JP2008127296A - Heat shock protein inducer, skin care preparation and food comprising the same and method for producing heat shock protein inducer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new HSP (heat shock protein) inducer having a high HSP induction activity. <P>SOLUTION: The HSP inducer comprises a plant extract to be an origin of a crude drug having properties corresponding to warm or neutral nature in four vital energies which are properties of the crude drug. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention is a plant-derived heat shock protein inducer that is a source of herbal medicine having a property corresponding to warm or flat among the four characteristics of herbal medicine, and a skin external preparation containing the same, food, and The present invention relates to a method for producing a heat shock protein inducer.

In cells, tissues or individuals, there is a group of proteins with a molecular weight range of 10-110 KDa measured by SDS-PAGE that is induced to synthesize as a defense reaction of the body when exposed to temperatures higher than 3 ° C. by physiological temperature. It is called heat shock proteins (hereinafter referred to as “HSP”).
HSP has a family formed by its molecular weight. For example, HSP90 family (molecular weight is 90 kDa or more and 110 kDa or less), HSP70 family (molecular weight is 70 kDa or more and less than 80 kDa), HSP60 family (molecular weight is 60 kDa or more and less than 70 kDa) and low molecules Classified as HSP family (molecular weight less than 60 kDa).
The functions of HSPs are diverse. For example, the HSP70 family and HSP60 family bind to denatured proteins and unwind them to natural folding (higher-order structure / folded structure). It has been clarified that it has functions called so-called molecular chaperones such as association, intracellular localization, and membrane permeation (Non-patent Documents 1 and 2).

The synthesis of HSP is induced not only at high temperatures but also when subjected to various external stresses such as heavy metals, oxygen deficiency, various chemicals, radiation, and ultraviolet rays. Stress, including high temperatures, denatures cellular proteins and forms insoluble precipitates that cause fatal damage to cells.
It has been clarified that abnormal protein folding in cells causes various diseases, and it is now called abnormal protein folding disease. For example, Alzheimer's disease is caused by aggregation caused by abnormal folding of the causal protein called β-amyloid, and neuronal cells are killed or lose function by the aggregate.

Therefore, research on HSP inducers that are effective for the cure and prevention of protein folding abnormal diseases is underway. For example, geranylgeranylacetone used as an anti-ulcer drug has an HSP-inducing action, and a method for preventing and treating viral infection using this has been proposed (Patent Document 1).
However, geranylgeranylacetone is a non-toxic heat shock protein inducer, but its induction ability is weak.

In addition, it has been found that an active extract (artemia extract) component extracted from a durable egg immediately before hatching of artemia, an aquatic plankton, induces the production of HSP70 in human skin cells, and a cosmetic using the same is proposed. (Patent Document 2).
However, Artemia extract is desirably 40 ° C. or less as a blending condition, and has restrictions such as a contraindication with a proteolytic enzyme.

Hendrick, J. et al. P. and Hartl, F.A. -U. (1993) Ann. Rev Biochem. 62, 349-384 Georgepoulos, C.I. and Welch. W. J. et al. (1993) Ann. Rev. Cell Biol. 9,601-634 International Publication WO2003 / 035052 Pamphlet JP 2004-238297 A

  This invention is made | formed based on the subject, and it aims at providing the novel HSP inducer with high HSP induction activity.

As a result of intensive studies in view of the above-described present situation, the present inventor has found that the above-described problems can be solved by the following means.
(1) A heat shock protein inducer containing a plant extract that is the origin of a herbal medicine having a property corresponding to warm or normal among the four characteristics of herbal medicine.
(2) A heat shock protein inducer containing a plant extract selected from the family Ginger and / or Compositae.
(3) A heat shock protein inducer containing a plant extract selected from plants belonging to the genus Ammomum or Hanaminga of the ginger family.
(4) A heat shock protein inducer containing a plant extract selected from the group consisting of Yishushsha, Shukusha, and Nankyo.
(5) A heat shock protein inducer containing a plant extract selected from plants belonging to the genus Hydrobana or Oguruma.
(6) A heat shock protein inducer comprising a plant extract selected from the group consisting of Sad budbird, Oguruma and Hosobagurum.
(7) The heat shock protein induction according to any one of (1) to (6), wherein the heat shock protein inducer induces a heat shock protein having a molecular weight measured by SDS-PAGE of 70 kDa or more and less than 80 kDa. Agent.
(8) The heat shock protein inducer according to any one of (1) to (6), wherein the heat shock protein inducer induces a heat shock protein having a molecular weight of 72 kDa as measured by SDS-PAGE.
(9) An external preparation for skin containing the heat shock protein inducer according to any one of (1) to (8).
(10) A food containing the heat shock protein inducer according to any one of (1) to (8).
(11) A method for producing a heat shock protein inducer, comprising extracting a plant that is a source of a herbal medicine having a property corresponding to warm or normal among the four spirits that are the properties of a herbal medicine.

  By employing the present invention, a heat shock protein inducer having a high HSP induction ability was obtained.

Hereinafter, the contents of the present invention will be described in detail. In the present specification, “to” is used to mean that the numerical values described before and after it are included as a lower limit value and an upper limit value.
The heat shock in the present invention means that when the temperature is higher by 3 ° C. or more than the physiological temperature, it is preferably higher by 6 ° C. or more than the physiological temperature.

The HSP inducer in the present invention effectively induces HSP having a molecular weight of 70 kDa or more and less than 80 kDa (hereinafter sometimes referred to as “HSP70 family”), and induces HSP having a molecular weight of 72 kDa more effectively. It also effectively induces HSP with a molecular weight of 70 kDa.
The molecular weight of HSP in the present invention refers to the molecular weight measured by SDS-PAGE. More specifically, the molecular weight is estimated by comparison with a standard protein band of known molecular weight, and Western blotting using an antibody. The value determined by.
Here, in the HSP70 family, HSP72 is synthesized in the cytoplasm, and can be detected in the cell nucleus immediately after synthesis and in the nucleolus thereafter. In addition, in cells in which the expression level increased dramatically due to heat stress and HSP72 increased, the rate of cell damage due to ischemia given thereafter significantly decreased depending on the concentration of HSP expression level. (Hutter, MM., Sievers, RE, Wolfe, CL (1994) Circulation, 89, 355-360).
However, no naturally-occurring material having HSP72-inducing activity and low toxicity without applying heat stress has been found so far. That is, a naturally-derived HSP inducer that induces HSP72 has been strongly demanded, but has not been obtained until the present invention.

  The HSP inducer of the present invention can be further blended at a temperature higher than 40 ° C., or can be blended with a proteolytic enzyme, and has a wide range of applications.

The HSP inducer of the present invention refers to a plant that is the source of a herbal medicine having a property corresponding to warm or normal among the four spirits that are the properties of the herbal medicine. Here, Shiki is a medicinal property used to express the properties of herbal medicines, and is basically based on four types: cold, heat, cool and warm. Cold and cool, heat and temperature are similar in nature, but cool is slightly cooler than cold and warm is slightly warmer than heat. In addition, herbal medicine that does not belong to either is expressed as flat, and because of its mild action, it is used for both cold and fever. In the present invention, for example, plants can be classified according to the description in the Chuo University Encyclopedia (Shogakukan).
Conventionally, it is not known at all that the plant extract, which is the origin of a herbal medicine having the properties corresponding to warm or normal, has the ability to induce HSP among the four spirits that are the characteristics of herbal medicine. It is very surprising that it was obtained.

In the present invention, a plant extract selected from the ginger family and / or asteraceae among the plants that are the origin of the herbal medicine having the properties corresponding to warm or flat among the four characteristics of the herbal medicine is preferable.
As the ginger family, plants belonging to the genus Ammomum or Hanaminga are preferred. As the plant belonging to the genus Ammum, Ioshunsha (Amoum viilosum Lour.) And Shukusha (shrinked sand; Amoum xanthioides Wall.) Are preferable. As the plant belonging to the genus Hanamyouga, Nanpiyou (Alpinia galanga (L.) Swartz) is preferable.
As plants of the family Asteraceae, plants belonging to the genus Hydrobana or Oguruma are preferred. As a plant belonging to the genus Hydrangea, Sawa hydrangea (Eupatorium Lindleyanum DC.) Is preferable. As the plant belonging to the genus Oguruma, Oguruma (Inula japonica Thunb.) And Hosobaogurma (Inula linariae folia Turcz.) Are preferable.

  In the present invention, a plant extract is used. The extraction method of the plant is not particularly defined as long as it has an extract fraction having HSP induction ability. In the extraction, the plant is preferably extracted after pulverization. In the case of using a dried product, it is preferable to return the dried product to water and pulverize it with a miller, then freeze-dry it, and pulverize it again with a miller. In particular, a method of adding an appropriate amount of an organic solvent (for example, ethanol) to the dried pulverized product and extracting it for 2 to 3 hours can be exemplified. Extraction may be performed by standing or stirring. The extracted liquid may be used as it is as an HSP inducer, but is preferably concentrated before use to enhance the effect. The degree of concentration varies depending on the use environment. For example, it may be used as a liquid agent with a concentration of about 10%, or the solvent may be removed until it becomes powdery.

  In the present invention, only one kind of the above may be adopted as the plant extract, or two or more kinds may be adopted. Furthermore, as another plant-derived material, a material having HSP induction ability is used in combination. You can also.

  The HSP inducer of the present invention can be used in various modes depending on the purpose. For example, it can be used safely and effectively as an external preparation for skin, food (including beverages), and pharmaceuticals.

 When used as an external preparation for skin, it can be provided in various dosage forms such as lotions, emulsions, gels, creams, ointments, powders, granules and the like. Specifically, skin cosmetics such as lotion, milky lotion, cream, cosmetic liquid, and pack, base cosmetics such as makeup base lotion and makeup base cream, emulsions, oils, solids, etc. It can be provided as makeup cosmetics such as foundation, eye color and cheek color, and body cosmetics such as hand cream, leg cream, neck cream and body lotion.

  When used for foods (including beverages), functional foods and functional beverages can be obtained by including them in various foods (including beverages). For example, it can be added to black tea, soft drinks, juice, candy, starchy food, various processed foods, and the like. The amount of the HSP inducer of the present invention can be set within a range of about 0.1 to 99% by weight. Moreover, you may improve food texture by adding a gelatinizer etc. as needed.

  When used as a pharmaceutical, various dosage forms can be selected depending on the administration route. The HSP inducer of the present invention can be administered orally or parenterally. For example, rectal administration, intranasal administration, buccal administration, sublingual administration, intravaginal administration, intramuscular administration, subcutaneous administration, and intravenous administration can be performed.

  As preparations suitable for oral administration, tablets, capsules, powders, fine granules, granules, solutions, syrups and the like can be mentioned. As preparations suitable for parenteral administration, injections, drops, suppositories , Inhalants, transdermal absorbents, transmucosal absorbents, patches and the like. The injection may be used for any of intravenous injection, intramuscular injection, subcutaneous injection, infusion and the like.

A pharmacologically and pharmaceutically acceptable additive can be added to the HSP inducer of the present invention as needed. For example, excipients, disintegrants or disintegration aids, binders, lubricants, coating agents, dyes, diluents, bases, solubilizers or solubilizers, isotonic agents, pH adjusters, stabilizers A propellant, an adhesive, a wetting agent, and the like can be used.
By using these additives in appropriate combinations, the HSP inducer of the present invention can have various additional functions. For example, the active ingredient can be designed so as to be released as required. It can also be designed so that the active ingredient is released intensively at the required location in the body. Such sustained-release preparations and drug delivery systems can be designed and manufactured according to methods well known in the pharmaceutical industry.

  In addition, an organic or inorganic carrier can be used for the HSP inducer of the present invention. Examples of such carriers include lactose, starch, vegetable and animal fats and oils. The HSP inducer of the present invention can be used within a range of 0.01 to 100% by weight.

  The dosage of the HSP inducer of the present invention is appropriately determined according to various conditions such as the purpose of treatment or prevention, patient's sex, body weight, age, type and degree of disease, dosage form, route of administration, number of administrations, and the like. . For example, in the case of oral administration, it can be administered at a dose of 0.1 μg to 100 mg (dry weight of active ingredient) / kg body weight / day once to several times a day. It is not limited.

  The present invention will be described more specifically with reference to the following examples. The materials, amounts used, ratios, processing details, processing procedures, and the like shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention is not limited to the specific examples shown below.

Example 1
(Production of plant extract)
In this example, the following plants were used.
(A) Oguruma, Hosoba Oguruma head flower (Sempukuka (turned flower)): (Manufacturer Eijin Shoji, product number B025)
(B) Whole plant of Sawa bully (Yabatsui (Nomaoi)): (Manufacturer, Eijin Shoji, product number B072)
(C) Nansou (Odaka Ryo) fruit (cow (red bean?)): (Manufacturer Eijin Shoji, product number C089)
(D) Yoshunsha (Hyunchun sand) husk (Shajinkaku (sand husk)): (Manufacturer Eijin Trading, product number D030)

Extraction was performed by the following method.
The raw plant was pulverized as it was, freeze-dried, and pulverized again with a miller. The dried product was returned to water, pulverized with a miller, freeze-dried, and then pulverized again with a miller.
10 g of ethanol was added to 1 g of the obtained dried pulverized product, and the mixture was stirred for 2 hours, followed by Buchner filtration, and the filtrate was recovered. This operation was repeated twice.
The obtained filtrate was concentrated under reduced pressure until it became pasty or dry solid to obtain a plant extract.

(Extraction of intracellular protein)
A human fibroblast cell line (NB1RGB) (obtained by: RIKEN BioResource Center) was seeded in a 35 mm culture dish so as to obtain a cell suspension of 2.0 × 10 ⁵ cells / 2 mL, and then in a CO ₂ incubator. The cells were cultured at 37 ° C. for 24 hours. To this, the plant extracts (A) to (D) were added so that the concentrations were as shown in Table 1, and further cultured at 37 ° C. for 4 hours in a CO ₂ incubator. Next, the supernatant medium was removed, the cells were washed with the medium, the cells were removed from the dish with trypsin, and the cells were collected. Add lysis buffer for cultured cells (RIPA buffer, 50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1% nonidet P-40, 0.5% sodium deoxysholate, 0.1% SDS) Thawing was repeated once to lyse the cells and the supernatant was collected by centrifugation. The obtained elution fraction was subjected to protein quantification by the Bradford method.

(Analysis of HSP72 expression level)
After the cell extract was subjected to polyacrylamide gel (SDS-PAGE) electrophoresis, the expression level of HSP72 was examined by Western blotting using an anti-HSP72 antibody (manufactured by Stressgen).
That is, SDS-PAGE gel was transferred and immobilized on a membrane to prepare a blot, and the blot was reacted with an antibody against HSP72 (primary antibody). Thereafter, the secondary antibody labeled with the HRP enzyme was reacted with the primary antibody, and the color developed with Super Signal West Dura Extended Duration Substrate was quantified by the chemifluorescence method. The intensity of the obtained band was quantified by NIH-Image 1.62 and digitized.

(Control group and positive control)
As a control group, the procedure was the same except that no plant extract was added. In addition, as a positive control, a human fibroblast cell line that was heat shocked at 43 ° C. for 1 hour without adding a plant extract was prepared, and the others were performed in the same manner.

(result)
The measured band intensity was corrected by the band intensity of actin that is constantly expressed in the cells, and was calculated as the relative hand intensity when the band intensity of the positive control was 1. The results are shown in Table 1.
In any case, induction of HSP72 was observed by the addition of the plant extract. In particular, with regard to (A), (B), and (C), by adjusting the addition concentration of the plant extract, the relative band intensity is sometimes seen to be 1 or more, and human fibroblasts subjected to heat shock It was shown that HSP72 expression by the plant extract was higher than that of the strain.

Example 2
In Example 1, the human fibroblast cell line (NB1RGB) was replaced with a human epidermis cell line (HaCaT) (obtained from: Cell lines service), and the rest was performed in the same manner to measure HSP72-inducing activity.

(result)
The measured band intensity was corrected by the band intensity of actin that is constantly expressed in the cells, and was calculated as the relative hand intensity when the band intensity of the positive control was 1. The results are shown in Table 2.
In any case, induction of HSP72 was observed by the addition of the plant extract. In particular, for (A), (B), and (C), depending on the concentration added, the relative band intensity may be 1 or more, and it is more dependent on the plant extract than the human epidermal cell line subjected to heat shock. HSP72 expression was shown to be high.

Claims (11)

A heat shock protein inducer containing a plant extract that is the origin of a herbal medicine having a property corresponding to warm or normal among the four characteristics of herbal medicine. A heat shock protein inducer containing a plant extract selected from the family Ginger and / or Compositae. A heat shock protein inducer comprising a plant extract selected from a plant belonging to the genus Ammomum or Hanaminga of the ginger family. A heat shock protein inducer comprising a plant extract selected from the group consisting of Yoshunsha, Shukusha, and Nankyo. A heat shock protein inducer comprising a plant extract selected from plants belonging to the genus Hydrobana or Oguruma. A heat shock protein-inducing agent comprising a plant extract selected from the group consisting of Sad eagle, Oguruma and Hosoba-Oguruma. The heat shock protein inducer according to any one of claims 1 to 6, wherein the heat shock protein inducer induces a heat shock protein having a molecular weight of 70 kDa or more and less than 80 kDa as measured by SDS-PAGE. The heat shock protein inducer according to any one of claims 1 to 6, wherein the heat shock protein inducer induces a heat shock protein having a molecular weight of 72 kDa as measured by SDS-PAGE. The external preparation for skin containing the heat shock protein inducer of any one of Claims 1-8. The foodstuff containing the heat shock protein inducer of any one of Claims 1-8. A method for producing a heat shock protein inducer, comprising extracting a plant that is a source of a herbal medicine having a property corresponding to warm or normal among the four characteristics of herbal medicine.