Compounds with antioxidant activity, methods of preparation and uses thereof
By optimizing culture conditions through fungal co-culture, compounds with antioxidant activity were prepared, solving the problems of scarce natural product resources and difficulty in reproducing active ingredients, and realizing the application and sustainable production of the compounds in the treatment of oxidative damage diseases.
Patent Information
- Application Number
- CN202410018904.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-05
- Publication Date
- 2025-11-21
- Estimated Expiration
- 2044-01-05
AI Technical Summary
Existing technologies struggle to effectively utilize natural products to develop antioxidants, as resources are scarce and active ingredients are difficult to reproduce. Furthermore, microbial co-culture methods have not been widely applied in antioxidant development.
By employing a fungal co-culture method and optimizing culture conditions, and through the co-culture of Penicillium and Alternaria, the fermentation products were used to extract metabolites and prepare compounds with antioxidant activity.
Compounds with concentration-dependent antioxidant activity were obtained, which are suitable for treating oxidative damage-related diseases such as neurodegenerative diseases, cardiovascular diseases and malignant tumors. The process is reproducible and sustainable, and does not cause waste of resources.
Smart Images
Figure CN117903079B_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of pharmaceutical technology, and in particular to compounds with antioxidant activity, their preparation methods, and their applications. Background Technology
[0002] When the body is subjected to various harmful stimuli, it often leads to the excessive production of highly reactive molecules such as reactive oxygen species (ROS) and reactive nitrogen species (RNS). The degree of oxidation exceeds the clearance of oxidants, resulting in an imbalance between the oxidation and antioxidant systems, thus causing tissue damage. Excessive ROS can directly or indirectly oxidize or damage DNA, proteins, and lipids, inducing gene mutations, protein denaturation, and lipid peroxidation. These reactions cause physiological and pathological responses in cells and tissues, leading to various diseases such as neurodegenerative diseases, cardiovascular and cerebrovascular diseases, diabetes, and malignant tumors.
[0003] Antioxidants combat oxidative stress and regulate the body's redox balance, playing a crucial role in maintaining human health. Endogenous antioxidant defense systems, such as enzymatic action, metal chelation, and free radical scavenging, are responsible for maintaining this dynamic balance. When exposed to pathogens or other harmful stimuli, the body's endogenous antioxidant defense system often fails to promptly remove excess reactive oxygen species (ROS), necessitating the timely removal of ROS by effective exogenous antioxidants.
[0004] Natural products are an important resource for small molecule drugs. However, mining natural products from traditional plants has many drawbacks, such as resource scarcity and minimal dose-response effects. Furthermore, the composition and content of secondary metabolites often vary significantly among the same plant species from different locations, making it difficult to reproduce the active ingredients. Since the 20th century, scientists have conducted extensive research on the secondary metabolites of microorganisms, discovering many bioactive metabolites. In natural environments, antagonistic and synergistic effects among different microorganisms are very common, resulting in a rich variety of metabolites. Therefore, microbial co-culture can recreate or simulate natural microbial growth conditions, stimulating the interaction of different microbial metabolic enzymes or metabolites, and activating silent gene clusters in individual microorganisms, thereby maximizing the extraction of their rich and diverse bioactive substances. This application employs a fungal co-culture method, optimizing culture conditions to mine the metabolites and bioactivity of fermentation products from co-cultured fungi, which is of great significance for developing novel drugs to treat diseases caused by oxidative damage. Summary of the Invention
[0005] In view of this, this application provides compounds with antioxidant activity, preparation methods and application technologies to address the aforementioned technical deficiencies to a certain extent.
[0006] Firstly, embodiments of this application disclose compounds with antioxidant activity, said compounds including, The compound represented by Formula I; or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof.
[0007] Secondly, embodiments of this application disclose an formulation for treating diseases caused by oxidative damage, characterized in that the formulation comprises the compound described in the first aspect, and pharmaceutically acceptable excipients.
[0008] The pharmaceutically acceptable excipients include at least one of diluents, carriers, and excipients.
[0009] "Excipient" refers to a pharmaceutically acceptable material, composition, or medium that contributes to imparting form or consistency to a pharmaceutical composition. Each excipient must be compatible with the other components of the pharmaceutical composition when mixed to avoid interactions that would significantly reduce the efficacy of the compound when administered to a patient, and to avoid interactions that would render the pharmaceutical composition pharmaceutically unacceptable. Furthermore, each excipient must have sufficiently high purity to be pharmaceutically acceptable.
[0010] Suitable pharmaceutically acceptable excipients will vary depending on the specific dosage form chosen. Additionally, suitable pharmaceutically acceptable excipients can be selected for their specific functions in the composition. For example, certain pharmaceutically acceptable excipients may be selected because they have the ability to promote the production of a homogeneous dosage form. Certain pharmaceutically acceptable excipients may be selected because they have the ability to produce a stable dosage form. Certain pharmaceutically acceptable excipients may be selected because they facilitate the carrying or transport of one or more compounds of this application from one organ or part of the body to another organ or part of the body once administered to a patient. Certain pharmaceutically acceptable excipients may be selected because they have the ability to enhance patient compliance.
[0011] Suitable pharmaceutically acceptable excipients include the following types: diluents, fillers, binders, disintegrants, lubricants, flow aids, granulators, coating agents, wetting agents, solvents, solubilizers, suspending agents, emulsifiers, sweeteners, flavoring agents, flavor masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, thickeners, antioxidants, preservatives, stabilizers, surfactants, and buffers. As those skilled in the art will understand, depending on the amount of excipient and other components present in the formulation, some pharmaceutically acceptable excipients can perform more than one function and can serve alternative functions.
[0012] The formulations provided in this application can be in forms suitable for use in the following ways: oral administration (e.g., as tablets, capsules, pouches, pills, lozenges, powders, syrups, elixirs, suspensions, solutions, emulsions, sachets, and capsules), topical administration (e.g., as creams, ointments, emulsions, solutions, pastes, sprays, foams, and gels), transdermal administration (e.g., via transdermal patches), inhalation administration (e.g., as dry powders, aerosols, suspensions, and solutions), inhalation administration (e.g., as fine powders), or parenteral administration (e.g., as sterile aqueous or oily solutions for intravenous, subcutaneous, intramuscular, intraperitoneal, or intramuscular administration, or as suppositories for rectal administration).
[0013] Thirdly, embodiments of this application disclose a method for preparing the compound described in the first aspect. The preparation method includes the following steps: obtaining activated colonies containing *Penicillium* sp. and *Alternaria* sp.; inoculating the colonies into a PDB fermentation medium containing 0.012 mg / mL of cork oxime acid for fermentation to obtain a fermentation broth; and extracting and separating the fermentation broth to obtain the compound; wherein *Penicillium* sp. HUBU0120 was deposited at the China Center for Type Culture Collection on April 19, 2021, accession number: CCTCC NO: M2021412; and *Alternaria* sp. HUBU0122 was deposited at the China Center for Type Culture Collection on April 19, 2021, accession number: CCTCC NO: M 2021414.
[0014] In the embodiments of this application, the activated colonies containing Penicillium and Alternaria were obtained by inoculating the strains into PDA medium and culturing at 25°C.
[0015] In this embodiment of the application, the fermentation medium is PDB.
[0016] In this embodiment of the application, the extraction and separation steps include: obtaining a crude extract, which is obtained by extracting the fermentation broth with ethanol; obtaining a refined extract, which is obtained by sequentially extracting the crude extract with ethyl acetate, methanol and petroleum ether; and obtaining the compound, which is obtained by purifying the refined extract by membrane filtration and reversed-phase high-performance preparative chromatography, wherein the compound is the compound described in the first aspect.
[0017] In this embodiment of the application, the steps of obtaining the compound specifically include: obtaining a first component, which is obtained by filtering the refined extract through a filter membrane; obtaining a second component, which is obtained by loading the first component onto a reversed-phase high-performance liquid chromatography column and collecting the eluent, wherein the second component includes a compound as shown in Formula I.
[0018] Fourthly, embodiments of this application disclose the use of the compounds described in the first aspect or the compounds prepared by the preparation method described in the second aspect in the preparation of drugs for diseases caused by oxidative damage.
[0019] Compared with the prior art, this application has at least the following beneficial effects:
[0020] This application relates to compounds with antioxidant activity, their preparation methods, and their applications. By employing a strategy of co-culturing two strains of bacteria, and by modifying the culture medium, culture conditions, and mixing ratios, epigenetic regulation of their silenced biosynthetic genes is achieved. This maximizes the potential of microorganisms to synthesize secondary metabolites, enabling scale-up cultivation based on reproducible and sustainable processes without resource waste.
[0021] Secondly, the compounds provided in this application all possess antioxidant activity, which is concentration-dependent. These compounds are expected to be developed into drugs for treating diseases caused by oxidative damage, such as neurodegenerative diseases, cardiovascular diseases, diabetes, and malignant tumors.
[0022] In addition, this application only requires reviving and subculturing the frozen strains according to the experimental protocol, and culturing them under optimized culture conditions to infinitely scale up the fermentation. Furthermore, the yield of compounds can be purposefully controlled by adjusting the total amount of fermentation products, ultimately obtaining the target product with antioxidant activity. Attached Figure Description
[0023] Figure 1 This is a relative comparison diagram of the liquid components provided in the embodiments of this application.
[0024] Figure 2 SPSS principal component factor plot provided for embodiments of this application.
[0025] Figure 3 The X-single crystal structure of the compound of Formula I provided in the embodiments of this application is shown in the figure.
[0026] Figure 4 The structure diagram of the compound shown in Formula I provided in the embodiments of this application is shown.
[0027] Figure 5 The free radical scavenging activity (DPPH method) of the PA.PDB.S group extracts provided in the embodiments of this application.
[0028] Figure 6 The free radical scavenging activity (ABTS method) of the PA.PDB.S group extracts provided in the embodiments of this application.
[0029] Figure 7Total antioxidant capacity (FRAP method) of PA.PDB.S group extracts provided in the embodiments of this application. Detailed Implementation
[0030] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.
[0031] Preparation of compounds as shown in Formula I
[0032] 1. Strains
[0033] The compounds shown in Formula I disclosed in the embodiments of this application are obtained by extracting and separating their secondary metabolites through fungal fermentation culture.
[0034] The bacterial strains involved in the embodiments of this application come from the following sources:
[0035] The fungus Penicillium sp. HUBU0120 was deposited at the China Center for Type Culture Collection on April 19, 2021, with accession number CCTCC NO: M2021412.
[0036] Alternaria sp. HUBU0122 was deposited at the China Center for Type Culture Collection on April 19, 2021, with accession number CCTCC NO: M2021414.
[0037] 2. Culture medium
[0038] PDA medium is short for Potato Dextrose Agar (Medium), which corresponds to the English words for potato, glucose, and agar, respectively. The formula is: 200 g / L potato, 20 g / L glucose, and 15–20 g / L agar.
[0039] PDB medium is a liquid PDA medium without agar, used for the liquid culture of fungi, etc.
[0040] 3. Activation of bacterial strains
[0041] Pour the prepared PDA culture medium into a blue-capped bottle, place it in an autoclave set to 121℃ for 30 minutes, irradiate with ultraviolet light for 30 minutes to sterilize, and wait for it to cool to about 50℃ before pouring it into plates.
[0042] 4. Fermentation culture
[0043] In one embodiment (P.PDA), activated Penicillium HUBU0120 was inoculated onto a PDA plate at a 10% inoculum and cultured at a constant temperature of 25°C for 5-7 days.
[0044] In one embodiment (P.PDB), activated Penicillium HUBU0120 was inoculated into PDB culture medium at a 10% inoculum and cultured in a constant temperature shaker at 25°C for 5-7 days at a rotation speed of 180 rpm.
[0045] In one embodiment (P.PDB.S), activated Penicillium HUBU0120 was inoculated at a 10% inoculum into a PDB culture medium containing 0.012 mg / mL cork oxime acid, and cultured at a constant temperature of 25°C with a shaking speed of 180 rpm for 5-7 days.
[0046] In one embodiment (P.PDB.5), activated Penicillium HUBU0120 was inoculated at a 10% inoculum into PDB culture medium containing 0.012 mg / mL 5-aza-2'-deoxycytidine and cultured at 25°C with a shaker at 180 rpm for 5-7 days.
[0047] In one embodiment (A.PDA), activated Alternaria HUBU0122 was inoculated onto a PDA plate at a 10% inoculum and cultured at a constant temperature of 25°C for 5-7 days.
[0048] In one embodiment (A.PDB), activated Alternaria HUBU0122 was inoculated into PDB culture medium at a 10% inoculum and cultured in a constant temperature shaker at 25°C at a rotation speed of 180 rpm for 5-7 days.
[0049] In one embodiment (A.PDB.S), activated Alternaria HUBU0122 was inoculated at a 10% inoculum into PDB culture medium containing 0.012 mg / mL cork oxime acid and cultured at 25°C in a shaker at 180 rpm for 5-7 days.
[0050] In one embodiment (A.PDB.5), activated Alternaria HUBU0122 was inoculated at a 10% inoculum into PDB culture medium containing 0.012 mg / mL 5-aza-2'-deoxycytidine and cultured at 25°C with a shaker at 180 rpm for 5-7 days.
[0051] In one embodiment (PA.PDA), activated Penicillium HUBU0120 and Alternaria HUBU0122 are inoculated onto a PDA plate at an inoculation rate of 5% each and incubated at a constant temperature of 25°C for 5-7 days.
[0052] In one embodiment (PA.PDB), activated Penicillium HUBU0120 and Alternaria HUBU0122 were inoculated into PDB culture medium at an inoculation rate of 5% each, and cultured in a constant temperature shaker at 25°C and 180 rpm for 5-7 days.
[0053] In one embodiment (PA.PDB.S), activated Penicillium HUBU0120 and Alternaria HUBU0122 were inoculated at a rate of 5% each into a PDB culture medium containing 0.012 mg / mL of cork oxime acid, and cultured at 25°C in a shaker at 180 rpm for 5-7 days.
[0054] In one embodiment (PA.PDB.5), activated Penicillium HUBU0120 and Alternaria HUBU0122 were inoculated at a rate of 5% each into PDB culture medium containing 0.012 mg / mL 5-aza-2'-deoxycytidine and cultured at 25°C with a shaking device at 180 rpm for 5-7 days.
[0055] Table 1. Culture methods and conditions for different groups
[0056] Serial Number Group Name Training methods strains culture medium additive Additive concentration (mg / mL) 1 P.PDA Individual cultivation Penicillium PDA none - 2 P.PDB Individual cultivation Penicillium PDB none - 3 P.PDB.S Individual cultivation Penicillium PDB Soft oxime 0.012 mg / mL 4 P.PDB.5 Individual cultivation Penicillium PDB 5-aza-2'-deoxycytidine 0.012 mg / mL 5 A.PDA Individual cultivation Alternaria PDA none - 6 A.PDB Individual cultivation Alternaria PDB none - 7 A.PDB.S Individual cultivation Alternaria PDB Soft oxime 0.012 mg / mL 8 A.PDB.5 Individual cultivation Alternaria PDB 5-aza-2'-deoxycytidine 0.012 mg / mL 9 PA.PDA Joint training Penicillium, Alternaria PDA none - 10 PA.PDB Joint training Penicillium, Alternaria PDB none - 11 PA.PDB.S Joint training Penicillium, Alternaria PDB Soft oxime 0.012 mg / mL 12 PA.PDB.5 Joint training Penicillium, Alternaria PDB 5-aza-2'-deoxycytidine 0.012 mg / mL
[0057] Note: The meanings of the group names are as follows: P represents Penicillium sp. HUBU0120, A represents Alternaria sp. HUBU0122, PA represents co-culture of Penicillium sp. HUBU0120 and Alternaria sp. HUBU0122, PDA represents PDA medium, PDB represents PDB medium, S represents cork oxime acid, and 5 represents 5-aza-2'-deoxycytidine.
[0058] 5. Extraction and separation
[0059] Cultured according to the methods described in Table 1, each group was extracted with ethanol after culture. Each extraction lasted 12 hours, for a total of 8 extractions. The extract was concentrated under reduced pressure to obtain the total extract. The total extract was then further extracted: first, with ethyl acetate and water in a 1:1 ratio for 7 extractions; then, the ethyl acetate fraction was extracted with 95% methanol and petroleum ether in a 1:1 ratio for 7 extractions to obtain the 95% methanol fraction.
[0060] 6. Extract Analysis
[0061] After filtration through a filter membrane, coarse materials from different culture conditions were passed through a preparative COSMOSIL Packed Column (C500-C50) by reverse-phase high-performance liquid chromatography. 18 MS-II (4.6 × 250 mm, 5 μm particle), mobile phase methanol:water = 80:20, flow rate 1 mL / min, relative proportions of each component liquid... Figure 1 As shown.
[0062] 7. SPSS Principal Component Factor Analysis
[0063] 1.00 mg of each of the purified extracts from the two strains under the 12 culture methods listed in Table 1 were weighed and diluted with methanol to a concentration of 1.00 × 10⁻⁶ mg. -9 Mass spectrometry (MS) analysis was performed on the samples at mg / mL using methanol:water = 80:20. The mass spectrometry data were saved in txt format, and principal component factor analysis was performed on the mass spectrometry data using SPSS software. The results are as follows: Figure 2 As shown.
[0064] The analysis results showed that different components existed among the groups, with the two components with the highest proportions being principal component 1 and principal component 2. Principal component 1 had a variance percentage of 98.44% and an accumulation of 98.44%, while principal component 2 had a variance percentage of 0.58 and an accumulation of 99.02%. Further analysis of these two principal components revealed the groups containing the most of these two components. The results showed that principal component 1 was the most abundant in the PA.PDB.S group, with a content of 0.997. In the A.PDA group, principal component 2 was the most abundant, with a content of 0.217. Therefore, the culture method and conditions for the PA.PDB.S group were selected for scale-up fermentation. After extraction, separation, and purification of the fermentation product, the compound shown in Formula I was obtained.
[0065] Structural identification of the compound shown in Formula I
[0066] The absolute configuration of the compound shown in Formula I was obtained by nuclear magnetic resonance (NMR), computational electron circular dichroism (ECD), and X-ray diffraction (X-Ray) methods, respectively.
[0067] 1. Nuclear magnetic resonance
[0068] Compound represented by Formula I: white powder; HRESIMS m / z 485.2477 [M+H] + (calcd for C 27 H 36 N2O4S + ,485.2469); [α] 2 D0=+61.9(c 0.14,CH3OH); UV(CH3OH)λ max(logε)=263(4.42),324(4.09)nm; IR(KBr)ν max 3445,2967,2843,2378,1869,1645,1541,1055,1032,1015,419cm –1 ; 1 H and 13 The C NMR data are shown in Table 2.
[0069] Table 2. Compounds shown in Formula I 1 H NMR and 13 C NMR (CD3OD, δin ppm, J in Hz).
[0070]
[0071] 2. X-ray single-crystal diffraction analysis
[0072] X-ray analysis of the copper target of the compound represented by Formula I determined the absolute configuration of the compound. The single-crystal structure diagram is shown below. Figure 3 Absolute configuration see Figure 4 .
[0073] Antioxidant activity test of fungal metabolites
[0074] 1. Antioxidant activity test of extracts from each group
[0075] The ethanol extracts of P.PDA, P.PDB, P.PDB.S, P.PDB.5, A.PDA, A.PDB, A.PDB.S, A.PDB.5, PA.PDA, PA.PDB, PA.PDB.S, and PA.PDB.5 were prepared into 100 μg / mL test solutions using 80% methanol. The antioxidant activity of each group's extracts was tested using a DPPH free radical scavenging ability kit (Nanjing Jiancheng Bioengineering Institute, catalog number A153-1-1).
[0076] The DPPH radical scavenging rate was calculated using the following formula based on the absorbance (OD) values (at 517 nm) of the control tube, test tube, and blank tube: DPPH radical scavenging rate (%) = [1 - (A... 测定 -A 对照 ) / A 空白 ×100%. The DPPH free radical scavenging rates of the extracts of each group are shown in Table 3. Among them, the PA.PDB.S group extract has the highest DPPH free radical scavenging rate.
[0077] Table 3. DPPH free radical scavenging rate of extracts from each group
[0078] Serial Number Group Name Clearance rate (%) 1 P.PDA 44.54％ 2 P.PDB 49.00％ 3 P.PDB.S 55.41％ 4 P.PDB.5 38.18％ 5 C.PDA 40.02％ 6 C.PDB 34.17％ 7 C.PDB.S 34.78％ 8 C.PDB.5 34.78％ 9 PC.PDA 32.16％ 10 PC.PDB 52.62％ 11 PC.PDB.S 55.74％ 12 PC.PDB.5 47.83％
[0079] 2. Antioxidant capacity test of PA, PDB, and S group extracts
[0080] Since the PA.PDB.S extract showed the highest scavenging rate of DPPH free radicals and the best antioxidant effect, the extract from this group was subsequently selected for further antioxidant capacity testing.
[0081] (1) DPPH free radical scavenging rate test
[0082] Take 2.00 mg of the component sample and dilute it with 80% methanol to concentrations of 3.91, 7.82, 15.64, 31.28, and 62.55 μg / mL. The DPPH free radical scavenging rate is obtained by using step 2 above.
[0083] Using 1,1-diphenyl-2-trinitrophenylhydrazine radical as a control, the results showed that the antioxidant capacity of this sample increased with increasing concentration. Figure 5 As shown. The IC50 of its free radical scavenging rate was fitted using SPSS software. 50 The value was 1.54 μg / mL.
[0084] (2) ABTS free radical scavenging rate test
[0085] Total antioxidant capacity (T-AOC) was measured using a kit (Nanjing Jiancheng Bioengineering Institute, catalog number A015-2-1). A standard curve was constructed with the OD values of the standards on the x-axis and the corresponding standard concentrations on the y-axis. The standard curve was generated using Excel. The measured OD values of the extract were substituted into the linear equation of the standard curve to obtain the concentration of the extract equivalent to the standard. Trolox was used as the standard for total antioxidant capacity determination. The antioxidant capacity of the extract was expressed as Trolox-Equivalent Anyioxidant Capacity (TEAC).
[0086] Take 4.00 mg of the extract from this group and dilute it with distilled water to concentrations of 4.00, 2.00, 1.00, 0.50, 0.25, 0.13, and 0.06 mg / mL. Detect the OD value at a wavelength of 405 nm and use the aforementioned calculation method to obtain the free radical scavenging rate of the sample at different concentrations.
[0087] Using TEAC as a positive control, the results showed that the antioxidant capacity of this sample increased with increasing concentration. Figure 6 As shown. The IC50 of its free radical scavenging rate was fitted using SPSS software. 50 The value was 1.69 mg / mL.
[0088] (3) FRAP method for antioxidant activity testing
[0089] Total antioxidant capacity (T-AOC) was measured using a kit (Nanjing Jiancheng Bioengineering Institute, catalog number A015-3-1). OD values were read from blank wells, standard wells, and assay wells at a wavelength of 593 nm, and the OD value of the blank well was subtracted from the OD value of each well. A standard curve was constructed with the OD value of the standard on the x-axis and the corresponding standard concentration on the y-axis, and the linear equation of the standard curve was obtained. The OD value of the extract after subtracting the blank was substituted into the linear equation to calculate the concentration of the sample equivalent to the standard.
[0090] Take 4.00 mg of the component sample and dilute it with distilled water to concentrations of 4.00, 2.00, 1.00, 0.50, 0.25, 0.13, 0.06, and 0.03 mg / mL. Detect the OD value at a wavelength of 405 nm and use the calculation method in 4.3 to obtain the antioxidant capacity of the sample at different concentrations.
[0091] Using FeSO4-7H2O as a positive control, the results showed that the antioxidant capacity of this sample increased with increasing concentration. Figure 7 As shown. The IC50 of its total antioxidant capacity was fitted using SPSS software. 50 The value was 1.06 mg / mL.
[0092] Comparison of antioxidant activity tests of different compounds
[0093] The antioxidant capacity of the compound shown in Formula I was tested using the three kits described above, and the IC50 of its antioxidant activity was calculated using SPSS analysis software. 50 Values. Table 4 shows the DPPH (ICP-values) of compound (Formula I), 2,6-di-tert-butyl-4-methylphenol (BHT), and quinoline dimethacrylate (Trolox), respectively. 50 μM), ABTS (IC 50 In the table, " / " indicates that it was not detected. As shown in Table 4, this compound (Formula I) has good antioxidant activity, while BHT did not show ABTS (IC50, μM) and FRAP (FeSO4 value), and Trolox did not show anti-DPPH activity.
[0094] Table 4. Results of antioxidant activity tests for compounds and standards.
[0095] compound <![CDATA[DPPH(IC 50 ,μM)]]> <![CDATA[ABTS(IC 50 ,μM)]]> <![CDATA[FRAP(FeSO4 value)]]> Compound Formula I 37.74 49.64 1.77 2,6-Di-tert-butyl-4-methylphenol (BHT) 91.35 / / Trolox (quinoline dimethacrylate) / 101.23 1.80
[0096] In summary, this application utilizes the co-culture of Penicillium sp. HUBU0120 and Alternaria sp. HUBU0122, and optimizes the culture conditions by adding cork oxime acid, thereby producing crude extracts and monomeric compounds with antioxidant activity. This application allows for process reproducibility and scale-up cultivation based on sustainable development, minimizing resource waste.
[0097] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.
Claims
1. A compound with antioxidant activity, characterized in that, The compound is The compound of Formula I or a pharmaceutically acceptable salt thereof.
2. An antioxidant preparation, characterized in that, The formulation comprises the compound of claim 1, and pharmaceutically acceptable excipients.
3. The formulation according to claim 2, characterized in that, The pharmaceutically acceptable excipients include at least one of diluents and excipients.
4. A method for preparing the compound according to claim 1, characterized in that, include: Activated colonies containing Penicillium and Alternaria were obtained, respectively. The colonies were inoculated into PDB fermentation medium containing 0.012 mg / mL cork oxime acid for fermentation to obtain fermentation broth; as well as The fermentation broth was extracted and separated to obtain the compound; Among them, Penicillium sp. HUBU0120 was deposited at the China Center for Type Culture Collection on April 19, 2021, with accession number: CCTCC NO: M 2021412. Alternaria sp. HUBU0122 was deposited at the China Center for Type Culture Collection on April 19, 2021, with accession number CCTCC NO: M 2021414.
5. The preparation method according to claim 4, characterized in that, Activated colonies containing Penicillium and Alternaria were obtained by inoculating the strains into PDA medium and culturing at 25°C.
6. The preparation method according to claim 4, characterized in that, The fermentation medium is PDB medium containing 0.012 mg / mL cork oxime acid.
7. The preparation method according to claim 4, characterized in that... The extraction and separation steps, involving the co-culturing of two bacterial strains, include: A crude extract is obtained by extracting a solid culture medium or fermentation broth with ethanol. A refined extract is obtained by sequentially extracting the crude extract with ethyl acetate, methanol, and petroleum ether; and The compound is obtained by purifying the refined extract through membrane filtration and reversed-phase high-performance preparative chromatography, and the compound is the compound as described in claim 1.
8. The preparation method according to claim 7, characterized in that, The steps for obtaining the compound specifically include: The first component is obtained by filtering the refined extract through a filter membrane; A second component is obtained by loading the first component onto a reversed-phase high-performance liquid chromatography column and collecting the eluent. The second component includes compounds as shown in Formula I.
9. The use of the compound of claim 1, or the compound prepared by the method of any one of claims 4 to 8, in the preparation of a drug for treating diseases caused by oxidative damage.