JP2012135244A - Citrus extract, and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a further technique relating to the use of parts of citrus other than fruit, squeezed juice residue, and the like.SOLUTION: A citrus extract is obtained by a process including the following processes (a) and (b): (a) a process for extracting citrus raw material by lower alcohol; and (b) a process for processing the lower alcohol extract, obtained by the process (a), by an aromatic synthetic adsorbent resin. A method for producing the citrus extract is also provided.

Description

  The present invention relates to a citrus extract obtained from a citrus raw material and a method for producing the citrus extract.

  Citrus citrus fruits such as orange, grapefruit, citrus orange, jujube, lemon, yuzu, lime, buntan, shikwasa, and tankan are used as beverages using fruit juice and other foods.

  However, citrus fruits have not been effectively used for parts other than fruits or juice residue obtained after squeezing fruit juice, and most of them have been discarded.

  Although some reports have been made on the use of parts other than citrus fruits and juice residues (Patent Documents 1 to 8), it was necessary to develop further techniques.

Japanese Patent Laid-Open No. 10-276578 JP 2001-46019 A JP 2002-153231 A JP 2004-18737 A JP 2005-229836 A JP 2008-214223 A JP 2009-17822 A JP 2009-67755 A

  This invention made it the subject to provide the further technique regarding utilization of site | parts other than a citrus fruit, squeezed residue, etc.

  As a result of diligent research to solve the above-mentioned problems, the present inventors obtained an extract having high collagenase inhibitory activity and elastase inhibitory activity by extracting a citrus raw material with a lower alcohol and then treating with a special synthetic adsorption resin. And found that this can be used for food and drink, cosmetics, etc., and completed the present invention.

That is, the present invention includes the following steps (a) and (b)
(A) Step of extracting citrus raw material with lower alcohol (b) Citrus extract obtained by a step including a step of treating the lower alcohol extract obtained in step (a) with an aromatic synthetic adsorption resin.

The present invention also includes the following steps (a) and (b)
(A) a step of extracting a citrus raw material with a lower alcohol (b) a method of producing a citrus extract comprising a step of treating the lower alcohol extract obtained in step (a) with an aromatic synthetic adsorption resin It is.

  The citrus extract of the present invention is excellent in fragrance derived from citrus, and is excellent in containing many functional substances such as various citrus-derived polyphenols, sphingolipids, and plant steroids.

FIG. 1 is a flowchart for obtaining a ceramide glycoside and a steroid glycoside from a citrus extract.

The citrus extract of the present invention (hereinafter referred to as “the present invention extract”) is prepared by the following steps (a) and (b)
(A) Extracting citrus raw material with lower alcohol (b) Obtained by a process including a process of treating the lower alcohol extract obtained in process (a) with an aromatic synthetic adsorption resin.

  Citrus raw materials used as the raw material of the extract of the present invention are orange (Citrus sinensis (L.) Osbeck), grapefruit (Citrus Xparadisi), citrus unshiu Marc., Jujube (Citrusnatsudaidai Hayata), lemon (Citrus limon (L. ) Burm.f.), Yuzu (Citrus junos Siebold ex. Taknaka), Lime (Citrus aurantifolia), Buntan (Citrus maxima), Sikhwasa (Citrus depressa Hayata), Tankan (Citrus tankan Hayata), Kumquat (Fortunella), Karatachi ( Poncirus trifoliata) is one or more selected from fruits, pericarps, seeds, sandbags and squeezed residues of plants selected from Citrus citrus (citrus). Among these citrus raw materials, squeezer juice residue is preferred. In addition, in this invention, a squeezed residue is a residue obtained after squeezing a fruit with a well-known squeezing machine etc., and contains a fruit skin, a seed, and a sandbag, but hardly contains fruit juice. Moreover, these citrus raw materials may be dried or pulverized by known means.

  The extraction of the citrus raw material with a lower alcohol is not particularly limited. For example, the citrus raw material is immersed in 1 to 99% by mass (hereinafter simply referred to as “%”), preferably 20 to 25% in the lower alcohol. Extraction may be performed at -100 ° C, preferably 20-30 ° C for 1 minute to 1 week, preferably 3-24 hours. Examples of the lower alcohol used for this extraction include methanol, ethanol, isopropyl alcohol, ethylene glycol, glycerin, 1,3-butylene glycol and the like. Of these lower alcohols, ethanol is preferred.

  The lower alcohol extract obtained by the extraction is preferably separated into an extract and an extraction residue by a known means such as centrifugation before the treatment with the aromatic synthetic adsorption resin described later. The extract may be concentrated by a known means.

  The extract is then treated with an aromatic synthetic adsorption resin. Examples of the aromatic adsorption resin used for this treatment include styrene-benzene resin. Examples of such commercially available resins include Diaion (registered trademark) HP-20, HP-21 (manufactured by Mitsubishi Chemical Corporation), and the like.

  The method of treating the extract with the aromatic synthetic adsorption resin is not particularly limited, but for example, the extract is brought into contact with the aromatic synthetic adsorption resin to adsorb the extract of the present invention to the resin, and then, Examples include a method of eluting the extract of the present invention adsorbed on a resin. Specifically, in order to adsorb the extract of the present invention in the extract onto the aromatic synthetic adsorption resin, it is 1 to 99%, preferably 10 to 90%, more preferably on a column packed with the aromatic synthetic adsorption resin. What is necessary is just to let an extract pass, after fully passing 20-60% ethanol aqueous solution. In order to elute the extract of the present invention adsorbed on the resin, a 2 to 100%, preferably 70 to 100% aqueous ethanol solution having a higher ethanol concentration than the aqueous ethanol solution used at the time of adsorption is passed through the column. Just do it.

  Further, after the above treatment, the extract of the present invention may be purified by a known purification means such as liquid-liquid distribution or column chromatography.

  The extract of the present invention obtained by the steps including the steps (a) and (b) is excellent in citrus-derived fragrance, and contains a large amount of functional substances such as various citrus-derived polyphenols, sphingolipids, and plant steroids. Therefore, the extract of the present invention can be used for cosmetics such as lotion, cosmetic cream and soap, foods and beverages such as beverages, candy and ice confectionery, fragrances and the like in which conventional plant-derived extracts are used.

The extract of the present invention has a collagenase inhibitory activity (IC ₅₀ ) of 1.0 to 120 μg / ml, preferably 1.0 to 12.0 μg / ml, and an elastase inhibitory activity (IC ₅₀ ) of 0.5 to ₅₀ μg. / Ml, preferably 0.5 to 5.0 μg / ml. Therefore, the extract of the present invention is particularly preferably used for cosmetics. In the present specification, collagenase inhibitory activity and elastase inhibitory activity are determined according to literature methods (Pharmaceutical Journal Vol. 118, pp 423-429, (1998) and experimental methods for developing functional cosmetic materials-in vitro / cell / tissue culture). It is a value measured by the method as described in Sakagawa Genso, CMC Publishing, pp 60-64, (2007)) and the examples described later.

The extract of the present invention includes a novel ceramide glycoside represented by the following formula (I) or (II). These ceramide glycosides can be obtained from the extract of the present invention by the method shown in FIG.

The physical properties of these ceramide glycosides are as follows.
・ Waxy white powder ・ Insoluble in water, ether and petroleum ether, soluble in pyridine, chloroform and hot ethanol ・ Relatively stable and can be stored for a long time ・ Celebron fraction has a melting point of 212 to 215 ° C., specific rotation [α ] ²² _D + 3.98 °
-Keracin fraction has a melting point of 185 to 187 ° C, specific rotation [α] ²² _D -3.82 °
・ Nelbon fraction has a melting point of 180 ° C. and specific rotation [α] ²² _D −3.70 °
・ Oxinerbon fraction has a melting point of 205 to 210 ° C., specific rotation [α] ²² _D + 3.80 °
・ Molecular weight 713 (mass spectrometry)

  These ceramide glycosides can be used in cosmetics such as lotions, cosmetic creams, and soaps, foods and drinks such as drinks, jellies, and supplements, pharmaceuticals such as paints and ointments, quasi drugs, and the like.

Further, the extract of the present invention includes a novel steroid glycoside represented by the following formula (III). This steroid glycoside can be obtained from the extract of the present invention by the method shown in FIG.

The physical properties of this steroid glycoside are as follows.
・ White powder ・ Easily soluble in chloroform and pyridine, soluble in methanol and ethanol, hardly soluble in water ・ Melting point: 293-294 ° C., specific rotation [α] ²⁵ _D -40.0 ° (c 2.6, pyridine)
・ Molecular weight 576 (mass spectrometry)

  This steroid glycoside can be used in cosmetics such as soap and shampoo, ointments, energy drinks, syrups, gargles and other pharmaceutical products, quasi drugs and the like.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples at all.

Example 1
Production of citrus extract:
15 kg of squeezer squeezed residue was put on a freeze dryer to obtain 3 kg of freeze-dried product. After 3 kg of this lyophilized product was pulverized, 12 l of 95% ethanol was added and extracted overnight (12 hours) at room temperature (20 ° C.). The extracted lyophilized product and ethanol mixture was separated into an extract and an extraction residue using a centrifuge, and 9 L of extract was obtained.

  500 mL of an aromatic synthetic adsorption resin (Diaion (registered trademark) HP-20: manufactured by Mitsubishi Chemical) previously immersed in 95% ethanol was packed in a 6 cm diameter column, and 3 L of 57% ethanol was permeated. After 6 L of distilled water was added to 9 L of the extract obtained above and stirred, the extract was adsorbed on an aromatic synthetic adsorbent (Diaion HP-20: manufactured by Mitsubishi Chemical) through the column prepared above. . Thereafter, 2000 mL of 57% ethanol was permeated to wash impurities in the column. Subsequently, the extract was eluted by passing 2.2 L of 83% ethanol through the column to obtain 2.2 L of an eluent. After adding 1.0 L of 1,3-butylene glycol to 2.2 L of the eluent and mixing, ethanol was removed by a rotary evaporator and concentrated under reduced pressure to obtain 1.0 kg of citrus extract.

Example 2
Determination of collagenase inhibitory activity of citrus extract:
(1) Preparation of Collagenase Inhibiting Activity Reagent Trisaminomethane (manufactured by Wako Pure Chemical Industries) and hydrochloric acid were adjusted to 0.1M, and 0.1M Tris-HCl buffer (containing 20 mM potassium chloride (manufactured by Wako Pure Chemical Industries)) ( pH 7.1) was prepared. 7.8 mg of Pz-peptide (Pz-Pro-Leu-Gly-Pro-D-Arg-OH: manufactured by Fukonashi) was weighed and dissolved in 20 mL of 0.1 M Tris-HCl buffer solution as a substrate solution. Further, a solution obtained by dissolving 5 mg of collagenase type IV (from Clostridium hystolyticum: Sigma) in 1 ml of distilled water was used as an enzyme solution. This was dispensed in 100 μl aliquots, stored at −20 ° C., and diluted 50 times with distilled water at the time of use. Furthermore, what adjusted citric acid (made by Wako Pure Chemical Industries) to 25 mM was used as the reaction stop liquid.

(2) Method for measuring collagenase inhibitory activity 5 mL of the citrus extract produced in Example 1 was concentrated until the solid content was 55.1 mg. This was dissolved in 2.5 mL of 50% dimethyl sulfoxide, and further diluted 400 times with distilled water to prepare a sample solution having a solid content of 55 μg / mL. 25 μL of the sample solution, 25 μL of the enzyme solution, and 200 μL of the substrate solution were mixed to adjust the solid content to 5.5 μg / mL, and then incubated at 37 ° C. for 30 minutes. After the incubation, the reaction was stopped with 0.5 ml of a reaction stop solution, and further 2.5 ml of ethyl acetate was added for extraction. Next, this was centrifuged at 3000 rpm for 10 minutes, and the absorbance at a wavelength of 320 nm of the ethyl acetate layer was measured using ethyl acetate as a control. The sample solution blank contains a sample solution containing distilled water and a substrate solution, the control solution contains distilled water and an enzyme solution and a substrate solution, and the control blank contains a substrate solution added to the distilled water. After the enzyme reaction, the absorbance of the ethyl acetate layer at a wavelength of 320 nm was measured.

The collagenase inhibition rate was calculated from the absorbance value according to the following formula.

The inhibition rate of collagenase was 51.6% with respect to the solid content of the citrus extract of 5.5 μg / mL. The results of converting the inhibition rate of this collagenase into IC ₅₀ and the values of other known collagenase inhibitory activities are shown in Table 1.

Example 3
Measurement of elastase inhibitory activity of citrus extract:
(1) Preparation of elastase inhibitory activity reagent Trisaminomethane (manufactured by Wako Pure Chemical Industries, Ltd.) and hydrochloric acid were adjusted to 0.2M to prepare 0.2M Tris-HCl buffer (pH 8.0). N-succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide weighed 4.52 mg, dissolved in 200 μL of dimethyl sulfoxide, and diluted 10-fold with 0.2 M Tris-HCl buffer at the time of use. A substrate solution was obtained. Further, human elastase (manufactured by Fukonashi) was diluted with distilled water to make 10 μg / mL as an enzyme solution.

(2) Method for Measuring Elastase Inhibitory Activity 5 mL of the citrus extract produced in Example 1 was concentrated until the solid content was 55.1 mg. This was dissolved in 2.5 mL of 50% dimethyl sulfoxide, and further diluted 4000 times with distilled water to prepare a sample solution having a solid content of 5.5 μg / mL. A sample solution (50 μL), an enzyme solution (100 μL), and a substrate solution (50 μL) were mixed on a microplate, adjusted to a solid content of 1.4 μg / mL, and then incubated at 37 ° C. for 1 hour. Immediately after the incubation, the absorbance (wavelength 405 nm) of p-nitroanilide, which is a degradation product of the sample solution, was measured using a microplate reader. The sample solution blank contains a sample solution containing distilled water and a substrate solution, the control solution contains distilled water and an enzyme solution and a substrate solution, and the control blank contains a substrate solution added to the distilled water. Was used to measure the absorbance (wavelength 405 nm) of p-nitroanilide after the enzyme reaction.

The elastase inhibition rate was calculated from the absorbance value according to the following formula.

The inhibition rate of elastase was 41.0% with respect to the solid content of the citrus extract of 1.4 μg / mL. Table 2 shows the results obtained by converting the inhibition rate of this elastase into IC ₅₀ and the values of other known elastase inhibitory activities.

Example 4
Lotion:
A lotion having the following formulation was produced by the following production method.
(Ingredient name) (Blending amount)
(1) Citrus extract 0.5%
(2) Glycerin 5%
(3) 1,3-butylene glycol 4%
(4) PEG-11 methyl ether dimethicone 1%
(5) EDTA-2Na Trace (6) Glycyrrhizic acid 2K Trace (7) Phenoxyethanol 1%
(8) Methylparaben 1%
(9) Purified water remaining

<Manufacturing method>
A: (2), (3) and (5) were dissolved in (9).
B: A mixture of (1), (4), (6) to (8) was added to A and solubilized to produce a lotion.

Example 5
Cosmetic cream (1):
A cosmetic cream having the following formulation was produced by the following production method.
(Ingredient name) (Blending amount)
(1) Tri (capryl / capric acid) glyceryl 7%
(2) Jojoba oil 5%
(3) Citrus extract 3%
(4) Dipropylene glycol 2%
(5) 2.25% behenyl alcohol
(6) Polyvinyl alcohol 3%
(7) Decaglyceryl pentastearate 0.95%
(8) 0.80% bead wax
(9) Sucrose stearate 1%
(10) Sodium stearoyl lactate 0.80%
(11) Laureth-7 0.15%
(12) Phenoxyethanol 1%
(13) Methylparaben 1%
(14) Purified water remaining

<Manufacturing method>
A: (3) and (4) were added to (14) and heated to 70 ° C.
B: (1), (2), (5) to (13) were added and heated at 70 ° C.
C: A and B were stirred with a homomixer until the emulsified particles were uniform, deaerated, filtered and cooled to obtain a cosmetic cream.

Example 6
Cosmetic cream (2):
A cosmetic cream having the following formulation was produced by the following production method.
(Ingredient name) (Blending amount)
(1) Tri (capryl / capric acid) glyceryl 12%
(2) Jojoba oil 4%
(3) Dipropylene glycol 4%
(4) Citrus extract 3%
(5) PEG-2 hydrogenated castor oil 3%
(6) Cetanol 1.5%
(7) Polyvinyl alcohol 2%
(8) Decaglyceryl pentastearate 1.33%
(9) Bead wax 1.20%
(10) 1% sucrose stearate
(11) Sodium stearoyl lactate 0.42%
(12) Quinsed gum 0.3%
(13) Arginine 0.20%
(14) Phenoxyethanol 1%
(15) Methylparaben 1%
(16) Remaining amount of purified water

<Manufacturing method>
A: (3) and (4) were added to (16) and heated to 70 ° C.
B: (1), (2), (5) to (15) were added and heated to 70 ° C.
C: A and B were stirred with a homomixer until the emulsified particles were uniform, deaerated, filtered and cooled to obtain a cosmetic cream.

Example 7
Soap (1):
Soap having the following formulation was manufactured by the following manufacturing method.
(Ingredient name) (Blending amount)
(1) Palm oil 22.0%
(2) Palm kernel oil 10.0%
(3) Castor oil 4.0%
(4) 4.0% olive oil
(5) Caustic soda 6.0%
(6) Ethyl alcohol 20.0%
(7) Purified water 20.0%
(8) Sugar 9.0%
(9) Glycerin 4.5%
(10) Citrus extract 0.5%

<Manufacturing method>
A: Saponification was performed while heating (1) to (7) on a hot water bath.
B: (8) to (10) were added to A, mixed, filtered through a sieve and put into a mold.
C: B was solidified in a refrigerator and then dried and aged for 40 days to obtain soap.

Example 8
Soap (2):
Soap having the following formulation was manufactured by the following manufacturing method.
(Ingredient name) (Blending amount)
(1) Palm oil 22.0%
(2) Palm kernel oil 10.0%
(3) Castor oil 7.0%
(4) 22.0% olive oil
(5) Caustic soda 6.0%
(6) Caustic potash 3.5%
(7) Jojoba seed oil 3.0%
(8) Hydrolyzed collagen 2.0%
(9) Titanium dioxide 1.0%
(10) Citrus extract 0.5%
(11) Purified water 23.0%

<Manufacturing method>
A: Saponification was performed while heating (1) to (6) and (11) on a hot water bath.
B: (7) to (10) were added to A, mixed, filtered through a sieve and put into a mold.
C: B was solidified in a refrigerator and then dried and aged for 40 days to obtain soap.

Example 9
Purification of ceramide glycosides and steroid glycosides:
Using the citrus extract obtained in Example 1, ceramide glycosides (SQC1 and SQC2) and steroid glycoside (SQSG1) were purified according to the method described in FIG. As a result of analyzing the structure of steroid glycoside and ceramide glycoside by mass spectrometry and proton nuclear magnetic resonance spectroscopy, it was found to have the following structural formula.

セラミド配糖体（ＳＱＣ２）

Ceramide glycoside (SQC1)

Ceramide glycoside (SQC2)

Steroid glycoside (SQSG1)

The citrus extract of the present invention is excellent in citrus-derived fragrance and contains many functional substances derived from various citrus fruits. Therefore, the citrus extract of the present invention can be used for cosmetics such as skin lotion, cosmetic cream, soap, and the like, beverages, candy and other foods such as ice confectionery, and fragrances.

Claims (9)

Next steps (a) and (b)
(A) The process of extracting a citrus raw material with a lower alcohol (b) The citrus extract obtained by the process including the process of processing the lower alcohol extract obtained at the process (a) with an aromatic synthetic adsorption resin.   The citrus extract according to claim 1, wherein the citrus raw material used in the step (a) is a squeezer juice residue. The citrus extract according to claim 1 or 2, wherein the collagenase inhibitory activity (IC ₅₀ ) is 1.0 to 120 µg / ml and the elastase inhibitory activity (IC ₅₀ ) is 0.5 to ₅₀ µg / ml. Next steps (a) and (b)
(A) a step of extracting a citrus raw material with a lower alcohol (b) a method of producing a citrus extract comprising a step of treating the lower alcohol extract obtained in step (a) with an aromatic synthetic adsorption resin .   The method for producing a citrus extract according to claim 4, wherein the citrus raw material used in the step (a) is a squeezer juice residue. 6. The citrus extract produced has a collagenase inhibitory activity (IC ₅₀ ) of 1.0 to 12.0 μg / ml and an elastase inhibitory activity (IC ₅₀ ) of 0.5 to 5.0 μg / ml. A method for producing a citrus extract. Ceramide glycoside represented by the following formula (I).

Ceramide glycoside represented by the following formula (II).

Steroid glycoside represented by the following formula (III).

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