JP4982337B2 - Use of effective products extracted from ginger rhizomes in treating diseases associated with Helicobacter pylori - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/906—Zingiberaceae (Ginger family)
- A61K36/9068—Zingiber, e.g. garden ginger
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
- A61K31/121—Ketones acyclic
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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Description
本発明は、胃炎、胃潰瘍または十二指腸潰瘍のようなヘリコバクター・ピロリに関連する疾患を治療する際に使用される、生姜の根茎から抽出された有効産物の使用に関する。 The present invention relates to the use of effective products extracted from ginger rhizomes used in treating diseases associated with Helicobacter pylori, such as gastritis, gastric ulcers or duodenal ulcers.
中国の生薬またはスパイス、すなわち生姜（Zingiber officinale）、クローブ（Eugeniacaryophyllata）、にんにく（Alliumsativum）は、薬や食物の味付けに用いられてきた。粗生姜は、制吐薬や去痰薬、咳止め、消化促進剤として用いられる。セミドライの古い粗生姜はまた、胃痛、胸痛、腰痛、咳、普通感冒に用いられ、また、「水浮腫（"stagnate of water".）」と呼ばれる浮腫の形態の治療用いられる。 Chinese herbal medicines or spices, namely ginger (Zingiber officinale), clove (Eugeniacaryophyllata), and garlic (Alliumsativum) have been used to season medicines and foods. Crude ginger is used as an antiemetic, expectorant, cough, and digestive enhancer. Semi-dry old crude ginger is also used for gastric pain, chest pain, low back pain, cough, common cold and is used to treat a form of edema called "stagnate of water".
ジンゲロン（Zingerone）は、生姜の辛味の特徴の主な原因となる主成分であり；ジンジャオール（gingerol）およびショーガオール（shogaol）は、生姜中の他の辛味成分である。ジンジャオールは、強心作用を有し、マウスにおける摘出された門脈（isolated portal veins）の収縮を抑制し、マウスおよびラットの血管のエイコサノイド−誘起収縮を緩和する。ジンジャオールおよびショーガオールはいずれも突然変異原性があって、生姜（ginger）およびジンゲロンは、抗変異原活性を示すことが分かってきた。 Zingerone is the main ingredient responsible for the characteristics of ginger pungent; gingerol and shogaol are other pungent ingredients in ginger. Ginjaol has a cardiotonic effect, suppresses contraction of isolated portal veins in mice and alleviates eicosanoid-induced contraction of mouse and rat blood vessels. Both gingerol and shogaol are mutagenic, and ginger and gingerone have been shown to exhibit antimutagenic activity.
ショーガオールは、カラゲニン浮腫および血小板凝集において阻害活性を有する（特許文献１；発明の背景）。特許文献２は、胃潰瘍用治療組成物およびそれを調整する工程について開示し、Aegle marmetosおよびWithania somnifraの植物部位から得られる抽出物および８つの植物からなる群から選択される少なくとも１つの部材の植物部位から得られる抽出物を含み、それの一つが生姜である。前記組成物の抽出物は、好ましくは、水性の抽出物である。 Shogaol has inhibitory activity in carrageenan edema and platelet aggregation (Patent Document 1; Background of the Invention). Patent Document 2 discloses a therapeutic composition for gastric ulcer and a process for preparing the same, and an extract obtained from plant parts of Aegle marmetos and Withania somnifra and a plant of at least one member selected from the group consisting of eight plants Contains the extract obtained from the site, one of which is ginger. The extract of the composition is preferably an aqueous extract.
特許文献３は、病気予防に有用な抗ヘリコバクターピロリの有効産物ならびに胃炎ならびに胃および十二指腸潰瘍治療を開示し、カッコウ、センブリ、オウゴン、シソシ、ウイキョウ、カンキョウ、ビャクジュツ、サンショウ、コロンボ、ショウキョウ、モッコウ、クジン、アセンヤク、アロエ及びシャクヤクからなる群から選択される一以上の本草薬の使用を含む病気予防に有用な抗ヘリコバクターピロリ有効産物ならびに胃炎ならびに胃および十二指腸潰瘍治療を開示している。 Patent Document 3 discloses an effective product of anti-Helicobacter pylori useful for disease prevention and treatment of gastritis and gastric and duodenal ulcers, including cuckoo, assembly, oxon, perilla, fennel, citrus, peanut, sansho, colombo, kyoto, Disclosed are anti-Helicobacter pylori effective products and gastritis and gastric and duodenal ulcer treatments useful for disease prevention including the use of one or more herbal medicines selected from the group consisting of mokko, kujin, asenyaku, aloe and peony.
ジンジャオールを含む生姜の根抽出物は、Ｈ．ｐｙｌｏｒｉＣａｇＡ＋ｓｔｒａｉｎｓの成長を抑制することが記載されている（非特許文献１）。
生姜の根茎から調製されるヘリコバクター・ピロリに関する疾患の治療に使用される有効産物を提供することを目的とする。 An object of the present invention is to provide an effective product for use in treating diseases related to Helicobacter pylori prepared from ginger rhizomes.
粗有機溶剤抽出物を、逆相クロマトグラフィーカラムにかけることで得られる水性の溶出液は、前記疾患を治療する際に非常に弱い効能を示し、比較的弱い極性を有する溶離剤の溶出液は向上した効能を有する。驚くべきことに、溶出液に６−ジンジャオールおよび６−ショーガオールのいずれもがなくなるような弱い極性を有する溶離剤による溶出液は、非常に強い効能を示すことを見出した。この知見は、６−ジンジャオールおよび６−ショーガオールは、辛味成分であり、比較的不安定であるため、有効なものである。 The aqueous eluate obtained by subjecting the crude organic solvent extract to a reverse phase chromatography column exhibits a very weak efficacy in treating the disease, and the eluent eluent having a relatively weak polarity is Has improved efficacy. Surprisingly, it has been found that eluates with weak eluents that are free of both 6-gingaol and 6-shogaol in the eluate are very potent. This finding is effective because 6-gingerol and 6-shogaol are pungent components and relatively unstable.
本発明において調製される有効産物は、生姜の根茎由来の水性の粗抽出物または有機溶剤粗抽出物と比較して顕著に有意な効果がある。 The effective product prepared in the present invention has a significantly significant effect as compared with an aqueous crude extract or organic solvent crude extract derived from ginger rhizome.
本発明を好適ないくつかの実施形態について詳細に説明する。 The invention will now be described in detail with reference to a few preferred embodiments.
本発明の好ましい実施形態は、これに制限されるものでないが、以下を含む。 Preferred embodiments of the present invention include, but are not limited to:
１． 生姜の根茎由来の、治療上有効な量の有効産物を含むヘリコバクター・ピロリが関連する疾患の治療用薬剤組成物であって、
前記有効産物が、下記段階を含む工程：
ａ）６−ジンジャオールおよび６−ショーガオールを含む、生姜の根茎由来の粗抽出物を調製する；
ｂ）粗抽出物を、逆相クロマトグラフィーカラムまたは順相クロマトグラフィーカラムに導入し、第１溶離剤、第２溶離剤の順でカラムから溶出し、この際、ｉ）逆相クロマトグラフィーカラムを使用するときは、前記第１溶離剤は、水よりも弱い極性を有し、および、前記第２溶離剤は、第１溶離剤よりも弱い極性を有し、または、ｉｉ）順相クロマトグラフィーカラムを用いるときは、前記第２溶離剤は、酢酸エチルおよびメタノールの混合物（１：１の質量比）よりも弱い極性を有し、および、前記第１溶離剤は、第２溶離剤よりも弱い極性を有し、第１溶離剤の溶出により第１溶出液および第２溶離剤の溶出により第２溶出液を得；さらに、
ｃ）エバポレーションにより、第１溶出液から第１溶離剤を除去し、第１濃縮溶出液を得、有効産物として使用し、または、エバポレーションにより、第２溶出液から第２溶離剤を除去し、第２濃縮溶出液を得、有効産物として使用し；
前記段階ａ）は、段階ｉ）〜ｉｖ）、または、段階Ｉ）、段階Ｉ’）もしくは段階Ｉ”）を含み、
前記段階ｉ）〜ｉｖ）は；
ｉ）新鮮な生姜の根茎をすりおろし、その結果生じた混合物をろ過し、ろ液および残渣を得；
ｉｉ）第１有機溶剤により、前記ろ液を抽出し、前記抽出溶液を回収し、前記抽出溶液から第１有機溶剤を蒸発させ、第１濃縮抽出溶液を得；
ｉｉｉ）第２有機溶剤により残渣を抽出し、前記抽出溶液を回収し、前記抽出溶液から第２有機溶剤を蒸発させ、第２濃縮抽出溶液を得；さらに、
ｉｖ）前記第１濃縮抽出溶液および前記第２濃縮抽出溶液を混ぜ、粗抽出物溶液を得；
前記段階Ｉ）は：
Ｉ）前記第２有機溶剤と同じ有機溶剤で、乾燥した生姜の根茎を抽出し、前記抽出溶液を回収し、前記抽出溶液から前記有機溶剤を蒸発させ、粗抽出物を得；
前記段階Ｉ’）は：
Ｉ’）乾燥した生姜の根茎を水蒸気蒸留し、エバポレーションにより生じる蒸留液を濃縮して、粗抽出物を得；
前記段階Ｉ”）は：
Ｉ”）超臨界ＣＯ２にて生姜の根茎の乾燥粉末を抽出し、前記抽出溶液を回収し、前記抽出溶液からＣＯ２をエバポレーションして、粗抽出物を得る、
により調製される、薬剤組成物。
1. A pharmaceutical composition for the treatment of diseases associated with Helicobacter pylori comprising a therapeutically effective amount of an effective product derived from ginger rhizomes,
The effective product comprises the following steps:
a) preparing a crude extract from ginger rhizomes containing 6-jinjaol and 6-shogaol;
b) The crude extract is introduced into a reverse phase chromatography column or a normal phase chromatography column and eluted from the column in the order of the first eluent and the second eluent, i) the reverse phase chromatography column When used, the first eluent has a weaker polarity than water and the second eluent has a weaker polarity than the first eluent, or ii) normal phase chromatography When using a column, the second eluent has a weaker polarity than a mixture of ethyl acetate and methanol (1: 1 mass ratio), and the first eluent is more than the second eluent. Having a weak polarity, elution of the first eluent provides a first eluate and a second eluent by elution of the second eluent;
c) The first eluent is removed from the first eluate by evaporation to obtain the first concentrated eluate and used as an effective product, or the second eluent is removed from the second eluate by evaporation. A second concentrated eluate is obtained and used as an effective product;
Said step a) comprises steps i) to iv) or step I), step I ′) or step I ″),
Said steps i) to iv) are:
i) grate fresh ginger rhizomes and filter the resulting mixture to obtain filtrate and residue;
ii) extracting the filtrate with a first organic solvent, recovering the extracted solution, evaporating the first organic solvent from the extracted solution to obtain a first concentrated extracted solution;
iii) extracting the residue with a second organic solvent, collecting the extracted solution, evaporating the second organic solvent from the extracted solution to obtain a second concentrated extracted solution;
iv) mixing the first concentrated extract solution and the second concentrated extract solution to obtain a crude extract solution;
Said step I) is:
I) Extracting dried ginger rhizomes with the same organic solvent as the second organic solvent, collecting the extracted solution, and evaporating the organic solvent from the extracted solution to obtain a crude extract;
Said step I ′) is:
I ′) steaming the dried ginger rhizome and concentrating the distillate produced by evaporation to obtain a crude extract;
Said step I ") is:
I ″) Extracting ginger rhizome dry powder with supercritical CO 2 , recovering the extracted solution, and evaporating CO 2 from the extracted solution to obtain a crude extract,
A pharmaceutical composition prepared by
２．前記疾患は胃潰瘍である、上記１に係る薬剤組成物。 2. 2. The pharmaceutical composition according to 1 above, wherein the disease is gastric ulcer.
３．前記粗抽出物は、段階ｉ）〜ｉｖ）を含む方法により調製される、上記１または２に係る薬剤組成物。 3. The pharmaceutical composition according to 1 or 2 above, wherein the crude extract is prepared by a method comprising steps i) to iv).
４．前記第１有機溶剤が、エチルエーテルで、前記第２有機溶剤が、アセトン、メタノール、エタノール、メタノールおよび水の混合物、エタノールおよび水の混合物またはこれらの組み合わせである、上記３に係る薬剤組成物。 4). 4. The pharmaceutical composition according to 3 above, wherein the first organic solvent is ethyl ether, and the second organic solvent is acetone, methanol, ethanol, a mixture of methanol and water, a mixture of ethanol and water, or a combination thereof.
５．前記第２有機溶剤が、アセトンである、上記４に係る薬剤組成物。 5). 4. The pharmaceutical composition according to 4 above, wherein the second organic solvent is acetone.
６．前記粗抽出物が、段階Ｉ）を含む方法により調製される、上記１または２に係る薬剤組成物。 6). The pharmaceutical composition according to 1 or 2 above, wherein the crude extract is prepared by a method comprising step I).
７．前記有機溶剤が、アセトン、メタノール、エタノール、メタノールおよび水の混合物、エタノールおよび水の混合物またはこれらの組み合わせである上記６に係る薬剤組成物。 7). 7. The pharmaceutical composition according to 6 above, wherein the organic solvent is acetone, methanol, ethanol, a mixture of methanol and water, a mixture of ethanol and water, or a combination thereof.
８．前記有機溶剤が、アセトン、エタノールまたはエタノールおよび水の混合物である、上記７に係る薬剤組成物。 8). 8. The pharmaceutical composition according to 7 above, wherein the organic solvent is acetone, ethanol or a mixture of ethanol and water.
９．前記逆相クロマトグラフィーカラムを用いるときに、第１溶離剤が、エタノール４０質量％を含むエタノールおよび水の混合物よりも弱い極性を有する、上記１に係る薬剤組成物。 9. The pharmaceutical composition according to 1 above, wherein when using the reverse phase chromatography column, the first eluent has a weaker polarity than a mixture of ethanol and water containing 40% by mass of ethanol.
１０．前記第１溶離剤が、メタノール、メタノールおよび水の混合物、エタノール、エタノールおよび水の混合物、アセトンおよび水の混合物またはこれらの組み合わせである、上記９に係る薬剤組成物。 10. 10. The pharmaceutical composition according to 9 above, wherein the first eluent is methanol, a mixture of methanol and water, ethanol, a mixture of ethanol and water, a mixture of acetone and water, or a combination thereof.
１１．前記第１溶離剤が、エタノールおよび水の混合物である、上記１０に係る薬剤組成物。 11. The pharmaceutical composition according to 10 above, wherein the first eluent is a mixture of ethanol and water.
１２．前記逆相クロマトグラフィーカラムが、第１溶離剤の前に水またはエタノール２０質量％を含むエタノールおよび水の混合物で溶出される、上記１１に係る薬剤組成物。 12 12. The pharmaceutical composition according to 11 above, wherein the reverse phase chromatography column is eluted with water or a mixture of ethanol and water containing 20% by mass of ethanol before the first eluent.
１３．前記有効産物は、前記第１濃縮溶出液であり、前記第１濃縮溶出液が、６−ジンジャオールおよび６−ショーガオールを含む、上記９に係る薬剤組成物。 13. 10. The pharmaceutical composition according to 9 above, wherein the effective product is the first concentrated eluate, and the first concentrated eluate contains 6-gingerol and 6-shogaol.
１４．逆相クロマトグラフィーカラムを用いるときに、前記有効産物は、前記第２濃縮溶出液であり、第１溶離剤が、エタノール４０質量％を含むエタノールおよび水の混合物よりも弱い極性を有する、上記１、１０、１１または１２に係る薬剤組成物。 14 When using a reverse-phase chromatography column, the effective product is the second concentrated eluent, and the first eluent has a weaker polarity than a mixture of ethanol and water containing 40% by mass of ethanol. A pharmaceutical composition according to 10, 11, or 12.
１５．前記第２溶離剤は、アセトン、アセトンおよび水の混合物、アセトンおよびメタノールの混合物、アセトンおよびエタノールの混合物、アセトンおよび炭素数４〜６のアルカンの混合物、炭素数４〜６のアルカン、メタノールおよび炭素数４〜６のアルカンの混合物、エタノールおよび炭素数４〜６のアルカンの混合物、またはこれらの組み合わせである、上記１４に係る薬剤組成物。 15. The second eluent is acetone, a mixture of acetone and water, a mixture of acetone and methanol, a mixture of acetone and ethanol, a mixture of acetone and an alkane having 4 to 6 carbon atoms, an alkane having 4 to 6 carbon atoms, methanol and carbon. The pharmaceutical composition according to 14 above, which is a mixture of alkanes having 4 to 6 carbon atoms, a mixture of ethanol and alkanes having 4 to 6 carbon atoms, or a combination thereof.
１６．前記第２溶離剤が、アセトンである、上記１５に係る薬剤組成物。 16. 16. The pharmaceutical composition according to 15 above, wherein the second eluent is acetone.
１７．前記第２濃縮溶出液に、ジンジャオールまたは６−ショーガオールが存在しない、上記１４に係る薬剤組成物。なお、「ジンジャオールまたは６−ショーガオールが存在しない」とは、「全く存在しない」との概念のみならず、「実質的にジンジャオールまたは６−ショーガオールが存在しない」との概念をも含む。具体的には、下記実施例で規定されているＨＰＬＣ分析の検出限界を超える値を意味する。 17. 14. The pharmaceutical composition according to 14 above, wherein no gingerol or 6-shogaol is present in the second concentrated eluate. Note that “there is no ginger or 6-shogaol” includes not only the concept of “not at all” but also the concept of “there is substantially no gingerol or 6-shogaol”. . Specifically, it means a value exceeding the detection limit of HPLC analysis defined in the following examples.
１８．前記第２濃縮溶出液に、６−ジンジャオールおよび６−ショーガオールいずれも存在しない、上記１４に係る薬剤組成物。なお、「６−ジンジャオールおよび６−ショーガオールいずれも存在しない」とは、上記と同様、「全く存在しない」との概念のみならず、「実質的にジンジャオールまたは６−ショーガオールが存在しない」との概念をも含む。具体的には、下記実施例で規定されているＨＰＬＣ分析の検出限界を超える値を意味する。 18. 14. The pharmaceutical composition according to 14 above, wherein neither the 6-gingaol nor 6-shogaol is present in the second concentrated eluate. “No 6-gingaol and 6-shogaol” is not only the concept of “not at all” as described above, but “substantially no gingerol or 6-shogaol is present”. Including the concept of "." Specifically, it means a value exceeding the detection limit of HPLC analysis defined in the following examples.
１９．前記順相クロマトグラフィーカラムを用いるときに、前記有効産物が、前記第１濃縮溶出液であり、前記第１溶離剤が、ｎ−ヘキサンおよび酢酸エチルの混合物（質量比６：４）よりも極性が弱い、上記１に係る薬剤組成物。 19. When the normal phase chromatography column is used, the effective product is the first concentrated eluent, and the first eluent is more polar than a mixture of n-hexane and ethyl acetate (mass ratio 6: 4). The pharmaceutical composition according to 1 above, which is weak.
２０．前記第１溶離剤が、ｎ−ヘキサンおよび酢酸エチルの混合物（質量比９：１）と近い極性を有する、上記１９に係る薬剤組成物。 20. 20. The pharmaceutical composition according to 19 above, wherein the first eluent has a polarity close to that of a mixture of n-hexane and ethyl acetate (mass ratio 9: 1).
２１．第１溶離剤が、ｎ−ヘキサンおよび酢酸エチルの混合物（質量比９：１）である、上記１９に係る薬剤組成物。 21. 20. The pharmaceutical composition according to 19 above, wherein the first eluent is a mixture of n-hexane and ethyl acetate (mass ratio 9: 1).
２２．前記第１濃縮溶出液に、６−ジンジャオールまたは６−ショーガオールが存在しない、上記１９に係る薬剤組成物。 22. 19. The pharmaceutical composition according to 19 above, wherein 6-gingerol or 6-shogaol is not present in the first concentrated eluate.
２３．前記第１濃縮溶出液に、６−ジンジャオールおよび６−ショーガオールのいずれも存在しない、上記１９に係る薬剤組成物。 23. 19. The pharmaceutical composition according to 19 above, wherein neither the 6-ginjaol nor 6-shogaol is present in the first concentrated eluate.
２４．前記順相クロマトグラフィーカラムを用いるときに、前記有効産物が、前記第２濃縮溶出液であり、前記第２溶離剤が、ｎ−ヘキサンおよび酢酸エチルの混合物（質量比６：４）の極性と近いまたは同じ、そして、前記第２濃縮溶出液が、６−ジンジャオールおよび６−ショーガオールを含む、上記１に係る薬剤組成物。 24. When using the normal phase chromatography column, the effective product is the second concentrated eluent, and the second eluent is the polarity of a mixture of n-hexane and ethyl acetate (mass ratio 6: 4). The pharmaceutical composition according to 1 above, wherein the second concentrated eluate is close or the same and comprises 6-gingerol and 6-shogaol.
２５．前記第２溶離剤は、ｎ−ヘキサンおよび酢酸エチルの混合物（質量比６：４）である、上記２４に係る薬剤組成物。 25. The pharmaceutical composition according to 24 above, wherein the second eluent is a mixture (mass ratio 6: 4) of n-hexane and ethyl acetate.
２６．前記薬剤組成物は、さらに、希釈剤、賦形剤または担体を含んでもよい。 26. The pharmaceutical composition may further comprise a diluent, excipient or carrier.
上記発明は、生姜の根茎由来の、治療上有効な量の有効産物を含むヘリコバクター・ピロリが関連する疾患の治療用薬剤組成物の製造方法としても、好ましい。 The above invention is also preferred as a method for producing a pharmaceutical composition for treating diseases associated with Helicobacter pylori comprising a therapeutically effective amount of an effective product derived from ginger rhizomes.
本願出願人は、イギリス特許第２３６６５６５号において、消炎、抗血小板凝集および抗真菌活性に有効な生姜由来の抽出物を調製する方法であって、下記段階を含むものを開示する：
有機溶剤もしくは超臨界ＣＯ２または蒸気による蒸留によって抽出された生姜の根茎由来の粗液を調製し；
前記粗液を、逆相クロマトグラフィーカラムに導入し、水、第１溶離剤、第１溶離剤よりも低い極性を有するがクロロホルムよりも高い極性を有する第２溶離剤により、カラムで溶出し、第１溶離剤の溶離により第１溶出液と、第２溶離剤の溶離により第２溶出液と、を得；
エバポレーションにて、第１溶出液および第２溶出液から、それぞれ、第１溶離剤および第２溶離剤を除去し、有効な抽出物として、第１濃縮溶出液および第２濃縮溶出液を得る。
Applicant discloses in GB 2366565 a method for preparing ginger-derived extracts effective for anti-inflammatory, antiplatelet aggregation and antifungal activity comprising the following steps:
Preparing a crude liquid derived from ginger rhizomes extracted by distillation with organic solvent or supercritical CO 2 or steam;
The crude liquid is introduced into a reverse phase chromatography column and eluted from the column with water, a first eluent, a second eluent having a polarity lower than that of the first eluent but higher than that of chloroform, Obtaining a first eluent by elution of the first eluent and a second eluent by elution of the second eluent;
By evaporation, the first eluent and the second eluent are removed from the first eluate and the second eluate, respectively, and the first concentrated eluate and the second concentrated eluate are obtained as effective extracts. .
本発明をさらに実施例を以って詳しく説明するが、これに制限されるものではない。なお、パーセントおよび他の量は、特に記載がなければ、すべて質量とする。また、全量は１００％とする。 The present invention will be described in more detail with reference to examples, but is not limited thereto. All percentages and other amounts are by weight unless otherwise specified. The total amount is 100%.
［実施例１］
スライスし陰干した生姜の根茎を粉砕し、篩の目開きメッシュＮｏ．１０の篩に通した。その結果生じた粉末 １質量部を、９５％エタノール ８質量部と混合し、その結果生じた混合物を、１時間、還流下で沸騰させ、ろ過し、ろ液Ａと残渣を得た。前記残渣を９５％エタノールと混ぜ（質量比１：８）、その結果生じた混合物を、１時間還流下で沸騰させ、ろ過し、ろ液Ｂを得た。
[Example 1]
Sliced and dried ginger rhizomes were crushed and sieved mesh No. Passed through 10 sieves. 1 part by weight of the resulting powder was mixed with 8 parts by weight of 95% ethanol, and the resulting mixture was boiled under reflux for 1 hour and filtered to obtain filtrate A and a residue. The residue was mixed with 95% ethanol (mass ratio 1: 8), and the resulting mixture was boiled under reflux for 1 hour and filtered to obtain filtrate B.
ろ液Ａおよびろ液Ｂを合わせ、７０℃のウォーターバスで濃縮し、「ＲｏｔａｖａｐｏｒＲ−２２０，ＢＵＣＨＩ，Ｓｗｉｔｚｅｒｌａｎｄ」にて真空にして濃縮した。その結果生じた濃縮物を、前記濃縮物の乾燥質量の２０倍の量のＨＰ−２０樹脂（Ｍｉｔｓｕｂｉｓｈｉ，Ｊａｐａｎ）Ｄｉａｉｏｎ（登録商標）を充填した逆相クロマトグラフィーカラム（７．１ｃｍ×９０ｃｍ）に導入し、「水 ５ベッドボリューム、４０％エタノール ４ベッドボリュームおよびアセトン ４ベッドボリューム」をこの順で抽出した。
The filtrate A and the filtrate B were combined, concentrated in a water bath at 70 ° C., and concentrated in a vacuum using “Rotavapor R-220, BUCHI, Switzerland”. The resulting concentrate was loaded onto a reverse phase chromatography column (7.1 cm × 90 cm) packed with HP-20 resin (Mitsubishi, Japan)
４０％エタノールで溶出した溶出液およびアセトンで溶出した溶出液を、それぞれ回収した。 The eluate eluted with 40% ethanol and the eluate eluted with acetone were collected respectively.
４０％エタノールで溶出した溶出液を、「Ｒｏｔａｖａｐｏｒ Ｒ−２２０，ＢＵＣＨＩ，Ｓｗｉｔｚｅｒｌａｎｄ」にて真空濃縮し、次いで、８０℃ウォーターバスで１時間再度濃縮し、１時間真空濃縮（４０ｍｂａｒ）し、サンプル１を得た。 The eluate eluted with 40% ethanol was concentrated in vacuo with “Rotavapor R-220, BUCHI, Switzerland”, then concentrated again in an 80 ° C. water bath for 1 hour, and concentrated in vacuo (40 mbar) for 1 hour. Got.
アセトンで溶出した溶出液を、「Ｒｏｔａｖａｐｏｒ Ｒ−２２０，ＢＵＣＨＩ，Ｓｗｉｔｚｅｒｌａｎｄ」にて真空濃縮し、８０℃ウォーターバスで再度濃縮し、１時間真空濃縮（４０ｍｂａｒ）し、サンプル２を得た。 The eluate eluted with acetone was concentrated in vacuo with “Rotavapor R-220, BUCHI, Switzerland”, concentrated again in an 80 ° C. water bath, and concentrated in vacuo (40 mbar) for 1 hour to obtain Sample 2.
［実施例２］
４０％エタノール溶離剤を７０％エタノールに置き換えた以外は、実施例１の手順と同様に行った。
[Example 2]
The procedure was as in Example 1, except that the 40% ethanol eluent was replaced with 70% ethanol.
サンプル３は、７０％エタノールで溶出することにより得、サンプル４は、アセトンで溶出することにより得た。
Sample 3 was obtained by eluting with 70% ethanol and
［実施例３］
４０％エタノールの溶離剤を９５％エタノールに置き換えた以外は、実施例１の手順と同様に行い、サンプル５を得た。
[Example 3]
Sample 5 was obtained in the same manner as in Example 1 except that the eluent of 40% ethanol was replaced with 95% ethanol.
［実施例４］
スライスし陰干した生姜の根茎を粉砕し、篩の目開きメッシュＮｏ．１０に通した。その結果生じた粉末 １質量部を、９５％エタノール ８質量部と混合し、その結果生じた混合物を、１時間還流下で沸騰させ、ろ過し、ろ液Ａと残渣を得た。
[Example 4]
Sliced and dried ginger rhizomes were crushed and sieved mesh No. 10 was passed. 1 part by weight of the resulting powder was mixed with 8 parts by weight of 95% ethanol, and the resulting mixture was boiled under reflux for 1 hour and filtered to obtain filtrate A and a residue.
前記残渣を、９５％エタノールに混合し（質量比１：８）、その結果生じた混合物を、１時間還流下で沸騰させ、ろ過し、ろ液Ｂを得た。 The residue was mixed with 95% ethanol (mass ratio 1: 8), and the resulting mixture was boiled under reflux for 1 hour and filtered to obtain filtrate B.
ろ液Ａおよびろ液Ｂを合わせ、７０℃ウォーターバスにて濃縮し、「Ｒｏｔａｖａｐｏｒ Ｒ−２２０，ＢＵＣＨＩ，Ｓｗｉｔｚｅｒｌａｎｄ」にて真空濃縮し、当初の１／５の質量になるようにした。 Filtrate A and filtrate B were combined, concentrated in a 70 ° C. water bath, and concentrated in vacuo with “Rotavapor R-220, BUCHI, Switzerland” to the original mass of 1/5.
その結果生じた濃縮物の４倍の質量の水を、当初の質量に戻るように加え、その結果生じた液体を、逆相クロマトグラフィーカラム（７．１ｃｍ×９０ｃｍ）にかけ、前記濃縮物の乾燥質量の２０倍に相当するＤｉａｉｏｎ（登録商標）を充填した逆相クロマトグラフィーカラム（７．１ｃｍ×９０ｃｍ）ＨＰ−２０樹脂（Ｍｉｔｓｕｂｉｓｈｉ，Ｊａｐａｎ）に導入し、「２０％ エタノール １ベッドボリュームおよび７０％エタノール ４ベッドボリューム」の順で溶離した。
Four times as much water as the resulting concentrate is added back to the original mass, and the resulting liquid is applied to a reverse phase chromatography column (7.1 cm × 90 cm) to dry the concentrate. It was introduced into a reverse phase chromatography column (7.1 cm × 90 cm) HP-20 resin (Mitsubishi, Japan) packed with Diaion® corresponding to 20 times the mass, “20% ethanol 1 bed volume and 70%. Elution was performed in the order of “
７０％エタノールで溶出した溶出液を回収し、「ＲｏｔａｖａｐｏｒＲ−２２０，ＢＵＣＨＩ，Ｓｗｉｔｚｅｒｌａｎｄ」にて真空濃縮し、８０℃ウォーターバスで再度濃縮し、１時間真空濃縮（４０ｍｂａｒ）し、サンプル６を得た。 The eluate eluted with 70% ethanol was collected, concentrated in vacuo with “Rotavapor R-220, BUCHI, Switzerland”, concentrated again in an 80 ° C. water bath, and concentrated in vacuo (40 mbar) for 1 hour to obtain Sample 6. .
［実施例５］
７０％エタノール溶離剤を９５％エタノールに置き換えた以外は、実施例４の手順と同様の方法を行い、サンプル７を得た。
[Example 5]
Sample 7 was obtained in the same manner as in Example 4 except that the 70% ethanol eluent was replaced with 95% ethanol.
［実施例６］
スライスし陰干した生姜の根茎を粉砕し、篩の目開きメッシュＮｏ．１０に通した。その結果生じた粉末 １質量部を、９５％エタノール ８質量部と混合させ、その結果生じた混合物を、ホモゲナイザー ２０００ｒｐｍ（ＴｙｐｅＸ５０／１０，Ｙｓｔｒａｌ，Ｇｅｒｍａｎｙ）で、１時間で攪拌し、ろ過し、ろ液Ａおよび残渣を得た。
[Example 6]
Sliced and dried ginger rhizomes were crushed and sieved mesh No. 10 was passed. 1 part by weight of the resulting powder was mixed with 8 parts by weight of 95% ethanol, and the resulting mixture was stirred at a
前記残渣を、９５％エタノールと混合し（質量比１：８）、その結果生じた混合物を、１時間、ホモゲナイザー ２０００ｒｐｍで攪拌し、ろ過し、ろ液Ｂを得た。 The residue was mixed with 95% ethanol (mass ratio 1: 8), and the resulting mixture was stirred for 1 hour with a homogenizer at 2000 rpm and filtered to obtain filtrate B.
ろ液Ａおよびろ液Ｂを合わせ、７０℃ウォーターバスにて濃縮し、「Ｒｏｔａｖａｐｏｒ Ｒ−２２０，ＢＵＣＨＩ，Ｓｗｉｔｚｅｒｌａｎｄ」にて真空濃縮し、当初の質量の１／５にした。その結果生じた濃縮物の質量の４倍の水を、当初の質量に戻るように加えた。 Filtrate A and filtrate B were combined, concentrated in a 70 ° C. water bath, and concentrated in vacuo with “Rotavapor R-220, BUCHI, Switzerland” to 1/5 of the initial mass. Four times the mass of the resulting concentrate was added to return to the original mass.
その結果生じた液体を、前記濃縮物の乾燥質量の２０倍の量のＤｉａｉｏｎ（登録商標）ＨＰ−２０（Ｍｉｔｓｕｂｉｓｈｉ，Ｊａｐａｎ）樹脂を充填した逆相クロマトグラフィーカラム（７．１ｃｍ×９０ｃｍ）に導入し、「２０％エタノール １ベッドボリュームおよび７０％エタノール ４ベッドボリューム」の順で溶離した。
The resulting liquid was introduced into a reverse phase chromatography column (7.1 cm × 90 cm) packed with Diaion® HP-20 (Mitsubishi, Japan)
７０％エタノールで溶出した溶出液を回収し、「Ｒｏｔａｖａｐｏｒ Ｒ−２２０，ＢＵＣＨＩ，Ｓｗｉｔｚｅｒｌａｎｄ」にて真空濃縮し、８０℃ウォーターバスで濃縮し、１時間真空濃縮（４０ｍｂａｒ）し、サンプル８を得た。 The eluate eluted with 70% ethanol was collected, concentrated in vacuo with “Rotavapor R-220, BUCHI, Switzerland”, concentrated in an 80 ° C. water bath, and concentrated in vacuo (40 mbar) for 1 hour to obtain Sample 8. .
［実施例７］
７０％エタノールの溶離剤を９５％エタノールに置き換えた以外は、実施例６の手順の同様の方法でサンプル９を得た。
[Example 7]
［実施例８］
スライスし陰干した生姜の根茎を粉砕し、篩の目開きメッシュＮｏ．１０の篩に通した。その結果生じた粉末 １質量部を、アセトン ８質量部と混合し、その結果生じた混合物を、１時間還流下で沸騰させ、ろ過し、ろ液Ａおよび残渣を得た。
[Example 8]
Sliced and dried ginger rhizomes were crushed and sieved mesh No. Passed through 10 sieves. 1 part by mass of the resulting powder was mixed with 8 parts by mass of acetone, and the resulting mixture was boiled under reflux for 1 hour and filtered to obtain filtrate A and a residue.
前記残渣を、アセトンに混合し（質量比 １：８）、その結果生じた混合物を、１時間還流下で、沸騰させ、ろ過し、ろ液Ｂを得た。 The residue was mixed with acetone (mass ratio 1: 8), and the resulting mixture was boiled under reflux for 1 hour and filtered to obtain filtrate B.
ろ液Ａおよびろ液Ｂを合わせ、７０℃ウォーターバスで濃縮し、「Ｒｏｔａｖａｐｏｒ Ｒ−２２０，ＢＵＣＨＩ，Ｓｗｉｔｚｅｒｌａｎｄ」にて真空濃縮した。 Filtrate A and filtrate B were combined, concentrated in a 70 ° C. water bath, and concentrated in vacuo with “Rotavapor R-220, BUCHI, Switzerland”.
その結果生じた濃縮物を、前記濃縮物の乾燥質量の２０倍の量のＤｉａｉｏｎ（登録商標）ＨＰ−２０樹脂（Ｍｉｔｓｕｂｉｓｈｉ，Ｊａｐａｎ）が充填した逆相クロマトグラフィーカラム（７．１ｃｍｘ９０ｃｍ）に導入し、「水 ５ベッドボリューム、４０％エタノール ４ベッドボリュームおよびアセトン ２ベッドボリューム」の順で溶離した。
The resulting concentrate was introduced into a reverse phase chromatography column (7.1 cm × 90 cm) packed with Diaion® HP-20 resin (Mitsubishi, Japan) 20 times the dry mass of the concentrate. , "Water 5 bed volume, 40
アセトンで溶出した溶出液を回収し、「Ｒｏｔａｖａｐｏｒ Ｒ−２２０，ＢＵＣＨＩ，Ｓｗｉｔｚｅｒｌａｎｄ」にて真空乾燥し、８０℃ウォーターバスで再度濃縮し、１時間真空濃縮（４０ｍｂａｒ）し、サンプル１０を得た。
The eluate eluted with acetone was collected, vacuum-dried with “Rotavapor R-220, BUCHI, Switzerland”, concentrated again with an 80 ° C. water bath, and vacuum concentrated (40 mbar) for 1 hour to obtain
［実施例９］
４０％エタノールの溶離剤を７０％エタノールに置き換えた以外は、実施例８の手順と同様に行いサンプル１１を得た。７０％エタノールで溶出した溶出液を回収し、「Ｒｏｔａｖａｐｏｒ Ｒ−２２０，ＢＵＣＨＩ，Ｓｗｉｔｚｅｒｌａｎｄ」にて真空濃縮し、８０℃ウォーターバスで再度濃縮し、１時間真空濃縮（４０ｍｂａｒ）し、サンプル１１を得た。
[Example 9]
Sample 11 was obtained in the same manner as in Example 8, except that the eluent of 40% ethanol was replaced with 70% ethanol. The eluate eluted with 70% ethanol is collected, concentrated in vacuo with “Rotavapor R-220, BUCHI, Switzerland”, concentrated again in an 80 ° C. water bath, and concentrated in vacuo (40 mbar) for 1 hour to obtain Sample 11. It was.
［実施例１０］
粉末および残渣を、アセトンの代わりに酢酸エチルで沸騰させた（還流させた）以外は、実施例８の手順と同様に行い、サンプル１２を得た。
[Example 10]
［実施例１１］
粉末および残渣を、アセトンの代わりに酢酸エチルで沸騰させた（還流させた）以外は、実施例９の手順と同様の方法を行い、サンプル１３を得た。
[Example 11]
Sample 13 was obtained in the same manner as in Example 9 except that the powder and the residue were boiled (refluxed) with ethyl acetate instead of acetone.
［実施例１２］
スライスし陰干した生姜の根茎を粉砕し、篩の目開きメッシュＮｏ．１０の篩に通した。その結果生じた粉末 １質量部を、９５％エタノール ８質量部と混合し、その結果生じた混合物を、１時間還流下で沸騰し、ろ過し、ろ液Ａおよび残渣を得た。前記残渣を、９５％エタノールに混合し（質量比１：８）、ろ液Ｂを得た。
[Example 12]
Sliced and dried ginger rhizomes were crushed and sieved mesh No. Passed through 10 sieves. 1 part by weight of the resulting powder was mixed with 8 parts by weight of 95% ethanol, and the resulting mixture was boiled under reflux for 1 hour and filtered to obtain filtrate A and a residue. The residue was mixed with 95% ethanol (mass ratio 1: 8) to obtain filtrate B.
ろ液Ａおよびろ液Ｂを合わせ、７０℃ウォーターバスで濃縮し、「Ｒｏｔａｖａｐｏｒ Ｒ−２２０，ＢＵＣＨＩ，Ｓｗｉｔｚｅｒｌａｎｄ」にて真空濃縮した。 Filtrate A and filtrate B were combined, concentrated in a 70 ° C. water bath, and concentrated in vacuo with “Rotavapor R-220, BUCHI, Switzerland”.
その結果生じた濃縮物を、前記濃縮物の質量の２０倍のシリカゲル６０（Ｍｅｒｃｋ，Ｇｅｒｍａｎｙ）を充填した順相クロマトグラフィーカラムに導入し、「ｎ−ヘキサン：酢酸エチルの混合溶離剤（９：１） ４ベッドボリューム、ｎ−ヘキサン：酢酸エチルの混合溶離剤（６：４） ３ベッドボリュームおよび酢酸エチル：メタノールの混合溶離剤（１：１） ２ベッドボリューム」をこの順で溶離した。 The resulting concentrate was introduced into a normal phase chromatography column packed with silica gel 60 (Merck, Germany) 20 times the mass of the concentrate and "n-hexane: ethyl acetate mixed eluent (9: 1) 4 bed volume, n-hexane: ethyl acetate mixed eluent (6: 4) 3 bed volume and ethyl acetate: methanol mixed eluent (1: 1) 2 bed volume ”were eluted in this order.
ｎ−ヘキサン：酢酸エチル（９：１）の溶離による溶出液を回収し、「Ｒｏｔａｖａｐｏｒ Ｒ−２２０，ＢＵＣＨＩ，Ｓｗｉｔｚｅｒｌａｎｄ」にて真空濃縮し、８０℃ウォーターバスにて再度濃縮し、１時間真空濃縮（４０ｍｂａｒ）し、サンプル１４を得た。
The eluate obtained by elution with n-hexane: ethyl acetate (9: 1) was collected, concentrated in vacuo with “Rotavapor R-220, BUCHI, Switzerland”, concentrated again in an 80 ° C. water bath, and concentrated in vacuo for 1 hour. (40 mbar) to obtain
サンプル１４を得るのと同様の手順により、ｎ−ヘキサン：酢酸エチル（６：４）、酢酸エチル：メタノール（１：１）により、サンプル１５およびサンプル１６を得た。
Sample 15 and sample 16 were obtained by n-hexane: ethyl acetate (6: 4) and ethyl acetate: methanol (1: 1) by the same procedure as that for obtaining
［実施例１３］
９５％エタノールをアセトンに置き換えた以外は、実施例１２の手順と同様に行った。
[Example 13]
The procedure was as in Example 12, except that 95% ethanol was replaced with acetone.
サンプル１７、サンプル１８およびサンプル１９は、「ｎ−ヘキサン：酢酸エチル＝９：１、ｎ−ヘキサン：酢酸エチル＝６：４，および酢酸エチル：メタノール＝１：１」でそれぞれ溶離して得た。 Samples 17, 18 and 19 were obtained by eluting with “n-hexane: ethyl acetate = 9: 1, n-hexane: ethyl acetate = 6: 4, and ethyl acetate: methanol = 1: 1”, respectively. .
［実施例１４］
スライスし陰干した生姜の根茎を粉砕し、篩の目開きメッシュＮｏ．１０に通した。その結果生じた粉末 １質量部を、９５％エタノール ８質量部と混合し、その結果生じた混合物を、１時間還流下で沸騰し、ろ過し、ろ液Ａおよび残渣を得た。
[Example 14]
Sliced and dried ginger rhizomes were crushed and sieved mesh No. 10 was passed. 1 part by weight of the resulting powder was mixed with 8 parts by weight of 95% ethanol, and the resulting mixture was boiled under reflux for 1 hour and filtered to obtain filtrate A and a residue.
前記残渣を、９５％エタノールと混合し（質量比１：８）、その結果生じた混合物を、還流下で沸騰し、ろ過し、ろ液Ｂを得た。 The residue was mixed with 95% ethanol (mass ratio 1: 8), and the resulting mixture was boiled under reflux and filtered to obtain filtrate B.
ろ液Ａおよびろ液Ｂを合わせ、薄膜バキュームエバポレーター（ＣＥＰ−Ｌ，ＯｋａｗａｒａＭｆｇ．Ｃｏ．，Ｊａｐａｎ）にて濃縮し、当初の質量の１／５にした。 The filtrate A and the filtrate B were combined and concentrated with a thin film vacuum evaporator (CEP-L, Okawara Mfg. Co., Japan) to make 1/5 of the initial mass.
その結果生じた濃縮物を、前記濃縮物の乾燥質量の２０倍の量のＤｉａｉｏｎ（登録商標）ＨＰ−２０樹脂（Ｍｉｔｓｕｂｉｓｈｉ，Ｊａｐａｎ）を充填した逆相クロマトグラフィーカラム（３４．５ｃｍ×１００ｃｍ）に導入し、「９５％エタノール ２ベッドボリュームおよびアセトン ２ベッドボリューム」を順に、溶離した。 The resulting concentrate was loaded onto a reverse phase chromatography column (34.5 cm × 100 cm) packed with Diaion® HP-20 resin (Mitsubishi, Japan) 20 times the dry mass of the concentrate. And 95% ethanol 2 bed volume and acetone 2 bed volume were sequentially eluted.
アセトンで溶出した溶出液を回収し、「Ｒｏｔａｖａｐｏｒ Ｒ−２２０，ＢＵＣＨＩ，Ｓｗｉｔｚｅｒｌａｎｄ」にて真空濃縮し、８０℃ウォーターバスで再度濃縮し、１時間真空濃縮（４０ｍｂａｒ）し、サンプル２０を得た。
The eluate eluted with acetone was collected, concentrated in vacuo with “Rotavapor R-220, BUCHI, Switzerland”, concentrated again in an 80 ° C. water bath, and concentrated in vacuo (40 mbar) for 1 hour to obtain
ＨＰＬＣ分析サンプル調製：
サンプルを秤量し、サンプルボトルに入れ、１０ｍｇ／ｍｌの濃度のサンプル溶液になるようにメタノールで希釈した。
Sample preparation for HPLC analysis:
The sample was weighed, placed in a sample bottle, and diluted with methanol to give a sample solution having a concentration of 10 mg / ml.
０．４５μｍろ紙で、サンプル溶液をろ過し、ＨＰＬＣでろ液を分析した。 The sample solution was filtered with 0.45 μm filter paper, and the filtrate was analyzed by HPLC.
上記に調製されたサンプル１〜サンプル２０のＨＰＬＣ分析の結果を、図１〜図２０に示す。 The results of the HPLC analysis of Sample 1 to Sample 20 prepared above are shown in FIGS.
サンプル中の６−ジンジャオールおよび６−ショーガオール成分（ｍｇ／ｇ）をＨＰＬＣ分析で算出し、サンプル調製の使用した抽出条件および溶離条件とともに表１にリストアップした。 The 6-gingerol and 6-shogaol components (mg / g) in the sample were calculated by HPLC analysis and listed in Table 1 along with the extraction and elution conditions used for sample preparation.
サンプル１〜１３および２０は、逆相クロマトグラフィーカラムを用いて調製した。 Samples 1-13 and 20 were prepared using reverse phase chromatography columns.
図１〜４に示すＨＰＬＣ分析の特徴と比較して、４０％エタノールで溶出した溶出液は、６−ジンジャオールよりも強い極性を有する化合物を含み（サンプル１）、７０％エタノールで溶出した溶出液は、６−ジンジャオールおよび６−ショーガオールよりも強い極性を有する化合物を含み（サンプル３）、アセトンで溶出した溶出液は、比較的弱い極性を有する残りの化合物であることが分かる（サンプル２およびサンプル４）。 Compared with the characteristics of the HPLC analysis shown in FIGS. 1 to 4, the eluate eluted with 40% ethanol contains a compound having a stronger polarity than 6-gingerol (sample 1) and eluted with 70% ethanol. The liquid contains a compound having a stronger polarity than 6-jinjaol and 6-shogaol (sample 3), and the eluate eluted with acetone is the remaining compound having a relatively weak polarity (sample) 2 and sample 4).
図６および図７を見ると、溶離剤として７０％エタノールを使用したものと比較して、９５％エタノールを使用したものは、より右側にピークが見られることが分かる。同様の傾向が、抽出条件は異なるものの、図６および７のように図８および９で観察できる。 6 and 7, it can be seen that a peak is seen on the right side when 95% ethanol is used as compared with the case where 70% ethanol is used as an eluent. A similar tendency can be observed in FIGS. 8 and 9 as in FIGS. 6 and 7, although the extraction conditions are different.
図１０および１２に示す特徴は、抽出条件は異なるが、溶離条件が同じである点、図２に示すそれと似ている。 The features shown in FIGS. 10 and 12 are similar to those shown in FIG. 2 in that the extraction conditions are different but the elution conditions are the same.
図１１および１３に示す特徴は、抽出条件は異なるが、溶離条件が同じである点、図３に示すそれと似ている。 The features shown in FIGS. 11 and 13 are similar to those shown in FIG. 3 in that the extraction conditions are different but the elution conditions are the same.
サンプル２０は、弱い極性を有する化合物を多く含み、図２０に示すように６−ジンジャオールおよび６−ショーガオールを観察することができず、それは、９５％エタノール溶離剤が、逆相クロマトグラフィーカラムから、６−ジンジャオールおよび６−ショーガオールを含む比較的強い極性を有するものを溶出したことを示唆する。
サンプル１３〜１５は、ＨＰＬＣ分析において、抽出条件が異なるが、順相クロマトグラフィーカラムの溶離条件が同じである点、サンプル１６〜１９と似ている（図１３〜１９）。 Samples 13 to 15 are similar to Samples 16 to 19 in that the extraction conditions differ in HPLC analysis, but the elution conditions of the normal phase chromatography column are the same (FIGS. 13 to 19).
ｎ−ヘキサン：酢酸エチル＝９：１溶離剤を使用すると、図１４（図１６）で示すように、サンプル１４（サンプル１６）の成分の殆どが無極性化合物であり、ｎ−ヘキサン：酢酸エチル＝６：４溶離剤を使用すると、サンプル１５（サンプル１７）は、かなりの量の６−ジンジャオールおよび６−ショーガオールを含む。 When n-hexane: ethyl acetate = 9: 1 eluent is used, as shown in FIG. 14 (FIG. 16), most of the components of Sample 14 (Sample 16) are nonpolar compounds, and n-hexane: ethyl acetate. Using = 6: 4 eluent, sample 15 (sample 17) contains significant amounts of 6-gingerol and 6-shogaol.
１．抗菌試験
ヘリコバクター・ピロリにおけるサンプルの最小（発育）阻止濃度（ＭＩＣ）は、寒天希釈法［Malanosk G.J. et al. Effect of pH variation on the susceptibility of Helicobacter pylori to three marcolide antimicrobial agents and temafloxacin. Eur. J. Clin. Microbial Infect Dis. 12: pp. 131-133, 1993.］により測定した。
1. Antibacterial test The minimum inhibitory concentration (MIC) of a sample in Helicobacter pylori is determined by the agar dilution method [Malanosk GJ et al. Effect of pH variation on the susceptibility of Helicobacter pylori to three marcolide antimicrobial agents and temafloxacin. Clin. Microbial Infect Dis. 12: pp. 131-133, 1993.].
前記検体を溶解し、ＤＭＳＯに連続的に希釈し、所望の濃度にした。 The specimen was dissolved and serially diluted in DMSO to the desired concentration.
それぞれの濃度試験のために、７％のウサギの脱線維素血液が培地されたＣｏｌｕｍｂｉａａｇａｒｂａｓｅ（Ｏｘｏｉｄ，Ｅｎｇｌａｎｄ）０．９９ｍｌ含む４８−ｗｅｌｌｐｌａｔｅに、アリコート０．０１ｍｌを加える。 For each concentration test, 0.01 ml aliquots are added to 48-well plates containing 0.99 ml of Columbia agarbase (Oxoid, England) in which 7% rabbit defibrinated blood is cultured.
ヘリコバクター・ピロリ（ＡＴＣＣ４３５０４）接種材料は、５×１０５ＣＦＵ／ｍｌの密度のブレインハートインフュージョン培養液に懸濁させることで、調製する。 Helicobacter pylori (ATCC 43504) inoculum is prepared by suspending in brain heart infusion culture medium at a density of 5 × 10 5 CFU / ml.
ＤＭＳＯの最終的な最大濃度は、１％であり、最初の検体濃度は、３００μｇ／ｍｌである。 The final maximum concentration of DMSO is 1% and the initial analyte concentration is 300 μg / ml.
前記プレートを、微好気的条件（混合ガスＮ２ ８５％，ＣＯ２ １０％およびＯ２ ５％）３５℃７２時間培養し、視診し、コロニー増殖の抑制を、「陽性（＋）」とスコア−し、コロニー増殖の影響がない場合、「陰性（−）」とした。
The plate was incubated for 72 hours at 35 ° C. under microaerobic conditions (mixed gas N 2 85%,
担体−対照およびゲンタマイシンを、それぞれブランクおよび陽性対照として使用した。 Carrier-control and gentamicin were used as blank and positive controls, respectively.
各濃度は、２回評価した。 Each concentration was evaluated twice.
結果を表２に示す。 The results are shown in Table 2.
２．ヘリコバクター・ピロリ誘起胃潰瘍
ヘリコバクター・ピロリ−誘起胃潰瘍におけるサンプルの影響は、先に示されている［[Marchetti, M., Arico, B., Burroni, D., Figura, N., Rappuoli, R. and Ghiara, P. Development of a mouse model of Helicobacter pylori infection that mimics human disease. Science 267: 1655-1658, 1995.］により評価する。
2. Helicobacter pylori-induced gastric ulcers The effects of samples in Helicobacter pylori-induced gastric ulcers have been shown previously [[Marchetti, M., Arico, B., Burroni, D., Figura, N., Rappuoli, R. and Ghiara, P. Development of a mouse model of Helicobacter pylori infection that mimics human disease. Science 267: 1655-1658, 1995.].
質量２４±２ｇのマウスで分けられた５つのオスのＣＤ−１（Ｃｒｌ．）の群は、「３．２×１０９コロニー形成ユニット／０．４ｍｌ／ｍｏｕｓｅ」の懸濁における臨床分離されたヘリコバクター・ピロリの胃内接種の前に１８時間絶食させた。 Groups of 5 male CD-1 (Crl.) Separated by mice with a mass of 24 ± 2 g were clinically isolated Helicobacter in suspension of “3.2 × 10 9 colony forming units / 0.4 ml / mouse”. • Fasted for 18 hours prior to intragastric inoculation of H. pylori.
ヘリコバクター・ピロリ接種の１時間後、７日連続で１日２回（１０：００ａｍおよび４：００ｐｍ）試験サンプルおよび担体（２％ＣＭＣ，１０ｍｌ／ｋｇ）が、それぞれ試験動物に経口投与された。 One hour after Helicobacter pylori inoculation, test samples and vehicle (2% CMC, 10 ml / kg) were orally administered twice daily for 7 consecutive days (10:00 am and 4:00 pm).
オメプラゾール（Ｏｍｅｐｒａｚｏｌｅ）（１ｍｇ／ｋｇ）と、クラリスロマイシン（ｃｌａｒｉｔｈｒｏｍｙｃｉｎ）（１０ｍｇ／ｋｇ）と、が組み合わされ、陽性の制御因子のために使用され、一日一回７日連続で同じスケジュールの下、試験動物に経口投与した。 Omeprazole (1 mg / kg) and clarithromycin (10 mg / kg) are combined and used for positive regulators, once a day for 7 consecutive days under the same schedule The test animals were orally administered.
感染８日後、すべての動物を夜通し絶食させ、殺し、大弯に沿って胃を解剖した。 Eight days after infection, all animals were fasted overnight, killed, and the stomach was dissected along the heel.
胃潰瘍性は、出血および潰瘍性病巣の重篤性の程度により４つのレベルにスコアした。 Gastric ulceration was scored on four levels according to the degree of bleeding and severity of ulcerative lesions.
０＝通常の外見
１＝緩やかな赤い点
２＝適度な赤い点および／または出血点
３＝著しい出血点
ヘリコバクター・ピロリ-誘起胃潰瘍における抑制性の割合は、下記の通り、算出することができる。
0 = normal appearance 1 = slow red spot 2 = moderate red spot and / or bleeding point 3 = significant bleeding point The percentage of inhibition in Helicobacter pylori-induced gastric ulcer can be calculated as follows:
（担体群スコア-試験群スコア）／（担体群スコア）×１００％
結果を表３に示す。
(Carrier group score−Test group score) / (Carrier group score) × 100%
The results are shown in Table 3.
上記実施例により調製されたサンプル中ヘリコバクター・ピロリに誘起された胃潰瘍治療において、サンプル１のみ６−ジンジャオールより効果が弱かった。 In the treatment of gastric ulcer induced by Helicobacter pylori in the sample prepared according to the above example, only sample 1 was less effective than 6-gingerol.
サンプル２０が、一番効果があり１００ｍｇ／ｋｇ、３０ｍｇ／ｋｇ、１０ｍｇ／ｋｇおよび３ｍｇ／ｋｇの投与においてヘリコバクター・ピロリ-誘起胃潰瘍の抑制性が、それぞれ８３％，７５％，６７％および５０％であった。
Claims (17)
前記有効産物が、下記段階を含む工程：
ａ）６−ジンジャオールおよび６−ショーガオールを含む、生姜の根茎由来の粗抽出物を調製する；
ｂ）粗抽出物を、逆相クロマトグラフィーカラムまたは順相クロマトグラフィーカラムに導入し、第１溶離剤、第２溶離剤の順でカラムから溶出し、この際、ｉ）逆相クロマトグラフィーカラムを使用するときは、前記第１溶離剤は、水よりも弱い極性を有し、および、前記第２溶離剤は、第１溶離剤よりも弱い極性を有し、または、ｉｉ）順相クロマトグラフィーカラムを用いるときは、前記第２溶離剤は、酢酸エチルおよびメタノールの混合物（１：１の質量比）よりも弱い極性を有し、および、前記第１溶離剤は、第２溶離剤よりも弱い極性を有し、第１溶離剤の溶出により第１溶出液および第２溶離剤の溶出により第２溶出液を得；さらに、
ｃ）順相クロマトグラフィーカラムを用いるときに、エバポレーションにより、第１溶出液から第１溶離剤を除去し、第１濃縮溶出液を得、有効産物として使用し、または、逆相クロマトグラフィーカラムを用いるときに、エバポレーションにより、第２溶出液から第２溶離剤を除去し、第２濃縮溶出液を得、有効産物として使用し；
前記段階ａ）は、段階ｉ）〜ｉｖ）、または、段階Ｉ）、段階Ｉ’）もしくは段階Ｉ”）を含み、
前記段階ｉ）〜ｉｖ）は；
ｉ）生姜の根茎をすりおろし、その結果生じた混合物をろ過し、ろ液および残渣を得；
ｉｉ）第１有機溶剤により、前記ろ液を抽出し、前記抽出溶液を回収し、前記抽出溶液から第１有機溶剤を蒸発させ、第１濃縮抽出溶液を得；
ｉｉｉ）第２有機溶剤により残渣を抽出し、抽出溶液を回収し、前記抽出溶液から第２有機溶剤を蒸発させ、第２濃縮抽出溶液を得；さらに、
ｉｖ）前記第１濃縮抽出溶液および前記第２濃縮抽出溶液を混ぜ、粗抽出物溶液を得；
前記段階Ｉ）は：
Ｉ）前記第２有機溶剤と同じ有機溶剤で、乾燥した生姜の根茎を抽出し、抽出溶液を回収し、前記抽出溶液から前記有機溶剤を蒸発させ、粗抽出物を得；
前記段階Ｉ’）は：
Ｉ’）乾燥した生姜の根茎を水蒸気蒸留し、エバポレーションにより生じる蒸留液を濃縮して、粗抽出物を得；
前記段階Ｉ”）は：
Ｉ”）超臨界ＣＯ２にて生姜の根茎の乾燥粉末を抽出し、抽出溶液を回収し、前記抽出溶液からＣＯ２をエバポレーションして、粗抽出物を得る、
により調製され、
前記有効産物に、６−ジンジャオールおよび６−ショーガオールのいずれも存在しない、薬剤組成物。 A pharmaceutical composition for the treatment of gastritis, gastric ulcer or duodenal ulcer comprising a therapeutically effective amount of an active product derived from a rhizome of ginger (Zingiber officinale),
The effective product comprises the following steps:
a) preparing a crude extract from ginger rhizomes containing 6-jinjaol and 6-shogaol;
b) The crude extract is introduced into a reverse phase chromatography column or a normal phase chromatography column and eluted from the column in the order of the first eluent and the second eluent, where i) the reverse phase chromatography column is When used, the first eluent has a weaker polarity than water and the second eluent has a weaker polarity than the first eluent, or ii) normal phase chromatography When using a column, the second eluent has a weaker polarity than a mixture of ethyl acetate and methanol (1: 1 mass ratio), and the first eluent is more than the second eluent. Having a weak polarity, elution of the first eluent provides a first eluate and a second eluent by elution of the second eluent;
c) When using a normal phase chromatography column, the first eluent is removed from the first eluate by evaporation to obtain a first concentrated eluate, used as an active product, or a reverse phase chromatography column when using, by evaporation, from the second eluate to remove the second eluant to obtain a second concentrated eluate was used as the active product;
Said step a) comprises steps i) to iv) or step I), step I ′) or step I ″),
Said steps i) to iv) are:
i) ginger rhizomes and grate the resulting mixture to obtain filtrate and residue;
ii) extracting the filtrate with a first organic solvent, recovering the extracted solution, evaporating the first organic solvent from the extracted solution to obtain a first concentrated extracted solution;
iii) extracting the residue with a second organic solvent, recovering the extracted solution, evaporating the second organic solvent from the extracted solution to obtain a second concentrated extracted solution;
iv) mixing the first concentrated extract solution and the second concentrated extract solution to obtain a crude extract solution;
Said step I) is:
I) Extracting dried ginger rhizomes with the same organic solvent as the second organic solvent, collecting the extracted solution, and evaporating the organic solvent from the extracted solution to obtain a crude extract;
Said step I ′) is:
I ′) steaming the dried ginger rhizome and concentrating the distillate produced by evaporation to obtain a crude extract;
Said step I ") is:
I ″) Extracting ginger rhizome dry powder with supercritical CO 2 , recovering the extract solution, and evaporating CO 2 from the extract solution to obtain a crude extract,
It is prepared by,
A pharmaceutical composition wherein neither the 6-gingaol nor 6-shogaol is present in the active product .
請求項１５に記載の薬剤組成物。 The first eluent is a mixture of n-hexane and ethyl acetate (mass ratio 9: 1),
The pharmaceutical composition of claim 1 5.
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| GB0720144A GB2453728B (en) | 2007-10-16 | 2007-10-16 | Use of a potent product extracted from rhizomes of zingiber offcinale in preparation of a medicament for treatment gastritis, gastric ulcer and duodenal ulcer |
| GB0720144.5 | 2007-10-16 |
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| CN110878014B (en) * | 2019-12-18 | 2022-06-24 | 保定冀中药业有限公司 | Method for rapidly extracting gingerol |
| KR102652214B1 (en) * | 2021-05-12 | 2024-04-01 | 주식회사 코시스바이오 | Polysaccharide fraction isolated from natural plant with immune-enhancing activity and composition including the same |
| KR102488117B1 (en) * | 2021-12-15 | 2023-01-13 | (주)에스디생명공학 | Composition for Prevention or Treatment of Infective Gastritis by Helicobacter pylori Infection Comprising Steamed Ginger Extract as an Active Ingredient |
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| US5804603A (en) | 1993-11-26 | 1998-09-08 | National Science Council | Synthesized β-adrenergic blockers derivatives of guaiacol |
| JP3547782B2 (en) * | 1993-12-28 | 2004-07-28 | ロート製薬株式会社 | Anti-Helicobacter pylori activator |
| JP3593357B2 (en) * | 1994-02-28 | 2004-11-24 | エーザイ株式会社 | Antibacterial agent |
| JPH08119872A (en) * | 1994-10-18 | 1996-05-14 | Otsuka Pharmaceut Co Ltd | Crude drug composition against helicobacter pylori |
| DE19716660C2 (en) * | 1997-04-22 | 2002-11-14 | Schwabe Willmar Gmbh & Co | Preparations, in particular pharmaceutical and pharmaceutical forms based on plants, for combating Helicobacter pylori infections |
| TWI290469B (en) * | 2000-07-12 | 2007-12-01 | Medical & Pharm Ind Tech & Dev | Method for manufacturing product with component capable of efficiently preventing inflammation and platelets agglutination form ginger and medical composition with the efficient component |
| US6855347B2 (en) | 2002-03-25 | 2005-02-15 | Council Of Scientific And Industrial Research | Composition for treating gastric ulcer and a process for preparing the same |
| JP2004115536A (en) | 2003-11-27 | 2004-04-15 | Rohto Pharmaceut Co Ltd | Anti-helicobacter pylori activator |
| US8158608B2 (en) * | 2006-03-30 | 2012-04-17 | Council Of Scientific And Industrial Research | Bioactive fraction from Zingier officinale and a process for the preparation thereof |
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