A caprylane-type diterpene compound and its preparation method and application

Abstract

The present invention discloses a styraxane-type diterpene compound and its preparation method and application, and relates to the fields of extraction and separation of traditional Chinese medicine, natural drug chemistry and medical technology. The chemical structural formula of the styraxane-type diterpene compound is as follows:; The preparation method includes methanol immersion extraction, vacuum rotary evaporation, extraction, and chromatography; Its application includes the preparation of drugs with neuroprotective activity, drugs for treating stroke, and drugs for treating ischemic stroke. The beneficial effect of the present invention is that a new compound is prepared, the preparation method is simple, the process parameters are simple and easy to control, and it is suitable for industrial production, and the compound shows significant neuroprotective activity in the glutamate-induced HT-22 cell model, and the compound can alleviate glutamate-induced neuronal cell death by inhibiting apoptosis pathways, and can be used to prepare drugs for neuroprotection.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine extraction and separation, natural drug chemistry and medical technology, and specifically relates to a caprylane-type diterpene compound and a preparation method and application thereof. Background Art

[0002] Zeqi is an annual herbaceous plant of the Euphorbiaceae family, with high medicinal value. TCM monographs such as "Compendium of Materia Medica" and "Shengji Zonglu" describe the various therapeutic effects of Zeqi, including promoting diuresis, reducing edema, relieving cough and expectoration, and having detoxification and insecticide effects.

[0003] Studies have found that lacquer contains a variety of chemical components including terpenes, flavonoids, polyphenols, lipids, volatile oils and amino acids. The most important bioactive substances are terpenes. Modern pharmacological studies have shown that lacquer has various biological activities such as p-glycoprotein regulation, anti-proliferation, anti-inflammatory and neuroprotective activities to varying degrees.

[0004] Ischemic stroke is an acute cerebrovascular disease caused by rupture or blockage of blood vessels in the brain, resulting in cerebral ischemia. It has the characteristics of high morbidity, high mortality, high disability, and high recurrence rate. Neuroprotective agents can reduce the stress response under pathological conditions of the brain, reduce inflammatory damage, promote nerve cell regeneration and repair, prevent and treat stroke, and improve the prognosis of stroke. Therefore, finding and developing drugs with neuroprotective effects has a good research prospect for the treatment of cerebral ischemia-reperfusion injury. Summary of the invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide a caprylane-type diterpene compound and a preparation method and application thereof.

[0006] The technical solution of the present invention is as follows:

[0007] The first aspect of the present invention provides a caprylane-type diterpene compound having the structural formula shown below:

[0008] .

[0009] The molecular formula is C 30 H 43 O7, the absolute configuration of the compound is (2 R , 3 S , 4 S , 7 R , 9 S , 11 R , 15 R , 6′ S ).

[0010] In a preferred embodiment, the caprylane-type diterpene compound is isolated from Euphorbia cerifera.

[0011] The second aspect of the present invention provides a method for preparing a caperane-type diterpene compound, comprising the following steps:

[0012] S1, extracting the lacquer by immersion in methanol, and concentrating by rotary evaporation under reduced pressure to obtain a crude extract;

[0013] S2, diluting the crude extract with water, extracting with ethyl acetate and n-butanol in sequence to obtain an extract, and concentrating under reduced pressure to obtain a concentrate;

[0014] S3, separating the concentrate by silica gel column chromatography, eluting with petroleum ether-ethyl acetate gradient, and combining to obtain fractions;

[0015] S4, purifying and separating the fractions to obtain caprylane-type diterpene compounds.

[0016] In one of the preferred embodiments, in step S1, the lacquer is crushed into 50-150 meshes, and then the crushed lacquer is immersed in methanol for extraction, and concentrated by vacuum rotary evaporation to obtain a crude extract; wherein the volume concentration of methanol is 92%-98%, and the amount of methanol is 14 times-16 times the weight of the lacquer; the temperature of vacuum rotary evaporation and concentration is 55 ℃-65 ℃.

[0017] More preferably, the ze lacquer is crushed into 100 mesh coarse powder, the volume concentration of methanol is 95%, the amount used is 15 times the weight of the ze lacquer, and the temperature of the reduced pressure rotary evaporation concentration is 58°C~62°C.

[0018] In one preferred embodiment, in step S2, the amount of ethyl acetate and n-butanol used is 40 L to 50 L, and the temperature for vacuum concentration is 35° C. to 45° C. More preferably, the temperature for vacuum concentration is 40° C.

[0019] In one preferred embodiment, in step S3, the volume ratio of petroleum ether and ethyl acetate used in gradient elution is gradually reduced. More preferably, it is gradually reduced from 100:0 to 50:50. The volume ratio of petroleum ether and ethyl acetate used in gradient elution is specifically 100:0, 90:10, 80:20, 70:30, 60:40, 50:50.

[0020] In one preferred embodiment, in step S4, the volume ratio of acetonitrile-water used in the mobile phase for purification and separation is adjusted from 47:53 to 70:30 in a gradient manner, the time is 23 min, and the volume flow rate is 3 mL / min.

[0021] The third aspect of the present invention provides the use of the above-mentioned caprylane-type diterpene compound or a pharmaceutically acceptable salt thereof in the preparation of a drug with neuroprotective activity.

[0022] The fourth aspect of the present invention provides the use of the above-mentioned caprylane-type diterpene compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cerebral stroke.

[0023] The fifth aspect of the present invention provides the use of the above-mentioned caprylane-type diterpene compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating ischemic stroke.

[0024] In a preferred embodiment, the drug is in the form of one or more of a tablet, a soft capsule, a hard capsule, a solution, and a suspension.

[0025] The present invention has at least one of the following beneficial effects:

[0026] 1. The present invention extracts and separates a novel caprylane-type diterpene compound from ze lacquer, and determines its molecular structure based on spectrum and spectral data.

[0027] 2. The present invention provides a method for extracting and separating the caprylic acid-type diterpene compound using zeqi as raw material through methanol immersion extraction, vacuum rotary evaporation, extraction, chromatography and other steps. The method is simple, the process parameters are simple and easy to control, and it is suitable for industrial production.

[0028] 3. The succinyl terpenoid compound of the present invention exhibited significant neuroprotective activity in the in vitro glutamate-induced HT-22 neural cell injury model. At a concentration of 5 μM, the cell survival rate was increased by 34.2%, which was equivalent to the positive control drug butylphthalide racemate. Further studies have shown that the compound can alleviate glutamate-induced neural cell death by inhibiting the apoptosis pathway and can be used to prepare neuroprotective drugs, such as drugs for treating stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the structural diagram of the caprylane-type diterpene compound in Example 1 of the present invention.

[0030] Figure 2 This is a high resolution mass spectrum (HRESIMS) of the caprylane-type diterpene compound in Example 1 of the present invention.

[0031] Figure 3 This is the ultraviolet (UV) spectrum of the caprylane-type diterpene compound in Example 1 of the present invention.

[0032] Figure 4 This is the infrared (IR) spectrum of the caprylane-type diterpene compound in Example 1 of the present invention.

[0033] Figure 5 This is the nuclear magnetic hydrogen spectrum of the caprylane-type diterpene compound in Example 1 of the present invention ( 1 H-NMR) chart.

[0034] Figure 6 The NMR carbon spectrum of the caprylane-type diterpene compound in Example 1 of the present invention ( 13 C-NMR) chart.

[0035] Figure 7 This is the nuclear magnetic DEPT 135 spectrum of the caprylane-type diterpene compound in Example 1 of the present invention.

[0036] Figure 8 The NMR of the caprylane-type diterpene compound in Example 1 of the present invention 1 H– 1 H COSY spectrum.

[0037] Fig. 9 This is the nuclear magnetic NOESY spectrum of the caprylane-type diterpene compound in Example 1 of the present invention.

[0038] Fig.10 This is the NMR HSQC spectrum of the caprylane-type diterpene compound in Example 1 of the present invention.

[0039] Fig.11 This is the HMBC NMR spectrum of the caprylane-type diterpene compound in Example 1 of the present invention.

[0040] Fig.12 The NMR of the caprylane-type diterpene compound in Example 1 of the present invention 1 H– 1 Interpretation of H COSY and HMBC spectra.

[0041] Fig.13 This is the ECD curve of the caprylane-type diterpene compound experiment in Example 1 of the present invention.

[0042] Fig.14 This is the experimental and calculated ECD curve diagram of the caprylane-type diterpene compound in Example 1 of the present invention.

[0043] Fig.15The present invention shows that the styraxane diterpenoid compound in Example 1 alleviates glutamate-induced cell death, wherein A is the experimental procedure outline; B is a CCK-8 experiment showing the dose-dependent glutamate-induced HT-22 cell death; C is a CCK-8 experiment showing that the styraxane diterpenoid compound in Example 1 can alleviate glutamate-induced HT-22 cell death; D is the evaluation of apoptotic cells using Annexin V-FITC / PI staining; E is a quantitative analysis of the apoptosis rate in D; F is a live (green) / death (red) experiment of HT-22 cells incubated with glutamate (5 mM, 24h) in Example 1, size bar, 50 µm; G is a quantitative analysis of cell viability; H is a protein immunoblot (Western Blot) experiment measuring the expression level of apoptosis regulatory proteins; I is a quantitative analysis of Bcl-2 concentration; J is a quantitative analysis of Bax concentration. DETAILED DESCRIPTION

[0044] The following is a further description of the technical solution of the present invention in conjunction with the specific embodiments of the present application to better understand the present invention, but the protection scope of the present invention is not limited to the embodiments, and all modifications and modifications based on the present invention should be within the protection scope. The experimental methods described in the following embodiments are conventional methods in the technical field unless otherwise specified.

[0045] The equipment used in the following examples include: Shimadzu LC-20AT high performance liquid chromatograph, Shanghai Instrument SGW-533 polarimeter, Jena SPECPRD PLUS-50 ultraviolet spectrophotometer, Waters Xevo G2-XS TOF high resolution mass spectrometer, Thermo Fisher Nicolet iS5 infrared spectrometer, JASCO J-1500 circular dichroism spectrometer, Bruker AV-400 MHz nuclear magnetic resonance instrument, etc.

[0046] Unless otherwise specified, the reagents and materials mentioned above can be obtained from commercial sources.

[0047] Example 1

[0048] Extraction and separation of caprylane-type diterpenoid compounds:

[0049] 1. Take 9.2 kg of dried whole herb of Zeqi, crush it into coarse powder of about 100 mesh, add 15 times the volume concentration of 95% methanol, soak and extract 3 times, and concentrate the extract under reduced pressure at 52 °C to obtain 1.2 kg of crude extract; add appropriate amount of deionized water to the obtained crude extract for suspension, extract with 45 L of ethyl acetate and 45 L of n-butanol in turn, and concentrate the ethyl acetate extract under reduced pressure at 40 °C to obtain 299.7 g of concentrate.

[0050] 2. Take the above ethyl acetate concentrate and separate it by silica gel column chromatography, and use petroleum ether-ethyl acetate (volume ratio 100:0, 90:10, 80:20, 70:30, 60:40, 50:50) gradient elution, 3 column volumes for each gradient, and collect one eluate for every 500 mL, a total of 101 portions. After TLC thin layer chromatography, each eluate was combined with the same components to obtain 6 fractions, numbered Fr.A1-A6.

[0051] 3. Take the above Fr.A6 fraction and use acetonitrile-water (volume ratio 47:53→70:30, running time 23 min, elution flow rate 3 mL / min) as the mobile phase and separate by semi-preparative HPLC (target compound t R = 21.2 min), a new caprylane-type diterpene compound (1.6 mg, purity 99.2%) was obtained, and its structural formula is as follows Figure 1 shown.

[0052] Example 2

[0053] Structural analysis and identification of caprylane-type diterpenoid compounds:

[0054] The caprylane-type diterpene compound obtained in Example 1 of the present invention is a colorless oil, which is easily soluble in DMSO; [α] +42.3 ( c 0.09, MeOH); UV (MeCN) λmax: (log ε) 197 (4.03), 263 (4.30) nm; IR νmax: 3430, 2923, 1736, 1709, 1644, 1615, 1453 cm -1 ; ECD (MeCN, mdeg) λmax: 199(+23.78), 231 (–29.64), 260 (+34.22) HRESIMS: m / z 515.3006 [M+H] + (calcd for C 30 H 43 O7, 515.3009), and combined with carbon and hydrogen spectrum data (Table 1), its molecular formula was determined to be C 30 H 43 O7.

[0055] like Figure 4 As shown, the infrared spectrum shows a hydroxyl group (3430 cm -1 ) and a carbonyl group (1738 cm -1 、1708 cm -1 、1644 cm-1 )’s characteristic absorption peak.

[0056] like Figure 5 and Fig.10 As shown, 1 H-NMR spectrum and HSQC showed 7 methyl signals δ H 1.85 (3H, overlapped), 1.57 (3H, s), 1.20 (3H, s), 1.10 (3H, s), 1.09 (3H, d, J = 6.9Hz), 0.95 (3H, t, J = 7.4 Hz), 1 acetyl signal δ H 2.05 (3H, s), 3 oxygenated methine signals δ H 4.89 (1H, dd, J = 11.6, 3.0 Hz), 4.19 (1H, q, J = 6.1 Hz), 3.83 (1H, dd, J = 6.9,4.2 Hz), 6 double bond signals δ H 6.04 (1H, dd, J = 10.9, 1.7 Hz), 6.58 (1H, dd, J = 11.5,1.4 Hz), 5.90 (1H, d, J = 15.4 Hz), 7.27 (1H, dd, J = 15.4, 11.0 Hz), 6.36 (1H,dd, J = 15.5, 11.2 Hz), 6.12 (1H, dd, J = 15.3, 5.8 Hz).

[0057] like Figure 6 and Figure 7 As shown, 13 C-NMR and DEPT spectra showed 30 carbon signals, including 1 carbonyl carbon. δ C 194.7, 2 ester carbonyl carbon signals δ C 169.7, 166.4, 1 acetyl carbon δ C 21.7, 4 double bond carbons δC 121.3,121.3, 127.5, 133.1, 143.3, 144.4, 145.2, 145.3, 6 methyl groups, 3 methylene groups, 7 methine groups (3 with oxygen), 1 tertiary carbon with oxygen ( δ C 96.2), 1 quaternary carbon δ C 24.3.

[0058] like Fig.11 As shown, in the HMBC spectrum, H3-16 ( δ H 1.09) and C-1 ( δ C 40.8)、C-2 ( δ C 40.6)、C-3 ( δ C 81.5) related, H3-17 ( δ H 1.57) and C-5 ( δ C 121.3), C-6 ( δ C 143.3)、C-7 ( δ C 76.9) related, H3-18 ( δ H 1.20) and C-9 ( δ C 30.2), C-10 ( δ C 24.3)、C-11 ( δ C 29.3)、C-19 ( δ C 16.2) related, H3-20( δ H 1.85) and C-12 ( δ C 145.3), C-13 ( δ C 133.1)、C-14 ( δ C194.7). These results confirm that the oxadiazole-type diterpenoid compound obtained in Example 1 has a similar planar structure to the known compound euphzycopia G (ZHANG Y, XIONG F, ZHANGJ-J, et al. Euphzycopias AI, macrocyclic diterpenes with NLRP3 inflammasomeinhibitory activity from Euphorbia helioscopia L [J]. Fitoterapia, 2022, 157:105139). The main difference between them is that the C-6′ ( δ C The methylene group at C-35.0 is replaced by the hydroxyl group at C-6′ ( δ C 73.2) substitution, H3-8′ ( δ H 0.95) and H-4′ ( δ H 6.36) is further verified by the correlation with C-6′. In addition, the data showed that the hydroxyl group at C-3 in the caprylane-type diterpene compound obtained in Example 1 replaced the acetoxy group ( δ C 170.1). The above speculation is supported by HRESIMS data.

[0059] like Fig. 9 As shown in the NOESY spectrum, CH3-16 is related to H-3 / H-4α / CH3-17 / H-7 / H-9 / H-11 and CH3-18. α Configuration. H-2′( δ H 5.90) / H-4′( δ H 6.36) / H-6′( δ H 4.19) and H-3′ ( δ H 7.27) / H-5′( δ H 8.12) is related, indicating that Δ 2′,3′ and Δ 4′,5′ It is E configuration. Through experiments and calculations 1 The linear correlation between the H NMR chemical shifts showed that 6′ S *(R 2=0.9934) has a better correlation coefficient.

[0060] The absolute configuration of the compound was finally determined by experimental and calculated ECD (2 R , 3 S , 4 S , 7 R , 9 S , 11 R , 15 R , 6′ S ).

[0061] The structural formula of the caprylane-type diterpene compound is as follows Figure 1 ; HRESIMS mass spectrum as shown Figure 2 ; UV spectrum as shown Figure 3 ; IR spectrum as shown Figure 4 ; 1 H-NMR, 13 C-NMR, DEPT 135, 1 H– 1 H COSY, NOESY, HSQC, HMBC spectra and NOESY and HMBC spectra analysis are shown in Figure 5-Figure 12 , the experimental ECD curve is shown in Fig.13 , experimental and calculated ECD curves are shown in Fig.14 The above spectrum confirmed the structure of the compound.

[0062] The caprylane diterpene compound 1 H-NMR and 13 The C-NMR data are summarized in Table 1 (“a” in the table refers to overlapping signals).

[0063] Table 1 Caprylane-type diterpene compounds 1 H-NMR and 13 C-NMR data

[0064]

[0065] Example 3

[0066] Bioactivity test tools

[0067] 1. Cell incubation and main reagents

[0068] Rat hippocampal neural cells HT-22 were purchased from Wuhan Life Science Co., Ltd. (Wuhan, China) and incubated in DMEM medium containing 10% FBS and 1% penicillin or streptomycin at 37°C with 5% CO humidity.

[0069] 2. Neuroprotection Experiment

[0070] In order to determine the neuroprotective activity of the caprylane-type diterpenoid compound obtained in Example 1, we determined the cell survival rate according to the previous method (Xu X, Chua CC, Kong J, Kostrzewa RM, Kumaraguru U, HamdyRC, Chua BH. Necrostatin-1 protects against glutamate-induced glutathionedepletion and caspase-independent cell death in HT-22 cells. Journal of neurochemistry. 2007 Dec;103(5):2004-14). The glutamate-induced toxicity model was used to simulate brain damage after stroke. Fig.15 As shown in A in (Compound 1 is the caprylane-type diterpene compound obtained in Example 1), HT-22 cells were seeded in 96-well plates at a density of 8,000 cells / well in 100 µL of culture medium and fed in an incubator overnight. Then, the cells were incubated with 5 mM glutamate (Sigma-Aldrich Trading Co., Ltd.) and various concentrations of compound 1 (concentrations were 5 µM, 10 µM, 15 µM and 20 µM) for 24 h, and butylphthalide racemate (NBP) was used as a control drug. Cell viability was determined by CCK-8 assay (CK04, Dojindo Chemical Research Institute, Kumamoto, Japan). Only DMSO was added to the control group. 6 wells were one group. The experiment was repeated at least 3 times.

[0071] CCK-8 results showed that incubation of mouse hippocampal neuron cells (HT-22) with glutamate resulted in a dose-dependent decrease in cell survival, with a median lethal dose (LD50) of 5 mM (e.g. Fig.15 In addition, Fig.15 As can be seen from Figure C and Table 2, when mouse hippocampal neuron cells (HT-22) were incubated with 5 mM glutamate, the cell survival rate was only 53.64 ± 2.59%; while under the condition of 5 mM glutamate, after adding 5 µM, 10 µM and 15 µM of compound 1, the cell survival rate increased, approaching the cell survival rate after the control group (5 mM glutamate + butylphthalide racemate) was treated, indicating that compound 1 prepared in Example 1 can alleviate glutamate-induced neuronal cell death (such as Fig.15 C and shown in Table 2).

[0072] Table 2 Protective effects of compounds on HT22 cells induced by glutamate

[0073]

[0074] 3. Apoptosis

[0075] Previous studies have indicated that apoptosis is crucial in glutamate-induced cell death. Therefore, the present invention uses a method of double staining followed by flow cytometry to detect fluorescence signals to assess cell apoptosis, specifically using FITC-labeled Annexin V (Annexin V-FITC) and propidium iodide (PI) for double staining to assess cell apoptosis rate. The present invention quantifies apoptotic cells using a cell apoptosis detection kit (Annexin V-FITC / PI kit, Jiangsu Keygen Biotechnology Co., Ltd., Nanjing, China). Under the same conditions, a blank control group (only complete cell culture medium, no drug treatment), an experimental group (5mM glutamate + 5μM compound 1), a control group 1 (5mM glutamate), a control group 2 (5μM compound 1), a positive control group 1 (5mM glutamate + 5μM NBP) and a positive control group 2 (5mM glutamate + 20 μM Z-VAD) were set up. After 24h of drug incubation, the cells were collected, washed twice with cold PBS, and centrifuged at 800 × g for 5min. Then, the cells were diluted with a combination buffer (500uL) containing 5uL of Annexin V and 5uL of PI for 10min at room temperature. The samples were analyzed using a BD FACS Canto II flow cytometer (BD Biosciences). The average apoptosis rate was calculated from three independent cell incubation experiments.

[0076] The results showed that the caprylane-type diterpene compound (Compound 1) obtained in Example 1 showed comparable anti-apoptotic effects to pan-caspase inhibitors (Z-VAD, N-[benzyloxycarbonyl]-L-valyl-N-[(1S)-3-fluoro-1-(2-methoxy-2-oxoethyl)-2-oxopropyl]-L-alaninamide) and butylphthalide racemate (NBP). Fig.15 D and E in the figure).

[0077] 4. Live / dead staining experiment

[0078] Live / dead cell staining reagent (Keygen Biotech, Nanjing, China) was used to distinguish live and dead cells according to the manual. The staining solution contained 2uM calcein AM reagent and 8uMPI reagent mixed in PBS. The samples were incubated for 30 min and then imaged using a 20x objective fluorescence microscope (Leica DM18, Germany). The total number of green fluorescent cells (live cells) and red fluorescent cells (dead cells) in the images collected from more than three independent repeated experiments was counted, and the cell survival was analyzed by comparing the ratio of dead cells to the total number of cells in different treatment groups.

[0079] The results showed that the caprylane-type diterpene compound obtained in Example 1 can improve the cell survival rate of HT-22 cells treated with glutamate without damaging normal cells (such as Fig.15 F and G in FIG.

[0080] 5. Western Blot Experiment Analysis

[0081] The Western Blotting experiment method is as follows: the cultured cells were divided into 5 groups, namely, blank control group (without any drug treatment, only complete cell culture medium), experimental group (5mM glutamate + 5μM compound 1), control group 1 (5mM glutamate), positive control group 1 (5mM glutamate + 5μM NBP) and positive control group 2 (5mM glutamate + 20 μM Z-VAD). After the drug and cells were incubated for 24 hours, protein lysis buffer containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium alkyl sulfate, 1mM EDTA, 1mM EGTA, 150mM NaCl, 10% glycerol, 1% Triton X-100, 100mM NaF, 1mM vanadate and protease inhibitors was added to the samples, and the collected protein samples were denatured at high temperature and used as samples for Western blotting. The primary antibodies were mouse anti-GAPDH (1:3000, HC301, Desheng Biotech, China), rabbit anti-Bax (1:1000, 50599-2-Ig, Proteintech, USA), mouse anti-Bcl-2 (1:1000, 68103-1-Ig, Proteintech, USA), and anti-rabbit HRP-conjugated secondary antibodies from The Jackson Laboratory (West Grove, PA, USA).

[0082] The present invention detects the expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax.

[0083] The results showed that after treatment with compound 1, the expression of apoptosis-related protein Bcl-2 increased and the expression of Bax decreased (e.g. Fig.15 H, I and J in FIG.

[0084] In conclusion, the above results indicate that the caprylane-type diterpene compound obtained in Example 1 has a neuroprotective effect on glutamate-induced cell death via an anti-apoptotic pathway.

[0085] In summary, the present invention focuses on finding compounds with neuroprotective effects in Zechus chinensis. In the present invention, the applicant isolated a cephalosporin-type diterpene compound from the whole plant of Zechus chinensis. The absolute configuration of the compound was determined by calculating NMR DP4+ and calculating ECD. The neuroprotective effect of compound 1 obtained in Example 1 was evaluated in the glutamate-induced neuronal cell HT-22 injury model. In particular, the cephalosporin-type diterpene compound obtained in Example 1 showed significant neuroprotective activity in this model, and at a concentration of 5 μM, the cell survival rate was increased by 34.2%, which is equivalent to the positive control drug butylphthalide. Further studies have shown that the cephalosporin-type diterpene compound obtained in Example 1 can alleviate glutamate-induced neuronal cell death by inhibiting the apoptosis pathway. In short, the cephalosporin-type diterpene compound obtained in Example 1 may be a potential neuroprotective agent and is worthy of further study.

[0086] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A caprylane-type diterpene compound, characterized in that: It has the following structural formula: 。 2. The caperane-type diterpene compound according to claim 1, characterized in that: The caperane-type diterpene compound is isolated from Euphorbia cerasifera.

3. A method for preparing a caperane-type diterpene compound as claimed in claim 1 or 2, characterized in that: The following steps are involved: S1, extracting the lacquer by immersion in methanol, and concentrating by rotary evaporation under reduced pressure to obtain a crude extract; S2, diluting the crude extract with water, extracting with ethyl acetate and n-butanol in sequence to obtain an extract, and concentrating under reduced pressure to obtain a concentrate; S3, separating the concentrate by silica gel column chromatography, eluting with petroleum ether-ethyl acetate gradient, and combining to obtain fractions; S4, purifying and separating the fractions to obtain caprylane-type diterpene compounds.

4. The method for preparing the caperane-type diterpene compound according to claim 3, characterized in that: In step S1, the lacquer is crushed into 50-150 meshes, and then the crushed lacquer is extracted by methanol immersion, and concentrated by vacuum rotary evaporation to obtain a crude extract; Among them, the volume concentration of methanol is 92%~98%, and the amount of methanol used is 14 times~16 times the weight of the lacquer; the temperature of the reduced pressure rotary evaporation concentration is 55 ℃~65 ℃.

5. The method for preparing the caperane-type diterpene compound according to claim 3, characterized in that: In step S2, the amounts of ethyl acetate and n-butanol used are both 40 L to 50 L, and the temperature for concentration under reduced pressure is 35° C. to 45° C.

6. The method for preparing the caperane-type diterpene compound according to claim 3, characterized in that: In step S3, the volume ratio of petroleum ether and ethyl acetate used in gradient elution is 100:0, 90:10, 80:20, 70:30, 60:40, and 50:

50.

7. The method for preparing the caperane-type diterpene compound according to claim 3, characterized in that: In step S4, the volume ratio of acetonitrile-water used in the purification separation was adjusted from 47:53 to 70:30 in a gradient manner, with a time of 23 min and a volume flow rate of 3 mL / min.

8. Use of the caprylane-type diterpene compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug having neuroprotective activity.

9. Use of the caprylane-type diterpene compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for treating cerebral stroke.

10. Use of the caprylane-type diterpene compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for treating ischemic stroke.

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