CN114410477B - Inhibitor of inducible NO synthetase, and production strain and preparation method thereof - Google Patents

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

An inhibitor of inducible NO synthase and its production strain and preparation method

Technical field

The invention relates to the field of biopharmaceuticals, and specifically relates to an inhibitor of inducible NO synthase, its production strain and preparation method.

Background technique

Inflammation is an important physiological cellular response activated by the invasion of foreign pathogens and harmful irritants and is considered an innate immune mechanism that helps repair damaged tissues and clear irritants and damaged cells. As a potential self-defense mechanism, inflammation is a complex physiological process involving the immune/vasculature system and molecular mediators, all of which are involved in sequestering inflammatory agents such as bacteria, viruses, and damaged cells. Activated macrophages mediate the inflammatory process by secreting a variety of pro-inflammatory mediators, including nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL- 1β) and interleukin-6 (IL-6). NO is a cell-derived signaling molecule produced by inducible nitric oxide synthase (iNOS). This important physiological molecule exhibits a variety of biological functions, such as vasodilation, by relaxing and reducing smooth muscle cells and platelet aggregation. Recruits white blood cells into tissues. However, abnormal production of NO can lead to the formation of superoxide anion (ONOO-), DNA damage, and cell death.

Regarding the mechanism of various inflammatory pathogenic effects, it is known that nitric oxide (NO) in the body is a toxic substance that mediates cellular immunity and inflammation. Its precursor is L-arginine (L-arg), which generates NO under the action of NO synthase (NOS). Three different types of NOS have been isolated so far, including endothelial NO synthase (eNOS), neuronal NO synthase (nNOS) and inducible NO synthase (iNOS). It is currently known that macrophages, hepatocytes, smooth muscle cells, adenocarcinoma cells and epithelial cells can all express iNOS. Certain inflammatory cytokines and microbial products such as lipopolysaccharide (LPS) can induce iNOS expression. Once induced, iNOS can express high activity and produce large amounts of NO. Excessive production of NO causes inflammation and even eventually causes cancer.

Therefore, for a long time, people have been committed to finding inhibitors of inducible NO synthase iNOS to treat inflammatory diseases related to it. However, most of these inhibitors are limited to chemically synthesized substances and have serious side effects.

Contents of the invention

Therefore, the technical problem to be solved by the present invention is to overcome the shortcoming of existing inhibitors of inducible NO synthase iNOS that are chemically synthesized, resulting in greater side effects, thereby providing an inducible NO that does no harm to cells. Synthase inhibitors, production strains and preparation methods thereof.

A deep-sea fungus (Trichoderma sp.SCSIOW21). The deep-sea fungus Trichoderma sp.SCSIOW21 is deposited in the China Type Culture Collection Center. The address is Wuhan University, China. The deposit number is CCTCC No: M20211395. The deposit date is November 2021. 11th.

An application of utilizing the above-mentioned deep-sea fungus to produce an inhibitor of inducible NO synthase.

An inhibitor of inducible NO synthase, including at least one compound of the following formula 1 to formula 7,

A method for preparing an inhibitor of inducible NO synthase, including: culturing the above-mentioned deep-sea fungus to obtain a culture, and isolating from the culture a product containing at least one compound of Formula 1 to Formula 7.

The culture process is as follows: after the deep-sea fungal strains are revived, the strains are inoculated into the seed culture medium, and cultured at 27-29°C for 47-49 hours to obtain the seed culture liquid; then the seed culture liquid is inoculated into the rice culture medium. , culture for 40 to 50 days, and obtain the culture.

In terms of mass percentage, the seed culture medium includes: glucose 1.9-2.1%, peptone 0.9-1.1%, yeast extract 0.4-0.6%, sea salt 2-4%, and the balance is water.

The rice culture medium includes sea salt, water and rice; the mass ratio of sea salt:water:rice is 7-8:240-260:140-160.

The preparation method of the rice culture medium is as follows: weigh the sea salt in proportion and dissolve it in deionized water. After the sea salt is completely dissolved, add the rice and soak it overnight.

The inoculum amount of deep-sea fungi in the rice culture medium is: inoculate 5 mL of seed culture solution per 140 to 160 g of rice.

The separation process is: sequentially perform liquid-liquid extraction of the culture with ethyl acetate and n-butanol, collect the n-butanol extract, and concentrate the n-butanol extract to obtain fermentation liquid extract; repeat the aforementioned extraction steps at least once, The n-butanol fraction is obtained by combining the fermentation broth extract; the n-butanol fraction is subjected to silica gel column chromatography to obtain eight components A-H with different polarities, and the B-D components are passed through reverse silica gel column chromatography and reverse HPLC. Any one or more compounds of Formula 1 to Formula 7 can be obtained by isolation and purification.

Components B and C were combined, and then passed through a medium-pressure ODS chromatography column using MeOH-water for gradient elution to obtain 5 subfractions, which were named subfractions 1-5. Purify and isolate sub-fraction 2 to obtain the compound represented by formula 1; purify and isolate sub-fraction 3 to obtain compounds represented by formula 2, formula 7 and formula 6; purify and isolate sub-fraction 5 to obtain the compound represented by formula 3.

The D component was separated on a medium-pressure liquid chromatography YMC-ODS-A C ₁₈ column, using MeOH-water for gradient elution to obtain 14 subfractions, which were named subfractions 1-14, among which the subfractions The compound represented by formula 5 is purified and separated in sub-fraction 7, and the compound represented by formula 4 is purified and separated in sub-fraction 9.

The technical solution of the present invention has the following advantages:

1. The deep-sea fungus provided by the present invention can effectively produce a compound that inhibits nitric oxide (NO). The compound not only has the effect of an inhibitor of inducible NO synthase iNOS, but is also non-toxic to cells. It can ensure that the cell survival rate reaches 100% without side effects.

2. The compounds in formulas 1 to 7 provided by the present invention have been tested and found to have inhibitory activity on nitric oxide (NO), and therefore can be used as inhibitors of inducible NO synthase; wherein, at the experimental concentration: At 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 both 100%, indicating that the two compounds of Formula 2 and Formula 7 are effective at this concentration. It has good NO inhibitory activity and is non-toxic to cells. The compound can be used to prepare drugs that inhibit the production of nitric oxide, that is, it can be used as an inhibitor of inducible NO synthase.

Description of the drawings

In order to more clearly explain the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings that need to be used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description The drawings illustrate some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without exerting any creative effort.

Figure 1 is an absolute configuration diagram obtained by X-ray single crystal diffraction of Formula 1 and Formula 2 in Example 1 of the present invention;

Figure 2 is a spectral curve obtained by performing ECD calculation on the compound in the present invention.

Figure 3 is a diagram showing the inhibitory effect of compounds of Formula 1 to Formula 7 of the present invention on nitric oxide (NO) in RAW264.7 cells.

Detailed ways

Example 1

A deep-sea fungus (Trichoderma sp.SCSIOW21). The deep-sea fungus Trichoderma sp.SCSIOW21 is deposited in the China Type Culture Collection Center. The address is Wuhan University, China. The deposit number is CCTCC No: M20211395. The deposit date is November 2021. 11th.

The process of using the above-mentioned deep-sea fungus Trichoderma sp.SCSIOW21 to carry out fermentation cultivation and isolating the inhibitor of inducible NO synthase is as follows:

1. Seed cultivation:

After reviving the cryopreserved deep-sea fungal strain Trichoderma sp.SCSIOW21, the strain was inoculated into the seed culture medium and cultured on a shaking table at 28°C and 200 r/min for 47 to 49 hours to obtain a seed culture medium. Among them, the formula of the seed culture medium is: 2% glucose, 1% peptone, 0.5% yeast extract, 3% sea salt, and the rest is water; it is autoclaved at 121°C for 20 minutes.

2. Fermentation and culture:

In an ultra-clean workbench, add 5 mL of seed culture solution into a 2000 mL Erlenmeyer flask containing rice culture medium, a total of 60 bottles. After static cultivation at room temperature for 40 days, a fermentation culture of the deep-sea fungus Trichoderma sp.SCSIOW21 was obtained.

Among them, the preparation process of rice culture medium is: weigh 450 grams of sea salt and dissolve it in 15 liters of deionized water. After the sea salt is completely dissolved, evenly distribute it into 60 2000mL Erlenmeyer flasks containing 150 grams of rice, and soak overnight.

3. Extraction and separation:

At the end of the culture, add 100 mL of ethyl acetate (EtOAc) to each Erlenmeyer flask and concentrate in a vacuum rotary evaporator below 50°C. After evaporating the remaining EtOAc, add 100 mL of water to each Erlenmeyer flask. Saturated n-butanol (BuOH) and concentrated 3 times on a vacuum rotary evaporator to give a BuOH fraction (12.9 g).

Stir the BuOH fraction into silica gel and install it into a silica gel column. Use chloroform (CH ₂ Cl ₂ )-methanol (MeOH)-water as the elution solvent for elution. Use 100% CH ₂ Cl ₂ in sequence, with 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 CH ₂ Cl ₂ -MeOH-water, and 2.0L each of 100% MeOH Perform elution to obtain eight components of AH.

Combine component B and component C and elute through a C ₁₈ chromatographic column under normal pressure, using a volume ratio of 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0. Elute with 1000 mL of MeOH-water each, and obtain 6 components at natural flow rate under normal pressure, which are named subfractions 1-6 respectively. Separate the No. 2 component through a semi-preparative YMC-ODS-A C ₁₈ (20×250mm, 5μm) HPLC column (acetonitrile-water volume ratio 40:60, 5.0ml/min, t _R 52.1min) to obtain formula 1 (9.0mg). Component No. 3 was purified through a semi-preparative YMC-ODS-A C ₁₈ (20×250mm, 5μm) HPLC column (acetonitrile-water volume ratio 47:53, 10ml/min) to obtain formula 2 (t _R 49.2min, 3.0mg), Formula 7 (t _R 32.8min, 0.3mg) and Formula 6 (t _R 34.1min, 0.4mg). Separate component No. 5 through a semi-preparative YMC-ODS-AC ₁₈ (20×250mm, 5μm) HPLC column (acetonitrile-water volume ratio 70:30, 10.0ml/min, t _R 15.9min) to obtain formula 3 (0.5mg).

Component D was eluted through YMC-ODS-A C ₁₈ chromatographic column under normal pressure, with a gradient of MeOH-water (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 and 100% MeOH) were eluted with 1000 mL each, and 10 components were obtained, which were named subfractions 1-10. The seventh component was purified through a semi-preparative YMC-ODS-A C ₁₈ (20×250mm, 5μm) HPLC column (acetonitrile-water volume ratio 18:82, 10ml/min, t _R 29.6min) to obtain formula 5 ( 1.6 mg). Component 9 was purified through a semi-preparative YMC-ODS-A C ₁₈ (20×250mm, 5μm) HPLC column (acetonitrile-water volume ratio 23:77, 10.0ml/min, t _R 41.5min) to obtain formula 4 (0.6 mg).

Example 2

The process of using the above-mentioned deep-sea fungus Trichoderma sp.SCSIOW21 to carry out fermentation cultivation and isolating the inhibitor of inducible NO synthase is as follows:

1. Seed cultivation:

After reviving the cryopreserved deep-sea fungal strain Trichoderma sp. SCSIOW21, the strain was inoculated into the seed culture medium and cultured on a shaking table at 27°C and 200 r/min for 47 to 49 hours to obtain a seed culture medium. Among them, in terms of mass percentage, the formula of the seed culture medium is: 1.9% glucose, 1.1% peptone, 0.6% yeast extract, 4% sea salt, and the rest is water; it is autoclaved at 121°C for 20 minutes.

2. Fermentation and culture:

In an ultra-clean workbench, add 5 mL of seed culture solution into a 2000 mL Erlenmeyer flask containing rice culture medium, a total of 60 bottles. After static cultivation at room temperature for 40 days, a fermentation culture of the deep-sea fungus Trichoderma sp.SCSIOW21 was obtained.

Among them, the preparation process of the rice culture medium is as follows: weigh 420 grams of sea salt and dissolve it in 15.6 liters of deionized water. After the sea salt is completely dissolved, distribute it evenly into 60 2000mL Erlenmeyer flasks containing 140 grams of rice, and soak overnight.

3. Extraction and separation: The extraction and separation process is the same as in Example 1.

Example 3

The process of using the above-mentioned deep-sea fungus Trichoderma sp.SCSIOW21 to carry out fermentation cultivation and isolating the inhibitor of inducible NO synthase is as follows:

1. Seed cultivation:

After reviving the cryopreserved deep-sea fungal strain Trichoderma sp. SCSIOW21, the strain was inoculated into the seed culture medium, and cultured on a shaking table at 29°C and 200 r/min for 47 to 49 hours to obtain a seed culture medium. Among them, the formula of the seed culture medium is: glucose 2.1%, peptone 0.9%, yeast extract 0.4%, sea salt 2%, and the rest is water; it is autoclaved at 121°C for 20 minutes.

2. Fermentation and culture:

In an ultra-clean workbench, add 5 mL of seed culture solution into a 2000 mL Erlenmeyer flask containing rice culture medium, a total of 60 bottles. After static cultivation at room temperature for 40 days, a fermentation culture of the deep-sea fungus Trichoderma sp.SCSIOW21 was obtained.

Among them, the preparation process of the rice culture medium is as follows: weigh 480 grams of sea salt and dissolve it in 14.4 liters of deionized water. After the sea salt is completely dissolved, distribute it evenly into 60 2000mL Erlenmeyer flasks containing 160 grams of rice, and soak overnight.

3. Extraction and separation: The extraction and separation process is the same as in Example 1.

Test Example 1: Relative structural identification of compounds of formulas 1-7

The compounds of Formulas 1-7 prepared in the above Example 1 were subjected to structural analysis and testing, and the following physical and chemical property data were obtained.

Formula 1: Colorless crystal; [α] 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/ 150MHz, DMSO-d ₆ , TMS is the internal standard, J in Hz, δin ppm) are shown in Table 1; HREIMS m/z: 317.2115[M+H] ⁺ (calculated as C ₂₀ H ₂₉ O ₃ ,317.2117).

Table 1

Formula 4: colorless gel; [α] ²⁵ _D +15.3 (c 0.28, MeOH); UV (MeOH) λ _max (logε) 256.0 (3.89) nm; ECD (0.14 mg/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 ; NMR data (600/150MHz, DMSO-d ₆ , TMS is the internal standard, J inHz, δin ppm) see Table 2; HREIMS m/z: 319.2278[M+H]+ (calculated C ₂₀ H ₃₁ O ₃ ,319.2273).

Table 2

^a Overlapping signals.

Formula 5: Colorless gel; [α] 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 and ¹³ C NMR data (600 /150MHz, DMSO-d ₆ , TMS is the internal standard, Jin Hz, δin ppm) are shown in Table 3; HREIMS m/z: 335.2229[M+H] ⁺ (calculated C ₂₀ H ₃₁ O ₄ ,335.2222).

table 3

Formula 6: Amorphous powder; [α] ²⁵ _D +10.1(c 0.18,MeOH); UV(MeOH)λ _max (logε)252.3(4.19)nm; ECD(0.18mg/ml,MeOH)λ _max (Δε ¹ H _NMR and ¹³ C NMR data (600/150MHz, DMSO-d ₆ , TMS as internal standard, J in Hz, δin ppm) are shown in Table 4; HREIMS m/z: 341.2089[M+Na] ⁺ (calculated C ₂₀ H ₃₀ NaO ₃ ,341.2093).

Table 4

Formula 7: Amorphous powder; [α] 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; ^1H NMR and ^13C NMR data (600/ 150MHz, DMSO-d ₆ , TMS is the internal standard, Jin Hz, δin ppm) are 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 Formula 1 and Formula 2 to obtain their absolute configuration, as shown in Figure 1, where a is the absolute configuration diagram of Formula 1 and b is the absolute configuration diagram of Formula 2. The other compounds obtained were subjected to ECD calculation to determine their absolute configuration. The experimental results are shown in Figure 2, where A is the experimental and calculated curve (2S, 5R, 6R, 8S, 13S, 14S) ECD formula 1 spectrum, and B is Experimental and calculated curves (2S, 5R, 6R, 13S, 14S) ECD formula 4 spectrum, C is the experimental and calculated curve (2S, 5R, 6R, 8S, 13S, 14S) ECD formula 5 spectrum, D is the experimental and calculated curve (2S, 5R, 6R, 8S, 13S, 14S) ECD formula 6 spectrum, E is the experimental and calculated curve (2S, 5R, 6R, 13S, 14S, 15S) ECD formula 7 spectrum.

Based on the above analysis of the physical and chemical data in Tables 1 to 5, combined with the ECD test calculation results of the absolute configurations of Formula 1 to Formula 7, it can be determined that the absolute structures of Formula 1 to Formula 7 are as follows:

Therefore, it can be proved that the deep-sea fungus Trichoderma sp. SCSIOW21 of the present invention can effectively produce and isolate the compounds represented by Formula 1 to Formula 7.

Test Example 2: Experiment on the anti-inflammatory activity of compounds represented by Formula 1 to Formula 7

1. Detection of cell culture

Mouse RAW264.7 macrophages were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) in RPMI-1640 medium supplied with 10% heat-inactivated fetal calf serum (Gibco) at 37°C. , culture in a humidified incubator with 5% _CO2 and 95% air. The culture medium was changed regularly every two days. After RAW264.7 cells reached confluence, they were digested with trypsin and passaged to obtain detection cells.

2. Nitric oxide inhibition test

The detection cells were seeded in a 96-well plate at a density of 1.0 × 10 ⁵ cells/well and grown for 2 hours to allow the cells to adhere to the plate. Dissolve the test sample in DMSO (dimethyl sulfoxide), and then perform a two-fold concentration gradient dilution in RPMI-1640 (Thermo Fisher Scientific, Carlsbad, CA, USA) medium, so that the sample in the medium is Equation 1 to Equation 7 The final concentrations were 100, 50, and 25 μM.

Detection cells were added simultaneously with lipopolysaccharide phosphate LPS (1.5g/mL, Sigma, St. Louis, MO, USA) and recombinant mouse interferon IFN-γ (10ng/mL, PeproTech, Rocky Hill, NJ, USA). in culture medium.

The cells were then incubated at 37°C for approximately 24 hours and subsequently cooled on ice.

Subsequently, 100 μL of cell culture supernatant was added into the wells of a 96-well plate in parallel in duplicate. To quantify NO, 50 μL of Griess reagent (1% sulfonamide, 5% H ₂ PO ₄ solution, and 0.1% N-1-naphthalenedicarboxamide dihydrochloride) was added to each well.

After 10 minutes, the reaction product was colorimetrically quantified at 550 nm using a microplate reader (BIO-RAD, Hercules, CA, USA). The concentration of NO was calculated using the standard calibration curve. The concentration of NO under different sample types and concentrations is shown in Figure 3.

It can be seen from Figure 3 that the compounds of Formula 1 to Formula 7 all have the activity of inhibiting the production of nitric oxide (NO). 2, 3, and 7 showed the strongest NO production inhibitory activity at 100 μM, with inhibition rates of 81.8%, 46.8%, and 50.5% respectively. The remaining compounds showed weak NO inhibitory activity at high concentrations.

Obviously, the above-mentioned embodiments are only examples for clear explanation and are not intended to limit the implementation. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. An exhaustive list of all implementations is neither necessary nor possible. The obvious changes or modifications derived therefrom are still within the protection scope of the present 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.