JPS63152322A - Solution containing fibronectin derived from placenta tissue - Google Patents

Abstract

PURPOSE:To obtain the titled solution useful as a drug and a cosmetic, by pretreating human or bovine placenta tissue fibrin by a specific method to give fibronectin and extracting the fibronectin by a specific method. CONSTITUTION:Human placenta after normal delivery or a bovine placenta tissue fibrin is immersed in a solution obtained by adding 5M urea to a buffer solution of 0.15M sodium chloride.0.06M tris hydrochloric acid and pretreated. The supernatant liquid thereof is used as a crude solution and the solution is treated by one method of saturated fractionation of <=0.4 aluminum sulfate, 50v/v% fractionation of ethanol and dialysis or a combination thereof to give fibronectin (hereinafter referred to FN). FN is dissolved in a buffer solution of 0.15 sodium chloride.0.06M tris hydrochloric acid containing 0.5-1.0M urea to give a solution containing FN derived from placenta tissue. Approximately 90% FN in placenta can be recovered by repeating the pretreatment operation three times or more.

Description

[Detailed Description of the Invention] [A] Object of the Invention The present invention relates to the placenta of a human or cow after normal delivery, or the placenta of a cow 3 to 4 months after pregnancy (hereinafter referred to as placenta).
The present invention relates to a solution containing a high amount of fibronectin (hereinafter abbreviated as FN) obtained from tissue fibrous material.

"Industrial Field of Application J The placental tissue-derived FN-containing solution or powder thereof according to the present invention can be used in cosmetics or medicines.

"Prior Art" FN has attracted attention as one of the physiologically active substances in living organisms, and is known to be 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 control substances, for example, liquid substances in the spaces between collagen fibers in skin tissue (such as mucopolysaccharides)
It is thought that it also has an important relationship with the maintenance of

In particular, in skin tissue, it is involved in the growth phase of fibroblasts, and in the therapeutic field, it has been used as a repair agent for tissue damage, as an anticancer agent, and as a cosmetic. It is progressing.

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

On the other hand, although we have investigated examples of extracting FN from placenta and applying it to chemical additives and medicines, we have not found any publications that specifically disclose this.

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

rTable 1, Main publications related to FN "Problems to be solved by the invention" 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.

[Mouth] Structure of the Invention The present invention is based on the placenta of humans or cows after normal delivery, or the placenta of cows up to 3 to 4 months after pregnancy.
．． The supernatant after immersion in a solution of 5MR element in a 15M sodium chloride/0.06M Tris/hydrochloric acid buffer solution was used as the crude solution, and then the saturated fraction of ammonium sulfate of 0.4 or less and 50% of ethanol were added. The present invention relates to a FN-containing solution characterized in that it is obtained by any one of v/v% fractionation, dialysis, or a combination of one or more treatments.

Specifically, the details will be described below.

Means for Solving Problem 1 4 (Experimental Example-1) The presence of FN in the placenta is naturally expected from the above-mentioned known publications. However, there is no precedent as to how much it should contain or what means should be used to achieve efficient extraction. Therefore, the present inventors first obtained a known water-soluble placenta extract,
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 or cosmetic purposes can be roughly divided into extraction methods or main components contained.When the low-temperature extraction method is used as the main ingredient, albumin, globulin, and alkaline are extracted. An extract containing water-soluble polymeric proteins such as phosphatase can be obtained. It can be divided into water-soluble peptides produced by hydrolysis, extracts mainly containing amino acids, and extracts containing other fat-soluble components. Among these extracts, extracts mainly produced by low-temperature extraction methods include In some cases, a very small amount of FN was confirmed. However, the extracted F
N is not primarily CFN, but migrates when blood removal and washing are insufficient during pretreatment to obtain the extract.
It was presumed to be PFN in the blood. In other words, in conventional placenta extracts, there is no established method for extracting FN as one of the main substances.Therefore, for effective use of placenta, extracts with high FN content or
Application of FN derived from placenta has been desired.

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

That is, what can be considered from Table 2 is that FN contained in placecenters is itself decomposed by the hydrolysis method. Therefore, it is considered that FN was not confirmed. On the other hand, some water-soluble extracts have been confirmed after being treated with ether, acetone, n-butanol, etc. based on the low-temperature extraction method, but in this case, FN is It was thought that the origin of PFN. On the other hand, FN could not be confirmed in the fat-soluble extract. Considering the above points, the FN in the placenter is more CFN derived from tissue cells than the PFN derived from plasma, and as a result, the FN in the placenter tissue residue after low-temperature extraction is It has been speculated that organic solvents such as ether, n-butanol, etc. may still remain unaffected.

=8- (Experimental Example-2) Based on the above-mentioned experimental results regarding the FN content of the known placenta extract, the present inventors investigated the extract obtained from the conventional known placenta extract by a low-temperature extraction method. It is assumed that FN remains in the residue, that is, the placecenter tissue fibers, in an adhesive or bound state without being eluted, and therefore we decided to set new FN extraction conditions. We have started experiments for this purpose. 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 the role, action, location, function, etc. of FN that has been known so far, the most necessary condition is that it be strongly adhered to or bonded to the tissue of the placenta. it was thought.

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 IM urea based on avian embryonic fibroblast cells. In this case, purification was performed by chromatography using a Sepharose CL-4B column and further treatment with a gelatin Sepharose column (Reference source: Yamada
, M. et al.: Ann, NY, Acad.

Sci., 312.256.1978).

On the other hand, in the method of obtaining PFN based on human plasma, a protease inhibitor is added to non-coagulated plasma in advance to obtain PFN.
By suppressing the decomposition of N, passing it through a gelatin affinity column at room temperature, washing it with neutral physiological saline, eluating it with 4M urea, and removing urea from the eluted fraction by dialysis. Habo Pure PFN is 1st in plasma! Hit 170
(Reference location: )Iayash
i, M et al.: J, Bio1. Chem,, 257.52
63.1982).

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 was confirmed that FN is also contained in the residue after low-temperature extraction in placental tissue fibers, but PF
Unlike N, it is insoluble in physiological saline and the like. In other words, C.F.
In order to efficiently recover N, one of the conditions was considered to be how to use a 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. is inefficient. In other words, using a gelatin affinity column is one of the best ways to obtain a small amount of FN during purification, but when the purpose is industrial mass production, there is an alternative to this. A simple method 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 is established, the result will be that the finally obtained FN can be immediately 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
M sodium chloride/0.06M Tris ψ hydrochloric acid buffer,
It was found that by using a solution to which 5M urea was added, FN in the placenter could be recovered as much as possible.

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

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 placenter of a cow during 3 to 4 months of pregnancy is approximately 2 to 3 times the value of the placenter of the 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 refine it.

Table 4 shows the results. In other words, the effect brought about by the pretreatment solubilizer (urea solution) was that the combination of ammonium sulfate fraction and ethanol fraction facilitated the purification of FN.In other words, FN could be efficiently extracted from the placenta. , Immediately apply the obtained FN to cosmetics or medicines,
In order to make it usable, the most important method was to elute FN from the tissue. By repeating this operation three or more times, FN in the placenter can be recovered with a good yield. Based on this, if ammonium sulfate fractionation is performed, a solution with a saturation level of 0.4 or less can be obtained. , it was found that nearly 90% of the amount was easily recovered.

However, the characteristic organ organs (fishy odor) that the crude liquid containing FN exhibits still remains. For this, next, by fractionation with a 50 v/v% solution of ethanol,
It was found that the stains were 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.

1 Table 3 "Urea concentration and FN amount for placenter (
μg/m11) "Table 4, Relationship between FN amount and odor in ammonium sulfate fraction and ethanol fraction (according to human placenta)" The reason why we were able to perform purification using such a method was because the combination conditions of the dissolving solution in the pretreatment were the best.Table 4 shows that human placenta The purification of FN obtained from bovine placenta has been shown in this paper.
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 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 group A finely cut material for convenience), or placecenter extract residue obtained mainly by water extraction based on this group A finely cut material (hereinafter referred to as group A delicately cut material) For convenience, it is referred to as Bi-woven pickles), or based on water extraction based on Group A delicate cuts, n-butanol, ether, acetone, ethanol, ethyl acetate, penzol,
Based on the residue obtained by extracting the placenta extract after treatment with an organic solvent such as hexane (hereinafter referred to as C tissue residue for convenience), or fresh human or bovine placenta, blood is removed and washed with water. After cutting into pieces with a mincing cutter without processing, the eluted water-soluble protein is extracted with physiological saline etc., and the residue is left (hereinafter referred to as D tissue residue for convenience). Any residue from which soluble substances have been removed or extracted (hereinafter referred to as E tissue residue for convenience) may be used. In other words, the starting material used in the present invention is the residue (I ) to (c), etc. may be used, or any of the delicate cut pieces of fresh plastic center (2 in Table 3) may be used.

Here, 5 kg of the grouped delicate cuts or tissue residues shown in A-E above are used, and 0.15 M sodium chloride/0.06 M Tris/hydrochloric acid buffer (pH 7.0 to 80) is added.
(hereinafter referred to as buffer A for convenience) was added to 20p,
Stir occasionally and leave at 4°C overnight. Next, this was centrifuged, and the resulting precipitate was added again to 20μ
Add Buffer A and incubate in a homomixer at 4℃ for 2~
Stir for 3 hours and centrifuge to remove the precipitate. By repeating this operation two or more times, the buffer A
Substances such as proteins that are soluble in water can be removed. Finally FN
In order to recover FN with high purity and efficiency, it is important to repeat the above operation using buffer A.

Next, buffer A containing twice the amount of 5M urea is added to the precipitate, and the mixture is stirred and extracted overnight at 4° C. using a blender, and then centrifuged to obtain an extract. On this occasion,
To one of the precipitates, twice the amount of buffer A containing 5M urea is added again, and the same operation is performed to obtain an extract. This extraction with buffer A containing 5M urea
Approximately 90% of FN is recovered by repeating the process twice.

The respective extracts obtained by repeating the above three times are mixed, and then ammonium sulfate is slowly added to dissolve the mixture to saturation. After incubating at 4°C for one night, centrifuge and collect the precipitate.

The recovered 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. 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 then freeze-dried to obtain a yield of 40 to 90 g, which can be immediately used as a pharmaceutical product. Or it can be used in cosmetics.

However, for immediate use in actual medicines or cosmetics,
It is desirable to formulate a solution 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 M urea 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, making it highly compatible with pharmaceuticals and cosmetics. In addition, when preparing this solution, the FN content should be 100 μg/m0 or more, preferably 30 μg/m0 or more.
Preparing to contain about 0 μg/mQ is one guideline for stability and for expecting the effect of FN.

[Example-2] As the starting material, either the fine cut pieces of A-E or the tissue residues used in Example-1 may be used, but here, B
The results are shown below using tissue residues. In other words, in the conventional extraction process for water-soluble placenta extract, when extracting a finely cut tissue with water, for example, n-butanol is used to separate it from a water reservoir, and the tissue residue that precipitates by itself is removed. Using 500 g of this removed material, add 212,710 g of buffer A (pH 8,0), and perform the following operations according to Example 1 to obtain an extract.

However, in the next ammonium sulfate fractionation, based on a solution in which ammonium sulfate was gradually added and dissolved so that it became 04 saturated,
The precipitate was collected, and buffer A containing 0.5-1.0M urea was added to the precipitate to dissolve it.
Dialysis was performed in buffer A containing urea, followed by addition of preservatives and filtration to obtain an FN content of 350 μg/m
A solution of eggplant A 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, it is also easy to incorporate into the formulation of cosmetics and external rubbing agents.

[Example 3] Here, FN-containing solution obtained by the operating means disclosed in Examples 1 and 2 (solution before filtration before addition of preservative/1i
2) to 4! To this liquid, add regenerated CM-cellulose or CM-Sephadex prepared in advance, stir at 4°C for about 10 to 20 minutes, and then perform suction filtration. A preservative was added to the obtained filtrate and filtered again to prepare an FN-containing solution.
1! Obtained.

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

The placecenter-derived FN according to the present invention is CFN,
It is 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 to deal with it.

Examples-1 to 3 regarding FN-containing liquid derived from placenta
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 value for maintaining stability of FN-containing liquid = 23
- On the other hand, when used in cosmetics and external preparations, the generation of specific odor from organs is a drawback in formulation, but Examples 1 to 3
When evaluating the odor (fishy smell) of each FN-containing solution obtained according to the method shown in
It was confirmed that the treatments using ammonium sulfate fractionation, ethanol fractionation, and ion exchanger were each effective.

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.

=24- "Table 6, Odor of FN-containing liquid and FN content in placental tissue (average amount of FN in 1 kg of placental tissue)
[Example-4] Regarding the application of FN for the purpose of external application to the skin, for example, No.
No. 6007) is disclosed. According to it, plasma-derived FN (PFN) is used, and 0.00001
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 inventor tested three healthy male volunteers (adults) at 6 locations on the upper and lower sides of both sides of the back, for a total of 122 locations on both sides. In this method, an aluminum plate with a round hole of a diameter of 2 cm made to accommodate the light source is placed on both sides of the back, and then a UV lamp is used to illuminate the light source for a certain period of time on both sides of the back. By creating an erythema of the same size and applying an FN-containing solution to the erythema site, we compared the number of days required for the erythema to disappear 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. was dissolved in a buffer A solution containing 0.5 M urea, adjusted to pH 7.5, and the FN content contained in the solution was adjusted to 10.25.40.50.100.
．． 150.200.250.300.500μg/mQ
A solution prepared as follows 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 10 CT 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/mQ or more. It shows a tendency to be particularly effective at a concentration of 100 to 300 μg/mQ. However, 300
It is difficult to judge the superiority of μg/mQ and FN containing 500 μg/ll111 due to the small number of cases.

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 some effect on hastening the disappearance of erythema.

In any case, when considering the FN content as a guideline for formulation, it seems to be advantageous to adjust the FN content to about 100 to 300 μg/mQ.

In addition, the application was carried out on 122 localized areas on the back using pharmacopoeial cotton (cut into 2 cm square pieces) impregnated with the test solution of each concentration.
The method is to apply a cloth to the cotton, tape it from above, and then remove the tape once the cotton is dry.
Treatment was performed twice.

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. However, combination with these known anti-inflammatory drugs will enhance 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 in animals, FN content is 5 μg/mQ.
It can be confirmed even below 0μg/mQ.

Next, the FN-containing liquid obtained in each example can be applied or rubbed directly onto the skin as long as it falls within the standard values in Table 5, but it can also be applied to known emulsions, creams, lotions, etc. It can be formulated into the following dosage forms.

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

[Formulation example-1) Skin inflammation relieving moisturizing protective liquid (%) (1)
FN-containing liquid (with standard values shown in Table 5) 1#-...1
...l11ヱ～70 (2) R3C liquid (cock's comb extract hyaluronic acid-containing solution) ...3-5 (3) Brasenand V (large placenta extract)...・ ・ ・ ・
・ ・ ・ ・ ・ ・ ・ ・ ・ ・ 3 to 5 (4
)BG (1,3-butylene glycol)・・
・ ・ Urn 1 ・ ・ ・ ・ Australia ψ ＃φ 5
~10(5) Sodium salicylate...0.1-0.
3 (a) [J1 liquid (solution containing 75% dextrin) 2-3 (7) Make up to 100 with purified water.

Table 7: Effect 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 cannot be applied to extracting PFN derived from plasma, it can be fully utilized for extracting CFN derived from other cells, and furthermore, processing operations are easy and production efficiency is high. In addition, in Example 1, one of the best methods was to use a solution of 5M urea in buffer A and stir it at 4°C overnight. Regardless of the method, the purpose can be sufficiently achieved by soaking at 4°C overnight and extracting with a homomixer the next day.

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.

In addition, the FN-containing liquid according to the present invention may contain moisturizing agents such as mucopolysaccharides such as hyaluronic acid, water-soluble peptides obtained by hydrolyzing silk (fibroin), keratin, collagen, elastin, etc., which are known as skin moisturizing agents, or plastics. Extracts extracted from various animal organs (e.g. blood, brain, liver, intestines, tiles, skin, etc. of cows, pigs, sheep, etc.), or fibroblasts extracted from the brain, skin, etc. It can also be used in combination with cell growth substances.

[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, the preservative has been removed.) Figure 1 Wavelength (ns) Procedural amendment (spontaneous) November 1, 1985 Day 1, Incident indication 1985 Patent application No. 234108 2, Title of the invention Plancenter tissue-derived fibronetin-containing solution

Claims (1)

[Claims] (1) After immersing human placenta after normal delivery or cow placenta tissue fibers in a solution of 0.15 M sodium chloride/0.06 M Tris/HCl buffer and 5 M urea, The clear liquid was used as a crude liquid, and fibronectin was obtained by combining one or more of the following treatments: ammonium sulfate 0.4 saturated fractionation, ethanol 50v/v% fractionation, and dialysis. 0.0M containing 0M urea.
A solution containing fibronectin derived from placental tissue, characterized in that it is dissolved in a 15M sodium chloride/0.06M Tris/hydrochloric acid buffer.