JP4877782B2 - Skin barrier function restoring agent evaluation method and evaluation kit - Google Patents

Description

  The present invention relates to a skin barrier function restoring agent evaluation method and an evaluation kit for evaluating whether a compound has a skin barrier function recovery effect.

  The skin is composed of epidermal cells and fibroblasts, and these epidermal cells and fibroblasts are supported by collagen or the like. Young skin in the 20s has high fibroblast activity and high collagen production. In this age, female hormones are actively secreted, and female hormones act on the cell membrane of fibroblasts, so that the activity of fibroblasts is maintained at a high level. For this reason, the skin is maintained in a fresh state with a tension and gloss.

  In contrast, aged skin over the 50s has low lipid synthesis ability and low production of collagen. In addition, since the secretion of female hormones is small at this age, it is difficult for female hormones to act on epidermal cells, spurring a decline in barrier function. As a result, the skin of this age has a low skin barrier function, and the skin is not stretched or glossy and exhibits roughness, wrinkles, dullness and the like.

  Thus, the decrease in the skin barrier function associated with the aging of the skin involves the low activation of the lipid synthesis ability and the low secretion of female hormones. Therefore, for example, it is considered that aging of the skin can be suppressed to some extent by administering a skin barrier function restoring agent capable of activating epidermal cells in place of female hormones.

In developing such a skin barrier function recovery agent, it is essential to evaluate the skin barrier function recovery effect of candidate compounds. As a conventional method for evaluating the effect of restoring the skin barrier function, an animal experiment using a hairless mouse or the like is known (see, for example, Patent Document 1).
JP 2004-175687 A JP 2002-291909 A JP 2005-2063 A

  However, animal experiments have the problem of labor and time, and there are other ethical problems with the use of animals, so the development of a method to easily evaluate the skin barrier function recovery effect without using animals. Was anxious.

  Therefore, an object of the present invention is to provide a novel skin barrier function restoring agent evaluation method and evaluation kit capable of simply and reliably evaluating whether a compound has a skin barrier function recovery effect.

  As a result of various studies to solve the above problems, there is a correlation between the size of the liposome in the liposome solution prepared by adding a compound that is a candidate for the skin barrier function recovery agent and the effect of the compound on restoring the skin barrier function. It was found that the smaller the size of the liposome, the higher the effect of restoring the skin barrier function, and the present invention was completed.

  That is, the skin barrier function recovery agent evaluation method according to claim 1 of the present invention uses the size of the liposome in the liposome solution prepared by adding a compound that is a candidate for the skin barrier function recovery agent as an index. It is characterized in that the smaller the thickness is, the higher the skin barrier function recovery effect is.

  Moreover, the skin barrier function restoration | recovery agent evaluation method of Claim 2 of this invention is the said liposome solution. The said liposome solution makes a film formation solution which consists of a phospholipid solution dry, creates a film, It is characterized by being prepared by adding a compound that is a candidate for a skin barrier function restoring agent after adding a buffer solution.

  Moreover, the skin barrier function restoration | recovery agent evaluation method of Claim 3 of this invention is a film formation formed by adding the compound used as the candidate for a skin barrier function restoration | recovery agent to the said lipolipid solution in the said liposome solution. A film is prepared by drying the solution, and a buffer solution is added to the film.

  Furthermore, the skin barrier function recovery agent evaluation kit according to claim 4 of the present invention is a skin barrier function recovery agent using as an index the size of the liposome in the liposome solution prepared by adding a compound that is a candidate for the skin barrier function recovery agent. A kit for evaluating a skin barrier function restoring agent for evaluating an effect, comprising a phospholipid and a buffer solution.

  According to the skin barrier function restoring agent evaluation method and the evaluation kit of the present invention, the liposome size in the liposome solution prepared by adding a compound that is a candidate for the skin barrier function restoring agent is used as an index. By evaluating that the smaller the skin barrier function recovery effect is, the smaller it is, it is possible to simply and reliably evaluate whether or not the compound has the skin barrier function recovery effect.

  The skin barrier function restoring agent evaluation method of the present invention uses the size of liposomes in a liposome solution prepared by adding a compound that is a candidate for skin barrier function restoring agent as an index, and the smaller the liposome size, the more the skin barrier function. It is evaluated that the recovery effect is high.

  The balance of substances having a steroid skeleton such as sex hormones changes due to aging, stress, etc., and skin barrier function is lowered. As an example of a compound to be evaluated in the present invention, there is a compound having a recovery effect against the skin barrier function reduction caused by such a substance having a steroid skeleton.

  Here, the preparation method of the liposome solution containing the candidate compound is not limited to a specific method, but can be prepared as follows, for example.

  The first method is a method of preparing a liposome solution not containing a candidate compound and adding the candidate compound thereto. That is, a film-forming solution composed of a phospholipid solution is dried to create a film, a buffer solution is added to the film, and then a compound that is a candidate for a skin barrier function restoring agent is added to prepare a liposome solution. .

  More specifically, first, a phospholipid solution is prepared by dissolving phospholipid in a solvent to obtain a film-forming solution. Preferably, a coloring pigment is added here. Next, the solvent is removed from the film-forming solution under reduced pressure in a container and dried to form a film. Then, a buffer solution is added to swell the film to obtain a uniform solution. And a liposome solution is created by adding the compound used as a skin barrier function recovery agent candidate here.

  The second method is a method of adding a candidate compound before preparing a film for preparing a liposome solution. That is, a film is formed by drying a film forming solution obtained by adding a compound that is a candidate for a skin barrier function restoring agent to a phospholipid solution, and a buffer solution is added to the film.

  More specifically, first, a phospholipid solution is prepared by dissolving phospholipid in a solvent, and a solution of a candidate compound prepared separately from this is added to obtain a film-forming solution. Preferably, a coloring pigment is added here. Next, the solvent is removed from the film-forming solution under reduced pressure in a container and dried to form a film. Then, a liposome solution is prepared by adding a buffer solution to swell the film into a uniform solution.

  The size of the liposome in the liposome solution can be measured by observing it using a fluorescence microscope. And it evaluates that the skin barrier function recovery effect is so high that the magnitude | size of a liposome is small. For example, when the average diameter of the liposome is 1 μm or less, it can be evaluated as having a skin barrier function recovery effect. Alternatively, the skin barrier function recovery effect can be evaluated by comparison with a control to which no compound is added. Further, the evaluation may be performed based on the average area of the liposome in the microscope visual field.

  As the phospholipid and buffer used in the present invention, those usually used for preparing a liposome solution can be used.

  Thus, according to the skin barrier function recovery agent evaluation method and evaluation kit of the present invention, the liposome size in the liposome solution prepared by adding a compound that is a candidate for the skin barrier function recovery agent is used as an index. By evaluating that the smaller the size of the skin, the higher the skin barrier function recovery effect, it is possible to easily and reliably evaluate whether the compound has the skin barrier function recovery effect without using animal experiments. Therefore, the method of the present invention can efficiently screen for compounds that are candidates for a skin barrier function restoring agent.

  Moreover, since evaluating the skin barrier function recovery effect against the skin barrier function decline due to aging will simultaneously evaluate the anti-aging effect, the method of the present invention is effective for the treatment of spots, wrinkles, gray hair, etc. It can also be used effectively for the evaluation of anti-aging agents against the skin barrier function decline due to aging.

  Moreover, the skin barrier function restoration | recovery agent evaluation kit of this invention is equipped with the phospholipid and buffer solution which are required for said method. Therefore, a kit capable of simply and reliably evaluating whether or not a compound has a skin barrier function recovery effect is provided.

  Note that the present invention is not limited to the above, and various modifications can be made without departing from the spirit of the present invention.

  The present invention will be described in more detail based on the following specific examples.

  13.2 mg of phospholipid (L-α-phosphatidylcholine derived from dried egg yolk (manufactured by Sigma)) is taken into an eggplant flask, and 13.2 ml of methanol: chloroform = 1: 2 solution is added as a solvent to form a 1 mg / ml film. Formed solution. A fluorescent dye (Nile Red) was added to a final concentration of 1 μM.

  Next, the solvent was removed under reduced pressure for 60 minutes with a rotary evaporator under reduced pressure, and a film was formed in the eggplant flask. Thereto, 13.2 ml of a buffer solution (2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) aqueous solution) was added and allowed to swell for 60 minutes to obtain a uniform solution. Moved to.

  And the compound (testosterone, (beta) -estradiol, etc.) used as evaluation object was added here, and the liposome solution was created. It observed using the fluorescence microscope 30 minutes after addition.

  As the compounds to be evaluated, testosterone, androsterone, progesterone, β-estradiol, polyoxyethylene-polyoxypropylene-dimethyl ether (Aquain Pool (registered trademark): AQP) were used. In addition, a liposome solution was prepared by adding testosterone, androsterone, progesterone, and β-estradiol to a final concentration of 100 μM and AQP to a final concentration of 5%.

  Fig. 1 shows a photo when β-estradiol was added (upper left), a photo when testosterone was added (upper right), a photo when β-estradiol and testosterone were added (lower right), and a control photo without any addition. (Lower left). When testosterone was added (upper right), it was observed that the liposome was destabilized and the diameter increased. On the other hand, when β-estradiol was further added in addition to testosterone (lower right), it was observed that the liposome was stabilized and the diameter was reduced. Thus, it was confirmed that the liposome size was reduced by adding β-estradiol even under conditions where testosterone for enlarging the liposomes was present.

  Moreover, the result of having measured the average area on the visual field of the microscope of a liposome to FIGS. FIG. 2 shows the case where testosterone alone, testosterone and β-estradiol, testosterone and AQP are added, and FIG. 3 shows the case where androsterone alone, androsterone and β-estradiol, androsterone and AQP are added, and FIG. 4 shows the case where progesterone alone, progesterone and β-estradiol, progesterone and AQP are added.

  As shown in FIGS. 2 and 3, it was confirmed that testosterone and androsterone have an action of enlarging liposomes, but β-estradiol counteracts the action. In addition, it was confirmed that AQP has an action of reducing the size of liposomes when added together with testosterone and androsterone, compared to the case of control or AQP alone.

  On the other hand, as shown in FIG. 4, β-estradiol hardly negated the action of enlarging the liposome of progesterone.

  When these results were compared with the measurement results of the skin barrier function recovery promoting effect (FIGS. 5 to 7) measured by the method described later, there was a correlation between the skin barrier function recovery promoting effect and the size of the liposome. That is, the liposomes when testosterone and androsterone are added are large, but these delay the skin barrier recovery. On the other hand, when β-estradiol is added to these, the liposomes are slightly reduced, but the delay in recovery of the skin barrier is alleviated. In addition, when AQP is added to androsterone, the liposomes become smaller, but the delay in recovery of the skin barrier is eliminated.

  Moreover, the liposome when progesterone is added is large, and even when β-estradiol is added, there is no change in size, but the effect of recovering the skin barrier is not improved even when β-estradiol is added.

  As described above, it was found that the smaller liposome size has a higher barrier recovery rate, and the larger liposome size has a smaller barrier recovery rate. Therefore, it was confirmed that it was possible to evaluate whether or not it had an effect of restoring the skin barrier function from simple liposome shape change without measuring the barrier recovery rate.

  Hereinafter, the measuring method of the skin barrier function recovery promotion effect used for confirming the method of the present invention will be described.

  The skin barrier function recovery promoting effect can be evaluated by various methods. For example, the skin barrier function destroyed by applying tape stripping to the skin of a mammal (eg, human, mouse, rat, rabbit). By evaluating the process of recovering to the above state using transdermal water transpiration (TEWL) as an index, it can be measured quantitatively or qualitatively. Such measurement can be performed, for example, as follows.

  1. The amount of transdermal moisture transpiration (TEWL) near the back of the hairless mouse is measured with a moisture transpiration measuring device. The value at this time is assumed to be a TEWL recovery rate of 100%.

  2. The skin barrier is destroyed by peeling off the horny layer of hairless mice using cellophane tape or the like. At this time, it is preferable to repeat this operation until the value of TEWL becomes about 800 to 900. The value obtained by subtracting the measured value before peeling the stratum corneum from the measured value after peeling the stratum corneum is the deepest damage state, that is, the recovery rate is 0%.

  3. The test sample is placed on a suitable base material such as a plastic wrap at an appropriate concentration (for example, 1 mM) at an appropriate amount (for example, 100 μl), applied to the back of a mammal, and after an appropriate time (for example, 5 minutes), Remove it.

  4). After an appropriate time (for example, 0, 2, 4, 6 hours), TEWL is measured by a moisture transpiration measuring device. As in the case of stratum corneum removal, the recovery rate is calculated by subtracting the TEWL value before stratum corneum removal from the measured value at each time.

  That is, the recovery rate can be determined according to the following formula:

  Here, “promoting the recovery of the skin barrier function” means that the value of transdermal moisture transpiration (TEWL) immediately after tape stripping of the skin is 0%, and the value before tape stripping is 100%. The value of TEWL clearly has a significant difference when compared with the control, meaning that it has the effect of promoting the recovery rate of TEWL. According to the method of Andrew et al. (JInvest Dermatol, 86; 598, 1986), 4 This is different from the so-called rough skin improvement preventing effect in which the skin is treated and judged with Cottonball soaked with a sodium dodecyl sulfate (SDS) aqueous solution.

  Phospholipid (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)) and testosterone or β-estradiol were dissolved in a chloroform-methanol solution, and a fluorescent dye (Nile red) was added. The solvent was removed under reduced pressure to prepare a film, and a buffer solution was added to swell (35 ° C., 1 hour) to prepare a liposome solution. Images were recorded using a fluorescence microscope and measured using analysis software (Cosmos32).

  The left side of FIG. 8 is an image of the liposome when testosterone is added. The size of the liposomes tended to increase, but the shape did not change. Similarly, the right side is an image when β-estradiol is added. Contrary to testosterone, the size decreased and the number of tube-shaped liposomes increased. These effects were also confirmed by examination using the roundness, which is an index of liposome diameter size distribution and shape change.

It is a fluorescence micrograph of a liposome when testosterone and β-estradiol are added. It is a graph which shows the average area of a liposome when testosterone, (beta) -estradiol, and AQP are added. It is a graph which shows the average area of a liposome when androsterone, (beta) -estradiol, and AQP are added. It is a graph which shows the average area of a liposome when progesterone, (beta) -estradiol, and AQP are added. It is a graph of the barrier recovery rate after 3 hours which shows the skin barrier function recovery promotion effect of androsterone, testosterone, and β-estradiol. It is a graph of the time-dependent change of the barrier recovery rate which shows the skin barrier function recovery promotion effect of androsterone and AQP. It is a graph of the time-dependent change of the barrier recovery rate which shows the skin barrier function recovery promotion effect of progesterone and (beta) -estradiol. It is a fluorescence micrograph of a liposome when testosterone and β-estradiol are added.

Claims (4)

Using the size of liposomes in a liposome solution created by adding a candidate compound for skin barrier function recovery agent as an index, the smaller the liposome size, the higher the skin barrier function recovery effect is evaluated. Skin barrier function recovery agent evaluation method. The liposome solution was prepared by drying a film-forming solution consisting of a phospholipid solution to prepare a film, adding a buffer solution to the film, and then adding a compound that is a candidate for a skin barrier function restoring agent. The method for evaluating a skin barrier function-restoring agent according to claim 1, wherein: The liposome solution is prepared by drying a film forming solution obtained by adding a compound that is a candidate for a skin barrier function restoring agent to a phospholipid solution, and then adding a buffer solution to the film. The method for evaluating a skin barrier function-restoring agent according to claim 1. A skin barrier function recovery agent evaluation kit for evaluating the skin barrier function recovery effect using the size of the liposome in a liposome solution prepared by adding a compound that is a candidate for the skin barrier function recovery agent as an index. And a buffer solution, a skin barrier function restoring agent evaluation kit.