KR950014458B1 - Process for preparing cerebroside from soybean - Google Patents

Abstract

1)extraction of soybean germs with isopropyl alcohol; 2)extraction with mixture of methanol and chloroform; 3)reducing pressure and drying the extractive mixture of (1) and (2); 4)separating polar lipids with ethanol and hexane, and drying; 5)reacting the (4)product in NaOH/methanol solution; 6)adding acetic acid/chloroform/water and separating the lower part; and 7)separating cerebrocide by silica column chromatography.

Description

How to Extract Cerebroside from Soybean

The present invention relates to a method for extracting cerebrosde from the soybeans, especially soybean embryos, which contains a large amount of sphingollpids, and can be used in the cosmetic and pharmaceutical industries.

The most critical role in preventing the leakage of body fluids and foreign substances through the skin and maintaining moisturizing and swelling functions is played by lipid membranes in the stratum corneum (P, M Ellas and D. Friend, J Cell B1 (1975)). The stratum corneum in the stratum corneum is largely composed of ceramide, fatty acid (Fatty acld), cholesterol (CholesteroI), cholesterol sulfate (ChoIestero1 sulfate), etc. (C. T Downlng et al, J. SOL. Invest.Dermato1 (1987) 88: 25S) Ceramides are made in the form of cerebromide during epidermal differentiation (epldermal dlfferentiatlon) and converted into ceramides by glycolysis enzymes present in the stratum corneum (R D. Peterson, Cosmetics) and To1Ietries (1992) 107 45-49) The external application of cerebromide to the skin has been reported to improve the moisturizing function and improve the aesthetic appearance and to recover the skin damage very quickly. There ryeojyeo (∫V P Smith et al, Cosmetles and Toiletrles (1991) 106 65-71). Therefore, studies have been actively conducted to supply the skin with lipids such as lipid membrane components in the stratum corneum and excellent skin improvement like cerebromide.

In nature, however, cerebrosides are present in animal brains in small amounts and rarely or in very small amounts in plants. Therefore, since the difficulty of obtaining raw materials is expensive, the use of more than the amount of actual effect has been very limited. In addition, cerebroside extracted from the animal brain has a complicated purification process and has a few odors, which makes it impossible to use in large quantities in cosmetics.

In order to solve the problem of animal-derived cereviside, pseudoceramide (pseudoceramide, Ceramdede HO3, manufactured by Serdema) has been synthesized and used.However, when the synthetic pseudoceramide is applied to a real cosmetic, its solubility is low, and the effect is natural. It is known to be inferior to ceramides, and the results of the present inventors show that it interferes with natural healing of damaged skin (Figures 1 and 2). In addition, since pseudoceramide is very expensive, it cannot be applied more than the amount which shows actual efficacy.

Recently, cerebroside extracted from plants has no difference in efficacy and animal-derived cerebroside, and no side effects or toxicity have been reported (P. Gossioux, Parfumes, Cosmetlques, Aromes (1992) 10355-56). Plant-derived cerebroside has the advantage of being able to purchase large quantities of raw materials without smell and color. However, since the amount is present in a very small amount, the disadvantage of the expensive price can not be improved, it was almost impossible to use more than the amount showing the actual efficacy.

Thus, the present inventors found that the content of cerebrose in soybeans, especially soybean embryos, is 10 to 20 times higher than that of conventional raw materials, and by presenting a method of extracting the same, it is a very inexpensive plant-derived blobbro. The side is provided so that it can be applied more than the amount that can be effective as a moisturizer for cosmetics and medicines.

That is, the soybean embryos are extracted with isopropyl alcohol, the mixture is extracted with a mixed solution of methanol and chloroform, and the combined extracts are dried under reduced pressure, and the polar lipids are separated and dried, followed by reaction with caustic soda / methane in a solution. The lower layer was separated, dried, and dried to extract the acetone fraction from Daelong Chromatography to extract the cerebroside.

The present invention will be described in detail with the extraction process as follows.

Grind a predetermined amount of dry soybean embryos, making lipid extracts according to the usual methods of lipid extraction. Dry soybeans may be used here, but it is more preferable to use dried soybean embryos and soybean meal, and in particular, soybean embryos contain 10 to 20 times more weight per cent of cerebroside and more food than multiplants. It is most preferred to extract the cerebrosides from soybean embryos since they can be obtained from by-products of. The extract of lipids was extracted with Dubin using a method of Nicols et al., Boiling isopropyl alcohol, and then extracted twice with a solution of methanol: chloroform 1: 1 (Nicols BW, Biochim. Bjiphys. Acta (1963)). 70 417-422).

All of the extracts were combined and dried under reduced pressure, the polar lipids and the nonpolar lipids were separated and the polar lipid layer was dried to give a yellow solid. Here, the separation of polar lipids and nonpolar lipids is carried out by dissolving the dried liquid under reduced pressure in 87% ethanol and then adding n-hexane to separate the nonpolar lipids into the n-hexane layer and drying the polar layer to obtain the polar lipid. At this time, the identification of lipid components from the solid obtained by drying the ethanol layer can be confirmed by thin membrane chromatography changing the solvent three times according to the method of Melnik et al. (B C. Melmik et al. J. Lipid Res. (L989) 3 (134-136)

To remove phospholipids and glycolipids other than sphingolipids from the sample obtained by drying the ethanol layer, sphingolipids having amide bonds and other phospholipids and glycolipids having ester bonds are used. That is, the sample obtained by drying the ethanol layer was added 0.5 M caustic soda / methane to the solution and reacted at 50 ° C. for 10 minutes to boil the ester bond (MA Wells and JC Dittmer, J. Chromatogr. (1965) 18: 503- 511), to which sphingolipids are obtained by adding chloroacetic acid 'water in a ratio of 1:40:15 and separating and drying the lower layer. As a method of boiling the ester bond, there is also a method of pyrolysis in an acid solution in addition to the above method. On the other hand, the presence of cerebroside from sphingolipids separated from the ester bonds was confirmed by thin membrane chromatography and then confirmed by densitometer (manufactured by Simadzu).

In order to obtain more purified cerebroside, the solid obtained by cleaving the ester bond was separated and dried in an acetone fraction in silica gel macro chromatography, thereby obtaining a pale yellow solid, that is, a more purified cerebroside. In this extract, the presence of sugar can be confirmed by Kushwaha et al. (S C Kushwaha and Mkates, Lipid (1981) 16 372-), and by quantifying ninhydrin, it can be confirmed that there is no unbound amino group. In addition, by quantifying ninhydrin after cleaving the amide bond according to the method of Kadowaki et al., A peak was obtained at an ultraviolet wavelength of 575 nm to confirm the existence of an amino group (H. Kadowaki et al J. Lipid Res (1983) 24 1389-1397). Again, this long chain base can be identified as a sphingo base by measuring its molecular weight in the mass spectrum.

Cerebroside can be extracted from soybeans as described above, and in particular, embryos of soybeans contain 10 to 20 times the weight of cerebroside per weight of wheat or millet as shown in the chart below. Since soybean embryos can be obtained as food by-products, it can be seen that it is much more economical to extract cerebrosides from soybean embryos.

(1) T. A Macmurray and lV. R. Morrlson, J Sci. Fd Agric. (1970) 21 520-528

(2) A U. Osagie and M. Kates Lipid (1984) 19 958-965

Cerebroside obtained from soybean embryos is much better than conventional synthetic pseudoceramides as in Test I and II, and can be supplied at a very low price. Can be.

[Production Example 1]

100 g of dried soybeans were finely ground, extracted twice with 150 ml of boiling isopropyl alcohol, and extracted again with 300 ml of a solution of methanol: chloroform 1: 2. The combined extracts were dried under reduced pressure to give 15 g of a yellow liquid, which was dissolved in 30 ml of 87% ethane, and 90 ml of n-hexane was added to separate nonpolar lipids into an n-hexane layer. The ethanol layer was dried to give 8 g of a yellow solid. . This was added to 60 ml of 0.5 M caustic soda / methanol solution and reacted at 50 ° C. for 10 minutes, and then 3 ml of acetic acid, 120 ml of chloroform, and 45 ml of water were added, and the lower layer was separated to obtain 0.8 g of a yellow solid. The azetone fraction was dried again on silica gel long chromatography to yield 0.20 g of a pale yellow solid. Qualitative and quantitative analysis showed that the presence of cerebroside was greater than 0.15w / w%.

[Production Example 2]

Cerebroside was extracted from 100 g of dried soybeans in the same manner as in Preparation Example 1, and quantitatively confirmed that less than 0.01 w / w% of serebroside was present.

[Manufacture example 3]

Cerebroside was extracted by quantifying 100 g of dried soybean meal (soybean jelly) in the same manner as in Preparation Example 1, and it was confirmed that cerebromide was present in 0.05 w / w% or less.

Example 1

A lotion containing 0.5% of cerebroside extracted according to Preparation Example 1.

Comparative Example 1

Lotions containing 0.5% of synthetic pseudoceramides

(Test I). Comparison of Efficacy of Cerebroside Extracted from Soybean Embryo

Skin evaporation at 7 weeks of age was measured by transvaporation (TransEpidermal Water Loss TEWL) using a water evaporation meter (Servo Med) and de-lipidized by acetone treatment. Again, the transdermal moisture evaporation rate was measured, followed by application of a commercially available conventional lotion or a lotion containing 0.5% pseudoceramide (Comparative Example 1), or a lotion containing 0.5% of cerebroside extracted from soybean embryos (Example 1). After the application to the skin 30 minutes after the transdermal moisture evaporation was investigated over time. Transdermal moisture evaporation was measured on 10 hairless mice and statistically processed. Transdermal moisture evaporation is directly inversely proportional to the blocking function of lipid membranes in each stratum corneum of the skin. Therefore, the transdermal moisture evaporation rate is 100% before acetone treatment and the transdermal moisture evaporation rate is 0% after acetone treatment. As a result, the relative recovery rate of skin barrier function is expressed in% and is shown in Table 1.

TABLE 1

* The standard deviations are all within 0.3.

According to this data, Comparative Example 1 has a faster skin penetration from 30 minutes to 3 hours after application, which is 38% and 45% more effective in compensating for damaged lipid membranes than natural recovery and Example 1. However, after three hours, the lotion prevents the restoring of the lipid membrane barrier rather than applying the lotion alone. Synthetic pseudoceramides are mixed with isomers and differ in structure from ceramides produced in vivo. Therefore, the infiltrated pseudoceramide makes the lipid membrane structure in the stratum corneum formed by natural recovery rather incomplete. However, the cerebroside extracted by the present inventors has a sugar, and the skin penetration is slightly slower than the synthetic similar ceramide, but within 3 hours, it recovers the blocking function much faster than the natural recovery rate of 10% and 27% by 27% and 44%. Let's do it. After 3 hours, it is much faster than pseudoceramide, especially faster than natural recovery rate, helping natural recovery.

Accordingly, it was found that the cerebroside extracted by the present inventors restores the damaged keratinocyte blocking function very quickly and forms the correct keratinous membrane like ceramide made in vivo.

(Test II). Comparison of the Effects of Cerebroside Extracted from Soybean Embryo on the Skin for Long Term

Ten hairless rats were applied to both sides of the rats, and the lotion, Example 1, and Comparative Example 1 were applied twice a day, and the transdermal moisture evaporation amount was examined every week.

TABLE 2

* Percentages in parentheses indicate the rate of decrease of transdermal moisture evaporation relative to the control

* The standard deviations are all within 0.3.

Comparative Example 1 showed a decrease in transdermal moisture evaporation of 3.2% and 5.3% after 1 and 2 weeks, respectively, compared to the control group. On the other hand, as in Example 1 of the present invention, when applying a lotion containing 0.5% of cerebroside extracted from soybean embryos, 10.0% and 20.2% were reduced, indicating that the effect was three times better than the synthesized pseudoceramide. have. In addition, when using the control group and Comparative Example 1, the feeling at the time of application was much better, the skin was very smooth, and the aesthetic appearance was greatly improved.

Claims (2)

Soybean embryos were extracted with isopropyl alcohol and extracted with a mixed solution of methanol and chloroform. The combined extracts were dried under reduced pressure, and the polar lipids were separated using ethanol and hexane and dried. Separating the lower layer by adding chloroform / water, and then dried to dry the acetone fraction in silica gel macron chromatography to extract cerebroside (cerebroside). Soybean embryos were extracted with isopropyl alcohol and extracted with a mixed solution of methanol and chloroform. The combined extracts were dried under reduced pressure and the polar lipids were separated using ethanol and hexane, and then dried in a caustic soda / methanol solution, followed by acetic acid / chloroform. A skin moisturizer containing cerebroside extracted by adding water and separating the lower layer, followed by drying to dry the acetone fraction in silica gel macro chromatography.