Preparation method of anti-inflammatory compound tetramethyl sweet heart alkali

Abstract

The invention discloses a preparation method of an anti-inflammatory compound tetramethyl sweet heart alkali. The invention relates to a cordyceps sinensis strain, which is obtained by separating from a fermentation culture of cordyceps sinensis (Ophiococcal sinensis). According to the method disclosed by the invention, the ethyl alcohol (95%) crude extract of the cordyceps sinensis mycelium is gradually separated, so that the tetramethyl sweet heart alkali is obtained for the first time; meanwhile, the in-vitro anti-inflammatory activity of the compound is measured by adopting Beas 2B cells, and the anti-inflammatory effect of the compound is proved. The chemical components of the cordyceps sinensis mycelium are enriched, and a scientific basis is provided for reasonable development and utilization of medicinal resources of the cordyceps sinensis mycelium.

Description

Technical Field

[0001] The invention belongs to the field of anti-inflammatory, and specifically relates to a preparation method of an anti-inflammatory compound tetramethyl betaine. Background Art

[0002] Cordyceps sinensis is a complex of sclerotia and stroma of the larvae and stroma of the fungus Cordyceps sinensis of the family Clavicipitaceae. It is distributed in the high-altitude alpine meadows of 3000-5000m above sea level in the Qinghai-Tibet Plateau, Nepal, Bhutan, India, etc. Cordyceps sinensis is a precious traditional Chinese medicine unique to the Qinghai-Tibet Plateau and a second-class protected medicinal plant in my country. In order to meet people's needs for food, health care, and medicine, and to achieve effective protection of 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. The main biological and pharmacologically active ingredients of the two are: 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 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 cause cor pulmonale and respiratory failure. This disease is related to abnormal inflammatory response to harmful gases and particles, and the global incidence rate of people over 40 years old has reached 9%-10%.

[0007] Treatments for COPD include antibiotics, bronchodilators, theophylline, glucocorticoids, expectorants, and phosphodiesterase-4 (PDE-4) inhibitors. Cordyceps sinensis has been used in the clinical treatment of COPD. However, it remains unclear which chemical components are responsible.

[0008] The invention discloses a compound tetramethyl betaine separated from cordyceps sinensis for the first time. It is found that the compound has the effect of resisting COPD inflammation and has good application potential for treating COPD. Summary of the invention

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

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

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

[0012] The mycelium of Cordyceps sinensis is prepared, and then extracted with alcohol or alcohol-water solution, the extract is concentrated to obtain an extract, the extract is extracted with petroleum ether, the petroleum ether extract is concentrated to obtain a petroleum ether extract, the petroleum ether extract is separated by a silica gel column, and gradient elution is performed with petroleum ether: ethyl acetate 100:1; 80:1; 50:1; 20:1; 10:1; 5:1, v / v, and a 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 ultrasonic extraction is completed, filtering the sample with 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 rotation speed of 80rpm to obtain an extract.

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

[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 tetramethyl betaine for the first time by gradually separating the crude ethanol (95%) extract of Cordyceps sinensis mycelium; at the same time, Beas 2B cells are used to measure its in vitro anti-inflammatory activity to prove its anti-inflammatory effect, in order to enrich the chemical composition of Cordyceps sinensis mycelium and provide a scientific basis for the rational development and utilization of its medicinal resources. BRIEF 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] Figure 3 , 4 is the mass spectrum of compound 5;

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

[0023] Figure 6 is the effect of tetramethyl betaine at different concentrations on the proliferation of Beas-2B cells;

[0024] Figure 7 is the in vitro anti-inflammatory activity graph of compound 5;

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

[0026] Fig. 9 is the effect of compounds 3, 4, and 5 on the production of IL-6 by Beas-2B cells induced by LPS;

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

[0028] 3. 4, wherein R is CH3. DETAILED DESCRIPTION

[0029] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0030] Embodiment 1:

[0031] 1. Experimental methods and results:

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

[0033] ① Cultivate the mycelium of Cordyceps sinensis KD1202 (Cordyceps sinensis KD1202 is disclosed in NCBI with 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 fungus, 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 available to the public within 20 years from the date of application. 6033 g of mycelium of Ophiocordyceps sinensis KD1202 was scooped out from 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, and extracted with ultrasound (40°C; 45 kHz) for 30 min. After the ultrasound was finished, the sample was filtered using a Buchner funnel, and the Cordyceps mycelium extract was poured into a 1000 mL rotary evaporator for reduced pressure concentration and rotary evaporation (50°C; rotation speed: 80 rpm), and extracted 3 times to obtain 177.80 g of extract; ② Use 20% methanol by volume to fully dissolve 177.80 g of the crude extract, add it to a separating funnel, and extract it 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), and each extract Repeat three times (until there is no color), and concentrate with a rotary evaporator to obtain 69.66g of petroleum ether extract, 11.24g of ethyl acetate extract, and 20.36g of n-butanol extract; ③ The petroleum ether extract was mixed with 60-80 mesh C18 reverse silica gel for silica gel column separation, and gradient elution was started with petroleum ether: ethyl acetate (100:1; 80:1; 50:1; 20:1; 10:1; 5:1, v / v), dichloromethane: methanol (20:1; 10:1; 5:1, v / v), and the fractions were collected.

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

[0035] The petroleum ether extract of Cordyceps sinensis mycelium was analyzed by TLC and HPLC and combined to obtain 44 fractions, among which fraction Frs9 eluted with a silica gel column (petroleum ether: ethyl acetate = 5:1, v / v) was separated from fraction Frs9 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 (151MHz, CDCl3) δ183.98,159.98,154.61,132.14,126.82,122.31,113.65,61.59,55.44( Figure 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 Hypoxylonfendleri BCC32408. Fitoterapia. 2019Oct; 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 below:

[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: Take out the cryovial containing F2 generation Beas-2B cells (purchased from Wuhan Punosai Life Science Technology Co., Ltd.) from the liquid nitrogen tank, immediately immerse it in a 37°C water bath, shake it from time to time to thaw it as quickly as possible within 1 min, and take out the cells (sterile); in the clean bench, aspirate the cryopreservation solution into a centrifuge tube, use a pipette to repeatedly and gently blow the cell cryopreservation suspension until it is uniform, centrifuge at 1000 rpm for 3 min, discard the supernatant, add 2 mL of fresh culture medium (DMEM culture medium (1×) / Thermo Fisher Suzhou Instrument Co., Ltd.) to resuspend the cells; inoculate into a culture bottle, place it in a constant temperature cell culture incubator at 37°C, 5% CO2 for static culture.

[0042] ②Beas-2B cell passage: 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 discard the culture medium. Gently add PBS along the side of the culture flask opposite to the adherent cell layer and wash with PBS (1 mL / time) twice, then remove the PBS. Add 1 mL of trypsin for digestion. When the cells are digested to a rounded and floating state under a microscope, add 2 mL of DMEM culture medium to terminate the 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, add 1 mL of DMEM complete culture medium to resuspend and mix the cells, inoculate them into a new cell bottle, add 3 mL of DMEM culture medium into the new cell culture bottle, shake and mix by the cross method, mark the relevant information (Beas-2B cells, F3 generation and time), and place the culture bottle in a 37°C, 5% CO2 incubator for culture until the cells adhere to the wall and grow.

[0043] ③ Cryopreservation of Beas-2B cells: Follow the above cell passaging operation, discard the upper liquid, add 1 mL of the prepared cryopreservation solution (DMEM culture medium, fetal bovine serum, and DMSO were freshly prepared in a volume ratio of 7:2:1, DMEM culture medium (1×) was purchased from Thermo Fisher 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.), gently pipette 10-15 times to mix, and then transfer to a cryopreservation tube. Label the cryopreservation tube containing Beas-2B cells and place it in a cryopreservation box, transfer it to a -80℃ refrigerator, and transfer it to a liquid nitrogen tank for storage after 48 hours.

[0044] ④ Detection of LPS-induced Beas-2B cell toxicity by CCK-8 method: F5 generation Beas-2B cells were seeded in 96-well plates, 1 bottle / plate, 96 plates in total. Cultured at 37°C, 5% CO2 to 80-90% density, 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 LPS stock solution with a final concentration of 1 mg / mL. Filter it with a 0.22 μm diameter filter in a sterile clean bench, dispense it into 1.5 mL EP tubes sterilized by high pressure, and store it at -20°C for later use.

[0047] Grouping: normal group (normal cells + DMEM culture medium), LPS stimulation group (LPS final concentration 0.1, 0.5, 1, 5, 10 μg / mL), each concentration was repeated 6 times, and the drug administration time was 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 multifunctional microplate reader to determine the cell mortality rate, and the concentration with the 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: Effects of LPS on the survival rate of Beas-2B cells Figure 5 As shown, with the increase of LPS concentration, compared with the normal group cells, the survival rates of cells treated with LPS at 0.1μg / mL, 0.5μg / mL, 1μg / mL, 5μg / mL, and 10μg / mL were 100%, 100%, 100%, 99.78%, and 99.17%, respectively. When the LPS concentration was 10μg / mL, the inhibitory effect on cells was the most obvious, and the difference was significant compared with the control group (P=0.02). The survival rate of cells treated with LPS at 1μg / mL was higher, and it can be used for subsequent modeling experiments. ( Figure 5 ).

[0049] ⑤ CCK-8 method was used 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 inoculated with cell suspension (100 μL / well) in a 96-well plate, with approximately 1×10 5 / mL cells (5 replicates per group), and add PBS to the outer circle of the 96-well plate for moisturizing. After the cells adhere to the wall, continue to culture until the confluence is about 70%-80%, discard the original culture medium, add DMEM culture medium to the normal group, add culture medium containing different concentrations of compounds to the sample group (1μL of compound solution, 99μL of DEME culture medium), continue to culture for 24h, and observe the cell growth state and culture medium changes under an inverted microscope. After the culture is completed, add 10μL of CCK-8 solution to each well, incubate at 37℃ in the dark for 2h, detect the absorbance value under the microplate reader (450nm), and 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, there was no significant difference in the results. When p<0.05 (*), p<0.01 (**) or p<0.001 (***), there was a significant difference, and the difference gradually increased.

[0053] Results: Figure 6 As shown in the figure, by one-way ANOVA, compared with the normal group cells, tetramethyl betaine had no significant effect on the viability of Beas-2B cells at three concentrations of 25 μM, 12.5 μM, and 5 μM. Therefore, 25 μM and 12.5 μM of tetramethyl betaine were selected as high and low doses.

[0054] ⑥ In vitro anti-inflammatory activity study: Beas-2B cells in the logarithmic growth phase (fusion degree 70-80%) were taken and the cell density was adjusted to 2×10 6 / mL, 500μL / well of cells were inoculated into 48-well plates, 3 replicates for each concentration, and after the cells adhered to the wall, they were cultured until the confluence was about 80%, and the DMEM medium was discarded. The normal group was added with 500μL / well of normal fresh DMEM medium, and the model group was added with culture medium containing 5μL 1mg / mL LPS and 495μL fresh DMEM medium (the final concentration of LPS was 1μg / mL, and an equal amount of sample solvent DMSO 1μL was added, with a total volume of 500μL / well). The sample group (compound 5 (tetramethyl betaine) 25μM and 12.5μM were high and low doses; compound 3 (cyclo(L-alanine-L-proline)) 100μM and 50μM were high and low doses, and compound 4 (linoleic acid glyceride) 1.25μM and 1μM were high and low doses, among which Figure 7 and Fig. 9100, 50, 1.25, 1, 25, 12.5 μM from top to bottom are the 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 different concentrations of sample solution (the final concentration of LPS was 1 μg / mL, and the final concentration of the sample was low / high concentration), and the positive group was added with 5 μL Fresh DMEM medium containing 1 mg / mL LPS and 1 μL 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 in each group, the average value was taken to calculate the value, n=3).

[0055] 1. Establishment of LPS-induced Beas-2B cell inflammation model: Figure 7 As shown, after one-way analysis of variance, the absorbance values ​​of the model group (LPS) and the normal group were significantly different (P=0.01), indicating that the cells in the model group secreted more inflammatory factors IL-6 under the action of LPS, and the model was successfully established. Compared with the model group, the absorbance value of compound 5 (12.5μM) was significantly reduced after dilution 10 times, with a significant difference (P<0.05), indicating that compound 5 has a certain activity in reducing inflammatory response at a low concentration of 12.5μM.

[0056] 2. Detection of IL-6 content in the culture supernatant of Beas-2B cells 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 abscissa and the IL-6 content as the ordinate. The quadratic curve equation is: y = -1.01429 + 0.71729x - 0.05135x 2 , R 2 =0.988885 (e.g. Figure 8 ).like Fig. 9 The IL-6 content in the normal group Beas-2B cells was low, and after LPS stimulation, the IL-6 content increased significantly. Compound 5 could significantly inhibit LPS-induced IL-6 production at a low concentration (12.5 μM), indicating that compound 5 could reduce the secretion of IL-6 in Beas-2B cells to a certain extent.

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

[0058] Absorbance IL-6 concentration (pg / mL) 2.438 200pg / mL 1.558 100pg / mL 1.086 50pg / mL 0.751 25pg / mL 0.570 12.5 pg / mL 0.339 6.25 pg / mL 0.204 3.12 pg / mL 0.158 0pg / mL

Claims

1. A method for preparing a compound tetramethyl betaine, characterized in that: It is isolated from the fermentation culture of Cordyceps sinensis; The structure of the tetramethyl betaine is shown below: Among them, 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: The mycelium of Cordyceps sinensis is prepared, and then extracted with alcohol or alcohol-water solution, the extract is concentrated to obtain an extract, the extract is extracted with petroleum ether, the petroleum ether extract is concentrated to obtain a petroleum ether extract, the petroleum ether extract is separated by a silica gel column, and the ratio of petroleum ether:ethyl acetate is 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, and separate the fraction Frs9 by Sephadex LH-20 gel methanol chromatography to obtain the compound tetramethyl betaine.

3. The preparation method according to claim 2, characterized in that: The alcohol aqueous solution is an ethanol aqueous solution with a volume fraction of 95%.

4. The preparation method according to claim 2, characterized in that: The alcohol or alcohol aqueous solution extraction is to soak in 95% ethanol aqueous solution for 48 hours, perform ultrasonic extraction at 40°C and 45kHz for 30 minutes, and after the ultrasonication is finished, filter the sample with a Buchner funnel, pour the Cordyceps sinensis mycelium extract into a rotary evaporator for reduced pressure concentration and rotary evaporation at 50°C and a rotation speed of 80rpm to obtain an extract.

5. The preparation method according to claim 2, characterized in that: The preparation of mycelium of Cordyceps sinensis is to inoculate Cordyceps sinensis into a rice culture medium for fermentation and culture to obtain mycelium, wherein the rice culture medium is a mixture of 8g of rice, 8g of wheat and 22mL of nutrient solution, wherein the nutrient solution components 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 per liter.

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

7. Application of Cordyceps sinensis (O. sinensis) in the preparation of tetramethyl betaine, the structure of which is shown below: Among them, R1, 2, 3, and 4 are all CH3.

8. The use according to claim 7, characterized in that: The cordyceps sinensis is cordyceps sinensis (O. sinensis) KD1202.

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