CN113402488A

CN113402488A - Compound in pteridium aquilinum, extraction, separation and purification method and application thereof

Info

Publication number: CN113402488A
Application number: CN202110690535.6A
Authority: CN
Inventors: 邹娟; 何康; 叶江海; 赵臣亮; 张敬杰; 袁航; 王蕾
Original assignee: Guizhou University of Traditional Chinese Medicine
Current assignee: Guizhou University of Traditional Chinese Medicine
Priority date: 2021-06-22
Filing date: 2021-06-22
Publication date: 2021-09-17
Anticipated expiration: 2041-06-22
Also published as: CN113402488B

Abstract

The invention relates to a compound in pteris spinosa, an extraction, separation and purification method and application thereof, wherein the compound is respectively as follows: 7 beta, 9-dihydroxy-15-oxo-ent-kauran-16-en-19,6 beta-olide, 5,11 beta, 12 beta-trihydroxyxy-15-oxo-ent-kuar-16-en-19-oic acid, 11 beta-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid, 7 alpha, 11 alpha-dihydroxy-15-oxo-16-methylene-ent-kaurane-19, 6 beta-lactone and 9-hydroxy-15-oxo-kauran-16-en-19-oic acid. The results of screening the antitumor activity of the compounds of the invention show that the 5 compounds have half inhibitory activity on the in vitro tumor growth of leukemia HL-60, lung cancer A549, liver cancer SMMC-7721, breast cancer MCF-7 and colon cancer SW480, and provide references for continuously strengthening the screening research on the chemical components and the activity of the plant and the research on the structural modification of the compound with better activity in the plant.

Description

Compound in pteridium aquilinum, extraction, separation and purification method and application thereof

Technical Field

The invention relates to the field of pharmacy, and particularly relates to a compound in pteris spinosa, and an extraction, separation and purification method and application thereof.

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.

Description of the drawings:

FIG. 1: visual map of the inhibition rate of the tested compound on human leukemia cells (HL-60);

FIG. 2: visual representation of the IC50 values of the test compounds against human leukemia cells (HL-60).

FIG. 3: positive control compound cell growth profile (human blood protein);

FIG. 4: visual map of inhibition rate of tested compound on human lung cancer cell (A549);

FIG. 5: visual plots of IC50 values for test compounds on human lung cancer cells (a 549);

FIG. 6: positive control compound cell growth profile (lung cancer cells);

FIG. 7: a visual chart of the inhibition rate of the tested compound on human liver cancer cells (SMMC-7721);

FIG. 8: visual chart of IC50 values of test compounds on human hepatoma cells (SMMC-7721);

FIG. 9: positive control compound cell growth profile (hepatoma cells);

FIG. 10: visual chart of inhibition rate of tested compound on human breast cancer cells (MCF-7);

FIG. 11: visual chart of IC50 values of test compounds on human hepatoma cells (SMMC-7721);

FIG. 12: positive control compound cell growth profile (breast cancer cells);

FIG. 13: visual map of inhibition rate of test compound on human colon cancer cells (SW 480);

FIG. 14: visual chart of IC50 values for test compounds on human colon cancer cells (SW 480);

FIG. 15: positive control compound cell growth profile (colon cancer cells).

The technical solution of the present invention will be further specifically described below by way of specific examples.

Structural formula of the Compound of example 1

example 27 Crystal Structure of beta, 9-dihydroxy-15-oxo-ent-kauran-16-en-19,6 beta-olide

Example 311 Crystal Structure of β -hydroxy-15-oxo-ent-kauran-16-en-19-oic acid

Example 49 Crystal Structure of hydroxy-15-oxo-ent-kauran-16-en-19-oic acid

Example 57 characterization of beta, 9-dihydroxy-15-oxo-ent-kauran-16-en-19,6 beta-olide

White strip crystal (pyridine), insoluble in chloroform and petroleum ether, relatively soluble in methanol, ethyl acetate and pyridine, slightly soluble in dichloromethane, with dichloromethane-methanol as developing agent at a ratio of 20: 1, TLC has fluorescence spot under UV 254nm, 10% sulfuric acid-absolute ethanol solution develops color: a blue circular spot;¹H-NMR(400MHz，C₅D₅N)δH 6.38(1H，s)，6.03(1H，s，H-17b)，5.75(1H，t，J＝6.1Hz)，5.18(2H，d，J＝13.9Hz，H-17)，4.71(1H，d，J＝6.0Hz)，3.62(1H，s)，3.23(1H，d，J＝6.3Hz)，3.01(1H，dd，J＝9.3，4.5Hz)，2.53(1H，dd，J＝12.1，4.7Hz)，1.12(3H，s，H-20)。¹³C-NMR(101M Hz，C₅D₅N)δC36.9(C-1)，19.2(C-2)，28.9(C-3)，42.0(C-4)，51.0(C-5)，85.5(C-6)，81.1(C-7)，62.2(C-8)，73.1(C-9)，46.2(C-10) 43.2(C-11), 30.7(C-12), 36.9(C-13), 34.2(C-14), 202.1(C-15), 156.2(C-16), 113.1(C-17), 26.5(C-18), 183.8(C-19), 22.1 (C-20); the crystal parameters are as follows: molecular formula C₂₀H₂₆O₅The crystal system is orthorhombic and the unit of the crystal lattice is

α＝90.00°，β＝90.00°，γ＝90.00°，

T296 (2) K, Z1, etc.;

example 65, characterization of 11 β,12 β -trihydoxy-15-oxo-ent-kuar-16-en-19-oic acid

Transparent needle crystal (methanol), compound is difficult to dissolve in dichloromethane, chloroform, petroleum ether, and easy to dissolve in methanol, pyridine, with dichloromethane-methanol as developing agent at ratio of 20: 1, Rf ═ 0.8, TLC has fluorescence spot at wavelength of 254nm, 10% sulfuric acid-absolute ethanol develops color: a light purple cross-bar-shaped spot,¹H-NMR(600MHz，CD₃OD)δH5.74(1H，s)，5.24(1H，s)，5.13(1H，t，J＝6.2Hz)，4.59(2H，s)，4.18(1H，d，J＝6.0Hz)，3.35(1H，s)，3.04(1H，dd，J＝9.5，4.6Hz)，2.79(1H，d，J＝6.3Hz)，2.27-2.20(1H，m)，2.14-2.06(3H，m)，2.00(1H，dd，J＝14.6，6.0Hz)，1.82(1H，td，J＝12.6，6.5Hz)，1.55(3H，dd，J＝10.7，4.9Hz)，1.38(2H，dd，J＝11.5，5.9Hz)，1.31(3H，s)，1.27-1.21(1H，m)，0.90(3H，s)。¹³C-NMR(150MHz，CD₃OD)δC 203.6(C-15)，184.9(C-19)，155.3(C-16)，113.4(C-17)，85.1(C-12)，80.5(C-5)，72.5(C-11)，61.5(C-8)，45.7(C-9)，43.1(C-4)，41.4(C-10)，36.6(C-13)，33.4(C-14)，31.8(C-1)，30.1(C-7)，28.0(C-3)，26.7(C-6)，25.6(C-18)，21.1(C-20)，18.5(C-2)；

example 711 characterization of β -hydroxy-15-oxo-ent-kauran-16-en-19-oic acid

Transparent needle crystal (methanol), the compound is difficult to dissolve in dichloromethane, chloroform, petroleum ether, and easy to dissolve in methanol and pyridine, dichloromethane-methanol with a ratio of 20: 1 is used as developing agent, Rf is 0.7, TLC has mauve fluorescence point under the wavelength of 254nm, 10% sulfuric acid-absolute ethyl alcohol is used for color development: peach round spots;¹H-NMR(600MHz，CD₃OD)δH5.72(1H，s)，5.49(1H，s)，5.24(1H，s)，4.02(1H，d，J＝4.3Hz)，3.03(1H，d，J＝3.5Hz)，2.44(1H，d，J＝11.8Hz)，2.16(1H，d，J＝12.4Hz)，2.09(1H，ddd，J＝14.3，4.7，3.1Hz)，1.97(2H，dd，J＝18.7，2.0Hz，)，1.94(2H，t，J＝4.9Hz)，1.91-1.87(1H，m)，1.86-1.81(2H，m)，1.48-1.43(1H，m)，1.41-1.34(3H，m)，1.23(3H，s)，1.22-1.18(2H，m)，1.14-1.06(2H，m)，0.99(3H，s)。¹³C-NMR(150MHz，CD₃OD) δ C212.0(C-15), 181.3(C-19), 152.2(C-16), 112.7(C-17), 66.7(C-11), 63.7(C-9), 57.5(C-5), 52.0(C-8), 44.6(C-4), 41.4(C-12), 40.9(C-1), 40.0(C-10), 39.0(C-3), 38.5(C-13), 37.8(C-7), 35.3(C-14), 29.5(C-18), 21.2(C-6), 20.1(C-2), 16.3 (C-20). The crystal parameters are as follows: molecular formula C₂₀H₂₈O₄The crystal system is monoclinic, and the unit of the crystal lattice is

α＝90.00°，β＝104.654°，γ＝90.00°，

T293 (2), K, Z2, etc.;

example 87 characterization of α,11 α -dihydroxy-15-oxo-16-methylene-ent-kaurane-19, 6 β -lactone

White strip crystal (methanol), insoluble in chloroform and petroleum ether, soluble in methanol and ethyl acetate, and slightly soluble in dichloromethane, to obtainDichloromethane-methanol at a ratio of 20: 1 as developing solvent, Rf 0.7, TLC has a fluorescent spot under UV 254nm, 10% sulfuric acid-absolute ethanol solution develops color: purple circular spots;¹H-NMR(600MHz，CD₃OD)δH5.84(1H，s)，5.39(1H，s)，5.28(1H，t，J＝6.0Hz)，4.43(1H，d，J＝6.0Hz)，4.27-4.24(1H，m)，3.35(1H，s)，3.13(1H，dd，J＝9.1，4.8Hz)，2.77(1H，d，J＝11.4Hz)，2.33-2.27(1H，m)，2.10(1H，dt，J＝14.4，5.7Hz)，2.04(1H，dd，J＝11.2，4.9Hz)，1.89(1H，d，J＝6.0Hz)，1.77(1H，dd，J＝7.5，4.3Hz)，1.75(1H，t，J＝5.9Hz)，1.65-1.57(1H，m)，1.54(1H，ddd，J＝11.3，10.1，5.7Hz)，1.45-1.38(1H，m)，1.31(3H，s)，1.29(1H，d，J＝3.3Hz)，1.13(3H，s)，1.12-1.09(1H，m)。¹³C-NMR(150MHz，CD₃OD)δC207.3(C-15)，184.9(C-19)，154.5(C-16)，116.3(C-17)，85.4(C-6)，71.5(C-7)，65.8(C-11)，56.8(C-9)，56.4(C-8)，53.7(C-5)，43.6(C-12)，42.8(C-4)，38.0(C-1)，37.6(C-10)，36.6(C-13)，29.9(C-14)，29.4(C-3)，26.2(C-18)，23.2(C-20)，18.4(C-2)；

example characterization of 99-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid

White square crystal (methanol), which is easily dissolved in dichloromethane and methanol, dichloromethane-methanol with a ratio of 20: 1 is used as developing agent, Rf is 0.8, TLC has mauve fluorescent point under the wavelength of 254nm, 10% sulfuric acid-absolute ethyl alcohol is used for color development: light pink circular spots;¹H-NMR(600MHz，CD₃OD)δH5.91(1H，s)，5.32(1H，s)，3.05(1H，s)，2.64(1H，d，J＝12.5Hz)，2.26-2.21(1H，m)，2.18(1H，dd，J＝15.4，6.0Hz)，2.10(1H，d，J＝13.2Hz)，2.00-1.95(1H，m)，1.92(1H，d，J＝9.7Hz)，1.91-1.88(1H，m)，1.88-1.79(1H，m)，1.79-1.76(1H，m)，1.74(1H，dd，J＝14.5，4.3Hz)，1.51-1.47(1H，m)，1.47-1.45(1H，m)，1.45(1H，dd，J＝7.2，2.6Hz)，1.28(1H，dd，J＝14.3，7.2Hz)，1.24-1.19(3H，m)，1.18(3H，d，J＝9.8Hz)，1.12(1H，dt，J＝13.5，3.0Hz)，1.08-0.99(1H，m)。¹³C-NMR(150MHz，CD₃OD)δC210.0(C-15)，181.8(C-19)，151.0(C-16)，115.2(C-17)，78.1(C-9), 58.2(C-8), 50.3(C-5), 46.2(C-10), 44.8(C-4), 38.9(C-3), 38.9(C-13), 38.4(C-1), 35.1(C-7), 32.6(C-14), 30.8(C-12), 30.6(C-11), 29.6(C-18), 21.4(C-6), 20.0(C-2), 17.8 (C-20). The crystal parameters are as follows: molecular formula C₂₀H₂₈O₄The crystal system is m onocolic, the unit of the crystal lattice is

α＝90.00°，β＝132.624°，γ＝90.00°，

T173K, Z8, etc.

Example 10 compound and extraction separation purification method:

1) extraction and crude separation of medicinal materials

Collecting 74.15kg of fresh medicinal materials of the pteris multifida, 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 for 5 times, each time is 5 days, 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 leaching solution is subjected to reduced pressure concentration at 55 ℃ to obtain 7.5kg of total extract (the density is 1.06), the total extract is diluted by 1.25 times of warm water, the diluted total extract is extracted by equal volume of ethyl acetate, after extraction is finished, the extraction solutions are combined and subjected to reduced pressure concentration at 55 ℃, and the concentration is carried out to obtain 348g of ethyl acetate part extract (the density is 1.06);

2) separation and purification of ethyl acetate fraction

348g of extract extracted by ethyl acetate is taken, the same amount of polyamide with 30-60 meshes is used for mixing samples, an MCI reverse phase column is adopted for elution, firstly, 50% methanol/water is used for replacing a solvent in the MCI column, then, 50% -100% methanol/water system is used for gradient elution and segmentation, and 154.349g of H part, 37.855g of I part and 53.323g of other part are obtained; wherein, the separation and purification of the H part are as follows: 154.349g of H part sample is taken, 150 meshes of equal silica gel is used for mixing samples, the samples are arranged in a dry method, dichloromethane-ethyl acetate and dichloromethane-methanol with the proportion of 20: 1 and 4: 1 are used for gradient elution in sequence, the fraction is subjected to thin layer detection, 4 parts of H-1, H-2, H-3 and H-4 are obtained by merging the same parts, the H-1 and the H-2 are separated by silica gel column chromatography again, dichloromethane-ethyl acetate with the proportion of 20: 1 and 10: 1 and dichloromethane-methanol with the proportion of 20: 1 and 10: 1 and 5: 1 are used for gradient elution in sequence, crystals are analyzed in the flow, and the compound 7 is 7 beta, 9-dihydroxy-15-oxo-ent-kauran-16-en-19 is obtained by recrystallization and purification, 17mg of 6 beta-olide, 11 beta, 12 beta-trihydroxy-15-oxo-ent-kuar-16-en-19-oic acid as compound 8, 11mg of 11 beta-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid as compound 9 and 1.17g of compound; 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-kaurane-19, 6 beta-lactone, 19mg) and a white square-crystal compound 11 is 9-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid, 5 mg); isolation and purification of fraction I was: separating by silica gel column chromatography, mixing 37.855g of part I sample with 100-mesh 200-mesh equivalent silica gel, performing gradient elution with dichloromethane-methanol at the ratio of 50: 1, 30: 1, 20: 1, 10: 1, 5: 1 and 1: 1 in sequence, performing flow analysis to separate crystals, and performing repeated recrystallization on the crystals for thin layer detection, wherein the compound 9 mainly comprises 11 beta-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid and 2 g.

The structural formula of the compound: the same as in example 1.

Crystal structure of the compound: the same as in examples 2, 3 and 4.

Characteristics of the compounds: the same as in examples 5, 6, 7, 8 and 9.

Example 11 compound and extraction separation purification method:

1) extraction and crude separation of medicinal materials

Collecting 74.15kg of fresh medicinal materials of the pteris multifida, naturally drying, crushing and sieving by a 30-mesh sieve to obtain 25kg of coarse powder, soaking and extracting the coarse powder by using 2 times of 80% industrial methanol at normal temperature for 4 times, wherein each time is 4 days, extracting by using 2 times of 80% methanol at normal temperature once, extracting by using 2 times of 60% methanol at normal temperature once, concentrating the extract at 50 ℃ under reduced pressure to obtain a total extract 5k g (the density is 1.06), diluting the total extract by using 1.25 times of warm water, extracting by using equal volume of ethyl acetate after dilution, merging the extract after extraction, concentrating at 50 ℃ under reduced pressure to obtain 300g of an ethyl acetate part extract (the density is 1.06);

2) separation and purification of ethyl acetate fraction

Taking 300g of extract extracted by ethyl acetate, mixing the extract with 30-mesh polyamide in the same amount, eluting by adopting an MCI reverse phase column, firstly replacing a solvent in the MCI column by 50% methanol/water, and then sequentially carrying out gradient elution and segmentation by using a 50% -100% methanol/water system to obtain 140g of an H part, 30g of an I part and 40g of other parts; wherein, the separation and purification of the H part are as follows: taking 140g of a H part sample, mixing the H part sample with 100-mesh equivalent silica gel, loading the sample into a column by a dry method, carrying out gradient elution by dichloromethane-ethyl acetate and dichloromethane-methanol according to the proportion of 20: 1 and 4: 1 and the proportion of 10: 1, 9:1 and 4: 1 in sequence, carrying out thin-layer detection on the fraction, 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 by 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 obtaining a compound 7 of 7 beta, 9-dihydroxy-15-oxo-ent-kauran-16-en-19 by recrystallization and purification, 17mg of 6 beta-olide, 11 beta, 12 beta-trihydroxy-15-oxo-ent-ku ar-16-en-19-oic acid as compound 8, 11mg of 11 beta-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid as compound 9 and 1.17g of compound; 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-kaurane-19, 6 beta-lactone, 19mg) and a white square-crystal compound 11 is 9-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid, 5 mg); isolation and purification of fraction I was: separating by silica gel column chromatography, mixing 37.855g of part I sample with 100-mesh 200-mesh equivalent silica gel, performing gradient elution with dichloromethane-methanol at the ratio of 50: 1, 30: 1, 20: 1, 10: 1, 5: 1 and 1: 1 in sequence, separating crystals by flow analysis, and repeatedly recrystallizing the crystals for thin layer detection to obtain the compound 9 mainly containing 11 beta-hydroxy-15-oxoent-kauran-16-en-19-oic acid and 2 g.

The structural formula of the compound: the same as in example 1.

Crystal structure of the compound: the same as in examples 2, 3 and 4.

Characteristics of the compounds: the same as in examples 5, 6, 7, 8 and 9.

Example 12 compound and extraction separation purification method:

1) extraction and crude separation of medicinal materials

Collecting 74.15kg of fresh medicinal materials of the pteris multifida, naturally drying, crushing and sieving with a 60-mesh sieve to obtain 35kg of coarse powder, soaking and extracting the coarse powder with 2 times of 98% industrial methanol at normal temperature for 6 times, wherein each time is 6 days, extracting with 2 times of 100% methanol at normal temperature once, extracting with 2 times of 80% methanol at normal temperature once, concentrating the extract at 60 ℃ under reduced pressure to obtain 10kg of total extract (the density is 1.06), diluting the total extract with 1.25 times of warm water, extracting with equal volume of ethyl acetate after dilution, merging the extract liquid at 60 ℃ after extraction is finished, concentrating under reduced pressure to obtain 400g of ethyl acetate part extract (the density is 1.06);

2) separation and purification of ethyl acetate fraction

Taking 400g of extract extracted by ethyl acetate, mixing the extract with 60-mesh polyamide in the same amount, eluting by adopting an MCI reverse phase column, firstly replacing a solvent in the MCI column by 50% methanol/water, and then sequentially carrying out gradient elution and segmentation by using a 50% -100% methanol/water system to obtain 160g of part H, 40g of part I and 70g of other part; wherein, the separation and purification of the H part are as follows: taking 160g of a H part sample, mixing the H part sample with 200-mesh equivalent silica gel, loading the sample into a column by a dry method, carrying out gradient elution by 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 detection 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 by 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 obtaining a compound 7 of 7 beta, 9-dihydroxy-15-oxo-ent-kauran-16-en-19 by recrystallization and purification, 17mg of 6 beta-olide, 11 beta, 12 beta-trihydroxy-15-oxo-ent-kuar-16-en-19-oic acid as compound 8, 11mg of 11 beta-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid as compound 9 and 1.17g of compound; 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-kaurane-19, 6 beta-lactone, 19mg) and a white square-crystal compound 11 is 9-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid, 5 mg); isolation and purification of fraction I was: separating by silica gel column chromatography, mixing 37.855g of part I sample with 100-mesh 200-mesh equivalent silica gel, performing gradient elution with dichloromethane-methanol at the ratio of 50: 1, 30: 1, 20: 1, 10: 1, 5: 1 and 1: 1 in sequence, separating crystals by flow analysis, and repeatedly recrystallizing the crystals for thin layer detection to obtain the compound 9 mainly containing 11 beta-hydroxy-15-oxoent-kauran-16-en-19-oic acid and 2 g.

The structural formula of the compound: the same as in example 1.

Crystal structure of the compound: the same as in examples 2, 3 and 4.

Characteristics of the compounds: the same as in examples 5, 6, 7, 8 and 9.

Example 13 taking any one of 7 β,9-dihydroxy-15-oxo-ent-kauran-16-en-19,6 β -olide, 5,11 β,12 β -trihydroxyxy-15-oxo-ent-kuar-16-en-19-oic acid, 11 β -hydroxy-15-oxo-16-en-19-oic acid, 7 α,11 α -dihydroxy-15-oxo-16-methylene-ent-kaurane-19, 6 β -lactone, 9-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid as a raw material, adding 20% microcrystalline cellulose and 20% powdered sugar to granulate, and (5) obtaining granules.

Example 14 taking any one of 7 β,9-dihydroxy-15-oxo-ent-kauran-16-en-19,6 β -olide, 5,11 β,12 β -trihydroxyxy-15-oxo-ent-kuar-16-en-19-oic acid, 11 β -hydroxy-15-oxo-16-en-19-oic acid, 7 α,11 α -dihydroxy-15-oxo-16-methylene-ent-kaurane-19, 6 β -lactone, 9-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid as a raw material, adding 10% of starch and 3% of magnesium stearate, mixing well, filling into capsules, and making into capsule.

Example 15 any one of the compounds of 7 beta, 9-dihydroxy-15-oxo-ent-kauran-16-en-19,6 beta-olide, 5,11 beta, 12 beta-trihydroxyxy-15-oxo-ent-kuar-16-en-19-oic acid, 11 beta-hydroxy-15-oxo-16-en-19-oic acid, 7 alpha, 11 alpha-dihydroxy-15-oxo-16-methylene-ent-kaurane-19, 6 beta-lactone and 9-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid is taken as a raw material, 10% of starch and 15% of honey are added to be mixed uniformly, making into pill.

Example 16A preparation method of a pharmaceutical composition comprising any one of 7 β,9-dihydroxy-15-oxo-ent-kauran-16-en-19,6 β -olide, 5,11 β,12 β -trihydroxyxy-15-oxo-ent-kuar-16-en-19-oic acid, 11 β -hydroxy-15-oxo-16-en-19-oic acid, 7 α,11 α -dihydroxy-15-oxo-16-methylene-ent-kaurane-19, 6 β -lactone, and 9-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid as a raw material, 10% of starch syrup and 0.3% of magnesium stearate were added to the raw material, followed by granulation, tabletting, and making into tablet.

Example 17 taking 7 β,9-dihydroxy-15-oxo-ent-kauran-16-en-19,6 β -olide, 5,11 β,12 β -trihydroxyxy-15-oxo-ent-kuar-16-en-19-oic acid, 11 β -hydroxy-15-oxo-ent-kauran-16-en-19-oic acid, any one compound of 7 alpha, 11 alpha-dihydroxy-15-oxo-16-methylene-ent-kaurane-19, 6 beta-lactone and 9-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid is used as a raw material drug, 12 times of injection water is added, and the injection is obtained by filtering and sterilizing.

Example 18 7 β,9-dihydroxy-15-oxo-ent-kauran-16-en-19,6 β -olide, 5,11 β,12 β -trihydroxyxy-15-oxo-ent-kuar-16-en-19-oic acid, 11 β -hydroxy-15-oxo-ent-kauran-16-en-19-oic acid, any one compound of 7 alpha, 11 alpha-dihydroxy-15-oxo-16-methylene-ent-kaurane-19, 6 beta-lactone and 9-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid is used as a raw material drug, 8 times of water for injection is added, and the mixture is filtered and freeze-dried to obtain freeze-dried powder.

Example 19 any one of 7 β,9-dihydroxy-15-oxo-ent-kauran-16-en-19,6 β -olide, 5,11 β,12 β -trihydroxyxy-15-oxo-ent-kuar-16-en-19-oic acid, 11 β -hydroxy-15-oxo-16-en-19-oic acid, 7 α,11 α -dihydroxy-15-oxo-16-methylene-ent-kaurane-19, 6 β -lactone, and 9-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid was used as a raw material, 12 times of purified water and 0.05% sodium benzoate were added and mixed uniformly, filtering, and sterilizing to obtain oral liquid.

In order to further verify the feasibility and the effectiveness of the invention, the inventor carries out a series of tests, which are as follows:

isolation and characterization of Compounds

1 apparatus, reagent and Material

1.1 instruments and reagents

Three color developing agents are involved in the experiment, namely 10% sulfuric acid-absolute ethyl alcohol solution, 5% phosphomolybdic acid-absolute ethyl alcohol solution and 2% iodine-silica gel.

1.2 materials

Experimental medicinal materials (pteridophyte): the original plant is collected in the terrace village of Li Ping county of Guizhou province in 7 months in 2018, is identified as pteridium aquilinum (Pteridaceae) pteridium (Pteris L.) of Pteridaceae (Pteris L.) by professor Zhao Junhua of Guizhou traditional Chinese medicine university, and the original plant voucher specimen is stored in the traditional Chinese medicine national medicine focus laboratory of Guizhou traditional Chinese medicine university.

Blood COAGULATION quality control plasma (COAGULATION quality control plasma) (batch No. 093B-J186A, manufacturer: TECO, Germany); TT test solution (batch No. 30002697, manufacturer: produced by Germany TECO company); Tris-HCl (batch No. 20160118, manufacturer: Amresco, USA); PT test solution (batch No. 10002706, manufacturer: TECO, Germany); APTT test solutions (batch No. 20002745, manufacturer: TECO, Germany); CaCl2 (batch number: 031N-J073A, manufacturer: TECO, Germany); enoxaparin (LMWH, Kurarin Schk, Lot: 4SH 69). Leukemia cells HL-60, lung cancer cells A549, liver cancer cells SMMC-7721, breast cancer cells MCF-7, colon cancer cells SW480, human normal lung epithelial cells BEAS-2B, and human normal liver cells LO2 were purchased from ATCC. Fetal bovine serum (lot: 1706126), RMPI-1640 medium (lot: 0023019), DMEM medium (lot: 0024719), pancreatin (lot: 0023518), double antibody (lot: 1936917), phosphate buffered saline PBS (lot: 0044818), and dimethyl sulfoxide (lot: B821BA0018) were purchased from BI. The positive drugs cisplatin (lot: N1001A) and paclitaxel (lot: D1106A) were purchased from Melam Biotech. MTS kit (lot: 0000219904) was purchased from Promega.

2 extraction and separation

2.1 extraction and crude separation of herbs

74.15kg of fresh medicinal materials of the pteris spinosa are collected, and 29.9kg of coarse powder with 30-60 meshes is obtained through natural drying and crushing. The coarse powder is first soaked and extracted with 2 times of 95% industrial methanol at normal temperature for 5 times, each time lasts for 5 days, then soaked and extracted with 2 times of 90% methanol at normal temperature, finally soaked and extracted with 2 times of 70% methanol at normal temperature, and the leaching liquor is decompressed and concentrated at 55 ℃ to obtain 7.5kg of total extract (the density is 1.06, and the extraction rate is about 25.1%). Diluting the total extract with 1.25 times of warm water, extracting with equal volume of ethyl acetate, mixing the extractive solutions, concentrating at 55 deg.C under reduced pressure to obtain 348g of ethyl acetate extract (density of 1.06)

2.2 separation and purification of the Ethyl acetate fraction

The ethyl acetate part sample amount is 348g, the same amount of polyamide (30-60 meshes) is used for sample mixing, an MCI reverse phase column is used for elution, firstly, 50% methanol/water is used for replacing a solvent in the MCI column, and then, 50% -100% methanol/water system is used for gradient elution and segmentation to obtain an H part (154.349g), an I part (37.855g) and other parts (53.323 g); and separating and purifying the 2 parts of H-I:

separation of H part: firstly, silica gel column chromatography is used for separation, TLC identification is carried out, two systems (dichloromethane-ethyl acetate and dichloromethane-methanol) are selected, 154.349g of H part sample is mixed with equal amount of silica gel (100 meshes and 200 meshes), the column is packed by a dry method, gradient elution is carried out in sequence on a dichloromethane-ethyl acetate system (20: 1 and 4: 1) and a dichloromethane-methanol system (10: 1, 9:1 and 4: 1), thin-layer identification is carried out on fractions, and 4 parts (H-1, H-2, H-3 and H-4) are obtained by combining the same parts. Separating H-1 and H-2 again by silica gel column chromatography, eluting with dichloromethane-ethyl acetate system (20: 1, 10: 1) and dichloromethane-methanol (20: 1, 10: 1, 5: 1) system as eluent, eluting to give crystals, and recrystallizing to obtain compound 7(PDY-11, 17mg), compound 8(PDY-11-1, 11mg) and compound 9(PDY-12, 1.17 g); h-3 and H-4 were again separated by silica gel column chromatography, and eluted with a dichloromethane-ethyl acetate system (20: 1, 15: 1, 10: 1) and a dichloromethane-methanol (20: 1, 10: 1, 8: 1, 5: 1) system as an eluent, to obtain white crystals 10(PDY-15, 19mg) and white cristobalite 11(PDY-16, 5 mg).

Separation of part I: separation was carried out by silica gel column chromatography, 37.855g of the sample of fraction I was mixed with an equal amount of silica gel (100-mesh 200), gradient elution was sequentially carried out with a methylene chloride-methanol system (50: 1, 30: 1, 20: 1, 10: 1, 5: 1, 1: 1), crystals were eluted by flow analysis, and thin layer chromatography was carried out by repeated recrystallization of the crystals to mainly contain the compound 9(PDY-12, 2 g).

2.3 structural identification of 5 chemical Components in Pteris spinosa

Compound 7: white strip crystal (pyridine) is insoluble in chloroform and petroleum ether, relatively easy to dissolve in methanol, ethyl acetate and pyridine, and slightly soluble in dichloromethane. Developing agent: dichloromethane-methanol (20: 1), TLC with fluorescent spot at UV 254nm, 10% sulphuric acid-ethanol solution as developer, and heating (115 deg.C) to develop blue circular spot. According to¹H-NMR(400MHz，C₅D₅N). delta.H 1.12(3H, s), 1.01(3H, s) are hydrogen on the methyl group.¹³C-NMR(101MHz，C₅D₅N) the data indicate that the compound contains 20 carbons, where δ C202.1 is a carbonyl signal, and 183.8 and 85.5 indicate the presence of a lactone; δ C156.2, 113.1 is an exocyclic double bond; δ C73.1 and 81.1 suggest two vicinal oxygen groups. In conclusion, it is concluded that the compound may be a diterpenoid. Verifying by an X-ray Diffraction Method to confirm the guess; according to the literature, molecular weight 346(EI-MS) of 6F, melting point (mp): 220-]. From the data, the compound is identified to be 7 beta, 9-dihydroxy-15-oxo-ent-kauran-16-en-19,6 beta-olide, and the structural formula is shown as follows; process for preparation of Compound 7¹³The C-NMR data are shown in Table 1.

TABLE 1 Compound 7 and references¹³C-NMR data comparison Table (delta in ppm)

Data¹were recorded in C₅D₅N on a Brucker 400MHz NMR spectrometer.

Structural formula (I)

Single crystal structure

Compound 8: the transparent needle crystal (methanol) is insoluble in dichloromethane, chloroform and petroleum ether, and is more soluble in methanol and pyridine. Developing agent: dichloromethane-methanol (20: 1, Rf ═ 0.8), TLC showed a fluorescent spot at 254nm, and a 10% sulfuric acid-ethanol solution was used as a developing reagent to develop a light purple circular spot upon heating (115 ℃). According to¹H NMR(600MHz，CD₃OD) δ H5.24, 5.74 is hydrogen on the exocyclic double bond; Δ H0.89(3H, s), 1.31(3H, s) is hydrogen on the methyl group.¹³C-NMR(150MHz，CD₃OD) data indicate that the compound contains 20 carbons, wherein the delta C203.6(C-15) indicates that the compound contains oneA carbonyl group; δ C184.9 has one carboxyl group; δ C155.3(C-16), 113.4(C-17) is an exocyclic double bond; δ C85.1, 80.5, 72.5 suggests three vicinal oxygens. In conclusion, it is concluded that it may be a diterpenoid of the ent-kaurane type. The above data and literature [6 ]]The report is consistent, so that the compound is determined to be 5,11 beta, 12 beta-trihydroxy-15-oxo-ent-kuar-16-en-19-oic acid. Carbon spectrum data of compound 8 and in the literature¹³The C-NMR data are shown in Table 2, and the structural formula is shown below.

TABLE 2 Compound 8 and references¹³C-NMR data comparison Table (delta in ppm)

Data^8a were recorded in CD₃OD on a Brucker 150MHz NMR spectrometer.

Data^8b were recorded in C₅D₅N on a Brucker 150MHz NMR spectrometer.

Structural formula (I)

Compound 9: the transparent needle crystal (methanol) is insoluble in petroleum ether, chloroform and dichloromethane, and soluble in methanol and pyridine. Developing agent: dichloromethane-methanol (20: 1, Rf ═ 0.7), TLC had a purple-red fluorescent spot at a wavelength of 254nm, and a 10% sulfuric acid-ethanol solution was used as a color developing agent, and heated (115 ℃) to develop a round pink spot. According to¹H-NMR(600MHz，CD₃OD) delta H5.24(H-17) vs. olefinic proton signals 0.99(3H, s), 1.23(3H, s) are hydrogen on methyl.¹³C-NMR(150MHz，CD₃OD) data showed that the compound contained 20 carbons, δ C212.5 suggested a carbonyl group; 181.3 has a carboxyl group; 152.2, 112.7 is an exocyclic double bond; 66.7 suggests an oxygen linking group. In conclusion, it is concluded that it may be a diterpenoid. The guess is verified by an X-ray Diffraction Method, the above spectral data and the literature [12 ]]The 5F data in the formula (I) are basically consistent, so that the formula (I) is identified as 11 beta-hydroxyThe structural formula of the-15-oxo-ent-kauran-16-en-19-oic acid is shown as follows; carbon spectrum data of compound 9 and in literature¹³The C-NMR data are shown in Table 3 below.

TABLE 3 Compound 9 and references¹³C-NMR data comparison Table (delta in ppm)

Data^9a were recorded in CD₃OD on a Brucker 150MHz NMR spectrometer.

Data^9b were recorded in C₅D₅N on a Brucker 400MHz NMR spectrometer.

Structural formula (I)

Single crystal structure

Compound 10: white strip crystal (methanol) is easy to dissolve in methanol and ethyl acetate, slightly soluble in dichloromethane and insoluble in chloroform and petroleum ether. Developing agent: dichloromethane-methanol (20: 1, Rf ═ 0.7), TLC showed a fluorescent spot at UV 254nm, and a 10% sulfuric acid-ethanol solution was used as a developing reagent, and heating (115 ℃) revealed a purple circular spot.¹H-NMR(600MHz，CD₃OD) Δ H5.84(H-17 β), 5.39(1H, s, H-17 α); 1.31(3H, s), (3H, s) are hydrogen on methyl. According to¹³C-NMR(150MHz，CD₃OD) data showed that the compound contained 20 carbons, where δ C207.3 is a carbonyl signal, 184.9 and 85.4 indicate the presence of a lactone; 154.5, 116.3 is an exocyclic double bond; 71.5 and 65.8 suggest two vicinal oxygen radicals. Combining the above data, it was concluded that the compound was likely a diterpenoid. The above spectral data and literature [16]The data of the 7 alpha, 11 alpha-dihydroxy-15-oxo-16-methylene-ent-kaurane-19, 6 beta-lactone in the formula are basically consistent, so that the 7 alpha, 11 alpha-dihydroxy-15-oxo-16-methylene-ent-kaurane-19, 6 beta-lactone is identified as the following structural formula; compound (I)10 carbon spectra data and in literature¹³The C-NMR data are shown in Table 4 below.

TABLE 4 Compound 10 and references¹³C-NMR data comparison Table (delta in ppm)

Data^10a were recorded in CD₃OD on a Brucker 150MHz NMR spectrometer.

Data^10b were recorded in CD₃OD on a Brucker 400MHz NMR spectrometer.

Structural formula (I)

Compound 11: white cristobalite (methanol), readily soluble in dichloromethane, methanol, developing solvent: dichloromethane-methanol (20: 1), TLC with mauve fluorescence spot at 254nm wavelength and Rf value of about 0.8, and 10% sulfuric acid-ethanol solution as developer, and heating (115 deg.C) to develop pink circular spot. According to¹H-NMR(600MHz，CD₃OD) δ H1.23(3H, s), 1.19(3H, s) are hydrogen on methyl.¹³C-NMR(150MHz，CD₃OD) data showed that the compound contained 20 carbons, where δ C209.9 is a carbonyl signal; 181.8 is a carboxyl signal; 150.9 and 115.2 are an exocyclic double bond; 78.1 indicates the attachment of a hydroxyl group. In conclusion, it is concluded that the compound may be a diterpenoid. Verified and inferred by an X-ray Diffraction Method, the above spectral data and literature [17 ]]The data in the formula are basically consistent, so that the acid is identified as 9-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid, and the structural formula is as follows; carbon spectrum data of compound 11 and in literature¹³The C-NMR data are shown in Table 5.

TABLE 5 Compound 11 and references¹³C-NMR data comparison Table (delta in ppm)

Data^11a were recorded in CD₃OD on a Brucker 150MHz NMR spectrometer.

Data^11b were recorded in CD₃OD on a Brucker 600MHz NMR spectrometer.

Structural formula (I)

Single crystal structure

2.4 physicochemical constants and spectral data of the Compound

7 beta, 9-dihydroxy-15-oxo-ent-kauran-16-en-19,6 beta-olide: white strip crystal (pyridine) is insoluble in chloroform and petroleum ether, relatively easy to dissolve in methanol, ethyl acetate and pyridine, and slightly soluble in dichloromethane. Developing agent: dichloromethane-methanol (20: 1), TLC has a fluorescent spot at UV 254nm, 10% sulfuric acid-absolute ethanol solution develops color: a blue circular spot.¹H-NMR(400MHz，C₅D₅N)δH 6.38(1H，s)，6.03(1H，s，H-17b)，5.75(1H，t，J＝6.1Hz)，5.18(2H，d，J＝13.9Hz，H-17)，4.71(1H，d，J＝6.0Hz)，3.62(1H，s)，3.23(1H，d，J＝6.3Hz)，3.01(1H，dd，J＝9.3，4.5Hz)，2.53(1H，dd，J＝12.1，4.7Hz)，1.12(3H，s，H-20)。¹³C-NMR(101MHz，C₅D₅N) δ C36.9(C-1), 19.2(C-2), 28.9(C-3), 42.0(C-4), 51.0(C-5), 85.5(C-6), 81.1(C-7), 62.2(C-8), 73.1(C-9), 46.2(C-10), 43.2(C-11), 30.7(C-12), 36.9(C-13), 34.2(C-14), 202.1(C-15), 156.2(C-16), 113.1(C-17), 26.5(C-18), 183.8(C-19), 22.1 (C-20). The crystal parameters are as follows: molecular formula C₂₀H₂₆O₅The crystal system is orthorhombic and the unit of the crystal lattice is

α＝90.00°，β＝90.00°，γ＝90.00°，

T296 (2) K, Z1, etc.

5,11 beta, 12 beta-trihydroxyoxy-15-oxo-ent-kuar-16-en-19-oic acid: the transparent needle crystal (methanol) is insoluble in dichloromethane, chloroform and petroleum ether, and is more soluble in methanol and pyridine. Developing agent: dichloromethane-methanol (20: 1, Rf ═ 0.8), TLC showed fluorescent spots at 254nm, 10% sulfuric acid-absolute ethanol developed: light purple horizontal stripe spots.¹H-NMR(600MHz，CD₃OD)δH5.74(1H，s)，5.24(1H，s)，5.13(1H，t，J＝6.2Hz)，4.59(2H，s)，4.18(1H，d，J＝6.0Hz)，3.35(1H，s)，3.04(1H，dd，J＝9.5，4.6Hz)，2.79(1H，d，J＝6.3Hz)，2.27-2.20(1H，m)，2.14-2.06(3H，m)，2.00(1H，dd，J＝14.6，6.0Hz)，1.82(1H，td，J＝12.6，6.5Hz)，1.55(3H，dd，J＝10.7，4.9Hz)，1.38(2H，dd，J＝11.5，5.9Hz)，1.31(3H，s)，1.27-1.21(1H，m)，0.90(3H，s)。¹³C-NMR(150MHz，CD₃OD)δC203.6(C-15)，184.9(C-19)，155.3(C-16)，113.4(C-17)，85.1(C-12)，80.5(C-5)，72.5(C-11)，61.5(C-8)，45.7(C-9)，43.1(C-4)，41.4(C-10)，36.6(C-13)，33.4(C-14)，31.8(C-1)，30.1(C-7)，28.0(C-3)，26.7(C-6)，25.6(C-18)，21.1(C-20)，18.5(C-2).

11 beta-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid: the transparent needle crystal (methanol) is insoluble in dichloromethane, chloroform and petroleum ether, and is more soluble in methanol and pyridine. Developing agent: dichloromethane-methanol (20: 1, Rf ═ 0.7), TLC showed a purple-red fluorescent spot at a wavelength of 254nm, 10% sulfuric acid-absolute ethanol developed: pink circular spot.¹H-NMR(600MHz，CD₃OD)δH5.72(1H，s)，5.49(1H，s)，5.24(1H，s)，4.02(1H，d，J＝4.3Hz)，3.03(1H，d，J＝3.5Hz)，2.44(1H，d，J＝11.8Hz)，2.16(1H，d，J＝12.4Hz)，2.09(1H，ddd，J＝14.3，4.7，3.1Hz)，1.97(2H，dd，J＝18.7，2.0Hz，)，1.94(2H，t，J＝4.9Hz)，1.91-1.87(1H，m)，1.86-1.81(2H，m)，1.48-1.43(1H，m)，1.41-1.34(3H，m)，1.23(3H，s)，1.22-1.18(2H，m)，1.14-1.06(2H，m)，0.99(3H，s)。¹³C-NMR(150MHz，CD₃OD) δ C212.0(C-15), 181.3(C-19), 152.2(C-16), 112.7(C-17), 66.7(C-11), 63.7(C-9), 57.5(C-5), 52.0(C-8), 44.6(C-4), 41.4(C-12), 40.9(C-1), 40.0(C-10), 39.0(C-3), 38.5(C-13), 37.8(C-7), 35.3(C-14), 29.5(C-18), 21.2(C-6), 20.1(C-2), 16.3 (C-20). The crystal parameters are as follows: molecular formula C₂₀H₂₈O₄The crystal system is monoclinic, and the unit of the crystal lattice is

α＝90.00°，β＝104.654°，γ＝90.00°，

T293 (2) K, Z2, etc.

7 α,11 α -dihydroxy-15-oxo-16-methylene-ent-kaurane-19, 6 β -lactone: white strip crystal (methanol), insoluble in chloroform and petroleum ether, soluble in methanol and ethyl acetate, and slightly soluble in dichloromethane. Developing agent: dichloromethane-methanol (20: 1, Rf ═ 0.7), TLC showed fluorescent spots at UV 254nm, 10% sulfuric acid-absolute ethanol solution developed: purple circular spots.¹H-NMR(600MHz，CD₃OD)δH5.84(1H，s)，5.39(1H，s)，5.28(1H，t，J＝6.0Hz)，4.43(1H，d，J＝6.0Hz)，4.27-4.24(1H，m)，3.35(1H，s)，3.13(1H，dd，J＝9.1，4.8Hz)，2.77(1H，d，J＝11.4Hz)，2.33-2.27(1H，m)，2.10(1H，dt，J＝14.4，5.7Hz)，2.04(1H，dd，J＝11.2，4.9Hz)，1.89(1H，d，J＝6.0Hz)，1.77(1H，dd，J＝7.5，4.3Hz)，1.75(1H，t，J＝5.9Hz)，1.65-1.57(1H，m)，1.54(1H，ddd，J＝11.3，10.1，5.7Hz)，1.45-1.38(1H，m)，1.31(3H，s)，1.29(1H，d，J＝3.3Hz)，1.13(3H，s)，1.12-1.09(1H，m)。¹³C-NMR(150MHz，CD₃OD)δC207.3(C-15)，184.9(C-19)，154.5(C-16)，116.3(C-17)，85.4(C-6)，71.5(C-7)，65.8(C-11)，56.8(C-9)，56.4(C-8)，53.7(C-5)，43.6(C-12)，42.8(C-4)，38.0(C-1)，37.6(C-10)，36.6(C-13)，29.9(C-14)，29.4(C-3)，26.2(C-18)，23.2(C-20)，18.4(C-2)。

9-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid: white cristobalite (methanol), readily soluble in dichloromethane, methanol, developing solvent: dichloromethane-methanol (20: 1, Rf ═ 0.8), TLC showed a purple-red fluorescent spot at a wavelength of 254nm, 10% sulfuric acid-absolute ethanol developed: light pink circular spot.¹H-NMR(600MHz，CD₃OD)δH5.91(1H，s)，5.32(1H，s)，3.05(1H，s)，2.64(1H，d，J＝12.5Hz)，2.26-2.21(1H，m)，2.18(1H，dd，J＝15.4，6.0Hz)，2.10(1H，d，J＝13.2Hz)，2.00-1.95(1H，m)，1.92(1H，d，J＝9.7Hz)，1.91-1.88(1H，m)，1.88-1.79(1H，m)，1.79-1.76(1H，m)，1.74(1H，dd，J＝14.5，4.3Hz)，1.51-1.47(1H，m)，1.47-1.45(1H，m)，1.45(1H，dd，J＝7.2，2.6Hz)，1.28(1H，dd，J＝14.3，7.2Hz)，1.24-1.19(3H，m)，1.18(3H，d，J＝9.8Hz)，1.12(1H，dt，J＝13.5，3.0Hz)，1.08-0.99(1H，m)。¹³C-NMR(150MHz，CD₃OD) δ C210.0(C-15), 181.8(C-19), 151.0(C-16), 115.2(C-17), 78.1(C-9), 58.2(C-8), 50.3(C-5), 46.2(C-10), 44.8(C-4), 38.9(C-3), 38.9(C-13), 38.4(C-1), 35.1(C-7), 32.6(C-14), 30.8(C-12), 30.6(C-11), 29.6(C-18), 21.4(C-6), 20.0(C-2), 17.8 (C-20). The crystal parameters are as follows: molecular formula C₂₀H₂₈O₄The crystal system is monoclinic, and the unit of the crystal lattice is

α＝90.00°，β＝132.624°，γ＝90.00°，

T173K, Z8, etc.

(II) study of biological Activity

1 study of antitumor Activity

Screening of

compounds

7, 8, 9, 10 and 11 for antitumor activity of human leukemia cell (HL-60), human lung cancer cell (A549), human liver cancer cell (SMMC-7721), human breast cancer cell (MCF-7) and human colon cancer cell (SW480) was performed by MTS method.

1.1 principle of the experiment

MTS method: the experimental principle is as follows: MTS is a novel MTT analogue, is called 3- (4, 5-dimethylthiozol-2-yl) -5- (3-carboxymethyloxyphenyl) -2- (4-sulfopheny) -2H-tetrazolium, and is a yellow dye. Succinate dehydrogenase in the mitochondria of living cells can metabolize and reduce MTS to generate soluble Formazan (Formazan) compounds, and the content of the Formazan can be measured at 490nm by using an enzyme labeling instrument. Since the formazan production amount is generally proportional to the number of living cells, the number of living cells can be estimated from the optical density OD value.

1.2 Experimental methods

1.2.1 Primary screening method

Inoculation of cells: preparing single cell suspension by using culture solution (DMEM or RMPI1640) containing 10% fetal calf serum, inoculating 3000-15000 cells in each hole to a 96-hole plate, wherein each hole volume is 100 mu l, and the cells are inoculated and cultured 12-24 hours in advance.

II, adding a solution of a compound to be detected: compounds were dissolved in DMSO and compounds were prescreened at a concentration of 40. mu.M, with a final volume of 200. mu.l per well, with 3 replicates per treatment.

III, color development: after culturing for 48 hours at 37 ℃, removing culture solution in the adherent cells, and adding 20 mu l of MTS solution and 100 mu l of culture solution in each hole; suspension cell HL-60 abandons 100. mu.l of culture supernatant, and 20. mu.l of MTS solution is added into each well; setting 3 blank multiple wells (mixed solution of 20 mul MTS solution and 100 mul culture solution), continuing incubation for 2-4 hours, and measuring the light absorption value after the reaction is fully performed.

IV, color comparison: the light absorption value of each well is read by a multifunctional microplate reader (MULTISKAN FC) with the wavelength of 492nm selected, the result is recorded, and after the data are processed, the inhibition rate of the cell is plotted by taking the number of the compound as the abscissa and the inhibition rate of the cell as the ordinate.

V. positive control compound: two positive compounds of cisplatin (DDP) and paclitaxel (Taxol) are arranged in each experiment,the concentration is used as the abscissa and the cell survival rate is used as the ordinate to draw a cell growth curve, and a two-point method (Reed and Muench method) is applied to calculate the IC of the compound₅₀The value is obtained.

1.2.2 rescreening method

Then the compound with half inhibition is re-screened, the methods I, III and V are the same as the primary screening,

II, adding a solution of a compound to be detected: compounds were dissolved in DMSO and compounds were rescreened at concentrations of 40. mu.M, 8. mu.M, 1.6. mu.M, 0.32. mu.M, 0.064. mu.M, with a final volume of 200. mu.l per well, with 3 replicates per treatment.

IV, color comparison: selecting 492nm wavelength, reading light absorption value of each hole with multifunctional microplate reader (MULTISKAN FC), recording result, drawing cell growth curve with concentration as abscissa and cell survival rate as ordinate after data processing, and calculating IC of compound by two-point method (Reed and Muench method)₅₀The value is obtained.

1.3 screening results for antitumor Activity

1.3.1 inhibition of human leukemia cells (HL-60) by Compounds

The test result shows that: the

compounds

7, 8, 9, 10 and 11 have half inhibition activity on human leukemia cells (HL-60) at the concentration of 40 mu M, the inhibition rates are 102.49 +/-0.65, 104.41 +/-0.03, 52.54 +/-1.45, 104.33 +/-0.63 and 103.98 +/-0.96 respectively, and the experimental results are shown in Table 6 and figure 1; IC for leukemia HL-60₅₀The experimental results are respectively 11.11 + -0.35, 5.452 + -0.222, 36.94 + -1.86, 3.590 + -0.021 and 3.523 + -0.092, which are shown in table 7 and fig. 2; the positive control compound cells were grown as shown in FIG. 3.

TABLE 6 results of the effect of test compounds on the proliferation of human leukemia cells (HL-60)

TABLE 7 IC of test Compounds on human leukemia cells (HL-60)₅₀Value of

1.3.2 inhibition of human Lung cancer cells (A549) by Compounds

The test 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, the inhibition rates are respectively 97.89 +/-0.35, 98.47 +/-0.12, 99.80 +/-0.11 and 98.84 +/-0.08, and the experimental results are shown in Table 8 and figure 4; IC of human Lung cancer cells (A549)₅₀15.69 +/-0.82, 15.15 +/-0.22, 4.602 +/-0.131 and 5.429 +/-0.114 respectively, and the experimental results are shown in a table 9 and a figure 5; the positive control compound cells were grown as shown in FIG. 6.

TABLE 8 results of the Effect of test Compounds on the proliferation of human Lung cancer cells (A549)

TABLE 9 IC of test Compounds on human Lung cancer cells (A549)₅₀Value of

1.3.3 inhibitory Activity of Compounds on human hepatoma cells (SMMC-7721)

The test result shows that: the

compounds

7, 8, 9, 10 and 11 have half inhibitory activity to human liver cancer cells (SMMC-7721) at the concentration of 40 mu M, the inhibition rates are 96.85 +/-0.47, 100.02 +/-0.09, 69.63 +/-1.69, 99.75 +/-0.16 and 99.39 +/-0.05 respectively, and the experimental results are shown in Table 10 and figure 7; 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, the experimental results are shown in Table 11 and FIG. 8; the positive control compound cell growth is shown in figure 9.

TABLE 10 results of the effect of test compounds on proliferation of human hepatoma cells (SMMC-7721)

Table 11 test compoundsIC of human liver cancer cell (SMMC-7721)₅₀Value of

1.4 inhibitory Activity of Compounds on human Breast cancer cells (MCF-7)

The test result shows that: the

compounds

7, 8, 10 and 11 have half inhibitory activity to human breast cancer cells (MCF-7) at the concentration of 40 mu M, the inhibition rates are 98.89 +/-0.96, 97.99 +/-1.78, 97.97 +/-0.16 and 96.67 +/-1.44 respectively, and the experimental results are shown in Table 12 and figure 10; IC of human breast cancer cells (MCF-7)₅₀The experimental results are 7.111 + -0.111, 5.809 + -0.097, 4.094 + -0.301 and 6.091 + -0.168 respectively, which are shown in Table 13 and FIG. 11; the positive control compound cells were grown as shown in FIG. 12.

TABLE 12 results of the Effect of test Compounds on the proliferation of human Breast cancer cells (MCF-7)

TABLE 13 IC of test Compounds on human Breast cancer cells (MCF-7)₅₀Value of

1.5 inhibitory Activity of Compounds on human Colon cancer cells SW480

The test 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, the inhibition rates are 91.75 +/-1.47, 97.16 +/-0.89, 85.88 +/-1.60 and 93.20 +/-0.80 respectively, and the experimental results are shown in Table 14 and figure 13; IC on human colon cancer cells (SW480)₅₀The experimental results are 4.237 + -0.111, 4.662 + -0.090, 1.882 + -0.168 and 4.683 + -0.298 respectively, which are shown in Table 15 and FIG. 14. The positive control compound cells were grown as shown in FIG. 15.

TABLE 14 results of the Effect of test Compounds on the proliferation of human Colon cancer cells (SW480)

TABLE 15 IC of test Compounds on human Breast cancer cells (SW480)₅₀Value of

(III) results

Chemical components of pteridophyte extracted at normal temperature by methanol and application thereof¹H-NMR，¹³Analyzing and identifying related spectrum data such as C-NMR and 2D-NMR, and 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 (7), 5,11 beta, 12 beta-dihydroxy-15-oxo-ent-kuar-16-en-19-oic acid (8), 11 beta-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid (9), 7 alpha, 11 alpha-dihydroxy-15-oxo-16-methylene-ent-kauran-19, 6 beta-lactone (10), 9-hydroxy-15-oxo-ent-kauran-16-en-19-oic acid (10) 11). And anti-tumor activity screening is carried out, and experimental results show that the

compounds

7, 8, 10 and 11 have half inhibition activity on the in vitro tumor growth of leukemia HL-60, lung cancer A549, liver cancer SMMC-7721, breast cancer MCF-7 and colon cancer SW 480; the compound 9 has half inhibition activity on the in vitro tumor growth of leukemia HL-60 and liver cancer SMMC-7721; the in vitro tumor growth median inhibitory concentrations of the compound 7 on leukemia HL-60, lung cancer A549, liver cancer SMMC-7721, breast cancer MCF-7 and colon cancer SW480 are respectively 11.11 +/-0.35, 15.69 +/-0.82, 9.541 +/-0.468, 7.111 +/-0.111 and 4.237 +/-0.111. The in vitro tumor growth median inhibitory concentrations of the compound 8 to leukemia HL-60, lung cancer A549, liver cancer SMMC-7721, breast cancer MCF-7 and colon cancer SW480 are respectively 5.452 +/-0.222, 15.15 +/-0.22, 7.177 +/-0.515, 5.809 +/-0.097 and 4.662 +/-0.090; the compound 9 has the inhibition concentration of 36.94 +/-1.86 on the in vitro tumor growth half of leukemia HL-60 and the inhibition concentration of 27.05 +/-0.34 on the in vitro tumor growth half of liver cancer SMMC-7721; compound 10 can inhibit in vitro tumor growth of leukemia HL-60, lung cancer A549, liver cancer SMMC-7721, breast cancer MCF-7 and colon cancer SW480The preparation concentrations are 3.590 + -0.021, 4.602 + -0.131, 4.005 + -0.165, 4.094 + -0.301 and 1.882 + -0.168 respectively; the in vitro tumor growth median inhibitory concentrations of the compound 11 to leukemia HL-60, lung cancer A549, liver cancer SMMC-7721, breast cancer MCF-7 and colon cancer SW480 are 3.523 +/-0.092, 5.429 +/-0.114, 4.160 +/-0.066, 6.091 +/-0.168 and 4.683 +/-0.298 respectively.

While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain changes and modifications may be made therein based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Reference to the literature

[1] Separation and purification of 6F as an anti-tumor component in Henan Bian Qi Ling, Limonici and Du Heng (J.Asia Pacific medicine), 2012,8(9):29-30.

[2] The chemical components of Zhang Qianyuyu, Banqi and Qianjin are studied [ D ]. Zhejiang, Industrial university of Zhejiang, 2009:11-17.

[3]Gou ZhanPing,LiangNianCi,HouJie,et al.Two new diterpene and sesquiterpene from Pteris dispar[J].Chinese Chemical Letters,2011,22(12):1451-1453.

[4]KYamasaki,HKohda,TKobayyashi,et al.Application of 13C-nuclear magnetic resonance speatroscopy to chemistry of glycosides:structures of paniculosides-I,-II,-III,-IV and Vditerpene glucosides of Stevia paniculata Lag[J].Chem Pharm,1977,25(11):2895-2899.

[5] Zhang Xiao, Li jin Hua, He Cheng Wei, et al, preliminary study on the phytochemical and antitumor activities of diterpenes in Banlang [ J ]. J.J.Pharmacology, 1999,34(8): 512-514).

[6]T Murakami,H Iida,N Tanaka,et al.Chemische und chemotaxonomis che Untersuchungen von Filices.XXXIII.Chemische Untersuchungen der Inhaltsstoffe von Pteris longipes Don[J].Chemical&Pharmaceutical Bulletin,1981,29(3):657-662.

Claims

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

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;

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.