Composition

Abstract

Provided is a composition comprising Chlamydomonas reinhardtii or an extract of Chlamydomonas reinhardtii, the Chlamydomonas reinhardtii or the extract of Chlamydomonas reinhardtii comprising an agonist of a progesterone receptor.

Description

TECHNICAL FIELD

[0001] The present application relates to a composition comprising Chlamydomonas reinhardtii or an extract of Chlamydomonas reinhardtii. BACKGROUND

[0002] In order to ensure healthy life for all people of all ages, to promote the well-being, the development of drugs is being carried out.

[0003] In Patent Literature 1, an isolated antibody which specifically binds to human LAG-3 is described. In addition, as a host expression system, Chlamydomonas reinhardtii which is infected with a recombinant virus expression vector containing an antibody coding sequence, or which is transformed with a recombinant plasmid expression vector is exemplified.

[0004] On the other hand, in Patent Literature 2, as an example of Chlamydomonas reinhardtii, Honda DREAMO strain (Deposition No. FERM BP-22306) is described.

[0005] [Prior Art Documents]

[0006] (Patent Literature)

[0007] Patent Literature 1: Japanese Patent Application Publication No. 2022-105535

[0008] Patent Literature 2: International Publication No. 2017 / 217116 SUMMARY

[0009] [Problems to be Solved by the Invention]

[0010] However, it is not known that Chlamydomonas reinhardtii or an extract of Chlamydomonas reinhardtii functions as an agonist of progesterone receptor.

[0011] The present application aims to provide a composition which contains Chlamydomonas reinhardtii or an extract of Chlamydomonas reinhardtii as an effective ingredient, which functions as an agonist of progesterone receptor.

[0012] [Technical Means to Solve the Problems]

[0013] (1) A composition comprising Chlamydomonas reinhardtii or an extract of Chlamydomonas reinhardtii, the aforementioned Chlamydomonas reinhardtii or the aforementioned extract of Chlamydomonas reinhardtii containing an agonist of progesterone receptor.

[0014] (2) The composition according to (1), wherein the aforementioned Chlamydomonas reinhardtii is Honda DREAMO strain (Deposition No. FERM BP-22306).

[0015] (3) The composition according to (1) or (2), which is a drug for treating menstrual disorder, a drug for treating prostatic hyperplasia, or a drug for treating prostate cancer.

[0016] (4) The composition according to any one of (1) to (3), wherein the aforementioned Chlamydomonas reinhardtii is a freshwater alga.

[0017] (EFFECTS OF THE INVENTION)

[0018] According to the present application, it is possible to provide a composition containing Chlamydomonas reinhardtii or an extract thereof, which functions as an agonist of progesterone receptor, as an effective ingredient. BRIEF DESCRIPTION OF DRAWINGS

[0019] None DETAILED DESCRIPTION

[0020] Hereinafter, the embodiments of the present application will be described.

[0021] The composition of the present embodiment contains Chlamydomonas reinhardtii or an extract thereof, which contains an agonist of progesterone receptor.

[0022] As Chlamydomonas reinhardtii, there is no particular limitation, and for example, UTEX 90 strain, Honda DREAMO strain (Depositor No. FERM BP-22306) can be listed. Here, UTEX 90 strain is a source strain of Honda DREAMO strain.

[0023] Chlamydomonas reinhardtii is preferably a freshwater alga. Thereby, it can be mass-cultured on land.

[0024] The composition of the present embodiment contains Chlamydomonas reinhardtii or an extract thereof, which functions as an agonist of progesterone receptor, as an effective ingredient, and thus, it can be applied to a drug for treating menstrual disorder, a drug for treating prostatic hyperplasia, a drug for treating prostate cancer, and the like.

[0025] The administration method of the drug can be any one of oral (internal) and non-oral (external, injection).

[0026] As the dosage form of the drug, there is no particular limitation, and for example, solid preparations such as tablets, granules, powders, and capsules, liquid preparations such as solutions, suspensions, and emulsions, and freeze-dried preparations can be listed.

[0027] The drug can be manufactured using a publicly known method.

[0028] The composition of the present embodiment can also be applied to, for example, a cosmeceutical, a cosmetic, and a food.

[0029] The composition of this embodiment is preferably used for humans, but can also be used for animals other than humans.

[0030] The method for culturing Chlamydomonas reinhardtii is not particularly limited, and examples thereof include static culture, shaking culture, submerged culture, and aeration culture.

[0031] Chlamydomonas reinhardtii can be cultivated outdoors for large-scale cultivation on land. There are no particular limitations on the method for cultivating Chlamydomonas reinhardtii outdoors, and examples include methods using a raceway-type bioreactor in an open pond format and outdoor cultivation using a flat-plate photobioreactor.

[0032] The culture medium used for culturing Chlamydomonas reinhardtii is not particularly limited, and examples thereof include TAP medium, urea medium, HSM medium, and BG11 medium.

[0033] The temperature during the culture of Chlamydomonas reinhardtii is not particularly limited, but is, for example, 2°C or higher and 38°C or lower.

[0034] The light-dark cycle during culturing Chlamydomonas reinhardtii is not particularly limited, and for example, the light period is a 24-hour cycle with a light period of 6 hours to 24 hours.

[0035] The photosynthetic effective light quantum flux density during the bright period is not particularly limited, and is, for example, 50 μmol / m 2 / s and above and 2000 μmol / m 2 / s or less.

[0036] The gas aeration rate per unit volume when culturing Chlamydomonas reinhardtii by the aeration culture method is not particularly limited, and is, for example, 0.001 VVM or more and 10 VVM or less. VVM indicates how many times the volume ratio of gas is aerated into the culture medium per minute.

[0037] When culturing Chlamydomonas reinhardtii using an aeration culture method, carbon dioxide is preferably supplied to the culture medium. The concentration of carbon dioxide in the introduced gas is not particularly limited, and for example, is 0.1% by volume or higher and 10% by volume or lower. Furthermore, the source of carbon dioxide is not particularly limited, and examples thereof include gas discharged from carbon dioxide cylinders, factories, and the like, and concentrated carbon dioxide recovered from the atmosphere using direct air capture (DAC) technology.

[0038] The method for recovering the cultured Chlamydomonas reinhardtii is not particularly limited, and examples thereof include centrifugation and filtration.

[0039] Furthermore, the recovered Chlamydomonas reinhardtii may be dried as needed.

[0040] As a method for drying Chlamydomonas reinhardtii, there is no particular limitation, and for example, freeze-drying method, spray-drying method, warm-air drying method, sun-drying method, and the like can be exemplified.

[0041] As an extraction method for extracting Chlamydomonas reinhardtii, there is no particular limitation, and for example, ultrasonic extraction method, and the like can be exemplified.

[0042] As a solvent for extracting Chlamydomonas reinhardtii, there is no particular limitation, and for example, lower alcohols such as methanol, ethanol, propanol, and isopropanol; lower aliphatic ketones such as acetone and methyl ethyl ketone; polyhydric alcohols such as 1,3-butanediol, propylene glycol, and glycerol; and hydrophilic organic solvents such as dimethyl sulfoxide (DMSO) can be exemplified. Among these, DMSO is preferred.

[0043] As a method for recovering the extract solution of Chlamydomonas reinhardtii, there is no particular limitation, and for example, centrifugal separation method, filtration method, and the like can be exemplified.

[0044] Further, the extract of Chlamydomonas reinhardtii recovered can also be dried as needed.

[0045] As a method for drying the extract of Chlamydomonas reinhardtii, there is no particular limitation, and for example, freeze-drying method, and the like can be exemplified.

[0046] Example

[0047] Hereinafter, an example of the present application will be described, but the present application is not limited to the example.

[0048] (Example 1)

[0049] [Cultivation of Chlamydomonas reinhardtii]

[0050] Using TAP medium 2 L, Honda DREAMO strain as Chlamydomonas reinhardtii was incubated for five days under the following culture conditions (seed culture).

[0051] Temperature: 25°C

[0052] Light-dark cycle: light period 12 hours, dark period 12 hours

[0053] Photosynthetically active photon flux density in light period: 60 to 80 μmol / m 2 / s

[0054] Light source: white light-emitting diode (LED)

[0055] Next, the seed culture 500 mL was centrifuged at 3000 rpm for 5 minutes, after which the culture supernatant was removed, and the Honda DREAMO strain of the seed culture was recovered.

[0056] Next, the recovered Honda DREAMO strain was stationary cultured for 7 days (pre-culture) using TAP medium 2 L under the same culture conditions as the seed culture.

[0057] Next, the pre-cultured Honda DREAMO strain was aerated for 7 days (main culture) using urea medium 5 L under the same culture conditions as the seed culture. In addition, the aeration conditions were as follows.

[0058] Gas aeration amount per unit volume: 0.15 VVM

[0059] Concentration of carbon dioxide in the gas: 5 vol%

[0060] Next, the main culture 5 L was centrifuged at 6000 rpm for 10 minutes, after which the culture supernatant was removed, and the Honda DREAMO strain of the main culture was recovered.

[0061] [Extraction]

[0062] The recovered Honda DREAMO strain was ultrasonically extracted for 5 minutes using an ultrasonic homogenizer with DMSO 100 mL. At this time, the ultrasonic extraction was performed on ice.

[0063] Next, the mixture of the extract and the residue was centrifuged at 3000 rpm for 5 minutes, after which the residue was removed, and the extract was recovered as about 100 mL.

[0064] Next, the extract was freeze-dried to obtain an extract of the Honda DREAMO strain 490.74 mg.

[0065] [Preparation of a specimen]

[0066] The extract of the Honda DREAMO strain 490 mg was dissolved in DMSO 4.9 mL using an ultrasonic homogenizer to obtain a DMSO solution of the extract of the Honda DREAMO strain at 100 mg / mL.

[0067] Next, the DMSO solution of the extract of the Honda DREAMO strain was centrifuged at 15000 rpm for 5 seconds, after which the supernatant was recovered to obtain a specimen.

[0068] Next, in order to determine the concentration of the specimen used in the assay (in vitro functional evaluation test), the following two tests were performed.

[0069] [Cell proliferation inhibition test]

[0070] A dose-dependent test was performed by adding the specimen at 13 concentrations (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, 500 μg / mL) to HEK293 cells (human embryonic kidney cell-derived cell line) cultured in D-MEM (High Glucose) + 10% FBS + 1% Penicillin-Streptomycin medium. The medium was changed 24 hours after the specimen was added to the cells, 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 Multi-Mode Microplate Reader (manufactured by Molecular Devices). Here, the above test was performed with n = 3.

[0071] As a result, in any concentration of the specimen, a decrease in the optical density was not confirmed, and it was judged that there was no effect due to the concentration.

[0072] [Effect of specimen on optical density]

[0073] A dose-dependent test was performed by adding the specimen at 13 concentrations (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, 500 μg / mL) to HEK293 cells (human embryonic kidney cell-derived cell line) cultured in D-MEM (High Glucose) + 10% FBS + 1% Penicillin-Streptomycin medium. The medium was changed 24 hours after the specimen was added to the cells, 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 Multi-Mode Microplate Reader (manufactured by Molecular Devices). Here, the above test was performed with n = 3.

[0074] As a result, in the case where the concentration of the specimen was 250 μg / mL and 500 μg / mL, an increase in optical density was confirmed. Therefore, it was found that, in the case where the specimen was added at 250 μg / mL and 500 μg / mL, the optical density was affected regardless of the cells. On the other hand, it was found that, in the case where the specimen was added at a concentration of 100 μg / mL or less, the optical density was not affected.

[0075] Based on the above results, 100 μg / mL, which is the maximum value of the concentration at which the cell proliferation inhibition and the effect of the specimen on the optical density were not confirmed, was set as the specimen concentration in the analysis.

[0076] [Analysis]

[0077] The analysis of the specimen with the progesterone receptor as the target molecule was performed at a specimen concentration of 100 μg / mL. Here, the number of repetitions of the analysis was set to 2. In addition, the analysis was entrusted to Eurofins Pharma Discovery Services, and was performed with two analysis systems in order to exclude suspected positives. Here, the target molecule with a work rate of 50% or more was judged to have an agonist effect.

[0078] The results of the analysis of the specimen are shown in Table 1.

[0079] [Table 1]

[0080] Target molecule Action Work rate [%] Efficacy of a drug targeting the target molecule Progesterone receptor Agonist 53 Menstrual disorder treatment drug, prostatic hyperplasia treatment drug, prostatic cancer treatment drug

[0081] As shown in Table 1, the extract of the Honda DREAMO strain contained in the specimen contained an agonist of the progesterone receptor, that is, functioned as an agonist of the progesterone receptor.

Claims

1. A composition comprising Chlamydomonas reinhardtii or an extract of Chlamydomonas reinhardtii, The Chlamydomonas reinhardtii or the extract of the Chlamydomonas reinhardtii contains a progesterone receptor agonist.

2. The composition according to claim 1, wherein The aforementioned Chlamydomonas reinhardtii was the Honda DREAMO strain (trusted number FERM BP-22306).

3. The composition according to claim 1 or 2, which is a drug for treating menstrual disorders, a drug for treating benign prostatic hyperplasia, or a drug for treating prostate cancer.

4. The composition according to claim 1 or 2, wherein The aforementioned Chlamydomonas reinhardtii is a freshwater algae.