JP2006151889A - Anti-mrsa composition - Google Patents
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- 239000000203 mixture Substances 0.000 title claims abstract description 78
- 206010041925 Staphylococcal infections Diseases 0.000 claims abstract description 100
- 208000015688 methicillin-resistant staphylococcus aureus infectious disease Diseases 0.000 claims abstract description 100
- 239000000284 extract Substances 0.000 claims abstract description 21
- 239000000126 substance Substances 0.000 claims abstract description 8
- 239000004480 active ingredient Substances 0.000 claims abstract description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 18
- 241000206759 Haptophyceae Species 0.000 abstract description 16
- 239000013543 active substance Substances 0.000 abstract description 11
- 241000722208 Pleurochrysis Species 0.000 abstract description 5
- 229930014626 natural product Natural products 0.000 abstract description 4
- 230000009545 invasion Effects 0.000 abstract description 3
- 210000004400 mucous membrane Anatomy 0.000 abstract description 3
- 238000005507 spraying Methods 0.000 abstract description 3
- 241001245608 Isochrysidales Species 0.000 abstract description 2
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 abstract description 2
- 241000191967 Staphylococcus aureus Species 0.000 abstract description 2
- 229960003085 meticillin Drugs 0.000 abstract description 2
- 230000037361 pathway Effects 0.000 abstract 1
- 238000000034 method Methods 0.000 description 23
- 230000000052 comparative effect Effects 0.000 description 22
- 230000000694 effects Effects 0.000 description 22
- 238000012360 testing method Methods 0.000 description 11
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 241000199264 Phaseolus carteri Species 0.000 description 9
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 9
- 238000004108 freeze drying Methods 0.000 description 8
- 238000000605 extraction Methods 0.000 description 7
- 239000000706 filtrate Substances 0.000 description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 241000195493 Cryptophyta Species 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 229940049954 penicillin Drugs 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000006150 trypticase soy agar Substances 0.000 description 3
- 241001536641 Gephyrocapsa Species 0.000 description 2
- 241001501885 Isochrysis Species 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- RMRCNWBMXRMIRW-BYFNXCQMSA-M cyanocobalamin Chemical compound N#C[Co+]N([C@]1([H])[C@H](CC(N)=O)[C@]\2(CCC(=O)NC[C@H](C)OP(O)(=O)OC3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)C)C/2=C(C)\C([C@H](C/2(C)C)CCC(N)=O)=N\C\2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O RMRCNWBMXRMIRW-BYFNXCQMSA-M 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
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- 239000011259 mixed solution Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- LMSDCGXQALIMLM-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;iron Chemical compound [Fe].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O LMSDCGXQALIMLM-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 229940046414 biotin 1 mg Drugs 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 229960002104 cyanocobalamin Drugs 0.000 description 1
- 235000000639 cyanocobalamin Nutrition 0.000 description 1
- 239000011666 cyanocobalamin Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 239000012134 supernatant fraction Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229960000344 thiamine hydrochloride Drugs 0.000 description 1
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 1
- 239000011747 thiamine hydrochloride Substances 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Abstract
Description
本発明は、微細藻から抽出された抽出物を有効成分として含有する抗ＭＲＳＡ組成物に関するものである。 The present invention relates to an anti-MRSA composition containing an extract extracted from microalgae as an active ingredient.
メチシリン耐性黄色ぶどう球菌（以下、ＭＲＳＡという）は、新たな抗生物質を開発しても、必ずその耐性菌が誕生し、更に悪い状況を作り出すことから、抗生物質が効かなくなったＭＲＳＡは、院内感染の原因細菌として、社会的な問題になっている。 Since methicillin-resistant Staphylococcus aureus (hereinafter referred to as MRSA) develops new antibiotics, the resistant bacteria are always born and create a worse situation. As a cause of bacteria, it has become a social problem.
しかし、天然由来の抗菌活性剤を用いた場合には、これまで耐性菌が作り出されたという報告は殆どなく、また、天然物由来であるため、天然由来の抗菌活性剤を用いたことによって生じる副作用が少ないという特徴がある。 However, when natural antibacterial active agents are used, there have been almost no reports that resistant bacteria have been created so far, and because they are derived from natural products, they are caused by using natural antibacterial active agents. It is characterized by fewer side effects.
そこで、天然由来の抗菌活性剤としては、例えば、ハプト藻綱（Haptophyceae）のイソクリシス目（Isochrysidales）に属するゲフィロカプサ属（Gephyrocapsa）又は、プレウロクリシス属（Pleurochrysis） の藻体から分離される多糖、またはこれを含有した抗菌・抗癌剤がある（特許文献１参照）。 Therefore, as the naturally-occurring antibacterial active agent, for example, a polysaccharide isolated from an alga of the genus Gephyrocapsa or Pleurochrysis belonging to the Isochrysidales of Haptophyceae, or this There is an antibacterial / anticancer agent containing the above (see Patent Document 1).
この特許文献１の公知技術においては、ハプト藻綱のイソクリシス目に属するゲフィロカプサ属又は、プレウロクリシス（プリュウロクリシス）属の藻体から分離された多糖類が抗菌活性を有しているというものである。 In the known technique of Patent Document 1, a polysaccharide isolated from an algal body belonging to the genus Gephyrocapsa belonging to the genus Isocrisis of the haptophyceae class or from the genus Preurocrisis (Pleuurocrisis) has antibacterial activity. .
しかしながら、前記特許文献１の公知技術においては、ハプト藻綱のイソクリシス目に属するゲフィロカプサ属又は、プレウロクリシス（プリュウロクリシス）属の藻体から分離された多糖類が抗菌活性を有しているというだけであり、ＭＲＳＡに対する抗菌活性については、未だ報告されていない。 However, in the known technique of Patent Document 1, the polysaccharide isolated from the algal body of the genus Gefilocapus or the genus Preurocrisis belonging to the genus Isocrisis of the haptophyceae class only has antibacterial activity. No antibacterial activity against MRSA has been reported yet.
従って、従来の抗菌活性剤においては、天然由来の抗菌活性剤であって、且つＭＲＳＡに対する抗菌活性を有している抗ＭＲＳＡの物質を得るということに解決しなければならない課題を有している。 Therefore, the conventional antibacterial active agent has a problem to be solved by obtaining an anti-MRSA substance which is a naturally occurring antibacterial active agent and has antibacterial activity against MRSA. .
上記した従来例の課題を解決するために、本発明者等は種々の微細藻について研究し実験した結果、ハプト藻綱（Haptopheceae）イソクリシス目（Isochrysisdales）のプリュウロクリシス属（Pleurochrysis）の微細藻の抽出物が抗ＭＲＳＡ活性を有していることを見出したのであり、そのプリュウロクリシス属（Pleurochrysis）の微細藻として、その代表的なものとしてP.carteraeまたはP.haptonemafera等を挙げることができる。 In order to solve the problems of the conventional examples described above, the present inventors have studied and experimented on various microalgae. As a result, the microalgae of the genus Pleurochrysis of the Haptopheceae isochrysis order (Isochrysisdales) It has been found that the extract of P. carterae or P. haptonemafera is a typical example of the microalga of the genus Pleurochrysis. it can.
そして、課題を解決する具体的手段として本発明に係る抗ＭＲＳＡ組成物は、ハプト藻類イソクリシス目のプリュウロクリシス属に属する微細藻の抽出物を有効成分として含有していることを最も主要な特徴とする。 As a specific means for solving the problem, the anti-MRSA composition according to the present invention contains, as an active ingredient, an extract of a microalga belonging to the genus Pleuurocrisis of the haptoalgae isocrisis. And
この発明において、前記抽出物は、水もしくは熱水またはアルコールにより抽出されること；前記抽出物は、分子量が略１００００以下の水溶性物質であること；を付加的な要件として含むものである。 In the present invention, the extract is extracted with water, hot water, or alcohol; the extract is a water-soluble substance having a molecular weight of about 10,000 or less as an additional requirement.
本発明に係る抗ＭＲＳＡ組成物は、ハプト藻類イソクリシス目のプリュウロクリシス属に属する微細藻の抽出物を有効成分として含有していることにより、天然物由来の微細藻から抽出された抗菌活性剤であるため、人体に対する安全性が高く、且つＭＲＳＡに対する抗菌活性を有している抗ＭＲＳＡの物質であるため、抗ＭＲＳＡ組成物をＭＲＳＡの侵入経路である喉等の粘膜や手指に直接噴霧してＭＲＳＡの体内への侵入を防ぐことができるという優れた効果を奏する。 The anti-MRSA composition according to the present invention contains, as an active ingredient, an extract of a microalga belonging to the genus Purourocrisis of the haptoalgae isocratic order, so that the antibacterial active agent extracted from the natural algae Therefore, since it is an anti-MRSA substance that is highly safe for the human body and has antibacterial activity against MRSA, the anti-MRSA composition is directly sprayed on the mucous membranes and fingers of the throat, which are MRSA penetration routes. Thus, the MRSA can prevent the MRSA from entering the body.
次に、本発明を具体的な実施の形態に基づいて詳しく説明する。
本発明の実施の形態に係る抗ＭＲＳＡ組成物を得る上で、まず、該抗ＭＲＳＡ組成物を得るために用いるハプト藻綱（Haptopheceae）イソクリシス目（Isochrysisdales）のプリュウロクリシス属（Pleurochrysis）の微細藻を生産または培養する方法について説明する。
Next, the present invention will be described in detail based on specific embodiments.
In obtaining the anti-MRSA composition according to the embodiment of the present invention, first, the fineness of the genus Pleurochrysis of the haptophyceae Isochrysis order (Isochrysisdales) used for obtaining the anti-MRSA composition. A method for producing or culturing algae will be described.
ハプト藻綱イソクリシス目のプリュウロクリシス属の微細藻を得る方法としては、例えば、天然に生育している微細藻を収穫して使用すること等ができるが、微細藻の安定した供給と品質保持との理由から、培養により増殖させた微細藻を使用することが好ましい。微細藻は、光合成を行って自らのエネルギーとしているため、培養は光照射下に藻類培養用の培地を用い、通常の培養方法により培養することができる。なお、ハプト藻綱イソクリシス目のプリュウロクリシス属の微細藻としては、代表的に、P.carteraeまたはP.haptonemafera等の微細藻を培養して用いることにした。 As a method for obtaining the microalga of the genus Purourocrisis of the haptophyceae isocrisis, for example, it is possible to harvest and use naturally grown microalgae. However, stable supply and quality maintenance of microalgae are possible. For this reason, it is preferable to use microalgae grown by culture. Since microalgae carry out photosynthesis and make their own energy, the culture can be performed by a normal culture method using a culture medium for algae under light irradiation. As the microalgae of the genus Pleuurocrisis of the Haptophyceae isocratic order, typically, microalgae such as P.carterae or P.haptonemafera were cultured and used.
（培養例１）
この培養例１においては、例えば、ハプト藻綱イソクリシス目のプリュウロクリシス属の微細藻としてP.carteraeを用いて該微細藻を培養する具体的な方法の一例について説明する。
(Culture Example 1)
In this culture example 1, for example, an example of a specific method for culturing the microalgae using P.carterae as a microalga of the genus Pleuurocrisis of the haptophyceae isocrisis will be described.
培地としては、一般的に用いられている海産性ハプト藻綱を培養する際に用いられるものであれば格別な制限はなく、この培養例１においては、Eppley's mediumを用いて培養を行った。 The medium is not particularly limited as long as it is used when cultivating marine haptophyceae that are generally used. In this culture example 1, culture was performed using Eppley's medium.
この培地においては、新鮮な濾過海水１０００ｍｌに対してＫＮＯ３ ５０．５ｍｇ及びＫ２ＨＰＯ４ ８．７ｍを添加した溶液に、ＣｕＳＯ４・５Ｈ２Ｏ １９．６ｍｇ／ｌ、ＺｎＳＯ４・７Ｈ２Ｏ ４４ｍｇ／ｌ、ＣｏＣｌ２・６Ｈ２Ｏ ２０ｍｇ／ｌ、ＭｎＣｌ２・４Ｈ２Ｏ ３６０ｍｇ／ｌ、Ｎａ２ＭｏＯ４・２Ｈ２Ｏ １２．６ｍｇ／ｌ及びＦｅ−ＥＤＴＡ １０ｇ／ｌからなる微量元素混合溶液１ｍｌを添加し、次いでチアミン塩酸塩２００ｍｇ／ｌ、ビオチン１ｍｇ／ｌ、シアノコバラミン０．２ｍｇ／ｌを含有するビタミン混合溶液１ｍｌを添加することにより培地を調整した。 In this medium, CuSO 4 · 5H 2 O 19.6 mg / l, ZnSO 4 · 7H 2 O was added to a solution obtained by adding 50.5 mg of KNO 3 and 8.7 m of K 2 HPO 4 to 1000 ml of fresh filtered seawater. 44 ml / l, CoCl 2 · 6H 2 O 20 mg / l, MnCl 2 · 4H 2 O 360 mg / l, Na 2 MoO 4 · 2H 2 O 12.6 mg / l and Fe-EDTA 10 g / l 1 ml trace element mixed solution Then, the medium was prepared by adding 1 ml of a vitamin mixed solution containing thiamine hydrochloride 200 mg / l, biotin 1 mg / l, and cyanocobalamin 0.2 mg / l.
培養は、P.carteraeの細胞数が１０−２０×１０５ cells／ｍｌになるように培地に接種し、２リットル容量のガラス扁平フラスコを用いて行った。培養期間は、５−７日間程度が適当であり、培養は適当な通気手段により空気が導入する好気的条件下で、且つ蛍光灯を光源として照度を４０μEinsteins／ｍ２／secに設定し、連続光照射下において行うのが好ましい。このときの培養温度は、２０−２５℃程度であり、２３℃程度の温度が好ましい。このような条件にして、前記P.carteraeに限らず、P.haptonemafera等のハプト藻綱イソクリシス目のプリュウロクリシス属の微細藻を培養することができる。 The culture was performed by inoculating the medium so that the number of P.carterae cells was 10-20 × 10 5 cells / ml, and using a 2-liter glass flat flask. The culture period is suitably about 5-7 days, the culture is performed under aerobic conditions where air is introduced by an appropriate ventilation means, and the illuminance is set to 40 μEinsteins / m 2 / sec using a fluorescent lamp as the light source. It is preferable to carry out under continuous light irradiation. The culture temperature at this time is about 20-25 ° C, and a temperature of about 23 ° C is preferable. Under such conditions, not only P.carterae, but also P. haptonemafera and other haptophyceae isocratic microalgae can be cultured.
（抗ＭＲＳＡ組成物を得る方法１）
次に、ハプト藻綱イソクリシス目のプリュウロクリシス属の微細藻から抗ＭＲＳＡ組成物を得る方法について説明する。この抗ＭＲＳＡ組成物を得る方法１においては、前記微細藻の藻体濃度が１−７０％程度になるように水を添加し、室温−９５℃程度の温度下で略１−２４時間程度抽出処理して抽出液を得る。次いで、その抽出液を濾過してその濾液を凍結乾燥させることにより抗ＭＲＳＡ組成物を得ることができる。なお、室温で抽出処理することは可能であるが、処理時間が長くなって工業生産的には適さないので、好ましくは８０℃以上の熱湯で抽出処理した方が処理時間が短くて済み工業的に適する。
(Method 1 of obtaining anti-MRSA composition)
Next, a method for obtaining an anti-MRSA composition from a microalga of the genus Purourocris of the haptophyceae Isocrisis will be described. In Method 1 for obtaining this anti-MRSA composition, water is added so that the algal body concentration of the microalgae is about 1-70%, and extraction is performed at a temperature of about room temperature-95 ° C. for about 1-24 hours. Process to obtain an extract. Next, the anti-MRSA composition can be obtained by filtering the extract and freeze-drying the filtrate. Although it is possible to perform extraction at room temperature, it is not suitable for industrial production because the processing time is long. Therefore, it is preferable to perform extraction with hot water of 80 ° C. or more because the processing time is shorter. Suitable for.
（抗ＭＲＳＡ組成物を得る方法２）
この抗ＭＲＳＡ組成物を得る方法２においては、前記微細藻の藻体濃度が１−７０％程度になるように略１０−９０％の濃度のエタノールまたはメタノール溶液を添加し、室温−７０℃程度の温度下で略１−２４時間程度抽出処理して抽出液を得る。次いで、その抽出液を濾過してその濾液を得た後、該濾液からエタノールまたはメタノールを除去してから凍結乾燥させることにより抗ＭＲＳＡ組成物を得ることができる。なお、エタノールまたはメタノールのアルコール濃度が低いと抽出処理時間が長くなるので、好ましくは３０−６０％の濃度で抽出処理した方が処理時間が短くて済み工業的に適する。
(Method 2 for obtaining anti-MRSA composition)
In Method 2 for obtaining this anti-MRSA composition, an ethanol or methanol solution having a concentration of about 10-90% is added so that the algal body concentration of the microalgae is about 1-70%, and room temperature is about -70 ° C. Extraction is performed for about 1-24 hours at a temperature of 1 to obtain an extract. Subsequently, the extract is filtered to obtain the filtrate, and then ethanol or methanol is removed from the filtrate, followed by lyophilization to obtain an anti-MRSA composition. In addition, when the alcohol concentration of ethanol or methanol is low, the extraction treatment time becomes long. Therefore, it is preferable to perform the extraction treatment at a concentration of 30 to 60% because the treatment time is short and industrially suitable.
（抗ＭＲＳＡ組成物を得る方法３）
この抗ＭＲＳＡ組成物を得る方法３においては、前記抗ＭＲＳＡ組成物を得る方法１で得られた抗ＭＲＳＡ組成物に水を加え、さらに最終濃度が略８０％になるようにエタノールを添加し、得られた上清画分のエタノールを除去してから凍結乾燥させることにより抗ＭＲＳＡ組成物を得ることができる。
(Method 3 for obtaining anti-MRSA composition)
In Method 3 for obtaining this anti-MRSA composition, water is added to the anti-MRSA composition obtained in Method 1 for obtaining said anti-MRSA composition, and ethanol is further added so that the final concentration is approximately 80%. The anti-MRSA composition can be obtained by removing ethanol from the obtained supernatant fraction and then freeze-drying it.
（抗ＭＲＳＡ組成物を得る方法４）
この抗ＭＲＳＡ組成物を得る方法４においては、前記抗ＭＲＳＡ組成物を得る方法２または抗ＭＲＳＡ組成物を得る方法３で得られた抗ＭＲＳＡ組成物を分子量１００００で限外濾過し、その濾液を凍結乾燥させることにより抗ＭＲＳＡ組成物を得ることができる。
(Method 4 of obtaining anti-MRSA composition)
In Method 4 for obtaining the anti-MRSA composition, the anti-MRSA composition obtained in Method 2 for obtaining the anti-MRSA composition or Method 3 for obtaining the anti-MRSA composition is ultrafiltered at a molecular weight of 10,000, and the filtrate is obtained. The anti-MRSA composition can be obtained by lyophilization.
（抗ＭＲＳＡ組成物を得る方法５）
この抗ＭＲＳＡ組成物を得る方法５においては、前記抗ＭＲＳＡ組成物を得る方法４で得られた抗ＭＲＳＡ組成物を水に溶解させ、等量ヘキサンを加えて分画し、得られた水溶性画分に等量クロロホルムを加えて分画し、得られた水溶性画分に等量の酢酸エチルを加え、得られた水溶性画分を凍結乾燥させることにより抗ＭＲＳＡ組成物を得ることができる。
(Method 5 of obtaining anti-MRSA composition)
In Method 5 for obtaining this anti-MRSA composition, the anti-MRSA composition obtained in Method 4 for obtaining said anti-MRSA composition was dissolved in water, and an equal amount of hexane was added for fractionation. It is possible to obtain an anti-MRSA composition by adding an equal volume of chloroform to the fraction, adding an equal volume of ethyl acetate to the obtained water-soluble fraction, and freeze-drying the obtained water-soluble fraction. it can.
このようにして抗ＭＲＳＡ組成物を得ることができるが、これら得られた抗ＭＲＳＡ組成物においては、抗ＭＲＳＡ活性が認められたが、濾過後の残渣や濾液分配後の有機層には抗ＭＲＳＡ活性が認められなかった。このことは、抗ＭＲＳＡ活性物質の分子量が小さく（低分子物質）、水に溶解しやすい（水溶性物質）物質であるものと推察される。 Although anti-MRSA compositions can be obtained in this manner, anti-MRSA activity was observed in the obtained anti-MRSA compositions, but the anti-MRSA activity was found in the residue after filtration and the organic layer after filtrate distribution. No activity was observed. This is presumed that the anti-MRSA active substance has a low molecular weight (low molecular weight substance) and is easily dissolved in water (water soluble substance).
そして、前記得られた抗ＭＲＳＡ組成物は、天然物由来の微細藻から抽出されたものであるから、人体に対する安全性が高く、また、ＭＲＳＡが薬剤耐性を持ち難い極めて有効な抗ＭＲＳＡ剤となることが期待できる。そのため、抗ＭＲＳＡ組成物をＭＲＳＡの侵入経路である喉等の粘膜や手指に直接噴霧することにより、ＭＲＳＡの脅威から開放され、更に、他人が触れる可能性のあるものに対しても、抗ＭＲＳＡ組成物を噴霧または塗布する等により、広範囲に利用することが可能である。 And since the obtained anti-MRSA composition is extracted from natural products-derived microalgae, it is highly safe for the human body, and MRSA is difficult to have drug resistance and is an extremely effective anti-MRSA agent. Can be expected. Therefore, by spraying the anti-MRSA composition directly on the mucous membranes and fingers of the throat, which is the MRSA invasion route, the MRSA threat is released and the anti-MRSA is also able to be touched by others. It can be used in a wide range by spraying or applying the composition.
次に、本発明の実施の形態に係る抗ＭＲＳＡ組成物をより具体的な実施例を挙げて説明する。この実施例１においては、ハプト藻綱イソクリシス目のプリュウロクリシス属の微細藻としてP.carteraeを用い、該微細藻の乾燥藻体略５ｇに略９０℃の熱水５００ｍｌを加え、室温にて１時間撹拌抽出した。その抽出液をガラス繊維ろ紙にて濾過後、その濾液を凍結乾燥させることにより実施例１の抗ＭＲＳＡ組成物を得た。 Next, the anti-MRSA composition according to the embodiment of the present invention will be described with reference to more specific examples. In this Example 1, P.carterae was used as a microalga of the genus Pleuurocrisis of the Haptophyceae isoclysis, 500 ml of hot water at about 90 ° C. was added to about 5 g of dry algae of the microalgae, and The mixture was extracted with stirring for 1 hour. The extract was filtered through glass fiber filter paper, and the filtrate was freeze-dried to obtain the anti-MRSA composition of Example 1.
この実施例２においては、ハプト藻綱イソクリシス目のプリュウロクリシス属の微細藻としてP.carteraeを用い、該微細藻の乾燥藻体略５ｇに略５０％のエタノール溶液５００ｍｌを加え、室温にて１時間撹拌抽出した。その抽出液をガラス繊維ろ紙にて濾過後、その濾液を減圧濃縮によってエタノールを除去してから凍結乾燥させることにより実施例２の抗ＭＲＳＡ組成物を得た。 In this Example 2, P.carterae was used as a microalga of the genus Pleuurocrisis of the Haptophyceae isocratic order, 500 ml of an approximately 50% ethanol solution was added to about 5 g of the dry alga body of the microalgae, and at room temperature. The mixture was extracted with stirring for 1 hour. The extract was filtered through glass fiber filter paper, and the filtrate was concentrated under reduced pressure to remove ethanol and then lyophilized to obtain the anti-MRSA composition of Example 2.
実施例３においては、ハプト藻綱イソクリシス目のプリュウロクリシス属の微細藻としてP.carteraeを用い、該微細藻の乾燥藻体略５ｇに略９０℃の熱水５００ｍｌを加え、室温にて１時間撹拌抽出した。このように熱水により抽出して得た抽出液に、最終濃度が略８０％になるようにエタノールを添加して、室温にて一晩（２４時間）放置後、エタノール可溶画分を分画して凍結乾燥させることにより実施例３の抗ＭＲＳＡ組成物を得た。なお、上記各実施例においては、ハプト藻綱イソクリシス目のプリュウロクリシス属の微細藻の代表としてP.carteraeを用いたが、他の同じプリュウロクリシスに属し同じ性質を有する例えば、P.haptonemafera等を用いることができることは当然のことである。 In Example 3, P. carterae was used as a microalga of the genus Pleuurocrisis of the Haptophyceae isoclysis, 500 ml of hot water at about 90 ° C. was added to about 5 g of dry algae of the microalga, and 1 at room temperature. Extracted with stirring for hours. Ethanol is added to the extract obtained by extraction with hot water in this way so that the final concentration is approximately 80%, and the mixture is allowed to stand overnight at room temperature (24 hours). And anti-MRSA composition of Example 3 was obtained by lyophilization. In each of the above examples, P. carterae was used as a representative of the microalga of the genus Pryurocrisis of the haptophyceae isocratic order, but belongs to other same pleuurolysis and has the same properties, for example, P. haptonemafera It is natural that the above can be used.
（比較例１）
この比較例１においては、前記実施例１のエタノール不溶画分を分画して凍結乾燥させることにより比較例１の組成物を得た。
(Comparative Example 1)
In Comparative Example 1, the ethanol-insoluble fraction of Example 1 was fractionated and lyophilized to obtain the composition of Comparative Example 1.
（試験例１）
この試験例１においては、前記実施例１、２、３により得られた抗ＭＲＳＡ組成物と、比較例１により得られた組成物とを用いると共に、従来から知られているペニシリンとを用い、これらにおける抗ＭＲＳＡ活性をペーパーディスク法にて試験した。
(Test Example 1)
In Test Example 1, the anti-MRSA composition obtained in Examples 1, 2, and 3 and the composition obtained in Comparative Example 1 were used, and a conventionally known penicillin was used. These were tested for anti-MRSA activity by the paper disk method.
まず、酵母抽出物を０．５％程度含むTryptic Soy寒天平板を予め作製しておき、前培養したＭＲＳＡ菌液を略１０６個／ｍｌになるように酵母抽出物を０．５％程度含むTryptic Soy液体培地で希釈し、これに軟寒天を加え、前記０．５％程度含むTryptic Soy寒天平板に撒いた。 First, including yeast extract 0.5%, as previously prepared Tryptic Soy agar plate containing yeast extract 0.5%, the pre-cultured MRSA bacterial suspension to approximately 10 6 cells / ml The solution was diluted with Tryptic Soy liquid medium, soft agar was added thereto, and the mixture was plated on a Tryptic Soy agar plate containing about 0.5%.
滅菌した直径１０ｍｍのペーパーディスクに前記実施例１、２、３により得られた抗ＭＲＳＡ組成物と、比較例１により得られた組成物とからなるそれぞれの試料溶液５０ｍｇ／ｍｌを７０μｌアプライし、同様にペニシリン１ｍｇ／ｍｌ、０．５ｍｇ／ｍｌ、０．２５ｍｇ／ｍｌをそれぞれアプライし、乾燥後に前記０．５％程度含むTryptic Soy寒天平板上にのせ、３７℃の条件下で２４時間倒置培養した。そして、ペーパーディスクの周囲に生じた阻止円により、抗ＭＲＳＡ活性を評価した。 70 μl of each sample solution 50 mg / ml consisting of the anti-MRSA composition obtained in Examples 1, 2, and 3 and the composition obtained in Comparative Example 1 was applied to a sterilized paper disk having a diameter of 10 mm, Similarly, penicillin 1 mg / ml, 0.5 mg / ml, and 0.25 mg / ml were applied, and after drying, placed on the Tryptic Soy agar plate containing about 0.5%, and incubated at 37 ° C. for 24 hours. did. Then, the anti-MRSA activity was evaluated by the inhibition circle generated around the paper disk.
表１に、この試験例１について、ペーパーディスクの周囲に生じた阻止円による抗ＭＲＳＡ活性の評価を示す。この表１においては、阻止円が大きいほど抗ＭＲＳＡ活性が高いことを示している。
Table 1 shows the evaluation of the anti-MRSA activity by the blocking circle generated around the paper disk in Test Example 1. In Table 1, the larger the inhibition circle, the higher the anti-MRSA activity.
この表１から明らかなように、比較例１により得られた組成物には抗ＭＲＳＡ活性が認められなかったが、前記実施例１、２、３により得られた抗ＭＲＳＡ組成物には高い抗ＭＲＳＡ活性が認められ、特に、ペニシリンと同等以上の抗ＭＲＳＡ活性が認められたことから、該実施例１、２、３により得られた抗ＭＲＳＡ組成物は、天然由来の抗菌活性剤であり、且つＭＲＳＡに対する抗菌活性を有している抗ＭＲＳＡの物質であることが理解できる。また、時間を要するがハプト藻綱イソクリシス目のプリュウロクリシス属の微細藻を室温（２５℃）で抽出処理した抽出物であっても、上記実施例１と略同等の抗菌活性を有していることが確認された。 As is clear from Table 1, the anti-MRSA activity was not observed in the composition obtained in Comparative Example 1, but the anti-MRSA composition obtained in Examples 1, 2, and 3 had a high anti-MRSA activity. MRSA activity was observed, and in particular, anti-MRSA activity equivalent to or higher than that of penicillin was observed. Therefore, the anti-MRSA compositions obtained in Examples 1, 2, and 3 are naturally derived antimicrobial active agents, It can also be understood that it is an anti-MRSA substance having antibacterial activity against MRSA. Further, although it takes time, even an extract obtained by subjecting a microalga of the genus Purourocrisis of the haptophyceae isocrisis to room temperature (25 ° C.) has substantially the same antibacterial activity as in Example 1 above. It was confirmed that
この実施例４においては、前記実施例２の熱水抽出物エタノール可溶画分について、限外濾過を行い、分子量１００００以下の画分を分画して凍結乾燥させることにより実施例３の抗ＭＲＳＡ組成物を得た。 In Example 4, the ethanol-soluble fraction of the hot water extract of Example 2 was subjected to ultrafiltration, and the fraction having a molecular weight of 10,000 or less was fractionated and freeze-dried, whereby the anti-antigen of Example 3 was obtained. An MRSA composition was obtained.
（比較例２）
この比較例２においては、前記実施例４における分子量１００００以上の画分を分画して凍結乾燥させることにより比較例２の組成物を得た。
(Comparative Example 2)
In Comparative Example 2, the fraction of molecular weight 10,000 or more in Example 4 was fractionated and lyophilized to obtain the composition of Comparative Example 2.
（試験例２）
この試験例２においては、これら実施例４により得られた抗ＭＲＳＡ組成物と、比較例２により得られた組成物とを用い、これらにおける抗ＭＲＳＡ活性を前記試験例１と同様にしてペーパーディスク法にて試験した。
(Test Example 2)
In Test Example 2, the anti-MRSA composition obtained in Example 4 and the composition obtained in Comparative Example 2 were used. Tested by the method.
表２に、この試験例２について、ペーパーディスクの周囲に生じた阻止円による抗ＭＲＳＡ活性の評価を示す。この表２においては、阻止円が大きいほど抗ＭＲＳＡ活性が高いことを示している。 Table 2 shows the evaluation of the anti-MRSA activity by the blocking circle formed around the paper disk in Test Example 2. Table 2 shows that the larger the inhibition circle, the higher the anti-MRSA activity.
この表２から明らかなように、比較例２により得られた組成物には抗ＭＲＳＡ活性が認められなかったが、前記実施例４により得られた抗ＭＲＳＡ組成物には高い抗ＭＲＳＡ活性が認められた。 As is apparent from Table 2, the composition obtained in Comparative Example 2 did not show anti-MRSA activity, but the anti-MRSA composition obtained in Example 4 showed high anti-MRSA activity. It was.
この実施例５においては、前記実施例４の抗ＭＲＳＡ組成物を水で溶解し、等量のヘキサンを加えて分画し、得られた水溶性画分に等量クロロホルムを加えて分画し、得られた水溶性画分に等量の酢酸エチルを加え、得られた水溶性画分を凍結乾燥させることにより実施例５の抗ＭＲＳＡ組成物を得た。 In this Example 5, the anti-MRSA composition of Example 4 was dissolved in water and fractionated by adding an equal amount of hexane, and the resulting water-soluble fraction was fractionated by adding an equal amount of chloroform. Then, an equivalent amount of ethyl acetate was added to the obtained water-soluble fraction, and the obtained water-soluble fraction was freeze-dried to obtain the anti-MRSA composition of Example 5.
（比較例３）
この比較例３においては、前記実施例４の抗ＭＲＳＡ組成物を水で溶解し、等量のヘキサンを加えて凍結乾燥させることにより比較例３の組成物を得た。
(Comparative Example 3)
In Comparative Example 3, the composition of Comparative Example 3 was obtained by dissolving the anti-MRSA composition of Example 4 with water, adding an equal amount of hexane and freeze-drying.
（比較例４）
この比較例４においては、前記実施例４の抗ＭＲＳＡ組成物を水で溶解し、等量のクロロホルムを加えて凍結乾燥させることにより比較例４の組成物を得た。
(Comparative Example 4)
In Comparative Example 4, the composition of Comparative Example 4 was obtained by dissolving the anti-MRSA composition of Example 4 in water, adding an equal amount of chloroform and freeze-drying.
（比較例５）
この比較例５においては、前記実施例３の抗ＭＲＳＡ組成物を水で溶解し、等量の酢酸エチルを加えて凍結乾燥させることにより比較例５の組成物を得た。
(Comparative Example 5)
In Comparative Example 5, the composition of Comparative Example 5 was obtained by dissolving the anti-MRSA composition of Example 3 with water, adding an equal amount of ethyl acetate and lyophilizing.
（試験例３）
この試験例３においては、これら実施例５により得られた抗ＭＲＳＡ組成物と、比較例３、４、５により得られた組成物とを用い、これらにおける抗ＭＲＳＡ活性を前記試験例１と同様にしてペーパーディスク法にて試験した。
(Test Example 3)
In Test Example 3, the anti-MRSA composition obtained in Example 5 and the compositions obtained in Comparative Examples 3, 4, and 5 were used, and their anti-MRSA activities were the same as in Test Example 1 described above. The paper disc method was used for testing.
表３に、この試験例３について、ペーパーディスクの周囲に生じた阻止円による抗ＭＲＳＡ活性の評価を示す。この表３においては、阻止円が大きいほど抗ＭＲＳＡ活性が高いことを示している。 Table 3 shows the evaluation of the anti-MRSA activity by the blocking circle generated around the paper disk in Test Example 3. In Table 3, the larger the inhibition circle, the higher the anti-MRSA activity.
この表３から明らかなように、比較例３、４、５により得られた組成物には抗ＭＲＳＡ活性が認められなかったが、前記実施例５により得られた抗ＭＲＳＡ組成物には抗ＭＲＳＡ活性が認められた。 As is apparent from Table 3, the anti-MRSA activity was not observed in the compositions obtained in Comparative Examples 3, 4, and 5, but the anti-MRSA composition obtained in Example 5 was anti-MRSA. Activity was observed.
いずれにしても、本発明においては、ハプト藻綱イソクリシス目のプリュウロクリシス属の微細藻からの抽出物が抗ＭＲＳＡ活性を有することを発見し、その抽出物を抗ＭＲＳＡ組成物として有効に広く利用できるようにしたものである。 In any case, in the present invention, it has been discovered that an extract from a microalga of the genus Pleuurocrisis of the Haptophyceae isocratic order has anti-MRSA activity, and the extract is effectively and widely used as an anti-MRSA composition. It is made available.
Claims (3)
を特徴とする抗ＭＲＳＡ組成物。 An anti-MRSA composition comprising, as an active ingredient, an extract of a microalga belonging to the genus Pleuurocrisis of the haptoalgae isocrisis.
水もしくは熱水またはアルコールにより抽出されること
を特徴とする請求項１に記載の抗ＭＲＳＡ組成物。 The extract is
The anti-MRSA composition according to claim 1, which is extracted with water, hot water or alcohol.
分子量が略１００００以下の水溶性物質であること
を特徴とする請求項１または２に記載の抗ＭＲＳＡ組成物。 The extract is
The anti-MRSA composition according to claim 1 or 2, which is a water-soluble substance having a molecular weight of about 10,000 or less.
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|---|---|---|---|---|
| JPH0725780A (en) * | 1993-07-08 | 1995-01-27 | Nippon N U S Kk | Bacteriostatic/carcinostatic agent |
| JPH11228437A (en) * | 1998-02-13 | 1999-08-24 | Micro Aruje Corporation Kk | Hyaluronidase inhibitor or antimicrobial agent and cosmetic containing the same |
| JP2002173437A (en) * | 2000-12-07 | 2002-06-21 | Kochi Medical School | Medicinal composition including extract of loquat core for controlling amount of lipid in humor |
| WO2004082696A1 (en) * | 2003-03-20 | 2004-09-30 | Laboratorios Dalmer S.A. | Pharmaceutical composition and method for the treatment and prevention of prostatic hyperplasia and prostatitis using roystonea regia (royal palm) fruits |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0725780A (en) * | 1993-07-08 | 1995-01-27 | Nippon N U S Kk | Bacteriostatic/carcinostatic agent |
| JPH11228437A (en) * | 1998-02-13 | 1999-08-24 | Micro Aruje Corporation Kk | Hyaluronidase inhibitor or antimicrobial agent and cosmetic containing the same |
| JP2002173437A (en) * | 2000-12-07 | 2002-06-21 | Kochi Medical School | Medicinal composition including extract of loquat core for controlling amount of lipid in humor |
| WO2004082696A1 (en) * | 2003-03-20 | 2004-09-30 | Laboratorios Dalmer S.A. | Pharmaceutical composition and method for the treatment and prevention of prostatic hyperplasia and prostatitis using roystonea regia (royal palm) fruits |
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