CN115611957A - Stilbene or flavonoid compound and preparation method and application thereof - Google Patents

Stilbene or flavonoid compound and preparation method and application thereof Download PDF

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CN115611957A
CN115611957A CN202211204346.4A CN202211204346A CN115611957A CN 115611957 A CN115611957 A CN 115611957A CN 202211204346 A CN202211204346 A CN 202211204346A CN 115611957 A CN115611957 A CN 115611957A
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李宁
陈刚
周地
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Shenyang Pharmaceutical University
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Abstract

The invention relates to a stilbene or flavonoid compound and a preparation method and application thereof, relating to the technical field of medicines. Stilbene or flavonoid compounds with the following structure and pharmaceutically acceptable salts thereof,

Description

Stilbene or flavonoid compound and preparation method and application thereof
Technical Field
The invention relates to a stilbene or flavonoid compound, a preparation method and application thereof, and relates to the technical field of medicines.
Background
Millettia pachyrhizus (Millettia pachycarpa Benth) is mainly distributed in southwest to Taiwan province of China, and is most prevalent in southwest. It is hot in nature and bitter in flavor, has the effects of counteracting toxic substances, relieving pain, killing parasites, and is mainly used for treating infantile malnutrition, abdominal pain, scabies, tinea, scabies, etc. At present, few chemical components and pharmacological activity research reports of the Millettia dielsiana are reported, modern pharmacological research shows that the Millettia dielsiana has an anti-inflammatory effect, and the chemical components mainly comprise stilbenes, flavones, lignans, triterpenes, phenylpropanoids and the like.
Disclosure of Invention
The invention aims to provide a series of stilbene and flavonoid compounds, a preparation method and a new medical application thereof.
The invention provides the following 2 specific compounds:
stilbene or flavonoid compounds with the following structure and pharmaceutically acceptable salts thereof,
Figure BDA0003873011230000011
a preparation method of stilbene or flavonoid compounds comprises the following steps:
heating and extracting root of Millettia pachyrhizus (Millettia pachycarpa Benth) to obtain crude extract; dissolving the crude extract with water, and extracting with organic solvent to obtain extracts with different polarities; separating the obtained extract by silica gel column chromatography, and gradient eluting with mixed solvent; separating the obtained flow by ODS column chromatography, and gradient eluting with methanol-water or acetonitrile-water mixed solvent as mobile phase; and further separating the obtained methanol-water or acetonitrile-water eluate by HPLC, and performing gradient elution by using a mixed solvent of methanol and water or a mixed solvent of acetonitrile and water as a mobile phase to obtain a compound 1 and a compound 2.
Preferably, the root of akebia trifoliata koidz is subjected to heating reflux ethanol extraction, heating reflux methanol extraction or heating ultrasonic extraction for 2-5 times to obtain a crude extract, wherein the volume concentration of ethanol is 70-95%, the volume concentration of methanol is 60-90%, and the ratio of material to liquid is 1.
Preferably, the obtained crude extract is dissolved by water, petroleum ether or cyclohexane, dichloromethane or chloroform, ethyl acetate and n-butyl alcohol are respectively used for sequentially extracting for 3-5 times according to the volume ratio of the water phase to the organic phase being 1.
Preferably, the obtained extract is separated by silica gel column chromatography, and eluted with a mixed solvent of petroleum ether and ethyl acetate, or a mixed solvent of petroleum ether and acetone, or a mixed solvent of chloroform and acetone, or a mixed solvent of dichloromethane and acetone, or a mixed solvent of chloroform and methanol, or a mixed solvent of dichloromethane and methanol in a gradient manner.
Preferably, the volume ratio of the petroleum ether to the ethyl acetate to the mixed solvent of the petroleum ether and the acetone is 100; the volume ratio of dichloromethane to acetone, a mixed solvent of chloroform and acetone, a mixed solvent of dichloromethane and methanol, or a mixed solvent of chloroform and methanol is (100).
Preferably, the obtained flow is subjected to ODS column chromatography separation, and is eluted by taking methanol-water or acetonitrile-water mixed solvent as a mobile phase gradient, wherein the volume ratio of the methanol-water mixed solvent is (2).
Preferably, the obtained methanol-water or acetonitrile-water eluate is further separated by HPLC, and is eluted by using a methanol and water mixed solvent or acetonitrile and water mixed solvent as a mobile phase gradient, wherein the volume ratio of the methanol and water mixed solvent is 2-7.
A pharmaceutical composition comprises stilbene or flavonoid compounds and pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers.
Stilbene or flavonoid compounds with the following structure and pharmaceutically acceptable salts thereof,
Figure BDA0003873011230000021
Figure BDA0003873011230000031
the invention also aims to provide application of the compound and the pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing medicaments for preventing or treating diseases such as lung epithelial injury and the like.
The invention has the beneficial effects that: the invention adopts mouse normal alveolar type II epithelial (MLE-12) cells, takes the cell survival rate as an index, and evaluates the lung epithelial protection effect of the prepared compound 1-2 by an MTT method. The results show that the novel compound 2 can significantly reverse NNK-induced lung cell damage. Therefore, the compound prepared by the invention can be applied to the development of medicaments for treating diseases such as lung epithelial injury and the like. The invention provides a method for preparing and identifying 2 new compounds by taking dry root of Millettia dielsiana as a raw material for the first time, systematically evaluates the activity of the Millettia dielsiana in the aspect of lung epithelium protection, and clarifies the application of the Millettia dielsiana in the aspect of developing and treating medicaments for treating diseases such as lung epithelium damage and the like.
Detailed Description
The following non-limiting examples will allow one of ordinary skill in the art to more fully understand the present invention, but will not limit the invention in any way.
The test methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
One of the specific implementation modes is as follows:
the invention provides a preparation method of the compound 1-2, which comprises the following steps:
(1) Extracting root of Millettia pachyrhizus (Millettia pachycarpa Benth) with 70-95% ethanol or 60-90% methanol under heating and refluxing, and recovering extractive solution to obtain crude extract;
(2) Dissolving the crude extract obtained in the step (1) with water, and sequentially extracting for 3-5 times with petroleum ether or cyclohexane, dichloromethane or chloroform, ethyl acetate and n-butyl alcohol according to the volume ratio of the water phase to the organic phase being 1-1;
(3) Separating the extract obtained in the step (2) by silica gel column chromatography, and eluting by a mixed solvent of petroleum ether and ethyl acetate 100;
(4) Separating the flow obtained in the step (3) by ODS column chromatography, and performing gradient elution by using a mixed solvent of methanol-water 2;
(5) Further separating methanol and water, acetonitrile and water eluates obtained in the step (4) by HPLC, and carrying out gradient elution by using a mixed solvent of methanol and water 2-7 or a mixed solvent of acetonitrile and water 1;
the preparation method of the compound 1-2 provided by the invention comprises the step (1) of heating reflux ethanol extraction, heating reflux methanol extraction or heating ultrasonic extraction for 2-5 times, wherein the used solvent is 70-95% of ethanol or 60-90% of methanol, preferably 75-85% of ethanol or 65-85% of methanol. The feed-liquid ratio is 1.
According to the preparation method of the compound 1-2 provided by the invention, in the organic solvent extraction method in the step (2), the crude extract is dissolved by water, and is sequentially extracted for 3-5 times, preferably 5 times, by using petroleum ether or cyclohexane, dichloromethane or chloroform, ethyl acetate and n-butanol according to the volume ratio of the water phase to the organic phase of 1.
According to the preparation method of the compound 1-2 provided by the invention, the volume ratio of the petroleum ether and the ethyl acetate or the mixed solvent of the petroleum ether and the acetone in the step (3) is 100-1, preferably 100-1; the volume ratio of dichloromethane and acetone, or chloroform and acetone, or dichloromethane and methanol, or the mixed solvent of chloroform and methanol is 100.
According to the preparation method of the compound 1-2 provided by the invention, the volume ratio of the mixed solvent of methanol and water in the step (4) is 2.
According to the preparation method of the compound 1-2 provided by the invention, the volume ratio of the mixed solvent of methanol and water in the step (5) is (2-7).
Example 1
(1) Extracting 1000g of dry root of Millettia speciosa with 80% ethanol under heating and refluxing for 3 times (dosage: 10L), and recovering the extractive solution under reduced pressure to obtain crude extract;
(2) Dissolving the 80% ethanol crude extract obtained in the step (1) with water, sequentially extracting with dichloromethane, ethyl acetate and n-butanol, extracting each organic phase for 3 times, wherein the volume ratio of the water phase to the organic phase is 1;
(3) In the step (2), the n-butanol extract is separated by silica gel column chromatography, and is sequentially eluted by dichloromethane and methanol mixed solution, wherein the ratio of the solvent to the solvent is 100, 10;
(4) Dichloromethane obtained in the above step (3): methanol 8 through 1-3 fraction through ODS chromatography, eluting with a mixed solvent gradient of methanol-water 2;
(5) The methanol-water (2-4: water =33, yielding compound 2 (t) R =15 min) (yield 0.00013 ‰).
(6) The methanol-water (6-8): water =45, yielding compound 1 (t) R =40 min) (yield 0.0035 ‰).
The structures of compounds 1-2 were identified based on their physicochemical properties and spectral data.
The structural identification data for compound 1 is as follows:
pale yellow powder (methanol). HR-ESI-MS gives the excimerIon Peak [ M + H] - m/z：567.2078(calcd. 567.2072for C 27 H 35 O 13 ) Determining the molecular formula as C 27 H 34 O 13 The unsaturation was calculated to be 11.
1 H NMR(600MHz,DMSO-d 6 ) Middle, delta H 9.44(1H,s,5'-OH) Is a characteristic hydrogen signal of phenolic hydroxyl; delta. For the preparation of a coating H 7.24 (1h, d, j =1.7hz, h-2), 7.08 (2h, m, h-5, 6) is a set of ABX coupled aromatic hydrogen signals; delta H 6.60 (1H, t, J =2.2Hz, H-2 '), 6.24 (1H, t, J =2.2Hz, H-4 '), 6.56 (1H, t, J =2.2Hz, H-6 ') is a meta-coupled aromatic hydrogen signal; delta. For the preparation of a coating H 7.06 (1H, m, H-7), 7.01 (1H, d, J =16.3Hz, H-8) is a set of trans double bond hydrogen signals. Delta. For the preparation of a coating H 4.87 (1H, d, J=7.5Hz, H-1') for glucose end group hydrogen signals, delta H 4.81 (1H, d, J =3.1Hz, H-1 "') is the cresyl terminal hydrogen signal. Delta H 3.83(3H,s,3-OCH 3 ),3.73(3H,s,3'-OCH 3 ) Two methoxy hydrogen signals. 13 C NMR(150MHz,DMSO-d 6 ) Giving 27 carbon signals, 14 of which are stilbene parent nuclear carbon signals: delta. For the preparation of a coating C 131.1 (C-1), 110.0 (C-2), 149.0 (C-3), 146.4 (C-4), 115.4 (C-5), 119.9 (C-6), 128.3 (C-7), 127.0 (C-8), 139.2 (C-1 '), 102.6 (C-2'), 160.6 (C-3 '), 100.7 (C-4'), 158.6 (C-5 '), 106.0 (C-6'); 11 glycosyl carbon signals, comprising a set of glucose carbon signals: delta. For the preparation of a coating C 100.1 (C-1 "), 73.2 (C-2"), 76.8 (C-3 "), 70.0 (C-4"), 75.6 (C-5 "), 67.8 (C-6") and a set of cresyl carbon signals: delta C 109.3 (C-1 "'), 75.8 (C-2" '), 78.7 (C-3 "'), 73.3 (C-4" '), 63.1 (C-5 "'); 2 methoxy carbon signals: delta C 55.7 (3-OCH 3 ),55.0(3'-OCH 3 ). From 1D NMR data, in combination with literature [1] The comparison can predict that the compound 1 has a stilbene structure, and the structure comprises 1 furanosyl group and 1 glucopyranosyl group. The hydrocarbon data is further attributed according to relevant information of HSQC spectrum (see Table 1).
In HMBC spectra, delta H 3.83(3-OCH 3 ) And delta C 149.0 (C-3) remote correlation; delta H 3.73(3'-OCH 3 ) And delta C 160.6(C-3') Remote correlation, suggesting that two methoxy groups are attached at the C-3 and C-3' positions, respectively. Delta H 9.44(5'-OH) And delta C 100.7(C-4')、δ C 106.0 (C-6 ') remote association, suggesting that the hydroxyl group is attached at the C-5' position. Delta. For the preparation of a coating H 4.87 (H-1') and delta C 146.4 (C-4) remote correlation, δ H 4.81 (H-1' ") and delta C 67.8 (C-6') remote correlation, the connection position between sugar fragments and the connection position of the sugar chain to the parent nucleus are determined. In the NOESY spectrum, δ H 3.83(3-OCH 3 ) And delta H 7.24 (H-2) related; delta H 3.73(3'-OCH 3 ) And delta H 6.61(H-2')、δ H 6.24 (H-4 ') further demonstrating the substitution of two methoxy groups at the C-3 and C-3' positions, respectively; delta. For the preparation of a coating H 9.44(5'-OH) And delta H 6.24(H-4')、δ H 6.56 (H-6 ') further demonstrates the substitution of the C-5' position by a hydroxy group.
Determining that the relative configuration is beta according to the hydrogen coupling constant of the glucose end group being 7.5 Hz; chemical shift value of 4.81 according to the hydrogen of the end group of the apiose, coupling constant of 3.7Hz and in NOESY spectrum, delta H 3.76 (H-2' ") and delta H 3.89 (H-4' ") and delta H 3.38 (H-5' ") relative, the apiosyl relative configuration was determined to be beta.
In conclusion, the compound is identified as 3,3'-dimethoxy-5' -hydroxystilbene-4-O-beta-D-iodofuranosyl- (1 → 6) -beta-D-glucopyranoside, and the compound is an unreported new compound by Sci-finder search.
TABLE 1 assignment of NMR data for Compound 1
Figure BDA0003873011230000061
The structural identification data for compound 2 is as follows:
white amorphous powder (methanol). HR-ESI-MS gives the peak of the excimer ion [ M + H ]] - m/z：595.1658 (calcd.595.1657for C 27 H 31 O 15 ) Determining the molecular formula as C 27 H 30 O 15 The unsaturation was calculated to be 13.
1 H NMR(600MHz,DMSO-d 6 ) Middle, delta H 12.94(1H,s,5-OH) Is a characteristic hydrogen signal of the 5-hydroxyl on the ring A of the isoflavone; delta. For the preparation of a coating H 8.42 (1H, s, H-2) is the characteristic hydrogen signal of 2-position of isoflavone; delta. For the preparation of a coating H 6.47 (1H, d, J =2.2Hz, H-6), 6.73 (1H, d, J =2.2Hz, H-8) is the coupling hydrogen signal at the meta position on the ring of isoflavone A; delta H 7.16 (1H, d, J =2.1Hz, H-2 '), 6.84 (1H, d, J =8.1Hz, H-5 '), 7.00 (1H, dd, J =8.1,2.1Hz, H-6 ') is the ABX spin coupling system hydrogen signal on the B ring of isoflavone; delta H 5.04 (1H, d, J =7.5Hz, H-1 ") is a glucose end group hydrogen signal; delta H 4.81 (1H, d, J =3.1Hz, H-1') is a hydrogen signal at the end of the apiose group; delta H 3.80(3H,s,3'-OCH 3 ) Is a methoxyhydrogen signal. 13 C NMR(150MHz,DMSO-d 6 ) Giving 27 carbon signals, 15 of which are isoflavone mother nucleus carbon signals: delta C 154.8 (C-2), 122.6 (C-3), 180.5 (C-4), 161.6 (C-5), 99.6 (C-6), 162.9 (C-7), 94.6 (C-8), 157.2 (C-9), 106.1 (C-10), 121.5 (C-1 '), 113.3 (C-2'), 147.3 (C-3 '), 146.8 (C-4'), 115.3 (C-5 '), 121.7 (C-6'); 11 glycosyl carbon signals, comprising a set of glucose carbon signals: delta C 99.8 (C-1 "), 73.0 (C-2"), 76.4 (C-3 "), 69.9 (C-4"), 75.6 (C-5 "), 67.7 (C-6") and a set of apiose carbon signals: delta. For the preparation of a coating C 109.4 (C-1 "'), 75.9 (C-2" '), 78.7 (C-3 "'), 73.3 (C-4" '), 63.2 (C-5 "'); 1 methoxy carbon signal: delta C 55.7 (3'-OCH 3 ). From 1D NMR data, in combination with literature [10] The compound is presumed to have an isoflavone structure, and the structure comprises 1 furanosyl and 1 glucopyranosyl. The hydrocarbon data is further attributed according to relevant information of HSQC spectrum (see Table 2).
In HMBC spectra, delta H 3.80(3'-OCH 3 ) And delta C 147.3 (C-3 ') remote association, suggesting that the methoxy group is at the C-3' position of the B ring. Delta H 5.04 (H-1') and delta C 162.9 (C-7) remote correlation, δ H 4.81 (H-1' ") and δ C 67.7 (C-6') remote correlation, the connection position between sugar fragments and the connection position of the sugar chain and the parent nucleus are determined. In the NOESY spectrum, δ H 3.80 (3'-OCH 3 ) And delta H 7.16 (H-2') further demonstrates methoxyThe substituent is at the C-3' position of the B ring; delta H 9.18(4'-OH) And delta H 6.84 (H-5 ') further demonstrates that the hydroxyl substitution is at the C-4' position of the B ring.
Determining that the relative configuration is beta according to the glucose end group hydrogen coupling constant of 7.5 Hz; chemical shift value of 4.81 according to the hydrogen of the end group of the apiose, coupling constant of 3.1Hz and in NOESY spectrum, delta H 3.76 (H-2' ") and delta H 3.93 (H-4' ") and delta H 3.38 (H-5' ") relative, the apiosyl relative configuration was determined to be beta.
In conclusion, the compound is identified to be 4',5-dihydroxy-3' -methoxyisoflavone-7-O-beta-D-furanosyl- (1 → 6) -beta-D-glucopyranoside [4',5-dihydroxy-3' -methoxyisoflavone-7-O-beta-D-arabinofuranosyl- (1 → 6) -beta-D-glucopyranoside ], and the compound is an unreported new compound after Sci-finder search.
TABLE 1 assignment of NMR data for Compound 2
Figure BDA0003873011230000071
Example 2
(1) Extracting 500g of Millettia speciosa with 95% ethanol under heating and refluxing for 2 times (dosage: 8L), and recovering the extractive solution under reduced pressure to obtain crude extract;
(2) Extracting the ethanol extract obtained in the step (1) by using an organic solvent, sequentially extracting by using petroleum ether, dichloromethane, ethyl acetate and n-butanol according to the volume ratio of a water phase to an organic phase of 1;
(3) Subjecting the n-butanol extract obtained in the step (2) to silica gel column chromatography, and sequentially eluting with a mixed solvent 100 of dichloromethane and acetone, wherein the ratio of the solvent;
(4) Dichloromethane obtained in the above step (3): acetone 10 through 1-3 fraction through ODS chromatography, eluting with a mixed solvent gradient of methanol-water 2;
(5) The methanol-water (2-4The phases are methanol: water =30, to give compound 2 (t) R =20 min) (yield 0.00014 ‰);
(6) The methanol-water (6 R =38 min) (yield 0.0036 ‰).
1-2 see example 1.
Example 3
(1) Extracting 1000g of Millettia speciosa with 70% ethanol under heating and refluxing for 3 times (dosage: 15L), and recovering the extractive solution under reduced pressure to obtain crude extract;
(2) Extracting the ethanol extract obtained in the step (1) by using an organic solvent, sequentially extracting by using cyclohexane, dichloromethane, ethyl acetate and n-butanol according to the volume ratio of a water phase to an organic phase of 1;
(3) Separating the n-butanol extract obtained in the step (2) by silica gel column chromatography, and sequentially eluting with a chloroform and methanol mixed solvent 100, 1, 8;
(4) Chloroform obtained in the step (3): methanol 8;
(5) The methanol-water (2: water =27, yielding compound 2 (t) R =29 min) (yield 0.00012 ‰).
(6) The methanol-water (5 to 8 R =35 min) (yield 0.0035 ‰).
1-2 see example 1.
Example 4
(1) Extracting Millettia speciosa (1500 g) with 60% methanol under reflux for 4 times (20L), and recovering the extractive solution under reduced pressure to obtain crude extract;
(2) Extracting the methanol extract obtained in the step (1) by using an organic solvent, sequentially extracting by using petroleum ether, ethyl acetate and n-butanol according to the volume ratio of a water phase to an organic phase of 1;
(3) Separating the n-butanol extract obtained in the step (2) by silica gel column chromatography, and sequentially eluting with a chloroform-acetone mixed solvent 100, 10;
(4) Chloroform obtained in the step (3): acetone 9 through 1-3 flow through ODS chromatography, eluting with a mixed solvent gradient of acetonitrile-water 1;
(5) The acetonitrile-water (1-3: water =18, yielding compound 2 (t) R =26 min) (yield 0.00014 ‰).
(6) The acetonitrile-water (5 to 7 R =40 min) (yield 0.0035 ‰).
1-2 see example 1.
Example 5
(1) Extracting Millettia speciosa (2000 g) with 80% methanol under reflux for 3 times (20L), and recovering the extractive solution under reduced pressure to obtain crude extract;
(2) Extracting the methanol extract obtained in the step (1) by using an organic solvent, sequentially extracting by using cyclohexane, chloroform and n-butanol according to the volume ratio of a water phase to an organic phase of 1;
(3) The n-butanol extract obtained in the above step (2) is subjected to silica gel column chromatography, and is sequentially eluted with a mixed solvent of dichloromethane and methanol 100;
(4) Dichloromethane obtained in the above step (3): methanol 9 through 1-5 fractions through ODS chromatography, eluting with a mixed solvent gradient of methanol-water 3;
(5) The methanol-water (3: water =35, yielding compound 2 (t) R =12 min) (yield 0.00013 ‰).
(6) The methanol-water (6 R =45 min) (yield 0.0035 ‰).
1-2 see example 1.
Example 6
(1) Extracting 2500g of Millettia speciosa with 90% methanol under heating and refluxing for 3 times (25L), and recovering the extractive solution under reduced pressure to obtain crude extract;
(2) Extracting the methanol extract obtained in the step (1) by using an organic solvent, sequentially extracting by using petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol according to a volume ratio of the water phase to the organic phase of 1;
(3) The n-butanol extract obtained in the above step (2) is subjected to silica gel column chromatography, and sequentially eluted with a mixed solvent of chloroform and methanol 100;
(4) Chloroform obtained in the above step (3): methanol 10 through 1-4 flow through ODS chromatography, eluting with a mixed solvent gradient of acetonitrile-water 2;
(5) The acetonitrile-water (2-3): water =25, yielding compound 2 (t) R =31 min) (yield 0.00013 ‰).
(6) The acetonitrile-water (4 R ＝35min)。
1-2 see example 1.
Example 7 pulmonary epithelial protective activity of the compounds 1-2 prepared in examples 1-6.
(1) The experimental principle is as follows: there are approximately 4000 compounds in smoke, of which 55 are identified as carcinogens by the international cancer research council. The lung cancer-causing factors in tobacco mainly include polycyclic aromatic carbohydrates (PAH) and tobacco-specific nitrosamines (TSNA). Among them, 4- (N-methyl-N-nitrosamine) -1- (3-pyridyl) -1-butanone (NNK) is the most potent carcinogen in TSNA. In-vitro and in-vivo research models for chemoprevention of lung cancer, NNK is mostly adopted as a chemical inducer for inducing lung epithelial cells and tissues to be damaged.
The experiment adopts an NNK-induced MLE-12 (mouse normal alveolar type II epithelial cells) cell model, and evaluates the lung epithelial cell protection effect of the separation and identification compound by taking the cell survival rate as an index.
(2) The experimental method comprises the following steps:
(1) mouse lung epithelial cell culture
DMEM/F-12 medium is used as a base to prepare a cell culture solution containing 10% fetal calf serum. Cells were cultured at a rate of about 2.5X 10 5 Density of cells/mL at 5% CO 2 Culturing in an incubator at 37 ℃ until the third day, wherein the adherent cells account for about 80-90% of the bottom area of the culture bottle, digesting the adherent cells with pancreatin, and passaging to another culture bottle.
(2) Method for preparing medicine
Test compounds were dissolved in DMSO. Stock solution (100 mM) was prepared and stored at-20 ℃. The cells were diluted with DMEM medium immediately before use, and then diluted to 100. Mu.M, 30. Mu.M, 10. Mu.M, and 1. Mu.M, respectively. The final concentration of DMSO is less than 1 ‰.
(3) MTT method for detecting influence of compound on MLE-12 cell survival rate
MLE-12 cells cultured in the logarithmic growth phase were taken and the cell density was adjusted to 2.5X 10 with fresh DMEM/F-12 containing 10% fetal bovine serum 5 cells/mL, seeded in 96-well plates, 100. Mu.L/well at 37 ℃ C. 5% CO 2 The incubator of (2) for cultivation. After the cells are cultured for 12 hours adherent, the cells are changed into fresh culture solution, and meanwhile, the cells are treated by adding medicine. Root of Manyflower MillettiaThe same dose, blank control was also set. The NNK induced cell damage experiment is performed as above, and after the supernatant is aspirated off the second day, 0.5mg/mL of NNK and 3 samples to be tested with different concentration gradients are added. After adding the drug, the cells were cultured for another 48 hours, then MTT solution, 50. Mu.L/well was added to the cell fluid, the cells were incubated with 2mg/mL MTT at 37 ℃ for 4 hours, the culture fluid was aspirated, then 100. Mu.L DMSO solution was added, and the absorbance OD value was measured. The OD values of 3 wells per sample were averaged, and the cell viability (CV%) was calculated from the average value as follows.
Cell survival% = (sample group OD value-blank group OD value)/(control group OD value-blank group OD value) × 100%
(4) Statistical method
All data were examined using GraphPad Prism 8.0 statistical software package. Results are expressed as mean ± standard error, and the global differences were evaluated, mean between groups was analyzed for homogeneity of variance using One-way ANOVA analysis and compared between groups using Dunnett's test analysis. The multiple sample homogeneity of variance test uses the Leven test, when p >0.05, the variance is homogeneous, dunnett's test two-sided T test is used to test the difference of the mean between groups, when p <0.05, the variance is not homogeneous, dunnett T3 is used to test the difference of the mean between groups.
⑤IC 50 Is calculated by a computer
Calculating IC by nonlinear regression fitting of parameters such as each dose and inhibition rate 50 。
(3) The experimental results are as follows:
the results are shown in Table 3.
TABLE 3-2 Effect on NNK-induced MLE-12 cell survival Experimental results
Figure BDA0003873011230000111
Note: * P is<0.05,**P<0.01,***P<0.001 compared to LPS-induced group; ### P<0.001 compared to the control group.
As a result, it was found that both of the novel compounds 1 and 2 prepared in examples 1 to 6 can improve the survival rate of the NNK-induced lung epithelial cells, and that the novel compound 2 can significantly improve the survival rate of the NNK-induced lung epithelial cells.

Claims (10)

1. Stilbene or flavonoid compounds having the following structure and pharmaceutically acceptable salts thereof,
Figure FDA0003873011220000011
2. a process for preparing stilbene or flavonoid compounds as claimed in claim 1, which comprises the steps of: the method comprises the following steps:
heating and extracting roots of millettia speciosa to obtain a crude extract; dissolving the crude extract with water, and extracting with organic solvent to obtain extracts with different polarities; separating the obtained extract by silica gel column chromatography, and gradient eluting with mixed solvent; separating the obtained flow by ODS column chromatography, and gradient eluting with methanol-water or acetonitrile-water mixed solvent as mobile phase; and further separating the obtained methanol-water or acetonitrile-water eluate by HPLC, and performing gradient elution by using a mixed solvent of methanol and water or a mixed solvent of acetonitrile and water as a mobile phase to obtain a compound 1 and a compound 2.
3. The method of claim 1, wherein: heating reflux ethanol extraction, heating reflux methanol extraction or heating ultrasonic extraction of the roots of the akebia fruit Millettia for 2-5 times to obtain a crude extract, wherein the volume concentration of ethanol is 70-95%, the volume concentration of methanol is 60-90%, and the material-liquid ratio is 1.
4. The method of claim 1, wherein: dissolving the obtained crude extract by water, sequentially extracting for 3-5 times by using petroleum ether or cyclohexane, dichloromethane or chloroform, ethyl acetate and n-butyl alcohol according to the volume ratio of the water phase to the organic phase of 1-1.
5. The method of claim 1, wherein: separating the obtained extract by silica gel column chromatography, and gradient eluting with mixed solvent of petroleum ether and ethyl acetate, or mixed solvent of petroleum ether and acetone, or mixed solvent of chloroform and acetone, or mixed solvent of dichloromethane and acetone, or mixed solvent of chloroform and methanol, or mixed solvent of dichloromethane and methanol.
6. The method of claim 5, wherein: the volume ratio of the petroleum ether to the ethyl acetate to the mixed solvent of the petroleum ether and the acetone is 100 to 1; the volume ratio of dichloromethane to acetone, a mixed solvent of chloroform and acetone, a mixed solvent of dichloromethane and methanol, or a mixed solvent of chloroform and methanol is (100).
7. The method of claim 1, wherein: and separating the obtained flow by ODS column chromatography, and carrying out gradient elution by using a methanol-water or acetonitrile-water mixed solvent as a mobile phase, wherein the volume ratio of the methanol-water mixed solvent to the acetonitrile-water mixed solvent is 2-8.
8. The method of claim 1, wherein: and further separating the obtained methanol-water or acetonitrile-water eluate by HPLC, and performing gradient elution by using a mixed solvent of methanol and water or a mixed solvent of acetonitrile and water as a mobile phase, wherein the volume ratio of the mixed solvent of methanol and water is 2-7.
9. A pharmaceutical composition comprising a compound of claim 1 and pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier.
10. The use of a compound of claim 1 and pharmaceutically acceptable salts thereof or a pharmaceutical composition of claim 9 for the manufacture of a medicament for the prevention or treatment of disorders associated with epithelial damage to the lung.
CN202211204346.4A 2022-09-29 2022-09-29 Stilbene or flavonoid compound and preparation method and application thereof Pending CN115611957A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106632390A (en) * 2015-11-03 2017-05-10 沈阳药科大学 Biflavonoids compounds, and preparation method and pharmaceutical applications thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106632390A (en) * 2015-11-03 2017-05-10 沈阳药科大学 Biflavonoids compounds, and preparation method and pharmaceutical applications thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KAENNAKAM, SUTIN, ET AL.: ""Dalvelutinoside, a new isoflavone glycoside from the methanol extract of Dalbergia velutina roots"", 《NATURAL PRODUCT RESEARCH》, vol. 30, no. 13, 31 December 2016 (2016-12-31), pages 1493 - 1498 *
TCHOUMTCHOUA, JOB, ET AL.: ""Estrogenic activity of isoflavonoids from the stem bark of the topical tree Amphimas pterocarpoides, a source of traditional medicines"", 《JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY》, vol. 158, 31 December 2016 (2016-12-31), pages 138 - 148 *

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