CN114796175A

CN114796175A - Application of butyric acid and its salt in preparing medicine for treating cervical cancer

Info

Publication number: CN114796175A
Application number: CN202210666477.8A
Authority: CN
Inventors: 郭君萍; 王方岩; 张珂; 季夏薇
Original assignee: Hangzhou Wuyunshan Hospital Hangzhou Institute Of Health Promotion
Current assignee: Hangzhou Wuyunshan Hospital Hangzhou Institute Of Health Promotion
Priority date: 2022-06-13
Filing date: 2022-06-13
Publication date: 2022-07-29

Abstract

The invention discloses application of butyric acid and a salt thereof in preparing a medicine for treating cervical cancer, and relates to the technical field of biological medicines. The invention claims the application of butyric acid or butyrate in preparing a medicine for treating cervical cancer. The invention also claims the use of butyric acid or butyrate in the preparation of a formulation inducing mitochondrial apoptosis. According to the invention, CCK8 experiments, EdU fluorescence experiments, Transwell experiments, scratch experiments, western blot analysis, cell flow analysis, ROS immunofluorescence, TUNEL experiments, CytC fluorescence experiments and ROS flow analysis show that butyric acid and salts thereof inhibit proliferation, invasion and migration of cervical cancer cell lines by activating mitochondria-dependent apoptosis pathways.

Description

Application of butyric acid and its salt in preparing medicine for treating cervical cancer

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of butyric acid and a salt thereof in preparing a medicine for treating cervical cancer.

Background

Cervical Cancer (CC) is the fourth most common cancer of women worldwide with a new 57 million cases per year, 31.1 million deaths, and a serious threat to the life and health of female patients. Since early symptoms of cervical cancer are not evident, most patients are diagnosed at the middle and late stages with a low 5-year survival rate. The current cervical cancer treatment is mainly based on operation, and is assisted by chemotherapy and other treatments, such as radiotherapy, immunization and targeted therapy, which can cause permanent damage to the body of a patient. The limited effectiveness of cervical cancer therapy is due to an incomplete understanding of the pathogenesis of the cancer.

The apoptosis way of the cervical cancer cell line is researched, and the medicine for inhibiting the proliferation, invasion and migration of the cervical cancer cell line is developed, so that the method has important practical significance for treating the cervical cancer.

Disclosure of Invention

The invention aims to provide application of butyric acid and a salt thereof in preparing a medicament for treating cervical cancer, so as to solve the problems in the prior art, and the butyric acid can inhibit the proliferation, invasion and migration of a cervical cancer cell line by activating a mitochondrion-dependent apoptosis pathway.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides application of butyric acid or butyrate in preparation of a medicine for treating cervical cancer.

Further, the butyric acid or butyrate inhibits proliferation, invasion and migration of cervical cancer cell lines by activating a mitochondria-dependent apoptosis pathway.

Further, the butyrate salt includes sodium butyrate.

The invention also provides application of butyric acid or butyrate in preparation of a preparation for inducing mitochondrial apoptosis.

Further, the mitochondria are endogenous mitochondria.

The invention also provides a medicament for inducing mitochondrial apoptosis or treating cervical cancer, which takes butyric acid or butyrate as a main component.

The invention discloses the following technical effects:

according to the invention, CCK8 experiments, EdU fluorescence experiments, Transwell experiments, scratch experiments, western blot analysis, cell flow analysis, ROS immunofluorescence, TUNEL experiments, CytC fluorescence experiments and ROS flow analysis show that butyric acid and salts thereof inhibit proliferation, invasion and migration of cervical cancer cell lines by activating mitochondria-dependent apoptosis pathways.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 shows the inhibition of proliferation of human cervical cancer cells by sodium butyrate, measured by cell counting kit of proliferation of Hela (A) and Caski (B) cells after 12, 24, 48 and 72 hours of action of 5mM sodium butyrate; EdU fluorescent staining to detect apoptotic cells in Hela (C) and Caski (E) cell lines; d is quantitative analysis of the red fluorescence intensity in C; f is quantitative analysis of the red fluorescence intensity in E; g and I are cell cycle distributions of Hela cells analyzed by flow cytometry, CON: a control group; NaB: group 48 hours with 5mM sodium butyrate; h is quantitative analysis of S-phase Hela cells; all the above data are expressed as mean ± standard deviation; p < 0.05, P < 0.001;

FIG. 2 is a graph showing that sodium butyrate reduces invasion and migration of cervical cancer cells, and a scratch test is used to determine migration of Hela (A) and Caski (C) cells after 12, 24, and 48 hours of treatment with 5mM sodium butyrate; b is quantitative analysis of the healing rate of the Hela group wound; d is quantitative analysis of wound healing rate of Caski group; e, detecting the influence of the sodium butyrate on the invasion and migration of Hela cells by adopting a Transwell method; f, detecting the influence of the sodium butyrate on the invasion and migration of the Caski cells by adopting a Transwell method; CON: control, NaB: group 48 hours with 5mM sodium butyrate; data are presented as mean ± standard deviation; p < 0.01, P < 0.001;

FIG. 3 shows that sodium butyrate promotes cervical cancer apoptosis, and the qualitative and quantitative analysis of Hela apoptosis is carried out by using TUNEL (A) and Annexin-V/PI (B is CON group, C is NaB group) staining; d is the expression of protein related to apoptosis detected by western blot analysis; e is quantitative analysis of CON and NaB histone expression; CON: control, NaB: group 48 hours with 5mM sodium butyrate; data are presented as mean ± standard deviation; NS indicates no significant difference, P < 0.001;

FIG. 4 shows that sodium butyrate activates the mitochondrial-dependent apoptosis pathway of cervical cancer; a is quantitative detection of Caspase 8, Caspase 9 and Caspase 12 by Western blot analysis; b is quantitative analysis of

Caspase

8, 9 and 12 protein expression; d is quantitative detection of Apaf-1, BCL-2 and BAX by western blot analysis; e is the quantitative analysis of Apaf-1, BCL-2 and BAX protein expression; c is the expression of cytochrome C detected by immunofluorescence; f is the expression of immunofluorescence detection active oxygen; G-J is the active oxygen content of 0-48h of quantitative determination of flow cytometry, wherein G-J is 0h, 12h, 24h and 48h in sequence; CON: control, NaB: group 48 hours with 5mM sodium butyrate; data are presented as mean ± standard deviation; NS indicates no significant difference, P < 0.01.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every intervening value, to the extent any stated value or intervening value in a stated range, and any other stated or intervening value in a stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1

Materials and methods

1.1 reagents and antibodies

Sodium butyrate (> 98% purity) was purchased from aladdin, prepared as a 100mM stock solution in media and filtered through a 0.22 μm filter membrane and stored at-20 ℃. Primary antibodies for Caspase 3(Caspase 3, AC0301) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, AB-P-R001) were purchased from Hangzhou Goodher and Shanghai Beyotime, Cleaved-Caspase 3(9661S), Caspase 9(#9508), and BCL-2 related X (BCL-2associated X, BAX, 2772), respectively, from Cell Signaling Technology, Caspase 8(ET1612-70), Caspase 12(HA500144), B-Cell lymphoma 2(B-Cell lymphoma 2, BCL-2, BAX-47), and Cytochrome C (Cytochrome C, CytC, R1510-41), protease activator 1 (apoptosis promoter, Apgene 1, Ab-P-R001), respectively, at a dilution ratio of ABgene 1 to ABgene 1, ABIO 1: 20. Secondary antibodies were purchased from martian proteintech, in a 1: diluting at 5000 proportion. All antibodies were stored at 4 ℃.

1.2 cell culture

Cervical cancer cells Hela and Caski used in the experiment were purchased from American Type Culture Collection (ATCC), the Hela cells were cultured in DMEM medium containing 10% Fetal Bovine Serum (FBS), and the Caski cells were cultured in 1640 medium containing 10% FBSBoth cells were incubated at 37 ℃ and 5% CO ₂ And (5) culturing under an environment.

1.3CCK8 experiment

The cell counting cassette (CK04, Japan institute of Hojindo chemistry) examined the proliferative capacity of cervical cancer cells. The cervical cancer cells Hela and Caski were inoculated in a 96-well plate, 3000 cells were counted per well and cultured for 24 hours for adherent growth, and incubated with a normal medium containing 5mM sodium butyrate for 12, 24, 48 and 72 hours, a 10% CCK8 working solution was prepared using a pure medium, 100. mu.L of the working solution was added per well, and absorbance at 450nm was measured by pipetting the supernatant two hours later. The absorbance-time growth curve was plotted.

1.4EdU fluorescence assay

The EdU fluorescent staining kit (C10310-1 kit, Bo et al) detects the DNA replication activity of cervical cancer cells. Hela cells and Caski cells were inoculated into 12-well plates containing cell slide, cultured for 24h, and cultured for 48h with a normal culture medium containing 5mM sodium butyrate, and no treatment was performed on the normal control group. Subsequently, EdU labeling, cell immobilization, Apollo staining, DNA staining were performed, and the red fluorescence intensity of each group was observed under a fluorescence microscope.

1.5Transwell experiment

Matrigel (CORNING)1 was mixed with Phosphate Buffer Saline (PBS): 20 dilution, spreading 200ul of each solution evenly in a cell chamber, placing the migration group in an incubator for 2h without treatment, and separating out redundant PBS. The Hela cells and Caski cells were digested with trypsin, resuspended in serum-free medium, the cell suspension was inoculated into a cell chamber, and the lower chamber was filled with medium containing 10% FBS and cultured in a cell incubator in which the Hela cells were cultured for 8 hours and the Caski cells were cultured for 17 hours, fixed with 4% paraformaldehyde for 30min at room temperature, stained with 0.1% crystal violet for 15min, and the field of view was photographed under a microscope.

1.6 scratch test

Hela cells and Caski cells were seeded in 6-well plates, and after the cells were fully grown, 2 scratches were scribed vertically in each well at a fixed position using a 1ml tip, and the blank control group and the drug-treated group were changed to 2% FBS medium and 5mM 2% FBS medium, respectively, and photographed at fixed positions of 0h, 12h, 24h, and 48 h.

1.7 Western blot analysis

Hela cells are inoculated in a 6-well plate, treated by 5mM sodium butyrate for 48h, sampled, added with RAPI lysate (Beyotime) and protease inhibitor for protein extraction, protein concentration is determined by using a dimethylaminopropionic acid protein determination kit (Beyotime), cell proteins are separated by electrophoresis of 12% sodium dodecyl sulfate polyacrylamide gel and transferred into polyvinylidene fluoride (BIO-RAD), immediately sealed by 5% skimmed milk powder at room temperature for 1.5h, washed three times by Tris buffered saline solution (Tris buffered saline with Tween, TBST), applied to a primary antibody 4 ℃ shaking table for overnight, and washed three times by TBST on the next day, and incubated with a secondary antibody at room temperature for 2 h. Scanning was performed using an Amersham Image 680 imaging system. Protein bands were quantified by densitometry using ImageJ software (national institute of health, beseisida, maryland).

1.8 cell flow analysis of cell cycle and apoptosis

Hela cells were seeded in a six-well plate, the control group was left untreated, the treated group was exposed to 5mM sodium butyrate for 48h, the cells were harvested, washed with PBS, fixed overnight with 75% ethanol, and analyzed using Propidium Iodide (PI) staining. The apoptosis is detected by double staining the cells with 5 μ L of fluorescein isothiocyanate labeled annexin V (annexin V) and 5 μ L of PI, and staining for 15min in dark place.

1.9ROS immunofluorescence

Mitochondrial superoxide fluorescence probe (YEASEN) was used to detect cellular mitochondrial ROS levels. Culturing the cells in a 12-well plate containing a slide for 48h, removing the culture medium, washing with PBS, adding 5 μ M ROS working solution prepared from PBS, incubating at 37 ℃ in the dark for 10min, blocking with DAPI anti-fluorescence quenching blocking solution (Solarbio), and observing under a fluorescence microscope (LEICA).

1.10TUNEL experiment

Apoptosis was detected using TUNEL apoptosis detection kit (40307ES20, YEASEN). The cells were cultured in a 12-well plate containing a slide for 24h, the experimental group was treated with 5mM sodium butyrate for 48h, the medium was discarded, PBS was washed, the cells were fixed with 4% paraformaldehyde for 30min, resuspended in 0.3% Triton X-100 PBS, incubated at room temperature for 5min, according to the instructions, a TUNEL assay solution was prepared, 50. mu.L of the TUNEL assay solution was added to the sample, incubated at 37 ℃ in the dark for 60 min, and mounted with DAPI-containing anti-fluorescence quenching mounting medium (Solarbio) and observed under a fluorescence microscope.

1.11CytC fluorescence assay

Hela cells are inoculated on a cell slide of a 12-well plate, when the cells grow to a certain density, a treatment group is acted for 48 hours by 5mM sodium butyrate, a culture solution is discarded, PBS is washed, 4% paraformaldehyde solution is added for fixation at room temperature for 15min, the PBS washing process is repeated, the cells are treated by 0.1% Triton X-100 for 5min and then washed again, 1% BSA is fixed at room temperature for 30min, CYTC antibody diluent (1: 500) is used for incubation, the cells are kept out of the sun overnight at 4 ℃, PBS is washed the next day, fluorescent 488 secondary antibodies (Thermo Fisher) are applied, the cells are kept out of the sun at room temperature for 2 hours, PBS is washed, and then the cells are detected under a fluorescent microscope.

1.12ROS flow assay

Detecting relative expression level of cell mitochondrial ROS by using a mitochondrial superoxide fluorescence probe (YEASEN), culturing cells in a six-well plate, carrying out no treatment on a control group, allowing a treatment group to act for 48h by 5mM sodium butyrate, adding 5 mu M ROS working solution prepared by PBS, incubating for 10min at 37 ℃ in the dark, collecting cells, and detecting on a computer.

1.13 statistical analysis

Data are shown as mean ± standard deviation. Differences between the two groups were assessed using the t-test and P values less than 0.05 were considered statistically significant.

Second, result in

2.1 butyric acid inhibits proliferation of cervical cancer

Hela cells and Caski cells were treated with 5mM sodium butyrate, and proliferation was observed at 12h, 24h, 48h, and 72h, and it was found that growth of Hela and Caski cells was inhibited as the drug time was extended (FIGS. 1A-B). To further verify the effect of sodium butyrate on inhibition of proliferation, EdU staining was used to detect the activity of Hela and Caski cell DNA replication. The drug-treated groups showed a significant reduction in red fluorescence compared to the normal control group (FIGS. 1C-D), with statistical differences between the two groups (FIGS. 1E-F). In addition, we found that the drug-treated cells were somewhat reduced in S phase, indicating that the cells were reduced in the number ratio in the DNA synthesis phase and that the proliferation of the cells was inhibited (FIGS. 1G-I).

2.2 butyric acid inhibits metastatic invasion of cervical carcinoma

Results of the scratch experiments on Hela cells and Caski cells showed that the healing capacity of the cells was significantly lower than that of the normal control group after the treatment with sodium butyrate, and the cells were statistically different (FIGS. 2A-D). To further explore the effect of sodium butyrate on cervical cancer migration invasion, we used Transwell to examine the migration and invasion capacity of cells. Both the migration and invasion rates of cervical cancer cells were significantly reduced in the drug-treated group compared to the normal control group (fig. 2E-F).

2.3 butyric acid-induced apoptosis of Hela cells

The results of TUNEL fluorescent staining showed a significant increase in green fluorescence in the drug-treated group, indicating that sodium butyrate promoted apoptosis of Hela (fig. 3A). We analyzed the level of apoptosis of Hela cells quantitatively by annexin V-PI staining, the rate of early apoptosis and late apoptosis of the normal control group of Hela cells was 12.1%, 6.10%, and the rate of early apoptosis and late apoptosis of the drug-treated group was increased to 22.6%, 22.7% (FIG. 3B, C). We further detected the expression of the key protein Caspase3 and clear-Caspase 3 protein in Hela cell apoptosis, and found that the clear-Caspase 3 protein in the drug treated group has higher expression and statistical difference (FIG. 3D, E).

2.4 butyric acid induces Hela apoptosis via the mitochondrial pathway

Results from Westernblot showed that Caspase 9 was significantly increased in the drug-treated group compared to the normal control group, while Caspase 8 and Caspase 12 were not significantly altered, suggesting that sodium butyrate may induce Hela apoptosis via the mitochondrial pathway (FIGS. 4A-B). Furthermore, the expression of Apaf-1 and CytC, two other important members of the mitochondrial apoptotic complex, increased significantly after sodium butyrate treatment (fig. 4C-E). Sodium butyrate up-regulated the synthesis of the pro-apoptotic protein BAX, and did not work against the apoptotic protein BCL-2 (FIGS. 4D-E). Both the ROS kit and flow assay showed a significant increase of ROS in Hela cells after sodium butyrate treatment (fig. 4F, G-J).

Butyric acid, an important component of intestinal microecology, has been shown to inhibit a variety of malignancies. The invention discovers that butyric acid can remarkably inhibit the proliferation of Hela cells by promoting apoptosis and reduce cell migration and invasion. Furthermore, butyrate-induced apoptosis is mediated by endogenous mitochondrial rather than exogenous death receptors or endoplasmic reticulum stress.

The results of the present invention confirmed that the growth of Hela cells was inhibited by sodium butyrate and arrested at stage G1. Since the migration and invasion of CC are the reasons which lead to the unfavorable prognosis and treatment result of patients, we further found that the migration and invasion of Hela cell line are significantly reduced by the treatment of sodium butyrate through Transwell experiments. The induction of apoptosis becomes an important mechanism for butyric acid to play an anticancer role, and the change of relevant apoptosis indexes after the treatment of sodium butyrate is further detected. Consistent with AnnexinV/PI staining results, TUNEL staining also showed that sodium butyrate treatment increased apoptotic cells. Western blot results show that apoptosis-related protein clear Caspase3 is increased, indicating that butyric acid may inhibit CC by inducing apoptosis.

Apoptosis is a "self-controlling" cell death that over time eliminates diseased and damaged cells, and is critical for tumor development and treatment. Apoptosis can be initiated by three different pathways: exogenous death receptors, endogenous mitochondrial and endoplasmic reticulum stress. Death receptors can activate Caspase 8, stimulate downstream Caspase factors, and lead to apoptosis. Caspase 12 triggers apoptosis when the endoplasmic reticulum is stressed. Caspase 9 is a key promoter for mitochondrion-dependent apoptosis. Our western blot results showed a significant increase in Caspase 9, but no change in

caspases

8 and 12, indicating that butyrate promotes apoptosis of human CC cells via the mitochondrial pathway. We also found that following sodium butyrate treatment, the key factors Apaf-1 and CytC mediating the mitochondrial apoptotic pathway were significantly upregulated. Previous studies have shown that butyric acid can increase synthesis of the pro-apoptotic protein BAX, but decrease anti-apoptotic BCL-2, thereby promoting mitochondrion-dependent apoptosis. However, our data show that sodium butyrate only regulates BAX protein expression in CC cells. The accumulation of ROS is believed to be an early event in mitochondrially mediated apoptosis, as evidenced by our findings. In human bladder and breast cancer, butyrate overproduces ROS, leading to cysteine protease-dependent apoptosis. Taken together, our data indicate that butyrate treatment activates mitochondrion-dependent apoptosis of human CC.

The "butyric paradox" describes the opposite effect of butyric acid on cancer cells and normal colon epithelium. Butyric acid, an essential nutrient, is used to repair damaged epithelial cells, maintain the integrity of luminal mucosa, but inhibit cancer cells. Previous authors have shown that small amounts of butyrate are metabolized to acetyl-coa to stimulate histone acetyltransferase, while high levels of butyrate accumulate in the nucleus and function as histone deacetylase inhibitors. Both of these mechanisms increase histone acetylation, thereby regulating the expression of different genes, resulting in a wide range of anti-tumor effects, including induction of apoptosis and inhibition of cell proliferation. Further research shows that butyric acid can reduce the expression of anti-apoptosis genes such as BCL-2 and BCL-xl and increase pro-apoptosis genes such as BAX and BAK, thereby promoting apoptosis. On the other hand, butyrate changes the expression of metabolism-related genes, which may explain the Warburg effect. The Warburg effect refers to the utilization of glycolysis by cancer cells to meet energy requirements under aerobic conditions, rather than oxidative phosphorylation. Butyrate significantly inhibits glucose metabolism by lowering mRNA levels of the glucose transporter GLUT1 and glucose-6-phosphate dehydrogenase, thereby inducing more apoptosis in colorectal cancer cells. In addition, butyric acid can interact with G protein-coupled receptor 41 (GPR 41), GPR43 and GPR109a, and exert anti-inflammatory and anti-tumor effects. GPR109a binds to ligand butyrate, inducing apoptosis of colon cancer cells. In breast cancer, butyrate can activate GPR109a, inhibiting the expression of anti-apoptotic genes and cell survival. However, the detailed mechanism of butyrate inhibition of human CC remains to be studied further in the future.

The Hela and Caski cell lines used in our study were HPV-18 and HPV-16 positive cells, respectively, suggesting that butyric acid has a potential therapeutic effect on HPV infected CC. Although HPV is an important causative factor for CC, whether the cervical epithelium of HPV-positive patients is cancerous depends on the vaginal microbiota. Studies have revealed unique microbial signatures associated with the development of cervical cancer. HPV-positive women are more complex in bacterial diversity and composition than HPV-negative women. Furthermore, studies have shown that cervical microbiota like clostridia may increase anti-inflammatory cytokines, thereby playing an important role in the development of immunosuppressive microenvironments associated with HPV infection and persistence. Chlamydia trachomatis has been shown to increase the persistence of HPV infection, and this co-infection appears to result in a higher risk of CC. Butyric acid has been found to correct the dysregulation of the gut microbiota and to increase the abundance of beneficial bacteria, including lactobacilli. It is well known that butyric acid is an important metabolite of lactobacilli and clostridia, suggesting that butyric acid may play a key role in the inhibition of CC by the vaginal microbiota.

In summary, the results of the present study show that butyrate inhibits the proliferation, invasion and migration of CC cell lines by activating a mitochondria-dependent apoptotic pathway. Given the inhibitory effect on HPV positive CC cells, butyrate may have potential therapeutic effects on HIV positive CC patients.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. Application of butyric acid or butyrate in preparing a medicament for treating cervical cancer.

2. The use according to claim 1, wherein butyric acid or butyrate inhibits proliferation, invasion and migration of cervical cancer cell lines by activating a mitochondria-dependent apoptotic pathway.

3. The use according to claim 1, wherein the butyrate salt comprises sodium butyrate.

4. Use of butyric acid or butyrate for the preparation of a formulation for inducing mitochondrial apoptosis.

5. The use of claim 4, wherein the mitochondria are endogenous mitochondria.

6. A drug for inducing mitochondrial apoptosis or treating cervical cancer, characterized in that butyric acid or butyrate is used as a main component.