Novel thiopeptide antibiotic mimicin B as well as preparation and application thereof

Abstract

The invention discloses a novel thiopeptide antibiotic mimicin B as well as preparation and application of the novel thiopeptide antibiotic mimicin B. The natriacin B is a secondary metabolite of micromonospora TMD166, is a novel thiopeptide antibiotic, has good antibacterial activity, has strong in-vitro antibacterial activity on gram-positive bacteria such as staphylococcus (a beta-lactam antibiotic-resistant strain) and enterococcus (a vancomycin-resistant strain), and has an MIC value in a range of 0.25-8 [mu] g / ml. As a novel anti-infective drug, the mimicin B has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a novel thiopeptide antibiotic xuanzangmicin B and a preparation and application thereof. Background Art

[0002] The crisis of infectious disease treatment caused by antibiotic resistance has become a medical and social issue of global concern. Gram-positive bacteria such as Staphylococcus, Enterococcus and Streptococcus are common pathogens of hospital-acquired and community-acquired infections, which can cause serious infections. A study on bacterial resistance surveillance in China published in 2023 showed that methicillin-resistant Staphylococcus aureus (MRSA) was the most common MRSA in Gram-positive bacteria. staphylococcus aureus , MRSA) and methicillin-resistant Staphylococcus epidermidis Staphylococcus epidermidis , MRSE) detection rates reached 36.4% and 79.9% respectively. Vancomycin, as the first choice drug for the treatment of resistant Gram-positive bacterial infections, has developed drug resistance. Even as a treatment for vancomycin-resistant Enterococcus bacteria (vancomycin-resistant enterococcus , VRE) have also developed resistance to linezolid and other oxazolidinone antibiotics. The increasingly serious multidrug-resistant and pan-drug-resistant pathogens have resulted in a decrease in the number of antibiotics that are effective in treating them clinically. The World Health Organization warns that if we do not increase the research and development of antibiotics, use antibiotics irrationally, and reduce the occurrence of drug resistance, the fatal threats that may be caused by common infections will become a reality. Therefore, it is urgent to develop new antibiotics with novel structures and unique antibacterial mechanisms, which are of great significance for the treatment of clinical drug-resistant bacterial infections.

[0003] Thiopeptide antibiotics are complex peptide secondary metabolites containing multiple thiazole rings produced by microorganisms. Since the discovery of the first thiopeptide antibiotic, micrococcin, in 1948, hundreds of such antibiotics have been reported. According to the differences in the degree of oxidation and substitution of the nitrogen six-membered heterocyclic ring in the structure, they are divided into five categories of thiopeptide antibiotics: piperidines, dehydropiperidines, dihydroimidazole piperidines, trisubstituted pyridines and hydroxylated pyridines; according to the number of atoms in the core macrocyclic structure including the pyridine ring, thiopeptide antibiotics are also divided into 26-, 29-, 32- and 35-membered ring thiopeptides. Thiopeptide antibiotics have diverse activities, including antibacterial, antitumor, antimalarial, and immunosuppressive activities. In particular, they have attracted widespread attention from academia and the pharmaceutical industry for their extremely strong anti-Gram-positive bacterial activity and novel mechanism of action that is different from existing clinically used antibiotics. In addition to thiostrepton and nosiheptide, which have been used as animal feed additives, some thiopeptide antibiotic derivatives have entered the clinical research stage, such as the 29-membered thiopeptide antibiotic GE2270A derivative LEF571 as a treatment for Clostridium difficile ( Clostridioides difficile ) has completed Phase II clinical trials. Another GE2270A analogue, NAI003, is effective against Propionibacterium acnes ( propionibacterium acnes ) has narrow-spectrum antibacterial activity and has completed Phase II clinical studies as a new drug for the treatment of acne. Studies have shown that thiopeptide antibiotics inhibit bacterial protein synthesis by acting on different sites of bacterial ribosomes. Thiopeptide antibiotics of different structural types have different targets. Therefore, the discovery of new structural thiopeptide antibiotics is very likely to develop novel therapeutic drugs to address human and animal related infections and bacterial resistance. Summary of the invention

[0004] The purpose of the present invention is to provide a new thiopeptide antibiotic Xuanzangmixin B and its preparation and application.

[0005] The inventors isolated a new thiopeptide antibiotic, Xuanzangmicin B, from the secondary metabolites of a Micromonospora strain TMD166 from a Taklimakan Desert plant. After careful analysis of spectral data such as ultraviolet spectroscopy, infrared spectroscopy, mass spectrometry and nuclear magnetic resonance, Xuanzangmicin B was determined to be a new type of thiopeptide antibiotic. The structure of Xuanzangmicin B is similar to that of Xuanzangmicin. It also has a dimethoxy-substituted pyranose attached to the hydroxyl group of hydroxylated pyridine. It is also an antibiotic of this type with four glycosyl side chains. The biggest feature of Xuanzangmicin B is that it loses the dehydroalanine at the end of the molecule. This is a rare antibiotic of this type that loses the dehydroalanine residue. The antibacterial activity of Xuanzangmicin B was evaluated and it was found that Xuanzangmicin B has strong inhibitory activity against Gram-positive bacteria such as Staphylococcus, Enterococcus faecalis, Enterococcus faecium (including strains resistant to β-lactam antibiotics and vancomycin) in vitro. As a new anti-infective lead compound, it has potential good development prospects.

[0006] In the first aspect, the present invention provides a new thiopeptide antibiotic, Xuanzangmixin B, whose structure is shown in formula (1): Formula (1) In the second aspect, the present invention provides a method for preparing Xuanzang rice star B, fermenting and culturing a microsporum with a preservation number of CGMCCNO. 21622 ( Micromonospora sp.) TMD166, harvest the fermentation broth; then extract and separate the Xuanzangmixing B from the fermentation broth. Micromonospora TMD166 has been disclosed in CN112899199A.

[0007] Furthermore, the fermentation culture comprises: inoculating the Micromonospora on a seed culture medium for culture, and then transferring to a fermentation culture medium for shaking culture; The conditions for culturing on the seed culture medium are 28-30°C, 150-200 r / min, and the culturing time is 48-72 hours; the conditions for culturing on the fermentation culture medium are 28-30°C, 150-200 r / min, and the culturing time is 96-120 hours; The seed culture medium and the fermentation culture medium have the same formula, specifically: 4 g glucose, 4 g yeast powder, 5 g malt extract powder, 0.001 g ZnSO4.7H2O, 0.001 g MnCl2.4H2O, 0.002 g FeSO4.7H2O, 0.001 g phenylalanine, 0.0003 g alanine, 0.001 g vitamin B1, 0.001 g vitamin B2, 0.001 g vitamin B6, 0.001 g niacin, 0.001 g biotin, 1.0 L sterile distilled water, pH 8.5.

[0008] Furthermore, the method for extracting and separating the Xuanzang Mixing B from the fermentation broth includes: after the fermentation broth is centrifuged, the supernatant is extracted with equal volume of ethyl acetate three times, and the organic solvent is removed by rotary evaporation at low temperature and reduced pressure to obtain a reddish-brown crude extract, the crude extract is dissolved in methanol, and Sephadex LH-20 gel column chromatography is performed, methanol is used as an eluent, and the light yellow band at the front is collected, and rotary evaporation is performed under reduced pressure to obtain a semi-pure product; the semi-pure product is dissolved in methanol, prepared by HPLC, and freeze-dried to obtain a slightly light yellow powder of Xuanzang Mixing B pure product.

[0009] Furthermore, the method of preparing by HPLC includes: the chromatographic column parameters are Capcell-Pak-C 18 MGⅡ5 μm, 10 mm × 250 mm; the mobile phase was 40% acetonitrile aqueous solution; the flow rate was 2 mL / min; the components with a retention time near 43 min were collected, the UV detection wavelength was 230 nm, and after combining the collected components, the methanol was removed by rotary evaporation, and the pure Xuanzangmixin B was obtained after freeze-drying.

[0010] In a third aspect, the present invention provides the use of the Xuanzang Mixing B in the preparation of antibacterial products.

[0011] Furthermore, the bacteria are Gram-positive bacteria, such as Staphylococcus, Enterococcus (such as Enterococcus faecalis and Enterococcus faecium), etc.

[0012] Furthermore, the Gram-positive bacteria include β-lactam antibiotic-resistant Staphylococci and vancomycin-resistant Enterococci.

[0013] The compound represented by the structural formula (I) provided by the present invention is a new type of thiopeptide antibiotic - Xuanzangmicin B. In vitro biological activity assays show that the compound has good antibacterial activity and has good application prospects as a new anti-infective drug.

[0014] The antibacterial activity of Xuanzang Mixing B was determined: the minimum inhibitory concentration (MIC) was determined according to the microbroth dilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS). The experimental results showed that Xuanzang Mixing B has bacterial inhibition activity: it has strong in vitro antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus (strains resistant to β-lactam antibiotics) and Enterococcus aureus (strains resistant to vancomycin), and the MIC value is in the range of 0.25-8μg / ml. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 In the preferred embodiment of the present invention Figure 1 The main 1 H- 1 Schematic diagram of the correlation between HCOSY, HMBC and ROE.

[0016] Figure 2 It is the ultraviolet spectrum of Xuanzang Mixing B in the preferred embodiment of the present invention.

[0017] Figure 3 This is the infrared spectrum of Xuanzang Mixing B in a preferred embodiment of the present invention.

[0018] Figure 4 This is the high resolution mass spectrum (HR-ESI-MS) of Xuanzang Mixing B in the preferred embodiment of the present invention.

[0019] Figure 5 This is the preferred embodiment of the present invention, Xuanzang Mixin B in CDCl3:CD3OD (9:1) 1 H-NMR spectrum.

[0020] Figure 6 This is the preferred embodiment of the present invention, Xuanzang Mixin B in CDCl3:CD3OD (9:1) 13 C-NMR spectrum.

[0021] Figure 7 This is the DEPT spectrum of Xuanzangmixin B in CDCl3:CD3OD (9:1) in a preferred embodiment of the present invention.

[0022] Figure 8 This is the preferred embodiment of the present invention, Xuanzang Mixin B in CDCl3 + CD3OD (9:1) 1 H- 1 H COSY spectrum.

[0023] Fig. 9 This is the HSQC spectrum of Xuanzangimicin B in CDCl3:CD3OD (9:1) in a preferred embodiment of the present invention.

[0024] Fig.10 This is the HMBC spectrum of Xuanzangmixin B in CDCl3:CD3OD (9:1) in a preferred embodiment of the present invention.

[0025] Fig.11 This is the ROESY spectrum of Xuanzangmixin B in CDCl3:CD3OD (9:1) in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0026] The present invention adopts the following technical solution: The present invention aims to provide a novel thiopeptide antibiotic, a preparation method of Xuanzangmixin B, and its application in the preparation of antibacterial infection drugs. The inventors isolated an endophytic micromonospora from the Taklimakan Desert in Xinjiang, my country ( Micromonospora taklimakanensis A compound with strong antibacterial activity, Xuanzangmixin B, was isolated from the secondary metabolites of the genus Xuanzangmixin sp. TMD166, and its structure was determined through a series of spectral analyses. A new antibacterial active compound Xuanzangmixin B with a structure shown in formula (1) is provided, and its chemical formula is shown in formula (1).

[0027] The compound has the core skeleton of thiopeptide antibiotics: containing multiple thiazole rings and having a characteristic nitrogen six-membered heterocyclic ring: hydroxylated pyridine, and its structure is similar to that of Xuanzang Mixing, with a dimethoxy-substituted pyranose connected to the hydroxyl group of the pyridine ring. It is also a thiopeptide antibiotic containing four glycosyl side chains, but Xuanzang Mixing B rarely loses dehydroalanine at the end of the molecular structure, and this structure is reported for the first time in the world. Further antibacterial activity determination shows that the compound has a broad spectrum of antibacterial activity, especially against Enterococcus faecalis and Enterococcus faecium (including vancomycin-resistant strains) in Gram-positive bacteria in vitro. The present invention provides new resources and new drug candidates for the development of antibacterial drugs.

[0028] The Micromonospora strain TMD166 ( Micromonospora taklimakanensis sp. TMD166), which was deposited on January 13, 2021 at the General Microbiology Center of China Microorganism Culture Collection Committee (referred to as CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), with the deposit number CGMCC No. 21622, and its classification name is Micromonas ( Micromonospora taklimakanensis sp.). Micromonospora TMD166 has been disclosed in CN112899199A.

[0029] The present invention provides a method for preparing the above-mentioned Xuanzangmixin B compound of natural origin, comprising the following steps: firstly, fermenting and culturing the Micromonospora strain with a preservation number of CGMCC No.21622 Micromonospora taklimakanensis sp. TMD166, harvesting the fermentation broth; and then extracting and separating the thiopeptide antibiotic compound from the fermentation broth.

[0030] The fermentation culture step is: the Micromonospora strain TMD166 (with a preservation number of CGMCC No. 21622) Micromonospora taklimakanensis sp. TMD166) were inoculated on seed culture medium and then transferred to fermentation medium for shaking culture. The fermentation liquid was harvested after the culture matured.

[0031] Among them, the culture conditions in the seed culture medium are 28-30 ℃, 150-200 r / min, and culture for 48-72 hours.

[0032] The culture conditions in the fermentation medium are 28-30°C, 150-200 r / min, and culture for 96-120 h.

[0033] The extraction and separation steps include: firstly, the fermentation liquid is centrifuged, and the supernatant is extracted with ethyl acetate three times with equal volume, and the crude extract is obtained after the organic solvent is removed by rotary concentration at low temperature and reduced pressure. The crude extract is chromatographed on a Sephadex LH-20 gel column, collected by volume, and separated to obtain a semi-pure product; the semi-pure product is dissolved in methanol, prepared by HPLC, and freeze-dried to obtain a pure compound.

[0034] The specific extraction and separation steps are as follows: the fermentation liquid is centrifuged in a large-capacity centrifuge at 4500 rpm to obtain a supernatant, an equal volume of ethyl acetate is used to extract the supernatant, the extracted organic phase is subjected to low-temperature and reduced-pressure rotary distillation, and after removing the organic solvent, a crude extract is obtained. The crude extract is dissolved in methanol, subjected to Sephadex LH-20 gel column chromatography, eluted with methanol, the light yellow band at the front is collected, and the semi-pure product is obtained by rotary evaporation under reduced pressure; the semi-pure product is dissolved in methanol, prepared by HPLC, and the methanol is removed by rotary distillation under low-temperature and reduced pressure. The aqueous solution is freeze-dried to obtain a pure compound in the form of a slightly light yellow powder.

[0035] The present invention also provides the use of Xuanzang Mixing B and its composition in the preparation of antibiotic drugs. The antibiotic is used to resist bacterial infection, and the bacterial infection is caused by Gram-positive bacteria Staphylococcus resistant to beta-lactam antibiotics and vancomycin-resistant enterococci.

[0036] The present invention also provides application of Micromonospora strain TMD166 in producing Xuanzangmixin B.

[0037] In order to obtain Xuanzangmixin B and carry out antibacterial activity determination, the present invention adopts the following technical routes and steps: Fermentation and culture of Xuanzangmixing B-producing bacteria: First, the Micromonospora strain TMD166 grown on the slant was inoculated into the seed medium and cultured on a rotary shaker for 72 h, then transferred to the fermentation medium and cultured on a shaking shaker, and the fermentation liquid was harvested after 7 days; The seed culture medium is medium No. 38 (glucose 4 g, yeast powder 4 g, malt extract powder 5 g, ZnSO4.7H2O 0.001 g, MnCl2.4H2O 0.001 g, FeSO4.7H2O 0.002 g, phenylalanine 0.001 g, alanine 0.0003 g, vitamin B1 0.001 g, vitamin B2 0.001 g, vitamin B6 0.001 g, niacin 0.001 g, biotin 0.001 g, 1.0L sterile distilled water, pH 8.5). The fermentation medium is consistent with the seed culture medium formula.

[0038] Extraction and separation of Xuanzang Mixing B: After the fermentation broth is centrifuged, the supernatant is extracted with equal volumes of ethyl acetate three times, and the organic solvent is removed by rotary evaporation at low temperature and reduced pressure to obtain a reddish-brown crude extract. The crude extract is dissolved in methanol and subjected to Sephadex LH-20 gel column chromatography with methanol as the eluent. The light yellow band at the front is collected and rotary evaporated to dryness under reduced pressure; the semi-pure product is dissolved in methanol, prepared by HPLC, and freeze-dried to obtain the target compound as a slightly light yellow powder.

[0039] Structural identification of Xuanzangmixin B: Based on UV, IR, HR-ESI-MS, 1 H-NMR, 13 C-NMR, DEPT and 1 H- 1 H correlation spectrum ( 1 H- 1 H COSY)、 1 H- 13 C correlation spectroscopy (HSQC), reverse detection remote 1 H- 13 C heteronuclear multiple bond correlation spectroscopy (HMBC) and rotating coordinate ROESY spectroscopy determined the structure of Xuanzangmi Star B.

[0040] Determination of antibacterial activity of Xuanzang Mixing B: The determination of minimum inhibitory concentration (MIC) was carried out according to the microbroth dilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS). The experimental results showed that Xuanzang Mixing B has bacterial inhibition activity: it has strong in vitro antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus (strains resistant to β-lactam antibiotics) and Enterococcus aureus (strains resistant to vancomycin), and the MIC value is in the range of 0.25-8μg / ml.

[0041] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0042] Example 1 Fermentation of Micromonospora strain TMD166 The Micromonospora strain TMD166 ( Micromonospora taklimakanensis sp. TMD166) was inoculated into seed culture medium, the seed culture medium was medium No. 38 (glucose 4.0 g, yeast powder 4.0 g, malt extract powder 5.0 g, ZnSO4.7H2O 0.001 g, MnCl2.4H2O 0.001 g, FeSO4.7H2O0.002 g, phenylalanine 0.001 g, alanine 0.0003 g, vitamin B1 0.001 g, vitamin B2 0.001 g, vitamin B6 0.001 g, niacin 0.001 g, biotin 0.001 g, 1.0 L sterile distilled water, pH 8.5). Each 500 mL shake flask was filled with 100 mL of No. 38 medium and cultured on a rotary shaker at 28-30 ℃ and 180 rpm for 72 h. Then, the fermentation medium (same as the seed medium) was transferred at a 10% inoculation rate. Each 5.0 L shake flask was filled with 1.0 L of fermentation medium and cultured on a rotary shaker at 28-30 ℃ and 180 rpm for 7 d, and the fermentation broth was harvested.

[0043] Example 2 Extraction of Myrica chinensis B The fermentation broth obtained in Example 3 was centrifuged (4500 rpm, 20 min), and the supernatant (300 L) was extracted with equal volumes of ethyl acetate three times, the aqueous layer was discarded, and the organic solvent was removed by rotary evaporation at low temperature and reduced pressure to obtain a reddish-brown crude extract (30 g).

[0044] Example 3 Isolation of Myricetin B The crude extract obtained in Example 4 was dissolved in methanol and loaded onto a Sephadex LH-20 gel chromatography column (column volume 700 mL), eluted with methanol, and the yellow ribbon at the front was collected and evaporated to dryness under reduced pressure to obtain a semi-pure product (1.9 g) containing the target compound. The semi-pure product was dissolved in 3.0 mL of methanol and prepared by HPLC: The column parameters were Capcell-Pak-C 18 MGⅡ5 μm, 10 mm × 250 mm; mobile phase is 40% acetonitrile aqueous solution; flow rate is 2 mL / min. Collect the components with a retention time of around 43 min (UV detection wavelength is 230 nm), combine the collected components, and remove methanol by rotary evaporation. After freeze drying, the pure Xuanzangmixin B is obtained as a slightly yellow powder (13 mg).

[0045] Example 4 Structural Identification of Xuanzang Mixing B The structure of the pure product obtained in Example 5 was identified. According to the UV spectrum ( Figure 2 )、IR spectrum( Figure 3 )、HR-ESI-MS( Figure 4 ), and the NMR spectrum with CDCl3:CD3OD (9:1) as solvent: 1 H-NMR( Figure 5 )、 13 C-NMR( Figure 6 )、DEPT spectrum( Figure 7 ), 1 H- 13 C correlation HSQC ( Figure 8 ), reverse detection remote 1 H- 13 C heteronuclear multiple bond correlation spectroscopy HMBC ( Fig. 9 ), 1 H- 1 HCOSY correlation spectrum ( Fig.10 ) and ROESY spectra ( Fig.11 ) determined the structure of Xuanzangmi Star B.

[0046] Xuanzang Mixing B is a slightly yellow powder, insoluble in water, and easily soluble in organic solvents such as methanol, chloroform, and dimethyl sulfoxide. Its ultraviolet absorption spectrum is similar to that of Xuanzang Mixing, with maximum absorption at 232 nm, 267 nm, 311 nm, and 355 nm ( Figure 2 ), indicating that Xuanzangmixin B has the structural characteristics of thiopeptide compounds. The infrared spectrum shows that at 3378 cm -1 , 2979 cm -1 ,2953 cm -1 , 1731 cm -1 , 1663 cm-1 , 1528 cm -1 , 1480 cm -1 , 1244 cm -1 , 1089 cm -1 , 794cm -1 , 750 cm -1 It has characteristic absorption peaks similar to those of Xuanzangmi Star. Its specific rotation is close to that of Xuanzangmi Star, [α]20 D = -20.8 (c 0.5, MeOH). According to HR-ESI-MS data ( Figure 4 ), [M+H] + The measured value is 1881.4800, the theoretical value is 1881.4809, and the molecular formula is C 80 H 96 N 12 O 29 S6, unsaturation is 39.

[0047] Through the Xuanzang Mixing B 1 H-NMR, 13 C-NMR, HSQC, and DEPT spectra showed that the molecule contained 80 carbon atoms, including 29 quaternary carbon atoms (δ C : 180.7, 170.4, 170.3, 168.1, 168.1, 164.5, 163.7,162.1, 161.4, 161.4, 160.8, 160.4, 159.7, 155.0, 154.1, 150.5, 150.5, 148.7,145.9, 145.6, 139.7, 137.5, 131.8, 131.4, 129.7, 129.5, 124.9, 117.9, 110.4), 32 tertiary carbons (δ C : 127.4, 125.8, 125.6, 125.6, 124.4, 124.4, 121.4, 114.6, 102.1,100.2, 98.5, 96.1, 88.4, 83.1, 83.0, 81.0, 80.2, 80.1, 77.2, 75.9, 71.7,70.8, 70.4, 70.1, 69.9, 69.9, 69.6, 68.9, 66.8,56.8 , 53.8, 49.5), 5 secondary carbons (δ C :66.9, 64.5, 40.1, 38.3, 29.6), 14 primary carbons (δ C: 61.0, 60.8, 59.6, 59.4, 58.9,57.8, 57.8, 55.9, 18.0, 17.9, 17.8, 17.7, 17.3, 13.7).

[0048] The chemical shifts of each C directly connected to H were determined by HSQC (Table 1~Table 2).

[0049] Table 1 NMR data of Xuanzangmixin B in CDCl3:CD3OD (9:1)

[0050] Table 2 NMR data of Xuanzangmixin B in CDCl3:CD3OD (9:1)

[0051] In Table 1 and Table 2, 1 H-NMR: 600 MHz; 13 C-NMR: 150 MHz.

[0052] Further analysis of the hydrogen and carbon spectra showed that Xuanzang Mixing B is a thiopeptide antibiotic containing five thiazole rings, including three carbonyl thiazole rings (Thz1, Thz2, Thz5), one fully substituted carbonyl thiazole ring (Thz3), one thiazole ring (Thz4), one tetrasubstituted hydroxylated pyridine (Pyr), one threonine (Thr), one methoxy-substituted dehydrothreonine (Dht), one 2,3,4-trisubstituted indole ring (Ind), one hydroxyglutamic acid (Glu) and one cysteine ​​(Cys). Comparison of the carbon and hydrogen spectra of Xuanzang Mixing B with those of Xuanzang Mixing and philipimycin revealed that the mother nucleus of Xuanzang Mixing B lacked one dehydroalanine (Deala) residue, and the rest of the carbon and hydrogen data were basically consistent. Combined with the molecular formula of Xuanzang Mixing B being C 80 H 96 N 12 O 29 S 6， Compared with Xuanzang Mixing, it lacks the C3H3NO group, which corresponds to the structural fragment of dehydroalanine. 1 H- 1 HCOSY signal ( Figure 1 ), which also further confirmed the parent nucleus structure of Xuanzangmi Star B.

[0053] In addition, 1 H- 1In HCOSY, δ5.82 (1H, d, J=1.8 Hz,S1-1H) and δ3.85 (1H, dd, J=3.6, 1.8 Hz,S1-2H); δ4.27 (1H, dd, 9.6, 3.6Hz, S1-3H) and δ3.85 (1H, dd, J=3.6,1.8 Hz,S1-2H) and δ3.16 (1H, dd,J=9.6, 9.0Hz, S1-4H); δ3.16 (1H, dd, J=9.6, 9.0Hz, S1-4H) and δ3.66 (1H, overlap,S1-5H); δ3.66 (1H, overlap,S1-5H) and δ1.30 (3H,d,J=6.6, In combination with HMBC, the two methoxy hydrogens δ3.57 (3H, S, S1-C2-OMe) and δ3.56 (3H, S, S1-C4-OMe) are correlated with C-2 and C-4, respectively. Based on this information, it can be determined that S1 is a 2,4-dimethoxy-substituted pyranose rhamnoside. The coupling constants of S1-2H, S1-1H, and S1-3H are 1.8Hz and 3.6Hz, respectively, and the coupling constant of S1-3H and S1-4H is 9.6Hz. In combination with ROESY, S1-1H is correlated with S1-2H and S1-C2-OMe, indicating that S1-1H and S1-2H are located in the flat bond direction on the pyranose ring, while S1-3H and S1-4H are located in the upright bond direction. In NOESY and ROESY, S1-4H and S1-C5-Me-H were correlated, indicating that S1-5H was also located in the vertical bond direction. Thus, glycoside S1 was determined to be a 2,4-dimethoxy-substituted α-rhamnopyranoside. In addition, in HMBC, S1-1H and Pyr-C3 were correlated, confirming the connection position of glycoside S1 and the pyridine ring.

[0054] The same comprehensive analysis 1 H- 1 H COSY and HMBC confirmed that S2 is a 2,3-dimethoxy-substituted rhamnoside, S3 is a 2-deoxyhexoside, and S4 is a 2,4-dimethoxy-substituted rhamnoside (related 1 H- 1 H COSY, HMBC Figure 1). The coupling constants of S2-2H and S2-1H, S2-3H are 1.8 and 3.0 Hz, respectively. In ROESY, S2-1H is correlated with S2-2H, S2-C2-OMe-H, and S2-2H is correlated with S2-C3-OMe-H, S2-C3-H, indicating that S2-1H and S2-2H are both located in the direction of the flat bond. In addition, in ROESY, S2-4H is correlated with S2-C3-OMe-H, S2-C5-Me, S3-1H, indicating that S2-4H, S2-3H and S2-5H are located in the direction of the upright bond. The carbon and hydrogen chemical shifts of S2 and S2 of philipimycin and S1 of philipimycin are almost consistent, indicating that S2 and S2 of philipimycin and S1 of philipimycin have the same configuration, and are all 2,3-dimethoxy-substituted α-rhamnosides. S3-1H and S3-2H a 、S3-2H b The coupling constants are 9.6 Hz and 1.8 Hz, indicating that S3-1H is located in the vertical bond direction; S3-2H a The coupling constants are 12.6, 5.4, 1.8 Hz, S3-2H b The coupling constants are 12.6, 12.0, 9.6 Hz, indicating that S3-2H a Located in the flat bond direction, S3-2H b and S3-3H are located in the vertical bond direction; while the coupling constants of S3-4H are 9.0 and 8.4 Hz, indicating that S3-4H and S3-5H are also located in the vertical bond direction on the pyran ring. In addition, in ROESY, S3-5H is correlated with S3-1H and S3-3H, further confirming that S3-1H, S3-3H, and S3-5H are all located in the vertical bond direction. Thus, glycoside S3 is determined to be a deoxy β-pyranoside. The coupling constants of S4-4H are 9.6 and 9.0 Hz, indicating that S4-3H, S4-4H, and S4-5H are located in the vertical bond direction on the pyranoside. Combined with the correlation peaks of S4-1H, S4-2H, S4-C2-OMe-H, S4-2H, and S4-3H in ROESY, S4 is determined to be a 2,4-dimethoxy-substituted α-rhamnoside. In HMBC, S2-1H and Thz3-C5, S2-4H and S3-C1, and S3-4H and S4-C1 were related, and the connection positions of glycoside S2 and thiazole ring-5, as well as the connection positions of glycosides S2 and S3, and S3 and S4 were determined.

[0055] The complete structure of Xuanzangmi Star B is deduced as shown in formula (1).

[0056] Example 5 Determination of the antibacterial activity of Xuanzangmixin B against bacteria The activity was determined using the compound obtained in Example 3. The minimum inhibitory concentration (MIC) was determined in accordance with the microbroth dilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS). The specific implementation steps are as follows: dissolve Xuanzang Mixing B in DMSO with a stock solution concentration of 1.0 mg / ml, and dilute the drug by half with a culture medium solution. After half dilution, different concentrations of antibacterial drug solutions were added to a sterilized 96-well polystyrene plate, with the drug solution added to the 1st to 11th wells, 100 μl per well, and no drug was added to the 12th well as a growth control. The bacterial activity assay culture medium used the Mueller-Hinton (MH) broth recommended by NCCLS, pH 7.2-7.4. Directly take the colonies cultured for 18-24 h and mix them with physiological saline to make a 0.5 McFarland turbidimetric standard (1×10 8 -2×10 8 CFU / ml) of bacterial suspension. Dilute the bacterial suspension to be tested with MH broth at 1:1000, add 100μl to each well, place in a 35℃ constant temperature incubator, and incubate for 20 hours to determine the result. Result determination: The lowest drug concentration that completely inhibits bacterial growth in the well is MIC, and the determination is repeated three times.

[0057] The results of the activity of Xuanzang Mixing B against common pathogenic bacteria are shown in Table 3. Xuanzang Mixing B has strong inhibitory activity against Gram-positive bacteria: including Staphylococcus (strains resistant to β-lactam antibiotics) and Enterococcus (vancomycin-resistant Enterococcus faecalis and Enterococcus faecium), with MIC values ​​ranging from 0.25 to 8 μg / ml.

[0058] Table 3 Antibacterial activity of Xuanzangmixin B

[0059] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Xuanzang rice star B, the structure is shown in formula (1): Formula (1).

2. The method for preparing Xuanzangmixing B according to claim 1, characterized in that: The fermentation culture preservation number is CGMCC NO.21622 Micromonospora ( Micromonospora sp.) TMD166, harvesting the fermentation broth; and then extracting and separating the Xuanzangmixing B from the fermentation broth.

3. The method according to claim 2, characterized in that The fermentation culture comprises: inoculating the micromonospora on a seed culture medium for culture, and then transferring to a fermentation culture medium for shaking culture; The conditions for culturing on the seed culture medium are 28-30°C, 150-200 r / min, and the culturing time is 48-72 hours; the conditions for culturing on the fermentation culture medium are 28-30°C, 150-200 r / min, and the culturing time is 96-120 hours; The seed culture medium and the fermentation culture medium have the same formula, specifically: 4 g glucose, 4 g yeast powder, 5 g malt extract powder, 0.001 g ZnSO4.7H2O, 0.001 g MnCl2.4H2O, 0.002 g FeSO4.7H2O, 0.001 g phenylalanine, 0.0003 g alanine, 0.001 g vitamin B1, 0.001 g vitamin B2, 0.001 g vitamin B6, 0.001 g niacin, 0.001 g biotin, 1.0 L sterile distilled water, pH 8.

5.

4. The method according to claim 3, characterized in that The method for extracting and separating the Xuanzang Mixing B from the fermentation broth comprises: after the fermentation broth is centrifuged, taking the supernatant and performing equal volume extraction with ethyl acetate three times, and removing the organic solvent by rotary evaporation at low temperature and reduced pressure to obtain a reddish brown crude extract, dissolving the crude extract in methanol, and performing Sephadex LH-20 gel column chromatography, using methanol as an eluent, collecting the light yellow band at the front end, and performing rotary evaporation under reduced pressure to obtain a semi-pure product; dissolving the semi-pure product in methanol, preparing it by HPLC, and freeze-drying it to obtain a pure Xuanzang Mixing B product in the form of a slightly light yellow powder.

5. The method according to claim 4, characterized in that The method of preparing by HPLC includes: the chromatographic column parameters are Capcell-Pak-C 18 MGⅡ 5 μm, 10 mm × 250 mm; the mobile phase was 40% acetonitrile aqueous solution; the flow rate was 2 mL / min; the component with a retention time of 43 min was collected, the UV detection wavelength was 230 nm, and after combining the collected components, the methanol was removed by rotary evaporation, and the pure Xuanzangmixin B was obtained after freeze-drying.

6. Use of the Xuanzangmixin B described in claim 1 in the preparation of antibacterial products.

7. The use according to claim 6, characterized in that: The bacteria are Gram-positive bacteria.

8. The use according to claim 7, characterized in that: The Gram-positive bacteria include Staphylococcus and Enterococcus.

9. The use according to claim 8, characterized in that: The Gram-positive bacteria include β-lactam antibiotic-resistant Staphylococci and vancomycin-resistant Enterococci.

10. The use according to claim 8 or 9, characterized in that: The enterococci include Enterococcus faecalis and Enterococcus faecium.

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