WO2013046813A1

WO2013046813A1 - Enhancer of expression of biological defense function factor

Info

Publication number: WO2013046813A1
Application number: PCT/JP2012/065011
Authority: WO
Inventors: 雅夫田北
Original assignee: 有限会社西原
Priority date: 2011-09-29
Filing date: 2012-06-12
Publication date: 2013-04-04
Also published as: JP2013071932A; JP5561647B2

Abstract

Provided is an enhancer of the expression of a biological defense function factor, which can be achieved as a novel use application of a licorice extract. The enhancer of the expression of a biological defense function factor comprises, as an active ingredient, a licorice extract that is soluble in water and ethanol and does not contain glycyrrhizin or glabridin, has an activity of enhancing the expression of BCL-XL, survivin, P53, SOD and HO-1 that are biological defense function factors, and also has an activity of inhibiting the expression of iNOS. The enhancer can be used as a BCL-XL expression enhancer, a survivin expression enhancer, a P53 expression enhancer, an SOD expression enhancer, an HO-1 expression enhancer, or an inhibitor of the expression of iNOS that has an oxidative stress stimulating activity.

Description

Agents that enhance the expression of biological defense function factors

The present invention relates to an agent for enhancing the expression of a biological defense function factor in an apoptosis inhibitory factor, a DNA repair factor or an oxidative stress inhibitory factor.

In general, licorice, which is well known as a herbal medicine, is used as a raw material for medicinal or sweeteners, and is also used as a component for external preparations for skin such as cosmetics. In addition, licorice leaf extract extracted with an aqueous solvent such as water or an alcohol aqueous solution is known to have an action of promoting collagen production of skin fibroblasts.

Further, the purity of the whitening component contained in the licorice by the inventors of the present application is purified and enhanced, in particular, a whitening component other than liquiritin is selected, that is, a whitening skin external preparation with an enhanced whitening effect by an action other than tyrosinase inhibitory activity. Is known (Patent Document 1).

By the way, humans have various functions as a body defense function, but apoptosis, which is a programmed cell death of skin and other tissues, causes various diseases and progresses disease symptoms. Therefore, a technique for adjusting apoptosis associated with a disease is demanded, and pharmaceuticals are being developed.

For example, when apoptosis starts due to DNA damage, an apoptosis inhibitor such as BCL-2 protein, BCL-XL protein, survivin protein or the like prevents cell death due to apoptosis.

Also, against DNA damage before apoptosis begins, the P53 protein exerts a biological defense function by stopping cell growth and gaining time for DNA repair by ATR or the like.

In addition, there is an enzyme called SOD that removes active oxygen as a biological defense function factor, and HO-1 protein protects the living body from active oxygen, while iNOS protein reduces nitric oxide (NO). Produce and dilate vascular smooth muscle, prevent hypertension and arteriosclerosis.

Incidentally, in the medical field, nitroglycerin that produces NO has become a specific drug for angina and has a biological defense function.

However, if NO increases excessively, it reacts with active oxygen and produces strong peroxynitrite (peroxynitrite; peroxynitrite), which damages DNA and causes mutations and carcinogenesis. .
Moreover, although the NO produced by the iNOS protein has a biological defense function in a small amount, an oxidative stress effect appears when the amount is large, and becomes a factor that impairs biological defense.

These proteins produced by the expression of P53, ATR, BCL-2, BCL-XL, survivin, SOD, HO-1, iNOS, etc. are all produced in the cell by the action of cellular DNA. . Adjustment to increase or suppress these proteins in vivo is also effective for suppressing various stresses and protecting the organism.

It is known that the component that enhances the expression of the protein serving as a biological defense function factor can be collected from plant origin, and safflower, carrot, amacha, parsley, litchi, onionis, fennel, bowie It is known that a component having an effect of increasing BCL-2 protein is contained (Patent Document 2).

In addition, carrots are known to have an effect of increasing production of BCL-XL protein (Patent Document 3), and basidiomycete cultures are known to suppress iNOS production (Patent Document 4). In addition, it is known that the ethanol-soluble and water-soluble fraction of licorice extract has an effect of increasing the amount of BCL-2 protein (Patent Document 5).

Also, glycyrrhizin and glabrizine contained in licorice are known to be anti-inflammatory components (Patent Document 6).

JP 2006-028157 A JP 2002-080382 A JP 2001-139483 A JP 2008-013550 A JP 2008-239573 A JP 2011-068574 A

However, among the background arts described above, Patent Document 5 using licorice extract describes the result that BCL-2 protein is increased. However, other proteins having biological defense functions are disclosed. However, there is a problem that the possibility of inhibiting the production of harmful proteins has not been elucidated, and the necessity of discovering new uses for them has been recognized.

In particular, it is considered that there are unknown properties of licorice extract components other than glycyrrhizin and grabrizine contained in conventional licorice extracts, and it is necessary to clarify their usefulness.

Thus, an object of the present invention is to solve the above-described problems and provide an agent for enhancing the expression of a biological defense function factor that can be implemented for a novel use for a licorice extract.

In order to solve the above problems, in the present invention, BCL-XL, survivin (survivin), which is a biological defense function factor, contains licorice extract that is soluble in water and ethanol and does not contain glycyrrhizin and glabrizine as an active ingredient. ), An expression enhancer of a biological defense function factor having an action of enhancing the expression of P53, SOD and HO-1, and also having an action of suppressing the expression of iNOS.

As described above, the expression enhancer of the biological defense function factor of the present invention configured as described above is a BCL-XL expression enhancer, a survivin expression enhancer, or a P53 expression enhancer. It has an effect that can be used as an SOD expression enhancer, HO-1 expression enhancer, and iNOS expression inhibitor, and water and ethanol-soluble licorice extract can be used as an active ingredient for these novel uses.

Further, when the above-mentioned biological defense function factor is an apoptosis inhibitory factor comprising BCL-XL protein or survivin protein, enhanced expression of the biological defense function factor comprising water and ethanol-soluble licorice extract as active ingredients It can also be used as an agent.

In addition, when the above-mentioned biological defense function factor is a DNA repair factor composed of P53 protein, it can also be used as an expression enhancer of the biological defense function factor containing water and ethanol-soluble licorice extract as active ingredients.

In the case where the above-described biological defense function factor has an action of promoting the expression of SOD and HO-1, which are oxidative stress inhibitors, or an oxidative stress inhibitor that suppresses the expression of iNOS that promotes oxidative stress. Furthermore, it can also be used as an agent for enhancing the expression of biological defense function factors comprising water and ethanol-soluble licorice extract as active ingredients.

Since the present invention contains water and ethanol-soluble licorice extract as active ingredients, an apoptosis inhibitory factor consisting of BCL-XL or survivin, a DNA repair factor consisting of P53, and SOD and HO which are oxidative stress inhibitors -1 has an expression promoting effect, or has an expression enhancing property of a biological defense function factor that is an oxidative stress suppressing factor that suppresses the expression of iNOS that promotes oxidative stress, a BCL-XL expression enhancing agent, survivin ( survivin) expression enhancer, P53 expression enhancer, SOD expression enhancer, HO-1 expression enhancer or iNOS expression suppressor has an advantage of becoming an expression enhancer of biological defense function factors.

The licorice used in this invention is a herbal medicine obtained by drying licorice, which is a medicinal plant of legumes in Spain, Siberia, China, etc., and has improved preservation, and its scientific name is Glycyrrhiza urarenlensis Fischer or glycyrrhiza It is a plant called Glycyrrhiza glabra l var.glandulifera REG.Et HERD or its related species.

Preferably, the root or rhizome (also referred to as stalk or stron) of this plant is in a skinned state or is pulverized or ground by removing cork skin, and these are water or ethanol or other aqueous solvent What was extracted in (3) can be used as an active ingredient extraction material. The safety of common licorice extract to the skin is well known.

The solvent used for extracting the active ingredient in the present invention is water (neutral, weak acid or weak alkaline) or other solvent (mainly organic solvent) with water as the main component. It is an aqueous extraction solvent to which is added), and an alcohol-containing aqueous solution can also be employed. Examples of the alcohol include well-known alcohol solvents such as methanol, ethanol, isopropanol, and t-butanol. The active ingredients extracted with these solvents are both soluble in water and ethanol (= ethyl alcohol).

Such an extraction operation with an aqueous solvent is preferably performed in a state of hot water or hot water in order to increase extraction efficiency. In particular, when the extraction operation is performed at 100 ° C. or higher, preferably 100 to 130 ° C., the components effective for enhancing the expression of the above-mentioned biological defense function factor are efficiently extracted.

In the extraction process, the extract is filtered by passing it through an adsorbent such as activated carbon, or solid-liquid separation is performed using the difference in specific gravity between solid and liquid, or the difference in sedimentation speed due to gravity or centrifugal force. It is preferred to remove turbid material.

The adsorbent may be any substance that provides an interface that causes a large amount of positive adsorption, such as activated carbon, and the transparent liquid obtained as described above is concentrated and again added with water or ethanol. After the dilution, it is preferable to separate and remove insoluble matters generated by concentration by filtration or the like, thereby making the purification more complete. Specific examples of the adsorbent include synthetic adsorbents (organic adsorbents) in addition to inorganic adsorbents made of a porous material such as activated carbon.

In this way, the solid-liquid separation and other operations for removing suspended substances include passing the extract through a container such as a column packed with activated carbon particles, or using an activated carbon powder (such as talc if necessary) in the extract. A method of filtering the mixture with a filter that captures the powder particles of the activated carbon such as filter paper and filter cloth can be employed. The solid-liquid separation means may be employed.

For the concentration operation on the aqueous solvent extract, known concentration means such as reduced pressure operation or heat concentration can be employed. The concentration ratio is preferably about 2 to 100 times, more preferably about 5 to 50 times, and usually about 10 times is preferable.

The obtained concentrate is preferably diluted again with the aforementioned aqueous solvent extract or an aqueous solvent corresponding thereto. Although there is little need to limit the dilution rate, for example, a dilution rate that returns to the volume before concentration is considered preferable, and is, for example, about 2 to 100 times. And it is preferable to repeat the concentration and dilution operations as one step and repeat two or more steps.

In order to further purify the liquid thus obtained, it is preferable to use anhydrous alcohol having an ethanol concentration of 99.5% or more as an extraction solvent after concentration.

In order to prepare water and ethyl alcohol-soluble components obtained by solid-liquid separation as agents for enhancing the expression of biological defense function factors, they can be prepared as external preparations, internal medicines, subcutaneous injections and the like.
For example, in order to prepare as a skin external preparation, it is mixed with a base for a skin external preparation that has an appropriate hydrophilicity / lipophilic property and has good skin adhesion, or an extract such as cream, ointment, milky lotion, etc. If mixed with a base material, it can be prepared into a pharmaceutical form that can be applied to the skin.

As will be described later, expression of biological defense function factor having enhanced expression of BCL-XL, survivin, P53, SOD, and HO-1 in water and ethanol-soluble licorice extract and suppressing iNOS expression The active ingredient as an enhancer should be formulated so that it is formulated at a concentration of 0.1 to 20% by weight with respect to the skin as a dry weight of the final concentrate (dry solid). Is preferred.

[Manufacturing method of licorice extract (T) actual production]
1) To 2000 kg of Tohoku daylily, 2000 liters of water (hereinafter, liters are indicated by L) was added, extracted at 95 ° C. for 2.5 hours, and the extract was filtered through a vibrating sieve (300 mesh, stainless steel). The obtained extract filtrate was concentrated (liquid temperature 60 ° C. or lower, reduced pressure), and the concentrated liquid (spray-dried stock solution) was spray-dried to obtain a dried extract. This was sieved (60 mesh, made of stainless steel) to obtain a yield of 20 kg of dried licorice (T raw).
2) To 20 kg of Tohoku licorice dry extract (T raw material) obtained in 1) was added 100 L of Pharmacopoeia ethanol, stirred for 6 hours, and filtered through No. 2 filter paper.
3) Pharmacopoeia ethanol was added to the filtrate to make 100 L, activated carbon (granular white birch KL) 4.5 kg was added and stirred for 3 hours, and filtered through No. 2 filter paper. This process was repeated three times.

4) Pharmacopoeia ethanol was added to the filtrate obtained in 3) to make 100 L, and this was concentrated to 10 L (50 ° C. or less, reduced pressure). Further, 400 g of activated carbon (granular white birch KL) was added, stirred for 2 hours, and filtered through No. 2 filter paper. This process was repeated 6 times.
5) Pharmacopoeia ethanol was added to the filtrate obtained in 4) to make 10 L, and the filtrate was concentrated to 2 L. To this was added 1 L of pharmacopoeia ethanol and 100 g of activated carbon (granular white birch KL), stirred for 3 hours, and filtered with No. 2 filter paper.
6) Pharmacopoeia ethanol was added to the filtrate obtained in 5) to make 2 L, activated carbon (granular white birch KL) 100 g was added, stirred for 3 hours, and filtered with No. 2 filter paper.
7) Pharmacopoeia ethanol was added to the filtrate obtained in 6) to make 2 L, and filtered with No. 5 filter paper.
8) The filtrate obtained in 7) was concentrated (40 ° C. or lower, reduced pressure) to dryness, extracted with 300 mL of water (shaking, room temperature), and filtered through No. 5 filter paper. This process was performed three times.
9) The filtrate obtained in 8) was 1 L.

99.5% anhydrous to the concentrate (37.1 g) obtained by heating 250 mL of the filtrate obtained in 9) to 95 ° C. or lower (room temperature to 95 ° C., preferably room temperature to less than 95 ° C., the same shall apply hereinafter). 1 L of ethanol (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the ethanol-soluble fraction was taken and heated to 95 ° C. or lower to obtain a concentrate (21.5 g).

To this, 250 mL of purified water and 50 g of activated carbon (manufactured by Wako Pure Chemical Industries, Ltd .: activated carbon powder) were added and stirred, 75 g of talc (manufactured by Maruishi Pharmaceutical Co., Ltd.) was added and further stirred, and then filtered through filter paper. 400 ml of 99.5% absolute ethanol (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the concentrate (14.7 g) obtained by heating the obtained filtrate to 95 ° C. or lower, and an ethanol-soluble fraction was taken. To the concentrate (11.7 g) obtained by heating, 250 mL of purified water and 30 g of activated carbon (manufactured by Wako Pure Chemical Industries, Ltd .: activated carbon powder) are added and stirred, and 50 g of talc (manufactured by Maruishi Pharmaceutical) is added. Further stirring was performed. Thereafter, the mixture was filtered through a filter paper, and the obtained filtrate was heated to 95 ° C. or lower, and 400 ml of 99.5% absolute ethanol (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the concentrate (8.6 g). And a concentrate (5.2 g) obtained by heating to 95 ° C. or lower. Purified water was added to 2.0 g of this concentrate to make a 12 g sample.

The active ingredient of the licorice extract is brought into contact with the skin in a concentration range of 0.1 to 20% by weight when converted to the blended weight% of the final concentrate (2.0 g) described above. This is considered effective for obtaining the effects of the present invention.

Evaluation experiment method [A: Examination using cultured cells]
1) The influence of the sample was examined using human oral squamous cell carcinoma cell HSC4. The experiment was divided into four groups under the following four conditions. Hereinafter, phosphate buffered saline is abbreviated as PBS.
Group 1: PBS treatment (24 hours)
Group 2: Sample processing (24 hours)
Group 3: PBS treatment before and after UV irradiation (24 hours from 1 hour before irradiation)
Group 4: Sample treatment before and after UV irradiation (24 hours from 1 hour before irradiation)

2) The treatment concentration was 1/200 of the stock solution.
3) For UV irradiation, the culture solution is removed, washed twice with PBS, then PBS is removed, and irradiation is performed with an ultraviolet ray generator (xx-15BLB, UVP, Co. Tokyo, Japan) at an ultraviolet intensity of 5 j / cm ² . Irradiated at a distance of 5 cm for 1.5 minutes. After irradiation, a culture solution was added (sample and PBS were added depending on conditions).

4) The treated cells were washed with PBS, peeled and collected, RNA was extracted with RNeasy kit (Qiagen, Germany), and cDNA was prepared using ReverTra Ace-a-RT kit (Toyobo, Japan). Quantitative RT-PCR reactions are performed by Step One Real-Time PCR Systems (Applied Biosystems, Foster City, CA, USA) using East SYBR Green Master Mix (Applied Biosystems) and semi-quantitatively by relativerestandard curve quantification method And ACTB mRNA was standardized as internal control.

Evaluation experiment method [B: Examination using mice]
1) 4 weeks old C56BL6 male mice (Japan SLC) were acclimated for 1 week and then grouped after shaving. The experimental groups were 4 groups from the 1st group to the 4th group, and 10 mice were used for each group. Group 1: Shaving + PBS application Group 2: Shaving + sample application Group 3: Shaving + before and after UV irradiation PBS application Group 4: Shaving + before and after UV irradiation sample application

2) The sample was stored at 4 ° C. and returned to room temperature before coating.
3) In ultraviolet irradiation, all wavelengths of ultraviolet light (UV-A, -B, -C) are irradiated to cause acute ultraviolet damage on the skin. At this time, the sample is exposed to mouse skin for 24 hours before and immediately after ultraviolet irradiation. The change of acute UV damage was examined.
4) The shaved area is 2 cm square centered at the intersection of the ribbed thoracic vertebra lower edge and the spine, and the shaved area is 4 cm ² . Shaving was performed using an electric clipper (National), and no razor was used.

5) For ultraviolet irradiation, an ultraviolet ray generator (xx-15BLB, UVP, Co. Tokyo, Japan) was used for 15 minutes with an ultraviolet intensity of 5 J / cm ² and an irradiation distance of 5 cm.
6) At the time of irradiation, the mouse was anesthetized with Nembutal (mg / mouse subcutaneous injection), and the limbs were fixed with tape to keep the body posture during irradiation.
7) 200 μl of the sample was applied to the shaved portion with a cotton swab while drying the entire amount.
8) Mice were killed by cervical dislocation 24 hours after UV irradiation, and the shaved skin was peeled off on the fascia including the surrounding non-shaved parts and frozen in liquid nitrogen.

9) The frozen specimen was pulverized with a homogenizer, RNA was extracted with RNeasy® kit® (Qiagen, Germany), and cDNA was prepared using ReverTra® Ace-a-RT® kit (Toyobo, Japan). Quantitative RT-PCR reactions were performed using Step Real-Time PCR Systems (Applied Biosystems, Foster City, CA, USA) using East SYBR Green Master Mix (Applied Biosystems), and semi-quantified by relative standard curve quantification method ACTB mRNA was standardized as internal control.

The following table summarizes the results of the above evaluation tests. In addition, P <0.05 in the table indicates that the probability that the numerical value to be compared is accidental is 5% or less, and in the table, the numerical value difference between the groups with the same lowercase letter is accidental. It is shown that the probability of being 5% or less. For example, regarding HSC4 cells in Table 1, e P <0.05 indicates that the probability that the difference between the numerical values of Group 1 and Group 4 is by chance is 5% or less.

Then, when a 5-fold diluted solution of the sample was analyzed by high performance liquid chromatography, glycyrrhizin was not detected with an accuracy of 0.005 g / kg for content measurement detection, and gravridine was detected with a content measurement detection limit of 0.00. It was not detected with an accuracy of 5 mg / 100 g.

As is clear from the results in Tables 1 to 8, when the first group and the third group are compared, the generation of P53, BCL-2, BCL-XL, survivin, SOD, HO-1, and iNOS is caused by ultraviolet irradiation. It can be seen that it has increased. This is because the biological defense function is activated by the strong stress of ultraviolet irradiation, and can be said to be a unified reaction. It should be noted that the increase in iNOS can be interpreted as being due to the action of activation of biological defense factors, because the product NO, that is, nitric oxide, is effective for biological defense when a small amount is produced.

In addition, when comparing Group 1 and Group 2 in Tables 1 to 8, the measured values of P53, BCL-2, BCL-XL, survivin, SOD, HO-1, and iNOS are almost the same and If there is no stress, it can be said that the sample had no gene expression effect to increase or decrease the specific protein.

However, when comparing the third group and the fourth group, the fourth group treated with the sample containing licorice extract containing no glycyrrhizin and glabrizine as an active ingredient was P53, BCL-2, BCL- It can be seen that the gene expression of XL, survivin, SOD, and HO-1 was promoted and the expression of the iNOS gene, which is an oxidative stress promoter, was suppressed.

As a result, the expression enhancer of biological defense function factor that is soluble in water and ethanol and contains licorice extract containing no glycyrrhizin and glabrizine as an active ingredient enhances the biological defense mechanism against stress inherent in the living body. Has a series of actions, enhances the expression of apoptosis-inhibiting factor consisting of BCL-2, BCL-XL and survivin, enhances the expression of DNA repair factor consisting of P53, and oxidizes consisting of SOD and HO-1 It can be seen that the expression of the stress inhibitory factor was enhanced and the expression of iNOS having oxidative stress was suppressed.

1) The expression enhancer of the biological defense function factor of the present invention can be used as a reagent for apoptosis research. Although anticancer drugs have a prominent effect at the beginning, cancer cells acquire drug resistance during use, and the effects of anticancer drugs are reduced and become ineffective. This is because cancer cells acquire anti-apoptotic ability. This drug has a wide range of anti-apoptotic effects, and if an antagonist against the cellular receptor, which is its action point, is developed, it can be expected that the cancer treatment will be completely cured by drug administration by using it together with the anticancer drug.

2) The anti-apoptotic effect is useful for preventing the onset of AIDS. The true symptom of AIDS is a decrease in CD4 cells. However, HIV-infected CD4 cells are less than 1 / 100,000 of all CD4 cells. Normally, no decrease in CD4 cells should occur. However, HIV-infected CD4 cells cause normal CD4 cells to apoptotic by some mechanism. Therefore, CD4 cells are decreased. Since this agent has an anti-apoptotic effect, it prevents the decrease of CD4 cells even if it is infected with HIV, thus preventing the onset of AIDS. And since this agent acts on the host (human) side, tolerance does not arise.

3) The main condition of Alzheimer's disease is that the central nerve cell is killed by apoptosis, and the insulin secretory cell of Type 2 diabetes is killed by apoptosis. Accordingly, anti-apoptotic agents are beneficial for the treatment of these diseases.
4) The agent for enhancing the expression of the biological defense function factor of the present invention is useful for the treatment of all diseases caused by apoptosis including AIDS, Alzheimer and diabetes.
5) P53 protein has the effect of preventing cancer development and extinguishing the cancer cells that have occurred. The enhancement of the expression of the P53 gene by the expression enhancer of the biological defense function factor of the present invention is useful for cancer treatment.

6) The expression enhancer of the biological defense function factor of the present invention promotes the expression of an oxidative stress inhibitor and suppresses the expression of iNOS that promotes oxidative stress. Oxidative stress is the cause of diseases such as Alzheimer's disease, Parkinson's disease, angina pectoris, myocardial infarction, and schizophrenia. If oxidative stress is suppressed, the onset of the above diseases can be prevented.
7) Depletion of tissue stem cells is considered to be a major cause of human aging, and the cause of depletion is apoptosis of tissue stem cells. Therefore, by using the agent for enhancing expression of the biological defense function factor of the present invention as an anti-apoptotic agent, tissue stem cells are increased, and as a result, prevention of aging and further rejuvenation effect can be expected. In addition, the increase in tissue stem cells enhances the tissue regeneration ability and enables the regeneration of lost tissue. Specifically, it can be expected to regenerate limbs lost due to accidents and war injury, complete healing of spinal cord injury, etc. .

Claims

Contains licorice extract which is soluble in water and ethanol and does not contain glycyrrhizin and glabrizine as an active ingredient, and enhances the expression of BCL-XL, survivin, P53, SOD and HO-1 An expression enhancer of a biological defense function factor having an iNOS expression suppressing action.
The agent for enhancing expression of a biological defense function factor according to claim 1, wherein the biological defense function factor is an apoptosis inhibitory factor consisting of BCL-XL or survivin.
The biological defense function factor expression enhancer according to claim 1, wherein the biological defense function factor is a DNA repair factor comprising P53.
The agent for enhancing expression of a biological defense function factor according to claim 1, wherein the biological defense function factor is an oxidative stress suppression factor comprising SOD and HO-1.
The biological defense function factor expression enhancer according to claim 1, wherein the biological defense function factor is an oxidative stress suppression factor that suppresses the expression of iNOS that promotes oxidative stress.