CN110627640B

CN110627640B - Preparation and medical application of streptomyces xanthioides acid and streptomyces xanthione

Info

Publication number: CN110627640B
Application number: CN201910730614.8A
Authority: CN
Inventors: 张治针; 陈梦宣; 柴卫云; 连晓媛
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2017-09-19
Filing date: 2017-09-19
Publication date: 2020-10-30
Anticipated expiration: 2037-09-19
Also published as: CN107619803A; CN107619803B; CN110627640A

Abstract

The invention provides preparation and medical application of dark yellow streptomycete and dark yellow streptomycete, the classification name of the dark yellow streptomycete is Streptomyces fulvissimus ZZ406, the dark yellow streptomycete is preserved by the China center for type culture Collection-Wuhan center, the preservation number is CCTCC NO: M2017435, the preservation date is as follows: 8/3/2017. The compound of the streptomyces darkoflavus (1) and the streptomyces darkoflavus (2) obtained by utilizing the streptomyces darkoflavus have anti-glioma activity, can obviously inhibit the proliferation of different glioma cells and obviously reduce the protein expression level of glycolytic metabolism key enzyme of the glioma cells, has a unique anti-tumor action mechanism, and can be applied to the preparation of drugs for treating the glioma. The structural formulas of the compounds of the dark yellow streptomycic acid (1) and the dark yellow streptomycone (2) are as follows:

Description

Preparation and medical application of streptomyces xanthioides acid and streptomyces xanthione

the present case is application number: 201710846707.8, filing date: 2017.9.19, title of the invention: preparation of Streptomyces xanthylifolius acid and Streptomyces xanthylifolius ketone and division application of medical application thereof are provided.

Technical Field

The invention belongs to the field of medicines, and relates to a method for preparing antineoplastic active compounds of endoxanthostreptomycin and endoxanthostreptomycin from marine actinomycete streptomyces fulvissimus ZZ406, and application of the antineoplastic active compounds of endoxanthostreptomycin and endoxanthostreptomycin in preparation of medicines for treating brain glioma.

Background

Gliomas are the most common brain tumors in the brain and have a high mortality rate. Since gliomas are mostly located in important functional areas of the brain, surgery is difficult and it is not easy to completely remove the tumor, so radiotherapy and drugs are more important for the treatment of gliomas. However, there is a serious deficiency in current anti-glioma drugs, mainly including a few anti-glioma drugs, including traditional carmustine (carmustine), lomustine (lomustine), procarbazine (procarbazine), and the second generation Temozolomide (TMZ), which is the only first-line drug of choice for glioma treatment alone. Moreover, most of the existing glioma treatment drugs including temozolomide are cytotoxic alkylating agents, and have the defects of limited curative effect, great toxic and side effects and serious drug resistance. Therefore, a novel anti-glioma drug with better curative effect, higher safety and unique action mechanism is urgently needed clinically. The secondary metabolite derived from marine microorganisms is an important resource for discovering the antitumor drug or antitumor drug lead compound. Reprogramming of tumor cell metabolism provides a new target for research and development of novel anti-glioma drugs.

The excessive Glycolysis (Glycolysis) of tumor cells to meet their rapid unrestricted proliferation is one of the prominent metabolic features of tumor cells as distinct from normal cells. A plurality of key enzymes which are specifically depended by glioma cells and are highly expressed by taking a large amount of glucose from a microenvironment by the tumor cells in a glycolysis link comprise hexokinase 2(HK2), phosphofructokinase/fructose 2, 6-diphosphatase (PFKFB3), pyruvate kinase M2(PKM2), lactate dehydrogenase 5(LDH5) and the like which are jointly involved in producing a large amount of intermediate products and final products of lactic acid, wherein the intermediate products are important starting materials for synthesizing biomacromolecules by the tumor cells, and the lactic acid is secreted out of the cells to inhibit the cleaning capacity of immune cells of the body to the tumor cells and promote the diffusion and radiation of the tumor cells, and the highly expressed glycolysis key enzyme HK2 can also improve the resistance of the tumor cells to radiotherapy and temozolomide chemotherapy. It is understood that it is the ability of tumor cells to highly utilize their metabolic intermediates to synthesize biomacromolecules, such as DNA, RNA, proteins and biofilms, to promote their rapid and unlimited proliferation, and that specific key enzymes that regulate glycolysis of tumor cells can effectively inhibit the proliferation of tumor cells. Therefore, a drug targeting a multi-target effect of a specific key enzyme of tumor metabolism may have a better anti-tumor efficacy. The Streptomyces fuscous acid and Streptomyces fuscous ketone are two new compounds separated from marine bacteria Streptomyces fuscissimusZZ 406, have obvious inhibition effect on the proliferation of various glioma cells, can obviously reduce the protein expression levels of important specific key enzymes HK2, PFKFB3, PKM2 and LDH5 of glycolysis of the glioma cells, and have unique anti-tumor action mechanisms, so the Streptomyces fuscous acid and the Streptomyces fuscous ketone have application prospects in the aspect of preparing anti-glioma medicaments.

Disclosure of Invention

The invention aims to provide Streptomyces fulvescens which is classified and named as Streptomyces fulvisimuZZ 406 and is preserved by the China center for type culture Collection, Wuhan center, with the preservation number of CCTCC NO: m2017435, date of deposit: 8/3/2017. The streptomyces aureofuscus is separated from marine animal malvastrum longissima (Haliplanella lineata) and obtained by separation and culture through the following steps:

(1) isolated culture of Streptomyces fulvescens (ZZ406)

A certain amount of fresh nervilia longissima (Haliplanella lineata) is washed with sterilized natural seawater for three times, and homogenized to obtain suspensions with different concentrations. Taking a certain amount of suspensions with different concentrations to be uniformly dispersed in a culture dish containing a solid culture medium, culturing for a certain time at room temperature, respectively transferring different bacterial colonies to another culture dish containing the solid culture medium, and continuously culturing for a certain time at room temperature. Finally, inoculating the well-grown single colony (ZZ406) to a slant culture medium for culture, and then storing the colony in a refrigerator at 4 ℃ for later use.

The sea anemone longissima (Haliplanella lineata) is obtained from rock cracks of the beach of Predada, Zhejiang, and the natural seawater is obtained from the east sea area near Zhoushan, Zhejiang; the different concentrations of the suspension are 1X 10^-4～1×10^-1g/mL; the sample suspension has a sampling quantity of 100 to300 mu L; the solid culture medium contained in the culture dish is a Gauss's agar culture medium or other solid culture media; the slant culture medium is a Gao's agar culture medium or other solid slant culture medium; the room temperature culture temperature is 22-28 ℃; the culture time is 5-15 days.

(2) Identification of Streptomyces fulvescens (ZZ406) species

The strain ZZ406 obtained by separation and culture in the step (1) is identified by a 16S rDNA sequence analysis method commonly used in the laboratory at present, is determined to be Streptomyces atrophaeofaciens, is classified and named as Streptomyces fulvisimusZZ 406, is preserved by China center for type culture Collection-Wuhan center, and has the preservation number of CCTCC NO: m2017435.

The second purpose of the invention is to provide two compounds of streptomyces xanthioic acid (1) and streptomyces xanthione (2) with anti-glioma activity, wherein the streptomyces xanthioic acid and streptomyces xanthione are new compounds, and the chemical structural formulas of the compounds are as follows:

the third purpose of the invention is to provide a preparation method of the streptomyces xanthyli acid (1) and the streptomyces xanthyli ketone (2), which is realized by the following steps:

(1) preparation of Streptomyces fulvescens (Streptomyces fulvissimus ZZ406) fermentation broth

A colony of Streptomyces fulvescens (ZZ406) is inoculated into a large triangular flask containing a certain amount of liquid strain culture medium, and the culture solution containing ZZ406 strain is subjected to shaking culture for a certain time at room temperature to obtain a strain solution. And finally transferring the strain liquid into a large triangular flask containing a certain amount of liquid fermentation medium, and carrying out shake culture for a certain time at room temperature to obtain a culture solution of ZZ406 with anti-tumor activity.

The strain culture medium is a liquid Gao's medium, and the dosage is 150-250 mL; the liquid fermentation medium is an SC liquid medium (10 g of soluble starch, 0.3 g of casein, 2 g of potassium nitrate, 0.5 g of magnesium sulfate, 2 g of dipotassium hydrogen phosphate, 0.02 g of calcium carbonate, 0.01 g of ferrous sulfate, 1L of natural seawater and 7.2 of pH value); the volume of the large triangular culture bottle is 500 mL; the room temperature culture temperature is 22-28 ℃; the rotating speed of the oscillation is 160-180 rpm; the culture time is 5-15 days.

(2) Extraction, separation and purification of streptomyces obscurus acid and streptomyces obscurus ketone

The fermented mycelium of strain ZZ406 was extracted with methanol, and the methanol extract was extracted with ethyl acetate to obtain an ethyl acetate extract. Separating the ethyl acetate extract by normal phase silica gel column chromatography, gradient eluting by mixed solvent of ethyl acetate and cyclohexane, and analyzing by thin layer chromatography to obtain nine components (Fr.1-Fr.9). And respectively separating the component Fr4 and the component Fr 8 by using a preparative High Performance Liquid Chromatography (HPLC) instrument to obtain pure compounds of the dark yellow streptomycic acid (1) and the dark yellow streptomycone (2).

The ratio of the normal phase silica gel amount of the column chromatography to the sample amount of the column chromatography is 30-50 g:1.0 g; the high performance liquid phase separation conditions comprise a novel constant CXTH-3000 high performance liquid chromatograph and Fuji C₁₈CT-30 chromatographic column (280X 30mm,10 μm), methanol and water as mobile phase, detection wavelength of 220nm, and flow rate of 10.0 mL/min.

(3) Structural identification of streptomyces xanthyli acid and streptomyces xanthyli ketone

The structures of the dark yellow streptomycic acid and the dark yellow streptomycone are determined according to the methods of one-dimensional and two-dimensional NMR spectra, high-resolution mass spectrum data, ECD calculation and the like of the dark yellow streptomycic acid and the dark yellow streptomycone.

The fourth purpose of the invention is to provide the application of the streptomyces xanthylifolinic acid and the streptomyces xanthylifolin in preparing the medicines for treating the brain glioma. The dark yellow streptomycic acid and the dark yellow streptomycone can obviously inhibit the proliferation of various glioma cells, reduce the protein level expression of a plurality of key enzymes in the glycolysis pathway of the glioma cells, and have unique anti-tumor action mechanism.

The drug is composed of the streptomyces flavus acid or the streptomyces flavus ketone alone or the combination of the two or the combination and other active substances together with a pharmaceutically acceptable carrier.

The Streptomyces fulvescens ZZ406 is separated and obtained from marine animal malva sylvestris (Haliplanella lineata). Research shows that the compounds of the streptomyces aureoflavus acid (1) and the streptomyces aureoflavus ketone (2) obtained by utilizing the streptomyces aureoflavus have anti-glioma activity, can obviously inhibit the proliferation of different glioma cells and obviously reduce the protein expression level of glycolytic metabolism key enzymes of the glioma cells, have a unique anti-tumor action mechanism, and can be applied to the preparation of medicines for treating the glioma.

Drawings

FIG. 1 is a colony map of Streptomyces fulvescens (ZZ 406).

FIG. 2 is a hydrogen spectrum of dark yellow streptomycic acid.

FIG. 3 is a carbon spectrum of dark yellow streptomycic acid.

FIG. 4 is an HMBC spectrum of dark yellow streptavidin.

FIG. 5 is a schematic representation of the main HMBC correlation of the xanthostreptomycic acid.

FIG. 6 is a high resolution mass spectrum of dark yellow streptavidin.

FIG. 7 is a hydrogen spectrum of Streptomyces xanthene.

FIG. 8 is a carbon spectrum of dark yellow streptavidin.

FIGS. 9-10 are of xanthostreptanone¹H-¹HCOSY spectra.

FIG. 11 is the HSQC spectra of dark yellow streptavidin.

FIG. 12 is an HMBC spectrum of dark yellow streptavidin.

FIG. 13 is a photograph of a xanthostreptanone base¹H-¹Schematic relating to H COSY and HMBC.

FIG. 14 is a high resolution mass spectrum of dark yellow streptavidin.

FIG. 15 is a graph of the effect of S.xanthans and S.xanthans on the protein expression levels of different metabolic key enzymes HK2, PFKFB3, PKM2, LDH5, ACO2 and CytoC in U87-MG cells.

Detailed Description

The invention is described in further detail below with reference to the figures and examples. However, the present invention is not limited to these examples.

Example 1

1. Isolated culture of Streptomyces fulvescens (ZZ406)

Taking 5 g of fresh nervilia longissima (Haliplanella lineata), washing with sterilized natural seawater for three times, homogenizing to obtain a slurry with a concentration of 1 × 10^-1g/mL of suspension. Will be 1 × 10^-1g/mL suspensions were diluted sequentially to a concentration of 1X 10^-2、1×10^-3And 1X 10^-4g/mL of suspension. mu.L of each concentration of the suspension was uniformly dispersed in a petri dish containing a solid medium of Gauss's agar, and after 5 days of culture at 28 ℃, different colonies were transferred to another petri dish containing a solid medium of Gauss's agar, respectively, and continued for 5 days at 28 ℃. Finally, a single colony (ZZ406) which grows well is inoculated to a Gao's agar solid slant culture medium for culture and then is stored in a refrigerator at 4 ℃ for later use.

2. Identification of Streptomyces fulvescens (ZZ406) species

The species of the strain ZZ406 obtained was identified using 16S rDNA sequence analysis.

2.1 Experimental reagents and instruments

PCR reagents: EX Taq enzyme (TaKaRa), dNTP (TaKaRa), primer (synthesized by Invitrogen), and primer sequence is:

SEQ ID No：1：AGAGTTTGATCCTGGCTCAG

SEQ ID No：2：GGTTACCTTGTTACGACTT

Marker：DL5000

an experimental instrument: centrifuge, electrophoresis apparatus, PCR apparatus, ABI 3730XL sequencer.

2.2 Experimental procedures

Bacterial genomic DNA extraction

Electrophoretic detection

PCR amplification

PCR reaction System

PCR reaction conditions

c. Electrophoretic detection

d. Sequencing, gel cutting, purifying and sequencing

e. And analyzing the result, namely the splicing sequence.

2.3 results of the experiment

The spliced sequence is SEQ ID No: 3:

tgcagtcgacgatgaagccgcttcggtggtggattagtggcgaacgggtgagtaacacgtgggcaatctgcccttcactctgggacaagccctggaaacggggtctaataccggataatactcctgcctgcatgggtgggggttgaaagctccggcggtgaaggatgagcccgcggcctatcagcttgttggtggggtaatggcctaccaaggcgacgacgggtagccggcctgagagggcgaccggccacactgggactgagacacggcccagactcctacgggaggcagcagtggggaatattgcacaatgggcgaaagcctgatgcagcgacgccgcgtgagggatgacggccttcgggttgtaaacctctttcagcagggaagaagcgcaagtgacggtacctgcagaagaagcgccggctaactacgtgccagcagccgcggtaatacgtagggcgcaagcgttgtccggaattattgggcgtaaagagctcgtaggcggcttgtcacgtcggatgtgaaagcccggggcttaaccccgggtctgcattcgatacgggctagctagagtgtggtaggggagatcggaaattcctggtgtagcggtgaaatgcgcagatatcaggaggaacaccggtggcgaaggcggatctctgggccattactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaaacgttgggaactaggtgttggcgacattccacgtcgtcggtgccgcagctaacgcattaagttccccgcctggggagtacggccgcaaggctaaaactcaaaggaattgacgggggcccgcacaagcagcggagcatgtggcttaattcgacgcaacgcgaagaaccttaccaaggcttgacatataccggaaagcatcagagatggtgccccccttgtggtcggtatacaggtggtgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgttctgtgttgccagcatgcctttcggggtgatggggactcacaggagactgccggggtcaactcggaggaaggtggggacgacgtcaagtcatcatgccccttatgtcttgggctgcacacgtgctacaatggccggtacaatgagctgcgatgccgtgaggcggagcgaatctcaaaaagccggtctcagttcggattggggtctgcaactcgaccccatgaagtcggagttgctagtaatcgcagatcagcattgctgcggtgaatacgttcccgggccttgtacacaccgcccgtcacgtcacgaaagtcggtaacacccgaagccggtggcccaaccccttgtgggaggga

the comparison of the 16S rDNA sequence obtained above with the NCBI GenBank database of NIH in USA revealed that the 16S rDNA sequence of strain ZZ406 has 99% similarity to the 16S rDNA sequence of Streptomyces fulvissimus DSM 40593 in the GenBank database (accession No.: NC 021177.1). Thus, the marine strain ZZ406 obtained according to the invention is designated Streptomyces fulvescens ZZ406 (FIG. 1). The obtained strain of Streptomyces fulvidrosis (ZZ406) has been preserved by China center for type culture Collection, Wuhan center, with the preservation number of CCTCCNO: m2017435, date of deposit: 8/3/2017.

Example 2 preparation of Streptomyces fulvescens (Streptomyces fulvissimus ZZ406) fermentation broth

A strain of Streptomyces aureofaciens (Streptomyces fulvissimuzz 406) in a solid slant medium of Gao-agar was inoculated into a 500mL Erlenmeyer flask containing 250mL of a liquid Gao-agar medium, and the culture broth containing the ZZ406 strain was subjected to shake culture at 28 ℃ for 5 days with rotation (180rpm) to obtain a strain liquid. 5mL of the strain solution was transferred to a 500mL Erlenmeyer flask containing 250mL of SC broth (10 g of soluble starch, 0.3 g of casein, 2 g of potassium nitrate, 0.5 g of magnesium sulfate, 2 g of dipotassium hydrogen phosphate, 0.02 g of calcium carbonate, 0.01 g of ferrous sulfate, 1L of natural seawater, pH 7.2), and cultured by shaking at 28 ℃ for 13 days with rotation (180rpm) to obtain ZZ406 strain-fermented solution having anti-tumor activity.

Example 3

1. Extraction, separation and purification of streptomyces obscurus acid and streptomyces obscurus ketone

The fermented broth (70.0L) obtained in example 2 was filtered to obtain mycelia. The mycelia were extracted three times with methanol to obtain methanol extract. The methanol extract was further extracted with ethyl acetate to obtain an ethyl acetate extract (8.0 g), the ethyl acetate extract was separated by column chromatography on silica gel (250 g), gradient-eluted with cyclohexane and ethyl acetate (4:1 to 1:1), each eluate was analyzed by thin layer chromatography, and fractions containing the same components were combined to obtain 9 fractions (fr.1 to fr.9) in total. Separating component Fr.4(30 mg) with semi-preparative high performance liquid chromatograph (instrument: Innovative constant CXTH-3000; chromatographic column: Fuji C)₁₈CT-30,280 is multiplied by 30mm,10 mu m; mobile phase: methanol/water, 75/25; the detection wavelength is 220nm, the flow rate is 10.0mL/min), and the compound of the streptomyces xanthus acid (1,9.3mg, t)_R31 min). Also, the same HPLC separation conditions (but different)Mobile phase methanol/water, 21/79) fraction Fr.8(20mg) was separated to give the pure compound, scouletin (2,8.0mg, t)_R35min)。

2. Structural identification of streptomyces xanthyli acid and streptomyces xanthyli ketone

Dark yellow streptomycic acid (1): a yellow powder; molecular formula C₁₆H₁₀O₆(ii) a UV absorption (MeOH) lambda_max(log)217(4.32),279(4.22),411(3.66) nm; high resolution mass spectrometry (HRESIMS) of M/z [ M-H ]]^-297.0407 (calculation C)₁₆H₉O₆297.0399). According to dark yellow streptomycic acid¹H spectrum (figure 2),¹³The C spectrum (figure 3), HMBC spectrum (figure 4, figure 5) and high resolution mass spectrum (figure 6) determine the structure of the dark yellow streptomycic acid, and the compound is a new compound¹³C and¹the H NMR signals are assigned in Table one.

Epi-and S-Streptomyces obscurus acid¹³C and¹h NMR data (solvent: deuterated dimethyl sulfoxide DMSO-d)₆)

Dark yellow streptomycone (2): a yellow powder; molecular formula C₁₆H₁₆O₆；[α]_D ²⁵-5.6(c 0.20, MeOH); UV absorption (MeOH) lambda_max(log)224(4.09),302(3.63) nm; high resolution mass spectrum (HRESIMS) is M/z [ M + H ]]⁺305.0998 (calculation C)₁₆H₁₇O₆305.1025). According to Streptomyces aureofaciens¹H spectrum (FIG. 7),¹³C spectrum (FIG. 8),¹H-¹H COSY spectrum (figure 9, figure 10, figure 13), HSQC spectrum (figure 11), HMBC spectrum (figure 12, figure 13), high resolution mass spectrum (figure 14) and ECD spectrum calculation determine the structure of the dark yellow streptomycone, which is a new compound, dark yellow streptomycone¹³C and¹the H NMR signals are assigned in Table II.

TABLE II, S.amboin (2)¹³C and¹h NMR data (solvent: deuterated dimethyl sulfoxide DMSO-d)₆)

Example 4 Activity of Streptomyces xanthylideyne and Streptomyces xanthenone

1. Effect of Streptomyces aureofaciens and Streptomyces aureofaciens on inhibiting glioma cell proliferation

Human brain glioma U251 cells were cultured in DMEM and 10% FBS, while glioma U87MG cells, SHG44 cells and normal human glial HA cells were cultured in MEM medium, RPMI-1640 medium and AM medium, respectively. All cells were cultured for the experimental study of the present invention at 37 ℃ and 5% carbon dioxide in an incubator for three generations.

The inhibition activity of the streptomyces fuscous acid and the streptomyces fuscous ketone on the proliferation of the tumor cells is measured by a sulforhodamine B (SRB) method, and the adriamycin is used for positive drug control. Inoculating cells in a 96-well plate, adding test compounds with different concentrations after adhering to the wall for 24h, culturing in an incubator for 72h, staining with SRB, measuring the absorbance at 515nm with a microplate reader, detecting the survival rate of tumor cells, and calculating the IC of each test compound for inhibiting the proliferation of glioma cells₅₀The value is obtained. The experimental results show that: the proliferation of three different glioma cells, IC, is significantly inhibited by the dark yellow streptomycic acid and dark yellow streptomycone₅₀The value is 4.7-25.8. mu.M, wherein the activity of the xanthostreptic acid is stronger, and the IC thereof is₅₀The values were 4.7-8.1. mu.M (Table III). The cytotoxicity of Streptomyces xanthanensis and Streptomyces xanthanensis on normal glial cells (HA), their CC, was also determined₅₀Values are all greater than 100. mu.M, selectivity index (CC) of scoxanthotenic acid₅₀/IC₅₀) Is greater than 12.3 to 21.3, and the selectivity index (CC) of the scotopic streptomycin ketone₅₀/IC₅₀) Greater than 3.8 to 4.6, similar to the positive control drug doxorubicin. The results show that: selectivity of Dixanthansetronic acid on glioma cellsThe use of doxorubicin, a more positive control, in other words, the toxicity of the xanthostreptic acid to normal glial cells was lower than that of the control doxorubicin (table three).

Epimeris, streptomyces obscurus acid and streptomyces obscurus ketone inhibit glioma cell and normal glial cell proliferation

2. Effect of Streptomyces aureofaciens and Streptomyces aureofaciens on glioma cell metabolizing enzymes

Preparation of protein samples: human glioma U87MG cells were cultured in MEM and 10% FBS medium at 37 ℃ in an incubator with 5% carbon dioxide, and the cells cultured for three generations were used for the experimental study of the present invention. Cells (1.5X 10)⁷) After 24 hours of attachment to the wall, they were incubated with either dark yellow streptomycic acid (1, 30. mu.M), dark yellow streptomycone (2, 60. mu.M) or 2-deoxyglucose (2-DG,6mM) for 48 hours, washed twice with ice-cold PBS buffer and then lysed with ice-cold lysis buffer (200. mu.L) for 15 minutes. The lysate was centrifuged at 4 ℃ at low temperature and high speed (11200rpm) and the supernatant was the protein sample.

Determination of protein content: protein content of each protein sample was determined using the BCA kit. Reagents a and B in the kit were mixed at a ratio of 50: 1, preparing a working reagent solution, and diluting a standard BCA (burst cutting protein) by using Bovine Serum Albumin (BSA) to prepare BCA standard solutions with different concentrations. 10 mu L of standard solution or protein sample solution and 200 mu L of working reagent solution are mixed uniformly and incubated for 30 minutes at 37 ℃, and the absorbance of the incubation solution is measured by a microplate reader at the wavelength of 562 nm. And drawing a standard curve by taking the BCA amount as an abscissa and the absorbance as an ordinate, and calculating a regression equation. The protein content of each protein sample solution was calculated from the regression equation.

Western Blotting: each sample containing an equivalent amount of protein (15. mu.g) was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were electrophoresed on the gelThe spectra were transferred to polyvinylidene fluoride (PVDF) membranes which were blocked with 0.1% TBST plus 5% nonfat milk for 2 hours at room temperature. Incubating the sealed PVDF membrane and the primary antibody of the test enzyme at 4 ℃ overnight, and washing the membrane by TBST; and then incubated with HRP-labeled secondary antibody at room temperature for 2 hours. After washing the membrane for three times by TBST, detecting immunoreactivity by using a chemiluminescence-enhancing reagent, developing, washing with water, fixing, washing with water, drying in the air, and observing an experimental result. Beta-actin (beta-actin) was used as an internal control.

The results of the experiment (fig. 15) show that: both the xanthostreptoc acid (1) and xanthostreptone (2) significantly reduced protein level expression of key enzymes HK2, PFKFB3, PKM2, and LDH5 of tumor cell glycolysis compared to the negative control group (U87 MG cells without drug treatment, CON). However, the effects of S.tabasheer and S.tabasheer on the protein expression levels of the TCA cycle enzymes aconitase 2(ACO2) and oxidative phosphorylase cytochrome C (CytoC) were insignificant. CON: U87-MG cell control group; CON: a negative control group; 1: dark yellow streptomycic acid treatment group; 2: a dark yellow streptomycon treatment group; 2-DG is 2-deoxyglucose; HK2 hexokinase 2: PFKFB3 phosphofructokinase/fructose 2, 6-bisphosphatase; PKM2 pyruvate kinase M2; LDH5 lactate dehydrogenase 5; ACO2, aconitase 2; CytoC is cytochrome C; β -actin internal reference). It is known that tricarboxylic acid cycle and oxidative phosphorylation are the normal cellular predominant metabolic pathways, while ACO2 and CytoC are one of the key enzymes of the tricarboxylic acid cycle pathway and oxidative phosphorylation pathway, respectively. The above results and analysis show that: the two active compounds of the dark yellow streptomycin acid and the dark yellow streptomycin ketone can generate anti-glioma activity through selectively influencing a plurality of key enzymes of glioma glycolysis, and have unique anti-tumor action mechanisms.

In conclusion, the invention separates a marine actinomycete Streptomyces fusciparum ZZ406 from marine animal malvastrum longissima (Haliplanella lineata), which can produce anti-glioma active compounds of Streptomyces fusciparum acid and Streptomyces fusciparum ketone. The invention provides a separation culture method of streptomyces aureoflavus, an extraction separation purification method of active compounds of streptomyces aureoflavus and streptomyces aureoflavus ketone, and anti-glioma activity of streptomyces aureoflavus and streptomyces aureoflavus ketone. Because the streptomyces xanthyli acid and the streptomyces xanthyli ketone have good glioma resistance activity and a unique anti-tumor action mechanism for regulating and controlling metabolic enzymes of tumor cells, the streptomyces xanthyli acid and the streptomyces xanthyli ketone have application prospects in the aspect of preparing medicines for treating brain glioma.

Sequence listing

<110> Zhejiang university

<120> preparation and medical application of streptomyces xanthioic acid and streptomyces xanthione

<160>3

<170>SIPOSequenceListing 1.0

<210>1

<211>20

<212>DNA

<213> Artificial sequence (Unknown)

<400>1

agagtttgat cctggctcag 20

<210>2

<211>19

<212>DNA

<213> Artificial sequence (Unknown)

<400>2

ggttaccttg ttacgactt 19

<210>3

<211>1390

<212>DNA

<213>Streptomyces fulvissimus ZZ406

<400>3

tgcagtcgac gatgaagccg cttcggtggt ggattagtgg cgaacgggtg agtaacacgt 60

gggcaatctg cccttcactc tgggacaagc cctggaaacg gggtctaata ccggataata 120

ctcctgcctg catgggtggg ggttgaaagc tccggcggtg aaggatgagc ccgcggccta 180

tcagcttgtt ggtggggtaa tggcctacca aggcgacgac gggtagccgg cctgagaggg 240

cgaccggcca cactgggact gagacacggc ccagactcct acgggaggca gcagtgggga 300

atattgcaca atgggcgaaa gcctgatgca gcgacgccgc gtgagggatg acggccttcg 360

ggttgtaaac ctctttcagc agggaagaag cgcaagtgac ggtacctgca gaagaagcgc 420

cggctaacta cgtgccagca gccgcggtaa tacgtagggc gcaagcgttg tccggaatta 480

ttgggcgtaa agagctcgta ggcggcttgt cacgtcggat gtgaaagccc ggggcttaac 540

cccgggtctg cattcgatac gggctagcta gagtgtggta ggggagatcg gaaattcctg 600

gtgtagcggt gaaatgcgca gatatcagga ggaacaccgg tggcgaaggc ggatctctgg 660

gccattactg acgctgagga gcgaaagcgt ggggagcgaa caggattaga taccctggta 720

gtccacgccg taaacgttgg gaactaggtg ttggcgacat tccacgtcgt cggtgccgca 780

gctaacgcat taagttcccc gcctggggag tacggccgca aggctaaaac tcaaaggaat 840

tgacgggggc ccgcacaagc agcggagcat gtggcttaat tcgacgcaac gcgaagaacc 900

ttaccaaggc ttgacatata ccggaaagca tcagagatgg tgcccccctt gtggtcggta 960

tacaggtggt gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1020

cgagcgcaac ccttgttctg tgttgccagc atgcctttcg gggtgatggg gactcacagg 1080

agactgccgg ggtcaactcg gaggaaggtg gggacgacgt caagtcatca tgccccttat 1140

gtcttgggct gcacacgtgc tacaatggcc ggtacaatga gctgcgatgc cgtgaggcgg 1200

agcgaatctc aaaaagccgg tctcagttcg gattggggtc tgcaactcga ccccatgaag 1260

tcggagttgc tagtaatcgc agatcagcat tgctgcggtg aatacgttcc cgggccttgt 1320

acacaccgcc cgtcacgtca cgaaagtcgg taacacccga agccggtggc ccaacccctt 1380

gtgggaggga 1390

Claims

1. The preparation method of the dark yellow streptomycic acid is characterized by comprising the following steps of:

(1) preparation of Streptomyces aureofaciens zymocyte liquid

Taking Streptomyces aureofaciens (A), (B)Streptomyces fulvissimusZZ406) is inoculated into a large triangular flask containing a liquid strain culture medium, the culture solution containing the strain is subjected to shake culture at room temperature to obtain a strain solution, and finally the strain solution is transferred into the large triangular flask containing the liquid fermentation culture medium and subjected to shake culture at room temperature to obtain a Streptomyces aureofaciens fermentation broth; the classification and the name of the streptomyces atrophaeoflavus areStreptomyces fulvissimusZZ406, preservation number: CCTCC NO: M2017435, preservation date: 8 months and 3 days 2017;

the strain culture medium is a liquid high-grade culture medium, and the liquid fermentation culture medium is an SC liquid culture medium: 10 g of soluble starch, 0.3 g of casein, 2 g of potassium nitrate, 0.5 g of magnesium sulfate, 2 g of dipotassium hydrogen phosphate, 0.02 g of calcium carbonate, 0.01 g of ferrous sulfate, 1L of natural seawater and 7.2 of pH value; the volume of the large triangular culture bottle is 500 mL; the room temperature culture temperature is 22-28 ℃; the oscillating rotating speed is 160-180 rpm; the culture time is 5-15 days;

(2) extraction, separation and purification of streptomyces obscurus acid

Extracting a fermentation mycelium of the strain ZZ406 with methanol, extracting the methanol extract with ethyl acetate to obtain an ethyl acetate extract, separating the ethyl acetate extract by using normal-phase silica gel column chromatography, performing gradient elution by using a mixed solvent of ethyl acetate and cyclohexane, and analyzing by using thin-layer chromatography to obtain nine components Fr.1-Fr. 9, and separating the component Fr.4 and the component Fr.8 by using a preparative high-performance liquid chromatograph respectively to obtain a pure compound of the dark yellow streptomycic acid;

the ratio of the normal phase silica gel amount of the column chromatography to the sample amount of the column chromatography is 30-50 g:1.0 g; the high performance liquid phase separation conditions comprise a novel constant CXTH-3000 high performance liquid chromatograph and Fuji C₁₈CT-30 column, 280

30 mm, 10μm，Methanol and water are used as mobile phases, the detection wavelength is 220nm, and the flow rate is 10.0 mL/min;

(3) structural identification of Streptomyces xanthus acid

The structure of the dark yellow streptomycic acid is determined according to one-dimensional and two-dimensional NMR spectra, high-resolution mass spectrum data and an ECD calculation method of the dark yellow streptomycic acid;

the chemical structural formula of the streptomyces xanthioii acid is as follows:

。