CN111321095B - Butenolide dimer with alpha-glucosidase inhibition effect and application thereof - Google Patents

Abstract

The invention discloses an aromatic butenolide dimer with an alpha-glucosidase inhibiting effect, and a preparation method and application thereof. The invention relates to an Aspergillus fungusAspergillus terreusAn aromatic butenolide dimer is obtained by separating a fermentation product of SCAU011 CGMCC No. 19270, has alpha-glycosidase inhibiting activity as shown in figure 1, and can be prepared into an alpha-glucosidase inhibitor with the function of reducing blood sugar, and can be used for preparing compositions, medicines and health products for preventing and/or treating diabetes, obesity and complications thereof.

Description

Butenolide dimer with alpha-glucosidase inhibition effect and application thereof

Technical Field

The invention belongs to the field of marine natural medicines, and particularly relates to a butenolide dimer and application thereof in preparation of an alpha-glucosidase inhibitor and preparation of a composition, a medicine and a health-care product for preventing or treating diabetes, obesity and complications thereof.

Background

With the influence of the great improvement of living standard, the aging of population, the change of environment and the like of people, diabetes becomes a chronic disease next to tumor diseases and cardiovascular diseases, the number and proportion of attack people are also increased year by year, and the public health is seriously harmed. The clinical classification of diabetes is divided into type I diabetes (insulin-dependent diabetes) and type II diabetes (non-insulin-dependent diabetes), wherein the type II diabetes accounts for more than 90% of diabetes. Alpha-glucosidase inhibitors can delay intestinal carbohydrate absorption and are considered to be an ideal way to control type II diabetes. At present, acarbose, miglitol, voglibose and the like are mainly marketed as alpha-glucosidase inhibitors, and acarbose, miglitol and voglibose are all metabolites derived from microorganisms. Therefore, the discovery of α -glucosidase inhibitors from microbial metabolites is of great significance for the prevention or treatment of type II diabetes.

Disclosure of Invention

The invention provides a butenolide dimer compound with alpha-glucosidase inhibition function separated from aspergillus fungi and fermentation products thereof 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 address: west road No. 1, north west of the morning area, beijing, 3, institute for microbiology, china academy of sciences, accession number: CGMCC number 19270, preservation date: 12 and 27 days 2019.

Aspergillus fungusAspergillus terreusSCAU011 and application of fermentation products thereof in producing alpha-glucosidase inhibitors. The Aspergillus fungusAspergillus terreusThe SCAU011 can produce fermentation product after culture, the fermentation product contains alpha-glucosidase inhibitory active substance, and the fermentation liquid can be used for producing drugs or additives with alpha-glucosidase inhibitory effect.

Aspergillus fungusAspergillus terreusThe butenolide dimer compound produced by SCAU011 has a structural formula shown in figure 1.

A preparation method of the butenolide dimer 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 a normal phase silica gel column, a rapid medium pressure chromatography column, a reverse phase C18 column high efficiency liquid phase and other methods.

The invention provides application of a compound containing the extract in the step (2) or shown in a figure 1 or salt thereof in preparing alpha-glucosidase inhibitor drugs and drug intermediates.

The invention provides an alpha-glucosidase inhibitory drug containing the extract in the step (2) or a compound shown in figure 1 or a salt thereof. The medicine with the alpha-glucosidase inhibition effect also comprises medically acceptable auxiliary materials. The drug with alpha-glucosidase inhibition effect can also comprise other effective components to enhance the effect of inhibiting alpha-glucosidase or reducing blood sugar.

The invention has the following advantages:

the butenolide dimers of the present invention may be prepared byAspergillus terreusThe SCAU011 is obtained by fermentation, extraction and separation, has alpha-glucosidase inhibitory activity, and has application potential in the aspect of preparing alpha-glucosidase inhibitors.

Drawings

FIG. 1 is a structural formula of butenolide dimer compound.

FIG. 2 is a drawing of the culture of strain SCAU011 on a plate.

FIG. 3 is a graph of hydrogen (600 MHz) and carbon (150 MHz) spectra (CDCl) data for the compound of FIG. 1₃）。

FIG. 4 shows the main components of the compound shown in FIG. 1¹H-¹H COSY, HMBC information.

FIG. 5 is an ECD spectrum of the compound shown in FIG. 1.

FIG. 6 shows the Mosher reagent derivative structure and delta of the compound shown in FIG. 1_H (Δδ = δ_S−δ_R) The difference value.

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.

The bacterial strain of the inventionAspergillus terreusSCAU011 is preserved in China general microbiological culture Collection center (CGMCC) with the address: west road No. 1, north west of the morning area, beijing, 3, institute for microbiology, china academy of sciences, accession number: CGMCC No19270, date of deposit: 12 and 27 days 2019.

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 terreusSCAU011, 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 preparation of butenolide dimer.

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 60g 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 fractions (Fr.1-Fr.7).

Fr.5 further subjected to normal phase silica gel (200-300 mesh) column chromatography using methylene chloride-acetone as an eluent, the fractions were combined according to the case of thin layer chromatography, and the elution solvent was recovered to obtain 6 fractions (Fr.5-1-Fr.5-6). The fraction Fr.5-4 was subjected to rapid medium pressure (reverse C-18) chromatography using methanol-water as an eluent, and the fractions were combined according to the case of thin layer chromatography, and the elution solvent was recovered to obtain 9 fractions (Fr.5-4-1-Fr.5-4-9). The component Fr.5-4-9 was isolated and purified by HPLC semi-preparative (column YMC-Pack ODS-A10 mm. times.250 mm, flow rate 3.0 mL/min, detection wavelength 254 nm, mobile phase methanol-water-acetic acid in volume ratio 76: 24: 0.05) at a peak-off time of 11.2 min to give the compound shown in FIG. 1 (6.3 mg).

The compound shown in figure 1 is light yellow colloid, is easily soluble in chloroform, methanol and DMSO, is hardly soluble in water, and has specific luminosity value [ alpha ]]²⁹ _D 77.1（c 0.36，MeOH）。

And (3) carrying out high resolution mass spectrum (HR-ESIMS), 1H NMR, 13C NMR, 2D 1H-1H COSY, HSQC and HMBC analysis on the separated compound to determine a planar structure, and determining the three-dimensional structure of the compound by using an ECD and improved Mosher method. The 1H and 13C NMR data are shown in FIG. 3, the main 1H-1H COSY and HMBC related information is shown in FIG. 4, and the ECD spectrum is shown in FIG. 5. The structure of the isolated compound is shown in FIG. 1.

Example 4 preparation of derivatives of the compounds shown in figure 1.

1.0 mg of the compound shown in FIG. 1 was weighed out and dissolved in 0.5 mL of anhydrous pyridine, and 10. mu.L of Mosher's reagent (R) -2-methoxy-2-trifluoromethylphenylacetyl chloride and 1.0 mg of DMAP (as a catalyst) were added to the solution to react at room temperature for 9 hours. Evaporating the solvent under reduced pressure, and semi-preparing the crude product with high performance liquid phase to obtain a compound a (figure 6); the same procedure, substituting Mosher reagent for (S) -2-methoxy-2-trifluoromethylphenylacetyl chloride, gave compound b (fig. 6).

Example 5 α -glucosidase inhibitory activity of the compound shown in figure 1.

Preparing a PNPG (p-nitrophenyl- β -D-glucoside) solution (0.4 mmol/L) with phosphate buffer (0.1 mol/L, pH = 6.9) for use; preparing a lyophilized enzyme powder (enzyme activity is 16U/mL) solution (0.2U/mL) with a phosphate buffer (0.1 mol/L pH = 6.9) for later use; the experiment is divided into an experimental group, a blank group and a control group, 2.0 mu L of DMSO solution containing samples with different concentrations, 25 mu L of 0.2U/mL enzyme solution and 98 mu L of PBS buffer solution are added into a 96-well plate to be mixed evenly, and incubation is carried out for 20 min at 37 ℃; then 25.0 μ L of 0.4 mM PNPG was added and incubated at 37 ℃ for 15 min; finally 50.0. mu.L of 0.2M Na was added₂CO₃The reaction was terminated. Measuring absorbance at 405 nm of microplate reader, and calculating inhibition rate and IC₅₀。

The experimental result shows that the compound shown in figure 1 has good alpha-glucosidase inhibitory activity and IC₅₀The value is 10.5 mu M, which is far better than the positive control acarbose (IC)₅₀ = 274.5 μM）。

Claims (3)

1. Aspergillus fungusAspergillus terreus SCAU011 preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 19270.

2. An aromatic butyrolactone dimer produced by an Aspergillus fungus according to claim 1 having the structure

3. An Aspergillus fungus as claimed in claim 1Aspergillus terreus Use of SCAU011 for preparing a compound according to claim 2, comprising the steps of:

(1) aspergillus fungiAspergillus terreus Fermenting 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 a normal phase silica gel column, a rapid medium pressure chromatography column, a reverse phase C18 column high efficiency liquid phase and other methods.

Images