Anthraquinone compound, extraction method and application thereof

Abstract

The application belongs to the technical field of traditional Chinese medicine extraction and medicine, and particularly relates to an anthraquinone compound and an extraction method and application thereof. The anthraquinone compound is extracted from Oldenlandia diffusa. The application provides an anthraquinone compound extracted from Oldenlandia diffusa. The compound has a novel structure and pharmacological activity, and provides a material basis for pharmacological action research of Oldenlandia diffusa. According to the data results of the embodiment of the application, the anthraquinone compound provided by the application has an anti-leukemia effect, and provides a strong basis for exploring potential new anti-leukemia drugs. In addition, the application further provides a method for preparing the compound from Oldenlandia diffusa. The whole preparation process is simple in operation, easy to control, and suitable for industrial production.

Description

Technical Field

[0001] This application belongs to the field of traditional Chinese medicine extraction and pharmaceutical technology, specifically relating to an anthraquinone compound, its extraction method, and its application. Background Technology

[0002] Hedyotis diffusa Willd. is the whole herb, including the roots, of the Rubiaceae family. It has a sweet and bitter taste and enters the heart, liver, and spleen meridians. It possesses the effects of clearing heat and detoxifying, promoting diuresis and reducing swelling, and invigorating blood and relieving pain. It was initially recorded as "Dwarf Hedyotis diffusa Willd." in the *Flora of Guangxi*.

[0003] Oldenlandia diffusa, a valuable anti-cancer herb, can treat various cancers. Besides its anti-tumor pharmacological effects, it also possesses antioxidant, anti-inflammatory, and immunomodulatory properties. The chemical components in Oldenlandia diffusa are complex and diverse. Currently, the main compounds isolated from it are flavonoids, anthraquinones, and terpenoids. These compounds exhibit good anti-tumor activity and are important anti-tumor active ingredients in Oldenlandia diffusa. Research has primarily focused on nasopharyngeal carcinoma, digestive system tumors, lung cancer, urinary system tumors, nervous system tumors, and gynecological tumors; however, there are no reports on the anti-leukocyte activity of its anthraquinone compounds. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide anthraquinone compounds, their extraction methods, and applications. Specifically, the following technical solution is adopted: In a first aspect, the present invention provides an anthraquinone compound, which is extracted from Hedyotis diffusa; the structural formula of the anthraquinone compound is shown below: .

[0005] This invention provides an anthraquinone compound extracted from Oldenlandia diffusa, which has a novel structure and pharmacological activity, providing a material basis for the study of the pharmacological effects of Oldenlandia diffusa.

[0006] Secondly, the present invention provides a method for extracting the above-mentioned anthraquinone compounds, comprising the following steps: S1. Using Hedyotis diffusa as raw material, after pulverizing, it is extracted by reflux with a mixed solution of sulfuric acid and 75% ethanol, and concentrated under reduced pressure to obtain alcohol-free dry extract. S2. The alcohol-free dry extract obtained in S1 is suspended in water to obtain a suspension, and then extracted with ethyl acetate and concentrated under reduced pressure to dryness to obtain ethyl acetate extract concentrate. S3. The ethyl acetate extract concentrate obtained in S2 was subjected to silica gel column chromatography with gradient elution using trichloromethane-methanol at volume ratios of 50:1, 20:1, 17:1, 15:1, 12:1, 10:1, 7:1, 5:1, and 3:1. The eluent was collected and examined sequentially by thin-layer chromatography to obtain 9 fractions, which were designated as F1 to F9. S4. Fraction F6 obtained in S3 was subjected to silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate in volume ratios of 8:1, 6:1, 4:1, 2:1, 1:1, and 0:1. The eluent was collected and examined sequentially by thin-layer chromatography to obtain four fractions, which were named F6-1 to F6-4. S5. Fraction F6-4 obtained in S4 is subjected to C18 medium-pressure column chromatography with gradient elution using methanol-water with volume ratios of 10:90, 30:70, 50:50, 60:40, 70:30, 80:20, and 100:0. The eluent is collected and then examined sequentially by high-performance liquid chromatography to obtain 5 fractions, which are designated as F6-4-1 to F6-4-5. S6. The fraction F6-4-4 obtained in S5 is separated and purified by reversed-phase high-performance preparative liquid chromatography to obtain the anthraquinone compound.

[0007] As a further preferred embodiment, the volume ratio of sulfuric acid to 75% ethanol in the mixed solution of sulfuric acid and 75% ethanol in S1 is 0.5%:1; The weight ratio of alcohol-free dry extract to water in S2 is 1:20.

[0008] As a further preferred embodiment, the elution time for each gradient volume ratio in S3 is 0.5 h; The elution time for each gradient volume ratio in S4 is 1.5 h. In S5, the elution time for each gradient volume ratio is 0.5 h.

[0009] Thirdly, the present invention provides the use of the above-mentioned anthraquinone compounds in the preparation of antitumor drugs.

[0010] As a further preferred embodiment, the tumor includes leukemia.

[0011] Fourthly, the present invention provides a medicament for treating leukemia, comprising the above-mentioned anthraquinone compounds.

[0012] As a further preferred embodiment, the drug further includes one or more pharmaceutically acceptable excipients.

[0013] As a further preferred embodiment, the excipient includes at least one of carbohydrates, polymers, lipids, or minerals.

[0014] As a further preferred embodiment, the dosage form of the drug includes one or more of tablets, capsules, powders, syrups, and injections.

[0015] The beneficial effects of this invention are as follows: (1) This invention provides an anthraquinone compound extracted from Oldenlandia diffusa, which has a novel structure and pharmacological activity, providing a material basis for the study of the pharmacological effects of Oldenlandia diffusa.

[0016] (2) The anti-leukemia effect of the anthraquinone compounds provided by this invention provides a strong basis for the discovery of potential new anti-leukemia drugs.

[0017] (3) The present invention provides a method for preparing the compound from Oldenlandia diffusa. The entire preparation process is simple to operate, easy to control, and suitable for industrial production. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a high-resolution mass spectrum of anthraquinone compounds.

[0020] Figure 2 This is the UV spectrum of anthraquinone compounds.

[0021] Figure 3 Anthraquinone compounds 1 H-NMR spectrum.

[0022] Figure 4 Anthraquinone compounds 13 C-NMR spectrum.

[0023] Figure 5 This is the HSQC spectrum of anthraquinone compounds.

[0024] Figure 6 This is the HMBC spectrum of anthraquinone compounds. Detailed Implementation

[0025] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0026] The specific conditions for reversed-phase high-performance preparative liquid chromatography in the following examples are as follows: A CAPCELL PAK C18 column (20 mm × 250 mm, 5 μm) with a detection wavelength of 210 nm and an injection volume of 200 μL. It should also be noted that other brands of columns are acceptable in actual experiments, with detection wavelengths ranging from 200 nm to 400 nm and injection volumes ranging from 50 μL to 500 μL.

[0027] Example 1 A method for extracting anthraquinone compounds, specifically comprising the following steps: (1) Take 9.5 kg of Oldenlandia diffusa, crush it, and extract it twice by reflux with a mixed solution of sulfuric acid and 75% ethanol (sulfuric acid accounts for 0.5% of the volume of ethanol), each time for 2 hours. Combine the extracts and concentrate under reduced pressure to obtain an alcohol-free dry extract. Add 20 times its weight of deionized water to the alcohol-free dry extract to obtain a suspension. Extract it three times with ethyl acetate. Combine the ethyl acetate extracts and concentrate under reduced pressure to dryness to obtain 213.2 g of ethyl acetate extract concentrate.

[0028] (2) The ethyl acetate extract concentrate was subjected to silica gel column chromatography (silica gel particle size 100-200 mesh) with a gradient elution using chloroform-methanol (volume ratios 50:1, 20:1, 17:1, 15:1, 12:1, 10:1, 7:1, 5:1, 3:1) as the eluent (elution time for each gradient volume ratio was 0.5 h). The eluent was collected in batches of 1 L and numbered sequentially. The collected eluents were examined sequentially by thin-layer chromatography, and similar components were combined to obtain 9 fractions, designated F1 to F9. Among them, fraction F6, weighing 38.42 g, was obtained by combining the 37th to 43rd eluents.

[0029] (3) The obtained fraction F6 was subjected to silica gel column chromatography (silica gel particle size 100-200 mesh) with gradient elution using petroleum ether-ethyl acetate (volume ratio 8:1, 6:1, 4:1, 2:1, 1:1, 0:1) as eluent (elution time for each gradient volume ratio was 1.5 h). The eluent was collected in 500 mL portions and numbered sequentially. The collected eluents were examined sequentially by thin-layer chromatography, and similar components were combined to obtain 4 fractions, designated F6-1 to F6-4; among them, 11.43 g of fraction F6-4 was obtained, which was obtained by combining the 28th to 35th eluents.

[0030] (4) The obtained fraction F6-4 was subjected to C18 medium-pressure column chromatography, using methanol-water (volume ratios of 10:90, 30:70, 50:50, 60:40, 70:30, 80:20, 100:0) as eluent for gradient elution (elution time for each gradient volume ratio was 2 h). The eluent was collected in 500 mL portions and numbered sequentially. The collected eluents were examined sequentially by high performance liquid chromatography, and similar components were combined to obtain 5 fractions, designated F6-4-1 to F6-4-5; among them, fraction F6-4-4, with a yield of 192.33 mg, was obtained by combining eluents from the 23rd to the 29th fractions.

[0031] (5) The obtained fraction F6-4-4 was subjected to reversed-phase high-performance preparative liquid chromatography with acetonitrile-0.01% trifluoroacetic acid (volume ratio 33:67, flow rate 20 mL / min) as the mobile phase to prepare anthraquinone compound (16.85 mg).

[0032] Example 2 The anthraquinone compound extracted in Example 1 was subjected to structural analysis and identification. The anthraquinone compound obtained in Example 1, which is a yellow powder soluble in methanol, was measured to have an mp of 227–229 °C using an XT5B micro melting point apparatus; UV (MeOH) was measured using a UV-260 ultraviolet spectrophotometer. l max (log e The characteristic UV absorbances of anthraquinone compounds are 202.2 nm, 217.2 nm, and 280.6 nm. HRESIMS was measured using a Waters ACQUITY UPLC / Xevo G2 QTOF analyzer. m / z 337.0698 [M+H] + (calcd for C 17 H 14 O6Na, 337.0688), its molecular formula was determined to be C 17 H 14 O6.

[0033] 1 H-NMR showed three singlet aromatic proton signals. d H 7.84 (1H, s), 7.68 (1H, s), and 7.49 (1H, s), two methoxy signals. d H 4.03 (3H, s), 3.87 (3H, s), and a methyl signal. d H 2.34 (3H, s). 13 C-NMR spectra revealed 17 carbon signals, including 12 aromatic carbons ( d C 156.7, 154.1, 153.5, 148.1, 133.0, 130.0, 129.6, 127.3, 127.2, 125.6, 113.3, 109.8), 2 carbonyl carbons ( d C 183.6, 183.5), 2 methoxy groups ( d C 62.1, 56.9) and one methyl signal d C 16.8, consistent with the characteristics of 9,10-anthraquinone.

[0034] Displayed in HMBC spectrum d H 7.68 and d C 129.6 d C 154.1 d C 183.6 related, d H 7.49 and d C 130.1 d C 153.5 d C 183.5 related, d H 4.03 (3H, s) and d C The 154.1 correlation indicates that one hydroxyl group is located at the C-6 position and one methoxy group is located at the C-7 position. d H 7.84 and d C 16.8, 125.6, 156.7, 183.5 are relevant. d H2.34 and d C Relevant values: 127.2, 133.0, 156.7 d H 3.87 (3H, s) and d C The correlation at 148.4 indicates that the methyl group is located at C-3, another hydroxyl group at C-2, and another methoxy group at C-1. Therefore, the anthraquinone compound was ultimately named 2,6-dihydroxy-1,7-dimethoxy-3-methylanthraquinone.

[0035] The UV spectrum of 2,6-dihydroxy-1,7-dimethoxy-3-methylanthraquinone is shown below. Figure 1 As shown; high-resolution mass spectrum as follows Figure 2 As shown; 1 H-NMR spectrum, 13 C-NMR spectrum, HSQC spectrum, HMBC spectrum, see respectively Figure 3-Figure 6 ,Depend on Figure 3-Figure 6 The structure of 2,6-dihydroxy-1,7-dimethoxy-3-methylanthraquinone of the present invention has been confirmed, and its structural formula is shown below: Table 1 shows the NMR data for 2,6-dihydroxy-1,7-dimethoxy-3-methylanthraquinone: 1 H-NMR (600 MHz) and 13 C-NMR (150 MHz) in CD3OD.

[0036] Table 1 lists 2,6-dihydroxy-1,7-dimethoxy-3-methylanthraquinone. 1 H-NMR, 13 C-NMR data Example 3 The anthraquinone compound prepared in Example 1 was subjected to in vitro antileukemia activity testing. Test Principle: MTT Assay: Living cell mitochondria contain dehydrogenases associated with NAPP (nicotinamide adenine dinucleotide phosphate, coenzyme II). Succinate dehydrogenase can reduce exogenous yellow thiazolyl blue MTT (3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide) to water-insoluble blue-purple crystals of formazan, which then deposit in the cells. In dead cells, this enzyme disappears, and MTT is not reduced. After dissolving formazan in dimethyl sulfoxide (DMSO), the absorbance can be measured at 570 nm and 630 nm using a microplate reader. The optical density value is directly proportional to the number of living cells.

[0037] The cell lines used were KASUMI-1 (human acute myeloid leukemia) and SKNO-1 (human acute myeloid leukemia).

[0038] Experimental method: MTT assay: Logarithmically grown cells were taken, digested, and thoroughly pipetted into a single-cell suspension. After counting, the suspension was diluted to 1×10⁻⁶ cells. 5 Cells were seeded at a density of 100 μL / mL into 96-well plates, with 100 μL of cell suspension added to each well. The plates were incubated at 37°C with 5% CO2 for 24 hours, after which the original culture medium was discarded. Six concentration gradients were designed for each sample, and 100 μL of culture medium containing each concentration gradient was added to each well, with six replicates per concentration. The 96-well plates were incubated at 37°C with 5% CO2 for 72 hours. Then, 20 μL of freshly prepared serum-free culture medium containing 5 mg / mL MTT was added to each well, and the plates were incubated at 37°C for another 4 hours, after which the supernatant was removed. 150 μL of DMSO was added to each well to dissolve the formazan precipitate, and the plates were shaken for 5 minutes. The absorbance at 570 nm and 630 nm was measured using a microplate reader to reflect the number of viable cells. The inhibitory concentration (IC50) of the drug was calculated using SPSS software. 50 )value.

[0039] The calculation formula is as follows: Tumor cell growth inhibition rate (%) = (1 - test well measurement value / control well measurement value) × 100%.

[0040] Table 2 shows the inhibitory effect of 2,6-dihydroxy-1,7-dimethoxy-3-methylanthraquinone on KASUMI-1 (human acute myeloid leukemia cells). Table 3 shows the inhibitory effect of 2,6-dihydroxy-1,7-dimethoxy-3-methylanthraquinone on SKNO-1 (human acute myeloid leukemia cells). It is evident that 2,6-dihydroxy-1,7-dimethoxy-3-methylanthraquinone has a significant inhibitory effect on both KASUMI-1 (human acute myeloid leukemia cells) and SKNO-1 (human acute myeloid leukemia cells), and can be used as a treatment or for research on drugs for treating leukemia.

[0041] The embodiments of this application have been described above with reference to the accompanying drawings. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the core ideas of this application. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. An anthraquinone compound, characterized in that, The anthraquinone compound was extracted from Hedyotis diffusa; the structural formula of the anthraquinone compound is shown below: ； The method for extracting the anthraquinone compound includes the following steps: S1. Using Hedyotis diffusa as raw material, after pulverizing, it is extracted by reflux with a mixed solution of sulfuric acid and 75% ethanol, and concentrated under reduced pressure to obtain alcohol-free dry extract. S2. The alcohol-free dry extract obtained in S1 is suspended in water to obtain a suspension, and then extracted with ethyl acetate and concentrated under reduced pressure to dryness to obtain ethyl acetate extract concentrate. S3. The ethyl acetate extract concentrate obtained in S2 was subjected to silica gel column chromatography with gradient elution using trichloromethane-methanol at volume ratios of 50:1, 20:1, 17:1, 15:1, 12:1, 10:1, 7:1, 5:1, and 3:

1. The eluent was collected and examined sequentially by thin-layer chromatography to obtain 9 fractions, which were designated as F1 to F9. S4. Fraction F6 obtained in S3 was subjected to silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate in volume ratios of 8:1, 6:1, 4:1, 2:1, 1:1, and 0:

1. The eluent was collected and examined sequentially by thin-layer chromatography to obtain four fractions, which were named F6-1 to F6-4. S5. Fraction F6-4 obtained in S4 is subjected to C18 medium-pressure column chromatography with gradient elution using methanol-water with volume ratios of 10:90, 30:70, 50:50, 60:40, 70:30, 80:20, and 100:

0. The eluent is collected and then examined sequentially by high-performance liquid chromatography to obtain 5 fractions, which are designated as F6-4-1 to F6-4-5. S6. The fraction F6-4-4 obtained in S5 is separated and purified by reversed-phase high-performance preparative liquid chromatography to obtain the anthraquinone compound.

2. The anthraquinone compound according to claim 1, characterized in that, In the mixed solution of sulfuric acid and 75% ethanol described in S1, the volume ratio of sulfuric acid to 75% ethanol is 0.5%:1; The weight ratio of alcohol-free dry extract to water in S2 is 1:

20.

3. The anthraquinone compound according to claim 1, characterized in that, The elution time for each gradient volume ratio in S3 is 0.5 h. The elution time for each gradient volume ratio in S4 is 1.5 h. In S5, the elution time for each gradient volume ratio is 0.5 h.

4. The use of the anthraquinone compound of claim 1 in the preparation of an anti-leukemia drug.

5. A drug for treating leukemia, characterized in that, Includes the anthraquinone compound of claim 1.

6. The drug according to claim 5, characterized in that, The drug also includes one or more pharmaceutically acceptable excipients.

7. The drug according to claim 6, characterized in that, The excipients include at least one of carbohydrates, polymers, lipids, or minerals.

8. The medicament according to claim 7, characterized in that, The dosage form of the drug includes one or more of tablets, capsules, powders, syrups, and injections.

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