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 an opioid receptor.

Description

Technical Field

[0001] The present invention relates to a composition comprising Chlamydomonas reinhardtii or an extract of Chlamydomonas reinhardtii. Background Art

[0002] Drug development is ongoing to ensure healthy lives and improve well-being for all people of all ages.

[0003] Patent Document 1 describes a drug that specifically binds to opioid receptors.

[0004] On the other hand, Patent Document 2 describes the Honda DREAMO strain (trusted number FERM BP-22306) as an example of Chlamydomonas reinhardtii.

[0005] [Prior Art Literature]

[0006] (Patent Document)

[0007] Patent Document 1: Japanese Patent Application No. 2017-506505

[0008] Patent Document 2: International Publication No. 2017 / 217116 Summary of the Invention

[0009] [Problems to be solved by the invention]

[0010] However, it is not known that Chlamydomonas reinhardtii or extracts of Chlamydomonas reinhardtii function as agonists of opioid receptors.

[0011] An object of the present invention is to provide a composition comprising Chlamydomonas reinhardtii or an extract of Chlamydomonas reinhardtii that functions as an opioid receptor agonist as an active ingredient.

[0012] [Technical means to solve the problem]

[0013] (1) A composition comprising Chlamydomonas reinhardtii or an extract of Chlamydomonas reinhardtii, wherein the Chlamydomonas reinhardtii or the extract of Chlamydomonas reinhardtii contains an opioid receptor agonist.

[0014] (2) The composition according to (1), wherein the Chlamydomonas reinhardtii is a Honda DREAMO strain (trusted number FERMBP-22306).

[0015] (3) The composition according to (1) or (2), which is an emetic, an antiperistaltic drug, or a therapeutic drug for diarrheal diseases.

[0016] (4) The composition according to any one of (1) to (3), wherein the opioid receptor is a μ1 opioid receptor.

[0017] (5) The composition according to any one of (1) to (4), wherein the Chlamydomonas reinhardtii is a freshwater alga.

[0018] (Effects of the Invention)

[0019] According to the present invention, there is provided a composition comprising Chlamydomonas reinhardtii or an extract of Chlamydomonas reinhardtii functioning as an opioid receptor agonist as an active ingredient. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] none DETAILED DESCRIPTION

[0021] Hereinafter, embodiments of the present invention will be described.

[0022] The composition of this embodiment includes Chlamydomonas reinhardtii or an extract of Chlamydomonas reinhardtii, and Chlamydomonas reinhardtii or the extract of Chlamydomonas reinhardtii includes an opioid receptor agonist.

[0023] Chlamydomonas reinhardtii is not particularly limited, and examples thereof include UTEX 90 strain and Honda DREAMO strain (trusted number FERM BP-22306). UTEX 90 strain is the source strain of Honda DREAMO strain.

[0024] Chlamydomonas reinhardtii is preferably a freshwater algae, so that it can be cultivated in large quantities on land.

[0025] Examples of opioid receptors include δ opioid receptors (δ1 opioid receptor and δ2 opioid receptor), κ opioid receptors (κ1 opioid receptor, κ2 opioid receptor, and κ3 opioid receptor), and μ opioid receptors (μ1 opioid receptor and μ2 opioid receptor). Among these, μ opioid receptors are preferred, and μ1 opioid receptors are more preferred.

[0026] Since the composition of this embodiment contains Chlamydomonas reinhardtii or an extract thereof that functions as an opioid receptor agonist as an active ingredient, it can be used as a drug for emetics, motility inhibitors, and therapeutics for diarrheal diseases. Examples of diarrheal diseases include acute diarrheal diseases.

[0027] The drug can be administered orally (for internal use) or parenterally (for external use, injection).

[0028] The dosage form of the drug is not particularly limited, and examples thereof include solid preparations such as tablets, granules, powders, and capsules; liquid preparations such as solutions, suspensions, and emulsions; and lyophilized preparations.

[0029] The drug can be produced using known methods.

[0030] The composition of this embodiment can also be used in, for example, cosmeceuticals, cosmetics, and foods.

[0031] The composition of this embodiment is preferably used for humans, but can also be used for animals other than humans.

[0032] The method for culturing Chlamydomonas reinhardtii is not particularly limited, and examples thereof include static culture, shaking culture, submerged culture, and aeration culture.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] The method for recovering the cultured Chlamydomonas reinhardtii is not particularly limited, and examples thereof include centrifugation and filtration.

[0041] Furthermore, the recovered Chlamydomonas reinhardtii may be dried as needed.

[0042] The method for drying Chlamydomonas reinhardtii is not particularly limited, and examples thereof include freeze drying, spray drying, warm air drying, and sun drying.

[0043] The extraction method for extracting Chlamydomonas reinhardtii is not particularly limited, and examples thereof include ultrasonic extraction.

[0044] The solvent used for extracting Chlamydomonas reinhardtii is not particularly limited. Examples include lower alcohols such as methanol, ethanol, propanol, and isopropanol; lower aliphatic ketones such as acetone and methyl ethyl ketone; polyols such as 1,3-butanediol, propylene glycol, and glycerol; and hydrophilic organic solvents such as dimethyl sulfoxide (DMSO). Among these, DMSO is preferred.

[0045] The method for recovering the Chlamydomonas reinhardtii extract is not particularly limited, and examples thereof include centrifugation and filtration.

[0046] Furthermore, the recovered Chlamydomonas reinhardtii extract may be dried as needed.

[0047] The method for drying the Chlamydomonas reinhardtii extract is not particularly limited, and examples thereof include freeze-drying.

[0048] Example

[0049] Hereinafter, examples of the present invention will be described, but the present invention is not limited to the examples.

[0050] (Example 1)

[0051] [Cultivation of Chlamydomonas reinhardtii]

[0052] Chlamydomonas reinhardtii strain Honda DREAMO was statically cultured using 2 L of TAP medium under the following culture conditions for five days (seed culture).

[0053] Temperature: 25℃

[0054] Light-dark cycle: 12 hours light period, 12 hours dark period

[0055] Photosynthetically effective light quantum flux density during the bright period: 60-80 μmol / m 2 / s

[0056] Light source: White light-emitting diode (LED)

[0057] Next, 500 mL of the seed culture was centrifuged at 3000 rpm for 5 minutes, and then the culture supernatant was removed to recover the seed-cultured Honda DREAMO strain.

[0058] Next, the recovered HondaDREAMO strain was statically cultured for 7 days using 2 L of TAP medium under the same culture conditions as for the seed culture (preculture).

[0059] Next, the pre-cultured HondaDREAMO strain was aerated in 5 L of urea medium under the same culture conditions as the seed culture for 7 days (main culture). The aeration conditions were as follows.

[0060] Gas ventilation per unit volume: 0.15 VVM

[0061] Carbon dioxide concentration in gas: 5% by volume

[0062] Next, 5 L of the main culture was centrifuged at 6000 rpm for 10 minutes, and the culture supernatant was removed to recover the main cultured Honda DREAMO strain.

[0063] [extract]

[0064] About 20 mL of the recovered 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.

[0065] Next, the mixture of the extract and the residue was centrifuged at 3000 rpm for 5 minutes, and then the residue was removed to recover about 100 mL of the extract.

[0066] Next, the extract was freeze-dried to obtain 490.74 mg of an extract of the Honda DREAMO strain.

[0067] [Preparation of specimens]

[0068] 490 mg of the Honda DREAMO strain extract was dissolved in 4.9 mL of DMSO using an ultrasonic homogenizer to obtain a 100 mg / mL DMSO solution of the Honda DREAMO strain extract.

[0069] Next, the DMSO solution of the Honda DREAMO strain extract was centrifuged at 15,000 rpm for 5 seconds, and the supernatant was recovered to obtain a sample.

[0070] Next, the following two tests were performed to determine the concentration of the sample used in the assay (in vitro functional evaluation test).

[0071] [Cell proliferation inhibition test]

[0072] A dose-dependent study was conducted by adding the sample to HEK293 cells (a human embryonic kidney cell line) cultured in D-MEM (high glucose) + 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin. The sample was added at 13 concentrations (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, and 500 μg / mL). Twenty-four hours after sample addition, the medium was replaced, and the cells were stained with WST-8 for four hours. The optical density (OD) at a wavelength of 450 nm was then measured using a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices). This experiment was conducted with n = 3 participants.

[0073] As a result, no decrease in optical density was observed at any sample concentration, and therefore it was determined that there was no influence due to concentration.

[0074] [Effect of specimen on optical density]

[0075] Specimens were added to D-MEM (High Glucose) + 10% FBS + 1% Penicillin-Streptomycin medium at 13 concentrations (0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, and 500 μg / mL) 24 hours later. The medium was replaced and WST-8 staining was performed for 4 hours. Optical density at a wavelength of 450 nm was then measured using a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices). This experiment was performed with n = 3 participants.

[0076] The results showed an increase in optical density at sample concentrations of 250 μg / mL and 500 μg / mL. Therefore, it was found that adding sample at concentrations of 250 μg / mL and 500 μg / mL affected the optical density regardless of the cells. On the other hand, adding sample at concentrations of 100 μg / mL or less did not affect the optical density.

[0077] Based on the above results, the maximum concentration of 100 μg / mL, at which no inhibition of cell growth or influence of the sample on optical density was observed, was set as the sample concentration used in the analysis.

[0078] [analyze]

[0079] Analysis of samples targeting the μ1 opioid receptor was performed at a concentration of 100 μg / mL. The number of replicates was set to two. The analysis was outsourced to Eurofins Pharma Discovery Services, and two analytical systems were used to exclude suspected positives. Target molecules with an activity rate of 50% or greater were considered to have agonist activity.

[0080] Table 1 shows the analysis results of the samples.

[0081] [Table 1]

[0082] target molecule effect Working rate [%] The efficacy of drugs targeting target molecules <![CDATA[μ1 opioid receptor]]> agonists 73 Emetics, peristaltic inhibitors, and drugs for treating diarrhea

[0083] As shown in Table 1, the extract of the Honda DREAMO strain contained in the specimen contains an agonist of the μ1 opioid receptor, that is, it functions as an agonist of the μ1 opioid 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 an agonist of an opioid receptor.

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

3. The composition according to claim 1 or 2, which is an emetic, an antiperistaltic drug, or a drug for treating diarrhea.

4. The composition according to claim 1 or 2, wherein The aforementioned opioid receptor is a μ1 opioid receptor.

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