Acanthopanax trifoliatus extract and preparation method thereof

Abstract

The invention belongs to the field of biological medicines, and discloses an Acanthopanax trifoliatus extract and a preparation method thereof, and the Acanthopanax trifoliatus extract comprises a plurality of acridone alkaloid analogues in ten compounds JBL-1-JBL-10. The Acanthopanax trifoliatus extract is simple in preparation path and high in extraction efficiency of acridone alkaloid analogues, the Acanthopanax trifoliatus raw material is fully utilized, and the Acanthopanax trifoliatus extract is expected to be used for inhibition treatment of prostatic cancer (LNCaP), neuroblastoma (SH SY5Y), hepatoblastoma (HepG2) and colorectal cancer (HT29).

Description

Technical Field

[0001] The invention belongs to the field of biological medicines, and particularly relates to a safflower extract and a preparation method thereof. Background Art

[0002] Atalantia buxifolia (Poir.) Oliv., also known as Dongfeng orange, is an evergreen shrub or small tree of the genus Atalantia in the Rutaceae family. According to Chinese medicine research, Atalantia buxifolia has the effects of dispelling wind and relieving exterior symptoms, resolving phlegm and relieving cough, regulating qi and relieving pain. It is pungent, bitter, slightly warm, and enters the lung, stomach, and spleen meridians. Modern pharmacological research shows that it has good pharmacological effects such as resolving phlegm, relieving cough, relieving asthma, anti-inflammatory, antibacterial, anti-tumor, and cytotoxicity. It is currently used clinically to treat chronic bronchitis. Chemical composition research has found that plants of the genus Rubus contain compounds of different structural types, including alkaloids, triterpenes, volatile oils, flavonoids, coumarins, amides, sterols, etc. Among them, the typical ingredient acridone alkaloids have good anti-tumor activity. Studies have shown that acridone alkaloids have very broad potential in inhibiting four types of cancer cells: prostate cancer (LNCaP), neuroblastoma (SH SY5Y), hepatoblastoma (HepG2), and colorectal cancer (HT29).

[0003] The wine cake taro also contains other acridone alkaloid analogs with anti-tumor activity. There is an urgent need for a chemical purification method that can prepare wine cake taro extracts containing multiple acridone alkaloids and their analogs with anti-tumor activity, so as to fully utilize the wine cake taro raw materials and achieve better yield and efficacy. Summary of the invention

[0004] The object of the present invention is to overcome at least one deficiency of the prior art and to provide a Herba Lycopodii extract comprising a plurality of acridone alkaloids and analogues thereof with anti-tumor activity.

[0005] The technical solution adopted by the present invention is:

[0006] A scutellaria baicalensis extract, comprising a plurality of acridone alkaloid analogs among ten compounds JBL-1 to JBL-10, and the scutellaria baicalensis extract satisfies at least one of the following characteristics:

[0007] 1) After separation through a Sephadex LH-20 gel column, the same components were combined after TLC detection to obtain 8 fractions B5-F1 to B5-F8, among which fractions B5-F6 and B5-F7 were eluted again through Sephadex LH-20 (methanol) to obtain components B5-F6-2 and B5-F7-2, which were then analyzed through high performance liquid chromatography to purify and obtain at least one of compounds JBL-1 to JBL-10;

[0008] 2) When passing through FLASH ODS column chromatography, the same components are combined after TLC detection to obtain 9 fractions B7-F1 to B7-F9, and B7-F5 is eluted with Sephadex LH-20 (methanol) to obtain B7-F5-5; then purified by high performance liquid chromatography analysis to obtain at least one of compounds JBL-1 to JBL-10.

[0009] In some examples of the extract of Herba Lycopodii, the compounds JBL-1 to JBL-10 at least include compounds with the following spectra:

[0010] 1H-NMR (400MHz, DMSO) δ: 7.98 (1H, d, J = 9.5Hz, H-4), 7.62 (1H, d, J = 8.6Hz, H-5), 6.26 (1H, d, J = 9.5Hz, H-3) ,7.06(1H,d,J=8.6Hz,H-6),3.84(3H,s,7-OCH3),3.9(H-1'),1.11(3H,s,H-4'),1.09(3H,s,H-5'),13C-N MR(125MHz,DMSO)δ:210.8(C-2'),160.5(C-2),160.5(C-7),153.1(C-8a),145.1(C-4),128.5(C-5),112. 9(C-4a),112.7(C-3),111.6(C-8),108.3(C-6),56.8(OCH3),39.9(C-3'),34.8(C-1'),18.6(C-4',C-5');

[0011] 1H-NMR (400MHz, DMSO) δ: 8.09 (1H, d, J = 2.4Hz, H-2'), 8.1 (1H, d, J = 2.4Hz, H-2'), 6.34 (1H, d, J =9.5Hz,H-3),7.39(1H,d,J=2.4Hz,H-3'),4,17(3H,s,8-OCH3),4.03(3H,s,5-OCH3); 13C-NMR( 125MHz, DMSO)δ:159.8(C-2),149.4(C-7),146.4(C-2'),144.3(C-5),143.9(C-9),139.8(C-4) ,127.2(C-8),114.4(C-6),112.6(C-3),106.8(C-10),105.7(C-3'),61.3(OCH3),60.8(OCH3);

[0012] 1H-NMR(400MHz,CDCl3)δ:7.94(1H,d,J=8.0,1.4Hz,H-5),7.51(1H,dt,J=8.0,1.4Hz,H-7),7.31(1H,d,J=7.8Hz,H-8),7.24(1H,t,J=7.8Hz,H-6),4.21(3H,s,4-OCH3),3.94(3H,s,3-OCH3),3.71(3H,s,N-CH3);13C-NMR(125MHz,CDCl3)δ:161.3(C=O),151.6(C-4),136.6(C-10),135.2(C-3),129.8(C-7),123.7(C-5),122.3(C-6),118.1(C-9),113.8(C-8),60.9(OCH3),60.6(OCH3),29.6(N-CH3);

[0013] 1H-NMR(400MHz,CDCl3)δ:7.77(1H,d,J=9.8Hz,H-4),7.66(1H,d,J=2.0Hz,H-2'),7.37(1H,s,H-5),6.82(1H,d,J=2.0Hz,H-3'),6.37(1H,d,J=9.8Hz,H-3),5.20(2H,s,H-1”),1.21(3H,d,J=6.8Hz,H-4”),1.21(3H,d,J=6.8Hz,H-5”);13C-NMR(125MHz,CDCl3)δ:210.1(C-2”),160.3(C-2),146.8(C-2'),144.5(C-4),131.2(C-7),126.2(C-5),116.7(C-8a),114.9(C-3),113.5(C-4a),106.9(C-3'),75.4(C-1”),37.1(C-3”),18.1(C-4”);

[0014] 1H NMR (400 MHz, CD3OD) δ 7.84 (1H, d, J = 9.4 Hz, H-4), 7.43 (1H, d, J = 8.6 Hz, H-5), 6.77 (1H, d, J = 8.5 Hz, H-6), 6.68 (1H, s, H-8), 6.15 (1H, d, J = 9.4 Hz, H-3); 13C-NMR (125 MHz, CD3OD) δ: 164.3 (C-2), 163.8 (C-7), 157.4 (C-9), 146.1 (C-4), 130.6 (C-5), 114.9 (C-6), 112.7 (C-3), 111.7 (C-10), 103.5 (C-8);

[0015] 1H NMR (400 MHz, CD3OD) δ: 7.87 (1H, d, J = 8.8 Hz, H-8), 6.86 (1H, d, J = 8.8 Hz, H-7), 6.18 (1H, s, H-2), 3.84 (3H, s, 5-OCH3), 3.79 (3H, s, N-CH3), 3.76 (3H, s, 4-OCH3); 13C-NMR (125 MHz, CD3OD) δ: 182.4 (C-9), 161.4 (C-1), 160.0 (C-3), 158.6 (C-6), 144.0 (C-10a), 143.5 (C-9a), 137.9 (C-5), 130.8 (C-4), 123.3 (C-8), 118.1 (C-7), 114.3 (C-8a), 106.1 (C-4a), 98.7 (C-2), 60.9 (OCH3), 60.8 (OCH3), 46.9 (N-CH3);

[0016] 1H NMR (400 MHz, CD3OD) δ: 7.73 (1H, d, J = 8.4 Hz, H-8), 7.20~7.12 (2H, m, H-6, H-7), 6.20 (1H, s, H-2), 3.83 (3H, s, OCH3), 3.76 (3H, s, N-CH3); 13C-NMR (100 MHz, CD3OD) δ: 183.3 (C-9), 162.7 (C-1), 160.1 (C-3), 149.7 (C-6), 143.8 (C-10a), 138.4 (C-5), 130.4 (C-4), 125.7 (C-9a), 123.9 (C-8), 120.6 (C-7), 117.0 (C-8a), 106.9 (C-4a), 98.1 (C-2), 60.3 (OCH3), 46.5 (N-CH3); and

[0017] 1H NMR (400MHz, CD3OD) δ: 7.71 (1H, d, J = 7.3Hz, H-8), 7.16 (2H, d, J = 8.3Hz, H-6, H-7), 6.41 (1H, s, H-2), 3.94 (3H, s, OCH3), 3.69 (3H, s, N-CH3).

[0018] In some examples of the Herba Lycopodiellae extract, the Herba Lycopodiellae extract is obtained by extracting the extract of the dried whole herb of Herba Lycopodiellae by soaking in ethyl acetate.

[0019] In some examples of the extract of the schisandra chinensis, when the extract of the schisandra chinensis is subjected to Sephadex LH-20 gel column detection, the mobile phase is a 1:1 mixture of dichloromethane and methanol solvent.

[0020] In some examples of the extract of the schisandra chinensis, when the extract of the schisandra chinensis is subjected to FLASH ODS column chromatography, the chromatographic conditions are:

[0021] Methanol(%) water(%) 5 95 10 90 15 85 20 80 25 75 30 70 50 50 0 100

[0022] The elution condition is gradient elution.

[0023] In some examples of the extract of Rhizoma Cistanches, the high performance liquid chromatography uses a reverse phase liquid chromatography column, wherein the stationary phase is C18 bonded silica gel; and the mobile phase is methanol-water or acetonitrile-water.

[0024] The second aspect of the present invention provides:

[0025] A method for preparing a sauerkraut extract, characterized in that it is used to prepare the sauerkraut extract according to any one of the above items, and the preparation method comprises:

[0026] S1: Crush the whole herb of the wine cake vine and dry it, add ten times the amount of n-hexane, ethyl acetate and formaldehyde, and soak for 24 hours;

[0027] S2: extract the soaking liquid, rotary evaporate, and concentrate under reduced pressure to obtain a hexane extract and an ethyl acetate extract;

[0028] S3: taking the ethyl acetate extract, separating it by FLASH silica gel column chromatography, and eluting and separating to obtain the Herba Lycopodii extract containing a plurality of acridone alkaloid analogs among the ten compounds JBL-1 to JBL-10.

[0029] In some examples of the preparation method, the FLASH silica gel column chromatography further comprises the following steps:

[0030] S31: Set the elution conditions to dichloromethane-methanol system, and the gradient elution conditions are:

[0031] Dichloromethane (%) Methanol(%) 100 0 90 10 80 20 70 30 60 40 50 50 0 100

[0032] obtaining a preliminary separation sample; and

[0033] S32: The preliminary separated sample is concentrated under reduced pressure and then transferred into a vial, detected by TLC, observed under ultraviolet light, and then developed using a universal color developer sulfuric acid-ethanol, and the same components are combined to obtain 8 fractions B1 to B8.

[0034] In some examples of the preparation method, the preparation method further comprises:

[0035] S4: taking the B5 fraction, separating it through a Sephadex LH-20 gel column, and combining the same components after TLC detection to obtain 8 fractions B5-F1 to B5-F8; and

[0036] S5: The B5-F6 fraction and the B5-F7 fraction were eluted with methanol through Sephadex LH-20 to obtain the B5-F6-2 fraction and the B5-F7-2 fraction, which are the extract of the Herba Lycopodii.

[0037] In some examples of the preparation method, the preparation method further comprises:

[0038] S6: taking the B7 fraction, separating it through a FLASH ODS chromatographic column, and performing gradient elution with a methanol-water system to obtain 9 fractions B7-F1 to B7-F9; and

[0039] S7: The B7-F5 fraction was eluted with methanol through Sephadex LH-20 to obtain the B7-F5-5 fraction, which is the Herba Lycopodii extract.

[0040] The beneficial effects of the present invention are:

[0041] The extracts of some examples of the present invention contain a variety of acridone alkaloid analogs, which simplify the extraction process, improve the utilization rate of the raw materials of the extracts, and are conducive to achieving better yield and anti-tumor activity and efficacy. They are expected to be applied to the inhibitory treatment of prostate cancer (LNCaP), neuroblastoma (SH SY5Y), hepatoblastoma (HepG2) and colorectal cancer (HT29).

[0042] The preparation method of the extract of some examples of the present invention can obtain the extract of the Glechoma longituba containing at most ten compounds JBL-1 to JBL-10 by extracting the Glechoma longituba with ethyl acetate, thereby improving the utilization rate of the Glechoma longituba raw material.

[0043] The preparation methods of the extracts of some examples of the present invention can improve the purity and yield of the extracts of the extracts of the extracts of the extracts of the invention.

[0044] The preparation methods of the extracts of some examples of the present invention can separate the extracts of ... BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a chemical formula structure diagram of components JBL-1 to JBL-8 of the Herba Lysimachiae extract of the present application. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described below in conjunction with exemplary embodiments.

[0047] Experiment 1: Separation and purification of extracts from the herbaceous plant:

[0048] 1. Experimental Materials and Methods

[0049] 1.1 Experimental Materials

[0050] 1.1.1 Medicinal Materials

[0051] The medicinal material of Jiubingra was collected from Maoming City, Guangdong Province in July 2023, and 500g of the medicinal material sample was retained.

[0052] 1.1.2 Reagents and consumables

[0053] The main reagents and consumables are shown in the following table.

[0054] Table 1 Experimental materials

[0055]

[0056]

[0057] 1.1.3 Experimental instruments

[0058] The main experimental instruments are listed in the following table.

[0059] Table 2 Experimental instruments

[0060]

[0061] 1.2 Experimental methods

[0062] 1.2.1 Thin layer chromatography (TLC) analysis

[0063] Dissolve the sample in a suitable solvent, use a thin-layer glass spotting capillary to absorb an appropriate amount of the sample, and spot the sample 0.5 cm from the bottom of the thin layer. Spot the sample a second time after the spot is dry to avoid spot diffusion; put the spotted thin layer plate into the developing cylinder with the prepared developing agent, pre-saturate for 15 minutes, and take out the thin layer plate when the solvent front is about 1 cm away from the top of the thin layer. Place the thin layer plate on a plate baking machine to dry, and then observe it under a UV lamp. Finally, use concentrated sulfuric acid-vanillin color developer to develop the color of the thin layer plate.

[0064] 1.2.2 Fast silica gel column chromatography (FLASH) separation method

[0065] Silica gel is the most widely used adsorbent in natural product chemistry research. Silica gel is a polar adsorbent rich in silanol groups, and the target molecules are adsorbed and retained by silica gel through hydrogen bonds and dipole-dipole interactions.

[0066] The samples were analyzed by TLC in different solvent systems, and the system with the best separation effect was selected. After the sample was dissolved, blank silica gel (200-300 mesh) was added to mix the sample. After a proper amount of blank silica gel (80-100 mesh) was loaded into the FLASH column, it was installed on the rapid purification chromatography system instrument, and the blank silica gel was rinsed with washing liquid to make the blank silica gel settle, compact, flat, without fractures and bubbles. The upper column sample was evenly filled into the FLASH silica gel column. When eluting, an appropriate eluent was selected, such as dichloromethane-methanol, and gradient elution was performed from small to large polarity. The eluent was concentrated under reduced pressure on a rotary evaporator and transferred to a penicillin bottle. After detection by TLC thin layer chromatography, the eluents containing the same R f Values ​​and components with the same color rendering conditions.

[0067] 1.2.3 Sephadex LH-20 column chromatography

[0068] Sephadex is made of dextran and glycerol cross-linked by ether bonds. The separation principle of Sephadex LH-20 is that small molecules have a longer retention time on gel filtration chromatography than large molecules according to their relative molecular weight.

[0069] Swell the gel in methanol solvent, stir appropriately to eliminate bubbles, slowly pour it into the glass column, and wait for it to settle naturally. Use as little eluent as possible to dissolve the sample, filter it, and use a long rubber-tipped dropper to absorb the sample solution. Slowly add it along the inner wall of the column vertically to about 1 cm above the column bed. Try to keep the gel surface as flat as possible when loading the sample. Open the column valve and slowly add the eluent (methanol) along the wall. Adjust the flow rate to 8-10 drops / min and pass the column at normal pressure. Collect the eluent in a vial, collecting 5-6 mL per bottle. Perform TLC analysis on the collected components and combine those containing the same R f The composition of the compound with different color values ​​and color development conditions.

[0070] 1.2.4 Analytical High Performance Liquid Chromatography

[0071] Filter the prepared mobile phase, ultrasonically degas, and place the sample in the injection bottle after filtering. Turn on the pump power and detector power, adjust the detector to the required wavelength, replace the required mobile phase, exhaust the bubbles, flush the system, and install the column. After balancing the chromatographic column, run according to the set method or sequence. After the HPLC analysis is completed, flush the chromatographic column and system, and finally turn off the detector power and pump power.

[0072] 1.2.5 Preparative High Performance Liquid Chromatography

[0073] Preparative HPLC has the advantages of large sample loading capacity, high separation, and good reproducibility. The stationary phase of reversed-phase preparative HPLC is generally C 18 Bonded silica gel; the mobile phase is generally methanol-water or acetonitrile-water, and the commonly used detector is a UV detector.

[0074] Filter the prepared mobile phase, ultrasonically degas, and filter the sample. Turn on the pump power and detector power of the HPLC and adjust the detector wavelength. Remove bubbles and flush the system with solvent before loading the column. Equilibrate the column before injection. After manual injection, collect samples according to the chromatographic peaks in the real-time chromatogram. After preparation, flush the column, remove the chromatographic column, flush the system, and then turn off the pump power and detector power.

[0075] 2 Experimental results

[0076] 2.1 Solvent extraction of wine cake

[0077] The dried whole herb of Rhizoma Cibotii (dry weight 5.0 kg) was crushed and soaked in 10 times amount of n-hexane, ethyl acetate and methanol for 24 hours, and the soaking liquid was concentrated under reduced pressure in a rotary evaporator at 80 rpm under heating in a 45°C water bath, and repeated 3 times. About 38.42 g of n-hexane extract and about 290.18 g of ethyl acetate extract were collected.

[0078] 2.2 Systematic separation of ethyl acetate extract from wine cake

[0079] The extract B (70.1 g) of the ethyl acetate part of the wine cake was initially separated by FLASH silica gel column chromatography, using a dichloromethane-methanol system (100:0, 90:10, 80:20, 70:30, 60:40, 1:1, 0:100) gradient elution. The sample obtained by the initial separation was concentrated under reduced pressure and transferred to a penicillin bottle. After TLC detection and observation under ultraviolet light, a universal color developer sulfuric acid-ethanol was used for color development. After combining the same components, 8 fractions B1 to B8 were obtained.

[0080] B5 (4.6107 g) was separated by Sephadex LH-20 gel column (dichloromethane: methanol = 1: 1) and the same components were combined after TLC detection to obtain 8 fractions (B5-F1 to B5-F8). B5-F6 (731.2 mg) and B5-F7 (167.4 mg) were eluted by Sephadex LH-20 (methanol) to obtain B5-F6-2 (595.2 mg) and B5-F7-2 (150.8 mg). Subsequently, the target compound was prepared by HPLC analysis and preparative HPLC, and finally compounds JBL-1 to JBL-4 ​​were obtained.

[0081] B7 (15.2866 g) was subjected to FLASH ODS column chromatography, gradient elution with methanol-water system (5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 1:1, 100:0), and then concentrated under reduced pressure and detected by TLC. After observation under ultraviolet light, sulfuric acid-ethanol was used as a universal color developer to develop the color. After combining the same components, 9 fractions B7-F1 to B7-F9 were obtained. B7-F5 (897.8 mg) was eluted with Sephadex LH-20 (methanol) to obtain B7-F5-5 (595.2 mg). After HPLC analysis, the target compound was prepared by preparative HPLC, and finally compounds JBL-5 to JBL-10 were obtained.

[0082] 3 Experimental Summary

[0083] Through various separation methods such as fast silica gel column chromatography (FLASH) separation, Sephadex LH-20 column chromatography separation, and preparative high performance liquid chromatography, a total of ten compounds (JBL-1~JBL-10) have been isolated from the ethyl acetate extraction part of the branches and leaves of J.

[0084] Experiment 2: Qualitative analysis of extracts from the herbaceous plant:

[0085] 1. Experimental Materials

[0086] 1.1 Experimental Reagents

[0087] Table 3 Experimental reagents

[0088]

[0089]

[0090] 1.2 Experimental instruments

[0091] Table 4 Experimental instruments

[0092]

[0093] Spectral data of acridone alkaloid analogs JBL-1 to JBL-8 in 2-JBL extract

[0094] JBL-1: Pale yellow square crystal, molecular formula is C 16 H 18 O3. 1 H-NMR(400MHz,DMSO)δ:7.98(1H,d,J=9.5Hz,H-4),7.62(1H,d,J=8.6Hz,H-5),6.26(1H,d,J=9.5Hz,H-3),7.06(1H,d,J=8.6Hz,H-6),3.84(3H,s,7-OC H 3),3.9(H-1'),1.11(3H,s,H-4'),1.09(3H,s,H-5'), 13 C-NMR(125MHz,DMSO)δ:210.8(C-2'),160.5(C-2),160.5(C-7),153.1(C-8a),145 .1(C-4),128.5(C-5),112.9(C-4a),112.7(C-3),111.6(C-8),108.3(C-6),56.8(O C H3),39.9(C-3'),34.8(C-1'),18.6(C-4',C-5').

[0095] Compound JBL-1 was identified as isohesperidone based on spectral data.

[0096] JBL-2: Yellow powder, molecular formula is C 13 H 10 O5. 1 H-NMR(400MHz,DMSO)δ:8.09(1H,d,J=2.4Hz,H-2'),8.1(1H,d,J=2.4Hz,H-2'),6.34(1H,d,J=9.5Hz,H-3),7.39(1H,d,J=2.4Hz,H-3'),4,17(3H,s,8-OC H 3),4.03(3H,s,5-OC H 3); 13 C-NMR(125MHz,DMSO)δ:159.8(C-2),149.4(C-7),146.4(C-2'),144.3(C-5),143.9(C-9) ,139.8(C-4),127.2(C-8),114.4(C-6),112.6(C-3),106.8(C-10),105.7(C-3'),61.3(O CH3),60.8(O C H3).

[0097] Compound JBL-2 was identified as isoanisole based on spectral data.

[0098] JBL-3: light yellow oil, molecular formula is C 12 H 13 NO3. 1 H-NMR (400MHz, CDCl3) δ: 7.94 (1H, d, J = 8.0, 1.4 Hz, H-5), 7.51 (1H, dt, J = 8.0, 1.4 Hz,H-7),7.31(1H,d,J=7.8Hz,H-8),7.24(1H,t,J=7.8Hz,H-6),4.21(3H,s,4-OC H 3),3.94(3H,s,3-OC H 3),3.71(3H,s,NC H 3); 13 C-NMR (125MHz, CDCl3) δ: 161.3 (C=O), 151.6 (C-4), 136.6 (C-10), 135.2 (C- 3),129.8(C-7),123.7(C-5),122.3(C-6),118.1(C-9),113.8(C-8),60.9(O C H3),60.6(O C H3),29.6(N- C H3).

[0099] Compound JBL-3 was identified as N-Methylswietenidine B based on spectral data.

[0100] JBL-4: light yellow amorphous powder, molecular formula is C 16 H 14 O5. 1 H-NMR (400MHz, CDCl3) δ: 7.77 (1H, d, J = 9.8 Hz, H-4), 7.66 (1H, d, J = 2.0 Hz, H-2'), 7.37 (1H, s, H-5), 6.82 (1H, d, J = 2. 0Hz,H-3'),6.37(1H,d,J=9.8Hz,H-3),5.20(2H,s,H-1”),1.21(3H,d,J=6.8Hz,H-4”),1.21(3H,d,J=6.8Hz,H-5”); 13C-NMR (125MHz, CDCl3) δ: 210.1 (C-2”), 160.3 (C-2), 146.8 (C-2’), 144.5 (C-4), 131.2 (C-7), 126.2 ( C-5),116.7(C-8a),114.9(C-3),113.5(C-4a),106.9(C-3'),75.4(C-1”),37.1(C-3”),18.1(C-4”).

[0101] Compound JBL-4 ​​was identified as herbage oxychloranone based on spectral data.

[0102] JBL-5: Pale yellow lamellae, molecular formula is C9H6O3. 1 HNMR (400MHz, CD3OD) δ7.84(1H,d,J=9.4Hz,H-4),7.43(1H,d,J=8.6Hz,H-5),6.77(1H,d,J=8.5Hz,H-6),6.68(1H,s,H-8),6.15(1H,d,J=9.4Hz,H-3); 13 C-NMR (125MHz, CD3OD) δ: 164.3(C-2), 163.8(C-7), 157.4(C-9), 146.1(C-4), 130.6(C-5), 114.9(C-6), 112.7(C-3), 111.7(C-10), 103.5(C-8).

[0103] Compound JBL-5 was identified as umbelliferone based on spectral data.

[0104] JBL-6: Yellow needle crystal, molecular formula is C 16 H 15 NO6. 1 H NMR(400MHz,CD3OD)δ:7.87(1H,d,J=8.8Hz,H-8),6.86(1H,d,J=8.8Hz,H-7),6.18(1H,s,H-2),3.84(3H,s,5-OC H 3),3.79(3H,s,NC H 3),3.76(3H,s,4-OC H 3); 13C-NMR(125MHz,CD3OD)δ:182.4(C-9),161.4(C-1),160.0(C-3),158.6(C-6),144.0(C-10a),143.5(C-9 a),137.9(C-5),130.8(C-4),123.3(C-8),118.1(C-7),114.3(C-8a),106.1(C-4a),98.7(C-2),60.9(O C H3),60.8(O C H3),46.9(N- C H3).

[0105] Compound JBL-6 was identified as buxifoliadine H based on spectral data.

[0106] JBL-7: Yellow powder, molecular formula is C 15 H 13 NO5. 1 HNMR(400MHz,CD3OD)δ:7.73(1H,d,J=8.4Hz,H-8),7.20~7.12(2H,m,H-6,H-7),6.20(1H,s,H-2),3.83(3H,s,OC H 3),3.76(3H,s,NC H 3); 13 C-NMR(100MHz,CD3OD)δ:183.3(C-9),162.7(C-1),160.1(C-3),149.7(C-6),143.8(C-10a),138.4(C-5 ),130.4(C-4),125.7(C-9a),123.9(C-8),120.6(C-7),117.0(C-8a),106.9(C-4a),98.1(C-2),60.3(O C H3),46.5(N- C H3).

[0107] Compound JBL-7 was identified as citrusinine II based on spectral data.

[0108] JBL-8: Yellow powder, molecular formula is C 15 H 13 NO5. 1 HNMR(400MHz,CD3OD)δ:7.71(1H,d,J=7.3Hz,H-8),7.16(2H,d,J=8.3Hz,H-6,H-7),6.41(1H,s,H-2),3.94(3H,s,OCH 3),3.69(3H,s,NC H 3).

[0109] Based on the spectral data, compound JBL-8 was identified as 1,5-dihydroxy-3,4-dimethoxy-10-acridin-9(10H)-one.

[0110] 3 Chemical structures of compounds JBL-1 to JBL-8 in the extract of 3-Dry-Dried-Wine-Cake

[0111] See also Figure 1 , Figure 1 The chemical structures of compounds JBL1 to JBL8 are shown, which have characteristic groups: keto, hydroxyl, amino, aromatic ring and other functional groups.

[0112] 4. Experimental Summary and Discussion

[0113] Combined with the results of liquid chromatography-mass spectrometry, 10 acridone alkaloid analogues have been extracted and separated from the raw materials of J. japonica vine. The structures of 8 compounds (JBL-1~JBL-8) have been identified, namely, isohesperidone, isoanisole, N-methylswietenidine B, umbelliferone, buxifoliadine H, citrusinine II, and 1,5-dihydroxy-3,4-dimethoxy-10-acridin-9(10H)-one.

[0114] Based on the acridone alkaloid analogs isolated so far, we can understand the detailed characteristic information (such as ultraviolet absorption, polarity, etc.) of acridone alkaloid analogs to a certain extent, which will help to continue to search for and discover more acridone alkaloid analogs.

[0115] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A wine cake extract, characterized in that The extract of the Herba Lycopodii contains a plurality of acridone alkaloid analogs among ten compounds JBL-1 to JBL-10, and the extract of the Herba Lycopodii satisfies at least one of the following characteristics: 1) After separation through a Sephadex LH-20 gel column, the same components were combined after TLC detection to obtain 8 fractions B5-F1 to B5-F8, among which fractions B5-F6 and B5-F7 were eluted again through Sephadex LH-20 (methanol) to obtain fractions B5-F6-2 and B5-F7-2, which were then analyzed through high performance liquid chromatography to purify and obtain at least one of compounds JBL-1 to JBL-10; 2) When the FLASH ODS column chromatography was performed, the same components were combined after TLC detection to obtain 9 fractions B7-F1 to B7-F9, and B7-F5 was eluted with Sephadex LH-20 (methanol) to obtain B7-F5-5; and then analyzed by high performance liquid chromatography, Purify to obtain at least one of Compounds JBL-1 to JBL-10.

2. The extract of claim 1, characterized in that The compounds JBL-1 to JBL-10 at least include compounds with the following spectra: 1 H-NMR(400MHz,DMSO)δ:7.98(1H,d,J=9.5Hz,H-4),7.62(1H,d,J=8.6Hz,H-5),6.26(1H,d,J=9.5Hz,H-3),7.06(1H,d,J=8.6Hz,H-6),3.84(3H,s,7-OC H 3),3.9(H-1'),1.11(3H,s,H-4'),1.09(3H,s,H-5'), 13 C-NMR(125MHz,DMSO)δ:210.8(C-2'),160.5(C-2),160.5(C-7),153.1(C-8a),145.1(C-4),128.5(C-5),112.9(C-4a),112.7(C-3),111.6(C-8),108.3(C-6),56.8(O C H3),39.9(C-3'),34.8(C-1'),18.6(C-4',C-5'); 1 H-NMR(400MHz,DMSO)δ:8.09(1H,d,J=2.4Hz,H-2'),8.1(1H,d,J=2.4Hz,H-2'),6.34(1H,d,J=9.5Hz,H-3),7.39(1H,d,J=2.4Hz,H-3'),4,17(3H,s,8-OC H 3),4.03(3H,s,5-OC H 3); 13 C-NMR(125MHz,DMSO)δ:159.8(C-2),149.4(C-7),146.4(C-2'),144.3(C-5),143.9(C-9),139.8(C-4),127.2(C-8),114.4(C-6),112.6(C-3),106.8(C-10),105.7(C-3'),61.3(O C H3),60.8(O C H3); 1 H-NMR(400MHz,CDCl3)δ:7.94(1H,d,J=8.0,1.4Hz,H-5),7.51(1H,dt,J=8.0,1.4Hz,H-7),7.31(1H,d,J=7.8Hz,H-8),7.24(1H,t,J=7.8Hz,H-6),4.21(3H,s,4-OC H 3),3.94(3H,s,3-OC H 3),3.71(3H,s,N-C H 3); 13 C-NMR(125MHz,CDCl3)δ:161.3(C=O),151.6(C-4),136.6(C-10),135.2(C-3),129.8(C-7),123.7(C-5),122.3(C-6),118.1(C-9),113.8(C-8),60.9(O C H3),60.6(O C H3),29.6(N- C H3); 1 H-NMR(400MHz,CDCl3)δ:7.77(1H,d,J9.8Hz,H-4),7.66(1H,d,J2.0Hz,H-2'),7.37(1H,s,H-5),6.82(1H,d,J2. 0Hz,H-3'),6.37(1H,d,J9.8Hz,H-3),5.20(2H,s,H-1”),1.21(3H,d,J6.8Hz,H-4”),1.21(3H,d,J6.8Hz,H-5”) 13 C-NMR(125MHz,CDCl3)δ: 210.1(C-2", 160.3(C-2), 146.8(C-2'), 144.5(C-4), 131.2(C-7), 126.2( C-5), 116.7(C-8a), 114.9(C-3), 113.5(C-4a), 106.9(C-3'), 75.4(C-1"), 37.1(C-3"), 18.1(C-4") 1 H NMR(400MHz,CD3OD)δ7.84(1H,d,J=9.4Hz,H-4),7.43(1H,d,J=8.6Hz,H-5),6.77(1H,d,J=8.5Hz,H-6),6.68(1H,s,H-8),6.15(1H,d,J=9.4Hz,H-3); 13 C-NMR(125MHz,CD3OD)δ:164.3(C-2),163.8(C-7),157.4(C-9),146.1(C-4),130.6(C-5),114.9(C-6),112.7(C-3),111.7(C-10),103.5(C-8); 1 H NMR(400MHz,CD3OD)δ:7.87(1H,d,J=8.8Hz,H-8),6.86(1H,d,J=8.8Hz,H-7),6.18(1H,s,H-2),3.84(3H,s,5-OC H 3),3.79(3H,s,N-C H 3),3.76(3H,s,4-OC H 3); 13 C-NMR(125MHz,CD3OD)δ:182.4(C-9),161.4(C-1),160.0(C-3),158.6(C-6),144.0(C-10a),143.5(C-9a),137.9(C-5),130.8(C-4),123.3(C-8),118.1(C-7),114.3(C-8a),106.1(C-4a),98.7(C-2),60.9(O C H3),60.8(O C H3),46.9(N- C H3); 1 1H NMR (400 MHz, CD3OD) δ: 7.73 (1H, d, J = 8.4 Hz, H-8), 7.20 - 7.12 (2H, m, H-6, H-7), 6.20 (1H, s, H-2), 3.83 (3H, s, OC H 3), 3.76 (3H, s, N-C H 3); 13 13C-NMR (100 MHz, CD3OD) δ: 183.3 (C-9), 162.7 (C-1), 160.1 (C-3), 149.7 (C-6), 143.8 (C-10a), 138.4 (C-5), 130.4 (C-4), 125.7 (C-9a), 123.9 (C-8), 120.6 (C-7), 117.0 (C-8a), 106.9 (C-4a), 98.1 (C-2), 60.3 (O C H3), 46.5 (N- C H3); and 1 H NMR(400MHz,CD3OD)δ:7.71(1H,d,J=7.3Hz,H-8),7.16(2H,d,J=8.3Hz,H-6,H-7),6.41(1H,s,H-2),3.94(3H,s,OC H 3),3.69(3H,s,N-C H 3)。 3. The extract of Rhizoma Cibotii according to claim 1, characterized in that The extract of the Herba Lycopodiellae is obtained by extracting the extract of the dried whole herb of the Herba Lycopodiellae by soaking it in ethyl acetate.

4. The extract of Rhizoma Cibotii according to claim 1, characterized in that When the extract of the schisandra chinensis is subjected to Sephadex LH-20 gel column detection, the mobile phase is a 1:1 mixture of dichloromethane and methanol solvent.

5. The extract of Rhizoma Cibotii according to claim 1, characterized in that When the extract of the Herba Lycopodii is subjected to FLASHODS column chromatography, the chromatographic conditions are: The elution condition is gradient elution.

6. The extract of Rhizoma Cibotii according to claim 1, characterized in that The high performance liquid chromatography uses a reverse phase liquid chromatography column, wherein the stationary phase is C 18 Bonded silica gel; mobile phase is methanol-water or acetonitrile-water.

7. A method for preparing a safflower extract, characterized in that: For preparing the Herba Lycopodiellae extract according to any one of claims 1 to 6, the preparation method comprising: S1: Crush the whole herb of the wine cake vine and dry it, add ten times the amount of n-hexane, ethyl acetate and formaldehyde, and soak for 24 hours; S2: extract the soaking liquid, rotary evaporate, and concentrate under reduced pressure to obtain a hexane extract and an ethyl acetate extract; S3: taking the ethyl acetate extract, separating it by FLASH silica gel column chromatography, and eluting and separating to obtain the Herba Lycopodii extract containing a plurality of acridone alkaloid analogs among the ten compounds JBL-1 to JBL-10.

8. The preparation method according to claim 7, characterized in that: The FLASH silica gel column chromatography also The following steps are involved: S31: Set the elution conditions to dichloromethane-methanol system, and the gradient elution conditions are: ， obtaining a preliminary separation sample; and S32: The preliminary separated sample is concentrated under reduced pressure and then transferred into a vial, detected by TLC, observed under ultraviolet light, and then developed using a universal color developer sulfuric acid-ethanol, and the same components are combined to obtain 8 fractions B1 to B8.

9. The preparation method according to claim 8, characterized in that: The preparation method further comprises: S4: taking the B5 fraction, separating it through a Sephadex LH-20 gel column, and combining the same components after TLC detection to obtain 8 fractions B5-F1 to B5-F8; and S5: The B5-F6 fraction and the B5-F7 fraction were eluted with methanol through Sephadex LH-20 to obtain the B5-F6-2 fraction and the B5-F7-2 fraction, which are the extract of the Herba Lycopodii.

10. The preparation method according to claim 8, characterized in that: The preparation method further comprises: S6: taking the B7 fraction, separating it through a FLASH ODS chromatographic column, and performing gradient elution with a methanol-water system to obtain 9 fractions B7-F1 to B7-F9; and S7: The B7-F5 fraction was eluted with methanol through Sephadex LH-20 to obtain the B7-F5-5 fraction, which is the Herba Lycopodii extract.

Images