CN105294720B - A kind of dimerization chromone alkaloid compound and its preparation method and application - Google Patents

Abstract

The invention discloses a dimeric chromone alkaloid compound and its preparation method and application. The dimeric chromone alkaloid compound is obtained from the dried tree of the medicinal plant Cassia fistula of the leguminous family Cassia It is isolated from skin, its molecular formula is C ₂₆ H ₂₁ NO ₆ , it is named sausagein A, its English name is Fistulain A, and it has the following structural formula: The preparation method is to use the dried bark of the medicinal plant Cassia fistula ( Cassia fistula ) of the genus Fabaceae as raw material, and obtain it through extract extraction, organic solvent extraction, MCI decolorization, silica gel column chromatography, and high-pressure liquid chromatography separation steps. . The compound of the present invention is the first dimer chromone alkaloid of the novel skeleton type connected by C-14-N found in medicinal plants, and has good biological activity, and can be used as anticancer drug and anti-tobacco The lead compound of mosaic virus has good application prospect.

Description

A dimeric chromone alkaloid compound and its preparation method and application

technical field

The invention belongs to the technical field of extraction and separation, structure identification and activity screening of active ingredients in medicinal plants with ethnic characteristics, and specifically relates to a dimeric chromone alkaloid compound and its preparation method and application.

Background technique

Cassia fistula , a leguminous plant, is the national flower of Thailand. It is native to southern South Asia, distributed in Myanmar, Sri Lanka, India, and the south and southwest of mainland China. It grows at an altitude of 1,000 meters. In China, sausage tree is a kind of Chinese herbal medicine commonly used by the Dai people. It is widely used by the Dai people for the treatment of skin infections, obesity, periodic fever and tumor diseases. The sausage tree is also called "Guolongliang" in Dai language. Its fruit, seeds, roots and bark can be used as medicine, which contains quinone compounds, which have a purging effect; the fruit contains tannic acid compounds, and the bark can be used as medicine. It is used to make leather and red dye, etc. The fruits, roots, branches, leaves and bark of the Dai people are widely used as medicinal plants. Among them, the leaves of the sausage tree are bitter, astringent, and cool in nature, and have the effects of reducing swelling and pain, reducing fire and detoxification, diuresis and stone, dispersing wind and relieving pain; the seeds of the sausage tree are used to treat liver and gallbladder diseases, and can also increase appetite; The root is mainly used to treat various skin diseases, leprosy, tuberculosis and syphilis; its fruit has certain curative effect on inflammation, liver disease, sore throat, bronchial asthma, rheumatism and rheumatoid arthritis. Different parts of the plant have been reported to have antidiabetic, antitumor, anti-inflammatory, antiviral, antibacterial, and antioxidant activities. In the previous research on the chemical components of the plant, some conventional flavonoids, triterpenes, naphthoquinones, sugars and lignans were mainly obtained. Different from the previous ones, the present invention isolates a novel C-14-N-linked dimer chromone alkaloid compound from sausage tree, and the compound has significant cytotoxic activity and antiviral activity.

Contents of the invention

The first purpose of the present invention is to provide a dimeric chromone alkaloid compound; the second purpose is to provide the preparation method of the dimeric chromone alkaloid compound; the third purpose is to provide the dimeric chromone alkaloid compound Application of base compound in preparation of anti-cancer and anti-tobacco mosaic virus drugs.

The first object of the present invention is achieved in that the dimeric chromone alkaloid compound is isolated from the bark of the dried leguminous Cassia medicinal plant Cassia fistula , and its molecular formula is C ₂₆ H ₂₁ NO ₆ has the following structure:

The compound is a yellow jelly, named Fistulain A, and its English name is Fistulain A.

The second object of the present invention is achieved in this way. The preparation method of the dimeric chromone alkaloid compound is to use the dry bark of the leguminous plant Cassia fistula as raw material, extract through the extract, and use an organic solvent Extraction, MCI decolorization, silica gel column chromatography, high pressure liquid chromatography separation steps, specifically:

A. Extraction: crush the bark of the leguminous plant Cassia fistula to 20-40 meshes, use organic solvents for ultrasonic extraction 2-5 times, each time for 30-60 minutes, or extract by cold soaking for 2-5 Once, for 3 days each time, combine the extracts, filter, concentrate the extracts under reduced pressure, let stand, filter out the precipitate, and concentrate into extract a;

B. Organic solvent extraction: add water with a weight ratio of 1 to 2 times the amount of extract a, extract 3 to 5 times with an organic solvent equal to the volume of water, combine the organic solvent extraction phase, and concentrate under reduced pressure to form extract b;

C. MCI decolorization: the extract b is dissolved in methanol water with a weight ratio of 3 to 5 times, put on the MCI column, elute with 80%-95% methanol water, combine the organic solvent extraction phase, and concentrate under reduced pressure to form extract c;

D. Silica gel column chromatography: silica gel column chromatography on the extract c, the column silica gel is 160~200 mesh, and the dosage is 6~10 times the weight of the extract c; the volume ratio is 1:0~0:1 Gradient elution with mixed organic solvent of chloroform and acetone, the gradient elution solution was collected, concentrated, monitored by TLC, and the same parts were combined;

E. High-performance liquid chromatography separation: the eluent obtained by eluting with a petroleum ether-acetone solution with a volume content of 50-90% is separated and purified by high-performance liquid chromatography to obtain the dimeric chromone alkaloid compound .

The structure of the dimeric chromone compound prepared by the above method is identified by the following method:

The compound of the present invention is yellow jelly; (1) UV spectrum (solvent is methanol), λ _max (log ε ): 220(4.46), 248 (3.68), 276 (3.84), 322 (3.48), 376 (2.92 ) nm; (2) Infrared spectrum (potassium bromide pellet) ν _max : 3446, 3158, 2938, 1730, 1647, 1605, 1574, 1442, 1385, 1338, 1276, 1164, 1076, 862, 751 cm ^–1 (3) HRESIMS shows that the quasi-molecular ion peak of the compound of the present invention is m/z 466.1261[M+Na] ⁺ (calculated value is 466.1261), combined with ¹³ C and ¹ H NMR spectra (Figure 1 and Figure 2, carbon spectrum hydrogen spectrum data See Table 1 for the assignment). Its molecular formula is C ₂₆ H ₂₁ NO ₆ , and its degree of unsaturation is 17. See Table 1 for ¹ H NMR (CDCl ₃ , 500 MHz) and ¹³ C NMR (CDCl ₃ , 125 MHz) data. ¹ H NMR spectrum (Figure 2) shows 3 methyl signals ( δ _H 2.26, s, H ₃ -13; 2.38, s, H ₃ -9'; 2.51, s, H ₃ -12'), 2 Methylene signal ( δ _H 3.45, s, H ₂ -14; 4.24, s, H ₂ -11), 5 unimodal aromatic proton signals ( δ _H 6.21, s, H-3; 6.53, s, H- 6'; 6.78, s, H-8'; 6.85, s, H-3'; 6.93, s, H-10') and a 1,2,3,5-tetrasubstituted benzene ring signal ( δ _H 6.59, d, J = 1.8 Hz, H-6; 6.66, d, J = 1.8 Hz, H-8). In the carbon spectrum and DEPT spectrum (Figure 1), 26 carbon atom signals were observed, including 3 methyl groups (including two aromatic methyl groups), 2 methylene groups, 7 aromatic methine groups and 14 quaternary carbons Signals (including 3 carbonyl and 5 oxygen-containing quaternary carbon signals). Among them, 3 carbonyl groups and 18 double-bonded carbons occupy 12 degrees of unsaturation, so the molecule is a highly aromatizing pentacyclic alkaloid. Combining the above spectral data with the compounds previously isolated from Cassia, it is preliminarily speculated that sausagein A is a heterodimeric alkaloid composed of two different C13 skeletons. It consists of A and B fragments.

According to the characteristic signal ( δ _C 166.4, 113.1, 180.1, 138.5, 121.1, 163.8, 103.6, 161.4, 115.9; δ _H 6.21, 6.59, 6.66) of the A fragment (O-1 to C-14), it is speculated that it is a C13 Skeleton of chromones (A and B rings). This speculation is confirmed by HMBC correlation, that is, H-3 is related to C-2/C-4/C-10, H-6 is related to C-8/C-10, and H-8 is related to C-6/C-10 related (Fig. 3). Second, there is also 1 acetonyl group and 1 hydroxyl group attached to C-5 and C-7, respectively, which are also related to C-6/C _- 10 by H2-11 in the HMBC spectrum, and 7- Correlation of OH with C-6/C-7/C-8 was confirmed. These data show that the A segment of the compound is a 5-acetonyl-7-hydroxyl-2-chromone derivative. The main difference is that the C-14 position has changed from the original methyl group to a methylene group. It is speculated that the A segment is passed through C-14 is linked to the B segment. The remaining 13 carbon atoms, including 2 methyl groups, 4 methine groups, and 7 quaternary carbon signals, belong to the B segment. HMBC correlation gives the approximate structure of the B fragment. In the HMBC spectrum, H-6' and C-5'/C-7'/C-8', H-8' and C-6'/C-7'/C-8a', H ₃ - 9' with C-2'/C-3'/C-8a', H-3' with C-2'/C-4a'/C-9' and H-6' with C-4a'/C- 5'/C-7'/C-8'/C-10', thus confirming the basic fragments of chromone (C and D rings) in the B fragment. At the same time, H-10' and C-4a'/C-5'/C-6'/C-11'/C-12' and H-12' and C-14/C-4 were also observed in the HMBC spectrum '/C-10'/C-11', which shows that C-4', C-4a', C-5', C-10', C-11', C-12' and N atoms together A 2-picoline derivative fragment (ring E) is formed, which is bound together with C and D rings via C-4', C-4a', and C-5'. Finally, through the HMBC correlation between H ₂ -14 and C-4'/C-11' and C-14 of H ₃ -12', the connection sequence of A and B fragments was determined. So far, the dimeric chromone alkaloid The structure of the compound was determined.

Table 1 ¹ H and ¹³ C NMR data of compounds (solvent is CDCl ₃ ) (125 and 500 MHz)

No. ^{13C 1} h 2 166.4 s 3 113.1 d 6.21, s 4 180.1 s 5 138.5 s 6 121.1 d 6. 59 d (1.8) 7 163.8 s 8 103.6 d 6.66 d (1.8) 9 161.4 s 10 115.9 s 11 50.5t 4.24 s 12 207.9 s 13 30.8q 2.26 s 14 58.2 tons 3.45 s 2' 168.3 s 3′ 98.2 d 6.85s 4′ 146.3 s 4a' 110.2 s 5′ 136.2 s 6′ 108.3 d 6.53s 7′ 175.1s 8' 105.3 d 6.78 s 8a' 155.9 s 9' 22.1q 2.38 s 10′ 116.8 d 6.93 s 11′ 141.6 s 12′ 21.6q 2.51 s 7-OH 10.21 s

The third object of the present invention is achieved in this way, the application of the dimeric chromone alkaloid compound in the preparation of anticancer and antiviral drugs.

The compound of the present invention is isolated from the bark of the sausage tree for the first time, and is determined to be a dimerchromone alkaloid compound by nuclear magnetic resonance and mass spectrometry methods, and its specific structure is characterized. Using the compound of the present invention as a raw material, the cytotoxic activity against acute promyelocytic leukemia NB4 cells, lung adenocarcinoma A549 cells, human bone marrow neuroblastoma SHSY5Y cells, human prostate cancer PC3 cells, and human breast cancer MCF7 cells respectively reached 5.5 , 4.6, and 8.8 μM . The compound of the present invention is through the experiment of resisting tobacco mosaic virus, and its relative inhibition rate reaches 32.8% at 20 μ M, surpasses the relative inhibition rate (30.5%) of positive control product Nanningmycin, and its _IC50 value is 43.8 μ M, The activity is better than that of the positive control substance Nanningmycin (IC ₅₀ =52.4 μ M), indicating that the compound has good activity against tobacco mosaic virus.

The compound of the invention has a novel structure and good activity, can be used as a leading compound of anticancer and antiviral drugs, and has good application prospects in drug discovery and biopesticide preparation.

Description of drawings

Figure 1 is the carbon nuclear magnetic resonance spectrum ( ¹³ C NMR) of the compound sausagein A;

Figure 2 is the hydrogen nuclear magnetic resonance spectrum ( ¹ H NMR) of the compound sausagein A;

Figure 3 Major HMBC correlations for compound sausagein A.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings, but the present invention is not limited in any way, and any changes or improvements based on the teaching of the present invention fall within the protection scope of the present invention.

The dimeric chromone alkaloid compound of the present invention is isolated from the dried bark of the leguminous plant Cassia fistula , and its molecular formula is C ₂₆ H ₂₁ NO ₆ , which has the following structure :

The compound is a yellow jelly, named Fistulain A, and its English name is Fistulain A.

The preparation method of the dimeric chromone alkaloid compound of the present invention is to use the dry bark of the leguminous plant Cassiafistula as raw material, extract through extract, organic solvent extraction, MCI decolorization, silica gel column chromatography, High-performance liquid chromatography preparative separation steps, specifically:

A. Extraction: crush the bark of the leguminous plant Cassia fistula to 20-40 meshes, use organic solvents for ultrasonic extraction 2-5 times, each time for 30-60 minutes, or extract by cold soaking for 2-5 Once, each time for 3 days, combine the extracts, filter, concentrate the extracts under reduced pressure, let stand, filter out the precipitate, and concentrate into extract a;

B. Organic solvent extraction: add water with a weight ratio of 1 to 2 times the amount of extract a, extract 3 to 5 times with an organic solvent equal to the volume of water, combine the organic solvent extraction phase, and concentrate under reduced pressure to form extract b;

C. MCI decolorization: the extract b is dissolved in methanol water with a weight ratio of 3 to 5 times, put on the MCI column, elute with 80%-95% methanol water, combine the organic solvent extraction phase, and concentrate under reduced pressure to form extract c;

D. Silica gel column chromatography: silica gel column chromatography on the extract c, the column silica gel is 160~200 mesh, and the dosage is 6~10 times the weight of the extract c; the volume ratio is 1:0~0:1 Gradient elution with mixed organic solvent of chloroform and acetone, the gradient elution solution was collected, concentrated, monitored by TLC, and the same parts were combined;

E. High-performance liquid chromatography separation: the eluent obtained by eluting with a petroleum ether-acetone solution with a volume content of 50-90% is separated and purified by high-performance liquid chromatography to obtain the dimeric chromone alkaloid compound .

The organic solvent in the step A is 90-100% methanol, 90-100% ethanol or 70-100% acetone.

The organic solvent of the B step is ethyl acetate, diethyl ether, petroleum ether, chloroform or dichloromethane.

The extract c in step D is dissolved in methanol or acetone with a weight ratio of 1.5 to 3 times before being subjected to silica gel column chromatography, and then mixed with 80 to 100 mesh silica gel 0.8 to 1.2 times the weight of the extract.

The volume ratio of the mixed organic solvent of chloroform and acetone in the D step is 20:1, 9:1, 8:2, 7:3, 6:4 and 1:1.

The high-performance liquid chromatography separation and purification of the E step is prepared with 40-70% methanol or 20-60% acetonitrile as mobile phase, flow rate 10-14ml/min, 21.2×250 mm, 5 μm Zorbax PrepHT GF reverse phase preparation The column is a stationary phase, and the detection wavelength of the ultraviolet detector is 202-280nm. Each injection is 10-100μL, and the chromatographic peaks are collected for 10-40min, and evaporated to dryness after repeated accumulation.

The application of the dimeric chromone alkaloid compound of the present invention in the preparation of anticancer drugs.

The application of the dimeric chromone compound of the present invention in the preparation of anti-tobacco mosaic virus drugs.

The Cassia plants described in the present invention are not limited by regions and varieties, and can realize the present invention.

Example 1

Take 2.2kg of dried bark of Cassia fistula ( Cassia fistula ), a leguminous plant, coarsely crush it to 40 mesh, extract it four times with 70% acetone ultrasonically, each time for 60 minutes, and combine the extracts; filter the extracts, reduce Concentrate under pressure to 1/4 of the volume; leave standstill, filter out the precipitate, and concentrate into 63.5g of extract a; add 130g of water to extract a, extract 5 times with chloroform equal to the volume of water, combine the extract phases, Concentrate under reduced pressure into 40g of extract b; extract b is packed in MCI column, add 120g of 80% methanol water to dissolve extract b, then put it on the column, elute with 1 to 4 liters of 90% methanol water, collect and elute liquid, concentrated under reduced pressure to obtain 31.2g of extract c; add 62g of acetone into extract c to dissolve extract c, then add 32g of 100 mesh silica gel to mix the sample, after mixing the sample, pack the column with 200 mesh silica gel 200g; 20:1, 9:1, 8:2, 7:3, 6:4 and 1:1 chloroform-acetone mixed organic solvents were used for gradient elution, the gradient eluate was collected, concentrated, monitored by TLC, and the same The part obtained 6 parts AF, wherein, for the collected sample B part 6.42g, repeat the silica gel column chromatography, and use the petroleum ether-acetone mixed organic solvent gradient elution with a volume ratio of 9:1-1:2, The gradient eluate was collected, concentrated, monitored by TLC, and the same parts were combined to obtain 6 parts B1-B6, of which the part B3, namely the 7:3 part, was about 650 mg, and then 58% methanol was used as the mobile phase, and the flow rate was 10 ml/min, 21.2×250mm, 5μm Zorbax PrepHT GF reverse-phase preparative column is used as stationary phase, the detection wavelength of UV detector is 254 nm, 50μL is injected each time, the chromatographic peaks are collected for 25 minutes, evaporated to dryness after repeated accumulation , to obtain the dimeric chromone alkaloid compound sausagein A.

Example 2

Take 10.0 kg of dry bark of Cassia fistula ( Cassia fistula ), a leguminous plant, coarsely crush it to 40 meshes, extract it by cold immersion in 80% methanol for 4 times, each time for 3 days, and combine the extracts; filter the extracts, Concentrate under reduced pressure to 1/4 of the volume; let stand, filter out the precipitate, and concentrate into 300g of extract a; add 350g of water to extract a, extract 5 times with ethyl acetate equal to the volume of water, and combine the extract phases , concentrated under reduced pressure into 210g extract b; extract b was loaded into a column with MCI, dissolved in 600g of 80% methanol water in extract b, then loaded on the column, eluted with 5 to 15 liters of 90% methanol water, collected and washed Deliquification, concentrated under reduced pressure to obtain 150g extract c; add 300g acetone to dissolve extract c, then add 150g of 100 mesh silica gel to mix the sample, pack the column with 1Kg of 200 mesh silica gel, and load the column after mixing the sample; 20:1, 9:1, 8:2, 7:3, 6:4 and 1:1 chloroform-acetone mixed organic solvent gradient elution, collected gradient eluate, concentrated, monitored by TLC, combined the same fraction , to obtain 6 parts AF, wherein, for the collected sample B part 32g, repeat the silica gel column chromatography, use the petroleum ether-acetone mixed organic solvent gradient elution with a volume ratio of 9:1-1:2, and collect the gradient elution Deliquified, concentrated, monitored by TLC, combined the same parts to obtain 6 parts B1-B6, of which the part B3, namely the 7:3 part, was about 2.8 g, and then 45% acetonitrile was used as the mobile phase, and the flow rate was 10 ml/ min, 21.2×250mm, 5μm ZorbaxPrepHT GF reversed-phase preparative column is used as stationary phase, the detection wavelength of ultraviolet detector is 254 nm, 80μL is injected each time, the chromatographic peaks are collected for 15min, evaporated to dryness after repeated accumulation, and the described The dimeric chromone alkaloid sausagein A.

Example 3

Get the compound sausage A prepared in Example 1, which is a yellow jelly;

The determination method is: use nuclear magnetic resonance, combined with other spectral techniques to identify the structure.

(1) Ultraviolet spectrum (the solvent is methanol), λ _max (log ε ): 220 (4.46), 248 (3.68), 276(3.84), 322 (3.48), 376 (2.92) nm;

(2) Infrared Spectrum (Potassium Bromide Tablet) ν _max : 3446, 3158, 2938, 1730, 1647, 1605, 1574, 1442, 1385, 1338, 1276, 1164, 1076, 862, 751 cm ^–1 ;

(3) HRESIMS shows that the quasi-molecular ion peak of the compound of the present invention is m/z 466.1261 [M+Na] ⁺ (calculated value is 466.1261), combined with ¹³ C and ¹ H NMR spectra (Figure 1 and Figure 2, carbon spectrum hydrogen spectrum data attribution See Table 1) giving its molecular formula as C ₂₆ H ₂₁ NO ₆ and its degree of unsaturation as 17. See Table 1 for ¹ H NMR (CDCl ₃ , 500 MHz) and ¹³ C NMR (CDCl ₃ , 125 MHz) data. ¹ H NMR spectrum (Figure 2) shows 3 methyl signals ( δ _H 2.26, s, H ₃ -13; 2.38, s, H ₃ -9'; 2.51, s, H ₃ -12'), 2 Methylene signal ( δ _H 3.45, s, H ₂ -14; 4.24, s, H ₂ -11), 5 unimodal aromatic proton signals ( δ _H 6.21, s, H-3; 6.53, s, H- 6'; 6.78, s, H-8'; 6.85, s, H-3'; 6.93, s, H-10') and a 1,2,3,5-tetrasubstituted benzene ring signal ( δ _H 6.59, d, J = 1.8 Hz, H-6; 6.66, d, J = 1.8 Hz, H-8). In the carbon spectrum and DEPT spectrum (Figure 1), 26 carbon atom signals were observed, including 3 methyl groups (including two aromatic methyl groups), 2 methylene groups, 7 aromatic methine groups and 14 quaternary carbons Signals (including 3 carbonyl and 5 oxygen-containing quaternary carbon signals). Among them, 3 carbonyl groups and 18 double-bonded carbons occupy 12 degrees of unsaturation, so the molecule is a highly aromatizing pentacyclic alkaloid. Combining the above spectral data with the compounds previously isolated from Cassia, it is preliminarily speculated that sausagein A is a heterodimeric alkaloid composed of two different C13 skeletons. It consists of A and B fragments.

According to the characteristic signal ( δ _C 166.4, 113.1, 180.1, 138.5, 121.1, 163.8, 103.6, 161.4, 115.9; δ _H 6.21, 6.59, 6.66) of the A fragment (O-1 to C-14), it is speculated that it is a C13 Skeleton of chromones (A and B rings). This speculation is confirmed by HMBC correlation, that is, H-3 is related to C-2/C-4/C-10, H-6 is related to C-8/C-10, and H-8 is related to C-6/C-10 related (Fig. 3). Second, there is also 1 acetonyl group and 1 hydroxyl group attached to C-5 and C-7, respectively, which are also related to C-6/C _- 10 by H2-11 in the HMBC spectrum, and 7- Correlation of OH with C-6/C-7/C-8 was confirmed. These data show that the A segment of the compound is a 5-acetonyl-7-hydroxyl-2-chromone derivative. The main difference is that the C-14 position has changed from the original methyl group to a methylene group. It is speculated that the A segment is passed through C-14 is linked to the B segment. The remaining 13 carbon atoms, including 2 methyl groups, 4 methine groups, and 7 quaternary carbon signals, belong to the B segment. HMBC correlation gives the approximate structure of the B fragment. In the HMBC spectrum, H-6' and C-5'/C-7'/C-8', H-8' and C-6'/C-7'/C-8a', H ₃ - 9' with C-2'/C-3'/C-8a', H-3' with C-2'/C-4a'/C-9' and H-6' with C-4a'/C- 5'/C-7'/C-8'/C-10', thus confirming the basic fragments of chromone (C and D rings) in the B fragment. At the same time, H-10' and C-4a'/C-5'/C-6'/C-11'/C-12' and H-12' and C-14/C-4 were also observed in the HMBC spectrum Correlation of '/C-10'/C-11', which shows that C-4', C-4a', C-5', C-10', C-11', C-12' and N atoms together A 2-picoline derivative fragment (ring E) is formed, which is bound together with C and D rings via C-4', C-4a', and C-5'.

Finally, through the HMBC correlation between H ₂ -14 and C-4'/C-11' and C-14 of H ₃ -12', the connection sequence of A and B fragments was determined. So far, the dimeric chromone alkaloid The structure of the compound was determined and named sausin A.

Table 1 ¹ H and ¹³ C NMR data of compounds (solvent is CDCl ₃ ) (125 and 500 MHz)

No. ^{13C 1} h 2 166.4 s 3 113.1 d 6.21, s 4 180.1 s 5 138.5 s 6 121.1 d 6. 59 d (1.8) 7 163.8 s 8 103.6 d 6.66 d (1.8) 9 161.4 s 10 115.9 s 11 50.5t 4.24 s 12 207.9 s 13 30.8q 2.26 s 14 58.2 tons 3.45 s 2' 168.3 s 3' 98.2 d 6.85s 4′ 146.3 s 4a' 110.2 s 5' 136.2 s 6' 108.3 d 6.53s 7' 175.1s 8' 105.3 d 6.78 s 8a' 155.9 s 9' 22.1q 2.38 s 10′ 116.8 d 6.93 s 11′ 141.6 s 12′ 21.6q 2.51 s 7-OH 10.21 s

Example 4

The compound prepared in Example 2 was a yellow jelly. The determination was the same as in Example 3, and it was confirmed that the compound prepared in Example 2 was the dimeric chromone alkaloid compound——sausin A. .

Example 5

Get any dimeric chromone alkaloid compound prepared by Examples 1 and 2 to carry out the cytotoxic activity detection test, and the test conditions are as follows:

Cell lines: leukemia cells (NB4), lung cancer cells (A549), human neuroblastoma cells (SHSY5Y), prostate cancer cells (PC3), breast cancer cells (MCF7) were provided by Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

Experimental design: The above cells were incubated with different concentrations of compounds for 72 hours. The experiment for each cell line was repeated once, and the results of the two experiments were used for data processing. The improved MTT method and SRB method were used to evaluate the inhibitory degree of the compound on cell proliferation, and the calculated Inhibition rate, according to the inhibition rate, the IC ₅₀ was calculated by the Logit method, and the antitumor activity of the compounds in vitro was compared.

Proliferation inhibition rate of cells = (OD value of blank control - OD value of drug-dosed well) / OD value of blank control × 100%.

(a) Modified MTT method

Take suspension cells in the logarithmic growth phase, adjust the cell concentration to 4×10 ⁴ /ml, add to 96-well culture plate, 90 µL /well. The positive control was cisplatin, which was dissolved in normal saline. Add 10 μl of samples of different concentrations (test solution No. 1-test solution No. 5) to each well. Four replicate wells were set up for the sample addition group and the control group, parallel wells for medium addition were also set up for the sample addition group and the high concentration group of the positive control group, and each plate was provided with 4 blank control wells (only the medium was added). ). The final concentrations of the samples were 10 ^-2 , 10 ^-1 , 1, 10 and 10 ² µg /mL, respectively, and the corresponding final concentrations of DMSO were 0.1%, 0.01%, 0.001%, 0.0001%, and 0.00001%, respectively. When the sample was at a final concentration of 10 ² µg /mL, 0.1% DMSO was used as a solvent control, and normal saline was used as a negative control for other concentrations. The final concentration of positive control drug cisplatin was 10 ^-1 , 1, 10 μ g/mL. After the cells were incubated for 48 h at 37°C in a 5% CO ₂ incubator, MTT (5 mg/ml, Sigma) was added at 10 µL /well. After continuing to culture for 4 h, add triplex solution [10% SDS – 5% isobutanol – 0.012mol /L HCL (w/v/v)], 100 µL/well, leave overnight and use a microplate reader at 570 nm , Measure the OD value of each well under the double wavelength of 630 nm.

(b) SRB method

Adherent cell lines in the logarithmic growth phase were taken and routinely digested with 25% trypsin, then the cell concentration was adjusted to 5×10 ⁴ /mL with 15% calf serum complete RPMI-1640 medium, and added to 96-well culture plate, 90 μL /well. The cells were incubated at 37°C in a 5% CO ₂ incubator for 24 hours, and then the positive control, negative control and test samples were added (each test concentration was the same as the MTT method above, 10 μ L/well), and the final concentration of the samples was 10 ^-2 , 10 ^-1 , 1, 10, 10 ² μg /mL, the corresponding final concentrations of DMSO were 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, respectively. When the final concentration of the sample was 10 ² μg /mL, 0.1% DMSO was used as the solvent control, and normal saline was used as the negative control for the rest of the concentrations. The final concentration of the positive control drug cisplatin was 10 ^-1 , 1, 10 μg/mL, and the negative control was an equal volume of normal saline. Both the sample addition group and the control group were set with 4 replicate wells, and the sample addition group and the high concentration group of the positive control group were also provided with parallel wells for the addition of the medium, and each plate was provided with 4 blank control wells (only the medium was added). . Place the 96-well culture plate in a 37°C, 5% CO ₂ incubator and incubate for 48 hours (the interaction between cells and samples), then add 50 μL /well of 50% TCA (trichloroacetic acid) at 4°C. After adding TCA, incubate the 96-well culture plate at 4°C for 1 hour, take out the culture plate, and pour off the liquid in the plate gently. Rinse gently with tap water 5 times (pour the tap water from the beaker into the board gently, shake it lightly and then pour the water out), and let it air dry until no water marks appear. Then add the prepared 0.4% SRB (diluted with 1% acetic acid), 50 μL /well, let stand at room temperature for 30 minutes for staining, then pour off the SRB solution, wash 4 times with 1% acetic acid to remove unbound proteins. of dyes. After air-drying in the air until there is no water trace, add 150 μL/well of 10 mM unbuffered Tris ( Trisine ) solution (PH10, prepared with triple distilled water), dissolve the dye, and shake on a shaker for 5 Read the OD value of each well with a microplate reader at a wavelength of 570 nm.

(c) Experimental results

The experimental results show that: through the cytotoxic activity experiments on leukemia acute promyelocytic NB4 cells, lung adenocarcinoma A549 cells, human bone marrow neuroblastoma SHSY5Y cells, human prostate cancer PC3 cells, and human breast cancer MCF7 cells, sausagein A has NB4, A549 and MCF7 cell lines have good cytotoxic activity, with IC ₅₀ values of 5.5, 4.6 and 8.8 μM , respectively.

Table 2 Cytotoxic activity of compound sausagein A

Compounds NB4 A549 SHSY5Y PC3 MCF7 Sausagein A 5.5 4.6 >10 >10 8.8 Taxol 0.02 0.02 0.1 0.1 0.05

Example 6

Get the dimeric chromone alkaloid compound prepared by embodiment 1 and 2 and carry out anti-tobacco mosaic virus activity test, test situation is as follows:

The anti-tobacco mosaic virus activity of the compound of the present invention was determined when the mass concentration of the medicament was 50 mg/L by half-leaf method. On the plants of flue-cured tobacco in the 5th to 6th age, select the leaves suitable for the test (the leaf row is normal, no disease and no insects), and the leaves are evenly sprinkled with fine emery, and the spare tobacco mosaic virus source (3.0 × 10 -3) Spread evenly on the leaves sprinkled with carborundum. After all the selected leaves have been inoculated with the poison, immediately place them in a petri dish containing the medicine solution for 20 minutes, take them out, wipe off the water drops and medicine solution on the leaves, The two half-leaves were recovered and placed in a glass jar covered with toilet paper and covered with a glass lid. The temperature was controlled at (23 ± 2) °C, and placed in the greenhouse under natural light. Blight spots could be seen after 2-3 days. For each treatment, the other half of the leaf was set as a control, and another group was set as a commercial Ningnanmycin treatment as a comparison, and the relative inhibition rate was calculated according to the formula.

XI%=(CK-T)/CK×100%

X: relative inhibition rate (%), CK: the number of dead spots (pieces) of half a poisoned leaf soaked in clear water, T the number of dead spots (pieces) of half a poisoned leaf soaked in medicinal liquid.

After the experiment against tobacco mosaic virus, its relative inhibition rate reached 32.8% at 20 μM, which exceeded the relative inhibition rate (30.5%) of the positive control product Nanningmycin, and its _IC50 value was 43.8 μM, and its activity was better than that of the positive control product Nanningmycin (IC ₅₀ =52.4μM), indicating that the compound has good activity against tobacco mosaic virus.

Claims (5)

1. A dimer chromone alkaloid compound, characterized in that said dimer chromone alkaloid compound has the following structure: . 2. a preparation method of dimeric chromone alkaloid compound described in claim 1, is characterized in that the dry bark of leguminous plant sausage tree (Cassia fistula) is used as raw material, extracted through extract, organic solvent extraction, MCI decolorization, silica gel column chromatography, high performance liquid chromatography preparation and separation steps, specifically: A. Extraction: crush the bark of the leguminous plant Cassia fistula into 20-40 meshes, use 70% acetone or 80% methanol to ultrasonically extract 2-5 times, each time for 30-60 minutes, or cool Extract 2-5 times, 3 days each time, combine the extracts, filter, concentrate the extracts under reduced pressure, let stand, filter out the precipitate, and concentrate into extract a; B. Organic solvent extraction: add water of 1 to 2 times the weight ratio to the extract a, extract 3 to 5 times with ethyl acetate or chloroform equal to the volume of water, combine the organic solvent extraction phase, and concentrate under reduced pressure to obtain the extract. ointment b; C. MCI decolorization: the extract b is dissolved in methanol water with a weight ratio of 3 to 5 times, put on the MCI column, elute with 80%-95% methanol water, combine the organic solvent extraction phase, and concentrate under reduced pressure to form extract c; D, silica gel column chromatography: silica gel column chromatography on the extract c, the packing silica gel is 160~200 mesh, the dosage is 6~10 times of the weight of extract c; the volume ratio is 20:1, 9:1, 8:2, 7:3, 6:4 and 1:1 chloroform-acetone mixed organic solvents were used for gradient elution, the gradient eluent was collected, concentrated, monitored by TLC, and the same fractions were combined to obtain 6 fractions A-F. The collected part B was subjected to silica gel column chromatography, and was eluted with a gradient elution of petroleum ether-acetone mixed organic solvent with a volume ratio of 9:1-1:2, and the gradient eluate was collected, concentrated, monitored by TLC, and the same parts were combined; E. High-performance liquid chromatography separation: the collected volume ratio of 7:3 petroleum ether-acetone mixed organic solvent elution part is separated and purified by high-performance liquid chromatography, and high-performance liquid chromatography is separated and purified with 40~70% methanol or 20~60% acetonitrile as mobile phase, flow rate 10~14ml/min, 21.2×250mm, 5μm Zorbax PrepHT GF reversed-phase preparation column as stationary phase, UV detector detection wavelength 202~280nm, each injection 10~ 100 μL, collect the chromatographic peaks for 10-40 minutes, add them up several times and evaporate to dryness to obtain the dimeric chromone alkaloid compound. 3. according to the preparation method of the described dimer chromone alkaloid compound of claim 2, it is characterized in that the extract c of described D step is before silica gel column chromatography, with the methanol of 1.5～3 times of weight ratio or Dissolve in acetone, and then mix the sample with 80-100 mesh silica gel 0.8-1.2 times the weight of the extract. 4. The application of the dimeric chromone alkaloid compound described in claim 1 in the preparation of anticancer drugs. 5. The application of the dimeric chromone alkaloid compound described in claim 1 in the preparation of anti-tobacco mosaic virus medicine.