Intestinal flora improvement components

Abstract

To provide a composition for improving intestinal bacterial flora that exhibits improved intestinal bacterial flora improving action.SOLUTION: A composition for improving intestinal bacterial flora contains a solvent extract of agarwood leaves as an active ingredient. The solvent extract may be a hot water extract.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a composition for improving the intestinal flora, which contains a solvent extract of Aquilaria leaves.

Background Art

[0002] It has become clear that the balance of bacterial species in the flora (intestinal flora) plays an important role in maintaining health. Although the flora is a relatively stable ecosystem, its composition and activity may change under the influence of various factors. If the balance of bacterial species in the flora is disrupted due to stress, aging, etc., there is a risk of affecting the health status. As one of the methods for preventing a wide range of diseases, maintaining and improving the intestinal environment has become increasingly important.

[0003] Conventionally, the dried leaves of Aquilaria of the Thymelaeaceae family, which is a functional material composed of natural ingredients derived from plants with high safety, are known. As disclosed in Patent Documents 1 and 2, the dried leaves of Aquilaria of the Thymelaeaceae family are known to have effects effective for the living body, such as a laxative effect exerted by promoting the ileum's automatic movement via the parasympathetic nerve and a detoxifying effect.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] The inventors of the present invention searched for new physiological actions of the dried leaves of Aquilaria of the Thymelaeaceae family. As a result, they found that the solvent extract of Aquilaria leaves can improve the action of improving the intestinal flora.

Means for Solving the Problems

[0006] This invention is based on the discovery that a solvent extract of agarwood leaves improves the bacterial flora. One embodiment of the present invention, which solves the above problems, is a composition for improving the intestinal microbiota, the gist of which is that it contains a solvent extract of agarwood leaves as an active ingredient.

[0007] In the aforementioned composition for improving the intestinal flora, the solvent extract may be a hot water extract. In the aforementioned composition for improving the intestinal microbiota, the improvement of the intestinal microbiota may be at least one selected from: an increase in the number of Bifidobacteria, a decrease in the proportion of Erysipelatoclostridium bacteria, a decrease in intestinal pH, and an increase in the proportion of propionic acid relative to the total amount of acetic acid, propionic acid, and butyric acid in the intestines. [Effects of the Invention]

[0008] The present invention's composition for improving the intestinal microbiota can enhance the effect of improving the intestinal microbiota. [Modes for carrying out the invention]

[0009] The following describes a first embodiment of the intestinal microbiota improvement composition of the present invention. The intestinal microbiota improvement composition contains a solvent extract of agarwood leaves as an active ingredient. (Agarwood leaf) Agarwood is a plant belonging to the genus Aquilaria in the family Thymelaeaceae, mainly found in Southeast Asia, including Thailand, Vietnam, Cambodia, and Malaysia. The leaves of agarwood contain genkwanin 5-O-β-primeveroside as their main medicinal component.

[0010] Agarwood leaves may be subjected to solvent extraction after drying, or roasted before solvent extraction. Roasting is carried out by dry roasting the agarwood leaves. Specifically, the agarwood leaves are placed in a container, and without adding water, they are heated from the outside of the container to a predetermined temperature. The roasting temperature of the agarwood leaves is preferably 130 to 170°C, more preferably 140 to 160°C. If the roasting temperature is below 130°C, a grassy aroma derived from agarwood may remain, and the aftertaste may become bitter. On the other hand, if the roasting temperature exceeds 170°C, a strong burnt odor may be produced.

[0011] The roasting time for agarwood leaves is not particularly limited. Since maintaining a high roasting temperature for a long time may cause the agarwood leaves to carbonize, it is preferable to place the agarwood leaves in a heated container and continue heating until a predetermined temperature is reached, at which point the heating is stopped, the agarwood leaves are quickly removed, and forced cooling is performed by blowing air.

[0012] (Solvent extraction) A method for producing a solvent extract of agarwood leaves includes the step of adding water, an organic solvent, or a mixture thereof as an elution solvent to agarwood leaves. Examples of elution solvents include water, an organic solvent, and a mixture of water and an organic solvent. Examples of organic solvents include hydrophilic organic solvents such as methanol and ethanol, and hexane. Among these solvents, water, ethanol, or a mixture of ethanol and water are preferred from the viewpoint of safety and ease of handling, water is more preferred, and hot water is particularly preferred. The concentration of ethanol in the ethanol / water mixture is preferably 55 to 95% by volume, more preferably 60 to 80% by volume, from the viewpoint of extraction efficiency of the main component, genkwanin 5-O-β-primeveroside.

[0013] The amount of solvent used for elution is preferably 1 to 500 times the mass of agarwood leaves, more preferably 5 to 200 times, and even more preferably 10 to 150 times. If the amount of solvent used for elution is less than 1 time, the extraction rate of the main component, genkwanin 5-O-β-primeveroside, is poor, which is undesirable. Conversely, if the amount of solvent used for elution exceeds 500 times, not only is an unnecessarily large apparatus required, but the concentration process and other steps take time, and work efficiency is significantly reduced, which is also undesirable. Furthermore, the concentration of the main component, genkwanin 5-O-β-primeveroside, decreases in the elution solvent, making it difficult to obtain an effective amount.

[0014] Furthermore, in order to improve the extraction efficiency of the main component, genkwanin 5-O-β-primeveroside, it is preferable to remove impurities such as debris mixed in during collection and to pulverize the agarwood leaves before the extraction process. The extraction temperature can preferably be applied in a wide temperature range of 30 to 100°C. If the extraction temperature is below 30°C, the extraction rate of the contained components is poor, which is undesirable. Conversely, if the extraction temperature exceeds 100°C, the extraction solvent evaporates, which is also undesirable. The extraction operation can be carried out at the above extraction temperature for several tens of seconds to several hours while stirring or standing. After sufficiently extracting the contained components under the above extraction conditions, the agarwood leaf extract can be obtained by performing a filtration process such as filter paper filtration or diatomaceous earth filtration. The agarwood leaf extract obtained as described above may be applied directly to food and beverages, pharmaceuticals, etc., using the extract obtained with an elution solvent. Alternatively, it may be concentrated by evaporating the solvent before application, or it may be dried and powdered before application.

[0015] (Improvement of gut microbiota) The intestinal microbiota improving composition of this embodiment has effects such as increasing the number of Bifidobacteria, decreasing the proportion of Erysipelatoclostridium bacteria, lowering the intestinal pH, and increasing the proportion of propionic acid relative to the total amount of acetic acid, propionic acid, and butyric acid in the intestines. The intestinal microbiota improving composition exerts effects such as improving the intestinal microbiota and mitigating its deterioration by increasing the number of Bifidobacteria.

[0016] (Application) The intestinal microbiota improving composition of this embodiment may be applied as a health food or supplement intended to improve the intestinal microbiota. It may also be applied to various pharmaceuticals and quasi-drugs intended to improve the intestinal microbiota.

[0017] When the intestinal microbiota improving composition of this embodiment is applied to food and beverages, it can be used in its original form or by adding it to various food ingredients. Alternatively, the intestinal microbiota improving composition may be applied in the form of tablets, capsules (hard capsules, soft capsules, etc.), granules, and powders for direct intake. The food and beverages may also contain other food additives such as sugars, flavorings, sweeteners, oils and fats, base materials, excipients, auxiliary materials, and bulking agents as appropriate. Furthermore, the use of the food and beverages is not particularly limited and can be applied to so-called general foods, health foods, functional foods, nutritional supplements, supplements, foods for specified health uses, foods with functional claims, and foods for the sick.

[0018] When indicating the uses of food and beverages, this includes not only indications on packaging such as boxes and containers, but also indications on advertising media such as brochures. The indications for the various uses of the intestinal microbiota improving composition of this embodiment include, in addition to indications such as the above-mentioned improvement of the intestinal microbiota, increase in the number of bifidobacteria, decrease in the proportion of Erysipelatoclostridium bacteria, decrease in intestinal pH, and increase in the proportion of propionic acid relative to the total amount of acetic acid, propionic acid, and butyric acid in the intestines, as well as indications suggesting improvement, prevention, prevention of worsening, or delay of the worsening of various symptoms such as diabetes, weakened immunity, stress, and obesity associated with the improvement of the intestinal microbiota.

[0019] The effects of the intestinal microbiota improvement composition of this embodiment will be described. (1) The composition for improving the intestinal flora of this embodiment contains a solvent extract of agarwood leaves as an active ingredient. Therefore, it particularly affects intestinal bacteria and exhibits an effect that improves the bacterial flora in a way that was previously unknown.

[0020] (2) When a hot water extract of Aquilariae Lignum Resinatum leaves is used as an active ingredient, the bioavailability can be further improved. In addition, the above embodiments may be modified as follows.

[0021] · The composition for improving the intestinal flora in the above embodiments can be applied not only to foods, beverages, pharmaceuticals, etc. ingested by humans, but also can be formulated as supplements, dietary supplements, pharmaceuticals, etc. in livestock feed.

[0022] · For the extraction method using an elution solvent, in order to reduce costs, the extract after solid-liquid separation treatment after extraction may be reused multiple times in the next extraction treatment.

Examples

[0023] Test examples are given below to further specifically explain the above embodiments, but the present invention is not limited thereto. <Test Example 1: Preparation of Solvent Extract of Aquilariae Lignum Resinatum Leaves> 15 L of water was added as an extraction solvent to 1 kg of dry pulverized Aquilariae Lignum Resinatum leaves as a raw material. Extraction was carried out with stirring at 90 °C for 60 minutes. Then, the extract was filtered to remove the residue, and a water extract was obtained. After the obtained water extract was concentrated to Brix 20-25°, 50% by mass of maltodextrin was added as an excipient and powdered while drying with a spray dryer to obtain a powder having a moisture content of 7% by mass or less.

[0024] <Test Example 2: Evaluation of Improvement of Intestinal Flora> (1) Human large intestine flora model As a human colon flora model, a test system is known that reproduces an individual's intestinal environment by culturing human feces in GAM medium ("A Single-Batch Fermentation System to Simulate Human Colonic Microbiota for High-Throughput Evaluation of Prebiotics," Takagi et al., PloS ONE (2016)). By using such a human colon flora model, it is possible to evaluate the effect of agarwood leaf solvent extract on the intestinal microbiota while eliminating individual differences and lifestyle factors (the intestinal microbiota being the same before the addition of the test substance).

[0025] (subject) We chose three men and five women in their 20s. (Culture method) A human intestinal model system (Kobe University Human Intestinal Model [KUHIM]) was used. Fecal samples were collected using BD BBL culture swabs (manufactured by Becton Dickinson, Inc.). After collection, the swabs were stored at 4°C and subjected to culture testing within 24 hours.

[0026] (a) 5.9 g of GAM medium (equivalent to 100 mL final volume) was measured and dissolved in 80 mL of ultrapure water. 5 μL of defoaming agent was added, the mixture was thoroughly stirred, and then autoclaved at 115°C for 15 minutes.

[0027] (b) Before starting the culture, the temperature was lowered to 37°C while performing gas replacement (N2:CO2=8:2). (c) The prescribed amount of sample was measured into a 50 mL centrifuge tube and dissolved in 20 mL of sterile water. Then it was added to the culture medium from (b).

[0028] (d) At the time of the test, the seeds were suspended in 2 mL of physiological saline containing 1% ascorbic acid and seeded in 100 μL in a culture tank. (Amount added) In Test Example 1, 300 mg or 600 mg of a solvent extract of powdered agarwood leaves was added as a sample.

[0029] (statistical analysis) One-way analysis of variance (ANOVA) and multiple comparison tests (Tukey) (2) Effect on pH The pH of the culture medium was measured after 48 hours of incubation. The pH value, mean, and standard error (SE) for each subject were determined. The results are shown in Table 1.

[0030] [Table 1] As shown in Table 1, a decrease in the culture medium pH was observed in all subjects with the addition of agarwood leaf solvent extract (control vs 300 mg p=0.30718, control vs 600 mg p=0.00908). The addition of 600 mg showed a statistically significant difference compared to the control. A change in the statistical test method to the Wilcoxon rank-sum test also showed a statistically significant difference with 300 mg intake (Wilcoxon matched-pairs signed rank test p=0.0078). It is thought that agarwood leaves affect the gut microbiota, and the pH decreases due to the production of organic acids, etc. Since components produced by harmful bacteria such as E. coli and Clostridium perfringens make the intestines alkaline, it was confirmed that the gut microbiota is changed in a direction that improves with agarwood leaf solvent extract, and the pH in the intestines decreases.

[0031] (3) Effects on organic acid production After 48 hours of incubation, the proportion (mass%) of propionic acid production relative to the total (100 mass%) of three organic acids (acetic acid, propionic acid, and butyric acid), which are metabolites of intestinal bacteria, was determined in the culture medium. The quantification of organic acids was performed using HPLC, and calibration curves were obtained from various standards. The proportion of each organic acid, along with the mean and standard error (SE), was determined for each subject. The results are shown in Table 2.

[0032] [Table 2] As shown in Table 2, a significant increase in the proportion of propionic acid was observed with solvent extract of agarwood leaves (control vs 300 mg, p=0.0234). An increase in propionic acid is known to have beneficial effects such as increased immune function and appetite suppression. In other words, it was confirmed that solvent extract of agarwood leaves improved the gut microbiota and promoted the production of propionic acid.

[0033] (4) Effects on the number of Bifidobacteria The number of Bifidobacterium species in the culture medium after 48 hours of incubation was measured. Quantitative analysis (PCR) of Bifidobacterium was performed using DNA extracted from the culture medium after each incubation period. The bacterial count (log10 notation), mean, and standard error (SE) of Bifidobacterium for each subject were calculated. The results are shown in Table 3.

[0034] [Table 3] As shown in Table 3, an increase in Bifidobacterium was observed in 7 out of 8 subjects after treatment with agarwood leaf solvent extract. In other words, it was confirmed that the agarwood leaf solvent extract improved the intestinal microbiota and influenced the increase in the number of Bifidobacteria, which are so-called beneficial bacteria.

[0035] (5) Influence on the proportion of the genus Erysipelatoclostridium Next-generation sequencing (NGS) analysis was performed on the gut microbiota in the culture medium after 48 hours of incubation (51,564 reads / sample). Bacterial DNA was extracted from the culture medium and performed next-generation sequencing (NGS) analysis to evaluate changes in the Erysipelatoclostridium genus in each test sample. The number of Erysipelatoclostridium reads for each subject was determined. The results are shown in Table 4.

[0036] [Table 4] As shown in Table 4, NGS analysis (two-group test: 0 (control) vs. 300 mg) revealed a significant decrease in the Erysipelatoclostridium genus (P=0.039). Since Erysipelatoclostridium is a harmful bacterium that increases, for example, during gastrointestinal inflammation, it was confirmed that solvent extracts of agarwood leaves improved the intestinal microbiota and influenced the proportion of Erysipelatoclostridium, which is a so-called harmful bacterium.

Claims

[Claim 1] A composition for improving the intestinal flora containing a solvent extract of agarwood leaves as an active ingredient, The solvent extract is a hot water extract. The aforementioned improvement of the intestinal microbiota is an intestinal microbiota composition, which is at least one selected from: an increase in the number of Bifidobacteria, a decrease in the proportion of Erysipelatoclostridium bacteria, and an increase in the proportion of propionic acid relative to the total amount of acetic acid, propionic acid, and butyric acid in the intestines.

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