Leucolane diterpenoid compound in daphne genkwa as well as preparation method and application of eucolane diterpenoid compound

Abstract

The invention discloses a euphorbia lathyris type diterpenoid compound in daphne genkwa and a preparation method and application thereof, and relates to the technical field of plant medicines.The preparation process of the diterpenoid compound comprises the following steps that daphne genkwa is subjected to cold soaking extraction with ethyl alcohol, vacuum concentration is conducted to obtain extract, then the extract is extracted with ethyl acetate, and the extract is concentrated to obtain the euphorbia lathyris type diterpenoid compound. The preparation method comprises the following steps: extracting ethyl acetate from the ethyl acetate layer, quickly separating the obtained ethyl acetate layer extract through reduced pressure silica gel column chromatography to obtain three fractions Frs.A1-Frs.A3, and separating the Frs.A3 through reduced pressure silica gel column chromatography, Sephadex LH-20 gel column chromatography, reversed-phase ODS column chromatography and reversed-phase high-performance liquid chromatography to obtain compounds 1-4. The compounds 1-4 provided by the invention have a protective effect on H2O2-induced nerve cells SHSY5Y, so that the compounds 1-4 can be used for preparing drugs with a neuroprotective effect.

Description

Technical Field

[0001] This invention relates to the field of plant medicine technology, specifically to the isolation method and structural identification of four diterpenoid compounds of the genus Daphne from Daphne genkwa, and their application in the preparation of neuroprotective drugs. Background Technology

[0002] Yellow daphne is a plant belonging to the genus Daphne in the family Thymelaeaceae. Wikstroemia chamaedaphne The dried flower buds of *Daphne genkwa* (Meigen) are a traditional Chinese medicine from Shanxi Province, first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), and subsequently included in the 1977 edition of the *Chinese Pharmacopoeia* and the 1987 edition of the *Shanxi Provincial Standards for Traditional Chinese Medicine*. In recent years, it has been widely used to treat acute and chronic hepatitis, infectious hepatitis, schizophrenia, epilepsy, and other diseases. It can also be used for induced labor. In agriculture, it is used as a plant-derived insecticide. *Daphne genkwa* mainly contains diterpenoids and flavonoids.

[0003] There are currently no clear reports on the activity of strychnine-type diterpenes in Daphne genkwa. Through research on the activity of strychnine-type diterpenes, it has been found that strychnine-type diterpenes have certain neuroprotective activities. Summary of the Invention

[0004] This invention provides a sucralose diterpenoid compound or a pharmaceutically acceptable salt thereof, wherein the sucralose diterpenoid compound in Daphne genkwa is selected from any one of the following compounds:

[0005] The method for preparing the diterpenoid compound of the sucralose type according to the present invention includes the following steps: (1) Extract the yellow daphne flowers with ethanol; (2) The extract was concentrated under reduced pressure to obtain the total extract; (3) The total extract was extracted with ethyl acetate to obtain the ethyl acetate extract; (4) The ethyl acetate extract was separated by vacuum silica gel column chromatography to obtain three crude fractions, which were denoted as Frs.A1-Frs.A3, respectively. (5) Frs. A3 was separated by reduced pressure silica gel column chromatography to obtain 4 fractions, which were denoted as Frs. A3-1-Frs. A3-4 respectively; (6) Frs. A3-2 was separated by Sephadex LH-20 gel column chromatography to obtain Frs. A3-2-T, Frs. A3-2-M and Frs. A3-2-H; (7) Frs. A3-2-T was separated by silica gel column chromatography to obtain 4 fractions, which were denoted as Frs. T-1-Frs. T-4; (8) Frs. T-2 were isolated and purified by reversed-phase ODS column chromatography and reversed-phase high performance liquid chromatography.

[0006] The preparation method of the above-mentioned homolanyl diterpenoids, wherein: In step (1), the yellow jasmine is extracted 2-3 times by cold soaking with ethanol; and the mass concentration of the ethanol is 70-98%.

[0007] In step (2), the temperature for the reduced-pressure concentration is 50-55 DEG C.

[0008] In step (4), the ethyl acetate layer is separated by gradient elution of the reduced-pressure silica gel column chromatography to obtain three crude fractions; and the gradient elution conditions of the reduced-pressure silica gel column chromatography are petroleum ether: ethyl acetate = 1:1-1:2.

[0009] In step (5), Frs. A3 are separated by gradient elution of the reduced-pressure silica gel column chromatography to obtain four fractions; and the gradient elution conditions of the reduced-pressure silica gel column chromatography are ethyl acetate: methanol = 5:1-1:1.

[0010] In step (6), the elution conditions of the Sephadex LH-20 gel column chromatography are methanol: water = 70:30-50:50.

[0011] In step (7), the gradient elution conditions of the silica gel column chromatography are dichloromethane: methanol = 50:1-1:1.

[0012] In step (8), the gradient elution conditions of the reversed-phase ODS column chromatography are methanol: water = 10:90-90:10; and the separation conditions of the reversed-phase high performance liquid chromatography are methanol: water = 30:70-50:50, the retention times of compounds 1 and 2 are 40 min and 42 min respectively, and the retention times of compounds 3 and 4 are 30 min and 36 min respectively.

[0013] The application further provides a pharmaceutical composition comprising one or more of the homolanyl diterpenoids 1-4 or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable excipients.

[0014] The pharmacological tests prove that the homolanyl diterpenoids 1-4 have certain degree of cell protection effect on H2O2-induced oxidative damage SH-SY5Y cells, and the protection activity of the high-concentration samples of compounds 1-3 is equivalent to that of the positive drug.

[0015] Based on the same, the application further provides the use of any one of the sequirine diterpenoids 1-4 or pharmaceutically acceptable salts thereof or a pharmaceutical composition containing the same in the preparation of a neuroprotective drug.

[0016] Advantages of the application: The compound has certain protective effect on H2O2-induced nerve cells SHSY5Y, provides an important material basis for developing a drug for preventing and treating oxidative stress-related nervous system diseases (such as Alzheimer's disease and Parkinson's disease), and has a good prospect of pharmaceutical development. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 (+)-HRESIMS spectrum of compound 1 Figure 2 (+)-HRESIMS spectrum of compound 1 1 H-NMR spectrum (600MHz, CD3OD) of compound 1 Figure 3 (+)-HRESIMS spectrum of compound 1 13 C-NMR spectrum (150MHz, CD3OD) of compound 1 Figure 4 HSQC spectrum (600MHz, CD3OD) of compound 1 Figure 5 HMBC spectrum (600MHz, CD3OD) of compound 1 Figure 6 ECD spectrum of compound 1 Figure 7 NOESY spectrum (600MHz, CD3OD) of compound 1 Figure 8 (+)-HRESIMS spectrum of compound 2 Figure 9 (+)-HRESIMS spectrum of compound 2 1 H-NMR spectrum (600MHz, CD3OD) of compound 2 Figure 10 ECD spectrum of compound 2 Figure 11 (+)-HRESIMS spectrum of compound 2 13 C-NMR spectrum (150MHz, CD3OD) of compound 2 Figure 12 HSQC spectrum (600MHz, CD3OD) of compound 2 Figure 13 HMBC spectrum (600MHz, CD3OD) of compound 2 Figure 14 NOESY spectrum (600MHz, CD3OD) of compound 2 Figure 15 (+)-HRESIMS spectrum of compound 3; Figure 16 Compound 3 1 H-NMR spectrum (600MHz, CD3OD); Figure 17 Compound 3 13 C-NMR spectrum (150MHz, CD3OD); Figure 18 HSQC spectrum of compound 3 (600MHz, CD3OD); Figure 19 HMBC spectrum of compound 3 (600MHz, CD3OD); Figure 20 NOESY spectrum of compound 3 (600MHz, CD3OD); Figure 21 ECD spectrum of compound 3; Figure 22 (+)-HRESIMS spectrum of compound 4; Figure 23 Compound 4 1 H-NMR spectrum (600MHz, CD3OD); Figure 24 Compound 4 13 C-NMR spectrum (150MHz, CD3OD); Figure 25 HSQC spectrum of compound 4 (600MHz, CD3OD); Figure 26 HMBC spectrum of compound 4 (600MHz, CD3OD); Figure 27 NOESY spectrum of compound 4 (600MHz, CD3OD); Figure 28 ECD spectrum of compound 4; Figure 29 The protective activity of compounds 1-4 against H2O2-induced oxidative damage in SH-SY5Y cells. Detailed Implementation

[0018] The present invention will be further described in conjunction with specific embodiments.

[0019] Example 1

[0020] Preparation of compounds 1-4: Selecting 50 kg of Huangyuhua, using 95% industrial ethanol to extract 3 times by cold soak method, 50 ℃ reduced pressure concentration to get total extract 5.4 kg, using ethyl acetate to extract, get ethyl acetate layer extract 498.1 g. Then the ethyl acetate layer extract is separated by reduced pressure silica gel column chromatography, in the separation process, with petroleum ether (PE) and ethyl acetate (EtOAc) as eluent, according to the gradient ratio PE:EtOAc=1:1, 1:2 (v / v), gradient elution, quickly divide it into 3 crude fractions Frs. A1-Frs. A3, of which Frs. A3 get 441 g. Then use reduced pressure silica gel column chromatography to remove color and segment Frs. A3, with EtOAc:CH3OH=5:1, 1:1, gradient elution, get 4 parts Frs. A3-1-Frs. A3-4. Then with CH3OH:H2O=60:40 as isocratic eluent, Frs. A3-2 (98 g) is separated by Sephadex LH-20 gel column chromatography to get fractions Frs. A3-2-T, Frs. A3-2-M and Frs. A3-2-H. Frs. A3-2-T (47.4 g) is separated by silica gel column chromatography, elution conditions are CH2Cl2:CH3OH=50:1, 30:1, 15:1, 8:1, 5:1, 3:1, 1:1, gradient elution, get fractions Frs. T-1-Frs. T-4. Frs. T-2 is separated and purified by reverse phase ODS column chromatography and reverse phase high performance liquid chromatography to get compounds 1-4. The gradient elution conditions of reverse phase ODS column chromatography are CH3OH:H2O=10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10.

[0021] The separation conditions of reverse phase high performance liquid chromatography are CH3OH:H2O=40:60, the retention time of compounds 1 and 2 is 40 min and 42 min respectively, and the retention time of compounds 3 and 4 is 30 min and 36 min respectively.

[0022] The above obtained compounds 1-4 are systematically identified, as shown in the following: Figures 1-28 The results are as follows: Compound 1: HRESIMS gives a quasi-molecular ion peak [M+Na] + 517.2413 (calcd for C 26 H 38 O9Na 517.2408), combined with 1 H NMR, 13 C NMR data determines its molecular formula as C 26 H38 O9, unsaturation calculated as 8.

[0023] In 1 H NMR (600MHz, CD3OD) spectrum (Table 1), δ H 6.01 (1H, s, H-5) presumed to be the signal of hydrogen on the double bond, 3.50 (1H, d, J =11.5 Hz, H-18a), 3.45 (1H, d, J =11.4 Hz, H-18b) presumed to be the signals of a set of magnetically nonequivalent methylene protons with oxygen, 1.22 (3H, m, H-16), 1.66 (3H, s, H-17), 1.12 (3H, s, H-19), 1.04 (3H, d, J =7.0 Hz, H-20) presumed to be the signals of four methyl protons, δ H 4.32 (1H, dd, J =7.9, 5.7 Hz, H-1') presumed to be the signal of the anomeric hydrogen.

[0024] In 13 C NMR (150MHz, CD3OD) showed 26 carbon signals, in the low field region there were δ c 213.3, 211.6, 170.0, 146.2, 141.5, 117.6, 101.7 seven carbon signals, among which δ c 213.3 (C-1), 211.6(C-14) presumed to be the carbon signals of two ketone carbonyl groups, δ c 117.6 (C-5), 146.2 (C-6), 170.0 (C-4), 141.5 (C-15) presumed to be the carbon signals of two sets of double bonds; δ c 79.1 (C-7), 63.9 (C-18) presumed to be the carbon signals of two oxygen- substituted groups, δ c 101.7 (C-1') presumed to be the carbon signal of the anomeric group; δ c 24.5 (C-19), 16.5 (C-16), 18.0 (C-17), 18.2 (C-20) presumed to be the carbon signals of four methyl groups. All carbon-hydrogen direct correlation signals were assigned by HSQC data.

[0025] In the HMBC spectrum, H-16 ( δ H 1.22) correlated with C-2 ( δc 41.3), C-3 ( δ c 37.6) has a correlation with H-3( δ H 2.93) with C-1 ( δ c 213.3), C-4 ( δ c 170.0) has a correlation with H-2 ( δ H 2.61) with C-1 ( δ c 213.3), C-16 ( δ c 16.5) has a correlation with H-17 ( δ H 1.66) with C-5 ( δ c 117.6), C-6 ( δ c 146.2) and C-7 ( δ c 79.1) has a correlation with H-1' ( δ H 4.32) with C-7 ( δ c 79.1) has a correlation with H-8 ( δ H 2.23) with C-6 ( δ c 146.2), C-7 ( δ c 79.1) and C-9 ( δ c 23.5) has a correlation with H-11 ( δ H 0.66) with C-10 ( δ c 23.5) has a correlation with H-18( δ H 3.45) with C-10 ( δ c 23.5) has a correlation with H-18 ( δ H 3.50) with C-10 ( δ c 23.5) has a correlation with H-19 ( δ H 1.12) with C-10 ( δ c 23.5) and C-18 ( δ c 63.9) has a correlation with H-20 ( δ H 1.04) with C-12 ( δ c 27.9), C-13 ( δ c 44.5), C-14 ( δ c 211.6) has a correlation with H-12 ( δ H 1.45) with C-13 (δ c 44.5), C-20 ( δ c18.2) have correlation with H-12 ( δ H 1.99) with C-20 ( δ c 18.2), C-13 ( δ c 44.5), C-14 ( δ c 211.6) have correlation. Based on the above, it is speculated that this compound is a clerodane type diterpenoid compound. At the same time, the planar structure of the compound is determined by hydrogen spectrum, carbon spectrum, HSQC and HMBC spectrum.

[0026] In the NOESY spectrum, H-11 ( δ H 0.66) with H3-19 ( δ H 1.12) / H-13 ( δ H 3.35) / H-9 ( δ H 1.07) have correlation, H-9 ( δ H 1.07) with H3-19 ( δ H 1.12) have correlation, H-7 ( δ H 4.66) with H3-9 ( δ H 1.07) / H-1'( δ H 4.32) have correlation. Combined with literature research, it is speculated that H-7, H-9, H-11, H-13, H3-19 are α orientation, H3-20, H-18 are β orientation. Through chemical shift δ c 41.3 (C-2) and literature carbon spectrum data comparison and calculation of hydrogen spectrum, it is speculated that H3-16 is β orientation.

[0027] The absolute configuration is determined by comparing the calculated and measured ECD. By comparing the calculated ECD spectrum and the measured ECD spectrum, the Cotton effect peak in the experimental CD spectrum of compound 1 is in good agreement with the Cotton effect peak of the calculated 1a (2 R ,7 S ,9 S ,10 R ,11 R ,13 S ) configuration, so the absolute configuration of compound 1 is (2R ,7 S ,9 S ,10 R ,11 R ,13 S )。

[0028] Compound 2: HRESIMS gave a pseudomolecular ion peak [M+Na] + C 26 H 38 O9Na 517.2408), confirming its molecular formula as C 26 H 38 O9, with an unsaturation count of 8.

[0029] The NMR data of compound 2 (Table 1) was very similar to that of compound 1, 13 The main difference in the C NMR spectra was that compound 1 δ C 41.3 (C-2) to compound 2 δ c 41.6 (C-2), 1 The difference in the H NMR spectra was that compound 1 δ H 2.93 (1H, ddd, J = 18.7, 6.8, 2.9 Hz, H-3a) and δ H 2.53 (1H, dq, J = 18.6, 2.7 Hz, H-3b) to compound 2 δ H 3.22 (1H, m, H-3a) and δ H 2.29 (1H, m, H-3b). This indicated that compound 2 was a secologanoid diterpene, and the planar structure of this compound was determined by 1H, 13C, HSQC and HMBC spectra, which was consistent with that of compound 1.

[0030] The retention time of compound 1 and compound 2 was 2 min apart, and the relative configuration of compound 2 was further verified by NOESY spectra, which was the same as that of compound 1 except for the configuration of C-2.

[0031] In the NOESY spectra, H-11 ( δ H 0.66) correlated with H3-19 ( δ H 1.12) / H-13 ( δH 3.35) / H-9 ( δ H 1.07) have correlation, H-9 ( δ H 1.07) have correlation, H-9 ( δ H 1.12) have correlation, H-7 ( δ H 4.66) have correlation, H-9 ( δ H 1.07) / H-1'( δ H 4.32) have correlation. It is speculated that H-7, H-9, H-11, H-13, H3-19 are α orientation, H3-20, H-18 are β orientation. By chemical shift δ c 41.6 (C-2) and literature carbon spectrum data comparison and calculation of hydrogen spectrum, it is speculated that H3-16 is α orientation.

[0032] Its absolute configuration is determined by comparing the calculated and measured ECD. By comparing the calculated ECD spectrum and the measured ECD spectrum, the Cotton effect peak in the experimental CD spectrum of compound 2 is in good agreement with the Cotton effect peak of the calculated 2a (2 S ,7 S ,9 S ,10 R ,11 R ,13 S ) configuration, so the absolute configuration of compound 2 is (2 S ,7 S ,9 S ,10 R ,11 R ,13 S ).

[0033] Table 1: Compound 1 and compound 2 1 H NMR (600MHz) and 13 C NMR (150MHz) chemical shift values (solvent is CD3OD)

[0034] Compound 3: HRESIMS gives a quasi-molecular ion peak [M+Na] + 517.2418 (calcd for C 26 H 38 O9Na 517.2408), combined1 H NMR, 13 C NMR data determined its molecular formula as C 26 H 38 O9, calculated unsaturation as 8.

[0035] In 1 H NMR (600MHz, CD3OD) spectrum (Table 2), δ H 6.01 (1H, s, H-5) presumed to be the signal of hydrogen on the double bond, δ H 3.24 (1H, m, H-18a), 3.32 (1H, m, H-18b) presumed to be the signals of methylene protons with one oxygen attached, δ H 1.21 (3H, d, J = 7.4 Hz, H-16), 1.67 (3H, d, J = 1.4 Hz, H-17), 0.94(3H, s, H-19), 1.03 (3H, d, J = 7.0 Hz, H-20) presumed to be the signals of four methyl protons, δ H 4.34(1H, d, J = 7.8 Hz, H-1¢) presumed to be the signal of the anomeric hydrogen.

[0036] In 13 C NMR (150MHz, CD3OD) showed 26 carbon signals, in the low field region there were δ c 213.3,211.7, 170.0, 146.1, 141.6, 117.6, 101.7 seven carbon signals, among which δ c 213.3 (C-1), 211.7(C-14) presumed to be the signals of two carbonyl carbons of ketones, δ c 117.6 (C-5), 146.1 (C-6), 170.0 (C-4),141.6 (C-15) presumed to be the signals of two sets of double bond carbons; δ c 79.3 (C-7), 73.4 (C-18) presumed to be the signals of two carbons with oxygen substitution, δ c 101.7 (C-1¢) presumed to be the signal of the anomeric carbon of sugar; δc 11.3 (C-19), 16.5 (C-16), 18.0 (C-17), 18.3 (C-20) carbon signals of four methyl groups. All carbon hydrogen direct correlations were assigned by HSQC data.

[0037] In the HMBC spectrum, H-16 ( δ H 1.21) correlates with C-2 ( δ c 41.3), C-3 ( δ c 37.6), C-1( δ c213.3) and H-3 ( δ H 2.54) correlates with C-4 ( δ c 170.0), C-15 ( δ c 141.6) and H-17 ( δ H 1.67) correlates with C-7 ( δ c 79.3) and H-1' ( δ H 4.34) correlates with C-7 ( δ c 79.3) and H-5 ( δ H 6.01) correlates with C-7 ( δ c 79.3) and C-17 ( δ c 18.0) and H-9 ( δ H 1.06) correlates with C-18 ( δ c 73.4) and H-11( δ H 0.66) correlates with C-18 ( δ c 73.4) and H-19 ( δ H 0.94) correlates with C-9 ( δ c 21.2), C-10 ( δ c 24.4) and C-18 ( δ c 73.4) and H-20 ( δ H 1.03) correlates with C-12 ( δ c 28.0), C-13 ( δ c 44.5), C-14 ( δ(c211.7) is relevant. In summary, it is speculated that this compound is a diterpenoid of the sucralose type. Furthermore, the planar structure of this compound was determined by proton, carbon, HSQC, and HMBC spectra.

[0038] In the NOESY spectrum, H-11 ( δ H 0.66) and H3-19 ( δ H 0.94) / H-13 ( δ H 3.33) / H-9 ( δ H 1.06) is related, H-9 ( δ H 1.06) and H3-19 ( δ H 0.94) is relevant, H-7 ( δ H 4.68) and H-9 ( δ H 1.06) / H-1'( δ H 4.34) is relevant. H-7 ( δ H 4.68) and H3-19 ( δ H 0.94) / H-1' ( δ H 4.34) / H-13 ( δ H 3.33) is related, H-11 ( δ H 0.66) and H-13 ( δ H 3.33) is relevant, H-18 ( δ H 3.24) and H-9 ( δ H 1.06) / H3-20 ( δ H 1.03) is related. It is speculated that H-7, H-11, H3-19, and H-13 are... α Orientation, H-9, H-18, H-20 are β Orientation. Through chemical shift δ C Comparison of 41.3 (C-2) and literature carbon spectrum data suggests that H3-16 is... β orientation.

[0039] By comparing the spectra, the difference between compound 1 and compound 3 is mainly reflected in the C-9 position configuration.

[0040] The absolute configuration of compound 3 is determined by comparing the calculated and measured ECD. By comparing the calculated ECD spectrum and the measured ECD spectrum, the Cotton effect peak in the experimental CD spectrum of compound 3 is in good agreement with the Cotton effect peak of 3a (2 R ,7 S ,9 R ,10 R ,11 R ,13 S ) configuration, so the absolute configuration of compound 3 is (2 R ,7 S ,9 R ,10 R ,11 R ,13 S ).

[0041] Compound 4: HRESIMS gives the quasi-molecular ion peak [M+Na] + 517.2408 (calcd for C 26 H 38 O9Na 517.2408), combined with 1 H NMR, 13 C NMR data determines its molecular formula as C 26 H 38 O9, the calculation of unsaturation is 8.

[0042] The NMR data of compound 4 (Table 2) is very similar to that of compound 3, 13 The main difference between the C NMR spectra of compound 3 δ C 41.3 (C-2) to compound 4 δ C 41.6 (C-2), 1 The difference between the H NMR spectra of compound 3 δ H 2.93 (1H, ddd, J = 18.6, 6.7, 2.7 Hz, H-3a) and δ H 2.54 (1H, dt, J = 18.6, 2.6 Hz, H-3b) to compound 4 δ H 3.21 (1H, m, H-3a) and 2.29 (1H, dt, J= 18.7, 2.6 Hz, H-3b). This indicates that compound 4 is a drimane type diterpenoid, and the planar structure of this compound was determined by 1H NMR, 13C NMR, HSQC and HMBC spectra, which is consistent with the planar structure of compound 3.

[0043] In the NOESY spectrum, H-7 ( δ H 4.68) correlated with H3-19 ( δ H 0.94) / H-1' ( δ H 4.33) / H-13 ( δ H 3.32) correlated, H-11 ( δ H 0.67) correlated with H-13 ( δ H 3.32) correlated, H-18 ( δ H 3.24) correlated with H-9 ( δ H 1.06) / H3-20 ( δ H 1.03) correlated. It is speculated that H-7, H3-11, H3-19, H-13 are in α orientation, H-9, H-18, H-20 are in β orientation. By comparing the chemical shift δ C 41.6 (C-2) and carbon spectrum data, it is speculated that H3-16 is in α orientation.

[0044] Compound 3 and compound 4 are 6 min apart in retention time, and the relative configuration of compound 4 is further verified by NOESY spectrum, which is the same as compound 3 except the configuration of C-2.

[0045] Its absolute configuration is determined by comparing the calculated and measured ECD. By comparing the calculated ECD spectrum and the measured ECD spectrum, the Cotton effect peak in the experimental CD spectrum of compound 4 is better matched with the Cotton effect peak of the calculated 4a (2 S ,7 S ,9 R ,10 R ,11 R ,13 S ) configuration, so the absolute configuration of compound 4 is (2 S ,7 S ,9 R ,10R ,11 R ,13 S )。

[0046] Table 2: in vitro protective activity of compound 3 and compound 4 against H2O2-induced oxidative damage in SH-SY5Y cells 1 H NMR (600MHz) and 13 C NMR (150MHz) chemical shift values (solvent is CD3OD)

[0047] Example 2 Evaluation of protective activity of compound 1-4 in vitro against H2O2-induced oxidative damage in SH-SY5Y cells.

[0048] WST-8 in CCK-8 reagent is reduced to water-soluble yellow formazan by dehydrogenase in mitochondria in the presence of 1-Methoxy PMS electron coupling agent, and the amount of formazan is positively correlated with the number of living cells. Therefore, the change in absorbance at 450 nm detected by a microplate reader can indirectly reflect the change in the number of living cells.

[0049] CCK8 method was used. SH-SY5Y cells in logarithmic growth phase were taken, and the cell density was adjusted to 1×10 5 cells / mL with DMEM medium containing 10% fetal bovine serum, and the cells were inoculated in a 96-well plate at 100 μL per well (1×10 4Cells were cultured in a 37℃, 5% CO2 incubator for 24 h until 70%-80% cell adhesion and confluence were achieved before further treatment. The experiment was divided into 5 groups, each with 6 replicates (to reduce experimental error): Control group: cells and culture medium only, no drug or H2O2 treatment; Model group: cells, culture medium, and H2O2, no drug pretreatment; Compound 1-4 intervention groups and the positive control group received the corresponding drugs, with specific concentrations shown in Table 3. The original culture medium in the 96-well plate was discarded. 100 μL of fresh culture medium was added to the control and model groups, and 100 μL of culture medium containing the corresponding drug concentration was added to each drug group. Pretreatment was performed in the incubator for 2 h. Except for the control group, 10 μL of H2O2 working solution (final concentration 200-400 μM) was added to each group to ensure uniform H2O2 distribution in the wells; 10 μL of culture medium was added to the control group as a control. The cells were cultured for another 24 h to induce oxidative damage. Discard the supernatant from each well. Add 100 μL of fresh culture medium to each well, followed by 10 μL of CCK-8 reagent to each well of a 96-well plate. Avoid generating air bubbles during the addition process, as this will affect the absorbance measurement. Return the plate to the incubator and incubate the cells with CCK-8 at 37°C for 3 h. After the reaction, measure the OD value (absorbance) at 450 nm using a microplate reader. Calculate cell viability (CV) using the following formula.

[0050] The dosage concentrations of the compounds used in this study and their corresponding group identifiers are shown in Table 3 below: Table 3: Drug Dosage Concentrations and Grouping

[0051] The formula for calculating cell survival rate is as follows:

[0052] Data are expressed as mean ± standard deviation (x ± s) and analyzed using GraphPad Prism 9.0 software. One-way ANOVA was used for comparisons among multiple groups, and LSD-t tests were used for pairwise comparisons between groups. P < 0.05 was considered statistically significant.

[0053] Activity results as follows δ δ δ δ δ δ δ Figure 29 As shown, the results indicate that compounds 1-3 can improve the survival rate of H2O2-induced oxidative damage SH-SY5Y cells to a certain extent.

[0054] Compounds 1-4 showed a certain degree of cytoprotective effect against H2O2-induced oxidative damage to SH-SY5Y cells. The protective activity of high concentration (50 µM) samples of compounds 1-3 was comparable to that of positive control drugs. Their extraction and separation methods are simple, which facilitates further pharmacological and pharmacodynamic studies and the development of their application in the preparation of neuroprotective drugs.

Claims

1. A diterpenoid compound of the genus Daphne type or a pharmaceutically acceptable salt thereof from Daphne genkwa, characterized in that, The diterpenoid compounds of the *Daphne genkwa* species are selected from any one of the following compounds: 。 2. The diterpenoid compound of the *Daphne genkwa* type or its pharmaceutically acceptable salt as described in claim 1, characterized in that, This diterpenoid compound is derived from *Daphne hainanensis*, a plant belonging to the genus *Daphne* in the family Thymelaeaceae. Wikstroemia chamaedaphne It was extracted and isolated from the dried flower buds of Meisn.

3. A method for preparing the strychnine-type diterpenoid compound from Daphne genkwa as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Extract the yellow daphne flowers with ethanol; (2) The extract was concentrated under reduced pressure to obtain the total extract; (3) The total extract was extracted with ethyl acetate to obtain an ethyl acetate layer; (4) The ethyl acetate layer was separated by vacuum silica gel column chromatography to obtain three crude fractions, denoted as Frs. A1-Frs. A3; (5) Frs. A3 was separated by reduced pressure silica gel column chromatography to obtain 4 fractions, denoted as Frs. A3-1-Frs. A3-4; (6) Frs. A3-2 was separated by Sephadex LH-20 gel column chromatography to obtain Frs. A3-2-T, Frs. A3-2-M and Frs. A3-2-H; (7) Frs. A3-2-T was separated by silica gel column chromatography to obtain 4 fractions, denoted as Frs. T-1-Frs. T-4; (8) Frs. T-2 was separated and purified by reversed-phase ODS column chromatography and reversed-phase high-performance liquid chromatography to obtain compounds 1-4.

4. The preparation method according to claim 3, characterized in that, In step (1), the yellow daphne flower is extracted with ethanol by cold soaking 2-3 times; wherein the mass concentration of ethanol is 70%-98%.

5. The preparation method according to claim 3, characterized in that, In step (4), the elution conditions for the ethyl acetate layer by vacuum silica gel column chromatography were petroleum ether: ethyl acetate = 1:1-1:2; in step (5), the elution conditions for Frs. A3 by vacuum silica gel column chromatography were ethyl acetate: methanol = 5:1-1:

1.

6. The preparation method according to claim 3, characterized in that, In step (6), the elution conditions for Frs. A3-2 by Sephadex LH-20 gel column chromatography were methanol:water = 70:30-50:

50.

7. The preparation method according to claim 3, characterized in that, In step (7), the elution conditions for Frs. A3-2-T by silica gel column chromatography are dichloromethane:methanol = 50:1-1:

1.

8. The preparation method according to claim 3, characterized in that, In step (8), the elution conditions for Frs. T-2 by reversed-phase ODS column chromatography were methanol:water = 10:90-90:10; and the separation conditions by reversed-phase high-performance liquid chromatography were methanol:water = 30:70-50:

50.

9. A pharmaceutical composition, characterized in that, It comprises one or more of the following: a diterpenoid compound of the genus Daphne as described in claim 1 or 2, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

10. The use of the strychnine-type diterpenoid compound from Daphne genkwa as described in claim 1 or 2, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described in claim 9, in the preparation of a neuroprotective drug.

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