KR19980039736A - Testosterone 5α Reductase Inhibitor Containing Gangjinhyang Extract - Google Patents

Abstract

The present invention relates to a testosterone 5α-reductase inhibitor containing an alcoholic extract of Kangjin-yang as an active ingredient. The present invention also relates to a testosterone 5α-reductase inhibitor containing, as an active ingredient, a beestitol, an isoflavan component, isolated from Kangjin-hyang extract. The alcoholic extract of Kangjin-hyang and its active ingredient Vestitol according to the present invention can inhibit the production of dihydrotestosterone in tissues by inhibiting the action of testosterone 5α-reducing enzyme and can be used to treat prostatic hyperplasia, acne, and alopecia areata. have.

Description

Testosterone 5α Reductase Inhibitor Containing Gangjinhyang Extract

The present invention relates to novel testosterone 5α-reductase inhibitors that can be used to treat prostatic hyperplasia, acne and androgenetic alopecia. More specifically, the present invention relates to testosterone 5α-reductase inhibitors containing an alcoholic extract of Kangjin-yang as the active ingredient. The present invention also relates to a testosterone 5α-reductase inhibitor containing, as an active ingredient, wasostolban, an isoflavan component, isolated from an alcoholic extract of Kangjin-yang as an active ingredient.

Testosterone secreted in the blood is converted to dihydrotestosterone by the action of testosterone 5α reductase in tissues such as prostate, hair follicle, sebaceous gland. However, even in the presence of congenital testosterone 5α reductase deficiency, the prostate gland is not enlarged, no male alopecia is present, and acne is not severe. It is known that Dennis A Holt, et al., J. Med. Chem. 33, 943 (1990). From this fact, it was found that the body of a substance that exhibits physiological functions in the prostate, hair follicle, sebaceous gland, etc. is not testosterone but dihydrotestosterone. Therefore, compounds such as finasteride, which inhibit the action of testosterone 5α reductase and inhibit the production of dihydrotestosterone in tissues, are used for the treatment of prostatic hyperplasia, and these compounds can also be used for the treatment and prevention of androgenetic alopecia and acne. .

Among diseases caused by excessive dehydrotestosterone production, prostatic hyperplasia is proportional to age in men after 50 years of age and suffers from difficulty urination due to urethra compression. In this case, the enlarged prostate tissue can be causally treated by excisional surgery. However, most patients are limited to the elderly and have problems such as postoperative side effects and recurrences. Is increasing.

Conventionally, anti-male hormone having a steroid ring structure has been mainly used for the treatment of prostatic hyperplasia, but undesirable side effects such as decreased libido, gynecomastia, and implants are problematic. Recently, alpha 1 receptor blockers have been developed to relax the smooth muscles of prostate tissues and to improve the symptoms of urination disorder. However, it is not possible to reduce the size of the enlarged prostate gland, so it is difficult to expect radical treatment. . Testosterone 5α reductase inhibitors selectively inhibit the production of dihydrotestosterone without affecting testosterone, which exhibits male hormone action. Therefore, this enzyme inhibitor is recognized as a safe and causative prostatic hypertrophy treatment because it can treat urination disorder by reducing the enlarged prostate without the side effects found in anti-male hormone.

Therefore, the present inventors conducted intensive studies on various substances to find inhibitors that can effectively inhibit testosterone 5α reductase without side effects. As a result of screening the inhibitory activity against testosterone 5α-reductase against the alcohol extracts obtained from each of the 200 herbal medicines, it was confirmed that the alcohol extract of Kangjin-flavor showed very strong testosterone 5α-reductase inhibitory activity, In addition, the present invention was completed by identifying specific active ingredients from this extract.

Accordingly, it is an object of the present invention to provide a novel testosterone 5α-reductase inhibitor having an excellent effect on the prevention and treatment of enlarged prostate.

More specifically, the present invention relates to a testosterone 5α-reductase inhibitor containing an alcoholic extract of Kangjin-yang as an active ingredient.

In addition, the present invention relates to a testosterone 5α-reductase inhibitor containing, as an active ingredient, beestitol isolated from the alcoholic extract of Gangjin-hyang.

Kangjin-hyang, which is the herbal medicine of the present invention, has been used as a core of roots of legumes (Dalbergia odorifera T. Chen.), Which has been used for the purpose of guerrillary blood (혈) in oriental medicine. Newly discovered by the invention. In addition, Kang Jin-hyang's extract and the testosterone 5α-reductase inhibitory activity of Vestitol, the main active ingredient, were first identified by the present inventors, and their potent testosterone 5α-reductase inhibitory activity prevented the treatment of prostatic hyperplasia, acne, and alopecia areata. It can be used as a drug.

In the present invention, each herbal medicine collected was extracted with 80% alcohol and the inhibitory activity against testosterone 5α reductase was measured. To identify its active substance against strong jinyang which showed strong inhibitory activity against this enzyme, the extract obtained by boiling hot gin (100 g) with methanol according to the conventional method and concentrated under reduced pressure was extracted with normal hexane, chloroform, ethyl acetate, and normal butanol. The testosterone 5α reductase inhibitory activity was compared with each fraction obtained by fractionation using a solvent such as distilled water. The chloroform fraction with the highest inhibitory activity was suspended on a silica gel column, eluted with a mixture of dichloromethane and ethyl acetate (4: 1) to collect the active fractions, and then mixed with dichloromethane and ethyl acetate (10: 1-10: 4). And silica gel column chromatography twice more with benzene-ethyl acetate mixture (4: 1). It is crystallized in a dichloromethane-normal hexane mixture with an active fraction to give a pale red crystalline compound (320 mg).

The physicochemical properties of the compound and the measurement results of various instrument spectra such as NMR, MS, UV, etc. were identified as Vestinol in accordance with the literature data of the isoflavan compound Vestinol isolated from the same plant. The IC ₅₀ values of the 5α-reductase extracted from the liver and epididymis of rats were 600 and 180 μM, respectively, which inhibited both type I and type II enzymes, and proved to be effective against acne and male alopecia, as well as prostatic hyperplasia. .

The invention is explained in more detail based on the following examples. However, the present invention is not limited in any way by these.

Example 1

Preparation of Type I Testosterone 5α Reductase

10-week-old SD females were dissected under diethyl ether anesthesia, and livers were extracted. Three-fold buffer solution (20 mM sodium phosphate, pH 6.5, 0.32 M white sugar, 1 mM dithiositol) was added to the cell disruptor. Homogenized with. The supernatant obtained by centrifugation (10,000 x g) of hepatocyte crushed solution was again subjected to super-centrifugation (105,000 x g) to obtain a microsomal fraction, suspended in the buffer and stored at -80 ° C.

Example 2

Preparation of Type II Testosterone 5α Reductase

10-week-old male rats were dissected under diethyl ether anesthesia to extract the epididymis, followed by homogenization of the cell disruptor (polytron) by adding three volumes of buffer solution (10 mM potassium phosphate, pH 6.5, 150 mM potassium chloride, 1 mM EDTA). It was. The cell lysate was ultracentrifuged (105,000 × g) to obtain a precipitate, homogenized by addition of the buffer solution and stored at -80 ° C.

Example 3

Inhibitory Activity of Testosterone 5α Reductase of Type I

5 µl of ethanol solution of plant extract, 20 µl of Type II enzyme solution and 10 µl of 2.5 mM NADPH solution were mixed with 450 µl of 40 mM sodium phosphate buffer (pH 6.5), followed by 5 µl of [ ¹⁴ C] -testosterone ethanol solution (0.005). μCi, 7.0 nanomolar) was added and reacted in a 37 ° C. water bath. After 15 minutes, 300 µl of an ethyl acetate solution containing 60 mg / ml of testosterone, dihydrotestosterone, and androstandioone were added thereto, followed by shaking to terminate the reaction and simultaneously extracting the reaction product. The ethyl acetate extract was concentrated and then dropped onto a silica gel plastic plate, and then the cyclohexane-ethyl acetate mixture (1: 1) was developed twice with a developing solvent. Observed under UV light, the testosterone and the dihydrotestosterone were cut off and the radioactivity of testosterone and dihydrotestosterone was measured with a liquid scintillation counter, and the inhibition rate of enzyme activity was calculated by the following equation.

% Inhibition = {(A-B) / A} × 100

A: The amount of dehydrotestosterone produced when no test sample was added

B: Dehydrotestosterone production amount when a sample for search was added

Example 4

Inhibitory Activity of Testosterone 5α Reductase of Type II

After adding 5 µl of ethanol solution of plant extract, 20 µl of type I enzyme and 2 µl of 2.5 mM NADPH solution to 68 µl of 10 mM triscitric acid buffer (pH 5.0), 5 µl of [ ¹⁴ C] -testosterone ethanol solution (0.005) was mixed. μCi, 0.086 nanomolar) was added and reacted in a 37 ° C water bath. After 15 minutes, 200 µl of an ethyl acetate solution containing 6 mg / ml of testosterone, dihydrotestosterone, and androstandioone were added and shaken to terminate the reaction and simultaneously extract the reaction product. The ethyl acetate extract was dropped onto a silica gel plastic plate, and then the cyclohexane-ethyl acetate mixture (1: 1) was developed twice with a developing solvent. Observation under ultraviolet light was performed to cut off the testosterone and dihydrotestosterone portions, and then measured the respective radioactivity with a liquid scintillation counter to calculate the inhibition rate of the enzyme activity in the same manner as in Example 3.

Example 5

Purification and structure determination of enzymatic inhibitor

The dried Kangjin flavor (100g) was pulverized to powder, and then methanol-added and extracted for 2 hours. Extraction of inhibitory activity of type 5α reductase isolated from rats was performed at 500 mg / mL. 81.7% showed a significant inhibition of enzyme activity than the control. The extract was concentrated under reduced pressure and solvent fractionation was carried out using hexane, chloroform, ethyl acetate, butanol and distilled water according to the conventional method, and 5α reductase inhibitory activity was measured using each fraction. Indicated. The chloroform fraction was subjected to silica gel column, chromatographed with ethyl acetate and dichloromethane mixed solvent (10: 1-10: 4), and divided into three fractions. After eluting with an acetate mixed solvent (4: 1), the active fraction was crystallized in a mixture of dichloromethane and normal hexane to obtain a crystalline compound. IC ₅₀ values of 600 μM and 180 μM, respectively, were determined by measuring the inhibitory activity of Type I and Type II testosterone 5α reductase.

This compound has a melting point of 157-158 ° C., and the results of ¹ H-NMR or ¹³ C-NMR and MS are compared with those of the literature [Ushio Sankawa, et al., Chem. Pharm. Bull., 40, 2542 (1992)] has been shown to be Vestinol, isolated and reported in Gangjin. Physical and chemical properties and instrumental analysis of this vestitol are as follows.

① Appearance: Light red needle crystal

② Melting Point: 157-158 ℃

③ TLC: Red color in cyclohexane-ethyl acetate (1: 1) with Rf value 0.45 anisealdehyde sulfate reagent

④ Mass Spectrum m / z (ret. Int.%): 272 (60), 150 (100), 137 (33), 135 (12), 123 (9), 107 (5)

⑤ ¹ H magnetic resonance spectrum (400 MHz, CDCL ₃ ) δ: 2.90-2.99 (2H, m, C4-H ₂ ), 3.49 (1H, m, C3-H), 3.77 (3H, s, OCH ₃ ), 4.04 -4.32 (2H, m, C2-H ₂ ), 4.64, 4.89 (1H, s, OH), 6.35 (3H, m, C6-H, C8-H, C3'-H), 6.48 (1H, d , J = 8.5, C5′-H), 6.94 (1H, d, J = 8.1, C5-H), 7.01 (1H, d, J = 8.6, C6′-H)

⑥ ¹³ C magnetic resonance spectrum (100MHz, CDCL ₃ ) δ: 30.76 (C-4), 32.11 (C-3), 55.77 (-OCH ₃ ), 70.31 (C-2), 102.56 (C-3´), 103.63 (C-8), 106.41 (C -5´), 108.31 (C-6), 115.09 (C-6´), 120.25 (C-5), 128.62 (C-10), 130.83 (C-1´ ), 154.62, 155.23, 155.58 (C-7, C-9, C-2´), 159.77 (C-4´)

⑦ Structural formula:

Example 6

Enzyme Inhibitory Activity of Kangjin-hyang Extract and Vestitol

The results of measuring the inhibitory activity against testosterone 5α-reducing enzymes of Kangjin-hyang methanol extract and Vestitol of type I and type II are as shown in Table 1 below.

Table 1.Inhibitory Activity of Testosterone 5α-Reducing Enzyme from Kangjin-Hyang Extract and Vestitol

Claims (3)

Testosterone 5α-reducing enzyme inhibitor containing an alcoholic extract of Gangjin-hyang as an active ingredient. Testosterone 5α-reductase inhibitor containing Vestitol represented by the following structural formula (I) as an active ingredient A pharmaceutical composition for treating prostatic hyperplasia, acne and androgenetic alopecia, containing an alcoholic extract of Gangjin-hyang and Vesitol of the above formula (I) as an active ingredient of Gangjin-yang.