A natural Rakicidins compound, Rakicidin K, and its fermentation extraction method.

Abstract

The application belongs to the technical field of natural compounds, and particularly relates to a natural Rakicidins compound Rakicidin K and a fermentation extraction method thereof. The Rakicidins analogue Rakicidin K is a novel compound isolated and extracted from fermentation liquor of a mutant strain FIM-R150103 of Micromonospora. The cytotoxic activity of the Rakicidins analogue Rakicidin K is further evaluated and verified. The activity results show that the natural compound Rakicidin K has strong in-vitro inhibition on human colon cancer cells HCT-8 and human pancreatic cancer cells PANC-1, has certain inhibition activity on various gram-positive bacteria, and has good biological activity and high medicinal value.

Description

Technical Field

[0001] This invention belongs to the field of natural compound technology, specifically relating to a natural Rakicidins compound, Rakicidin K, and its fermentation extraction method. Background Technology

[0002] Secondary metabolites of marine microorganisms contain compounds of various structural types, many of which possess excellent biological activity and potential clinical applications, making them a major source for new drug development. Among these, rakicidins are an important class of compounds with potent inhibitory activity against tumor cells. Their skeletal structure is a cyclic peptide, typically composed of four specific amino acids, possessing a unique 4-amino-2,4-pentadienoic acid (APD) structural unit.

[0003] In recent years, a large number of rakicidins analogues with good biological activity have been discovered in the metabolites of Micromonospora and Streptomyces. For example, in 2000, Hu Jin-Feng et al. discovered rakicidin C from Streptomyces sp. GT61042. This compound was inactive against both Gram-positive and Gram-negative strains and had no cytotoxicity against various tumor cells. In 2010, Yasuhiro Igarashi et al. discovered rakicidin D from the metabolite of Streptomyces sp. MWW064 and verified that it could inhibit the invasion of mouse colon cancer 26-L5 cells, but had no inhibitory effect on the cancer cells at 10 μg / mL. In 2014, Naoya Oku et al. discovered rakicidin E from the metabolite of Micromonospora, which showed strong hypoxia-selective cytotoxicity against HCT-8 and PANC-1 tumor cells. In 2017, the literature reported the discovery of rakicidin F in Streptomyces, which had growth inhibitory activity against Bacillus subtilis and Escherichia coli. From 2016 to 2018, the research group at the Fujian Institute of Microbiology isolated rakicidin B1 and rakicidin G, H, and I from the metabolites of Micromonospora FIM02-523, and verified that they have strong cytotoxic activity against HCT-8 and PANC-1 human tumor cell lines, and also have good inhibitory activity against Gram-positive anaerobic bacteria.

[0004] Therefore, it remains essential to develop new rakicidins, which will provide more possibilities for clinical drug discovery and screening as novel anticancer drugs. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a natural Rakicidins compound, Rakicidin K, and further disclose its fermentation extraction method.

[0006] To solve the above-mentioned technical problems, the present invention provides a natural Rakicidins compound, Rakicidin K, and its pharmaceutically acceptable salt, wherein the compound Rakicidin K has the structure shown in formula (Ⅰ):

[0007]

[0008] The present invention also discloses a method for preparing the natural Rakicidins compound Rakicidin K, comprising the steps of fermentation and extraction using micromonospora strain FIM-R150103;

[0009] The micromonospora strain FIM-R150103 is classified as Micromonospora sp. and has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 14822. The deposit date was October 16, 2017, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0010] Specifically, the method for preparing the natural Rakicidins compound Rakicidin K includes the following steps:

[0011] (1) Ferment the preserved micromonospora strain FIM-R150103, collect the fermentation broth and separate the solid and liquid to obtain mycelium; soak the obtained mycelium in methanol or ethanol and collect the soaking liquid;

[0012] (2) The chromatography was performed using an HP20 macroporous resin adsorption column, and gradient elution was performed with 63% and 72% ethanol-water, respectively. The 72% ethanol-water eluent was collected.

[0013] (3) The collected eluent was subjected to chromatography using an HZ816 resin adsorption column, and gradient elution was performed with 65%, 68% and 70% ethanol-water solutions, respectively, and the 70% ethanol eluent containing Rakicidin K was collected.

[0014] (4) The eluent obtained by extraction with ethyl acetate or dichloromethane is concentrated to obtain crude product, which is then dissolved in methanol, separated by semi-preparative liquid chromatography, and eluted with acetonitrile-water at a volume ratio of 715:285. The fractions are collected to obtain the crude product.

[0015] Specifically, in the method for preparing the natural Rakicidins compound Rakicidin K, in step (2), the diameter-to-height ratio of the HP20 macroporous resin adsorption column is 1:3-1:8, the column volume is 2.0-3.5L, and the adsorption flow rate is 30-40ml / min.

[0016] Specifically, in the method for preparing the natural Rakicidins compound Rakicidin K, in step (3), the diameter-to-height ratio of the HZ816 resin adsorption column is 1:3-1:6, the column volume is 2.0-2.7L, and the adsorption flow rate is 25-32ml / min.

[0017] Specifically, in the method for preparing the natural Rakicidins compound Rakicidin K, in step (4), the semi-preparative liquid chromatography is Welch Material, 5μm, 250mm×10mm, and the eluent flow rate is controlled at 7ml / min.

[0018] The present invention also discloses the use of the natural Rakicidins compound Rakicidin K and its pharmaceutically acceptable salts in the preparation of drugs with antitumor activity and / or antigram-positive bacterial activity.

[0019] Specifically, the tumors include human colon cancer and human pancreatic cancer;

[0020] Specifically, the Gram-positive bacteria include methicillin-resistant Staphylococcus aureus, vancomycin-sensitive Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, or Clostridium difficile.

[0021] Specifically, the daily dose of the natural Rakicidins compound Rakicidin K and its pharmaceutically acceptable salt is 1-5000 mg / day, and the dose may be used beyond this range depending on the dosage form and the severity of the disease.

[0022] The present invention also discloses a drug having antitumor activity and / or anti-Gram-positive bacterial activity, wherein the natural Rakicidins compound Rakicidin K and its pharmaceutically acceptable salt are used as active ingredients, and a pharmaceutically acceptable carrier is added.

[0023] The drug may be in the conventional pharmaceutical formulations such as ordinary tablets or capsules, sustained-release tablets or capsules, controlled-release tablets or capsules, oral liquids, or injections.

[0024] This invention also discloses the use of the micromonospora strain FIM-R150103 for the fermentation preparation of the natural Rakicidins compound Rakicidin K. The micromonospora strain FIM-R150103 is classified as Micromonospora sp. and has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 14822, deposit date of October 16, 2017, and address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0025] The present invention isolates and extracts a new Rakicidins analog Rakicidin K from the fermentation broth of the marine micromonospora mutant strain FIM-R150103. The structure of the Rakicidins analog Rakicidin K described in this invention is different from that of the known Rakicidin B1 by removing an H2O molecule at position 2.

[0026] This invention further evaluated and verified the cytotoxic activity of the Rakicidins analogue Rakicidin K. The activity results showed that Rakicidin K exhibited inhibitory activities of 109.541 μg / mL and 101.85 μg / mL against HCT-8 and PANC-1 cancer cells under hypoxic conditions, respectively, and 785.872 ng / mL and 722.803 ng / mL under normoxic conditions, respectively. Its activity against hypoxic-cultured HCT-8 tumor cells was 7.17 times stronger than that against normoxic conditions, and its activity against hypoxic-cultured PANC-1 tumor cells was 7.10 times stronger than that against normoxic conditions. This demonstrates that the natural compound Rakicidin K has a strong in vitro inhibitory effect on human colon cancer cells HCT-8 and human pancreatic cancer cells PANC-1, and that 2-OH has a significant impact on the hypoxia-selective cytotoxicity of rakicidins.

[0027] This invention further evaluated and verified the antibacterial activity of the Rakicidins analogue Rakicidin K. The activity results showed that Rakicidin K exhibited weak inhibitory activity against 10 strains of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, vancomycin-sensitive Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, and Clostridium difficile, demonstrating its inhibitory activity against various Gram-positive bacteria.

[0028] The Rakicidins analog Rakicidin K described in this invention is a novel secondary metabolite with good activity. It not only enriches the variety of rakicidins compounds, but also has good biological activity and high medicinal value. It will be further studied as a candidate compound for new drug development to explore its medicinal value. Detailed Implementation

[0029] Example 1: Preparation of Rakicidin K

[0030] Fermentation was carried out using the preserved Micromonospora strain FIM-R150103 (accession number CGMCC No. 14822), with specific fermentation conditions following the fermentation method disclosed in Chinese Patent CN108130284B.

[0031] Freshly cultured marine micromonospora FIM-R150103 slant spores were scraped and prepared into a bacterial suspension, which was then inoculated into a shake flask seed culture. The culture was carried out at 32℃ and 250rpm for 48 hours. Then, 5% of the culture was inoculated into the shake flask fermentation medium and cultured at 30℃ and 250rpm for 120 hours. The culture was then removed from the flask and the fermentation products were determined by HPLC.

[0032] Seed culture medium formula (mass fraction): soluble starch 2.0%, glucose 1.0%, yeast powder 2.0%, peptone 1.0%, MgSO4·7H2O 0.05%, FeSO4·7H2O 0.005%, CuSO4·5H2O 0.005%, CoCl2·6H2O 0.0005%, CaCO3 0.2%, prepared with tap water, adjusted to pH 7.0-7.5.

[0033] Fermentation medium formula (mass fraction): soluble starch 4.0%, sucrose 1.0%, soybean meal 3.0%, ammonium sulfate 0.5%, valine 0.1%, MgSO4·7H2O 0.04%, FeSO4·7H2O 0.005%, CuSO4·5H2O 0.005%, CoCl2·6H2O 0.0005%, CaCO3 0.5%, prepared with tap water, pH adjusted to 7.5.

[0034] After fermentation, the fermentation broth was collected and centrifuged at 4500 rpm for 15 min to obtain mycelial residue. The obtained mycelial residue was soaked twice overnight in 2 times the volume of 90% ethanol. The mycelial residue containing alcohol was centrifuged again at 4500 rpm for 15 min, and the supernatants were combined to obtain the fermentation extract.

[0035] Take 65 L of fermentation extract and dilute it to an ethanol concentration of 55%. Then, perform adsorption chromatography on an HP20 macroporous resin column (diameter-to-height ratio 1:6, column volume 3.0 L) at an adsorption flow rate of 35 mL / min. Elute with 63% and 72% ethanol and water for 4 BV and 8 BV (column volumes), respectively. Analyze the elution using HPLC (YMC ODS C). 18The column (5 μm, 250 mm × 4.6 mm) was used to detect the eluent (acetonitrile-water 700:300, column temperature 40 °C, flow rate 1.0 mL / min, wavelength 262 nm), and the 72% ethanol eluent containing Rakicidin K (35.2 L) was collected.

[0036] The collected eluent was diluted to an ethanol concentration of 60% and subjected to chromatography on an HZ816 resin adsorption column (diameter-to-height ratio 1:5, column volume 2.3L). The adsorption flow rate was controlled at 28 mL / min. Elution was performed with 65%, 68%, and 70% ethanol-water solutions, yielding 3 BV, 3 BV, and 5.5 BV respectively. The eluent was analyzed by HPLC (YMC ODS C). 18 The eluent (acetonitrile-water 700:300, column temperature 40℃, flow rate 1.0 mL / min, wavelength 262 nm) was monitored using a 5 μm column (250 mm × 4.6 mm). A 70% aqueous ethanol solution (17.9 L) containing Rakicidin K was collected.

[0037] The collected eluent was concentrated to about 1 / 3 of its volume, extracted three times with an equal volume of ethyl acetate, concentrated under reduced pressure to obtain the crude product, and dissolved in methanol. The crude product was then eluted using semi-preparative liquid chromatography (5 μm, 250 mm × 10 mm, Welch Material) with acetonitrile-water at a volume ratio of 715:285, at a flow rate of 7 ml / min. The collected solution was analyzed by HPLC to obtain the sample Rakicidin K.

[0038] The compound Rakicidin K prepared in this example was determined to be a white amorphous powder, soluble in DMSO, methanol, ethanol, and chloroform.

[0039] High-resolution mass spectrometry (HR-ESI-MS) showed that its molecular ion peak was [M+H]. + is 603.4154, [M+Na] + The value is 625.3952, suggesting its molecular formula is C. 33 H 54 N4O6 (UN=9).

[0040] 1 1H NMR results showed that the compound has 5 double protons [δ] H 8.09 (1H,d,J=15.0Hz), 6.93 (1H,d,J=15.0Hz), 6.27 (1H,d,J=15.0Hz), 6.21 (1H,s), 5.42 (1H,s)], 3 exchangeable protons [δ H 8.68 (1H,s), 7.79 (1H,s), 7.36 (1H,s)], 5 methyl protons [δH 3.02(3H,s),1.11(3H,d,J=6.8Hz), 0.91(3H,d,J=6.0Hz), 0.85(3H,t,J=6.6Hz), 0.82(3H,d,J=6.6Hz)].

[0041] 13 12C NMR and DEPT 135 assays showed that the molecule contained 33 carbon signals, including 5 quaternary carbons (δ¹²C). C 172.4, 172.3, 167.9, 166.5, 165.2), 6 double-bonded carbons [including one sp] 2 Methylene carbon (δ C 116.7), three sps 2 Methylene carbon (δ) C [139.2,129.9,119.9], 4 sps 3 Methylene carbon [contains one hydroxyl carbon atom (δ)] C 81.4), 13 sp 3 Methylene carbon and 5 methyl carbon atoms (δ C 36.3, 19.6, 15.9, 14.7, 11.7).

[0042] All hydrogen protons were detected by HSQC spectrum. 1 H– 13 C is related to identification. 1 H– 1 The H COSY correlation spectrum and proton coupling constants indicate that this compound contains four independent spin-coupled systems: NH-2–C-2–C-3–OH-3, C-9–C-10, C-31–C-14–C-15–C-16(C-32)–C-17–C-18, and C-27–C-28(C-33)–C-29–C-30. 1 H– 1 According to H COSY and HMBC, H-3 is associated with C-2 / C-4, Ha-6 with C-5 / C-7, H3-7 with C-6 / C-8, H-9 with C-8 / C-10, H-10 with C-8 / C-11, Hb-12 with C-10 / C-11, H-14 with C-13 / C-15 / C-31, H3-30 with C-28 / C-29, H3-31 with C-13 / C-14 / C-15, H3-32 with C-15 / C-16 / C-17, and H3-33 with C-27 / C-28 / C-29. Based on the analysis of the above NMR data, unsaturation, molecular formula, and chemical shifts, the chemical shifts of hydrogen and carbon are listed in Table 1 below.

[0043] Table 1 Chemical shifts of hydrogen and carbon

[0044]

[0045]

[0046] Analysis of the above NMR data, unsaturation degree, molecular formula, and chemical shift yielded the chemical structure of compound 1 as shown in formula (Ⅰ). It is evident that the structure of compound Rakicidin K prepared in this embodiment is correct.

[0047] Example 2: Preparation of Rakicidin K

[0048] Fermentation was carried out using the preserved Micromonospora strain FIM-R150103, following the same fermentation process as in Example 1. The fermentation broth was collected and centrifuged at 4500 rpm for 15 min to obtain mycelial residue. The obtained mycelial residue was soaked twice overnight in 2 times the volume of 90% ethanol. The mycelial residue containing alcohol was centrifuged again at 4500 rpm for 15 min, and the supernatants were combined to obtain the fermentation extract.

[0049] Take 50 L of fermentation extract and dilute it to an ethanol concentration of 50%. Then perform HP20 macroporous resin adsorption column chromatography (diameter-to-height ratio 1:4, column volume 2.0 L) at an adsorption flow rate of 30 mL / min. Elute with 63% and 72% ethanol and water, respectively, for 4 BV and 5.5 BV, respectively. Analyze the elution using HPLC (YMC ODS C). 18 The column (5 μm, 250 mm × 4.6 mm) was used to detect the eluent (acetonitrile-water 700:300, column temperature 40 °C, flow rate 1.0 mL / min, wavelength 262 nm), and the 72% ethanol eluent containing Rakicidin K (18.5 L) was collected.

[0050] The collected eluent was diluted to an ethanol concentration of 55% and subjected to chromatography on an HZ816 resin adsorption column (diameter-to-height ratio 1:5, column volume 2.2 L). The adsorption flow rate was controlled at 25 mL / min. Elution was performed with 65%, 68%, and 70% ethanol-water solutions, yielding 2.5 BV, 3 BV, and 6 BV eluents, respectively. The eluent was analyzed by HPLC (YMC ODS C). 18 The eluent (acetonitrile-water 700:300, column temperature 40℃, flow rate 1.0 mL / min, wavelength 262 nm) was monitored using a 5 μm column (250 mm × 4.6 mm). A 70% aqueous ethanol solution (14.4 L) containing Rakicidin K was collected.

[0051] The collected eluent was concentrated to approximately 1 / 3 of its volume, extracted three times with an equal volume of ethyl acetate, and concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in methanol and prepared using a semi-preparative liquid chromatography (5 μm, 250 mm × 10 mm, Welch Material) with acetonitrile-water at a volume ratio of 715:285, at a flow rate of 7 ml / min. The eluent was then analyzed by HPLC (YMC ODS C). 18 The sample Rakicidin K was obtained by detecting and collecting the solution using a column (5 μm, 250 mm × 4.6 mm).

[0052] The product Rakicidin K prepared in this embodiment has the correct structure, as confirmed by testing.

[0053] Example 3: Preparation of Rakicidin K

[0054] Fermentation was carried out using the preserved Micromonospora strain FIM-R150103, following the same fermentation process as in Example 1. The fermentation broth was collected and centrifuged at 4500 rpm for 15 min to obtain mycelial residue. The obtained mycelial residue was soaked twice overnight in 2 times the volume of 90% ethanol. The mycelial residue containing alcohol was centrifuged again at 4500 rpm for 15 min, and the supernatants were combined to obtain the fermentation extract.

[0055] Take 80 L of fermentation extract and dilute it to an ethanol concentration of 60%. Then, perform adsorption chromatography on an HP20 macroporous resin column (diameter-to-height ratio 1:8, column volume 3.5 L) at an adsorption flow rate of 40 mL / min. Elute with 63% and 72% ethanol and water, respectively, for 3 BV and 7.5 BV, respectively. Detection was performed by HPLC (YMC ODS C). 18 The column (5 μm, 250 mm × 4.6 mm) was used as the eluent (acetonitrile-water 700:300, column temperature 40 °C, flow rate 1.0 mL / min, wavelength 262 nm), and 42.2 L of ethanol containing Rakicidin K was collected.

[0056] The collected eluent was diluted to an ethanol concentration of 60% and subjected to chromatography on an HZ816 resin adsorption column (diameter-to-height ratio 1:6, column volume 2.7 L). The adsorption flow rate was controlled at 32 mL / min. Elution was performed with 65%, 68%, and 70% ethanol-water solutions, yielding 3.5 BV, 3 BV, and 7.5 BV eluents, respectively. The eluent was analyzed by HPLC (YMC ODS C). 18 The eluent (acetonitrile-water 700:300, column temperature 40℃, flow rate 1.0 mL / min, wavelength 262 nm) was monitored using a 5 μm column (250 mm × 4.6 mm). A 70% aqueous ethanol solution (21.6 L) containing Rakicidin K was collected.

[0057] The collected eluent was concentrated to approximately 1 / 3 of its volume, extracted three times with an equal volume of dichloromethane, and concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in methanol and prepared using a semi-preparative liquid chromatography (5 μm, 250 mm × 10 mm, Welch Material) with acetonitrile-water at a volume ratio of 715:285, at a flow rate of 7 ml / min. The eluent was then analyzed by HPLC (YMC ODS C). 18 The sample Rakicidin K was obtained by detecting and collecting the solution using a column (5 μm, 250 mm × 4.6 mm).

[0058] Testing confirmed that the product Rakicidin K prepared in this embodiment has the correct structure.

[0059] Experimental Example

[0060] I. Antitumor activity test

[0061] The purified and collected Rakicidin K samples were dissolved in DMSO to a concentration of 1 μg / ml, and then diluted to final concentrations of 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, 0.0625 μg / ml, 0.03125 μg / ml, and 0.015625 μg / ml.

[0062] Routine 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 concentration: HCT-8 5.0 × 10⁻⁶). 4 cells / ml, PANC-14.0×10 4 Add 100 μl / well (drugs / ml, 100 μl / well) and incubate for 24 hours to allow adherence. Add 100 μl / well of fresh culture medium containing the drug. Set up 3 replicates for each concentration. Set up blank control wells (culture medium only) as negative controls, also with 3 replicates. Continue incubation for 48 hours, then terminate the culture.

[0063] Rakicidin K samples were dissolved in DMSO to a concentration of 1 μg / ml, and then diluted to final concentrations of 0.4444 μg / ml, 0.148148 μg / ml, 0.0493827 μg / ml, 0.0164609 μg / ml, 0.00548697 μg / ml, and 0.00182899 μg / ml.

[0064] Hypoxic 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 concentration: HCT-8 5.0 × 10⁻⁶). 4 cells / ml, PANC-14.0×10 4(100 μl / well), aerate for 30 minutes under hypoxic conditions, then close the aeration valve and incubate at 37°C for 24 hours to allow adhesion. Add 100 μl / well of fresh culture medium containing the drug, with 3 replicates for each concentration. Include blank control wells (culture medium only) as a negative control, also with 3 replicates. Aerate for 30 minutes under hypoxic conditions, then close the aeration valve and incubate at 37°C for another 48 hours, then terminate the culture.

[0065] MTT assay: After terminating cell culture, add 10 μl of CCK-8 to each well and incubate for 2 hours. Discard the supernatant, add 150 μl of DMSO solution to each well, shake gently for 10 minutes, and read the OD value of each well at 450 nm using a microplate reader. Calculate the inhibition rate. Based on the conversion of the inhibition rate, calculate the IC50 using SPSS software. 50 The values ​​are shown in Tables 2 and 3 below.

[0066] Inhibition rate (%) = (OD value of negative control group - OD value of experimental group) / OD value of negative control group × 100%.

[0067] Table 2. Inhibitory activity of Rakicidin K against human colon cancer cells HCT-8 under hypoxic conditions.

[0068]

[0069] Table 3. Inhibitory activity of Rakicidin K against human pancreatic cancer cells PANC-1 under hypoxic conditions.

[0070]

[0071] The above results indicate that the compound Rakicidin K of the present invention has strong antitumor activity, especially against hypoxic tumor cells. Its activity against hypoxic cultured HCT-8 tumor cells is 7.17 times stronger than that against normoxic cultured HCT-8 tumor cells, and its activity against hypoxic cultured PANC-1 tumor cells is 7.10 times stronger than that against normoxic cultured HCT-8 tumor cells.

[0072] II. Antimicrobial activity test

[0073] In vitro antibacterial test methods for Staphylococcus and Enterococcus

[0074] Sample preparation: Dissolve compound Rakicidin K in DMSO to achieve the following final test concentrations: 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03, 0.015, 0.008 μg / ml.

[0075] Inoculum preparation: Prepare a suspension with a concentration equivalent to 0.5 McFarland standard turbidity tubes, dilute with broth (Staphylococcus: MH broth; Enterococcus: brain heart extract broth), then add 100 μl of the test bacterial solution to each well (200 μl per well), the final bacterial concentration of which is approximately 10. 5 CFU / ml. After sealing, incubate in a 35-37℃ incubator for 18-24 hours.

[0076] MIC determination: The lowest drug concentration that completely inhibits bacterial growth in a small well is the minimum inhibitory concentration. The results are shown in Table 4 below.

[0077] In vitro antibacterial activity test method of Clostridium difficile

[0078] Inoculum preparation and inoculation: Prepare a suspension with a concentration equivalent to 0.5 McFarland standard turbidimetric tubes. Using a multi-point inoculator, aspirate 1-2 μl of the prepared bacterial suspension [using Bourg's medium supplemented with 5% (v / v) defibrinated sheep blood, heme chloride (5 mg / L), and vitamin K1 (1 mg / L)] and inoculate onto an agar plate. (Add different serially diluted concentrations of antibacterial drugs to autoclaved and sterilized Bourg's broth agar, equilibrated in a 60°C water bath, mix thoroughly, and pour onto a plate to a thickness of 3-4 mm. Prepare drug agar plates at a 1:9 ratio, setting 12 drug concentration gradients from 0.008 to 16 mg / L). The inoculation amount at each point is approximately 10... 5 CFU was used to form bacterial plaques with a diameter of 5-8 mm. After inoculation, the cells were incubated in an anaerobic environment at 35°C for 48 hours, and the results were observed.

[0079] MIC determination: The plate was placed on a dark, non-reflective surface to determine the endpoint of the experiment. The lowest drug concentration that inhibited bacterial growth was defined as the MIC. The lowest drug concentration that inhibited bacterial growth by more than 80% compared with the growth control was defined as the endpoint concentration. The results are shown in Table 4 below.

[0080] Table 4 Antibacterial activity of Rakicidin K

[0081]

[0082] The above antibacterial test results show that the compound Rakicidin K has weak inhibitory activity against 10 strains of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, vancomycin-sensitive Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, and Clostridium difficile.

[0083] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A natural Rakicidins compound, Rakicidin K, and its pharmaceutically acceptable salt, characterized in that, The structural formula of the compound Rakicidin K is shown in formula (Ⅰ): 。 2. A method for preparing the natural Rakicidins compound Rakicidin K as described in claim 1, characterized in that, This includes the steps of fermentation and isolation / extraction using the micromonospora strain FIM-R150103; The micromonosporal strain FIM-R150103 is classified as Micromonospora. Micromonospora sp. It has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 14822.

3. The method for preparing the natural Rakicidins compound Rakicidin K according to claim 2, characterized in that, Includes the following steps: (1) Ferment the preserved micromonospora strain FIM-R150103, collect the fermentation broth and separate the solid and liquid to obtain mycelium; soak the obtained mycelium in methanol or ethanol and collect the soaking liquid; (2) Chromatography was performed using an HP20 macroporous resin adsorption column, and gradient elution was performed with 63% and 72% ethanol-water, respectively. The 72% ethanol-water eluent fraction was collected. (3) The collected eluent was subjected to chromatography using an HZ816 resin adsorption column, and gradient elution was performed with 65%, 68% and 70% ethanol-water solutions, respectively, and the 70% ethanol eluent containing Rakicidin K was collected. (4) The eluent obtained by extraction with ethyl acetate or dichloromethane is concentrated to obtain crude product, which is then dissolved in methanol, separated by semi-preparative liquid chromatography, and eluted with acetonitrile-water at a volume ratio of 715:

285. The fractions are collected to obtain the final product.

4. The method for preparing the natural Rakicidins compound Rakicidin K according to claim 3, characterized in that, In step (2), the diameter-to-height ratio of the HP20 macroporous resin adsorption column is 1:3-1:8, the column volume is 2.0-3.5L, and the adsorption flow rate is 30-40ml / min.

5. The method for preparing the natural Rakicidins compound Rakicidin K according to claim 3 or 4, characterized in that, In step (3), the HZ816 resin adsorption column diameter-to-height ratio is 1:3-1:6, the column volume is 2.0-2.7L, and the adsorption flow rate is 25-32ml / min.

6. The method for preparing the natural Rakicidins compound Rakicidin K according to claim 5, characterized in that, In step (4), the semi-preparative liquid chromatography is Welch Material, 5μm, 250mm×10mm, and the eluent flow rate is controlled at 7ml / min.

7. The use of the natural Rakicidins compound Rakicidin K of claim 1 and its pharmaceutically acceptable salts for the preparation of drugs having antitumor activity and / or antigram-positive bacterial activity; The tumors described are human colon cancer and human pancreatic cancer; The Gram-positive bacteria are methicillin-resistant Staphylococcus aureus, vancomycin-sensitive Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, or Clostridium difficile.

8. A drug having antitumor activity and / or anti-Gram-positive bacterial activity, characterized in that, The active ingredient is the natural Rakicidins compound Rakicidin K and its pharmaceutically acceptable salt as described in claim 1, with the addition of a pharmaceutically acceptable carrier.

9. The use of micromonosporal strain FIM-R150103 for fermentation preparation of the natural Rakicidins compound Rakicidin K as described in claim 1, characterized in that, The micromonosporal strain FIM-R150103 is classified as Micromonospora. Micromonospora sp. It has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 14822.

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