JP5110676B2 - Singlet oxygen scavenger, hyaluronidase inhibitor and hexosaminidase release inhibitor - Google Patents

Description

  The present invention relates to a singlet oxygen scavenger, a hyaluronidase inhibitor, and a hexosaminidase release inhibitor containing a plant extract as an active ingredient.

In recent years, active oxygen has attracted attention as a factor that particularly oxidizes biological components, and its adverse effect on living organisms has become a problem. Active oxygen is generated in the process of energy metabolism in living cells, and is superoxide (that is, superoxide anion (· O ₂ ⁻ ) generated by one-electron reduction of oxygen molecules), hydrogen peroxide (H ₂ O ₂ ). , Singlet oxygen ( ¹ O ₂ ), hydroxy radical (.OH), and the like. These active oxygens are indispensable for the phagocytic sterilization mechanism and play an important role in removing viruses and cancer cells. However, excessively generated active oxygen is in vivo that forms membranes and tissues in the body. Attacks molecules and induces various diseases.

  For example, active oxygen is thought to decompose, denature or crosslink biological tissues such as collagen, or oxidize fats and oils to produce lipid peroxides that damage cells, and is caused by active oxygen These disorders are considered to cause skin aging symptoms such as skin wrinkle formation and reduced elasticity (see Non-Patent Document 1).

  Therefore, it is considered that aging symptoms of skin can be prevented, treated or ameliorated by eliminating such active oxygen. As what has an active oxygen scavenging action, an ogi extract (refer patent document 1), a wisteria tea extract (refer patent document 2), a rape blossom extract (refer patent document 3), etc. are known.

  There are various causes and mechanisms of inflammatory diseases such as contact dermatitis (rash), psoriasis, acne vulgaris, and various other skin diseases with rough skin, but hyaluronidase activation, histamine Release etc. are known. The activation of hyaluronidase promotes histamine degranulation from mast cells, resulting in inflammation. Therefore, it is considered that the above-mentioned diseases can be prevented, treated or ameliorated by inhibiting the activation of hyaluronidase. As a thing which has a hyaluronidase inhibitory effect, the Japanese maple extract (refer patent document 4) etc. are known.

Histamine is present in the mast cell, and the substance released by the degranulation reaction acts as a flammable substance. Since histamine in the cells is released and hexosaminidase is released at the same time, histamine release inhibitory action can be evaluated using hexosaminidase release as an index. Therefore, it is considered that by suppressing the release of hexosaminidase, the release of histamine can also be suppressed at the same time, thereby preventing, treating or improving inflammatory diseases and the like. As what has a hexosaminidase release inhibitory effect, the Fuji tea extract (refer patent document 5) etc. are known.
JP-A-6-65074 JP 2001-97873 A JP 2003-81848 A JP 2003-1113068 A JP 2003-12532 A "Fragrance Journal Extra Number", 1995, No.14, p.156

  The present invention finds a singlet oxygen scavenging action, hyaluronidase inhibitory action or hexosaminidase release inhibitory action from natural products, and a singlet oxygen scavenger, hyaluronidase inhibitor and hexide containing it as an active ingredient. An object is to provide a sosaminidase release inhibitor.

  The singlet oxygen scavenger, the hyaluronidase inhibitor, and the hexosaminidase release inhibitor of the present invention are characterized by containing an extract from uranium and / or kanran as an active ingredient.

  ADVANTAGE OF THE INVENTION According to this invention, the singlet oxygen scavenger which contains the extract from uranium and / or kanran which has the outstanding singlet oxygen scavenging action, hyaluronidase inhibitory action, or hexosaminidase release inhibitory action as an active ingredient, hyaluronidase inhibition Agents and hexosaminidase release inhibitors can be provided.

The present invention will be described below.
The singlet oxygen scavenger, hyaluronidase inhibitor, and hexosaminidase release inhibitor of the present invention contain an extract from uranium and / or kanran as an active ingredient.

  Here, in the present invention, the “extract” means an extract obtained by using uranium or lanthanum as an extraction raw material, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or these Either a crude product or a purified product is included.

  The extraction raw materials used in the present invention are uranium (scientific name: Canarium pimela) and kanran (scientific name: Canarium album). Uranium (Canarium pimela) and kanran (Canarium album) are evergreen trees belonging to the family Orchidaceae, and are cultivated widely in tropical regions such as southern China and Taiwan, and are easily available from these regions. The constituent parts of the plant used as the raw material for extraction are not particularly limited, and examples thereof include flowering parts, leaf parts, branch parts, seed parts, bark parts, root parts, etc., among these, especially leaf parts. Is preferably used as an extraction raw material. In addition to the complete leaf, the leaf part includes a part of the leaf (for example, a leaf blade, a petiole, a bamboo leaf, etc.).

  Details of substances having singlet oxygen scavenging action, hyaluronidase inhibitory action or hexosaminidase release inhibitory action contained in uranium extract and kanran extract are unknown, but extraction methods commonly used for plant extraction Thus, an extract having a singlet oxygen scavenging action, a hyaluronidase inhibitory action, or a hexosaminidase release inhibitory action can be obtained from uranium or kanran.

  For example, an extract having a singlet oxygen scavenging action, hyaluronidase inhibitory action, or hexosaminidase release inhibitory action after drying uranium or lanthanum, pulverized as it is or using a crusher, and subjected to extraction with an extraction solvent Can be obtained. Drying may be performed in the sun or using a commonly used dryer. Moreover, after performing pretreatment, such as degreasing, with a nonpolar solvent such as hexane, it may be used as an extraction raw material. By performing pretreatment such as degreasing, extraction with a polar solvent can be performed efficiently.

  As the extraction solvent, it is preferable to use a polar solvent, and examples thereof include water and hydrophilic organic solvents. These are used alone or in combination of two or more at room temperature or a temperature below the boiling point of the solvent. It is preferable.

  Examples of water that can be used as the extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, and those subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, and the like. Therefore, the water that can be used as the extraction solvent in the present invention includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.

  Examples of hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene. Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycol, propylene glycol and glycerin.

  When using the liquid mixture of 2 or more types of polar solvents as an extraction solvent, the mixing ratio can be adjusted suitably. For example, when using a liquid mixture of water and a lower aliphatic alcohol, it is preferable to mix 1 to 90 parts by weight of a lower aliphatic alcohol with respect to 10 parts by weight of water. When using a mixed solution, it is preferable to mix 1 to 40 parts by mass of a lower aliphatic ketone with 10 parts by mass of water, and when using a mixed solution of water and a polyhydric alcohol, water 10 It is preferable to mix 1-90 mass parts of polyhydric alcohol with respect to mass parts.

  The extraction treatment is not particularly limited as long as the soluble component contained in the extraction raw material can be eluted in the extraction solvent, and can be performed according to a conventional method. For example, the extraction raw material is immersed in 5 to 15 times the extraction solvent (mass ratio) of the extraction raw material, the soluble components are eluted at room temperature or under reflux, and then filtered to remove the extraction residue. A liquid can be obtained. The obtained extract is diluted, concentrated, dried, purified, etc. according to a conventional method in order to obtain a diluted or concentrated solution of the extract, a dried product of the extract, or a crude purified product or a purified product thereof. Processing may be performed.

  Purification can be performed by, for example, activated carbon treatment, adsorption resin treatment, ion exchange resin treatment, or the like. The obtained extract can be used as an active ingredient of a singlet oxygen scavenger, a hyaluronidase inhibitor or a hexosaminidase release inhibitor as it is, but a concentrated solution or a dried product is easier to use.

  The uranium extract and the kanran extract have a specific odor and taste, and thus it is possible to perform purification for the purpose of decolorization, deodorization, etc. within a range that does not cause a decrease in the physiological activity. When blended into skin cosmetics, etc., it is not used in large quantities, so there is no practical problem even if it is not purified.

  The uranium extract and kanran extract obtained as described above have a singlet oxygen scavenging action, a hyaluronidase inhibitory action, or a hexosaminidase release inhibitory action. It can be used as an active ingredient of a quencher, hyaluronidase inhibitor or hexosaminidase release inhibitor.

  The singlet oxygen scavenger, hyaluronidase inhibitor or hexosaminidase release inhibitor of the present invention may contain a uranium extract or a kanran extract, or a mixture of a uranium extract and a kanran extract. May be included. When a uranium extract and a kanran extract are mixed and contained, the mixing ratio may be adjusted as appropriate. In addition, the singlet oxygen scavenger, hyaluronidase inhibitor or hexosaminidase release inhibitor of the present invention may be composed only of uranium extract and / or kanran extract, and / or uranium extract and / or The kanran extract may be formulated.

  The uranium extract and / or kanran extract can be used in any dosage form such as powder, granule, liquid, etc. according to a conventional method using a pharmaceutically acceptable carrier such as dextrin and cyclodextrin and any other auxiliary agent. Can be formulated. In this case, as an auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring agent and the like can be used. Further, the uranium extract and / or kanran extract can be used by blending with other compositions (for example, skin cosmetics, etc.), and can also be used as an ointment, a liquid for external use, a patch and the like. it can.

  The singlet oxygen scavenger of the present invention can be used as an active ingredient by blending with other natural extracts having a singlet oxygen scavenging action, if necessary. Moreover, the hyaluronidase inhibitor of this invention can use the other natural extract which has a hyaluronidase inhibitory effect as an active ingredient as needed. Furthermore, the hexosaminidase release inhibitor of the present invention can use, as necessary, another natural extract having a hexosaminidase release inhibitory action as an active ingredient.

  The singlet oxygen scavenger of the present invention can scavenge singlet oxygen generated in the living body through the singlet oxygen scavenging action of the uranium extract and / or kanran extract, Generation can be suppressed. As a result, skin aging symptoms such as wrinkle formation and reduced elasticity, skin pigmentation, and the like can be prevented, treated, or improved. However, the singlet oxygen scavenger of the present invention can be used for all purposes other than these uses that are meaningful for exerting a singlet oxygen scavenging action.

  The hyaluronidase inhibitor of the present invention can inhibit the activity of hyaluronidase through the hyaluronidase inhibitory action of the uranium extract and / or kanran extract, thereby preventing, treating or improving inflammatory diseases. it can. However, the hyaluronidase inhibitor of the present invention can be used for all purposes other than these uses that are meaningful for exerting a hyaluronidase inhibitory action.

  The hexosaminidase release inhibitor of the present invention can suppress the release of hexosaminidase through the hexosaminidase release inhibitory action of the uranium extract and / or kanran extract. Sexual diseases can be prevented, treated or ameliorated. However, the hexosaminidase release inhibitor of the present invention can be used for all purposes that are meaningful for exerting a hexosaminidase release inhibitory action in addition to these uses.

  The singlet oxygen scavenger, the hyaluronidase inhibitor and the hexosaminidase release inhibitor of the present invention are preferably applied to humans, but as long as the respective effects are exerted, other than humans. It can also be applied to other animals.

  Hereinafter, although a manufacture example, a test example, and a compounding example are shown and this invention is demonstrated concretely, this invention is not limited to each following example at all.

[Production Example 1] Production of uranium extract 10 g of coarsely pulverized uranium leaf was added to 100 mL of extraction solvent, heated and extracted for 2 hours at 80 ° C. with gentle stirring, and filtered while hot. . The obtained filtrate was concentrated under reduced pressure at a temperature of 40 ° C., and further dried with a vacuum dryer to obtain a uranium extract. Table 1 shows the yield of each extract when water, 50% by mass ethanol (mass ratio of water to ethanol = 1: 1), and ethanol were used as the extraction solvent.

[Production Example 2] Production of kanran extract 10 g of coarsely pulverized kanran leaves were put into 100 mL of extraction solvent, heated and extracted for 2 hours at 80 ° C. with gentle stirring, and filtered while hot. . The obtained filtrate was concentrated under reduced pressure under a temperature condition of 40 ° C., and further dried with a vacuum dryer to obtain a kanran extract. Table 1 shows the yield of each extract when water, 50% by mass ethanol (mass ratio of water to ethanol = 1: 1), and ethanol were used as the extraction solvent.

[Table 1]
Sample extraction raw material extraction solvent extract yield (%)
1 Uranium Water 22.1
2 Uranium 50% ethanol 27.0
3 Uranium Ethanol 10.2
4 Kanran Water 29.4
5 Kanran 50% ethanol 35.6
6 Kanran Ethanol 10.3

[Test Example 1] Singlet oxygen scavenging action test The uranium extract (Samples 1 to 3) obtained in Production Example 1 and the kanran extract (Samples 4 to 6) obtained in Production Example 2 were as follows. The singlet oxygen scavenging action was tested.

5 mL of 2% red blood cell suspension in a clear glass bottle (10 mL volume), 5 mL of an isotonic phosphate buffer solution of pH 7.4 containing a sample (Samples 1 to 6) at a predetermined concentration, and a photosensitizer (10 mM hematoporphyrin- 0.01 mL of 20 mM sodium hydroxide solution) was mixed. The resulting solution was uniformly irradiated on a merry-go-round with a 7.5 W halogen lamp for 35 minutes to generate singlet oxygen ( ¹ O ₂ ), thereby causing red blood cell hemolysis. 1 mL of this reaction solution was collected, 2 mL of isotonic phosphate buffer was added and mixed, and then centrifuged at 4 ° C. and 3000 rpm for 5 minutes.

  Next, the supernatant was collected and the absorbance at a wavelength of 540 nm was measured. Separately, 1 mL of the above reaction solution in which red blood cells were partially hemolyzed was taken, and 2 mL of distilled water was added thereto to completely hemolyze it, and the absorbance was measured in the same manner. From the measured absorbance, the singlet oxygen elimination rate (%) was calculated by the following formula.

Singlet oxygen elimination rate (%) = (1−B / A) × 100
In the formula, A represents “the absorbance of the control”, and B represents “the absorbance of the reaction solution supernatant”.

For each sample (sample 1-6), the sample concentration stepwise decreased calculated singlet oxygen scavenging rate above a (%), sample concentration IC ₅₀ for singlet oxygen erasing rate (%) is 50% ( μg / mL) was determined by interpolation.
The results are shown in Table 2.

[Table 2]
Sample IC ₅₀ (μg / mL)
1 240.4
2 165.9
3 284.6
4 173.6
5 109.6
6 228.4

  As shown in Table 2, it was confirmed that the uranium extract and kanran extract have an excellent singlet oxygen scavenging action. It was also confirmed that the degree of singlet oxygen scavenging action of uranium extract and cannula extract can be adjusted by the concentration of the extract.

[Test Example 2] Hyaluronidase Inhibitory Action Test With respect to the uranium extract obtained in Production Example 1 (Samples 1 to 3) and the kanran extract obtained in Production Example 2 (Samples 4 to 6), hyaluronidase was treated as follows. Inhibitory effects were tested.

  0.1 mL of a hyaluronidase solution (Type IV-S (from bovine testis, manufactured by SIGMA, 400 U / mL)) was added to 0.2 mL of 0.1 mol / L acetate buffer (pH 3.5) in which the sample was dissolved. The reaction was carried out for 20 minutes under the temperature condition of ° C. Further, 0.2 mL of 2.5 mmol / L calcium chloride was added as an activator, and the mixture was reacted for 20 minutes at a temperature of 37 ° C. To this was added 0.5 mL of 0.4 mg / mL potassium hyaluronate solution (from rooster comb), and the mixture was reacted at 37 ° C. for 40 minutes.

  Thereafter, 0.2 mL of 0.4 mol / L sodium hydroxide was added to stop the reaction and cooled, and then 0.2 mL of 0.8 mol / L boric acid solution (pH 9.1) was added to each reaction solution for 3 minutes. Boiled. After cooling with ice for 20 minutes, 6.0 mL of p-DABA reagent (10 g of p-dimethylaminobenzaldehyde dissolved in a mixture of 12.5 mL of 10N hydrochloric acid and 87.5 mL of acetic acid and diluted 10-fold with acetic acid) was added. , And reacted for 20 minutes at a temperature of 37 ° C. Thereafter, the absorbance at a wavelength of 585 nm was measured. The same operation and absorbance measurement were performed when no enzyme solution was added, and the same operation and absorbance measurement were performed when no sample solution was added. From the obtained results, the hyaluronidase inhibition rate (%) was calculated by the following formula.

Hyaluronidase inhibition rate (%) = {1− (St−Sb) / (Ct−Cb)} × 100
In the formula, St represents “absorbance when enzyme solution is added / sample solution added”, Sb represents “absorbance when enzyme solution is not added / sample solution is added”, and Ct is “when enzyme solution is added / sample solution is not added” "Cb" represents "absorbance when no enzyme solution is added / sample solution is not added".

For each sample (samples 1 to 6), the concentration of the sample solution was decreased stepwise to calculate the hyaluronidase inhibition rate (%), and the sample concentration IC ₅₀ (μg / g) at which the hyaluronidase inhibition rate (%) was 50%. mL) was determined by interpolation.
The results are shown in Table 3.

[Table 3]
Sample IC ₅₀ (μg / mL)
1 886.2
2 804.4
3 974.4
4 238.2
5 221.6
6 368.0

  As shown in Table 3, it was confirmed that the uranium extract (samples 1 to 3) and the kanran extract (samples 4 to 6) have a hyaluronidase inhibitory action. In particular, it was confirmed that the kanran extract has an excellent hyaluronidase inhibitory action. It was also confirmed that the degree of hyaluronidase inhibitory action of uranium extract and kanran extract can be adjusted by the concentration of the extract.

[Test Example 3] Hexosaminidase release inhibitory action test About the uranium extract obtained in Production Example 1 (Samples 1 to 3) and the kanran extract obtained in Production Example 2 (Samples 4 to 6), the following Thus, the inhibitory effect on hexosaminidase release was tested.

S-MEM medium containing 15% FBS was seeded with 1.0 × 10 ⁶ rat basophil leukemia cells (RBL-2H3) and cultured at 37 ° C. and 5% CO ₂ -95% air for 4 days. The cells were cultured and cells were collected by trypsin treatment. The collected cells are diluted to a cell density of 4.0 × 10 ⁵ cells / mL so that the final concentration of mouse monoclonal anti-dinitrophenyl group IgE (DNP-specific IgE) is 0.5 μg / mL. After adding DNP-specific IgE, 100 μL per well was seeded in a 96-well plate and cultured overnight at 37 ° C. and 5% CO ₂ -95% air. After completion of the culture, the medium was removed and washing was performed twice with 500 μL of Silaganian buffer.

  Next, 30 μL of the Siraganian buffer and 10 μL of the sample solution in which the sample (Samples 1 to 6) was dissolved were added to the same buffer, and the mixture was allowed to stand at 37 ° C. for 10 minutes. Thereafter, 10 μL of a 100 ng / mL dinitrophenylated bovine serum albumin (DNP-BSA) solution was added, and the mixture was allowed to stand at 37 ° C. for 15 minutes to release hexosaminidase. Thereafter, the release of hexosaminidase was stopped by allowing the 96-well plate to stand on ice. 10 μL of cell supernatant in each well and 10 μL of 1 mmol / L p-nitrophenyl-N-acetyl-α-D-glucosaminide (p-NAG) solution were added to a new 96-well plate, and the temperature was maintained at 37 ° C. For 1 hour.

After completion of the reaction, 250 μL of a 0.1 mol / L Na ₂ CO ₃ —Na ₂ HCO ₃ solution was added, and the absorbance was measured at 415 nm using a microplate reader with 650 nm as a control (the absorbance measured at this time is A). ). Further, the same treatment and absorbance measurement were performed on the cell supernatant to which the Siraganian buffer was added instead of the sample solution (the absorbance measured at this time is B). Absorbance measurement was also performed on the cell supernatant and 0.1 mol / L Na ₂ CO ₃ —Na ₂ HCO ₃ solution reacted in the same manner (the absorbance measured at this time is C). In addition, the same treatment and absorbance measurement were also performed on a sample in which a silaganian buffer was added instead of DNP-BSA (the absorbance measured at this time is D).

From the obtained results, the hexosaminidase release inhibition rate (%) was calculated by the following formula.
Inhibition rate of hexosaminidase release (%) = {1− (A−C−D) / (B−D)} × 100

For each sample (samples 1 to 6), the concentration of the sample solution was decreased stepwise to calculate the hexosaminidase release inhibition rate (%), and the hexosaminidase release inhibition rate (%) was 50%. The value of the sample concentration IC ₅₀ (μg / mL) was calculated by interpolation.
The results are shown in Table 4.

[Table 4]
Sample IC ₅₀ (μg / mL)
1 515.2
2 462.4
3 550.6
4 401.0
5 356.6
6 456.4

  As shown in Table 4, it was confirmed that the uranium extract (samples 1 to 3) and the kanran extract (samples 4 to 6) have an excellent hexosaminidase release inhibitory action. In addition, it was confirmed that the degree of hexosaminidase release inhibitory action of the uranium extract and kanran extract can be adjusted by the concentration of the extract.

The singlet oxygen scavenger of the present invention is useful for prevention, treatment or improvement of skin aging symptoms such as wrinkle formation and reduced elasticity, skin pigmentation, etc., and the hyaluronidase inhibitor and hexosaminidase of the present invention The release inhibitor is useful for prevention, treatment or improvement of various inflammatory diseases.

Claims (2)

A hyaluronidase inhibitor (excluding anti-inflammatory use) characterized by containing an extract from the leaves of mandarin as an active ingredient. A hexosaminidase release inhibitor (excluding anti-inflammatory use) characterized by containing an extract from the leaves of mandarin as an active ingredient.