JPS63201110A - Cosmetic containing fibronectin originated from placenta tissue - Google Patents

Abstract

PURPOSE:To provide a cosmetic containing fibronectin produced by treating a tissue fiber of human or bovine placenta obtained after normal delivery with a buffer liquid containing urea and subjecting the obtained crude liquid to treatments such as fractionation with ammonium sulfate, dialysis, etc. CONSTITUTION:Tissue fiber of human or bovine placenta obtained after normal delivery or bovine placenta of 3-4 month after conception is treated with a buffer liquid containing urea and the obtained crude liquid is subjected to one or more treatments selected from fractionation with 0.4 saturated ammonium sulfate, fractionation with 50V/V% ethanol and dialysis. The obtained placenta- originated fibronectin is used as a component of a cosmetic or drug. Fibronectin participates in the proliferation stage of fibroblast cell in dermatic tissue and can be applied as a restoration agent for damaged tissue or as a carcinostatic agent. The starting raw material may be placenta of man and cattle as well as of swine or sheep, etc., or dermatic tissue of domestic animals except for human.

Description

[Detailed Description of the Invention] [A] Object of the Invention The present invention is directed to the placenta of a human or cow after normal delivery, or the placenta of a cow that is three or four months old after pregnancy (hereinafter referred to as placenta).
The present invention relates to the application of fibronectin (hereinafter abbreviated as FN) obtained from tissue fibers to cosmetics.

'Industrial Application Field' The solution containing FN derived from placental tissue or its powder according to the present invention can be used in cosmetics or medicines.

2. Prior Art FN has attracted attention as one of the physiologically active substances in living organisms, and it is known that it is distributed in plasma, on fibroblast membranes, in the basement membrane of the epidermis, and the like.

Plasma-derived FN and cellular FN differ in molecular weight, and plasma-derived FN (abbreviated as PFN) has a molecular weight of 22 to 2 when reduced.
50,000 to form a dimer, and therefore the number is estimated to be about 460,000. On the other hand, cellular FN (abbreviated as CFN) is known to be a dimer or more.

However, immunochemical reactions indicate that they have the same or closely related effects. One of the roles of FN in vivo is to maintain adhesion or bonding between cells or tissues.
Or, it is considered to be a substance that acts as a blood clot, for example, a liquid substance in the space between collagen fibers in skin tissue* (such as mucopolysaccharide)
It is thought that it also has an important relationship with the maintenance of

In particular, in skin tissue, it is involved in the proliferation (growth) phase of fibroblasts, and in the therapeutic field, it has applications such as as a repair agent for tissue damage, as an anticancer agent, and in cosmetics. It is progressing.

As publications related to FN, there are, for example, the documents shown in Table 1.

I tried to research examples of extracting FN from placenta and applying it to cosmetics and medicine, but I couldn't find any publications specifically disclosing it.

In addition, 1 to 2 in Table 1 are recent research on FN,
Its application fields are explained in an easy-to-understand manner. Also, 3~
No. 10 shows published patent publications in Japan as well-known publications regarding specific FN-related applications.

r Table 1 J Main publications related to FN "Problems to be solved by the invention j" In applying FN to cosmetics or medicine, the present inventor sought the starting material from the placenta of humans or cows, The aim is to develop technical means that can be used in cosmetics and medicine.

C] Structure of the invention The present invention uses human or bovine placenta as a starting material,
After obtaining the crude solution with a buffer containing urea, ammonium sulfate 0.4 saturated fraction, ethanol 50 v/v
This cosmetic is characterized by containing placenta-derived FN obtained by a combination of one or more of % fractionation and dialysis.

Specifically, the details will be described below.

"Means for Solving Problems" (Experimental Example-1) It is naturally expected from the above-mentioned known publications that FN is located in the placenta. However, there is no precedent as to how much it contains and what means can be used to extract it efficiently.Therefore, the present inventors first investigated the known water-soluble placenta extract. obtain it,
Research began by quantifying the content of FN contained in these extracts. The results were as shown in Table 2.

In other words, placecenter extracts used for conventional medicinal purposes or chemical M uses can be broadly classified based on the extraction method or the main components they contain. Extracts containing water-soluble polymeric proteins such as phosphatase can be obtained, and can be classified into water-soluble peptides produced by hydrolysis, extracts mainly containing amino Nl, and other extracts containing fat-soluble components. However, among these extracts, there were some that were mainly extracted using low-temperature extraction methods, in which very small amounts of FN were confirmed. However, the extracted FN was not primarily CFN, but was presumed to be PFN in the blood, which was transferred when blood removal and washing were insufficient during pretreatment to obtain the extract. In other words, there is no established method for extracting FN as one of the main substances in placenta extracts using conventional methods.Therefore, for effective use of placenta, extracts with high FN content or placenta-derived Application of FN was desired.

"Table 2" Based on the results of the FN content (Table 2) of conventional placecenter extracts, the present inventors determined that FN in placecenter extracts
I have added a few considerations regarding the extraction conditions.

In other words, what can be considered from Table 2 is that the FN contained in placenta would be degraded by the hydrolysis method, so it is thought that FN was not confirmed. Originally, some water-soluble extracts were identified after being treated with ether, acetone, butanol, etc., but the FN in this case is thought to be blood-derived PFN. On the other hand, FN was not confirmed in the fat-soluble extract. Considering the above points, it seems that the FN in the placenta is more likely to be CFN derived from assemblages than plasma-derived PFN. As a result, we have come to the conclusion that 1f1 may still remain in the placecenter tissue residue after low-temperature extraction, unaffected by organic solvents such as ether and n-butanol. Ta.

(Experimental Example-2) Based on the experimental results regarding the FN content of the above-mentioned known placenta extracts, the present inventors obtained extract residues obtained from conventional known placenta extracts by a low-temperature extraction method,
In other words, it is assumed that FN mostly remains in the placental tissue fibers in an adhesive or bound state without being eluted, and we conducted an experiment to establish new FN extraction conditions. started. In other words, it was considered that the conventional well-known method of extracting placenta extract was completely useless as a means for obtaining FN.

Considering FN's previously known role, action, site of existence, II function, etc., the most necessary condition is that it is strongly adhered to or bonded to the tissue of the placenta. It was considered.

Therefore, it was thought that finding an eluent or an eluent for separating this would be the first barrier to solving the object of the present invention. Of course, at this time, hydrolysis and high heat treatment are
This is not a good idea as it will lead to the decomposition of N.

Therefore, we developed an optimal eluent for placental tissue. When extracting FN, it is known to use guanidine hydrochloride or urea as known means.

For example, CFN has been obtained using the LM technique based on avian embryonic amblasts. In this case, purification is performed by chromatography using a Sepharose CL-4B column and then treated with a gelatin Sepharose column (Reference source: Yamada
, M. et al.: Ann, NY, Acad.

Sci., 312.256.1978) - On the other hand, in a method for obtaining PFN from human plasma, a protease inhibitor is added to non-coagulated plasma in advance to suppress the decomposition of PFN, and then it is mixed with gelatin at room temperature.・After passing through an affinity column and washing with neutral physiological saline,
By eluting with 4Mm and removing urea from the eluted fraction by dialysis, pure Habo PFN can be extracted from plasma. Hit 170■
(Reference source: Hayashi,
M et al.: J. Biol. Chew, 257.5263
．． 1982).

Based on this precedent, the present inventor attempted to extract FN from placenta.

As a result, the elution of FN to placental tissue fibers (
It has been found that using urea for elution is one method. However, the difference in starting materials complicates the yield and treatment operating conditions, and several improvements are required for industrial large-scale production.

In other words, it has been confirmed that FN is also contained in the pickles left after low-temperature extraction in 1 am of placenta tissue;
Unlike FN, it is insoluble in physiological saline and the like. In other words, C
In order to efficiently recover FN, one of the conditions was considered to be how to use a dissolving solution that can replace physiological saline in combination with urea.

Furthermore, although the above-mentioned two known methods are suitable for use as means for small-scale extraction and purification at the laboratory level and as various research reagents, they are not suitable for immediate use in pharmaceuticals or cosmetics. In other words, using a gelatin affinity column is one of the best methods to obtain a small amount of FN during purification, but it is not suitable for industrial mass production. In some cases, a simple alternative is needed. For this purpose, what kind of eluate should be prepared in the pretreatment?

This was considered an important condition for mass production. If this condition can be established, the result will be that the finally obtained FN can be supplied to pharmaceuticals or cosmetics at low cost.
I thought we could make great progress. Therefore, the present inventor has repeatedly conducted FN extraction experiments to find an optimal dissolving solution based on the placecenter structure, and has continued to make comparisons based on the results.

Finally, for the placenta tissue, 0.15
It has been found that by using a solution prepared by adding 5M urea to M sodium chloride/0.06M Tris/hydrochloric acid buffer, FN in the placenter can be recovered as much as possible.

Table 3 shows the concentration of the solution obtained by adding urea to the buffer solution and the concentration contained in the crude solution (mother solution) eluted by the solution. It shows the relationship between the amount of FN.

In Table 3, the starting material indicated as cow indicates the amount of FN obtained from the placenta of the cow after normal delivery. In addition, the value of the placenta of a cow at 3 to 4 months of pregnancy is about 2 to 3 times the value of the placenta of a cow after normal delivery.

(Experiment Example-3) Having passed the barrier for eluting FN from the placenter, the inventor next determined that FN could be eluted by any simple means.
I tried to solve this problem by asking if it was possible to purify and transfer it.

Table 4 shows the results. That is, the effect brought about by the pretreatment solubilizer (urine JIF solution) was that the combination of the ammonium sulfate fraction and the ethanol fraction facilitated the purification of FN. In other words, F from the plastic center
In order to efficiently extract N and make it possible to immediately use the obtained FN in cosmetics or medicine, the most important method was to elute FN from the tissue. By repeating this operation three or more times using a solution containing 5M urea in a 0.06M 1-Lys-Salt buffer, FN in the placenta can be recovered with a good yield. Based on this, if ammonium sulfate fractionation is performed, 0.
It was found that nearly 90% of the solution could be easily recovered in a solution with less than 4 saturation.

However, the characteristic organ organs (fishy odor) that the crude liquid containing FN exhibits still remains. For this, next, by fractionating a 50 v/v% hot solution of ethanol,
It turns out that it can be completely removed. It was also found that the recovery rate of FN is about 90%, which is good when the concentrations of ethanol are 30 and 50 v/v%.

Table 4 shows the relationship between the concentrations of solutions in fractions.

'Table 3' Urea concentration and FN amount for placenter (
μg/ml) 'Table 4' Relationship between FNlt and odor in ammonium sulfate fraction and ethanol fraction (according to human placenta) The reason why we were able to carry out purification by means of this method was that the conditions for the combination of lysis solutions in the pretreatment were optimal, which led to good results. Table 4 shows the purification of FN obtained from human placenta, but the purification of FN obtained from cow placenta is
Most of the odor was removed by treatment with either the ammonium sulfate fraction or the ethanol fraction.

The problems and solutions for obtaining FN from placenta have been disclosed above, and now they will be described in more detail based on examples.

[Example-1] As a starting material, freshly frozen human or cow placenta after normal delivery is thawed under running water, washed with water, cut into pieces using a mince cutter, etc., and cut into pieces using a centrifuge. Blood removal,
Dehydrated group finely crushed material (hereinafter referred to as Affi delicate cut material for convenience) or a placecenter extract residue obtained mainly by water extraction based on this group A delicate cut material (hereinafter referred to as Affi delicate cut material)
(hereinafter referred to as B tissue residue for convenience), or water extraction based on Group A delicate cut tissue, using organic solvents such as n-butanol, ether, acetone, ethanol, ethyl acetate, penzole, hexane, etc. Based on the residue from which the placenta extract was extracted after processing (hereinafter referred to as C tissue residue for convenience) or fresh human or bovine placenta, without removing blood or washing with water, After cutting into pieces with a mince cutter and extracting the eluted water-soluble proteins with physiological saline etc., the remaining pickled material (hereinafter referred to as D tissue residue for convenience) is used to remove fat-soluble substances from the placenta. Either the removed or extracted residue (hereinafter referred to as E tissue residue for convenience) may be used. In other words, the starting material used in the present invention is a water-soluble active ingredient extracted from placenta in advance, or The residues left after extracting fat-soluble active ingredients, such as those shown in (a) to (c) in the diagram (Table 3), may also be used, or fresh placenta may be used. You may use any of the group delicate cuts (Table 3, 2).

Here, 5 kg of the grouped delicate cuts or tissue residues shown in A to E above were used, and 5 kg of them were mixed with 0.15 M sodium chloride, 0.06 M) solution, and hydrochloric acid buffer (pH 7.0-8.
0 ) (hereinafter referred to as buffer A for convenience) is 20
Add 1, stir occasionally and leave overnight at 4°C.
Next, this was centrifuged, 209 Buffer A was added to the obtained precipitate again, stirred in a homomixer at 4°C for 2 to 3 hours, and centrifuged. Collect the precipitate. By repeating this operation two or more times, substances such as proteins that are soluble in buffer A can be completely removed. In order to recover FN, the purity and efficiency of FN are the easiest. Repeating the above operation using buffer A:
This is a big point.

Next, buffer A containing twice the amount of 5M urea was added to the precipitate, and the mixture was extracted overnight at 4° C. using a blender, and then centrifuged to obtain an extract. At this time, for one of the precipitates, again use two units of 5MJIR.
Add Buffer A containing the element and perform the same operation to obtain an extract. Approximately 90% of FN can be recovered by repeating this extraction with buffer A containing 5MrR three times. The respective extracts obtained by repeating the above three times are mixed, and then ammonium sulfate is slowly added to dissolve at 0.4 saturation. At 4°C,
- After setting up at night, centrifuge to collect the precipitate.

The collected precipitate was added to buffer A containing 0.5M urea.
To this solution, add cold ethanol to 50 v/v%, stir slowly while cooling in an ice room, leave at 4°C overnight, and centrifuge 1a. machine to collect the precipitate. This precipitate is dispersed in purified water, centrifuged to remove ammonium sulfate or ethanol, further dispersed in purified water, and freeze-dried to obtain a yield of 40 to 90 g. Therefore, it can be used in medicine or cosmetics.

However, for immediate use in actual medicines or cosmetics,
It is desirable to formulate it as a solution, and in particular, in order to incorporate it into the formulation of external preparations such as cosmetics and use it immediately, buffer A4ff containing 0.5 to 1.0 Mm element is recommended.
1, and if necessary, add a preservative and filter to obtain an FN-containing liquid.
It maintains a stable solution state for a long period of time and is highly compatible with pharmaceuticals or cosmetics, and when preparing this solution, the FN content is 100 μg/ml or more, preferably 300 μg.
Preparation so as to contain about /- is one guideline for stability and further the effect of FN can be expected.

[Example 2] As the starting material, either the fine cut pieces of A-H or tissue residues, which are the same as in Example-Process, may be used, but here, B
The results are shown below for the results obtained using tissue residues. In other words, in the conventionally known extraction process for water-soluble placenta extracts, for example, n-butanol is was used to separate the aqueous layer from the aqueous layer, thereby removing precipitated tissue residue. Using 500 g of this removed material, add 22 kg of buffer A (pH 8,0) and perform the following operations. The procedure was carried out according to Example 1 to obtain an extract.

However, in the next ammonium sulfate fractionation, ammonium sulfate is gradually added and dissolved in the solution so that the saturation is 0.4, and the precipitate is collected, and this precipitate is added with a concentration of 0.5-1.0. M.R.
Add and dissolve Pamphur A containing 0.5 to 1
．． Dialysis was carried out in buffer A containing 0M urea, followed by addition of preservative and filtration until the FN content was 350.
A solution of jig/mQ was prepared.

This product can be used as it is as an agent for applying to the skin, such as external application, and has good stability.

Of course, cosmetics and topical i! ! It is also easy to incorporate into the formulation of rubbing agents.

[Example 3] Here, FN-containing 1B solution (filtered moe solution before addition of preservative) obtained by the operating means disclosed in Examples 1 and 2
Take 4p of the liquid, add the regenerated CM-cellulose or CM-Sephadex prepared in advance, stir at 4°C for about 10 to 20 minutes, and then filter by suction. A preservative was added to the obtained filtrate, which was then filtered again to prepare a FN-containing solution, and about 4i was obtained.

At this time, the FN content was adjusted to 280 μg/l1jI.

The placecenter-derived FN according to the present invention is CFN,
Insoluble in saline. Therefore, as a stable dissolving solution, 0.0.
The FN-containing solution was stabilized by using buffer A containing 5 to 1.0 M urea. This stabilization is
Now that the FN extraction conditions of the present invention have been established, this is an appropriate means that corresponds to them.

Regarding the FN-containing liquid derived from placenta, Examples = 1 to 3
Looking at the stability and other physical and chemical values obtained based on the following, for example, when compounding into skin cosmetics or skin external preparations, the following table (Table 5) shows that It is practical to prepare a FN-containing liquid that is above the numerical value.

In addition, since the amount of FN contained in bovine placenta is lower than that in human placenta, a concentration step is added to prepare it in each example. In other words, as long as it meets the standard values shown in Table 5, it can be added to any external preparation or cosmetic formulation.
FN rarely precipitates in lotions and the like.

"Table 5, Numerical values for maintaining stability of FN-containing liquids,"
When used in cosmetics and external preparations, the generation of specific organ odor is a drawback in formulation, but each FN-containing solution obtained according to the method shown in Examples 1 to 3 The odor (fishy smell) was evaluated as shown in the following table (Table 6), and it was confirmed that the treatments using ammonium sulfate fractionation, ethanol fractionation, and the use of ion exchangers were each effective. Ta.

Table 6 shows this, and at the same time, the amount of FN contained in 1 kg of the delicately cut pieces of placenter and tissue residues A to E used was determined.

[Example N-4] Regarding the application of FN for the purpose of external application to the skin, for example, see Table 1 Chuyo 4 (Publication Patent Publication: 1987-76007).
No.) has a disclosing fin. According to this, plasma-derived FN
(PFN) is used, o, o o o o t
It has been shown that by incorporating ~0.1% into cosmetics, it is expected to promote wound healing, hair growth, and whitening.

In order to confirm the action or effect of the FN-containing solution shown in the Examples, the present inventors tested three healthy male volunteers (adults) on both sides of the back, six places on the top and bottom, and a total of 12 places on both sides. , by placing an aluminum plate with a round hole of a diameter of 2 CIm made in advance on both sides of the back so that the light source can hit it, and then irradiating the light source from above with an ultraviolet lamp for a certain period of time, approximately 2 cm on both sides of the back. The number of days required for the erythema to disappear can be reduced by creating an erythema with a diameter of
I decided to compare it with the application of a control solution.

That is, an image of skin inflammation (erythema) caused by ultraviolet rays was created, and an attempt was made to determine the effectiveness of the FN-containing liquid based on its healing effect.

At that time, the relationship with the concentration (FN amount) in the FN-containing liquid was also determined. However, unlike plasma-derived FN (PFN), the FN-containing solution according to the present invention is insoluble in physiological saline. It was dissolved in a buffer A solution containing 0.5M urea and adjusted to pH 7.5, and the FN content contained in the solution was 10.25.40.50.10.
0.150.200.250.300.500μg/+
A solution prepared to give am was used.

Note that as control solutions, physiological saline and a buffer A solution containing 0.5M urea were used.

For erythema formation, use Toshiba fluorescent lamps FL-20SE and FL.
It was artificially created by connecting three -20BLB lights in parallel and irradiating from IQctn on the epidermis.

Table 7 shows the results, and it is clear that there is a difference in the disappearance of erythema between the control solution and the FN-containing solution, and the concentration of the FN-containing solution is 50 μg/1lj1 or more. There is a tendency for it to be particularly effective at a concentration of 100 to 300 μgod. However, 300μ
It is difficult to judge the superiority of FN containing g/ILQ and FN containing soo11 g/- due to the small number of examples.

In addition, physiological saline was used as a control solution, and 0, which was used for dissolving FN,
The 5M urea-containing buffer solution also contains the latter solution itself.
It seemed to have the effect of accelerating the disappearance of erythema.

In any case, when considering the F'N content as a guideline for formulation, it seemed advantageous to prepare the F'N content to about 100 to 300 μg/-.

In addition, for application, a method of applying localized cotton (cut into 2cm + square pieces) impregnated with each concentration of the test solution to 12 localized areas on the back and fixing it with tape from above was adopted, and when the cotton was dry. The treatment was performed twice, once in the morning and once in the evening, for one week by removing the tape.

On the other hand, erythema formation on the skin can be considered as a type of inflammation, but for example, known anti-inflammatory drugs include steroid drugs and non-steroid drugs.
The anti-inflammatory effect of N itself is weak compared to these drugs, but its combined use with these known anti-inflammatory drugs increases the effect. For example, adding a small amount of salicylic acid or the like to an FN-containing solution accelerates the disappearance of erythema. In that case, the FN content is 50 or more, 100 to 30
However, for carrageenan edema using animals, the FN content is 5011
g/ml! If the FN-containing liquid obtained in each example is within the standard values in Table 5, it can be applied or rubbed directly onto the skin, It can be incorporated into dosage forms such as milky lotions, creams, and lotions.

The formulation shown below is a clear aqueous solution (lotion type).
This is an example of a formulation that can be used for the purpose of alleviating erythema or stinging caused by sunlight on the skin, alleviating swelling, and preventing dryness.

[Formulation example-1] Skin inflammation relieving moisturizing protective liquid (%) (1)
FN-containing liquid (standard (!) shown in Table 5)...
・・・・・・・・・1～70(2) R3C liquid (
Cockscomb extract hyaluronic acid-containing solution)
...3-5 (3) Brasenando V (human placenter extract) ...3-5 (4) B
G (1,3-budelene glycol)...
・・・・・・・・・5～10(5) Sodium salicylate・・
...0.1-0.3(6)U, lJ Kid (solution containing 75% dextrin)...
2-3 (7) Make 100 with purified water.

1 Table 7 "Effects of application of FN-containing solution on ultraviolet erythema (inflammation) [C] Effects of the invention The present invention obtains FN from placenta and makes it possible to use it in cosmetics or medicines. is one of its major effects.

In the present invention, the starting material is limited to the placenta of humans or cows, but if the technical means shown in the Examples or Experimental Examples are adopted, the starting material is not limited to these organs, but can be used as the placenta of pigs, sheep, etc. In addition, it can be extracted from the skin tissue and other tissues of other livestock animals other than humans, and it has great effects.

According to the present invention, although it is not applicable to the extraction of PFN derived from plasma, it can be fully utilized for the extraction of CFN derived from other cells, and the processing operation is easy and the production efficiency is high. In Example 1, one of the best methods was to use a solution of 5M diluted buffer A and stir it at 4°C overnight, but ξ et al. Without being particular about this, a method of soaking overnight at 4°C and extracting with a homomixer the next day will also satisfactorily achieve the purpose.

The FN-containing liquid obtained by the present invention can be used immediately as it is as a cosmetic or external preparation, and can also be optionally incorporated into their formulations.

Furthermore, the FN-containing liquid according to the present invention may contain moisturizing agents such as mufu polysaccharides such as hyaluronic acid, water-soluble peptides obtained by hydrolysis of silk (fibroin), keratin, collagen, elastin, etc., which are known as skin moisturizing agents, or plastics. Extracts extracted from the internal organs of various animals (e.g., blood, brain, liver, intestines, tiles, skin, etc. of cows, pigs, sheep, etc.), or fibers extracted from the brain, skin, etc. It can also be used in combination with a sprout proliferation substance.

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption of a solution containing FN derived from placer tissue according to the present invention diluted 50 times with purified water. (However, preservatives have been removed.)

Claims (1)

[Claims] (1) After obtaining a crude solution from human placenta after normal delivery or cow placenta tissue fibers with a buffer containing urea, ammonium sulfate 0.4 saturated fraction and ethanol 50%
A cosmetic product characterized by containing placenta-derived fibronectin obtained by a combination of one or more of v/v% fractionation and dialysis.