Method for preparing Anti-inflammatory compound tetramethyl atromentin

Abstract

Disclosed in the present invention is a method for preparing an anti-inflammatory compound tetramethyl atromentin. The compound is isolated from a fermentation culture of Ophiocordyceps sinensis. In the present invention, an ethanol (95%) crude extract of Ophiocordyceps sinensis mycelium is subjected to stepwise isolation to obtain tetramethyl atromentin for the first time, and the in-vitro anti-inflammatory effect activity of the tetramethyl atromentin is determined using Beas 2B cells, thus proving the anti-inflammatory effect thereof. The compound is expected to enrich the chemical components of Ophiocordyceps sinensis mycelium, thereby providing a scientific basis for reasonable development and use of medicinal resources thereof.

Description

A preparation method of anti-inflammatory compound tetramethyl betaine Technical field:

[0001] The present invention belongs to the field of anti-inflammatory, and in particular relates to a method for preparing an anti-inflammatory compound, tetramethyl betaine. Background technology:

[0002] Cordyceps sinensis (Ophiocordyceps sinensis) is a fungus of the genus Cordyceps in the family Clavicipitaceae. It parasitizes the larvae of insects belonging to the family Bat-Moth in the order Lepidoptera. It is distributed in alpine meadows at altitudes of 3,000-5,000 meters, including the Qinghai-Tibet Plateau in my country, Nepal, Bhutan, and India. Cordyceps sinensis is a valuable traditional Chinese medicinal herb unique to the Qinghai-Tibet Plateau and a Class II protected medicinal plant in my country. To meet the needs of food, healthcare, and medicinal uses, and to effectively protect Cordyceps sinensis resources, Cordyceps sinensis mycelium has been obtained through liquid fermentation and solid culture. Cordyceps sinensis and Cordyceps sinensis mycelium have similar chemical compositions and pharmacological effects. Their main biologically and pharmacologically active ingredients include nucleosides, mannitol, alkaloids, amino acids, unsaturated fatty acids, vitamins, polysaccharides, and sterols. These active substances have the effects of enhancing and regulating immune function, treating lung and kidney diseases, anti-aging, and anti-tumor, and are widely used to treat various diseases such as respiratory tract, kidney, heart, liver, etc.

[0003] Tetramethyl betaine is disclosed in the document Intaraudom C, Punyain W, Bunbamrung N, Dramae A, Boonruangprapa T, Pittayakhajonwut P. Antimicrobial drimane-phthalide derivatives from Hypoxylon fendleri BCC32408. Fitoterapia. 2019 Oct; 138: 104353., which is compound 20 in the document.

[0004] The structure of tetramethyl betaine is shown in the figure below:

[0005] Among them, R1, 2, 3, and 4 are all CH3.

[0006] Chronic obstructive pulmonary disease (COPD) is a common chronic disease characterized by chronic bronchitis and / or emphysema with airflow obstruction, which can lead to cor pulmonale and respiratory failure. This disease is associated with an abnormal inflammatory response to harmful gases and particles, and its global prevalence has reached 9%-10% among people over 40 years old.

[0007] Treatments for chronic obstructive pulmonary disease include antibiotics, bronchodilators, theophylline-based drugs, glucocorticoids, expectorants, and phosphodiesterase-4 (PDE-4) inhibitors. Cordyceps sinensis has been used clinically to treat COPD. However, the specific chemical components responsible remain unclear.

[0008] The invention discloses a compound tetramethyl betaine isolated from Cordyceps sinensis for the first time. It is found that the compound has an anti-COPD inflammation effect and has good application potential in treating COPD. Summary of the invention:

[0009] The purpose of the present invention is to provide a method for preparing the anti-inflammatory compound tetramethyl betaine.

[0010] The preparation method of the compound tetramethyl betaine of the present invention is to separate it from the fermentation culture of Cordyceps sinensis.

[0011] Preferably, the specific preparation method is as follows:

[0012] The mycelium of Cordyceps sinensis is prepared, and then extracted with alcohol or an alcohol-water solution. The extract is concentrated to obtain an extract, the extract is extracted with petroleum ether, and the petroleum ether extract is concentrated to obtain a petroleum ether extract. The petroleum ether extract is separated on a silica gel column and eluted with a petroleum ether: ethyl acetate ratio of 100:1; 80:1; 50:1; 20:1; 10:1; 5:1, v / v gradient. The fraction Frs9 eluted with petroleum ether: ethyl acetate = 5:1, v / v is collected, and the fraction Frs9 is separated by Sephadex LH-20 gel methanol chromatography to obtain compound 5.

[0013] Preferably, the alcohol aqueous solution is an ethanol aqueous solution with a volume fraction of 95%.

[0014] Preferably, the extraction with alcohol or alcohol aqueous solution is performed by soaking in 95% ethanol aqueous solution for 48 hours, performing ultrasonic extraction at 40°C and 45kHz for 30 minutes, and after the ultrasonication is completed, filtering the sample using a Buchner funnel, pouring the Cordyceps sinensis mycelium extract into a rotary evaporator for reduced pressure concentration and rotary evaporation at 50°C and a speed of 80rpm to obtain an extract.

[0015] Preferably, the mycelium of Cordyceps sinensis is prepared by inoculating Cordyceps sinensis into a rice culture medium for fermentation to obtain mycelium, wherein the rice culture medium is a mixture of 8g of rice and 8g of wheat and 22mL of nutrient solution, and the nutrient solution components per liter are 20g of glucose, 5g of peptone, 2g of potassium dihydrogen phosphate, 1g of magnesium sulfate, 1g of ammonium citrate, 20mg of vitamin B1, 18.5g of silkworm pupa powder, and 1L of water.

[0016] Preferably, the Cordyceps sinensis (O. sinensis) is Cordyceps sinensis (O. sinensis) KD1202.

[0017] The second object of the present invention is to provide the use of Cordyceps sinensis (O. sinensis) in the preparation of tetramethyl betaine.

[0018] The present invention obtains tetramethylbetaine for the first time by progressively separating it from a crude ethanol (95%) extract of Cordyceps sinensis mycelia. Furthermore, its in vitro anti-inflammatory activity was determined using Beas 2B cells, demonstrating its anti-inflammatory effect. This approach aims to enrich the chemical composition of Cordyceps sinensis mycelia and provide a scientific basis for the rational development and utilization of its medicinal resources. Description of the drawings:

[0019] Figure 1 is the H spectrum of compound 5;

[0020] Figure 2 is the C spectrum of compound 5;

[0021] Figures 3 and 4 are the mass spectra of compound 5;

[0022] Figure 5 shows the effect of LPS on Beas-2B cell viability;

[0023] FIG6 shows the effect of tetramethyl betaine at different concentrations on the proliferation of Beas-2B cells;

[0024] FIG7 is a graph showing the in vitro anti-inflammatory activity of compound 5;

[0025] Figure 8 is the quadratic curve regression of the inflammatory factor IL-6 ELISA Calc (background value deducted);

[0026] Figure 9 shows the effects of compounds 3, 4, and 5 on IL-6 production by LPS-induced Beas-2B cells;

[0027] The structural formulas of compounds 3 and 4 are shown below:

[0028] Wherein R is CH3. Specific implementation method:

[0029] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0030] Example 1:

[0031] 1. Experimental methods and results

[0032] (1) Extraction and separation methods of compounds:

[0033] ① Mycelia of Ophiocordyceps sinensis KD1202 (Ophiocordyceps sinensis KD1202 is disclosed in NCBI under the accession number KM205217 and is also disclosed in the literature Cao L, Ye Y, Han R. Fruiting body production of the medicinal Chinese caterpillar mushroom, Ophiocordyceps sinensis (Ascomycetes), in artificial medium. International Journal of Medicinal Mushrooms, 2015, 17(11): 1017–1022. The applicant also holds the strain and guarantees that it will be made available to the public within 20 years from the date of application. 6033 g of mycelium of Ophiocordyceps sinensis KD1202 was scooped out of a blue-capped bottle with a spoon (dried at 30°C for 3 h and weighed), placed in a 5 L conical flask, and soaked in 95% ethanol (solid-liquid ratio 1:8 g / mL) for 48 h. Ultrasonic extraction (40°C; 45 kHz) was performed for 30 min. After the ultrasonic treatment, the sample was filtered using a Buchner funnel, and the Cordyceps sinensis mycelium extract was poured into a 1000 mL rotary evaporator for vacuum concentration and rotary evaporation (50°C; speed: 80 rpm). The extract was extracted three times to obtain 177.80 g of extract. ② 177.80 g of crude extract was fully dissolved in 20% methanol by volume, added to a separating funnel, and extracted with petroleum ether (1:1, v / v), ethyl acetate (1:1, v / v) and n-butanol (1:1, v / v) in sequence (extraction 30 min). Each extract Repeat three times (until there is no color), and concentrate on a rotary evaporator to obtain 69.66 g of petroleum ether extract, 11.24 g of ethyl acetate extract, and 20.36 g of n-butanol extract; ③ The petroleum ether extract is mixed with 60-80 mesh C18 reverse silica gel for silica gel column separation, and gradient elution is started with petroleum ether: ethyl acetate (100:1; 80:1; 50:1; 20:1; 10:1; 5:1, v / v), and dichloromethane: methanol (20:1; 10:1; 5:1, v / v), and the fractions are collected.

[0034] (2) Isolation and purification of compounds:

[0035] The petroleum ether extract of Cordyceps sinensis mycelia was analyzed by TLC and HPLC, and 44 fractions were obtained. Among them, fraction Frs9 was eluted with a silica gel column (petroleum ether: ethyl acetate = 5:1, v / v). Fraction Frs9 was separated by Sephadex LH-20 gel methanol chromatography to obtain compound 5 (6.4 mg).

[0036] (3) Structural identification of compound 5:

[0037] Brown solid, C 22 H 21 O6, insoluble in methanol, soluble in dichloromethane and ethyl acetate, 1 H NMR (600MHz, CDCl3) δ7.30(d,J=8.6Hz,3H),6.97(d,J=8.4Hz,4H),3.85(s,7H),3.82(s,7H),3.50(s,8H),3.49(s,8H). 13 C NMR (151 MHz, CDCl3) δ 183.98, 159.98, 154.61, 132.14, 126.82, 122.31, 113.65, 61.59, 55.44 (Figures 1-4) The above NMR data are basically consistent with the data reported in the literature [Intaraudom C, Punyain W, Bunbamrung N, Dramae A, Boonruangprapa T, Pittayakhajonwut P. Antimicrobial drimane-phthalide derivatives from Hypoxylon fendleri BCC32408. Fitoterapia. 2019 Oct; 138: 104353.], which is compound 20 in the literature, so the compound was identified as tetramethyl betaine.

[0038] The structure of tetramethyl betaine is shown in the figure:

[0039] Among them, R1, 2, 3, and 4 are all CH3.

[0040] (4) Study on the anti-inflammatory activity of compounds in Cordyceps sinensis mycelium

[0041] ① Beas-2B cell recovery and culture: The cryovial containing F2 generation Beas-2B cells (purchased from Wuhan Punosai Life Science Technology Co., Ltd.) was removed from the liquid nitrogen tank and immediately immersed in a 37°C water bath. The cells were shaken from time to time to thaw as quickly as possible within 1 min, and the cells were removed (sterile); in an ultra-clean workbench, the cryopreservation solution was aspirated into a centrifuge tube, and the cell cryopreservation suspension was repeatedly and gently blown with a pipette until it was uniform. After centrifugation at 1000 rpm for 3 min, the supernatant was discarded, and 2 mL of fresh culture medium (DMEM medium (1×) / Thermo Fisher Suzhou Instrument Co., Ltd.) was added to resuspend the cells; the cells were inoculated into culture flasks and placed in a constant temperature cell culture incubator at 37°C and 5% CO2 for static culture.

[0042] ②Beas-2B cell passaging: After 30 minutes of UV exposure, observe the cell density under a microscope. When it reaches 80%-90%, place the cells in a clean bench and aspirate the culture medium. Gently add PBS along the side of the culture flask opposite the adherent cell layer and wash with PBS (1 mL / time) twice. Aspirate the PBS. Add 1 mL of trypsin for digestion. When the cells are digested to a round and floating state under a microscope, add 2 mL of DMEM culture medium to stop digestion and mix the liquid thoroughly. Collect the cell suspension into a 15 mL centrifuge tube and centrifuge (1000 r / 3 min). Discard the supernatant and add 1 mL of DMEM complete culture medium to resuspend and mix the cells. Inoculate into a new cell flask. Add 3 mL of DMEM culture medium to the new cell culture flask, shake and mix thoroughly by cross-shaking. Mark the relevant information (Beas-2B cells, F3 generation and time). Place the culture flask in a 37°C, 5% CO2 incubator and culture until the cells adhere to the wall and grow.

[0043] ③ Cryopreservation of Beas-2B cells: Follow the above-mentioned cell passaging procedure, discard the supernatant liquid, and add 1 mL of the prepared freezing solution (DMEM medium, fetal bovine serum, and DMSO were freshly prepared in a volume ratio of 7:2:1. DMEM medium (1×) was purchased from Thermo Fisher Scientific Suzhou Instrument Co., Ltd., deuterated dimethyl sulfoxide (DMSO) was purchased from Beijing Biolab Technology Co., Ltd., and fetal bovine serum was purchased from Sangon Biotech Shanghai Co., Ltd.). Mix thoroughly by gently pipetting 10-15 times and transfer the mixture to a cryovial. Label the cryovial containing Beas-2B cells and place it in a cryopreservation box. Transfer the cryovial to a -80°C freezer and store it in a liquid nitrogen tank after 48 h.

[0044] ④ Detection of LPS-induced Beas-2B cytotoxicity by CCK-8 assay: F5 generation Beas-2B cells were seeded into 96-well plates, 1 vial per plate, for a total of 96 plates. Cultured at 37°C, 5% CO2, to a density of 80-90% and set aside.

[0045] LPS solution preparation

[0046] LPS stock solution: Weigh 1 mg of LPS powder and dissolve it in 1 mL of RPIM-1640 culture medium to obtain a final concentration of 1 mg / mL LPS stock solution. Filter the solution using a 0.22 μm filter in a sterile laminar flow hood. Aliquot into autoclaved 1.5 mL EP tubes and store at -20°C until needed.

[0047] Grouping: normal group (normal cells + DMEM culture medium), LPS stimulation group (LPS final concentration 0.1, 0.5, 1, 5, 10 μg / mL), 6 replicates for each concentration, administration time for 24 hours (3 groups in parallel); at the detection time point, CCK-8 reagent was added to the corresponding wells to give drug stimulation, PBS was added to the outermost circle of the 96-well plate for moisturizing, 10 μL per well, and cultured for 2 hours at 37°C, 5% CO2; OD450 was detected by a multi-function microplate reader to determine the cell mortality rate, and the concentration with appropriate LPS induction effect and the lowest mortality rate was selected as the optimal model parameter (the maximum and minimum values ​​of the absorbance value of each group were removed).

[0048] Experimental Results and Analysis: The effect of LPS on the survival rate of Beas-2B cells is shown in Figure 5. As LPS concentrations increased, the survival rates of cells treated with LPS at 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, and 10 μg / mL were 100%, 100%, 100%, 99.78%, and 99.17%, respectively, compared to cells in the normal control group. The inhibitory effect on cells was most pronounced at a concentration of 10 μg / mL, with a significant difference compared to the control group (P=0.02). Cells treated with 1 μg / mL of LPS had a higher survival rate and can be used for subsequent modeling experiments (Figure 5).

[0049] ⑤ CCK-8 assay to determine the effect of compound 5 in Cordyceps sinensis mycelium on Beas-2B cell viability:

[0050] F6 generation Beas-2B cells (80-90% confluence) in the logarithmic growth phase were seeded in a 96-well plate with a cell suspension (100 μL / well), with approximately 1×10 cells per well. 5 Cells were plated at 500 μg / mL (5 replicates per group) and PBS was added to the periphery of the 96-well plate to keep it moist. After the cells adhered, they were cultured until the confluence was approximately 70%-80%. The original culture medium was discarded, and DMEM culture medium was added to the normal group. The sample group was added with culture medium containing different concentrations of compounds (1 μL of compound solution and 99 μL of DEME medium). The cells were cultured for 24 hours and the cell growth status and culture medium changes were observed under an inverted microscope. After the culture was completed, 10 μL of CCK-8 solution was added to each well and incubated at 37°C in the dark for 2 hours. The absorbance value was measured at 450 nm using a microplate reader to calculate the cell growth rate.

[0051] Cell inhibition rate = 1-(drug-added well-background value) / (control well-background value) × 100%

[0052] Data processing: GraphPad Prism 8.0.2 263 software was used for experimental data processing. When p>0.05, the results were not significantly different. When p<0.05 (*), p<0.01 (**), or p<0.001 (***), there was a significant difference, and the difference gradually increased.

[0053] Results: As shown in Figure 6, a one-way ANOVA analysis showed that tetramethyl betaine at 25 μM, 12.5 μM, and 5 μM concentrations had no significant effect on Beas-2B cell viability compared to the normal control group. Therefore, 25 μM and 12.5 μM of tetramethyl betaine were selected as the high and low doses.

[0054] ⑥ In vitro anti-inflammatory activity study: Beas-2B cells in the logarithmic growth phase (70-80% confluence) were taken and the cell density was adjusted to 2×10 6Cells were seeded into 48-well plates at a concentration of 500 μL / well, and three replicates were made for each concentration. After the cells adhered to the wall, they were cultured until the confluence was about 80%. The DMEM medium was discarded, and 500 μL / well of normal fresh DMEM medium was added to the normal group. The model group was added with a culture medium containing 5 μL of 1 mg / mL LPS and 495 μL of fresh DMEM medium (the final concentration of LPS was 1 μg / mL, and an equal amount of sample solvent DMSO was added). 1 μL, total volume 500 μL / well), sample group (compound 5 (tetramethyl betaine) 25 μM and 12.5 μM as high and low doses; compound 3 (cyclo(L-alanine-L-proline)) 100 μM and 50 μM as high and low doses, compound 4 (linoleic acid glyceride) 1.25 μM and 1 μM as high and low doses, wherein 100, 50, 1.25, 1, 25, 12.5 μM from top to bottom in Figures 7 and 9 are high and low concentrations of compounds 3, 4, and 5, respectively) were added with 5 μL of 1 mg / mL LPS and 1 μL of sample solutions of different concentrations (the final concentration of LPS was 1 μg / mL, and the final concentration of the sample was low / high concentration), the positive group was added with 5 μL of Fresh DMEM medium containing 1 mg / mL LPS and 1 μL of roflumilast solution (final LPS concentration was 1 μg / mL, final roflumilast concentration was 2 μM, final sample concentration was low / high concentration, total volume was 500 μL / well) was added and cultured for 24 h. After the culture was completed, the supernatant of each well was collected and centrifuged in a 2 mL centrifuge tube (1000 g / 20 min). 100 μL of the supernatant was transferred to another 2 mL centrifuge tube and stored at -20°C for ELISA detection (avoid repeated freezing and thawing). The IL-6 content was detected according to the instructions of the ELISA kit (two replicate wells per group, the average value was calculated, n=3).

[0055] 1. Establishment of an LPS-induced Beas-2B cell inflammation model: As shown in Figure 7, a one-way ANOVA analysis showed a significant difference in absorbance between the model group (LPS) and the normal group (P = 0.01), indicating that LPS-induced IL-6 secretion in the model group increased, indicating successful modeling. Comparing the sample group with the model group, the absorbance of compound 5 (12.5 μM) decreased significantly after a 10-fold dilution, with a significant difference (P < 0.05), indicating that compound 5 has a certain ability to reduce inflammatory responses at a low concentration of 12.5 μM.

[0056] 2. Detection of IL-6 content in the supernatant of Beas-2B cell culture induced by LPS by compound 5-tetramethyl betaine: According to the instructions of the IL-6 enzyme-linked immunosorbent assay kit, the obtained ELISA experimental standard solution and the corresponding average absorbance value (Table 1) were used to draw a standard curve with the absorbance value as the horizontal axis and the IL-6 content as the vertical axis. The quadratic curve equation is: y = -1.01429 + 0.71729x - 0.05135x 2 , R 2 =0.988885 (as shown in Figure 8). As shown in Figure 9, the IL-6 content in Beas-2B cells in the normal group was low. After LPS stimulation, the IL-6 content increased significantly. Compound 5 at a low concentration (12.5 μM) significantly inhibited LPS-induced IL-6 production, indicating that compound 5 can reduce IL-6 secretion in Beas-2B cells to a certain extent.

[0057] Table 1 ELISA experimental standard solutions and corresponding average absorbance values

Claims

1. A preparation method of a compound tetramethyl sweet alkaloid, characterized in that, It is isolated from the fermentation culture of Ophiocordyceps sinensis; The structure of the tetramethyl sweet alkaloid is shown as follows: wherein R1, 2, 3, and 4 are all CH3.

2. The preparation method according to claim 1, characterized in that, The preparation method is as follows: Prepare the mycelium of Ophiocordyceps sinensis, then extract it with alcohol or an alcohol aqueous solution. The extract is concentrated to obtain an extract paste. The extract paste is extracted with petroleum ether. After the petroleum ether extract is concentrated, a petroleum ether extract paste is obtained. The petroleum ether extract paste is separated by a silica gel column, using petroleum ether: ethyl acetate 100:1; 80:1; 50:1; 20：1； 10：1； 5:1, v / v gradient elution. Collect the fraction Frs9 eluted with petroleum ether: ethyl acetate = 5:1, v / v. The fraction Frs9 is separated by Sephadex LH-20 gel methanol chromatography to obtain the compound tetramethyl sweet alkaloid.

3. The preparation method according to claim 2, characterized in that, The alcohol aqueous solution is a 95% (v / v) ethanol aqueous solution.

4. The preparation method according to claim 2, characterized in that The extraction with alcohol or an alcohol aqueous solution is to soak with a 95% ethanol aqueous solution for 48 h, perform ultrasonic extraction at 40 °C and 45 kHz for 30 min. After the ultrasonic extraction is completed, filter the sample using a Buchner funnel. Pour the Ophiocordyceps sinensis mycelium extract into a rotary evaporation flask and perform rotary evaporation under reduced pressure at 50 °C with a rotation speed of 80 rpm to obtain an extract paste.

5. The preparation method according to claim 2, characterized in that, The preparation of the mycelium of Ophiocordyceps sinensis is to inoculate Ophiocordyceps sinensis into a rice medium for fermentation culture to obtain the mycelium. The rice medium is a mixture of 8 g of rice, 8 g of wheat, and 22 mL of nutrient solution. The components of the nutrient solution per liter are 20 g of glucose, 5 g of peptone, 2 g of potassium dihydrogen phosphate, 1 g of magnesium sulfate, 1 g of ammonium citrate, 20 mg of vitamin B1, 18.5 g of silkworm pupa powder, and 1 L of water.

6. The preparation method according to claim 2, characterized in that, The Ophiocordyceps sinensis is Ophiocordyceps sinensis (O. sinensis) KD1202.

7. Use of Ophiocordyceps sinensis (O. sinensis) in the preparation of tetramethyl sweet alkaloid, the structure of the tetramethyl sweet alkaloid is shown as follows: wherein R1, 2, 3, and 4 are all CH3.

8. The application according to claim 7, wherein The Ophiocordyceps sinensis is Ophiocordyceps sinensis (O. sinensis) KD1202.

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