CN110959862B - Composition with functions of removing trimethylamine oxide and resisting inflammation and preparation method and application thereof - Google Patents
Composition with functions of removing trimethylamine oxide and resisting inflammation and preparation method and application thereof Download PDFInfo
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- CN110959862B CN110959862B CN201911114374.5A CN201911114374A CN110959862B CN 110959862 B CN110959862 B CN 110959862B CN 201911114374 A CN201911114374 A CN 201911114374A CN 110959862 B CN110959862 B CN 110959862B
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- eha2
- lactobacillus reuteri
- tmao
- curcumin
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Images
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- A—HUMAN NECESSITIES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a composition with functions of removing trimethylamine oxide and resisting inflammation, and a preparation method and application thereof. The composition comprises curcumin and/or lactobacillus reuteri EHA2 strain. The research of the invention finds that after the interaction between the curcumin and/or the fermentation supernatant of the Lactobacillus reuteri EHA2 strain and the TMAO, the content of the TMAO in the solution can be reduced, and meanwhile, the expression level of inflammatory factors can be obviously reduced by the curcumin and/or the fermentation supernatant of the Lactobacillus reuteri EHA2 strain. The composition can eliminate TMAO and inflammation caused by TMAO, has the effect of relieving atherosclerotic diseases, and has good social and economic benefits.
Description
Technical Field
The invention relates to the technical field of biological medicines, and in particular relates to a composition with functions of removing trimethylamine oxide and resisting inflammation, and a preparation method and application thereof.
Background
Atherosclerotic disease (AS) is one of the diseases with high worldwide morbidity and mortality, and since atherosclerosis has long been considered to be a disease caused by lipid deposition on the arterial wall, conventional treatments have been directed to lowering blood lipid and blood pressure. However, cardiovascular disease due to atherosclerosis remains high, causing millions of deaths worldwide each year. Today, the atherosclerotic concept has been gradually replaced by a lipid-driven, chronic inflammatory disease of the arterial wall. More and more studies have confirmed that there is a close relationship between the intestinal flora and cardiovascular disease. The intestinal flora can metabolize food containing trimethylamine structure to produce Trimethylamine (TMA), and further be oxidized by flavin-monooxygenase 3 (FMO 3) to produce trimethylamine oxide (TMAO) on the liver, and the TMAO can promote vascular inflammation and accelerate the development process of AS through various signal pathways. Therefore, blocking the production of TMAO and down-regulating this inflammatory signaling pathway may be regarded as an effective strategy to intervene in cardiovascular inflammation and thus prevent and treat atherosclerosis.
Only chinese patent 201380041723.X discloses a pharmaceutical composition for reduction of trimethylamine N-oxide levels in humans comprising 3- (2, 2, 2-trimethylhydrazinium) propionate (meldonium) as an active ingredient, and a pharmaceutically acceptable carrier; chinese patent 201380069004.9 discloses a composition comprising a microorganism, preferably archaea, which expresses TMA methyltransferase and TMA methyl group receptor corrinoid protein, which is capable of undergoing Trimethylamine (TMA) metabolism in a human compartment in the presence of hydrogen, such as the gut or vagina, as a medicament for treating, reducing or eliminating TMA in the human compartment; chinese patent 201810889757.9 discloses the application of berberine in preparing medicine for preventing and treating vascular endothelial dysfunction. The few compositions which are reported to reduce the level of trimethylamine oxide in human body are still available, and especially the compositions which directly remove the trimethylamine oxide and have anti-inflammatory function are not reported.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings in the prior art and provide a composition with functions of removing trimethylamine oxide and resisting inflammation.
It is another object of the present invention to provide a method for preparing said composition.
It is a further object of the present invention to provide the use of said composition.
The above object of the present invention is achieved by the following technical solutions:
the research of the invention finds that after the interaction between the curcumin and/or the fermentation supernatant of the Roy's lactobacillus EHA2 strain and the TMAO, the content of the TMAO in the solution can be reduced, and particularly, the content of the TMAO in the solution can be obviously reduced by the composition of the curcumin and the fermentation supernatant of the Roy's lactobacillus EHA2 strain which are mixed according to a specific proportion. And simultaneously, after the human vascular smooth muscle cells are pretreated by fermenting the supernatant of the curcumin and/or the Lactobacillus reuteri EHA2 strain, the human vascular smooth muscle cells are intervened by TMAO. The results show that compared with the method of directly using TMAO to intervene human vascular smooth muscle cells without using curcumin or fermentation supernatant of Lactobacillus reuteri, the expression level of the inflammatory factors of the human vascular smooth muscle cells after the pre-drying prognosis of the curcumin or fermentation supernatant of the Lactobacillus reuteri EHA2 strain is remarkably reduced, and particularly the expression level of the inflammatory factors of the pre-drying prognosis of the fermentation supernatant of the curcumin and the Lactobacillus reuteri EHA2 strain is most remarkably reduced. The results show that the fermentation supernatant of curcumin and/or lactobacillus reuteri EHA2 strain has functions of removing trimethylamine oxide and resisting inflammation.
Therefore, the invention firstly protects the application of the curcumin and/or the lactobacillus reuteri EHA2 strain in preparing the pharmaceutical preparation with functions of removing the trimethylamine oxide and resisting inflammation.
A composition with functions of eliminating trimethylamine oxide and anti-inflammatory comprises flavin and/or Lactobacillus reuteri EHA2 strain; the lactobacillus reuteri EHA2 strain is preserved in Guangdong province microorganism strain preservation center in 2019, 9, 2, with the preservation number of GDMCC NO: 60758.
preferably, the composition comprises a flavin and/or a fermentation supernatant of lactobacillus reuteri EHA2 strain.
Preferably, the composition comprises the following components in volume ratio: the curcumin solution and fermentation supernatant of lactobacillus reuteri EHA2 strain are 1-3: 1 to 3.
More preferably, the composition comprises the following components in volume ratio: curcumin solution and lactobacillus reuteri EHA2 strain fermentation supernatant ═ 1: 3; the curcumin solution and the lactobacillus reuteri composition in the proportion have the best effect of removing TMAO.
Preferably, the concentration of the curcumin solution is 40-300 mM.
The fermentation liquor of the strain of lactobacillus reuteri EHA2 is prepared according to the conventional preparation method of the fermentation liquor of lactobacillus reuteri in the field. Preferably, the preparation method of the fermentation supernatant of the lactobacillus reuteri EHA2 strain comprises the steps of activating the strain of lactobacillus reuteri EHA2, inoculating the activated strain into a fermentation medium, fermenting for 40-50 h (preferably 48h) at 30-37 ℃ (preferably 37 ℃), adjusting OD of the fermented bacterial liquid to 1.6-1.8 (preferably 1.8), centrifuging, and reserving the supernatant to obtain the fermentation supernatant of the lactobacillus reuteri EHA2 strain.
Preferably, the activation medium is MRS solid medium, and the strain activation time is 37 ℃ for culturing for 48 h; the fermentation medium is MRS broth.
Preferably, the centrifugation is 5000-7000 r/min, 0-4 ℃, and 10-15 min (preferably 6000r/min, 4 ℃, and 15 min).
The invention also claims the application of the composition in preparing a preparation with functions of removing trimethylamine oxide and resisting inflammation.
The application of the composition in preparing a preparation for preventing and treating atherosclerotic diseases and/or cardiovascular diseases.
Preferably, the formulation is a tablet, capsule, powder, oral liquid, soft capsule, pill, tincture, syrup, suppository, gel, spray, umbilical patch, effervescent tablet or injection.
A preparation for eliminating trimethylamine oxide and anti-inflammatory function comprises fermentation supernatant of flavin and/or Lactobacillus reuteri EHA2 strain.
Preferably, the preparation method of the preparation comprises the following steps: weighing the raw material components according to the volume ratio, and dissolving the raw material components in water with the volume multiple of 80-100 to obtain the preparation with functions of removing trimethylamine oxide and resisting inflammation. Curcumin is a chemical component extracted from rhizomes of some plants in Zingiberaceae and Araceae, is a pigment with diketone which is rare in plant world, and is a diketone compound. Curcumin is orange yellow crystal powder, slightly bitter in taste and insoluble in water, is mainly used for coloring sausage products, cans, sauced and marinated products and other products in food production, and has the effects of reducing blood fat, resisting tumors, benefiting gallbladder, resisting oxidation and the like. The lactobacillus reuteri is intestinal probiotics which can improve the functions of human bodies and improve the immunity, so that the human safety of the preparation can be ensured.
The composition can eliminate TMAO and inflammation caused by TMAO, can be used for relieving atherosclerotic diseases and cardiovascular diseases, and has good social and economic benefits.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a composition with functions of removing trimethylamine oxide and resisting inflammation, and after the composition interacts with TMAO, the content of TMAO in a solution can be reduced, and the expression level of inflammatory factors can be obviously reduced. The composition can eliminate TMAO and inflammation caused by TMAO, has the effect of relieving atherosclerotic diseases, and has good social and economic benefits.
Drawings
FIG. 1 is a standard curve for TMAO content analysis established by the present invention.
FIG. 2 shows that TMAO is used to intervene the expression effect of human vascular smooth muscle cells 6h on inflammatory factors in human vascular smooth muscle cells after the human vascular smooth muscle cells are intervened in advance for 6h in different pretreatment modes.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 isolated culture of Lactobacillus reuteri EHA2 and preparation of fermentation broth
1. Media preparation
MRS solid medium: 10.0g of peptone, 8.0g of beef powder, 4.0g of yeast powder, 20.0g of glucose, 2.0g of dipotassium phosphate, 5.0g of sodium acetate, 2g of diammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 801 g of tween, 15g of agar, 1L of water and high-pressure sterilization at 121 ℃ for 15 min;
MRS broth culture medium: 10.0g of peptone, 8.0g of beef powder, 4.0g of yeast powder, 20.0g of glucose, 2.0g of dipotassium phosphate, 5.0g of sodium acetate, 2g of diammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 1L of water in 801 g of Tween, and autoclaving at 121 ℃ for 15 min.
2. Isolated culture of Lactobacillus reuteri EHA2 strain
(1) Preparation of bacterial suspension
Firstly, stirring human intestinal excrement uniformly, sucking 10mL of a sample by using a sterile pipette, adding the sample into a triangular flask containing 90mL of sterile water, and fully shaking for 20 minutes on a vortex homogenizer to uniformly disperse the sample. A1 mL sterile pipette is used for sucking 1mL of the fecal bacterial liquid suspension from the tube, injecting the fecal bacterial suspension into a 9mL test tube of sterile water, and blowing the tube for three times to ensure that the fecal bacterial suspension is fully and uniformly mixed. Then using a 1mL sterile pipette to draw 1mL of the solution from the test tube and inject the solution into another test tube containing 9mL of sterile water, and so on to obtain 10-1、10-2、10-3、10-4、10-5、10-6、10-7Various dilutions of the bacterial suspension (7 mL tubes were taken).
(2) Inverted plate
Taking 12 sterile plates, numbering 10-1、10-5、10-6、10-7Three sets of the components; cooling MRS solid culture medium sterilized at 121 deg.C for 30min to about 65 deg.CPouring 12 plates according to the aseptic technique, wherein each dish is about 15 mL; the culture medium is evenly distributed at the bottom of the dish by flat placement and is to be solidified.
3. Separation method
(1) Marking on a position close to flame, holding the bottom of the dish by the left hand and holding the inoculating ring by the right hand, and marking the flat plate according to aseptic operation.
(2) And (3) selecting a first bacterial liquid ring by using an inoculating ring in an aseptic operation, firstly carrying out primary parallel lineation on one side of a plate culture medium for 3-4 lines, then rotating a culture dish for about 70 degrees, burning the remainder on the inoculating ring, cooling, then carrying out secondary parallel lineation on a first lineation part, and then carrying out third parallel lineation on a second parallel lineation part and carrying out fourth parallel lineation on a third parallel lineation part by using the same method. After the streaking, the dish cover is closed, and the dish cover is placed upside down and cultured in a constant temperature incubator at 37 ℃ for 48 hours. Respectively picking and inoculating single colonies grown after culture to a new MRS solid culture medium, checking whether pure culture is carried out or not, and if not, continuing isolated culture until pure culture is obtained to obtain a pure culture strain EHA 2; gram-positive, rod-shaped, no spore, no movement, milky round, neat edge, and smooth and glossy surface. The optimum growth temperature is 37 ℃, catalase is invisible, and lactose, galactose, maltose, rhamnose and mannose can be hydrolyzed. The 16S rRNA gene of the strain EHA2 is extracted and sequenced, the sequence of the gene is shown as SEQ ID NO.1, and BLAST analysis is carried out on the gene and an NCBI website database, and the result analysis shows that the similarity of the gene and a model strain of the strain Lactobacillus reuteri is the highest, wherein the sequence similarity with the Lactobacillus reuteri strain LQ2 is more than 99%. By combining the physiological and biochemical characteristics and the 16S rRNA gene sequence results, the strain EH132 is identified to belong to Lactobacillus reuteri, the strain is named as Lactobacillus reuteri EHA2(Lactobacillus reuteri EHA2), and the strain is preserved in Guangdong province microbial culture Collection (GDMCC) in 2019, 9 and 2 days, and the address: guangzhou city, Xielizhonglu No. 100 college No. 59 building No. 5, with the preservation number GDMCC NO: 60758.
4. lactobacillus reuteri EHA2 culture method and preparation of fermentation supernatant
Activating strains by adopting an MRS solid culture medium, and culturing for 48 hours in an incubator at 37 ℃; then 2 rings of the activated strain are inoculated into MRS broth and fermented for 48h at 37 ℃. Adjusting the OD of the fermented bacterial liquid to 1.8; centrifuging at 4 deg.C for 15min at 6000r/min, and collecting supernatant.
Example 2 the Effect of curcumin and Lactobacillus reuteri EHA2 fermentation broths on TMAO content
Method and device
1. Preparation of the solution:
(1) TMAO solution: 75.11mg of anhydrous trimethylamine oxide powder was weighed, 10mL of PBS was added, and the mixture was mixed by sonication to obtain 100mM TMAO solution, which was dispensed into 10 EP tubes (1 mL each) each having a concentration of 1.5mL and placed in a refrigerator at-20 ℃.
(2) Curcumin solution: 552.57mg of curcumin powder is weighed, 5mL of DMSO is added and mixed evenly to obtain a curcumin solution with the concentration of 300mM, and the curcumin solution is stored in a refrigerator at the temperature of minus 20 ℃ after being subpackaged.
(3) Preparation of a sample: taking 50 mu L of TMAO solution in the step (1), 200 mu L of fermentation supernatant of the Lactobacillus reuteri EHA2 strain in the example 1 and curcumin solution in the step (2) according to the proportion of 1:1, 1:2, 1:3, 2:1 and 3:1, putting the mixture into a 2mL EP tube together, and reacting for 12 h.
(4) Preparing a TMAO standard solution: TMAO powder was precisely weighed at 5.0mg, and placed in a 50mL EP tube, dissolved and diluted with methanol to a concentration of 100. mu.g/mL, and stored at-20 ℃ after being dispensed.
(5) Preparation of internal standard solution: dissolving d9-TMAO standard product in methanol to obtain 5 μ M methanol solution, packaging, labeling, and storing at-20 deg.C.
(6) And (4) taking 50 mu L of the mixed sample in the step (3) to be detected.
2. And (3) establishing a standard curve, and preparing TMAO standard solutions with the concentrations of 0, 7.8125, 15.625, 31.625, 125, 250, 500, 1000 and 2000ng/mL by using methanol solutions respectively. And (5) sampling to determine peak area and calculating a regression equation of the curve.
3. And substituting the measurement peak area of each plasma sample into a standard regression equation to obtain the plasma TMAO concentration of each sample.
Second, result in
The established standard curve is shown in fig. 1, the detection result is shown in table 1, the curcumin or the fermentation supernatant of the lactobacillus reuteri EHA2 strain has a good effect of removing TMAO, and particularly, after the curcumin, the fermentation supernatant of the lactobacillus reuteri EHA2 strain and the TMAO react together for 12 hours, the ratio of the curcumin to the bacteria supernatant is 1: the maximum cleaning effect on TMAO is achieved at the time of 3.
TABLE 1 curcumin and Lactobacillus reuteri EHA2 fermentation supernatants effects on TMAO levels (triplicates)
| Composition (v/v) | TMAO(ng/mL) | TMAO(ng/mL) | TMAO(ng/mL) |
| Blank group | 244.0 | 243.5 | 244.2 |
| Curcumin (4) | 75.5 | 76.1 | 76.4 |
| Curcumin: mushroom supernatant (2:1) | 73.7 | 71.1 | 73.3 |
| Mushroom supernatant (4) | 70.0 | 68.2 | 68.9 |
| Curcumin: mushroom supernatant (3:1) | 62.8 | 62.5 | 61.6 |
| Curcumin: mushroom supernatant (1:2) | 59.9 | 61.4 | 59.0 |
| Curcumin: mushroom supernatant (1:3) | 55.8 | 53.4 | 53.3 |
Example 3 the Effect of curcumin and Lactobacillus reuteri EHA2 fermentation broths on the expression of smooth muscle cell inflammatory factors
1. Cell pretreatment
(1) Human vascular Smooth Muscle Cells (Human Vasc μ Lar Smooth Muscle Cells, HVSMCs) were sourced from the cardiovascular laboratory gift of the zhajiang hospital, southern medical university.
(2) Culture of HVSMCs
Adherent growth in RPMI1640 medium containing 10% fetal bovine serum at 37 deg.C under 5% CO2And culturing in a cell culture box with fully saturated humidity, and changing the liquid every other day to maintain a good growth state.
(3) And (3) pre-intervening human vascular smooth muscle cells for 6h with or without curcumin or curcumin plus Lactobacillus reuteri supernatant, and intervening human vascular smooth muscle cells for 6h with TMAO.
2. Trizol method for extracting total RNA of vascular smooth muscle cells in each group
Human Vascular Smooth Muscle Cells (HVSMCs) were grown adherent to RPMI1640 medium containing 10% fetal bovine serum at 37 deg.C and 5% CO2And culturing in a cell culture box with completely saturated humidity, and changing the culture solution every 1 day to maintain a good growth state. Cells in good state are put into a 6-well plate for culture, and the medicine is added when the cells are fused to 80-90%. With or without curcumin (concentration 40mM, volume 3. mu.L) or curcumin (concentration 40mM, volume 3. mu.L) plus 9. mu.L (OD) of Lactobacillus reuteri EHA2 supernatant600The nm value is 1.8), intervening human vascular smooth muscle cells for 6 hours in advance, sucking out culture solution in a 6-hole plate, and washing the cells for 2-3 times by using PBS; adding 500 mu L of Trizol into each hole, standing for 5min on ice, repeatedly blowing and beating the bottom of the dish by using an enzyme-free gun head to fully and uniformly mix the cells with the Trizol, and facilitating further cell lysis. The cell lysate was then transferred to a 1.5mL EP tube. If the time is not enough for subsequent operation, the EP tube filled with the cell lysate can be placed in a refrigerator at minus 80 ℃ for short-term storage and standby. If RNA in blood vessels is extracted, 20-40 mg of tissue is weighed and placed into a 1.5mL EP tube, 1mL of Trizol is added after shearing, and 60HZ lysis is carried out on a homogenizer for 120 s.
(2) To each 1.5mL of EP tube was added 200. mu.L of chloroform (chloroform), the lid of the EP tube was closed, and the solution was mixed well to pink by vigorous shaking on a shaker for 1 min. After standing on ice for 10min, the mixture was centrifuged at 12000g for 15min at 4 ℃.
(3) After the centrifugation is finished, the centrifuge tube is taken out, and a sample is divided into three layers, namely an upper colorless water phase, a white middle layer and a bottom organic phase, wherein RNA is mainly located in the upper water phase, and the volume of the water phase is about 40% of the dosage of Trizol. Carefully sucking 100-200 mu L of upper-layer aqueous phase liquid, and avoiding contacting with the middle-layer liquid and the lower-layer liquid in the sucking process. If the operation carelessly sucks the liquid in the lower two layers, the centrifugation is carried out once under the same condition, and the liquid in the upper layer is carefully and slowly sucked. The supernatant liquid was transferred to a new enzyme-free 1.5mL EP tube.
(4) Adding isopropanol with the volume equal to that of the liquid absorbed in the step (3), slightly inverting the EP tube from top to bottom and from left to right to mix the liquid uniformly, moving the liquid to ice, standing for 5min, and centrifuging for 10min in a centrifuge with the conditions of 4 ℃ and 12000 g.
(5) The supernatant was decanted and the EP tube was inverted on a paper towel to leave no residual isopropanol on the tube wall and white RNA precipitate was visible at the bottom of the tube.
(6) And respectively adding 500 mu L of precooled 75% ethanol into each tube, and slightly reversing the EP tube from top to bottom and from left to right for 8-10 times so as to facilitate the ethanol to dissolve the residual isopropanol and achieve the purpose of washing RNA precipitates. Centrifuge at 7500g for 5min at 4 ℃, then discard the supernatant and reverse-thread the EP tube on a paper towel so that no liquid remains on the tube wall.
(7) The EP tube was removed from the hood, the lid opened to allow ethanol to evaporate sufficiently, and the RNA pellet dried. After 5min, adding a proper amount of sterile enzyme-free water to dissolve the RNA precipitate, and before measuring the RNA concentration, blowing and stirring the mixed liquid.
(8) The RNA concentration was determined by first zeroing the SimpliNano detector with 2. mu.L of sterile, enzyme-free water and repeating 2 times.
(9) After the zero calibration is completed, the genome RNA concentration of each sample is sequentially detected, each sample is repeatedly measured for 2 times, and the recorded data is averaged. The purity of each sample is referred to by the ratio of EHA260 to EHA280, and the ratio is 1.8-2.0, which represents that the RNA purity of the sample is high. The detected RNA sample can be stored in a refrigerator at-80 ℃ for several weeks for reverse transcription. To avoid RNA degradation, reverse transcription should be performed as early as possible after the sample RNA concentration is determined.
2. Reverse transcription of genomic RNA into cDNA for each sample
(1) Mu.g of RNA are taken from each group and reverse transcribed into cDNA, the required sample volume can be as VVolume of RNA(μL)＝1000ng/CRNA concentration(ng/. mu.L) calculation.
(2) The composition of the reverse transcription 20. mu.L reaction system is shown in Table 2 below:
TABLE 2
(3) The conditions of reverse transcription on the computer are as follows: 15min at 37 ℃; 5s at 85 ℃; 4 ℃ and infinity.
(4) After the reverse transcription reaction is finished, each sample is diluted by sterile enzyme-free water according to the proportion of 1:4 and is uniformly mixed for use. The cDNA can be stored in a refrigerator at-20 ℃.
3. Real-time fluorescent quantitative PCR reaction for detecting expression quantity of inflammatory factor
(1) Primer sequences for inflammatory factors are shown in table 3 below:
TABLE 3
(2) The composition of the RT-PCR 20. mu.L reaction system is shown in Table 4:
TABLE 4
(4) The RT-PCR on-machine reaction conditions are as follows in sequence: pre-denaturation at 95 ℃ for 15 min; denaturation at 95 ℃ for 15 s; annealing, extending at 60 ℃, and performing 40-50 cycles.
(5) Calculation method
And (3) obtaining CT values of each group by RT-PCR, taking beta-actin as a reference gene, and carrying out relative quantification on the expression quantity of each group of target genes by a Livak method. The calculation method is as follows:
Δ CT control group ═ CT (control group, target gene) -CT (control group, reference gene)
Δ CT experimental group ═ CT (experimental group, target gene) -CT (experimental group, internal reference gene)
The Δ CT values for the experimental groups were normalized by the Δ CT values for the control group:
Δ Δ CT ═ Δ CT experimental group- Δ CT control group
Ratio (experimental/control) 2-ΔΔCT
And calculating the obtained value according to a formula, namely the change of the expression level before and after the intervention of the target gene.
4. Results
The experimental result is shown in fig. 2, and the result shows that after the intervention of TMAO on smooth muscle cells, the expression level of inflammation-related factors IL-1, IL-6 and TNF-alpha is increased, the expression level of the inflammation factors of human vascular smooth muscle cells is obviously reduced after the pretreatment of fermentation supernatant of curcumin or Lactobacillus reuteri EHA2 strain alone, and especially the intervention of curcumin and Lactobacillus reuteri supernatant compound obviously can reduce the expression level of the inflammation factors IL-1, IL-6 and TNF-alpha better than the intervention of curcumin alone.
The results show that the curcumin and/or the Lactobacillus reuteri EHA2 strain can eliminate TMAO and inflammation caused by TMAO, has the function of relieving atherosclerotic diseases, and has good social and economic benefits.
Sequence listing
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Claims (5)
1. The application of the composition containing the curcumin solution and the fermentation supernatant of the lactobacillus reuteri EHA2 strain in the preparation of the preparation with functions of removing trimethylamine oxide and resisting inflammation is characterized in that the lactobacillus reuteri EHA2 strain is preserved in Guangdong province microbial strain preservation center in 2019, 9 and 2 days, and the preservation number is GDMCC NO: 60758.
2. the use according to claim 1, wherein the composition comprises the following components in the following volume ratios: the curcumin solution and fermentation supernatant of lactobacillus reuteri EHA2 strain = 1-3: 1 to 3.
3. The use according to claim 2, wherein the composition comprises the following components in the following volume ratios: curcumin solution and lactobacillus reuteri EHA2 strain fermentation supernatant = 1: 3.
4. the use according to claim 3, wherein the curcumin solution is at a concentration of 40 to 300 mM.
5. The application of any one of claims 2 to 4, wherein the fermentation liquid of the Lactobacillus reuteri EHA2 strain is prepared by activating strains, inoculating the activated strains into a fermentation medium, fermenting at 30-37 ℃ for 40-50 h, adjusting the OD of the fermented liquid to 1.6-1.8, centrifuging, and taking the supernatant to obtain the fermentation supernatant of the Lactobacillus reuteri EHA2 strain.
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