JP7575883B2 - Composition for suppressing blood sugar level increase - Google Patents

Description

Patent Law Article 30, Paragraph 2 applied ▲1▼Publication name: Pharmacology and Treatment Vol. 47, No. 10, 1665-1675 (2019) Publication date: October 20, 2019

The present invention relates to a composition for suppressing an increase in blood glucose level and a method for suppressing an increase in postprandial blood glucose level.

In recent years, diabetes has been widely recognized as a serious health problem. However, the proportion of people with diabetes in Japan is high, accounting for 18.1% of adult men and 10.5% of adult women, according to Non-Patent Document 1. Furthermore, according to Non-Patent Document 2, it is estimated that there were 425 million diabetic patients worldwide in 2017, making diabetes a global problem. The onset and progression of diabetes are closely related to eating habits, and repeated rapid increases in postprandial blood glucose levels in particular lead to increases in fasting blood glucose levels and are thought to be one of the factors that worsen diabetes (Non-Patent Document 3). A rapid increase in postprandial blood glucose levels is considered a risk factor for lifestyle-related diseases because it promotes fat synthesis, causes insulin resistance, and vascular inflammation due to the production of glycated proteins (Non-Patent Documents 4 and 5).

Non-patent literature 6 reports that the ingestion of vegetables results in a more gradual rise in postprandial blood glucose levels. Patent literature 1 describes that the inclusion of tomatoside A, a type of saponin found in large amounts in tomato seeds, or a physiologically acceptable salt thereof in food and drink can suppress blood glucose rise. Patent literature 2 describes that β-cryptoxanthin contained in citrus pulp is effective in improving cholesterol metabolism, glucose tolerance, and insulin resistance.

JP 2017-192312 A Patent No. 5613953

Ministry of Health, Labor and Welfare. National Health and Nutrition Survey Report 2019 International Diabetes Federation. IDF Diabetes Atlas 8th Edition:2017 Monnier L, Colette C, Dunseat GJ, Owens DR. The loss of postprandial glycemic control precedes stepwise deterioration of fasting with worthening diabetes. e 2007;30:263-9. Ludwig D. S. Dietary glycemic index and obesity. J. Nutr. 130, p. 280S-283S (2000) Gerich J.E. Clinical significance, pathology, and management of postprandial hyperglycemia. Arch. Intern. Med. 163, p. 1306-1316 (2003) Inhibitory effect of food intake order on postprandial blood glucose rise in diabetic patients. Diabetes 2010; 53: 112-5.

The problem that the present invention aims to solve is to provide a novel composition for suppressing blood glucose elevation.

In light of the above, the present inventors have conducted intensive research and discovered that dietary fiber derived from at least one of tomatoes and citrus fruits has the function of suppressing an increase in blood glucose level. In other words, by ingesting dietary fiber derived from at least one of tomatoes and citrus fruits, the uptake of glucose into cells is inhibited, or the diffusion of glucose is inhibited, thereby suppressing an increase in blood glucose level. From this perspective, the present invention is defined as follows.

The contributing component of the composition for suppressing an increase in blood glucose level according to the present invention is dietary fiber derived from at least one of tomatoes and citrus fruits. The tomato-derived dietary fiber is preferably derived from tomato pulp. The citrus-derived dietary fiber is derived from citrus fruits, and is preferably derived from at least one of lemon and lime fruits. The target for which the increase suppression effect is exerted is blood glucose level, preferably postprandial blood glucose level. The mechanism of suppression of an increase in blood glucose level is at least one of inhibiting the uptake of glucose into cells or inhibiting the diffusion of glucose.

The dietary fiber is composed of at least sugar (hereinafter referred to as "constituent sugar"). The constituent sugar contains at least xylose and glucose, and the total content of xylose and glucose in the constituent sugar is 50 mol% or more. Preferably, the content of xylose in the constituent sugar is 7.0 mol% or more, and the content of glucose is 40 mol% or more. The content ratio of glucose and xylose in the constituent sugar (glucose (mol)/xylose (mol)) is 3.5 to 8.5. The constituent sugar further contains mannose, and the content of mannose in the constituent sugar is 6.0 mol% or more. The content ratio of glucose and mannose in the constituent sugar (glucose (mol)/mannose (mol)) is 4.0 to 10.0. In addition, the dietary fiber contained in the composition may be 1.0 g or more per intake, and it is preferable that the dietary fiber derived from tomatoes is 1.0 g or more.

The contributing component of the composition for inhibiting glucose uptake into cells according to the present invention is dietary fiber derived from at least one of tomatoes and citrus fruits.

The contributing component of the glucose diffusion inhibition composition of the present invention is dietary fiber derived from at least one of tomatoes and citrus fruits.

The method of the present invention for suppressing postprandial blood glucose rise comprises at least a first intake and a second intake. In the first intake, dietary fiber derived from at least one of tomatoes or citrus fruits is ingested. In the second intake, a food or drink containing carbohydrates is ingested. The second intake is simultaneous with the first intake or within 10 minutes after the start of the first intake. In the first intake, the amount of dietary fiber ingested is preferably 1.0 g or more.

The present invention makes it possible to provide a novel composition for suppressing an increase in blood glucose level. In other words, by ingesting dietary fiber derived from at least one of tomatoes and citrus fruits, the uptake of glucose into cells and the diffusion of glucose are inhibited, thereby suppressing an increase in blood glucose level.

Schematic diagram of a method for suppressing postprandial blood glucose rise A diagram showing the schedule for test food intake and blood glucose measurement FIG. 1 shows the results of efficacy evaluation items when each test beverage was ingested in Example 1. FIG. 1 shows the results of efficacy evaluation items when each test beverage was ingested in Example 2. FIG. 1 shows the results of a stratified analysis of subjects with maximum blood glucose levels of 140-199 mg/dL when taking a placebo beverage (P2) in Example 2. Figure showing the effect of tomato-derived dietary fiber on blood glucose levels following glucose administration Figure showing the effect of tomato-derived dietary fiber on 2-DG uptake Figure showing the effect of tomato-derived dietary fiber on glucose diffusion Diagram showing the effect of carrot-derived dietary fiber on glucose diffusion

<Composition for suppressing increase in blood glucose level>
The composition for suppressing an increase in blood glucose level according to the present invention is characterized in that it contains dietary fiber derived from at least one of tomatoes and citrus fruits as a contributing component. The amount of the composition per intake is not particularly limited as long as it is an amount that can be ingested by a human within 10 minutes, but is preferably 500 g or less, more preferably 350 g or less, and particularly preferably 265 g or less. The lower limit is not particularly limited.

<Dietary fiber derived from tomatoes>
The tomato-derived dietary fiber used in the present invention may be any fiber that contributes to the suppression of blood glucose level elevation, and is not particularly limited, but is preferably derived from tomato pulp. Dietary fiber extracted from tomatoes or their juice may be used, or tomato juice containing tomato-derived dietary fiber may be used as is. Examples of tomato juice include tomato juice, tomato puree, tomato paste, tomato pulp, etc., and may be used as liquid, but concentrated or dried versions of these may also be used.

The constituent sugars of tomato-derived dietary fiber are glucose, galacturonic acid, xylose, mannose, galactose, arabinose, rhamnose, glucuronic acid, etc. The total content of glucose and xylose in the constituent sugars is 50 mol% or more, preferably 60 mol% or more, more preferably 69 mol% or more. The content of xylose in the constituent sugars is 7.0 mol% or more, preferably 8.0 mol% or more, more preferably 9.0 mol% or more. The content of glucose in the constituent sugars is 40 mol% or more, preferably 50 mol% or more, more preferably 60 mol% or more, and is the main constituent sugar. The content ratio of glucose to xylose in the constituent sugars (glucose (mol)/xylose (mol)) is 3.5 to 8.5, preferably 5.0 to 7.5. The content of mannose in the constituent sugars is 6.0 mol% or more, preferably 6.5 mol% or more, more preferably 7.0 mol% or more. The glucose to mannose content ratio (glucose (mol)/mannose (mol)) in the constituent sugars is 4.0 or more and 10.0 or less, preferably 6.0 or more and 9.0 or less. It has been suggested that the constituent sugar composition of the above tomato-derived dietary fiber is different from the constituent sugar composition of dietary fiber derived from other vegetables, and it is presumed that the characteristic constituent sugar composition of tomato-derived dietary fiber is related to its effect of suppressing an increase in blood glucose level.

The amount of tomato-derived dietary fiber contained in the composition for suppressing an increase in blood glucose level is not particularly limited, and may be an amount sufficient to exert an effect as a contributing component of the blood glucose level increase suppression effect. For example, the amount of tomato-derived dietary fiber contained in the composition per intake may be 1.0 g or more, preferably 1.3 g or more, and more preferably 1.6 g or more. By setting the amount in the above range, a composition for suppressing an increase in blood glucose level with excellent effect of suppressing an increase in blood glucose level can be provided.

<Dietary fiber derived from citrus fruits>
The citrus-derived dietary fiber used in the present invention is not particularly limited as long as it has an effect of suppressing an increase in blood glucose level, but is derived from citrus fruits. Among citrus fruits, dietary fiber derived from at least one of lemon and lime is preferable.

The citrus-derived dietary fiber can be the residue obtained by squeezing the juice from the citrus fruits. More specifically, it can be obtained, for example, by drying and pulverizing the residue generated when extracting pectin from the fruits. Such dietary fiber is commercially available, and an example of a dietary fiber that is preferably used in the present invention is Herbacell AQ Plus (DSP Gokyo Food & Chemical Co., Ltd.), but is not particularly limited thereto.

The amount of citrus-derived dietary fiber contained in the composition for suppressing an increase in blood glucose level is not particularly limited, but for example, the amount of citrus-derived dietary fiber contained in the composition per intake may be 1.0 g or more, preferably 2.0 g or more, and more preferably 2.9 g or more. By keeping the amount in the above range, a composition for suppressing an increase in blood glucose level that has an excellent effect of suppressing an increase in blood glucose level can be provided.

<Other active ingredients or contributing ingredients>
The composition for suppressing an increase in blood glucose level of the present invention may contain any ingredients such as other active ingredients, contributing ingredients, additives, etc., as long as the effect of suppressing an increase in blood glucose level of the tomato-derived dietary fiber and citrus fruit-derived dietary fiber is not impaired.

Specific examples of other active ingredients or contributing ingredients include, for example, resistant dextrin, barley β-glucan, guar gum hydrolyzate, α-cyclodextrin, inulin, salacinol derived from Salacia, gallic acid derived from Terminalia bellirica, corosolic acid derived from banaba leaves, proanthocyanidin derived from acacia bark, epigallocatechin gallate, chlorogenic acid derived from Peucedanum japonicum, luteolin, isomaltodextrin, cellulose, apple fiber, potato dextrose, psyllium, beet fiber, gum arabic, etc. Furthermore, these active ingredients or contributing ingredients may be blended alone or in combination of two or more.

<Additives>
The additives contained in the composition for suppressing an increase in blood glucose level of the present invention are not particularly limited, and examples thereof include pharma- ceutically acceptable bases, carriers, excipients, binders, disintegrants, lubricants, colorants, pH regulators, buffers, stabilizers, preservatives, etc. These additives may be blended alone or in combination of two or more kinds.

Pharmaceutically acceptable bases are not particularly limited, but examples include water, polar solvents such as ethanol, and oily bases.

The carriers and excipients are not particularly limited, and examples thereof include sugar alcohols such as maltitol, xylitol, sorbitol, and erythritol, crystalline cellulose, dextrin, glucose, white sugar, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, and calcium sulfate.

Binders are not particularly limited, but examples include starch, gelatin, syrup, tragacanth gum, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, and carboxymethyl cellulose.

The disintegrant is not particularly limited, but examples include starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, sodium alginate, sodium carboxymethylcellulose, and calcium carboxymethylcellulose.

The lubricant is not particularly limited, but examples include magnesium stearate, hydrogenated vegetable oil, talc, macrogol, etc.

The coloring agent is not particularly limited, but examples include cochineal, carmine, curcumin, riboflavin, annatto, titanium oxide, iron oxide, talc, calcined silica, magnesium carbonate, etc.

The pH adjuster is not particularly limited, but examples include citric acid, gluconic acid, succinic acid, potassium carbonate, and lactic acid.

The buffering agent is not particularly limited, but examples include phosphate, arginine, histidine, etc.

The stabilizer is not particularly limited, but examples include arginine, polysorbate 80, macrogol 4000, etc.

Preservatives are not particularly limited, but examples include benzoic acid, phenoxyethanol, and thimerosal.

Other additives include dissolution aids, surfactants, emulsifiers, antioxidants, gloss agents, foaming agents, moisture-proofing agents, preservatives, sweeteners, flavoring agents, cooling agents, flavoring agents, fragrances, flavorings, and disintegration aids.

These additives may be used alone or in combination of two or more.

<Suppression of blood sugar rise>
In the present invention, "suppression of blood glucose rise" refers to suppressing or alleviating the rise in blood glucose concentration mainly after a meal. The blood glucose rise suppression effect of dietary fiber derived from at least one of tomatoes and citrus fruits is thought to be due to multiple mechanisms. For example, inhibition of glucose uptake into cells and inhibition of glucose diffusion. "Uptake of glucose into cells" refers to the uptake of glucose from outside the cells into the cells by a glucose transporter (SGLT1) present on the cell membrane of the intestinal epithelium. By inhibiting the uptake of glucose into cells, the absorption of sugar in the intestine is suppressed and the rise in blood glucose level is suppressed. In addition, "diffusion of glucose" refers to the diffusion of glucose in the digestive tract. By inhibiting the diffusion of glucose, the movement speed of glucose in the digestive tract is delayed and the rise in blood glucose level is gradual.

<Medicines and quasi-drugs>
The form of the medicine or quasi-drug containing the composition for suppressing an increase in blood glucose level of the present invention is not particularly limited, and examples of the oral administration form include tablets such as sugar-coated tablets, buccal tablets, coated tablets, and chewable tablets, troches, pills, powders, capsules including soft capsules, granules, suspensions, emulsions, syrups including dry syrups, and liquids such as elixirs, and the parenteral administration form includes intraperitoneal administration, enteral administration, and oral administration. From the viewpoint of convenience and versatility, the form of the medicine or quasi-drug is preferably an oral administration form.

The dosage of the pharmaceutical or quasi-drug containing the composition for suppressing blood glucose level rise of the present invention is not particularly limited. For example, for a 60 kg human, the dietary fiber derived from at least one of tomato and citrus fruits is 0.017 g/kg (body weight) or more per administration. The tomato-derived dietary fiber is preferably 0.022 g/kg (body weight) or more per administration, more preferably 0.027 g/kg (body weight) or more. The citrus-derived dietary fiber is preferably 0.033 g/kg (body weight) or more per day, more preferably 0.048 g/kg (body weight) or more. When the subject is a non-human animal, the dosage can be appropriately changed depending on the type of animal.

The number of times that a pharmaceutical or quasi-drug containing the composition for suppressing an increase in blood glucose level of the present invention is administered is not particularly limited, and may be, for example, once a day, twice a day, or three times a day. The administration period is not particularly limited, and may be, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, or may be used continuously without a set administration period. In addition, administration during the administration period is not particularly limited, and may be, for example, daily administration, or every other day, every third day, or every third day.

<Food, drink, feed>
The form of the food, drink, or feed containing the composition for suppressing an increase in blood glucose level of the present invention is not particularly limited, and examples include processed foods, health foods (nutritional supplements, nutritional functional foods, foods for the sick, foods for specified health uses, foods with functional claims, etc.), supplements, foods for the sick (hospital food, food for sick people, nursing care food, etc.), confectionery, oils and fats, dairy products, retort foods, microwave foods, frozen foods, seasonings, dietary supplements, beverages, nutritional drinks, etc.

The shape and properties of the food, drink, and feed containing the food composition of the present invention are not particularly limited, and examples include solid, semi-solid, gel, liquid, and powder. In particular, when used as a beverage, a vegetable and fruit beverage can be suitably selected.

Food, beverage, and feed containing the food composition of the present invention may be labeled with a notice indicating that it may have a beneficial effect on suppressing an increase in blood glucose level. Such a notice may be attached to the container packaging means by a known method, and this clearly indicates that the food, beverage, and feed containing the composition of the present invention are used to suppress an increase in blood glucose level, making it possible to clearly distinguish them from ordinary food, beverage, and feed.

The intake amount of food, drink, or feed containing the food composition of the present invention is not particularly limited. For example, for a 60 kg human, the dietary fiber derived from at least one of tomato and citrus fruits is 0.017 g/kg (body weight) or more per intake. The tomato-derived dietary fiber is preferably 0.022 g/kg (body weight) or more per intake, more preferably 0.027 g/kg (body weight) or more per intake. The citrus-derived dietary fiber is preferably 0.033 g/kg (body weight) or more per intake, more preferably 0.048 g/kg (body weight) or more per intake. When targeting non-human animals, the intake amount can be appropriately changed depending on the type of animal.

The number of times that the food, drink, or feed containing the composition of the present invention is ingested is not particularly limited, and may be, for example, once a day, twice a day, or three times a day. The period of ingestion is not particularly limited, and may be, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, or may be used continuously without a set period of ingestion. In addition, the period of ingestion is not particularly limited, and may be, for example, daily ingestion, ingestion every other day, ingestion every third day, or ingestion every third day.

<Outline of methods for suppressing postprandial blood glucose rise>
1 shows the flow of a method for suppressing postprandial blood glucose rise (hereinafter referred to as "the method"). The method mainly comprises a first intake (S10) and a second intake (S20).

<First intake (S10)>
The purpose of the first intake is to suppress an increase in postprandial blood glucose level. Here, the dietary fiber to be ingested is derived from at least one of tomatoes and citrus fruits. The amount of dietary fiber to be ingested in the first intake is preferably 1.0 g or more, more preferably 1.3 g or more, and particularly preferably 1.6 g or more.

<Second intake (S20)>
The second intake is a general meal or snack, and its purpose is to replenish nutrition. The food or drink ingested here is a carbohydrate-containing food or drink. The type, form, and amount of the food or drink are not particularly limited, but it is preferable that the food or drink is capable of providing nutrition for a general meal, and staple foods such as white rice, bread, noodles, and potatoes are particularly preferable. The second intake is simultaneous with the first intake or within 10 minutes after the start of the first intake.

<Carbohydrates>
Carbohydrates are defined in the Food Nutrition Labeling Standards (Ministry of Health, Labour and Welfare Notification No. 176, 2003). Examples of carbohydrates include monosaccharides, disaccharides, trisaccharides to decasaccharides (so-called oligosaccharides), starch, etc. Examples of monosaccharides include glucose, fructose, galactose, mannose, etc. Examples of disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, etc. Examples of oligosaccharides include stachyose, maltotriose, maltotetraose, maltopentaose, etc. Examples of starches include tapioca starch, potato starch, corn starch, wheat starch, sweet potato starch, rice starch, modified starch, dextrin, etc.

<Human testing>
A placebo-controlled crossover comparative study was conducted according to the method described below.

<Test subjects>
Healthy adult men and women aged 20 years or older were selected as study subjects, and those who did not fall under the following exclusion criteria were selected as study subjects.
(1) Those who have been diagnosed with diabetes at or above the standard value (fasting blood glucose level of 126 mg/dL or higher, or HbAlc level of 6.5% or higher) in their most recent health check. (2) Those who have a food allergy to the test food. (3) Those who are so sensitive to alcohol that they have difficulty using alcohol disinfectants. (4) Those who plan to become pregnant or breastfeed during the study period. (5) Those who are participating in other human studies at the start of this study. <Restrictions on study subjects>
(1) Refrain from excessive exercise the day before the test, and on the day of the test, limit your activities to quiet standing or sitting until the end of the test.
(2) Do not consume any food or drink anything other than water or hot water from 9:00 p.m. on the day before the test until the start of the test (9:00 a.m.) on the day of the test.
(3) On the day of the test, fluid intake other than the test drink until the blood glucose measurement is completed is limited to a specified amount (200 ml in Example 1, and no intake in Example 2).
(4) Women should undergo testing outside of their menstrual period.
(5) Follow any other instructions given by the principal investigator, etc.

<Test beverage>
Different beverages were prepared and used in the tests in Examples 1 and 2. The ingredients and volumes of the test beverages in each test are shown in Table 1, and the nutritional components are shown in Table 2.

Example 1
<Test beverage>
Four types of test beverages were used: water (W), placebo beverage (P1), beverage with high tomato-derived dietary fiber content (T1), and beverage with high citrus-derived dietary fiber content (C1). The volume of each beverage was 200 mL. P1 uses tomato, carrot, and lemon juice as raw materials (dietary fiber content: 1.5 g/200 ml), and T1 uses tomato pulp (FIT F tomato pulp, Kagome Co., Ltd.) with a higher dietary fiber content than P1, thereby increasing the content of tomato-derived dietary fiber (dietary fiber content: 3.1 g/200 ml). The tomato pulp mentioned above was obtained by washing and crushing tomatoes produced in Portugal, preheating them at 65°C, removing the seed coat, and centrifuging the precipitated fraction. C1 was made by replacing part of the tomato pulp in P1 with dietary fiber derived from citrus fruits (lemon and lime) (Helbacell AQ Plus; DSP Gokyo Food & Chemical Co., Ltd.), to increase the dietary fiber content (dietary fiber content: 4.4/200 ml). The energy, carbohydrate, protein, and lipid values of the placebo drink, the tomato-derived high dietary fiber drink, and the citrus-derived high dietary fiber drink were almost the same (Table 2). The difference in dietary fiber between P1 and T1 was 1.6 g, and the difference in dietary fiber between P1 and C1 was 2.9 g, but these differences were due to the difference in the amount of tomato-derived dietary fiber and citrus-derived dietary fiber, respectively.

<How to measure blood glucose level>
On the morning of the test day, test subjects collected a small amount of fasting blood from their fingertips using a lancing device (Medisafe Fine Touch; Terumo Corporation), and their blood glucose levels before ingesting the test food (0 minutes) were measured using a self-blood glucose meter (Glutest Eye; Sanwa Kagaku Kenkyusho) and a dedicated sensor (Glutest Neo Sensor; Sanwa Kagaku Kenkyusho).

After that, the subjects promptly consumed the test beverage, and then ingested 150 g (48.8 g carbohydrates) of pre-cooked, sterilely packaged cooked rice (Sato Foods Industry) as the challenge food. The total time spent ingesting the test beverage and the challenge food was within 10 minutes. Blood glucose levels were measured 15, 30, 45, 60, 90, 120, and 180 minutes after starting to ingest the test beverage, using the same method as at 0 minutes. The schedule for test food intake and blood glucose measurement is shown in Figure 2.

<Efficacy evaluation>
The following four items were used as efficacy evaluation items.
(1) Blood glucose level at each time point (2) The change in blood glucose level at each time point minus the blood glucose level at 0 minutes (Δ blood glucose level)
(3) ΔMaximum blood glucose value (ΔC _max )
(4) ΔIncremental area under the curve (IAUC) calculated from the change in blood glucose level using the trapezoidal rule. Two areas were calculated: the entire test time (IAUC _{180 min} ) and the time when blood glucose level increased significantly (IAUC _{60 min} ).

<Statistical analysis>
The above evaluation items are expressed as mean ± standard deviation (Figures 1 to 3). EZR (Ver. 1.40) was used for statistical analysis software. As a result of the Smirnoff-Grubbs test, subjects whose blood glucose levels in any test were critical (p < 0.05) were excluded from the analysis. Comparisons of each evaluation item between those who consumed the placebo drink and those who consumed the other test drinks were performed using paired t-tests. The significance level for all tests was set at 5% on both sides.

<Results>
Fourteen subjects were selected as study subjects. The profile of the study subjects is shown in Table 3. No subjects dropped out of the study, and all 14 subjects completed the study. No adverse events such as abdominal discomfort due to the intake of the test beverage were observed. As a result of the rejection test using the Smirnoff-Grubbs test, 2 subjects were excluded from the analysis, as their blood glucose levels were rejected in either test.

Figure 3 shows the results of each evaluation item when each test beverage was ingested. Blood glucose levels were significantly lower at 0 and 60 minutes after T1 ingestion, and at 60 minutes after C1 ingestion, compared to P1 ingestion. W ingestion showed significantly higher values at 90 minutes after P1 ingestion (Figure 3a). For delta blood glucose levels, there was no significant difference when T1 was ingested compared to P1 ingestion, but C1 ingestion showed significantly lower values at 60 minutes after ingestion, and W ingestion showed significantly higher values at 90 minutes after ingestion (Figure 3b).

Example 2
Except for the test beverage described below, the test was carried out in the same manner as in Example 1. Although not carried out in Example 1, in the evaluation of efficacy, a stratified analysis was also carried out targeting only those whose postprandial blood glucose levels were slightly high when ingesting the placebo beverage (those with a maximum postprandial blood glucose level of 140-199 mg/dL).

<Test beverage>
Three types of test beverages were used: a placebo beverage (P2), a beverage with high tomato-derived dietary fiber content (T2), and a beverage with high citrus-derived dietary fiber content (C2). The volume of each beverage was 265 g. P2 uses tomato, carrot, and lemon juice as raw materials (dietary fiber content: 0.5 g/265 ml), and T2 uses tomato pulp with a higher dietary fiber content than P2, thereby increasing the tomato-derived dietary fiber content (dietary fiber content: 2.1 g/265 ml). C2 is a beverage with increased dietary fiber content (dietary fiber content: 5.8/265 ml) by replacing part of the tomato pulp of P2 with citrus (lemon and lime)-derived dietary fiber (Helbacell AQ Plus, DSP Gokyo Food & Chemical Co., Ltd.). The energy, carbohydrate, protein, and lipid contents of the placebo beverage, the tomato-derived dietary fiber-rich beverage, and the citrus-derived dietary fiber-rich beverage were almost the same (Table 2). The difference in dietary fiber amount between P2 and T2 was 1.6 g, and the difference in dietary fiber amount between P2 and C2 was 5.3 g, but these differences were due to the difference in the amount of tomato-derived dietary fiber and citrus fruit-derived dietary fiber, respectively.

<Results>
Thirty-three subjects were selected as study subjects. The profile of the study subjects is shown in Table 3. No subjects dropped out of the study, and all 33 subjects completed the study, with no adverse events such as abdominal discomfort due to the ingestion of the test beverage. As a result of the rejection test using the Smirnoff-Grubbs test, 4 subjects were found to have rejection values for blood glucose levels in any of the tests, and 29 subjects were selected for analysis. In the stratified analysis, analysis was performed on 26 subjects whose maximum blood glucose levels when ingesting P2 were 140-199 mg/dL.

Figure 4 shows the results of efficacy evaluation items when each test beverage was ingested. When either T2 or C2 was ingested, blood glucose levels and Δ blood glucose levels at 15 and 30 minutes were significantly lower than when P2 was ingested (Figure 4 a, b). In addition, ΔC _max and IAUC _{60 min} were also significantly lower when T2 or C2 was ingested compared to when P2 was ingested (Figure 4 c, e). Stratified analysis of subjects with maximum blood glucose levels of 140-199 mg/dL when P2 was ingested also showed similar results to the overall subjects (Figure 5 a, b, c, d, e).

The results of Examples 1 and 2 confirmed that dietary fiber derived from tomatoes or citrus fruits has the effect of suppressing the rise in blood glucose level after a meal, and particularly suppressed the rise in blood glucose level 60 minutes or more after the start of ingestion of the test beverage, suggesting that it inhibits or delays sugar absorption, especially in the short period after a meal.

Example 3: Animal testing
In Example 3, the effect of tomato-derived dietary fiber on glucose absorption in rats was examined.

<Preparation of test samples>
Tomato pulp containing tomato-derived dietary fiber (FIT F tomato pulp, Kagome Co., Ltd.) was freeze-dried and powdered for use. The dietary fiber content of the freeze-dried powder was 43.1 g/100 g. The freeze-dried powder of tomato pulp was suspended in an aqueous glucose solution (15 g/100 mL) so that the final dietary fiber concentration was 3% (3 g/100 mL), and this was used as a test sample for the tomato-derived dietary fiber group described below.

<Test Method>
Eight 6-week-old male SD rats (CLEA Japan) were purchased and used in the study. The rats were individually housed in plastic cages with a 12-hour light-dark cycle (7:00-19:00: light, 19:00-7:00: dark). The rats were given MF solid feed (Oriental Yeast Co., Ltd.) and tap water ad libitum. After 5 days of acclimation, the rats were divided into two groups, a control group and a tomato-derived dietary fiber group, with four rats each, so that the weights of the groups were approximately equal.

Fasting began at 5 p.m. on the day before the test, and after a 16-hour fast, the control group was administered an aqueous glucose solution (15 g/100 mL), while the tomato-derived dietary fiber group was administered the above-mentioned test sample. The dosage was 10 mL/kg (body weight), with the glucose dosage being 1.5 g/kg (body weight) and the dietary fiber dosage being 0.3 g/kg (body weight). Blood glucose levels were then measured.

<Blood Glucose Measurement>
At 0 min (before administration of the test sample), and 15 and 30 min after administration of the test sample, the rats were immobilized in an immobilization device ICN-5A (ICM Co., Ltd.). The tail vein was punctured with a scalpel, and blood glucose levels were measured using a portion of the blood (approximately 5 μL) with Glutest Ace R and Glutest Sensor (both from Sanwa Kagaku Kenkyusho).

<Statistical processing>
The blood glucose levels and changes in blood glucose levels were compared between the control group and the tomato-derived dietary fiber group using a Student t-test.

<Results>
The results are shown in Figure 6. In the tomato-derived dietary fiber group, the blood glucose level and the change in blood glucose level 15 minutes after administration were significantly lower than those in the control group (p<0.05 and p<0.01, respectively).

Example 4: Cellular testing
In Example 4, the effect of tomato-derived dietary fiber on sodium-dependent glucose transporter (SGLT) 1, which is an intestinal epithelial glucose transporter, was examined.

The effect on SGLT1 was examined by using cells in which a human SGLT1 expression vector was transfected into Chinese hamster ovary-derived CHO-K1 cells (a human SGLT1 stable high-expressing cell line) and evaluating the amount of uptake of the glucose analog 2-deoxyglucose (2-DG) into the cells.

<Construction of a stable cell line expressing high levels of human SGLT1>
Human SGLT1 gene was cloned to construct an expression vector for mammalian cells. After gene transfer of the human SGLT1 expression vector into CHO-K1 cells, selection was performed with antibiotics, and single clones were obtained by limiting dilution. Among them, the clone with the highest sodium-dependent glucose uptake activity was isolated and used as a stable high-expression cell line of human SGLT1.

<Cell culture>
The human SGLT1 stable high-expressing cell line was cultured in a 100 mm plastic petri dish for cell culture at 37°C in an incubator containing 5% _CO2 . The medium was replaced every 1 to 2 days. The cell culture medium used was 500 mL of Ham's F-12 medium to which 56 mL of FBS, 2 mL of penicillin-streptomycin solution (final penicillin concentration: 40 U/mL, final streptomycin concentration: 40 μg/mL), and G418 (final concentration: 2 mg/mL) were added.

<Passage>
When the cells cultured in a 100 mm Petri dish reached 70-90% confluence, the medium was removed and washed with about 10 mL of PBS, after which 1 mL of trypsin-EDTA (Trypsin-EDTA (0.05%), phenol red, Thermo Fisher Scientific) solution was added and spread over the entire Petri dish, and the cells were left in an incubator for about 5 minutes to detach the cells, and 9 mL of medium was added to collect the cells while suspending them. A portion (about 40 μL) was used to count the number of cells using a cell counting board. The remaining cell suspension was centrifuged at 1,000 rpm at room temperature for 5 minutes, the supernatant was removed, and an appropriate amount of medium was added and the cells were uniformly suspended by pipetting, and then seeded on a 100 mm Petri dish so that the number of cells per Petri dish was about 1.0 × 10 ⁵ cells.

<Preparation of test samples>
5 g of freeze-dried tomato pulp was collected and placed in an Erlenmeyer flask and covered with aluminum foil. 250 mL of 0.08 M phosphate buffer (pH 6.0) was added thereto, and the pH was adjusted to 6.0 ± 0.1 with 0.275 M sodium hydroxide aqueous solution. 250 μL of heat-resistant amylase (total dietary fiber analysis kit, Japan Biocon Co., Ltd.) was added, gently stirred, and incubated for 30 minutes with stirring in a thermostatic water bath heated to 98 to 100 ° C. After that, it was cooled to room temperature, and about 50 mL of 0.275 M sodium hydroxide aqueous solution was added to adjust the pH to 7.5 ± 0.1. 500 μL of protease was added, gently stirred, and incubated for 30 minutes with stirring in a thermostatic water bath heated to 60 ° C. After that, it was cooled to room temperature, and about 50 mL of 0.325 M hydrochloric acid was added to adjust the pH to 4.5 ± 0.2. 1000 μL of amyloglucosidase (total dietary fiber analysis kit, Japan Biocon Co., Ltd.) was added, and the mixture was stirred gently, and then incubated for 30 minutes in a thermostatic water bath heated to 60° C. with stirring. Then, 1400 mL of 95% ethanol heated to 60° C. was added, and the mixture was left to stand at room temperature for 60 minutes. After standing, the mixture was filtered by suction using 5A filter paper to obtain a residue. The residue was washed five times with 60 mL of 78% ethanol, twice with 50 mL of 95% ethanol, and twice with 50 mL of acetone. The washed residue was vacuum-dried in a desiccator overnight or more, and the obtained dried product was crushed into powder to obtain a tomato-derived dietary fiber fraction.

The prepared tomato-derived dietary fiber fraction was suspended in KRH (Krebs-Ringer-HEPES) buffer at concentrations of 25, 12.5, and 6.25 mg/mL, and sonicated for 20 minutes. It was then centrifuged at 3,000 rpm for 10 minutes, and the supernatant was collected.

Uptake of 2-DG into cells
A human SGLT1 stable expression cell line was seeded in a 24-well plate at a cell density of about 2.0 x ¹⁰⁵ cells/well and cultured in serum-free medium for 24 hours. The cells were washed twice with KRH buffer and then subjected to a 2-DG uptake test. In the study of simultaneous treatment, 2-DG was dissolved at a concentration of 1 mM in the above-mentioned test sample or KRH buffer not containing tomato-derived dietary fiber, and 500 μL was added to each well, and a 2-DG uptake test was performed at 37°C for 10 minutes. In the study of pretreatment, 500 μL of the above-mentioned test sample was added to each well and pretreated at 37°C for 10 minutes, followed by washing twice with KRH buffer, adding 500 μL of 1 mM 2-DG to each well, and performing 2-DG uptake at 37°C for 10 minutes.

After the uptake test was completed, the cells were washed twice with KRH buffer, and 300 μL of 0.1 M aqueous sodium hydroxide solution was added and allowed to stand at 37°C for 60 minutes to solubilize the cells. After that, 300 μL of 0.1 M hydrochloric acid was added to neutralize the cells, which were then collected and used as samples for 2-DG measurement.

<Measurement of 2-DG uptake in cells>
2-DG was measured using Promega's Glucose Uptake-Glo ^™ Assay. 2DG6P detection reagent was prepared by mixing 10 mL of luciferase reagent, 100 μL of NADP+, 250 μL of G6PDH, 50 μL of reductase, and 6.25 μL of reductase substrate per 100 samples. 25 μL of 2-DG measurement sample and 25 μL of 2DG6P detection reagent collected in the cell test were placed in each well of a 96-well plate, and incubated at room temperature for 30 minutes in the dark. Thereafter, the amount of 2-DG uptake was measured by measuring the luminescence intensity of luciferase using a plate reader (Tristar LB941, Berthold Technologies).

<Results>
When tomato-derived dietary fiber and 2-DG were simultaneously treated, 2-DG uptake was significantly suppressed at concentrations of 12.5 mg/mL or higher. Pretreatment with tomato-derived dietary fiber for 10 minutes also significantly suppressed 2-DG uptake at concentrations of 12.5 mg/mL or higher, similar to the simultaneous treatment (Figure 7).

Example 5: Glucose diffusion test
In Example 5, the effect of tomato-derived dietary fiber on glucose diffusion was examined.

<Test sample>
The tomato-derived dietary fiber fraction prepared in Example 4 was used. A carrot-derived dietary fiber fraction prepared from concentrated carrot juice (Bolthouse Farms) in the same manner was also used in the test.

<Diffusion test using dialysis membrane>
Dietary fiber fractions derived from tomatoes or carrots were suspended in a solution of 0.22 M glucose dissolved in 0.15 M saline at concentrations of 50, 25, 12.5, and 6.25 mg/mL. The dietary fiber suspension was packed into a dialysis tube (Spectra/Pore 7, MWCO 2,000, φ11.5 mm×18 mm, REPLIGEN) cut to a length of about 8 cm, and the top and bottom were sealed with closures. The dialysis tube was immersed in 45 mL of 0.15 M saline solution placed in a 100 mL centrifuge tube, and the 100 mL centrifuge tube was stirred with a shaker. 100 μL of 0.15 M saline solution was collected from the centrifuge tube 30, 60, 90, 120, 150, and 180 minutes after the start of stirring, and the concentration of glucose leached from the dialysis membrane was measured.

<Measurement of glucose amount>
The concentration of glucose leached from the dialysis membrane was calculated by measuring the absorbance at 570 nm using a glucose measurement kit EnzyChrom ^™ Glucose Assay Kit (EBGL-100, BioAssay Systems) according to the method described in the accompanying instruction manual. The absorbance was measured using a Corona Multigrating Microplate Reader SH-9000Lab (Hitachi High-Tech Science).

<Results>
The time course of the data showed that the diffusion of glucose was inhibited more as the concentration of tomato-derived dietary fiber increased (Fig. 8a). The area under the curve (AUC) was calculated from the time course of the data, and the AUC was significantly lower at 50 mg/mL (Fig. 8b).

Furthermore, carrot-derived dietary fiber did not inhibit glucose diffusion (Figure 9).

Example 6: Measurement of sugar components of dietary fiber derived from tomatoes and carrots
In Example 6, the constituent sugars produced by acid hydrolysis of dietary fiber fractions derived from tomatoes and carrots were analyzed by high performance liquid chromatography (HPLC). The preparation method of each dietary fiber fraction was the same as that in Examples 4 and 5.

<Acid hydrolysis of dietary fiber derived from tomatoes>
0.3 g of dietary fiber fraction derived from tomato or carrot was precisely weighed, 4 mL of 72% sulfuric acid was added, and the mixture was stirred at room temperature for 1 hour, then washed into an Erlenmeyer flask with 112 mL of water and heated in an autoclave at 121°C for 1 hour. After that, the mixture was ice-cooled to neutralize, and then the volume was adjusted to 200 mL with water and filtered using filter paper (No. 5B [Toyo Roshi Kaisha, Ltd.]) and a membrane filter with a pore size of 0.45 μm. The filtered solution was diluted 2-fold with water to prepare the test solution.

<HPLC conditions>
The HPLC operating conditions for rhamnose, ribose, mannose, arabinose, fucose, galactose, xylose, and glucose were as follows:

Model: LC-20AD (Shimadzu Corporation)
Detector: Fluorescence spectrophotometer RF-20Axs (Shimadzu Corporation)
Column: TSKgel SUGAR AXI, φ4.6 mm × 150 mm (Tosoh Corporation)
Column temperature: 60℃
Mobile phase: 0.5 mol/L borate buffer (pH 8.7)
Flow rate: 0.4 mL/min
Injection volume: 20 μL
Fluorescence excitation wavelength: 320 nm
Fluorescence measurement wavelength: 430 nm
Post-column: Reaction solution; 1% L-arginine solution
Reaction solution flow rate: 0.7 mL/min
Reaction temperature: 150 °C
The HPLC operating conditions for galacturonic acid and glucuronic acid were as follows:

Model: ICS-5000+ (Thermo Fisher Scientific Co., Ltd.)
Detector: Pulsed amperometric detector ED
(Thermo Fisher Scientific Japan Co., Ltd.)
Column: CarboPac PAI, φ4.0 mm x 250 mm
(Thermo Fisher Scientific Japan Co., Ltd.)
Column temperature: 32 °C
Mobile phase: 0.1 mol/L sodium hydroxide solution and 0.1 mol/L sodium acetate solution
Mixture (1:1)
Flow rate: 1 mL/min
Injection volume: 5 μL
<Results>
The results of quantification of the constituent sugars of tomato-derived dietary fiber by HPLC analysis are shown in Table 4. Moreover, a comparison of the results of the constituent sugar analysis of tomato-derived and carrot-derived dietary fiber is shown in Table 5. The values in Table 5 represent the peak area of each constituent sugar when the peak area of the constituent sugar glucose is taken as 100. Note that galacturonic acid and glucuronic acid are not shown in Table 5 because the analytical method and the method of calculating the peak area are different from those of the other sugars.

The ratio of glucose to the sugar components of carrot-derived dietary fiber is significantly lower than that of tomato-derived dietary fiber, with galactose and arabinose being the main sugar components, which is significantly different from the sugar composition of tomato-derived dietary fiber. Therefore, it is presumed that the characteristic sugar composition of tomato-derived dietary fiber is related to its effect of suppressing blood glucose levels.

The fields in which this invention is useful include the provision of pharmaceuticals, health foods, supplements, food and beverages, and feed.

Claims (12)

A composition for suppressing an increase in blood glucose level, comprising:
Contains dietary fiber as a contributing component,
The dietary fiber is derived from tomato pulp,
The dietary fiber contained as a contributing component is derived from tomato pulp,
The dietary fiber is composed of at least sugar (hereinafter referred to as "constituent sugar");
The constituent sugars include at least xylose and glucose,
The total content of xylose and glucose in the constituent sugars is 50 mol% or more,
The amount of dietary fiber contained in each intake of the composition is 1.0 g or more . 2. The composition of claim 1 ,
The blood glucose level is a postprandial blood glucose level. 3. The composition of claim 1 or 2,
What the dietary fiber inhibits is the uptake of glucose into cells. 4. The composition of claim 1, further comprising:
What dietary fiber inhibits is the diffusion of glucose. 6. The composition of any one of claims 1 to 5,
The content of xylose in the constituent sugars is 7.0 mol % or more. 6. The composition of any one of claims 1 to 5,
The glucose content in the constituent sugars is 40 mol % or more. 7. The composition of any one of claims 1 to 6,
The content ratio of glucose to xylose in the constituent sugars (glucose (mol)/xylose (mol)) is 3.5 or more and 8.5 or less. 8. The composition of any one of claims 1 to 7,
The sugar components further include mannose,
The mannose content in the constituent sugars is 6.0 mol % or more. 9. The composition of any one of claims 1 to 8,
The content ratio of glucose to mannose in the constituent sugars (glucose (mol)/mannose (mol)) is 4.0 or more and 10.0 or less. 10. The composition of any one of claims 1 to 9,
The dietary fiber contained in one intake of the composition is 1.0 g or more of tomato-derived dietary fiber. A composition for inhibiting glucose uptake into a cell, comprising:
Contains dietary fiber as a contributing component,
The dietary fiber is derived from tomato pulp,
The dietary fiber contained as a contributing component is derived from tomato pulp,
The dietary fiber is composed of at least sugar (hereinafter referred to as "constituent sugar");
The constituent sugars include at least xylose and glucose,
The total content of xylose and glucose in the constituent sugars is 50 mol% or more,
The amount of dietary fiber contained in each intake of the composition is 1.0 g or more . A composition for inhibiting the diffusion of glucose, comprising:
Contains dietary fiber as a contributing component,
The dietary fiber is derived from tomato pulp,
The dietary fiber contained as a contributing component is derived from tomato pulp,
The dietary fiber is composed of at least sugar (hereinafter referred to as "constituent sugar");
The constituent sugars include at least xylose and glucose,
The total content of xylose and glucose in the constituent sugars is 50 mol% or more,
The amount of dietary fiber contained in each intake of the composition is 1.0 g or more .

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