Food and drink composition and use thereof

Abstract

The invention relates to a food and drink composition and a use thereof. The present invention addresses the problem of providing a novel technique for suppressing aging. [Solution] The present invention provides, for example, a food and drink composition comprising longan aril and pyrroloquinoline quinone.

Description

Technical Field

[0001] The present invention relates to a food and beverage composition, an anti-aging agent, a mitochondrial dysfunction improving agent, a nicotinamide phosphoribosyltransferase production promoter, a nicotinamide adenine dinucleotide production promoter, a sirtuin 1 production promoter, and a melanin production inhibitor. Background Art

[0002] With the recent development of an aging society, the importance of technologies for suppressing aging is increasing.

[0003] For example, Patent Document 1 focuses on nicotinamide phosphoribosyltransferase (NAMPT), an enzyme associated with aging inhibition, and proposes an NAMPT activator comprising one or more active ingredients selected from the group consisting of grape seed, milk thistle, Korean ginseng, houttuynia cordata, guava, pomegranate, HMB-Ca, bilberry, cinnamon bark, gardenia fruit, mulberry leaf, longan pulp, jujube, NMN, and Peucedanum chinense.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application Laid-Open No. 2024-022503 Summary of the Invention

[0007] Problems to be solved by the invention

[0008] However, there is a further need for technologies for inhibiting aging.

[0009] The present invention has been made in view of the above-mentioned actual situation, and its object is to provide a new aging-inhibiting technology.

[0010] Solutions for solving problems

[0011] The present inventors conducted research and found that the above-mentioned problems can be solved by combining longan pulp and pyrroloquinoline quinone, thereby completing the present invention. More specifically, the present invention provides the following.

[0012] (1) A food or beverage composition comprising longan pulp and pyrroloquinoline quinone.

[0013] (2) The food and drink composition according to (1), wherein the mass ratio of the component derived from the longan pulp to the pyrroloquinoline quinone is 5:1 to 30:1.

[0014] (3) An anti-aging agent comprising longan pulp and pyrroloquinoline quinone.

[0015] (4) A mitochondrial dysfunction improving agent comprising longan pulp and pyrroloquinoline quinone.

[0016] (5) A nicotinamide phosphoribosyltransferase production promoter comprising longan pulp and pyrroloquinoline quinone.

[0017] (6) A nicotinamide adenine dinucleotide production promoter comprising longan pulp and pyrroloquinoline quinone.

[0018] (7) A Sirtuin1 production promoter comprising longan pulp and pyrroloquinoline quinone.

[0019] (8) A melanin production inhibitor comprising longan pulp and pyrroloquinoline quinone.

[0020] Effects of the Invention

[0021] According to the present invention, a new aging suppression technology is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph showing the results of the effects on the amount of melanin production in Examples.

[0023] Figure 2 This is a graph showing the results of the effects on the amount of NAMPT produced in Examples.

[0024] Figure 3 It represents the NAD in the embodiment + A graph showing the results of the effect of volume is generated.

[0025] Figure 4 This is a graph showing the results of the effects on IL-1β production in Examples.

[0026] Figure 5 This is a graph showing the results of the effects on the amount of GDF-15 produced in Examples.

[0027] Figure 6 This is a graph showing the results of the effects on NLRP3 production in Examples.

[0028] Figure 7 This is a diagram showing the results of the influence on the Sirt1 production amount in the examples.

[0029] Figure 8 It is a graph showing the results of the influence on the amount of Per2 produced in the Examples.

[0030] Figure 9 It represents the NAD in the embodiment + A graph showing the results of the effect of volume is generated.

[0031] Figure 10 This is a graph showing the results of the effects on ATP production in Examples.

[0032] Figure 11 This is a graph showing the results of the effects on lactic acid values ​​in Examples.

[0033] Figure 12 This is a graph showing the results of the influence on the oxygen consumption rate in Examples.

[0034] Figure 13 This is a graph showing the results of the effects on cell viability in Examples.

[0035] Figure 14 This is a graph showing the results of the influence on SPiDER-βGal in Examples. DETAILED DESCRIPTION

[0036] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited thereto.

[0037] In addition, the following abbreviations may be used below.

[0038] Pyrroloquinoline quinone: PQQ

[0039] Nicotinamide phosphoribosyltransferase: NAMPT

[0040] Nicotinamide adenine dinucleotide: NAD +

[0041] Niacinamide: NAM

[0042] β-Nicotinamide mononucleotide: NMN

[0043] Interleukin-1β: IL-1β

[0044] Growth Differentiation Factor 15: GDF-15

[0045] Pyrin domain-containing protein 3: NLRP3

[0046] Adenosine triphosphate: ATP

[0047] Oxygen consumption rate: OCR

[0048] 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide: MTT

[0049] Sirtuin1: Sirt1

[0050] Clock gene Period2: Per2

[0051] Intracellular retention β-galactosidase detection fluorescent probe: SPiDER-βGal

[0052] (1) Food and Beverage Composition

[0053] The food and drink composition of the present invention contains longan pulp and pyrroloquinoline quinone.

[0054] As described above, it has been reported that longan meat and the like independently have an NAMPT-activating effect.

[0055] Meanwhile, the present inventors have found the following surprising finding: by combining longan pulp with pyrroloquinoline quinone, not only can the effect of promoting NAMPT production be exerted, but also various effects related to the inhibition of aging can be exerted.

[0056] In the present invention, the "aging-inhibiting effect" includes promoting the production of substances known to be involved in aging inhibition, inhibiting the production of substances known to be involved in aging acceleration, and various phenomena resulting from these results.

[0057] For example, in one embodiment of the present invention, the "aging-inhibiting effect" includes the following effects, the anti-aging effect exhibited by the anti-aging agent of the present invention described later, and the like.

[0058] ・Inhibitory effect on melanin production and the resulting skin whitening effect.

[0059] · Promotes the production of NAMPT, and the resulting skin whitening effects, antioxidant effects, life extension effects, damage repair effects, etc.

[0060] · Promote NAD + The effects produced, as well as the subsequent effects of improving mitochondrial dysfunction, skin whitening effects, antioxidant effects, life extension effects, damage repair effects, etc.

[0061] Hereinafter, the constitution of the food and drink composition of the present invention will be described in detail.

[0062] (1-1) Longan meat

[0063] Longan meat (scientific name: Euphoria longana Lamarck (Sapindaceae), or Dimocarpus longan L.) is a fruit tree of the genus Dimocarpus in the family Sapindaceae, also known as "longan."

[0064] Regarding longan pulp, its aril (the skin covering the seed coat produced by the thickening of the funicle and placenta) and its extracts have been used in foods and drinks.

[0065] In the present invention, any longan pulp that has been used conventionally can be utilized.

[0066] The form of longan pulp is not particularly limited and may be in powder, liquid, solid, paste, or the like.

[0067] In a preferred embodiment of the present invention, the longan pulp is a water extract of aril.

[0068] The aqueous extract can be appropriately subjected to filtration, concentration, sterilization, powderization, etc. Furthermore, an excipient (such as dextrin) can be blended as needed.

[0069] In a preferred embodiment of the present invention, longan pulp is a powder obtained by the following steps.

[0070] The aril of longan pulp was used as the raw material, and it was processed in the order of water extraction, filtration, concentration (accompanied by pectinase treatment) and sterilization (125±10℃).

[0071] Next, after optionally adding an excipient, spray drying and granulation are performed, and the obtained powder is recovered as longan pulp extract powder.

[0072] As the longan pulp, commercially available products (such as the longan pulp extract powder used in the Examples) can be used.

[0073] The content of the longan pulp in the food and drink composition is not particularly limited and can be appropriately adjusted depending on the desired effect.

[0074] The lower limit of the content of longan pulp (dry mass of components derived from longan pulp) is preferably 0.2% by mass or more, and more preferably 1.0% by mass or more, based on the food and drink composition.

[0075] The upper limit of the content of longan pulp (dry mass of components derived from longan pulp) is preferably 10.0% by mass or less, and more preferably 5.0% by mass or less, based on the food and drink composition.

[0076] In the present invention, the “dry mass of components derived from longan flesh” refers to the total amount of components excluding water in the longan flesh blended in the food-drink composition of the present invention.

[0077] (1-2)Pyrroloquinoline quinone

[0078] PQQ(C 14 H6N2O8, CAS registration number: 72909-34-3) functions as a redox coenzyme in vivo, and its effects include antioxidant and neuroprotective effects.

[0079] However, the present inventors have discovered for the first time that a combination of PQQ and longan pulp can exert a good aging-inhibiting effect.

[0080] The PQQ in the present invention includes not only free forms of PQQ but also salts of PQQ. These free forms and salts may be used alone or in combination of two or more.

[0081] The salt of PQQ includes any salt that can be used as a food or beverage.

[0082] Preferred salts include pyrroloquinoline quinone disodium salt and the like.

[0083] The content of PQQ in the food or beverage composition is not particularly limited and can be appropriately adjusted depending on the desired effect.

[0084] The lower limit of the PQQ content (amount calculated as free PQQ) is preferably 0.00044% by mass or more, and more preferably 0.02200% by mass or more, based on the food or drink composition.

[0085] The upper limit of the PQQ content (amount calculated as free PQQ) is preferably 0.44000% by mass or less, and more preferably 0.22000% by mass or less, based on the food or drink composition.

[0086] In the present invention, the "amount calculated as free PQQ" refers to the amount of PQQ added to the food and drink composition of the present invention calculated as free PQQ.

[0087] The amount of free PQQ in the food or drink composition is calculated based on the content of the blended pyrroloquinoline quinone and the molecular weight of pyrroloquinoline quinone or a salt thereof.

[0088] (1-3) Total amount and ratio of longan pulp and pyrroloquinoline quinone

[0089] The total amount and ratio of longan pulp and PQQ to be added to the food and beverage composition are not particularly limited and can be appropriately set depending on the desired effect and the like.

[0090] From the viewpoint of more easily exhibiting the effects of the present invention, it is preferred to increase the ratio of longan pulp to PQQ.

[0091] The mass ratio of the longan pulp-derived component (dry mass of the longan pulp-derived component) to PQQ (as free PQQ) in the food and drink composition is preferably 5:1 to 30:1, more preferably 10:1 to 20:1, and even more preferably 12.5:1 to 15:1.

[0092] According to the present invention, compared with the case where longan pulp or PQQ is added alone, an effective anti-aging effect can be achieved even when the total amount of longan pulp and PQQ is lower.

[0093] From this viewpoint, the lower limit of the total amount of longan pulp-derived components (dry mass of longan pulp-derived components) and PQQ (equivalent amount of free PQQ) is preferably 0.5% by mass or more, more preferably 1.0% by mass or more, based on the food and drink composition.

[0094] The upper limit of the total amount of the longan pulp-derived component (dry mass of the longan pulp-derived component) and PQQ (amount calculated as free PQQ) is preferably 10.0% by mass or less, more preferably 2.0% by mass or less, based on the food or drink composition.

[0095] (1-4) Other ingredients

[0096] The food and drink composition may or may not contain ingredients other than longan pulp and PQQ within a range not impairing the effects of the present invention.

[0097] Ingredients other than longan pulp and PQQ can be appropriately selected according to the form of the food and drink composition, and any ingredients used as food and drink raw materials are included.

[0098] Examples of such components include pigments, antioxidants, excipients, colorants, surfactants, preservatives, and pH adjusters.

[0099] One embodiment of the present invention includes an embodiment in which the food and drink composition consists only of longan pulp and PQQ.

[0100] (1-5) Form of food and beverage composition

[0101] The form of the food and drink composition is not particularly limited, and any form is encompassed.

[0102] Examples of the form of the food and drink composition include powder, liquid, solid, granular, particulate, paste, and gel.

[0103] Specific examples of food and beverage compositions include nutritional supplements, cooked foods, seasonings, soft drinks, jelly drinks, and confectionery (eg, soft candies, candies, Japanese confectionery, and Western confectionery).

[0104] The amount and frequency of intake of the food and beverage composition are not particularly limited and can be appropriately set depending on the desired effect and the like.

[0105] (1-6) Method for producing food and beverage composition

[0106] The method for producing the food-drink composition is not particularly limited, and any method can be employed depending on the type of the food-drink composition.

[0107] (2) Anti-aging agents

[0108] The anti-aging agent of the present invention comprises longan pulp and pyrroloquinoline quinone.

[0109] In the present invention, the "anti-aging (effect)" includes a change (increase or decrease) in an aging marker, suppression of a change in an aging marker, and the like.

[0110] As an aging marker whose increase shows anti-aging, NAD can be listed. + , ATP, OCR, cell viability (MTT), Sirt1, etc.

[0111] Examples of aging markers whose reduction indicates anti-aging include lactic acid, SPiDER-βGal, GDF-15, IL-1β, and NLRP3.

[0112] It should be noted that among the above-mentioned aging markers, changes in Sirt1 may affect the expression of Per2 (Nakahata Y, Kaluzova M (2008) Cell 134:329-340, etc.). For example, an increase in Sirt1 leads to a decrease in Per2, which can result in the formation of a circadian rhythm. Therefore, Per2 can also be used as an indirect indicator of anti-aging.

[0113] The presence or absence and degree of the anti-aging effect can be evaluated by culturing using a three-dimensional skin model containing melanocytes as described in the Examples or by culturing using human skin fibroblasts.

[0114] (2-1) Ingredients contained in anti-aging agents

[0115] As the components contained in the anti-aging agent, the same constitution as that of the above-mentioned food and drink composition can be adopted.

[0116] The anti-aging agent may or may not contain ingredients other than longan pulp and PQQ within a range not inhibiting the effects of the present invention.

[0117] One embodiment of the present invention includes an embodiment in which the anti-aging agent consists only of longan pulp and PQQ.

[0118] (2-2) Form of Anti-aging Agent

[0119] The form of the anti-aging agent is not particularly limited, and includes any form (food and drink compositions, etc.) (excluding pharmaceuticals).

[0120] When the anti-aging agent is a food or beverage composition, the same configuration as that of the above-mentioned food or beverage composition can be adopted.

[0121] (2-3) Method for producing anti-aging agent

[0122] The method for producing the anti-aging agent is not particularly limited, and any method can be employed according to the form of the preparation.

[0123] (3) Mitochondrial dysfunction improvers

[0124] The mitochondrial dysfunction improving agent of the present invention comprises longan pulp and pyrroloquinoline quinone.

[0125] Mitochondria are organelles that play a vital role in living organisms, including producing ATP. In recent years, reports have linked mitochondrial dysfunction to cellular aging.

[0126] According to the present invention, since the effect of improving mitochondrial dysfunction in a living body is exerted, an effect of suppressing aging by improving mitochondrial function can be expected.

[0127] In the present invention, "improvement of mitochondrial dysfunction (effect)" includes improvement of mitochondrial function in a living body compared to a case where the mitochondrial dysfunction-improving agent of the present invention is not taken.

[0128] Examples of markers showing improvement in mitochondrial dysfunction when the levels thereof increase include ATP and OCR.

[0129] Examples of markers showing improvement in mitochondrial dysfunction when the levels thereof are reduced include lactic acid and GDF-15.

[0130] The presence and extent of the effect of improving mitochondrial dysfunction can be evaluated by culturing using a three-dimensional skin model containing melanocytes as described in the Examples or by culturing using human skin fibroblasts.

[0131] (3-1) Components contained in mitochondrial dysfunction improving agents

[0132] As the components contained in the mitochondrial dysfunction-improving agent, the same configuration as that of the above-mentioned food and drink composition can be adopted.

[0133] The mitochondrial dysfunction-improving agent may or may not contain ingredients other than longan pulp and PQQ, as long as the effects of the present invention are not inhibited.

[0134] One embodiment of the present invention includes an embodiment in which the mitochondrial dysfunction improving agent consists only of longan pulp and PQQ.

[0135] (3-2) Forms of Mitochondrial Dysfunction Improvement Agents

[0136] The form of the mitochondrial dysfunction-improving agent is not particularly limited, and includes any form (food and drink compositions, etc.) (excluding pharmaceuticals).

[0137] When the mitochondrial dysfunction-improving agent is a food or beverage composition, the same configuration as that of the above-mentioned food or beverage composition can be employed.

[0138] (3-3) Method for producing a mitochondrial dysfunction improving agent

[0139] The method for producing the mitochondrial dysfunction-improving agent is not particularly limited, and any method can be employed according to the form of the preparation.

[0140] (4) Nicotinamide phosphoribosyltransferase production promoter

[0141] The NAMPT production promoter of the present invention comprises longan pulp and pyrroloquinoline quinone.

[0142] NAMPT is an enzyme that converts NAM to NMN and controls the production of NAD (described below). It is suggested that the activation of NAMPT is useful for the treatment and improvement of skin diseases and conditions through anti-aging, life extension, and damage repair.

[0143] According to the present invention, since the effect of promoting NAMPT production in the body is exerted, an effect of suppressing aging can be expected through improvement of various biological functions by NAMPT.

[0144] For example, promoting NAD + It can inhibit the production of melanin.

[0145] In the present invention, the "effect of promoting NAMPT production" includes enhancement of NAMPT gene expression, enhancement of the NAMPT production mechanism, etc., such as enhancement of the amount of NAMPT produced in the body compared to the case where the NAMPT production promoter of the present invention is not ingested.

[0146] The presence or extent of the effect of promoting NAMPT production can be evaluated by culturing using a three-dimensional skin model containing melanocytes as described in the Examples or by culturing using human skin fibroblasts.

[0147] (4-1) Components contained in the NAMPT production promoter

[0148] As the components contained in the NAMPT production promoter, the same configuration as that of the above-mentioned food and drink composition can be adopted.

[0149] The NAMPT production promoter may or may not contain ingredients other than longan pulp and PQQ as long as the effects of the present invention are not inhibited.

[0150] One embodiment of the present invention includes an embodiment in which the NAMPT production promoter consists only of longan pulp and PQQ.

[0151] (4-2) Form of NAMPT production promoter

[0152] The form of the NAMPT production promoter is not particularly limited, and includes any form (food and drink compositions, etc.) (excluding pharmaceuticals).

[0153] When the NAMPT production promoter is a food or beverage composition, the same configuration as that of the above-mentioned food or beverage composition can be employed.

[0154] (4-3) Method for producing NAMPT production promoter

[0155] The method for producing the NAMPT production promoter is not particularly limited, and any method can be employed according to the form of the preparation.

[0156] (5) Nicotinamide adenine dinucleotide production promoter

[0157] NAD of the present invention + The production promoter comprises longan pulp and pyrroloquinoline quinone.

[0158] NAD + It is an important coenzyme that exists in all parts of the body and plays a role in energy metabolism, cell metabolism, gene expression, DNA repair, etc. + Since it has a large molecular weight, it is difficult to be taken in even if it is administered externally. In addition, there are reports of problems with degradation in the intestine.

[0159] According to the present invention, due to the promotion of NAD + The effect produced can be expected through NAD + It improves various biological functions and inhibits the effect of aging.

[0160] For example, promoting NAD + It can inhibit the production of melanin.

[0161] In the present invention, "promoting NAD + The effects produced include: + Hyperexpression of genes, NAD + The enhancement of the production mechanism, etc., and not taking the NAD of the present invention + Compared with the case of producing promoters, NAD + Production volume is promoted, etc.

[0162] Boosting NAD + The presence or absence and extent of the effect can be evaluated by culturing using a three-dimensional skin model containing melanocytes as described in the Examples or by culturing using human skin fibroblasts.

[0163] (5-1)NAD + Ingredients contained in the production accelerator

[0164] As NAD + The components contained in the production promoter can have the same configuration as that of the above-mentioned food and drink composition.

[0165] NAD + The production promoter may or may not contain ingredients other than longan pulp and PQQ within a range not impairing the effects of the present invention.

[0166] One embodiment of the present invention comprises NAD + The production enhancer consists only of longan pulp and PQQ.

[0167] (5-2)NAD + Form of the promoter

[0168] NAD + The form of the production promoter is not particularly limited, and includes any form (food and drink compositions, etc.) (excluding pharmaceuticals).

[0169] NAD + When the production promoter is a food or beverage composition, the same configuration as that of the above-mentioned food or beverage composition can be employed.

[0170] (5-3)NAD + Production accelerator manufacturing method

[0171] NAD + The method for producing the production accelerator is not particularly limited, and any method can be employed according to the form of the preparation.

[0172] (6) Sirtuin1 production promoter

[0173] The Sirtuin1 production promoter of the present invention comprises longan pulp and pyrroloquinoline quinone.

[0174] Sirtuin 1 is NAD + One of the β-dependent deacetylases, also known as the longevity gene.

[0175] In the present invention, the "effect of promoting Sirtuin 1 production" includes an increase in Sirtuin 1 gene expression, an increase in the amount of Sirtuin 1 produced in a living body compared to a case where the Sirtuin 1 production promoter of the present invention is not ingested, and the like.

[0176] The presence and extent of the effect of promoting Sirtuin 1 production can be evaluated by culturing the three-dimensional skin model containing melanocytes described in the Examples or by culturing human skin fibroblasts.

[0177] (6-1) Components of Sirtuin1 Production Promoters

[0178] As the components contained in the Sirtuin 1 production promoter, the same configuration as that of the above-mentioned food and drink composition can be adopted.

[0179] The sirtuin 1 production promoter may or may not contain ingredients other than longan pulp and PQQ, as long as the effects of the present invention are not inhibited.

[0180] One embodiment of the present invention includes an embodiment in which the sirtuin 1 production promoter consists only of longan pulp and PQQ.

[0181] (6-2) Morphology of Sirtuin1 Production Promoter

[0182] The form of the sirtuin 1 production promoter is not particularly limited, and includes any form (food and drink compositions, etc.) (excluding pharmaceuticals).

[0183] When the Sirtuin 1 production promoter is a food or beverage composition, the same configuration as the above-mentioned food or beverage composition can be employed.

[0184] (6-3) Method for producing a sirtuin1 production promoter

[0185] The method for producing the sirtuin 1 production promoter is not particularly limited, and any method can be employed depending on the form of the preparation.

[0186] (7) Melanin production inhibitors

[0187] The melanin production inhibitor of the present invention contains longan pulp and pyrroloquinoline quinone.

[0188] Melanin (C 18 H 10 N2O4 (CAS No.: 8049-97-6) is a colored substance produced by melanocytes (pigment cells) present in the skin and other parts of the body and may contribute to aging of the skin and other parts of the body.

[0189] In the present invention, the "effect of inhibiting melanin production" includes: reducing the amount of melanin produced in the body compared to the case where the melanin production inhibitor of the present invention is not taken, such as through reducing anti-inflammatory effects, reducing melanin gene expression, inhibiting the melanin production mechanism in melanocytes, etc.

[0190] In the present invention, the "effect of suppressing melanin production" includes a change (increase or decrease) in a melanin production marker, suppression of a change in a melanin production marker, and the like.

[0191] Examples of melanin production markers whose reduction shows an effect of suppressing melanin production include IL-1β and NLRP3.

[0192] The presence or absence and extent of the effect of suppressing melanin production can be evaluated by culturing the three-dimensional skin model containing melanocytes as described in the Examples.

[0193] (7-1) Ingredients contained in melanin production inhibitors

[0194] As the components contained in the melanin production inhibitor, the same configuration as that of the above-mentioned food and drink composition can be adopted.

[0195] The melanin production inhibitor may or may not contain ingredients other than longan pulp and PQQ as long as the effects of the present invention are not inhibited.

[0196] One embodiment of the present invention includes an embodiment in which the melanin production inhibitor consists only of longan pulp and PQQ.

[0197] (7-2) Forms of Melanin Production Inhibitors

[0198] The form of the melanin production inhibitor is not particularly limited, and includes any form (food and drink compositions, etc.) (excluding pharmaceuticals).

[0199] When the melanin production inhibitor is a food or beverage composition, the same configuration as that of the above-mentioned food or beverage composition can be adopted.

[0200] (7-3) Method for producing a melanin production inhibitor

[0201] The method for producing the melanin production inhibitor is not particularly limited, and any method can be employed according to the form of the preparation.

[0202] Example

[0203] Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[0204] <Test 1 using a three-dimensional skin model containing melanin>

[0205] Evaluations were performed under various addition conditions using a three-dimensional skin model containing melanin by the following method.

[0206] (1) Sample preparation

[0207] The following three types of samples were prepared and used for the culture test described below.

[0208] (1-1) Longan meat sample

[0209] Longan pulp extract powder (manufactured by Matsuura Pharmaceutical Co., Ltd.) was prepared.

[0210] The sample is a powder sample obtained by the following operation: using the aril of longan pulp (scientific name: Euphoria longana (Sapindaceae)) as the base material, it is processed in the order of water extraction, filtration, concentration (accompanied by pectinase treatment) and sterilization (125±10℃), and then dextrin is added, spray-dried and granulated (sieve: opening 0.5mm, accompanied by heavy metal removal using magnet treatment).

[0211] It should be noted that the longan pulp extract powder used in this example contains components derived from longan pulp and an excipient (dextrin). Therefore, the concentration of longan pulp below represents the amount (dry weight) converted into components derived from longan pulp.

[0212] (1-2) Pyrroloquinoline quinone sample

[0213] Pyrroloquinoline quinone disodium salt (purity 99.0% or more, manufactured by Ryusendo Co., Ltd.) was prepared. In addition, pyrroloquinoline quinone disodium salt is also described as "PQQ" hereinafter.

[0214] This sample is a powder sample obtained through fermentation production using microbial fermentation and purification steps (extraction with aqueous ethanol, concentration, drying, sieving, etc.).

[0215] Hereinafter, the concentration of the pyrroloquinoline quinone sample (sodium salt) indicates the amount converted to the free form of pyrroloquinoline quinone.

[0216] (1-3) Positive control sample

[0217] As a positive control substance, L-(-) ascorbic acid (powder sample, manufactured by Kanto Chemical Co., Ltd.) was prepared. In addition, L-(-) ascorbic acid is also described as "VC" hereinafter.

[0218] (2) Culture test

[0219] In this example, the following method (system using a three-dimensional skin model) was commonly employed as a culture method.

[0220] The culture was performed in an incubator at 37°C and 5% CO2 for 3 weeks using a three-dimensional skin model containing melanocytes ("MEL-300", manufactured by KURABO INDUSTRIES LTD.) under the conditions of adding specified ingredients.

[0221] EPI-100NMM113 was used as the culture medium. This medium contains bFGF, α-MSH, and KGF as melanin-stimulating factors. These components function as melanin-forming factors, inflammatory factors, and external stimuli in the main culture system.

[0222] The culture medium was replaced every other day, and the test substance was dissolved in the culture medium each time. The biological activity of the test substance was evaluated from the bottom side of the skin model, which was assumed to be used for food.

[0223] The conditions for adding components to the culture medium are as follows.

[0224] Control: culture medium only

[0225] VC(100): Positive control sample 100 μg / ml

[0226] Longan meat (30): Longan meat sample 30μg / ml

[0227] Longan meat (100): Longan meat sample 100μg / ml

[0228] PQQ(5): Pyrroloquinoline quinone sample 5 μg / ml

[0229] PQQ(10): Pyrroloquinoline quinone sample 10 μg / ml

[0230] Longan meat (50) + PQQ (5): Longan meat sample 50 μg / ml and pyrroloquinoline quinone sample 5 μg / ml

[0231] Longan meat (15) + PQQ (2.5): Longan meat sample 15μg / ml and pyrroloquinoline quinone sample 2.5μg / ml

[0232] Four wells (n=4) were set for each sample.

[0233] The statistical analysis of the obtained results used Bonferroni multiple comparisons or Dunnett test as multiple comparison tests.

[0234] In either method, whether or not a significant improvement was shown compared to the control group was indicated by *p<0.05, **p<0.01. Note that when 0.05≤p<0.1, a significant trend was determined, and the p value was indicated.

[0235] (3) Test 1: Effect on melanin production

[0236] Three-dimensional skin models containing melanocytes were cultured under various addition conditions, and the amount of melanin produced was measured.

[0237] The amount of melanin production was measured according to the following method.

[0238] The cultured skin model was washed with PBS(-), extracted with 0.1N NaOH at 60°C overnight, and then centrifuged to remove the membrane and insoluble components contained in the skin model as a precipitate to obtain a solution fraction.

[0239] The absorbance (405 nm) of the obtained solution fraction was measured to determine the amount of melanin production. Next, the melanin inhibition rate in each group was calculated using the following formula, assuming the melanin production amount of the "control" was 100%.

[0240] Melanin inhibition rate (%) = 100-100*(absorbance of each group / absorbance of control)

[0241] The results are shown in Figure 1 The higher the melanin inhibition rate, the better the melanin inhibition effect.

[0242] like Figure 1 As shown, the melanin-inhibiting effect was confirmed by the single administration of the positive control (VC), longan pulp, and pyrroloquinoline quinone.

[0243] However, this effect was significantly enhanced by the combination of longan pulp and pyrroloquinoline quinone.

[0244] In particular, it was found that the combination of longan pulp and pyrroloquinoline quinone exhibited a superior melanin-inhibiting effect compared to adding a larger amount of longan pulp or pyrroloquinoline quinone alone, even when the total amount was low. This was an extremely unexpected discovery.

[0245] Although the data are not shown, when the positive control sample was combined with longan pulp or pyrroloquinoline quinone, the synergistic effect at small amounts as described above was not observed, and the effects were equivalent to those of the same amounts of each administered alone.

[0246] (4) Test 2: Effect on the production of nicotinamide phosphoribosyltransferase (NAMPT)

[0247] Three-dimensional skin models containing melanocytes were cultured under various addition conditions, and the amount of NAMPT produced was measured.

[0248] The amount of NAMPT produced was measured according to the following method.

[0249] The culture medium of the cultured skin model was treated using an adsorption column ("10 kD Spin Columns", manufactured by Abcam) to remove proteins, and the eluate containing low molecular weight fractions was recovered.

[0250] The amount of NAMPT produced in the obtained low-molecular fraction was quantified using "NAD / NADH Assay Kit" (manufactured by Abcam).

[0251] The results are shown in Figure 2 The higher the amount of NAMPT produced, the greater the anti-aging effect on the skin can be expected.

[0252] like Figure 2 As shown in the results, the effect of increasing the amount of NAMPT produced was confirmed by administering longan pulp and pyrroloquinoline quinone independently.

[0253] However, this effect was significantly enhanced by the combination of longan pulp and pyrroloquinoline quinone.

[0254] In particular, it was found that the combination of longan pulp and pyrroloquinoline quinone exhibited a significant effect of increasing NAMPT production compared to adding a larger amount of longan pulp or pyrroloquinoline quinone alone, even when the total amount was low. This was an extremely unexpected discovery.

[0255] (5) Test-3: Nicotinamide adenine dinucleotide (NAD + ) The impact of production volume

[0256] A three-dimensional skin model containing melanocytes was cultured under various addition conditions to measure NAD + Production volume.

[0257] NAD + The production amount was measured according to the following method.

[0258] The culture medium of the cultured skin model was treated using an adsorption column ("10 kD Spin Columns", manufactured by Abcam) to remove proteins, and the eluate containing low molecular weight fractions was recovered.

[0259] The obtained low molecular weight fraction was analyzed by using "NAD / NADH Assay Kit" (manufactured by Colorimetric Co., Ltd.). + Quantify the amount of production.

[0260] The results are shown in Figure 3 NAD + The higher the production, the greater the anti-aging effect on the skin can be expected.

[0261] like Figure 3As shown in the results, it was confirmed that the NAD + The effect of quantity.

[0262] However, this effect was significantly enhanced by the combination of longan pulp and pyrroloquinoline quinone.

[0263] In particular, the combination of longan meat and pyrroloquinoline quinone showed a good effect in increasing NAD levels compared to adding a larger amount of longan meat or pyrroloquinoline quinone alone, even when the total amount was low. + The effect of production volume is an extremely unexpected discovery.

[0264] <Test 2 using a three-dimensional skin model containing melanin>

[0265] Evaluations were performed under various addition conditions using a three-dimensional skin model containing melanin by the following method.

[0266] (1) Sample preparation

[0267] The following three types of samples were prepared and used for the culture test described below.

[0268] (1-1) Longan meat sample

[0269] Longan pulp extract powder (manufactured by Matsuura Pharmaceutical Co., Ltd.) was prepared.

[0270] The sample is a powder sample obtained by the following operation: using the aril of longan pulp (scientific name: Euphoria longana (Sapindaceae)) as the base material, it is processed in the order of water extraction, filtration, concentration (accompanied by pectinase treatment) and sterilization (125±10℃), and then dextrin is added, spray-dried and granulated (sieve: opening 0.5mm, accompanied by heavy metal removal using magnet treatment).

[0271] It should be noted that the longan pulp extract powder used in this example contains components derived from longan pulp and an excipient (dextrin). Therefore, the concentration of longan pulp below represents the amount (dry weight) converted into components derived from longan pulp.

[0272] (1-2) Pyrroloquinoline quinone sample

[0273] Pyrroloquinoline quinone disodium salt (purity 99.0% or more, manufactured by Ryowato Co., Ltd.) was prepared. In addition, pyrroloquinoline quinone disodium salt is also described as "PQQ" hereinafter.

[0274] This sample is a powder sample obtained through fermentation production using microbial fermentation and purification steps (extraction with aqueous ethanol, concentration, drying, sieving, etc.).

[0275] Hereinafter, the concentration of the pyrroloquinoline quinone sample (sodium salt) indicates the amount converted to the free form of pyrroloquinoline quinone.

[0276] (1-3) Positive control sample

[0277] As a positive control substance, L-(-) ascorbic acid (powder sample, manufactured by Kanto Chemical Co., Ltd.) was prepared. In addition, L-(-) ascorbic acid is also described as "VC" hereinafter.

[0278] (2) Culture test

[0279] In this example, the following method (system using a three-dimensional skin model) was commonly employed as a culture method.

[0280] The culture was performed in an incubator at 37°C and 5% CO2 for 3 weeks using a three-dimensional skin model containing melanocytes ("MEL-300", manufactured by KURABO INDUSTRIES LTD.) under the conditions of adding specified ingredients.

[0281] EPI-100NMM113 was used as the culture medium. This medium contains bFGF, α-MSH, and KGF as melanin-stimulating factors. These components function as melanin-forming factors, inflammatory factors, and external stimuli in the main culture system.

[0282] The culture medium was replaced every other day, and the test substance was dissolved in the culture medium each time. The biological activity of the test substance was evaluated from the bottom side of the skin model, which was assumed to be used for food.

[0283] The conditions for adding components to the culture medium are as follows.

[0284] Control: culture medium only

[0285] VC(100): Positive control sample 100 μg / ml

[0286] Longan meat (15): Longan meat sample 15μg / ml

[0287] Longan meat (50): Longan meat sample 50μg / ml

[0288] Longan meat (100): Longan meat sample 100μg / ml

[0289] PQQ (2.5): Pyrroloquinoline quinone sample 2.5 μg / ml

[0290] PQQ(3): Pyrroloquinoline quinone sample 3 μg / ml

[0291] PQQ(5): Pyrroloquinoline quinone sample 5 μg / ml

[0292] PQQ(10): Pyrroloquinoline quinone sample 10 μg / ml

[0293] Longan meat (50) + PQQ (5): Longan meat sample 50 μg / ml and pyrroloquinoline quinone sample 5 μg / ml

[0294] Longan meat (15) + PQQ (2.5): Longan meat sample 15μg / ml and pyrroloquinoline quinone sample 2.5μg / ml

[0295] Four wells (n=4) were set for each sample.

[0296] The results were statistically analyzed using the Dunnett test for multiple comparisons. Significant improvement compared to the control group was determined using *p<0.05 and **p<0.01. A significant trend was determined when 0.05≤p<0.1, and the p value was indicated.

[0297] (3) Experiment 1: Effect on IL-1β production

[0298] Three-dimensional skin models containing melanocytes were cultured under various addition conditions, and the amount of IL-1β produced was measured.

[0299] The IL-1β production amount was measured according to the following method.

[0300] 100 μl of the culture medium was collected and the amount of IL-1β produced was measured using “IL-1β, ELISA kit, Human” (ENZ).

[0301] The results are shown in Figure 4 The lower the IL-1β production value, the higher the effect of suppressing melanin production.

[0302] like Figure 4 As shown in the figures, the IL-1β inhibitory effect was confirmed by the administration of longan pulp and pyrroloquinoline quinone alone.

[0303] However, this effect was significantly enhanced by the combination of longan pulp and pyrroloquinoline quinone.

[0304] (4) Experiment 2: Effects on the production of GDF-15 and NLRP3

[0305] Three-dimensional skin models containing melanocytes were cultured under various addition conditions, and the production of GDF-15 and NLRP3 was measured.

[0306] The production levels of GDF-15 and NLRP3 were measured according to the following method.

[0307] The cultured skin model was washed with PBS (-), and then 250 μL of "T-PER (trademark) Tissue Protein Extraction Reagent buffer" (Thermo Scientific) was added and ground and extracted at 4°C to prepare a lysate.

[0308] The pulverization extraction was performed as follows: a skin model and two glass beads were placed in a polypropylene tube, and "T-PER Buffer" was added. The cells were then pulverized using a pulverization extractor (MicroSmash MS-100R, manufactured by TOMY SEIKO CO., LTD.) at 4°C and 2000 rpm for 5 minutes.

[0309] One aliquot (20 μL) of the lysate was used to quantify the total protein concentration by comparing it with BSA (bovine serum albumin) using a conventional BCA protein assay method (measured at OD562 nm).

[0310] The lysate (100 μL) was subjected to "Quantikine Human GDF-15 ELISA Kit" and "Human NLRP3 ELISA Kit" (ab274401, Abcam Limited), and the amounts of GDF-15 protein and NLRP3 protein per unit protein amount were quantified.

[0311] The results of GDF-15 are shown in Figure 5 The lower the GDF-15 production, the better the anti-aging effect can be expected.

[0312] like Figure 5 As shown, the GDF-15 inhibitory effect was confirmed by the single administration of longan pulp and pyrroloquinoline quinone.

[0313] However, this effect was significantly enhanced by the combination of longan pulp and pyrroloquinoline quinone.

[0314] In particular, it was found that the combination of longan pulp and pyrroloquinoline quinone exhibited a superior GDF-15 inhibitory effect compared to the addition of a larger amount of longan pulp or pyrroloquinoline quinone alone, even when the total amount was low. This was a very unexpected discovery.

[0315] The results for NLRP3 are shown in Figure 6 The lower the NLRP3 production, the greater the effect of suppressing melanin production can be expected.

[0316] like Figure 6 As shown in the results, the effects of suppressing NLRP3 were confirmed by administering longan pulp and pyrroloquinoline quinone independently.

[0317] However, this effect was significantly enhanced by the combination of longan pulp and pyrroloquinoline quinone.

[0318] In particular, it was found that the combination of longan pulp and pyrroloquinoline quinone exhibited a superior NLRP3 inhibitory effect compared to the addition of a larger amount of longan pulp or pyrroloquinoline quinone alone, even when the total amount was low. This was an extremely unexpected discovery.

[0319] <Test using human skin fibroblasts-1>

[0320] Evaluations were performed under various addition conditions using human skin fibroblasts by the following method.

[0321] (1) Sample preparation

[0322] In the same manner as in the above-mentioned "Test using a three-dimensional skin model containing melanin", longan pulp samples and pyrroloquinoline quinone samples were prepared.

[0323] The standard for labeling the concentration of each sample is the same as that in the above-mentioned "Test using a three-dimensional skin model containing melanin".

[0324] (2) Culture test

[0325] In this example, the following method (system using human skin fibroblasts) was commonly used as a culture method.

[0326] (2-1) Preparation of human skin fibroblasts

[0327] Human skin fibroblasts were subcultured in DMEM medium containing 5% FBS, and cells that had reached the population doubling level (PDL) specified in each experiment were used.

[0328] Hereinafter, the PDL in this example is determined by the following method.

[0329] At the end of each subculture, the total number of cells in the culture was measured, and the cell proliferation rate and population doubling time were calculated using the least squares method.

[0330] Based on these values, PDL was estimated for each cell passage.

[0331] The cell proliferation rate and population doubling time were calculated based on the following formulas.

[0332] Proliferation rate μ = log(N(t)) - log(N(t0)) / (t - t0)

[0333] Population doubling time x = log2 / μ

[0334] (t: the most recent subculture time

[0335] t0: culture time before one passage

[0336] t-t0: culture time)

[0337] In normal human skin fibroblasts (cultured at a PDL of approximately 20, indicating no signs of cellular aging), the number of cells grown from 13,000 seeded cells to 55,000 recovered cells over 72 hours of culture. Based on this, the following formula was derived, defining the proliferation rate from the early to mid-stage of culture as "34.6 hours," and calculating the PDL (population doubling level) based on this culture time.

[0338] μ=log(55000)-log(N(13000) / (72-0))

[0339] x = log² / 0.0087 = 34.6 hours

[0340] It should be noted that it was confirmed that as the number of passages increases, the population doubling time is delayed and the cell proliferation rate decreases with the increase in the number of passages, thereby causing cell aging.

[0341] (2-2) Tracking of Per2 and Sirt1 expression

[0342] At PDL levels of 50 to 60, an experiment was performed to track the expression levels of Per2 and Sirt1.

[0343] Per2 is known as a clock gene, and its gene expression cycle generally fluctuates over a 24-25 hour period. It is believed that the expression level of Per2 protein is also affected by its gene expression cycle.

[0344] Therefore, in this example, before adding PQQ and longan meat, cells were treated for 2 hours under high serum concentration conditions (40% horse serum, Heat-Inactivated Horse Serum, Gibco HI Horse Serum) to synchronize the expression cycle of clock genes. Then, PQQ and longan meat were added, and the changes in Per2 production were tracked.

[0345] Specifically, human skin fibroblasts were subcultured, and 100,000 cells / well were seeded in a 6-well plate at a stage close to PDL50. After cell colonization, the cells were treated with 40% horse serum to synchronize the expression status related to the circadian rhythm. After 2 hours, the horse serum was temporarily removed and replaced with serum-free culture medium (Gibco D-MEM, Fujifilm Wako Pure Chemical Industries, Ltd.), and PQQ and longan meat were added.

[0346] After adding PQQ and longan pulp, the cells were cultured at 37°C in a 5% CO2 incubator for 72 hours.

[0347] After the culture was completed, part of the NAD was measured. + The culture medium was removed from the remaining culture medium, washed with PBS(-), and cells were collected with a scraper. The cell suspension was centrifuged to prepare a cell pellet, which was then solubilized with T-PER buffer (Tissue Protein Extraction Reagent (Thermofisher)) to prepare a cell extract (lysate).

[0348] A portion (20 μL) of the cell extract was used to quantify protein (total protein amount) using a conventional BCA protein assay method (measured at OD562 nm), and the total protein concentration was quantified by relative comparison with BSA (bovine serum albumin).

[0349] The remaining cell extract (100 μL) was used to quantify the protein expression levels of Per2 and Sirt1 using a commercially available ELISA kit.

[0350] The ELISA kits used are as follows:

[0351] ·Per2: human Per2 ELISA kit Human PER2 (Period circadian proteinhomolog 2) ELISA Kit (Assay Genie)

[0352] ·Sirt1:human Sirt1 ELISAkit Human SIRT1 ELISAKit(ab171573)(Abcam)

[0353] The conditions for adding components to the culture medium are as follows.

[0354] Control: culture medium only

[0355] Longan meat (100): Longan meat sample 100μg / ml

[0356] Longan meat (300): Longan meat sample 300μg / ml

[0357] PQQ(3): Pyrroloquinoline quinone sample 3 μg / ml

[0358] PQQ(10): Pyrroloquinoline quinone sample 10 μg / ml

[0359] PQQ(30): Pyrroloquinoline quinone sample 30 μg / ml

[0360] Longan meat (150) + PQQ (5): Longan meat sample 150 μg / ml and pyrroloquinoline quinone sample 5 μg / ml

[0361] Four wells (n=4) were set for each sample.

[0362] The results were statistically analyzed using the Dunnett test for multiple comparisons. Significant improvement compared to the control group was determined using *p<0.05 and **p<0.01. A significant trend was determined when 0.05≤p<0.1, and the p value was indicated.

[0363] The results of Sirt1 are shown in Figure 7 The results of Per2 are shown in Figure 8 .

[0364] The higher the Sirt1 production, the better the anti-aging effect can be expected.

[0365] In addition, it is known that the expression level of Per2 changes with the fluctuation of Sirt1 and can form a circadian rhythm.

[0366] like Figure 7 and 8 As shown, the Sirt1-increasing effect and the Per2-decreasing effect were confirmed by administering pyrroloquinoline quinone alone. However, these effects were more pronounced by the combination of longan pulp and pyrroloquinoline quinone.

[0367] (2-3)NAD + Determination of production

[0368] The culture solution collected in the above (2-2) was deproteinized using an adsorption column ("10 kD Spin Columns", manufactured by Abcam), and the eluate containing low molecular weight fractions was recovered.

[0369] The obtained low molecular weight fraction was analyzed by using "NAD / NADH Assay Kit" (manufactured by Colorimetric Co., Ltd.). + Quantify the amount of production.

[0370] The results were statistically analyzed using the Dunnett test for multiple comparisons. Significant improvement compared to the control group was determined using *p<0.05 and **p<0.01. A significant trend was determined when 0.05≤p<0.1, and the p value was indicated.

[0371] NAD + The results are shown in Figure 9 NAD + The higher the production amount, the better the anti-aging effect can be expected.

[0372] like Figure 9 As shown, by administering longan meat and pyrroloquinoline quinone alone, NAD + The production volume has barely changed.

[0373] However, the combination of longan meat and pyrroloquinoline quinone makes NAD + The production volume increased significantly.

[0374] <Test using human skin fibroblasts-2>

[0375] Evaluations were performed under various addition conditions using human skin fibroblasts by the following method.

[0376] (1) Sample preparation

[0377] In the same manner as in the above-mentioned "Test using a three-dimensional skin model containing melanin", longan pulp samples and pyrroloquinoline quinone samples were prepared.

[0378] The standard for labeling the concentration of each sample is the same as that in the above-mentioned "Test using a three-dimensional skin model containing melanin".

[0379] (2) Culture test

[0380] In this example, the following method (system using human skin fibroblasts) was commonly used as a culture method.

[0381] (2-1) Preparation of human skin fibroblasts

[0382] Human skin fibroblasts were subcultured in DMEM medium containing 5% FBS, and cells that had reached the population doubling level (PDL) specified in each experiment were used.

[0383] (2-2) Tracking of Cell Metabolism

[0384] An experiment to track cell metabolism was conducted at a PDL of 80 to 90. ATP, lactate, cell viability, oxygen consumption rate, and cell senescence (SPiDER-βGal) were selected as cell metabolism indicators.

[0385] Furthermore, cells with PDL levels of 80 to 90 are considered to be in a state of cellular senescence (also called replicative senescence) in which cell proliferation is significantly reduced.

[0386] Specifically, human skin fibroblasts with a PDL of 80 to 90 were seeded at 20,000 cells / well on a 96-well plate, and PQQ and longan meat were added after the cells were colonized.

[0387] After adding PQQ and longan pulp, the cells were cultured at 37° C. in a 5% CO 2 incubator for the culture time set for each measurement item.

[0388] The conditions for adding components to the culture medium are as follows.

[0389] Control: culture medium only

[0390] Longan meat (100): Longan meat sample 100μg / ml

[0391] PQQ(10): Pyrroloquinoline quinone sample 10 μg / ml

[0392] Longan meat (50) + PQQ (0.5): Longan meat sample 50 μg / ml and pyrroloquinoline quinone sample 0.5 μg / ml

[0393] Longan meat (50) + PQQ (2.5): Longan meat sample 50 μg / ml and pyrroloquinoline quinone sample 2.5 μg / ml

[0394] Longan meat (50) + PQQ (5): Longan meat sample 50 μg / ml and pyrroloquinoline quinone sample 5 μg / ml

[0395] Longan meat (150) + PQQ (5): Longan meat sample 150 μg / ml and pyrroloquinoline quinone sample 5 μg / ml

[0396] Four wells (n=4) were set for each sample.

[0397] The results were statistically analyzed using the Dunnett test for multiple comparisons. Significant improvement compared to the control group was determined using *p<0.05 and **p<0.01. A significant trend was determined when 0.05 ≤ p<0.1, and the p value was indicated.

[0398] (2-2-1) ATP production

[0399] After adding PQQ and longan pulp, the cells were cultured for 2 hours, the culture medium was removed, and the cells were washed with PBS(-).

[0400] Next, ATP chemiluminescence reagent ("Cellular" ATP Assay Reagent Ver. 2 (TOYO B-NET)) was added to the cells in each well at 50 μL / well, and reacted at room temperature for 10 minutes under light-shielding conditions. Chemiluminescence was quantified using a microplate reader (SYNERGY H1, BioTek).

[0401] The results are shown in Figure 10 The higher the ATP production, the better the anti-aging effect can be expected.

[0402] like Figure 10 As shown in the results, the effect of increasing ATP production was confirmed by the combination of longan pulp and pyrroloquinoline quinone.

[0403] (2-2-2) Lactic acid value

[0404] After adding PQQ and longan pulp, the cells were cultured for 2 hours and the culture medium was recovered.

[0405] The resulting culture solution was added to a new 96-well plate at 20 μL / well. The absorbance (OD450 nm) was quantified using the Lactate Assay Kit-WST (Dojin) in a 96-well plate reader. The lactate concentration in the culture solution was quantified using a standard curve based on the quantification results.

[0406] The results are shown in Figure 11 The lower the lactic acid value, the better the anti-aging effect can be expected.

[0407] like Figure 11 As shown in the results, the combination of longan pulp and pyrroloquinoline quinone was found to have a lactic acid lowering effect.

[0408] (2-2-3) Oxygen consumption rate

[0409] Immediately after the addition of PQQ and longan pulp, the cellular respiration of oxidative phosphorylation, that is, the oxygen consumption rate from the mitochondrial reaction, was measured over time using the "Extracellular OCR Plate Assay Kit" (Dojin).

[0410] From 0 to 3 hours after addition, the fluorescence intensity at Ex=500 nm / Em=650 nm was kinetically analyzed using a fluorescence microplate reader (SYNERGY H1, BioTek) and quantified as the extracellular oxygen consumption rate (OCR) (pmol / min).

[0411] The results are shown in Figure 12The higher the oxygen consumption rate, the better the anti-aging effect can be expected.

[0412] like Figure 12 As shown in the results, no decrease in oxygen consumption rate was found when longan pulp and pyrroloquinoline quinone were administered alone.

[0413] However, the combination of longan pulp and pyrroloquinoline quinone significantly increased the oxygen consumption rate.

[0414] (2-2-4) Cell viability (MTT assay)

[0415] After adding PQQ and longan pulp, the cells were cultured for 72 hours, the culture medium was removed, and the cells were washed with PBS(-).

[0416] Next, 0.5 mg / ml MTT (NACALAI TESQUE, INC) was added, and a formazan reaction was carried out at 37°C for 2 hours. After the reaction, the solution was removed, the cells were washed with PBS(-), and 100 μL / well of 2-propanol was added. The formazan formed by the MTT reaction was quantified using an absorbance plate reader at OD570nm, and the cell survival rate was calculated as a ratio to the control value.

[0417] The results are shown in Figure 13 The higher the cell survival rate, the better the anti-aging effect can be expected.

[0418] like Figure 13 As shown, almost no change in cell viability was found by administering longan pulp and pyrroloquinoline quinone alone.

[0419] However, the combination of longan pulp and pyrroloquinoline quinone significantly improved cell survival.

[0420] (2-2-5) Cell aging (SPiDER-βGal assay)

[0421] After culturing for 72 hours from the addition of PQQ and longan pulp, the degree of cell senescence was quantified using the "Cellular Senescence Plate Assay Kit-SPiDER-βGal" (Dojin).

[0422] Specifically, first, the following two reagents were prepared in advance according to the instructions of the kit.

[0423] SPiDER-βGal DMSO stock solution: Add DMSO (125 μL) to the tube containing SPiDER-βGal and dissolve using a vortex mixer.

[0424] "SPiDER-βGal working solution": Dilute "SPiDER-βGal DMSO stock solution" 10-fold with "Assay Buffer".

[0425] Next, the supernatant after 72 hours of culture was removed by aspiration, and the cells were washed once with PBS. Then, 50 μl / well of "Lysis Buffer" was added, and the cells were incubated at room temperature for 10 minutes.

[0426] The obtained lysate was added to each well of a 96-well clear-bottom plate, and then 50 μL of “SPiDER-βGalworking solution” was added to each well, followed by incubation at 37°C for 30 minutes.

[0427] Next, 100 μL of "Stop Solution" was added to each well, and the amount of β-galactosidase derived from senescent cells was quantified by measuring the fluorescence using a fluorescence microplate reader under the conditions of Ex: 530 nm, Em: 580 nm.

[0428] The results are shown in Figure 14 The lower the SPiDER-βGal value, the better the anti-aging effect can be expected.

[0429] like Figure 14 As shown, the changes in SPiDER-βGal were hardly observed by the administration of longan pulp and pyrroloquinoline quinone alone.

[0430] However, the value of SPiDER-βGal was significantly decreased by the combination of longan pulp and pyrroloquinoline quinone.

Claims

A food and beverage composition comprising longan pulp and pyrroloquinoline quinone.

2. The food and drink composition according to claim 1, wherein The mass ratio of the component derived from the longan pulp to the pyrroloquinoline quinone is 5:1 to 30:

1.

3. Use of a composition comprising longan pulp and pyrroloquinoline quinone as an anti-aging agent.

4. Use of a composition comprising longan pulp and pyrroloquinoline quinone as an agent for improving mitochondrial dysfunction.

5. Use of a composition comprising longan pulp and pyrroloquinoline quinone as a nicotinamide phosphoribosyltransferase production promoter.

6. Use of a composition comprising longan pulp and pyrroloquinoline quinone as a nicotinamide adenine dinucleotide production promoter.

7. Use of a composition comprising longan pulp and pyrroloquinoline quinone as a Sirtuin 1 production promoter.

8. Use of a composition comprising longan pulp and pyrroloquinoline quinone as a melanin production inhibitor.

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