JPH09235293A - New triterpene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new triterpene through extraction from mushroom basidiocarp, which has collagenase inhibitory action, thus is useful for treating diseases attributable to collagenase activity sthenia such as arthrosis, bone resorption disorder, periodontosis, corneal ulcer and bullous epidermolysis. SOLUTION: This new triterpene is expressed by the formula, having a molecular formula: C30 H40 O5 and a molecular weight of 484, having inhibitory activity against collagenase as an enzyme involving connective tissue matrix metabolism, therefore being useful for e.g. treating various diseases attributable to collagenase activity sthenia such as arthrosis, bone resorption disorder, periodontosis, corneal ulcer and bullous epidermolysis. This compound is obtained by the following process: the basidiocarp of a kind of mushroom such as Trametes mushroom is lyophilized and then subjected to extraction with 85% ethanol, and the resultant extract liquid is concentrated and then further subjected to extraction with ethyl acetate, and the resultant extract is put to a silica get column and eluted with chloroform-acetone (8:2), and the resultant elute is subjected to high-performance liquid chromatography and purified.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel triterpene having a collagenase inhibitory action and useful as a treatment for various diseases such as joint diseases, bone resorption diseases, periodontal diseases, corneal ulcers and epidermolysis bullosa.

[0002]

BACKGROUND OF THE INVENTION Collagenase is an enzyme involved in metabolism of connective tissue matrix, and enhancement of this enzyme activity is a main cause of various diseases such as joint diseases, bone resorption diseases, periodontal diseases, corneal ulcers, epidermolysis bullosa. It is believed that.

For example, in joint diseases such as rheumatoid arthritis and osteoarthritis, enhanced collagenase activity was reported by EM Gravallese et al. [Arthritis Rheum,
34 (9), 1076 (1991)].

Bone resorption disease is a disease due to the fact that the bone remodeling process consisting of bone formation and bone resorption is biased to bone resorption, and hypercalcemia, osteoporosis and the like are known. Regarding the enhancement of bone resorption in these diseases, the involvement of cysteine proteases such as cathepsin L has been conventionally noted, but in recent years, a report of V. Everts et al. [J. Cell. Physiol. 150, 221] that collagenase is also involved. (1992)].

Therefore, a compound having an inhibitory activity against collagenase may be a therapeutic agent for a disease in which the collagenase activity is enhanced, and therefore various compounds having a collagenase inhibitory activity have been studied.

[0006] For example, there are Japanese Patent Publication No. 1-146896, US Pat. No. 7,774,491, etc., which are peptide-type inhibitors having hydroxamic acid as a ligand, and non-peptidic ones are gallic acid derivatives (Japanese Patent Laid-Open No. Hei. -290
819), tetracyclines [Lorne M. Golub,
NSRamamurthy, Thomas F. McNamara, Critical Reviews
in Oral Biology and Medicine, 2 (2): 297-322 (1991))
Etc. are known.

However, the activity of these compounds is still insufficient, and there is a demand for new non-peptide lead compounds which are more active and have higher bioavailability (stability in vivo).

[0008]

In view of the above circumstances, the inventors of the present invention have made a diligent search for compounds having collagenase inhibitory activity from natural products, and as a result, have shown that a novel triterpene compound separated and purified from spinach mushroom inhibits human collagenase activity. The present invention has been completed and the present invention has been completed, and the purpose thereof is to have a collagenase inhibitory action and to treat various diseases such as joint diseases, bone resorption diseases, periodontal diseases, corneal ulcers, epidermolysis bullosa. The present invention provides a novel non-peptidic triterpene useful as

[0009]

The above-mentioned object is to provide a novel triterpene 20-carboxy-16-hydrox represented by the following formula (I).
Achieved by y-21-nor-5 α-7,9 (11) -lanostadien-3,24-dione.

[0010]

Embedded image

[0011]

DETAILED DESCRIPTION OF THE INVENTION The constitution of the present invention will be described in detail below.

The novel triterpene (I) used in the present invention can be prepared by synthesis, but it can also be prepared from natural products, specifically, mushroom fruit bodies and the like.

Preparation from mushroom fruiting bodies and the like can be carried out by appropriately combining ordinary chemical operations such as extraction, filtration and various chromatographies.

Examples of mushrooms used for extraction include
Daedalea dickinsi(Porcelain),Fomitopsis pi
nicola(Tsugasarunokeshi),Fomitopsis rosea
(Balairo Sarnos moss),Fomitopsis nigra(Ku
Losarno moss),Polyporu s betulinusEqual octopus
Kind mushrooms,Ganoderma valesiacum(Tsuganoman
Mushrooms such as Ganoderma lucidum
You.

In the freeze-dried product of fruiting bodies of these mushrooms,
5 to 30 times by weight of extraction solvent is added, usually 15 to 50
The extract of each mushroom is obtained by infiltration at 24 ° C. for 24 hours to 1 week. Further, the extract is filtered through filter paper or 100
An insoluble matter can be removed by centrifugation at 0 to 3000 × g, and then a concentrated extract can be obtained by an ordinary concentration means, for example, concentration under reduced pressure.

Further, a dried powder can be obtained by a usual drying means such as vacuum drying, spray drying or freeze drying.

By extracting the concentrated extract or dried powder with an organic solvent and purifying by a separating means such as a column,
The novel triterpenes (I) of the present invention can be isolated.

Examples of the organic solvent for extracting the novel triterpene of the present invention from a concentrated extract or dried powder include water, water-soluble organic solvents such as ethanol, ethyl acetate,
Examples include organic solvents such as chloroform and acetone, and mixed solvents thereof.

Further, examples of the column used for purifying the organic solvent extract of mushrooms include a silica gel column and a high performance liquid chromatography column in a reverse phase mode.

The form of the preparation containing the novel triterpene (I) as an active ingredient is a tablet, a powder or a fine powder prepared by using a commonly used carrier, excipient or other additive for the preparation. Granules, granules, capsules, pills,
Liquid preparations, injections, suppositories, ointments, patches and the like can be mentioned.

The content of the novel triterpene of the present invention in the preparation is 0.001 to 1 based on the total amount of the preparation composition.
0.0% by weight is preferable, and 0.005 to 1.0% by weight is particularly preferable.

[0022]

INDUSTRIAL APPLICABILITY The novel triterpene (I) of the present invention has a collagenase inhibitory action, and is thought to be caused by the increased activity of collagenase, such as joint diseases, bone resorption diseases, periodontal diseases,
It is useful for treatment of various diseases such as corneal ulcer and epidermal blisters 23.

[0023]

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

Example 1 Fruit bodies of spinach mushrooms were freeze-dried to obtain 7.36k thereof.
g was extracted with 15 l of 85% ethanol to give a spinach extract. The extract was concentrated, extracted with ethyl acetate, applied to a silica gel column (BW-127ZH), and eluted with chloroform: acetone (8: 2). Furthermore, the silica gel column (Klesselgel 6
0) and eluting with chloroform: acetone (82:18) were applied to an ODS column for HPLC under conditions of methanol: water (86:14) to elute 20.2 mg of the novel triterpene (I) (Example 1). Obtained.

(1) Molecular weight of novel triterpene (I) (Example 1) The molecular weight of novel triterpene (I) (Example 1) is shown by mass spectrum (JEOL-DX303HF manufactured by JEOL Ltd.).
Was measured by As a result, the molecular weight of the novel triterpene (I) (Example 1) was 484.

(2) ¹ H and ¹³ C-NMR of the novel triterpene (I) (Example 1) The chemical structure of the novel triterpene (I) (Example 1) is ¹ H.
And ¹³ C-NMR (JEOL-LAMB manufactured by JEOL Ltd.)
DA 500). As a result, the following spectrum values were obtained, and 20-carboxy-16-hydroxy-21-n
It was identified to be or-5 α-7,9 (11) -lanostadien-3,24-dione.

¹ H-NMR (CDCl ₃ ) δ: 0.60 (1 H, s), 1.07
(1H, s), 1.07 (1H, d, J = 6.9Hz), 1.08 (1H, s), 1.08 (1H, d, J =
7.1Hz), 1.10 (1H, s), 1.15 (1H, s), 1.48 (1H, br.d, J = 13.4H
z), 1.52 (1H, dd, J = 11.9,3.7Hz), 1.72 (1H, ddd, J = 14.0,13.
9,4.6Hz), 1.77 (1H, dd, J = 17.4,6.1Hz), 1.85 (1H, m), 2.05
(1H, ddd, J = 13.7,6.6,3.7Hz), 2.08 (1H, dd, J = 13.7,11.9H
z), 2.22 (3H, m), 2.28 (1H, m), 2.33 (1H, ddd, J = 14.7,4.4,3.
4Hz), 2.46 (1H, ddd, J = 11.1,10.8,3.5Hz), 2.55 (1H, m) 2.57
(1H, m), 2.74 (1H, ddd, J = 14.7,14.0,5.8Hz), 4.21 (1H, dd, J
= 6.4,7.9Hz), 5.33 (1H, d, J = 6.1Hz), 5.49 (1H, d, J = 6.6Hz)

¹³ C-NMR (CDCl ₃ ) δ: 17.3, 18.2, 18.3,
22.0,22.4,23.6,25.3,25.3,26.0,34.7,35.5,36.3,37.2,
37.3,41.0,43.3,44.6,45.4,47.5,48.7,50.6,56.3,76.5,
116.5,120.8,141.7,144.4,179.8,214.4,216.7

Test Example 1 1. Preparation of Human Collagenase As human collagenase, anchorage-independent cells derived from human fibrosarcoma cells were supplemented with human procollagenase produced in a serum-free protein-free medium by CM Sepharose ^™ (Pharmacia). company, Ltd.) and zinc chelating Sepharose ^TM
Purified by (Pharmacia) and dissolved in buffer,
Trypsin (Activated by Sigma, Type)
e12) was added and incubated at 35 ° C. for 5 minutes, and then soybean trypsin inhibitor (manufactured by Merck) was added to inactivate trypsin.

2. Measurement of Human Collagenase Inhibitory Activity The inhibitory activity of the novel triterpene (I) of Example 1 against human collagenase was measured as follows.

First, a novel triterpene (I) (Example 1)
10 mg was dissolved in 200 μl of dimethylsulfoxide, and this was used as a measurement buffer [0.2 M sodium chloride, 5 mM calcium chloride, 0.05 vol% Brij-35 (polyoxyethylene manufactured by ICI).
(23) lauryl ether), 50 mM Tris-HCl buffer containing 0.02% by volume sodium azide, pH 7.5] and diluted 2.5 times.

An equal amount of the diluted solution and a known amount (0.4 unit; 1 unit represents the amount of enzyme degrading 1 μg of type I collagen per minute at 35 ° C.) were mixed in equal amounts to prepare bovine I. Using type collagen (manufactured by Cosmo Bio Inc.) as a substrate, reaction was carried out at 35 ° C. for 16 hours. An equal amount of sample buffer [3.2% by volume sodium n-dodecyl sulfate was added to this solution.
(SDS), 8% by volume β-mercaptoethanol, 16% by volume BPB solution (0.05% by volume bromphenol blue, 70% by volume glycerin, 0.0625M Tris hydrochloric acid solution, pH 6.8) containing 0.1M Tris hydrochloric acid solution, pH 6 .8] was added and boiled for 3 minutes to prepare a sample.

This sample was prepared by the method of Laemmli (Natur
e, 227, 680, 1970), and after 1 hour staining with CBB staining solution (0.25 vol% Coomassie Brilliant Blue R-250, 45 vol% methanol, 10 vol% acetic acid) Decolorization was carried out with a decolorizing solution (25% by volume methanol, 8% by volume acetic acid).

After the electrophoretic gel was dried, the collagen band of the electrophoretic pattern and the intensity of the cleaved band were used as an index of inhibition of human collagenase activity.

3. Test Results The results are shown in FIG. The novel triterpene (I) (Example 1) inhibited human collagenase activity at 5 mg / ml.

[Brief description of drawings]

FIG. 1 shows that the novel triterpene (I) (Example 1) inhibits the activity of collagenase.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area A61K 35/84 ADA A61K 35/84 ADAA // A61K 7/16 A61K 7/16 C12N 9/99 C12N 9/99 C07M 7:00

Claims (1)

[Claims] 1. A novel triterpene represented by the following formula (I): Embedded image