JP5008973B2 - 5α-reductase inhibitor - Google Patents

Description

  The present invention relates to a 5α-reductase inhibitor that has a 5α-reductase inhibitory action and is effective in the prevention and treatment of prostate cancer, prostatic hypertrophy, and male pattern baldness.

It is known that diseases such as prostate cancer, prostatic hypertrophy, androgenetic alopecia, acne vulgaris, and seborrhea are caused by increased male hormone stimulation.
In various diseases involving these male hormones, testosterone, which is the main body of the male hormone, has an important role. 5α-reductase is an enzyme that converts this testosterone into dihydrotestosterone, and its inhibitor is effective for the prevention and treatment of the above-mentioned diseases.
Therefore, it is considered that the symptoms of these diseases can be reduced or prevented by inhibiting the activity of 5α-reductase, and various inhibitors that specifically inhibit 5α-reductase have been found and synthesized. It has been.

In particular, prostatic hypertrophy is one of the most important senile diseases in the urological field, but the disease has not been fully elucidated. Surgical excision is said to be the most effective for the treatment, but patients with this disease are elderly people, and in many cases they refuse to operate or are unable to operate themselves. Other treatments are desirable for those with no early symptoms. In addition, conservative therapy is more desirable if renal impairment or numbness due to dysuria is eliminated.
In recent years, with the arrival of an aging society, the need for better treatments and preventive agents for benign prostatic hypertrophy is increasing.

Conventionally, as a drug for improving dysuria associated with benign prostatic hyperplasia, several kinds of plants such as L-glutamic acid, amino acid preparation containing L-alanine and aminoacetic acid, pollen preparation containing sernitine pollen extract, and giant grasshopper In addition to oral preparations such as preparations containing extracts, intramuscular injections containing porcine prostate extract are also widely used.
Recently, it has been revealed that the proliferated hypertrophic nodules have androgen dependence, and as antiandrogen agents that can be expected to directly suppress prostate hypertrophy, oral agents such as chlormadinone acetate, oxedron and Intramuscular injections such as caprolate guestnolone have been developed and are commercially available.
However, conventional inhibitors have a steroid structure or a steroid-like structure, and there are many cases where side effects such as hormone-like effects are problematic, and are not practical from the viewpoint of safety. There are various problems that it causes pain and may cause local induration, and the treatment requires a relatively long period of time, resulting in the restriction of going to hospital.

  On the other hand, a number of medicinal effects have been passed down to Mannentake (Reishi), and it is known as a mushroom that is particularly effective against cancer, but it has been proved that there are substances that have various physiological effects. And the present inventors found out that the extract extracted with the alcohol etc. of Ganoderma lucidum has a 5 (alpha) -reductase activity inhibitory effect and suppresses the enlargement of a prostate, and applied for earlier. However, it is known that there are a large number of compounds in the extract of Ganoderma lucidum (Reishi), and various compounds have been identified, but any of them has an activity of inhibiting 5α-reductase activity. Or was not known.

Toru Kikuchi, Research on physiologically active components of Reishi, Pharma Medica, 1988, Vol. 6, special issue, pp. 110-125 FS Wang, H Cai, JS Yang, YM Zhang, CY Hou, JQ Liu, MJ Zhao (1997), Syudies on the ganoderic acid, a new constituents from the fruiting body of Ganoderma lucidum (Fr) Karst, Acta Pharmceutica Sinica, 32 (6): 447-450 Antonio G. Gonzales et al., J. Nat. Prod., 62, 1700-1701 (1999) Jiyuan Ma et al., J. Nat. Prod., 65, 1700-1701 (2002)

  Therefore, the object of the present invention is to search for various compounds in the extract of Ganoderma lucidum and extract from these compounds prostate cancer, prostatic hypertrophy, androgenetic alopecia, acne vulgaris, seborrhea, etc. It is an object of the present invention to find effective compounds for the prevention and treatment of diseases involving male hormones and to provide them as a 5α-reductase inhibitor and a prophylactic / therapeutic agent for benign prostatic hyperplasia.

  In order to solve the above-mentioned problems, the present inventors searched for extracts from nanentake using α-reductase inhibitory activity as an index. As a result, among the compounds extracted from mannentake, the present inventors inhibited extremely high 5α-reductase action. The present inventors have found that there is a substance to be completed, and have completed the present invention.

That is, this invention is specified by the matter described in the following [1]-[7].
[1] A 5α-reductase inhibitor containing lanostane-type triterpenes as an active ingredient.
[2] The 5α-reductase inhibitor according to [1], wherein the lanostane-type triterpenes are derived from Mannentake.
[3] Lanostane type triterpenes are ganoderic acid DM (ganoderic acid DM), 15α-hydroxy-3-oxolanosta-7,9 (11), 24-triene-26-oic acid (15a-hydroxy-3- oxolanosta-7,9 (11), 24-trien-26-oic acid), 5-lanosta-7,9 (11), 24-triene-15α, 26-dihydroxy-3-one (5-lanosta-7, The 5α-reductase inhibitor according to [1] or [2], which is 9 (11), 24-triene-15a, 26-dihydroxy-3-one) or tyromycic acid.

[4] 15α-hydroxy-3-oxolanosta-7,9 (11), 24-trien-26-oic acid (15a-hydroxy-3-oxolanosta-7,9 (11), 24-trien-26-oic acid).
[5] A prophylactic / therapeutic agent for benign prostatic hyperplasia containing lanostane-type triterpenes as an active ingredient.
[6] The prophylactic / therapeutic agent for benign prostatic hyperplasia according to [5], wherein the lanostane-type triterpenes are derived from Mannentake.
[7] Lanostane-type triterpenes are ganoderic acid DM, 15α-hydroxy-3-oxolanosta-7,9 (11), 24-triene-26-oic acid (15a-hydroxy-3- oxolanosta-7,9 (11), 24-trien-26-oic acid), 5-lanosta-7,9 (11), 24-triene-15α, 26-dihydroxy-3-one (5-lanosta-7, The prophylactic / therapeutic agent for prostatic hypertrophy according to [5] or [6], which is 9 (11), 24-triene-15a, 26-dihydroxy-3-one) or tyromycic acid.

  The 5α-reductase inhibitor of the present invention is effective for the prevention and treatment of diseases involving male hormones such as prostate cancer, benign prostatic hyperplasia, androgenetic alopecia, acne vulgaris, seborrhea and the like, and synthetic steroids Since it is not a compound but a natural product, it is excellent in safety. Also, 15α-hydroxy-3-oxolanosta-7,9 (11), 24-trien-26-oic acid (15a-hydroxy-3-oxolanosta-7,9 (11), 24-trien-26-oic acid ) Is a novel compound and has particularly excellent 5α-reductase inhibitory activity.

It is a UV-VIS absorption spectrum of ganoderic acid DM. It is a density | concentration dependence curve of 5 alpha-reductase inhibitory activity (inhibition activity) by ganoderic acid DM. It is a density | concentration dependence curve of 5 (alpha) -reductase inhibitory activity (inhibition activity) by the banyan extract before fractionation. It is a fractionation scheme of the garlic mushroom extract performed in Example 3. Ethanol extract fraction Fr. It is a HPLC chromatogram of G-5. Ethanol extract fraction Fr. It is the UV-VIS absorption spectrum of each component of retention time 19.8 minutes and 20.5 minutes in HPLC analysis in G-5. FIG. 4 is a correlation diagram obtained from an HMBC spectrum of the novel compound obtained in Example 4. 15- (R) - (+) - MTPA ester (a) and 15- (S) - (-) - MTPA is a diagram showing the difference in the chemical shift of ¹ H-NMR of ester (b).

Hereinafter, the present invention will be described in detail.
The 5α-reductase inhibitor and the prostatic hypertrophy prophylactic / therapeutic agent of the present invention contain lanostane-type triterpenes as active ingredients. The lanostane-type triterpenes according to the present invention have a lanostane skeleton, and include not only ganoderic acids such as ganoderic acid DM but also 24-triene-15α, 26-dihydroxy-3-one (5-lanosta-7). , 9 (11), 24-triene-15a, 26-dihydroxy-3-one). Among them, ganoderic acid DM (ganoderic acid DM), 15α-hydroxy-3-oxolanosta-7,9 (11), 24-triene-26-oic acid (15a-hydroxy-3-oxolanosta-7,9 ( 11), 24-trien-26-oic acid), 5-lanosta-7, 9 (11), 24-triene-15α, 26-dihydroxy-3-one (5-lanosta-7, 9 (11), 24 -triene-15a, 26-dihydroxy-3-one) and tyromycic acid have high 5α-reductase inhibitory activity and prophylactic / therapeutic effects on benign prostatic hyperplasia.

The lanostane-type triterpenes in the present invention can be obtained from various fungi, but can be obtained from an extract obtained from Ganoderma lucidum , which is generally referred to as ganoderma.
Ganoderma lucidum ( Ganoderma lucidum ) used in the present invention is a basidiomycete belonging to the genus Sarnia mushroom, and has been called Ganoderma or turfgrass in China, and has been called Ganoderma or Saiwaitake in Japan. it is a mushroom of eye ganodermataceae belonging to the (Ganodermatales) (Ganodermataceae) Ganoderma genus in (Ganoderma).

For the extraction of banyan mushroom used in the present invention, an organic solvent such as methanol, n-hexane, ethanol, ether, ethyl acetate, or water is used. The organic solvent extraction method and Soxhlet extraction are performed by impregnating them and leaving them to stand. A Soxhlet extraction method by heating reflux using a vessel is applicable, but is not limited to these methods.
For example, the methanol extraction method is a method in which 5.0 g of ganoderma fruit bodies are pulverized, 60 ml of methanol is added, left at room temperature for one day, the extract is taken out, and methanol is further added to the residue for extraction. is there. This operation is performed three times in total, and the resulting extract is combined and concentrated and dried to prepare an extract.

  The isolation and purification of lanostane-type triterpenes in the present invention can be performed by various chromatographies (silica gel column chromatography, reverse phase preparative liquid chromatography, etc.) using the 5α-reductase inhibitory activity as an index by the method described in the Examples. This can be achieved by repeating the fractionation operation, but is not limited to these methods. By fractionating, isolating and purifying in this way, ganodermic acids having 5α-reductase activity, such as ganoderic acid DM and ganoderenic acid, 15α- Hydroxy-3-oxolanosta-7,9 (11), 24-trien-26-oic acid, 15-hydroxy-3-oxolanosta-7,9 (11), 24-trien-26-oic acid Acids such as (tyromycic acid), 5-lanosta-7,9 (11), 24-triene-15α, 26-dihydroxy-3-one (5-lanosta-7,9 (11), 24-triene-15a, Various lanostane type triterpenes including alcohols such as 26-dihydroxy-3-one), ganodermadiol, lucidumol B, and ganodermanontriol are obtained.

In particular, ganoderic acid DM (15a-hydroxy-3-oxolanosta-7,9 (11), 15α-hydroxy-3-oxolanosta-7,9 (11), 24-triene-26-oic acid (15a-hydroxy-3-oxolanosta-7,9 (11) ), 24-trien-26-oic acid), 5-lanosta-7,9 (11), 24-triene-15α, 26-dihydroxy-3-one (5-lanosta-7,9 (11), 24- triene-15a, 26-dihydroxy-3-one) and tyromycic acid show extremely high 5α-reductase inhibitory activity, and their IC ₅₀ are 10.6 μM, 8.5 μM, 41.9 μM, 251 μM. By the way, ganoderic acid DM shows equivalent 5α-reductase inhibitory activity at a concentration of 1/19 compared with the ethanol extract as the raw material.
Of these compounds, 15α-hydroxy-3-oxolanosta-7,9 (11), 24-triene-26-oic acid (15a-hydroxy-3-oxolanosta-7,9 (11), 24- trien-26-oic acid) is a novel compound not described in the literature.

  The content of the lanostane type triterpenes in the 5α-reductase inhibitor of the present invention may be appropriately selected as necessary, and can be, for example, 1 μg to 100 mg per unit dosage form.

  The 5α-reductase inhibitor according to the present invention may contain an additive such as a pharmaceutically acceptable carrier. Specifically, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used. Excipients, lubricants, binders, disintegrants, thickeners in solid preparations; solvents, dispersants, dissolution agents in liquid preparations It is added as an adjuvant, suspending agent, tonicity agent, buffering agent, soothing agent and the like. If necessary, additives such as preservatives, antioxidants, colorants, sweeteners and the like can be used. Preferable examples of the excipient include lactose, sucrose, D-mannitol, starch, crystalline cellulose, light anhydrous silicic acid and the like. Preferable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like. Preferable examples of the binder include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone and the like. Preferable examples of the disintegrant include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium and the like. Preferable examples of the thickener include natural gums, cellulose derivatives, acrylic acid polymers and the like. Preferable examples of the solvent include water for injection, alcohol, propylene glycol, mark gol, sesame oil, corn oil and the like. Preferable examples of the dispersing agent include, for example, Tween 80, HCO 60, polyethylene glycol, carboxymethyl cellulose, sodium alginate and the like. Preferable examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like. Suitable examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and xericerin monostearate; for example, polyvinyl alcohol, Examples thereof include hydrophilic polymers such as polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose. Preferable examples of the isotonic agent include sodium chloride, glycerin, D-mannitol and the like. Preferable examples of the buffer include buffer solutions such as phosphate, acetate, carbonate and citrate. Preferable examples of the soothing agent include benzyl alcohol. Preferable examples of the preservative include paraoxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like. Preferable examples of the antioxidant include sulfite and ascorbic acid.

  The 5α-reductase inhibitor of the present invention is, for example, oral administration of tablets, capsules, granules, drinks, etc., administration by systemic routes such as intravenous infusion or injection, or parenteral administration such as intramuscular injection. Enteral administration, topical administration. Specific examples of the pharmaceutical preparation are shown below.

(1) Tablets, powders, granules, capsules: The pharmaceutical composition of the present invention is compression-molded by adding, for example, an excipient, a disintegrant, a binder or a lubricant, and then, if necessary, It can be produced by coating for masking, enteric or persistent purposes.

(2) Injection: The inhibitor of the present invention is dissolved in a vegetable oil such as olive oil, sesame oil, cottonseed oil, corn oil, propylene glycol, etc. as an aqueous injection together with a dispersing agent, preservative, tonicity agent, etc. It can be produced by suspending or emulsifying and molding it as an oily injection.

(3) External preparation: produced by making the inhibitor of the present invention into a solid, semi-solid or liquid composition. For example, the solid composition can be produced by using the inhibitor as it is, or by adding and mixing an excipient, a thickener and the like into a powder form.
The liquid composition is almost the same as in the case of an injection, and is produced by making it an oily or aqueous suspension. The semi-solid composition is preferably an aqueous or oily gel or ointment. In addition, any of these compositions may contain a buffering agent, a preservative, and the like.

(4) Suppository: manufactured by making the inhibitor of the present invention into an oily or aqueous solid, semi-solid or liquid composition. Examples of the oily base used in such a composition include glycerides of higher fatty acids (for example, cocoa butter, vitepsols), intermediate fatty acids (for example, miglyols), or vegetable oils (for example, sesame oil, soybean oil, Cottonseed oil, etc.). Examples of the aqueous gel base include natural gums, cellulose derivatives, vinyl polymers, and acrylic acid polymers.

  The 5α-reductase inhibitor of the present invention may be used in combination with other therapeutic agents including known antitumor agents conventionally used in the art. Suitable examples of such conventionally used antitumor agents include adriamycin, cisplatin, colchicine, CCNU (Lomastine), BCNU (Carmustine), actinomycin D, 5-fluorouracil, thiotepa, cytosine arabinoside, cyclophosphine One kind or a mixture of two or more kinds such as famide and mitomycin C may be mentioned.

  The 5α-reductase inhibitor of the present invention is generally administered at a daily dose of 1.0 μg to 100 mg per kg of body weight, divided into 1 to several times. The pharmaceutically effective amount and the administration method or administration means, The treatment period depends on the pathological state of the target patient and animal.

  Since the ganoderic acids of the present invention have a 5α-reductase inhibitory action, diseases caused by excessive production of dihydrotestosterone by 5α-reductase in mammals, particularly humans, such as prostate cancer, benign prostatic hyperplasia, and male pattern hair loss It can be suitably used for the prevention and treatment of symptoms, acne vulgaris, seborrhea and the like. In addition, it can be applied to other pharmaceuticals, quasi-drugs, cosmetics, and the like.

Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1
Extraction with ethanol 15 kg of Manntake fruit body was immersed in 126 L of EtOH (95%) and stirred for 16 hours. After the stirring, advantec Toyo filter paper No. And the filtrate was concentrated under reduced pressure using a rotary evaporator. 5 L of pure water was added to 1 L of the concentrated liquid and lyophilized. 571.1 g was obtained as a solid extract.

Measurement of 5α-reductase inhibitory activity A microsomal fraction was prepared from a homogenized SD female rat liver, and the one frozen and stored at −80 ° C. was used as a 5α-reductase crude enzyme solution.
The reaction system was a system with a total volume of 300 μl. 10 μl of 4.5 mM ethanol solution containing [4- ¹⁴ C] -testosterone (8 nCi, 0.7 μM, about 0.5% of the total testosterone is a radioactive substrate) and cold testosterone (the final concentration of the reaction system is 0.15 mM) 220 μl of 20 mM phosphate buffer (pH 6.5) containing 1 mM dithiothreitol and 10 μl of DMSO solution of the sample were added and incubated at 37 ° C. for 10 minutes. As a control, only DMSO was added.
After the incubation, the reaction was started by adding 50 μl of 1 mM NADPH-buffer solution and 10 μl of the 5α-reductase crude enzyme solution prepared previously. After incubation at 37 ° C. for 10 minutes, the reaction was stopped by adding 10 μl of 3M NaOH aqueous solution. Steroids were extracted by adding 900 μl of ether to the reaction system and vortexing, and 200 μl of this was applied onto a TLC plate. The mixture was developed twice with a mixed solvent of ethyl acetate: n-hexane (7: 3) as a developing solvent, and the radioactivity on the TLC plate was measured with an imaging analyzer (FRA-5000RF, Fuji Film Co. Ltd.).
In addition, description with mM and M represents mmol / L and mol / L here.
The control (no inhibitor) activity was defined as 100% of the enzyme activity, and calculated as in the following formulas (1) and (2), and IC ₅₀ was determined.
5α-reductase activity (%) = (radioactivity of the spot of dihydrotestosterone / radioactivity of the entire TLC plate from the applied position to the upper limit where the solvent was developed) × 100 (1)
5α-reductase inhibition activity (inhibition activity) (%) = {(control activity−sample activity) / control activity} × 100 (2)

Fractionation of Mannentake extract The ethanolic extract of Mannentake obtained above (571.1 g) was fractionated on a silica gel column (100 × 10 cm). The eluent was performed in a hexane-ethyl acetate gradient system. Further, using a 5α-reductase inhibitory activity as an index, fractionation was performed by repeating silica gel column chromatography in a hexane-ethyl acetate gradient system, and a 5α-reductase active component (170 mg) was isolated. When this component was developed on a silica gel TLC plate with n-hexane: ethyl acetate = 70: 30, the Rf value was 0.14 (colored with iodine).
The IR, NMR, and melting point of this component are shown below.
Melting point: 203-205 ° C
IR: 3402 (OH), 1711, 1679, 1660 (C = O), 1587 cm ^−1
1 H and ¹³ C NMR data (δ (ppm), solvent: CDCl ₃ ) are shown in Table 1.

以上の分析値を文献１）の値と比較し、本成分をのガノデリックアシッドＤＭ（ganoderic acid）と同定した。
１）FS Wang, H Cai, JS Yang, YM Zhang, CY Hou, JQ Liu, MJ Zhao (1997), Studies on the ganoderic acid, a new constituents from the fruiting body of Ganoderma lucidum (Fr.) Karst, Acta Pharmaceutica Sinica, 32 (6): 447-450

The above analysis value was compared with the value of the literature 1), and this component was identified as ganoderic acid DM (ganoderic acid).
1) FS Wang, H Cai, JS Yang, YM Zhang, CY Hou, JQ Liu, MJ Zhao (1997), Studies on the ganoderic acid, a new constituents from the fruiting body of Ganoderma lucidum (Fr.) Karst, Acta Pharmaceutica Sinica, 32 (6): 447-450

Moreover, in the HPLC chromatographic analysis measured on condition of the following, Ganodermic Acid DM had a retention time of 20.7 minutes. The UV-VIS spectrum is shown in FIG. Ganodermic Acid DM had maximum absorption wavelengths at 218 and 254 nm.
HPLC measurement condition column: Intersil ODS3 (4.6 mm × 150 mm)
Fluid phase: methanol: water (80:20)
Flow rate: 1 ml / min
Detector: PDA (photodiode array) detector

Inhibition of 5α-reductase activity of ganodermic acid DM and ethanol extract
activity)
Using various concentrations of Ganodermic Acid DM, 5α-reductase inhibitory activity was measured, and a concentration-dependent curve was prepared. The results are shown in FIG. From the concentration dependency curve, IC ₅₀ was 10.6 μM (5 ppm). For comparison, FIG. 3 shows a concentration-dependent curve of 5α-reductase inhibitory activity (inhibition activity) of the extract of Mannentake before fractionation. The IC ₅₀ of the garlic mushroom extract was 93.6 ppm. _{IC 50} of Ganoderma extract showed 19-fold greater than the _{IC 50} of Ganoderma derrick Acid DM.

Example 2
Suppressive effect of ganodermic acid DM on prostatic hypertrophy model animals
In order to verify whether ganodermic acid DM, which exhibits high 5α-reductase inhibitory activity in in vitro experiments, suppresses prostate hypertrophy in vivo, experiments were performed using prostate hypertrophy model rats.
Three weeks old SD rat male (Nippon Charles River Co., Ltd.) was preliminarily raised for 1 week, then the testis was removed, and 4 days later, testosterone was administered subcutaneously at 100 μg / a / 0.2 ml daily for 7 days (prostatic hypertrophy control group). ). In the experimental group, ganodermic acid DM was suspended in a 0.5% methylcellulose solution and administered by oral gavage with a sonde simultaneously with testosterone administration.
Here, the rats were reared at room temperature 23.0 + 0.5 ° C. and humidity 55 + 5% under a light-dark cycle of 12 hours from 7:00 am to 7:00 pm, and were freely fed food and water.
After fasting for 18 hours, blood was collected under pentobarbital anesthesia, and the prostate was removed. The prostate weight was measured and expressed as a body weight ratio.
The results are shown in Table 2.

表２よりガノデリックシッドＤＭは０．００１および０．０１ｍｇ／ｋｇ投与で有意に前立腺肥大を抑制した。
in vitroでの強い５α−リダクターゼ阻害活性を示すガノデリックシッドＤＭは、in vivoで前立腺肥大抑制効果を示した。

From Table 2, ganodermic sid DM significantly inhibited prostatic hypertrophy at doses of 0.001 and 0.01 mg / kg.
Ganodermic sid DM, which exhibits strong 5α-reductase inhibitory activity in vitro, showed an effect of suppressing prostate hypertrophy in vivo.

Example 3
The ethanol extract (571.1 g) obtained in Example 1 was fractionated by silica gel column chromatography (100 × 10 cm). Each fraction was grouped by TLC (n-hexane: ethyl acetate = 70: 30, iodine color development), and Fr. A (fatty acid fraction), Fr. B (having α-reductase inhibitory activity: fraction containing ganoderic acids) and Fr. C (no activity) fraction was obtained. Fr. In B, 5α-reductase inhibitory activity and prostate hypertrophy suppression effect were observed. Fr. B was further fractionated by silica gel column chromatography using ethyl acetate and hexane as elution solvents according to FIG. G (4.1 g) was obtained. Fr. G-5 showed 49% 5α-reductase inhibitory activity at a concentration of 200 ppm. In addition, ganodermic acid DM of Example 1 is Fr. It was contained in H-5 (2.7 g).
Fr. FIG. 5 shows a chromatogram obtained by HPLC analysis of G-5 under the conditions described in Example 1. Fr. G-5 contained two components at retention times of 19.8 and 20.5 minutes, similar to those of Ganodermic Acid DM. The UV-VIS absorption spectrum of each component is shown in FIG. All the components exhibited a UV-VIS absorption spectrum characteristic of Ganodermic Acid DM, and had maximum absorption wavelengths at 218 and 254 nm.

Example 4
Fr. By further fractionating G-5, three types of compounds were isolated. As a result of NMR and FABMS analysis, two of these compounds had the same data as the literature values. Therefore, 5-lanosta-7,9 (11), 24-triene-15α, 26-dihydroxy-3, respectively. -On (5-lanosta-7,9 (11), 24-triene-15a, 26-dihydroxy-3-one) (Gonzalez, Antonio G. et al., J. Nat. Prod., 65 (3) , 417-421 (2002)) and tyromycic acid (Quang, Dang Ngoc et al., Chem. & Pharmaceutical Bull. 51 (12) (2003)). Moreover, IC ₅₀ of 5α-reductase inhibitory activity of these compounds measured in the same manner as in Example 1 was 41.9 μM and 251 μM, respectively.
The other white crystalline compounds were analyzed by UV, 1D and 2D NMR, FABMS, optical rotation, and new Mosher method. As a result, the molecular weight was 468 from FABMS data, and it had a characteristic absorption of α, β-unsaturated carbonyl at 253 nm. From the NMR data (shown in Table 3), 7 methyl groups, 1 hydroxyl group and diene structure were observed.

ＨＭＢＣのスペクトルからＣ１５とＣ２６の相関を図７に示した。Ｃ１５位の水酸基の絶対配置は、以下のとおり、新Ｍｏｓｈｅｒ法を用いて測定した（図８）。この化合物と（Ｒ）−（＋）−ＭＴＰＡ及び（Ｓ）−（−）−ＭＴＰＡの試薬を６時間反応させ、分取ＴＬＣで生成して、１５−（Ｒ）−（＋）−ＭＴＰＡエステル（ａ）と１５−（Ｓ）−（−）−ＭＴＰＡエステル（ｂ）を得た。^１Ｈ−ＮＭＲで（Ｓ）−（−）−ＭＴＰＡエステルのＨ−１６、Ｈ−１７プロトンシグナルは、（Ｒ）−（＋）−ＭＴＰＡエステルより高磁場で観察されるに対して、（Ｓ）−（−）−ＭＴＰＡエステルのＨ−１４及びＨ−６は、（Ｒ）−（＋）−ＭＴＰＡエステルより低磁場で観察された（図８）。よって、１５位の水酸基はαであった。
以上の結果より、 この化合物の構造は、１５α−ヒドロキシ−３−オキソラノスタ−７，９（１１），２４−トリエン−２６−オイックアシッド（15a-hydroxy-3-oxolanosta-7,9(11),24-trien-26-oic acid）と決定した。
実施例１と同様にして測定したこの化合物の５α−リダクターゼ阻害活性のＩＣ_５０は、８．５μＭであった。

FIG. 7 shows the correlation between C15 and C26 from the spectrum of HMBC. The absolute configuration of the hydroxyl group at the C15 position was measured using the new Mosher method as follows (FIG. 8). This compound was reacted with (R)-(+)-MTPA and (S)-(−)-MTPA reagents for 6 hours and produced by preparative TLC to produce 15- (R)-(+)-MTPA ester (A) and 15- (S)-(−)-MTPA ester (b) were obtained. ^{In 1} H-NMR, H-16 and H-17 proton signals of (S)-(−)-MTPA ester are observed in a higher magnetic field than (R)-(+)-MTPA ester, whereas (S) )-(−)-MTPA ester H-14 and H-6 were observed in a lower magnetic field than (R)-(+)-MTPA ester (FIG. 8). Therefore, the hydroxyl group at the 15-position was α.
From the above results, the structure of this compound is 15α-hydroxy-3-oxolanosta-7,9 (11), 24-triene-26-oic acid (15a-hydroxy-3-oxolanosta-7,9 (11) , 24-trien-26-oic acid).
The IC ₅₀ of the 5α-reductase inhibitory activity of this compound measured in the same manner as in Example 1 was 8.5 μM.

Example 5
Fr. Inhibitory effect by administration of G-5 fraction As in Example 3, Fr. G-5 was forcibly administered orally with a sonde, and rats were raised in the same manner as in Example 3, and food and water were freely given.
After fasting for 18 hours, blood was collected under pentobarbital anesthesia, and the prostate was removed. Prostate weight was measured.
The results are shown in Table 3.

表４よりＦｒ．Ｇ−５画分０．０１ｍｇ／ｋｇ投与で有意に前立腺肥大を抑制した。
in vitroで５α−リダクターゼ阻害活性を示すｒ．Ｇ−５画分は、in vivoで前立腺肥大抑制効果を示した。

From Table 4, Fr. Prostatic hypertrophy was significantly suppressed by administration of 0.01 mg / kg of the G-5 fraction.
r. showing 5α-reductase inhibitory activity in vitro The G-5 fraction showed an effect of suppressing prostate hypertrophy in vivo.

Claims (5)

Ganoderic acid DM (15a-hydroxy-3-oxolanosta-7,9 (11), 15α-hydroxy-3-oxolanosta-7,9 (11), 24-triene-26-oic acid 24-trien-26-oic acid) or 5-lanosta-7,9 (11), 24-triene-15α, 26-dihydroxy-3-one (5-lanosta-7,9 (11), 24-triene- 5α-reductase inhibitor containing 15a, 26-dihydroxy-3-one) as an active ingredient. Ganoderic acid DM (15a-hydroxy-3-oxolanosta-7,9 (11), 15α-hydroxy-3-oxolanosta-7,9 (11), 24-triene-26-oic acid 24-trien-26-oic acid) and 5-lanosta-7,9 (11), 24-triene-15α, 26-dihydroxy-3-one (5-lanosta-7,9 (11), 24-triene- The 5α-reductase inhibitor according to claim 1, wherein 15a, 26-dihydroxy-3-one) is derived from Mannentake.   15α-hydroxy-3-oxolanosta-7,9 (11), 24-trien-26-oic acid (15a-hydroxy-3-oxolanosta-7,9 (11), 24-trien-26-oic acid). Ganoderic acid DM (15a-hydroxy-3-oxolanosta-7,9 (11), 15α-hydroxy-3-oxolanosta-7,9 (11), 24-triene-26-oic acid 24-trien-26-oic acid) or 5-lanosta-7,9 (11), 24-triene-15α, 26-dihydroxy-3-one (5-lanosta-7,9 (11), 24-triene- A prophylactic / therapeutic agent for benign prostatic hyperplasia containing 15a, 26-dihydroxy-3-one) as an active ingredient. Ganoderic acid DM (15a-hydroxy-3-oxolanosta-7,9 (11), 15α-hydroxy-3-oxolanosta-7,9 (11), 24-triene-26-oic acid 24-trien-26-oic acid) and 5-lanosta-7,9 (11), 24-triene-15α, 26-dihydroxy-3-one (5-lanosta-7,9 (11), 24-triene- The prophylactic / therapeutic agent for prostatic hypertrophy according to claim 4, wherein 15a, 26-dihydroxy-3-one) is derived from Mannentake.

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