CN103739660A - Compound, extraction method thereof, application thereof to preparation of antitumor drugs, and antitumor drugs prepared by using compound - Google Patents

Abstract

The invention relates to the technical field of medicinal chemistry, in particular to a compound, an extraction method of the compound, application of the compound to the preparation of antitumor drugs, and the antitumor drugs prepared by using the compound. The compound is extracted from a camellia oleifera abel root; structure identification results show that the compound is a novel compound, and the structural formula is shown in the formula I; an in vitro cell test shows that after the compound shown in the formula I acts for 24 h, the compound has a remarkable inhibiting effect (P is less than 0.05) on the growth of human lung cancer A549 cells, B16 murine melanoma cells, human liver cancer BEL-7402 cells and human breast cancer MCF-7 cells; a murine transplantation tumor model test shows that the compound has a remarkable inhibiting effect (P is less than 0.05) on the growth of murine liver cancer H22 transplantation tumors and murine S180 sarcomas, and the dose-effect relationship is remarkable.

Description

A compound, its extraction method, its application in the preparation of anti-tumor drugs and the prepared anti-tumor drugs

technical field

The invention relates to the technical field of medicinal chemistry, in particular to a compound, its extraction method, its application in the preparation of anti-tumor drugs and the prepared anti-tumor drugs.

Background technique

Camellia oleifera (CameLLia oLeifera AbeL) belongs to the family Theaceae (Theaceae) Camellia (CameLLiaLinn), is a small evergreen tree. It is named Camellia oleifera because its seeds can be squeezed for oil (tea oil) for consumption. Camellia oleifera is mainly distributed in tropical and subtropical regions, and is produced in the southwest and southeast of my country, covering 17 provinces. The unsaturated fatty acid content of camellia oil is as high as 90%, which is much higher than that of vegetable oil, peanut oil and soybean oil, and the content of vitamin E is twice as high as that of olive oil. It has extremely high nutritional value, so camellia oil has attracted much attention.

In addition, Camellia oleifera also has high medicinal value. It is recorded in "Compendium of Materia Medica": "Tea seed, bitter cold poison (saponin), mainly treats asthma and cough, removes disease scale"; "Chinese Materia Medica" also records that Camellia oleifera The root and its root bark have the effects of clearing away heat and detoxifying, regulating qi and relieving pain, promoting blood circulation and reducing swelling. But the medicinal value of camellia oleifera has not been paid enough attention.

In recent years, people have studied the chemical composition and biological activity of Camellia oleifera. At present, saponins, flavonoids, fatty acids, tannins, Aromatic glycosides, alkaloids and other compounds. Studies have found that some saponins have gastrointestinal protection, blood lipid lowering, weight loss, anti-allergic or anti-tumor effects. Ma Liyuan found that more than 95% of the total saponins of camellia oleifera in camellia oleifera tea seed cake have significant antitumor activity. Therefore, Camellia oleifera saponins have high medicinal value. Camellia oleifera saponins are mainly extracted from camellia oleifera, but previous studies on camellia oleifera focused on tea seed heart, tea seed cake, camellia oleifera, and camellia oil, and less research on camellia oleifera root, and there may be undiscovered biologically active substances in camellia oleifera root. Camellia oleifera saponins, therefore, the study of saponins in Camellia oleifera root has important significance.

Contents of the invention

In view of this, the present invention provides a compound, its extraction method, its application in the preparation of anti-tumor drugs and the prepared anti-tumor drugs. The present invention finds through in vitro cell test and animal xenograft tumor test that the compound represented by formula I has significant inhibitory effect on the growth of various cancer cells in human body.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides a compound with the structure shown in formula I:

The present invention also provides a method for extracting compounds of the structure shown in formula I, comprising the following steps:

Step 1: Pulverize Camellia oleifera root, extract with the first solvent, and concentrate to obtain liquid extract;

Step 2: Take the liquid extract and mix it with water, filter, collect the filtrate, centrifuge, collect the supernatant, and then separate it through a D101 macroporous resin column, take the ethanol-water system for gradient elution, collect ethanol and water volume ratio of 70 : 30～95: 5 to elute the obtained components, separate and purify;

The first solvent is ethanol and water in a volume ratio of 0:100˜95:5.

Preferably, the mesh size of the camellia oleifera root is 10 mesh to 60 mesh.

Preferably, the first solvent is ethanol and water in a volume ratio of 20:80-80:20.

More preferably, the first solvent is ethanol and water in a volume ratio of 70:30.

Preferably, in step 2, in the step of mixing the liquid extract with water, the volume ratio of the liquid extract to water is 1:1-10, and the amount of water used is sufficient to dissolve the liquid extract.

The effect is preferable, and the mesh number of the filter cloth used for filtering is 100-300 mesh.

Preferably, the separation and purification techniques are any one or more of precipitation techniques, crystallization techniques or chromatography techniques, but are not limited to the above techniques.

Preferably, the separation and purification techniques are chromatographic techniques.

As a preference, the separation and purification are specifically as follows: the components are separated by a silica gel column, and the gradient elution of the chloroform-methanol system is used to collect the eluted components with a volume ratio of chloroform and methanol of 80:20 to 60:40; and then passed through an ODS column Separation, take methanol-water system gradient elution, collect methanol and water volume ratio of 70:30 ~ 90:10 eluted components; then separate through dynamic axial compression column, take methanol-water system gradient elution, collect The volume ratio of methanol and water is 75:25 to elute the obtained components; finally, it is separated by high performance liquid chromatography, using C ₁₈ chromatographic column, eluted with the second solvent, the flow rate is 2mL/min, and the components are collected for 37.5min to obtain the product;

The second solvent is: a mixed solution composed of methanol and water at a volume ratio of 72:28 and a mixture obtained by mixing with formic acid accounting for 0.2% by mass of the mixed solution.

The specification of the C ₁₈ chromatographic column is 250mm×10mm, and the packing particle size is 5μm.

Preferably, the silica gel column is a decompression silica gel column.

Preferably, the mesh number of the silica gel in the silica gel column is 60-100 mesh.

As a preference, the solvent for the gradient elution of the chloroform-methanol system is chloroform and methanol in a volume ratio of 90:10→80:20→70:30→60:40→50:50→35:65→20:80→0: 100.

Preferably, the ODS column is a medium-pressure reversed-phase ODS column.

Preferably, the specification of the medium-pressure reversed-phase ODS column is: 460mm×26mm.

Preferably, the ODS column gradient elution solvent is methanol and water with a volume ratio of 50:50-100:0.

Preferably, the ODS column gradient elution solvent is methanol and water with a volume ratio of 50:50→60:40→70:30→80:20→90:10.

Preferably, the ODS column flow rate is 25mL/min.

Preferably, the detection wavelength of the ODS column is 203nm.

Preferably, the dynamic axial compression column is an ODS column.

Preferably, the specification of the dynamic axial compression column is: 650mm×100mm, 30μm, 1500g; NewstyLe.

Preferably, the solvent for gradient elution of the dynamic axial compression column is methanol and water in a volume ratio of 60:40→75:25→80:20→90:10.

Preferably, the flow rate of the dynamic axial compression column is 150 mL/min.

Preferably, the detection wavelength of the dynamic axial compression column is 203nm.

Preferably, the high performance liquid chromatography is semi-preparative high performance liquid chromatography.

Preferably, the detection wavelength of the semi-preparative high performance liquid chromatography is 203nm.

Preferably, the compound detection method includes thin layer plate analysis and high performance liquid chromatography analysis.

Preferably, the detection wavelength of the compound represented by formula I is 203nm.

Preferably, the solvent for gradient elution in step 2 is ethanol and water in a volume ratio of 0:100→30:70→50:50→60:40→70:30→80:20→95:5.

Preferably, the extraction temperature in step 1 is 30°C-100°C, and the extraction time in step 1 is 8.5h-33h.

Preferably, the extraction temperature in step 1 is 50° C. to 85° C., and the extraction time in step 1 is 1.0 h to 2.5 h.

Preferably, the extraction temperature in step 1 is 70°C-85°C, and the extraction time in step 1 is 2h-2.5h.

Preferably, the extraction includes the steps of soaking, reflux extraction, filtering and collecting the filtrate.

Preferably, in the soaking step, the mass volume ratio of camellia oleifera root to the first solvent is 1:3-20 in g/mL.

More preferably, in the soaking step, the mass volume ratio of camellia oleifera root to the first solvent is 1:5-15 in g/mL.

More preferably, in the soaking step, the mass volume ratio of camellia oleifera root to the first solvent is 1:10 in g/mL.

In the extraction process of step 1, as long as the first solvent is in excess, a similar effect can be achieved, all of which are within the protection scope of the present invention.

Preferably, the soaking time is 8h-24h, and the soaking temperature is 30°C-80°C.

Preferably, the temperature of reflux extraction is 70°C to 100°C.

More preferably, the temperature of reflux extraction is 80°C.

Preferably, the time for reflux extraction is 0.5h-3h.

More preferably, the time of reflux extraction is 2.0h.

Preferably, the frequency of reflux extraction is 1 to 3 times.

More preferably, the number of times of reflux extraction is 2 times.

The filtering step can be carried out after each reflux extraction, or after 2 to 3 times of reflux extraction, and all the filtrates are collected to be the extract.

Preferably, the filtering step is performed after each reflux extraction, and the filtered residue is mixed with the first solvent for the next reflux extraction.

Preferably, in g/mL, the mass volume ratio of the filter residue to the first solvent is 1:3-20.

Preferably, the concentration temperature in step 1 is 70° C. to 100° C., and the concentration time in step 1 is 2.0 h to 6.0 h.

Preferably, the concentration temperature in step 1 is 80°C to 100°C, and the concentration time in step 1 is 3h to 5h.

Preferably, the concentration is concentration under reduced pressure.

Preferably, the concentration ratio is 6-10 times.

The present invention also provides the application of the compound with the structure shown in formula I in the preparation of antitumor drugs. In the present invention, the inhibitory effect of the drug on the tumor cell line is first determined by the MTT method. MTT assay, also known as MTT colorimetric assay, is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan (Formazan) and deposit in the cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value is measured at a wavelength of 490nm with an enzyme-linked immunosorbent detector, which can indirectly reflect the number of living cells. Within a certain cell number range, the amount of MTT crystal formation is proportional to the cell number. This method has been widely used in the activity detection of some biologically active factors, large-scale antitumor drug screening, cytotoxicity test and tumor radiosensitivity determination, etc. It is characterized by high sensitivity and economy. Therefore, this test uses the MTT method to screen the tested drugs. In this study, human lung cancer A549 cells, B16 mouse melanoma cells, human liver cancer BEL-7402 cells and human breast cancer cells MCF-7 were used as test cells, and the action time of the test drugs was set to 24h. The test results show that the compound represented by formula I provided by the present invention acts on human lung cancer A549 cells, B16 mouse melanoma cells, human liver cancer BEL-7402 cells and human breast cancer cells MCF- ₇ cells after 24 hours of action. The values are all less than 20 μg/mL. It can be seen that the compound represented by formula I has a significant inhibitory effect on the growth of the above four kinds of tumor cells.

The present invention also tested the anti-tumor effect of the compound through a mouse transplanted tumor model test, and the results showed that the compound represented by formula I had a significant (P<0.05) effect on the growth of mouse liver cancer _H22 transplanted tumor and mouse _S180 sarcoma. The inhibitory effect has an obvious dose-effect relationship, and the inhibitory effect on the growth of mouse _S180 sarcoma is significantly better than that of the positive control CTX. Therefore, the present invention provides the application of the compound with the structure shown in formula I in the preparation of antitumor drugs.

Preferably, the tumor is lung cancer, liver cancer, melanoma or breast cancer. Tumors can also be other types of tumors that are not disclosed in the present invention but have inhibitory effects.

The present invention also provides an antineoplastic drug, which comprises the compound with the structure shown in formula I and pharmaceutically acceptable auxiliary materials.

The dosage form of the antitumor drug of the present invention can be any dosage form that can be realized in the art, the adjuvant used is the conventional adjuvant of the dosage form used, and the preparation method is the conventional preparation method of the corresponding dosage form.

Preferably, the dosage form of the antineoplastic drug is powder, tablet, granule, capsule, solution, emulsion, suspension, injection, powder injection or spray.

The present invention provides a compound with the structure shown in formula I, which is extracted from camellia oleifera root, and the structure identification results show that the compound is a new compound named: 21β,22α-O-diangelyl-15α, 16α,28-Trihydroxyolean-12-en-23-aldehyde 3β-O-β-D-xylose-(1→2)-β-D-galactose-(1→3)-[β- D-galactose-(1→2)]-β-D-glucuronide. The present invention uses the MTT method to carry out in vitro cell tests, and the results show that the compound shown in formula I provided by the present invention acts on human lung cancer A549 cells, B16 mouse melanoma cells, human liver cancer BEL-7402 cells and human breast cancer cells after acting for 24 hours. The IC ₅₀ values of MCF-7 cells were all less than 20 μg/mL, and the inhibitory effect on the growth of human lung cancer A549 cells and human liver cancer BEL-7402 cells was significantly better than (P<0.05) the positive control 5-FU. It can be seen that the compound represented by formula I has significant inhibitory effect on the growth of the above four kinds of tumor cells. The present invention also tests the antitumor effect of the compound through a mouse transplanted tumor model test. The test results show that the compound shown in formula I has a significant (P<0.05) effect on the growth of mouse liver cancer _H22 transplanted tumor and mouse S180 sarcoma. The inhibitory effect has an obvious dose-effect relationship, and the inhibitory effect on the growth of mouse S180 sarcoma is significantly better than that of the positive control CTX. Therefore, the compound represented by formula I provided by the present invention can also be used to prepare antitumor drugs.

Description of drawings

Figure 1 is 21β,22α-O-diangeloyl-15α,16α,28-trihydroxyolean-12-ene-23-aldehyde 3β-O-β-D-xylose-(1→2)-β -High resolution mass spectrum of D-galactose-(1→3)-[β-D-galactose-(1→2)]-β-D-glucuronide;

Figure 2 is 21β,22α-O-diangeloyl-15α,16α,28-trihydroxyolean-12-ene-23-aldehyde 3β-O-β-D-xylose-(1→2)-β -H NMR spectrum of D-galactose-(1→3)-[β-D-galactose-(1→2)]-β-D-glucuronide;

Figure 3 is 21β,22α-O-diangeloyl-15α,16α,28-trihydroxyolean-12-ene-23-aldehyde 3β-O-β-D-xylose-(1→2)-β -D-galactose-(1→3)-[β-D-galactose-(1→2)]-β-D-glucuronide H NMR spectrum (enlarged view);

Figure 4 is 21β,22α-O-diangeloyl-15α,16α,28-trihydroxyolean-12-ene-23-aldehyde 3β-O-β-D-xylose-(1→2)-β -C NMR spectrum of D-galactose-(1→3)-[β-D-galactose-(1→2)]-β-D-glucuronide;

Figure 5 is 21β,22α-O-diangeloyl-15α,16α,28-trihydroxyolean-12-ene-23-aldehyde 3β-O-β-D-xylose-(1→2)-β -D-galactose-(1→3)-[β-D-galactose-(1→2)]-β-D-glucuronide carbon NMR spectrum (enlarged view);

Figure 6 is 21β,22α-O-diangeloyl-15α,16α,28-trihydroxyolean-12-en-23-aldehyde 3β-O-β-D-xylose-(1→2)-β -D-galactose-(1→3)-[β-D-galactose-(1→2)]-β-D-glucuronide carbon NMR spectrum (enlarged view);

Figure 7 is 21β,22α-O-diangeloyl-15α,16α,28-trihydroxyolean-12-ene-23-aldehyde 3β-O-β-D-xylose-(1→2)-β HSQC diagram of -D-galactose-(1→3)-[β-D-galactose-(1→2)]-β-D-glucuronide;

Figure 8 is 21β,22α-O-diangeloyl-15α,16α,28-trihydroxyolean-12-ene-23-aldehyde 3β-O-β-D-xylose-(1→2)-β -HMBC diagram of D-galactose-(1→3)-[β-D-galactose-(1→2)]-β-D-glucuronide;

Figure 9 is 21β, 22α-O-diangeloyl-15α, 16α, 28-trihydroxyolean-12-ene-23-aldehyde 3β-O-β-D-xylose-(1→2)-β ¹ H- ¹ H COZY diagram of -D-galactose-(1→3)-[β-D-galactose-(1→2)]-β-D-glucuronide;

Figure 10 is 21β, 22α-O-diangeloyl-15α, 16α, 28-trihydroxyolean-12-ene-23-aldehyde 3β-O-β-D-xylose-(1→2)-β - NOESY plot of D-galactose-(1→3)-[β-D-galactose-(1→2)]-β-D-glucuronide.

Detailed ways

The invention provides a compound, its extraction method, its application in the preparation of antitumor drugs and the prepared antitumor drugs. Those skilled in the art can refer to the content of this article to appropriately improve the process parameters to achieve. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.

The materials and reagents used in the embodiments of the present invention can be purchased from the market. In the embodiments of the present invention, the sources of raw materials are as follows:

Nuclear magnetic resonance instrument 500Hz (Varian Inc., Palo Alto, CA, USA); high-resolution mass spectrometer Q-TOF2 (UK Micromass company); electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); polarimeter 241 ( PerkinElmer Inc., Waltham, MA, USA); semi-preparative high performance liquid chromatography (LC-20AT, SPD-20A, Shimadzu Corporation, Japan); C18 semi-preparative chromatographic column (250mm×10mm, 5μm, Kromsil Corporation, USA); Medium pressure preparative liquid chromatography (Büchi chromatography system, C-650 pump, medium pressure column (460mm×26mm i.d., Büchi Corp., Flawil, Swiss); dynamic axial column chromatography (NP7000 pump (Newstyle), NU3000UV-VIS detection Newstyle apparatus (Newstyle) and ODS column (650mm×100mm, 30μm, 1500g; Newstyle); rotary evaporator (Tokyo Physical and Chemical Instruments sole proprietorship factory); chemical reagents (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); thin-layer chromatography silica gel plate ( HSGF254, produced by Yantai Zhifu Huangwu Silica Gel Development and Experimental Factory); various silica gels for column chromatography are produced by Qingdao Ocean Chemical Factory.

Human lung cancer A549 cells, B16 mouse melanoma cells, human liver cancer BEL-7402 cells and human breast cancer cells MCF-7 were purchased from Shanghai Institute of Cells, Chinese Academy of Sciences.

H22 mouse liver cancer (sarcoma) cells: purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

S180 mouse liver cancer (sarcoma) cells: purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

ICR mice: clean grade, male, weighing 18-22 g, provided by Shanghai Slack Experimental Animal Co., Ltd.

Mice: The breed is ICR, purchased from Shanghai Slack Experimental Animal Co., Ltd., and the experimental animal production license number is SCXK (Shanghai) 2007-0005.

Complete medium: the formula is RPMI1640 medium containing 10% inactivated fetal bovine serum, and the manufacturer is GIBCOBRL Company of the United States.

The rest of the reagents and materials were purchased from the market.

Below in conjunction with embodiment, further set forth the present invention:

The extraction of embodiment 1 compound

Take 2kg of dry Camellia oleifera root, grind it into 10-40 mesh wood chips with a grinder, soak in 6L water at 40°C for 8h, heat and reflux at 100°C for 0.5h, filter, add 3L water to the filter residue, and heat to reflux at 100°C 3.0h, filter, the filter residue was mixed with 3L of water, heated to reflux at 100°C for 2h, filtered, combined the filtrate obtained from three times of filtration, and concentrated under reduced pressure at 80°C for 6h to obtain 2000mL of liquid extract.

Take the prepared liquid extract and mix it with 20000mL water, filter it with a 100-mesh filter cloth, centrifuge the filtrate, take the supernatant and separate it through a D101 macroporous resin column, and then use water, 30% ethanol, 50% ethanol, 60% ethanol, 70 % ethanol, 80% ethanol, and 95% ethanol were eluted, and the components eluted with 70% ethanol to 95% ethanol were collected and concentrated under reduced pressure to obtain total saponins of camellia oleifera.

The obtained total saponins of camellia oleifera were first separated by a decompression silica gel column of 60 mesh silica gel, followed by chloroform and methanol with a volume ratio of 90:10→80:20→70:30→60:40→50:50→40:60 →20:80→0:100 gradient elution to obtain components 1 to 8, which were analyzed by TLC and combined with chloroform and methanol in a volume ratio of 80:20 to 60:40 to elute the components (developing solvent When the system is a BAW system, the Rf value is 0.4-0.7), and then separated by a medium-pressure reversed-phase ODS column, 50%-100% gradient elution in the methanol-water system, the flow rate is 25mL/min, and the detection wavelength is 203nm, and 6 Components, after high-efficiency analytical liquid phase analysis, gradient elution with methanol and water at a volume ratio of 60:40 to 90:10 for 30 minutes, combined components with a volume ratio of methanol and water of 70:30 to 90:10 (the peak at 20 minutes -28min), separated by a dynamic axial compression column, eluted with 60%, 75%, 80% and 90% methanol respectively, with a flow rate of 150mL/min, and a detection wavelength of 203nm, a total of five components were obtained. For phase analysis, the components eluted with 75% methanol (peak at 13.5-15.0min) were separated by semi-preparative high-efficiency liquid phase, methanol-water (72:28, v/v)-0.2% formic acid was eluted and eluted, set With a constant flow rate of 2 mL/min and a detection wavelength of 203 nm, 150 mg of white amorphous powder was obtained at 37.5 min.

Take the obtained white amorphous powder, test the color development characteristics and hydrolysis characteristics, and then analyze the spectrum by ¹ H-NMR, ¹³ C-NMR, HMBC, HSQC, ¹ H- ¹ H COZY, NOESY and HR-ESI-MS , the spectral data diagrams are shown in Figures 1 to 10, and the NMR data are shown in Table 1. The structural analysis process is as follows: after color analysis, ethanol sulfate showed purple spots, the acetic anhydride-concentrated sulfuric acid reaction was positive, and the MoLish reaction was positive. It is suggested that the compound may be a saponin compound. The mass spectrum shown in Fig. 1 shows that the quasi-ion peak of the HR-ESI-MS of this compound is m/z1315.5968[MH] ^- , shows that the molecular formula of this compound is C ₆₃ H ₉₆ O ₂₉ (calculated value [MH] ^- ,m/z1315.5959, the calculated value is based on the molecular formula, calculated from the most accurate value of abundance). Monosaccharides were obtained by complete acid hydrolysis with 2N TFA. The sugars were derivatized and analyzed by GC, and the presence of D-glucuronic acid, D-galactose, and D-xylose was detected.

From the ¹³ C-NMR spectrum of the compound shown in Figure 4, Figure 5, and Figure 6, it can be seen that this compound has 63 carbon signals (as shown in Table 1), and the HSQC spectrum of the compound shown in Figure 7 shows that the compound It contains 10 methyl carbons, 10 methylene carbons, 31 methine carbons and 12 quaternary carbons. Further analysis of the ¹³ C-NMR spectrum, the spectrum shows four sugar end group carbon signals δ104.3, 101.8, 107.9 and 103.1, which are D-glucuronic acid, D-galactose, D-xylose and another D- Terminal carbon of galactose. In addition, there is an aldehydic carbon signal located at δ210.3 in the downfield region. From the hydrogen spectra of the compounds shown in Figure 2 and Figure 3, it can be seen that there are six obvious triterpenoid saponin angle methyl hydrogen signals in the high field region of the ¹ H-NMR spectrum δ0.88(Me-25), 1.04(Me-25) 26), 1.18(Me-29), 1.41(Me-30), 1.53(Me-24) and 1.90(Me-27); a oxymethylene δ3.57(H-28,d,J=11.0 Hz) and 3.82 (H-28, d, J=11.0Hz); five oxymethine δ4.09 (1H, dd, J=11.0, 4.5Hz, H-3), 4.26 (1H, brs, H-15), 4.54 (1H, brs, H-16), 6.78 (1H, d, J=10.5Hz, H-21) and 6.41 (1H, d, J=10.5Hz, H-22); a ene Hydrogen proton δ5.58 (1H, brs, H-12) and an aldehyde hydrogen proton δ9.94 (1H, brs, H-23). In addition, ^{the 1} H-NMR spectrum of the compound also showed two groups of angeloyl group signals δ[6.07(1H,dq,J=7.5,1.5Hz,21-O-Ang-3'),2.18(3H,d,J= 7.5Hz,21-O-Ang-4') and 2.09(3H,s,21-O-Ang-5')]; δ[5.88(1H,dq,J=7.5,1.5Hz,22-O-Ang -3″), 2.05 (3H, d, J=7.5Hz, 22-O-Ang-4″) and 1.83 (3H, s, 22-O-Ang-5″)].

From the ¹ H- ¹ H COZY spectrum shown in Figure 9, it can be seen that the methine δ _H 4.26 (1H, brs) adjacent to the oxymethine proton is related to δ _H 4.54 (1H, brs), indicating that they In the ortho position and δ _H 4.54 is H-16 signal, δ _H 4.26 is H-15 signal; δ _H 6.78(1H,d,J=10.5Hz) and δ _H 6.41(1H,d,J=10.5Hz) The correlation signals between them indicate that they are in the ortho position and δ _H 6.78 is the signal of H-21, and δ _H 6.41 is the signal of H-22.

Through the analysis of the HMBC spectrum of the compound shown in Figure 8, it is known that the two angelica acyl groups are respectively connected to the 21 and 22 positions of the compound, because in HMBC δ _H 6.78 (1H, d, J=10.5Hz, H-21) Correlates to δ _C 168.0 (21-O-Ang-1'), δ _H 6.41 (1H, d, J=10.5Hz, H-22) correlates to δ _C 168.3 (22-O-Ang-1''). Combined with ¹³ C-NMR spectrum and ¹ H-NMR spectrum data for comprehensive analysis, and compared with the NMR data of known aglycones reported in the literature "Helvetica Chimica Acta2013.Vol.96", the aglycon structure of the compound was determined to be 21β, 22α-O-Diangeloyl-15α,16α,28-trihydroxyolean-12-en-23-aldehyde.

Analyze the HSQC spectrum of the compound as shown in Figure 7, and attribute the terminal carbon and hydrogen signals of the sugar connected to the saponin. The results are as follows: δ _H 4.80 (1H, d, J=6.5Hz), 5.80 (1H, d, J=7.5Hz), 5.09(1H, d, J=7.5Hz) and 5.95(1H, d, J=7.5Hz), connected at δ _C 104.3(glucuronic acid-C-1), 101.8( Galactose-C-1), 107.9 (xylose-C-1), 103.1 (galactose'-C-1). In addition, the 3-position of the compound aglycone is located at 12.6ppm to the downfield, indicating that the sugar chain is connected to the 3-position of the aglycon, and in the HMBC spectrum, the glucuronic acid terminal group proton δ _H 4.80 is related to the δ _C 84.7 of the 3-position of the aglycone, further It was confirmed that the sugar chain was connected to the 3-position of the aglycon. Further review of the literature found that the sugar chain of the compound was similar to the sugar chain data of camellenodiol reported in the literature "Bioorganic & medicinal chemistry letters 2010, 20, 7435-7439", thus inferring that the sugar chain of the compound was β-D-xylose-(1→2) -β-D-galactose-(1→3)-[β-D-galactose-(1→2)]-β-D-glucuronic acid. The connection order of the sugar chain can also be confirmed by analyzing the HMBC spectrum. In HMBC, the glucuronic acid anomeric proton δ _H 4.80 is related to the carbon δ _C 84.7 at the 3-position of the aglycone, and the galactose anomeric proton δ _H 5.80 is related to the 3-position of the glucuronic acid. Carbon δ _C 84.9 is related, xylose end group proton δ _H 5.09 is related to galactose 2 carbon δ _C 84.1, galactose 2 hydrogen δ _H 4.57 is related to xylose end group carbon δ _C 107.9, another galactose end base The sub δ _H 5.95 is related to the carbon δ _C 78.2 at the 2-position of glucuronic acid, and the hydrogen δ _H 4.57 at the 2-position of glucuronic acid is related to the carbon δ _C 103.1 of the terminal group of galactose. In addition, the anomeric proton coupling constants ³ J _H-1 and _H-2 of the four sugars are relatively large, indicating that the four sugars are all in the β-configuration.

The NOESY spectrum shown in Figure 10 shows that δ _H 6.78 (1H, d, J=10.5Hz, H-21) is related to δ _H 1.18 (3H, s, H-29), suggesting that H-21 is in the α configuration; δ _H 4.09(1H,dd,J=4.5,11.0Hz,H-3) is related to δ _H 9.94(1H,brs,H-23), suggesting that H-3 is in α configuration; δ _H 4.54(1H,brs ,H-16) and δ _H 3.57(1H,d,J=11.0Hz,H-28), 3.82(1H,d,J=11.0Hz,H-28) suggest that H-16 is the β configuration, δ _H 6.41 (1H, d, J=10.5Hz, H-22) is related to δ _H 1.41 (3H, s, H-30), suggesting that H-22 is in the β configuration.

Therefore, the structure of this compound was named 21β,22α-O-diangeloyl-15α,16α,28-trihydroxyolean-12-ene-23-aldehyde 3β-O-β-D-xylose-(1→ 2) -β-D-galactose-(1→3)-[β-D-galactose-(1→2)]-β-D-glucuronide. The structure of the compound is shown in formula I:

NMR (pyridine-d5, 500MHz) data of the compound shown in Table 1 Formula I

The chemical shifts in the table are in ppm, and the coupling constants (J) are in Hz.

The extraction of embodiment 2 compound

Take 2kg of dried Camellia oleifera root, crush it into 10-mesh sawdust with a pulverizer, soak it in 40L of 95% ethanol at 40°C for 24h, heat and reflux at 80°C for 0.5h, filter, add 2L of 95% ethanol to the filter residue, and heat it at 80°C Heat to reflux for 3.0 h, filter, combine the filtrates obtained from two filtrations, and concentrate under reduced pressure at 90° C. for 2 h to obtain 4,200 mL of liquid extract.

Take the obtained liquid extract and mix it with 4200mL water, filter it with a 200-mesh filter cloth, centrifuge the filtrate, take the supernatant and separate it through a D101 macroporous resin column, and then use water, 30% ethanol, 50% ethanol, 60% ethanol, and 70% ethanol in sequence. % ethanol, 80% ethanol, and 95% ethanol were eluted, and the components eluted with 70% ethanol to 95% ethanol were collected and concentrated under reduced pressure to obtain total saponins of camellia oleifera.

The obtained total saponins of camellia oleifera were first separated by a 100-mesh silica gel decompression silica gel column, followed by chloroform and methanol with a volume ratio of 90:10→80:20→70:30→60:40→50:50→40:60 →20:80→0:100 gradient elution to obtain components 1 to 8, which were analyzed by TLC and combined with chloroform and methanol in a volume ratio of 80:20 to 60:40 to elute the components (developing solvent When the system is a BAW system, the Rf value is 0.4-0.7), and then separated by a medium-pressure reversed-phase ODS column, 50%-100% gradient elution in the methanol-water system, the flow rate is 25mL/min, and the detection wavelength is 203nm, and 6 Components, after high-efficiency analytical liquid phase analysis, gradient elution with methanol and water at a volume ratio of 60:40 to 90:10 for 30 minutes, combined components with a volume ratio of methanol and water of 70:30 to 90:10 (the peak at 20 minutes -28min), separated by a dynamic axial compression column, eluted with 60%, 75%, 80% and 90% methanol respectively, with a flow rate of 150mL/min, and a detection wavelength of 203nm, a total of five components were obtained. For phase analysis, the components eluted with 75% methanol (peak at 13.5-15.0min) were separated by semi-preparative high-efficiency liquid phase, methanol-water (72:28, v/v)-0.2% formic acid was eluted and eluted, set The constant flow rate was 2mL/min, and the detection wavelength was 203nm. At 37.5min, 180mg of white amorphous powder was obtained. The hydrogen nuclear magnetic resonance spectrum detection showed that the obtained data was consistent with the hydrogen spectrum data of the compound obtained in Example 1. Therefore, it was determined that this compound was 21β ,22α-O-Diangeloyl-15α,16α,28-Trihydroxyolean-12-en-23-aldehyde 3β-O-β-D-xylose-(1→2)-β-D-semi Lactose-(1→3)-[β-D-galactose-(1→2)]-β-D-glucuronide, the compound structure is shown in Formula I.

The extraction of embodiment 3 compound

Take 2kg of dried camellia oleifera root, grind it into 10-60 mesh sawdust with a grinder, soak it in 20L of 70% ethanol at 80°C for 8h, heat and reflux at 85°C for 3h, filter, collect the filtrate, and concentrate under reduced pressure at 70°C After 6 hours, 2000 mL of liquid extract was obtained.

Take the prepared liquid extract and mix it with 6000mL water, filter it with a 300-mesh filter cloth, centrifuge the filtrate, take the supernatant and separate it through a D101 macroporous resin column, and then use water, 30% ethanol, 50% ethanol, 60% ethanol, and 70% ethanol in sequence. % ethanol, 80% ethanol, and 95% ethanol were eluted, and the components eluted with 70% ethanol to 95% ethanol were collected and concentrated under reduced pressure to obtain total saponins of camellia oleifera.

The obtained total saponins of camellia oleifera were first separated by a decompression silica gel column of 80 mesh silica gel, followed by chloroform and methanol with a volume ratio of 90:10→80:20→70:30→60:40→50:50→35:65 →20:80→0:100 gradient elution to obtain components 1 to 8, which were analyzed by TLC and combined with chloroform and methanol in a volume ratio of 80:20 to 60:40 to elute the components (developing solvent When the system is a BAW system, the Rf value is 0.4-0.7), and then separated by a medium-pressure reversed-phase ODS column, 50%-100% gradient elution in the methanol-water system, the flow rate is 25mL/min, and the detection wavelength is 203nm, and 6 Components, after high-efficiency analytical liquid phase analysis, gradient elution with methanol and water at a volume ratio of 60:40 to 90:10 for 30 minutes, combined components with a volume ratio of methanol and water of 70:30 to 90:10 (the peak at 20 minutes -28min), separated by a dynamic axial compression column, eluted with 60%, 75%, 80% and 90% methanol respectively, with a flow rate of 150mL/min, and a detection wavelength of 203nm, a total of five components were obtained. For phase analysis, the components eluted with 75% methanol (peak at 13.5-15.0min) were separated by semi-preparative high-efficiency liquid phase, methanol-water (72:28, v/v)-0.2% formic acid was eluted and eluted, set The constant flow rate is 2mL/min, the detection wavelength is 203nm, and 160mg of white amorphous powder is obtained at 37.5min. After the proton nuclear magnetic resonance spectrum detection, the obtained data are consistent with the proton spectrum data of the compound obtained in Example 1. Therefore, it is determined that this compound is 21β ,22α-O-Diangeloyl-15α,16α,28-Trihydroxyolean-12-en-23-aldehyde 3β-O-β-D-xylose-(1→2)-β-D-semi Lactose-(1→3)-[β-D-galactose-(1→2)]-β-D-glucuronide, the compound structure is shown in Formula I.

The extraction of embodiment 4 compound

Take 2 kg of dry Camellia oleifera root, crush it into 10-40 mesh wood chips with a grinder, soak in 20L of 80% ethanol at 30°C for 8h, heat and reflux at 75°C for 0.5h, heat and reflux at 80°C for 3.0h, and heat at 80°C Heat to reflux at ℃ for 2h, filter, collect the filtrate, concentrate under reduced pressure at 100°C for 3h, and obtain 2000 mL of liquid extract.

Take the prepared liquid extract and mix it with 6000mL water, filter it with a 250-mesh filter cloth, centrifuge the filtrate, take the supernatant and separate it through a D101 macroporous resin column, and then use water, 30% ethanol, 50% ethanol, 60% ethanol, and 70% ethanol in sequence. % ethanol, 80% ethanol, and 95% ethanol were eluted, and the components eluted with 70% ethanol to 95% ethanol were collected and concentrated under reduced pressure to obtain total saponins of camellia oleifera.

The obtained total saponins of camellia oleifera were first separated by a decompression silica gel column of 60 mesh silica gel, followed by chloroform and methanol with a volume ratio of 90:10→80:20→70:30→60:40→50:50→40:60 →20:80→0:100 gradient elution to obtain components 1 to 8, which were analyzed by TLC and combined with chloroform and methanol in a volume ratio of 80:20 to 60:40 to elute the components (developing solvent When the system is a BAW system, the Rf value is 0.4-0.7), and then separated by a medium-pressure reversed-phase ODS column, methanol-water system 50:50→60:40→70:30→80:20→90:10 gradient elution , the flow rate is 25mL/min, the detection wavelength is 203nm, and 6 components are obtained. After high-efficiency analysis liquid phase analysis, the gradient elution with methanol and water volume ratio of 60:40 to 90:10 is used for 30 minutes, and the volume ratio of methanol and water is combined. Components of 70:30～90:10 (peak at 20min-28min) were separated by dynamic axial compression column, eluted with 60%, 75%, 80% and 90% methanol respectively, flow rate 150mL/min, detection The wavelength is 203nm, and a total of five components are obtained. After HPLC analysis, the components eluted with 75% methanol (peak at 13.5-15.0min) are separated by semi-preparative HPLC, methanol-water (72:28, v/v)-0.2% formic acid elution and elution, the set flow rate is 2mL/min, the detection wavelength is 203nm, and 230mg of white amorphous powder is obtained in 37.5min, and the obtained data is the same as that obtained in Example 1 through the detection of proton nuclear magnetic resonance. The hydrogen spectrum data of the compound are consistent, therefore, this compound is determined to be 21β,22α-O-diangeloyl-15α,16α,28-trihydroxyolean-12-ene-23-aldehyde 3β-O-β-D- Xylose-(1→2)-β-D-galactose-(1→3)-[β-D-galactose-(1→2)]-β-D-glucuronide, the compound structure is as follows: I show.

Example 5 in vitro cell test

Take human lung cancer A549 cells, B16 mouse melanoma cells, human liver cancer BEL-7402 cells, and human breast cancer cells MCF-7, respectively, with RPMI1640 medium containing 10% inactivated NBS (plus 0.03% Glu, Hepes0 .06%, NaHCO ₃ 0.2%) were cultured at 37°C and 5% CO ₂ , subcultured for 3 days, and the cells in the logarithmic growth phase were taken for experiments.

Use the complete medium to adjust the concentration of the cells in the logarithmic production phase so that the cell concentration is 5×10 ⁴ cells/mL, inoculate in a 96-well culture plate, 100 μL/well, and store at 37°C and 5% CO ₂ After culturing for 24 hours, they were divided into the following groups:

The test group was added with the compound shown in formula I prepared in Example 1, solubilized with 1% DMSO and 99% PBS, and the final concentrations were adjusted to 6.25 μg/mL, 9.375 μg/mL, 12.5 μg/mL, 18.25 μg/mL, and 25.00 μg/mL, respectively. μg/mL, 10 μL/well.

Add 5-FU to the positive control group, add solvent 1% DMSO and 99% PBS, adjust the concentration to 50 μg/mL, 10 μL/well.

Negative control group was added with complete medium, 10 μL/well.

Each group was set up with 3 replicate wells and cultured for 24 hours respectively. Add MTT at a concentration of 5 mg/mL 4 hours before terminating the culture, 10 μL/well, add 100 μL of DMSO to each well after the culture, place on a shaker for 10 min, and detect the absorbance A value at a wavelength of 490 nm with a microplate reader. Calculate the tumor cell growth inhibition rate (Inhibition Rate), the calculation formula is as follows:

Inhibition Rate (%)=[(A negative control group-A test group)/A negative control group]×100%

The IC ₅₀ (half inhibitory concentration) was calculated according to the tumor cell growth inhibition rate, and the test results are shown in Table 2:

Table 2 The _IC50 (μg/mL) value of the compound prepared in Example 1 to 4 kinds of tumor cells for 24h

From the test results shown in Table 2, it can be seen that the compound shown in formula I prepared in Example 1 has a 24h effect on human lung cancer A549 cells, B16 mouse melanoma cells, human liver cancer BEL-7402 cells and human breast cancer cells MCF-7. The final IC ₅₀ values were all less than 20 μg/mL, indicating that the compound represented by formula I prepared in Example 1 had a significant inhibitory effect on the growth of the above four tumor cells.

During the test, it was found that after the positive control 5-FU acted at a concentration of 50 μg/mL for 24 hours, the inhibitory rate to human lung cancer A549 tumor cells was only 32%, while the compound shown in formula I prepared in Example 1 had a concentration of 14.95 After ±0.40 μ g/mL acted for 24 hours, the inhibitory rate to human lung cancer A549 tumor cells reached 50%, thus it can be seen that the inhibitory effect of the compound shown in formula I prepared in Example 1 on human lung cancer A549 tumor cells was significantly better than (P <0.05) positive control 5-FU.

During the test, it was also found that the positive control 5-FU had an inhibitory rate of only 24% on human liver cancer BEL-7402 tumor cells after 24 hours of action at a concentration of 50 μg/mL, while the compound shown in formula I prepared in Example 1 After acting for 24 hours at a concentration of 15.76 ± 0.33 μg/mL, the inhibitory rate to human liver cancer BEL-7402 tumor cells reached 50%. It can be seen that the compound shown in formula Ⅰ prepared in Example 1 is effective against human liver cancer BEL-7402 tumor cells. The inhibitory effect of cells was significantly better than (P<0.05) the positive control 5-FU.

During the test, it was also found that the positive control 5-FU had an inhibitory rate of 49% on B16 mouse melanoma cells after acting at a concentration of 50 μg/mL for 24 hours. It can be seen that the formula I prepared in Example 1 The results showed that the inhibitory effect of the compound on B16 mouse melanoma cells was better than that of the positive control 5-FU.

During the test, it was also found that the positive control 5-FU had an inhibitory rate of 43% on human breast cancer cell MCF-7 after acting at a concentration of 50 μg/mL for 24 hours. It can be seen that the formula I prepared in Example 1 The results showed that the inhibitory effect of the compound on human breast cancer cell MCF-7 was better than that of the positive control 5-FU.

The compounds shown in Formula I prepared in Examples 2 to 4 of the present invention were subjected to in vitro cell tests, the test method was the same as in Example 5, and the results obtained were similar to those in Example 5, that is, the compounds of formula I provided in Examples 2 to 4 of the present invention The compound shown in Ⅰ has a good inhibitory effect on the growth of human lung cancer A549 cells, B16 mouse melanoma cells, human liver cancer BEL-7402 cells and human breast cancer cells MCF-7 cells. BEL-7402 cell, breast cancer MCF-7 cell growth inhibitory effect is better.

Based on the above test results, it can be seen that the compounds shown in Formula I provided by Examples 1 to 4 of the present invention have the effect on human lung cancer A549 cells, B16 mouse melanoma cells, human liver cancer BEL-7402 cells and human breast cancer cells MCF-7 cells. It has a good growth inhibitory effect, especially for human lung cancer A549 cells, human liver cancer BEL-7402 cells, and breast cancer MCF-7 cells.

Example 6 Mouse transplanted tumor model test

Take H ₂₂ and S ₁₈₀ mouse liver cancer (sarcoma) cell cryopreservation tubes respectively, place them in a constant temperature water bath at 37°C to thaw, collect the cells by centrifugation, wash twice with PBS, resuspend the cells in PBS, and pass through ICR mice Intraperitoneal passage for more than 3 generations, the mice with obviously enlarged abdomen were taken, killed by dislocation, the abdomen was disinfected with alcohol, milky white ascites was extracted with a disposable sterile syringe, injected into a sterilized centrifuge tube, counted by a cell counter, and the cell density was adjusted with normal saline To 5×10 ⁶ cells/mL, each mouse was inoculated with 0.2 mL of cell suspension in the right armpit for modeling, and the mice were randomly divided into the following groups the next day:

Blank control group: 8 mice inoculated with transplanted tumors were intraperitoneally injected with 0.2 mL of 0.9% normal saline, once a day, for 10 days.

Positive control group: 8 mice inoculated with transplanted tumors were intraperitoneally injected with cyclophosphamide (CTX), 20 mg/kg, once every other day, for 10 days.

Experimental group 1: 8 mice inoculated with transplanted tumors were intraperitoneally injected with the compound represented by formula I prepared in Example 1, 1.5 mg/kg, once a day for 10 consecutive days.

Test group 2: 8 mice inoculated with transplanted tumors were intraperitoneally injected with the compound shown in Formula I prepared in Example 1, 3.0 mg/kg, once a day for 10 consecutive days.

2 hours after the last administration, the tumor body was peeled off, the tumor weight was weighed, and the tumor inhibition rate was calculated. The calculation formula is as follows:

Wherein the administration group is positive control group, test group 1 or test group 2.

The test results are shown in Table 3 and Table 4:

Table 3 The effect of the compound shown in formula I prepared in Example 1 on the weight of mouse liver cancer _H22 transplanted tumor

*P<0.05, **P<0.01, vs blank control group

From the experimental data shown in Table 3, it can be known that the compound shown in Formula I prepared in Example 1 of the present invention has a significant effect on mouse liver cancer _H22 transplanted tumors when the doses are respectively 1.5 mg/kg and 3.0 mg/kg, and the drug is continuously administered for 10 days. The inhibitory rates of the drugs reached 32.1% and 43%, respectively. Compared with the blank control group, the tumor inhibitory effect was significant (P<0.05), and with the increase of the dose, the tumor inhibitory rate was significantly enhanced; when the positive control group was 20mg/kg, the The inhibition rate of mouse liver cancer _H22 transplanted tumor was 57.1%. Compared with the positive control group, the inhibitory effect of the compound represented by formula I prepared in Example 1 on mouse liver cancer _H22 transplanted tumor was not as good as that of the positive control group.

It can be seen that the compound represented by formula I prepared in Example 1 has a significant (P<0.05) inhibitory effect on the growth of mouse liver cancer _H22 xenografts, and the dose-effect relationship is obvious.

Effect of the compound shown in formula I prepared in Table 4 Example 1 on the tumor weight of mouse _S180 sarcoma

*P<0.05, **P<0.01, vs blank control group

From the test data shown in Table 4, it can be known that the compound shown in Formula I prepared in Example 1 of the present invention has a dose of 1.5 mg/kg and 3.0 mg/kg respectively, and continuous medication for 10 days has an inhibitory effect on mouse _S180 sarcoma. The tumor inhibition rate reached 30.3% and 55% respectively. Compared with the blank control group, the tumor inhibition effect was significant (P<0.05), and with the increase of the dose, the tumor inhibition rate was significantly enhanced; when the positive control group was 20mg/kg, the The inhibition rate of S ₁₈₀ sarcoma was only 37.1%, and the inhibitory effect of the compound represented by formula I prepared in Example 1 on mouse S ₁₈₀ sarcoma was significantly better (P<0.05) than the positive control CTX.

It can be seen that the compound represented by formula I prepared in Example 1 has a significant (P<0.05) inhibitory effect on the growth of mouse S ₁₈₀ sarcoma, and the tumor inhibitory effect is significantly better than that of the positive control CTX, and the dose-effect relationship is obvious.

The compound shown in Formula I prepared in Examples 2 to 4 was subjected to a mouse xenograft tumor model test, and the test method was the same as that in Example 6, and the obtained test results were similar to those in Example 6, that is, the compounds of formula I prepared in Examples 2 to 4. The compound shown in Ⅰ has a significant (P<0.05) inhibitory effect on the growth of mouse liver cancer H ₂₂ transplanted tumor, and the dose-effect relationship is obvious; it has a significant (P<0.05) inhibitory effect on the growth of mouse S ₁₈₀ sarcoma, and the inhibitory effect The tumor effect was significantly better than the positive control CTX, and the dose-effect relationship was obvious.

In summary, the compounds represented by formula I prepared in Examples 1 to 4 of the present invention have significant (P<0.05) inhibitory effects on the growth of mouse liver cancer _H22 transplanted tumors, and the dose-effect relationship is obvious; The growth of S ₁₈₀ sarcoma has a significant (P<0.05) inhibitory effect, and the tumor inhibitory effect is significantly better than that of the positive control CTX, and the dose-effect relationship is obvious.

The preparation of embodiment 7 medicine

Get the compound shown in the formula I prepared in Example 1 and add the conventional auxiliary materials of the powder, and make the powder according to the conventional method.

The preparation of embodiment 8 medicine

Get the compound of the structure shown in formula I prepared in Example 2 and add the conventional adjuvant of the tablet, and make a tablet according to the conventional method.

The preparation of embodiment 9 medicine

The compound with the structure shown in Formula I prepared in Example 3 was added with conventional adjuvants for granules, and granules were prepared according to conventional methods.

The preparation of embodiment 10 medicine

Get the compound of the structure shown in formula I prepared in Example 4 and add the conventional adjuvant of the capsule, and make the capsule according to the conventional method.

The preparation of embodiment 11 medicine

Take the compound of formula I prepared in Example 2 and add the conventional auxiliary materials of the solution, and prepare the solution according to the conventional method.

The preparation of embodiment 12 medicine

Take the compound of the formula I shown in Example 1 and add the conventional auxiliary materials of the emulsion, and prepare the emulsion according to the conventional method.

The preparation of embodiment 13 medicine

Take the compound of formula I prepared in Example 3 and add the conventional auxiliary materials of the suspension, and prepare the suspension according to the conventional method.

The preparation of embodiment 14 medicine

The compound with the structure shown in Formula I prepared in Example 4 was added with conventional adjuvants for injections, and injections were prepared according to conventional methods.

The preparation of embodiment 15 medicine

The compound with the structure shown in Formula I prepared in Example 2 was added with conventional adjuvants for powder injections, and powder injections were prepared according to conventional methods.

The preparation of embodiment 16 medicine

Take the compound of formula I prepared in Example 1 and add the conventional adjuvant of the spray, and prepare the spray according to the conventional method.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (10)

1. the compound suc as formula structure shown in I:

2. an extracting method for compound as claimed in claim 1, is characterized in that, comprises following steps:

Step 1: oil tea root is pulverized, got the first solvent extraction, concentrated, obtain fluid extract;

Step 2: get described fluid extract and mix with water, filter, collect filtrate, centrifugal, collect supernatant liquor, then separate through D101 type macroporous resin column, get ethanol-water system gradient elution, collecting ethanol and water volume ratio is 70:30～95:5 wash-out obtained component, and separation, purifying, obtain;

Described the first solvent is that ethanol and water volume ratio are 0:100～95:5.

3. extracting method according to claim 2, it is characterized in that, described separation, purifying are specially: get described component and separate through silicagel column, get chloroform-methanol system gradient elution, collecting chloroform and methyl alcohol volume ratio is 80:20～60:40 wash-out obtained component; Through ODS post, separate, get methanol-water system gradient elution, collecting methyl alcohol and water volume ratio is 70:30～90:10 wash-out obtained component; Through dynamic axial compression column, separate, get methanol-water system gradient elution, collecting methyl alcohol and water volume ratio is 75:25 wash-out obtained component; Finally by high performance liquid chromatography, separate, adopt C ₁₈chromatographic column, the second solvent elution, flow velocity is 2mL/min, collects 37.5min component, obtains;

Described the second solvent is: first alcohol and water by volume for the mixing solutions of 72:28 composition again with the mixture that accounts for the formic acid mixing gained that described mixing solutions quality percentage composition is 0.2%.

4. extracting method according to claim 2, is characterized in that, the solvent of gradient elution is followed successively by ethanol and water volume ratio is 0:100 → 30:70 → 50:50 → 60:40 → 70:30 → 80:20 → 95:5 described in step 2.

5. extracting method according to claim 2, is characterized in that, the temperature of extracting described in step 1 is 30 ℃～100 ℃, and the time of extracting described in step 1 is 8.5h～33h.

6. extracting method according to claim 2, is characterized in that, concentrated temperature is 70 ℃～100 ℃ described in step 1, and the concentrated time is 2.0h～6.0h described in step 1.

One kind as claimed in claim 1 compound in the application of preparing in antitumor drug.

8. application according to claim 7, is characterized in that, described tumour is lung cancer, liver cancer, melanoma or mammary cancer.

9. an antitumor drug, is characterized in that, it comprises compound and pharmaceutically acceptable auxiliary material described in claim 1.

10. antitumor drug according to claim 9, is characterized in that, its formulation is powder, tablet, granule, capsule, solution, emulsion, suspensoid, injection liquid, powder injection or sprays.

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