CN115677520B - Diterpene compound and preparation method and application thereof - Google Patents
Diterpene compound and preparation method and application thereof Download PDFInfo
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- -1 Diterpene compound Chemical class 0.000 title claims abstract description 21
- 229930004069 diterpene Natural products 0.000 title claims description 8
- 238000002360 preparation method Methods 0.000 title abstract description 13
- 150000001875 compounds Chemical class 0.000 claims abstract description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 9
- 238000004440 column chromatography Methods 0.000 claims abstract description 8
- 238000011894 semi-preparative HPLC Methods 0.000 claims abstract description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 23
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- 239000000284 extract Substances 0.000 claims description 16
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- 239000000463 material Substances 0.000 claims description 7
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- Steroid Compounds (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention relates to the technical field of medical biology, in particular to a diterpenoid compound in Dolichos glabra, a preparation method and application thereof, wherein alcohol extraction, silica gel column chromatography, MCI column chromatography and semi-preparative HPLC are adopted for separation and purification, so that a novel diterpenoid compound strophiofbrins is successfully obtained, the operation method is simple and quick, the survival rate of model damaged nerve cells PC12 can be obviously improved, the PC12 nerve cell apoptosis can be well inhibited, and the compound has obvious neuroprotective activity and can be applied to preparation of novel neuroprotective medicines.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to a diterpenoid compound in Dolichos glabra, a preparation method thereof and application thereof in neuroprotective drug development.
Background
The family Euphorbiaceae, genus Euphorbia (Strophioblachia fimbricalyx Boerl.) is a perennial shrub plant. Mainly distributed in Hainan, yunnan, guangxi and Thailand. The Thailand folk medicine is used for treating cancers, fever, migraine and the like. However, few reports about chemical components of the eriocalyx mori are made at home and abroad. At present, more than thirty compounds such as phenanthrene deformation, terpenes, flavonoid glycoside and the like are mainly reported to be separated from the phenanthrene deformation, terpene deformation, flavonoid glycoside and the like, and the material basis is not clear. Therefore, the chemical components of the eriocalyx mori are needed to be studied in depth, active components of the eriocalyx mori are separated and prepared, and novel medicines are developed.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a novel strophanthin diterpenoid compound extracted and separated from the glabrous stump, a preparation method and application thereof, and researches show that the novel skeleton structure compound has a good inhibition effect on PC12 cell apoptosis. Meanwhile, the invention provides a simple, convenient, rapid and high-purity extraction and separation method aiming at the novel compound.
The invention aims at realizing the following technical scheme, namely a diterpene compound, the general formula of which is shown as the formula (I):
wherein R is 1 -H or = O; r is R 2 is-OH, -OCH 3 or-NH 2 ，R 3 is-OH or-OCH 3 。
Novel 5/6/7 tricyclic compounds of the invention and derivatives thereof having neuroprotective activity are also included in the present invention are acid addition salts, hydrates or prodrugs of the compounds of formula (I).
Preferably, the diterpene compound wherein R 1 Is =o, R 2 is-NH 2 ，R 3 is-OCH 3 The structural formula is shown as formula 1:
，
the chemical name of formula 1 is strophiofimbrin A.
Preferably, the diterpene compound wherein R 1 is-H, R 2 is-OCH 3 ，R 3 is-OCH 3 The structural formula is shown as formula 2:
the chemical name of formula 2 is strophiofimbrin B.
The preparation method of the strophiofimbrin A diterpenoid compound comprises the following steps:
step a, shearing the glaucocalyxa medicinal material, reflux-extracting with ethanol, recovering the extract, concentrating under reduced pressure, and drying to obtain total extract;
dispersing the total extract with distilled water, and sequentially extracting with petroleum ether and ethyl acetate with equal volume to obtain petroleum ether part and ethyl acetate part;
step c, subjecting the ethyl acetate extract to silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate with volume ratio of 30:1, 15:1, 8:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:5, 0:1, and mixing to obtain 11 fractions;
subjecting the fraction 11 to silica gel column chromatography, gradient eluting with dichloromethane-methanol with volume ratio of 1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 5:1, 1:1, 0:1, and mixing to obtain 9 fractions;
subjecting the fraction 1 to MCI column chromatography, eluting with methanol-water gradient with volume ratio of 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 1:0, and mixing to obtain 10 fractions;
the fraction 9 was purified by semi-preparative high performance liquid chromatography eluting with acetonitrile-water at a volume ratio of 41:59 and collecting the main peak to give compound strophiofimbrin A.
Preferably, the ethanol in the step a is reflux-extracted for 4 times at 85 ℃ by adopting an ethanol water solution with the volume fraction of 95%.
The preparation method of the strophiofimbrin B diterpenoid compound comprises the following steps:
step S1: shearing the glaucocalyxa medicinal material, reflux-extracting with ethanol, recovering the extract, concentrating under reduced pressure, and drying to obtain total extract;
step S2: dispersing the total extract with distilled water, sequentially extracting with petroleum ether and ethyl acetate with equal volume to obtain petroleum ether part and ethyl acetate part;
step S3: subjecting the ethyl acetate part extract to silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate with volume ratio of 30:1, 15:1, 8:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:5, 0:1, and mixing to obtain 11 fractions;
subjecting the fraction 11 to silica gel column chromatography, gradient eluting with dichloromethane-methanol with volume ratio of 1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 5:1, 1:1, 0:1, and mixing to obtain 9 fractions;
subjecting the fraction 1 to MCI column chromatography, eluting with methanol-water gradient with volume ratio of 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 1:0, and mixing to obtain 10 fractions;
the fraction 8 was purified by semi-preparative high performance liquid chromatography eluting with methanol-water at a volume ratio of 3:2 and collecting the main peak to give compound strophiofimbrin B.
Preferably, in the step S3, the ethanol is reflux-extracted for 4 times at 85 ℃ by using an ethanol aqueous solution with a volume fraction of 95%.
The diterpenoid compound or tautomer, regional isomer, stereoisomer, enantiomer, diastereoisomer and the structure or pharmaceutically acceptable salt containing the same or the framework of the diterpenoid compound are applied to the preparation of neuroprotective medicaments.
The term "pharmaceutically acceptable salts" refers to those salts which, within the scope of sound medical judgment, are suitable for contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in detail in Berge et al, J.pharmaceutical Sciences (1977) 66:1-19.
The compounds of the invention may include one or more asymmetric centers and thus may exist in a variety of stereoisomeric forms, for example, enantiomeric and/or diastereomeric forms. For example, the compounds of the invention may be individual enantiomers, diastereomers, or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. The isomers may be separated from the mixtures by methods known to those skilled in the art, including: chiral High Pressure Liquid Chromatography (HPLC), formation and crystallization of chiral salts; alternatively, preferred isomers may be prepared by asymmetric synthesis.
The invention has the following beneficial effects: (1) The invention provides a novel compound strophiofibins diterpenoid compound derived from Dolicarpa glabra and an extraction, separation and purification method aiming at the novel compound, which adopts alcohol extraction, silica gel column chromatography, MCI column chromatography and semi-preparative HPLC for separation and purification to successfully obtain the novel compound, the operation method is simple, convenient and quick, and the purity of the compound obtained by separation by the method is higher than 95 percent.
(2) The novel compound strophiofbrins diterpenoid compound disclosed by the invention can obviously improve the survival rate of model injured nerve cells PC12, and has a better inhibition effect on apoptosis of the nerve cells PC12, so that the compound disclosed by the invention has a more obvious neuroprotective activity, can be applied to preparation of novel neuroprotective medicaments, and can be used for treating cognitive dysfunction and bradykinesia of patients with depression, anxiety, cerebral apoplexy and improving neurodegenerative diseases, for example.
Drawings
FIG. 1 is a two-dimensional correlation diagram of HMBC for Compound 1 and Compound 2;
fig. 2 shows the structures of compound 1 and compound 2 and their neuroprotective effect on OGD-damaged PC12 cells (mean ± SD, n=4). In comparison with the control group, ### p<0.001，*p<0.05，**p<0.01，***p<0.001. positive control edaravone (Eda);
FIG. 3 is an experimental and calculated ECD spectra of Compound 1 and Compound 2;
FIG. 4 is a diagram of Compound 1 1 H-NMR spectrum;
FIG. 5 is a diagram of Compound 1 13 C-NMR spectrum;
FIG. 6 is a HMBC spectrum of Compound 1;
FIG. 7 is a HSQC spectrum of Compound 1;
FIG. 8 is a diagram of Compound 1 1 H- 1 H COSY profile;
FIG. 9 is a NOESY spectrum of Compound 1
FIG. 10 is a high resolution mass spectrum of Compound 1
FIG. 11 is single crystal data for Compound 1
FIG. 12 is a diagram of Compound 2 1 H-NMR spectrum;
FIG. 13 is a diagram of Compound 2 13 C-NMR spectrum;
FIG. 14 is a HMBC spectra of Compound 2;
FIG. 15 is a HSQC spectrum of Compound 2;
FIG. 16 is a diagram of Compound 2 1 H- 1 H COSY profile;
FIG. 17 is a NOESY spectrum of Compound 2;
fig. 18 is a high resolution mass spectrum of compound 2.
Detailed Description
The following examples are provided to further illustrate the invention and should not be construed as limiting the scope of the invention.
A diterpenoid compound has a general formula shown in a formula (I):
wherein R is 1 -H or = O; r is R 2 is-OH, -OCH 3 or-NH 2 ，R 3 is-OH or-OCH 3 。
Example 1: preparation of Compound 1 (strophiofimbrin A)
18kg of the glaucocalyxa medicinal material is sheared, reflux-extracted for 4 times at the temperature of 95 percent ethanol and 85 ℃, the extracting solutions are combined, and the total extract (472 g) is obtained after decompression concentration and drying. The total extract was dispersed with 10 times of distilled water, and sequentially extracted with an equal volume of petroleum ether, ethyl acetate, to obtain a petroleum ether fraction (123 g) and an ethyl acetate fraction (55 g). Subjecting the ethyl acetate part extract to silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate with volume ratio of 30:1, 15:1, 8:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:5, 0:1, and mixing to obtain 11 fractions; subjecting the fraction 11 to silica gel column chromatography, gradient eluting with dichloromethane-methanol with volume ratio of 1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 5:1, 1:1, 0:1, and mixing to obtain 9 fractions; subjecting the fraction 1 to MCI column chromatography, eluting with methanol-water gradient with volume ratio of 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 1:0, and mixing to obtain 10 fractions; the fraction 9 was purified by semi-preparative high performance liquid chromatography eluting with acetonitrile-water at a volume ratio of 41:59 and collecting the main peak to give the compound 1.
Example 2: preparation of Compound 2 (strophiofimbrin B)
18kg of the glaucocalyxa medicinal material is sheared, reflux-extracted for 4 times at the temperature of 95 percent ethanol and 85 ℃, the extracting solutions are combined, and the total extract (472 g) is obtained after decompression concentration and drying. The total extract was dispersed with 10 times of distilled water, and sequentially extracted with an equal volume of petroleum ether, ethyl acetate, to obtain a petroleum ether fraction (123 g) and an ethyl acetate fraction (55 g). Subjecting the ethyl acetate part extract to silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate with volume ratio of 30:1, 15:1, 8:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:5, 0:1, and mixing to obtain 11 fractions; subjecting the fraction 11 to silica gel column chromatography, gradient eluting with dichloromethane-methanol with volume ratio of 1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 5:1, 1:1, 0:1, and mixing to obtain 9 fractions; subjecting the fraction 1 to MCI column chromatography, eluting with methanol-water gradient with volume ratio of 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 1:0, and mixing to obtain 10 fractions; and purifying the fraction 8 by semi-preparative high performance liquid chromatography, eluting with methanol-water with a volume ratio of 3:2, and collecting a main peak to obtain the compound 2.
The structural formulas of compound 1 and compound 2 are as follows:
the structure analysis process comprises the following steps:
compound 1: the novel compound prepared in example 1 of the present invention is yellow needle crystals. High resolution mass spectrum gives an excimer ion peak m/z 350.1346[ M+Na ]]++ (calculated 350.1363), combined with 1 H-NMR 13 C-NMR (Table 1) confirmed the compoundThe molecular formula of the compound is C 19 H 21 NO 4 The calculated unsaturation was 10. 1 H、 13 C-NMR and HSQC showed three methyl groups (two methoxy groups), three methylene groups (one of which is an alkene), four methine groups (two of which are alkenes) and nine quaternary carbons (three carbonyl groups and five of which are alkenes). According to the analysis, from Me-18 (delta) H 1.87 From C-4 (delta) C 143.5)、C-19(δ C 114.2 And C-5 (delta) C 49.4 From terminal olefins methylene H-19 to Me-18, C-4 (. Delta.) C 147.1)、C-19(δ C 114.2 And C-5 (delta) C 52.3 From OMe-3 (delta) H 3.81,3H, s) to C-3 (delta) C 177.4 HMBC-related data (fig. 1), it can be confirmed that typical isopropenyl (δ) is present H 4.89,5.07，2H，s；δ C 114.8,147.1；δ H 1.87，3H，s；δ C 24.1 And methyl ester carbonyl (delta) C 175.6,52.3). Quaternary carbon of the remaining six olefins (delta) C 197.4, 172.1, 160.9, 158.5,136.5 and 137.9) and two alkenylmethines form three further double bonds and a conjugated carbonyl group (. Delta.) C 177.5). The above functional groups account for 7 out of 10 unsaturations. Thus, the remaining 3 unsaturation requirement is a tricyclic system in the molecule.
In HMBC spectra (FIG. 1), the correlation originates from Me-20 (delta) H 1.19 From C-1, C-9, C-10, me-20, from Me-18 and H-19 to C-5, from H-2 (. Delta.) of typical isopropenyl groups H 4.25 From C-1, C-11 and C-3, and continuous correlation 1 H- 1 The H-5/H-6α/H-7α/H-7β in the H COSY spectrum creates segment C-5 of six-membered carbocyclic ring B substituted with methyl and typically isopropenyl at C-10, which is part of a tricyclic structure. 1 H- 1 The H COSY spectrum also shows the presence of a spin system H-2/H-1α/H-1β, and the presence of a spin system from H-2 (δ H 4.25 HMBC-related 3 methyl formate groups to C-1, C-11 and C-3, from Me-20 to C-1 and C-9, from H-1 (delta) H 2.28,2.14) to C-20, C-9, C-11 and C-3, a five-membered ring A is constructed, substituted at C-2 with a methyl ester carbonyl group, which is bonded to ring B at the common C-9 and C-10 positions. Then, by reacting alpha, beta unsaturated ketone groups (delta C 160.9，C-9；δ C 137.9，C-11；δ C 158.5, C-17) from two olefinic methines H-13 (delta) H 6.89 To C-8, C-12, and H-14 (delta) H 7.23 From C-9 to C-17, and a spin system H-13/H-14, to construct a seven-membered ring C. The HMBC cross-peaks of H-14/C-7, H-7/C-14 and H-2/C-17 further indicate that ring C shares C-8 and C-9 with ring B and C-9 and C-11 with ring A. Thus, a planar structure of strophiofimbrin A with a novel 5/6/7 ring system is illustrated. Furthermore, the chiral nature of its molecular structure was found by calculation of circular dichroism (fig. 2) to determine its configuration as (2 r,5r,10 r). We also performed single crystal tests on this compound, the crystal structure of which is shown in the figure (fig. 11), and on the whole, the structure of this compound was determined and named strophiofimbrin A.
Compound 2: high resolution mass spectrum gives excimer ion peak m/z 351.1572[ M+Na ]]++ (calculated 351.1567), combined with 1 H-NMR 13 C-NMR (Table 1) confirmed that the molecular formula of the compound was C 20 H 24 O 4 The calculated unsaturation was 9. 1 H、 13 C-NMR and HSQC showed four methyl groups (two methoxy groups), four methylene groups (one of which is an alkene), four methine groups (two of which are alkenes) and eight quaternary carbons (two carbonyl groups and five of which are alkenes). According to the analysis, from Me-18 (delta) H 1.87 From C-4 (delta) C 147.1)、C-19(δ C 114.2 And C-5 (delta) C 51.8 From terminal olefins methylene H-19 to Me-18, C-4 (. Delta.) C 147.1)、C-19(δ C 114.2 And C-5 (delta) C 51.8 From OMe-3 (delta) H 3.70,3H, s) to C-3 (delta) C 177.4 HMBC-related data, it was confirmed that typical isopropenyl (δ) H 4.85,5.00，2H，s；δ C 114.2,147.1；δ H 1.87，3H，s；δ C 24.2 And methyl ester carbonyl (delta) C 177.4,52.5). The remaining five olefins quaternary carbon (delta) C 177.5, 164.3, 160.8, 146.5 and 138.4) and two olefinic methines (delta) H 7.05 and 7.23,2H, d, j=10.2 Hz) constitute three further pairs of double bonds and one conjugated carbonyl group (δ) C 177.5). The above functional groups account for 6 out of 9 unsaturations. Thus, the remaining 3 unsaturation requirement is a tricyclic system in the molecule.
In HMBC spectra (FIG. 1), the correlation originates from Me-20 (delta) H 1.11 From Me-18 and H-19 from typical isopropenyl groups to C-5, C-9, from H-5 (. Delta.) H 2.44 From H-7 (delta) to C-4, C-10 and C-20 H 3.11,2.92) to C-5, C-6, C-8 and C-9, and continuous correlation 1 H- 1 The H-5/H-6α/H-7α/H-7β in the H COSY spectrum creates segment C-5 of six-membered carbocyclic ring B substituted with methyl and typically isopropenyl at C-10, which is part of a tricyclic structure. 1 H- 1 The H COSY spectrum also shows the presence of a spin system H-2/H-1α/H-1β, and the presence of a spin system from H-2 (δ H 4.15 HMBC-related 3 methyl formate groups to C-1, C-11 and C-, from Me-20 to C-1 and C-9, from H-1 (delta) H 2.25,2.01) to C-20, C-9, C-11 and C-3, a five-membered ring A is constructed, substituted at C-2 with a methyl ester carbonyl group, which is bonded to ring B at the common C-9 and C-10 positions. Then, by reacting alpha, beta unsaturated ketone groups (delta C 160.8，C-9；δ C 146.5，C-11；δ C 177.5, C-17 or delta C 115.1，C-13；δ C 164.3,C-12；δ C 177.5, C-17) and from other OMe-12 to C-12 (. Delta.) C 164.3 From two olefinic methines H-13 (delta) H 7.05 To C-8, C-12 and C-17, and H-14 (delta) H 7.23 From C-9 to C-12, and a spin system H-13/H-14, to construct a seven-membered ring C. The HMBC cross-peaks of H-14/C-7, H-7/C-14 and H-2/C-17 further indicate that ring C shares C-8 and C-9 with ring B and C-9 and C-11 with ring A. Thus, a planar structure with a novel 5/6/7 ring system is illustrated. We additionally determined by calculation of circular dichroism (fig. 2) that its configuration was likewise (2 r,5r,10 r). In summary, the structure of this compound was determined and named strophiofimbrin B.
Table 1: compound 1 and compound 2 1 H NMR 13 C NMR data
a Recorded in CDCl 3 ； b Recorded in MeOD.
Active part:
experimental materials: cell culture medium DMEM, air permeable cell culture flasks, 96-well cell culture plates, PC12 cells, fetal bovine serum, trypsin, CCK8 kit, penicillin-streptomycin diabody solution (penicillin working concentration 100U/mL, streptomycin working concentration 0.1 mg/L), strophiofimbrin A, strophiofimbrin B, edaravone, phosphate buffer PBS (0.03 mol/L, pH =7.2), microporous filter, syringe, ethanol, DMSO, centrifuge tube, EP tube, 1mL gun, 200ul gun, glove, mask, etc.
Purchased PC12 cells were incubated at 37℃with 5% CO 2 And (5) carrying out adaptive culture in an incubator. The culture medium was DMEM medium (containing 5% fetal bovine serum and 1% diabody). At 37℃with 5% CO 2 Culturing in a saturated humidity incubator, pouring out the culture solution after the cells grow at the bottom of the culture flask, washing for 2 times by PBS, adding trypsin for digestion for 2min, adding DMEM culture solution into the culture solution to stop digestion, gently blowing, centrifuging at 1200r/min for 5min, resuspending cell sediment, inoculating the cell sediment in the culture flask, observing and photographing under a microscope, and obtaining the cell-free culture flask after the cells grow at a monolayer.
Establishing experimental models, grouping, modeling and drug administration:
establishing oxygen glucose deprivation/reoxygenation (Oxygen Glucose Deprivation-reoxygenation, OGD/R) model, diluting the treated cells with DMEM medium to 5×10 4 The cell suspension of each mL was inoculated into a 96-well plate, 200. Mu.L of the cell suspension was added to each well, and the cells were subjected to adaptive culture for 24 hours. The experiments were performed in normal control group (control group), model group (OGD group), experimental group (strophiofimbrin A, strophiofimbrin B drug group), positive drug control group (edaravone group): the normal control group is subjected to full-volume liquid exchange to be DMEM culture solution (containing 10% of fetal calf serum), and the culture is carried out in a constant-temperature incubator; the model group, the experimental group and the positive drug control group are completely replaced by DMEM sugar-free culture solution containing 1%O 2 、94％N 2 And 5% CO 2 Hypoxia treatment for 6h in a three-gas incubator, adding samples with final concentrations of 6.25 mu M, 12.5 mu M, 25 mu M, 50 mu M and 100 mu M respectively into each hole of the experimental group, and adding the positive control groupEdatavone with corresponding concentration is recovered, and then normal oxygen is recovered for 24 hours. mu.L of CCK8 was added to each well and incubated at 37℃for 2 hours, and the absorbance (OD) at 450nm was measured for each cell group by a microplate reader.
The calculation formula is as follows:
cell viability = [ (OD dosing group-OD blank)/(OD control group-OD blank) ]x100%.
The protective effect was evaluated on cell viability, which was 80.23.+ -. 3.14% and 81.25.+ -. 3.83% for compounds 1 (50. Mu.M) and 2 (50. Mu.M), respectively, compared to the OGD control (DMSO, cell viability 50.82.+ -. 1.99%), and 85.95.+ -. 1.07% for the positive control, edoavanone (50. Mu.M) (FIG. 2). Experimental results show that the compounds 1-2 have neuroprotective effect on the PC12 cells damaged by OGD.
The novel compound disclosed by the invention can obviously improve the survival rate of the model damaged nerve cells PC12, has a good inhibition effect on the apoptosis of the nerve cells PC12, and can be applied to the preparation of novel neuroprotective medicaments, such as treatment of cognitive dysfunction and bradykinesia of patients with depression, anxiety, cerebral apoplexy and improvement of neurodegenerative diseases. The above description of the embodiments of the invention has been presented in connection with the drawings but these descriptions should not be construed as limiting the scope of the invention, which is defined by the appended claims, and any changes based on the claims are intended to be covered by the invention.
Claims (5)
1. A diterpenoid compound has a structural formula shown in formula 1:
，
the chemical name of formula 1 is strophiofimbrin A.
2. The process for producing a diterpene compound according to claim 1, which comprises the steps of:
step a, shearing the glaucocalyxa medicinal material, reflux-extracting with ethanol, recovering the extract, concentrating under reduced pressure, and drying to obtain total extract;
dispersing the total extract with distilled water, and sequentially extracting with petroleum ether and ethyl acetate with equal volume to obtain petroleum ether part and ethyl acetate part;
step c, performing silica gel column chromatography on the ethyl acetate part, performing gradient elution by using petroleum ether-ethyl acetate, and combining to obtain 11 fractions; subjecting the fraction 11 to silica gel column chromatography, gradient eluting with dichloromethane-methanol, and mixing to obtain 9 fractions; subjecting the fraction 1 to MCI column chromatography, gradient eluting with methanol-water, and mixing to obtain 10 fractions; fraction 9 was purified by semi-preparative hplc eluting with acetonitrile-water and collecting the main peak to give compound strophiofimbrin A.
3. The method for preparing diterpene compound according to claim 2, wherein the specific step of step c is as follows:
subjecting the ethyl acetate part to silica gel column chromatography, gradient eluting with petroleum ether-ethyl acetate with volume ratio of 30:1, 15:1, 8:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:5, 0:1, and mixing to obtain 11 fractions;
subjecting the fraction 11 to silica gel column chromatography, gradient eluting with dichloromethane-methanol with volume ratio of 1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 5:1, 1:1, 0:1, and mixing to obtain 9 fractions;
subjecting the fraction 1 to MCI column chromatography, eluting with methanol-water gradient with volume ratio of 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 1:0, and mixing to obtain 10 fractions;
the fraction 9 was purified by semi-preparative high performance liquid chromatography eluting with acetonitrile-water at a volume ratio of 41:59 and collecting the main peak to give compound strophiofimbrin A.
4. The method for preparing diterpene compounds according to claim 2, wherein the ethanol in the step a is extracted 4 times by reflux with a 95% ethanol aqueous solution at 85 ℃.
5. Use of a diterpenoid compound of claim 1 in the manufacture of a medicament for neuroprotection.
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| CN101360506A (en) * | 2005-11-25 | 2009-02-04 | 派普林研究股份有限公司 | Methods for wound healing |
| CN105152921A (en) * | 2015-10-22 | 2015-12-16 | 云南民族大学 | Tricyclic diterpenoid compound and preparation method and application thereof |
| CN113683507A (en) * | 2021-08-24 | 2021-11-23 | 沈阳药科大学 | Guaiane sesquiterpene derivative in regachia and application thereof |
| CN114031579A (en) * | 2021-11-11 | 2022-02-11 | 沈阳药科大学 | Preparation and application of daphnane diterpenoid compounds in lilac daphne flower buds |
| CN114874098A (en) * | 2022-06-15 | 2022-08-09 | 扬州大学 | Compound extracted and separated from persistent calyx wood as well as preparation method and application thereof |
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| CN101360506A (en) * | 2005-11-25 | 2009-02-04 | 派普林研究股份有限公司 | Methods for wound healing |
| CN105152921A (en) * | 2015-10-22 | 2015-12-16 | 云南民族大学 | Tricyclic diterpenoid compound and preparation method and application thereof |
| CN113683507A (en) * | 2021-08-24 | 2021-11-23 | 沈阳药科大学 | Guaiane sesquiterpene derivative in regachia and application thereof |
| CN114031579A (en) * | 2021-11-11 | 2022-02-11 | 沈阳药科大学 | Preparation and application of daphnane diterpenoid compounds in lilac daphne flower buds |
| CN114874098A (en) * | 2022-06-15 | 2022-08-09 | 扬州大学 | Compound extracted and separated from persistent calyx wood as well as preparation method and application thereof |
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