CN117343866A - Streptomyces bobili KQ-K6 anthraquinone compound and application thereof - Google Patents
Streptomyces bobili KQ-K6 anthraquinone compound and application thereof Download PDFInfo
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- CN117343866A CN117343866A CN202311208751.8A CN202311208751A CN117343866A CN 117343866 A CN117343866 A CN 117343866A CN 202311208751 A CN202311208751 A CN 202311208751A CN 117343866 A CN117343866 A CN 117343866A
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- PYKYMHQGRFAEBM-UHFFFAOYSA-N anthraquinone Natural products CCC(=O)c1c(O)c2C(=O)C3C(C=CC=C3O)C(=O)c2cc1CC(=O)OC PYKYMHQGRFAEBM-UHFFFAOYSA-N 0.000 title claims abstract description 64
- -1 anthraquinone compound Chemical class 0.000 title claims abstract description 64
- 241000958260 Streptomyces bobili Species 0.000 title claims abstract description 36
- 230000000259 anti-tumor effect Effects 0.000 claims abstract description 37
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 36
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C07C2603/42—Ortho- or ortho- and peri-condensed systems containing four condensed rings containing only six-membered rings
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Abstract
The invention discloses an anthraquinone compound with antineoplastic activity, which is prepared by fermenting Streptomyces bobiliKQ-K6 strain, separating Streptomyces bobili KQ-K6 strain from Streptomyces solani, fermenting and culturing the strain,repeatedly extracting with ethyl acetate to obtain crude extract, purifying the crude extract with silica gel column by high performance liquid chromatography to obtain anthraquinone compound with antitumor activity, and performing antitumor activity experiment to prove that the compound has inhibition rates of 88.63% and 80.96% on human colon cancer cell HCT-116 and breast cancer cell strain 2316, and IC 50 The compounds obtained by separation and purification of the invention are respectively 9.83 mu M and 12.61 mu M, which shows that the compounds can be used for preparing antitumor drugs for inhibiting colon cancer and breast cancer cell lines.
Description
Technical Field
The invention belongs to the field of microbial medicaments, and particularly relates to Streptomyces bobili KQ-K6 for producing anthraquinone compounds with antitumor activity, and preparation and application thereof.
Background
Anthraquinone compounds are a common class of compounds found in natural environments, where the yield of anthraquinone compounds in plant rhizosphere is relatively high, in terms of biological activity: most anthraquinone compounds have antibacterial and antitumor activities, are also clinically used for treating bacterial infection, and at present, the research of the anthraquinone compounds has become a research hotspot.
Global primary tumor drugs were sold in dollars in 1430 billions in 2019, accounting for 20% of the global drug market, with five major disease categories of breast cancer, lung cancer, prostate cancer, lymphoma, and myeloma being sold in 900 billions. The next 5 years predicted a 12% increase in the composite year, with 2024 reaching a 2500 billion dollar scale and the five-tumor drug market above reaching 1410 billion dollars. There are 1700 tumor drugs in clinical research stage, which account for 30% of all clinical drugs, so the importance and acceptance of tumors by society is undoubted. The success rate of clinical research and development of the tumor medicine is 13%, the average time is 9.5 years, and the anthraquinone compound is one of main natural products with remarkable biological activity and is mainly used as an anti-tumor and antibiotic medicine.
The streptomyces microorganism can synthesize secondary metabolites with various structures such as alkaloid, anthraquinone, lactone, flavone, phenols and the like; meanwhile, the metabolites also have various biological activities of antagonizing pathogenic bacteria, resisting tumors, inhibiting kinase and the like. However, with the advent of the new strain source "golden age" of streptomyces, genomic analysis and information technology improvement screening model are performed on the secondary metabolite gene cluster of streptomyces based on the original research, compound structure is improved, metabolic pathway screening is performed, separation and purification technology is improved, expression of the strain silencing gene is activated by genetic engineering means to mine new secondary metabolites, and the method becomes a current research hot spot. Streptomyces scab is a plant pathogenic Streptomyces scab causing potato, which is isolated from potato tubers growing with typical scab spots or potato rhizosphere soil in which it grows, and this particular growth metabolic environment creates a basis for the later screening of active Streptomyces scab strains with novel structures. At present, research on Streptomyces solani is concentrated on the level of plant pathogenic toxin Thaxtomin and species diversity, and research on other metabolites and biological activity is not reported yet, and research on the digging and biological activity of natural products is further clarified.
Disclosure of Invention
Aiming at the biological activity of anthraquinone compounds and the fact that the existing anthraquinone compounds are mainly obtained by chemical synthesis and streptomycete fermentation and separation, the microbial strain capable of efficiently producing anthraquinone compounds is few, and no literature or no document is foundThe patent reports that the anthraquinone compound is obtained from potato streptomyces scab through separation and purification, the invention aims to provide Streptomyces bobili KQ-K6 for producing the anthraquinone compound with anti-tumor activity, the preparation and the application thereof, the invention separates Streptomyces bobili KQ-K6 from potato tubers and plant rhizosphere soil in Xinjiang region, the anthraquinone compound with anti-tumor activity is obtained through fermentation culture by using a special liquid culture medium, extraction, separation and purification by ethyl acetate, and the anti-tumor activity experiment shows that the anthraquinone compound has inhibitory activity on proliferation of colon cancer cell strains HCT-116 and breast cancer cell strain 2133, and the tumor cell inhibition rates are 88.63% and 80.96%, respectively, and IC 50 The method is 9.83 mu M and 12.61 mu M respectively, which shows that the anthraquinone compound is obtained by adopting the Streptomyces bobili KQ-K6 crude extract to separate and purify, and the anthraquinone compound is used for preparing medicines of colon cancer cells and breast cancer cells, has wide application prospect, and provides natural anthraquinone compounds for treating tumors.
In order to achieve the technical effects, the invention is realized by the following technical scheme.
The invention provides a strain Streptomyces bobili KQ-K6 for high yield of anthraquinone compounds with antitumor activity, which is preserved in China general microbiological culture Collection center with a strain preservation number of CGMCC No.27670.
The Streptomyces bobili KQ-K6 provided by the invention is screened and separated from potato tubers and plant rhizosphere soil in Uygur autonomous region in Xinjiang, and the strain KQ-K6 belongs to streptomyces scab through 16Sr DNA sequence phylogenetic analysis and morphological analysis. By sequencing the strain gene, the obtained sequence was analyzed by BLAST alignment at NCBI website, and the 16SrDNA gene sequence of strain KQ-K6 showed the highest similarity with Streptomyces bobili strain NBRC 16166 (NR_ 112584.1) of 99.86%. And (3) selecting a sequence with higher homology to construct a 16SrDNA phylogenetic tree, wherein the genetic relationship between the strain KQ-K6 and Streptomyces bobili strain NBRC 16166 (NR_ 112584.1) is nearest. The strain KQ-K6 is determined to be Streptomyces bobili through serial multiphase classification and identification, and the strain Streptomyces bobili with the number KQ-K6 belongs to the strain in the streptomyces scab as is known in the art.
The Streptomyces bobili KQ-K6 provided by the invention is subjected to molecular level identification of a strain system, morphological identification, physiological and biochemical characteristics and other multiphase classification system identification analysis by combining the molecular level identification of the strain system, the invention relates to a plurality of differences between the strain KQ-K6 and a standard mode strain with the closest homology of the strain KQ-K6, and proves that the strain KQ-K6 belongs to a conventional strain of Streptomyces scab, and is named Streptomyces bobili KQ-K6 and is preserved in China general microbiological culture collection center with the strain preservation number of CGMCC NO.27670 and the preservation date: 2023, 06, 21.
The gene sequence of the Streptomyces bobili KQ-K6 is shown as SEQ ID NO: 1.
The invention provides an anthraquinone compound with antitumor activity produced by Streptomyces bobili KQ-K6, and the structure of the compound is shown as a formula (I):
the preparation method of the anthraquinone compound with anti-tumor activity produced by Streptomyces bobili KQ-K6 specifically comprises the following steps:
(1) Inoculating the preserved strain Streptomyces bobili KQ-K6 into 500mL conical flasks, wherein each flask contains 200mL of special liquid culture medium, and performing shake culture at 28 ℃ and 180rpm for 3-4d to obtain seed liquid of the strain;
(2) Inoculating 2mL of the strain seed solution obtained in the step (1) into a special liquid culture medium, and performing shake culture at 28 ℃ and 180rpm for 10d to obtain a liquid fermentation broth of the strain;
(3) Repeatedly extracting the supernatant of the liquid fermentation broth 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 eluting the crude extract obtained in the step (3) by a silica gel column, and adopting silica gel 100-200 meshes of 1:1.5 to mix samples, wherein 300-400 meshes of silica gel 1:20 are filled with the silica gel column, wherein an eluting system is petroleum ether-ethyl acetate (PE: EA) solution and methylene dichloride-methanol (D: M) solution, and the volume ratio of PE to EA is 10:1,5:1 and 1:1; and D, the volume ratio of M is 40:1,30:1,20:1,10:1,5:1 and 0:1, and the anthraquinone compound with anti-tumor activity is obtained through preparative high performance liquid chromatography purification according to analysis liquid chromatography conditions.
In the preparation method of the anthraquinone compound with anti-tumor activity, the special liquid culture medium (g/L) is as follows: 20g of soluble starch, 0.5g of magnesium sulfate, 0.5g of sodium chloride, 0.5g of dipotassium hydrogen phosphate, 1.0g of potassium nitrate, 0.01g of ferrous sulfate, 1000mL of water, pH=7.2 and sterilization at 121 ℃ for 20 min.
In the preparation method of the anthraquinone compound with anti-tumor activity, in the purification of the high performance liquid chromatography, the mobile phase condition is 70% -100% methanol to water, the elution time is 40min, the flow rate is 10mL/min, and the detection wavelength lambda=230 nm; repeatedly washing the bottle with methanol, collecting fraction of 31min, steaming, and dripping appropriate amount of methanol to dissolve thoroughly to obtain monomer compound K6-6 of 0.9mg, tR=31 min, and monomer compound purity of 91%.
Meanwhile, the invention provides application of the anthraquinone compound with anti-tumor activity in preparing anti-tumor drugs.
Further, the anthraquinone compound is applied to the preparation of antitumor drugs, and the anthraquinone compound is applied to the preparation of drugs for inhibiting colon cancer and breast cancer.
The invention also provides application of the strain Streptomyces bobili KQ-K6 for producing the anthraquinone compounds in preparing the anthraquinone compounds with anti-tumor activity.
Through the technical scheme, the invention has the following technical effects:
(1) The anthraquinone compound with anti-tumor activity is produced by fermenting streptomyces potato scab with a special liquid culture medium, repeatedly extracting liquid fermentation culture with ethyl acetate, and separating and purifying with silica gel column chromatography and preparative high performance liquid chromatography.
(2) The invention provides a strain Streptomyces bobili KQ-K6 for preparing anthraquinone compounds with anti-tumor activity, which is produced by liquid fermentation, wherein the anthraquinone compounds with anti-tumor activity are obtained by extracting liquid fermentation culture with ethyl acetate, separating and purifying with silica gel column chromatography and preparative high performance liquid chromatography, and the structure identification shows that the anti-tumor activity of the anthraquinone compounds is 4-deoxypyrrolomycin ketone, the inhibition rate of the anthraquinone compounds on proliferation of human colon cancer cell lines HCT116 and breast cancer 2316 cell lines is 88.63% and 80.96%, respectively, and the IC is 50 9.83 mu M and 12.61 mu M, which shows that the compound obtained by the separation of the invention can be used for preparing medicines for inhibiting colon cancer and breast cancer.
Drawings
FIG. 1 shows a phylogenetic tree constructed as Streptomyces bobili KQ-K6 based on the 16SrDNA gene.
FIG. 2 shows a colony characterization of Streptomyces bobili KQ-K6, wherein A is the colony morphology of the strain and B is an electron micrograph of spores thereof at 10 Xmagnification.
FIG. 3 is a spectrum of an anthraquinone compound obtained by the present invention, wherein A is 1 H NMR, B is 13 C NMR chart, C HMQC chart, D HMBC chart.
FIG. 4 shows an MS chart of the anthraquinone compound obtained by the present invention.
Detailed Description
Example 1: isolation and purification and identification of Streptomyces bobili KQ-K6
(one) separation and purification
The plate dilution method is adopted. Sampling from potato tubers and plant rhizosphere soil in Uygur autonomous region of Xinjiang, naturally airing for 7-10d, weighing a certain amount of air-dried soil sample, performing high-temperature drying treatment at 85 ℃ for 3-4 h, placing the soil sample in a triangular flask containing 90mL of sterile water, shaking for 2d at 120rpm and 30 ℃ to obtain soil suspension, then injecting into a test tube containing 9mL of sterile water, sequentially carrying out gradient dilution, and diluting to 10 ℃ by using sterile water -4 Actinomycetes are separated by a dilution coating plate method and a plant tissue separation method. 200. Mu.L of each concentration gradient of the dilution was aspirated,respectively connecting the strain with 3 solid plate culture mediums, uniformly coating each gradient with a coating rod, standing at 30 ℃ for inversion culture, picking single bacterial colony, streaking and purifying on the solid culture mediums by streak separation to obtain purified bacterial strains, and preserving at 4 ℃ inclined plane; the isolation medium was: 20g of soluble starch, 0.5g of magnesium sulfate, 0.5g of sodium chloride, 0.5g of dipotassium hydrogen phosphate, 1.0g of potassium nitrate, 0.01g of ferrous sulfate, 1000mL of water, pH 7.2 and high-temperature high-pressure sterilization at 121 ℃ for 20 min; the purified medium was: 20g of soluble starch, 0.5g of magnesium sulfate, 0.5g of sodium chloride, 0.5g of dipotassium hydrogen phosphate, 1.0g of potassium nitrate, 0.01g of ferrous sulfate, 1000mL of water, pH 7.2 and high-temperature high-pressure sterilization at 121 ℃ for 20 min.
(II) 16SrDNA Gene identification
Extraction of DNA
Streptomyces bobili KQ-K6 is inoculated in Gaoshi No. 1 liquid culture medium, cultured for 4 days at 28 ℃ and 180rpm, bacterial cells are obtained, and the novel plant genome DNA rapid extraction kit is adopted for genome DNA extraction.
PCR amplification
The PCR reaction system is 20 mu L by adopting a streptomycete 16S rDNA universal amplification primer: 10 μL Mix; 2. Mu.L template, 1. Mu.L each of the upstream and downstream primers, dd H 2 O6.4. Mu.L. The PCR products were detected by 1% agarose gel electrophoresis. The universal primer is PA/PH. Is contained by Dingguo Chang the company performs the measurement. Sequencing results were aligned at NCBI and phylogenetic tree was constructed using Mega7.0. The primer is as follows:
table 1: PCR reaction system
Table 2: strain identification and related primer sequences
3. Sequencing
The PCR amplified product is sequenced after electrophoresis detection and purification, the sequence length is 1409bp, and the sequencing result is shown in SEQ ID No:1, BLAST homologous sequence search is carried out on NCBI, a phylogenetic tree (repeated sampling 1000 times) is established by utilizing MEGA7.0 software commonly adopted in the field through a Neighbor-Joining method, the result is shown in figure 1, the obtained sequence is compared and analyzed on NCBI website, the homology of the 16Sr DNA gene sequence of a strain KQ-K6 with Streptomyces bobili strain NBRC 16166 (NR-112584.1) is highest, the similarity is 99.86%, and in the phylogenetic tree constructed by the 16Sr DNA gene sequence, the relationship between the 16Sr DNA sequence of the strain KQ-K6 and Streptomyces bobili strain NBRC 16166 (NR-112584.1) strain is nearest, which indicates that the strain KQ-K6 is Streptomyces bobili conventional strain and can be purchased through the well-known channels such as China general microbiological culture collection center.
Colony morphology characterization of (III) Streptomyces bobili KQ-K6
The strain KQ-K6 to be observed is inoculated on a Gao's first plate, shake cultivation is carried out for 4 days at 28 ℃ and 180rpm, the colony is observed to be full of the whole plate, the characteristic record of the colony is observed, the record is photographed and stored, meanwhile, the scanning electron microscope photograph of the colony is recorded, and the record result is shown in the figure 2.
As shown in the figure 2, the strain KQ-K6 grows on a solid culture medium of Gaoshi No. 1, the colony is round, the surface is convex and is provided with folds, the surface can generate off-white spores to cover the colony after the colony grows for a certain time, and the strain is further identified as a conventional strain of Streptomyces scab Streptomyces bobili by combining with 16Sr DNA molecular biology, and can be purchased through the common microorganism center and other well-known channels of China Committee for culture Collection of microorganisms.
Example 2: preparation of anthraquinone compound with antitumor activity
The anthraquinone compound is obtained by separating and purifying a fermentation culture of the strain Streptomyces bobiliKQ-K6 provided in the example 1, and the preparation method comprises the following steps:
(1) Inoculating the preserved strain Streptomyces bobili KQ-K6 into 500mL conical flasks, wherein each flask contains 200mL of special liquid culture medium, and performing shake culture at 28 ℃ and 180rpm for 3-4d to obtain seed liquid of the strain;
(2) Inoculating 2mL of the strain seed solution obtained in the step (1) into a special liquid culture medium, and performing shake culture at 28 ℃ and 180rpm for 10d to obtain a liquid fermentation broth of the strain;
(3) Repeatedly extracting the supernatant of the liquid fermentation broth 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 eluting the crude extract obtained in the step (3) by a silica gel column, and adopting silica gel 100-200 meshes of 1:1.5 to mix samples, wherein 300-400 meshes of silica gel 1:20 are filled with the silica gel column, wherein an eluting system is petroleum ether-ethyl acetate (PE: EA) solution and methylene dichloride-methanol (D: M) solution, and the volume ratio of PE to EA is 10:1,5:1 and 1:1; and D, the volume ratio of M is 40:1,30:1,20:1,10:1,5:1 and 0:1, and the anthraquinone compound with anti-tumor activity is obtained through preparative high performance liquid chromatography purification according to analysis liquid chromatography conditions. In the preparation method of the anthraquinone compound with anti-tumor activity, the special liquid culture medium (g/L) is as follows: 20g of soluble starch, 0.5g of magnesium sulfate, 0.5g of sodium chloride, 0.5g of dipotassium hydrogen phosphate, 1.0g of potassium nitrate, 0.01g of ferrous sulfate, 1000mL of water, pH=7.2 and sterilization at 121 ℃ for 20 min.
The chromatographic conditions of the high performance liquid elution of the compound are as follows: adopts octadecyl bonded silica gel as filler, and the gradient elution condition is as follows: 70% -100% of methanol and water (no acid is added), 40min, the flow rate is 10mL/min, and the detection wavelength lambda=230 nm. Repeatedly washing bottle with methanol, collecting fraction of 31min, steaming, adding appropriate amount of methanol dropwise to dissolve thoroughly, detecting by liquid chromatography, collecting monomer compound with purity of 91% to obtain monomer compound K6-6 (0.9 mg, t) R ＝31min)。
Example 3: isolation and identification of Compounds
In this example, based on example 2, the supernatant of the liquid fermentation broth containing the anthraquinone compound was repeatedly extracted 3 times with ethyl acetate, and the recovered solvent phase was concentrated to obtain a crude product. Separating the crude material by silica gel column chromatography, wherein the elution system is petroleum ether and ethyl acetate=1:0, 10:1,5:1,1:1; dichloromethane: methanol=40:1, 30:1,20:1,10:1,1:0, tlc analysis of the fractions combined with similar compounds. The obtained component containing the specific compound is subjected to preparative high performance liquid chromatography (LC-3000 (10 μm, 21.2X1250 mm) column, detection wavelength lambda=230nm), gradient elution of 70% -100% methanol/water (no acid addition) mobile phase, 40min, flow rate: the solvent is recovered at 10mL/min to obtain a compound 1, the structure of the compound 1 is identified, and a specific test result is shown in figure 3.
As can be seen from the data of FIG. 3, the anthraquinone compound obtained by the invention is brown powder (MeOH), and the excimer ion peak is M/z413.1235[ M+H ] according to mass spectrum] + (C 22 H 20 O 8 ，413.1236)，m/z435.1058[M+Na] + From this, it was determined that the molecular formula was C 22 H 20 O 8 。 1 H NMR(CD 3 OD,400 Hz) data display: delta 7.65 (s, 1H), 7.33-7.29 (m, 2H), 3.95 (s, 1H), 3.72 (s, 4H), 3.02 (d, j=19.0 hz, 1H), 2.83 (t, j=18.6 hz, 1H), 2.27-2.21 (m, 1H), 1.94 (d, j=13.8 hz, 1H), 1.73-1.67 (m, 1H), 1.60-1.53 (m, 2H), 1.28 (s, 3H), 1.07 (t, j=7.5 hz, 4H), 0.91-0.87 (m, 1H); 13 C NMR(CD 3 OD100 Hz) data display: delta 186.04, 173.92, 161.87, 158.90, 143.69, 131.65, 130.47, 130.22, 121.21, 113.90, 74.94, 71.02, 66.40, 59.51, 56.78, 54.86, 52.68, 33.53, 30.55, 28.74, 22.05, 20.85,6.97, compound K6-6 is an anthraquinone compound 4-Deoxy-epsilon-pyrromycin.
Example 4: application of anthraquinone compounds
In the embodiment, on the basis of the embodiment 1-3, the anthraquinone compounds after separation and purification are applied to the preparation of medicines for inhibiting the growth of colon cancer cells and breast cancer cells, and the inhibition effect on the growth of colon cancer cells HCT116 and breast cancer cells 2316 is examined.
The test cells were colon cancer cells HCT116 and breast cancer cells 2316. The compounds were dissolved in DMSO and configured 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 antitumor activity of the compound is detected by a Sulfonyl Rhodamine B (SRB) colorimetric method, and the specific operation is as follows: colon cancer HCT116 cells were configured to 8×10 3 Cell suspension with concentration of each mL is taken, 100 mu L is injected into a 96-well plate, two repeats are arranged, the cell suspension is placed in an incubator containing 5% carbon dioxide for culturing for 24 hours at 37 ℃, and the cell suspension is dosed, doxorubicin is taken as a positive drug, 2 auxiliary holes are arranged for each concentration, and the culture is continued for 72 hours. After 72h, the 96-well plate was removed, 100. Mu.L of 10% aqueous trichloroacetic acid was added to each well after the medium was discarded, and the wells were placed in a refrigerator at 4℃and fixed for 2h or more. After the fixation is completed, the 96-well plate is taken out, the trichloroacetic acid solution is discarded, the solution is washed for at least 4 times by water and dried, then 80 mu L of 0.4% SRB solution prepared by 1% acetic acid is added for dyeing for 20min, 1% acetic acid aqueous solution is prepared during the dyeing, after the dyeing is finished, the solution is washed for 4 times by 1% acetic acid aqueous solution to remove unbound dye, and the solution is dried again. After the 96-well plate was completely dried, 100. Mu.L of 10mM Tris solution was added to each well, and the protein-bound dye was completely dissolved by shaking, and absorbance was measured at 515nm in a microplate reader, with specific results shown in tables 3 and 4.
Table 3: compound 1 of the invention (10 mug/mL) has inhibition ratio of proliferation of colon cancer cell strain HCT116 (ADR is positive control doxorubicin)
| Compounds of formula (I) | Inhibition rate |
| 1 | 88.63±0.72 |
| ADR | 98.31±0.92 |
As shown in the experimental data of Table 3, the inhibition rate of the obtained compound 1 of the invention on the proliferation of colon cancer cells HCT116 cells is 88.63%, and the inhibition rate of the positive drug doxorubicin on the proliferation of colon cancer cells HCT116 cells is 98.31%, which indicates that the obtained compound 1 of the invention has potential inhibition activity on colon cancer cells HCT 116.
Table 4: inhibition of proliferation of breast cancer cell line 2316 by Compound 1 of the present invention (10. Mu.g/mL) (ADR is positive control doxorubicin)
| Compounds of formula (I) | Inhibition rate |
| 1 | 80.96±1.44 |
| ADR | 97.41±1.23 |
As shown in the experimental data of Table 4, the inhibition rate of the compound 1 isolated by the invention on the proliferation of the breast cancer cells 2316 is 80.96%, and the inhibition rate of the doxorubicin, a positive drug, on the proliferation of the breast cancer cells 2316 is 97.41%, which indicates that the compound 1 isolated by the invention has potential inhibition activity on the breast cancer cells 2316.
Further proved by experimental verification, the anthraquinone compound with anti-tumor activity obtained by adopting the Streptomyces bobili KQ-K6 fermentation, separation and purification provided by the invention is an anthraquinone compound 4-oxygen-epsilon-pyrromycin which has 88.63% and 80.96% inhibition rate on proliferation of a human colon cancer cell strain HCT116 and a breast cancer cell strain 2316 respectively through structural identification, and the anti-tumor activity experiment shows that the compound obtained by the separation of the invention can be used for preparing medicaments for inhibiting human colon cancer and breast cancer and provides natural anthraquinone compounds for treating tumors.
The above examples are only illustrative of the invention and are not intended to be limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. All embodiments need not be nor can be exemplified here. While remaining within the scope of the invention, obvious variations or modifications thereof are contemplated.
Claims (8)
1. The strain Streptomyces bobili KQ-K6 for high yield of anthraquinone compounds with antitumor activity is characterized in that the strain Streptomyces bobili KQ-K6 is preserved in China general microbiological culture collection center (CGMCC) with the strain preservation number of CGMCC NO.27670, and the gene sequence of the strain Streptomyces bobiliKQ-K6 is shown as SEQ ID NO: 1.
2. An anthraquinone compound with anti-tumor activity, characterized in that the compound produced by using the strain Streptomyces bobili KQ-K6 of the anthraquinone compound with high yield and anti-tumor activity as set forth in claim 1 has the structure shown in the formula (I):
3. the method for preparing an anthraquinone compound with antitumor activity according to claim 2, comprising the following steps:
(1) Inoculating the preserved strain Streptomyces bobili KQ-K6 into 500mL conical flasks, wherein each flask contains 200mL of special liquid culture medium, and performing shake culture at 28 ℃ and 180rpm for 3-4d to obtain seed liquid of the strain;
(2) Inoculating 2mL of the strain seed solution obtained in the step (1) into a special liquid culture medium, and performing shake culture at 28 ℃ and 180rpm for 10d to obtain a liquid fermentation broth of the strain;
(3) Repeatedly extracting the supernatant of the liquid fermentation broth 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 eluting the crude extract obtained in the step (3) by a silica gel column, and adopting silica gel 100-200 meshes of 1:1.5 to mix samples, wherein 300-400 meshes of silica gel 1:20 are filled with the silica gel column, wherein an eluting system is petroleum ether-ethyl acetate (PE: EA) solution and methylene dichloride-methanol (D: M) solution, and the volume ratio of PE to EA is 10:1,5:1 and 1:1; and D, the volume ratio of M is 40:1,30:1,20:1,10:1,5:1 and 0:1, and the anthraquinone compound with anti-tumor activity is obtained through preparative high performance liquid chromatography purification according to analysis liquid chromatography conditions.
4. The method for preparing anthraquinone compounds with antitumor activity as set forth in claim 3, wherein said special liquid medium is: 20g of soluble starch, 0.5g of magnesium sulfate, 0.5g of sodium chloride, 0.5g of dipotassium hydrogen phosphate, 1.0g of potassium nitrate, 0.01g of ferrous sulfate, 1000mL of water, pH=7.2 and sterilization at 121 ℃ for 20 min.
5. The method for preparing an anthraquinone compound with antitumor activity according to claim 3, wherein in the purification by high performance liquid chromatography, the mobile phase condition is 70% -100% methanol/water, the elution time is 40min, the flow rate is 10mL/min, and the detection wavelength λ=230 nm; repeatedly washing bottle with methanol, collecting fraction of 31min, steaming, and dripping appropriate amount of methanol to dissolve thoroughly to obtain monomer compound K6-6 of 0.9mg, t R =31 min, monomer compound purity was 91%.
6. The use of an anthraquinone compound with antitumor activity as claimed in claim 2 for the preparation of antitumor drugs.
7. The application of the anthraquinone compound with anti-tumor activity in preparing anti-tumor drugs according to claim 6, wherein the application of the anthraquinone compound in preparing drugs for inhibiting colon cancer or breast cancer provides natural anthraquinone compounds for treating tumors.
8. The use of the strain Streptomyces bobiliKQ-K6 of anthraquinone compounds with antitumor activity according to claim 1 for preparing anthraquinone compounds with antitumor activity.
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