア)の背部皮内に、800倍希釈した抗DNP−回虫(DNP-Ascaris)IgE(生化学工業、東京)0.1mLを注射した。48時間後、前記テレホール酸又はクロモグリク酸ナトリウムを静脈内注射(1mg/kg量)し、その1分後、最終濃度0.3mg/mLのDNP−回虫(DNP-Ascaris)(コスモ・バイオ)溶液と最終濃度5mg/mLのエバンス青溶液(シグマ・アルドリッチ、東京)との混合液を静脈内注射(1mL/匹)した。30分後、ラットをエーテル麻酔下で放血致死し、注射部位周囲の皮膚を剥離して色素漏出部をはさみで切り出した。これを2mol/L水酸化カリウム水溶液2mL中で37℃にて21時間処理し、7.4%リン酸・アセトン混液(1:3)6mLを加えて激しく混和させた。3,000rpmで15分間遠心分離した後、回収した上清100μLの630nmにおける吸光度を分光光度計で測定し、検量線より色素濃度を算出した。なお、前記検量線は、エバンス青を0、3.125、6.25、12.5、25、50、100、及び200μg/mL濃度になるように、2mol/L水酸化カリウム水溶液2mLを加え、無処置対照のラット皮膚を加えて溶解して調整した色素標準液の630nm吸光度を測定することにより作成した。 Into the back skin of 8-week-old male SD rat (Claire Japan), 0.1 mL of anti-DNP-DNP-Ascaris IgE (Seikagaku Corporation, Tokyo) diluted 800-fold was injected. 48 hours later, the telephoric acid or cromoglycate sodium was intravenously injected (1 mg / kg amount), and after 1 minute, a DNP-Ascaris (Cosmo Bio) solution having a final concentration of 0.3 mg / mL And a final concentration of 5 mg / mL of Evans blue solution (Sigma Aldrich, Tokyo) was intravenously injected (1 mL / animal). After 30 minutes, the rats were exsanguinated under ether anesthesia, the skin around the injection site was peeled off, and the pigment leakage part was cut out with scissors. This was treated in 2 mL of 2 mol / L aqueous potassium hydroxide solution at 37 ° C. for 21 hours, and 6 mL of a 7.4% phosphoric acid / acetone mixture (1: 3) was added and mixed vigorously. After centrifuging at 3,000 rpm for 15 minutes, the absorbance at 630 nm of 100 μL of the collected supernatant was measured with a spectrophotometer, and the dye concentration was calculated from a calibration curve. The calibration curve was obtained by adding 2 mL of 2 mol / L potassium hydroxide aqueous solution so that Evans blue had a concentration of 0, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μg / mL. This was prepared by measuring the absorbance at 630 nm of a dye standard solution prepared by adding and dissolving rat skin of an untreated control.
結果を表3に示す。テレホール酸投与により、受身PCA反応は抑制された。すなわち、テレホール酸は抗アレルギー活性を有することが示唆された。 The results are shown in Table 3. The passive PCA reaction was suppressed by administration of teleholic acid. That is, it was suggested that teleholic acid has antiallergic activity.
本発明の抗アレルギー剤は、アレルギーの治療又は予防の用途に適用することができる。
以上、本発明を特定の態様に沿って説明したが、当業者に自明の変形や改良は本発明の範囲に含まれる。
The antiallergic agent of the present invention can be applied to the use for treatment or prevention of allergy.
As mentioned above, although this invention was demonstrated along the specific aspect, the deformation | transformation and improvement obvious to those skilled in the art are included in the scope of the present invention.
Claims (2)
で表されるテレホール酸化合物若しくはその塩を有効成分として含有することを特徴とする抗アレルギー剤。 The following general formula (I):
Antiallergic agents you characterized by containing Terehoru acid compound or a salt thereof as an active ingredient represented in.
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Free format text: JAPANESE INTERMEDIATE CODE: R250 |
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LAPS | Cancellation because of no payment of annual fees |