CN110698537B - A kind of natural Rakicidins compound Rakicidin B1-2 and its fermentation extraction method - Google Patents

Abstract

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 the new Rakicidin component of Rakicidin B1-2 from the marine micromonospora fermentation broth, and compared with the known Rakicidin B1, the structure of the compound is characterized in that 11 and 14 open loops are formed, and ethoxy 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 normoxic and hypoxic conditions, has a certain inhibition activity on 10 anaerobic bacteria such as methicillin-resistant staphylococcus aureus, clostridium difficile and vancomycin-resistant enterococcus faecalis, is a secondary metabolite with novel structure and better activity, and has higher medicinal value.

Description

A natural rakicidins compound Rakicidin B1-2 and its fermentation extraction method

Technical Field

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

Background Art

At present, hundreds of bioactive substances have been isolated from the metabolites of marine microorganisms, some of which are antibiotics with antitumor or antibacterial activities that may have clinical application value. Rakicidins compounds are one of the important compounds. It has been reported that a series of Rakicidins compounds with antitumor or antibacterial activities have been found from Micromonospora and Streptomyces.

In recent years, the literature has reported that the marine Micromonospora sp.FIM 02-523 can produce a series of lipopeptide compounds with anti-tumor activity, including Rakicidin A, B, B1, etc. In 2006, our research group was the first in China to report the isolation of compounds Rakicidin A and B from this strain of Micromonospora. Studies have found that Rakicidin A has excellent hypoxia-selective anti-tumor cell activity, and its anti-colon cancer HCT-8 cell activity under hypoxia is 17.5 times that under normoxic conditions. It is considered by many peers to be a promising anti-hypoxic tumor cell and anti-CSC drug; and the in vitro anti-tumor activity of Rakicidin B also showed that it has a significant inhibitory effect on the growth of tumor cell lines K562 and L929. In 2016, our research group obtained a new compound, RakicidinB1. The experiment used a zebrafish model transplanted with human colon cancer HCT-8 tumors to test the in vivo tumor inhibition activity of Rakicidin A, B, and B1. The results showed that Rakicidin A, B, and B1 all had inhibitory activity against HCT-8 cell transplanted tumors in zebrafish, and preliminary experiments showed that its anti-hypoxic tumor cells were based on its ability to effectively inhibit the expression of HIF-1 (hypoxia-inducible factor-1). The previous study further determined the cytotoxic activity of Rakicidin A, B, and B1 compounds against five human tumor cell lines, HCT-8, MGC803, A549, A375, HepG2, and CASKI. The results showed that the three isolated compounds had significant inhibitory activity against these tumor cell lines. In 2018, our research group isolated and purified three new compounds, Rakicidin G, H, and I, from the metabolites of the marine Micromonospora sp. FIM 02-523 strain. The activity test results showed that Rakicidin G, H, and I compounds all had strong cytotoxic activity against human colon cancer cells HCT-8 and human pancreatic cancer cells PANC-1, and were stronger under hypoxic conditions. In addition, the three compounds also had good inhibitory activity against a variety of Gram-positive anaerobic bacteria. It can be seen that Rakicidins compounds have excellent clinical application prospects.

Summary of the invention

Therefore, the technical problem to be solved by the present invention is to provide a natural Rakicidins compound Rakicidin B1-2, and further disclose a fermentation extraction method thereof.

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

The invention also discloses a method for preparing the natural Rakicidins compound Rakicidin B1-2. The Rakicidin B1-2 is obtained by fermenting and separating the marine micromonospora strain Micromonosporas p.FIM-R160609 with a preservation number of CGMCC No.14823.

Specifically, the marine micromonospora strain Micromonospora sp.FIM-R160609 described in the present invention is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC No.14823, and the deposit date is October 16, 2017.

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

(1) fermenting the preserved marine micromonospora strain Micromonospora sp.FIM-R160609, collecting the fermentation liquid for solid-liquid separation to obtain mycelium; soaking the obtained mycelium in methanol or ethanol, and collecting the soaking liquid;

(2) performing chromatography using a D3502 macroporous resin adsorption column and gradient eluting with 67% and 75% ethanol-water solutions, respectively, and collecting the 75% ethanol-water eluate fraction;

(3) The collected eluate was chromatographed on a HZ816 resin adsorption column and gradient eluted with 66%, 69% and 73% ethanol-water solutions, respectively, to collect the 73% ethanol-water eluate fraction containing Rakicidin B1-2;

(4) extracting the obtained eluate with ethyl acetate or dichloromethane, concentrating the obtained crude product, dissolving it with methanol, performing semi-preparative liquid chromatography separation, and eluting it with acetonitrile-water in a volume ratio of 710:290, collecting the fractions, and obtaining the product.

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

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

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

The invention also discloses the use of the natural Rakicidins compound Rakicidin B1-2 and its pharmaceutically acceptable salt for preparing drugs with anti-tumor activity and anti-clinical pathogenic anaerobic bacteria activity.

Specifically, the tumors include human colon cancer and human pancreatic cancer; the pathogenic anaerobic bacteria include methicillin-resistant Staphylococcus aureus, vancomycin-intermediately sensitive Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis and Clostridium difficile.

Specifically, the daily dosage of the natural Rakicidins compound Rakicidin B1-2 and its pharmaceutically acceptable salt is 1-5000 mg/day, and the dosage may exceed this range depending on the dosage form and the severity of the disease.

The invention also discloses a medicine for treating pathogenic anaerobic bacteria infection diseases, which uses the natural Rakicidins compound Rakicidin B1-2 and its pharmaceutically acceptable salt as active ingredients, and is added with a pharmaceutically acceptable carrier.

Specifically, the drug can be in the form of conventional pharmaceutical preparations such as ordinary tablets or capsules, sustained-release tablets or capsules, controlled-release tablets or capsules, oral solutions, and injections.

The invention also discloses the use of marine micromonospora strain Micromonospora sp.FIM-R160609 for fermenting and preparing natural Rakicidins compound Rakicidin B1-2. The marine micromonospora strain Micromonospora sp.FIM-R160609 has been deposited in the General Microbiological Center of China Microbiological Culture Collection Committee, and its deposit number is CGMCC No.14823.

The present invention separates a new Rakicidins component Rakicidin B1-2 from a fermentation broth of marine micromonospora. Compared with the known Rakicidin B1, the structure of the compound is open at positions 11 and 14, and an ethoxy group is connected at position 11. The natural compound Rakicidin B1-2 of the present invention has a certain in vitro inhibitory effect on human colon cancer cells HCT-8 and human pancreatic cancer cells PANC-1 cultured under normoxic and hypoxic conditions, and has a certain inhibitory activity on 10 anaerobic bacteria such as methicillin-resistant Staphylococcus aureus, Clostridium difficile and vancomycin-resistant Enterococcus faecalis. It is a secondary metabolite with a novel structure and good activity, and has a high medicinal value.

Experimental example

1. Anti-tumor activity test

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

Normal culture: Human colon cancer cells HCT-8 and human pancreatic cancer cells PANC-1 in the exponential growth phase were seeded in 96-well plates (cell concentrations were 5.0×10 ⁴ cells/ml for HCT-8 and 4.0×10 ⁴ cells/ml for PANC-1, 100ul/well), cultured for 24 hours to allow them to adhere to the wall, and 100ul/well of fresh medium with drugs was added. Three replicate wells were set for each concentration, and blank control wells (only medium was added) were set as negative controls, also with three replicate wells. Culture was continued for 48 hours, and then the culture was terminated.

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

Hypoxic culture: Human intestinal cancer cells HCT-8 and human pancreatic cancer cells PANC-1 in the exponential growth phase were planted in 96-well plates (cell concentrations were HCT-8 5.0×10 ⁴ cells/ml, PANC-1 4.0×10 ⁴ cells/ml, 100ul/well), hypoxic ventilation for 30 minutes, the ventilation valve was closed, and the plates were placed in an incubator at 37°C for 24 hours to adhere to the wall, and 100ul/well of fresh medium with drugs was added. Three replicate wells were set for each concentration, and blank control wells (only medium was added) were set as negative controls, and three replicate wells were also set. Hypoxic ventilation for 30 minutes, the ventilation valve was closed, and the plates were placed in an incubator at 37°C for 48 hours, and the culture was terminated.

MTT assay: Add 10ul of CCK-8 to each well of the terminated cells, continue to culture in an incubator for 2 hours, discard the supernatant, add 150ul of DMSO solution to each well, shake for 10 minutes and gently mix, read the OD value of each well with a microplate reader at a wavelength of 450nm, and calculate the inhibition rate. By converting the inhibition rate, the IC ₅₀ value was calculated using SPSS software, and the results are shown in Tables 1 and 2 below. Inhibition rate (%) = (OD value of negative control group - OD value of experimental group) / OD value of negative control group × 100%.

Table 1 Inhibitory activity of Rakicidin B1-2 on human colon cancer cells HCT-8 under hypoxia

Table 2 Inhibitory activity of Rakicidin B1-2 on human pancreatic cancer cells PANC-1 under hypoxia

The above results show that the compound Rakicidin B1-2 of the present invention has certain anti-tumor activity both under normoxic and hypoxic conditions.

2. Anti-pathogenic anaerobic bacteria activity test

(1) In vitro antibacterial test methods for Staphylococcus and Enterococcus

Sample preparation: Compound Rakicidin B1-2 was dissolved in DMSO to make the final test 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: Prepare a suspension with a concentration equivalent to 0.5 McFarland standard turbidimetric tube, dilute with broth (Staphylococcus: MH broth; Enterococcus: Brain Heart Infusion Broth), then add 100 μl of the test bacterial solution to each well (200 μl per well), the final concentration of the bacterial solution is about 10 ⁵ CFU/ml. After sealing, place in a 35-37℃ incubator for 18-24 hours.

MIC determination: The lowest drug concentration that completely inhibits bacterial growth in the small hole is taken as its minimum inhibitory concentration. The test results are shown in Table 3 below.

(2) In vitro antibacterial activity test method for Clostridium difficile

Preparation of inoculum and inoculation: Prepare a suspension with a concentration equivalent to 0.5 McFarland standard turbidimetric tube, and use a multi-point inoculator to draw the prepared bacterial solution (1-2μl) [using Brucella medium supplemented with 5% (V/V) defibrinated sheep blood, hemin (5mg/L) and vitamin K1 (1mg/L)] and inoculate it on the surface of the agar plate (add the antimicrobial drugs of different concentrations that have been diluted in multiples to the fortified Brucella broth agar that has been sterilized by high pressure dissolution and balanced in a 60℃ water bath, mix well, pour the plate, and the agar thickness is 3-4mm. Prepare the drug agar plate at a ratio of 1:9, set 12 drug concentration gradients from 0.008-16mg/L), the amount of bacteria inoculated at each point is about 10 ⁵ CFU, and a plaque with a diameter of 5-8mm is formed. After inoculation, place it in an anaerobic environment at 35℃ for 48h and observe the results.

MIC determination: Place the plate on a dark, non-reflective surface to determine the test endpoint, and the lowest drug concentration that inhibits bacterial growth is the MIC. The lowest drug concentration that inhibits bacterial growth by more than 80% compared with the growth control is the endpoint concentration. The test results are shown in Table 3 below.

Table 3 Antibacterial activity of Rakicidin B1-2

The above antibacterial test results show that the compound Rakicidin B1-2 has certain inhibitory activity against 10 anaerobic bacteria including methicillin-resistant Staphylococcus aureus, Clostridium difficile and vancomycin-resistant Enterococcus faecalis.

DETAILED DESCRIPTION

Example 1 Preparation of Rakicidin B1-2

The preserved marine micromonospora strain Micromonospora sp.FIM-R160609 (deposit number is CGMCC No.14823) was fermented. The specific fermentation conditions can refer to the fermentation method disclosed in Chinese patent CN108300672B, which specifically includes: scraping the slant spores of freshly cultured marine micromonospora FIM-R160609 to make a bacterial suspension, inoculating it into a shake flask seed culture, culturing it at 32°C and 250rpm for 48 hours, and then inoculating it into a shake flask fermentation medium at a 5% inoculum amount, culturing it at 30°C and 250rpm for 120 hours, and then releasing the bottle, and determining the fermentation product by HPLC.

Seed culture medium formula (mass fraction): maltodextrin 2.0%, glucose 1.0%, soybean cake powder 1.0%, yeast powder 2.0%, MgSO ₄ ·7H ₂ O 0.05%, KH ₂ PO ₄ 0.05%, CaCO ₃ 0.3%, tap water, pH value adjusted to 7.0-7.5.

Formula of fermentation medium (mass fraction): 5.0% maltodextrin, 1.0% glucose, 2.0% soybean cake powder, 1.5% yeast powder, 0.8% ammonium sulfate, 0.05% MgSO ₄ ·7H ₂ O, 0.5% NaCl, 0.5% CaCO ₃ 0.5%, tap water, adjusted to pH 7.5.

After the fermentation is completed, the fermentation liquid is collected and centrifuged at 4500rpm for 15 minutes to obtain mycelial residue, which is then soaked twice in 2 times the volume of 90% ethanol overnight. The mycelial residue containing alcohol is centrifuged again at 4500rpm for 15 minutes, and the supernatants are combined to obtain the fermentation extract.

Take 45L of the fermentation extract and dilute it to an ethanol concentration of 55%, then chromatograph on a D3502 macroporous resin adsorption column (diameter-to-height ratio of 1:8, column volume of 3.0L), with an adsorption flow rate of 33ml/min, and elute 3BV and 8.5BV (column volumes) with 67% and 75% ethanol water, respectively. Detect the eluate with HPLC (YMCODS _C18 column, 5μm, 250mm×4.6mm) (acetonitrile-water 700:300, column temperature 40°C, flow rate 1.0mL/min, wavelength 262nm), and collect the 75% ethanol eluate (23L) containing Rakicidin B1-2.

The collected eluate was diluted to an ethanol concentration of 60%, and chromatographed on a HZ816 resin adsorption column (diameter-to-height ratio of 1:6, column volume of 2.5 L). The adsorption flow rate was controlled at 30 ml/min, and eluted with 66%, 69% and 73% ethanol-water solutions, eluting 3 BV, 2.5 BV and 7 BV respectively. The eluate was tracked and detected by HPLC (YMC ODS C ₁₈ column, 5 μm, 250 mm×4.6 mm) (acetonitrile-water 700:300, column temperature 40°C, flow rate 1.0 mL/min, wavelength 262 nm), and 73% ethanol-water solution (13.8 L) containing Rakicidin B1-2 was collected.

The collected eluate was concentrated to about 1/3 of the volume, extracted three times with an equal volume of dichloromethane, and concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in methanol and eluted using semi-preparative liquid chromatography (5 μm, 250 mm×10 mm, Welch Material) with acetonitrile-water in a volume ratio of 710:290. The flow rate was controlled at 8 ml/min, and the collected liquid was detected by HPLC to obtain the sample Rakicidin B1-2.

Through observation and detection, the compound Rakicidin B1-2 prepared in this example is a white amorphous powder, soluble in chloroform, methanol, and DMSO, but insoluble in water.

The results of high-resolution mass spectrometry (HR-ESI-MS) showed that the molecular ion peaks [M+H] ⁺ were 669.4433 and [M+Na] ⁺ were 691.4248. It was inferred that the molecular formula was C ₃₄ H ₆₀ N ₄ O ₉ and the degree of unsaturation was 7.

¹ HNMR detection results showed that the compound had 2 double bond protons [δ _H 7.44 (1H, d, J = 15.5 Hz), 6.57 (1H, d, J = 15.5 Hz)], 6 exchangeable protons including [δ _H 7.40 (1H, d, J = 9.0 Hz), 6.84 (1H, s), 6.81 (1H, s), 5.79 (1H, s)], and 6 methyl protons [δ _H 3.04 (3H, s), 1.25 (3H, t, J = 6.0 Hz), 0.99 (3H, d, J = 7.0 Hz), 0.85 (3H, t, J = 6.9 Hz), 0.83 (3H, d, J = 6.6 Hz), 0.82 (3H, d, J = 6.6 Hz)].

¹³ CNMR and DEPT135 detection results showed that the molecule contained 34 carbon signals, including 6 quaternary carbons (δ _C 176.1, 169.6, 169.4, 168.5, 165.3, 164.7), 2 double bond carbons (δ _C 134.9, 130.6), 6 sp ³ methyl carbons [including four oxygen-connected carbon atoms (δ _C 78.6, 71.6, 55.4, 42.0)], 14 sp ³ methylene carbons and 6 methyl carbon atoms (δ _C 36.6, 19.6, 14.8, 14.5, 13.8, 11.7).

All hydrogen protons were assigned by ¹ H- ¹³ C correlation of HSQC spectrum. ¹ H- ¹ H COSY correlation spectrum and proton coupling constants showed that the compound contained four independent spin coupling systems: NH-2-C-2-C-3-OH-3, C-9-C-10, C-32-C-15-C-16-C-17(C-33)-C-18-C-19 and C-28-C-29(C-34)-C-30-C-31. Combining ¹ H- ¹ H COSY and HMBC, it can be seen that H-2 is related to C-1/C-5, H-3 is related to C-4, OH-3 is related to C-2/C-3, Ha-6 is related to C-5/C-7, H-7 is related to C-6/C-8, H-9 is related to C-8/C-11, H-10 is related to C-8/C-11, H-12 is related to C-11/C-13, H-16 is related to C-1, H-32 is related to C-14/C-15/C-16, H-33 is related to C-16/C-17/C-18, and H-31 is related to C-29/C-30, and H-34 is related to C-29/C-30. Through the analysis of the above NMR data, unsaturation, molecular formula and chemical shift, the chemical shifts of hydrogen and carbon are listed in Table 4.

Table 4 Chemical shifts of hydrogen and carbon

By analyzing the above NMR data, unsaturation, molecular formula and chemical shift, the chemical structure of compound 1 is shown in formula (I). It can be seen that the structure of the compound Rakicidin B1-2 prepared by fermentation extraction in this example is correct.

Example 2 Preparation of Rakicidin B1-2

The preserved Micromonospora sp. FIM-R160609 was fermented in the same manner as in Example 1. The fermentation broth was collected and centrifuged at 4500 rpm for 15 min to obtain mycelial residues. The mycelial residues were soaked twice overnight with 2 times the volume of 90% ethanol. The mycelial residues containing alcohol were centrifuged again at 4500 rpm for 15 min, and the supernatants were combined to obtain a fermentation extract.

Take 30L of the fermentation extract and dilute it to an ethanol concentration of 50%, then perform D3502 macroporous resin adsorption column chromatography (diameter-to-height ratio of 1:5, column volume 2.5L), the adsorption flow rate is 45ml/min, 2.5BV and 6BV are eluted with 67% and 75% ethanol water, respectively, and the eluate is detected by HPLC (YMC ODS _C18 column, 5μm, 250mm×4.6mm) (acetonitrile-water 700:300, column temperature 40°C, flow rate 1.0mL/min, wavelength 262nm), and the 75% ethanol eluate (12.5L) containing RakicidinB1-2 is collected.

The collected eluate was diluted to an ethanol concentration of 55%, and chromatographed on a HZ816 resin adsorption column (diameter-to-height ratio of 1:8, column volume of 3.0 L). The adsorption flow rate was controlled at 35 ml/min, and eluted with 66%, 69% and 73% ethanol-water solutions, eluting 3 BV, 2 BV and 4.5 BV respectively. The eluate was tracked and detected by HPLC (YMC ODS C ₁₈ column, 5 μm, 250 mm×4.6 mm) (acetonitrile-water 700:300, column temperature 40°C, flow rate 1.0 mL/min, wavelength 262 nm), and 73% ethanol-water solution (12 L) containing Rakicidin B1-2 was collected.

The collected eluate was concentrated to about 1/3 of the volume, extracted three times with an equal volume of ethyl acetate, and concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in methanol and eluted using a semi-preparative liquid phase preparation (5 μm, 250 mm×10 mm, Welch Material) with acetonitrile-water in a volume ratio of 710:290. The flow rate was controlled at 8 ml/min, and the collected liquid was detected by HPLC (YMC ODS C ₁₈ column, 5 μm, 250 mm×4.6 mm) to obtain a sample of Rakicidin B1-2.

After testing, the product structure obtained by the scheme of this embodiment is correct.

Example 3 Preparation of Rakicidin B1-2

The preserved Micromonospora sp. FIM-R160609 was fermented in the same manner as in Example 1. The fermentation broth was collected and centrifuged at 4500 rpm for 15 min to obtain mycelial residues. The mycelial residues were soaked twice overnight with 2 times the volume of 90% ethanol. The mycelial residues containing alcohol were centrifuged again at 4500 rpm for 15 min, and the supernatants were combined to obtain a fermentation extract.

Take 55L of the fermentation extract and dilute it to an ethanol concentration of 60%, then perform D3502 macroporous resin adsorption column chromatography (diameter-to-height ratio of 1:8, column volume 4.0L), the adsorption flow rate is 30ml/min, 3.5BV and 8BV are eluted with 67% and 75% ethanol water, respectively, and the eluent is detected by HPLC (YMC ODSC ₁₈ column, 5μm, 250mm×4.6mm) (acetonitrile-water 700:300, column temperature 40℃, flow rate 1.0mL/min, wavelength 262nm), and collect the 75% ethanol eluate (29.4L) containing Rakicidin B1-2.

The collected eluate was diluted to an ethanol concentration of 60%, and chromatographed on a HZ816 resin adsorption column (diameter-to-height ratio of 1:5, column volume of 2.2 L). The adsorption flow rate was controlled at 25 ml/min, and eluted with 66%, 69% and 73% ethanol-water solutions, eluting 2.5 BV, 3 BV and 6 BV respectively. The eluate was tracked and detected by HPLC (YMC ODS C ₁₈ column, 5 μm, 250 mm×4.6 mm) (acetonitrile-water 700:300, column temperature 40°C, flow rate 1.0 mL/min, wavelength 262 nm), and 73% ethanol-water solution (11.5 L) containing Rakicidin B1-2 was collected.

The collected eluate was concentrated to about 1/3 of the volume, extracted three times with an equal volume of dichloromethane, and concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in methanol and eluted using a semi-preparative liquid phase preparation (5 μm, 250 mm×10 mm, Welch Material) with acetonitrile-water in a volume ratio of 710:290. The flow rate was controlled at 8 ml/min, and the collected liquid was detected by HPLC (YMC ODS C ₁₈ column, 5 μm, 250 mm×4.6 mm) to obtain a sample of Rakicidin B1-2.

After testing, the product structure obtained by the scheme of this embodiment is correct.

Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the protection scope of the invention.

Claims (9)

1. A natural Rakicidin compound Rakicidin B1-2 and pharmaceutically acceptable salts thereof are characterized in that the structure of the Rakicidin B1-2 is shown as the following formula (I):

2. a method for preparing the natural Rakicidins B1-2 of claim 1, wherein the Rakicidins B1-2 are obtained by fermenting and separating a marine Micromonospora sp.FIM-R160609 strain with a preservation number of CGMCC No.14823.

3. A process for the preparation of said natural Rakicidins, rakicidin B1-2, as claimed in claim 2, comprising the steps of:

(1) Fermenting the preserved marine Micromonospora sp.FIM-R160609 strain, and collecting fermentation liquor for solid-liquid separation to obtain mycelium; soaking the mycelium with methanol or ethanol, and collecting soaking solution;

(2) Carrying out chromatography by using a D3502 macroporous resin adsorption column, carrying out gradient elution by using 67% and 75% ethanol-water solutions respectively, and collecting 75% ethanol-water eluent components;

(3) Subjecting the collected eluate to HZ816 resin adsorption column chromatography, and gradient eluting with 66%, 69% and 73% ethanol-water solution, respectively, to collect 73% ethanol-water eluate component containing Rakicidin B1-2;

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

4. A process for the preparation of said natural Rakicidins group compound Rakicidin B1-2 as claimed in claim 3 wherein in step (2) the D3502 macroporous resin adsorption column has an aspect ratio of 1:5-1:10, the column loading volume is 2.5-4.0L, and the adsorption flow rate is 30-40ml/min.

5. The method for preparing Rakicidin B1-2 as claimed in claim 3 or 4, wherein in step (3), the HZ816 resin column aspect ratio is 1:5-1:8, the packed volume is 2.2-3L, and the adsorption flow rate is 25-35ml/min.

6. The method for preparing Rakicidin B1-2 as claimed in claim 3 or 4, wherein in step (4), the semi-preparative liquid chromatography is Welch Material,5 μm,250mm×10mm, and eluent flow rate is controlled to 8ml/min.

7. The use of the natural Rakicidins of claim 1, rakicidin B1-2, and pharmaceutically acceptable salts thereof, for the preparation of a medicament having activity against clinically pathogenic anaerobic bacteria; wherein,,

the anti-clinical pathogenic anaerobic bacteria are methicillin-resistant staphylococcus aureus MRSA15-1, methicillin-resistant staphylococcus aureus MRSA15-2, methicillin-sensitive staphylococcus aureus MSSA15-1, methicillin-sensitive staphylococcus aureus MSSA15-2, vancomycin-resistant enterococcus faecalis ATCC51575 and vancomycin-resistant enterococcus faecalis ATCC700802.

8. A medicament for treating pathogenic anaerobic bacterial infection diseases, which is characterized in that natural Rakicidins B1-2 and pharmaceutically acceptable salts thereof as active ingredients in claim 1 are added with pharmaceutically acceptable carriers.

9. The use of a marine Micromonospora sp.fim-R160609 strain for the fermentative preparation of natural Rakicidins of type Rakicidin B1-2 according to claim 1, characterized in that said marine Micromonospora sp.fim-R160609 strain has been deposited in the China general microbiological culture collection center with the accession number CGMCC No.14823.