JP2008266265A - Antiviral agent - Google Patents
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- JP2008266265A JP2008266265A JP2007114807A JP2007114807A JP2008266265A JP 2008266265 A JP2008266265 A JP 2008266265A JP 2007114807 A JP2007114807 A JP 2007114807A JP 2007114807 A JP2007114807 A JP 2007114807A JP 2008266265 A JP2008266265 A JP 2008266265A
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本発明は、原料多糖が有する水酸基が全てまたは部分的に硫酸エステル化された硫酸化多糖または該硫酸化多糖を部分構造として有する化合物、特に硫酸化セルロースもしくは硫酸化グルコマンナンを含む抗ウイルス剤に関する。 The present invention relates to a sulfated polysaccharide in which the hydroxyl groups of the raw material polysaccharide are all or partially sulfated, or a compound having the sulfated polysaccharide as a partial structure, in particular, an antiviral agent containing sulfated cellulose or sulfated glucomannan. .
従来より硫酸化多糖による抗ウイルス作用はヘパリンを初めとして数多く研究されてきている（例えば非特許文献１、特許文献１）。しかしながら硫酸化多糖の有する各種活性がこれらを薬剤として用いる場合に、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)。
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る。
Therefore, development of safe and inexpensive antiviral agents is desired.
本発明の課題は、細胞毒性の少ない抗ウイルス剤を安価に提供することにある。 An object of the present invention is to provide an antiviral agent with low cytotoxicity at low cost.
本発明者らは、上記課題を解決するために鋭意研究を行った。その結果、植物由来の多糖を原料として調製した硫酸化多糖が、細胞毒性の少ない抗ウイルス作用を有することを見出した。すなわち、安価な植物由来の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少な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 inventors of the present invention have intensively studied to solve the above problems. As a result, it was found that a sulfated polysaccharide prepared using plant-derived polysaccharide as a raw material has an antiviral action with little cytotoxicity. That is, the present inventors have found that a sulfated polysaccharide obtained by sulfating an inexpensive plant-derived polysaccharide by an existing method has an antiviral action with little cytotoxicity, and thus completed the present invention.
本発明は下記の構成を有する。
（１）植物由来の多糖を原料とし、該原料多糖が有する水酸基の６〜１００％が硫酸エステル化された硫酸化多糖または該硫酸化多糖を部分構造として有する化合物を含有する抗ウイルス剤。
（２）原料多糖が有する水酸基の２５〜８５％が硫酸エステル化された硫酸化多糖または該硫酸化多糖を部分構造として有する化合物を含有する、前記（１）記載の抗ウイルス剤。
（３）原料多糖がセルロースもしくはグルコマンナンである前記（１）または（２）に記載の抗ウイルス剤。
（４）硫酸化多糖の重量平均分子量が１ＫＤａ〜１０００ＫＤａである、前記（１）〜（３）のいずれかに記載の抗ウイルス剤。
（５）硫酸化多糖の重量平均分子量が５ＫＤａ〜５００ＫＤａである、前記（１）〜（３）のいずれかに記載の抗ウイルス剤。
（６）抗ヒトレトロウイルス剤である、前記（１）〜（５）のいずれかに記載の抗ウイルス剤。
（７）抗ＨＩＶ剤である、前記（６）に記載の抗ウイルス剤。
なお、本明細書においては、硫酸エステル化を硫酸化と、水酸基が全てまたは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。
The present invention has the following configuration.
(1) An antiviral agent comprising a plant-derived polysaccharide as a raw material, and a sulfated polysaccharide in which 6 to 100% of hydroxyl groups of the raw material polysaccharide are sulfated or a compound having the sulfated polysaccharide as a partial structure.
(2) The antiviral agent according to (1) above, comprising a sulfated polysaccharide in which 25 to 85% of the hydroxyl groups of the raw material polysaccharide are sulfated or a compound having the sulfated polysaccharide as a partial structure.
(3) The antiviral agent according to (1) or (2) above, wherein the raw material polysaccharide is cellulose or glucomannan.
(4) The antiviral agent according to any one of (1) to (3), wherein the sulfated polysaccharide has a weight average molecular weight of 1 KDa to 1000 KDa.
(5) The antiviral agent according to any one of (1) to (3), wherein the sulfated polysaccharide has a weight average molecular weight of 5 KDa to 500 KDa.
(6) The antiviral agent according to any one of (1) to (5), which is an antihuman retroviral agent.
(7) The antiviral agent according to (6) above, which is an anti-HIV agent.
In the present specification, sulfated esterification is sometimes referred to as sulfated, and a polysaccharide in which hydroxyl groups are all or partially sulfated is sometimes referred to as sulfated polysaccharide.
以上のように、本発明によって、細胞毒性の少ない安価な抗ウイルス剤を提供できるようになった。 As described above, according to the present invention, an inexpensive antiviral agent with little cytotoxicity can be provided.
以下、本発明を詳細に説明する。
本発明の硫酸化多糖の硫酸化率は、原料多糖の種類によっても異なるが、例えば多糖としてセルロースを用いた場合は、該原料多糖が有する水酸基に対し、硫酸基が平均して６から１００％となることが好ましく、２５から８５％がより好ましい。
硫酸化した多糖誘導体の重量平均分子量は、多糖の種類によっても異なるが、一般に、１ＫＤa〜２０００ＫＤaが好ましく、５ＫＤa〜５００ＫＤaがより好ましい。
Hereinafter, the present invention will be described in detail.
Although the sulfation rate of the sulfated polysaccharide of the present invention varies depending on the type of raw material polysaccharide, for example, when cellulose is used as the polysaccharide, the average of sulfate groups is 6 to 100% with respect to the hydroxyl groups of the raw material polysaccharide. Preferably, 25 to 85% is more preferable.
Although the weight average molecular weight of the sulfated polysaccharide derivative varies depending on the type of polysaccharide, it is generally preferably 1 KDa to 2000 KDa, more preferably 5 KDa to 500 KDa.
本発明の抗ウイルス剤を構成する硫酸化多糖の原料として使用される植物由来の多糖とは、グルコース、マンノース、タロース、ガラクトース、ソルボース、タガトース、フルクトース、プシコース、グルクロン酸、ガラクツロン酸等の単糖が、グリコシド結合で連結した多糖のうち、被子植物、裸子植物、地衣類、緑藻、褐藻等の植物より抽出等の操作で得られたものをいう。具体的には、アガロース、アガロペクチン、アラビアガム、アルギン酸、カラギーナン、カロース、キシラン、グアーガム、ゾウゲヤシマンナン、コンニャクマンナン、アラビナン、セルロース、タマリンドシードガム、トラガガントガム、プスツラン、フノラン、ペクチン、ローカストビーンガム、デンプン、リケナン、レンチナン等が挙げられる。なかでも、好ましいものとして、安価にかつ容易に入手可能な、セルロース、グルコマンナンが挙げられる。 The plant-derived polysaccharide used as a raw material for the sulfated polysaccharide constituting the antiviral agent of the present invention is a monosaccharide such as glucose, mannose, talose, galactose, sorbose, tagatose, fructose, psicose, glucuronic acid, galacturonic acid, etc. However, among the polysaccharides linked by glycosidic bonds, those obtained by operations such as extraction from plants such as angiosperms, gymnosperms, lichens, green algae, brown algae and the like. Specifically, agarose, agaropectin, gum arabic, alginic acid, carrageenan, callose, xylan, guar gum, elephant palm mannan, konjac mannan, arabinan, cellulose, tamarind seed gum, tragagant gum, pustulan, funolan, pectin, locust bean gum, Examples include starch, lichenan, and lentinan. Among these, preferred are cellulose and glucomannan, which are inexpensive and easily available.
硫酸化の方法は特に限定されないが、例えば、多糖をピリジン、ジメチルスルホキシド、ジメチルホルムアミド等の溶媒で膨潤させ、ここにクロルスルホン酸、ピペリジン−Ｎ−硫酸、無水硫酸−ジメチルホルムアミド錯体、三酸化硫黄−ピリジン錯体、三酸化硫黄−トリメチルアミン錯体等の硫酸化剤を滴下する方法が挙げられる。硫酸化剤の使用量は、糖水酸基に対し、１．２〜３当量用いるのが適当である。多糖と硫酸化剤は、溶媒、硫酸化剤によっても異なるが、不活性ガス中で、好ましくは０〜１００℃、より好ましくは２０〜８５℃で、好ましくは０．５〜２４時間、より好ましくは０．５〜１０時間、反応させる。その後、エタノール、イソプロパノール、アセトン等を加えて、ポリマーを沈殿させるか、あるいはエタノール、イソプロパノール、アセトン等の中に反応液を滴下し、ポリマーを沈殿させることができる。また、蒸留水を加えて反応を停止し、次いで、アルカリ、例えば水酸化ナトリウムで中和してもよい。これをろ過または遠心分離し、蒸留水に溶解し、再度エタノール、イソプロパノール、アセトン等を加えて、ポリマーを沈殿させるか、エタノール、イソプロパノール、アセトン等の中に滴下し、ポリマーを沈殿させ、乾燥することによって硫酸化多糖を得ることができる。あるいはイオン交換樹脂カラムを用いて余剰の無機塩を除去し、エタノール、イソプロパノール、アセトン等を加えて、ポリマーを沈殿させてもよい。あるいは蒸留水に溶解後透析し、濃縮乾燥することによって硫酸化多糖を得ることもできる。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 of sulfation is not particularly limited. For example, a polysaccharide is swollen with a solvent such as pyridine, dimethyl sulfoxide, dimethylformamide, and then chlorosulfonic acid, piperidine-N-sulfuric acid, anhydrous sulfuric acid-dimethylformamide complex, sulfur trioxide. -The method of dripping sulfating agents, such as a pyridine complex and a sulfur trioxide-trimethylamine complex, is mentioned. The amount of sulfating agent used is suitably 1.2 to 3 equivalents relative to the sugar hydroxyl group. The polysaccharide and the sulfating agent differ depending on the solvent and the sulfating agent, but in an inert gas, preferably 0 to 100 ° C., more preferably 20 to 85 ° C., preferably 0.5 to 24 hours, more preferably Is allowed to react for 0.5 to 10 hours. Thereafter, ethanol, isopropanol, acetone or the like can be added to precipitate the polymer, or the reaction solution can be dropped into ethanol, isopropanol, acetone or the like to precipitate the polymer. Alternatively, the reaction may be stopped by adding distilled water and then neutralized with an alkali such as sodium hydroxide. This is filtered or centrifuged, dissolved in distilled water, ethanol, isopropanol, acetone, etc. are added again to precipitate the polymer, or it is dropped into ethanol, isopropanol, acetone, etc., to precipitate the polymer, and then dried. Thus, a sulfated polysaccharide can be obtained. Alternatively, excess inorganic salt may be removed using an ion exchange resin column, and ethanol, isopropanol, acetone, or the like may be added to precipitate the polymer. Alternatively, the sulfated polysaccharide can be obtained by dissolving in distilled water, dialyzing, and concentrating to dryness. 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、アミノ基を導入した多糖、及びアミノ基を有するカチオン性ポリマーなどである。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の具体例はアミノ化セルロースである。アミノ基を有するカチオン性ポリマーの具体例はポリエチレンイミンである。その他、本発明の硫酸化多糖の抗ウイルス活性を失活しな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, a polysaccharide having an amino group introduced therein, and a cationic polymer having an amino group. 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. A specific example of the cationic polymer having an amino group is polyethyleneimine. In addition, there is no problem even if it is combined with any compound as long as the antiviral activity of the sulfated polysaccharide of the present invention is not inactivated.
硫酸化多糖を上記の基質に導入する方法の例は、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とを結合する方法、硫酸化多糖の還元末端アルデヒド基と基質のアミノ基とを弱アルカリ条件下で反応させた後に、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）で処理する方法などである。その他、本発明の硫酸化多糖の抗ウイルス活性を失活しなければその導入方法は特に限定されない。 Examples of a method for introducing a sulfated polysaccharide into the above-mentioned substrate include a method of binding a carboxyl group of a sulfated polysaccharide and an amino group of the substrate using water-soluble carbodiimide (WSC) as a catalyst, and a reducing terminal of the sulfated polysaccharide. For example, the aldehyde group and the amino group of the substrate are reacted under a weak alkaline condition and then treated with a reducing agent (such as sodium tetrahydroborate or dimethylamine borane). In addition, the introduction method is not particularly limited as long as the antiviral activity of the sulfated polysaccharide of the present invention is not inactivated.
調製された抗ウイルス剤の対象疾患は、特に限定されないが、例えばヒトレトロウイルス疾患、特にＨＩＶ（Human Immunodeficiency virus）疾患の治療に、抗ヒトレトロウイルス剤として（例えば、ＨＩＶの場合、抗ＨＩＶ剤として）用いることができる。この疾患は重篤な免疫不全症で、ＨＩＶの感染力は弱いものの、感染が成立し発症した後の死亡率は極めて高い。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 target disease of the prepared antiviral agent is not particularly limited. For example, in the treatment of human retroviral disease, particularly HIV (Human Immunodeficiency virus) disease, as an anti-human retroviral agent (for example, in the case of HIV, anti-HIV agent) As). Although this disease is a serious immunodeficiency and HIV has a weak infectivity, the mortality rate after the onset of infection and onset is extremely high. The administration method of the antiviral agent should be appropriately performed according to the disease state, and the main methods are appropriate administration such as subcutaneous administration by injection, intravenous administration, oral administration in tablet form, intestinal administration as suppository, etc. As long as an administration method corresponding to the form is selected as the form, there is no problem with any administration method or pharmaceutical form.
以下、本発明について実施例およ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。実施例において使用する用語の定義および測定方法は以下の通りである。
（１）重量平均分子量（ＫＤａ）：合成した硫酸化多糖を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によるゲルろ過法により重量平均分子量を測定した。ゲルろ過のためのカラムはＳｈｏｄｅｘ Ｉｏｎｐａｋ ＫＳ−８０４およびＳｈｏｄｅｘ Ｉｏｎｐａｋ ＫＳ−Ｇ（昭和電工（株）製）を使用し、溶出物の検出は示差屈折率検出器により測定した。別途測定した分子量既知のプルラン（Ｓｈｏｄｅｘ ＳＴＡＮＤＡＲＤ Ｐ−８２）により溶出時間と分子量の検量線を作成し、検量線に当てはめることで合成した硫酸化多糖の重量平均分子量を決定した。
（２）水酸基の硫酸化率（％）：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量から水酸基の硫酸化率を算出した。
（３）ＭＯＬＴ４細胞を用いた細胞障害抑制試験：抗ウイルス剤の細胞毒性と抗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穴に、１０容量％ＦＣＳ加ＲＰＭＩ１６４０培地（Ｓｉｇｍａ社製）で所定の濃度に希釈した試料溶液を２００μｌ加えた。残りの８８穴には前記培地を１００μｌずつ入れ、左端の穴から８連ピペットで１００μｌで２倍段階希釈を１１穴まで行い、１２穴目は抗ウイルス剤の濃度を０として細胞増殖及びＨＩＶ感染のコントロールとした。
抗ウイルス剤１種類につき、細胞毒性測定用のプレートと抗ＨＩＶ活性測定用プレートのそれぞれの２列を使用した。抗ウイルス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で再浮遊させ、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℃で１時間感染させた後、培地１０ｍｌで再浮遊させて、抗ＨＩＶ活性測定プレートのすべての穴に１００μｌ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）と細胞毒性とを観察した。細胞障害や細胞毒性があれば、細胞変性が起きるため、細胞は丸くなって縮み、顕微鏡下では、細胞が黒ずんで見える。細胞の検鏡により細胞障害抑制効果を判定した。
（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細胞による巨細胞抑制試験を行った。測定濃度は、最大有効濃度から５倍段階希釈により実施した。
具体的には、２４穴平底プレートに所定の濃度に希釈した試料溶液５００μｌを加えた。培地に懸濁した持続感染Ｍｏｌｔ４／ＨＩＶ細胞浮遊液１×１０６個/ｍｌを１穴あたり２５０μｌ加え、さらに非感染Ｍｏｌｔ４細胞浮遊液１×１０６個/ｍｌを２５０μｌ加え、攪拌し、ＣＯ２存在下、３７℃で混合培養した。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った。検鏡により試料の巨細胞形成抑制効果を判定した。
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 used in the examples and measurement methods are as follows.
(1) Weight average molecular weight (KDa): The synthesized sulfated polysaccharide 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 dissolved into the eluent. The weight average molecular weight was measured by gel filtration by HPLC. As columns for gel filtration, Shodex Ionpak KS-804 and Shodex Ionpak KS-G (manufactured by Showa Denko KK) were used, and the detection of eluate was measured by a differential refractive index detector. A calibration curve of elution time and molecular weight was prepared using pullulan (Shodex STANDARD P-82) with a known molecular weight measured separately, and the weight average molecular weight of the synthesized sulfated polysaccharide was determined by applying the calibration curve to the calibration curve.
(2) Sulfation rate of hydroxyl group (%): The ratio of sulfate esterification of the hydroxyl group 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 by ICP, and the amount of free S released from the sulfated polysaccharide main body was measured by ion chromatography. The sulfation rate of the hydroxyl group was calculated from the bound S amount obtained by subtracting the free S amount from the total S amount.
(3) Cytotoxicity inhibition test using MOLT4 cells: Two types of plates for measuring cytotoxicity and anti-HIV activity of antiviral agents 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 vol% FCS-added RPMI 1640 medium (manufactured by Sigma) was added to the leftmost 8 holes. In the remaining 88 wells, add 100 μl of the above medium, and double-dilute serially to 11 wells with 100 μl of 8 pipettes from the leftmost hole. In the 12th hole, the concentration of antiviral agent is 0 and cell growth and HIV infection The control was.
For each type of antiviral agent, two rows of a cytotoxicity measurement plate and an anti-HIV activity measurement plate were used. Collect 2 × 10 6 logarithmically growing MOLT4 cells per plate in a plate diluted with antiviral agent, resuspend in 10 ml of medium on the cytotoxicity plate, and add 100 μl of each well. Added to. 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 to 100 TCID 50 and infected at 37 ° C. for 1 hour. The suspension was 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 culture, the cytotoxic effect (CPE) and cytotoxicity due to HIV were observed with a microscope. If there is cell damage or cytotoxicity, cell degeneration occurs, so the cells become round and shrink, and under the microscope, the cells appear dark. Cytotoxicity was determined by cell microscopy.
(4) Giant cell formation inhibition test: About the sample whose effectiveness was confirmed, the giant cell inhibition test by Molt4 cell was done. Measurement concentration was carried out by 5-fold serial dilution from the maximum effective concentration.
Specifically, 500 μl of a sample solution diluted to a predetermined concentration was added to a 24-well flat bottom plate. Medium persistent infection Molt4 / HIV cell suspension of 1 × 10 6 cells / ml was added 250 [mu] l per well suspended, further uninfected Molt4 cell suspension of 1 × 10 6 cells / ml was added 250 [mu] l, stirring, CO 2 Mixed culture was performed at 37 ° C. in the presence. Giant cell formation was observed several hours after the mixed culture, and became prominent after 20 hours. The giant cell formation inhibitory effect of the sample was determined by microscopy.
[実施例１]
温度計、滴下ロート、攪拌装置、及び窒素ガス導入管を備えた５００ｍｌのセパラブルフラスコに、セルロース（アビセル ＰＨ-１０１）４．００ｇ及びジメチルホルムアミド２０ｍｌを入れ、３０分間攪拌した。次に、氷冷下で１８％無水硫酸−ジメチルホルムアミド溶液１８０．０２ｇを３０分かけて投入した後、反応容器を２４℃の恒温槽に浸け、２０分かけて昇温させ２４℃で６時間反応した。
反応終了後、反応容器を５℃まで氷冷し、氷冷しながら水１８０ｍｌを徐々に投入し、続いて、水酸化カルシウムを反応液のｐＨが７になるまで投入した。このとき中和に要した水酸化カルシウムは１６．４７ｇであった。生じた沈殿をろ別し、ろ液を１０００ｍｌのイソプロパノール中に投入してポリマーを析出させ、再びろ別し、ろ液を４０℃で８時間減圧乾燥し、セルロース硫酸カルシウムを得た。
収量：３．７５ｇ、重量平均分子量：７７ＫＤa、硫酸化率：４１．７％。
[Example 1]
In a 500 ml separable flask equipped with a thermometer, a dropping funnel, a stirrer, and a nitrogen gas inlet tube, 4.00 g of cellulose (Avicel PH-101) and 20 ml of dimethylformamide were added and stirred for 30 minutes. Next, 180.02 g of 18% anhydrous sulfuric acid-dimethylformamide solution was added over 30 minutes under ice-cooling, and then the reaction vessel was immersed in a constant temperature bath at 24 ° C., and the temperature was raised over 20 minutes, at 6 ° C. for 6 hours. Reacted.
After completion of the reaction, the reaction vessel was ice-cooled to 5 ° C., 180 ml of water was gradually added while cooling with ice, and then calcium hydroxide was added until the pH of the reaction solution reached 7. At this time, the calcium hydroxide required for neutralization was 16.47 g. The resulting precipitate was filtered off, the filtrate was put into 1000 ml of isopropanol to precipitate a polymer, filtered again, and the filtrate was dried under reduced pressure at 40 ° C. for 8 hours to obtain calcium calcium sulfate.
Yield: 3.75 g, weight average molecular weight: 77 KDa, sulfation rate: 41.7%.
この硫酸化セルロースは１．９μｇ/ｍｌ以上で細胞変異抑制効果が認められた。また、少なくとも１０００μｇ/ｍｌにおいても細胞毒性は認められなかった。さらに、８μｇ/ｍｌ以上で巨細胞抑制効果が認められた。 This sulfated cellulose was found to have a cell mutation inhibitory effect at 1.9 μg / ml or more. Further, no cytotoxicity was observed even at least at 1000 μg / ml. Furthermore, a giant cell inhibitory effect was observed at 8 μg / ml or more.
[実施例２] 硫酸化低分子グルコマンナンの性質
荻野商店製造グルコマンナンを１重量％塩酸水溶液に投入し、５０℃で５時間酸分解を行なった。反応後、反応液をイソプロパノール中に滴下して沈殿をろ過し、ろ過物を真空乾燥し、低分子グルコマンナンを得た。
温度計、滴下ロート、攪拌装置、及び窒素ガス導入管を備えた２００ｍｌのセパラブルフラスコに、上記低分子グルコマンナン０．１５ｇ及びジメチルホルムアミド２０ｍｌを入れ、一晩攪拌した。次に、氷冷下で１８％無水硫酸−ジメチルホルムアミド溶液１２．３５ｇを徐々に滴下し、３０±２℃で６時間反応させた。反応終了後、反応液を氷冷下で、冷却したイソプロパノール中に滴下し、硫酸化グルコマンナンを析出させ、ろ過分別した。ろ過物をイオン交換水に完全に溶解させた後、飽和塩化カルシウム溶液０．５１ｇを加えカルシウム化し、グルコマンナン硫酸カルシウムを析出させた。析出物をろ過分別後、イオン交換水５ｍｌに溶解させ、イソプロパノール２０ｍｌを用いてグルコマンナン硫酸カルシウムを析出させた。ろ過分別し、ろ過物をイオン交換水に溶解させ、この水溶液が中性になるまで、溶解、析出操作を繰り返した。ｐＨ試験紙で中性であることを確認後、得られたグルコマンナン硫酸カルシウム水溶液を凍結乾燥し、目的物を得た。
収量：０．２２ｇ、重量平均分子量：２０ＫＤa、硫酸化率：３３．５％。
Example 2 Properties of Sulfated Low-Molecular Glucomannan Glunomannan manufactured by Hadano Shoten was charged into a 1% by weight hydrochloric acid aqueous solution and subjected to acid decomposition at 50 ° C. for 5 hours. After the reaction, the reaction solution was dropped into isopropanol, the precipitate was filtered, and the filtrate was vacuum-dried to obtain a low molecular glucomannan.
In a 200 ml separable flask equipped with a thermometer, a dropping funnel, a stirrer, and a nitrogen gas inlet tube, 0.15 g of the low molecular glucomannan and 20 ml of dimethylformamide were placed and stirred overnight. Next, 12.35 g of 18% anhydrous sulfuric acid-dimethylformamide solution was gradually added dropwise under ice cooling, and the mixture was reacted at 30 ± 2 ° C. for 6 hours. After completion of the reaction, the reaction solution was added dropwise to cooled isopropanol under ice cooling to precipitate sulfated glucomannan and separated by filtration. After the filtrate was completely dissolved in ion-exchanged water, 0.51 g of a saturated calcium chloride solution was added to form calcium, and calcium glucomannan sulfate was precipitated. The precipitate was separated by filtration, dissolved in 5 ml of ion-exchanged water, and calcium glucomannan sulfate was precipitated using 20 ml of isopropanol. After filtration and separation, the filtrate was dissolved in ion-exchanged water, and dissolution and precipitation operations were repeated until the aqueous solution became neutral. After confirming neutrality with pH test paper, the obtained calcium glucomannan sulfate aqueous solution was lyophilized to obtain the desired product.
Yield: 0.22 g, weight average molecular weight: 20 KDa, sulfation rate: 33.5%.
この硫酸化グルコマンナンは１．９μｇ/ｍｌ以上で細胞変異抑制効果が認められた。また、少なくとも１０００μｇ/ｍｌにおいても細胞毒性は認められなかった。さらに、８μｇ/ｍｌ以上で巨細胞抑制効果が認められた。 This sulfated glucomannan was found to have a cell mutation inhibitory effect at 1.9 μg / ml or more. Further, no cytotoxicity was observed even at least at 1000 μg / ml. Furthermore, a giant cell inhibitory effect was observed at 8 μg / ml or more.
[比較例１] 硫酸化されていない低分子グルコマンナンの性質
実施例２で作製した分子量３０ＫＤa〜１００ＫＤaの低分子グルコマンナンの細胞毒性は１０００μｇ/ｍｌ以上であったが、細胞変異抑制効果は５００μｇ/ｍｌ以上と高く、また巨細胞抑制効果は認められなかった。
[Comparative Example 1] Properties of non-sulfated low molecular glucomannan The cytotoxicity of the low molecular glucomannan having a molecular weight of 30 KDa to 100 KDa prepared in Example 2 was 1000 μg / ml or more, but the cell mutation inhibitory effect was 500 μg. / ml or higher, and no giant cell inhibitory effect was observed.
このように、硫酸化セルロース及び硫酸化グルコマンナンは１．９μｇ/ｍｌで、ＨＩＶによる細胞障害を抑制することができ、８μｇ/ｍｌでウイルス感染で生じる巨細胞の形成も抑制された。従って、これらの硫酸化多糖は抗ウイルス剤として有用であることがわかる。また、両者とも、１０００μｇ/ｍｌの濃度でも細胞毒性は観察されず、抗ウイルス作用を発揮する濃度よりかなりの高濃度にしても、細胞毒性が生じなかった。 Thus, sulfated cellulose and sulfated glucomannan were 1.9 μg / ml, which could suppress cell damage due to HIV, and at 8 μg / ml, the formation of giant cells caused by virus infection was also suppressed. Therefore, it can be seen that these sulfated polysaccharides are useful as antiviral agents. In both cases, cytotoxicity was not observed even at a concentration of 1000 μg / ml, and cytotoxicity did not occur even at a concentration much higher than the concentration at which antiviral action was exerted.
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| WO2022217763A1 (en) * | 2021-04-14 | 2022-10-20 | 大连工业大学 | Sulfated abalone polysaccharide and application thereof in inhibiting sars-cov-2 |
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