JPS5892617A - Polysaccharide ps-b derived from plant belonging to epimedium genus, its preparation, and prophylactic agent containing polysaccharide ps-b as active component - Google Patents
Polysaccharide ps-b derived from plant belonging to epimedium genus, its preparation, and prophylactic agent containing polysaccharide ps-b as active componentInfo
- Publication number
- JPS5892617A JPS5892617A JP56190202A JP19020281A JPS5892617A JP S5892617 A JPS5892617 A JP S5892617A JP 56190202 A JP56190202 A JP 56190202A JP 19020281 A JP19020281 A JP 19020281A JP S5892617 A JPS5892617 A JP S5892617A
- Authority
- JP
- Japan
- Prior art keywords
- reaction
- water
- polysaccharide
- molecular weight
- precipitate
- 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
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/29—Berberidaceae (Barberry family), e.g. barberry, cohosh or mayapple
- A61K36/296—Epimedium
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
Abstract
Description
【発明の詳細な説明】
本発明は、メギ科イカリソウ属（Ｅｐｉｍｅｄｉｔｔｍ
ｓｐ、）植物の全草から得られる多糖体（ＰＳ−Ｂと命
名）及びその製造方法並びにそれを有効成分とする感染
防禦剤に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the use of a plant of the genus Epimeditum of the family Berberaceae (Epimedittm).
The present invention relates to a polysaccharide (named PS-B) obtained from the whole plant of sp.
イカリソウ属と称される植物としては、イカリソウ（Ｅ
ｐｉｍｅｄｉｗｍ ｍａｃｒａｎｔｈｕｍ、 Ｍ、ｅｔ
、Ｄ、 ｖａｒ。A plant called Epimedium genus is Epimedium (E
pimediwm macranthum, M, et
, D, var.
ｖｉｏｌａｃｅｒｂｍ、 Ｆｒ、）、ホザキノイ力。リ
ソウ（１：ｐ＊ｍｅｄｉｔｂｙｙｒ ｓａｇｉｔｔａｔ
ｕ、ｍ、 Ｂａｋ、）、シロバナイカリソウ（Ｅｐｉｍ
ｔｄｉｕｍ ｍａｃｒａｎｔｈｔＬｍ、 Ｍ、ｅｔ、Ｄ
、）、キバナイカリソウ（Ｅｐｉｍｔｄｉｔｂｍ ｋｏ
ｒｅａｒＬｌＬｍ、 Ｎａｋ、 ）等が知られている。violacerbm, Fr,), Hozakinoi force. Risou (1:p*meditbyr sagittat
u, m, Bak,), Epim.
tdium macranthtLm, M, et, D
), Epimtditbm ko
rearLlLm, Nak, ), etc. are known.
これらのイカリソウ属植物は、日本、中国、朝鮮等に自
生する多年草であり、その茎葉根は漢方では淫羊舊と命
名され、全草を煎じて強壮、強精の目的に用いられてい
る。なお、これらのイカリソウ属植物は、種の違いによ
ってもその効果には大差なく、生薬として市販されて・
いるものは、これらの単独種または混合′働程をイカリ
ソウと称している。このイカリソウの成分研究は古くか
ら行われており、例えば光弁等〔薬学雑誌５５ ５３７
，７０５，７１９゜７８８．１１５９（１９５５））、
富田等〔薬学雑誌７７ １１４、２１２（１９５７））
などの報告があり、地上部からイカリイン、地下部から
デス−０−メチルイカリイン、マグノフロリンなる物質
が見出され、夫々化学構造が決定されている。また薬理
学的研究については、前日〔東京医事新語２１５５、２
７９５（１９５２））、三宅〔岡山医学会雑誌４９ ４
４．２０４３（１９５７））、平部等〔基礎と臨床４
１３９（１９７０））などにより、その抽出エキスに精
液分易促進作用、血圧降下作用、制糖作用のあることが
報告されている。しかし、その詳細については依然不明
な点が多い。These plants of the genus Epimedium are perennial plants that grow naturally in Japan, China, Korea, and other countries, and their stems, leaves, and roots are named Inyangfu in Chinese medicine, and the whole plant is infused and used for the purpose of tonicity and tonicity. The effects of these plants of the genus Epimedium do not differ much depending on the species, and they are not commercially available as herbal medicines.
Some people refer to these single species or mixed processes as epimedium. Research on the components of Epimedium has been conducted for a long time, such as Koben et al. [Pharmacy Journal 55 537
, 705, 719° 788.1159 (1955)),
Tomita et al. [Pharmaceutical Journal 77 114, 212 (1957)]
There have been reports such as icariin in the aboveground parts, and des-0-methylicariin and magnoflorin in the underground parts, and the chemical structures of each have been determined. Regarding pharmacological research, the previous day [Tokyo Medical Shingo 2155, 2
795 (1952)), Miyake [Okayama Medical Society Journal 49 4
4.2043 (1957)), Hirabe et al. [Basic and Clinical 4
139 (1970)), etc., it has been reported that its extract has semen separation promoting effects, blood pressure lowering effects, and antiglycemic effects. However, many details remain unclear.
本発明者らは先に特願昭５５−２１１５２号、特願昭５
５−５７４２４号、特願昭５５−８５５３．９号にて、
イカリソウ属植物より免疫増強作用を有する抽出物を見
出したが、更に研究の結果感染防禦作用を有する多糖体
ＰＳ−Ｂの抽出、単離に成功し本発明を完成した。The present inventors previously filed Japanese Patent Application No. 55-21152,
No. 5-57424, patent application No. 55-8553.9,
An extract having an immune-enhancing effect was discovered from a plant of the genus Epimedium, and as a result of further research, the present invention was completed by successfully extracting and isolating polysaccharide PS-B, which has an infection-preventing effect.
以下に、本発明の詳細な説明する。The present invention will be explained in detail below.
まず、本発明の多糖体ＰＳ−Ｈの理化学的性質は次のと
おりである。First, the physicochemical properties of the polysaccharide PS-H of the present invention are as follows.
１ 元素分析： Ｃ：５７．６９ Ｈ：６．ＯＯ灰分
：微１２ 分子量：ｓｏ、ｏｏｏ±２５，０００（平均
分子量）５ 物質の性状：晶色〜微かっ色不定形粉末４
分解点：２００℃
５ 紫外部吸収スペクトル：２４０〜ａａａｎｙｎに極
大吸収は認めらＡない。1 Elemental analysis: C: 57.69 H: 6. OO ash content: Fine 12 Molecular weight: so, ooo ± 25,000 (average molecular weight) 5 Property of substance: Crystal color to faint brown amorphous powder 4
Decomposition point: 200°C 5 Ultraviolet absorption spectrum: No maximum absorption observed between 240 and aaanyn.
６ 光外部吸収スベ久トルコ第１図のスペクトルを示す
。ν九：二（副−１）
５３５０、２９２０．１６３０．１５５０゜１４２０、
１１００゜
７７）Ｈ：６．９±０．１ （ＰＳ−Ｂ１００シを水５
０屑ｇに溶解するとき）
８ 溶解性：（ａ）可溶・・・水
（ｈ）不溶・・・メタノール、エタノール、アセトン、
酢酸エチル、
ジエチルエーテル、ヘキサン、
クロロホルム。6 The spectrum of the optical external absorption substrate shown in Figure 1 is shown. ν9:2 (sub-1) 5350, 2920.1630.1550°1420,
1100°77) H: 6.9±0.1 (PS-B100 water 5
8 Solubility: (a) Soluble...water (h) Insoluble...methanol, ethanol, acetone,
Ethyl acetate, diethyl ether, hexane, chloroform.
９ 比旋光度：〔α躍＝ −ｏ、 ５°（Ｈ２Ｏ中、Ｃ
＝０．７５６ｔｓ）
１０ 呈色反応：
次の諸反応に陽性を示す。9 Specific rotation: [α = −o, 5° (in H2O, C
=0.756ts) 10 Color reaction: The following reactions are positive.
（α）アンスロン硫酸反応 （ｂ）モーリッシュ反応
（Ｃ）スカトール反応 （ｄ）ビアル反応次の諸反応に
陰性を示す。(α) Anthrone sulfuric acid reaction (b) Molish reaction
(C) Skatole reaction (d) Vial reaction The following reactions are negative.
（α）ニンヒドリン反応 （Ａ） ２．４−ＤＮＰ反応
（Ｃ）セリヴアノフ反応 （ｄ）ナフトレゾルシン反応
（ｇｌカルバゾール硫酸反応
１１ 構成糖：
多糖体ＰＳ−Ｂ２０″Ｉ９に１規定硫酸５罰を加え、２
時間１００℃で加熱、還流する。今後、水酸化バリウム
で中和し、沈殿を濾別し、得られた濾液をペーパークロ
マトグラフィーにかけるとアラビノースとガラクトース
の存在が確認される。(α) Ninhydrin reaction (A) 2.4-DNP reaction (C) Selivanov reaction (d) Naftresorcin reaction (gl carbazole sulfuric acid reaction 11 Constituent sugar: Add 1 N sulfuric acid 5 times to polysaccharide PS-B20″I9, 2
Heat and reflux at 100°C for an hour. In the future, the presence of arabinose and galactose will be confirmed by neutralizing with barium hydroxide, filtering out the precipitate, and subjecting the resulting filtrate to paper chromatography.
１２ 〜均一性： （α）超遠心 平衡密度勾配遠心（１２０，０ＯＯｐＸ７２時間。12 ~ Uniformity: (α) Ultracentrifugation Equilibrium density gradient centrifugation (120,000p x 72 hours.
ＣａＣ１２）−”Ｃ単一
（ｈ）電気泳動
酢酸セルロース膜（２０〜２５ ”１ｃｍ警 ２時間）
で単一
（Ｃ）ゲル濾過
セファデックスＧ１００で単−
次に、本発明の多糖体ＰＳ＝Ｂの抽出、゛単離、精製方
法について述べる。CaC12)-"C single (h) electrophoresis cellulose acetate membrane (20-25" 1cm length 2 hours)
Single (C) Gel filtration Single using Sephadex G100 Next, the extraction, isolation, and purification methods of the polysaccharide PS=B of the present invention will be described.
第１工程
イカリソウ属植物を水に混和する有機溶媒と水との混合
溶媒または水のいずれかで抽出し、得られた抽出液を減
圧下濃縮する。この際、水に混和する有機溶媒としては
メタノール、エタノール、イソブーロバノール等の低級
アルコール類、アセトン等のケトン類、ジオキサン等の
水に混和するエーテル類等を用いる。またこれらの混合
有機溶媒を用いてもよい。また抽出溶媒として、１規定
以下の濃度の酢酸等の低級脂肪酸類、１モル以下の濃度
のエタノールアミン等の水と混和する低級アミン類、１
規定以下の濃度の水酸化ナトリウム等の水に易溶且つア
ルカリ性を示す水酸化物も用いることができる。水性抽
出液を得るこの操作は、濃縮操作のことを考えると、水
に混和する有機溶媒と水との混合溶媒を用い有機溶媒の
容量が５０チ以下の方が好ましい。また本発明に使用す
るイカリソウは、市販の全草をそのまま用いてもよいが
、細切を用いる方が好ましい。First step: A plant of the genus Epimedium is extracted with either a mixed solvent of an organic solvent and water that is miscible with water, or water, and the resulting extract is concentrated under reduced pressure. In this case, as the organic solvent that is miscible with water, lower alcohols such as methanol, ethanol, and isobutanol, ketones such as acetone, and ethers that are miscible with water such as dioxane are used. Further, a mixed organic solvent of these may also be used. In addition, as an extraction solvent, lower fatty acids such as acetic acid with a concentration of 1N or less, lower amines that are miscible with water such as ethanolamine with a concentration of 1M or less, 1
Hydroxides that are easily soluble in water and exhibit alkalinity, such as sodium hydroxide at a concentration below the specified level, can also be used. In this operation for obtaining an aqueous extract, considering the concentration operation, it is preferable that a mixed solvent of a water-miscible organic solvent and water be used and the volume of the organic solvent be 50 ml or less. Furthermore, although commercially available whole plants of Epimedium used in the present invention may be used as they are, it is preferable to use finely chopped pieces.
第２工程・
こうして得た水性抽出液を脱脂する。この操作は省略す
ることができるが、製剤上の面を考慮すると行う方が好
ましい。2nd step: The aqueous extract thus obtained is defatted. Although this operation can be omitted, it is preferable to perform it in consideration of formulation aspects.
脱脂操作は、水性抽出液に酢酸エチル等の低級脂肪酸エ
ステル類、クロロホルム等のハロゲン化炭化水素類、ジ
エチルエーテル等の水１混和しにくいエーテル類、ルー
へキサン等の脂肪族炭化水素類等の有機溶媒の１種また
は２種以上の混合溶媒を加え、激しく振盪した後、水層
のみ分取する。得られた水層は同様、の操作を３〜讐回
行った後、水浴上で加温してわずかに残っている有機溶
媒を除き、濾過することにより脱脂した抽出液となる。The degreasing operation involves adding lower fatty acid esters such as ethyl acetate, halogenated hydrocarbons such as chloroform, ethers that are difficult to miscible with water such as diethyl ether, and aliphatic hydrocarbons such as roohexane to the aqueous extract. After adding one or more organic solvents and shaking vigorously, only the aqueous layer is separated. The obtained aqueous layer is subjected to the same operation three times or more, heated on a water bath to remove a slight amount of remaining organic solvent, and filtered to obtain a defatted extract.
なお最初に脱脂操作を行った後、水に混和する有機溶媒
と水との混合溶媒または水のいずれかで抽出してもよい
。Note that after first performing a degreasing operation, extraction may be performed with either a mixed solvent of water and an organic solvent that is miscible with water, or water.
第５工程
この脱脂した水性抽出液に水と任意に混和する有機溶媒
を加え、沈殿を析出させる。生じた沈殿を濾取後、水と
任意に混オロする有機溶媒にて洗浄する。次いで、この
沈殿を水の中に注いだ後、水と任意に混和する有機溶媒
を加え、再度沈殿を析出させる。生じた沈殿を濾取し、
減圧下乾燥させると抽出物が得られる。この際、水と任
意に混和する有機溶媒としては、メタノール、エタノー
ル等の低級アルコール類もしくはアセトン等のケトン類
を用いる。またこれらの混合溶媒を用いてもよい。こう
して得られた抽出物は、水で再度抽出し精製してもよい
。即ち、室温下、水を加え、十分攪拌後、濾過する。Fifth step: An organic solvent miscible with water is added to the defatted aqueous extract to precipitate a precipitate. The resulting precipitate is collected by filtration and washed with an organic solvent that is optionally miscible with water. Next, after pouring this precipitate into water, an organic solvent that is optionally miscible with water is added to precipitate again. The resulting precipitate was collected by filtration,
An extract is obtained by drying under reduced pressure. At this time, lower alcohols such as methanol and ethanol, or ketones such as acetone are used as the organic solvent that is optionally miscible with water. A mixed solvent of these may also be used. The extract thus obtained may be extracted again with water and purified. That is, water is added at room temperature, thoroughly stirred, and then filtered.
この濾液を減圧下、濃縮乾固することにより精製できる
。Purification can be achieved by concentrating this filtrate to dryness under reduced pressure.
第４工程
第３工程で得られた抽出物にセチルトリメチルアンモニ
ウム塩、セチルピリジニウム塩等長鎖を有する第４級ア
ンモニウム塩を加え、生じた複合体を濾過又は遠心分離
で除去する。濾液を分取し、水と任意に混和する有機溶
媒を加え、生じた沈殿を濾取する。Fourth step A quaternary ammonium salt having a chain of equal length such as cetyltrimethylammonium salt or cetylpyridinium salt is added to the extract obtained in the third step, and the resulting complex is removed by filtration or centrifugation. The filtrate is separated, an organic solvent miscible with water is optionally added, and the resulting precipitate is collected by filtration.
第５工程
第４工程で得られた沈殿に水を加え、３５〜４５℃加温
下、十分に攪拌し溶解させる。この溶液を限外濾過し、
濾過膜を通過する低分子化合物を除去する。数回、この
操作を行い膜を通過する低分子化合物を完全に除去する
。膜上に捕捉された濃縮液を分取し、水を加え、希釈し
、水と任意に混和する有機溶媒を加えると沈殿が得られ
る。Fifth step Water is added to the precipitate obtained in the fourth step, and the mixture is sufficiently stirred and dissolved while heating at 35 to 45°C. This solution is ultrafiltered,
Removes low molecular weight compounds that pass through the filtration membrane. This operation is repeated several times to completely remove low-molecular compounds passing through the membrane. A precipitate is obtained by separating the concentrated liquid captured on the membrane, diluting it by adding water, and adding an organic solvent that is optionally miscible with water.
第６エ程
第５工程で得られた沈殿をクロマトグラフィーに付すこ
とにより目的とする多糖体ＰＳ−Ｂを得ることができる
。クロマトグラフィーとしては吸着剤を用いたクロマト
グラフィーまたは分子ふるい効果を有する担体を用いた
クロマトグラフィーを利用するとよい。The desired polysaccharide PS-B can be obtained by subjecting the precipitate obtained in the fifth step of the sixth process to chromatography. As the chromatography, it is preferable to use chromatography using an adsorbent or chromatography using a carrier having a molecular sieving effect.
以下に操作の具体例を２例示す。Two specific examples of operations are shown below.
第１例：第５工程で得られた沈殿を吸着クロマトグララ
イ−に付し、水で溶出させ、糖の定性反応例えばａ−ナ
フトール反応で陽性を示す水溶出画分のみを分取する。First example: The precipitate obtained in the fifth step is subjected to adsorption chromatography, eluted with water, and only the water-eluted fraction showing positive in a qualitative sugar reaction, such as an a-naphthol reaction, is collected.
この溶出画分を減圧上濃縮乾固することにより本発明多
糖体ＰＳ−Ｂを得ることができる。吸着剤としては、ポ
リアミド、陰イオン交換樹脂等を用いることができ、ポ
リアミドを用いるのが好ましい。なお、ポリアミドを里
いた吸着クロマトグラフィーにおいては、本発明多糖体
ＰＳ−Ｂを溶出させたあとさらに続けて、溶出溶媒を揮
発性の弱アルカリ性水溶液、例えばアンモニア水溶液に
変えて溶出し、糖の定性反応に陽性を示す溶出画分を分
取し、減圧下濃縮乾燥又は凍結乾燥すると抽出物にを得
ることができる。この抽出物には免疫増強作用を有する
。The polysaccharide PS-B of the present invention can be obtained by concentrating this elution fraction to dryness under reduced pressure. As the adsorbent, polyamide, anion exchange resin, etc. can be used, and polyamide is preferably used. In addition, in adsorption chromatography using polyamide, after eluting the polysaccharide PS-B of the present invention, the elution solvent is changed to a volatile weakly alkaline aqueous solution, such as an ammonia aqueous solution, and elution is performed to qualitatively characterize the sugar. An extract can be obtained by separating the eluted fraction showing a positive reaction and concentrating and drying it under reduced pressure or freeze-drying it. This extract has immunoenhancing properties.
第２例：第５工程で得られた沈殿を水に溶解後、ゲル濾
過等の分子ふるい効果を有す、る担体を用いたクロマト
グラフィーに付し、添付図面第２図のＦｒ−２に該当す
る画分を分取する。この溶出画分を減圧下濃縮乾固する
ことにより本発明、多糖体ＰＳ−Ｂを得ることカーでき
る。2nd example: After dissolving the precipitate obtained in the 5th step in water, it was subjected to chromatography using a carrier that has a molecular sieving effect such as gel filtration, and was converted to Fr-2 in Figure 2 of the attached drawing. Collect the relevant fraction. The polysaccharide PS-B of the present invention can be obtained by concentrating this elution fraction to dryness under reduced pressure.
本発明多糖体ＰＳ−Ｂの有用性を確認するため、以下に
示す試験を行った。In order to confirm the usefulness of the polysaccharide PS-B of the present invention, the following tests were conducted.
（１）感染−防禦効果判定試験
（１）大腸菌
多糖体ＰＳ−ＢをＩＣＲ／ＪＣＬ系５週令雄マウス（体
重的２５Ｆ）、１群１０匹に１日１回５日間連日計５回
背部皮下投与した。(1) Infection-prevention effect determination test (1) Escherichia coli polysaccharide PS-B was administered to ICR/JCL 5-week-old male mice (weight 25F), once a day for 5 days in a group of 10 mice, for a total of 5 times on the back. Administered subcutaneously.
対照群には生理食塩水（以下ＰＳと略す）。Physiological saline (hereinafter abbreviated as PS) was used as a control group.
を投与した。was administered.
６日目に大腸菌（Ｅ、 Ｃｏ ｌ ｉＭＬ’４７０７株
）を腹腔内投与した。菌接種後７′Ｂ目における生存菌
数を観察した。結果を第１表に示す。On the 6th day, Escherichia coli (E, ColiML'4707 strain) was administered intraperitoneally. The number of surviving bacteria was observed at 7'B after inoculation. The results are shown in Table 1.
多糖体ＰＳ−Ｂ投与−より、大腸菌に対する感染防禦作
用が認められた。Administration of polysaccharide PS-B was found to have a protective effect against Escherichia coli infection.
（１１）緑膿菌、肺炎桿菌
（１）と同様の方法で緑膿菌（ｐＳ、 ａｅｒＬＬｇｉ
ｎｏ、？ａ癌患者、腎透析患者、癌患者等の感染症に抗
生物質と併用させて用いる等の用途が期待できる。(11) Using the same method as Pseudomonas aeruginosa and Klebsiella pneumoniae (1), Pseudomonas aeruginosa (pS, aerLLgi)
No? It can be expected to be used in combination with antibiotics to treat infectious diseases such as cancer patients, renal dialysis patients, and cancer patients.
本発明抽出物は、いずれの抗菌物質とも併用、配合する
ことができ、例えば、アンピシリン、アモキシシリン、
ピペラジリン、カルベニシリン、シクラシリン、ヘタシ
リン、プロピシリン、フルクロキサンリン、クロキサシ
リン、オキサシリン等のペニシリン系抗生物質、セファ
レキンン、セファロチン、セファロリジン、セファゾリ
ン、セファログリシン等のセファロスポリン系抗生物質
、チェナマイシン等のβ−ラクタム系抗生物質、ジョサ
マイシン、エリスロマイシン、オレアンドマイシン、キ
タサマイシン、スピラマイシン等のマクロライド系抗生
物質、ミノサイクリン、ドキシサイクリン、メタサイク
、リン、テトラサイクリン、クロルテトラサイクリン、
オキシテトラサｊ久リン、デメチルクロルテトラサイク
°リン、ピロリジノメチルテトラサイクリン等のテトラ
サイクリン系抗生物質、ストレプトマイシン、リファマ
イシン、ゲンタマイシン、パロモマイシン、カナマイ７
ン、フラジオマイシン等のアミノグリコ７ド系抗生物質
、グラミシジン、バシトラシン、コリスチン、ポリミキ
シンＢ等のポリペプチド系抗生物質、バリオチン、アン
ピシリンＢ、）リコ÷イシン、ペンタマイシン、ビマシ
リン、ナイスクチン等のポリエン系抗生物質、リファマ
イシン、リンコマイシン、ナリジキス酸等がめげられる
。The extract of the present invention can be combined with or blended with any antibacterial substance, such as ampicillin, amoxicillin,
Penicillin antibiotics such as piperagiline, carbenicillin, cyclacillin, hetacillin, propicillin, flucloxanrin, cloxacillin, oxacillin, cephalosporin antibiotics such as cephalequin, cephalothin, cephaloridine, cefazolin, cephaloglycin, and β- Lactam antibiotics, macrolide antibiotics such as josamycin, erythromycin, oleandomycin, kitasamycin, spiramycin, minocycline, doxycycline, methacycline, phosphorus, tetracycline, chlortetracycline,
Tetracycline antibiotics such as oxytetracycline, demethylchlortetracycline, pyrrolidinomethyltetracycline, streptomycin, rifamycin, gentamicin, paromomycin, Kanamai 7
Aminoglyco7d antibiotics such as N, fradiomycin, polypeptide antibiotics such as gramicidin, bacitracin, colistin, polymyxin B, polyenes such as variotin, ampicillin B,) lyco÷isin, pentamycin, bimacillin, and nyscutin. Antibiotics such as rifamycin, lincomycin, and nalidikisic acid are recommended.
本発明多糖体ＰＳ−Ｂを人体に投与するに当っては経口
投与、注射（皮下、筋肉、静脈）その他の方法が用いら
れる。When administering the polysaccharide PS-B of the present invention to the human body, oral administration, injection (subcutaneous, intramuscular, intravenous) and other methods are used.
経口投与の際、固形製剤として用いる場合は錠剤、顆粒
剤、散剤、カプセル剤等にすることができ、製剤上一般
に使用される糖類、セルロース調合物のような賦形剤、
でんぷんペースト、メチルセルロースのような結合剤、
増量剤、崩壊剤等の添加物を傾含してもよい。また経口
用液体製剤として用いる場合は、内用水剤、懸濁液剤、
乳剤、シロップ剤などの形態であっても良く、また使用
する前に再溶解させる乾燥生成物の形態であってもよい
。When used as a solid preparation for oral administration, it can be made into tablets, granules, powders, capsules, etc., and excipients such as sugars and cellulose preparations commonly used in the preparation,
binders such as starch pastes, methylcellulose,
Additives such as fillers and disintegrants may also be included. When used as oral liquid preparations, oral solutions, suspensions,
It may be in the form of an emulsion, syrup, etc., or as a dry product which is redissolved before use.
注射の場合は、水溶液、懸濁剤、油性又は水溶性乳剤の
形態であっても良いが、通常滅菌水又は生理食塩液など
水性液体媒体に溶解又は懸濁することにより調整される
。必要に応じて一般に使用される溶解剤、安定化剤、保
存剤、等張化剤などを加えても良い。In the case of injection, it may be in the form of an aqueous solution, suspension, oil-based or water-soluble emulsion, but it is usually prepared by dissolving or suspending it in an aqueous liquid medium such as sterile water or physiological saline. If necessary, commonly used solubilizers, stabilizers, preservatives, tonicity agents, etc. may be added.
このようにして得られた注射液剤は筋肉注射、皮下注射
等適当な方法で投与される。The injection solution thus obtained is administered by an appropriate method such as intramuscular injection or subcutaneous injection.
本発明多糖体ＰＳ−Ｂを成人に投与する場合は１日０．
０２５〜１０ ”Ｆ／Ａ９の範囲で用いることができる
。この際、用量は症状、年令、剤型等により適宜増減さ
れる。When administering the polysaccharide PS-B of the present invention to adults, the polysaccharide PS-B is administered at 0.00% per day.
It can be used within the range of 0.025 to 10"F/A9. At this time, the dose is adjusted as appropriate depending on the symptoms, age, dosage form, etc.
次に、本発明の実施例を示して更に詳細に説明するが、
本発明はこれに限定されるものではない。Next, examples of the present invention will be shown and explained in more detail.
The present invention is not limited to this.
実施例１
市販のイカリソウ（キバナイカリソウ、韓国産）細切２
Ｋｇに５０％エタ／−ル（Ｖ／Ｖ） １５／。Example 1 Commercially available epimedium (Kibanaicou, produced in Korea) shredded 2
Kg with 50% ethanol (V/V) 15/.
を加え、還流冷却器を付し、水浴上５０℃で６時間加温
抽出した。抽出後、温時濾過し、残渣は新たに５０％エ
タノール（Ｖ／ｖ ） １５ ｔずつで同様な抽出、濾
１尚操作を３回行った。途濾液を合わせ、減圧下４５℃
で濃縮し、水性抽出液１０ｔを得た。この抽出液を分液
ロートに入れ、酢酸エチル５ｔを加えて激しく振盪し、
水層のみ分取した。この水層は新たに、酢酸エチル５ｔ
ずつで同様な抽出を３回行った。水層を減圧下濃縮し、
”残存する酢酸エチルを留去した後、濾過した。濾液に
エタノールｓａｔを添加、攪拌後、−夜装置し、析出す
る沈殿を濾取した。was added, a reflux condenser was attached, and the mixture was heated and extracted on a water bath at 50°C for 6 hours. After extraction, the mixture was filtered while still warm, and the residue was subjected to the same extraction and filtration procedure three times using 15 t each of 50% ethanol (V/v). Combine the filtrate and heat at 45°C under reduced pressure.
The mixture was concentrated to obtain 10 tons of an aqueous extract. Put this extract into a separating funnel, add 5 t of ethyl acetate, and shake vigorously.
Only the aqueous layer was separated. This aqueous layer was newly added with 5 t of ethyl acetate.
Similar extractions were performed three times. The aqueous layer was concentrated under reduced pressure.
After distilling off the remaining ethyl acetate, it was filtered. Ethanol sat was added to the filtrate, stirred, and then left overnight, and the precipitate was collected by filtration.
沈殿をエタノール２００ ｗｔｌで洗浄した後、水２ｔ
に溶解し、次いでエタノール８ｔを添加した後沈殿を濾
取した。この沈殿を水１ｔにて抽出後、濾過し、濾液を
減圧下濃縮乾燥し茶褐色粉末状の抽出物１６ｙを得た。After washing the precipitate with 200 wtl of ethanol, add 2t of water.
After adding 8 tons of ethanol, the precipitate was collected by filtration. This precipitate was extracted with 1 t of water, filtered, and the filtrate was concentrated and dried under reduced pressure to obtain extract 16y in the form of a brown powder.
この抽出物１６ＰＫ［１０２モル硫酸ナトリウム水溶液
１１を加え、４０℃で加温渾拌後、温時濾過した。３０
〜４０℃加温下、この濾液に１０チーセチルトリメチル
アンモニウムブロマイト水溶液１００ｄを加え、−夜５
７℃にて保温、静置後、遠心分離（５，００Ｏｒ、 ｐ
、’ ｍ、、１０分間）し、上澄液１ｔを得た。この上
澄液に３ｔのエタノールを加え生じた沈殿（３，Ｏｆ
）を濾取した。This extract 16PK [102M aqueous sodium sulfate solution 11] was added, stirred while heating at 40°C, and then filtered while still warm. 30
To this filtrate was added 100 d of an aqueous solution of 10-thiacetyltrimethylammonium bromite while heating at ~40°C, and at night 5
After keeping warm at 7℃ and leaving it still, centrifugation (5,00 Or, p
,' m,, for 10 minutes) to obtain 1 t of supernatant. 3 tons of ethanol was added to this supernatant, resulting in a precipitate (3, Of
) was collected by filtration.
この沈殿（５，Ｏｙ ）に水１ｔを加え、４０℃で加温
、攪拌後、減圧下残存エタノールを留去し濾過した。こ
の濾液は更にミリポアフィルタ−ＡＰ２０（径１４２１
１１１１１、日本ミリポアリミッテッド社製）、ミリポ
アメンブランフィルタ−Ｉ（Ａ型（径１４２１１！１１
１％孔径０．４５μｍ、日本ミリポアリミッテッド社製
）を用いて濾過した。この濾液をミリポアベリコン限外
濾過膜ＰＳＡＣ（公称分子量限外値１，０００、日本ミ
リボアリミツテツド社製）を用いて限外濾過し、膜通過
部を除去した。次に、膜不通過部に水５００ｇＡｆを加
え、希釈後、再度限外濾過した。この操作を３回行なっ
た後、膜不通過部を分取した。□そして、膜不通一部を
水にて２５０ ｍｌに希釈し工ダノール１ｔを加え、沈
殿２．０ｙを得た。1 ton of water was added to this precipitate (5, Oy), heated at 40° C., stirred, and then the remaining ethanol was distilled off under reduced pressure and filtered. This filtrate was further filtered using a Millipore filter-AP20 (diameter 1421
11111, manufactured by Japan Millipore Limited), Millipore Membrane Filter-I (Type A (diameter 14211!11
It was filtered using 1% pore size 0.45 μm (manufactured by Japan Millipore Limited). This filtrate was ultrafiltered using a Millipore Vericon ultrafiltration membrane PSAC (nominal molecular weight limit value 1,000, manufactured by Nippon Millipore Limited) to remove the membrane passing portion. Next, 500 g of water was added to the part that did not pass through the membrane, and after dilution, ultrafiltration was performed again. After performing this operation three times, the portion that did not pass through the membrane was collected. □Then, the part that did not pass through the membrane was diluted with water to 250 ml, and 1 t of Danol was added to obtain 2.0 y of precipitate.
この沈殿に水５００ ｔｕｔを加え約４０℃に加温攪拌
し、溶解させた後温時濾過し、得られた濾液をポリアミ
ドＣ−２００（和光紬薬社製）を用いたカラムクロマト
グラフィー（径×長さ＝４５ ｍｍ Ｘ ４００ｍｍ
）に付し、水２ｔで溶出し、α−ナフトール反応で陽性
を示す水溶出部を減圧下濃縮乾燥し、目的とする多糖体
ＰＳ−Ｂ １． Ｏ’１を得た。その理化学的性質は前
述のとおりであった。サラに、１．４％アンモニア水水
溶溶液３ｔ溶出し、α−ナフトール反応で陽性を示すア
ンモニア溶出部を減圧下濃縮乾燥し、次の理化学的性質
を有する抽出物Ｎ０１８りを婦だ。500 tut of water was added to this precipitate, heated to about 40°C, stirred, dissolved, and then filtered while still warm.The resulting filtrate was subjected to column chromatography (diameter x length = 45mm x 400mm
), eluted with 2 tons of water, and the water-eluted portion showing positive in the α-naphthol reaction was concentrated and dried under reduced pressure to obtain the desired polysaccharide PS-B 1. Obtained O'1. Its physicochemical properties were as described above. Then, 3 tons of 1.4% ammonia aqueous solution was eluted, and the ammonia eluted portion showing positive in α-naphthol reaction was concentrated and dried under reduced pressure to obtain an extract N018 having the following physical and chemical properties.
１ 物質の性状：微褐色〜褐色粉末つ
２ 紫外部吸収スペクトル：２４０〜４００ ｎｍに極
太吸収は認められない。1. Properties of substance: slightly brown to brown powder. 2. Ultraviolet absorption spectrum: no extremely thick absorption is observed in the range of 240 to 400 nm.
６ 赤外部吸′収スペクトル：第３図のスペクトルを示
す。6 Infrared absorption spectrum: Shows the spectrum in Figure 3.
１／、”、；（ｃｒｎ−”） ３２００．１６００．１
４００゜１１００゜
４ ＰＨニア、０±（１２（抽出物７Ｖ１００’７を
７７、５０ ｄに溶解するとき）
５ 溶解性：可溶・・・水
不溶・・・メタノール、エタノール、
アセトン、酢酸エチル、ジ
エチルエーテル、ヘキサン、
グロロホルム。1/,”,;(crn-”) 3200.1600.1
400゜1100゜4 PH near, 0±(12 (when extract 7V100'7 is dissolved in 77,50 d) 5 Solubility: Soluble...Water-insoluble...Methanol, ethanol, acetone, ethyl acetate , diethyl ether, hexane, gloloform.
６ 呈色反応： 次の諸反応に陽性を示す。6 Color reaction: The following reactions are positive.
（α）アンスロン硫酸反応（ｈ）モーリッシュ反応（Ｃ
）スカトール反応 （ｄ）ビアル反応次の諸反応に
陰性を示す。(α) Anthrone sulfuric acid reaction (h) Molish reaction (C
) Skatole reaction (d) Vial reaction Shows negative results for the following reactions.
（ａ） ＝ ７ ヒドリン反応 （ｂ） ２．４−Ｄ
ＮＰ反応（Ｃ）セリヴアノフ反応 （ｄ）ナフトレゾ
ルシン反応（−１力ルバゾール硫酸反応
７ 構成糖：
抽出物に２０１１Ｌｙに１規定硫酸５１１１１を加え、
２時間加熱還流する。今後、水酸化バリウムで中和し、
沈殿を濾別後、得られる濾液をペーパークロマトグラフ
ィーにかけるとアラビノースとガラクトースの存在が確
認される。(a) = 7 Hydrin reaction (b) 2.4-D
NP reaction (C) Selivanov reaction (d) Naftresorcin reaction (-1% Rubazole sulfuric acid reaction 7 Constituent sugars: Add 1N sulfuric acid 51111 to 2011Ly to the extract,
Heat to reflux for 2 hours. From now on, it will be neutralized with barium hydroxide,
After filtering off the precipitate, the resulting filtrate is subjected to paper chromatography to confirm the presence of arabinose and galactose.
この抽出物にの有用性を確認するため、以下に示す試験
を行った。In order to confirm the usefulness of this extract, the following tests were conducted.
（１）網内系機能試験
７週令のＩＣＲ／ＪＣＬ系雄マウス（体重約３０））、
１群１０匹に、抽出物Ａ’ ０．７２”９７Ｋｇ体重／
日を１日１回連日５日間計５回ｊ復腔内投、与した。対
照群にはリン酸塩緩衝生理食塩水を授与した。(1) Reticuloendothelial system function test 7-week-old ICR/JCL male mice (body weight approximately 30),
Extract A'0.72"97Kg body weight per group of 10 animals
The drug was administered intracavitally once a day for 5 days in total, 5 times in total. The control group received phosphate buffered saline.
網内系の食作用活性に対する影響をみるため、抽出物Ａ
′の最終投与２４時間後に、抽出物Ｎ投与群及び対照群
の各マウスの尾静脈より、コロイド性炭素（ｐｅｌｉｋ
ａｎａｃｔｉｒＬｇ ｃａｒｂｏｎＣｏ／ １４５１
ａ、ギュンタ・バーブナ−（（３ｎｔｈｅｒＷａ夕ｎｅ
ｒ ）社、***〕を注入し、血中からのクリアランスを
調べた。即ち、コロイド性炭素を３％−ゼラチン生理食
塩水で２倍希−釈した液を、１０Ｉｌ！ｊ／９体重とな
るよう尾静脈から注入し、眼窩穿刺法により、ヘパリン
処理したマイクロピペットで血液［１０１０ｍ７！を採
取し、直ちに０．１ ％ ＮＩＺＩＩＣＯ３２ｔｄに移
入して溶血させ、日立ダブルビーム１２４型（日立製作
新製）を用い６５０ｎｍにおける吸光度を測定した。食
作用係数はコロイド性炭素希釈液を静脈内注射して、２
分後（’ｌ）及び２０分後（ｔｚ ） Ｋ 採血Ｌ、そ
の炭素濃度（２分後の濃度Ｃ１，２０分後の濃度Ｃ２）
を下式に挿入して算出した。To examine the effect on the phagocytic activity of the reticuloendothelial system, extract A
24 hours after the final administration of colloidal carbon (pelik) from the tail vein of each mouse in the extract N administration group and control group.
anactirLg carbonCo/1451
a, Günter Bärbner ((3ntherWa evening
R), West Germany] was injected, and its clearance from the blood was examined. That is, a solution obtained by diluting colloidal carbon twice with 3% gelatin saline was diluted to 10 Il! Blood was injected through the tail vein to give a weight of J/9, and blood [1010 m7! was collected, immediately transferred to 0.1% NIZIICO32td for hemolysis, and the absorbance at 650 nm was measured using Hitachi Double Beam Model 124 (manufactured by Hitachi Seisakusho). The phagocytosis coefficient was determined by intravenous injection of diluted colloidal carbon solution.
Minutes later ('l) and 20 minutes later (tz) K Blood collection L, its carbon concentration (concentration C1 after 2 minutes, concentration C2 after 20 minutes)
It was calculated by inserting into the formula below.
結果を第４表に示す。The results are shown in Table 4.
第４表 網内系機能試験
（注）上記表中の数値は平均信士標準偏差を示す抽出物
にの静脈経路のＬ Ｄ ｓｏ ＜”ｆ／”？ ）はリッ
テフイールドーウィルコクラン法（Ｊ、 ｐｈａｒｍ、
ｅｘｐ。Table 4: Reticuloendothelial system function test (Note) The values in the table above indicate the average standard deviation of the intravenous route of the extract. ) is the Rittefield-Wil-Cochran method (J, pharm,
exp.
Ｔｈｅｒ、 ９６９９ （１９４９）記屋〕で行ったと
ころ６００以上であった。Ther, 9699 (1949) Kikiya] and the number was over 600.
以上のことから、抽出物には、免疫増強作用を有するの
で、免疫能の低下している患者又は高齢者、例えば、癌
患者に抗癌剤と併用させる、感染症の予防と阻止、重症
の細菌感染症患者に化学療法剤と併用させる、等に用い
たり、あるいは肝機能が低下して異物（例えば薬剤）の
排出困難な患者に投与して、その機能を元通させる等の
用途が期待できる。From the above, the extract has an immune-enhancing effect, so it can be used in combination with anticancer drugs for patients with reduced immune function or the elderly, such as cancer patients, for the prevention and prevention of infectious diseases, and for severe bacterial infections. It is expected to be used in combination with chemotherapeutic agents for patients with liver disease, or administered to patients with impaired liver function who have difficulty excreting foreign substances (such as drugs) to restore their function.
実施例２
市販のイカリソウ（キバナイカリソウ、韓国産）細切２
Ｋｙに５０％エタノール（■／■）１ｓｔを加え、還
流冷却器を付し、水溶上５０℃で６時間加温抽出した。Example 2 Commercially available epimedium (Kibanaicou, produced in Korea) shredded 2
1 st of 50% ethanol (■/■) was added to Ky, a reflux condenser was attached, and the mixture was heated and extracted at 50° C. for 6 hours.
抽出後、温時濾過し、残渣は新たに５０％エタノール（
Ｖ／ｖ）１５１ずつで同様な抽出、濾過操作を５回行っ
た。全濾液を会わせ、減圧下４５℃で濃縮し、水性抽出
液１０２を得た。この抽出液を分液ロートに入れ、′酢
酸エチル“５ｔを力１１ｉえて激しく振盪し、水層のみ
分取した。この水層は新たに、酢酸エチル５ｔずつで同
様な抽出を６回行った。水層を減圧下濃縮し、残存する
酢酸エチルを留去した後、濾過した。濾液にエタノール
５０ｔを添加、攪拌後、−夜装置し、析出する沈殿を濾
取□した。After extraction, it is filtered while hot, and the residue is freshly dissolved in 50% ethanol (
Similar extraction and filtration operations were performed five times using 151 (V/v) each. All filtrates were combined and concentrated under reduced pressure at 45°C to obtain aqueous extract 102. This extract was placed in a separating funnel and shaken vigorously with 5 tons of ethyl acetate at a force of 11 degrees, and only the aqueous layer was separated. This aqueous layer was extracted in the same manner six times with 5 tons of ethyl acetate each. The aqueous layer was concentrated under reduced pressure, and the remaining ethyl acetate was distilled off, followed by filtration. 50 t of ethanol was added to the filtrate, stirred, and left overnight, and the precipitate was collected by filtration.
沈殿をエタノール２００ｍ／で洗浄した後、水２ｔ′に
溶解し、次いでエタノール８ｔを添加した後沈殿を濾取
した。この沈殿を水１ｔにて抽出後、濾過し、濾液を減
圧下濃縮乾燥し抽出物１６ノを得た。The precipitate was washed with 200 m/g of ethanol, dissolved in 2 t' of water, and then 8 t of ethanol was added thereto, and the precipitate was collected by filtration. This precipitate was extracted with 1 t of water, filtered, and the filtrate was concentrated and dried under reduced pressure to obtain 16 extracts.
この抽出物１６ｙに０．０２モル硫酸ナトリウム水溶液
１ｔを加え、４０℃で加温攪拌後、温時濾過した。３０
〜４０℃加温下、この濾液に１０チーセチルトリメチル
アンモニウムブロマイド水溶液１００ｘ／を加え、−夜
５７℃にて保温、静置後、遠心分離（５，０００γ、
ｐ、 ｍ、、１０分間）し、上澄液１ｔを得た。この上
澄液に３ｔのエタノールを加え生じた沈殿（５，０１）
を濾取した。1 t of 0.02M sodium sulfate aqueous solution was added to this extract 16y, and after heating and stirring at 40°C, it was filtered while still warm. 30
To this filtrate was added 100x of a 10-thicetyltrimethylammonium bromide aqueous solution while heating at ~40°C, kept warm at 57°C overnight, allowed to stand, and then centrifuged (5,000γ,
p, m, 10 minutes) to obtain 1 t of supernatant. Adding 3 tons of ethanol to this supernatant resulted in a precipitate (5,01)
was collected by filtration.
この沈殿＜ｓａｐ＞に水１ｔを加え、４０℃で加温、攪
拌後、減圧下残存エタノールを留去し濾過した。この濾
液は更にミリポアフィルタ−ＡＰ２０（径１４２ｍ、日
本ミリポアリミッテッド社製）、ミリポアメンブランフ
ィルタ−ＨＡ型（径１４２１１１ｍ、孔径０．４５μｍ
２日本ミリボアリミッテッド社製）を用いて濾過した。1 ton of water was added to this precipitate <sap>, and after heating and stirring at 40°C, residual ethanol was distilled off under reduced pressure and filtered. This filtrate was further filtered into Millipore filter-AP20 (diameter 142 m, manufactured by Japan Millipore Limited), Millipore membrane filter-HA type (diameter 142111 m, pore size 0.45 μm).
2 (manufactured by Nippon Millibore Limited).
この濾液をミリボアペリコン限外濾過膜ＰＳＡＣ（公称
分子量限外値１．０００、日本ミリボアリミッテッド社
製）を用いて限外濾過し、嘆通過部を除去した。次に、
膜不通過部に水５００Ｗｌｌを加え、希釈後、再度限外
濾過した。この操作を３回行なった後、膜不通過部を分
取した。そして、膜不通過部を水にて２５０ ｔｔｔｌ
に希釈しエタノール１ｔを加え、沈殿２、ＯＦを得た。This filtrate was ultrafiltered using a Millibore Pericone ultrafiltration membrane PSAC (nominal molecular weight limit value 1.000, manufactured by Japan Millibore Limited) to remove the membrane passage. next,
500 Wll of water was added to the part that did not pass through the membrane, and after dilution, ultrafiltration was performed again. After performing this operation three times, the portion that did not pass through the membrane was collected. Then, soak the part that does not pass through the membrane with water at 250 tttl.
1 t of ethanol was added to obtain precipitate 2, OF.
この沈殿２．ＯＦを水２０ｍ／に溶解後、セファデック
スＧ−１５０カラム（径５０ｍｍ）？長さ９００ＩＩＩ
Ｉ＋、ファルマシアファインケミカル社製）に付し、水
２０ｍ１ずつで溶出し１５０の画分を得た。各々の溶出
画分の一部（（１２ｍｇ程度）を順次フェノール硫酸法
にて発色させ、４９０ｎｍの吸光度（ＯＤ）を測定した
。第２図のＦｒ−２に該５．当する両分（溶出画分番号
５４〜８８）を選択、集収し、減圧濃縮ついで凍結乾燥
することにより本発明多糖体ＰＳ−Ｂ８００”ｐを得た
。その理化学的性質は前述のとおりであ・りた。This precipitation 2. After dissolving OF in 20ml of water, Sephadex G-150 column (diameter 50mm)? Length 900III
I+ (manufactured by Pharmacia Fine Chemicals) and eluted with 20 ml of water to obtain 150 fractions. A portion (about 12 mg) of each elution fraction was sequentially developed using the phenol-sulfuric acid method, and the absorbance (OD) at 490 nm was measured. Fraction numbers 54 to 88) were selected and collected, concentrated under reduced pressure, and then freeze-dried to obtain the polysaccharide of the present invention PS-B800''p. Its physicochemical properties were as described above.
第１図は本発明ＰＳ−Ｈの光外部吸収スペクトル、第２
図は第５工程で得られた沈殿を分子ふるい効果を有する
担体を用いたクロマトグラフィーに付した際のクロマト
グラム、第３図は実施例１で得た抽出物Ｘ゛の光外部吸
収スペクトルを示す。
特許出願人
全薬工業株式會社Figure 1 shows the optical external absorption spectrum of PS-H of the present invention, Figure 2 shows
The figure shows the chromatogram obtained when the precipitate obtained in the fifth step was subjected to chromatography using a carrier having a molecular sieving effect. Figure 3 shows the optical external absorption spectrum of the extract X obtained in Example 1. show. Patent applicant Zenyaku Kogyo Co., Ltd.
Claims (1)
得られる多糖体ＰＳ−Ｂ １ 元素分析： Ｃ：５７．６９ Ｈ：６．００
灰分：微量２ 分子量： ５０，０００±２５，００
０（平均分子量）５ 物質の性状：白色〜徽かっ色不定
形粉末４ 分解点：２００℃ ５ 紫外部吸収スペクトル：２４０−４００ ｎｕＨに
極大吸収は認められない。 ６ 赤外部吸収スペクトル：第１図のスペクトルを示す
。 ７’ ｐＨ： ６．９±α１ （ＰＳ−Ｂ１００５を
水５０１ＩＬｌに溶解するとき） ８ 溶解性：可溶・・・水 不溶・・・メタノール、エタ／−ル、 アセトン、酢酸エチル、 ジエチルエーテル、ヘキ サン、クロロホルム ９ 比旋光度：〔α〕１９＝−１５°（Ｂ２０中、Ｃ＝
［１，７５６チ） １０ 呈色反応： 次の諸反応に陽性を示す。 （α）アンスロン硫酸反応 （ｈ）モーリッシュ反応
（Ｃｌｌストール反応 （ｄ）ビアル反応次の諸反応に
陰性を示す。 （α）ニンヒドリン反応 （ｂ）２．４−ＤＮＰ反応（
Ｃ）セリヴアノフ反応 （ｄ）ナフトレゾルシン反応
（ｅ）カルバゾール硫酸反応 １１ 構成糖： アラビノースとガラクトース。 １２ 均一性： 超遠心、電気泳動、ゲル濾過で均一。 ２）イカリソウ属植物に、水に混和する有機溶媒と水と
の混合溶媒または水のいずれかを加え水性抽出液を得、
脱脂後、水性抽出液に水と任意に混和する有機溶媒を加
え、沈殿を生成させ、この沈殿を水に溶解させ、長鎖を
有する第４級アンモニウム塩を加え、生じた複合体を除
去し得られる濾液からさらに低分子化合物のみ除去した
後、クロマトグラフィーに付し、下記の理化学的性質を
有する多糖体ＰＳ−Ｂ倉製造する方法。 １ 元素分析： Ｃ： ５７．６９ Ｈ： ６．
００ 灰分：微量２ 分子量：５０，０００±２５，０
００（平均分子量）５ 物質の性状：白色〜微かっ色不
定形粉末４ 分解点：２００℃ ５ 紫外部吸収スペクトル：２４０〜４００ ｎｍに極
大吸収は認められない。 ６ 光外部吸収スペクトル：第１図のスペクトルを示す
。 ７ｐＨ：６．９±０．１ （ＰＳ−Ｂ１００清７を水５
０ｍ１に溶解するとき） ８ 溶解性：可溶・・・水 不溶・・・メタノール、エタン−、ル、アセトン、酢酸
エチル、 ジエチルエーテル、ヘキ サン、クロロホルム。 ９ 比旋光度：〔α〕１９＝−α５°（Ｂ２０中、Ｃ＝
０．７５６％） １０ 呈色反応： 次の諸反応に陽性を示す。 （α）アンスワン硫酸反応 （ｂｌモーリッシュ反応
（Ｃ）スカトール反応 （ｄ）ビアル反応次の諸反応に
陰性を示す。 （α）ニンヒドリン反応 （ｂ）′″〜２，４−ＤＮＰ
〜２，４−ＤＮＰ反応反応 （ｄＪナフトレゾルシン反
応 （ｇ）カルバゾール硫酸反応 １１ 構成糖二 ′ アラビノースとガラクトース。 １２ 均一性： 超遠心、電気泳動、ゲル濾過で均一。 ３）り・↓トグラしイーとして、ポリアミドによる吸着
クロマトグラフィーまたはゲル濾過によるクロマトグラ
フィーを用いる特許請求の範囲第２）項記載あ製造方法
。 ４）下記の理化学的性質を有するイカリソウ属植物から
得られる多糖体ＰＳ−Ｂを有効成分とする感染防禦剤。 １ 元素分析： Ｃ’、５７．６９ Ｈ：６．（
ｔｏ灰分：微量２ 分子量：５０，０００±２５，００
０（平均分子量）５ 物質の性状：白色〜微かっ色不定
形粉末４ 分解点＝２００℃ ５ 紫外部吸収スペクトル＝２４０〜４００ｎｍに極大
吸収は認められない。 ６ 光外部吸収スペクトル：第１図のスペクトルを示す
。 ７ｐＨ：６．９±（Ｌｌ （ＰＳ−Ｂ１００”ｐを水５
０ｍ／にに溶解するとき） ８ 溶解性：可溶・・・水 不溶・・・メタノール、エタノール、 アセトン、酢酸エチル、 ジエチルエーテル、ヘキ サン、クロロホルム。 ９ 比旋光度：〔α躍＝ −０，５°（Ｂ２０中、Ｃ＝
０７５６チ） １０ 呈色反応： 次の諸反応に陽性を示す。 （（Ｚｌアンスロン硫酸反応（ｈ）モーリッシュ反応
（Ｃ）スカトール反応 （ｄ）ピアル反応次の諸反応に
陰性を示す。 （α）ニンヒドリン反応 （Ａ） ２．４−ＤＮＰ反応
（Ｃｌセリヴアノフ反応 （ｄ）ナフトレゾルシン
・反応 （ｅ）カルバゾール硫酸反応 １１ 構成糖： アラビノースとガラクトース。 １２ 均一性： 超遠心、電気泳動、ゲル濾過で均一。[Claims] 1) Polysaccharide PS-B obtained from a plant of the genus Epimedium having the following physicochemical properties Elemental analysis: C: 57.69 H: 6.00
Ash content: Trace 2 Molecular weight: 50,000±25,00
0 (average molecular weight) 5 Properties of substance: white to dark brown amorphous powder 4 Decomposition point: 200°C 5 Ultraviolet absorption spectrum: 240-400 No maximum absorption is observed at nuH. 6 Infrared absorption spectrum: Shows the spectrum in Figure 1. 7' pH: 6.9±α1 (When dissolving PS-B1005 in 501 ILl of water) 8 Solubility: Soluble...Water-insoluble...methanol, ethanol, acetone, ethyl acetate, diethyl ether, Hexane, chloroform 9 Specific optical rotation: [α] 19 = -15° (in B20, C =
[1,756chi] 10 Color reaction: The following reactions are positive. (α) Anthrone sulfuric acid reaction (h) Molish reaction
(Cll stall reaction (d) Vial reaction The following reactions are negative. (α) Ninhydrin reaction (b) 2.4-DNP reaction (
C) Selivanov reaction (d) Naftresorcin reaction
(e) Carbazole sulfate reaction 11 Constituent sugars: arabinose and galactose. 12 Uniformity: Uniform by ultracentrifugation, electrophoresis, and gel filtration. 2) Adding either a mixed solvent of a water-miscible organic solvent and water or water to a plant of the genus Epimedium to obtain an aqueous extract;
After defatting, an organic solvent that is optionally miscible with water is added to the aqueous extract to form a precipitate, this precipitate is dissolved in water, a quaternary ammonium salt having a long chain is added, and the resulting complex is removed. A method for producing a polysaccharide PS-B having the following physical and chemical properties by further removing only low molecular weight compounds from the obtained filtrate and subjecting it to chromatography. 1 Elemental analysis: C: 57.69 H: 6.
00 Ash content: Trace 2 Molecular weight: 50,000±25,0
00 (average molecular weight) 5 Properties of substance: White to faintly brown amorphous powder 4 Decomposition point: 200°C 5 Ultraviolet absorption spectrum: No maximum absorption is observed between 240 and 400 nm. 6 Optical external absorption spectrum: Shows the spectrum in Figure 1. 7pH: 6.9±0.1 (PS-B100 clear 7 to water 5
0ml) 8 Solubility: Soluble...Water-insoluble...methanol, ethane, acetone, ethyl acetate, diethyl ether, hexane, chloroform. 9 Specific rotation: [α]19=-α5° (in B20, C=
0.756%) 10 Color reaction: The following reactions are positive. (α) Ansuwan sulfuric acid reaction (bl Molish reaction
(C) Skatole reaction (d) Vial reaction The following reactions are negative. (α) Ninhydrin reaction (b)′″~2,4-DNP
~2,4-DNP reaction (dJ Naftresorcin reaction (g) Carbazole sulfuric acid reaction 11 Constituent sugars di′ arabinose and galactose. 12 Homogeneity: Uniform by ultracentrifugation, electrophoresis, and gel filtration. 3) Reaction and ↓ toggle The manufacturing method according to claim 2), wherein adsorption chromatography using polyamide or chromatography using gel filtration is used as E. 4) An infection preventive agent containing as an active ingredient polysaccharide PS-B obtained from a plant of the genus Epimedium having the following physicochemical properties. 1 Elemental analysis: C', 57.69 H:6. (
to ash content: trace amount 2 molecular weight: 50,000±25,00
0 (average molecular weight) 5 Properties of substance: white to faintly brown amorphous powder 4 Decomposition point = 200°C 5 Ultraviolet absorption spectrum = No maximum absorption observed in the range of 240 to 400 nm. 6 Optical external absorption spectrum: Shows the spectrum in Figure 1. 7pH: 6.9±(Ll (PS-B100”p with water 5
8 Solubility: Soluble...Water-insoluble...methanol, ethanol, acetone, ethyl acetate, diethyl ether, hexane, chloroform. 9 Specific rotation: [α = −0,5° (in B20, C =
0756) 10 Color reaction: The following reactions are positive. ((Zl anthrone sulfuric acid reaction (h) Molish reaction
(C) Skatole reaction (d) Pial reaction The following reactions are negative. (α) Ninhydrin reaction (A) 2.4-DNP reaction (Cl Selivanov reaction (d) Naftresorcin
-Reaction (e) Carbazole sulfate reaction 11 Constituent sugars: arabinose and galactose. 12 Uniformity: Uniform by ultracentrifugation, electrophoresis, and gel filtration.
Priority Applications (10)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP56190202A JPS5892617A (en) | 1981-11-27 | 1981-11-27 | Polysaccharide ps-b derived from plant belonging to epimedium genus, its preparation, and prophylactic agent containing polysaccharide ps-b as active component |
| DE19823242672 DE3242672T1 (en) | 1981-04-30 | 1982-04-20 | POLYSACCHARID PS-A RECEIVE PLANTS OF THE GENERATION EPIMEDIUM, METHOD FOR THE PRODUCTION THEREOF AND PHYLACTIC AND IMMUNOSTIMULATING AGENTS WHICH CONTAIN THIS POLYSACCHARIDE PS-A AS AN ACTIVE COMPONENT |
| AU83346/82A AU8334682A (en) | 1981-04-30 | 1982-04-20 | Polysaccharide ps=a isolated from the plant of genus epimedium violaceium morr. et decne., process for its preparation, and infection-preventing agent and immunostimulating agent containing the ploysaccharide as effective ingredient |
| PCT/JP1982/000132 WO1982003771A1 (en) | 1981-04-30 | 1982-04-20 | Polysaccharide ps-a isolated from the plant of genus epimedium violaceum morr,et decne.,process for its preparation,and infection-preventing agent and immunostimulating agent containing the polysaccharide as effective ingredient |
| EP82901157A EP0086233A1 (en) | 1981-04-30 | 1982-04-20 | Polysaccharide ps-a isolated from the plant of genus epimedium violaceum morr. et decne., process for its preparation, and infection-preventing agent and immunostimulating agent containing the polysaccharide as effective ingredient |
| GB838314489A GB8314489D0 (en) | 1981-04-30 | 1982-04-20 | Polysaccharide ps-a |
| US06/456,027 US4528188A (en) | 1981-04-30 | 1982-04-20 | Polysaccharide PS-A obtained from barrenwort deriving from plants belonging to the genus Epimedium, process for preparation thereof and phylactic and immunostimulating agents comprising said polysaccharide PS-A effective component |
| CA000401970A CA1188687A (en) | 1981-04-30 | 1982-04-29 | Polysaccharide ps-a obtained from plants of genus epimedium sp., process for preparation thereof and phylactic and immunostimulating agents comprising said polysaccharide ps-a as effective component |
| IT2100782A IT1209459B (en) | 1981-04-30 | 1982-04-29 | Polysaccharide PS-A - with infection prevention and immunostimulating effects is isolated from Epimedium sp. |
| KR1019820001917A KR830009778A (en) | 1981-11-27 | 1982-04-30 | Method for preparing plysaccharide PS-A |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP56190202A JPS5892617A (en) | 1981-11-27 | 1981-11-27 | Polysaccharide ps-b derived from plant belonging to epimedium genus, its preparation, and prophylactic agent containing polysaccharide ps-b as active component |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS5892617A true JPS5892617A (en) | 1983-06-02 |
Family
ID=16254149
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP56190202A Pending JPS5892617A (en) | 1981-04-30 | 1981-11-27 | Polysaccharide ps-b derived from plant belonging to epimedium genus, its preparation, and prophylactic agent containing polysaccharide ps-b as active component |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPS5892617A (en) |
| KR (1) | KR830009778A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013541625A (en) * | 2010-11-05 | 2013-11-14 | インスティテュート オブ ファーマコロジー アンド トキシコロジー アカデミー オブ ミリタリー メディカル サイエンシズ ピー．エル．エー．チャイナ | Epimedium polysaccharide, its fractions, and its use as a vaccine adjuvant |
-
1981
- 1981-11-27 JP JP56190202A patent/JPS5892617A/en active Pending
-
1982
- 1982-04-30 KR KR1019820001917A patent/KR830009778A/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013541625A (en) * | 2010-11-05 | 2013-11-14 | インスティテュート オブ ファーマコロジー アンド トキシコロジー アカデミー オブ ミリタリー メディカル サイエンシズ ピー．エル．エー．チャイナ | Epimedium polysaccharide, its fractions, and its use as a vaccine adjuvant |
Also Published As
| Publication number | Publication date |
|---|---|
| KR830009778A (en) | 1983-12-23 |
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