JPS6225355B2 - - Google Patents
Info
- Publication number
- JPS6225355B2 JPS6225355B2 JP12459482A JP12459482A JPS6225355B2 JP S6225355 B2 JPS6225355 B2 JP S6225355B2 JP 12459482 A JP12459482 A JP 12459482A JP 12459482 A JP12459482 A JP 12459482A JP S6225355 B2 JPS6225355 B2 JP S6225355B2
- Authority
- JP
- Japan
- Prior art keywords
- glycyrrhetinic acid
- epi
- acid
- glycyrrhizin
- glycyrrhetinic
- 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.)
- Expired
Links
- MPDGHEJMBKOTSU-YKLVYJNSSA-N 18beta-glycyrrhetic acid Chemical compound C([C@H]1C2=CC(=O)[C@H]34)[C@@](C)(C(O)=O)CC[C@]1(C)CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CC[C@H](O)C1(C)C MPDGHEJMBKOTSU-YKLVYJNSSA-N 0.000 claims description 94
- 229960003720 enoxolone Drugs 0.000 claims description 84
- MPDGHEJMBKOTSU-UHFFFAOYSA-N Glycyrrhetinsaeure Natural products C12C(=O)C=C3C4CC(C)(C(O)=O)CCC4(C)CCC3(C)C1(C)CCC1C2(C)CCC(O)C1(C)C MPDGHEJMBKOTSU-UHFFFAOYSA-N 0.000 claims description 47
- 230000000968 intestinal effect Effects 0.000 claims description 21
- 239000004378 Glycyrrhizin Substances 0.000 claims description 20
- LPLVUJXQOOQHMX-UHFFFAOYSA-N glycyrrhetinic acid glycoside Natural products C1CC(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2=O)C(O)=O)C)(C)CC2)(C)C2C(C)(C)C1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O LPLVUJXQOOQHMX-UHFFFAOYSA-N 0.000 claims description 20
- 229960004949 glycyrrhizic acid Drugs 0.000 claims description 20
- UYRUBYNTXSDKQT-UHFFFAOYSA-N glycyrrhizic acid Natural products CC1(C)C(CCC2(C)C1CCC3(C)C2C(=O)C=C4C5CC(C)(CCC5(C)CCC34C)C(=O)O)OC6OC(C(O)C(O)C6OC7OC(O)C(O)C(O)C7C(=O)O)C(=O)O UYRUBYNTXSDKQT-UHFFFAOYSA-N 0.000 claims description 20
- 235000019410 glycyrrhizin Nutrition 0.000 claims description 20
- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 claims description 16
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims 1
- 239000002207 metabolite Substances 0.000 description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 14
- 239000000243 solution Substances 0.000 description 11
- 239000002253 acid Substances 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 239000012530 fluid Substances 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 6
- 238000001819 mass spectrum Methods 0.000 description 6
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 5
- 239000013078 crystal Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- LPLVUJXQOOQHMX-MOGLOQIBSA-N (2s,3s,4s,5r,6r)-6-[(2r,3r,4s,5s,6s)-2-[[(3s,4ar,6ar,6bs,8as,11s,12ar,14ar,14bs)-11-carboxy-4,4,6a,6b,8a,11,14b-heptamethyl-14-oxo-2,3,4a,5,6,7,8,9,10,12,12a,14a-dodecahydro-1h-picen-3-yl]oxy]-6-carboxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-c Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-MOGLOQIBSA-N 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000002844 melting Methods 0.000 description 4
- 230000008018 melting Effects 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 238000004809 thin layer chromatography Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 210000003608 fece Anatomy 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 241000607534 Aeromonas Species 0.000 description 1
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 description 1
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000053187 Glucuronidase Human genes 0.000 description 1
- 108010060309 Glucuronidase Proteins 0.000 description 1
- 240000004670 Glycyrrhiza echinata Species 0.000 description 1
- 235000001453 Glycyrrhiza echinata Nutrition 0.000 description 1
- 235000006200 Glycyrrhiza glabra Nutrition 0.000 description 1
- 235000017382 Glycyrrhiza lepidota Nutrition 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 208000019025 Hypokalemia Diseases 0.000 description 1
- 102000004195 Isomerases Human genes 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- 208000026709 Liddle syndrome Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 206010037113 Pseudoaldosteronism Diseases 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000003266 anti-allergic effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000767 anti-ulcer Effects 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- KRVSOGSZCMJSLX-UHFFFAOYSA-L chromic acid Substances O[Cr](O)(=O)=O KRVSOGSZCMJSLX-UHFFFAOYSA-L 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000036267 drug metabolism Effects 0.000 description 1
- 238000000921 elemental analysis Methods 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- AWJWCTOOIBYHON-UHFFFAOYSA-N furo[3,4-b]pyrazine-5,7-dione Chemical compound C1=CN=C2C(=O)OC(=O)C2=N1 AWJWCTOOIBYHON-UHFFFAOYSA-N 0.000 description 1
- 125000002168 glycyrrhetinic acid group Chemical group 0.000 description 1
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 229940010454 licorice Drugs 0.000 description 1
- 239000012907 medicinal substance Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 125000000896 monocarboxylic acid group Chemical group 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 208000024896 potassium deficiency disease Diseases 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 210000002268 wool Anatomy 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Description
【発明の詳細な説明】
本発明は、３−エピ−グリチルレチン酸の製造
法に関する。更に詳しくは、グリチルリチン又は
グリチルレチン酸を用いてヒト腸内フローラによ
る３−エピ−グリチルレチン酸の生化学的製造法
に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing 3-epi-glycyrrhetinic acid. More specifically, the present invention relates to a biochemical method for producing 3-epi-glycyrrhetinic acid using human intestinal flora using glycyrrhizin or glycyrrhetinic acid.
甘草中のグリチルリチンは、抗潰瘍作用、抗炎
症作用、抗アレルギー作用等を有する物質として
知られているが、近年グリチルリチンの有効成分
はそのアグリコンであるグリチルレチン酸である
ことが明らかにされている。 Glycyrrhizin in licorice is known as a substance that has anti-ulcer, anti-inflammatory, and anti-allergic effects, but in recent years it has been revealed that the active ingredient of glycyrrhizin is its aglycone, glycyrrhetinic acid.
そのため、上記薬効作用物質としてのグリチル
レチン酸を製造する試みがこれまで主として有機
合成的手段によつて種々なされてきたが最近生化
学的手段によつてもグリチルレチン酸の製造がな
された。例えば特開昭56−137898にはグリチルリ
チン酸よりアエロモナス属バクテリアの産生する
酵素を用いてグリチルレチン酸を製造することが
記載されている。 Therefore, various attempts have been made to produce glycyrrhetinic acid as a medicinal substance, mainly by organic synthetic means, but recently, glycyrrhetinic acid has also been produced by biochemical means. For example, JP-A-56-137898 describes the production of glycyrrhetinic acid from glycyrrhetinic acid using an enzyme produced by bacteria belonging to the genus Aeromonas.
しかしながら、グリチルレチン酸には、高血
圧、低カリウム血症、浮腫等の症状のいわゆる偽
アルドステロン症の副作用が発現する問題点が指
摘されている（特開昭56−139416）。 However, it has been pointed out that glycyrrhetinic acid has the problem of causing side effects of so-called pseudoaldosteronism, such as hypertension, hypokalemia, and edema (Japanese Patent Application Laid-Open No. 139416/1983).
本発明者らは、従来より腸内細菌の薬物代謝に
関する研究を行なつており、たまたまグリチルリ
チンの代謝についての研究中に、グリチルリチン
が腸内細菌により、グリチルレチン酸に水解さ
れ、かつ生成したグリチルレチン酸が３−エピ−
グリチルレチン酸に変換されることを見出した。
そして鋭意検討した結果、グリチルレチン酸と同
様の薬効を示し、かつ又グリチルレチン酸のもつ
副作用を有しないことが予想される３−エピ−グ
リチルレチン酸の生化学的製造手段を見出し本発
明を完成せしめたものである。 The present inventors have been conducting research on drug metabolism by intestinal bacteria, and it happened that during the research on the metabolism of glycyrrhizin, glycyrrhizin was hydrolyzed by intestinal bacteria to glycyrrhetinic acid, and the produced glycyrrhetinic acid is 3-epi-
It was found that it was converted to glycyrrhetinic acid.
As a result of intensive studies, the inventors discovered a biochemical means of producing 3-epi-glycyrrhetinic acid, which is expected to exhibit the same medicinal efficacy as glycyrrhetinic acid and not have the side effects of glycyrrhetinic acid, and completed the present invention. It is something.
即ち、本発明は、ヒト腸内フローラをグリチル
リチンを含む培地で嫌気的に培養することによつ
て培養液中のグリチルリチンを水解してグリチル
レチン酸を生成させ、かつ生成したグリチルレチ
ン酸を３−エピ−グリチルレチン酸に変換せしめ
た後、又はヒト腸内フローラをグリチルレチン酸
を含む培地で嫌気的に培養することによつて、培
養液中のグリチルレチン酸を３−エピ−グリチル
レチン酸に変換せしめた後、該培養液より３−エ
ピ−グリチルレチン酸を採取することを特徴とす
る３−エピ−グリチルレチン酸の製造法である。 That is, the present invention involves culturing human intestinal flora anaerobically in a medium containing glycyrrhizin, hydrolyzing glycyrrhizin in the culture solution to produce glycyrrhetinic acid, and converting the produced glycyrrhetinic acid into 3-epi- After converting the glycyrrhetinic acid into 3-epi-glycyrrhetinic acid, or by culturing human intestinal flora anaerobically in a medium containing glycyrrhetinic acid, the glycyrrhetinic acid in the culture solution is converted to 3-epi-glycyrrhetinic acid. This is a method for producing 3-epi-glycyrrhetinic acid, which is characterized by collecting 3-epi-glycyrrhetinic acid from a culture solution.
本発明は腸内フローラ液を有効に利用すること
特色とするものであるが以下にその調製法につい
て述べる。 The present invention is characterized by the effective use of intestinal flora fluid, and the preparation method thereof will be described below.
新鮮なヒト糞便を炭酸ガスを満たしたビニール
袋にとり、内容をよく混合し、その一部をとり、
５倍容のGAMブイヨン又はPGPYブイヨン（い
ずれも日水製薬製）等の嫌気性菌培地に懸濁せし
め、ゆつくり遠心（16×ｇ、１分間）し、残渣を
除いた上清区分を腸内フローラ液として用いる。
腸内フローラ液中の嫌気性菌の構成は、ヒトによ
り若干異なることも考えられるが、本発明者らの
実験結果からは、異なつた糞便の腸内フローラ液
を用いてもグリチルリチン又はグリチルレチン酸
からいずれも３−エピ−グリチルレチン酸を多量
生成することが確められているので、健康なヒト
糞便であれば、容易にいつでも本発明に使用でき
る腸内フローラ液を調製することができる。 Place fresh human feces in a plastic bag filled with carbon dioxide gas, mix the contents well, and take a portion of it.
Suspend in 5 times the volume of anaerobic bacterial culture medium such as GAM broth or PGPY broth (both manufactured by Nissui Pharmaceutical Co., Ltd.), slowly centrifuge (16 x g, 1 minute), remove the residue, and transfer the supernatant to the intestines. Used as an internal flora liquid.
Although the composition of anaerobic bacteria in the intestinal flora fluid may differ slightly depending on the person, the experimental results of the present inventors indicate that even if different fecal intestinal flora fluids are used, glycyrrhizin or glycyrrhetinic acid can be removed. Since it has been confirmed that both of them produce a large amount of 3-epi-glycyrrhetinic acid, an intestinal flora solution that can be used in the present invention can be easily prepared at any time using healthy human feces.
つぎに、上記腸内フローラ液を用いての３−エ
ピ−グリチルレチン酸の製造法について述べる。
グリチルリチン又はグリチルレチン酸を含む嫌気
性菌培養培地、例えばGAMブイヨン、PGPYブ
イヨン等の培地に腸内フローラ液を加え、反応温
度は20〜45℃、好ましくは30〜40℃、反応時間は
10〜80時間、好ましくは24〜48時間嫌気的に培養
し、培養液中のグリチルリチンは、まず水解され
てグリチルレチン酸となり、ついで生成したグリ
チルレチン酸もしくは、はじめから培地に添加さ
れたグリチルレチン酸は同じ腸内フローラ液によ
り３−デヒドログリチルレチン酸を介して３−エ
ピ−グリチルレチン酸に変換される。この腸内フ
ローラ液によるグリチルリチン又はグリチルレチ
ン酸からの代謝産物である３−エピ−グリチルレ
チン酸の生成が事実であることを以下の試験例に
よつて具体的に説明する。 Next, a method for producing 3-epi-glycyrrhetinic acid using the above-mentioned intestinal flora fluid will be described.
The intestinal flora liquid is added to an anaerobic bacterial culture medium containing glycyrrhizin or glycyrrhetinic acid, such as GAM broth or PGPY broth, and the reaction temperature is 20 to 45 °C, preferably 30 to 40 °C, and the reaction time is
Cultivate anaerobically for 10 to 80 hours, preferably 24 to 48 hours, and the glycyrrhizin in the culture solution is first hydrolyzed to glycyrrhetinic acid, and then the glycyrrhetinic acid produced or the glycyrrhetinic acid added to the medium from the beginning is the same. It is converted to 3-epi-glycyrrhetinic acid via 3-dehydroglycyrrhetinic acid by intestinal flora fluid. The fact that 3-epi-glycyrrhetinic acid, which is a metabolite from glycyrrhizin or glycyrrhetinic acid, is produced by this intestinal flora fluid will be specifically explained using the following test examples.
試験例 １
ヒト糞便をとりGAMブイヨン培地に懸濁し、
軽く遠心分離し、その上清を菌液とし、次いでこ
の菌液をグリチルリチンを含むGAMブイヨン培
地に培養し、嫌気的に48時間培養し、このものを
酢酸エチルで抽出し、TLCスキヤナーで調べた
ところ、Rf0.28（代謝産物）とRf0.34（代謝産
物）の２つの代謝産物が得られた。Test Example 1 Human feces was taken and suspended in GAM broth medium,
Lightly centrifuged, the supernatant was used as a bacterial suspension, this bacterial suspension was then cultured in GAM broth containing glycyrrhizin, cultured anaerobically for 48 hours, extracted with ethyl acetate, and examined using a TLC scanner. However, two metabolites, Rf0.28 (metabolite) and Rf0.34 (metabolite), were obtained.
まず代謝産物と標準品のグリチルレチン酸と
を比較したところ、TLC、NMR、IR、マススペ
クトル、UV及び融点の全てにおいて一致したの
で代謝産物はグリチルレチン酸であることが確
認された。一方代謝産物は、Rf値が標準品の
グリチルレチン酸と３−デヒドログリチルレチン
酸との中間にあること、マススペクトルが標準の
グリチルレチン酸と同一スペクトルであること、
さらには、NMRに関してグリチルレチン酸は
3.04ppmに、代謝産物は3.18ppmにそれぞれピ
ークがあること（これはグリチルレチン酸の方は
ダブルダブレツトで、３αのブロトンと２位のプ
ロトンとのカツプリングによるものであり、代謝
産物の方はグリチルレチン酸と比較して半値巾
の狭いブロードシーグレツトとして現れているこ
とによる）等のデータから代謝産物は、グリチ
ルレチン酸の３位の水酸基がα化されたものであ
ることが推察された。そこでそのことを更に確か
めるために代謝産物とグリチルレチン酸のそれ
ぞれをアセトン中でクロム酸酸化した生成物につ
いてTLC、IR、UV、マススペクトル、NMRを
調べたところ、いずれの生成物も３−デヒドログ
リチルレチン酸であつた（即ちグリチルレチン酸
と代謝産物とは異性体である。）。以上のことか
ら代謝産物は３位の水酸基がα位のグリチルレ
チン酸即ち、３−エピ−グリチルレチン酸である
と決定されたのである。そして又代謝産物と３
α−ヒドロキシステロイドデヒドロゲナーゼとは
定量的に反応するがグリチルレチン酸と該酵素と
は全く反応しないことによつて生化学的にも代謝
産物が３−エピ−グリチルレチン酸であること
が確認されたのである。次に反応液から３−エピ
−グリチルレチン酸を単離するに際して、まず３
−エピ−グリチルレチン酸含有培養液を酸性とし
た後、クロロホルム、メタノール、アセトン、酢
酸エステル等の有機溶媒にて３−エピ−グリチル
レチン酸を抽出し、得られた抽出液を減圧濃縮
し、濃縮液中に含まれる３−エピ−グリチルレチ
ン酸をシリカゲルカラムに吸着せしめ、ついでク
ロロホルム−メタノール等の溶媒で吸着された３
−エピ−グリチルレチン酸を溶出し、３−エピ−
グリチルレチン酸を含む溶出液を減圧濃縮又はこ
の溶出液を更に薄層クロマトグラフイーで展開せ
しめ、３−エピ−グリチルレチン酸に相当するス
ポツトを集め、溶剤で抽出し、溶剤を減圧濃縮
後、得られた結晶残渣をクロロホルム石油エーテ
ルにて結晶化して３−エピ−グリチルレチン酸の
純品を得た。ここでグリチルリチンよりグリチル
レチン酸を経て、３−エピ−グリチルレチン酸に
変換する一連の生化学的な変化を図示すれば第１
図のとおりである。グリチルリチンよりグリチル
レチン酸の水解は或る種の腸内細菌、例えばオイ
バクテリウム属およびクロストリジウム属等の菌
株の生産するβ−グルクロニダーゼの作用による
ことが判明しているが、グリチルレチン酸から３
−デヒドログリチルレチン酸を経て３−エピ−グ
リチルレチン酸への変換については、腸内細菌な
どの生菌作用によるものであるが、未だその酸化
還元酵素もしくは異性化酵素は証明するに至つて
いない。いずれにしても化学的合成法では困難な
グリチルリチンより３−エピ−グリチルレチン酸
の製造が、腸内フローラを用いる生化学的手段に
よつて、容易に行いうることがわかつたのであ
る。 First, when the metabolite was compared with the standard glycyrrhetinic acid, it was confirmed that the metabolite was glycyrrhetinic acid because they matched in TLC, NMR, IR, mass spectrum, UV, and melting point. On the other hand, the metabolite has an Rf value that is between the standard glycyrrhetinic acid and 3-dehydroglycyrrhetinic acid, and a mass spectrum that is the same as that of the standard glycyrrhetinic acid.
Furthermore, regarding NMR, glycyrrhetinic acid is
There is a peak at 3.04 ppm, and a peak at 3.18 ppm for the metabolite (this is a double doublet for glycyrrhetinic acid, due to the coupling between the 3α broton and the proton at the 2nd position, and the metabolite has a peak at 3.18 ppm). It was inferred from the data that the metabolite was a gelatinized hydroxyl group at the 3-position of glycyrrhetinic acid (because it appeared as a broad siglet with a narrower half-width than that of the acid). To further confirm this, we investigated TLC, IR, UV, mass spectra, and NMR for the products obtained by chromic acid oxidation of the metabolites and glycyrrhetinic acid in acetone, and found that all of the products were 3-dehydroglycyrrhetinic acid. acid (i.e., glycyrrhetinic acid and the metabolite are isomers). From the above, it was determined that the metabolite was glycyrrhetinic acid with the hydroxyl group at the 3-position at the α-position, that is, 3-epi-glycyrrhetinic acid. And also metabolites and 3
It was biochemically confirmed that the metabolite was 3-epi-glycyrrhetinic acid, as it quantitatively reacted with α-hydroxysteroid dehydrogenase but did not react with glycyrrhetinic acid at all. . Next, when isolating 3-epi-glycyrrhetinic acid from the reaction solution, first
- After making the epi-glycyrrhetinic acid-containing culture solution acidic, 3-epi-glycyrrhetinic acid is extracted with an organic solvent such as chloroform, methanol, acetone, or acetate, and the resulting extract is concentrated under reduced pressure to form a concentrated solution. The 3-epi-glycyrrhetinic acid contained in the 3-epi-glycyrrhetinic acid was adsorbed on a silica gel column, and then the 3-epi-glycyrrhetinic acid was adsorbed with a solvent such as chloroform-methanol.
-Epi-glycyrrhetinic acid is eluted, 3-epi-
The eluate containing glycyrrhetinic acid is concentrated under reduced pressure, or this eluate is further developed by thin layer chromatography, the spot corresponding to 3-epi-glycyrrhetinic acid is collected, extracted with a solvent, and the solvent is concentrated under reduced pressure. The resulting crystal residue was crystallized from chloroform petroleum ether to obtain pure 3-epi-glycyrrhetinic acid. Here, the series of biochemical changes in which glycyrrhizin is converted to glycyrrhetinic acid and then to 3-epi-glycyrrhetinic acid is illustrated as follows:
As shown in the figure. It has been found that the hydrolysis of glycyrrhetinic acid from glycyrrhizin is due to the action of β-glucuronidase produced by certain intestinal bacteria, such as strains of the genus Oibacterium and Clostridium.
The conversion of -dehydroglycyrrhetinic acid to 3-epi-glycyrrhetinic acid is due to the action of living bacteria such as intestinal bacteria, but the oxidoreductase or isomerase has not yet been demonstrated. In any case, it has been found that 3-epi-glycyrrhetinic acid can be easily produced from glycyrrhizin by biochemical means using intestinal flora, which is difficult to produce by chemical synthesis methods.
次に実施例にて３−エピ−グリチルレチン酸の
製造法を具体的に説明する。 Next, a method for producing 3-epi-glycyrrhetinic acid will be specifically explained in Examples.
実施例 １
グリチルリチン250mgをGAM培地225mlに溶か
し、次に腸内フローラ液25mlを加え、37℃48時
間、スチールウール法で嫌気培養した。培養液を
1NHClでPH１に調整後、クロロホルム又は酢酸エ
チル250mlで４回抽出した。抽出液を合わせ、飽
和食塩水で洗滌後、無水硫酸ソーダで乾燥し、溶
媒を減圧留去後、濃縮液をシリカゲルカラム
（2.4×44cm）にかけ３−エピ−グリチルレチンを
吸着せしめた後、カラムをクロロホルム800mlで
充分洗滌後、クロロホルム−メタノール（100：
１）で溶出した。溶出液はフラクシヨンコレクタ
ーを用い約100mlづつ分取し、薄層クロマトグラ
フイーにかけ、Rf0.28およびRf0.34を示すフラク
シヨンを集め、それぞれ減圧濃縮後、クロロホル
ム石油エーテルで再結晶し、前者からグリチルレ
チン酸44mg、後者から３−エピ−グリチルレチン
酸59mgの収量を得た。こうして得られたグリチル
レチン酸及び３−エピ−グリチルレチン酸の理化
学的性質を以下に記す。Example 1 250 mg of glycyrrhizin was dissolved in 225 ml of GAM medium, then 25 ml of intestinal flora fluid was added, and anaerobic culture was performed at 37° C. for 48 hours using the steel wool method. culture solution
After adjusting the pH to 1 with 1NHCl, the mixture was extracted four times with 250 ml of chloroform or ethyl acetate. The extracts were combined, washed with saturated brine, dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the concentrated solution was applied to a silica gel column (2.4 x 44 cm) to adsorb 3-epi-glycyrrheti. After washing thoroughly with 800ml of chloroform, chloroform-methanol (100:
1) was eluted. The eluate was collected in approximately 100ml portions using a fraction collector, subjected to thin layer chromatography, and the fractions showing Rf0.28 and Rf0.34 were collected, concentrated under reduced pressure, recrystallized with chloroform petroleum ether, and separated from the former. A yield of 44 mg of glycyrrhetinic acid and from the latter 59 mg of 3-epi-glycyrrhetinic acid were obtained. The physicochemical properties of glycyrrhetinic acid and 3-epi-glycyrrhetinic acid thus obtained are described below.
グリチルレチン酸：
(1) 結晶の色及び形状：無色プリズム晶
(2) 融点：294〜296℃
(3) 紫外吸収：250nｍ
(4) 赤外吸収（Ｋ Br）（cm-1）：3430（OH）、
1700（Ｃ＝０）、1661（conjugated Ｃ＝
０）、1615（conjugated Ｃ＝Ｃ）cm-1
(5) マススペクトル：ｍ／Z470（Ｍt、10％）、
303（93％）、262（82％）、216（17％）、175
（52％）、135（100％）
(6) 旋光度：〔α〕２６ Ｄ＝＋153.8（Ｃ＝1.3、クロ
ロ
ホルム−メタノール（19：１））
(7) NMR：Ｃ−CH3（3H、Ｓ）δ0.70、0.71、
0.92、1.04、1.05、1.11、1.37、−CH−OH
（1H、ｍ）δ3.04、Ｃ＝CH（ Ｓ）δ5.44
３−エピ−グリチルレチン酸：
(1) 結晶の形状及色：無色プリズム晶
(2) 融点：300℃以上
(3) 元素分析値：C30H46O4
分析値（％）Ｃ、76.55；Ｈ、9.85
実測値（％）Ｃ、76.75；Ｈ、9.83
(4) 紫外部吸収：250nｍ（ε＝13600）
(5) 赤外吸収（Ｋ Br）（cm-1）：3500（OH）、
1717（Ｃ＝０）、1641（conjugated Ｃ＝
０）、1615（conjugated Ｃ＝Ｃ）
(6) マススペクトル：ｍ／Ｚ 470（Ｍt、25
％）、303（96％）、26（84％）、216（16％）、
175（76％）、135（100％）
(7) 旋光度：〔α〕２４ Ｄ＝＋146.9（Ｃ＝1.3クロロ
ホ
ルム−メタノール（19：１））
(8) NMR：Ｃ−CH3（3H、Ｓ）δ0.80、0.88、
1.07、1.14、1.40、CH−OH（1H br、Ｓ）
δ3.21、Ｃ＝CH（1H、Ｓ）δ5.46
実施例 ２
GAM培地（４）にグリチルレチン酸（272
mg）を均一に懸濁させ、これに腸内フローラ液
（200ml）を加え、37℃、48時間、嫌気的に培養
後、培養液を1N塩酸でPH１に調整し、酢酸エチ
ル又はクロロホルム（２）で３回抽出し、抽出
液を２％食塩水で洗滌後、減圧濃縮し、これをシ
リカゲルカラムにかけ３−エピ−グリチルレチン
酸を吸着せしめ、ついでカラムをクロロホルム
（５）で洗滌した。この溶出液中には３−デヒ
ドログリチルレチン酸を含み、再結晶により２mg
を得た。さらにカラムをクロロホルム−メタノー
ル（100：１）で溶出させ、前部フラクシヨンよ
り３−エピ−グリチルレチン酸25mg（収率９
％）、後部フラクシヨンよりグリチルレチン酸175
mgを得た。Glycyrrhetinic acid: (1) Crystal color and shape: Colorless prismatic crystal (2) Melting point: 294-296℃ (3) Ultraviolet absorption: 250nm (4) Infrared absorption (K Br) (cm -1 ): 3430 (OH ),
1700 (C=0), 1661 (conjugated C=
0), 1615 (conjugated C=C) cm -1 (5) Mass spectrum: m/Z470 (M t , 10%),
303 (93%), 262 (82%), 216 (17%), 175
(52%), 135 (100%) (6) Optical rotation: [α] 26 D = +153.8 (C = 1.3, chloroform-methanol (19:1)) (7) NMR: C-CH 3 (3H , S) δ0.70, 0.71,
0.92, 1.04, 1.05, 1.11, 1.37, −CH−OH
(1H, m) δ3.04, C=CH (S) δ5.44 3-epi-glycyrrhetinic acid: (1) Crystal shape and color: Colorless prismatic crystal (2) Melting point: 300℃ or higher (3) Elemental analysis Value: C 30 H 46 O 4 Analytical value (%) C, 76.55; H, 9.85 Actual value (%) C, 76.75; H, 9.83 (4) Ultraviolet absorption: 250 nm (ε=13600) (5) Infrared Absorption (K Br) (cm -1 ): 3500 (OH),
1717 (C=0), 1641 (conjugated C=
0), 1615 (conjugated C=C) (6) Mass spectrum: m/Z 470 (M t , 25
%), 303 (96%), 26 (84%), 216 (16%),
175 (76%), 135 (100%) (7) Optical rotation: [α] 24 D = +146.9 (C = 1.3 chloroform-methanol (19:1)) (8) NMR: C-CH 3 (3H , S) δ0.80, 0.88,
1.07, 1.14, 1.40, CH-OH (1H br, S)
δ3.21, C=CH (1H, S) δ5.46 Example 2 Glycyrrhetinic acid (272
mg) was homogeneously suspended, intestinal flora fluid (200 ml) was added thereto, and cultured anaerobically at 37°C for 48 hours. The culture solution was adjusted to pH 1 with 1N hydrochloric acid, and ethyl acetate or chloroform (200 ml) ), the extract was washed with 2% saline, concentrated under reduced pressure, applied to a silica gel column to adsorb 3-epi-glycyrrhetinic acid, and then the column was washed with chloroform (5). This eluate contains 3-dehydroglycyrrhetinic acid, and by recrystallization, 2 mg
I got it. The column was further eluted with chloroform-methanol (100:1), and 25 mg of 3-epi-glycyrrhetinic acid (yield 9) was extracted from the front fraction.
%), glycyrrhetinic acid 175 from the rear fraction
I got mg.
ここで得られた中間体の３−デヒドログリチル
レチン酸の理化学的性質を以下に記す。 The physicochemical properties of the intermediate 3-dehydroglycyrrhetinic acid obtained here are described below.
(1) 融点：300℃以上
(2) 紫外吸収：入max 250nｍ
(3) マススペクトル：ｍ／Ｚ 468（Ｍt、29
％）、453（15％）、440（15％）、442（17
％）、303（73％）、262（75％）、216（12
％）、135（100％）
(4) 赤外吸収（Ｋ Br）cm-1：3310（COOH）、
1726（Ｃ＝０）、1682（Ｃ＝０）、1645
（conjugate Ｃ＝０）1615（conjugated Ｃ＝
Ｃ）(1) Melting point: 300℃ or higher (2) Ultraviolet absorption: Max. 250nm (3) Mass spectrum: m/Z 468 (M t , 29
%), 453 (15%), 440 (15%), 442 (17
%), 303 (73%), 262 (75%), 216 (12
%), 135 (100%) (4) Infrared absorption (K Br) cm -1 : 3310 (COOH),
1726 (C=0), 1682 (C=0), 1645
(conjugate C=0) 1615 (conjugated C=
C)
第１図は、腸内フローラによるグリチルリチン
の３−エピ−グリチルレチン酸への変換を示す図
である。図中において(1)の構造式はグリチルリチ
ンを、(2)の構造式はグリチルレチン酸を、(3)の構
造式は３−デヒドログリチルレチン酸を、(4)の構
造式は３−エピ−グリチルレチン酸をそれぞれ示
すものである。
FIG. 1 is a diagram showing the conversion of glycyrrhizin to 3-epi-glycyrrhetinic acid by intestinal flora. In the figure, the structural formula (1) is glycyrrhizin, the structural formula (2) is glycyrrhetinic acid, the structural formula (3) is 3-dehydroglycyrrhetinic acid, and the structural formula (4) is 3-epi-glycyrrhetinic acid. Each indicates an acid.
Claims (1)
地で嫌気的に培養し培養液中に含まれるグリチル
リチンを水解してグリチルレチン酸を生成させ、
かつ生成グリチルレチン酸を３−エピ−グリチル
レチン酸に変換せしめた後又はヒト腸内フローラ
をグリチルレチン酸を含む培地で嫌気的に培養し
培養液中に含まれるグリチルレチン酸を３−エピ
−グリチルレチン酸に変換せしめた後、該培養液
より３−エピ−グリチルレチン酸を採取すること
を特徴とする３−エピ−グリチルレチン酸の製造
法。1 Human intestinal flora is anaerobically cultured in a medium containing glycyrrhizin, and glycyrrhizin contained in the culture solution is hydrolyzed to produce glycyrrhetinic acid,
After converting the produced glycyrrhetinic acid to 3-epi-glycyrrhetinic acid, or by culturing human intestinal flora anaerobically in a medium containing glycyrrhetinic acid, the glycyrrhetinic acid contained in the culture solution is converted to 3-epi-glycyrrhetinic acid. A method for producing 3-epi-glycyrrhetinic acid, which comprises collecting 3-epi-glycyrrhetinic acid from the culture solution after incubation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12459482A JPS5914799A (en) | 1982-07-16 | 1982-07-16 | Preparation of 3-epi-glycyrrhetic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12459482A JPS5914799A (en) | 1982-07-16 | 1982-07-16 | Preparation of 3-epi-glycyrrhetic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5914799A JPS5914799A (en) | 1984-01-25 |
| JPS6225355B2 true JPS6225355B2 (en) | 1987-06-02 |
Family
ID=14889313
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP12459482A Granted JPS5914799A (en) | 1982-07-16 | 1982-07-16 | Preparation of 3-epi-glycyrrhetic acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5914799A (en) |
-
1982
- 1982-07-16 JP JP12459482A patent/JPS5914799A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5914799A (en) | 1984-01-25 |
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