1. A compound in the pteris spinosa is characterized in that the structural formula of the compound is respectively as follows:

1) the structural formula of the 7 beta, 9-dihydroxy-15-oxo-ent-kauran-16-en-19,6 beta-olide is as follows:

2) the structural formula of 5,11 beta, 12 beta-trihydroxy-15-oxo-ent-kuar-16-en-19-oic acid:

3) the structural formula of 11 beta-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid is as follows:

4) the structural formula of 7 alpha, 11 alpha-dihydroxy-15-oxo-16-methylene-ent-kaurane-19, 6 beta-lactone:

5) the structural formula of the 9-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid is as follows:

2. the compound of claim 1, wherein the compound is for use in the preparation of a therapeutic anti-tumor agent.

3. The compound of claim 2, wherein the compound is for use in the preparation of a medicament for the treatment of leukemia in a human, lung cancer in a human, liver cancer in a human, breast cancer in a human, or colon cancer in a human.

4. The compound of claim 2, wherein the drug can be formulated with pharmaceutically acceptable excipients into pharmaceutically acceptable solid or liquid formulations.

5. The compound of claim 4, wherein the solid formulation is a granule, a capsule, a tablet, a pill, a powder, or a lyophilized powder for injection.

6. The compound of claim 4, wherein the liquid formulation is an injectable formulation or an oral liquid.

7. The compound according to any one of claims 1 to 6, wherein the compound extraction, separation and purification method comprises the following steps:

1) extraction and crude separation of medicinal materials

Collecting 74.15kg of fresh medicinal materials of the pteris spinosa, naturally drying and crushing the medicinal materials, and sieving the medicinal materials by a 30-60-mesh sieve to obtain 25-35 kg of coarse powder, wherein the coarse powder is firstly subjected to normal-temperature immersion extraction by using 80-98% industrial methanol at 2 times, then subjected to normal-temperature immersion extraction by using 80-100% methanol at 2 times, and finally subjected to normal-temperature immersion extraction by using 60-80% methanol at 2 times, the extract liquid is subjected to reduced-pressure concentration at 50-60 ℃ to obtain an extract with the density of 1.0-1.1, diluted by using warm water at 1.25 times, diluted and extracted by using ethyl acetate with the same volume, and after extraction is finished, the extract liquid is combined and subjected to reduced-pressure concentration at 50-60 ℃ to obtain an ethyl acetate part extract with the density of 1-1.1;

2) separation of ethyl acetate fraction

Taking an extract extracted by ethyl acetate, mixing the extract with 30-60-mesh polyamide in the same amount, eluting by adopting an MCI (methanol-toluene-ethylene) reverse phase column, firstly replacing a solvent in the MCI column by 50% of methanol/water, and then sequentially carrying out gradient elution by using 50-100% of methanol/water system and segmenting to obtain a part H, a part I and other parts;

3) purification of

Taking a part H of a sample, mixing the sample with 100-200 meshes of equivalent silica gel, filling the sample into a column by a dry method, carrying out gradient elution on dichloromethane-ethyl acetate and dichloromethane-methanol according to the proportion of 20: 1 and 4: 1 and 10: 1, 9:1 and 4: 1 in sequence, carrying out thin-layer chromatography on fractions, merging the same parts to obtain 4 parts of H-1, H-2, H-3 and H-4, separating the H-1 and H-2 by silica gel column chromatography again, carrying out gradient elution on dichloromethane-ethyl acetate according to the proportion of 20: 1 and 10: 1 and dichloromethane-methanol according to the proportion of 20: 1 and 10: 1 and 5: 1 in sequence, carrying out flow analysis on crystals, and carrying out recrystallization and purification to obtain a compound 7 of 7 beta, 9-dihydroxy-15-oxo-ent-kauran-16-en-19,6 beta-olide, a compound 8 is 5,11 beta, 12 beta-trihydroxyxy-15-oxo-ent-kuar-16-en-19-oic acid, and a compound 9 is 11 beta-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid; separating H-3 and H-4 again by silica gel column chromatography, wherein the ratio of the H-3 to the H-4 is 20: 1, 15: 1 and 10: 1, performing gradient elution by using dichloromethane-ethyl acetate, and then performing elution by using a dichloromethane-methanol system with the proportion of 20: 1, 10: 1, 8: 1 and 5: 1, wherein a white strip-shaped crystalline compound 10 is 7 alpha, 11 alpha-dihydroxy-15-oxo-16-methylene-ent-kauran-19, 6 beta-lactone, and a white square-crystal compound 11 is 9-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid;

and (3) taking a part I of sample, mixing the part I with 100-200 meshes of equal silica gel, performing gradient elution sequentially by dichloromethane-methanol according to the proportion of 50: 1, 30: 1, 20: 1, 10: 1, 5: 1 and 1: 1, performing flow analysis to obtain crystals, repeatedly recrystallizing the crystals for thin layer identification, and obtaining the compound 9 which is 11 beta-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid.

8. The compound of claim 7, wherein the extraction and crude separation of the crude drug material in step 1) are: collecting 74.15kg of fresh medicinal materials of the pteris spinosa, naturally drying and crushing the medicinal materials, and sieving the medicinal materials by a 30-60-mesh sieve to obtain 29.9kg of coarse powder, wherein the coarse powder is firstly soaked and extracted by 2 times of 95% industrial methanol at normal temperature, then soaked and extracted by 2 times of 90% methanol at normal temperature, finally soaked and extracted by alcohol at normal temperature, the extract liquid is subjected to reduced pressure concentration at 55 ℃ to obtain an extract with the density of 1.06, then diluted by 1.25 times of warm water, and extracted by equal volume of ethyl acetate, after extraction is finished, the extract liquid is combined at 55 ℃ for reduced pressure concentration, and concentrated to obtain an ethyl acetate part extract with the density of 1.06.

9. The compound of claim 7, wherein the coarse powder in step 1) is first extracted by 2 times of 80-98% industrial methanol at normal temperature, the times of extraction are 4-6 times, and each time of extraction is 4-6 days.

10. The compound of claim 8, wherein said meal of step 1) is first extracted by 2-fold soaking with 95% industrial methanol at room temperature for 5 times and 5 days.

Background

The Pteris davidii (Willd.) Ohwi is derived from whole plant of Pteris davidii P.dispar Kunze of Pteris of Pteridaceae, also called Pteris davidii, and grows under forest, under bush, hillside, and ditch; the resources are distributed in Sichuan, Chongqing, Hunan, Hubei, etc. Has the effects of clearing away heat and toxic materials, cooling blood and removing blood stasis, and is often used for treating enteritis, dysentery, sore, and the like.

At present, tumors are the first disease in the world, the morbidity is very high, research and development of anti-tumor drugs are imminent, researches on the pteris spinosa have been reported from the last 90 years, but few students do not research deeply, the former separates about 30 diterpenoid compounds from the plant, and most of the diterpenoid compounds have good biological activity, such as anti-tumor, anti-bacterial and anti-inflammatory activities; the research does not see reports about the chemical components and biological activities of the pteris spinosa planted in Guizhou, so that systematic and deep research on the chemical components of the pteris spinosa planted in Guizhou is very necessary and meaningful.

Aiming at the problems, the team of the invention has the same or similar chemical components of plants with similar relatives according to the principle of the chemical taxonomy of the medicinal plants. Research shows that the plant contains abundant diterpene compounds, wherein the diterpene compounds have good inhibition effect on tumor cells. Therefore, the pteridophyte produced in Guizhou probably contains a plurality of new diterpene compounds with potential medicinal values, so that the research on screening the chemical components and the activity of the plant and the research on structural modification of the compound with better activity in the plant can be continuously enhanced subsequently, and more monomer compounds with better biological activity are expected to be found to provide better antitumor drugs.

Disclosure of Invention

The invention aims to provide a compound in the pteris spinosa.

The invention also aims to provide a method for extracting, separating and purifying compounds in the pteris spinosa.

Another object of the present invention is to provide a compound application in Pteris spinosa.

The compound of the invention has the following structural formula:

1) the structural formula of the 7 beta, 9-dihydroxy-15-oxo-ent-kauran-16-en-19,6 beta-olide is as follows:

2) the structural formula of the 5,11 beta, 12 beta-trihydroxy-15-oxo-ent-kuar-16-en-19-oic acid is as follows:

3) the structural formula of the 11 beta-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid is as follows:

4) the structural formula of the 7 alpha, 11 alpha-dihydroxy-15-oxo-16-methylene-ent-kaurane-19, 6 beta-lactone is as follows:

5) the structural formula of the 9-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid is as follows:

the compound disclosed by the invention is applied to the preparation of anti-tumor drugs.

The compound of the invention is applied to the preparation of medicaments for treating human leukemia, human lung cancer, human liver cancer, human breast cancer and human colon cancer.

The medicine can be added with pharmaceutically acceptable auxiliary materials to prepare pharmaceutically acceptable solid preparations or liquid preparations.

The solid preparation is granules, capsules, tablets, pills, powder and freeze-dried powder injection;

the liquid preparation provided by the invention is an injection preparation and an oral liquid.

The method for extracting, separating and purifying the compound comprises the following steps:

1) extraction and crude separation of medicinal materials

Collecting thorns, performing thin-layer chromatography on fractions, combining the fractions to obtain 4 parts of H-1, H-2, H-3 and H-4, separating the H-1 and the H-2 by silica gel column chromatography, performing gradient elution by using dichloromethane-ethyl acetate and dichloromethane-methanol in the proportions of 20: 1, 10: 1 and 5: 1 in sequence, performing flow analysis to obtain crystals, performing recrystallization and purification to obtain a compound 7 of 7 beta, 9-dihydroxy-15-oxo-ent-kauran-16-en-19,6 beta-olide, a compound 8 of 5,11 beta, 12 beta-trihydroxy y-15-oxo-ent-kuar-16-en-19-oic acid, a compound 9 of 11 beta-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid oic a cid; separating H-3 and H-4 again by silica gel column chromatography, wherein the ratio of the H-3 to the H-4 is 20: 1, 15: 1 and 10: 1, performing gradient elution by using dichloromethane-ethyl acetate, and then performing elution by using a dichloromethane-methanol system with the proportion of 20: 1, 10: 1, 8: 1 and 5: 1, wherein a white strip-shaped crystalline compound 10 is 7 alpha, 11 alpha-dihydroxy-15-oxo-16-methylene-ent-kauran-19, 6 beta-lactone, and a white square-crystal compound 11 is 9-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid;

and (3) taking a part I of sample, mixing the part I with 100-200 meshes of equal silica gel, performing gradient elution sequentially by dichloromethane-methanol according to the proportion of 50: 1, 30: 1, 20: 1, 10: 1, 5: 1 and 1: 1, performing flow analysis to obtain crystals, repeatedly recrystallizing the crystals for thin layer identification, and obtaining the compound 9 which is 11 beta-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid.

Preferably, the extraction and crude separation method of the medicinal materials comprises the following steps: collecting 74.15kg of fresh medicinal materials of the pteris spinosa, naturally drying and crushing the medicinal materials, and sieving the medicinal materials by a 30-60-mesh sieve to obtain 29.9kg of coarse powder, wherein the coarse powder is firstly soaked and extracted by 2 times of 95% industrial methanol at normal temperature, then soaked and extracted by 2 times of 90% methanol at normal temperature, finally soaked and extracted by 2 times of 70% methanol at normal temperature, the extract liquid is subjected to reduced pressure concentration at 55 ℃ to obtain an extract with the density of 1.06, then diluted by 1.25 times of warm water, extracted by equal volume of ethyl acetate after dilution, and after extraction is finished, the extract liquid is combined and subjected to reduced pressure concentration at 55 ℃ to obtain an ethyl acetate part extract with the density of 1.06;

preferably, the normal-temperature dipping extraction in the step 1) of the invention specifically comprises: soaking and extracting for 4-6 times (each time for 4-6 days), and mixing extractive solutions;

further preferably, the normal-temperature dipping extraction in step 1) of the invention specifically comprises: soaking and extracting for 5 times (each time for 5 days), and mixing extractive solutions.

The invention has the beneficial effects that:

1. the inventor uses the principle of medicinal plant chemical taxonomy, and plants with similar relatives have the same or similar chemical components. Researches show that the plant contains abundant diterpene compounds, and some compounds also have good inhibitory effect on certain tumor cell lines. Therefore, the pteridophyte produced in Guizhou probably contains a plurality of novel diterpene compounds with potential medicinal values, so that the research on screening the chemical components and the activity of the plant and the research on structural modification of the compound with better activity in the plant can be continuously strengthened subsequently.

2. The invention relates to a chemical component of methanol normal temperature lixiviation of Pteris spinosa and application thereof¹H-NMR，¹³Analyzing and identifying related spectrum data such as C-NMR and 2D-NMR, separating 5 compounds from the pteris odonta, wherein the compounds are respectively 7 beta, 9-dihydroxy-15-oxo-ent-kauran-16-en-19,6 beta-olide, 5,11 beta, 12 beta-dihydroxy-15-oxo-ent-kuar-16-en-19-oic acid, 11 beta-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid and 7 alpha, 11 alpha-dihydroxy-15-oxo-16-methylene-enantiomorphous kaurane-19, 6 beta-lactone and 9-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid.

3. The invention researches the test of the inhibition effect of the compound on human leukemia cells (HL-60), and the result shows that: the compounds 7, 8, 9, 10 and 11 have half inhibitory activity on human leukemia cells (HL-60) at 40 μ M concentration, and the inhibitory rates are 102.49 + -0.65, 104.41 + -0.03, 52.54 + -1.45, 104.33 + -0.63, 103.98 + -0.96, and IC for leukemia HL-60₅₀11.11 +/-0.35, 5.452 +/-0.222, 36.94 +/-1.86, 3.590 +/-0.021 and 3.523 +/-0.092 respectively.

4. The invention researches the inhibition effect test of the compound on human lung cancer cells (A549), and the result shows that: the compounds 7, 8, 10 and 11 have half inhibition activity on human lung cancer cells (A549) at the concentration of 40 mu M, and the inhibition rates are 97.89 +/-0.35, 98.47 +/-0.12, 99.80 +/-0.11 and 98.84 +/-0.08 respectively; IC of human Lung cancer cells (A549)₅₀Respectively 15.69 + -0.82, 15.15 + -0.22, 4.602 + -0.131 and 5.429 + -0.114.

5. The invention researches the inhibitory activity test of the compound on human liver cancer cells (SMMC-7721), and the result shows that: the compounds 7, 8, 9, 10 and 11 have half inhibitory activity on human liver cancer cells (SMMC-7721) at a concentration of 40 μ M, and the inhibitory rates are 96.85 + -0.47, 100.02 + -0.09 and 69.63 + -respectively1.69, 99.75 plus or minus 0.16 and 99.39 plus or minus 0.05; IC of human hepatoma cells (SMMC-7721)₅₀9.541 + -0.468, 7.177 + -0.515, 27.05 + -0.34, 4.005 + -0.165, 4.160 + -0.066, respectively.

6. The invention researches the inhibitory activity test of the compound on human breast cancer cells (MCF-7), and the result shows that: the compounds 7, 8, 10 and 11 have half inhibition activity on human breast cancer cells (MCF-7) at the concentration of 40 mu M, and the inhibition rates are 98.89 +/-0.96, 97.99 +/-1.78, 97.97 +/-0.16 and 96.67 +/-1.44 respectively; IC of human breast cancer cells (MCF-7)₅₀7.111 + -0.111, 5.809 + -0.097, 4.094 + -0.301 and 6.091 + -0.168.

7. The invention researches the inhibition activity test of the compound on human colon cancer cells SW480, and the result shows that: the compounds 7, 8, 10 and 11 have half inhibition activity on human colon cancer cells (SW480) at the concentration of 40 mu M, and the inhibition rates are 91.75 +/-1.47, 97.16 +/-0.89, 85.88 +/-1.60 and 93.20 +/-0.80 respectively; IC on human colon cancer cells (SW480)₅₀4.237 + -0.111, 4.662 + -0.090, 1.882 + -0.168 and 4.683 + -0.298 respectively.