CN115991692A - Preparation method and application of spirodienone lignan compound in Isatis tinctoria - Google Patents
Preparation method and application of spirodienone lignan compound in Isatis tinctoria Download PDFInfo
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- -1 lignan compound Chemical class 0.000 title claims abstract description 16
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 9
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- 208000015122 neurodegenerative disease Diseases 0.000 claims abstract description 8
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- HBENZIXOGRCSQN-VQWWACLZSA-N (1S,2S,6R,14R,15R,16R)-5-(cyclopropylmethyl)-16-[(2S)-2-hydroxy-3,3-dimethylpentan-2-yl]-15-methoxy-13-oxa-5-azahexacyclo[13.2.2.12,8.01,6.02,14.012,20]icosa-8(20),9,11-trien-11-ol Chemical compound N1([C@@H]2CC=3C4=C(C(=CC=3)O)O[C@H]3[C@@]5(OC)CC[C@@]2([C@@]43CC1)C[C@@H]5[C@](C)(O)C(C)(C)CC)CC1CC1 HBENZIXOGRCSQN-VQWWACLZSA-N 0.000 claims description 9
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- 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
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Abstract
The invention relates to a preparation method of spirodienone lignan compounds in Isatis tinctoria and application thereof, belongs to the technical field of medicines, and in particular relates to 3 spirodienone lignan compounds extracted and separated from Isatis tinctoria (Isatis indigotica Fortune) which is a plant of Isatis tinctoria in Brassicaceae, which have the same spirodienone lignan parent nucleus, a preparation method of the novel compounds and novel application thereof in preparation of medicines for treating neurodegenerative diseases. The preparation method is simple and easy to implement, and has better reproducibility and higher purity. The obtained compound has good neuroprotective activity.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a method for preparing spirodienone lignan compounds from plant Isatis tinctoria and application of the compounds in the aspect of neuroprotection.
Background
Isatis tinctoria: isatis tinctoria [ Isatis indigotica Fortune ], isatis tinctoria of Brassicaceae, is a biennial herb, and is distributed and cultivated in both the north and south of China. Isatis tinctoria leaf is a well-known traditional Chinese medicine for clearing heat and detoxicating, is widely used for treating pestilence and influenza, and is originally recorded in Miniatrogen; the dry leaves and dry roots of Isatis tinctoria are called "dyers woad leaf" and "Isatis root" in the Pharmacopeia of the people's republic of China in 2020 edition, respectively, and the dried She Zuoda green leaves and the dried roots are used as the Isatis root for medicine. Cold nature and bitter taste.
With the progress of global population aging, neurodegenerative diseases are receiving increasing attention, wherein Alzheimer's Disease (AD) and Parkinson's disease and the like are becoming main killers causing death of middle aged and elderly people. Recent studies have shown that active compounds that inhibit active oxygen-induced cell damage may be useful in the prevention and treatment of AD. Therefore, the exploration of natural components with potential neuroprotection has great significance and has good research prospect for treating neurodegenerative diseases.
Disclosure of Invention
The primary purpose of the invention is to provide 3 spirodienone lignan compounds separated from Isatis tinctoria [ Isatis indigotica Fortune ] of Isatis tinctoria of Cruciferae, and the structures are shown in the figure:
the preparation technical scheme of the invention comprises the following steps:
extracting dried Isatis tinctoria whole herb with 70-80% industrial ethanol, mixing extractive solutions, concentrating to obtain extract, extracting with dichloromethane, subjecting the obtained component to silica gel column chromatography, performing isocratic gradient elution with dichloromethane-methanol system 100:1-3:1, and collecting total 4 fractions 1-4.
Subjecting the fraction 3 to polyamide column chromatography, and gradient eluting with ethanol-water system 0:100-90:10 to obtain three components A-C. And (3) performing gradient elution on the component A by using an HP20 column chromatography in an ethanol-water system of 0:100-90:10 to obtain three subfractions A1-A3.
The obtained subfraction A2 is subjected to gradient elution by an ODS column in an ethanol-water system of 10:90-90:0, and two subfractions A2.1-A2.2 are obtained.
The use of a methanol-water mobile phase to separate a2.1 on preparative and semi-preparative reverse phase high performance liquid chromatography gives compound 1a/1b-2. Wherein compound 1 is resolved using a chiral chromatography column to give a pair of enantiomers 1a/1b.
The preparation method comprises the steps of heating reflux extraction for 2-3 times, each time for 3-4 hours.
The preparation method comprises using Isatis tinctoria of Isatis genus of Brassicaceae as Isatis tinctoria [ Isatis indigotica Fortune ].
The preparation method uses a mobile phase of methanol-water (20:80, v/v) to separate A2.1 on preparative and semi-preparative reverse phase high performance liquid chromatography to obtain the compound 1-2.
The results of the system structure identification of the obtained compound are as follows:
structural identification of compounds 1-2 by high resolution mass spectrometry, one-dimensional NMR, two-dimensional NMR and computational ECD was performed as shown in FIGS. 1-17.
sibiricumin 1a/1b yellow oil (methanol).
HRESI-MS gives an excimer ion peak of 607.2158[ M+Na ]] + (calcd for C 31 H 36 O 11 Na 607.2150), combined with 1 H, 13 C NMR data confirm that the compound has formula C 31 H 36 O 11 The calculated unsaturation of the compound was 14.
1 H-NMR(600MHz,DMSO-d 6 ) The spectra show proton signal delta for two groups of ABX coupling systems H 7.07 (1 h, d, j=1.4 hz, h-2), 6.76 (1 h, overlap, h-5), 6.88 (1 h, overlap, h-6) and δ H 6.87 (1H, overlap, H-2 "), 6.77 (1H, overlap, H-5"), 6.72 (1H, d, J=8.2 Hz, H-6 "), and also shows the signal delta of a trans double bond H 6.39(1H,d,J＝15.9Hz,H-7”),6.19(1H,dt,J＝15.9,5.3Hz,H-8”)；δ H 6.14 (1H, s, H-2 '), 6.11 (1H, s, H-6') shows two double bond hydrogen signals, δ H 5.00 (1 h, d, j=9.5 hz, h-7), 2.72 (1 h, m, h-8), 4.67 (1 h, overlap, h-7 '), 4.34 (1 h, m, h-8') was suggested as four methine proton signals, δ H 3.22(1H,dd,J＝10.5,5.2Hz,H-9),3.16(1H,dd,J＝10.5,5.0Hz,H-9),δ H 3.72 (1H, m, H-9 ') and 3.57 (1H, m, H-9 '), 4.07 (2H, br s, H-9 ') indicate three aliphatic methylene signals; delta H 8.91 (1H, brs, OH-4), 4.37 (1H, t, J=4.4 Hz, OH-9), 4.67 (1H, overlap, OH-9 '), 4.78 (1H, t, J=5.1 Hz, OH-9') are suggested as hydroxyl signals, δ H 3.80(3H,s,OCH 3 -3),3.54(3H,s,OCH 3 -3′),3.55(3H,s,OCH 3 -5'),3.64(3H,s,OCH 3 -3 ") cue as methoxy signal. 13 C-NMR(100MHz,DMSO-d 6 ) The spectra give a total of 31 carbon signals, with the low field region comprising 12 benzene ring carbon signals, 6 double bond carbon signals and 1 carbonyl carbon signal; the high field region includes 4 methines, 3 methylenes, 4 methoxy groups, and 1 quaternary carbon signal. The hydrocarbon direct correlation attribution is carried out on the compound according to the HSQC spectrum, and the compound is further analyzed according to the HMBC spectrum.
In HMBC spectra, δ H 7.07 (1 h, d, j=1.4 hz, h-2), 6.88 (1 h, overlap, h-6) and a carbon signal δ C 81.9 (C-7) remote correlation, delta H 5.00 (1H, d, J=9.5 Hz, H-7) and delta C 58.1 (C-9), 83.8 (C-7'), and delta H 4.37 (1H, t, J=4.4 Hz, OH-9) and delta C 59.8 (C-8) related, showing that the compound has a similar structure to tetrahydrofuran type lignans, and contains a structural fragment of a benzotetrahydrofuran ring; delta H 6.14 (1H, s, H-2') and delta C 151.0 (C-3 '), 175.4 (C-4 '), 119.0 (C-6 '), delta H 6.11 (1H, s, H-6') and delta C 175.4 (C-4 '), 150.4 (C-5'), and delta H 2.72 (1H, m, H-8), 4.67 (1H, overlap, H-7') are all equal to delta C 113.1 (C-2 '), and 119.0 (C-6 '), which indicates that the compound has a cyclohexadienone fragment attached to the C-1' position of the tetrahydrofuran ring; delta H 6.87 (1H, overlap, H-2 "), 6.72 (1H, d, J=8.2 Hz, H-6"), and delta 128.8 (C-7 "), delta H 6.39(1h, d, j=15.9 hz, h-7 ") and δ C 61.7 (C-9') correlation, delta H 6.19 (1 h, dt, j=15.9, 5.3hz, h-8 ") and δ C 129.4 (C-1 '), 61.7 (C-9') indicating the presence of a fragment of cinnamyl alcohol in the structure; from delta H 4.67 (1H, overlap, H-7') and delta C 60.8 (C-9') correlation, delta H 4.34 (1H, m, H-8') and delta C 51.9 (C-1 ') correlating to determine the C-7' -C-9 ' structural unit; delta H 4.34 (1H, m, H-8') and delta C 146.4 (C-4 ') suggests that the cinnamyl alcohol fragment is linked to C-8' through an oxygen atom, thus defining the planar structure of compound 1a/1b.
The relative configuration of the compounds was determined by NOESY spectra in combination with coupling constants, the presence of signals associated with H-7 and H-9,H-2', the presence of signals associated with H-8 and H-6', H-7', the presence of signals associated with H-8' and H-2', and the coupling constants between H-7' and H-8' (J H-7',H-8' =8.7 Hz) is suggested as the trans configuration, indicating that H-7,H-2', H-8' is on the same side of the plane and H-8,H-6', H-7' is on the other side of the plane. Thus, the relative configuration of the compound 1a/1b was determined.
Further separation of compound 1 using chiral chromatography column results in a pair of enantiomers
And->
The ratio is about 1:2. From the positive Cotton effect at 292nm presented by 1a, the negative Cotton effect at 333nm presented by 1b, and the negative Cotton effect at 292nm presented by positive Cotton effect at 333nm, the configurations of 1a and 1b were determined to be 7S,8R,7'S,8' R and 7R,8S,7'R,8' S configurations, respectively.
In summary, the structures of compounds 1a and 1b were determined and all hydrocarbon signals were assigned, and through systematic literature search, both are novel compounds not reported in the literature, named (+) isatispironol a and (-) isatispironol a, respectively.
TABLE 1 Compounds 1a/1b 1 H-NMR (600 MHz) and 13 C-NMR (100 MHz) data (DMSO-d 6 )
Isatisfroneol B yellow oil (methanol).
HRESIMS gives an excimer ion peak of 607.2167[ M+Na ]] + (calcd.for C 31 H 36 O 11 Na, 607.2150), combined with 1 H, 13 C-NMR data confirm that compound formula is C 31 H 36 O 11 The calculated unsaturation of the compound was 14. 1 H-NMR(600MHz,DMSO-d 6 ) In the spectrum, delta H 7.05 (1 h, d, j=1.4 hz, h-2), 6.76 (1 h, d, j=8.1 hz, h-5), 6.91 (1 h, dd, j=1.4, 8.1hz, h-6) and δ H 6.99 (1 h, d, j=1.3 hz, h-2 "), 6.84 (1 h, d, j=8.4 hz, h-5"), 6.82 (1 h, dd, j=1.3, 8.4hz, h-6 ") suggests the presence of benzene rings of two ABX coupling systems in the structure, δ H 6.42 (1 h, d, j=15.9 hz, h-7 "), 6.22 (1 h, overlapped, h-8") suggests a hydrogen signal of the trans double bond. Delta H 6.24 (1H, s, H-2 '), 6.18 (1H, s, H-6') suggests two double bond hydrogen signals. Delta H 3.24 (2 h, m, h-9), 3.40 (2 h, m, h-9 '), 4.07 (2 h, d, j=5.2 hz, h-9') suggests the presence of three methylene groups. Delta H 5.01 (1 h, d, j=9.4 hz, h-7), 2.77 (1 h, m, h-8), 4.70 (1 h, d, j=4.9 hz, h-7 '), 4.10 (1 h, overlapped, h-8') indicate the presence of four methines. Delta H 3.79(3H,s,3-OCH 3 ),3.48(3H,s,3′-OCH 3 ),3.59(3H,s,5′-OCH 3 ),3.74(3H,s,3”-OCH 3 ) The cue is methoxy hydrogen signal. Delta H 8.94 (1H, br s, 4-OH) suggests a hydroxyl signal. 13 C-NMR(100MHz,DMSO-d 6 ) The spectrum gives a total of 31 carbon signals, including 12 benzene ringsCarbon signal, 6 double bond carbon signal, 1 carbonyl carbon signal, 4 methine groups, 3 methylene groups, 4 methoxy groups, and 1 quaternary carbon signal.
In the HMBC spectra, H-2 is associated with C-7, H-7 is associated with C-6, C-9, and H-8 is associated with C-1, suggesting that the compound is an analogue of tetrahydrofuran type lignan, and a benzo tetrahydrofuran ring fragment exists. H-2 'is associated with C-3', C-4', C-6', H-6 'is associated with C-4', C-5', H-8 is associated with C-2', C-6', H-7' is associated with C-2', H-6' is associated with C-7', and a cyclohexadienone fragment is also present in the structure which fragment is linked to the tetrahydrofuran ring via C-1'. H-2 "is associated with C-7", H-7 "is associated with C-6", C-9", and H-8" is associated with C-1", suggesting the presence of cinnamyl alcohol fragments in the structure. H-8 'is associated with C-4", suggesting that the cinnamyl alcohol fragment is linked to C-8' through an oxygen atom. Thus, the planar structure of the compound was determined.
The partial relative configuration of compound 2 was determined by NOESY spectra in combination with coupling constants. There is a correlation between H-7 and H-9,H-2', and a correlation between H-6' and H-7', indicating that H-7,H-2', H-9 are on the same side of the plane and H-6', H-7' are on the other side of the plane. Because the side chain of the chiral center C-8' has stronger flexibility, more conformations and larger calculation workload, other methods are adopted to further confirm the relative and absolute configuration of the compound. Because of the chiral carbon present in the structure, we attempted to resolve it using a variety of chiral columns and different conditions, but did not give the corresponding isomer, presumably as an optically pure compound. Thus, the partial relative configuration of the compound is determined.
In summary, the planar structure of the compound 2 was determined and the entire hydrocarbon signal was attributed, and after systematic literature search, the compound was found to be a novel compound which was not reported in the literature and was named isatispironol B.
TABLE 2 Compound 2 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) data (DMSO-d 6 )
para-H of the 3 novel compounds of the present invention by MTT method 2 O 2 The neuroprotection effect of the induced SH-SY5Y cell oxidative damage is examined, and the neuroprotection effect of the compound 1a/1b-2 is equivalent to that of a Trolox positive control, so that the spirodiene lignin has a powerful neuroprotection effect on SH-SY5Y cells, and has research value and application prospect for treating neurodegenerative diseases.
A pharmaceutical composition is prepared by mixing a spirodienone lignan compound or pharmaceutically acceptable salt thereof in Isatis tinctoria as an active ingredient with a pharmaceutically acceptable excipient, and preparing a clinically acceptable dosage form, wherein the excipient refers to a diluent, an auxiliary agent or a carrier which can be used in the pharmaceutical field, and the dosage form is an injection, a tablet or a capsule.
The spirodienone lignan compound in the Isatis tinctoria or the pharmaceutically acceptable salt thereof or the pharmaceutical composition is applied to the preparation of medicines for treating neurodegenerative diseases.
The invention has the advantages that:
1. provides a novel medical application of spirodiene lignin compound in Isatis tinctoria in preparing medicines for treating neurodegenerative diseases, and has wide application prospect.
2. Isatis tinctoria is cultivated in all regions of the country, and has rich sources, low cost and easy obtainment.
3. The compounds are novel compounds, have novel structures, and have strong neuroprotective activity and further development values.
Drawings
FIG. 1 Compound 1 1 H-NMR spectrum (DMSO-d) 6 ,400MHz)；
FIG. 2 Compound 1 13 C-NMR spectrum (DMSO-d) 6 ,100MHz)；
FIG. 3 HSQC spectrum of Compound 1 (600 MHz, DMSO-d 6 )；
FIG. 4 HMBC spectra of Compound 1 (600 MHz, DMSO-d 6 )；
FIG. 5 NOESY spectra of Compound 1 (600 MHz, DMSO-d 6 )；
FIG. 6 HR-ESIMS spectrum of Compound 1;
FIG. 7 UV spectrum of Compound 1;
FIG. 8 ECD spectra of Compound 1 a;
FIG. 9 ECD spectra of Compound 1 b;
FIG. 10 Compound 2 1 H-NMR spectrum (DMSO-d) 6 ,400MHz)；
FIG. 11 Compound 2 13 C-NMR spectrum (DMSO-d) 6 ,100MHz)；
FIG. 12 HSQC spectrum of Compound 2 (600 MHz, DMSO-d 6 )；
FIG. 13 HMBC spectra of Compound 2 (600 MHz, DMSO-d 6 )；
FIG. 14 NOESY spectrum of Compound 2 (600 MHz, DMSO-d 6 )；
FIG. 15 HR-ESIMS spectrum of Compound 2;
FIG. 16 UV spectrum of Compound 2;
figure 17 ECD spectrum of compound 2.
Detailed Description
The examples set forth below are presented to aid one skilled in the art in a better understanding of the present invention and are not intended to limit the invention in any way.
Example 1
Preparation of spirodienone lignan Compound 1a/1b-2 in Isatis tinctoria.
Reflux-extracting dried Isatis tinctoria whole herb (50 kg) with 80% industrial ethanol for 2 times each for 4 hr, mixing the extractive solutions, concentrating to obtain extract, extracting the extract with dichloromethane, subjecting the obtained component to silica gel column chromatography, and subjecting the obtained component to isocratic gradient elution with dichloromethane-methanol system 100:1-3:1, and collecting 4 fractions 1-4 (Fr.1:270.1 g, fr.2:290.6g, fr.3:289.3g, fr.4:281.8 g).
The fraction 3 was subjected to polyamide column chromatography and eluted with a gradient of ethanol-water system 0:100-90:10 to give three fractions A-C (Fr.A: 88.9g, fr.B:81.4g, fr.C:90.1 g). Component A was eluted with a gradient of ethanol-water system 0:100-90:10 using HP20 column chromatography to give three subfractions A1-A3 (Fr.A 1:20.6g, fr.A2:22.8g, fr.A3:21.7 g).
The resulting subfraction A2 was subjected to gradient elution with an ODS column in an ethanol-water system of 10:90-90:0 to give two subfractions A2.1-A2.2 (Fr. A2.1:17.5g, fr. A2.2:18.7 g).
The mobile phase of methanol-water (20:80, v/v) was used to isolate A2.1 on preparative and semi-preparative reverse phase high performance liquid chromatography to give compound 1a/1b-2 (1 a:2.4mg,1b:4.9mg,2:5.7 mg). Wherein compound 1 is resolved using a chiral chromatography column to give a pair of enantiomers.
Example 2
Neuroprotective Activity investigation of the Compound 1a/1b-2.
The human neuroblastoma SH-SY5Y cell line was taken from the ATCC center and cultured in DMEM medium (Hyclone) containing 5% CO at 37 DEG C 2 10% fetal bovine serum (FBS, gibco) was supplemented with humid air, and all experiments used log-grown cells. SH-SY5Y cells were pretreated with test compounds at various concentrations (12.5, 25, 50. Mu.M) for 1H and then H was further treated 2 O 2 (200. Mu.M) for 4h. Then, 20. Mu.L of MTT (5 mg/mL) was added to each well and the mixture was treated for 4 hours, so that the crystals were dissolved in DMSO, and the optical density at 490nm was measured using a flat-panel microscope. With different concentrations (12.5, 25 and 50. Mu.M) of H 2 O 2 All compounds showed moderate neuroprotection in vitro compared to the treatment group.
TABLE 3 neuroprotective Activity of Compounds 1a/1b-2
1a/1b-2 pair H 2 O 2 Neuroprotection induced (200. Mu.M) of SH-SY5Y cell damage from human neuroblastoma. All data are expressed as mean ± SD of three independent experiments. Trolox as positive control with H 2 O 2 In comparison, p<0.05、**p<0.01 and p<0.001。
Claims (8)
1. A spirodienone lignan compound in Isatis tinctoria, characterized in that the spirodienone lignan compound has any one of the following structures:
2. a method for preparing a spirodienone lignan compound in Isatis tinctoria according to claim 1, comprising the steps of:
extracting dried Isatis tinctoria whole herb with 70-80% industrial ethanol, mixing extractive solutions, concentrating to obtain extract, extracting the extract with dichloromethane, subjecting the obtained components to silica gel column chromatography, performing isocratic gradient elution with dichloromethane-methanol system 100:1-3:1, and collecting 4 fractions 1-4;
subjecting the component 3 to gradient elution by a polyamide column chromatography with an ethanol-water system of 0:100-90:10 to obtain three components A-C, and subjecting the component A to gradient elution by an HP20 column chromatography with an ethanol-water system of 0:100-90:10 to obtain three sub-components A1-A3;
the obtained subfraction A2 is subjected to gradient elution by an ODS column according to an ethanol-water system of 10:90-90:0 to obtain two subfractions A2.1-A2.2;
separating A2.1 on preparative and semi-preparative reverse phase high performance liquid chromatography using a methanol-water mobile phase to give compound 1a/1b-2, wherein compound 1a/1b is resolved using a chiral chromatographic column.
3. The method for preparing spirodienone lignan compound in Isatis tinctoria according to claim 2, wherein the extraction is a heat reflux extraction for 2-3 times each for 3-4 hours.
4. The method for producing a spirodienone lignan compound in Isatis tinctoria according to claim 2, wherein Isatis tinctoria is used as a plant Isatis tinctoria [ Isatis indigotica Fortune ] of Isatis tinctoria of Brassicaceae.
5. The method for preparing spirodienone lignan compound in Isatis tinctoria according to claim 2, wherein the compound 1-2 is obtained by separating a2.1 on preparative and semi-preparative reversed phase high performance liquid chromatography using a mobile phase of methanol-water (20:80).
6. A pharmaceutical composition, characterized in that a spirodienone lignan compound in Isatis tinctoria of claim 1 or a pharmaceutically acceptable salt thereof is used as an active ingredient, and is mixed with a pharmaceutically acceptable excipient to prepare a composition, and is prepared into a clinically acceptable dosage form, wherein the excipient refers to a diluent, an auxiliary agent or a carrier which can be used in the pharmaceutical field, and the dosage form is an injection, a tablet or a capsule.
7. Use of a spirodienone lignan compound in Isatis tinctoria according to claim 1 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of neurodegenerative diseases.
8. The use of the pharmaceutical composition of claim 6 for the preparation of a medicament for the treatment of neurodegenerative diseases.
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| CN116768912A (en) * | 2023-07-03 | 2023-09-19 | 沈阳药科大学 | Spiroindolone alkaloid compound in Isatis tinctoria as well as preparation method and application thereof |
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| CN112999254A (en) * | 2019-12-18 | 2021-06-22 | 沈阳药科大学 | Isatis tinctoria leaf total alkaloid and extraction method and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116768912A (en) * | 2023-07-03 | 2023-09-19 | 沈阳药科大学 | Spiroindolone alkaloid compound in Isatis tinctoria as well as preparation method and application thereof |
| CN116768912B (en) * | 2023-07-03 | 2024-04-26 | 沈阳药科大学 | Spiroindolone alkaloid compound in Isatis tinctoria as well as preparation method and application thereof |
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