JP2016011259A - Collagenase activity and/or elastase activity inhibitor containing phytoene and/or phytofluene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel collagenase activity and/or elastase activity inhibitor containing phytoene and/or phytofluene which are novel collagenase activity and/or elastase activity inhibition substances.SOLUTION: The invention relates to a collagenase activity and/or elastase activity inhibitor containing phytoene and/or phytofluene.

Description

  The present invention relates to a collagenase activity and / or an elastase activity inhibitor containing phytoene and / or phytofluene.

  Extracellular matrices such as elastin and collagen that support the skin structure are important for maintaining moisture in the skin, maintaining flexibility, maintaining elasticity, etc. When elastin and collagen decrease with age, skin wrinkle formation and skin elasticity It causes a decrease. Development of an elastase inhibitor and a collagenase inhibitor that suppress the decrease in elastin and collagen has been carried out (Patent Documents 1 and 2).

  On the other hand, in living organisms such as plants, algae such as green algae, bacteria, and yeast, phytoene, the first carotenoid, is synthesized in the carotenoid synthesis pathway, starting from an appropriate carbon source for that species such as glucose. And then phytofluene is known to be synthesized. Of the carotenoids synthesized in the carotenoid synthesis pathway, phytoene and phytofluene are substances that lack a conjugated double bond in part of their structure. In particular, phytoene is known to have a lipid peroxidation inhibitory effect on cells.

  Phytoene is abundant in Dunaliella algae. For the purpose of preventing cancer and lifestyle-related diseases, foods containing this Dunaliella alga body itself, foods containing phytoene obtained by culturing Dunaliella alga bodies, and the like are known (Patent Document 3). Furthermore, based on the knowledge that phytoene and phytofluene reduce the melanin content in skin cells, have antioxidant properties, absorb ultraviolet rays, etc., skin bleach containing them, oxidized or ultraviolet rays Cosmetics or pharmaceuticals for the prevention of skin damage caused by the skin are also known (Patent Documents 4 and 5).

JP 2012-001457 A JP 2003-055245 A JP 2003-183130 A JP 2008-13541 A JP 2008-1721 A

Although phytoene and phytofluene have been known to have an antioxidant function and the like, it has not been known to have an effect of inhibiting collagenase activity and / or elastase activity.
The present invention has been made under such circumstances, and a novel collagenase activity and / or elastase activity inhibitor containing a new collagenase activity and / or elastase activity inhibitor phytoene and / or phytofluene. Offering is an issue.

As a result of intensive studies, the present inventors have found that the following novel collagenase activity and / or elastase activity inhibitors can solve the above-mentioned problems, and have completed the present invention. That is, the present invention is as follows.
[1] A collagenase activity and / or elastase activity inhibitor containing phytoene and / or phytofluene.
[2] An anti-wrinkle agent comprising the inhibitor according to [1].

  ADVANTAGE OF THE INVENTION According to this invention, the novel collagenase activity and / or elastase activity inhibitor containing the new collagenase activity and / or elastase activity inhibitory substance phytoene and / or phytofluene can be provided.

It is a figure which shows the relationship between the phytoene density | concentration and the elastase activity inhibition rate in one embodiment of this invention. It is a figure which shows the relationship between the phytoene density | concentration and collagenase activity inhibition rate in one embodiment of this invention.

  The present invention includes a first embodiment relating to a collagenase activity and / or elastase activity inhibitor containing phytoene and / or phytofluene, and a second embodiment relating to an anti-wrinkle agent containing the inhibitor.

<1. Collagenase activity and / or elastase activity inhibitor containing phytoene and / or phytofluene>
The first embodiment of the present invention is a collagenase activity and / or elastase activity inhibitor containing phytoene and / or phytofluene.

<1-1. Phytoen>
Phytoene according to this embodiment is a known substance whose structure is represented by the following chemical formula.

The method for obtaining the phytoene is not particularly limited, and it may be extracted from plants or microorganisms, recovered by production by microorganisms, or produced by chemical synthesis.
When extracting from a plant, the type of the plant is not particularly limited, but since it contains a large amount of phytoene, for example, a plant such as tomato and orange Chinese cabbage is preferable, and a tomato powder obtained by processing tomato is also preferable. When extracting from a microorganism, the type of the microorganism is not particularly limited, and examples thereof include photosynthesis or non-photosynthesis bacteria, yeast, and mold. Among them, since it contains a large amount of phytoene, for example, algae such as green algae (Dunariella, Haematococcus) and non-photosynthetic bacterium Algibacter sp. (Algibacter sp.) Is preferred. When phytoene is produced and recovered by a microorganism, Paracoccus sp. Mutated to produce phytoene is used. (Paracoccus sp.) FA1 or FA3 or genetically modified Escherichia coli introduced with a foreign gene so as to generate phytoene may be used. As the phytoene of this embodiment, one obtained by any one of the methods described above may be used, or one obtained by two or more methods may be used.

The extraction method and extraction conditions of phytoene from plants and microorganisms are not particularly limited, and for example, known extraction methods such as water extraction, hot water extraction, hot water extraction, alcohol extraction, supercritical extraction and the like can be used.
When performing solvent extraction, examples of the solvent include water; lower alcohols such as methanol, anhydrous ethanol, and ethanol; and alcohols such as polyhydric alcohols such as propylene glycol and 1,3-butylene glycol (anhydrous or water-containing). Not); ketones such as acetone; esters such as diethyl ether, dioxane, acetonitrile, and ethyl acetate; xylene, benzene, chloroform, and the like. Preferred are water, ethanol, and the like. Any one of these solvents may be used, or two or more thereof may be used in combination.

  Moreover, if extracting from tomato, you may follow the method of Unexamined-Japanese-Patent No. 2008-13541, etc. In the case of extraction from algae such as Dunaliella, JP 2003-180387 A, non-photosynthetic bacterium Algibacter sp. In the case of extracting from (Algibacter sp.), The method described in JP 2008-541697 A may be followed. The mutant Paracoccus sp. (Paracoccus sp) When producing by fermentation using FA1 or FA3, the method described in the pamphlet of WO2009-115629 may be followed. Furthermore, in the case of producing and recovering a recombinant E. coli introduced with a foreign gene so as to generate phytoene, J. Agric. Food Chem., 59 (18), 9996-10005 (2011), Appl. Microbiol. Botechnol., 81 (5), 915-925 (2009) may be used.

  The obtained extract can be used as it is, but a dried powder may be used. Moreover, you may refine | purify, a concentration process, etc. to the extract obtained as needed. As the purification treatment, treatment such as filtration or adsorption or decolorization using an ion exchange resin or activated carbon column can be performed. As the concentration treatment, a conventional method such as an evaporator can be used.

  In addition, a method of subjecting the obtained extract (or purified product or concentrate) to lyophilization and pulverization, adding excipients such as dextrin, corn starch, gum arabic, etc., and pulverizing by spray-drying You may powder according to well-known methods, such as a method. Further, it may be used after being dissolved in pure water, ethanol or the like as required.

<1-2. Phytofluene>
Phytofluene according to this embodiment is a known substance whose structure is represented by the following chemical formula.

  The method for obtaining the phytofluene is not particularly limited, and is the same as that for the phytoene described above. Further, the extraction method, extraction conditions, and subsequent processing of the obtained extract are the same as in the case of phytoene described above.

<3. Collagenase activity inhibitor>
The collagenase activity inhibitor which concerns on this embodiment inhibits the activity of collagenase by competitive inhibition. For example, from the viewpoint of anti-wrinkle, the collagenase activity is inhibited by the mechanism, and the decrease in collagen is suppressed, so that an anti-wrinkle effect is realized as a result.
Therefore, the elastase activity inhibitor according to this embodiment is preferably used for anti-wrinkle use.

  As the collagenase activity inhibitor according to this embodiment, phytoene and / or phytofluene can be used alone, but in addition to the above components, known excipients, fragrances, coloring agents, emulsifiers, stabilizers, thickening agents. Agents, enzymes, preservatives, lubricants, surfactants, disintegrants, disintegration inhibitors, binders, absorption enhancers, adsorbents, moisturizers, solubilizers, preservatives, flavoring agents, sweeteners, etc. It can mix | blend as needed in the range which does not impair the effect of this embodiment.

  The content of the phytoene and / or phytofluene with respect to the total amount of the collagenase activity inhibitor according to this embodiment is not particularly limited as long as the desired effect of this embodiment is exhibited, but is generally 0.00001 to 20 mass in total. %, Preferably 0.0001 to 3% by mass, more preferably 0.001 to 1% by mass.

<4. Elastase activity inhibitor>
The elastase activity inhibitor according to this embodiment inhibits the activity of elastase. For example, from the viewpoint of anti-wrinkle, the activity of elastase is inhibited and the decrease in elastin is suppressed, so that an anti-wrinkle effect is realized as a result.
Therefore, the elastase activity inhibitor according to this embodiment is preferably used for anti-wrinkle use.

  As the elastase activity inhibitor according to this embodiment, phytoene and / or phytofluene can be used alone, but in addition to the above components, known excipients, fragrances, coloring agents, emulsifiers, stabilizers, thickening agents. Agents, enzymes, preservatives, lubricants, surfactants, disintegrants, disintegration inhibitors, binders, absorption enhancers, adsorbents, moisturizers, solubilizers, preservatives, flavoring agents, sweeteners, etc. It can mix | blend as needed in the range which does not impair the effect of this embodiment.

  The content of the phytoene and / or phytofluene with respect to the total amount of the elastase activity inhibitor according to the present embodiment is not particularly limited as long as the desired effect of the present embodiment is exhibited, but the total amount is usually 0.00001 to 20 mass. %, Preferably 0.001 to 3% by mass, more preferably 0.001 to 1% by mass.

<5. Anti-wrinkle agent>
The second embodiment of the present invention is an anti-wrinkle agent comprising the inhibitor.
The anti-wrinkle agent according to this embodiment may contain the above-mentioned collagenase activity and / or elastase activity inhibitor alone, but in addition to the above-mentioned components, known excipients, flavors, colorants, emulsifiers, stabilization Agent, thickener, enzyme, preservative, lubricant, surfactant, disintegrant, disintegration inhibitor, binder, absorption enhancer, adsorbent, moisturizer, solubilizer, preservative, flavoring agent, sweetness An agent etc. can be mix | blended as needed in the range which does not impair the effect of this embodiment.
The anti-wrinkle agent according to this embodiment can be used as a material for cosmetics, pharmaceuticals, foods, and the like according to the embodiment.

<5-1. Cosmetics>
When the anti-wrinkle agent according to this embodiment is used as a cosmetic material, the anti-wrinkle agent is a liquid such as an aqueous solution, lotion, spray liquid, suspension and emulsion; a solid state such as a powder, granule and block; It can be prepared into cosmetics in various desired dosage forms such as semi-solid forms such as creams and pastes; gels and the like. Such cosmetics include facial cleansers, milks, creams, gels, essences (beauty serums), basic cosmetics such as packs and masks, makeup cosmetics such as foundations and lipsticks, oral cosmetics, aromatic cosmetics and hair cosmetics. It is useful as various cosmetics such as cosmetics and body cosmetics.

  The cosmetic containing the anti-wrinkle agent according to this embodiment can be produced according to a conventional method. Further, the blending amount, blending method, and blending time of the anti-wrinkle agent according to this embodiment for cosmetics can be appropriately selected. Furthermore, it can be enclosed in appropriate containers, such as a bottle, a bag, a can, a spray can, a spray container, a box, and a pack, as needed.

  When the anti-wrinkle agent according to this embodiment is used as a cosmetic material, the content of the anti-wrinkle agent with respect to the total amount of the cosmetic is not particularly limited as long as the desired effect of this embodiment is exhibited. Or as a phytofluene, it is 0.00001-10 mass% normally with a total amount, Preferably it is 0.0001-3 mass%, More preferably, it is 0.0003-1 mass%.

When the anti-wrinkle agent according to this embodiment is used as a cosmetic material, it can be used by appropriately adding commonly used known components.
For example, anionic surfactants (fatty acid soaps, sulfonate type anionic surfactants, sulfate ester type anionic surfactants, phosphate ester type anionic surfactants, acylmethyl taurine salts, monoalkyl phosphates , Acyl glutamate, isethionate, etc.), cationic surfactant (amine salt type cationic surfactant, quaternary ammonium type cationic surfactant (tetraalkylammonium type, pyridinium type)), nonionic Surfactant (glycerin fatty acid ester, propylene glycol fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, tetraoleic acid polyoxyethylene sorbit, polyoxyethylene alkyl ether, polyoxyethylene polyoxypropylene Recall, polyoxyethylene polyoxypropylene alkyl ether, polyethylene glycol fatty acid ester, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyglycerin fatty acid ester, etc.), amphoteric surfactant (imidazoline type, betaine type, amino acid type) , Natural surfactants such as fluorosurfactants and silicone surfactants, synthetic surfactants,
Sodium alginate, propylene glycol alginate, gum arabic, xanthan gum, pectin, tragacanth, sodium carboxymethylcellulose, methylcellulose, carboxyvinyl polymer, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, cationized cellulose, cationized dextran, cationized dextrin, chitosan, Water-soluble polymers such as cationized vinylpyrrolidone polymer, N, N-dimethyl-3,5-methylenepiperidinium chloride polymer, milk protein, soybean protein, gelatin, egg protein, sodium caseinate, whey protein,
Ginkgo biloba, pearl oyster, carrot, shikopi, kaika, inchkou, yashajitsu, licorice fraction, gokahi, sempukuka, hikai, yuzuuriha, chamomile, maronier, escin, terminaria, ruscogenin, butcher bloom, cola, guarana, mate, coffee, Cacao, Plectrantus, Tanjin, Bisnaga, Silymarin, Leucocyanin, Hypericum Grass, Bear Goose, Perilla, Ogon, Keigai, Rosemary, Sage, Thyme, Mugwort, Chinese Artemisia, Sow Jutsu, Achillea Millefolium, Sicon, Fennel, Pepper, Pepper , Plant components such as Toki, Senkyu, Chimbu, Kanokosou, Bakushi, Spruce, Glaze, safflower, camellia, Bukkuri, mint
Succinic acid, fumaric acid, citric acid, pyruvic acid, glucuronic acid, 2-hydroxybutyric acid, lactic acid, malic acid, tartaric acid, tartronic acid, methyl pyruvate, ethyl pyruvate, vitamin A acid, vitamin C derivative, vitamin D, vitamin E, active ingredients such as oligopeptide, tranexamic acid ester,
Moisturizers such as polyhydric alcohols, amino acids, mucopolysaccharides, proteins, biological extracts, fermented metabolites, polysaccharides, plant extracts, phospholipids, ceramides,
Fats and oils (soybean oil, nutka oil, jojoba oil, avocado oil, almond oil, cacao oil, olive oil, sesame oil, persic oil, castor oil, coconut oil, mink oil, beef fat, pork fat, and other natural fats and oils Hydrogenated hardened oil and synthetic triglycerides such as myristic acid glyceride and 2-ethylhexanoic acid glyceride, diglyceride etc.), waxes (carnauba wax, whale wax, beeswax, lanolin etc.), hydrocarbons (liquid paraffin, Petrolatum, paraffin, microcrystalline wax, ceresin, squalane, pristane, etc.), higher fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, lanolinic acid, isostearic acid, etc. ), Higher alcohols (lauryl alcohol, Alcohol, stearyl alcohol, oleyl alcohol, cholesterol, 2-hexyldecanol, etc.), esters (cetyl octoate, myristyl lactate, cetyl lactate, isopropyl myristate, myristyl myristate, isopropyl palmitate, isopropyl adipate, butyl stearate, Decyl oleate, Colesteel isostearate, etc., essential oils (mint oil, jasmine oil, cypress brain oil, cypress oil, spruce oil, ryu oil, turpentine oil, cinnamon oil, bergamot oil, mandarin oil, shibu oil, pine Oil, Lavender oil, Bay oil, Clove oil, Hiba oil, Rose oil, Eucalyptus oil, Lemon oil, Peppermint oil, Thyme oil, Rose oil, Sage oil, Menthol, Cineol, Eugenol, Citral, Citronellal, Borne Lumpur, linalool, geraniol, camphor, thymol, spilanthol, pinene, limonene, terpene compounds), fats and oils such as silicone oils (emollient component),
Sodium carbonate, sodium bicarbonate, sodium sesquicarbonate, borax, sodium sulfate, sodium sulfide, sodium nitrate, sodium thiosulfate, sodium polyphosphate, sodium phosphate, potassium chloride, potassium sulfide, calcium oxide, magnesium oxide, calcium carbonate, Inorganic salts such as magnesium carbonate,
Inorganic acids such as boric acid, metasilicic acid, silicic anhydride,
Yellow No. 4, Blue No. 1, Yellow No. 202, pigments such as chlorophyll, riboflavin, safflower, crocin, anthraquinone,
Fragrances,
Polymers such as acrylic resin, styrene resin, epoxy resin, nylon, polyethylene, polypropylene, polyvinyl chloride, polyethylene terephthalate resin, polytetrafluoroethane, copolymers of these polymers, silicic acid, calcium silicate, natural aluminum silicate , Fine powders such as synthetic aluminum silicate, zeolite, titanium oxide, talc, kaolin, mica, bentonite,
Sulfur, Yunohana, mineral sand, mica powder, neutral white clay, rice cake, fungicide, preservative,
And other ingredients necessary for the preparation.

<5-2. Pharmaceuticals>
When the anti-wrinkle agent according to this embodiment is used as a raw material for pharmaceutical products, the dosage form can be selected according to the use form, administration route, and the like. Examples include tablets, coated tablets, pills, capsules, granules, powders, solutions, suspensions, emulsions, syrups, injections, suppositories, dip agents, decoction, tinctures and the like. These various preparations are prepared according to conventional methods as necessary with respect to the active ingredient, such as fillers, extenders, excipients, binders, humectants, disintegrants, surfactants, lubricants, coloring agents, flavoring agents. The pharmaceutical composition can be formulated using known adjuvants that can be usually used in the pharmaceutical preparation technical field, such as solubilizing agents, suspension agents, and coating agents. Moreover, you may contain a coloring agent, a preservative, a fragrance | flavor, a flavoring agent, a sweetening agent, etc. and other pharmaceuticals in this pharmaceutical formulation.

  When the anti-wrinkle agent according to the present embodiment is used as a raw material for a pharmaceutical product, the content of the anti-wrinkle agent with respect to the total amount of the pharmaceutical agent is not particularly limited as long as the desired effect of the present embodiment is exhibited, but phytoene and / or phyto The total amount of fluorene is usually 0.0001 to 20% by mass, preferably 0.001 to 5% by mass, and more preferably 0.003 to 1% by mass.

<5-3. Food>
When the anti-wrinkle agent according to this embodiment is used as a food material, it can be used as a food for specified health use, a dietary supplement, a functional food, a food for a sick person, a food additive, etc. in addition to a general food. As a form of food, soft drinks containing the anti-glycating agent according to the present invention, milk, pudding, jelly, rice cake, gum, gummy, yogurt, chocolate, soup, cookies, snacks, ice cream, popsicles, bread, cakes, Examples include cream puffs, ham, meat sauce, curry, stew, cheese, butter, dressing and the like.

  The anti-wrinkle agent according to this embodiment can use water, protein, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors and the like as main components. Examples of the protein include whole milk powder, skim milk powder, partially skim milk powder, casein, soybean protein, chicken egg protein, meat protein, and other animal and vegetable proteins, and their hydrolysates and butter. Examples of the saccharide include saccharides, processed starch (in addition to dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like. Examples of lipids include lard, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, hydrogenated oil, and vegetable oils such as transesterified oil. Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. And minerals include, for example, calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey minerals. Examples of the organic acid include malic acid, citric acid, lactic acid, and tartaric acid. Two or more of these components may be used in combination, or a synthetic product and / or a food containing a large amount thereof may be used.

The food containing the anti-wrinkle agent according to this embodiment can be produced according to a conventional method. Moreover, the compounding quantity of the anti-wrinkle agent to a foodstuff, a compounding method, and a compounding time can be selected suitably. Furthermore, it can be enclosed in appropriate containers, such as a bottle, a bag, a can, a box, and a pack, as needed.
When the anti-wrinkle agent according to the present embodiment is used as a food material, the content of the anti-wrinkle agent with respect to the total amount of food is not particularly limited as long as the desired effect of the present embodiment is achieved, but phytoene and / or phyto The total amount of fluorene is generally 0.00001 to 3% by mass, preferably 0.0001 to 1% by mass, and more preferably 0.001 to 0.3% by mass.

  Hereinafter, the present invention will be described in more detail with reference to specific examples, but the present invention is not limited to these examples.

[Phytoene concentration calculation method]
The phytoene in each sample separated and purified in “1. Production of phytoene” below was quantified using high performance liquid chromatography (HPLC) under the following conditions. Under certain conditions, the phytoene isomers have retention times of 43 and 46 minutes.

(Analysis conditions)
Analytical column: Nova-Pak C18, 3.9 × 300 mm (Waters)
Detection wavelength: 286 nm, 350 nm, 400 nm, 450 nm
Mobile phase: acetonitrile / methanol / isopropanol = 90/6/4
Column temperature: 30 ° C
Flow rate: 1mL / min

The phytoene concentration in the standard solution is ε (molecular extinction coefficient) (mM ⁻¹ cm ⁻¹ ) = 50 in the n-hexane solution at a detection wavelength of 286 nm of phytoene, the absorbance (A) of the standard solution at 286 nm, and the phytoene molecular weight = 544. It was calculated by the following formula (1) using the area percentage% (B) of the phytoene peak when the HPLC analysis was conducted under .94 and the above analysis conditions. (Calculation formula for phytoene concentration in standard solution)
Phytoene concentration of standard solution (mg / L) = A / (50 × B × 544.94) (1)

The phytoene concentration of the sample was calculated by the following method.
(Sample preparation and detection)
In the measurement, 20 μL of the sample solution in the solution state obtained in each step is directly or diluted with n-hexane (dilution ratio X) and injected into the analytical column, and detection of phytoene in the column eluent is performed with a wavelength. Performed at 286 nm.

(Sample phytoene concentration calculation formula)
Measurement sample phytoene concentration (mg / L) = (area percentage of phytoene peak in sample (%) × dilution ratio (X) × phytoene concentration of standard solution (mg / L)) / area of phytoene peak in standard solution Percentage (%) (2)

<1. Manufacture of Phytoene>
[Step 1: Extraction step]
As a result of analyzing the phytoene concentration in tomato powder (manufactured by Kagome Co., Ltd.), it was 0.11% (concentration per dry weight). 60 g of water, 158 g of ethanol and 10 g of 25% NaOH aqueous solution are added to 20 g of this tomato powder (dry weight), and saponification treatment and carotenoids of fats and oils are stirred for 30 minutes at room temperature (20 to 30 ° C.) in a nitrogen atmosphere. Was extracted. Thereafter, 132 g of n-hexane was added, and low polarity carotenoids containing phytoene were extracted while stirring at room temperature (20 to 30 ° C.) for 30 minutes in a nitrogen atmosphere. This extract was transferred to a separatory funnel, and after separating by standing, the lower layer was removed to obtain 102 g (116 mg / L) of a reddish brown n-hexane extract containing about 18 mg of phytoene.

[Step 2: Extract solution concentration, column charge solution preparation step]
The extract obtained in step 1 was concentrated to dryness at 40 ° C. or lower using a rotary evaporator. Thereafter, the column was re-dissolved with 6 mL of an eluent of n-hexane / ethyl acetate = 99/1 (vol / vol) to obtain a phytoene-containing column charge solution.

[Step 3: Silica gel column sorting step]
The phytoene-containing charge liquid obtained in Step 2 was subjected to silica gel column chromatography packed with Wakosil C300, and fractionation was performed, and the phytoene-containing fraction was recovered. The solvent was eluted using n-hexane / ethyl acetate = 99/1 (vol / vol), and 5 mL fractions were cut to obtain each fraction. As a result of fractionation, the obtained fraction eluate was subjected to HPLC analysis to obtain a yellow solution containing 15.6 mg of phytoene.

[Step 4: Activated carbon treatment decolorization step]
80 mg of activated carbon (strong white egret) was added to 13.2 mL of the yellow phytoene-containing fraction obtained in Step 3, and the mixture was stirred at room temperature (20 to 30 ° C.) for 15 minutes in a nitrogen atmosphere. Thereafter, the activated carbon was removed, and concentrated to dryness at 40 ° C. or lower using a rotary evaporator to obtain 11.4 mg of phytoene as a colorless carotenoid.

<2-1. Measurement of elastase activity inhibition rate>
[Test Example 1]
A composition solution containing 200 mM Tris-HCl buffer (pH 8.5) (containing 1 M NaCl), 1 μg / mL neutrophil-derived elastase (manufactured by Sigma-Aldrich), and each concentration of phytoene as a test sample is 96-well micro Added to the plate and incubated at 37 ° C. for 20 minutes. Phytoene dissolved in DMSO was added. Then 0.1 mM N-methoxysuccinyl-L-alanyl-L-alanyl-L-prolyl-L-valine 4-methylcoumaryl-7-amide (Suc (OMe) -Ala-Ala-Pro-Val-MCA) (peptide 50 μL of (Ala-Ala-Pro-Val: SEQ ID NO: 1) was added to prepare a 200 μL reaction solution, and an enzyme reaction was performed at 37 ° C. for 30 minutes. By measuring the fluorescence intensity of this reaction solution at an excitation wavelength of 360 nm and a fluorescence wavelength of 460 nm, 7-amino-4-methylcoumarin released from Suc (OMe) -Ala-Ala-Pro-Val-MCA by enzymatic reaction. The amount produced was measured.

[Comparative Test Example 1]
Moreover, what was carried out similarly to the said Test example 1 except not adding a test sample was used as control.

The elastase activity inhibition rate was calculated by the following formula (3).
Elastase activity inhibition rate (%) = {1− (A−B) / (C−D)} × 100 (3)
A: Fluorescence intensity 30 minutes after adding the test sample B: Fluorescence intensity at 0 minutes after adding the test sample C: Fluorescence intensity 30 minutes after the control D: Fluorescence intensity at 0 minutes of the control

  A graph showing the relationship between the phytoene concentration in the reaction solution and the elastase activity inhibition rate is shown in FIG. As can be seen from FIG. 1, it was confirmed that phytoene has an effect of inhibiting elastase activity in a concentration-dependent manner and suppressing the degradation of elastin.

<2-2. Measurement of collagenase activity inhibition rate>
[Test Example 2]
A composition solution containing 50 mM Tris-HCl buffer (pH 7.3), 40 μg / mL collagenase Type V (manufactured by Wako Pure Chemical Industries, Ltd.) and each concentration of phytoene as a test sample was added to a 96-well microplate at 37 ° C. for 20 minutes. Incubated. Phytoene dissolved in DMSO was added. Then, 5 μM MOCAc-Pro-Leu-Gly-Leu-A ₂ pr (Dnp) -Ala-Arg-NH ₂ (manufactured by Peptide Institute) (Pro-Leu-Gly-Leu-A ₂ pr (Dnp) -Ala- A reaction solution of 200 μL was prepared by adding 50 μL of Arg: SEQ ID NO: 2), and an enzyme reaction was performed at 37 ° C. for 30 minutes. The reaction, excitation wavelength 320 nm, by measuring the fluorescence intensity at the fluorescence wavelength of 405 nm, is liberated by enzymatic reaction from _{MOCAc-Pro-Leu-Gly-} Leu-A 2 pr (Dnp) -Ala-Arg-NH 2 The amount of decomposition products was measured.

[Comparative Test Example 2]
Moreover, what was carried out similarly to the said test example 2 except not adding a test sample was used as control.

Collagenase activity inhibition rate was calculated by the following formula (4).
Collagenase activity inhibition rate (%) = {1− (A−B) / (C−D)} × 100 (4)
A: Fluorescence intensity 30 minutes after adding the test sample B: Fluorescence intensity at 0 minutes after adding the test sample C: Fluorescence intensity 30 minutes after the control D: Fluorescence intensity at 0 minutes of the control

  A graph showing the relationship between the phytoene concentration in the reaction solution and the collagenase activity inhibition rate is shown in FIG. As can be seen from FIG. 2, it was confirmed that phytoene has an effect of inhibiting collagenase activity in a concentration-dependent manner and suppressing collagen degradation.

<3. Sensory test>
(1) Sensory test 1 of cosmetic cream
[Example 1, Comparative Example 1]
A sensory test was conducted using a cosmetic cream prepared based on the formulation described in Table 1.

  Panelists were 16 female panelists in their late 30s and 40s who were aware of fine wrinkles and skin tension. After face washing twice a day in the morning and evening, the cosmetic cream of Example 1 was applied around the right eye corner. The cosmetic cream of Comparative Example 1 was used around the left eye corner for 3 weeks. Then, using a mirror, the “wrinkle depth” and “wrinkle depth” of both eye corners were compared. The results are shown in Table 2.

  From the results in Table 2, it was found that the use of the cosmetic cream of Example 1 containing the phytoene-containing tomato extract improves the “wrinkle depth” and “wrinkle depth” on the skin surface of the outer corner of the eye. It was.

(2) Sensory test 2 of cosmetic cream
[Example 2]
It implemented like Example 1 except the compounding quantity of the phytoene containing tomato extract having been 0.5 weight%. The results are shown in Table 3.

  From the results in Table 3, the cosmetic cream of Example 2 with 0.5% by weight of the phytoene-containing tomato extract in the amount of 0.03% by weight is more effective for the skin surface of the outer corner of the eyes. It was found that “wrinkle depth” and “wrinkle depth” were further improved.

(3) Sensory test 1 of cosmetic lotion
[Example 3, Comparative Example 3]
A sensory test was performed using a cosmetic lotion prepared based on the formulation described in Table 4.

  The panelists were 14 female panelists in their 40s who were aware of wrinkles and skin tension. After washing their face twice a day in the morning and evening, they used only the cosmetic lotion of Example 3 in the right half of their face. Only the cosmetic lotion of Comparative Example 3 was used in the left half. After one month, the “smoothness” of the left and right cheek skin was compared by palpation. The results are shown in Table 5.

  From the results of Table 5, it was found that the “smoothness” of the cheek skin surface was improved by using the cosmetic lotion of Example 3 containing a phytoene-containing tomato extract.

(4) Sensory test of cosmetic lotion 2
[Example 4, Comparative Example 4]
A sensory test was performed using a cosmetic lotion prepared based on the formulation described in Table 4.

  Panelists were 16 female panelists in their 40s who were aware of wrinkles and skin firmness. After sleeping at night, washing their face, and using only the cosmetic lotion of Example 4 on the right half of their face. Only the cosmetic lotion of Comparative Example 4 was used in the left half. After 1 month, the left and right cheek skin conditions were compared by palpation. The results are shown in Table 7.

  From the results in Table 7, it was found that the “smoothness” of the cheek skin surface was improved by using the cosmetic lotion of Example 4 containing a phytoene-containing tomato extract.

<4. Formulation Example>
Tables 8 to 11 show formulation examples of tablets, beverages (soft drinks), and foods (candy and tomato jelly), respectively.

  The present invention can be applied to formulation techniques for cosmetics, medicines, foods and the like.

Claims (2)

  Collagenase activity and / or elastase activity inhibitor containing phytoene and / or phytofluene.   An anti-wrinkle agent comprising the inhibitor according to claim 1.