CN114276339B - Sesquilignans compound, and separation method and application thereof - Google Patents
Sesquilignans compound, and separation method and application thereof Download PDFInfo
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 33
- 238000000926 separation method Methods 0.000 title abstract description 10
- 238000010828 elution Methods 0.000 claims abstract description 27
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 17
- 206010061218 Inflammation Diseases 0.000 claims abstract description 8
- 239000003814 drug Substances 0.000 claims abstract description 8
- 230000004054 inflammatory process Effects 0.000 claims abstract description 8
- 239000003960 organic solvent Substances 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- 235000017524 noni Nutrition 0.000 claims description 36
- 244000131360 Morinda citrifolia Species 0.000 claims description 33
- 239000012046 mixed solvent Substances 0.000 claims description 20
- 239000003480 eluent Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 239000003208 petroleum Substances 0.000 claims description 12
- 238000000605 extraction Methods 0.000 claims description 9
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims description 8
- HWJHWSBFPPPIPD-UHFFFAOYSA-N ethoxyethane;propan-2-one Chemical compound CC(C)=O.CCOCC HWJHWSBFPPPIPD-UHFFFAOYSA-N 0.000 claims description 8
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 8
- 239000000284 extract Substances 0.000 claims description 7
- 238000010898 silica gel chromatography Methods 0.000 claims description 7
- 238000004440 column chromatography Methods 0.000 claims description 6
- 239000000499 gel Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 5
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 238000010298 pulverizing process Methods 0.000 claims description 4
- 239000003643 water by type Substances 0.000 claims description 4
- 208000004232 Enteritis Diseases 0.000 claims description 2
- 208000007882 Gastritis Diseases 0.000 claims description 2
- 206010035664 Pneumonia Diseases 0.000 claims description 2
- 206010051379 Systemic Inflammatory Response Syndrome Diseases 0.000 claims description 2
- 206010006451 bronchitis Diseases 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims description 2
- 230000006837 decompression Effects 0.000 claims 1
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 9
- 229930013686 lignan Natural products 0.000 abstract description 5
- 235000009408 lignans Nutrition 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 4
- -1 lignan compound Chemical class 0.000 abstract description 3
- 230000000144 pharmacologic effect Effects 0.000 abstract description 3
- 238000004811 liquid chromatography Methods 0.000 abstract description 2
- 238000004821 distillation Methods 0.000 abstract 1
- 238000002386 leaching Methods 0.000 abstract 1
- FKLJPTJMIBLJAV-UHFFFAOYSA-N Compound IV Chemical compound O1N=C(C)C=C1CCCCCCCOC1=CC=C(C=2OCCN=2)C=C1 FKLJPTJMIBLJAV-UHFFFAOYSA-N 0.000 description 9
- 238000001228 spectrum Methods 0.000 description 9
- NLFBCYMMUAKCPC-KQQUZDAGSA-N ethyl (e)-3-[3-amino-2-cyano-1-[(e)-3-ethoxy-3-oxoprop-1-enyl]sulfanyl-3-oxoprop-1-enyl]sulfanylprop-2-enoate Chemical compound CCOC(=O)\C=C\SC(=C(C#N)C(N)=O)S\C=C\C(=O)OCC NLFBCYMMUAKCPC-KQQUZDAGSA-N 0.000 description 8
- 238000005481 NMR spectroscopy Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 4
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 4
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 241000157491 Morinda Species 0.000 description 3
- 235000008898 Morinda citrifolia Nutrition 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 239000002158 endotoxin Substances 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 3
- 150000005692 lignans Chemical class 0.000 description 3
- 229920006008 lipopolysaccharide Polymers 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- XILIYVSXLSWUAI-UHFFFAOYSA-N 2-(diethylamino)ethyl n'-phenylcarbamimidothioate;dihydrobromide Chemical compound Br.Br.CCN(CC)CCSC(N)=NC1=CC=CC=C1 XILIYVSXLSWUAI-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 2
- 235000011613 Pinus brutia Nutrition 0.000 description 2
- 241000018646 Pinus brutia Species 0.000 description 2
- 241001107098 Rubiaceae Species 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 238000005100 correlation spectroscopy Methods 0.000 description 2
- 239000002024 ethyl acetate extract Substances 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
- 150000002338 glycosides Chemical class 0.000 description 2
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 2
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 2
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 150000002995 phenylpropanoid derivatives Chemical class 0.000 description 2
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 2
- 238000005728 strengthening Methods 0.000 description 2
- FDKRLXBXYZKWRZ-UWJYYQICSA-N 3-[(21S,22S)-16-ethenyl-11-ethyl-4-hydroxy-12,17,21,26-tetramethyl-7,23,24,25-tetrazahexacyclo[18.2.1.15,8.110,13.115,18.02,6]hexacosa-1,4,6,8(26),9,11,13(25),14,16,18(24),19-undecaen-22-yl]propanoic acid Chemical compound CCC1=C(C2=NC1=CC3=C(C4=C(CC(=C5[C@H]([C@@H](C(=CC6=NC(=C2)C(=C6C)C=C)N5)C)CCC(=O)O)C4=N3)O)C)C FDKRLXBXYZKWRZ-UWJYYQICSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 125000003016 chromanyl group Chemical group O1C(CCC2=CC=CC=C12)* 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229930004069 diterpene Natural products 0.000 description 1
- 150000004141 diterpene derivatives Chemical class 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229930003944 flavone Natural products 0.000 description 1
- 150000002212 flavone derivatives Chemical class 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 1
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 1
- 201000001881 impotence Diseases 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 231100000535 infertility Toxicity 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 150000007965 phenolic acids Chemical class 0.000 description 1
- 229930015704 phenylpropanoid Natural products 0.000 description 1
- NSFSLUUZQIAOOX-LDCXZXNSSA-N pheophorbide a Chemical compound N1C(C=C2[C@H]([C@H](CCC(O)=O)C(=N2)C2=C3NC(=C4)C(C)=C3C(=O)[C@@H]2C(=O)OC)C)=C(C)C(C=C)=C1C=C1C(C)=C(CC)C4=N1 NSFSLUUZQIAOOX-LDCXZXNSSA-N 0.000 description 1
- BOTWFXYSPFMFNR-PYDDKJGSSA-N phytol Chemical class CC(C)CCC[C@@H](C)CCC[C@@H](C)CCC\C(C)=C\CO BOTWFXYSPFMFNR-PYDDKJGSSA-N 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 150000003648 triterpenes Chemical class 0.000 description 1
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a lignan compound, a separation method and application thereof, which mainly obtain sesquilignan compounds I and II of the invention through multi-stage separation steps such as ethyl acetate leaching, reduced pressure distillation, organic solvent gradient elution, liquid chromatography separation and the like. Pharmacological activity experiments show that the compound has obvious anti-inflammatory activity, which shows that the compound has good application prospect in preparing medicines for preventing or treating inflammation.
Description
Technical Field
The invention relates to a sesquilignan compound, and a separation method and application thereof, belonging to the technical field of phytochemistry.
Background
Noni (Morinda Citrifolia) is also called Morinda citrifolia, and is a evergreen shrub or small arbor of Morinda (Morinda) of Rubiaceae (Rubiaceae), and is concentrated in tropical and subtropical regions, and is mainly distributed in Hainan island, sisha island, etc. in China. The "Morinda citrifolia" has the functions of strengthening body resistance, eliminating pathogenic factors, nourishing yin, tonifying kidney, strengthening yang, dispelling wind-damp, treating infertility due to cold womb and treating impotence "recorded in Ben Cao gang mu.
Noni fruit is a fig with unique health care function, and natural juice (noni ferment) produced by fermenting the fig is a popular health care drink, and has the effects of reducing blood sugar, blood pressure, blood fat, resisting tumor and the like. Noni enzyme in 2003 was listed by the U.S. national Food and Drug Administration (FDA) in the book "doctor guidelines for medicine" (Physicians Desk Reference).
The chemical composition research of noni reports that the noni mainly contains various compounds such as flavone (glycoside), phenylpropanoid (glycoside), diterpene, triterpene, iridoid, phenolic acid and the like, and most of noni has better biological activity. Noni seeds contain a large amount of bioactive compounds, especially partial lignans have good anti-inflammatory effect, but have better research reports.
Patent CN101489396 relates to a method of health-beneficial extraction of bioactive compounds or compositions from noni fruits (Morinda cirtrifolia). Specifically, one or more of the following are used: noni leaf extract, noni leaf juice or baked leaf. In particular, it relates to the administration of one or more of the following: pyropheophorbide a, pheophorbide a, purpin7 or pheophorbide phytol esters, all of which may be derived from noni leaf extract, noni leaf juice or baked leaf. In addition, the above preparation can relieve pain and inflammation. However, the patent does not disclose the active ingredients in noni that help to reduce inflammation and are derived primarily from noni leaves.
Patent CN109206394A discloses a sesquilignan compound, a preparation method and application thereof, and belongs to the technical field of extraction, separation and purification of traditional Chinese medicine components. The sesquilignan compound is derived from Chinese medicinal cortex Illici, has a chromane ring structure, and can be used for preparing anticancer drugs, can be used as a lead compound for synthesizing other compounds, or can be used as a raw material for developing new drugs. However, the use of sesquilignans for the preparation of a medicament for the prevention and/or treatment of inflammation is not disclosed.
The invention aims to research chemical components of noni seeds so as to obtain lignan compounds with a novel structure and obvious anti-inflammatory effect, and fully dig the medicinal value of noni health-care fruits.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention provides highly oxidized sesquilignans with novel structures, has remarkable anti-inflammatory effect, and provides a separation method of target compounds.
The technical scheme of the invention is as follows:
a sesquilignan compound, said compound having the following structural formula:
wherein R is 1 、R 2 、R 3 OH or H;
wherein R is 1 、R 2 、R 3 、R 4 OH or H.
Preferably, said R 1 、R 2 、R 3 Is OH, named rel- (7 'beta, 8' alpha, 7 'beta, 8' alpha) -3,3', 4",9' -pentahydroxy-1-propenal-7 ',8' -benzodihydrofuran-3, 7 ':7,9' -dioxacyclosesqui-neolignans, having the structure (III):
the R is 1 、R 2 、R 3 、R 4 Is OH, named rel- (7β,8β,7β,8β,8α) -3',3",4',4",9 "-pentahydroxy-3, 7":7,9 '-dioxan-7', 9-lactone-4, 8 '-epoxy-8, 8' -sesqui-neolignans having the structure (IV):
a method for separating sesquilignans, comprising the following steps:
(1) Pulverizing dried noni seeds, sequentially extracting noni seed powder with organic solvent by ultrasonic for multiple times, mixing the extractive solutions, and concentrating under reduced pressure to obtain extract;
(2) Performing gradient elution on the extract obtained by the extraction in the step (1) by using a silica gel column chromatography and using a petroleum ether-acetone mixed solvent as an eluent to obtain 7 components, namely Fr.1-Fr.7, according to the polarity;
(3) Subjecting Fr.5 to normal phase silica gel column chromatography, gradient eluting with chloroform-methanol mixed solvent as eluent, concentrating under reduced pressure, separating with ODS reversed phase column, gradient eluting with methanol-water mixed solvent as eluent to obtain 7 components (Fr.5a-Fr.5g) according to polarity; fr.5d is taken, eluted by Sephadex LH-20 gel column chromatography, decompressed and concentrated, and then prepared by high performance liquid chromatography HPLC, and the compounds of the general formula I and II are obtained in sequence.
Preferably, in the step (1), the organic solvent is petroleum ether or ethyl acetate, the dissolution is analytically pure, and the dosage is 3-6L of solvent for each kilogram of noni seed powder; the extraction times are 3-5 times, and each time of extraction is 1-3 hours; the ultrasonic frequency is 30-50kHz, and the ultrasonic time is 15-30min each time.
Preferably, in the step (2), the elution gradient is (100:1) to (1:100).
Preferably, in the step (2), the elution gradient of the petroleum ether-acetone mixed solvent is 100:1, 80:1, 20:1, 10:1, 5:1, 1:1, 1:10, 3 column volumes are collected for each gradient, and 7 components, namely Fr.1-Fr.7, are obtained for each gradient.
Preferably, in the step (3), the chloroform-methanol mixed solvent has an elution gradient of (20:1) to (1:10), and the methanol-water mixed solvent has an elution gradient of 1:90 (MeOH: H) by volume ratio 2 O，V:V)-100:1(MeOH:H 2 O，V:V)。
Preferably, in the step (3), the elution gradient of the chloroform-methanol mixed solvent is 20:1, 10:1, 5:1, 1:1, and each gradient elutes 2 to 5 column volumes; the reverse elution proportion of ODS is 10:90, 30:70, 50:50, 60:40, 70:30, 80:20 and 100:1 of MeOH to H2O (V: V), and each gradient is eluted for 3-5 column volumes; sephadex LH-20 gel column chromatography, wherein the eluent is MeOH, and the elution is carried out for 3-6 column volumes; the chromatographic column of high performance liquid chromatography is Waters C 18 The mobile phase is MeOH to H with volume ratio of 15:85 2 O, flow rate was 2mL/min.
Preferably, noni seeds are provided by the commercial technology company of Hainan pine Ji Yun.
The invention also confirms that the obtained compounds III and IV (the compounds I and II in the general formula) have good anti-inflammatory effect through pharmacological activity experiments, and the compounds III and IV have good application prospects in preparing medicines for preventing and/or treating inflammation. The inflammation is systemic inflammatory response syndrome, bronchitis, pneumonia, gastritis or enteritis.
Compared with the prior art, the invention has the beneficial effects that:
the invention extracts and separates highly oxidized sesquilignans I and II from noni seeds, which are formed by combining 3 phenylpropanoid fragments and have a plurality of chiral centers. The effective components with anti-inflammatory effect in noni seeds are clarified. Specifically, the multi-chiral center highly oxidized sesquilignans are obtained from noni seeds by multistage separation and extraction methods such as polar solvent extraction, solvent gradient elution, liquid chromatography separation and the like.
Drawings
Fig. 1: compound III 1 H-NMR spectrum (MeOD-d) 4 )
Fig. 2: compound III 13 C-NMR spectrum (MeOD-d) 4 )
Fig. 3: DEPT (135 DEG) spectrum of Compound III (MeOD-d 4 )
Fig. 4: compound III 1 H- 1 H COSY spectrum (MeOD-d) 4 )
Fig. 5: HSQC spectra of Compound III (MeOD-d 4 )
Fig. 6: HMBC spectra of Compound III (MeOD-d 4 )
Fig. 7: NOESY spectrum of Compound III (MeOD-d 4 )
Fig. 8: HRESIMS spectrum of Compound III
Fig. 9: compound IV 1 H-NMR spectrum (MeOD-d) 4 )
Fig. 10: compound IV 13 C-NMR spectrum (MeOD-d) 4 )
Fig. 11: DEPT (135 ℃) spectrum of Compound IV (MeOD-d 4 )
Fig. 12: compound IV 1 H- 1 H COSY spectrum (MeOD-d) 4 )
Fig. 13: HSQC spectra of Compound IV (MeOD-d 4 )
Fig. 14: HMBC spectra of Compound IV (MeOD-d 4 )
Fig. 15: NOESY spectra of Compound IV (MeOD-d 4 )
Fig. 16: HRESIMS spectrum of Compound IV
Detailed Description
In order to better understand the technical content of the present invention, the following provides specific examples to further illustrate the present invention.
The experimental methods used in the embodiment of the invention are conventional methods unless otherwise specified.
Materials, reagents, and the like used in the examples of the present invention are commercially available unless otherwise specified.
The experimental material noni is provided by the Hainan pine Ji Yun commercial technology Co., ltd, and the part used is seeds.
EXAMPLE 1 preparation of sesquilignans
The method comprises the following steps:
(1) Pulverizing the dried noni seeds, sequentially extracting with 4L petroleum ether and ethyl acetate solvent for 3 times (each time for 3 hr), mixing the extractive solutions, and concentrating under reduced pressure to obtain ethyl acetate extract (about 110 g);
(2) Taking ethyl acetate part extractum (about 60 g) in the step (1), performing gradient elution by using a petroleum ether-acetone mixed solvent as an eluent through silica gel column chromatography, wherein the elution gradient of the petroleum ether-acetone mixed solvent is 100:1, 80:1, 20:1, 10:1, 5:1, 1:1 and 1:10, collecting 2 column volumes for each gradient, and obtaining 7 components, namely Fr.1-Fr.7;
(3) Subjecting Fr.5 to normal phase silica gel column chromatography, and eluting with chloroform-methanol mixed solvent with gradient of 20:1, 10:1, 5:1, and 1:1, wherein each gradient is 2 column volumes; concentrating the product under reduced pressure, eluting with ODS in reverse phase to obtain MeOH H 2 O (V: V) 10:90, 30:70, 50:50, 60:40, 70:30, 80:20, 100:1, 4 column volumes per gradient elution; subjecting the product to Sephadex LH-20 gel column chromatography, eluting with MeOH as eluent, eluting 3 column volumes, concentrating under reduced pressure, and subjecting to high performance liquid chromatography under the following conditions: chromatographic column Waters C 18 The flow rate is 2mL/min, the mobile phase is MeOH/H with the volume ratio of 15:85 2 O, to give compounds III and IV.
EXAMPLE 2 preparation of sesquilignans
The method comprises the following steps:
(1) Pulverizing dried noni seeds, sequentially extracting with 4L petroleum ether and ethyl acetate solvent for 4 times under ultrasonic treatment, extracting for 2 hr each time, mixing extractive solutions, and concentrating under reduced pressure to obtain ethyl acetate extract (about 120 g);
(2) Taking ethyl acetate part extractum (about 60 g) in the step (1), performing gradient elution by using a petroleum ether-acetone mixed solvent as an eluent, wherein the elution gradient of the petroleum ether-acetone mixed solvent is 100:1, 80:1, 20:1, 10:1, 5:1, 1:1 and 1:10, collecting 3 column volumes for each gradient, and obtaining 7 components, namely Fr.1-Fr.7;
(3) Subjecting Fr.5 to normal phase silica gel column chromatography, eluting with chloroform-methanol mixed solvent with gradient of 20:1, 10:1, 5:1, and 1:1, wherein each gradient is used for eluting 3 column volumes; concentrating the product under reduced pressure, eluting with ODS in reverse phase to obtain MeOH H 2 O (V: V) 10:90, 30:70, 50:50, 60:40, 70:30, 80:20, 100:1, 5 column volumes per gradient elution; subjecting the product to Sephadex LH-20 gel column chromatography, eluting with MeOH as eluent, and eluting for 4 column volumes; the conditions of high performance liquid chromatography after the product is decompressed and concentrated are as follows: chromatographic column Waters C 18 The flow rate is 2mL/min, the mobile phase is MeOH/H with the volume ratio of 15:85 2 O, to give compounds III and IV.
EXAMPLE 3 structural identification of sesquilignans
Spectrum of application 1 H NMR， 13 C NMR, HSQC, HMBC, NOESY) and MS, and the chemical structures of the compounds iii and IV obtained in example 1 and example 2.
The structural identification data are as follows:
compound iii: is yellow oil and is easy to dissolve in methanol. HRESI (-) MS (m/z 491.1345[ M-H)] - Theoretical value 491.1342) determines that its molecular formula is C 27 H 24 O 9 The method comprises the steps of carrying out a first treatment on the surface of the According to 1 H， 13 C and two-dimensional nuclear magnetic resonance data determine the structure, the skeleton type is sesquilignans, namely rel- (7 ' beta, 8' alpha, 7' beta, 8' alpha) -3,3', 4', 9'Pentahydroxy-1-propenal-7 ',8' -benzodihydrofuran-3, 7 ':7,9' -dioxacyclosesqui-new lignans, designated morictan A, which 1 H and 13 the C NMR data are shown in Table 1. [600MHz ] 1 H)，150 MHz( 13 C) Solvent: meOD-d 4 ]。
Compound IV: as a yellow oil, readily soluble in methanol. HRESI (-) MS (m/z 507.1291[ M-H)] - Theoretical value 507.1291) determines that its molecular formula is C 27 H 24 O 10 The method comprises the steps of carrying out a first treatment on the surface of the According to 1 H， 13 C and two-dimensional nuclear magnetic resonance data determine the structure, the framework type is sesquilignans, namely rel- (7β,8β,7β,8α) -3',3",4',4",9 "-pentahydroxy-3, 7":7,9 '-Dioxacyclo-7', 9-lactone-4, 8 '-epoxy-8, 8' -sesqui-new lignans, designated Moricitan B. Which is a kind of 1 H and 13 the C NMR data are shown in Table 1. [400MHz ] 1 H)， 100MHz( 13 C) Solvent: meOD-d 4 ]。
TABLE 1 Compounds III and IV 1 H-NMR 13 C-NMR(MeOD-d 4 ) Data
a1 H-NMR 13 C-NMR(600,150MHz)
b1 H-NMR 13 C-NMR(400,100MHz)
From the above analysis, it was determined that the structures of compounds III and IV were:
EXAMPLE 4 pharmacological Activity assay
Experimental materials:
and (3) cells: mouse mononuclear macrophage raw264.7.
Cell culture fluid: DMEM medium containing 10% Fetal Bovine Serum (FBS), lipopolysaccharide (LPS) carbohydrate.
NO detection kit: priley (APPLYGEN).
The experimental method comprises the following steps:
induction: raw264.7 cells were cultured with DMEM medium containing 10% FBS at 37deg.C and 5% CO 2 Culturing in an incubator conventionally. Cell count 1X 10 5 Inoculating 200 μL/well of the sample into 96-well plate, respectively setting blank control group, positive control group, LPS induction group, and high, medium and low (50,25,12.5 μM) dosage groups of the tested drug, placing at 37deg.C, 5% CO 2 After 24h of adherence in the cell incubator.
And (3) detection: 50 mu L of supernatant is taken as a liquid to be detected in a 96-well plate, 50 mu L of reagent A and 50 mu L of reagent B are sequentially added according to a detection method of a kit instruction, and an OD value is detected at 540nm by adopting an enzyme-labeled instrument.
The anti-inflammatory activity of the compounds is shown in Table 2, from which it is clear that both compounds III and IV exhibit significant anti-inflammatory effects, IC 50 2.52.+ -. 0.22 and 1.83.+ -. 0.31. Mu.M, respectively.
TABLE 2 anti-inflammatory Activity of Compounds III and IV against RAW264.7 cellsn＝3).
The foregoing is a further detailed description of the invention in connection with specific embodiments, and it is not intended that the invention be limited to such description. It will be apparent to those skilled in the art that several simple deductions or substitutions can be made without departing from the spirit of the invention.
Claims (9)
1. A sesquilignan compound, which is characterized by being named rel- (7 ' beta, 8' alpha, 7' beta, 8' alpha) -3,3', 4', 9',9' -pentahydroxy-1-propenal-7 ',8' -benzodihydrofuran-3, 7' ':7,9' -dioxacyclosesquilignan and having a structure of (iii):
(III); or the compound name rel- (7β,8β,7β,8α) -3',3' ',4',4' ',9' ' -pentahydroxy-3, 7' ':7,9' -dioxa-7 ', 9-lactone-4, 8' ' -epoxy-8, 8' -sesqui-neolignans, structure (IV):
（IV）。
2. the method for separating a sesquilignan compound according to claim 1, comprising the steps of:
(1) Pulverizing dried noni seeds, sequentially extracting noni seed powder with organic solvent by ultrasonic for multiple times, mixing the extractive solutions, and concentrating under reduced pressure to obtain extract;
(2) Performing gradient elution on the extract obtained by the extraction in the step (1) by using a silica gel column chromatography and using a petroleum ether-acetone mixed solvent as an eluent to obtain 7 components, namely Fr.1-Fr.7, according to the polarity;
(3) Subjecting Fr.5 to normal phase silica gel column chromatography, performing gradient elution by using chloroform-methanol mixed solvent as an eluent, concentrating under reduced pressure, separating by using ODS reverse phase column, performing gradient elution by using methanol-water mixed solvent as the eluent, and obtaining 7 components, namely Fr.5a-Fr.5g, according to the polarity; fr.5d is taken, and is eluted by SephadexLH-20 gel column chromatography, and is prepared by high performance liquid chromatography HPLC after decompression concentration, thus obtaining the compounds of the general formula I and II in sequence.
3. The method for separating sesquilignans according to claim 2, wherein in the step (1), the organic solvent is petroleum ether or ethyl acetate, the solvents are all analytically pure, and the amount of the solvents is 3-6L per kg noni seed powder; the extraction times are 3-5 times, and each time of extraction is 1-3 hours; the ultrasonic frequency is 30-50kHz, and the ultrasonic time is 15-30min each time.
4. The method for separating sesquilignans according to claim 2, wherein in the step (2), the elution gradient is 100:1 to 1:100.
5. The method according to claim 2, wherein in the step (2), the elution gradient of the petroleum ether-acetone mixed solvent is 100:1, 80:1, 20:1, 10:1, 5:1, 1:1, 1:10, and 3 column volumes are collected for each gradient, and each gradient gives 7 components, i.e., fr.1 to fr.7.
6. The method for separating sesquilignans according to claim 2, wherein in the step (3), the elution gradient of the chloroform-methanol mixed solvent is 20:1-1:10, and the elution gradient of the methanol-water mixed solvent is 1:90 by volume.
7. The method for separating sesquilignans according to claim 2, wherein in the step (3), the gradient of elution of chloroform-methanol mixed solvent is 20:1, 10:1, 5:1, 1:1, each gradient eluting 2 to 5 column volumes; ODS reverse phase elution MeOH H 2 The volume ratio of O is 10:90, 30:70, 50:50, 60:40, 70:30, 80:20 and 100:1, and each gradient elution is 3-5 column volumes; sephadexLH-20 gel column chromatography, wherein the eluent is MeOH, and the elution is carried out for 3-6 column volumes; the chromatographic column of the high performance liquid chromatography is Waters C18, and the mobile phase is MeOH/H with the volume ratio of 15:85 2 O, flow rate was 2mL/min.
8. The use of a sesquilignan compound according to claim 1 for the preparation of a medicament for the prevention and/or treatment of inflammation.
9. The use according to claim 8, wherein the inflammation is systemic inflammatory response syndrome, bronchitis, pneumonia, gastritis or enteritis.
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