New compound in acanthopanax gracilistylus, preparation method and medical application

Abstract

The invention discloses a novel compound in acanthopanax gracilistylus, a preparation method and medical application. According to the invention, a compound with a novel structure is separated from acanthopanax gracilistylus; activity studies show that the compound has an obvious inhibition effect on colon cancer cells and liver cancer cells. Therefore, the compound provided by the invention has an application prospect of being developed into medicines for treating colon cancer or liver cancer.

Description

Technical Field

[0001] The invention belongs to the field of chemistry and relates to the discovery and application of natural products, and in particular to a new compound in Acanthopanax gracilis, a preparation method and medical use thereof. Background Art

[0002] Acanthopanax gracilistylus W.W. Smith, a plant of the Araliaceae family, is a dried root bark that has the effects of dispelling wind and dampness, nourishing the liver and kidneys, strengthening tendons and bones, and promoting diuresis and reducing swelling. It can be used to treat rheumatic arthritis, weakness of tendons and bones, delayed walking in children, physical weakness, edema, beriberi, etc. The root bark and aerial parts of Acanthopanax gracilistylus contain a variety of chemical components such as flavonoids, triterpenes, diterpenes, and some chemical components have pharmacological effects such as anti-tumor, anti-inflammatory, liver protection, lipid-lowering and blood sugar-lowering, and immunomodulation.

[0003] The present invention is specially proposed based on a new compound separated from the dry aerial part of Acanthopanax gracilis and its preparation method and medical use. Summary of the invention

[0004] The first purpose of the present invention is to provide a new compound separated from Acanthopanax gracilis, the second purpose is to provide a preparation method of the new compound, and the third purpose is to provide medical use of the new compound.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] A compound represented by formula I or a pharmaceutically acceptable salt or solvate thereof;

[0007]

[0008] A method for preparing a compound represented by formula I comprises the following steps:

[0009] Step S1: collecting the aerial parts of Acanthopanax gracilis, drying, crushing, taking an appropriate amount, adding an appropriate amount of ethanol aqueous solution, extracting under hot reflux, concentrating the extract under reduced pressure to obtain a fluid extract, extracting with petroleum ether, ethyl acetate, and n-butanol in sequence, collecting extracts with different organic solvents, and concentrating them under reduced pressure to obtain extracts, respectively, to obtain extraction parts with different organic solvents;

[0010] Step S2: taking an appropriate amount of extract from the n-butanol extraction part, loading it onto a silica gel chromatography column, gradient eluting it with a chloroform-methanol mixed solvent with a volume ratio of 15:1, 8:1, and 4:1 for 4, 4, and 3 column volumes, respectively, and concentrating under reduced pressure to obtain an extract;

[0011] Step S3: Take an appropriate amount of the elution portion with chloroform-methanol 8:1, load it onto a Sephadex LH-20 gel chromatography column, elute with methanol for 2 column volumes, collect 30 equal volumes of fractions per column volume, and collect fractions No. 37-45;

[0012] Step S4: The gel column chromatography elution product was separated and purified by preparative high performance liquid chromatography, and the chromatographic column was Thermo BDS HYPERSLL-C 18 The chromatographic column specifications were 250 mm × 10 mm, 5 μm, the mobile phase was acetonitrile-0.5% formic acid aqueous solution in a volume ratio of 37:63, and the eluent corresponding to the chromatographic peak at a retention time of 12.59 min was collected and concentrated and dried.

[0013] Preferably, the ethanol aqueous solution used for extraction in step S1 is an ethanol aqueous solution with a volume fraction of 80%.

[0014] Preferably, in step S1, 85° C. hot reflux extraction is used.

[0015] More preferably, the hot reflux extraction is performed three times, each time for 1.5 hours.

[0016] Preferably, in step S1, petroleum ether, ethyl acetate and n-butanol are sequentially used for extraction 5 times with equal volumes.

[0017] Preferably, the silica gel in step S2 has a mesh size of 100 to 200.

[0018] Preferably, in step S3, the Sephadex LH-20 gel chromatography column is repeatedly purified three times.

[0019] Preferably, the detection wavelength of the preparative high performance liquid chromatography separation and purification in step S4 is 254 nm.

[0020] The compound or its pharmaceutically acceptable salt or solvate is used for preparing medicine for treating colon cancer or liver cancer.

[0021] Beneficial effects:

[0022] The present invention separates a novel compound from Acanthopanax gracilis and provides a separation and preparation method thereof. Activity studies show that the compound has a significant inhibitory effect on colon cancer cells and liver cancer cells. Therefore, the compound provided by the present invention has an application prospect of being developed into a drug for treating colon cancer or liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 For compound 1 1 HNMR spectrum;

[0024] Figure 2 , Figure 3is the DEPT spectrum of compound 1;

[0025] Figure 4 is the HSQC spectrum of compound 1;

[0026] Figure 5 is the HMBC spectrum of compound 1;

[0027] Figure 6 It is a bar graph showing the inhibition rate of compound 1 at different concentrations on gastric adenocarcinoma BGC823 cells;

[0028] Figure 7 It is a bar graph showing the inhibition rate of compound 1 at different concentrations on osteosarcoma 143B cells;

[0029] Figure 8 It is a bar graph of the inhibition rate of different concentrations of compound 1 on breast cancer MDA-MB-231 cells;

[0030] Fig. 9 It is a bar graph showing the inhibition rate of compound 1 at different concentrations on colon cancer SW620 cells;

[0031] Fig.10 It is a bar graph showing the inhibition rate of compound 1 at different concentrations on liver cancer HegG2 cells. DETAILED DESCRIPTION

[0032] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0033] Example 1: Isolation, preparation and structural confirmation of compounds

[0034] The aerial parts of Acanthopanax gracilistylus WWSmith were collected, naturally air-dried, crushed, 10 kg was taken, 200 kg of 80% volume fraction ethanol aqueous solution was added, and the extraction was performed under 85°C hot reflux for 3 times, each time for 1.5 hours, the 3 extracts were combined, and the extracts were concentrated under reduced pressure to obtain 10 L of fluid extract, which was extracted 5 times with equal volumes of petroleum ether, ethyl acetate, and n-butanol in sequence, and the extracts of different organic solvents were collected, and concentrated under reduced pressure to extracts to obtain the extraction parts of different organic solvents.

[0035] 480 g of extract from the n-butanol extraction part was loaded onto a 100-200 mesh silica gel chromatographic column, and gradient eluted with a chloroform-methanol mixed solvent with a volume ratio of 15:1, 8:1, and 4:1 for 4, 4, and 3 column volumes, respectively, and concentrated under reduced pressure to obtain an extract;

[0036] Take 3.5 g of the eluted portion of chloroform-methanol 8:1, load it on a Sephadex LH-20 gel chromatography column, elute with methanol for 2 column volumes, collect 30 equal volumes of fractions per column volume, collect fractions 37-45, and repeat this process 3 times; separate and purify the eluted product of the gel column chromatography by preparative high performance liquid chromatography [chromatographic column: Thermo BDS HYPERSLL-C 18 (250 mm×10 mm, 5 μm); mobile phase: acetonitrile-0.5% formic acid aqueous solution (37:63); 254 nm], the eluate corresponding to the chromatographic peak with a retention time of 12.59 min was collected, concentrated and dried to obtain compound 1.

[0037] After analyzing the physical and chemical properties, nuclear magnetic resonance spectrum, mass spectrum and other data of the compound, it was determined that the compound was 5-O-β-D-(6′-caffeoyl)glucosyl-6-methyl-2-pyrone, and the chemical structure was as follows. After searching professional databases such as Scifinder, compound 1 was identified as a new compound with a purity of ≥95%.

[0038]

[0039] Compound 1 is a white powder (methanol) and is easily soluble in methanol, ethanol and aqueous alcohol solutions. Thin layer chromatography identified the compound as having dark spots at 254 nm and showing reddish purple spots after heating 10% sulfuric acid ethanol solution. HR-ESI / MS m / z: 449.1161[MH] - (C 21 H 21 O 11 ), indicating that the molecular formula of the compound is C 21 H 22 O 11 . 1 In the HNMR spectrum, δ6.39 (1H, d, J = 5.6 Hz, H-3) and 8.00 (1H, d, J = 5.6 Hz, H-4) are a group of cis-substituted olefin H signals, δ2.29 (3H, br s, H-6) is the olefinic methyl H signal, δ4.76 (1H, d, J=7.6 Hz, H-1′) is the sugar terminal proton signal, 7.04 (1H, d, J=2.1 Hz, H-2″), 6.76 (1H, d, J=8.0 Hz, H-5″) and 6.99 (1H, dd, J=2.1, 8.0 Hz, H-6″) are the ABX coupled proton signals of trisubstituted benzene ring, 7.43 (1H, d, J=15.8 Hz, H-7″) and 6.22 (1H, d, J=15.8 Hz, H-8″) are the trans olefinic hydrogen signals. 13There are 21 C signals in the C NMR spectrum. After HSQC and literature data comparison, it is easy to determine that δ103.4 (C-1′), 73.9 (C-2′), 76.1 (C-3′), 70.0 (C-4′), 74.2 (C-5′), and 63.0 (C-6′) are glucose carbon signals, δ125.4 (C-1″), 114.8 (C-2″), 145.6 (C-3″), 148.5 (C-4″), 115.8 (C-5″), 121.4 (C-6″), 145.3 (C-7″), 113.7 (C-8″), and 166.4 (C-9″) are caffeoyl carbon signals, and the remaining C signals δ174.1 (C-2), 116.1 (C-3), 155.7 (C-4), 141.6 (C-5 5), 161.4 (C-6) and 15.0 (C-7) can be inferred to be methylpyrone structural fragments. In the HMBC data, δ6.39 (H-3) is correlated with 155.7 (C-4) and 141.6 (C-5), δ8.00 (H-4) is correlated with 174.1 (C-2), 116.1 (C-3) and 161.4 (C-6), and δ2.29 (H-6) is correlated with 141.6 (C-5) and 161.4 (C-6), which further clarifies that the structure contains 6-methyl-2-pyrone structural fragments. The sugar end group Hδ4.76 is correlated with 141.6 (C-5), indicating that the sugar substitution is located at the C-5 position of pyrone, and δ4.35 (H-6′) is correlated with 166.4 (C-9″), indicating that the glucose C-6′ position is substituted with caffeoyl. In summary, the structure of compound 1 was inferred to be 5-O-β-D-(6′-caffeoyl)glucosyl-6-methyl-2-pyrone. 1 H NMR spectrum Figure 1 As shown, the DEPT spectrum is as follows Figure 2 , 3 As shown, the HSQC spectrum is Figure 4 As shown, the HMBC spectrum is Figure 5 shown. 1 H. 13 C NMR data are assigned in the following table.

[0040] Pos. <![CDATA[ 13 CNMR]]> DEPT <![CDATA[ 1 H NMR]]> HMBC C-2 174.18 C C-3 116.18 CH 6.39(d,5.6) 141.4,155.7 C-4 155.71 CH 8.00(d,5.6) 174.1,161.4,116.1 C-5 141.66 C C-6 161.44 C C-7 15.05 <![CDATA[CH 3 ]]> 2.29 141.6,161.4 C-1′ 103.44 CH 4.76(d,7.6) 141.6 C-2′ 73.89 CH 3.18(m) 76.13,103.42 C-3′ 76.13 CH 3.23(m) 70.03,73.89 C-4′ 70.03 CH 3.16(m) 62.99,74.36 C-5′ 74.21 CH 3.42(m) 70.03,63.21,103.44 C-6′ 63.09 <![CDATA[CH 2 ]]> 4.35(d,11.5),4.22(dd,6.9,11.5,) 166.4 C-1″ 125.46 C C-2″ 114.87 CH 7.04(d,2.1) 121.42,145.30,148.54 C-3″ 145.65 C C-4″ 148.54 C C-5″ 115.84 CH 6.76(d,8.0) 125.46,145.65 C-6″ 121.42 CH 6.99(dd,2.1,8.0) 114.87,148.54,145.30 C-7″ 145.3 CH 7.43(d,15.8) 114.87,121.42,166.40 C-8″ 113.71 CH 6.22(d,15.8) 125.46 C-9″ 166.4 C

[0041] Example 2: Activity of Compounds

[0042] 1. Experimental Materials

[0043] 1. Instrument

[0044]

[0045]

[0046] 2. Reagents

[0047]

[0048] 3. Cells

[0049] Human breast cancer cells MDA-MB-231, human gastric adenocarcinoma cells BGC823, human liver cancer cells HepG2, human osteosarcoma cells 143B, and human colon cancer cells SW620 were all from Jiangsu KeyGene Biotechnology Co., Ltd.

[0050] 2. Experimental Methods

[0051] 1. Cell culture and passaging

[0052] Each tumor cell line was taken out of the liquid nitrogen tank and quickly thawed in a 37°C water bath. The cell suspension was transferred to a 15 mL centrifuge tube, supplemented with 4 mL of complete medium, centrifuged at 700 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in 5 mL of complete medium. The cell suspension was pipetted into a T25 culture flask and placed at 37°C and 5% CO. 2 cultured in a cell culture incubator.

[0053] When the cell density in the culture flask reaches 90%, the cells are subcultured. The original culture medium is discarded, the cells are washed twice with PBS, and 1 mL of 0.25% trypsin is added for digestion. The cells are observed under a microscope. MDA-MB-231, BGC823, HepG2, 143B, and SW620 cells are cultured at 37°C and 5% CO 2 Under the conditions, the cells were subcultured in DMEM high-glucose medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 mg / L streptomycin.

[0054] 2. MTT method to determine cell inhibition rate

[0055] Take cells in the logarithmic growth phase and prepare a single cell suspension with complete culture medium at a concentration of 1×10 4 200 μL of cells were inoculated into 96-well plates for MTT experiments. After starvation overnight, the original culture medium was discarded, and the experimental group was replaced with 200 μL of complete culture medium (containing 0.1% DMSO) with a final drug concentration of 1, 2.5, 5, 10, 20, 40, 60, 80, and 100 μM, and the control group (Con) was replaced with 200 μL of complete culture medium containing 0.1% DMSO, and culture was continued for 24 hours. After the culture was completed, 10 μL of MTT solution was added to each well according to the instructions of the kit, and incubated at 37°C for 4 hours, the culture was terminated, and the culture supernatant in the well was aspirated and discarded. 100 μL of DMSO was added to each well and shaken at room temperature for 10 minutes. The wavelengths of 490 nm and 630 nm (reference) were selected, and the absorbance (OD) of each well was measured on an enzyme-linked immunosorbent monitor, the results were recorded, and the cell inhibition rate was calculated according to the following formula.

[0056]

[0057] Among them, OD 490t OD 630t are the absorbance values ​​of the experimental groups at 490 and 630 nm, OD 490c OD 630c They are the absorbance values ​​of the control group at 490 and 630 nm, respectively.

[0058] A bar graph was drawn with drug concentration as the horizontal axis and cell inhibition rate as the vertical axis.

[0059] 3. Data processing and statistical analysis

[0060] The results are expressed as mean ± standard deviation (SD). One-way ANOVA was performed using GraphPad Prism 8 for statistical comparison among groups.

[0061] 3. Experimental Results

[0062] The cell inhibition rates of different tumor cells after treatment with different concentrations of compound 1 are shown in Tables 1 and 2. Figures 6 to 10 As shown (the data are expressed as mean ± SD of at least six independent experiments; **p<0.01, ***p<0.001 vs. control group). IC of compound 1 against different tumor cells 50 The values ​​are also listed in Tables 1 and 2.

[0063] Table 1

[0064]

[0065] Table 2

[0066]

[0067] It is generally believed that the IC value of synthetic compounds or pure plant extracts 50 When the concentration of the compound 1 is less than 10 μg / mL, the sample is judged to have a killing effect on tumor cells in vitro. After conversion, the IC 50 When the concentration is less than 22.20 μM, it is judged that it has a killing effect on the tumor cells in vitro. Therefore, the above experimental results show that the compound 1 of the present invention has a killing and inhibitory effect on colon cancer cells SW620 and liver cancer cells HepG2.

[0068] In summary, the present invention separates a novel compound from Acanthopanax gracilis; activity studies show that the compound has a significant inhibitory effect on colon cancer cells and liver cancer cells. Therefore, the compound provided by the present invention has an application prospect of being developed into a drug for treating colon cancer or liver cancer.

[0069] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. A compound represented by formula I or a pharmaceutically acceptable salt or solvate thereof; 2. A method for preparing a compound represented by formula I, characterized in that: The steps include: Step S1: collecting the aerial parts of Acanthopanax gracilis, drying, crushing, taking an appropriate amount, adding an appropriate amount of ethanol aqueous solution, extracting under hot reflux, concentrating the extract under reduced pressure to obtain a fluid extract, extracting with petroleum ether, ethyl acetate, and n-butanol in sequence, collecting extracts with different organic solvents, and concentrating them under reduced pressure to obtain extracts, respectively, to obtain extraction parts with different organic solvents; Step S2: taking an appropriate amount of extract from the n-butanol extraction part, loading it onto a silica gel chromatography column, gradient eluting it with a chloroform-methanol mixed solvent with a volume ratio of 15:1, 8:1, and 4:1 for 4, 4, and 3 column volumes, respectively, and concentrating under reduced pressure to obtain an extract; Step S3: Take an appropriate amount of the elution portion with chloroform-methanol 8:1, load it onto a Sephadex LH-20 gel chromatography column, elute with methanol for 2 column volumes, collect 30 equal volumes of fractions per column volume, and collect fractions No. 37-45; Step S4: Separate and purify the gel column chromatography elution product by preparative high performance liquid chromatography, the chromatographic column is ThermoBDS HYPERSLL-C 18 The chromatographic column specifications were 250 mm × 10 mm, 5 μm, the mobile phase was acetonitrile-0.5% formic acid aqueous solution in a volume ratio of 37:63, and the eluent corresponding to the chromatographic peak at a retention time of 12.59 min was collected and concentrated and dried.

3. The preparation method according to claim 1, characterized in that: The ethanol aqueous solution used for extraction in step S1 is an ethanol aqueous solution with a volume fraction of 80%.

4. The preparation method according to claim 1, characterized in that: In step S1, 85° C. hot reflux extraction is adopted.

5. The preparation method according to claim 4, characterized in that: The extraction was performed under hot reflux for 3 times, each time for 1.5 hours.

6. The preparation method according to claim 1, characterized in that: In step S1, petroleum ether, ethyl acetate and n-butanol were used in sequence for extraction 5 times with equal volumes.

7. The preparation method according to claim 1, characterized in that: The silica gel in step S2 is 100-200 meshes.

8. The preparation method according to claim 1, characterized in that: In step S3, the Sephadex LH-20 gel chromatography column was used for purification three times.

9. The preparation method according to claim 1, characterized in that: The detection wavelength of the preparative high performance liquid chromatography separation and purification in step S4 is 254 nm.

10. Use of the compound according to claim 1 or its pharmaceutically acceptable salt or solvate for preparing a drug for treating colon cancer or liver cancer.

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