CN117069788A - Furosestanol type steroid saponin compounds, preparation method and application thereof - Google Patents
Furosestanol type steroid saponin compounds, preparation method and application thereof Download PDFInfo
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- -1 steroid saponin compounds Chemical class 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 93
- 102100038280 Prostaglandin G/H synthase 2 Human genes 0.000 claims abstract description 13
- 150000001875 compounds Chemical class 0.000 claims abstract description 13
- 101710113864 Heat shock protein 90 Proteins 0.000 claims abstract description 11
- 102100029438 Nitric oxide synthase, inducible Human genes 0.000 claims abstract description 11
- 101710089543 Nitric oxide synthase, inducible Proteins 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 10
- 102100034051 Heat shock protein HSP 90-alpha Human genes 0.000 claims abstract description 9
- 239000000287 crude extract Substances 0.000 claims abstract description 9
- 239000003814 drug Substances 0.000 claims abstract description 5
- 108050003267 Prostaglandin G/H synthase 2 Proteins 0.000 claims abstract 3
- 238000010828 elution Methods 0.000 claims description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 34
- 229940125904 compound 1 Drugs 0.000 claims description 24
- 229940125782 compound 2 Drugs 0.000 claims description 24
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 22
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 21
- 238000000605 extraction Methods 0.000 claims description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- 239000012071 phase Substances 0.000 claims description 18
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 16
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 11
- 238000004809 thin layer chromatography Methods 0.000 claims description 11
- 238000004440 column chromatography Methods 0.000 claims description 9
- 239000003480 eluent Substances 0.000 claims description 8
- 239000003208 petroleum Substances 0.000 claims description 8
- 238000010829 isocratic elution Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
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- 229920005989 resin Polymers 0.000 claims description 6
- 238000001179 sorption measurement Methods 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 5
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 4
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 4
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- ZEEBGORNQSEQBE-UHFFFAOYSA-N [2-(3-phenylphenoxy)-6-(trifluoromethyl)pyridin-4-yl]methanamine Chemical compound C1(=CC(=CC=C1)OC1=NC(=CC(=C1)CN)C(F)(F)F)C1=CC=CC=C1 ZEEBGORNQSEQBE-UHFFFAOYSA-N 0.000 claims description 3
- 239000002246 antineoplastic agent Substances 0.000 claims description 3
- 239000008346 aqueous phase Substances 0.000 claims description 3
- 238000003810 ethyl acetate extraction Methods 0.000 claims description 3
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- 229940041181 antineoplastic drug Drugs 0.000 claims description 2
- 239000002021 butanolic extract Substances 0.000 claims description 2
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 2
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- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 description 19
- 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 19
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- ADHFZEPOBOTKSO-QLTVFDSQSA-N (1R,2S,4R,5'R,6R,7S,8R,9S,12S,13S)-5',7,9,13-tetramethylspiro[5-oxapentacyclo[10.8.0.02,9.04,8.013,18]icosane-6,2'-oxane]-3-ol Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)CCCCC4CC[C@H]3[C@@H]2C1O)C)[C@@H]1C)[C@]11CC[C@@H](C)CO1 ADHFZEPOBOTKSO-QLTVFDSQSA-N 0.000 description 2
- 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
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- GMBQZIIUCVWOCD-UQHLGXRBSA-N Isosarsasapogenin Natural products O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)CC[C@H](O)C[C@H]4CC[C@H]3[C@@H]2C1)C)[C@@H]1C)[C@]11CC[C@@H](C)CO1 GMBQZIIUCVWOCD-UQHLGXRBSA-N 0.000 description 2
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- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 2
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- JUJWROOIHBZHMG-RALIUCGRSA-N pyridine-d5 Chemical compound [2H]C1=NC([2H])=C([2H])C([2H])=C1[2H] JUJWROOIHBZHMG-RALIUCGRSA-N 0.000 description 2
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- 206010007572 Cardiac hypertrophy Diseases 0.000 description 1
- 101710163595 Chaperone protein DnaK Proteins 0.000 description 1
- 108010058432 Chaperonin 60 Proteins 0.000 description 1
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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- 239000008280 blood Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
- C07J71/0005—Oxygen-containing hetero ring
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Rheumatology (AREA)
- Pain & Pain Management (AREA)
- Steroid Compounds (AREA)
Abstract
The invention relates to the technical field of natural medicines, in particular to a furostanol type steroid saponin compound in the rhizome of the arrow, and a separation preparation method and application thereof. According to the invention, 2 new furostanol type steroid saponin compounds are obtained by separating and purifying methanol reflux crude extract of the medicinal plant arrow. Molecular docking experiments prove that the 2 compounds have better binding activity with iNOS, COX-2, HSP90 and GR and can be potential inhibitors of iNOS, COX-2, HSP90 and GR. The method is relatively simple and has good practical value, and has a reference function for continuously finding out the components from the open arrow, thereby having important significance for the comprehensive development and application of the open arrow.
Description
Technical Field
The invention relates to the technical field of natural medicines, in particular to a furostanol type steroid saponin compound in the rhizome of the arrow, and a separation preparation method and pharmaceutical application thereof.
Background
The Kaikou arrow (Tupistra chinensis Baker) is a plant of the genus Kaikou of the family Liliaceae (Liliaceae), and is also called Kaikou arrow, lophatherum gracile, rohdea japonica, etc., and is mainly produced in the middle and southwest regions of China, hubei Sanxia ku region and Shenlong Jianlin region. The open arrow is perennial herb, the beard and root are removed, and the dry rhizome is used as medicine, so that the open arrow is cold in nature, sweet and slightly bitter in taste, and is used for folk medicine of minority. The ancient medical books record that the open arrow has the effects of clearing heat and detoxicating, dispelling blood stasis and relieving pain and is commonly used for treating sore throat, diphtheria, traumatic injury, venomous snake bite and the like. Modern pharmacological research also shows that the arrow has a plurality of pharmacological activities such as good anti-tumor, anti-inflammatory, antibacterial, antioxidant, myocardial hypertrophy resistance and the like. The most important chemical components in the arrow are steroid saponins, which are also main active components in the arrow, and the steroid saponins are classified into spirostanol type steroid saponins, isosteronol type steroid saponins, furostanol type steroid saponins and deformed spirostanol type steroid saponins according to the parent nuclear structure of aglycone.
Disclosure of Invention
The invention provides a furostanol type steroid saponin compound separated from an arrow and a preparation method thereof.
The furostanol-type steroid saponin compounds provided by the invention are all obtained by extracting and separating from rhizome of the arrow, and the structural formula of the furostanol-type steroid saponin compounds (1-2) is as follows:
the preparation method of the steroid saponin compound comprises the following steps:
s1, weighing dried rhizome of the arrow, slicing, crushing, adding methanol, refluxing for 3 times at 65+/-3 ℃ for 1-2 hours each time, combining the extracting solutions after 3 times of refluxing, and concentrating the filtrate under reduced pressure after suction filtration to obtain a methanol crude extract;
s2, dissolving the methanol crude extract in water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, discarding petroleum ether and ethyl acetate extraction parts, and concentrating the rest extraction liquid under reduced pressure to obtain an n-butanol extraction part and a water phase extraction part;
s3, performing gradient elution on the water phase extraction part by using D101 macroporous adsorption resin column chromatography and using ethanol-water as a mobile phase, detecting by using a thin-layer G plate chromatography, and combining similar components to obtain 8 components Fr.A-Fr.H;
purifying the component Fr.D by high performance liquid chromatography, and performing isocratic elution to obtain a compound 1;
s4, performing gradient elution on the n-butanol extraction part by using D101 macroporous adsorption resin column chromatography and using ethanol-water as a mobile phase, and detecting and combining similar components by TLC to obtain 9 components Fr.A-Fr.I;
subjecting Fr.F components to normal phase silica gel column chromatography, performing gradient elution by using dichloromethane-methanol as mobile phase, detecting and combining similar components by TLC to obtain 6 components Fr.HI-Fr.VI;
subjecting Fr.FV component to reverse phase ODS column chromatography, gradient eluting with methanol-water as mobile phase, detecting by TLC, mixing similar components to obtain 7 components Fr.FV 1-Fr.FV 7;
purifying the component Fr.FV 6 by high performance liquid chromatography, and performing gradient elution to obtain a compound 2;
further, in the step S1, the liquid-solid ratio of the methanol to the open arrow is 3:1-6:1 (mL: g);
further, in the step S2, the volumes of the extracting solvents of petroleum ether, ethyl acetate and n-butanol are respectively 1-2 times of the volume of the aqueous solution of the methanol extract;
further, in step S3, conditions for gradient elution of the aqueous phase extraction portion are as follows: gradient elution is carried out according to the volume ratio of ethanol to water of 0:1, 1:9, 2:8, 3:7, 1:1, 6:4, 7:3, 9:1 and 1:0;
further, in step S3, the conditions for isocratic elution of the component fr.d are: isocratic elution is carried out at a flow rate of 3mL/min according to the volume ratio of acetonitrile to water of 18:82;
further, in step S4, the conditions for the gradient elution of the n-butanol extract portion are as follows: gradient elution is carried out according to the volume ratio of ethanol to water of 0:1, 1:9, 2:8, 3:7, 1:1, 6:4, 7:3, 9:1 and 1:0;
further, in step S4, conditions for gradient elution of fr.f component are: gradient elution is carried out according to the volume ratio of dichloromethane to methanol of 20:1, 15:1, 10:1, 9:1, 8:2, 7:3, 6:4, 1:1 and 0:1;
further, in step S4, conditions for gradient elution of fr.fv component are: gradient elution is carried out according to the volume ratio of methanol to water of 0:1, 1:9, 2:8, 3:7, 1:1, 6:4, 7:3, 9:1 and 1:0;
further, in step S4, conditions for gradient elution of fr.fv6 component are: gradient elution was carried out at a flow rate of 3mL/min at a ratio of acetonitrile to water of 23:77 to 30:70 by volume for 0-50 min.
The technical scheme provided by the invention has the following beneficial effects:
according to the invention, 2 new compounds are obtained by separating and purifying methanol reflux crude extract of the medicinal plant arrow, and are determined to be furostanol type steroid saponin compounds by combining and applying a plurality of modern spectrum technologies; the method for extracting the new furostanol type steroid saponin compounds from the arrow is relatively simple and has good practical value, and meanwhile, the method has an important significance for the comprehensive development and application of the arrow for continuously finding out the components from the arrow.
Drawings
FIG. 1 is a flow chart showing the extraction and separation of a furostanol-type steroid saponin compound prepared in example 1 of the present invention;
FIG. 2 shows a compound 1 prepared in example 1 of the present invention 1 H-NMR(600MHz,Pyridine-d 5 ) A spectrogram;
FIG. 3 shows a compound 1 prepared in example 1 of the present invention 13 C-NMR(150MHz,Pyridine-d 5 ) A spectrogram;
fig. 4 is a DEPT (θ=135°) spectrum of the compound 1 prepared in example 1 of the present invention;
FIG. 5 is a HSQC spectrum of Compound 1 obtained in example 1 of the present invention;
FIG. 6 is a HMBC spectrum of the compound 1 prepared in example 1 of the present invention;
FIG. 7 shows a compound 1 prepared in example 1 of the present invention 1 H- 1 H COSY profile;
FIG. 8 is a ROESY spectrum of Compound 1 obtained in example 1 of the present invention;
FIG. 9 is a UV spectrum of compound 1 prepared in example 1 of the present invention;
FIG. 10 is a spectrum of HR-ESI-MS of compound 1 prepared in example 1 of the present invention;
FIG. 11 is a diagram showing the compound 2 produced in example 1 of the present invention 1 H-NMR(500MHz,Pyridine-d 5 ) A spectrogram;
FIG. 12 shows a compound 2 prepared in example 1 of the present invention 13 C-NMR(125MHz,Pyridine-d 5 ) A spectrogram;
fig. 13 is a DEPT (θ=135°) spectrum for compound 2 prepared in example 1 of the present invention;
FIG. 14 is a HSQC spectrum of Compound 2 obtained in example 1 of the present invention;
FIG. 15 is a HMBC spectrum of Compound 2 prepared in example 1 of the present invention;
FIG. 16 shows a compound 2 prepared in example 1 of the present invention 1 H- 1 H COSY profile;
FIG. 17 is a ROESY spectrum of Compound 2 prepared in example 1 of the present invention;
FIG. 18 is a UV spectrum of compound 2 prepared in example 1 of the present invention;
FIG. 19 is a HR-ESI-MS spectrum of compound 2 prepared in example 1 of the present invention;
FIG. 20 is a three-dimensional effect of the docking of aglycone of Compound 1 prepared in example 1 of the present invention with iNOS;
FIG. 21 is a three-dimensional effect of the docking of aglycone of Compound 2 prepared in example 1 of the present invention with iNOS;
FIG. 22 is a three-dimensional effect of the docking of aglycone of Compound 1 prepared in example 1 of the present invention with COX-2;
FIG. 23 is a graph showing the three-dimensional effect of the docking of aglycone of Compound 2 with COX-2 produced in example 1 of the present invention;
FIG. 24 is a three-dimensional effect of the docking of aglycone of Compound 1 prepared in example 1 of the present invention with HSP 90;
FIG. 25 is a three-dimensional effect of the docking of aglycone of Compound 2 prepared in example 1 of the present invention with HSP 90;
FIG. 26 is a three-dimensional effect of the aglycone of Compound 1 prepared in example 1 of the present invention in butt joint with GR;
FIG. 27 is a three-dimensional effect of the aglycone of Compound 2 prepared in example 1 of the present invention on GR docking.
Detailed Description
For a clearer demonstration of the objects, technical solutions and advantages of the present invention, embodiments of the present invention will be further described below with reference to the accompanying drawings and examples of embodiments.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1:
the embodiment 1 provides a preparation method of a furostanol type steroid saponin compound separated from an arrow, which comprises the following specific steps:
step S1, weighing 500g of dried rhizome of the open arrow, slicing, crushing into powder, placing into a three-necked flask, adding absolute methanol (Shanghai Taitan technology Co., ltd.), placing into an electrothermal sleeve, heating, refluxing for three times at 65 ℃, wherein the volume of each methanol addition is 3L, 1.5L and 1.5L, and the refluxing time is set to be 2h, 1h and 1h. After the reflux is finished, combining the crude extracts after three times of reflux, and concentrating the filtrate under reduced pressure after suction filtration to obtain 140.4g of methanol crude extract; the rhizome of the open arrow used in this example 1 was obtained from Yichang city of Hubei province, and was identified as the rhizome of the open arrow (Tupistra chinensis Baker.) of the plant of the Convallaria family of Liliaceae by the university of ethnic group of medicine, south China;
and S2, adding 2L of water into the methanol crude extract for dissolution, sequentially extracting with 2.3L of petroleum ether, 4.0L of ethyl acetate and 3.7L of n-butanol, wherein the volume of an extraction solvent is 1-2 times that of the aqueous solution of the extract, discarding petroleum ether and ethyl acetate extraction parts, concentrating the rest of the extraction liquid under reduced pressure to recover the solvent, and obtaining 56.3g of n-butanol extraction part and 81.1g of aqueous phase extraction part.
Step S3, taking 30g of water phase extraction part to carry out D101 macroporous adsorption resin column chromatography, using ethanol and water as eluent, carrying out gradient elution according to the volume ratio of 0:1, 1:9, 2:8, 3:7, 1:1, 6:4, 7:3, 9:1 and 1:0, detecting and combining similar components by TLC (thin-layer chromatography), and obtaining 8 components Fr.A-Fr.H according to the eluting sequence, wherein the developing agent is chloroform: methanol: water=7:3:1 (v/v/v, mixed, allowed to stand for delamination, lower layer taken as developing agent);
the fraction Fr.D (fraction eluted at se:Sup>A volume ratio of ethanol to water of 1:1, mass of 1.1 g) was purified by reversed phase HPC using se:Sup>A semi-preparative column YMC-Pack ODS-A (250X 10mm,5 μm), and isocratic elution was performed with acetonitrile and water as mobile phases at se:Sup>A volume ratio of 18:82 at se:Sup>A flow rate of 3 m/min for se:Sup>A retention time t R Component =32.4 min, compound 1, 5.2mg;
step S4, carrying out D101 macroporous adsorption resin column chromatography on 56.3g of n-butanol extraction part, using ethanol and water as eluent, and carrying out gradient elution according to the volume ratio of 0:1, 1:9, 2:8, 3:7, 1:1, 6:4, 7:3, 9:1 and 1:0 in sequence, wherein the similar components are combined through TLC detection, and 9 components Fr.A-Fr.I are obtained according to the elution sequence, and the developing agent is chloroform: methanol: water=7:3:1 (v/v/v, mixed, allowed to stand for delamination, lower layer taken as developing agent);
subjecting a component Fr.F (31.5 g, combining eluting parts with the volume ratio of ethanol to water being 1:1 and the volume ratio of ethanol to water being 7:3) to normal phase silica gel column (200-300 meshes) chromatography, sequentially carrying out gradient elution by taking methylene dichloride to methanol as an eluent according to the volume ratio of 20:1, 15:1, 10:1, 9:1, 8:2, 7:3, 6:4, 1:1 and 0:1, detecting the combined similar components by TLC, and obtaining 6 components Fr.FI-Fr.VI according to the eluting sequence, wherein the developing agent is chloroform: methanol: water=7:3:1 (v/v/v, mixed, allowed to stand for delamination, lower layer taken as developing agent);
subjecting a component Fr.FV (16.61 g, eluting component when the volume ratio of dichloromethane to methanol is 8:2) to reversed-phase ODS column chromatography, sequentially performing gradient elution by using methanol-water as an eluent according to the volume ratio of 0:1, 1:9, 2:8, 3:7, 1:1, 6:4, 7:3, 9:1 and 1:0, detecting by TLC to combine similar components, and obtaining 7 components Fr.FV 1-Fr.FV 7 according to the eluting sequence, wherein the developing agent is chloroform: methanol: water=7:3:1 (v/v/v, mixed, allowed to stand for delamination, lower layer taken as developing agent);
the component Fr.FV 6 (10.6 g, the eluting part with the volume ratio of methanol-water being 1:1 and the volume ratio of methanol-water being 7:3) is purified by HPC by utilizing se:Sup>A semi-preparative column YMC-Pack ODS-A (250X 10mm,5 μm), acetonitrile and water are taken as mobile phases, gradient elution is carried out in 0-50min according to the volume ratio of the acetonitrile and the water from 23:77 to 30:70 and the flow rate of 3 m/min, and the retention time t is obtained R Component=35.7 min, compound 2, 1.3mg;
the flow chart of the above steps is shown in fig. 1.
Example 2: the structure of compounds 1-2 was identified as follows:
the structure of the compound 1-2 was determined by subjecting the compound 1-2 obtained in example 1 to high resolution mass spectrometry, ultraviolet spectrometry, and nuclear magnetic resonance analysis.
Physicochemical data and spectroscopic data for compounds 1-2 are as follows:
compound 1: white amorphous powder; HR-ESI-MS m/z 755.42120[ M+H ]] + (calcd for C 39 H 63 O 14 ,755.42123)；UV(MeOH)λ max (log ε) 210 (3.81), 250 (3.44); the structural formula is as follows:
the Chinese and English designations of the compound 1 are shown in the table 1, the nuclear magnetic resonance data are shown in the tables 2 and 3, 1 H-NMR(600MHz,Pyridine-d 5 ) The spectrogram is shown in figure 2, 13 C-NMR(150MHz,Pyridine-d 5 ) The spectrogram is shown in fig. 3, the dept (θ=135°) spectrogram is shown in fig. 4, the hsqc spectrogram is shown in fig. 5, the hmbc spectrogram is shown in fig. 6, 1 H- 1 the H COSY spectrogram is shown in the figure7, the ROESY spectrum is shown in FIG. 8, the UV spectrum is shown in FIG. 9, and the HR-ESI-MS spectrum is shown in FIG. 10.
Compound 2: white amorphous powder; HR-ESI-MS m/z 775.38774[ M+Na ]] + (calcd for C 39 H 60 O 14 Na,775.38753)；UV(MeOH)λ max (log ε) 210 (3.93); the structural formula is as follows:
the Chinese and English designations of the compound 2 are shown in the table 1, the nuclear magnetic resonance data are shown in the tables 2 and 3, 1 H-NMR(500MHz,Pyridine-d 5 ) The spectrogram is shown in figure 11, 13 C-NMR(125Hz,Pyridine-d 5 ) The spectrum is shown in fig. 12, the dept (θ=135°) spectrum is shown in fig. 13, the hsqc spectrum is shown in fig. 14, the hmbc spectrum is shown in fig. 15, 1 H- 1 the H COSY spectrum is shown in FIG. 16, the ROESY spectrum is shown in FIG. 17, the UV spectrum is shown in FIG. 18, and the HR-ESI-MS spectrum is shown in FIG. 19.
Table 1: chinese and english naming of compound 1, compound 2
Table 2: compounds 1 to 2 13 C-NMR data (δin ppm)
b:Measured in Pyridine-d 5 .
Table 3: compounds 1 to 2 1 H-NMR data (δin ppm, J in Hz)
b:Measured in Pyridine-d 5 .
Example 3
Since glycoside compounds are decomposed in vivo, the binding mode of aglycone of the 2 novel compounds obtained in example 1 to inducible nitric oxide synthase, cyclooxygenase 2, glucocorticoid receptor and heat shock protein 90 was verified and explained using a molecular docking method:
the database used in the step 1, experiments was PDB database (http:// www.rcsb.org /), and the software was SYBYL-X1.10 (Tripos, USA), discovery Studio 4.1Client (DS, accelrys, USA), chemOffice.
Step 2, the aglycone structural formulae of 2 compounds were plotted using ChemDraw software and saved as mol2 format. And (3) introducing a mol2 format file into SYBYL-X1.10 software, adopting a minimum module to perform structure optimization, performing energy minimization calculation based on a Tripos force field on the compound, loading Gasteiger-Huckel charges, storing the optimized structure as a mol2 format, and establishing a ligand small molecule compound library to prepare for molecular docking.
Step 3, downloading nitric oxide synthase (PDB ID:3E 6T) from the PDB database (http:// www.rcsb.org) 1 Cyclooxygenase 2 (PDB ID:4 OTJ) 2 Heat shock protein 90 (PDB ID:7 KRJ) 3 And glucocorticoid receptor (PDB ID:6 NWK) 4 The target protein is modified, hydrogenated and loaded with AMBER FF99 charge and its active site is located in the SYBYL software using the locking module definition function in Application.
And 4, after the treatment, the ligand small molecule compound library of the step 2 is also used for butt joint with the target protein under a locking module, and small molecules in the small molecule compound library are combined with the target protein in sequence and stored as PDF files.
And 5, analyzing the molecular docking result by using a Receptor-Ligand Interactions module in Discovery Studio software, and manufacturing a three-dimensional effect graph.
iNOS is one of three isoforms of nitric oxide synthase, and is present in cardiomyocytes, vascular smooth muscle cells, fibroblasts, endothelial cells and inflammatory cells, and is a calcium ion-independent enzyme. Once iNOS is induced, it will induce a large amount of NO to mediate the occurrence of various diseases, and thus it will be of great importance to find potential inhibitors of iNOS. iNOS molecular docking experiments are referred to reference 1 below.
The aglycone of compound 1 is stabilized with iNOS by forming hydrogen bond interaction with Arg260, ala276, glu371, and hydrophobic interaction with residues Ala276, pro344, val346, arg 375.
The aglycone of compound 2 is stabilized with iNOS by forming a hydrogen bond interaction with Arg260, ala276 and a hydrophobic interaction with residues Pro344, arg 375.
Cyclooxygenase 2 (COX-2) is a key enzyme in prostaglandin biosynthesis and acts as both a dioxygenase and a peroxidase, mediating arachidonic acid to form prostaglandins, playing a special role in inflammatory reactions. Targeted delivery of chemotherapeutic agents to tumors has been explored as a means, and a related approach is to target intracellular proteins by binding ligands of anticancer drugs. An attractive target for this approach is COX-2, which is highly expressed in a range of malignancies. COX-2 molecular docking experiments reference is made to reference 2 below.
The aglycone of compound 1 is stabilized with COX-2 by forming a hydrogen bond interaction with Val349, and forming a hydrophobic interaction with residues Val89, val116, val349, leu352, tyr355, leu359, phe518, val523, ala 527.
The aglycone of compound 2 is stabilized with COX-2 by hydrophobic interactions with residues Pro86, val89, tyr115, val116, val349, tyr355, ala 527.
Heat shock proteins are classified into five types, HSP100, HSP90, HSP70, HSP60, and small molecule heat shock proteins small Heat Shock Proteins (sHSPs). HSP90 is one of the most abundant proteins in the human body and also one of the essential chaperones in cells, HSP90 is associated with numerous diseases in the human body, and its synthesis enhancement and release are beneficial for maintaining cell homeostasis. HSP90 molecular docking experiments refer to reference 3 below.
The aglycone of compound 1 is stabilized with HSP90 by forming a hydrogen bond interaction with Asn564, gln570, met604, arg611, and a hydrophobic interaction with residues Met560, met601, met604, phe623, met646, leu732, tyr735, cys 736.
The aglycone of compound 2 is stabilized with HSP90 by forming a hydrogen bond interaction with Met560, leu563, leu732, thr739, and a hydrophobic interaction with residues Leu563, leu566, met601, met604, ala605, leu608, phe623, cys 736.
Glucocorticoid receptor (Glucocorticoid receptor, GR) is a ligand-regulated transcription factor that plays a key role in inflammation, metabolism and immunity. GR consists of an n-terminal domain, a dna binding domain, a flexible hinge region, and a c-terminal Ligand Binding Domain (LBD). GR molecular docking experiments are referred to below in reference 4.
The aglycone of compound 1 is stabilized with GR by hydrogen bond interaction with gin 111 and hydrophobic interaction with residues Leu32, leu35, gin 39, met70, met73, leu77, phe92, cys 205.
The aglycone of compound 2 is stabilized with GR by hydrogen bond interaction with Thr208, and hydrophobic interaction with residues Leu32, asn33, leu35, met70, met73, leu77, phe92, cys 205.
These hydrogen bond interactions with hydrophobic bonds play an important role in the binding of these 2 compounds to the four above targets, demonstrating that these 2 compounds can stably bind to the four above proteins and can act as potential inhibitors of these four targets in the future, according to the binding conformation and docking scores in fig. 20-27.
Table 4: butt score for Compounds 1-2
Reference is made to:
1.Garcin,E.D.；Arvai,A.S.；Rosenfeld,R.J.；Kroeger,M.D.；Crane,B.R.；Andersson,G.；Andrews,G.；Hamley,P.J.；Mallinder,P.R.；Nicholls,D.J.；St-Gallay,S.A.；Tinker,A.C.；Gensmantel,N.P.；Mete,A.；Cheshire,D.R.；Connolly,S.；Stuehr,D.J.；Aberg,A.；Wallace,A.V.；Tainer,J.A.；Getzoff,E.D.,Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase.Nat Chem Biol 2008,4(11),700-707.
2.Uddin,M.J.；Crews,B.C.；Xu,S.；Ghebreselasie,K.；Daniel,C.K.；Kingsley,P.J.；Banerjee,S.；Marnett,L.J.,Antitumor Activity of Cytotoxic Cyclooxygenase-2Inhibitors.ACS Chem Biol 2016,11(11),3052-3060.
3.Noddings,C.M.；Wang,R.Y.；Johnson,J.L.；Agard,D.A.,Structure of Hsp90-p23-GR reveals the Hsp90 client-remodelling mechanism.Nature 2022,601(7893),465-469.
4.Liu,X.；Wang,Y.；Ortlund,E.A.,First High-Resolution Crystal Structures of the Glucocorticoid Receptor Ligand-Binding Domain-Peroxisome Proliferator-Activated gamma Coactivator 1-alpha Complex with Endogenous and Synthetic Glucocorticoids.Mol Pharmacol 2019,96(4),408-417.
Claims (10)
1. the furostanol type steroid saponin compounds have the structure shown in a compound 1 or a compound 2:
2. the furostanol type steroid saponin compound according to claim 1, wherein the structural formula of the compound 1-2 is as follows:
3. a process for the preparation of a compound as claimed in claim 1 or 2, comprising the steps of:
s1, weighing dried rhizome of the arrow, slicing, crushing, leaching with methanol, filtering the extracting solution, and concentrating the filtrate under reduced pressure to obtain a methanol crude extract;
s2, dissolving the methanol crude extract with water to obtain a methanol extract aqueous solution, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, discarding petroleum ether and ethyl acetate extraction parts, and concentrating the rest extraction liquid under reduced pressure to obtain an n-butanol extraction part and a water phase extraction part;
s3, carrying out D101 macroporous adsorption resin column chromatography on the water phase extraction part, and carrying out gradient elution to obtain 8 components Fr.A-Fr.H;
purifying the component Fr.D by high performance liquid chromatography, and performing isocratic elution to obtain a compound 1;
s4, carrying out D101 macroporous adsorption resin column chromatography on the n-butanol extraction part, and carrying out gradient elution to obtain 9 components Fr.A-Fr.I;
subjecting the Fr.F components to normal phase silica gel column chromatography, and performing gradient elution to obtain 6 components Fr.HI-Fr.VI;
subjecting the Fr.FV component to reverse phase ODS column chromatography, and gradient eluting to obtain 7 components Fr.FV 1-Fr.FV 7;
and (3) purifying the component Fr.FV 6 by high performance liquid chromatography, and carrying out gradient elution to obtain the compound 2.
4. The method according to claim 3, wherein in the step S1, the liquid-solid ratio of methanol to the rhizome of the open arrow is 3:1-6:1, and the ratio is mL/g;
the leaching method comprises the following steps: adding methanol into crushed rhizome of Ophioglossoides at a temperature of 65+ -3deg.C for 1-2 hr, and mixing the extractive solutions.
5. The method according to claim 3, wherein in step S2, the volumes of petroleum ether, ethyl acetate and n-butanol are each 1-2 times the volume of the aqueous solution of methanol extract.
6. The method according to claim 3, wherein in step S3, the conditions for the gradient elution of the aqueous phase extraction fraction are: ethanol-water is used as an eluent, gradient elution is carried out according to the volume ratio of ethanol to water of 0:1, 1:9, 2:8, 3:7, 1:1, 6:4, 7:3, 9:1 and 1:0, and similar components are combined by TLC detection, wherein the steps are as follows: methanol: preparing a mixed solvent with the volume ratio of water being 7:3:1, standing for layering after mixing, and taking the lower layer as a developing agent;
the conditions for isocratic elution of fr.d. component were: ODS column, in which the volume ratio of acetonitrile to water was 18:82, was eluted at a flow rate of 3 mL/min.
7. The method according to claim 3, wherein in step S4, the conditions for the gradient elution of the n-butanol extract fraction are: ethanol-water is used as an eluent, gradient elution is carried out according to the volume ratio of ethanol to water of 0:1, 1:9, 2:8, 3:7, 1:1, 6:4, 7:3, 9:1 and 1:0, and similar components are combined by TLC detection, wherein the steps are as follows: methanol: preparing a mixed solvent with the volume ratio of water being 7:3:1, standing for layering after mixing, and taking the lower layer as a developing agent;
the conditions for gradient elution of the component fr.f were: dichloromethane-methanol is used as an eluent, gradient elution is carried out according to the volume ratio of dichloromethane to methanol of 20:1, 15:1, 10:1, 9:1, 8:2, 7:3, 6:4, 1:1 and 0:1, and similar components are combined by TLC detection, wherein, according to chloroform: methanol: preparing a mixed solvent with the volume ratio of water being 7:3:1, standing for layering after mixing, and taking the lower layer as a developing agent;
the conditions for gradient elution of the component Fr.FV are as follows: methanol-water is used as eluent, gradient elution is carried out according to the volume ratio of methanol to water of 0:1, 1:9, 2:8, 3:7, 1:1, 6:4, 7:3, 9:1 and 1:0, and the similar components are combined by TLC detection, wherein, according to chloroform: methanol: preparing a mixed solvent with the volume ratio of water being 7:3:1, standing for layering after mixing, and taking the lower layer as a developing agent;
the conditions for gradient elution of the component Fr.FV6 are as follows: ODS chromatographic column, gradient elution is carried out at a flow rate of 3mL/min at a ratio of acetonitrile to water of 23:77-30:70 by volume.
8. Use of a furostanol-type steroid saponin compound as defined in claim 1 or 2 in the preparation of a medicament for inhibiting iNOS, COX-2, HSP90 and/or GR.
9. Use of the furostanol-type steroid saponin compound as defined in claim 1 or 2 in the preparation of anti-inflammatory drugs.
10. The use of the furostanol-type steroid saponin compound as defined in claim 1 or 2 in the preparation of antitumor drugs.
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