CN111909856B

CN111909856B - Preparation method and antibacterial application of ring-opened aromatic butenolide

Info

Publication number: CN111909856B
Application number: CN202010840435.2A
Authority: CN
Inventors: 张华�; 鲍洁; 贺菲; 李秀秀
Original assignee: University of Jinan
Current assignee: University of Jinan
Priority date: 2020-08-20
Filing date: 2020-08-20
Publication date: 2022-02-01
Anticipated expiration: 2040-08-20
Also published as: CN111909856A

Abstract

The invention discloses a ring-opening aromatic butenolide compound, a preparation method thereof and application thereof in antibacterial drugs. In the present invention, the open-ring aromatic crotonolide compound shown in FIG. 1 with antibacterial activity is isolated from the fermentation product of an Aspergillus fungusAspergillus terreus SCAU011 CGMCC No: 19270, which can inhibit the growth of Staphylococcus aureus(Staphylococcus aureus). The IC₅₀value 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. aureusDrug resistance, in particular emergence of methicillin-resistant Staphylococcus aureus (MRSA)Now, the treatment of Staphylococcus aureus infection has become complicated and difficult, and therefore, the development of drugs having inhibitory activity against Staphylococcus aureus is urgent.

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.

Aspergillus fungusAspergillus 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 appropriate amount of fungus with sterile bamboo stickAspergillus 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 was inoculated into LB medium and cultured on a shaker (180 rpm) at 37 ℃ for 24 hours, then the strain concentration of the culture broth was diluted with LB medium to 104-105 cfu/mL by McLeod's turbidimetry, inoculated into a 96-well plate containing 200. mu.L (containing the dilution of the sample to be tested) of the culture broth per well, left at 37 ℃ for 12 hours, and incubated withAnd detecting the absorbance A value of each hole at 600 nm by using a microplate reader, and calculating the bacterial growth inhibition rate. 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

1. An open-ring aromatic butenolide produced by Aspergillus fungus and having the following structural formula

2. An antibacterial agent comprising an effective amount of the ring-opened aromatic butenolide according to claim 1 as an active ingredient, and a pharmaceutically acceptable carrier.