A marine polyketide compound with anti-tumor activity, its preparation method and application

Abstract

The present invention provides a seaweed polyketide compound with anti-tumor activity, its preparation method and application. The compound is a polyketide compound, and its raw materials are collected from Sargassum thunbergii in the waters of Nanji Island, Zhejiang Province, and obtained after separation and purification. When the polyketide compound is applied to drugs, it has proliferation inhibitory activity against human cervical cancer cells (HeLa), human renal clear cell adenocarcinoma cells (786-O), human gallbladder cancer cells (GBC-SD) and human thyroid cancer cells (CAL-62), and the half-maximal inhibitory concentration (IC50) is 9.4 μM, 9.1 μM, 3.2 μM, 6.1 μM, and it can be used to prepare anti-tumor drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of extraction of marine natural products, and particularly relates to a seaweed polyketide compound with anti-tumor activity, a preparation method and an application thereof. Background Art

[0002] Seaweeds can produce secondary metabolites with rich chemical structures and remarkable biological activities, and are an important source of drug lead compounds. Sargassum thunbergii is a kind of seaweed, the thallus is dark brown, the main stem is short and thick, and it is mainly distributed in the intertidal zone. Among the secondary metabolites of seaweeds, polyketide compounds have anti-tumor, antibacterial and antioxidant effects, and are widely used in the medical field.

[0003] Due to the variety of secondary metabolites of seaweeds, when applied, various metabolites may affect each other, making it difficult to grasp the uncertainty of the use effect. Especially in the application of anti-tumor drugs, due to different types of tumors and different treatment mechanisms, the effects of seaweed drugs are not significant.

[0004] It can be seen that the separation and purification of the secondary metabolites of seaweeds is of great significance for their application in drugs. Summary of the Invention

[0005] In view of the above deficiencies of the prior art, the present invention provides a seaweed polyketide compound with anti-tumor activity, a preparation method and an application thereof. The compound is a polyketide compound, and its raw materials are collected from Sargassum thunbergii in the waters of Nanji Island, Zhejiang Province, and obtained after separation and purification. When the polyketide compound is applied to drugs, it has proliferation inhibitory activity against human cervical cancer cells (HeLa), human renal clear cell adenocarcinoma cells (786-O), human gallbladder cancer cells (GBC-SD) and human thyroid cancer cells (CAL-62), and the half-maximal inhibitory concentration (IC50) is 9.4 μM, 9.1 μM, 3.2 μM, 6.1 μM, and it can be used to prepare anti-tumor drugs.

[0006] The technical solution of the present invention is as follows:

[0007] A seaweed polyketide compound with anti-tumor activity has the following structural formula:

[0008]

[0009] The seaweed polyketide compound is a white amorphous powder, (c 0.20, CH3OH); UV (MeOH) λmax (logε) 207 (0.23); high-resolution ESI mass spectrometry m / z 185.0804 [M + H] + , C 9 H 13 O 4The calculated value is 185.0808; through retrieval by SciFinder, this compound is a new compound.

[0010] Preferably, the above compound is obtained by extraction and purification of Sargassum thunbergii collected from the waters of Nanji Island, Zhejiang.

[0011] A preparation method of a seaweed polyketide compound, comprising sampling, extraction, rough fractionation and purification;

[0012] Among them, rough fractionation is carried out by silica gel column separation, and the polarity of mobile phase A is set from small to large;

[0013] Purification is successively carried out by gel column chromatography, silica gel column chromatography and thin layer chromatography;

[0014] Mobile phase A, mobile phase B for gel column chromatography, mobile phase C for silica gel column chromatography and mobile phase D for thin layer chromatography are all composed of dichloromethane and methanol.

[0015] Preferably, the preparation method of the above seaweed polyketide compound is as follows:

[0016] (1) After drying and crushing the Sargassum thunbergia sample collected from the waters of Nanji Island, Zhejiang, it is extracted three times with 95% (v / v) ethanol, the extract is collected, and the extract is distilled under reduced pressure to obtain extract A;

[0017] (2) Add water to extract A, and the weight ratio of extract A to water is 1:5 - 10 to obtain a suspension;

[0018] The suspension is extracted with petroleum ether, and the volume ratio of petroleum ether to the suspension is 1:1 - 2. The extraction liquids are combined to obtain aqueous phase A; aqueous phase A is extracted with ethyl acetate, and the volume ratio of ethyl acetate to aqueous phase A is 1:1 - 2. The ethyl acetate phases are combined; the ethyl acetate phase is distilled under reduced pressure to obtain extract B for standby;

[0019] (3) Dissolve extract B with a mixed solution of dichloromethane and methanol, mix it with silica gel, and after drying, load it into a silica gel column, wherein the volume ratio of dichloromethane to methanol is 1:1;

[0020] Elute with mobile phase A of different polarities. Mobile phase A is composed of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 65 - 20:1;

[0021] When the volume ratio of dichloromethane to methanol is 20:1 of dichloromethane and methanol, collect eluate 10;

[0022] (4) The eluate 10 was subjected to Sephadex LH-20 gel column chromatography and eluted with mobile phase B; mobile phase B was composed of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol was 1:1. The eluate 10-1 was collected;

[0023] (5) The eluate 10-1 was subjected to silica gel column chromatography and eluted with mobile phases C of different polarities;

[0024] Mobile phase C was composed of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol was 100 - 20:1;

[0025] When the volume ratio of dichloromethane to methanol was 20:1, the eluate 10-2 was collected;

[0026] (6) The eluate 10-2 was purified by preparative thin-layer chromatography and developed with mobile phases D of different polarities. Mobile phase D was composed of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol was 100 - 50:1;

[0027] When the volume ratio of dichloromethane to methanol was 50:1, the eluate 10-3 was collected, which was the solution of seaweed polyketides.

[0028] In the above preparation method, by adjusting the polarities of mobile phase A, mobile phase C, and mobile phase D, the impurities in the solution can be reduced and the content of seaweed polyketides in the eluate 10-3 can be increased.

[0029] Preferably, in step (2), extraction was performed 3 times with petroleum ether and 3 times with ethyl acetate.

[0030] Preferably, in step (3), in the mobile phases A of different polarities, the volume ratios of dichloromethane to methanol were 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1;

[0031] In the mobile phases C of different polarities, the volume ratios of dichloromethane to methanol were 100:1, 70:1, 50:1,

[0032] 40:1, 30:1, 20:1; in the mobile phases D of different polarities, the volume ratios of dichloromethane to methanol were 100:1, 80:1, 60:1, 50:1.

[0033] An application of a seaweed polyketide compound, specifically the application of the above polyketide compound in the preparation of anti-tumor drugs.

[0034] Preferably, the above application is the application of the polyketide compound in the preparation of anti-tumor drugs for the treatment of human cervical cancer, human renal clear cell adenocarcinoma, human gallbladder cancer, and human thyroid cancer.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] The new compound provided by the present invention is derived from Sargassum thunbergia in the waters of Nanji Island, Zhejiang; the Sargassum thunbergia is obtained through extraction, separation and purification; through anti-tumor activity experiments, it is found that this compound has proliferation inhibitory activity against human cervical cancer cells (HeLa), human renal clear cell adenocarcinoma cells (786-O), human gallbladder cancer cells (GBC-SD) and human thyroid cancer cells (CAL-62), and the half inhibitory concentration (IC50) is 9.4 μM, 9.1 μM, 3.2 μM, 6.1 μM, indicating that this compound can be used to prepare anti-tumor drugs. Description of the Drawings

[0037] Figure 1 Mass spectrum of the polyketide compound in Example 1;

[0038] Figure 2 1H nuclear magnetic resonance spectrum of the polyketide compound in Example 1;

[0039] Figure 3 13C nuclear magnetic resonance spectrum of the polyketide compound in Example 1. Detailed Embodiments

[0040] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0041] Example 1

[0042] A kind of algal polyketide compound, the preparation method is as follows:

[0043] (1) After drying the Sargassum thunbergia sample collected from the waters of Nanji Island, Zhejiang, weighing 2.5 kg, crushing it, and then extracting it three times with 95% (v / v) ethanol, collecting the extract, and subjecting the extract to reduced pressure distillation to obtain extract A;

[0044] (2) Add water to extract A, and the weight ratio of extract A to water is 1:7 to obtain a suspension;

[0045] The suspension was extracted with petroleum ether at a volume ratio of petroleum ether to suspension of 1:1 for 3 times, and the extraction solutions were combined to obtain aqueous phase A; aqueous phase A was extracted with ethyl acetate at a volume ratio of ethyl acetate to aqueous phase A of 1:1 for 3 times, and the ethyl acetate phases were combined; the ethyl acetate phase was distilled under reduced pressure to obtain 8.0 g of extract B for standby;

[0046] (3) Extract B was dissolved in a mixed solution of dichloromethane and methanol, silica gel was added and stirred, and after evaporation to dryness, it was loaded onto a silica gel column, where the volume ratio of dichloromethane to methanol was 1:1;

[0047] It was eluted with mobile phase A of different polarities. Mobile phase A was composed of dichloromethane and methanol. In mobile phase A of different polarities, the volume ratios of dichloromethane to methanol were 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1 respectively;

[0048] When the volume ratio of dichloromethane to methanol was 20:1, 102.1 g of eluate was collected;

[0049] (4) The eluate 10 was further purified by Sephadex LH-20 gel column chromatography and eluted with mobile phase B; mobile phase B was composed of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol was 1:1, and eluate 10-1 was collected;

[0050] (5) The eluate 10-1 was subjected to silica gel column chromatography and eluted with mobile phase C of different polarities; mobile phase C was composed of dichloromethane and methanol. In mobile phase C of different polarities, the volume ratios of dichloromethane to methanol were 100:1, 70:1, 50:1, 40:1, 30:1, 20:1 respectively;

[0051] When the volume ratio of dichloromethane to methanol was 20:1, eluate 10-2 was collected;

[0052] (6) The eluate 10-2 was purified by preparative thin-layer chromatography and developed with mobile phase D of different polarities. Mobile phase D was composed of dichloromethane and methanol. In mobile phase D of different polarities, the volume ratios of dichloromethane to methanol were 100:1, 80:1, 60:1, 50:1 respectively;

[0053] When the volume ratio of dichloromethane to methanol was 50:1, eluate 10-3 was collected. After drying, 5.9 mg of the seaweed polyketide compound was obtained; the mass spectrum of this polyketide compound is shown in Figure 1 , the nuclear magnetic resonance hydrogen spectrum is shown in Figure 2 , and the nuclear magnetic resonance carbon spectrum of the polyketide compound is shown in Figure 3 ;

[0054] This seaweed polyketide compound is a white amorphous powder, (c 0.20, CH3OH); UV (MeOH) λmax (logε) 207 (0.23); 1H and 13C NMR spectra are shown in Table 1, HR-ESI-MS m / z 185.0804 [M + H] + , C 9 H 13 O 4 Calculated value is 185.0808; Through SciFinder search, this compound is a new compound, and its structural formula is as follows:

[0055]

[0056] Table 1 1H and 13C NMR Data for Compound 2 in DMSO-d6 (1H at 500 MHz, 13C at 125 MHz)

[0057]

[0058] Example 2

[0059] The difference from Example 1 is that in step (2): water was added to Extract A, and the weight ratio of Extract A to water was 1:5 to obtain a suspension;

[0060] The suspension was extracted with petroleum ether, the volume ratio of petroleum ether to the suspension was 1:2, and the extraction was carried out 3 times. The extraction liquids were combined to obtain aqueous phase A; Aqueous phase A was extracted with ethyl acetate, the volume ratio of ethyl acetate to aqueous phase A was 1:2, and the extraction was carried out 3 times. The ethyl acetate phases were combined; The ethyl acetate phase was distilled under reduced pressure to obtain 8.5 g of Extract B for standby.

[0061] Example 3

[0062] The difference from Example 1 is that in step (2): water was added to Extract A, and the weight ratio of Extract A to water was 1:10 to obtain a suspension;

[0063] The suspension was extracted with petroleum ether, the volume ratio of petroleum ether to the suspension was 1:1.5, and the extraction was carried out 3 times. The extraction liquids were combined to obtain aqueous phase A; Aqueous phase A was extracted with ethyl acetate, the volume ratio of ethyl acetate to aqueous phase A was 1:1.5, and the extraction was carried out 3 times. The ethyl acetate phases were combined; The ethyl acetate phase was distilled under reduced pressure to obtain 8.3 g of Extract B for standby.

[0064] Example 4

[0065] The anti-tumor activity of the compound obtained in Example 1 was tested as follows:

[0066] 1. Materials and Reagents

[0067] PBS phosphate buffer (PB180327), Wuhan Punosai Life Science Co., Ltd.; fetal bovine serum (FBS) 4-001-1acs), Biological industries; penicillin-streptomycin sulfate double antibody mixture (100×) (P1400), Solarbio life sciences; RPMI1640 culture medium (PM150110), Wuhan Punosai Life Science Co., Ltd.; CCK-8 reagent, Biosharp life sciences; trypsin-EDTA digestion solution (05200-056), GIBCO; cell-grade DMSO (D2650-100ML), SIGMA; 96-well cell culture plate, Corning Life Sciences (Wujiang) Co., Ltd.; 25, 75 cm 2 Cell culture flasks, Corning Life Sciences (Wujiang) Co., Ltd.; blood cell counting chamber, Shanghai Qiujing Biochemical Reagent Instrument Co., Ltd.; doxorubicin hydrochloride (D8740), Solarbio life sciences.

[0068] 2. Instruments and equipment

[0069] Clean bench (SW-CJ-2FD), Sujing Group Antai Company; microscope (NIB-100), Ningbo Yongxin Optics Co., Ltd.; carbon dioxide cell culture incubator (MCO-18AC), PHcbi Company; electric constant temperature water bath (HWS-24), Shanghai Yiheng Technology Co., Ltd.; multifunctional microplate reader (MULTISKAN MK3), Thermo Company; one-hundred-thousandth balance (MS105DU), Mettler Company of Switzerland; centrifuge (TD4N), Changsha Yingtai Instrument Co., Ltd.; mixer (SCI-VS), Selo Czech, USA.

[0070] 3. Sample preparation

[0071] The sample of Example 1 was weighed, and cell-grade DMSO was added to dissolve and mix well, with the stock solution concentration being 50 mM; and the basal medium was diluted to the detection concentration.

[0072] 4. Operating procedures

[0073] 4.1 Cytotoxicity (CCK-8 method) detection principle:

[0074] The detection principle is that the CCK-8 reagent contains WST-8, which is reduced by dehydrogenase in the cell mitochondria to a highly water-soluble yellow formazan product (Formazan) under the action of the electron carrier 1-methoxy-5-methylphenazine dimethyl sulfate (1-Methoxy PMS). The amount of formazan generated is proportional to the number of living cells.

[0075] 4.2 Experimental methods:

[0076] (1) Cell seeding: The cells were resuspended in a culture medium containing 10% fetal bovine serum to form a single-cell suspension. 100 μL of cells at a concentration of 5×10 4 / mL were seeded into each well of a 96-well plate and pre-cultured at 37 °C in 5% CO 2 2 for 24 h;

[0077] (2) Addition of test sample solution: The sample was dissolved in DMSO and diluted with basal medium. The old culture medium in the wells was aspirated (for suspended cells, 10 μL of 10-fold concentrated sample solution was directly added). 100 μL of sample solution was added to each well. For primary screening, one concentration was set for each sample, and for IC50 determination, eight concentration gradients were set for each sample, with three replicates for each concentration. The plate was placed in an incubator and cultured for 48 h;

[0078] (3) Color development: The old culture medium was aspirated (for suspended cells, 10 μL of the original CCK-8 solution was directly added). 100 μL of 10-fold diluted CCK-8 solution was directly added to each well and further cultured at 37 °C in 5% CO 2 2 for 2 - 3 h (operate under light protection and observe in real time);

[0079] (4) Colorimetry: The absorbance at 450 nm was measured using a microplate reader, and the original data results were recorded;

[0080] (5) The original data was normalized using Excel software. For primary screening, the cell viability was calculated from the OD value of each well using the formula: Cell viability = (Oddrug - Odblank) / (ODcountrol - Odblank) * 100%; The inhibition rate was statistically analyzed; The IC50 value of the sample was calculated using GraphPad Prism 8 (depending on the linearity of the XY data, the calculation formula: Y = 100 / (1+(IC50 / X)^HillSlope) or Y = Bottom+(Top - Bottom) / (1+(IC50 / X)^HillSlope)), and the experimental results were expressed as ±SD;

[0081] (6) Positive control sample: Doxorubicin hydrochloride (prepared freshly before use).

[0082] 5. Sample test results

[0083] The cells used in the experiment included human gastric cancer cells (MKN-45), human lung cancer cells (A549), human colon cancer cells (HCT 116), human cervical cancer cells (HeLa), human chronic myelogenous leukemia cells (K-562), human renal clear cell adenocarcinoma cells (786-O), human esophageal cancer cells (TE-1), human bladder cancer cells (5637), human gallbladder cancer cells (GBC-SD), human normal liver cells (L-02), human breast cancer cells (MCF7), human liver cancer cells (HepG2), human brain tumor cells (SF126), human prostate cancer cells (DU145), human thyroid cancer cells (CAL-62), human pancreatic cancer cells (PATU8988T), human osteosarcoma cells (HOS), human malignant melanoma cells (A-375), human rhabdomyosarcoma cells (A-673), and human embryonic kidney cells (293T).

[0084] During the experiment, the compound of Example 1 was the experimental compound, and the positive control was Doxorubicin hydrochloride.

[0085] Table 2 Detection results of cell inhibition rate 1

[0086]

[0087] Table 3 Detection results of cell inhibition rate 2

[0088]

[0089] Table 4 Detection results of cell inhibition rate 3

[0090]

[0091] Table 5 Detection results of cell inhibition rate 4

[0092]

[0093] Table 6 Detection results of cell inhibition rate 5

[0094]

[0095] Table 7 IC50 results

[0096]

[0097] As can be seen from Tables 2 - 7, the compound obtained in Example 1 has proliferation inhibitory activity against human cervical cancer cells (HeLa), human renal clear cell adenocarcinoma cells (786 - O), human gallbladder cancer cells (GBC - SD), and human thyroid cancer cells (CAL - 62), and the half - inhibitory concentration (IC50) is 9.4 μM, 9.1 μM, 3.2 μM, and 6.1 μM, indicating that this compound can be used to prepare corresponding anti - tumor drugs.

[0098] Although the present invention has been described in detail by referring to the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, those of ordinary skill in the art can make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions should all be within the scope of the present invention. / Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A kind of seaweed polyketide compound with anti-tumor activity, Characterized in that, The structural formula is as follows: 。 2. A preparation method of the seaweed polyketide compound as described in claim 1, Characterized in that, It includes sampling, extraction, rough fractionation and purification; Among them, for rough fractionation, silica gel column separation is adopted, and the polarity of mobile phase A is set from small to large; Purification is successively carried out by gel column chromatography, silica gel column chromatography and preparative thin layer chromatography; Mobile phase A, mobile phase B for gel column chromatography, mobile phase C for silica gel column chromatography and mobile phase D for thin layer chromatography are all composed of dichloromethane and methanol; The preparation method of the seaweed polyketide compound is specifically as follows: (1) After drying and crushing the Sargassum thunbergii ([ Sargassum thunbergia ]) samples collected from the waters of Nanji Island, Zhejiang Province, extract them three times with 95% (v / v) ethanol, collect the extract, and perform vacuum distillation on the extract to obtain extract A; Sargassum thunbergia ) (2) Add water to extract A, and the weight ratio of extract A to water is 1:5 - 10 to obtain a suspension; Extract the suspension with petroleum ether, and the volume ratio of petroleum ether to the suspension is 1:1 - 2. Combine the extraction solutions to obtain aqueous phase A; Extract aqueous phase A with ethyl acetate, and the volume ratio of ethyl acetate to aqueous phase A is 1:1 - 2. Combine the ethyl acetate phases; Distill the ethyl acetate phase under reduced pressure to obtain extract B for standby; (3) Dissolve extract B with a mixed solution of dichloromethane and methanol, mix it with silica gel, and load it into a silica gel column after drying. Among them, the volume ratio of dichloromethane to methanol is 1:1; Elute with mobile phase A of different polarities. Mobile phase A is composed of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 65 - 20:1; When the volume ratio of dichloromethane to methanol is 20:1 of dichloromethane and methanol, collect eluate 10; (4) Subject eluate 10 to Sephadex LH-20 gel column chromatography and elute with mobile phase B; Mobile phase B is composed of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 1:

1. Collect eluate 10-1; (5) Subject eluate 10-1 to silica gel column chromatography and elute with mobile phase C of different polarities; Mobile phase C is composed of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 100 - 20:1; When the volume ratio of dichloromethane to methanol is 20:1, collect eluate 10-2; (6) Purify eluate 10-2 by preparative thin layer chromatography and develop with mobile phase D of different polarities. Mobile phase D is composed of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 100 - 50:1; When the volume ratio of dichloromethane to methanol is 50:1, collect eluate 10-3.

3. The preparation method of the seaweed polyketide compound as described in claim 2, Characterized in that, In step (2), extract with petroleum ether 3 times and extract with ethyl acetate 3 times.

4. The preparation method of the seaweed polyketide compound as described in claim 2, Characterized in that, In step (3), in mobile phase A with different polarities, the volume ratios of dichloromethane to methanol are 65:1, 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1 respectively; in step (5), in mobile phase C with different polarities, the volume ratios of dichloromethane to methanol are 100:1, 70:1, 50:1, 40:1, 30:1, 20:1 respectively; in step (6), in mobile phase D with different polarities, the volume ratios of dichloromethane to methanol are 100:1, 80:1, 60:1, 50:1 respectively.

5. Use of a polyketide compound of seaweed as claimed in claim 1, characterized in that the polyketide compound is used in the preparation of anti-tumor drugs for treating human cervical cancer, human clear cell renal carcinoma, human gallbladder cancer and human thyroid cancer.

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