CN111909856A - Preparation method and antibacterial application of ring-opened aromatic butenolide - Google Patents

Preparation method and antibacterial application of ring-opened aromatic butenolide Download PDF

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CN111909856A
CN111909856A CN202010840435.2A CN202010840435A CN111909856A CN 111909856 A CN111909856 A CN 111909856A CN 202010840435 A CN202010840435 A CN 202010840435A CN 111909856 A CN111909856 A CN 111909856A
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aspergillus
scau011
butenolide
ring
aromatic
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CN111909856B (en
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张华�
鲍洁
贺菲
李秀秀
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University of Jinan
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    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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Abstract

The invention discloses a ring-opening aromatic butenolide compound, a preparation method thereof and application thereof in antibacterial drugs. The invention relates to an Aspergillus fungusAspergillus terreus The open-loop aromatic butenolide compound shown in figure 1 with antibacterial activity and capable of inhibiting golden yellow grape is obtained by separating from fermentation product of SCAU011 CGMCC No:19270Coccus (A)Staphylococcus aureus) Growth of, IC50The value is 21.3 mu M, and the antibacterial agent has good application prospect in the aspect of preparing antibacterial drugs.

Description

Preparation method and antibacterial application of ring-opened aromatic butenolide
Technical Field
The invention belongs to the field of marine natural medicines, and particularly relates to a ring-opening aromatic butenolide compound and application thereof in preparing antibacterial medicines.
Background
Staphylococcus aureus (1)Staphylococcus aureus) Is a common clinical bacterium, belongs to gram-positive pathogenic bacteria, can cause various diseases of human beings and animals such as gastroenteritis, mastitis, wound infection, bacteremia, pneumonia, endocarditis, septicemia and the like, and can seriously endanger life.S. aureusThe requirement for the growth environment is low, so that the staphylococcus aureus is widely existed in nature, and the staphylococcus aureus accounts for 20% -25% of the population for a long time and is mainly existed on the nasal cavity and the skin of a human. In the last 40 th century, penicillin had a good therapeutic effect in the treatment of infectious diseases caused by Staphylococcus aureus, but due to the large use of antibiotics, a large number of antibiotics appearedS. aureusThe emergence of resistance, particularly methicillin-resistant staphylococcus aureus (MRSA), complicates and makes difficult the treatment of staphylococcus aureus infections, and the development of drugs with staphylococcus aureus inhibitory activity is therefore imminent.
Disclosure of Invention
The invention provides a fungus of Aspergillus and a method for separating the fungus from the fermented product and inhibiting staphylococcus aureus (S. aureus) Growth-active ring-opening aromatic butenolide compounds and a preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme.
Aspergillus fungusAspergillus terreusSCAU011, preserved in China general microbiological culture Collection center (CGMCC), with the preservation number: CGMCC number 19270, preservation date: 12 and 27 days 2019.
Aspergillus fungusAspergillus terreusSCAU011 and application of fermentation products thereof in producing antibacterial drugs. The Aspergillus fungusAspergillus terreusThe SCAU011 can produce fermentation product after culture, the fermentation product contains active substances for inhibiting the growth of S. aureus, and the fermentation liquid can be used for producing medicaments or additives with antibacterial activity.
KojiFungi of the genus mildewsAspergillus terreusThe open-loop aromatic butenolide compound produced by SCAU011 has the structural formula shown in figure 1.
A preparation method of the ring-opening aromatic butenolide compound comprises the following steps:
(1) aspergillus fungiAspergillus terreusFermenting SCAU 011;
(2) extracting the fermented product obtained in the step (1) to obtain a fermented extract;
(3) and (3) separating the extract in the step (2) by using methods such as normal-phase silica gel column chromatography, rapid medium-pressure chromatography, reverse-phase C18 column chromatography, high-performance liquid chromatography and the like.
The invention provides application of a compound containing the extract in the step (2) or shown in figure 1 or salt thereof in preparing antibacterial drugs and drug intermediates.
The invention provides an antibacterial drug containing the extract in the step (2) or the compound shown in figure 1 or the salt thereof. The antibacterial medicine also comprises medically acceptable auxiliary materials. The antibacterial drug can also comprise other effective components to enhance the bacteriostatic effect.
The invention has the following advantages:
the ring-opened aromatic butenolide compound of the invention can be prepared byAspergillus terreusSCAU011 obtained by fermentation, extraction and separation, and has effect in inhibiting Staphylococcus aureus (S.), (S. aureus) The growth activity has application potential in the preparation of antibacterial drug.
Drawings
FIG. 1 shows the structural formula of ring-opened aromatic butenolide compounds.
FIG. 2 is a drawing of the culture of strain SCAU011 on a plate.
FIG. 3 is a high resolution mass spectrum of the compound of FIG. 1.
FIG. 4 is a hydrogen spectrum of the compound shown in FIG. 1.
FIG. 5 is a carbon spectrum of the compound shown in FIG. 1.
FIG. 6 is a two-dimensional COSY spectrum of the compound shown in FIG. 1.
FIG. 7 is a two-dimensional HSQC spectrum of the compound shown in FIG. 1.
FIG. 8 is a two-dimensional HMBC spectrum of the compound shown in FIG. 1.
Figure 9 is an experimental and theoretical calculated ECD profile for the compound shown in figure 1.
FIG. 10 is a hydrogen (600 MHz) and carbon (150 MHz) spectra data (CDCl) for the compound shown in FIG. 13）。
And (3) strain preservation information: according to the inventionAspergillus terreusSCAU011 is preserved in China general microbiological culture Collection center (CGMCC), and the address is as follows: western road No. 1, north zhou yang ward, beijing, institute for microbiology, china academy of sciences, accession number: CGMCC number 19270, preservation date: 12 and 27 days 2019.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples. The experimental procedures in the examples, unless otherwise specified, were carried out by conventional techniques in the art and the experimental reagents were all purchased commercially.
Example 1 isolation, purification and characterization of the species.
Separating and purifying the sediments in the mangrove forest protected area of Zhanjiang province to obtain strainsAspergillus terreus SCAU011, a typical culture picture of which is shown in FIG. 2: the colony is round, white hypha and earthy yellow spore. The product is identified by comparing with Gene Bank data through ITS rDNA detectionAspergillus terreusAnd (3) fungi.
Example 2 preparation of fermentates.
The preparation method of the seed culture medium comprises the following steps: 200 mL of potato leachate, 20 g of glucose and 30 g of sea salt, and the volume is adjusted to 1L by using water. PDB medium was charged into 20 500 mL Erlenmeyer flasks, about 150 mL per flask, and autoclaved at 121 ℃ for 25 minutes for future use.
The rice culture medium configuration method comprises the following steps: putting 80 g of rice sold in market, 0.4 g of yeast extract, 0.4 g of glucose, 120 mL of sterile water and 3.6 g of sea salt into a 1L triangular flask, and putting 60 bottles in total. Sterilizing with high pressure steam at 121 deg.C for 25 min.
Picking up proper amount of the extract by using sterile bamboo sticksFungiAspergillus terreusInoculating SCAU011 strain into seed culture medium, culturing at 28 deg.C for 3 days in shaking table (180 rpm) to obtain seed solution, inoculating 10 mL seed solution into 1L triangular flask containing rice culture medium with liquid transfer gun, standing at 28 deg.C for 30 days, and collecting fermented culture medium.
Soaking fermented rice culture medium with 95% ethanol, recovering ethanol from the extractive solution, collecting the residual water phase, extracting with ethyl acetate, and concentrating under reduced pressure to obtain ethyl acetate extract as crude extract.
Example 3 FIG. 1 preparation of ring-opened aromatic butenolide.
The method of example 2 was followed to obtain 4.0 kg of fermented rice medium, which was soaked in 95% ethanol, the ethanol was recovered from the extract, and the remaining aqueous phase was extracted with ethyl acetate, and concentrated under reduced pressure to obtain 60 g of ethyl acetate extract. The ethyl acetate extract was subjected to column chromatography using normal phase silica gel (100-200 mesh), gradient elution was carried out from a volume ratio of 100:0 to 0:100 using methylene chloride-methanol as an eluent, and the respective fractions were combined according to the case of thin layer chromatography, and the elution solvent was recovered to obtain 7 elution fractions (Fr.1-Fr.7).
Fr.4 further subjected to normal phase silica gel (200-300 mesh) column chromatography using methylene chloride-acetone as an eluent, and the respective fractions were combined according to the case of thin layer chromatography to obtain 9 fractions (Fr.4-1-Fr.4-9). Fraction Fr.4-4 was separated by flash medium pressure (reverse phase C18 silica gel) chromatography using methanol-water as eluent, and the fractions were combined according to thin layer chromatography to obtain 9 fractions (Fr.4-4-1-Fr.4-4-9). The component Fr.4-4-5 was purified by chiral chromatography (xylonite AD-H, flow rate 1.0 mL/min, detection wavelength 254 nm, mobile phase 15% isopropanol-n-hexane), the component with peak time 9.5 min was collected, and the solvent was removed to obtain pure compound (2.1 mg) shown in FIG. 1.
The compound shown in figure 1 is a light yellow colloidal solid, is easily soluble in chloroform, methanol and dimethyl sulfoxide, is hardly soluble in water, and has specific optical rotation value [ alpha ]]29 D +16.1（c 0.18，MeOH）。
Subjecting the separated sample to high resolution mass spectrometry (HR-ESIMS),1H NMR、13C NMR、2D 1H-1H COSY, HSQC, HMBC analysis (fig. 3-8), determined their planar structure, and by comparing their experimental ECD spectra and calculating the ECD spectra (fig. 9), determined their absolute configuration as shown in fig. 1. It is composed of1H and13the C NMR data are shown in FIG. 10.
Example 4 antibacterial activity of the compound shown in figure 1.
The compounds shown in fig. 1 were tested for antibacterial activity using 96-well plates. Respectively treating Staphylococcus aureusStaphylococcus aureusATCC25923 and Vibrio splendidusVibrio splendidusMCCC1A04069 is inoculated in LB culture medium and cultured for 24 hours at 37 ℃ by a shaking table (180 rpm), then the strain concentration of the culture solution is diluted to 104-105 cfu/mL by the LB culture medium through a Mach-Zehnder method, the culture solution is inoculated in a 96-well plate, each well comprises 200 mu L (including a sample to be detected which is diluted by a multiple proportion) of the culture solution, the culture solution is placed for 12 hours at 37 ℃, the absorbance A value of each well at 600 nm is detected by a microplate reader, and the bacterial growth inhibition rate is calculated. Bacterial growth inhibition = 1- (experimental OD-blank OD) ÷ (control OD-blank OD) × 100%.
The experimental result shows that the open-loop aromatic butenolide compound shown in figure 1 can inhibit staphylococcus aureusStaphylococcus aureus ATCC25923, IC thereof50The value was 21.3. mu.M.

Claims (5)

1. Aspergillus fungusAspergillus terreusSCAU011, preserved in China general microbiological culture Collection center (CGMCC), with the preservation number of CGMCC No. 19270 and the preservation date: 12 and 27 days 2019.
2. An open-ring aromatic butenolide produced by the Aspergillus fungus of claim 1, which has the structural formula shown in FIG. 1.
3. An Aspergillus fungus as claimed in claim 1Aspergillus terreusUse of SCAU011 for preparing a compound of FIG. 1 according to claim 2, comprising the steps of：
(1) Aspergillus fungiAspergillus terreusFermenting SCAU 011;
(2) extracting the fermented product obtained in the step (1) to obtain a fermented extract;
(3) and (3) separating the extract in the step (2) by using methods such as normal-phase silica gel column chromatography, rapid medium-pressure chromatography, reverse-phase C18 column chromatography, high-performance liquid chromatography and the like.
4. An Aspergillus fungus as described in the above step (2)Aspergillus terreusUse of the SCAU011 fermentation product, or the ring-opened aromatic butenolide compound or the salt thereof according to claim 2 for the preparation of an antibacterial agent or as an antibacterial agent intermediate.
5. An antibacterial agent characterized by comprising an effective amount of the fungus of the genus Aspergillus according to the above step (2) as an active ingredientAspergillus terreusThe SCAU011 fermentation product, or the ring-opened aromatic butenolide compound or the salt thereof according to claim 2, and a pharmaceutically acceptable carrier.
CN202010840435.2A 2020-08-20 2020-08-20 Preparation method and antibacterial application of ring-opened aromatic butenolide Active CN111909856B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108165500A (en) * 2018-02-26 2018-06-15 广东海洋大学深圳研究院 One plant of marine fungi Aspergillus terreus C23-3, zymotic fluid activity extract and its preparation method and application
CN111334438A (en) * 2020-03-25 2020-06-26 济南大学 Preparation method of aromatic butenolide dimer and application of aromatic butenolide dimer in preparation of antibacterial drugs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108165500A (en) * 2018-02-26 2018-06-15 广东海洋大学深圳研究院 One plant of marine fungi Aspergillus terreus C23-3, zymotic fluid activity extract and its preparation method and application
CN111334438A (en) * 2020-03-25 2020-06-26 济南大学 Preparation method of aromatic butenolide dimer and application of aromatic butenolide dimer in preparation of antibacterial drugs

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BAO, J;等: "Asperbutenolide A, an unusual aromatic butenolide dimer with diverse", 《TETRAHEDRON LETTERS》 *
NUCLEAR, P等: "Butenolide and Furandione from an Endophytic Aspergillus terreus", 《 CHEMICAL & PHARMACEUTICAL BULLETIN 》 *

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