CN111254083B

CN111254083B - Antamimetic acid A and preparation method and application thereof

Info

Publication number: CN111254083B
Application number: CN202010185304.5A
Authority: CN
Inventors: 丁传波; 郑毅男
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-08-15
Filing date: 2017-07-27
Publication date: 2023-08-25
Anticipated expiration: 2037-07-27
Also published as: CN106282033A; CN107488594A; CN107488594B; CN111254083A

Abstract

The application relates to a strain of penicillium Z08 from soil and a metabolite of the same, namely antacid A, wherein the strain is separated from the soil where corn is planted, ITS sequences are obtained by amplification of ITS primers, and the strain is penicillium (Penicilillium flavonim) Z08 which is preserved in the China general microbiological culture Collection center with the preservation number of China general microbiological culture Collection center: CGMCC No.12761, date of preservation: 2016, 6 and 29 days. The penicillium Z08 has a strong phosphate solubilizing effect. The new light yellow compound antamimetic acid A is obtained by separating the water-soluble metabolite thereof, and the metabolite antamimetic acid A has the effects of resisting inflammation, treating diabetes and the like and is expected to be widely applied in the field of medicines.

Description

Antamimetic acid A and preparation method and application thereof

The application is a divisional application of patent application (application number 201710624795.7, application name "a strain of new penicillium and metabolite antamimetic acid A thereof") submitted to the national intellectual property agency in 2017, 7 and 27 days.

Technical Field

The application relates to penicillium Z08 which is derived from soil and can produce new natural product ambamoxicillin and application thereof in medicine, forestry and agriculture.

Background

Soil microorganisms spend all or part of their life cycle in the soil and play an important role in various processes inside the soil, which affects the social and economic development of humans in many ways. Many soil microorganisms secrete secondary metabolites, such as rhizobium to secrete rhizobium nodulation factors, lipochitooligosaccharidesPlays an important role in the symbiotic nodulation process of rhizobia and plants. Fungal natural products are also one of the important sources of natural drugs ^1，2 Such as ergot alkaloids, diketopiperazines, quinolines, quinazolines, diazepams, polyketides, and the like. The completion of large-scale fungal genome sequence sequencing has shown that fungi have the potential to produce abundant secondary metabolites that have become a hotspot for research and the direction of development ^3，4，5 . The relationship between microorganisms and soil fertility was noted at the beginning of the 20 th century, and it was found that some poorly soluble complexes were applied to the soil and could be used as phosphorus source, and that more than 50 strains of bacteria were screened from the soil, several strains of penicillium forming macroscopic phosphorus-dissolving rings on the plate ⁶ . Along with the increasing serious industrial pollution and environmental destruction, the incidence of malignant tumors is increasing year by year, and the conventional anticancer drugs have great side effects, so the research and development of the antitumor drugs are important subjects for research in the field of pharmaceutical chemistry research. The natural products of fungi exhibit a very wide range of biological activities including anti-tumor, antibacterial, antiviral, immunosuppressive, etc.

Reportedly, it is reported that ⁷ The penicillium oxalicum P8 has the effect of dissolving various indissolvable phosphorus in soil, is suitable for various soil types such as red soil in south, black soil in north, brown soil, wet soil and the like, and has various functions of improving crop yield, activating the indissolvable phosphorus in soil, preventing the chemical fixation of the soil of phosphate fertilizer and improving the utilization efficiency of the phosphate fertilizer. Ma Zhongyan, etc ⁸ In the disclosed application patent, the paecilomyces lilacinus bacterial agent is broadcasted around the root of the tomato seedling for treatment, so that the growth of the tomatoes can be obviously promoted, the effect is stable, and the method has good popularization and application prospects. The phoxim degradation microbial inoculum prepared by using paecilomyces lilacinus strain FJAT-9041 can effectively degrade pesticide phoxim ⁹ . The field test shows that the inoculation of the lysophosphate-penicillium can increase the corn yield ¹⁰ . The penicillium oxalicum NJDL-03 can repair lead pollution in soil and improve the utilization rate of calcareous soil phosphorus, and has remarkable effect in promoting crop growth ¹¹ . Paecilomyces tenuipes strain culture product for regulating immunity, resisting tumor and virus, protecting liver and improvingHas nourishing, antiaging, anoxia resisting, tranquilizing, and analgesic effects ¹² 。

Therefore, the penicillium has wide application prospect in agriculture, medicine and other aspects. Therefore, further studies on penicillium and its metabolites are necessary.

Disclosure of Invention

The application aims to provide a new penicillium (Penicillium flavum) Z08 from soil, which is registered and preserved in China general microbiological culture Collection center (CGMCC) on the day of the year 2016, 6 and 29, wherein the preservation place is China institute of microorganisms, national academy of sciences, no. 3, north Chen West Lu 1, the region of the Korean of Beijing, and the preservation number is CGMCC No.12761.

The application separates a fungus from the soil where corn is planted, the fungus uses ITS primer to amplify to obtain ITS sequence, and BLAST identification shows that the sequence has the highest similarity with Penicillium sp.1JJK-2011 (GENE Bank ID: HM 469397) and reaches 98.9%, thus identifying the fungus belonging to penicillium Z08 of penicillium (Penicilillium flavonim).

The microbiological characteristics of the bacteria are: after 4 days of incubation at 28℃on PDA plates, the colonies appeared white, mostly vegetative hyphae. The nutrient body is colorless or pale yellow mycelium, and a diaphragm is arranged between cells of the mycelium. After 7 days, the aerial hyphae are seen to generate simple long upright conidiophores, the top ends of the aerial hyphae are branched in a special symmetrical broom shape, meanwhile, bacterial colonies are light green, and the clustered hyphae are seen by microscopic examination.

It is another object of the present application to provide a novel compound, antamimetic acid A (antanic acid A), isolated from the fungus, having the chemical designation: 2- {2- [ (2E) -3- [ (2-aminophenyl) carboxamido]Prop-2-enamido]Ethyl } malonic acid; the molecular formula is C ₁₅ H ₁₇ N ₃ O ₆ The molecular weight is 335.1117; the chemical structure is as follows:

the compound is light yellow amorphous powder, and is easy to dissolve in water, dimethyl sulfoxide, etc.

The application has the advantages and positive effects that:

(1) The new penicillium Z08 separated in the soil has good phosphorus dissolving effect, can dissolve various indissolvable phosphorus in the soil, is used in the soil for planting crops, can activate the indissolvable phosphorus in the soil, prevent the soil chemical fixation of phosphate fertilizer and improve the crop yield.

(2) According to the application, a new compound, namely the antamimetic acid A, is extracted from a fermentation product of the penicillium neoformans Z08, and can inhibit proliferation of NIH3T3 cells, and the greater the concentration of the antamimetic acid A is, the more obvious the inhibition effect on proliferation of the NIH3T3 cells is; meanwhile, the antacid A has a strong inhibition effect on the expression of the NIH3T3 cell TGF-beta 1 gene, and the compound is expected to be applied to the medicines for treating diabetes and provides a new product for the treatment of diabetes patients.

(3) The antamimetic acid A extracted by the application can inhibit lipopolysaccharide-induced macrophage from secreting inflammatory factors, and is also expected to be applied to anti-inflammatory medicines.

Drawings

FIG. 1 is a 1H nuclear magnetic resonance spectrum of an antamimetic acid A compound.

FIG. 2 is a 13C profile of an antamimetic acid A compound.

FIG. 3 is a chart of the HMQC profile of the antamimetic acid A compound 1H-13C.

FIG. 4 is a chart of HMBC pattern of the compound 1H-13C of antamimetic acid A.

FIG. 5 is a chart of the COSY pattern of the antamimetic acid A compound 1H-1H.

FIG. 6 is a high resolution mass spectrum of the antamimetic acid A compound.

FIG. 7 is a graph showing a dynamic analysis of phosphorus dissolution in soil by Penicillium Z08.

FIG. 8 is a graph of total RNA quality assay extracted from NIH3T3 cells.

FIG. 9 is a graph showing the effect of three concentrations of antamimetic acid A on RAW264.7 cell activity.

FIG. 10 is a graph showing the generation of NO by LPS-induced mouse cells with three doses of antamimetic acid A.

Detailed Description

The application is further described below in connection with examples to be more clearly understood by those skilled in the art, but is not to be construed as limiting the application in any way.

Example 1

1. Isolation and identification of Penicillium Z08 Strain

21 strains of fungi were isolated from the soil of the farm of Jiu Zhang Jia Zhang, jilin province using PDA+penicillin and streptomycin plates, one strain of which was Penicillium Z08, and Penicillium Z08 produced pale yellow compounds on PDA agarose plates. The strain was identified as penicillium by taxonomic studies and molecular biology studies (Penicilillium flavonim).

1. Preparation of Penicillium Z08 Strain

The following culture media were used for the purification of penicillium: 40 g of potato culture medium and 20 g of glucose and 20 g of agar were added to 1000 ml of water, sterilized at 121℃under a pressure of 1kg/cm2 for 20 minutes, cooled and then 30. Mu.g/ml of each of sterile penicillin and streptomycin were added. Weighing about l g of a soil sample under a sterile state, adding 10ml of sterile water, shaking and uniformly mixing, respectively diluting 10 times, 100 times and 100 times to prepare 4 concentration gradient samples, respectively taking 0.25 ml of each gradient and coating on a solid culture medium, when an isolated strain enters a vigorous growth period on the culture medium, picking spores of the strain, carrying out streak purification on a fungus purification culture medium, carrying out inversion culture under the conditions of relative humidity of 90% at 28 ℃, and constant temperature and humidity, continuously streaking according to the method until a colony with a single form on a single plate is obtained, picking the single colony on the plate for pure culture, and storing the pure strain after the colony grows.

2. Genomic DNA extraction of Penicillium Z08

The strain was first cultured in a shaker (150 r/min) at 28℃for 7 days, then 100mg of the freeze-dried mycelium was taken, and was sufficiently ground in a mortar with an appropriate amount of liquid nitrogen, and 2mL of buffer (50 mM Tris-HCl pH 7.2, 50mM EDTA,2.5%SDS,1% beta-mercaptoethanol) was added, mixed well, transferred to a 10mL test tube, and 2mL phenol was added: chloroform (1:1) extract, extracting, centrifuging the extract, adding 50. Mu.l RNase to the supernatant, standing at 55deg.C for 1 hr, and adding protease. Then, the mixture was washed twice with 70% cold ethanol, dried at room temperature for 30 minutes, dissolved in 500. Mu.l of water, and stored at-80℃for further use.

3. Identification of Penicillium Z08 Strain

By primer ITS58A1F, 5'-GCATCGATGAAGAACGC-3' and

ITSNLB3:5'-GGATTCTCACCCTCTATGA-3' amplification of Penicillium Z08 genomic DNA, transformation of the amplified fragment into DH5a with T-loaded clones and sequencing of the positive clones, the ITS sequences of Penicillium Z08 were obtained as follows:

BLAST analysis of the ITS sequence of Penicillium Z08 shows that the sequence has the highest similarity with Penicillium sp.1JJK-2011 (GENE Bank ID: HM 469397) up to 98.9%. The strain was identified as belonging to Penicillium (Penicillium flavum) Penicillium Z08. The strain is registered and preserved in China general microbiological culture Collection center (CGMCC) at the date of year 6 and 29 of 2016, wherein the preservation place is China academy of sciences of China, national academy of sciences of China, no.1 and No. 3 of the Korean area of Beijing, and the preservation number is CGMCC NO.12761.

2. Separation and identification of antacid A in penicillium Z08 fermentation product

1. Preparation and separation method of penicillium Z08 fermentation product

(1) Preparation of the fermentate

Spore streaks from Penicillium Z08 are listed on agar plates (g/l) of 8 l PDA: yeast extract (available from Sigma) 0.5 g, glucose 10.0 g, calcium phosphate 5.0 g, ammonium sulfate 0.5 g, potassium chloride 0.2 g, magnesium sulfate 0.1 g, manganese sulfate 0.0001 g, ferrous sulfate 0.0001 g, agar 15.0 g, and plates were incubated at room temperature (20-22 ℃) for 14 days. When the agar plate was observed to turn yellow in color while the spores turned green, the spores were removed from the agar plate with water, and then the agar was extracted with water again. The resulting agar slurry was stirred in 10 liters of water for 5 hours and filtered through a metal (100 mesh) to give a fermentation product of Penicillium Z08.

(2) Reversed phase liquid chromatography separation and purification

The fermentation product solution is decompressed and evaporated to a volume of 2L, then the fermentation product solution is extracted for 3 times by using ethyl acetate in an equivalent amount, the extract is decompressed and evaporated to dryness, the dried extract is dissolved in water, cyclohexane and methanol are sequentially used for extraction, and the methanol part is taken for HPLC preparation.

Instrument: HPLC-ESI-MS (Agilent 1100HPLC system, amaZon spot ion trap mass spectrometer of ESI mode Broker, equipped with 160-600nm variable UV detector, phenominex Luna 5 μm C18 (250 mm. Times.15 mm, 4.6 μm), flow rate (10 ml/min);

chromatographic conditions: a (acetonitrile containing 0.1% formic acid) and B (water containing 0.1% formic acid) solvent gradient, 0-5 min, 5%A;5-15.5 min, linear gradient to 60% a;15.5-16 minutes, linear gradient to 100% a;16-17.5 minutes, 100% A isocratic; 17.5-18 min, linear to 5% A,18-21 min, 5%A.

(3) Results

The fermentation product of Penicillium Z08 (about 200 g) is extracted 3 times with ethyl acetate (2L) to obtain 1.5 g of extract, the extract is dissolved in water, the extract is sequentially extracted with cyclohexane and methanol, the methanol part is taken for HPLC preparation, 30 parts are collected, and a 13.5 minute sample peak is collected to obtain a bright yellow powder, 5 mg. For detection by Thermo Scientific LTQ, orbitrap XL high resolution mass spectrometry and Bruker, avance 600MHz nuclear magnetic resonance spectrometer.

2. Identification of antacid A in Penicillium Z08 fermentation product

Taking 1 mg of the bright yellow powder obtained above, analyzing by using Thermo Scientific LTQ and Orbitrap to obtain high-resolution mass spectrum with molecular weight of335.1117 (see FIG. 6); dissolving 3 mg of the bright yellow powder obtained in the above in DMSO-d ₆ In (1) was detected by Bruker, avance 600MHz nuclear magnetic resonance spectrometer (see Table 1 and FIG. 2).

¹ H NMR(600MHz，DMSO-d ₆ )δ：7.34(d,1H,J＝8.5，H-2)，8.51(t，1H，J＝8.5,1.5,H-3)，7.01(t，1H,J＝8.5,H-4)，8.01(d,1H,J＝8.5，H-5)，7.35(d,1H,J＝8.5，H-8)，5.21(d,J＝10.2，H-9)，3.06(m,1H,H-11)，2.13(m,1H,H-12a)，1.98(m,1H,H-12b)，2.51(m,1H,H-13)。

¹³ C NMR(150MHz，DMSO-d ₆ )δ:140.7(s，C-1)，131.6(d，C-2)，119.3(d，C-3)，122.4(d,C-4)，131.6(d,C-5)，124.2(s,C-6)，170.7(s，C-7)，135.4(d,C-8)，101.2(d，C-9)，166.3(s,C-10),58.7(d,C-11)，28.2(t,C-12)，32.6(t，C-13)，176.1(s,C-14)，176.1(s,C-15)。

TABLE 1 Nuclear magnetic resonance Spectrometry data

Deriving its molecular formula as C from HRESIMS ₁₅ H ₁₇ N ₃ O ₆ ：m/z＝336.1191[M+H] ⁺ (calculation 336.1196) at the same time according to ¹³ C NMR and other nuclear magneto-optical spectrum data, which is deduced to require 9 unsaturations. ¹ H- ¹ Analysis of the H COSY (fig. 5) and HMQC (fig. 3) spectra indicated the presence of the following spin systems: H2-H3-H4-H5, H8-H9, H11-H12-H13. The hydrocarbon correlations observed in the HMBC spectra (fig. 4 and table 1) are as follows: from C-1 to H-2, H-5; from C-6 to H-4, H-2; from C-7 to H-8,H-5; from C-10 to H-8,H-7; from C-14 to H-12; from C-15 to H-12; from C-13 to H-11. HMBC-related to antarnic a:

after searching and all the above data are finally determined, the compound is a novel compound with the chemical name: 2- {2- [ (2E) -3- [ (2-aminophenyl) carboxamido]Prop-2-enamido]Ethyl } malonic acid, named antamimetic a; the chemical formula is:the compound is light yellow amorphous powder, and is easy to dissolve in water, dimethyl sulfoxide, etc.

EXAMPLE 2 phosphorus solubilizing efficacy test of Penicillium Z08

1. Quantitative determination and result of phosphorus-dissolving capability of penicillium Z08

A phosphorus standard curve was prepared according to the study method of the former (reference 13), a regression equation was obtained, and the phosphorus dissolving amount of bacteria was calculated. After the stored endophytes were cultured overnight (28 ℃ C., 120 r/min), 100uL was inoculated into 50mL of PKO inorganic phosphorus culture solution, the reaction was repeated 3 times, the inoculated endophytes were subjected to sterile water as a control, and after the endophytes were cultured for 12d in a shaking table (120 r/min) at 28 ℃ C., the supernatant was taken and the phosphate solubilizing amount was determined according to the method of reference [13 ]. The calculation formula of the phosphorus dissolving amount of the strain is as follows:

bacterial strain phosphate solubilizing amount (mg/mL) =colorimetric solution (mg/mL) × (100/5) ×usage multiple

TABLE 2 quantitative determination of phosphorus-solubilizing amount of Penicillium Z08 Strain

2. Soil phosphorus dissolving capacity measurement of penicillium Z08 and result weighing 5g of sieved soil, placing the soil in a 150mL triangular flask, and performing 2 treatments of sterilization and non-sterilization respectively, wherein each treatment is divided into 3 groups: (1) 1mL of the culture solution of Penicillium Z08 strain overnight (28 ℃ C., 120 r/min) and 9mL of sterile water are added into a triangular flask, and 1mL of the culture solution of Penicillium Z08 strain overnight and 9mL of sterile water are added to be CK;

(2) 5mL of the overnight culture solution of the penicillium Z08 strain and 5mL of sterile water are added, and 5mL of the overnight culture solution of the sterilized penicillium Z08 strain and 5mL of sterile water are added as CK;

(3) 10mL of the penicillium Z08 strain overnight culture solution is added, and 10mL of the sterilized penicillium Z08 strain overnight culture solution is added as CK; the resulting mixture was incubated at 28℃and the amount of phosphorus to be dissolved was measured by the method for measuring soil quick-acting phosphorus in reference [13] on days 3,5,10,15,20,25 and 30, respectively, and the results were shown in FIG. 7.

As can be seen from FIG. 7, as the inoculum size increased, the phosphate solubilizing amount increased, and the phosphate solubilizing amount of the three culture groups significantly increased at day 5 of cultivation, the phosphate solubilizing tendency was retarded during the period of 5-15 days, and the phosphate solubilizing amount of 10mL of Penicillium Z08 was increased at the period of 15-30 days. The result shows that the inoculation amount is large, the cardinality of the penicillium Z08 thalli is large, the phosphate dissolving capability is strong, and the accumulation of soluble phosphorus is large; the penicillium Z08 can be in reciprocal cooperation with indigenous microorganisms in soil, and the indigenous microorganisms can enhance the phosphate dissolving capacity of the penicillium Z08.

Example 3 Effect of antamimetic acid A on NIH3T3 cells and TGF-beta 1 Gene expression

1. Experimental materials

1.1 cells

NIH3T3 cells were donated by the Jilin agricultural university student bioreactor and the department of drug development education engineering research center.

1.2 Experimental drugs

DEME high sugar medium, hyclone company; fetal Bovine Serum (FBS), hyclone company; antibiotics, gibco company; dimethylthiazole (MTT), amerco corporation; recombinant basic fibroblast growth factor (rbFGF), oryzogen; high-purity total RNA rapid extraction kit, beijing Baitaike Biotechnology Co., ltd; bioTake super RT Kit Beijing Baitaike Biotechnology Co., ltd; 2 x Power Taq PCR Master Mix, beijing Baitaike Biotechnology Co., ltd; 2 XSYBP real-time PCR premixture, beijing Baitaike Biotechnology Co.

1.3 laboratory apparatus

TGL-20B high speed bench centrifuge (Shanghai Anpavilion scientific instruments), inverted microscope (Olympus Co.), SW-CJ-1F ultra clean bench (Suzhou purification Equipment Co.), electrophoresis apparatus (Beijing Liuyi instruments), gel imager (BIORAD Co., USA), RCR apparatus (SBP Co., USA), applide Biosystems real-time fluorescence quantitative RCR apparatus (ABI Co., USA).

2. Experimental method

2.1 cell pretreatment

Cell resuscitation: taking out a centrifuge tube containing NIH3T3 cells from liquid nitrogen, resuscitating in a water bath at 37deg.C for 1min, adding 5mL of culture medium (DEME) in a super clean bench, centrifuging for 5min, discarding supernatant, sucking cells into a cell culture bottle, adding 4mL of culture medium, adding 10% serum (FBS) and 1%o antibiotic, shaking gently, standing at 37deg.C and CO concentration of 5% ² Culturing in an incubator, and the culture bottle cap is not required to be screwed tightly, so that the cell respiration is facilitated.

Passage of cells: observing the cells in the cell culture flask under a microscope, flushing the cells with PBS buffer solution for 2 times when the cells grow to 80-90%, discarding the PBS, adding 5mLDEME with a pipette, blowing the cells down, and carrying out passage.

2.2 determination of cell viability by MTT method

Transferring NIH3T3 cells growing to logarithmic phase into centrifuge tube, and adjusting cell concentration to 1×10 ⁵ And each mL. Then spotted into 96 Kong Xi plates, 100 μl per well, wells around 96 well plates, to prevent edge effects. Then it is placed at 37℃with 5% CO ² The cells were allowed to adhere to the wall after culturing in an incubator for 24 hours. The experiment is divided into 5 groups, namely a blank control group (equal culture solution is added), an rbFGF group (5 ng/mL), an antacid A group (0.4,2, 10, 50, 250 mug/mL), 3 groups of the antacid A group are arranged in parallel, each group of the antacid A group is cultured in a carbon dioxide incubator at 37 ℃ for 12, 24 and 48 hours, 20 mu.l of MTT is added into each hole by a pipetting gun, and the mixture is put into the incubator for incubation for 4 hours, and the culture is terminated. 150 μl DMSO was added to each well and placed on a shaker for 10min to allow complete dissolution of the DMSO. The light absorption value of each well was measured at 570nm on an ELISA monitor.

2.3 fluorescent real-time quantitative PCR detection of TGF-beta 1

Transferring NIH3T3 cells growing to logarithmic phase into centrifuge tube, and adjusting cell concentration to 4×10 ⁵ NIH3T3 cells were seeded in 6-well plates at a rate of 2 mL/mL, and the cells were incubated in a 5% carbon dioxide incubator at 37℃for 24 hours with the medium removed. Adding DMEM culture solution into blank group, adding DEME containing 5ng/mL rbFGF into positive control group, adding DEME containing 0.4,2, 10, 50, 250 μg/mL antamimetic acid A into experimental group, and adding 5% CO at 37deg.C ₂ Culturing for 24h, and stopping culturing. The culture solution is discarded, the cells are washed for 2 to 3 times by PBS, RNA in the cells is extracted by the high-purity total RNA rapid extraction kit, and cDNA is reversely transcribed by the BioTake super RT Kit kit.

TABLE 3 primer sequences

3. Results

3.1 determination of cell viability by MTT method

After MTT colorimetric detection of absorbance of the NIH3T3 cells by the antacid A and rbFGF, the results show that the OD value of the blank control group cells without any drug intervention is gradually increased after the blank control group cells are cultured for 12, 24 and 48 hours, which indicates that the cells are gradually propagated in the culture medium. NIH3T3 cells proliferated significantly more under rbFGF (P < 0.05). As shown in Table 4, the inhibition of proliferation of NIH3T3 cells was more pronounced with increasing concentration of antamimetic acid A. Compared with the blank group, the OD value of the 250 mug/mL group after 12 hours of culture has a significant difference (P < 0.05), the 10 mug/mL group at 24 hours has a significant difference (P < 0.05), the 50 mug/mL group and the 250 mug/mL group have a significant difference (P < 0.01), and the 10, 50 and 250 mug/mL groups after 48 hours of culture have significant differences (P < 0.01), so that the inhibition effect at 24 hours is most obvious.

TABLE 4MTT assay for OD values of NIH3T3 cells after action of antamimetic acid A

Note that: in comparison with the blank group, ^# P<0.05， ^## P<0.01

3.2 Effect of AF on expression of the NIH3T3 cell TGF- β1 Gene

Total RNA extracted from NIH3T3 cells was detected by agarose gel electrophoresis, and clear bands of 18S and 28S were observed on the UV detector (FIG. 8, A: blank; B: rbFGF; C: 0.4. Mu.g/mL; D: 2. Mu.g/mL; E: 10. Mu.g/mL; F: 50. Mu.g/mL; G: 250. Mu.g/mL). As shown in fig. 8, table 4 and table 5, the total RNA obtained by extraction has better integrity, and it can be seen that the more significant the inhibitory effect of the amamimetic acid a on NIH3T3 cell TGF- β1 gene expression is with increasing concentration.

TABLE 5 relative expression levels of TGF-. Beta.1 Gene in NIH3T3 cells

Note that: in comparison with the blank group, ^# P<0.05， ^## P<0.01

example 4 Effect of antamimetic acid A on lipopolysaccharide-induced secretion of inflammatory factors by macrophages

1. Material

1.1 cell line Source

RAW264.7 mononuclear macrophages from mice were purchased from Shanghai institute of life sciences cell bank, china academy of sciences.

1.2 pharmaceutical products and reagents

The antamimetic acid A is prepared by a laboratory, the purity is more than 90 percent, and purified water is used for preparing the antamimetic acid A into the required concentration of high, medium and low doses when the antamimetic acid A is used; the Nitric Oxide (NO) determination kit is ordered in Nanjing to build a bioengineering institute; interleukin-1 beta (IL-1 beta) enzyme-linked immunoassay kit and interleukin-6 (IL-6) enzyme-linked immunityEpidemic assay kit, tumor necrosis factor-alpha (TNF-alpha) enzyme linked immunoassay kit and prostate E ₂ (PGE ₂ ) ELISA kit, lot number: 201111, all purchased from U.S. R&Company D; calf serum (FBS) was purchased from Hyclone company; lipopolysaccharide (LPS) was purchased from Sigma company; dimethylthiazole (MTT) was purchased from amesco corporation; dimethyl sulfoxide (DMSO) was purchased from Sigma; dexamethasone (DMX) was purchased from Sigma; green chain diabodies were purchased from Gibco company; distilled water, self-made in laboratory.

1.3 major instrumentation

TGL-20B high-speed bench centrifuge (Shanghai Anting scientific instruments Co.); micro-oscillator (Jiangsu Jian-Taan Honghua Instrument works, inc.); -70 ℃ refrigerator (Haier); CO ₂ Incubator (Forma Scientific company, usa); inverted microscope (Olympus corporation, japan); electronic balance (mertrer-tolidol instruments limited, shanghai); SW-CJ-1F ultra clean bench (su zhou purification plant); MK3 microplate reader (Thermo Electron Co., USA); an electric heating constant temperature water bath pot (medical instruments factory in gold altar); pipetting gun (Thermo); 48 well cell plates (COSTER); LC-20AT Shimadzus HPLC (detector SPD-20A, shimadzu corporation); FD-1D-50 vacuum freeze dryer (Beijing Bo Yikang laboratory instruments Co., ltd.).

2. Method of

2.1 cell culture

Mouse macrophage cell line RAW264.7 was cultured in 50mL of RPMI1640 medium at 5% CO ₂ Culturing at 37deg.C in incubator.

2.2 administration group

Blank control group: no drug intervention or stimulation by lipopolysaccharide is added; LPS model group: without drug intervention, only 10 mug.L ^-1 Establishing an inflammation model by LPS treatment; positive control group: dexamethasone DMX0.5 mug.L ^-1 After 2h of dry period, 10 mug.L is added ^-1 LPS stimulation of (b); drug administration group: adding different concentrations (5 mg.L) ^-1 、10mg·L ^-1 、20mg·L ^-1 ) Is subjected to dry prognosis for 2h with 10. Mu.g.L ^-1 Is stimulated by LPS.

2.3 detection method

2.3.1 Effect on RAW264.7 cell viability

Taking cells in logarithmic growth phase, and digesting with 0.25% trypsin to obtain 1×10 ⁵ Individual mL ^-1 Single cell suspension was spotted at 100. Mu.L per well in 48 well plates and placed at 37℃in 5% CO ₂ Culturing in incubator for 24 hr, adding different concentrations (5 mg.L) ^-1 、10mg·L ^-1 、20mg·L ^-1 ) After 2h pretreatment of the antamimetic acid A, 10. Mu.g.L was added ^-1 The LPS-stimulated cells of (C) were further cultured for 24 hours, and 20. Mu.L, 5 mg.L, was added to each well cell of each treatment group 4 hours before the termination of the cell culture ^-1 Continues incubation at 37 ℃. After the cell culture was terminated, 150. Mu.L of DMSO was added to each well, and the mixture was shaken until the intracellular crystals were completely dissolved, and the absorbance (OD value) of each well was measured at a wavelength of 530nm using an enzyme-labeled instrument.

2.3.2 detection of LPS-induced RAW264.7 cell NO

Will be adjusted to 1X 10 ⁵ Individual mL ^-1 Single cell suspension was spotted at 100. Mu.L per well in 48 well plates, and after grouping treatment according to the procedure described in 4.2.2, 37℃and 5% CO ₂ Culturing was continued in the incubator for 24 hours, and the supernatant was aspirated, and the NO content in the cell culture broth was measured according to the instructions of the NO measurement kit (Griess method).

2.3.3 LPS-induced RAW264.7 cells IL-1 beta, IL-6, TNF-alpha and PGE ₂ Is detected by (a)

Taking logarithmic phase cell to make cell suspension concentration 1×10 ⁵ Individual mL ^-1 100. Mu.L of each well was spotted in 48-well plates, and after grouping treatment according to the procedure described in 4.2.2, the procedure was performed at 37℃with 5% CO ₂ After cell culture was continued for 24 hours, the supernatant was collected and assayed for IL-1. Beta., IL-6, TNF-. Alpha.and PGE, respectively, according to the methods described in ELISA kit ₂ Is a secretion amount of (a).

2.4 statistical analysis

Statistical analysis was performed using SPSS17.0 software, and the detection results for each group were as followsThe representation, P, compares the differences between groups using one-way anova<0.05 isThe differences are statistically significant.

3. Results

3.1 Effect on RAW264.7 cell viability

The MTT results are shown in fig. 9, and after 24h of cell intervention with three concentrations of amastatin a, all three doses of amastatin a were statistically not significant (P > 0.05) compared to the blank, indicating that amastatin a had no significant effect on cell activity.

3.2NO detection

As shown in FIG. 10, the effect of NO production was significantly increased in the model group compared with the blank group, and the difference was statistically significant (P<0.01). Compared with the model group, the different doses of the antamimetic acid A treatment group have significantly reduced NO generation, and the difference has statistical significance (P<0.01 Low, medium and high doses of antamimetic acid a were compared to the model group and the NO content was gradually reduced with inhibition rates of 22.2%, 30.3% and 50.6% for the low, medium and high doses, respectively. In fig. 10, compared with the blank group ^## P<0.01; compared with the model group ^** P<0.01。

3.3 detection of IL-1 beta, IL-6

The model group showed a significant increase in both IL-1β and IL-6 (P < 0.01) compared to the control group, while the secretion of IL-1β and IL-6 by the treatment with antamimetic A was significantly reduced (P < 0.01) compared to the model group, wherein the secretion of IL-1β by the treatment with low and medium doses of antamimetic A was statistically significant (P > 0.05) compared to the positive control group; the difference in IL-1 beta secretion after high dose treatment of antamimetic A compared to the positive control group was statistically significant (P < 0.01). The results are shown in Table 6.

TABLE 6 three doses of antamimetic acid A for LPS-induced IL-1 beta and IL-6 production in mouse cells

Compared with blank group ^## P<0.01; compared with the model group ^** P<0.01; compared with the positive group ^Δ P<0.05， ^ΔΔ P<0.01

3.4 detection of TNF-alpha, PGE2

TNF-alpha and PGE ₂ The generation of TNF-. Alpha.and PGE was compared with the model control group and the blank group as shown in Table 7 ₂ Is significantly increased (P)<0.01 Three doses of antamimetic A were compared to model groups, TNF-. Alpha.and PGE ₂ Is significantly reduced in the production of (P)<0.01). Among them, in the production of TNF-alpha, the three doses of the A-ambroxol, namely, the low, medium and high doses, gradually decrease, and the 20mg/L dose of the A-ambroxol is most effective. PGE treated with medium and high doses of antamimetic acid A ₂ The secretion amount is lower than that of the low-dose group of the ambroxol A, and has no obvious difference with the positive control group (P>0.05)。

TABLE 7 three doses of antamimetic acid A for LPS-induced production of mouse cells TNF- α, PGE2

Compared with blank group ^## P<0.01; compared with the model group ^** P<0.01; compared with the positive group ^ΔΔ P<0.01。

Reference is made to:

1)Nishikori S,Takemoto K,Kamisuki S,Nakajima S,Kuramochi K,Tsukuda S,Iwamoto M,Katayama Y,Suzuki T,Kobayashi S,Watashi K,Sugawara F.Anti-hepatitis C Virus NaturalProduct from a Fungus,Penicilillium herquei.Journal of Natural Product,2016,79,442-446.

2)Olesya I.Zhuravleva,Maria P.Sobolevskaya,Elena V.Leshchenko,Natalya N.Kirichuk,Vladimir A.Denisenko,Pavel S.Dmitrenok,Sergey A.Dyshlovoy,Alexander M.Zakharenko,Natalya Yu.Kim,and Shamil Sh.Afiyatullov.Meroterpenoids from the Alga-Derived FungiPenicilillium thomii Maire and Penicilillium lividum Westling.Journal of Natural Product,

2014,77,1390–1395.

3) Li Chunyuan, ding Weijia, shao Changlun, she Zhigang, lin Yongcheng. Research on active metabolites of mangrove penicillium fungi (No 12556). Chinese medicinal materials 2008, 31, 960-962.

4) Pengfei, left Wen Jian, guo Zhikai, mei Wenli. Fu. Chenopodium triquetrum FJ-1 chemical composition study [ J ].

Pharmaceutical report 2013,48 (11): 1688-1691.

5) Raw henna; aging; bai Jiao; hua Huiming; xu Wenfeng; pei Yuehu. Isolation and identification of the secondary metabolite of Penicillium oxalicum HSY-P-17. University of Shenyang pharmacy report 2014,31;34-36.

6)Steven A.Wakelin,Rosemary A.Warren,Paul R.Harvey,Maarten H.Ryder.Phosphatesolubilization by Penicilillium spp.closely associated with wheat roots.Biologyand Fertility of Soils.2004,40(1):36-43

7) Mingbo A Penicillium phosphate-dissolving mould P8, chinese patent 200810118076.9

8) Ma Zhongyan Liang Xiaowen Wang Genhao Chen Xiaoyan Zhou Lifeng Li Xiaoyu Zeng Fanfu Paecilomyces lilacinus and application thereof, chinese patent 201310644028.4

9) Liu Bo, huang Sufang, shi Huai, li Fang, zhu Yojing, tang Jianyang. Paecilomyces lilacinus strain for degrading phoxim and application thereof, chinese patent 201210276405.9

10)M.LEGGETT,N.K.NEWLANDS,D.GREENSHIELDS,L.WEST,S.INMAN,and M.E.

KOIVUNEN，Maize yield response to a phosphorus-solubilizing microbial inoculant in fieldtrials，J Agric Sci.2015；153(8)：464–1478

11 Xu Yangchun Shen Jirong of Wang Shimei Li Zhendai Le Tianwei, a strain of Penicillium oxalicum NJDL-03 and its use, chinese patent 201510713680.6

12 Chen Zhouan Tan Youjiu Li Chengwei Ning Sunchang peptide, a paecilomyces gracilomyces strain and application thereof, chinese patent 201210062125.8

13 Soil agrochemistry analysis [ M ] Beijing: agricultural press, 1980.

<110> Zheng Yinan

<120> antamimetic acid A, preparation method and application thereof

<160> 1

<210> 1

<211> 520bp

<212> DNA

<213> Penicillium sp.

<400> 1

GCATCGATGA AGAACGCAGC GAAATGCGAT AACTAATGTG AATTGCAGAA TTCAGTGAAT 60

CATCGAGTCT TTGAACGCAC ATTGCGCCCC CTGGTATTCC GGGGGGCATG CCTGTCCGAG 120

CGTCATTGCT GCCCTCAAGC ACGGCTTGTG TGTTGGGCCT CCGTCCCCCC GCCCCGGGGG 180

GACGGGCCCG AAAGGCAGCG GCGGCACCGT GTCCGGTCCT CGAGCGTATG GGGCTTTGTC 240

ACCCGCTCTG TAGGCCCGGC CGGCGCCTGC CGACCCCCAA TCATCCTTTT TTTTCAGGTT 300

GACCTCGGAT CAGGTAGGGA TACCCGCTGA ACTTAAGCAT ATCAATAAGC GGAGGAAAAG 360

AAACCAACAG GGATTGCCCC AGTAACGGCG AGTGAAGCGG CAAGAGCTCA AATTTGAAAG 420

CTGGCTCCTT CGGGGTCCGC ATTGTAATTT GCAGAGGATG CTTCGGGAGC GGTCCCCATC 480

TAAGTGCCCT GGAACGGGCT GTCATAGAGG GTGAGAATCC 520

Claims

1. The antamimetic acid A is characterized by comprising the following chemical name: 2- {2- [ (2E) -3- [ (2-aminophenyl) carboxamido]Prop-2-enamido]Ethyl } malonic acid; the molecular formula is C ₁₅ H ₁₇ N ₃ O ₆ The molecular weight is 335.1117; the chemical structure is as follows:

the compound is light yellow amorphous powder, is easy to dissolve in water, and is dimethyl sulfoxide.

2. Use of antamimetic acid a according to claim 1 in the manufacture of an anti-inflammatory medicament.