A polyoxo-benzene compound, and a preparation method and use thereof

Abstract

The application discloses a polyoxo-phenol compound and a preparation method and application thereof, and the compound is obtained by separating and purifying a fermentation product of a cave fungus. The preparation method is as follows: adopting the cave fungus Aspergillus fumigatus GZWMJZ-152, using a solid-state fermentation mode, and obtaining the compound after fermentation, extraction, and monomer separation and purification. The polyoxo-phenol compound has a new skeleton. Pharmacological experiments show that the compound has a strong antioxidation effect, and can be applied to preparation of an antioxidation stress pharmaceutical preparation.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a polyoxophenol compound, its preparation method, and its uses. Background Technology

[0002] Cave ecosystems possess unique microbial ecosystems. Due to darkness, high humidity, low oxygen concentration, and minimal nutrients, the survival environment of microorganisms in caves is more extreme than that on the surface. This extreme environment may have fostered unique metabolic mechanisms in cave microorganisms, potentially leading to the production of novel bioactive compounds. Previously, the inventors isolated six thiolated compounds with antioxidant activity from the fermentation extract of the cave fungus *Aspergillus fumigatus* GZWMJZ-152 (J.Nat.Prod.2022,85,433-440; Chinese Invention Patent ZL 202210061429.6). Building upon this foundation, this invention further investigates the fermentation extract of this strain, isolating and identifying two novel polyoxyphenolic compounds with strong antioxidant activity. Oxidative stress is a negative effect produced by free radicals in the body, which can lead to a variety of diseases, such as damaging pancreatic β cells and causing insulin resistance, leading to the development of diabetes. It can also accelerate atherosclerosis in cardiovascular diseases. Therefore, the discovery of novel antioxidant molecules is of great significance in combating diseases caused by oxidative stress. Summary of the Invention

[0003] The purpose of this invention is to extract, isolate, and purify polyoxophenolic compounds from the fermentation products of cave fungi, which possess antioxidant activity and can be used in anti-oxidative stress drugs. This invention provides a polyoxophenolic compound, its preparation method, and its uses. The objective of this invention and the solution to its main technical problem are achieved by the following technical solution: a polyoxophenolic compound, which is isolated and purified from the fermentation products of cave fungi, and its structural formulas are (1) and (2):

[0004]

[0005] The preparation method of compounds 1 and 2 includes the following steps:

[0006] A. Preparation of the strain: The purified culture medium used consists of 200-300 parts by weight of potato, 20-25 parts by weight of glucose, and 15-20 parts by weight of agar. Add the mixture to 1L of purified water, adjust the pH to 6.5-7.5, prepare a slant, and inoculate it with the mycelium of Aspergillus fumigatus GZWMJZ-152 at room temperature. Incubate at 28-35℃ for 3 days to obtain the strain.

[0007] B. Inoculation: Using solid-state fermentation, each conical flask is filled with 50-100 parts rice and 50-100 parts purified water. The strain obtained in step A is inoculated, and the fermentation medium is obtained after static culture at 28℃ for 30 days.

[0008] C. Extraction: Soak the fermentation medium described in step B in ethyl acetate for 48-72 hours, then extract with a stirrer 3-5 times, 30-40 minutes each time. Combine the supernatants and recover ethyl acetate in a rotary evaporator to obtain crude extract.

[0009] D. Monomer separation and purification: The crude extract obtained in step C is separated by column chromatography to obtain compounds with new structures of formula (1) and formula (2); the chromatography separation method includes silica gel column chromatography, gel column chromatography, and semi-preparative high performance liquid chromatography.

[0010] The application of the aforementioned polyoxyphenolic compounds in the preparation of drugs against oxidative stress.

[0011] A pharmaceutical composition comprising a polyoxophenolic compound and pharmaceutically acceptable excipients.

[0012] The pharmaceutical composition contains 0.1-99% by mass of polyoxyphenolic compounds, with the remainder being pharmaceutical carriers or excipients.

[0013] Through ongoing and in-depth research on the cave fungus *Fumigatus* GZWMJZ-152, the inventors obtained two polyoxophenolic compounds, 1 and 2. These prepared polyoxophenolic compounds possess antioxidant properties and can be used in anti-oxidative stress drugs. Attached Figure Description

[0014] Figure 1 The structural formulas of compounds 1 and 2 of this invention are as follows.

[0015] Figure 2 This is the high-resolution mass spectrum of compound 1 of the present invention.

[0016] Figure 3 This is the high-resolution mass spectrum of compound 2 of the present invention.

[0017] Figure 4 This is the 1H NMR spectrum of compound 1 of the present invention.

[0018] Figure 5 This is the 1H NMR spectrum of compound 2 of the present invention.

[0019] Figure 6 This is the carbon NMR spectrum of compound 1 of the present invention.

[0020] Figure 7 This is the carbon NMR spectrum of compound 2 of the present invention. Detailed Implementation

[0021] Example 1:

[0022] (I) Preparation of compounds 1 and 2

[0023] (1) Preparation of strains

[0024] The purified culture medium used consisted of 200 parts potato, 20 parts glucose, 20 parts agar, and 1 L of purified water. The medium was sterilized at high temperature, prepared as slant culture, and placed at room temperature. When no other microorganisms grew, the slant culture was inoculated with mycelia of the fungus *Aspergillus fumigatus* GZWMJZ-152 and incubated statically at 28°C.

[0025] (2) Vaccination

[0026] Using solid-state fermentation, 100 conical flasks were prepared, each containing 100 parts rice and 100 parts purified water. After sterilization at 121°C for 30 minutes, the above-mentioned bacterial strain was inoculated and cultured statically at 28°C for 30 days.

[0027] (3) Extraction

[0028] The fermentation broth and mycelium obtained from the above fermentation were soaked in ethyl acetate for 48 hours, and then extracted in batches using a stirrer, with each batch extracted three times, each extraction lasting 30 minutes. After standing, the supernatants were combined and the ethyl acetate was recovered in a rotary evaporator to obtain 620.1 g of crude extract.

[0029] (4) Monomer separation and purification

[0030] After dissolving the crude extract, the sample was mixed with 100-200 mesh silica gel and subjected to silica gel column chromatography. Elution was performed using a gradient of petroleum ether / ethyl acetate (0-100%) and dichloromethane / methanol (0-50%). TLC detection was performed, and identical fractions were combined, yielding a total of 7 fractions. Fraction 2 was further subjected to silica gel column chromatography with a petroleum ether:ethyl acetate gradient elution from 20:1 to 5:1, yielding another 7 fractions. Fractions 2-2 were further separated by semi-preparative high-performance liquid chromatography (65% methanol / water) to obtain compound 2 (12.3 mg). Fraction 6 was further subjected to silica gel column chromatography with a dichloromethane:methanol gradient elution from 50:1 to 2:1, yielding 8 fractions. Fractions 6-6 were further separated by preparative thin-layer chromatography with dichloromethane:methanol at a 25:1 eluent. The concentrate was then further separated by semi-preparative high-performance liquid chromatography (55% methanol / water) to obtain compound 1 (15.7 mg).

[0031] (II) Structural identification of compounds 1 and 2

[0032] The structures of compounds 1 and 2 were determined through comprehensive analysis using high-resolution mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, and nuclear magnetic resonance. Their physicochemical properties are as follows:

[0033] Compound 1: Colorless crystals; Molecular formula C 26 H 28 O 13 Molecular weight 548; UV(MeOH)λmax(logε)202(5.92),286(0.99)nm; IR(KBr)vmax 3425,2923,2856,2357,2333,1731,1631,1602,1540,1403,1342,1219,1112,1055,898,669cm -1 HRESIMS m / z 571.1422[M+Na] + The specific rotation value is close to 0, and compound 1 is a racemic mixture. Its 1H NMR and 1C NMR data are shown in Table 1.

[0034] Compound 2: Purple powder; Molecular formula C 18 H 20 O8; Molecular weight 364; UV(MeOH)λmax(logε) 210(3.39), 280(1.65)nm; IR(KBr)νmax 3476, 1735, 1672, 1610, 1497, 1441, 1352, 1191, 1104, 1071, 1034, 1017, 903, 885, 832cm -1 HRESIMS m / z 387.1047[M+Na] + The specific rotation value is close to 0, and compound 2 is a racemic mixture. Its 1H NMR and 1C NMR data are shown in Table 1.

[0035] Table 1. 1H and 1C NMR spectra of compounds 1 and 2

[0036]

[0037] Example 2

[0038] To further verify the beneficial effects of the compounds described in this invention, ORAC antioxidant activity tests were conducted on compounds 1 and 2. The specific experiments are as follows:

[0039] The main solutions used in this experiment included 0.153 mol / L 2,2'-azobisisobutylamidine hydrochloride solution (AAPH), 81.6 nmol / L fluorescein solution (FL), five series of positive control drugs Trolox (concentrations of 50, 25, 12.5, 6.25, and 3.125 μmol / L), and the test samples. All solvents were dissolved in 75 mmol / L phosphate buffer (pH 7.4) (PBS buffer). The experimental method involved setting up a negative control group, a blank control group, and a positive control group in a 96-well plate. For each of the test sample groups, three replicates were prepared. 25 μL of PBS buffer, Trolox, and the test sample were added to each group. Then, 150 μL of FL was added to each well. The 96-well plate was incubated at 37°C for 10 min. After incubation, 25 μL of PBS buffer was added to the negative control group, and 25 μL of AAPH was added to the remaining groups. The plates were immediately placed in a multi-mode microplate reader for fluorescence detection. The instrument was set to excitation wavelength of 485 nm and emission wavelength of 530 nm, with one detection per minute for approximately 1 hour. The relative fluorescence intensity f is equal to the ratio of the measured fluorescence value to the initial fluorescence value. The area under the fluorescence decay curve (AUC) was calculated using an approximate integral method based on the relative fluorescence intensity. The formula is: AUC = 0.5 + f1 + ... + f59 + 0.5 * f60, where f... i Represents the relative fluorescence intensity measured at time i minutes. Net AUC is the difference between the sample AUC and the blank AUC. A regression equation is performed on net AUC using the time t of Trolox fluorescence measurement, Y( netAUC ) = aX( μM The final ORAC value was calculated using a regression equation between Trolox concentration and net AUC, and expressed as Trolox equivalents per micromole of sample, in μmol TE / μmol. The formula is as follows:

[0040] Relative ORAC value = [(AUC) 样品 -AUC 空白 ) / (AUC Trolox -AUC 空白 *(Trolox molar concentration μmol / L) / (sample molar concentration μmol / L)

[0041] The test results showed that the ORAC values ​​of compounds 1 and 2 were 0.88 and 2.82 μM TE / μM, respectively. Under these experimental conditions, the antioxidant capacity of compound 1 was comparable to that of vitamin E, while the antioxidant capacity of compound 2 was 2.82 times that of vitamin E.

[0042] These embodiments should be understood as illustrative only and not as limiting the scope of protection of the invention. After reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent changes and modifications also fall within the scope defined by the claims of this invention.

Claims

1. A polyoxyphenolic compound, characterized in that, The compound has the structure shown in either Formula 1 or 2: 。 2. The use of a polyoxophenolic compound as described in claim 1 in the preparation of drugs against oxidative stress.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the polyoxophenolic compound of claim 1 and pharmaceutically acceptable excipients.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutical composition contains 0.1-99% by mass of the polyoxyphenolic compound of claim 1, with the remainder being a pharmaceutical carrier or excipient.

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