JPH04330008A - 5alpha-reductase inhibitor - Google Patents

Abstract

PURPOSE:To obtain a 5alpha-reductase inhibitor useful for the treatment and/or prevention of diseases caused by excessive formation of dihydrotestosterone by using an extract of pericarp of duku or langsat as an active component. CONSTITUTION:The objective agent contains the extract of the pericarp of duku (Lansium domesticum Jack var. Duku) or langsat (Lansium domesticum Jack var. Langsat) as an active component. The main components of the extract is lansic acid of formula I, lansioside A of formula II (R is N-acetyl-beta-D- glucosamine), lansioside B (R is beta-D-glucose) and lansioside Q (R is beta-D-xylose).

Description

[Detailed description of the invention]

The present invention relates to a novel 5α-reductase inhibitor. To be more specific, Zuku (randium
Domesticum jack barduke, Lansium
domesticum Jack var. Duku
) or Lansium (Lansium domesticum J)
ack var. The present invention relates to a 5α-reductase inhibitor containing an extract obtained from the pericarp of A. Langsat as an active ingredient.

[0002] Traditionally, the causes of androgenetic alopecia are as follows:
(1) Hormone imbalance theory, (2) Genetic theory,
Many theories have been proposed, including (3) blood circulation failure theory and (4) nutritional theory, but it has long been suggested that the male hormone testosterone plays an important role in hair development. Ta. Adachi et al.'s theory [Biochem. Biophy
s. Res. Commun. , 41, 884 (1970
), the testosterone biosynthesized in the testicles is transferred to the 5α-α found in hair follicles, fatty glands, etc. in the head.
-Dihydrotestosterone (5α-reductase)
sterone), and this dihydrotestosterone is converted into adenyl cyclase (adenyl cyclase).
As a result, the intracellular cyclic-AMP level is reduced by significantly reducing the activity of the hair, thereby inducing a reduction in energy production in and around the hair and suppression of protein synthesis. Therefore, it is thought that due to a series of these phenomena, the hair in the growth phase shifts to the resting phase, and while this state is repeated, the hair progresses from terminal hair to vellus hair, and finally to male-pattern baldness. To support this theory, Schweikert [H. V. Schwei
[Kert] et al. found that the hair follicles of male-pattern baldness contain larger amounts of 5α-reductase metabolites, such as dihydrotestosterone, compared to female hair follicles and hair follicles of non-bald people. reported [J. Clin. En
docr. , 38, 811 (1974)]
.

In addition to androgenetic alopecia, dihydrotestosterone, which is produced from testosterone by 5α-reductase, is known to play an important physiological role in the occurrence and aggravation of acne (acne, acne, etc.). It has been reported. That is, J. B. Hay et al. compared the metabolic rate of testosterone between the affected skin and normal skin of acne patients and reported that the metabolism of testosterone by 5α-reductase was accelerated in the acne affected area [Br. J. Dermatol. ,91,
123 (1974)]. Also G. Sansone et al. found that the ability to synthesize dihydrotestosterone from testosterone in the affected skin of acne patients was 2 to 20 times higher than that of normal people.
found that dihydrotestosterone, which is produced by 5α-reductase, is significantly involved in the occurrence and exacerbation of acne [J. Invest. Dermatol. ,56,3
66 (1971)].

[0004] Based on these findings, the present inventors have
5α-5α, which strongly inhibits the action of α-reductase and is useful for the treatment and/or prevention of diseases caused by excessive production of dihydrotestosterone, such as alopecia, acne, etc.
As a result of intensive research aimed at finding a reductase inhibitor, the present invention was completed after discovering that an extract obtained from the pericarp of a plant belonging to the Meliaceae family achieved the objective. Accordingly, the present invention relates to a 5α-reductase inhibitor containing an extract obtained from the pericarp of Zuku as an active ingredient.

[0005] Zuku and Langsa are shrubs of the Meliaceae family that are widely distributed throughout Southeast Asia, especially Indonesia, and bear fruit during the rainy season from January to April. In particular, the fruit of Zuku is widely loved as a fruit, and its pulp is delicious, but the thick skin of the fruit contains a large amount of milky fluid and has traditionally been considered poisonous [E. J. H. Corner
, Kiyohiko Watanabe, “Illustrated Tropical Plant Collection”, p. 403 (196
9) Hirokawa Shoten Reference].

Although not all of the components of these pericarp have been elucidated, the main ones have been isolated and their structures determined. The following four types of compounds have been reported as main extracts.

[Chemical formula 1]

[Case 2]

[0007] Lansic Acid
id) is the most abundant component in the dried pericarp, and its isolation and structure were determined from 1967 to 1968 [Tetrahedron Letter
s, 37, 3571 (1967) and the same magazine, 34,
3731 (1968)]. lungioside (
Lansiosides) are included in the more polar fraction than lansic acids, and because they are glycosides, they were named lansiosides A, B, and C, respectively [Tetr
ahedron Letters, 23, 1349 (1
982) and J. Org. Chem. ,48,44
62 (1983)].

[0008] The extract included in the present invention includes all components extracted from the dried pericarp of Zuku and Langsa and mixtures thereof, and may also include components whose structures have not yet been isolated or determined. be. This is also supported by the experimental examples described later. That is, in Experimental Example 1, the ethanol extract from Zuku (presumed to be a mixture of several types of structurally known and structurally unknown components) has a strong 5α-reductase inhibitory effect, and this fact indicates that the structurally unknown component This suggests the possibility that there are also strong inhibitory effects. A preferred extract is an ethanol extract.

[0009] Up to now, no reports have been made regarding the pharmacological effects of the extract included in the present invention. For some of the isolated components, landioside, landoside A inhibits the contraction of guinea pig ileal tissue induced by leukotriene D4, whereas landoside B and C inhibit approximately 10% of landoside A. It has only been reported that the inhibitory activity is only one-fold lower [J. Org
．． Chem. , 48, 4462 (1983)]. The 5α-reductase inhibitory effect of the present invention has a completely different mechanism of action from the inhibitory effect on leukotriene D4, and therefore, the 5α-reductase inhibitory effect of the extract of the present invention was discovered for the first time by the present inventors. This is a useful pharmacological effect.

[0010] The extracts included in the invention, in particular the extraction of randic acids and randiosides A, B and C;
Isolation and purification methods are described in the aforementioned literature, Tetr
ahedron Letters and J. Org. C
hem. However, the extracts of the present invention are not limited to those obtained by the methods described therein. Also, in the literature, extraction is performed using zuku as a raw material, but it can also be extracted from the peel of langsa in the same way.

[0011] To briefly explain the extraction, isolation and purification method, first, the dried peel of Zuku or Langsa is crushed;
It is cooled in an appropriate solvent for several hours to several weeks, or extracted using a Soxhlet extractor. Isolating the extract into fractions enriched in each component using a suitable separation means, such as silica gel, column or high performance liquid chromatography, and optionally repeating the above chromatography and/or crystallization method, Specific components can be isolated and purified. The solvent used in each operation may be any solvent as long as it is suitable for the respective operation, such as aliphatic hydrocarbons such as pentane, hexane, heptane, and cyclohexane, aromatic hydrocarbons such as benzene, toluene, and xylene, Examples include halogenated hydrocarbons such as methylene chloride, chloroform, and carbon tetrachloride, alcohols such as methanol and ethanol, water, diethyl ether, petroleum ether, ethyl acetate, acetone, or a mixed solvent of two or more of these. .

[0012] The extract contained in the present invention has 5α-reductase inhibitor action and is therefore useful for the treatment and/or prevention of diseases caused by excessive production of dihydrotestosterone by 5α-reductase in mammals, especially humans. be. Such diseases include, for example, alopecia including androgenetic alopecia, and acne.

[0013] To use the extract included in the present invention for the above-mentioned purposes, it is usually administered systemically or locally, orally or parenterally. The dosage varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but for example, for the treatment and/or prevention of alopecia and acne, it is usually 10 μg to 50 mg per adult. , preferably in the range of 100 μg to 5 mg, is administered transdermally once to several times a day. Of course, as mentioned above, the dosage varies depending on various conditions, so there are cases where it is sufficient to use an amount smaller than the above-mentioned dosage range, and there are also cases where it is necessary to administer the dosage beyond the range.

Solid compositions for oral administration according to the present invention include tablets, powders, granules, and the like. In such solid compositions, one or more active substances are present in at least one inert diluent, such as lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, aluminum metasilicate. mixed with magnesium acid. The compositions may contain additives other than inert diluents, such as lubricants such as magnesium stearate and disintegrants such as fibrin calcium gluconate, in accordance with conventional methods. Tablets or pills may be coated with a film of gastric or enteric substances such as sucrose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose phthalate, etc., or may be coated with two or more layers, if necessary. Also included are capsules of absorbable materials such as gelatin.

Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, etc., including commonly used inert diluents, Examples include purified water and ethanol. In addition to inert diluents, the compositions may also contain adjuvants such as wetting agents and suspending agents, sweetening agents, flavoring agents, aromatic agents, and preservatives.

Other compositions for oral administration include sprays containing one or more active substances and formulated in a manner known per se.

Injections for parenteral administration according to the present invention include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of aqueous solutions and suspensions include distilled water for injection and physiological saline. Examples of non-aqueous solutions and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, and polysorbate 8.
There is 0 etc. Such compositions may further contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. These include, for example, filtration through a bacteria retention filter;
Sterilization is achieved by adding disinfectants or by irradiation. They can also be prepared as sterile solid compositions and dissolved in sterile water or sterile injectable solvents before use.

Other compositions for parenteral administration include topical solutions, liniments, such as ointments, containing one or more active substances and formulated in a manner known per se, and for rectal administration. This includes suppositories for intravaginal administration and pessaries for intravaginal administration.

Compositions for transdermal administration include external solutions such as lotions, tonics, sprays, solutions, suspensions, and emulsions, and liniments such as creams, gels, and creams. In such compositions, one or more active substances are present in at least one inert diluent, such as distilled water, lower alcohols such as ethanol, higher alcohols such as cetanol, polyethylene glycol, propylene glycol, etc. polyhydric alcohols such as, celluloses such as hydroxypropyl cellulose, animal and vegetable fats, petrolatum, wax,
Contains silicone, vegetable oils like olive oil, surfactants, zinc oxide, etc. In addition to the diluent mentioned above, this composition also contains
It may also contain adjuvants such as wetting agents, suspending agents, flavoring agents, and preservatives.

The present invention will be explained in detail below using reference examples, working examples, and experimental examples, but the present invention is not limited to these examples.

Reference Example 1 Extraction with Ethanol 50g of dried apricot peel was ground and extracted with ethanol 200g.
ml and left at room temperature for one week. Filter the mixture;
The filtrate was concentrated under reduced pressure to obtain 5.5 g of an oily substance.

Reference Example 2 Extraction of Landsic Acid (Method A) 950 g of dried zucchini pericarp was crushed and extracted continuously with n-hexane for 5 hours using a Soxhlet extractor.
The extract was concentrated under reduced pressure to obtain 120 g of an oily substance. The obtained oily substance was purified by silica gel column chromatography. Ethyl acetate and n-hexane (2
: 8) was used to elute 65 g of the part containing randic acid, and the elution solvent was added with ethyl acetate and n
- Gradually increase the proportion of ethyl acetate from a mixture of hexane (1:1) and finally change it to ethyl acetate alone, and prepare 40 g of a portion containing landiosides A, B, and C (
total) was eluted. n the part containing rangic acid.
The crude crystals thus obtained were recrystallized from a mixture of ethanol and water (3:1) to obtain 23 g of Rangic acid as white crystals having the following physical properties.

NMR (CDCl3 solution): δ=5.5
7 (1H, m), 4.48, 4.86, 4.82, 4.
79, 4.69 and 4.65 (1H, bs each), 1
．． 81, 1.77 and 1.73 (3H, bs each),
0.80 and 0.73 (3H, s each); IR (KB
r tablet method): ν=3300-2500, 1705, 16
30cm-1; Mass: m/e=470 (M+), 455, 4
52, 397.

Reference Example 3 Extraction of Landic Acid (Method B) 5.5 kg of dried and ground Zucrus fruit peel was mixed with ethyl acetate (12 kg).
1 night (3 times), and ethyl acetate was distilled off from 36 liters of the combined solution to obtain 920 g of an oily substance. The resulting oil was dissolved in n-hexane, extracted with 20% aqueous potassium hydroxide (3 times), the aqueous layer was washed with n-hexane (2 times) to remove neutral substances, and then extracted with water The layer was acidified with concentrated hydrochloric acid and then extracted with ethyl acetate. The extract was concentrated under reduced pressure, and the resulting residue was crystallized using n-hexane to obtain 110 g of crude crystals of randic acid. The mother liquor was purified by silica gel column chromatography (eluent: ethyl acetate:n-hexane = 1:9) to obtain 200 g of an oil containing randic acid. The obtained oil was crystallized using n-hexane to obtain 15 g of crude crystals of randic acid. Combined coarse crystals 125
g was recrystallized from a mixture of ethanol and water (3:1) to obtain 105 g of Landic acid having the same physical properties as Reference Example 2.

Reference Example 4 Extraction of Landiosides A, B and C 40 g of the portion containing Landiosides A, B and C obtained in Reference Example 2 was extracted by J. Org. Chem. ,48,44
62 (1983) to obtain landiosides A, B and C.

Example 1 Preparation of Tablets Containing Ethanol Extract 5 g of ethanol extract of starch, 200 mg of cellulose calcium gluconate (disintegrant), 100 mg of magnesium stearate (lubricant) and 4.7 g of microcrystalline cellulose
100 tablets each containing 50 mg of the active ingredient were obtained by mixing and tableting in a conventional manner.

Example 2 Preparation of a lotion containing ethanol extract 0.1 g of ethanol extract of Cucumber, 1.1 g of hydroxypropyl cellulose (HPC-M: registered trademark, manufactured by Nippon Soda) and several drops of fragrance were mixed with 80% by a conventional method. % ethanol to make a total volume of 100 ml to obtain the desired lotion.

Example 3 Preparation of cream containing ethanol extract Polyethylene glycol-400 and polyethylene glycol-4
000 (all registered trademarks, manufactured by NOF) 4.
After heating and dissolving a mixture of 0 g and 0.5 g of cetanol at 80° C., 0.1 g of ethanol extract of Zuku was added and sufficiently dissolved, and the mixture was cooled to room temperature. Add a few drops of fragrance to the mixture;
Add purified water and stir thoroughly to bring the total volume to 10
The desired cream was obtained as g.

Reference Example 5 Production of lotion containing Rangic acid 0.1 g of Rangic acid, 1.1 g of hydroxypropyl cellulose (HPC-M: registered trademark, manufactured by Nippon Soda) and several drops of fragrance were mixed with 80% ethanol in a conventional manner. The target lotion was obtained by dissolving the lotion in a total volume of 100 ml.

Reference Example 6 Production of cream containing randic acid Polyethylene glycol-400 and polyethylene glycol-
4000 (all registered trademarks, manufactured by NOF) 4 each
．． After heating and dissolving a mixture of 0 g and 0.5 g of cetanol at 80° C., 0.1 g of Rangic acid was added and sufficiently dissolved, and the mixture was cooled to room temperature. Add a few drops of fragrance and purified water to the mixture and stir thoroughly to make a total amount of 10g.
The desired cream was obtained.

Experimental example 1 Inhibitory effect on 5α-reductase

(1) Experimental method J. Shimazaki et al.'s method [Endocrin
ol, Japan. , 18, 179 (1971)]. That is, 4g of male rat prostate was mixed with 0.1g containing 3 times the volume of 0.25M sucrose.
The mixture was homogenized with M HEPES (pH 7.4) and centrifuged (3000 rpm for 10 minutes). The precipitate was suspended in 10 ml of the above buffer and centrifuged again (300 ml).
0 rpm for 5 minutes) and add the above buffer solution 3 to the resulting sediment.
ml was added and suspended to obtain an enzyme solution.

[0033] Enzyme activity was measured using [4-14C]-testosterone (1.5 nmol, 1.5 x 105 cpm).
), NADPH (0.5 μmol), the above enzyme solution (0.03 ml) and various concentrations of the specimen.
．． 1 ml of the reaction solution was incubated at 37°C for 60 minutes. The enzyme reaction was performed using chloroform and methanol (1:2
) and then centrifuged (2000 rpm for 3 minutes).
μl was spotted on a silica gel thin layer plate and mixed with chloroform, methanol and acetic acid (99.2:0.6:
0.2) was used for separation. The plate was subjected to autoradiography, the radioactivity of the generated dihydrotestosterone was measured using a TLC scanner, and the enzyme activity inhibition rate was calculated. The results are shown in Table 1.

(2) Results

[Table 1]

[0035] From the above results, the ethanol extract and Landic acid have a very strong 5α-reductase inhibitory effect, while Landiosides A, B and C do not have the same effect as Landic acid. However, 500 μg/ml showed a strong inhibitory effect of 20-60%. Based on this result, we concluded that extracts from the pericarp of Zuku and Langsa have 5α-reductase inhibitory activity, and then we used rangic acid as a representative example of the active ingredient of the extract to inhibit 5α-reductase. We investigated its effectiveness in the following animal experimental models and clinical trials, in which inhibiting this drug can be expected to improve or prevent disease conditions.

Experimental Example 2 Effect of hair growth on mice with genetic alopecia Three weeks old genetic alopecia mice (HRS/J-hr-
) A solution containing 1% Rangic acid in 5 cases (0.1 g Rangic acid and 0.15 HPC-M
G was dissolved in 80% ethanol to make a total volume of 10 ml, and 0.1 ml of the solution was applied to the head once a day. From about the 20th day after the start of application, white vellus hair began to grow in all cases, and from about the 30th day, numerous black terminal hairs were formed. However, solutions containing no randic acid (HP
Dissolve 0.15g of C-M in 80% ethanol,
When 0.1 ml of the solution prepared to a total volume of 10 ml was applied in the same manner, no such change was observed. The results are summarized in Table 2.

[0037]

[Table 2] From the above, it was proven that the extract contained in the present invention exerts excellent hair growth and hair growth effects on genetically alopecia mice.

Experimental Example 3 Hair Growth Effect on Humans Using a lotion containing 0.1% rangic acid (manufactured in Reference Example 5), eight volunteer patients with androgenetic alopecia (28 to 40 years old) ), the effects on hair growth and growth were investigated. The administration method was to sprinkle about 1 ml of the above lotion on the thinning hair area of the head and massage lightly once or twice a day. The effectiveness was evaluated by evaluating dandruff, dandruff, and
The subjects were asked to record symptoms such as hair loss and itching on a daily basis, and the bald areas were observed in detail and objectively once a month. As a result, symptoms such as dandruff, hair loss, and itchiness were significantly reduced in all subjects from about 2 to 3 days after administration of the lotion, and these effects were sustained throughout the administration period. Furthermore, as a result of periodic observation, 2 out of 8 subjects showed a remarkable hair growth effect (i.e., an increase in the number of hairs, a change from soft hair to terminal hair (a phenomenon in which the hair becomes thicker and harder) 4 months after administration. ) and hair growth promotion), and a significant hair thickening effect (i.e., hair growth promotion) was observed in 4 patients, but no effect was observed in the remaining 2 patients.

[0039] From the above, the extract contained in the present invention is not only effective against alopecia mice, but also prevents dandruff, hair loss, itching, etc., which is noticeable in human male pattern alopecia, and has an effect on hair growth. It has been proven that it has a hair thickening effect.

Experimental Example 4 Effect on acne in humans The effect of Rangic acid on acne was investigated on 15 healthy men and women (15 to 25 years old) suffering from acne. When an appropriate amount of a cream containing 1% Rangic acid (manufactured in Reference Example 6) was applied topically to the acne-affected area twice a day, in the morning and evening, the symptoms persisted for 1 to 3 days after application in all cases. Almost complete disappearance of acne was observed in the eyes.

From the above, it has been proven that the extract contained in the present invention has a dramatic therapeutic effect on human acne as well.

Experimental Example 5 Acute toxicity test 5-week old JCL-ICR male mice (body weight: 24-2
8g), 10% ethanol and 0.4% Tween
Rangic acid dissolved in physiological saline containing 80 (registered trademark) was administered into the tail vein, and the presence or absence of death was determined after 7 days.
Observed for days. As a result, Rangic acid 60mg
/kg, there were no deaths among the 10 cases, and therefore the LD50 value was 60 mg/kg or more.

[0043] From the above, it was found that the toxicity of the extract included in the present invention is extremely low. Furthermore, in Experimental Examples 3 and 4, there were no side effects (for example, irritation to the skin such as itching or swelling), proving that the extract contained in the present invention can be used safely as a medicine.

Claims (4)

[Claims] 1. A 5α-reductase inhibitor containing as an active ingredient an extract obtained from the pericarp of Zuku or Langsa. 2. The 5α- according to claim 1, wherein the active ingredient is an extract extracted with ethanol from the peel of Zuku root.
Reductase inhibitor. 3. The 5α-reductase inhibitor according to claim 1 or 2, wherein the disease caused by 5α-reductase is alopecia. 4. The 5α-reductase inhibitor according to claim 1 or 2, wherein the disease caused by 5α-reductase is acne.