Application of Peniazathilone A as alpha-glucosidase inhibitor and preparation method of Peniazathilone A

Abstract

The name of the invention is application of Peniazathilone A as an alpha-glucosidase inhibitor and a preparation method of the Peniazathilone A. The invention belongs to the technical field of medicines, and relates to application of an azone compound as the alpha-glucosidase inhibitor. The compound has the following structure: # imgabs0 #. The compound Peniazathilone A is obtained by extracting, separating and purifying an ethyl acetate extract phase of a fermented product of Penicillium sp from soil, and the alpha-glucosidase inhibition activity of the compound is evaluated by adopting a pNPG method. Experimental results show that the IC50 value of the compound Peniazathilone A is 86.8 [mu] M (the IC50 value of a positive control acarbose is 5.97 mM), and the inhibition rate reaches 92.4% when the concentration of the compound is 0.466 mM. The compound has obvious activity of inhibiting alpha-glucosidase, is expected to be developed into a novel hypoglycemic drug, and has potential medical and health-care values.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and specifically relates to the application of Peniazaphilone A as an alpha-glucosidase inhibitor and a preparation method thereof. Background Art

[0002] Diabetes is a chronic disease caused by pancreatic islet dysfunction and characterized by high blood sugar, of which type 2 diabetes is the most common. Diabetic patients are prone to chronic lesions or functional impairment of various tissues and organs in the body, and in severe cases, organ failure. Globally, the prevalence of diabetes is on the rise, seriously affecting the health and life of patients. Drugs used clinically to treat type 2 diabetes include biguanides, sulfonylureas, α-glucosidase inhibitors, thiazolidinediones and other drugs. Alpha-glucosidase inhibitors, represented by acarbose, inhibit α-glucosidase in the intestines, slow down the digestion and absorption of carbohydrates, and cause slow intestinal glucose absorption, thereby achieving the effect of controlling postprandial blood sugar levels. However, the α-glucosidase inhibitors commonly used in clinical practice usually have obvious gastrointestinal side effects, so seeking new or new skeleton α-glucosidase inhibitor molecules is particularly important for the development of hypoglycemic drugs.

[0003] Fungal secondary metabolites are one of the important sources for new drug research and development. There are more than 300 species of Penicillium. Through studying the chemical composition of Penicillium fermentation broth, people have discovered various compounds with good biological activity, including polyketides, alkaloids, terpenes, steroids, etc., which show antibacterial activity, antiplatelet activity, antitumor activity, hypoglycemic activity and many other biological activities.

[0004] Peniazaphilone A belongs to the class of azoquinone compounds, which belongs to the polyketide class of natural products. Azoquinones are common secondary metabolites of fungi, characterized by a highly oxidized pyranoquinone bicyclic nucleus and a quaternary carbon center, and the pyranoquinone oxygen atom is easily replaced by a nitrogen atom to form quinoline quinone. The molecular structure of Peniazaphilone A is shown below:

[0005]

[0006] Peniazaphilone A was first reported in 2021. It was also isolated from the fermentation broth of Penicillium fungi and was shown to have LPS-mediated nitric oxide release inhibition activity and cancer cell inhibition activity. According to research, no studies have been reported on the inhibition of α-glucosidase by Peniazaphilone A. Summary of the invention

[0007] The present invention provides a natural azophilic ketone compound Peniazaphilone A for use as an α-glucosidase inhibitor, wherein the compound has a rare isoquinoline quinone mother nucleus, and activity evaluation shows that the compound has strong α-glucosidase inhibitory activity. The structure of the compound is shown below:

[0008]

[0009] The invention also provides a preparation method of Peniazaphilone A.

[0010] In summary, according to the technical solution, the value or effect that the present invention can produce is:

[0011] 1. The results of α-glucosidase inhibitory activity evaluation showed that Peniazaphilone A had a strong inhibitory effect on α-glucosidase activity. The IC 50 The value was 86.8 μM (positive control acarbose IC 50 value is 5.97 mM), which is expected to further develop Peniazaphilone A into a new glucose-lowering drug or related health care product for the treatment of type 2 diabetes.

[0012] 2. The Peniazaphilone A studied in the present invention can be obtained in large quantities from the fermentation broth of Penicillium. The materials required for the fermentation are simple and easy to obtain, the steps are relatively easy, and many harmful substances will not be produced during the preparation process, which makes up for the shortcomings of complicated steps and high costs of reagents and consumables in artificial synthesis. It can utilize microorganisms to expand fermentation for systematic large-scale production, which is of great significance for the development of hypoglycemic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The concentration-inhibition rate curve of Peniazaphilone A and acarbose DETAILED DESCRIPTION

[0014] The purpose, technical route and technical effects of the present invention are described in detail below in conjunction with the embodiments. The described embodiments are only some embodiments of the present invention, not all embodiments. Unless otherwise specified in the embodiments, the methods, instruments and reagents are all conventional means in the art.

[0015] Example 1 Preparation of Peniazaphilone A

[0016] 1. Strain Isolation

[0017] (1) Take 100 g of soil from the bottom of a stream located in a bamboo forest area in Sichuan Province and put it into a conical flask. Add 100 ml of sterile water to the conical flask in a clean bench, then place the prepared soil sample in a shaker and shake it at 160 r / min overnight. Let it stand and wait for the fungi to be isolated.

[0018] (2) Take 1 mL of the soil supernatant in (1) and place it in a sterile test tube containing 9 mL of sterile water. Mix by pipetting. Dilute to a concentration of 10% of (1). -1 .

[0019] (3) Take 1 mL of the soil supernatant in (2) and place it in a sterile test tube containing 9 mL of sterile water. Mix by pipetting. Dilute to a concentration of 10% of (1). -2 .

[0020] (4) Take 1 mL of the soil supernatant in (3) and place it in a sterile test tube containing 9 mL of sterile water. Mix by pipetting. Dilute to a concentration of 10% of (1). -3 .

[0021] (5) Pipette 0.2 mL of the supernatant from (1), (2), (3), and (4) onto the prepared plates, spread evenly, and culture in a 28°C incubator. When visible colonies grow in the culture medium, use an inoculation needle to pick up hyphae from the edge of a single colony and transfer it to a new PDA culture medium. Follow this process to purify the fungus and obtain a Penicillium strain, which was identified as Penicillium sp. by PCR amplification and sequencing.

[0022] 2. Fermentation:

[0023] (1) Weigh 20 g of PDA solid into a beaker, add 500 mL of distilled water, heat to boil in a water bath, and stir until the solid is completely dissolved to obtain PDA culture medium. When the temperature drops to a slight heat, pour the plate. After the agar solidifies, use a sterile bamboo stick to pick up the mycelium and inoculate it into the plate. Seal it and culture it at 28°C for three days.

[0024] (2) Considering the cost of reagents, potatoes, glucose and agar are used to prepare PDB culture medium. Weigh 600g of potatoes cut into small pieces and put them into a pot, then add clean water to cover them. Cook until the potatoes are soft and filter with gauze. Take the filtrate and add 60g of glucose, then add clean water to a total volume of 3L, stir evenly until the solids are completely dissolved, and then divide into conical flasks at 250mL each. Use an inoculation skewer to pick up the mycelium of the strain that has been cultured for three days and transfer it together with the agar block into PDB to obtain seed liquid. Put it in a constant temperature shaker, set the temperature to 28℃, the speed to 160rpm, and culture for three days.

[0025] (3) Weigh 100 mL of rice and add it to a 500 mL conical flask together with the prepared PDB. Sterilize it in a high pressure steam sterilizer at 121°C. After sterilization, wait for cooling and transfer it to the clean bench. Take out the 57 bottles of seed liquid from the shaker. It can be observed that the liquid surface is covered with mycelium and spherical bacteria grow in the liquid. In the clean bench, pour each bottle of seed liquid into the rice solid culture medium in four even amounts. After sealing, place all the rice solid culture medium in a cool place and ferment at room temperature for three months.

[0026] All microbiological tests in step 1 are carried out in a clean bench, and all materials used must be sterilized by high-pressure steam.

[0027] 3. Extraction:

[0028] The solvent impregnation method is used to transfer the fermented solid from the conical flask to a barrel, and methanol is added to cover the fermentation product and soak for three days. After three days, the methanol extract is red. The solid residue is filtered with gauze to obtain a methanol extract. The remaining solid filter residue continues to be soaked in methanol, and the above operation is repeated three times to collect the extract. The extract is transferred into a rotary evaporator, the temperature is set to 35°C, and the extract is concentrated under reduced pressure while recovering methanol. Since the extract still contains a large amount of water, it is dissolved with a small amount of solvent and transferred to a separatory funnel. An appropriate amount of ethyl acetate is added to the concentrated extract for extraction. The upper layer is the ethyl acetate phase and the lower layer is the aqueous phase. The extraction operation is repeated three times, the ethyl acetate phases are combined, and the crude extract is concentrated under reduced pressure.

[0029] 4. Compound separation:

[0030] (1) The crude extract was gradient eluted with a system of petroleum ether, ethyl acetate, and methanol. The eluate was concentrated under reduced pressure and combined for TLC detection to obtain 14 components (Fr.A~Fr.N).

[0031] (2) The Fr.J fraction was subjected to silica gel column chromatography to obtain 13 fractions from Fr.J-1 to Fr.J-13. The eluent ratio was changed to petroleum ether: ethyl acetate: methanol (100:0:0→98:2:0→95:5:0→90:10:0→85:15:0→80:20:0→60:40:0→50:50:0→0:100:0→0:90:10→0:80:20→0:0:100).

[0032] (3) The Fr.J-2 component was separated by dextran gel chromatography with the eluent ratio of dichloromethane:methanol = 1:1. Five components, Fr.J-2-1 to Fr.J-2-5, were obtained.

[0033] (4) The Fr.J-2-3 component was separated by silica gel column chromatography and eluted with a polarity ratio of dichloromethane:methanol = 40:1 to obtain three components, Fr.J-2-3-1 to Fr.J-2-3-3. Among them, the Fr.J-2-3-1 component was separated and purified by dextran gel chromatography (eluent: methanol) to obtain compound Peniazaphilone A.

[0034] (5) The purity of Peniazaphilone A was tested by high performance liquid chromatography. The mobile phase composition was methanol: water (0.01-8.00min: methanol gradient change from 1% to 100%; 8.01-14.00min: 100% methanol elution; 14.01-19.00min: methanol gradient change from 100% to 0%). Chromatographic column conditions: injection volume 1μL; flow rate 1mL / min; column temperature 25℃; filler material C-18 alkyl bonded silica gel. The purity was measured by area integration method and was 98%.

[0035] (6) The NMR data of compound Peniazaphilone A are shown in Tables 1 and 2:

[0036] Table 1. 1 HNMR (500MHz, CDCl3)

[0037]

[0038] Table 2. 13 CNMR (126MHz, CDCl3)

[0039]

[0040] Example 2 Determination of the inhibitory activity of Peniazaphilone A on α-glucosidase

[0041] 1. Reagent preparation:

[0042] (1) 0.1 M phosphate buffer: Dissolve 0.8709 g of potassium dihydrogen phosphate and 0.6805 g of potassium dihydrogen phosphate in deionized water and make up to 100 mL. Prepare and use immediately.

[0043] (2) 2.5 mM p-NPG (4-nitrophenol-α-D-pyranoglucopyranoside) solution: Dissolve 7.53 mg of p-NPG in 10.0 mL of water.

[0044] (3) 0.2 U / mL α-glucosidase solution: dilute 2.0 mg of α-glucosidase with 100 mL of buffer and dilute to 0.04 U / mL solution before use.

[0045] (4) 0.2 mol / L sodium carbonate inactivation solution: 2.12 g of anhydrous sodium carbonate dissolved in 100 mL of deionized water.

[0046] 2. Activity determination:

[0047] (1) Sample reaction solution: In a 1.5 mL centrifuge tube, add 100 μL of 0.1 M phosphate buffer, 50 μL of sample solution (10% DMSO as solvent), and 50 μL of 2.5 mM p-NPG solution, mix well, and keep warm at 37°C for 20 min. Add 50 μL of 0.04 U / ml α-glucosidase solution, mix well, and react at 37°C for 30 min. Finally, add 250 μL of 0.2 mol / L Na2CO3 to terminate the reaction. Detect the absorbance (A) at 405 nm using a UV spectrophotometer.

[0048] (2) Sample blank (without enzyme): In a 1.5 mL centrifuge tube, add 100 μL of 0.1 M phosphate buffer, 50 μL of sample solution (10% DMSO as solvent), and 50 μL of 2.5 mM p-NPG solution, mix well, and keep warm at 37°C for 20 min. Add 50 μL of 0.1 M phosphate buffer, mix well, and react at 37°C for 30 min. Finally, add 250 μL of 0.2 mol / L Na2CO3 to terminate the reaction, and measure the absorbance (A0) at 405 nm using a UV spectrophotometer.

[0049] (3) Negative control (without inhibitor): In a 1.5 mL centrifuge tube, add 100 μL of 0.1 M phosphate buffer, 50 μL of 10% DMSO, and 50 μL of 2.5 mM p-NPG solution, mix well, and keep warm at 37°C for 20 min. Add 50 μL of 0.04 U / ml α-glucosidase solution, mix well, and react at 37°C for 30 min. Finally, add 250 μL of 0.2 mol / L Na2CO3 to terminate the reaction. Detect the absorbance at 405 nm using a UV spectrophotometer (B).

[0050] (4) Negative blank (without inhibitors and enzymes): In a 1.5 mL centrifuge tube, add 100 μL of 0.1 M phosphate buffer, 50 μL of 10% DMSO, and 50 μL of 2.5 mM p-NPG solution, mix well, and keep warm at 37°C for 20 min. Add 50 μL of 0.1 M phosphate buffer, mix well, and react at 37°C for 30 min. Finally, add 250 μL of 0.2 mol / L Na2CO3 to terminate the reaction. Detect the absorbance at 405 nm using a UV spectrophotometer (B0).

[0051] The inhibition rate was calculated as shown in formula (1), where A is the absorbance of the sample reaction solution; A0 is the absorbance of the sample blank; B is the absorbance of the negative control; and B0 is the absorbance of the negative blank. The inhibition rate of Peniazaphilone A at different concentrations was determined. Three parallel tests were performed for each concentration. The logarithm of the concentration was used as the abscissa and the inhibition rate was used as the ordinate. The obtained curve was linearly fitted to obtain the equation. The inhibition rate = 50% was substituted into the equation to obtain the half inhibition concentration IC50. Acarbose was used as the positive control to compare the inhibitory activities of the two.

[0052] (1)

[0053] 3. Measurement results:

[0054] The inhibition rate results of acarbose and Peniazaphilone A are shown in Table 3, and the concentration-inhibition rate curves of the two are shown in the attached Figure 1 As shown, the IC of Peniazaphilone A was calculated. 50 The value was 86.8 μM, while the positive control acarbose IC 50 The value is 5.97 mM, which shows that the inhibitory activity of Peniazaphilone A on α-glucosidase is significantly higher than that of acarbose.

[0055] Table 3. Inhibition rate of acarbose and Peniazaphilone A

[0056]

Claims

1. Use of Peniazaphilone A as an α-glucosidase inhibitor, characterized in that: Peniazaphilone A belongs to the class of azotoxin compounds with an isoquinoline quinone structure, and its structure is shown below: 。 2. The method for preparing the compound Peniazaphilone A according to claim 1, characterized in that: The compound is isolated from soil Penicillium sp., and specifically comprises the following steps: (1) Isolation and purification of soil fungi 100 g of soil from the bottom of a stream was placed in a conical flask. The stream was located in a bamboo forest growing area in Sichuan. 100 ml of sterile water was added to the conical flask in a clean bench. The prepared soil sample was then placed in a shaker and shaken overnight at 160 r / min. The sample was then left to stand for fungal separation. The supernatant was cultured on a PDA medium plate. When colonies visible to the naked eye grew, the hyphae at the edge of a single colony was picked up with an inoculation needle and transferred to a new PDA medium. After multiple stages of purification, the purified strain was finally transferred to a PDA slant. (2) Penicillium fermentation The bacteria are cultured on plates → in liquid form → in solid form to obtain the fermented product. (3) Extraction, separation and purification of fungal metabolites S1. Soak the fermented product with methanol, concentrate the supernatant to remove the methanol to obtain a metabolite concentrate, extract the metabolite extract with an ethyl acetate-water system, remove the aqueous phase, and concentrate the organic phase to obtain a crude extract. S2. The crude extract was mixed with silica gel, loaded by dry method, and subjected to silica gel column chromatography. Gradient elution was performed using a system of petroleum ether, ethyl acetate, and methanol. The eluate was concentrated under reduced pressure and combined by TLC detection to obtain 14 components (Fr.A~Fr.N). S3, component Fr.J was purified by silica gel column chromatography to obtain 13 components from Fr.J-1 to Fr.J-13. The eluent ratio was changed to petroleum ether: ethyl acetate: methanol (100:0:0→98:2:0→95:5:0→90:10:0→85:15:0→80:20:0→60:40:0→50:50:0→0:100:0→0:90:10→0:80:20→0:0:100). The S4 and Fr.J-2 components were separated by dextran gel chromatography, with the eluent ratio of dichloromethane: methanol = 1:1, and five components Fr.J-2-1 to Fr.J-2-5 were obtained. The Fr.J-2-3 component was separated by silica gel column chromatography, with dichloromethane: methanol = 40:1 polarity elution, and three components Fr.J-2-3-1 to Fr.J-2-3-3 were obtained, among which the Fr.J-2-3-1 component was purified by gel chromatography with methanol as the eluent to obtain the compound Peniazaphilone A.

3. The preparation method according to claim 2, characterized in that PDA medium was used for plate culture, PDB medium was used for liquid culture, and a medium prepared from rice and PDB was used for solid culture.

4. The preparation method according to claim 2, characterized in that When extracting the fermentation product in step (3), the extraction with methanol is repeated three times, and the extract is concentrated under reduced pressure at 35° C. to recover methanol to obtain a metabolite concentrate. The metabolite concentrate is extracted with ethyl acetate-water to obtain an organic phase, and the extraction is repeated three times. The ethyl acetate phases are combined and the extract is concentrated under reduced pressure to obtain a crude extract.

5. Use of the compound according to claim 1 in the preparation of α-glucosidase inhibitors or hypoglycemic drugs or health foods.

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