JP5196465B2 - MMP gene expression promoter and collagen degradation promoter - Google Patents

Description

  The present invention relates to an MMP gene expression promoter, a collagen metabolism promoter, a cosmetic and a medicine, and more particularly, to an MMP gene expression promoter, a collagen metabolism promoter, a cosmetic and a medicine containing a Coriaceae plant root extract.

  Excessive fibrosis and accumulation of fiber components (collagen) in vivo cause collagen diseases such as scleroderma, cirrhosis, nephrosclerosis, and pulmonary fibrosis. These phenomena are also observed in forty shoulders and fifty shoulders where shoulder arthritis has progressed. Although autoimmune disease is thought to be involved as a cause of fibrosis, details have not been elucidated yet. Therefore, despite the large number of patients with these diseases, no effective treatment has been established so far for fibrosis, a type of collagen disease.

  In recent years, it has been reported that an experimental attempt to introduce a gene (MMP1) involved in collagen degradation into cells using adenovirus is effective in improving fibrosis (Non-patent Document 1: Iimuro). The degradation of collagen in the diseased part and the promotion of collagen metabolism by this are expected as an effective means for the above collagen disease. However, gene therapy using viruses still has problems in terms of safety, for example, cases of leukemia have been reported so far. In Japan, according to the guidelines of the Ministry of Health, Labor and Welfare, the target diseases for gene therapy are limited to “diseases that are life threatening” and “diseases whose efficacy is sufficiently predicted by conducting gene therapy clinical research”. For this reason, gene therapy using adenovirus has a narrow application range and low versatility.

  Here, as a special crop of Tokyo, plants belonging to the genus Cericaceae such as Ashitaba have been used for food since ancient times. Patent Document 1 (Japanese Patent Laid-Open No. 2005-255527) discloses that granulation is promoted by administering a mixture of a paffia extract and an Ashitaba extract to an experimental animal, and the content of fudroxyproline increases. From this, it is said that a mixture of the paffia extract and the ashitaba extract has an action of promoting collagen synthesis.

JP 2005-255527 A Iimuro Y, Nishio T, Morimoto T, Nitta T, Stefanovic B, Choi SK, Brenner DA, Yamaoka Y., "Delivery of matrix metalloproteinase-1 attenuates established liver fibrosis in the rat.", Gastroenterology, 2003 Feb; 124 (2 ): 445-58. Robert Visse, Hideaki Nagaseya, "Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases", Circulation Research, 2003 May, 92, pp827-839

  An object of the present invention is to provide a highly safe MMP gene expression promoter, collagen metabolism promoter, cosmetics and medicine.

  According to one aspect of the present invention, there is provided an MMP gene expression promoter comprising an effective amount of an Aceraceae plant root extract. According to another aspect of the present invention, there is provided a collagen metabolism promoter comprising an effective amount of an Aceraceae plant root extract. According to another aspect of the present invention, there is provided a cosmetic comprising a root extract of the genus Cericaceae. According to another aspect of the present invention, there is provided a medicament for treating a fibrotic condition comprising an effective amount of an Aceraceae plant root extract.

  According to another aspect of the present invention, an MMP gene expression promoter, a collagen metabolism promoter, a cosmetic, comprising the steps of contacting a sorghum genus plant root with an extraction solvent to obtain a sericidae plant root extract. Alternatively, a method for producing a medicament for treating a fibrotic condition is provided.

  As will be described in detail below, according to the present invention, an MMP gene expression promoter, collagen metabolism promoter, cosmetics and pharmaceuticals derived from a plant extract belonging to the genus Cericaceae that are kind to the living body are provided.

  Embodiments of the present invention will be described below. However, the following description is for specifically explaining the present invention, and is not intended to limit the technical scope of the present invention.

  When the action of Ashitaba root extract on cultured human cells was examined, an action on cell morphology was observed. Therefore, as a result of analysis at the gene level using a DNA microarray, it was confirmed that the gene (MMP1) involved in the promotion of collagen degradation was expressed about 500 times that of the control group by the extract of the root of Ashitaba. (See Examples).

  As described above, although it has been reported that an experimental attempt to introduce the MMP1 gene into cells using adenovirus is effective in improving fibrosis (Non-patent Document 1: Iimuro), it has been reported against fibrotic conditions. At present, no effective treatment has been established. Based on the above findings, the present inventors can use the root extract of Ashitaba as an MMP gene expression promoter or a collagen metabolism promoter, and thus a cosmetic or fiber having a collagen metabolism promoting effect or a fibrotic condition improving effect. The present invention has been found out that it can be used as a medicine for treating a chemical condition.

  Thus, according to the present invention, utilizing the physiological activity of Ashitaba root extract, it is possible to induce the expression of its own MMP gene and promote the degradation of collagen. Since Ashitaba, which has been edible since ancient times, is used as a raw material and does not involve gene transfer, the present invention is highly safe and does not cause pain.

  In the present invention, an extract from the root part of the Apiaceae plant is used as an active ingredient. In addition to Ashitaba (Angelica keiskei), Acerium (Angelica edulis), Ishizuchi-Chinodake (Angelica saxicola), Ishizuchi-shibofu (Angelica hikoki), Angelica hikoskei Iwanin ginseng (Angelica hakonensis), Ubata ginseng (Angelica ubatakensis), Ezonoyu (Angelica ursina), Ezonoyorogusa (Angelica anomala), Pterodactyl ginseng (Angelica ubatakensis), Obasenkyu (Angelica genuflexa) Angelica tenuisecta, Angelica mayebarana, Angelica cartilaginomarginata, Angelica pubescens, Shiranesenkyu (Angelica polymorpha), Angelica longeradiata, Angelica pseudosuki (hiko) , Angelica yoshinagae, Angelica acutiloba, Angelica decursiva, Angelica hakonensis, Angelica inaequalis, Angelica japonica, Angelica japonica, Giica lag , Angelica stenoloba, Angelica matsumurae, Angelica acutiloba, Angelica yakusimensis, Angelica dahurica, Angelica archangelica, etc. In particular, it is preferable to use Ashitaba as a Ciliaceae plant.

  In the present invention, the extract includes not only an extract directly obtained by extraction from the root, but also a diluted solution, a concentrated solution, a purified solution, a dried product thereof, and the like. The extract is obtained by bringing the root of Ashitaba into contact with the extraction solvent, purifying the extract obtained as necessary, and further diluting, concentrating or drying the extract or purified solution obtained as necessary. It is done.

  The extraction can be performed either continuously or batchwise, and can be performed with stirring or standing. For the extraction, for example, a Soxhlet extractor can be used. For the extraction, the root can be used as a raw plant or a dried product thereof (lyophilized product or the like) as it is or after being crushed or cut. Examples of extraction solvents include water (distilled water and ion-exchanged water), hydrophilic organic solvents (alcohols such as methanol, ethanol, isopropyl alcohol, and butanol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; dimethyl sulfoxide. Etc.) or a hydrophobic organic solvent (chloroform, dichloromethane, etc.) can be used alone or in admixture at any ratio. In particular, it is preferable to use ethyl acetate as the extraction solvent. The extraction can be performed at room temperature or under heating, preferably at 0 to 50 ° C, more preferably at 10 to 30 ° C. The extraction time can be arbitrarily set according to the temperature, the type and amount of the solvent, and can be set to 1 hour, for example. The extraction conditions such as the solvent, the amount of solvent, temperature, pH, pressure, and extraction time can be determined from the viewpoint of extraction efficiency and cost. It is preferable to remove root residues such as plant fibers from the extract by a commonly used method such as filtration, pressing, and centrifugation.

  If necessary, it is preferable to purify the obtained extract and remove inactive components such as pigments. Purification can be performed by a commonly used method such as liquid-liquid partition extraction or various chromatographies.

  Further, the obtained extract or purified solution can be used as it is, and can be diluted, concentrated or dried as necessary. For example, the obtained extract or purified solution can be diluted with a solvent such as water or alcohol, concentrated under heating and / or under reduced pressure, and dried under reduced pressure, freeze-dried, spray-dried, etc. Can be dried. The obtained root extract can be made into a desired composition and form by further adding an additive depending on the application.

  As described above, the plant root extract can be used as an MMP gene expression promoter and a collagen metabolism promoter. Here, the MMP gene includes MMP3 in addition to MMP1. MMP1 (matrix metalloproteinase) is also called interstitial collagenase and is known to be involved in collagen degradation (Non-patent document 1: Iimuro, Non-patent document 2: Visse et al.). MMP3 (matrix metalloproteinase) is also called stromelysin and is known to be involved in degradation of fibronectin and laminin in addition to collagen (Non-patent document 2: Visse et al.).

  Furthermore, the plant root extract can be used for cosmetics, in particular, for cosmetics having a collagen metabolism promoting action or a fibrosis treatment action. Further, the root extract of the genus Sisius can be used as a medicine for treating a fibrosis state. According to the present invention, collagen metabolism in a fibrotic state can be led to a normal state by promoting the degradation of collagen. Thus, according to the present invention, it is possible to promote collagen metabolism and to treat a fibrosis state by the root extract of the genus Sisius. Here, treatment includes treatment, improvement, maintenance (prevention of deterioration) and prevention.

  In the present specification, the fibrosis state refers not only to a fibrosis pathology but also a state widely associated with collagen accumulation, and includes a state where excessive fibrosis is observed and a state where fiber components are excessively accumulated. As used herein, conditions include diseases, disorders, and abnormal conditions. More specifically, the fibrosis state is scleroderma, cirrhosis, liver fibrosis, nephrosclerosis, pulmonary fibrosis, myelofibrosis caused by excessive fibrosis and accumulation of fiber components (collagen) in vivo. Forty-shoulder, fifty-shoulder, etc. in which collagen disease such as fibrosis of each organ / tissue such as pancreatic fibrosis and shoulder arthritis progressed. Furthermore, it includes scars (burns, trauma, ulcers, acne scars, etc.) and keloids in which fibrotic foci are formed by the growth of fibrous connective tissue. The present invention can be applied to mammals including humans.

  The content of Ashitaba root extract is an amount effective to promote collagen metabolism and / or an amount effective to treat fibrotic conditions. The presence or absence of these effects can be tested by the method described in Non-Patent Document 1 (Iimuro). More specifically, when used as a cosmetic, it should be set according to the dosage form, etc., but in general, the content of the root extract is 0.01% as a root converted amount of the original plant based on the total amount of the cosmetic. It is preferably ˜10000 wt%, more preferably 0.1 to 6500 wt%, and further preferably 1.0 to 5000 wt%. When used as a medicine, it should be set according to the subject of administration, age of administration subject, administration route, dosage form, fibrosis state, etc. Generally, the dose of plant root extract is 0.1 It is preferably ˜200 g / kg body weight / day, more preferably 0.1 to 100 g / kg body weight / day, and further preferably 0.1 to 50 g / kg body weight / day.

  Cosmetics of the present invention are cosmetics for skin such as lotion, milky lotion, cream, makeup base lotion, makeup base cream, liquid or cream or ointment type foundation, eye color, and cheek color. Hair cosmetics such as shampoos, rinses and hair treatments, and body cosmetics such as hand creams, leg creams and body lotions. The medicament of the present invention is preferably an external preparation, and particularly preferably a skin external preparation. Specifically, the medicament of the present invention can be used as a skin external preparation in the form of a lotion, emulsion, gel, cream, ointment or the like. The medicament of the present invention can also be used as an internal preparation. Specifically, the medicament of the present invention is in a solid or liquid form of tablets, pills, capsules, powders, fine granules, granules, emulsions, solutions, suspensions, syrups, etc. It can be used as an agent.

  The cosmetics and medicaments of the present invention are binders, stabilizers, emulsifiers, suspending agents, dispersing agents, lubricants, preservatives, extenders, surfactants, viscous agents, powders according to their use and dosage form. , Pigments, colorants, bactericides, fragrances, whitening agents, UV protection agents, antioxidants, humectants, blood circulation promoters, and the like.

  Ceramaceae plants have been edible since ancient times, and no toxicity is observed.

  Embodiments of the present invention will be described below with reference to the accompanying drawings. However, the following description is for explaining the present invention more specifically, and is not intended to limit the technical scope of the present invention.

[Materials and methods]
[Preparation of Ashitaba root extract, etc.]
In FIG. 1, the typical flowchart of the preparation method of an Ashitaba root part extract is shown. About 1 kg of Ashitaba root washed with running water was cut into small pieces and pre-frozen at -30 ° C, and then freeze-dried. The freeze-dried Ashitaba root was pulverized with a cyclone mill and passed through a 1 mm mesh sieve. 50 g of ethyl acetate was added to 1 g of crushed Ashitaba root, and extraction was performed at room temperature for 1 hour. The suction filtered extract was dried under reduced pressure using a rotary evaporator to obtain an Ashitaba root extract.

[Separation of mesenchymal cells]
Mesenchymal cells were isolated from surplus fingers resected by surgery. First, the periosteum covering the bone material was removed with a scalpel from the excess finger washed with PBS, and washed again with PBS. Excess finger bone marrow was refluxed (over 10 times) with Dulbecco's modified Eagle medium (DMEM) using a syringe on a 10 cm petri dish, and DMEM containing bone marrow accumulated in the petri dish was transferred to a test tube through a 40 μm cell filter. Subsequently, the supernatant was removed by centrifugation at 1,000 rpm for 5 minutes to obtain mesenchymal cells. The obtained mesenchymal cells were stored frozen at −196 ° C. until used for experiments.

[Cell culture]
The cryopreserved surplus finger-derived mesenchymal cells are quickly thawed at 37 ° C., suspended in DMEM supplemented with 0.5% inactivated fetal bovine serum (FBS), insulin and transferrin, and 1 × 10 ^{5 in a} petri dish. The cells were seeded in a 10 cm petri dish so as to contain individual cells and cultured under conditions of 37 ° C. and 5% CO ₂ . One week later, the medium was removed, washed with PBS, and the cells were detached and collected using trypsin. The collected cells are suspended in a medium containing 0.3 to 0.4% Ashitaba root extract and leaf extract, and each well contains 0.3 × 10 ⁴ cells and 800 μl of medium. In a 24-well plate. The culture period was 2 weeks, and the medium was changed once after 1 week.

[Extraction of total RNA]
Total RNA was collected from cultured human mesenchymal cells using RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's recommended protocol.

[Microarray analysis]
Comprehensive analysis of genes expressed in human mesenchymal cells using Human Genome U133A Probe array (manufactured by Affymetrix) for understanding genome-wide effects of Ashitaba root extract on cells went. Experiments were performed using the total RNA obtained above according to the manufacturer's recommended protocol. The obtained data was analyzed using Genespring software version 5 (manufactured by Silicon Genetics), and the expression level was obtained as a relative numerical value.

[RT-PCR]
(Synthesis of cDNA)
To 5 μg of the total RNA obtained above, 1 μl of 50 μM oligo-dT primer and 1 μl of 10 mM dNTP mix were added, reacted at 65 ° C. for 5 minutes, and rapidly cooled on ice for 1 minute. Thereafter, 4 μl of 5 × first strand reaction buffer, 1 μl of 0.1 M DTT, RNase out Recombinant Ribonuclease Inhibitor (40 U / μl), 1 μl of reverse transcriptase Super Script III (200 U / μl) were added and mixed gently. Treated at 50 ° C. for 1 hour followed by 70 ° C. for 15 minutes. Thereafter, 1 μl of RNase H was added and treated at 37 ° C. for 20 minutes to degrade all RNA and purify cDNA. The volume of the reaction composition liquid was as follows.

<Reaction composition liquid>
(1) 5 μg of total RNA
(2) 50 ng / μl oligo-dT primer (manufactured by Invitrogen) 1 μl
(3) 1 μl of 10 mM dNTP mix (manufactured by Invitrogen)
The total volume was adjusted to 13 μl with (1) + (2) + (3) + distilled water.
(4) 5 × 1st strand reaction buffer (manufactured by Invitrogen) 4 μl
(5) 0.1 M DTT (manufactured by Invitrogen) 1 μl
(6) RNase out Recombinant RNase Inhibitor (Invitrogen) 1 μl
(7) Enzyme Super ScriptIII RT (manufactured by invitrogen)
(8) RNase H (Invitrogen) 1 μl

(PCR reaction)
500 ng of cDNA, 2 μl of 10 × Ex Tag Buffer (20 mM Mg2 + plus), 1 μl of dNTP mix (2.5 mM each), 0.5 μl of 10 μM sense primer, 0.5 μl of 10 μM antisense primer, 0.1 μl of TaKaRa A reaction solution was prepared by adding Ex Taq Polymerase (5 U / μl). The reaction conditions, reaction composition solution and primer sequence were as follows.

<Reaction conditions>
MMP-1; 30 cycles of 94 ° C for 30 seconds, 57 ° C for 1 minute, 72 ° C for 1 minute 18S ribosomal RNA; 30 cycles of 94 ° C for 30 seconds, 50 ° C for 1 minute, 72 ° C for 1 minute

<Reaction composition liquid>
(1) cDNA 500 ng
(2) 10 × Ex Tag Buffer (Takara Bio) 2 μl
(3) 1 μl of dNTP mix,
(4) 10 mM sense primer 0.5 μl
(5) 10 mM antisense primer 0.5 μl
(6) TaKaRa Ex Taq Polymerase (5 U / μl) (Takara Bio) 0.1 μl

<Primer sequence>
MMP-1-sense CTGAAGGTGATGAAGCAGCC
MMP-1-antisense AGTCCAAGAGAATGGCCGAG

(Electrophoresis)
10 μl of the PCR product was electrophoresed using a 0.8% agarose gel containing ethidium bromide at a concentration of 1 μg / ml. The markers used are as follows.

<Electrophoretic marker>
φX174DNA Haelll Digest (50 ng / μl) (manufactured by BioLabs)
OneSTEP Ladder 50 (0.05-2 kbp) (Nippon Gene)
OneSTEP Ladder 100 (0.1-2kbp) (Nippon Gene)

[result]
[About microarray analysis]
FIG. 2 shows the results of expression analysis using a microarray for the expression of the MMP1 gene. As shown in FIG. 2, the expression levels of MMP-1 in human mesenchymal cells in the non-added group, the DMSO-added group, and the DMSO-dissolved Ashitaba leaf extract extract group after 2 weeks of culture were 50.2, 6, and 8, respectively. In contrast, it was 2801.3 in the group cultured with the DMSO-dissolved Ashitaba root extract added, which was about 500 times the value when DMSO alone was added. Thus, it was confirmed that significant expression of the gene MMP1 in cells was induced by adding Ashitaba root extract to human cultured cells.

  FIG. 3 shows the results of expression analysis using a microarray for the expression of the MMP3 gene. As shown in FIG. 3, the expression levels of MMP-3 in human mesenchymal cells in the non-added group, DMSO-added group, and DMSO-dissolved Ashitaba leaf extract extract group after 2 weeks of culture were 84, 75, and 56, respectively. On the other hand, in the group cultured with DMSO-dissolved Ashitaba root extract, it was 714, which was a value corresponding to about 10 times that when DMSO alone was added. Thus, it was confirmed that significant expression of the gene MMP3 in cells was induced by adding Ashitaba root extract to human cultured cells.

[About RT-PCR]
FIG. 4 shows the results of expression analysis by electrophoresis. As shown in FIG. 3, as a result of electrophoresis, a band of 428 bp was obtained with the MMP-1 primer set and a band of 186 bp was obtained with the 18S ribosomal RNA primer set. A clear band appeared at 428 bp in the DMSO-dissolved Ashitaba root extract addition group, confirming that the gene MMP-1 was expressed. In other additive-free sections, the expression of gene MMP-1 was not confirmed.

In FIG. 1, the typical flowchart of the preparation method of an Ashitaba root part extract is shown. FIG. 2 shows the results of expression analysis using a microarray for the expression of the MMP1 gene. FIG. 3 shows the results of expression analysis using a microarray for the expression of the MMP3 gene. FIG. 4 shows the results of expression analysis by electrophoresis.

Claims (2)

An MMP gene expression promoter comprising an effective amount of Angelica keiskei root extract. A collagen degradation accelerator comprising an effective amount of Angelica keiskei root extract.

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