KR102359029B1 - Composition for improving intestinal microbiome comprising an aged Platycodon grandiflorus extract as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for alleviating intestinal flora containing a Platycodon grandiflorus extract manufactured by steaming and drying Platycodon grandiflorus as an active ingredient. When treating the Platycodon grandiflorus extract of the present invention, it is possible to prevent or alleviate various diseases by controlling the balance of intestinal microbes, and in particular, exhibits an effect of enhancing the reduced immune function.

Description

Composition for improving intestinal microbiome comprising an aged Platycodon grandiflorus extract as an active ingredient

The present invention relates to a composition for improving intestinal flora containing a red bellflower extract as an active ingredient.

A close correlation between human microbes, especially intestinal microbes, and human diseases has been continuously reported. Accordingly, studies are underway to improve human health by improving the human intestinal microflora in a beneficial direction, and the most representative examples include probiotics, lactic acid bacteria, and prebiotics. Prebiotics in the traditional sense refer to ingredients such as dietary fiber and oligosaccharides, but many researchers are making great efforts to develop natural prebiotics using plant-derived natural products. The group of bacteria present in the intestine is called the intestinal flora, and beneficial bacteria and harmful bacteria are in balance in this intestinal flora, and this balance is very closely related to human health.

The intestinal flora fluctuates depending on various factors such as host physiological conditions, food, drugs, and stress, and the balance of the intestinal flora depends on the habitat and nutrients in the intestine under strong anaerobic conditions, intestinal peristalsis, and continuous excretion. It has been reported that the balance of the intestinal flora is achieved in the process of competing with each other for pH, redox potential, bile acid, produced bacteriocin, fatty acid, hydrogen emulsion, etc. (Yazawa K. and Tamura Z. Bifidobacteria Microflora) 1(1): 39-44, 1982).

It has been reported in the preceding literature that changes in the intestinal flora are related to various diseases. In the case of inflammatory bowel diseases including Crohn's disease and ulcerative colitis, it has been reported that the diversity of microorganisms in the feces is lacking, and in particular, the diversity of the intestinal flora decreases proportionally according to the severity of inflammatory bowel disease ( Halfvarson et al, Nat Microbiol, 13;2:17004, 2017). In addition, there is a report that the short-chain fatty acid producing strain is significantly reduced in atopic skin patients in children, resulting in a change in the concentration of short-chain fatty acid, resulting in atopy (Reddel et al, Sci Rep, 21;9(1):4996, 2019) . In addition, it has been suggested that persistent inflammation caused by intestinal bacteria may act as a cause of diabetes (Muccioli et al, Mol Syst Biol, 6:392, 2010). In addition, there is a report that the cause of obesity is the intestinal flora, not the type or amount of food, and that skinny people have more different types of intestinal bacteria than obese people (Ley et al, Nature, 444:1200-1023) , 2006). Dementia is also likely to be affected by intestinal bacteria, and systemic inflammation caused by intestinal imbalance causes infiltration of peripheral and central nervous system microbes, resulting in increased inflammation and amyloid production, which may lead to increased amyloidization of the central nervous system. (Hill and Lukiw, Front Aging Neurosci, 10;7:9, 2015).

Bellflower (Platycodon grandiflorus) is a perennial plant of the Campanulaceae family. The main root is about 10-15 cm, the diameter is 1-3 cm, and the upper part has irregular stem spots, grayish-brown or milky-white, with deep vertical wrinkles, and horizontally There are skins (皮目) and wrinkles. The quality is hard but non-fibrous, so it is easy to break, has a slight odor, and has a bitter taste. The main pharmacological component of bellflower known is triterpenoid-based saponin, which accounts for about 3% of the root. and gastric juice secretion inhibitory action, anticholinergic action to lower blood pressure by dilating blood vessels, blood sugar lowering action, cholesterol metabolism improvement action, etc. (Chem. Phram. Bull, 20,1952, 1972; Chem. Pharm. Bull, 23 , 2965, 1975; J. Chem Soc, Perkin trans I, 661, 1984; J. Pharm. Soc. Kor, 19, 164, 1975). In addition, steroid compounds such as alpha-spinasterol, stigmast-7-enol, alpha-spinasterol glucoside, and polysaccharides such as inulin and betulin are also found in bellflower. Antidiabetic activity (Journal of the Korean Society for Food Science and Technology, 39:701-707, 2007), growth inhibitory activity of lung cancer cells (Journal of Dong-Eui Physiological Pathology 17: 183-189, 2003), and immune enhancing activity (Journal of Pharmaceutical Sciences 42) : 382-387, 1998), oxidative stress protective effect on hepatocytes (Journal of Pharmacy 46: 466-471, 2002), and mutation inhibitory effect (Journal of Food Science and Technology 33: 651-655, 2001), etc. have been reported.

On the other hand, if the process of steaming and drying bellflower is repeated several times, the white bellflower gradually changes to yellow, red, and black to produce red and black bellflower. An increase in functionality is expected. Browning substances produced during repeated steaming and drying of bellflower exhibit antioxidant, anti-mutagenic and anti-aging effects (Lee Sang-Hoon et al., 2013. Journal of the Korean Society for Food and Nutrition 42: 1405-1411; Lee Su-jin et al., 2013. Korea Food Storage and Distribution) Journal, 20: 510-517), it is reported that the antioxidant and antidiabetic activity is increased in red bellflower and black bellflower than in bellflower (Jongho Park, 2011, Jungbu University Master's thesis, "Preparation of extract from red bellflower by extraction and aging conditions") and quality characteristics"; Eunjoo Kim, 2006. Gyeongsang National University Master's thesis, "Physicochemical properties and antidiabetic activity of Jangsaeng red bellflower"; Sujin Lee et al., 2013. Journal of the Korean Society for Food Storage and Distribution, 20: 510-517).

In particular, the content of phenolic compounds significantly increased according to the number of repetitions of steaming and the content of platycodin D, a major functional substance, increased by 2.6 times, so it is predicted that the immune enhancing effect will be superior to that of general bellflower.

However, to date, there have been no reports of literature disclosing the effect of improving the intestinal flora of mature bellflower such as red bellflower and black bellflower.

Accordingly, the present inventors completed the present invention by confirming that the bellflower extract and the red bellflower extract prepared by steaming and drying bellflower restores intestinal flora in a state of reduced immunity.

It is an object of the present invention to provide a composition for improving intestinal flora containing an extract of red bellflower (aged Platycodon grandiflorus) as an active ingredient.

In order to achieve the above object, the present invention provides a composition for improving intestinal flora containing an extract of red bellflower (aged Platycodon grandiflorus) as an active ingredient.

The red bellflower extract of the present invention affects the diversity of intestinal microflora in a state of reduced immunity, restores a flora similar to that of a normal person, and exhibits an immune enhancing effect in various diseases, particularly by controlling microorganisms that are correlated with immunity This has the effect of improving the weakened immune function.

1 is a diagram showing a manufacturing process of a red bellflower extract.
2 is a diagram showing changes in the intestinal flora according to the treatment of red bellflower extract.
Nor (normal group): normal mice administered with distilled water without CPA (cyclophosphamide) treatment
NC (negative control): mice treated with CPA and administered with distilled water
PC (positive control): mice treated with CPA and administered with β-glucan
PGS1: Mice treated with CPA and administered with red bellflower extract (150 mg/kg BW)
PGS2: Mice treated with CPA and administered with red bellflower extract (300 mg/kg BW)
PG1: Mice treated with CPA and administered with general bellflower extract (150 mg/kg BW)
PG2: Mice treated with CPA and administered with general bellflower extract (300 mg/kg BW)
Figure 3a is a diagram showing the division of microorganisms that change according to the treatment of red bellflower extract into phylums.
Figure 3b is a diagram divided into genus (genus) the microorganisms that are changed according to the treatment of the red bellflower extract.
Figure 4a is a diagram showing the change in body weight according to the treatment of red bellflower extract.
Figure 4b is a diagram showing the change in the expression of Immunoglobulin A (IgA) according to the treatment of red bellflower extract.
Figure 4c is a diagram showing the change in the expression of Immunoglobulin M (IgM) according to the treatment with a red bellflower extract.
Figure 4d is a diagram showing the correlation coefficient (Corr) of the intestinal microorganisms showing a significant correlation with body weight and the amount of the intestinal microorganisms according to the treatment with red bellflower extract.
Figure 5 is a diagram predicting the function of intestinal microorganisms according to the treatment of red bellflower extract.

Hereinafter, the present invention will be described in detail.

When a person is born, microorganisms begin to inhabit the intestine, and when the population reaches equilibrium, a stable intestinal flora is formed. The intestinal flora fluctuates according to a number of factors, such as the physiological conditions of the host, food, drugs, and stress. The number and activity of microorganisms constituting the normal flora are regulated by aloogenic factors and endogenous factors (Yazawa T., Letters in applied Mocrobiology 10: 229-232, 1990), the former being the host's It is caused by the environment or diet, and the latter occurs mainly among microorganisms in the flora.

Normal intestinal bacteria rarely harm people, but rather have various beneficial effects on the human body, and there are many important bacteria that are indispensable for human survival. On the other hand, as you get older and your health deteriorates, the balance of intestinal bacteria changes. For example, the dominant beneficial bacteria gradually weakens, and instead the putrefactive bacteria begin to increase rapidly. If this happens, the intestinal environment becomes more decomposed and odors occur. Furthermore, putrefactive bacteria in the gut bacteria change a part of proteins or amino acids to produce harmful substances such as ammonia, indole, and hydrogen sulfide that emit odors. These harmful substances affect intestinal muscle movement, causing diarrhea or constipation, and promoting headache, stiff shoulders, rough skin, and aging. Of course, most of these harmful substances go through detoxification in the liver, but if the amount of toxic substances increases, liver function deteriorates and causes fatigue and various adult diseases.

Improving the intestinal flora refers to promoting the growth or growth of beneficial intestinal bacteria and suppressing the growth or growth of harmful intestinal bacteria, while maintaining a balance between beneficial intestinal bacteria and harmful bacteria. Microorganisms begin to inhabit the organ and form the intestinal flora as the population reaches equilibrium. Individual microorganisms constituting the intestinal flora are involved in vitamin supply, infection prevention, and intestinal function, and thus, the composition of the flora is known to have a deep relationship with the occurrence of constipation and intestinal-related diseases.

Intestinal beneficial bacteria may refer to microorganisms that live in the intestine and exert beneficial effects on the human body. For example, beneficial intestinal bacteria may include probiotics. For example, the beneficial intestinal bacteria are not limited thereto, and Bifidobacterium, Lactobacillus, Lactococcus, Akkermansia, Faecalibacterium , or Enterococcus genus (Enterococcus). In particular, Akkermansia is a bacterium that promotes mucus secretion and makes the barrier mechanism stronger. In a diabetic mouse model in which the intestinal mucosal barrier is disrupted, the symptoms of disease are reduced by administration of the genus Akkermansia. has been reported and is known to modulate host immune function by influencing T cell responses.

Intestinal harmful bacteria may refer to microorganisms that live in the intestine and have harmful effects on the human body, such as enteritis. For example, the intestinal harmful bacteria are not limited thereto, and Escherichia coli, Fusobacterium, Clostridium, Staphylococcus, or Porphyromonas. ) may be included. In particular, the genus Staphylococcus is well known to cause an immune response such as food poisoning or skin allergy by toxins.

Despite the various effects of bellflower, consumer preference is divided due to the strong bitter taste of bellflower itself, so it can be consumed by more people through a cooking process such as peeling the skin and brewing the bitter taste with cold water to remove it or cook it. In order to overcome this problem, it has been reported that red bellflower made by steaming and fermenting raw bellflower, hydrolyzed saponin contained in the bellflower, not only improved the taste and smell of bellflower, but also improved the functionality. In particular, the total polyphenol content and the saponin content significantly increased according to the number of repetitions of red bellflower steaming, which resulted in higher antioxidant activity in red bellflower than general bellflower.

The present invention provides a composition for improving intestinal flora comprising, as an active ingredient, a red bellflower extract prepared by steaming and drying bellflower (Playtycodon grandiflorus).

The red bellflower extract may be prepared by a manufacturing method comprising the following steps:

1) preparing red bellflower by steaming and drying the bellflower;

2) extracting by adding an extraction solvent to red bellflower;

3) filtering the extract of step 2); and

4) The filtered extract of step 3) was concentrated under reduced pressure and dried

preparing the extract.

The bellflower (Playtycodon grandiflorus) of step 1) can be used without limitation, such as cultivated or commercially available ones.

The bellflower in step 1) may be any one or more selected from the group consisting of flowers, leaves, stems, fruits and roots, but is not limited thereto.

According to a specific embodiment of the present invention, it is preferably the root of a bellflower.

The steaming in step 1) may be performed at 60 to 120 ℃, 70 to 110 ℃, or 80 to 100 ℃, and the steaming time may be made for 40 to 160 minutes, 50 to 140 minutes, or 60 to 120 minutes.

The temperature and the steaming time of the steaming may be changed and applied according to the amount or state of the bellflower.

The term steamed means to cook by steaming. Steaming may be performed using steam, simple heating, or other methods of cooking by applying heat. By the above process, the quality of the bellflower can be softened.

The drying in step 1) may be made at a temperature of 20 to 80 ℃, 30 to 70 ℃, or 40 to 60 ℃, and the drying time may be made for 4 to 28 hours, 6 to 26 hours, or 8 to 24 hours. . The drying temperature and drying time may be changed and applied according to the amount or state of the bellflower. The temperature and drying time can be adjusted to reach the target moisture content.

The red bellflower in step 1) may be prepared by repeating the cycle of steaming and drying 1 to 4 times, or 2 to 4 times, and is preferably manufactured by repeating 3 to 4 times.

In addition, the extraction solvent of step 2) may be water, alcohol, or a mixed solvent thereof. The alcohol may be a C1 to C4 lower alcohol, and specifically, the alcohol may be ethanol or methanol. According to a specific embodiment of the present invention, extraction with ethanol is preferable.

In addition, the ethanol is 30% (v / v) to 100% ethanol, 40% to 100% ethanol, 40% to 90% ethanol, 40% to 80% ethanol, 40% to 70% ethanol, 40% to 60% Ethanol, 45% to 60% ethanol, 40% to 55% ethanol, or 45% to 50% ethanol, or 50% ethanol can be used, and 50% ethanol is preferable.

In addition, the extraction method of step 2) may be shaking extraction, Soxhlet extraction, reflux extraction, or ultrasonic extraction. At this time, the extraction temperature is preferably 40 to 100 ℃, more preferably 50 to 80 ℃, but is not limited thereto. In addition, the extraction time is preferably 2 to 10 hours, more preferably 4 to 8 hours, but is not limited thereto. In addition, the number of extractions is preferably 1 to 5 times, more preferably 1 to 2 times, but is not limited thereto. In a range outside the conditions of the extraction temperature, extraction time, or number of times of extraction, extraction may not be performed sufficiently, so that the content of active ingredients in the red bellflower extract may be insignificant. Alternatively, the extraction yield may no longer increase and the efficiency of the extraction operation may decrease.

The vacuum concentration in step 4) may use a vacuum vacuum concentrator or a vacuum rotary evaporator. In addition, the drying may be reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying.

In order to determine what effect the red bellflower extract has on the changes in the intestinal flora, the changes in the intestinal flora were checked after the red bellflower extract was treated. It was confirmed that the intestinal flora, which was different from the normal group due to CPA (cyclophosphamide) treatment, was recovered close to that of the normal group by the red bellflower extract (FIG. 2). In order to discover microorganisms that change according to the treatment of red bellflower extract, microorganisms were classified into phylums or genus. As a result of classifying and comparing microorganisms by phyllotaxis, it was confirmed that the diversity of microorganisms in the red bellflower treated group was improved similarly to that of the normal group (FIG. 3a). When the data was classified at the genus level, it was found that the microorganisms of the genus Akkermansia and Staphylococcus, which are known to be particularly related to immunity, change similarly to the normal group by the treatment with the red bellflower extract. was confirmed (FIG. 3b). In addition, it was confirmed that body weight, IgA, and IgM, which are major immune-related indicators, increased in the intestines of mice treated with the red bellflower extract ( FIGS. 4a , 4b and 4c ), and the intestines of the mice treated with the red bellflower extract It was confirmed that the microorganisms (Akkermansia, Lactobacillaceae, Gemellates and Staphylococcus) correlated with body weight were regulated in the (Fig. 4b). In addition, it was predicted that the red bellflower extract would enhance the functions related to the carbon fixation pathways through the KEGG pathway (FIG. 5).

Hereinafter, the present invention will be described in detail by way of Examples and Experimental Examples.

However, the following Examples and Preparation Examples only illustrate the present invention, and the content of the present invention is not limited by the following Examples and Preparation Examples.

<Example 1> Preparation of red bellflower extract

When the bellflower is steamed with moist heat at a high temperature and dried repeatedly, the white bellflower changes to yellow, red, and black, which is called red bellflower or black bellflower. In the present invention, using bellflower grown in Geumsan, Chungcheongnam-do, red bellflower was prepared as follows.

<1-1> Production of red bellflower through steaming and drying of bellflower

The bellflower (Platycodon grandiflorus) was washed twice with water to clean the soil or dirt. The washed bellflower was steamed at 80-100° C. for 60-120 minutes in a steamer, and the steamed bellflower was dried in a dryer at a temperature of 40-60° C. for 8-24 hours. After repeating the steaming and drying cycle a total of 3-4 times, additional drying was performed for 1-3 days (FIG. 1).

<1-2> Preparation of red bellflower extract

After the primary treatment has been completed, extract the bellflower raw material with 50% ethanol twice at 60-100°C for 4-10 hours, filter it with a filter press, and after concentration under reduced pressure, add the same amount of excipient relative to the solid content, and dry was used. The content of platycoside E used as an indicator component of the bellflower extract was quantified using the HPLC method. As a standard solution (platycoside E), 0.4 mL of DMSO and 50% MeOH were added to about 5 mg of the standard, followed by ultrasonic extraction, and dissolved, and used as MeOH. For the test solution, 1.5 g of bellflower extract powder was placed in a 20 mL flask, 0.4 mL of DMSO and 5 mL of distilled water were added, followed by ultrasonic extraction for 10 minutes. Then, 5 mL of MeOH was added, followed by ultrasonic extraction for 10 minutes, followed by purification using 50% MeOH, and filtration with a 0.20 μm nylon syringe filter (Whatman, Clifton, NJ, USA). This was used as the final HPLC test solution, and the analysis conditions are shown in Table 1.

As a result, as shown in Table 2, in red doraji (RD) analyzed by the internal standard method, the index component platycoside E content was 0.6 mg/g.

<Experimental Example 1> Changes in intestinal flora by the extract of red bellflower in a mouse model with reduced immunity

Since the decrease in immunity causes an imbalance in the interaction of intestinal microbes and intestinal cells, changes in the intestinal flora by the treatment of red bellflower extract were confirmed.

For the experiment, 72 6-week-old male C57BL/6 mice (8 mice per experimental group) in Specificpathogen free (SPF) condition were purchased from Samtaco and used. During the breeding period, general solid feed and water were freely ingested, and the temperature was 23±2 ^o C, humidity 50±10%, and the light-dark cycle was adjusted in units of 12 hours. After acclimatization to the experimental environment for 1 week, mice were intraperitoneally administered with 150 mg/kg BW of cyclophosphamide (CPA) 3 days before oral administration, and 110 mg/kg BW of 1 day before oral administration, thereby creating an immune lowering model. The body weight of the experimental animals was measured 24 hours after the last CPA administration was finished and classified as follows according to the randomized complete block design. Experimental groups were normal group (Nor), negative control group (NC), positive control group (PC), red bellflower PGS1 group and general bellflower PG1 group (150 mg/kg BW), PGS2 group and PG2 group (300 mg/kg BW) was composed of Distilled water in which the extract was dissolved was used in the Nor group and NC group, and in the PC group, β-glucan (50 mg/kg BW, Sigma-Aldrich Co.), which is known to be effective in enhancing immunity, was dissolved and used, and all experimental groups were administered orally for 14 days. was administered (once/day). Intestinal samples were collected from each mouse and used for DNA extraction using the AccuPrep Stool DNA Extraction Kit according to the manufacturer's instructions. After quality control (QC) was performed, the qualified samples proceeded to library building. The V3 and V4 regions of the 16S rRNA gene were PCR amplified from microbial genomic DNA, and the DNA quality was determined by PicoGreen and Nanodrop. Input gDNA (10ng) was PCR amplified using primers 341F/805R (341F:5' CCTACGGGNGGCWGCAG 3, 805R:5'GACTACHVGGGTATCTAATCC 3'), and the final purified product was qPCR quantification protocol guide (KAPA library quantification for Illumina sequencing platform). kit) and validated using the LabChip GX HT DNA High Sensitivity Kit (Perkin Elmer, Mass.). 300 paired-end sequencing reactions were performed on the MiSeq™ platform (Illumina, San Diego, USA), and the sequencing data generated for this study was obtained from the NCBI (http://www.ncbi.nlm.nih.gov/) of BioProject PRJNA700675. sra) in the SRA (Sequence Read Archive).

All sequence data were sparsed to a sampling depth of 12,361 sequences per sample prior to calculation of alpha and beta diversity analysis using QIIME2 plugin diversity such as observed OTU, Shannon and Weighted Unifrac. Weighted Unifrac distance matrices were used for nonparametric permutation multivariate analysis of variance (PERMANOVA) and analysis of principle coordinates (PCoA) plots. PERMANOVA was performed with 999 permutations of the weighted UniFrac distance matrix using the Adonis function from the R package 'vegan'.

As a result of showing the intestinal microbial diversity between individuals through a PCoA (Principal coordinates analysis) plot (FIG. 2), it was confirmed that the normal group (Nor) showed a significant difference from the negative control group (NC). In the group (PGS1 and PGS2) ingesting red bellflower, the intestinal flora was different from the negative control group, but the positive control group and the PG2 group showed similar intestinal flora diversity to the negative control group. In particular, the experimental group (PGS1 and PGS2) ingesting red bellflower showed a closer intestinal flora diversity to that of the normal group compared to the experimental group (PG1) ingesting general bellflower. difference could be identified.

Therefore, it was confirmed that the red bellflower extract restored the diversity of the intestinal flora changed in the immunocompromised state similarly to that of the normal group.

<Experimental Example 2> Excavation of microorganisms that change according to the intake of red bellflower extract

Since it was confirmed that the intestinal flora is changed by the red bellflower extract, the following experiment was performed to confirm which specific microorganisms are actually changed.

Specifically, the analysis of the pretreated samples was processed using QIIME2 version 2020.02. Illumina sequence FASTA2Q files were denoised and corrected by using the DADA2 software package implemented in QIIME2 and removing the chimeric sequence using the "consensus" method. Based on the quality plot of DADA2, the readings were trimmed using the following parameters. Naive Bayes classifiers were processed in the GreenGenes 97% (version 13.8) operational taxonomic unit (OTU) database using the QIIME2 plug-in functional classifier and reference sequences trimmed to the V3-V4 regions.

As a result, as in FIG. 3A , when microorganisms were classified by phylum among classification units, the most dominant microorganism was Bacteroidetes. It was confirmed that the ratio of Bacteroidetes in the negative control group was decreased compared to the normal group. On the other hand, in the group treated with red bellflower (PGS1 and PGS2), the ratio of Bacteroidetes increased compared to the negative control group, and it was confirmed that it was improved similarly to the normal group. . In the case of the general bellflower treated group (PG1 and PG2), compared to the negative control group, it was confirmed that the diversity of the intestinal flora was not improved, such as a decrease in the ratio of Bacteroidetes. When the microorganisms were classified into more subdivided genus, the microorganisms related to the red bellflower extract were found to be Akkermansia and Staphylococcus (Fig. 3b). In particular, it was confirmed that the genus Akkermansia, known to regulate immunity with beneficial intestinal bacteria, was increased in the normal group and the group treated with red bellflower extract, and the genus Staphylococcus, known as harmful intestinal bacteria, was decreased. In the case of the general bellflower extract treated group (PG1), the genus Accomencia showed a tendency to increase similarly to that of the normal group, but the Staphylococcus genus, which decreased in the normal group, increased similarly to the negative control group. It was confirmed that the microflora could not be improved.

Therefore, it was confirmed that the red bellflower extract improved the intestinal flora similar to that of the normal group by increasing beneficial intestinal bacteria and reducing intestinal harmful bacteria.

<Experimental Example 3> Correlation between immunity-related indicators and microorganisms according to the treatment of red bellflower extract

Since the intestinal flora was changed by the treatment with the red bellflower extract in immune-suppressed mice, an experiment was performed to confirm the correlation between the red bellflower extract and immunity.

Specifically, the body weight and serum immunoglobulin concentration of experimental mice were measured and a total of 26 samples (3 or 4 mice in each of 7 groups) similar to the average group weight for performance analysis were selected. Body weights were monitored once a week, and upon sacrifice, all animals were fasted overnight (without water limitation) prior to initial test substance administration. Mice were then sacrificed after anesthesia with CO2 using a rodent inhalation anesthesia apparatus (Surgivet, Waukesha, WI, USA). Serum concentrations of immunoglobulin A (IgA) and immunoglobulin M (IgM) were determined using an enzyme-linked immunosorbent assay kit (ELISA, Abcam, USA) according to the manufacturer's instructions. All analyzes were performed in two replicates, and all animals were treated in accordance with international regulations on the use and welfare of laboratory animals.

Trimmed Mean of M values (TMM) of M values were adjusted for different library sizes using edgeR. Then, statistical tests were performed in a generalized linear model (GLM) by considering the number of OTUs as a negative binomial distribution. To compare the fit of the two models, log likelihood ratio statistics were calculated and the false discovery rate (FDR) was used to adjust for multiple test errors with a significance level of 5%. Another statistical test for differentially enriched microbial taxa was evaluated using the QIIME2 Microbiomes Composition Analysis (ANCOM) plugin to identify features that differed in abundance in each study group. Differentially rich functions at each phylogenetic level were calculated by ANCOM in the DADA2 function table. Student's t-test was used to compare biomarkers (weight, IgA and IgM) between groups. We also used the Spearman correlation coefficient (r) from the corplot R package to determine the correlation between microbiome abundance and immune-related biomarkers. The correlation was confirmed by visualizing the abundance of the genus that showed a significant correlation (p-value <0.05) with the corresponding phenotype as a line graph. All R packages are implemented in RStudio version 4.0.1.

As a result, it was confirmed that the weight decreased in the negative control group by CPA treatment was recovered by the red bellflower extract, but it was confirmed that the general bellflower extract treated group (PG2) had a value similar to that of the negative control group (FIG. 4a). The expression of immune indicators IgA and IgM was also increased in the red bellflower extract treated group compared to the negative control group. In contrast, in the case of IgA, it was confirmed that the bellflower extract treated group (PG1) expressed lower than the negative control group ( FIGS. 4b and 4c ). The red bellflower extract treated group could confirm significant improvement and increase in immune markers in both groups (PGS1 and PGS2), but the bellflower extract treated group (PG1 and PG2) did not show a consistent improvement effect. As shown in Figure 4d, the red bellflower extract was found to affect the Akkermansia genus (genus), Lactobacillaceae family (family), Gemellates order (order) and Staphylococcus genus (genus), which are known to be related to body weight. Akkermansia and Lactobacillaceae showed a positive correlation with body weight, and Gemellates and Staphylococcus showed a negative correlation with body weight. Akkermansia and Lactobacillaceae, which showed a positive correlation with body weight, decreased in the negative control group, but increased in the red bellflower extract treated group (PGS1 and PGS2). Gemellates and Staphylococcus, which showed a negative correlation with body weight, increased in the negative control group, but decreased in the group treated with red bellflower extract. In contrast, in the general bellflower extract treatment group (PG2), it was confirmed that Akkermansia and Lactobacillaceae, which were positively correlated with body weight, decreased, and Gemellates and Staphylococcus, which had a negative correlation, increased. This means that the red bellflower extract is closely related to the weight-related microorganisms in the immune lowering model, unlike the general bellflower extract, and it shows that the intestinal microbes are involved in the weight control caused by the treatment of the red bellflower extract.

Therefore, the red bellflower extract increases the body weight, IgA and IgM, which are immune-related indicators, by regulating the intestinal flora, and thus the reduced immune function is enhanced.

<Experimental Example 4> Functions of intestinal microorganisms predicted by treatment with red bellflower extract

The function of intestinal microbes by the treatment of red bellflower extract was predicted and statistically analyzed how different the predicted function was compared with the negative control group.

Specifically, a phylogenetic survey of communities by reconstruction of unobserved states 2 (PICRUSt2) was used to predict the Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO) genes from 16S rRNA data. The tree with the most near-sequenced taxon index (NSTI) cutoff of 2 generated in PICRUSt2 was supplemented with a feature table generated by the QIIME2 plugin dada2 for hidden state prediction (hsp) using the maximal succinct method. Following the prediction of the KO gene, the KEGG pathway was mapped to the KO gene using the PICRUSt2 package. Significant pathways were then performed by calculating multi-group comparisons using the analysis of variance (ANOVA) statistical test (p-value <0.05) in the STAMP software package. In addition, functions related to human disease were obtained using White's nonparametric t-test of the STAMP software package.

When the function of microorganisms was statistically predicted in consideration of the amount of intestinal microbes in the red bellflower and the negative control group, it was predicted that the carbon fixation pathways of prokaryotes related to the metabolism in the red bellflower treated group were improved (FIG. 5).

Claims (11)

A composition for improving intestinal flora comprising an extract of red bellflower produced by steaming and drying bellflower (Playtycodon grandiflorus) as an active ingredient. The composition for improving intestinal flora according to claim 1, wherein the red bellflower is prepared by steaming the bellflower at 60 to 120° C. for 40 to 160 minutes. The composition for improving intestinal flora according to claim 1, wherein the red bellflower is prepared by drying the bellflower at 20 to 80° C. for 4 to 24 hours. The composition for improving intestinal flora according to claim 1, wherein the red bellflower is prepared by repeating the cycle of steaming and drying the bellflower one to four times. The composition for improving intestinal flora according to claim 1, wherein the red bellflower extract is an extract extracted from red bellflower extract using water, C₁ to C₄ lower alcohol or a mixture thereof as a solvent. The composition for improving intestinal flora according to claim 5, wherein the alcohol is ethanol or methanol. The composition for improving intestinal flora according to claim 1, wherein the composition for improving intestinal flora promotes the growth of beneficial intestinal bacteria or inhibits the growth of intestinal harmful bacteria. The composition for improving intestinal flora according to claim 1, wherein the composition for improving intestinal flora enhances immunity. The composition for improving intestinal flora according to claim 8, wherein the immune enhancement increases body weight. [Claim 10] The composition for improving intestinal flora according to claim 9, wherein the increase in weight increases the genus Akkermansia and the Lactobacillaceae family, which are intestinal microorganisms that are positively correlated with body weight. [Claim 10] The composition for improving intestinal flora according to claim 9, wherein the increase in weight reduces the genera of Gemellates and Staphylococcus, which are intestinal microorganisms that have a negative correlation with body weight.

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