CN114410477B - Inhibitor of inducible NO synthetase, and production strain and preparation method thereof - Google Patents
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Abstract
The invention discloses an inhibitor of inducible NO synthase, a production strain and a preparation method thereof; wherein, the deep sea fungus is preserved in China Center for Type Culture Collection (CCTCC) No. M20211395; the preservation time is as follows: 2021, 11/11. The deep sea fungi can effectively produce the compound with the inhibitory activity on Nitric Oxide (NO), and the compound not only has the efficacy of an inhibitor of inducible NO synthase (iNOS), but also has NO toxicity on cells, and can ensure that the cell survival rate reaches 100 percent without side effects.
Description
Technical Field
The invention relates to the field of biological pharmacy, in particular to an inhibitor of inducible NO synthase, a production strain thereof and a preparation method thereof.
Background
Inflammation is an important physiological cellular response activated by invasion of foreign pathogens and harmful stimuli, and is thought to be an innate immune mechanism that helps repair damaged tissues and to clear both the stimuli and the damaged cells. Inflammation is a complex physiological process involving the immune/vascular system and molecular mediators, all of which are involved in sequestering inflammatory factors such as bacteria, viruses and damaged cells, as a potential self-defense mechanism. Activated macrophages mediate inflammatory processes by secreting a variety of pro-inflammatory mediators, including Nitric Oxide (NO), prostaglandin E2 (PGE 2), tumor necrosis factor- α (TNF- α), interleukin-1 β (IL-1 β), and interleukin-6 (IL-6). Where NO is a cell-derived signaling molecule, produced by Inducible Nitric Oxide Synthase (iNOS), an important physiological molecule that exhibits various biological functions, such as vasodilation, recruiting leukocytes into tissues by relaxing and reducing smooth muscle cells and platelet aggregation. However, abnormal production of NO leads to the formation of superoxide anions (ONOO-), DNA damage and cell death.
Regarding the mechanism of various inflammatory pathogenic actions, nitric Oxide (NO) in vivo is known to be a toxic substance that mediates cellular immunity and inflammation. The precursor is L-arginine (L-arg), which generates NO under the action of NO synthase (NOS). Three different types of NOS have been isolated, including endothelial NO synthase (eNOS), neuronal NO synthase (nNOS) and inducible NO synthase (iNOS). It is known that macrophages, hepatocytes, smooth muscle cells, adenocarcinoma cells and epithelial cells all express iNOS. Some inflammatory cytokines and microbial products such as Lipopolysaccharide (LPS) induce expression of iNOS. iNOS, once induced, expresses a high level of activity, producing large amounts of NO. Excessive production of NO causes an increase in inflammation and even eventually causes cancer.
Thus, there has been a long effort to find inhibitors of the inducible NO synthase iNOS for the treatment of inflammatory diseases associated therewith. However, these inhibitors are mostly limited to chemically synthesized substances, and have a large side effect.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the defect that the chemical synthesis is adopted in the inhibitor of the inducible NO synthase iNOS in the prior art, which leads to larger side effect, thereby providing an inhibitor of the inducible NO synthase which has NO harm to cells, and a production strain and a preparation method thereof.
A deep sea fungus (Trichoderma sp.SCSIOW21) is deposited with China center for type culture Collection, with the accession number of CCTCC No. M20211395, and the preservation time of 2021, 11 months and 11 days.
An application of the inhibitor for producing inducible NO synthetase by using the deep sea fungi.
An inhibitor of inducible NO synthase comprising at least one compound of the following formulas 1 to 7,
a method of preparing an inhibitor of inducible NO synthase, comprising: culturing the above deep sea fungus to obtain a culture, and separating from the culture to obtain a product containing at least one compound of formula 1-formula 7.
The culture process comprises the following steps: after resuscitating the deep sea fungus strain, inoculating the strain into a seed culture medium, and culturing for 47-49 hours at the temperature of 27-29 ℃ to obtain a seed culture solution; inoculating the seed culture solution into a rice culture medium, and culturing for 40-50 days to obtain a culture.
The seed culture medium comprises the following components in percentage by mass: glucose 1.9-2.1%, peptone 0.9-1.1%, yeast extract 0.4-0.6%, sea salt 2-4%, and water in balance.
The rice culture medium comprises sea salt, water and rice; the sea salt: water: the mass ratio of the rice is 7-8:240-260:140-160.
The preparation method of the rice culture medium comprises the following steps: weighing sea salt according to a proportion, dissolving the sea salt in deionized water, adding rice after the sea salt is completely dissolved, and soaking overnight.
The inoculation amount of the deep sea fungi in the rice culture medium is as follows: 5mL of seed culture solution is inoculated into every 140-160 g of rice.
The separation process comprises the following steps: sequentially extracting the culture with ethyl acetate and n-butanol, collecting n-butanol extract, and concentrating n-butanol extract to obtain fermentation broth extract; repeating the extraction steps at least once, and mixing the fermented liquid extractum to obtain n-butanol fraction; the n-butanol fraction adopts silica gel column chromatography to obtain eight components A-H with different polarities, and any one or more compounds in formulas 1-7 are obtained from the components B-D by reverse silica gel column chromatography and reverse HPLC separation and purification.
The B and C fractions were combined and then subjected to gradient elution with MeOH-water through a medium pressure ODS column to give 5 subfractions, designated subfractions 1-5, respectively. Purifying and separating the sub fraction 2 to obtain a compound shown in a formula 1; purifying and separating the sub fraction 3 to obtain compounds shown in formulas 2, 7 and 6; purifying and separating the sub-fraction 5 to obtain the compound shown in the formula 3.
Subjecting the D component to medium pressure liquid chromatography YMC-ODS-A C 18 Separating by chromatography column, gradient eluting with MeOH-water to obtain 14 subfractions, respectively named subfractions 1-14, wherein subfraction 7 is purified and separated to obtain compound shown in formula 5, and subfraction 9 is purified and separated to obtain compound shown in formula 4.
The technical scheme of the invention has the following advantages:
1. the deep sea fungus provided by the invention can effectively produce the compound with the inhibitory activity on Nitric Oxide (NO), has the efficacy of an inhibitor of inducible NO synthase (iNOS), is non-toxic to cells, can ensure that the cell survival rate reaches 100%, and has NO side effect.
2. The compounds in the formulas 1-7 provided by the invention are detected to have inhibitory activity on Nitric Oxide (NO), so that the compounds can be used as an inhibitor of inducible NO synthase; wherein, at an experimental concentration of 100 μm, the inhibition rates of the two compounds of formula 2 and formula 7 can reach 81.8% and 50.5%, respectively, and the corresponding cell survival rates are 100%, which indicates that the two compounds of formula 2 and formula 7 have good NO inhibition activity at such concentrations, and are nontoxic to cells, and the compounds can be used for preparing drugs for inhibiting nitric oxide production, i.e. can be used as inhibitors of inducible NO synthase.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is an absolute configuration diagram obtained by X-ray single crystal diffraction of the formulae 1 and 2 in example 1 of the present invention;
FIG. 2 is a graph of spectra obtained by ECD calculation of compounds according to the present invention.
FIG. 3 is a graph showing the Nitric Oxide (NO) inhibitory effect of the compounds of formulae 1 to 7 on RAW264.7 cells according to the present invention.
Detailed Description
Example 1
A deep sea fungus (Trichoderma sp.SCSIOW21) is deposited with China center for type culture Collection, with the accession number of CCTCC No. M20211395, and the preservation time of 2021, 11 months and 11 days.
The process of fermenting and culturing the deep sea fungus Trichoderma sp.SCSIOW21 and separating the inhibitor of the induced NO synthase comprises the following steps:
1. seed culture:
resuscitates the deep sea fungus strain Trichoderma sp.SCSIOW21, inoculates the strain into seed culture medium, and shake cultures at 28deg.C and 200r/min for 47-49 hr to obtain seed culture solution. Wherein, the formula of the seed culture medium is as follows: glucose 2%, peptone 1%, yeast extract 0.5%, sea salt 3% and water in balance; the mixture was autoclaved at 121℃for 20 minutes.
2. Fermentation culture:
in an ultra clean bench, 5mL of seed culture broth was inoculated into 2000mL conical flasks containing rice medium, 60 total flasks. Standing and culturing at room temperature for 40 days to obtain fermentation culture of deep sea fungus Trichoderma sp.SCSIOW21.
Wherein, the preparation process of the rice culture medium comprises the following steps: 450 g of sea salt is weighed and dissolved in 15L of deionized water, and after the sea salt is completely dissolved, the sea salt is split into 60 conical flasks of 2000mL containing 150 g of rice on average and soaked overnight.
3. And (3) extracting and separating:
at the end of the incubation, 100mL of ethyl acetate (EtOAc) was added to each conical flask, concentrated in a vacuum rotary evaporator at less than 50℃and after evaporation of the remaining EtOAc, 100mL of water-saturated n-butanol (BuOH) was added to each conical flask and concentrated 3 times under a vacuum rotary evaporator to give a BuOH fraction (12.9 g).
The BuOH fraction was stirred with silica gel and packed in a silica gel column with chloroform (CH 2 Cl 2 ) Methanol (MeOH) -water as eluting solvent, followed by 100% CH 2 Cl 2 CH in a volume ratio of 50:1:0, 20:1:0, 10:1:0, 5:1:0.1, 3:1:0.1, 1:1:0.1 2 Cl 2 Elution with 2.0L each of MeOH-water, and 100% MeOH, provided eight components A-H.
Combining component B and component C and passing through component C at atmospheric pressure 18 Eluting with MeOH-water at volume ratio of 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0 respectively at 1000mL, and naturally flowing at normal pressure to obtain 6 components named as sub-fractions 1-6. By semi-prepared YMC-ODS-A C 18 (20X 250mm,5 μm) HPLC column (acetonitrile-water volume ratio 40:60,5.0ml/min, t) R 52.1 min) to isolate component No. 2, to give formula 1 (9.0 mg). By semi-prepared YMC-ODS-A C 18 (20X 250mm,5 μm) HPLC column (acetonitrile-water volume ratio 47:53,10 ml/min) to purify fraction 3 to give formula 2 (t) R 49.2min,3.0 mg), 7 (t) R 32.8min,0.3 mg) and formula 6 (t R 34.1min,0.4 mg). By semi-prepared YMC-ODS-AC 18 (20X 250mm,5 μm) HPLC column (acetonitrile-water volume ratio 70:30,10.0ml/min, t) R 15.9 min) component No. 5 was isolated to give formula 3 (0.5 mg).
Passing component D through YMC-ODS-A C under normal pressure 18 Chromatography column elution with a gradient of 1000mL each of MeOH-water (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 and 100% MeOH) gave 10 fractions, designated subfractions 1-10, respectively. By semi-prepared YMC-ODS-A C 18 (20X 250mm,5 μm) HPLC column (acetonitrile-water volume ratio 18:82, 10ml/min, t) R 29.6 min) purification of the 7 th fraction to give formula 5 (1.6 mg). By semi-prepared YMC-ODS-A C 18 (20X 250mm,5 μm) HPLC column (acetonitrile-water volume ratio 23:77, 10.0ml/min, t) R 41.5 min) purification of fraction 9 to give formula 4 (0.6 mg).
Example 2
The process of fermenting and culturing the deep sea fungus Trichoderma sp.SCSIOW21 and separating the inhibitor of the induced NO synthase comprises the following steps:
1. seed culture:
resuscitates the deep sea fungus strain Trichoderma sp.SCSIOW21, inoculates the strain into seed culture medium, and shake cultures at 27deg.C and 200r/min for 47-49 hr to obtain seed culture solution. Wherein, the formula of the seed culture medium is as follows by mass percent: glucose 1.9%, peptone 1.1%, yeast extract 0.6%, sea salt 4% and water in balance; the mixture was autoclaved at 121℃for 20 minutes.
2. Fermentation culture:
in an ultra clean bench, 5mL of seed culture broth was inoculated into 2000mL conical flasks containing rice medium, 60 total flasks. Standing and culturing at room temperature for 40 days to obtain fermentation culture of deep sea fungus Trichoderma sp.SCSIOW21.
Wherein, the preparation process of the rice culture medium comprises the following steps: 420 g of sea salt is weighed and dissolved in 15.6L of deionized water, and after the sea salt is completely dissolved, the sea salt is split into 60 conical flasks of 2000mL containing 140 g of rice on average and soaked overnight.
3. And (3) extracting and separating: the extraction and separation process was the same as in example 1.
Example 3
The process of fermenting and culturing the deep sea fungus Trichoderma sp.SCSIOW21 and separating the inhibitor of the induced NO synthase comprises the following steps:
1. seed culture:
resuscitates the deep sea fungus strain Trichoderma sp.SCSIOW21, inoculates the strain into seed culture medium, and shake cultures at 29 deg.C and 200r/min for 47-49 hr to obtain seed culture solution. Wherein, the formula of the seed culture medium is as follows: glucose 2.1%, peptone 0.9%, yeast extract 0.4%, sea salt 2% and water in balance; the mixture was autoclaved at 121℃for 20 minutes.
2. Fermentation culture:
in an ultra clean bench, 5mL of seed culture broth was inoculated into 2000mL conical flasks containing rice medium, 60 total flasks. Standing and culturing at room temperature for 40 days to obtain fermentation culture of deep sea fungus Trichoderma sp.SCSIOW21.
Wherein, the preparation process of the rice culture medium comprises the following steps: 480 g of sea salt was weighed and dissolved in 14.4 l of deionized water, and after the sea salt was completely dissolved, the mixture was split into an average of 60 conical flasks of 2000mL containing 160g of rice and soaked overnight.
3. And (3) extracting and separating: the extraction and separation process was the same as in example 1.
Test example 1: identification of the relative Structure of Compounds of formulas 1-7
The compounds of formulas 1 to 7 prepared in example 1 above were subjected to structural analysis test to obtain the following physicochemical property data.
Formula 1: colorless crystals; [ alpha ]] 25 D +64.1(c 0.36,MeOH);UV(MeOH)λ max (logε)252.6(3.77)nm;ECD(0.12mg/ml,MeOH)λ max (Δε)245(-36.7),292(+7.4),351(+10.6)nm;IR(KBr)v max 3402,2927,1734,1695,1541,1435,1338,1190,1043cm-1; NMR data (600/150 MHz, DMSO-d) 6 TMS is an internal standard, J in Hz, δin ppm) is shown in Table 1; HREIMS m/z 317.2115[ M+H ]] + (calculated C) 20 H 29 O 3 ,317.2117)。
TABLE 1
Formula 4: colorless gel; [ alpha ]] 25 D +15.3(c 0.28,MeOH);UV(MeOH)λ max (logε)256.0(3.89)nm;ECD(0.14mg/ml,MeOH)λ max (Δε)247(-36.7),349(+2.7)nm;IR(KBr)v max 3402,2927,1734,1695,1541,1435,1338,1190,1043cm -1 The method comprises the steps of carrying out a first treatment on the surface of the NMR data (600/150 MHz, DMSO-d) 6 TMS is an internal standard, J in Hz, δin ppm) is shown in Table 2; HREIMS m/z 319.2278[ M+H ]]++ (calculated C) 20 H 31 O 3 ,319.2273)。
TABLE 2
a The signals are superimposed.
Formula 5: colorless gel; [ alpha ]] 25 D +14.1(c 0.15,MeOH);UV(MeOH)λ max (logε)251.0(4.07)nm;ECD(0.15mg/ml,MeOH)λ max (Δε)245(-8.2),358(+4.8)nm;IR(KBr)v max 3360,2922,1732,1668,1541,1435,1338,1122,1024cm-1; 1 H NMR 13 C NMR data (600/150 MHz, DMSO-d) 6 TMS is an internal standard, jin Hz, δin ppm) is shown in Table 3; HREIMS m/z 335.2229[ M+H ]] + (calculated C) 20 H 31 O 4 ,335.2222)。
TABLE 3 Table 3
Formula 6: an amorphous powder; [ alpha ]] 25 D +10.1(c 0.18,MeOH);UV(MeOH)λ max (l ogε)252.3(4.19)nm;ECD(0.18mg/ml,MeOH)λ max (Δε)220(+1.7),245(-4.9),353(+2.4)nm;IR(KBr):v max 3379,2924,1734,1647,1558,1456,1435,1153,1049cm-1; 1 H NMR 13 C NMR data (600/150 MHz, DMSO-d) 6 TMS is an internal standard, J in Hz, δin ppm) is shown in Table 4; HREIMS m/z 341.2089[ M+Na ]] + (calculated C) 20 H 30 NaO 3 ,341.2093)。
TABLE 4 Table 4
Formula 7: an amorphous powder; [ alpha ]] 25 D +12.0(c 0.14,MeOH);UV(MeOH)λ max (logε)256.3(4.11)nm;ECD(0.14mg/ml,MeOH)λ max (Δε)255(-1.8),340(+1.1)nm;IR(KBr)v max 3360,2922,1718,1660,1558,1465,1338,1147,1058cm-1; 1 H NMR 13 C NMR data (600/150 MHz, DMSO-d) 6 TMS is an internal standard, jin Hz, δin ppm) is shown in Table 5; HREIMS m/z 319.2269[ M+H ]] + (calculated C) 20 H 31 O 3 ,319.2273)。
TABLE 5
X-ray single crystal diffraction was performed on formulas 1 and 2 to obtain absolute configurations thereof, as shown in FIG. 1, wherein a is an absolute configuration diagram of formula 1 and b is an absolute configuration diagram of formula 2. The absolute configuration of the obtained other compound was determined by ECD calculation, and the experimental result is shown in FIG. 2, wherein A is the experimental and calculation curve (2S, 5R,6R,8S,13S, 14S) ECD formula 1 spectrum, B is the experimental and calculation curve (2S, 5R,6R,13S, 14S) ECD formula 4 spectrum, C is the experimental and calculation curve (2S, 5R,6R,8S,13S, 14S) ECD formula 5 spectrum, D is the experimental and calculation curve (2S, 5R,6R,8S,13S, 14S) ECD formula 6 spectrum, and E is the experimental and calculation curve (2S, 5R,6R,13S,14S, 15S) ECD formula 7 spectrum.
From the above physicochemical data analysis of tables 1 to 5, in combination with the calculation results of the ECD test of the absolute configuration of formulas 1 to 7, it can be determined that the absolute structures of formulas 1 to 7 are as follows:
thus, it was confirmed that the deep sea fungus Trichoderma sp.SCSIOW21 of the present invention can efficiently produce and isolate the compounds represented by formulas 1 to 7.
Test example 2: experiments on anti-inflammatory Activity of Compounds of formulas 1-7
1. Detection of cell culture
Mouse RAW264.7 macrophages from American type culture Collection (ATCC, manassas, va., USA) were cultured in RPMI-1640 medium supplied with 10% heat-inactivated fetal bovine serum (Gibco) at 37℃with 5% CO 2 And a humidified incubator with 95% air. The medium was changed periodically every two days. After RAW264.7 cells reached confluence, test cells were obtained by trypsinization passaging.
2. Nitric oxide inhibition assay
The cells were assayed at 1.0X10 5 The density of individual cells/wells was seeded in 96-well plates and grown for 2 hours to allow cells to adhere to the plates. Test samples were dissolved in DMSO (dimethyl sulfoxide) and then subjected to double concentration gradient dilution in RPMI-1640 (Thermo Fisher Scientific, carlsbad, calif., USA) medium to give final concentrations of 100, 50, and 25. Mu.M for sample formulas 1-7 in the medium.
The test cells were added to the medium simultaneously with LPS (1.5 g/mL, sigma, st.Louis, MO, USA) and recombinant mouse interferon IFN-gamma (10 ng/mL, peproTech, rocky Hill, NJ, USA).
The cells were then incubated at 37℃for about 24 hours, followed by cooling on ice.
Subsequently, 100 μl of cell culture supernatant was added in duplicate to wells of a 96-well plate. To quantify NO, 50 μl Griess reagent (1% sulfanilamide, 5%H 2 PO 4 Solution and 0.1% n-1-naphthalamide dihydrochloride) was added to each well.
After 10 minutes, the reaction products were colorimetrically quantified using a microplate reader (BIO-RAD, hercules, calif., USA) at 550 nm. The concentration of NO was calculated using a standard calibration curve, and the concentration of NO at different sample types and concentrations is shown in fig. 3.
As can be seen from FIG. 3, the compounds of formulas 1 to 7 all have the activity of inhibiting the production of Nitric Oxide (NO). 2. 3, 7 showed the strongest NO generation inhibitory activity at 100 μm, with inhibition rates of 81.8%, 46.8% and 50.5%, respectively, and the remaining compounds showed weak NO inhibitory activity at high concentrations.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (4)
1. An inhibitor of inducible NO-synthase produced by deep sea fungi, characterized by comprising at least one compound of the following formula 1-formula 7,
the deep sea fungi are preserved in China Center for Type Culture Collection (CCTCC) No. M20211395; the preservation time is as follows: 2021, 11/11.
2. A method for preparing an inhibitor of inducible NO synthase, comprising: culturing the deep sea fungus of claim 1 to obtain a culture, and isolating the culture to obtain a product comprising at least one compound of formulae 1-7; the culture process comprises the following steps: after resuscitating the deep sea fungus strain, inoculating the strain into a seed culture medium, and culturing at 27-29 ℃ for 47-49 hours to obtain a seed culture solution; inoculating the seed culture solution into a rice culture medium, and culturing for 40-50 days to obtain a culture;
the seed culture medium comprises the following components in percentage by mass: 1.9-2.1% of glucose, 0.9-1.1% of peptone, 0.4-0.6% of yeast extract, 2-4% of sea salt and the balance of water;
the rice culture medium comprises sea salt, water and rice; the sea salt: water: the mass ratio of the rice is 7-8, 240-260 and 140-160.
3. The preparation method according to claim 2, wherein the preparation method of the rice culture medium comprises the following steps: weighing sea salt according to a proportion, dissolving the sea salt in deionized water, adding rice after the sea salt is completely dissolved, and soaking overnight.
4. A method according to claim 2 or 3, wherein the deep sea fungi are inoculated in the rice culture medium in an amount of: and inoculating 5mL of seed culture solution into every 140-160 g of rice.
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Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102952756A (en) * | 2012-08-23 | 2013-03-06 | 浙江工业大学 | Marine fungi altemaria MNP801 and application thereof |
CN103074233A (en) * | 2012-12-25 | 2013-05-01 | 浙江工业大学 | Marine fungus penicillium chrysogenum and application thereof to preparation of anti-tumor medicines |
CN104031844A (en) * | 2014-05-19 | 2014-09-10 | 广东省微生物研究所 | Cladosporium sp. strain, and extract and application thereof |
CN105503531A (en) * | 2015-12-04 | 2016-04-20 | 深圳大学 | Extract of fungus culture as well as preparation method and application of extract |
CN106497797A (en) * | 2016-11-01 | 2017-03-15 | 深圳大学 | A kind of extract of fungal cultures and its preparation method and application |
CN108265007A (en) * | 2017-12-18 | 2018-07-10 | 国家海洋局第三海洋研究所 | The application of deep-sea fungi 3A00421 and its fermented cpds |
CN108277164A (en) * | 2017-12-28 | 2018-07-13 | 中山大学 | A kind of indene derivative in sea paint endogenetic fungus source and its application in preparing anti-inflammatory drug |
CN108795774A (en) * | 2017-04-28 | 2018-11-13 | 华中科技大学 | The separation application of new steroid compound in a kind of Phomopsis and its secondary metabolite |
CN111602662A (en) * | 2020-06-22 | 2020-09-01 | 深圳大学 | Preparation method and application of tricobactin |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1076703B2 (en) * | 1998-05-15 | 2010-12-15 | Genentech, Inc. | Therapeutic uses of il-17 homologous polypeptides |
-
2021
- 2021-11-29 CN CN202111434433.4A patent/CN114410477B/en active Active
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102952756A (en) * | 2012-08-23 | 2013-03-06 | 浙江工业大学 | Marine fungi altemaria MNP801 and application thereof |
CN103074233A (en) * | 2012-12-25 | 2013-05-01 | 浙江工业大学 | Marine fungus penicillium chrysogenum and application thereof to preparation of anti-tumor medicines |
CN104031844A (en) * | 2014-05-19 | 2014-09-10 | 广东省微生物研究所 | Cladosporium sp. strain, and extract and application thereof |
CN105503531A (en) * | 2015-12-04 | 2016-04-20 | 深圳大学 | Extract of fungus culture as well as preparation method and application of extract |
CN106497797A (en) * | 2016-11-01 | 2017-03-15 | 深圳大学 | A kind of extract of fungal cultures and its preparation method and application |
CN108795774A (en) * | 2017-04-28 | 2018-11-13 | 华中科技大学 | The separation application of new steroid compound in a kind of Phomopsis and its secondary metabolite |
CN108265007A (en) * | 2017-12-18 | 2018-07-10 | 国家海洋局第三海洋研究所 | The application of deep-sea fungi 3A00421 and its fermented cpds |
CN108277164A (en) * | 2017-12-28 | 2018-07-13 | 中山大学 | A kind of indene derivative in sea paint endogenetic fungus source and its application in preparing anti-inflammatory drug |
CN111602662A (en) * | 2020-06-22 | 2020-09-01 | 深圳大学 | Preparation method and application of tricobactin |
Non-Patent Citations (3)
Title |
---|
Novel Harziane Diterpenes from Deep-Sea Sediment Fungus Trichoderma sp. SCSIOW21 and Their Potential Anti-Inflammatory Effects;Hongxu Li等;《Marine Drugs》;第19卷(第12期);第1-11页 * |
分离自木霉菌Trichoderma sp.的新型化合物Trichoderol A及绝对构型的确定;许兰兰等;《高等学校化学学报》;第37卷(第11期);第1972-1976页 * |
翟欣.内生真菌Trichoderma atroviride D16促进丹参酮合成的机制及其栽培应用.2019,(第10期),第D047-6页. * |
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