Patent Application
20220273007
The present invention pertains to a functional supplement, particularly one for preventing or improving voiding dysfunction.
Generally, symptoms associated with voiding dysfunction, or difficulty urinating, appear with increased age due to one cause or another, including genetic factors, diet, obesity, high blood pressure, high blood sugar, dyslipidemia, and so forth. Voiding dysfunction is an impairment of the functioning of the lower urinary tract, which consists of the bladder and urethra (including the prostate in men) and the urethral sphincters. The two main causes of voiding dysfunction are overactive bladder and benign prostatic hyperplasia, with the severity of the symptoms thereof depending upon the individual.
Amidst these circumstances, saw palmetto has been the subject of attention in recent years as an easily consumable dietary supplement that is effective for voiding dysfunction. Saw palmetto fruit extract is well known in Japan as well as in Western countries to be effective for urinary tract symptoms, chronic pelvic pain, bladder dysfunction, loss of libido, hair loss, hormonal imbalance, and prostate cancer, and has been used to treat these conditions.
However, even as domestic demand for saw palmetto increases, bad weather and other factors have resulted in poor harvests for four consecutive years in the Florida peninsula and in Mexico, the main growing regions for saw palmetto, which has destabilized the supply to Japan. This, in turn, has resulted in problems such as rising price.
The present invention provides a novel functional supplement/dietary supplement that is inexpensive, abundant, easy to obtain and consume, is capable of preventing and improving voiding dysfunction, and has few side effects.
In the course of testing a wide variety of natural materials in order to achieve the abovementioned goal, the inventors focused on akamoku (Sargassum horneri), a type of seaweed, as a material that is inexpensive, abundant, and easy to obtain and consume.
Akamoku, like hijiki (Sargassum fusiforme) and the like, is a perennial brown alga in genus Sargassum, family Sargassaceae, order Fucales, that is widely distributed along the entire coast of Japan apart from the eastern part of Hokkaido, and from the Korean peninsula to China and northern Vietnam. In particular, algae of genus Sargassum are so abundant that there is a region referred to as the “great Atlantic Sargassum belt”.
Akamoku has long been consumed in the local cuisine of the Tōhoku region of northern Japan. Akamoku is known to contain various nutrients, including polysaccharides such as fucoidan and alginic acid, minerals, fucoxanthin, polyunsaturated fatty acids, and polyphenols, and to therefore have pharmacological effects and functions that are beneficial for beauty and health.
Focusing on the abovementioned abundance and multiple functions of akamoku, the inventors theorized that seaweeds, including akamoku, have the potential to exhibit functions that would contribute to the prevention or improvement of voiding dysfunction, and, through careful experimentation, identified the preconditions necessary for such functions to manifest, thereby arriving at the present invention.
Specifically, a primary aspect of the present invention provides the following.
(1) A functional supplement for preventing or improving voiding dysfunction, characterized by containing a seaweed extract.
(2) The supplement according to (1), wherein the voiding dysfunction is caused by benign prostatic hyperplasia or overactive bladder.
(3) The supplement according to (1), wherein the seaweed is one seaweed selected from the group consisting of aosa (Ulva), aonori (green laver), kombu, arame (Eisenia bicyclis), kajime (Ecklonia cava), wakame (Undaria pinnatifida), mekabu (root of the wakame), hijiki (Sargassum fusiforme), mozuku (Nemacystus decipiens), tengusa (red algae in family Gelidiaceae), dulse (Palmaria palmata), iwanori (various species of Pyropia), and akamoku (Sargassum horneri).
(4) The supplement according to (1), wherein the seaweed extract is extracted from a specific seaweed in at least 50% ethanol solution.
(5) The supplement according to (4), wherein the seaweed extract is extracted from a specific seaweed in at least 95% ethanol solution.
(6) The supplement according to (1), wherein the seaweed extract has an extract concentration of 300 μg/mL or greater.
(7) The supplement according to (6), wherein the seaweed extract has an extract concentration of 1 mg/mL or greater.
(8) The supplement according to (1), wherein the seaweed extract has a fucoxanthin concentration of at least 0.5 mg/Kg, an eicosapentaenoic acid concentration of at least 71 μg/mL, and a stearidonic acid concentration of at least 47 μg/mL.
(9) The supplement according to (1), wherein the seaweed extract is water extracted or hot water extracted from a specific seaweed.
(10) The supplement according to (1), wherein the seaweed extract has an extract concentration of 50 mg/mL or greater.
In accordance with the features described above, it is possible to suppress excessive contractions from overactive bladder, inhibit 5α-reductase activity, which is a cause of benign prostatic hyperplasia, and inhibit androgen receptor binding.
As a result of these effects, it is possible to suppress excessive contractions from overactive bladder, and benign prostatic hyperplasia. This yields the effect of making it possible to prevent or improve voiding dysfunction.
In addition, as an effect of the abundance and inexpensiveness of seaweeds such as akamoku, it is possible to mass produce a functional supplement that is safe and free of side effects.
Other characteristics of the present invention will be made apparent in the descriptions of the embodiment of the present invention described below.
This specification refers to several documents, the entire contents of which are incorporated herein by reference.
An embodiment of the present invention will be described below with reference to the drawings and tables.
In the course of testing a wide variety of natural materials in order to achieve the abovementioned goal, as discussed above, the inventors focused on akamoku (Sargassum horneri), a type of seaweed, as a material that is inexpensive, abundant, and easy to obtain and consume.
Akamoku, like hijiki (Sargassum fusiforme) and the like, is a perennial brown alga in genus Sargassum, family Sargassaceae, order Fucales, that is widely distributed along the entire coast of Japan apart from the eastern part of Hokkaido, and from the Korean peninsula to China and northern Vietnam. In particular, algae of genus Sargassum are so abundant that there is a region referred to as the “great Atlantic Sargassum belt”.
Akamoku is known to contain various nutrients, including polysaccharides such as fucoidan and alginic acid, minerals, fucoxanthin, polyunsaturated fatty acids, and polyphenols, and to have pharmacological effects and functions that are good for beauty and health.
Focusing on the abovementioned abundance and multiple functions of akamoku, the inventors theorized that seaweeds, including akamoku, have the potential to exhibit functions that would contribute to the prevention or improvement of voiding dysfunction—in particular, the potential to act upon overactive bladder and benign prostatic hyperplasia, two major causes of voiding dysfunction—and, through careful experimentation, identified the preconditions necessary for such functions to manifest, thereby arriving at the present invention.
In order to explain the features and functions of the present invention, the two major causes of voiding dysfunction will first be explained.
As discussed above, overactive bladder is one of two major causes of voiding dysfunction.
Voiding dysfunction caused by overactive bladder can be neuropathic or non-neuropathic, i.e., arising from non-neurological causes. In the former case, problems arise in the circuits of the nerves of the brain and the muscles of the bladder (urethra), potentially leading to voiding dysfunction, as a result of neuropathies of the brain such as cerebrovascular disease, Parkinson's disease, multiple system atrophy, or dementia, or neuropathies of the spinal cord such as spinal cord injury, multiple sclerosis, or spinocerebellar degeneration. Voiding dysfunction may also develop as a complication of the abovementioned benign prostatic hyperplasia, loss of pelvic floor muscle strength due to childbirth or the like, and so forth.
Anti-cholines, β3 adrenergic receptor agonists, and the like, which control the contraction and relaxation of the bladder, are generally used to treat these conditions. However, anti-cholines have side effects such as dry mouth, constipation, and accommodative dysfunction, and β3 adrenergic receptor agonists have age restrictions that contraindicate administration to patients of reproductive age.
Benign prostatic hyperplasia is another of the two major causes of voiding dysfunction. Because the prostate encircles the urethra, enlargement of the prostate as a result of benign prostatic hyperplasia constricts the urethra, leading to voiding dysfunction. Testosterone, one of the male hormones, is converted to dihydrotestosterone by 5α-reductases, which is more potent and causes enlargement of the prostate, and the binding of this dihydrotestosterone to androgen receptors (ARs) can cause enlargement of the prostate through repeated excessive prostate cell proliferation.
Therefore, research is currently underway into 5α-reductase-inhibiting therapeutic agents, such as 5α-reductase inhibitors, and therapeutic agents, such as anti-androgens, and methods that work to inhibit binding between androgen receptors and dihydrotestosterone (DHT). At present, however, these therapeutic agents must be taken internally for extended periods, and are recognized as having side effects such as impaired sexual function caused by reduced serum testosterone levels.
In response to these circumstances, the inventors focused on specific abundant and inexpensively obtainable seaweeds, discovered that extracts thereof exhibit the function of inhibiting bladder contraction in specific conditions and have the 5α-reductase-inhibiting function of 5α-reductase inhibitors and the like, and empirically confirmed the same, thereby arriving at the present invention.
Specifically, the present invention is a functional supplement for preventing or improving voiding dysfunction, characterized by containing a seaweed extract.
In accordance with a first embodiment of the present invention, the seaweed extract is obtained through extraction from seaweed using an ethanol solution of a specific concentration. This seaweed is preferably akamoku. The concentration of the ethanol solution used to perform extraction is 50%, more preferably 90% or higher, and the extract concentration is preferably 300 μg/mL, more preferably 1 mg/mL.
The functional supplement according to this first embodiment may be produced, for example, as follows.
In this embodiment, an example in which Japanese akamoku is used will be described with reference to the flowchart of
First, 3.6 kg of Japanese akamoku is immersed overnight (16 hours) in 72 L of tap water, then desalinated while being rinsed with tap water. The seaweed is then fan-dried at room temperature to a water content of 10% or less.
Next, the dried akamoku is submerged in a fivefold volume of 95% ethanol solution or a twentyfold volume of 50% ethanol solution, and extraction is performed through maceration or agitation. Extraction is performed at room temperature for one to sixteen hours. The volume of the recovered extract solution is then reduced fiftyfold or more using a vacuum concentrator, after which the solvent is removed using a centrifugal vacuum concentrator to recover an ethanol extract of akamoku.
In this embodiment, the recovered ethanol extract of akamoku is sealed without further modification in a container to create a functional supplement.
However, the functional supplement is not limited to such a form, and the ethanol extract of akamoku produced as described above may be diluted and dissolved in vegetable oil or the like, and worked into a form such as a softgel to create a functional supplement.
A second embodiment of the present invention may be produced as follows.
Japanese akamoku is submerged in a twentyfold volume of tap water, and extraction is performed via maceration or agitation. Extraction is performed in water (room temperature) or hot water (70-90° C.) from one hour to overnight. The volume of the recovered extract solution is reduced fiftyfold or more using a vacuum concentrator, after which the solution is dried using a centrifugal vacuum concentrator, a freeze dryer, or a spray dryer, and an aqueous (hot water) extract of akamoku is recovered.
In this embodiment, the recovered aqueous (hot water) extract of akamoku is sealed without further modification in a container to create a functional supplement.
However, the functional supplement is not limited to such a form, and the water (hot water) extract of akamoku produced as described above may be worked into tablets or capsules, or, taking advantage of the water-soluble properties of the extract, into a form such as a soft drink, jelly, or the like to create a functional supplement.
Experiments conducted in order to determine whether a functional supplement containing the akamoku extract produced as described above is effective against overactive bladder and benign prostatic hyperplasia, as well as the results of said experiments, will be described below.
In experiment 1, a 95% EtOH (ethanol solution) extract of akamoku (akamoku extract) was first obtained according to the flowchart shown in
For the sake of the further extract analysis to be described below, lipid-soluble n-Hex (n-hexane), MeCN (acetonitrile), and CHCL3 (chloroform) recovery (redissolves insolubles) fractions were obtained via partial purification.
[Experiment 2] Organ Bath Assay (1 mg/mL Akamoku Extract): 1 mM ACh Contractile Inhibition Assay
In experiment 2, an in vitro assay was performed using an organ bath for evaluating contractile/relaxant action to investigate the effects of akamoku extract upon acetylcholine (ACh) induced contraction.
As shown in
In the two graphs on the left in
From the results, it was found that 30 minutes of 1 mg/mL of the akamoku extract obtained in experiment 1 most significantly inhibited 1 mM ACh (acetylcholine) contraction.
[Experiment 3] Organ Bath Assay Results (1 mg/mL Akamoku Extract): 80 mM KCl Contractile Inhibition Assay, 10 mM Carbachol Contractile Inhibition Assay
Next, 1 mg/mL akamoku extract was added to the organ bath tank, and 80 mM KCl and 10 mM carbachol, which promotes further acetylcholine induction, were separately added after five minutes and thirty minutes, respectively, to induce contraction in order to investigate the suppressant effects of the akamoku extract in the presence of stronger contraction.
As shown in
[Experiment 4] Organ Bath Assay Results (300 μg/mL Akamoku Extract Fraction): 80 mM KCl Contractile Inhibition Assay
In experiment 4, the contractile-inhibitory action of the lipid-soluble n-Hex (n-hexane), MeCN (acetonitrile), and CHCL3 (chloroform) recovery (redissolves insolubles) fractions (respective concentrations: 300 μg/mL) partially purified from the akamoku extract in experiment 1 was investigated.
In
The results show that, out of the abovementioned fractions of the akamoku extract, the MeCN (acetonitrile) fraction significantly inhibits contraction.
[Experiment 5] Organ Bath Assay Results (300 μg/mL Akamoku Extract Fraction): 1 mM ACh Contractile Inhibition Assay
In experiment 5, the n-Hex (n-hexane), MeCN (acetonitrile), and CHCL3 (chloroform) recovery (redissolves insolubles) fractions (respective concentrations: 300 μg/mL) of the akamoku extract were observed for fixed periods, in addition to the inquiry performed in experiment 4, to investigate in which phase in particular there is effective contractile-inhibitory action.
In
The results show that contraction was more significantly inhibited in the plateau (tonic) phase in particular than in the early phase, as shown in the graphs in
[Experiment 6] Organ Bath Assay Results (100 μg/mL Akamoku Extract Fraction): 1 mM ACh Contractile Inhibition Assay
For experiment 6, the same experiment as in experiment 5 was performed, apart from altering the concentration of the akamoku extract fraction in experiment 5 from 300 μg/mL to 100 μg/mL to investigate what sort of differences would occur in the plateau phase.
As a result, as in experiment 5, plateau (tonic) phase contraction was significantly inhibited, as shown in the graphs in
On the basis of the results from experiments 5 and 6, a comparison was performed of the MeCN (acetonitrile) fraction, which most significantly inhibited contraction among the three fractions, at concentrations of 100 μg/mL, 300 μg/mL, and 1 mg/mL.
As shown in
On the basis of an in vitro assay using an organ bath for evaluating contractile/relaxant action similar to that described above, akamoku extracts (95% ethanol extract, 50% ethanol extract, and aqueous extract, respectively) were added to the organ bath tank in concentrations of 100 μg/mL, 300 μg/mL, and 1,000 μg/mL, and, after 30 minutes, 80 mM KCl, a contraction inducer, was added to induce contraction. Subsequently, the tension (contractile force) of slices of rat bladder smooth muscle was measured, and contraction-inhibiting effects were evaluated by respective ethanol concentration and extract concentration to investigate the optimal concentration at which contraction was significantly suppressed.
As shown in
As shown in
This experiment investigates the action of other seaweed extracts upon overactive bladder through in vitro organ bath assays.
Along with akamoku, the seaweeds that were tested are aosa and aonori, which are green algae; kombu, arame, kajime, wakame, mekabu, hijiki, and mozuku, which are brown algae; and tengusa, dulse, and iwanori (susabinori; Neopyropia yezoensis, asakusanori; Neopyropia tenera), which are red algae. 1 mg/mL extracts of the various seaweeds in 95% ethanol or water were used. 80 mM KCl, a contraction inducer, was also added to induce contraction. The tension (contractile force) of slices of rat bladder smooth muscle was then measured to evaluate contraction inhibition effects.
As shown in
As shown in
From this, it can be seen that the 95% ethanol extracts of all 12 seaweed types significantly inhibited contraction.
Next, the effects upon overactive bladder of each of various fatty acids (EPA, arachidonic acid, stearidonic acid, α-linolenic acid) present in akamoku was investigated via in vitro assay using an organ bath. Specifically, each individual component was analyzed to test whether the component had greater effects upon overactive bladder.
The concentration of each fatty acid was set according to the amount thereof present in a 95% ethanol extract of akamoku (Japan Food Research Laboratories; JFRL quantitative analysis). Specifically, an EPA content of 71 μg/mL, an arachidonic acid content of 44 μg/mL, a stearidonic acid content of 47 μg/mL, and an α-linolenic acid content of 36 μg/mL were set. Ethanol was used as a control. As in the example described above, 80 mM KCl, a contraction inducer, was also added to induce contraction. The tension (contractile force) of slices of rat bladder smooth muscle was then measured to evaluate contraction inhibition effects.
As shown in
Next, the effects upon overactive bladder of combinations of the EPA, arachidonic acid, and α-linolenic acid, out of the fatty acids present in akamoku (EPA, arachidonic acid, stearidonic acid, α-linolenic acid), were investigated. Specifically, the additive/synergistic effects of these substances were investigated using different combinations.
The concentration of each fatty acid was set according to the amount thereof present in a 95% ethanol extract of akamoku (JFRL quantitative analysis). A combination of EPA, arachidonic acid, and α-linolenic acid, a combination of EPA and arachidonic acid, and a combination of EPA and α-linolenic acid were compared. Unadulterated EPA was used as a control. As in the examples described above, 80 mM KCl, a contraction inducer, was also added to induce contraction. The tension (contractile force) of slices of rat bladder smooth muscle was then measured to evaluate contraction inhibition effects.
As seen in
This experiment was a repeat of experiment 10 described above with adjusted concentrations of EPA and stearidonic acid, which, among the fatty acids present in akamoku (EPA, arachidonic acid, stearidonic acid, and α-linolenic acid), yielded significant inhibition in that experiment.
The maximum concentration of each fatty acid was set according to the amount thereof present in a 95% ethanol extract of akamoku (JFRL quantitative analysis). Concentrations of 7.1 μg/mL EPA, 21.3 μg/mL EPA, 71 μg/mL EPA, 4.7 μg/mL stearidonic acid, 14.1 μg/mL stearidonic acid, and 47 μg/mL stearidonic acid were set. As in the examples described above, 80 mM KCl, a contraction inducer, was also added to induce contraction. The tension (contractile force) of slices of rat bladder smooth muscle was then measured to evaluate contraction inhibition effects.
As seen in
In this experiment, 0.1% acetic acid diluted with normal saline was directly injected into the bladders of urethane-anesthetized rats to induce bladder hypersensitivity and create model rats having symptoms of acute (or chronic) pollakiuria, and in vivo assays were performed using these acetic-acid-induced pollakiuria model rats. As seen in the schematic illustration in
Voiding function before and after akamoku extract administration (single dose) was compared.
The administration samples were:
control (vehicle)=0.5% methyl cellulose (MC); and
akamoku extract=50 mg/mL MC solution of akamoku extract.
Cystometry parameters were as follows.
In
In
Using the model rats with symptoms of acute (chronic) pollakiuria created in experiment 13, a 50 mg/mL MC solution of 50% ethanol extract of akamoku was orally administered to the model rats (n=7). In the graphs in
In
Using the model rats with symptoms of acute (chronic) pollakiuria created in experiment 13, a 50 mg/mL aqueous solution of aqueous akamoku extract was orally administered to the model rats. In the graphs in
In
The ethanol extracts of akamoku described above showed efficacy in organ bath assays and cystometric testing; the mechanism of action is hypothesized to be inhibition of bladder smooth muscle contraction mediated by muscarinic receptors present on the cells that make up bladder smooth muscle, or by membrane-depolarizing properties.
By contrast, the aqueous akamoku extract demonstrated no effects in an organ bath assay (
In other words, the aqueous akamoku extract is hypothesized to have demonstrated improvement of pollakiuria in in vivo cystometric testing through a different mechanism of action than that of the ethanol extract.
There is also the effect that water or hot water extracts can generally be produced more cheaply and easily than ethanol extract. It is hypothesized that effects will be stronger in hot water (approx. 70° C. to 90° C.) than in (unheated) water.
Using the model rats with symptoms of acute (chronic) pollakiuria created in experiment 13, 0.5 mg/kg akamoku-derived fucoxanthin Fx (MC solution) was orally administered to the model rats. The 0.5 mg/kg of akamoku-derived fucoxanthin Fx (MC solution) is equivalent to 50 mg/kg of 95% ethanol extract of akamoku. In the graphs in
As seen in
[Experiment 17] In Vivo Assay (50 mg/kg 95% Ethanol Extract of Akamoku) Using CYP-Induced Pollakiuria (Cystitis) Model Rats.
Cyclophosphamide (CYP) was intraperitoneally injected to create CYP-induced pollakiuria (cystitis) model rats, which received 25 mg/kg of orally administered MC solution of 95% ethanol extract of akamoku twice daily (50 mg/kg/day of MC solution of 95% ethanol extract of akamoku). In the graphs in
In
As discussed above, an in vitro assay was performed using high-performance liquid chromatography (HPLC) to observe the inhibitory action of akamoku extract upon 5α-reductase, which convert the prostate-enlarging male hormone testosterone to dihydrotestosterone.
Akamoku extract concentrations of 10, 5, 2.5, 1.25, 0.63, and 0.32 mg/ml, which were extracted with 100% ethanol solution, had high inhibition rates. In particular, the 0.32 mg/ml akamoku extract concentration yielding about 18% 5α-reductase inhibition, the 0.63 mg/ml akamoku extract concentration yielding about 39% 5α-reductase inhibition, the 1.25 mg/ml akamoku extract concentration yielding about 61% 5α-reductase inhibition, the 2.5 mg/ml akamoku extract concentration yielding about 78% 5α-reductase inhibition, the 5.0 mg/ml akamoku extract concentration yielding about 91% 5α-reductase inhibition, and the 10.0 mg/ml akamoku extract concentration yielding about 96% 5α-reductase inhibition, showing that inhibition rate increased as akamoku extract concentration increased.
Next, as described above, an in vitro assay was performed in order to observe akamoku extract inhibitory action upon AR binding, which causes dihydrotestosterone converted by 5α-reductase to effect additional prostate cellular proliferation and hyperplasia.
The AR-EcoScreen Assay system developed by Otsuka Pharmaceutical Factory in order to evaluate AR-mediated antagonistic action was used. Dihydrotestosterone (DHT) that has been converted from testosterone emits chemiluminescence upon binding to AR; thus, a reduction in fluorescent intensity when DHT and a test substance are added in tandem suggests the presence of AR binding inhibitory action. Luciferase activity in a 95% ethanol extract of akamoku containing 0.2 nM DHT and 0.1% DMSO was investigated.
An in vitro assay was performed to observe cellular proliferation suppressant action in human prostate cancer LNCaP.FGC cells. Specifically, the wells of a 96-well plate were inoculated with human prostate cancer LNCaP.FGC cells to a volume of 1×104 cells/well·100 μL using RPMI 1640 medium containing 10% fetal bovine serum (FBS) and the cells were cultured for 24 hours, after which the medium was replaced with 1% FBS-containing RPMI 1640 containing 0.1 nM, 0.5 nM, 1 nM, 5 nM, 10 nM, 50 nM, and 100 nM dihydrotestosterone (DHT), and containing mixtures of each of these amounts of DHT with 12.5 μg/mL of 95% ethanol extract of akamoku, and culturing was performed for three days, after which the absorbance (measurement wavelength; 450 nm, calibration wavelength; 630 nm) of the culture in the plate in these various conditions was measured using a plate reader.
As shown in the graph in
An experiment for in vivo drug efficacy in a rat benign prostatic hyperplasia model was performed using model rats in states of benign prostatic hyperplasia. The model rats received 60 mg/kg/day of 95% ethanol extract of akamoku. For the control, the model rats received a 0.5% methyl cellulose solution. This regimen was continued for 28 days, after which the prostates were removed from the rats and measured.
While the foregoing has been a description of an embodiment of the present invention, the present invention is not limited thereto, and various modifications may be made thereto to the extent that they do not depart from the gist of the invention.
While akamoku extract is used in the functional supplement of the embodiment described above, a seaweed other than akamoku may be used, as it has been confirmed that any seaweed extract that is abundant and easily utilized will exhibit effects comparable of those of akamoku, arame, hijiki, mozuku, and tengusa are particularly well-suited in terms of availability and abundance.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2020/031358 | 8/19/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62889106 | Aug 2019 | US |
