JP6487613B1 - Method for producing IL-33 expression inhibitor - Google Patents

Abstract

A method for producing an IL-33 expression inhibitor is provided. A method for producing an IL-33 expression inhibitor includes a step of preparing bamboo chips, a step of roasting the prepared bamboo chips at 50 to 89 ° C, and a step of drying the roasted bamboo chips. And a step of extracting the active ingredient from the bamboo chip by immersing the dried bamboo chip in an extraction solvent at 5 to 39 ° C. [Selection] Figure 1

Description

  The present invention relates to a method for producing an IL-33 (interleukin 33) expression inhibitor, and particularly relates to a method for producing an IL-33 expression inhibitor used for atopic dermatitis.

  Japanese Patent Laying-Open No. 2005-200393 (Patent Document 1) discloses a cream-like composition containing bamboo vinegar. Patent Document 1 states that “bamboo vinegar has antibacterial and antioxidant functions, therapeutic effects on atopic dermatitis and athlete's foot, blood glucose level and liver function, osteoporosis, periodontal disease and stomatitis. It is a material that has been expected from its unique effects such as anti-deodorant effect, deodorizing effect, etc., and it has been used by mixing bamboo vinegar liquid with water and alcohol. There is a disadvantage that it is uncomfortable when applied to the skin, and it is difficult to apply to the skin because it is liquid, making it inconvenient to carry. "(Paragraph 0002)," Bamboo vinegar is produced by dry distillation of bamboo. It can also be obtained as a liquid that accumulates at the bottom of the autoclave when it is pulverized into fibers by repeatedly pressing and releasing the pressure with hot steam. They are described that can be. "(See paragraph 0009).

  Japanese Patent Application Laid-Open No. 2003-221786 (Patent Document 2) discloses an external preparation for skin containing an extract of bamboo as an active ingredient. In Patent Document 2, “a pulverized product obtained by cutting these bamboos into chips is used as a raw material of the present invention. The pulverized product is used in a powdery form in consideration of the extraction operation. (Paragraph 0006) and "in the present invention, the bamboo pulverized product is treated with water or an organic solvent and the extract is used" (paragraph 0007).

  Japanese Translation of PCT International Publication No. 2010-505944 (Patent Document 3) discloses a composition for treating atopic dermatitis containing a bamboo extract and a Koganebana extract. Patent Document 3 describes the production of bamboo extract as follows: “To 20 kg of dried bamboo, 200 L of 25% ethanol was added and the mixture was extracted by heating for 6 hours at 80 ° C. The extract was filtered and extracted. The ethanol was removed by concentration until the volume of the liquid was about 5 L. The concentrated extract was then cooled to room temperature and the precipitate was collected and dried to give 390 g of bamboo extract. 0031), “To 20 kg of dried bamboo, 10 times the weight of water was added and the mixture was extracted by heating for 4 hours at 100 ° C. The extract was filtered and concentrated under reduced pressure. 10 L of ethanol was added to the liquid, stirred for 2 hours at 70 ° C., and then cooled to room temperature.The precipitate was filtered and concentrated under reduced pressure to obtain 350 g of bamboo extract. ”(Paragraph 0032). Yes.

  JP 2010-116377 A (Patent Document 4) discloses an antibacterial composition. The antibacterial composition is an antibacterial agent against methicillin-resistant Staphylococcus aureus (MRSA) and contains a bamboo extract. Patent Document 4 states that “a bamboo extract can be obtained by stirring a powdered Moso bamboo bamboo shoot portion in an aqueous solvent such as ethanol and filtering the supernatant obtained by centrifugation. Also, the bamboo extract may be extracted with an extraction solvent containing ethanol after the bamboo plant is steamed at 120 to 180 ° C. in the presence of steam and cooled. (Paragraph 0021).

  JP 2011-46636 A (Patent Document 5) discloses an antiviral composition. Patent Document 5 states that “An antiviral composition contains a bamboo extract. And, the bamboo extract is obtained by (a) obtaining a liquid extract by extracting a bamboo plant with an aqueous solvent; (B) obtained by a method comprising: removing the aqueous solvent from the extract to obtain a dried product; and (c) obtaining a water-soluble fraction by dissolving the dried product in water. (Paragraph 0033).

  On the other hand, the involvement of IL-33 (interleukin 33), which is a kind of cytokine, has been pointed out in the onset of atopic dermatitis (for example, WO2014 / 178392A1 (Patent Document 6), WO2015 / 099175A1 (Patent Document) 7), Japanese Unexamined Patent Application Publication No. 2018-118965 (Patent Document 8)). That is, keratinocytes of the skin produce IL-33, and as a result, it is considered that in addition to atopic dermatitis, inflammatory diseases such as hay fever, asthma, allergic rhinitis and inflammatory bowel disease are caused. .

Japanese Patent Laid-Open No. 2005-200393 JP 2003-212786 A Special table 2010-505944 JP 2010-116377 A JP 2011-46636 A WO2014 / 178392A1 publication WO2015 / 099175A1 publication JP 2018-118965 A

  As described above, bamboo vinegar used as an external preparation for atopic dermatitis has a unique smoked odor, so that some people may feel that the odor is unpleasant. Then, this inventor examined the improvement effect of atopic dermatitis using the bamboo extract instead of the bamboo vinegar which has a smoked smell. As a result, the present inventors have obtained a new finding that an active ingredient that suppresses the expression of IL-33 can be extracted from bamboo under specific production conditions.

  An object of the present invention is to provide a method for producing an IL-33 expression inhibitor.

  The method for producing an IL-33 expression inhibitor according to the present invention includes a step of preparing bamboo chips, a step of roasting the prepared bamboo chips at 50 to 89 ° C., and a step of drying the roasted bamboo chips. And a step of extracting the active ingredient from the bamboo chip by immersing the dried bamboo chip in an extraction solvent at 5 to 39 ° C.

FIG. 1 is a graph showing test results for verifying the IL-33 expression suppression effect of each sample shown in Table 1. FIG. 2 is a graph showing test results for verifying the IL-33 expression suppression effect of each sample shown in Table 2.

  Embodiments of the present invention will be described below.

  The method for producing an IL-33 expression inhibitor according to the present embodiment includes (1) a step of preparing bamboo chips, (2) a step of roasting the prepared bamboo chips at 50 to 89 ° C., and (3 ) Drying the roasted bamboo chips, and (4) extracting the active ingredients from the bamboo chips by immersing the dried bamboo chips in an extraction solvent at 5 to 39 ° C.

[Bamboo chips]
Bamboo chips are prepared by crushing bamboo to an appropriate size. The type of bamboo used is not particularly limited.For example, the genus Mosochiku, Madoake, Hachiku, Hosochiku, Shihochiku, Shudosasa, Sasamorpha, Narihirada, Tochiku, Okamesa, Sasa, Azumasa, Bamboo belonging to the genus Yadatake, Medaka, Kanchiku, Horaichiku and the like can be used. The part of the bamboo used is not particularly limited, and an epidermis or bamboo can be used.

  In addition, you may ferment bamboo chip | tip before roasting mentioned later.

[Roasting]
When the roasting temperature is 50 to 89 ° C. (lower than the general roasting temperature), an active ingredient having an excellent IL-33 expression inhibitory effect can be extracted as shown in Examples described later. The roasting temperature should just be lower than 90 degreeC, a preferable upper limit is 89 degreeC, and a more preferable upper limit is 85 degreeC, 80 degreeC, 75 degreeC, and 70 degreeC. Moreover, the minimum with a preferable roasting temperature is 50 degreeC, and a more preferable minimum is 55 degreeC and 60 degreeC. Although roasting time is not specifically limited, For example, it is 5 to 60 minutes, Preferably it is 25 to 35 minutes.

[Dry]
The drying is not particularly limited, but if it is sun-dried, an active ingredient having an excellent IL-33 expression inhibitory effect can be extracted as shown in Examples described later.

  The weather at the time of sun drying is not particularly limited as long as the sunlight reaches the bamboo chip. Moreover, the bamboo chip may be behind the container containing it. Although the sun drying time is not particularly limited, it is, for example, half a day to 10 days, preferably 1 to 5 days. The average sunshine time per day is about 12 hours. Moreover, you may irradiate a bamboo chip | tip with an ultraviolet-ray instead of sun-drying.

[Extract]
The extraction solvent is not particularly limited, and is, for example, an organic solvent, preferably an alcohol such as ethyl alcohol or polyhydric alcohol, and more preferably butylene glycol (BG) or hexylene glycol (HG). The extraction solvent may be water.

  If the extraction temperature (the temperature of the extraction solvent at the time of extracting the active ingredient) is 5 to 39 ° C., an active ingredient that is excellent in the IL-33 expression inhibitory effect can be extracted as shown in the Examples described later. The extraction temperature should just be lower than 40 degreeC, a preferable upper limit is 39 degreeC, and a more preferable upper limit is 38 degreeC, 36 degreeC, 34 degreeC, 32 degreeC, 30 degreeC. Moreover, the minimum with preferable extraction temperature is 5 degreeC, and a more preferable minimum is 10 degreeC, 15 degreeC, 20 degreeC, 25 degreeC, and 30 degreeC.

  Although extraction time (time which extracts an active ingredient) is not specifically limited, For example, it is 1 to 25 days, Preferably it is 5 to 15 days.

  The extraction pressure (pressure applied to the extraction solvent when extracting the active ingredient) is not particularly limited, but is preferably normal pressure (1013 hPa).

[filtration]
After extraction, it is preferable to remove bamboo chips by filtering the bamboo extract containing the active ingredient.

[Bamboo chip size]
In the above, since the size of the prepared bamboo chips varies, it is preferable to sort the bamboo chips by size before extraction. Bamboo chips may be selected after extraction, after roasting or after drying, or before roasting. The size of the bamboo chip is not particularly limited, but preferred upper limits are about 10 mm, about 8 mm, about 6 mm, and about 4 mm. In particular, if the size of the bamboo chip is 4 mm or less, it is considered that an effective ingredient having an excellent IL-33 expression suppression effect can be easily extracted. On the other hand, if the bamboo chip is too large, it will be difficult to extract the active ingredient. The lower limit of the size of the bamboo chip is not particularly limited, but if the bamboo chip is too small (for example, less than 2 mm), the bamboo chip (or bamboo powder) will absorb the extraction solvent and expand, which is effective. It is thought that it becomes difficult to extract an ingredient.

  For example, a bamboo chip of less than 2 mm is a bamboo chip that has passed through a sieve having an opening of 2 mm. Bamboo chips of less than 4 mm are bamboo chips that have passed through a sieve having an opening of 4 mm. Bamboo chips of less than 10 mm are bamboo chips that have passed through a sieve having an opening of 10 mm. Bamboo chips of 10 mm or more are bamboo chips that did not pass through a sieve having an opening of 10 mm. However, since bamboo chips may pass through the sieve almost upright, bamboo chips of 2 mm or more pass through a sieve having a 2 mm opening, or bamboo chips of 4 mm or more have a 4 mm opening. In some cases, bamboo chips of 10 mm or more pass through a sieve having an opening of 10 mm. Also, bamboo chips of less than 2 mm do not pass through a sieve having an opening of 2 mm, bamboo chips of less than 4 mm do not pass through a sieve having an opening of 4 mm, or bamboo chips of less than 10 mm have an opening of 10 mm Or may not pass through a sieve having. Therefore, regarding the size of the bamboo chip, there is no strict meaning such as “less than”, “more than”, “less than”, etc., and generally only means such a size.

  A 2-4 mm bamboo chip is a bamboo chip that has passed through a sieve having a 4 mm opening but has not passed through a sieve having a 2 mm opening. Bamboo chips of 2 to 10 mm are bamboo chips that have passed through a sieve having an opening of 10 mm but have not passed through a sieve having an opening of 2 mm. A 4-10 mm bamboo chip is a bamboo chip that has passed through a sieve having an opening of 10 mm but has not passed through a sieve having an opening of 4 mm. Thus, by properly using one or two or more types of sieves, bamboo chips having a desired size can be selected.

[Density adjustment]
Finally, water or the like may be added to the obtained bamboo extract to adjust the concentration of the bamboo extract.

[Usage]
The IL-33 expression inhibitor (bamboo extract containing an active ingredient having an IL-33 expression inhibitory effect) obtained by the above production method is preferably used for atopic dermatitis.

  Examples of the agent for atopic dermatitis include an external preparation, an internal preparation (internal use), an injection, and the like that relieve symptoms such as skin pruritus caused by atopic dermatitis. A topical preparation for atopic dermatitis is produced, for example, in a cream, lotion or powder form.

  The IL-33 expression inhibitor obtained by the above production method is also used in a preparation for alleviating inflammatory diseases such as hay fever, asthma, allergic rhinitis and inflammatory bowel disease.

  As mentioned above, embodiment mentioned above is only the illustration for implementing this invention. Therefore, the present invention is not limited to the above-described embodiment, and can be implemented by appropriately modifying the above-described embodiment without departing from the spirit thereof.

[Example 1]
As shown in the following Table 1, test samples 1 to 7 which are bamboo extracts and reference samples 1 to 3 for relatively comparing the expression levels of these IL-33 were prepared.

[Evaluation]
Using human epidermal keratinocytes, the IL-33 expression inhibitory effect of test samples 1 to 7 was verified.

<Used cells>
Adult-derived normal human epidermal keratinocytes (Kurabo, KK-4109)

<Medium>
Add 500 mL medium (Kurabo, HuMedia-KG2) with growth additives (10 μg / m insulin, 0.67 μg / m hydrocortisone) and antibacterial agent (50 μg / mL gentamicin, 50 ng / mL amphotericin) Prepared.

<Test method>
Normal human epidermal cells derived from pre-cultured adult cells were put to sleep in a T-75 flask using HuMedia-KG2 (medium) and cultured in a CO ₂ incubator (5% CO ₂ , 37 ° C., wet). The medium was changed every other day, and when the cells reached 80% confluence, the cells were collected and used for the test.

IL-33 expression induction test cells were obtained at 2 × 10 ⁴ cells / 0. The medium was adjusted to 1 mL / well, seeded in a 96-well plate, and cultured in a CO ₂ incubator (5% CO ₂ , 37 ° C.). Thereafter, TNFα and IFNγ were added to the medium to a final concentration of 10 ng / mL (reference sample 2), RNA was extracted after 6 hours, and the expression of IL-33 was measured. RNA extraction and real-time PCR were performed by known methods. The experiment was performed with n = 2.

The test cells were prepared in a medium so as to be 2 × 10 ⁴ cells / 0.1 mL / well and seeded in a 96-well plate. The cells were cultured in a CO ₂ incubator (5% CO ₂ , 37 ° C.). Thereafter, TNFα and IFNγ were adjusted to a final concentration of 10 ng / mL with respect to the medium, and samples 1 to 8 were cultured for 6 hours to a final concentration of 0.01% to extract RNA. The test was performed at n = 3. Recovery of total RNA from cells and gene expression analysis by real-time PCR were performed.

<Reference sample>
Reference sample 1: nothing was added to the medium.
Reference sample 2: TNFα and IFNγ were added to the medium to a final concentration of 10 ng / mL.
Reference sample 3: TNFα and IFNγ were added to the medium to a final concentration of 10 ng / mL, and an aqueous solution of butylene glycol having a concentration of 50% was added to the medium.

<Test sample>
Test samples 1 to 7 prepared under the production conditions shown in Table 1 were added to the medium so that the final concentration was 0.1%. As the extraction solvent, an aqueous solution of butylene glycol having a concentration of 50% was used. The aqueous solution of butylene glycol was prepared by mixing 50 g of water and 50 g of butylene glycol. Then, 25 g of bamboo chips were added to 100 g of the resulting aqueous solution of butylene glycol.

Calculation method of relative quantification Ct value (number of PCR cycles) is calculated from the intersection of the amplification curve and the threshold line of each gene. Ct (target gene) −Ct (GAPDH) = ΔCt value obtained by subtracting the Ct value of the internal standard GAPDH gene from the Ct value of the target gene. Further, ΔCt (sample processing section) −ΔCt (blank section) = ΔΔCt value obtained by subtracting the average ΔCt value of blanks from the ΔCt value. The 2- ^ΔΔCt value obtained by substituting the ΔΔCt value into the multiplier term is the relative expression level.

<Test results>
The results of this test are shown in FIG. Reference sample 2 (in which only TNFα and IFNγ were added to the medium) increased the expression level of IL-33 to about 6 times that of reference sample 1 (in which nothing was added to the medium). Reference sample 3 (added only with a butylene glycol aqueous solution) was found to have an effect of suppressing the increase in IL-33 expression by TNFα + IFNγ to about one half.

  In particular, since the expression of IL-33 was lower than that of the reference sample 3 in the test samples 1, 5, and 7, the test samples 1, 5, and 7 have an effect of suppressing the increase in the expression of IL-33 by TNFα + IFNγ. It was considered. Since the expression of IL-33 was significantly increased in the non-added group and the group added with only TNFα + IFNγ, the cell response was normal, and this test was considered to have been performed without problems.

  However, in test samples 2, 3, 4 and 6, a sufficient IL-33 expression inhibitory effect was not observed. If the bamboo chips are not roasted as in test sample 2, it is considered that the active ingredient that suppresses the expression of IL-33 cannot be sufficiently extracted. In addition, when the roasting temperature is too high as in test sample 3 (for example, 90 ° C. or higher), bamboo protein begins to denature, and as a result, an active ingredient that suppresses the expression of IL-33 is sufficiently extracted. It is considered impossible. Therefore, it was found that the roasting temperature is preferably lower than 90 ° C.

  The roasting temperature was measured without contact with the temperature of the bamboo chips during roasting using an infrared radiation thermometer. In test samples 1 and 4 to 7, the roasting temperature was adjusted to be slightly higher than 60 ° C, but not to exceed 70 ° C. In test sample 3, the roasting temperature was adjusted to be slightly higher than 90 ° C but not to exceed 100 ° C.

  Moreover, it is thought that the active ingredient which suppresses expression of IL-33 cannot fully be extracted unless the bamboo chip is dried as in test sample 4. In addition, when the extraction temperature is too high as in the test sample 6 (for example, 40 ° C. or higher), it is considered that components that interfere with the IL-33 expression suppression effect are also extracted. Therefore, it was found that the extraction temperature is preferably lower than 40 ° C.

[Example 2]
Example 1 above used an alcohol extraction method in which the active ingredient is extracted from the bamboo chip by immersing the bamboo chip in the aqueous solution of butylene glycol, but instead of this, from the bamboo chip by immersing the bamboo chip in water. You may use the water extraction method which extracts an active ingredient.

  As shown in the following Table 2, test sample 8 which is a bamboo extract and reference samples 4 to 6 for relatively comparing the expression levels of IL-33 were prepared.

  Using human epidermal keratinocytes, the IL-33 expression inhibitory effect of test sample 8 was verified. Reference sample 4 is obtained by adding TNFα and IFNγ to the medium and corresponds to reference sample 2 of Example 1 above. The reference sample 5 is obtained by adding TNFα and IFNγ and water to the medium, and corresponds to the reference sample 3 of Example 1 except that water is added to the medium. The reference sample 6 corresponds to the reference sample 3 of the first embodiment. Test sample 8 is the same as test sample 1 of Example 1 above, except that it is a bamboo extract obtained by the water extraction method and that water is added to the medium.

  However, in Example 2, TNFα and IFNγ were added to the medium to a final concentration of 10 ng / mL. Moreover, the extraction pressure of the test sample 8 was 100 MPa. Moreover, the test sample 8 was added to the culture medium so that it might become final concentration 0.1%.

  The results of this test are shown in FIG. Reference sample 5 (with only water added) was found to have an effect of suppressing the increase in IL-33 expression by TNFα + IFNγ to about one third. Reference sample 6 (added with only a butylene glycol aqueous solution) was found to have an effect of suppressing the increase in IL-33 expression by TNFα and IFNγ to about 3/5. On the other hand, since the expression of IL-33 in test sample 8 was significantly lower than that of reference sample 5, it is considered that test sample 8 has the effect of significantly suppressing the increase in IL-33 expression caused by TNFα + IFNγ. It was.

Claims (4)

Preparing bamboo chips;
Roasting the prepared bamboo chips at 50-89 ° C .;
Drying the roasted bamboo chips;
And a step of extracting an active ingredient from the bamboo chip by immersing the dried bamboo chip in an extraction solvent at 5 to 39 ° C. The manufacturing method according to claim 1,
The said extraction solvent is a manufacturing method of the IL-33 expression inhibitor containing a butylene glycol aqueous solution or water. The manufacturing method according to claim 1 or 2,
The drying step is a method for producing an IL-33 expression inhibitor, wherein the bamboo chips are sun-dried. It is a manufacturing method given in any 1 paragraph of Claims 1-3,
The said IL-33 expression inhibitor is a manufacturing method of the IL-33 expression inhibitor used for atopic dermatitis.

Images