A seaweed lactone compound with anti-tumor activity and its preparation method and application

Abstract

The present invention relates to a seaweed lactone compound, its preparation method, and application, belonging to the field of marine natural products. This seaweed lactone compound is extracted and isolated from Sargassum thunbergii. Anti-tumor activity experiments have shown that the compound has a half-maximal inhibitory concentration (IC50) of 8.4, 4.6, and 6.2 μM for human renal clear cell adenocarcinoma cells (786-O), human gallbladder carcinoma cells (GBC-SD), and human embryonic kidney cells (293T). The compound also exhibits proliferation inhibition activity against human gastric cancer cells (MKN-45) and human cervical cancer cells (HeLa). It can be used to prepare anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of marine natural products, and in particular relates to an algae lactone compound with anti-tumor activity and a preparation method and application of the compound. Background Art

[0002] Seaweed can produce secondary metabolites with rich chemical structure diversity and significant biological activity, and is an important source of drug lead compounds.

[0003] Cancer is a major threat to human health. Currently, there is no effective treatment for most cancers. While chemotherapy drugs have some efficacy, they also have significant side effects. Traditional Chinese medicines (TCMs) are generally less effective, resulting in less than ideal results.

[0004] Lactone compounds have anti-tumor and antibacterial effects and are widely used in the medical field. Summary of the Invention

[0005] The present invention obtains a new algal lactone compound by extracting and isolating Sargassum thunbergia collected from the waters of Nanji Island, Zhejiang Province. Currently, there are no reports on the chemical structure and anti-tumor activity of the compound, and there are no related drugs on the market. The purpose of the present invention is to provide a new algal lactone compound, a preparation method and application thereof.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A seaweed lactone compound with anti-tumor activity, characterized in that the seaweed lactone compound has the following structure: .

[0008] Preferably, the algal lactone compound is a white amorphous powder, [α] +11.4 (c 0.70, CH3OH); UV (MeOH) λ max (logε)211(3.86); high resolution ESI mass spectrum m / z251.0896[M+Na] + ,C 11 H 16 The calculated value of O5Na is 251.0895.

[0009] As a preferred method, the seaweed lactone compound is extracted by:

[0010] Step 1: drying and crushing the Sargassum thunbergii sample, soaking it in a 95% by volume ethanol solution, and extracting the ethanol solution after soaking three times to obtain an extract;

[0011] Step 2: Evaporate the extract under reduced pressure, extract with petroleum ether and ethyl acetate three times each, and concentrate the ethyl acetate extract under reduced pressure to obtain an extract;

[0012] Step 3: The extract was initially separated by silica gel column chromatography, first with a petroleum ether-ethyl acetate gradient elution, then with a dichloromethane-methanol gradient elution, to obtain 14 fractions (Frs. 1-14), and the fractions containing the target compound were detected and combined based on thin layer chromatography analysis;

[0013] Step 4: elute fraction Frs.10 with dichloromethane-methanol, collect the eluate, and further purify the eluate by Sephadex LH-20 gel column chromatography;

[0014] Step 5: Silica gel column chromatography with DCM-MeOH gradient elution to obtain subfraction Frs. 10-1;

[0015] Step 6: Further purify Frs. 10-1 using a preparative thin layer chromatography system to obtain algal lactone compounds.

[0016] Preferably, in step three, a gradient elution of petroleum ether-ethyl acetate with a volume ratio of 50:1 to a volume ratio of 1:1 is used; a gradient elution of dichloromethane-methanol with a volume ratio of 50:1 to a volume ratio of 1:1 is used, and the elution is carried out in the order of increasing polarity of the eluent to remove impurities with low polarity.

[0017] Preferably, in step 4, the volume ratio of dichloromethane to methanol is 20:1; and in the eluent, the volume ratio of dichloromethane to methanol is 10:1.

[0018] Preferably, in step 4, the volume ratio of dichloromethane to methanol in the eluent is 10:1.

[0019] Preferably, in step 4, when fraction Frs.10 is purified by Sephadex LH-20 gel column chromatography, the volume ratio of dichloromethane to methanol used is 1:1.

[0020] Preferably, in step 5, dichloromethane-methanol is used as the eluent for gradient elution in a volume ratio of 100:1 to 20:1.

[0021] Preferably, in step six, the volume ratio of dichloromethane to methanol during thin layer chromatography purification is 50:1.

[0022] Preferably, the algal lactone compound is used in the preparation of anticancer drugs.

[0023] Compared with the prior art, the advantages and positive effects of the present invention are:

[0024] 1. The present invention extracts and separates new algal lactone compounds from Sargassum thunbergii. Anti-tumor activity experiments show that the half-maximal inhibitory concentration (IC50) of the compound on human renal clear cell adenocarcinoma cells (786-O), human gallbladder carcinoma cells (GBC-SD), and human embryonic kidney cells (293T) is 50 ) were 8.4, 4.6, and 6.2 μM, respectively. The compound also exhibited antiproliferative activity against human gastric cancer cells (MKN-45) and human cervical cancer cells (HeLa). Therefore, the algal lactone compound represented by Formula I can be used to prepare anti-tumor drugs.

[0025] 2. The new algal lactone compounds of the present invention are extracted from Sargassum thunbergii, and the preparation process is simple. The raw materials are cheap and easily available. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0027] Figure 1 The diagram below shows the structure and carbon atom numbering of algal lactone compounds;

[0028] Figure 2 Schematic diagram of key related signals of 1H-1H COSY (thick line) and HBMC (arrow) of algal lactone compounds; DETAILED DESCRIPTION

[0029] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described below in conjunction with the accompanying drawings and embodiments. It should be noted that, in the absence of conflict, the embodiments of the present application and the features therein can be combined with each other.

[0030] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0031] Example 1, Preparation, Structural Identification and Anti-tumor Activity Testing of Algae Lactone Compounds:

[0032] Extraction and separation of seaweed lactone compounds: Step 1: After drying the Sargassum thunbergia seaweed sample collected from the waters of Nanji Island, Zhejiang Province, 2.5 kg of the sample was taken and crushed, and extracted three times with a 95% by volume ethanol-water solution.

[0033] Step 2: Collect the ethanol solution after extraction, evaporate under reduced pressure, extract three times with petroleum ether (PE), ethyl acetate (EtOAc) and n-butanol (n-Bu), respectively, and take the ethyl acetate extract and concentrate under reduced pressure to obtain 8.0 g of extract.

[0034] Step 3: The extract was initially separated by silica gel column chromatography, using a gradient elution of petroleum ether (PE)-ethyl acetate (EtOAc) from a volume ratio of 50:1 to a volume ratio of 1:1, followed by a gradient elution of dichloromethane (DCM)-MeOH from a volume ratio of 50:1 to a volume ratio of 1:1. Specifically, elution was performed in order of increasing eluent polarity to remove impurities of low polarity, yielding 14 fractions (Frs. 1-14). Fraction Frs. 10, weighing 2.1 g, was selected and analyzed by thin-layer chromatography, and the fractions containing the target compound were detected and combined.

[0035] Step 4: Elute fraction Frs.10 with DCM-MeOH in a volume ratio of 20:1, collect the eluate, wherein the volume ratio of DCM-MeOH in the eluate is 10:1, and then further purify the eluate on Sephadex LH-20 gel column chromatography, using DCM-MeOH in a volume ratio of 1:1 as the eluent.

[0036] Step 5: Silica gel column chromatography was performed again, and DCM-MeOH was used for gradient elution with a volume ratio of 100:1 to 20:1 to obtain subfraction Frs.10-1.

[0037] Step 6: The subfraction Frs.10-1 was subjected to preparative thin layer chromatography using a DCM-MeOH developing system with a volume ratio of 50:1 for further purification to obtain 5.1 mg of the new algal lactone compound.

[0038] The structure, physicochemical properties and spectral data of algal lactone compounds Figure 1 、 Figure 2 As shown, the structure of the lactone compound of the present invention is shown below,

[0039]

[0040] The physicochemical data of algae lactone compounds are as follows: algae lactone compounds are white amorphous powders, [α] +11.4 (c 0.70, CH3OH); UV (MeOH) λ max (logε)211(3.86); high resolution ESI mass spectrum m / z251.0896[M+Na] + ,C 11 H 16 The calculated value of O5Na is 251.0895.

[0041] Compound 1 was found to be a new compound by searching SciFinder.

[0042] Table 1 H and C NMR spectra of algal lactone compounds in DMSO-d (1H at 500 MHz, 13Cat1 at 25 MHz)

[0043]

[0044] Tumor Activity Test of Compounds 1) Materials and Reagents Used

[0045] PBS phosphate buffered saline (PB180327) was provided by Wuhan Punosai Life Science Technology Co., Ltd.

[0046] Fetal bovine serum (FBS) 04-001-1acs), Biological industries;

[0047] Penicillin-streptomycin sulfate double antibody mixture (100×) (P1400), Solarbio life sciences;

[0048] RPMI1640 culture medium (PM150110), Wuhan Punosai Life Science Technology Co., Ltd.;

[0049] CCK-8 reagent, Biosharp life sciences;

[0050] Trypsin-EDTA digestion solution (05200-056), GIBCO;

[0051] Cell-grade DMSO (D2650-100mL), SIGMA;

[0052] 96-well cell culture plates, Corning Life Sciences (Wujiang) Co., Ltd.;

[0053] 25, 75cm 2 Cell culture flasks, Corning Life Sciences (Wujiang) Co., Ltd.;

[0054] Hemocytometer, Shanghai Qiujing Biochemical Reagent Instrument Co., Ltd.

[0055] Doxorubicin hydrochloride (D8740), Solarbio life sciences;

[0056] 2) Instruments and equipment used

[0057] Clean bench (SW-CJ-2FD), Antai Company of Sujing Group;

[0058] Microscope (NIB-100), Ningbo Yongxin Optics Co., Ltd.;

[0059] Carbon dioxide cell culture incubator (MCO-18AC), PHcbi;

[0060] Electric constant temperature water bath (HWS-24), Shanghai Yiheng Technology Co., Ltd.;

[0061] Multifunctional microplate reader (MULTISKAN MK3), Thermo Scientific;

[0062] 100,000th balance (MS105DU), Mettler, Switzerland;

[0063] Centrifuge (TD4N), Changsha Yingtai Instrument Co., Ltd.;

[0064] SCI-VS mixer (SCI-VS), manufactured by Selotech, USA.

[0065] 3) Sample configuration

[0066] Weigh 1 mg of sample and dissolve in cell-grade DMSO to a stock concentration of 50 mM. Dilute with basal culture medium to the assay concentration.

[0067] 4) Principles and methods of standard operating procedures

[0068] ① Cytotoxicity (CCK-8 method) detection principle:

[0069] The test works by reducing WST-8, contained in the CCK-8 reagent, to a highly water-soluble yellow formazan product (formazan) via the action of the electron carrier 1-methoxy-5-methylphenazine methylsulfate (1-Methoxy PMS). The amount of formazan produced is proportional to the number of viable cells.

[0070] ②Experimental methods:

[0071] (1) Inoculation of cells: Prepare cells into a single cell suspension using culture medium containing 10% fetal bovine serum. Inoculate 100 μL of 5×104 / mL cells per well of a 96-well plate and pre-culture at 5% CO2 and 37°C for 24 h.

[0072] (2) Add the sample solution to be tested: dissolve the sample with DMSO, dilute the sample with basal medium, aspirate the old medium in the well (add 10 μL of 10-fold concentration sample solution directly to the suspended cells), add 100 μL of sample solution to each well, set 1 concentration for each sample in the initial screening, IC 50 For each sample, eight concentration gradients were set up, with three replicate wells for each concentration; the samples were cultured in an incubator for 48 hours.

[0073] (3) Color development: Aspirate the old culture medium (add 10 μL of CCK-8 solution directly to the suspended cells), add 100 μL of CCK-8 solution diluted ten times directly to each well, and continue to culture at 37°C, 5% CO2 for 2-3 hours (keep away from light and observe in real time).

[0074] (4) Colorimetry: Measure the absorbance at 450 nm using an enzyme-labeled instrument and record the raw data.

[0075] (5) Positive control sample: Doxorubicin hydrochloride (prepared immediately before use).

[0076] 5) Sample test results and analysis

[0077] The original data were normalized using Excel software, and the cell viability was calculated by the OD value of each well in the initial screening (formula = (Oddrug-Odblank) / ODcountrol-Odblank)*100%). The inhibition rate was statistically calculated, and the experimental results were expressed as ±SD. The inhibition rates of the algae lactone compounds of the present invention on cancer cells in various parts are shown in Table 2.

[0078] Table 2 Inhibitory effects of algal lactone compounds and doxorubicin hydrochloride on various cancer cells

[0079]

[0080] Analysis of the data in Table 2 shows that the algal lactone compounds of the present invention have a high inhibitory effect on MKN-45 human gastric cancer cells, HeLa human cervical cancer cells, 786-O human renal clear cell adenocarcinoma cells, GBC-SD human gallbladder cancer cells, and 293T human embryonic kidney cells.

[0081] The IC values ​​of the samples were calculated using GraphPad Prism 8. 50 Value (Calculated according to the linearity of XY data using the formula: Y=100 / (1+(IC 50 / X)^HillSlope) or Y=Bottom+(Top-Bottom) / (1+(IC 50 / X)^HillSlope)), the results are shown in Table 3,

[0082] Table 3 Toxicity test results of algal lactone compounds and doxorubicin hydrochloride on several cancer cells

[0083]

[0084] The sample test results were analyzed. Compared with doxorubicin hydrochloride, the half-maximal inhibitory concentration (IC50) of the compound on human renal clear cell adenocarcinoma cells (786-O), human gallbladder cancer cells (GBC-SD), and human embryonic kidney cells (293T) was obtained through anti-tumor activity experiments. 50 ) were 8.4, 4.6, and 6.2 μM, respectively. The compound also exhibited proliferation inhibition activity against human gastric cancer cells (MKN-45) and human cervical cancer cells (HeLa). It can be used to prepare anti-tumor drugs.

[0085] The above description is merely a preferred embodiment of the present invention and does not limit the present invention in any other form. Any technician familiar with the present invention may use the technical content disclosed above to change or modify it into an equivalent embodiment with equivalent changes for application in other fields. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A seaweed lactone compound with anti-tumor activity, characterized in that: The algae lactone compound has the structure shown below: .

2. The method for preparing a seaweed lactone compound with anti-tumor activity according to claim 1, characterized in that: The method for extracting the seaweed lactone compounds is as follows: Step 1: drying and crushing the Sargassum thunbergii sample, soaking it in a 95% by volume ethanol solution, and extracting the ethanol solution after soaking three times to obtain an extract; Step 2: Evaporate the extract under reduced pressure, extract with petroleum ether and ethyl acetate three times each, and concentrate the ethyl acetate extract under reduced pressure to obtain an extract; Step 3: The extract was initially separated by silica gel column chromatography, first with a petroleum ether-ethyl acetate gradient elution, then with a dichloromethane-methanol gradient elution, to obtain 14 fractions, and the fractions containing the target compound were detected and combined based on thin layer chromatography analysis; Step 4: elute fraction Frs.10 with dichloromethane-methanol, collect the eluate, and further purify the eluate by Sephadex LH-20 gel column chromatography; Step 5: Silica gel column chromatography with DCM-MeOH gradient elution to obtain subfraction Frs. 10-1; Step 6: Further purify Frs. 10-1 using a preparative thin layer chromatography system to obtain algal lactone compounds.

3. The method for preparing a seaweed lactone compound with anti-tumor activity according to claim 2, characterized in that: In step 3, the mixture was eluted with a gradient of petroleum ether-ethyl acetate in a volume ratio of 50:1 to 1:1; and with a gradient of dichloromethane-methanol in a volume ratio of 50:1 to 1:

1.

4. The method for preparing a seaweed lactone compound with anti-tumor activity according to claim 2, characterized in that: In step 4, the volume ratio of dichloromethane to methanol is 20:

1.

5. The method for preparing a seaweed lactone compound with anti-tumor activity according to claim 2, characterized in that: In step 4, the volume ratio of dichloromethane to methanol in the eluent is 10:

1.

6. The method for preparing a seaweed lactone compound with anti-tumor activity according to claim 2, characterized in that: In step 4, the eluent used in Sephadex LH-20 gel column chromatography purification is dichloromethane-methanol at a volume ratio of 1:

1.

7. The method for preparing a seaweed lactone compound with anti-tumor activity according to claim 2, characterized in that: In step 5, dichloromethane-methanol is used as the eluent for gradient elution from a volume ratio of 100:1 to 20:

1.

8. The method for preparing a seaweed lactone compound with anti-tumor activity according to claim 2, characterized in that: In step 6, the volume ratio of dichloromethane to methanol during thin layer chromatography purification is 50:

1.

9. The use of a seaweed lactone compound with anti-tumor activity according to any one of claims 1 to 8, characterized in that: The algal lactone compound is used in preparing medicines for resisting human renal clear cell adenocarcinoma cells, human gallbladder cancer cells, human embryonic kidney cells, human gastric cancer cells and human cervical cancer cells.

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