Application of a secondary metabolite of Penicillium fasciatum in antibacterial compounds and extraction and purification method thereof

Abstract

The present invention discloses an application of a secondary metabolite of Penicillium scalycopersicum in antibacterial compounds and a method for extracting and purifying the same. The present invention separates a secondary metabolite having the activity of inhibiting methicillin-resistant Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus from the Penicillium scalycopersicum species for the first time. The secondary metabolite is used to prepare a drug having the activity of inhibiting methicillin-resistant Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus, and has good clinical prospects. The present invention also provides a method for isolating secondary metabolites from the Penicillium scalycopersicum species, which has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of antibacterial compounds, and particularly relates to an application of a secondary metabolite of Penicillium fasciatum in antibacterial compounds and an extraction and purification method thereof. Background Art

[0002] The vast number of marine biological species provides valuable resources for obtaining a large number of natural products with diverse structures and excellent activities.

[0003] A large number of studies have reported that some secondary metabolites of marine organisms have significant biological activities, including antibacterial, antiviral, anti-cardiovascular, anti-tumor, etc. However, the problem of drug resistance has intensified in recent years, making it necessary to develop antibiotics with high safety, good activity and low resistance. With the development of life sciences and technology, secondary metabolites derived from microorganisms have attracted more and more attention.

[0004] Pathogenic bacteria such as Staphylococcus aureus and Staphylococcus epidermidis are important pathogens in hospitals and communities, and can cause various life-threatening infections, including necrotizing pneumonia and subcutaneous abscesses. Due to the overuse of antibiotics in clinical treatment, the antibiotic resistance of Staphylococcus aureus and Staphylococcus epidermidis has increased, and the development and discovery of new antibiotics has become an urgent problem to be solved. Summary of the invention

[0005] The extracted and purified Penicillium crustosum secondary metabolite of the present invention is extracted from Penicillium crustosum S14 strain (Latin name: Penicillium crustosum), which is derived from South China Sea mud and has a preservation number of CCTCCNO: M 20221447. The molecular formula of the Penicillium crustosum secondary metabolite of the present invention is: 15 H 11 NO3, the structural formula is as follows:

[0006]

[0007] The Penicillium S14 strain of the present invention is as follows Figure 1 and Figure 2 As shown, the mycelium is dark green, the edge area is white, pigment is produced, there is no exudate, the back of the colony is yellow, and cotton blue staining observation shows that the morphological characteristics are similar to those of Penicillium fungi. By extracting the mycelium genomic DNA and performing sequence comparison, it was identified as the species of Penicillium scaly.

[0008] As one aspect of the present invention, the present invention provides an application of a secondary metabolite of Penicillium fasciatum in an antibacterial compound.

[0009] Preferably, the secondary metabolites of Penicillium fasciatum can inhibit the activity of methicillin-resistant Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus.

[0010] Specifically, the antibacterial compounds include antibacterial drugs and salts thereof.

[0011] As another aspect of the present invention, the present invention provides a method for extracting and purifying the secondary metabolites of Penicillium fasciatum, which comprises the following steps:

[0012] S1. Preparation of crude extract: soaking the Penicillium scabra obtained by fermentation culture in ethanol, filtering to obtain a filtrate, rotary evaporating the obtained filtrate to 1 / 4 of the original volume, adding ethyl acetate to extract at a volume ratio of 1:1, and then rotary evaporating again to obtain a fermentation extract, adding methanol to dissolve the fermentation extract to obtain a crude extract; S2. Separation and purification: separating the crude extract using a silica gel G chromatography column, using 100-200 mesh silica gel G, the elution gradient is: dichloromethane; dichloromethane: methanol = 80: 1; dichloromethane: methanol = 60: 1; dichloromethane: methanol = 40: 1; dichloromethane ∶methanol=20∶1;、dichloromethane:methanol=10∶1;collect and combine the elution products obtained by the elution gradients of dichloromethane:methanol=20∶1 and dichloromethane:methanol=10∶1, and purify them by ODS column chromatography, the elution gradients are: water; water:methanol=80∶20; water:methanol=60∶40; water:methanol=40∶60; water:methanol=20∶80; methanol, purify the elution products obtained by the elution gradient of water:methanol=20∶80 by high performance liquid chromatography, the mobile phase is: methanol:water=1∶1, the flow rate is 2ml / min, and lavender crystals are obtained.

[0013] As a preferred embodiment of the method for extracting and purifying the secondary metabolites of Penicillium sphaerocephala described in the present invention: the Penicillium sphaerocephala strain is the Penicillium sphaerocephala strain S14.

[0014] As a preferred embodiment of the method for extracting and purifying the secondary metabolites of Penicillium fasciatum described in the present invention: the fermentation culture is to inoculate the seed culture solution of Penicillium fasciatum S14 strain into rice solid fermentation medium, and culture it in an incubator under a dark environment for thirty days at a culture temperature of 28 degrees Celsius.

[0015] As a preferred scheme of the method for extracting and purifying the secondary metabolites of Penicillium odoratum described in the present invention: the seed culture solution of the Penicillium odoratum S14 strain is prepared by inoculating the Penicillium odoratum S14 strain on a PDA culture medium plate, placing it in an incubator for 3 days in a dark environment, and the culture temperature is 28 degrees Celsius. When a single colony appears on the PDA culture medium plate, the spores and hyphae are scraped into 200 ml of PDB liquid culture medium, and placed in an incubator for 3 days in a dark environment, and the culture temperature is 28 degrees Celsius.

[0016] As a preferred embodiment of the method for extracting and purifying the secondary metabolites of Penicillium rotundum described in the present invention: the preparation method of the rice solid fermentation medium is as follows: 1 kg of rice is added to 1 liter of distilled water, 0.075 g of magnesium sulfate and 33 g of sea crystal, mixed evenly, the pH value is adjusted to about 7.0, packaged according to 50 g / can, and sterilized under high pressure at 121 degrees Celsius.

[0017] Beneficial effects of the present invention: The present invention is the first to isolate secondary metabolites with the activity of inhibiting methicillin-resistant Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus from the species of Penicillium fasciatum, and the secondary metabolites are used to prepare drugs with the activity of methicillin-resistant Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus, which has good clinical prospects. The present invention also provides a method for isolating secondary metabolites from the species of Penicillium fasciatum, which has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a picture of mycelium stained with cotton wool observed under a 40x microscope.

[0019] Figure 2 This is a photo of the Penicillium colony of the present invention.

[0020] Figure 3 The invention discloses a high performance liquid chromatography of the secondary metabolites of Penicillium fasciatum separated and purified by the present invention.

[0021] Figure 4 The present invention discloses a hydrogen nuclear magnetic resonance spectrum of the secondary metabolites of Penicillium fasciatum separated and purified by the present invention.

[0022] Figure 5 The present invention discloses a carbon nuclear magnetic resonance spectrum of the secondary metabolites of Penicillium fasciatum separated and purified by the present invention. DETAILED DESCRIPTION

[0023] The following is an explanation of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0024] Embodiment 1:

[0025] The extraction and purification method of the secondary metabolites of Penicillium fasciatum of the present invention:

[0026] 1. Seed liquid preparation: Inoculate the Penicillium S14 strain on a PDA medium plate and culture it in an incubator in a dark environment for 3 days at a temperature of 28 degrees Celsius. When a single colony appears on the PDA medium plate, scrape the spores and hyphae into 200 ml of PDB liquid culture medium and culture it in an incubator in a dark environment for three days at a temperature of 28 degrees Celsius.

[0027] 2. Bacteria fermentation culture: 4 kg of rice solid fermentation medium inoculated with seed liquid was loaded into 80 fermentation tanks respectively, the seed liquid obtained in step 1 was inoculated into the rice solid fermentation medium, 100 microliters of seed liquid was added to each gram of rice solid fermentation medium, and the solid fermentation medium was placed in an incubator for 30 days under a dark environment, and the culture temperature was 28 degrees Celsius. (The preparation method of the rice solid fermentation medium: 1 kg of rice was added to 1 liter of distilled water, 0.075 grams of magnesium sulfate and 33 grams of sea crystal were added, mixed evenly, autoclaved at 121 degrees Celsius, and the pH value was adjusted to 7.0).

[0028] 3. Prepare a crude extract: add 150 ml / tank of CH3CH2OH to the mixed fermentation product of the rice solid fermentation medium and the Penicillium S14 strain obtained by fermentation culture, soak for 10 hours, filter and obtain a filtrate, rotary evaporate the filtrate to 1 / 4 of the original volume, add ethyl acetate for extraction, wherein the volume ratio of the added ethyl acetate to the volume of the filtrate is 1:1, and then rotary evaporate again to obtain a total of 35 grams of fermentation extract, add 20 ml of CH3OH to dissolve the fermentation extract to obtain a crude extract.

[0029] 4. Separation and purification:

[0030] (1) Silica gel chromatography column separation: The crude extract was separated using a silica gel G chromatography column, using 100-200 mesh silica gel G, eluent: dichloromethane-methanol, the eluent gradient and the elution product numbers obtained under each elution gradient are shown in Table 1. The elution products obtained under each elution gradient are numbered F1-F6 respectively.

[0031] Table 1 Eluent gradient and elution product number obtained by each elution gradient

[0032] Product No. Eluent volume ratio (v:v) F1 Dichloromethane F2 Dichloromethane: methanol = 80: 1 (v: v) F3 Dichloromethane: methanol = 60: 1 (v: v) F4 Dichloromethane: methanol = 40: 1 (v: v) F5 Dichloromethane: methanol = 20: 1 (v: v) F6 Dichloromethane: methanol = 10: 1 (v: v)

[0033] Using methicillin-resistant Staphylococcus aureus (MRSA) as the indicator bacteria, the products F1-F6 obtained under each elution gradient were screened for active components;

[0034] The screening method for active components is as follows: MHB culture solution of methicillin-resistant Staphylococcus aureus (indicator bacteria concentration: OD 600nm=0.1) was spread on an MHB medium plate, and holes were punched on the plate with a hole puncher, each hole having a diameter of 6 mm; 30 μl of the eluted products of each group of F1-F6 were added to each well as the experimental group, 30 μl of chloramphenicol (concentration of 100 μg / ml) was added to each well as the positive control, and 30 μl of methanol was added to each well as the negative control. The cells were cultured at 37 degrees Celsius for 18 hours, and the diameter (cm) of the inhibition zone of each experimental group was calculated.

[0035] The above antibacterial activity screening results showed that components F5 and F6 had antibacterial activity. Components F5 and F6 were combined and counted as component A for the next separation.

[0036] (2) Component A separated in step (1) was purified by ODS column chromatography, eluent: water-methanol; the eluent gradient and the elution product numbers obtained under each elution gradient are shown in Table 2. The elution products obtained under each elution gradient are numbered A1-A6 respectively.

[0037] Table 2 Eluent gradient and elution product number obtained by each elution gradient

[0038] Product No. Eluent volume ratio (v:v) A1 water A2 Water: methanol = 80: 20 (v: v) A3 Water: methanol = 60: 40 (v: v) A4 Water: methanol = 40: 60 (v: v) A5 Water: methanol = 20: 80 (v: v) A6 Methanol

[0039] Using methicillin-resistant Staphylococcus aureus (MRSA) as the indicator bacteria, the active components of the products A1-A6 obtained under each elution gradient were screened respectively according to the method described in step (1).

[0040] The antibacterial activity screening results showed that component A5 had antibacterial activity.

[0041] (3) A small amount of the active component A5 obtained in step (2) was subjected to thin layer chromatography (TLC) to analyze the specific material composition of the active component A5. The developing solvent was: dichloromethane: methanol = 20: 1 (v: v). The separated bands were detected under ultraviolet light (254 nm) to provide a reference for the subsequent purification steps.

[0042] (4) The active component A5 obtained in step (2) was purified by HPLC with a mobile phase of 50% methanol:50% water (volume ratio) at a flow rate of 2 ml / min to obtain lavender crystals. The antibacterial activity of the component was tested according to the method described in step (1). After the antibacterial activity test, it was confirmed that the antibacterial active component was the target compound. 1 H-NMR and 13 C-NMR identification ( Figure 3-Figure 5 ), whose molecular formula is: C 15 H 11 NO3, the structural formula is:

[0043]

[0044] The yield of the secondary metabolites of Penicillium fasciatum of the present invention is calculated to be 15 mg / kg rice.

[0045] Embodiment 2:

[0046] Application of Penicillium fasciatum secondary metabolites as antimicrobial compounds:

[0047] Minimum inhibitory concentration (MIC) test of secondary metabolites of Penicillium fasciatum:

[0048] Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), Klebsiella pneumoniae, Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa, Acinetobacter baumannii were used as indicator bacteria. The above indicator bacteria were cultured in MHB liquid culture medium at 37 degrees Celsius. The concentration of indicator bacteria in the indicator bacteria solution was 1×10 5 CFU / ml;

[0049] The above-mentioned indicator bacteria solutions were added to a 96-well plate, and the secondary metabolite compound I of Penicillium fasciatum was added in each concentration gradient. Specifically, the concentration gradient of the secondary metabolite compound I of Penicillium fasciatum was set to: 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, and 50 μg / mL of chloramphenicol was added to each bacterial solution as a positive control. The 96-well plate was placed in a constant temperature incubator and cultured at 37 degrees Celsius for 18 hours. For each of the above-mentioned indicator bacteria, the lowest administration concentration that makes the bacterial solution clear is the minimum inhibitory concentration of the compound for the indicator bacteria.

[0050] Experimental results:

[0051] The MIC of Staphy / ococcus aureus is 25μg / mL; the MIC of Methicillin-resistant Staphy / ococcus epidermidis (MRSA) is 25μg / mL; the MIC of Methicillin-resistant Staphy / ococcus epidermidis (MRSE) is 50μg / mL; the MIC of Klebsiella aerogenes, Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa, and Acinetobacter baumannii are all 200μg / mL.

[0052] The above experimental results show that the secondary metabolites of Penicillium fasciatum of the present invention have good antibacterial activity and can be used as antibacterial compounds, especially for drug-resistant strains: methicillin-resistant Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus. They have very excellent antibacterial effects and can be used to solve the technical problem that drug-resistant bacterial infections are difficult to treat clinically.

[0053] Research example:

[0054] The fermentation medium for the fermentation culture of the strain in step 2 of Example 1 was selected as rice solid fermentation medium or corn solid medium, and the fermentation culture conditions were a dark environment or a fluorescent light illumination environment, respectively. The antibacterial effects of the crude extracts obtained under different culture conditions were studied, and the experimental results are shown in Table 3.

[0055] Experimental method: According to the method of steps 1-3 of Example 1, the strains were fermented and cultured under various experimental conditions to prepare crude extracts. The culture solutions of the indicator bacteria shown in Table 3 (indicator bacteria concentration: OD 600nm =0.1) were spread on MHB culture medium plates, and holes were punched on each culture medium plate with a hole puncher, each hole had a diameter of 6 mm; 30 μl of crude extract was added to each well as the experimental group, 30 μl of chloramphenicol (concentration of 100 μg / ml) was added to each well as the positive control, and 30 μl of methanol was added to each well as the negative control. The cells were cultured at 37 degrees Celsius for 18 hours, and the diameter of the inhibition zone (cm) of each experimental group was calculated. The antibacterial effects of the crude extracts obtained under different culture conditions are shown in Table 3.

[0056] Table 3 Antibacterial effects of crude extracts obtained under different culture conditions

[0057]

[0058] From the above experimental results, it can be seen that the antibacterial activity of the crude extract obtained by fermentation with rice solid medium is better than that with corn solid medium, so rice solid medium is preferably used for fermentation.

[0059] It should be noted that the above are only preferred embodiments of the present invention, and any simple modification or equivalent replacement made according to the technical solution of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. An application of a secondary metabolite of Penicillium fasciatum in the preparation of an antibacterial compound, characterized in that: The preparation method of the secondary metabolite of Penicillium fasciatum comprises: soaking the Penicillium fasciatum obtained by fermentation and culture in ethanol, filtering to obtain a filtrate, rotary evaporating the obtained filtrate to 1 / 4 of the original volume, adding ethyl acetate to extract at a volume ratio of 1:1, and then rotary evaporating again to obtain a fermentation extract, and adding methanol to dissolve the fermentation extract to obtain a crude extract; The fermentation culture is to inoculate the seed culture liquid of the Penicillium species into the rice solid fermentation medium, and place it in an incubator for 30 days in a dark environment at a culture temperature of 28 degrees Celsius; the preparation method of the rice solid fermentation medium is: add 1 kg of rice to 1 liter of distilled water, add 0.075 grams of magnesium sulfate and 33 grams of sea crystal, mix evenly, adjust the pH value to 7.0, and sterilize under high pressure at 121 degrees Celsius; The antibacterial agent is anti-Escherichia coli ( Escherichiacoli ) or against Acinetobacter baumannii ( Acinetobacter baumannii ).

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