CN111235042A - Butenolactone dimer with COX-2 inhibitory and antioxidant effects and its application - Google Patents

Abstract

The invention discloses an aromatic butenolide dimer, a preparation method thereof and application thereof in COX-2 inhibitors and anti-oxidation drugs. The invention relates to an Aspergillus fungusAspergillus terreusSCAU011 CGMCC No:19270 to obtain the compound with COX-2 inhibitory activity and antioxidant activity (shown in figure 1), and has good application prospect in the aspect of preparing COX-2 inhibitors and antioxidant drugs.

Description

Butenolactone dimer with COX-2 inhibitory and antioxidant effects and its application

technical field

The invention belongs to the field of marine natural medicines, in particular to an aromatic crotonic acid lactone dimer and its application in the preparation of COX-2 inhibitors and antioxidant medicines.

Background technique

Cyclooxygenase (COX) is the key enzyme that catalyzes the oxidation of arachidonic acid to synthesize prostaglandins. COX-1 mainly exists in normal cells and catalyzes the production of prostaglandins that maintain normal physiological functions; COX-2 is inducibly expressed in most tissues, is an important inducible enzyme in the process of inflammation, and is the role of non-steroidal anti-inflammatory drugs However, the currently marketed non-steroidal anti-inflammatory drugs can cause gastrointestinal and renal adverse reactions after long-term use. At the same time, COX-2 is highly expressed in a variety of tumor tissues, and is involved in tumor development, invasion and metastasis, and angiogenesis. It has played an important role and has become one of the new targets for tumor prevention and treatment. Therefore, the development of new COX-2 inhibitors is of great significance for the treatment of both inflammation and cancer.

Oxidative damage is not only related to aging and tumors, but also has a close relationship with many other diseases, such as coronary heart disease, heart failure, Alzheimer's disease, chronic inflammation, etc. Antioxidants can scavenge oxygen free radicals, improve oxidative damage, and improve oxidative stress. The treatment of stress-related diseases is of great significance.

SUMMARY OF THE INVENTION

The invention provides an aromatic crotonic acid lactone dimer compound with COX-2 inhibitory activity and antioxidant activity isolated from Aspergillus fungus and its fermentation and a preparation method thereof.

In order to achieve the above objects, the present invention adopts the following technical solutions.

A fungus of the genus Aspergillus terreus SCAU011, preserved in the China General Microorganism Culture Collection and Management Center (CGMCC), address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, preservation number: CGMCC No. 19270, deposit date: December 27, 2019.

The application of an Aspergillus terreus SCAU011 and its fermentation products in the production of COX-2 inhibitors and antioxidants. The Aspergillus terreus SCAU011 can be cultured to produce a fermentation product, the fermentation product contains COX-2 inhibitory activity and antioxidant active substances, and the fermentation broth can be used to produce a drug with COX-2 inhibitory activity and antioxidant activity or additives.

An aromatic crotonic acid lactone dimer compound produced by Aspergillus terreus SCAU011, the structural formula of which is shown in Figure 1.

A preparation method of the above-mentioned aromatic crotonic acid lactone dimer compound, comprising the following steps:

(1) Fermentation of Aspergillus terreus SCAU011;

(2) extracting a fermented extract from the fermented product obtained in step (1);

(3) The extract in step (2) is separated by normal phase silica gel column, fast medium pressure chromatography column, reverse phase C18 column high performance liquid phase and other methods.

The present invention provides a use of the extract in step (2), or the compound shown in Figure 1 or a salt thereof in the preparation of a COX-2 inhibitor or antioxidant, a medicine or a medicine intermediate.

The present invention provides a medicament containing a COX-2 inhibitor or antioxidant containing the extract in step (2), or the compound shown in Figure 1 or a salt thereof. The COX-2 inhibitor and antioxidant drugs also include medically acceptable excipients. The COX-2 inhibitor and antioxidant drug may further include other active ingredients to enhance COX-2 inhibition or antioxidant effect.

The present invention has the following advantages:

The aromatic crotonic acid lactone dimer of the present invention can be obtained by fermentation, extraction and separation of Aspergillus terreus SCAU011, has the activity of inhibiting COX-2 and the activity of scavenging oxygen free radicals, and is useful in the preparation of COX-2 inhibitors and antioxidants. Has application potential.

Description of drawings

Fig. 1 is the structural formula of the aromatic crotonic acid lactone dimer compound.

Figure 2 is a picture of the strain SCAU011 plate culture.

Figure 3 is the hydrogen spectrum (600 MHz) and carbon spectrum (150 MHz) data (CDCl ₃ ) of the compound shown in Figure 1 .

Fig. 4 is the main ¹ H- ¹ H COSY and HMBC information of the compound shown in Fig. 1 .

Figure 5 is the ECD spectrum of the compound shown in Figure 1.

Figure 6 shows the structure of the Mosher reagent derivative of the compound shown in Figure 1 and its δ _H (Δδ = δ _S − δ _R ) difference.

Detailed ways

The present invention will be further described below with reference to the embodiments and the accompanying drawings, but the present invention is not limited by the following embodiments. The experimental methods in the examples, unless otherwise specified, are all conventional techniques in the art, and the experimental reagents are all purchased commercially.

The strain Aspergillus terreus SCAU011 of the present invention is preserved in China General Microorganism Culture Collection and Management Center (CGMCC), address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, preservation number: CGMCC No. 19270 , preservation date: December 27, 2019.

Example 1 Isolation, purification and identification of bacterial species.

The bacterial species Aspergillus terreus SCAU011 was isolated and purified from the sediments of the Mangrove Reserve in Techeng Island, Zhanjiang. The typical culture picture is shown in Figure 2: the colonies are round, the hyphae are white, and the spores are khaki. By ITS rDNA detection, compared with Gene Bank data, identified as Aspergillus terreus fungus.

Example 2 Preparation of Fermentation.

Preparation method of seed medium: 200 mL of potato extract, 20 g of glucose, 30 g of sea salt, and make up to 1 L with water. Put the PDB medium into 20 500 mL Erlenmeyer flasks, each with about 150 mL, and sterilize them by autoclaving at 121 °C for 25 minutes.

Rice culture medium configuration method: put 80 g of commercially available rice, 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 1 L conical flask, a total of 60 bottles. Sterilize by autoclaving at 121°C for 25 minutes.

Pick an appropriate amount of fungus Aspergillus terreus SCAU011 with a sterile bamboo stick and inoculate it into the seed medium, inoculate it on a shaker (180 rpm) at 28°C for 3 days to get the seed solution, and then inoculate 10 mL of the seed solution with a pipette to 1 L The fermented medium was collected after 30 days of static culture at 28°C in the Erlenmeyer flask containing the rice medium.

The fermented rice culture medium was soaked with 95% ethanol, the ethanol was recovered from the leaching solution and the remaining aqueous phase was extracted with ethyl acetate, and concentrated under reduced pressure to obtain an ethyl acetate extract as a crude extract.

Example 3 Preparation of aromatic crotonic acid lactone dimer.

4.0 kg of fermented rice culture medium was obtained according to the method of Example 2, the rice culture medium was soaked with 95% ethanol, the remaining aqueous phase was extracted with ethyl acetate after the ethanol was recovered from the leaching solution, and the ethyl acetate extract was obtained by concentrating under reduced pressure 60g. The ethyl acetate extract was subjected to column chromatography on normal phase silica gel (100-200 mesh), with dichloromethane-methanol as the eluent, and the gradient elution was carried out from 100:0 to 0:100 in a volume ratio. According to the analysis situation, each fraction was combined, and the elution solvent was recovered to obtain 7 fractions (Fr.1-Fr.7).

Fr.5 was further carried out with normal phase silica gel (200-300 mesh) column chromatography with dichloromethane-acetone as the eluent, combined each fraction according to the thin-layer chromatography situation, recovered the elution solvent, and obtained 6 fractions (Fr. .5-1-Fr.5-6). Component Fr.5-4 was separated by fast medium pressure (reverse C-18) chromatography with methanol-water as the eluent. According to the thin layer chromatography, each fraction was combined, and the elution solvent was recovered to obtain 9 fractions. (Fr.5-4-1-Fr.5-4-9). Component Fr.5-4-9 was semi-prepared by high performance liquid phase (the chromatographic column was YMC-Pack ODS-A 10 mm × 250 mm, the flow rate was 3.0 mL/min, the detection wavelength was 254 nm, and the mobile phase was a volume ratio of 76 : 24: 0.05 methanol-water-acetic acid) was separated and purified when the peak time was 11.2 min to obtain the compound shown in Figure 1 (6.3 mg).

The compound shown in Figure 1 is a pale yellow gel, easily soluble in chloroform, methanol, DMSO, and insoluble in water, with a specific optical rotation value [α] ²⁹ _D 77.1 ( c 0.36, MeOH).

The isolated compounds were analyzed by high-resolution mass spectrometry (HR-ESIMS), ¹ H NMR, ¹³ C NMR, 2D ¹ H- ¹ HCOSY, HSQC, HMBC, and the planar structure was confirmed. The three-dimensional structure of the compound. The ¹ H and ¹³ C NMR data are shown in Figure 3, the main ¹ H- ¹ H COSY and HMBC related information is shown in Figure 4, and the ECD spectrum is shown in Figure 5. The structures of the isolated compounds are shown in Figure 1 .

Example 4 Preparation of derivatives of compounds shown in Figure 1 .

Weigh 1.0 mg of the compound shown in Figure 1 and dissolve it in 0.5 mL of anhydrous pyridine, add Mosher reagent 10 μL ( R )-2-methoxy-2-trifluoromethylphenylacetyl chloride and 1.0 mg DMAP (as catalyst) for 9 hours at room temperature. After the solvent was evaporated to dryness under reduced pressure, the crude product was semi-prepared by high performance liquid phase to obtain compound a (Figure 6); the Mosher reagent was replaced with ( S )-2-methoxy-2-trifluoromethylphenylacetyl chloride, the same as method to obtain compound b (Figure 6).

Example 5 COX-2 inhibitory activity of the compounds shown in Figure 1 .

The COX-2 inhibitory activity of the compounds shown in Figure 1 was tested using a cyclooxygenase (COX-2) inhibitor screening kit (Biyuntian), and the celecoxib provided by the kit was used as a positive control.

The experimental results showed that when the test concentration was 20 nM, the inhibition rate of the compound shown in Figure 1 on COX-2 was 76.47%, which was better than the 56.7% inhibition rate of the positive control celecoxib.

Example 6 Antioxidative activity of the compounds shown in Figure 1 .

Determination of the antioxidant activity of compounds by DPPH method: add 100 μL, 200 μM DPPH ethanol solution and 100 μL ethanol solution of the test sample to a 96-well plate, incubate at 25 °C for 30 min, and measure the reaction at 517 nm The absorbance A _S of the solution, the mixture of 100 μL, 200 μM DPPH ethanol solution and 100 μL ethanol solution was used as the negative control A _C , DPPH free radical scavenging rate (%) = [(A _C - A _S )/ A _C ]×100%. Curcumin was used as a positive control.

The experimental results show that the compound shown in Figure 1 has the activity of scavenging oxygen free radicals with an IC ₅₀ of 16.6 μM, which is comparable to the inhibitory activity of the positive control curcumin (IC ₅₀ of 16.2 μM).

Claims (6)

1. Aspergillus fungusAspergillus terreusSCAU011 preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 19270.

2. An aromatic butenolide dimer produced by the Aspergillus fungus of claim 1, having a structural formula shown in FIG. 1.

3. An Aspergillus fungus as claimed in claim 1Aspergillus terreusUse of SCAU011 for preparing a compound according to claim 2 and represented in FIG. 1, 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 a normal phase silica gel column, a rapid medium pressure chromatography column, a reverse phase C18 column high efficiency liquid phase and other methods.

A COX-2 inhibitor drug comprising an effective amount of the Aspergillus fungus of the above-mentioned step (2) as an active ingredientAspergillus terreusSCAU011 fermentation extract, or aromatic butyrolactone dimer compound of claim 2, or a salt thereof, and a pharmaceutically acceptable carrier.

5. An antioxidant agent comprising an effective amount of the fungus of the genus Aspergillus of the above step (2) as an active ingredientAspergillus terreusThe SCAU011 fermented extract is prepared by fermenting SCAU011 fermented extract,or the aromatic butyrolactone dimer compound or salt thereof according to claim 2, and a pharmaceutically acceptable carrier.

6. The Aspergillus fungus of the step (2)Aspergillus terreusUse of a SCAU011 fermentation extract, or an aromatic butyrolactone dimer compound of claim 2, or a salt thereof, in the preparation of COX-2 inhibitors and antioxidant drugs or pharmaceutical intermediates.

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