JP2005170839A - Anti-leishmania agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an inexpensive antiprotozoal agent or anti-leishmania agent having low adverse effect. <P>SOLUTION: The antiprotozoal agent or anti-leishmania agent comprises a substance that is derived from Chyi Thee or Thinwin and exhibits antiprotozoal activity or anti-leishmania activity as an active ingredient. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an antiprotozoal agent or an anti-Leishmania agent that contains, as an active ingredient, a substance exhibiting antiprotozoal activity or anti-Leishmania activity, derived from Chii Thee or Thinwin.

  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 the wound at the time of 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.

  Chi Shie (Chyi Thee, Semecarpus anacardium L., English name Marking Nut Tree) is a plant of the Urushi family that is distributed in Myanmar. In Myanmar, Chi Si fruits are used for the treatment of leprosy, infections, psoriasis, gonorrhea, dyspepsia, etc., but there are many unclear parts regarding the relationship between their ingredients and activities.

  In addition, Millettia pendula (Chinwin) is a tree belonging to the genus Leguminosae, which is distributed in Thailand and Myanmar. The heartwood is dark purple or brown, but is known to fade when exposed to sunlight. Chinwin is mainly used for building materials, and there are few reports of using it for medicinal purposes. As components of Chinwin, equol, (-)-Makiain ((-)-maackiain), claussequinone, and pendulone, which are already reported by Hayashi et al. Patent Document 1). Of these, penjuron is a component obtained mainly from heartwood.

JP 2001-226369 A JP 2003-146877 A Journal of the Wood Society (Vol. 24, No. 12, pages 898-901, 1978, Hayashi et al.)

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 intensive studies, the present inventors have discovered that a substance derived from Chi Chi or Chin Win exhibits antiprotozoal activity or anti-Leishmania activity.

The present invention for solving the above problems includes the following inventions.
(1) An antiprotozoal agent containing, as an active ingredient, a substance derived from Chi-Chi or Chinwin and exhibiting antiprotozoal activity.
(2) An anti-Leishmania agent containing as an active ingredient a substance derived from Chi-Chi or Chinwin and exhibiting anti-Leishmania activity.
(3) An anti-Leishmania agent containing an extract of chi-shi or chinwin or a processed product thereof as an active ingredient.

(4) The anti-Leishmania agent according to (3), containing an extract of chi-shi or chinwin heartwood or a processed product thereof as an active ingredient.
(5) An anti-Leishmania agent containing urushiol.
(6) Urushiol is the following formula:

で表されるビラワノールである（５）記載の抗リーシュマニア剤。

The anti-Leishmania agent according to (5), wherein

で表されるペンジュロンを含有する抗リーシュマニア剤。 (7) The following formula:

An anti-Leishmania agent containing penjuron represented by

(8) The antiprotozoal agent or anti-Leishmania agent according to any one of (1) to (7), which is used for the treatment of leishmaniasis.
(9) A method for producing an anti-Leishmania agent, comprising a step of pulverizing chi-shi or chinwin, a step of extracting the crushed chi-shi or chinwin, and 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 or anti-Leishmania agent of the present invention is inexpensive because the material is inexpensive and easy to manufacture.

  The antiprotozoal agent or anti-Leishmania agent of the present invention contains a substance derived from natural ingredients such as Chi-Chi or Chinwin as an active ingredient, and has few problems of side effects.

(1) Chi-Shi The antiprotozoal agent or anti-Leishmania agent of the present invention is derived from Chi-Shi and contains a substance exhibiting antiprotozoal activity or anti-Leishmania activity as an active ingredient.

  There are no particular restrictions on the production area or variety of chi-shi used in the present invention. Chi-shi may be obtained by squeezing the fruit, pulverized, pulverized, or the like, but is preferably used as an extract or a processed product thereof.

  When used as an extract, hexane, dichloromethane, chloroform, ethanol, preferably methanol can be used as the extraction solvent. Usually, 3-10 L of extraction solvent is used per 1 kg of Chi-Chi.

  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 anti-leishmania 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 used as a processed product with enhanced activity.

  The authors further searched for the active ingredient of Chi-Chi. It has been found that the anti-Lyusmania activity of the hexane fraction is particularly high when the methanol-extracted chi-see is partitioned with hexane, chloroform, butanol and water. Therefore, when the active component was separated and purified by chromatography for the hexane fraction, three types of urushiol-related compounds were successfully isolated and identified. Of these, the main compound in the silica gel column fraction has the following formula:

で表されるビラワノール（Bhilawanol）であった。ビラワノールの抗リーシュマニア活性は、抗リーシュマニア剤として知られるアンフォテリシンＢの抗リーシュマニア活性（ＩＣ₅₀；０．２μｇ／ｍｌ）と比較しても、非常に強い。

Bhilawanol represented by The anti-Leishmania activity of bilawanol is very strong even when compared to the anti-Leishmania activity of amphotericin B (IC ₅₀ ; 0.2 μg / ml) known as an anti-Leishmania agent.

  The remaining two urushiol-related compounds were Anacardol and Cardanoldiene, but their anti-Lyusmania activity was weak.

  In addition, the present inventors have isolated and identified tetrahydroamentoflavone, which is a biflavonoid, as the main compound in the butanol fraction, but the anti-Lyushumania activity of this compound was also weak.

(2) Chinwin The antiprotozoal agent or anti-Leishmania agent of the present invention contains a substance that is derived from Chinwin and exhibits antiprotozoal activity or anti-Leishmania activity as an active ingredient.

There are no particular restrictions on the production area or variety of chinwin used in the present invention. Chinwin may be obtained by squeezing its fruit and seeds, pulverized, pulverized, or the like, but is preferably used as an extract or a processed product thereof.
The extract and treatment method are the same as those used in the above-mentioned CHC.

  The present inventors further searched for the active ingredient of Chinwin. Chinwin extracted with methanol was subjected to separation and purification of active ingredients by chromatography, and two types of compounds were successfully isolated and identified. Of these, the following formula:

で表されるペンジュロンは強い抗リューシュマニア活性を示した。ペンジュロンの抗リーシュマニア活性は、抗リーシュマニア剤として知られるアンフォテリシンＢの抗リーシュマニア活性（ＩＣ₅₀；０．２μｇ／ｍｌ）と比較しても、非常に強い。

Penjuron represented by the formula showed strong anti-Lyusmania activity. The anti-Leishmania activity of penjuron is very strong even compared to the anti-Leishmania activity (IC ₅₀ ; 0.2 μg / ml) of amphotericin B, known as an anti-Leishmania agent.

  As another compound, 2,3-dimethoxy-9-hydroxypterocarpan was isolated and identified, but its anti-Leishmania activity was weak.

(3) Formulation Further, the antiprotozoal agent or anti-leishmania agent of the present invention can be formulated alone or in combination with a known pharmaceutical carrier.

  The dosage form is not particularly limited and is appropriately selected as necessary. In general, 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 patient's age, body weight, degree of disease, and administration route.For example, in oral administration, Usually, it is 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.
Example 1 Preparation of Chi-Chi extract 2.6 kg of Chy Thee obtained in Myanmar was pulverized and then extracted twice with 5 L of methanol for 12 hours each. After filtering the extract with filter paper, the filtrate was concentrated under reduced pressure to obtain 240 g of a methanol extract. The minimum lethal concentration of this methanol extract was 6.3 μg / ml. The methanol extract was further partitioned with hexane, chloroform, butanol, and water to obtain each fraction. The hexane fraction had strong anti-Leishmania activity.

Example 2 Isolation and identification of bilawanol The hexane fraction obtained in Example 1 was purified by silica gel column chromatography (developing solvent: gradient elution with a hexane-ethyl acetate mixed solvent or gradient elution with a hexane-chloroform mixed solvent). Repeatedly, bilawanol, anacardol and cardanoldiene were isolated. In addition, the butanol fraction was subjected to silica gel column chromatography (developing solvent: gradient elution with chloroform-methanol mixed solvent), medium pressure liquid chromatography (reverse layer silica gel, developing solvent: gradient elution with methanol-water mixed solvent, silica gel, developing solvent). : Gradient elution with a chloroform-methanol mixed solvent) and tetrahydroamentene flavone as a dimer was isolated.
Bilawanol data:
¹³ C-NMR (δ (ppm) in CDCl ₃ ): 143.0 (s), 141.8 (s), 130.1 (d), 129.9 (d), 129.3 (s), 122.1 (d), 120.1 (d), 112.9 (d), 31.9 (t), 31.8 (t), 29.7 (t), 29.5 (t), 29.4 (t), 29.2 (t), 28.9 (t), 27.2 (t), 26.9 (t), 22.6 (t), 22.3 (t), 14.1 (q).

Example 3 Purification and isolation of penjuron 68 g of Chinwin obtained in Myanmar was pulverized and extracted twice with 1 L of methanol every 12 hours. After the extract was filtered through filter paper, the filtrate was concentrated under reduced pressure to obtain 4.8 g of a methanol extract. The minimum lethal concentration of this methanol extract was 3.1 μg / ml. The methanol extract thus obtained was subjected to silica gel column chromatography (first developing solvent: chloroform-methanol mixed solvent: gradient elution, second developing solvent: chloroform-ethyl acetate mixed solvent gradient elution), medium pressure preparative chromatography (silica gel, Developing solvent: hexane-chloroform = 1: 3 mixed solvent) and Sephadex LH-20 (developing solvent: methanol) to obtain 85 mg of pendulum.
Penduron data:
¹ H-NMR δ (ppm) in CDCl ₃ : 6.87 (1H, d, J = 8.3 Hz), 6.38 (1H, dd, J = 8.3, 2.5 Hz), 6.34 (1H, d, J = 1.1 Hz), 6.29 (1H, d, J = 2.5 Hz), 4.21 (1H, ddd, J = 9.8, 3.0, 1.1 Hz), 4.01 (1H, dd, J = 9.8, 1.2 Hz), 3.99 (3H, s), 3.98 (3H, s), 3.40 (1H, dddd, J = 6.7, 6.0, 3.0, 1.2 Hz), 2.99 (1H, dd, J = 16.0, 6.0 Hz), 2.67 (1H, dd, J = 16.0, 6.7 Hz) );
¹³ C-NMR (δ (ppm) in CDCl ₃ ): 184.1 (s), 183.5 (s), 155.4 (s), 154.6 (s), 146.6 (s), 145.1 (s), 144.6 (s), 131.0 (d), 130.3 (d), 112.1 (s), 108.8 (d), 103.4 (d), 68.1 (d), 61.3 (q), 61.2 (q), 30.8 (t), 28.9 (d).

Example 4 Measurement of anti-Leishmania activity The anti-Leishmania activity of bilawanol, anacardol, cardanol diene and bipendurone obtained in Example 2 was measured.

First, 1 to 5 mg of each sample was dissolved in dimethyl sulfoxide (DMSO) to prepare a DMSO solution (10 mg / ml). Next, this solution was diluted with Medium 199 medium (800 μg / ml) and passed through a membrane filter. The sample solution was adjusted to 9 concentrations, and each microtiter plate was inoculated with 50 ml of each concentration of sample solution and 50 ml of Leishmania culture solution prepared so that the final concentration was 1 × 10 ⁵ / ml. The total volume was 100 ml. After incubating at 27 ° C. under 5% CO ₂ for 72 hours, 10 μl of Tetracolor ONE reagent was added and incubated for 6 hours, and then the OD value (450-630 nm) was measured with a microplate reader. The measurement was performed for each sample and each concentration at 3 wells, and the average value and average error were obtained and shown in FIG. IC ₅₀ value was determined from the graph.

From FIG. 1, IC ₅₀ of bilawanol, anacardol, cardanol diene, tetrahydroamente flavone, penduron and amphotericin B was determined. The IC ₅₀ of bilawanol was 0.44 μg / ml, and the IC ₅₀ of pendurone was 0.066 μg / ml. Met. This is a very strong activity even when compared with the anti-Leishmania activity IC _{50 of} 0.2 mg / ml of amphotericin B known as an anti-Leishmania agent.

Meanwhile, each of the IC ₅₀ of Anakarudoru and cardanol diene 68μg / ml, was 69μg / ml, anti-Leishmania activity was weak.

FIG. 1 shows the IC ₅₀ for bilawanol, anacardol, cardanoldiene, tetrahydroamentoflavone, penduron and amphotericin B.

Claims (9)

  An antiprotozoal agent comprising a substance that exhibits antiprotozoal activity derived from Chi-Chi or Chinwin as an active ingredient.   An anti-Leishmania agent containing as an active ingredient a substance that exhibits anti-Leishmania activity derived from Chi-Chi or Chinwin.   An anti-Leishmania agent containing an extract of chi-shi or chin-win or a processed product thereof as an active ingredient.   The anti-Leishmania agent according to claim 3, comprising an extract of chi-cy or chi-win heartwood or a processed product thereof as an active ingredient.   Anti-Leishmania agent containing urushiol. Urushiol has the following formula:

The anti-Leishmania agent of Claim 5 which is bilavanol represented by these. The following formula:

An anti-Leishmania agent containing penjuron represented by   The antiprotozoal agent or anti-Leishmania agent according to any one of claims 1 to 7, which is used for the treatment of leishmaniasis.   A method for producing an anti-Leishmania agent, comprising a step of pulverizing chi-shi or chinwin, a step of extracting the crushed chi-shi or chinwin, and a step of purifying as necessary.

Images