A preparation method of bousigonine D

Abstract

The invention relates to a preparation method of bousigonine D, and belongs to the technical field of alkaloid preparation. The invention separates and prepares bousigonine D from bousigonine fruit for the first time, extracts bousigonine D from bousigonine fruit by alcohol-water extraction and petroleum ether and ethyl acetate extraction, dissolves the target compound bousigonine D to the maximum extent, and then sequentially performs silica gel column chromatography separation, gel column chromatography separation and reverse phase column chromatography separation, and the yield of the prepared bousigonine D is as high as 0.25% and the purity is as high as 98%, which is conducive to further exploring more biological functions of drug molecule lead compound bousigonine D, and provides a selection template for developing small molecules for clinical drug use.

Description

Technical Field

[0001] The invention belongs to the technical field of alkaloid preparation, and particularly relates to a preparation method of bousigonine D. Background Art

[0002] The natural product bousigonine D belongs to the eburnane-aspidosperma type monoterpene indole alkaloid dimer. Its structure has a complex ring system and multiple chiralities. It is not practical to prepare it in the laboratory or factory by total synthesis or biomimetic synthesis. Therefore, the extraction and preparation of bousigonine D has broad application prospects in the alternative utilization of plant resources and the discovery of drug leads.

[0003] At present, the extraction and preparation method of bousigonine D is to separate it from Bousigonia mekongensis. For example, the prior art (Zhi-Wei Wang, et al. Monoterpene indole alkaloids from the roots of Bousigonia mekongensis and their anti-diabetic nephropathy activity, Fitoterapia, 2021, 153, 104964) discloses a method for separating bousigonine D from Bousigonia mekongensis. However, the yield of bousigonine D in the above-mentioned prior art is low (only 0.000183%), resulting in its activity research only staying at the cellular level. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for preparing bousigonine D. The yield of bousigonine D prepared by the preparation method provided by the present invention is high.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] The present invention provides a method for preparing bousigonine D, comprising the following steps:

[0007] Mixing the milk fruit with alcohol-water for extraction and concentrating to obtain an alcohol extract paste; the volume fraction of alcohol in the alcohol-water is 90-100%;

[0008] Dispersing the alcohol extract paste in water, extracting with petroleum ether and ethyl acetate in sequence to obtain an ethyl acetate extraction phase;

[0009] The ethyl acetate extract phase is subjected to silica gel column chromatography to obtain component Fr.2; the eluent used in the silica gel column chromatography separation includes dichloromethane-methanol; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 50:1 to 1:1;

[0010] The component Fr.2 is subjected to gel column chromatography to obtain an alkaloid component; the eluent used in the gel column chromatography is a dichloromethane-methanol mixture; the volume fraction of dichloromethane in the dichloromethane-methanol mixture is 50%;

[0011] The alkaloid components are separated by reverse phase column chromatography to obtain bousigonine D; the eluent used in the reverse phase column chromatography separation is methanol-water, and the volume fraction of methanol in the methanol-water is 20-30%, 40-50%, 60-70% and 85-100% in sequence;

[0012] The structure of bousigonine D is shown in Formula I:

[0013]

[0014] Preferably, the solid-liquid ratio of the dry weight of the milk fruit to the alcohol-water used for a single mixed extraction is 1g:4-10mL;

[0015] The alcohol in the alcohol-water includes methanol and / or ethanol.

[0016] Preferably, the temperature of the mixed extraction is 45-50° C.; the number of mixed extractions is 3-4 times, and the time of a single mixed extraction is 3-4 hours.

[0017] The ratio of the mass of the extract obtained by the mixed extraction to the mass of the alcohol extract paste is 40 to 80:1.

[0018] Preferably, the mass of the dispersion water is 2-5% of the mass of the alcohol extract paste.

[0019] Preferably, the petroleum ether extraction is performed 3 to 5 times, and the ratio of the mass of the alcohol-extracted paste to the volume of petroleum ether used for a single petroleum ether extraction is 1 g: 3 to 10 mL.

[0020] Preferably, the number of ethyl acetate extractions is 3 to 5 times, and the ratio of the mass of the alcohol-extracted paste to the volume of ethyl acetate used for a single ethyl acetate extraction is 1 g: 3 to 10 mL.

[0021] Preferably, during the silica gel column chromatography separation process, the volume ratios of dichloromethane and methanol in dichloromethane-methanol are 50:1, 30:1, 20:1, 10:1, 5:1 and 1:1, respectively.

[0022] Preferably, the eluent used in the silica gel column chromatography separation further comprises one or more of petroleum ether-acetone, dichloromethane-acetone and petroleum ether-ethyl acetate.

[0023] Preferably, after the reverse phase column chromatography separation, the method further comprises: collecting an eluate having a methanol volume fraction of 85-100% for crystallization to obtain bousigonine D.

[0024] The present invention provides a preparation method of bousigonine D. The present invention separates and prepares bousigonine D from the fruit of the man's milk for the first time, and the target compound bousigonine D is dissolved to the maximum extent by subjecting the fruit of the man's milk to alcohol-water extraction and petroleum ether and ethyl acetate extraction, and then silica gel column chromatography separation, gel column chromatography separation and reverse phase column chromatography separation are sequentially performed, and the yield of the prepared bousigonine D is as high as 0.25% and the purity is as high as 98%, which is conducive to further exploring more biological functions of the drug molecule lead compound bousigonine D, and provides a selection template for the development of small molecules for clinical drug use. Moreover, the preparation method provided by the present invention is simple, low in cost, and suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0026] Figure 1 is a process flow chart of the preparation of bousigonine D in Example 1;

[0027] Figure 2 Bousigonine D prepared in Example 1 1 H spectrum (500 MHz, DMSO-d 6 );

[0028] Figure 3 Bousigonine D prepared in Example 1 13 C spectrum (125MHz, DMSO-d 6 ). DETAILED DESCRIPTION

[0029] The present invention provides a method for preparing bousigonine D, comprising the following steps:

[0030] Mixing the milk fruit with alcohol-water for extraction and concentrating to obtain an alcohol extract paste; the volume fraction of alcohol in the alcohol-water is 90-100%;

[0031] Dispersing the alcohol extract paste in water, extracting with petroleum ether and ethyl acetate in sequence to obtain an ethyl acetate extraction phase;

[0032] The ethyl acetate extract phase is subjected to silica gel column chromatography to obtain component Fr.2; the eluent used in the silica gel column chromatography separation includes dichloromethane-methanol; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 50:1 to 1:1;

[0033] The component Fr.2 is subjected to gel column chromatography to obtain an alkaloid component; the eluent used in the gel column chromatography is a dichloromethane-methanol mixture; the volume fraction of dichloromethane in the dichloromethane-methanol mixture is 50%;

[0034] The alkaloid components are separated by reverse phase column chromatography to obtain bousigonine D; the eluent used in the reverse phase column chromatography separation is methanol-water, and the volume fraction of methanol in the methanol-water is 20-30%, 40-50%, 60-70% and 85-100% in sequence;

[0035] The structure of bousigonine D is shown in Formula I:

[0036]

[0037] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0038] The invention extracts the milk fruit by mixing with alcohol-water and then concentrates the mixture to obtain an alcohol extract paste; the volume fraction of alcohol in the alcohol-water is 90-100%.

[0039] In the present invention, the milk-sucking fruit is preferably dried milk-sucking fruit branches and / or fruits; the milk-sucking fruit is preferably from Yunnan Province. In the present invention, the milk-sucking fruit is preferably crushed to a particle size of 1 to 10 mm, more preferably 3 to 7 mm, and most preferably 4 to 6 mm before use.

[0040] In the present invention, the alcohol in the alcohol-water preferably includes methanol and / or ethanol, more preferably methanol. In the present invention, the volume fraction of the alcohol in the alcohol-water is 90-100%, preferably 91-98%, more preferably 92-96%, and most preferably 93-95%.

[0041] In the present invention, the temperature of the mixed extraction is preferably 45-50°C, more preferably 46-49°C, and most preferably 47-48°C; the number of mixed extractions is preferably 3-4 times; the time for a single mixed extraction is preferably 3-4h, more preferably 3.2-3.8h, and most preferably 3.4-3.6h.

[0042] In the present invention, the solid-liquid ratio of the dry weight of the milk fruit to the alcohol-water for single mixed extraction is preferably 1g:4-10mL, more preferably 1g:5-8mL, and most preferably 1g:6-7mL.

[0043] In the present invention, the ratio of the mass of the extract obtained by the mixed extraction to the mass of the alcohol extract paste is preferably 40 to 80:1, more preferably 50 to 70:1, and most preferably 55 to 65:1. In the present invention, the concentration temperature is preferably 35 to 45°C, more preferably 38 to 42°C, and most preferably 39 to 40°C; the concentration is preferably reduced pressure concentration; the pressure of the reduced pressure concentration is preferably 0.03 to 0.06 MPa, and more preferably 0.04 to 0.05 MPa. The present invention does not specifically limit the time of the concentration, as long as the mass ratio of the alcohol extract paste to the extract is within the above range.

[0044] After obtaining the alcohol extract paste, the present invention disperses the alcohol extract paste in water, and sequentially extracts with petroleum ether and ethyl acetate to obtain an ethyl acetate extraction phase.

[0045] In the present invention, the dispersion water is preferably distilled water; the mass of the dispersion water is preferably 2-5% of the mass of the alcohol extract paste, more preferably 3-4%.

[0046] In the present invention, the number of petroleum ether extractions is preferably 3 to 5 times, more preferably 3 to 4 times, and most preferably 3 times; the ratio of the mass of the alcohol extract paste to the volume of petroleum ether used for a single petroleum ether extraction is preferably 1 g: 3 to 10 mL, more preferably 1 g: 4 to 8 mL, and most preferably 1 g: 5 to 6 mL.

[0047] In the present invention, the number of ethyl acetate extractions is preferably 3 times, more preferably 3 to 4 times, and most preferably 3 times; the ratio of the mass of the alcohol extract paste to the volume of ethyl acetate used for a single ethyl acetate extraction is preferably 1g:3 to 10mL, more preferably 1g:4 to 8mL, and most preferably 1g:5 to 6mL.

[0048] After obtaining the ethyl acetate extraction phase, the present invention performs silica gel column chromatography separation on the ethyl acetate extraction phase to obtain component Fr.2; the eluent used in the silica gel column chromatography separation includes dichloromethane-methanol; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 50:1 to 1:1.

[0049] In the present invention, in the silica gel column chromatography separation process, the volume ratio of dichloromethane to methanol in dichloromethane-methanol is preferably 50:1, 30:1, 20:1, 10:1, 5:1 and 1:1, respectively.

[0050] In the present invention, the eluent used in the silica gel column chromatography separation preferably also includes one or more of petroleum ether-acetone, dichloromethane-acetone and petroleum ether-ethyl acetate. In the present invention, when the eluent used in the silica gel column chromatography separation also includes petroleum ether-acetone, the silica gel column chromatography separation preferably uses petroleum ether-acetone and dichloromethane-methanol for elution in sequence. In the present invention, during the silica gel column chromatography separation, the volume ratio of dichloromethane and methanol in dichloromethane-methanol is preferably the same as above, and no further details are given here. In the present invention, during the silica gel column chromatography separation, the volume ratio of petroleum ether and acetone in petroleum ether-acetone is preferably 1:0 to 0:1, and more preferably 1:0, 20:1, 10:1 and 5:1 in sequence.

[0051] In the present invention, the silica gel particle size used in the silica gel column chromatography separation is preferably 100-300 mesh, more preferably 100-200 mesh, and most preferably 200 mesh. In the present invention, when the silica gel column chromatography separation uses petroleum ether-acetone and dichloromethane-methanol for elution in sequence, the silica gel particle size used in the silica gel column chromatography separation is preferably 100-200 mesh and 200-300 mesh in sequence. In the present invention, the silica gel column chromatography separation process preferably also includes thin layer chromatography detection, and the elution parts with similar color development are combined to obtain 5 components, which are successively recorded as component Fr.1, component Fr.2, component Fr.3, component Fr.4 and component Fr.5, and the component Fr.2 is further separated.

[0052] After obtaining component Fr.2, the present invention performs gel column chromatography separation on the component Fr.2 to obtain an alkaloid component; the eluent used in the gel column chromatography separation is a dichloromethane-methanol mixture; the volume fraction of dichloromethane in the dichloromethane-methanol mixture is 50%.

[0053] In the present invention, the chromatographic column used in the gel column chromatography separation is preferably a Sephadex LH-20 gel chromatography column. In the invention, before the component Fr.2 is subjected to gel column chromatography separation, it is preferred to use a dichloromethane-methanol mixed solution to dissolve the component Fr.2. In the present invention, the volume ratio of dichloromethane and methanol in the dichloromethane-methanol mixed solution is preferably 1:1 to 3, more preferably 1:2. In the present invention, the gel column chromatography separation process preferably also includes thin layer chromatography detection.

[0054] After obtaining the alkaloid components, the present invention performs reverse phase column chromatography separation on the alkaloid components to obtain bousigonine D; the eluent used in the reverse phase column chromatography separation is methanol-water, and the volume fraction of methanol in the methanol-water is 20-30%, 40-50%, 60-70% and 85-100% in sequence.

[0055] In the present invention, the chromatographic column used for the reverse phase column chromatography separation is preferably a reverse phase-C18 chromatographic column. After the reverse phase column chromatography separation, the present invention preferably further comprises collecting an eluent having a methanol volume fraction of 85 to 100% for crystallization to obtain bousigonine D. In the present invention, the collected eluent is preferably an eluent having a methanol volume fraction of 90%. In the present invention, the crystallization is preferably crystallized by standing at room temperature; the crystallization time is preferably 24 to 48 hours, more preferably 30 to 45 hours, and most preferably 35 to 40 hours. In the present invention, the eluent is allowed to stand for crystallization, and the methanol aqueous solution evaporates in a standing state to obtain crystallized bousigonine D.

[0056] In order to further illustrate the present invention, the preparation method of bousigonine D provided by the present invention is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0057] Example 1

[0058] Figure 1 The preparation process flow chart of bousigonine D in this embodiment is as follows:

[0059] 8.0 kg of dried twigs and leaves of the milk fruit were crushed to a particle size of 1 to 10 mm, and then hot-extracted with 95% methanol-water (v / v) for 3 times, each time for 4 hours, and then the methanol-water was recovered after vacuum concentration to obtain a methanol extract paste. The hot extraction temperature was 45°C; the solid-liquid ratio of the dry weight of the milk fruit branches and leaves to the methanol-water used for a single hot extraction was 1g:6mL; the temperature of the vacuum concentration was 40°C and the pressure was 0.05MPa.

[0060] The methanol extract paste was dispersed with water, extracted with petroleum ether and ethyl acetate three times in sequence to obtain the ethyl acetate extraction phase. Among them, the mass of water is 3% of the mass of the methanol extract paste; the ratio of the mass of the methanol extract paste to the volume of petroleum ether used for a single extraction is 1g:5mL; the ratio of the mass of the methanol extract paste to the volume of ethyl acetate used for a single extraction is 1g:5mL.

[0061] The ethyl acetate extract was mixed with silica gel and loaded on a column. The two-phase system of petroleum ether-acetone and dichloromethane-methanol was used for elution. The eluted fractions were detected by TLC, and the same fractions were combined and recorded as components Fr.1, Fr.2, Fr.3, Fr.4 and Fr.5. Among them, the mass ratio of the ethyl acetate extract and silica gel was 1:1.5; when petroleum ether-acetone was used for elution, the volume ratio of petroleum ether and acetone was 1:0, 20:1, 10:1 and 5:1; when dichloromethane-methanol was used for elution, the volume ratio of dichloromethane and methanol was 50:1, 30:1, 20:1, 10:1, 5:1 and 1:1.

[0062] The obtained component Fr.2 was dissolved in dichloromethane-methanol, loaded on Sephadex LH-20 gel, eluted with dichloromethane-methanol, detected by thin layer chromatography (TLC), and non-alkali was removed to obtain the alkaloid component. The volume ratio of dichloromethane to methanol in dichloromethane-methanol was 1:1.

[0063] The alkaloid components were separated by reverse phase-C18 chromatographic column (RP-C18), and after column treatment, methanol-water was used for elution, and the volume fraction of methanol in methanol-water was 30%, 50%, 70%, 90%, and 100% in sequence. The eluate with a volume fraction of methanol of 90% was collected, slowly evaporated at room temperature, and crystallized for 36 hours to obtain 2.0g of pure bousigonine D in the form of colorless transparent needle crystals, with a yield of 0.25% and a purity of 98%.

[0064] The structure of the pure bousigonine D prepared above was identified as follows:

[0065] The ultraviolet determination was carried out using a Shimazu UV2401PC ultraviolet-visible spectrophotometer. Using TLC, the product showed strong fluorescence at 254 nm. The potassium bismuth iodide color developer showed orange-yellow color, proving that the extract was an alkaloid.

[0066] Bruker Avance-600 MHz NMR spectrometer was used for NMR determination (with TMS as internal standard). Figure 2 Bousigonine D 1 H spectrum (500 MHz, DMSO-d 6 ); Figure 3 Bousigonine D prepared in Example 1 13 C spectrum (125MHz, DMSO-d 6 ). The specific NMR results are as follows:

[0067] 1 H-NMR (DMSO-d 6,500MHz)δ:5.62(1H,br s,1-NH),3.35(1H,br s,H-2),2.17(1H,m,H-3a),3.01(1H,m,H-3b),1.98(1H,m,H-5a),2.93(1H,m,5b),1.00(1H,m,H-6a),1.89(1H,m,H-6b),6.53(1H,d,J=10.0Hz,H-9),6.48(1H,br s,H-10),8.91(1H,br s,12-OH),1.41(1H,m,H-14a),1.91(1H,m,H-14b),1.56(1H,m,H-15a),2.04(1H,m,H-15b),1.41(1H,m,H-16a),1.75(1H,m,H-16b),1.01(1H,m,H-17a),1.89(1H,m,H-17b),0.74(3H,t,like,H-18),1.08(1H,m,H-19a),1.87(1H,m,H-19b),2.88(1H,s,H-21),2.28(1H,t,J=11.5Hz,H-3'a);2.48(1H,m,H-3'b),3.16(2H,m,H-5'),2.44(1H,m,H-6'a),2.88(1H,m,H-6'b),7.31(1H,d,J=10.0Hz,H-9'),6.86(1H,t,J=5.0Hz,H-10'),6.64(1H,t,J=5.0Hz,H-11'),6.02(1H,br s,H-12'),1.32(1H,m,H-14'a),1.61(1H,m,H-14'b),1.17(1H,m,H-15'a),1.32(1H,m,H-15'b),5.22(1H,dd,J=5.0,10.0Hz,H-16),1.70(1H,m,H-17'a),1.95(1H,m,H-17'b),0.82(3H,t,J=5.0Hz,H-18'),1.40(1H,m,H-19'a),2.07(1H,m,H-19'b),3.91(1H,s,H-21');

[0068] 13 C-NMR(DMSO-d 6, 125MHz) δ: 64.4(C-2), 52.4(C-3), 53.6(C-5), 34.4(C-6), 53.1(C-7), 118.6(C-8), 111.9(C-9), 125.8(C-10), 114.4(C-11), 149.7 (C-12), 153.1(C-13), 21.4(C-14), 35.9(C-15), 28.6(C-16), 22.2(C-17), 6.7(C-18), 27.7(C-19), 35.2(C-20), 68.3(C-21), 134.1 (C-2'), 44.2(C-3'), 50.4(C-5'), 16.7(C-6'), 103.5(C-7'), 128.2(C-8'), 117.3(C-9'), 118.5(C-10'), 119.2(C-11'), 107.2(C-1 2'), 136.1(C-13'), 20.4(C-14'), 24.2(C-15'), 49.0(C-16'), 42.5(C-17'), 7.5(C-18'), 28.3(C-19'), 34.6(C-20'), 58.8(C-21').

[0069] Depend on Figure 2 , Figure 3 The NMR results show that the product prepared by the present invention is pure bousigonine D.

[0070] Example 2

[0071] 8.0 kg of dried leaves and branches of the milk fruit were crushed to a particle size of 4-6 mm, and then hot-extracted with anhydrous methanol for 3 times, each time for 3 hours. After vacuum concentration, methanol-water was recovered to obtain a methanol extract paste. The hot extraction temperature was 45°C; the solid-liquid ratio of the dry weight of the milk fruit branches and leaves to the anhydrous methanol used for a single hot extraction was 1g:6mL; the vacuum concentration temperature was 40°C and the pressure was 0.05MPa.

[0072] The methanol extract paste was dispersed in water, extracted with petroleum ether and ethyl acetate three times in sequence, and the ethyl acetate extraction phase was obtained. Among them, the mass of water was 4% of the mass of the methanol extract paste; the mass of the methanol extract paste and the volume of petroleum ether used for a single extraction were 1g:5mL; the mass of the methanol extract paste and the volume of ethyl acetate used for a single extraction were 1g:5mL.

[0073] The ethyl acetate extract was mixed with silica gel and loaded into the column. The two-phase system of dichloromethane-methanol was used for elution. The eluted fractions were detected by TLC, and the same fractions were combined and recorded as components Fr.1, Fr.2, Fr.3, Fr.4 and Fr.5. The mass ratio of the ethyl acetate extract to the silica gel was 1:1.5; the volume ratio of dichloromethane to methanol during the elution process was 50:1, 30:1, 20:1, 10:1, 5:1, and 1:1.

[0074] The obtained component Fr.2 was dissolved in dichloromethane-methanol, loaded on Sephadex LH-20 gel, eluted with dichloromethane-methanol, detected by thin layer chromatography (TLC), and non-alkali was removed to obtain the alkaloid component. The volume ratio of dichloromethane to methanol in dichloromethane-methanol was 1:1.

[0075] The alkaloid components were separated by reverse phase-C18 chromatographic column (RP-C18), and eluted with methanol-water, wherein the volume fraction of methanol in the methanol-water was 30%, 50%, 70%, 90%, and 100% in sequence. The eluate with a volume fraction of methanol of 90% was collected, slowly evaporated at room temperature, and crystallized for 36 hours to obtain 2.0 g of pure bousigonine D, with a yield of 0.25% and a purity of 98%.

[0076] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing bousigonine D, characterized in that: The following steps are involved: The milk fruit is mixed with alcohol-water for extraction and then concentrated to obtain an alcohol extract paste; the volume fraction of alcohol in the alcohol-water is 90-100%; the alcohol in the alcohol-water is methanol; Dispersing the alcohol extract paste in water, extracting with petroleum ether and ethyl acetate in sequence to obtain an ethyl acetate extraction phase; The ethyl acetate extract phase is subjected to silica gel column chromatography to obtain component Fr.2; the eluent used in the silica gel column chromatography separation includes dichloromethane-methanol; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol is 50:1 to 1:1; The component Fr.2 is subjected to gel column chromatography to obtain an alkaloid component; the eluent used in the gel column chromatography is a dichloromethane-methanol mixture; the volume fraction of dichloromethane in the dichloromethane-methanol mixture is 50%; The alkaloid components are separated by reverse phase column chromatography to obtain bousigonine D; the eluent used in the reverse phase column chromatography separation is methanol-water, and the volume fraction of methanol in the methanol-water is 20-30%, 40-50%, 60-70% and 85-100% in sequence; The structure of bousigonine D is shown in Formula I:

2. The preparation method according to claim 1, characterized in that: The solid-liquid ratio of the dry weight of the milk fruit to the alcohol-water used for single mixed extraction is 1g:4-10mL.

3. The preparation method according to claim 1 or 2, characterized in that: The temperature of the mixed extraction is 45-50° C.; the number of mixed extractions is 3-4 times, and the time of a single mixed extraction is 3-4 hours.

4. The preparation method according to claim 1 or 2, characterized in that: The ratio of the mass of the extract obtained by the mixed extraction to the mass of the alcohol extract paste is 40 to 80:

1.

5. The preparation method according to claim 1, characterized in that: The mass of the dispersing water is 2-5% of the mass of the alcohol extraction paste.

6. The preparation method according to claim 1 or 5, characterized in that: The petroleum ether extraction is performed 3 to 5 times, and the ratio of the mass of the alcohol-extracted paste to the volume of the petroleum ether used for a single petroleum ether extraction is 1 g: 3 to 10 mL.

7. The preparation method according to claim 1 or 5, characterized in that: The number of ethyl acetate extractions is 3 to 5 times, and the ratio of the mass of the alcohol-extracted paste to the volume of ethyl acetate used in a single ethyl acetate extraction is 1 g: 3 to 10 mL.

8. The preparation method according to claim 1, characterized in that: During the silica gel column chromatography separation process, the volume ratios of dichloromethane to methanol in dichloromethane-methanol are 50:1, 30:1, 20:1, 10:1, 5:1 and 1:1, respectively.

9. The preparation method according to claim 1, characterized in that: The eluent used in the silica gel column chromatography separation also includes any one of petroleum ether-acetone, dichloromethane-acetone and petroleum ether-ethyl acetate.

10. The preparation method according to claim 1, characterized in that: After the reverse phase column chromatography separation, the method further comprises: collecting an eluate having a methanol volume fraction of 85-100% for crystallization to obtain bousigonine D.

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