JPH0819149B2 - Method for extracting cerebroside from soybean germ - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention confirms that soybean, especially soybean germ, contains a large amount of cerebroside.
It relates to a method for extracting cerebroside from this. The extracted cerebroside can be used as a moisturizer in cosmetics or pharmaceuticals.

[0002]

2. Description of the Related Art The most decisive role in preventing the outflow of body fluids and the penetration of external substances through the skin and maintaining the moisturizing and swelling functions is played by the lipid membrane in the stratum corneum of the skin (PM Elias and DS Friend, J Cell Biol. (197
5), 65: 180-191). The lipid membrane in the stratum corneum is large and is composed of ceramide, fatty acid, and cholesterol.
(Cholesterol), cholesterol sulfate (Cholesterol su
lfate) etc. (CT Downing et al.,
J. Soc. Invest. Dermatol. (1987) 88: 25S). Ceramide is formed in the form of cerebroside during epidermal differentiation, and is converted to ceramide by glycolytic enzymes present in the stratum corneum to form a lipid membrane (R.
D. Perterson. Cosmetics and Toiletries (1992) 107: 4
5-49). Topical application of cerebroside to the skin has been reported to have excellent heat retention and aesthetic improvement effects, and is known to restore skin damage quite quickly (WP Smith et al., Cosmetics and T
oiletries (1990) 106: 65-72). Therefore, as with cerebroside, the skin-improving effect is excellent, and studies have been actively conducted to supply the same lipid to the skin as the lipid membrane component in the stratum corneum.

However, in the natural world, cerebroside is present in a small amount in the brain of animals, and is rarely present in plants or in very small amounts. Therefore, since it is difficult and expensive to obtain raw materials, the use of more than the amount that actually shows the effect has been extremely limited. In addition, cerebroside extracted from animal brain has a complicated purification process, is odorous and has a color, and cannot be used in a large amount in cosmetics. In order to solve such problems of animal-derived cerebroside, similar ceramide (Pseudoceramide: Ceramide HO ₃ ,
Serdema's product) has been synthesized and used, but when the synthesized similar ceramide is applied to cosmetics, it is known that its solubility is low and its stability is reduced, and its effect is inferior to that of natural ceramide. According to the effect of research conducted by the inventor, it hinders the natural healing of rather damaged skin (Tables 2 and 3). In addition, since similar ceramide is also very expensive, it cannot be applied in an amount exceeding the actual efficacy.

Recently, it has been reported that cerebroside extracted from plants has no difference in efficacy and animal cerebroside, and has no inactivity or toxicity (P. Gossioux. Pr.
efumes, Cosmetiques, Aromes (1992) 103: 55-56). In addition, plant cerebroside has no odor or color,
It has the advantage of being able to purchase large amounts of raw materials. However, since its content is present in an extremely small amount, the disadvantage of being expensive cannot be improved, and it has been almost impossible to use more than the amount showing the actual efficacy.

Studies on cerebrosides of plants have been
Although it was announced for wheat etc., its content was low and there was no merit for commercial use (see Table 4). Also, JP
The application for a mixture of sphingolipids and triterpenes extracted from rice bran, wheat bran and jade corn 62-187404A is completely distinct from the present invention using soybean sprouts as the extraction material. Also, in JP63-255212, a plant extract containing saponin and cerebroside was claimed to have an anti-aging effect, but the present invention does not contain saponin, has a good skin moisturizing effect, and is a soybean germ in particular. Is the first time to use.

[0006]

The present inventor has clarified that soybean, particularly soybean germ obtained as a by-product of food production, contains a high concentration of cerebroside and a method for extracting cerebroside. By presenting, cerebroside derived from a very inexpensive food was provided and used in an amount more than that which can be effective as a moisturizer for cosmetics or pharmaceuticals.

[0007]

[Table 1]

As shown in Table 1, soybean and soybean germ differ greatly in the contents of lipid and nitrogen-free (N-free) extract + fibrin. In addition, soybean germ is a material that has a very high content of saponin, has a bitter taste, has digestive problems, and is a large amount generated as a by-product of soybean oil and then discarded. Therefore,
Soybean and soybean germ are different materials whose components and uses are completely distinguished. Therefore, the present inventor has completed the present invention by starting the world's first research on soybean germ.

[0009]

To explain the extraction method of the present invention, first, a known method for extracting lipids is used to extract polar alcohols, that is, methanol, ethanol, or a mixed solvent of butanol and water, followed by drying. Then, dissolve in ethanol and saponify with alkali. Focusing on the fact that cerebroside cannot be saponified, water is added thereto, lipids are separated with n-hexane or ether, and then dried and recrystallized from acetone to extract cerebroside.

The present invention will be described in detail below together with the extraction process. A certain amount of dried soybeans is finely crushed into powder and a lipid extract is prepared by a general lipid extraction method. The dried soybeans may be used as they are, but especially the soybean germ contains a higher concentration of cerebroside than other plants and is obtained from a by-product of the plant, so cerebroside is extracted from the soybean germ. Is most desirable. The lipid extract is extracted by a method such as Nicols, that is, extracted twice with boiling isopropyl alcohol, then extracted twice with a solution of methanol: chloroform mixed 1: 2, and extracted together. Method of making liquid (Nicols BW, Biochem. Biophys. Act (1963) 70:
417-422) or n-butanol, ethanol, methanol and a mixture of water and extraction.

After all of the above extracts are dried under reduced pressure, polar lipids and non-polar lipids are separated and the lipid layer is dried to obtain a yellow solid. Here, the polar lipid and the non-polar lipid are separated by dissolving a liquid dried under reduced pressure in 87% ethanol, and then adding n-hexane to separate the non-polar lipid into an n-nucleic acid layer, and drying the ethanol layer which is the polar layer. To obtain polar lipids. At that time, the lipid component in the solid obtained by drying the ethanol layer was Melnik.
It can be confirmed by thin layer chromatography (TLC) while changing the solvent three times (BC
Melnik et al., J. Lipid Res. (1989) 3: 134-136).

In order to remove phospholipids and glycolipids other than sphingolipids from the sample obtained by drying the ethanol layer, the properties of sphingolipids having an amide bond and other phospholipids and glycolipids having an ester bond were determined. To use. That is, a sample obtained by drying the ethanol layer was put into a 0.5 M caustic soda / ethanol solution and reacted at 70 ° C. for 1 hour to break the ester bond, and then water was added thereto to add lipids with n-hexane or ether. Extract. Acetone is added to the dried lipid, and the dye is removed through repeated recrystallization to obtain cerebroside from the remaining precipitate. The presence of cerebroside can be confirmed by thin layer chromatography (TLC) and then by a densitometer (Densitometer, Simadzu product).

In order to obtain a more purified cerebroside, the ester bond is removed, and the separated solid is fractionated with acetone by silica gel column chromatography and dried. Thereby, a pale yellow solid, that is, a more purified cerebroside can be obtained. The presence of sugar can be confirmed from this extract by a method such as Kushwaha (SC Kushwaha and M. Ka-tes, Lipid.
(1981) 16: 372-) and ninhydrin quantification can confirm that there is no unbound amino group. When the amide bond is broken by a method such as Kodowaki and then ninhydrin is quantified, a peak at an ultraviolet wavelength of 575 nm can be obtained to confirm the presence of a long-chain base (H. Kodowaki et al., J. Lipid Res. (19
83) 24: 1389-1397). The molecular weight of this long-chain base can be measured again by an actual spectrum to confirm that it is a sphingo base.

Example 1 100 g of dried soybean germ was finely crushed to extract 1 L of methanol: water (85:15, v / v). After the extracts were combined and dried under reduced pressure, 30 g of a yellow liquid was obtained, which was dissolved in 60 ml of 87% ethanol,
180 ml of n-hexane was added to separate the non-polar lipid into an n-hexane layer, and then the ethanol layer was dried to obtain 15 g of a yellow solid. 0.5M caustic soda / 95%
After putting in 200 ml of ethanol solution and reacting at 78 ° C. for 1 hour, water was added to n-hexane or ether 50.
The lipid was separated with 0 ml to obtain 2.0 g of a yellow solid. This was again separated by silica gel column chromatography to obtain 0.2 g of a white solid. As a result of qualitative and quantitative analysis, cerebroside was 0.10 w / w per soybean germ.
It was confirmed that more than%.

Example 2 100 g of dried soybean germ was mixed with ethanol: water (80:
As a result of extraction and quantification with 20 v / v), it was confirmed that cerebroside was present at 0.10 w / w% or more.

Example 3 Cerebroside was extracted from 100 g of dried soybeans in the same manner as in Example 1 and quantified. As a result, it was confirmed that cerebroside was present in an amount of 0.05 w / w% or less.

Comparative Example 1 A lotion containing 0.5% cerebroside extracted according to Example 1 was used.

Comparative Example 2 A lotion containing 0.5% of the synthesized similar ceramide was used.

Test 1 Efficacy comparison of cerebroside extracted from soybean embryos Transepidermal W evaporation from the skin of 7-week-old nude mice using a water evaporation measurement device (Servo Med product)
ater Loss: TEWL), and then the skin of nude mice was treated with acetone to be lipidated. After measuring the transepidermal water evaporation amount again, each lotion, lotion containing 0.5% similar ceramide (Comparative Example 2), or lotion containing 0.5% cerebroside extracted from soybean germ (Comparative Example 1) ) Was applied to the skin, and the amount of transepidermal water evaporation was examined over 30 minutes. Transepidermal water loss is 10 each
The measurement was carried out on nude mice and then statistically processed. Since the transepidermal water evaporation amount is directly inversely proportional to the blocking function of the lipid membrane in the stratum corneum of the skin, the transepidermal water evaporation amount before the acetone treatment is taken as the standard with the recovery rate as 100%, and the transepidermal water evaporation immediately after the acetone treatment. Based on the amount of recovery as 0%, the relative recovery rate of the skin blocking function is shown in Table 2 in Table 2 as the transepidermal water loss is reduced.

[0020]

[Table 2]

According to this material, Comparative Example 2 is 30 after coating.
From the minute to 3 hours, skin penetration is fast, and the effect of complementing the damaged lipid membrane is 38% and 45%, which is larger than that of lotion (spontaneous recovery) and Comparative Example 1.

However, after 3 hours, the recovery of the blocking function of the lipid membrane is disturbed rather than the case where only the lotion is applied. The synthesized similar ceramide has a mixture of isomers, and has a structure different from that of ceramide produced in vivo.
Therefore, the infiltrated similar ceramides rather incomplete the lipid membrane structure within the stratum corneum formed by spontaneous recovery. However, the cerebroside extracted by the present inventor (Comparative Example 1) has sugar, and the skin permeation is slightly slower than that of the synthesized similar cerebroside, but 27% or 4% within 3 hours.
4%, 10% of lotion (natural recovery) and 27
Recovers the blocking function much faster than%. Also, after 3 hours, rather, healed much faster than similar ceramides,
In particular, it promotes natural recovery faster.

From the above, it has been found that cerebroside extracted by the present inventor restores the damaged corneal barrier function very quickly and forms the corneal cornea as well as the ceramide produced in vivo.

Test 2 Comparison of the effect of cerebroside extracted from soybean embryo on the skin for a long period of time: 10 nude mice each, lotion on both sides of the rat, Comparative Example 1 or Comparative Example 2 twice a day After each application, the transepidermal water evaporation amount was examined every week.

[0025]

[Table 3]

Comparative Example 2 showed a decrease in transepidermal water evaporation of 3.2% and 5.3% after 1 week and 2 weeks, respectively, compared with the control group. On the other hand, when a lotion containing 0.5% of soybean germ extract was applied as in Comparative Example 1, it was 1
It can be seen that the reductions are 0.0% and 20.2%, respectively, and the effect is three times or more superior to that of the synthesized similar ceramide. In addition, the feeling of use during application was much better than when the control group and Comparative Example 1 were used, the skin was extremely smooth, and the aesthetic appearance was greatly improved.

[0027]

EFFECTS OF THE INVENTION As described above, cerebroside can be extracted from soybean, and in particular, soybean germ contains 10 times or more cerebroside by weight as compared with wheat or black sorghum, as shown in Table 4 below. ing. Also, since soybean germ is obtained as a by-product of food production, it is much more economical to extract cerebroside from soybean germ.

[0028]

[Table 4]

Further, cerebroside obtained from soybean germ has far superior efficacy as shown in Tests 1 and 2 as compared with existing synthetic similar ceramide, and is supplied at a very low price. Therefore, it is possible to apply more than the amount showing the actual efficacy to the product.

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Claims (4)

[Claims] 1. A method of extracting soybean embryo with a polar solvent, drying it, saponifying it with an alkali, obtaining an unsaponifiable product with n-hexane or ether, and then recrystallizing the solvent to extract cerebroside. 2. The method according to claim 1, wherein the polar solvent is ethanol, methanol or a mixture of n-butanol and water. 3. The method of claim 1, wherein the solvent is acetone. 4. A moisturizing product containing cerebroside extracted from soybean germ by the method according to claim 1.
Agent .