KR20230116557A - Xylosylated flavonoid derivatives and method thereof - Google Patents
Xylosylated flavonoid derivatives and method thereof Download PDFInfo
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- KR20230116557A KR20230116557A KR1020220013630A KR20220013630A KR20230116557A KR 20230116557 A KR20230116557 A KR 20230116557A KR 1020220013630 A KR1020220013630 A KR 1020220013630A KR 20220013630 A KR20220013630 A KR 20220013630A KR 20230116557 A KR20230116557 A KR 20230116557A
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- South Korea
- Prior art keywords
- xylopyranoside
- baicalein
- xylosylated
- xyl
- derivative
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- 150000002214 flavonoid derivatives Chemical class 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 19
- 229930003935 flavonoid Natural products 0.000 claims abstract description 16
- 150000002215 flavonoids Chemical class 0.000 claims abstract description 16
- 235000017173 flavonoids Nutrition 0.000 claims abstract description 16
- -1 pentose saccharide Chemical class 0.000 claims abstract description 16
- 238000004519 manufacturing process Methods 0.000 claims abstract description 13
- UDFLTIRFTXWNJO-UHFFFAOYSA-N baicalein Chemical compound O1C2=CC(=O)C(O)=C(O)C2=C(O)C=C1C1=CC=CC=C1 UDFLTIRFTXWNJO-UHFFFAOYSA-N 0.000 claims description 124
- 150000001875 compounds Chemical class 0.000 claims description 87
- LKOJGSWUMISDOF-UHFFFAOYSA-N oroxylin A Chemical compound C=1C(=O)C2=C(O)C(OC)=C(O)C=C2OC=1C1=CC=CC=C1 LKOJGSWUMISDOF-UHFFFAOYSA-N 0.000 claims description 44
- FXNFHKRTJBSTCS-UHFFFAOYSA-N Baicalein Natural products C=1C(=O)C=2C(O)=C(O)C(O)=CC=2OC=1C1=CC=CC=C1 FXNFHKRTJBSTCS-UHFFFAOYSA-N 0.000 claims description 38
- 229940015301 baicalein Drugs 0.000 claims description 38
- IPQKDIRUZHOIOM-UHFFFAOYSA-N Oroxin A Natural products OC1C(O)C(O)C(CO)OC1OC(C(=C1O)O)=CC2=C1C(=O)C=C(C=1C=CC=CC=1)O2 IPQKDIRUZHOIOM-UHFFFAOYSA-N 0.000 claims description 29
- AQHDANHUMGXSJZ-UHFFFAOYSA-N baicalin Natural products OC1C(O)C(C(O)CO)OC1OC(C(=C1O)O)=CC2=C1C(=O)C=C(C=1C=CC=CC=1)O2 AQHDANHUMGXSJZ-UHFFFAOYSA-N 0.000 claims description 29
- 229960003321 baicalin Drugs 0.000 claims description 29
- 241000995749 Polyporus brumalis Species 0.000 claims description 28
- BJBKXYIIWYIZCX-UHFFFAOYSA-N Trigraecum Natural products C1=C(O)C(OC)=CC(C(C=2)=O)=C1OC=2C1=CC=CC=C1 BJBKXYIIWYIZCX-UHFFFAOYSA-N 0.000 claims description 22
- 239000001963 growth medium Substances 0.000 claims description 19
- RTIXKCRFFJGDFG-UHFFFAOYSA-N chrysin Chemical compound C=1C(O)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=CC=C1 RTIXKCRFFJGDFG-UHFFFAOYSA-N 0.000 claims description 16
- 238000012258 culturing Methods 0.000 claims description 13
- 238000007069 methylation reaction Methods 0.000 claims description 10
- NYCXYKOXLNBYID-UHFFFAOYSA-N 5,7-Dihydroxychromone Natural products O1C=CC(=O)C=2C1=CC(O)=CC=2O NYCXYKOXLNBYID-UHFFFAOYSA-N 0.000 claims description 8
- 229940043370 chrysin Drugs 0.000 claims description 8
- 235000015838 chrysin Nutrition 0.000 claims description 8
- 230000033444 hydroxylation Effects 0.000 claims description 8
- 238000005805 hydroxylation reaction Methods 0.000 claims description 8
- LGQKSQQRKHFMLI-SJYYZXOBSA-N (2s,3r,4s,5r)-2-[(3r,4r,5r,6r)-4,5,6-trihydroxyoxan-3-yl]oxyoxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)CO[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)OC1 LGQKSQQRKHFMLI-SJYYZXOBSA-N 0.000 claims description 6
- LGQKSQQRKHFMLI-UHFFFAOYSA-N 4-O-beta-D-xylopyranosyl-beta-D-xylopyranose Natural products OC1C(O)C(O)COC1OC1C(O)C(O)C(O)OC1 LGQKSQQRKHFMLI-UHFFFAOYSA-N 0.000 claims description 6
- SQNRKWHRVIAKLP-UHFFFAOYSA-N D-xylobiose Natural products O=CC(O)C(O)C(CO)OC1OCC(O)C(O)C1O SQNRKWHRVIAKLP-UHFFFAOYSA-N 0.000 claims description 6
- 238000005906 dihydroxylation reaction Methods 0.000 claims description 6
- 239000001257 hydrogen Substances 0.000 claims description 6
- 229910052739 hydrogen Inorganic materials 0.000 claims description 6
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 6
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- 125000003860 C1-C20 alkoxy group Chemical group 0.000 claims description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 2
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 27
- 101100541106 Aspergillus oryzae (strain ATCC 42149 / RIB 40) xlnD gene Proteins 0.000 description 21
- 238000005481 NMR spectroscopy Methods 0.000 description 13
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- 235000000346 sugar Nutrition 0.000 description 11
- 241000221198 Basidiomycota Species 0.000 description 10
- 150000002972 pentoses Chemical group 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 230000011987 methylation Effects 0.000 description 9
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- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 8
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- 229940097043 glucuronic acid Drugs 0.000 description 5
- HMUNWXXNJPVALC-UHFFFAOYSA-N 1-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)C(CN1CC2=C(CC1)NN=N2)=O HMUNWXXNJPVALC-UHFFFAOYSA-N 0.000 description 4
- 241001520612 Neolentinus lepideus Species 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 4
- 235000019439 ethyl acetate Nutrition 0.000 description 4
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 2
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- 108010022337 Leucine Enkephalin Proteins 0.000 description 2
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- ZRSNZINYAWTAHE-UHFFFAOYSA-N p-methoxybenzaldehyde Chemical compound COC1=CC=C(C=O)C=C1 ZRSNZINYAWTAHE-UHFFFAOYSA-N 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
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- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 description 2
- JYKYYPPZLPVIBY-UHFFFAOYSA-N 1-isothiocyanato-2-methylbenzene Chemical compound CC1=CC=CC=C1N=C=S JYKYYPPZLPVIBY-UHFFFAOYSA-N 0.000 description 1
- LDXJRKWFNNFDSA-UHFFFAOYSA-N 2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]ethanone Chemical compound C1CN(CC2=NNN=C21)CC(=O)N3CCN(CC3)C4=CN=C(N=C4)NCC5=CC(=CC=C5)OC(F)(F)F LDXJRKWFNNFDSA-UHFFFAOYSA-N 0.000 description 1
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- 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
- 230000001939 inductive effect Effects 0.000 description 1
- URLZCHNOLZSCCA-UHFFFAOYSA-N leu-enkephalin Chemical compound C=1C=C(O)C=CC=1CC(N)C(=O)NCC(=O)NCC(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=CC=C1 URLZCHNOLZSCCA-UHFFFAOYSA-N 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
- C07H17/07—Benzo[b]pyran-4-ones
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- Zoology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
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- Saccharide Compounds (AREA)
Abstract
플라보노이드 유도체 및 이의 제조 방법에 관한 것으로, 상기 방법에 의하면, 미생물의 생물 전환을 이용하여 물질의 구조적 변화를 유도할 수 있는 바, 전구 물질로부터 다양한 유도체의 제조가 가능하다. 특히, 플라보노이드의 자일로실화 산물들을 선택적으로 수득할 수 있으므로, 5탄당 당화 효소를 경제적으로 제조할 수 있다.It relates to a flavonoid derivative and a method for producing the same. According to the method, a structural change of a substance can be induced using biotransformation of microorganisms, and various derivatives can be prepared from precursors. In particular, since xylosylation products of flavonoids can be selectively obtained, pentose saccharide enzymes can be economically produced.
Description
생물전환법을 이용하여 자일로실화된 플라보노이드 유도체를 생산하는 방법에 관한 것이다.It relates to a method for producing xylosylated flavonoid derivatives using bioconversion.
천연물 또는 천연물 유래 의약품은 오랜 기간의 경험을 근거로 이미 안정성 및 유효성이 검증되어 왔다. 또한, 합성 의약품 개발에 비해 상대적으로 구조적, 생물학적 효능이 다양할 뿐만 아니라 개발 성공률이 높고 비용이 적게 소요된다는 장점이 있다. 한편, 생체 변환(biotransformation)은 미생물이 가지고 있는 효소적 기능을 이용하여 전구물질로부터 다양한 산물을 제조하는 방법으로서, 미생물 발효나 효소 처리 등의 생물학적 방법을 통해 물질의 구조적 변화를 유도한다. The stability and efficacy of natural products or drugs derived from natural products have already been verified based on long-term experience. In addition, compared to the development of synthetic drugs, they have relatively diverse structural and biological efficacy, as well as a high development success rate and low cost. On the other hand, biotransformation is a method of producing various products from precursors by using the enzymatic function of microorganisms, and induces structural changes in substances through biological methods such as microbial fermentation or enzyme treatment.
플라보노이드는 야채, 과일 등에서 발견되는 식물 유해 2차 대사산물로서 항산화, 심장 보호 및 신경 보호와 같은 인체 건강에 유익한 효과를 나타낼 뿐만 아니라, 항균, 항바이러스 등을 포함한 약리학적 활성을 나타내는 것으로 보고되고 있다. 상기 플라보노이드는 C6-C3-C6의 기본 구조로 이루어져 있으며, 기본 구조의 어느 위치에 어떤 작용기가 결합되어 있는지 여부에 따라 물리화학적, 생물학적 및 대사적 특성을 조절하는데 중요한 역할을 한다. Flavonoids, as secondary metabolites found in vegetables and fruits, are reported to have beneficial effects on human health such as antioxidant, heart protection and neuroprotection, as well as pharmacological activities including antibacterial and antiviral activities. . The flavonoid has a basic structure of C6-C3-C6, and plays an important role in regulating physicochemical, biological and metabolic properties depending on which functional group is attached to which position of the basic structure.
따라서, 미생물을 이용하여 플라보노이드의 생체 변환을 유도함으로써 새로운 화합물을 생산하는 방법을 개발할 필요가 있다. Therefore, it is necessary to develop a method for producing new compounds by inducing biotransformation of flavonoids using microorganisms.
일 양상은 자일로실화된 플라보노이드 유도체(Xylosylated flavonoid derivatives)를 제공하는 것이다. One aspect is to provide xylosylated flavonoid derivatives.
다른 양상은 폴리포러스 브루말리스(Polyporus brumalis) 균주 및 플라보노이드를 배양하는 단계를 포함하는 자일로실화된 플라보노이드 유도체의 제조방법을 제공하는 것이다. Another aspect is to provide a method for preparing a xylosylated flavonoid derivative comprising culturing a Polyporus brumalis strain and a flavonoid.
또 다른 양상은 상기 방법에 의해 제조된 자일로실화된 플라보노이드 유도체를 제공하는 것이다. Another aspect is to provide a xylosylated flavonoid derivative prepared by the above method.
일 양상은 자일로실화된 플라보노이드 유도체(Xylosylated flavonoid derivatives)를 제공한다. One aspect provides xylosylated flavonoid derivatives.
일 구체예에 있어서, 상기 자일로실화된 플라보노이드 유도체는 플라보노이드가 O-자일로스화(O-xylosylation) 또는 O-글루코스화(O-glucoxylation)된 화합물인 것일 수 있다. In one embodiment, the xylosylated flavonoid derivative may be a compound obtained by O-xylosylation or O-glucosylation of flavonoids.
다른 구체예에 있어서, 상기 바이칼린 유도체는 하기 화학식 10으로 표시될 수 있다;In another embodiment, the baicalin derivative may be represented by Formula 10;
[화학식 10][Formula 10]
상기에서, From above,
R1, R4 및 R5는 각각 독립적으로 수소(H) 또는 하이드록시(OH)이고, R 1 , R 4 and R 5 are each independently hydrogen (H) or hydroxy (OH);
R2는 -O-자일로피라노사이드(xyropyranoside), -O-자일로퓨라노사이드(Xylopuranoside), -O-자일로피라노사이드(xyropyranoside), -O-자일로퓨라노사이드(xylopuranoside), -O-라이보퓨라노사이드(ribofuranoside), -O-아라비노피라노사이드(arabinopyranoside), -O-릭소피라노사이드(lyxopyranoside), -O-리보피라노사이드(ribopyranoside), 및 -O-자일로바이오스(xylobiose)로 구성된 군에서 선택된 것이고, R 2 is -O-xyropyranoside, -O-xylopuranoside, -O-xyropyranoside, -O-xylopuranoside , -O-ribofuranoside, -O-arabinopyranoside, -O-lyxopyranoside, -O-ribopyranoside, and -O- It is selected from the group consisting of xylobiose,
R3은 수소(H), 하이드록시(OH), 치환 또는 비치환된 C1-C20 알콕시, -O-자일로피라노사이드(xyropyranoside), -O-자일로퓨라노사이드(xylopuranoside), -O-라이보퓨라노사이드(ribofuranoside), -O-아라비노피라노사이드(arabinopyranoside), -O-릭소피라노사이드(lyxopyranoside), -O-리보피라노사이드(ribopyranoside), 및 -O-자일로바이오스(xylobiose)로 구성된 군에서 선택된 것이다. 상기 R2는 D-형(D-form)의 다당류인 것일 수 있다. R 3 is hydrogen (H), hydroxy (OH), substituted or unsubstituted C 1 -C 20 alkoxy, -O-xyropyranoside, -O-xylopuranoside, -O-ribofuranoside, -O-arabinopyranoside, -O-lyxopyranoside, -O-ribopyranoside, and -O-xyl It is selected from the group consisting of xylobiose. The R 2 may be a D-form polysaccharide.
또 다른 구체예에 있어서, 상기 자일로실화된 플라보노이드 유도체는 바이칼린(bicalin) 또는 바이칼레인(bicalein)인 것일 수 있다. 본 명세서에서 용어, "바이칼레인(bicalein)"은 황금 (Scutellaria baicalensis Georgi)의 뿌리에서 추출되는 주요 생리 활성 화합물로서, B-고리에 4'-하이드록시(OH)가 없는 특이적인 구조를 가지는 플라보노이드이다. In another embodiment, the xylosylated flavonoid derivative may be bicalin or bicalein. As used herein, the term "bicalein" is a major physiologically active compound extracted from the root of Scutellaria baicalensis Georgi, a flavonoid having a specific structure without 4'-hydroxy (OH) in the B-ring. am.
본 명세서에서 용어, "바이칼린(bicalin)"은 바이칼레인의 7-하이드록시기(OH)에 β-글루쿠론산(β-glucuronic acid)이 결합된 유도체이다. As used herein, the term "bicalin" is a derivative in which β-glucuronic acid is bound to the 7-hydroxyl group (OH) of baicalin.
상기 플라보노이드는 항산화, 심장 보호 및 신경 보호와 같은 인체 건강에 유익한 효과를 가질 뿐만 아니라, 항균, 항바이러스 등을 포함한 약리학적 활성을 나타낼 수 있다. 따라서, 일 구체예에 따른 바이칼린 또는 바이칼레인 유도체는 항산화, 심장보호, 신경 보호, 항균, 항바이러스의 활성을 가질 수 있다. The flavonoids may have beneficial effects on human health such as antioxidant, cardioprotective and neuroprotective, as well as pharmacological activities including antibacterial and antiviral. Accordingly, baicalin or baicalein derivatives according to one embodiment may have antioxidant, cardioprotective, neuroprotective, antibacterial, and antiviral activities.
상기 바이칼레인의 자일로실화된 유도체는 (a) 바이칼레인 7- O-β-D-자일로피라노사이드 [Baicalein 7-O-β-D-xylopyranoside]; (b) 크리신 7-o-β-D-자일로피라노사이드 [Chrysin 7-O-β-D-xylopyranoside]; (c) 바이칼레인 6,7-O-β-D-디자일로피라노사이드 [Baicalein 6,7-O-β-D-dixylopyranoside]; (d) 오록실린 A 7-O-β-D-자일로피라노사이드 [Oroxylin A 7-O-β-D-xylopyranoside]; (e) 오록실린 A 7-O-β-D-자일로피라노실-(1→3)-β-D-자일로피라노사이드 [Oroxylin A 7-O-β-D-xylopyranosyl-(1→3)-β-D-xylopyranoside]; (f) 5.8-디하이드록시-6-메톡시플라본 7-O-β-D-자일로피라노사이드 [5,8-dihydroxy-6-methoxyflavone 7-O-β-D-xylopyranoside]; (g) 5,8-디하이드록시-6-메톡시플라본 7-O-β-D-자일로피라노실-(1→3)-β-d-자일로피라노사이드 [5,8-dihydroxy-6-methoxyflavone 7-O-β-D-xylopyranosyl-(1→3)-β-d-xylopyranoside]; 및 (h) 오록실린 A [Oroxylin A] 로 구성된 군에서 선택되는 것일 수 있다. The xylosylated derivatives of baicalein include (a) Baicalein 7-O-β-D-xylopyranoside; (b) Chrysin 7-O-β-D-xylopyranoside [Chrysin 7-O-β-D-xylopyranoside]; (c) Baicalein 6,7-O-β-D-dixylopyranoside; (d) Oroxylin A 7-O-β-D-xylopyranoside [Oroxylin A 7-O-β-D-xylopyranoside]; (e) Oroxylin A 7-O-β-D-xylopyranosyl-(1→3)-β-D-xylopyranoside [Oroxylin A 7-O-β-D-xylopyranosyl-(1→3) 3) -β-D-xylopyranoside]; (f) 5,8-dihydroxy-6-methoxyflavone 7-O-β-D-xylopyranoside [5,8-dihydroxy-6-methoxyflavone 7-O-β-D-xylopyranoside]; (g) 5,8-dihydroxy-6-methoxyflavone 7-O-β-D-xylopyranosyl-(1→3)-β-d-xylopyranoside [5,8-dihydroxy -6-methoxyflavone 7-O-β-D-xylopyranosyl-(1→3)-β-d-xylopyranoside]; And (h) may be selected from the group consisting of oroxylin A [Oroxylin A].
본 명세서에서 용어, "생물 전환(biotransformation)"은 미생물이 가지고 있는 효소적 기능을 이용하여 전구 물질로부터 다양한 산물을 제조하는 방법을 의미하며, 미생물 발효나 효소 처리 등의 생물학적 방법을 통해 물질의 구조적 변화를 유도하는 것이다. 일 실시예에서는 폴리포러스 브루말리스 균주와 바이칼린을 배양한 배양액에서 신규한 바이칼린 유도체를 수득함으로써, 상기 균주에 의한 신규 화합물을 제조하였다. 따라서, 일 구체예에 있어서, 상기 바이칼레인 유도체는 폴리포러스 브루말리스(Polyporus brumalis) 균주의 생물 전환(biotransformation)에 의해 제조된 것일 수 있다. As used herein, the term "biotransformation" refers to a method of producing various products from precursors using the enzymatic function of microorganisms, and the structural transformation of substances through biological methods such as microbial fermentation or enzymatic treatment is to induce change. In one embodiment, a novel compound by the strain was prepared by obtaining a novel baicalin derivative from a culture medium in which the Polyporus brumalis strain and baicalin were cultured. Therefore, in one embodiment, the baicalein derivative may be prepared by biotransformation of a Polyporus brumalis strain.
다른 구체예에 있어서, 상기 바이칼린 유도체는 바이칼린 화합물의 글루쿠로닐(glucuronyl) 결합이 분해되어 바이칼레인(baicalein)이 생성되는 단계; 및 상기 바이칼레인의 6-OH 및 7-OH 위치로 구성된 군에서 선택되는 어느 하나 이상의 위치가 자일로실화(xylosylation), 하이드록실화(hydroxylation), 디하이드록실화(dihydroxylation), 또는 메틸화(methylation) 반응이 이루어지는 단계에 의해 생성되는 것일 수 있다. 상기 바이칼레인은 상기 폴리포러스 브루말리스 및 바이칼린을 배양한 후, 0 내지 2일 이내에 상기 글루코시드 결합이 분해됨으로써 생성되는 것일 수 있다. 구체적으로, 상기 바이칼린의 7-OH 위치에서 글루코시드 결합이 분해되어 글루쿠론산(glucuronic acid)이 제거되는 것일 수 있다. 따라서, 상기 균주는 β-글루코시다제(β-glucosidase) 활성을 갖는 것일 수 있다. In another embodiment, the baicalin derivative comprises the steps of decomposing the glucuronyl bond of the baicalin compound to produce baicalein; And at least one position selected from the group consisting of the 6-OH and 7-OH positions of the baicalein is xylosylation, hydroxylation, dihydroxylation, or methylation ) may be produced by the step in which the reaction takes place. The baicalein may be produced by cleavage of the glucosidic bond within 0 to 2 days after culturing the Polyporus brumalis and baicalin. Specifically, the glucuronic acid may be removed by cleavage of the glucosidic bond at the 7-OH position of the baicalin. Therefore, the strain may have β-glucosidase activity.
또한, 상기 바이칼레인이 자일로실화, 하이드록실화, 디하이드록실화, 또는 메틸화반응이 이루어지는 단계는 바이칼레인의 6-OH 및/또는 7-OH 위치가 자일로실화, 하이드록실화 또는 메틸화 되어 1차 바이칼레인 유도체를 생성하는 단계; 상기 1차 바이칼레인 유도체의 6-OH 및/또는 7-OH 위치가 자일로실화 또는 하이드록실화 되어 2차 바이칼레인 유도체를 생성하는 단계를 포함할 수 있다. 상기 자일로실화는 바이칼레인의 6-OH 및/또는 7-OH 위치에 5탄당 또는 6탄당이 결합하는 것일 수 있다. In addition, in the step of xylosylation, hydroxylation, dehydroxylation, or methylation of baicalein, the 6-OH and/or 7-OH positions of baicalein are xylosylated, hydroxylated, or methylated. producing a primary baicalein derivative; A step of generating a secondary baicalein derivative by xylosylating or hydroxylating the 6-OH and/or 7-OH positions of the primary baicalein derivative may be included. The xylosylation may be a pentose or hexose bonded to the 6-OH and/or 7-OH positions of baicalein.
도 12b는 폴리포러스 브루말리스 균주의 생체 촉매 반응 따른 화합물의 생성 과정을 나타낸 개략도이다. 도 12b를 참조하여 설명하면, 화학식 1 내지 화학식 3으로 표시되는 화합물은 바이칼레인의 7-OH 위치가 자일로실화 됨으로써 생성되는 것일 수 있다. 구체적으로, 상기 화학식 1로 표시되는 화합물은 바이칼레인의 7-OH 위치가 자일로실화 되어 생성되는 것일 수 있다. 또한, 상기 화학식 2로 표시되는 화합물은 바이칼레인의 7-OH 위치가 자일로실화 되어 화학식 1로 표시되는 화합물을 생성하는 단계; 및 상기 화학식 1로 표시되는 화합물의 6-OH 위치가 디하이드록실화 되는 단계에 의해 생성되는 것일 수 있다. 또한, 상기 화학식 3으로 표시되는 화합물은 바이칼레인의 7-OH 위치가 자일로실화되어 화학식 1로 표시되는 화합물을 생성하는 단계; 및 상기 화학식 1로 표시되는 화합물의 6-OH 위치가 자일로실화 되는 단계에 의해 생성되는 것일 수 있다. Figure 12b is a schematic diagram showing the production process of the compound according to the biocatalytic reaction of Polyporus brumalis strain. Referring to FIG. 12B, the compounds represented by Chemical Formulas 1 to 3 may be produced by xylosylation of the 7-OH position of baicalein. Specifically, the compound represented by Formula 1 may be produced by xylosylation of the 7-OH position of baicalein. In addition, the compound represented by Formula 2 is xylosylated at the 7-OH position of baicalein to produce a compound represented by Formula 1; And it may be produced by dehydroxylating the 6-OH position of the compound represented by Formula 1. In addition, the compound represented by Formula 3 is xylosylated at the 7-OH position of baicalein to produce a compound represented by Formula 1; And it may be produced by xylosylating the 6-OH position of the compound represented by Formula 1.
또한, 상기 화학식 4 내지 8로 표시되는 화합물은 바이칼레인의 6-OH 위치가 메틸화 됨으로써 생성되는 것일 수 있다. 구체적으로, 상기 화학식 8로 표시되는 화합물은 바이칼레인의 6-OH 위치가 메틸화 되어 생성된 것일 수 있다. 또한, 상기 화학식 4로 표시되는 화합물은 바이칼레인의 6-OH 위치가 메틸화 되어 화학식 8로 표시되는 화합물을 생성하는 단계; 및 상기 화학식 8로 표시되는 화합물의 7-OH가 자일로실화 되는 단계에 의해 생성되는 것일 수 있다. 또한, 화학식 5로 표시되는 화합물은 바이칼레인의 6-OH 위치가 메틸화 되어 화학식 8로 표시되는 화합물을 생성하는 단계; 상기 화학식 8로 표시되는 화합물의 7-OH가 자일로실화 되어 화학식 4로 표시되는 화합물을 생성하는 단계; 및 상기 화학식 4로 표시되는 화합물의 자일로실기의 2-OH, 3-OH 또는 4-OH 위치가 자일로실화 되는 단계에 의해 생성되는 것일 수 있다. In addition, the compounds represented by Chemical Formulas 4 to 8 may be produced by methylation of the 6-OH position of baicalein. Specifically, the compound represented by Chemical Formula 8 may be produced by methylation of the 6-OH position of baicalein. In addition, the compound represented by Chemical Formula 4 is methylated at the 6-OH position of baicalein to produce a compound represented by Chemical Formula 8; And it may be produced by xylosylating 7-OH of the compound represented by Formula 8. In addition, the compound represented by Formula 5 is methylated at the 6-OH position of baicalein to produce a compound represented by Formula 8; generating a compound represented by Chemical Formula 4 by xylosylating 7-OH of the compound represented by Chemical Formula 8; And it may be produced by xylosylation of the 2-OH, 3-OH or 4-OH position of the xylosyl group of the compound represented by Formula 4.
또한, 상기 화학식 6으로 표시되는 화합물은 바이칼레인의 6-OH 위치가 메틸화 되어 화학식 8로 표시되는 화합물을 생성하는 단계; 상기 화학식 8로 표시되는 화합물의 7-OH가 자일로실화 되어 화학식 4로 표시되는 화합물을 생성하는 단계; 및 상기 화학식 4로 표시되는 화합물의 8-H 위치가 하이드록실화 되는 단계에 의해 생성되는 것일 수 있다. 또한, 상기 화학식 7로 표시되는 화합물은 바이칼레인의 6-OH 위치가 메틸화 되어 화학식 8로 표시되는 화합물을 생성하는 단계; 상기 화학식 8로 표시되는 화합물의 7-OH가 자일로실화 되어 화학식 4로 표시되는 화합물을 생성하는 단계; 상기 화학식 4로 표시되는 화합물의 6-CH3 위치가 하이드록실화 되어 화학식 6으로 표시되는 화합물을 생성하는 단계; 및 상기 화학식 6으로 표시되는 화합물의 자일로실기의 2-OH, 3-OH 또는 4-OH 위치가 자일로실화 되는 단계에 의해 생성되는 것일 수 있다. In addition, the compound represented by Chemical Formula 6 is methylated at the 6-OH position of baicalein to produce a compound represented by Chemical Formula 8; generating a compound represented by Chemical Formula 4 by xylosylating 7-OH of the compound represented by Chemical Formula 8; And it may be produced by hydroxylating the 8-H position of the compound represented by Formula 4. In addition, the compound represented by Chemical Formula 7 is methylated at the 6-OH position of baicalein to produce a compound represented by Chemical Formula 8; generating a compound represented by Chemical Formula 4 by xylosylating 7-OH of the compound represented by Chemical Formula 8; hydroxylating the 6-CH3 position of the compound represented by Chemical Formula 4 to produce a compound represented by Chemical Formula 6; And it may be produced by xylosylation of the 2-OH, 3-OH or 4-OH position of the xylosyl group of the compound represented by Formula 6.
다른 양상은 폴리포러스 브루말리스(Polyporus brumalis) 균주를 플라보노이드와 배양하는 단계를 포함하는 자일로실화된 플라보노이드 유도체의 제조방법을 제공한다. 상기 플라보노이드 유도체의 구체적인 내용은 전술한 바와 같다. Another aspect provides a method for producing a xylosylated flavonoid derivative comprising culturing a Polyporus brumalis strain with a flavonoid. Details of the flavonoid derivative are as described above.
일 구체예에 있어서, 상기 방법은 폴리포러스 브루말리스 균주를 플라보노이드와 배양하여 배양액을 획득하는 단계; 및 상기 배양액으로부터 자일로실화된 플라보노이드 유도체를 수득하는 단계를 포함할 수 있다. In one embodiment, the method comprises culturing a Polyporus brumalis strain with flavonoids to obtain a culture medium; and obtaining a xylosylated flavonoid derivative from the culture medium.
상기 폴리포러스 브루말리스 균주를 플라보노이드와 배양하는 단계는 1 내지 10일 동안 수행될 수 있다. 상기 배양은 예를 들어, 1 내지 10일, 1 내지 8일, 1 내지 6일, 2 내지 9일, 3 내지 8일 또는 4 내지 7일 동안 수행될 수 있다. 이때, 상기 배양 기간이 상기 범위 미만인 경우, 플라보노이드가 자일로실화된 플라보노이드 유도체로 충분히 전환될 수 없으며, 상기 범위를 초과하는 경우, 목적으로 하는 플라보노이드 유도체 외에 부산물(by-product)이 추가로 생성되는 문제점이 있다. The step of culturing the Polyporus brumalis strain with flavonoids may be performed for 1 to 10 days. The culturing may be performed for, for example, 1 to 10 days, 1 to 8 days, 1 to 6 days, 2 to 9 days, 3 to 8 days, or 4 to 7 days. At this time, when the culture period is less than the above range, flavonoids cannot be sufficiently converted into xylosylated flavonoid derivatives, and when it exceeds the above range, by-products are additionally produced in addition to the target flavonoid derivative. There is a problem.
또한, 상기 폴리포러스 브루말리스 균주와 플라보노이드를 배양하는 단계는 플라보노이드의 글루코시드 결합이 분해되는 바이칼레인이 생성되는 단계; 및 상기 플라보노이드의 6-OH 및 7-OH 위치로 구성된 군에서 선택되는 어느 하나 이상의 위치가 자일로실화, 하이드록실화, 디하이드록실화 또는 메틸화 반응에 의해 자일로실화된 플라보노이드 유도체를 생성하는 단계를 포함할 수 있다. 상기 자일로실화된 플라보노이드 유도체를 생성하는 단계의 구체적인 내용은 전술한 바와 같다. In addition, the step of culturing the Polyporus brumalis strain and the flavonoids may include producing baicalein in which the glucosidic bond of the flavonoid is broken down; and generating a flavonoid derivative in which at least one position selected from the group consisting of 6-OH and 7-OH positions of the flavonoid is xylosylated by xylosylation, hydroxylation, dehydroxylation or methylation. can include Details of the step of generating the xylosylated flavonoid derivative are as described above.
상기 배양액으로부터 자일로실화된 플라보노이드 유도체를 수득하는 단계는 상기 배양액을 물, C1 내지 C4의 저급 알코올 또는 이들의 혼합물을 용매로 추출하여 배양액의 추출물을 제조하는 단계; 상기 추출물을 크로마토그래피(chromatography)를 수행하여 활성 분획을 수거하는 단계; 및 상기 활성 분획을 크로마토그래피로 분리하여 목적 화합물을 얻는 단계를 포함할 수 있다. Obtaining a xylosylated flavonoid derivative from the culture medium may include preparing an extract of the culture medium by extracting the culture medium with water, C1 to C4 lower alcohol, or a mixture thereof as a solvent; Collecting an active fraction by performing chromatography on the extract; and obtaining a target compound by separating the active fraction by chromatography.
또 다른 양상은 상기 방법에 의해 제조된 자일로실화된 플라보노이드 유도체를 제공한다. 상기 자일로실화된 플라보노이드 유도체의 구체적인 내용은 전술한 바와 같다. Another aspect provides a xylosylated flavonoid derivative prepared by the above method. Details of the xylosylated flavonoid derivative are as described above.
일 양상에 따른 방법에 의하면, 미생물의 생물 전환을 이용하여 물질의 구조적 변화를 유도할 수 있는 바, 전구 물질로부터 다양한 유도체의 제조가 가능하다. 또한, 자일로실화 산물들을 선택적으로 수득할 수 있으므로, 5탄당 당화 효소를 경제적으로 제조할 수 있다. According to the method according to one aspect, a structural change of a substance can be induced using biotransformation of microorganisms, and various derivatives can be prepared from precursors. In addition, since xylosylation products can be selectively obtained, the pentose saccharide enzyme can be economically produced.
도 1은 담자균 14종을 분류한 그림이다.
도 2a는 대사 산물의 변화를 나타낸 4종의 담자균에서 바이칼린 첨가에 따라 생성된 바이칼레인 유도체 피크(A-I)를 확인한 결과이다.
도 2a는 대사 산물의 변화를 나타낸 4종의 담자균에서 바이칼린 첨가에 따라 생성된 바이칼린 유도체 피크(A-I)를 확인한 결과이다.
도 2b는 대사 산물의 변화를 나타낸 4종의 담자균에서 바이칼레인 검출을 확인한 결과이다.
도 3은 폴리포러스 브루말리스(Polyporus brumalis) 균주에 의해 생물 전환되는 바이칼레인 유도체 구조를 확인한 결과이다.
도 4a는 일 구체예에 따른 바이칼레인 유도체(화합물 1)의 1H NMR 스펙트럼 결과이다.
도 4b는 일 구체예에 따른 바이칼레인 유도체(화합물 1)의 13C NMR 스펙트럼 결과이다.
도 4c는 일 구체예에 따른 바이칼레인 유도체(화합물 1)의 HSQC 스펙트럼 결과??.
도 4d는 일 구체예에 따른 바이칼레인 유도체(화합물 1)의 HMBC 스펙트럼 결과이다.
도 4e는 일 구체예에 따른 바이칼레인 유도체(화합물 1)의 1H-1H COSY 스펙트럼 결과이다.
도 5a는 일 구체예에 따른 바이칼레인 유도체(화합물 2)의 1H NMR 스펙트럼 결과이다.
도 5b는 일 구체예에 따른 바이칼레인 유도체(화합물 2)의 13C NMR 스펙트럼 결과이다.
도 5c는 일 구체예에 따른 바이칼레인 유도체(화합물 2)의 HSQC 스펙트럼 결과이다.
도 5d는 일 구체예에 따른 바이칼레인 유도체(화합물 2)의 HMBC 스펙트럼 결과이다.
도 5d는 일 구체예에 따른 바이칼레인 유도체(화합물 2)의 1H-1H COSY 스펙트럼 결과이다.
도 6a는 일 구체예에 따른 바이칼레인 유도체(화합물 3)의 1H NMR 스펙트럼 결과이다.
도 6b는 일 구체예에 따른 바이칼레인 유도체(화합물 3)의 13C NMR 스펙트럼 결과이다.
도 6c는 일 구체예에 따른 바이칼레인 유도체(화합물 3)의 HSQC 스펙트럽 결과이다.
도 6d는 일 구체예에 따른 바이칼레인 유도체(화합물 3)의 HMBC 스펙트럼 결과이다.
도 6e는 일 구체예에 따른 바이칼레인 유도체(화합물 3)의 1H-1H COSY 스펙트럼 결과이다.
도 7a는 일 구체예에 따른 바이칼레인 유도체(화합물 4)의 1H NMR 스펙트럽 결과이다.
도 7b는 일 구체예에 따른 바이칼레인 유도체(화합물 4)의 13C NMR 스펙트럼 결과이다.
도 7c는 일 구체예에 따른 바이칼레인 유도체(화합물 4)의 HSQC 스펙트럼 결과이다.
도 7d는 일 구체예에 따른 바이칼레인 유도체(화합물 4)의 HMBC 스펙트럼 결과이다.
도 7e는 일 구체예에 따른 바이칼레인 유도체(화합물 4)의 1H-1H COSY 스펙트럼 결과이다.
도 8a는 일 구체예에 따른 바이칼레인 유도체(화합물 5)의 1H NMR 스펙트럼 결과이다.
도 8b는 일 구체예에 따른 바이칼레인 유도체(화합물 5)의 13C NMR 스펙트럼 결과이다.
도 8c는 일 구체예에 따른 바이칼레인 유도체(화합물 5)의 HSQC 스펙트럼 결과이다.
도 8d는 일 구체예에 따른 바이칼레인 유도체(화합물 5)의 HMBC스펙트럼 결과이다.
도 8e는 일 구체예에 따른 바이칼레인 유도체(화합물 5)의 1H-1H COSY 스펙트럼 결과이다.
도 9a는 일 구체예에 따른 바이칼레인 유도체(화합물 6)의 1H NMR 스펙트럼 결과이다.
도 9b는 일 구체예에 따른 바이칼레인 유도체(화합물 6)의 13C NMR 스펙트럼 결과이다.
도 9c는 일 구체예에 따른 바이칼레인 유도체(화합물 6)의 HSQC스펙트럼 결과이다.
도 9d는 일 구체예에 따른 바이칼레인 유도체(화합물 6)의 HMBC 스펙트럼 결과이다.
도 9e는 일 구체예에 따른 바이칼레인 유도체(화합물 6)의 1H-1H COSY 스펙트럼 결과이다.
도 10a는 일 구체예에 따른 바이칼레인 유도체(화합물 7)의 1H NMR 스펙트럼 결과이다.
도 10b는 일 구체예에 따른 바이칼레인 유도체(화합물 7)의 13C NMR 스펙트럼 결과이다.
도 10c는 일 구체예에 따른 바이칼레인 유도체(화합물 7)의 HSQC 스펙트럼 결과이다.
도 10d는 일 구체예에 따른 바이칼레인 유도체(화합물 7)의 HMBC 스펙트럼 결과이다.
도 10e는 일 구체예에 따른 바이칼레인 유도체(화합물 7)의 1H-1H COSY 스펙트럼 결과이다.
도 11a는 일 구체예에 따른 바이칼레인 유도체(화합물 8)의 1H NMR 스펙트럼 결과이다.
도 11b는 일 구체예에 따른 바이칼레인 유도체(화합물 8)의 13C NMR 스펙트럼 결과이다.
도 11c는 일 구체예에 따른 바이칼레인 유도체(화합물 8)의 HSQC 스펙트럼 결과이다.
도 11d는 일 구체예에 따른 바이칼레인 유도체(화합물 8)의 HMBC 스펙트럼 결과이다.
도 11e는 일 구체예에 따른 바이칼레인 유도체(화합물 8)의 1H-1H COSY 스펙트럼 결과이다.
도 12a는 일 구체예에 따른 화합물의 MS 데이터 피크 면적에 의한 히트맵을 나타낸다.
도 12b는 폴리포러스 브루말리스 균주의 생체 촉매 반응에 따른 화합물의 생성 과정을 나타낸 개략도이다.1 is a picture showing the classification of 14 species of basidiomycetes.
Figure 2a is a result of confirming the baicalin derivative peak (AI) generated according to the addition of baicalin in four species of basidiomycetes showing changes in metabolites.
Figure 2a is a result of confirming the baicalin derivative peak (AI) generated according to the addition of baicalin in four species of basidiomycetes showing changes in metabolites.
Figure 2b is the result of confirming the detection of baicalein in four species of basidiomycetes showing changes in metabolites.
3 is a result of confirming the structure of a baicalein derivative bioconverted by a Polyporus brumalis strain.
4a is a 1 H NMR spectrum result of a baicalein derivative (Compound 1) according to one embodiment.
Figure 4b is a 13 C NMR spectrum of a baicalein derivative (Compound 1) according to one embodiment.
4c is an HSQC spectrum result of a baicalein derivative (Compound 1) according to one embodiment.
Figure 4d is an HMBC spectrum result of a baicalein derivative (Compound 1) according to one embodiment.
4e is a 1H-1H COZY spectrum result of a baicalein derivative (Compound 1) according to one embodiment.
5a is a 1 H NMR spectrum result of a baicalein derivative (Compound 2) according to one embodiment.
Figure 5b is a 13 C NMR spectrum of a baicalein derivative (Compound 2) according to one embodiment.
Figure 5c is a HSQC spectrum result of a baicalein derivative (Compound 2) according to one embodiment.
Figure 5d is an HMBC spectrum result of a baicalein derivative (Compound 2) according to one embodiment.
Figure 5d is a 1 H- 1 H COZY spectrum of a baicalein derivative (Compound 2) according to one embodiment.
6a is a 1 H NMR spectrum result of a baicalein derivative (Compound 3) according to one embodiment.
Figure 6b is a 13 C NMR spectrum result of a baicalein derivative (Compound 3) according to one embodiment.
Figure 6c is a HSQC spectrum result of a baicalein derivative (Compound 3) according to one embodiment.
Figure 6d is an HMBC spectrum result of a baicalein derivative (Compound 3) according to one embodiment.
6e is a 1 H- 1 H COZY spectrum result of a baicalein derivative (Compound 3) according to one embodiment.
7a is a 1 H NMR spectrum result of a baicalein derivative (Compound 4) according to one embodiment.
Figure 7b is a 13 C NMR spectrum of a baicalein derivative (Compound 4) according to one embodiment.
Figure 7c is a HSQC spectrum result of a baicalein derivative (Compound 4) according to one embodiment.
Figure 7d is an HMBC spectrum result of a baicalein derivative (Compound 4) according to one embodiment.
Figure 7e is a 1 H- 1 H COZY spectrum of a baicalein derivative (Compound 4) according to one embodiment.
8a is a 1 H NMR spectrum result of a baicalein derivative (Compound 5) according to one embodiment.
Figure 8b is a 13 C NMR spectrum of a baicalein derivative (Compound 5) according to one embodiment.
Figure 8c is a HSQC spectrum result of a baicalein derivative (Compound 5) according to one embodiment.
Figure 8d is an HMBC spectrum result of a baicalein derivative (Compound 5) according to one embodiment.
Figure 8e is a 1 H- 1 H COZY spectrum of a baicalein derivative (Compound 5) according to one embodiment.
9a is a 1 H NMR spectrum result of a baicalein derivative (Compound 6) according to one embodiment.
Figure 9b is a 13 C NMR spectrum of a baicalein derivative (Compound 6) according to one embodiment.
Figure 9c is a HSQC spectrum result of a baicalein derivative (Compound 6) according to one embodiment.
Figure 9d is an HMBC spectrum result of a baicalein derivative (Compound 6) according to one embodiment.
Figure 9e is a 1 H- 1 H COZY spectrum of a baicalein derivative (Compound 6) according to one embodiment.
10a is a 1 H NMR spectrum result of a baicalein derivative (Compound 7) according to one embodiment.
Figure 10b is a 13 C NMR spectrum result of a baicalein derivative (Compound 7) according to one embodiment.
Figure 10c is a HSQC spectrum result of a baicalein derivative (Compound 7) according to one embodiment.
10d is an HMBC spectrum result of a baicalein derivative (Compound 7) according to one embodiment.
Figure 10e is a 1 H- 1 H COZY spectrum of a baicalein derivative (Compound 7) according to one embodiment.
11a is a 1 H NMR spectrum result of a baicalein derivative (Compound 8) according to one embodiment.
Figure 11b is a 13 C NMR spectrum of a baicalein derivative (Compound 8) according to one embodiment.
Figure 11c is a HSQC spectrum result of a baicalein derivative (Compound 8) according to one embodiment.
Figure 11d is an HMBC spectrum result of a baicalein derivative (Compound 8) according to one embodiment.
Figure 11e is a 1 H- 1 H COZY spectrum of a baicalein derivative (Compound 8) according to one embodiment.
Figure 12a shows a heat map by MS data peak area of a compound according to one embodiment.
Figure 12b is a schematic diagram showing the production process of the compound according to the biocatalytic reaction of the Polyporus brumalis strain.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.
[실시예][Example]
실시예 1. 생체 촉매를 선별하기 위한 담자균(Basidiomycetes)의 스크리닝Example 1. Screening of Basidiomycetes to Select Biocatalysts
미생물을 이용한 생물 전환법의 적절한 생체 촉매를 선별하기 위하여, 한반도진균자원은행에서 담자균을 분양 받은 후, 다양한 분류군 중에서 14종의 균주를 선택하여 종간 비교를 수행하였다(도 1 참조). 구체적으로, 상기 균주 14종을 포테이토 덱스트로스 아가(potato dextrose agar, PDA) 배지에 각각 접종하여 27℃에서 7일 동안 배양하였다. 이후, 포테이토 덱스트로스 브로스(potato dextrose broth, PDB) 배지 100 ㎖를 250 mL 플라스크에 넣고 27℃, 130 rpm의 회전 믹서(rotatory shaker)에서 7일 동안 상기 균주를 추가로 배양하였다. 이후, DMSO에 용해한 바이칼린을 상기 배양액에 첨가하여 배양하였다. 일정 시간(0일, 1일, 2일, 4일, 6일 및 8일) 경과 후, 배양 배지 1 ㎖를 회수한 뒤, 아세트산에틸(EtOAc) 용매(1㎖ x 2)로 배양액 추출물을 제조하였다. 이후, 증발농축기를 사용하여 상기 추출물을 농축한 뒤, LC-MS/MS 분석으로 추출물 내 대사산물을 확인하였다. 구체적으로, 상기 추출물을 50% MeOH/H2O에 1 ㎎/㎖의 농도로 녹여 PTFE 필터(0.22 ㎛, 13 ㎜)로 여과한 후 분석용 시료를 제조하였다. 이후, 0.1% 포름산이 포함된 H2O (A)와 MeCN (B)를 이동상으로 하여 10-100% B (0-12 분), 100-100% B (12-15 분), 10-10% B (15-18 분)의 농도 구배 조건으로 분석을 진행하였다. 유속은 0.3 ㎖/min으로 설정하였으며 컬럼의 온도는 40 ℃로 유지하였다. 질량 분석은 data-independent acquisition (DIA) mode로 MSe mode를 활용하였고, mass range는 50-1500 Da로 분석하였다. 또한, 질량 분석의 정확도를 위하여 측정 과정에서 leucine enkephalin (Tyr-Gly-Gly-Phe-Leu), m/z 554.2615 (in negative mode), 556.2771 (in positive mode)을 활용하는 LockSprayTM을 사용하였다. In order to select an appropriate biocatalyst for the bioconversion method using microorganisms, basidiomycetes were received from the Korean Peninsula Fungal Resource Bank, and 14 strains were selected from various taxonomic groups and compared between species (see FIG. 1). Specifically, each of the 14 strains was inoculated into a potato dextrose agar (PDA) medium and cultured at 27° C. for 7 days. Thereafter, 100 ml of potato dextrose broth (PDB) medium was added to a 250 ml flask, and the strain was further cultured for 7 days in a rotatory shaker at 27° C. and 130 rpm. Thereafter, baicalin dissolved in DMSO was added to the culture medium and cultured. After a certain period of time (0 days, 1 day, 2 days, 4 days, 6 days and 8 days), 1 ml of the culture medium is recovered, and then a culture medium extract is prepared with ethyl acetate (EtOAc) solvent (1 ml x 2) did Then, after concentrating the extract using an evaporator, metabolites in the extract were confirmed by LC-MS/MS analysis. Specifically, the extract was dissolved in 50% MeOH/H 2 O at a concentration of 1 mg/ml and filtered through a PTFE filter (0.22 μm, 13 mm) to prepare a sample for analysis. Then, 10-100% B (0-12 minutes), 100-100% B (12-15 minutes), 10-10 with H 2 O (A) and MeCN (B) containing 0.1% formic acid as a mobile phase The analysis was performed under a concentration gradient condition of % B (15-18 minutes). The flow rate was set to 0.3 ml/min and the temperature of the column was maintained at 40 °C. For mass spectrometry, MSe mode was used as a data-independent acquisition (DIA) mode, and the mass range was analyzed at 50-1500 Da. In addition, for the accuracy of mass spectrometry, LockSpray TM was used that utilizes leucine enkephalin (Tyr-Gly-Gly-Phe-Leu), m/z 554.2615 (in negative mode), and 556.2771 (in positive mode) in the measurement process.
도 1은 담자균 14종을 분류한 그림이다. 1 is a picture showing the classification of 14 species of basidiomycetes.
그 결과, 14종의 균주 중에서 4종의 균주(Polyporus brumalis, Fomes fomentarius, Postia stiptica, Neolentinus lepideus)는 바이칼린 유무에 따른 대사 산물의 변화를 나타내었으나, 나머지 10종의 균주는 바이칼린 유무에 따른 대사 산물의 변화가 나타내지 않았다. As a result, among the 14 strains, 4 strains ( Polyporus brumalis, Fomes fomentarius, Postia stiptica, Neolentinus lepideus ) showed changes in metabolites depending on the presence or absence of baicalin, but the remaining 10 strains showed changes in metabolites according to the presence or absence of baicalin. No metabolite changes were indicated.
실시예 2. 바이칼레인 유도체의 확인Example 2. Identification of baicalein derivatives
상기 실시예 1에서 대사 산물의 변화를 나타낸 4종의 균주에 대하여 LC-MS/MS 분석을 통하여 바이칼레인 유도체를 확인하였다. Baicalein derivatives were confirmed through LC-MS/MS analysis for the four strains showing metabolite changes in Example 1.
도 2a는 대사 산물의 변화를 나타낸 4종의 담자균에서 바이칼린 첨가에 따라 생성된 바이칼린 유도체 피크(A-I)를 확인한 결과이다. Figure 2a shows the results of confirming the peaks (A-I) of baicalin derivatives generated by the addition of baicalin in four species of basidiomycetes showing changes in metabolites.
바이칼린 첨가에 따라 생성된 바이칼린 유도체 A 내지 I 피크의 이온화된 단편 이온의 패턴 특성을 고려하여 바이칼레인 유도체의 구조를 유추하고, 그 결과를 하기 표 1에 나타내었다.The structure of the baicalin derivative was inferred by considering the pattern characteristics of the ionized fragment ions of the baicalin derivatives A to I peaks generated by the addition of baicalin, and the results are shown in Table 1 below.
MS [M-H]-precursor ion
MS [MH]-
* a: 바이칼레인 피크, m/z 268 또는 269* a: baicalein peak, m/z 268 or 269
표 1에 나타낸 바와 같이, 피크의 단편 이온 패턴을 통하여 대부분의 화합물에서 당이 존재하였으며, 특히 5탄당이 존재하는 것을 확인할 수 있었다. 특히, 4종의 담자균 중에서도 폴리포러스 브루말리스(Polyporus brumalis)에서 가장 다양한 피크가 나타나는 것을 확인할 수 있었다. 즉, 폴리포러스 브루말리스(Polyporus brumalis)는 바이칼린을 바이칼레인으로 비배당체화(aglyconation)한 후, 5탄당을 붙이는 활성을 가지는 것을 알 수 있다. As shown in Table 1, sugar was present in most of the compounds through the fragment ion pattern of the peak, and in particular, it was confirmed that pentose sugar was present. In particular, it was confirmed that the most diverse peaks appeared in Polyporus brumalis among the four types of basidiomycetes. That is, it can be seen that Polyporus brumalis has an activity of attaching a pentose sugar after aglyconation of baicalin to baicalin.
도 2b는 대사 산물의 변화를 나타낸 4종의 담자균에서 바이칼레인 검출을 확인한 결과이다. Figure 2b is the result of confirming the detection of baicalein in four species of basidiomycetes showing changes in metabolites.
그 결과, 도 2b에 나타낸 바와 같이, 4종의 균주 모두에서 배양 0일차부터 순차적으로 m/z 269.04 [M-H]-에서 신호가 검출되는 것을 확인할 수 있었다. 구체적으로, 4종의 균주 배양액에서는 바이칼린에서 글루쿠론산(glucuronic acid)이 떨어진 형태인 바이칼레인(baiclaein) 화합물이 검출되었으며, 이후, 다른 화합물로 다양하게 변화하는 것을 확인할 수 있었다. 즉, 폴리포러스 브루말리스(Polyporus brumalis), 포메스 포멘타리우스(Fomes fomentarius), 포스티아 스티프리카(Postia stiptica), 및 네오렌티누스 레피데우스(Neolentinus lepideus) 균주의 배양액에 바이칼린을 첨가하여 배양하는 경우, 배양 초기에 베타-글루코시다아제(b-glucuronidase) 활성이 나타나는 것을 알 수 있다. As a result, as shown in Figure 2b, it was confirmed that signals were detected at m/z 269.04 [MH] - sequentially from day 0 of culture in all four strains. Specifically, baicalein compound, which is a form in which glucuronic acid is removed from baicalin, was detected in the culture medium of the four strains, and then variously changed to other compounds. That is, Polyporus brumalis , Fomes fomentarius , Postia stiptica , and Neolentinus lepideus Cultured by adding baicalin to the culture medium of the strains In this case, it can be seen that beta-glucuronidase activity appears at the beginning of the culture.
실시예 3. 바이칼레인 유도체의 구조 확인Example 3. Structure confirmation of baicalein derivatives
3-1. 바이칼레인 유도체 분획3-1. Baicalein derivative fraction
상기 실시예 2의 결과를 바탕으로 바이칼린의 생물 전환에 사용할 생체 촉매로서, 폴리포러스 브루말리스(Polyporus brumalis) 균주를 선정한 후, 대량 배양(large-scale fermentation)을 통해 바이칼린 유도체의 구조를 확인하였다. 구체적으로, 포테이토 덱스트로스 아가(potato dextrose agar, PDA) 배지를 500 mL의 플라스크 20개에 200 ㎖씩 넣고 폴리포러스 브루말리스(Polyporus brumalis) 균주를 접종하여 7일 동안 배양하였다. DMSO에 용해된 바이칼린을 배양액에 첨가하여 1차 배양하였다. 이후, 실시예 1과 동일한 방법으로 배양 배지 1㎖를 회수하여 추출 및 농축하여 배양액 추출물을 제조하고, LC-MS 분석으로 추출물 내 대사 산물을 분석하였다. 그 결과, 바이칼린 첨가 후 배양 4일 및 6일 사이에 바이칼레인 유도체 피크가 새롭게 생성되거나 사라지는 것을 확인하였다. 따라서, 배양 5일차의 배양액을 회수하여 동일한 양의 EtOAc로 2회 추출한 후 농축하여 최종 추출물을 제조하고, 폴리포러스 브루말리스(Polyporus brumalis) 균주에 의해 생물 전환되는 바이칼레인 유도체의 구조를 확인하였다. 먼저, preparative HPLC (Hector C18, 5micron)를 이용하여 상기 배양액 추출물의 EtOAc 분획 426 ㎎의 분리를 진행하였다. 0.1 % 포름산(formic acid)이 포함된 H2O(A)와 MeCN(B)를 이동상으로 하여 20-50% B (0-15 분), 50-70% B (15-20 분), 70-100% B (20-25 분), 100-100% B (25-30 분)의 조건으로 분리하였다. Based on the results of Example 2, a Polyporus brumalis strain was selected as a biocatalyst to be used for bioconversion of baicalin, and the structure of the baicalin derivative was confirmed through large-scale fermentation. did Specifically, 200 ml of potato dextrose agar (PDA) medium was added to 20 500 ml flasks, and Polyporus brumalis strain was inoculated and cultured for 7 days. Baicalin dissolved in DMSO was added to the culture medium for primary culture. Thereafter, in the same manner as in Example 1, 1 ml of the culture medium was recovered, extracted and concentrated to prepare a culture medium extract, and metabolites in the extract were analyzed by LC-MS analysis. As a result, it was confirmed that the baicalin derivative peak was newly generated or disappeared between the 4th and 6th days of culture after the addition of baicalin. Therefore, the culture solution on the 5th day of culture was recovered, extracted twice with the same amount of EtOAc, concentrated to prepare a final extract, and the structure of the baicalein derivative bioconverted by the Polyporus brumalis strain was confirmed. First, 426 mg of the EtOAc fraction of the culture extract was separated using preparative HPLC (Hector C18, 5micron). 20-50% B ( 0-15 minutes), 50-70% B (15-20 minutes), 70 It was separated under conditions of -100% B (20-25 minutes) and 100-100% B (25-30 minutes).
그 결과, 화합물 8(9.5 mg, tR 28.0 min)이 분리된 것을 확인할 수 있었다. 이후, 추가 분리를 시도하였으나 추출물의 양이 충분하지 않았다. 따라서, 플라스크 40개에서 배양하였다는 점을 제외하고는 상기와 동일한 방법으로 폴리포러스 브루말리스(Polyporus brumalis) 균주를 배양한 뒤, 화합물의 분리를 계속하였다. 이전의 분리에서 표적으로 하는 플라보노이드 유도체가 특정 부분에 모여 존재함을 확인하였고, 이후의 분리에서는 이 부분을 중심으로 분리를 시도하였다. 상기 배양액 추출물의 EtOAc 분획 859.7 ㎎를 35-35% B (0-5 분), 35-40% B (5-25 분), 40-100% B (25-28 분), 100-100% B (28-32 분)의 조건으로 분리하였다는 점을 제외하고는 상기와 동일한 방법으로 화합물을 분리하였다. As a result, it was confirmed that compound 8 (9.5 mg, t R 28.0 min) was isolated. Thereafter, further separation was attempted, but the amount of extract was not sufficient. Therefore, after culturing the Polyporus brumalis strain in the same manner as above except that it was cultured in 40 flasks, the separation of the compound was continued. In the previous separation, it was confirmed that the target flavonoid derivatives were gathered in a specific part, and in the subsequent separation, separation was attempted around this part. 859.7 mg of the EtOAc fraction of the culture extract was 35-35% B (0-5 minutes), 35-40% B (5-25 minutes), 40-100% B (25-28 minutes), 100-100% B (28-32 minutes), the compound was separated in the same manner as above except that it was separated.
그 결과, 11개의 소획물을 얻을 수 있었으며, 소획물 9에서 화합물 5(24.4 mg, tR 21.5 min)가 분리된 것을 확인할 수 있었다. 한편, 소획물 3, 4, 7, 8에서는 유기 용매의 용해도 차이로 인해 MeOH에 용해되지 않은 물질을 따로 분리하였으며, 그 결과 화합물 1(3.2 mg), 4(0.8 mg), 6(1.5 mg) 이 분리된 것을 확인할 수 있었다. 이후, semipreparative HPLC (Spursil C18 EP, 4 ㎖/min)을 이용하여 나머지 소획물에 대한 분리를 진행하였다. 0.1 % 포름산이 포함된 H2O (A)와 MeCN (B)를 이동상으로 하여 소획물 10을 37% B isocratic의 조건으로 분리하였다. 그 결과, 화합물 2(1.2 mg, tR 10.5 min)가 분리된 것을 확인할 수 있었다. 또한, 소분획 3을 25-35 % B의 조건에서 분리하여 화합물 3(3.9 mg, tR 21.5 min)을 분리하였으며, 마지막으로 소획물 5를 25-34 % B의 조건에서 분리하여 화합물 7(0.6 mg, tR 23.0 min)을 수득하였다.As a result, 11 fractions were obtained, and it was confirmed that compound 5 (24.4 mg, t R 21.5 min) was separated from fraction 9. On the other hand, in fractions 3, 4, 7, and 8, substances that were not dissolved in MeOH were separately separated due to the difference in solubility of organic solvents, and as a result, compounds 1 (3.2 mg), 4 (0.8 mg), and 6 (1.5 mg) This separation could be confirmed. Thereafter, the remaining fractions were separated using semipreparative HPLC (Spursil C18 EP, 4 ml/min). Small fraction 10 was separated under conditions of 37% B isocratic using H 2 O (A) and MeCN (B) containing 0.1% formic acid as mobile phases. As a result, it was confirmed that Compound 2 (1.2 mg, t R 10.5 min) was isolated. In addition, compound 3 (3.9 mg, t R 21.5 min) was isolated by separating small fraction 3 under the condition of 25-35% B, and finally, compound 7 ( 0.6 mg, t R 23.0 min).
도 3은 폴리포러스 브루말리스(Polyporus brumalis) 균주에 의해 생물 전환되는 바이칼레인 유도체 구조를 확인한 결과이다. 3 is a result of confirming the structure of a baicalein derivative bioconverted by a Polyporus brumalis strain.
그 결과, 폴리포러스 브루말리스(Polyporus brumalis) 균주에 의해 생물 전환되는 바이칼린 유도체로서 총 8개의 화합물을 분리하였다. 바이칼린에서 글루쿠론산이 제거된 바이칼레인이 생성된 후, 자일로실화(xylosylation), 하이드록실화(hydroxylation), 메틸화(methylation) 등의 다양한 변화가 일어난 것을 확인할 수 있었다. 특히, 화합물 1 내지 7은 5탄당인 자일로스가 결합된 구조로서, 이전에 알려지지 않은 신규 화합물에 해당한다. As a result, a total of 8 compounds were isolated as baicalin derivatives bioconverted by Polyporus brumalis strain. After the production of baicalin in which glucuronic acid was removed from baicalin, it was confirmed that various changes such as xylosylation, hydroxylation, and methylation occurred. In particular, compounds 1 to 7 have a structure in which xylose, a pentose sugar, is bound, and correspond to previously unknown new compounds.
3-2. 바이칼레인 유도체의 구조 동정3-2. Structural identification of baicalein derivatives
상기 실시예 3-1에서 분리 정제된 화합물의 구조를 동정하기 위하여, 핵자기공명(NMR)을 수행하고, 당의 결합 위치를 확인하기 위하여, HMBC를 수행하였다. 그 결과, 하기 화학식 1 내지 8의 구조를 갖는, 바이칼레인 유도체를 확인하였다. 특히, 화합물 1 내지 7은 천연에서 처음으로 분리된 신규 구조의 자일로실화된 바이칼레인 유도체 화합물임을 확인하였다. Nuclear magnetic resonance (NMR) was performed to identify the structure of the compound isolated and purified in Example 3-1, and HMBC was performed to confirm the binding site of the sugar. As a result, baicalein derivatives having structures represented by Chemical Formulas 1 to 8 were identified. In particular, it was confirmed that Compounds 1 to 7 are xylosylated baicalein derivative compounds having a novel structure isolated for the first time in nature.
(1) 화합물 1: 바이칼레인 7-(1) Compound 1: Baicalein 7- OO -b-D-자일로피라노사이드[Baicalein 7--b-D-xylopyranoside [Baicalein 7- OO -b-D-xylopyranoside]-b-D-xylopyranoside]
[화학식 1][Formula 1]
; ;
- 무정형 고체;- amorphous solid;
- C20H18O9;- C 20 H 18 O 9 ;
- = - 9.5 (c 0.01, MeOH); - = -9.5 ( c 0.01, MeOH);
- HRESIMS m/z 401.0861 [M - H]- (calcd for C20H17O9, 401.0873);- HRESIMS m/z 401.0861 [M - H] - (calcd for C 20 H 17 O 9 , 401.0873);
- 1H NMR (DMSO-d 6, 500 MHz) δ: 12.57 (1H, s, 5-OH), 8.11 (2H, H-3’), 7.60 (3H, H-2’, 4’), 7.01 (2H, s, H-3, 8), 5.15 (2H, s, OH), 5.02 (1H, d, J =7.5 Hz, Xyl-1), 3.82 (1H, d, J =5.9Hz, Xyl-5), 3.29-3.42 (4H, m, Xyl-2, 3, 4, 5); - 1H NMR (DMSO- d 6 , 500 MHz) δ: 12.57 (1H, s, 5-OH), 8.11 (2H, H-3'), 7.60 (3H, H-2', 4'), 7.01 (2H, s, H-3, 8), 5.15 (2H, s, OH), 5.02 (1H, d, J =7.5 Hz, Xyl-1), 3.82 (1H, d, J =5.9Hz, Xyl-1) 5), 3.29-3.42 (4H, m, Xyl-2, 3, 4, 5);
- 13C NMR (DMSO-d 6, 125 MHz) δ: 182.6 (C-4), 163.5 (C-2), 151.5 (C-7), 149.2 (C-5), 146.7 (C-9), 132.1 (C-4’), 130.8 (C-1’), 130.7 (C-6), 129.2 (C-3’), 126.5 (C-2’), 106.1 (C-10), 104.7 (C-3), 101.3 (Xyl-1), 94.0 (C-8), 75.7 (Xyl-3), 72.9 (Xyl-2), 69.3 (Xyl-4), 65.9 (Xyl-5).- 13 C NMR (DMSO- d 6 , 125 MHz) δ: 182.6 (C-4), 163.5 (C-2), 151.5 (C-7), 149.2 (C-5), 146.7 (C-9), 132.1 (C-4'), 130.8 (C-1'), 130.7 (C-6), 129.2 (C-3'), 126.5 (C-2'), 106.1 (C-10), 104.7 (C- 3), 101.3 (Xyl-1), 94.0 (C-8), 75.7 (Xyl-3), 72.9 (Xyl-2), 69.3 (Xyl-4), 65.9 (Xyl-5).
(2) 화합물 2: 크리신 7-(2) Compound 2: Chrysin 7- OO -β-D-자일로피라노사이드[Chrysin 7--β-D-xylopyranoside [Chrysin 7- OO -β-D-xylopyranoside]-β-D-xylopyranoside]
[화학식 2][Formula 2]
; ;
- 무정형 고체;- amorphous solid;
- C20H18O8;- C 20 H 18 O 8 ;
- = - 4.1 (c 0.01, MeOH); - = -4.1 ( c 0.01, MeOH);
- HRESIMS m/z 385.0917 [M - H]- (calcd for C20H17O8, 385.0923);- HRESIMS m/z 385.0917 [M - H] - (calcd for C 20 H 17 O 8 , 385.0923);
- 1H NMR (DMSO-d 6, 500 MHz) δ: 8.12 (2H, d, J =7.3 Hz, H-2’), 7.61 (3H, m, H-3’, 4’), 7.07 (1H, s, H-3), 6.89 (1H, s, H-8), 6.45 (1H, s, H-6), 5.09 (1H, d(s), J =5.3 Hz, Xyl-1), 3.77 (1H, dd, J =8.6, 2.7 Hz, H-5``), 3.26-3.39 (4H, m, Xyl-2, 3, 4, 5);- 1 H NMR (DMSO- d 6 , 500 MHz) δ: 8.12 (2H, d, J =7.3 Hz, H-2'), 7.61 (3H, m, H-3', 4'), 7.07 (1H , s, H-3), 6.89 (1H, s, H-8), 6.45 (1H, s, H-6), 5.09 (1H, d(s), J =5.3 Hz, Xyl-1), 3.77 (1H, dd, J =8.6, 2.7 Hz, H-5``), 3.26-3.39 (4H, m, Xyl-2, 3, 4, 5);
- 13C NMR (DMSO-d 6, 125 MHz) δ: 182.2 (C-4), 163.8 (C-2), 163.0 (C-5), 161.1 (C-7), 132.2 (C-4’), 130.6 (C-1’), 129.2 (C-3’), 126.6 (C-2’), 105.4 (C-3), 100.2 (Xyl-1), 99.7 (C-6), 94.8 (C-8), 76.3 (Xyl-3), 72.9 (Xyl-2), 69.2 (Xyl-4), 65.9 (Xyl-5).- 13 C NMR (DMSO- d 6 , 125 MHz) δ: 182.2 (C-4), 163.8 (C-2), 163.0 (C-5), 161.1 (C-7), 132.2 (C-4') , 130.6 (C-1'), 129.2 (C-3'), 126.6 (C-2'), 105.4 (C-3), 100.2 (Xyl-1), 99.7 (C-6), 94.8 (C- 8), 76.3 (Xyl-3), 72.9 (Xyl-2), 69.2 (Xyl-4), 65.9 (Xyl-5).
(3) 화합물 3: 바이칼레인 6,7-O-β-D-디자일로피라노사이드[Baicalein 6,7-(3) Compound 3: Baicalein 6,7-O-β-D-disylopyranoside [Baicalein 6,7- OO -β-D-dixylopyranoside]-β-D-dixylopyranoside]
[화학식 3][Formula 3]
; ;
- 무정형 고체;- amorphous solid;
- C25H26O13;- C 25 H 26 O 13 ;
- = +3.6 (c 0.01, MeOH); - = +3.6 ( c 0.01, MeOH);
- HRESIMS m/z 533.1285 [M - H]- (calcd for C25H25O13, 533.1295); - HRESIMS m/z 533.1285 [M - H] - (calcd for C 25 H 25 O 13 , 533.1295);
- 1H NMR (DMSO-d 6, 500 MHz) δ: 8.12 (2H, m, H-2’), 7.62 (3H, m, H-3’, 4’), 7.06 (2H, s, H-3, 8), 5.08 (1H, d, J =7.2 Hz, Xyl-1'), 4.93 (1H, d, J =6.6 Hz, Xyl-1), 3.83 (1H, d, J =5.9 Hz, Xyl-5'), 3.78 (1H, dd, J =11.6, 5.0 Hz, Xyl-5), 3.23-3.42 (1H, m, Xyl-2, 2’, 3, 3’, 4, 4’, 5’), 3.03 (1H, dd, J =11.5, 9.0 Hz, Xyl-5);- 1 H NMR (DMSO- d 6 , 500 MHz) δ: 8.12 (2H, m, H-2'), 7.62 (3H, m, H-3', 4'), 7.06 (2H, s, H- 3, 8), 5.08 (1H, d, J =7.2 Hz, Xyl-1′), 4.93 (1H, d, J =6.6 Hz, Xyl-1), 3.83 (1H, d, J =5.9 Hz, Xyl -5'), 3.78 (1H, dd, J =11.6, 5.0 Hz, Xyl-5), 3.23-3.42 (1H, m, Xyl-2, 2', 3, 3', 4, 4', 5' ), 3.03 (1H, dd, J = 11.5, 9.0 Hz, Xyl-5);
- 13C NMR (DMSO-d 6, 125 MHz) δ: 182.5 (C-4), 163.7 (C-2), 156.0 (C-7), 152.7 (C-9), 132.2 (C-1’), 130.6 (C-1’), 129.2 (C-3’), 126.6 (C-2’), 128.9 (C-6), 106.2 (C-10), 105.0 (C-3), 103.7 (Xyl-1), 101.3 (Xyl-1’), 94.49 (C-8), 75.6 (Xyl-3’), 75.4 (Xyl-3), 73.3 (Xyl-2), 73.0 (Xyl-2’), 69.4 (Xyl-4), 69.2 (Xyl-4’), 65.9 (Xyl-5’), 65.6 (Xyl-5).- 13 C NMR (DMSO- d 6 , 125 MHz) δ: 182.5 (C-4), 163.7 (C-2), 156.0 (C-7), 152.7 (C-9), 132.2 (C-1') , 130.6 (C-1'), 129.2 (C-3'), 126.6 (C-2'), 128.9 (C-6), 106.2 (C-10), 105.0 (C-3), 103.7 (Xyl- 1), 101.3 (Xyl-1'), 94.49 (C-8), 75.6 (Xyl-3'), 75.4 (Xyl-3), 73.3 (Xyl-2), 73.0 (Xyl-2'), 69.4 ( Xyl-4), 69.2 (Xyl-4'), 65.9 (Xyl-5'), 65.6 (Xyl-5).
(4) 화합물 4: 오록실린 A 7-(4) Compound 4: Oroxylin A 7- OO -β-D-자일로피라노사이드[Oroxylin A 7--β-D-xylopyranoside [Oroxylin A 7- OO -b-D-xylopyranoside]-b-D-xylopyranoside]
[화학식 4][Formula 4]
; ;
- 무정형 고체;- amorphous solid;
- C21H20O9;- C 21 H 20 O 9 ;
- = - 26.1 (c 0.01, MeOH); - = -26.1 ( c 0.01, MeOH);
- HRESIMS m/z 415.1022 [M - H]- (calcd for C21H19O9, 415.1029);- HRESIMS m/z 415.1022 [M - H] - (calcd for C 21 H 19 O 9 , 415.1029);
- 1H NMR (DMSO-d 6, 500 MHz) δ: 12.79 (1H, s, 5-OH), 8.11 (2H, d, J =7.3 Hz, H-2’), 7.60 (3H, m, H-3’, 4’), 7.06 (1H, s, H-8), 7.04 (1H, s, H-3), 5.48 (1H, s, OH), 5.19 (1H, s, OH), 5.15 (1H, d, J =7.3 Hz, Xyl-1), 3.81 (1H, d, J =5.9 Hz, Xyl-5), 3.77 (3H, s, 6-OCH3), 3.43 (2H, m, Xyl-4, 5), 3.35 (1H, m, Xyl-2), 3.30 (1H, m, Xyl-3);- 1H NMR (DMSO- d 6 , 500 MHz) δ: 12.79 (1H, s, 5-OH), 8.11 (2H, d, J =7.3 Hz, H-2'), 7.60 (3H, m, H -3', 4'), 7.06 (1H, s, H-8), 7.04 (1H, s, H-3), 5.48 (1H, s, OH), 5.19 (1H, s, OH), 5.15 ( 1H, d, J =7.3 Hz, Xyl-1), 3.81 (1H, d, J =5.9 Hz, Xyl-5), 3.77 (3H, s, 6-OCH 3 ), 3.43 (2H, m, Xyl- 4, 5), 3.35 (1H, m, Xyl-2), 3.30 (1H, m, Xyl-3);
- 13C NMR (DMSO-d 6, 125 MHz) δ: 182.5 (C-4), 163.7 (C-2), 156.4 (C-7), 152.5 (C-5), 152.3 (C-9), 132.7 (C-6), 132.2 (C-4’), 130.6 (C-1’), 129.2 (C-3’), 126.5 (C-2’), 106.1 (C-10), 104.9 (C-3), 100.5 (Xyl-1), 94.3 (C-8), 76.4 (Xyl-3), 72.9 (Xyl-2), 69.2 (Xyl-4), 65.9 (Xyl-5), 60.3 (C-OCH3).- 13 C NMR (DMSO- d 6 , 125 MHz) δ: 182.5 (C-4), 163.7 (C-2), 156.4 (C-7), 152.5 (C-5), 152.3 (C-9), 132.7 (C-6), 132.2 (C-4'), 130.6 (C-1'), 129.2 (C-3'), 126.5 (C-2'), 106.1 (C-10), 104.9 (C-10) 3), 100.5 (Xyl-1), 94.3 (C-8), 76.4 (Xyl-3), 72.9 (Xyl-2), 69.2 (Xyl-4), 65.9 (Xyl-5), 60.3 (C-OCH 3 ).
(5) 화합물 5: 오록실린 A 7-O-β-D-자일로피라노실-(1→3)-β-D-자일로피라노사이드[Oroxylin A 7-O-β-D-xylopyranosyl-(1→3)-β-D-xylopyranoside](5) Compound 5: Oroxylin A 7-O-β-D-xylopyranosyl-(1→3)-β-D-xylopyranoside [Oroxylin A 7-O-β-D-xylopyranosyl- (1→3)-β-D-xylopyranoside]
[화학식 5][Formula 5]
; ;
- 무정형 고체;- amorphous solid;
- C26H28O13;- C 26 H 28 O 13 ;
- = +25.4 (c 0.005, MeOH);- = +25.4 ( c 0.005, MeOH);
- HRESIMS m/z 547.1448 [M - H]- (calcd for C26H27O13, 547.1452);- HRESIMS m/z 547.1448 [M - H] - (calcd for C 26 H 27 O 13 , 547.1452);
- 1H NMR (DMSO-d 6, 500 MHz) δ: 12.82 (1H, s, 5-OH), 8.13 (2H, m, H-2’), 7.62 (3H, m, H-3’, 4’), 7.07 (2H, s, H-3, 8), 5.65 (1H, d, J =4.7 Hz, OH), 5.25 (1H, d, J =7.3 Hz, Xyl-1), 5.23(1H, d, J =3.6 Hz, OH), 5.05 (1H, d, J =4.7 Hz, OH), 5.01 (1H, d, J =5.0 Hz, OH), 4.92 (1H, d, J =3.2 Hz, OH), 4.48 (1H, d, J =7.4 Hz, Xyl-1’), 3.88 (1H, dd, J =10.1, 3.7 Hz, Xyl-5), 3.78 (1H, m, Xyl-5’), 3.77 (1H, s, 6-OCH3), 3.59 (1H, m, Xyl-2), 3.52 (2H, m, Xyl-3, 4), 3.32-3.49 (2H, m, Xyl-5, 4’), 3.16 (1H, m, Xyl-3’), 3.11 (2H, m, Xyl-2’, 5’);- 1H NMR (DMSO- d 6 , 500 MHz) δ: 12.82 (1H, s, 5-OH), 8.13 (2H, m, H-2'), 7.62 (3H, m, H-3', 4 '), 7.07 (2H, s, H-3, 8), 5.65 (1H, d, J =4.7 Hz, OH), 5.25 (1H, d, J =7.3 Hz, Xyl-1), 5.23(1H, d, J =3.6 Hz, OH), 5.05 (1H, d, J =4.7 Hz, OH), 5.01 (1H, d, J =5.0 Hz, OH), 4.92 (1H, d, J =3.2 Hz, OH) ), 4.48 (1H, d, J =7.4 Hz, Xyl-1'), 3.88 (1H, dd, J =10.1, 3.7 Hz, Xyl-5), 3.78 (1H, m, Xyl-5'), 3.77 (1H, s, 6-OCH 3 ), 3.59 (1H, m, Xyl-2), 3.52 (2H, m, Xyl-3, 4), 3.32-3.49 (2H, m, Xyl-5, 4′) , 3.16 (1H, m, Xyl-3'), 3.11 (2H, m, Xyl-2', 5');
- 13C NMR (DMSO-d 6, 125 MHz) δ: 182.6 (C-4), 163.8 (C-2), 156.2 (C-7), 152.6 (C-5), 152.6 (C-9), 132.6 (C-6), 132.2 (C-4’), 130.6 (C-1’), 129.2 (C-3’), 126.5 (C-2’), 106.2 (C-10), 105.1 (C-3), 104.4 (Xyl-1’), 99.9 (Xyl-1), 94.4 (C-8), 85.4 (Xyl-3), 76.1 (Xyl-3’), 73.8 (Xyl-2’), 72.1 (Xyl-2), 69.4 (Xyl-4’), 67.5 (Xyl-4), 65.8 (Xyl-5’), 65.4 (Xyl-5), 60.3 (6-OCH3).- 13 C NMR (DMSO- d 6 , 125 MHz) δ: 182.6 (C-4), 163.8 (C-2), 156.2 (C-7), 152.6 (C-5), 152.6 (C-9), 132.6 (C-6), 132.2 (C-4'), 130.6 (C-1'), 129.2 (C-3'), 126.5 (C-2'), 106.2 (C-10), 105.1 (C-10) 3), 104.4 (Xyl-1'), 99.9 (Xyl-1), 94.4 (C-8), 85.4 (Xyl-3), 76.1 (Xyl-3'), 73.8 (Xyl-2'), 72.1 ( Xyl-2), 69.4 (Xyl-4'), 67.5 (Xyl-4), 65.8 (Xyl-5'), 65.4 (Xyl-5), 60.3 (6-OCH 3 ).
(6) 화합물 6: 5.8-디하이드록시-6-메톡시플라본 7-(6) Compound 6: 5.8-dihydroxy-6-methoxyflavone 7- OO -β-D-자일로피라노사이드[5,8-dihydroxy-6-methoxyflavone 7--β-D-xylopyranoside [5,8-dihydroxy-6-methoxyflavone 7- OO -β-D-xylopyranoside]-β-D-xylopyranoside]
[화학식 6][Formula 6]
; ;
- 무정형 고체;- amorphous solid;
- C21H20O10;- C 21 H 20 O 10 ;
- + 70.2 (c 0.01, MeOH); - +70.2 ( c 0.01, MeOH);
- HRESIMS m/z 431.0973 [M - H]- (calcd for C21H19O10, 431.0978); - HRESIMS m/z 431.0973 [M - H] - (calcd for C 21 H 19 O 10 , 431.0978);
- 1H NMR (DMSO-d 6, 500 MHz) δ: 12.75 (1H, s, 5-OH), 8.24 (2H, m, H-2’), 7.59 (2H, m, H-3’, 4’), 7.02 (1H, s, H-3), 5.07 (1H, d, J =5.2 Hz, OH), 4.73 (1H, d, J =7.5 Hz, Xyl-1), 3.82 (1H, dd, J =11.4, 5.2 Hz, Xyl-5), 3.77 (1H, s, 6-OCH3), 3.46 (1H, m, Xyl-4), 3.44 (1H, m, Xyl-2), 3.25 (1H, t, J =8.8 Hz, Xyl-3), 3.14 (1H, dd, J =11.4, 5.2 Hz, Xyl-5); - 1H NMR (DMSO- d 6 , 500 MHz) δ: 12.75 (1H, s, 5-OH), 8.24 (2H, m, H-2'), 7.59 (2H, m, H-3', 4 '), 7.02 (1H, s, H-3), 5.07 (1H, d, J =5.2 Hz, OH), 4.73 (1H, d, J =7.5 Hz, Xyl-1), 3.82 (1H, dd, J = 11.4, 5.2 Hz, Xyl-5), 3.77 (1H, s, 6-OCH 3 ), 3.46 (1H, m, Xyl-4), 3.44 (1H, m, Xyl-2), 3.25 (1H, t, J =8.8 Hz, Xyl-3), 3.14 (1H, dd, J =11.4, 5.2 Hz, Xyl-5);
- 13C NMR (DMSO-d 6, 125 MHz) δ: 182.3 (C-4), 162.9 (C-2), 152.3 (C-6), 149.2 (C-5), 145.5 (C-9), 132.1 (C-4’), 131.6 (C-8), 130.7 (C-1’), 129.1 (C-3’), 126.6 (C-2’), 125.3 (C-7), 106.4 (Xyl-1), 104.4 (C-3), 102.8 (C-10), 75.9 (Xyl-3), 73.9 (Xyl-2), 69.3 (Xyl-4), 66.2 (Xyl-5) 60.1 (6-OCH3).- 13 C NMR (DMSO- d 6 , 125 MHz) δ: 182.3 (C-4), 162.9 (C-2), 152.3 (C-6), 149.2 (C-5), 145.5 (C-9), 132.1 (C-4'), 131.6 (C-8), 130.7 (C-1'), 129.1 (C-3'), 126.6 (C-2'), 125.3 (C-7), 106.4 (Xyl- 1), 104.4 (C-3), 102.8 (C-10), 75.9 (Xyl-3), 73.9 (Xyl-2), 69.3 (Xyl-4), 66.2 (Xyl-5) 60.1 (6-OCH 3 ).
(7) 화합물 7: 5,8-디하이드록시-6-메톡시플라본 7-(7) Compound 7: 5,8-dihydroxy-6-methoxyflavone 7- OO -b-D-자일로피라노실-(1→3)-b-d-자일로피라노사이드[5,8-dihydroxy-6-methoxyflavone 7--b-D-xylopyranosyl-(1→3)-b-d-xylopyranoside [5,8-dihydroxy-6-methoxyflavone 7- OO -b-D-xylopyranosyl-(1®3)-b-d-xylopyranoside]-b-D-xylopyranosyl-(1®3)-b-d-xylopyranoside]
[화학식 7][Formula 7]
; ;
- 무정형 고체;- amorphous solid;
- C26H28O14;- C 26 H 28 O 14 ;
- = +92.8 (c 0.005, MeOH); - = +92.8 ( c 0.005, MeOH);
- HRESIMS m/z 563.1388 [M - H]- (calcd for C26H27O14, 563.1406); - HRESIMS m/z 563.1388 [M - H] - (calcd for C 26 H 27 O 14 , 563.1406);
- 1H NMR (DMSO-d 6, 500 MHz) δ: 12.62 (1H, s, 5-OH), 8.14 (2H, m, H-2’), 7.51 (3H, m, H-3’, 4’), 6.53 (1H, s, H-3), 4.53 (1H, d, J =7.4 Hz, Xyl-1’). 4.32 (1H, d, J =7.4 Hz, Xyl-1), 3.84 (1H, dd, J =11.3, 5.4 Hz, Xyl-5), 3.73 (1H, dd, J =11.3, 5.2 Hz, Xyl-5’), 3.64 (1H, s, 6-OCH3), 3.48 (1H, m, Xyl-4), 3.41 (2H, m, Xyl-2, 3), 3.29 (1H, ddd, J =10.0, 8.6, 5.3 Hz, Xyl-5’), 3.20 (1H, m, Xyl-5), 3.14 (1H, m, Xyl-3’), 3.06 (1H, dd, J =6.3, 5.2 Hz, Xyl-2’);- 1 H NMR (DMSO- d 6 , 500 MHz) δ: 12.62 (1H, s, 5-OH), 8.14 (2H, m, H-2'), 7.51 (3H, m, H-3', 4 '), 6.53 (1H, s, H-3), 4.53 (1H, d, J = 7.4 Hz, Xyl-1'). 4.32 (1H, d, J =7.4 Hz, Xyl-1), 3.84 (1H, dd, J =11.3, 5.4 Hz, Xyl-5), 3.73 (1H, dd, J = 11.3, 5.2 Hz, Xyl-5 '), 3.64 (1H, s, 6-OCH 3 ), 3.48 (1H, m, Xyl-4), 3.41 (2H, m, Xyl-2, 3), 3.29 (1H, ddd, J = 10.0, 8.6 , 5.3 Hz, Xyl-5'), 3.20 (1H, m, Xyl-5), 3.14 (1H, m, Xyl-3'), 3.06 (1H, dd, J =6.3, 5.2 Hz, Xyl-2');
- 13C NMR (DMSO-d 6, 125 MHz) δ: 179.6 (C-4), 159.9 (C-2), 148.8 (C-9), 134.4 (C-6), 131.7 (C-1’), 131.0 (C-4’), 128.8 (C-3’), 126.0 (C-2’), 128.5 (C-7), 109.3 (Xyl-1), 104.8 (Xyl-1’), 103.4 (C-3), 96.1 (C-10), 85.36 (Xyl-3), 76.4 (Xyl-3’), 74.4 (Xyl-2’), 73.6 (Xyl-2), 69.5 (Xyl-4’), 67.6 (Xyl-4), 66.2 (Xyl-5), 65.7 (Xyl-5’), 58.6 (6-OCH3).- 13 C NMR (DMSO- d 6 , 125 MHz) δ: 179.6 (C-4), 159.9 (C-2), 148.8 (C-9), 134.4 (C-6), 131.7 (C-1') , 131.0 (C-4'), 128.8 (C-3'), 126.0 (C-2'), 128.5 (C-7), 109.3 (Xyl-1), 104.8 (Xyl-1'), 103.4 (C -3), 96.1 (C-10), 85.36 (Xyl-3), 76.4 (Xyl-3'), 74.4 (Xyl-2'), 73.6 (Xyl-2), 69.5 (Xyl-4'), 67.6 (Xyl-4), 66.2 (Xyl-5), 65.7 (Xyl-5′), 58.6 (6-OCH 3 ).
(8) 화합물 8: 오록실린 A[Oroxylin A](8) Compound 8: Oroxylin A [Oroxylin A]
[화학식 8][Formula 8]
; ;
- 무정형 고체;- amorphous solid;
- C16H12O5; - C 16 H 12 O 5 ;
- HRESIMS m/z 283.0605 [M - H]- (calcd for C16H11O5, 283.0607); - HRESIMS m/z 283.0605 [M - H] - (calcd for C 16 H 11 O 5 , 283.0607);
- 1H NMR (DMSO-d 6, 500 MHz) δ: 12.93 (1H, s, 5-OH), 8.07 (2H, m, H-2’), 7.60 (3H, m, H-3’, 4’), 6.98 (1H, s, H-3), 6.64 (1H, s, H-8), 3.76 (3H, s, 6-OCH3);- 1H NMR (DMSO- d 6 , 500 MHz) δ: 12.93 (1H, s, 5-OH), 8.07 (2H, m, H-2'), 7.60 (3H, m, H-3', 4 '), 6.98 (1H, s, H-3), 6.64 (1H, s, H-8), 3.76 (3H, s, 6-OCH 3 );
- 13C NMR (DMSO-d 6, 125 MHz) δ: 182.3 (C-4), 163.2 (C-2), 157.7 (C-7), 152.7 (C-9), 152.6 (C-5), 132.0 (C-4’), 131.5 (C-6), 130.8 (C-1’), 129.2 (C-3’), 126.4 (C-2’), 104.7 (C-3), 104.3 (C-10), 94.42 (C-8), 60.0 (6-OCH3).- 13 C NMR (DMSO- d 6 , 125 MHz) δ: 182.3 (C-4), 163.2 (C-2), 157.7 (C-7), 152.7 (C-9), 152.6 (C-5), 132.0 (C-4'), 131.5 (C-6), 130.8 (C-1'), 129.2 (C-3'), 126.4 (C-2'), 104.7 (C-3), 104.3 (C- 10), 94.42 (C-8), 60.0 (6-OCH 3 ).
3-2. 당의 종류 확인3-2. Check the type of sugar
상기 실시예 3-1에서 분획하여 얻은 8개 화합물의 당의 종류를 분석하였다. 구체적으로, 화합물 8을 제외한 모든 화합물 0.5 ㎎을 1N HCl (1.0 ㎖)에 녹인 뒤, 80℃에서 2시간 동안 방치하여 산 가수분해 하였다. EtOAc (2 x 0.5 ㎖)을 이용하여 반응물 내 아글리콘(aglycone)을 제거하였다. 이후, 물층을 분리 및 농축하여 검액으로 하고, 5탄당을 포함하는 화합물 1 내지 7을 D-라이보스(D-ribose), L-아라비노스(L-arabinose) 및 D-자일로스(D-xylose)와 함께 박층 크로마토그래피(thin-layer Chromatography, TLC)에 점적 하였다. 이후, n-BuOH*acetone*pyridine*H2O (2:2:1:1)을 전개 용매로 하여 전개한 뒤, 박층 판을 건조하였다. p-아니살데하이드(p-anisaldehyde)을 사용하여 표준품과 검액의 Rf값을 비교하였다. The types of sugars of the 8 compounds obtained by fractionation in Example 3-1 were analyzed. Specifically, after dissolving 0.5 mg of all compounds except for compound 8 in 1N HCl (1.0 ml), acid hydrolysis was performed by standing at 80° C. for 2 hours. EtOAc (2 x 0.5 ml) was used to remove aglycone in the reaction mixture. Thereafter, the water layer was separated and concentrated to form a test solution, and compounds 1 to 7 containing pentose sugars were prepared with D-ribose, L-arabinose, and D-xylose. ) and spotted on thin-layer chromatography (TLC). Thereafter, n -BuOH*acetone*pyridine*H 2 O (2:2:1:1) was developed as a developing solvent, and then the thin-layer plate was dried. R f values of the standard product and the sample solution were compared using p -anisaldehyde .
그 결과, 화합물 1 내지 7은 5탄당의 D-자일로스(D-xylose)로 확인되었다. As a result, compounds 1 to 7 were identified as pentose D-xylose.
3-3. 당의 입체 구조 확인3-3. Identification of sugar conformation
상기 실시예 3-1에서 분획하여 얻은 8개 화합물의 당의 입체 구조(D, L-isomer)를 확인하였다. 구체적으로, 상기 실시예 3-2와 동일한 방법으로 화합물을 산 가수분해 한 후, 가수분해물을 건조하였다. 이후, L-시스테인 메틸 에스터 하이드로클로라이드(L-cysteine methyl ester hydrochloride) 0.5 ㎎을 포함한 피리딘(pyridine) 0.1 ㎖에 상기 건조된 가수분해물을 녹여 60 ℃에서 1시간 동안 방치한 뒤, o-톨릴 이소티오시아네이트(o-Tolyl isothiocyanate) 10 ㎕를 첨가하여 다시 60 ℃에서 1시간 동안 방치하였다. 이후, 반응물을 바로 analytical HPLC로 분석하여 표준품의 반응물과 머무름 시간(retention time, tR)을 비교하였다. Analytical HPLC 측정 시 0.1 % 포름산(formic acid)이 포함된 H2O (A)와 MeCN (B)를 이동상으로 하여 20-40% B (0-30 분), 100-100% B (30-35 분), 20-20% B (35-40 분)의 농도 구배 조건으로 분석을 진행하였으며, 컬럼의 온도는 30 ℃로 유지하였다. The three-dimensional structures (D, L-isomer) of the sugars of the eight compounds obtained by fractionation in Example 3-1 were confirmed. Specifically, after acid hydrolysis of the compound in the same manner as in Example 3-2, the hydrolyzate was dried. Thereafter, the dried hydrolyzate was dissolved in 0.1 ml of pyridine containing 0.5 mg of L-cysteine methyl ester hydrochloride, left at 60 ° C. for 1 hour, and o- tolyl isothio After adding 10 μl of cyanate ( o -Tolyl isothiocyanate), the mixture was left at 60° C. for 1 hour. Thereafter, the reaction was directly analyzed by analytical HPLC, and the retention time (t R ) was compared with that of the standard reaction. For Analytical HPLC measurement, 20-40% B (0-30 minutes), 100-100% B ( 30-35 min), the analysis was performed under the conditions of a concentration gradient of 20-20% B (35-40 min), and the temperature of the column was maintained at 30 °C.
그 결과, 검액의 D-자일로스 유도체(D-xylose derivative) 의 머무름 시간은 21.28 분으로 측정되었으며, 표준품의 D-자일로스 유도체(D-xylose derivative) 의 머무름 시간은 20.59분으로 측정되었다. 따라서, 화합물 1 내지 7의 5탄당은 모두 D-자일로스(D-xylose) 임을 확인하였다. As a result, the retention time of the D-xylose derivative of the test solution was measured to be 21.28 minutes, and the retention time of the D-xylose derivative of the standard product was measured to be 20.59 minutes. Accordingly, it was confirmed that all pentose sugars of Compounds 1 to 7 were D-xylose.
실시예 4. 바이칼레인 유도체의 변화 과정 유추Example 4. Analogy of change process of baicalein derivatives
상기 실시예 3-1에 의할 때, 폴리포러스 브루말리스(Polyporus brumalis) 균주에 바이칼린을 첨가하여 배양할 경우, b-글루쿠로니다제(b-glucuronidase)가 활성화되어 바이칼린에 결합된 글루쿠론산이 떨어지면서 바이칼레인이 생성되고, 이후 히드록실화, 메틸화 등을 통해 다양한 화합물로의 변화가 이루어지는 것을 알 수 있다. 따라서, 본 실시예에서는 LC-MS/MS 데이터에서 분리된 화합물 각각의 날짜 별 피크 넓이를 확인하여 넓이 크기와 날짜에 따른 히트맵을 통해 이들 산물의 생성이 언제 일어나는지, 시간의 변화함에 따라 물질의 대략적인 정도를 확인하였다. According to Example 3-1, when cultured by adding baicalin to Polyporus brumalis strain, b-glucuronidase is activated and bound to baicalin It can be seen that baicalein is produced as glucuronic acid falls, and then changed into various compounds through hydroxylation, methylation, and the like. Therefore, in this example, the peak area of each compound separated from the LC-MS/MS data was checked, and through the heat map according to the area size and date, when the production of these products occurred, and the The approximate degree was confirmed.
도 12a는 일 구체예에 따른 화합물의 MS 데이터 피크 면적에 의한 히트맵을 나타낸다. Figure 12a shows a heat map by MS data peak area of a compound according to one embodiment.
도 12b는 폴리포러스 브루말리스 균주의 생체 촉매 반응에 따른 화합물의 생성 과정을 나타낸 개략도이다. Figure 12b is a schematic diagram showing the production process of the compound according to the biocatalytic reaction of the Polyporus brumalis strain.
그 결과, 도 13a에 나타낸 바와 같이, 바이칼린 유도체는 폴리포러스 브루말리스와 바이칼린의 배양 후, 최소 0일 및 최대 8일 내에 생성되는 것을 확인할 수 있었다. 또한, 도 13b에 나타낸 바와 같이, 바이칼린은 반응 후, 0일 이내에 7-OH 위치의 글루쿠론산이 분해되어 바이칼레인이 생성되었으며, 바이칼레인의 6-OH 및/또는 7-OH 위치가 자일로실화화, 하이드록실화, 디하이드록실화 및/또는 메틸화 됨으로써 바이칼레인 유도체가 순차적으로 생성되는 것을 확인할 수 있었다. 구체적으로, 바이칼레인은 1 내지 2일 내에 자일로실화, 하이드록시화, 디하이드록시화 및/또는 메틸화 과정을 거쳐 최종 산물이 생성되는 것을 확인할 수 있었다. As a result, as shown in FIG. 13a, it was confirmed that baicalin derivatives were produced within a minimum of 0 days and a maximum of 8 days after culturing Polyporus brumalis and baicalin. In addition, as shown in FIG. 13B, after the reaction of baicalin, glucuronic acid at the 7-OH position was decomposed within 0 days to produce baicalein, and the 6-OH and/or 7-OH positions of baicalin were xyl. It was confirmed that baicalein derivatives were sequentially produced by rosylation, hydroxylation, dehydroxylation and/or methylation. Specifically, it was confirmed that baicalein undergoes xylosylation, hydroxylation, dehydroxylation and/or methylation within 1 to 2 days to produce the final product.
즉, 폴리포러스 브루말리스 균주에 바이칼린 기질을 추가하여 배양함으로써 배양 후, 10일 이내에 신규 바이칼레인 유도체를 제조할 수 있다. 따라서, 일 양상에 따른 방법은 바이칼레인 유도체를 지속적이고, 경제적으로 합성할 수 있다. That is, by adding and culturing a baicalin substrate to a Polyporus brumalis strain, a novel baicalin derivative can be produced within 10 days after culturing. Thus, the method according to one aspect can continuously and economically synthesize a baicalein derivative.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.
Claims (11)
[화학식 10]
상기에서,
R1, R4 및 R5는 각각 독립적으로 수소(H) 또는 하이드록시(OH)이고,
R2는 -O-자일로피라노사이드(xyropyranoside), -O-자일로퓨라노사이드(Xylopuranoside), -O-자일로피라노사이드(xyropyranoside), -O-자일로퓨라노사이드(xylopuranoside), -O-라이보퓨라노사이드(ribofuranoside), -O-아라비노피라노사이드(arabinopyranoside), -O-릭소피라노사이드(lyxopyranoside), -O-리보피라노사이드(ribopyranoside), 및 -O-자일로바이오스(xylobiose)로 구성된 군에서 선택된 것이고,
R3은 수소(H), 하이드록시(OH), 치환 또는 비치환된 C1-C20 알콕시, -O-자일로피라노사이드(xyropyranoside), -O-자일로퓨라노사이드(xylopuranoside), -O-라이보퓨라노사이드(ribofuranoside), -O-아라비노피라노사이드(arabinopyranoside), -O-릭소피라노사이드(lyxopyranoside), -O-리보피라노사이드(ribopyranoside), 및 -O-자일로바이오스(xylobiose)로 구성된 군에서 선택된 것이다. Xylosylated flavonoid derivatives represented by Formula 10 below;
[Formula 10]
From above,
R 1 , R 4 and R 5 are each independently hydrogen (H) or hydroxy (OH);
R 2 is -O-xyropyranoside, -O-xylopuranoside, -O-xyropyranoside, -O-xylopuranoside , -O-ribofuranoside, -O-arabinopyranoside, -O-lyxopyranoside, -O-ribopyranoside, and -O- It is selected from the group consisting of xylobiose,
R 3 is hydrogen (H), hydroxy (OH), substituted or unsubstituted C 1 -C 20 alkoxy, -O-xyropyranoside, -O-xylopuranoside, -O-ribofuranoside, -O-arabinopyranoside, -O-lyxopyranoside, -O-ribopyranoside, and -O-xyl It is selected from the group consisting of xylobiose.
(a) 바이칼레인 7- O-β-D-자일로피라노사이드 [Baicalein 7-O-β-D-xylopyranoside];
(b) 크리신 7-o-β-D-자일로피라노사이드 [Chrysin 7-O-β-D-xylopyranoside];
(c) 바이칼레인 6,7-O-β-D-디자일로피라노사이드 [Baicalein 6,7-O-β-D-dixylopyranoside];
(d) 오록실린 A 7-O-β-D-자일로피라노사이드 [Oroxylin A 7-O--D-xylopyranoside];
(e) 오록실린 A 7-O-β-D-자일로피라노실-(1→3)-β-D-자일로피라노사이드 [Oroxylin A 7-O-β-D-xylopyranosyl-(1→3)-β-D-xylopyranoside];
(f) 5.8-디하이드록시-6-메톡시플라본 7-O-β-D-자일로피라노사이드 [5,8-dihydroxy-6-methoxyflavone 7-O-β-D-xylopyranoside]; 및
(g) 5,8-디하이드록시-6-메톡시플라본 7-O-β-D-자일로피라노실-(1*β-d-자일로피라노사이드 [5,8-dihydroxy-6-methoxyflavone 7-O-β-D-xylopyranosyl-(1→3)-β-d-xylopyranoside]; 로 구성된 군에서 선택되는 것인 바이칼린 유도체.The method according to claim 1, wherein the flavonoid derivative
(a) Baicalein 7-O-β-D-xylopyranoside [Baicalein 7-O-β-D-xylopyranoside];
(b) Chrysin 7-O-β-D-xylopyranoside [Chrysin 7-O-β-D-xylopyranoside];
(c) Baicalein 6,7-O-β-D-dixylopyranoside;
(d) Oroxylin A 7-O-β-D-xylopyranoside [Oroxylin A 7-O--D-xylopyranoside];
(e) Oroxylin A 7-O-β-D-xylopyranosyl-(1→3)-β-D-xylopyranoside [Oroxylin A 7-O-β-D-xylopyranosyl-(1→3) 3) -β-D-xylopyranoside];
(f) 5,8-dihydroxy-6-methoxyflavone 7-O-β-D-xylopyranoside [5,8-dihydroxy-6-methoxyflavone 7-O-β-D-xylopyranoside]; and
(g) 5,8-dihydroxy-6-methoxyflavone 7-O-β-D-xylopyranosyl-(1*β-d-xylopyranoside [5,8-dihydroxy-6- methoxyflavone 7-O-β-D-xylopyranosyl-(1→3)-β-d-xylopyranoside];
상기 바이칼레인의 6-OH 및 7-OH 위치로 구성된 군에서 선택되는 어느 하나 이상의 위치가 자일로실화(xylosayltion), 하이드록실화(hydroxylation), 디하이드록실화(dehydroxylation) 또는 메틸화(methylation) 반응이 이루어지는 단계에 의해 생성되는 것인 자일로실화된 플라보노이드 유도체. The method according to claim 1, wherein the flavonoid derivative is produced by decomposing a glucuronyl bond of a baicalin compound to produce baicalein; and
At least one position selected from the group consisting of the 6-OH and 7-OH positions of the baicalein is xylosayltion, hydroxylation, dehydroxylation or methylation reaction A xylosylated flavonoid derivative produced by the step comprising the above.
상기 배양액으로부터 자일로실화된 플라보노이드 유도체를 수득하는 단계를 포함하는 자일로실화된 플라보노이드 유도체(Xylosylated flavonoid derivatives) 의 제조 방법. Polyporus brumalis ( Polyporus brumalis ) Obtaining a culture medium by culturing the strain with a flavonoid; and
A method for producing xylosylated flavonoid derivatives comprising obtaining a xylosylated flavonoid derivative from the culture medium.
상기 추출물을 크로마토그래피(chromatography)를 수행하여 활성 분획을 수거하는 단계; 및
상기 활성 분획을 크로마토그래피로 분리하여 목적 화합물을 얻는 단계를 포함하는 것인 자일로실화된 플라보노이드 유도체의 제조방법.The method according to claim 6, wherein the step of obtaining the xylosylated flavonoid derivative from the culture medium comprises: preparing an extract of the culture medium by extracting the culture medium with water, C1 to C4 lower alcohol, or a mixture thereof as a solvent;
Collecting an active fraction by performing chromatography on the extract; and
A method for producing a xylosylated flavonoid derivative comprising the step of obtaining a target compound by separating the active fraction by chromatography.
[화학식 10]
상기에서,
R1, R4 및 R5는 각각 독립적으로 수소(H) 또는 하이드록시(OH)이고,
R2는 -O-자일로피라노사이드(xyropyranoside), -O-자일로퓨라노사이드(Xylopuranoside), -O-자일로피라노사이드(xyropyranoside), -O-자일로퓨라노사이드(xylopuranoside), -O-라이보퓨라노사이드(ribofuranoside), -O-아라비노피라노사이드(arabinopyranoside), -O-릭소피라노사이드(lyxopyranoside), -O-리보피라노사이드(ribopyranoside), 및 -O-자일로바이오스(xylobiose)로 구성된 군에서 선택된 것이고,
R3은 수소(H), 하이드록시(OH), 치환 또는 비치환된 C1-C20 알콕시, -O-자일로피라노사이드(xyropyranoside), -O-자일로퓨라노사이드(xylopuranoside), -O-라이보퓨라노사이드(ribofuranoside), -O-아라비노피라노사이드(arabinopyranoside), -O-릭소피라노사이드(lyxopyranoside), -O-리보피라노사이드(ribopyranoside), 및 -O-자일로바이오스(xylobiose)로 구성된 군에서 선택된 것이다. The method for preparing a xylosylated flavonoid derivative according to claim 6, wherein the flavonoid derivative is represented by Formula 10 below;
[Formula 10]
From above,
R 1 , R 4 and R 5 are each independently hydrogen (H) or hydroxy (OH);
R 2 is -O-xyropyranoside, -O-xylopuranoside, -O-xyropyranoside, -O-xylopuranoside , -O-ribofuranoside, -O-arabinopyranoside, -O-lyxopyranoside, -O-ribopyranoside, and -O- It is selected from the group consisting of xylobiose,
R 3 is hydrogen (H), hydroxy (OH), substituted or unsubstituted C 1 -C 20 alkoxy, -O-xyropyranoside, -O-xylopuranoside, -O-ribofuranoside, -O-arabinopyranoside, -O-lyxopyranoside, -O-ribopyranoside, and -O-xyl It is selected from the group consisting of xylobiose.
(a) 바이칼레인 7- O-β-D-자일로피라노사이드 [Baicalein 7-O-β-D-xylopyranoside];
(b) 크리신 7-o-β-D-자일로피라노사이드 [Chrysin 7-O-β-D-xylopyranoside];
(c) 바이칼레인 6,7-O-β-D-디자일로피라노사이드 [Baicalein 6,7-O-β-D-dixylopyranoside];
(d) 오록실린 A 7-O--D-자일로피라노사이드 [Oroxylin A 7-O--D-xylopyranoside];
(e) 오록실린 A 7-O-β-D-자일로피라노실-(1→3)-β-D-자일로피라노사이드 [Oroxylin A 7-O-β-D-xylopyranosyl-(1→3)-β-D-xylopyranoside];
(f) 5.8-디하이드록시-6-메톡시플라본 7-O-β-D-자일로피라노사이드 [5,8-dihydroxy-6-methoxyflavone 7-O-β-D-xylopyranoside];
(g) 5,8-디하이드록시-6-메톡시플라본 7-O--D-자일로피라노실-(1→3)-β-d-자일로피라노사이드 [5,8-dihydroxy-6-methoxyflavone 7-O-β-D-xylopyranosyl-(1→3)-β-d-xylopyranoside]; 및
(h) 오록실린 A [Oroxylin A]로 구성된 군에서 선택되는 것인 자일로실화된 플라보노이드 유도체의 제조 방법.The method according to claim 6, wherein the flavonoid derivative is
(a) Baicalein 7-O-β-D-xylopyranoside [Baicalein 7-O-β-D-xylopyranoside];
(b) Chrysin 7-O-β-D-xylopyranoside [Chrysin 7-O-β-D-xylopyranoside];
(c) Baicalein 6,7-O-β-D-dixylopyranoside;
(d) Oroxylin A 7-O- -D-xylopyranoside [Oroxylin A 7-O--D-xylopyranoside];
(e) Oroxylin A 7-O-β-D-xylopyranosyl-(1→3)-β-D-xylopyranoside [Oroxylin A 7-O-β-D-xylopyranosyl-(1→3) 3) -β-D-xylopyranoside];
(f) 5,8-dihydroxy-6-methoxyflavone 7-O-β-D-xylopyranoside [5,8-dihydroxy-6-methoxyflavone 7-O-β-D-xylopyranoside];
(g) 5,8-dihydroxy-6-methoxyflavone 7-O--D-xylopyranosyl-(1→3)-β-d-xylopyranoside [5,8-dihydroxy- 6-methoxyflavone 7-O-β-D-xylopyranosyl-(1→3)-β-d-xylopyranoside]; and
(h) A method for producing a xylosylated flavonoid derivative selected from the group consisting of oroxylin A [Oroxylin A].
Xylosylated flavonoid derivatives prepared by the method of claim 6.
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