CN100395241C - A kind of degrading furan sesquiterpene cutin spongin and its preparation method and application - Google Patents

Abstract

The present invention relates to a novel furan sesquiterpene compound sclerotidin, which is isolated from Sarcotragus sp animal and is represented by formula (1). The invention also relates to the preparation method of the compound and its application in treating cancer, especially human lung cancer, human uterine cancer, human central nervous system cancer, human skin cancer and human colorectal cancer. Experiments have proved that the compound of the present invention has moderate cytotoxicity, and the maximum inhibitory rate to human lung cancer, human uterine cancer, human central nervous system cancer, human skin cancer, and human colorectal cancer cell lines is usually greater than or equal to the positive drugs cisplatin and adriamycin The IC ₅₀ values of these proteins are all around 10 μg/mL, which shows that they have a clear inhibitory effect on the growth of cancer cells.

Description

A kind of degrading furan sesquiterpene cutin spongin and its preparation method and application

technical field

The invention relates to a new degraded furan sesquiterpene compound, specifically a cutin spongin, a preparation method of the compound and its application in inhibiting the growth of cancer cells.

Background technique

At present, cyclic alcohol derivatives, quinone compounds and furan sesquiterpene compounds have been found from Sarcotragus sponges. Most of these compounds have furan rings and hydroxybutyrolactone groups, and furan sesquiterpene Such compounds usually have antibacterial, antiviral, antiinfective, anti-tremor and other activities.

Contents of the invention

The object of the present invention is to isolate a new degraded furan sesquiterpene compound that has a strong inhibitory effect on cancer cells from the genus Sarcotragus (Sarcotragus sp), and another object is to provide a preparation method for the compound , and the further object is to provide the application of the compound in the preparation of anticancer drugs.

The present invention separates and obtains a new degraded furan sesquiterpene compound cutin spongin from cutin sponge (Sarcotragus) cutin sponge (Sarcotragus sp), has α, β dihydroxy γ butanone group, and this compound has moderate cytotoxicity , has relatively definite growth inhibitory effect on human lung cancer, human uterine cancer, human central nervous system cancer, human skin cancer and human colorectal cancer cell lines, thereby achieving the purpose of the present invention.

Degraded furan sesquiterpene cutin spongin of the present invention is represented by formula (1):

Formula 1)

The degraded furan sesquiterpene keratin spongin of the present invention is isolated from Sarcotragus (Sarcotragus sp) animals, and its preparation method is to use the Sarcotragus sp animals as raw materials, chopping and using methanol Or ethanol extraction, methanol or ethanol extract with dichloromethane and water solution to obtain dichloromethane layer and water layer, dichloromethane layer concentrated, then extracted with n-hexane and aqueous methanol, respectively to obtain n-hexane layer and aqueous methanol layer , the aqueous methanol layer was subjected to reverse flash chromatography, eluted with methanol-water with a volume ratio of 70:30, then reverse-eluted with acetonitrile-water with a volume ratio of 60:40, and then reverse-phase HPLC preparative chromatography Separation, separation and purification with methanol-water with a volume ratio of 75:25.

Experiments have proved that the present invention's degraded furan ssquiterpene keratinosaponin has moderate cytotoxicity, and the maximum inhibitory rate to human lung cancer, human uterine cancer, human central nervous system cancer, human skin cancer, and human colorectal cancer cell lines is usually greater than or It is equal to the positive drugs cisplatin and doxorubicin, and its IC ₅₀ value is about 10 μg/mL, which shows that it has a clear growth inhibitory effect on cancer cells, and can be used in the treatment of cancer, especially human lung cancer, human uterine cancer, and human central nervous system. Systemic cancer, human skin cancer, and human colorectal cancer.

Detailed ways

The following examples are further illustrations of the present invention, but the present invention is not limited to the following examples.

Embodiment 1: the preparation of cutin spongin

Get 7 kg of horny sponge wet weight, extract 3 times with 10 liters of methanol at room temperature, suspend 800 grams of methanol extract with 5 liters of water, add 5 liters of dichloromethane for liquid separation, dichloromethane is partially concentrated to obtain 200 grams of extract, and then use 3 Methyl alcohol and 3 liters of normal hexane liquid separation of 1 volume fraction 90%, obtain 90% methanol extract 54 grams and normal hexane extract 13 grams, 90% methanol extract is through C-18 reverse flash chromatography (600 grams Reverse silica gel, YMC Gel ODS-A, particle size 10 microns), was eluted with 5 liters of methanol-water with a volume ratio of 70:30, combined to obtain 7.4 g, and then reversed with 5 liters of acetonitrile-water with a volume ratio of 60:40 To elution 5.7g, with reverse HPLC preparative chromatographic separation (Gilson Lab-Prep preparation system, flow rate: 5 milliliters/min, column temperature: room temperature, column type YMC ODS-H80, column length 250 millimeters, diameter 20 mm, particle size 4 microns), separated by methanol-water with a volume ratio of 75:25, and purified by semi-preparative reverse HPLC preparative chromatography (Gilson Lab-Prep preparation system, flow rate: 2 ml/min, column temperature: At room temperature, the column model is YMC ODS-H80, the column length is 250 mm, the diameter is 10 mm, the particle size is 4 microns, and the methanol-water volume ratio is 70:30), and 5 mg of yellow oil is obtained.

According to FAB-MS, the characteristic ion peak m/z[M+Na] ⁺ of this compound is 410, combined with ¹ H and ¹³ C NMR spectra, the molecular formula is C ₂₄ H ₃₆ O ₄ . It can be seen from ^{the 1} H and ¹³ C NMR spectra that the compound has a β-substituted furan ring and three broad single peaks at δ _H 7.37, 7.25 and 6.29. Two methyl singlets at δ _H 1.72 and 1.76 (δ _C are 16.5 and 24.3, respectively) and a methyl doublet at δ _H 1.01 (J = 7.0 Hz, δ _C 21.4) are present at the linking furan ring group on the carbon chain. ¹ H and ¹³ C NMR spectra showed the occurrence of trisubstituted ethylene hydrogens (δ _H 5.27) and 1,1,4 trisubstituted dienes (δ _H 6.17, 5.76, 5.40). ^{The 13} C NMR spectrum is consistent with the carbon spectrum data of other similar types of compounds (see Table 1).

The trisubstituted double bond of the E configuration is assigned based on the high field resonance effect of the methyl carbon (δ _C 16.5, C-9), while the double substituted double bond is assigned to the E configuration based on the coupling of the larger hydrogen often Count (J=15.0Hz). The chemical shift value (24.3) for C-19 shows the fact that this trisubstituted double bond is in the Z configuration. α, β dihydroxy γ-butanone groups replace hydroxybutyrolactone groups, two oxymethylene (δ 4.06, δ _C 72.0; δ 3.95, δ _C 80.1) signals and one methyl ketone signal be observed. The HMBC spectrum found propylene vinyl methylene (δ2.42, dd, J = 14.0, 6.5Hz and δ2.28, dd, J = 14.0, 7.0Hz) and _δC 129.5, 132.5, 72.0, and 80.1 These signals are coupled . Therefore this compound is identified as the compound of formula (1), chemical formula is (6Z, 10S, 11E, 13E)-17-(3-furyl)-3,4-dihydroxyl-6,10,14-trimethyl 17 Alkyl-6,11,13-trien-2-one[(6Z,10S,11E,13E)-17-(furan-3-yl)-3,4-dihydroxy-6,10,14-trimethylheptadeca- 6, 11, 13-trien-2-one}], the compound is a new compound after searching by CA, which belongs to the compound of degrading furan sesquiterpene, named as Sarcotin P.

Embodiment 2: In vitro pharmacodynamic cell experiment of keratin spongin P

The keratospongein P obtained in Example 1 was dissolved in DMSO, and the final concentration used was 1, 3, 10, 30, 100 μg/mL. Positive control drugs cisplatin and doxorubicin were prepared in PBS solution with final concentrations of 25, 50, 100, 200, 400 μg/mL and 2.5, 5, 10, 20, 40 μg/mL, respectively. The negative control was the corresponding complete cell culture solution containing 1% DMSO.

The in vitro pharmacodynamic cell experiment of keratinosaponin P took 5 kinds of human-derived tumor cell lines (human lung cancer, human uterine cancer, human central nervous system cancer, human skin cancer, and human colorectal cancer) as research objects, using MTT tumor cell growth inhibition assay. The specific experimental method is as follows: each cell line was subcultured with the corresponding complete cell culture medium to the logarithmic growth phase, digested with trypsin-EDTA solution, adjusted the final cell concentration to 5×10 ⁴ /mL, and inoculated in a 96-well plate , 200 μL per well. After culturing in a 37°C, 5% _CO2 incubator for 24 hours, the original liquid in each well on the plate was discarded and replaced with a complete culture solution containing the corresponding concentration of drugs, and 6 parallel wells were set for each concentration of each drug. After 48 hours, 20 μL of MTT solution with a concentration of 5 mg/mL was added to each well. After continuing to incubate for 4 hours, the original liquid in each well of the 96-well plate was discarded, and 200 μL of DMSO was added to each well to dissolve the formazan precipitate. After standing at room temperature for 0.5 h, the absorbance of each well was measured with a microplate reader at a wavelength of 570 nm. The inhibition rate of cell growth was calculated according to the following formula: cell growth inhibition rate (%)=(absorbance value of control group−absorbance value of experimental group)/(absorbance value of control group)×100%.

Statistical processing The half inhibitory concentration (IC ₅₀ ) of keratinosaponin on the growth of various tumor cell lines was calculated by Logit regression method. The results of in vitro MTT assay show that keratin spongin P of the present invention has obvious inhibitory effect on the growth of five types of tumor cell strains tested. The maximum inhibitory rate on tumor cells is usually equal to or slightly weaker than the positive drugs cisplatin and doxorubicin. The IC ₅₀ values were all around 10 μg/mL (2.3 μg/mL for cisplatin and 0.1 μg/mL for doxorubicin). It is suggested that it has a clear growth inhibitory effect on the above five types of tumor cell lines.

Table 1 ¹ H, ¹³ C NMR data of Sarcotin P

Claims (4)

1. Compounds of the following formula (1): Formula 1). 2. The preparation method of the compound of claim 1 is characterized in that taking cutin sponge (Sarcotragus sp) animal as raw material, extracting with methanol or ethanol after chopping, methyl alcohol or ethanol extract use dichloromethane and moisture liquid, obtain dichloromethane The methane layer and the water layer, the dichloromethane layer were concentrated, and then extracted with n-hexane and aqueous methanol to obtain the n-hexane layer and the aqueous methanol layer respectively. - elution with water, then reverse elution with acetonitrile-water with a volume ratio of 60:40, separation with reverse HPLC, separation with methanol-water with a volume ratio of 75:25, and purification. 3. The application of the compound of claim 1 in the preparation of a drug for treating cancer. 4. The application according to claim 3, wherein said cancer is human lung cancer, human uterine cancer, human central nervous system cancer, human skin cancer or human colorectal cancer.