Disubstituted propane natural product as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of natural pharmaceutical chemistry, and particularly relates to a disubstituted propane natural product as well as a preparation method and application thereof. The disubstituted propane natural product extracted from aquilaria sinensis Yunnan has good anti-tumor activity, especially has remarkable inhibitory activity on breast cancer cells and lung cancer cells, provides a new choice for clinically treating the breast cancer and the lung cancer, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural medicinal chemistry, and in particular relates to a disubstituted propane natural product, a preparation method and application thereof. Background Art

[0002] A tumor is a mass of tissue formed when the body's cells lose normal regulation and proliferate abnormally, driven by factors such as gene mutations, epigenetic changes, or abnormal microenvironment. Based on their biological behavior, they can be divided into benign tumors (limited growth, no invasion or metastasis) and malignant tumors (i.e., cancer, which is invasive, metastatic, and highly lethal). Existing tumor treatment methods mainly include surgical treatment, radiotherapy, chemotherapy, hormone therapy, etc., but they have certain limitations, such as many side effects, high treatment costs, high toxicity, and easy to induce drug resistance. Therefore, there is an urgent need to find a new anti-tumor drug to provide a more effective treatment for tumors.

[0003] The disubstituted propane natural product is a component extracted from Yunnan Aquilaria sinensis. There is no report on whether it can be used as an anti-tumor drug for the treatment of tumors. Summary of the Invention

[0004] In response to the problems of the prior art, the present invention provides a disubstituted propane natural product and its preparation method and use.

[0005] A Yunnan agarwood extract, comprising a compound represented by formula I:

[0006]

[0007] Preferably, the preparation method of the Yunnan Aquilaria wood extract comprises the following steps:

[0008] Step 1, extracting Yunnan Aquilaria sinensis with solvent 1 to obtain a crude extract; the solvent 1 is selected from an alcohol solution;

[0009] Step 2, extracting the crude extract obtained in step 1 with solvent 2 to obtain an extract; the solvent 2 is selected from an ester solution;

[0010] Step 3, dissolving the extract in solvent 3, and gradient eluting with a mixed solvent of dichloromethane and methanol to obtain an elution component A with a volume ratio of dichloromethane to methanol of 10-30:1; the solvent 3 is at least one of dichloromethane and methanol;

[0011] Step 4, the eluted component A is dissolved in solvent 4, adsorbed on a reverse C18 chromatographic column, and gradient eluted with methanol as the eluent to obtain an eluted component B with a volume percentage of 75-90% methanol; the solvent 4 is selected from an alcohol solution;

[0012] Step 5, the eluted component B is separated by thin layer chromatography, and then the components in the silica gel are extracted by ultrasonication to obtain.

[0013] Preferably, in step 3, the elution component A is obtained by elution at a volume ratio of dichloromethane to methanol of 20:1;

[0014] And / or, in step 4, the elution component B is obtained by eluting with 85% by volume of methanol.

[0015] Preferably, in step 1, the alcohol solution is selected from at least one of a methanol aqueous solution and an ethanol aqueous solution; the extraction method is heating under reflux, the extraction times are 1 to 3 times, the reaction temperature is 50 to 85° C., and the reaction time is 2 to 6 hours per time;

[0016] And / or, in step 2, the solvent 2 is selected from at least one of ethyl acetate and n-butanol;

[0017] And / or, in step 3, the solvent 3 is dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is 1:0 to 1:5;

[0018] And / or, in step 3, the elution gradient of the gradient elution is a volume ratio of dichloromethane to methanol of 1:0, 50:1, 20:1, 10:1, and 1:1 respectively;

[0019] And / or, in step 4, the solvent 4 is selected from at least one of methanol and ethanol; the reverse C18 chromatographic column is an RP-18 chromatographic column; the gradient elution is 40%, 60%, and 85% methanol by volume, respectively;

[0020] And / or, in step 5, the thin layer chromatography uses ultraviolet light to identify compounds, and takes blue fluorescent band silica gel with an Rf value of 0.4-0.6; the solvent of the ultrasonic extraction is selected from at least one of dichloromethane and methanol.

[0021] Preferably, in step 1, the volume concentration of the methanol aqueous solution in the solvent 1 is 70-100%, and the volume concentration of the ethanol aqueous solution is 90-100%;

[0022] And / or, the volume ratio of each gradient elution in step 3 is 10:10:10:10:(5-10).

[0023] Preferably, in step 4, the volume ratio of each elution gradient is 1:(1-2):1.

[0024] And / or, in step 5, the wavelength of the ultraviolet lamp is 254-365 nm; the solvent for ultrasonic extraction is selected from dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 10:1-1:5.

[0025] The present invention also provides a method for preparing the Yunnan Aquilaria wood extract, comprising the following steps: Step 1, extracting the Yunnan Aquilaria wood with a solvent 1 to obtain a crude extract; the solvent 1 is selected from an alcohol solution;

[0026] Step 2, extracting the crude extract obtained in step 1 with solvent 2 to obtain an extract; the solvent 2 is selected from an ester solution;

[0027] Step 3, dissolving the extract in solvent 3, and gradient eluting with a mixed solvent of dichloromethane and methanol to obtain an elution component A with a volume ratio of dichloromethane to methanol of 10-30:1; the solvent 3 is at least one of dichloromethane and methanol;

[0028] Step 4, the eluted component A is dissolved in solvent 4, adsorbed on a reverse C18 chromatographic column, and gradient eluted with methanol as the eluent to obtain an eluted component B with a volume percentage of 75-90% methanol; the solvent 4 is selected from an alcohol solution;

[0029] Step 5, the eluted component B is separated by thin layer chromatography, and then the components in the silica gel are extracted by ultrasonication to obtain.

[0030] The present invention also provides use of the Yunnan Aquilaria wood extract in preparing a medicament for treating tumors.

[0031] Preferably, the tumor is at least one of breast cancer, lung cancer, cervical cancer, colon cancer, and lymphocytoma.

[0032] Preferably, the tumor is breast cancer or lung cancer.

[0033] The present invention also provides a pharmaceutical composition for treating tumors, which is a preparation prepared by taking the Yunnan Aquilaria wood extract as an active ingredient and adding pharmaceutically acceptable excipients.

[0034] The disubstituted propane natural product extracted from Yunnan agarwood has good anti-tumor activity, especially significant inhibitory activity against breast cancer cells and lung cancer cells, providing a new choice for clinical drugs for the treatment of breast cancer and lung cancer and having good application prospects.

[0035] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0036] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the disubstituted propane natural product of the present invention is ( 1 H NMR spectrum).

[0038] Figure 2 The carbon NMR spectrum of the disubstituted propane natural product of the present invention is ( 13 C NMR and DEPT spectra). DETAILED DESCRIPTION

[0039] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available.

[0040] Unless otherwise specified, the ratios in the present invention are volume ratios and the concentrations are volume percentages.

[0041] Example 1 Disubstituted propane natural product and preparation method thereof

[0042] 1. The molecular formula of disubstituted propane natural product is C 24 H 34 O8, molecular weight is 450, and its structure is as follows:

[0043]

[0044] 2. Preparation method

[0045] 18.0 kg of dried Yunnan Aquilaria sinensis powder was ground and extracted three times with 90% methanol under reflux (70°C; each extraction time was 4 hours). The solvent was removed by distillation under reduced pressure, and the extracts were combined to obtain a crude methanol extract. The crude extract was suspended in 15.5 L of water and extracted with ethyl acetate (15 L x 3). The ethyl acetate was removed by distillation under reduced pressure to obtain 67.3 g of a brown extract. The ethyl acetate extract was dissolved in 3.0 L of a mixed solvent of dichloromethane and methanol (volume ratio 1:1) and adsorbed onto 0.3 kg of 200-300 mesh silica gel. The column was dry packed and gradient elution was performed with a volume ratio of dichloromethane:methanol (1:0; 50:1; 20:1; 10:1; 1:1, with volumes of 10 L, 10 L, 10 L, 10 L, and 5 L, respectively). The elution was complete at a rate of 1 drop / second. After removing the solvent by distillation under reduced pressure, the corresponding elution samples Fr.1 (8.0 g), Fr.2 (6.3 g), Fr.3 (7.1 g), Fr.4 (10.3 g), and Fr.5 (11.3 g) were obtained. Sample Fr.3 (7.1 g) was dissolved in methanol and adsorbed onto RP-18. Dry column chromatography was performed, sequentially eluting with 40% methanol (8.0 L), 60% methanol (8.0 L), and 85% methanol (8.0 L) at a rate of 1 drop / second. The solvent was removed by distillation under reduced pressure to yield the corresponding eluted samples Fr.3a (2.6 g), Fr.3b (2.7 g), and Fr.3c (3.2 g). Fr.3c (60 mg) was extracted from silica gel with a blue fluorescent band exhibiting an Rf value of 0.4-0.6 at 254 nm under a UV lamp using dichloromethane:methanol (10:1, by volume) as the solvent. Ultrasonic extraction of the disubstituted propane natural product from the silica gel was performed using dichloromethane:methanol (10:1, by volume). The solvent was removed under reduced pressure to yield a light yellow powder, the disubstituted propane natural product (6.5 mg).

[0046] 3. Structural characterization

[0047] High-resolution Fourier transform mass spectrometry (HR-FT-MS) showed that its quasi-molecular ion peak was m / z 473.2144 [M+Na] + (C 24 H 34 8Na + ,[M+Na] + calcd.for 473.2146), indicating that the molecular formula of the compound is C 24 H 34 O8, degree of unsaturation is 8. 1 δ in H-NMR spectra H 6.82 (d, J = 7.3 Hz, 4H) and δ H 7.12 (d, J = 7.3 Hz, 4H) indicates that there are two para-substituted benzene rings in this compound. Another characteristic signal δH 1.62 (s, 6H) and δ C 41.90(s) suggests the presence of two methyl groups in the compound (see Table 1), which is consistent with the diphenyl-substituted propane skeleton. 13 C-NMR and DEPT spectra showed that the compound had 24 carbon signals, including 2 methyl groups: δ C 41.90 (q, C-α), 41.90 (q, C-γ), 6 methylene groups: δ C 67.09 (t, C-7), 63.87 (t, C-9), 68.90 (t, C-7'), 69.39 (t, C-9'), 71.49 (t, C-10'), 63.87 (t, C-12'), 11 methine groups: δ C 114.15 (d, C-2, 6, 2', 6'), 127.95 (d, C-3, 5, 3', 5'), 70.59 (d, C-8), 69.00 (d, C-8'), 69.32 (d, C-11') and 5 quaternary carbons: δ C 156.46 (s, C-1,1'), 143.92 (s, C-4,4'), 41.90 (s, C-β) (see Table 2). These signals suggest that the compound is a 2,2-bis[4-(2,3-dihydroxypropoxy)phenyl]propane homologue, a compound containing a disubstituted phenylpropane skeleton; the chemical structure of 2,2-bis[4-(2,3-dihydroxypropoxy)phenyl]propane is shown below:

[0048]

[0049] Comparing the chemical shift of this compound with that of the carbon of 2,2-[bis-4-(2,3-dihydroxypropoxy)phenyl]propane, this compound has one more signal of glycerol structural unit than the known compound: δ C 71.49 (t), 69.32 (d) and 63.87 (t); at the same time, the chemical shift of C-9' position changes from δ C 62.8(t) shifts downfield to δ C 69.39 (t). These information suggest that the glycerol structural unit of this compound is connected at the C-9' position. In the HMBC spectrum, δ H 3.55 (m, 1H, H-10') and δ C 69.39 (t, C-9') is correlated, further suggesting that the glycerol structural unit of this compound is connected at the C-9' position. H1.62(s,6H,H-α,γ) and δ C 41.90 (s, C-β) and δ C 143.68(s,C-4,4') is also relevant. 1 H- 1 In the H COSY spectrum, δ H 7.06 (d, J = 8.5 Hz) and δ H A significant correlation was also observed at 7.26 (d, J = 8.5 Hz). Therefore, the compound was identified as β-[1-(8,9-dihydroxypropoxy)phenyl]-β-9'-11',12'-dihydroxypropoxy[1'-(8',9'-dihydroxypropoxy)phenyl]propane.

[0050] Table 1 Hydrogen chemical shifts of disubstituted propane natural products (δ in ppm, J in Hz)

[0051]

[0052] Table 2 Carbon chemical shifts (δ in ppm) of disubstituted propane natural products

[0053]

[0054] Known compound: recordedin MeOD; disubstituted propane natural product: recordedin CDCl3.

[0055] Example 2 Antitumor activity of disubstituted propane natural products

[0056] 1. Experimental Methods

[0057] 1. Cell lines: malignant triple-negative breast cancer (TNBC) cell line HCC1806, human lung cancer cell line (A549), cervical cancer cells (Hela cells), colon cancer cells (CT-26 cells), and human B lymphoma cells (ST486 cells).

[0058] 2. MTT method

[0059] 1) Tumor cells (HCC1806, A549, Hela, CT-26, ST486 cells) were cultured in RPMI1640 medium containing 10% fetal bovine serum. Cells in the logarithmic growth phase were taken and cultured at 5.0×10 3The number of cells was added to the culture plate, 100 μL / well, and plated in a 96-well culture plate. To avoid edge effect, the edge wells were filled with an equal volume of PBS.

[0060] 2) Incubate the cells in 5% CO2, 37°C for 24 h and treat the cells with 2.5, 5, 10, 20, 40, and 80 μM disubstituted propane natural products. The positive control groups consisted of cisplatin and paclitaxel dissolved in DMSO. 100 μL / well of each drug concentration was added as a blank control. Three replicate wells were set for each concentration, and the average value was calculated.

[0061] 3) After incubation in an incubator at 5% CO2 and 37°C for 48 hours, observe cell growth. Weigh MTT and prepare a 5 mg / mL solution in PBS, then repeatedly shake to mix.

[0062] 4) Add 20 μL / well of MTT solution and incubate at 37°C with 5% CO2 for 4 hours.

[0063] 5) Gently remove the supernatant culture medium from each well until it is completely removed, add 150 μL / well of DMSO, and shake evenly for a few seconds on a microplate shaker to ensure uniform dissolution of MTT-formazan.

[0064] 6) Turn on the microplate reader, measure the absorbance at 570 nm, and calculate the half-maximal inhibitory concentration (IC) of the compound using Origin software (www.originlab.com). 50 .

[0065] 2. Experimental Results

[0066] As shown in Table 3, the IC values of disubstituted propane natural products against triple-negative breast cancer cells HCC1806 were 50 The IC value for human lung cancer cell line A549 is (0.0077±0.0085)μM. 50 was (5.62±1.89)μM, which was superior to the positive drugs paclitaxel and cisplatin; it showed weak inhibitory activity against colon cancer cells CT-26, IC 50 The results showed that the disubstituted propane natural product has good anti-tumor activity, especially against breast cancer cells and lung cancer cells.

[0067] Table 3 Cytotoxicity test results of disubstituted propane natural products

[0068]

[0069] a IC 50 Data represent three replicates. express;

[0070] b Positive controls were used to assess cytotoxic activity.

[0071] In summary, the disubstituted propane natural product extracted from Yunnan Aquilaria wood in the present invention has good anti-tumor activity, especially significant inhibitory activity against breast cancer cells and lung cancer cells, providing a new choice for clinical drugs for the treatment of breast cancer and lung cancer and has good application prospects.

Claims

1. A Yunnan Aquilaria wood extract, characterized in that: The Yunnan Aquilaria sinensis extract includes the compound shown in Formula I:

2. The Yunnan Aquilaria wood extract according to claim 1, characterized in that The preparation method of the Yunnan Aquilaria wood extract comprises the following steps: Step 1, extracting Yunnan Aquilaria sinensis with solvent 1 to obtain a crude extract; the solvent 1 is selected from an alcohol solution; Step 2, extracting the crude extract obtained in step 1 with solvent 2 to obtain an extract; the solvent 2 is selected from an ester solution; Step 3, dissolving the extract in solvent 3, and gradient eluting with a mixed solvent of dichloromethane and methanol to obtain an elution component A with a volume ratio of dichloromethane to methanol of 10-30:1; the solvent 3 is at least one of dichloromethane and methanol; Step 4, the eluted component A is dissolved in solvent 4, adsorbed on a reverse C18 chromatographic column, and gradient eluted with methanol as the eluent to obtain an eluted component B with a volume percentage of 75-90% methanol; the solvent 4 is selected from an alcohol solution; Step 5, the eluted component B is separated by thin layer chromatography, and then the components in the silica gel are extracted by ultrasonication to obtain.

3. The Yunnan Aquilaria wood extract according to claim 2, characterized in that In step 1, the alcohol solution is selected from at least one of a methanol aqueous solution and an ethanol aqueous solution; the extraction method is heating under reflux, the extraction times are 1 to 3 times, the reaction temperature is 50 to 85° C., and the reaction time is 2 to 6 hours per time; And / or, in step 2, the solvent 2 is selected from at least one of ethyl acetate and n-butanol; And / or, in step 3, the solvent 3 is dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is 1:0 to 1:5; And / or, in step 3, the elution gradient of the gradient elution is a volume ratio of dichloromethane to methanol of 1:0, 50:1, 20:1, 10:1, and 1:1 respectively; And / or, in step 4, the solvent 4 is selected from at least one of methanol and ethanol; the reverse C18 chromatographic column is an RP-18 chromatographic column; the gradient elution is 40%, 60%, and 85% methanol by volume, respectively; And / or, in step 5, the thin layer chromatography uses ultraviolet light to identify compounds, and takes blue fluorescent band silica gel with an Rf value of 0.4-0.6; the solvent of the ultrasonic extraction is selected from at least one of dichloromethane and methanol.

4. The Yunnan Aquilaria wood extract according to claim 3, characterized in that In step 1, the volume concentration of the methanol aqueous solution in the solvent 1 is 70-100%, and the volume concentration of the ethanol aqueous solution is 90-100%; And / or, the volume ratio of each gradient elution in step 3 is 10:10:10:10:(5-10).

5. The Yunnan Aquilaria wood extract according to claim 3, characterized in that In step 4, the volume ratio of each elution gradient is 1:(1-2):

1. And / or, in step 5, the wavelength of the ultraviolet lamp is 254-365 nm; the solvent for ultrasonic extraction is selected from dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 10:1-1:

5.

6. The method for preparing the Yunnan Aquilaria wood extract according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: step 1, extracting Yunnan agarwood with solvent 1 to obtain a crude extract; the solvent 1 is selected from an alcohol solution; Step 2, extracting the crude extract obtained in step 1 with solvent 2 to obtain an extract; the solvent 2 is selected from an ester solution; Step 3, dissolving the extract in solvent 3, and gradient eluting with a mixed solvent of dichloromethane and methanol to obtain an elution component A with a volume ratio of dichloromethane to methanol of 10-30:1; the solvent 3 is at least one of dichloromethane and methanol; Step 4, the eluted component A is dissolved in solvent 4, adsorbed on a reverse C18 chromatographic column, and gradient eluted with methanol as the eluent to obtain an eluted component B with a volume percentage of 75-90% methanol; the solvent 4 is selected from an alcohol solution; Step 5, the eluted component B is separated by thin layer chromatography, and then the components in the silica gel are extracted by ultrasonication to obtain.

7. Use of the Yunnan Aquilaria wood extract according to any one of claims 1 to 5 in the preparation of a medicament for treating tumors.

8. The use according to claim 7, characterized in that The tumor is at least one of breast cancer, lung cancer, cervical cancer, colon cancer, and lymphocytoma.

9. The use according to claim 8, characterized in that The tumor is breast cancer or lung cancer.

10. A pharmaceutical composition for treating tumors, characterized in that: The preparation is prepared by taking the Yunnan agarwood extract as claimed in any one of claims 1 to 5 as an active ingredient and adding pharmaceutically acceptable excipients.

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