Novel bisbenzylisoquinoline alkaloid compound as well as preparation method and application thereof

Abstract

According to the invention, two bisbenzylisoquinoline alkaloid compounds with novel structures are obtained through separation, and further research finds that the two bisbenzylisoquinoline alkaloid compounds have the effects of resisting nerve injury, inhibiting acetylcholin esterase and inhibiting butyrylcholin esterase; therefore, the two bisbenzylisoquinoline alkaloid compounds have the prospect of being developed into medicines for inhibiting acetylcholin esterase and butyrylcholin esterase and medicines for resisting nerve injury.

Description

Technical field:

[0001] The present invention relates to alkaloid compounds, in particular to a bisbenzylisoquinoline alkaloid compound and a preparation method and application thereof. Background technology:

[0002] The applicant discovered and isolated two new bisbenzylisoquinoline alkaloid compounds from lotus seed core and conducted pharmacological activity studies. Summary of the invention:

[0003] The present invention provides a bisbenzylisoquinoline alkaloid compound, a preparation method and an application thereof.

[0004] A bisbenzylisoquinoline alkaloid compound, the chemical structure of which is one of the following structural formulas:

[0005]

[0006] A method for preparing the above-mentioned bisbenzylisoquinoline alkaloid compound of formula I comprises the following steps:

[0007] The total alkaloid fraction is obtained by concentrating the ethanol extract or methanol extract of the lotus seed core to obtain an extract, preparing the extract into an aqueous suspension, adjusting the pH value of the aqueous suspension to 2-3 to obtain an acidic mixed solution; then, extracting the acidic mixed solution with ethyl acetate; after extraction, adjusting the pH value of the remaining aqueous solution to 9-10 to obtain an alkaline solution; then, extracting the alkaline solution with dichloromethane to obtain the total alkaloid fraction;

[0008] Separation of the total alkaloid fraction: The total alkaloid fraction was separated using HP-20 macroporous resin using 80% CH3OH-H2O as the eluent. The eluted fraction was further separated by column chromatography on a silica gel column using dichloromethane-methanol as the eluent. The separated fractions were combined by thin-layer chromatography to obtain six fractions: Fraction A, Fraction B, Fraction C, Fraction D, Fraction E, and Fraction F.

[0009] Isolation and purification of the bis-benzylisoquinoline alkaloid compound of formula I: Fraction D was eluted by ODS C18 reverse-phase column chromatography using 100% CH3OH-H2O as the eluent, and the obtained fraction was evaporated to dryness. It was then separated and purified by semi-preparative liquid chromatography (C18 reverse-phase column) using MeCN-H2O or CH3OH-H2O as the mobile phase to obtain the bis-benzylisoquinoline alkaloid compound of formula I.

[0010] Preferably, the step of obtaining the total alkaloid fraction comprises concentrating an 80% ethanol extract of lotus seed core to obtain an extract, preparing the extract into an aqueous suspension, adjusting the pH value of the aqueous suspension to 2-3 using a 2% sulfuric acid aqueous solution to obtain an acidic mixed solution; then, extracting the acidic mixed solution twice with ethyl acetate; after extraction, retaining the aqueous solution, and adjusting the pH value of the aqueous solution to 9-10 using ammonia water to obtain an alkaline solution; then, extracting the alkaline solution three times with dichloromethane to obtain the total alkaloid fraction;

[0011] Preferably, in the separation step of the total alkaloid portion, the total alkaloid portion is separated by HP-20 macroporous resin using eluents of 30% CH3OH-H2O, 50% CH3OH-H2O, 80% CH3OH-H2O, and 100% CH3OH-H2O; the portion obtained by the 80% CH3OH-H2O eluent is further subjected to column chromatography on a silica gel column with 200-300 mesh silica gel, using dichloromethane-methanol with volume ratios of 20:1, 15:1, 10:1, 5:1, and 1:1 as eluents, respectively; the separated fractions are combined by thin layer chromatography to obtain six fractions, namely fraction A, fraction B, fraction C, fraction D, fraction E, and fraction F.

[0012] Preferably, in the step of separating and purifying the bisbenzylisoquinoline alkaloid compound of formula I, fraction D is subjected to ODS C18 reverse phase column chromatography using 20% CH3OH-H2O, 30% CH3OH-H2O, 50% CH3OH-H2O, 60% CH3OH-H2O, 70% CH3OH-H2O, 90% CH3OH-H2O, and 100% CH3OH-H2O as eluents; the fraction obtained by eluting with 100% CH3OH-H2O as the eluent is evaporated to dryness, and then semi-preparative liquid chromatography (C18 reverse phase column) is used, the mobile phase is MeCN-H2O with a volume ratio of 65:35, the flow rate is 2 mL / min, and 0.02% NH3H2O is added to the mobile phase water to separate and purify to obtain the bisbenzylisoquinoline alkaloid compound of formula I.

[0013] A method for preparing the above-mentioned bisbenzylisoquinoline alkaloid compound of formula II, characterized in that it comprises the following steps:

[0014] The total alkaloid fraction is obtained by concentrating the ethanol extract or methanol extract of the lotus seed core to obtain an extract, preparing the extract into an aqueous suspension, adjusting the pH value of the aqueous suspension to 2-3 to obtain an acidic mixed solution; then, extracting the acidic mixed solution with ethyl acetate; after extraction, adjusting the pH value of the remaining aqueous solution to 9-10 to obtain an alkaline solution; then, extracting the alkaline solution with dichloromethane to obtain the total alkaloid fraction;

[0015] Separation of the total alkaloid fraction: The total alkaloid fraction was separated using HP-20 macroporous resin using 80% CH3OH-H2O as an eluent. The eluted fraction was further separated by column chromatography on a silica gel column using dichloromethane-methanol as an eluent. The separated fractions were combined by thin layer chromatography to obtain nine fractions: Fraction 1, Fraction 2, Fraction 3, Fraction 4, Fraction 5, Fraction 6, Fraction 7, Fraction 8, and Fraction 9.

[0016] Isolation and purification of the bisbenzylisoquinoline alkaloid compound of formula II: Fraction 7 was separated and purified using semi-preparative liquid chromatography (C18 reverse phase column) with a mobile phase of MeCN-H2O or CH3OH-H2O to obtain the bisbenzylisoquinoline alkaloid compound of formula II.

[0017] Preferably, the step of obtaining the total alkaloid fraction comprises concentrating an 80% ethanol extract of lotus seed core to obtain an extract, preparing the extract into an aqueous suspension, adjusting the pH value of the aqueous suspension to 2-3 using a 2% sulfuric acid aqueous solution to obtain an acidic mixed solution; then, extracting the acidic mixed solution twice with ethyl acetate; after extraction, retaining the aqueous solution, and adjusting the pH value of the aqueous solution to 9-10 using ammonia water to obtain an alkaline solution; then, extracting the alkaline solution three times with dichloromethane to obtain the total alkaloid fraction;

[0018] Preferably, in the separation step of the total alkaloid portion, the total alkaloid portion is separated by HP-20 macroporous resin using eluents of 30% CH3OH-H2O, 50% CH3OH-H2O, 80% CH3OH-H2O, and 100% CH3OH-H2O; the portion obtained by the 80% CH3OH-H2O eluent is further separated by column chromatography on a silica gel column with 200-300 mesh silica gel, using dichloromethane-methanol with volume ratios of 20:1, 15:1, 10:1, 5:1, and 1:1 as eluents, respectively; the separated fractions are combined by thin layer chromatography to obtain 9 fractions, namely fraction 1, fraction 2, fraction 3, fraction 4, fraction 5, fraction 6, fraction 7, fraction 8, and fraction 9.

[0019] Preferably, in the step of separating and purifying the bisbenzylisoquinoline alkaloid compound of formula II, fraction 7 is subjected to semi-preparative liquid chromatography (C18 reverse phase column) with a mobile phase of MeCN-H2O in a volume ratio of 51:49 at a flow rate of 2 mL / min, and 0.02% NH3H2O is added to the mobile phase water for separation and purification to obtain the bisbenzylisoquinoline alkaloid compound of formula II.

[0020] Application of the bisbenzylisoquinoline alkaloid compound of formula I in inhibiting acetylcholinesterase.

[0021] Application of bisbenzylisoquinoline alkaloid compounds of formula II in inhibiting acetylcholinesterase.

[0022] Application of bisbenzylisoquinoline alkaloid compounds of formula I in inhibiting butyrylcholinesterase.

[0023] Application of bisbenzylisoquinoline alkaloid compounds of formula II in inhibiting butyrylcholinesterase.

[0024] Application of the bisbenzylisoquinoline alkaloid compounds of formula I in preparing drugs for resisting nerve damage.

[0025] Application of the bisbenzylisoquinoline alkaloid compound of formula II in preparing drugs for resisting nerve damage.

[0026] Two novel bisbenzylisoquinoline alkaloid compounds isolated and obtained by the present invention have been found to exhibit anti-nerve damage effects, which may be related to their ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Therefore, these two bisbenzylisoquinoline alkaloid compounds have the potential to be developed into drugs that inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), as well as drugs that treat nerve damage. Description of the drawings:

[0027] Attachment Figure 1 This is the (+)-HR-ESI-MS spectrum of the bisbenzylisoquinoline alkaloid compound of formula I.

[0028] Attachment Figure 2 It is a bisbenzylisoquinoline alkaloid compound of formula I 1 H-NMR spectrum.

[0029] Attachment Figure 3 It is a bisbenzylisoquinoline alkaloid compound of formula I 13 C-NMR spectrum.

[0030] Attachment Figure 4 It is the HSQC spectrum of the bisbenzylisoquinoline alkaloid compound of formula I.

[0031] Attachment Figure 5 It is a bisbenzylisoquinoline alkaloid compound of formula I 1 H- 1 H COSY spectrum.

[0032] Attachment Figure 6 It is the HMBC spectrum of the bisbenzylisoquinoline alkaloid compound of formula I.

[0033] Attachment Figure 7This is the (+)-HR-ESI-MS spectrum of the bisbenzylisoquinoline alkaloid compound of formula II.

[0034] Attachment Figure 8 It is a compound of formula II bisbenzylisoquinoline alkaloids 1 H-NMR spectrum.

[0035] Attachment Figure 9 It is a compound of formula II bisbenzylisoquinoline alkaloids 13 C-NMR spectrum.

[0036] Attachment Figure 10 It is the HSQC spectrum of the bisbenzylisoquinoline alkaloid compound of formula II.

[0037] Attachment Figure 11 It is a compound of formula II bisbenzylisoquinoline alkaloids 1 H- 1 H COSY spectrum.

[0038] Attachment Figure 12 It is the HMBC spectrum of the bisbenzylisoquinoline alkaloid compound of formula II.

[0039] Attachment Figure 13 The chemical structural formulas are the bisbenzylisoquinoline alkaloid compounds of formula I and the bisbenzylisoquinoline alkaloid compounds of formula II.

[0040] Attachment Figure 14 The invention relates to the inhibitory activity of the bisbenzylisoquinoline alkaloid compounds of formula I and formula II on butyrylcholinesterase.

[0041] Attachment Figure 15 The invention relates to the inhibitory activity of the bisbenzylisoquinoline alkaloid compounds of formula I and formula II on acetylcholinesterase.

[0042] Attachment Figure 16 The cell survival rates were determined under different drug concentrations. Specific implementation method:

[0043] The lotus seed core mentioned by the applicant is the young leaves and radicle in the lotus seeds.

[0044]

[0045] (1) Separation and preparation of bisbenzylisoquinoline alkaloid compounds of formula I.

[0046] The alcohol solvent extract of the lotus seed core is concentrated to obtain an extract. The alcohol solvent is ethanol or methanol, and the optimal extract is an 80% ethanol extract of the lotus seed core. The extract is prepared into an aqueous suspension, and the pH of the aqueous suspension is adjusted to 2-3 using a 2% aqueous sulfuric acid solution to obtain an acidic mixed solution. The acidic mixed solution is then extracted twice with ethyl acetate. After extraction, the aqueous solution is retained and the pH of the aqueous solution is adjusted to 9-10 using aqueous ammonia to obtain an alkaline solution. The alkaline solution is then extracted three times with dichloromethane to obtain the total alkaloid fraction.

[0047] The total alkaloid fraction was separated by HP-20 macroporous resin using 30% CH3OH-H2O, 50% CH3OH-H2O, 80% CH3OH-H2O, and 100% CH3OH-H2O as eluents.

[0048] The fraction obtained with 80% CH3OH-H2O eluent was further separated by column chromatography on a silica gel column using eluents of dichloromethane-methanol (20:1), dichloromethane-methanol (15:1), dichloromethane-methanol (10:1), dichloromethane-methanol (5:1), and dichloromethane-methanol (1:1). The eluent ratio of dichloromethane-methanol is a volume ratio, for example, dichloromethane-methanol (15:1) is 15 ml of dichloromethane and 1 ml of methanol; the silica gel in the silica gel column is 200-300 mesh, and the separated fractions are combined by thin layer chromatography to obtain six fractions, namely fraction A, fraction B, fraction C, fraction D, fraction E, and fraction F.

[0049] Fraction D was chromatographed on an ODS C18 reverse phase column using eluents of 20% CH3OH-H2O, 30% CH3OH-H2O, 50% CH3OH-H2O, 60% CH3OH-H2O, 70% CH3OH-H2O, 90% CH3OH-H2O, and 100% CH3OH-H2O to obtain different fractions.

[0050] The fraction obtained by eluting with the 100% CH3OH-H2O eluent was evaporated to dryness and then separated and purified using semi-preparative liquid chromatography (C18 reverse phase column) with a mobile phase of MeCN-H2O or CH3OH-H2O at a flow rate of 2 mL / min. 0.02% NH3H2O was added to the mobile phase water to obtain the compound of Formula I. The MeCN-H2O mobile phase had a volume ratio of 65:35, i.e., 65 mL of MeCN and 35 mL of H2O.

[0051] (2) Structural identification of the bisbenzylisoquinoline alkaloid compounds of formula I.

[0052] The compound is a brown oily substance, and the reaction of potassium bismuth iodide is positive, indicating that it is an alkaloid compound.

[0053] As attached Figure 1 As shown, HR-ESI-MS (m / z 611.3116 [M+H] + ; The calculated value is 611.3116, C 37 H 43 N2O6, the molecular formula is inferred to be C based on mass spectrometry data 37 H 42 N2O6, its saturation is 18. (c 0.10, MeOH).

[0054] As attached Figure 2 As shown, 1 H NMR (500 MHz, CDCl3) showed 11 benzene ring proton signals, δ H 6.90 (2H, d, J = 8.4 Hz, H-11, 15), 6.70 (2H, d, J = 8.5 Hz, H-12, 14), 6.82 (1H, d, J = 8.1 Hz, H-14'), 6.74 (1H, dd, J = 8.1 Hz, 2.1, H-15'), 6.64 (1H, s, H-5), 6.46 (2H, s, H-11', 5'), 6.32 (1H, s, H-8') and 6.28 (1H, s, H-8), where δ H 6.82 (1H, d, J = 8.1 Hz, H-14') and 6.74 (1H, dd, J = 8.1 Hz, 2.1, H-15') prove the existence of ABX benzene ring system, while δ H 6.90 (2H, d, J = 8.4 Hz, H-11, 15) and 6.70 (2H, d, J = 8.5 Hz, H-12, 14) showed a para-substituted benzene ring. In addition, there were three methoxy proton signals δ H 3.80 (3H, s, 13'-OCH3), 3.79 (3H, s, 6-OCH3) and 3.72 (3H, s, 13-OCH3), two nitrogen methyl signals δ H 2.51 (3H, s, 2-NCH3) and 2.39 (3H, s, 2'-NCH3).

[0055] As attached Figure 3 As shown, the carbon spectrum shows 37 carbon signals, combined with the attached Figure 4 The HSQC shown in the figure shows 24 benzene ring carbon signals: δ C157.95(C-13),149.08(C-6),145.34(C-13'),145.29(C-6'),144.57(C-7'),143.59(C-12 '),142.97(C-7),131.99(C-10'),131.52(C-10),130.72(C-4a),130.60(C-8a'),130.56(C -11,15),130.24(C-8a),125.73(C-15'),125.32(C-4a'),120.01(C-8),119.21(C-11'),115.56(C-14'),113.90(C-8'),113.66(C-12,14),112.56(C-5),110.76(C-5'), 2 methine carbon signals: δ C 64.64 (C-1') and 64.46 (C-1), 3 methoxy carbon signals: δ C 56.08 (6-OCH3), 55.92 (13'-OCH3) and 55.29 (13-OCH3), 6 methylene signals: δ C 47.50 (C-3'), 46.98 (C-3), 40.73 (C-9'), 39.44 (C-9), 26.15 (C-4) and 25.66 (C-4'), and 2 nitrogen methyl carbon signals: δ C 42.80 (2'-NCH3) and 42.72 (2-NCH3).

[0056] As attached Figure 5 As shown, 1 H- 1 The H COSY spectrum shows that H-11 is correlated with H-12, H-1 is correlated with H-9, H-1' is correlated with H-9', H-3 is correlated with H-4, and H-3' is correlated with H4'. Figure 6 As shown, the HMBC spectrum shows that H-1 is correlated with C-9 and C-8, H-8 is correlated with C-4a and C-6, H-5 is correlated with C-4 and C-7, and H-11, 15 are correlated with C-9 and C-13, proving that there is a monobenzylisoquinoline alkaloid containing a para-substituted benzene ring structure. Combined with the ABX benzene ring system, the following correlations are found in the HMBC spectrum: H-1' is correlated with C-8' and C-9', H-5' is correlated with C-4' and C-7', H-8' is correlated with C-6' and C-4a', H-15' is correlated with C-9' and C13', and H-14' is correlated with C-12' and C-10'. In addition, in the HMBC spectrum, δ H 3.79 (6-OCH3) is related to C-6, δ H3.72(13-OCH3) is related to C-13, δ H 3.80 (13'-OCH3) is related to C-13', and it is inferred that the three methoxy groups are related to C-6, C-13 and C-13' respectively. According to relevant literature reports and mass spectrometry, combined with the optical rotation value, its configuration can be determined to be 1R, 1'R. The compound structure is as follows Figure 13 As shown in the formula I, the bisbenzylisoquinoline alkaloid compound of formula I was found to be a new compound through Scifinder search and was named 6'-demethyl-13'-O-methylisoliensinine.

[0057] (3) Separation and preparation of bisbenzylisoquinoline alkaloid compounds of formula II.

[0058] The alcohol solvent extract of the lotus seed core is concentrated to obtain an extract. The alcohol solvent is ethanol or methanol, and the optimal extract is an 80% ethanol extract of the lotus seed core. The extract is prepared into an aqueous suspension, and the pH of the aqueous suspension is adjusted to 2-3 using a 2% aqueous sulfuric acid solution to obtain an acidic mixed solution. The acidic mixed solution is then extracted twice with ethyl acetate. After extraction, the aqueous solution is retained and the pH of the aqueous solution is adjusted to 9-10 using aqueous ammonia to obtain an alkaline solution. The alkaline solution is then extracted three times with dichloromethane to obtain the total alkaloid fraction.

[0059] The total alkaloid fraction was separated by HP-20 macroporous resin using 30% CH3OH-H2O, 50% CH3OH-H2O, 80% CH3OH-H2O, and 100% CH3OH-H2O as eluents.

[0060] The fraction obtained by 80% CH3OH-H2O eluent was further separated by column chromatography on a silica gel column using eluents of dichloromethane-methanol (20:1), dichloromethane-methanol (15:1), dichloromethane-methanol (10:1), dichloromethane-methanol (5:1), and dichloromethane-methanol (1:1). The eluent ratio of dichloromethane-methanol is a volume ratio. For example, dichloromethane-methanol (15:1) is 15 ml of dichloromethane and 1 ml of methanol. The silica gel in the silica gel column is 200-300 mesh. The separated fractions were combined by thin layer chromatography to obtain 9 fractions, namely fraction 1, fraction 2, fraction 3, fraction 4, fraction 5, fraction 6, fraction 7, fraction 8, and fraction 9.

[0061] Fraction 7 was separated and purified using semi-preparative liquid chromatography (C18 reverse-phase column) with a mobile phase of MeCN-H2O or CH3OH-H2O at a flow rate of 2 mL / min. 0.02% NH3H2O was added to the water mobile phase to obtain the compound of Formula II. The MeCN-H2O mobile phase had a volume ratio of 51:49, i.e., 51 mL of MeCN and 49 mL of H2O.

[0062] (4) Structural identification of the bisbenzylisoquinoline alkaloid compound of formula II:

[0063] Brown oily substance, soluble in chloroform, the compound reacts positively with potassium bismuth iodide, indicating that it is an alkaloid compound. Figure 7 As shown, HR-ESI-MS (m / z 611.3116 [M+H] + ; The calculated value is 611.3116, C 37 H 43 N2O6), the molecular formula is inferred to be C based on the mass spectrometry data. 37 H 42 N2O6, its saturation is 18. (c 0.10, MeOH).

[0064] As attached Figure 8 As shown, 1 The H NMR (500 MHz, CDCl3) spectrum showed 11 benzene ring proton signals, δ H 6.91(2H,d,J=8.6Hz,H-11,15),6.82(1H,d,J=8.2Hz,H-14'),6.73(1H,dd,J=8.3Hz,2.0,H-15'),6.70(2H,d,J=8.7Hz ,H-12,14), 6.63(1H,s,H-5'), 6.48(1H,d,J=1.9Hz,H-11'), 6.45(1H,s,H-5), 6.34(1H,s,H-8) and 6.30(1H,s,H-8'), where δ H 6.82 (1H, d, J = 8.2 Hz, H-14'), 6.73 (1H, dd, J = 8.3, 2.0 Hz, H-15'), 6.48 (1H, d, J = 1.9 Hz, H-11') prove the existence of ABX benzene ring system, and δ H 6.91 (2H, d, J = 8.6 Hz, H-11, 15) and 6.70 (2H, d, J = 8.7 Hz, H-12, 14) showed a para-substituted benzene ring. In addition, there were three methoxy proton signals δ H3.81 (3H, s, 13'-OCH3), 3.80 (3H, s, 6'-OCH3) and 3.73 (3H, s, 13-OCH3), two nitrogen methyl signals δ H 2.51 (3H, s, 2-NCH3) and 2.39 (3H, s, 2'-NCH3).

[0065] As attached Figure 9 As shown, the carbon spectrum shows 37 carbon signals, combined with the attached Figure 10 The HSQC spectrum shown shows that there are 24 benzene ring carbon signals: δ C 157.95(C-13),149.14(C-6'),145.32(C-13'),145.27(C-7),144.61(C-6),143.61(C-12' ),142.98(C-7'),131.99(C-10'),131.52(C-10),130.79(C-4a'),130.57(C-11,15),130.4 4(C-8a'),130.29(C-8a),125.79(C-4a),125.33(C-15'),120.11(C-8'),119.22(C-11'),115.46(C-14'),113.90(C-8),113.65(C-12,14),112.54(C-5'),110.72(C-5), 2 methine carbon signals: δ C 64.64 (C-1') and 64.48 (C-1), 3 methoxy carbon signals: δ C 56.07 (13'-OCH3), 55.91 (6'-OCH3) and 55.28 (13-OCH3), 6 methylene signals: δ C 47.59 (C-3'), 47.03 (C-3), 42.85 (2'-NCH3), 42.75 (2-NCH3), 40.75 (C-9'), 39.45 (C-9), 26.20 (C-4') and 25.74 (C-4), and there are 2 nitrogen methyl carbon signals: δ C 42.85 (2'-NCH3) and 42.75 (2-NCH3).

[0066] As attached Figure 11 As shown, 1 H- 1 The H COSY spectrum shows that H-11 is correlated with H-12, H-14' is correlated with H-15', H-1 is correlated with H-9, H-1' is correlated with H-9', H-3 is correlated with H-4, and H-3' is correlated with H-4'. Figure 12As shown, the HMBC spectrum shows that H-1 is correlated with C-9 and C-8, H-8 is correlated with C-1, C-4a and C-6, H-5 is correlated with C-4, C-8a and C-7, H-11, 15 are correlated with C-9 and C-13, and 13-OCH3 is correlated with C-13, proving that there is a monobenzylisoquinoline alkaloid containing a para-substituted benzene ring structure. Combined with the ABX benzene ring system, the following correlations are found in the HMBC spectrum: H-1' is correlated with C-10', H-5' is correlated with C-4', C-8a' and C-7', H-8' is correlated with C-1', C-6' and C-4a', H-15' is correlated with C-9', C-11' and C13', and H-14' is correlated with C-12' and C-10'. In addition, in the HMBC spectrum, δ H 3.81(13'-OCH3) is related to C-13', δ H 3.80 (6'-OCH3) is related to C-6', δ H 3.73 (13-OCH3) is related to C-13, and it is inferred that the three methoxy groups are related to C-6', C-13 and C-13' respectively. According to relevant literature reports and mass spectrometry, combined with the optical rotation value, its configuration can be determined to be 1R, 1'R. The compound structure is as follows Figure 13 As shown in formula II, the bisbenzylisoquinoline alkaloid compound of formula II was found to be a new compound through Scifinder search and was named 6-demethyl-13'-O-methylisoliensinine.

[0067] (5) Determination of enzyme inhibition activity of bisbenzylisoquinoline alkaloid compounds of formula I and formula II

[0068] The compounds 6'-demethyl-13'-O-methylisoliensinine (a bis-benzylisoquinoline alkaloid compound of formula I) and 6-demethyl-13'-O-methylisoliensinine (a bis-benzylisoquinoline alkaloid compound of formula II) are prepared by the applicant from lotus seed core extract, and their purity is not less than 95% as determined by HPLC.

[0069] 1. Experimental Reagents

[0070] Table 1-1 Experimental reagents and manufacturers

[0071]

[0072] 2. Experimental Methods

[0073] 2.1 Preparation of experimental reagents

[0074] Tris-HCl buffer: Accurately weigh 302.85 mg of Tris base, add distilled water to dissolve it, and then dilute to 50 mL. Concentrated hydrochloric acid is added to adjust the pH to 7.8 to obtain a buffer solution.

[0075] Acetylcholinesterase (AChE) solution: Dissolve 50 mg (200 U / g) of lyophilized AChE powder in 1 mL of Tris-HCl (0.05 M, pH 7.8) buffer to a storage concentration of 10 U / mL. Add 1 mg of bovine serum albumin to stabilize the enzyme and mix well. Dilute to 0.3 U / mL with PBS buffer, distribute into EP tubes, and freeze at -20°C.

[0076] Butyrylcholinesterase (BuChE) solution: Dissolve 100 U of lyophilized butyrylcholinesterase powder in 10 mL of Tris-HCl (0.05 M, pH 7.8) buffer to a storage concentration of 10 U / mL. Add 10 mg of bovine serum albumin to stabilize the enzyme and mix well. Dilute to 0.3 U / mL with PBS and dispense into EP tubes and freeze at -20°C.

[0077] Thioacetylcholine (ATCI M=289.18): Weigh 21.67 mg of ATCI, dissolve it in distilled water, and then dilute to 5 mL. Store in the dark.

[0078] Thiobutyrylcholine (BTCI M = 317.23): Weigh 23.79 mg of BTCI, dissolve it in distilled water, and then dilute to 5 mL. Store in the dark.

[0079] 5,5'-Dithio-2-nitrobenzoic acid (DTNB M = 396.35): Weigh 3.96 mg of DTNB and dissolve it in 500 μL of anhydrous ethanol. Then add 2 mg of sodium bicarbonate and shake well. Finally, dilute to 5 mL with PBS and store in dark.

[0080] 2.2 Experimental grouping and drug addition

[0081] Weigh at least 1 mg of 6-demethyl-13'-O-methylisoliensinine (a bisbenzylisoquinoline alkaloid compound of Formula II) and 6'-demethyl-13'-O-methylisoliensinine (a bisbenzylisoquinoline alkaloid compound of Formula I) accurately. Dissolve both compounds in DMSO to a 20 mM stock solution and store frozen at -20°C. Then, dilute the appropriate amount of the stock solution with PBS to the desired concentration. The inhibitory activities of the compounds and the positive drug against acetylcholinesterase and butyrylcholinesterase, respectively, were determined using a modified Ellman method. To the experimental group, add 140 μL of PBS buffer, 20 μL of 0.3 U / mL acetylcholinesterase or butyrylcholinesterase solution, and 20 μL of the test sample solution, mix well, and incubate at 4°C for 20 minutes. Subsequently, 15 μL of 2 mM DTNB solution and 15 μL of 15 mM thioacetylcholine solution or thiobutyrylcholine solution were added. The mixture was incubated at 37°C in the dark for 20 min, and the absorbance at 412 nm was measured using a microplate reader. In the blank group, 20 μL of PBS buffer was used instead of the 20 μL sample. The inhibition rate at different concentrations was calculated based on the measured absorbance according to the following formula.

[0082] Inhibition rate = (1-A experimental group / A blank) * 100%

[0083] For the sample group with an inhibition rate of more than 60%, the corresponding inhibition rate was measured under a series of concentration gradients, and the IC value of the sample was calculated. 50 value.

[0084] 2.3 Experimental Results

[0085] 2.3.1 Inhibitory activity against butyrylcholinesterase

[0086] like Figure 14 -A shows the inhibitory activity of donepezil, a positive drug, on butyryl esterase, and its IC 50 The value is 15.64μM; Figure 14 -B shows the inhibitory activity of 6'-demethyl-13'-O-methylisoliensinine (bisbenzylisoquinoline alkaloid compound of formula I) on butyrylcholinesterase, and its IC 50 The value is 23.26μM; Figure 14 -C shows the inhibitory activity of 6-demethyl-13'-O-methylisoliensinine (bisbenzylisoquinoline alkaloid compound of formula II) on butyrylcholinesterase, and its IC 50 The value was 5.44 μM.

[0087] It can be seen that the inhibitory activity of compound 6'-demethyl-13'-O-methylisoliensinine (bisbenzylisoquinoline alkaloid compound of formula I) on butyrylcholinesterase is weaker than that of the positive drug donepezil.

[0088] The inhibitory activity of 6-demethyl-13'-O-methylisoliensinine (a bisbenzylisoquinoline alkaloid compound of formula II) on butyrylcholinesterase is stronger than that of the positive drug donepezil.

[0089] 2.3.2 Inhibitory activity against acetylcholinesterase

[0090] At a test concentration of 47.62 μM, the inhibition rates of 6'-demethyl-13'-O-methylisoliensinine (a bisbenzylisoquinoline alkaloid compound of Formula I) and 6-demethyl-13'-O-methylisoliensinine (a bisbenzylisoquinoline alkaloid compound of Formula II) on acetylcholinesterase were 44.48% and 31.64%, respectively. The inhibition rate of the positive drug donepezil was 16.23%.

[0091] At a test concentration of 23.81 μM, the inhibition rates of 6'-demethyl-13'-O-methylisoliensinine (a bisbenzylisoquinoline alkaloid compound of Formula I) and 6-demethyl-13'-O-methylisoliensinine (a bisbenzylisoquinoline alkaloid compound of Formula II) on acetylcholinesterase were 26.02% and 18.79%, respectively. The inhibition rate of the positive drug donepezil was 11.93%.

[0092] The results are as attached Figure 15 As shown, it can be seen that the inhibition rates of the two compounds on acetylcholinesterase at the tested concentrations are higher than that of the positive drug donepezil.

[0093] In summary, the inhibitory activity of the bisbenzylisoquinoline alkaloid compounds of Formula I against butyrylcholinesterase is weaker than that of the positive drug donepezil; and the inhibition rate against acetylcholinesterase is higher than that of the positive drug donepezil. The inhibitory activity of the bisbenzylisoquinoline alkaloid compounds of Formula II against butyrylcholinesterase is stronger than that of the positive drug donepezil; and the inhibition rate against acetylcholinesterase is higher than that of the positive drug donepezil. Overall, the bisbenzylisoquinoline alkaloid compounds of Formula I and Formula II have stronger butyrylesterase inhibition activity than acetylcholinesterase inhibition activity, indicating that these two compounds have selective inhibition of cholinesterase.

[0094] (6) Determination of neuroprotective activity of the bisbenzylisoquinoline alkaloid compounds of formula I and formula II.

[0095] The compounds 6'-demethyl-13'-O-methylisoliensinine (a bis-benzylisoquinoline alkaloid compound of formula I) and 6-demethyl-13'-O-methylisoliensinine (a bis-benzylisoquinoline alkaloid compound of formula II) are prepared by the applicant from lotus seed core extract, and their purity is not less than 95% as determined by HPLC.

[0096] 1. Experimental Materials

[0097] Table 1-1 Drugs and reagents

[0098]

[0099] Table 1-2 Main instruments and equipment

[0100]

[0101]

[0102] 2. Drug Configuration

[0103] 2.1 Preparation of corticosterone

[0104] Calculate the amount of corticosterone and DMSO in advance according to the required concentration, accurately weigh the corresponding amount of corticosterone, add DMSO, and mix it on a vortex mixer to fully dissolve it to make a 200mM stock solution. Add the corresponding amount of culture medium to a centrifuge tube of appropriate specifications, use a pipette to draw up the corticosterone mother solution, insert the pipette tip below the liquid surface of the culture medium and add it evenly to the culture medium, cover the lid tightly, and mix it on a vortex mixer until the two are fully miscible. Prepare a 1mM corticosterone intermediate solution for use. Note that it should be prepared as soon as possible before use.

[0105] 2.2 Preparation of MTT solution

[0106] Accurately weigh 75 mg of MTT and dissolve it in 15 mL of PBS to make a 5 mg / mL solution. Filter the solution through a 0.22 μm filter membrane to remove bacteria and impurities. Keep the solution away from light throughout the process and store at -20°C.

[0107] 2.3 Preparation of mother liquor of lotus seed core alkaloid monomer compound

[0108] The bisbenzylisoquinoline alkaloid compounds of formula I and formula II from lotus seed core were accurately weighed and dissolved in DMSO to prepare a 20 mM stock solution. The corresponding amount of culture medium was added to a 2 mL centrifuge tube. The mother solutions of the bisbenzylisoquinoline alkaloid compounds of formula I and formula II were respectively aspirated with a pipette. The pipette tip was inserted below the liquid surface of the culture medium and evenly added to the culture medium. The lid was tightly closed and the mixture was placed on a vortex mixer to mix until the two were fully miscible. The mother solutions of the bisbenzylisoquinoline alkaloid compounds of formula I and formula II were prepared to be 1 mM and stored at -20°C.

[0109] 3. Experimental Methods

[0110] 3.1 Cell recovery

[0111] Remove the cell cryovial from the -80°C freezer, preheat the culture medium for half an hour, place under ultraviolet light on the clean bench, turn on the centrifuge and water bath, adjust the water bath temperature to 37°C, hold the upper end of the cell cryovial, immerse the lower end in a 37°C water bath, and shake continuously for about 2 minutes. After the ice melts, move to the clean bench and use a pipette to transfer the cell suspension to a centrifuge tube. Centrifuge at 1000 rpm for 5 minutes. After the cell centrifugation is completed, move to the clean bench, open the lid and pour out the supernatant. With the pipette held flat, add 1000 μL of culture medium and pipette through the cells. After pipetting evenly, inoculate the cells into a culture dish with 4400 μL of 1640 medium and 600 μL of serum-containing medium. Shake the culture dish using a cross-shake method to evenly disperse the cells. Place in a 37°C incubator with 5% CO2 and culture.

[0112] 3.2 Cell culture medium replacement

[0113] Take out the culture medium in advance and preheat it for half an hour. Illuminate the clean bench with UV light, pour out the old culture medium in the culture dish, hold the pipette level, slowly add 2mL PBS toward the side wall of the culture dish, gently shake the culture dish to rinse the cells, pour out the PBS after rinsing, repeat this process twice, slowly add 5400μL of culture medium and 600μL of serum toward the side wall of the culture dish, shake the culture dish with a cross shake method, and place it in a 37℃ incubator containing 5% CO2 for static culture.

[0114] 3.3 Cell passaging

[0115] Take out the culture medium in advance and preheat it for half an hour. Expose the clean bench to UV light, turn on the centrifuge, and observe the cells in the culture dish under a microscope. When the cells grow to 70% to 80% of the culture dish, they can be passaged. Pour out the old culture medium in the dish, hold the pipette flat, slowly add 2mL PBS toward the side wall of the culture dish, gently shake the culture dish, rinse the cells with PBS, and pour out the PBS after rinsing. Repeat this process twice, add 600μL of trypsin, gently shake the culture dish, so that the trypsin evenly covers the cells on the bottom of the culture dish. After 1 to 2 minutes, the digestion is basically completed. Under the microscope, it can be observed that the cells are separated from each other and are round. Add 1800μL of culture medium and 200μL of serum to terminate the digestion. Hold the pipette flat and blow the cells from the bottom of the dish to make them fall off and disperse. After blowing evenly, transfer the cell suspension to a 2mL centrifuge tube and centrifuge it at 1000rpm for 5 minutes. Add 4400 μL of 1640 medium and 600 μL of serum to each of the two culture dishes, using a 1:2 cell culture ratio. After centrifugation, move the cells to a clean bench, open the lid and discard the supernatant. Add 2000 μL of culture medium and pipette the cells to disperse. Once dispersed, pipette 1 mL of the cell suspension into each of the two prepared culture dishes. Shake the dishes using a cross-shaking technique. Observe under a microscope to ensure that the cells are in a single, suspended state. Continue to incubate in a 37°C incubator with 5% CO2.

[0116] 3.4 Cell cryopreservation

[0117] Take out the culture medium in advance and preheat it for half an hour. Put the ultraviolet light on the clean bench and turn on the centrifuge in advance. Observe the cells in the culture dish under a microscope. When the cells grow to 70% to 80% of the culture dish, they can be passaged. Pour out the old culture medium in the dish, hold the pipette flat, slowly add 2mL PBS towards the side wall of the culture dish, shake the culture dish gently, rinse the cells with PBS, and pour out the PBS after rinsing. Repeat this process twice, add 800μL of trypsin, shake the culture dish gently, so that the trypsin evenly covers the cells at the bottom of the culture dish. After 1 to 2 minutes, the digestion is basically completed, and then add 18 00μL of culture medium and 100μL of serum to terminate digestion. Hold the pipette level and blow the cells from the bottom of the dish to make them fall off and disperse. After blowing evenly, transfer the cell suspension to a 2mL centrifuge tube and centrifuge it at 1000rpm for 5 minutes. After the cell centrifugation is completed, move it to the clean bench, open the lid and pour out the supernatant, add 1000μL of freezing solution, blow evenly, and aspirate the cell suspension into the freezing tube. Label the cell type and freezing time, seal it with sealing film, place it at 4℃ for 30 minutes, -20℃ for 4 hours, and then place it in a -80℃ ultra-low temperature refrigerator.

[0118] 3.5 Cell Counting

[0119] Soak the counting plate with 75% alcohol in advance. Wipe the counting plate and cover glass clean before use. Align the cover glass with the counting plate and cover tightly. Be careful not to have bubbles. Digest the cells with trypsin, then add 1800 μL of culture medium and 200 μL of serum to terminate the digestion. Hold the pipette flat and blow the cells from the bottom of the dish to make them fall off and disperse. After blowing evenly, transfer the cell suspension to a 2 mL centrifuge tube and centrifuge it at 1000 rpm for 5 minutes. After the cell centrifugation is completed, move it to the clean bench, open the lid and pour out the supernatant. , add 1mL complete culture medium, gently blow the cells, aspirate 10μL cell suspension, and add it to the gap between the counting plate and the coverslip. At this time, the cell suspension will be quickly absorbed into the counting groove and placed under a microscope for counting. Count the number of cells in the 16-type squares on the four corners respectively. The counting should follow the principle that the cell above the pressure line does not count the cell below, and the cell on the left does not count the cell on the right. When there are two or more cell clusters, they should be counted as 1 cell. After the cell counting is completed, calculate the average value m of the cells in the four 16-type squares.

[0120] m×V1=n×V2 (1)

[0121] V1 is the required volume of cell suspension stock, mL

[0122] V2 is the total volume of cell suspension required for plating, mL

[0123] 3.6 Cell plating

[0124] The poorly differentiated PC12 cells were cultured to the logarithmic growth phase. After counting, the volumes of cell suspension stock solution and complete culture medium required for plating were calculated according to formula (1) to prepare a cell suspension of a certain concentration (3*10 4 200 μL of cell suspension was added to each well, and the cells were placed in a 37°C incubator containing 5% CO2 for 24 hours.

[0125] 3.7 Grouped Dosing

[0126] After 24 hours, the cells were divided into a blank group (only 1640 culture medium was added), a model group (corticosterone concentration was 500 μM), and an experimental group (corticosterone concentration was 500 μM + monomer compound formula II bisbenzylisoquinoline alkaloid compound concentrations were 2.5 μM, 5 μM, and 10 μM, and formula I bisbenzylisoquinoline alkaloid compound concentrations were 5 μM, 10 μM, and 20 μM). After the administration was completed, the cells were placed in a 37°C incubator containing 5% CO2 and cultured for 24 hours.

[0127] 3.8 Determination of cell viability by MTT assay

[0128] After the cells were cultured for 24 h, 20 μL of MTT solution was added to each well and the cells were placed in a 37°C incubator containing 5% CO2 for static culture. After 4 h, the cells were taken out and the culture medium was aspirated before adding 150 μL of DMSO to each well to fully dissolve the formazan crystals in the cells. The cells were placed in a microplate reader to measure the OD value and the cell survival rate was calculated according to formula (2).

[0129]

[0130] 4 Experimental results

[0131] like Figure 16 As shown, dark blue represents the blank control group, light blue represents the model group, red columns represent the bisbenzylisoquinoline alkaloid compound of Formula I, and orange columns represent the bisbenzylisoquinoline alkaloid compound of Formula II. The average cell viability in the model group was 77.45%. In the experimental groups, the average cell viability at concentrations of 2.5 μM, 5 μM, and 10 μM of the bisbenzylisoquinoline alkaloid compound of Formula II was 86.39%, 97.23%, and 101.27%, respectively. The average cell viability at concentrations of 5 μM, 10 μM, and 20 μM of the bisbenzylisoquinoline alkaloid compound of Formula I was 86.70%, 95.45%, and 75.46%, respectively.

[0132] The bisbenzylisoquinoline alkaloid compound of formula II exhibited good anti-cell damage activity at three concentrations of 2.5 μM, 5 μM, and 10 μM, and was dose-dependent within this concentration range.

[0133] The two concentrations of 5μM and 10μM of the bisbenzylisoquinoline alkaloid compound of formula I showed good anti-cell damage activity, and were dose-dependent within this concentration range with good significance. However, the cell survival rate of the 20μM compound was lower than that of the model group, which may be because this concentration exceeded the anti-cell damage concentration range and had certain cytotoxicity, thereby causing cell damage.

[0134] Therefore, the bisbenzylisoquinoline alkaloid compounds of formula II have a neuroprotective effect in the concentration range of 2.5-10 μM, that is, good anti-cell damage activity, while the bisbenzylisoquinoline alkaloid compounds of formula I have a neuroprotective effect in the concentration range of 5-10 μM, that is, good anti-cell damage activity.

Claims

1. A bisbenzylisoquinoline alkaloid compound, characterized in that: The chemical structural formula is one of the following:

2. A method for preparing the bisbenzylisoquinoline alkaloid compound of formula I according to claim 1, characterized in that: The following steps are involved: The total alkaloid fraction is obtained by concentrating the ethanol extract or methanol extract of the lotus seed core to obtain an extract, preparing the extract into an aqueous suspension, adjusting the pH value of the aqueous suspension to 2-3 to obtain an acidic mixed solution; then, extracting the acidic mixed solution with ethyl acetate; after extraction, adjusting the pH value of the remaining aqueous solution to 9-10 to obtain an alkaline solution; then, extracting the alkaline solution with dichloromethane to obtain the total alkaloid fraction; Separation of the total alkaloid fraction: The total alkaloid fraction was separated using HP-20 macroporous resin using 80% CH3OH-H2O as the eluent. The eluted fraction was further separated by column chromatography on a silica gel column using dichloromethane-methanol as the eluent. The separated fractions were combined by thin-layer chromatography to obtain six fractions: Fraction A, Fraction B, Fraction C, Fraction D, Fraction E, and Fraction F. Isolation and purification of the bis-benzylisoquinoline alkaloid compound of formula I: Fraction D was eluted by ODS C18 reverse-phase column chromatography using 100% CH3OH-H2O as the eluent, and the obtained fraction was evaporated to dryness. It was then separated and purified by semi-preparative liquid chromatography (C18 reverse-phase column) using MeCN-H2O or CH3OH-H2O as the mobile phase to obtain the bis-benzylisoquinoline alkaloid compound of formula I.

3. The method for preparing the bisbenzylisoquinoline alkaloid compound of formula (I) as claimed in claim 2, wherein: The step of obtaining the total alkaloid fraction comprises the following steps: concentrating an 80% ethanol extract of lotus seed core to obtain an extract, preparing the extract into an aqueous suspension, adjusting the pH value of the aqueous suspension to 2-3 using a 2% sulfuric acid aqueous solution to obtain an acidic mixed solution; then, extracting the acidic mixed solution twice with ethyl acetate; after the extraction, retaining the aqueous solution, adjusting the pH value of the aqueous solution to 9-10 using ammonia water to obtain an alkaline solution; then, extracting the alkaline solution three times with dichloromethane to obtain the total alkaloid fraction; The separation step of the total alkaloid portion comprises the following steps: using 30% CH3OH-H2O, 50% CH3OH-H2O, 80% CH3OH-H2O, and 100% CH3OH-H2O eluents to separate the total alkaloid portion via HP-20 macroporous resin; further subjecting the portion obtained with the 80% CH3OH-H2O eluent to column chromatography on a 200-300 mesh silica gel column using dichloromethane-methanol with volume ratios of 20:1, 15:1, 10:1, 5:1, and 1:1 as eluents, respectively; and combining the separated fractions via thin layer chromatography to obtain six fractions, namely, fraction A, fraction B, fraction C, fraction D, fraction E, and fraction F.

4. The method for preparing the bisbenzylisoquinoline alkaloid compound of formula (I) as claimed in claim 2, wherein: In the separation and purification step of the bis-benzylisoquinoline alkaloid compound of formula I, fraction D is subjected to ODS C18 reverse-phase column chromatography using eluents of 20% CH3OH-H2O, 30% CH3OH-H2O, 50% CH3OH-H2O, 60% CH3OH-H2O, 70% CH3OH-H2O, 90% CH3OH-H2O, and 100% CH3OH-H2O; the fraction obtained by eluting with 100% CH3OH-H2O as the eluent is evaporated to dryness, and then separated and purified using semi-preparative liquid chromatography (C18 reverse-phase column) with a mobile phase of MeCN-H2O in a volume ratio of 65:35 at a flow rate of 2 mL / min, and 0.02% NH3H2O is added to the mobile phase water to obtain the bis-benzylisoquinoline alkaloid compound of formula I.

5. A method for preparing the bisbenzylisoquinoline alkaloid compound of formula II according to claim 1, characterized in that: The following steps are involved: The total alkaloid fraction is obtained by concentrating the ethanol extract or methanol extract of the lotus seed core to obtain an extract, preparing the extract into an aqueous suspension, adjusting the pH value of the aqueous suspension to 2-3 to obtain an acidic mixed solution; then, extracting the acidic mixed solution with ethyl acetate; after extraction, adjusting the pH value of the remaining aqueous solution to 9-10 to obtain an alkaline solution; then, extracting the alkaline solution with dichloromethane to obtain the total alkaloid fraction; Separation of the total alkaloid fraction: The total alkaloid fraction was separated using HP-20 macroporous resin using 80% CH3OH-H2O as an eluent. The eluted fraction was further separated by column chromatography on a silica gel column using dichloromethane-methanol as an eluent. The separated fractions were combined by thin layer chromatography to obtain nine fractions: Fraction 1, Fraction 2, Fraction 3, Fraction 4, Fraction 5, Fraction 6, Fraction 7, Fraction 8, and Fraction 9. Isolation and purification of the bisbenzylisoquinoline alkaloid compound of formula II: Fraction 7 was separated and purified using semi-preparative liquid chromatography (C18 reverse phase column) with a mobile phase of MeCN-H2O or CH3OH-H2O to obtain the bisbenzylisoquinoline alkaloid compound of formula II.

6. The method for preparing the bisbenzylisoquinoline alkaloid compound of formula II as claimed in claim 5, characterized in that: The step of obtaining the total alkaloid fraction comprises the following steps: concentrating an 80% ethanol extract of lotus seed core to obtain an extract, preparing the extract into an aqueous suspension, adjusting the pH value of the aqueous suspension to 2-3 using a 2% sulfuric acid aqueous solution to obtain an acidic mixed solution; then, extracting the acidic mixed solution twice with ethyl acetate; after the extraction, retaining the aqueous solution, adjusting the pH value of the aqueous solution to 9-10 using ammonia water to obtain an alkaline solution; then, extracting the alkaline solution three times with dichloromethane to obtain the total alkaloid fraction; The separation step of the total alkaloid portion comprises the following steps: using 30% CH3OH-H2O, 50% CH3OH-H2O, 80% CH3OH-H2O, and 100% CH3OH-H2O eluents to separate the total alkaloid portion via HP-20 macroporous resin; further subjecting the portion obtained with the 80% CH3OH-H2O eluent to column chromatography on a 200-300 mesh silica gel column using dichloromethane-methanol with volume ratios of 20:1, 15:1, 10:1, 5:1, and 1:1 as eluents, and combining the separated fractions via thin layer chromatography to obtain nine fractions, namely, fraction 1, fraction 2, fraction 3, fraction 4, fraction 5, fraction 6, fraction 7, fraction 8, and fraction 9.

7. The method for preparing the bisbenzylisoquinoline alkaloid compound of formula II as claimed in claim 5, characterized in that: The separation and purification step of the bisbenzylisoquinoline alkaloid compound of formula II is as follows: fraction 7 is subjected to semi-preparative liquid chromatography (C18 reverse phase column) with a mobile phase of MeCN-H2O in a volume ratio of 51:49 at a flow rate of 2 mL / min, and 0.02% NH3H2O is added to the mobile phase water for separation and purification to obtain the bisbenzylisoquinoline alkaloid compound of formula II.

8. Use of the bisbenzylisoquinoline alkaloid compound according to claim 1 in inhibiting acetylcholinesterase.

9. Use of the bisbenzylisoquinoline alkaloid compound according to claim 1 in inhibiting butyrylcholinesterase.

10. Use of the bisbenzylisoquinoline alkaloid compound according to claim 1 for preparing drugs for resisting nerve damage.

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