JP5295301B2 - Matrix metalloproteinase inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new effective component inhibiting a matrix metalloprotease to stimulate applications of the matrix metalloprotease inhibitor in various fields and a development of a new supplying source thereof. <P>SOLUTION: The matrix metalloprotease inhibitor is characterized by including a dimethyl allyl caffeic acid ester. The matrix metalloprotease inhibitor can be utilized as compositions for drink and food or oral compositions containing the matrix metalloprotease inhibitor. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

The present invention relates to a poplar extract, an extract of propolis originating from poplar, or a matrix metalloprotease (matrix) containing these components as an active ingredient.
metalloproteinase: hereinafter referred to as MMP).

MMP is an enzyme group that degrades extracellular matrix, and 19 types of human MMPs have been reported [Yoshinori Okada, Obstetrics and Gynecology 9 (7): 1115-1124, 2000 (Non-patent Document 1)]. For example, gelatinase MMP2 is an enzyme that degrades gelatin, laminin, aggrecan, collagen, fibronectin and elastin, which are components of the extracellular matrix. By inhibiting the action of this enzyme, invasion and metastasis of cancer cells are suppressed. [Imren S. et al., Cancer Res 56: 2891-2895, 1996 (Non-patent Document 2)]. MMP2 is particularly deeply involved in breast cancer metastasis [Motoo Nakajima Journal of Gynecologic Oncology 16: 2 1998 (Non-patent Document 3)]. MMP7 called matrilysin is produced specifically for cancer cells and, like MMP2, degrades gelatin, laminin, collagen, fibronectin and elastin. In gastric cancer and colorectal cancer, a correlation between the expression of MMP7 and cancer metastasis has been reported. MT1-MMP that binds to cell membrane is known to degrade proteoglycan, gelatin, laminin, collagen and fibronectin and cause migration of colon cancer cells and breast cancer cells [Koshikawa N. et al., J.
Cell Biol 148: 615-624,2000 (Non-Patent Document 4)].

  The extracellular matrix is a biopolymer that fills the gaps between cells in mammalian tissues, but the metabolism of this extracellular matrix is a balance between these MMPs and TMP inhibitors (Tissue inhibitors of matrix metalloproteinase). It is adjusted by. Structural abnormalities of extracellular matrix components and disruption of synthesis / degradation metabolic balance include tumor metastasis, allergy (eg, arthritic disease, rheumatoid arthritis), bone resorption disease (eg, osteoporosis), periodontal disease, corneal ulcer, It causes diseases such as skin ulceration and collagen breakdown associated with diabetes. Therefore, a substance that inhibits MMP activity may be used as a therapeutic or prophylactic agent for these six diseases.

  Several substances that inhibit MMP activity have been reported so far. Examples include hydroxam derivatives [JP 08-81443 (Patent Document 1)], flavonoids [JP 8-104628 (Patent Document 2)], JP 2000-80035 (Patent Document 3), esculetin derivatives [JP 08-183785 (Patent Document 4)], sulfonyl amino acid derivatives [JP 09-309875 (Patent Document 5)], terpenes [JP 11-139947 (Patent Document 6)], tannins [JP 2000-344672 Patent Document 7)] and the like are known. In addition, although an active ingredient has not been specified, it has been reported that plant extracts such as eucalyptus have an inhibitory action on MMP [JP 2001-64192 (Patent Document 8), JP 2001-192316 (Patent Document). 9)].

  Poplar is a plant inhabiting East Asia and its buds are used as an expectorant in folk remedies [Noro Seio et al. Medicinal Botany p106 1992 revised 4th edition (Non-patent Document 5)]. The components involved are saponin, tannin, and essential oil. However, there is no report that poplar extracts have MMP inhibitory activity. In addition, there is no report that it is effective for alleviation of tumor metastasis, allergy, bone resorption disease, periodontal disease, corneal ulcer, skin ulceration, collagen collapse associated with diabetes, which is a disease related to MMP inhibition.

  On the other hand, propolis is a substance that bees are constructed by mixing resin and sap and saliva such as leaves, shoots and berries of plants such as Aleklin (Baccharis dracunculifolia) [Shigemi Tazawa et al. Natural Medicines 54 (6): 306-313, 2000 (Non-patent Document 6)], which is excellent in antibacterial properties and antioxidative effects, and has been used in folk remedies especially for cancer treatment and prevention. In animal studies using mice, it has been reported that propolis extract suppresses metastasis of rectal cancer cells to the lung [Naruyuki Arai Bee Science 15 (4): 155-162, 1994 (Non-patent Document 7)] . However, the mechanism has not been fully elucidated, and it is only speculated that the propolis extract is due to the activation of immune cells mainly composed of macrophages.

  Propolis is also known to have antiallergic action, anti-inflammatory action, and periodontal disease prevention action, but its action mechanism has many unclear points. For antiallergic and anti-inflammatory effects, propolis extract suppresses hyaluronidase activity [Kazutoshi Yanami Food Research 165: 35-39, 2000 (Non-patent Document 8)], for prevention of periodontal disease, antimicrobial of propolis extract It was thought to be due to its action and antioxidant effect [Dental Science Report: 97 (6) 649-653, 1997 (Non-patent Document 9)]. Therefore, it was not thought that propolis extract and its components would significantly inhibit MMP activity.

The propolis used as experimental material in these reports is from Brazil (its plant origin is mainly alectrin and does not include poplar at all). Essentially different. In fact, there are differences in the types and contents of active ingredients contained in propolis depending on the country or region where they are collected. The reason is considered to be because the origin of propolis is different [Shigenori Kumazawa: Bee Science, 22 (1): 1-8, 2001 (Non-patent Document 10)].
JP 08-81443 JP-A-8-104628 JP2000-80035 JP 08-183785 JP-A-11-139947 JP-A-11-139947 JP2000-344672 JP2001-64192 JP2001-192316 Yasunori Okada Obstetrics and Gynecology 9 (7): 1115-1124,2000 Imren S. et al., Cancer Res56: 2891-2895,1996 Motoo Nakajima Journal of Gynecological Oncology 16: 2 1998 Koshikawa N. et al., J Cell Biol148: 615-624,2000 Noro Seio et al. Medicinal Botany p106 1992 revised 4th edition Tazawa Shigeru et al. Natural Medicines 54 (6): 306-313, 2000 Arai Naruyuki Bee Science 15 (4): 155-162, 1994 Kazutoshi Yanami Food Research 165: 35-39,2000 Dentistry Report: 97 (6) 649-653, 1997 Shigenori Kumazawa: Bee Science, 22 (1): 1-8, 2001

  An object of the present invention is to develop a new active ingredient that inhibits MMP and its source in order to promote the utilization of MMP inhibitors in various fields.

  As a result of investigating the degree of inhibition of MMP activity, the present inventor discovered that the poplar extract is an excellent MMP inhibitor, and propolis originating from poplar, particularly from China. It was discovered that propolis also has an equivalent MMP inhibitory activity. Furthermore, the extract is purified to identify the components involved and determine the structure, find that it is a caffeic acid ester represented by the following formula (I), and use a sample that is actually chemically synthesized. It was also demonstrated that these participating components have MMP inhibitory activity.

Thus, the present invention provides an MMP inhibitor characterized by containing a poplar extract or an extract of propolis (especially Chinese propolis) originating from poplar. Furthermore, this invention also provides the MMP inhibitor characterized by containing the dimethylallyl caffeic acid ester which is a compound shown by said structural formula (I).
In addition, according to the present invention, as a use of the MMP inhibitor of the present invention as described above, a food or drink composition or oral cavity composition containing the inhibitor is provided.

  The present invention enables an unrecognized poplar extract, a propolis extract derived from poplar, and a new type of MMP inhibitor comprising dimethylallyl caffeic acid ester and various compositions containing the same. It is to make.

  Hereinafter, the present invention will be described in detail. The matrix metalloprotease inhibitor of the present invention includes poplar or an extract of propolis originating from poplar, or a component substance thereof. Propolis is collected in various countries such as Brazil, China, Australia, Europe, etc., but there are differences in the types and amounts of ingredients contained in propolis because the source plant of propolis growing in that region is different (Non-Patent Document 10 described above). The propolis used in the present invention is limited to those containing poplar as a source plant. This kind of propolis is mainly collected in China.

  In order to obtain a poplar extract or propolis extract (an extract of propolis originating from poplar) that constitutes the MMP inhibitor of the present invention, poplar (particularly, its sprouts) or propolis is subjected to an extraction operation. Although this extraction operation is not particularly limited, it is generally an alcohol such as ethanol or glycerol or an aqueous alcohol solution (a mixture of water and alcohol) or a hydrophobic organic solvent (eg, hexane) or a supercritical state (carbon dioxide). The carbon dioxide in a state exceeding the critical temperature and critical pressure of the gas) is efficiently extracted by using it as an extractant. In addition, when using a material composed of an extract obtained using a hydrophobic organic solvent for pharmaceutical food, if the solvent remains, there is a problem in terms of safety and health, so purify and completely remove the solvent. There is a need.

  The poplar extract can be purified, concentrated or diluted, or used as it is as a food. It can be used by emulsifying in water using a surfactant, or it can be used alone or mixed with an auxiliary raw material to be included in a capsule or tablet. Food and drink compositions obtained by mixing poplar extracts (eg, confectionery, bread, rice cake, gum, drinking water, etc.) can be used for the purpose of preventing diseases such as cancer metastasis. An oral composition obtained by mixing poplar extract with toothpaste or gargle can also be used for preventing periodontal disease.

  Extracts of propolis originating from poplar (especially Chinese propolis) can also be used with similar specifications and shapes.

When using the oral composition obtained by mixing poplar extract with toothpaste etc. for periodontal disease prevention, the content rate of poplar and propolis extract with respect to toothpaste is not specifically limited. However, in order to expect a sufficient effect, the poplar extract concentration is desirably 3% (W / W) or more.
EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Measurement of MMP inhibitory activity of poplar extract The inhibition rate against two types of MMPs [membrane-bound MT1-MMP and gelatinase (MMP-2)] was measured. These active proteins of MMP were prepared according to a known method (Patent Document 7: Japanese Patent Laid-Open No. 2000-344672) in the following manner.
First, these two cDNAs were incorporated into an expression vector, infected with E. coli, and expressed in large quantities in the E. coli. The expressed protein was purified with an affinity column and reconstituted (refolded). MT1-MMP was reacted with trypsin to prepare an active protein. On the other hand, for MMP-2, p-AMPA (p-aminophenylmercuric
acetate: mercuric acetate p-aminophenyl acetate) to prepare an active protein.

  One gram of dried shoots of poplar (Populus lassiocarpa) was pulverized to remove debris such as wood chips, then mixed with 3 ml of 95% ethanol or an aqueous ethanol solution and stirred at room temperature for 3 days. The mixture was squeezed and filtered to obtain a poplar extract (No. 1 squeeze). Further, ethanol was added to the squeezed residue and extracted in the same manner (second squeezing). Finally, these extracts were mixed into a poplar extract for testing.

  Poplar extract stock solution prepared with 100% ethanol to 50 mg / ml, 70 μl is injected into HPLC (column: CAPCELLPAKC18 10 mm × 250 mm) and fraction collected (elution conditions: 70% acetonitrile / 30 mn, UV 280 nm, Flow rate 3.0) ml / min). The aliquot sample was freeze-dried, the dry weight was measured, and it was adjusted to 1 mg / ml with 50% ethanol.

20 μl of each of these fraction samples and appropriately diluted propolis extract (extract concentration: 0.1-10000 μg / ml), assay buffer 20 μl (Tris-HCl pH 7.5 500 mM, NaCl 1.5 M, CaCl ₂ 100 mM, ZnSO ₄ 500 μM) , NaN ₃ 30mM, Brij35 0.05%), MT1-MMP, preincubation for 15 minutes at 37 ° C, then add MOCAc / DNP peptide 120µl (4.16µM) and keep at 37 ° C for fluorescence microplate reader (detection conditions) : Excitation wavelength 320 nm, fluorescence wavelength 390 nm) every 15 minutes and measured over time for 120 minutes. After the measurement, 20 μl of 20 mM EDTA was added to stop the reaction, and 5 μl was injected into HPLC with fluorescent UV detector to detect the peptide (detection conditions: 70% acetonitrile / 0.1% TFA / 30mn, UV280 nm, Flow rate 1.0 ml / min). The inhibition rate was calculated according to the following calculation method.
Inhibition rate: Inhibition rate calculated from HPLC analysis results
Inhibition rate (%) = (AB) / A × 100
A = MT1 peak height
B = peak height of each sample

Furthermore, in order to quantitatively evaluate the degree of MMP inhibitory activity, IC ₅₀ was calculated from the inhibition rate of each sample and used as an evaluation index. IC ₅₀ is the “concentration of the sample necessary to inhibit the activity of MMP by 50% under a certain condition”, and the smaller this value, the higher the inhibitory activity.

The IC ₅₀ (mg / ml) of Poplar extract against MT1-MMP was measured and found to be 0.031. Therefore, it was revealed that the poplar extract has a high MMP inhibitory activity.

Measurement of MMP inhibitory activity of Chinese propolis extract Chinese propolis is a substance that bees are constructed from the leaves, shoots, and berries of plants such as poplar. On the other hand, the main plant of Brazilian propolis is Aleklin. Therefore, 3 ml of 80% ethanol was added to 1 gram of each propolis to prepare a propolis extract for testing in the same manner as in the preparation method of the poplar extract of Example 1. The inhibition rate of MMP activity was measured, and IC ₅₀ (mg / ml) against MT1-MMP was calculated. As a result, the IC ₅₀ (mg / ml) of the Chinese propolis extract was 0.034. Chinese propolis extract was found to have MMP inhibitory activity almost the same as poplar.

Measurement of MMP inhibitory activity of dimethylallyl caffeic acid ester Since Chinese propolis contains more caffeic acid derivatives than Brazilian propolis, the MMP inhibitory activity of one of them, dimethylallyl caffeic acid ester, was measured.
In addition, commercially available caffeic acid was purchased for testing as a comparative control. On the other hand, dimethylallyl caffeic acid ester was synthesized in-house by the following procedure. 1.8 grams of 3,4-dihydroxycinnamic acid was dissolved in pyridine and 2.2 grams of acetic anhydride was added dropwise. Pyridine is distilled off under reduced pressure, dissolved in 40 ml of ethyl acetate, washed with 0.1N hydrochloric acid to remove pyridine. After drying ethyl acetate, the residue was recrystallized with acetone to obtain 3,4-diacetoxycinnamic acid.

Next, 1.32 grams of 3,4-diacetoxycinnamic
acid dissolved in CH ₂ Cl ₂ and 1.53 grams of 2-chloro-N-methylpyridinium
Chloride was added and Et3N was added dropwise with stirring. CH ₂ Cl ₂ was washed with hydrochloric acid, 10% sodium hydrogen carbonate and water, and the CH ₂ Cl ₂ layer was dried over anhydrous magnesium sulfate. After evaporation under reduced pressure, the residue was treated with silica gel column chromatography, prenyl 3,4 -Obtained diacetoxycinnamate. This was dissolved in a 1: 1 mixture of CH ₂ Cl ₂ and methanol, 10% sodium hydrogen carbonate was added, and after stirring, CH ₂ Cl ₂ was added, and the CH ₂ Cl ₂ layer was separated and washed with water. The extract was dried over anhydrous magnesium sulfate and distilled under reduced pressure to obtain dimethylallyl caffeic acid ester. The isolated compound was analyzed by NMR to confirm the structure. The results are shown in Table 1. Dimethylallyl caffeic acid ester specifically contained in poplar and Chinese propolis was confirmed to have higher inhibitory activity than caffeic acid on two types of MMPs.

According to the present invention, it has become possible to inhibit the activity of matrix metalloprotease using a poplar extract, an extract of propolis originating from poplar, or dimethylallyl caffeic acid ester. Ingestion or application of these inhibitors can prevent or prevent matrix metalloprotease related diseases such as tumor metastasis, allergy, bone resorption disease, periodontal disease, corneal ulcer, skin ulceration, collagen breakdown associated with diabetes Can be treated.

Claims (2)

A matrix metalloprotease MT1-MMP inhibitor characterized by containing dimethylallyl caffeic acid ester, which is a compound represented by the following structural formula (I), and having MT1-MMP inhibitory activity.
An oral composition for inhibiting a matrix metalloproteinase comprising the matrix metalloprotease MT1-MMP inhibitor according to claim 1.