Method for producing a composition containing Job's tears components, and Job's tears component-containing composition

Abstract

The present invention provides a method for producing a composition containing Job's tears components that enables the favorable expression of UCP1 in brown adipose tissue, and a Job's tears component-containing composition. [Solution] The method for producing a Job's tears component-containing composition is a method for producing a Job's tears component-containing composition to suppress the inhibition of UCP1 (Uncoupling protein 1) expression, and includes extracting components contained in Job's tears by soaking unroasted Job's tears in water. In the Job's tears component-containing composition, the components extracted from Job's tears suppress at least one of the transcriptional activity of NFκB (Nuclear factor kappa B) and the transcriptional activity of AP1 (Activator protein 1). Soaking Job's tears in water may include soaking the hulls or whole grains of Job's tears in water. The temperature of the water in which the Job's tears are soaked may be 60°C or lower, preferably 50°C or lower.

Description

Technical Field

[0001] The present invention relates to a method for producing a composition containing adlay components and a composition containing adlay components.

Background Art

[0002] Uncoupling protein 1 (UCP1) expressed in brown adipocytes has a function of dissipating energy as heat by converting neutral lipids into heat. Therefore, it is considered that promoting the expression of UCP1 can suppress obesity by efficiently converting neutral lipids into heat. For example, Patent Document 1 discloses a technique of combining black ginger with specific components to promote the expression of UCP1.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] By the way, the expression of UCP1 in brown adipocytes may be inhibited by inflammatory cytokines. Therefore, in order to preferably express UCP1 in brown adipocytes, it is required to suppress the inhibition of UCP1 expression by inflammatory cytokines.

Means for Solving the Problems

[0005] A method for producing a Job's tears component-containing composition to solve the above problems is a method for producing a Job's tears component-containing composition to suppress the inhibition of UCP1 (Uncoupling protein 1) expression, and includes extracting components contained in Job's tears by immersing unroasted Job's tears in water. In the above production method, the components extracted from Job's tears in the Job's tears component-containing composition suppress at least one of the transcriptional activity of NFκB (Nuclear factor kappa B) and the transcriptional activity of AP1 (Activator protein 1). According to the above production method, the inhibition of UCP1 expression in brown adipose tissue can be suppressed by the suppression of inflammatory cytokine expression by the Job's tears component-containing composition.

[0006] In the method for producing the above-mentioned adlay component-containing composition, soaking the adlay in water may include soaking the adlay hulls or the whole grains of the adlay in water. By using the hulls or whole grains including the hulls of adlay seeds as raw materials, the transcriptional activity of NFκB can be suitably suppressed.

[0007] In the method for producing the above-mentioned adlay component-containing composition, the temperature of the water in which the adlay is immersed may be 60°C or lower. By setting the extraction temperature to 60°C or lower, the transcriptional activity of NFκB and AP1 can be suitably suppressed. That is, the expression of inflammatory cytokines can be more suitably suppressed, and therefore the inhibition of UCP1 expression caused by inflammatory cytokines can be more suitably suppressed.

[0008] In the method for producing the above-mentioned adlay component-containing composition, the temperature of the water in which the adlay is immersed may be 50°C or lower. If the extraction temperature is 50°C or lower, the inhibitory effect on the transcriptional activity of NFκB can be further enhanced, and thus the inhibition of UCP1 expression caused by inflammatory cytokines can be more effectively suppressed.

[0009] The adlay component-containing composition for solving the above problem is a composition for suppressing the inhibition of UCP1 expression, and the composition contains components found in unroasted adlay. [Effects of the Invention]

[0010] According to the present invention, UCP1 can be suitably expressed in brown adipose tissue. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 is a graph showing the results of the cytotoxicity evaluation of unroasted whole-grain hot water extract of Job's tears in the preliminary test of Test Example 1. [Figure 2] Figure 2 is a graph showing the results of the cytotoxicity evaluation of the hot water extract of unroasted Job's tears husks in the preliminary test of Test Example 1. [Figure 3] Figure 3 is a graph showing the results of the cytotoxicity evaluation of the hot water extract of unroasted Job's tears granules in the preliminary test of Test Example 1. [Figure 4] Figure 4 is a graph showing the results of the cytotoxicity evaluation of the whole grain hot water extract of roasted Job's tears in the preliminary test of Test Example 1. [Figure 5] Figure 5 is a graph showing the results of the evaluation of NFκB transcription activity of unroasted whole-grain hot water extract of Job's tears in Test Example 1. [Figure 6] Figure 6 is a graph showing the results of the evaluation of NFκB transcription activity of unroasted Job's tears husk hot water extract in Test Example 1. [Figure 7] Figure 7 is a graph showing the results of the evaluation of NFκB transcription activity of unroasted Job's tears refined grain hot water extract in Test Example 1. [Figure 8] Figure 8 is a graph showing the results of the evaluation of NFκB transcription activity of roasted Job's tears whole grain hot water extract in Test Example 1. [Figure 9] Figure 9 is a graph showing the results of the cytotoxicity evaluation of unroasted whole-grain cold water extract of Job's tears in the preliminary test of Test Example 2. [Figure 10]Figure 10 is a graph showing the results of cytotoxicity evaluation of the cold water extract of unroasted adlay husk in the preliminary test of Test Example 2. [Figure 11] Figure 11 is a graph showing the results of cytotoxicity evaluation of the cold water extract of unroasted adlay polished grains in the preliminary test of Test Example 2. [Figure 12] Figure 12 is a graph showing the results of cytotoxicity evaluation of the cold water extract of roasted whole adlay in the preliminary test of Test Example 2. [Figure 13] Figure 13 is a graph showing the results of NFκB transcriptional activity evaluation of the cold water extract of unroasted whole adlay in Test Example 2. [Figure 14] Figure 14 is a graph showing the results of NFκB transcriptional activity evaluation of the cold water extract of unroasted adlay husk in Test Example 2. [Figure 15] Figure 15 is a graph showing the results of NFκB transcriptional activity evaluation of the cold water extract of unroasted adlay polished grains in Test Example 2. [Figure 16] Figure 16 is a graph showing the results of NFκB transcriptional activity evaluation of the cold water extract of roasted whole adlay in Test Example 2. [Figure 17] Figure 17 is a graph showing the results of AP1 transcriptional activity evaluation of the hot water extract of adlay in Test Example 3. [Figure 18] Figure 18 is a graph showing the results of AP1 transcriptional activity evaluation of the cold water extract of adlay in Test Example 4. [Figure 19] Figure 19 is a graph showing the results of cytotoxicity evaluation of the adlay whole grain extract with an extraction temperature of 4°C in the preliminary test of Test Example 5. [Figure 20] Figure 20 is a graph showing the results of cytotoxicity evaluation of the adlay whole grain extract with an extraction temperature of 15°C in the preliminary test of Test Example 5. [Figure 21] Figure 21 is a graph showing the results of cytotoxicity evaluation of the adlay whole grain extract with an extraction temperature of 25°C in the preliminary test of Test Example 5. [Figure 22]Figure 22 is a graph showing the results of cytotoxicity evaluation of whole-grain adlay extract with an extraction temperature of 40°C in the preliminary test of Test Example 5. [Figure 23] Figure 23 is a graph showing the results of cytotoxicity evaluation of whole-grain adlay extract with an extraction temperature of 50°C in the preliminary test of Test Example 5. [Figure 24] Figure 24 is a graph showing the results of cytotoxicity evaluation of whole-grain adlay extract with an extraction temperature of 60°C in the preliminary test of Test Example 5. [Figure 25] Figure 25 is a graph showing the results of cytotoxicity evaluation of whole-grain adlay extract with an extraction temperature of 80°C in the preliminary test of Test Example 5. [Figure 26] Figure 26 is a graph showing the results of cytotoxicity evaluation of whole-grain adlay extract with an extraction temperature of 90°C in the preliminary test of Test Example 5. [Figure 27] Figure 27 is a graph showing the results of NFκB transcriptional activity evaluation using a test solution containing 5 μg / mL of whole-grain adlay extract in Test Example 5. [Figure 28] Figure 28 is a graph showing the results of NFκB transcriptional activity evaluation using a test solution containing 100 μg / mL of whole-grain adlay extract in Test Example 5. [Figure 29] Figure 29 is a graph showing the results of AP1 transcriptional activity evaluation of whole-grain adlay extract in Test Example 6. [Figure 30] Figure 30 is a graph showing the results of UCP1 expression level evaluation of whole-grain adlay extract in Test Example 7. [Embodiments for Carrying out the Invention]

[0012] Hereinafter, a method for producing a composition containing adlay components and an embodiment of the composition containing adlay components will be described. [Composition Containing Adlay Components] The composition containing adlay components is a composition for suppressing the inhibition of UCP1 expression in brown adipose cells. The composition containing adlay components contains components extracted from adlay.

[0013] In this embodiment, from the viewpoint of suitably suppressing the inhibition of UCP1 expression, unroasted Job's tears seeds are used as the raw material. The Job's tears used as the raw material may be in the form of a powder or granules, for example. From the viewpoint of efficiently extracting the components contained in Job's tears, it is preferable that the Job's tears used as the raw material be in the form of a powder. Furthermore, the variety of Job's tears used as the raw material is not particularly limited. Examples of Job's tears varieties used as the raw material include Akishizuku, Hatokirara, Hatojiro, and Nakazato Zairai.

[0014] The part of the Job's tears used is, for example, at least one selected from the whole grain, hull, and polished grain of the Job's tears seed. Whole Job's tears refers to the entire seed. Whole Job's tears includes the involucre, glumes, pericarp, and endosperm. Hull of Job's tears includes the involucre and glumes. Polished Job's tears includes the endosperm.

[0015] The form of the Job's tears component-containing composition is not particularly limited. The Job's tears component-containing composition can take various forms, such as liquid, lotion, gel, emulsion, suspension, cream, ointment, aerosol, spray, powder, granules, tablets, paste, and capsules. As an example, the Job's tears component-containing composition of this embodiment is in powder form.

[0016] The composition containing Job's tears extract may be an oral composition or a parenteral composition. Oral compositions include, for example, foods, beverages, and supplements. Parenteral compositions include, for example, cosmetic compositions. Cosmetic compositions include, for example, lotions, creams, emulsions, masks, hair tonics, hair creams, shampoos, hair rinses, treatments, facial cleansers, foundations, hair growth products, aqueous ointments, and sprays.

[0017] [Method for producing a composition containing Job's tears components] A method for producing a composition containing adlay components includes a step of extracting components contained in adlay, which is the raw material. A method for extracting components contained in adlay includes a step of immersing the adlay, which is the raw material, in a solvent.

[0018] The solvent used for extraction may be water, a lower alcohol, a lower ester, acetone, or a mixture of these with water. Examples of lower alcohols include ethanol, methanol, isopropanol, and butanol. Examples of lower esters include ethyl acetate and methyl acetate. For extraction, for example, a solvent in an amount between 2 and 20 times the mass of the raw material, Job's tears, is used.

[0019] The solvent used for extraction is preferably water, considering the possibility of oral ingestion of the adlay component-containing composition. The temperature of the water is not particularly limited, but from the viewpoint of effectively suppressing the inhibition of UCP1 expression in brown adipose tissue by the adlay component-containing composition, a temperature of 60°C or lower is preferred, and 50°C or lower is more preferred.

[0020] The soaking time for the Job's tears in water is not particularly limited, but is, for example, 10 minutes or more, preferably 60 minutes or more. The soaking time may be adjusted as appropriate depending on the water temperature. During soaking, the water in which the Job's tears are soaked may be left to stand or stirred.

[0021] After immersion is complete, the Job's tears can be removed from the water by filtration or centrifugation to obtain a Job's tears extract containing the water-soluble components of the Job's tears. The Job's tears extract is one example of a composition containing Job's tears components. The Job's tears removed from the water by filtration or centrifugation may be used again for extraction.

[0022] The adlay extract obtained as described above can be used as is, but it may be concentrated or purified as needed. The adlay component-containing composition may also be a dried product obtained by drying the adlay extract. The drying method is not particularly limited; for example, spray drying, freeze-drying, vacuum drying, or fluidized bed drying can be used. The dried product can also be used in powder form.

[0023] [Effect of the Embodiment] UCP1 expression in brown adipocytes may be inhibited by inflammatory cytokines such as TNFα (Tumor necrosis factor alpha) and IL1β (Interleukin 1 beta). The expression levels of these inflammatory cytokines are regulated, for example, by the transcription factors NFκB (Nuclear factor-kappa B) or AP1 (Activator protein 1). NFκB is involved in the NFκB pathway, while AP1 is involved in the MAPK (Mitogen-activated protein kinase) pathway.

[0024] In the adlay component-containing composition, components extracted from adlay suppress the transcriptional activity of NFκB and AP1. In other words, the adlay component-containing composition suppresses the inhibition of UCP1 expression in brown adipose tissue by inflammatory cytokines by suppressing the expression of inflammatory cytokines. The higher the expression level of UCP1, the greater the potential for improvement in obesity through increased heat production, etc. Therefore, the adlay component-containing composition has an anti-obesity effect by suppressing the inhibition of UCP1 expression by inhibiting the transcriptional activity of NFκB and AP1 in macrophages.

[0025] By keeping the water temperature below 60°C when extracting components from Job's tears, the transcriptional activity of NFκB and AP1 is suitably suppressed by the Job's tears component-containing composition. In particular, by keeping the water temperature below 50°C when extracting components from Job's tears, the transcriptional activity of NFκB is more suitably suppressed by the Job's tears component-containing composition.

[0026] [Example Test] Test Examples 1 to 7 will be described below with reference to Figures 1 to 30. Note that the test conditions used in each test example are examples or comparative examples used to illustrate the effects of this embodiment and do not limit the present invention.

[0027] [Test Example 1] Refer to Figures 1 to 8 to explain Test Example 1. In Test Example 1, the inhibitory effect on NFκB transcription activity of a Job's tears component-containing composition extracted using hot water from one of the following raw materials: unroasted whole Job's tears, hulls, polished grains, or roasted whole Job's tears. When roasting the whole Job's tears, the grains were heated in a constant temperature incubator at 230°C for 95 minutes.

[0028] In Test Example 1, a composition containing Job's tears components was prepared using the following procedure. First, unroasted whole Job's tears, hulls, polished grains, and roasted whole Job's tears were each ground in a mill. Then, to the ground material of each part of Job's tears, pure water was added so that the mass of Job's tears was 6% of the sum of the mass of Job's tears and the mass of pure water. Extraction was then carried out in a constant temperature bath at 90°C for 1 hour. Next, the supernatant was collected by centrifugation at 8000 rpm, 20°C, and 30 minutes. The Job's tears precipitate was also collected and subjected to the same procedure again. That is, pure water was added to the collected Job's tears precipitate, and after extraction in a constant temperature bath at 90°C, the supernatant was collected by centrifugation. Then, the collected supernatant was subjected to suction filtration to obtain an extract of Job's tears components. Subsequently, the extract was freeze-dried to obtain a sample of the powdered Job's tears component-containing composition. Following the above procedure, four levels of hot water extracts were obtained as samples of the adlay component-containing composition, using one of the following as raw materials: whole adlay grains, hulls, polished adlay grains, and roasted adlay whole grains.

[0029] [Example Exam 1: Preliminary Exam] In Test Example 1, a preliminary cytotoxicity evaluation was performed using the following procedure. First, a test solution was obtained by adding a composition containing Job's tears components to a culture medium for the test solution. Specifically, several levels of test solutions were obtained by adjusting the concentration of the Job's tears component-containing composition for each type of Job's tears raw material. The concentrations of the Job's tears component-containing composition in the test solutions were set to six levels: 1 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL. For the test solution culture medium, MEM Alpha (manufactured by Nacalai Tesque Co., Ltd.) containing 10% FBS (manufactured by Biowest Co., Ltd.) by volume, and Penicillin (manufactured by Nacalai Tesque Co., Ltd.) with a final concentration of 100 u / mL was used.

[0030] Next, mouse macrophage-like cells, RAW264.7 cells (obtained from ATCC), were seeded and cultured in a 96-well cell culture plate. The number of passages for RAW264.7 cells was not particularly limited. After confirming cell adhesion, the culture medium was removed from the cell culture plate and replaced with a test solution containing a Job's tears component composition and a test medium. For comparison, a control level was prepared in which the test medium containing the Job's tears component composition was replaced with a test medium without the Job's tears component composition. After 24 hours of culture, cell viability was calculated using Cell Counting Kit-8 (manufactured by Dojin Chemical Laboratories Co., Ltd.).

[0031] The viability of cells to which the test solution containing the adlay component composition was added was calculated with the viability of cells at the control level (without the adlay component composition) set as 100%. Cells with a viability higher than 85% were judged to have no cytotoxicity. The evaluation results of the preliminary test in Test Example 1 are shown in Figures 1 to 4. In the evaluation results of cytotoxicity, the control level is indicated as CTL.

[0032] As shown in Figures 1-4, the cell viability of hot water extracts derived from any one of the following raw materials—unroasted whole grains, hulls, polished grains, or roasted whole grains of Job's tears—was higher than 85% compared to the control at all concentrations. In other words, no cytotoxicity was observed in hot water extracts derived from any one of the following raw materials—unroasted whole grains, hulls, polished grains, or roasted whole grains of Job's tears—at concentrations between 1 μg / mL and 100 μg / mL.

[0033] [Test Example 1: Evaluation of NFκB Transcriptional Activity] In Test Example 1, the inhibitory effect of four levels of hot water extracts, each derived from either unroasted whole grains, hulls, polished grains, or roasted whole grains of Job's tears, on the transcriptional activity of NFκB was evaluated. Specifically, NFκB transcriptional activity was measured using RAW264.7 (RAW / NFκB-luc) cells, which are mouse macrophage-like cells containing a vector into which the luciferase protein gene was introduced downstream of the response element to which NFκB binds. NFκB transcriptional activity was measured using the luminescence intensity induced by the luciferase protein as an indicator. Specifically, the evaluation was performed using the following procedure.

[0034] First, for each type of adlay raw material, a test solution was obtained by adding the adlay component-containing composition to the same type of test solution culture medium as in the preliminary test. Two concentrations of the adlay component-containing composition in the test solution were selected from the concentration range in which no cytotoxicity was observed in the preliminary test: 50 μg / mL and 100 μg / mL.

[0035] Next, RAW264.7 (RAW / NFκB-luc) cells (obtained from ATCC) were seeded and cultured in 96-well cell culture plates. The number of passages for RAW264.7 (RAW / NFκB-luc) cells was not particularly limited. MEM Alpha (manufactured by Nacalai Tesque Co., Ltd.) was used as the culture medium in the cell culture plates. After confirming cell adhesion, the culture medium was removed from the cell culture plates and replaced with a test solution containing a composition of Job's tears components and a test medium. For comparison, a control level was also prepared in which the culture medium was removed from the cell culture plates and replaced with a test medium that did not contain the Job's tears component composition.

[0036] Twenty-one hours after the culture medium was changed, an LPS-treated solution, which was prepared by adding lipopolisaccharaide (LPS, manufactured by Nacalai Tesque Co., Ltd.), an inflammation-inducing stimulant, to the same level of test solution used during the culture medium change, was added to the cultured cells. In the LPS-treated solution, the concentration of LPS was adjusted so that the final concentration of LPS was 100 ng / mL. For the comparative level, which did not contain the adlay component composition, two samples were prepared: one in which LPS was added to the cultured cells 21 hours after the culture medium change to achieve a final concentration of 100 ng / mL, and another in which the cells were cultured without LPS addition. In the comparative level, the sample with added LPS was added to the cultured cells using the test medium with added LPS, rather than the LPS-treated solution with LPS already added to the test solution. In the comparative level, the same procedure was performed using the test medium without added LPS. After adding LPS, the cells were cultured for a further three hours.

[0037] After 3 hours, the culture medium containing the Job's tears component and LPS was removed, and a 5-fold diluted solution of cell lysis buffer (product name: 5X Cells lysis buffer, manufactured by PROMEGA Corporation) was added and mixed. Then, luciferin (manufactured by PROMEGA Corporation) was added, and luciferase activity was measured based on the luminescence intensity. A higher luminescence intensity indicates higher luciferase activity. Furthermore, in Test Example 1, high luciferase activity means that LPS activated the intracellular NFκB pathway, thereby promoting the expression of the luciferase gene. In other words, high luciferase activity means high NFκB transcriptional activity.

[0038] Luciferase activity when a test solution containing a Job's tears component composition was added was calculated using the control level of luciferase activity (without Job's tears component composition and with LPS added) as 100%. If the luciferase activity was greater than 90% but less than 100% of the control level, it was considered that an effect of suppressing NFκB transcriptional activity was observed. Furthermore, if the luciferase activity was 90% or less of the control level, it was considered that an effect of suitably suppressing NFκB transcriptional activity was observed.

[0039] Figures 5 to 8 show the evaluation results of luciferase activity in Test Example 1. In the evaluation results of NFκB transcription activity, the level without the addition of the Job's tears component composition and without the addition of LPS is denoted as LPS-. Furthermore, in the evaluation results of NFκB transcription activity, the level of the control without the addition of the Job's tears component composition and with the addition of LPS is denoted as CTL.

[0040] As shown in Figure 5, in the hot water extract made from unroasted whole grains of Job's tears, the luciferase activity was 90% or less of the control level regardless of whether the test solution concentration was 50 μg / mL or 100 μg / mL. In other words, the hot water extract made from unroasted whole grains of Job's tears showed a favorable inhibitory effect on NFκB transcriptional activity. At a test solution concentration of 100 μg / mL, NFκB transcriptional activity was favorably inhibited compared to at a test solution concentration of 50 μg / mL, indicating a concentration-dependent effect where the inhibitory effect on NFκB transcriptional activity increases with higher concentrations of the Job's tears component-containing composition.

[0041] As shown in Figure 6, in the hot water extract made from unroasted Job's tears hulls, the luciferase activity was 90% or less of the control level regardless of whether the test solution concentration was 50 μg / mL or 100 μg / mL. In other words, the hot water extract made from unroasted Job's tears hulls showed a favorable inhibitory effect on NFκB transcriptional activity. At a test solution concentration of 100 μg / mL, NFκB transcriptional activity was favorably suppressed compared to at a test solution concentration of 50 μg / mL, indicating a concentration-dependent effect where the inhibitory effect on NFκB transcriptional activity increased with increasing concentration of the Job's tears component-containing composition.

[0042] As shown in Figure 7, in the hot water extract made from unroasted, polished Job's tears grains, the luciferase activity was 90% or less of the control level when the test solution concentration was 50 μg / mL. Furthermore, when the test solution concentration was 100 μg / mL, the luciferase activity was between 90% and 100% of the control level. In other words, the hot water extract made from unroasted, polished Job's tears grains showed an effect of suppressing the transcriptional activity of NFκB.

[0043] From the above results, it was confirmed that in hot water extracts obtained from unroasted Job's tears at 90°C, using whole grains or hulls of Job's tears as the raw material allowed for more effective suppression of NFκB transcription activity than using polished Job's tears as the raw material. Furthermore, it was confirmed that hot water extracts using unroasted whole grains of Job's tears as the raw material suppressed NFκB transcription activity more effectively than hot water extracts using unroasted hulls of Job's tears as the raw material.

[0044] As shown in Figure 8, in the hot water extract made from roasted whole grains of Job's tears, the luciferase activity was 90% or less of the control level regardless of whether the test solution concentration was 50 μg / mL or 100 μg / mL. In other words, the hot water extract made from roasted whole grains of Job's tears showed a favorable effect in suppressing NFκB transcription activity. Comparing the results with and without roasted Job's tears, it was confirmed that the hot water extract made from unroasted whole grains of Job's tears, as shown in Figure 5, had a higher effect in suppressing NFκB transcription activity.

[0045] [Test Example 2] Refer to Figures 9 to 16 to explain Test Example 2. In Test Example 2, the inhibitory effect on NFκB transcriptional activity of a Job's tears component-containing composition extracted with cold water from one of the following raw materials: unroasted whole Job's tears, hulls, polished grains, or roasted whole Job's tears. When roasting the whole Job's tears, the grains were heated in a constant temperature incubator at 230°C for 95 minutes.

[0046] In Test Example 2, a composition containing Job's tears components was prepared using the following procedure. First, unroasted whole Job's tears, hulls, and polished grains were each ground in a mill. Then, five times the mass of pure water relative to the mass of Job's tears was added to the ground material of each part of Job's tears. After stirring with a stirrer, extraction was carried out overnight at 4°C using a constant temperature incubator. Next, after stirring again, the supernatant was collected by centrifugation at 8000 rpm, 20°C, and 30 minutes. The Job's tears precipitate was also collected and subjected to the same procedure again. That is, pure water was added to the collected Job's tears precipitate, stirred with a stirrer, extracted at 4°C using a constant temperature incubator, stirred again with a stirrer, and then centrifuged to collect the supernatant. The collected supernatant was then filtered by suction to obtain an extract of Job's tears components. Subsequently, the extract was freeze-dried using a freeze-dryer to obtain a sample of the powdered Job's tears component-containing composition. Following the above procedure, four levels of cold water extracts were obtained as samples of the adlay component-containing composition, using one of the following as raw materials: whole adlay grains, hulls, polished adlay grains, and roasted adlay whole grains.

[0047] [Example Exam 2: Preliminary Exam] In Test Example 2, a preliminary cytotoxicity assessment was performed using the same procedure as in Test Example 1. Cells with a viability rate higher than 85% were judged to be free of cytotoxicity. The results of the preliminary assessment in Test Example 2 are shown in Figures 9 to 12.

[0048] As shown in Figure 9, in the cold water extract made from unroasted whole Job's tears, the cell viability relative to the control was higher than 85% at test solution concentrations between 1 μg / mL and 5 μg / mL. On the other hand, at test solution concentrations between 10 μg / mL and 100 μg / mL, the cell viability relative to the control was 85% or less. In other words, in the cold water extract made from unroasted whole Job's tears, no cytotoxicity was observed when the test solution concentration was between 1 μg / mL and 5 μg / mL, but cytotoxicity was observed at test solution concentrations of 10 μg / mL or higher.

[0049] As shown in Figure 10, in the cold water extract made from unroasted Job's tears hulls, the cell viability relative to the control was higher than 85% at test solution concentrations between 1 μg / mL and 10 μg / mL. On the other hand, at test solution concentrations between 25 μg / mL and 100 μg / mL, the cell viability relative to the control was 85% or less. In other words, in the cold water extract made from unroasted Job's tears hulls, no cytotoxicity was observed at test solution concentrations between 1 μg / mL and 10 μg / mL, but cytotoxicity was observed at test solution concentrations of 25 μg / mL or higher.

[0050] As shown in Figure 11, in the cold water extract made from unroasted, polished Job's tears, the cell viability was higher than 85% compared to the control at test solution concentrations between 1 μg / mL and 100 μg / mL. In other words, no cytotoxicity was observed at any concentration in the cold water extract made from unroasted, polished Job's tears.

[0051] As shown in Figure 12, the cold water extract made from roasted whole Job's tears showed a cell viability rate higher than 85% compared to the control at test solution concentrations between 1 μg / mL and 100 μg / mL. In other words, no cytotoxicity was observed at any concentration of the cold water extract made from roasted whole Job's tears.

[0052] [Test Example 2: Evaluation of NFκB Transcriptional Activity] In Test Example 2, the inhibitory effect of a Job's tears component-containing composition on NFκB transcriptional activity was evaluated for four levels of cold water extracts using one of the following raw materials: unroasted whole grains, hulls, polished grains, and roasted whole grains of Job's tears, using the same procedure as in Test Example 1. The concentration of the Job's tears component-containing composition in the test solution was selected from the concentration range in which no cytotoxicity was observed in preliminary tests for each Job's tears raw material. For cold water extracts using unroasted whole grains or hulls of Job's tears as the raw material, two levels of test solution concentration were adopted: 2.5 μg / mL and 5 μg / mL. For cold water extracts using unroasted polished grains of Job's tears or roasted whole grains of Job's tears as the raw material, two levels of test solution concentration were adopted: 50 μg / mL and 100 μg / mL. The evaluation criteria were the same as in Test Example 1. The evaluation results of luciferase activity in Test Example 2 are shown in Figures 13 to 16.

[0053] As shown in Figure 13, in the cold water extract made from unroasted whole Job's tears, the luciferase activity was 90% or less of the control level regardless of whether the test solution concentration was 2.5 μg / mL or 5 μg / mL. In other words, the cold water extract made from unroasted whole Job's tears showed a favorable inhibitory effect on NFκB transcription activity. At a test solution concentration of 5 μg / mL, NFκB transcription activity was favorably inhibited compared to at a test solution concentration of 2.5 μg / mL, indicating a concentration-dependent effect where the inhibitory effect on NFκB transcription activity increased with increasing concentration of the Job's tears component-containing composition. Furthermore, compared with the results of Test Example 1 shown in Figure 5, it was confirmed that in the case of a Job's tears component-containing composition made from unroasted whole Job's tears, the cold water extract could inhibit NFκB transcription activity to the same or greater extent than the hot water extract, even at a smaller amount and lower concentration.

[0054] As shown in Figure 14, in the cold water extract made from unroasted Job's tears hulls, the luciferase activity was 90% or less of the control level regardless of whether the test solution concentration was 2.5 μg / mL or 5 μg / mL. In other words, the cold water extract made from unroasted Job's tears hulls showed a favorable inhibitory effect on NFκB transcription activity. Furthermore, at a test solution concentration of 5 μg / mL, NFκB transcription activity was favorably inhibited compared to the test solution concentration of 2.5 μg / mL, indicating a concentration-dependent effect where the inhibitory effect on NFκB transcription activity increased with increasing concentration of the Job's tears component-containing composition. Moreover, compared with the results of Test Example 1 shown in Figure 6, it was confirmed that in the case of a Job's tears component-containing composition made from unroasted Job's tears hulls, the cold water extract could inhibit NFκB transcription activity to the same or greater extent than the hot water extract, even at smaller amounts and lower concentrations.

[0055] As shown in Figure 15, in cold water extracts using unroasted, polished Job's tears as the raw material, luciferase activity was 90% or less of the control level regardless of whether the test solution concentration was 50 μg / mL or 100 μg / mL. In other words, cold water extracts obtained by extracting Job's tears components using polished Job's tears as the raw material at an extraction temperature of 4°C showed a favorable effect in suppressing NFκB transcription activity. Furthermore, compared with the results of Test Example 1 shown in Figure 7, it was confirmed that in the case of Job's tears component-containing compositions using unroasted, polished Job's tears as the raw material, cold water extracts can favorably suppress NFκB transcription activity more effectively than hot water extracts.

[0056] From these results, it was confirmed that, in cold water extracts obtained from unroasted Job's tears at 4°C, using whole grains or hulls of Job's tears as the raw material allowed for the effective suppression of NFκB transcription activity even at smaller amounts and lower concentrations than when using polished Job's tears as the raw material. Furthermore, it was confirmed that cold water extracts using unroasted whole grains of Job's tears as the raw material suppressed NFκB transcription activity more effectively than cold water extracts using unroasted hulls as the raw material.

[0057] As shown in Figure 16, in the cold water extract made from roasted whole grains of Job's tears, the luciferase activity was 99% of the control level when the test solution concentration was 50 μg / mL. Furthermore, when the test solution concentration was 100 μg / mL, the luciferase activity was 90% or less of the control level. Comparing the results with and without roasted Job's tears, it was confirmed that the cold water extract made from unroasted whole grains of Job's tears, as shown in Figure 13, was more effective in suppressing NFκB transcriptional activity, even at smaller amounts and lower concentrations than the cold water extract made from roasted whole grains of Job's tears.

[0058] [Test Example 3] Refer to Figure 17 to explain Test Example 3. In Test Example 3, the inhibitory effect on AP1 transcriptional activity of a Job's tears component-containing composition extracted using hot water from unroasted whole Job's tears or hulls was evaluated. In Test Example 3, two levels of hot water extracts were obtained using unroasted whole Job's tears or hulls as raw materials, following the same procedure as in Test Example 1, as samples for the Job's tears component-containing composition. Note that preliminary tests were omitted in Test Example 3.

[0059] In Experimental Example 3, AP1 transcriptional activity was measured using RAW264.7 (RAW / AP1-luc) cells, which are mouse macrophage-like cells containing a vector into which the luciferase protein gene was introduced downstream of the response element to which AP1 binds. AP1 transcriptional activity was measured using the luminescence intensity induced by the luciferase protein as an indicator. Specifically, the evaluation was performed using the following procedure.

[0060] First, for each part of the Job's tears used as raw material, test solutions were obtained by adding the Job's tears component-containing composition to the same type of test solution culture medium as in the preliminary test of Test Example 1. The concentration of the Job's tears component-containing composition in the test solution was set at a level of 100 μg / mL, which was within the concentration range in which no cytotoxicity was observed in the preliminary test of Test Example 1.

[0061] Next, RAW264.7 (RAW / AP1-luc) cells (obtained from ATCC) were seeded and cultured in 96-well cell culture plates. The number of passages for RAW264.7 (RAW / AP1-luc) cells was not particularly limited. MEM Alpha was used as the culture medium in the cell culture plates. After confirming cell adhesion, the medium was removed from the cell culture plates and replaced with a test solution containing a composition of Job's tears components and a test medium. For comparison, a control level was also prepared in which the medium was removed from the cell culture plates and replaced with a test medium that did not contain the Job's tears component composition.

[0062] Twenty-one hours after the culture medium was changed, an LPS-treated solution, prepared by adding LPS to the same level of test solution used during the medium change, was added to the cultured cells. In the LPS-treated solution, the concentration of LPS was adjusted so that the final concentration of LPS was 100 ng / mL. For the comparative level, which did not contain the adlay component composition, two samples were prepared: one in which LPS was added to the cultured cells 21 hours after the culture medium was changed to achieve a final concentration of 100 ng / mL, and another in which the cells were cultured without LPS addition. In the comparative level, the sample with added LPS was added to the cultured cells using test medium with added LPS, rather than the LPS-treated solution with LPS already added to the test solution. In the comparative level, the same procedure was performed using test medium without added LPS. After adding LPS, the cells were cultured for a further three hours.

[0063] After 3 hours, the medium containing the Job's tears component composition and LPS was removed, and a 5-fold diluted solution of cell lysis buffer (5X Cells lysis buffer) was added and mixed. Then, luciferin was added, and luciferase activity was measured based on the luminescence intensity. In Test Example 3, high luciferase activity means that LPS activated the intracellular MAPK pathway, thereby promoting the expression of the luciferase gene. In other words, high luciferase activity means that AP1 transcription activity is high.

[0064] Luciferase activity when a test solution containing a Job's tears component composition was added was calculated using the control level of luciferase activity (without Job's tears component composition and with LPS added) as 100%. If the luciferase activity was greater than 90% but less than 100% of the control level, it was considered that an effect of suppressing AP1 transcriptional activity was observed. Furthermore, if the luciferase activity was 90% or less of the control level, it was considered that an effect of suitably suppressing AP1 transcriptional activity was observed.

[0065] Figure 17 shows the evaluation results of luciferase activity in Test Example 3. In the evaluation results of AP1 transcriptional activity, the level without the addition of the Job's tears component composition and without the addition of LPS is indicated as LPS-. Furthermore, in the evaluation results of AP1 transcriptional activity, the level of the control without the addition of the Job's tears component composition and with the addition of LPS is indicated as CTL.

[0066] As shown in Figure 17, when a test solution containing a hot water extract made from whole Job's tears at a concentration of 100 μg / mL was used, the luciferase activity was 90% or less of the control level. In other words, the hot water extract made from whole Job's tears showed a favorable effect in suppressing the transcriptional activity of AP1.

[0067] Furthermore, when a test solution containing a hot water extract made from Job's tears hulls at a concentration of 100 μg / mL was used, the luciferase activity was between 90% and 100% of the control level. In other words, the hot water extract made from Job's tears hulls was found to have an effect in suppressing AP1 transcriptional activity. In addition, it was confirmed that the hot water extract using whole Job's tears grains as the raw material could suppress AP1 transcriptional activity more effectively than the hot water extract using Job's tears hulls as the raw material.

[0068] [Test Example 4] Refer to Figure 18 to explain Test Example 4. In Test Example 4, the inhibitory effect on AP1 transcriptional activity of a Job's tears component-containing composition extracted from unroasted whole Job's tears or hulls using cold water was evaluated. In Test Example 4, two levels of cold water extract were obtained as samples of the Job's tears component-containing composition, using unroasted whole Job's tears or hulls as raw materials, following the same procedure as in Test Example 2. Note that preliminary tests were omitted in Test Example 4.

[0069] In Test Example 4, the inhibitory effect of a composition containing Job's tears extract on the transcriptional activity of AP1 was evaluated using the same procedure as in Test Example 3. The concentration of the Job's tears extract composition in the test solution was set at 5 μg / mL, selected from the concentration range in which no cytotoxicity was observed in the preliminary test. The evaluation criteria were the same as in Test Example 3. Figure 18 shows the evaluation results of luciferase activity in Test Example 4.

[0070] As shown in Figure 18, when a test solution containing a cold water extract made from whole grains or hulls of Job's tears at a concentration of 5 μg / mL was used, the luciferase activity was 90% or less of the control level. In other words, a cold water extract made from whole grains or hulls of Job's tears was found to effectively suppress AP1 transcription activity. Furthermore, it was confirmed that a cold water extract using whole grains of Job's tears as the raw material could suppress AP1 transcription activity more effectively than a cold water extract using hulls of Job's tears as the raw material.

[0071] Furthermore, comparing the results with those of Test Example 3 shown in Figure 17, it was confirmed that in the case of a Job's tears component-containing composition made from whole grains or hulls of Job's tears, cold water extracts can suppress AP1 transcriptional activity to the same or greater extent than hot water extracts, even at smaller amounts and lower concentrations.

[0072] [Test Example 5] Refer to Figures 19 to 28 to explain Test Example 5. In Test Example 5, the inhibitory effect of multiple levels of Job's tears component-containing compositions, with different extraction temperatures, on the transcriptional activity of NFκB was evaluated using whole Job's tears as the raw material.

[0073] In Test Example 5, a composition containing Job's tears components was prepared using the following procedure. First, whole grains of unroasted Job's tears were ground in a mill. Then, five times the mass of Job's tears was added to the ground material in pure water. Subsequently, extraction was carried out for 1 hour at a predetermined extraction temperature using a constant temperature bath or incubator. Eight levels of extraction temperatures were used: 4°C, 15°C, 25°C, 40°C, 50°C, 60°C, 80°C, and 90°C. Next, the supernatant was collected by centrifugation using a high-speed refrigerated centrifuge under the conditions of 8000 rpm, 20°C, and 30 minutes. The collected supernatant was then subjected to suction filtration to obtain an extract of Job's tears components. Subsequently, the extract was freeze-dried to obtain a sample of the powdered Job's tears component-containing composition. By the above procedure, samples of Job's tears component-containing compositions at eight different extraction temperatures were obtained using whole grains of unroasted Job's tears as the raw material.

[0074] [Example Exam 5: Preliminary Exam] In Test Example 5, a preliminary cytotoxicity assessment was performed using the same procedure as in Test Example 1. Cells with a viability rate higher than 85% were judged to have no cytotoxicity. The results of the preliminary assessment in Test Example 5 are shown in Figures 19 to 26.

[0075] As shown in Figure 19, in the adlay component-containing composition extracted from whole adlay grains at 4°C, the cell viability compared to the control was higher than 85% at test solution concentrations of 1 μg / mL to 5 μg / mL. On the other hand, at test solution concentrations of 10 μg / mL to 100 μg / mL, the cell viability compared to the control was 85% or less. In other words, in the adlay component-containing composition extracted from whole adlay grains at 4°C, no cytotoxicity was observed when the test solution concentration was between 1 μg / mL and 5 μg / mL, but cytotoxicity was observed at test solution concentrations of 10 μg / mL or higher.

[0076] As shown in Figure 20, in the adlay component-containing composition extracted from whole adlay grains at 15°C, the cell viability compared to the control was higher than 85% at test solution concentrations between 1 μg / mL and 50 μg / mL. On the other hand, at a test solution concentration of 100 μg / mL, the cell viability compared to the control was 85% or less. In other words, in the adlay component-containing composition extracted from whole adlay grains at 15°C, no cytotoxicity was observed at test solution concentrations between 1 μg / mL and 50 μg / mL, but cytotoxicity was observed at a test solution concentration of 100 μg / mL.

[0077] As shown in Figure 21, in the adlay component-containing composition extracted from whole adlay grains at 25°C, the cell viability compared to the control was higher than 85% at test solution concentrations between 1 μg / mL and 25 μg / mL. On the other hand, at test solution concentrations between 50 μg / mL and 100 μg / mL, the cell viability compared to the control was 85% or less. In other words, in the adlay component-containing composition extracted from whole adlay grains at 25°C, no cytotoxicity was observed when the test solution concentration was between 1 μg / mL and 25 μg / mL, but cytotoxicity was observed when the test solution concentration was 50 μg / mL or higher.

[0078] As shown in Figure 22, in the adlay component-containing composition extracted from whole adlay grains at 40°C, the cell viability compared to the control was higher than 85% at test solution concentrations of 1 μg / mL to 10 μg / mL. On the other hand, at test solution concentrations of 25 μg / mL to 100 μg / mL, the cell viability compared to the control was 85% or less. In other words, in the adlay component-containing composition extracted from whole adlay grains at 40°C, no cytotoxicity was observed when the test solution concentration was between 1 μg / mL and 10 μg / mL, but cytotoxicity was observed at test solution concentrations of 25 μg / mL or higher.

[0079] As shown in Figures 23 to 26, in the adlay component-containing compositions extracted from whole adlay grains at temperatures between 50°C and 90°C, the cell viability compared to the control was higher than 85% at test solution concentrations between 1 μg / mL and 100 μg / mL. In other words, no cytotoxicity was observed at any concentration in the adlay component-containing compositions extracted from whole adlay grains at temperatures between 50°C and 90°C.

[0080] [Test Example 5: Evaluation of NFκB Transcriptional Activity] In Test Example 5, eight levels of Job's tears component-containing compositions, each extracted at a different temperature using unroasted whole Job's tears as the raw material, were evaluated for their inhibitory effect on NFκB transcriptional activity using the same procedure as in Test Example 1. The concentration of the Job's tears component-containing composition in the test solution was set at 5 μg / mL, selected from the concentration range in which no cytotoxicity was observed in preliminary tests. The evaluation criteria were the same as in Test Example 1. Figure 27 shows the evaluation results of luciferase activity in Test Example 5.

[0081] As shown in Figure 27, in a composition containing adlay components made from whole adlay grains, at extraction temperatures between 4°C and 60°C, luciferase activity was 90% or less of the control level, even at a test solution concentration of 5 μg / mL. In other words, in an adlay component-containing composition extracted from whole adlay grains at extraction temperatures between 4°C and 60°C, a suitable inhibitory effect on NFκB transcription activity was observed even at a test solution concentration of 5 μg / mL. In particular, at extraction temperatures between 4°C and 50°C, luciferase activity was 60% or less of the control level, indicating a more suitable inhibitory effect on NFκB transcription activity compared to extraction temperatures above 60°C.

[0082] On the other hand, in a composition containing adlay components made from whole adlay grains, when the extraction temperature was between 80°C and 90°C, the luciferase activity was 100% or more of the control level when the test solution concentration was 5 μg / mL. In other words, in a composition containing adlay components extracted from whole adlay grains at an extraction temperature between 80°C and 90°C, no effect of suppressing NFκB transcription activity was observed when the test solution concentration was 5 μg / mL.

[0083] In Test Example 5, an additional evaluation of the inhibitory effect on NFκB transcription activity was conducted at two extraction temperatures, 80°C and 90°C, where no inhibitory effect on NFκB transcription activity was observed when the test solution concentration was 5 μg / mL. In the additional evaluation, the concentration of the adlay component-containing composition in the test solution was changed to 100 μg / mL. The results of the additional evaluation are shown in Figure 28.

[0084] As shown in Figure 28, among the adlay component-containing compositions made from whole adlay grains, when the extraction temperature was 80°C or 90°C and the test solution concentration was 100 μg / mL, the luciferase activity was 90% or less of the control level. In other words, in adlay component-containing compositions made from whole adlay grains and extracted at an extraction temperature of 80°C to 90°C, a test solution concentration of 100 μg / mL was found to effectively suppress NFκB transcriptional activity.

[0085] In Test Example 5, the effect of extraction temperature on NFκB transcription activity when whole Job's tears grains were used as the raw material was evaluated. In Test Examples 1 and 2, even when Job's tears hulls were used as the raw material, it was confirmed that cold water extracts could suitably suppress NFκB transcription activity at smaller amounts and lower concentrations than hot water extracts. Therefore, even when Job's tears hulls are used as the raw material, it is considered that NFκB transcription activity can be more suitably suppressed if the extraction temperature is 60°C or lower.

[0086] [Test Example 6] Refer to Figure 29 to explain Test Example 6. In Test Example 6, the inhibitory effect on AP1 transcriptional activity of multiple levels of Job's tears component-containing compositions, with different extraction temperatures, was evaluated using unroasted whole Job's tears as the raw material. In Test Example 6, eight levels of Job's tears component-containing compositions were obtained using unroasted whole Job's tears as the raw material, with different extraction temperatures, following the same procedure as in Test Example 5. Note that preliminary tests were omitted in Test Example 6.

[0087] In Test Example 6, the inhibitory effect of a composition containing Job's tears components on the transcriptional activity of AP1 was evaluated using the same procedure as in Test Example 3. The concentration of the Job's tears component-containing composition in the test solution was set at 5 μg / mL, selected from the concentration range in which no cytotoxicity was observed in the preliminary test. The evaluation criteria were the same as in Test Example 3. Figure 29 shows the evaluation results of luciferase activity in Test Example 6.

[0088] As shown in Figure 29, in a composition containing Job's tears components made from whole Job's tears grains, at extraction temperatures between 4°C and 100°C, the luciferase activity was 90% or less of the control level, even at a test solution concentration of 5 μg / mL. In other words, in a composition containing Job's tears components extracted from whole Job's tears grains, a suitable inhibitory effect on AP1 transcription activity was observed regardless of the extraction temperature, even at a test solution concentration of 5 μg / mL. In particular, at extraction temperatures between 4°C and 60°C, the luciferase activity was 70% or less of the control level, indicating a more suitable inhibitory effect on AP1 transcription activity compared to extraction temperatures above 80°C.

[0089] In Test Example 6, the effect of extraction temperature on AP1 transcription activity was evaluated when whole Job's tears grains were used as the raw material. In Test Examples 3 and 4, even when Job's tears hulls were used as the raw material, it was confirmed that cold water extracts could suitably suppress AP1 transcription activity at smaller amounts and lower concentrations than hot water extracts. Therefore, even when Job's tears hulls are used as the raw material, it is considered that AP1 transcription activity can be more suitably suppressed if the extraction temperature is 60°C or lower.

[0090] [Test Example 7] Refer to Figure 30 to explain Test Example 7. In Test Example 7, the effect of multiple levels of Job's tears component-containing compositions with different extraction temperatures on inhibiting UCP1 expression was evaluated, using unroasted whole Job's tears as the raw material. In Test Example 7, following the same procedure as in Test Example 5, samples of three levels of Job's tears component-containing compositions were obtained using unroasted whole Job's tears as the raw material, with extraction temperatures of 4°C, 50°C, or 90°C. Note that preliminary tests were omitted in Test Example 7.

[0091] In Experimental Example 7, the first treatment was performed to promote the differentiation of 10T1 / 2 preadipocytes, which are precursors to brown adipocytes. Specifically, 10T1 / 2 preadipocytes (obtained from ATCC) were first cultured in 10% FBS / DMEM high-glucose medium (Nacalai Tesque Co., Ltd.) in a 6-well multi-well plate. To induce G1 arrest, which halts the cell cycle, the 10T1 / 2 preadipocytes were cultured for more than 24 hours without changing the medium even after reaching confluence. Subsequently, the medium was changed to differentiation induction medium, and differentiation induction stimulation was performed by culturing under conditions of 24 to 48 hours. The differentiation induction medium used was a 10% FBS / DMEM high-glucose medium containing 0.5 mM 3-isobutyl-1-methylxanthine (Nacalai Tesque Co., Ltd.), 0.25 μM dexamethasone (Nacalai Tesque Co., Ltd.), 10 μg / mL insulin (Nacalai Tesque Co., Ltd.), and 1 μM pioglitazone (Sigma Aldrich). After differentiation induction stimulation, the medium was replaced with a differentiation-promoting medium and cultured for 5 days. The differentiation-promoting medium used was a 10% FBS / DMEM high-glucose medium containing 5 μg / mL insulin.

[0092] Next, the second treatment was performed. Specifically, for each extraction temperature of Job's tears, a test solution was obtained by adding a Job's tears component-containing composition to the culture medium for the test solution. In Test Example 7, a 10% FBS / DMEM high-glucose medium was used as the culture medium for the test solution. For Job's tears component-containing compositions extracted at 4°C or 50°C, a concentration of 5 μg / mL was adopted as the concentration in the test solution from the concentration range in which no cytotoxicity was observed in the preliminary test of Test Example 5. For Job's tears component-containing compositions extracted at 90°C, a concentration of 100 μg / mL was adopted as the concentration in the test solution from the concentration range in which no cytotoxicity was observed in the preliminary test of Test Example 5.

[0093] Next, mouse macrophage-like cells, RAW264.7 cells, were cultured in 10% FBS / DMEM medium (Nacalai Tesque Co., Ltd.) in 100 mm dishes. When the RAW264.7 cells occupied approximately 60% to 70% of the adhesion surface of the culture vessel, the medium was replaced with a test solution containing the adlay component composition and the test solution medium. For comparison, a control level was also prepared in which the medium was removed from the cell culture plate and replaced with a test solution medium that did not contain the adlay component composition.

[0094] Eighteen hours after the medium change, 1 mg / mL of LPS was added to the cultured cells to a 1000-fold dilution, i.e., to a final LPS concentration of 1 μg / mL. Six hours after initiating inflammation induction with LPS, i.e., 24 hours after adding the adlay component-containing composition, the medium was removed and replaced with serum-free DMEM high-glucose medium. After another 24 hours, the culture supernatant was collected, and any contaminating cells were removed by centrifugation at 800G for 5 minutes. The culture supernatant after centrifugation was mixed with an equal volume of 20% FBS / DMEM high-glucose medium to obtain four levels of culture supernatant for addition, including a comparative level.

[0095] Next, a third treatment was performed. In the third treatment, the culture medium of 10T1 / 2 preadipocytes cultured in differentiation-promoting medium for 5 days in the first treatment was removed and replaced with four levels of culture supernatant prepared in the second treatment. Twenty hours after the replacement with the culture supernatant, isopreterenol (Sigma Aldrich), an adrenaline receptor activator, was added to the culture medium of the 10T1 / 2 preadipocytes to a final concentration of 10 μM. Here, 10 mM isopreterenol was added at a 1000-fold dilution. Four hours after initiating stimulation with isopreterenol, i.e., 24 hours after adding the culture supernatant, the medium was removed and total RNA was extracted using Sepazol (Nacalai Tesque Co., Ltd.) according to the prescribed protocol. cDNA was synthesized using the extracted RNA by reverse transcription. The expression level of the UCP1 gene was quantified by real-time PCR on the obtained cDNA. The evaluation results of the UCP1 gene expression level in Test Example 7 are shown in Figure 30.

[0096] Furthermore, when using the culture supernatant to which the test solution containing the Job's tears component composition was added, the UCP1 expression level was calculated using the control level, which used the culture supernatant without the Job's tears component composition, as 100% for comparison. In the evaluation results of UCP1 expression levels, the control level is indicated as CTL.

[0097] The control group represents a level where UCP1 expression is inhibited by inflammation induced by LPS. Furthermore, since the control group does not contain the adlay component composition, it represents a level where LPS-induced inflammation is not suppressed by the adlay component composition. Therefore, if the UCP1 expression level is greater than 100%, it can be considered that the adlay component composition is suppressing the inhibition of UCP1 expression by LPS-induced inflammation. In Test Example 7, a UCP1 expression level of 200% or more above the control level was considered to indicate a favorable suppression of the inhibition of UCP1 expression by inflammation.

[0098] In the third treatment, for reference, a level was also prepared in which the culture medium of 10T1 / 2 preadipocytes cultured for 5 days in differentiation-promoting medium in the first treatment was removed and replaced with 20% FBS / DMEM high-glucose medium without the added culture supernatant. At the reference level, 20 hours after the medium change, samples were prepared in which isopreterenol was added to the culture medium of 10T1 / 2 preadipocytes to a final concentration of 10 μM, and in which no isopreterenol was added. In the evaluation results of UCP1 expression levels, the sample with isopreterenol added at the reference level is indicated as ISO+, and the sample without isopreterenol at the reference level is indicated as ISO-. Since these reference levels do not contain the added culture supernatant, LPS and the adlay component-containing composition are not added.

[0099] As shown in Figure 30, when whole Job's tears grains were used as the raw material and the extraction temperature was 4°C or 50°C, a test solution concentration of 5 μg / mL resulted in a UCP1 expression level of more than 200% compared to the control level. Furthermore, when whole Job's tears grains were used as the raw material and the extraction temperature was 90°C, a test solution concentration of 100 μg / mL also resulted in a UCP1 expression level of more than 200% compared to the control level. Therefore, at these levels, an effect of suppressing the inhibition of UCP1 expression in brown adipose tissue was confirmed. Additionally, it was confirmed that a Job's tears component-containing composition extracted at an extraction temperature of 4°C to 50°C could suppress the inhibition of UCP1 expression in brown adipose tissue at a smaller amount and lower concentration than the Job's tears component-containing composition extracted at 90°C.

[0100] In Test Example 7, the expression level of UCP1 was evaluated when whole Job's tears grains were used as the raw material. In Test Examples 1-4, it was confirmed that, similar to when whole Job's tears grains were used as the raw material, the transcriptional activity of NFκB and AP1 could be suitably suppressed when Job's tears hulls were used as the raw material. Therefore, it is considered that even when Job's tears hulls are used as the raw material, the effect of suppressing the inhibition of UCP1 expression in brown adipose tissue can be obtained. Similarly, in Test Examples 1 and 2, it was confirmed that even when polished Job's tears grains were used as the raw material, the transcriptional activity of NFκB and AP1 could be suppressed. Therefore, it is considered that even when polished Job's tears grains are used as the raw material, the effect of suppressing the inhibition of UCP1 expression in brown adipose tissue can be obtained.

[0101] [Effects of the Embodiment] (1) According to the adlay component-containing composition produced by the manufacturing method of this embodiment, the components extracted from adlay suppress the transcriptional activity of NFκB and AP1 in macrophages. Therefore, according to the adlay component-containing composition of this embodiment, the inhibition of UCP1 expression caused by inflammatory cytokines in brown adipocytes can be suppressed by suppressing the expression of inflammatory cytokines in macrophages.

[0102] (2) By using the hull or whole grain of Job's tears as a raw material for the Job's tears component-containing composition, the transcriptional activity of NFκB can be more effectively suppressed than when polished Job's tears grains are used as a raw material. In other words, by using the hull or whole grain of Job's tears as a raw material for the Job's tears component-containing composition, the inhibition of UCP1 expression caused by inflammatory cytokines can be more effectively suppressed than when polished Job's tears grains are used as a raw material.

[0103] (3) By using whole grains of Job's tears as a raw material for the Job's tears component-containing composition, the transcriptional activity of NFκB and AP1 can be suppressed more effectively than when using Job's tears hulls as a raw material. In other words, by using whole grains of Job's tears as a raw material for the Job's tears component-containing composition, the inhibition of UCP1 expression caused by inflammatory cytokines can be suppressed more effectively than when using Job's tears hulls as a raw material.

[0104] (4) In the method for producing a composition containing Job's tears components, by setting the temperature of the water in which the Job's tears are immersed (i.e., the extraction temperature) to 60°C or lower, the transcriptional activity of NFκB and AP1 can be suitably suppressed. That is, the expression of inflammatory cytokines can be more suitably suppressed, and therefore the inhibition of UCP1 expression caused by inflammatory cytokines can be more suitably suppressed. In particular, if the extraction temperature is 50°C or lower, the inhibitory effect on the transcriptional activity of NFκB can be further enhanced, and therefore the inhibition of UCP1 expression caused by inflammatory cytokines can be even more suitably suppressed.

[0105] (5) By using unroasted adlay as the raw material, the effect of suppressing NFκB transcription activity can be further enhanced compared to when roasted adlay is used as the raw material. [Example of changes] The above embodiment can be implemented with the following modifications. Furthermore, the following modifications can be combined to the extent that they do not contradict the technical standards.

[0106] • In the above test example, the cytotoxicity evaluation conducted as a preliminary test is one example of a method for determining the appropriate concentration when conducting cell tests in the test example. Therefore, the concentration range determined by the cytotoxicity evaluation in the above test example does not limit the concentration of the coix seed extract-containing composition in each form of use of the coix seed extract-containing composition. In other words, the concentration of the coix seed extract-containing composition in each form of use of the coix seed extract-containing composition may be adjusted as appropriate.

[0107] • Compositions containing Job's tears extract may also contain additives such as excipients, binders, thickeners, emulsifiers, colorants, and flavorings.

Claims

1. A method for producing a composition containing Job's tears components to suppress the inhibition of UCP1 (Uncoupling protein 1) expression, This includes extracting components contained in unroasted Job's tears by soaking them in water. A method for producing a composition containing Job's tears components.

2. In the aforementioned adlay component-containing composition, the component extracted from adlay inhibits at least one of the transcriptional activity of NFκB (Nuclear factor kappa B) and AP1 (Activator protein 1). A method for producing the adlay component-containing composition according to claim 1.

3. Soaking the Job's tears in the water includes soaking the hulls or the whole grains of the Job's tears in the water. A method for producing a composition containing Job's tears components according to claim 1 or 2.

4. The temperature of the water in which the adlay is immersed is 60°C or lower. A method for producing a composition containing Job's tears components according to claim 1 or 2.

5. The temperature of the water in which the Job's tears are immersed is 50°C or lower. A method for producing a composition containing Job's tears components according to claim 1 or 2.

6. A composition for suppressing the inhibition of UCP1 expression, The aforementioned composition contains components found in unroasted Job's tears. Composition containing Job's tears extract.

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