Anthraquinone compound with anti-tobacco mosaic virus activity and its preparation method and application

Abstract

The present invention discloses an anthraquinone compound having anti-tobacco mosaic virus activity, a preparation method and an application thereof. The molecular formula of the compound is: C 19 H 14 O4, the compound is named: 2',5-dimethyl-6-hydroxymethylanthracene[2,3- b ]furan-9,10-dione, English name: 6-hydroxymethyl-2',5-dimethylanthra[2,3- b Furan-9,10-dione has the following structure: This compound was isolated for the first time from the Dai medicinal plant, the genus Ligusticum chuanxiong. It was identified as an anthraquinone compound by nuclear magnetic resonance and mass spectrometry, and its specific structure was characterized. This anthraquinone compound exhibits excellent anti-tobacco mosaic virus activity: in experiments against tobacco mosaic virus, its relative inhibition rate reached 43.2%, significantly higher than the relative inhibition rate of the reference substance, ningnanmycin (32.4%). This compound has promising application prospects in the preparation of anti-tobacco mosaic virus drugs and can serve as a lead compound for such drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural product chemistry, and particularly relates to an anthraquinone compound with anti-tobacco mosaic virus activity extracted from the Cassia tree of the Leguminosae family, and its application in the preparation of an anti-tobacco mosaic virus drug. Background Art

[0002] Plant viruses are second only to fungi in the list of pathogens found in nature. The difficulty in finding drugs that inhibit viruses without harming their hosts makes plant viral diseases extremely challenging to control. Virtually every agricultural and commercial crop is susceptible to viral diseases, causing significant losses to agricultural production. Take tobacco mosaic virus (TMV), for example. TMV has a wide host range, strong resistance, and significant production damage. It can infect over 260 plant species across 38 families. The high incidence of mosaic disease causes significant losses to agriculture, severely impacting both agricultural development and farmers' incomes.

[0003] In recent years, research on the antiviral activity of natural plants has been increasing both domestically and internationally, and they play a crucial role in preventing diseases in humans, animals, and crops. Research progress in this field has been reviewed numerous times by scholars both domestically and internationally. It is also expected that the development of highly effective and safe antiviral inhibitors from natural active substances derived from plants, microorganisms, and animals will remain a hot topic in natural product research for a considerable period of time. There are over 600 species of Cassia L. in the Leguminosae family, widely distributed throughout tropical and subtropical regions of the Earth, particularly in tropical America and tropical Africa. There are 25 species in my country, widely distributed across southern provinces, all of which are found in Yunnan. Several Cassia species are used medicinally, and five of these (Ironwood, Sausage Tree, Winged Pod Cassia, Mingwangjiangnan, and Cassia Seed) are commonly used in folk medicine by the Dai people of Yunnan. Traditional folk medicines demonstrate that these plants possess the properties of clearing heat and detoxifying, promoting diuresis and dissolving stones, reducing swelling and relieving pain, dispelling wind and relieving pain, and killing insects and alleviating itching. The representative chemical components of Cassia plants are chromones and anthraquinone compounds, which exhibit various activities such as anti-cancer, anti-HIV, anti-hepatitis, insecticide, anxiolytic and sedative.

[0004] Anthraquinone is a quinone chemical. Anthraquinone complexes exist naturally and can also be synthesized artificially. Anthraquinones include products and dimers with different degrees of reduction, such as anthraquinone, oxidized anthraquinone, anthrone, etc., as well as glycosides of these compounds. Among natural products, anthraquinones are often found in the metabolites of higher plants and lower plants, lichens and fungi, and have hemostatic, antibacterial, detoxifying, laxative and diuretic effects. The present invention discovered a new anthraquinone compound from the plant Cassia tomentosa of the Leguminosae family. It is worth noting that the compound is a rare new anthraquinone skeleton molecule with a 2-methylfuran structural fragment. Its structure is highly novel, and the compound has significant anti-tobacco mosaic virus activity. Summary of the Invention

[0005] The first purpose of the present invention is to provide an anthraquinone compound with anti-tobacco mosaic virus activity; the second purpose is to provide a preparation method of the anthraquinone compound with anti-tobacco mosaic virus activity; and the third purpose is to provide an application of the anthraquinone compound with anti-tobacco mosaic virus activity.

[0006] The first object of the present invention is achieved as follows: the anthraquinone compound having anti-tobacco mosaic virus activity is prepared from the branches of the Dai medicine sausage tree through pretreatment, extract extraction, MCI decolorization, silica gel column chromatography, gel column chromatography and high performance liquid chromatography separation. The molecular formula of the anthraquinone compound having anti-tobacco mosaic virus activity is: C 19 H 14 O4, named: 2',5-dimethyl-6-hydroxymethylanthracene[2,3- b ] Furan-9,10-dione, English name: 6-hydroxymethyl-2',5-dimethylanthra[2,3- b ]furan-9,10-dione, having the following structure:

[0007] .

[0008] The second object of the present invention is achieved by using Dai medicine sausage tree branches as raw materials, and preparing the product through pretreatment, extract extraction, MCI decolorization, silica gel column chromatography, gel column chromatography and high performance liquid chromatography separation steps, specifically comprising:

[0009] A. Pretreatment: Grind the sausage tree branches through a 20-60 mesh sieve to obtain material a;

[0010] B. Extraction: Add 2 to 6 times the mass of organic extraction solvent to material a, soak and extract 2 to 5 times at room temperature, each extraction time is 12 to 20 hours, combine the extracts and filter, concentrate the filtrate, add an equal volume of ethyl acetate and extract 2 to 4 times, combine the extracts to obtain sample extract b;

[0011] C. MCI decolorization: concentrate the sample extract b and decolorize it on an MCI column, collect the effluent and concentrate it under reduced pressure to obtain extract c;

[0012] D. Silica gel column chromatography:

[0013] 1) Add 3-10 times the mass of extract C to a 200-300 mesh silica gel column and load the column with a gradient elution using a chloroform-methanol solution with a volume ratio of 20:1 to 1:1. Monitor by TLC and combine the same fractions.

[0014] 2) The eluate obtained by eluting with a chloroform-methanol solution in a volume ratio of 8:2 was loaded onto a column with 200-300 mesh silica gel in an amount equivalent to 3-10 times the mass of extract C. The column was further separated by silica gel column chromatography, and then gradient eluted with a chloroform-acetone solution in a volume ratio of 4:1 to 1:9. The eluates were collected, monitored by TLC, and the same fractions were combined;

[0015] E. Gel column chromatography purification: The eluate obtained by eluting with a 2:8 ratio of chloroform-acetone solution was concentrated, then dissolved with methanol, and purified again by column chromatography using Sephadex LH-20 dextran gel with methanol as the mobile phase to obtain the target crude anthraquinone compound with anti-tobacco mosaic virus activity;

[0016] F. High performance liquid chromatography separation: The crude product of the target anthraquinone compound having anti-tobacco mosaic virus activity is separated and purified by high performance liquid chromatography to obtain the target anthraquinone compound having anti-tobacco mosaic virus activity.

[0017] The structure of the anthraquinone compound with anti-tobacco mosaic virus activity prepared in the above steps can be identified by the following method:

[0018] Appearance observation showed that the compound of the present invention was a light red colloid; the UV-visible absorption spectrum showed that it had maximum absorption at 215, 264, 286, and 410 nm, proving that the compound had an aromatic ring structure; the infrared spectrum (potassium bromide tablet) showed that the compound had a hydroxyl group (3396 cm -1 ), keto carbonyl (1668 cm -1 ), aromatic ring (1615, 1576, 1438cm -1) characteristic functional groups; high resolution mass spectrometry (HRESIMS) gave a quasi-molecular ion peak of 329.0787 [M+Na] + , the molecular formula of the compound can be determined to be C 19 H 14 O4.

[0019] The presence of ortho-coupled aromatic protons was determined by H NMR spectra. d H 7.51 (d, 7.8 Hz, H-7) and 7.72 (d, 7.8 Hz, H-8), three aromatic singlet protons in d H 8.11 (s, H-1), 7.94 (s, H-4) and 7.40 (s, H-1'), one hydroxymethyl d H 4.60 (s, H2-5'), two methyl groups d H 2.04 (s, H3-3') and 2.30 (s, H3-4'). Combined 13 C NMR, DEPT, HSQC spectral analysis, the 19 carbon atoms in the compound include: 2 carbonyl groups d C 181.0 (C-9) and 179.9 (C-10), five aromatic tertiary carbons, nine aromatic aprotic quaternary carbons, and three aliphatic carbon signals (two methyl carbons and one hydroxymethyl carbon). In the HMBC spectrum, H-1 correlates with C-4a / C-3 / C-9 and C-2'; H-4 correlates with C-1a / C-10 and C-2, H-7 correlates with C-5 and C-9a, and H-8 correlates with C-6 / C-10a and C-9; and H-1' correlates with C-2 and C-3, further indicating anthraquinone-[2,3- b ] and the parent nucleus of furan-9,10-dione exists.

[0020] In addition, the HMBC correlations of H-3' with C-1' / C-2' and C-2, H-4' with C-5 / C-6 and C-10a, and H-5' with C-5 / C-6 and C-7 showed that two methyl groups and one hydroxymethyl group were located at C-2', C-5 and C-6, respectively. It was thus inferred that the compound was an anthraquinone compound and named: 2',5-dimethyl-6-hydroxymethylanthracene[2,3- b ] Furan-9,10-dione, English name is 6-hydroxymethyl-2',5-dimethylanthra[2,3- b ]furan-9,10-dione.

[0021] Table 1. Compounds 1 H NMR and 13 C NMR data (CDCl3)

[0022]

[0023] Spectral data of the compound: UV (methanol) l max (log e ) 215 (4.32), 264 (3.52), 286 (3.60), 410 (3.46) nm; IR (KBr) n max 3396, 3042, 2960, 2812, 1668, 1615, 1576,1438, 1342, 1160, 1024, 862 cm -1 ; 1 H and 13 C NMR data are shown in Table 1; HRESIMS (positive ion mode) m / z 329.0787 [M+Na] + (Calculated value 329.0790, C 19 H 14 NaO4).

[0024] According to literature search, the compound of the present invention is a new anthraquinone skeleton molecule with a 2-methylfuran structural fragment that is discovered in natural products for the first time, and its structure has a high degree of novelty.

[0025] The third object of the present invention is achieved by using the anthraquinone compounds with anti-tobacco mosaic virus activity in the preparation of anti-tobacco mosaic virus drugs.

[0026] The third object of the present invention, the anti-tobacco mosaic virus activity test of the compound, is achieved in this way.

[0027] The anti-mosaic virus activity test was conducted using the half-leaf method. The anti-tobacco mosaic virus activity of the compounds of the present invention was determined at a mass concentration of 20 μM. From 5 to 6 flue-cured tobacco plants, leaves suitable for testing (normal leaves, free of disease and insects) were selected. The leaves were first evenly sprinkled with fine emery. A spare tobacco mosaic virus source (3.0×10 -3) was evenly applied to leaves sprinkled with corundum. After all selected leaves were inoculated, they were immediately placed in a petri dish containing the drug solution for 20 minutes. The leaves were then removed, and the water droplets and liquid on the leaves were sprinkled away. The two halves of the leaves were then placed in a glass-covered enameled container covered with moisturizing toilet paper. The temperature was controlled at (23±2)°C and placed in a greenhouse under natural light. Necrosis spots appeared within 2–3 days. For each treatment, the other half of the leaf served as a control, and a separate group was treated with the commercial product Ningnanmycin for comparison. The relative inhibition rate was calculated according to the following formula.

[0028] XI%=(CK-T) / CK×100%

[0029] X: relative inhibition rate (%), CK: number of necrotic spots on half of the infected leaf immersed in clean water, T: number of necrotic spots on half of the infected leaf immersed in drug solution.

[0030] The results showed that the relative inhibition rate of the compound was 43.2%, which was higher than the relative inhibition rate of the control Ningnanmycin of 32.4%, indicating that the compound had good anti-tobacco mosaic virus activity.

[0031] The advantages of the present invention are:

[0032] 1. The present invention is the first to isolate a new compound from the Dai medicine sausage tree. It was determined to be an anthraquinone compound by nuclear magnetic resonance and mass spectrometry, and its specific structure was characterized. This compound is a new anthraquinone compound with a 2-methylfuran structural fragment found for the first time in natural products. The compound also has good anti-tobacco mosaic virus activity: experiments against tobacco mosaic virus showed that the relative inhibition rate of the anthraquinone compound reached 43.2%, which was higher than the relative inhibition rate of the reference substance Ningnanmycin (32.4%). The above results reveal that the compound of the present invention has good application prospects in the preparation of anti-tobacco mosaic virus drugs. The compound of the present invention has a simple structure and good activity, and can be used as a lead compound for anti-tobacco mosaic virus drugs.

[0033] 2. Since the raw materials of the compound of the present invention are readily available, the extraction method of the compound is simple, the compound is easily separated, and industrial preparation is easily achievable.

[0034] 3. The compound of the present invention is easy to realize artificial synthesis, and subsequent industrialization can also be realized through artificial synthesis.

[0035] 4. The preparation method combines conventional column chromatography and high performance liquid chromatography. The compound preparation operation process is simple, the obtained compound of the present invention has high purity, and the quality and purity of the compound in subsequent industrial production are guaranteed.

[0036] 5. The compounds of the present invention are safe and non-toxic, exhibit good anti-tobacco mosaic virus activity, and can provide an ideal new skeleton-type drug-derived molecule for the prevention and treatment of tobacco mosaic disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The carbon-NMR spectrum of the isopentenyl anthraquinone compounds of the present invention ( 13 C NMR);

[0038] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of the isopentenyl anthraquinone compound of the present invention ( 1 H NMR);

[0039] Figure 3 Key HMBC correlation diagram of the isopentenyl anthraquinone compounds of the present invention. DETAILED DESCRIPTION

[0040] The present invention is further described below with reference to the embodiments and drawings, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention fall within the scope of protection of the present invention.

[0041] The anthraquinone compound with anti-tobacco mosaic virus activity of the present invention is prepared from the branches of the Dai medicine sausage tree through the steps of pretreatment, extract extraction, MCI decolorization, silica gel column chromatography, gel column chromatography and high performance liquid chromatography separation. The molecular formula of the anthraquinone compound with anti-tobacco mosaic virus activity is: C 19 H 14 O4, named: 2',5-dimethyl-6-hydroxymethylanthracene[2,3- b ] Furan-9,10-dione, English name: 6-hydroxymethyl-2',5-dimethylanthra[2,3- b ]furan-9,10-dione, having the following structure:

[0042] .

[0043] The preparation method of the anthraquinone compound having anti-tobacco mosaic virus activity of the present invention is to use the branches of the Dai medicine sausage tree as raw materials, and to prepare the compound through the steps of pretreatment, extract extraction, MCI decolorization, silica gel column chromatography, gel column chromatography and high performance liquid chromatography separation, and specifically comprises:

[0044] A. Pretreatment: Grind the sausage tree branches through a 20-60 mesh sieve to obtain material a;

[0045] B. Extraction: Add 2 to 6 times the mass of organic extraction solvent to material a, soak and extract 2 to 5 times at room temperature, each extraction time is 12 to 20 hours, combine the extracts and filter, concentrate the filtrate, add an equal volume of ethyl acetate and extract 2 to 4 times, combine the extracts to obtain sample extract b;

[0046] C. MCI decolorization: concentrate the sample extract b and decolorize it on an MCI column, collect the effluent and concentrate it under reduced pressure to obtain extract c;

[0047] D. Silica gel column chromatography:

[0048] 1) Add 3-10 times the mass of extract C to a 200-300 mesh silica gel column and load the column with a gradient elution using a chloroform-methanol solution with a volume ratio of 20:1 to 1:1. Monitor by TLC and combine the same fractions.

[0049] 2) The eluate obtained by eluting with a chloroform-methanol solution in a volume ratio of 8:2 was loaded onto a column with 200-300 mesh silica gel in an amount equivalent to 3-10 times the mass of extract C. The column was further separated by silica gel column chromatography, and then gradient eluted with a chloroform-acetone solution in a volume ratio of 4:1 to 1:9. The eluates were collected, monitored by TLC, and the same fractions were combined;

[0050] E. Gel column chromatography purification: The eluate obtained by eluting with a 2:8 ratio of chloroform-acetone solution was concentrated, then dissolved with methanol, and purified again by column chromatography using Sephadex LH-20 dextran gel with methanol as the mobile phase to obtain the target crude anthraquinone compound with anti-tobacco mosaic virus activity;

[0051] F. High performance liquid chromatography separation: The crude product of the target anthraquinone compound having anti-tobacco mosaic virus activity is separated and purified by high performance liquid chromatography to obtain the target anthraquinone compound having anti-tobacco mosaic virus activity.

[0052] The organic extraction solvent in step B is a 70-100% by mass methanol aqueous solution, a 70-100% by mass ethanol aqueous solution, or a 65-100% by mass acetone aqueous solution.

[0053] Before silica gel column chromatography, step D also includes dissolving the sample with acetone or methanol in an amount of 1.5 to 3 times the mass of extract c, and then mixing the sample with 80-100 mesh silica gel in an amount of 0.8 to 2.0 times the mass of extract c.

[0054] In step D1), the volume ratios of chloroform and methanol in the chloroform-methanol solution are 20:1, 9:1, 8:2, 7:3, 6:4 and 5:5.

[0055] D. In step 2), the volume ratios of chloroform and acetone in the chloroform-acetone solution are 4:1, 3:2, 2:3, 1:4 and 1:9.

[0056] In step F, the high-pressure liquid chromatography separation and purification uses a methanol-water solution with a volume concentration of 50-65% as the mobile phase at a flow rate of 12 mL / min, a 2.12×250 mm, 5 μm Zorbax PrepHT GF reverse-phase preparative column as the stationary phase, and a UV detector with a detection wavelength of 405 nm. 0.5-1.0 mL is injected each time, and the chromatographic peak is collected for 25-30 minutes. After multiple accumulations, the target anthraquinone compounds are evaporated to dryness to obtain them.

[0057] The application of the anthraquinone compound with anti-tobacco mosaic virus activity of the present invention is the application of the anthraquinone compound with anti-tobacco mosaic virus activity in the preparation of anti-tobacco mosaic virus drugs.

[0058] The specific steps of the preparation method are as follows:

[0059] A. Sample Extraction and Purification: Sun-dry twigs of the Chinese sausage tree and crush them to a size of 20-60 mesh. Extract them 2-5 times with a solvent, using 2-6 times the solvent of the starting material for each extraction. The extraction time is 12-20 hours. The precipitate is filtered to obtain a sample extract, which is then concentrated under reduced pressure to obtain an extract. The prepared ethanol extract is partitioned between ethyl acetate and an aqueous solution. The ethyl acetate layer is concentrated under reduced pressure to obtain a crude extract. After filtering the crude extract to remove the precipitate, the extract is decolorized on an MCI column. The effluent is collected and concentrated under reduced pressure to obtain an extract for column chromatography purification. The fraction enriched in the anthraquinone compounds described herein is used for preparative chromatographic separation.

[0060] B. Primary silica gel column chromatography: Dilute the resulting extract with 1.5-3 times the weight of acetone or methanol, then mix the sample with 0.8-2.0 times the weight of the extract's silica gel, using a 100-150 mesh silica gel. Roughly fractionate the sample by silica gel column chromatography, using 200-300 mesh silica gel at a weight of 3-10 times the weight of the extract. Gradient elution was performed using a mixed organic solvent of chloroform and methanol in a volume ratio of 20:1 to 1:1. The gradient eluates were collected and concentrated. After monitoring by TLC, the same fractions were combined to obtain five fractions (AE).

[0061] C. Silica gel column chromatography again: Component b (chloroform-methanol 8:2 elution fraction) in step B was further subjected to silica gel column chromatography: the column was packed with 200-300 mesh silica gel, the weight of the silica gel used was 3-10 times the weight of the extract, and gradient elution was performed with a mixed organic solvent of chloroform and acetone in a volume ratio of 1:0-1:2. The gradient eluate was collected and concentrated.

[0062] D. Gel purification: Component b (eluted with a chloroform-acetone 8:2 mixed organic solvent) from step C was subjected to gel purification and separated by column chromatography using Sephadex LH-20 dextran gel with methanol as the mobile phase.

[0063] E. High Performance Liquid Chromatography Separation: The enriched portion of the anthraquinone compounds of the present invention finally obtained in step D is then separated and purified by high performance liquid chromatography to obtain the pure anthraquinone compounds of the present invention.

[0064] Furthermore, preferably, the solvent in step A is an acetone aqueous solution with a volume concentration of 70-100%, an ethanol aqueous solution with a volume concentration of 70-100%, or a methanol aqueous solution with a volume concentration of 70-100%.

[0065] Furthermore, preferably, in step C, the extract is diluted with acetone or methanol 1.5 to 3 times the weight of the extract before silica gel column chromatography, and then mixed with 80 to 100 mesh silica gel 0.8 to 2.0 times the weight of the extract before loading the sample.

[0066] Furthermore, preferably, in step C, during gradient elution, the volume ratios of the chloroform and methanol mixed organic solvents used are 20:1, 8:2, 7:3, 6:4, and 1:1, respectively. (Each elution should be performed until no component flows out, i.e., no component remains in the distillation flask after the solvent is evaporated, and then the next gradient is started).

[0067] Furthermore, preferably, the high performance liquid chromatography separation and purification in step E uses a methanol aqueous solution with a volume concentration of 50-65% as the mobile phase, a flow rate of 12 mL / min, a 2.12×250 mm, 5 μm Zorbax PrepHT GF reverse phase preparative column as the stationary phase, and a UV detector with a detection wavelength of 405 nm. 0.5~1.0 mL is injected each time, and the chromatographic peak is collected for 25~30 min. The compound can be obtained by evaporating after multiple accumulation.

[0068] The raw materials used in the present invention are not limited by regions and varieties, and can be used to implement the present invention.

[0069] The present invention is further described below using sausage tree raw materials from different production areas in Yunnan:

[0070] Example 1

[0071] This embodiment provides a method for preparing anthraquinone compounds with anti-tobacco mosaic virus activity according to the present invention. The method comprises the steps of extract extraction, silica gel column chromatography, and high performance liquid chromatography separation. The twigs of the sausage tree are used as raw materials. The specific operations are as follows:

[0072] The raw material used was 5.2 kg of twigs from the Dai medicinal sausage tree, which is grown at the mouth of the Honghe River in Yunnan Province. After crushing to 30 mesh, the sample was extracted four times with a 70% acetone solution, each for 15 hours. The combined extracts were then extracted three times with equal volumes of ethyl acetate. The combined extracts were combined. Then the extract was decolorized on an MCI column, and the eluate was concentrated under reduced pressure to obtain an extract; the extract was dissolved with methanol 2 times the mass of the extract, and then 90 mesh silica gel was added to mix the sample, and the column was loaded with 220 mesh silica gel. After mixing the sample, the column was loaded; gradient elution was performed with chloroform-methanol eluents with volume ratios of 20:1, 8:2, 7:3, 6:4 and 1:1, respectively. The gradient eluents were collected and concentrated, and monitored by TLC. The same fractions were combined to obtain 6 fractions AF, among which 196 g of the collected sample B (8:2) fraction was added with 8 times the amount of 220 mesh silica gel to load the column, and gradient elution was performed with chloroform-acetone eluents with volume ratios of 4:1, 3:2, 2:3, 1:4, and 1:9, respectively. The gradient eluents were collected and concentrated, and monitored by TLC. The same fractions were combined to obtain 5 fractions, among which 29.1 g of the 8:2 fraction was used with methanol as the mobile phase and was again purified by Sephadex chromatography. The anthraquinone compounds enriched in the fraction separated by gel column chromatography were then separated using 65% methanol as the mobile phase at a flow rate of 12 ml / min, 2.12×250 mm, 5 µ A Zorbax PrepHT GF reverse phase preparative column of m was used as the stationary phase, the detection wavelength of the UV detector was 405 nm, 0.8 mL was injected each time, and the chromatographic peak of 27.6 min was collected. After multiple accumulations, the anthraquinone compounds with anti-tobacco mosaic virus activity of the present invention were obtained by evaporation.

[0073] The structures of the prepared isopentenyl anthraquinone compounds with antibacterial activity were identified by the following method:

[0074] Appearance observation showed that the compound of the present invention was a light red colloid; the UV-visible absorption spectrum showed that it had maximum absorption at 215, 264, 286, and 410 nm, proving that the compound had an aromatic ring structure; the infrared spectrum (potassium bromide tablet) showed that the compound had a hydroxyl group (3396 cm -1 ), keto carbonyl (1668 cm -1 ), aromatic ring (1615, 1576, 1438cm -1 ) characteristic functional groups; high resolution mass spectrometry (HRESIMS) gave a quasi-molecular ion peak of 329.0787 [M+Na] + , the molecular formula of the compound can be determined to be C 19 H 14 O4.

[0075] The presence of ortho-coupled aromatic protons was determined by H NMR spectra. d H 7.51 (d, 7.8 Hz, H-7) and 7.72 (d, 7.8 Hz, H-8), three aromatic singlet protons in d H 8.11 (s, H-1), 7.94 (s, H-4) and 7.40 (s, H-1'), one hydroxymethyl d H 4.60 (s, H2-5'), two methyl groups d H 2.04 (s, H3-3') and 2.30 (s, H3-4'). Combined 13 C NMR, DEPT, HSQC spectral analysis, the 19 carbon atoms in the compound include: 2 carbonyl groups d C 181.0 (C-9) and 179.9 (C-10), five aromatic tertiary carbons, nine aromatic aprotic quaternary carbons, and three aliphatic carbon signals (two methyl carbons and one hydroxymethyl carbon). In the HMBC spectrum, H-1 correlates with C-4a / C-3 / C-9 and C-2'; H-4 correlates with C-1a / C-10 and C-2, H-7 correlates with C-5 and C-9a, and H-8 correlates with C-6 / C-10a and C-9; and H-1' correlates with C-2 and C-3, further indicating anthraquinone-[2,3- b ] and furan-9,10-dione parent nucleus exists ( Figure 2 ).

[0076] In addition, the HMBC correlations of H-3' with C-1' / C-2' and C-2, H-4' with C-5 / C-6 and C-10a, and H-5' with C-5 / C-6 and C-7 showed that two methyl groups and one hydroxymethyl group were located at C-2', C-5 and C-6, respectively. It was thus inferred that the compound was an anthraquinone compound and named: 2',5-dimethyl-6-hydroxymethylanthracene[2,3- b ] Furan-9,10-dione, English name is 6-hydroxymethyl-2',5-dimethylanthra[2,3- b ]furan-9,10-dione.

[0077] Example 2

[0078] This embodiment provides a method for preparing anthraquinone compounds with anti-tobacco mosaic virus activity according to the present invention. The method comprises the steps of extract extraction, silica gel column chromatography, and high performance liquid chromatography separation. The medicinal plant Ligusticum wallichii is used as the raw material. The specific operations are as follows:

[0079] The medicinal plant, the Medicinal Sausage Tree, is grown in Yuanjiang, Yuxi, Yunnan. The raw material was 6.5 kg of twigs from the Dai medicinal Sausage Tree, crushed to 30 mesh. The sample was then extracted three times with a 75% acetone solution, each for 12 hours. The combined extracts were then extracted three times with equal volumes of ethyl acetate. The combined extracts were then combined. Then the extract was decolorized on an MCI column, and the eluate was concentrated under reduced pressure to obtain an extract; the extract was dissolved with methanol 2 times the mass of the extract, and then 100 mesh silica gel was added to mix the sample, and the column was loaded with 200-300 mesh silica gel. After mixing the sample, the column was loaded; gradient elution was performed with chloroform-methanol eluents with volume ratios of 20:1, 8:2, 7:3, 6:4 and 1:1, respectively. The gradient eluents were collected and concentrated, and monitored by TLC. The same fractions were combined to obtain 6 fractions AF, among which 235 g of the collected sample B (8:2) fraction was added with 6 times the amount of 250 mesh silica gel to load the column, and gradient elution was performed with chloroform-acetone eluents with volume ratios of 4:1, 3:2, 2:3, 1:4, and 1:9, respectively. The gradient eluents were collected and concentrated, and monitored by TLC. The same fractions were combined to obtain 5 fractions, among which 35.4 g of the 4:1 fraction was used with methanol as the mobile phase and was again separated by Sephadex chromatography. The anthraquinone compounds enriched in the fraction separated by gel column chromatography were then separated using 65% methanol as the mobile phase at a flow rate of 12 ml / min, 2.12×250 mm, 5 µ A Zorbax PrepHT GF reverse phase preparative column of m was used as the stationary phase, the detection wavelength of the UV detector was 405 nm, 1.5 mL was injected each time, and the chromatographic peak of 27.6 min was collected. After multiple accumulations, the anthraquinone compounds with anti-tobacco mosaic virus activity of the present invention were obtained by evaporation.

[0080] Example 3

[0081] This embodiment provides a method for preparing anthraquinone compounds with anti-tobacco mosaic virus activity according to the present invention. The method comprises the steps of extract extraction, silica gel column chromatography, and high performance liquid chromatography separation. The medicinal plant Ligusticum wallichii is used as the raw material. The specific operations are as follows:

[0082] The medicinal plant, the Medicinal Sausage Tree, is grown in Menghai, Xishuangbanna, Yunnan Province. The raw material was 8.5 kg of twigs from the Dai medicinal Sausage Tree. After crushing to 50 mesh, the sample was extracted four times with a 70% acetone solution, each time for 15 hours. The combined extracts were then extracted three times with an equal volume of ethyl acetate. The combined extracts were then applied to an MCI column for decolorization. The eluate was concentrated under reduced pressure to obtain an extract. The extract was dissolved in methanol (2 times the extract mass), then mixed with 80-mesh silica gel. The column was packed with 200-300 mesh silica gel, mixed, and loaded onto the column. Gradient elution was performed using chloroform-methanol ratios of 20:1, 8:2, 7:3, 6:4, and 1:1, respectively. The gradient eluates were collected, concentrated, and monitored by TLC. Identical fractions were combined to yield six fractions, A to F. Among them, 322 g of the collected sample B (8:2) portion was added to a 200-300 mesh silica gel column with 7 times the amount of the extract, and gradient elution was performed with chloroform-acetone eluents of volume ratios of 4:1, 3:2, 2:3, 1:4, and 1:9. The gradient eluents were collected and concentrated, and monitored by TLC. The same fractions were combined to obtain 5 fractions, of which 42.6 g of the 4:1 fraction was further separated by column chromatography using Sephadex LH-20 dextran gel with methanol as the mobile phase. The fraction enriched in anthraquinone compounds obtained by gel column chromatography was again separated by 65% ​​methanol as the mobile phase at a flow rate of 12 ml / min, 2.12×250 mm, 5 µ A Zorbax PrepHT GF reverse phase preparative column of m was used as the stationary phase, the detection wavelength of the UV detector was 405 nm, 0.8 mL was injected each time, and the chromatographic peak at 27.6 min was collected. After multiple accumulations, the anthraquinone compounds with anti-tobacco mosaic virus activity of the present invention were obtained by evaporation.

[0083] Example 4

[0084] Any of the compounds prepared in Examples 1-3 was obtained as a light red colloid. The determination method was the same as in Example 1, confirming that the compound prepared in Example 1-3 was the aforementioned 2',5-dimethyl-6-hydroxymethylanthracen[2,3-b]furan-9,10-dione.

[0085] Example 5

[0086] The anti-mosaic virus activity test was conducted using the half-leaf method. The anti-tobacco mosaic virus activity of the compounds of the present invention was determined at a mass concentration of 20 μM. From 5 to 6 flue-cured tobacco plants, leaves suitable for testing (normal leaves, free of disease and insects) were selected. The leaves were first evenly sprinkled with fine emery. A spare tobacco mosaic virus source (3.0×10 -3) was evenly applied to leaves sprinkled with corundum. After all selected leaves were inoculated, they were immediately placed in a petri dish containing the drug solution for 20 minutes. The leaves were then removed, and the water droplets and liquid on the leaves were sprinkled away. The two halves of the leaves were then placed in a glass-covered enameled container covered with moisturizing toilet paper. The temperature was controlled at (23±2)°C and placed in a greenhouse under natural light. Necrosis spots appeared within 2–3 days. For each treatment, the other half of the leaf served as a control, and a separate group was treated with the commercial product Ningnanmycin for comparison. The relative inhibition rate was calculated according to the following formula.

[0087] XI%=(CK-T) / CK×100%

[0088] X: relative inhibition rate (%), CK: number of necrotic spots on half of the infected leaf immersed in clean water, T: number of necrotic spots on half of the infected leaf immersed in drug solution.

[0089] The results showed that the relative inhibition rate of the compound was 43.2%, which was higher than the relative inhibition rate of the control Ningnanmycin of 32.4%, indicating that the compound has good anti-tobacco mosaic virus activity.

Claims

1. An anthraquinone compound having anti-tobacco mosaic virus activity, characterized in that: The anthraquinone compound with anti-tobacco mosaic virus activity is prepared from the branches of the Dai medicine sausage tree through pretreatment, extract extraction, MCI decolorization, silica gel column chromatography, gel column chromatography and high performance liquid chromatography separation. The molecular formula of the anthraquinone compound with anti-tobacco mosaic virus activity is: C 19 H 14 O4, named: 2',5-dimethyl-6-hydroxymethylanthracene[2,3- b ] Furan-9,10-dione, English name: 6-hydroxymethyl- 2',5-dimethylanthra[2,3- b ] furan-9,10-dione, having the following structure: 。 2. A method for preparing anthraquinone compounds with anti-tobacco mosaic virus activity according to claim 1, which is prepared from branches of Dai medicine sausage trees through pretreatment, extract extraction, MCI decolorization, silica gel column chromatography, gel column chromatography and high performance liquid chromatography separation, specifically comprising: A. Pretreatment: Grind the sausage tree branches through a 20-60 mesh sieve to obtain material a; B. Extraction: Add 2 to 6 times the mass of material a to material a, soak and extract 2 to 5 times at room temperature, each extraction time is 12 to 20 hours, combine the extracts and filter, concentrate the filtrate, add an equal volume of ethyl acetate and extract 2 to 4 times, combine the extracts to obtain sample extract b; the organic extraction solvent is 70 to 100% by mass methanol aqueous solution, 70 to 100% by mass ethanol aqueous solution or 65 to 100% by mass acetone aqueous solution; C. MCI decolorization: concentrate the sample extract b and decolorize it on an MCI column, collect the effluent and concentrate it under reduced pressure to obtain extract c; D. Silica gel column chromatography: 1) Add 3-10 times the mass of extract C to a 200-300 mesh silica gel column and load the column with a gradient elution using a chloroform-methanol solution with a volume ratio of 20:1 to 1:

1. Monitor by TLC and combine the same fractions. 2) The eluate obtained by eluting with a chloroform-methanol solution in a volume ratio of 8:2 was loaded onto a column with 200-300 mesh silica gel in an amount equivalent to 3-10 times the mass of extract C. The column was further separated by silica gel column chromatography, and then gradient eluted with a chloroform-acetone solution in a volume ratio of 4:1 to 1:

9. The eluates were collected, monitored by TLC, and the same fractions were combined; E. Gel column chromatography purification: The eluate obtained by eluting with a 2:8 ratio of chloroform-acetone solution was concentrated, then dissolved with methanol, and purified again by column chromatography using Sephadex LH-20 dextran gel with methanol as the mobile phase to obtain the target crude anthraquinone compound with anti-tobacco mosaic virus activity; F. High-performance liquid chromatography separation: The crude product of the target anthraquinone compound having anti-tobacco mosaic virus activity is separated and purified by high-performance liquid chromatography to obtain the target anthraquinone compound having anti-tobacco mosaic virus activity; the high-performance liquid chromatography separation and purification is carried out using a methanol aqueous solution with a volume concentration of 50-65% as the mobile phase at a flow rate of 12 mL / min, with a 2.12×250 mm, 5 µ A Zorbax PrepHT GF reverse-phase preparative column with a diameter of 100 μm was used as the stationary phase, the UV detector was used at a wavelength of 405 nm, 0.5-1.0 mL was injected each time, and the chromatographic peak was collected for 25-30 min. After multiple accumulation, the target anthraquinone compounds were obtained by evaporation to dryness.

3. The preparation method according to claim 2, characterized in that Before silica gel column chromatography, step D also includes dissolving the sample with acetone or methanol in an amount of 1.5 to 3 times the mass of extract c, and then mixing the sample with 80-100 mesh silica gel in an amount of 0.8 to 2.0 times the mass of extract c.

4. The preparation method according to claim 2, characterized in that In step D1), the volume ratios of chloroform and methanol in the chloroform-methanol solution are 20:1, 9:1, 8:2, 7:3, 6:4 and 5:

5.

5. The preparation method according to claim 2, characterized in that D. In step 2), the volume ratios of chloroform and acetone in the chloroform-acetone solution are 4:1, 3:2, 2:3, 1:4 and 1:

9.

6. Use of the anthraquinone compound having anti-tobacco mosaic virus activity according to claim 1, characterized in that: The invention relates to an application of the anthraquinone compound with anti-tobacco mosaic virus activity in the preparation of anti-tobacco mosaic virus medicine.

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