WO2024181024A1 - Composition - Google Patents

Abstract

Provided is a composition containing Chlamydomonas reinhardtii or an extract of Chlamydomonas reinhardtii, wherein the Chlamydomonas reinhardtii or the extract of the Chlamydomonas reinhardtii contains an agonist of a 5-hydroxytryptamine receptor.

Description

Composition

The present invention relates to a composition containing the green alga Chlamydomonas or an extract of the green alga Chlamydomonas.

Medicines are developed to ensure healthy lives and promote well-being for all people at all ages.

Patent Document 1 describes a light-activated cation channel (LACC) protein that includes a channelrhodopsin-2 (ChR2) protein or a portion of that protein. Here, the ChR2 protein is a rhodopsin derived from the unicellular green alga Chlamydomonas reinhardtii.

On the other hand, Patent Document 2 describes the Honda DREAMO strain (accession number FERM BP-22306) as an example of the green alga Chlamydomonas reinhardtii.

Special Publication No. 2009-502140 International Publication No. 2017/217116

However, it is not known that the green alga Chlamydomonas reinhardtii or extracts of the green alga Chlamydomonas reinhardtii function as agonists of the 5-hydroxytryptamine (serotonin) receptor.

The present invention aims to provide a composition containing as an active ingredient the green alga Chlamydomonas reinhardtii or an extract of the green alga Chlamydomonas reinhardtii, which functions as an agonist of the 5-hydroxytryptamine receptor.

(1) A composition comprising the green alga Chlamydomonas reinhardtii or an extract of the green alga Chlamydomonas reinhardtii, the green alga Chlamydomonas reinhardtii or the extract of the green alga Chlamydomonas reinhardtii containing an agonist of the 5-hydroxytryptamine receptor.

(2) The composition described in (1), in which the green alga Chlamydomonas is the Honda DREAMO strain (accession number FERM BP-22306).

(3) The composition according to (1) or (2), which is a migraine drug or a vasoconstrictor.

(4) The composition according to any one of (1) to (3), wherein the 5-hydroxytryptamine receptor is 5-hydroxytryptamine receptor _1B or 5-hydroxytryptamine receptor _1D .

(5) The composition according to any one of (1) to (4), wherein the green alga Chlamydomonas is a freshwater alga.

The present invention provides a composition containing as an active ingredient the green alga Chlamydomonas or an extract of the green alga Chlamydomonas that functions as an agonist of the 5-hydroxytryptamine receptor.

The following describes an embodiment of the present invention.

The composition of this embodiment contains the green algae Chlamydomonas reinhardtii or an extract of the green algae Chlamydomonas reinhardtii, and the green algae Chlamydomonas reinhardtii or an extract of the green algae Chlamydomonas reinhardtii contains an agonist of the 5-hydroxytryptamine receptor.

The green alga Chlamydomonas is not particularly limited, but examples thereof include the UTEX 90 strain and the Honda DREAMO strain (accession number FERM BP-22306). Here, the UTEX 90 strain is the parent strain of the Honda DREAMO strain.

The green alga Chlamydomonas is preferably a freshwater alga, which allows it to be cultivated in large quantities on land.

Examples of the 5-hydroxytryptamine receptor include 5-hydroxytryptamine receptor ₁ (5-hydroxytryptamine receptor _1A , 5-hydroxytryptamine receptor _1B , and 5-hydroxytryptamine receptor _1D ), 5-hydroxytryptamine receptor ₂ (5-hydroxytryptamine receptor _2A , 5-hydroxytryptamine receptor _2B , and 5-hydroxytryptamine receptor _2C ), 5-hydroxytryptamine receptor ₃ , 5-hydroxytryptamine receptor ₄ , 5-hydroxytryptamine receptor ₅ , 5-hydroxytryptamine receptor ₆ , and 5-hydroxytryptamine receptor _7. Among these, 5-hydroxytryptamine receptor ₁ is preferred, and 5-hydroxytryptamine receptor _1B and 5-hydroxytryptamine receptor _1D are more preferred.

The composition of this embodiment contains as an active ingredient the green alga Chlamydomonas reinhardtii or an extract of the green alga Chlamydomonas reinhardtii, which functions as an agonist of the 5-hydroxytryptamine receptor, and can therefore be used in medicines such as migraine medications and vasoconstrictors. Examples of diarrhea include acute diarrhea.

The dosage form of the medicine may be either oral (internal) or parenteral (external, injection).

The dosage form of the pharmaceutical product is not particularly limited, but examples include solid preparations such as tablets, granules, powders, and capsules, liquid preparations such as solutions, suspensions, and emulsions, and freeze-dried preparations.

The pharmaceutical product can be manufactured using known methods.

The composition of this embodiment may be applied to, for example, quasi-drugs, cosmetics, and foods.

The composition of this embodiment is preferably for use in humans, but may also be for use in animals other than humans.

The method for culturing the green alga Chlamydomonas is not particularly limited, and examples include static culture, shaking culture, submerged culture, and aeration culture.

The green alga Chlamydomonas can be cultivated in large quantities on land by culturing it outdoors. Methods for culturing the green alga Chlamydomonas outdoors are not particularly limited, but examples include a method of culturing it in an open pond using a raceway-type bioreactor, and a method of culturing it in a flat-panel photobioreactor installed outdoors.

The medium used to culture the green alga Chlamydomonas is not particularly limited, but examples include TAP medium, urea medium, HSM medium, BG11 medium, etc.

The temperature at which the green alga Chlamydomonas is cultured is not particularly limited, but is, for example, between 2°C and 38°C.

The light-dark cycle for culturing the green alga Chlamydomonas is not particularly limited, but for example, is a 24-hour cycle with the light period being 6 hours or more and 24 hours or less.

The photosynthetically active photon flux density during the light period is not particularly limited, but is, for example, 50 μmol/m ² /s or more and 2000 μmol/m ² /s or less.

The amount of gas aeration per unit volume when culturing the green alga Chlamydomonas using the aeration culture method is not particularly limited, but is, for example, 0.001 VVM or more and 10 VVM or less. Note that VVM indicates how many times the volume of gas is aerated relative to the culture medium in one minute.

When culturing the green alga Chlamydomonas by aeration culture, it is preferable to supply carbon dioxide to the medium. The concentration of carbon dioxide in the gas to be aerated is not particularly limited, but is, for example, 0.1% by volume or more and 10% by volume or less. The source of carbon dioxide is not particularly limited, but examples include carbon dioxide cylinders, gas discharged from factories, and concentrated carbon dioxide captured from the atmosphere by DAC (Direct Air Capture).

　Methods for recovering the cultured green alga Chlamydomonas are not particularly limited, but include, for example, centrifugation, filtration, etc.

The recovered Chlamydomonas may be dried if necessary.

　Methods for drying the green alga Chlamydomonas are not particularly limited, but examples include freeze-drying, spray-drying, hot air drying, and sun drying.

The extraction method used to extract the green alga Chlamydomonas is not particularly limited, but examples include ultrasonic extraction.

The solvent used for extraction of the green alga Chlamydomonas is not particularly limited, but examples include lower alcohols such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; polyhydric alcohols such as 1,3-butylene glycol, propylene glycol, and glycerin; and hydrophilic organic solvents such as dimethyl sulfoxide (DMSO). Of these, DMSO is preferred.

The method for recovering an extract of the green alga Chlamydomonas is not particularly limited, but examples include centrifugation and filtration.

The recovered extract of the green alga Chlamydomonas may be dried if necessary.

The method for drying the extract of the green alga Chlamydomonas is not particularly limited, but examples include freeze-drying.

The following describes examples of the present invention, but the present invention is not limited to these examples.

[Example 1]
[Cultivation of the green alga Chlamydomonas]
Honda DREAMO strain of Chlamydomonas reinhardtii was statically cultured (seed culture) for 5 days in 2 L of TAP medium under the culture conditions shown below.

Temperature: 25°C
Light/dark cycle: 12 hours light, 12 hours dark Photosynthetic photon flux density during light period: 60-80 μmol/m ² /s
Light source: White LED
Next, 500 mL of the seed culture was centrifuged at 3,000 rpm for 5 minutes, and the culture supernatant was removed to recover the seed-cultured Honda DREAMO strain.

Next, the recovered Honda DREAMO strain was statically cultured (precultured) for 7 days using 2 L of TAP medium under the same culture conditions as the seed culture.

Next, the precultured Honda DREAMO strain was aerated (main culture) for 7 days using 5 L of urea medium under the same culture conditions as those for the seed culture. The aeration conditions were as follows:
Gas flow rate per unit volume: 0.15 VVM
Concentration of carbon dioxide in gas: 5% by volume

Next, 5 L of the main culture was centrifuged at 6,000 rpm for 10 minutes, the culture supernatant was removed, and the cultured Honda DREAMO strain was recovered.

[extraction]
About 20 mL of the collected Honda DREAMO strain was subjected to ultrasonic extraction with 100 mL of DMSO for 5 minutes using an ultrasonic homogenizer. The ultrasonic extraction was performed on ice.

The mixture of extract and residue was then centrifuged at 3000 rpm for 5 minutes, the residue was removed, and approximately 100 mL of extract was recovered.

The extract was then freeze-dried to obtain 490.74 mg of Honda DREAMO strain extract.

[Preparation of specimen]
Using an ultrasonic homogenizer, 490 mg of the extract of Honda DREAMO strain was dissolved in 4.9 mL of DMSO to obtain a 100 mg/mL DMSO solution of the extract of Honda DREAMO strain.

Next, the DMSO solution of the Honda DREAMO strain extract was centrifuged at 15,000 rpm for 5 seconds, and the supernatant was collected to obtain the sample.

Next, in order to determine the concentration of the sample to be used in the assay (in vitro functionality evaluation test), the following two types of tests were carried out.
[Cytoinhibition test]
The sample was added at 13 concentrations (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, 500 μg / mL) to HEK293 cells (human fetal kidney cell-derived cell line) grown in D-MEM (High Glucose) + 10% FBS + 1% Penicillin-Streptomycin medium, and a dose-dependency test was performed. 24 hours after adding the sample to the cells, the medium was replaced, and the cells were stained with WST-8 for 4 hours. Next, the optical density (O.D.) at a wavelength of 450 nm was measured using a FlexStation 3 multimode microplate reader (Molecular Devices). Here, the above test was performed with n = 3.

As a result, no decrease in optical density was observed at any sample concentration, so it was determined that there was no effect due to concentration.

Effect of sample on optical density
The samples were added to D-MEM (High Glucose) + 10% FBS + 1% Penicillin-Streptomycin medium at 13 different concentrations (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, 500 μg/mL) and the medium was replaced 24 hours later, and stained with WST-8 for 4 hours. Next, the optical density at a wavelength of 450 nm was measured using a FlexStation 3 multimode microplate reader (Molecular Devices). Here, the above test was performed with n=3.

As a result, an increase in optical density was observed when the sample concentration was 250 μg/mL and 500 μg/mL. This shows that adding a sample at 250 μg/mL and 500 μg/mL has an effect on optical density, regardless of the cells. On the other hand, adding a sample at a concentration of 100 μg/mL or less had no effect on optical density.

Based on these results, the maximum concentration at which there was no evidence of inhibition of cell proliferation or an effect on the optical density of the sample, 100 μg/mL, was determined as the sample concentration in the assay.

[Assay]
Assays of samples targeting 5-hydroxytryptamine receptor _1B (5-HT _1B ) and 5-hydroxytryptamine receptor _1D (5-HT _1D ) were carried out at a sample concentration of 100 μg/mL. The assay was repeated twice. The assay was outsourced to Eurofins Pharma Discovery Services, and was carried out using two types of assay systems to eliminate false positives. Target molecules with an activation rate of 50% or more were determined to act as agonists.

Table 1 shows the assay results for the samples.

From Table 1, it can be seen that the extract of Honda DREAMO strain contained in the sample contains a 5-HT _1B agonist and a 5-HT _1D agonist, that is, it functions as a 5-HT _1B agonist and a 5-HT _1D agonist.

Claims (5)

A composition comprising the green alga Chlamydomonas reinhardtii or an extract of the green alga Chlamydomonas reinhardtii,
A composition, wherein the green alga Chlamydomonas or an extract of the green alga Chlamydomonas comprises an agonist of the 5-hydroxytryptamine receptor. The composition according to claim 1, wherein the green alga Chlamydomonas is the Honda DREAMO strain (accession number FERM BP-22306). The composition according to claim 1 or 2, which is a migraine medication or a vasoconstrictor. The composition according to claim 1 or 2, wherein the 5-hydroxytryptamine receptor is 5-hydroxytryptamine receptor _1B or 5-hydroxytryptamine receptor _1D . The composition according to claim 1 or 2, wherein the green algae Chlamydomonas is a freshwater algae.