WO2022210522A1

WO2022210522A1 - Method for manufacturing oil extract component, whitening agent, hyperpigmentation improving agent, oral composition for whitening, and oral composition for improving hyperpigmentation

Info

Publication number: WO2022210522A1
Application number: PCT/JP2022/014955
Authority: WO
Inventors: こず枝坂尾; 雅史山本; 徳興侯
Original assignee: 国立大学法人鹿児島大学
Priority date: 2021-03-29
Filing date: 2022-03-28
Publication date: 2022-10-06
Also published as: JPWO2022210522A1

Abstract

This method for manufacturing an oil extract component includes a crushing step for crushing fruit in vegetable oil, and a separation step for separating an extract oil from the mixture obtained in the crushing step. The fruit is at least one type of fruit selected from the group consisting of mandarins, mandarin hybrids, citrons, and yuzus.

Description

Method for producing oil extract component, whitening agent, anti-pigmentation agent, oral composition for whitening, and oral composition for improving hyperpigmentation

The present invention relates to a method for producing an oil extract component, a whitening agent, an anti-pigmentation agent, an oral composition for whitening, and an oral composition for improving hyperpigmentation.

Citrus fruits are known to contain functional ingredients that have a whitening effect. For example, Patent Document 1 shows that tyramine, which is also contained in mandarins, reduces the amount of melanin. Patent Document 2 discloses a whitening agent containing an extract obtained by extracting citrus fruit with alcohol.

In addition to highly volatile essential oils extracted from citrus peel, polymethoxyflavonoids such as hesperidin, tangeretin and nobiletin, which are hydrophobic components, have been reported as components contained in many citrus fruits (Patent Document 3, Non-Patent Document 3). See Document 1 and Non-Patent Document 2). As a method for extracting polymethoxyflavonoids, extraction using alcohols such as ethanol is common. As a method for extracting essential oils, a compression method, a distillation method, a microwave extraction method, and the like are known. Non-Patent Document 3 discloses a cold-pressed oil method in which citrus flavedo (exocarp) is pressed to extract the essential oil present in the oil sac.

Japanese Patent Publication No. 2004-506677 JP 2005-132793 A JP 2018-16693 A JP 2009-29989 A

In extraction using alcohol, essential oils, which are volatile components, volatilize during the concentration stage, so it is not possible to efficiently obtain essential oils that may be contained in citrus fruits. In the distillation method, the essential oil may change in quality due to heat. Oil yield is low in the pressing method. In Patent Document 3, the essential oil is separated from the fruit of Shikwasa by pressing, but only 800 g of the essential oil is obtained from 2,800 kg of fruit. In addition, microwave extraction, supercritical carbon dioxide extraction, and ultrahigh pressure treatment are known, but all of them require special equipment, and it is difficult to say that essential oils can be easily extracted.

Patent Document 4 discloses a method of obtaining olive oil containing citrus fruit components by simultaneously pressing citrus fruits and olive fruits in a mixed state. However, in Patent Document 4, limonene and total polyphenols are quantified as components of citrus fruits transferred to olive oil, but the function of the olive oil is not examined. Therefore, it is unknown whether other useful citrus components can be extracted by this method.

The present invention has been made in view of the above circumstances, and provides a method for producing an oil-extracted component that can easily and efficiently extract components useful for skin whitening, and a skin whitening agent and pigmentation ameliorator using the useful components. , an oral composition for whitening and an oral composition for improving hyperpigmentation.

The method for producing an oil extract component according to the first aspect of the present invention comprises:
a comminution step of comminuting the fruit in vegetable oil;
a separation step of separating the extracted oil from the mixture obtained in the grinding step;
including
The fruit is
It is at least one selected from the group consisting of mandarins, mandarin hybrids, citrons and citrons.

In the pulverizing step,
grinding 1 part by weight of said fruit in 1-0.7 parts by weight of said vegetable oil;
You can do it.

The fruit is
Kozu B (Citrus depressa Hayata), Citrus medica L., Shikunin (Citrus depressa Hayata), Kunenbo (Citrus nobilis Lour.), Citrus sp. Bergamot (Citrus bergamia Poit. et Turp.), Rokugatsu mandarin orange (Citrus rokugatsu Hort. ex Tanaka) or Yuzu (Citrus junos Sieb. ex Tanaka),
You can do it.

The vegetable oil is
corn oil, olive oil, rice oil, castor oil or canola oil;
You can do it.

The whitening agent or anti-pigmentation agent according to the second aspect of the present invention is
Contains fruit oil extract ingredients,
The fruit is
It is at least one selected from the group consisting of mandarins, mandarin hybrids, citrons and citrons.

The oral composition for whitening or the oral composition for improving pigmentation according to the third aspect of the present invention is
Contains fruit oil extract ingredients,
The fruit is
It is at least one selected from the group consisting of mandarins, mandarin hybrids, citrons and citrons.

According to the present invention, ingredients useful for whitening can be extracted simply and efficiently. Also provided are a whitening agent, an anti-pigmentation agent, an oral composition for whitening, and an oral composition for improving hyperpigmentation using the useful ingredient.

1 is a diagram showing a spectrum of ultraviolet-visible spectroscopy (UV-vis) of extracted oil extracted from thinned or mature fruits of mandarins in Example 1. FIG. 1 shows the UV-vis spectrum of the extracted oil extracted from the mandarin fruits pulverized in a predetermined amount of vegetable oil in Example 1. FIG. 1 is a diagram showing a UV-vis spectrum of extracted oil extracted from thinned fruits such as mandarins in Example 1. FIG. 1 is a diagram showing a UV-vis spectrum of an extracted oil extracted from mature fruits such as mandarins in Example 1. FIG. 1 is a diagram showing a chromatogram of extracted oil extracted from thinned fruits such as mandarins obtained by liquid chromatography-mass spectrometry (LC-MS) in Test Example 1. FIG. 1 is a chromatogram of oil extracted from mature fruits such as mandarins obtained by LC-MS in Test Example 1. FIG. FIG. 1 shows polymethoxyflavonoids presumed to be contained in extracted oil. 2 is a diagram showing a chromatogram of oil extracted from thinned fruits such as mandarins obtained by gas chromatography-mass spectrometry (GC-MS) in Test Example 2. FIG. 2 is a chromatogram of oil extracted from mature fruits such as mandarins obtained by GC-MS in Test Example 2. FIG. FIG. 2 is a diagram showing a chromatogram of extracted oil extracted from thinned fruits of Kozu B obtained by high performance liquid chromatography (HPLC) in Test Example 3. FIG. 2 is a diagram showing a chromatogram of extracted oil extracted from mature fruits of Kozu B obtained by HPLC in Test Example 3. FIG. 2 is a diagram showing the relative concentrations of nobiletin in extracted oils such as mandarins according to Test Example 3. FIG. FIG. 10 is a diagram showing relative values of melanin content in mouse cells exposed to the extracted oil according to Test Example 4. FIG. FIG. 10 is a diagram showing relative values of melanin content in mouse cells exposed to ethanol extract and extracted oil according to Test Example 4. FIG. FIG. 10 is a diagram showing relative values of melanin content in human cells exposed to the extracted oil according to Test Example 4. FIG. FIG. 10 shows the relative inhibition of intracellular tyrosinase activity in human cells exposed to the extracted oil according to Test Example 4; FIG. 4 shows the relative viability of human cells exposed to extracted oil according to Test Example 4; FIG. 10 is a diagram showing relative values of melanin content in mouse cells exposed to the extracted oil according to Test Example 5. FIG. FIG. 10 is a diagram showing a UV-vis spectrum according to Test Example 6;

An embodiment according to the present invention will be described with reference to the drawings. The present invention is not limited by the following embodiments and drawings. In the following embodiments, expressions such as "have", "include" and "contain" also include the meaning of "consisting of" or "consisting of". In this specification, mandarins, mandarin hybrids, citrons and yuzu may be collectively referred to as "mandarins and the like", and mandarins and the like are mandarins, mandarin hybrids, citrons and yuzu. means at least one of

(Embodiment 1)
The method for producing an oil extract component such as mandarins according to the present embodiment includes a pulverization step and a separation step. The crushing step crushes the fruit in vegetable oil.

A vegetable oil is an oil extracted and refined from a plant. Plants that are raw materials for vegetable oil are not particularly limited, and include corn, olive, rice, castor, canola, soybean, rapeseed, cottonseed, sunflower seed, peanut, palm fruit, coconut, sesame and linseed. Vegetable oils include, for example, corn oil, olive oil, rice oil, castor oil, canola oil, and the like. Preferably, the vegetable oil is corn oil. Corn oil itself has a skin-whitening effect, and a synergistic effect with mandarins and the like is expected to provide a more skin-whitening effect.

The fruit is at least one selected from the group consisting of mandarins, mandarin hybrids, citrons and citrons.

Mandarins are any variety or strain belonging to the genus Citrus mandarins. Mandarins include Kunenbo, Tokunin, King Mandarin (Citrus nobilis Lour.), Unshu Mandarin Log, Aoshima Unshu, Jutaro Unshu, Shirakawa Unshu, Juman Unshu, Otsu No. 4, Okitsu Wase, Miyagawa Wase, Kagoshima Wase ( Citrus unshu Marc.), Citrus keraji Hort. ex Tanaka, Kikai mandarin orange, Natsukunin, Kabuchii (Citrus keraji var. Kabuchii Hort. ex Tanaka), Citrus oto Hort. ex Y. Tanaka , Yoshida Ponkan, Sashu (Citrus reticulata Blanco), Mediterranean Mandarin (Citrus deliciosa Ten.), Genshokan (Citrus genshokan Hort. ex Tanaka), Red mandarin orange (Citrus tangerina Hort. ex Tanaka), Clementine (Citrus mandarin) ex Tanaka), Tachibana (Citrus tachibana (Mak.) Tanaka), Kinokuni, Sakurajima Comics, Uskawa (Citrus kinokuni Hort. ex Tanaka), Sunki (Citrus sunki Hort. ex Tanaka), Cleopatra (Citrus Tanak resh. ), Shikwasa, Sekunin, Seek Rib (Citrus depressa Hayata), Koji (Citrus leiocarpa Hort. ex Tanaka), Kimikan and Kozu, and Kuroshima Mikan (Citrus sp.). It should be noted that some coz are classified as Citrus depressa Hayata.

Mandarin hybrids are varieties developed by crossing mandarins and varieties other than mandarins in the genus Citrus, such as limes, lemons, buntans, sour oranges, sweet oranges, or kumquats. Mandarin hybrids include, for example, Sweet Spring, which is a hybrid of Unshu mandarin and Hassaku, Kiyomi, which is a hybrid of Unshu mandarin and Sweet Orange, Southern Yellow, which is a hybrid of Buntan and Kinokuni, and Orlando, which is a hybrid of Dancy Tangerine and Grapefruit. , Seminole and Minneola, Ariake, a cross between Navel Orange and Clementine, Shiranui, a cross between Kiyomi and Ponkan, Amakusa, a cross between Kiyomi and Okitsuwase and Page, and Marcot, a cross between Kiyomi and Angkor. and Tsunoka, which is a cross between Kiyomi and Okitsuwase. Although Buntan belongs to the genus Citrus, it is neither a mandarin nor a mandarin hybrid.

Citrons are, for example, citron and barotine bergamot. Citrons are cultivars that arose independently of mandarin. Examples of citrons include yuzu, hanayu (Citrus hanayu Hort. ex Shirai), kabosu (Citrus sphaerocarpa Hort. ex Tanaka) and sudachi (Citrus sudachi Hort. ex Shirai). Yuzu is a cultivar that arose independently of mandarin.

Preferable fruits include Unshu mandarin orange, Kikai mandarin orange, Kunenbo, Kerajimikan (Citrus keraji Hort. ex Tanaka) such as Otsu No. 4, Kozu B, Sekunin, Shimamikan (also called Chinazekunin or Kuroshimamikan), Tunugekunin (Citrus sp.), Citron, Kimikan, ballotin bergamot, and citrus mandarin are exemplified. Particularly preferably, the fruit is Kozu B, Citron, Shikunin, Kunenbo, Shimamikan, Kimmikan, Barotine Bergamot, Rokugatsumikan or Yuzu.

Fruits such as mandarins may be small, green fruit that has been thinned, or mature fruit that has been matured. Preferably, the fruit is used together with flavedo containing oil sacs. As the fruit, a fruit that has been frozen and thawed after harvesting may be used, or a freeze-dried fruit may be used. In addition, mandarins and the like may be used singly or in combination of plural kinds.

Fruits such as mandarins may be pulverized in vegetable oil by a known method, for example, pulverized with a mixer. Grinding may be performed in stages, and tumble mixing may be performed between grindings to uniformly mix the vegetable oil and the pulverized fruit pieces.

The ratio of the mass of fruit and the mass of vegetable oil in the grinding step is preferably 1:1 to 3:2. For example, in the grinding step, 1 part by weight of fruit in 1 to 0.7 parts by weight, preferably 1 to 0.8 parts by weight, more preferably 1 to 0.9 parts by weight, particularly preferably 1 part by weight of vegetable oil pulverize.

A mixture of fruit pieces and vegetable oil is obtained through a pulverization step. The separation step separates the extracted oil from the mixture obtained in the grinding step. The extracted oil can be separated from the mixture by known methods. For example, in the separation step, the upper layer obtained by centrifugation may be recovered as extracted oil. In the separation step, the upper layer obtained by centrifugation may be further filtered to remove solids.

The components contained in the extracted oil obtained in the separation step (oil extract components) include flavonoids and carotenoids, as well as essential oils (fragrant components), as shown in the examples below. The oil extract component contains a functional substance having a skin whitening effect.

According to the method for producing an oil-extracted component according to the present embodiment, components useful for whitening can be simply and efficiently extracted from mandarins and the like without using a special device or the like. In the pulverization step in the method for producing an oil extract component according to the present embodiment, fruits such as mandarins in vegetable oil are pulverized. By pulverizing in vegetable oil, it is possible to extract aromatic components together with components useful for skin whitening.

It should be noted that in the pulverization step, in addition to the vegetable oil, the fruit may be pulverized together with an oil agent other than the vegetable oil. The oil agent is not particularly limited as long as it is, for example, an oil other than the above vegetable oils, and includes animal oils, synthetic oils, semi-solid oils, liquid oils, volatile oils, fats and oils, fatty acids, silicone oils, fluorine-based oils, and lanolin. derivatives and the like. Specific examples of oil agents include fatty acids such as isostearic acid and oleic acid; low polymerization degree dimethylpolysiloxane, methylphenylpolysiloxane, decamethylcyclopentasiloxane, octamethylcyclotetrasiloxane, crosslinked organopolysiloxane, and fluorine-modified silicone oils such as polysiloxane; fluorine-based oils such as perfluorodecane and perfluorooctane;

Also, in the crushing step, the fruit may be crushed in the presence of the additive. Additives include, for example, excipients, lubricants, binders, disintegrants, solvents, solubilizers, suspending agents, tonicity agents, buffers, soothing agents, preservatives, antioxidants, It is a coloring agent and a sweetening agent.

Excipients include lactose, sucrose, D-mannitol, D-sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, low-substituted hydroxypropylcellulose, sodium carboxymethylcellulose, gum arabic, pullulan, soft anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminometasilicate, xylitol, sorbitol and erythritol;

Lubricants include, for example, magnesium stearate, calcium stearate, talc, colloidal silica and polyethylene glycol.

Binders include pregelatinized starch, sucrose, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropylcellulose, hydroxypropylmethylcellulose and polyvinyl. Pyrrolidone and the like are exemplified.

Disintegrants include, for example, lactose, sucrose, starch, carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, carboxymethylstarch sodium, low-substituted hydroxypropylcellulose, soft anhydrous silicic acid and calcium carbonate.

Solvents include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil and cottonseed oil. Solubility aids include, for example, polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, tris(hydroxymethyl)aminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate and acetic acid. sodium and the like.

Suspending agents include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride and glyceryl monostearate; Hydrophilic polymers such as sodium methyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose; polysorbates, polyoxyethylene hydrogenated castor oil and the like.

Isotonic agents include, for example, sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose, xylitol and fructose. Buffers include, for example, phosphate, acetate, carbonate and citrate buffers. Analgesics include, for example, propylene glycol, lidocaine hydrochloride and benzyl alcohol.

Preservatives include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid and sorbic acid. Examples of antioxidants include sulfites and ascorbates. Coloring agents include water-soluble colored tar pigments, lake pigments, natural pigments, and the like. Sweeteners are, for example, sodium saccharin, dipotassium glycyrrhizinate, aspartame and stevia.

(Embodiment 2)
The whitening agent according to the present embodiment contains an oil extract component of fruits such as mandarins. The oil extract component is preferably produced by the production method according to the first embodiment. Skin lightening agents may also be included as diluents and concentrates of oil extracts, as well as dried and powdered products thereof.

The whitening agent according to the present embodiment is produced by a known method, and contains 0.000001 to 99.9% by mass, 0.00001 to 99.8% by mass, 0.0001 to 99.7% by mass, 0.001-99.6% by weight, 0.01-99.5% by weight, 0.1-99% by weight, 0.5-60% by weight, 1-50% by weight or 1-20% by weight of oil extraction Contains ingredients. A whitening agent may be a solid formulation or a liquid formulation. The whitening agent may be administered orally or topically, but is preferably used as an external preparation. When the whitening agent is an external preparation, the whitening agent is applied to the skin in such a manner that the oil extract component is percutaneously absorbed. Examples of external preparations include ointments, creams, milky lotions, liniments, lotions and patches.

The whitening agent may contain any pharmacologically acceptable ingredient in addition to the oil extract ingredient. Optional ingredients are, for example, the above excipients, lubricants, binders, disintegrants, solvents, solubilizers, suspending agents, tonicity agents, buffers and soothing agents. Additives such as the above-mentioned preservatives, antioxidants, coloring agents and sweetening agents may also be added to the whitening agent, if necessary.

The dosage of the whitening agent is appropriately determined according to the subject's gender, age, weight, symptoms, etc. The skin lightening agent is administered in an effective amount of oil extract. An effective amount is that amount of the oil extract necessary to achieve the desired result and is that amount necessary to slow, inhibit, prevent, reverse or cure the progression of skin darkening and darkening. .

The dosage of the whitening agent is, for example, 0.01 mg/kg to 1000 mg/kg, preferably 0.1 mg/kg to 200 mg/kg, more preferably 0.2 mg/kg to 20 mg/kg, once a day. When the whitening agent is administered in divided doses, the whitening agent is administered from 1 to 4 times a day. Also, the whitening agent may be administered at various dosing frequencies such as daily, every other day, once a week, every other week and once a month. Amounts outside the above ranges can also be used, if desired.

The administration target of the whitening agent is preferably a vertebrate, more preferably a mammal. Mammals include, for example, humans, chimpanzees and other primates, domestic animals such as dogs, cats, rabbits, horses, sheep, goats, cows, pigs, rats, mice and guinea pigs, pet animals and laboratory animals. mentioned. Most preferably, the mammal is human.

The fruit oil extract component such as mandarins contained as an active ingredient in the whitening agent according to the present embodiment can suppress the amount of melanin in melanoma cells, as shown in the following examples. In addition, the oil extract component inhibits the activity of intracellular tyrosinase. Therefore, the whitening agent according to the present embodiment has a whitening effect by suppressing melanin production.

Since the production of melanin causes hyperpigmentation such as age spots, freckles and dark skin, another embodiment provides a hyperpigmentation improving agent containing a fruit oil extract component such as mandarins. In addition, the above-mentioned whitening agents and anti-pigmentation agents can also be used as cosmetics. When used as cosmetics, the whitening agent and antipigmentation agent are preferably applied to the skin as an ointment, cream, milky lotion, liniment, lotion, patch, or the like.

In another embodiment, there is provided an oral composition for whitening or an oral composition for improving hyperpigmentation, containing an oil extract component of fruits such as mandarins as an active ingredient. Specific examples of oral compositions include supplements, food compositions, food and drink, functional foods and food additives.

The form of the supplement is not particularly limited, and may be in any form such as tablets, powders, granules, capsules, sugar-coated tablets, films, lozenges, chewables, solutions, emulsions, and suspensions. The supplement may contain any component commonly used as a supplement, in addition to the fruit oil extract component such as mandarins. Examples of such components include amino acids, peptides; vitamins such as vitamin E, vitamin C, vitamin A, vitamin B and folic acid; minerals; sugars; inorganic salts; citric acid or salts thereof; Nutrient tonic ingredients such as royal jelly and taurine; crude drug extracts such as ginger extract and ginseng extract; herbs;

Mandarins and other fruit oil extract ingredients are mixed into various foods or beverages to make functional foods so that the oil extract ingredients of fruits such as mandarins can be easily orally ingested on a daily basis. Fruit oil extract components can be ingested for a long period of time. “Functional food” means food or beverage that is ingested for the purpose of maintaining health, and includes food with health claims, food for specified health use, food with nutrient function claims, health food, nutritional supplement, and the like. Among these, food with specified health uses or food with nutrient function claims, which are foods with health claims, are preferable. In addition, when commercializing as a functional food, various additives used in food, specifically, coloring agents, preservatives, thickening stabilizers, antioxidants, bleaching agents, antibacterial antifungal agents , acidulants, seasonings, emulsifiers, enhancers, manufacturing agents and flavoring agents may be added.

There are no particular restrictions on foods and beverages that are subject to functional foods. Forms of functional foods include, for example, nutritional drinks, soft drinks, beverages such as black tea and green tea; sweets such as candy, cookies, tablets, chewing gum and jelly; processed grain products such as noodles, bread, rice and biscuits; These include paste products such as sausage, ham and fish paste; dairy products such as butter and yogurt; furikake; The functional food may contain additives such as sweeteners, flavoring agents and coloring agents.

The ratio of the fruit oil extract component such as mandarins to be added to the functional food is arbitrary, but the ratio is selected within a range that contributes to at least one of promotion of whitening, suppression, prevention and improvement of pigmentation. . In addition, fruit oil extract components such as mandarins can also be used as food additives.

In another embodiment, there is provided a method of treating hyperpigmentation by administering a fruit oil extract component such as mandarins to a subject. Yet another embodiment is the use of fruit oil extracts such as mandarins to treat hyperpigmentation. In another embodiment, fruit oil extracts such as mandarins are provided for use as skin lightening agents or anti-pigmentation agents. Yet another embodiment is the use of fruit oil extract components such as mandarins for the manufacture of a medicament for promoting skin whitening or for inhibiting hyperpigmentation.

In addition, in order to evaluate the function of the oil extract component of fruits such as mandarins, for example, the inhibitory activity of melanin production in cultured cells, separation of the extracted oil produced from fruits such as mandarins and an aqueous culture medium In order to suppress this, it is necessary to make the extracted oil an O/W (Oil in Water) emulsion liquid. In this case, casein is preferred as the emulsifier. Casein may be alkali metal salts such as lithium, sodium and potassium salts.

For the O/W emulsion liquid containing extracted oil, for example, water, extracted oil and emulsifier may be mixed and sufficiently stirred. Stirring may be performed by a known method, but is preferably agitated by ultrasonication in order to emulsify in a short period of time. The mixing ratio of water, extracted oil and emulsifier is 10 to 15 v/v%, preferably 11 to 14 v/v%, more preferably 12 v/v%. parts is 0.004 to 0.007, preferably 0.005 to 0.006, more preferably 0.0053 to 0.0057.

The present invention will be explained more specifically by the following examples, but the present invention is not limited by the examples.

[Example 1: Extraction of hydrophobic components with vegetable oil]
Corn oil, olive oil, rice oil, castor oil and canola oil were used as vegetable oils for extraction. As mandarins and the like, fruits such as Unshu mandarin "Otsu No. 4", Kikai mandarin, Kunenbo, Kerajimikan, Kozu B, Sekunin, Chinazekunin and Tunugekunin were used together with their peels. As fruits, thinned fruits and mature fruits were used. A sample frozen at −20° C. was naturally thawed, and about 50 g was weighed into a mixer cup. A predetermined mass ratio of vegetable oil to the mass of the fruit was weighed into the same cup, and ground in oil in stages of 30 seconds→10 seconds→20 seconds using a mixer. Inversion mixing was also performed appropriately during milling. It was centrifuged at 4°C and 2330 xg for 5 minutes using a refrigerated centrifuge. The uppermost layer (oil layer) was collected by filtration through a two-layered coffee filter (FP102, manufactured by Kalita).

(Measurement of UV-vis)
UV absorption spectra were measured at full spectrum (190-840 nm) using a Thermo Scientific NanoDrop™ 2000c ultratrace spectrophotometer. Blanks were taken with corn oil and then measured with 2 μl of each extracted oil extracted with a given mass ratio of vegetable oil to fruit mass.

(result)
FIG. 1 shows the absorbance of the extracted oil extracted from the thinned or mature fruit of Unshu mandarin "Otsu No. 4". Peaks at 300-400 nm corresponding to flavonoids and 400-500 nm corresponding to carotenoids were confirmed for all vegetable oils. Although the extraction efficiency differs depending on the type of oil, it was shown that extraction was possible with all the vegetable oils used.

Fig. 2 shows the absorbance of extracted oil extracted from mature fruit at a mass ratio of sequinin to corn oil of 1:10, 1:1 or 3:2. It was shown that more flavonoids and carotenoids were extracted at a mass ratio of fruit to vegetable oil ranging from 1:1 to 3:2.

Fig. 3 shows the absorbance obtained by measuring the extracted oil extracted from the thinned fruit at a mass ratio of 1:1 between each mandarin, etc. and corn oil. FIG. 4 shows the absorbance obtained by measuring the extracted oil extracted from the mature fruit at a mass ratio of 1:1 between the mass of each mandarin and the like and the corn oil. As shown in FIGS. 3 and 4, hydrophobic components including flavonoids and carotenoids were extracted from both thinned and mature fruits.

[Test Example 1: LC-MS measurement]
Hydrophobic components were analyzed by LC-MS measurement under the following conditions.
LC section conditions (Apparatus) LC-20 high pressure gradient system (manufactured by Shimadzu Corporation)
(Analysis column) COSMOSIL, πNAP (4.6 ID x 250 mm, manufactured by Nacalai Tesque)
(Mobile phase flow rate) 1.0 ml/min (Injection volume) 5 μl
(Column temperature) 40°C
(Gradient) A: H ₂ O (100%) B: Tetrahydrofuran (THF) (100%), 30% B (0 min) → 30% B (2 min) → 70% B (20 min) linear total run time : 25 minutes (detection UV wavelength) 330 nm
Conditions for MS part (Apparatus) 3200QTRAP LC/MS/MS system (manufactured by AB SCIEX)
(Ion source parameters) Curtain Gas: 20 psi, Collision Gas: High, Ion Spray Voltage: 5500 V, Temperature: 600° C., Ion Source Gas1: 50 psi, Ion Source Gas2: 80 psi, Interface Heater: ON

(result)
FIG. 5 shows the measured strength of the extracted oil extracted from the thinned fruit at a mass ratio of 1:1 between the mass of each mandarin and the like and the corn oil. FIG. 6 shows the measured strength of the extracted oil extracted from the mature fruits at a mass ratio of 1:1 between the mass of each mandarin and the like and the corn oil. As shown in FIGS. 5 and 6, it was confirmed that flavonoids or glycosides were contained in the extracted oil regardless of whether the fruit was thinned or matured.

Fig. 7 shows examples of polymethoxyflavonoids presumed to be contained in each mandarin, etc. based on their molecular weights (○ means presumed containment). It was suggested that the extracted oil obtained from mandarins and the like contains a functional ingredient having a whitening effect.

[Test Example 2: GC-MS measurement]
Analysis of essential oils (aroma components) by GC-MS measurement was performed as follows. 1 ml of each sample was sealed in a 2 ml vial and measured with an Agilent 5975C series GC/MSD system (manufactured by Agilent Technologies). As conditions for GC analysis, the inlet temperature was set to 250° C., the injection conditions were set to split mode, and the split ratio was set to 1:20. HP-5MS (length 30 m, inner diameter 0.25 mm, film thickness 0.25 μm, manufactured by Agilent J&W) was used as a separation column, and the average linear velocity of helium carrier gas was set to 1 ml/min. The column temperature was maintained at 40° C. for 5 minutes, raised at 3° C./min to 150° C., then raised at 9° C./min to 240° C., and held at a final temperature of 240° C. for 5 minutes (Total run time: 60 minutes).

The measurement conditions for the mass detector were an ionization voltage of 70 eV (EI) in the scan range of m/z 35-450, and a transfer temperature of 240°C. Estimation of aroma components was performed using mass spectra (NIST08 library, similarity >80%).

(result)
FIG. 8 shows the absorbance obtained by measuring the extracted oil extracted from the thinned fruit at a mass ratio of 1:1 between the mass of each mandarin and the like and the corn oil. FIG. 9 shows the absorbance measured for the extracted oil extracted from the mature fruit at a mass ratio of 1:1 between the mass of each mandarin and the like and the corn oil. As shown in FIGS. 8 and 9, it was confirmed that the extracted oil contained aromatic components regardless of whether the fruit was thinned or matured.

GC-MS measurement revealed 14 aldehydes, 20 monoterpenes, 2 diterpenes, 17 sesquiterpenes, 3 alcohols, 11 monoterpene derivatives, 6 diterpene derivatives, 6 sesquiterpenes, It was presumed that 1 type of terpene derivative and 8 types of other aroma components were detected. In addition, from mature fruits, 10 kinds of aldehydes, 17 kinds of monoterpenes, 1 kind of diterpenes, 10 kinds of sesquiterpenes, 1 kind of alcohols, 9 kinds of monoterpene derivatives, 4 kinds of diterpene derivatives, sesquiterpene derivatives It was presumed that 1 type and 3 other scent components were detected. From the above, it was suggested that the extracted oil may contain aromatic components having a whitening effect derived from mandarins and the like.

[Test Example 3: HPLC measurement]
The component analysis of the extracted oil was performed by HPLC measurement under the following conditions. As a measurement sample, the extracted oil was diluted with an equal amount of THF.
(Apparatus) LC-900 series (manufactured by JASCO Corporation)
(Analysis column) COSMOSIL, πNAP (4.6 ID × 250 mm, manufactured by Nacalai Tesque)
(Mobile phase flow rate) 1.0 ml/min (Injection volume) 5 µl
(Column temperature) 40°C
(Gradient) A: H ₂ O (100%) B: THF (100%), 30% B (0 min) → 30% B (2 min) → 70% B (20 min) linear Total run time: 25 min (Detection UV wavelength) 330 nm

Regarding the content of nobiletin in the Coz B extracted oil, the concentration was determined from the ratio of the calibration curve of the standard product and the UV absorption value of the measurement sample.

(result)
FIGS. 10 and 11 show HPLC chromatograms of the oil extracted from the thinned and mature fruits of Kozu B, respectively. FIG. 12 shows the concentrations of nobiletin in extracted oils such as mandarins. Nobiletin tended to be contained more in thinned fruit than in mature fruit. A particularly large amount of nobiletin was found in thinned fruits of sequnin, coz B and chinazekunin.

[Test Example 4: Evaluation experiment 1 using cells]
(Preparation of O/W emulsion liquid)
In order to evaluate the skin-whitening effect of the extracted oil in cultured cells, we first examined the conditions under which the extracted oil and water would not separate in the cell culture medium. As a result of examining the food additives trisodium citrate, lecithin and sodium caseinate as emulsifiers, the stability of the O/W emulsion liquid prepared with sodium caseinate was the highest, and even in a 48-hour aqueous experiment, the emulsion form was stable. was found to be maintained.

The O/W emulsion liquid was prepared by the method shown below. 988 µl of sterilized water, 12 µl of corn oil or citrus extract oil, and 0.005496 g of sodium caseinate were placed in a 1.5 ml tube to prepare a mixed solution. A 1.5 ml tube containing the mixed solution was placed in an ultrasonic cleaner (ASU series, manufactured by AS ONE), and subjected to ultrasonic treatment at 43 kHz for 10 minutes under cold water a total of two times.

(cell culture)
C57BL/6 mouse melanoma (B16 melanoma cells) and human malignant melanoma (HMV-II cells) were obtained from RIKEN CELL BANK. Using DMEM (containing 1% PSG and 10% FBS) medium and Ham'F12 (containing 10% FBS) medium, they were cultured at 37° C. and 5% CO ₂ .

(Measurement of intracellular melanin content)
B16 melanoma cells and HMV-II cells were seeded at 1.4×10 ⁵ cells/well and HMV-II cells at 3.0×10 ⁵ cells/well in a 6-well plate, respectively, and cultured at 37° C., 5% CO ₂ for 24 hours. did. After adding the O/W emulsion containing the extracted oil, the cells were cultured at 37° C. and 5% CO ₂ for 48 hours. After adding 500 μl of trypsin and incubating at 37° C. for 3 minutes, the cells were collected. After centrifugation at 300 G for 5 min at 4° C., the supernatant was removed and washed once with phosphate buffered saline (PBS). After adding 120 μl of 1 M aqueous sodium hydroxide solution and heating at 80° C. for 30 minutes, 100 μl of each well was spread on each well of a 96-well plate, and the absorbance at a wavelength of 405 nm was measured with a plate reader spectrophotometer. Cell supernatants other than controls were added with 0.050 mM theophylline as a melanogenesis inducer along with the samples. Also, kojic acid, which has a high whitening effect, was used as a positive control.

(Measurement of intracellular tyrosinase activity inhibition)
B16 melanoma cells were plated on a 6-cm dish at 1.4×10 ⁵ cells/well and cultured at 37° C., 5% CO ₂ for 24 hours. After adding the O/W emulsion containing the extracted oil, the cells were cultured at 37° C. and 5% CO ₂ for 48 hours. The cells were scraped off with a scraper and collected in a tube together with the medium. It was centrifuged at 300G for 5 minutes at 4°C and the supernatant was removed. After centrifugation at 300G for 5 minutes at 4°C, the supernatant was aspirated and washed once with PBS. Add 300 μl of 1 M phosphate buffer containing 0.1% Triton X-100 to the cell pellet, and sonicate for 12.5 minutes (ice water, High power, 1 cycle of crushing for 10 seconds and resting for 20 seconds). 25 cycles), followed by centrifugation at 15,000 rpm for 30 minutes at 4° C., and 200 μl of the supernatant was recovered to obtain a protein solution.

To quantify the protein, 10 μl of the protein solution was added to 90 μl of 1×PBS and diluted, and 15 μl each of the diluted protein solution and BSA aqueous solution were placed in a 96-well plate. 1× Dye Reagent (RC DC protein assay, manufactured by Bio-Rad) was added in an aqueous solution of 135 μl each, shaken for 1 minute and allowed to stand for 4 minutes under light-shielding conditions, and the absorbance at a wavelength of 595 nm was measured with a plate reader spectrophotometer. .

On the other hand, 150 μl of 1M phosphate buffer and 150 μl of MBTH (3-methyl-2-benzothiazolinone hydrazone, manufactured by Tokyo Kasei Kogyo Co., Ltd.) aqueous solution were added to 150 μl of the protein solution and warmed at 37° C. for 10 minutes. Furthermore, 150 μl of L-DOPA (L-3-(3,4-Dihydroxyphenyl) alanine, manufactured by Nacalai Tesque) solution was added, reacted at 37° C. for 30 minutes, 150 μl was placed in each well of a 96-well plate, and a spectrometer for a plate reader was added. Absorbance was measured at a wavelength of 450 nm with a photometer.

(Cytotoxicity experiment)
The toxicity of the extracted oil to cells was measured using the MTT assay. An O/W emulsion solution containing extracted oil was added to HMV-II cells precultured for 24 hours, and after culturing for 48 hours, 10 μl of MTT solution (5 mg/ml) was added and further cultured for 4 hours. After removing the supernatant and adding DMSO (500 μl/well) to completely dissolve MTT formazan, absorbance at a wavelength of 570 nm was measured with a plate reader spectrophotometer (manufactured by Thermo Scientific). Cell viability (%) was calculated based on the decrease in absorbance due to sample addition.

(result)
FIG. 13 shows relative values of melanin content per cell in B16 melanoma cells. In this test, an oil extracted from a freeze-dried fruit sample was also examined in the same manner as in Example 1 above. Each mandarin and the like inhibited melanin production induced by theophylline more than kojic acid (0.7 mM).

Fig. 14 shows the results of comparing the effect on the amount of melanin in B16 melanoma cells with an ethanol extract extracted in the same manner as in Example 1 with 70% ethanol instead of vegetable oil. The corn oil extract significantly suppressed the melanin production induced by theophylline compared to the ethanol extract.

Fig. 15 shows relative values of melanin amount per cell in HMV-II cells. Extracted oil, especially coz B, significantly suppressed the theophylline-induced melanin production even in human-derived cells.

FIG. 16 shows relative inhibition of intracellular tyrosinase activity. Extracted oil inhibited tyrosinase activity, the rate-limiting enzyme for melanogenesis.

Figure 17 shows the viability of HMV-II cells. The extracted oil showed no toxicity to human-derived cells.

[Test Example 5: Evaluation experiment 2 using cells]
Extracted oils were prepared from yuzu and sour pomelo as in Example 1 using corn oil. Using B16 melanoma cells in the same manner as in Test Example 4, the effect of the extracted oil on the amount of melanin per cell in the presence of theophylline was evaluated. Although sour pomelo belongs to the genus Citrus, it does not belong to the mandarin family.

(result)
FIG. 18 shows relative values of melanin content per cell. Extracted oil from yuzu inhibited melanin production induced by theophylline. On the other hand, the extract oil extracted from sour pomelo hardly inhibited melanin production induced by theophylline.

[Test Example 6: Comparison of extraction methods]
Corn oil was used as the vegetable oil for extraction, and extracted oil was extracted from Mandarin orange in the same manner as in Example 1 (Example A). The mass ratio of fruit to vegetable oil was 1:1. On the other hand, in order to compare the case of crushing the fruit in corn oil and the case of mixing the fruit with corn oil after crushing, the fruit was crushed in a mixer in the same manner as in Example 1 without being mixed with corn oil. A comparative example was prepared by mixing corn oil with the extract obtained by centrifugation followed by filtration. The masses of fruits and corn oil used in the preparation of Comparative Examples are the same as in Example A, respectively.

(result)
In the comparative preparation, the corn oil and mandarin orange were difficult to mix and remained separated. Similar to Test Example 1, the UV absorption spectra of Example A and Comparative Example were measured, and the results are shown in FIG. It was shown that almost no hydrophobic components, especially carotenoids, could be extracted by mixing with corn oil after crushing the fruit.

The above-described embodiments are for explaining the present invention and do not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims rather than the embodiments. Various modifications made within the scope of the claims and within the meaning of the invention equivalent thereto are considered to be within the scope of the present invention.

This application is based on Japanese Patent Application No. 2021-55807 filed on March 29, 2021. The entire specification, claims, and drawings of Japanese Patent Application No. 2021-55807 are incorporated herein by reference.

The present invention is useful for manufacturing pharmaceuticals, functional foods, beauty products, and the like.

Claims

a comminution step of comminuting the fruit in vegetable oil;
a separation step of separating the extracted oil from the mixture obtained in the grinding step;
including
The fruit is
At least one selected from the group consisting of mandarins, mandarin hybrids, citrons and citrons,
A method for producing an oil extract.
In the pulverizing step,
grinding 1 part by weight of said fruit in 1-0.7 parts by weight of said vegetable oil;
The manufacturing method according to claim 1.
The fruit is
Kozu B (Citrus depressa Hayata), Citrus medica L., Shikunin (Citrus depressa Hayata), Kunenbo (Citrus nobilis Lour.), Citrus sp. Bergamot (Citrus bergamia Poit. et Turp.), Rokugatsu mandarin orange (Citrus rokugatsu Hort. ex Tanaka) or Yuzu (Citrus junos Sieb. ex Tanaka),
The manufacturing method according to claim 1 or 2.
The vegetable oil is
corn oil, olive oil, rice oil, castor oil or canola oil;
The manufacturing method according to any one of claims 1 to 3.
Contains fruit oil extract ingredients,
The fruit is
At least one selected from the group consisting of mandarins, mandarin hybrids, citrons and citrons,
Whitening agent or pigmentation improving agent.
Contains fruit oil extract ingredients,
The fruit is
At least one selected from the group consisting of mandarins, mandarin hybrids, citrons and citrons,
An oral composition for whitening or an oral composition for improving hyperpigmentation.