JP6339426B2 - Method for producing composition containing glyceroglycolipid and glyceroglycolipid-containing composition - Google Patents

Description

  The present invention relates to a composition containing a glyceroglycolipid.

Glycolipid refers to a group of substances containing both sugar and lipid in the molecule. Glycolipids are further classified into glycosphingolipids, glyceroglycolipids, and others. Glyceroglycolipid is a general term for a group of glycolipids having a carbohydrate as a hydrophilic group and diacylglycerol, alkylacylglycerol, or alkenylacylglycerol as a fat-soluble (nonpolar) group (Non-patent Document 1).
Glyceroglycolipids are widely distributed from animals and plants to microorganisms. Since hydrophilic groups and fat-soluble groups exist as described above, they are used as natural surfactants. In addition, physiological activities such as brain function improvement are known. For this reason, it is used for pharmaceuticals, foods, hair care, skin cosmetics, and the like, and new uses are being developed.
Patent Document 1 describes the interleukin 4 production inhibitory action and melanin production inhibitory effect of kale-derived glyceroglycolipid, which is a cruciferous plant.
In order to obtain glyceroglycolipid, a method of synthesizing a specific compound by chemical reaction (Patent Document 2) and a method of extracting from a plant are known. As a method for extracting from a plant, for example, a method is known in which water is added to tea leaves and an organic solvent such as ethanol is added to tea leaves generated after extraction of water-soluble components to extract glyceroglycolipid (Patent Document). 3).
However, the method of extracting from a plant has a low extraction efficiency (solvent partition) because glyceroglycolipid is an amphiphilic compound, and it is difficult to obtain a glyceroglycolipid extract with a high content. In addition, when extraction is performed with a solvent such as ethanol without removing water-soluble components in advance by water extraction or the like, reducing sugars such as glucose and fructose behave in the same manner as the target glyceroglycolipid. Extract is obtained. The reducing sugar in the extract browns the extract by the Maillard reaction.
In addition, when formulating glyceroglycolipid, a technique of encapsulating in a soft capsule formulation made of gelatin or the like has become widespread. However, the reducing sugar is easily crystallized in the capsule, and is precipitated on the inner surface of the capsule film by the crystallization. And it is pointed out that it becomes a factor which causes the capsule disintegration delay. Therefore, when extracting glyceroglycolipid from a plant, it is calculated | required to reduce reducing sugar content as much as possible.
Furthermore, since the content of glyceroglycolipid in plants is extremely small, a technique for efficient extraction is required.

Japanese Patent No. 4749696 Japanese Patent No. 2939649 Japanese Patent No. 4308714

Biochemical Dictionary, Tokyo Chemical Doujinshi, 1989, 363 pages, 860 pages

  An object of the present invention is to provide a glyceroglycolipid-containing composition having a low reducing sugar content from plants and a method for producing the same. Moreover, this invention makes it a subject to provide the soft capsule formulation which encloses the glyceroglycolipid by which the brown change during a preservation | save is suppressed and decay | disintegration delay does not occur easily.

(1) A method for producing a glyceroglycolipid-containing composition by extracting glyceroglycolipid from a plant body by a two-stage supercritical extraction method, wherein supercritical carbon dioxide is used as an extraction solvent in the first stage of extraction. only extraction residue after extracting Te was recovered, as extracted for this extraction residue of the second stage, and extracts as a extraction solvent supercritical carbon dioxide and ethanol, glyceroglycolipid-containing composition A method of manufacturing things.
(2) The method according to (1), wherein the plant is one or more plants selected from kale, barley young leaves, mugwort, and komatsuna.
( 3 ) The method according to (1) or (2), wherein the composition is a glyceroglycolipid-containing composition comprising a reducing sugar extracted from a plant and a glyceroglycolipid, wherein the content of the reducing sugar is 1% or less. A glyceroglycolipid-containing composition comprising the extract obtained in 1 .
( 4 ) A soft capsule formulation containing the glyceroglycolipid-containing composition according to (3) .

The present invention provides a method for producing a glyceroglycolipid-containing composition having a low reducing sugar content from a plant. Since the method of the present invention does not use the organic solvent used in the prior art, a safe composition can be obtained. And a manufacturing process is simple and does not require complicated operation like a solvent extraction method.
In addition, the glyceroglycolipid-containing composition of the present invention has a low reducing sugar content, suppresses browning due to the Maillard reaction, and makes it difficult to delay the collapse of the soft capsule preparation.

The chart of the HPLC chromatography which analyzed the extract obtained in Example 1 is shown. The chart of the HPLC chromatography which analyzed the extract obtained in Example 2 is shown. The chart of the HPLC chromatography which analyzed the extract obtained in comparative example 1 is shown. The chart of the HPLC chromatography which analyzed the extract obtained by the comparative example 2 is shown. The chart of the HPLC chromatography which analyzed the extract obtained by the comparative example 3 is shown.

  The present invention relates to a method for producing a glyceroglycolipid-containing composition by extracting glyceroglycolipid from a plant body by a supercritical extraction method, characterized by using supercritical carbon dioxide as an extraction solvent, A method for producing a lipid-containing composition. The present invention also relates to a glyceroglycolipid-containing composition comprising a reducing sugar extracted from a plant and a glyceroglycolipid, wherein the reducing sugar content is 10% or less. is there.

(1) Raw material As a raw material of the method of the present invention, any plant containing glyceroglycolipid can be used, but a plant that is preferably edible or medicinal is preferable. The following plants can be exemplified.
Almond, Aosa, Aonori, Akaza, Acacia, Akane, Aka grape, Red pine (including pine crab, cocoon, copal), Agaricus, Akinonogeshi, Akebi, Asagao, Azalea, Hydrangea, Ashitaba, Azuki, Asparagus Gas, Acerola, Asenyaku, Anise, Avocado, Achacha, Achacharu, Amaryllis, Altea, Arnica, Aloe, Angelica, Apricot, Ansocco, Igusa, Izayoi Rose, Ichii, Fig, Ginkgo, Iran Iran, Fritillaria, Ukrainian, Wolves , Udo, Ume, Vulture, Wenzhou mandarin, Ages, Shallot, Ezokogi, Enishi, Elderflower, Pea, Orchid, Plantain, Great white thistle, Barley, Okra, O La, Osmanthus, Hypericum, Odoricho, Onidokoro, Olive, Oregano, Orange (including orange peel), Carnation, Cacao, Oyster, Oyster, Cuckoo, Kashiwa, Katakuri, Pumpkin, Chamomile, Camcam, Chamomile, Oxalis, Larch, Karin, Garcinia, Cardamom, Raspberry, Kiwi, Koki, Cabbage (including Kale), Caraway, Cucumber, Kumquat, Ginnan, Guava, Cuco, Kudzu, Gardenia, Cumin, Cranberry, Walnut, Grapefruit, Clove, Black Pine, Black Bean, Kale, Ketsumeishi, Gennoshoko, Cowberry, Pepper, Cosmos, Burdock, Wheat (including wheat germ), Sesame, Komatsuna, Rice (Including rice bran), Coriander, Konjac koji (Konjac powder) ), Kombu, salmon berry, cypress, pomegranate, sweet potato, sugarcane, sugarcane, sugar beet, saffron, pomelo, hawthorn, salamander, cyclamen, perilla, shimeji, jaguar, peony, jasmine, juzudama, shungiku, ginger , Shabu, Syringe, Ginkgo biloba, Cinnamon, Watermelon, Sweet pea, Horsetail, Star anise, Star apple, Sudachi, Stevia, Plum, Sage (Salvia), Zenia mushroom, Celery, Cyprus, Assembly, Buckwheat, Broad bean, Daikon, Soybean (Okara) Daidai, thyme, bamboo shoot, onion, tarragon, taro, tanjin, dandelion, chicory, camellia, camellia, camellia, camellia, clover, clover, tuna, camellia, dill, pearl oyster (gelani) Um), chili, capsicum, cypress, corn, dodami, tocon, eucommia, ash, Chinese yam, natsuna, nutmeg, nanten, bitter gourd, mugwort, leek, carrot, garlic, leek, yarrow, saw palmetto, nobil, paver, palm , Hibiscus, Octopus, Basil, Parsley, Hadakamugi, Hakka, Barley, Banana, Banaba, Vanilla, Paprika, Hamelis, Beet, Bell Pepper, Hydrangea, Hashi, Pistachio, Hyssop (Ceramic), Daisies, Daisies, Hinoki Sunflower, loquat, phalaenopsis, phenegrigree, cypress, blackberry, plum, blueberry (including bilberry), prunes, loofah, safflower, belladonna, bergamot, spinach Noodles, physalis, body bream, buttons, hops, jojoba, maou, maca, macadamia nuts, matatabi, marigold, mango, honey bees, mimosa, mioga, myrrh, murasaki, mace, melissa, merilot, melon, men ), Bean sprouts, cornflower, mountain bream, mountain lily, porcupine, eucalyptus, yukinoshita, yuzu, lily, yokuinin, yomena (aster), mugwort, lime, rye, lilac, raspberry, peanut, radish, apple, gentian, lettuce, lemon , Lotus root, lotus root, rose hips, rosemary, bay leaf, bamboo grass, wasabi (including horseradish). The whole of these plants, leaves, or fruits can be used. Of these, kale, young barley leaves, mugwort and komatsuna are preferred, and kale is particularly preferred.
The plant body is preferably dried and then pulverized. In kale, barley young leaves, mugwort, and komatsuna, it may be a squeezed residue squeezed after harvesting for a green juice drink.

(2) Supercritical extraction operation The present invention is not particularly limited as long as it is a supercritical extraction apparatus.
In the extraction operation, the raw material is stored in an extraction tank in a supercritical extraction apparatus, and carbon dioxide is used as an extraction solvent to perform the extraction operation under each extraction condition. Suitable extraction conditions are, for example, a carbon dioxide flow rate of 10-100 g / min, an extraction pressure of 10-100 MPa, and an extraction temperature of 32-100 ° C. More preferably, the carbon dioxide flow rate is 65 g / min, the extraction pressure is 25 MPa, and the extraction temperature is 40 ° C. The longer the extraction time, the more glyceroglycolipid can be obtained, but preferably 0.5 to 1 hour. Moreover, it is preferable to use polar organic solvents, such as ethanol and methanol, as an entrainer. A particularly preferred polar solvent is ethanol. By using a polar solvent as an entrainer, a glyceroglycolipid-containing composition having a lower reducing sugar content can be obtained.
The extract composition thus obtained is in the form of a grease, contains glyceroglycolipid, and has a reducing sugar content of 10% or less. This composition can be subjected to column chromatography or preparative high performance liquid chromatography (HPLC) to obtain a raw material having a further increased glyceroglycolipid content.

(3) Confirmation method of glyceroglycolipid The glyceroglycolipid in the obtained extract can be confirmed by analyzing by the following method.
1) Pretreatment method of glyceroglycolipid-containing extract
i) Weigh the appropriate amount of the extract obtained, add dimethyl sulfoxide and dissolve.
ii) The solution obtained in i) is appropriately diluted with dimethyl sulfoxide according to the component concentration to prepare a sample for analysis.
2) Analysis method
i) Analysis by high performance liquid chromatograph mass spectrometer (LC-MS)

LC-MS analysis conditions are as follows. Detector: LCMS-2000 (Shimadzu Corporation)
Ionization condition: (+/-)-ESI
Mobile phase A: 0.5 mM ammonium formate, 0.1% formic acid aqueous solution Mobile phase B: 0.5 mM ammonium formate, 0.1% formic acid acetonitrile: methanol = 1: 1 solution Analytical column: Merck Select B 250 × 4 mm, 5 μm
Injection Volume: 10μL
Column oven: 25 ° C
Analysis time: 100 minutes Flow rate: 0.5 mL / min Gradient conditions are as shown below
(1) 0-30 minutes
Mobile phase A: 85% → 0%, mobile phase B: 15% → 100%
(2) 30-80 minutes
Mobile phase A: 0%, mobile phase B: 100%
(3) 80-90 minutes
Mobile phase A: 0 → 85%, mobile phase B: 100% → 15%
(4) 90-100 minutes
Mobile phase A: 85%, mobile phase B: 15%
The presence or absence of a peak showing the following ionization pattern is confirmed according to the analysis conditions. Here, the peak of kale-derived glyceroglycolipid of the following IUPAC name in Patent Document 1 can be confirmed and used as an index. Since glyceroglycolipids having other structures also exhibit the same behavior, inclusion of a compound having the following IUPAC name indicates that glyceroglycolipids having other structures are also contained.
(+)-ESI: 699 [M + NH ₄ ] ⁺ , 515 [M-Gal + H] ⁺ , 353 [M-2Gal + H] ⁺ ,
261 [C ₁₈ H ₂₉ O] ⁺
(-)-ESI: 721 [M + HCOO] ^- , 675 [MH] ^- , 277 [C ₁₈ H ₂₉ O ₂ ] ^-

IUPAC name
(9Z, 12Z, 15Z) -2-hydroxy-3-((2R, 3R, 4S, 5R, 6R) -3,4,5-trihydroxy-6-(((2R, 3R, 4S, 5R, 6R) -3,4,5-trihydroxy-6- (hydroxymethyl) -tetrahydro-2H-pyran-2-yloxy) methyl) -tetrahydro-2H-pyran-2-yloxy) propyl octadeca-9,12,15-trienoate


ii) Analysis by HPLC
HPLC analysis conditions are as follows Analyzer: Ultimate 3000 HPLC System (Thermo Fisher Scientific Co., Ltd.)
Detector: Charged particle detector (Thermo Fisher Scientific Co., Ltd.)
Other conditions are the same as those for LC-MS

(4) Method for Confirming Reducing Sugar The confirmation and content of the reducing sugar in the composition of the present invention can be analyzed by the following method.
i) Analysis by LC-MS Ionization patterns of glucose, fructose and sucrose standards and each extract were compared under the above conditions. In the extract containing the reducing sugars glucose and fructose, peaks with a mass-to-charge ratio of 225,297,367 are detected.

ii) Analysis by HPLC Under the above conditions, the standard products of glucose, fructose and sucrose are compared with chromatographs of each extract.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples.
Example 1
About 1 g of dry kale powder was sampled and transferred to a pressure-resistant container (extraction tank, 0.5 L container) of a supercritical extraction device (Mitsubishi Chemical Corporation). Next, using carbon dioxide as the extraction solvent, the temperature is increased and the pressure is increased. The extraction operation is performed by passing supercritical carbon dioxide through the extraction tank for 1 hour under the conditions of 40 ° C. and 25 MPa (CO ₂ flow rate: 65 g / min). Went.
The extract was transferred to a collection tank and weighed. About 8% of the weighed raw materials were extracted by the extraction operation under these conditions.

(Example 2)
The supercritical extraction residue obtained in Example 1 was subjected to an extraction operation by passing supercritical carbon dioxide through an extraction tank under the conditions of 40 ° C. and 25 MPa for 1 hour (CO ₂ flow rate: 65 g / min). . At this time, ethanol was used as an entrainer (flow rate: 3 mL / min).
The extract was transferred to a collection tank and weighed. About 35% of the weighed raw materials were extracted by the extraction operation under these conditions.

(Comparative Example 1)
About 1 g of dry kale powder was collected, about 50 mL of ethanol was added, and shake extraction was performed at room temperature for 1 hour. After separating the extract from the extraction residue using filter paper, the solvent was removed by vacuum treatment to obtain an extract. About 10% of the weighed raw materials were extracted by the extraction operation under these conditions.

(Comparative Example 2)
About 1 g of the dry kale powder was collected, about 50 mL of methanol was added, and shake extraction was performed at room temperature for 1 hour. After separating the extract from the extraction residue using filter paper, the solvent was removed by vacuum treatment to obtain an extract. About 30% of the weighed raw materials were extracted by the extraction operation under these conditions.

(Comparative Example 3)
About 1 g of the dry kale powder was collected, and about 50 mL of 2: 1 = chloroform: methanol mixture was added, followed by extraction with shaking at room temperature for 1 hour. After separating the extract from the extraction residue using filter paper, the solvent was removed by vacuum treatment to obtain an extract. About 20% of the weighed raw materials were extracted by the extraction operation under these conditions.

[Evaluation of extraction efficiency]
Table 1 shows the recovery rates of the extracted compositions of Examples and Comparative Examples with respect to raw materials.

  There was no big difference between the extraction amount by solvent extraction and supercritical extraction.

[Evaluation of glyceroglycolipid-containing composition]
Table 2 shows the results of confirming the presence of glyceroglycolipid in the compositions obtained in Examples and Comparative Examples, and the approximate value of the content of reducing sugar. In addition, the reducing sugar content rate in a composition is an estimated value from the peak area of a chromatograph.

  It was confirmed that the compositions of Examples 1 and 2 of the present invention had a low reducing sugar content as compared with Comparative Examples 1 to 3.

(Example 3)
Using the composition of Example 2 and Comparative Example 1, a soft capsule formulation was prepared by encapsulating a solution in 10% of a medium chain fatty acid triglyceride containing gelatin in a gelatin soft capsule by a conventional method. The preparation was stored for 1 month in a constant temperature and humidity chamber adjusted to a temperature of 40 ° C. and a humidity of 75%, and then the browned state of the contents and a disintegration test were performed.
In the soft capsule preparation containing the glyceroglycolipid-containing composition of Example 2, neither discoloration of contents nor delay of disintegration time was observed.
On the other hand, in the soft capsule preparation encapsulating the glyceroglycolipid-containing composition of Comparative Example 2, the content changed brown and the disintegration time was further delayed.

Claims (4)

A method of manufacturing a glyceroglycolipid-containing composition to extract glyceroglycolipid from plant by two stages of supercritical extraction, the extraction of the first step was extracted by the extraction solvent supercritical carbon dioxide extraction residue only was recovered after, as extracted for this extraction residue of the second stage, and extracts as a extraction solvent supercritical carbon dioxide and ethanol, producing a glyceroglycolipid-containing composition how to.   The method according to claim 1, wherein the plant is one or more plants selected from kale, barley young leaves, mugwort, and komatsuna. A glyceroglycolipid-containing composition comprising a reducing sugar extracted from a plant and a glyceroglycolipid , wherein the extract obtained by the method according to claim 1 or 2 having a reducing sugar content of 1% or less . A glyceroglycolipid-containing composition comprising:   A soft capsule preparation containing the glyceroglycolipid-containing composition according to claim 3.