JP5561711B2 - Urinary dysfunction improving agent including saw palmetto - Google Patents

Description

  The present invention relates to a urination disorder-improving agent containing saw palmetto, and particularly to a urination disorder-improving agent having an excellent urination disorder-improving effect.

The prostate is the organ that surrounds the posterior urethra just below the male bladder. Usually, the prostate is about 20 grams of walnut, but the majority of older men have enlarged prostate. It is thought that the prostatic hypertrophy is caused by a decrease in male hormones due to aging and a loss of hormone balance due to a relative increase in female hormones. When the balance of sex hormones is lost in old age, the portion close to the urethra (periurethral gland) inside the prostate grows and enlarges (mechanical obstruction), and dihydrotestosterone (DHT) accumulates in the prostate As a result, benign prostate hyperplasia (BPH) is thought to occur. BPH is known to increase the tension of the smooth muscle at the prostate tissue, the urethra of the prostate, or the outlet of the bladder. Symptom excitation when urine accumulates causes these muscles to contract via functional receptors (functional obstruction).
Symptoms of BPH include lower urinary tract symptoms (hereinafter referred to as LUTS) such as difficulty in producing urine, lack of urine momentum, and increased frequency of urination. BPH treatment methods are roughly classified into drug therapy, minimally invasive treatment (surgery), surgical treatment, urethral indwelling catheter, and the like.

Standard drug therapies used for BPH include α ₁ blockers, 5α-reductase inhibitors, and botanical formulations. Here, 5α-reductase is an enzyme that converts testosterone into the most active DHT.
Among these, botanical preparations are common in Europe and the United States and account for about 80% of all drugs prescribed for the treatment of BPH. There are about 30 types of plant preparations, but the most commonly used is a saw palmetto extract (hereinafter referred to as SPE). SPE is a fruit extract of a low shrub (Serenoa repens) belonging to the palm family that grows naturally in the southeastern United States. Saw palmetto fruit matures in tufts from October to December. Saw palm fruit has been used by American Indians since the early 1700s to testicular atrophy, erectile dysfunction, prostate enlargement, improvement of mucosal inflammation, and breast enlargement. For BPH LUTS patients, Permixon ^™ (a fat-soluble fraction of saw palmetto fruit) is known to be almost as effective as tamsulosin, an α ₁ blocker (Non-patent Document 1). SPE is also known to inhibit both type 1 and type 2 isozymes of 5α-reductase in in vitro experiments (Non-patent Document 2).
On the other hand, the main components of SPE depend on the extraction method, but Sabalselect ^™ (trade name of Indena), which is a supercritical CO ₂ extract of Serenoa repens, has 93.5% fatty acid and 0.2% fatty alcohol. %, And sterol is known to be 0.32%.

Bayne C.W. et al., Prostate, 40 (1999): 232-241 Iehle C et al., J Steroid Biochem Mol Biol, 54 (1995): 273-279

However, it has not been clarified so far which component in SPE is effective for BPH treatment. In addition, there were many patients who asked for a preparation with a better effect of improving urination disorder.
The present invention has been made in view of the above prior art, and a problem to be solved is to provide a urination disorder improving agent having an excellent urination disorder improvement effect.

  As a result of intensive studies conducted by the present inventors in order to solve the above problems, it is possible to enhance the urination disorder improving effect of SPE by adjusting SPE to a specific fatty acid composition, which is very excellent. The present inventors have found that it is possible to produce a dysuria improving agent having an effect of improving dysuria, and have completed the present invention.

That is, the urination disorder improving agent according to the present invention contains a saw palmetto fruit extract, and is characterized by containing oleic acid and myristic acid at 1: 0 to 2: 3.
Moreover, the urination disorder improving agent according to the present invention comprises a saw palm fruit extract,
extraction with n-hexane / methanol-water,
The aqueous phase (methanol-water) is extracted with diethyl ether,
The diethyl ether phase was purified by silica gel chromatography with n-hexane-diethyl ether (composition 4: 1),
The active fraction is produced by fractionating by reverse phase chromatography;
It contains oleic acid and myristic acid at 1: 0 to 2: 3.

  ADVANTAGE OF THE INVENTION According to this invention, the urination disorder improving agent which can implement | achieve the outstanding urination disorder improvement effect can be manufactured by adjusting the compounding ratio of oleic acid and myristic acid including SPE.

¹ H NMR spectrum (500 MHz) of SPE in CDCl ₃ . The flowchart which showed the fractionation method of the active ingredient from SPE. For each fraction obtained by reverse phase chromatography (A) [ ³ H] prazosin (0.25 nM), (B) [ ³ H] NMS (0.25 nM), (C) (+)-[ ³ H] PN The inhibition rate of specific binding of 200-110 (0.3 nM) was measured. (A) [ ³ H] prazosin, (B) [ ³ H] NMS, (C) (+)-[ ³ H] PN 200-110 of oleic acid and lauric acid isolated from SPE and their respective preparations Inhibition curve of specific binding. (A) [ ³ H] prazosin, (B) [ ³ H] NMS, (C) (+)-by SPE and the main fatty acids (lauric acid, oleic acid, myristic acid, palmitic acid, linoleic acid) in SPE Inhibition curve of specific binding of [ ³ H] PN 200-110. (A) Selected ion chromatograms of dexamethasone and (B) DHT which are internal standard substances. Inhibition curve of 5α-reductase activity of SPE and major fatty acids (lauric acid, oleic acid, myristic acid, palmitic acid, linoleic acid) in SPE. 5α-reductase activity of SPE (116 μg / mL) and three fatty acid mixed samples (lauric acid (34.8 μg / mL), oleic acid (33.6 μg / mL), myristic acid (13.9 μg / mL)). Black bars indicate 5α-reductase activity of SPE (116 μg / mL). White bars represent 5α-reductase activity of fatty acid mixed samples remixed at concentrations present in SPE. Gray bars represent 5α-reductase activity (calculated value) taking into account the sum of the inhibitory effects theoretically calculated by each of the three fatty acids contained in 116 μg / mL SPE. Specificity of [ ³ H] NMS by SPE at different concentrations, fatty acid simple substance samples (lauric acid, oleic acid, myristic acid), two fatty acid mixed samples (oleic acid and myristic acid), and the three fatty acid mixed samples Bond rate. The inhibition rate (%) of specific binding of [ ³ H] NMS when the composition of three kinds of fatty acids (lauric acid, oleic acid, myristic acid) in total 50 μg / mL was changed is shown in the circle.

In the present invention, first, which component in SPE is effective in improving urination disorder was examined, and then, an excellent urination disorder improving preparation was examined based on the result.
First, the results of isolation and structural analysis of active ingredients in SPE are shown.

FIG. 1 shows a ¹ H NMR spectrum of SPE (Sabalselect ^™ , manufactured by Indena) measured by Bruker Avance500 (manufactured by Karlsruhe). Aliphatic proton signals were detected in the spectrum at δ0.8-2.4, and olefin proton signals were detected at δ5.4. From these signals, it became clear that saturated and unsaturated fatty acids are the main components contained in SPE. Further investigations by the present inventors confirmed that SPE contains glycerol esters of fatty acids in addition to free fatty acids. From the intensity of the methylene signal at δ 2.35, the ratio of free fatty acid to glycerol ester was considered to be about 3: 1.

Next, the extraction method for isolating the active ingredient in SPE is shown below.
As shown in FIG. 2, first, 2 mL of SPE was diluted with 50 mL of methanol, and 100 mL of purified water was added. The operation of extracting with 100 mL of n-hexane was repeated three times. The operation of extracting the aqueous layer with 100 mL of diethyl ether (hereinafter Et ₂ O) was repeated twice. Each organic layer was distilled off the solvent under reduced pressure. About 200 mg of Et ₂ O residue, it was subjected to silica gel column chromatography. The active fraction 3 (18 mg) was subjected to reverse phase chromatography using a C8 column and fractionated every minute. An Agilent 1100 system (Agilent 1100 autosampler, binary pump, photodiode array detector) was used as a high performance liquid chromatography (hereinafter, HPLC) apparatus. Each fraction was dried in a nitrogen stream, redissolved in ethanol, and subjected to receptor binding evaluation.
Were subjected Et ₂ O extracts SPE to preparative HPLC to purify the lauric acid and oleic acid. LC-8A (manufactured by Shimadzu Corporation) was used for preparative HPLC.

Fraction 3 was subjected to reverse phase chromatography, further fractionated, and the results of evaluation of the binding activity for each receptor by the method shown below are shown in FIG.
Here, each receptor (α ₁ adrenergic, muscarinic, 1,4-DHP Ca channel antagonist) binding activity, which serves as an index for evaluating the urination disorder improving agent according to the present invention, will be described.

(Α ₁ adrenergic receptor binding activity)
The α ₁ adrenergic receptor is a G protein-coupled receptor activated by catecholamines, and is mainly present in the synapse after smooth muscle. Contraction through sympathetic alpha ₁ adrenergic receptors in prostate and urethral smooth muscles causes urethral obstruction (functional occlusion or dynamic occlusion). Therefore, substances exhibiting α ₁ adrenergic receptor binding activity are effective in urethral obstruction due to excessive sympathetic nervous system tension in the prostate and bladder neck, relax prostate smooth muscle and reduce urethral resistance I can expect that.
(Muscarinic receptor binding activity)
The main neurotransmitter of bladder contraction is via the parasympathetic pelvic nerve and via the muscarinic receptor of the bladder smooth muscle, causes the contraction response of the bladder and relaxation of the internal urethral sphincter, an involuntary muscle. Therefore, a substance exhibiting muscarinic receptor binding activity can be expected to suppress bladder contraction.
(1,4-DHP Ca channel antagonist receptor binding activity)
When the concentration of Ca ⁺ taken up into the cell via the 1,4-DHP Ca channel receptor present in the bladder smooth muscle increases, the bladder smooth muscle contracts. Therefore, a substance exhibiting 1,4-DHP Ca channel antagonist receptor binding activity can be expected to suppress bladder smooth muscle contraction.

Receptor binding activity evaluation method alpha ₁ adrenergic, muscarinic, in the measurement of each receptor of l, 4-DHP Ca channel antagonists, as a label, ^{respectively, [7-methoxy- 3 H]} prazosin (2.979 TBq / mmol ) (Hereinafter, [ ³ H] prazosin), [N-Methyl- ³ H] scopolamine methyl chloride (2.997 TBq / mmol) (hereinafter, [ ³ H] NMS), (+)-[ ³ H] PN 200-110 Radioreceptor assay using (3.180 TBq / mmol) (hereinafter (+)-[ ³ H] PN 200-110) (both manufactured by PerkinElmer) was followed.
Rats were laparotomized under ether anesthesia, and blood was collected from an abdominal descending aorta with a syringe treated with heparin and sacrificed. After refluxing the physiological saline cooled from the artery, the brain was removed. A 19-fold volume of cooled 50 mM Tris buffer (pH 7.4) was added to the brain from which the cerebellum had been removed, homogenized, and centrifuged at 40,000 × g for 20 minutes at 4 ° C. After removing the supernatant, 19-fold volume of cooled 50 mM Tris buffer (pH 7.4) was added again to the sediment, and after suspension, the mixture was centrifuged at 40,000 × g for 20 minutes at 4 ° C. After removing the supernatant, 29-fold volume of cooled 50 mM Tris buffer (pH 7.4) was added to the sediment and suspended to prepare a receptor preparation.
As a tissue amount, 50 mM Tris buffer (pH 7.4), fatty acid and 0.25 nM [ ³ H] prazosin were added to 10 mg of the receptor preparation to make a final volume of 1 mL. This reaction solution was incubated at 25 ° C. for 30 minutes. As a tissue quantity, add 50 mM N- (2-hydroxyethyl) piperazine-N'-ethanesulfonic acid (HEPES) buffer (pH 7.4), fatty acid, 0.25 nM [ ³ H] NMS to 3 mg of the receptor preparation, and a final volume of 500 μL. It was. This reaction solution was incubated at 25 ° C. for 60 minutes. As a tissue amount, 50 mM Tris buffer (pH 7.4), fatty acid, and 0.3 nM (+)-[ ³ H] PN 200-110 were added to 5 mg of the receptor preparation to make a final volume of 500 μL. This reaction solution was incubated in a dark room at 25 ° C. for 60 minutes with a sodium lamp lit.
Each reaction solution was subjected to rapid suction filtration on glass fiber filter paper (Whatman GF / B) using Cell Harvester (manufactured by Brandel) after completion of incubation. The filter paper was immediately washed with 3 mL of a cooled 50 mM phosphate buffer (pH 7.4). Toluene scintillator (toluene 2L, Triton-X 1L, 2,5-diphenylloxazole 15g, 1,4-bis [2- (5-phenyloxazolyl)] benzene 0.3g) is added to the filter paper and left at room temperature for 6 hours or more. The radioactivity was measured using a liquid scintillation counter. As a displacer, 10 μM phentolamine ([ ³ H] prazosin), 1 μM atropin ([ ³ H] NMS), 1 μM nifedipine ((+)-[ ³ H] PN 200-110) was used in the absence of the displacer and The radioactivity obtained in the presence was defined as total binding and nonspecific binding, respectively, and the difference between the two was defined as specific binding to the receptor.

From FIG. 3, fractions 3-6, 3-7, 3-9, 3-10, 3-12, 3-13 are [ ³ H] prazosin, (+)-[ ³ H] PN 200-110, [ ³ It can be seen that the specific binding of H] NMS is strongly suppressed. Fraction 3-11 strongly suppresses the specific binding of (+)-[ ³ H] PN 200-110. From LC / MS analysis, the main components of each fraction are lauric acid (fractions 3-6, 3-7), myristic acid (fractions 3-8, 3-9), linoleic acid (fractions 3-10), palmitic Estimated as acid (fractions 3-11) and oleic acid (fractions 3-12, 3-13), respectively. The Et ₂ O extract was subjected to preparative chromatography to isolate lauric acid and oleic acid. Oleic acid was obtained with a purity of 93% and a yield of 14.2%, and lauric acid with a purity of 95% and a yield of 16.7%.

  Oleic acid and lauric acid were isolated as the main active ingredients in SPE. The results of detailed examination of the receptor binding activities of these two types of fatty acids are shown in FIG. Here, the standard oleic acid (purity 99%) and lauric acid (purity 99%) were manufactured by Tokyo Chemical Industry Co., Ltd. and Nacalai Tesque, respectively. In the following, the values in the table are expressed as mean ± S.E. (Standard error).

FIG. 4 shows that oleic acid (10-80 μg / mL) and lauric acid (50-150 μg / mL) isolated from SPE inhibited the specific binding of [ ³ H] prazosin in a concentration-dependent manner. Recognize. Similarly, oleic acid (30-200 μg / mL) and lauric acid (100-300 μg / mL) suppress the specific binding of [ ³ H] NMS in a concentration-dependent manner. In addition, the specific binding of (+)-[ ³ H] PN 200-110 is also suppressed in a concentration-dependent manner (oleic acid: 30-100 μg / mL, lauric acid: 10-150 μg / mL). In addition, oleic acid and lauric acid samples also inhibited specific binding of [ ³ H] prazosin, [ ³ H] NMS, (+)-[ ³ H] PN 200-110 in a concentration-dependent manner. 1 revealed that their IC ₅₀ values were almost equivalent to those of oleic acid and lauric acid isolated from SPE.

Based on the above, oleic acid and lauric acid isolated from SPE showed specific concentrations of [ ³ H] prazosin, [ ³ H] NMS, and (+)-[ ³ H] PN 200-110 in a concentration-dependent manner. It became clear to suppress. That is, it was suggested that oleic acid and lauric acid isolated from SPE show each receptor binding activity of α ₁ adrenergic, muscarinic, 1,4-DHP Ca channel antagonist.
From the comparison of IC ₅₀ values, the binding activity of the oleic acid and lauric acid preparations was almost the same as that of oleic acid and lauric acid isolated from SPE. Furthermore, the IC ₅₀ value revealed that the binding activity of oleic acid to each receptor was higher than that of lauric acid. The oleic acid and lauric acid contents in SPE were almost equal, suggesting that oleic acid contributes greatly to the development of pharmacological action of SPE.

Next, for myristic acid, palmitic acid, and linoleic acid, fatty acids other than oleic acid and lauric acid contained in SPE, each receptor binding of α ₁ adrenergic, muscarinic, 1,4-DHP Ca channel antagonist The activity was examined, and the effects of the unsaturation degree and chain length on the activity of the main five fatty acids were considered. Here, myristic acid and linoleic acid were manufactured by Sigma-Aldrich, and palmitic acid was manufactured by Wako Pure Chemical Industries. The results are shown in FIG.

From FIG. 5, SPE inhibited the specific binding of [ ³ H] prazosin in a concentration-dependent manner (10-200 μg / mL), and its IC ₅₀ value was 106 ± 11 μg / mL (Table 2). The above five fatty acids suppressed the specific binding of [ ³ H] prazosin in a concentration-dependent manner (10-300 μg / mL), and these IC ₅₀ values ranged from 23.8-136 μg / mL. From the IC ₅₀ values shown in Table 2, when the inhibitory action of specific binding of [ ³ H] prazosin by these five fatty acids was compared, linoleic acid was the highest, 4.5 times that of SPE. Hereinafter, oleic acid was 2.3 times and myristic acid was 1.7 times, and lauric acid and palmitic acid were almost equivalent to SPE.
As shown in the figure, the four fatty acids excluding SPE and palmitic acid inhibited the specific binding of [ ³ H] NMS in a concentration-dependent manner (10-200 μg / mL). The IC ₅₀ value of SPE was 185 ± 8 μg / mL, and the IC ₅₀ values of the four fatty acids excluding palmitic acid were in the range of 56.4-169 μg / mL (Table 2). Comparing the inhibitory action of SPE and the specific binding of [ ³ H] NMS of four fatty acids, oleic acid is 2.6 times higher, myristic acid and linoleic acid are 1.8 times higher and 3.3 times higher than SPE, respectively. The order of inhibitory action of these fatty acids was the same as that of the inhibitory action of specific binding of [ ³ H] prazosin.
Furthermore, as shown in the figure, SPE and each fatty acid suppressed the specific binding of (+)-[ ³ H] PN 200-110 in a concentration-dependent manner (3-200 μg / mL), and their IC ₅₀ values Was 24.5-61.3 μg / mL (Table 2). Similarly to the other receptors, when the binding inhibitory action of five types of fatty acids and SPE was compared, linoleic acid and oleic acid showed an inhibitory action 2.4 and 1.8 times higher than SPE.

Therefore, the binding activity of linoleic acid containing two unsaturated double bonds is the highest in any of α ₁ -adrenergic, muscarinic, and 1,4-DHP Ca channel antagonist receptors, and then double bonds become 1 It became clear that the binding activity of oleic acid containing one was high. From this, the binding activity of unsaturated fatty acids tended to be higher than the binding activity of saturated fatty acids. As an example of a report of the relationship between the degree of unsaturation and the activity, there is an example of evaluation of activity inhibition of α7 nicotinic receptor (Vijayaraghavan S. et al., J Neurosci, 15 (1995): 3679-3687). The inhibitory effect on α7 nicotinic receptor activity by linolenic acid (C18: 3), linoleic acid (C18: 2), and oleic acid (C18: 1) was enhanced as the degree of unsaturation increased. Also in the present invention, it could be presumed that the binding activity to each receptor is enhanced by increasing the degree of unsaturation. Of the saturated fatty acids, myristic acid (C14: 0) has the highest binding activity, and for α ₁ adrenergic and muscarinic receptors, lauric acid (C12: 0) is better than palmitic acid (C16: 0). it was high. For saturated fatty acids, there was no correlation between chain length and binding activity.

Next, oleic acid and lauric acid, which are the main components in SPE, were used for the kinetic analysis of the binding to each receptor.
In this analysis, the concentration of each labeled ligand was determined using [ ³ H] prazosin (0.03-0.5 nM), [ ³ H] NMS (0.06-1.0 nM), (+)-[ ³ H] PN 200-110 (0.03- 1.0 nM) based on the receptor binding evaluation test.
The maximum number of binding sites (Bmax) and the apparent dissociation constant (Kd) of each labeled ligand measure the specific binding of each labeled ligand at various concentrations. Graph Pad PRISM 4.01 Using a pad regression software). The significant difference test was performed using Dunnett's test after the analysis of variance of Fischer, and a risk rate of less than 5% was determined to be significant. Asterisks indicate significant differences from control values, * P <0.05, ** P <0.01.
Table 3 shows the results of kinetic analysis of the binding of oleic acid and lauric acid to each receptor.

According to Table 3, in vitro, oleic acid (52.7 μg / mL) and lauric acid (73.5 μg / mL) each had a B max value of [ ³ H] prazosin-specific binding in the rat brain of 39 compared to the control value, respectively. %, 33% significantly decreased. Similarly, oleic acid (72.8 μg / mL) and lauric acid (163 μg / mL) significantly reduced the B max value of [ ³ H] NMS-specific binding in rat brain by 49% and 24%, respectively, compared to the control value. I let you. In addition, oleic acid (33.3μg / mL) and lauric acid (82.3μg / mL) showed a Bmax value for the specific binding of (+)-[ ³ H] PN 200-110 in the rat brain compared to the control value, respectively. 34% and 42% significantly decreased.

The presence of lauric acid and oleic acid, ^[3 H] prazosin and ^[3 H] where the specific binding of NMS was determined in rat brain, ^[3 H] prazosin and ^[3 H] Bmax value of the specific binding of NMS Decreased. Previous studies by the inventors have shown that SPE decreases the Bmax value of the specific binding of [ ³ H] prazosin in the rat prostate and [ ³ H] NMS in the bladder and is non-competitively suppressed It is considered to be. Inhibition of [ ³ H] prazosin binding by oleic acid and lauric acid is accompanied by a decrease in Bmax value, similar to SPE, and is considered to be suppressed non-competitively.
In addition, oleic acid and arachidonic acid (C20: 4) have been reported to decrease the Bmax value of specific binding of [ ³ H] QNB to canine heart membrane (Rauch B. et al., J Mol Cell Cardiol , 21 (1989), 495-506). Preincubation of free fatty acid with the receptor membrane preparation changes the conformation of the muscarinic receptor and renders the radiolabeled ligand unable to bind. As a result, Rauch et al. Considered that the Bmax value decreased.
Based on this study by Rauch et al., It is considered that oleic acid and lauric acid decreased the Bmax value of [ ³ H] NMS binding due to a change in the conformation of the muscarinic receptor.

In addition, oleic acid and lauric acid decreased the Bmax value of specific binding of (+)-[ ³ H] PN 200-110 in rat brain.
In experiments using rat cardiomyocytes, docosahexaenoic acid (C22: 6) has been reported to attenuate the effects of Ca channel agonists (BAY K 8644) and antagonists (nitrendipine) (Kjome JR et al., J Mol Neurosci , 10 (1998), 209-217). This mechanism is thought to be due to the fact that docosahexaenoic acid binds to the 1,4-DHP binding site or its vicinity and changes the protein-lipid environment of the membrane Ca channel and the environment between lipids.
Therefore, considering the conformational change of muscarinic receptors, (+)-[ ³ H] because oleic acid and lauric acid changed the conformation of 1,4-DHP Ca channel antagonist receptors. It was suggested that the Bmax value of PN 200-110 binding decreased.

  Next, the 5α-reductase activity of five major fatty acids (lauric acid, oleic acid, myristic acid, palmitic acid, linoleic acid) contained in SPE was evaluated. The results are shown in FIG.

Here, the measurement of 5α-reductase inhibitory activity was performed with reference to a known method (Liu J. et al., Biol Pharm Bull, 29 (2006), 392-395). The protein concentration was also measured by a known method (Bradford MM, Anal Biochem, 72 (1976), 248-254) and stored at −80 ° C. until activity evaluation.
The evaluation with an inhibitor was performed under the following conditions. Add 20 mM phosphate buffer (pH 6.5) containing 1 mM dithiothreitol, 50 μM testosterone, ethanol solution of fatty acid, 167 μM nicotinamide adenine dinucleotide phosphate (NADPH), 0.2 mg protein of female rat liver microsomes, and a final volume of 300 μL. did. Ethanol was used instead of fatty acid as a control for activity. The reaction solution excluding NADPH and microsomes was preincubated for 10 minutes at 37 ° C., NADPH and microsomes were added, and after incubation at 37 ° C. for 10 minutes, the reaction was stopped by adding 10 μL of 2M sodium hydroxide. After adding 10 μL of 30 μM dexamethasone as an internal standard substance, extraction was performed with 600 μL of ethyl acetate. The ethyl acetate layer was dried in a nitrogen stream and then dissolved in 50 μL of methanol. For LC / MS measurement, Waters Alliance 2790 and Waters ZQ 2000 were used. The control DHT / dexamethasone area ratio was taken as 100% of the 5α-reductase activity, and compared with the 5α-reductase activity in the presence of fatty acids. The selected ion chromatogram of DHT and dexamethasone is shown in FIG.

From FIG. 6, SPE inhibited 5α-reductase activity in a concentration-dependent manner, and its IC ₅₀ value was 101 ± 2 μg / mL (Table 4). Four fatty acids except palmitic acid also inhibited 5α-reductase activity in a concentration-dependent manner (10-200 μg / mL). As shown in Table 4, their IC ₅₀ values were in the range of 42.1-67.6 μg / mL, and the enzyme activity inhibitory action of the four fatty acids excluding palmitic acid was higher than that of SPE. Four types of linoleic acid, oleic acid, myristic acid, and lauric acid were suggested to be possible 5α-reductase activity inhibiting components in SPE.

SPE, oleic acid, lauric acid, myristic acid and linoleic acid inhibited 5α-reductase activity in a concentration-dependent manner. There was no significant difference in the activity-inhibiting action between the fatty acids. From the IC ₅₀ value, it can be seen that the activity inhibitory action of each fatty acid is 1.4-2.4 times higher than SPE. Therefore, it was found that oleic acid, lauric acid, myristic acid, and linoleic acid showed a 5α-reductase activity inhibitory action, and palmitic acid did not show an inhibitory action.

FIG. 8 shows the results of comparing the 5α-reductase activity of three fatty acid mixed samples mixed based on SPE (116 μg / mL) near the IC ₅₀ value and the content ratio at that concentration.
As shown in the figure, the 5α-reductase activity of SPE near the IC ₅₀ value was 41 ± 2.0% compared to the control. When the concentrations of lauric acid, oleic acid and myristic acid at SPE (116 μg / mL) were calculated from the Indena website (http://www.indena.com/pdf/sabalselect.pdf), 34.8 μg / mL, They were 33.6 μg / mL and 13.9 μg / mL. As shown in FIG. 6, lauric acid 40 μg / mL and myristic acid 20 μg / mL showed no 5α-reductase inhibitory activity. On the other hand, the 5α-reductase activity in the presence of 33.6 μg / mL oleic acid was calculated as 83.6% of the control from the inhibition curve.
Therefore, as shown in FIG. 8, the theoretical value of 5α-reductase activity in the presence of a mixed sample of 34.8 μg / mL lauric acid, 33.6 μg / mL oleic acid, and 13.9 μg / mL myristic acid is 83.6% of the control. Calculated. On the other hand, as shown in the figure, the measured value of 5α-reductase activity of the mixed sample was 23 ± 1.5% of the control. Compared with the addition of the inhibitory action of fatty acids alone, the inhibitory action was enhanced by the fatty acid mixture.
Here, when lauric acid 34.8 μg / mL, oleic acid 33.6 μg / mL, and myristic acid 13.9 μg / mL are converted to molar concentrations, they are 152 μM, 119 μM, and 69 μM, respectively, for a total of 341 μM. The theoretical value of 5α-reductase activity in the presence of 341 μM oleic acid (96.2 μg / mL) was calculated to be 11% of the control. Compared to the inhibitory action of mixed fatty acids, oleic acid alone had a higher inhibitory action on enzyme activity.

The concentrations of lauric acid, oleic acid, and myristic acid contained in SPE (116 μg / mL) are 34.8, 33.6, and 13.9 μg / mL, respectively, according to the Indena website as described above. About 80% of the theoretical value of 5α-reductase activity when lauric acid, oleic acid, and myristic acid are mixed at each concentration remains compared to the control. On the other hand, the actually measured value of 5α-reductase activity in the presence of the fatty acid mixed at the above concentration showed 23% activity compared to the control, and about 80% of the activity was suppressed. This fact cannot be explained by the sum of the 5α-reductase inhibitory activities of the three fatty acids. From this, it is considered that the action of oleic acid by addition of lauric acid and myristic acid is enhanced, that is, there is a pharmacodynamic interaction. In addition, the theoretical value of 5α-reductase activity in the presence of oleic acid (96.2 μg / mL) corresponding to the total concentration of the three fatty acids was calculated to be 11% of the control ratio. It was suggested that the inhibitory action may be higher than the measured value when lauric acid and myristic acid were mixed.
From the above, it was clarified that the inhibitory action of the fatty acid mixed sample is higher than the inhibitory action of SPE on 5α-reductase activity. This is probably because SPE contains an ester that does not exhibit an inhibitory action on 5α-reductase activity, and therefore, the inhibitory action is considered to be lower than that of mixed fatty acids containing only free fatty acids.

Furthermore, in order to confirm that the three types of fatty acids are effective for improving urination, SPE, fatty acid alone (lauric acid, oleic acid, myristic acid), two types of fatty acid samples (oleic acid and myristic acid), Using the three types of fatty acid mixed samples, the binding activity to the muscarinic receptor was examined. The test method was measured using the above method.
Here, each sample was adjusted at a ratio (lauric acid: 30.2%, oleic acid: 26.5%, myristic acid: 12.1%) contained in SPE.
That is, for the 100 μg / mL sample, SPE (100 μg / mL), lauric acid (30.2 μg / mL), oleic acid (26.5 μg / mL), myristic acid (12.1 μg / mL), oleic acid and myristic acid (26.5 μg / mL (oleic acid) + 12.1 μg / mL (myristic acid)), fatty acid mixed sample (30.2 μg / mL (lauric acid) + 26.5 μg / mL (oleic acid) + 12.1 μg / mL (myristic acid)) The test was carried out using a sample having a concentration. The results are shown in Table 5 and FIG.

From Table 5 and FIG. 9, it can be seen that in most samples, the [ ³ H] NMS-specific binding rate decreases with increasing sample concentration. That is, it became clear that the binding activity to the muscarinic receptor increased as the concentration of the sample increased. Fatty acid alone did not show very high binding activity, but the high concentration oleic acid sample showed significant binding activity.
The mixed sample of oleic acid and myristic acid also showed high muscarinic receptor binding activity.
In addition, similar to the results of the inhibitory action of 5α-reductase activity, it was revealed that the three fatty acid mixed samples had higher binding activity to muscarinic receptors than SPE. Therefore, it was suggested that an urinary dysfunction-improving agent equivalent to or better than SPE can be produced by including these components having not only a 5α-reductase activity inhibitory action but also a high muscarinic receptor binding activity.

  Next, based on these results, the above three fatty acids (lauric acid, oleic acid, myristic acid) were blended to produce a urination disorder improving agent equivalent to or better than SPE. The fatty acid composition which can expect the effect was examined.

  The binding activity to the muscarinic receptor when the composition of three kinds of fatty acids (lauric acid, oleic acid, myristic acid) in total 50 μg / mL was changed was measured using the above test method. The results are shown in Table 6 and FIG.

From Table 6 and FIG. 10, each fatty acid alone had a higher [ ³ H] NMS specific binding inhibition rate in the order of oleic acid, lauric acid, and myristic acid, and in particular, the inhibition rate of oleic acid was high. That is, it can be seen that the binding activity of oleic acid to the muscarinic receptor is the highest. However, since the inhibition rate was highest in the mixture of oleic acid and myristic acid of sample No. 2 (blending ratio 4: 1), the addition of myristic acid gave muscarinic receptor binding activity of oleic acid. It was found that the effect was enhanced. However, adding lauric acid, which has a slightly higher binding activity than myristic acid alone, to oleic acid did not enhance the muscarinic receptor binding activity of oleic acid.

  From these results, it is important for the urination disorder improving agent according to the present invention to add a specific ratio of myristic acid to oleic acid. That is, the urination disorder improving agent according to the present invention is characterized by containing oleic acid and myristic acid at 1: 0 to 2: 3. It is preferable that oleic acid and myristic acid are contained at 4.5: 0.5 to 1: 1. Further, it is particularly preferable to contain oleic acid and myristic acid in a ratio of 4: 1 to 3: 2. When the content ratio of myristic acid is too small, it becomes impossible to enhance the muscarinic receptor binding activity by adding myristic acid. When the content ratio of myristic acid is too large, the content ratio of oleic acid is relatively decreased, and therefore tends to be inferior to the muscarinic receptor binding activity.

  In the agent for improving dysuria according to the present invention, a method for adjusting the oleic acid and myristic acid to the above-mentioned composition containing a saw palm fruit extract is not particularly limited. Examples thereof include a method of changing the fruit extraction method and a method of adding oleic acid or myristic acid to the saw palmetto fruit extract.

Claims (2)

Including saw palmetto fruit extract,
An agent for improving urination disorder , wherein a blending mass ratio of three components of myristic acid (X), oleic acid (Y), and lauric acid (Z) is within a range surrounded by the following coordinates in a three-phase diagram .
(X, Y, Z) = (0, 100, 0), (0, 80, 20), (20, 60, 20), (40, 60, 0) Saw palm fruit extract,
extraction with n-hexane / methanol-water,
The aqueous phase (methanol-water) is extracted with diethyl ether,
The diethyl ether phase was purified by silica gel chromatography with n-hexane-diethyl ether (composition 4: 1),
The active fraction is produced by fractionating by reverse phase chromatography;
An agent for improving urination disorder , wherein a blending mass ratio of three components of myristic acid (X), oleic acid (Y), and lauric acid (Z) is within a range surrounded by the following coordinates in a three-phase diagram .
(X, Y, Z) = (0, 100, 0), (0, 80, 20), (20, 60, 20), (40, 60, 0)

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