JP4842507B2 - Anti-influenza virus activator - Google Patents
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本発明はマイタケ由来の抗インフルエンザウイルス活性化物質並びに該抗ウイルス活性化物質を含有する食品若しくは医薬品に関する。 The present invention relates to an anti-influenza virus activator derived from maitake and a food or pharmaceutical product containing the anti-virus activator.
インフルエンザはインフルエンザウイルス(A型,B型,C型)の感染によって生ずる急性炎症であり、上気道よりさらに気管支などの下気道の炎症に及ぶことが多く、又気道の症状と共に高熱、倦怠感、頭痛、筋肉痛、関節痛などの全身症状が著明なことである。A型インフルエンザは他の2型に比べ世界的大流行を惹起することで知られている。近年は両型インフルエンザが混在して流行することも少なくないといわれ、A型,B型インフルエンザは肺炎、気管支炎と合併することも少なくなく、又5才以下の年少者では脳炎・脳症を併発する例も報告されている。一方、わが国においては老齢者の増加に伴い,A型,B型インフルエンザの流行の影響は大きく、心・肺疾患保有者も同様に重症化する危険が高い。インフルエンザワクチンの効果についても、インフルエンザウィルスの抗原性が変わりやすいため、効果に揺れを生じ必ずしも所期効果が期待できない場合もあると言われている。 Influenza is an acute inflammation caused by infection with influenza viruses (types A, B, and C), which often affects inflammation of the lower respiratory tract, such as the bronchi, rather than the upper respiratory tract. Systemic symptoms such as headache, muscle pain, and joint pain are prominent. Influenza A is known to cause a global pandemic compared to the other two types. In recent years, it has been reported that both types of influenza are prevalent, and influenza A and B are often associated with pneumonia and bronchitis, and encephalitis and encephalopathy occur in younger children under 5 years old Some examples have been reported. On the other hand, in Japan, with the increase in the number of elderly people, the effects of influenza A and B influenza are significant, and the risk of heart disease and pulmonary disease carriers is also increased. Regarding the effects of influenza vaccines, the antigenicity of influenza viruses is likely to change, and it is said that there may be cases where the desired effects cannot be expected due to fluctuations in the effects.
従って、安全かつ入手が容易な天然物若しくはその抽出物から抗インフルエンザウイルス活性物質が得られることが切望されているところである。 Accordingly, it is highly desired that an anti-influenza virus active substance can be obtained from a natural product that is safe and easily available or an extract thereof.
本発明は、天然物若しくはその抽出物から抗インフルエンザウイルス活性化物質を取得することを課題とする。 An object of the present invention is to obtain an anti-influenza virus activator from a natural product or an extract thereof.
本発明者等は上記課題を解決するために、各種天然物の抽出物について検討を試み、最近入手が容易になったキノコの一種であるマイタケに注目した。 In order to solve the above-mentioned problems, the inventors of the present invention have tried to examine various natural product extracts, and have focused attention on maitake, which is a kind of mushroom that has recently become easy to obtain.
マイタケ抽出物については、本出願人等の開発努力により多彩な作用が知られており、例えばAIDS症改善効果については特開平7−69913号公報(特許文献1)抗腫瘍作用については、特開平9−238697号公報(特許文献2)に、活性酸素消去活性については特開2000−119650号公報(特許文献3)に、NO産生誘導作用については特開2001−172194号公報(特許文献4)等で報告されている。
As for the maitake extract, various actions are known due to the development efforts of the present applicants. For example, regarding the improvement effect of AIDS disease, JP-A-7-69913 (Patent Document 1) to 9-238697 (Patent Document 2), to the active oxygen
本発明者等は、鋭意研究の結果、マイタケの抽出物に抗インフルエンザウイルス活性があることを見出し本発明を完成した。 As a result of intensive studies, the present inventors have found that an extract of maitake has anti-influenza virus activity and completed the present invention.
即ち本発明は
(1) 生マイタケ、乾燥マイタケ及びマイタケ燥乾粉末のいずれか1以上を水乃至熱水で抽出して得られる抽出物からなるマクロファージ存在下における抗インフルエンザウイルス活性化物質、
(2) 生マイタケ、乾燥マイタケ及びマイタケ乾燥粉末のいずれか1以上を水乃至熱水で抽出して得られる抽出液に、アルコールを加え生成する沈殿からなるマクロファージ存在下における抗インフルエンザウイルス活性化物質、
(3) 生マイタケ、乾燥マイタケ及びマイタケ乾燥粉末のいずれか1以上を水乃至熱水で抽出して得られる抽出液に、沈殿が生じない程度にアルコールを加え、放置後液面若しくは液中に浮遊又は容器の壁面に付着する物質を取り除き、次いで該溶液を乾燥したものからなるマクロファージ存在下における抗インフルエンザウイルス活性化物質、
(4) 生マイタケ、乾燥マイタケ及びマイタケ乾燥粉末のいずれか1以上を水乃至熱水で抽出して得られる抽出液に、沈殿が生じない程度にアルコールを加え、放置後液面若しくは液中に浮遊又は容器の壁面に付着する物質を取り除き、次いで更にアルコールを加え生成する沈殿からなるマクロファージ存在下における抗インフルエンザウイルス活性化物質、
(5) 活性発現の機序がTNF-α産生誘導を介してなるものであることを特徴とする(1)乃至(4)記載のマクロファージ存在下における抗インフルエンザウイルス活性化物質、
(6)(1)乃至(5)のいずれか記載の抗インフルエンザウイルス活性化物質を配合してなる食品、
(7) (1)乃至(5)のいずれか記載の抗インフルエンザウイルス活性化物質を配合してなる医薬品
に関する。
That is, the present invention
(1) an anti-influenza virus activator in the presence of macrophages comprising an extract obtained by extracting one or more of raw maitake, dried maitake and maitake dry powder with water or hot water,
(2) Anti-influenza virus activator in the presence of macrophages consisting of a precipitate formed by adding alcohol to an extract obtained by extracting one or more of raw maitake, dried maitake and dried maitake with water or hot water ,
(3) Add alcohol to the extract obtained by extracting one or more of raw maitake, dried maitake and dried maitake with water or hot water to the extent that precipitation does not occur. An anti-influenza virus activator in the presence of macrophages, consisting of a material that floats or adheres to the wall of the container and then dries the solution;
(4) Add alcohol to the extract obtained by extracting one or more of raw maitake, dried maitake and dried maitake with water or hot water to the extent that precipitation does not occur. An anti-influenza virus activator in the presence of macrophages consisting of a precipitate formed by removing floating or adhering substances on the wall of the container, and then adding more alcohol;
(5) The anti-influenza virus activator in the presence of macrophages according to (1) to (4), wherein the mechanism of activity expression is through TNF-α production induction,
(6) A food comprising the anti-influenza virus activator according to any one of (1) to (5),
(7) A pharmaceutical product comprising the anti-influenza virus activator according to any one of (1) to (5).
本発明により、安全かつ容易に入手可能なマイタケ抽出物を用いて、インフルエンザ等感冒の予防或いは罹患後の症状を軽減することができ、食品若しくは薬品として用いることが可能となる。 According to the present invention, a maitake extract that can be obtained safely and easily can be used to prevent or prevent symptoms of the common cold such as influenza, and can be used as a food or a medicine.
以下本発明について詳述する。 The present invention is described in detail below.
本発明において、マイタケは(Grifola frondosa)、白マイタケ(Grifola albicans)、チョレイマイタケ(Dendropolyporus umbellatus)、トンビマイタケ(Grifola gigantia)等いづれも用いることが出来る。又これらマイタケ類の子実体、菌糸体いずれも用いることが出来るが、最近ではマイタケの子実体の人工栽培が可能となり、安定した原料確保の面から該マイタケの子実体を使用することが好ましい。 In the present invention, maitake ( Grifola frondosa ), white maitake ( Grifola albicans ), choreimaitake ( Dendropolyporus umbellatus ), tombii maitake ( Grifola gigantia ), etc. can be used. Both fruit bodies and mycelium of these maitakes can be used, but recently, fruit bodies of maitake can be artificially grown, and it is preferable to use the fruit bodies of maitake from the viewpoint of securing a stable raw material.
生マイタケは収穫後、出来るだけ新鮮なものを使用することが好ましいが、食に供する状態であれば用いることができる。使用部位は特に特定されることなく茎部以上すべて使用しうる。乾燥マイタケとしては天日、熱風乾燥或いは凍結乾燥したもの等いずれも用いることが出来る。マイタケ乾燥粉末は粒子の粗いものから微細なものまで使用することが出来る。 Raw maitake is preferably as fresh as possible after harvesting, but can be used as long as it is ready for food. The use site is not particularly specified and can be used above the stem. As the dried maitake, any of sun, hot air dried or freeze dried can be used. Maitake dry powder can be used from coarse to fine particles.
抽出の方法は水乃至熱水で行う。「水乃至熱水」とは水から熱水に至るまでを意味し、温水も当然包含される。従って抽出は、常温、加温乃至加熱下、常圧乃至加圧下、常法に準じて適宜行いうる。 The extraction method is performed with water or hot water. “Water or hot water” means from water to hot water, and naturally includes hot water. Therefore, extraction can be appropriately performed according to a conventional method at room temperature, under heating or heating, under normal pressure or under pressure.
例えば常温〜120℃で5分〜数時間行う。短時間で効率よく行うためには、圧力下、100℃以上、例えば圧力釜を用いて加圧下120℃前後で30分〜1時間前後抽出を行うのが好ましい。 For example, it is performed at room temperature to 120 ° C. for 5 minutes to several hours. In order to perform efficiently in a short time, it is preferable to perform extraction at about 100 ° C. or higher under pressure, for example, at about 120 ° C. under pressure for about 30 minutes to about 1 hour under pressure.
水としては蒸留水、イオン交換水、逆浸透膜(RO)水、水道水、天然水いずれも使用しうる。乾燥マイタケ若しくは乾燥マイタケ粉末1重量に対して水を4〜30倍重量程度使用する。生マイタケを使用する場合は1重量に対して2〜10倍重量程度の水を使用する。 Distilled water, ion exchange water, reverse osmosis membrane (RO) water, tap water, and natural water can be used as water. About 4 to 30 times the weight of water is used per 1 weight of dry maitake or dry maitake powder. When using raw maitake, use about 2 to 10 times the weight of water.
以上の様に水乃至は熱水抽出液はそのまま、更に濃縮して濃縮エキス若しくは濃縮エキスを乾燥して乾燥抽出エキス末として使用することが出来る。 As described above, the water or hot water extract can be used as a dry extract powder by further concentrating it and drying the concentrated extract or concentrated extract.
水乃至熱水抽出液はアルコール沈殿法により精製することが可能である。例えばアルコールを抽出液に加え沈殿する物質を採取する。沈殿物はマイタケ中に含まれる高分子多糖体β−グルカンを主体にした画分であり、このまま利用できる。また必要に応じて更に精製することも可能である。例えばアルコールを水乃至熱水抽出液に沈殿が生じない程度に加え、望ましくは1〜25℃で、放置し生ずる液面、液中に浮遊する物質或いは容器の壁に付着する物資を除去して精製することができる。除去は濾過、吸引或いは網状のもので掬うなど適宜行いうる。 Water or hot water extract can be purified by alcohol precipitation. For example, an alcohol is added to the extract to collect a precipitated substance. The precipitate is a fraction mainly composed of high-molecular polysaccharide β-glucan contained in maitake and can be used as it is. Further purification can be performed as necessary. For example, alcohol is added to such an extent that precipitation does not occur in water or hot water extract, and preferably at 1 to 25 ° C., the liquid surface left standing, the substance floating in the liquid or the material adhering to the wall of the container is removed. Can be purified. The removal can be appropriately performed by filtration, suction, or scrubbing with a net-like material.
沈殿が生じない程度のアルコールの添加量は、抽出液の濃度や温度により異なり、一概には決めがたいが、アルコールの最終容量濃度が比較的低濃度、即ち約30〜60容量%を目安にする。
尚アルコールとしては低級アルコールが使用しうるが、特にエタノールが好ましい。
The amount of alcohol added to such an extent that precipitation does not occur differs depending on the concentration and temperature of the extract, and although it is generally difficult to determine, the final volume concentration of alcohol is relatively low, that is, about 30-60% by volume. To do.
In addition, although lower alcohol can be used as alcohol, especially ethanol is preferable.
この様にして得られた溶液は乾燥しても良いし、必要に応じて、アルコールを比較的高濃度、即ち最終容量濃度が60容量%以上になるよう加え、沈殿する物質を採取する様な手段をとることにより精製することもできる。尚上記乾燥は噴霧乾燥、濃縮乾燥若しくは凍結乾燥等何れの手段も用いうるが、特に噴霧乾燥が好ましい。 The solution thus obtained may be dried, or if necessary, alcohol is added to a relatively high concentration, that is, the final volume concentration is 60% by volume or more, and the precipitated substance is collected. It can also be purified by taking measures. In addition, although any means, such as spray drying, concentration drying, or freeze drying, can be used for the said drying, spray drying is especially preferable.
以上のように得られた乾燥物若しくは沈殿物は水に可溶で、多糖体であるβ-グルカンを含有する。水溶液とし上述の様にアルコール沈殿法を繰り返すことにより更に精製することも出来るし、更にクロマトグラフイーにより、β-グルカンの純度を高めることも可能である。例えば ゲル濾過法、イオンクロマト法、アフィニティクロマト法等使用することが出来る。 The dried product or precipitate obtained as described above is soluble in water and contains β-glucan which is a polysaccharide. It can be further purified by using an aqueous solution and repeating the alcohol precipitation method as described above, and the purity of β-glucan can be increased by chromatography. For example, gel filtration, ion chromatography, affinity chromatography and the like can be used.
以上のようにして得られた特定のマイタケ抽出物を、インフルエンザウイルスを接種したイヌ腎臓由来のMDCK細胞に、マクロファージ非存在下直接種々の濃度で加え、培養後インフルエンザウイルスの定量を行ったが増殖抑制は殆ど認められなかった。 The specific maitake extract obtained as described above was added to MDCK cells derived from canine kidney inoculated with influenza virus at various concentrations directly in the absence of macrophages. Almost no inhibition was observed.
しかしながら、マクロファージ由来のRAW細胞とマイタケ抽出物を種々時間培養後採取した培養上清を、インフルエンザウイルスを接種したMDCK細胞に加え培養を行ったところ、時間経過とともに、インフルエンザウイルスが濃度依存的に減少していることが認められた。 However, when the culture supernatant collected after various times of culturing macrophage-derived RAW cells and maitake extract was added to MDCK cells inoculated with influenza virus, the influenza virus decreased in a concentration-dependent manner over time. It was recognized that
そして更にマイタケ抽出物で、RAW細胞を刺激したのちTNF-αmRNA発現がマイタケ抽出物の濃度依存的に誘発されていることが、RT-PCR法で明らかになった。また実際にTNF-αmRNAが翻訳されTNF-αが経時的に増加していることがELISA kit を用いて検討して判明した。 Furthermore, it was revealed by RT-PCR that TNF-α mRNA expression was induced in the concentration of maitake extract after stimulating RAW cells with maitake extract. In addition, the fact that TNF-α mRNA was actually translated and TNF-α increased over time was found using an ELISA kit.
以上の結果より、マイタケ抽出物がマクロファージを刺激し、TNF-αを誘発し、抗インフルエンザウイルス活性が惹起されていることが判明した。従って、インフルエンザ等感冒の予防或いは罹患後の症状軽減のための食品若しくは薬品としての応用が可能である。 From the above results, it was found that the maitake extract stimulated macrophages, induced TNF-α, and induced anti-influenza virus activity. Therefore, it can be applied as a food or medicine for prevention of colds such as influenza or reduction of symptoms after illness.
本発明のマイタケ抽出物質は、毒性がないことが確認されており新しい形の食品として利用できるし又医薬品としても応用できる。 The maitake extract of the present invention has been confirmed to be non-toxic and can be used as a new form of food or as a pharmaceutical product.
その経口摂取量は成人1日当たり(濃縮乾燥粉末で)50〜500mgを目安とするが、必要に応じ増減することができる。 The oral intake is generally 50 to 500 mg per day (concentrated dry powder) for adults, but can be increased or decreased as necessary.
例えば食品として応用する場合は、健康食品、サプリメント、機能性食品として用いることができるが、一般的な食品、例えば菓子・パン類、麺類、調味料、香辛類、食用粉類、乳製品、肉製品、加工水産物、加工果実・野菜、各種飲料・ジュース類、お茶、インスタント食品等に配合することができる。 For example, when applied as food, it can be used as health foods, supplements, functional foods, but general foods such as confectionery / breads, noodles, seasonings, spices, edible powders, dairy products, meat It can be blended into products, processed marine products, processed fruits and vegetables, various beverages and juices, tea, and instant foods.
健康食品、サプリメント、機能性食品又は医薬品として用いる場合は適宜、賦形剤、増量剤を加え錠剤、カプセル剤、顆粒剤、粉末剤、丸剤、液剤、懸濁液剤等各種製剤に加工することができる。
以下に実施例を示すが、本発明はそれらによって限定されるものではない。
When used as health foods, supplements, functional foods or pharmaceuticals, excipients and extenders are added as appropriate, and processed into various preparations such as tablets, capsules, granules, powders, pills, liquids, suspensions, etc. Can do.
Examples are shown below, but the present invention is not limited thereto.
抗インフルエンザウイルス活性物質の製造
(1) 乾燥マイタケ子実体の粉末2.5kgにイオン交換水30Lを加圧釜に入れ、加圧下115〜120℃で約0.5時間処理し、その後濾過して褐色液(A)約20Lを得た。該褐色液(A)5Lを取り、減圧下濃縮してBrix濃度20%の濃縮液を得、該濃縮液をスプレードライ装置を用いて噴霧乾燥して褐色乾燥粉末(マイタケ抽出物[1])約152gを得た。
(2)上記(1)で得られた褐色液(A)3Lを取り、減圧下、0.5Lまで濃縮し室温で99%エタノールを同容量加え、約10時間放置して褐色沈殿を回収し、それを減圧乾燥して褐色物質(マイタケ抽出物[2])5gを得た。
(3)上記(1)で得られた褐色液(A)を10Lをとり減圧下約1Lまで濃縮し、室温で99%エタノールを同容量加え、10℃以下で約10時間放置すると液面、液中に浮遊及び容器の壁面に付着する茶褐色の物質が生成した。これら物質を除去し、褐色の溶液を得る。該褐色の液約1.9Lを減圧下エタノールを除去し、更に濃縮してBrix濃度35%の濃縮液を得た。該濃縮液をスプレードライ装置を用いて噴霧乾燥して褐色乾燥粉末(マイタケ抽出物[3])約230gを得た。
Manufacture of anti-influenza virus active substance (1) 2.5 kg of dried maitake fruit body powder is charged with 30 L of ion exchange water in a pressure kettle, treated under pressure at 115-120 ° C. for about 0.5 hours, and then filtered to brown About 20 L of liquid (A) was obtained. 5 L of the brown liquid (A) is taken and concentrated under reduced pressure to obtain a concentrated liquid having a Brix concentration of 20%. The concentrated liquid is spray-dried using a spray drying apparatus to obtain a brown dry powder (Mitake extract [1]) About 152 g was obtained.
(2) Take 3 L of the brown liquid (A) obtained in (1) above, concentrate to 0.5 L under reduced pressure, add the same volume of 99% ethanol at room temperature, and leave it for about 10 hours to recover the brown precipitate. It was dried under reduced pressure to obtain 5 g of a brown substance (Maitake extract [2]).
(3) Take 10 L of the brown liquid (A) obtained in (1) above, concentrate to about 1 L under reduced pressure, add the same volume of 99% ethanol at room temperature, and leave it at 10 ° C. or less for about 10 hours to leave the liquid level. A brownish substance floating in the liquid and adhering to the wall of the container was formed. These materials are removed to give a brown solution. About 1.9 L of the brown liquid was subjected to removal of ethanol under reduced pressure and further concentrated to obtain a concentrated liquid having a Brix concentration of 35%. The concentrated solution was spray-dried using a spray-drying apparatus to obtain about 230 g of a brown dry powder (Mitake extract [3]).
マイタケ抽出物[3]についてラットを用いた急性経口毒性試験において、LD50値は2g/kg以上を示し、毒性を示さなかった。
又Salmonella typhimurium及びEscherichia coli 等細菌を用いた復帰突然変異試験を行った結果、全く変異原性を有しないことが確認された。
In the acute oral toxicity test using rats on the maitake extract [3], the LD50 value was 2 g / kg or more, indicating no toxicity.
Further, as a result of a reverse mutation test using bacteria such as Salmonella typhimurium and Escherichia coli, it was confirmed that there was no mutagenic activity.
(1)材料の調整
抗インフルエンザウイルス活性検討の実験に用いた材料は以下の通りである。
被験物刺激細胞:マウスマクロファージ由来RAW264.7細胞〔10%非働化牛胎仔血清加Dulbecco’s MEMで培養したもの〕(以下RAW細胞と称す)を使用。
インフルエンザウイルス感染細胞:MDCK細胞〔8%非働化牛胎仔血清加MEMで培養したもの〕を用いた。
(1) Preparation of materials The materials used in the experiment for examining the anti-influenza virus activity are as follows.
Test-subject stimulating cells: Mouse macrophage-derived RAW264.7 cells (cultured in Dulbecco's MEM with 10% inactivated fetal bovine serum) (hereinafter referred to as RAW cells) were used.
Influenza virus-infected cells: MDCK cells (cultured in 8% inactivated fetal bovine serum-added MEM) were used.
インフルエンザウイルス:infulenza virus A/Aich/2/68(H3N2)〔発育鶏卵漿尿膜腔に接種し、得られた感染漿尿液を活性ウイルス液としてPBSで適宜希釈したもの〕を使用。
被験物質: 実施例1で製造したマイタケ抽出物[1]乃至マイタケ抽出物[3]
以下マイタケ抽出物[3]についての結果を例示する。
Influenza virus: Influenza virus A / Aich / 2/68 (H3N2) [inoculated into the chorioallantoic cavity of the developing chicken egg and the obtained infected chorioallantoic fluid diluted appropriately with PBS as an active viral fluid] was used.
Test substance: Maitake extract [1] to Maitake extract [3] produced in Example 1
The results for the maitake extract [3] are illustrated below.
(2)抗インフルエンザウイルス活性
Conditioned mediumとして、6 センチデイシュにコンフルエントに培養したRAW細胞を1回serum free DMEDにて洗浄後、種々の濃度のマイタケ抽出物[3](以下、マイタケ抽出物と省略。)含有培地で培養後、適宜採取した培養上清をConditioned medium(以下CMと省略)として用いた。
(2) Anti-influenza virus activity
As conditioned medium, RAW cells cultured confluently in 6 centimeters were washed once with serum free DMED, and then cultured in medium containing various concentrations of maitake extract [3] (hereinafter abbreviated as maitake extract). Thereafter, the culture supernatant appropriately collected was used as a conditioned medium (hereinafter abbreviated as CM).
増殖試験は、24穴プレートに培養したMDCK細胞に、インフルエンザウイルス液をMOI 5 PFU/cellで接種し、室温にて60分吸着させた後、PBS で3回洗浄し未吸着ウイルスを洗浄除去した。その後種々濃度のマイタケ抽出物を添加して培養したCMを加え37℃で培養を開始した。
此の培養開始時を感染0時間とし培養12時間後のウイルス量をplaque法で定量した。
In the proliferation test, influenza virus solution was inoculated with MOI 5 PFU / cell on MDCK cells cultured in 24-well plates, adsorbed at room temperature for 60 minutes, and then washed 3 times with PBS to remove unadsorbed virus. . Thereafter, CM cultured with various concentrations of maitake extract was added, and the culture was started at 37 ° C.
The start of this culture was defined as 0 hours of infection, and the amount of virus after 12 hours of culture was quantified by the plaque method.
試験に先立ちマイタケ抽出物のRAW細胞への細胞毒性をMTT法にて検討した。その結果は図1に示す。グラフに示すとおり、RAW細胞をマイタケ抽出物1mg/mlで12時間刺激した場合、コントロールのserum free DMEMに比し、細胞毒性が認められた。しかしながらマイタケ抽出物300μg/ml以下の濃度では細胞毒性は認められなかった。又図には示さないが、マイタケ抽出物300μg/mlはMDCK細胞にも細胞毒性を示さなかった。これらの結果より、以後実験ではマイタケ抽出物の濃度として300μg/ml以下を用いることにした。 Prior to the test, the cytotoxicity of maitake extract to RAW cells was examined by MTT method. The result is shown in FIG. As shown in the graph, when RAW cells were stimulated with 1 mg / ml of maitake extract for 12 hours, cytotoxicity was observed compared to serum free DMEM as a control. However, cytotoxicity was not observed at concentrations of 300 or less maitake extract. Although not shown in the figure, 300 μg / ml of maitake extract did not show cytotoxicity to MDCK cells. From these results, it was decided to use 300 μg / ml or less as the concentration of the maitake extract in the subsequent experiments.
結果を図2及び図3に示す。
図2の左側のグラフで示される様に12時間刺激後のCMを用いた群では、マイタケ抽出物300μg/mlのCM添加のウイルス量は47.7×104PFU/ml、100μg/mlのCM添加のウイルス量は2.5×106PFU/mlであり、無刺激対照CMのウイルス量を100%とした場合、それぞれ12%, 64%とウイルス量は濃度依存的に減少を示した。
The results are shown in FIGS.
As shown in the graph on the left side of FIG. 2, in the group using CM after stimulation for 12 hours, the virus amount of CM addition of 300 μg / ml of maitake extract was 47.7 × 10 4 PFU / ml, CM addition of 100 μg / ml CM The amount of virus was 2.5 × 10 6 PFU / ml. When the amount of virus in the unstimulated control CM was taken as 100%, the amount of virus decreased by 12% and 64%, respectively, depending on the concentration.
これに対して、右側のグラフは感染したMDCK細胞に直接マイタケ抽出物を添加し、培養後のウイルス量をplaque法で定量した結果で、マイタケ抽出物が300μg/ml、100μg/mlともにDMEMのみのコントロールのウイルス量に比し有意な差は認められなかった。 On the other hand, the graph on the right shows the result of adding the maitake extract directly to the infected MDCK cells and quantifying the amount of virus after culturing by the plaque method. The maitake extract was only DMEM for both 300 μg / ml and 100 μg / ml. There was no significant difference compared to the amount of virus in the control.
図3はマイタケ抽出物300μg/mlで刺激し、時間経過に従って採取したCMで同様に増殖実験を行った結果を示す。
FIG. 3 shows the results of a similar proliferation experiment performed on CM collected with
この場合、30分及び1時間後に採取したCMを用いた場合、対照コントロールCMに対して、有意なウイルス増殖抑制は認められなかった。しかしながら、刺激時間が長くなるにつれて徐々にウイルス量は減少して行き、刺激後20時間のCMを用いたときにはそのウイルス量は50×104PFU/mlとなり、対照の0.6%にまで減少していた。 In this case, when CM collected after 30 minutes and 1 hour was used, no significant virus growth inhibition was observed with respect to the control control CM. However, the viral load gradually decreased as the stimulation time became longer, and when using CM for 20 hours after stimulation, the viral load was 50 × 10 4 PFU / ml, decreasing to 0.6% of the control. It was.
(3)TNF-αmRNA発現
マイタケ抽出物300μg/ml及び100μg/ml で12時間刺激後のTNF-αmRNA発現の誘発をRT-PCR法で見たところ、図4に示される様に、マイタケ抽出物は濃度依存性にTNF-αmRNAの発現を誘発していることが明らかになった。
(3) Expression of TNF-α mRNA When induction of TNF-α mRNA expression after stimulation with
更に、マイタケ抽出物の濃度を300μg/mlと固定してRAW細胞を刺激した時のTNF-αmRNA発現レベルの経時的変化を検討した。
結果を示すと図5の様になる。グラフに示されるように、TNF-αmRNA発現レベルを内部標準として使用したGAPDHにおけるmRNAの発現レベルとの比で見ると、刺激後30分のRAW細胞では、TNF-αmRNA発現は認められなかったが、1時間後より急激に増加し、2時間目でピークに達し、その後徐々にその発現は減少した。
Furthermore, changes in TNF-α mRNA expression level over time when RAW cells were stimulated with the concentration of the maitake extract fixed at 300 μg / ml were examined.
The result is shown in FIG. As shown in the graph, TNF-α mRNA expression level was not observed in
(4)TNF-αの産生
次に、実際にTNF-αmRNAが翻訳され、TNF-αが産生されているかELISA Kitを用いて検討した。
結果を示すと図6のようになる。
(4) Production of TNF-α Next, it was examined using ELISA Kit whether TNF-α mRNA was actually translated and TNF-α was produced.
The result is shown in FIG.
グラフ一番右の対照コントロールであるマイタケ抽出物300μg/mlのみの場合において、TNF-α量は僅かながら増加を示した。これに対してマイタケ抽出物300μg/mlでRAW細胞を刺激したCM中のTNF-α量は経時的に増加しており、最大800pg/ml迄増加している。
この結果は先に示したTNF-αmRNAの発現の経時的変化に一致している。
The amount of TNF-α slightly increased in the case of only 300 μg / ml of maitake extract, which is the control control on the rightmost side of the graph. In contrast, the amount of TNF-α in CM stimulated with RAW cells with 300 μg / ml of maitake extract increased with time, and increased to a maximum of 800 pg / ml.
This result is consistent with the change over time in the expression of TNF-α mRNA shown above.
すなわち、本願発明のマイタケ抽出物がRAW細胞を刺激することによって、TNF-αの産生を誘導し、ひいては抗インフルエンザウイルスの活性を高めることが認められた。 That is, it was confirmed that the maitake extract of the present invention stimulates RAW cells to induce the production of TNF-α and thus enhance the activity of anti-influenza virus.
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