Isopentenyl flavonoid compound as well as preparation method and application thereof

Abstract

The invention discloses an isopentenyl flavonoid compound as well as a preparation method and application thereof, belongs to the technical field of natural medicinal chemistry, and particularly relates to five isopentenyl flavonoid compounds, namely daphne D, (3 ''R)-daphne F, (3'' S)-daphne F, daphne E and daphne H, which are separated from daphne giraldii Nitsche which is a daphne giraldii plant and are subjected to silica gel column chromatography, macroporous resin, ODS column chromatography and HPLC (High Performance Liquid Chromatography), and the inhibitory activity of the five isopentenyl flavonoid compounds on HepG2 and Hep3B cells is tested. Finally, the compound plays an anti-tumor role by inhibiting the growth activity of tumor cells. The preparation method disclosed by the invention is simple and good in reproducibility, and the obtained compound is high in purity and has obvious anti-tumor activity. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of natural medicine chemistry, and particularly relates to five new prenylated flavonoid compounds isolated from the root bark and stem bark of Daphne giraldii Nitsche, their preparation methods, and their uses in anti-tumor activities. Background Art

[0002] Daphne giraldii Nitsche is a plant of the genus Daphne in the family Thymelaeaceae. Its root bark and stem bark are used as medicine and are one of the important source plants of Zushima. It is mainly used to treat symptoms such as traumatic injuries, rheumatism, and rheumatoid arthritis, and has significant curative effects. It is a commonly used traditional Chinese medicine in folk. The root bark and stem bark of Daphne giraldii Nitsche are rich in chemical components, including coumarins, flavonoids, diterpenoids, etc., and thus have various pharmacological activities. Summary of the Invention

[0003] The purpose of the present invention is to provide a series of prenylated flavonoid compounds isolated from the root bark and stem bark of Daphne giraldii Nitsche, their preparation methods, and their uses in the preparation of anti-tumor drugs.

[0004] In the first aspect, the structures of the prenylated flavonoid compounds of the present invention are as follows:

[0005]

[0006] In the second aspect, the preparation method of the prenylated flavonoid compounds of the present invention includes the following steps:

[0007] The dried root bark and stem bark of Daphne giraldii Nitsche are extracted with ethanol, the extract is concentrated under reduced pressure, and the combined extract is concentrated to obtain an extract. The extract is successively extracted with ethyl acetate and n-butanol. The ethyl acetate extract is separated by various chromatographic methods such as silica gel column chromatography, HP-20 macroporous resin, ODS, and HPLC, and finally the above 5 new compounds are obtained.

[0008] The specific operations are as follows:

[0009] (1) Take the dried root bark and stem bark of Daphne giraldii Nitsche and extract them with ethanol. The combined extract is concentrated to obtain an extract. The obtained extract is successively extracted with ethyl acetate and n-butanol. The ethyl acetate part is separated by silica gel column chromatography, and a total of 4 fractions, Fr.A–Fr.D, are collected;

[0010] (2) Fraction Fr.A is separated by HP-20 and ODS column chromatography and eluted with an ethanol-water gradient. A total of 7 fractions, Fr.A1–Fr.A7, are collected;

[0011] (3) Fraction Fr.A6 obtained after elution is separated by semi-preparative HPLC and further purified through an acetonitrile-water system to obtain the above 5 prenylated flavonoid compounds.

[0012] Among them:

[0013] In step (1), the gradient of dichloromethane - methanol used in silica gel column chromatography is: 100:1 - 1:1; the ethanol is 70% industrial ethanol, and reflux extraction is carried out 3 times, 2 hours each time.

[0014] In step (2), the gradient of ethanol - water is: 20% - 90%.

[0015] Structural analysis of the new prenylflavonoid compounds (daphengiratriprenylone D, (3"'R)-daphnegiravone F, (3"'S)-daphnegiravone F, daphnegiravone E, daphnegiralin H).

[0016] Compound 1: Yellow amorphous powder (methanol). HRESIMS gives the quasi - molecular ion peak [M + H] + m / z 521.2567 (calcd for C 31 H 37 O7, 521.2573), combined with 1 1H - NMR, 13 13C - NMR spectra, the molecular formula of this compound can be determined as C 31 H 36 O7, the calculated degree of unsaturation is 14, and the 1 1H - NMR, 13 13C - NMR signals were assigned, and at the same time, the planar structure was determined by HMBC spectrum. Finally, this compound was determined to be a prenylflavonoid structure. And through Scifinder search, it was found that this compound is a new compound not reported before, and it was named daphengiratriprenylone D. Its 1 1H NMR, 13 13C NMR signal assignments are shown in Table 1 and Table 2 respectively, and the relevant spectra are shown in Figures 1 - 6 .

[0017] Compound 2a / 2b: Yellow amorphous powder (methanol); HRESIMS gives the quasi - molecular ion peak [M + H] + m / z 439.1762 (calcd for C 25 H 27 O7, 439.1751), combined with 1 1H - NMR, 13 13C - NMR spectra, the molecular formula of this compound can be determined as C 25 H 26O7, the calculated degree of unsaturation is 13, and for 1 H-NMR, 13 the signals of 13C-NMR were assigned. Meanwhile, the planar structure was determined by HMBC spectrum. Finally, the planar structure of this compound was determined as isopentenyl flavonoid structure. Through Scifinder search, it was found that this compound is a new compound not reported before, and it was named (3"'R)-daphnegiravone F and (3"'S)-daphnegiravone F respectively. Its 1 1H NMR, 13 the signal assignments of 13C NMR are shown in Table 1 and Table 2 respectively, and the relevant spectra are shown in Figures 7 - 12 .

[0018] Compound 3: Yellow amorphous powder (methanol); HRESIMS gave the quasi-molecular ion peak [M + H]+ + m / z 435.1817 (calcd for C 26 25H 27 O6, 435.1802). Combining 1 1H-NMR, 13 13C-NMR and HSQC NMR, the molecular formula of this compound can be determined as C 25 25H 28 O3, the calculated degree of unsaturation is 14, and for 1 1H-NMR, 13 the signals of 13C-NMR were assigned. Meanwhile, the planar structure was determined by HMBC spectrum. Finally, the planar structure of this compound was determined as isopentenyl flavonoid structure. Through Scifinder search, it was found that this compound is a new compound not reported before, and it was named daphnegiravone E. Its 1 1H NMR, 13 the signal assignments of 13C NMR are shown in Table 1 and Table 2 respectively, and the relevant spectra are shown in Figures 13 - 18 .

[0019] Compound 4: Brown amorphous powder (methanol). HRESIMS gave the quasi-molecular ion peak [M–H2O + Na]+ + m / z 447.1814 (calcd for C 25 25H 28 O6Na, 447.1814). Combining 1 1H-NMR, 13 13C-NMR spectra, the molecular formula of this compound can be determined as C 25 25H 30 O7, the calculated degree of unsaturation is 11, and for 1 1H-NMR, 13The 13C-NMR signals were assigned, and the planar structure was determined by the HMBC spectrum. Finally, the compound was identified as a prenylated flavonoid structure. Through a search in Scifinder, it was found that this compound is a new compound that has not been reported before, and it was named daphnegiralin H. Its 1 1H NMR, 13 The signal assignments of the 13C NMR spectra are shown in Table 1 and Table 2 respectively. The relevant spectra are shown in Figures 19 - 24 .

[0020] Table 1 1H NMR (600 MHz) spectral data (δ in ppm) of 1, 2a / 2b, 3, 4 1

[0021]

[0022] Table 2 13C NMR (150 MHz) spectral data (δ in ppm) of 1, 2a / 2b, 3, 4 13

[0023]

[0024]

[0025] In the third aspect, the present invention provides a pharmaceutical composition comprising the prenylated flavonoid compound described in the first aspect above and a pharmaceutically acceptable carrier or excipient.

[0026] In the fourth aspect, the present invention provides the use of the prenylated flavonoid compound described in the first aspect above or the pharmaceutical composition described in the third aspect above in the preparation of an anti-tumor drug.

[0027] Preferably, in the said use, the tumor is liver cancer.

[0028] Preferably, the prenylated flavonoid compound or the pharmaceutical composition achieves the anti-tumor purpose by inhibiting the growth of tumor cells.

[0029] In the fifth aspect, the present invention provides an extract of the cortex and stem bark of Daphne giraldii Nitsche, which contains the prenylated flavonoid compound and is applied in the preparation of an anti-tumor drug.

[0030] Advantages of the present invention:

[0031] The compound involved in the present application has a better inhibitory effect on tumor cells compared with the positive drug sorafenib, and can be prepared from the traditional Chinese medicine Daphne giraldii Nitsche. This application enriches the chemical research of Daphne giraldii Nitsche, improves the pharmacological research of Daphne giraldii Nitsche, and provides a research basis for subsequent structure modification work targeting activity. ​​Brief Description of the Drawings

[0032] Figure 1 UV spectrum of Compound 1;

[0033] Figure 2 HRESIMS spectrum of Compound 1;

[0034] Figure 3 of Compound 1 1 1H NMR spectrum (DMSO-d6, 600 MHz);

[0035] Figure 4 of Compound 1 13 13C NMR spectrum (DMSO-d6, 150 MHz);

[0036] Figure 5 HSQC spectrum of Compound 1 (DMSO-d6, 600 MHz);

[0037] Figure 6 HMBC spectrum of Compound 1 (DMSO-d6, 600 MHz);

[0038] Figure 7 UV spectrum of Compound 2a / 2b;

[0039] Figure 8 HRESIMS spectrum of Compound 2a / 2b;

[0040] Figure 9 of Compound 2a / 2b 1 1H NMR spectrum (DMSO-d6, 600 MHz);

[0041] Figure 10 of Compound 2a / 2b 13 13C NMR spectrum (DMSO-d6, 150 MHz);

[0042] Figure 11 HSQC spectrum of Compound 2a / 2b (DMSO-d6, 600 MHz);

[0043] Figure 12 HMBC spectrum of Compound 2a / 2b (DMSO-d6, 600 MHz);

[0044] Figure 13 UV spectrum of Compound 3;

[0045] Figure 14 HRESIMS spectrum of Compound 3;

[0046] Figure 15 of Compound 3 11H NMR spectrum (DMSO-d6, 600 MHz);

[0047] Figure 16 For compound 3 13 13C NMR spectrum (DMSO-d6, 150 MHz);

[0048] Figure 17 HSQC spectrum of compound 3 (DMSO-d6, 600 MHz);

[0049] Figure 18 HMBC spectrum of compound 3 (DMSO-d6, 600 MHz);

[0050] Figure 19 UV spectrum of compound 4;

[0051] Figure 20 HRESIMS spectrum of compound 4;

[0052] Figure 21 For compound 4 1 1H NMR spectrum (DMSO-d6, 600 MHz);

[0053] Figure 22 For compound 4 13 13C NMR spectrum (DMSO-d6, 150 MHz);

[0054] Figure 23 HSQC spectrum of compound 4 (DMSO-d6, 600 MHz);

[0055] Figure 24 HMBC spectrum of compound 4 (DMSO-d6, 600 MHz). Detailed implementation mode

[0056] Example 1

[0057] Preparation of isopentenyl flavonoid compounds 1, 2a / 2b, 3, 4

[0058] (1) Take the dried root bark and stem bark of Daphne giraldii Nitsche (100 kg), grind them into fragments, extract with 70% industrial ethanol by heating under reflux for 3 times, 3 hours each time. Filter the crude extract and concentrate it under reduced pressure to obtain an extract. Add water to the obtained extract and extract it successively with ethyl acetate and n-butanol. Subject the ethyl acetate layer (1200 g) to silica gel column chromatography separation (200 - 300 mesh), elute with dichloromethane - methanol (100:1 - 1:1) gradient to obtain fractions Fr.A - Fr.D.

[0059] (2) The fraction Fr.A was eluted with ethanol-water (20%-90%) on HP-20 macroporous resin, and then subjected to ODS column chromatography and eluted with the same ethanol-water gradient. After analysis, 7 fractions Fr.A1-Fr.A7 were obtained.

[0060] (4) The fraction Fr.A6 obtained after elution was further purified by semi-preparative HPLC using an acetonitrile-water system, and isopentenyl flavonoids daphengiratriprenylone D (4.2 mg), (3"'R)-daphnegiravone F (1.3 mg), (3"'S)-daphnegiravone F (0.8 mg), daphnegiravone E (14.2 mg), and daphnegiralin H (5.9 mg) were obtained.

[0061] Experimental Example 2

[0062] The inhibitory effects of compounds daphengiratriprenylone D, daphnegiravone F, daphnegiravone E, and daphnegiralin H on HepG2 and Hep3B cells were determined by in vitro experiments to further explore their anti-tumor activities.

[0063] (1) Cell culture

[0064] Human hepatoma cells HepG2 and Hep3B were placed in DMEM medium containing 10% fetal bovine serum and 1% double antibody and cultured in an incubator at 37°C and 5% CO2. Cells in stable passage and logarithmic growth were used for the experiment.

[0065] (2) Cell grouping

[0066] Blank group: Cultured with DMEM complete culture medium without any drugs.

[0067] Compound 1 group: After HepG2 and Hep3B cells were cultured in DMEM complete culture medium, different concentrations (5 μM, 12.5 μM, 25 μM, 50 μM) of compound daphengiratriprenylone D were added and cultured for 48 hours, and then 20 μL of MTT was added to each well and cultured for another 4 hours.

[0068] Compound 2 group: After HepG2 and Hep3B cells were cultured in DMEM complete culture medium, different concentrations (5 μM, 12.5 μM, 25 μM, 50 μM) of compound daphnegiravone F were added and cultured for 48 hours, and then 20 μL of MTT was added and cultured for another 4 hours.

[0069] Compound 3 group: After HepG2 and Hep3B cells were cultured in complete DMEM medium, different concentrations (5 μM, 12.5 μM, 25 μM, 50 μM) of compound daphnegiravone E were added and cultured for 48 hours, then 20 μL of MTT was added and the culture was continued for 4 hours.

[0070] Compound 4 group: After HepG2 and Hep3B cells were cultured in complete DMEM medium, different concentrations (5 μM, 12.5 μM, 25 μM, 50 μM) of compound daphnegiralin H were added and cultured for 48 hours, then 20 μL of MTT was added and the culture was continued for 4 hours.

[0071] Positive drug group: After HepG2 and Hep3B cells were cultured in complete DMEM medium, different concentrations (5 μM, 12.5 μM, 25 μM, 50 μM) of the positive drug sorafenib were added and cultured for 48 hours, then 20 μL of MTT was added and the culture was continued for 4 hours.

[0072] Add 150 μL / well of DMSO, shake on a thermostatic shaker for 10 min, and measure the absorbance value of each well at 490 nm using an enzyme-linked immunosorbent assay instrument (Thermo Scientific Multiskan MK3, Shanghai, China).

[0073] Cell survival rate (%) = [A (给药组) -A (空白对照) / [A (阴性对照) -A (空白对照) ×100%

[0074] (3) Experimental results

[0075] Data on the inhibitory effects of compounds 1, 2, 3, and 4 on tumor cells (IC 50 ) are shown in Table 3 below, with the unit of μM.

[0076] Table 3

[0077]

[0078] The results are expressed as the mean ± standard deviation of IC 50 , with the unit of μM. The experiment was repeated three times.

[0079] The experimental results showed that compound 2 had the best inhibitory activity against HepG2 and Hep3B cells, and its IC 50 values were IC 50 = 11.63 ± 0.22 μM and IC 50 = 0.10 ± 0.02 μM.

[0080] Obviously, the above embodiments are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. Those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and its equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. An isopentenyl flavonoid compound, characterized in that: It is any one of the compounds shown below; 2. The prenylated flavonoid compound according to claim 1, characterized in that: The isopentenyl flavonoid compound is extracted and separated from the dried root bark and stem bark of Daphne koreana.

3. The method for preparing the isopentenyl flavonoid compound according to claim 1 or claim 2, characterized in that: The preparation method comprises the following steps: (1) The dried root bark and stem bark of Daphne koreana were extracted with ethanol, and the combined extracts were concentrated to obtain an extract. The obtained extract was extracted with ethyl acetate and n-butanol in turn. The ethyl acetate fraction was subjected to silica gel column chromatography to collect a total of 4 fractions Fr.A-Fr.D; (2) Fraction Fr.A was subjected to HP-20 column chromatography and then to ODS column chromatography with ethanol-water gradient elution, and a total of 7 fractions Fr.A1–Fr.A7 were collected; (3) The fraction Fr.A6 obtained after elution was subjected to semi-preparative HPLC and further purified by acetonitrile-water system to obtain the five compounds described in claim 1.

4. The method for preparing the isopentenyl flavonoid compound according to claim 3, characterized in that: In the step (1), the gradient of dichloromethane-methanol used in silica gel column chromatography is: 100:1-1:1; the ethanol is 70% industrial ethanol, and reflux extraction is performed 3 times, each time for 2 hours.

5. The method for preparing the isopentenyl flavonoid compound according to claim 3, characterized in that: In the step (2), the gradient of ethanol-water is 20%-90%.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the prenylated flavonoid compound according to claim 1 or claim 2 and a pharmaceutically acceptable carrier or excipient.

7. Use of the prenylated flavonoid compound according to claim 1 or claim 2 or the pharmaceutical composition according to claim 6 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that The tumor is liver cancer.

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