Butylphthalide dimer and its preparation method and application

Abstract

The present invention relates to a butylphthalide dimer and a preparation method and application thereof, and the structural formula thereof is as follows: The present invention isolates butylphthalide dimer enantiomers from Lycopodiella vulgaris. 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 activity of the positive control drug 3-n-Butylphthalide. Compound (-)-2 can alleviate glutamate-induced neuronal cell death by inhibiting the apoptosis pathway.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly 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 summary, 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 remedy for rheumatism, contusions, strains, and swelling. Previous studies on the phytochemical constituents of Lycopodium truncatum have primarily focused on lycopodium alkaloids. Furthermore, these compounds have been reported to exhibit significant acetylcholinesterase inhibitory activity. Previous studies on the phytochemical constituents of Lycopodium truncatum have yielded fatty acids, enantiobistane diterpenes, and Lycopodium-type triterpenes. However, butylphthalide compounds have not yet 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 vulgaris 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 Lycopodiella family.

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

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

[0012] S2. After adjusting the pH value of the crude extract to 2-3, the crude extract was extracted with ethyl acetate to obtain an ethyl acetate fraction; the ethyl acetate fraction was 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 respectively purified by semi-preparative high performance liquid phase 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 cuneata are crushed into coarse powder of 80-120 mesh.

[0016] In one preferred embodiment, in step S1, 60-85% ethanol aqueous solution is refluxed 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-time elution in step S2 is: gradient elution with dichloromethane-methanol solution to obtain eight components AH, including component B; the volume ratios of dichloromethane and methanol in the dichloromethane-methanol solution are 1:0, 100:1, 80:1, 40:1, 20:1, 10:1, 5:1, and 0:1, respectively; each concentration gradient elution is 3 column volumes; and each column volume is 5-15 L.

[0018] In one 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 a dichloromethane-methanol solution to obtain six subfractions 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; and each column volume is 300-600 mL.

[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); the volume ratios of petroleum ether and ethyl acetate in the petroleum ether-ethyl acetate solution 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 an ethanol-water solution to obtain seven components B2-4-1 to B2-4-7, including component B2-4-2; the volume ratios of ethanol and water in the ethanol-water solution 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.

[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 protection for the use of the butylphthalide dimer or a pharmaceutically acceptable salt thereof in the preparation of 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] A third object of the present invention is to provide a pharmaceutical composition comprising at least one of the above-mentioned butylphthalide dimers or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients thereof.

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

[0029] The present invention isolated neuroprotective enantiomers of butylphthalide from Lycopodiella truncatula. The neuroprotective effects of the compounds of the present invention were evaluated in a glutamate-induced HT-22 neuronal cell injury model. In particular, (-)-(6S,7R,6'R,7'S)-lycocasuarolide B(-)-2 exhibited significant neuroprotective activity in this model, increasing cell survival by 29.3%, 25.45%, 21.96%, and 21.57% at concentrations of 5μM, 10μM, 15μM, and 20μM, respectively, comparable to the positive control drug 3-n-Butylphthalide. Further studies demonstrated that (-)-2 alleviated glutamate-induced neuronal cell death by inhibiting apoptotic 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] Figure 9 is the HRESIMS of compound (±)-1;

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

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

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

[0042] Figure 13 Compound (±)-2 1 H NMR spectrum (500 MHz, CD3OD);

[0043] Figure 14 Compound (±)-2 13 C NMR spectrum (125 MHz, CD3OD);

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

[0045] Figure 16 Compound (±)-2 1 H- 1 H COSY spectrum;

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

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

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

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

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

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

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

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

[0054] Figure 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 23 nm);

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

[0056] Figure 27 Experimental ECD spectra of (+)-2 (blue dashed line) and (-)-2 (red dashed line); calculated ECD spectra of 2a (pink solid line), 2b (red solid line), 2c (green solid line) and 2d (blue solid line);

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

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

[0059] Figure 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] Figure 31 The CCK-8 experiment shows that compound (-)-2 can alleviate glutamate-induced HT-22 cell death, where 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] Figure 32 The results of apoptosis analysis are shown in Figure 5. E shows the quantitative analysis of apoptosis; F shows the live (green) / death (red) experiment of HT-22 cells incubated with glutamate (5 mM, 24 h). Bar: 50 μm; G shows the quantitative analysis of cell survival.

[0062] Figure 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 will be 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 new embodiments.

[0064] The UV spectra of the compounds of the present invention were measured on a Shimadzu UV-2600 spectrometer (Shimadzu Corporation, Tokyo, Japan); the optical rotation of the compounds was measured on a JASCO model 1020 polarimeter (Japan Spectroscopy Corporation, Tokyo, Japan); the electronic circular dichroism spectra of the compounds were measured on a JASCO J-815 circular dichroism spectrometer; the high-resolution mass spectra of the compounds were measured on an Agilent 6520Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, California, USA); and the one-dimensional and two-dimensional NMR spectra of the compounds were measured on Bruker AV-400, AV-500, and AV-600 MHz spectrometers (Bruker, Karlsruhe, Germany). The present invention uses an Agilent 1200 liquid chromatograph with a DAD detector and a Green Baicao ODS C18 column (250 mm × 4.6 mm, 5 μm; Beijing, China) for compound analysis. An Agilent 1260 semi-preparative HPLC with a VWD detector and a Green Baicao ODS C18 column (250 mm × 10.0 mm, 5 μm) are 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 (20-45 μm, Nakale Tesko), 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 × 4.6 mm, 5 μm) 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 analyses were 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 compounds

[0069] 20.0 kg of dried aerial parts of Lycopodium truncatum were ground into a coarse powder of approximately 100 mesh. Ten times the weight of 75% ethanol was added and refluxed at 80°C for three 2-hour cycles. The extract was then concentrated under reduced pressure at 60°C to yield 1.8 kg of a crude extract. This crude extract was suspended in 7.0 L of water, adjusted to pH 2-3 with 3% tartaric acid, and extracted with 40 L of ethyl acetate to yield the ethyl acetate fraction. The ethyl acetate fraction (248.0 g) was chromatographed on a silica gel column using a gradient elution system using dichloromethane:methanol (dichloromethane:methanol volume ratios of 1:0, 100:1, 80:1, 40:1, 20:1, 10:1, 5:1, and 0:1, respectively; each concentration gradient was eluted over 3 column volumes; each column volume ranged from 5 to 15 L). Fractions A and H were obtained. Component B (20.8 g) was chromatographed on a silica gel column with a dichloromethane-methanol gradient elution (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 subfractions B1-B6. Component B2 (5.7 g) was chromatographed on a silica gel column with a gradient of petroleum ether-ethyl acetate (15:1, 8:1, 4:1 2:1, 1:1, 1:2, 1:50) and 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 with ethanol-water (5:95, 20:80, 35:65, 50:50, 65:35, 80:15, 95:5) and 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 (88 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 give compound (±)-2 (4.5 mg, t R =27.7min). The mobile phase acetonitrile: water: acetic acid volume ratio was adjusted from 30:70:0.01 to 90:10:0.01 in a gradient over 40 min at a flow rate of 3 mL / min. Finally, racemic compounds (±)-1 and 2 were separated and purified on a Daicel chiral column AD-H to obtain their enantiomers (Table 1). Liquid phase analysis of their enantiomers revealed the following results: Figure 28 、 Figure 29 shown.

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

[0071]

[0072]

[0073] Example 2

[0074] Structural analysis and identification of caperane-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 indicates that compound (±)-1 is 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 connected at C-7. In addition, the C-7 chemical shift of compound (±)-1 shifts to the downfield (δ C 63.9→66.9), which also confirms this conclusion. Analysis of NOESY data shows that Δ 8 Double bond configuration 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 ( Figure 24 , Figure 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 spectrum analysis are shown in Figure 2-Figure 8 ; HRESIMS mass spectrum see Figure 9 ; UV spectrum see Figure 10 ; Experimental ECD curve see Figure 11 and 12 , calculate the ECD curve see Figure 24 and 25 The chiral HPLC high performance liquid chromatogram of compound (+)-1 / (-)-1 is shown in Figure 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 (100 MHz) data

[0081]

[0082] Compound (±)-2 is a white powder, 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 7.8 ppm. The HMBC relationship of H-7(7') to C-7'(7) indicates that the two butylphthalide units are 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 group and the 7(6')-hydroxyl group 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 the 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. Figure 26 , Figure 27 Finally, the structures of compounds (+)-2 and (-)-2 are shown in the figure and named (+)-(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 spectrum analysis are shown in Figures 13-19 ; HRESIMS mass spectrum see Figure 20 ; UV spectrum see Figure 21 ; Experimental ECD curve see Figure 22 and 23 , calculate the ECD curve see Figure 26 and 27 The chiral HPLC chromatogram of compound (+)-2 / (-)-2 is shown in Figure 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 (150 MHz)

[0086]

[0087]

[0088] Example 3

[0089] Biological activity 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 DMEM complete medium containing 10% FBS and 1% penicillin and streptomycin.

[0092] 2 Neuroprotection Experiment

[0093] To determine the neuroprotective activity of the compound, we determined cell viability according to a 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. Specifically, HT-22 cells in good growth condition were seeded into 96-well plates at a density of 8,000 cells / 100 μL of culture medium, transferred to a cell culture incubator, and cultured overnight for 24 hours. Subsequently, 5 mM glutamate was added to the normal culture medium and treated 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 consisted of untreated cells. The glutamate toxicity group consisted of cells treated with 5 mM glutamate for 24 hours. The experimental groups were treated with 5 mM glutamate and various concentrations of the compounds for 24 hours. Cell viability was assessed using a CCK-8 assay (CK04, Dojindo Chemical Laboratories, Japan). Six replicate wells were set up in a 96-well plate, and the experiment was repeated at least three 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 ( Figure 30 A and Figure 30 B). In addition, compound (-)-2 can alleviate glutamate-induced neuronal cell death ( Figure 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 have indicated that glutamate-induced cell death is mainly apoptosis. Therefore, the present invention uses Annexin V-FITC and PI double staining to assess the cell apoptosis rate. The present invention quantifies apoptotic cells by using the Annexin V-FITC / PI kit (Jiangsu Keyi Biotechnology Co., Ltd., Nanjing, China). That is, the cells after drug incubation are collected, washed twice with pre-cooled PBS, and then centrifuged at a centrifugal force of 800g for 5 minutes. Then, the cells are incubated with a combined buffer (500uL) containing 5uL of Annexin V and 5uL of PI at room temperature for 10 minutes. The samples are analyzed by flow cytometry using a BD FACSCanto II flow cytometer (BD Bioscience Division). The average cell 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) ( Figure 31 D and Figure 31 E).

[0101] 4. Live / dead staining experiment

[0102] To more intuitively observe cell viability, we distinguished live from dead cells by observing the color of cells stained with a live / dead assay reagent (Keygen Biotech, Nanjing, China) under a fluorescence microscope. Specifically, a mixture of 2 μM calcein AM and 8 μM PI in PBS was added to the sample to be tested. The sample was incubated at 37°C for 30 minutes, and images were captured using a fluorescence microscope (Leica DM18, Germany) with a 20x objective. Data analysis was performed by counting the total number of green fluorescent cells (live cells) and red fluorescent cells (dead cells) in images collected from more than three independent replicates. Cell viability was analyzed by comparing the proportion of dead cells to 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 ( Figure 32 F and 32G).

[0104] 5. Western Blot 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.). Samples were dissolved in a buffer containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium alkyl sulfate, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 100 mM NaF, 1 mM vanadate, and protease inhibitors. Primary antibodies included mouse anti-GAPDH (1:3000, HC301, Quanshijin Biotechnology, China), rabbit anti-Bax (1:1000, 50599-2-Ig, Proteintech, USA), and mouse anti-Bcl-2 (1:1000, 68103-1-Ig, Proteintech, USA), as well as HRP-conjugated secondary antibodies against mouse and rabbit from The 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 and the expression of Bax decreased ( Figure 33 HJ).

[0108] Together, these results suggest that (-)-2 exerts neuroprotective effects against glutamate-induced cell death via an anti-apoptotic pathway.

[0109] In summary, this work focused on identifying neuroprotective enantiomers of butylphthalide from Lycopodiola truncatula. In this study, we obtained two pairs of butylphthalide dimer enantiomers [(±)-1, (±)-2] from the aerial parts of Lycopodiola truncatula. These enantiomers were further resolved by chiral separation to obtain optically pure products. The absolute configurations of these optically pure products were determined by calculating spin-spin coupling constants, NMR DP4+, and ECD. The neuroprotective effects of all compounds were evaluated in the glutamate-induced HT-22 neuronal cell injury model. In particular, (-)-(6S,7R,6'R,7'S)-lycocasuarolide B(-)-2 exhibited significant neuroprotective activity in this model. At concentrations of 5μM, 10μM, 15μM, and 20μM, cell survival rates increased by 29.3%, 25.45%, 21.96%, and 21.57%, respectively, comparable to the neuroprotective activity of the positive control drug 3-n-Butylphthalide. Further studies revealed that (-)-2 could alleviate glutamate-induced neuronal cell death by inhibiting the apoptotic pathway. In summary, compound (-)-2 may be a potential neuroprotective agent and warrants further investigation.

[0110] The above 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 replacements made by technicians in this field on the basis of the present invention 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 a butylphthalide dimer according to claim 1, wherein The following steps are involved: S1. Grind the aerial parts of Lycopodiella truncatula, and then reflux extract with 60-85% ethanol aqueous solution at 70-90° C. to obtain a crude extract; S2. After adjusting the pH value of the crude extract to 2-3, the crude extract was extracted with ethyl acetate to obtain an ethyl acetate fraction; the ethyl acetate fraction was 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 respectively purified by semi-preparative high performance liquid phase 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 refluxed 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 primary elution in step S2 is: gradient elution with a dichloromethane-methanol solution to obtain component B; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution is 1:0, 100:1, 80:1, 40:1, 20:1, 10:1, 5:1, and 0:1 respectively; Each concentration gradient elution was performed for 3 column volumes; each column volume was 5-15 L.

5. The preparation method according to claim 2, characterized in that The secondary elution in step S3 is as follows: gradient elution with a dichloromethane-methanol solution to obtain component B2; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution 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; the volume ratios of petroleum ether and ethyl acetate in the petroleum ether-ethyl acetate solution 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 an ethanol-water solution to obtain component B2-4-2; the volume ratios of ethanol and water in the ethanol-water solution 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 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 pharmaceutically acceptable excipients thereof.

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