CN108486011B - Terphenyl compound, preparation method and application thereof - Google Patents
Terphenyl compound, preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 9
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- YJTKZCDBKVTVBY-UHFFFAOYSA-N 1,3-Diphenylbenzene Chemical group C1=CC=CC=C1C1=CC=CC(C=2C=CC=CC=2)=C1 YJTKZCDBKVTVBY-UHFFFAOYSA-N 0.000 claims abstract description 13
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- 238000000855 fermentation Methods 0.000 claims abstract description 9
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- 239000007787 solid Substances 0.000 claims description 11
- KDCIHNCMPUBDKT-UHFFFAOYSA-N hexane;propan-2-one Chemical compound CC(C)=O.CCCCCC KDCIHNCMPUBDKT-UHFFFAOYSA-N 0.000 claims description 8
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- MPDDTAJMJCESGV-CTUHWIOQSA-M (3r,5r)-7-[2-(4-fluorophenyl)-5-[methyl-[(1r)-1-phenylethyl]carbamoyl]-4-propan-2-ylpyrazol-3-yl]-3,5-dihydroxyheptanoate Chemical compound C1([C@@H](C)N(C)C(=O)C2=NN(C(CC[C@@H](O)C[C@@H](O)CC([O-])=O)=C2C(C)C)C=2C=CC(F)=CC=2)=CC=CC=C1 MPDDTAJMJCESGV-CTUHWIOQSA-M 0.000 description 1
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to a terphenyl compound, a preparation method and application thereof, wherein the terphenyl compound 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonyl cyclohexadienone is extracted from fermentation liquor of a yellow-handled-mud-bee symbiotic actinomycete, the yellow-handled-mud-bee symbiotic actinomycete belongs to endophytic streptomyces, and 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonyl cyclohexadienone has a certain inhibition effect on tumor cells, so that the 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonyl cyclohexadienone can be used as an antitumor drug lead compound. The 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone has activity inhibiting effect on osteosarcoma cells U2OS, osteosarcoma cells Saos2, oral epidermoid carcinoma cells, and vinblastine resistant cells.
Description
Technical Field
The invention relates to the technical field of microbial engineering, in particular to a novel terphenyl compound 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonyl cyclohexadienone extracted from fermentation liquor of an actinomycete strain, a preparation method and application thereof.
Background
In nature, p-Terphenyls (p-Terphenyls) are rare natural products, p-Terphenyls are important natural products mainly distributed in lichens and fungi, and the Terphenyls have multiple biological activities, including inhibition of tumor cell proliferation, specific inhibition of 5-lipoxygenase, antibacterial property, antioxidation property and the like.
Most of the terphenyl compounds are separated from fungi so far, and the number of the terphenyl compounds separated from streptomyces is less than 10, so that the streptomyces is a good source which is not fully utilized for the discovery of novel terphenyl.
The symbiotic actinomycetes exist in plant bodies and animal bodies, most symbiotic actinomycetes are obtained by separating from the plant bodies at present, the number of symbiotic actinomycetes discovered from insect bodies is small, the living environment of insects is complex and changeable, factors such as eating different types of food and the like endow the characteristics of diversity and complexity of microorganisms in insect intestinal tracts, so that the actinomycetes are separated from the insect bodies, and the natural product is found from new actinomycetes.
Disclosure of Invention
Aiming at the problems in the prior art, the symbiotic actinomycete obtained by separating the yellow-handled mud bee from the body is prepared by using a mixture of n-hexane and acetone in different proportions as an eluent to obtain a terphenyl compound (2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone), the activity test of the anti-tumor cells of the terphenyl compound (2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone) is carried out, and the half inhibition concentration of a tumor cell strain is determined to study the anti-tumor effect of the terphenyl compound (2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone).
One of the purposes of the invention is to provide a symbiotic actinomycete of the yellow-handled mudagrass containing a terphenyl compound, namely Streptomyces 1510-2(Streptomyces sp.1510-2), wherein the strain is preserved in China center for type culture Collection (the address is Wuhan Luo Gashan university in Wuhan) in 2018, 03 and 11 days, and the preservation number is CCTCC NO: M2018111.
The streptomyces 1510-2 used in the invention is a streptomyces isolated from the body of the yellow horned mud bee obtained from the suburb of texas, shandong in 2015, and the strain number is N1510-2. It is grey white in air silk and yellowish brown in basal silk on Gao's first agar culture medium.
The separation and purification method of streptomyces 1510-2 in the yellow horned mud bees comprises the following steps:
under the aseptic condition, disinfecting the body surface of the clitocybe huangliana by 60-80% alcohol, putting the wasps in an aseptic mortar, adding a small amount of sterile water for grinding, carrying out gradient dilution on the grinding liquid by the sterile water, respectively taking each gradient dilution liquid in a Gao-shi culture medium and a YMS culture medium, putting the obtained product in a thermostat at 34-40 ℃ for culture, and after bacterial colonies grow out, selecting spores for purification and culture to obtain the in-vivo symbiotic actinomycetes N1510-2 of the clitocybe huangliana.
Preferably, the alcohol concentration is 70%; the temperature of the incubator was 37 ℃.
The second object of the present invention is to provide a method for fermentation culture of the symbiotic actinomycetes, the method comprising: the streptomycete 1510-2 is obtained by separating and purifying in vivo of the yellow-handled mudbee, and the thallus is inoculated on a Gao-shi No. one solid culture medium and cultured in an incubator;
specifically, the Gao's No. one solid culture medium is prepared from soluble starch and KNO3、K2HPO4、MgSO4·7H2O、NaCl、FeSO4·7H2O, agar and distilled water.
Preferably, the temperature of the incubator is 26-30 ℃, and the culture time is 5-7 days.
The invention also aims to provide a terphenyl compound with anti-tumor activity: 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone, the structural formula of which is:
the terphenyl compound is separated and extracted from a solid culture medium of streptomyces 1510-2.
The fourth purpose of the invention is to provide a preparation method of a terphenyl compound 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonyl cyclohexadienone, which comprises the following specific steps:
collecting and cutting solid culture medium of streptomycete 1510-2, extracting with ethyl acetate, distilling under reduced pressure at 42-47 deg.C under 0.07-0.11MPa, drying, and concentrating to obtain extract; separating ethyl acetate soluble part by silica gel column chromatography, using n-hexane-acetone as eluent, distilling the eluted solution under reduced pressure at 42-47 deg.C under 0.07-0.11MPa to collect the fraction containing terphenyl compound 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone, and recrystallizing the collected fraction to obtain 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone, wherein the n-hexane-acetone ratio is 4-6: 1.
Preferably, the pressure and temperature of reduced pressure distillation after extraction is 0.09MPa and 45 ℃;
preferably, the pressure and temperature of reduced pressure distillation after elution are 0.09MPa and 45 ℃;
the terphenyl compound 2, 5-diphenyl-3-methoxyl-6-hydroxyl-6-acetonyl cyclohexadienone is applied to the development of lead compounds of antitumor drugs.
The tumor cells are osteosarcoma cells (U2OS), osteosarcoma cells (Saos2), oral epidermoid carcinoma cells (KB), and vinblastine resistant (KB) cells (KB/VCR).
An antineoplastic medicine is prepared from terphenyl compound 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone and auxiliary materials including starch and sodium carboxymethylcellulose talc.
The anti-tumor medicine comprises an active component and an auxiliary material; the active component terphenyl compound accounts for 0.01-99.9%.
The invention has the beneficial effects that:
1) the invention separates a streptomycete from the body of yellow-handled mudbee for the first time, and separates and extracts a terphenyl compound (2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonyl cyclohexadienone) from the fermentation liquor of the streptomycete, wherein the 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonyl cyclohexadienone is a compound with a novel structure, can be used as an anti-tumor lead compound, and can be prepared into an anti-tumor medicament;
2)2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonyl cyclohexadienone can be produced by solid fermentation with microorganism, and has simple process and short period;
3) in the preparation method of the terphenyl compound biological 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone, five eluents of n-hexane-acetone are used for eluting products separated by silica gel column chromatography in a ratio of 20:1-1:1, and the elution result is compared with a ratio of the n-hexane-acetone of 4-6: the elution effect is best when 1 is used;
4) the identification of the structure of the terphenyl compound (2, 5-diphenyl-3-methoxyl-6-hydroxyl-6-acetonylcyclohexadienone) of the invention utilizes mass spectrum and nuclear magnetic resonance spectrum (1H-NMR、13C-NMR, DEPT, HMQC, HMBC, COSY, NOE, X-RAY) to obtain the terphenyl compound (2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone) with the spectroscopic data as follows: 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone, yellow crystal, easily soluble in acetone, and having fluorescence absorption under 254 and 365nm ultraviolet;
5) the terphenyl compound (2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone) is used for determining the half inhibitory concentration of four tumor cells, wherein the four tumor cells are respectively: osteosarcoma cells (U2OS), osteosarcoma cells (Saos2), oral epidermoid carcinoma cells (KB) and vinblastine resistant (KB) cells (KB/VCR) are provided by Shandong university (Weihai) laboratories, and the results show that the terphenyl compound has strong inhibitory activity on four tumor cells.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.
FIG. 1 is a graphical representation of the morphological characteristics of Streptomyces 1510-2 on the Gao's No. one medium;
FIG. 2 is a microscopic morphological feature of spores of Streptomyces 1510-2;
FIG. 3 is a phylogenetic tree of Streptomyces 1510-2 strain.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, the conditions are generally as usual or as recommended by the reagents company; reagents, consumables and the like used in the following examples are commercially available unless otherwise specified.
The invention relates to streptomyces flavomarginatus 1510-2, in particular to separation and identification of endophytic streptomyces, solid fermentation, extraction and separation of terphenyl compounds and activity test of antitumor cells.
The present invention provides the following embodiments:
(1) the streptomyces flavomarginatus 1510-2 of the invention is separated from the body of the flavomarginitides through strict surface disinfection.
(2) The streptomyces flavomarginatus 1510-2 is in a gas silk powder shape and a gray brown color on a bacterial colony of a Gao's No. I solid culture medium. Primarily identified as Streptomyces.
(3) The streptomyces flavomarginatus 1510-2 disclosed by the invention is characterized in that air filaments are grey white, basal filaments are yellow brown, spore filaments are long and straight, and form clusters on a Gao's I agar culture medium. The spores are nearly spherical to elliptical and have smooth surfaces. Identified as Streptomyces.
(4) The Streptomyces flavomarginatus 1510-2 is preserved in China center for type culture Collection (address: Wuhan university in mountain of Luo Gashan of Wuhan) in 2018, 03 and 11 days, and the preservation number is CCTCC NO: M2018111.
Example 1
Isolation and purification of Streptomyces flavomarginatus 1510-2
The streptomyces used in the invention is a symbiotic actinomycete obtained by separating in vivo the yellow-handle mud bee obtained from suburbs of texas in 2015, and the strain number is N1510-2.
Under the aseptic condition, disinfecting the body surface of the clitocybe huangliana with 70% alcohol, placing the wasps in an aseptic mortar, adding a small amount of sterile water for grinding, carrying out gradient dilution on the grinding liquid with the sterile water into 10-1,10-2 and 10-3, respectively taking 100 microliters of each gradient dilution liquid in a Gauss culture medium and a YMS culture medium, placing the gradient dilution liquid in a 37 ℃ incubator for culture, picking spores for purification culture after bacterial colonies grow out, and obtaining symbiotic actinomycetes in the clitocybe huangliana.
Example 2: identification of Streptomyces flavomarginatus 1510-2
(1) And (3) morphological identification:
the colony surface of the streptomyces 1510-2 on the Gao's No. I culture medium is gray white, and the circle of the colony is shown in figure 1. And (4) performing microscopic examination by using an optical microscope, wherein spore filaments grow and are straight and clustered. Spores were nearly spherical to elliptical with smooth surfaces and were initially identified as Streptomyces as shown in FIG. 2.
(2) And (3) molecular identification: extracting DNA from the mycelium according to the instruction of a rapid extraction kit for genome DNA of the bacteria, wherein a forward primer is Fd 2: 5' -GAGTTTGATCATGGCTCAG-3, and reverse primer 16 Sr: 5' -TTGCGGGACTTAACCCAACAT-3, performing PCR amplification on a 16SrDNA sequence, purifying a PCR product, sequencing the PCR product by using a Cissima corporation, performing BLAST comparison analysis on a sequencing result, selecting a similar sequence, constructing a phylogenetic tree by using MEGA5.0 software, and determining the genus classification status of the 1512-16 strain. After performing Blast alignment in the GenBank database, strains having homology higher than 97% were selected for phylogenetic analysis (FIG. 3), and it was found that 1510-2 was clustered in the same branch with Streptomyces antibioticus FJ883742.1 and Streptomyces griseoruber FJ919750.1, and the sequence similarity reached 99%. Accordingly, symbiotic actinomycete 1510-2 was identified as Streptomyces.
By combining the characteristics, the strain 1510-2 of the invention is determined to be streptomyces according to streptomyces identification handbook and molecular identification. The strain is preserved in China center for type culture Collection in 2018, 03 and 11 months, and the preservation number is CCTCC NO: M2018111.
Example 3
Solid fermentation of Streptomyces flavomarginatus 1510-2
Activated Streptomyces 1510-2 was streaked out by inoculating with an inoculating loop on a solid culture medium of Kao-shi No. one, and cultured in an incubator at 28 ℃ for 5 to 7 days.
Example 4
Extraction and separation of 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone
Collecting and shearing the solid agar in the embodiment 3, extracting with ethyl acetate, carrying out reduced pressure distillation, concentrating and recovering a solvent to obtain a total extract, carrying out chromatographic separation on an ethyl acetate soluble part by using a silica gel column, collecting a fraction containing a terphenyl compound 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone at a section of 4-6:1 of n-hexane-acetone by using n-hexane-acetone (20:1-1:1) as an eluent, and recrystallizing to obtain a pure product of the 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone.
Example 5
Structural identification of 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone
The structure of 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone is determined according to mass spectrum and nuclear magnetic resonance spectrum (1H-NMR、13C-NMR, DEPT, HMQC, HMBC, COSY, NOE, X-RAY).
The spectroscopic data for 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone were as follows:
2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone, yellow crystal, easily soluble in acetone, fluorescent absorption under 254 and 365nm ultraviolet,1h and13C-NMR data are shown in Table 1
TABLE 11H and13C-NMR data
Example 6
Activity test of 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone against tumor cells
Osteosarcoma cells (U2OS), osteosarcoma cells (Saos2), oral epidermoid carcinoma cells (KB), vinblastine resistant (KB) cells (KB/VCR) were provided by the university of Shandong (Wiegand) laboratories. Culturing cells in 199 culture medium, adding 10% calf serum, and culturing at 37 deg.C under 5% CO2In the incubator. The monomer compound is terphenyl compound 2, 5-diphenyl-3-methoxyl-6-hydroxy-6-acetonyl cyclohexadienone identified by chemical structure.
Test method MTT colorimetric method is adopted to determine half inhibitory concentration IC of the compound50. Collecting cells in logarithmic phase according to cell growth rate, adjusting cell suspension concentration to 3-5 × 104Perml, inoculated in 96-well plates, 100ul per well, 5% CO2Culturing at 37 deg.C for 24 hr, adding medicine with concentration gradient, setting 3 multiple wells, incubating for 48 hr, adding 20ul 0.5% MTT solution into each well, culturing for 4 hr, carefully removing culture solution from the wells, washing with PBS, adding 150ul dimethyl sulfoxide into each well, shaking on a shaker at low speed for 10min to dissolve the crystal, and detecting with ELISA OD570Measuring absorbance of each well at nm, and calculating IC50。
TABLE 2 inhibition of tumor cells by 2, 5-Diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone
IC50(μ M): indicating that the half effective inhibitory concentration of the terphenyl compound of the invention.
The results show that the 2, 5-diphenyl-3-methoxy-6-hydroxy-6-acetonylcyclohexadienone has stronger inhibitory activity on four tumor cells, particularly oral epidermoid carcinoma cells (KB).
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
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