Flavonoid compound with tumor cytotoxicity and anti-inflammatory effect, and preparation method and application thereof

Abstract

The application discloses a flavonoid compound with tumor cytotoxicity and anti-inflammatory effect, and a preparation method and application thereof, and belongs to the field of phytochemistry. The application discloses a new compound (2', 4', 5, 7-tetrahydroxy-5'-isopentenyl dihydroflavone) obtained through the following process steps: taking medicinal material of Cudrania tricuspidata as raw material, ethanol reflux extraction, ethyl acetate extraction, silica gel column chromatography separation, and preparation type HPLC system preparation. The compound 2', 4', 5, 7-tetrahydroxy-5'-isopentenyl dihydroflavone (formula I) has significant tumor cytotoxic activity on various cancer cells of human body, also has anti-inflammatory and anti-aging activities, and has the application prospect of developing anticancer, anti-inflammatory and / or anti-aging drugs.

Description

Technical Field

[0001] This invention belongs to the field of phytochemistry, specifically relating to a flavonoid compound with tumor cell toxicity and anti-inflammatory properties, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Cancer is a prevalent and common disease that seriously threatens human health. Factors such as population aging, smoking, excessive alcohol consumption, lack of exercise, unhealthy dietary habits, stress, and psychological tension may lead to a surge in cancer cases, severely endangering human life and quality of life. Therefore, finding effective anti-cancer drugs is a crucial research topic in global medicine. Traditional Western chemotherapy further damages bone marrow hematopoietic function, causing cancer cells to lose immune control and accelerate growth and spread. The significant toxic side effects of traditional chemotherapy cause systemic toxicity; many patients do not die from cancer itself, but from the toxic reactions and comprehensive damage to immune function during cancer treatment. Compared to Western medicine, traditional Chinese medicine (TCM) has higher safety, and its vast resource pool provides favorable conditions for exploring anti-tumor active molecules. Addressing the shortcomings of existing anti-tumor drugs, such as significant toxic side effects and some damage to immune function, developing a highly effective and safe TCM anti-tumor active molecule holds significant social and economic benefits for human health.

[0004] Cudrania tricuspidata (Carr.) Bur., a plant belonging to the genus Cudrania in the family Moraceae, is a dried root and vine. It is sweet, warm in nature, and non-toxic. It possesses the effects of clearing heat, cooling blood, and unblocking meridians, and is mainly used to treat pulmonary tuberculosis and damp-heat jaundice. Cudrania tricuspidata contains a variety of components, mainly including flavonoids, xanthones, organic acids, polysaccharides, phenylpropanoids, alkaloids, amino acids, steroidal compounds, and glycosides, among which flavonoids and xanthones are the most abundant. Modern pharmacological studies have shown that Cudrania tricuspidata has various pharmacological activities, inhibiting the growth, migration, and metastasis of various cancer cells. It can also promote apoptosis and induce autophagy, thus exhibiting good anti-tumor activity. It has heat-clearing effects, showing analgesic, anti-inflammatory, and antibacterial activities. It can act on the body's immune system, having a certain enhancing effect on decreased immune function. It can prevent liver damage caused by oxidative stress and also has the potential to prevent and treat cardiovascular diseases and delay aging. Therefore, further elucidating the pharmacodynamic material basis of the antitumor and anti-inflammatory effects of Cudrania tricuspidata is of great significance for the development of anticancer and anti-inflammatory drugs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a flavonoid compound with tumor cytotoxicity and anti-inflammatory properties, its preparation method, and its application. This invention extracts a flavonoid compound with anti-tumor, anti-inflammatory, and anti-aging functions from *Cudrania tricuspidata*, providing more treatment options for clinical practice.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a flavonoid compound with tumor cell toxicity, the structural formula of which is shown in Formula I:

[0008]

[0009]

[0010] The compound is a white solid. Electrospray ionization mass spectrometry (ESI-MS) showed: positive ion 357.11 [M+H]+, negative ion 355.10 [MH]-, indicating a molecular weight of 356 and a molecular formula of C2. 20 H 20 O6 confirmed that the compound was a new compound and named 2',4',5,7-tetrahydroxy-5'-isopentenyl dihydroflavone according to systematic nomenclature.

[0011] A second aspect of the present invention provides a method for preparing the flavonoid compound with tumor cytotoxicity and anti-inflammatory properties described in the first aspect, comprising the following steps:

[0012] S1. Extract the mulberry wood by ethanol reflux, collect the extract and concentrate it, extract, dry it to obtain an extract;

[0013] S2. Perform chromatography separation on the extract and collect the eluent;

[0014] S3. The eluent is concentrated to dryness under reduced pressure to obtain a solid. After redissolving the solid with petroleum ether-ethyl acetate, methanol is added dropwise to the solution, and the mixture is filtered to obtain a filtrate.

[0015] S4. The filtrate is concentrated and then subjected to chromatographic separation to collect the eluent. The eluent is separated by preparative high-performance liquid chromatography to obtain the flavonoids with tumor cell toxicity and anti-inflammatory properties described in the first aspect.

[0016] This invention uses *Cudrania trifoliata* as raw material and obtains a new medicinal compound with pharmacological activity through processes such as ethanol extraction, ethyl acetate extraction, silica gel column chromatography separation, and preparation using a preparative HPLC system. The preparation method is simple and easy to control.

[0017] In some embodiments of the present invention, in S1, 80%-95% ethanol is used as the extraction solvent for reflux extraction of Cudrania tricuspidata, and the extraction is performed 4-5 times, with each extraction lasting 1.8-2.2 hours;

[0018] The reflux extraction temperature is 80–90°C, preferably 85°C;

[0019] For the first extraction, the material-to-liquid ratio is 5-7:1, and for subsequent extractions it is 2-4:1.

[0020] In some embodiments of the present invention, in S1, the extract is filtered and concentrated under reduced pressure. The resulting concentrate is extracted with petroleum ether and ethyl acetate, respectively. The ethyl acetate extract is collected, concentrated, and dried to obtain an extract.

[0021] In some embodiments of the present invention, in step S2, silica gel column chromatography is used for separation, and gradient elution is performed sequentially with petroleum ether-ethyl acetate at volume ratios of 9:1, 10:2, 10:4, 10:8, and 0:1. The eluent fraction at a volume ratio of 10:2 is collected. When collecting the eluent fraction, seven portions of equal volume are collected according to the collection time and labeled as eluent part one, eluent part two, eluent part three, eluent part four, eluent part five, eluent part six, and eluent part seven, respectively. The eluent part three is used for processing in steps S3 and S4.

[0022] In some embodiments of the present invention, in step S3, after redissolving in petroleum ether-ethyl acetate at a volume ratio of 10:2 to 4, methanol is added dropwise to the solution until a precipitate forms, and then the solution is filtered to obtain the filtrate.

[0023] In some embodiments of the present invention, in S4, the filtrate is concentrated and then separated by silica gel column chromatography, with isocratic elution using petroleum ether-ethyl acetate at a volume ratio of 10:3, and the eluent is collected.

[0024] In some embodiments of the present invention, in S4, the preparative high-performance liquid chromatography uses a UniSil 5-120C18 Ultra column, 21.2×250mm, 5μm; a flow rate of 8-12mL / min; a mobile phase of acetonitrile-0.1% formic acid aqueous solution (v / v); and a detection wavelength of 278-282nm. The flavonoids with tumor cytotoxicity and anti-inflammatory properties described in the first aspect are collected during the peak elution time period.

[0025] A third aspect of the present invention provides the use of the flavonoid compound described in the first aspect, which has tumor cytotoxicity and anti-inflammatory properties, in the preparation of antitumor, anti-inflammatory and / or anti-aging drugs.

[0026] In some embodiments of the present invention, the tumor includes liver cancer, cervical cancer, gastric cancer, or lung cancer.

[0027] The beneficial effects of this invention are as follows:

[0028] Compared with existing technologies, the flavonoids with tumor cytotoxicity and anti-inflammatory properties provided by this invention have the structure shown in Formula I. This invention uses *Cudrania tricuspidata* as raw material and obtains new compounds through processes such as ethanol reflux extraction, ethyl acetate extraction, silica gel column chromatography separation, and preparative HPLC system preparation. The preparation method is simple and easy to control. The flavonoids provided by this invention exhibit significant tumor cytotoxic activity against various types of human cancer cells, and also possess anti-inflammatory and anti-aging activities, showing promise for the development of anticancer, anti-inflammatory, and / or anti-aging drugs. Detailed Implementation

[0029] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0030] Example 1

[0031] A method for extracting compounds isolated from *Cudrania tricuspidata*, comprising the following steps:

[0032] (1) Take the medicinal material of mulberry tree, use 80%-95% ethanol as the extraction solvent, reflux at 85℃ for extraction, extract four times, with material-liquid ratios of 6:1, 3:1, 3:1 and 3:1 respectively, and each extraction time is 2 hours.

[0033] (2) Take the extract obtained in step (1), filter it, concentrate it under reduced pressure, extract the concentrate with petroleum ether and ethyl acetate respectively, collect the ethyl acetate extract, concentrate it, dry it to obtain the extract for later use.

[0034] (3) Take the extract obtained in step (2) and separate it by silica gel column chromatography. Elute it sequentially with petroleum ether-ethyl acetate at ratios of 9:1, 10:2, 10:4, 10:8, and 0:1 (v / v). Collect the eluent fraction of 10:4 (v / v) petroleum ether-ethyl acetate. When collecting the eluent fraction, collect seven portions according to the collection time. Each portion has an equal volume. The seven eluent fractions are labeled according to the collection time as the first eluent fraction (i.e., the first eluent fraction obtained), the second eluent fraction, the third eluent fraction, the fourth eluent fraction, the fifth eluent fraction, the sixth eluent fraction, and the seventh eluent fraction (i.e., the last eluent fraction obtained), and set them aside for later use.

[0035] (4) Take the third part of the eluent obtained in step (3) and remove most of the main components by antisolvent precipitation. The specific operation is as follows: concentrate the third part of the eluent under reduced pressure until dry to obtain a light brown solid. Dissolve the solid with an appropriate amount of petroleum ether-ethyl acetate at a ratio of 10:2 to 4 (v / v). Continue to add methanol dropwise to the solution until a large amount of white precipitate is precipitated. Filter to remove the white precipitate and obtain a brownish-red mother liquor.

[0036] (5) Take the brownish-red mother liquor obtained in step (4), concentrate it, and separate it by silica gel column chromatography. Elute it isocratically with petroleum ether-ethyl acetate at a ratio of 10:3 (v / v). The eluent is labeled as A1.

[0037] (6) Take A1 obtained in step (5) and separate it using preparative HPLC. The chromatographic column was UniSil5-120C18Ultra (21.2×250mm, 5μm, Suzhou Nanomicro Technology Co., Ltd.), the flow rate was 10mL / min, the mobile phase was 55:45 (v / v) acetonitrile-0.1% formic acid water, the detection wavelength was 280nm, and the compound was collected during the peak elution period (15-16min).

[0038] Structural identification of the obtained compounds:

[0039] The compound was a white solid. Electrospray ionization mass spectrometry (ESI-MS) showed a positive ion at 357.11 [M+H]. + Negative ions: 355.10 [MH] - That is, the molecular weight of this compound is 356, and its molecular formula is C. 20 H 20 O6, chemical structural formula as shown in Formula I:

[0040]

[0041] This compound is a novel compound, named according to systematic nomenclature as 2',4',5,7-tetrahydroxy-5'-isopentenyl dihydroflavonoid.

[0042] 1H-NMR(400MHz,DMSO-d6)δ:12.16(s,1H,5-OH),7.01(s,1H,H-6'),6.39(s,1H,H-3'),5.86(s,2H,H-6,H-8),5.57(dd,J=12.6,2.9Hz,1H,H-2),5.22(t,J=7.2Hz,1H,H-2"),3.26(dd,J=17.2,12.7Hz,1H,H-3a),3.10(d,J=7.0Hz,2H,H-1"),2.60(dd,J=17.1,3.0Hz,1H,H-3b),1.66(s,3H,H-4"),1.63(s,3H,H-5")。

[0043] 13 C-NMR(100MHz,DMSO-d6)δ:197.27(C-4),167.14(C-7),164.00(C-9),163.86(C-5),156.24(C-4'),153.98(C-2'),131.06(C-3"),128.41(C-6'),123.92(C-2"),118.69(C-5'),115.33(C-1'),102.82(C-3'),102.15(C-10),96.11(C-6),95.35(C-8),74.47(C-2),41.58(C-3),

[0044] 27.93(C-1"),25.98(C-4"),18.07(C-5")。

[0045]

[0046] The 1H and 1C spectra of the compound obtained in Example 1 are very similar to those of Leachianone G (the chemical structure of Leachianone G is shown in Formula II), the difference being the substitution position of the isopentenyl group. From the 1H spectrum, the two methyl substituents show δH values ​​of 1.63 and 1.66 (each 3H, s); in the low-field region, proton signals of four benzene rings are shown at δH values ​​of 5.86 (2H, s), 6.39 (1H, s), and 7.01 (1H, s). Carbon and hydrogen assignments were performed using HSQC. HMBC revealed correlations between δH 7.01 (H-6′) and δC 27.93 (C-1″), 74.47 (C-2), 153.98 (C-2′), and 156.24 (C-4′); simultaneously, δH 6.39 (H-3′) was correlated with δC 118.69 (C-2), 115.33 (C-4′), 153.98 (C-2′), and 156.24 (C-4′). Furthermore, δH 3.10 (H-1″) was correlated with δC 118.69 (C-5′), 123.92 (C-2″), 128.41 (C-6′), 131.06 (C-3″), and 156.24 (C-4′), indicating that the isopentenyl group is attached to C-5′. By comparing the 1H NMR spectral data and coupling constants of the compound with those of Leachianone G, it was found that the C-2 configuration in the A ring of both compounds is the same, i.e., the S-type. Literature review indicates that this compound is a novel compound, systematically named 2',4',5,7-tetrahydroxy-5'-isopentenyl dihydroflavonoid.

[0047] HMBC (Hypermagnetic Resonance Cognition):

[0048]

[0049] Experimental Example 1

[0050] Pharmacological experiments

[0051] 1. Antitumor activity experiment

[0052] The MTT assay was used, with cisplatin as a positive control. Human HepG2 liver cancer cells, HeLa cervical cancer cells, SGC7901 gastric cancer cells, and A549 lung cancer cells in logarithmic growth phase were collected and adjusted to a cell concentration of 5 × 10⁻⁶ cells using serum-free culture medium. 4Cells / mL were seeded into 96-well plates, 200 μL per well, and incubated at 37°C and 5% CO2 for 12 hours. The culture medium was aspirated. The experimental groups were treated with compounds obtained in Example 1 (60 μM, 30 μM, 15 μM, 7.5 μM, 3.75 μM), and the control groups were treated with positive control drugs (50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM), 200 μL per well, with three replicates for each concentration. Incubation continued for 24 hours. The drug-containing culture medium was aspirated, and 100 μL of serum-free culture medium and 20 μL of 5 mg / mL LMT solution were added to each well. The plates were incubated for 4 hours, the culture medium was discarded, and 100 μL of DMSO was added to each well. After thorough shaking, the absorbance (OD) value was read at 570 nm. All compounds were tested in triplicate for each concentration, and the inhibitory rate of the tested drugs on tumor cells was calculated.

[0053] The half-maximal inhibitory concentration (IC50) was calculated using SPSS software. 50 )value.

[0054] Inhibition rate = (A 对照 -A 给药 ) / A 对照 ×100%

[0055] Results: The in vitro antitumor activity of the new compound (i.e., the compound obtained in Example 1) is shown in Table 1. The results indicate that the new compound 2',4',5,7-tetrahydroxy-5'-isopentenyl dihydroflavonoid exhibited strong inhibitory effects on human hepatocellular carcinoma HepG2 cells, human cervical carcinoma HeLa cells, human gastric carcinoma SGC7901 cells, and human lung cancer A549 cells, suggesting that this compound has the potential for application in the preparation of antitumor drugs.

[0056] Table 1. Inhibitory effect of the new compound on tumor cells.

[0057]

[0058] 2. Anti-inflammatory activity experiment

[0059] RAW264.7 mouse macrophage cell line was obtained, and after resuscitation, adherent cells in the logarithmic growth phase were selected. After trypsin digestion, RPMI 1640 medium containing 10% fetal bovine serum was prepared to a concentration of 5 × 10⁶ cells / mL. 4Cell suspensions were seeded into 96-well plates at 100 μL per well and cultured at 37°C for 24 h in 5% CO2. Anti-inflammatory experiments included a blank control group, a lipopolysaccharide (LPS) group, a drug-treated group, and a positive control group containing dexamethasone (DEX). The blank control group received only culture medium; the LPS group received only 1 μg / mL LPS; the drug-treated group received 1 μg / mL LPS and five different concentrations of the compound prepared in Example 1 (60 μM, 30 μM, 15 μM, 7.5 μM, 3.75 μM); and the positive control group containing DEX received 1 μg / mL LPS and five different concentrations of dexamethasone (50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM). Each group was in triplicate and cultured at 37°C for 48 h in 5% CO2. Discard the supernatant from the culture plate, wash twice with PBS, add 100 μL of freshly prepared 0.5 mg / mL LMT T medium to each well, and continue incubation at 37°C for 4 h. Carefully discard the supernatant again, add 150 μL of DMSO, mix well with a micro-shaker for 10 min, and then measure the optical density (OD value) at 492 nm using a microplate reader and calculate the IC50. 50 value.

[0060] Results: The inhibitory effects of the new compound and dexamethasone on LPS-induced NO release from RAW264.7 mouse macrophages were determined using the MTT assay. The IC50 of the positive control drug dexamethasone was [not specified in the original text]. 50 The IC50 value of the new compound 2',4',5,7-tetrahydroxy-5'-isopentenyl dihydroflavonoid was 4.7 μM. 50 The value was 10.1 μM. The results indicate that the new compound 2',4',5,7-tetrahydroxy-5'-isoprenyl dihydroflavonoid possesses good anti-inflammatory activity.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a flavonoid compound with tumor cytotoxicity and anti-inflammatory properties, characterized in that, Includes the following steps: S1. Extract the mulberry wood by ethanol reflux, collect the extract and concentrate it, extract, dry it to obtain an extract; S2. The extract is separated by silica gel column chromatography, and eluted sequentially with petroleum ether-ethyl acetate at volume ratios of 9:1, 10:2, 10:4, 10:8, and 0:

1. The eluent fraction at a volume ratio of 10:2 is collected. When collecting the eluent fraction, seven equal volumes are collected according to the collection time and labeled as eluent fraction 1, eluent fraction 2, eluent fraction 3, eluent fraction 4, eluent fraction 5, eluent fraction 6, and eluent fraction 7, respectively. The eluent fraction 3 is used for steps S3 and S4. S3. The eluent is concentrated to dryness under reduced pressure to obtain a solid. After redissolving the solid with petroleum ether-ethyl acetate, methanol is added dropwise to the solution, and the mixture is filtered to obtain a filtrate. S4. The filtrate is concentrated and separated by silica gel column chromatography with isocratic elution using petroleum ether-ethyl acetate at a volume ratio of 10:

3. The eluent is collected. The eluent is then separated by preparative high-performance liquid chromatography to obtain flavonoids with tumor cell toxicity and anti-inflammatory properties. The structural formula of the flavonoid compound is: .

2. The preparation method according to claim 1, characterized in that, In S1, 80%-95% ethanol was used as the extraction solvent for reflux extraction of Cudrania tricuspidata, and the extraction was performed 4-5 times, with each extraction lasting 1.8-2.2 hours. The reflux extraction temperature is 80~90℃; For the first extraction, the material-to-liquid ratio is 5-7:1, and for subsequent extractions it is 2-4:

1.

3. The preparation method according to claim 1, characterized in that, In S1, the extract is filtered and concentrated under reduced pressure. The resulting concentrate is extracted with petroleum ether and ethyl acetate, respectively. The ethyl acetate extract is collected, concentrated, and dried to obtain the extract.

4. The preparation method according to claim 1, characterized in that, In S3, after redissolving in petroleum ether-ethyl acetate at a volume ratio of 10:2~4, methanol is added dropwise to the solution until a precipitate forms. The solution is then filtered to obtain the filtrate.

5. The preparation method according to claim 1, characterized in that, In S4, the preparative high-performance liquid chromatography uses a UniSil 5-120C18 Ultra column (21.2 × 250 mm, 5 μm), a flow rate of 8–12 mL / min, a mobile phase of acetonitrile-0.1% formic acid aqueous solution (v / v), and a detection wavelength of 278–282 nm. The flavonoids with tumor cytotoxicity and anti-inflammatory properties are collected during the peak elution period.

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