JPH11279072A - Skin preparation for external use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a skin preparation for external use excellent in improvement of roughened skin, skin moisture-retaining effect and aging-preventing and improving effect of skin by making the preparation include a protein containing lectin of the genus Eucheuma or genus Kappaphycus as an active ingredient. SOLUTION: This skin preparation for external use contains a protein containing lectin of the genus Eucheuma or genus Kappaphycus obtained from seaweed [e.g. Eucheuma serra] belonging to the genus Eucheuma or the genus Kappaphycus as an active ingredient. A lectin having about 25,000 molecular weight by gel permeation method, about 29,000 molecular weight by SDS electrophoresis, p14.5 to 5.7 isoelectric point measured by polyacrylamide gel isoelectric point electrophoresis, not having sugar chain and having an amino acid sequence in N terminal region represented by the formula Gly-Arg-Tyr-Thr-Val-X-Asn-Gln- Trp-gly (X is Gln or Lys) is preferably used as the lectin of the genus Eucheuma or genus Kappaphycus and formulating amount of the lection is preferably used in an amount of 0.001-5 wt.% based on the objective preparation for external use.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for improving skin roughness,
The present invention relates to an external preparation for skin having excellent skin moisturizing effects and skin aging prevention / improvement effects.

[0002]

2. Description of the Related Art In recent years,
Skin troubles are increasing due to decreased skin barrier function and allergic diseases due to drying, improving rough skin,
There is a strong demand for an external preparation having an excellent skin moisturizing effect. Therefore, for dryness and allergic diseases of the skin,
Many proposals have been made to use anti-inflammatory agents such as glycyrrhetinic acid and various moisturizing agents for the purpose of enhancing the barrier function of the skin and keeping the skin healthy, but they cannot be said to be sufficient in terms of effect. Further, as the skin ages, the skin becomes less perceived as having reduced flexibility and elasticity, increased wrinkles, and is dry and rough without rough skin. These factors that cause skin aging include internal factors of cells, that is, genetic factors, and other factors such as nutrition, endocrine, stress, and environmental factors. In particular, skin directly contacts the outside world. Yes,
It is known that it is strongly affected by ultraviolet rays, drying, cold and the like. For the purpose of improving such aging symptoms, drugs such as α-tocopherol, α-hydroxy acid, and retinol have been proposed, but are not sufficient as antiaging effects,
There are problems such as skin irritation.

[0003] Accordingly, an object of the present invention is to provide an external preparation for skin having the above-mentioned problems, which is excellent in improving skin roughness, moisturizing the skin, and preventing and improving skin aging.

[0004]

Means for Solving the Problems and Embodiments of the Invention The present inventors have conducted intensive studies in order to achieve the above object, and as a result, have found that proteins containing Kirinsai lectin, especially W
The lectin as described in O95 / 18149 has been found to provide excellent effects on improving skin roughness, moisturizing the skin, preventing and improving skin aging, and completed the present invention.

Now, the present invention will be described in further detail. The external preparation for skin of the present invention contains a protein containing a Ginseng lectin as an active ingredient.

[0006] The protein containing the lectin of the genus Giraffe is a genus of the genus Giraffe (Euchema or Kappa).
aphycus), and as the seaweeds belonging to the genus Kirinsai, there are E. serra and Eucumacotney (E. c).
o. totonii), E. gela
tinae) and E. amakurinsai (E.
saensis), E.C. denticulatum,
E. FIG. muricatum, E.C. cupresoiseu
m, E.C. spinosum, E .; arnoldii,
E. FIG. alvarezii, E. et al. E such as stratum
ucheuma and K. procrusteanu
m, K. cottonii, K .; striatum,
K. alvarezii, K. et al. arnoldii et al.
Seaweed belonging to the genus appaphycus is exemplified. Judging from the lectin content and availability, etc., it is preferable to use E. serra, E. cottonii, E. gelatinae, and E. akumasaensis. , E .; dentic
uratum, E .; spinosum, E .; alvar
ezii, K .; procrusteanum and K. et al. c
ottonii, K .; alvarezii, particularly preferred are E. serra, E. cottonii, E. gelatinae and E. amakusaensis.

[0007] As the protein containing the genus Kirinsai lectin, seaweed belonging to the genus Kirinsai is used as a raw material, extracted with an extraction solvent selected from a buffer solution and a solvent-water mixture, and then subjected to solvent precipitation and dialysis from the extract. Lectin-containing proteins can be formulated as crude lectins. The crude lectin can be blended as a purified lectin separated and purified by gel filtration and, if necessary, fractionated by ion exchange chromatography.

In this case, it is preferable to use, as the lectin of the genus Kirinsai, one having at least one of the following properties (a) to (c) and having the following properties (d) to (g). (A) The molecular weight by gel filtration is about 25,000. (B) molecular weight of about 29,000 by SDS electrophoresis
It is. (C) When the isoelectric point is measured by a polyacrylamide gel isoelectric focusing method, it is in the range of pI 4.5 to pI 5.7, preferably pI4 by the polyacrylamide gel isoelectric focusing method. .75, pI 4.95, pI 5.0
5, at least one isoelectric point selected from pI 5.20 and pI 5.50. (D) It has no sugar chain. (E) The amino acid sequence of the N-terminal region is Gly-Arg-T
yr-Thr-Val-X-Asn-Gln-Trp-
Gly (X is Gln or Lys). (F) It has a specific agglutinating activity on trypsin-treated rabbit erythrocytes, sheep erythrocytes and trypsin-treated sheep erythrocytes. (G) Shows binding specificity for fetin, asialofetin, thyroglobulin, and yeast mannan.

Such lectins are described, for example, in WO 95/18
149 can be obtained.

The amount of these lectins varies depending on the form of the product, the frequency of use, and the physiological activity. Usually, one or a mixture of two or more of the lectins is used in a total amount of 0.1% of the total external preparation.
The range of 001 to 5% by weight is appropriate.

The external preparation for skin of the present invention includes, in addition to the above-mentioned essential components, compounding agents usually used for external preparation for skin, for example,
Contains surfactants, oils, alcohols, humectants, thickeners, preservatives, antioxidants, chelating agents, pH adjusters, fragrances, pigments, UV absorbers / scatterers, vitamins, amino acids, water, etc. can do. The optional components are not limited to these.

The external preparation for skin of the present invention can be prepared in the form of a lotion, emulsion, cream, ointment and the like, and is applied as a cosmetic according to the purpose of use. In the present invention, the skin includes the scalp.

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in more detail with reference to embodiments. However, the present invention is not limited to the following examples. The compositions of the skin external preparations in Examples and Comparative Examples are shown by weight%. In the composition of each example, the total amount of all components is 100%.

[Production Example] The desired lectin was obtained according to the lectin production method described in WO95 / 18149. That is, the horned giraffe, the eucouma cotney, the katamen giraffe and the amasaki giraffe were collected, washed well with seawater, freeze-dried, and pulverized with a pulverizer to obtain a seaweed powder.

Each seaweed powder has a 10-fold amount of 20 to 2
Add a 5% aqueous ethanol solution and extract twice at low temperature.
An extract containing lectin was obtained. Next, in order to recover lectins from these extracts, an appropriate amount of cold ethanol was added to the extracts to a final concentration of 80% or more, and proteins containing lectins were precipitated as precipitates. The obtained precipitate was suspended in a 50 mM phosphate buffer (pH 7.2), and the buffer was dialyzed to remove ethanol to obtain crude lectin solutions.

Next, the crude lectin solution was subjected to gel filtration chromatography, and lectin ES0 was obtained from the crude solution derived from the rhododendron rhizome, lectin EC0 was obtained from the crude solution derived from Eucoma cotney, lectin EG0 was obtained from the crude solution derived from the rhododendron rhinoceros, and the crude solution was derived from the crude solution derived from the rhododendron. Lectin EA0 was each purified.

Further, the purified lectin was subjected to DEAE ion-exchange chromatography, and two fractions of lectins ES0 to ES1 and ES2 derived from Rhododendron were extracted from lectins EC0 to EC1 and EC1 derived from Eucoma cotney.
Two fractions of C2 were used as the lectin E derived from
The three fractions of G0 to EG1, EG2 and EG3 were further divided into lectins EA0 to EA1 from A.
Three fractions, EA2 and EA3, were obtained.

The lectin obtained by the above method has the following (a) to (4) regardless of the type of seaweed used as a starting material.
(G). (A) A molecular weight of about 25.0 as determined by gel filtration
00 or (b) a molecular weight of about 29,000 as measured by SDS electrophoresis or (c) an isoelectric focusing method,
shows one or more isoelectric points in the range of pI4.5 to pI5.7, (d) has no sugar chain, and (e) the amino acid sequence of the N-terminal region is Gly-Arg-Tyr- Th
r-Val-X-Asn-Gln-Trp-Gly (X
Is Gln or Lys), (f) has a specific agglutinating activity on trypsin-treated rabbit erythrocytes, sheep erythrocytes and trypsin-treated sheep erythrocytes, and (g) has a function on fetin, asialofetin, thyroglobulin and yeast mannan. The binding specificity is shown.

As a result of isoelectric point measurement by polyacrylamide gel isoelectric point electrophoresis, it was confirmed that it has an isoelectric point shown in Table 1 below.

[0020]

[Table 1]

Further, it was confirmed that each lectin has blastogenic activity on mouse and human lymphocytes, respectively.

In the following examples, as shown above, 20 powders of the seaweed powders of the rhododendron, the eucouma cotney, the phalaenopsis and the
Extracted with ~ 25% ethanol aqueous solution, added cold ethanol to the extract to a final concentration of 80% or more, precipitated lectin-containing protein as a precipitate, and dialyzed the obtained precipitate against pure water Was used as a crude lectin.

In the following examples, the effects of improving rough skin and moisturizing the skin were examined by the following methods. Acetone: ether = 1: 1 for the inner forearm of 30 male and female panelists
The mixture was degreased with a mixed solvent to prepare rough skin. Thereafter, each sample was applied twice a day, and three days later, the following two evaluations were performed. (1) Improvement effect of rough skin; evaluation of surface shape observation The symptoms of rough skin were observed by direct visual observation and replica method. The symptoms of rough skin referred to here are horny layer peeling,
It shows erythema and edema, skin sulcus and skin ridges are not clear, and skin texture is not uniform.

The rough skin improving effect was rated and evaluated according to the following criteria. (Score) Excellent Efficacy: The symptoms of rough skin disappeared. Efficacy: The symptoms of rough skin weakened. Eight subjects who evaluated significant, effective, and somewhat effective
0% or more ○: 5 subjects who evaluated excellent, effective, and somewhat effective
0% or more and less than 80% △: 3 subjects who evaluated excellent, effective and slightly effective
0% or more and less than 50% ×: 3 subjects who evaluated significant, effective and slightly effective
Less than 0%

(2) Skin moisturizing effect; measurement of skin keratin water content The skin keratin water content at the evaluation site was measured using an impedance meter.

The skin moisturizing effect was rated and evaluated according to the following criteria. Here, the base is a cream containing neither lectin nor comparative components. (Score) Excellent: The measured value is 200% or more compared to the base application site. Effective: The measured value is 150% or more and less than 200% compared to the base application site. Is 110% or more and less than 150% Ineffective: The measured value is less than 110% as compared with the base application site (Evaluation) ：: 8 subjects who evaluated excellent, effective, and slightly effective
0% or more ○: 5 subjects who evaluated excellent, effective, and somewhat effective
0% or more and less than 80% △: 3 subjects who evaluated excellent, effective and slightly effective
0% or more and less than 50% ×: 3 subjects who evaluated significant, effective and slightly effective
Less than 0%

Further, the effect of preventing and improving skin aging was examined by the following method. (3) Evaluation of skin aging prevention / improvement effect 70 male and female panelists (35 to 60 years old) were grouped into 10 persons, and the face of each group was used three times a day blindly for three consecutive months. Was. After the evaluation was completed, the panelists evaluated the skin condition as “improved”, “slightly improved”, and “no change” for “effects on fine wrinkles”, “effect on skin texture”, and “effect on gloss and beam”, respectively. The evaluation was made in three stages.

Examples 1 to 20, Comparative Examples 1 to 5
A cream having the composition shown in No. 3 was produced. The production method is oil,
An oil phase composed of an activator and an aqueous phase composed of purified water were mixed and dissolved at 70 ° C. respectively, and the oil phase was gradually added while stirring the aqueous phase to carry out preliminary emulsification. Thereafter, an emulsifier (homomixer) treatment was performed to homogenize the emulsified particles, and after degassing and cooling, lectins derived from Kirinsai were added and dispersed at room temperature to finish.

Here, the effects of improving rough skin and improving moisture retention were evaluated by the tests (1) and (2) described above.

[0030]

[Table 2]

[0031]

[Table 3]

Examples 21 to 26, Comparative Example 6 Creams having the compositions shown in Table 4 were produced. The production method is to convert an oil phase, an oil phase composed of an activator, and an aqueous phase composed of purified water into 70 parts each.
The mixture was dissolved at a temperature of ° C., and the oil phase was gradually added while stirring the aqueous phase to carry out preliminary emulsification. After this, an emulsifier (homomixer)
After the treatment, the emulsified particles were made uniform, and after degassing and cooling, lectins derived from Kirinsai were added and dispersed at room temperature to finish.

Here, the effect of preventing and improving aging of the skin was evaluated by the test of the above (3). The results are shown in Table 4 by the number of panelists indicating each evaluation.

[0034]

[Table 4]

Hereinafter, examples of the formulation will be further described.

[0036]

[Table 5]

[0037]

[Table 6]

[0038]

[Table 7]

[0039]

[Table 8]

[0040]

[Table 9]

[0041]

[Table 10]

[0042]

[Table 11]

[0043]

[Table 12]

[0044]

[Table 13]

[0045]

[Table 14]

[0046]

[Table 15]

[0047]

[Table 16]

[0048]

[Table 17]

[0049]

[Table 18]

[0050]

[Table 19]

[0051]

[Table 20]

[0052]

[Table 21]

[0053]

[Table 22]

[0054]

[Table 23]

[0055]

The external preparation for skin of the present invention is excellent in improving rough skin, moisturizing skin, and preventing and improving skin aging.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 38/36 AGZ A61K 37/46 AGZ

Claims (2)

[Claims] 1. An external preparation for skin containing a protein containing a ginseng lectin. 2. The ginseng lectin according to the following (a)-
2. The external preparation for skin according to claim 1, which has at least one property of (c) and has the following properties (d) to (g). (A) The molecular weight by gel filtration is about 25,000. (B) molecular weight of about 29,000 by SDS electrophoresis
It is. (C) The isoelectric point obtained by isoelectric focusing is pI 4.5 to pI.
I5.7. (D) It has no sugar chain. (E) The amino acid sequence of the N-terminal region is Gly-Arg-T
yr-Thr-Val-X-Asn-Gln-Trp-
Gly (X is Gln or Lys). (F) It has a specific agglutinating activity on trypsin-treated rabbit erythrocytes, sheep erythrocytes and trypsin-treated sheep erythrocytes. (G) Shows binding specificity for fetin, asialofetin, thyroglobulin, and yeast mannan.