JP2022157082A - Viral respiratory infection preventing or treating agent - Google Patents
Viral respiratory infection preventing or treating agent Download PDFInfo
- Publication number
- JP2022157082A JP2022157082A JP2021061114A JP2021061114A JP2022157082A JP 2022157082 A JP2022157082 A JP 2022157082A JP 2021061114 A JP2021061114 A JP 2021061114A JP 2021061114 A JP2021061114 A JP 2021061114A JP 2022157082 A JP2022157082 A JP 2022157082A
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- Prior art keywords
- glucosylceramide
- viral respiratory
- treating agent
- influenza
- acid
- 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.)
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Abstract
Description
本発明は、ウイルス性呼吸器感染予防、治療剤に関する。これらは、医薬品、医薬部外品、健康食品、一般商品等に広く利用される。 TECHNICAL FIELD The present invention relates to agents for preventing and treating viral respiratory infections. These are widely used in pharmaceuticals, quasi-drugs, health foods, general merchandise, and the like.
ウイルスの感染による流行性の急性呼吸器感染症にはいろいろあるが、例えば、インフルエンザ、コロナウイルス感染症等がある。インフルエンザはA型またはB型インフルエンザウイルスの感染による流行性の急性呼吸器感染症である。その臨床像は、上気道炎症状に加えて、突然の高熱と全身倦怠感、頭痛、筋肉痛、関節痛などの全身症状を特徴とし、数日の臥床を強いられるような重症感を伴う。インフルエンザは毎年冬季に流行を繰り返し、短期間に集中して数百万人規模で多数の患者が発生する。さらに65歳以上の高齢者、乳幼児、妊婦やさまざまな基礎疾患をもつハイリスク群がインフルエンザに罹患すると、肺炎の合併や入院、死亡の危険が健康成人に比べて数100倍も高くなる。したがってインフルエンザは、患者の健康被害の大きさとその流行規模から社会・経済的にも大きな影響をもたらす重要な疾患として認識されている。 There are various types of epidemic acute respiratory infections caused by viral infections, such as influenza and coronavirus infections. Influenza is a prevalent acute respiratory infection caused by infection with influenza A or B viruses. The clinical picture is characterized by systemic symptoms such as sudden high fever, general malaise, headache, muscle pain, and joint pain, in addition to symptoms of upper respiratory tract inflammation, accompanied by a severe feeling of bed rest for several days. Influenza epidemics repeat every winter season, and many patients occur on the scale of millions in a short period of time. Furthermore, when high-risk people aged 65 and older, infants, pregnant women, and other high-risk groups with various underlying diseases contract influenza, the risks of complication of pneumonia, hospitalization, and death are several hundred times higher than those of healthy adults. Therefore, influenza is recognized as an important disease that exerts a great impact on society and economy because of the magnitude of the health damage to patients and the scale of its epidemic.
インフルエンザの予防方法として従来よりインフルエンザワクチンが使用されている。しかし、ワクチン株と流行株とが異なったり、株が一致したとしても不十分な生体防御惹起能であったり、必ずしもその予防効果は満足いくものではない。さらに、ワクチン接種者に発赤、腫脹、疼痛、発熱、頭痛、悪寒、倦怠感などの副作用が起こることがある。また、卵アレルギーの人には蕁麻疹、発疹、口腔のしびれ、アナフィラキシーショックなどの可能性もあり、ワクチンに安定剤として含まれているゼラチンに対するゼラチンアレルギーも報告されている。その他ギランバレー症候群、急性脳症、痙攣、紫斑などの副作用報告もある。そのため、ワクチンを接種するためには、医師の管理指導の下行われている(特許文献1)。
そこで、副作用が起きにくく、食品としても摂取することができるインフルエンザの予防、治療剤が求められている。
Influenza vaccines have been conventionally used as methods for preventing influenza. However, the vaccine strain and epidemic strain are different, and even if the strain matches, the ability to induce biological defense is insufficient, and the preventive effect is not always satisfactory. In addition, vaccinees may experience side effects such as redness, swelling, pain, fever, headache, chills, and malaise. In addition, people with egg allergy may experience hives, rashes, numbness in the mouth, anaphylactic shock, etc. Gelatin allergy to gelatin, which is included as a stabilizer in vaccines, has also been reported. There are also reports of side effects such as Guillain-Barre syndrome, acute encephalopathy, convulsions, and purpura. Therefore, vaccination is performed under the supervision and guidance of a doctor (Patent Document 1).
Therefore, there is a demand for a prophylactic and therapeutic agent for influenza that is less likely to cause side effects and that can be ingested as food.
このような背景の下、本発明者は、食品素材として知られているグルコシルセラミド含有米抽出物、及びβ-シトステロールグルコシドにインフルエンザ等のウイルス性の呼吸器感染症の予防、治療作用を有することを見出し、本発明を完成させた。
すなわち、本発明は、副作用が少ない食品素材を有効成分とする新規なウイルス性呼吸器感染予防、治療剤を提供することを目的とする。
Against this background, the present inventor discovered that glucosylceramide-containing rice extract and β-sitosterol glucoside, which are known as food materials, have preventive and therapeutic effects on viral respiratory infections such as influenza. and completed the present invention.
That is, it is an object of the present invention to provide a novel preventive and therapeutic agent for viral respiratory infection, which contains food materials with few side effects as active ingredients.
上記課題を解決するための本発明の特徴は以下の通りである。
1. グルコシルセラミド及び/又はグルコシルセラミド含有米抽出物を有効成分とするウイルス性呼吸器感染症予防、治療剤。
2.β-シトステロール配糖体を有効成分とするウイルス性呼吸器感染症予防、治療剤。
3.上記ウイルス性呼吸器感染症はインフルエンザであることを特徴とする上記1.又は上記2.に記載のウイルス性呼吸器感染症予防、治療剤。
The features of the present invention for solving the above problems are as follows.
1. A prophylactic or therapeutic agent for viral respiratory infections containing glucosylceramide and/or a glucosylceramide-containing rice extract as an active ingredient.
2. A preventive and therapeutic agent for viral respiratory infections containing β-sitosterol glycoside as an active ingredient.
3. The method of claim 1, wherein the viral respiratory infection is influenza. or the above 2. 3. The agent for preventing and treating viral respiratory infections according to .
以下、本発明を詳細に説明する。
本発明は、グルコシルセラミド及び/又はグルコシルセラミド含有米抽出物を有効成分とすることを特徴とする。
本発明の有効成分であるグルコシルセラミドはセラミドに糖(主にガラクトース)がグリコシルエーテル結合したものである。
The present invention will be described in detail below.
The present invention is characterized by using glucosylceramide and/or a glucosylceramide-containing rice extract as an active ingredient.
Glucosylceramide, which is the active ingredient of the present invention, is obtained by glycosylether-bonding sugar (mainly galactose) to ceramide.
上記グルコシルセラミドは、特に限定されないが、下記化学式(1)で示されたものであることが好ましい。
グルコシルセラミド質含有米抽出物は、米ヌカから多量に抽出することができる。原料米は、品種を限定しないが、甘味を呈する日本型(japonica)が好適である。特に、コシヒカリ、ササニシキなどで代表される主食用米品種が好ましい。通常、玄米の最外層にある果皮、種皮および糊粉層が精米機で取り除かれてヌカとなる。 A large amount of the glucosylceramide-containing rice extract can be extracted from rice bran. Although the variety of rice used as the raw material is not limited, japonica, which exhibits sweetness, is suitable. In particular, staple rice varieties such as Koshihikari and Sasanishiki are preferred. Usually, the pericarp, seed coat and aleurone layer, which are the outermost layers of brown rice, are removed by a rice polishing machine to form bran.
米ヌカからグルコシルセラミド質含有米抽出物を抽出する方法としては特に限定されないが、下記工程A~Cによるのが望ましい。
A. 米ヌカを有機溶媒で抽出する工程、
B.前記抽出物に水と共に酸を加え、この酸溶液中にガム質を沈殿させる工程、
C.前記ガム質をアルコールで抽出し、このアルコール抽出物からスフィンゴ脂質を精製する工程。
The method for extracting the glucosylceramide-containing rice extract from rice bran is not particularly limited, but the following steps A to C are preferable.
A. A step of extracting rice bran with an organic solvent,
B. adding acid with water to the extract and precipitating gums in the acid solution;
C. A step of extracting the gum substance with alcohol and purifying sphingolipids from the alcohol extract.
前記工程AおよびBは、米ヌカ油の製造工程を利用することができる。一般に、米ヌカ油を製造する場合、原料の米ヌカをヘキサンで抽出し、次いで、リン酸処理により、油分を分離し、ガム質を得る。従って、このような米ヌカ油の製造工程で生じるガム質に、前記工程Cを適用することにより、グルコシルセラミドを効率よく抽出することができる。 The steps A and B can utilize the production process of rice bran oil. In general, when producing rice bran oil, the raw material rice bran is extracted with hexane, and then treated with phosphoric acid to separate the oil and obtain a gum. Therefore, glucosylceramide can be efficiently extracted by applying the above step C to the gum produced in the production process of such rice bran oil.
前記有機溶媒は、ヘキサンの他、ヘプタン、石油エーテル、エーテル、クロロホルム、アセトン等を用いることができる。前記有機溶媒の抽出物を処理する酸は、有機酸としては酢酸、シュウ酸、クエン酸、蟻酸等を、無機酸としてはリン酸、硫酸、塩酸、硝酸等を用いることができる。望ましくは、リン酸を用いるとよい。リン酸によれば、以下に示すような利点があるためである。
a.米ヌカ油の製造工程をそのまま利用することができる。
b.強酸ではないので、タンク、パイプ等が腐食しにくく、製造設備の耐久性が向上する。
c.酢酸、蟻酸のような刺激臭がない。
d.有機溶剤の抽出物に含まれるリン脂質を取り除きやすいため、沈殿物(ガム質)に含まれるグルコシルセラミドの濃度が高まる。
As the organic solvent, besides hexane, heptane, petroleum ether, ether, chloroform, acetone, and the like can be used. As the acid for treating the organic solvent extract, organic acids such as acetic acid, oxalic acid, citric acid and formic acid can be used, and inorganic acids such as phosphoric acid, sulfuric acid, hydrochloric acid and nitric acid can be used. Desirably, phosphoric acid is used. This is because phosphoric acid has the following advantages.
a. The manufacturing process of rice bran oil can be used as it is.
b. Since it is not a strong acid, tanks and pipes are less likely to corrode, improving the durability of manufacturing equipment.
c. No irritating odor like acetic acid and formic acid.
d. Since the phospholipids contained in the organic solvent extract can be easily removed, the concentration of glucosylceramide contained in the sediment (gum) increases.
前記アルコールは、エタノール、メタノール、イソプロピルアルコール、ブタノールおよびこれらの含水アルコール等を用いることができる。グルコシルセラミドを飲食品に使用する場合には、エタノールを使用するとよい。前記工程Cでガム質のアルコール抽出物からグルコシルセラミドを精製する手段としては、カラムクロマトグラフィーその他の適当な精製手段を用いることができる。 As the alcohol, ethanol, methanol, isopropyl alcohol, butanol, hydrous alcohols thereof, and the like can be used. When using glucosylceramide for food and drink, it is good to use ethanol. As means for purifying glucosylceramide from the alcoholic extract of gum in step C, column chromatography or other suitable purification means can be used.
また、前記グルコシルセラミド含有米抽出物は市販品を用いても良く、例えば、オリザ油化株式会社製のオリザセラミド(登録商標)を用いてもよい。 Moreover, the glucosylceramide-containing rice extract may be a commercially available product, for example, Oryza Ceramide (registered trademark) manufactured by Oryza Oil & Chemicals Co., Ltd. may be used.
本発明は、β-シトステロール配糖体を有効成分とすることを特徴とする。
β-シトステロール配糖体は特に限定されず、例えば、β-シトステロールグルコシド、β-シトステロールルチノイド、β-シトステロールサンブビオシド等が挙げられるが、特にβ-シトステロールグルコシドが好ましい。尚、β-シトステロールグルコシドは下記化学式(2)に示される化合物である。
The present invention is characterized by using β-sitosterol glycoside as an active ingredient.
The β-sitosterol glycoside is not particularly limited, and examples thereof include β-sitosterol glucoside, β-sitosterol rutinoid, β-sitosterol sambubioside and the like, with β-sitosterol glucoside being particularly preferred. β-sitosterol glucoside is a compound represented by the following chemical formula (2).
β-シトステロールグルコシドを得る方法は特に限定されず、市販品のものを使用してもよいし、植物から抽出してもよい。市販品としては富士フイルム和光純薬工業株式会社製の「シトステロール-3-O-グルコシド, β- (AHP Verified)」を用いることができる。 The method for obtaining β-sitosterol glucoside is not particularly limited, and commercially available products may be used, or they may be extracted from plants. As a commercially available product, "Sitosterol-3-O-glucoside, β- (AHP Verified)" manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd. can be used.
β-シトステロールグルコシドを得るための植物原料は特に限定されないが、例えば、コメ、小麦、ビート、こんにゃく、トウモロコシ等が挙げられる。これらのうちコメが好ましい。
コメからβ-シトステロールグルコシドを得るために使用する部位は特に限定されないが、特に米糠、米胚芽を用いることが好ましい。
Plant raw materials for obtaining β-sitosterol glucoside are not particularly limited, and examples thereof include rice, wheat, beet, konjac, corn, and the like. Of these, rice is preferred.
The site used to obtain β-sitosterol glucoside from rice is not particularly limited, but it is particularly preferable to use rice bran and rice germ.
米糠や米胚芽からβ-シトステロールグルコシドを得る方法は特に限定されないが、例えば、米糠や米胚芽を発酵させて発酵物を得て、その後、この発酵物を非極性溶媒で抽出し発酵物抽出液を得て、その後、この発酵物抽出液を分配することにより得ることができる。また、オリザ油化株式会社製の「オリザセラミド」(登録商標)から、抽出することができる。 The method of obtaining β-sitosterol glucoside from rice bran or rice germ is not particularly limited. and then partitioning this fermented extract. Alternatively, it can be extracted from “Oryza Ceramide” (registered trademark) manufactured by Oryza Oil & Chemical Co., Ltd.
本発明のウイルス性呼吸器感染症予防剤は、各種飲食品の素材として使用することができる。飲食品としては、例えば、菓子類(ガム、キャンディー、キャラメル、チョコレート、クッキー、スナック、ゼリー、グミ、錠菓等)、麺類(そば、うどん、ラーメン等)、乳製品(ミルク、アイスクリーム、ヨーグルト等)、調味料(味噌、醤油等)、スープ類、飲料(ジュース、コーヒー、紅茶、茶、炭酸飲料、スポーツ飲料等)をはじめとする一般食品や、健康食品(錠剤、カプセル等)、栄養補助食品(栄養ドリンク等)が挙げられる。これらの飲食品に本発明ウイルス性呼吸器感染症予防剤を適宜配合するとよい。 The agent for preventing viral respiratory infections of the present invention can be used as a material for various foods and drinks. Examples of food and drink include confectionery (gum, candy, caramel, chocolate, cookies, snacks, jelly, gummies, tablets, etc.), noodles (soba, udon, ramen, etc.), dairy products (milk, ice cream, yogurt, etc.). ), seasonings (miso, soy sauce, etc.), soups, beverages (juice, coffee, black tea, tea, carbonated drinks, sports drinks, etc.), general foods, health foods (tablets, capsules, etc.), nutrition Supplementary foods (energy drinks, etc.) can be mentioned. The preventive agent for viral respiratory infections of the present invention may be appropriately added to these foods and drinks.
これら飲食品には、その種類に応じて種々の成分を配合することができ、例えば、ブドウ糖、果糖、ショ糖、マルトース、ソルビトール、ステビオサイド、コーンシロップ、乳糖、クエン酸、酒石酸、リンゴ酸、コハク酸、乳酸、L-アスコルビン酸、dl-α-トコフェロール、エリソルビン酸ナトリウム、グリセリン、プロピレングリコール、グリセリン脂肪酸エステル、ポリグリセリン脂肪酸エステル、ショ糖脂肪酸エステル、ソルビタン脂肪酸エステル、プロピレングリコール脂肪酸エステル、アラビアガム、カラギーナン、カゼイン、ゼラチン、ペクチン、寒天、ビタミンB類、ニコチン酸アミド、パントテン酸カルシウム、アミノ酸類、カルシウム塩類、色素、香料、保存剤の食品素材を使用することができる。 Various ingredients can be blended into these foods and drinks depending on their type, for example, glucose, fructose, sucrose, maltose, sorbitol, stevioside, corn syrup, lactose, citric acid, tartaric acid, malic acid, and succinate. acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, gum arabic, Food materials such as carrageenan, casein, gelatin, pectin, agar, B vitamins, nicotinamide, calcium pantothenate, amino acids, calcium salts, pigments, flavors and preservatives can be used.
具体的な製法としては、本発明の剤を粉末セルロースとともにスプレードライまたは凍結乾燥し、これを粉末、顆粒、打錠または溶液にすることで容易に飲食品(インスタント食品等)に含有させることができる。また、前記本発明の剤を、例えば、油脂、エタノール、グリセリンあるいはこれらの混合物に溶解して液状にし、飲料に添加するか、固形食品に添加することが可能である。必要に応じてアラビアガム、デキストリン等のバインダーと混合して粉末状あるいは顆粒状にし、飲料に添加するか固形食品に添加することも可能である。 As a specific production method, the agent of the present invention is spray-dried or freeze-dried together with powdered cellulose, and this is made into powder, granules, tablets, or a solution, so that it can be easily incorporated in foods and drinks (instant foods, etc.). can. Also, the agent of the present invention can be dissolved in, for example, fat, ethanol, glycerin, or a mixture thereof to form a liquid, which can be added to beverages or solid foods. If necessary, it can be mixed with a binder such as gum arabic or dextrin to form powder or granules, which can be added to beverages or solid foods.
本発明のウイルス性呼吸器感染症予防剤を飲食品に適用する場合の添加量としては、病気予防や健康維持が主な目的であるので、飲食品に対して有効成分の含量が合計1~20wt%以下であるのが好ましい。 When the agent for preventing viral respiratory infections of the present invention is applied to food and drink, the main purpose is to prevent disease and maintain health. It is preferably 20 wt% or less.
本発明のウイルス性呼吸器感染症予防剤は、薬品(医薬品および医薬部外品を含む。)の素材として用いてもよい。薬品製剤用の原料に、本発明の剤を適宜配合して製造することができる。本発明の剤に配合しうる製剤原料としては、例えば、賦形剤(ブドウ糖、乳糖、白糖、塩化ナトリウム、デンプン、炭酸カルシウム、カオリン、結晶セルロース、カカオ脂、硬化植物油、カオリン、タルク等)、結合剤(蒸留水、生理食塩水、エタノール水、単シロップ、ブドウ糖液、デンプン液、ゼラチン溶液、カルボキシメチルセルロース、リン酸カリウム、ポリビニルピロリドン等)、崩壊剤(アルギン酸ナトリウム、カンテン、炭酸水素ナトリウム、炭酸カルシウム、ラウリル硫酸ナトリウム、ステアリン酸モノグリセリド、デンプン、乳糖、アラビアゴム末、ゼラチン、エタノール等)、崩壊抑制剤(白糖、ステアリン、カカオ脂、水素添加油等)、吸収促進剤(第四級アンモニウム塩基、ラウリル硫酸ナトリウム等)、吸着剤(グリセリン、デンプン、乳糖、カオリン、ベントナイト、硅酸等)、滑沢剤(精製タルク、ステアリン酸塩、ポリエチレングリコール等)などが挙げられる。 The agent for preventing viral respiratory infections of the present invention may be used as a material for drugs (including pharmaceuticals and quasi-drugs). The agent of the present invention can be appropriately blended with raw materials for drug formulations for production. Pharmaceutical raw materials that can be incorporated into the agent of the present invention include, for example, excipients (glucose, lactose, sucrose, sodium chloride, starch, calcium carbonate, kaolin, crystalline cellulose, cacao butter, hydrogenated vegetable oil, kaolin, talc, etc.), Binders (distilled water, physiological saline, ethanol water, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.), disintegrants (sodium alginate, agar, sodium bicarbonate, carbonate) Calcium, sodium lauryl sulfate, monoglyceride stearate, starch, lactose, gum arabic powder, gelatin, ethanol, etc.), disintegration inhibitors (sucrose, stearin, cocoa butter, hydrogenated oil, etc.), absorption enhancers (quaternary ammonium base) , sodium lauryl sulfate, etc.), adsorbents (glycerin, starch, lactose, kaolin, bentonite, silicic acid, etc.), lubricants (purified talc, stearate, polyethylene glycol, etc.).
投与量は、投与方法、病状、患者の年齢等によって変化し得るが、大人では、通常、1日当たり有効成分として0.5~5000mg、子供では通常0.5~3000mg程度投与することができる。
本発明のウイルス性呼吸器感染症予防剤の配合比は、剤型によって適宜変更することが可能であるが、通常、経口または粘膜吸収により投与される場合は約0.3~15.0wt%、非経口投与による場合は、0.01~10wt%程度にするとよい。なお、投与量は種々の条件で異なるので、前記投与量より少ない量で十分な場合もあるし、また、範囲を超えて投与する必要のある場合もある。
The dosage may vary depending on the administration method, disease state, age of the patient, etc., but for adults the dosage is usually 0.5-5000 mg of the active ingredient per day, and for children it is usually 0.5-3000 mg.
The compounding ratio of the agent for preventing viral respiratory infections of the present invention can be appropriately changed depending on the dosage form, but is usually about 0.3 to 15.0 wt% when administered orally or by mucosal absorption. In the case of parenteral administration, it should be about 0.01 to 10 wt%. Since the dose varies depending on various conditions, there are cases where a dose smaller than the above dose is sufficient, and there are cases where it is necessary to administer a dose exceeding the above range.
以下、本発明の実施例を説明する。なお、以下に示す実施例は、本発明によって得られる剤の各種作用・効果等の確認のために説明するもので、本発明の範囲は、これらの製品および製法に限定されるものではない。 Examples of the present invention will be described below. The examples shown below are for confirmation of various functions and effects of the agents obtained by the present invention, and the scope of the present invention is not limited to these products and manufacturing methods.
試験例:グルコシルセラミド含有米抽出物、グルコシルセラミド及びβ-シトステロールグルコシド(以下、単に「BSG」する)におけるインフルエンザ予防作用、治療作用の評価
1.被験物質および調製方法
1‐1 被験物質1
名称:オリザセラミド(登録商標)(30%グルコシルセラミド含有米抽出物)
入手先:オリザ油化株式会社
1‐2.被験物質2
名称:上記オリザセラミドから単離された下記化学式(1)で示されるグルコシルセラミド
名称:BSG(β-シトステロールグルコシド)
1‐4 溶媒
名称:0.5w/v% メチルセルロース400溶液
1-5.調製方法
秤量済みの被験物質に溶媒を混合し、乳鉢と乳棒を用いて調製した。
なお、被験物質は3日分まとめて調製し、調製後は使用するまで冷蔵で保存した。
Test Example: Evaluation of Influenza Prophylaxis and Treatment of Rice Extract Containing Glucosylceramide, Glucosylceramide and β-Sitosterol Glucoside (hereinafter referred to simply as “BSG”) 1. Test substance and preparation method 1-1 Test substance 1
Name: Oryza Ceramide (registered trademark) (rice extract containing 30% glucosylceramide)
Source: Oryza Petrochemical Co., Ltd. 1-2.
Name: Glucosylceramide represented by the following chemical formula (1) isolated from the above oryzaceramide
Name: BSG (β-sitosterol glucoside)
1-4 Solvent name: 0.5 w/v% methyl cellulose 400 solution 1-5. Preparation method A solvent was mixed with the weighed test substance and prepared using a mortar and pestle.
The test substance was prepared for 3 days at a time and stored in a refrigerator after preparation until use.
2.使用ウイルスおよび接種ウイルスの調製
2-1.使用ウイルス
ATCCから購入後、ハムリー株式会社にてマウス高感受性ウイルスに変異させたウイルスを用いた。
ウイルス名:Influenza virus
株名:A/PR/8/34
ATCC. No.:VR-1469
BSL:2
ウイルス力価:1.58×108 TCID50/mL
保存:ディープフリーザー(CLN-51UD1, 日本フリーザー株式会社)で保存
2.2 接種ウイルスの調製
凍結保存したウイルス液を解凍し、PBSを用いて一般状態観察用は2 × 105 TCID50/mL(1 × 104 TCID50/50 μL)に、肺中ウイルス量測定用には6 × 104 TCID50/mL(3 × 103 TCID50/50 μL)に調製した。
2. Preparation of virus to be used and virus to be inoculated 2-1. Virus used
A virus that was purchased from ATCC and mutated into a mouse hypersusceptible virus at Hamley Co., Ltd. was used.
Virus name: Influenza virus
Strain name: A/PR/8/34
ATCC No.: VR-1469
BSL: 2
Viral titer: 1.58×10 8 TCID 50 /mL
Storage: Store in a deep freezer (CLN-51UD1, Nihon Freezer Co., Ltd.)
2.2 Preparation of virus inoculum Thaw the cryopreserved virus solution, and use PBS to make 2 × 10 5 TCID 50 /mL (1 × 10 4 TCID 50 /50 μL) for general condition observation, and for lung viral load measurement. was prepared to 6×10 4 TCID 50 /mL (3×10 3 TCID 50 /50 μL).
3.使用動物
動物種 :マウス
系統 :BALB/c
入手先 :日本チャールス・リバー株式会社
性別 :雌
週齢 :4週齢(入荷時)
使用匹数:48匹(入荷時50匹)
動物種選択理由:マウス感受性インフルエンザウイルスを用いることから、マウスの選択は試験系として適切と考えられる。
3. Animal species used: Mouse strain: BALB/c
Source: Charles River Japan Co., Ltd. Gender: Female Age: 4 weeks (at the time of arrival)
Number of animals used: 48 (50 at the time of arrival)
Reason for selection of animal species: Since mouse-susceptible influenza virus is used, mouse selection is considered appropriate as a test system.
4.動物管理条件
4‐1. 飼育条件
室温22.0~25.0%(設定24 ± 3 ℃)、湿度43.29~58.18%(設定50 ± 20 %)、換気(10~25回/1時間)、照明12時間(8:00~20:00)
4-2.飼料
MF(オリエンタル酵母工業株式会社、Lot 200617)を自由摂取。
4-3.飲料水
オートクレーブ滅菌した市水を自由摂取。
4-4.ケージ
オートクレーブ滅菌した耐熱性ポリサルフォン製ケージ(207W×365D×140H mm, TECNIPLAST Ltd)を使用。動物は1-5匹/ケージで飼育した。
4-5.検疫および馴化
動物入荷後から検疫および馴化終了日まで、一般状態を毎日観察した。また入荷日ならびに検疫および馴化終了日に体重を測定した。
4-6.群分け
検疫馴化終了時の体重を用いて、体重層別化無作為抽出法により各群に動物を振り分けた。
4. Animal care conditions 4-1. Breeding conditions Room temperature 22.0-25.0% (setting 24 ± 3 ℃), humidity 43.29-58.18% (setting 50 ± 20%), ventilation (10-25 times/hour),
4-2. feed
Ad libitum intake of MF (Oriental Yeast Co., Ltd., Lot 200617).
4-3. Drinking water Autoclave-sterilized city water was available ad libitum.
4-4. Cage Autoclave-sterilized heat-resistant polysulfone cage (207W×365D×140H mm, TECNIPLAST Ltd) is used. Animals were housed 1-5/cage.
4-5. From the arrival of quarantine and acclimatization animals until the end of quarantine and acclimatization, general conditions were observed daily. Body weights were also measured on the day of arrival and on the end of quarantine and acclimatization.
4-6. Grouping Animals were assigned to each group by weight-stratified random sampling using body weights at the end of quarantine acclimation.
5.試験群構成
試験群構成は下記表1に示されるとおりとした。
6.ウイルスの接種
イソフルラン(マイライン製薬株式会社、Lot 196AR0)麻酔下で接種ウイルス液を50 μL/head経鼻接種した。
6. Inoculation of Virus Under anesthesia with isoflurane (Lot 196AR0, Myline Pharmaceutical Co., Ltd.), 50 μL/head of the inoculated virus solution was nasally inoculated.
7.被験物質の投与
ウイルス接種7日前から観察終了前日まで、1日1回、シリンジとゾンデを用いて10mL/kgで経口投与した。ウイルス接種日はウイルス接種前に被験物質を投与した。
7. Administration of Test Substance From 7 days before virus inoculation to the day before the end of observation, 10 mL/kg of test substance was orally administered once a day using a syringe and sonde. On the day of virus inoculation, the test substance was administered before virus inoculation.
8.標本採取(5~8群)
チアミラールナトリウム(日医工株式会社、Lot LU1200、イソゾール(登録商標)注射用0.5 g)の過剰投与により安楽死後、肺を採取して重量を測定した。ウイルス量測定用として50-100mgの肺を採取し、RNAlater(Thermo Fisher Scientific、Lot 00874638)に浸漬し、冷蔵にて1日置いた後に使用するまでディープフリーザーに保管した。残りの肺もディープフリーザーで保管した。
8. Specimen collection (5-8 groups)
After euthanasia by overdose of thiamylal sodium (Nichi-Iko Co., Ltd., Lot LU1200, Isosol® injection 0.5 g), lungs were collected and weighed. 50-100 mg of lungs were harvested for viral load measurement, immersed in RNAlater (Thermo Fisher Scientific, Lot 00874638), refrigerated for 1 day, and then stored in a deep freezer until use. The remaining lungs were also stored in a deep freezer.
9.観察・測定項目
9‐1.一般状態
1日1回以上、一般状態を観察した。
9‐2.体重測定
群分け時(初回投与時:Day -7)、Day 0, 3, 7, 10およびDay 14
9. Observation/measurement items 9-1. General Condition General condition was observed at least once a day.
9-2. At the time of body weight measurement grouping (first administration: Day -7),
10.統計解析
試験データとして得られた計量データは平均±標準偏差を算出した。
一般状態観察用のマウスは平均生存日数及び生存率を算出後、溶媒群に対してLog-Rank検定を行った。
10. Statistical Analysis Weighing data obtained as test data was calculated as mean ± standard deviation.
After calculating the average survival days and survival rate of the mice for general condition observation, the Log-Rank test was performed on the vehicle group.
11.結果
図1 および下記表 2 に生存率を示した。
下記表3 に平均生存日数および観察終了時の生存率を示した。
1群(溶媒群)、2群(オリザセラミド群)、3群(グルコシルセラミド群)および4群(β-シトステロールグルコシド群)の平均生存日数はそれぞれ7.1日、8.5日、7.8日および8.9日であり、統計解析の結果、2群および4群は1群にくらべ有意に生存日数が延長した。
Table 3 below shows the average survival days and the survival rate at the end of observation.
The average survival days in Group 1 (vehicle group), Group 2 (oryzaceramide group), Group 3 (glucosylceramide group) and Group 4 (β-sitosterol glucoside group) were 7.1 days, 8.5 days, 7.8 days and 8.9 days, respectively. Statistical analysis showed that
12.考察及び実施例の効果
インフルエンザ感染マウスモデルを用いてオリザセラミドおよび含有成分の効果を確認することを目的として、ウイルス接種後のマウスの生存率・平均生存日数を評価した。
その結果、オリザセラミドおよびBSGは溶媒群とくらべ有意に生存日数が延長された。また、グルコシルセラミド主成分も生存日数が延長される傾向が確認された。
以上により、グルコシルセラミド及びそれを含有する米抽出物、並びにBSGはインフルエンザ予防、治療剤として有用であることが確認された。
12. Discussion and Effect of Examples For the purpose of confirming the effects of oryzaceramide and its ingredients using an influenza-infected mouse model, the survival rate and average survival days of mice after virus inoculation were evaluated.
As a result, oryzaceramide and BSG significantly extended the survival time compared to the vehicle group. In addition, it was confirmed that the main component of glucosylceramide also tended to prolong survival.
From the above, it was confirmed that glucosylceramide, the rice extract containing it, and BSG are useful as agents for preventing and treating influenza.
本発明による剤(グルコシルセラミド含有米抽出物)の配合例を示す。尚、以下の配合例は本発明を限定するものではない。また、下記配合例ではグルコシルセラミド含有米抽出物のみの例を示すが、BSG及び上記グルコシルセラミドにおいても同様に配合することができる。
配合例1:チューインガム
砂糖 53.0wt%
ガムベース 20.0
グルコース 10.0
水飴 16.0
香料 0.5
グルコシルセラミド含有米抽出物 0.5
100.0wt%
Fig. 2 shows a formulation example of the agent (glucosylceramide-containing rice extract) according to the present invention. In addition, the following formulation examples do not limit the present invention. In the formulation examples below, only the glucosylceramide-containing rice extract is used, but BSG and the above-mentioned glucosylceramide can also be blended in the same manner.
Formulation Example 1: Chewing gum
Sugar 53.0 wt%
gum base 20.0
glucose 10.0
Starch syrup 16.0
Perfume 0.5
Glucosylceramide-containing rice extract 0.5
100.0 wt%
配合例2:グミ
還元水飴 40.0wt%
グラニュー糖 20.0
ブトウ糖 20.0
ゼラチン 4.7
水 9.68
ユズ果汁 4.0
ユズフレーバー 0.6
色素 0.02
グルコシルセラミド含有米抽出物 1.0
100.0wt%
Formulation Example 2: Gummy
Reduced starch syrup 40.0 wt%
Granulated sugar 20.0
Glucose 20.0
gelatin 4.7
Water 9.68
Yuzu Juice 4.0
Yuzu flavor 0.6
Pigment 0.02
Glucosylceramide-containing rice extract 1.0
100.0 wt%
配合例3:キャンディー
砂糖 50.0wt%
水飴 33.0
水 14.4
有機酸 2.0
香料 0.2
グルコシルセラミド含有米抽出物 0.4
100.0wt%
Formulation Example 3: Candy
Sugar 50.0 wt%
Starch syrup 33.0
Water 14.4
Organic acid 2.0
Perfume 0.2
Glucosylceramide-containing rice extract 0.4
100.0 wt%
配合例4:ヨーグルト(ハード・ソフト)
牛乳 41.5wt%
脱脂粉乳 5.8
砂糖 8.0
寒天 0.15
ゼラチン 0.1
乳酸菌 0.005
グルコシルセラミド含有米抽出物 0.4
香料 微量
水 残余
100.0wt%
Formulation Example 4: Yogurt (hard/soft)
Milk 41.5 wt%
Skimmed milk powder 5.8
Sugar 8.0
Agar 0.15
gelatin 0.1
Lactic acid bacteria 0.005
Glucosylceramide-containing rice extract 0.4
Fragrance trace amount
water residue
100.0 wt%
配合例5:清涼飲料
果糖ブドウ糖液糖 30.0wt%
乳化剤 0.5
グルコシルセラミド含有米抽出物 0.3
香料 適量
精製水 残余
100.0wt%
Formulation Example 5: Soft drink
Fructose glucose liquid sugar 30.0 wt%
emulsifier 0.5
Glucosylceramide-containing rice extract 0.3
Perfume Appropriate amount
purified water residue
100.0 wt%
配合例6:錠菓
砂糖 76.4wt%
グルコース 19.0
ショ糖脂肪酸エステル 0.2
グルコシルセラミド含有米抽出物 0.5
精製水 3.9
100.0wt%
Formulation Example 6: Tablet candy
Sugar 76.4 wt%
Glucose 19.0
Sucrose fatty acid ester 0.2
Glucosylceramide-containing rice extract 0.5
Purified water 3.9
100.0 wt%
配合例7:ソフトカプセル
玄米胚芽油 47.0wt%
ユズ種子油 40.0
乳化剤 12.0
グルコシルセラミド含有米抽出物 1.0
100.0wt%
Formulation Example 7: Soft capsule
Brown rice germ oil 47.0 wt%
Yuzu seed oil 40.0
Emulsifier 12.0
Glucosylceramide-containing rice extract 1.0
100.0 wt%
配合例8:錠剤
乳糖 54.0wt%
結晶セルロース 30.0
澱粉分解物 10.0
グリセリン脂肪酸エステル 5.0
グルコシルセラミド含有米抽出物 1.0
100.0wt%
Formulation Example 8: Tablet
Lactose 54.0 wt%
Crystalline cellulose 30.0
Starch hydrolyzate 10.0
Glycerin fatty acid ester 5.0
Glucosylceramide-containing rice extract 1.0
100.0 wt%
以上により、本発明は、新規なウイルス性呼吸器感染予防、治療剤を提供することができる。 As described above, the present invention can provide a novel preventive and therapeutic agent for viral respiratory infections.
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