CN114920721A - Polyketone compound with antitumor activity and preparation method and application thereof - Google Patents
Polyketone compound with antitumor activity and preparation method and application thereof Download PDFInfo
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- CN114920721A CN114920721A CN202210407315.2A CN202210407315A CN114920721A CN 114920721 A CN114920721 A CN 114920721A CN 202210407315 A CN202210407315 A CN 202210407315A CN 114920721 A CN114920721 A CN 114920721A
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- compound
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- methanol
- polyketides
- liquid
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- 230000000259 anti-tumor effect Effects 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 150000001875 compounds Chemical class 0.000 title claims description 54
- 229920001470 polyketone Polymers 0.000 title claims description 25
- 229930001119 polyketide Natural products 0.000 claims abstract description 40
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 36
- 238000000855 fermentation Methods 0.000 claims abstract description 22
- 230000004151 fermentation Effects 0.000 claims abstract description 22
- 241000187747 Streptomyces Species 0.000 claims abstract description 21
- 229940125904 compound 1 Drugs 0.000 claims abstract description 19
- 125000000830 polyketide group Chemical group 0.000 claims abstract description 19
- 229940125782 compound 2 Drugs 0.000 claims abstract description 17
- 239000001963 growth medium Substances 0.000 claims abstract description 17
- UQZSVIGPZAULMV-DKWTVANSSA-N (2s)-2,4-diamino-4-oxobutanoic acid;propane-1,2,3-triol Chemical compound OCC(O)CO.OC(=O)[C@@H](N)CC(N)=O UQZSVIGPZAULMV-DKWTVANSSA-N 0.000 claims abstract description 15
- KVQIZFQVDNJTQH-UHFFFAOYSA-N 4,5,7-trihydroxy-1-(4-hydroxy-6-oxopyran-2-yl)-2-methylanthracene-9,10-dione Chemical compound CC1=CC(O)=C2C(=O)C3=C(O)C=C(O)C=C3C(=O)C2=C1C1=CC(O)=CC(=O)O1 KVQIZFQVDNJTQH-UHFFFAOYSA-N 0.000 claims abstract description 13
- HSOLPAFROQCEQW-UHFFFAOYSA-N Antibiotic SEK 15 Natural products Cc1cc(O)cc(O)c1C(=O)c2c(O)cc(O)cc2CC3=CC(=O)C=C(O)O3 HSOLPAFROQCEQW-UHFFFAOYSA-N 0.000 claims abstract description 13
- 238000009630 liquid culture Methods 0.000 claims abstract description 11
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- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 7
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 75
- 238000010828 elution Methods 0.000 claims description 28
- 239000007788 liquid Substances 0.000 claims description 27
- 238000001514 detection method Methods 0.000 claims description 14
- 239000003814 drug Substances 0.000 claims description 14
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 13
- -1 polyketide compounds Chemical class 0.000 claims description 13
- 239000000741 silica gel Substances 0.000 claims description 13
- 229910002027 silica gel Inorganic materials 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000000746 purification Methods 0.000 claims description 11
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 206010060862 Prostate cancer Diseases 0.000 claims description 9
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- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 6
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- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 5
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
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- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 claims description 3
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- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 4
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- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 4
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Abstract
The invention discloses two polyketides and a preparation method and application thereof, wherein the two polyketides are obtained by fermenting and culturing actinomycetes produced in salt lake Xinjiang by using a glycerol-asparagine liquid culture medium, extracting by using ethyl acetate, and separating and purifying by using silica gel column chromatography and high performance liquid chromatography. The strain streptomyces longispororuberber provided by the invention is adoptedTwo polyketides with anti-tumor activity are obtained from DBC5 CGMCC No.23972 fermentation liquor, and through identification, the compound 1 (formula I) is a novel polyketide derived from SEK-15, and the compound 2 (formula II) has an inhibition rate of 88% on the proliferation of a human prostate cell strain PC-3.
Description
Technical Field
The invention belongs to the field of microbial medicines, and particularly relates to two polyketone compounds with antitumor activity, a preparation method and application thereof, and a producing bacterium of the compounds.
Background
Polyketides are produced by repeated claisen decarboxylation and condensation of carboxylic acids under the catalysis of polyketide synthases (PKS). To date, there are 3 types of PKS known: the type I PKS is mainly used for catalyzing and synthesizing macrolide, polyene and polyether compounds. Type II PKSs are mainly used to catalyze the biosynthesis of aromatic polyketide compounds. Type III PKSs are primarily responsible for the biosynthesis of monocyclic or bicyclic aromatic polyketides. Polyketides have activity in inhibiting bacteria (e.g., erythromycin, tetracycline), fungi (e.g., griseofulvin, amphotericin), parasites (e.g., avermectin, nemadectin), tumors (e.g., doxorubicin, endidiynes), etc., and some antifungal polyketides also have immunosuppressive activity (e.g., rapamycin, FK506), and are widely used in medicine, livestock and agriculture.
Antibiotic resistance (AMR) has become a major threat to global public health, especially in developing countries, where 1000 million deaths are expected to result each year, with a reduction in total domestic production (GDP) of 2% to 3.5% by 2050. Polyketides are one of the main natural products with significant biological activity, mainly used as antitumor, antibiotic and antiparasitic agents.
The streptomyces microorganism can synthesize secondary metabolites with various structures such as alkaloid, anthraquinone, lactone, flavone and phenols; meanwhile, the metabolites also have various biological activities such as antagonistic microorganisms, anti-tumor, enzyme inhibition and the like. However, in the face of the advent of a bottleneck era of developing a new strain source for streptomyces, the development of a new secondary metabolite by re-starting the metabolism of streptomyces on the basis of the original research, integrating a molecular technology and an information technology to improve a screening model, modifying a metabolic pathway, improving a separation and purification technology and developing the new secondary metabolite becomes a research hotspot of the streptomyces. The salt lake actinomycetes live in extreme environments such as high salt, low temperature and malnutrition, are considered to be rich sources of natural products with novel structures and biological activities, and currently, research on the salt lake actinomycetes is mostly focused on community structures, and the problems of discovery and activity of the natural products need to be further clarified.
Disclosure of Invention
The invention aims at the technical current situation that no literature and patent report exists for separating and purifying the polyketone compound from the actinomycetes in the salt lake and preparing the anti-tumor medicament by using the polyketone compound. The invention aims to provide a novel polyketide, a polyketide with antitumor activity and a preparation method and application thereof, wherein a Streptomyces longispororber DBC5 CGMCC No.23972 strain is separated from a mud sample of Xinjiang salt lake, two polyketides with antitumor activity are obtained by utilizing a glycerol-asparagine liquid culture medium for fermentation culture and ethyl acetate extraction, separation and purification, one compound is identified to be a novel SEK-15 derived polyketide, the inhibition rate of the compound 2 on the proliferation of a human prostate cell strain PC-3 is 88%, and experiments prove that the compound separated by the invention can be used for preparing a medicament for inhibiting prostate cancer and providing a natural polyketide for treating tumors.
The invention specifically provides a polyketide compound with anti-tumor activity, which has the structure shown in formula (I) and formula (II):
the polyketide compound with the antitumor activity is obtained by separating and purifying Streptomyces longispororuber DBC5 CGMCC No.23972 fermentation culture species.
The strain Streptomyces longisporulus CGMCC No.23972 is numbered DBC5, has been preserved in China general microbiological culture Collection center (CGMCC) at 11 and 25 months of 2021, and has the address: beijing, Haizuan, Zhongguancun, No. 13, institute of microbiology, academy of sciences of China, zip code: 100080 with the deposition number: CGMCC No. 23972. According to the source of recipient bacteria, the DBC5 strain is Streptomyces longisporuber obtained by screening, breeding and domesticating in Xinjiang special ecological environment and microbiological identification according to the eighth edition of Bergey's system bacteriology manual.
The polyketone compounds with antitumor activity provided by the invention are prepared by the following preparation method:
(1) streptomyces longisporuber DBC5 CGMCC No.23972 preserved in China general microbiological culture Collection center (CGMCC) is inoculated into a 500mL conical flask, each flask contains 200mL of glycerol-asparagine liquid culture medium, and the mixture is subjected to shake cultivation at 22-35 ℃ and 100-250 rpm for 2-6 days to obtain a fermentation seed solution of the strain.
(2) Inoculating 5ml of the strain fermentation seed liquid obtained in the step (1) into a glycerol-asparagine liquid culture medium, and performing shake culture at the temperature of 22-35 ℃ and the rpm of 100-250 for 7-18 days to obtain liquid fermentation liquid of the strain.
(3) And (3) extracting the supernatant of the liquid fermentation liquor obtained in the step (2) with ethyl acetate for 3 times, combining ethyl acetate layers, and concentrating under reduced pressure to obtain a crude extract.
(4) Performing gradient elution on the crude extract obtained in the step (3) through a silica gel column chromatography, filling the silica gel column by adopting silica gel 300-400 meshes, wherein eluents are a petroleum ether-ethyl acetate solution and a methanol-dichloromethane solution, and the volume ratio of the petroleum ether to the ethyl acetate is 1:0,10:1 and 1: 1; the volume ratio of alcohol to dichloromethane is 40:1,30:1,20:1,10:1 and 1:0, and two polyketone compounds with antitumor activity are obtained by high performance liquid chromatography.
In the preparation method of the polyketone compound with the antitumor activity, the glycerol-asparagine liquid culture medium (g/L) is as follows: 10-20 parts of glycerol, 0-5 parts of dipotassium phosphate, 0-2 parts of trace salt, 0.1-0.5 part of ferric sulfate, 0.1-0.5 part of manganese chloride, 0.1-0.5 part of zinc sulfate, 7.2 parts of pH and 20min of sterilization at 121 ℃.
Further, the invention provides a novel SEK-15 derived polyketide compound, which has a structure shown in a formula (I):
the invention provides a preparation method of a novel SEK-15 derived polyketone compound (formula I), wherein the chromatographic conditions of high performance liquid elution are as follows: octadecyl bonded silica gel is adopted as a filler, methanol and water are used as mobile phases, the elution time is 40min, the flow rate is 10mL/min, and the detection wavelength is 210 nm. Collecting 10-30min fraction with a cyclone bottle rinsed with chromatographic methanol, concentrating under reduced pressure, adding appropriate amount of methanol dropwise for dissolving, and performing secondary purification by high pressure preparative liquid chromatography under gradient elution conditions: the mobile phase adopts 50% -100% methanol water solution, the elution time is 40min, the flow rate is 10mL/min, and the detection wavelength is 210nm, so as to obtain the monomeric compound (2.3mg, tR ═ 23 min).
In the preparation method of the polyketone compound (formula II) with the anti-tumor activity, the chromatographic conditions of high performance liquid elution are as follows: octadecyl bonded silica gel is adopted as a filler, methanol and water are used as mobile phases, the elution time is 40min, the flow rate is 10mL/min, and the detection wavelength is 210 nm. Collecting 15-30min fractions with a rotary bottle rinsed with chromatographic methanol, concentrating under reduced pressure, adding methanol dropwise for dissolving, and performing secondary purification by high pressure preparative liquid chromatography under gradient elution conditions: the mobile phase adopts 60-100% methanol aqueous solution, the elution time is 40min, the flow rate is 10mL/min, and the detection wavelength is 210nm, so as to obtain the monomer compound (5.1mg, tR is 23 min).
The invention provides application of polyketone compounds with antitumor activity in preparation of antitumor drugs.
The invention provides application of polyketone compounds with antitumor activity in preparation of antitumor drugs, wherein the antitumor drugs are anti-prostate cancer drugs.
Meanwhile, the polyketone compound 1 (formula I) and the polyketone compound 2 (formula II) with anti-tumor activity provided by the invention are applied to preparation of anti-tumor drugs, the compound synergy of the compound 1 (formula I) and the compound 2 (formula II) is obvious, and a preparation formulation formed by compounding the compound 1 and the compound 2 is applied to preparation of drugs for inhibiting prostatic cancer, so that the natural polyketone compound for treating tumors is provided.
The application of the Streptomyces longispororuber DBC5 CGMCC No.23972 in preparing the polyketide with anti-tumor activity.
Through the technical scheme, the invention achieves the following technical effects:
(1) the novel SEK-15 derived polyketide compound and the compound with anti-tumor activity are produced by performing liquid fermentation on actinomycetes produced in salt lake of Xinjiang, can be obtained by performing ethyl acetate extraction on a liquid fermentation culture and then performing separation and purification by using silica gel column chromatography and reversed phase chromatography, and provide data support for developing actinomycetes secondary metabolites in extreme environments.
(2) The invention provides a bacterial strain Streptomyces longispororber DBC5 CGMCC No.23972 for preparing a polyketide compound with anti-tumor activity, which is produced by liquid fermentation, and two polyketide compounds with anti-tumor activity are obtained by performing separation and purification on a liquid fermentation culture by silica gel column chromatography and reverse phase chromatography after ethyl acetate extraction, wherein one compound is identified as a novel SEK-15 derived polyketide compound, and the inhibition rate of the compound 2 on the proliferation of a human prostate cell strain PC-3 is 88 percent, which indicates that the compound obtained by separation can be used for preparing a medicament for inhibiting prostate cancer and providing a natural polyketide compound for treating tumors.
(3) The strain Streptomyces longisporulus DBC5 CGMCC No.23972 provided by the invention is adopted to prepare two polyketone compounds with anti-tumor activity, and through identification, the compound 1 is a new SEK-15 derived polyketone compound, and the inhibition rate of the compound 2 on the proliferation of a human prostate cell strain PC-3 is 88%.
Drawings
FIG. 1 is a characteristic diagram of the colony of Streptomyces longisporuber DBC5 CGMCC No.23972 on a glycerol-asparagine solid medium.
FIG. 2 is a spectrum diagram of polyketone compound of the compound 1 (streptoketals D), in which A is the polyketone compound of the compound 1 1 H NMR chart, B for Compound 1 13 C NMR chart, C is HSQC chart of compound 1, D is HMBC chart of compound 1, E is MS chart of compound 1)
FIG. 3 shows polyketides as compound 2 (arabimycin)The spectrum of the compound, in FIG. 3, A is that of compound 2 1 H NMR chart, B for Compound 2 13 C NMR chart.
Detailed Description
Example 1: separation, purification and identification of Streptomyces longispororuber CGMCC No.23972
The strain is separated from soil sample of Dalbavancin, Xinjiang and identified as Streptomyces longispororuber by 16S rRNA sequencing. Naturally air drying the soil sample collected from the salt lake for 7-10 days, weighing a certain amount of air-dried soil sample, drying at 70 ℃ for 2h, placing the soil sample in a 18mL sterile water triangular flask, shaking at 120 rpm at 30 ℃ for 1-2 days to prepare a soil suspension, injecting the soil suspension into a test tube containing 9mL sterile water, and preparing into a 10-mL sterile water suspension -2 And 10 -4 Two concentration gradients of soil suspension.
Adding 5-10% NaCl into the separating culture medium, and separating actinomycetes by using a dilution coating plate method. Suction 10 -2 And 10 -4 0.2mL of each dilution with concentration is respectively connected to 4 plate culture media, each dilution is repeated for 2 times, the plate culture media are evenly coated by a coating rod and then placed under the constant temperature of 30 ℃ and 37 ℃ for inverted culture, a single colony is picked up, the glycerol-asparagine culture media are subjected to zone streaking by adopting a streaking separation method, purified actinomycetes are obtained, and the actinomycetes are stored on a 4 ℃ inclined plane.
The strain is inoculated into a 500mL conical flask, each flask contains 200mL of glycerol-asparagine liquid culture medium, shaking culture is carried out for 4 days at 28 ℃ and 180rpm, and the record of the characteristics of the bacterial colony is observed when the bacterial colony grows over the whole plate, and the bacterial colony is photographed and stored. The recorded results are shown in FIG. 1.
As shown in the result of the attached figure 1, the strain Streptomyces longispooruber DBC5 CGMCC No.23972 grows on a glycerol-asparagine plate culture medium, the bacterial colony is circular, has a convex surface and folds, produces brown pigment, generates white spores to cover the bacterial colony when the bacterial colony grows for a certain time, and is further identified as the strain Streptomyces longispooruber DBC5 CGMCC No.23972 in actinomycetes.
Example 2: preparation of polyketides with antitumor activity
The polyketide compound is obtained by separating and purifying the fermentation culture of the strain Streptomyces longispooruber DBC5 CGMCC No.23972 provided by the embodiment 1, and the preparation method comprises the following steps:
(1) the strain Streptomyces longispooruber DBC5 CGMCC No.23972 of the China general microbiological culture Collection center provided in the embodiment 1 is inoculated into a 500mL conical flask, each flask contains 200mL of glycerol-asparagine liquid culture medium, and the mixture is subjected to shake culture at the temperature of 22-35 ℃ and the rpm of 100-250 for 2-6 days to obtain the fermentation seed liquid of the strain.
(2) Inoculating 5mL of the strain fermentation seed liquid obtained in the step (1) into a glycerol-asparagine liquid culture medium, and performing shake culture at 22-35 ℃ and 100-250 rpm for 7-18 days to obtain a liquid fermentation liquid of the strain.
(3) And (3) extracting the supernatant of the liquid fermentation liquor obtained in the step (2) with ethyl acetate for 3 times, combining ethyl acetate layers, and concentrating under reduced pressure to obtain a crude extract.
(4) Performing gradient elution on the crude extract obtained in the step (3) through a silica gel column chromatography, filling the silica gel column by adopting silica gel 300-400 meshes, wherein eluents are a petroleum ether-ethyl acetate solution and a methanol-dichloromethane solution, and the volume ratio of the petroleum ether to the ethyl acetate is 1:0,10:1 and 1: 1; the volume ratio of methanol to dichloromethane is 40:1,30:1,20:1,10:1 and 1:0, and two polyketone compounds with antitumor activity are obtained by high performance liquid chromatography.
Wherein the glycerol-asparagine liquid culture medium (g/L) is: 10-20 parts of glycerol, 0-5 parts of dipotassium hydrogen phosphate, 0-2 parts of trace salt, 0.1-0.5 part of ferric sulfate, 0.1-0.5 part of manganese chloride, 0.1-0.5 part of zinc sulfate, 7.2 parts of pH and 20min of sterilization at 121 ℃; the chromatographic conditions of the high performance liquid elution of the compound (formula I) are as follows: octadecyl bonded silica gel is adopted as a filler, methanol and water are used as mobile phases, the elution time is 40min, the flow rate is 10mL/min, and the detection wavelength is 210 nm. Collecting 10-30min fractions with a rotary bottle rinsed with chromatographic methanol, concentrating under reduced pressure, adding methanol dropwise for dissolving, and performing secondary purification by high pressure preparative liquid chromatography under gradient elution conditions: the mobile phase adopts 50-100% methanol aqueous solution, the elution time is 40min, the flow rate is 10mL/min, and the detection wavelength is 210nm, so as to obtain the monomer compound (2.3mg, tR is 23 min).
The chromatographic conditions of the high performance liquid elution of the compound (formula II) are as follows: octadecyl bonded silica gel is adopted as a filler, methanol and water are used as mobile phases, the elution time is 40min, the flow rate is 10mL/min, and the detection wavelength is 210 nm. Collecting 15-30min fraction with cyclone bottle rinsed with chromatographic methanol, concentrating under reduced pressure, adding appropriate amount of methanol dropwise for dissolving, preparing liquid chromatography under high pressure for secondary purification, and gradient eluting: the mobile phase adopts 60% -100% methanol water solution, the elution time is 40min, the flow rate is 10mL/min, and the detection wavelength is 210nm, so as to obtain the monomeric compound (5.1mg, tR ═ 23 min).
Example 3: isolation and identification of Compounds
In this example, based on examples 1 to 2, a liquid fermentation product containing two polyketides of the present invention was extracted 3 times with ethyl acetate, and the solvent was recovered and concentrated to obtain a crude product. Separating the crude product with silica gel column chromatography, wherein the elution system comprises petroleum ether and ethyl acetate in a ratio of 1:0,10:1 and 1: 1; methanol-dichloromethane solution, eluting with methanol-dichloromethane ═ 40:1,30:1,20:1,10:1,1:0, TLC analysis of fractions of the group incorporating the specific compound.
The obtained components containing the specific compounds are separated by high performance liquid preparative chromatography (Agilent Pursuit C-18(10 μm,21.2 multiplied by 250mm) chromatographic column, the detection wavelength is 292nm), the mobile phase is a 50-100% methanol (+ 0.05% TFA)/water (+ 0.05% TFA) system with the volume percentage for 40min, the elution is carried out by a gradient of 10mL/min, the solvent is recovered, the compound 1 is obtained, the structure of the compound is identified and tested, the specific test result is shown in Table 1 and figure 2, and the structure of the compound 1 is deduced to be shown in formula I.
Table 1: 1 h NMR data (solvent CD) 3 OD)
Brown yellow solid (MeOH), obtained according to mass spectrometry as an excimer ion peak of M/z 749.1865[ M + H ]] + (C 41 H 33 O 14 749.1865), from which it was determined that the formula was C 41 H 32 O 14 。 1 H NMR (CD3OD, 400Hz) indicated a methyl signal: δ H1.78 (s,1-CH3), two methylene signals: Δ H3.65 (s, H-15), Δ 0H 3.27(s, H-21) and five aromatic region signals: δ H5.95 (s, H-3), δ H6.13 (d, H-5, J ═ 2.2Hz), δ H6.79 (d, H-11, J ═ 8.2Hz), δ H7.23 (t, H-12, J ═ 8.0Hz), δ H6.85 (d, H-13, J ═ 7.6 Hz). The first two signals δ H5.95 and δ H6.13 are derived from the tetra-substituted aromatic system in compound 1. Further analysis of the two-dimensional HMBC spectrum can deduce the structure of compound 1. The first cyclic structure in compound 1 was established based on the correlation of HMBC mapping, Me-1 is associated with C-3, C-7 and C-2; h-3 is related to C-7; h-6 is related to C-3, C-7, C-4 and C-6; me-1, 4-OH and 6-OH are related to C-2, C-4 and C-6, respectively; according to the HMBC, the correlation of 4-OH with C-4 and C-6 is deduced respectively; 6-OH is related to C-5 and C-6; the structure of the compound 1 determines the existence of a second benzene ring according to HMBC mapping, namely H-12 is related to 1C-14 and C-10 respectively; h-13 is related to C-11, C-9 and C-15; h-11 is related to C-13 and C-9. The pyrones are linked to the benzene ring by a methylene moiety, based on the key HMBC correlation between the methylene carbon, delta C35.6, and H-13. Finally, to satisfy the requirement of unsaturation and molecular formula, benzene ring A and benzene ring B are linked through a keto carbon (C-8). These data indicate that Compound 1 may be another dimeric form of SEK-15 linked by a C-21 bond. Furthermore, the above conclusion is also confirmed by the correlation of H2-21 with HMBC at C-18, C-19, C-20, C-18 ', C-19 ', C-20 '. Careful comparison of the given data with the reported data reveals the polyketide nature of Compound 1. Thus, compound 1 was identified as strepelyketides D, a novel compoundA compound (I) is provided.
The obtained component containing the specific compound is separated by adopting a high performance liquid preparative chromatography (Agilent Pursuit C-18(10 mu m,21.2 multiplied by 250mm) chromatographic column, the detection wavelength is 292nm), the adopted mobile phase is a methanol/water system with the volume percentage of 60-100% for 40min, the gradient elution is carried out at 10mL/min, the solvent is recovered, the compound 2 is obtained, the data for identifying the compound 2 are shown in a table 2 and a figure 3, and the structure of the compound is deduced to be as formula (II).
Table 2: 1 h NMR data (solvent CD3OD)
| no. | δ C | δ H (JinHz) |
| 1 | 119.0,CH | 7.45,d(7.2) |
| 2 | 137.3,CH | 7.20,d(8.3) |
| 3 | 125.3,CH | 7.61,t(7.6) |
| 4 | 163.6,qC | |
| 4a | 117.2,qC | |
| 5 | 191.7,qC | |
| 5a | 191.7,qC | |
| 6 | 169.9,qC | |
| 6a | 125.1,qC | |
| 7 | 44.9,CH2 | 3.05,d(16.5)；2.98,d(16.5) |
| 8 | 72.4,qC | |
| 9 | 54.4,CH2 | 2.92,d(15.0)；2.79,d(15.0) |
| 10 | 198.9,qC | |
| 10a | 151.4,qC | |
| 11 | 124.8,CH | 6.85,s |
| 11a | 141.4,qC | |
| 12 | 187.2,qC | |
| 12a | 126.2,qC | |
| CH3 | 29.5,CH3 | 1.38,s |
Red yellow solid (MeOH), with strong UV absorption at 268 and 432nm, and the absorption band here indicates that the compound structure contains an anthraquinone core. The peak of the excimer ion obtained by mass spectrometry was m/z 339.0864[M+H] + (C 19 H 15 O 6 ，339.0863)，m/z361.0683[M+Na] + (C 19 H 14 NaO 6 361.0683), judging that the molecular formula of the compound is C 19 H 14 O 6 。 1 H NMR (CD3OD, 400Hz) data showed the presence of 4 aromatic hydrogen signals: δ 7.60(1H, t, J ═ 7.9Hz), δ 7.45(1H, d, J ═ 7.2Hz), δ 7.19(1H, d, J ═ 8.1Hz), δ 6.85(1H, s); 2 methylene groups, four doublets: δ 3.06 (1H, d, J ═ 16.4Hz), δ 2.98(1H, d, J ═ 16.5Hz), δ 2.92(1H, d, J ═ 15.0Hz), δ 2.78 (1H, d, J ═ 15.0 Hz); 1 methyl hydrogen signal: δ 1.38(1H, s, H-CH 3); 13 c NMR (CD3OD, 100MHz) data showed: delta 119.9(C-1), delta 141.4(C-2), delta 0125.1(C-3), delta 1163.9(C-4), delta 2117.3(C-5), delta 3191.7(C-6), delta 4119.1(C-7), delta 5169.9(C-8), delta 6126.2(C-9), delta 745.0(C-10), delta 872.4(C-11), delta 54.4(C-12), delta 198.9(C-13), delta 163.6(C-14), delta 124.8(C-15), delta 151.4(C-16), delta 187.2(C-17), delta 137.3 (C-18), a methyl carbon, delta 29.5(C-CH3) which, in comparison with the reference, have substantially the same signal, and thus compound 1 is determined to be arabomycin.
Example 4: application of polyketone compounds
In the embodiment, on the basis of the embodiments 1 to 3, two separated and purified polyketides are applied to preparation of a prostate cancer medicament, and the inhibition effect of the polyketides on the growth of prostate cancer cell PC-3 is examined.
(1) Test protocol
The tested cell is human prostate cancer cell PC-3. Compounds were prepared in DMSO in 6 concentration gradients of 10. mu.g/mL, 5. mu.g/mL, 2.5. mu.g/mL, 1.25. mu.g/mL, 0.625. mu.g/mL, 0.3125. mu.g/mL, respectively.
The SRB method is adopted, and the specific operation is as follows: preparing A549 cells into cell suspension with the concentration of 8 × 103/mL, injecting 100 μ L of the cell suspension into a 96-well plate, setting two times, placing the cell suspension in an incubator containing 5% carbon dioxide, culturing at 37 ℃ for 24 hours, adding medicines, setting 2 auxiliary wells for each concentration by taking adriamycin as a positive medicine, and continuing culturing for 72 hours. And taking out the 96-well plate after 72h, removing the culture medium, adding 100 mu L of 10% trichloroacetic acid aqueous solution into each well, placing in a refrigerator at 4 ℃, and fixing for more than 2 h. And (3) taking out the 96-well plate after the fixation is finished, discarding the trichloroacetic acid solution, washing with slow flowing water for at least 4 times, drying, adding 80 mu L of 0.4% SRB solution prepared from 1% acetic acid, dyeing for 20min, preparing 1% acetic acid aqueous solution, washing with 1% acetic acid aqueous solution for 4 times after the dyeing is finished to remove the unbound dye, and drying again. After the 96-well plate is completely dried, 100 mu L of 10mM Tris solution is added into each well, the solution is shaken to completely dissolve the dye combined with the protein, and the solution is placed in a microplate reader to detect the absorbance at 515 nm.
And (3) calculating the inhibition rate of the drugs on cell proliferation according to the OD value of each hole: inhibition rate [ 1- (OD) 515 medicine feeding hole /OD 515 control well )]×100％。
Table 3: the inhibition rate of the compound 2(10mg/mL) on the proliferation of the prostate cancer cell line PC-3 (ADR is positive drug)
| Compound (I) | |
| 2 | 88 |
| ADR | |
| 100％ |
From the data in table 3, the inhibition rate of compound 2 on PC-3 cell proliferation was 88%, indicating that compound 2 has potential inhibitory activity on tumor cells.
Further experimental verification proves that:
the application of the novel SEK-15 derived polyketide (formula I) in preparing the antitumor drug has obvious potential inhibition activity on tumor cells.
Two polyketone compounds with anti-tumor activity are prepared by adopting the Streptomyces longisporuber DBC5 CGMCC No.23972, and the compound 1 is identified to be a novel polyketone compound derived from SEK-15, the proliferation inhibition rate of the compound 2 to the human prostate cell strain PC-3 is 88 percent.
The above examples are merely examples for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications can be made while remaining within the scope of the present invention.
Claims (10)
1. Polyketone compounds with antitumor activity are characterized in that the structures of the compounds are shown as a formula (I) and a formula (II):
2. polyketide with antitumour activity according to claim 1, characterised in that it is isolated and purified from species of fermentation culture of the strain Streptomyces longisporuber DBC5 CGMCC No. 23972.
3. Polyketides with antitumor activity according to claim 1, wherein the compounds of formula (i) and (ii) are obtained by a process comprising:
(1) inoculating Streptomyces longispooruber DBC5 CGMCC No.23972 preserved in the common microorganism center of China Committee for culture of microorganisms into a 500mL conical flask, wherein each flask contains 200mL of glycerol-asparagine liquid culture medium, and carrying out shake culture at the temperature of 22-35 ℃ and the rpm of 100-250 for 2-6 days to obtain a fermentation seed solution of the Streptomyces longispooruber;
(2) inoculating 5mL of the strain fermentation seed liquid obtained in the step (1) into a glycerol-asparagine liquid culture medium, and performing shake culture at the temperature of 22-35 ℃ and the rpm of 100-250 for 7-18 days to obtain liquid fermentation liquid of the strain;
(3) extracting the supernatant of the liquid fermentation liquor obtained in the step (2) with ethyl acetate for 3 times, combining ethyl acetate layers, and concentrating under reduced pressure to obtain a crude extract;
(4) gradient elution is carried out on the crude extract obtained in the step (3) through a silica gel column chromatography column, silica gel 300-400-mesh silica gel is adopted to fill the silica gel column, eluent is petroleum ether-ethyl acetate solution and methanol-dichloromethane solution, and the volume ratio of petroleum ether to ethyl acetate is 1:0,10:1 and 1: 1; the volume ratio of alcohol to dichloromethane is 40:1,30:1,20:1,10:1 and 1:0, and two polyketone compounds with antitumor activity are obtained by high performance liquid chromatography.
4. The method for preparing polyketides with anti-tumor activity according to claim 3, wherein the glycerol-asparagine liquid medium (g/L) is: 10-20 parts of glycerol, 0-5 parts of dipotassium phosphate, 0-2 parts of trace salt, 0.1-0.5 part of ferric sulfate, 0.1-0.5 part of manganese chloride, 0.1-0.5 part of zinc sulfate, 7.2 parts of pH and 20min of sterilization at 121 ℃.
5. A novel SEK-15 derived polyketide, wherein the compound has the structure shown in formula (I):
6. the process for the preparation of the novel SEK-15 derived polyketides (formula i) as claimed in claim 5, which is obtainable by high performance liquid chromatography, using the process as provided in claim 3, wherein the chromatographic conditions for the high performance liquid elution are: octadecyl bonded silica gel is adopted as a filler, methanol and water are used as mobile phases, the elution time is 40min, the flow rate is 10mL/min, and the detection wavelength is 210 nm. Collecting 15-30min fraction with cyclone bottle rinsed with chromatographic methanol, concentrating under reduced pressure, adding appropriate amount of methanol dropwise for dissolving, preparing liquid chromatography under high pressure for secondary purification, and gradient eluting: the mobile phase adopts 60% -100% methanol water solution, the elution time is 40min, the flow rate is 10mL/min, and the detection wavelength is 210nm, so as to obtain the monomeric compound (5.1mg, tR ═ 23 min).
7. The use of the novel SEK-15 derived polyketides of formula i as claimed in claim 5 for the preparation of antitumor medicaments.
8. The application of the polyketide compounds 1 (formula I) and 2 (formula II) with antitumor activity in preparing antitumor drugs according to claim 1, characterized in that the compound 1 (formula I) and 2 (formula II) have obvious synergistic effect, and the preparation formulation formed by compounding the compound 1 and 2 is applied to preparing drugs for inhibiting prostate cancer, so that the natural polyketide compounds are provided for treating tumors.
9. The process for preparing polyketides (formula II) with antitumor activity according to claim 3, wherein the chromatographic conditions of the high performance liquid elution are: octadecyl bonded silica gel is adopted as a filler, methanol and water are used as mobile phases, the elution time is 40min, the flow rate is 10mL/min, and the detection wavelength is 210 nm. Collecting 10-30min fractions with a rotary bottle rinsed with chromatographic methanol, concentrating under reduced pressure, adding methanol dropwise for dissolving, and performing secondary purification by high pressure preparative liquid chromatography under gradient elution conditions: the mobile phase adopts 50% -100% methanol water solution, the elution time is 40min, the flow rate is 10mL/min, and the detection wavelength is 210nm, so as to obtain the monomeric compound (2.3mg, tR ═ 23 min).
10. Use of the strain Streptomyces longispooruber DBC5 CGMCC No.23972 for the preparation of polyketides with antitumor activity according to claim 1.
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| CN112592350A (en) * | 2020-12-18 | 2021-04-02 | 广东省微生物研究所（广东省微生物分析检测中心） | Polyketide lithocarpin E-G and preparation method and application thereof |
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| CN108358946A (en) * | 2018-04-11 | 2018-08-03 | 浙江大学 | A kind of anthraquinone analog compound and preparation method thereof and the application in preparing treating cancer drug |
| CN110357788A (en) * | 2019-06-17 | 2019-10-22 | 宁波大学 | A kind of polyketides and its preparation method and application |
| CN111233694A (en) * | 2020-03-05 | 2020-06-05 | 浙江大学 | Linear polyketone compound with blood fat reducing effect and preparation method and application thereof |
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