Composition for improving neuroinflammation, composition for improving brain function, composition for improving lipid metabolism, composition for adjusting compositional ratio of intestinal bacterial flora, and use of plant charcoal

Abstract

This composition for improving neuroinflammation contains plant charcoal.

Description

Use of composition for improving neuroinflammation, composition for improving brain function, composition for improving lipid metabolism, composition for adjusting intestinal flora composition ratio, and plant charcoal

[0001] The present invention relates to a composition for improving neuroinflammation, a composition for improving brain function, a composition for improving lipid metabolism, a composition for adjusting the composition ratio of intestinal microflora, and the use of plant charcoal.

[0002] Diabetes is a disease in which high blood sugar levels persist. Diabetes is broadly divided into type 1 diabetes, which is caused by a lack or deficiency of insulin to absorb blood sugar, and type 2 diabetes, which is caused by insulin resistance. 90-95% of diabetic patients in Japan have type 2 diabetes. Type 2 diabetes is thought to be related to genetic factors as well as lifestyle habits such as overeating and lack of exercise.

[0003] Type 2 diabetes can be improved or prevented from worsening by lifestyle changes or the use of medication. However, as the condition progresses, complications such as diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy may occur, and in some cases, dialysis or limb amputation may be necessary.

[0004] The relationship between diabetes and intestinal microbiota is becoming clear. For example, Non-Patent Document 1 reports that the intestinal microbiota of type 1 and type 2 diabetes patients has a decrease in bacteria of the phylum Firmicutes and a decrease in bacteria of the phylum Bacteroidetes. Non-Patent Document 2 reports that the intestinal microbiota of type 2 diabetes patients has a decrease in bacteria of the phylum Proteobacteria. Non-Patent Document 3 reports that the intestinal microbiota of type 1 diabetes patients has a decrease in bacteria of the phylum Verrucomicrobia and Actinobacteria.

[0005] In recent years, the effects of activated charcoal on obesity and type 2 diabetes have become known (Non-Patent Document 4). Patent Document 1 discloses a functional food for reducing obesity, which contains a charcoal composition such as bamboo charcoal or binchotan charcoal. Patent Document 1 also describes that ingestion of this functional food maintains a healthy composition of the intestinal bacterial flora.

[0006] Japanese Patent Application Laid-Open No. 2019-208461

[0007] Zaky, A. , et al., The Role of the Gut Microbiome in Diabetes and Obesity-Related Kidney Disease. , Int. J. Mol. Sci. , 2021, 22, 9641 Ahmad, A. , et al, Analysis of gut microbiota of obese individual with type 2 diabetes and health individual. , PLoS ONE, 2019, 14(12): e0226372 Murri, M. , et al., Gut microbiota in children with type 1 diabetes differs from that in healthy children: a case-control study. , BMC Med. , 2013, 11:46 Zhang, X. , et al., Acidic Activated Charcoal Prevents Obesity and Insulin Resistance in High-Fat Diet-Fed Mice. , Front. Nutr. , 2022, 9:852767

[0008] Because charcoal, such as wood charcoal and bamboo charcoal, has a high adsorption capacity, it has long been used for decontaminating oral toxicants, treating renal failure, etc. However, the function and mechanism of action of charcoal in the body, including the mechanism of action of charcoal on diabetes, have not yet been clarified.

[0009] The present invention has been made in view of the above circumstances, and aims to provide a composition for ameliorating neuroinflammation, a composition for improving brain function, a composition for improving lipid metabolism, and a composition for adjusting the composition ratio of intestinal microbiota, based on the novel function of plant charcoal. The present invention also provides the use of plant charcoal for producing the composition for ameliorating neuroinflammation, etc.

[0010] The composition for ameliorating neuroinflammation according to the first aspect of the present invention contains plant charcoal.

[0011] A composition for improving brain function according to a second aspect of the present invention includes plant charcoal.

[0012] A composition for improving lipid metabolism according to a third aspect of the present invention includes vegetable charcoal.

[0013] A composition for adjusting the composition ratio of intestinal bacterial flora according to a fourth aspect of the present invention comprises plant charcoal and is intended to increase the ratio of Firmicutes / Bacteroidetes in the intestinal bacterial flora.

[0014] A composition for adjusting the composition ratio of intestinal bacterial flora according to a fifth aspect of the present invention comprises plant charcoal and is intended to increase the proportion of Proteobacteria in the intestinal bacterial flora.

[0015] A composition for adjusting the composition ratio of intestinal bacterial flora according to a sixth aspect of the present invention comprises plant charcoal and is intended to increase the proportion of Actinobacteria in the intestinal bacterial flora.

[0016] A use according to a seventh aspect of the present invention is use of plant charcoal for producing a composition for improving neuroinflammation.

[0017] An eighth aspect of the present invention is the use of plant charcoal for the production of a composition for improving brain function.

[0018] A use according to a ninth aspect of the present invention is use of plant charcoal for producing a composition for improving lipid metabolism.

[0019] A use according to a tenth aspect of the present invention is use of plant charcoal for the production of a composition for adjusting the composition ratio of intestinal microbiota, in order to increase the ratio of Firmicutes / Bacteroidetes in the intestinal microbiota.

[0020] A use according to an eleventh aspect of the present invention is use of plant charcoal for producing a composition for adjusting the composition ratio of intestinal microbiota, in order to increase the proportion of Proteobacteria in the intestinal microbiota.

[0021] A use according to a twelfth aspect of the present invention is use of plant charcoal for producing a composition for adjusting the composition ratio of intestinal microbiota, in order to increase the proportion of Actinobacteria in the intestinal microbiota.

[0022] According to the present invention, a composition for improving neuroinflammation, a composition for improving brain function, a composition for improving lipid metabolism, and a composition for adjusting the composition ratio of intestinal bacterial flora are provided based on the new function of plant charcoal.

[0023] 1 is a diagram showing the ratio of the number of bacteria of the phylum Firmicutes to the number of bacteria of the phylum Bacteroidetes in the intestinal flora of rats according to Example 2.

[0034] FIG. 1 is a diagram showing the proportion of bacteria of the phylum Verrucomicrobia in the intestinal flora of rats in the normal control group (Group I) and the diabetic model control group (Group II) according to Example 2.

[0035] FIG. 1 is a diagram showing the proportion of bacteria of the phylum Verrucomicrobia in the intestinal flora of rats in the diabetic model control group, the low-temperature-treated bamboo charcoal-administered group (Group III), and the high-temperature-treated bamboo charcoal-administered group (Group IV) according to Example 2.

[0036] FIG. 1 is a diagram showing the proportion of bacteria of the phylum Proteobacteria in the intestinal flora of rats according to Example 2.

[0037] FIG. 1 is a diagram showing the proportion of bacteria of the phylum Actinobacteria in the intestinal flora of rats according to Example 2.

[0038] FIG. 2 is a diagram showing an image of liver tissue of a rat according to Example 3.

[0039] FIG. 3 is a diagram showing the area of ​​liver tissue stained with Oil Red staining of a rat according to Example 3.

[0039] FIG. 4 is a diagram showing the concentration of IL-1β in the plasma of a rat according to Example 4.

[0039] FIG. 5 is a diagram showing the concentration of IL-1β in the spleen tissue of a rat according to Example 4.

[0039] FIG. 6 is a diagram showing the concentration of IL-1β in the intestinal tissue of a rat according to Example 4. FIG. 1 is a diagram showing the concentration of IL-1β in the liver tissue of a rat according to Example 4. FIG. 2 is a diagram showing immunostained intestinal epithelial cells of a rat according to Example 4. FIG. 3 is a diagram showing the relative fluorescence intensity of inducible nitric oxide synthase (iNOS) obtained from FIG. 8. FIG. 4 is a diagram showing the percentage of iNOS-positive neurons in the hippocampus tissue of a rat according to Example 4. FIG. 5 is an image of immunostained intestinal epithelium of a rat according to Example 5. FIG. 6 is a diagram showing the relative fluorescence intensity of adiponectin obtained from FIG. 11. FIG. 7 is an image of the hippocampus of a rat immunostained for brain-derived neurotrophic factor (BDNF) and adiponectin according to Example 5. FIG. 8 is a diagram showing the relative fluorescence intensity of BDNF obtained from FIG. 13. FIG. 9 is a diagram showing the relative fluorescence intensity of adiponectin obtained from FIG. 13. FIG. 10 is a diagram showing the concentration of adiponectin in the plasma of a rat according to Example 5. FIG. 11 is a diagram showing the amount of adiponectin in the liver tissue of a rat according to Example 5. 16 shows images of rat hippocampus immunostained for Iba-1 and GFAP (glial fibrillary acidic protein) according to Example 5. FIG. 17 shows the percentage of GFAP-positive astrocytes obtained from FIG.16 is a diagram showing the percentage of Iba-1 positive microglial cells obtained from FIG. 16. FIG. 17 is a diagram showing the blood concentration of total cholesterol (T-CHO) in rats according to Example 6. FIG. 18 is a diagram showing the blood concentration of LDL-cholesterol (LDL-C) in rats according to Example 6. FIG. 19 is a diagram showing the blood concentration of triglyceride (TG) in rats according to Example 6. FIG. 20 is a diagram showing the blood concentration of alanine aminotransferase (ALT) in rats according to Example 6. FIG. 21 is a diagram showing the blood concentration of indoxyl sulfate in rats according to Example 6. FIG. 22 is a diagram showing the tube before and after centrifugation of a sample containing coconut charcoal according to Example 7. FIG. 23 is a diagram showing the tube before and after centrifugation of a sample containing pine charcoal according to Example 7. FIG. 24 is a diagram showing absorbance for quantifying endotoxin according to Example 8. FIG. 25 is a diagram showing electron microscope images of low-temperature treated bamboo charcoal, high-temperature treated bamboo charcoal, pine charcoal, and coconut charcoal according to Example 9.

[0024] Hereinafter, embodiments of the present invention will be described with reference to the drawings. In each drawing, the same or equivalent parts are designated by the same reference numerals. It should be noted that the present invention is not limited to the following embodiments and drawings. It should be noted that in the following embodiments, the expressions "have," "include," or "contain" also include the meaning of "consisting of" or "consisting of."

[0025] (Composition) The composition according to the present embodiment includes vegetable charcoal. The vegetable charcoal can be obtained by a known method. The type of plant is not particularly limited, and any type and any part of the plant can be used. The plant may be a plant immediately after harvesting, or a plant that has been harvested for several days. Examples of vegetable charcoal include bamboo charcoal, pine charcoal, binchotan charcoal, plum charcoal, plum seed charcoal, birch charcoal, maple charcoal, etc., as well as activated carbon such as coconut charcoal produced from coconut shells.

[0026] Preferably, the vegetable charcoal is bamboo charcoal. The type of bamboo is not limited, and any plant belonging to the order Poaceae, family Poaceae, subfamily Bambusoideae may be used. Not only one type of bamboo but also multiple types of bamboo may be used. Examples of bamboo include Madake (Phyllostachys bambusoides), Moso bamboo (Phyllostachys heterocycla f. pubescence), Hachiku (Phyllostachys nigra), Hoteichiku (Phyllostachys aurea), Kikkouchiku (Phyllostachys heterocycla f. heterocycla), Horaichiku (Bambusa multiplex), Narihiradake (Semiarundinaria fastuosa), Kurile bamboo (Sasa kurilensis), Touchiku (Sinobambusa Examples of such bamboo include Chimonobambusa quadrangularis, Chimonobambusa marmorea, Pseudosasa japonica, and Pleioblastus simonii.

[0027] A method for producing vegetable charcoal will be described using bamboo as an example of the plant. Bamboo charcoal can be obtained by heat-treating bamboo. For example, the bamboo is maintained at 200-2000°C, 250-1500°C, 300-1300°C, or 400-1200°C. The heat-treating temperature is preferably 500°C. Heat-treating may be performed in an air atmosphere, in a relatively low air atmosphere by supplying argon gas or the like, or in a vacuum. It is particularly preferable to heat the bamboo in a state similar to steaming under vacuum. The heat-treating time is set appropriately, but is typically 60-120 minutes, 70-110 minutes, 80-100 minutes, or 85-95 minutes. The bamboo charcoal obtained by heat treatment is cooled to room temperature using a known method. If the heat-treating is performed under vacuum, cooling under vacuum is also possible.

[0028] Before the heat treatment, the bamboo may be divided, cut or crushed into pieces of a size that is easy to heat treat, or after the heat treatment, the bamboo may be divided, cut or crushed.

[0029] The composition according to the present embodiment is produced by a known method and contains vegetable charcoal, for example, in an amount of 0.1 to 99% by weight, 1 to 50% by weight, and preferably 1 to 20% by weight, of vegetable charcoal as an active ingredient.

[0030] As shown in the examples below, the bamboo charcoal contained as an active ingredient in the composition has at least the effects of improving neuroinflammation, brain function, lipid metabolism, and adjusting the composition ratio of intestinal microbiota. More specifically, with regard to the neuroinflammation improving effect, bamboo charcoal suppresses inflammation of neurons in the brain, particularly in the hippocampus. Furthermore, bamboo charcoal suppresses neuroinflammation in the hippocampus, i.e., the abnormal activity of microglial cells and astrocytes. With regard to the brain function improving effect, bamboo charcoal increases the expression of BDNF (brain-derived neurotrophic factor) in the hippocampus. BDNF is a neurogenesis factor and memory enhancing factor.

[0031] Regarding its lipid metabolism-improving effect, bamboo charcoal significantly reduces liver fat content. Furthermore, bamboo charcoal lowers blood total cholesterol, triglycerides, and LDL-cholesterol. Regarding its intestinal microbiota-regulating effect, bamboo charcoal increases the Firmicutes / Bacteroidetes ratio, the Proteobacteria ratio, and the Actinobacteria ratio in the intestinal microbiota. Furthermore, as shown in the examples below, bamboo charcoal suppresses inflammatory cytokines, thereby exhibiting anti-inflammatory effects. The Firmicutes / Bacteroidetes ratio refers to the number of bacteria belonging to the Firmicutes phylum:the number of bacteria belonging to the Bacteroidetes phylum. Furthermore, the Proteobacteria ratio and the Actinobacteria ratio refer to the ratio of the number of bacteria belonging to the Proteobacteria phylum and the Actinobacteria phylum, respectively, to the number of bacteria constituting the intestinal microbiota.

[0032] Furthermore, as shown in the following examples, pine charcoal and coconut charcoal adsorb phenol red and endotoxin. Therefore, not only bamboo charcoal but also plant charcoal produced from other plants can have the same effects as bamboo charcoal.

[0033] Due to the various effects of the plant charcoal described above, the composition according to the present embodiment can be used for a variety of purposes, such as a composition for improving neuroinflammation, a composition for improving brain function, a composition for improving lipid metabolism, a composition for adjusting the composition ratio of intestinal microflora, and an anti-inflammatory composition.

[0034] The composition according to the present embodiment may be, for example, an oral composition or pharmaceutical composition such as a supplement, a food composition, a food or drink, a functional food, or a food additive. The form of the supplement is not particularly limited and may be any form such as a tablet, powder, granules, capsule, sugar-coated tablet, film, lozenge, chewable tablet, solution, emulsion, suspension, etc. The supplement may contain any component typically used in supplements.

[0035] The term "functional food" refers to food or beverages taken for the purpose of maintaining health, and includes foods with health claims, foods with specified health uses, foods with functional claims, foods with nutrient functions, health foods, and nutritional supplements. Functional foods are preferably foods with health claims or foods with nutrient functions. When commercializing a functional food, various additives used in foods, specifically colorants, preservatives, thickening agents, antioxidants, bleaching agents, antibacterial and antifungal agents, acidulants, sweeteners, seasonings, emulsifiers, strengthening agents, manufacturing agents, flavorings, etc., may be added to the oral composition.

[0036] Functional foods may be either foods or beverages, and are not particularly limited as long as they can be taken orally. Examples of functional foods include beverages, confectioneries, processed grain products, paste products, dairy products, seasonings, etc. Examples of beverages include nutritional drinks, soft drinks, black tea, green tea, etc. Examples of confectioneries include candy, cookies, tablet candy, chewing gum, jelly, etc. Examples of processed grain products include noodles, bread, cooked rice, biscuits, etc. Examples of paste products include sausages, ham, kamaboko, etc. Examples of dairy products include butter, yogurt, etc.

[0037] The oral composition may be added to food as a food additive. In this case, the food additive may be in the form of a paste, gel, powder, liquid, suspension, emulsion, granule, or the like to facilitate addition to food. The oral composition may contain water, vitamins, minerals, organic acids, organic bases, fruit juice, flavors, functional ingredients, food additives, and the like. The oral composition can be produced by known methods.

[0038] The oral composition may be divided and stored in one or more containers so that the daily intake amount is the above-mentioned intake amount, and in this case, preferably, one container contains one day's worth of the oral composition.

[0039] The oral composition is provided in a manner that allows it to be distinguished from other products in that it contains vegetable charcoal and is used for the above-mentioned purposes. For example, at least one of the packaging, instructions, and promotional materials for the oral composition indicates that it has the above-mentioned various effects.

[0040] When the composition according to the present embodiment is used as a pharmaceutical composition, the pharmaceutical composition may be, for example, a liquid, tablet, granule, fine granule, powder, tablet, capsule, etc. The pharmaceutical composition may also be an injection, oral preparation, rectal suppository, vaginal suppository, nasal absorbent, transdermal absorbent, pulmonary absorbent, or buccal absorbent. In addition to the vegetable charcoal, the pharmaceutical composition preferably contains a pharmacologically acceptable carrier. The pharmacologically acceptable carrier may be any of various organic or inorganic carrier substances. The pharmacologically acceptable carrier may be, for example, an excipient, lubricant, binder, or disintegrant in a solid formulation, or a solvent, solubilizer, suspending agent, isotonicity agent, buffer, or soothing agent in a liquid formulation. Additives such as preservatives, antioxidants, colorants, and sweeteners may also be used as needed.

[0041] Examples of excipients include lactose, sucrose, D-mannitol, starch, crystalline cellulose, light anhydrous silicic acid, etc. Examples of lubricants include magnesium stearate, calcium stearate, talc, colloidal silica, etc. Examples of binders include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, etc. Examples of disintegrants include starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, carboxymethylstarch sodium, etc.

[0042] Examples of solvents include water for injection, alcohol, propylene glycol, macrogol, etc. Examples of solubilizing agents include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, etc. Examples of suspending agents include surfactants, hydrophilic polymers, etc., such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate, polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, etc.

[0043] Examples of isotonic agents include sodium chloride, glycerin, D-mannitol, etc. Examples of buffers include phosphate, acetate, carbonate, citrate buffer solutions, etc. Examples of soothing agents include benzyl alcohol, etc. Examples of preservatives include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, etc. Examples of antioxidants include sulfites, ascorbic acid, etc.

[0044] The composition of this embodiment is administered to humans and non-human animals, preferably mammals, more specifically dogs, cats, cows, pigs, horses, sheep, deer, etc.

[0045] The dosage of the pharmaceutical composition of this embodiment is determined appropriately depending on the gender, age, weight, symptoms, etc. of the recipient. The pharmaceutical composition is administered so that the plant charcoal is in a therapeutically effective amount. An effective amount is the amount of plant charcoal necessary to achieve the desired result, i.e., the amount necessary to delay, inhibit, prevent, reverse, or cure the condition being treated or treated. The pharmaceutical composition can be administered once a day or in divided doses or more. The pharmaceutical composition may be administered at various dosing frequencies, such as daily, every other day, once a week, once every two weeks, and once a month.

[0046] The composition of this embodiment may be administered to or ingested by a subject with a neurodegenerative disease, a disease accompanied by neuroinflammation, a disease accompanied by decreased brain function, a disease accompanied by dyslipidemia, a disease accompanied by an abnormal composition ratio of intestinal microbiota, or an inflammatory disease. Preferably, the subject has diabetes, particularly type 2 diabetes.

[0047] In another embodiment, there is provided use of plant charcoal for the manufacture of a composition for improving neuroinflammation, a composition for improving brain function, or a composition for improving lipid metabolism. In other embodiments, there is provided use of plant charcoal for the manufacture of a composition for adjusting the composition ratio of intestinal microbiota to increase the ratio of Firmicutes / Bacteroidetes in the intestinal microbiota, a composition for adjusting the composition ratio of intestinal microbiota to increase the proportion of Proteobacteria in the intestinal microbiota, or a composition for adjusting the composition ratio of intestinal microbiota to increase the proportion of Actinobacteria in the intestinal microbiota.

[0048] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0049] [Example 1: Bamboo Charcoal Production] Bamboo charcoal (Marugoto Bamboo Charcoal, manufactured by Nippon Aim Co., Ltd.) carbonized at 1200°C was used as high-temperature-treated bamboo charcoal. Low-temperature-treated bamboo charcoal was produced as follows: Harvested bamboo (Moso bamboo) was cut into lengths of approximately 3 m and left to stand for approximately two weeks to remove moisture. The dehydrated bamboo was cut into 65 cm lengths and carbonized in a shin-gama kiln under the following conditions 1 to 5. Step 1: The temperature was raised to 500°C in a vacuum for approximately one hour. Step 2: The bamboo was maintained at 500°C and steamed for approximately one hour and a half. Step 3: The bamboo was cooled to below 100°C with nitrogen gas for approximately two hours under vacuum. Step 4: The bamboo was cooled to below 60°C under vacuum. Step 5: When the temperature inside the kiln dropped to 59.9°C, the vacuum was released and air was introduced into the kiln.

[0050] The bamboo charcoal was removed from the kiln and allowed to stand until it reached room temperature, and then crushed to obtain bamboo charcoal powder with a particle size of approximately 10 μm, which was used as low-temperature treated bamboo charcoal.

[0051] Example 2: Changes in intestinal bacterial flora due to bamboo charcoal ingestion Twenty-four 7-week-old male ZDF rats (strain name: ZDF-Lepr<fa> / CrlCrlj) (purchased from KBT Oriental Co., Ltd.) were used. This strain is a model that develops symptoms similar to those of adult human type 2 diabetes and its complications. After preliminary breeding, the rats were randomly assigned to the following three groups. Wistar rats were used as normal controls. Group I: Normal control group (n=8) Group II: Diabetic model control group (n=8) Group III: Diabetic model, low-temperature treated bamboo charcoal 30 mg / kg administration group (n=8) Group IV: Diabetic model, high-temperature treated bamboo charcoal 30 mg / kg administration group (n=8)

[0052] Rats in groups I to IV were pre-bred for 13 weeks until they reached 20 weeks of age. For the next four weeks, rats in group III were given low-temperature treated bamboo charcoal prepared in Example 1, and rats in group IV were given high-temperature treated bamboo charcoal prepared in Example 1, five times a week. Each rat was given a daily dose of 30 mg / kg, with the sample adjusted to within 1 ml per administration, administered orally via stomach tube. Because some rats were excluded due to abnormalities during breeding, the number of valid cases was n=8 for group I, n=6 for group II, n=8 for group III, and n=6 for group IV.

[0053] The intestinal flora of rats in groups I to IV was analyzed to determine whether the intestinal flora was affected by the ingestion of bamboo charcoal. Cosmo Bio Co., Ltd. was commissioned to analyze the intestinal flora by 16sRNA sequencing of feces.

[0054] (Results) Figure 1 shows the ratio of the number of bacteria in the phylum Firmicutes to the number of bacteria in the phylum Bacteroidetes in the intestinal flora of rats in groups I to IV. The ratio of the number of bacteria in the phylum Firmicutes to the number of bacteria in the phylum Bacteroidetes was significantly lower in group II compared to group I. Furthermore, this ratio was significantly higher in groups III and IV compared to group II. This indicates that administration of bamboo charcoal increases this ratio in the intestinal flora of the diabetes model. It also indicates that low-temperature treated bamboo charcoal is more effective than high-temperature treated bamboo charcoal in increasing this ratio.

[0055] Figure 2A shows the proportion of verrucomicrobia bacteria in the intestinal microbiota of rats in groups I and II. The proportion of verrucomicrobia bacteria was significantly lower in group II than in group I, indicating that the proportion of verrucomicrobia bacteria was significantly reduced in the diabetic model. Figure 2B shows the proportion of verrucomicrobia bacteria in the intestinal microbiota of rats in each of groups II to IV. The proportion of verrucomicrobia bacteria tended to be higher in group III compared to group II.

[0056] Figure 3 shows the proportion of Proteobacteria bacteria in the intestinal flora of rats in groups I to IV. The proportion of Proteobacteria bacteria in group II was shown to be higher than in group I. The proportion of Proteobacteria bacteria was significantly higher in group IV compared to group II.

[0057] Figure 4 shows the proportion of Actinobacteria bacteria in the intestinal flora of rats in groups I to IV. The proportion of Actinobacteria bacteria in group II was shown to be higher than in group I. The proportion of Actinobacteria bacteria in group IV was significantly higher than in group II. These results suggest that bamboo charcoal has an anti-diabetic effect by altering the intestinal flora.

[0058] Analysis of changes in the composition of the intestinal microbiota by order showed that the number of Lactobacillales bacteria increased with the intake of low-temperature treated bamboo charcoal, while the number of Bifidobacteriales bacteria increased with the intake of high-temperature treated bamboo charcoal. Bifidobacteria are reduced in diabetic patients, and selectively increasing their numbers is known to have an antidiabetic effect (Cani, P.D., et al., "Selective increases of bifidobacteria in gut microflora improve high-fat-diet-induced diabetes in mice through a mechanism associated with endotoxaemia." Diabetologia, 2007, 50:2374-2383). Clostridiales bacteria were reduced by both low-temperature and high-temperature treated bamboo charcoal intake.

[0059] The number of bacteria in the order Burkholderiales was reduced by the ingestion of low-temperature treated bamboo charcoal, while the number of bacteria in the orders Enterobacteriales and Erysipelotrichales was increased by the ingestion of both low-temperature and high-temperature treated bamboo charcoal. A reduction in Erysipelothrix order bacteria is considered a risk factor for diabetes (Hu, Y., et al., Evaluation of different mucosal microbiota leads to gut microbiota-based prediction of type 1 diabetes in NOD mice., Sci. Rep., 2018, 8:15451), so the beneficial effects of bamboo charcoal have been demonstrated.

[0060] Example 3: Changes in liver fat content due to bamboo charcoal intake After the administration period in Example 2, three rats from each group (I to IV) were subjected to oil red staining of liver tissues, and the stained area was measured.

[0061] (Results) Figure 5 shows images of liver tissues of rats in groups I to IV stained with oil red. ６ μm ２ The figures show the area of ​​liver tissue stained with oil red per 1000 mg / kg of liver tissue. As shown in Figures 5 and 6, the stained area was larger and the staining density was stronger in the liver tissue of Group II than in Group I, indicating significantly greater accumulation of fat. The stained area in the liver tissue of Groups III and IV was significantly smaller than that of Group I. This suggests that administration of bamboo charcoal can reduce fat in the liver tissue of diabetic models.

[0062] Example 4: Changes in inflammatory markers due to bamboo charcoal ingestion After the administration period in Example 2, the concentrations of interleukin-1β (IL-1β) in plasma, spleen tissue, and ulcer tissue were measured for rats in groups I to IV (BMS630, Invitrogen). Furthermore, the expression of iNOS (inducible nitric oxide synthase) in intestinal epithelial cells and hippocampal tissue neurons (NeuN-positive cells) was analyzed by immunostaining. Insulin resistance in type 2 diabetes is thought to be related to inflammatory cytokines, and both IL-1β and iNOS are inflammatory markers.

[0063] For tissue immunostaining, frozen tissues were sectioned at 20 μm thickness using a microtome. The sections were fixed in 4% paraformaldehyde and boiled in citrate buffer (microwaved at 600W for 10 minutes) to recover antigens. The sections were then stained with antibodies diluted 1:100 in TBST buffer (overnight at room temperature). After washing with TBST buffer, the sections were stained with fluorescently conjugated secondary antibodies (FITC for green and TRITC for red). DAPI was used to stain cell nuclei. Immunostained sections were photographed under a fluorescence microscope (Zeiss Axioskop 2). An anti-NOS2 antibody (PAI-036, Invitrogen) was used to detect iNOS.

[0064] (Results) Figures 7A, 7B, 7C, and 7D show the concentrations of IL-1β in plasma, spleen, intestinal, and liver tissues, respectively, in rats from groups I to IV. In group II, the concentrations of IL-1β in plasma and spleen were significantly higher than in group I, consistent with the finding that IL-1β expression is increased in type 2 diabetes patients. In groups III and IV, the concentrations of IL-1β in plasma, spleen, and intestinal tissues were significantly lower than in group II.

[0065] Figure 8 shows immunostained intestinal epithelial cells from rats in groups I to IV. In this immunostaining, iNOS expression is observed as fluorescence. DAPI is a fluorescent dye that binds to DNA. Figure 9 quantifies the iNOS fluorescence intensity in Figure 8 and shows the relative fluorescence intensity for each group, with the fluorescence intensity in group I set to 1 (n = 5 per group). Significant iNOS expression was observed in group II. In groups III and IV, the iNOS expression level was significantly lower than in group II.

[0066] Figure 10 shows the percentage of iNOS-positive cells quantified by immunostaining NeuN-positive neurons in the hippocampal tissues of rats in groups I to IV (n = 3 per group). Significant iNOS expression was observed in group II. In groups III and IV, the iNOS expression levels were significantly lower than in group II.

[0067] These results indicate that administration of bamboo charcoal can reduce inflammation in plasma, spleen tissue, intestinal tissue, intestinal epithelial cells, and hippocampal neurons. The anti-inflammatory effects of bamboo charcoal are suggested to be related to its anti-diabetic effects.

[0068] Example 5: Changes in Adiponectin, Brain-Derived Neurotrophic Factor, and Brain Neuroinflammation Following Bamboo Charcoal Intake. After the administration period in Example 2, rats from Groups I to IV were analyzed for adiponectin and BDNF expression in intestinal epithelial cells, brain hippocampal tissue, plasma, and liver tissue. Adiponectin is a hormone secreted by adipocytes and is an antidiabetic hormone that increases insulin resistance and suppresses inflammation. Adiponectin levels are known to be low in patients with type 2 diabetes, and high blood adiponectin levels are said to lower the risk of developing type 2 diabetes. Furthermore, adiponectin is believed to have protective effects against hypertension and arteriosclerosis. Among neurotrophic factors, BDNF is known to be particularly low in diabetic patients, and a decrease in its vascular protective function has been suggested to promote complications such as diabetic retinopathy. An anti-adiponectin antibody (MA1-054, Invitrogen) was used to detect adiponectin. BDNF was detected using an anti-BDNF antibody (ANT-006, manufactured by Alomone Labs), Iba-1 was detected using an anti-Iba-1 antibody (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 019-19741), and GFAP was detected using an anti-GFAP antibody (manufactured by Sigma-Aldrich, c9205).

[0069] (Results) Figure 11 shows images of immunostained intestinal epithelium from rats in groups I to IV. Figure 12 shows the relative fluorescence intensity of rats in each group, where the fluorescence intensity in group I was set to 1 (n = 5 per group). Adiponectin expression was significantly lower in group II compared to group I, consistent with the finding that adiponectin expression is reduced in diabetes. Furthermore, adiponectin expression was significantly higher in groups III and IV compared to group II, demonstrating that administration of bamboo charcoal increases adiponectin expression in the intestines of diabetic models.

[0070] Figure 13 shows images of the immunostained hippocampus of rats in groups I to IV for BDNF and adiponectin. Figures 14A and 14B show the relative fluorescence intensity of rats in each group, where the fluorescence intensity in group I is set to 1 (n = 5 per group). Compared to group I, group II also had significantly lower levels of BDNF and adiponectin expression in the hippocampus. Furthermore, compared to group II, groups III and IV had significantly higher levels of BDNF and adiponectin expression, demonstrating that bamboo charcoal administration increases BDNF and adiponectin expression in the hippocampus of diabetic models.

[0071] 15A and 15B show the adiponectin concentration in plasma and the amount of adiponectin in liver tissue, respectively. As in the hippocampus, the adiponectin concentration in plasma and the amount of adiponectin in liver tissue were significantly decreased compared to Group I, but were significantly increased in Groups III and IV. Low-molecular-weight adiponectin is known to cross the blood-brain barrier (Schon, M., et al., Effects of running on adiponectin, insulin, and cytokines in cerebrospinal fluid in healthy young individuals., Sci. Rep., 2019, 9:1959), and it has been suggested that increasing intestinal adiponectin by bamboo charcoal increases brain adiponectin.

[0072] Figure 16 shows images of the hippocampus immunostained for Iba-1 and GFAP in rats from groups I to IV. GFAP is an intermediate filament protein and a marker specifically expressed in astroglia (astrocytes). Figures 17A and 17B show the percentages of GFAP-positive astrocytes and Iba-1-positive microglial cells, respectively, in Figure 16. Compared to group I, group II showed a higher proportion of astrocytes and microglial cells in the hippocampus, indicating neuroinflammation and abnormal activity. Meanwhile, compared to group II, abnormal neuroinflammation was significantly suppressed in groups III and IV.

[0073] These results suggest that increased adiponectin expression may suppress inflammation in the hippocampus. Furthermore, the increased expression of BDNF (memory-enhancing and neurogenesis factor) in the hippocampus suggests that bamboo charcoal intake may be effective in improving memory in patients with neurodegenerative diseases, including Alzheimer's disease.

[0074] Example 6: Changes in Blood Biochemical Indices After the administration period in Example 2, blood samples were collected from the rats in Groups I to IV, and the blood biochemical indices were measured by requesting Oriental Yeast Co., Ltd.

[0075] (Results) Figures 18A, 18B, and 18C show the blood concentrations of T-CHO, LDL-C, and TG in rats in each group, respectively. T-CHO was statistically significantly reduced in groups III and IV compared to group II. LDL-C and TG were significantly reduced in group III compared to group II.

[0076] Figures 19A and 19B show the blood concentrations of ALT and indoxyl sulfate in rats in each group, respectively. ALT, a marker of fatty liver, was statistically significantly lower in Group IV compared to Group II. Indoxyl sulfate, a uremic substance, was significantly lower in Group III compared to Group II. Bamboo charcoal intake reduced blood T-CHO, TG, LDL-C, and ALT, indicating significant improvement in the lipid metabolism disorders and fatty liver associated with diabetes. One possible mechanism for this is thought to be that increased adiponectin due to bamboo charcoal intake reduced systemic inflammation.

[0077] Example 7: Phenol Red Removal Test The removal effect of phenol red in culture medium by pine charcoal and coconut charcoal was evaluated. Pine charcoal was prepared by carbonizing dried red pine logs and pulverizing them (particle size: average 300 mesh or less, Ina Akamatsu Taetan (trademark) powder, manufactured by Sumi Plus Lab). Coconut charcoal was prepared by carbonizing dried coconut shells, activating them, sterilizing them, and then pulverizing them (particle size: average 300 mesh or less, functional coconut shell activated carbon powder, manufactured by Sumi Plus Lab).

[0078] A predetermined amount of pine charcoal or coconut charcoal was mixed into DMEM medium containing 0.03% by weight of phenol red in a tube, and the tube was rotated for 36 hours at 4° C. Then, the sample was centrifuged at 10,000 rpm for 10 minutes.

[0079] (Results) Figure 20 shows tubes containing coconut charcoal before and after centrifugation. Coconut charcoal effectively removed phenol red at a concentration of 0.5%. Figure 21 shows tubes containing pine charcoal before and after centrifugation. Pine charcoal effectively removed phenol red at a concentration of 2%.

[0080] Example 8: Endotoxin Removal Test The endotoxin removal effects of the high-temperature and low-temperature treated bamboo charcoal used in Example 1, and the pine charcoal and coconut charcoal used in Example 7, were evaluated using the Pierce Chromogenic Endotoxin Quant Kit (A39552, Thermo Scientific). Endotoxin was dissolved in toxin-free water at a concentration of 0.5 U / ml. The endotoxin solution was mixed with 0.5% or 2% (wt / vol) activated charcoal. After mixing, the tube containing the sample was rotated for 36 hours at 4°C. The sample was then centrifuged at 10,000 rpm for 10 minutes. The dye absorbance of the resulting supernatant was measured using an ELISA plate reader with a 405 nm filter lens according to the kit's instructions, and the endotoxin concentration was quantified.

[0081] (Results) Figure 22 shows the absorbance. High-temperature treated bamboo charcoal was more effective at removing endotoxins than low-temperature treated bamboo charcoal. Coconut charcoal showed a strong endotoxin removal effect even at a concentration of 0.5%. Pine charcoal showed a higher endotoxin removal effect than bamboo charcoal.

[0082] Example 9: Observation under an electron microscope High-temperature treated bamboo charcoal, low-temperature treated bamboo charcoal, pine charcoal, and coconut charcoal were observed under an electron microscope.

[0083] (Results) Figure 23 shows electron microscope images. Many small particles were observed in the coconut charcoal, and these small particles had more pores than the other charcoals. Pine charcoal had thinner layers than the other charcoals. This suggests that the surface area of ​​pine charcoal is larger, especially compared to bamboo charcoal. High-temperature treated bamboo charcoal and low-temperature treated bamboo charcoal were shown to be relatively larger and have fewer pores than coconut charcoal.

[0084] The above-described embodiments are intended to explain the present invention and are not intended to limit the scope of the present invention. That is, the scope of the present invention is defined by the claims, not by the embodiments. Various modifications made within the scope of the claims and within the scope of the meaning of the invention equivalent thereto are considered to be within the scope of the present invention.

[0085] This application is based on Japanese Patent Application No. 2023-069368, filed on April 20, 2023. The entire specification, claims, and drawings of Japanese Patent Application No. 2023-069368 are incorporated herein by reference.

[0086] The present invention is useful for producing oral compositions, pharmaceutical compositions, and the like.

Claims

1. (delete)

2. Contains vegetable charcoal, A composition for increasing brain-derived neurotrophic factor expression.

3. (delete)

4. (delete)

5. (delete)

6. (delete)

7. (delete)

8. Use of plant charcoal for the manufacture of a composition for increasing brain-derived neurotrophic factor expression.

9. (delete)

10. (delete)

11. (delete)

12. (delete)

13. The plant charcoal is It is bamboo charcoal, The composition for increasing brain-derived neurotrophic factor expression according to claim 2.

14. The plant charcoal is It is bamboo charcoal, 9. The use according to claim 8.