ENDOTHELIN-CONVERTING ENZYME INHIBITOR

Abstract

Provision of a material, which has activity of inhibiting endothelin-converting enzyme and is effective for skin whitening or antihypertension. An endothelin-converting enzyme inhibitor comprising, as an active ingredient, at least one selected from the group consisting of Hiptage candicans, Piper sarmentosum, Solanum stramonifolium, Anaxagorea luzonensis, Smilax corbularia, Ardisia elliptica, Erythrina suberosa, Dactyloctenium aegyptium, Hopea ferrea and extracts thereof.

Claims

1-16. (canceled)

17. A method of inhibiting endothelin-converting enzyme in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of at least one selected from the group consisting of Hiptage candicans, Piper sarmentosum, Solanum stramonifolium, Anaxagorea luzonensis, Smilax corbularia, Ardisia elliptica, Erythrina suberosa, Dactyloctenium aegyptium, Hopea ferrea and extracts thereof.

18. A method of inhibiting melanogenesis in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of at least one selected from the group consisting of Hiptage candicans, Piper sarmentosum, Solanum stramonifolium, Anaxagorea luzonensis, Smilax corbularia, Ardisia elliptica, Erythrina suberosa, Dactyloctenium aegyptium, Hopea ferrea and extracts thereof.

19. A method of whitening skin in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of at least one selected from the group consisting of Hiptage candicans, Piper sarmentosum, Solanum stramonifolium, Anaxagorea luzonensis, Smilax corbularia, Ardisia elliptica, Erythrina suberosa, Dactyloctenium aegyptium, Hopea ferrea and extracts thereof.

20. A method of preventing or ameliorating hypertension in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of at least one selected from the group consisting of Hiptage candicans, Solanum stramonifolium, Anaxagorea luzonensis, Smilax corbularia, Ardisia elliptica, Erythrina suberosa, Dactyloctenium aegyptium, Hopea ferrea and extracts thereof.

21-36. (canceled)

Description

EXAMPLES

[0133] (Preparation of Plant Extracts)

[0134] Dried and crushed plant materials were each extracted with any of the following four types of solvent: (A) water, (B) 50% (v/v) ethanol aqueous solution, (C) 95% (v/v) ethanol aqueous solution, and (D) hexane. Approximately 50 g of each plant material was immersed in 500 mL of water at 60 C. for 4 hours, or in 500 mL of 50% (v/v) ethanol aqueous solution, 95% (v/v) ethanol aqueous solution, ethanol or hexane at 30 C. for 7 days. Thereafter, the resultants were filtrated to obtain crude extract solutions, and the crude extract solutions were then concentrated to dryness to obtain various extract solids. The extract solids were each dissolved in various types of solvents (A: 10% EtOH, B: 50% EtOH, C: 95% EtOH, and D: 99.5% EtOH), so that the solid concentrations became 1% (w/v), 0.5% (w/v), and 0.25% (w/v). The plant extract solutions were each preserved at 20 C., until they were used in the after-mentioned ECE inhibitory assay. The yields of the extract solids are shown in Table 1.

TABLE-US-00001 TABLE 1 Yield of extract (dry weight, g) 50% 95% Water EtOH EtOH Hexane Plant species Extract Extract Extract Extract Hiptage candicans Wood 1.04 5.03 4.39 Piper sarmentosum Whole 5.02 3.80 0.40 plant Solanum Root 0.08 stramonifolium Anaxagorea Wood or 3.80 7.46 8.16 luzonensis bark Smilax corbularia Rhizome 10.36 9.52 0.08 Ardisia elliptica Root or 1.96 2.00 0.55 stem Erythrina suberosa Leaves 5.08 2.50 1.22 Dactyloctenium Whole 5.02 0.48 aegyptium plant Hopea ferrea Bark 4.30 3.79 0.19

[0135] (Crude Endothelin-Converting Enzyme Protein)

[0136] In accordance with the method described in Non-Patent Document 4, a crude ECE-1 protein was newly prepared by extracting it from a fresh human vascular endothelial cell line EA.hy926. The crude ECE-1 protein was preserved at 18 C., until it was used.

Example 1

ECE Inhibitory Assay

[0137] An enzyme reaction was carried out by the procedures described in Non-Patent Document 4. Briefly speaking, an each plant extract (final concentration: 0.01, 0.005, or 0.0025% (w/v)) and a crude ECE-1 protein were pre-incubated in a BigET-1 buffer (0.1 M sodium phosphate buffer (pH 6.8), 0.5 M NaCl) at 37 C. for 15 minutes. Thereafter, a 0.2 M BigET-1 substrate (Enzo Life Sciences, Cat. # ALX-152-001-PC05) in a BigET-1 buffer (final concentration: 0.1 M) was added to the reaction solution, and the obtained mixture was then incubated at 37 Cr for 2 hours. After 5 mM EDTA had been added to the reaction solution to terminate the enzyme reaction, the mixed solution was transferred to ET-1 ELISA kit (Enzo Life Sciences, Cat. # ADI-900-020A), and ET-1 was then quantified according to the method described in the production manual. Briefly speaking, ET-1 in the mixed solution was captured by an anti-human ET-1 antibody immobilized on a 96-well microplate and was then labeled with a HRP-labeled anti-human ET-1 antibody. The absorbance of the labeled ET-1 at 450 nm was measured using a plate reader. The amount of ET-1 in the mixed solution was quantified based on a comparison made with the absorbance of a standard, and then, relative ECE-1 inhibitory activity (% relative to a control) was calculated according to the following formula.

Relative ECE-1 inhibitory activity (%)=(1X/Y)*100 [0138] (X; the amount of ET-1 in a liquid containing a plant extract, Y; the amount of ET-1 in a liquid containing a control) [0139] Phosphoramidon (PD) was used as a positive control of the ECE-1 inhibitor. 10% (v/v) ethanol aqueous solution, 50% (v/v) ethanol aqueous solution, 95% (v/v) ethanol aqueous solution or 100% ethanol was used as a control.

[0140] The results obtained by quantifying ECE-1 inhibitory activity are shown in Table 2. All of the extracts from Hiptage candicans, Piper sarmentosum, Solanum stramonifolium, Anaxagorea luzonensis, Smilax corbularia, Ardisia elliptica, Erythrina suberosa, Dactyloctenium aegyptium and Hopea ferrea had ECE-1 inhibitory activity. The activities of these plant extracts were higher than the activity of burnet (Sanguisorba officinalis L.) extract (Reference Example 1) which is a known ECE-1 inhibitor described in Non-Patent Document 4. As described in Non-Patent Document 4, the burnet extract as an ECE-1 inhibitor suppresses skin pigmentation. In addition, Non-Patent Document 4 describes that a plant extract which inhibits ET-1 suppresses the growth of melanocytes and prevents human skin pigmentation after UVB irradiation. Therefore, Hiptage candicans, Piper sarmentosum, Solanum stramonifolium, Anaxagorea luzonensis, Smilax corbularia, Ardisia elliptica, Erythrina suberosa, Dactyloctenium aegyptium, Hopea ferrea and extracts thereof are also suggested to have activity of inhibiting melanogenesis on skin and preventing pigmentation. Moreover, the above described plants and extracts thereof suppress vasoconstriction based on the ECE inhibitory activity and are effective as antihypertensive agents.

TABLE-US-00002 TABLE 2 Relative ECE-1 inhibitory activity (%) extraction 0.01% (w/v) 0.005% (w/v) 0.0025% (w/v) Plant species solvent Extract Extract Extract Hiptage candicans Water 79.4 50% EtOH 91.3 90.8 88.7 95% EtOH 90.3 90.8 88.9 Piper sarmentosum 50% EtOH 87.0 72.2 67.0 95% EtOH 91.7 89.1 85.3 Hexane 88.2 87.5 82.0 Solanum stramonifolium Hexane 87.6 86.5 80.8 Anaxagorea luzonensis Water 85.7 50% EtOH 90.1 91.7 89.6 95% EtOH 90.5 90.9 85.9 Smilax corbularia 50% EtOH 87.2 95% EtOH 88.2 Hexane 88.0 87.5 86.1 Ardisia elliptica 50% EtOH 89.7 95% EtOH 93.3 90.9 80.5 Hexane 92.4 89.1 89.4 Erythrina suberosa 50% EtOH 80.3 95% EtOH 90.1 84.7 82.0 Hexane 85.7 Dactyloctenium aegyptium 95% EtOH 88.5 82.6 70.4 Hexane 88.2 88.1 87.7 Hopea ferrea 50% EtOH 71.89 33.86 17.03 95% EtOH 84.87 82.40 77.05 Hexane 71.92 52.02 14.05 Reference Example 1 80.3 Extract of Sanguisorba officinalis L. (purchased from Maruzen Pharmaceutical Co. Ltd.)