An antitumor active compound, extraction and separation method, drug and use

Abstract

The present invention relates to the technical field of anti-tumor compounds, and discloses an anti-tumor active compound, an extraction and separation method, a drug and uses thereof. The compound has a chemical structure of formula I, or a pharmaceutical composition of a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug of the compound of formula I. The present invention can extract and prepare an anti-tumor active compound from Leucosceptrum canum, and this compound has an inhibitory effect on human liver cancer cells.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor compounds, and in particular to an anti-tumor active compound, an extraction and separation method, a drug and uses thereof. Background Art

[0002] Leucosceptrum canum is the leaf or root bark of Leucosceptrum canum Smith of the genus Leucosceptrum in the Lamiaceae family. The plant Leucosceptrum canum is distributed in Sichuan, Yunnan and Tibet, and has the effects of clearing heat and detoxifying, promoting diuresis and detumescence, and setting bones and stopping bleeding. It is mainly used to treat high fever without sweating, unknown swelling toxins, blain sores, osteomyelitis, traumatic fractures, external bleeding, etc. At present, more than 60 chemical components have been isolated from plants of the genus Leucosceptrum, including sesterterpenoids, flavonoids, phenyl ethanol glycosides, acyl glycosides and phenols, etc., among which sesterterpenoids and flavonoids are the main structural types.

[0003] As described above, in the prior art, Leucosceptrum canum is mainly used to treat diseases such as high fever without sweating, unknown swelling toxins, blain sores, osteomyelitis, traumatic fractures, external bleeding, etc. There has been no report on using the extract of Leucosceptrum canum as an anti-tumor drug.

[0004] For example, the patent with the application number CN02149759.1 proposed an extraction method and application of Leucosceptrum canum pigment. The extraction method is: extracting Leucosceptrum canum with hot water, then concentrating the extract, adding ethanol for precipitation, centrifuging or filtering, and concentrating the supernatant to obtain an aqueous solution of Leucosceptrum canum pigment. If it is further dried, a powder of Leucosceptrum canum pigment can be obtained. This extraction method has a simple process and a high extraction rate. The patent scheme mainly extracts pigments from Leucosceptrum canum for use in the food, pharmaceutical, cosmetic and chemical industries. Although it mentions using the pigment extracted from Leucosceptrum canum for drugs, it only extracts the pigment for use in drugs, but does not mention what chemical substance is extracted and what disease the drug is used to treat in this technical scheme.

[0005] Therefore, there is currently no relevant research on extracting anti-tumor active compounds from Leucosceptrum canum. This application extracts relevant active compounds from Leucosceptrum canum for anti-tumor use. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide an anti-tumor active compound, an extraction and separation method, a drug and uses thereof, which can extract and prepare an anti-tumor active compound from Leucosceptrum canum, and this compound has an inhibitory effect on human liver cancer cells.

[0007] The present invention solves the above technical problems through the following technical means:

[0008] In a first aspect, the present invention provides an anti-tumor active compound Leucosceptrum C, with the chemical name of 12-hydroxy-8,11,13,15-abietatetraene-2,7-dione, and its structure is shown in the following formula I:

[0009] The present invention also provides a pharmaceutical composition of the compound of formula I in the form of a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug.

[0010] In a second aspect, the present invention further provides a method for extracting and separating the above anti-tumor active compound, adopting the following technical solution:

[0011] Step (1): Using the air-dried branches of Leucosceptrum canum as raw materials, after pulverization, extract with an organic solvent, and concentrate under reduced pressure at low temperature to obtain an extract.

[0012] Step (2): Mix the extract with water to obtain a suspension, add ethyl acetate for extraction, and concentrate under reduced pressure at low temperature to obtain an ethyl acetate extraction phase.

[0013] Step (3): Separate the ethyl acetate extraction phase by silica gel column chromatography, and perform gradient elution using petroleum ether - ethyl acetate as the eluent. The volume ratio of petroleum ether to ethyl acetate is successively 9.8 - 10.2:1, 4.8 - 5.2:1, 2.8 - 3.2:1, 0.8 - 1.2:1. Collect the eluate corresponding to the volume ratio of petroleum ether to ethyl acetate of 0.8 - 1.2:1, and then analyze by thin-layer chromatography, and combine similar components to obtain different initial components F41 - F44.

[0014] Step (4): Separate the initial component F42 by preparative liquid chromatography, and perform isocratic elution using acetonitrile and water as the eluent to obtain the anti-tumor active compound Leucosceptrum C shown in formula I.

[0015] Further, in step (1), the organic solvent is ethanol with a volume fraction ≥ 90%, and the volume-mass ratio with the Leucosceptrum canum branches is 10 - 20 mL:1 g; the extraction temperature is 65 - 75 °C, extract 2 - 3 times, and each time for 4 - 6 h.

[0016] Further, in steps (1) and (2), the temperature for concentration under reduced pressure at low temperature is 40 - 50 °C, and the vacuum degree is 0.07 - 0.09 MPa.

[0017] Further, in step (2), the mass ratio of the extract to water is 1:1 - 3, the volume ratio of ethyl acetate to the suspension is 0.8 - 1:1, and extract 2 - 4 times.

[0018] Further, in step (4), the volume fraction of acetonitrile in the eluent is 35 - 45%, and the rest is water.

[0019] The anti-tumor active compound Leucosceptrum C of the present invention is obtained from the branches of Leucosceptrum canum by crushing, extraction, concentration under reduced pressure, extraction, and multiple separation and purification steps. During the preparation, dry Leucosceptrum canum branches are selected or the branches are dried before use, which facilitates the crushing of the branches. An eluent with equal volumes of petroleum ether and ethyl acetate is used as the target separation system. At the same time, due to the large number of impurity components in Leucosceptrum canum, a single-concentration elution cannot obtain the anti-tumor active compound Leucosceptrum C; if the concentration gradient is set unreasonably, too large or too small, it may cause the anti-tumor active compound Leucosceptrum C to not be fully eluted. In the absence of reference materials providing indications for extracting the anti-tumor active compound Leucosceptrum C, there is great uncertainty in processing Leucosceptrum canum branches and obtaining Leucosceptrum C from them.

[0020] In a third aspect, the present invention also provides an anti-tumor drug, which comprises a pharmaceutically acceptable carrier and the above-mentioned compound, or a pharmaceutical composition of a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug of the compound.

[0021] In a fourth aspect, the present invention also provides the use of a pharmaceutical composition containing the above anti-tumor active compound, or a pharmaceutically acceptable salt, stereoisomer, hydrate, solvate or prodrug thereof, in the preparation of an anti-tumor drug.

[0022] Further, the tumor cells corresponding to the tumor are human liver cancer SMMC7721 cells.

[0023] The beneficial effects of the present invention are as follows:

[0024] The anti-tumor active compound Leucosceptrum C of the present invention is a natural anti-tumor compound extracted from the branches of Leucosceptrum canum, and has the biological activity of inhibiting tumor cells. For example, it has an obvious inhibitory effect on human liver cancer SMMC7721 cells, and the half-maximal inhibitory concentration IC 50 value is smaller than that of the broad-spectrum anti-tumor drug cisplatin on the market, indicating that it has strong anti-tumor activity and has great application prospects in the preparation of anti-tumor drugs, and plays an important role in enhancing the medical and economic value of Leucosceptrum canum. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a process flow chart for the preparation of the anti-tumor active compound Leucosceptrum C of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention will be described in detail below with reference to the examples and the drawings:

[0027] Unless otherwise specified, the materials used in the present invention are all commercially available or commonly used in the art. Unless otherwise specified, the methods in the following examples are all conventional methods in the art.

[0028] Example 1: Physicochemical Property Testing of Anti-tumor Active Compound Leucosceptrum C

[0029] 1.1 Determination of Molecular Structural Formula

[0030] The nuclear magnetic resonance instrument used was a Bruker AVANCE III 500 type nuclear magnetic resonance instrument manufactured by Bruker Corporation. Deuterated CDCl3 (deuterated chloroform) reagent was used for nuclear magnetic resonance. The hydrogen spectrum and carbon spectrum data of the anti-tumor active compound Leucosceptrum C are shown in Table 1 below.

[0031] Table 1 Hydrogen Spectrum of Leucosceptrum C 1 H-NMR (500 MHz) and Carbon Spectrum 13 C-NMR (125 MHz) Data

[0032]

[0033]

[0034] Note: Chemical shift is represented by δ; s represents singlet; br s represents broad singlet; m represents multiplet; dd represents doublet of doublets.

[0035] 1.2 Molecular Weight Testing

[0036] High-resolution mass spectrometry HR-ESI-MS was used to determine the exact molecular weight of the compound. HR-ESI-MS [M-H]-m / z was 313.1804. According to the NMR data and mass spectrometry data in Table 1, the molecular formula of the anti-tumor active compound Leucosceptrum C is C 20 H 26 O3, with a molecular weight of 314, an unsaturation degree of 8, and the chemical name of 12-hydroxy-8,11,13,15-abietatetraene-2,7-dione, and the structure is shown in Formula I.

[0037] 1.3 Melting Point Testing

[0038] The melting point instrument used was a WRS-1C type melting point instrument manufactured by Shanghai Physical Optics. After testing, the melting point of the anti-tumor active compound Leucosceptrum C was m.p. 259-261 °C.

[0039] Example 2: Preparation of Anti-tumor Active Compound Leucosceptrum C

[0040] The method for preparing compound Leucosceptrum C according to the present invention comprises the following steps:

[0041] (1) Air-dry and crush 5 kg of the branches of Leucosceptrum canum, add them to ethanol with a volume fraction of 95%, add ethanol according to the ratio of the mass of Leucosceptrum canum to the volume of ethanol of 1 g:10 mL, carry out extraction at a temperature of 70 °C for 3 times, 5 hours each time; filter the insoluble matters and combine the filtrates, and then carry out low-temperature vacuum evaporation to dryness at a vacuum degree of 0.08 MPa and a temperature of 45 °C to obtain an extract;

[0042] (2) Mix the obtained extract with water according to a mass ratio of 1:2 to make a suspension, extract with an equal volume of ethyl acetate 3 times, combine the extraction liquids, and carry out low-temperature vacuum evaporation to dryness at a vacuum degree of 0.08 MPa and a temperature of 45 °C to obtain an ethyl acetate extraction phase;

[0043] (3) Mix the obtained ethyl acetate extraction phase with silica gel of equal mass evenly, then place it in the middle layer of a silica gel column for chromatography, use petroleum ether - ethyl acetate as an eluent for gradient elution, the volume ratios of each gradient elution of petroleum ether to ethyl acetate are 10:1, 5:1, 3:1, 1:1, collect the eluent when the volume ratio of petroleum ether to ethyl acetate is 1:1, analyze by thin-layer chromatography and combine similar components to obtain different primary components F41 - F44;

[0044] (4) Separate the collected primary component F42 by preparative liquid chromatography, use acetonitrile:water with a volume ratio of 40:60 as an eluent for isocratic elution to obtain the diterpenoid compound C (8.0 mg) with antitumor activity shown in Formula I.

[0045] This process is as Figure 1 shown.

[0046] Example 3: Preparation of antitumor active compound Leucosceptrum C

[0047] The method for preparing compound Leucosceptrum C according to the present invention comprises the following steps:

[0048] (1) Air-dry and crush 5 kg of the branches of Leucosceptrum canum, add them to ethanol with a volume fraction of 90%, the addition amount is 20 mL / g of Leucosceptrum canum, carry out extraction at a temperature of 65 °C for 3 times, 4 hours each time; filter the insoluble matters and combine the filtrates, and then carry out low-temperature vacuum evaporation to dryness at a vacuum degree of 0.07 MPa and a temperature of 50 °C to obtain an extract;

[0049] (2) Mix the obtained extract with water at a mass ratio of 1:1 to make a uniform suspension, extract with ethyl acetate of equal volume 4 times, combine the extracts, and evaporate to dryness under reduced pressure at a vacuum of 0.07 MPa and a temperature of 50 °C to obtain the ethyl acetate extraction phase;

[0050] (3) Mix the obtained ethyl acetate extraction phase with silica gel of equal mass evenly, then place it in the middle layer of a silica gel column for chromatography, use petroleum ether - ethyl acetate as the eluent for gradient elution, and the volume ratios of petroleum ether to ethyl acetate in each gradient elution are 9.8:1, 4.8:1, 2.8:1, 0.8:1. Collect the eluent when the volume ratio of petroleum ether to ethyl acetate is 0.8:1, analyze by thin-layer chromatography and combine similar components to obtain different preliminary components F41 - F44;

[0051] (4) Separate the preliminary component F42 collected by column chromatography by preparative liquid chromatography, use acetonitrile: water (volume ratio 35:65) as the eluent for isocratic elution to obtain the diterpenoid compound C (6.5 mg) with antitumor activity shown in formula I.

[0052] Example 4: Preparation of the antitumor active compound Leucosceptrum C

[0053] Use the method described in the present invention to prepare the compound Leucosceptrum C, including the following steps:

[0054] (1) Air-dry and crush 5 kg of the branches of Leucosceptrum canum, add them to ethanol with a volume fraction of 95%, with an addition amount of 15 mL of ethanol per gram of Leucosceptrum canum, extract at a temperature of 75 °C for 2 times, 4 hours each time; filter the insoluble matter and combine the filtrates, then evaporate to dryness under reduced pressure at a vacuum of 0.09 MPa and a temperature of 40 °C to obtain an extract;

[0055] (2) Mix the obtained extract with water at a mass ratio of 1:3 to make a uniform suspension, extract with ethyl acetate of equal volume 2 times, combine the extracts, and evaporate to dryness under reduced pressure at a vacuum of 0.09 MPa and a temperature of 40 °C to obtain the ethyl acetate extraction phase;

[0056] (3) Mix the obtained ethyl acetate extraction phase with silica gel of equal mass evenly, then place it in the middle layer of a silica gel column for chromatography, use petroleum ether - ethyl acetate as the eluent for gradient elution, and the volume ratios of petroleum ether to ethyl acetate in each gradient elution are 10.2:1, 5.2:1, 3.2:1, 1.2:1. Collect the eluent when the volume ratio of petroleum ether to ethyl acetate is 1.2:1, analyze by thin-layer chromatography and combine similar components to obtain different preliminary components F41 - F44;

[0057] (4) The initially collected chromatographic fraction F42 was separated by preparative liquid chromatography, and isocratic elution was performed using acetonitrile: water (45:55) as the eluent to obtain 5.0 mg of the diterpenoid compound C with antitumor activity shown in Formula I.

[0058] Example 5: Antitumor Performance Test of Antitumor Active Compound Leucosceptrum C

[0059] The antitumor active compound Leucosceptrum C prepared in Example 2 was used for the antitumor ability test, and the process was as follows:

[0060] In vitro tumor cell proliferation inhibition test: Tumor cells in the logarithmic growth phase were taken, the cell suspension concentration was adjusted (50,000 - 100,000 cells / ml), 100 μl of the cell suspension was inoculated into a 96-well cell culture plate per well. After 24 h of inoculation, drugs were administered (100 μl / well). A blank control group, a cell control group, and six concentration groups (3.12, 6.25, 12.5, 25, 50, 100 μmol / L) of the test drug were set respectively. The drug in the cell control group was cisplatin. After continued culture for 72 h, 100 μl of MTT (1 mg / ml, dissolved in DMEM culture medium) was added to each well, incubated at 37 °C for 4 h. After discarding the liquid in each well, 150 μl of acidified isopropanol (containing 0.04 mol / L HCl) was added, and it was placed in the dark for 30 min. The absorbance at 570 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the proliferation inhibition rate of the test drug on tumor cells was calculated. The half-maximal inhibitory concentration (IC 50 ) of the test substance on tumor cell proliferation (72 h) was calculated. To reduce the influence of experimental error, three parallels were performed for each experiment, and the results were as follows.

[0061] 5.1 Human hepatocellular carcinoma SMMC7721 cells

[0062] (1) The inhibitory effect of cisplatin in the control group on SMMC7721 cells is shown in Tables 2, 3, and 4.

[0063] Table 2 Results of the first parallel test of the inhibitory effect of cisplatin on SMMC7721 cells

[0064]

[0065] The IC 50 obtained by Graphpad Prism 6.0 was 21.97.

[0066] Table 3 Results of the second parallel test of the inhibitory effect of cisplatin on SMMC7721 cells

[0067]

[0068] The IC value obtained by Graphpad Prism 6.0 50 = 22.68.

[0069] Table 4 Results of the third parallel test on the inhibitory effect of cisplatin on SMMC7721 cells

[0070]

[0071]

[0072] The IC value obtained by Graphpad Prism 6.0 50 = 18.72.

[0073] From the above data, it can be obtained that the IC value of cisplatin 50 = 21.12 ± 2.11.

[0074] (2) The inhibitory effect of compound Leucosceptrum C on SMMC7721 cells is shown in Tables 5, 6, and 7.

[0075] Table 5 Results of the first parallel test on the inhibitory effect of compound Leucosceptrum C on SMMC7721 cells

[0076]

[0077] The IC value obtained by Graphpad Prism 6.0 50 = 17.92.

[0078] Table 6 Results of the second parallel test on the inhibitory effect of compound Leucosceptrum C on SMMC7721 cells

[0079]

[0080] The IC value obtained by Graphpad Prism 6.0 50 = 18.37.

[0081] Table 7 Results of the third parallel test on the inhibitory effect of compound Leucosceptrum C on SMMC7721 cells

[0082]

[0083]

[0084] The IC value obtained by Graphpad Prism 6.0 50 = 14.40.

[0085] From the above data, the IC of Leucosceptrum C can be obtained as follows: 50 = 16.90 ± 2.17.

[0086] 5.2 Half inhibitory concentration IC of Leucosceptrum C and cisplatin on SMMC7721 cells for 72 hours 50 (μmol / L) is shown in Table 8.

[0087] Table 8 Half inhibitory concentration IC50 (μmol / L) of Leucosceptrum C and cisplatin on SMMC7721 cells for 72 hours

[0088]

[0089] As can be seen from Tables 5 - 8 above, compounds of different concentrations of Leucosceptrum C have inhibitory effects on human liver cancer SMMC7721. The IC of Leucosceptrum C after 72h 50 value reaches 16.90 ± 2.17 μmol / L, and its IC 50 value is lower than that of the cisplatin control group (21.12 ± 2.11 μmol / L), indicating that Leucosceptrum C has a very significant inhibitory effect on SMMC7721 cells.

[0090] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention. The technologies, shapes, and structures not described in detail in the present invention are all well-known technologies.

Claims

1. A method for extracting and separating an antitumor active compound, characterized in that: The compound is a compound of formula I, including the following steps, Step (1): Using the air-dried branches of Leucosceptrum canum as raw materials, after pulverization, extraction is carried out with an organic solvent, and the extract is concentrated under reduced pressure at low temperature to obtain an extract; Step (2): Mix the extract with water to obtain a suspension, add ethyl acetate for extraction, and concentrate under reduced pressure at low temperature to obtain an ethyl acetate extraction phase; Step (3): Separate the ethyl acetate extraction phase by silica gel column chromatography, and perform gradient elution with petroleum ether - ethyl acetate as the eluent. The volume ratios of petroleum ether to ethyl acetate are successively (9.8 - 10.2):1, (4.8 - 5.2):1, (2.8 - 3.2):1, (0.8 - 1.2):1; collect the eluate corresponding to the volume ratio of petroleum ether to ethyl acetate of (0.8 - 1.2):1, and then analyze by thin layer chromatography, and combine similar components to obtain different initial components F41 - F44; Step (4): Separate the initial component F42 by preparative liquid chromatography, and perform isocratic elution with acetonitrile and water as the eluent to obtain the anti-tumor active compound; The organic solvent in step (1) is ethanol with a volume fraction ≥ 90%, the temperature of concentration under reduced pressure at low temperature in steps (1) and (2) is 40 - 50 °C, the volume fraction of acetonitrile in the eluent in step (4) is 35 - 45%, and the rest is water.

2. The preparation method of an anti-tumor active compound according to claim 1, characterized in that: In step (1), the volume-mass ratio of the organic solvent to the air-dried branches of Leucosceptrum canum is 10 - 20 mL:1 g; the extraction temperature is 65 - 75 °C, and extraction is carried out 2 - 3 times, 4 - 6 h each time.

3. The preparation method of an anti-tumor active compound according to claim 2, wherein: The vacuum degree of concentration under reduced pressure at low temperature in steps (1) and (2) is 0.07 - 0.09 MPa.

4. The extraction and separation method of an anti-tumor active compound according to claim 3, characterized in that: In step (2), the mass ratio of the extract to water is 1:1 - 3, the volume ratio of ethyl acetate to the suspension is 0.8 - 1:1, and extraction is carried out 2 - 4 times.

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