JP2003335648A - Protease inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a protease inhibitor which inhibits the actions of proteases and is useful for preventing and treating periodontal diseases. <P>SOLUTION: The protease inhibitor is characterized by containing one or more plants selected from the group consisting of Engelhardtia chrysolepis H. leaves, green tea, Artemisia princeps, Pseudocydonia sinensis, Rosa roxburghii Tratt., Gymnema sylvestre R. Br., Aspalathus linears tea, Crataegus cuneata Sieb. et Zucc., turmeric, Monomordica grosvenori, silymarin, Lychium chinense, unpolished violet rice, Eleuthero ginseng, Alpinia speciosa leaves, Houttuynia cordata, Zizyphi fructus, and Ganoderma lucidum, or the extracts of the plants as an active ingredient. The protease inhibitor exhibits inhibiting action on both arginine gingipain and lysine gingipain enzymes which are cysteine proteases produced by Porphyromonas gingivalis. Since the active ingredients are natural substances, the protease inhibitor not having a side effect and containing excellent safety can be obtained. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to novel protease inhibitors. More specifically, it relates to a novel protease inhibitor which is useful for the prevention or treatment of gingivitis, alveolar pyorrhea and the like, among cysteine proteases produced by periodontopathic bacteria, which inhibits arginine and lysine dipain activities.

[0002]

2. Description of the Related Art Periodontal disease is a disease occurring in periodontal tissues, and its main condition is the destruction of periodontal tissues existing relatively in the superficial layers such as gingival epithelium and connective tissue, and the subsequent destruction of alveolar bone. There is. It is considered that various destructive factors derived from the host and oral bacteria are involved in the development and progression of such pathological conditions. Among them, proteases derived from periodontal disease-causing bacteria are particularly important as factors that execute periodontal tissue destruction. ing.

The gram-negative anaerobic bacterium Porphyromonas gingivalis is considered to be the most important pathogenic bacterium in adult periodontitis and rapidly progressive periodontitis, and the trypsin-like cysteine protease produced by this bacterium ( Gindipain) has a strong trypsin-like activity as a catalytic activity, and due to its peptide bond cleavage specificity, arginine gindipain (Arg-gindipain) specific to the C-terminal side of arginine residues and C-terminal side of lysine residues are present. It is divided into specific lysine gindipine (Lys-gindipine). It is believed that both enzymes, individually or in cooperation with each other, cause the degradation of host proteins and the damage of host cells, resulting in various pathological conditions associated with periodontal disease. In addition, both enzymes, in the survival strategy for this bacterium to grow and grow in the periodontal pocket, that is, in the mechanism such as coaggregation / adhesion, hemagglutination, hemoglobin binding, amino acid and heme acquisition,
Plays an essential role (Shi, Y. et al .: J. Biol.
Chem. 274, 17955-17960, 1999). Therefore, as a means of preventing or treating periodontal disease, Arg-gingipain and
Inhibiting the action of both Lys-gingipain proteases appears to be a very effective means. Several kinds of plants or their solvent-extracted extracts have been shown as inhibitors against Arg-gingipain (JP-A-6-25000). However, there is no description or suggestion that these several types of plants or their solvent-extracted extracts have an inhibitory effect on both Arg-gingipain and Lys-gingipain enzymes.

Known protease inhibitors such as leupeptin, ethylenediaminetetraacetic acid and tosylphenylalanyl chloromethyl ketone inhibit these proteases but are highly toxic and therefore cannot be used for the prevention or treatment of periodontal disease.

[0005]

At the present time, there is a demand for a suitable preventive and therapeutic drug which is effective against periodontal disease and has no side effects. Therefore, it is useful for the prevention and treatment of periodontal disease to provide a highly safe substance that exhibits an inhibitory effect on both Arg-gindipain and Lys-gindipain enzymes.

[0006]

[Means for Solving the Problems] As a result of earnestly researching the present invention to obtain a drug having a high safety and a truly excellent protease inhibitory effect, a certain plant extract has a protease inhibitory activity. It was found that the present invention has been accomplished.

[0007] That is, the present invention, yellow tea leaves, green tea, turmeric, mugwort, karin, pear, gymnema, rooibos tea, hawthorn, lakanka, silymarin, lacquer, purple brown rice, eleutherocoque, moon peach leaf, dokudami, oju and spirits. The present invention provides a protease inhibitor, which comprises a plant or a plant extract selected from one or more species selected from the group consisting of turf as an active ingredient.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The plant used in the present invention is not particularly limited in its part and form, and it is usually preferable that the plant itself is crushed or dried powder. In addition, the plant extract used in the present invention is not particularly limited in the site or form to be extracted, and may be performed according to a known extraction method. For example, it can be obtained by extracting the plant or its dry powder with water or alcohol, concentrating and drying,
It is not limited to these. Moreover, a commercially available plant extract can also be used.

The protease inhibitor of the present invention is a chewing gum, candy, chocolate, chewable tablet, toothpaste, tooth powder, liquid toothpaste, mouthwash, cream, mouthwash, troche, ointment, patch, according to a conventional method. It can be a composition such as a tablet. In the present invention, the plant or its extract can be used alone or in combination of two or more kinds, and the blending amount thereof may be arbitrary, but may be appropriately determined according to the purpose of use, and is usually 0. By blending 0001 to 90% by weight, preferably 0.001 to 40% by weight, and more preferably 0.01 to 10% by weight, the desired effect can be obtained. However, when ingested for the purpose of health or maintenance of health over a long period of time, the amount may be smaller than the above range, and since the present active ingredient has no problem in safety, It can be used even if used in a larger amount. Also,
The other compounding ingredients are not particularly limited, and any ingredient usually used in this type of composition can be compounded.

[0010]

EXAMPLES The present invention will be described in more detail with reference to Examples and Test Examples.

[Example 1] Preparation of plant extract Addition of 10% by weight of plant extract to water was performed at 16000 rpm.
The supernatant after centrifugation for 10 minutes was obtained as a plant extract.

Example 2 A hard candy was manufactured according to a conventional method according to the following formulation. Hard candy

[Table 1]

Example 3 A chewing gum was produced according to a conventional method according to the following formulation. Chewing gum

[Table 2]

Example 4 A polishing agent was produced according to a conventional method according to the following formulation. Training agent

[Table 3]

[Test Example]Protease activity of Porphyromonas gingivalis
Search for substances that inhibit (1) Preparation of protease Porphyromonas gingivalis JCM8525 strain (Pg) EG
Anaerobic culture at 37 ℃ for 72 hours on agar medium, collect the bacteria, and use PBS.
After washing, PBS was added and ultrasonication was performed. Then, centrifuge (140
(00 rpm, 10 minutes), and the culture supernatant is treated with protease enzyme.
Was subjected to the following test.

(2) Preparation of substrate solution benzoyl-L-arginine-7-amido-4-methylcoumarin (Bz-Arg-MCA) and t-butyloxycarboyl-valine-leucine-lysine-7-amide-4 -Methylcoumarin (B
oc-Val-Leu-Lys-MCA) 50 μM in PBS containing 1 mM cysteine
The diluted solution was used as a substrate solution. Bz-Arg-MCA was used as a substrate for Arg-gingipain and Boc-Val-Leu-Lys-MCA was used as a substrate for Lys-gingipain.

(3) Preparation of sample such as plant extract Follow Example 1.

(4) Measurement of protease activity (method 1) Sample extract of the prepared plant extract or the food additive used as a target was appropriately diluted with PBS containing 1 mM cysteine and added to a 96-well plate at 200 μl each. , Protease was added to 1.28 μg / ml, the mixture was allowed to stand at room temperature for 2 hours, and finally 50 μl of fluorescent solution was added, and immediately the fluorescence intensity per minute (Ex 365 nm, Em 460 nm) was measured (n =
3). Enzyme activity when adding a sample such as plant extract a,
Enzyme activity without addition of sample such as plant extract
Then, the protease inhibition rate (%) of each food additive and plant extract was calculated by the following formula.

(5) Measurement of Protease Activity (Method 2) With respect to a plant extract or the like which showed an inhibition rate of 50% or more in Method 1, the protease activity was measured by changing the concentration added. Samples such as prepared food additives and plant extracts are appropriately diluted with PBS containing 1 mM cysteine, 200 μl each is added to a 96-well plate, 50 μl of fluorescent solution is added, and finally protease is adjusted to 1.28 μg / ml. Immediately after the addition, the fluorescence intensity per minute was measured (n = 3).

[0020]

[Equation 1] The results are shown in Tables 4-11.

Effect on Arg-gingipain

[Table 4]

Effect on Lys-gingipain

[Table 5]

Concentration dependence on Arg-gingipain

[Table 6]

Concentration dependence on Arg-gingipain

[Table 7]

Concentration dependence on Arg-gingipain

[Table 8]

Lys-gingipain inhibition rate

[Table 9]

Concentration dependence on Lys-gingipain

[Table 10]

Concentration dependence on Lys-gingipain

[Table 11]

[0029]

From the results shown in Tables 4 to 11, it was found that among the food additives and plants, the plant extract has protease inhibitory activity. It was found that the aqueous plant extract solutions shown in Tables 6 to 11 had protease inhibitory activity in a concentration-dependent manner and were used as active ingredients of these inhibitors.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 9/10 A61K 9/10 9/68 9/68 35/78 35/78 C H J M R S T U 35/84 35/84 A A61P 1/02 A61P 1/02 43/00 111 43/00 111 (72) Inventor Haruhisa Hirata 1-3-5 Nihombashi Muromachi, Chuo-ku, Tokyo Wakamoto Pharmaceutical Co., Ltd. F-term (reference) 4B014 GB01 GB06 GB13 GG09 GG18 GK12 4B018 LB01 MD59 MD61 MD62 MD84 ME09 MF01 4C076 AA16 AA69 BB23 CC16 CC18 CC31 DD37 DD38 DD61 DD67 EE58 FF12 DD31 DD21 AD21 CC41 AC32 AC41 AC2 EE33 FF01 FF05 4C088 AA04 AB16 AB19 AB26 AB45 AB47 AB48 AB51 AB59 AB74 AB81 AC01 BA09 BA10 CA05 CA06 CA11 MA28 MA47 MA57 ZA67 ZC20 ZC41

Claims (3)

[Claims] 1. Yellow-green leaf, green tea, turmeric, mugwort, karin,
Sashimi, Gymnema, rooibos tea, hawthorn, rockfish, silymarin, 杸 杞, purple brown rice, eleutherocoque, moon peach leaf, dokudami, oju and reishi
A protease inhibitor characterized by inhibiting arginine ginger dipain and lysine gine dipain containing a plant selected from one or more species or a plant extract thereof as an active ingredient. 2. An oral composition comprising at least one protease inhibitor according to claim 1. 3. A composition according to claim 2, wherein the oral composition is in the form of chewing gum, candy, chocolate, chewable tablets or toothpaste.