JP7239400B2 - Determination method of skin antioxidant capacity - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for determining skin antioxidant capacity using biophotons.

Sun exposure causes various types of damage to the skin. In particular, light in the ultraviolet region (290-400 nm) has a harmful effect on the skin, and is deeply involved in erythema and pigmentation formation caused by short-term exposure, and photoaging and carcinogenesis caused by long-term exposure. It is known to do (Non-Patent Documents 1 and 2).

As a technique for protecting and improving the skin from damage caused by ultraviolet rays, there is a method of applying a sunscreen agent or the like. When ultraviolet rays are blocked, the production of reactive oxygen species (ROS), which are important for the initial skin response immediately after irradiation, and biological oxides produced by reactions with ROS are suppressed. On the other hand, a method for controlling skin damage caused by ultraviolet rays has also been suggested by controlling ROS after ultraviolet irradiation and biological oxides produced by reactions with ROS. Application to the skin, such as antioxidants, corresponds to this.

It has been known for a long time that ROS production triggers various biological reactions such as degradation of extracellular matrix (photoaging), induction of inflammation, and induction of apoptosis. However (Non-Patent Document 3), there are still many unclear points about the detailed role of ROS in vivo, as represented by human skin, and the relevance to subsequent skin disorders that occur along the time axis. The reason for this is that there are almost no methods for noninvasively evaluating ROS, oxidative stress, and skin antioxidant capacity.

As a method for evaluating the oxidative stress and antioxidant capacity of human skin, there is a method using biopsy skin, but it is not widely used because it is invasive. Although evaluation of oxidized proteins and antioxidants by stratum corneum tape stripping is minimally invasive, it only evaluates the stratum corneum and cannot be said to reflect the state inside the skin (Non-Patent Documents 4 and 5). .

As a non-invasive evaluation method, there is a report of skin carotenoid measurement by Raman spectroscopy, but it is an evaluation of a single antioxidant substance, and it is difficult to say that this also reflects the response of the entire skin. (Non-Patent Document 6).

Under such circumstances, biophoton detection technology is attracting attention as a technology that can noninvasively evaluate the oxidation state of living organisms. A biophoton is an extremely weak spontaneous luminescence emitted by living organisms as part of their life activities. Singlet oxygen and excited carbonyl compounds are presumed to be the origin of the luminescence, and the luminescence is considered to be caused by the oxidation reaction of living organisms. Biophotons are observed in various living organisms such as plants, microorganisms, and animals.In human skin, biophotons are measured after irradiation with ultraviolet A (UVA) radiation, and the emission intensity varies depending on the skin color. Differently (Non-Patent Document 7), it has been reported that luminescence is reduced by applying antioxidant cream (Non-Patent Document 8).

However, in these reports, the evaluation is based on the integrated value for several minutes immediately after UVA irradiation, and details of the oxidative stress response that changes every moment are not evaluated over time. It has been suggested that the biophotons detected immediately after UV irradiation represent the amount of ROS produced. It is difficult from the integrated value. In addition, as mentioned above, biophotons are affected by skin color, so it is difficult to accurately evaluate skin oxidative stress and antioxidant capacity between different people and between different parts from the integrated values.

Photodermatol. Photoimmunol. Photomed. 18, 75-81 (2002) Toxicology 189, 21-39 (2003) J. Invest. Dermatol. 126, 2565-75 (2006) Skin Res. Technol. 13, 84-90 (2007) J. Invest. Dermatol. 110, 756-61 (1998) J. Invest. Dermatol. 115, 441-48 (2000) Photodermatol. Photoimmunol. Photomed. 25, 65-70 (2009) Skin Pharmacol. Physiol. 24, 300-4 (2011)

The present invention relates to providing a technique for noninvasively determining the skin antioxidant capacity of a subject using biophotons.

The present inventors calculated parameters related to skin antioxidant capacity from the attenuation curve of biophotons detected after irradiating human skin with an electromagnetic wave within a specific wavelength range such as ultraviolet rays, and used the skin as an index. It was found that the antioxidant capacity can be evaluated with high accuracy.

That is, the present invention is a method for determining the skin antioxidant capacity of a subject, comprising the step of irradiating the subject's skin with an electromagnetic wave having a wavelength of 285 nm to 1 mm and measuring the amount of biophotons detected after the irradiation, It relates to a method for determining skin antioxidant capacity by calculating parameters from a biophoton decay curve.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to determine a test subject's skin antioxidant ability and protective ability against photoaging in a noninvasive, simple, and early stage.

Biophoton decay curve. The figure which shows the chain reaction after ROS generation after ultraviolet irradiation.

The electromagnetic wave having a wavelength of 285 nm to 1 mm, which is irradiated to the skin in the method of the present invention, includes, for example, electromagnetic waves including ultraviolet rays, visible rays, and infrared rays, preferably light rays having wavelengths in the ultraviolet region. include UV-B waves with wavelengths of 285-320 nm, or UV-A waves with wavelengths of 320-400 nm. In the present invention, mixed ultraviolet rays of A wave and B wave are preferable, and the light intensity ratio (A wave/B wave) is preferably 6 to 20, more preferably 7 to 15, and 8 to 12. More preferably.

^The intensity ^{of the} electromagnetic wave to be irradiated ^is not particularly limited. It is more preferably 170 mW/cm ² or less, more preferably 150 mW/cm ² or less. Also, it is preferably 10 to 200 mW/cm ² , more preferably 20 to 170 mW/cm ² , and more preferably 30 to 150 mW/cm ² . In the present invention, the intensity of electromagnetic waves means, for example, the intensity of ultraviolet rays in a wavelength range (285 to 400 nm) combining UV-B waves and UV-A waves.
The intensity of electromagnetic waves can be measured using commercially available measuring instruments, such as Solarmeter Model 5.0 (UVA+B) (Solartech Inc.), multipurpose spectroradiometer MSR-7000N (Opto Research), etc. mentioned.

The irradiation time varies depending on the intensity of the electromagnetic wave to be irradiated, but may be, for example, 5 to 120 seconds, preferably 5 to 60 seconds, more preferably 5 to 45 seconds.

The irradiation dose (irradiation energy) determined by the irradiation electromagnetic wave intensity and the irradiation time is preferably 300 to 7000 mJ/cm ² , more preferably 500 to 6000 mJ/cm ² , still more preferably 600 to 5000 mJ/cm ² . be.

The irradiation device for irradiating electromagnetic waves is not particularly limited as long as it has a light source capable of emitting light in the wavelength range described above. lamps, metal halide lamps, Wood lamps, fluorescent inspection lamps, etc., preferably xenon lamps.
The irradiation wavelength is adjusted by combining such a light source with a filter that selectively transmits electromagnetic waves in the target wavelength range, if necessary.

The skin part of the subject to which electromagnetic wave irradiation is performed includes, for example, the skin of the inner upper arm, outer forearm, inner forearm, outer upper arm, neck, back, etc. Electromagnetic irradiation and biophoton measurement are easy. Therefore, the outer forearm or the inner upper arm is preferable.
The area to be irradiated with the electromagnetic wave is not particularly limited, but is, for example, 1 to 13 cm ² , preferably 1 to 8 cm ² .

Biophoton measurement is performed, for example, for 10 minutes immediately after irradiation, preferably for 5 minutes immediately after irradiation, more preferably for 4 minutes immediately after irradiation.

Biophoton detection is performed by optical detection equipped with a detection unit such as a high-sensitivity, low-noise photomultiplier tube or a high-sensitivity CCD that can convert extremely weak light energy of biophotons into electrical energy and detect it. performed by the device. As the optical detection device, for example, a weak emission intensity detection device (for example, CLA-IDFsk, manufactured by Tohoku Denshi Sangyo Co., Ltd.) equipped with a photomultiplier tube (for single photon counting) or a detection device equipped with an ultra-sensitive cooled CCD camera. (eg, CLA-IMG, manufactured by Tohoku Denshi Sangyo Co., Ltd.). In the present invention, a weak emission intensity detector equipped with a photomultiplier tube is preferably used. Although the wavelength of the detected emitted light depends on the photomultiplier tube of the detector, the device detects biophotons between 300 and 850 nm. In order to minimize the influence of light originating from the measurement environment, the measurement of biophotons is preferably performed in a space shielded from light as much as possible, for example, in a darkroom.
That is, it is preferable to irradiate the site to be measured with an electromagnetic wave using the electromagnetic wave irradiation apparatus in a dark room, and then measure biophotons emitted from the site irradiated with the electromagnetic wave using a weak emission intensity detector. In addition, the electromagnetic wave emitting unit in the electromagnetic wave irradiation device and the detection unit in the weak emission intensity detection device may be separate, but from the viewpoint that electromagnetic wave irradiation and biophoton detection can be performed without changing the device, the electromagnetic wave emitting unit (specifically Specifically, it is preferable that the light irradiation fiber extending from the electromagnetic wave irradiation device and the detection unit are integrated so that the device to be used can be changed by switching the optical path.

For biophotons generated by electromagnetic wave irradiation, the luminescence intensity at rest before electromagnetic wave irradiation is measured in advance, then the luminescence intensity within a predetermined period after electromagnetic wave irradiation is measured, and the luminescence intensity at rest is subtracted from that value. can be calculated as the luminescence increment.

Biophotons generated when the skin is irradiated with electromagnetic waves exhibit the maximum luminous intensity immediately after the electromagnetic wave irradiation, and then the luminescence attenuates over time. Therefore, by plotting the measured data of the biophoton intensity generated after electromagnetic wave irradiation against time and fitting the attenuation curve represented by the following formula (1) to it (for example, FIG. 1), four types of coefficients (A ₀ , k ₁ , k ₂ , k ₃ ) can be calculated.

[In the formula, A ₀ : initial concentration of ROS, k ₁ : production rate constant of lipid oxidation primary products, k ₂ : production rate constant of lipid oxidation secondary products, k ₃ : production rate constant of other oxidation products , indicates. ]

The introduction of formula (1) was made with reference to a document describing a biological oxidation reaction by ultraviolet irradiation (H. Masaki. J. Dermatol. Sci. 58, 85-90 (2010)). Specifically, when exposed to ultraviolet rays, reactive oxygen species and radical species are generated first, and then biomolecules such as lipids and proteins are oxidized (see Fig. 2). The species is A, the primary product of lipid oxidation represented by lipid hydroperoxide is B, the lipid oxidation secondary product generated from B is C, and the oxidation product such as protein generated from A is D ( See the reaction formula below), these reactions are defined by first-order reaction rate constants (k ₁ , k ₂ and k ₃ , respectively), and the concentration formula is calculated as follows.

Let the concentrations of A and B after electromagnetic wave irradiation be A ₀ , A ₀ * k ₁ / (k ₁ + k ₃ - k ₂ ) * e ^{- k2t,} respectively, and the biophoton intensity is the concentration of A and B at which light emission occurs during the reaction process Based on what is thought to be reflected in , the concentration equation of A+B (equation (1)) is fitted to the biophoton measurement data.

Then, as shown in the examples below, the difference between the low-exposure group and the high-exposure group grouped based on the site differences (inner arm, outer forearm) and annual average light exposure time for four types of coefficients was examined, k ₂ / (A ₀ * k ₁ ) was high in the exposed area (Table 1) and tended to be high in the high exposure group (Table 2). A statistically significant positive correlation was observed with the 4-week exposure time (Table 3).
A high k ₂ /(A ₀ *k ₁ ) value indicates that the primary product B of lipid oxidation is less likely to be generated or is easily converted to the secondary product C in the biooxidation reaction by electromagnetic irradiation. That is, it means that the primary product B is in a state where it is difficult to accumulate. The state in which the primary product B is difficult to accumulate refers to a qualitative change in B, in which the proportion of highly reactive substances relatively increases and the oxidation reaction to the next secondary product C easily proceeds. , i.e., associated with reduced antioxidant capacity.
Therefore, the k ₂ /(A ₀ *k ₁ ) value can be used as an index for determining skin oxidative stress elimination response, that is, skin antioxidant capacity. Here, the term "skin antioxidant capacity" refers to the ability of the skin to protect against oxidative stress. In addition, the k ₂ /(A ₀ *k ₁ ) value was found to have a positive correlation with the amount of transepidermal water loss, indicating a relationship with the skin barrier function. It has been reported that a decrease in skin barrier function increases UV sensitivity (M. Mildner. J. Invest. Dermatol. 130, 2286-94 (2010)), so the k ₂ / (A ₀ * k ₁ ) It can be said that the value can also be an index for judging protective ability against photoaging. Here, "photoaging" means changes in the skin caused by accelerated internal aging processes of the skin due to chronic exposure to sunlight, particularly ultraviolet radiation.

As a specific determination method, for example, for each age or age group, for each gender, indoor group / outdoor group, biophoton intensity detected after electromagnetic wave irradiation in the present invention is measured in advance and acquired as basic data. , from the average value and standard deviation of the k ₂ / (A ₀ * k ₁ ) values calculated from them, calculate the subject's age (age), gender, indoor / outdoor group, and calculate these various numerical values It is possible to appropriately set the reference value for skin antioxidant capacity determination based on. For example, when the average value is set as the reference value, if the k ₂ / (A ₀ * k ₁ ) value calculated from the biophoton intensity of the subject is lower than the average value, it can be determined that the skin antioxidant capacity is high. .
Alternatively, regarding the evaluation index of skin antioxidant capacity, such as high skin antioxidant capacity, slightly high skin antioxidant capacity, standard, slightly low skin antioxidant capacity, and low skin antioxidant capacity, the range of deviation values from these Appropriate criteria for correlation can be created, and the subject's skin antioxidant capacity against UV rays can be determined based on the subject's deviation value. When the deviation value is used to set the reference value, for example, the value of k ₂ / (A ₀ * k ₁ ) corresponding to each of the deviation values 40, 45, 55, and 60 is set as the reference value, and these multiple reference values Criteria can be developed that relate the numerical ranges based on the above evaluations of skin antioxidant capacity.

The method for determining the skin antioxidant capacity described above can be determined in a very short period of time and places little burden on the subject. The information on the skin antioxidant capacity obtained by the determination method of the present invention can be used for physical protection measures against ultraviolet rays, for measures against ultraviolet rays such as sunscreens and the like, and for the purchase of ultraviolet protective skin preparations for external use. It can be used as an index for product recommendation in product selection and recommended sales of UV protection skin external preparations. In addition to ultraviolet rays, near-infrared radiation and air pollutants are also related to photoaging, so it can be used to evaluate near-infrared protective agents, air pollutant adhesion inhibitors, and the like.
The method for determining skin antioxidant capacity of the present invention corresponds to a method for collecting various materials from the human body by measuring the structure or function of each organ of the human body, and is used for the above purpose. be. In other words, it is not intended for medical purposes to determine the physical or mental condition of a person, such as a medical condition or health condition. In this sense, the method for determining skin antioxidant capacity of the present invention can also be referred to as a method for measuring skin antioxidant capacity or a method for testing skin antioxidant capacity.

EXAMPLES The present invention will be described in more detail below based on examples, but the present invention is not limited to these.

Grouping by subjects and light exposure history The subjects were 43 healthy women in their twenties, and were grouped as follows. Based on a questionnaire survey on lifestyle habits and outdoor leisure activities, we estimated the standard time that subjects were exposed to sunlight in a certain age range, and calculated the cumulative light exposure time by considering their chronological age. The question items of the questionnaire were created based on the questionnaire regarding light exposure history published by the American Cancer Center (Arch. Dermatol. 144, 217-22 (2008)). Next, the average annual light exposure time of the subjects was obtained, and the subjects were divided into two groups, a low exposure group and a high exposure group, based on the length of the time, so as to be approximately equal.
In addition, the sunlight exposure time for the most recent 4 weeks was calculated for each subject from the hours of exposure to sunlight on standard weekdays/holidays for the most recent 4 weeks.

Test method Subjects washed the inside of the upper arm and the outside of the forearm with a commercially available makeup remover and facial cleanser, and after acclimatization for 20 minutes in a variable environment room (room temperature 21 ± 1 ° C, humidity 50% RH), transepidermal water transpiration. amount was measured. Next, the amount of biophotons was measured in a dark room.

Method for measuring transepidermal water loss The transepidermal water loss was measured using a TEWA meter probe MPA580 (manufactured by Courage + Khazaka).

The light source used was a 300W type xenon light source (MAX-302, Asahi Spectro) with a WG-320 filter (thickness 1 mm, Shibuya Optics) attached (ultraviolet A wave (UVA)/ultraviolet B wave (UVB) ratio : 10.8).

Biophoton amount measurement method Measurement was performed in a dark room using a weak luminescence intensity detector (CLA-IDFsk, Tohoku Denshi Sangyo Co., Ltd.). After 10 minutes of acclimation to the dark room, the subject was placed in a resting sitting position and brought the detection part attachment of the device into close contact with the measurement site (inside upper arm or outer forearm). A fiber for light irradiation from a light source is connected to the detection unit attachment and integrated, and has a structure in which ultraviolet irradiation and biophoton detection can be switched by switching the optical path. Before UV irradiation, the luminescence intensity at rest was measured for 2 minutes. Subsequently, ultraviolet irradiation (47.6 mW/cm ² , 30 seconds, irradiation area 1.8 cm ² ) was performed, and the emission intensity was measured for 4 minutes immediately after. Data were acquired every 0.1 seconds.

By fitting the measurement data obtained by analysis of the decay curve of weak luminescence to the following formula using Excel (manufactured by Microsoft) so that the square of the error is minimized, four types of coefficients (A ₀ , k ₁ , k ₂ , k ₃ ) were calculated. FIG. 1 shows an example of measured data and an attenuation curve fitted to it.

[In the formula, A ₀ : initial concentration of ROS, k ₁ : production rate constant of lipid oxidation primary products, k ₂ : production rate constant of lipid oxidation secondary products, k ₃ : production rate constant of other oxidation products , indicates. ]

Exploration of parameters related to light exposure When examining the site differences (inner arm, outer forearm) and inter-group differences between the low-exposure group and the high-exposure group for 4 types of coefficients, A ₀ is the exposed part, the outer forearm Although it was significantly decreased, no difference was observed between groups. It has been reported that skin antioxidant enzyme activity decreases when light exposure is high (summer) (J. Invest. Dermatol. 120, 434-39 (2003)). We searched for parameters that change with time. As a result, k ₂ / (A ₀ * k ₁ ) was high in the exposed area (Table 1) and tended to be high in the high exposure group, suggesting that it is an index related to light exposure (Table 2). . Furthermore, when the correlation coefficient (R) with the exposure time in the last 4 weeks was calculated, the statistically significant positive correlation with the exposure time in the last 4 weeks was inside the upper arm (R = 0.330), outside the forearm (R = 0.329) were observed at both sites (Table 3). In addition, a statistically significant positive correlation between k ₂ /(A ₀ *k ₁ ) and transepidermal water loss was observed on the inner side of the upper arm (Table 4), indicating a relationship with skin barrier function.

Claims (7)

A method for determining the skin antioxidant capacity of a subject, comprising the step of irradiating the subject's skin with an electromagnetic wave having a wavelength of 285 nm to 1 mm and measuring the amount of biophotons detected after the irradiation, wherein the biophoton decay curve determining skin antioxidant capacity by calculating parameters from 2. The method according to claim 1, wherein the biophoton decay curve is calculated from the biophoton emission intensity. 3. The method according to claim 1 or 2, wherein the biophoton attenuation curve is represented by the following formula (1).

[In the formula, A ₀ : initial concentration of ROS, k ₁ : production rate constant of lipid oxidation primary products, k ₂ : production rate constant of lipid oxidation secondary products, k ₃ : production rate constant of other oxidation products , indicates. ] 4. The method of claim 3, wherein the parameter calculated from equation (1) is _k2 /( _A0 * _k1 ). The method according to claim 4, wherein the skin antioxidant capacity is evaluated by comparing k ₂ /(A ₀ *k ₁ ) calculated from the subject's biophoton intensity with a reference value. 6. The method according to any one of claims 1 to 5, wherein the electromagnetic wave irradiation is irradiation of mixed ultraviolet rays of A wave and B wave. The method according to any one of claims 1 to 6, wherein the skin antioxidant ability is the ability to protect against photoaging.

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