CN115466268B - Oxaanthraquinone compound with antibacterial and anti-inflammatory activity, preparation method and application thereof, and pharmaceutical composition - Google Patents
Oxaanthraquinone compound with antibacterial and anti-inflammatory activity, preparation method and application thereof, and pharmaceutical composition Download PDFInfo
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Abstract
The invention provides an oxaanthraquinone compound with antibacterial and anti-inflammatory activity, a preparation method and application thereof, and a pharmaceutical composition, and belongs to the technical field of pharmaceutical preparation. The oxaanthraquinone compound disclosed by the invention is an oxaanthraquinone compound with a furopyranyl ring structure, so that the oxaanthraquinone compound has remarkable antibacterial and anti-inflammatory activity, can be applied to preparation of antibacterial and anti-inflammatory drugs, enriches the diversity of the oxaanthraquinone compound, and provides a new choice for development of antibacterial and anti-inflammatory drugs. The antibacterial activity screening test of the oxaanthraquinone compound shows that the oxaanthraquinone compound provided by the invention has obvious inhibition activity on candida albicans and has better inhibition effect on NO generation.
Description
Technical Field
The invention relates to the technical field of medicine preparation, in particular to an oxaanthraquinone compound with antibacterial and anti-inflammatory activities, a preparation method and application thereof, and a pharmaceutical composition.
Background
Oxaanthraquinone compounds, griseofulvin analogs and their derivatives, are a large class of natural products with a high diversity, and their derivatives have been developed as important drugs for the treatment of cancer, bacterial infections and various other human diseases. The oxaanthraquinone compounds are mostly secondary metabolites of microorganisms. The compounds have various biological activities such as antibiosis, cytotoxin resistance, antitumor, antibacterial and the like. In order to further popularize the oxaanthraquinone compounds, a new antibacterial strategy is provided, and the method has important significance for solving the current situation of abuse of the existing antibiotics.
Disclosure of Invention
The invention aims to provide an oxaanthraquinone compound with antibacterial and anti-inflammatory activity, and a preparation method, application and a pharmaceutical composition thereof; the oxaanthraquinone compound disclosed by the invention is novel in structure and has excellent antibacterial and anti-inflammatory activities.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an oxaanthraquinone compound, which has a structure shown in a formula I:
the invention provides a preparation method of the oxaanthraquinone compound, which comprises the following steps:
fermenting the penicillium gracile to obtain a penicillium gracile fermentation product;
mixing the penicillium gracile fermentation product with alcohol, and performing ultrasonic extraction to obtain a crude extract;
and (3) carrying out chromatographic purification on the crude extract to obtain the oxaanthraquinone compound with the structure shown in the formula I.
Preferably, the fermentation mode is solid fermentation, and the fermentation medium used for the fermentation is prepared from potato or rice; the fermentation temperature is 20-30 ℃ and the fermentation time is 25-35 d.
Preferably, the ratio of the mass of the penicillium gracile ferment to the volume of the alcohol is (30-50) g (50-120) mL.
Preferably, the chromatographic purification comprises silica gel column chromatography elution and gel column chromatography purification of the crude extract sequentially.
Preferably, the eluting solvent used for the silica gel column chromatography is petroleum ether-acetone solution; the volume ratio of the petroleum ether to the acetone is (5-100): 0-1.
Preferably, the silica gel column chromatography elution includes:
dissolving the crude extract in a chloroform-methanol mixed solution to obtain a crude extract solution;
mixing the crude extract solution with silica gel, removing solvent, and sequentially performing gradient elution by using petroleum ether-acetone solutions with the volume ratios of petroleum ether to acetone of 100:0, 100:1, 50:1, 10:1 and 5:1 to obtain petroleum ether-acetone solution elution parts with the volume ratios of petroleum ether to acetone of 100:0, 100:1, 50:1, 10:1 and 5:1 respectively; taking a petroleum ether-acetone solution elution part with the volume ratio of petroleum ether to acetone being 100:1, and eluting by silica gel column chromatography with petroleum ether-acetone with the volume ratio of petroleum ether to acetone being 120:1 as a solvent to obtain an eluent; the mass of the silica gel is 0.8-1.5 times of the dry weight of the crude extract.
Preferably, the gel column used for the gel column chromatography purification is a sephadex column, and the solvent used is methanol.
The invention provides the application of the oxaanthraquinone compound in the preparation of antibacterial drugs or the oxaanthraquinone compound prepared by the preparation method in the technical scheme.
The invention provides a pharmaceutical composition, which comprises the oxaanthraquinone compound according to the technical scheme or the oxaanthraquinone compound prepared by the preparation method according to the technical scheme; the content of the oxaanthraquinone compound in the pharmaceutical composition is 1-99 wt%.
The invention provides an oxaanthraquinone compound which has a furopyranum ring structure, contains a plurality of alpha, beta unsaturated ketone structures and is a classical Michael acceptor, thus having better antibacterial and anti-inflammatory activity, being applicable to the preparation of antibacterial and anti-inflammatory drugs, enriching the diversity of the oxaanthraquinone compound and providing a new choice for the development of antibacterial and anti-inflammatory drugs. The antibacterial activity screening test of the oxaanthraquinone compound shows that the oxaanthraquinone compound provided by the invention has obvious inhibition activity on candida albicans and has better inhibition effect on NO generation.
The invention provides a preparation method of the oxaanthraquinone compound, which adopts microorganism to ferment and change the gray penicillium to prepare the oxaanthraquinone compound, and has the advantages of simplicity, easiness, practicability, rapidness, mild culture condition, few byproducts, strong stereoselectivity, low cost and easiness in realizing industrialization, thereby not only meeting the requirements of modern environmental protection and low-carbon economy, but also providing a new way for the mass production of the oxaanthraquinone compound.
Drawings
FIG. 1 shows the oxaanthraquinone compounds provided by the present invention 1 H-NMR spectrum;
FIG. 2 shows the oxaanthraquinone compounds provided by the present invention 13 C-NMR spectrum;
FIG. 3 shows the oxaanthraquinone compounds provided by the present invention 1 H- 1 HCOSY profile;
FIG. 4 is a HMBC spectrum of the oxaanthraquinone provided by the invention;
FIG. 5 is a HSQC spectrum of the oxaanthraquinone provided by the invention;
FIG. 6 is a ROESY spectrum of the oxaanthraquinone provided by the invention;
FIG. 7 is an HR-ESI-MS spectrum of the oxaanthraquinone provided by the present invention;
FIG. 8 shows the single crystal structure of the oxaanthraquinone compound provided by the invention.
Detailed Description
The invention provides an oxaanthraquinone compound, which has a structure shown in a formula I:
the invention provides a preparation method of the oxaanthraquinone compound, which comprises the following steps:
fermenting the penicillium gracile to obtain a penicillium gracile fermentation product;
mixing the penicillium gracile fermentation product with alcohol, and performing ultrasonic extraction to obtain a crude extract;
and (3) carrying out chromatographic purification on the crude extract to obtain the oxaanthraquinone compound with the structure shown in the formula I.
In the present invention, the desired materials or reagents are commercially available as known to those skilled in the art unless otherwise specified.
The invention ferments the penicillium gracile to obtain the penicillium gracile ferment.
The penicillium gracile (Penicillium canescens) of the invention is derived from radix aconiti kusnezoffii, is an endophytic fungus of radix aconiti kusnezoffii (Aconitum vilmorinianum kom.), and is more specifically isolated from the root of radix aconiti kusnezoffii. The invention preferably carries out fermentation after the penicillium graying is activated; the activation method is preferably to inoculate the penicillium gracile on PDA slant culture medium, incubate for 5-7 d at 28 deg.C, store in refrigerator at 5 deg.C. The present invention is not particularly limited to other processes of the activation, and may be carried out according to processes well known in the art.
In the invention, the fermentation mode is preferably solid fermentation, the metabolism time of the penicillium gracilii in liquid fermentation is long, and the fermentation time can be shortened by adopting the solid fermentation mode.
The invention preferably inoculates penicillium gracilii in the fermentation medium, the inoculation amount is preferably 1-5 wt%. In the present invention, the fermentation medium used for fermentation is preferably prepared from potato or rice, more preferably from potato. The specific components of the fermentation medium are not particularly limited, and the penicillium griseum can be grown and propagated. In an embodiment of the invention, the preparation of the fermentation medium is specifically: taking cleaned potatoes, dividing the potatoes into potato blocks with the diameter of 1cm, placing the potato blocks into a tissue culture bottle with the specification of 45 g/bottle, capping the tissue culture bottle, sterilizing at the high temperature of 120 ℃ for 30min, and cooling to obtain a fermentation culture medium; or soaking 45g of rice in 40mL of water for 12h, taking out the rice, placing the rice in a tissue culture bottle, sealing the tissue culture bottle at 50 g/bottle, sterilizing at 120 ℃ for 30min, and cooling to obtain the fermentation medium.
In the present invention, the temperature of the fermentation is preferably 20 to 30 ℃, more preferably 26 to 28 ℃; the time is preferably 25 to 35 days, more preferably 28 to 30 days.
After the penicillium gracile fermentation product is obtained, the invention mixes the penicillium gracile fermentation product with alcohol and then carries out ultrasonic extraction to obtain a crude extract.
In the present invention, the alcohol is preferably methanol, ethanol, propanol or isopropanol. The invention adopts alcohol as solvent to extract from oxaanthraquinone compound penicillium griseum fermentation product.
In the present invention, the ratio of the mass of the fermentation product of Penicillium gracile to the volume of the alcohol is (30-50) g (50-120) mL, more preferably (40-45) g (70-80) mL, still more preferably 45g:80 mL.
In the present invention, the time of the ultrasonic extraction is preferably 20 to 40min, more preferably 30min; the ultrasonic power is preferably 250W to 350W, more preferably 300W.
After the ultrasonic extraction is completed, the obtained product is preferably filtered, and the solvent is removed from the obtained filtrate to obtain a crude extract. The invention preferably adopts a reduced pressure distillation mode to remove the solvent in the filtrate, and more preferably, the filtrate is distilled under reduced pressure until no alcohol smell exists; the pressure of the reduced pressure distillation is preferably 10 to 15kPa, more preferably 12kPa; the temperature of the reduced pressure distillation is preferably 45 to 55 ℃, more preferably 50 ℃.
After the crude extract is obtained, the crude extract is subjected to chromatographic purification to obtain the oxaanthraquinone compound with the structure shown in the formula I.
In the present invention, the chromatographic purification preferably includes silica gel column chromatography elution and gel column chromatography purification of the crude extract sequentially.
In the invention, the eluting solvent used for the silica gel column chromatography elution is preferably petroleum ether-acetone solution; the volume ratio of the petroleum ether to the acetone is preferably (5-100): 0-1, more preferably (10-50): 1.
In the present invention, the silica gel column chromatography elution preferably includes:
dissolving the crude extract in a chloroform-methanol mixed solution to obtain a crude extract solution;
mixing the crude extract solution with silica gel, removing solvent, and sequentially performing gradient elution by using petroleum ether-acetone solutions with the volume ratios of petroleum ether to acetone of 100:0, 100:1, 50:1, 10:1 and 5:1 to obtain petroleum ether-acetone solution elution parts with the volume ratios of petroleum ether to acetone of 100:0, 100:1, 50:1, 10:1 and 5:1 respectively; and (3) taking a petroleum ether-acetone solution elution part with the volume ratio of petroleum ether to acetone being 100:1, and eluting by silica gel column chromatography by taking petroleum ether-acetone with the volume ratio of petroleum ether to acetone being 120:1 as a solvent to obtain an eluent.
In the present invention, the eluting solvent used for dissolving the crude extract is preferably a chloroform-methanol mixed solution; the volume ratio of chloroform to methanol in the chloroform-methanol mixed solution is preferably 1:1; the concentration of the crude extract solution is preferably 0.1g/mL.
In the present invention, the particle size of the silica gel is preferably 200 to 400 mesh; the mass of the silica gel is preferably 0.8 to 1.5 times, more preferably 1 to 1.23 times the dry weight of the crude extract. The process of mixing the crude extract solution with silica gel is not particularly limited in the present invention, and may be carried out according to a process well known in the art.
In the present invention, the mode of removing the solvent is preferably distillation under reduced pressure; the temperature of the reduced pressure distillation is preferably 48 to 55 ℃, preferably 50 ℃.
After the solvent removal is finished, the obtained material is preferably loaded into a column for gradient elution; the specific process of the gradient is not particularly limited in the present invention, and may be performed according to a process well known in the art.
The specific process of the silica gel column chromatography elution is not particularly limited, and the elution can be carried out according to the process well known in the art.
After the eluent is obtained, the eluent is preferably subjected to gel column chromatography purification; the gel column used for the gel column chromatographic purification is preferably a sephadex column, and the solvent used is preferably methanol; the flow rate of the methanol is preferably 0.4 to 0.6mL/min, more preferably 0.5mL/min.
After the gel column chromatography purification is completed, the obtained product is preferably dried to obtain the oxaanthraquinone compound. The specific process of the drying is not particularly limited in the present invention, and may be performed according to a process well known in the art.
The invention provides the application of the oxaanthraquinone compound in the preparation of antibacterial drugs or the oxaanthraquinone compound prepared by the preparation method in the technical scheme.
The invention provides a pharmaceutical composition, which comprises the oxaanthraquinone compound according to the technical scheme or the oxaanthraquinone compound prepared by the preparation method according to the technical scheme; the content of the oxaanthraquinone compound in the pharmaceutical composition is 1-99 wt%, more preferably 55-90 wt%. The pharmaceutical composition preferably further comprises pharmaceutical excipients, the auxiliary materials to be used are not particularly limited, and the pharmaceutical excipients which are conventional in the art are selected.
The application method of the oxaanthraquinone compound is not particularly limited, and the oxaanthraquinone compound can be prepared into a required dosage form, such as a powder or a spray, by adopting a preparation method well known in the art.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
1. Activating strains: inoculating penicillium gracile on PDA slant culture medium, culturing at 28deg.C for 5d, and storing in refrigerator at 5deg.C;
2. preparation of fermentation medium: taking cleaned potatoes, dividing the potatoes into potato blocks with the diameter of 1cm, placing the potato blocks into a tissue culture bottle, placing the tissue culture bottle in 45 g/bottle, capping the tissue culture bottle, sterilizing the tissue culture bottle at the high temperature of 120 ℃ for 30min, and cooling to obtain a fermentation culture medium;
3. inoculating the activated penicillium gracile in the step 1 to the fermentation culture medium prepared in the step 2 according to the inoculation amount of 1wt%, and culturing for 30 days at the constant temperature of 28 ℃ after capping to obtain penicillium gracile fermentation product;
4. mixing 45g of penicillium gracile fermentation product with 80mL of methanol, carrying out ultrasonic treatment on the obtained mixed solution for 30min under the condition of 300W, filtering, taking filtrate, carrying out reduced pressure distillation until no alcohol smell exists, wherein the pressure of the reduced pressure distillation is 12kPa, the temperature is 50 ℃, and obtaining 30.0g of crude extract;
5. dissolving 30.0g of the crude extract with 300mL of chloroform-methanol solution with the volume ratio of 1:1, mixing the obtained crude extract with 30.0g of silica gel (200 meshes), distilling under reduced pressure at 50 ℃ to remove the solvent, and loading to a column; sequentially carrying out gradient elution by petroleum ether-acetone solutions with volume ratios of 100:0, 100:1, 50:1, 10:1 and 5:1 to obtain petroleum ether-acetone solution elution parts with volume ratios of 100:0, 100:1, 50:1, 10:1 and 5:1 respectively; taking petroleum ether-acetone solution elution part with the volume ratio of 100:1, and eluting by silica gel column chromatography with petroleum ether-acetone with the volume ratio of 120:1 as a solvent to obtain eluent;
and (3) taking methanol as a solvent, wherein the flow rate of the methanol is 0.5mL/min, purifying the eluent by using a sephadex column chromatography, and drying to obtain the oxaanthraquinone compound.
Example 2
1. Activating strains: inoculating penicillium gracile on PDA slant culture medium, culturing at 28deg.C for 5d, and storing in refrigerator at 5deg.C;
2. preparation of fermentation medium: soaking 45g of rice in 40mL of water for 12h, taking out the rice, placing the rice in a tissue culture bottle, sealing the tissue culture bottle at 50 g/bottle, sterilizing at 120 ℃ for 30min, and cooling to obtain a fermentation culture medium;
3. inoculating the activated penicillium gracile in the step 1 to the fermentation culture medium prepared in the step 2 according to the inoculation amount of 1wt%, and culturing at the constant temperature of 26 ℃ for 25 days after capping to obtain penicillium gracile fermentation product;
4. mixing 45g of penicillium gracile fermentation product with 120mL of methanol, carrying out ultrasonic treatment on the obtained mixed solution for 30min under the condition of 300W, filtering, taking filtrate, carrying out reduced pressure distillation until no alcohol smell exists, wherein the pressure of the reduced pressure distillation is 12kPa, and the temperature is 50 ℃, so as to obtain a crude extract;
5. 30.0g of the crude extract was dissolved in 300mL of a chloroform-methanol solution having a volume ratio of 1:1, the obtained crude extract solution was mixed with 37.0g of silica gel (200 mesh), the solvent was distilled off under reduced pressure at 50℃and packed in a column; sequentially carrying out gradient elution by petroleum ether-acetone solutions with volume ratios of 100:0, 100:1, 50:1, 10:1 and 5:1 to obtain petroleum ether-acetone solution elution parts with volume ratios of 100:0, 100:1, 50:1, 10:1 and 5:1 respectively; taking petroleum ether-acetone solution elution part with the volume ratio of 100:1, and eluting by silica gel column chromatography with petroleum ether-acetone with the volume ratio of 120:1 as a solvent to obtain eluent;
and (3) taking methanol as a solvent, wherein the flow rate of the methanol is 0.5mL/min, purifying the eluent by using a sephadex column chromatography, and drying to obtain the oxaanthraquinone compound.
Characterization and performance testing
1) Structural identification
The structure of the oxaanthraquinone compound prepared in example 1 was identified by 1D/2DNMR (one-dimensional nuclear magnetic resonance spectrum and two-dimensional nuclear magnetic resonance spectrum) and HR-ESI-MS (high resolution electrospray ionization mass spectrometry) (FIGS. 1 to 7).
As can be obtained from the HSQC spectrum in combination with the carbon spectrum, the chemical shift δ of H of the oxaanthraquinone compound and C linked thereto is shown in table 1:
TABLE 1 oxaanthraquinone compound prepared in example 1 13 C (150 MHz) and 1 h (600 MHz) NMR data, CDCl 3 Is a solvent
In FIGS. 1 to 7, HR-ESI-MSm/z of oxaanthraquinone compounds: [ M+H ]] + The excimer ion peak of (C) is 469.1025 (FIG. 7) 23 H 23 ClO 7 Na[M+H] + Calculated values: 469.1025 Indicated by the formula C 23 H 23 ClO 7 Contains 12 unsaturations. By reacting compounds 1 H (figure 1), 13 Analysis of C (FIG. 2) and HSQC (FIG. 5) spectra revealed that the oxaanthraquinone compound contained 5 methyl [ delta ] H 0.86(H 3 -14),1.62(H 3 -13),1.74(H 3 -7'),3.33(H 3 -16,oxygenated),3.92(H 3 -15, oxygenated)；δ C 15.3(C-14),23.0(C-13),16.4(C-7'),56.2(C-16,oxygenated), 60.9(C-15,oxygenated)]Two methylene [ delta ] H 2.81(H-8α),2.87(H-8β),3.40(H-11α, oxygenated),4.01(H-10β,oxygenated)；δ C 19.2(C-8),74.4(C-11,oxygenated)]4 methines [ delta ] H 1.93(H-10),1.95(H-9),5.98(H-3',aromatized),6.41(H-5', aromatized)；δ C 47.0(C-9),74.4(C-11),104.4(C-3',aromatized),129.8(C-5', aromatized)]12 quaternary carbons [ delta ] C 89.0(C-2),102.5(C-3a,aromatized),108.8(C-5, aromatized),106.9(C-7,aromatized),110.3(C-12,oxygenated),147.2(C-6', olefinic),152.4(C-4,aromatized),164.3(C-6,aromatized),167.9(C-2',olefinic), 169.4(C-7a,aromatized),186.0(C-4',carbonyl),189.9(C-2,carbonyl)]。
The planar structure of the oxaanthraquinone compound is determined by nuclear magnetic data comparison and 2DNMR spectrum, H 3 -6/C-2' and H 3 The HMBC correlation of 7'/C-2, C-5' and C-6 '(FIG. 4) indicates that an oxymethyl group and a methyl group are located in C-2' and C-6', H-3/C-2, C-2', C-4', C-5' and H-5'/C-2, C-3', C-4', C-6' respectively, and the HMBC correlation of 3, 5-dimethoxyycyclohexa-2, 5-dien-1-one ring is present in the oxaanthraquinone. The HMBC related data combined with nuclear magnetic resonance data indicate that the oxaanthraquinone compound has a lipopeptidyl benzophenone structural fragment, H-8/H-9/H-10/H-11, H-10/H-14 1 H- 1 H COSY (FIG. 3) correlation and H 3 -13/C-12, C-9 and H 2 HMBC correlation of-8/C-4, C-5, C-6 (FIG. 4) indicates the presence of a particular dimethylethylene hydro-4H-furo [2,3-b ] in the oxaanthraquinone type compounds]Structural fragment (B) of pyran, and this fragment is linked to fragment A by C-4 and C-5. Thus, the planar structure of the oxaanthraquinone compound provided by the invention is determined as that of an oxaanthraquinone compound containing a furopyranyl ring structure, and the correlation (figure 6) of H-9/H-13 and H-14 in the NOESY spectrum of the compound shows that the relative configuration is 2S,9R,10R and 12R, and the absolute configuration is 2S,9R,10R and 12R as determined by single crystal X-ray diffraction.
In summary, the structural formula of the compound prepared in example 1 can be determined as follows:
2) Antibacterial activity screening of oxaanthraquinone compounds
1. Antibacterial activity of oxaanthraquinone compounds:
(1) PDB (Potato Dextrose Broth ) medium: cleaning potato, peeling, weighing 200g, cutting into pieces, adding water, boiling until the pieces can be broken by a glass rod (10-15 minutes), filtering by using gauze to obtain filtrate, heating the filtrate, adding 20g of glucose, stirring uniformly until the glucose is completely dissolved, adding water to 1000mL, packaging into a 250mL conical flask, sealing, placing a high-temperature sterilization pot for sterilization at 120 ℃ for 30 minutes, taking out, cooling and storing for later use.
(2) LB (Luria-Bertani) medium: respectively taking 10g of Tryptone (Tryptone), 5g of yeast extract and 10g of sodium chloride (NaCl), dissolving to 1000mL, sterilizing at 121 ℃ for 30min, cooling and storing for later use.
(3) Activating strains: the fungi candida albicans (c.albicans, ATCC 10231) and 3 strains of bacteria [ e.coli, ATCC 25922), bacillus subtilis (b.subtilis, ATCC 6633) ] were inoculated into PDB medium and LB medium, respectively, the fungi were cultured at 28 ℃ for 3 days, and the bacteria were cultured at 37 ℃ for 18h.
(4) The antibacterial test was performed by a two-fold dilution method: dissolving an oxaanthraquinone compound to be detected and a positive control drug (bacteria: kanamycin Kanamycin; fungi: nystatin made mycin) respectively with DMSO to prepare working solution of 10.24mg/mL, adding 5 mu L of the working solution into a 96-well plate, setting 3 compound holes for each sample, adding 85 mu L of blank culture medium (fungi: PDB culture medium and bacteria: LB culture medium), diluting each sample into different concentrations (512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0.125 mu g/mL) by adopting a double dilution method, diluting 200 mu L of the activated bacterial solution to 10mL by adopting the corresponding culture medium, adding 10 mu L of the diluted bacterial solution into a sample hole and a control hole to enable the final volume of each hole to be 100 mu L, and adding no bacterial solution into the blank. After the bacterial liquid is fully mixed, the 96-well plate is placed in a constant temperature shaking table for culture (fungi: 28 ℃,3d; bacteria: 37 ℃,18 h), the absorbance value of each well under visible light is measured by an enzyme-labeled instrument, and the concentration of the compound in the corresponding well when the ratio of the absorbance value to a blank control is 1 is the minimum inhibitory concentration (Minimal Inhibit Concentration, MIC) of the sample on the corresponding strain, and the result is shown in Table 2.
2. Anti-inflammatory Activity of oxaanthraquinone Compounds:
nitric oxide has wide and important biological regulation function and has important effects on inflammation, tumor, cardiovascular system and the like. When immune cells are stimulated by microbial endotoxins, inflammatory mediators, etc., a large amount of inducible nitric oxide synthase is produced, which produces NO for an immune response. Thus, inhibition of NO production is a direct indicator of the anti-inflammatory activity of the compounds.
(1) The log phase mouse mononuclear macrophage RAW264.7 (purchased from Shanghai cell bank of Chinese sciences) was induced and stimulated with 1. Mu.g/mLLPS, and the final concentrations of the oxaanthraquinone compound prepared in example 1 and positive control L-NMMA were 40.0. Mu.M, three replicates were set for each sample, the average value was calculated, and cultured at 25 ℃.
Blank control: setting a drug-free group (comprising mouse mononuclear macrophage RAW264.7 and 1 μg/mL LPS); the positive drug group without L-NMMA (Total NOS inhibitor) was treated as a control in the same manner as described above.
(2) After overnight (10 h) incubation of the cells, NO production was measured at 570nm for each group; MTS was added to the culture broth obtained by overnight culture to conduct cell viability assay, excluding toxic effects of the compounds on cells.
NO generation inhibition (%) = (NO drug group OD 570nm Drug group OD 570nm ) Drug-free group OD 570nm ×100％；
TABLE 2 screening for antibacterial and NO production-inhibiting Activity of oxaanthraquinone compounds prepared in EXAMPLE 1 (IC 50 )
The results in Table 2 show that canecine A showed good inhibitory activity against Candida albicans in screening test for the antibacterial activity of the oxaanthraquinone compounds in vitro (positive control: kanamycin and nystatin) (Table 2), and that the oxaanthraquinone compounds showed more excellent inhibitory activity against the production of Nitric Oxide (NO) than the positive control in screening test for the NO inhibitory activity of the oxaanthraquinone compounds (positive control: L-NMMA) (Table 2). Therefore, the oxaanthraquinone compound provided by the invention has research value for developing antibacterial and anti-inflammatory medicaments as a lead compound. The method for producing the antibacterial anti-inflammatory oxaanthraquinone compound simply based on a large amount of microbial fermentation not only can realize the requirements of environmental protection and low carbon economy, but also lays a solid foundation for further research and development of the industrial mass production of the antibacterial anti-inflammatory compound in the later period, and has remarkable application value.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. An oxaanthraquinone compound is characterized by having a structure shown in a formula I:
2. the method for preparing the oxaanthraquinone compound according to claim 1, comprising the steps of:
fermenting the penicillium gracile to obtain a penicillium gracile fermentation product;
mixing the penicillium gracile fermentation product with alcohol, and performing ultrasonic extraction to obtain a crude extract;
and (3) carrying out chromatographic purification on the crude extract to obtain the oxaanthraquinone compound with the structure shown in the formula I.
3. The preparation method according to claim 2, wherein the fermentation mode is solid fermentation, and the fermentation medium used for the fermentation is prepared from potato or rice; the fermentation temperature is 20-30 ℃ and the fermentation time is 25-35 d.
4. The process according to claim 2 or 3, wherein the ratio of the mass of the fermentation product of Penicillium gracilii to the volume of the alcohol is (30-50) g (50-120) mL.
5. The method according to claim 2, wherein the chromatographic purification comprises silica gel column chromatography elution and gel column chromatography purification of the crude extract sequentially.
6. The preparation method according to claim 5, wherein the eluting solvent used for the silica gel column chromatography is petroleum ether-acetone solution; the volume ratio of the petroleum ether to the acetone is (5-100): 0-1.
7. The method of preparing according to claim 6, wherein the silica gel column chromatography elution comprises:
dissolving the crude extract in a chloroform-methanol mixed solution to obtain a crude extract solution;
mixing the crude extract solution with silica gel, removing solvent, and sequentially performing gradient elution by using petroleum ether-acetone solutions with the volume ratios of petroleum ether to acetone of 100:0, 100:1, 50:1, 10:1 and 5:1 to obtain petroleum ether-acetone solution elution parts with the volume ratios of petroleum ether to acetone of 100:0, 100:1, 50:1, 10:1 and 5:1 respectively; taking a petroleum ether-acetone solution elution part with the volume ratio of petroleum ether to acetone being 100:1, and eluting by silica gel column chromatography with petroleum ether-acetone with the volume ratio of petroleum ether to acetone being 120:1 as a solvent to obtain an eluent; the mass of the silica gel is 0.8-1.5 times of the dry weight of the crude extract.
8. The method according to claim 5, wherein the gel column used for the gel column chromatography purification is a sephadex column and the solvent used is methanol.
9. The application of the oxaanthraquinone compound as claimed in claim 1 or the oxaanthraquinone compound prepared by the preparation method as claimed in any one of claims 2 to 8 in the preparation of antibacterial drugs.
10. A pharmaceutical composition comprising an oxaanthraquinone compound according to claim 1 or an oxaanthraquinone compound prepared by the method of any one of claims 2 to 8; the content of the oxaanthraquinone compound in the pharmaceutical composition is 1-99 wt%.
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| CN108660082A (en) * | 2018-06-15 | 2018-10-16 | 河北大学 | A kind of marine aspergillus source oxa- anthraquinone analog compound and preparation method thereof and the application in preparing antiseptic |
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| CN108660082A (en) * | 2018-06-15 | 2018-10-16 | 河北大学 | A kind of marine aspergillus source oxa- anthraquinone analog compound and preparation method thereof and the application in preparing antiseptic |
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| 变灰青霉固态发酵降解植酸的初步研究;褚西宁;微生物学通报;第23卷(第4期);217-220 * |
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