WO2020045647A1 - Agent for suppressing increment of blood glucose level, diabetes preventing agent, and food composition - Google Patents

Abstract

Provided are an agent for suppressing the increment of a blood glucose level, a diabetes preventing agent and a food composition, which are novel uses of a carotenoid mixture derived from a microalga. An agent for suppressing the increment of a blood glucose level, a diabetes preventing agent and a food composition, each of which contains a carotenoid mixture derived from a microalga as an active ingredient, wherein the carotenoid mixture contains diatoxanthin as the main component thereof. The carotenoid mixture derived from a microalga is, for example, a carotenoid mixture derived from euglena, which contains diatoxanthin as the main component thereof, and also contains zeaxanthin and alloxanthin. The agent can be used for suppressing the increment of a blood glucose level through the ingestion together with, for example, a high-GI food.

Description

Blood sugar rise inhibitor, diabetes inhibitor, and food composition

The present invention relates to a novel blood glucose elevation inhibitor, a diabetes inhibitor, and a food composition, in particular, a blood glucose elevation inhibitor, a diabetes inhibitor, and a food composition containing a carotenoid mixture derived from microalgae as an active ingredient. About.

Normally, the glucose concentration in blood in a living body is adjusted to be within a certain range by the action of insulin.In diabetes, the glucose-controlling function of the blood glucose does not work normally, and the glucose concentration in blood is reduced. It is a disease that rises abnormally.
Diabetes is roughly classified into type 1 diabetes and type 2 diabetes. Type 1 diabetes is a disease in which insulin is not secreted from the pancreas due to destruction of β cells of the pancreas.
Type 2 diabetes is said to be one of the lifestyle-related diseases, and causes abnormalities in the immune system due to environmental factors such as obesity, overeating, lack of exercise, and stress, resulting in inhibition of insulin function. It is a disease that tends to develop and progress in relatively elderly obese people thereafter.
More specifically, it is said that abnormalities in the accumulation of triglycerides in the blood and organs occur, resulting in abnormal secretion of insulin and insulin resistance.

Under these circumstances, although many therapeutic agents have been developed for diabetes, particularly type 2 diabetes, which is said to account for more than 90% of the Japanese diabetic patients, clinical results are sufficiently obtained. Many did not, or caused mental symptoms or side effects on the heart.
Therefore, there has been a demand for the development of a blood sugar rise inhibitor and a diabetes inhibitor containing a naturally derived substance as an active ingredient, which has both safety and efficacy.

On the other hand, carotenoids are useful as natural pigments and have various effective actions such as antioxidant action, so that their use value is increasing in the fields of pharmaceuticals, foods, cosmetics and the like.
As a diabetes suppressant using a carotenoid, for example, Patent Document 1 discloses a carotenoid mixture containing fucoxanthin as a main component. Specifically, it discloses that fucoxanthin is an extract derived from microalgae and exhibits antidiabetic properties, and is desirable when combined with other carotenoids of nutritional value.
Non-Patent Document 1 discloses a preventive effect of lifestyle-related diseases by ingestion of astaxanthin derived from microalgae. Specifically, it has been reported that administration of astaxanthin to a mouse fed a high-fat diet suggested improvement in visceral fat mass, fasting blood glucose level, and insulin level of the mouse. .

Among the above carotenoids, as a characteristic carotenoid other than fucoxanthin and astaxanthin, diatoxanthin (CAS registration number: 31063-73-7) contained in microalgae such as phytoplankton, brown algae, and diatoms Exists.
Diatoxanthine is a type of xanthophyll and is a carotenoid having a unique structure having a triple bond in a polymer.
Diatoxanthin, like other carotenoids, is expected to have antioxidant activity, but it is relatively difficult to obtain in large quantities and it is very expensive, so its functionality is not well understood. Had not been.
Therefore, as carotenoids other than fucoxanthin and astaxanthin, it has been required to elucidate the function of a carotenoid mixture containing diatoxanthine as a main component and to elucidate the mechanism of functional expression, and further develop a method of using these substances.

JP-T-2018-512432

Yinhua Ni et al., “Astaxanthin prevents and reverses diet-induced insulin resistance and steatohepatitis in mice: A comparison with vitamin E '', Scientific Reports, 5: 17192 (2015)

The present invention has been made in view of the above problems, and an object of the present invention is to provide a novel blood glucose level increase inhibitor, a diabetes inhibitor, and a food composition.
Another object of the present invention is to provide a blood sugar increase inhibitor, a diabetes inhibitor, and a food composition, which are novel uses of a carotenoid mixture derived from microalgae containing diatoxanthin as a main component.

As a result of intensive studies, the present inventors have found that a carotenoid mixture derived from microalgae containing xanthophyll, particularly diatoxanthin as a main component, has an action of suppressing an increase in blood glucose level.

Therefore, according to the present invention, the object is to provide a carotenoid mixture derived from microalgae as an active ingredient, wherein the carotenoid mixture contains xanthophyll (excluding fucoxanthin and astaxanthin) as a main component. It is solved by a rise inhibitor.
At this time, it is preferable that the xanthophyll is diatoxanthin, and that the microalgae is present even if it has euglena.
In addition, the carotenoid mixture preferably contains diatoxanthin as a main component and one or more compounds selected from the group consisting of zeaxanthin and alloxanthin, and contains 5.0 to 80.0% of diatoxanthin. And good.
The carotenoid mixture preferably contains 10.0 to 80.0% of diatoxanthin, 2.0 to 20.0% of zeaxanthin, and 0.1 to 6.0% of alloxanthin.

In addition, when taken together with a high GI food, it may be used to suppress an increase in blood sugar level after ingesting the high GI food. In addition, it may be used to suppress an increase in blood glucose level by ingesting continuously for at least 6 weeks.
In the above, by ingesting the present carotenoid mixture together with a high GI food, it is possible to effectively suppress a rise in blood sugar level when the high GI food is ingested.
Here, “high GI food” means a food having a high GI (Glycemic Index), and “GI” is an index of a blood sugar level increasing speed after carbohydrate intake.
The higher the GI, the more rapidly the blood sugar level increases after ingestion of food. If the blood sugar level sharply increases, a large amount of insulin is rapidly secreted from the pancreas. The insulin converts blood sugar into energy while storing excess carbohydrates in adipose tissue. Therefore, high GI foods should be avoided as much as possible from the viewpoint of lifestyle-related diseases including diabetes.

The problem is also solved by a diabetes inhibitor comprising a carotenoid mixture derived from microalgae as an active ingredient, wherein the carotenoid mixture is mainly composed of xanthophylls (excluding fucoxanthin and astaxanthin). .
Further, the object is to contain a carotenoid mixture derived from microalgae as an active ingredient, and the carotenoid mixture contains xanthophyll (excluding fucoxanthin and astaxanthin) as a main component, for suppressing an increase in blood glucose level or suppressing diabetes. It is also solved by a food composition for use.
Further, the object is a method for producing a carotenoid mixture containing xanthophyll (excluding fucoxanthin and astaxanthin) as a main component for suppressing an increase in blood sugar level or suppressing diabetes, and has an ability to produce the carotenoid mixture. The present invention is also solved by a production method comprising a culturing step of culturing microalgae and an obtaining step of obtaining the carotenoid mixture from the microalgae obtained in the culturing step.

ADVANTAGE OF THE INVENTION According to this invention, a novel blood glucose level increase inhibitor, a diabetes inhibitor, and a food composition can be provided.
In addition, it is possible to provide a blood sugar increase inhibitor, a diabetes inhibitor, and a food composition, which are novel methods of using a carotenoid mixture derived from microalgae containing diatoxanthin as a main component.

It is a chromatogram of the HPLC quantitative analysis in the carotenoid mixture derived from Euglena (the 1). It is a chromatogram of the HPLC quantitative analysis in the carotenoid mixture derived from Euglena (the 2). It is a TLC analysis result of neutral lipid in a carotenoid mixture. It is a TLC analysis result of a polar lipid in a carotenoid mixture. It is the graph which compared the weight change at the time of giving the food of each group to a mouse for 6 weeks. It is the graph which compared the liver weight after giving the food of each group to a mouse for 6 weeks. It is the graph which compared the total cholesterol level in the blood after giving the food of each group to a mouse for 6 weeks. It is the graph which compared the glucose level in blood after giving the food of each group to a mouse for 6 weeks.

Hereinafter, embodiments of the present invention will be described with reference to FIGS.
The present embodiment contains a carotenoid mixture derived from microalgae as an active ingredient, and the carotenoid mixture contains xanthophyll (excluding fucoxanthin and astaxanthin) as a main component. , And food compositions.
Also, a method for producing a blood sugar rise inhibitor comprising a carotenoid mixture containing xanthophyll as a main component as an active ingredient, comprising: a culturing step of culturing microalgae having an ability to produce a carotenoid mixture; and a microalgae obtained. And a carotenoid mixture obtaining step of obtaining a carotenoid mixture from the product.

<Carotenoid mixture derived from microalgae>
“Microalgae” are single-cell or multicellular plants having a size of about several μm to several tens of μm that live mainly by performing photosynthesis in water.
Microalgae include cyanobacteria, green algae, Euglena, axle algae, flame algae, yellow algae, brown algae, red algae, diatoms, cobblestone algae, dinoflagellates, true eye algae and golden algae , Cyanobacteria, or treboxia.
In the present embodiment, it is desirable to use Euglena (Euglena), but it is not particularly limited.

“Euglena” includes all microorganisms, variants, and variants thereof classified into the genus Euglena according to the classification of zoology and botany.
Here, the microorganism of the genus Euglena refers to the euglenoids of the order Euglenida belonging to the order Euglenida belonging to the flagellate class (Mastigophorea) of the protozoa (Protozoa) and the subfamily Phytomastigophorea in zoology. It is a microorganism belonging to the order of the eye (Euglenoidina). On the other hand, in botany, it belongs to the order Euglenales belonging to the class Euglenaphyceae of the Euglena phylum (Euglenophyta).

As a microorganism of the genus Euglena (Euglena cells), E. gracilis (E. gracilis), in particular, E. gracilis Z strain, E. gracilis NIES-49 strain and the like can be used. it can. In addition, E. gracilis (E. gracilis) Z strain, a mutant strain of SM-ZK (chloroplast-deficient strain), a variant of var. β-1,3-glucanase may be used, and Euglena intermedia, Euglena piride, and other euglena may be used.
The genus Euglena is widely distributed in fresh water such as ponds and swamps, and may be used separately from them, or any genus Euglena that has already been isolated may be used.
In the present embodiment, it is preferable to use Euglena gracilis (E. gracilis) as Euglena, and it is particularly preferable to use Euglena gracilis Z strain or NIES-49 strain, but it is particularly limited. Not something.

"Euglena cell culture" can be performed using a culture solution. A carbon source is added to the culture solution as a nutrient source, and the carbon source includes an inorganic carbon source (CO ₂ , NaHCO ₃ , Na ₂ CO _3, etc.) and an organic carbon source (glucose, etc.). As the culture solution used in the present embodiment, it is preferable to use an autotrophic medium containing no organic carbon source such as glucose as a nutrient source, but it is not limited thereto. For example, a culture solution to which nutrients such as a nitrogen source, a phosphorus source, and minerals are added, for example, a Cramer-Myers medium or a modified Cramer-Myers medium ((NH ₄ ) ₂ HPO ₄ 1.0 g / L, KH ₂ PO _{4 1.0g / L, MgSO 4 · 7H} 2 O 0.2g / l, CaCl 2 · 2H 2 O 0.02g / l, Fe 2 (SO 2) 3 · 7H 2 O 3mg / l, MnCl 2 · 4H 2 O 1.8 _{mg / l, CoSO 4 · 7H} 2 O 1.5mg / l, ZnSO 4 · 7H 2 O 0.4mg / l, Na 2 MoO 4 · 2H 2 O 0.2mg / l, CuSO 4 · 5H 2 O 0.02g / l, Thiamine hydrochloride (vitamin B1) 0.1 mg / l, cyanocobalamin (vitamin B12), (pH 3.5) can be used. Note that (NH ₄ ) ₂ HPO ₄ can be converted to (NH ₄ ) ₂ SO ₄ or NH ₃ aq.

The pH of the culture solution is preferably 2 or more, and the upper limit thereof is preferably 6 or less, more preferably 4.5 or less. By setting the pH to the acidic side, the photosynthetic microorganism can grow more dominantly than other microorganisms, so that contamination can be suppressed.
Culture of Euglena cells may be performed by an open pond method using sunlight directly, or a light collecting method of sending sunlight condensed by a light condensing device through an optical fiber and irradiating it in a culture tank and using it for photosynthesis.
Further, the cultivation of Euglena cells can be performed using, for example, a feed batch method. However, cultivation using a flask culture or a fermenter, batch culture method, semi-batch culture method (fed-batch culture method), continuous culture method (perfusion) Culturing method) or any other liquid culturing method.
Separation of Euglena cells is performed, for example, by centrifugation or simple sedimentation of the culture solution.

The “carotenoid mixture” is a mixture containing one type of xanthophyll as a main component and one or more compounds selected from the group consisting of other compounds in xanthophyll as an auxiliary component.
"Xanthophyll" is a general term for pigments containing oxygen in the form of a hydroxyl group, a carbonyl group or an epoxide group among carotenoids, specifically, anthraxanthin, astaxanthin, canthaxanthin, citranaxanthin, β-cryptoxanthin , Diazinoxanthin, diatoxanthin, dinoxanthine, flavoxanthin, fucoxanthin, lutein, neoxanthine, rhodoxanthin, rubixanthin, violaxanthin, and zeaxanthin are included in the xanthophyll.

The carotenoid mixture of the present embodiment is, specifically, a mixture containing diatoxanthin as a main component and one or more compounds selected from the group consisting of zeaxanthin and alloxanthin as a subcomponent, more preferably It is preferable that the mixture contains both zeaxanthin and alloxanthin as accessory components.
Note that the carotenoid mixture may further contain other carotenoids (xanthophylls).

“Diatoxanthin” is a carotenoid having a structure having a triple bond in a polymer as shown in the following formula (Chem. 1) (Diathoxanthin, CAS registration number: 31063-73-7, chemical formula C ₄₀ H) ₅₄ O ₂ , molecular weight: 566.87).
Diatoxanthine is contained in microalgae such as phytoplankton, brown algae, and diatoms, and is obtained from Euglena in the present embodiment.

"Zeaxanthin" is a carotenoid having a structure represented by the following formula (Chemical Formula 2) (Zeaxanthin, CAS registration number: 144-68-3, chemical formula C ₄₀ H ₅₄ O ₂ , molecular weight: 568.89), and is edible. It is contained in bacteria and algae such as Spirulina in addition to vegetables, and is a component that is consumed in large quantities in daily diet.
Zeaxanthin is used for coloring foods, and is said to have an antiobesity effect, an anticancer effect, and an antioxidant effect of its derivatives.

“Alloxanthin” is a carotenoid having a structure having two triple bonds in a polymer as shown in the following formula (Formula 3) (Alloxanthin, CAS Registry Number: 28380-31-6, Chemical Formula C) ₄₀ H ₅₂ O ₂ , molecular weight: 564.85), contained in cryptoalgae.

In the carotenoid mixture of the present embodiment, as a ratio of a peak area in a chromatogram in a liquid chromatography quantitative analysis using a detector having a detection wavelength of 450 nm, diatoxanthine is determined based on 100% of the total weight of the carotenoid mixture. It is preferable to contain 5.0 to 80.0%, zeaxanthin 1.0 to 20.0%, and alloxanthin 0.1 to 10.0%.
Further, it is preferable that diatoxanthin is contained in 10.0 to 80.0%, zeaxanthin is contained in 2.0 to 20.0%, and alloxanthin is contained in 0.1 to 6.0%.

<Method for producing carotenoid mixture>
The carotenoid mixture of the present embodiment is a `` culturing step '' for culturing Euglena under the above culture conditions, a `` separation step '' for separating Euglena, a `` crushing step '' for crushing the separated Euglena, and an extraction solvent for crushing Euglena. A "dispersion step" to extract the carotenoid extract from the dispersed euglena, an "extraction step", and an "acquisition step" to obtain a carotenoid fraction containing diatoxanthine (a carotenoid mixture) from the carotenoid extract, It is manufactured by a manufacturing method of performing.

First, in the culture step (step S1), Euglena is cultured under the above culture conditions. Specifically, Euglena is cultured under independent culture conditions.
Next, in the separation step (Step S2), the Euglena cultured in the above-described culture step is separated. The separation of Euglena is specifically performed by a known separation method such as centrifugation or simple sedimentation of a culture solution, or membrane filtration, but is not particularly limited.
After the separated Euglena is washed, it may be dried by a known drying method (vacuum freeze-drying, spray drying, heating vacuum drying, etc.) to prepare a dried alga body of Euglena.

Next, in the pulverizing step (step S3), the Euglena separated in the separating step is pulverized by a known pulverizing method. Specifically, a method of adding an extraction solvent to the separated Euglena and pulverizing it with an ultrasonic crusher or the like can be mentioned, but is not particularly limited.
Next, in the dispersing step (step S4), the Euglena cells crushed in the crushing step are dispersed in an extraction solvent to obtain a dispersion.
As the extraction solvent used in the dispersion step, specifically, a lipophilic polar solvent such as acetonitrile can be used.

Next, in the extraction step (Step S5), a carotenoid extract is extracted from the euglena dispersed in the extraction solvent in the dispersion step.
The extraction conditions are not particularly limited, but it is also possible to promote the extraction of carotenoids by heating, applying an external stimulus such as ultrasonic waves, or shaking.

Finally, in the obtaining step (Step S6), a carotenoid fraction containing diatoxanthin (a carotenoid mixture) is obtained from the carotenoid extract obtained in the extraction step.
As a method for fractionation, specifically, by a suitable separation means (for example, reverse or normal phase high performance liquid chromatography partition extraction, gel filtration, silica gel chromatography, etc.), the diatoxanthine content is high. Examples of the method include fractionation of fractions. Alternatively, there is a method in which a carotenoid extract containing diatoxanthine is filtered, separated into an extract and a residue, and the extract is concentrated and fractionated by liquid chromatography.

<Use, usage and dosage>
The carotenoid mixture of the present embodiment can be used as a therapeutic agent for diabetes (particularly type 2 diabetes) by being administered to diabetic patients and animals other than humans who have developed diabetes.
In addition, a diabetes preventive agent (particularly a type 2 diabetes preventive agent) targeting humans before the onset of diabetes, humans with reserve diabetes, humans with fasting hyperglycemia, humans with postprandial hyperglycemia, and non-human animals, It can also be used as a diabetes inhibitor (particularly a type 2 diabetes inhibitor).
In addition, it can be used as a gestational diabetes preventive agent or a gestational diabetes inhibitor for pregnant women.
In addition, it can be used as a preventive agent for complications associated with diabetes, an inhibitor for complications, or a therapeutic agent for complications.

The carotenoid mixture of the present embodiment can be used as a blood sugar rise inhibitor.
Preferably, it can be used as a blood sugar level rise inhibitor that suppresses a rise in blood sugar level by ingesting it simultaneously with food (particularly GI food).
In addition, preferably, it can be used as a blood sugar level rise inhibitor that suppresses a rise in blood sugar level by ingesting continuously for a certain period of time.

The carotenoid mixture of the present embodiment can be used as a composition such as a pharmaceutical composition, a food composition, or the like for suppressing an increase in blood sugar level or diabetes.
In the field of medicine, a pharmaceutical composition having the action is provided by mixing a carotenoid mixture in an amount capable of effectively exerting the action with a pharmaceutically acceptable carrier or additive. The pharmaceutical composition may be a drug or a quasi drug.
The pharmaceutical composition may be applied internally or externally, and may be applied internally, such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection and / or intraperitoneal injection, transmucosal application, and the like. It can be used in the form of a formulation such as a transdermal application agent.
Examples of the dosage form of the pharmaceutical composition include solid preparations such as tablets, granules, capsules, powders, and powders, and semi-solid preparations such as liquid preparations, ointments, and gels.

In the field of food, an effective amount of a carotenoid mixture capable of exhibiting a blood sugar level-inhibiting action or a diabetes-inhibiting action in vivo is incorporated into various foods as a food material to provide a food composition having the action. it can.
That is, the present invention can provide a food composition indicated as a blood sugar level increase inhibitory action or the like in the food field. Examples of the food composition include general foods, foods for specified health use, nutritional functional foods, functionally labeled foods, foods for hospital patients, supplements, and the like. It can also be used as a food additive.
Examples of the food composition include seasonings, processed meat, processed agricultural products, drinks (soft drinks, alcoholic drinks, carbonated drinks, milk drinks, fruit drinks, tea, coffee, nutritional drinks, etc.), powdered drinks (powder Juices, powdered soups, etc., concentrated beverages, confectionery (candy (throat candy), cookies, biscuits, gum, gummy, chocolate, etc.), bread, cereals and the like. In the case of a food for specified health use, a nutritionally functional food, a functionally labeled food, and the like, the form may be a capsule, a troche, a syrup, a granule, a powder, or the like.

“Specified health foods” are foods that contain health functional ingredients that affect physiological functions, etc., and that can be indicated to be suitable for specific health uses with the permission of the Commissioner of the Consumer Affairs Agency. It is.
“Nutrient functional food” is a food used for replenishment of nutritional components (vitamin and mineral), and indicates the function of the nutritional component.
“Functionally labeled food” is a food product that claims its functionality based on scientific evidence at the responsibility of the business operator. Prior to sale, information on the basis of safety and functionality was notified to the Commissioner of Consumer Affairs Agency.

The dose of the blood sugar increase inhibitor and the diabetes inhibitor according to the present embodiment is, for example, that a human or non-human animal is orally ingested so as to have a predetermined intake at the same time as food (especially GI food). good.

<Example 1>
A carotenoid mixture from Euglena was prepared by the following procedure.
1) 20 L of acetonitrile was added to 20 kg of Euglena gracilis powder (manufactured by Euglena Co., Ltd.), and the mixture was crushed with an ultrasonic crusher to obtain a crushed extract mixture.
2) The crushed extract mixture was filtered to separate into extract and residue.
3) The extract was concentrated using a rotary evaporator and fractionated by medium pressure liquid chromatography. The fractionation was performed at a detection wavelength of 270 nm using a liquid chromatography apparatus, an ODS column (manufactured by GL Sciences Inc., InertStain C18) as the column, and using acetonitrile as an eluent.
4) A carotenoid fraction containing diatoxanthine was collected from each fractionated fraction using HPLC (high performance liquid chromatography), and concentrated with an evaporator.
By the above method, 0.4 g of a reddish brown powder which was a carotenoid fraction containing diatoxanthin was obtained. The powder was used as a carotenoid mixture.

<Test 1: HPLC quantitative analysis of carotenoid mixture>
Quantitative analysis of the carotenoid mixture of Example 1 was performed using HPLC under the following conditions.
0.1 g of the carotenoid mixture was dissolved in acetonitrile, and the solution was filtered and used as a measurement sample solution.
A high performance liquid chromatography apparatus was used, a pump used was LC-6AD (manufactured by Shimadzu Corporation), a detector was a PDA detector (SPD-M20A), and a column was an ODS column (Tosoh Corporation, TSKgel ODS-80Ts).
As a mobile phase, the liquid A is a mixed solvent of acetonitrile, methanol and water (a mixed solvent mixed at a volume ratio of 75:15:10), and the liquid B is a mixed solvent of ethyl acetate and methanol (a mixed solvent of 70:30 by volume). Mixed solvent) was used.
The measurement was performed at a column temperature of 40 degrees, a flow rate of 1.0 ml / min, and a detection wavelength of 450 nm.

As test results of Test 1, chromatograms of Example 1 are shown in FIGS. 1 and 2 (n = 2).
The presence of zeaxanthin and alloxanthin in addition to diatoxanthin was estimated from the measurement of the ultraviolet-visible absorption spectrum, the mass spectrometry, and the retention time in the chromatogram.
The ratio (%) of each peak area corresponding to diatoxanthine, zeaxanthin and alloxanthine in the chromatogram in the quantitative analysis was determined.
As the ratio (%) of each peak area, the ratio (%) to the total 100% of the peak areas of all the detected components (all estimated to be carotenoids) was determined.
The chromatogram in FIG. 1 (Example 1-1) was as follows.
Peak number 1: Alloxanthin 0.4%
Peak number 2: diatoxanthine 13.3%
Peak number 3: Zeaxanthin 2.1%
The chromatogram in FIG. 2 (Example 1-2) was as follows.
Peak number 1: Alloxanthin 5.6%
Peak number 2: diatoxanthine 75.9%
Peak number 3: Zeaxanthin 18.4%
In the following tests, the carotenoid mixture of Example 1-1 was used.

<Test 2: TLC analysis of carotenoid mixture (neutral lipid)>
Using TLC (thin layer chromatography), TLC analysis of neutral lipids in the carotenoid mixture of Example 1 was performed in the following procedure.
A carotenoid mixture (0.01 mg / ml, 0.1 mg / ml, 1 mg / ml) and a standard (oleic acid, triolein, cholesterol palmitate, cholesterol, diatoxanthin, zeaxanthin) were spotted on silica gel plates, respectively.
It was developed using a mixed solvent of hexane, diethyl ether and acetic acid (a mixed solvent mixed at a volume ratio of 65: 35: 1).
After the photograph was taken with a digital camera, the silica gel plate was immersed in a copper sulfate coloring solution, and heated at 180 ° C. for 5 to 10 minutes using a hot plate to develop a color.
In addition, the mass of the carotenoid mixture was quantified by comparing with a known amount of a standard diatoxanthine standard, and the mass (mg / ml) of diatoxanthin per 1 L of a liquid medium was used.

The TLC analysis result of Test 2 is shown in FIG. From the TLC analysis results before and after the coloring of the copper sulfate in each sample, it was found that there was no neutral lipid contamination in the carotenoid mixture.

<Test 3: TLC analysis of carotenoid mixture (polar lipid)>
Using TLC (thin layer chromatography), TLC analysis of polar lipids in the carotenoid mixture of Example 1 was performed in the following procedure.
A carotenoid mixture (0.01 mg / ml) and a standard (phosphatidylcholine, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, diatoxanthin, zeaxanthin) were respectively spotted on a silica gel plate.
It was developed using a mixed solvent of chloroform, methanol and water (a mixed solvent mixed at a volume ratio of 64: 16: 2).
After the photograph was taken with a digital camera, the silica gel plate was immersed in a copper sulfate coloring solution, and heated at 180 ° C. for 5 to 10 minutes using a hot plate to develop a color.

The TLC analysis result of Test 3 is shown in FIG. From the TLC analysis results before and after the coloring of copper sulfate in each sample, it was found that there was no contamination of the carotenoid mixture with polar lipids.
The chromatogram of Example 1 is shown in FIG.

<Test 4: Effect of carotenoid mixture using high fat diet mice>
When the carotenoid mixture was orally ingested with the feed composition shown in Table 1 below, the “weight change”, “liver weight”, “total cholesterol in blood”, and “amount of glucose in blood” were measured. A test to measure was performed.
In this test, 18 C57BL / 6J mice (male, 6 weeks old, weight: 20-23 g) were used (n = 6). The breeding conditions were set at a room temperature of 24 ± 2 degrees and a light-dark cycle of 12 hours.
The mice were first allowed free access to solid feed and water, and were adapted to the rearing environment by preliminary rearing for 10 days. After completion of the preliminary breeding, 1) AIN93G basic diet group (hereinafter, “control group”), 2) “high fat diet group” containing 30% by weight of lipid in the basic diet, according to the feed composition shown in Table 1 below, 3) The carotenoid mixture was divided into a total of three groups of “high fat diet + carotenoid group” containing 0.04% by weight of the carotenoid mixture.

※合計の値は、四捨五入することで１０００．００とした。
　飼料中のカロテノイド混合物は、以下の手順で大豆油に溶解させて配合した。
　カロテノイド混合物をナスフラスコにエタノールとともに入れて溶解させた。７０度に加熱した精製大豆油に第３ブチルヒドロキノンを溶解させ、常温に戻した後、エタノールに溶解させたカロテノイド混合物とよく混合させた。ロータリーエバポレーターを用いてエタノールを留去し、ナスフラスコに付着したカロテノイドをエタノールで再融解し、カロテノイドが完全に精製大豆油に混ざるまで繰り返した。更に、精製大豆油を窒素ガスでバブリングしてエタノールを完全に除去し、飼料にカロテノイド混合物を混ぜ込んだ。

* The total value was rounded off to 1000.00.
The carotenoid mixture in the feed was dissolved in soybean oil and blended in the following procedure.
The carotenoid mixture was dissolved in an eggplant flask together with ethanol. Tertiary butyl hydroquinone was dissolved in purified soybean oil heated to 70 ° C., returned to room temperature, and then mixed well with a carotenoid mixture dissolved in ethanol. Ethanol was distilled off using a rotary evaporator, and the carotenoid attached to the eggplant flask was redissolved with ethanol, and the process was repeated until the carotenoid was completely mixed with the purified soybean oil. Further, the refined soybean oil was bubbled with nitrogen gas to completely remove ethanol, and the feed was mixed with a carotenoid mixture.

Body weight was measured daily during the breeding period. At the end of the 6-week test period, the mice that had been fasted overnight were laparotomized under isoflurane anesthesia, the liver was collected, and blood was collected from the inferior vena cava.
After washing the collected liver with physiological saline, the liver weight was measured.
In addition, the blood obtained was centrifuged (2000 rpm, 15 minutes, 4 degrees), and the obtained blood was collected and stored at 80 degrees. Then, the total cholesterol level and glucose level in blood were quantified using a quantification kit (manufactured by Wako Pure Chemical Industries, Ltd.).

As a test result, "weight change", "liver weight", "total blood cholesterol level" and "blood glucose level" were compared between the control group, the high fat diet group, and the high fat diet + carotenoid group, respectively. The resulting graphs are shown in FIGS. 5, 6, 7, and 8. In addition, each numerical value is shown in Table 2 below.
The unit “mg / dL” means total cholesterol value (mg) and glucose value (mg) per 1 dL of blood.

Regarding the body weight during the breeding period, the mice in the “high-fat diet group” and the “high-fat diet + carotenoid group” tended to have a higher body weight than the “control group” for almost the entire period. The body weight of the “high fat diet + carotenoid group” tended to be lower than that of the “high fat diet group”.
In addition, regarding the body weight after the breeding period of 6 weeks, it was confirmed that the weight of the mice in the “high fat diet group” and the “high fat diet + carotenoid group” was significantly increased as compared with the “control group”. (P <0.05). The body weight of the “high fat diet + carotenoid group” tended to be lower than that of the “high fat diet group”.

肝 臓 Regarding the liver weight after the breeding period of 6 weeks, the liver weight of the mice in the “high fat diet group” tended to be higher than that in the “control group”. On the other hand, the liver weight of the “high-fat diet + carotenoid group” did not increase as compared to the “control group”, and tended to be equivalent.

Regarding the blood total cholesterol level after 6 weeks of breeding, the total blood cholesterol level of mice in the “high fat diet group” and the “high fat diet + carotenoid group” tended to be higher than in the “control group”. It was observed. In addition, the total cholesterol level in the blood of the “high fat diet + carotenoid group” tended to be lower than that of the “high fat diet group”.

Regarding the blood glucose level after the breeding period of 6 weeks, it was found that the blood glucose level of the mice in the “high fat diet group” was significantly higher than that in the “control group” (P <0. 05). On the other hand, the blood glucose level of the “high fat diet + carotenoid group” was not increased as compared with the “control group”, and was found to be equivalent (P <0.05).

From the above, based on the change in body weight during the breeding period, and the test results of body weight, liver weight, and total blood cholesterol level after 6 weeks of the breeding period, mice were fed a high fat diet (eg, high GI food). Orally ingesting the carotenoid mixture of Example 1 to suppress the increase in body weight (particularly body fat), liver weight (particularly liver fat), and blood cholesterol level after ingesting a high fat diet. .

Also, from the test results of the blood glucose level after the breeding period of 6 weeks, it was found that the carotenoid mixture of Example 1 was orally ingested together with the high-fat diet to the mice to increase the blood glucose level after the high-fat diet. It was found that there was an inhibitory action.
The “blood glucose level” means the glucose concentration in blood, and is a value indicating how many mg of glucose is contained in 1 dL of blood, and corresponds to the blood glucose level in this test.

Claims (11)

Contains a carotenoid mixture derived from microalgae as an active ingredient,
The carotenoid mixture contains xanthophyll (excluding fucoxanthin and astaxanthin) as a main component, and a blood sugar rise inhibitor. 剤 The blood sugar rise inhibitor according to claim 1, wherein the xanthophyll is diatoxanthin. The blood sugar level increase inhibitor according to claim 1 or 2, wherein the microalgae is euglena. The blood sugar level according to any one of claims 1 to 3, wherein the carotenoid mixture contains diatoxanthin as a main component and one or more compounds selected from the group consisting of zeaxanthin and alloxanthin. Rise inhibitor. 剤 The blood sugar increase inhibitor according to any one of claims 1 to 4, wherein the carotenoid mixture contains 5.0 to 80.0% of diatoxanthin. The carotenoid mixture contains 10.0 to 80.0% of diatoxanthin, 2.0 to 20.0% of zeaxanthin, and 0.1 to 6.0% of alloxanthin. 6. The blood sugar level increase inhibitor according to any one of 5. The blood sugar level increase inhibitor according to any one of claims 1 to 6, which is used for suppressing a rise in blood sugar level when taken together with a high GI food. 剤 The blood sugar level increase inhibitor according to any one of claims 1 to 7, which is used to suppress an increase in blood sugar level by ingesting continuously for at least 6 weeks. Contains a carotenoid mixture derived from microalgae as an active ingredient,
A diabetes suppressant, wherein the carotenoid mixture contains xanthophyll (excluding fucoxanthin and astaxanthin) as a main component. Contains a carotenoid mixture derived from microalgae as an active ingredient,
A food composition for suppressing an increase in blood sugar level or suppressing diabetes, wherein the carotenoid mixture contains xanthophyll (excluding fucoxanthin and astaxanthin) as a main component. Xanthophyll (fucoxanthin, excluding astaxanthin) as a main component, a method for producing a carotenoid mixture for suppressing blood sugar rise or diabetes,
A culturing step of culturing microalgae having the ability to produce the carotenoid mixture,
An obtaining step of obtaining the carotenoid mixture from the microalgae obtained in the culturing step.

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