Composition comprising L-tryptophan and chitosan for improving sleep disorders

Abstract

The present invention discloses a composition for improving the bioavailability of tryptophan containing chitosan as an active ingredient, and a composition for preventing, improving, and / or treating sleep disorders containing chitosan and tryptophan as active ingredients. The chitosan is chitosan powder or soluble chitosan, and the composition increases the permeability of tryptophan to the tight junction of the blood-brain barrier, thereby enhancing the sleep disorder improving effect of tryptophan.

Description

Technical Field

[0001] The present invention relates to a composition for improving the bioavailability of tryptophan containing chitosan as an active ingredient, and a composition for preventing, improving, and / or treating sleep disorders containing chitosan and tryptophan as active ingredients.

Background Art

[0002] Lack of sleep and anxiety are one of the problems that often occur in many people. Lack of sleep leads to decreased activity, increased stress, decreased productivity, psychological problems, etc. One of the bioactive compounds used to reduce such lack of sleep and improve sleep hormones is L-tryptophan (TRP).

[0003] In the brain, TRP, which is a precursor of melatonin, a sleep-inducing hormone, is one of the most promising amino acids in promoting sleep. However, during the transport of TRP to the brain, it experiences losses and has to pass through the tight-junction of the Blood-Brain Barrier (BBB), so the plant intake of TRP and its bioavailability in the brain are very different. Due to the lack of bioactive carrier molecules for TRP transport, TRP has not been able to successfully cross the tight-junction to stimulate serotonin-melatonin biosynthesis. The low bioavailability of TRP and the resulting decrease in melatonin levels induce decreased sleep, irregular sleep, and affect the quality of sleep. Therefore, due to its low bioavailability, it is actually difficult for TRP to substantially solve sleep disorders.

Disclosure of the Invention

Problems to be Solved by the Invention

[0004] Health supplements containing TRP have low bioavailability. The object of the present invention is to increase the bioavailability of TRP so that the active ingredient can improve or induce sleep in patients with sleep disorders or lack of sleep.

[0005] Another object of the present invention is to provide a composition for improving the bioavailability of tryptophan containing chitosan as an active ingredient.

[0006] Another object of the present invention is to provide a composition for preventing, improving, and / or treating sleep disorders containing chitosan and tryptophan as active ingredients.

Means for Solving the Problems

[0007] In one aspect, the present invention provides a composition for improving the bioavailability of tryptophan containing chitosan as an active ingredient.

[0008] The chitosan is chitosan powder or soluble chitosan. The chitosan powder contains LCH (large molecular weight chitosan) and SCH (small molecular weight chitosan) at a weight ratio of 0.5 to 0.7:0.3 to 0.5. The LCH is chitosan powder with an average molecular weight exceeding 50,000 Da, and the SCH is chitosan powder with an average molecular weight of less than 10,000 Da. The soluble chitosan contains LSCH (large molecular weight soluble chitosan), MSCH (medium molecular weight soluble chitosan), and SSCH (small molecular weight soluble chitosan) at a weight ratio of 0.1 to 0.3:0.5 to 0.8:0.1 to 0.3. The LSCH is soluble chitosan with an average molecular weight exceeding 50,000 Da, the MSCH is soluble chitosan with an average molecular weight of 10,000 to 50,000 Da, and the SSCH is soluble chitosan with an average molecular weight of less than 10,000 Da.

[0009] The composition is used in combination with tryptophan to enhance the effect of tryptophan in improving sleep disorders.

[0010] The composition is used in combination with tryptophan to enhance the permeability of tryptophan to the tight junction of the blood-brain barrier.

[0011] The composition is used in combination with tryptophan to reduce the expression of one or more tight junction proteins selected from the group consisting of Occludin, Claudin 5, and ZO-1.

[0012] The chitosan is used in combination with tryptophan at a weight ratio of 0.3 to 0.7:0.3 to 0.7.

[0013] The composition is used in combination with tryptophan by a method of simultaneous or sequential administration.

[0014] The composition is used in combination with tryptophan by a method of administering tryptophan within 1 hour after administration.

[0015] The composition is a pharmaceutical composition or a food composition.

[0016] In another aspect, the present invention provides a pharmaceutical composition for preventing or treating sleep disorders, containing chitosan and tryptophan as active ingredients.

[0017] The chitosan is chitosan powder or soluble chitosan. The chitosan powder contains LCH and SCH at a weight ratio of 0.5 to 0.7:0.3 to 0.5. The LCH is chitosan powder with an average molecular weight exceeding 50,000 Da, and the SCH is chitosan powder with an average molecular weight less than 10,000 Da. The soluble chitosan contains LSCH, MSCH, and SSCH at a weight ratio of 0.1 to 0.3:0.5 to 0.8:0.1 to 0.3. The LSCH is soluble chitosan with an average molecular weight exceeding 50,000 Da, the MSCH is soluble chitosan with an average molecular weight of 10,000 to 50,000 Da, and the SSCH is soluble chitosan with an average molecular weight less than 10,000 Da.

[0018] The chitosan and tryptophan are mixed at a weight ratio of 0.3 to 0.7:0.3 to 0.7.

[0019] The composition enhances the permeability of tryptophan to the tight junction of the blood-brain barrier.

[0020] In another aspect, the present invention provides a food composition for improving sleep disorders, comprising chitosan and tryptophan as active ingredients.

[0021] The chitosan is chitosan powder or soluble chitosan. The chitosan powder contains LCH and SCH in a weight ratio of 0.5 - 0.7:0.3 - 0.5. The LCH is chitosan powder with an average molecular weight exceeding 50,000 Da, and the SCH is chitosan powder with an average molecular weight less than 10,000 Da. The soluble chitosan contains LSCH, MSCH, and SSCH in a weight ratio of 0.1 - 0.3:0.5 - 0.8:0.1 - 0.3. The LSCH is soluble chitosan with an average molecular weight exceeding 50,000 Da, the MSCH is soluble chitosan with an average molecular weight of 10,000 - 50,000 Da, and the SSCH is soluble chitosan with an average molecular weight less than 10,000 Da.

[0022] The chitosan and tryptophan are mixed in a weight ratio of 0.3 - 0.7:0.3 - 0.7.

[0023] The composition enhances the permeability of tryptophan to the tight junction of the blood-brain barrier.

Advantages of the Invention

[0024] According to the present invention, a composition for improving the bioavailability of tryptophan containing chitosan as an active ingredient can be provided.

[0025] Also, according to the present invention, a composition for preventing, improving, and / or treating sleep disorders containing chitosan and tryptophan as active ingredients can be provided.

[0026] Furthermore, according to the present invention, by using chitosan as a bioactive carrier compound, the biological availability of TRP for the amplified production of serotonin and / or melatonin can be increased by the efficient passage of TRP molecules through the BBB.

[0027] The composition of the present invention increases the ability of TRP to cross the blood-brain barrier using chitosan, increases the bioavailability of TRP, increases the production of serotonin and melatonin, and is excellent in the effect of improving the quality of sleep or inducing sleep without any side effects or further chemical modifications.

Brief Description of the Drawings

[0028]

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Mode for Carrying Out the Invention

[0029] Hereinafter, the present invention will be described in detail.

[0030] In one aspect, the present invention provides a composition for improving the bioavailability of tryptophan containing chitosan as an active ingredient.

[0031] In another aspect, the present invention provides a pharmaceutical composition for preventing or treating sleep disorders containing chitosan and tryptophan as active ingredients.

[0032] In still another aspect, the present invention provides a food composition for improving sleep disorders containing chitosan and tryptophan as active ingredients.

[0033] In the present invention, an active ingredient means a component that exhibits the intended activity alone or, together with an inactive carrier or the like, can exhibit the intended activity.

[0034] In the present invention, bioavailability means the proportion of the administered drug that reaches the systemic circulation, and improving the bioavailability of tryptophan means increasing the permeability of tryptophan to the tight junction of the blood-brain barrier and increasing the amount of tryptophan reaching the brain.

[0035] In the present invention, tryptophan means L-tryptophan. Tryptophan is a precursor for the biosynthesis of serotonin, melatonin hormone, and / or neurotransmitters that have a mood-changing or sleep-inducing effect, and is an essential neutral amino acid required by the body for normal physiological processes.

[0036] In the present invention, improving sleep disorders means all actions that increase sleep-related mediator variables or increase the biosynthesis pathways of serotonin and melatonin to disclose a good sleep state.

[0037] The present invention provides an effect of inducing better sleep by increasing the bioavailability of TRP along with an increase in the biosynthesis rate of serotonin and melatonin. The composition of the present invention is suitable for people with insufficient sleep duration, low sleep quality, irregular sleep cycles with intermittent sleep-wake cycles, low sleep quality due to stress or situations, people with tryptophan deficiency, people with a tryptophan-deficient diet, insomnia, restless legs syndrome, narcolepsy, and people who are expected to face or be threatened by sleep disorders such as sleep apnea. The composition of the present invention has the effect of increasing the bioavailability of TRP and the levels of serotonin and melatonin, thereby inducing sleep or positively changing the mood.

[0038] As an embodiment, the chitosan is chitosan powder or soluble chitosan. The powder means a form in which chitosan is dehydrated on particles, and soluble means a form in which chitosan is dissolved in an appropriate solvent such as acetic acid or water with a decreased pH.

[0039] As another embodiment, the chitosan powder contains LCH (large molecular weight chitosan) and SCH (small molecular weight chitosan) in a weight ratio of 0.5 to 0.7:0.3 to 0.5, preferably 0.6 to 0.7:0.3 to 0.4, and more preferably 0.7:0.3. The LCH is chitosan powder with an average molecular weight exceeding 50,000 Da, and the SCH is chitosan powder with an average molecular weight of less than 10,000 Da.

[0040] To obtain LCH and SCH in a desired ratio in the powder form of chitosan, chitosan must be treated with HCl or an organic acid. For example, treat it at a weight ratio of HCl or organic acid:chitosan = 0.2 to 0.5:0.5 to 0.8, and the optimal mixing weight ratio is 0.3:0.7.

[0041] As an embodiment, the soluble chitosan contains LSCH (large molecular weight soluble chitosan), MSCH (medium molecular weight soluble chitosan), and SSCH (small molecular weight soluble chitosan) in a weight ratio of 0.1 to 0.3:0.5 to 0.8:0.1 to 0.3, preferably 0.1:0.6:0.3. The LSCH is soluble chitosan with an average molecular weight exceeding 50,000 Da, the MSCH is soluble chitosan with an average molecular weight of 10,000 to 50,000 Da, and the SSCH is soluble chitosan with an average molecular weight of less than 10,000 Da.

[0042] The soluble form of the chitosan can be produced by treating LSCH with a chitosan-degrading enzyme such as chitosanase. The time-controlled treatment of chitosan produces a mixture of LSCH, MSCH, and SSCH molecules.

[0043] Using high molecular weight chitosan alone is not sufficient for efficient transport of TRP into the BBB. Therefore, the present invention provides a composition that enhances the ability of TRP to cross the BBB and increases its bioavailability through chitosans of various molecular weights without side effects or toxicity.

[0044] As described above, TRP combined with chitosan powder and soluble forms in other molecular weight ranges can increase the biosynthesis of serotonin and / or melatonin that improve or stimulate sleep in the brain. The composition of the present invention is particularly useful for people with low, irregular, or intermittent sleep-wake cycles.

[0045] The chitosan powder is mixed with tryptophan powder, and the soluble chitosan is mixed with soluble tryptophan.

[0046] In one embodiment, the composition is used in combination with tryptophan to enhance the sleep disorder improving effect of tryptophan.

[0047] In one embodiment, the composition is used in combination with tryptophan to enhance the permeability of tryptophan to the tight junction of the blood-brain barrier.

[0048] In one embodiment, the composition is used in combination with tryptophan to reduce the expression of one or more tight junction proteins selected from the group consisting of occludin, claudin 5, and ZO-1.

[0049] In one embodiment, the chitosan is used in combination with tryptophan at a weight ratio of 0.3 to 0.7:0.3 to 0.7, preferably 0.6 to 0.7:0.3 to 0.4, more preferably 0.6:0.4.

[0050] In one embodiment, the composition is used in combination with tryptophan by a method of simultaneous or sequential administration.

[0051] In one embodiment, the composition is used in combination with tryptophan by a method of administering tryptophan within 1 hour after administration.

[0052] Tryptophan hydroxylase (TPH) is a tetrahydrobiopterin-dependent monooxygenase and is the rate-limiting enzyme in serotonin biosynthesis. This enzyme is located in specific neurons and secretory cells of peripheral organs including the pineal gland and gastrointestinal tract. An intracellular calcium mobilizing reagent stimulates the production of tryptophan hydroxylase (TPH) (Hasegawa et al., 1996). When the conversion time of TPH decreases, (faster conversion) tryptophan is converted to serotonin in a shorter time. An intracellular increase in Ca 2+ concentration simultaneously triggers related signal transduction cascades to increase TPH and vesicle secretion.

[0053] Chitosan activates the ARHGAP6 protein and is presumed to be an intracellular calcium mobilizing substance that activates signal transduction and affects TPH production and conversion time. This effect exhibits a synergistic effect when chitosan and tryptophan are both administered. It is increased by the fast enzymatic action of TPH to improve serotonin biosynthesis and provide a synergistic effect. 2+ Therefore, in order to increase the bioavailability of TRP and increase the production of serotonin and melatonin, the chitosan shows excellent effects when used in combination with tryptophan by a method of simultaneous administration. For example, chitosan is used as a carrier or capsule.

[0054]

[0055] In one embodiment, in the pharmaceutical composition for preventing or treating sleep disorders and / or the food composition for improving sleep disorders, chitosan has a form in which amino acids are encapsulated.​

[0056] In one embodiment, the composition for improving the bioavailability of tryptophan is a pharmaceutical composition.

[0057] In one embodiment, the composition for improving the bioavailability of tryptophan is a food composition.

[0058] In one embodiment, the food composition is a health functional food composition.

[0059] In one embodiment, the composition for improving the bioavailability of tryptophan is an adjuvant composition for a composition containing tryptophan.

[0060] In one embodiment, the pharmaceutical composition may further contain pharmaceutical adjuvants such as preservatives, stabilizers, hydrating agents, or emulsification promoters, salts for osmotic pressure adjustment, and / or buffer systems, and other therapeutically useful substances, and can be formulated into various oral dosage forms or parenteral dosage forms by conventional methods.

[0061] Examples of the oral dosage forms include tablets, pills, hard and soft capsules, solutions, suspensions, emulsions, syrups, powders, powders, fine granules, granules, pellets, etc. These dosage forms contain surfactants, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine), lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and polyethylene glycol). Tablets also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidine, and optionally contain pharmaceutical additives such as disintegrants, absorbents, coloring agents, flavoring agents, and sweetening agents such as starch, agar, alginic acid, or its sodium salt. The tablets are manufactured by conventional mixing, granulating, or coating methods.

[0062] In addition, the parenteral administration form is a transdermal administration dosage form, for example, dosage forms such as injections, infusions, ointments, lotions, gels, creams, sprays, suspensions, emulsions, patches, etc.

[0063] In one embodiment, the pharmaceutical composition can be administered orally, parenterally, rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, subcutaneously, etc. by the intended method, and the effective component of the pharmaceutical composition varies depending on the age, sex, weight, pathological condition, and its severity of the subject to be administered, the administration route, or the judgment of the prescriber. Determination of the appropriate dosage based on such factors is within the level of those skilled in the art, and the daily dosage volume is, for example, 0.001 mg / kg / day to 100 mg / kg / day, more specifically, 0.5 mg / kg / day to 50 mg / kg / day.

[0064] In one embodiment, the food composition is in a liquid or solid dosage form, for example, various foods, drinks, gums, teas, vitamin complexes, health supplements, etc., and is used in the form of powders, granules, tablets, capsules, or drinks. The food composition of each dosage form can be appropriately formulated by those skilled in the art by selecting the commonly used components in the field according to the dosage form or the purpose of use without difficulty.

[0065] In one embodiment, there are no special restrictions on the liquid components contained in the food composition, and various flavoring agents or natural carbohydrates can be included as additional components like ordinary drinks. Examples of the natural carbohydrates include monosaccharides, disaccharides such as glucose and fructose, polysaccharides such as maltose and sucrose, ordinary sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents (such as thaumatin and stevia extract) and synthetic flavoring agents (such as saccharin and aspartame) can be advantageously used. The proportion of the natural carbohydrates is generally about 0.01 to 0.04 parts by weight, preferably 0.02 to 0.03 parts by weight per 100 parts by weight of the composition disclosed in the present invention.

[0066] In one embodiment, the food composition includes, on one hand, various nutritional agents, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, and fillers (such as cheese and chocolate), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. On the other hand, it includes pulp for the production of natural fruit juices and vegetable beverages. The above components can be used independently or in combination. The proportions of the additives vary, but generally, they are selected in the range of about 0.001 to 20 parts by weight per 100 parts by weight of the composition disclosed in the present invention.

[0067] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are for illustrative purposes only, and it is self-evident to those with ordinary knowledge in the industry that the scope of the present invention is not construed as being limited by these examples.

[0068] <Example 1. Chitosan Production> Powder-shaped chitosan Powder-shaped chitosan contains LCH (average molecular weight range exceeding 50,000 Da) and SCH (average molecular weight range less than 10,000 Da). The powder-shaped chitosan preparation contains LCH:SCH at a weight ratio of 0.5 - 0.7:0.3 - 0.5, and the best results were obtained when used at a weight ratio of 0.7:0.3.

[0069] Soluble-shaped chitosan The soluble form of chitosan contains LSCH (average molecular weight range exceeding 50,000 Da), MSCH (average molecular weight range of 10,000 - 50,000 Da), and SSCH (average molecular weight range less than 10,000 Da). Soluble chitosan with a weight ratio of LSCH:MSCH:SSCH of 0.1 - 0.3:0.5 - 0.8:0.1 - 0.3 was used. The best mixing weight ratio was 0.1:0.6:0.3.

[0070] Soluble chitosan containing other molecular weight particles (LSCH, MSCH, and SSCH) was produced by enzymatic hydrolysis according to the protocol described by the known Chang et al. (2015).

[0071] Cellulase or chitosanase, or other chitosan-degrading enzymes are used.

[0072] 1% chitosan (300 kDa) in a 0.4% acetic acid solution was prepared.

[0073] The enzyme was added to the chitosan solution efficiently and stored at 55 °C for degradation.

[0074] The supernatant of the hydrolyzate was collected at each time point, specifically at 1, 3, 6, 9, 18, and 24 hours respectively.

[0075] The collected supernatant was filtered using ultrafiltration membrane filters with molecular weight cut-off values of 50 kDa (MWCO50), 30 kDa (MWCO30), 10 kDa (MWCO10), and 3 kDa (MWCO3).

[0076] For LSCH, the desired chitosan arrangement was prepared using MWCO50, for MSCH using MWCO50 and MWCO10, and for LSCH using MWCO10 and MWCO3.

[0077] <Example 2. Production of Chitosan Nanoparticles> Chitosan nanoparticles were produced by ionic gelation using tripolyphosphate (TPP) cross-linking according to the method described by the known Calvo and Remunan-Lopez, 1997.

[0078] 10 mg of chitosan was dispersed in 1 L of distilled water and solubilized with acetic acid in molar excess with respect to the amino groups of the chitosan used.

[0079] A 5 mg / mL concentrated TPP stock solution was prepared with secondary distilled water.

[0080] All solutions were filter sterilized using membrane filters with pore sizes of 0.45 μm (chitosan) and 0.22 μm (TPP), respectively.

[0081] The TPP solution was added dropwise to the chitosan solution at room temperature (25 °C) under magnetic stirring (75 rpm) to obtain chitosan nanoparticles.

[0082] <Example 3. Preparation of TRP (L-Tryptophan) Stock Solution> A 100x stock solution of TRP was prepared.

[0083] The solvent used was physiological saline.

[0084] 5 g of TRP powder (100%) was dissolved in 1 L of physiological saline at pH 7.4 at room temperature (25 °C).

[0085] The final concentration was 5 g / L.

[0086] Thereafter, the solution was filter sterilized.

[0087] The sterilized solution was diluted 1x using sterilized physiological saline.

[0088] <Example 4. Preparation of Chitosan-TRP Mixture> The powdered form of chitosan containing LCH and SCH was mixed with the powdered form of TRP, and the soluble form of chitosan containing LSCH, MSCH, and SSCH was mixed with the soluble form of TRP in physiological saline. Chitosan and TRP were mixed at a weight ratio of 0.3 - 0.7:0.3 - 0.7, and the best results were obtained when chitosan:TRP was combined at a weight ratio of 0.6:0.4.

[0089] Powder-shaped chitosan and TRP mixture Chitosan and L-tryptophan (TRP) with different molecular weights required in the market were obtained from Shaanxi Pioneer Biotech Co., Ltd, China.

[0090] As an example, in the case of 100 g of a powder composition, 40 g of chitosan powder, 40 g of TRP powder, and 20 g of any organic acid such as citric acid or maleic acid (in powder form) were placed in a sterile container or plastic bag and mixed using a mechanical bag mixer or manually.

[0091] Proper mixing of the product can be confirmed by uniformly colored powder granules within the mixture.

[0092] All procedures were carried out aseptically to avoid contamination problems.

[0093] Liquid-form chitosan and TRP mixture

[0094] As an example, to prepare 100 mL of a soluble form, 60 mL of a chitosan soluble form, or nanoparticles were gently mixed with 40 mL of a TRP solution at room temperature manually using a glass rod or using a mechanical stirrer at low rpm. The mixture was initially produced under acidic conditions and after being completely dissolved, the pH was adjusted to neutral once.

[0095] Three other molecular weights (LSCH, MSCH, and SSCH) of chitosan in 60 mL of soluble form were each mixed with 20 mL.

[0096] <Experimental Example 1. Evaluation of the effect of chitosan treatment period on the BBB (Blood - Brain Barrier) integrity of hBEC - 5i monolayers> To confirm the treatment time for chitosan molecules to increase the permeability of tight junctions of the BBB, the following experiments were conducted. The main tight junction proteins (occludin, claudin - 5, ZO - 1) were treated with chitosan molecules for 1 hour, 18 hours, and 24 hours, and the mRNA transcription levels were measured.

[0097] Experiments were conducted using hBEC-5i (Human Brain Micro-Endothelial Cells) obtained from ATCC. The chitosan treatment group and the control group were divided into three flasks of serum-free cell culture medium (DMEM / F-12 50:50 + ECGS + 1% PSA). Commercial chitosan powder was dissolved in 0.1% acetic acid (AA) solution and vortexed overnight to produce a chitosan solution (1 mg / mL). The 1 mg / mL chitosan solution was combined with 4 mL of serum-free cell culture medium and the pH was adjusted to produce 200 μg of chitosan. Before introduction into the cells, all final solutions were syringe-filtered using a 0.22 μm filter. Before adding the treatment solution, the cell monolayer was pretreated with serum-free medium. After washing twice with 1xDPBS and pretreatment, the treatment solution was applied to three flasks of 80%-90% confluent hBEC-5i monolayer. Thereafter, the cells were incubated at 37 °C and 5% CO2 for 48 hours. Each flask was sampled at 1, 18, and 24 hours compared to the control group.

[0098] After the treatment, single washing was performed using DPBS, and all cells were collected. The total RNA content was extracted by the TRIzol method according to the manufacturer's protocol. The abundance of tight junction protein (occludin, claudin 5, and ZO-1) mRNA was quantified using one-step reverse transcription real-time quantitative PCR using the QuantiTect SYBR Green RT-PCR Kit (Qiagen, Germany) on a Rotor-Gene Q Real-Time system (Qiagen, Germany). Gene primers were those used by the known Verma et al., 2009 (claudin 5 & occludin), and Jiang et al., 2017 (ZO-1 & GAPDH). Each reaction consisted of 50 ng of total RNA, 0.5 pmol of each primer, 5 μL of the one-step reaction mixture, and 0.1 μL of Quantiscript reverse transcriptase, and all were placed in a 10 μL reaction system to carry out the reaction. The reaction was carried out for 40 cycles at 50 °C for 30 minutes, 95 °C for 15 minutes, and 94 °C for 15 seconds, 58 °C for 30 seconds. Each sample analysis was performed 3 times to determine the average threshold cycle (Ct) value. The target gene expression was normalized with the GAPDH gene, and the relative amount of target gene mRNA was expressed as 2 -△△ct as described (Voge et al., 2004; Lagaly et al., 2008; Grado-Ahuir et al., 2011). Non-template control groups and non-reverse transcriptase control groups were used in each PCR run to confirm the absence of genomic DNA contamination in the samples and mastermixes.

[0099] The mRNA transcription levels of the three major tight junction proteins, occludin, claudin-5, and ZO-1, were quantified using qPCR. The occludin, claudin-5, and ZO-1 mRNA levels were found to be 61%, 74%, and 51% lower, respectively, than the corresponding control group levels 1 hour after treatment (see Figures 1 - 3). However, the occludin expression increased by 27% compared to the control group after 18 hours of incubation and further increased by 10% after 24 hours. In contrast, the ZO-1 expression returned to normal levels after 18 hours and showed a tendency to decrease as observed in the experimental group incubated for 1 hour. In the case of claudin-5, the relative expression level was low and decreased after chitosan treatment. Also, the decrease in mRNA levels was stably maintained even 1 hour after treatment. This confirmed that the tight junction opening effect of the BBB was the most excellent 1 hour after chitosan treatment.

[0100] <Experimental Example 2. Evaluation of the expression program file of three gene mRNAs from the tight junctions (TJ) of brain endothelial cells> The following experiments were conducted to confirm whether chitosan (Chit) and the combination of chitosan and tryptophan (ChitTRP) affect the TJ protein expression level in brain microvascular endothelial cells. If the TJ protein expression level decreases or changes from the normal level, it means that chitosan and ChitTRP molecules enter the TJ and cause changes in the expression of these proteins.

[0101] Experiments were conducted using hBEC-5i (Human Brain Micro-Endothelial Cells) obtained from ATCC. The experiments consisted of three treatment groups (Chitosan (Chit), Tryptophan (TRP), Chitosan-Tryptophan (ChitTRP)), and one control group (serum-free cell culture medium (DMEM / F-12 50:50 + ECGS + 1% PSA)). Substances were treated in the serum-free cell culture medium, and the pH was adjusted if necessary. As shown in Figure 4, each treatment solution was prepared by dissolving 1 mg / mL of chitosan solution in 0.1% acetic acid (AA), 1 mg / mL of TRP solution directly dissolved in the complete medium, and a ChitTRP solution obtained by mixing them. All solutions were filtered using a 0.22 μm filter before being added to the cells. Before adding the treatment solution, the cell monolayer was pretreated with the serum-free medium. After washing twice with 1xDPBS and pretreatment, the treatment solution was added to the 80%-90% confluent hBEC-5i monolayer. Thereafter, the cells were incubated at 37 °C under 5% CO2 conditions for 1 hour (see Figure 4).

[0102] All cells were collected after a single wash with DPBS after 1 h treatment. Total RNA content was extracted by the TRIzol method according to the manufacturer's protocol. The abundance of tight junction proteins (occludin, claudin 5, and ZO-1) mRNA was quantified using one-step reverse transcription real-time quantitative PCR using the QuantiTect SYBR Green RT-PCR Kit (Qiagen, Germany) on a Rotor-Gene Q Real-Time system (Qiagen, Germany). Gene primers were those used in the known Verma et al., 2009 (claudin 5 & occludin), and Jiang et al., 2017 (ZO-1 & GAPDH). Each reaction consisted of 50 ng of total RNA, 0.5 pmol of each primer, 5 μL of the one-step reaction mixture, and 0.1 μL of Quantiscript reverse transcriptase, and all were placed in a 10 μL reaction system for reaction. The reaction was carried out for 40 cycles at 50 °C for 30 min, 95 °C for 15 min, and 94 °C for 15 s, 58 °C for 30 s. Each sample analysis was performed 3 times to determine the average threshold cycle (Ct) value. Target gene expression was normalized with the GAPDH gene, and the relative amount of target gene mRNA was expressed as 2 -△△ct as described by the relative comparative threshold cycle method of the prior art (Voge et al., 2004; Lagaly et al., 2008; Grado-Ahuir et al., 2011). Non-template control groups and non-reverse transcriptase control groups were used in each PCR run to confirm the absence of genomic DNA contamination in the samples and mastermixes.

[0103] The expression of occludin (OCLN), a tight junction protein that plays an important role in determining the permeability of tight junctions, was evaluated. An increased expression of OCLN is known to lead to a tighter and less permeable tight junction (Hirase et al., 1997; Al-Sadi et al., 2011). The experimental results showed a higher expression level in all treatment groups compared to the control group, but a decrease in occludin expression was observed in the ChitTRP-treated experimental group compared to the experimental groups treated with chitosan and TRP (see Figure 5). Such a decrease in tight junction permeability means that it facilitates the absorption or passage of ChitTRP, and when TRP is combined with chitosan, it can penetrate the tight junctions of cells and increase the solubility by the plasma membrane.

[0104] Claudin 5 (CLDN5) is an important component of tight junctions that affects permeability. The expression of CLDN5 is known to be particularly important in brain endothelial cells of the blood-brain barrier (BBB) because it determines the molecules passing through the gate with a molecular mass greater than 800 Da (Lieu et al., 2012). It is known that the expression of CLDN5 in the ChitTRP-treated group is even lower compared to the groups treated with chitosan and TRP alone, which represents an increase in the permeability and ability of ChitTRP molecules to cross the BBB (see Figure 6). The high expression of CLDN5 in the TRP-alone treatment group suggests a decrease in permeability. Such results mean that chitosan and TRP interact to regulate the expression of CLDN5 in hBEC cells and improve the permeability of ChitTRP. It also means that the combination of chitosan and TRP can improve the availability of TRP to cross the plasma membrane by passing through the tight junctions.

[0105] The expression of ZO-1, a major auxiliary protein of blood-brain barrier tight junctions, depends on the expression of major proteins such as occludin and claudin 5. Changes in the expression of such proteins can affect ZO-1 expression. In this experimental example, hBEC-5i was treated with 200 μg of chitosan, TRP, and ChitTRP for 1 hour. The results show that the expression level of ZO-1 decreased due to changes in claudin 5 expression. In particular, the ChitTRP treatment group showed a decrease in ZO-1 mRNA level compared to the chitosan or TRP alone treatment groups (see Figure 7). However, this expression pattern was shown not to be affected by occludin expression.

[0106] Upon ChitTRP treatment, the permeability of the BBB increased, which was shown to be due to a decrease in the expression of the major tight junction proteins occludin, claudin-5, and ZO-1 compared to the control group. The temporary decrease in tight junction resistance and subsequent return to normal levels after ChitTRP treatment indicate that no permanent damage to integrity occurred and that the maximum possible availability of TRP in the BBB was demonstrated. As described above, it was confirmed that the BBB was more effectively penetrated by ChitTRP molecules compared to TRP alone, increasing the bioavailability of TRP to the brain.

[0107] Also, using BAC (Bovine Articular Chondrocytes), the relative expression concentrations of OCLN mRNA were measured and compared for the 10 treatment groups shown in Fig. 8 in the same manner as described above. As a result, it was confirmed that even when chitosan and TRP were mixed, there were differences in the expression levels of OCLN mRNA depending on the molecular weight of chitosan and the mixing weight ratio of chitosan and TRP. Specifically, a mixture of powdered chitosan and TRP showed the highest OCLN expression reduction rate when a chitosan powder containing LCH and SCH in a weight ratio of 0.5 to 0.7:0.3 to 0.5 was mixed with TRP powder in a weight ratio of 0.3 to 0.7:0.3 to 0.7. Also, a mixture of soluble chitosan and TRP showed the highest OCLN expression reduction rate when soluble chitosan containing LSCH, MSCH, and SSCH in a weight ratio of 0.1 to 0.3:0.5 to 0.8:0.1 to 0.3 was mixed with soluble TRP in a weight ratio of 0.3 to 0.7:0.3 to 0.7. Thus, it was confirmed that within the composition range of the present invention, when a chitosan-TRP mixture was produced, the expression of OCLN was significantly reduced, and by having further permeability with respect to tight junctions, the bioavailability of TRP was further increased.

[0108] Experimental Example 3. In vitro TEER (transendothelial electrical resistance) and immunofluorescence evaluation on human brain endothelial cells In vitro TEER evaluation

[0109] The in vitro BBB model was established using a Transwell insert (PTFE, 0.4 μm). hBEC-5i cells were seeded on the upper end of the filter and placed in a 24-well plate filled with 700 μL of fresh DMEM / F-12 medium containing 40 μM ECGS, 10% FBS, and 1% antibiotic solution. An additional 200 - 300 μL of the same medium was added to the insert culture area. After the cells reached confluence, they were cultured for 2 - 3 days. TEER (transendothelial electrical resistance) was measured using an EVOM2 (ERS-2, Millipore, Burlington, MA, USA) and STX2 chopstick electrodes. The TEER value of each hBEC monolayer was calculated by subtracting the control resistance (Ω, insert without cells) from the tested barrier resistance and then multiplying by the planting area of the insert in the 24-well plate (0.33 cm 2 ). When the TEER value reached the plateau phase, the treatment experiment was conducted (Wang et al. 2010 and Wu et al. 2020).

[0110] To evaluate the effect of ChitTRP molecules on the permeability of hBEC monolayers (BBB), the inserts were divided into four treatment groups, each replicated twice. The groups were as follows: Control group, containing only the medium; Chitosan group, hBEC-5i was exposed to 200 μg of chitosan; TRP group, hBEC-5i was exposed to 200 μg of TRP; ChitTRP group, hBEC-5i was exposed to 200 μg of ChitTRP. The treatments were applied for 1 hour, 6 hours, 24 hours, 48 hours, and 96 hours at 37 °C in a 5% CO2 environment. TEER values were recorded after each time point to determine hBEC cell permeability. The data were statistically analyzed and visually represented using Origin Pro2022 software.

[0111] As a BBB model, to evaluate the effect of ChitTRP (CTRP) on hBEC cells, TEER was measured in cells treated with chitosan, TRP, and ChitTRP compared to the control group. The experiment was repeated twice at other passages using hBEC cells, and slightly different TEER values were generated. After the cells were pretreated with serum-free medium, stable TEER values of 22.11 - 22.44 ± 0.23 Ωxcm 2 were shown, and then each formulation was treated.

[0112] In the first experiment, the control group initially showed a TEER value range of 21.12 - 22.44 ± 0.23 Ωxcm 2 and dropped to 15.18 - 16.5 ± 0.93 by 96 hours after treatment. When treated with chitosan and CTRP, the resistance decreased within 1 hour. Chitosan showed values of 12.87, powdered CTRP showed values of 14.52 - 16.5 ± 1.4, and liquid-form CTRP showed values of 13.86 - 16.17 ± 1.63. However, the tight junction integrity sequentially recovered to values similar to those of the control group, 15.51 - 18.81 ± 1.07 (see Figure 9). In the second experiment, the same trend was observed, and the initial TEER value was 38.78 - 42.74 ± 1.66 Ωxcm 2 . The group treated with TRP alone did not show a significant difference in TEER value compared to the control group (see Figure 10). Such results indicate that chitosan temporarily disassembles the BBB and increases permeability to improve the absorption rate of TRP. The said effect lasts for up to 6 hours with a peak at 1 hour, which means that the composition according to the present invention is safe for the BBB.

[0113] Immunofluorescence evaluation This experimental example was conducted using hBEC-5i seeded on a 4-well chamber slide coated with 1x adhesion factor protein (ThermoFisher Scientific). The hBEC-5i cells were cultured in a medium consisting of 1 mL of DMEM / F-12 50:50 supplemented with 10% FBS, ECGS, and 1% PSA, and incubated at 37 °C in a humidified atmosphere of 5% CO2 until confluence was reached. Thereafter, the cells were pretreated with a serum-free medium and then treated with various substances including medium alone (control group), 200 μg of chitosan, 200 μg of TRP, and 200 μg of ChitTRP for 1 hour.

[0114] After treatment, the cultures were fixed with 4% paraformaldehyde at 4 °C for 10 minutes and permeabilized with 0.1% Triton-X-100 in PBS for 10 minutes. Thereafter, the cells were blocked with 1% bovine serum albumin (BSA) for 30 minutes, incubated with primary antibodies against occludin, claudin-5, and ZO-1, and then incubated with Alexa Fluor 488-conjugated anti-rabbit secondary antibody in the dark for 1 hour. To ensure accuracy, the cells were counterstained with 1 μg / mL Hoechst 33342 in PBS according to a strict washing protocol between steps and mounted in a fluorescence mounting medium (Abcam). Observation was carried out using an Olympus BX53 surface fluorescence microscope.

[0115] The expression patterns of major tight junction proteins (occludin, claudin-5, and ZO-1) in hBEC-5i cells were analyzed using immunofluorescence images (see Figures 11-14). Figures 11 and 12 show the results of experiments with other passage hBEC cells. After detecting the target proteins bound to the primary antibody using Alexa Fluor-488-conjugated secondary antibody, they were observed with green fluorescence. Also, the cells were stained with Hoechst-33342 that stains the nucleus and observed with blue fluorescence. The results show that while the BBB structure is disrupted when treated with chitosan or chitosan-containing TRP formulations, cells treated with only TRP show a fluorescence pattern similar to the control group. The expression of occludin and claudin-5 is similar, and when treated with chitosan or chitosan-containing TRP (CTRP), the signal intensity decreases, indicating that chitosan can alter the expression of such major tight junction proteins and increase the permeability of the BBB. The expression of ZO-1 was higher in the control group compared to other proteins, but showed decreased expression when treated with chitosan-containing formulations. This confirmed that chitosan-containing TRP formulations can alter the BBB tight junction permeability and that TRP can improve the bioavailability of TRP in the brain for serotonin-melatonin synthesis towards the target site across the BBB.

[0116] As described above, specific parts of the present invention have been described in detail. It is clear that for those with ordinary knowledge in the art, such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention thereby. Therefore, it can be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A composition for improving the bioavailability of tryptophan, characterized by containing chitosan as an active ingredient.

2. The chitosan is chitosan powder or soluble chitosan, The chitosan powder contains LCH and SCH in a weight ratio of 0.5 to 0.7:0.3 to 0.

5. The LCH is chitosan powder with an average molecular weight exceeding 50,000 Da, and the SCH is chitosan powder with an average molecular weight less than 10,000 Da. The soluble chitosan contains LSCH, MSCH, and SSCH in a weight ratio of 0.1 to 0.3:0.5 to 0.8:0.1 to 0.

3. The LSCH is soluble chitosan with an average molecular weight exceeding 50,000 Da, the MSCH is soluble chitosan with an average molecular weight of 10,000 to 50,000 Da, and the SSCH is soluble chitosan with an average molecular weight less than 10,000 Da. The composition for improving the bioavailability of tryptophan according to claim 1, characterized by the above.

3. The composition according to claim 1, characterized in that it is used in combination with tryptophan to enhance the effect of tryptophan in improving sleep disorders.

4. The composition according to claim 3, characterized in that it is used in combination with tryptophan to enhance the permeability of tryptophan to the tight junction of the blood-brain barrier.

5. The composition according to claim 3, characterized in that it is used in combination with tryptophan to reduce the expression of one or more tight junction proteins selected from the group consisting of occludin, claudin 5, and ZO-1.

6. The chitosan is used in combination with tryptophan in a weight ratio of 0.3 to 0.7:0.3 to 0.

7. The composition for improving the bioavailability of tryptophan according to claim 3, characterized by the above.

7. The composition according to claim 3, characterized in that it is used in combination with tryptophan by simultaneous or sequential administration.

8. The composition according to claim 7, characterized in that it is used in combination with tryptophan by administering tryptophan within 1 hour after administration.

9. The composition according to claim 1, wherein the composition is a pharmaceutical composition or a food composition for improving the bioavailability of tryptophan.

10. A pharmaceutical composition for preventing or treating sleep disorders, characterized by containing chitosan and tryptophan as active ingredients.

11. The chitosan is chitosan powder or soluble chitosan, The chitosan powder contains LCH and SCH in a weight ratio of 0.5 to 0.7:0.3 to 0.

5. The LCH is chitosan powder with an average molecular weight exceeding 50,000 Da, and the SCH is chitosan powder with an average molecular weight of less than 10,000 Da. The soluble chitosan contains LSCH, MSCH, and SSCH in a weight ratio of 0.1 to 0.3:0.5 to 0.8:0.1 to 0.

3. The LSCH is soluble chitosan with an average molecular weight exceeding 50,000 Da, the MSCH is soluble chitosan with an average molecular weight of 10,000 to 50,000 Da, and the SSCH is soluble chitosan with an average molecular weight of less than 10,000 Da. The pharmaceutical composition for preventing or treating sleep disorders according to claim 10.

12. The pharmaceutical composition for preventing or treating sleep disorders according to claim 10, characterized in that the chitosan and tryptophan are mixed in a weight ratio of 0.3 to 0.7:0.3 to 0.

7.

13. The pharmaceutical composition for preventing or treating sleep disorders according to claim 10, characterized in that the composition enhances the permeability of tryptophan to the tight junction of the blood-brain barrier.

14. A food composition for improving sleep disorders, characterized by containing chitosan and tryptophan as active ingredients.

15. The chitosan is chitosan powder or soluble chitosan, The chitosan powder contains LCH and SCH at a weight ratio of 0.5 to 0.7:0.3 to 0.

5. The LCH is a chitosan powder with an average molecular weight exceeding 50,000 Da, and the SCH is a chitosan powder with an average molecular weight of less than 10,000 Da. The soluble chitosan contains LSCH, MSCH, and SSCH at a weight ratio of 0.1 to 0.3:0.5 to 0.8:0.1 to 0.

3. The LSCH is a soluble chitosan with an average molecular weight exceeding 50,000 Da, the MSCH is a soluble chitosan with an average molecular weight of 10,000 to 50,000 Da, and the SSCH is a soluble chitosan with an average molecular weight of less than 10,000 Da. The food composition for improving sleep disorders according to claim 14, characterized in that it is as described above.

16. The food composition for improving sleep disorders according to claim 14, characterized in that the chitosan and tryptophan are mixed at a weight ratio of 0.3 to 0.7:0.3 to 0.

7.

17. The food composition for improving sleep disorders according to claim 14, characterized in that the composition enhances the permeability of tryptophan to the tight junction of the blood-brain barrier.

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