JP5578599B2 - Th1 immune function enhancer - Google Patents

Description

  The present invention relates to an immune balance regulator containing a solvent extract from Hokkaido larch.

  Japanese larch (Larix leptolepis) is a plant belonging to the genus Larix, also called Larix kaempferi. In Japan, it is mainly used as a plantation tree species in relatively cold regions such as the Chubu mountainous region, the sub-alpine mountain region, Tohoku, and Hokkaido.

  The main use of Japanese larch is wood for housing or furniture. Hokkaido larch planted and grown especially in Hokkaido has characteristics not found in Honshu larch, which is excellent in strength and good quality wood by spending several times in the cold season.

On the other hand, regarding the physiological use of Japanese larch itself or an extract thereof, an invention relating to a skin whitening agent having a whitening effect including a solvent extract of larch bark (Patent Document 1), a moisturizing property and anti-resistance containing a larch extract component The invention relating to cosmetics having an oxidizing action (Patent Document 2) is just to the public, and so far it has not attracted attention.
Japanese Patent Laid-Open No. 10-25238 JP 2006-52197 A

  An object of this invention is to provide the new use of the extract of Hokkaido larch.

  The present inventors administer a solvent extract from Hokkaido larch to mice, thereby adjusting the immune balance in addition to infection protection and antitumor activity by the type 1 immunostimulatory effect. The inventors have found that it has an effect of improving allergic diseases caused by immune responses, and has completed the following inventions.

  (1) An immune balance regulator containing a solvent extract from Larix leptolepis from Hokkaido.

  (2) The immune balance regulator according to (1), which is for anti-infection.

  (3) The immune balance regulator according to (1), which is for antitumor use.

  (4) The immune balance regulator according to (1), which is for enhancing Th1 immune function.

  (5) The immune balance regulator according to (4), which is for promoting production of TNFα and / or interleukin 12.

  (6) The immune balance regulator according to any one of (1) to (5), wherein the solvent is water.

  The immune balance regulator of the present invention is a composition having a natural ingredient called a solvent extract of Hokkaido larch as an active ingredient and having an effect of normalizing by adjusting the immune balance of a living body with extremely high safety. .

  The present invention is an immune balance regulator containing a solvent extract from Hokkaido larch (Larix leptolepis). In Japan, Japanese larch (Japanese larch) is a plant grown or planted from the mountainous region of the Chubu mountainous area to the subalpine zone, Tohoku, Hokkaido, etc. The larch used in the present invention is planted and grown in Hokkaido. Hokkaido larch.

  The composition of the present invention comprises a solvent extract from Hokkaido larch. As the extraction solvent, water or a solvent miscible with water such as lower alcohols such as methanol and ethanol, polyhydric alcohols such as propylene glycol and 1,3-butylene glycol, etc. are used alone, or two or more kinds of mixed solvents. Can be mentioned. Preferred extraction solvents are water, alcohols, and mixtures thereof, with water being particularly preferred. The use of such a solvent has the advantage of being excellent in operability and safety during extraction.

  Solvent extraction can be done by adding water to pulverized Hokkaido larch to a suitable size of large sawdust and stirring at 4-50 ° C., normal pressure, preferably at room temperature for several minutes to several hours, or press machine Extraction extraction may be performed using. Water may be added in an amount that can agitate the pulverized product of Hokkaido larch after addition or more. The extract may be further filtered and concentrated for use, or may be dried by spray drying or freeze drying and used as a powder.

  The immune balance regulation in the present invention, that is, the action of regulating the immune balance is to eliminate one of the Th1 immune system function and Th2 immune system function, in particular, the state in which the Th2 immune system function is enhanced, and to function both immune system functions. Means an action that leads to a regulated state. The adjustment of the immune balance referred to in the present invention is used interchangeably with the adjustment of the immune balance.

  In general, the Th1 immune system is understood as cellular immunity involving Th1 cells (T helper 1 cells) induced by the presentation of antigen peptides from dendritic cells and macrophages that are antigen presenting cells and the action of IL-12. ing. Th1 cells produce IL-2, TNF-α, etc. in addition to cytokines that suppress the production of IgE antibodies through inhibition of Th2 cell differentiation and B cell maturation inhibition, such as IFN-γ. It has a function to activate and enhance the activity of antigen-presenting cells such as dendritic cells and macrophages. One Th2 immune system is understood as humoral immunity involving Th2 cells (T helper 2 cells) induced by the presentation of antigen peptides from macrophages that are antigen presenting cells and the action of IL-4. Th2 cells produce IL-5, IL-6, and IL-10 in addition to cytokines that increase IgE antibody production through B cell maturation, such as IL-4 and IL-13.

  IL-4 and IL-10 produced from Th2 cells are known to regulate each other's action so as to suppress production of IFN-γ from Th1 cells, and Th2 immune system functions If it is dominant, cellular immunity is suppressed, infection is likely to become severe, IgE antibody production through B cell maturation increases, and it is thought that it is easy to fall into an allergic constitution. Therefore, the disruption of the balance between the Th1 immune system function and the Th2 immune system function, in particular, the enhancement or superiority of the excessive Th2 immune system function, is not necessarily preferable for the living body.

  The solvent extract of Hokkaido larch used in the present invention has an action of promoting TNF-α production, an action of promoting the production of IL-12, and an action of promoting the macrophage aggregation to the same extent as LPS. Show. Therefore, by administering the immune balance regulator of the present invention to an individual with particularly enhanced Th2 immune function, the Th1 immune function can be enhanced, and as a result, the immune balance can be regulated. . As described above, the solvent extract of Hokkaido larch used in the present invention can be used as a Th1 immune system function enhancer that enhances the Th1 immune system function, and is also a TNF-α production promoter and IL-12 production. It can also be used as an accelerator.

  In addition, the physiological activity of the solvent extract of Hokkaido larch used in the present invention is surprisingly confirmed by the solvent extract from North American larch, which is a plant belonging to the same genus Larix. I can't.

  In addition, the solvent extract of Hokkaido larch used in the present invention can relieve a state in which the Th2 immune system function is superior, for example, allergies, by strengthening the Th1 immune function, or infectious diseases or malignant diseases. It is effective for treatment of diseases such as tumors that require enhanced cellular immunity.

  In addition, the solvent extract of Hokkaido larch used in the present invention can maintain the balance between the Th1 immune system function and the Th2 immune system function from the normal time by taking this, and external foreign substances such as infectious diseases can be maintained. It can be expected that it has an action to alleviate the invasion of humans, and to alleviate allergies and autoimmune diseases which are excessive immune response reactions.

  Specifically, the immune balance regulator of the present invention includes infectious diseases such as viruses and bacteria, inflammation, atopic dermatitis, rough skin, sensitive skin, hay fever, asthma, bronchial asthma, rhinitis, urticaria, etc. It can be expected to prevent, treat or improve symptoms of allergic diseases.

  The solvent extract of Hokkaido larch in the present invention can be used as an immune balance regulator as it is, and it can be combined with general excipients to give a composition. It is also possible to formulate and use in various dosage forms.

  The amount of the solvent extract of Hokkaido larch in the composition or dosage form is not particularly defined and varies slightly depending on the type, quality, and expected effect of the dosage form, but in the total amount of the composition or formulation, The dry solid content may be 1% to 99% by weight, preferably 10 to 99% by weight, and more preferably 50 to 99% by weight.

  The above-mentioned composition or various dosage forms include vitamins, herbal medicines, anti-inflammatory agents, antihistamines and other medicines as active ingredients in addition to Hokkaido larch extract, as necessary. There is no problem as a form.

  Excipients used in the formulation include, for example, oral solid preparations such as tablets and capsules, internal liquids such as aqueous solutions and suspensions, ointments, patches, lotions, creams, sprays, suppositories, Components that are widely known and used by those skilled in the art for each dosage form can be used in appropriate combination.

  The immune balance regulator of the present invention can be produced and used as a food, health food or supplement by combining with an appropriate food material.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated further in detail, this invention is not limitedly limited to this Example.

<Example 1>
840 L of water was added to a ground material of 149 kg of Hokkaido larch core (water content: about 45%), and after 180 minutes immersion extraction, the ground material was squeezed with a screw press to obtain 800 L of a water extract. This water extract was filtered through an MF membrane and then freeze-dried to obtain 2.8 kg of a water extract (dried) of Hokkaido larch.

<Comparative example>
A water extract (dried) of 2.5 kg of North American larch was obtained in the same manner as in Example 1 except that the Hokkaido larch of Example 1 was replaced with North American larch (Larix occidentalis).

<Test example>
(1) Promoting TNF-α production Intraperitoneal cells were collected from C57BL / 6 mice and added to a 96-well plate at 1 × 10 ⁵ cells / well. Non-adherent cells were removed after 3 hours incubation at 37 ° C. with 5% CO ₂ . A water extract of Hokkaido larch prepared in Example 1 (hereinafter referred to as the extract of Example 1) and a comparative example at a final concentration of 0.2 mg / mL or 2 mg / mL for adherent intraperitoneal cells A water extract of North American larch prepared in (1) was added (hereinafter referred to as a comparative extract). After culturing at 37 ° C. for 48 hours under the condition of 5% CO ₂ , TNF-α produced in the culture supernatant was measured using a commercially available ELISA kit together with the non-added condition (control).

  As a result, the TNF-α level was not enhanced in the group to which the extract of the comparative example was added as compared with the control, but in the group to which the extract of Example 1 was added, a significant TNF-α level was confirmed. A production promoting action was confirmed (FIG. 1).

(2) Peritoneal exudate cells (PEC) Macrophage aggregation action C57BL / 6 mice were intraperitoneally administered with thioglycolate, and peritoneal exudate cells (PEC) collected after 5 days were transferred to a 96-well plate at 1 × 10 ⁵ cells / After adding to wells and incubating at 37 ° C. under 5% CO ₂ for 3 hours, non-adherent cells were removed. The extract of Example 1 having a final concentration of 20 mg / mL was added to the obtained macrophages. A control group was prepared by adding lipopolysaccharide (LPS) to a final concentration of 1 μg / mL, and after 48 hours, cell morphology was observed using a phase contrast microscope.

  As a result, compared to the non-added group (control), significant cell aggregation was confirmed in the group to which the extract of Example 1 was added, but not in the LPS control group (FIG. 2).

(3) Spleen cell aggregation action Spleen cells collected from C57BL / 6 mice were added to a 96-well plate so as to be 5 × 10 ⁵ cells / well, and the extract of Example 1 having a final concentration of 20 mg / mL was further added. did. A control group was prepared by adding lipopolysaccharide (LPS) so that the final concentration was 1 μg / mL. After 48 hours, cell morphology was observed using a phase contrast microscope.

  As a result, cell aggregation was confirmed in the group to which the extract of Example 1 was added as compared to the non-addition group (control), but more marked cell aggregation was confirmed in the LPS control group (FIG. 3). .

(4) IL-12 and TNF-α production-inducing action from bone marrow-derived dendritic cells (BMDC) Bone marrow cells collected from C57BL / 6 mice were added to a ^6- well plate so as to be 1 × 10 ⁶ cells / well. CD11c positive dendritic cells were induced to differentiate by culturing in the presence of GM-CSF for 6 days. The extract of Example 1 was added to the induced CD11c-positive dendritic cells so that the final concentrations were 0.5, 2, and 10 mg / mL. After 6 hours, BFA was added, and the cells were collected after further culturing for 6 hours. IL-12 and TNF-α production from CD11c positive dendritic cells was evaluated by flow cytometry using an intracellular staining method.

  As a result, in the group to which the extract of Example 1 was added, the IL-12 and TNF-α production promoting action from dendritic cells was confirmed as compared to the non-added group (control) (FIG. 4).

(5) TNF-α production inducing action from BMDC via TLR2 and TLR4 Wild type, Toll-like receptor 2 knockout (TLR2KO, Oriental Bio Service Co., Ltd.), Toll-like receptor 4 knockout (TLR4KO, Oriental Bio Service Co., Ltd.) And bone marrow cells collected from each mouse (C57BL / 6 mouse) of Toll-like receptor 9 knockout (TLR9KO, Oriental Bio Service Co., Ltd.) were added to a ^6- well plate at 1 × 10 ⁶ cells / well, Each CD11c positive dendritic cell was induced to differentiate by culturing in the presence of GM-CSF for 6 days. After the extract of Example 1 was added to the induced CD11c positive dendritic cells at a final concentration of 10 mg / mL or 20 mg / mL and cultured for 14 hours, the level of TNF-α produced in the culture supernatant was Was evaluated by ELISA.

  As a result, when TLR2KO- and TLR4KO-derived CD11c-positive dendritic cells were stimulated, the production of TNF-α was attenuated as compared to the wild type, and the extract of Example 1 was mediated by the BMDC TLR2 and TLR4 molecules. It was confirmed that TNF-α production was induced (FIG. 5).

(6) IL-12 and TNF-α production-inducing action from BMDC by the extract of Example 1 from which LPS has been removed Bone marrow cells collected from C57BL / 6 mice were placed in a 6-well plate at 1 × 10 ⁶ cells / well. In addition, CD11c positive dendritic cells were induced to differentiate by culturing for 6 days in the presence of GM-CSF. For the induced CD11c positive dendritic cells, the extract of Example 1 in which LPS was removed using a commercially available endotoxin removal kit (Detoxi-Gel AffinityPak Columns Takara Bio Inc.) was 0.45 μm pore size or 0.22 μm pore size. After each filter sterilized with a filter, the final concentration was adjusted to 10 mg / mL and added. After culturing for 24 hours, IL-12 and TNF-α levels produced in the culture supernatant were evaluated by ELISA.

  As a result, production of IL-12 (FIG. 6a) and TNF-α (FIG. 6b) was confirmed even when BMDC was stimulated using the extract of Example 1 from which LPS was removed.

(7) Inducibility of IL-12 and TNF-α production from BMDC by the extract of Example 1 from which LPS was removed Bone marrow cells recovered from C57BL / 6 mice were transferred to 1 × 10 ⁶ cells / well in a ^6- well plate. In addition, CD11c positive dendritic cells were induced to differentiate by culturing for 6 days in the presence of GM-CSF. Induced CD11c positive dendritic cells were collected and incubated in ice for 40 minutes. Thereafter, the FITC-labeled extract of Example 1 was added to a concentration of 0.1 mg / mL, and incubated in ice or at 37 ° C. for 15 minutes. After the FITC-labeled extract of Example 1 in the free state was washed away, the level of the extract of Example 1 incorporated into dendritic cells was determined by flow cytometry using FITC fluorescence intensity as an index. evaluated.

  As a result, it was confirmed that the extract of Example 1 was directly taken into BMDC (FIG. 7).

(8) Induction of antigen-specific cytotoxic T cells A group in which ovalbumin (OVA) protein was administered alone at 200 μg / mouse to C57BL / 6 mice, and 5 mg / mouse of the extract of Example 1 was administered in addition to OVA Groups, a group to which BMDC ⁵ × 10 ⁵ cells / mouse was administered in addition to OVA, and a group to which OVA, the extract of Example 1 and BMDC were all administered in the aforementioned amounts were prepared. Administration was performed by intradermal injection into a mouse foot pad. Seven days after administration, repeated administration was performed under the same conditions. Five days after repeated administration, the regional lymph nodes in the vicinity of the administered foot pad were collected, and the inducibility of CD8 + T cells (OVA antigen-specific cytotoxic activity T cells) having a T cell receptor capable of binding to the OVA peptide antigen was determined. Evaluated by FACS analysis.

  As a result, it was confirmed that the antigen-specific cytotoxic activity T cells were efficiently induced by using the extract of Example 1 in combination as compared with the administration of OVA alone. Furthermore, in the combined administration with BMDC, the use of the extract of Example 1 confirmed efficient induction of antigen-specific cytotoxic activity T cells (FIG. 8).

(9) Anti-tumor activity by immunostimulatory action Cancer cells (EG7) 2 × 10 ⁶ cells that produce OVA as a virtual tumor antigen are intradermally inoculated into C57BL / 6 mice on the 5th, 8th and 11th days In addition, 200 μg of OVA, 5 × 10 ⁵ cells of BMDC and 10 mg / mouse or 20 mg / mouse of the extract of Example 1 were intradermally administered in the vicinity of the lymph node of the tumor. Mice treated with nothing were used as controls, and the volume of cancer tissue formed was measured from day 5 to day 21 after intradermal inoculation of cancer cells.

  As a result, it was confirmed that the increase in the volume of the cancer tissue was significantly suppressed in the group administered with the extract of Hokkaido larch of Example 1 as compared to the control (FIG. 9).

(10) Antitumor activity by immunostimulatory action Cancer cells (EG7) 2 × 10 ⁶ cells that produce OVA as a virtual tumor antigen are inoculated intradermally into C57BL / 6 mice on the 5th, 8th and 11th days 200 μg of OVA and 2 × 10 ⁶ cells of BMDC, 2 × 10 ⁶ cells of BMDC and 20 mg / mouse of the extract of Example 1, 200 μg of OVA and 20 mg / mouse of the extract of Example 1, or 200 μg of OVA and extraction of Example 1 20 mg / mouse and 2 × 10 ⁶ cells of BMDC were subcutaneously administered in the vicinity of the tumor lymph nodes. Mice that received no treatment were used as controls, and the volume of cancer tissue formed was measured from day 5 to day 27 after intradermal inoculation of cancer cells.

  As a result, it was confirmed that the increase in the volume of the cancer tissue was significantly suppressed in the group administered with OVA, BMDC and the Hokkaido larch extract of Example 1 as compared with the control or other groups (FIG. 10). , FIG. 11).

It is a graph which shows the TNF- (alpha) production promotion effect of the extract of Example 1. The vertical axis represents TNF-α production. It is a photograph which shows the peritoneal exudate cell (PEC) macrophage aggregation action of the extract of Example 1. In the figure, the upper left is the control, the upper upper is the extract of Example 1, the upper right is LPS (control group), and the lower photographs are obtained by changing the magnification of the upper photographs. 2 is a photograph showing the spleen cell aggregation action of the extract of Example 1. In the figure, the upper left is the control, the upper upper is the extract of Example 1, the upper right is LPS (control group), and the lower photographs are obtained by changing the magnification of the upper photographs. It is a graph which shows the result of having measured the IL-12 and TNF- (alpha) production induction effect with respect to the bone marrow origin dendritic cell (BMDC) of the extract of Example 1 by flow cytometry. It is a graph which shows that the extract of Example 1 induces production of TNF-α via TLR2 and TLR4 molecules of BMDC. It is a graph which shows the IL-12 production induction effect from BMDC by the extract of Example 1 which removed LPS. It is a graph which shows the TNF- (alpha) production induction effect from BMDC by the extract of Example 1 which removed LPS. It is a graph which shows that the extract of Example 1 labeled with FITC was taken up into dendritic cells and the result of flow cytometry measurement using the fluorescence intensity of FITC as an index. The top of the graph is incubation in ice, and the bottom of the graph is incubation at 37 ° C. FIG. 3 is a graph showing the results of FACS analysis showing the antigen-specific cytotoxic T cell inducing action of the extract of Example 1. FIG. It is a graph which shows that the volume increase of the cancer tissue in a cancer cell transplant mouse | mouth is suppressed by administration of the extract of Example 1. FIG. It is a graph which shows that the volume increase of the cancer tissue in a cancer cell transplant mouse | mouth is suppressed by administration of the extract of OVA, BMDC, and Example 1. FIG. It is a photograph which shows the mode of the cancer tissue (21st day after cancer cell transplantation) of the cancer cell transplantation mouse | mouth which administered OVA, BMDC, and the extract of Example 1. FIG. Photographs from the left are control (no treatment group), BMDC and the extract of Example 1, BMDC and OVA, OVA and the extract of Example 1, BMDC, OVA and the extract of Example 1, respectively. It is an organization.

Claims (4)

Th1 immune function enhancer containing a solvent extract extracted from Hokkaido larch (Larix leptolepis) with water (except for anti-infectives).   The Th1 immune function-enhancing agent according to claim 1, which is for antitumor use.   The Th1 type | system | group immune function enhancing agent of Claim 1 or Claim 2 which is an object for promotion of TNF (alpha) and / or interleukin 12 production. An antitumor agent comprising a solvent extract extracted from water from Hokkaido larch (Larix leptolepis).