JP2022077034A - Hibiscus hamabo extract, cosmetics containing the hibiscus hamabo extract and functional food - Google Patents

Abstract

To reduce aging troubles: rise in a blood glucose level, excessive ingestion of lipid, pigmentation of a skin and lost resilience of the skin, with a single plant-derived extract.SOLUTION: A Hibiscus hamabo extract comprises a component group showing solubility in an aqueous solvent made chiefly of water or a nonaqueous specific solvent composed of a liquid mixture of one or more of dimethyl sulfoxide, ethanol, methanol, and hexane, which at least comprises a hyperglycemia inhibitory component, a fat absorption inhibitory component, a melanogenesis inhibitory component and a collagen production promoting component. In some embodiments, the component group comprises a component group showing solubility in the aqueous solvent and further comprises a hypertension inhibitory component.SELECTED DRAWING: Figure 1

Description

The present invention relates to a Hibiscus hamabo extract, a cosmetic containing the Hibiscus hamabo extract, and a functional food.

Conventionally, various products such as medicines, cosmetics, and functional foods have been provided in order to lead a healthy and beautiful daily life.

Especially in recent years, in response to the growing health and beauty consciousness of consumers, each manufacturer is searching for new materials that can be used as raw materials for products.

Above all, plant resources still have ample room for development as a search target for new materials, and are considered to continue to contribute significantly to our lives in terms of health and beauty (see, for example, Patent Document 1). .).

Japanese Unexamined Patent Publication No. 2019-201594

By the way, as humans get older, various changes appear in their physical condition. Above all, chronic increase in blood glucose level leads to diabetes and causes complications such as neuropathy and retinopathy, so it is desirable to keep in mind in daily eating habits.

In addition, when blood lipids are high, it causes dyslipidemia, which increases the risk of causing ischemic heart disease and cerebral infarction.

In addition, as the age increases, the skin quality of the face and hands also changes. For example, pigmentation is likely to occur, minor skin problems may cause blemishes, and skin elasticity (tension) may be lost.

Therefore, in order to deal with these problems, various plant-derived materials and extracts (hereinafter, also simply referred to as plant materials) have been proposed so far.

However, for the plant materials known so far, in order to realize each function such as blood sugar, blood lipids, pigmentation and improvement of skin tension, plant materials suitable for each purpose are added. I needed it.

The present invention has been made in view of such circumstances, and extracts blood glucose levels and lipids, which are often anxious with aging, as well as skin pigmentation and improvement of elasticity from a single plant. It was completed to be realized by things.

In order to solve the above-mentioned conventional problems, in the present invention, the present invention comprises a water-based solvent containing water as a main component or a non-mixed solution consisting of any one or a mixture of two or more selected from dimethylsulfoxide, ethanol, methanol and hexane. A Hamabou extract was prepared by comprising a group of components exhibiting solubility in a specific aqueous solvent and containing at least a component for suppressing increase in blood glucose level, a component for suppressing fat absorption, a component for suppressing melanin production, and a component for promoting collagen production.

Further, the Hibiscus hamabo extract according to the present invention is also characterized in that the component group comprises a component group exhibiting solubility in an aqueous solvent and further contains a component for suppressing an increase in blood pressure.

The present invention also provides cosmetics and functional foods containing the above-mentioned Hibiscus hamabo extract.

In addition, the functional food according to the present invention contains 1.0 x 10 ⁵ lactic acid bacteria cells of at least one genus selected from the genus Lactobacillus, Lactococcus, Enterococcus, and Bifidobacterium per 0.1 g of the dried product of the Hamabou extract. It is also characteristic that it is added as described above.

Further, in the present invention, the solubility is made in an aqueous solvent containing water as a main component or a non-aqueous specific solvent composed of a mixture of any one or two or more selected from dimethylsulfoxide, ethanol, methanol and hexane. The Hamabou extract consisting of the indicated component group acts or has an effect on at least one event selected from the suppression of blood glucose level increase, the suppression of fat absorption, the suppression of melanin production, the promotion of collagen production, and the suppression of blood pressure increase derived from the Hamabou extract. In the functional food for the purpose of including, it was decided to use the constituent component of the Hamabou extract corresponding to the occurrence of the event as a raw material of the functional food containing the health functional component (participating component).

Further, in the present invention, the solubility is made in an aqueous solvent containing water as a main component or a non-aqueous specific solvent composed of a mixture of any one or two or more selected from dimethylsulfoxide, ethanol, methanol and hexane. Expression of the above-mentioned event in a cosmetic for the purpose of comprising at least one event of suppressing melanin production or promoting collagen production derived from the Hamabou extract as an action or effect of the Hamabou extract consisting of the indicated component group. It was decided to use it as a raw material for cosmetics containing the constituent component of the Hamabou extract corresponding to the above as an active ingredient.

Further, in the present invention, in order to sensually reverse the astringency and sweetness of the Hibiscus hamabo extract, lactic acid bacteria cells were used as an additive for improving the taste of the Hibiscus hamabo extract.

According to the present invention, it is soluble in an aqueous solvent containing water as a main component or a non-aqueous specific solvent consisting of one or a mixture of two or more selected from dimethylsulfoxide, ethanol, methanol and hexane. The Hamabou extract contains at least a blood glucose level increase inhibitory component, a fat absorption inhibitory component, a melanin production inhibitory component, and a collagen production promoting component. Ingestion of lipids and improvement of skin pigmentation and elasticity can be achieved with a single plant-derived extract called Hamabou.

Further, the component group is composed of a component group exhibiting solubility in an aqueous solvent, and if it further contains a component for suppressing an increase in blood pressure, the blood pressure can be further realized by a single plant-derived extract called Hamabou. Can be done.

In addition, if these Hibiscus hamabo extracts are included in cosmetics and functional foods, the use of these extracts can suppress the above-mentioned blood glucose level, lipids, and skin pigmentation and improve elasticity.

Further, by adding lactic acid bacteria cells to the Hibiscus hamabo extract, it is possible to provide a functional food with improved taste.

It is explanatory drawing which shows the α-glucosidase inhibitory activity. It is explanatory drawing which shows the lipase production inhibitory activity. It is explanatory drawing which shows the ACE inhibitory activity rate. It is explanatory drawing which shows the melanin production rate per cell. It is explanatory drawing which shows the collagen production ratio.

The present invention relates to a Hibiscus hamabo extract, and is particularly characteristic of a Hibiscus hamabo extract in that it contains a component extracted with a predetermined solvent.

As used herein, Hamabou means a plant whose scientific name is known as Hibiscus hamabo. It should be noted that although Hibiscus tiliaceus (scientific name: Talipariti tiliaceum) has a similar name, it is a completely different plant from the Hibiscus tiliaceae, which is the subject of the present invention. Hibiscus tiliaceus is a deciduous shrub with a height of about 1 to 5 m, while Hibiscus tiliaceus is an evergreen small tree with a height of 4 to 12 m. ..

The Hamabou extract according to the present embodiment is an extract extracted with an aqueous solvent or a specific non-aqueous solvent (also referred to as a non-aqueous specific solvent).

The water-based solvent is a solvent containing water as a main component, and in addition to water itself, those to which alcohols or salts showing compatibility with water are added are also water-based as described in the present specification if the ratio of water is 50% or more. Corresponds to the solvent.

As the non-aqueous specific solvent, a mixed solution of any one or any two or more selected from dimethyl sulfoxide, ethanol, methanol and hexane occupies 80 w / w% or more, more preferably 90 w / w% or more of the composition. A solvent, more preferably a solvent consisting of any one or a mixture of any two or more.

The extraction target site of Hibiscus hamabo is not particularly limited, and for example, leaves, branches, stems, seeds, flowers, roots, other sites, or a mixture of any part or all of these sites can be targeted.

The Hamabou extract according to the present embodiment has a solubility in the above-mentioned aqueous solvent-soluble component group (hereinafter, also referred to as an aqueous component group) or a non-aqueous specific solvent among the constituent components of Hamabou. It consists of the indicated component groups (hereinafter, also referred to as non-aqueous component groups), and in each case, it contains at least a blood glucose level increase inhibitory component, a fat absorption inhibitory component, a melanin production inhibitory component, and a collagen production promoting component. It is characteristic in terms of points.

The component for suppressing the increase in blood glucose level is preferably, for example, a component that inhibits the activity of α-glucosidase in the aqueous or non-aqueous component group, particularly a component that functions as an inhibitor of α-glucosidase present in small intestinal epithelial cells. Specifically, 100 μl of a 0.1 M sucrose aqueous solution at 37 ° C was prepared as a substrate, and 0.5 U (1 U is an active unit capable of digesting 1 μmol of the substrate into glucose at 30 ° C for 30 minutes) with α-glucosidase. , A solution of a dried product of an aqueous component group extracted from sucrose with a final concentration of 0.002, 0.02, 0.2, 0.4 mg / ml in water (hereinafter referred to as a water extract solution), or a non-aqueous component. When the concentration of sucrose, which is a substrate, can be maintained or suppressed when a solution in which the dried product of the group is dissolved in a predetermined solvent (hereinafter referred to as a non-aqueous extract solution) is added and reacted at 37 ° C. for 30 minutes. In addition, it can be interpreted that the aqueous or non-aqueous component group contains a component for suppressing an increase in blood glucose level.

The fat absorption inhibitor is, for example, a component that inhibits the activity of lipase. Specifically, 50 μl of a 0.1 mM 4-methylumbelliferyl oleate (4-MUO) solution at 37 ° C was prepared as a substrate, and 3.75 μg (1 U was 1 mmol 100% triolein at 37 ° C, pH 8.0, 2 hours). Water (or a given buffer) of pancreatic-derived lipase (an active unit that can be hydrolyzed and digested into diolein or oleic acid) and a dried product of an aqueous component group having a final concentration of 0.5, 5, 10 mg / ml. A solution in which a solution dissolved in water (hereinafter referred to as a water extract solution) or a solution obtained by dissolving a dried product of a non-aqueous component group in a predetermined solvent (hereinafter referred to as a non-aqueous extract solution) is added and 30 ° C. If the fluorescence intensity of 4-methylumbelliferone, which is the substrate when reacted for 1 minute, can be equal to or lower than that of orlistat (final concentration 0.005 mg / mL) used as a positive control, the aqueous or non-aqueous component is concerned. It can be interpreted that the group contains a fat absorption inhibitor.

The melanin production inhibitory component is a component capable of suppressing the production of melanin pigment in the cells constituting the skin. Specifically, the amount of melanin produced by melanin cells cultured in the presence of an aqueous extract solution or a non-aqueous extract solution and the amount of melanin in the absence of the extract solution are measured by absorbance at 405 nm. In comparison, when the amount of melanin produced is smaller than that in the absence, it can be interpreted that the aqueous or non-aqueous component group contains a melanin production inhibitory component.

The collagen production promoting component is a component capable of promoting collagen production in the cells constituting the skin. Specifically, the amount of collagen produced by human skin fibroblasts cultured in the presence of a water extract solution or a non-aqueous extract solution is compared with the amount of collagen in the absence of the extract solution. When the amount of collagen produced is larger than that in the absence, it can be interpreted that the aqueous or non-aqueous component group contains a collagen production promoting component.

According to the Hamabou extract according to the present embodiment having such a structure, the intake of blood sugar level and lipid, which are often anxious with aging, and the improvement of skin pigmentation and elasticity are single plants. It can be realized with the extract of origin.

Further, in the Hamabou extract according to the present embodiment, the component group comprises a component group exhibiting solubility in an aqueous solvent, that is, an aqueous component group, and may further contain a component for suppressing an increase in blood pressure.

Here, the blood pressure increase inhibitory component is a component capable of inhibiting the activity of angiotensin converting enzyme (ACE). Specifically, a method for detecting 3-Hydroxybutyric acid liberated from 3-Hydroxybutyryl-Gly-Gly-Gly by an enzymatic method, for example, using ACE Kit-WST manufactured by Dojin Chemical Research Institute, in the presence of a water extract solution. When the ACE activity is compared with the ACE activity in the absence of ACE, and the ACE activity is suppressed as compared with the absence, it can be interpreted that the aqueous component group contains a blood pressure increase inhibitory component.

According to the Hamabou extract according to the present embodiment having such a configuration, blood pressure can be further realized by a single plant-derived extract called Hamabo.

Further, the present application provides a cosmetic containing the Hibiscus hamabo extract according to the present embodiment. The present application also proposes the use of the Hamabou extract in cosmetics as a raw material for cosmetics in realizing such cosmetics.

Here, cosmetics generally include cosmetics with mild efficacy and effects used for the purpose of cleaning, beautifying, increasing attractiveness, keeping healthy, and also quasi-drugs such as so-called medicated cosmetics. include.

The cosmetic of the present application is a cosmetic for the purpose of including at least one of suppression of melanin production or promotion of collagen production derived from Hibiscus hamabo extract as an action or effect. In addition, when it is clearly stated that the purpose of the action or effect is to suppress melanin production or to promote collagen production in the cosmetic, it is described as an effect effect such as medicated cosmetics. Of course, even if it is not specified in this way, if a part of the process or an expression of the result is described, such as whitening the skin or giving tension to the skin. If so, it corresponds to the cosmetics of the present application.

The Hibiscus hamabo extract used in cosmetics consists of the above-mentioned aqueous or non-aqueous component group.

In addition, the use of the Hibiscus hamabo extract as a raw material for cosmetics is the use of the constituent components of the Hibiscus hamabo extract corresponding to the above-mentioned expression of suppression of melanin production or promotion of collagen production as an active ingredient. The active ingredient may be a part or all of the constituents of the Hibiscus hamabo extract. That is, the active ingredient may be an identified component or a plurality of identified components constituting the Hibiscus hamabo extract, or the Hibiscus hamabo extract itself may be the active ingredient.

With such a configuration, it is possible to provide a cosmetic that can realize skin pigmentation and improvement of elasticity with a single plant-derived extract called Hibiscus hamabo.

Further, the present application provides a functional food containing the Hibiscus hamabo extract according to the present embodiment. The present application also proposes the use of Hamabou extract as a raw material for functional foods in functional foods when realizing such functional foods.

Here, functional food is a concept that includes all foods that do not contain pharmaceutical ingredients and are considered to contribute to the maintenance and promotion of health. It also includes general foods, foods for specified health use, foods with nutritional function, and foods with health claims such as foods with functional claims.

In addition, the above-mentioned explanation about cosmetics and functional foods is a list of examples of articles and the like corresponding thereto for the understanding of the present invention, and the interpretation of each term is limited to these listed articles and the like. It is not something that will be done. However, it does not prevent the applicant from limiting the present invention to these articles, etc. in order to acquire the rights of the present application.

The functional food of the present application is intended to include at least one of suppression of blood glucose level increase, fat absorption suppression, melanin production suppression, collagen production promotion, and blood pressure increase suppression derived from Hamabou extract as an action or effect. It is a functional food. As for the purpose to be included as an action or effect, as in the case of the previous cosmetics, not only the case where these matters are described as the effect and efficacy, but also a part of the progress and the expression of the result are described. Including the case where it has been done.

The Hibiscus hamabo extract used in functional foods consists of the above-mentioned aqueous or non-aqueous component group.

In addition, the use of Hamabou extract as a raw material for functional foods is a component of Hamabou extract that corresponds to the above-mentioned suppression of blood glucose level increase, fat absorption suppression, melanin production suppression, collagen production promotion, and blood pressure increase suppression. It is used as a health functional ingredient (participating ingredient). The health functional component (participating component) may be a part or all of the components constituting the Hibiscus hamabo extract. That is, the health functional component (participating component) may be an identified component or a plurality of identified components constituting the Hibiscus hamabo extract, or the Hibiscus hamabo extract itself may be the active ingredient.

With such a configuration, it is possible to realize the intake of blood sugar level and lipid, suppression of blood pressure increase, and improvement of skin pigmentation and elasticity with a single plant-derived extract called Hamabou. Sex foods can be provided.

By the way, the Hamabou extract feels astringent and sweet when it is eaten, regardless of whether the extraction solvent is an aqueous solvent or a non-aqueous specific solvent. Although the two have different tastes, the astringent one feels a little stronger and the sweet one feels a little weaker.

Although the Hibiscus hamabo extract has such a sensual aspect, the present inventors have conducted diligent research so far to add lactic acid bacteria cells to the Hibiscus hamabo extract according to the present embodiment to obtain this astringency and sweetness. We have found that it is possible to reverse the strength of.

This will be explained later with reference to the experimental results, but it is not just that the astringency is unilaterally less likely to be felt and sensually reversed, but of course the astringency is less likely to be felt and the sweetness is strongly felt. It is known that they are sensually reversed from each other.

That is, the present application also proposes a hamabo extract preparation containing lactic acid bacteria cells in which the astringency and sweetness of the hamabo extract are sensually reversed.

The lactic acid bacterium added here may be a lactic acid bacterium that does not cause a hygienic problem even when ingested orally, and it is more desirable that the lactic acid bacterium is contained in conventional foods. Examples of such lactic acid bacteria include lactic acid bacteria belonging to the genus Lactobacillus, the genus Lactococcus, the genus Enterococcus, and the genus Bifidobacterium.

Lactobacillus属に属する乳酸菌としては、例えば、Lactobacillus acidophilus、Lactobacillus brevis、Lactobacillus casei、Lactobacillus delbrueckii subsp. bulgaricus、Lactobacillus delbrueckii subsp. delbrueckii、Lactobacillus delbrueckii subsp. lactis、Lactobacillus gasseri、Lactobacillus helveticus、Lactobacillus kimchii、Lactobacillus paracasei、Lactobacillus Examples include, but are not limited to, plantarum, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius.

Examples of lactic acid bacteria belonging to the genus Lactococcus include, but are not limited to, Lactococcus lactis (Lactococcus lactis subsp. Cremoris, Lactococcus lactis subsp. Lactis, etc.).

Examples of lactic acid bacteria belonging to the genus Enterococcus include, but are not limited to, Enterococcus faecalis and Enterococcus faecium.

Examples of lactic acid bacteria belonging to the genus Bifidobacterium include, but are not limited to, Bifidobacterium animalis subsp. Lactis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. Longum, and Bifidobacterium longum subsp. Infantis. ..

These lactic acid bacteria may be live bacteria or dead bacteria, may be a single strain or strain, or may be mixed-cultured (for example, a symbiotic relationship or a non-competitive relationship). It may be a culture of a plurality of lactic acid bacteria in the above.). In addition, it does not prevent the addition of another orally ingestible bacterium as long as the taste improving effect of the lactic acid bacterium is not lost.

Further, it is desirable that these lactic acid bacteria are contained in an amount of 1.0 × 10 ⁵ or more, more preferably 1.0 × 10 ⁶ or more, based on 0.1 g of the dried product of Hibiscus hamabo extract. The raw material of such a lactic acid bacterium may be, for example, one cultivated by milk fermentation, that is, yogurt or the like as a raw material, or may be cultivated and collected using another medium.

Then, a product obtained by adding lactic acid bacteria to the Hibiscus hamabo extract according to the present embodiment and sensually reversing the astringency and sweetness of the Hibiscus hamabo extract can be used as a food or a functional food.

In addition, the present application also provides a method of using the Hibiscus hamabo extract as a taste improving agent by adding lactic acid bacteria cells to sensually reverse the astringency and sweetness of the Hibiscus hamabo extract. It can be said that.

And, of course, the astringency of the Hibiscus hamabo extract is not so astringent that it can be ingested as a food, but according to this technology for adding lactic acid bacteria to the Hibiscus hamabo extract, it continues widely and continuously from children to the elderly. It is useful for ingesting foods containing Hamabou extract.

Hereinafter, the Hibiscus hamabo extract according to the present embodiment will be described with reference to a test on its production method and its influence on each viewpoint of blood glucose level, fat absorption, blood pressure, melanin production, and collagen production.

[1. Preparation of test sample]
Water extraction or ethanol (EtOH) extraction is performed on the leaves, branches, seeds, seed shells, and flowers of Hamabou collected from Hamabou that grows naturally on the coast, and further drying is performed to obtain a water extract or a water extract of each part. An ethanol extract was obtained.

Each part of Hibiscus hamabo was freeze-dried, crushed, and subjected to shaking extraction (200 rpm) for 24 hours using water or ethanol as an extraction solvent. After 24 hours, those using water as an extraction solvent were subjected to centrifugation, and the supernatant was freeze-dried to obtain a powder obtained as a water extract. Ethanol as an extraction solvent was concentrated in a rotary evaporator and then dried in a desiccator to obtain an ethanol extract. The water content, water extraction rate, and ethanol extraction rate of each part are shown in Table 1 below.

[2. α-Glucosidase activity inhibition test]
Diabetes is a disease in which hyperglycemia continues due to abnormal glucose metabolism, and it also causes various diseases. Lifestyle-related habits have a great influence on hyperglycemic conditions, and daily control of blood glucose levels is weight. α-Glucosidase is a complex of enzymes that hydrolyzes α-glucosidic bonds of sugars in small intestinal epithelial cells and is involved in the digestion and absorption of disaccharides. Here, in order to evaluate the blood glucose level suppressing effect, the α-glucosidase activity of each sample was measured.

The evaluated samples were 8 kinds of water extract and ethanol extract of leaves, water extract and ethanol extract of branches, water extract and ethanol extract of seeds, water extract and ethanol extract of seed shell. be.

The α-glucosidase activity was measured as follows. A 0.1 M sucrose solution, a 5 U / ml α-glucosidase solution, and a sample solution were separated by 100 μl each, mixed, and reacted at 37 ° C. for 30 minutes. Then, the reaction was stopped by heating for 10 minutes in a block incubator set at 100 ° C. Then, it was allowed to stand for 3 minutes, returned to room temperature, stirred, and then flushed with a centrifuge to collect droplets, and the glucose concentration was measured with a biosensor.

For all of the above eight types of sample solutions, prepare solutions with concentrations of 0.002 mg / ml, 0.02 mg / ml, 0.2 mg / ml, and 0.4 mg / ml, and the inhibitory activity (%) of α-glucosidase for each. Asked. The inhibitory activity of the water extract is shown in FIG. 1 (a), and the inhibitory activity of the ethanol extract is shown in FIG. 1 (b). DNJ (1-deoxynojirimycin) was provided as a positive control. DNJ is a component peculiar to morus alba and is known to strongly inhibit α-glucosidase and suppress sugar absorption.

As shown in FIG. 1 (a), as a result of measuring the α-glucosidase activity of the Hamabou water extract, in the Hamabou (leaves, branches, seeds, seed shells) water extract 0.2 mg / ml and 0.4 mg / ml. It showed a strong α-glucosidase inhibitory activity. Furthermore, it was confirmed that the α-glucosidase inhibitory activity was 64.2, 67.9 or 76.5% even with 0.02 mg / ml of a water extract of Hibiscus hamabo (leaves, branches, seed shells).

In addition, as shown in FIG. 1 (b), as a result of measuring the α-glucosidase activity of the Hamabou ethanol extract, the ethanol extract of Hamabou (leaves, branches, seeds, seed shells) was 0.2 mg / ml and 0.4. A strong α-glucosidase inhibitory activity was confirmed at mg / ml.

From the above results, it was shown that the water extract and ethanol extract of Hamabou (leaves, branches, seeds, seed shells) had α-glucosidase inhibitory activity and could be expected as a material for antidiabetes.

[3. Lipase activity inhibition test]
Lipase is an enzyme that breaks down fat and is secreted from the pancreas into the duodenum. Lipase is greatly involved in the digestion and absorption of fat.

Currently, the number of people suffering from diseases such as metabolic diseases such as diabetes and vascular diseases is rapidly increasing due to obesity, and it is urgent to solve obesity. Obesity has many factors such as environment and heredity, but the cause of obesity is that it consumes more energy than it consumes. Since pancreatic lipase is involved in fat absorption, substances that inhibit lipase activity are considered as one of the effective approaches to reduce energy intake and exert an anti-obesity effect.

In this test, the anti-lipase effect of each sample will be examined by evaluating the enzyme inhibitory activity of each sample against lipase.

The test method is to mix 25 μl of the test solution with 50 μl of 0.1 mM 4-Methylumbelliferyl oleate (4-MUO) (/ 13 mM Tris-HCl, 150 mM NaCl, 1.3 mM CaCl ₂ buffer) solution to a concentration of 150 μg / ml. 25 μl of porcine pancreas-derived lipase (Lipase from Porcine Pancreas L0057 manufactured by Tokyo Kasei Kogyo Co., Ltd.) was further added, and the reaction was carried out at 25 ° C. for 30 minutes.

After the reaction, 100 μl of 0.1 M citrate buffer (pH 4.2) was added to stop the reaction, and lipase acted on 4-MUO at an excitation wavelength of 360 nm to release 4-methylumbelliferone fluorescence (465 nm). ) Was measured, and the effect of the test solution on lipase activity was evaluated.

There are eight test solutions: water extract and ethanol extract of Hamabou leaves, water extract and ethanol extract of branches, water extract and ethanol extract of seeds, water extract and ethanol extract of seed shells. Therefore, solutions having concentrations of 0.5 mg / ml, 5 mg / ml, and 10 mg / ml were prepared, and the lipase production inhibition rate (%) was determined. Orlistat (final concentration 0.005 mg / ml) was used as a positive control. Orl is a kind of obesity drug and is known to act on lipase in the intestine and eventually inhibit the absorption of fat from the intestinal tract.

FIG. 2A is a graph showing the lipase production inhibition rate (%) of the water extract. As can be seen from FIG. 2 (a), in any of the leaves, branches, seeds, and seed shells, the high concentration sample at a concentration of 10 mg / ml has a strong inhibition of lipase production equal to or higher than that of orlistat. The rate was confirmed. In particular, the water extract of the branch was confirmed to have a lipase inhibition rate higher than that of orlistat at any of the concentrations of 0.5 mg / ml, 5 mg / ml, and 10 mg / ml. In addition, with respect to the water extract of seeds, a lipase inhibition rate similar to that of orlistat was confirmed at any of the tested concentrations.

FIG. 2B is a graph showing the lipase production inhibition rate (%) of the ethanol extract. Similar to the water extract, except for the ethanol extract of seeds, lipase production inhibition rate (%) similar to that of orlistat was confirmed at high concentrations of 5 mg / ml and 10 mg / ml.

From these results, it was shown that the water extract and the ethanol extract of the leaves, branches, seeds, seed shells, all had the effect of anti-lipase activity.

[4. Angiotensin converting enzyme activity inhibition test]
Angiotensin converting enzyme (ACE) produces angiotensin II, which has a pressor effect, from angiotensin I in the renin-angiotensin system, which is one of the blood pressure regulation mechanisms. At the same time, it is an enzyme that greatly contributes to the increase in blood pressure, such as degrading bradykinin, which is a hypotensive peptide.

In this study, the antihypertensive effect of each sample will be examined by evaluating the enzyme inhibitory activity of each sample against ACE.

The ACE activity test was performed using ACE Kit-WST (Dojin Kagaku Kenkyusho). This kit is a method for detecting 3-Hydroxybutyric acid (3HB) released from 3-Hydroxybutyryl-Gly-Gly-Gly (3HBGGG) by an enzymatic method.

The water extract was dissolved in ultrapure water to prepare a test solution, and the ethanol extract was dissolved in DMSO (Dimethyl sulfoxide) to prepare a test solution, which was subjected to an ACE activity test. The sample test solutions were prepared at concentrations of 125 μg / ml, 625 μg / ml, and 3125 μg / ml, respectively. As a positive control, Val-Tyr (sardine peptide) dissolved in ultrapure water or DMSO was used.

A 96-well plate was used for the measurement. 20 μl of test solution, 20 μl of Substrate buffer, and 20 μl of Enzyme working solution were added to the measurement wells of each sample. In addition, 20 μl of ultrapure water or DMSO, 20 μl of Substrate buffer, and 20 μl of Enzyme working solution were added to the wells of the blank (blank 1) without ACE inhibition. In addition, 40 μl of ultrapure water or DMSO and 20 μl of Substrate buffer were added to the wells of the reagent blank (blank 2).

It was then incubated at 37 ° C. for 60 minutes, then 200 μl of Indicator working solution was added and incubated at room temperature for 10 minutes.

Then, the absorbance at 450 nm was measured with a microplate reader, the ACE inhibitory activity rate (%) was obtained from the value, and the 50% inhibitory concentration (IC ₅₀ ) was further calculated. The ACE inhibitory activity rate (%) was determined according to the following formula (I).
ACE inhibitory activity rate (%) = [(A _blank1 -A _sample ) / (A _blank1 -A _blank2 )] × 100 (I)

FIG. 3A shows the ACE inhibitory activity rate (%) of the water extract. As shown in FIG. 3 (a), the ACE inhibitory activity rate at the highest concentration (3125 μg / ml) tested was 99.9% for Val-Tyr (positive control), 73.0% for leaf water extract, and branch water. The extract was 66.8%, the water extract of seeds was 86.6%, and the water extract of seed shells was 54.8%.

In Val-Tyr (positive control), the IC ₅₀ for ACE calculated from the absorbance value at 450 nm was 0.73 μg / ml. On the other hand, the water extract of leaves was 891.3 μg / ml, the water extract of branches was 952.1 μg / ml, the water extract of seeds was 194.9 μg / ml, and the water extract of seed shells was 2022.5 μg / ml. .. It was suggested that the enzyme inhibitory activity on ACE was highest in seeds and lowest in seed shells in water extract.

FIG. 3 (b) shows the ACE inhibitory activity rate (%) of the ethanol extract. As shown in FIG. 3 (b), the ACE inhibitory activity rate at the highest concentration (3125 μg / ml) tested was 90.0% for Val-Tyr (positive control), 26.3% for leaf ethanol extract, and branch ethanol. The extract was 26.3%, the seed ethanol extract was 15.0%, and the seed shell ethanol extract was 0%. Therefore, it was suggested that the inhibitory activity of the ethanol extract on ACE was weaker than that of the water extract.

From these results, it was suggested that the water extract of Hibiscus hamabo contains a component showing ACE inhibitory activity that can be expected to have an antihypertensive effect.

[5. Melanin production evaluation test]
Melanin is a general term for substances in which phenolic substances are polymerized into pigments. Spots are areas where melanin accumulates and looks darker than the background color. When the skin is stimulated by ultraviolet rays or the like, melanin pigment cells (melanocytes) that synthesize melanin inside the skin are activated, producing excessive melanin and causing spots.

On the other hand, the color of human hair is determined by the type and amount of melanin. For example, black hair contains a large amount of melanin, and white hair contains almost no melanin. Melanin is produced from tyrosine as a raw material, and melanin pigment is supplied to nearby hair matrix cells. If melanin is not supplied from melanocytes, the hair will become gray, so it is important for melanocytes to function normally in order to maintain healthy hair.

In this study, mouse melanoma cell line B16 was used to evaluate the effect of sample addition on melanin production.

The samples to be evaluated were water extract and ethanol extract of Hibiscus hamabo flower. Mouse melanoma cell lines (B16) were precultured with DMEM (low glucose, 1% penicillin-streptomycin and 10% fetal bovine serum (FBS)) in 75 cm ² flasks until confluent. Then, the seeds were sown in a 24-well plate at a concentration of 1.0 × 10 ⁵ cells / ml, and cultured overnight in a CO ₂ incubator (37 ° C, 5% CO ₂ ).

Next, water extract or ethanol extract of flowers was added to the mouse melanoma cell line after overnight culture so that the final concentration was 10 μg / ml, and the cells were cultured in a CO ₂ incubator for 72 hours. .. At the same time, DMSO was added as a control, and arbutin, which has the property of inhibiting melanin synthesis as a positive control, was added at a final concentration of 50 μg / ml.

In this test, cell viability was also measured. Cell viability was measured using the Cell Counting Kit-8 (Dojin Kagaku). To measure extracellular melanin production, 500 μl of DMEM (low glucose, 1% penicillin-streptomycin) was added to a 24-well plate from which the cell supernatant had been removed. Then, 50 μl of CCK-8 solution was added to each well, and the cells were statically cultured for 1 hour in a CO ₂ incubator. The cell supernatant was transferred to a 1.5 ml tube, mixed well, and then 100 μl each was transferred to a 96-well well plate, and the absorbance at 450 nm was measured with a microplate reader to calculate the cell viability.

For the measurement of intracellular melanin production, a 24-well plate from which the cell supernatant had been removed was washed with PBS, the cells were detached with a 0.25% trypsin-EDTA solution, and the cells were collected from each well. After centrifugation and washing, the cells were transferred to a 1.5 ml tube, centrifuged again, the supernatant was removed, and 120 μl of 1M NaOH solution was added. After reacting at 100 ° C. for 10 minutes, 100 μl of the suspension was transferred to a 96-well plate, and the absorbance at 405 nm was measured with a plate reader.

FIG. 4 is a graph showing the melanin production rate after adding a water extract or an ethanol extract of Hibiscus hamabo flowers. As can be seen from FIG. 4, a tendency to suppress melanin production was observed by adding a water extract of flowers as compared with the control (water). Also, in the case of the ethanol extract of flowers, a tendency to suppress melanin production was observed as compared with the control (DMSO).

From the above results, it was suggested that the water extract and the ethanol extract contain a component that suppresses melanin production and can be effective in preventing skin spots.

[6. Collagen production evaluation test]
Skin Fibroblasts in the dermis produce important constituents of the skin, such as hyaluronic acid and collagen. Collagen is a structural protein that forms connective tissues such as animal skin. Collagen is important for maintaining skin strength and keeping youthful, wrinkle-free skin.

If it is possible to promote collagen production in fibroblasts, it is considered that fresh and firm skin can be maintained. In this test, a test substance is added to human fibroblasts and changes in collagen production ability are measured to evaluate the skin's water retention effect and wrinkle prevention in the test substance.

The test used adult human skin fibroblasts (NHDF-Ad) as model cells. NHDF-Ad cells were precultured in a φ10 cm dish with Dulbecco's Modified Eagle Medium (DMEM) (high glucose) (including 1% penicillin-streptomycin and 10% fetal bovine serum (FBS)) until confluent. Then, it was washed with phosphate buffered saline (PBS), resuspended in the medium, and then seeded on a 96-well plate at a concentration of 0.5 × 10 ⁴ cells / well in a CO ₂ incubator (37 ° C, 5% CO ₂ ). Was cultured overnight in.

After overnight culture, the cells were replaced with a serum-free medium containing a sample (containing 1% penicillin-streptomycin) and cultured in a CO ₂ incubator for 72 hours. Samples were added at final concentrations of 10, 100 and 200 μg / ml.

The culture supernatant 72 hours after the sample addition was collected, and the amount of collagen produced was measured with a commercially available ELISA kit (Human Collagen Type I ELISA kit manufactured by Acel Co., Ltd.).

FIG. 5A is a graph showing the collagen production ratio 72 hours after the addition of the water extract of flowers. As shown in FIG. 5 (a), a tendency of increased collagen production was observed by adding the water extract of the flower as compared with the control (water). This tendency was particularly observed in samples with concentrations of 10 μg / ml and 100 μg / ml.

FIG. 5B is a graph showing the collagen production ratio 72 hours after the addition of the flower ethanol extract. As can be seen from FIG. 5 (b), the addition of 10 μg / ml, 100 μg / ml, and 200 μg / ml showed a tendency to increase collagen production as compared with the control (DMSO).

From the above results, it was suggested that the water extract and ethanol extract of Hibiscus hamabo contain components that enhance collagen production and contribute to skin moisturization.

[7. Cosmetics]
Next, the cosmetics containing the Hibiscus hamabo extract according to the present embodiment will be described in terms of their production and effects on the skin.

(7-1. Manufacture of cosmetics)
Here, as an example of the cosmetics according to the present embodiment, a lotion containing a Hibiscus hamabo extract was prepared. Specifically, 8.0 parts by weight of 1,3-butanediol, 6.0 parts by weight of dipropylene glycol, 20.0 parts by weight of glycerin, 0.5 parts by weight of sorbitol, and 0.3 parts by weight of lysine dilauroyl glutamate Na. , The extract of Hamabou flower having a final concentration of 10 μg / ml and water were mixed and stirred at room temperature until they were transparently and uniformly dissolved to prepare 100 parts by weight of lotion. Two kinds of flower extracts of Hibiscus hamabo were prepared, one using a water extract and the other using an ethanol extract.

(7-2. Confirmation of effect)
We confirmed the effect of suppressing melanin production and the effect of promoting collagen production by letting 14 men and women in their 40s and 60s use lotion for one month. The subjects were allowed to apply about 3 to 5 ml of lotion to their arms three times a day in the morning, noon, and before going to bed. One arm was made to use a lotion containing an extract, and the other arm was made to use a lotion containing no extract as a control. The subjects were randomly divided into two groups, and the first group used a lotion containing a water extract, and the second group used a lotion containing an ethanol extract. The effect was confirmed by comparing with the state before the start of the test by the subject's own visual and tactile sensation. The evaluation was performed in four stages of "0: no change", "1: slightly improved", "2: slightly improved", and "3: greatly improved", and when the average score was 1 or more, it was judged to be effective. The results are shown in Table 2.

As shown in Table 2, regarding the effect of suppressing melanin production, the water extract tended to be slightly more effective than the ethanol extract, but the production of melanin was effectively suppressed in both cases. The result was.

In addition, good results were obtained for both the water extract and the ethanol extract for skin tension, suggesting that the cosmetic water according to the present embodiment can promote collagen production.

[8. Taste improvement test by adding lactic acid bacteria]
(8-1. Preparation of lactic acid bacteria powder)
Preparation of lactic acid bacteria powder for addition to test foods: Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii subsp. Bulgaricus, Lactobacillus delbrueckii subsp. Two types of subsp. cremoris and Lactococcus lactis subsp. Lactis, two types of lactic acid bacteria belonging to the genus Enterococcus, Enterococcus faecalis and Enterococcus faecium, and two types of lactic acid bacteria belonging to the genus Bifidobacterium, Bifidobacterium bifidum and Bifidobacterium longum subsp. Longum.

Degas the liquid medium suitable for each bacterial species while sterilizing it in an autoclave, inoculate after cooling, and culture under the optimum temperature under the vapor phase conditions and shaking / standing conditions suitable for each bacterial species. rice field.

Next, a predetermined amount was collected from the cultured culture solution, diluted appropriately, seeded on an agar medium, cultured for a predetermined time, and then the colonies were counted to measure the viable cell count. During that period, the culture broth was subjected to centrifugation to recover the cells, washed several times with sterile physiological saline and centrifuged, and then dried and pulverized.

The number of viable bacteria contained in the entire culture solution was calculated from the calculated number of viable bacteria per unit liquid volume and the amount of culture solution before centrifugation, and after considering the recovery efficiency based on experience, per 1 g of lactic acid bacteria powder. The number of bacterial cells contained in was calculated.

Similarly, yeast (Saccharomyces cerevisiae) and Bacillus subtilis (Bacillus subtilis var. Natto) were also prepared in the same manner for the preparation of the test food to be used as a control.

(8-2. Preparation of test food)
In conducting the sensory test described later, granular test foods were prepared in which the extraction solvent, the presence or absence or type of bacterial cell powder, and the amount of bacterial cell powder added were different from each other. The test food used crystalline cellulose, which has little effect on taste, as an excipient, and was unified in a package of 2 g.

Specifically, a test food (hereinafter referred to as additive-free test food) as a comparative control, which contains a water extract or an ethanol extract of leaves or branches of Hamabou but does not contain any bacterial powder, and Hamabo. A test food containing a water extract or an ethanol extract of leaves and containing lactic acid bacteria (hereinafter referred to as a test food containing lactic acid bacteria) and a water extract or ethanol extract of Hamabou leaves, and a fungus of yeast or natto bacteria. A test food containing a body (hereinafter referred to as a yeast-containing test food or a natto bacterium-containing test food) was prepared.

The additive-free test food contained an amount (about 0.1 g) of water extract powder or ethanol extract powder in which the presence of astringency and sweetness derived from Hibiscus hamabo extract was clearly apparent when 2 g of one packet was ingested.

The lactic acid bacterium-containing test food contains the same water extract powder or ethanol extract powder as the additive-free test food, and is prepared for each of the above-mentioned 11 types of lactic acid bacterium powder, and the amount of each lactic acid bacterium powder added is adjusted. 1.0 × 10 ⁴ pieces / packet, 1.0 × 10 ⁵ pieces / package, 1.0 × 10 ⁶ pieces / package were prepared.

Like the test food containing lactic acid bacteria, the test food containing yeast or the test food containing natto bacteria is also prepared according to the bacterial cell powder after containing the same water extract powder or ethanol extract powder as the additive-free test food. In addition, the amount of each bacterial cell powder added was 1.0 × 10 ⁴ / packet, 1.0 × 10 ⁵ / packet, 1.0 × 10 ⁶ / packet.

(8-3. Sensory test)
In the sensory evaluation test, five people who have been involved in product trial production and sensory evaluation for many years were selected as panelists in the business establishment that manufactures health foods on an OEM basis, and the balance between astringency and sweetness was evaluated.

The contents of the test foods were disclosed to the panelists only for the additive-free foods, and the contents of the other test foods were not disclosed. The panelists evaluated whether the ingested test food had a change in the balance between astringency and sweetness compared to the additive-free food. When it was necessary to confirm the criteria for the balance between astringency and sweetness, each panelist was allowed to ingest additive-free foods as appropriate. In addition, each of the five panelists was allowed to sample an aqueous solution containing various balances of astringent and sweet ingredients in a preliminary sensory test conducted prior to the sensory test so that the degree of evaluation would be roughly the same. Training is being carried out.

As a result, the yeast-containing test food or the natto-containing test food did not affect the balance of astringency and sweetness derived from the Hamabou extract.

On the other hand, for the test food containing lactic acid bacteria, 1.0 x 10 ⁴ pieces / pack did not affect the balance of astringency and sweetness derived from the Hamabou extract, but 1.0 x 10 ⁶ pieces / pack. There was a tendency for the balance between astringency and sweetness to reverse. In addition, the balance between astringency and sweetness tended to be reversed for the 1.0 x 10 ⁵ pieces / pack of test food, although not as much as the 1.0 x 10 ⁶ pieces / pack of test food. No difference was observed depending on the type of lactic acid bacteria and the extraction solvent.

In addition, when we asked the panelists for their opinions, it was not just that the astringency was unilaterally felt and sensually reversed, but that the astringency was less likely to be felt and that the sweetness was strongly felt. The common opinion was that they would sensually reverse each other.

From these results, the number of lactic acid bacteria of at least one genus selected from the genus Lactobacillus, Lactococcus, Enterococcus, and Bifidobacterium is 1.0 × 10 ⁵ or more per 0.1 g of the dried product of the Hamabou extract, more preferably. It was shown that the balance of astringency and sweetness derived from Hamabou extract was reversed and the taste was improved in the functional food to which 1.0 × 10 ⁶ or more were added. In addition, extracts derived from other than leaves also exhibit similar tastes, suggesting that similar taste improvement is expected.

As described above, according to the Hamabou extract according to the present embodiment, an aqueous solvent containing water as a main component or a mixture of any one or two or more selected from dimethylsulfoxide, ethanol, methanol and hexane. This is a Hamabou extract that consists of a group of components that are soluble in a specific non-aqueous solvent consisting of a liquid and contains at least a blood glucose level increase inhibitory component, a lipid absorption inhibitory component, a melanin production inhibitory component, and a collagen production promoting component. Ingestion of blood glucose levels and lipids, which are often anxious with aging, and improvement of skin pigmentation and elasticity can be realized with a single plant-derived extract called Hamabou.

Finally, the description of each embodiment described above is an example of the present invention, and the present invention is not limited to the above-described embodiment. Therefore, it goes without saying that various changes can be made according to the design and the like as long as the technical idea of the present invention is not deviated from the above-described embodiments.

Claims (8)

It consists of a group of components exhibiting solubility in a non-aqueous specific solvent consisting of an aqueous solvent containing water as a main component or a mixed solution of any one or two or more selected from dimethylsulfoxide, ethanol, methanol, and hexane. , Hamabou extract containing at least a component for suppressing increase in blood glucose level, a component for suppressing fat absorption, a component for suppressing melanin production, and a component for promoting collagen production. The Hamabou extract according to claim 1, wherein the component group comprises a component group exhibiting solubility in an aqueous solvent and further contains a component for suppressing an increase in blood pressure. A cosmetic containing the Hibiscus hamabo extract according to claim 1. A functional food containing the Hibiscus hamabo extract according to claim 1 or 2. The fourth aspect of claim 4, wherein 1.0 × 10 ⁵ or more lactic acid bacteria cells of at least one genus selected from the genus Lactobacillus, Lactococcus, Enterococcus, and Bifidobacterium are added per 0.1 g of the dried product of the Hamabou extract. Functional food. It consists of a group of components exhibiting solubility in a non-aqueous specific solvent consisting of an aqueous solvent containing water as a main component or a mixed solution of any one or two or more selected from dimethylsulfoxide, ethanol, methanol and hexane. For the purpose of including at least one event selected from the Hamabou extract, that is, the suppression of blood glucose level increase, the suppression of fat absorption, the suppression of melanin production, the promotion of collagen production, and the suppression of blood pressure increase derived from the Hamabou extract as an action or effect. Use as a raw material for functional foods in which the constituents of the Hamabou extract corresponding to the occurrence of the above-mentioned events are used as health functional ingredients (participating ingredients) in the functional foods of the above. It consists of a group of components exhibiting solubility in a non-aqueous specific solvent consisting of an aqueous solvent containing water as a main component or a mixed solution of any one or two or more selected from dimethylsulfoxide, ethanol, methanol and hexane. The Hamabou corresponding to the manifestation of the event in a cosmetic for the purpose of comprising at least one event of suppressing melanin production or promoting collagen production derived from the Hamabou extract as an action or effect. Use as a raw material for cosmetics containing the constituents of the extract as active ingredients. Use as a taste-improving additive for Hamabou extract of lactic acid bacteria cells to sensually reverse the astringency and sweetness of Hamabou extract.

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