JP4961144B2 - Antiviral agent - Google Patents
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- JP4961144B2 JP4961144B2 JP2006030248A JP2006030248A JP4961144B2 JP 4961144 B2 JP4961144 B2 JP 4961144B2 JP 2006030248 A JP2006030248 A JP 2006030248A JP 2006030248 A JP2006030248 A JP 2006030248A JP 4961144 B2 JP4961144 B2 JP 4961144B2
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本発明は、原料多糖が有する水酸基が部分的に硫酸エステル化された硫酸化多糖類または該硫酸化多糖類を部分構造として有する化合物を含む新規な抗ウイルス剤に関する。 The present invention relates to a novel antiviral agent comprising a sulfated polysaccharide in which a hydroxyl group of a raw material polysaccharide is partially sulfated or a compound having the sulfated polysaccharide as a partial structure.
従来から硫酸化多糖による抗ウイルス作用については、ヘパリンを初めとして数多くの硫酸化多糖が研究されてきた（例えば非特許文献１、特許文献１参照）。しかしながらこれらを薬剤として用いる場合、硫酸化多糖の有する各種活性が逆に副作用となり問題となっている(例えば非特許文献１参照)。
このため安全でかつ安価な抗ウイルス剤の開発が望まれていた。このような事情に鑑み、本発明は、細胞毒性の低い抗ウイルス剤を安価に提供することを課題としてなされた。 Therefore, development of a safe and inexpensive antiviral agent has been desired. In view of such circumstances, an object of the present invention is to provide an antiviral agent having low cytotoxicity at a low cost.
本発明者は、上記課題を解決するために鋭意研究を行った。その結果、ジェランの水酸基を１０％以上硫酸化した硫酸化ジェランが、HIV感染に対する細胞障害抑制作用を有すること、細胞毒性が極めて低いこと、巨細胞形成抑制作用を有することを見出した。こうして、安価な多糖を既存の方法で硫酸化することで得られる硫酸化多糖に細胞毒性の低い抗ウイルス作用があることを見出し、本発明の完成に至った。 The present inventor has intensively studied to solve the above problems. As a result, the present inventors have found that sulfated gellan in which the hydroxyl group of gellan is sulfated by 10% or more has a cytotoxicity-inhibiting action against HIV infection, has extremely low cytotoxicity, and has an inhibitory action on giant cell formation. Thus, the sulfated polysaccharide obtained by sulfating an inexpensive polysaccharide by an existing method was found to have an antiviral action with low cytotoxicity, and the present invention was completed.
本発明は下記の構成を有する。
［１］グルコースとグルクロン酸とラムノースからなり、糖を構成する水酸基の一部又は全部が硫酸エステル化されている硫酸化多糖を有効成分として含有することを特徴とする抗ウイルス剤。
［２］前記硫酸化多糖が、グルコース２分子とグルクロン酸１分子とラムノース１分子からなる構成単位の繰り返しによって構成されていることを特徴とする前記［１］項記載の抗ウイルス剤。
［３］前記構成単位が、下記式（１）で示される単位であることを特徴とする前記［１］項記載の抗ウイルス剤。
［４］前記硫酸化多糖が硫酸化ジェランであることを特徴とする前記［３］項記載の抗ウイルス剤。
［５］ヒトレトロウイルスに効果があることを特徴とする前記［１］〜［４］のいずれか１項記載の抗ウイルス剤。
［６］前記ヒトレトロウイルスがＨＩＶであることを特徴とする前記［５］記載の抗ウイルス剤。
［７］前記硫酸化多糖の平均分子量が５〜５００ｋＤａであることを特徴とする前記［１］〜［６］のいずれか１項記載の抗ウイルス剤。
［８］前記水酸基の１０％以上が硫酸化されていることを特徴とする前記［１］〜［７］のいずれか１項記載の抗ウイルス剤。
The present invention has the following configuration.
[1] An antiviral agent comprising a sulfated polysaccharide consisting of glucose, glucuronic acid, and rhamnose, wherein a part or all of hydroxyl groups constituting the sugar is sulfated as an active ingredient.
[2] The antiviral agent according to [1], wherein the sulfated polysaccharide is composed of repeating structural units composed of two molecules of glucose, one molecule of glucuronic acid, and one molecule of rhamnose.
[3] The antiviral agent according to [1], wherein the structural unit is a unit represented by the following formula (1).
[4] The antiviral agent according to [3], wherein the sulfated polysaccharide is sulfated gellan.
[5] The antiviral agent according to any one of [1] to [4], which is effective for human retroviruses.
[6] The antiviral agent according to [5], wherein the human retrovirus is HIV.
[7] The antiviral agent according to any one of [1] to [6], wherein the sulfated polysaccharide has an average molecular weight of 5 to 500 kDa.
[8] The antiviral agent according to any one of [1] to [7], wherein 10% or more of the hydroxyl group is sulfated.
本発明により、細胞毒性の低い抗ウイルス剤を安価に提供することができるようになった。 According to the present invention, an antiviral agent having low cytotoxicity can be provided at low cost.
以下、本発明を詳細に説明する。なお、本明細書においては、硫酸エステル化を硫酸化と、水酸基が部分的に硫酸エステル化された多糖を硫酸化多糖と称することがある。 Hereinafter, the present invention will be described in detail. In the present specification, sulfate esterification may be referred to as sulfation, and a polysaccharide having a hydroxyl group partially sulfated may be referred to as a sulfated polysaccharide.
本願発明にかかる抗ウイルス剤は、グルコースとグルクロン酸とラムノースからなり、糖を構成する水酸基の一部又は全部が硫酸エステル化されている硫酸化多糖を有効成分として含有する抗ウイルス剤である。特に、原料多糖において、グルコースとグルクロン酸とラムノースの存在比が２：１：１である構成単位の繰り返しによって構成されていることが好ましいが、この存在比であれば、構成単位内における、上記単糖の結合順序、単糖間の結合様式（例えばα結合、β結合）、単糖間の結合位置は特に限定されるものではない。なお、本発明において、硫酸化率とは、原料多糖が有する水酸基のうち、硫酸エステル化された割合を百分率で表した値をいう。本発明に用いられる硫酸化多糖の硫酸化率は、１０％以上であり、１０〜８０％が好ましく、２０〜５０％がより好ましい。硫酸化多糖の平均分子量は５〜５００ｋＤａが好ましく、５〜３０ｋＤａがより好ましい。 The antiviral agent according to the present invention is an antiviral agent comprising, as an active ingredient, a sulfated polysaccharide that is composed of glucose, glucuronic acid, and rhamnose and in which part or all of the hydroxyl groups constituting the sugar are sulfated. In particular, in the raw material polysaccharide, it is preferable that the abundance ratio of glucose, glucuronic acid, and rhamnose is constituted by repeating structural units of 2: 1: 1. The binding order of monosaccharides, the binding mode between monosaccharides (for example, α bond, β bond), and the binding position between monosaccharides are not particularly limited. In addition, in this invention, a sulfation rate means the value which represented the ratio by which sulfate esterification was carried out in percentage among the hydroxyl groups which raw material polysaccharide has. The sulfation rate of the sulfated polysaccharide used in the present invention is 10% or more, preferably 10 to 80%, and more preferably 20 to 50%. The average molecular weight of the sulfated polysaccharide is preferably 5 to 500 kDa, more preferably 5 to 30 kDa.
本発明の抗ウイルス剤を構成する硫酸化多糖の原料の多糖は、化学合成されたものであっても、天然由来の微生物の発酵産物や海藻抽出物などであってもよく、特に起源は限定されるものではない。また、それらは塩酸、硫酸、トリフルオロ酢酸またはその他の酸および水酸化ナトリウムなどのアルカリ化合物などによる加水分解により、あらかじめ低分子量化してから硫酸化反応に用いることができる。 The raw material polysaccharide of the sulfated polysaccharide constituting the antiviral agent of the present invention may be a chemically synthesized product, a fermented product of a naturally occurring microorganism, a seaweed extract, or the like, and its origin is particularly limited. Is not to be done. In addition, they can be used in the sulfation reaction after the molecular weight has been reduced in advance by hydrolysis with hydrochloric acid, sulfuric acid, trifluoroacetic acid or other acid and an alkali compound such as sodium hydroxide.
原料多糖の具体例として、式（２）の構成単位を有する多糖をあげることができる。
原料多糖としてさらに具体的には、式（２）の構成単位からなる多糖、すなわち、シュードモナス エロデア（Pseudomonas elodea）が生産する多糖類を脱アシル化処理後精製したジェラン（gellan CAS 71010-52-1）があげられる。ジェランはグルコース、グルクロン酸、ラムノースが主成分である多糖類で、安価に大量に入手することが可能であるから、本発明に好ましく使用することができる。 More specifically, the raw material polysaccharide is gellan (gellan CAS 71010-52-1) obtained by deacylating and purifying a polysaccharide comprising the structural unit of formula (2), that is, a polysaccharide produced by Pseudomonas elodea. ). Gellan is a polysaccharide mainly composed of glucose, glucuronic acid, and rhamnose, and can be preferably used in the present invention because it can be obtained in large quantities at low cost.
多糖の硫酸化の方法は特に限定されず、通常知られている方法が利用できる。例えば、ジメチルホルムアミド（ＤＭＦ）中でクロロスルホン酸を作用させる方法（インターナショナル オブ ジャーナル オブ バイオロジカル マクロモレキュルズ（International Journal of Biological Macromolecules）２８，３８１（２００１））や、ＤＭＦ中でＤＭＦ／ＳＯ３複合体を作用させる方法（人工臓器 ２６,１（１９９７））や、脱水ピリジン中で三酸化硫黄錯体を作用させる方法（Journal of the Chemical Society ８１（１９５９））である。また他にジオキサン−ＳＯ３複合体、トリメチルアミン−ＳＯ３複合体、ピリジン−ＳＯ３複合体などの無水硫酸複合体を作用させる方法が使用可能である。原料の多糖の分子量、反応条件を変えることにより、任意の分子量と硫酸化度を有する硫酸化多糖を得ることができる。 The method for sulfating polysaccharides is not particularly limited, and generally known methods can be used. For example, a method of reacting chlorosulfonic acid in dimethylformamide (DMF) (International Journal of Biological Macromolecules 28, 381 (2001)) or DMF / SO 3 in DMF. These include a method of causing a complex to act (artificial organ 26, 1 (1997)) and a method of causing a sulfur trioxide complex to act in dehydrated pyridine (Journal of the Chemical Society 81 (1959)). In addition, a method in which a sulfuric anhydride complex such as a dioxane-SO 3 complex, a trimethylamine-SO 3 complex, or a pyridine-SO 3 complex is allowed to act can be used. By changing the molecular weight of the raw material polysaccharide and the reaction conditions, a sulfated polysaccharide having an arbitrary molecular weight and degree of sulfation can be obtained.
本発明の抗ウイルス剤には上記の硫酸化多糖が好ましく用いられるが、これら硫酸化多糖を部分構造として有する化合物も同様に用いることができる。硫酸化多糖を導入する基質の例は、アミノ基を有する多糖、アミノ基を有するポリアミノ酸、およびアミノ基を導入した多糖類などである。アミノ基を有する多糖の具体例はキトサンである。アミノ基を有するポリアミノ酸の具体例はポリリジンである。アミノ基を導入した多糖類の具体例はアミノ化セルロースである。その他、本発明の硫酸化多糖の抗ウイルス活性を失する物で無ければどのような化合物と結合しても問題ない。 The above-mentioned sulfated polysaccharides are preferably used for the antiviral agent of the present invention, but compounds having these sulfated polysaccharides as partial structures can also be used. Examples of the substrate into which the sulfated polysaccharide is introduced include a polysaccharide having an amino group, a polyamino acid having an amino group, and a polysaccharide having an amino group introduced therein. A specific example of a polysaccharide having an amino group is chitosan. A specific example of the polyamino acid having an amino group is polylysine. A specific example of the polysaccharide into which an amino group has been introduced is aminated cellulose. In addition, there is no problem even if it is combined with any compound as long as it does not lose the antiviral activity of the sulfated polysaccharide of the present invention.
硫酸化多糖を上記基質に導入する方法は、本発明の抗ウイルス剤の抗ウイルス活性を失わなければ、特に限定されないが、水溶性カルボジイミド（ＷＳＣ）を触媒に用いて、硫酸化多糖のカルボキシル基と基質のアミノ基を結合する方法、硫酸化多糖の還元末端アルデヒド基と基質のアミノ基を弱アルカリ条件下で反応させ還元剤（テトラヒドロホウ酸ナトリウムやジメチルアミンボラン等）で処理して結合させる方法等を挙げることができる。 The method for introducing the sulfated polysaccharide into the substrate is not particularly limited as long as the antiviral activity of the antiviral agent of the present invention is not lost, but the carboxyl group of the sulfated polysaccharide is obtained using water-soluble carbodiimide (WSC) as a catalyst. A method of binding the amino group of the substrate with the reducing terminal aldehyde group of the sulfated polysaccharide and the amino group of the substrate are reacted under a weak alkaline condition and then treated with a reducing agent (sodium tetrahydroborate, dimethylamine borane, etc.) The method etc. can be mentioned.
調製された抗ウイルス剤は、種々のウイルス疾患に用いることが可能であるが、特にＨＩＶ（Human Immunodeficiency virus）疾患などのヒトレトロウイルスなどの感染によるウイルス疾患に対し、感染前に投与することにより予防に用いることができ、感染後に投与することにより治療に用いることができる。なお、これら抗ウイルス剤の投与方法は病態に応じて適切に行われる必要があり、主な手法としては注射による皮下投与や静脈投与、錠剤化しての経口投与、坐薬としての腸管投与など、薬剤を適切な形態としてその形態に応じた投与方法を選択するのであればいかなる投与方法、薬剤形態としても何ら問題ない。 The prepared antiviral agent can be used for various viral diseases. In particular, it is administered before infection for viral diseases caused by infections such as human retroviruses such as HIV (Human Immunodeficiency virus) diseases. It can be used for prevention and can be used for treatment by administration after infection. In addition, the administration method of these antiviral agents needs to be appropriately performed depending on the disease state, and the main methods are drugs such as subcutaneous administration by injection, intravenous administration, oral administration in tablet form, intestinal administration as suppository, etc. As long as the administration method according to the form is selected as an appropriate form, there is no problem with any administration method or pharmaceutical form.
以下、本発明について実施例及び比較例を用いて詳細に説明するが、本発明はこれらの実施例に限定されるものではない。実施例、比較例において使用する用語の定義及び測定方法は以下の通りである。 Hereinafter, although the present invention is explained in detail using an example and a comparative example, the present invention is not limited to these examples. Definitions of terms and measurement methods used in Examples and Comparative Examples are as follows.
（１）平均分子量（ｋＤａ）：合成した硫酸化ジェランを０．２ｍｏｌ／ｌ−ＮａＣｌ水溶液（イオン交換水にて調製）に１．０ｍｇ／ｍｌの濃度で溶解し、同じＮａＣｌ水溶液を溶出液としたＨＰＬＣによってゲルろ過した。ここで、カラムはShodex（登録商標） Ionpak KS-804（昭和電工社）及びKS-G（昭和電工社）を使用し、溶出物は示差屈折率検出器により検出した。予め、同じ条件で測定した分子量既知のプルラン（Shodex STANDARD P-82（昭和電工社））をゲル濾過して溶出時間と分子量の検量線を作成し、合成した硫酸化ジェランの溶出時間を検量線に当てはめることにより、硫酸化ジェランの平均分子量を決定した。 (1) Average molecular weight (kDa): The synthesized sulfated gellan was dissolved in 0.2 mol / l-NaCl aqueous solution (prepared with ion-exchanged water) at a concentration of 1.0 mg / ml, and the same NaCl aqueous solution was used as the eluent. Gel filtration by HPLC. Here, Shodex (registered trademark) Ionpak KS-804 (Showa Denko) and KS-G (Showa Denko) were used for the column, and the eluate was detected by a differential refractive index detector. Pre-run pullulan (Shodex STANDARD P-82 (Showa Denko)) measured under the same conditions is gel filtered to create a calibration curve of elution time and molecular weight, and the elution time of synthesized sulfated gellan is Was applied to determine the average molecular weight of the sulfated gellan.
（２）硫酸化率（％）：原料多糖が有する水酸基のうち硫酸エステル化された割合を百分率で表示した。合成した硫酸化多糖の全Ｓ量をＩＣＰ発光分析装置による元素分析により測定し、イオンクロマトグラフィーにより硫酸化多糖本体から遊離した遊離Ｓ量を測定した。全Ｓ量から遊離Ｓ量を差し引いた結合Ｓ量から硫酸化率を算出した。なお、イオンクロマトグラフィーの条件は以下の通りである。
分離カラム：ICS-A23（横河製）
カラム温度：４０℃
溶離液：３mmol/l 炭酸ナトリウム水溶液 流量 １ml/min
除去液：１５mmol/l 硫酸水溶液 流量 １ml/min
(2) Sulfation rate (%): The ratio of sulfate esterification of the hydroxyl groups of the raw material polysaccharide was expressed as a percentage. The total amount of S in the synthesized sulfated polysaccharide was measured by elemental analysis using an ICP emission spectrometer, and the amount of free S released from the sulfated polysaccharide main body was measured by ion chromatography. The sulfation rate was calculated from the amount of bound S obtained by subtracting the amount of free S from the total amount of S. The conditions for ion chromatography are as follows.
Separation column: ICS-A23 (Yokogawa)
Column temperature: 40 ° C
Eluent: 3mmol / l Sodium carbonate aqueous solution Flow rate 1ml / min
Remover: 15mmol / l sulfuric acid aqueous solution, flow rate 1ml / min
（３）Ｍｏｌｔ４細胞を用いた細胞障害抑制試験及び細胞毒性試験：抗ＨＩＶ物質の細胞毒性と抗ＨＩＶ活性を測定する２種類のプレートを用意する。両プレートとも９６穴平底培養プレートを使用し、左端８穴（ウエル）に１０％ＦＣＳ含有ＲＰＭＩ１６４０培地で所定の濃度に希釈した試料溶液２００μｌを加えた。残りの穴には培地を１００μｌずつ入れ、左端の穴から１００μｌずつ隣の穴に溶液を移すことにより２倍段階希釈を１１穴まで行い、１２穴目は薬剤濃度を０として細胞増殖及びＨＩＶ感染のコントロールとした。検査薬剤１種類につき細胞毒性と抗ＨＩＶ活性測定プレートのそれぞれ２列を使用した。検査薬剤を希釈したプレートに１プレート当たり２×１０６の対数増殖期にあるＭｏｌｔ４細胞を遠心分離により集め、細胞毒性測定用のプレートには培地１０ｍｌで懸濁し１００μｌずつすべての穴に加えた。一方、抗ＨＩＶ測定用のプレートには遠心分離により集めた２×１０６のＭｏｌｔ４細胞に１００ＴＣＩＤ５０となるようにＨＩＶ（ＨＴＬＶ-IIIＢ）のストック溶液を加え、３７℃１時間感染させた後、遠心して沈殿した細胞を培地１０ｍｌで再懸濁し、抗ＨＩＶ活性測定プレートのすべての穴に１００μｌずつ加えた。
培養５日目に、顕微鏡によりＨＩＶによる細胞障害効果（ＣＰＥ）と細胞毒性を観察した。細胞障害や細胞毒性があれば、細胞変性がおきるため、細胞は丸くなってちぢみ、顕微鏡下では、細胞が黒ずんだ色に見えた。
(3) Cytotoxicity inhibition test and cytotoxicity test using Molt4 cells: Two types of plates for measuring cytotoxicity and anti-HIV activity of anti-HIV substances are prepared. Both plates used 96-well flat-bottom culture plates, and 200 μl of a sample solution diluted to a predetermined concentration with 10% FCS-containing RPMI 1640 medium was added to the leftmost 8 wells. Place 100 μl of medium in the remaining holes and transfer the solution from the leftmost hole to the next hole 100 μl at a time to perform 2-fold serial dilution up to 11 holes. In the 12th hole, the drug concentration is 0 and cell growth and HIV infection The control was. Two rows of cytotoxicity and anti-HIV activity measurement plates were used for each type of test drug. Molt4 cells in the logarithmic growth phase of 2 × 10 6 per plate were collected by centrifugation on a plate diluted with a test agent, suspended in 10 ml of medium on a plate for cytotoxicity measurement, and 100 μl was added to all wells. On the other hand, a stock solution of HIV (HTLV-IIIB) was added to 2 × 10 6 Molt4 cells collected by centrifugation on a plate for anti-HIV measurement so as to be 100 TCID 50, and the plate was infected at 37 ° C. for 1 hour. The cells precipitated by centrifugation were resuspended in 10 ml of medium, and 100 μl was added to all holes of the anti-HIV activity measurement plate.
On the fifth day of the culture, the cytotoxic effect (CPE) and cytotoxicity due to HIV were observed with a microscope. If there was cell damage or cytotoxicity, cell degeneration occurred, and the cells were rounded and squeezed. Under the microscope, the cells looked dark.
（４）巨細胞形成抑制試験：有効性の確認されたサンプルについてはＭｏｌｔ４細胞による巨細胞形成抑制試験を行った。平底２４穴プレートに所定の濃度に希釈した試料溶液５００μｌを加える。培地に懸濁した持続感染Ｍｏｌｔ４／ＨＩＶ細胞浮遊液１×１０６個／ｍｌを２５０μｌ／穴加え、さらに非感染Ｍｏｌｔ４細胞浮遊液１×１０６個／ｍｌを２５０μｌ／穴加え、攪拌し、ＣＯ２存在下、３７℃で混合培養する。混合培養後数時間で巨細胞形成は認められ、２０時間後には顕著となる。検鏡により試料の巨細胞形成抑制効果を判定する。 (4) Giant cell formation inhibition test: A sample with confirmed effectiveness was subjected to a giant cell formation inhibition test using Molt 4 cells. Add 500 μl of a sample solution diluted to a predetermined concentration to a flat bottom 24-well plate. Persistent infection Molt4 / HIV cell suspension suspended in the medium 1 × 10 6 cells / ml to 250 [mu] l / Anakuwae further uninfected Molt4 cell suspension of 1 × 10 6 cells / ml to 250 [mu] l / Anakuwae, stirring, CO 2 and mixed culture at 37 ° C. Giant cell formation is observed several hours after the mixed culture, and becomes significant after 20 hours. Determine the giant cell formation inhibitory effect of the sample by microscopy.
＜実施例１＞
ジェラン（和光純薬製）２ｇをあらかじめ０．５ｍｏｌ／ｌ−トリフルオロ酢酸水溶液２００ｍｌに添加し８０℃下で３０分間反応させ加水分解した。得られた低分子量化ジェランを窒素ガスシール下でＤＭＦ５ｇに添加し１０時間、室温で攪拌し膨潤させた。その後、温度を４０℃に上げてＤＭＦ／ＳＯ３錯体（ＳＯ３ １８質量％）を１４ｇ添加し６時間反応させた。反応終了後氷冷し０．３ｇの水を添加して未反応のＤＭＦ／ＳＯ３錯体を分解、反応停止した。続いて２倍容量のエタノールを添加し反応物を沈殿させ濾過により回収した。回収した沈殿を２０ｍｌのイオン交換水に溶解し１ｍｏｌ／ｌ−ＮａＯＨにて中和し、再び２倍容量のエタノールにて沈殿させ回収した。その後、回収した沈殿を純水に添加し２倍容量のエタノールで沈殿させる手法で精製・洗浄を計３回行い、５０℃の減圧乾燥により１日乾燥し、硫酸化ジェランの粉末１．７ｇ（収率６１質量％）を得た。硫酸化ジェランの平均分子量を前述の手法で測定したところ５．６ｋＤａであった。又、水酸基の硫酸化率は２４．４％であった。
<Example 1>
2 g of gellan (manufactured by Wako Pure Chemical Industries, Ltd.) was added in advance to 200 ml of 0.5 mol / l-trifluoroacetic acid aqueous solution and reacted at 80 ° C. for 30 minutes for hydrolysis. The obtained low molecular weight gellan was added to 5 g of DMF under a nitrogen gas seal and stirred for 10 hours at room temperature to swell. Thereafter, the temperature was raised to 40 ° C., 14 g of DMF / SO 3 complex (SO 3 18% by mass) was added, and the mixture was reacted for 6 hours. After completion of the reaction, the reaction mixture was ice-cooled, 0.3 g of water was added to decompose the unreacted DMF / SO 3 complex, and the reaction was stopped. Subsequently, twice the volume of ethanol was added to precipitate the reaction product and collected by filtration. The recovered precipitate was dissolved in 20 ml of ion-exchanged water, neutralized with 1 mol / l-NaOH, and precipitated again with 2 volumes of ethanol. Thereafter, the recovered precipitate was added to pure water and purified with a double volume of ethanol to perform purification and washing three times in total, followed by drying at 50 ° C. under reduced pressure for one day, and 1.7 g of sulfated gellan powder ( The yield was 61% by mass. It was 5.6 kDa when the average molecular weight of sulfated gellan was measured by the above-mentioned method. The hydroxyl group sulfation rate was 24.4%.
この硫酸化ジェランは３．８μｇ/ｍｌ以上で細胞障害抑制効果が認められた。また、少なくとも１０００μｇ/ｍｌにおいて、細胞毒性は認められなかった。さらに、２００μｇ/ｍｌ以上で巨細胞形成抑制効果が認められた。 This sulfated gellan was found to have a cytotoxic effect at 3.8 μg / ml or more. Further, no cytotoxicity was observed at least at 1000 μg / ml. Furthermore, a giant cell formation inhibitory effect was observed at 200 μg / ml or more.
＜実施例２＞
使用原料として低分子量化していない通常のジェランを２ｇ用い、硫酸化剤にクロロスルホン酸を３．６ｇ使用し、反応温度を５０℃で行った以外は実施例１に準じた方法で硫酸化したところ、分子量が２３ｋＤａ、水酸基の硫酸化率が３６．６％の硫酸化ジェランが得られた。
<Example 2>
Sulfated in the same manner as in Example 1 except that 2 g of normal gellan not reduced in molecular weight was used as a raw material, 3.6 g of chlorosulfonic acid was used as a sulfating agent, and the reaction temperature was 50 ° C. However, a sulfated gellan having a molecular weight of 23 kDa and a hydroxyl group sulfation rate of 36.6% was obtained.
この硫酸化ジェランは１．９μｇ/ｍｌ以上で細胞障害抑制効果が認められた。また、少なくとも１０００μｇ/ｍｌにおいて、細胞毒性は認められなかった。さらに、２００μｇ/ｍｌ以上で巨細胞形成抑制効果が認められた。 This sulfated gellan was found to have a cytotoxic effect at 1.9 μg / ml or more. Further, no cytotoxicity was observed at least at 1000 μg / ml. Furthermore, a giant cell formation inhibitory effect was observed at 200 μg / ml or more.
＜実施例３＞
使用原料として実施例１に準じた手法で低分子量化したジェランを使用した他は実施例２と同じ条件でジェランを硫酸化したところ、分子量が１３ｋＤａ、水酸基の硫酸化率が３９．８％の硫酸化ジェランを得た。
<Example 3>
Gellan was sulfated under the same conditions as in Example 2 except that gellan reduced in molecular weight by the method according to Example 1 was used as a raw material. As a result, the molecular weight was 13 kDa and the hydroxylation rate was 39.8%. Sulfated gellan was obtained.
この硫酸化ジェランは１．９μｇ/ｍｌ以上で細胞障害抑制効果が認められた。また、少なくとも１０００μｇ/ｍｌにおいて、細胞毒性は認められなかった。さらに、４０μｇ/ｍｌ以上で巨細胞形成抑制効果が認められた。 This sulfated gellan was found to have a cytotoxic effect at 1.9 μg / ml or more. Further, no cytotoxicity was observed at least at 1000 μg / ml. Furthermore, a giant cell formation inhibitory effect was observed at 40 μg / ml or more.
＜比較例１＞
使用原料として低分子量化していない通常のジェランを２ｇ用い、実施例１に準じた方法で硫酸化したところ、分子量が４．２ｋＤａ、水酸基の硫酸化率が９．０％の硫酸化ジェランを得た。
<Comparative Example 1>
When 2 g of normal gellan not reduced in molecular weight was used as a starting material and sulfated by the method according to Example 1, a sulfated gellan having a molecular weight of 4.2 kDa and a hydroxylation rate of 9.0% was obtained. It was.
この硫酸化ジェランの細胞毒性は少なくとも１０００μｇ/ｍｌ以上であったが、細胞障害抑制効果は５００μｇ/ｍｌ以上と高く、また巨細胞形成抑制効果は認められなかった。 The cytotoxicity of this sulfated gellan was at least 1000 μg / ml or more, but the cytotoxic effect was as high as 500 μg / ml or more, and no giant cell formation inhibitory effect was observed.
＜結論＞
表１に、実施例及び比較例の結果をまとめる。
<Conclusion>
Table 1 summarizes the results of the examples and comparative examples.
このように、平均硫酸化率の高い（約２０％以上）硫酸化ジェランを用いた場合、低い濃度で細胞障害抑制効果が得られ、細胞毒性は検出できず、巨細胞形成抑制効果が得られた。それに対し、平均硫酸化率の低い（約１０％未満）硫酸化ジェランを用いた場合（比較例１）、細胞毒性は検出できなかったが、細胞障害抑制効果は、より高濃度でしか検出できず、巨細胞形成抑制効果は検出されなかった。このことから、１０％以上の平均硫酸化率を有する硫酸化ジェランは、安価に製造でき、高い抗ウイルス活性を有し、細胞毒性が低いことが示された。
なお、硫酸化ジェランの細胞障害抑制効果は、ウイルスが感染した細胞に対して効果があるため、抗ウイルス剤としては、予防（感染前に投与）にも治療（感染後に投与）にも有効に用いることができることがわかった。
Thus, when sulfated gellan with a high average sulfation rate (about 20% or more) is used, a cytotoxic effect can be obtained at a low concentration, cytotoxicity cannot be detected, and a giant cell formation inhibitory effect can be obtained. It was. In contrast, when sulfated gellan with a low average sulfation rate (less than about 10%) was used (Comparative Example 1), cytotoxicity could not be detected, but the cytotoxic effect could be detected only at a higher concentration. No giant cell formation inhibitory effect was detected. This indicates that sulfated gellan having an average sulfation rate of 10% or more can be produced at low cost, has high antiviral activity, and low cytotoxicity.
In addition, since the cytotoxic effect of sulfated gellan is effective on cells infected with viruses, antiviral agents are effective for both prevention (administration before infection) and treatment (administration after infection). It was found that it can be used.
Claims (6)
前記硫酸化多糖は、下記式（１）で示される構成単位の繰り返しによって構成され、前記硫酸化多糖に含まれる水酸基の一部又は全部が硫酸エステル化されていることを特徴とする抗ウイルス剤。
The sulfated polysaccharide is constituted by a repeating structure unit represented by the following formula (1), antiviral agents in which a part or all of the hydroxyl groups contained in said sulfated polysaccharide is characterized in that it is sulfated .
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