Butylphthalide dimer as well as preparation method and application thereof

Abstract

The invention relates to a butylphthalide dimer as well as a preparation method and application thereof. The structural formula of the butylphthalide dimer is as follows: # imgabs0. According to the invention, a butylphthalide dimer enantiomer is separated from lycopodium clavatum. In a glutamic acid-induced nerve cell HT-22 injury model, the neuroprotective effect of the compound of the present invention was evaluated. Particularly, (-)-(6S, 7R, 6 'R, 7' S)-lycocasuaralide B (-)-2 shows remarkable neuroprotective activity in the model, and the survival rates of cells are respectively increased by 29.3%, 25.45%, 21.96% and 21.57% under the concentrations of 5 mu M, 10 mu M, 15 mu M and 20 mu M, which is equivalent to the activity of a positive control drug 3-n-Butylphthalide. And the compound (-)-2 can relieve nerve cell death induced by glutamic acid by inhibiting an apoptosis pathway.

Description

Technical Field

[0001] The invention relates to the field of medicine, and relates to a butylphthalide dimer and a preparation method and application thereof. Background Art

[0002] Butylphthalide is a class of complex plant-specific metabolites derived from the malonic acid pathway. Hundreds of butylphthalide monomers, dimers, and trimers have been reported from plants, fungi, mosses, and lichens. Most plant-derived butylphthalide is isolated from Chuanxiong and Angelica sinensis of the Apiaceae family. At the same time, pharmacological experiments have shown that butylphthalide has a wide range of biological activities, including antioxidant, antiplatelet aggregation, neuroprotection, inhibition of NO production, and anti-inflammatory activity. It is particularly noteworthy that in 2002, 3-n-butylbutylphthalide (NBP) was approved by the China Food and Drug Administration as a drug for the treatment of ischemic stroke. The structural formula of NBP is

[0003] In addition, Z-ligustilide isolated from Chuanxiong showed multiple pharmacological activities, including neuroprotection, anti-inflammatory, antioxidant and anticancer effects. In conclusion, butylphthalide has become a research hotspot due to its complex chemical structure and significant pharmacological activities.

[0004] Lycopodium truncatum (Lycopodiaceae) is widely distributed in tropical and subtropical Asia. In China, Lycopodium truncatum, also known as "Shujincao", is used as a folk herbal medicine to treat rheumatism, contusions, strains and swelling. Previous studies on the phytochemical components of Lycopodium truncatum were mostly on Lycopodium alkaloids. In addition, these compounds were reported to have significant acetylcholinesterase inhibitory activity. In the past, fatty acids, enantioboretic acid diterpenes and Lycopodium-type triterpenes were obtained in the process of studying the phytochemical components of Lycopodium truncatum. However, butylphthalide substances have not been isolated from Lycopodium truncatum. Summary of the invention

[0005] The purpose of the present invention is to provide a butylphthalide dimer with a new structure separated from Lycopodiella truncatula and a preparation method and application thereof.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] Butylphthalide dimer, its structural formula is as follows:

[0008]

[0009] The present invention separates new butylphthalide dimers (+)-1, (-)-1, (+)-2 and (-)-2 from Lycopodiella truncatula for the first time, and this is the first time that these butylphthalide compounds are found in the Lycopodiaceae family.

[0010] Based on the same invention, the present invention also protects the preparation method of the butylphthalide dimer, which comprises the following steps:

[0011] S1. The aerial part of Lycopodiella truncatula is crushed, and then extracted with 60-85% ethanol aqueous solution at reflux at 70-90° C. to obtain a crude extract;

[0012] S2, after adjusting the pH value of the crude extract to 2-3, extract with ethyl acetate to obtain the ethyl acetate fraction; the ethyl acetate fraction is subjected to silica gel column chromatography and eluted once to obtain component B;

[0013] S3, component B is subjected to silica gel column chromatography and eluted twice to obtain component B2; component B2 is subjected to silica gel column chromatography and eluted three times to obtain B2-4, and B2-4 is further subjected to polyamide column chromatography and eluted four times to obtain B2-4-5 and B2-4-6;

[0014] S4, components B2-4-5 and B2-4-6 were purified by semi-preparative HPLC to obtain compound (±)-1 and compound (±)-2; compound (±)-1 and compound (±)-2 were purified by chiral column to obtain compound (+)-1, compound (-)-1, compound (+)-2 and compound (-)-2.

[0015] In one preferred embodiment, the aerial parts of Lycopodiella cernua are crushed into coarse powder of 80-120 mesh.

[0016] In one preferred embodiment, in step S1, 60-85% ethanol aqueous solution is used for reflux extraction at 70-90° C. for 3-4 times, each time for 2-3 hours, to obtain a crude extract.

[0017] In one preferred embodiment, the one elution in step S2 is: gradient elution with dichloromethane-methanol solution to obtain eight components AH, including component B; in the dichloromethane-methanol solution, the volume ratio of dichloromethane to methanol is 1:0, 100:1, 80:1, 40:1, 20:1, 10:1, 5:1, 0:1, respectively; each concentration gradient elution is 3 column volumes; each column volume is 5-15L.

[0018] In a preferred embodiment, in step S2, 3% tartaric acid is used to adjust the pH value to 2-3.

[0019] In one preferred embodiment, the secondary elution in step S3 is: gradient elution with dichloromethane-methanol solution to obtain six subcomponents B1-B6, including component B2; in the dichloromethane-methanol solution, the volume ratio of dichloromethane to methanol is 100:1, 80:1, 70:1, 60:1, 50:1, and 40:1, respectively; each concentration gradient elution is 3 column volumes; each column volume is 300-600mL.

[0020] In one preferred embodiment, the three elutions in step S3 are: gradient elution with petroleum ether-ethyl acetate solution to obtain B2-4 (1.0 g); in the petroleum ether-ethyl acetate solution, the volume ratios of petroleum ether and ethyl acetate are 15:1, 8:1, 4:1, 2:1, 1:1, 1:2, and 1:50, respectively; each concentration gradient elution is 3 column volumes; and each column volume is 100-300 mL.

[0021] In one preferred embodiment, the four elutions in step S3 are: elution with ethanol-water solution to obtain seven components B2-4-1 to B2-4-7, including component B2-4-2; in the ethanol-water solution, the volume ratios of ethanol and water are 5:95, 20:80, 35:65, 50:50, 65:35, 80:15, and 95:5, respectively; each concentration gradient elution is 3 column volumes; each column volume is 50-100 mL.

[0022] In one preferred embodiment, in step S4, the mobile phase of the semi-preparative HPLC is acetonitrile-water-acetic acid solution, and the volume ratio of acetonitrile, water and acetic acid in the acetonitrile-water-acetic acid solution is gradually adjusted from 30:70:0.01 to 90:10:0.01, the time is 40-50 min, and the volume flow rate is 2-3 mL / min.

[0023] The third object of the present invention is to provide the use of the butylphthalide dimer or a pharmaceutically acceptable salt thereof in the preparation of a drug for protecting nerve cells.

[0024] The present invention also claims to protect the use of the butylphthalide dimer or a pharmaceutically acceptable salt thereof in preparing a drug for treating cerebral stroke.

[0025] The present invention also claims to protect the use of the butylphthalide dimer or a pharmaceutically acceptable salt thereof in preparing a drug for treating ischemic stroke.

[0026] In a preferred embodiment, the preparation is one or more of a tablet, a soft capsule, a hard capsule, a solution, and a suspension.

[0027] The third object of the present invention is to provide a pharmaceutical composition comprising at least one of the above-mentioned butylphthalide dimer or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient thereof.

[0028] The beneficial effects of the present invention are:

[0029] The present invention separates neuroprotective enantiomers of butylphthalide from Lycopodiella truncatula. The neuroprotective effect of the compound of the present invention is evaluated in a glutamate-induced neuronal cell HT-22 injury model. In particular, (-)-(6S,7R,6'R,7'S)-lycocasuarolide B(-)-2 exhibits significant neuroprotective activity in the model. At concentrations of 5μM, 10μM, 15μM, and 20μM, the cell survival rates are increased by 29.3%, 25.45%, 21.96%, and 21.57%, respectively, which is equivalent to the positive control drug 3-n-Butylphthalide. Further studies show that (-)-2 can alleviate glutamate-induced neuronal cell death by inhibiting apoptosis pathways. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the structural formula of the enantiomers of compound (±)-1 and (±)-2;

[0031] Figure 2 is compound (±)-1 1 H NMR spectrum;

[0032] Figure 3 is compound (±)-1 13 C NMR spectrum (100 MHz, CD3OD);

[0033] Figure 4 DEPT135 spectrum (100 MHz, CD3OD) of compound (±)-1;

[0034] Figure 5 is compound (±)-1 1 H- 1 H COSY spectrum;

[0035] Figure 6 is the HSQC spectrum of compound (±)-1;

[0036] Figure 7 is the HMBC spectrum of compound (±)-1;

[0037] Figure 8 is the NOESY spectrum of compound (±)-1;

[0038] Fig. 9 is the HRESIMS of compound (±)-1;

[0039] Fig.10 is the UV spectrum of compound (±)-1;

[0040] Fig.11 is the experimental ECD spectrum (MeOH) of compound (+)-1;

[0041] Fig.12 is the experimental ECD spectrum (MeOH) of compound (-)-1;

[0042] Fig.13 For compound (±)-2 1 H NMR spectrum (500 MHz, CD3OD);

[0043] Fig.14 For compound (±)-2 13 C NMR spectrum (125 MHz, CD3OD);

[0044] Fig.15 DEPT135 spectrum (125 MHz, CD3OD) of compound (±)-2;

[0045] Fig.16 For compound (±)-2 1 H- 1 H COSY spectrum;

[0046] Fig.17 is the HSQC spectrum of compound (±)-2;

[0047] Fig.18 is the HMBC spectrum of compound (±)-2;

[0048] Fig.19 is the NOESY spectrum of compound (±)-2;

[0049] Fig. 20 is the HRESIMS spectrum of compound (±)-2;

[0050] Fig.21 is the UV spectrum (MeOH) of compound (±)-2;

[0051] Fig. 22 is the experimental ECD spectrum (MeOH) of compound (+)-2;

[0052] Fig.23 is the experimental ECD spectrum (MeOH) of compound (-)-2;

[0053] Fig.24 There are two possible isomers of compound (±)-1;

[0054] Fig.25 Experimental ECD spectra of (+)-1 (red dashed line) and (-)-1 (blue dashed line); calculated ECD spectra of 1a (blue solid line) and 1b (red solid line) (blue shift 23nm);

[0055] Fig.26 There are four possible isomers of compound (±)-2;

[0056] Fig. 27 The experimental ECD spectra of (+)-2 (blue dashed line) and (-)-2 (red dashed line); the calculated ECD spectra of 2a (pink solid line), 2b (red solid line), 2c (green solid line) and 2d (blue solid line);

[0057] Fig.28 It is the chiral HPLC high performance liquid chromatogram of compound (+)-1 / (-)-1;

[0058] Fig.29 It is the chiral HPLC high performance liquid chromatogram of compound (+)-2 / (-)-2;

[0059] Fig.30 Compound (-)-2 alleviates glutamate-induced cell death, wherein A is compound (-)-2 alleviates glutamate-induced cell death; B is CCK-8 experiment showing that glutamate-induced HT-22 cell death is dose-dependent; C is CCK-8 experiment showing that 5 mM glutamate treatment-induced HT-22 cell death is time-dependent;

[0060] Fig.31 The CCK-8 experiment shows that compound (-)-2 can alleviate glutamate-induced HT-22 cell death, wherein D is the CCK-8 experiment showing that compound (-)-2 can alleviate glutamate-induced HT-22 cell death; E is the apoptotic cells determined by Annexin V-FITC / PI staining;

[0061] Fig.32 The results of apoptosis analysis; E is the quantitative analysis of apoptosis; F is the live (green) / death (red) experiment of HT-22 cells incubated with glutamate (5mM, 24h). Size bar, 50μm; G is the quantitative analysis of cell survival rate;

[0062] Fig.33 is the expression level of apoptosis regulatory proteins of the compound; H is the expression level of apoptosis regulatory proteins measured by Western blot experiment; I is the quantitative analysis of Bcl-2 concentration; J is the quantitative analysis of Bax concentration; DETAILED DESCRIPTION

[0063] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0064] The ultraviolet spectrum of the compound of the present invention is tested on a Shimadzu UV-2600 spectrometer (Shimadzu Corporation, Tokyo, Japan); the optical rotation of the compound is tested on a JASCO model 1020 polarimeter (Japan Spectrophotometer, Tokyo, Japan); the electronic circular dichroism spectrum of the compound is tested on a JASCO J-815 circular dichroism spectrometer; the high-resolution mass spectrum of the compound is tested on an Agilent 6520Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, California, the United States); and the one-dimensional and two-dimensional NMR spectra of the compound are tested on a Bruker AV-400, AV-500, AV-600 MHz spectrometer (Bruker, Karlsruhe, Germany). When the present invention performs high performance liquid chromatography analysis of compounds, an Agilent 1200 liquid chromatograph with a DAD detector is used, and a Green Baicao ODS C18 chromatographic column (250mm×4.6mm, 5μm; Beijing, China) is used; an Agilent 1260 semi-preparative high performance liquid phase with a VWD detector is used, and a Green Baicao ODS C18 (250mm×10.0mm, 5μm) chromatographic column is used for compound separation. Silica gel (200-300, 300-400 mesh, Qingdao Ocean Chemical Co., Ltd., Qingdao, China), dextran LH-20 (GE Healthcare, Sweden), ODS silica gel (20-45μm, Nakale Teske) and polyamide (80-100 mesh, Shanghai, China) are used for open column chromatography separation. The present invention uses an Agilent 1260 liquid chromatograph with a Daicel chiral column AD-H (250×4.6mm, 5μm) for normal phase separation. All reagents were of analytical grade.

[0065] Statistical analysis of all data:

[0066] Data were obtained from at least three independent experiments (mean ± SD). One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test was used to compare three or more groups. Statistical analysis was performed using GraphPad Prism software (version 4.00) (GraphPad Software Inc., San Francisco, CA, USA).

[0067] Example 1

[0068] Extraction and separation of butylphthalide dimer

[0069] 20.0 kg of the dried aerial part of Lycopodiella serrata was taken and crushed into a coarse powder of about 100 meshes, and 75% ethanol with a volume concentration of 10 times its weight was added. The extract was refluxed and extracted 3 times at 80°C for 2 hours each time. The extract was concentrated under reduced pressure at 60°C to obtain 1.8 kg of crude extract. The crude extract was suspended in water (7.0 L), and 3% tartaric acid was added to adjust the pH value to 2-3, and then extracted with 40 L of ethyl acetate to obtain the ethyl acetate part. The ethyl acetate part (248.0 g) was subjected to silica gel column chromatography and gradient eluted with dichloromethane:methanol (in the dichloromethane-methanol solution, the volume ratio of dichloromethane to methanol was 1:0, 100:1, 80:1, 40:1, 20:1, 10:1, 5:1, 0:1 respectively; each concentration gradient elution was 3 column volumes; each column volume was 5-15 L) to obtain eight components AH. Component B (20.8 g) was chromatographed on a silica gel column with a gradient elution of dichloromethane-methanol (100:1, 80:1, 70:1, 60:1, 50:1, 40:1) with 3 column volumes per gradient (1 column volume is 450 ml) to obtain six subcomponents B1-B6. Component B2 (5.7 g) was chromatographed on a silica gel column and eluted with a petroleum ether-ethyl acetate gradient (15:1, 8:1, 4:1 2:1, 1:1, 1:2, 1:50) with 3 column volumes for each gradient (1 column volume is 200 ml) to obtain 7 components B2-1-B2-7, of which component B2-4 (1.0 g) was further chromatographed on a polyamide column and eluted with ethanol-water (5:95, 20:80, 35:65, 50:50, 65:35, 80:15, 95:5) with 3 column volumes for each gradient (1 column volume is 80 ml) to obtain seven components B2-4-1 to B2-4-7. Component B2-4-5 (127 mg) was further purified by semi-preparative HPLC (mobile phase acetonitrile-water-acetic acid, 30:70:0.01-90:10:0.01, 40 min) to obtain compound (±)-1 (4.0 mg, t R =30.5min). Component B2-4-6 (88mg) was further subjected to semi-preparative HPLC (mobile phase acetonitrile-water-acetic acid, 30:70:0.01-90:10:0.01, 40min) to obtain compound (±)-2 (4.5mg, t R =27.7min). The volume ratio of the mobile phase acetonitrile: water: acetic acid was adjusted from 30:70:0.01 to 90:10:0.01 in a gradient manner, with a time of 40 min and a volume flow rate of 3 mL / min. Finally, the racemic compounds (±)-1 and 2 were separated and purified by a Daicel chiral column AD-H to obtain their enantiomers (Table 1). The liquid phase analysis of their enantiomers was as follows Fig.28 , Fig.29 shown.

[0070] Table 1 Chiral separation of racemic compounds (±)-1, 2

[0071]

[0072]

[0073] Example 2

[0074] Structural analysis and identification of caprylane-type diterpenoid compounds:

[0075] The butylphthalide dimer compounds (±)-1, 2 and their enantiomers obtained in Example 1 of the present invention.

[0076] Compound (±)-1 is a white powder, easily soluble in methanol; UV(MeOH)λ max :(logε)275(2.63)nm; HRESIMS m / z 435.1789[M+Na] + (calcd for C 24 H 28 O6Na,435.1784).Compound (+)-1:[α] 20 D +6.4(c 0.10,MeOH); ECD(MeOH,mdeg)λ max :206(+4.71),240(-2.64),266(-1.63)nm.Compound (-)-1:[α] 20 D –6.8(c 0.10,MeOH); ECD(MeOH,mdeg)λ max :204(-4.54),238(+3.30),271(+1.70)nm.

[0077] High resolution mass spectrometry data: ESI-HRMS m / z: 435.1789 [M+Na] + (C 24 H 28 O6 calculated value 435.1784), the molecular formula is estimated to be C 24 H 28 O6. Compound (±)-1 1 H NMR was assigned to the four o-phenyl protons [δ H 7.48(1H,dd,J=7.6,1.0Hz,H-4'), 7.67(1H,td,J=7.6,1.2Hz,H-5'), 7.58(1H,td,J=7.6,1.2Hz,H-6'), and δ H 7.92 (1H, dd, J = 7.6, 1.0 Hz, H-7')]; two methyl groups [δH 1.00 (3H, t, J = 7.4 Hz, CH3-11) and 0.93 (3H, t, J = 7.3 Hz, CH3-11')]; one olefin proton [δ H 5.61 (1H, t, J = 7.9 Hz, H-8)] and two oxygen-containing methines [δ H 4.21 (1H, ddd, J = 6.1, 3.7, 2.6 Hz, H-6) and 5.74 (1H, d, J = 3.7 Hz, H-7)]. 13 C NMR and DEPT spectra showed the presence of 24 carbon signals, including 6 aromatic carbons (δ C 126.3, 128.0, 129.8, 129.8, 132.3, and 142.7); three carbonyl carbons [one keto carbonyl (δ C 206.4) and two ester carbonyl groups (δ C 165.8 and 168.2)], two methyl groups (δ C 12.7 and 12.9), seven methylene groups (δ C 16.9, 21.8, 21.8, 24.8, 27.8, 28.5 and 42.0), three methines [one olefin (δ C 114.6)] and two oxygen-containing methines (δ C 66.4 and 66.9), three quaternary carbons (δ C 121.3, 148.3 and 156.4). The complete analysis of the one-dimensional and two-dimensional NMR spectra of compound (±)-1 indicated that compound (±)-1 was a butylphthalide dimer composed of (unit A, blue) and 2-(1-oxopentyl)-benzoic acid (unit B, red). In the HMBC spectrum, H-7 (δ H 5.74) to C-1'(δ C 165.8) and H-7'(δ H 7.92) to C-1', indicating that unit B is attached at the C-7 position. In addition, the C-7 chemical shift of compound (±)-1 is shifted downfield (δ C 63.9→66.9), which also confirms this conclusion. Analysis of NOESY data shows that Δ 8 The double bond configuration is Z-form. Coupling constant J 6,7 (3.7 Hz) indicated that H-6 and H-7 were cis-configured. Subsequently, compound (±)-1 was chirally resolved into two enantiomers (+)-1 ([α] 20D +6.4,t R =34.5min) and (-)-1([α] 20 D -6.8,t R =30.1min). In the ECD spectrum, the theoretically calculated ECD spectrum of (6R,7S)-(+)-1 and (6S,7R)-(-)-1 is consistent with the experimental ECD spectrum ( Fig.24 , Fig.25 ). Therefore, the (+)-1 and (-)-1 structures are shown in the figure and named as (+)-(6R,7S)-lycocasuarolide A and (-)-(6S,7R)-lycocasuarolide A, respectively.

[0078] The structural formula of compound (±)-1 is as follows Figure 1 ; 1 H-NMR, 13 C-NMR, DEPT 135, 1 H- 1 H COSY, NOESY, HSQC, HMBC and NOESY spectra are analyzed in Figure 2-Figure 8 ; HRESIMS mass spectrum see Fig. 9 ; UV spectrum see Fig.10 ; Experimental ECD curve see Fig.11 and 12 , calculate the ECD curve see Fig.24 and 25 The chiral HPLC chromatogram of compound (+)-1 / (-)-1 is shown in Fig.28 ; The above spectrum confirmed the structure of the compound.

[0079] Compound (±)-1 1 H-NMR and 13 The C-NMR data are summarized in Table 2.

[0080] Table 2 Compound (±)-1 1 H NMR (400 MHz) and 13 C NMR (100MHz) data

[0081]

[0082] Compound (±)-2, which is a white powder, is easily soluble in methanol; UV(MeOH)λ max :(logε)276(2.13)nm; HRESIMS m / z 453.1897[M+Na] + (calcd for C 24 H30 O7Na,453.1889).Compound (+)-5:[α] 20 D +4.5(c 0.05,MeOH); ECD(MeOH,mdeg)λ max :212(-3.30),235(+2.69),260(-0.46),285(+1.21).Compound (-)-5:[α] 20 D –9.0(c 0.10,MeOH); ECD(MeOH,mdeg)λ max :203(+8.60),234(-1.63),260(+4.27),286(-7.92). High resolution mass spectrometry data:ESI-HRMS m / z:453.1897[M+Na] + (C 24 H 28 O6 calculated value 453.1889), the molecular formula is estimated to be C 24 H 30 O7. Compound (±)-2 1 H NMR suggests a methyl proton δ H 0.96 (3H, t, J = 7.3 Hz, CH3-11 (11')), one olefin proton δ H 5.48 (1H, t, J = 7.9 Hz, H-8 (8')), two oxygen-containing methine protons δ H 4.40 (1H, m, H-6 (6')) and 4.28 (1H, d, J = 2.3 Hz, H-7 (7')). 13 C NMR showed 12 different carbon signals, including an ester carbon group (δ C 169.4, C-1(1')), one methyl group (δ C 12.7, C-11(11')), four methylene groups (δ C 16.3, 21.9, 23.5, and 27.7), three methines [one olefin (δ C 113.7, C-8 (8')) and two oxygen-containing carbons (δ C 66.4 and 71.7)], three quaternary carbon centers (δ C123.3, 148.6 and 154.8), indicating that the compound is a 6,7-dihydroxybutylphthalide derivative. 1D NMR and HRESIMS of compound (±)-2 revealed that compound (±)-2 is a homologous dimer structure. In addition, the chemical shift of C-7 of compound (±)-2 shifted to the high field by 7.8ppm. The HMBC relationship of H-7(7') to C-7'(7) indicated that the two butylphthalide units were connected by C-7-OC-7'. H-4(4')(δ H 2.51) and H-8(8')(δ H 5.48) shows that Δ 8 The double bond is Z-type. In addition, J 6,7 (2.3 Hz) showed that the 6(6')-hydroxyl and 7(6')-hydroxyl groups were on the same side. Interestingly, the optical rotation and Cotton effect of compound (±)-2 were close to 0, indicating that compound (±)-2 was a racemic mixture. Subsequently, based on the comparison of experimental and calculated ECD curves, the absolute configurations of (+)-2 and (-)-2 were determined to be 6R, 7S, 6'S, 7'R and 6S, 7R, 6'R, 7'S, respectively. Fig.26 , Fig. 27 Finally, the structures of compounds (+)-2 and (-)-2 are shown in the figure and named as (+)-(6R,7S,6'S,7'R)-lycocasuarolide B and (-)-(6S,7R,6'R,7'S)-lycocasuarolide B, respectively.

[0083] The structural formula of compound (±)-2 is as follows Figure 1 ; 1 H-NMR, 13 C-NMR, DEPT 135, 1 H- 1 H COSY, NOESY, HSQC, HMBC and NOESY spectra are analyzed in Figure 13-Figure 19 ; HRESIMS mass spectrum see Fig. 20 ; UV spectrum see Fig.21 ; Experimental ECD curve see Fig. 22 and 23 , calculate the ECD curve see Fig.26 and 27 The chiral HPLC chromatogram of compound (+)-2 / (-)-2 is shown in Fig.29 ; The above spectrum confirmed the structure of the compound.

[0084] Compound (±)-2 1 H-NMR and 13 The C-NMR data are summarized in Table 3.

[0085] Table 3 Compound (±)-2 1 H NMR (600 MHz) and 13 C NMR (150MHz)

[0086]

[0087]

[0088] Example 3

[0089] Bioactivity test tools

[0090] 1. Cell incubation and main reagents

[0091] Mouse hippocampal neural cells HT-22 were purchased from Wuhan Pronocell Life Science Co., Ltd., China. The cells were cultured overnight in a cell culture incubator (Thermo, HERAcell 240i) at 37°C and 5% CO2 in a complete DMEM medium containing 10% FBS and 1% penicillin and streptomycin.

[0092] 2 Neuroprotection Experiment

[0093] To determine the neuroprotective activity of the compounds, we determined the cell survival rate according to the previous method (Xu X, Chua CC, Kong J, Kostrzewa RM, Kumaraguru U, Hamdy RC, Chua BH. Necrostatin-1 protects against glutamate-induced glutathione depletion and caspase-independent cell death in HT-22 cells. Journal of neurochemistry. 2007 Dec; 103 (5): 2004-14). A glutamate-induced toxicity model was used to simulate brain damage after stroke. That is, HT-22 cells with good growth status were seeded into 96-well plates at 8000 cells / 100 μL culture medium, transferred to a cell culture incubator and cultured overnight for 24 hours, and then 5 mM glutamate was added to the normal culture medium to treat the normal cells for 24 hours to construct a glutamate-induced toxicity model. In the later experimental process, we randomly divided the cultured cells into a blank control group, a glutamate toxicity group, and an experimental group according to the requirements of the model construction. The blank control group was composed of cells without any treatment, the glutamate toxicity group was treated with 5mM glutamate for 24h, and the experimental group was treated with 5mM glutamate and various concentrations of compounds for 24h. The cell viability was detected by CCK-8 test (CK04, Dojin Chemical Research Institute, Japan). During the test, 6 replicate wells were set in the 96-well plate, and the experiment was repeated more than 3 times.

[0094] CCK-8 results showed that incubation of HT-22 cells with glutamate at a moderate lethal dose (5 mM) resulted in a decrease in cell viability over time ( Fig.30 A and Fig.30 B). In addition, compound (-)-2 can alleviate glutamate-induced neuronal cell death ( Fig.30 C and Table 4).

[0095] Table 4 Protective effect of compounds on HT22 cells induced by glutamate

[0096]

[0097]

[0098] 3. Apoptosis

[0099] Previous studies indicate that glutamate-induced cell death is mainly apoptosis. Therefore, the present invention uses Annexin V-FITC and PI double staining to evaluate the apoptosis rate. The present invention quantifies apoptotic cells by Annexin V-FITC / PI kit (Jiangsu Keygen Biotechnology Co., Ltd., Nanjing, China). That is, the cells after drug incubation are collected, washed twice with pre-cooled PBS, and centrifuged for 5 minutes under a centrifugal force of 800g. Then, the cells are co-incubated with a combined buffer (500uL) containing 5uL of Annexin V and 5uL of PI at room temperature for 10min. The sample is analyzed by flow cytometry using a BD FACSCanto II flow cytometer (BD Bioscience Division). The average apoptosis rate is obtained by calculating the results of three independent cell experiments.

[0100] The results showed that (-)-2 showed comparable anti-apoptotic effects to Z-VAD-FMK and NBP (positive control) ( Fig.31 D and Fig.31 E).

[0101] 4 Live / dead staining experiment

[0102] In order to observe the survival of cells more intuitively, we distinguished live and dead cells by observing the color of cells stained with live / dead test reagent (Keygen Biotech, Nanjing, China) under a fluorescence microscope. The specific steps are to add PBS reagent containing 2uM calcein AM reagent and 8uM PI reagent to the sample to be tested, incubate with the sample at 37°C for 30 minutes, and then use a fluorescence microscope (Leica DM18, Germany) to collect pictures under a 20x objective. In the data analysis, we counted the total number of green fluorescent cells (live cells) and red fluorescent cells (dead cells) in the pictures collected from more than 3 independent repeated experiments, and analyzed the survival of cells by comparing the proportion of dead cells in the total number of cells in different treatment groups.

[0103] The results showed that (-)-2 could enhance the cell survival rate of HT-22 cells treated with glutamate without damaging normal cells ( Fig.32 F and 32G).

[0104] 5. Western Blot Experiment Analysis

[0105] The Western Blotting experimental method was as described previously (references Liu L, Liu KJ, Cao JB, et al. A Novel Netrin-1-Derived Peptide Enhances Protection against Neuronal Death and Mitigates of Intracerebral Hemorrhage in Mice[J]. International journal of molecular sciences, 2021, 22(9): 4829 and Hu JW, Wang Q, Liu L, Hu YM, XieM, Zheng DK, Xie Z, Liu Y. Abietane diterpenoids from Phlegmariurus carinatus and their biological activities. Phytochemistry. 2022 Sep 28; 204: 113457.). The samples were dissolved in a 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. The primary antibodies were mouse anti-GAPDH (1:3000, HC301, Quanshijin Biotechnology, China), rabbit anti-Bax (1:1000, 50599-2-Ig, Proteintech, USA), mouse anti-Bcl-2 (1:1000, 68103-1-Ig, Proteintech, USA) and anti-mouse and rabbit HRP-conjugated secondary antibodies from Jackson Laboratory, USA.

[0106] The present invention detects the expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax.

[0107] The results showed that after treatment with (-)-2, the expression of apoptosis-related protein Bcl-2 increased, while the expression of Bax decreased ( Fig.33 HJ).

[0108] Taken together, these results suggest that (-)-2 has a neuroprotective effect against glutamate-induced cell death via an anti-apoptotic pathway.

[0109] In summary, this work focuses on finding neuroprotective enantiomers of butylphthalide in Lycopodiola. In the present invention, we obtained two pairs of butylphthalide dimer enantiomers [(±)-1, (±)-2)] from the aerial part of Lycopodiola. These enantiomers were further separated by chirality to obtain optically pure products. The absolute configuration of these optically pure products was determined by calculating the spin-spin coupling constant, calculating NMR DP4+ and calculating ECD. The neuroprotective effects of all compounds were evaluated in the glutamate-induced neuronal cell HT-22 injury model. In particular, (-)-(6S,7R,6'R,7'S)-lycocasuarolide B(-)-2 showed significant neuroprotective activity in this model. At concentrations of 5μM, 10μM, 15μM, and 20μM, the cell survival rates increased by 29.3%, 25.45%, 21.96%, and 21.57%, respectively, which is equivalent to the neuroprotective activity of the positive control drug 3-n-Butylphthalide. Further studies have shown that (-)-2 can alleviate glutamate-induced neuronal cell death by inhibiting the apoptotic pathway. In short, compound (-)-2 may be a potential neuroprotective agent and deserves further study.

[0110] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. Butylphthalide dimer, characterized in that Its structural formula is as follows:

2. The method for preparing butylphthalide dimer according to claim 1, characterized in that: The following steps are involved: S1. The aerial part of Lycopodiella truncatula is crushed, and then extracted with 60-85% ethanol aqueous solution at reflux at 70-90° C. to obtain a crude extract; S2, after adjusting the pH value of the crude extract to 2-3, extract with ethyl acetate to obtain the ethyl acetate fraction; the ethyl acetate fraction is subjected to silica gel column chromatography and eluted once to obtain component B; S3, component B is subjected to silica gel column chromatography and eluted twice to obtain component B2; component B2 is subjected to silica gel column chromatography and eluted three times to obtain B2-4, and B2-4 is further subjected to polyamide column chromatography and eluted four times to obtain B2-4-5 and B2-4-6; S4, components B2-4-5 and B2-4-6 were purified by semi-preparative HPLC to obtain compound (±)-1 and compound (±)-2; compound (±)-1 and compound (±)-2 were purified by chiral column to obtain compound (+)-1, compound (-)-1, compound (+)-2 and compound (-)-2.

3. The preparation method according to claim 2, characterized in that: In the step S1, 60-85% ethanol aqueous solution is used for reflux extraction at 70-90° C. for 3-4 times, each time for 2-3 hours, to obtain a crude extract.

4. The preparation method according to claim 2, characterized in that: The first elution in step S2 is: gradient elution with dichloromethane-methanol solution to obtain component B; in the dichloromethane-methanol solution, the volume ratio of dichloromethane to methanol is 1:0, 100:1, 80:1, 40:1, 20:1, 10:1, 5:1, 0:1 respectively; Each concentration gradient elution was 3 column volumes; each column volume was 5-15L.

5. The preparation method according to claim 2, characterized in that: The secondary elution in step S3 is as follows: gradient elution with dichloromethane-methanol solution to obtain component B2; in the dichloromethane-methanol solution, the volume ratio of dichloromethane to methanol is 100:1, 80:1, 70:1, 60:1, 50:1, and 40:1, respectively; each concentration gradient elution is 3 column volumes; and each column volume is 300-600 mL.

6. The preparation method according to claim 2, characterized in that: The three elutions in step S3 are as follows: gradient elution with petroleum ether-ethyl acetate solution to obtain B2-4; in the petroleum ether-ethyl acetate solution, the volume ratios of petroleum ether and ethyl acetate are 15:1, 8:1, 4:1, 2:1, 1:1, 1:2, and 1:50, respectively; each concentration gradient elution is 3 column volumes; and each column volume is 100-300 mL.

7. The preparation method according to any one of claims 2 to 6, characterized in that: The four elutions in step S3 are: elution with ethanol-water solution to obtain component B2-4-2; in the ethanol-water solution, the volume ratios of ethanol and water are 5:95, 20:80, 35:65, 50:50, 65:35, 80:15, and 95:5, respectively; each concentration gradient elution is 3 column volumes; and each column volume is 50-100 mL.

8. Use of the butylphthalide dimer or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for protecting nerve cells.

9. Use of the butylphthalide dimer or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for treating cerebral stroke.

10. A pharmaceutical composition, characterized in that The invention comprises at least one butylphthalide dimer or a pharmaceutically acceptable salt thereof as claimed in claim 1, and a pharmaceutically acceptable auxiliary material thereof.

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