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US20170165310A1 - Citrus seed extract-containing composition, food, drug, and method for producing citrus seed extract-containing composition - Google Patents

Citrus seed extract-containing composition, food, drug, and method for producing citrus seed extract-containing composition Download PDF

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Publication number
US20170165310A1
US20170165310A1 US15/118,132 US201415118132A US2017165310A1 US 20170165310 A1 US20170165310 A1 US 20170165310A1 US 201415118132 A US201415118132 A US 201415118132A US 2017165310 A1 US2017165310 A1 US 2017165310A1
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Prior art keywords
citrus
seed extract
obacunone
containing composition
citrus seed
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Taro HORIBA
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Kikkoman Corp
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Kikkoman Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/752—Citrus, e.g. lime, orange or lemon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction

Definitions

  • the present invention relates to a citrus seed extract-containing composition, a food, a drug, and a method for producing the citrus seed extract-containing composition.
  • the present invention relates to a citrus seed extract-containing composition, a food and a drug all of which have a novel application, and a method for producing such a citrus seed extract-containing composition.
  • a citrus fruit (e.g., belonging to genus Citrus , genus Poncirus and genus Fortunella of family Rutaceae of order Sapindales) is widely applied around the world to a food for eating raw, juice, a confectionery material, and seasoning for cooking. Further, a citrus fruit is recognized as a food for promoting health because a citrus contains quantities of vitamin C and dietary fibers.
  • Patent Document 1 discloses that a Yuzu extract has a function for promoting productivity of hyaluronic acid. Further, Patent Document 2 discloses that a single-celled product of Yuzu has a function for peeling keratin.
  • Patent Documents 1 and 2 disclose applications of Yuzu extracts (i.e., for promoting productivity of hyaluronic acid, and peeling keratin).
  • Patent Document 1 Japanese Unexamined Patent Application Publication No. 2001-158728
  • Patent Document 2 Japanese Unexamined Patent Application Publication No. 2004-10480
  • the demand includes identifying an active ingredient which is contained inside a citrus (i.e., particularly, inside a citrus seed) and greatly exerts the novel function, and eventually discovering a method for collecting more quantities of such an active ingredient.
  • an object of the present invention is to provide a citrus seed extract-containing composition, a food and a drug all of which sufficiently exert the novel function of a citrus (particularly, a citrus seed), and a method for producing the citrus seed extract-containing composition.
  • a method for producing a citrus seed extract-containing composition including the steps of heating citrus seeds at 60° C. or more, and extracting a citrus seed extract from the citrus seeds thus heated.
  • a ratio of the obacunone content to the nomilin content is set to at least a predetermined value, whereby a much quantity of obacunone is included therein. Accordingly, the citrus seed extract-containing composition of the present invention may sufficiently exert novel functions (e.g., lowering a blood-glucose level, increasing a skeletal muscle quantity and reducing a body fat).
  • a food and a drug of the present invention may sufficiently exert novel functions (e.g., lowering a blood-glucose level, increasing a skeletal muscle quantity and reducing a body fat) by containing a citrus seed extract-containing composition having a much quantity of obacunone.
  • a method for producing a citrus seed extract-containing composition of the present invention may sufficiently exert novel functions (e.g., lowering a blood-glucose level, increasing a skeletal muscle quantity and reducing a body fat) by including the step of heating citrus seeds at a predetermined temperature or more, allowing a much quantity of obacunone to be included in the citrus seed extract-containing composition.
  • novel functions e.g., lowering a blood-glucose level, increasing a skeletal muscle quantity and reducing a body fat
  • FIG. 1 shows measured data illustrating changes in blood-glucose levels of KKAy mice.
  • FIG. 2 shows measured data illustrating changes in HbA1c levels of KKAy mice.
  • FIG. 3 shows measured data illustrating changes in blood-glucose levels of KKAy mice.
  • FIG. 4 shows measured data illustrating changes in HbA1c levels of KKAy mice.
  • FIG. 5 shows measured data illustrating a weight ratio of a gastrocnemius muscle to a body of KKAy mice at the day 28 after initiating the rearing experiment.
  • FIG. 6A shows measured data illustrating a weight ratio of a perirenal fat to a body of KKAy mice at the day 28 after initiating the rearing experiment.
  • FIG. 6B shows measured data illustrating a weight ratio of a mesenterium fat to a body of KKAy mice at the day 28 after initiating the rearing experiment.
  • FIG. 6C shows measured data illustrating a weight ratio of an epididymis fat to a body of KKAy mice at the day 28 after initiating the rearing experiment.
  • FIG. 6D shows measured data illustrating a weight ratio of a subcutaneous fat to a body of KKAy mice at the day 28 after initiating the rearing experiment.
  • FIG. 7 shows LC-MS analytical data of a limonoid aglycone (i.e., deacetyl nomilin).
  • FIG. 8 shows LC-MS analytical data of a limonoid aglycone (i.e., limonin).
  • FIG. 9 shows LC-MS analytical data of a limonoid aglycone (i.e., nomilin).
  • FIG. 10 shows LC-MS analytical data of a limonoid aglycone (i.e., obacunone).
  • FIG. 11 shows measured data illustrating changes in blood-glucose levels of KKAy mice at the day 28 after starting a rearing experiment.
  • FIG. 12 shows measured data illustrating changes in HbA1c levels of KKAy mice.
  • FIG. 13 shows data illustrating how a heating-temperature influences a relative quantity of obacunone as well as a ratio of obacunone content to nomilin content.
  • FIG. 14 shows data illustrating how a heating-period influences a relative quantity of obacunone as well as a ratio of obacunone content to nomilin content.
  • FIG. 15 shows HPLC analytical data of lemon seeds.
  • FIG. 16 shows HPLC analytical data of grapefruit seeds.
  • FIG. 17 is a flowchart describing a method for producing a citrus seed extract-containing composition of the present invention.
  • a citrus seed extract-containing composition of the present embodiment (hereinafter, appropriately referring to as the “composition”) includes obacunone and nomilin both derived from a citrus seed extract, in which a ratio of a obacunone content to a nomilin content is set to at least a predetermined value.
  • a citrus seed is a seed of plant belonging to genus Citrus , genus Poncirus and genus Fortunella of family Rutaceae of order Sapindales.
  • a citrus seed extract is an extract obtained via extraction from the above seeds.
  • a citrus includes Yuzu of genus Citrus of family Rutaceae (botanical name: Citrus junos ), Lemon of genus Citrus of family Rutaceae (botanical name: Citrus limon ), Grapefruit of genus Citrus of family Rutaceae (botanical name: Citrus X paradisi ).
  • compositions in various parts of a plurality of fruits belonging to a citrus it was confirmed that considerable quantities of a limonoid aglycone, which enhances functions for lowering a blood-glucose level, increasing a skeletal muscle quantity and reducing a body fat, were included in a citrus, especially, in an extract of the citrus seeds.
  • limonoid aglycone considerable quantities of the limonoid aglycone can be obtained from a small quantity of a raw material (e.g., Yuzu seed, Lemon seed, and Grapefruit seed etc.).
  • a raw material e.g., Yuzu seed, Lemon seed, and Grapefruit seed etc.
  • use of the limonoid aglycone in the citrus seed extract can suppress a quantity of a raw material thus used, leading to cost and time reduction of the extraction in the production process.
  • a novel application of a citrus seed extract-containing composition of the present embodiment includes the following ones.
  • An application to lowering of a blood-glucose level is that the composition is used for lowering a blood-glucose and an HbA1c level, more specifically, for a hypoglycemic agent, a hypoglycemic food (i.e., health food) and a hypoglycemic drug.
  • HbA1c represents a value calculated by a ratio of a quantity of hemoglobin stably bonding with glucose at the N-terminus valine in the ⁇ -chain to a total quantity of hemoglobin.
  • An application to increase in a skeletal muscle quantity is that the composition is used for increasing a quantity of a skeletal muscle, more specifically, a skeletal muscle increasing agent, a skeletal muscle increasing food (i.e., health food) and a skeletal muscle increasing drug.
  • the skeletal muscle is one class of muscles, representing a muscle for moving a skeleton.
  • the skeletal muscle includes, for example, a gastrocnemius muscle.
  • An application to reduction of a body fat is that the composition is used for reducing a quantity of a body fat, more specifically, for a body fat reducing agent, a body fat reducing food (i.e., health food) and a body fat reducing drug.
  • the body fat i.e., adipose tissue
  • the body fat includes, for example, a perirenal fat, a mesenterium fat, an epididymis fat and a subcutaneous fat or the like.
  • a composition of the present embodiment includes a limonoid aglycone and a limonoid glycoside both derived from a citrus seed extract.
  • a limonoid aglycone is a group of chemical substances derived from a citrus seed extract (i.e., substances included in citrus seed extract) and having a chemical structure of a furanolactone skeleton.
  • limonoid of the limonoid aglycone is a phytochemical largely included in a citrus plant, and a chemical substance having a chemical structure of a furanolactone.
  • an aglycone of the limonoid aglycone represents a moiety of a glycoside formed by removing glucose from the glycoside.
  • the limonoid aglycone is limonin, nomilin, deacetylnomilin or obacunone described as follows.
  • Limonin (C 26 H 30 O 8 ) is represented by the following structural formula.
  • Nomilin (C 28 H 34 O 9 ) is represented by the following structural formula.
  • Deacetylnomilin (C 26 H 32 O 8 ) is represented by the following structural formula.
  • Obacunone (C 26 H 30 O 7 ) is represented by the following structural formula.
  • a ratio of the obacunone content to the nomilin content is set to at least 0.020.
  • obacunone may exert the functions for lowering a blood-glucose level, increasing a skeletal muscle quantity and reducing a body fat quantity, even with the extremely small amount thereof.
  • the content ratio of obacunone in the composition of the present embodiment is at least 0.020, such obacunone may sufficiently exert the respective functions described above.
  • the content ratio of obacunone is preferably at least 0.100, most preferably at least 0.300 in order to surely exert the respective functions described above.
  • an upper limit of the content ratio of obacunone is not particularly limited. However, the content ratio is 5.000 or less according to the method for producing the composition of the present embodiment described hereinafter.
  • obacunone may be included at preferably 0.001-10 mass %, more preferably 0.005-5 mass % in a solid substance of the citrus seed extract-containing composition.
  • a content ratio of obacunone and an obacunone content of the present embodiment may be controlled by a treatment in the heating step described hereinafter.
  • a limonoid glycoside is a group of chemical substances derived from a citrus seed extract (i.e., substances included in a citrus seed extract).
  • glucose is bonded to the above described limonoid aglycone (i.e., via glycosidic bond).
  • composition of the present embodiment may include a saccharide as an extraction residue besides the above described limonoid aglycone and limonoid glycoside.
  • composition of the present embodiment may be prepared in a powder form, a granule from, a fine granule form, a paste form, a gel form, a solid form, a capsule form or a liquid form.
  • composition of the present embodiment may be simply a composition containing a citrus extract.
  • composition of the present embodiment may be composed of a citrus extract alone.
  • a dosage of the composition of the present embodiment may be appropriately set depending on a target application, an age and weight of target.
  • a typical dosage of obacunone as a dried material may be in the range of 0.1-2,000 mg/kg, preferably 2-300 mg/kg per day per adult human.
  • obacunone may be administered in a single dosage or in dosage divided several times.
  • a food of the present embodiment includes the above described composition and is usable for a health food for lowering a blood-glucose level, increasing a skeleton muscle quantity, and reducing a body fat.
  • a food of the present embodiment includes, for example, confectionary, bread, milk, various kinds of beverage, udon (i.e., Japanese wheat noodle), soba (i.e., buckwheat noodle), pasta, cooked rice, seasoning, spice, daily dish, fat-containing food, liquor, soft drink or the like.
  • udon i.e., Japanese wheat noodle
  • soba i.e., buckwheat noodle
  • pasta cooked rice, seasoning, spice, daily dish, fat-containing food, liquor, soft drink or the like.
  • a food of the present embodiment is produced by blending various compositions known to a skilled person in the art depending on a type of the food described above.
  • a drug of the present embodiment includes the above described composition as an active ingredient, and is usable for lowering a blood-glucose level, increasing a skeleton muscle quantity and reducing a body fat.
  • the above described composition may be preferably formulated in combination with a pharmaceutical carrier such as a suitable excipient following a conventional method in order to improve drug controllability, or absorbability when the drug is administered to a living body.
  • a pharmaceutical carrier such as a suitable excipient following a conventional method in order to improve drug controllability, or absorbability when the drug is administered to a living body.
  • an oral administration drug is produced in a capsule form, a tablet form, a granule form, a fine granule form, a syrup form, a dry syrup form or the like.
  • a parenteral administration drug includes a percutaneous absorption agent such as an ointment, a percutaneous tape, an injection agent, a suppository, a pessary, an aerosol agent or the like.
  • a method for producing a citrus seed extract-containing composition includes a heating step S 1 and an extraction step S 2 . Further, the method for producing the composition of the present embodiment may include a grinding step as described hereinafter.
  • a heating step S 1 is the step of heating citrus seeds.
  • a heating temperature in the heating step S 1 is at 60° C. or more, preferably 100° C. or more.
  • obacunone may be generated in the citrus sees, thereby increasing the obacunone content.
  • the composition containing an extract thus extracted from the citrus seeds may sufficiently exert the functions for lowering a blood-glucose level, increasing a skeletal muscle quantity and reducing a body fat.
  • An upper limit of the heating temperature in the heating step S 1 is not specifically limited. However, when the temperature reaches more than 150° C., a quantity of a limonoid aglycone having obacunone is decreased, causing a disadvantage.
  • the heating temperature is preferably set at 150° C. or less, most preferably 140° C. or less.
  • the heating step S 1 is not specifically limited as long as S 1 is the step of heating citrus seeds.
  • S 1 may be also the step of drying citrus seeds via heat, namely, the drying step.
  • S 1 may be the step of heat-drying citrus seeds without performing humidification.
  • the heating step S 1 when the citrus seeds become dry by heating the citrus seeds at a predetermined temperature, a condition (i.e., state) of the citrus seeds thus heated may be kept substantially constant.
  • the extraction condition in the extraction step S 2 described hereinafter may be easily set, and simultaneously quality of the composition finally obtained may be kept stable.
  • the heating temperature described above is specifically a temperature measured in the heating environment (i.e., in the environment atmosphere) of the heating step S 1 .
  • the temperature of the targeted citrus seeds is almost the same as in the heating environment.
  • a heating-period in the heating step S 1 is set to at least 5 min, preferably at least 10 min, more preferably at least 30 min, the effect of increasing the obacunone content inside the citrus seeds may be sufficiently exerted.
  • the heating-period may be set within 1440 min, preferably within 720 min, most preferably within 360 min.
  • a treatment method in the heating step S 1 may be performed by known methods such as a method for placing the citrus seeds in the environment at the predetermined temperature or more thus described above (e.g., in dry furnace), and a method for blowing warm air onto the citrus seeds.
  • a grinding step is the step of grinding the citrus seeds.
  • a grinding method in the grinding step is not specifically limited, and may be performed by known methods using a mill or the like.
  • the grinding step is conducted after the heating step S 1 and before the extraction step S 2 as described hereinafter. However, the grinding step may be conducted before the heating step S 1 .
  • An extraction step S 2 is the step of extracting an extract from the citrus seeds.
  • An extraction treatment in the extraction step S 2 is a treatment for extracting an extract from the citrus seeds obtained after the heating step S 1 (or after grinding step) via using a polar solvent.
  • a temperature of extraction using a polar solvent in the extraction step S 2 is from about 0° C. to a boiling point of the polar solvent, typically an ambient temperature.
  • the extraction temperature is preferably set in the range from 0° C. to less than 70° C., more preferably from 5° C. to 60° C., in view of the extraction efficiency.
  • the extraction temperature is preferably set in the range from 0° C. to 100° C., more preferably from 25° C. to 100° C., in view of the extraction efficiency.
  • An extraction period in the extraction step S 2 is preferably from 1 min to 24 hr. However, the extraction may be performed for a period longer than 24 hr. Further, the extraction may be performed by any procedure of leaving at rest, stirring or the like.
  • a citrus seed extract may be obtained by adding a 1 to 50-fold volume, preferably a 5 to 20-fold volume of a polar solvent to the citrus seeds, to perform the extraction. Then, the resulting citrus seed extract may be diluted, condensed, dried and purified appropriately following a conventional method. Accordingly, a citrus seed extract-containing composition may be obtained.
  • a polar solvent used in the extraction step S 2 is not specifically limited.
  • the polar solvent may include one kind of solvent or a plurality kinds of solvents selected from, for example, acetone, ethanol, ethyl methyl ketone, glycerin, ethyl acetate, methyl acetate, diethyl ether, cyclohexane, dichloromethane, editable fat and oil, 1,1,1,2-tetrafluoroethane, 1,1,2-trichloroethene, 1-butanol, 2-butanol, 1-propanol, 2-propanol, propylene glycol, hexane, water, methanol or the like.
  • a preferably usable solvent may include one selected from the group of ethanol (from 10 volume % to 100 volume %), methanol (from 10 volume % to less than 99.5 volume %), 1-butanol (from 30 volume % to less than 99.5 volume %), hexane (from 5 volume % to less than 70 volume %) and water. More preferably, such a usable solvent is ethanol or water, most preferably ethanol.
  • an ethanol-containing solvent in the range from 10 volume % to 100 volume % may be used as a polar solvent.
  • such an ethanol-containing solvent in the range from. 50 volume % to 100 volume %, and more preferably at about 70 volume % may be used.
  • the ethanol-containing solvent at 70 volume % means a mixture of water and ethanol set at the volume ratio of 3:7.
  • a citrus seed extract-containing composition of the present embodiment is preferably an organic solvent extract from the citrus seeds as describe above, more preferably an alcohol extract from the citrus seeds, and most preferably an ethanol extract from citrus seeds.
  • a method for producing a food and a drug of the present embodiment may include the steps of mixing the citrus seed extract-containing composition thus prepared as above with materials of the food and the drug, and subsequently producing the food and the drug following a conventional method for producing a food and a drug known by a skilled person in the art.
  • KKAy mice at 3 weeks of age were purchased from CLEA Japan, Inc. as diabetes onset mode mice, and experimentally reared after preliminary rearing for 7 days.
  • the conditions of rearing the mice were as follows. Temperature: 23+1° C.; Humidity: 55+10%; Lighting: during 7 am to 7 pm (lights out for other period); Free intake of water and feed.
  • AIN-93G was used for base experimental feed. 1 mass % or 3 mass % of the Yuzu seed extract, or 0.03 mass % of pioglitazone was mixed to AIN-93G, and the mixture was administered to the mice.
  • the Yuzu seed extract used in Experiment 1 was prepared by heat-drying the Yuzu seeds at 100° C. for 720 min, subsequently grinding the dried Yuzu seeds so that the particle size (i.e., diameter) became about 1 mm or less, and performing extraction at an ambient temperature for 10 min using 10-fold volume of 100% ethanol to the ground Yuzu seeds.
  • mice were preliminarily reared for 7 days, and subsequently reared with the experimental feed totally for 28 days.
  • blood was collected through a tail vain at the time from 9 am to 11 am at a day after the predetermined days passed. Then, the collected blood was analyzed by blood-glucose analyzers (HORIBA, Ltd. Antsence III and Bayer DCA2000) to measure a blood-glucose level and an HbA1c level. The measured blood-glucose and HbA1c levels were processed to calculate the mean value of each group.
  • HORIBA, Ltd. Antsence III and Bayer DCA2000 blood-glucose analyzers
  • FIG. 1 shows changes in blood-glucose levels of the KKAy mice administered with experimental feed.
  • FIG. 2 shows changes in HbA1c levels of the KKAy mice administered with the experimental feed.
  • the ordinate of FIG. 1 represents blood-glucose levels (mg/dl) and the ordinate of FIG. 2 represents HbA1c levels, while the abscissas of FIGS. 1 and 2 represent the number of days (day) having passed after the rearing experiment was initiated.
  • P ⁇ 0.01 vs control means that the p value is smaller than 0.01 against the control group.
  • the data of the Yuzu seed extract (3% mass) added group demonstrated that the blood-glucose levels at the days 14 and 27 were lower than the levels at the day 0, and further the effect of lowering a blood-glucose level of the Yuzu seed extract (3% mass) added group was equal to or greater than the effect of the pioglitazone administered group.
  • the citrus seed (i.e., Yuzu seed) extract-containing composition of the present invention exerted not only the effect of lowering a blood-glucose level under an ordinary condition but also the effect of lowering a blood-glucose level even under a chronic hyperglycemia condition.
  • mice used in each group were the same as in Experiment 1 (but 8 or 9 mice used in each group)
  • the limonoid aglycone and the limonoid glycoside were obtained by fractionation of the Yuzu seed extract thus prepared in the same method as in Experiment 1.
  • FIG. 3 shows changes in blood-glucose levels of the KKAy mice administered with experimental feed
  • FIG. 4 shows changes in HbA1c levels of the KKAy mice administered with the experimental feed.
  • the ordinate of FIG. 3 represents blood-glucose levels (mg/dl)
  • the ordinate of FIG. 4 represents HbA1c levels
  • the abscissas of FIGS. 3 and 4 represent the number of days (day) having passed after the rearing experiment was initiated.
  • the blood-glucose levels were greatly lowered compared to those in the control group after the day 7. Further, in the limonoid aglycone added group, the blood-glucose levels were almost equal to those in the Yuzu seed extract added group (i.e., total in FIG. 3 ).
  • the citrus seed i.e., Yuzu seed
  • the limonoid aglycone exerted the effect of lowering a blood-glucose level in animal blood, and simultaneously the effect of lowering a blood-glucose level under a chronic hyperglycemia condition.
  • mice In each of a control group, a limonoid glycoside added group, a limonoid aglycone added group and a Yuzu seed extract added group, 8 or 9 mice were preliminarily reared for 7 days, and reared totally for 28 days with experimental feed.
  • a weight ratio of a “gastrocnemius muscle” to a mouse body of the respective mice at the day 28 after initiation of the rearing experiment i.e., gastrocnemius muscle weight/mouse body weight.
  • the weights of the above described muscle and fats were measured by removing the respective tissues, washing the removed tissues with saline, and excluding water therefrom with sanitary cotton.
  • FIG. 5 shows a weight ratio of a gastrocnemius muscle to a mouse body of the mice at the day 28 after initiation of the rearing experiment.
  • FIGS. 6A, 6B, 6C and 6C show respective weight ratios of a perirenal fat, a mesenterium fat, an epididymis fat and a subcutaneous fat to a mouse body of the mice at the day 28 after initiation of the rearing experiment.
  • the limonoid aglycone thus obtained in Experiment 3 was fractionated by a silica-gel column (daisogel 1002A IR60-40/63A), and four types of limonoid aglycones were obtained. Those samples were subjected to LC-MS analysis following a conventional method. The resulting data are shown in FIGS. 7-10 .
  • the limonoid aglycone of the citrus seed (i.e., Yuzu seed) extract-containing composition of the present invention included deacetyl nomilin, limonin, nomilin and obacunone.
  • mice were used, and water and feed were not fed in free feeding procedure but fed in pair feeding one.
  • AIN-93G containing obacunone (0.035 mass %) was used for the experimental feed.
  • obacunone used in Experiment 5 was prepared by heat-drying Yuzu seeds at 100° C. for 720 min, and subsequently grinding the dried seeds so that the particle size (diameter) became almost 1 mm or less, performing extraction at an ambient temperature for 10 min with 10-fold volume of 100% ethanol to the Yuzu seeds, and fractionating the resulting extract by silica gel column (daisogel 1002A IR60-40/63A).
  • FIG. 11 shows blood-glucose levels of the KKAy mice administered with the experimental feed (i.e., at the day 28 after initiation of the rearing experiment).
  • FIG. 12 shows changes in HbA1c levels of the KKAy mice administered with the experimental feed.
  • the ordinate of FIG. 11 represents blood-glucose levels (mg/dl) and the ordinate of FIG. 12 represents HbA1c levels, while the abscissas of FIGS. 11 and 12 represent the number of days (day) having passed after the rearing experiment was initiated.
  • the blood-glucose level of the obacunone added group was about 50 mg/dl lower than that of the control group. Further, as shown in the data of FIG. 12 , the HbA1c levels of the obacunone added group was lower than those of the control group.
  • the citrus seed (i.e., Yuzu seed) extract-containing composition including obacunone surely exerted the effects of lowering the blood-glucose and HbA1c levels.
  • Yuzu seeds were used as citrus seeds, and subjected to dry-heating. Then, the dried Yuzu seeds were ground so that the particle size (i.e., diameter) became substantially 1 mm or less. Extraction from the ground Yuzu seeds was conducted at an ambient temperature for 10 min using 10-fold volume of 100% ethanol, thereby providing the respective samples.
  • the term of “dry-heating” in Experiment 6 means a process that the Yuzu seeds were placed in a dry furnace for a predetermined period, at a predetermined temperature.
  • a nomilin standard and an obacunone standard (ChromaDex, Inc.) were subjected to HPLC analysis following a conventional method, and a nomilin quantity and an obacunone quantity thereof were determined. Based on the measurement values, a “relative quantity of obacunone” and a ratio of “obacunone content/nomilin content” were calculated.
  • a relative quantity of obacunone means a relative value.
  • a relative quantity of obacunone at 100° C. in the dry-heating is defined to 1.0000.
  • a relative quantity of obacunone at 30 min in the dry-heating is defined to 1.0000.
  • an “obacunone content/nomilin content” means a ratio calculated based on a “nomilin content” in a sample calculated by comparing the peak of the nomilin standard to the nomilin peak shown by the sample, and the “obacunone content” in the sample calculated by comparing the peak of the obacunone standard to the obacunone peak shown by the sample.
  • FIG. 13 and Table.1 show a “relative quantity of obacunone” and an “obacunone content/nomilin content” measured when a dry-heating period is fixed to 24 hr and a dry-heating temperature is varied.
  • FIG. 14 and Table.2 show a “relative quantity of obacunone” and an “obacunone content/nomilin content” measured when a dry-heating temperature is fixed to 150° C. and a dry-heating period is varied.
  • obacunone represents a “relative quantity of obacunone”
  • obacunone/nomilin represents a ratio of “obacunone content/nomilin content”.
  • the method for producing a citrus seed (i.e., Yuzu seed) extraction-containing composition of the present invention produced the composition in which a ratio of “obacunone content/nomilin content” became at least 0.020.
  • the method of the present invention definitely produced the composition surely exerting the effects of lowering a blood-glucose level, increasing a skeletal muscle quantity and reducing a body fat, by including the heating step of heating the citrus seeds at 60° C. or more.
  • “Lemon seeds” and “Grapefruit Seeds” were used as citrus seeds, and the respective seeds were subjected to freeze-drying or dry-heating (i.e., 100° C., 24 hr). After that, the respective seeds were ground so that the particle size (i.e., diameter) became substantially 1 mm or less. Then, extraction was conducted at an ambient temperature for 10 min by using a 10-fold volume of 100% ethanol to the ground Yuzu seeds, thereby to prepare the respective samples.
  • the “freeze-drying” in Experiment 7 was conducted by a freeze-dryer RLE-103 (KYOWA VACUUM ENGINEERING CO., LTD.), and drying the frozen samples at ⁇ 30° C. for 96 hr. Further, the “dry-heating” in Experiment 7 was conducted by placing the respective seeds in a dry furnace for 24 hr at the atmospheric temperature of 100° C.
  • FIGS. 15 and 16 show the data obtained by the HPLC analysis conducted under the same conditions as in Experiment 6 on the samples prepared by the above described method and an obacunone standard (ChromaDex, Inc.)
  • the data in FIG. 15 show the absorption spectra of a freeze-dried lemon seed sample, a dry-heated lemon seed sample and an obacunone standard, respectively.
  • the data in FIG. 16 show the absorption spectra of a freeze-dried grapefruit seed sample, a dry-heated grapefruit seed sample and an obacunone standard, respectively.
  • a peak of obacunone was not substantially observed at the retention time shown by the obacunone standard, in both spectra of the freeze-dried seed samples of FIGS. 15 and 16 . This result indicated that little obacunone was included in those samples. On the contrary, a large peak of obacunone was observed at the retention time shown by the obacunone standard, in both spectra of the dry-heated seed samples of FIGS. 15 and 16 . This result clearly indicated that obacunone was included in those samples.
  • the method for producing a citrus seed extract-containing composition of the present invention definitely produced a composition containing obacunone via including the heating step of heating the citrus seeds in the method, thereby to surely exert the effects of lowering a blood-glucose level, increasing a skeletal muscle quantity and reducing a body fat.

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Cited By (4)

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US20210138019A1 (en) * 2018-04-10 2021-05-13 Stardust Hd, Inc. Composition comprising yuzu seed extract and brown rice extract
US20220296615A1 (en) * 2019-08-12 2022-09-22 Zhejiang Yangshengtang Institute Of Natural Medication Co., Ltd. Combination product containing limonoid compound and sulfonylurea compound
CN115252630A (zh) * 2022-09-02 2022-11-01 深圳技术大学 黄柏酮在制备预防、改善或治疗非酒精性脂肪肝的药物中的应用
US11679097B2 (en) 2017-08-08 2023-06-20 Zhejiang Yangshengtang Institute Of Natural Medication Co., Ltd. Combination product containing limonoid compound and biguanide compound

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CN110314233B (zh) * 2019-08-12 2021-07-23 浙江养生堂天然药物研究所有限公司 包含柠檬苦素类化合物和dpp-4抑制剂的组合产品
CN110279866B (zh) * 2019-08-12 2021-06-29 浙江养生堂天然药物研究所有限公司 包含柠檬苦素类化合物和噻唑烷二酮类药物的组合产品
CN110327355B (zh) * 2019-08-12 2021-06-04 浙江养生堂天然药物研究所有限公司 包含柠檬苦素类化合物和sglt-2抑制剂的组合产品
CN110327356B (zh) * 2019-08-12 2021-06-04 浙江养生堂天然药物研究所有限公司 包含柠檬苦素类化合物和α-糖苷酶抑制剂的组合产品
KR102411718B1 (ko) * 2022-04-12 2022-06-22 주식회사 서울화장품 유자씨 추출물의 제조방법 및 이를 함유하는 화장료 조성물

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JP2001158728A (ja) 1999-12-01 2001-06-12 Shiseido Co Ltd ヒアルロン酸産生促進剤および皮膚外用剤
JP2004010480A (ja) 2002-06-03 2004-01-15 Shiseido Co Ltd 角層剥離剤
US20060116509A1 (en) * 2004-11-29 2006-06-01 The United States Of America, As Represented By The Secretary Of Agriculture Manufacture of limonoid compounds
WO2007118143A2 (fr) 2006-04-06 2007-10-18 The Texas A & M University System Procédé d'isolation de glucosides limonoïdes provenant d'agrumes
JP2007297343A (ja) * 2006-05-01 2007-11-15 Oriza Yuka Kk 脂質代謝改善剤
JP2008156294A (ja) * 2006-12-25 2008-07-10 Lion Corp 筋芽細胞活性化剤

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Publication number Priority date Publication date Assignee Title
US11679097B2 (en) 2017-08-08 2023-06-20 Zhejiang Yangshengtang Institute Of Natural Medication Co., Ltd. Combination product containing limonoid compound and biguanide compound
US20210138019A1 (en) * 2018-04-10 2021-05-13 Stardust Hd, Inc. Composition comprising yuzu seed extract and brown rice extract
US11850272B2 (en) * 2018-04-10 2023-12-26 Stardust Hd, Inc. Composition comprising yuzu seed extract and brown rice extract
US20220296615A1 (en) * 2019-08-12 2022-09-22 Zhejiang Yangshengtang Institute Of Natural Medication Co., Ltd. Combination product containing limonoid compound and sulfonylurea compound
CN115252630A (zh) * 2022-09-02 2022-11-01 深圳技术大学 黄柏酮在制备预防、改善或治疗非酒精性脂肪肝的药物中的应用

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