JP4590525B2 - Anti-leishmania - Google Patents

Description

  The present invention relates to an antiprotozoal agent, particularly an anti-Leishmania agent, which contains a plant-derived substance exhibiting antiprotozoal activity or anti-Leishmania activity as an active ingredient.

  Conventionally, various protozoa such as Leishmania protozoa, malaria protozoa, vaginal Trichomonas, trypanosoma, and Pneumocystis carini are known to cause parasitic diseases.

  In particular, leishmaniasis is a parasitic disease peculiar to the tropics including South America caused by a leishmania protozoa (Leishmania protozoa), and is one of the six major tropical diseases designated by WHO. The total number of patients in Africa, the Middle East, Latin America and Asia is approximately 12 million, with 400,000 being infected each year. The infection route is a fly sucker that is a blood-sucking insect, and infection is established when a leishmania protozoa in the body of the fly fly invades from a wound during blood sucking. Protozoa parasitize macrophages and exhibit visceral, dermal and mucocutaneous pathologies. In particular, the visceral type is a dangerous pathological condition that can lead to death in severe cases.

  There are four Leishmania protozoa, Leishmania donovani, L. tropica, L. mexicana, and L. braziliensis, and the pathology that occurs depends on each. It is caused by parasitizing the macrophages. Visceral leishmania is caused by L. donovani parasitizing macrophages such as liver, spleen and bone marrow, and reticuloendothelial cells. The main symptoms are liver and spleen enlargement, anemia, white blood cell loss, fever and lymphadenopathy. Skin leishmania can be divided into Old World and New World types. Old World Skin Leishmania is caused by L. tropica, and New World Skin Leishmania is caused by L. mexicana, both of which parasitize skin macrophages and form skin ulcers. L. braziliensis causes mucocutaneous leishmania that causes lesions in the mucosa and skin, but can also cause cutaneous leishmania.

  Thus, Leishmania protozoa are rich in diversity, and there are differences in antigenicity from region to region even in similar pathological conditions from an immunological standpoint. This makes vaccine development difficult and the need for chemotherapy is high.

  Currently, pentavalent antimony drugs (trade names: Pentostam, Glucantime) are used as first-line drugs for the treatment of leishmaniasis, and if they are not effective, pentamidine, amphotericin B (Amphotericin B) or the like is used. However, these drugs may have strong side effects and require physician attention when used. Another problem is that the antimony agent is expensive.

As other therapeutic agents, Patent Document 1 discloses a germanacranolide-type or guaianolide-type sesquiterpenoid compound and a drug containing the same, and Patent Document 2 discloses a triazole derivative effective for the treatment of leishmaniasis. Yes.
However, even now, it is desired to develop a therapeutic agent that is less expensive and has fewer side effects.

  Several types of triterpenoids have been extracted from the heartwood of Cedrela plant (Non-patent Document 1). Two types of limonoids have been isolated from C. toona seeds (Non-patent Document 2), but it is not known that these limonoids have antiprotozoal activity. Further, as other components of the plant of Cedrela species, casinane-type sesquiterpenes and coumarin derivatives have been reported (Non-patent Documents 1 and 3), but their antiprotozoan activity is not known.

  Several benzoquinone and hydrodoquinone derivatives have been isolated from several Cordia species plants. These components have a p-benzoquinone or p-hydroquinone moiety (Non-Patent Documents 4 to 7). There have been many reports on the isolation of components from plants of the Cordia species, but there have been no reports of isolation of chemicals from C. fragrantissima, and the presence of compounds with antiprotozoal activity in the plants has also been reported. Not.

  Plants of the genus Dalbergia usually contain neoflavonoids. This plant heartwood has been reported to contain 4-arylcoumarin derivatives, p-hydroquinone derivatives and diphenylpropene derivatives (Non-patent Document 8). However, there are many unclear points regarding these pharmacological activities.

  Calanolide A isolated from a plant of the Calophyllum species is known as a powerful anti-HIV coumarin and is a promising candidate drug as an anti-HIV agent (Non-patent Document 9). However, it is not known that components derived from C. Kunstleri have antiprotozoal activity.

JP 2001-226369 A JP 2003-146877 A Campos, A.M., Oliveira, F.S., Machado, M.I.L., Braz-Filho, R., Matosa, F.J.A., 1991, Phytochemistry 30, 1225-1229. Chatterjee, A., Chakrabortty, T., Chandrasekharan, S., 1971, Phytochemistry 10, 2533-2535. Nagasampagi, B.A., Sriraman, M.C., Yankov, L., Dev, S., 1975, Phytochemistry 14, 1673. Ioset, J.-R., Marston, A., Gupta, M.P., Hostettmann, K., 2000, Phytochemistry 53, 613-617. Ioset, J.-R., Marston, A., Gupta, M.P., Hostettmann, K., 1998, Phytochemistry 47, 729-734. Bieber, L.W., Messana, I., Lins, S.C.N., Filho, A. A.S., Chiappeta, A.A., Mello, J.F.D., 1990, Phytochemistry 29, 1955-1959. Stevens, K.L., Jurd, L. Manners, G., 1973, Tetrahedron Letters 14, 2955-2958. Donnelly, D.M.X., O'Reilly, J., Thompson, J., 1972, Phytochemistry 11, 823-826. Xu, Z.-Q., Hollingshead, M.G., Borgel, S., Elder, C., Khilevich, A., Flavin, M.T., 1999, Bioorganic & Medicinal Chemistry Letters 9, 133-138.

An object of the present invention is to provide an antiprotozoal agent that is inexpensive and has few side effects.
An object of the present invention is to provide an anti-Leishmania agent that is inexpensive and has few side effects.

  As a result of screening for the antiprotozoan activity of extracts of plants inhabiting Myanmar, the present inventors have found that a substance derived from a specific plant exhibits antiprotozoal activity, particularly anti-Leishmania activity, and the present invention. It came to complete.

The present invention for solving the above problems includes the following inventions.
(1) Dipterocarpus tuberculatus Roxb .; Grevillea robusta A-Cun .; Xylocarpus moluccensis (Lam.) Roem .; Tectona grandis Linn.f .; Lagerstroemia tomentosa Presl .; Terminalia chebula Retz .; Haplophragma adenophyllum (X) Dolabriformis Benth .; Cananga odorata Hook.f. &. T.; Michelia champaca Linn.; Cordia fragrantissima Kurz.; Acacia catechu Willd.; Albizzia procera Benth.; Dalbergia oliveri Gamble.; Terminalia tomentosa W &A.; Cedrelale. Calophyllum kunstleri King .; Cedrela toona Roxb .; Albizzia odoratissima Benth .; Dalbergia cultrata Grah .; Chukrasia tabularis A. Juss .; Anogeissus acuminata Wall .; Lagerstroemia villosa Wall. An antiprotozoal agent containing a substance derived from a plant of a species and exhibiting antiprotozoal activity as an active ingredient.
(2) The antiprotozoal agent according to (1), which is an anti-Leishmania agent.

(3) Dipterocarpus tuberculatus Roxb .; Grevillea robusta A-Cun .; Xylocarpus moluccensis (Lam.) Roem .; Tectona grandis Linn.f .; Lagerstroemia tomentosa Presl .; Terminalia chebula Retz .; Haplophragma adenophyllum (X) Dolabriformis Benth .; Cananga odorata Hook.f. &. T.; Michelia champaca Linn.; Cordia fragrantissima Kurz.; Acacia catechu Willd.; Albizzia procera Benth.; Dalbergia oliveri Gamble.; Terminalia tomentosa W &A.; Cedrelale. Calophyllum kunstleri King .; Cedrela toona Roxb .; Albizzia odoratissima Benth .; Dalbergia cultrata Grah .; Chukrasia tabularis A. Juss .; Anogeissus acuminata Wall .; Lagerstroemia villosa Wall. An antiprotozoal agent containing an extract of a seed plant or a processed product thereof as an active ingredient.
(4) The antiprotozoal agent according to (3), which contains an extract of the plant heartwood or a processed product thereof as an active ingredient.
(5) The antiprotozoal agent according to (3) or (4), which is an anti-Leishmania agent.

(6) Dipterocarpus tuberculatus Roxb .; Grevillea robusta A-Cun .; Xylocarpus moluccensis (Lam.) Roem .; Tectona grandis Linn.f .; Lagerstroemia tomentosa Presl .; Terminalia chebula Retz .; Haplophragma adenophyllum (X) Dolabriformis Benth .; Cananga odorata Hook.f. &. T.; Michelia champaca Linn.; Cordia fragrantissima Kurz.; Acacia catechu Willd.; Albizzia procera Benth.; Dalbergia oliveri Gamble.; Terminalia tomentosa W &A.; Cedrelale. Calophyllum kunstleri King .; Cedrela toona Roxb .; Albizzia odoratissima Benth .; Dalbergia cultrata Grah .; Chukrasia tabularis A. Juss .; Anogeissus acuminata Wall .; Lagerstroemia villosa Wall. A method for producing an antiprotozoal agent, comprising a step of pulverizing a seed plant, a step of extracting the obtained pulverized product, and a step of performing a purification treatment as necessary.

  The antiprotozoal agent of the present invention is effective in the treatment of parasitic diseases caused by various protozoa such as Leishmania protozoa, malaria protozoa, vaginal Trichomonas, trypanosoma, and Pneumocystis carini.

The anti-leishmania agent of the present invention is effective for the treatment of leishmaniasis.
The antiprotozoal agent of the present invention is inexpensive because it is inexpensive and easy to manufacture.

  The antiprotozoal agent of the present invention contains a substance derived from a natural component as an active ingredient and has few problems of side effects.

  The antiprotozoal agent of the present invention includes Dipterocarpus tuberculatus Roxb .; Grevillea robusta A-Cun .; Xylocarpus moluccensis (Lam.) Roem .; Tectona grandis Linn.f .; Lagerstroemia tomentosa Presl .; Terminalia chebula Retz. .) Dep; Xylia dolabriformis Benth .; Cananga odorata Hook.f. &. T .; Michelia champaca Linn .; Cordia fragrantissima Kurz .; Acacia catechu Willd .; Albizzia procera Benth .; Dalbergia oliveri Gamble .; Terminalia tomentosa W & A. Cedrela serrata Royle .; Calophyllum kunstleri King .; Cedrela toona Roxb .; Albizzia odoratissima Benth .; Dalbergia cultrata Grah .; Chukrasia tabularis A. Juss .; Anogeissus acuminata Wall .; A substance derived from at least one selected plant and having antiprotozoal activity, particularly anti-Leishmania activity, is contained as an active ingredient.

  Preferably, a substance derived from at least one plant selected from the group consisting of Cordia fragrantissima Kurz and Cedrela serrata Royle. Contains a substance exhibiting antiprotozoal activity, particularly anti-Leishmania activity, as an active ingredient.

  There are no particular restrictions on the production area or variety of the plant used in the present invention. The fruit may be squeezed, pulverized, pulverized, etc., but the extract or processed product thereof, in particular, the heartwood, bark and leaf extract or processed product thereof should be used. Is preferred.

  A known method can be used as a method for extracting an active ingredient from the raw material, and examples thereof include a solvent extraction method and a supercritical extraction method using carbon dioxide gas. A combination of supercritical extraction and solvent extraction may be used.

  When used as an extract, extraction solvents used include water, acetone, dioxane, hexane, methyl ethyl ketone, acetonitrile, ethyl acetate, butyl methyl ketone, diethyl ether, dichloromethane, xylene, trichloroethylene, carbon tetrachloride, benzene, chloroform, toluene and Examples of the alcohol include lower alcohols such as methanol, ethanol, propyl alcohol, isopropyl alcohol, butanol and isobutanol, and polyhydric alcohols such as propylene glycol and 1,3-butylene glycol. Preferably, alcohol, more preferably lower alcohol, particularly preferably methanol is used. The above solvents may be used alone, or two or more kinds may be mixed and used, or water and an organic solvent may be used in combination. Usually, 3 to 10 L of extraction solvent is used per 1 kg of plant.

  The extraction temperature is not particularly limited, and may be within the range of the melting point of the solvent to the boiling point of the solvent, and supercritical extraction may be performed. The supercritical extraction is preferably performed by using carbon dioxide gas as a main solvent and adding an alcohol such as methanol or ethanol as an entrainer. The extraction is usually performed under normal pressure, but may be performed under pressure or under reduced pressure. The extraction time varies depending on the extraction temperature, the amount used, etc., but is usually 5 minutes to 1 day.

  The extract obtained as described above is filtered with a cloth, a stainless steel filter, a filter paper or the like to remove impurities and the like, whereby the target extract can be obtained. Moreover, you may give processes, such as a spray-dry process, a freeze-dry process, a supercritical process, to the extract after filtration. The extract thus obtained can be used as it is as an active ingredient of the antiprotozoal agent of the present invention.

  Furthermore, the extract can be further processed by various purification means such as ion exchange chromatography, gel filtration chromatography, dialysis, etc., and a processed product with further enhanced activity can be used. In the present invention, it is preferable to use a processed product subjected to purification treatment.

  The antiprotozoal agent of the present invention is effective for the treatment of parasitic diseases caused by protozoa such as Leishmania protozoa, malaria protozoa, vaginal Trichomonas, trypanosoma, and Pneumocystis carini. In particular, the antiprotozoal agent of the present invention can be preferably used as an anti-Leishmania agent in one embodiment of the present invention because it is effective for the treatment of leishmaniasis caused by Leishmania protozoa.

  Leishmaniasis can be classified into four types: visceral leishmaniasis (Kala Azar), old world cutaneous leishmaniasis, new world cutaneous leishmaniasis, mucocutaneous leishmaniasis, and the anti-leishmania agent of the present invention is It is particularly effective in the treatment of Old World Skin Leishmaniasis and New World Skin Leishmaniasis.

  Examples of Leishmania protozoa causing Leishmaniasis include Leishmania aethiopica, Leishmania braziliensis, Leishmania donovani, Leishmania furunculosa, Leishmania major, Leishmania mexicana, Leishmania peruviana, Leishmania pifanoi, Leishmania tropica, etc. Anti-Leishmania drugs are particularly effective in the treatment of leishmaniasis caused by Leishmania major.

  Further, the antiprotozoal agent of the present invention may be derived from a single type of plant or may be derived from a plurality of types of plants. The antiprotozoal agent of the present invention can be formulated by combining a substance derived from the above plant and having antiprotozoal activity, or an extract of the above plant or a processed product thereof with a known pharmaceutical carrier.

  The dosage form is not particularly limited and is appropriately selected as necessary. Generally, tablets, capsules, granules, fine granules, powders, solutions, syrups, suspensions, emulsions, elixirs, extracts It is used as an oral preparation such as an injection, or a parenteral preparation such as an injection, infusion, suppository, inhalation, transdermal absorption agent, transmucosal absorption agent, patch, ointment and the like. Among these, an extract is an agent obtained by concentrating a crude drug leachate, and there are two types, a soft extract and a dry extract. An extract is usually produced by pulverizing a plant containing an active ingredient, and then adding a leaching agent and cooling or digestion for a certain time, or leaching by a percolation method, and concentrating the leaching solution.

  The dose of the antiprotozoal agent or anti-leishmania agent of the present invention varies depending on the age, body weight, degree of disease and route of administration of the patient. For example, in the case of oral administration, the extract of the present invention or an extract of the processed product thereof The dry powder is usually 10 to 3000 mg per day, and the frequency of administration is usually 1 to 3 times per day for oral administration.

  Oral preparations are produced according to a conventional method using excipients such as starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, and inorganic salts.

  In this type of preparation, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a corrigent, a colorant, a fragrance and the like can be appropriately used in addition to the above-mentioned excipients.

  Specific examples of the binder include crystalline cellulose, crystalline cellulose / carmellose sodium, methylcellulose, hydroxypropylcellulose, low-substituted hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carmellose sodium , Ethylcellulose, carboxymethylethylcellulose, hydroxyethylcellulose, wheat starch, rice starch, corn starch, potato starch, dextrin, pregelatinized starch, partially pregelatinized starch, hydroxypro W pill starch, pullulan, polyvinylpyrrolidone, aminoalkyl methacrylate copolymer E, amino Alkyl meta Relay copolymers RS, methacrylic acid copolymer L, methacrylic acid copolymer, polyvinyl acetal diethylamino acetate, polyvinyl alcohol, gum arabic, gum arabic powder, agar, gelatin, white shellac, tragacanth, purified sucrose, macrogol.

  Specific examples of the disintegrant include crystalline cellulose, methylcellulose, low-substituted hydroxypropylcellulose, carmellose, carmellose calcium, carmellose sodium, croscarmellose sodium, wheat starch, rice starch, corn starch, potato starch, and partially pregelatinized. Starch, hydroxypropyl starch, sodium carboxymethyl starch, tragacanth can be mentioned.

  Specific examples of surfactants include soybean lecithin, sucrose fatty acid ester, polyoxyl stearate, polyoxyethylene hydrogenated castor oil, polyoxyethylene polyoxypropylene glycol, sorbitan sesquioleate, sorbitan trioleate, sorbitan monostearate Sorbitan monopalmitate, sorbitan monolaurate, polysorbate, glyceryl monostearate, sodium lauryl sulfate, lauromacrogol.

  Specific examples of lubricants include wheat starch, rice starch, corn starch, stearic acid, calcium stearate, magnesium stearate, hydrous silicon dioxide, light anhydrous silicic acid, synthetic aluminum silicate, dry aluminum hydroxide gel, talc, Examples thereof include magnesium aluminate metasilicate, calcium hydrogen phosphate, anhydrous calcium hydrogen phosphate, sucrose fatty acid ester, waxes, hydrogenated vegetable oil, and polyethylene glycol.

  Specific examples of the fluidity promoter include hydrous silicon dioxide, light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, and magnesium silicate.

  In addition, the antiprotozoal agent or anti-leishmania agent of the present invention may contain a flavoring agent and a coloring agent when administered as a solution, syrup, suspension, emulsion, or elixir.

  The antiprotozoal agent or anti-Lyusmania agent of the present invention can be added to foods, chewing gums, beverages and the like to make so-called foods for specific health (for example, anti-Lyusmania foods).

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to these examples.

Leishmania promastigote culture
Leishmania major promastigotes (MHOM / SU / 73 / 5ASKH) were cultured using Medium 199 medium (Life Technologies Co. Ltd.). Promastigotes were cultured at 27 ° C. under 5% CO ₂ in a medium supplemented with heat inactivated (30 minutes at 56 ° C.) fetal calf serum (10%).

Preparation of extract and measurement of anti-Leishmania activity 30-50 g of 75 species of Myanmar plants were extracted 3 times at 50 ° C for 4 hours with 100 ml of methanol, combined with the extract and concentrated under reduced pressure. And a methanol extract was obtained.

  The anti-Leishmania activity of the methanol extract was measured using WST-8 (2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H- Tetrazolium monosodium salt) and 1-methoxy PMS (1-methoxy-5-methylphenazinium methyl sulfate) mixture (Tetracolor One reagent) was used to assay as follows.

40 mg of the extract was dissolved in 1 ml of DMSO, diluted with Medium 199 medium (Life Technologies Co. Ltd.), and passed through a membrane filter. Prepare sample solutions at 9 concentrations, inoculate a microtiter plate with 50 μl of each concentration of sample solution and 50 μl of Leishmania culture solution prepared to a final concentration of 2 × 10 ⁵ / ml. The total volume was 100 μl.

After incubation for 48 hours at 27 ° C. and 5% CO ₂ , Tetracolor One reagent (Seikagaku Corporation) was added, and after incubation for 6 hours at 27 ° C., the OD value (450 ~ 630nm) was measured. The test was carried out for 3 wells for each sample and each concentration, and MLC (minimum lethal concentration) and MIC (minimum inhibitory concentration) were determined from the average values. The results are shown in Table 1.

  From the results in Table 1, Dipterocarpus tuberculatus Roxb .; Grevillea robusta A-Cun .; Xylocarpus moluccensis (Lam.) Roem .; Tectona grandis Linn.f .; Lagerstroemia tomentosa Presl .; Terminalia chebula Retz .; Haplophragma adenophyllum Dep; Xylia dolabriformis Benth .; Cananga odorata Hook.f. &. T .; Michelia champaca Linn .; Cordia fragrantissima Kurz .; Acacia catechu Willd .; Albizzia procera Benth .; Dalbergia oliveri Gamble .; Terminalia tomentosa W & la. Calophyllum kunstleri King .; Cedrela toona Roxb .; Albizzia odoratissima Benth .; Dalbergia cultrata Grah .; Chukrasia tabularis A. Juss .; Anogeissus acuminata Wall .; Lagerstroemia villosa Wall. Either MLC value or MIC value was less than 400, and it was revealed that extracts of these plants and components derived from these plants have anti-Leishmania activity.

Claims (5)

An antiprotozoal agent comprising an extract of C ordia fragrantissima Kurz. Or a processed product thereof as an active ingredient. Cordia fragrantissima Kurz extract. Heartwood or antiprotozoal according to claim 1, further comprising a processed product thereof as an active ingredient. The antiprotozoal agent according to claim 1 or 2 , which is an anti-Leishmania agent. C ordia fragrantissima Kurz. Comprising the step of extracting process, and the obtained pulverized material grinding method as antiprotozoal agents. Furthermore, the manufacturing method of the antiprotozoal agent of Claim 4 including the process of performing a refinement | purification process.