A composition having the function of regulating intestinal flora

Abstract

The present invention provides a composition having the function of regulating intestinal flora, and belongs to the field of biomedicine. The composition having the function of regulating intestinal flora contains sesamin and α-linolenic acid in a mass ratio of 1-2:1-2. Through animal experiments and cell experiments, it is found that the combination of sesamin and α-linolenic acid can significantly improve the function of regulating intestinal flora, has the advantage of synergistically enhancing physiological activity, can be used to prepare health foods and medicines for regulating intestinal flora, and can improve intestinal dysfunction caused by intestinal flora imbalance.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to a composition that has the effect of regulating intestinal flora. Background Technology

[0002] Functional intestinal disorder, also known as functional gastrointestinal disorder, is a chronic, recurrent digestive tract disease, such as functional dyspepsia, irritable bowel syndrome, and functional constipation. The etiology is not fully understood, but it is closely related to gut microbiota imbalance, as well as factors such as emotions, diet, underlying diseases, and even has a certain genetic predisposition. Treatment includes lifestyle and dietary adjustments, regular living habits, and emotional regulation. Some medications, such as gastrointestinal motility drugs, probiotics, antacids, and gastric mucosal protectants, may be added. However, there are still no definitive and effective prevention and treatment methods. Therefore, developing health foods with active ingredients derived from food and traditional Chinese medicine (food and medicine homology) is expected to have a good preventive and therapeutic effect on patients with long-term intestinal disorders. These products can be taken alone for mild cases and can be used long-term; they can also be used in conjunction with chemical drugs, reducing the dosage and minimizing toxic side effects. Therefore, developing such products is essential.

[0003] The gut microbiota effectively promotes the host's intestinal barrier function, establishes the immune system, and facilitates nutrient metabolism, forming a mutually dependent and harmonious symbiotic whole. The intestinal biological barrier, formed by the close integration of gut microbiota and the intestinal mucosa, confines pathogenic bacteria and toxins within the intestine, preventing inflammation. During metabolic fermentation, gut microbiota produces a large amount of short-chain fatty acids, providing energy for the intestinal mucosal epithelium. Therefore, regulating the gut microbiota plays a crucial role in promoting human intestinal health.

[0004] Sesame seeds have been used in traditional Chinese medicine for a long time to improve gastrointestinal function and relieve constipation. Sesamin is the main functional component of sesame seeds, with the molecular formula C6H2O. 20 H 18 O6 belongs to the lignan class. Sesamin has a variety of physiological activities, including eliminating free radicals in the body, lowering serum cholesterol, protecting liver function, promoting alcohol metabolism, and antibacterial activity. It can be applied in the fields of health food and medicine.

[0005] Linolenic acid oil is extracted from flaxseed through pressing. It is rich in alpha-linolenic acid, which has effects such as regulating blood lipids, lowering cholesterol, and reducing blood viscosity. Alpha-linolenic acid is an important component of human tissue cells and an essential fatty acid for maintaining good health. It participates in the synthesis, metabolism, and conversion of phospholipids in the body, and is an essential bioactive factor for the human body, along with DHA and EPA. Alpha-linolenic acid cannot be synthesized in the human body and must be obtained from external sources. It has effects such as enhancing intelligence, protecting eyesight, lowering blood cholesterol, delaying aging, and anti-allergy properties.

[0006] There is a lack of compositions in the current technology that are derived from food and medicinal herbs and have a good effect on regulating intestinal flora. Summary of the Invention

[0007] The purpose of this invention is to provide a composition that regulates intestinal flora, which is derived from food and traditional Chinese medicine that is both food and medicine and has a good effect on regulating intestinal flora.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A composition having the effect of regulating intestinal flora contains sesamin and α-linolenic acid in a mass ratio of 1 to 2: 1 to 2.

[0010] In this invention, the sesamin is extracted from sesame meal; the α-linolenic acid is extracted from flaxseed.

[0011] In this invention, the sesamin content in the sesamin extract is greater than 50%; the α-linolenic acid content in the α-linolenic acid extract is greater than 50%.

[0012] In this invention, the preparation method of sesamin extract is as follows: sesame meal powder is defatted with n-hexane to obtain defatted sesame meal; the defatted sesame meal is extracted with aqueous ethanol solution to obtain sesamin extract.

[0013] In this invention, flaxseed is placed in an extraction vessel and extracted from flaxseed powder using supercritical carbon dioxide extraction to obtain α-linolenic acid extract.

[0014] Pharmacological tests show that the sesamin powder and alpha-linolenic acid oil composition of the present invention can reduce the number of harmful bacteria and increase the number of probiotics in the feces of mice on normal and high-fat diets, thus regulating the intestinal flora. Furthermore, sesamin and alpha-linolenic acid can promote the adhesion of Lactobacillus acidophilus and Bifidobacterium bifidum to NCM460 colonic epithelial cells, which is beneficial for the colonization of probiotics on colonic epithelial cells. The combination of sesamin and alpha-linolenic acid is significantly superior to using either alone, exhibiting a synergistic effect. Toxicological tests show that the composition of the present invention is safe for use. Therefore, the composition of the present invention can be used to prepare health foods and drugs that regulate intestinal flora, and can improve intestinal dysfunction caused by intestinal flora imbalance.

[0015] Beneficial effects: Animal and cell experiments have shown that the combination of sesamin and α-linolenic acid can significantly enhance the regulation of intestinal flora, and has the advantage of synergistic enhancement of physiological activity. It can be used to prepare health foods and drugs that regulate intestinal flora, and can improve intestinal dysfunction caused by intestinal flora imbalance. Detailed Implementation

[0016] The present invention will be further described below with reference to embodiments.

[0017] Example 1: Preparation of sesamin and α-linolenic acid

[0018] A composition having the effect of regulating intestinal flora is obtained by uniformly mixing sesamin extract and α-linolenic acid extract in a mass ratio of 1:1.

[0019] (1) Preparation method of sesamin extract

[0020] The preparation method of sesamin is as follows: Sesame meal is ground into powder, and hexane with twice the volume of the sesame meal powder is added. The mixture is stirred for 3 hours to defatted the sesame meal. After filtration, the same method is used for a second defatting. After filtration, the sesame meal is air-dried to obtain defatted sesame meal. 95% ethanol aqueous solution is added to the defatted sesame meal at a ratio of 8.5 ml per gram of defatted sesame meal. The mixture is stirred in a 55°C water bath for 2 hours and extracted. After filtration, the extract and defatted sesame meal are separated. The same method is then used to repeat the extraction once more. The two extracts are combined, filtered, and the filtrate is partially evaporated using a rotary evaporator to remove ethanol. The filtrate is concentrated under reduced pressure and dried under vacuum at 60°C to obtain the sesamin extract, which contains 652.39 mg / g of sesamin. The sesamin extract is a light yellow powder, odorless, tasteless, hygroscopic, soluble in alcohol, and insoluble in water.

[0021] The method for determining the sesamin content in the sesamin extract is as follows: HPLC was used. The concentration of the sesamin standard solution was between 20-100 μg / ml, and the corresponding peak area showed a significant linear relationship with the sesamin mass concentration; therefore, a standard curve was prepared. Sesamin powder was dissolved in anhydrous ethanol (chromatographic grade) and filtered. The sesamin content in the product was determined under the following conditions: column temperature: room temperature; mobile phase: methanol-water (8:2, v / v); flow rate: 0.8 ml / min; injection volume: 20 μl; detection wavelength: 290 nm; analysis time: 15 min. Qualitative analysis was performed based on the retention time of the standard, and the sesamin content was calculated using the external standard method. The sesamin content of this product was 652.39 mg / g.

[0022] (2) Preparation method of α-linolenic acid extract

[0023] The preparation method of α-linolenic acid extract is as follows: Flaxseed powder is placed in an extraction vessel and extracted using supercritical carbon dioxide extraction. The extraction temperature is 50℃, the extraction pressure is 30MPa, and the extraction time is 4h to obtain α-linolenic acid extract. The content of α-linolenic acid in the extract is 681.57mg / g.

[0024] The method for determining the α-linolenic acid content in the extract is as follows: GC-MS was used to analyze the α-linolenic acid content in linolenic acid oil. 50 mg of supercritical fluid-extracted linolenic acid oil was accurately weighed into a 25 mL stoppered test tube for methylation treatment: 1.5 mL of a pre-prepared 1 mol / L KOH CH3OH solution was added to the test tube, and the mixture was shaken in a 40℃ constant temperature water bath for 25 min to ensure complete methylation of α-linolenic acid. After removing the test tube, 4.5 mL of n-hexane was added, and the mixture was shaken for 2 min to ensure homogeneity. After standing for 3 min, 2 mL of the supernatant was collected into a new, dry, clean 10 mL centrifuge tube, and a small amount of anhydrous sodium sulfate was added to remove excess water. Finally, the supernatant was collected using a disposable syringe through a filter membrane into a 2 mL gas chromatograph vial. Gas chromatographic conditions for GC-MS analysis of linolenic acid oil: capillary column: DB-5MS (30m × 250μm, 0.25μm); carrier gas: helium; constant flow rate: 0.8mL·min -1 The split ratio was 10:1, and the injection port temperature was 250℃. The temperature program was: initial temperature 120℃, hold for 6 min, then increase at 5℃ / min. -1 Heat to 200℃, hold for 20 minutes, and finally reduce to 10℃·min. -1 The temperature was raised to 230℃ and held for 10 min; the injection volume was 1 μL. Mass spectrometry conditions: electron impact (EI) ion source, electron energy 70 eV, interface temperature 230℃, ion source temperature 150℃, mass scan range 30-500 amu. Sample analysis method: The peaks of the obtained total ion chromatogram were scanned by mass spectrometry and compared with the NIST database standard library to determine the relative content of α-linolenic acid in the oil.

[0025] Example 2: The regulatory effect of sesamin and α-linolenic acid on intestinal flora

[0026] This embodiment uses the sesamin extract and α-linolenic acid extract prepared in Example 1 for animal and cytological experiments to investigate the regulatory effect of the combined action of sesamin and α-linolenic acid on the intestinal flora. In this embodiment, sesamin is added in the form of a sesamin extract, and the mass of the sesamin extract used is calculated based on a sesamin content of 652.39 mg / g. Similarly, α-linolenic acid is added in the form of an α-linolenic acid extract, and the mass of the α-linolenic acid extract used is calculated based on an α-linolenic acid content of 681.57 mg / g.

[0027] (1) Animal experiments were conducted to investigate the regulatory effect of sesamin and α-linolenic acid on gut microbiota.

[0028] Use 6-week-old SPF grade C 57BL / 6J mice were randomly divided into four groups of 12 mice each, with half males and half females. They were fed either a normal diet or a high-fat diet. The regulatory effects of sesamin (ZMS), α-linolenic acid (YMS), and sesamin + α-linolenic acid (ZMS+YMS) on the intestinal flora were observed. Animal model based on normal diet: A normal control group, a ZMS group, a YMS group, and a ZMS+YMS group were established. The ZMS group was given 40 mg / kg of sesamin. -1 ·d -1 The YMS group was given α-linolenic acid 40 mg / kg. -1 ·d -1 The ZMS+YMS group was given sesamin 20 mg / kg. -1 ·d -1 and α-linolenic acid 20 mg·kg -1 ·d -1 (See Table 1). High-fat diet animal model: Normal control group, high-fat control group, ZMS group, YMS group, and ZMS+YMS group were established. The ZMS group was given sesamin 40 mg / kg. -1 ·d -1 The YMS group was given α-linolenic acid 40 mg / kg. -1 ·d -1 The ZMS+YMS group was given sesamin 20 mg / kg. -1 ·d -1 and α-linolenic acid 20 mg·kg -1 ·d -1 (See Table 2). After 6 weeks, the intestinal flora of each mouse was counted using the conventional plate culture method, and the number of intestinal flora was detected according to the following experimental method. 1g of feces from rats on a normal or high-fat diet was taken, and 9mL of sterile physiological saline was added under aseptic conditions. The mixture was vortexed until homogenized, and then filtered through 4 layers of sterile gauze to obtain 10g of feces. -1 Intestinal flora solution. Take 1 mL of 10 -1 Intestinal bacterial culture, diluted with 9 mL of sterile physiological saline to obtain 10 -2 Prepare intestinal bacterial culture. Inoculate 1 mL of the corresponding dilution onto the culture media of Lactobacillus, Bifidobacterium, Escherichia coli, and Enterococcus, making two replicates for each dilution. Use LBS agar and BS agar for Lactobacillus, and incubate in an anaerobic incubator for 24 hours. Use EMB agar and Enterococcus faecalis agar for Escherichia coli, and incubate in a light incubator for 48 hours. After incubation, calculate the colony count, expressing the results logarithmically (Log(CFU / mL sample)). Calculate the mean (x), and the final colony count is expressed as x ± s, where s is the standard deviation.

[0029] As shown in Table 1, compared with the normal control group, the intervention with sesamin, α-linolenic acid, and a combination of sesamin and α-linolenic acid significantly reduced the number of Enterococci in the feces of mice on a normal diet, while significantly increasing the number of Lactobacillus and Bifidobacterium. This indicates that sesamin and α-linolenic acid can reduce the number of harmful bacteria and increase the number of beneficial bacteria in the feces of mice on a normal diet, thus regulating the intestinal flora. The combination of sesamin and α-linolenic acid is significantly better than using either alone, therefore sesamin and α-linolenic acid have a synergistic effect.

[0030] Table 1. Regulatory effects of sesamin and α-linolenic acid on gut microbiota in mice on a normal diet

[0031]

[0032] Note: Bacterial count is expressed as x±s; compared with the normal control group, " * " indicates P < 0.05, ** "" indicates that P < 0.01.

[0033] As shown in Table 2, compared with the normal control group, the number of Lactobacillus and Bifidobacterium in the feces of mice in the high-fat control group was significantly reduced, while the number of Escherichia coli was significantly increased. Compared with the high-fat control group, the number of Escherichia coli in the feces of mice treated with sesamin, α-linolenic acid, and sesamin + α-linolenic acid was significantly reduced, while the number of Lactobacillus and Bifidobacterium was significantly increased. These results indicate that sesamin and α-linolenic acid can reduce the number of harmful bacteria and increase the number of beneficial bacteria in the intestines of mice on a high-fat diet, thus regulating the intestinal flora. However, the effect of the combination of sesamin and α-linolenic acid is significantly better than that of either alone, showing a synergistic effect.

[0034] Table 2. Regulatory effects of sesamin and α-linolenic acid on gut microbiota in mice on a high-fat diet

[0035]

[0036] Note: In Table 2, the bacterial count is expressed as x ± s; compared with the normal control group, " * " indicates P < 0.05, ** "" indicates P < 0.01; compared with the high-fat control group, " # " indicates P < 0.05, ## " indicates that P < 0.01.

[0037] (2) Cellular experiments were conducted to investigate the regulatory effect of sesamin and linolenic acid oil on the intestinal flora.

[0038] Activation of intestinal probiotics: *Bifidobacterium bifidum* was cultured in TPY broth, and *Lactobacillus acidophilus* was cultured in MRS broth, and passaged to activate the strains. Preparation of probiotic suspensions: The activated *Bifidobacterium bifidum* and *Lactobacillus acidophilus* were inoculated separately into sterilized liquid culture media. *Lactobacillus acidophilus* was cultured in an anaerobic environment for 16 hours, and *Bifidobacterium bifidum* for 20 hours. The cultured probiotics were centrifuged at 4℃ and 4000 rpm for 10 min to collect the bacterial cells. The cells were then washed three times with sterile PBS. Counting was performed using a hemocytometer, and the bacterial suspension was resuspended in high-glucose medium (DMEM) to adjust the concentration to 1×10⁻⁶. 8 CFU / mL.

[0039] The effects of sesamin and α-linolenic acid on the adhesion of Bifidobacterium bifidum to NCM460 colonic epithelial cells were investigated: Well-grown NCM460 cells were routinely digested, centrifuged, resuspended, and then counted. The cell suspension was diluted to 1×10⁻⁶. 6 Cells / mL. The diluted cell suspension was seeded into six-well plates and incubated overnight. After incubation, the supernatant was aspirated, and the cells were washed twice with PBS. 50 μg / mL sesamin solution, 50 μg / mL α-linolenic acid solution, or a solution containing 25 μg / mL sesamin and 25 μg / mL α-linolenic acid were added to each well, with three parallel wells for each reagent. The plates were incubated at 37°C in a 5% CO2 incubator. After 24 hours, the cell plates were removed, the supernatant was discarded, and the cells were washed three times with PBS. A concentration of 1×10⁻⁶ cells / mL was then added to each well. 8 CFU / mL of Bifidobacterium bifidum was added to the cells treated with each agent, with 1 mL of bacterial culture added to each well. Additionally, 1 mL of LDM was added to each well, and the cells were incubated for 2 hours. The incubated cell culture plates were then washed 3-5 times with sterile PBS, followed by the addition of an appropriate amount of trypsin and incubation in an incubator for complete digestion. Colony counts were performed on the digested liquid using the plate count method, and the number of probiotics adhering to each cell was calculated.

[0040] The effects of sesamin and α-linolenic acid on the adhesion of Lactobacillus acidophilus to NCM460 colonic epithelial cells were investigated using the same method described above, the only difference being that the concentration was 1×10 8 CFU / mL of Lactobacillus acidophilus was added to the cells treated with each agent. After co-incubation with the cells, colony counts were performed on the digested liquid using the plate count method, and the number of probiotics adhering to each cell was calculated.

[0041] The results (see Tables 3 and 4) showed that, compared with the normal control group, intervention with sesamin, α-linolenic acid, and sesamin + α-linolenic acid significantly increased the number of probiotics (Lactobacillus acidophilus and Bifidobacterium bifidum) adhering to colonic epithelial cells. This indicates that sesamin and α-linolenic acid can promote the adhesion of Lactobacillus acidophilus and Bifidobacterium bifidum to NCM460 colonic epithelial cells, which is beneficial for the colonization of probiotics on colonic epithelial cells. The combination of sesamin and α-linolenic acid was significantly better than either of them, exhibiting a synergistic effect.

[0042] Table 3. Effects of sesamin and α-linolenic acid on the adhesion of Lactobacillus acidophilus to NCM460 cells.

[0043] Group Colony count lg (CFU / mL) Adhesion count (bacteria / cell) Normal control 5.96±0.12 3.64±0.29 Sesamin 5.52±0.14 <![CDATA[3.87±0.13 * ]]> α-Linolenic acid 5.83±0.11 <![CDATA[4.01±0.39 * ]]> Sesamin + Alpha-linolenic acid 6.05±0.29 <![CDATA[5.49±0.25 ** ]]>

[0044] Note: Compared with the normal control group, " * " indicates p < 0.05, " ** " indicates p < 0.01.

[0045] Table 4. Effects of sesamin and α-linolenic acid on the adhesion of Bifidobacterium bifidum to NCM460 cells.

[0046] Group Colony count lg (CFU / mL) Adhesion count (bacteria / cell) Normal control 5.73±0.06 2.49±0.32 Sesamin 5.84±0.23 <![CDATA[3.44±0.68 * ]]> α-Linolenic acid 6.01±0.27 <![CDATA[3.11±0.52 * ]]> Sesamin + Alpha-linolenic acid 6.14±0.19 <![CDATA[5.49±0.60 ** ]]>

[0047] Note: Compared with the normal control group, " * " indicates p < 0.05, " ** " indicates p < 0.01.

[0048] Acute toxicity tests of this invention showed that, in mice administered sesamin and α-linolenic acid by gavage at a mass ratio of 1:1, the maximum daily dose (MTD) was 19.6 g / kg, equivalent to 120 times the effective dose. This indicates that the product of this invention is safe and has virtually no toxic side effects.

[0049] Example 3

[0050] Various dosage forms containing sesamin and alpha-linolenic acid were prepared using conventional methods. Sesamin was added as a sesamin extract, and the mass of the sesamin extract used was calculated based on a sesamin content of 652.39 mg / g. Alpha-linolenic acid was added as an alpha-linolenic acid extract, and the mass of the alpha-linolenic acid extract used was calculated based on an alpha-linolenic acid content of 681.57 mg / g.

[0051] Capsules containing sesamin and alpha-linolenic acid: Each capsule contains 40mg of sesamin and 40mg of alpha-linolenic acid.

[0052] Tablets containing sesamin and alpha-linolenic acid, each tablet containing 20mg sesamin and 40mg alpha-linolenic acid.

[0053] Granules containing sesamin and alpha-linolenic acid, each sachet contains 40mg of sesamin and 20mg of alpha-linolenic acid.

[0054] Soft capsules containing sesamin and alpha-linolenic acid, each capsule containing 40mg of sesamin and 40mg of alpha-linolenic acid.

[0055] The daily oral dose, based on the effective component composition, is 320 mg of sesamin and α-linolenic acid. It is used to regulate intestinal flora and improve intestinal function (such as intestinal dysfunction caused by functional dyspepsia, irritable bowel syndrome, functional constipation, etc.). The dosage for granules is 1-2 sachets twice daily, dissolved in warm water; the dosage for tablets is 1-2 tablets twice daily; and the dosage for capsules and soft capsules is 2 capsules twice daily. One month constitutes one course of treatment.

Claims

1. Use of a composition in the preparation of a drug for regulating intestinal flora, characterized in that The composition contains sesamin and α-linolenic acid in a mass ratio of 1:1, and the sesamin is added in the form of a sesamin extract, and the α-linolenic acid is added in the form of an α-linolenic acid extract; the sesamin content in the sesamin extract is greater than 50%; the α-linolenic acid content in the α-linolenic acid extract is greater than 50%; the preparation method of the sesamin extract is as follows: sesame meal powder is defatted by using n-hexane to obtain defatted sesame meal, and the defatted sesame meal is extracted by using an ethanol aqueous solution to obtain a sesamin extract; the preparation method of the α-linolenic acid extract is as follows: flax seeds are loaded into an extraction kettle, and supercritical carbon dioxide extraction is used to extract from the flax seed powder to obtain an α-linolenic acid extract.

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