Natural Rakicidins compound Rakicidin J and fermentation extraction method thereof
阅读说明：本技术 一种天然Rakicidins类化合物Rakicidin J及其发酵提取方法 (Natural Rakicidins compound Rakicidin J 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 J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 J,该化合物结构相比于已知的Rakicidin B1,在脂肽侧链的长度不同,多两个亚甲基。本发明天然化合物Rakicidin J对人结肠癌细胞HCT-8和人胰腺癌细胞PANC-1具有较好的体外抑制作用,对乏氧培养的肿瘤细胞HCT-8活性较常氧的强4.93倍,对乏氧培养的肿瘤细胞PANC-1活性较常氧的强6.08倍,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优良的次级代谢产物,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 J and a fermentation extraction method thereof. The invention separates new Rakicidins component Rakicidin J from marine micromonospora fermentation liquor, and compared with the known Rakicidin B1, the compound has the structure that the length of a lipopeptide side chain is different and two methylene groups are added. The natural compound Rakicidin J has better in-vitro inhibition effect on human colon cancer cells HCT-8 and human pancreatic cancer cells PANC-1, has 4.93 times stronger activity on hypoxic cultured tumor cells HCT-8 than normal oxygen, has 6.08 times stronger activity on hypoxic cultured tumor cells PANC-1 than normal oxygen, has 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 novel structure and excellent activity, and has higher medicinal value.)
1. A natural Rakicidins compound Rakicidin J and pharmaceutically acceptable salts thereof, wherein the compound Rakicidin J has a structure shown as the following formula (I):
2. a method for preparing the natural Rakicidins compound Rakicidin J as claimed in claim 1, which is prepared from Micromonosporasp.FIM-R150103 of Micromonosporas marinum with the preservation number of CGMCC No.14822 through fermentation and separation.
3. The process for the preparation of the natural Rakicidins compound Rakicidin J according to claim 2, characterized in that it comprises the following steps:
(1) fermenting the preserved Micromonospora sp.FIM-R150103, collecting the fermentation liquor, and performing solid-liquid separation to obtain 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 70% and 78% ethanol-water solution respectively, and collecting 78% ethanol-water eluate;
(3) carrying out chromatography on the collected eluent by using an HZ816 resin adsorption column, carrying out gradient elution by using 72% and 76% ethanol-water solutions respectively, and collecting 76% ethanol-water eluent components containing Rakicidin J;
(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 730: 270, eluting with acetonitrile-water, and collecting by sections to obtain the product.
4. The method for preparing the natural Rakicidins compound Rakicidin J 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 30-45 ml/min.
5. The method for preparing the natural Rakicidins compound Rakicidin J according to claim 3 or 4, wherein in the step (3), the height ratio of the HZ816 resin adsorption column diameter to the height ratio of 1: 5-1: 8, the column volume is 2.2-3L, and the adsorption flow rate is 25-35 ml/min.
6. The process for preparing Rakicidin J according to any one of claims 3-5, wherein in step (4), the semi-preparative liquid chromatography is Welch Material, 5 μm,250mm x 10mm, and the flow rate of the eluate is controlled to 8 ml/min.
7. Use of the natural Rakicidins compound Rakicidin J 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 J 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 J and pharmaceutically acceptable salts thereof in claim 1 are used as active ingredients, and pharmaceutically acceptable carriers are added.
10. The application of Micromonospora sp.FIM-R150103 in preparing natural Rakicidin compounds Rakicidin J by fermentation is characterized in that the Micromonospora sp.FIM-R150103 is preserved in China general microbiological culture Collection center with the preservation number of CGMCCNo.14822.
Technical Field
The invention belongs to the technical field of natural compounds, and particularly relates to a natural Rakicidins compound Rakicidin J and a fermentation extraction method thereof.
Background
At present, hundreds of bioactive substances have been isolated from metabolites of marine microorganisms, some of which are antibiotics with antitumor or bacteriostatic activity that may have clinical application value,
rakicidins compounds are an important class of compounds, and a series of Rakicidins compounds with anti-tumor activity or bacteriostatic activity are reported to be found in micromonospora and streptomyces at present.
In recent years, the literature reports that Micromonospora sp.FIM 02-523, a strain of Micromonospora sp.marinum, is capable of metabolizing to produce a series of lipopeptide compounds with antitumor activity, including Rakicidin A, B, B1 and the like. In 2006, the subject group first reported the isolation of the compounds Rakicidin A and B from this strain of Micromonospora domestica domestically. Research finds that Rakicidin A has excellent hypoxia-selective anti-tumor cell activity, the anti-colon cancer HCT-8 cell activity under the hypoxia condition is 17.5 times that under the normoxic condition, and the Rakicidin A is considered as an anti-hypoxia tumor cell and anti-CSC drug with extremely good development prospect by many colleagues; 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 and B, B1 both have inhibition activity on HCT-8 cell transplanted tumor in zebra fish, and a preliminary experiment shows that the hypoxia tumor cell is based on the fact that the hypoxia tumor cell can effectively inhibit the expression of HIF-1 (hypoxia inducible factor-1). The prior 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 J and further discloses a fermentation extraction method thereof.
In order to solve the technical problems, the invention provides a natural Rakicidins compound Rakicidin J and pharmaceutically acceptable salts thereof, wherein the compound Rakicidin J has a structure shown as the following formula (I):
the invention also discloses a method for preparing the natural Rakicidins compound Rakicidin J, which is characterized in that the Rakicidin J is obtained by fermenting and separating Micromonospora sp.FIM-R150103 of the marine Micromonospora strain with the collection number of CGMCC No. 14822.
Specifically, the marine Micromonospora sp.FIM-R150103 is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms with the preservation address: the No. 3 Xilu No.1 of Beijing, Chaoyang, the area of Chaoyang has a preservation number of CGMCC No.14822 and a preservation date of 2017, 10 months and 16 days.
Preferably, the method for preparing the natural Rakicidins compound Rakicidin J comprises the following steps:
(1) fermenting the preserved Micromonospora sp.FIM-R150103, collecting the fermentation liquor, and performing solid-liquid separation to obtain 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 70% and 78% ethanol-water solution respectively, and collecting 78% ethanol-water eluate;
(3) carrying out chromatography on the collected eluent by using an HZ816 resin adsorption column, carrying out gradient elution by using 72% and 76% ethanol-water solutions respectively, and collecting 76% ethanol-water eluent components containing Rakicidin J;
(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 730: 270, eluting with acetonitrile-water, and collecting by sections to obtain the product.
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 J and pharmaceutically acceptable salts thereof in preparing medicaments with antitumor activity and clinical pathogenic anaerobic bacteria activity resistance.
Specifically, the tumor comprises human colon cancer and human pancreatic cancer; the pathogenic anaerobic bacteria include Clostridium difficile, Streptococcus digestus, Porphyromonas gingivalis, and Propionibacterium acnes.
Specifically, the daily dose of the natural Rakicidins compound Rakicidin J and pharmaceutically acceptable salts thereof is 1-5000 mg/day, and the dose can exceed the range according to different dosage forms and the severity of diseases.
The invention also discloses a medicine for treating pathogenic anaerobic bacteria infection diseases, which takes the natural Rakicidins compound Rakicidin J and pharmaceutically acceptable salts thereof as active ingredients and adds pharmaceutically acceptable carriers.
The medicine can be common tablets or capsules, sustained-release tablets or capsules, controlled-release tablets or capsules, oral liquid, injection and other preparation forms which are conventional in pharmaceutics.
The invention also discloses application of Micromonospora sp.FIM-R150103 of the marine Micromonospora strain in preparing Rakicidin J of natural Rakicidin compounds by fermentation, wherein the Micromonospora sp.FIM-R150103 of the marine Micromonospora strain is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC NO. 14822.
The invention separates new Rakicidins component Rakicidin J from marine micromonospora fermentation liquor, and compared with the known Rakicidin B1, the compound has the structure that the length of a lipopeptide side chain is different and two methylene groups are added. The natural compound Rakicidin J has better in-vitro inhibition effect on human colon cancer cells HCT-8 and human pancreatic cancer cells PANC-1, has 4.93 times stronger activity on hypoxic cultured tumor cells HCT-8 than normal oxygen, has 6.08 times stronger activity on hypoxic cultured tumor cells PANC-1 than normal oxygen, has 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 novel structure and excellent activity, and has higher medicinal value.
Examples of the experiments
1. Antitumor Activity test
The extracted Rakicidin J samples were dissolved in DMSO to a solubility of 1ug/ml and then diluted individually to final concentrations of 0.5ug/ml, 0.25ug/ml, 0.125ug/ml, 0.0625ug/ml, 0.03125ug/ml, 0.015625 ug/ml.
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 (only adding culture medium) as negative control, and setting 3 multiple wells. The culture was continued for 48hr, and the culture was terminated.
Rakicidin J 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 into each well of the cells after the culture is stopped, continuously culturing for 2 hours in an incubator, discarding the supernatant, adding 150ul of DMSO solution into each well, gently mixing the mixture evenly 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 rate and 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 J against HCT-8 human Colon cancer cells in hypoxic states
TABLE 2 inhibitory Activity of Rakicidin J in hypoxic State on human pancreatic cancer cells PANC-1
The results show that the compound Rakicidin J has strong anti-tumor activity, particularly anti-hypoxic tumor cell activity, the activity of the compound Rakicidin J on tumor cells HCT-8 cultured by hypoxic is 4.93 times stronger than that of normoxic tumor cells HCT-8 cultured by hypoxic, and the activity of the compound Rakicidin J on tumor cells PANC-1 cultured by hypoxic is 6.08 times stronger than that of normoxic tumor cells PANC-1.
2. Activity test against pathogenic anaerobes
(1) In-vitro antibacterial test method for staphylococcus and enterococcus
Preparing a sample: compound Rakicidin B1-1 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 meat 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 test results are shown in Table 3 below, with the lowest drug concentration that completely inhibited bacterial growth in the wells being the lowest inhibitory concentration.
(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 on the surface of agar plate (adding diluted antibacterial agent with different concentrations at multiple times into reinforced Brucella meat which has been autoclaved, sterilized, and equilibrated in water bath at 60 deg.CMixing with soup agar, and pouring into plate with agar thickness of 3-4 mm. According to the following steps: 9 proportion preparing drug agar plate, setting 0.008-16 mg/L12 drug concentration gradient), each point inoculating bacterial quantity about 105CFU, forming plaque with a diameter of 5-8 mm. After inoculation, the mixture is placed in an anaerobic environment and incubated 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 3Rakicidin J antibacterial Activity
The antibacterial test result shows that the compound Rakicidin J 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