CN112961170B

CN112961170B - Sponge source actinomycetes and preparation method and application of sulfur-containing alkaloid produced by sponge source actinomycetes

Info

Publication number: CN112961170B
Application number: CN202110191829.4A
Authority: CN
Inventors: 张长生; 张海波; 张鑫雅; 陈思强; 张丽萍; 张庆波; 张文军
Original assignee: South China Sea Institute of Oceanology of CAS; Southern Marine Science and Engineering Guangdong Laboratory Guangzhou
Current assignee: South China Sea Institute of Oceanology of CAS; Southern Marine Science and Engineering Guangdong Laboratory Guangzhou
Priority date: 2021-02-19
Filing date: 2021-02-19
Publication date: 2022-03-11
Anticipated expiration: 2041-02-19
Also published as: CN112961170A

Abstract

The invention discloses a sponge source actinomycete, a preparation method and application of a sulfur-containing alkaloid produced by the sponge source actinomycete. Biological activity tests show that the compound 1 and the compound 2 which are separated from the fermentation product of sponge source actinomycetes Nocardiopsis dassvilli SCSIO 40065 have antibacterial and antitumor activities, so that the compound 1 and the compound 2 can be applied to preparation of antibacterial drugs and antitumor drugs.

Description

Sponge source actinomycetes and preparation method and application of sulfur-containing alkaloid produced by sponge source actinomycetes

Technical Field

The invention belongs to the technical field of microbial medicines, and particularly relates to sponge-derived actinomyces nocardioides and a preparation method and application of two sulfur-containing alkaloids dassonmycins A (1) and B (2) produced by the same.

Background

Sponge (Marine sponge) is the most primitive multicellular animal on earth, and the main components of the sponge cells are calcium carbonate or silicon carbonate and a large amount of collagen. Although the sponge is an animal, the sponge cannot walk by itself, and only can be attached to a reef fixed on the sea bottom to obtain food from seawater flowing around. A plurality of flagella and a sieve-shaped ring are grown in small holes which are distributed on the whole body of the sponge, seawater can be absorbed by using the swing of ocean currents and the flagella, oxygen, bacteria, micro algae and other organic debris are brought in, and the seawater is filtered by the ring to become the nutrient of the sponge. The sponge has unique cavity structure and filter feeding life style, so that the sponge has the reputation of 'microbial resource library'. In recent years, more and more researches show that various bioactive substances are separated from sponge-associated microbial fermentation products and can be used for preparing medicines and treating diseases such as tumors, cardiovascular diseases, respiratory diseases and the like.

Gram-positive bacteria generally refer to bacteria with purple gram-stain reaction, most pyococcus belong to gram-positive bacteria, can produce exotoxin to cause diseases for people, and are represented by staphylococcus aureus. Clinically common gram-positive pathogenic bacteria are Staphylococcus aureus (Staphylococcus aureus), methicillin-resistant Staphylococcus aureus (methicillin-resistant Staphylococcus aureus). Enterococcus faecalis (Enterococcus faecalis) is a commensal bacterium in the intestinal tract, and some Enterococcus faecalis can cause widespread infection. Due to the long-term and large-scale unregulated use of antibiotics, the acquired resistance of enterococcus faecalis is increasing and it is increasingly difficult to treat enterococcus faecalis infections. Micrococcus luteus (Micrococcus luteus) is a conditioned pathogen, causes local tissue infection such as wounds and can also cause serious infection such as endocarditis and other diseases. In the aspect of aquaculture, gram-negative bacteria Vibrio alginolyticus (Vibrio algicidalus) are common pathogenic bacteria of marine culture animals, and shell animals such as cultured shellfish and shrimps are more easily infected with the bacteria than cultured fishes. The vibrio alginolyticus is pathogenic bacteria causing the black gill and brown spot syndrome of the prawns. The bacteria are also pathogenic bacteria in Meretrix meretrix Linnaeus and Pinctada martensii, and can also cause death of sea bream, Epinephelus akaara, Pagrus major, etc.

Disclosure of Invention

It is a first object of the present invention to provide two sulfur-containing alkaloids, dassonomycins A (1) and B (2), which have antibacterial and antitumor activity.

The sulfur-containing alkaloids dassonemycins A (1) and B (2) have the following structural formulas:

the two sulfur-containing alkaloids are dapsone mycins A (1) and B (2), and the molecular formula of Dassomycin A is C₁₇H₁₆N₂O₄S (molecular weight 345.0904) and the molecular formula of dassonomycin B is C₁₇H₁₄N₂O₄S (molecular weight 343.0747).

Compounds

1 and 2 are the first naphthoquinones from nature with [2,3-e ]]Piperazine [1,2-c ]]Thiomorpholine (naphthoquinone [2,3-e ]]piperazine[1,2-c]thiomorpholine) skeleton compound.

The second objective of the present invention is to provide an actinomyces Nocardiopsis dassonvillei SCSIO 40065 with the deposit number GDMCC No.61460 for producing the sulfur-containing alkaloid.

It is a third object of the present invention to provide a process for producing sulfur-containing alkaloids dassonomycins A (1) and B (2) using the above actinomycetes.

Preferably, the specific steps are as follows:

preparing a fermentation culture of Nocardiaopsis dassonvillei SCSIO 40065, separating a supernatant and a mycelium, adsorbing the supernatant with resin, eluting with acetone, and concentrating to obtain a supernatant extract; extracting mycelium with methanol, concentrating the extract to remove methanol to obtain mycelium extract, mixing the supernatant extract and the mycelium extract, re-extracting with butanone, and concentrating the extract to obtain extract;

performing gradient elution on the extract by medium-pressure reversed-phase column chromatography, and performing gradient elution on the extract by using water in a volume ratio of: acetonitrile 100: 0 to 0: 100 gradient elution, collecting fractions F14 and F15; fraction F14 was purified to give compound 1; fraction F15 was purified to give compound 2.

Preferably, the extract is subjected to gradient elution by medium-pressure reverse-phase column chromatography, and the volume ratio of water: acetonitrile 100: 0 to 0: 100 gradient elution, fraction F14 and fraction F15 were collected by medium pressure reverse phase column chromatography gradient elution from water: acetonitrile 100: 0 to 0: the fraction F14 obtained by eluting with 60% by volume acetonitrile-water and the fraction F15 obtained by eluting with 65% by volume acetonitrile-water were collected by 100 gradient elution.

Preferably, the fraction F14 is purified to obtain the compound 1, and the fraction F15 is purified to obtain the compound 2 by using a high performance liquid chromatograph.

Preferably, the Nocardiopsis dassvillii SCSIO 40065 is prepared by fermenting N4 liquid medium as fermentation medium.

The fourth purpose of the invention is to provide the application of the sulfur-containing alkaloids dassonomycins A (1) and B (2) in the preparation of antibacterial or antitumor drugs.

The antibacterial drug is a drug for inhibiting micrococcus luteus, bacillus subtilis, staphylococcus aureus, methicillin-resistant staphylococcus aureus, enterococcus faecalis or vibrio alginolyticus.

The anti-tumor drug is a drug for resisting human nerve cancer, human breast cancer, human liver cancer or human non-small cell lung cancer.

The sulfur-containing alkaloid compound 1 and the sulfur-containing alkaloid compound 2 are separated from Nocardiaopsis dassvellii SCSIO 40065, and bioactivity screening finds that the two compounds have the activities of resisting bacteria and tumor cell strains, so that the two compounds can be applied to antibacterial and antitumor drugs.

Nocardiopsis dassonvillei SCSIO 40065 was deposited at 21.1.2021 at the collection of microorganisms and cell cultures of the guangdong province (GDMCC) address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5, zip code: 510070, accession number: GDMCC No: 61460.

description of the drawings:

FIG. 1 is a strain morphology map of Nocardiaopsis dassonvillei SCSIO 40065;

FIG. 2 is a Nocardiaopsis dassonvillei SCSIO 40065 phylogenetic tree;

FIG. 3 is a NMR spectrum (deuterated DMSO, 700 MHz) of Compound 1 (Dassonomycin A);

FIG. 4 is a carbon nuclear magnetic resonance spectrum (deuterated DMSO, 175 MHz) of Compound 1 (Dassonomycin A);

FIG. 5 is an HR-ESI-MS analysis of Compound 1 (Dassonomycin A) (positive ion mode, [ M + H ]]⁺)；

FIG. 6 is an X-ray single crystal diffractogram of Compound 1 (Dassonomycin A);

FIG. 7 is a NMR spectrum (deuterated DMSO, 700 MHz) of Compound 2 (Dassonomycin B);

FIG. 8 is a carbon nuclear magnetic resonance spectrum (deuterated DMSO, 175 MHz) of Compound 2 (Dassonomycin B);

FIG. 9 is an analysis graph of HR-ESI-MS of Compound 2 (Dassonomycin B) (positive ion mode, [ M + H ]]⁺)；

FIG. 10 is an X-ray single crystal diffractogram of Compound 2 (Dassonomycin B).

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1:

1. the inventor collects a Petrosia sp sponge sample from a shallow sea bottom (-20 m) near the Yongxing island in the south China sea, uses a homogenizer to grind the sample after the surface of the sample is disinfected, and inoculates a part of the ground sample on a sterile ISP2 agar plate (each 1 liter of culture medium contains 4.0g of yeast extract powder, 4.0g of glucose, 5.0g of malt extract powder, 500 microliters of compound vitamin, 30.0g of sea salt, 20.0g of agar powder, pH 7.0 and is sterilized for 30 minutes at 115 ℃); another portion of the ground sample was washed with sterile water and the supernatant was applied by dilution to sterile ISP2 agar plates. ISP2 agar plates were then placed in a 28 ℃ incubator for incubation. From the colonies grown on the plate, a strain having an actinomycete morphology was picked up for further isolation and purification, thereby obtaining Nocardiopsis dassvilli SCSIO 40065 (FIG. 1). Extracting the genome DNA of the strain, obtaining a 16S rDNA sequence through Polymerase Chain Reaction (PCR) amplification, and establishing a phylogenetic tree through sequence comparison. The results show that: the similarity of this strain to Nocardiopsis dassonvillei HZNU N1 (fig. 2) reached 100%, indicating that the strain SCSIO 40065 is actinomycetes Nocardiopsis, named Nocardiopsis dassonvillei SCSIO 40065, which was deposited at the guangdong province collection of microorganisms (GDMCC) at 21 days 1/2021, address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5, zip code: 510070, accession number: GDMCC No: 61460.

2. nocardia Nocardiopsis dassvilli SCSIO 40065 was inoculated from a storage tube onto an ISP2 agar plate for activation, and then spores were picked and inoculated into 250 ml Erlenmeyer flasks containing 50 ml of N4 liquid medium (containing 15 g of soluble starch, 8 g of fish peptone, 5 g of bacteriological peptone, 7.5 g of glycerol, 2g of calcium carbonate, 0.2g of potassium bromide, 30g of sea salt, and the balance water per 1 l of medium), the components were mixed uniformly, adjusted to pH 7.0, sterilized at 115 ℃ for 30 minutes, and 30 bottles (1.5 l) were used in total. Placing on a shaking table at 28 ℃, and performing shaking culture at 200 rpm for 2 days to obtain fermented seeds. 25 ml of the fermented seed solution was poured into a 1-liter Erlenmeyer flask containing 250 ml of N4 liquid medium (each 1 liter of the medium contains 15 g of soluble starch, 8 g of fish peptone, 5 g of bacteriological peptone, 7.5 g of glycerol, 2g of calcium carbonate, 0.2g of potassium bromide, 30g of sea salt, and the balance water, the components were mixed uniformly, pH was adjusted to 7.0, and sterilization was carried out at 115 ℃ for 30 minutes), and 64 bottles (16 liters) were used. The mixture was placed on a shaker at 28 ℃ and cultured for 7 days with shaking at 200 rpm to obtain a fermentation culture. The supernatant and the mycelia of the fermentation culture were separated by centrifugation at 4000 rpm. Adsorbing the supernatant with macroporous resin XAD-16, washing with acetone to obtain extractive solution, concentrating under reduced pressure, and recovering organic solvent to obtain supernatant extractive solution; the mycelium fraction is extracted four times with methanol, and the organic solvent is recovered by concentration under reduced pressure to obtain a mycelium extract. The supernatant extract and the mycelium extract are combined, extraction is carried out for four times by using butanone of the same volume, and the extract is decompressed and concentrated to obtain 12.69g of extract.

The crude Nocardiopsis dassonvillei SCSIO 40065 extract (12.69g) was subjected to gradient elution by medium pressure reverse phase column chromatography (gradient elution was performed with a volume ratio of water to acetonitrile of 100: 0 to 0: 100), and fraction F14 obtained by elution with 60% by volume of acetonitrile water and fraction F15 obtained by elution with 65% by volume of acetonitrile-water were collected.

Fraction F14 was further subjected to semi-preparative HPLC (acetonitrile B phase-water A phase; gradient elution (elution procedure: 0-26min, A: 95% -0%, B: 5% -100%, 26-30min, A: 0%; B: 100%, 30-32min, A: 95%; B: 5%, flow rate 2.5ml/min), Kinetex C-18 column, 250X 10mm, flow rate 2.5ml/mim) to yield Compound 1(20 mg, retention time 16 min). Further, compound 1 was left to stand at room temperature, and then precipitated red crystals in a methanol solution.

Fraction F15 was further subjected to semi-preparative HPLC (acetonitrile B phase-water A phase; isocratic elution (elution procedure: A: 65%, B: 35%, flow rate 2.5ml/min), Kinetex C-18 column, 250X 10mm, flow rate 2.5ml/mim) to obtain Compound 2(13.8mg, retention time 6 minutes). Further, compound 2 was left to stand at room temperature and precipitated red crystals in a methanol solution.

The NMR spectrum (deuterated DMSO, 700 MHz) of Compound 1 is shown in FIG. 3, the NMR spectrum (deuterated DMSO, 175 MHz) is shown in FIG. 4, and the HR-ESI-MS analysis chart (positive ion mode, [ M + H ], [ M ] is shown in FIG. 4]⁺) As shown in fig. 5. Compound 1 (dassnomycin a, X-ray single crystal diffractogram shown in fig. 6, compound 1: red crystal,¹H-NMR、¹³C-NMR data are consistent with the crystal structure alignment, and high-resolution mass spectrum (LR-ESI-MS) gives an excimer ion peak M/z 345.0904([ M + H)]⁺) The data relate to the formula C of Dassonomycin A₁₇H₁₆N₂O₄And S is consistent. Thus identified as Dassonomycin A.

The NMR spectrum (deuterated DMSO, 700 MHz) of Compound 2 is shown in FIG. 7, the NMR spectrum (deuterated DMSO, 175 MHz) is shown in FIG. 8, and the HR-ESI-MS analysis chart (positive ion mode, [ M + H ], [ M ] is shown in FIG. 8]⁺) As shown in fig. 9. Compound 2 (dassnomycin B, X-ray single crystal diffractogram shown in fig. 10, compound 2: red crystal,¹H-NMR、¹³the C-NMR data are consistent with the crystal structure alignment, and LR-ESIMS gives an excimer ion peak M/z 343.0747([ M + H ]]⁺) The data relate to the formula C of Dassonomycin B₁₇H₁₄N₂O₄And S is consistent. And thus identified as dassonomycin B.

TABLE 1 NMR data for

Compounds

1 and 2

Identifying that the compound 1 is Dassonomycin A, and the structural formula of the compound is shown as 1 in the formula I;

identifying that the compound 2 is Dassonomycin B, and the structural formula of the compound is shown as 2 in the formula I;

example 2: determination of antibacterial Activity of

Compounds

1 and 2

The inhibitory activity of

compounds

1 and 2 against 6 indicator bacteria, Micrococcus luteus (Micrococcus luteus SCSIO ML01), Bacillus subtilis (Bacillus subtilis 1064), Staphylococcus aureus (Staphylococcus aureus ATCC 29213), methicillin-resistant Staphylococcus aureus (methicillin-resistant Staphylococcus aureus shHsA1), Enterococcus faecalis (Enterococcus faecalis ATCC 29212), Vibrio alginolyticus (Vibrio alginate), was determined by the microculture medium dilution method. Culturing 6 indicator bacteria at 37 deg.C and 200rmp for 16h, diluting with sterile culture medium to OD (600nm) of 0.04-0.06, diluting by 10 times, and adding into 96-well plate; after the addition of the sample, the resulting solution was diluted in an equal volume to a final concentration of 64-0.125. mu.g mL ^-13 replicates per concentration; culturing at 37 ℃ for 18h, measuring the absorption value of each well by using a microplate reader, and determining the Minimum Inhibitory Concentration (MIC) of each compound. Trimethoxybenzidine (TMP) was used as a positive control, and the results are shown in Table 2.

The results are shown in table 2:

TABLE 2 test results of minimum inhibitory concentrations of

Compounds

1 and 2 against six indicator bacteria (unit: μ g/ml)

From Table 2, it can be found that the compound 1 and the compound 2 have inhibitory effects on the above six indicator bacteria, wherein the inhibitory activity of the compound 1 on Bacillus subtilis is stronger (8 μ g mL)^-1)。

Example 3: determination of antitumor Activity of

Compounds

1 and 2

The SRB method is adopted to determine the inhibitory activity of the

compounds

1 and 2 on 4 tumor cell strains SF-268, HepG2, MCF-7 and A549. 4 tumor cell lines were cultured in RPMI medium, and 180. mu.L of the culture (concentration: 3X 10)⁴Cells per mL) were added to 96-well plates at 37 ℃ with 5% CO₂Culturing for 18 h; mu.L of the sample to be tested (

final concentrations

1, 10 and 100. mu.M, solvent DMSO) was added to the corresponding well of a 96-well plateDMSO is used as a negative control, each concentration is performed in 3 parallels, and the culture is continued for 72 hours; adding 50 μ L of 50% trichloroacetic acid, mixing, adding 0.4% SRB (dissolved in 1% acetic acid), and standing for 30 min; the supernatant was removed, the dye-bound proteins were dissolved in 200. mu.L of 10mM Tris buffer, the OD (570nm) of each well was measured using a microplate reader, and the corresponding IC was calculated using non-linear curve fitting in SigmaPlut 14.0 software₅₀(ii) a Doxorubicin (Adriamycin) was used as a positive control. The results are shown in Table 3 and show the IC's of Dassonomycin A (1) and Dassonomycin B (2) against 4 indicator cell lines₅₀The range is 11.94 to 34.39. mu.M.

TABLE 3 IC of

Compounds

1 and 2 against four tumor cell lines₅₀Inhibitory concentration test results (Unit: μ M)

From Table 3, it can be found that Compound 1 and Compound 2 have inhibitory effects on the above four tumor cell lines, and Compound 2 has strong tumor cell line inhibitory activity (IC) against human neural cancer cell line SF-268₅₀＝11.94μM)。

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.

Claims

1. Any one of the sulfur-containing alkaloids shown as formula I or pharmaceutically acceptable salt thereof

2. The use of the sulfur-containing alkaloid of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an antibacterial or antitumor medicament.

3. The use of claim 2, wherein the antibacterial agent is an agent that inhibits Micrococcus luteus, Bacillus subtilis, Staphylococcus aureus, enterococcus faecalis, or Vibrio alginolyticus.

4. The use of claim 3, wherein said Staphylococcus aureus is methicillin-resistant Staphylococcus aureus.

5. The use of claim 2, wherein the anti-neoplastic agent is an anti-human neuro-cancer, human breast cancer, human liver cancer or human non-small cell lung cancer.

6. A process for the preparation of a sulphur-containing alkaloid according to claim 1, wherein the sulphur-containing alkaloid is isolated from a fermentation culture of the strain Nocardiaopsis dassonvillei SCSIO 40065.

7. The preparation method according to claim 6, comprising the following steps:

preparing a fermentation culture of Nocardiaopsis dassonvillei SCSIO 40065, separating a supernatant and mycelia, adsorbing the supernatant with resin, eluting with acetone, and concentrating to obtain a supernatant extract; extracting mycelium with methanol, concentrating the extract to remove methanol to obtain mycelium extract, mixing the supernatant extract and the mycelium extract, extracting with butanone, and concentrating the extract to obtain extract;

8. The process according to claim 7, wherein the extract is eluted by medium pressure reverse phase column chromatography gradient from water: acetonitrile 100: 0 to 0: 100 gradient elution, fraction F14 and F15 were collected by medium pressure reverse phase column chromatography gradient elution from water: acetonitrile 100: 0 to 0: the fraction F14 obtained by eluting with 60% by volume acetonitrile-water and the fraction F15 obtained by eluting with 65% by volume acetonitrile-water were collected by 100 gradient elution.

9. The process of claim 7, wherein the fraction F14 is purified to obtain Compound 1 and the fraction F15 is purified to obtain Compound 2, and the fractions are purified by HPLC.

10. The process according to claim 7, wherein the Nocardiaopsis dassonvillei SCSIO 40065 is prepared by fermenting N4 liquid medium.

Nocardiaopsis dassonvillei SCSIO 40065 with accession number GDMCC No. 61460.