JP2018203667A - Antitumor agent having peptide hetero polysaccharide separated from crushed cell wall of chlorella pyrenoidosa as active ingredient - Google Patents
Antitumor agent having peptide hetero polysaccharide separated from crushed cell wall of chlorella pyrenoidosa as active ingredient Download PDFInfo
- Publication number
- JP2018203667A JP2018203667A JP2017111293A JP2017111293A JP2018203667A JP 2018203667 A JP2018203667 A JP 2018203667A JP 2017111293 A JP2017111293 A JP 2017111293A JP 2017111293 A JP2017111293 A JP 2017111293A JP 2018203667 A JP2018203667 A JP 2018203667A
- Authority
- JP
- Japan
- Prior art keywords
- peptide
- cell wall
- molecular weight
- heteropolysaccharide
- chlorella pyrenoidosa
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
Description
本発明は、クロレラ・ピレノイドサの細胞壁破砕物の熱水抽出液から精製分離される特定の糖組成を有する高分子量のペプチドヘテロ多糖体を活性成分とする抗腫瘍剤に関する。 The present invention relates to an antitumor agent comprising, as an active ingredient, a high molecular weight peptide heteropolysaccharide having a specific sugar composition that is purified and separated from a hot water extract of cell wall crushed material of Chlorella pyrenoidosa.
クロレラ・ピレノイドサの細胞壁破砕物から分離された、糖組成がマンノースを主構成成分とし、更にグルコースを他の構成成分とする多糖体や、糖成分がラムノースを主構成成分とし、更にグルコース、ガラクトース、マンノース及びキシロースを他の構成成分とする多糖体に、免疫調節作用や免疫賦活作用のあることが報告されている（例えば非特許文献１及び２参照）。またクロレラ・ピレノイドサの細胞壁破砕物の熱水抽出液から精製分離した高分子量のペプチドヘテロ多糖体に、補体第３成分の活性化作用のあることが報告されている（例えば特許文献１及び２参照）。 Polysaccharides having a saccharide composition of mannose as a main component and glucose as another component, separated from a cell wall crushed product of Chlorella pyrenoidosa, and a sugar component having rhamnose as a main component, and glucose, galactose, It has been reported that polysaccharides containing mannose and xylose as other constituents have an immunomodulatory action and an immunostimulatory action (for example, see Non-Patent Documents 1 and 2). Further, it has been reported that a high molecular weight peptide heteropolysaccharide purified and separated from a hot water extract of a cell wall disrupted product of Chlorella pyrenoidosa has an effect of activating the third component of complement (for example, Patent Documents 1 and 2). reference).
本発明が解決しようとする課題は、クロレラ・ピレノイドサの細胞壁破砕物の熱水抽出液から精製分離した高分子量のペプチドヘテロ多糖体の新たな用途を提供する処にある。 The problem to be solved by the present invention is to provide a new use of a high molecular weight peptide heteropolysaccharide purified and separated from a hot water extract of cell wall crushed material of Chlorella pyrenoidosa.
本発明者らは、前記の課題を解決するべく研究した結果、クロレラ・ピレノイドサの細胞壁破砕物を熱水抽出処理し、その熱水抽出液から低分子物質を除去した後、精製処理して得られる、特定の糖組成を有する高分子量のペプチドヘテロ多糖体に優れた抗腫瘍活性があることを見出した。 As a result of researches to solve the above-mentioned problems, the present inventors obtained a cell wall crushed material of Chlorella pyrenoidosa by hot water extraction treatment, and after removing low molecular weight substances from the hot water extract, it was obtained by purification treatment. It was found that the high molecular weight peptide heteropolysaccharide having a specific sugar composition has excellent antitumor activity.
すなわち本発明は、クロレラ・ピレノイドサの細胞壁破砕物を熱水抽出処理し、その熱水抽出液から分子量１００００以下の低分子物質を除去した後、精製処理して得られる、糖組成がガラクトースを主構成成分とし、更にグルコース、マンノース、キシロース、ラムノース、アラニノース及びフラクトースを他の構成成分とする高分子量のヘプチドヘテロ多糖体を含有することを特徴とするクロレラ・ピレノイドサの細胞壁破砕物から分離されるペプチドヘテロ多糖体を活性成分とする抗腫瘍剤に係る。 That is, the present invention mainly comprises galactose having a sugar composition obtained by subjecting a chlorella pyrenoidosa cell wall fragment to a hot water extraction treatment, removing a low molecular weight material having a molecular weight of 10,000 or less from the hot water extract, and then purifying it. Peptide heteroseparated from cell wall debris of Chlorella pyrenoidosa characterized by containing a high molecular weight peptide heteropolysaccharide containing glucose, mannose, xylose, rhamnose, alaninose and fructose as other components The present invention relates to an antitumor agent comprising a polysaccharide as an active ingredient.
一般にクロレラとしては、クロレラ・ピレノイドサ、クロレラ・エリプソイデア、クロレラ・ブルガリス、クロレラ・レギラリス等が挙げられるが、本発明では、なかでもクロレラ・ピレノイドサを用いる。 In general, examples of chlorella include chlorella pyrenoids, chlorella ellipsoida, chlorella bulgaris, chlorella regilaris, and the like. Among them, chlorella pyrenoids are used in the present invention.
本発明において、クロレラ・ピレノイドサの細胞壁破砕物は、例えば、クロレラ・ピレノイドサの粉体の１０〜２５質量％水懸濁液を、１０℃以下に冷却して湿式粉砕機に供し、細胞壁破砕物のスラリーの温度が４０℃以下となるよう微粉砕した後、直ちに１０℃以下に冷却することにより得られる。必要に応じて、更に真空乾燥し、粉砕することもできる。用いる湿式粉砕機としては、外周に冷媒の流通可能なジャケットを備えるボールミルや振動ミル等が挙げられる。 In the present invention, the chlorella pyrenoidosa cell wall crushed material is, for example, a 10-25 mass% aqueous suspension of chlorella pyrenoidosa powder cooled to 10 ° C. or lower and supplied to a wet pulverizer, After pulverization so that the temperature of the slurry is 40 ° C. or less, the slurry is immediately cooled to 10 ° C. or less. If necessary, it can be further vacuum-dried and pulverized. Examples of the wet pulverizer to be used include a ball mill and a vibration mill provided with a jacket capable of circulating a refrigerant on the outer periphery.
本発明において、クロレラ・ピレノイドサの細胞壁破砕物の熱水抽出処理には、７０℃以上の熱水を用いるのが好ましく、９５〜１００℃の熱水を用いるのが好ましい。通常、クロレラ・ピレノイドサの細胞壁破砕物の５〜２０質量％熱水懸濁液を撹拌下に１〜５時間抽出処理する。 In the present invention, it is preferable to use hot water of 70 ° C. or higher, and preferably hot water of 95 to 100 ° C. for the hot water extraction treatment of the cell wall crushed material of Chlorella pyrenoidosa. Usually, a 5-20 mass% hot water suspension of cell wall crushed material of Chlorella pyrenoidosa is extracted for 1-5 hours with stirring.
前記のように熱水抽出処理したものを、例えば遠心分離し、その上澄液を透析濾過や限外濾過等に供して、分子量１００００以下の低分子物質を除去する。尚、本発明において分子量は、ゲル濾過法により測定されるプルラン換算の数平均分子量である。 What was subjected to the hot water extraction treatment as described above is centrifuged, for example, and the supernatant is subjected to diafiltration or ultrafiltration to remove low molecular weight substances having a molecular weight of 10,000 or less. In the present invention, the molecular weight is a number average molecular weight in pullulan conversion measured by a gel filtration method.
前記のように分子量１００００以下の低分子物質を除去したものを精製処理に供する。精製処理は、エタノール沈殿処理、イオン交換クロマト処理及びゲル濾過処理から選ばれる少なくとも一つで行なうのが好ましく、二つ以上を組合わせて行なうのがより好ましい。例えば、前記のように分子量１００００以下の低分子物質を除去したものに、３容量倍程度の無水エタノールを加え、沈殿物を遠心分離し、遠心分離した沈殿物を無水エタノールで洗浄し、乾燥した後、かくしてエタノール沈殿処理したものを、イオン交換クロマト処理及びゲル濾過処理に供する。イオン交換クロマト処理は、例えば和光純薬工業社製の商品名ＤＥＡＥ−Ｃｅｌｌｕｌｏｓｅ（Ｃｌ−）等を用いて行なうことができ、またゲル濾過処理は、例えば和光純薬工業社製の商品名Ｓｅｐｈａｄｅｘ Ｇ−５０等を用いて行なうことができる。 A material from which a low molecular weight material having a molecular weight of 10,000 or less is removed as described above is subjected to a purification treatment. The purification treatment is preferably carried out by at least one selected from ethanol precipitation treatment, ion exchange chromatography treatment and gel filtration treatment, more preferably in combination of two or more. For example, as described above, low-molecular substances having a molecular weight of 10,000 or less were removed, and about 3 volumes of absolute ethanol was added, the precipitate was centrifuged, and the centrifuged precipitate was washed with absolute ethanol and dried. Thereafter, the ethanol-precipitated material is subjected to ion exchange chromatography and gel filtration. Ion-exchange chromatography treatment, for example, manufactured by Wako Pure Chemical Industries, Ltd. trade name DEAE-Cellulose (Cl -) and the like can be performed using, also gel filtration, for example manufactured by Wako Pure Chemical Industries, Ltd. trade name Sephadex G -50 etc. can be used.
以上説明したように、クロレラ・ピレノイドサの細胞壁破砕物を熱水抽出処理し、その熱水抽出液から分子量１００００以下の低分子物質を除去した後、精製処理すると、特定の糖組成を有する高分子量のペプチドヘテロ多糖体が得られる。本発明では、かかるペプチドヘテロ多糖体を抗腫瘍活性成分として用いる。 As described above, a cell wall crushed material of Chlorella pyrenoidosa is subjected to hot water extraction treatment, and after removing low molecular weight substances having a molecular weight of 10,000 or less from the hot water extract, a high molecular weight having a specific sugar composition is obtained. The peptide heteropolysaccharide is obtained. In the present invention, such a peptide heteropolysaccharide is used as an antitumor active ingredient.
前記のペプチドヘテロ多糖体は、糖組成がガラクトースを主構成成分とし、更にグルコース、マンノース、キシロース、ラムノース、アラビノース及びフラクトース等を構成成分とする、分子量が１００万〜５００万程度の高分子量のものであり、糖組成がガラクトースを主構成成分とするという特徴を有し、また分子量が極めて高いという特徴を有する。 The peptide heteropolysaccharide has a high molecular weight with a molecular weight of about 1,000,000 to 5,000,000, the saccharide composition comprising galactose as a main constituent and glucose, mannose, xylose, rhamnose, arabinose, fructose, etc. And the sugar composition has a characteristic that galactose is a main constituent and has a very high molecular weight.
詳しくは実施例の欄で後述するように、前記のペプチドヘテロ多糖体は、優れた抗腫瘍活性を示し、５−フルオロラウシルと併用すると、更により優れた抗腫瘍活性を示す。 As will be described in detail in the Examples section, the peptide heteropolysaccharide exhibits excellent antitumor activity, and when used in combination with 5-fluorolauryl, exhibits further superior antitumor activity.
本発明で用いる、クロレラ・ピレノイドサの細胞壁破砕物の熱水抽出液から精製分離される特定の糖組成を有する高分子量のペプチドヘテロ多糖体は、優れた抗腫瘍活性を示す。 The high molecular weight peptide heteropolysaccharide having a specific sugar composition that is purified and separated from a hot water extract of a cell wall crushed product of Chlorella pyrenoidosa used in the present invention exhibits excellent antitumor activity.
以下、本発明を実施例に基づいて更に詳しく説明するが、本発明がかかる実施例に限定されるというものではない。 EXAMPLES Hereinafter, although this invention is demonstrated in more detail based on an Example, this invention is not limited to this Example.
試験区分１（ペプチドヘテロ多糖体の分離）
クロレラ・ピレノイドサの２０質量％水懸濁液を、外周に冷媒の流通可能なジャケットを有し且つ粉砕筒内に粉砕媒体としてグラスビーズを装填したボールミルに供して、品温を４０℃以下に保持しつつ２時間、湿式粉砕した後、湿式粉砕したスラリーを取り出し、真空乾燥して粉砕し、細胞壁を破砕したクロレラ・ピレノイドサの乾燥粉砕物４００ｇを得た。
Test Category 1 (Peptide heteropolysaccharide separation)
A 20% by mass aqueous suspension of Chlorella pyrenoidosa is supplied to a ball mill having a jacket capable of circulating a refrigerant on the outer periphery and loaded with glass beads as a grinding medium in the grinding cylinder to keep the product temperature at 40 ° C. or lower. Then, after wet pulverization for 2 hours, the wet pulverized slurry was taken out, vacuum-dried and pulverized to obtain 400 g of a dry pulverized product of Chlorella pyrenoidosa with crushed cell walls.
前記のクロレラ・ピレノイドサの乾燥粉砕物に９５〜１００℃の熱水を加えて、１５質量％熱水懸濁液とし、煮沸還流下に２時間、撹拌して熱水抽出処理した。室温に冷却後、沈殿物上の上澄液を分画分子量１００００のフィルターを用いて限外濾過し、濾液を廃棄して、１回目の抽出残渣を得た。別に、前記の沈殿物についても同様の熱水抽出処理を行ない、同様に限外濾過して、２回目の抽出残渣を得た。 Hot water at 95 to 100 ° C. was added to the dried pulverized product of Chlorella pyrenoidosa to prepare a 15% by mass hot water suspension, which was stirred for 2 hours under boiling reflux and subjected to hot water extraction treatment. After cooling to room temperature, the supernatant on the precipitate was ultrafiltered using a filter with a molecular weight cut off of 10,000, and the filtrate was discarded to obtain a first extraction residue. Separately, the same hot water extraction treatment was performed on the precipitate, and ultrafiltration was performed in the same manner to obtain a second extraction residue.
前記の１回目の抽出残渣と２回目の抽出残渣の混合物を３℃で１２時間静置した後、室温下に１００００ｒｐｍで２０分間遠心分離処理し、上澄液と沈殿物に分けた。上澄液を回収し、３℃で分画分子量１００００のフィルターを用いて限外濾過して、非濾過画分を得た。 The mixture of the first extraction residue and the second extraction residue was allowed to stand at 3 ° C. for 12 hours, and then centrifuged at 10,000 rpm for 20 minutes at room temperature to separate into a supernatant and a precipitate. The supernatant was collected and ultrafiltered using a filter with a molecular weight cut off of 10,000 at 3 ° C. to obtain a non-filtered fraction.
前記の非濾過画分に、３容量倍の無水エタノールを加えて撹拌し、室温下に１００００ｒｐｍで遠心分離処理して、上澄液と沈殿物に分けた。この沈殿物を回収し、無水エタノールで３回洗浄した後、更に無水エーテルで１回洗浄した。その後、４０℃で真空乾燥してＦＡ（粗多糖）を得た。 The unfiltered fraction was added with 3 volumes of absolute ethanol and stirred, and centrifuged at 10000 rpm at room temperature to separate the supernatant and the precipitate. This precipitate was collected, washed with anhydrous ethanol three times, and further washed once with anhydrous ether. Then, it vacuum-dried at 40 degreeC and obtained FA (crude polysaccharide).
前記のＦＡを水に溶解させ、イオン交換クロマトグラフィー｛和光純薬工業社製の商品名ＤＥＡＥ−Ｃｅｌｌｕｌｏｓｅ（Ｃｌ−）の使用による勾配溶離（ｇｒａｄｉｅｎｔ ｅｌｕｔｉｏｎ ｗｉｔｈ ０→１．０ＭＮａＣｌ）｝に供して、画分ＦＡ−１、ＦＡ−２、ＦＡ−３に分画した。画分ＦＡ−１を、ゲル濾過法｛和光純薬工業社製の商品名Ｓｅｐｈａｄｅｘ Ｇ−５０の使用による勾配溶離（ｇｒａｄｉｅｎｔ ｅｌｕｔｉｏｎ ｗｉｔｈ ０→２．０ＭＮａＣｌ）｝により精製して、ＦＡ−１ａ（ペプチドヘテロ多糖体）を得た。クロレラ・ピレノイドサの乾燥粉末物４００ｇからのＦＡ（粗多糖）及びＦＡ−１ａ（ペプチドヘテロ多糖体）の収量は、真空凍結乾燥粉末でそれぞれ１５．６３ｇ及び２５３．２７ｍｇであった。 Said the FA was dissolved in water, ion-exchange chromatography - is subjected to {manufactured by Wako Pure Chemical Industries, Ltd. trade name DEAE-Cellulose gradient elution (gradient elution with 0 → 1.0MNaCl) by use of (Cl)}, Fractions were fractionated into FA-1, FA-2 and FA-3. Fraction FA-1 was purified by a gel filtration method {gradient elution with 0 to 2.0 M NaCl using a brand name Sephadex G-50 manufactured by Wako Pure Chemical Industries, Ltd.} to obtain FA-1a (peptide Heteropolysaccharide) was obtained. The yields of FA (crude polysaccharide) and FA-1a (peptide heteropolysaccharide) from 400 g of a dry powder of chlorella pyrenoidosa were 15.63 g and 253.27 mg, respectively, as a vacuum lyophilized powder.
前記のＦＡ−１ａを分析した結果、次のような理化学的性質（ａ）〜（ｅ）を有するペプチドヘテロ多糖体であった。
（ａ）平均分子量：１００万
（ｂ）比旋光度：［ａ］Ｄ−１１．６（測定温度２５℃）
（ｃ）窒素含量５．３９％、蛋白質含量２９．５％、多糖含量７０．３％（各質量％）
（ｄ）糖組成（モル％）：ガラクトース（３２）／グルコース（２５）／マンノース（１３）／キシロース（８）／ラムノース（７）／アラビノース（５）／フラクトース（３）
（ｅ）アミノ酸組成（モル％）：グルタミン酸（１５．１）／アスパラギン酸（１３．８）／アラニン（９．９）／ロイシン（８．９）／トレオニン（６．５）／リシン（６．２）／バリン（５．９）／グリシン（５．８）／プロリン（５．５）／セリン（５．４）／アルギニン（４．８）／フェニルアラニン（３．６）／イソロイシン（３．１）／チロシン（２．１）／ヒスチジン（１．７）／メチオニン（１．６）
As a result of analyzing the FA-1a, it was a peptide heteropolysaccharide having the following physicochemical properties (a) to (e).
(A) Average molecular weight: 1 million (b) Specific rotation: [a] D -11.6 (measurement temperature 25 ° C.)
(C) Nitrogen content 5.39%, protein content 29.5%, polysaccharide content 70.3% (each mass%)
(D) Sugar composition (mol%): Galactose (32) / glucose (25) / mannose (13) / xylose (8) / rhamnose (7) / arabinose (5) / fructose (3)
(E) Amino acid composition (mol%): glutamic acid (15.1) / aspartic acid (13.8) / alanine (9.9) / leucine (8.9) / threonine (6.5) / lysine (6. 2) / valine (5.9) / glycine (5.8) / proline (5.5) / serine (5.4) / arginine (4.8) / phenylalanine (3.6) / isoleucine (3.1) ) / Tyrosine (2.1) / histidine (1.7) / methionine (1.6)
試験区分２（抗腫瘍活性の試験その１）
５週齢のマウス｛日本エスエルシー社製のＣ５７ＢＬ／６ＣｒＳＬＣ（ＳＰＦ）｝を購入し、これらのうちで、７日間予備飼育した後、一般的症状観察及び尿検査で異常が認められなかったマウスを試験に供した。またルイス肺癌細胞は、愛知ガンセンターより供与され、三重大学医学部にて本発明者の一人である伊藤均が継代しているものを用いた。以上のマウス及び血行性に容易に肺転移を起こすルイス肺癌細胞等を用いて、次のように、試験区分１で分離したＦＡ−１ａ（ペプチドヘテロ多糖体）の抗腫瘍活性の試験を行なった。
Test Category 2 (Anti-tumor activity test 1)
After purchasing a 5-week-old mouse {C57BL / 6CrSLC (SPF)} manufactured by Japan SLC Co., Ltd.], pre-bred for 7 days, and then no abnormalities were observed in general symptom observation and urinalysis Were subjected to the test. Lewis lung cancer cells were used from Aichi Cancer Center, which was passed by Hitoshi Ito, one of the inventors of the present invention at Mie University School of Medicine. Using the above mice and Lewis lung cancer cells that readily cause hematogenous lung metastases, the antitumor activity of FA-1a (peptide heteropolysaccharide) isolated in Test Category 1 was tested as follows. .
１群１０匹のマウスとし、試験１では、対照群と、合計３群（第１群、第２群及び第３群）の投与群で試験した。マウスは、温度２３±２℃、相対湿度５５±５％のバリアシステムのプラスチックケージで、固形飼料（日本クレア社製の商品名クレアＣＥ−７）と水道水とを自由に摂取させて、１４日間飼育した（飼育は試験２でも、また試験３でも同じ）。 Each group consisted of 10 mice. In Test 1, the control group and a total of 3 groups (Group 1, Group 2 and Group 3) were tested. The mouse is a plastic cage of a barrier system having a temperature of 23 ± 2 ° C. and a relative humidity of 55 ± 5%, and is allowed to freely ingest solid feed (trade name CLEA CE-7, manufactured by CLEA Japan) and tap water. The animals were reared for the day (same as test 2 and test 3).
試験１では、試験開始日に、対照群及び合計３群の投与群（第１群、第２群及び第３群）の各マウスの足蹠部皮下に、ルイス肺癌細胞を１０５個となるよう移植した。第１群のマウスは、試験開始後１０日目に、ＦＡ−１ａを５ｍｇ／ｋｇとなるよう腹腔内に投与し、前記のように１４日間飼育した。また第２群のマウスは同様にＦＡ−１ａを１０ｍｇ／ｋｇとなるよう投与し、更に第３群のマウスは同様にＦＡ−１ａを２０ｍｇ／ｋｇとなるよう投与して、１４日間飼育した。そして対照群のマウスはＦＡ−１ａに代えて生理食塩水を同様に投与し、１４日間飼育した。 In Test 1, 10 5 Lewis lung cancer cells were subcutaneously subcutaneously in the footpads of each mouse in the control group and a total of 3 administration groups (Group 1, Group 2 and Group 3) on the test start day. Transplanted as follows. The first group of mice was administered intraperitoneally with FA-1a at 5 mg / kg on day 10 after the start of the test, and was reared for 14 days as described above. Similarly, mice in Group 2 were similarly administered FA-1a at 10 mg / kg, and mice in Group 3 were similarly administered FA-1a at 20 mg / kg and reared for 14 days. And the mouse | mouth of the control group administered the physiological saline similarly instead of FA-1a, and was raised for 14 days.
１４日間飼育した対照群及び合計３群の投与群の各マウスについて、肺転移結節数をＷｅｘｌｅｒの方法（Ｊ Ｎａｔｌ Ｃａｎｃｅｒ Ｉｎｓｔ １９６６年、３６巻、６４１〜６４５頁）により測定し、結果を平均値±標準誤差で、表１にまとめて示した。対照群と投与群との間でスチューデントのｔ検定を行ない、危険率５％で有意と判断して、表１中に＊印で示した。 The number of lung metastasis nodules was measured by the method of Wexler (J Natl Cancer Inst 1966, 36, 641-645) for each mouse of the control group and the total of 3 groups administered for 14 days, and the results were averaged. Table 1 shows the standard error. Student's t-test was performed between the control group and the administration group, and it was judged to be significant at a risk rate of 5%.
１群１０匹のマウスとし、試験２では、対照群と、合計２群の投与群で試験した。試験２では、試験開始日（０日目）に、対照群及び合計２群の投与群（第４群及び第５群）の各マウスの足蹠部皮下に、ルイス肺癌細胞を１０５個となるよう移植した。第４群のマウスは、試験開始後１０日目、１２日目及び１４日目に、ＦＡ−１ａを１０ｍｇ／ｋｇとなるよう腹腔内に投与し、前記のように１４日間飼育した。また第５群のマウスは同様にＦＡ−１ａを２０ｍｇ／ｋｇとなるよう投与して、１４日間飼育した。そして対照群のマウスはＦＡ−１ａに代えて生理食塩水を同様に投与し、１４日間飼育した。 Each group consisted of 10 mice, and in Test 2, the control group and a total of 2 administration groups were tested. In Test 2, 10 5 Lewis lung cancer cells were subcutaneously injected on the footpad of each mouse in the control group and a total of 2 administration groups (Group 4 and Group 5) on the test start day (Day 0). It was transplanted to become. The mice in Group 4 were administered intraperitoneally with FA-1a at 10 mg / kg on the 10th, 12th and 14th days after the start of the test, and were reared for 14 days as described above. Similarly, mice in Group 5 were similarly fed with FA-1a at 20 mg / kg and bred for 14 days. And the mouse | mouth of the control group administered the physiological saline similarly instead of FA-1a, and was raised for 14 days.
１４日間飼育した対照群及び合計２群の投与群の各マウスについて、試験１と同様に、肺転移結節数を測定し、結果を平均値±標準誤差で、表１にまとめて示した。また対照群と投与群との間でスチューデントのｔ検定を行ない、危険率５％で有意と判断して、表１中に＊印で示した。 The number of lung metastasis nodules was measured for each mouse in the control group and the total of 2 administration groups bred for 14 days in the same manner as in Test 1, and the results are shown in Table 1 as mean values ± standard error. In addition, Student's t-test was performed between the control group and the administration group, and it was judged to be significant at a risk rate of 5%.
１群１０匹のマウスとし、試験３では、対照群と、第６群の投与群で試験した。試験３では、試験開始日（０日目）に、対照群及び第６群の投与群の各マウスの足蹠部皮下に、ルイス肺癌細胞を１０５個となるよう移植した。第６群のマウスは、試験開始後３日目、５日目、７日目、９日目、１０日目、１２日目及び１４日目に、ＦＡ−１ａを１０ｍｇ／ｋｇとなるよう腹腔内に投与し、前記のように１４日間飼育した。そして対照群のマウスはＦＡ−１ａに代えて生理食塩水を同様に投与し、１４日間飼育した。 Each group consisted of 10 mice. In Test 3, the control group and the sixth group were tested. In Test 3, on the day when the test was started (Day 0), Lewis lung cancer cells were transplanted to 10 5 subcutaneously in the footpads of each mouse in the control group and Group 6 administration group. The mice of group 6 were abdominal cavity of FA-1a at 10 mg / kg on the 3rd, 5th, 7th, 9th, 10th, 12th and 14th days after the start of the test. And bred for 14 days as described above. And the mouse | mouth of the control group administered the physiological saline similarly instead of FA-1a, and was raised for 14 days.
１４日間飼育した対照群及び第６群の投与群の各マウスについて、試験１と同様に、肺転移結節数を測定し、結果を平均値±標準誤差で、表１にまとめて示した。対照群と投与群との間でスチューデントのｔ検定を行ない、危険率５％で有意と判断して、表１中に＊印で示した。 The number of lung metastasis nodules was measured in the same manner as in Test 1 for each mouse in the control group and the sixth group administered for 14 days, and the results are shown in Table 1 as mean values ± standard error. Student's t-test was performed between the control group and the administration group, and it was judged to be significant at a risk rate of 5%.
試験区分３（抗腫瘍活性の試験その２）
試験区分２と同様のマウス及びルイス肺癌細胞等を用いて、次のように、試験区分１で分離したＦＡ−１ａ（ペプチドヘテロ多糖体）及び５−フルオロラウシルについての抗腫瘍活性の試験を行なった。
Test category 3 (anti-tumor activity test 2)
Using the same mice and Lewis lung cancer cells as in test category 2, the antitumor activity test for FA-1a (peptide heteropolysaccharide) and 5-fluorolauryl separated in test category 1 was conducted as follows. I did it.
１群１０匹のマウスとし、試験４では、対照群と、合計３群の投与群（第７群、第８群及び第９群）で試験した。試験４では、試験開始日に、対照群及び合計３群の投与群（第７群、第８群及び第９群）の各マウスの足蹠部皮下に、ルイス肺癌細胞を１０５個となるよう移植した。第７群のマウスは、試験開始後１０日目、１２日目及び１４日目に、ＦＡ−１ａを１０ｍｇ／ｋｇとなるよう腹腔内に投与し、前記のように１４日間飼育した。また第８群のマウスは同様に５−フルオロラウシルを３０ｍｇ／ｋｇとなるよう投与し、更に第９群のマウスは同様にＦＡ−１ａを１０ｍｇ／ｋｇ及び５−フルオロラウシルを３０ｍｇ／ｋｇとなるよう投与して、１４日間飼育した。そして対照群のマウスはＦＡ−１ａや５−フルオロラウシルに代えて生理食塩水を同様に投与し、１４日間飼育した。 Each group consisted of 10 mice, and in Test 4, the control group and a total of 3 administration groups (Group 7, Group 8 and Group 9) were tested. In Test 4, 10 5 Lewis lung cancer cells were subcutaneously subcutaneously in the footpads of each mouse of the control group and the total of 3 groups administered (Group 7, Group 8 and Group 9) on the test start day. Transplanted as follows. The mice in Group 7 were administered FA-1a intraperitoneally at 10 mg / kg on the 10th, 12th and 14th days after the start of the test, and were reared for 14 days as described above. Similarly, mice in Group 8 were similarly administered 5-fluorolauryl at 30 mg / kg, and mice in Group 9 were similarly 10 mg / kg FA-1a and 30 mg / kg 5-fluorolauryl. And was bred for 14 days. And the mouse | mouth of the control group administered the physiological saline similarly instead of FA-1a and 5-fluoro lauryl, and was raised for 14 days.
１４日間飼育した対照群及び合計３群の投与群の各マウスについて、試験１と同様に、肺転移結節数を測定すると共に、肺転移率を求め、結果を平均値±標準誤差で、表２にまとめて示した。対照群と投与群との間でスチューデントのｔ検定を行ない、危険率５％で有意と判断した場合を表２中に＊印で示すと共に、危険率１％で有意と判断した場合を表２中に＊＊印で示した。
For each mouse of the control group and the total of 3 groups administered for 14 days, the number of lung metastases was measured and the rate of lung metastasis was determined in the same manner as in Test 1. Table 2 Are summarized in Student's t-test was performed between the control group and the administration group, and the case where it was judged significant at a risk rate of 5% is indicated by * in Table 2, and the case where it was judged significant at a risk rate of 1% is shown in Table 2. Indicated by **.
表１の結果からも明らかなように、本発明によると、優れた抗腫瘍活性を示し、またかかる抗腫瘍活性は、表２の結果からも明らかなように、５−フルオロラウシルと併用すると、一段と顕著になる。ＦＡ−１ａは、直接的肺癌細胞増殖阻害作用を示さない。しかし、ＦＡ−１ａの投与により得られた粘着性腹腔浸出細胞は、それ自体がルイス肺癌細胞の増殖阻害作用を有する。その結果としてルイス肺癌細胞の肺転移抑制効果を示し、かかる結果は養子免疫療法に応用できることを示唆している。 As is clear from the results in Table 1, according to the present invention, excellent antitumor activity is exhibited, and when the antitumor activity is used in combination with 5-fluorolauryl, as is clear from the results in Table 2. , Become even more prominent. FA-1a does not show a direct lung cancer cell growth inhibitory effect. However, the adherent peritoneal exudate cells obtained by the administration of FA-1a itself have an inhibitory effect on the growth of Lewis lung cancer cells. As a result, the lung lung cancer cells showed an inhibitory effect on lung metastasis, which suggests that the results can be applied to adoptive immunotherapy.
Claims (4)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2017111293A JP2018203667A (en) | 2017-06-06 | 2017-06-06 | Antitumor agent having peptide hetero polysaccharide separated from crushed cell wall of chlorella pyrenoidosa as active ingredient |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2017111293A JP2018203667A (en) | 2017-06-06 | 2017-06-06 | Antitumor agent having peptide hetero polysaccharide separated from crushed cell wall of chlorella pyrenoidosa as active ingredient |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2018203667A true JP2018203667A (en) | 2018-12-27 |
Family
ID=64955545
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2017111293A Pending JP2018203667A (en) | 2017-06-06 | 2017-06-06 | Antitumor agent having peptide hetero polysaccharide separated from crushed cell wall of chlorella pyrenoidosa as active ingredient |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2018203667A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06183981A (en) * | 1992-12-24 | 1994-07-05 | Kurorera Kogyo Kk | Antiulcer agent containing muco polysaccharide extracellularly produced by chlorella bacterium |
| JPH06248003A (en) * | 1993-03-01 | 1994-09-06 | Kurorera Kogyo Kk | Polysaccharide and its production |
| JP2004505925A (en) * | 2000-08-10 | 2004-02-26 | オーシャン、ニュートリッション、カナダ、リミテッド | Chlorella preparations exhibiting immunomodulatory properties |
-
2017
- 2017-06-06 JP JP2017111293A patent/JP2018203667A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06183981A (en) * | 1992-12-24 | 1994-07-05 | Kurorera Kogyo Kk | Antiulcer agent containing muco polysaccharide extracellularly produced by chlorella bacterium |
| JPH06248003A (en) * | 1993-03-01 | 1994-09-06 | Kurorera Kogyo Kk | Polysaccharide and its production |
| JP2004505925A (en) * | 2000-08-10 | 2004-02-26 | オーシャン、ニュートリッション、カナダ、リミテッド | Chlorella preparations exhibiting immunomodulatory properties |
Non-Patent Citations (2)
| Title |
|---|
| 医学と生物学, 2012, VOL.156, NO.1, PP.26-34, JPN6021004963, ISSN: 0004571819 * |
| 静岡大学農学部研究報告, 1987, NO.37, PP.37-46, JPN6021004962, ISSN: 0004571818 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5048673B2 (en) | A kind of extract that prevents or treats thrombotic diseases | |
| JP6755980B2 (en) | High-purity heparin and its manufacturing method | |
| JPH09238697A (en) | Antitumor substance extracted from grifola frondosa | |
| JP2014501777A (en) | Aloe polysaccharide composition and method | |
| JPH0539305A (en) | Immuno suppressive polysaccharide extracted from astragalus membranaceous and pharma- ceutical composition containing same | |
| JP6681466B2 (en) | Homogeneous polysaccharide having immunomodulatory activity and method for preparing the same | |
| CN110105430B (en) | Ginkgo nut protein peptide with angiotensin converting enzyme inhibitory activity and preparation method thereof | |
| JP2018203667A (en) | Antitumor agent having peptide hetero polysaccharide separated from crushed cell wall of chlorella pyrenoidosa as active ingredient | |
| CN110183516B (en) | Preparation method of blood pressure lowering ginkgo nut protein peptide | |
| CN117820437A (en) | Jerusalem artichoke peptide, preparation method thereof and application thereof in kidney-protecting auxiliary diabetes treatment antioxidant product | |
| KR101949889B1 (en) | Method for preparing anticancer or immunactive polysaccharide from residues of immature citrus extract or immature citrus peel | |
| CN107915772B (en) | Novel sweet potato glycoprotein and sweet potato glycoprotein extract containing the same | |
| JP3051073B2 (en) | Antitumor active substance | |
| JPH02208301A (en) | Production of physiologically active polysaccharide ron derived from rice bran | |
| JP3129751B2 (en) | Bunashimeji fruit body hot water extract | |
| KR101497506B1 (en) | The Pharmaceutical composition and functional food for inhibiting angiotensin-I converting enzyme | |
| JP4480204B2 (en) | Anti-tumor fraction of Kawariharatake | |
| JPH115745A (en) | Anti-hiv active substance and its production | |
| JP2006028128A (en) | Thrombosis ameliorating agent | |
| EP2889037B1 (en) | Plant extract and the process for treating hepatic fibrosis and liver cancer | |
| KR20130012417A (en) | Composition for immune boosting having radioprotective effect comprising fucoidane from ecklonia cava extracts | |
| CA2730607A1 (en) | Process for producing glycosaminoglycans | |
| JPS6153221A (en) | Novel polysaccharide rin substance, its preparation, and antitumor agent, immuno-regulating agent and preventive and remedy for infectious diseases, containing said substance as active component | |
| EP4059509A1 (en) | Agrimonia eupatoria extract preparation method, and composition for improving liver functions or treating liver diseases, containing extract | |
| CN113491760A (en) | Medicine for treating respiratory system diseases |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20170606 |
|
| RD02 | Notification of acceptance of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7422 Effective date: 20171110 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20200204 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20210216 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20210817 |