阅读说明：本技术 一种暗黄链霉酸和暗黄链霉酮的制备及其医药用途 (Preparation and medical application of streptomyces xanthioides acid and streptomyces xanthione ) 是由 张治针 陈梦宣 柴卫云 连晓媛 于 2017-09-19 设计创作，主要内容包括：本发明提供的一种暗黄链霉酸和暗黄链霉酮的制备及其医药用途,所述极暗黄链霉菌的分类命名为Streptomyces fulvissimus ZZ406,已被中国典型培养物保藏中心-武汉中心保藏,保藏编号:CCTCC NO:M 2017435,保藏日：2017年8月3日。利用该极暗黄链霉菌获得的化合物暗黄链霉酸(1)和暗黄链霉酮(2)具有抗胶质瘤活性,可显著地抑制不同脑胶质瘤细胞的增殖和明显降低胶质瘤细胞糖酵解代谢关键酶的蛋白表达水平,具有独特的抗肿瘤作用机制,可在制备治疗脑胶质瘤药物中的应用。化合物暗黄链霉酸(1)和暗黄链霉酮(2)的结构式如下：<Image he="199" wi="700" file="DDA0002160415350000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention provides preparation and medical application of dark yellow streptomycete and dark yellow streptomycete, the classification of the Streptomyces obscurus is named as Streptomyces fulvissimus ZZ406, and the Streptomyces obscurus is protected by the China center for type culture Collection-Wuhan centerPreservation, preservation number is CCTCC NO: M2017435, 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:)

1. The streptomyces xanthioic acid and streptomyces xanthione are characterized in that the chemical structural formulas of the streptomyces xanthioic acid (1) and the streptomyces xanthione (2) are respectively as follows:

2. the process for preparing Streptomyces xanthanensis and Streptomyces xanthanensis ketone as claimed in claim 1, which comprises the steps of:

(1) preparation of Streptomyces aureofaciens zymocyte liquid

Inoculating a bacterial colony of Streptomyces fulvescens (ZZ406) into a large triangular flask containing a liquid strain culture medium, carrying out shake culture on a culture solution containing the strain at room temperature to obtain a strain liquid, transferring the strain liquid into the large triangular flask containing the liquid fermentation culture medium, and carrying out shake culture at room temperature to obtain a Streptomyces fulvescens zymocyte liquid; the Streptomyces atrophaeofuscus is classified and named as Streptomyces fulvissimus ZZ406, and has the preservation number of CCTCC NO: M2017435, and the preservation date is as follows: 8 months and 3 days 2017;

the strain culture medium is a liquid Gao's medium, the dosage is 150-250mL, 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 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 a mixed solvent of ethyl acetate and cyclohexane, analyzing by thin layer chromatography to obtain nine components Fr.1-Fr.9, and separating the component Fr4 and the component Fr8 by a preparative high performance liquid chromatograph to obtain pure compounds of dark yellow streptomycic acid and dark yellow streptomycone;

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 C₁₈A CT-30 chromatographic column with 280 mm multiplied by 30mm and 10 mu m, methanol and water as mobile phases, the detection wavelength of 220nm and the 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.

3. Use of the xanthostreptic acid and xanthostreptone according to claim 1 for the preparation of a medicament for the treatment of brain glioma.

4. The use of claim 3, wherein the drug is prepared from the combination of the two of the Streptomyces avermitilis acid and the Streptomyces avermitilis ketone, and the pharmaceutically acceptable carrier.

5. The use according to claim 3, wherein the medicament is prepared from the combination of the xanthosteptaenic acid and the xanthosteptaenone alone or in combination with other active substances and pharmaceutically acceptable carriers.

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 fulvidrosus ZZ406 which is classified and named as Streptomyces fulvissimus ZZ406 and has been preserved by 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 sampling amount of the sample suspension is 100-300 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 atrophaeofuscus, is classified and named as Streptomyces fulvissimus ZZ406, 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 Fr8 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 (2)80X 30mm,10 μm), methanol and water as mobile phases, a detection wavelength of 220nm and a 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.