阅读说明：本技术 一种天然Rakicidins类化合物Rakicidin B1-2及其发酵提取方法 (Natural Rakicidins compound Rakicidin B1-2 and fermentation extraction method thereof ) 是由 陈丽 林风 江红 赵薇 周剑 江宏磊 连云阳 于 2019-08-12 设计创作，主要内容包括：本发明属于天然化合物技术领域,具体涉及一种天然Rakicidins类化合物Rakicidin B1-2及其发酵提取方法。本发明从海洋小单孢菌发酵液中分离得到新的Rakicidins组分Rakicidin B1-2,该化合物结构与已知的Rakicidin B1相比,在于11、14位开环,且在11位接入乙氧基。本发明天然化合物Rakicidin B1-2对常氧和乏氧条件下培养的人结肠癌细胞HCT-8和人胰腺癌细胞PANC-1均具有一定的体外抑制作用,对甲氧西林耐药金黄色葡萄球菌、艰难梭菌和万古霉素耐药粪肠球菌等10株厌氧菌均具有一定的抑制活性,是一种结构新颖活性较好的次级代谢产物,具有较高的药用价值。(The invention belongs to the technical field of natural compounds, and particularly relates to a natural Rakicidins compound Rakicidin B1-2 and a fermentation extraction method thereof. The invention separates new Rakicidins component Rakicidin B1-2 from marine micromonospora fermentation liquor, and compared with the known Rakicidin B1, the structure of the compound is characterized in that 11 and 14 positions are opened, and an ethoxy group is connected to the 11 position. The natural compound Rakicidin B1-2 has a certain in-vitro inhibition effect on human colon cancer cells HCT-8 and human pancreatic cancer cells PANC-1 cultured under the conditions of normal oxygen and hypoxia, has a certain inhibition activity on 10 anaerobic bacteria such as methicillin-resistant staphylococcus aureus, clostridium difficile, vancomycin-resistant enterococcus faecalis and the like, is a secondary metabolite with a novel structure and good activity, and has a high medicinal value.)

1. A natural Rakicidins compound Rakicidin B1-2 and pharmaceutically acceptable salts thereof, wherein the compound Rakicidin B1-2 has a structure shown as the following formula (I):

2. a method for preparing the natural Rakicidins compound Rakicidin B1-2 as claimed in claim 1, wherein the Rakicidin B1-2 is obtained by fermenting and separating Micromonospora sp.FIM-R160609, a marine Micromonospora strain with the collection number of CGMCC No. 14823.

3. The process for the preparation of the natural Rakicidins compound Rakicidin B1-2 according to claim 2, comprising the steps of:

(1) fermenting the preserved Micromonospora sp.FIM-R160609, collecting fermentation liquor, and performing solid-liquid separation to obtain a mycelium; soaking the mycelium in methanol or ethanol, and collecting the soaking solution;

(2) performing chromatography with D3502 macroporous resin adsorption column, performing gradient elution with 67% and 75% ethanol-water solution respectively, and collecting 75% ethanol-water eluate;

(3) carrying out chromatography on the collected eluent by using an HZ816 resin adsorption column, carrying out gradient elution by using 66%, 69% and 73% ethanol-water solutions respectively, and collecting 73% ethanol-water eluent components containing Rakicidin B1-2;

(4) extracting the obtained eluent with ethyl acetate or dichloromethane, concentrating to obtain crude product, dissolving with methanol, performing semi-preparative liquid chromatography, and separating by volume ratio of 710: 290, eluting with acetonitrile-water, and collecting by stages.

4. The method for preparing the natural Rakicidins compound Rakicidin B1-2 according to claim 3, wherein in the step (2), the diameter-height ratio of the D3502 macroporous resin adsorption column is 1: 5-1: 10, the column volume is 2.5-4.0L, and the adsorption flow rate is 30-40 ml/min.

5. The method for preparing the natural Rakicidins compound Rakicidin B1-2 according to claim 3 or 4, wherein in the step (3), the height ratio of the HZ816 resin adsorption column diameter is 1: 5-1: 8, the column volume is 2.2-3L, and the adsorption flow rate is 25-35 ml/min.

6. The method for preparing Rakicidin B1-2 as a compound in the natural Rakicidin family of compounds according to any one of claims 3-5, wherein in the step (4), the semi-preparative liquid chromatography is Welch Material, 5 μm,250mm x 10mm, and the flow rate of the eluent is controlled to be 8 ml/min.

7. Use of the natural Rakicidins compound Rakicidin B1-2 and pharmaceutically acceptable salts thereof as claimed in claim 1 for the preparation of medicaments with antitumor activity and anti-clinical pathogenic anaerobic bacteria activity.

8. Use according to claim 7, wherein the natural Rakicidins compound Rakicidin B1-2 and pharmaceutically acceptable salts thereof is administered in a daily dose of 1-5000 mg/day.

9. A medicine for treating pathogenic anaerobic bacteria infection diseases, which is characterized in that the natural Rakicidins compound Rakicidin B1-2 and pharmaceutically acceptable salts thereof in claim 1 are used as active ingredients, and pharmaceutically acceptable carriers are added.

10. Use of Micromonospora sp.FIM-R160609 in fermentation preparation of natural Rakicidin compounds Rakicidin B1-2 is characterized in that the Micromonospora sp.FIM-R160609 has been preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 14823.

Technical Field

The invention belongs to the technical field of natural compounds, and particularly relates to a natural Rakicidins compound Rakicidin B1-2 and a fermentation extraction method thereof.

Background

At present, hundreds of bioactive substances are separated from metabolites of marine microorganisms, wherein part of the bioactive substances are antibiotics with antitumor activity or antibacterial activity which have possible clinical application value, and Rakicidins compounds are important compounds, and a series of Rakicidins compounds with antitumor activity or antibacterial activity are discovered from micromonospora and streptomyces.

In recent years, the literature has reported that marine Micromonospora sp.fim 02-523 can produce a series of lipopeptide compounds with antitumor activity including Rakicidin a, B, B1 and the like. In 2006, the subject group first reported in China that the compounds Rakicidin A and B were isolated from this strain of Micromonospora species. Research finds that Rakicidin A has excellent hypoxia-selective anti-tumor cell activity, the anti-colorectal cancer HCT-8 cell activity under the hypoxia condition is 17.5 times that under the normoxic condition, and the Rakicidin A is considered by many peers to be an anti-hypoxic tumor cell and anti-CSC drug with extremely good development prospects; in-vitro anti-tumor activity investigation of Rakicidin B also shows that Rakicidin B has obvious inhibition effect on the growth of tumor cell strains K562 and L929. In 2016, a new compound Rakicidin B1 was obtained from the subject group, and the experiment adopts a zebra fish model transplanted with human colon cancer HCT-8 tumor to test the in vivo tumor inhibition activity of Rakicidin A and B, B1, and the result shows that Rakicidin A, Rakicidin B and Rakicidin B1 all have inhibition activity on HCT-8 cell transplanted tumor in zebra fish, and a preliminary experiment shows that the hypoxia tumor cell can effectively inhibit the expression of HIF-1 (hypoxia inducible factor-1). The previous research further determines the cytotoxic activity of Rakicidin A and B, B1 compounds on five human tumor cell lines, namely HCT-8, MGC803, A549, A375, HepG2 and CASKI, and the result shows that the three separated compounds have obvious inhibitory activity on the tumor cell lines. In 2018, the subject group separates and purifies other three new compounds Rakicidin G and H, I from metabolites of marine Micromonospora sp.FIM 02-523 strains, and the activity experiment results show that the Rakicidin G and the Rakicidin H, I compounds have stronger cytotoxic activity on human colon cancer cells HCT-8 and human pancreatic cancer cells PANC-1 and are stronger under the anoxic condition, and the three compounds also have better inhibitory activity on various gram-positive anaerobic bacteria. Therefore, the Rakicidins compound has excellent clinical application prospect.

Disclosure of Invention

Therefore, the invention aims to provide a natural Rakicidins compound Rakicidin B1-2 and further discloses a fermentation extraction method thereof.

In order to solve the technical problems, the invention provides a natural Rakicidins compound Rakicidin B1-2 and pharmaceutically acceptable salts thereof, wherein the compound Rakicidin B1-2 has a structure shown as the following formula (I):

the invention also discloses a method for preparing the natural Rakicidins compound Rakicidin B1-2, wherein the Rakicidin B1-2 is obtained by fermenting and separating Micromonospora sp.FIM-R160609 of marine Micromonospora strain with the collection number of CGMCC No. 14823.

Specifically, the marine Micromonospora sp.FIM-R160609 is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms, and the preservation addresses are as follows: the No. 3 Xilu No.1 of Beijing, Chaoyang, the area of Chaoyang has a preservation number of CGMCC No.14823 and a preservation date of 2017, 10 months and 16 days.

Preferably, the method for preparing the natural Rakicidins compound Rakicidin B1-2 comprises the following steps:

(1) fermenting the preserved Micromonospora sp.FIM-R160609, collecting fermentation liquor, and performing solid-liquid separation to obtain a mycelium; soaking the mycelium in methanol or ethanol, and collecting the soaking solution;

(2) performing chromatography with D3502 macroporous resin adsorption column, performing gradient elution with 67% and 75% ethanol-water solution respectively, and collecting 75% ethanol-water eluate;

(3) carrying out chromatography on the collected eluent by using an HZ816 resin adsorption column, carrying out gradient elution by using 66%, 69% and 73% ethanol-water solutions respectively, and collecting 73% ethanol-water eluent components containing Rakicidin B1-2;

(4) extracting the obtained eluent with ethyl acetate or dichloromethane, concentrating to obtain crude product, dissolving with methanol, performing semi-preparative liquid chromatography, and separating by volume ratio of 710: 290, eluting with acetonitrile-water, and collecting by stages.

Specifically, in the step (2), the diameter-height ratio of the D3502 macroporous resin adsorption column is 1: 5-1: 10, the column volume is 2.5-4.0L, and the adsorption flow rate is 30-45 ml/min.

Specifically, in the step (3), the height ratio of the HZ816 to the adsorption column diameter is 1: 5-1: 8, the column volume is 2.2-3L, and the adsorption flow rate is 25-35 ml/min.

Specifically, in the step (4), the semi-preparative liquid chromatography is Welch Material, 5 μm and 250mm × 10mm, and the flow rate of the eluent is controlled to be 8 ml/min.

The invention also discloses application of the natural Rakicidins compound Rakicidin B1-2 and pharmaceutically acceptable salts thereof in preparing medicines with antitumor activity and clinical pathogenic anaerobic bacteria activity.

Specifically, the tumor comprises human colon cancer and human pancreatic cancer; the pathogenic anaerobic bacteria comprise methicillin-resistant staphylococcus aureus, vancomycin intermediate-sensitive staphylococcus aureus, vancomycin-resistant enterococcus faecalis and clostridium difficile.

Specifically, the daily dosage of the natural Rakicidins compound Rakicidin B1-2 and pharmaceutically acceptable salts thereof is 1-5000 mg/day, and the dosage can be out of the range according to different dosage forms and disease severity.

The invention also discloses a medicine for treating pathogenic anaerobic bacteria infection diseases, which takes the natural Rakicidins compound Rakicidin B1-2 and pharmaceutically acceptable salts thereof as active ingredients and adds pharmaceutically acceptable carriers.

Specifically, the drug can be in the form of ordinary tablets or capsules, sustained-release tablets or capsules, controlled-release tablets or capsules, oral liquid, injection and other preparations which are conventional in pharmaceutics.

The invention also discloses application of Micromonospora sp.FIM-R160609 in preparing a natural Rakicidin compound Rakicidin B1-2 by fermentation, wherein Micromonospora sp.FIM-R160609 is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms, and the preservation number is CGMCC No. 14823.

The invention separates new Rakicidins component Rakicidin B1-2 from marine micromonospora fermentation liquor, and compared with the known Rakicidin B1, the structure of the compound is characterized in that 11 and 14 positions are opened, and an ethoxy group is connected to the 11 position. The natural compound Rakicidin B1-2 has a certain in-vitro inhibition effect on human colon cancer cells HCT-8 and human pancreatic cancer cells PANC-1 cultured under the conditions of normal oxygen and hypoxia, has a certain inhibition activity on 10 anaerobic bacteria such as methicillin-resistant staphylococcus aureus, clostridium difficile, vancomycin-resistant enterococcus faecalis and the like, is a secondary metabolite with a novel structure and good activity, and has a high medicinal value.

Examples of the experiments

1. Antitumor Activity test

The extracted Rakicidin B1-2 sample was dissolved in DMSO to reach a solubility of 1ug/ml, and then diluted to final concentrations of 0.5ug/ml, 0.25ug/ml, 0.125ug/ml, 0.0625ug/ml, 0.03125ug/ml, and 0.015625ug/ml, respectively.

And (3) ordinary culture: respectively inoculating human colon cancer cell HCT-8 and human pancreatic cancer cell PANC-1 in 96-well plate (cell concentration is HCT-85.0 × 10)⁴Per ml, PANC-14.0X 10⁴100 ul/ml), culturing for 24hr, adding 100 ul/well fresh culture medium with drug, setting 3 multiple wells for each concentration, setting blank control well (adding culture medium only) as negative control, and setting 3 multiple wells. The culture was continued for 48hr, and the culture was terminated.

Rakicidin B1-2 samples were each dissolved in DMSO to a solubility of 1ug/ml and then diluted individually to final concentrations of 0.4444ug/ml, 0.148148ug/ml, 0.0493827ug/ml, 0.0164609ug/ml, 0.00548697ug/ml, 0.00182899 ug/ml.

Hypoxic culture: respectively inoculating human intestinal cancer cell HCT-8 and human pancreatic cancer cell PANC-1 in 96-well plate (cell concentration is HCT-85.0 × 10)⁴Per ml, PANC-14.0X 10⁴100 ul/ml, 100 ul/hole), aerating for 30 minutes with oxygen, closing the aeration valve, placing in an incubator at 37 ℃, culturing for 24 hours to allow the cells to adhere to the wall, adding 100 ul/hole of fresh culture medium with drugs, setting 3 multiple holes for each concentration, setting a blank control hole (only adding culture medium) as a negative control, and setting 3 multiple holes in the same way. Ventilating with oxygen for 30 min, closing the ventilation valve, placing into incubator at 37 deg.C, culturing for 48hr, and terminating culture.

And (3) detection by an MTT method: adding CCK-810ul to each well of the cells after the culture is stopped, continuously culturing the cells in an incubator for 2 hours, discarding the supernatant, adding 150ul of DMSO solution to each well, gently mixing the cells uniformly by a shaker for 10 minutes, reading the OD value of each well by an enzyme-labeling instrument under the wavelength of 450nm, and calculating the inhibition rate. Calculating IC by conversion of inhibition ratio using SPSS software₅₀The results are shown in tables 1 and 2 below. The inhibition ratio (%) was (negative control OD value-experimental OD value)/negative control OD value × 100%.

TABLE 1 inhibitory Activity of Rakicidin B1-2 against HCT-8 human Colon cancer cells in hypoxic states

TABLE 2 inhibitory Activity of Rakicidin B1-2 in hypoxic State on human pancreatic cancer cells PANC-1

The results show that the compound Rakicidin B1-2 has certain antitumor activity under both normoxic and hypoxic states.

2. Activity test against pathogenic anaerobes

(1) In-vitro antibacterial test method for staphylococcus and enterococcus

Preparing a sample: compound Rakicidin B1-2 was dissolved in DMSO to test final concentrations: 16. 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03, 0.015, 0.008 ug/ml.

Preparation of inoculum: a suspension corresponding to a concentration of 0.5M standard turbidimetric tubes was prepared, diluted with broth (Staphylococcus: MH broth; enterococcus: brain-heart infusion broth) and 100. mu.l of the test bacterial suspension (200. mu.l per well) was added to each well to give a final concentration of about 10⁵CFU/ml. Sealing, and culturing in 35-37 deg.C incubator for 18-24 hr.

MIC determination: the lowest inhibitory concentration was the lowest drug concentration that completely inhibited bacterial growth in the wells, and the test results are shown in table 3 below.

(2) Method for testing in-vitro antibacterial activity of clostridium difficile

Inoculum preparation and inoculation: preparing a suspension with a concentration corresponding to 0.5 McLeod standard turbidimetric tube, preparing a bacterial solution (1-2 μ L) by pipetting with a multi-point inoculator (using a Brookfield medium supplemented with 5% (V/V) defibrinated sheep blood, hemin (5mg/L) and vitamin K1(1 mg/L))]Inoculating to the surface of agar plate (prepared by dissolving antibacterial agent with different concentrations diluted at multiple times into reinforced Brookfield agar dissolved and sterilized under high pressure, balancing in water bath at 60 deg.C, mixing, pouring to obtain agar plate with thickness of 3-4mm, preparing medicinal agar plate at ratio of 1: 9, setting concentration gradient of 0.008-16mg/L for 12 medicines), and inoculating to the surface of agar plate with bacterial amount of about 10⁵CFU, forming bacteria with diameter of 5-8mmAnd (4) spots. After inoculation, the mixture is placed in an anaerobic environment for incubation for 48 hours at 35 ℃, and the result is observed.

MIC determination: the plate was placed on a dark, non-reflective object surface to determine the end of the test, with the lowest drug concentration inhibiting bacterial growth being the MIC. The lowest drug concentration that inhibited bacterial growth by more than 80% compared to the growth control was used as the endpoint concentration and the results are shown in table 3 below.

TABLE 3 Rakicidin B1-2 antibacterial Activity

The antibacterial test result shows that the compound Rakicidin B1-2 has certain inhibitory activity to 10 anaerobic bacteria such as methicillin-resistant staphylococcus aureus, clostridium difficile, vancomycin-resistant enterococcus faecalis and the like.

Detailed Description