CN113402391B - Diterpenoid compound derived from Balanophora japonica, preparation method and application thereof - Google Patents
Diterpenoid compound derived from Balanophora japonica, preparation method and application thereof Download PDFInfo
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- -1 Diterpenoid compound Chemical class 0.000 title claims abstract description 38
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 241000358234 Balanophora japonica Species 0.000 title description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 96
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 93
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 35
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims abstract description 33
- 150000004141 diterpene derivatives Chemical class 0.000 claims abstract description 31
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 22
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000004440 column chromatography Methods 0.000 claims abstract description 19
- 239000003208 petroleum Substances 0.000 claims abstract description 11
- 239000000287 crude extract Substances 0.000 claims abstract description 9
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 8
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 8
- 239000011259 mixed solution Substances 0.000 claims abstract description 8
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229920005654 Sephadex Polymers 0.000 claims abstract description 4
- 239000012507 Sephadex™ Substances 0.000 claims abstract description 4
- 239000004576 sand Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 25
- 238000010828 elution Methods 0.000 claims description 20
- 239000007864 aqueous solution Substances 0.000 claims description 17
- 238000000746 purification Methods 0.000 claims description 13
- 238000000926 separation method Methods 0.000 claims description 13
- 238000010829 isocratic elution Methods 0.000 claims description 9
- 241001116415 Balanophora Species 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 7
- 241000124001 Alcyonacea Species 0.000 claims description 4
- 229940125782 compound 2 Drugs 0.000 claims description 4
- 239000003112 inhibitor Substances 0.000 claims description 4
- 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
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 3
- 229930004069 diterpene Natural products 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 241000588807 Bordetella Species 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 abstract description 36
- 241001072256 Boraginaceae Species 0.000 abstract description 5
- 239000000284 extract Substances 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 2
- 229930000044 secondary metabolite Natural products 0.000 abstract description 2
- 230000005764 inhibitory process Effects 0.000 abstract 1
- 239000012046 mixed solvent Substances 0.000 abstract 1
- 238000005481 NMR spectroscopy Methods 0.000 description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 235000014653 Carica parviflora Nutrition 0.000 description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- 239000012230 colorless oil Substances 0.000 description 6
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 6
- 238000001228 spectrum Methods 0.000 description 6
- 241000243321 Cnidaria Species 0.000 description 5
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 4
- 230000000844 anti-bacterial effect Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 4
- PKDBCJSWQUOKDO-UHFFFAOYSA-M 2,3,5-triphenyltetrazolium chloride Chemical compound [Cl-].C1=CC=CC=C1C(N=[N+]1C=2C=CC=CC=2)=NN1C1=CC=CC=C1 PKDBCJSWQUOKDO-UHFFFAOYSA-M 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000242757 Anthozoa Species 0.000 description 2
- 229940125904 compound 1 Drugs 0.000 description 2
- 229940126214 compound 3 Drugs 0.000 description 2
- NNBZCPXTIHJBJL-UHFFFAOYSA-N decalin Chemical compound C1CCCC2CCCCC21 NNBZCPXTIHJBJL-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 238000009631 Broth culture Methods 0.000 description 1
- 241000628997 Flos Species 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 241001337904 Gordonia <angiosperm> Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 229940125898 compound 5 Drugs 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000009189 diving Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000012259 ether extract Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 238000013206 minimal dilution Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 229930004725 sesquiterpene Natural products 0.000 description 1
- 150000004354 sesquiterpene derivatives Chemical class 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- FGQOOHJZONJGDT-UHFFFAOYSA-N vanillin Natural products COC1=CC(O)=CC(C=O)=C1 FGQOOHJZONJGDT-UHFFFAOYSA-N 0.000 description 1
- 235000012141 vanillin Nutrition 0.000 description 1
- PXXNTAGJWPJAGM-UHFFFAOYSA-N vertaline Natural products C1C2C=3C=C(OC)C(OC)=CC=3OC(C=C3)=CC=C3CCC(=O)OC1CC1N2CCCC1 PXXNTAGJWPJAGM-UHFFFAOYSA-N 0.000 description 1
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- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
- C07C69/73—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids
- C07C69/732—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids of unsaturated hydroxy carboxylic acids
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Abstract
The invention discloses diterpenoid compounds derived from Boraginaceae scholaria, a preparation method and application thereof, which are characterized in that the diterpenoid compounds are separated from secondary metabolites generated by the West sand Boraginaceae scholaria, and the preparation steps are as follows 1) the frozen Boraginaceae scholaria extract is concentrated and then extracted to obtain an extract; 2) subjecting the crude extract to Sephadex column chromatography, isocratic eluting with mixed solvent of petroleum ether, dichloromethane and methanol as mobile phase, and collecting 9 fractions; performing normal phase vacuum column chromatography on the No. 2 fraction, taking a mixed solution of petroleum ether and ethyl acetate as a mobile phase and a mixed solution of acetonitrile and water as a mobile phase, and collecting fractions; respectively carrying out reverse-phase reduced-pressure column chromatography on the No. 2, No. 5 and No. 6 fractions, and collecting the corresponding fractions; finally, the corresponding fractions are prepared and separated and purified by high performance liquid chromatography to obtain six diterpenoid new compounds, and the compounds 1-4 and 6 have the inhibition effect on staphylococcus aureus and bacillus subtilis.
Description
Technical Field
The invention relates to a diterpenoid compound, in particular to a diterpenoid compound derived from Boraginaceae scholaria, a preparation method and application thereof.
Background
The south China sea is in tropical and subtropical areas, has wide sea areas and abundant marine biological resources, and is one of the most widely distributed sea areas of corals in the world. Since the 80 s in the 20 th century, researchers studied the chemical compositions of various corals collected from the south sea, and many compounds with novel structures, such as steroids, terpenoids, alkaloids, etc., have been isolated. Wherein,Lemnaliabelongs to new terpenoid compounds with various structures of the soft coral, mainly comprising sesquiterpene and diterpenoid glycoside compounds with good biological activity.
In the process of exploring bioactive secondary metabolites of marine coral, the coral is collected in the sea area (10 m deep) of West sand Islands by the aid of the hydropneumoniae diving technologyLemnaliasp. (accession number XSSC 201907). 6 new diterpenoid compounds are obtained from the acetone extract, and further activity screening results show that the compounds 1-4 and 6 have inhibitory effects on staphylococcus aureus and bacillus subtilis. At present, the chemical structures and the antibacterial activity of the compounds are not reported, so that the related medicines are not seen in the market.
Disclosure of Invention
The invention aims to solve the technical problem of providing a diterpenoid compound with biological activity of resisting staphylococcus aureus and bacillus subtilis and derived from gordonia galaxoides, a preparation method and application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows: diterpene compound derived from Boraginaceae flos Farfarae, and its preparation methodLemnaliaExtracted and separated from the soft coral, and the chemical structural formula is at least one of the following formulas:
the preparation method of the diterpenoid compound derived from the Balanophora japonica comprises the following steps:
(1) extracting frozen Bordetella fuliginosa with mixed solution of dichloromethane and methanol with equal volume for 5 times, concentrating under reduced pressure, extracting with mixed solution of ether and water with equal volume for 4 times, mixing ether extractive solutions, and concentrating under reduced pressure to obtain crude extract;
(2) dissolving the crude extract with 20-30mL of dichloromethane, and performing sephadex column chromatography by using a solvent with a volume ratio of 2: 1:1, eluting by using petroleum ether/dichloromethane/methanol solution as an eluent, collecting fractions tube by tube, arranging the fractions from large to small according to molecular weight, and combining to obtain 4 components;
(3) performing medium-pressure normal phase column chromatography on the 2 nd component obtained in the step (2), performing gradient elution by using petroleum ether/ethyl acetate solution with a volume ratio of (50: 1) - (1: 1) as a mobile phase, and combining similar fractions in sequence to obtain 9 components in total;
(4) performing medium-pressure reversed-phase column chromatography on the 3 rd component obtained in the step (3), performing gradient elution by using acetonitrile aqueous solution with acetonitrile content of 50-100% as a mobile phase, and combining similar fractions in sequence to obtain 6 components in total; separating and purifying the 5 th component by high performance liquid chromatography, and isocratic eluting with 97% methanol aqueous solution as mobile phase to obtain diterpene compound 4 (bioloranate D);
(5) performing medium-pressure reversed-phase column chromatography on the 5 th component obtained in the step (3), eluting by using acetonitrile water solution with acetonitrile content of 40-100% as a mobile phase, and combining similar fractions in sequence to obtain 4 components;
(6) performing high performance liquid chromatography separation and purification on the 2 nd component obtained in the step (5), and performing isocratic elution by using a methanol-water solution with the methanol content of 87% as a mobile phase to obtain diterpenoid compounds 5 (euplexarene D) and diterpenoid compounds 6(cNeorubin K);
(7) separating and purifying the 3 rd component obtained in the step (5) by high performance liquid chromatography, and carrying out isocratic elution by using a methanol aqueous solution with the methanol content of 92% as a mobile phase to obtain diterpenoid compound 2 (bioloranate B), wherein;
(8) performing medium-pressure reversed-phase column chromatography on the 6 th component obtained in the step (3), performing gradient elution by using an acetonitrile aqueous solution with acetonitrile content of 40-100 as a mobile phase, and combining similar fractions in sequence to obtain 3 components in total; separating and purifying the fraction 2 by high performance liquid chromatography, and performing isocratic elution by using an aqueous methanol solution with the methanol content of 88% as a mobile phase to obtain diterpenoid 1 (bioloranate A) and diterpenoid 3 (bioloranate C), wherein the chemical structural formulas of diterpenoid 1-4 (bioloranate A-D), diterpenoid 5 (eulexarene D) and diterpenoid 6 (cNeorubicin K) are shown as follows:
preferably, the petroleum ether/ethyl acetate solution in the step (3) has an elution gradient volume ratio of 50: 1. 20: 1. 10: 1. 5: 1. 2: 1 and 1: 1.
preferably, the elution gradient of acetonitrile in the acetonitrile water solution in the step (4) has the volume percentages of 50%, 60%, 70%, 80%, 90% and 100% in sequence; the flow rate of the high performance liquid chromatography separation and purification is 3 mL/min, the isocratic time is 45 min, the column temperature is room temperature, and the collection wavelength is 200 nm.
Preferably, the elution gradient of acetonitrile in the aqueous acetonitrile solution in the step (5) has a volume percentage of 40%, 50%, 60%, 70%, 80%, 90% and 100% in sequence.
Preferably, the flow rate of the separation and purification by high performance liquid chromatography in the step (6) is 3 mL/min, the isocratic time is 50 min, the column temperature is room temperature, and the collection wavelength is 200 nm.
Preferably, the flow rate of the separation and purification by high performance liquid chromatography in the step (7) is 3 mL/min, the isocratic time is 45 min, the column temperature is room temperature, and the collection wavelength is 200 nm.
Preferably, the flow rate of the separation and purification by high performance liquid chromatography in the step (8) is 3 mL/min, the isocratic time is 48 min, the column temperature is room temperature, and the collection wavelength is 200 nm.
The diterpenoid compound 1, the diterpenoid compound 2, the diterpenoid compound 3, the diterpenoid compound 4 and the diterpenoid compound 6 are used for preparing a bacillus subtilis inhibitor and a staphylococcus aureus inhibitor.
Compared with the prior art, the invention has the advantages that: the invention relates to diterpenoid compounds derived from Balanophora glabra and a preparation method and application thereof, wherein the Balanophora glabra is soaked in mixed liquor consisting of dichloromethane and methanol with the same volume and extracted by diethyl ether to obtain crude extract, the crude extract is subjected to reduced pressure silica gel column chromatography, medium pressure column chromatography and high performance liquid chromatography for separation and purification to obtain 6 diterpenoid compounds, including four decalin type bicyclic diterpenoid bifloranate A-D (1-4), one fine dentate diterpenoid euplexarene D (5) and one vanillin diterpenoid K (6), the biological activity screening result shows that the compounds 1-4 and 6 have an inhibiting effect on staphylococcus aureus and bacillus subtilis, the MIC range is 4-64 mu g/mL, and the diterpenoid compounds can be used as lead compounds to lay a foundation for developing new drugs.
Drawings
FIG. 1 is a NMR spectrum of the compound biflorate A;
FIG. 2 is a NMR carbon spectrum of compound biflorate A;
FIG. 3 is a NMR spectrum of the compound biflorate B;
FIG. 4 is a NMR carbon spectrum of compound biflorate B;
FIG. 5 is a NMR spectrum of the compound biflorate C;
FIG. 6 is a NMR carbon spectrum of compound biflorate C;
FIG. 7 is a NMR spectrum of the compound biflorate D;
FIG. 8 is a NMR carbon spectrum of compound biflorate D;
FIG. 9 is a NMR spectrum of the compound euplexarene D;
FIG. 10 is a NMR carbon spectrum of the compound euplexarene D;
FIG. 11 is a nuclear magnetic resonance hydrogen spectrum of compound cNeorubin K;
FIG. 12 is a carbon nuclear magnetic resonance spectrum of compound cNeorubin K.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Example 1
From south to westLemnaliaDiterpenoid compounds extracted and separated from coral softpanel: bioloranate A-D (1-4), euplexine D (5) and cNeorubin K (6), having the following chemical structures:
example 2
From south to westLemnaliaThe preparation method of six diterpenoid compounds extracted and separated from coral softgel comprises the following steps:
1. mixing the frozen south sea west sandLemnaliaCutting coral (wet weight 1.9 Kg) into small pieces, freeze-drying (dry weight 480 g), extracting with mixed solution of dichloromethane and methanol at equal volume under ultrasound for five times, concentrating under reduced pressure to obtain extract 33.8 g, extracting with mixed solution of ether and water at equal volume for four times, mixing the ether extracts, and concentrating under reduced pressure to obtain crude extract 25.5 g; (ii) a
2. Dissolving the crude extract with 20-30mL of dichloromethane, and performing sephadex column chromatography by using a solvent with a volume ratio of 2: 1:1, eluting by using petroleum ether/dichloromethane/methanol solution as an eluent, collecting fractions tube by tube, arranging the fractions from large to small according to molecular weight, and combining to obtain 4 components;
3. performing medium-pressure normal phase column chromatography on the 2 nd component obtained in the step 2, performing gradient elution by using petroleum ether/ethyl acetate solution with a volume ratio of (50: 1) - (1: 1) as a mobile phase, and combining similar fractions in sequence to obtain 9 components in total; wherein the elution gradient volume ratio of the petroleum ether/ethyl acetate solution is 50: 1. 20: 1. 10: 1. 5: 1. 2: 1 and 1: 1;
4. performing medium-pressure reversed-phase column chromatography on the 3 rd component obtained in the step 3, performing gradient elution by using acetonitrile aqueous solution with acetonitrile content of 50-100% as a mobile phase, and combining similar fractions in sequence to obtain 6 components in total; separating and purifying the 5 th component by high performance liquid chromatography, and performing isocratic elution by using a methanol aqueous solution with the methanol content of 97% as a mobile phase, wherein the flow rate is 3 mL/min, the isocratic time is 45 min, the column temperature is room temperature, the collection wavelength is 200 nm, and the diterpenoid compound 4 (bioloranate D) is obtained, wherein the elution gradient volume percentages of acetonitrile in the acetonitrile aqueous solution are 50%, 60%, 70%, 80%, 90% and 100% in sequence;
5. performing medium-pressure reverse phase column chromatography on the 5 th component obtained in the step 3, eluting by using acetonitrile aqueous solution with acetonitrile content of 40-100% as a mobile phase, and combining similar fractions in sequence to obtain 4 components in total;
6. performing high performance liquid chromatography separation and purification on the 2 nd component obtained in the step 5, and performing isocratic elution by using a methanol-water solution with the methanol content of 87% as a mobile phase, wherein the flow rate is 3 mL/min, the isocratic time is 50 min, the column temperature is room temperature, the collection wavelength is 200 nm, and diterpenoid compounds 5 (euplexine D) and diterpenoid compounds 6(cNeorubin K) are obtained;
7. performing high performance liquid chromatography separation and purification on the 3 rd component obtained in the step 5, and performing isocratic elution by using a methanol aqueous solution with the methanol content of 92% as a mobile phase, wherein the flow rate is 3 mL/min, the isocratic time is 45 min, the column temperature is room temperature, the collection wavelength is 200 nm, and the diterpenoid compound 2 (bioloranate B) is obtained, wherein the elution gradient volume percentages of acetonitrile in the acetonitrile aqueous solution are 40%, 50%, 60%, 70%, 80%, 90% and 100% in sequence;
8. performing medium-pressure reversed-phase column chromatography on the 6 th component obtained in the step 3, performing gradient elution by using an acetonitrile aqueous solution with acetonitrile content of 40-100 as a mobile phase, and combining similar fractions in sequence to obtain 3 components in total; separating and purifying the component 2 by high performance liquid chromatography, and performing isocratic elution by using a methanol aqueous solution with the methanol content of 88% as a mobile phase, wherein the flow rate is 3 mL/min, the isocratic time is 48 min, the column temperature is room temperature, the collection wavelength is 200 nm, and diterpenoid compound 1 (bioloranate A) and diterpenoid compound 3 (bioloranate C) are obtained, wherein the acetonitrile elution gradient volume percentages in the acetonitrile/aqueous solution are 40%, 50%, 60%, 70%, 80%, 90% and 100% in sequence; the chemical structures of diterpenoids 1-4 (bioloranate A-D), diterpenoid 5 (euplexine D) and diterpenoid 6(cNeorubin K) are shown below:
example 3
Structural identification and nuclear magnetic signal assignment of compounds:
diterpenoid 1(Biofloranate a): colorless oil; [ alpha ] to]25 D﹢34（c 0.1，MeOH)；HRESIMS m/z 357.2400 [M + Na]+ (calcd for C21H34O3357.2508), of the compound1H and13the C NMR data are shown in FIGS. 1 and 2, Table 1 and Table 2.
Diterpenoid 2(Biofloranate B): colorless oil; [ alpha ] to]25 D﹢33（c 0.1，MeOH)；HRESIMS m/z 357.2395 [M + Na]+ (calcd for C21H34O3357.2508), of the compound1H and13the C NMR data are shown in FIGS. 3 and 4, Table 1 and Table 2.
Diterpenoid 3 (Biofloranate C): colorless oil; [ alpha ] to]25 D-105（c 0.1，MeOH)；HRESIMS m/z 355.2251 [M + Na]+ (calcd for C21H32O3355.2351), of the compound1H and13the C NMR data are shown in FIGS. 5 and 6, Table 1 and Table 2.
Diterpenoid 4 (Biofloranate D): colorless oil; [ alpha ] to]25 D﹢30（c 0.1，MeOH)；HRESIMS m/z 355.2251 [M + Na]+ (calcd for C20H34O, 316.2610) of this compound1H and13the C NMR data are shown in FIGS. 7 and 8, Table 1 and Table 2.
Diterpenoid compound 5 (euplexurene D): colorless oil; [ alpha ] to]25 D﹢21（c 0.1，MeOH)；HREIMS m/z 290.2604 [M]+ (calcd for C20H34O, 290.2610) of this compound1H and13c NMR data are shown in FIGS. 9 and 10,Table 1 and table 2.
Diterpenoid compound 6(Cneorubin K): colorless oil; [ alpha ] to]25 D-24（c 0.1，MeOH)；HREIMS m/z 270.2334 [M-H2O]+ (calcd for C20H32290.2348), of the compound1H and13the C NMR data are shown in FIGS. 11 and 12, Table 1 and Table 2. Of Compounds 1 to 61The H NMR data are shown in Table 1.
TABLE 1 preparation of Compounds 1 to 61H NMR(600MHz, CDCl3)
TABLE 2 preparation of Compounds 1 to 613C NMR(150MHz, CDCl3)
Example 4
The detection and application of the antibacterial activity of the diterpenoid compounds bifloranate A-D (1-4), euplexine D (5) and cNeorubin K (6).
1. Experimental sample
Preparing a solution of a sample to be detected: the test samples were monomeric compounds of the compounds 1 to 6 isolated and purified in the above example 1, and appropriate amounts of the samples were precisely weighed and prepared into 1.28 mg/mL solutions in DMSO, respectively. The indicator bacteria used in the experiment are bacillus subtilis, and gentamicin is a positive control.
2. Experimental methods
Compounds 1-6 were tested for in vitro antibacterial activity using minimal dilutions of medium in 96-well microplates. The designated glycerol stock culture strains are respectively absorbed by 10 mu L of the glycerol stock culture strains and put into a broth culture medium, and the glycerol stock culture strains are activated by shaking culture. 100 mu L of MH culture medium containing 2,3, 5-triphenyltetrazolium chloride (TTC) developer is added into each well of a sterile 96-well microplate, 10 mu L of the compound and 90 mu L of MH culture medium containing TTC are added into the first row of wells, the mixture is sequentially diluted, the final concentration of the corresponding test compound is 256, 125, 64, 32, 16, 8, 4, 2, 1 and 0.5 mu g/mL, 50mL of MH culture medium is added into 10 mu L of seed liquid, the mixture is fully mixed, 100 mu L of bacterial liquid is added into each well, the culture is carried out in an incubator at 37 ℃, and the color is observed after 18h and 36 h.
3. Results of the experiment
TABLE 3 antibacterial Activity screening results for Compounds 1-6
As can be seen from Table 3, compounds 1-4 and 6 have inhibitory effects against Staphylococcus aureus and Bacillus subtilis with MIC ranging from 4-64 μ g/mL.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.
Claims (9)
1. Diterpenoid compounds derived from Balsamina boreale and characterized by being extracted from the south sea of West sandLemnaliaExtracted and separated from the soft coral, and the chemical structural formula is at least one of the following formulas:
2. a preparation method of diterpenoid compounds derived from Balanophora grandiflora is characterized by comprising the following steps:
(1) extracting frozen Bordetella fuliginosa with mixed solution of dichloromethane and methanol with equal volume for 5 times, concentrating under reduced pressure, extracting with mixed solution of ether and water with equal volume for 4 times, mixing ether extractive solutions, and concentrating under reduced pressure to obtain crude extract;
(2) dissolving the crude extract with 20-30mL of dichloromethane, and performing sephadex column chromatography by using a solvent with a volume ratio of 2: 1:1, eluting by using petroleum ether/dichloromethane/methanol solution as an eluent, collecting fractions tube by tube, arranging the fractions from large to small according to molecular weight, and combining to obtain 4 components;
(3) performing medium-pressure normal phase column chromatography on the 2 nd component obtained in the step (2), performing gradient elution by using petroleum ether/ethyl acetate solution with a volume ratio of (50: 1) - (1: 1) as a mobile phase, and combining similar fractions in sequence to obtain 9 components in total;
(4) performing medium-pressure reversed-phase column chromatography on the 3 rd component obtained in the step (3), performing gradient elution by using acetonitrile aqueous solution with acetonitrile content of 50-100% as a mobile phase, and combining similar fractions in sequence to obtain 6 components in total; separating and purifying the 5 th component by high performance liquid chromatography, and isocratic eluting with 97% methanol aqueous solution as mobile phase to obtain diterpene compound 4;
(5) performing medium-pressure reversed-phase column chromatography on the 5 th component obtained in the step (3), eluting by using acetonitrile water solution with acetonitrile content of 40-100% as a mobile phase, and combining similar fractions in sequence to obtain 4 components;
(6) performing high performance liquid chromatography separation and purification on the 2 nd component obtained in the step (5), and performing isocratic elution by using a methanol-water solution with the methanol content of 87% as a mobile phase to obtain a diterpenoid compound 6;
(7) separating and purifying the 3 rd component obtained in the step (5) by high performance liquid chromatography, and carrying out isocratic elution by using a methanol aqueous solution with the methanol content of 92% as a mobile phase to obtain the diterpenoid compound 2;
(8) performing medium-pressure reversed-phase column chromatography on the 6 th component obtained in the step (3), performing gradient elution by using an acetonitrile aqueous solution with acetonitrile content of 40-100% as a mobile phase, and combining similar fractions in sequence to obtain 3 components in total; separating and purifying the 2 nd component by high performance liquid chromatography, and isocratic eluting with 88% methanol water solution as mobile phase to obtain diterpenoid 1 and diterpenoid 3, wherein the diterpenoid has the following chemical structural formula:
3. the method for preparing diterpenoid compounds derived from Balanophora grandiflora as claimed in claim 2, wherein the elution gradient volume ratio of the petroleum ether/ethyl acetate solution in step (3) is 50: 1. 20: 1. 10: 1. 5: 1. 2: 1 and 1: 1.
4. the method for preparing diterpenoid compounds derived from Balanophora glabra according to claim 2, wherein the elution gradient of acetonitrile in the aqueous acetonitrile solution in the step (4) is 50%, 60%, 70%, 80%, 90% and 100% by volume; the flow rate of the high performance liquid chromatography separation and purification is 3 mL/min, the isocratic time is 45 min, the column temperature is room temperature, and the collection wavelength is 200 nm.
5. The method for preparing diterpenoid compounds derived from Ballon scale flower soft coral according to claim 2, wherein the elution gradient of acetonitrile in the aqueous acetonitrile solution in the step (5) is 40%, 50%, 60%, 70%, 80%, 90% and 100% by volume.
6. The method for preparing diterpenoid compounds derived from Balanophora glabra according to claim 2, wherein the flow rate of the separation and purification by high performance liquid chromatography in the step (6) is 3 mL/min, the isocratic time is 50 min, the column temperature is room temperature, and the collection wavelength is 200 nm.
7. The method for preparing diterpenoid compounds derived from Balanophora glabra according to claim 2, wherein the flow rate of the separation and purification by high performance liquid chromatography in the step (7) is 3 mL/min, the isocratic time is 45 min, the column temperature is room temperature, and the collection wavelength is 200 nm.
8. The method for preparing diterpenoid compounds derived from Balanophora glabra according to claim 2, wherein the acetonitrile elution gradient volume percentage in the acetonitrile/water solution in the step (8) is 40%, 50%, 60%, 70%, 80%, 90% and 100% in sequence, the flow rate of the high performance liquid chromatography separation and purification is 3 mL/min, the isocratic time is 48 min, the column temperature is room temperature, and the collection wavelength is 200 nm.
9. Use of diterpenoid 1, diterpenoid 2, diterpenoid 3, diterpenoid 4 and diterpenoid 6 according to claim 1 for the preparation of a bacillus subtilis inhibitor and a staphylococcus aureus inhibitor.
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| CN101265248A (en) * | 2007-03-14 | 2008-09-17 | 中国科学院上海药物研究所 | Cembrene lactone type diterpenoid lobophilides A and B, and preparation method and use thereof |
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